KR20100003823A - Peptides for binding specifically to membrane embedded with aquaporin-2 and uses thereof - Google Patents
Peptides for binding specifically to membrane embedded with aquaporin-2 and uses thereof Download PDFInfo
- Publication number
- KR20100003823A KR20100003823A KR1020080063831A KR20080063831A KR20100003823A KR 20100003823 A KR20100003823 A KR 20100003823A KR 1020080063831 A KR1020080063831 A KR 1020080063831A KR 20080063831 A KR20080063831 A KR 20080063831A KR 20100003823 A KR20100003823 A KR 20100003823A
- Authority
- KR
- South Korea
- Prior art keywords
- membrane
- peptide
- polypeptide
- aqp2
- present
- Prior art date
Links
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 129
- 239000012528 membrane Substances 0.000 title claims abstract description 98
- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 34
- 230000027455 binding Effects 0.000 title claims description 36
- 102000011899 Aquaporin 2 Human genes 0.000 title description 81
- 108010036221 Aquaporin 2 Proteins 0.000 title description 80
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 53
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 41
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 41
- 208000030159 metabolic disease Diseases 0.000 claims abstract description 24
- 238000000034 method Methods 0.000 claims abstract description 24
- 238000001514 detection method Methods 0.000 claims abstract description 11
- 238000003384 imaging method Methods 0.000 claims abstract description 10
- 238000012377 drug delivery Methods 0.000 claims abstract description 8
- 239000000203 mixture Substances 0.000 claims description 27
- 229920001184 polypeptide Polymers 0.000 claims description 20
- 239000008194 pharmaceutical composition Substances 0.000 claims description 17
- 150000001413 amino acids Chemical group 0.000 claims description 15
- 239000004480 active ingredient Substances 0.000 claims description 14
- 239000002245 particle Substances 0.000 claims description 14
- 208000016097 disease of metabolism Diseases 0.000 claims description 13
- 239000013598 vector Substances 0.000 claims description 12
- 239000003795 chemical substances by application Substances 0.000 claims description 11
- 201000010064 diabetes insipidus Diseases 0.000 claims description 11
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 10
- 102100026383 Vasopressin-neurophysin 2-copeptin Human genes 0.000 claims description 9
- 201000010099 disease Diseases 0.000 claims description 8
- 239000000463 material Substances 0.000 claims description 8
- 230000005298 paramagnetic effect Effects 0.000 claims description 8
- 201000008284 inappropriate ADH syndrome Diseases 0.000 claims description 6
- 108091033319 polynucleotide Proteins 0.000 claims description 5
- 102000040430 polynucleotide Human genes 0.000 claims description 5
- 239000002157 polynucleotide Substances 0.000 claims description 5
- CGIGDMFJXJATDK-UHFFFAOYSA-N indomethacin Chemical compound CC1=C(CC(O)=O)C2=CC(OC)=CC=C2N1C(=O)C1=CC=C(Cl)C=C1 CGIGDMFJXJATDK-UHFFFAOYSA-N 0.000 claims description 4
- 239000002773 nucleotide Substances 0.000 claims description 4
- 125000003729 nucleotide group Chemical group 0.000 claims description 4
- 102000003979 Mineralocorticoid Receptors Human genes 0.000 claims description 3
- 108090000375 Mineralocorticoid Receptors Proteins 0.000 claims description 3
- 102000008873 Angiotensin II receptor Human genes 0.000 claims description 2
- 108050000824 Angiotensin II receptor Proteins 0.000 claims description 2
- 229940127291 Calcium channel antagonist Drugs 0.000 claims description 2
- 239000000480 calcium channel blocker Substances 0.000 claims description 2
- 229960000905 indomethacin Drugs 0.000 claims description 2
- 238000002156 mixing Methods 0.000 claims description 2
- 239000002571 phosphodiesterase inhibitor Substances 0.000 claims description 2
- 239000002089 prostaglandin antagonist Substances 0.000 claims description 2
- 239000003087 receptor blocking agent Substances 0.000 claims 2
- 229940099471 Phosphodiesterase inhibitor Drugs 0.000 claims 1
- 206010012601 diabetes mellitus Diseases 0.000 claims 1
- 230000003020 moisturizing effect Effects 0.000 claims 1
- 125000003259 prostaglandin group Chemical group 0.000 claims 1
- 239000002464 receptor antagonist Substances 0.000 claims 1
- 229940044551 receptor antagonist Drugs 0.000 claims 1
- 102000034573 Channels Human genes 0.000 abstract description 14
- 108091006146 Channels Proteins 0.000 abstract description 14
- 238000003745 diagnosis Methods 0.000 abstract description 8
- 102000010637 Aquaporins Human genes 0.000 abstract description 5
- 108010063290 Aquaporins Proteins 0.000 abstract description 4
- 230000004060 metabolic process Effects 0.000 abstract description 4
- 230000002265 prevention Effects 0.000 abstract description 3
- 125000003275 alpha amino acid group Chemical group 0.000 abstract 1
- 210000000170 cell membrane Anatomy 0.000 description 66
- 210000004027 cell Anatomy 0.000 description 39
- 235000018102 proteins Nutrition 0.000 description 37
- 230000003834 intracellular effect Effects 0.000 description 30
- 210000003734 kidney Anatomy 0.000 description 23
- 239000000523 sample Substances 0.000 description 23
- 108010004977 Vasopressins Proteins 0.000 description 19
- 102000002852 Vasopressins Human genes 0.000 description 19
- KBZOIRJILGZLEJ-LGYYRGKSSA-N argipressin Chemical compound C([C@H]1C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CSSC[C@@H](C(N[C@@H](CC=2C=CC(O)=CC=2)C(=O)N1)=O)N)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCN=C(N)N)C(=O)NCC(N)=O)C1=CC=CC=C1 KBZOIRJILGZLEJ-LGYYRGKSSA-N 0.000 description 19
- 229960003726 vasopressin Drugs 0.000 description 19
- 239000003446 ligand Substances 0.000 description 18
- 210000002472 endoplasmic reticulum Anatomy 0.000 description 16
- MLSVJHOYXJGGTR-IFHOVBQLSA-N acetic acid;(2s)-n-[(2r)-1-[(2-amino-2-oxoethyl)amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]-1-[(4r,7s,10s,13s,16s)-7-(2-amino-2-oxoethyl)-10-(3-amino-3-oxopropyl)-13-benzyl-16-[(4-hydroxyphenyl)methyl]-6,9,12,15,18-pentaoxo-1,2-dithia-5,8,11,14,1 Chemical compound CC(O)=O.C([C@H]1C(=O)N[C@H](C(N[C@@H](CC(N)=O)C(=O)N[C@@H](CSSCCC(=O)N[C@@H](CC=2C=CC(O)=CC=2)C(=O)N1)C(=O)N1[C@@H](CCC1)C(=O)N[C@H](CCCNC(N)=N)C(=O)NCC(N)=O)=O)CCC(=O)N)C1=CC=CC=C1 MLSVJHOYXJGGTR-IFHOVBQLSA-N 0.000 description 14
- 239000011324 bead Substances 0.000 description 11
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 11
- 108090000643 Vasopressin Receptors Proteins 0.000 description 10
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 10
- 239000002953 phosphate buffered saline Substances 0.000 description 10
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 9
- 102000004136 Vasopressin Receptors Human genes 0.000 description 9
- 238000003119 immunoblot Methods 0.000 description 9
- 230000005291 magnetic effect Effects 0.000 description 9
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 8
- 241000699666 Mus <mouse, genus> Species 0.000 description 8
- 241000700159 Rattus Species 0.000 description 8
- GXBMIBRIOWHPDT-UHFFFAOYSA-N Vasopressin Natural products N1C(=O)C(CC=2C=C(O)C=CC=2)NC(=O)C(N)CSSCC(C(=O)N2C(CCC2)C(=O)NC(CCCN=C(N)N)C(=O)NCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(CCC(N)=O)NC(=O)C1CC1=CC=CC=C1 GXBMIBRIOWHPDT-UHFFFAOYSA-N 0.000 description 8
- 102000004888 Aquaporin 1 Human genes 0.000 description 7
- 108090001004 Aquaporin 1 Proteins 0.000 description 7
- 239000000499 gel Substances 0.000 description 7
- 210000002700 urine Anatomy 0.000 description 7
- 108020004414 DNA Proteins 0.000 description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 6
- DNIAPMSPPWPWGF-UHFFFAOYSA-N Propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 6
- 229940098773 bovine serum albumin Drugs 0.000 description 6
- 210000000805 cytoplasm Anatomy 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 6
- 238000002347 injection Methods 0.000 description 6
- 239000007924 injection Substances 0.000 description 6
- 238000010186 staining Methods 0.000 description 6
- QFVHZQCOUORWEI-UHFFFAOYSA-N 4-[(4-anilino-5-sulfonaphthalen-1-yl)diazenyl]-5-hydroxynaphthalene-2,7-disulfonic acid Chemical compound C=12C(O)=CC(S(O)(=O)=O)=CC2=CC(S(O)(=O)=O)=CC=1N=NC(C1=CC=CC(=C11)S(O)(=O)=O)=CC=C1NC1=CC=CC=C1 QFVHZQCOUORWEI-UHFFFAOYSA-N 0.000 description 5
- 241000283973 Oryctolagus cuniculus Species 0.000 description 5
- 239000000969 carrier Substances 0.000 description 5
- 230000008859 change Effects 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 5
- 239000003814 drug Substances 0.000 description 5
- 238000002823 phage display Methods 0.000 description 5
- 239000011780 sodium chloride Substances 0.000 description 5
- BCFXQBXXDSEHRS-FXQIFTODSA-N Cys-Ser-Arg Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O BCFXQBXXDSEHRS-FXQIFTODSA-N 0.000 description 4
- 241000588724 Escherichia coli Species 0.000 description 4
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 4
- 241000699670 Mus sp. Species 0.000 description 4
- 108010067902 Peptide Library Proteins 0.000 description 4
- 229920002472 Starch Polymers 0.000 description 4
- 238000003556 assay Methods 0.000 description 4
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 4
- 210000000234 capsid Anatomy 0.000 description 4
- OSASVXMJTNOKOY-UHFFFAOYSA-N chlorobutanol Chemical compound CC(C)(O)C(Cl)(Cl)Cl OSASVXMJTNOKOY-UHFFFAOYSA-N 0.000 description 4
- 150000001875 compounds Chemical class 0.000 description 4
- 229940079593 drug Drugs 0.000 description 4
- 230000001404 mediated effect Effects 0.000 description 4
- 238000000926 separation method Methods 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 241001515965 unidentified phage Species 0.000 description 4
- MKMKILWCRQLDFJ-DCAQKATOSA-N Cys-Lys-Arg Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O MKMKILWCRQLDFJ-DCAQKATOSA-N 0.000 description 3
- AHZNUGRZHMZGFL-GUBZILKMSA-N Met-Arg-Ser Chemical compound CSCC[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](CO)C(O)=O)CCCNC(N)=N AHZNUGRZHMZGFL-GUBZILKMSA-N 0.000 description 3
- 206010028980 Neoplasm Diseases 0.000 description 3
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 3
- 229930006000 Sucrose Natural products 0.000 description 3
- HEJJDUDEHLPDAW-CUJWVEQBSA-N Thr-His-Cys Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)N[C@@H](CS)C(=O)O)N)O HEJJDUDEHLPDAW-CUJWVEQBSA-N 0.000 description 3
- YQYFYUSYEDNLSD-YEPSODPASA-N Val-Thr-Gly Chemical compound CC(C)[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(O)=O YQYFYUSYEDNLSD-YEPSODPASA-N 0.000 description 3
- 235000001014 amino acid Nutrition 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 201000011510 cancer Diseases 0.000 description 3
- 208000028235 central diabetes insipidus Diseases 0.000 description 3
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 3
- 235000018417 cysteine Nutrition 0.000 description 3
- 239000003937 drug carrier Substances 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000001378 electrochemiluminescence detection Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 3
- 230000003993 interaction Effects 0.000 description 3
- 230000037361 pathway Effects 0.000 description 3
- 230000035699 permeability Effects 0.000 description 3
- 108010054624 red fluorescent protein Proteins 0.000 description 3
- 230000001105 regulatory effect Effects 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 230000028327 secretion Effects 0.000 description 3
- 239000008107 starch Substances 0.000 description 3
- 235000019698 starch Nutrition 0.000 description 3
- 239000005720 sucrose Substances 0.000 description 3
- 230000001225 therapeutic effect Effects 0.000 description 3
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 2
- 108091006112 ATPases Proteins 0.000 description 2
- 102000057290 Adenosine Triphosphatases Human genes 0.000 description 2
- HULHGJZIZXCPLD-FXQIFTODSA-N Arg-Ala-Cys Chemical compound C[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CCCN=C(N)N)N HULHGJZIZXCPLD-FXQIFTODSA-N 0.000 description 2
- FOQFHANLUJDQEE-GUBZILKMSA-N Arg-Pro-Cys Chemical compound C1C[C@H](N(C1)C(=O)[C@H](CCCN=C(N)N)N)C(=O)N[C@@H](CS)C(=O)O FOQFHANLUJDQEE-GUBZILKMSA-N 0.000 description 2
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- 244000063299 Bacillus subtilis Species 0.000 description 2
- 235000014469 Bacillus subtilis Nutrition 0.000 description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- QADHATDBZXHRCA-ACZMJKKPSA-N Cys-Gln-Asn Chemical compound C(CC(=O)N)[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)[C@H](CS)N QADHATDBZXHRCA-ACZMJKKPSA-N 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- KKBWDNZXYLGJEY-UHFFFAOYSA-N Gly-Arg-Pro Natural products NCC(=O)NC(CCNC(=N)N)C(=O)N1CCCC1C(=O)O KKBWDNZXYLGJEY-UHFFFAOYSA-N 0.000 description 2
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 2
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 2
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 2
- 206010062767 Hypophysitis Diseases 0.000 description 2
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 2
- 208000003450 Neurogenic Diabetes Insipidus Diseases 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 2
- 241000589516 Pseudomonas Species 0.000 description 2
- BKOKTRCZXRIQPX-ZLUOBGJFSA-N Ser-Ala-Cys Chemical compound C[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CO)N BKOKTRCZXRIQPX-ZLUOBGJFSA-N 0.000 description 2
- 241000191940 Staphylococcus Species 0.000 description 2
- 241000187747 Streptomyces Species 0.000 description 2
- 239000007983 Tris buffer Substances 0.000 description 2
- 239000013504 Triton X-100 Substances 0.000 description 2
- 229920004890 Triton X-100 Polymers 0.000 description 2
- 239000000443 aerosol Substances 0.000 description 2
- 239000011543 agarose gel Substances 0.000 description 2
- 239000001045 blue dye Substances 0.000 description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 2
- 239000001913 cellulose Substances 0.000 description 2
- 235000010980 cellulose Nutrition 0.000 description 2
- 229920002678 cellulose Polymers 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 229960004926 chlorobutanol Drugs 0.000 description 2
- 230000021615 conjugation Effects 0.000 description 2
- 238000011109 contamination Methods 0.000 description 2
- 108010082025 cyan fluorescent protein Proteins 0.000 description 2
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 description 2
- 239000008121 dextrose Substances 0.000 description 2
- 208000035475 disorder Diseases 0.000 description 2
- 239000002612 dispersion medium Substances 0.000 description 2
- 238000004043 dyeing Methods 0.000 description 2
- 210000003027 ear inner Anatomy 0.000 description 2
- 238000004520 electroporation Methods 0.000 description 2
- 108010048367 enhanced green fluorescent protein Proteins 0.000 description 2
- 239000012091 fetal bovine serum Substances 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 2
- 239000005090 green fluorescent protein Substances 0.000 description 2
- 210000003016 hypothalamus Anatomy 0.000 description 2
- 230000001771 impaired effect Effects 0.000 description 2
- 238000001990 intravenous administration Methods 0.000 description 2
- 239000008101 lactose Substances 0.000 description 2
- 239000002502 liposome Substances 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000011068 loading method Methods 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 238000000520 microinjection Methods 0.000 description 2
- 230000005012 migration Effects 0.000 description 2
- 238000013508 migration Methods 0.000 description 2
- 238000010369 molecular cloning Methods 0.000 description 2
- 201000005119 neurohypophyseal diabetes insipidus Diseases 0.000 description 2
- LXCFILQKKLGQFO-UHFFFAOYSA-N p-hydroxybenzoic acid methyl ester Natural products COC(=O)C1=CC=C(O)C=C1 LXCFILQKKLGQFO-UHFFFAOYSA-N 0.000 description 2
- 239000008188 pellet Substances 0.000 description 2
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 2
- 150000003904 phospholipids Chemical class 0.000 description 2
- 210000003635 pituitary gland Anatomy 0.000 description 2
- 229920001223 polyethylene glycol Polymers 0.000 description 2
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 2
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 2
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 239000003755 preservative agent Substances 0.000 description 2
- QELSKZZBTMNZEB-UHFFFAOYSA-N propylparaben Chemical compound CCCOC(=O)C1=CC=C(O)C=C1 QELSKZZBTMNZEB-UHFFFAOYSA-N 0.000 description 2
- 238000011002 quantification Methods 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- GEHJYWRUCIMESM-UHFFFAOYSA-L sodium sulfite Chemical compound [Na+].[Na+].[O-]S([O-])=O GEHJYWRUCIMESM-UHFFFAOYSA-L 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 238000012453 sprague-dawley rat model Methods 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 210000002536 stromal cell Anatomy 0.000 description 2
- 238000007920 subcutaneous administration Methods 0.000 description 2
- 235000000346 sugar Nutrition 0.000 description 2
- 230000008685 targeting Effects 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 2
- 108091005957 yellow fluorescent proteins Proteins 0.000 description 2
- LNAZSHAWQACDHT-XIYTZBAFSA-N (2r,3r,4s,5r,6s)-4,5-dimethoxy-2-(methoxymethyl)-3-[(2s,3r,4s,5r,6r)-3,4,5-trimethoxy-6-(methoxymethyl)oxan-2-yl]oxy-6-[(2r,3r,4s,5r,6r)-4,5,6-trimethoxy-2-(methoxymethyl)oxan-3-yl]oxyoxane Chemical compound CO[C@@H]1[C@@H](OC)[C@H](OC)[C@@H](COC)O[C@H]1O[C@H]1[C@H](OC)[C@@H](OC)[C@H](O[C@H]2[C@@H]([C@@H](OC)[C@H](OC)O[C@@H]2COC)OC)O[C@@H]1COC LNAZSHAWQACDHT-XIYTZBAFSA-N 0.000 description 1
- VZQHRKZCAZCACO-PYJNHQTQSA-N (2s)-2-[[(2s)-2-[2-[[(2s)-2-[[(2s)-2-amino-5-(diaminomethylideneamino)pentanoyl]amino]propanoyl]amino]prop-2-enoylamino]-3-methylbutanoyl]amino]propanoic acid Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](C(C)C)NC(=O)C(=C)NC(=O)[C@H](C)NC(=O)[C@@H](N)CCCNC(N)=N VZQHRKZCAZCACO-PYJNHQTQSA-N 0.000 description 1
- DDMOUSALMHHKOS-UHFFFAOYSA-N 1,2-dichloro-1,1,2,2-tetrafluoroethane Chemical compound FC(F)(Cl)C(F)(F)Cl DDMOUSALMHHKOS-UHFFFAOYSA-N 0.000 description 1
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 description 1
- MUSGYEMSJUFFHT-UWABRSFTSA-N 2-[(4R,7S,10S,13S,19S,22S,25S,28S,31S,34R)-34-[[(2S,3S)-2-[[(2R)-2-amino-3-(4-hydroxyphenyl)propanoyl]amino]-3-methylpentanoyl]amino]-4-[[(2S,3S)-1-amino-3-methyl-1-oxopentan-2-yl]-methylcarbamoyl]-25-(3-amino-3-oxopropyl)-7-(3-carbamimidamidopropyl)-10-(1H-imidazol-5-ylmethyl)-19-(1H-indol-3-ylmethyl)-13,17-dimethyl-28-[(1-methylindol-3-yl)methyl]-6,9,12,15,18,21,24,27,30,33-decaoxo-31-propan-2-yl-1,2-dithia-5,8,11,14,17,20,23,26,29,32-decazacyclopentatriacont-22-yl]acetic acid Chemical compound CC[C@H](C)[C@H](NC(=O)[C@H](N)Cc1ccc(O)cc1)C(=O)N[C@H]1CSSC[C@H](NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](Cc2cnc[nH]2)NC(=O)[C@H](C)NC(=O)CN(C)C(=O)[C@H](Cc2c[nH]c3ccccc23)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](Cc2cn(C)c3ccccc23)NC(=O)[C@@H](NC1=O)C(C)C)C(=O)N(C)[C@@H]([C@@H](C)CC)C(N)=O MUSGYEMSJUFFHT-UWABRSFTSA-N 0.000 description 1
- 101150047512 AQP2 gene Proteins 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 241000589158 Agrobacterium Species 0.000 description 1
- 241000589155 Agrobacterium tumefaciens Species 0.000 description 1
- QUIGLPSHIFPEOV-CIUDSAMLSA-N Ala-Lys-Ala Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(O)=O QUIGLPSHIFPEOV-CIUDSAMLSA-N 0.000 description 1
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 1
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 1
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 1
- 102000004363 Aquaporin 3 Human genes 0.000 description 1
- 108090000991 Aquaporin 3 Proteins 0.000 description 1
- 102000012002 Aquaporin 4 Human genes 0.000 description 1
- 108010036280 Aquaporin 4 Proteins 0.000 description 1
- MUXONAMCEUBVGA-DCAQKATOSA-N Arg-Arg-Gln Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCC(N)=O)C(O)=O MUXONAMCEUBVGA-DCAQKATOSA-N 0.000 description 1
- RWWPBOUMKFBHAL-FXQIFTODSA-N Arg-Asn-Cys Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CS)C(O)=O RWWPBOUMKFBHAL-FXQIFTODSA-N 0.000 description 1
- AMIQZQAAYGYKOP-FXQIFTODSA-N Arg-Ser-Asn Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(O)=O AMIQZQAAYGYKOP-FXQIFTODSA-N 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- OBMZMSLWNNWEJA-XNCRXQDQSA-N C1=CC=2C(C[C@@H]3NC(=O)[C@@H](NC(=O)[C@H](NC(=O)N(CC#CCN(CCCC[C@H](NC(=O)[C@@H](CC4=CC=CC=C4)NC3=O)C(=O)N)CC=C)NC(=O)[C@@H](N)C)CC3=CNC4=C3C=CC=C4)C)=CNC=2C=C1 Chemical compound C1=CC=2C(C[C@@H]3NC(=O)[C@@H](NC(=O)[C@H](NC(=O)N(CC#CCN(CCCC[C@H](NC(=O)[C@@H](CC4=CC=CC=C4)NC3=O)C(=O)N)CC=C)NC(=O)[C@@H](N)C)CC3=CNC4=C3C=CC=C4)C)=CNC=2C=C1 OBMZMSLWNNWEJA-XNCRXQDQSA-N 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 206010010071 Coma Diseases 0.000 description 1
- 238000011537 Coomassie blue staining Methods 0.000 description 1
- 229920002261 Corn starch Polymers 0.000 description 1
- IVOMOUWHDPKRLL-KQYNXXCUSA-N Cyclic adenosine monophosphate Chemical compound C([C@H]1O2)OP(O)(=O)O[C@H]1[C@@H](O)[C@@H]2N1C(N=CN=C2N)=C2N=C1 IVOMOUWHDPKRLL-KQYNXXCUSA-N 0.000 description 1
- AMRLSQGGERHDHJ-FXQIFTODSA-N Cys-Ala-Arg Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O AMRLSQGGERHDHJ-FXQIFTODSA-N 0.000 description 1
- XGIAHEUULGOZHH-GUBZILKMSA-N Cys-Arg-Val Chemical compound CC(C)[C@@H](C(=O)O)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CS)N XGIAHEUULGOZHH-GUBZILKMSA-N 0.000 description 1
- VIRYODQIWJNWNU-NRPADANISA-N Cys-Glu-Val Chemical compound CC(C)[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CS)N VIRYODQIWJNWNU-NRPADANISA-N 0.000 description 1
- XTHUKRLJRUVVBF-WHFBIAKZSA-N Cys-Gly-Ser Chemical compound SC[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O XTHUKRLJRUVVBF-WHFBIAKZSA-N 0.000 description 1
- HBHMVBGGHDMPBF-GARJFASQSA-N Cys-Leu-Pro Chemical compound CC(C)C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CS)N HBHMVBGGHDMPBF-GARJFASQSA-N 0.000 description 1
- LHMSYHSAAJOEBL-CIUDSAMLSA-N Cys-Lys-Asn Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(O)=O LHMSYHSAAJOEBL-CIUDSAMLSA-N 0.000 description 1
- HMWBPUDETPKSSS-DCAQKATOSA-N Cys-Pro-Lys Chemical compound C1C[C@H](N(C1)C(=O)[C@H](CS)N)C(=O)N[C@@H](CCCCN)C(=O)O HMWBPUDETPKSSS-DCAQKATOSA-N 0.000 description 1
- MHYHLWUGWUBUHF-GUBZILKMSA-N Cys-Val-Arg Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)NC(=O)[C@H](CS)N MHYHLWUGWUBUHF-GUBZILKMSA-N 0.000 description 1
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 1
- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 241000006271 Discosoma sp. Species 0.000 description 1
- 206010014418 Electrolyte imbalance Diseases 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 239000004386 Erythritol Substances 0.000 description 1
- UNXHWFMMPAWVPI-UHFFFAOYSA-N Erythritol Natural products OCC(O)C(O)CO UNXHWFMMPAWVPI-UHFFFAOYSA-N 0.000 description 1
- 206010061126 Escherichia infection Diseases 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 208000018522 Gastrointestinal disease Diseases 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- ZFADFBPRMSBPOT-KKUMJFAQSA-N Gln-Arg-Phe Chemical compound N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](Cc1ccccc1)C(O)=O ZFADFBPRMSBPOT-KKUMJFAQSA-N 0.000 description 1
- 108010024636 Glutathione Proteins 0.000 description 1
- POJJAZJHBGXEGM-YUMQZZPRSA-N Gly-Ser-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)CN POJJAZJHBGXEGM-YUMQZZPRSA-N 0.000 description 1
- 101001027128 Homo sapiens Fibronectin Proteins 0.000 description 1
- 101000619708 Homo sapiens Peroxiredoxin-6 Proteins 0.000 description 1
- 206010021036 Hyponatraemia Diseases 0.000 description 1
- 108020005350 Initiator Codon Proteins 0.000 description 1
- 241000087799 Koma Species 0.000 description 1
- 239000012741 Laemmli sample buffer Substances 0.000 description 1
- 241000880493 Leptailurus serval Species 0.000 description 1
- OYQUOLRTJHWVSQ-SRVKXCTJSA-N Leu-His-Ser Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CO)C(O)=O OYQUOLRTJHWVSQ-SRVKXCTJSA-N 0.000 description 1
- QMKFDEUJGYNFMC-AVGNSLFASA-N Leu-Pro-Arg Chemical compound CC(C)C[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCCN=C(N)N)C(O)=O QMKFDEUJGYNFMC-AVGNSLFASA-N 0.000 description 1
- GDBQQVLCIARPGH-UHFFFAOYSA-N Leupeptin Natural products CC(C)CC(NC(C)=O)C(=O)NC(CC(C)C)C(=O)NC(C=O)CCCN=C(N)N GDBQQVLCIARPGH-UHFFFAOYSA-N 0.000 description 1
- VHFFQUSNFFIZBT-CIUDSAMLSA-N Lys-Ala-Cys Chemical compound C[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CCCCN)N VHFFQUSNFFIZBT-CIUDSAMLSA-N 0.000 description 1
- KNKHAVVBVXKOGX-JXUBOQSCSA-N Lys-Ala-Thr Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O KNKHAVVBVXKOGX-JXUBOQSCSA-N 0.000 description 1
- CFVQPNSCQMKDPB-CIUDSAMLSA-N Lys-Cys-Cys Chemical compound C(CCN)C[C@@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CS)C(=O)O)N CFVQPNSCQMKDPB-CIUDSAMLSA-N 0.000 description 1
- TVOOGUNBIWAURO-KATARQTJSA-N Lys-Thr-Cys Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CCCCN)N)O TVOOGUNBIWAURO-KATARQTJSA-N 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- DLAFCQWUMFMZSN-GUBZILKMSA-N Met-Arg-Ala Chemical compound CSCC[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](C)C(O)=O)CCCN=C(N)N DLAFCQWUMFMZSN-GUBZILKMSA-N 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- YBAFDPFAUTYYRW-UHFFFAOYSA-N N-L-alpha-glutamyl-L-leucine Natural products CC(C)CC(C(O)=O)NC(=O)C(N)CCC(O)=O YBAFDPFAUTYYRW-UHFFFAOYSA-N 0.000 description 1
- 201000005118 Nephrogenic diabetes insipidus Diseases 0.000 description 1
- 239000000020 Nitrocellulose Substances 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 101710176384 Peptide 1 Proteins 0.000 description 1
- 108010033276 Peptide Fragments Proteins 0.000 description 1
- 102000007079 Peptide Fragments Human genes 0.000 description 1
- 102100022239 Peroxiredoxin-6 Human genes 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- DIFXZGPHVCIVSQ-CIUDSAMLSA-N Pro-Gln-Ser Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(O)=O DIFXZGPHVCIVSQ-CIUDSAMLSA-N 0.000 description 1
- 108010076504 Protein Sorting Signals Proteins 0.000 description 1
- 241000588769 Proteus <enterobacteria> Species 0.000 description 1
- 241000588770 Proteus mirabilis Species 0.000 description 1
- 241000700157 Rattus norvegicus Species 0.000 description 1
- FCRMLGJMPXCAHD-FXQIFTODSA-N Ser-Arg-Asn Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(N)=O)C(O)=O FCRMLGJMPXCAHD-FXQIFTODSA-N 0.000 description 1
- GZSZPKSBVAOGIE-CIUDSAMLSA-N Ser-Lys-Ala Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(O)=O GZSZPKSBVAOGIE-CIUDSAMLSA-N 0.000 description 1
- FPCGZYMRFFIYIH-CIUDSAMLSA-N Ser-Lys-Ser Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(O)=O FPCGZYMRFFIYIH-CIUDSAMLSA-N 0.000 description 1
- BEBVVQPDSHHWQL-NRPADANISA-N Ser-Val-Glu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O BEBVVQPDSHHWQL-NRPADANISA-N 0.000 description 1
- DWAQJAXMDSEUJJ-UHFFFAOYSA-M Sodium bisulfite Chemical compound [Na+].OS([O-])=O DWAQJAXMDSEUJJ-UHFFFAOYSA-M 0.000 description 1
- 244000061458 Solanum melongena Species 0.000 description 1
- 235000002597 Solanum melongena Nutrition 0.000 description 1
- 108020005038 Terminator Codon Proteins 0.000 description 1
- GSEJCLTVZPLZKY-UHFFFAOYSA-N Triethanolamine Chemical compound OCCN(CCO)CCO GSEJCLTVZPLZKY-UHFFFAOYSA-N 0.000 description 1
- KXIQQAWIPDDVOE-BPUTZDHNSA-N Trp-Pro-Cys Chemical compound O=C([C@H](CC=1C2=CC=CC=C2NC=1)N)N1CCC[C@H]1C(=O)N[C@@H](CS)C(O)=O KXIQQAWIPDDVOE-BPUTZDHNSA-N 0.000 description 1
- 206010048302 Tubulointerstitial nephritis Diseases 0.000 description 1
- IVOMOUWHDPKRLL-UHFFFAOYSA-N UNPD107823 Natural products O1C2COP(O)(=O)OC2C(O)C1N1C(N=CN=C2N)=C2N=C1 IVOMOUWHDPKRLL-UHFFFAOYSA-N 0.000 description 1
- MANXHLOVEUHVFD-DCAQKATOSA-N Val-His-Cys Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)N[C@@H](CS)C(=O)O)N MANXHLOVEUHVFD-DCAQKATOSA-N 0.000 description 1
- UKEVLVBHRKWECS-LSJOCFKGSA-N Val-Ile-Gly Chemical compound CC[C@H](C)[C@@H](C(=O)NCC(=O)O)NC(=O)[C@H](C(C)C)N UKEVLVBHRKWECS-LSJOCFKGSA-N 0.000 description 1
- DIOSYUIWOQCXNR-ONGXEEELSA-N Val-Lys-Gly Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)NCC(O)=O DIOSYUIWOQCXNR-ONGXEEELSA-N 0.000 description 1
- TVXBFESIOXBWNM-UHFFFAOYSA-N Xylitol Natural products OCCC(O)C(O)C(O)CCO TVXBFESIOXBWNM-UHFFFAOYSA-N 0.000 description 1
- 230000003187 abdominal effect Effects 0.000 description 1
- 230000005856 abnormality Effects 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- DPXJVFZANSGRMM-UHFFFAOYSA-N acetic acid;2,3,4,5,6-pentahydroxyhexanal;sodium Chemical compound [Na].CC(O)=O.OCC(O)C(O)C(O)C(O)C=O DPXJVFZANSGRMM-UHFFFAOYSA-N 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 229940023476 agar Drugs 0.000 description 1
- 235000010419 agar Nutrition 0.000 description 1
- 235000010443 alginic acid Nutrition 0.000 description 1
- 239000000783 alginic acid Substances 0.000 description 1
- 229920000615 alginic acid Polymers 0.000 description 1
- 229960001126 alginic acid Drugs 0.000 description 1
- 150000004781 alginic acids Chemical class 0.000 description 1
- 230000000172 allergic effect Effects 0.000 description 1
- WNROFYMDJYEPJX-UHFFFAOYSA-K aluminium hydroxide Chemical compound [OH-].[OH-].[OH-].[Al+3] WNROFYMDJYEPJX-UHFFFAOYSA-K 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 239000003146 anticoagulant agent Substances 0.000 description 1
- 229940127219 anticoagulant drug Drugs 0.000 description 1
- 229940121375 antifungal agent Drugs 0.000 description 1
- 239000003429 antifungal agent Substances 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 108010069926 arginyl-glycyl-serine Proteins 0.000 description 1
- 235000010323 ascorbic acid Nutrition 0.000 description 1
- 239000011668 ascorbic acid Substances 0.000 description 1
- 229960005070 ascorbic acid Drugs 0.000 description 1
- 239000012298 atmosphere Substances 0.000 description 1
- 208000010668 atopic eczema Diseases 0.000 description 1
- 239000013602 bacteriophage vector Substances 0.000 description 1
- 239000000022 bacteriostatic agent Substances 0.000 description 1
- 229960000686 benzalkonium chloride Drugs 0.000 description 1
- CADWTSSKOVRVJC-UHFFFAOYSA-N benzyl(dimethyl)azanium;chloride Chemical compound [Cl-].C[NH+](C)CC1=CC=CC=C1 CADWTSSKOVRVJC-UHFFFAOYSA-N 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 229960000074 biopharmaceutical Drugs 0.000 description 1
- 230000036772 blood pressure Effects 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 229960004424 carbon dioxide Drugs 0.000 description 1
- 239000001768 carboxy methyl cellulose Substances 0.000 description 1
- 229940082638 cardiac stimulant phosphodiesterase inhibitors Drugs 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 239000002738 chelating agent Substances 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 239000008120 corn starch Substances 0.000 description 1
- 229940099112 cornstarch Drugs 0.000 description 1
- 239000013601 cosmid vector Substances 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 229940095074 cyclic amp Drugs 0.000 description 1
- 238000012303 cytoplasmic staining Methods 0.000 description 1
- 210000003104 cytoplasmic structure Anatomy 0.000 description 1
- 230000001086 cytosolic effect Effects 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- 238000002716 delivery method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 1
- UMNKXPULIDJLSU-UHFFFAOYSA-N dichlorofluoromethane Chemical compound FC(Cl)Cl UMNKXPULIDJLSU-UHFFFAOYSA-N 0.000 description 1
- 229940099364 dichlorofluoromethane Drugs 0.000 description 1
- 229940087091 dichlorotetrafluoroethane Drugs 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 229940042399 direct acting antivirals protease inhibitors Drugs 0.000 description 1
- 239000007884 disintegrant Substances 0.000 description 1
- 238000002224 dissection Methods 0.000 description 1
- 208000002173 dizziness Diseases 0.000 description 1
- 239000008298 dragée Substances 0.000 description 1
- 210000001755 duct epithelial cell Anatomy 0.000 description 1
- 230000004064 dysfunction Effects 0.000 description 1
- 239000003995 emulsifying agent Substances 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 210000001062 endolymphatic sac Anatomy 0.000 description 1
- JPGQOUSTVILISH-UHFFFAOYSA-N enflurane Chemical compound FC(F)OC(F)(F)C(F)Cl JPGQOUSTVILISH-UHFFFAOYSA-N 0.000 description 1
- 229960000305 enflurane Drugs 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 201000010063 epididymitis Diseases 0.000 description 1
- 206010015037 epilepsy Diseases 0.000 description 1
- 210000002919 epithelial cell Anatomy 0.000 description 1
- UNXHWFMMPAWVPI-ZXZARUISSA-N erythritol Chemical compound OC[C@H](O)[C@H](O)CO UNXHWFMMPAWVPI-ZXZARUISSA-N 0.000 description 1
- 235000019414 erythritol Nutrition 0.000 description 1
- 229940009714 erythritol Drugs 0.000 description 1
- 208000020612 escherichia coli infection Diseases 0.000 description 1
- BEFDCLMNVWHSGT-UHFFFAOYSA-N ethenylcyclopentane Chemical compound C=CC1CCCC1 BEFDCLMNVWHSGT-UHFFFAOYSA-N 0.000 description 1
- 230000029142 excretion Effects 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000000799 fluorescence microscopy Methods 0.000 description 1
- 108010021843 fluorescent protein 583 Proteins 0.000 description 1
- 108091006047 fluorescent proteins Proteins 0.000 description 1
- 102000034287 fluorescent proteins Human genes 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 239000012737 fresh medium Substances 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 239000007903 gelatin capsule Substances 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 102000034356 gene-regulatory proteins Human genes 0.000 description 1
- 108091006104 gene-regulatory proteins Proteins 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 229960003180 glutathione Drugs 0.000 description 1
- 108010089804 glycyl-threonine Proteins 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 239000003906 humectant Substances 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 239000001866 hydroxypropyl methyl cellulose Substances 0.000 description 1
- 235000010979 hydroxypropyl methyl cellulose Nutrition 0.000 description 1
- 229920003088 hydroxypropyl methyl cellulose Polymers 0.000 description 1
- UFVKGYZPFZQRLF-UHFFFAOYSA-N hydroxypropyl methyl cellulose Chemical compound OC1C(O)C(OC)OC(CO)C1OC1C(O)C(O)C(OC2C(C(O)C(OC3C(C(O)C(O)C(CO)O3)O)C(CO)O2)O)C(CO)O1 UFVKGYZPFZQRLF-UHFFFAOYSA-N 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 238000003125 immunofluorescent labeling Methods 0.000 description 1
- 239000003547 immunosorbent Substances 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 210000001439 intercalated cell Anatomy 0.000 description 1
- 201000006334 interstitial nephritis Diseases 0.000 description 1
- 210000004020 intracellular membrane Anatomy 0.000 description 1
- 230000010189 intracellular transport Effects 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- FZWBNHMXJMCXLU-BLAUPYHCSA-N isomaltotriose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1OC[C@@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@@H](OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O)O1 FZWBNHMXJMCXLU-BLAUPYHCSA-N 0.000 description 1
- 239000007951 isotonicity adjuster Substances 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- GDBQQVLCIARPGH-ULQDDVLXSA-N leupeptin Chemical compound CC(C)C[C@H](NC(C)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C=O)CCCN=C(N)N GDBQQVLCIARPGH-ULQDDVLXSA-N 0.000 description 1
- 108010052968 leupeptin Proteins 0.000 description 1
- 210000005229 liver cell Anatomy 0.000 description 1
- 239000000314 lubricant Substances 0.000 description 1
- 210000004937 luminal membrane Anatomy 0.000 description 1
- 239000000845 maltitol Substances 0.000 description 1
- VQHSOMBJVWLPSR-WUJBLJFYSA-N maltitol Chemical compound OC[C@H](O)[C@@H](O)[C@@H]([C@H](O)CO)O[C@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O VQHSOMBJVWLPSR-WUJBLJFYSA-N 0.000 description 1
- 235000010449 maltitol Nutrition 0.000 description 1
- 229940035436 maltitol Drugs 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000002483 medication Methods 0.000 description 1
- HEBKCHPVOIAQTA-UHFFFAOYSA-N meso ribitol Natural products OCC(O)C(O)C(O)CO HEBKCHPVOIAQTA-UHFFFAOYSA-N 0.000 description 1
- 229920000609 methyl cellulose Polymers 0.000 description 1
- 235000010270 methyl p-hydroxybenzoate Nutrition 0.000 description 1
- 239000004292 methyl p-hydroxybenzoate Substances 0.000 description 1
- 239000001923 methylcellulose Substances 0.000 description 1
- 235000010981 methylcellulose Nutrition 0.000 description 1
- 229960002216 methylparaben Drugs 0.000 description 1
- 239000011325 microbead Substances 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 210000001589 microsome Anatomy 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 210000003470 mitochondria Anatomy 0.000 description 1
- 239000003068 molecular probe Substances 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 239000002105 nanoparticle Substances 0.000 description 1
- 239000006199 nebulizer Substances 0.000 description 1
- 230000002988 nephrogenic effect Effects 0.000 description 1
- 229920001220 nitrocellulos Polymers 0.000 description 1
- 230000009871 nonspecific binding Effects 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 239000003921 oil Substances 0.000 description 1
- 229940094443 oxytocics prostaglandins Drugs 0.000 description 1
- 238000004091 panning Methods 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 1
- 238000010647 peptide synthesis reaction Methods 0.000 description 1
- 239000002304 perfume Substances 0.000 description 1
- 102000013415 peroxidase activity proteins Human genes 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 229960003742 phenol Drugs 0.000 description 1
- 108010083476 phenylalanyltryptophan Proteins 0.000 description 1
- 239000006187 pill Substances 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 230000008488 polyadenylation Effects 0.000 description 1
- 229920005862 polyol Polymers 0.000 description 1
- 150000003077 polyols Chemical class 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 210000001883 posterior pituitary gland Anatomy 0.000 description 1
- 229920001592 potato starch Polymers 0.000 description 1
- 229940116317 potato starch Drugs 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000002335 preservative effect Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 239000003380 propellant Substances 0.000 description 1
- 235000010232 propyl p-hydroxybenzoate Nutrition 0.000 description 1
- 239000004405 propyl p-hydroxybenzoate Substances 0.000 description 1
- 229960003415 propylparaben Drugs 0.000 description 1
- 150000003180 prostaglandins Chemical class 0.000 description 1
- 238000000159 protein binding assay Methods 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 239000000941 radioactive substance Substances 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- YVSWPCCVTYEEHG-UHFFFAOYSA-N rhodamine B 5-isothiocyanate Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=C(N=C=S)C=C1C(O)=O YVSWPCCVTYEEHG-UHFFFAOYSA-N 0.000 description 1
- 229940100486 rice starch Drugs 0.000 description 1
- 230000007727 signaling mechanism Effects 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 235000010413 sodium alginate Nutrition 0.000 description 1
- 239000000661 sodium alginate Substances 0.000 description 1
- 229940005550 sodium alginate Drugs 0.000 description 1
- 235000019812 sodium carboxymethyl cellulose Nutrition 0.000 description 1
- 229920001027 sodium carboxymethylcellulose Polymers 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 235000010267 sodium hydrogen sulphite Nutrition 0.000 description 1
- 235000010265 sodium sulphite Nutrition 0.000 description 1
- 235000010199 sorbic acid Nutrition 0.000 description 1
- 239000004334 sorbic acid Substances 0.000 description 1
- 229940075582 sorbic acid Drugs 0.000 description 1
- 235000010356 sorbitol Nutrition 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 239000012192 staining solution Substances 0.000 description 1
- 210000000130 stem cell Anatomy 0.000 description 1
- 239000008227 sterile water for injection Substances 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 239000000375 suspending agent Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000002459 sustained effect Effects 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- 239000006188 syrup Substances 0.000 description 1
- 235000020357 syrup Nutrition 0.000 description 1
- 239000003826 tablet Substances 0.000 description 1
- 229940124597 therapeutic agent Drugs 0.000 description 1
- 239000002562 thickening agent Substances 0.000 description 1
- RTKIYNMVFMVABJ-UHFFFAOYSA-L thimerosal Chemical compound [Na+].CC[Hg]SC1=CC=CC=C1C([O-])=O RTKIYNMVFMVABJ-UHFFFAOYSA-L 0.000 description 1
- 229940033663 thimerosal Drugs 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 102000035160 transmembrane proteins Human genes 0.000 description 1
- 108091005703 transmembrane proteins Proteins 0.000 description 1
- 230000032258 transport Effects 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- CYRMSUTZVYGINF-UHFFFAOYSA-N trichlorofluoromethane Chemical compound FC(Cl)(Cl)Cl CYRMSUTZVYGINF-UHFFFAOYSA-N 0.000 description 1
- 229940029284 trichlorofluoromethane Drugs 0.000 description 1
- 229960004418 trolamine Drugs 0.000 description 1
- 230000002485 urinary effect Effects 0.000 description 1
- 108010073969 valyllysine Proteins 0.000 description 1
- 235000015112 vegetable and seed oil Nutrition 0.000 description 1
- 239000008158 vegetable oil Substances 0.000 description 1
- 239000003981 vehicle Substances 0.000 description 1
- 239000013603 viral vector Substances 0.000 description 1
- 235000012431 wafers Nutrition 0.000 description 1
- 229940100445 wheat starch Drugs 0.000 description 1
- 239000000811 xylitol Substances 0.000 description 1
- 235000010447 xylitol Nutrition 0.000 description 1
- HEBKCHPVOIAQTA-SCDXWVJYSA-N xylitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)CO HEBKCHPVOIAQTA-SCDXWVJYSA-N 0.000 description 1
- 229960002675 xylitol Drugs 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/34—Genitourinary disorders
- G01N2800/347—Renal failures; Glomerular diseases; Tubulointerstitial diseases, e.g. nephritic syndrome, glomerulonephritis; Renovascular diseases, e.g. renal artery occlusion, nephropathy
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Hematology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Organic Chemistry (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- Epidemiology (AREA)
- Microbiology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Pharmacology & Pharmacy (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Genetics & Genomics (AREA)
- Animal Behavior & Ethology (AREA)
- Biophysics (AREA)
- Food Science & Technology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Peptides Or Proteins (AREA)
Abstract
본 발명은 제2형 수분통로단백이 포배된 막과 특이적으로 결합하는 펩타이드 및 이의 용도에 관한 것으로, 보다 상세하게는 체내 수분대사에 중요한 작용을 하는 신장 집합관 주세포에서 제2형 수분통로단백이 포배된 막에 선택적으로 결합하는 펩타이드 및 이를 이용하여, 상기 막의 검출 및 수분 대사질환의 진단, 예방, 치료방법에 관한 것이다. 본 발명의 서열번호 1 내지 서열번호 16로 표시되는 아미노산 서열을 가지는 펩타이드는 제2형 수분통로단백이 포배된 막과 특이적으로 결합할 수 있다. 따라서, 본 발명의 펩타이드는 제2형 수분통로단백이 포배된 막의 검출, 더 나아가 수분 대사 질환의 진단, 영상화 및 표적 약물전달 등에 다양하게 사용될 수 있다.The present invention relates to a peptide that specifically binds a membrane cultured with a type 2 moisture channel protein and its use, and more particularly, a type 2 water channel protein in renal collecting duct main cells that play an important role in the body's water metabolism. Peptide that selectively binds to the membrane membrane, and using the same, the present invention relates to a method for the detection of the membrane and the diagnosis, prevention, treatment of water metabolic diseases. Peptides having an amino acid sequence represented by SEQ ID NO: 1 to SEQ ID NO: 16 of the present invention can specifically bind to the membrane cultivated type 2 water passage protein. Therefore, the peptide of the present invention can be used for various purposes such as detection of membranes in which the type 2 moisture channel protein is cultivated, furthermore, diagnosis of water metabolism disorders, imaging, and targeted drug delivery.
Description
본 발명은 제2형 수분통로단백이 포배된 막과 특이적으로 결합하는 펩타이드 및 이의 용도에 관한 것으로, 보다 상세하게는 체내 수분대사에 중요한 작용을 하는 신장 집합관 주세포에서 제2형 수분통로단백이 포배된 막에 선택적으로 결합하는 펩타이드 및 이를 이용하여, 상기 막의 검출 및 수분 대사질환의 진단, 예방, 치료방법에 관한 것이다.The present invention relates to a peptide that specifically binds a membrane cultured with a type 2 moisture channel protein and its use, and more particularly, a type 2 water channel protein in renal collecting duct main cells that play an important role in the body's water metabolism. Peptide that selectively binds to the membrane membrane, and using the same, the present invention relates to a method for the detection of the membrane and the diagnosis, prevention, treatment of water metabolic diseases.
파지디스플레이 기법은 타깃 (예, 단백질, 세포, 조직, 등)에 선택적인 결합능을 가지는 펩타이드 리간드를 빠르게 쉽게 분리하기 위해 고안된 혁신적인 기술로서 최근에 널리 이용되고 있다(Hoogenboom HR, Methods Mol Biol 178: 1-37, 2002.; Smith GP, Curr Opin Biotechnol 2: 668-673, 1991. ). 파지디스플레이 기술은 광범위한 펩타이드 라이브러리가 표면에 발현되는 박테리오파지를 이용하며, 리간드 펩티도믹스(ligand peptidomics), 단백질 간의 새로운 상호작용 발견, 항체 공학, 치료제 전달방법, 이미징 프로브 개발과 같이 진단과 치료기술 개발 등의 영역에서 선택적인 결합력을 가지는 펩타이드 리간드를 분리하기 위해 사용된다(Lee SM et al., Mol Cancer Res 5: 11-19, 2007.; Pasqualini R and Ruoslahti E. Nature 380: 364-366, 1996.; Petty NK et al., Trends Biotechnol 25: 7-15, 2007.; Rothe A et al., FASEB J 20: 1599-1610, 2006.). 파지 디스플레이는 in vitro에서 높은 특이성 및 결합능을 가지는 광범위한 펩티드 리간드를 발현할 수 있도록 대장균에서 증식이 가능한 박테리오파지를 사용한다. 파지 파티클의 표면에 표시되는 이러한 리간드는 어떠한 목표에 대해서도 선별할 수 있다(Petty NK et al., Trends Biotechnol 25: 7-15, 2007.; Rothe A et al., FASEB J 20: 1599-1610, 2006.).Phage display technology has recently been widely used as an innovative technique designed to quickly and easily isolate peptide ligands with selective binding capacity to targets (eg, proteins, cells, tissues, etc.) (Hoogenboom HR, Methods Mol Biol 178: 1 -37, 2002 .; Smith GP, Curr Opin Biotechnol 2: 668-673, 1991.). Phage display technology utilizes bacteriophages with a wide range of peptide libraries on the surface and develops diagnostic and therapeutic technologies such as ligand peptidomics, discovery of new interactions between proteins, antibody engineering, therapeutic delivery methods, and imaging probe development. It is used to separate peptide ligands having selective binding ability in the region of the back (Lee SM et al., Mol Cancer Res 5: 11-19, 2007 .; Pasqualini R and Ruoslahti E. Nature 380: 364-366, 1996 .; Petty NK et al., Trends Biotechnol 25: 7-15, 2007 .; Rothe A et al., FASEB J 20: 1599-1610, 2006.). Phage display uses bacteriophages capable of propagation in E. coli to express a wide range of peptide ligands with high specificity and binding capacity in vitro. Such ligands displayed on the surface of phage particles can be screened for any target (Petty NK et al., Trends Biotechnol 25: 7-15, 2007 .; Rothe A et al., FASEB J 20: 1599-1610, 2006.).
신장의 집합관 주세포 내강면막 (apical plasma membrane)의 수분투과도 조절은 신장에서 수분배출, 요농축, 그리고 체내 수분 균형 조절에 필수적인 요소이다. 수분통로단백은 생체세포막에서 수분의 선택적인 투과를 담당하는 중요한 단백질이다(Knepper MA et al., Semin Nephrol 14: 302-321, 1994.; Nielsen S et al., Physiol Rev 82: 205-244, 2002.). 이중 제 2형 수분통로단백 (aquaporin-2: AQP2)은 신장의 집합관 주세포 내강면막에서 발현되며 항이뇨호르몬(vasopressin)에 의해 조절되고 있다(Fushimi K et al., Nature 361: 549-552, 1993.). 항이뇨호르몬은 집합관 상피세포의 수분투과도를 빠르게 증가시킨다. 이 반응은 항이뇨 호르몬이 주세포 기저외측막 (basolateral plasma membrane)에 발현하는 V2-수용체와 결 합하여 집합관 주세포의 세포질에서 cAMP에 의존적으로 AQP2가 발현되는 소포체가 내강면막으로 이동됨으로서 매개된다(Knepper MA et al., Semin Nephrol 14: 302-321, 1994.; Nielsen S et al., Proc Natl Acad Sci U S A 92: 1013-1017, 1995.; Nielsen S et al., Physiol Rev 82: 205-244, 2002.). Moisture permeability control of the apical plasma membrane of the renal ducts is essential for renal excretion, urinary concentration, and body balance. Moisture pathway proteins are important proteins responsible for the selective permeation of moisture in living cell membranes (Knepper MA et al., Semin) . Nephrol 14: 302-321, 1994 .; Nielsen S et al., Physiol Rev 82: 205-244, 2002.). The type 2 water channel protein (aquaporin-2: AQP2) is expressed in the renal tubular main cell lumen and is regulated by vasopressin (Fushimi K et al., Nature 361: 549-552, 1993. Antidiuretic hormone rapidly increases the water permeability of the collecting duct epithelial cells. This reaction is mediated by the binding of the antidiuretic hormone to the V2-receptor, which is expressed on the basolateral plasma membrane, and the transport of vesicles expressing AQP2 to the luminal membrane in the cytoplasm of the collecting duct's main cell (Knepper MA). et al., Semin Nephrol 14: 302-321, 1994 .; Nielsen S et al., Proc Natl Acad Sci USA 92: 1013-1017, 1995 .; Nielsen S et al., Physiol Rev 82: 205-244, 2002.).
이러한 과정을 보다 상세히 설명하면, 바소프레신(vasopressin)으로 불리우는 항이뇨 호르몬(antidiuretic hormone, ADH)은 혈장 삼투질 농도의 증가 또는 혈장량 감소(volume depletion)에 반응하여 뇌하수체 후엽에서 분비된다. 바소프레신 수용체는 V1 수용체와 V2 수용체로 나뉘며 V1 수용체는 혈관 등에 분포하여 혈압 상승 등에 관여하는 반면, V2 수용체는 집합관의 기저외측막에 분포하여 물의 흡수와 소변 농축에 관여한다. 바소프레신이 집합관의 기저외측막의 V2 수용체에 결합하면 세포내 cyclic AMP의 농도가 증가하고 수분통로 AQP2가 인산화(phsophorylation)된다. 인산화된 AQP2-함유 소포(AQP2-containing vesicle)가 내강면막에 삽입되면 수분 통로가 열리고 물이 집합관 세포 내로 들어온 후 다시 기저외측막의 AQP3와 AQP4를 통해 속수질 간질로 흡수된다. 바소프레신의 자극이 없어지면 AQP2 함유 소포는 다시 내강면막 아래의 세포질로 되돌아간다. In more detail, the antidiuretic hormone (ADH), called vasopressin, is secreted from the posterior pituitary gland in response to an increase in plasma osmolality or a decrease in plasma volume. Vasopressin receptors are divided into V1 and V2 receptors, and V1 receptors are distributed in blood vessels and the like to increase blood pressure, while V2 receptors are distributed on the basal outer membrane of the collecting duct and are involved in water absorption and urine concentration. When vasopressin binds to the V2 receptor of the basolateral membrane of the collecting duct, the concentration of intracellular cyclic AMP is increased and water channel AQP2 is phosphorylated (phsophorylation). When phosphorylated AQP2-containing vesicles are inserted into the lumen, the water passages open, water enters the collecting duct cells, and is then absorbed into the stromal epilepsy via AQP3 and AQP4 in the basolateral membrane. After vasopressin stimulation disappears, AQP2-containing vesicles revert back to the cytoplasm below the lumen.
비록 이러한 현상은 이미 잘 알려져 있지만, AQP2를 발현하는 소포체와 세포막에서 이러한 AQP2 세포내 수송을 조절하는 조절 단백질이나 관여 단백질의 동정 및 단백질 간의 상호작용은 잘 알려져 있지 않다.Although this phenomenon is well known, the identification and interaction of regulatory proteins or involved proteins that regulate this AQP2 intracellular transport in cell membranes expressing AQP2 are not well known.
이에 본 발명자들은 AQP2가 포배된 막에 특이적으로 결합하는 새로운 단백질 또는 그 단편을 탐색하고자 연구한 결과, 서열번호 1 내지 서열번호 16으로 표시되는 아미노산 서열을 가지는 펩타이드가 이에 특이적으로 표적됨을 확인함으로써 본 발명을 완성하였다. Accordingly, the present inventors have studied to search for a new protein or fragment thereof that specifically binds to the membrane in which AQP2 is entrapped. As a result, the peptide having the amino acid sequence represented by SEQ ID NO: 1 to SEQ ID NO: 16 is specifically identified. By this, the present invention was completed.
따라서 본 발명의 목적은 제2형 수분통로단백이 포배된 막을 특이적으로 표적하는 펩타이드 및 이의 용도를 제공하는 것이다.It is therefore an object of the present invention to provide peptides and their use which specifically target membranes in which the type 2 water passage protein is cultivated.
상기와 같은 목적을 달성하기 위하여, 본 발명은 서열번호 1 내지 서열번호 16으로 이루어진 군에서 선택된 아미노산 서열을 가지며, 제2형 수분통로단백(aquaporin-2, AQP2)이 포배된(embeded) 막을 특이적으로 표적하는 펩타이드를 제공한다.In order to achieve the above object, the present invention has an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 16, specific for a membrane embedded with a type 2 water passage protein (aquaporin-2, AQP2) To provide a targeting peptide.
본 발명의 다른 목적을 달성하기 위하여, 본 발명은 상기 펩타이드를 암호화하는 폴리뉴클레오타이드를 제공한다.In order to achieve another object of the present invention, the present invention provides a polynucleotide encoding the peptide.
본 발명의 또다른 목적을 달성하기 위하여, 본 발명은 상기 펩타이드를 유효성분으로 포함하는 제2형 수분통로단백이 포배된 막 검출용 조성물을 제공한다.In order to achieve another object of the present invention, the present invention provides a composition for detecting a membrane cultivated type 2 water passage protein containing the peptide as an active ingredient.
본 발명의 또다른 목적을 달성하기 위하여, 본 발명은 상기 펩타이드를 유효성분으로 포함하는 약물 전달용 조성물을 제공한다.In order to achieve another object of the present invention, the present invention provides a drug delivery composition comprising the peptide as an active ingredient.
본 발명의 또다른 목적을 달성하기 위하여, 본 발명은 상기 펩타이드 및 이와 결합된 수분 대사 질환 제제를 유효성분으로 포함하는 수분 대사 질환 예방 및 치료용 약학적 조성물을 제공한다.In order to achieve another object of the present invention, the present invention provides a pharmaceutical composition for preventing and treating water metabolic disease comprising the peptide and a water metabolic disease agent coupled thereto as an active ingredient.
본 발명의 또다른 목적을 달성하기 위하여, 본 발명은 상기 펩타이드를 유효 성분으로 포함하는 수분 대사 질환 부위의 영상화용 조성물을 제공한다.In order to achieve another object of the present invention, the present invention provides a composition for imaging a water metabolic disease site comprising the peptide as an active ingredient.
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명자는 파지디스플레이 기법을 이용하여 실험쥐(Sprague-Dawley rat)의 신장에서 AQP2가 발현하는 집합관 주세포 세포막 혹은 주세포내 소포체막에 선택적으로 결합하는 펩타이드 리간드를 발굴하고자 하였다. 또한 비록 짧은 펩타이드 시퀀스로 인해 무작위 매칭 (random match) 가능성이 높지만, 발굴한 선택적 펩타이드 리간드의 시퀀스를 토대로 시퀀스 상동성 가지는 단백질들을 검색하였다. 이러한 단백질들은 항이뇨호르몬에 의한 AQP2의 세포막으로의 이동과 AQP2의 세포내 단백 발현조절에 단백질간의 상호작용 또는 다른 신호전달 기전을 통해 유기적으로 조절하고 있을 것으로 기대된다. The present inventors attempted to find a peptide ligand that selectively binds to a collection tube main cell membrane or intracellular vesicle membrane expressing AQP2 in the kidney of Sprague-Dawley rat using phage display technique. In addition, although the likelihood of random matching is high due to the short peptide sequence, the proteins having sequence homology were searched based on the sequence of the selected selective peptide ligand. These proteins are expected to be organically regulated through the interaction of proteins or other signaling mechanisms in the migration of AQP2 into the cell membrane by antidiuretic hormone and in the regulation of intracellular protein expression of AQP2.
이러한 목적으로 본 발명자들은 1) 무작위로 광범위한 펩타이드 라이브러리가 표현된 박테리오파지를 이용하여 실험쥐 신장에서 AQP2를 발현하는 세포막과 세포내 소포체막에 결합시켜, 선택적이며 특이적으로 결합하는 펩타이드 리간드 7 종을 발현하는 파지 클론을 발굴하였으며, 2) 다시 이들 파지클론들이 과연 선택적 결합을 이루어지는지를 검증하기 위해, 박테리오파지 표면에 펩타이드 라이브러리가 표현되지 않은 T7 파지와 비교하여 AQP2가 발현하는 세포막 또는 세포내 소포체막에 상대적인 선택 결합력을 비교하였고, 3) 또한 실험 쥐 신장의 집합관에서 주 세포가 아닌 또 다른 세포인 사이세포 (intercalated cell)에만 발현하는 H+-ATPase(B1-subunit)가 발현되는 세포막과 소포체막을 분리하고 결합시켜 결합능을 비교 조사하는 대조군 실험을 실시하였다. 4) 발굴한 펩타이드 리간드의 세포내 위치를 알아보기 위해 형광물질 (FITC)을 결합시킨 인공적인 펩타이드를 합성하고 실험쥐 신장에서 일차배양한 속수질 집합관세포에서 염색해 보았고, 항이뇨호르몬인 dDAVP (10-8M, 20분)를 단기간 처리하여 염색의 세포내 위치변화가 유도되는지를 살펴보았다. 그리고 5) 발굴한 선택적 펩타이드 리간드 7종에 각각 시퀀스 상동성을 가진 단백질들을 라이브러리 분석을 통해 선별하였다. To this end, the present inventors 1) using a bacteriophage with a randomly expressed peptide library to bind to AQP2 expressing cell membranes and intracellular vesicle membranes in mouse kidneys, and to selectively and specifically bind 7 peptide ligands. Phage clones expressing were excavated, and 2) in order to verify whether these phage clones are indeed selective-binding, compared to T7 phages without peptide libraries expressed on the surface of the bacteriophage, on the cell membrane or intracellular vesicle membrane expressing AQP2. The relative selective binding ability was compared. 3) In addition, the cell membrane and the vesicle membrane expressing H + -ATPase (B1-subunit) expressing only intercalated cells, which are not main cells, were separated from the collecting rat kidney. A control experiment was conducted in which the binding capacity was compared and examined. . 4) To find the intracellular location of the discovered peptide ligand, we synthesized an artificial peptide conjugated with fluorescent material (FITC) and stained the medullary collecting tube cells primaryly cultured in the kidneys of mice, and dDAVP (antidiuretic hormone) 10 -8 M, 20 min) was examined for a short period of time to see if the change in the intracellular location of the stain is induced. And 5) proteins with sequence homology to each of the seven selected selective peptide ligands were selected by library analysis.
따라서, 본 발명은 서열번호 1 내지 서열번호 16, 특히 서열번호 1 내지 서열번호 7로 표시되는 아미노산을 가지는 폴리펩타이드가 제2형 수분통로단백이 포배된 막에 특이적으로 결합한다는 점에 착안하여 서열번호 1 내지 서열번호 16으로 이루어진 군에서 선택된 아미노산을 가지는, 새로운 서열의 폴리펩타이드와 이의 용도로서 상기 폴리펩타이드를 포함하는 제2형 수분통로단백이 포배된 막 검출용 조성물 등을 제공하는 것을 특징으로 한다.Accordingly, the present invention focuses on the fact that a polypeptide having an amino acid represented by SEQ ID NO: 1 to SEQ ID NO: 16, in particular SEQ ID NO: 1 to SEQ ID NO: 7, specifically binds to a membrane in which a type 2 moisture channel protein is cultivated. It provides a polypeptide of the new sequence having an amino acid selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 16 and the composition for detecting the membrane cultivated type 2 moisture passage protein containing the polypeptide as the use thereof. It is done.
본 발명의 펩타이드는 제2형 수분통로단백이 포배된 막에 특이적으로 결합하며, 서열번호 1 내지 서열번호 16으로 이루어진 군에서 선택된 아미노산 서열을 가지는 펩타이드를 말한다. 상기 펩타이드는 바람직하게는 서열번호 1 내지 서열번호 7로 이루어진 군에서 선택된 아미노산 서열을 가질 수 있다. 본 발명의 펩타이드 단편은 모든 종류의 펩타이드, 단백질, 펩타이드 모조물, 화합물 및 생물제제를 포함하며, 제2형 수분통로단백이 포배된 막에 특이적으로 결합할 수 있는 활성을 갖는 것을 말한다. 본 발명의 펩타이드는 천연으로부터 유래될 수도 있으며, 공지의 펩타이드 합성 방법을 이용하여 합성될 수 있다. The peptide of the present invention specifically refers to a peptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 16 specifically binds to the membrane of the type 2 water passage protein. The peptide may preferably have an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 7. The peptide fragment of the present invention includes all kinds of peptides, proteins, peptide replicas, compounds, and biologics, and refers to a type 2 water passage protein having an activity that can specifically bind to an entrapped membrane. The peptide of the present invention may be derived from nature, and may be synthesized using known peptide synthesis methods.
상기에서 제2형 수분통로단백이 포배된 막은 제2형 수분통로단백이 막(membrane)에 관통된 형태로 함유하는 것을 의미한다. 제2형 수분통로단백은 그 구조상 막관통 단백질(transmembrane protein)이므로 수용액상에서 막관통부위가 소수성 물질과 잘 결합하게 되며, 인지질 층과 같은 막구조를 형성하는 구조체의 내부에 포배되어 안정한 형태를 유지하게 된다. 상기 막은 바람직하게는 인지질 층으로 된 생체막이며, 더 바람직하게는 세포막, 원형질막, 세포내막, 세포내 소포체막일 수 있다.The membrane in which the type 2 moisture channel protein is cultivated means that the type 2 moisture channel protein is penetrated into the membrane. Since the type 2 moisture pathway protein is a transmembrane protein in its structure, the membrane-penetrating portion binds well with a hydrophobic substance in an aqueous solution, and it is trapped inside a structure forming a membrane structure such as a phospholipid layer, thereby maintaining a stable form. Done. The membrane is preferably a biofilm consisting of a phospholipid layer, more preferably a cell membrane, a plasma membrane, an intracellular membrane, or an intracellular vesicle membrane.
아울러, 본 발명은 본 발명의 펩타이드를 암호화하는 염기서열을 가지는 폴리뉴클레오타이드를 제공한다. 상기 폴리뉴클레오타이드는 결과적으로 본 발명의 펩타이드를 암호화할 수 있는 어떤 조합의 염기서열도 가능하다.In addition, the present invention provides a polynucleotide having a nucleotide sequence encoding the peptide of the present invention. As a result, the polynucleotide may be any combination of base sequences capable of encoding the peptide of the present invention.
아울러, 본 발명은 본 발명의 펩타이드를 암호화하는 염기서열을 가지는 벡터 및 상기 벡터로 형질전환된 형질전환체를 제공한다. In addition, the present invention provides a vector having a nucleotide sequence encoding the peptide of the present invention and a transformant transformed with the vector.
본 발명의 벡터는 플라스미드 벡터, 코즈미드 벡터, 박테리오파아지 벡터 및 바이러스 벡터 등을 포함하나 이에 제한되지 않는다. 본 발명의 벡터는 통상의 클로닝 벡터 또는 발현벡터일 수 있으며, 발현벡터는 프로모터, 오퍼레이터, 개시코돈, 종결코돈, 폴리아데닐화 시그널 및 인핸서(촉진유전자) 같은 발현 조절 서열 외에도 막 표적화 또는 분비를 위한 시그널 서열 또는 리더 서열을 포함하며 목적에 따라 다양하게 제조될 수 있다. 또한 상기 벡터는 벡터를 함유하는 숙주 세포를 선택하기 위한 선택 마커를 포함하고, 복제 가능한 벡터인 경우 복제 기원을 포함한다. Vectors of the invention include, but are not limited to, plasmid vectors, cosmid vectors, bacteriophage vectors, viral vectors, and the like. Vectors of the present invention may be conventional cloning vectors or expression vectors, which may be used for membrane targeting or secretion in addition to expression control sequences such as promoters, operators, initiation codons, termination codons, polyadenylation signals and enhancers (promoter). It includes a signal sequence or leader sequence and can be prepared in various ways depending on the purpose. The vector also includes a selection marker for selecting a host cell containing the vector and, in the case of a replicable vector, the origin of replication.
상기 벡터로 형질전환하는 것은 당업자에게 공지된 형질전환기술에 의해 수행될 수 있다. 바람직하게는 미세사출법(microprojectile bombardment), 전기충격유전자전달법(electroporation), 인산 칼슘(CaPO4) 침전, 염화 칼슘(CaCl2) 침전, PEG-매개 융합법(PEG-mediated fusion), 미세주입법(microinjection) 및 리포좀 매개법(liposome-mediated method)를 이용할 수 있으며, 상기 형질전환체는 대장균(Escherichia coli), 바실러스 서브틸리스(Bacillus subtilis), 스트렙토마이세스(Streptomyces), 슈도모나스(Pseudomonas), 프로테우스 미라빌리스(Proteus mirabilis), 스타필로코쿠스( Staphylococcus), 아그로박테리움 투메파시엔스(Agrobacterium tumefaciens)일 수 있으나, 이로 제한되는 것은 아니다.Transformation with the vector can be carried out by transformation techniques known to those skilled in the art. Preferably, microprojectile bombardment, electroporation, electroporation, calcium phosphate (CaPO 4 ) precipitation, calcium chloride (CaCl 2 ) precipitation, PEG-mediated fusion, microinjection (microinjection) and liposome-mediated methods can be used, and the transformant is Escherichia coli ), Bacillus Subtilis ( Bacillus subtilis), Streptomyces (Streptomyces), Pseudomonas (Pseudomonas), Proteus Mira Billy's (Proteus mirabilis), Staphylococcus (Staphylococcus), Agrobacterium Tome Pacific Enschede (Agrobacterium tumefaciens ), but is not limited thereto.
본 발명자들은 제2형 수분통로단백이 포배된 막에 특이적으로 결합하는 것으로 선별된 상기 펩타이드의 기능을 확인하고자 다양한 실험을 수행한 결과, 본 발명의 펩타이드가 제2형 수분통로단백이 포배된 막에 특이적으로 인식하여 결합한다는 사실을 확인하였다. 따라서, 본 발명의 펩타이드를 제2형 수분통로단백이 포배된 막의 검출용 조성물로 이용할 수 있음을 알 수 있었으며, 더 나아가 수분 대사 질환의 진단 또는 치료추적용 제제, 또는 별도의 수분 대사 질환 치료용 제제와 더불어 수분 대사 질환 예방 및 치료용 약학적 조성물 등으로서 사용할 수 있음을 알 수 있었다.The present inventors conducted various experiments to confirm the function of the peptide selected as specifically binding to the membrane culturing the type 2 moisture channel protein, the peptide of the present invention is cultured type 2 moisture channel protein It was confirmed that the specific recognition and binding to the membrane. Therefore, it was found that the peptide of the present invention can be used as a composition for detecting a membrane cultivated with the type 2 moisture channel protein, and furthermore, for the diagnosis or treatment of water metabolic diseases, or for the treatment of separate water metabolic diseases. In addition to the formulation, it can be seen that it can be used as a pharmaceutical composition for preventing and treating water metabolic diseases.
보다 구체적으로 본 발명의 일 실시예에서는 상용의 T7 파지 라이브러리를 이용하여 제2형 수분통로단백이 포배된 막과 특이적으로 결합하는 파지를 각각 스크리닝하였다. 그 결과, 스크리닝을 통해 상기 막과 특이적으로 결합하는 파지를 각각 선별할 수 있었으며, 이의 서열을 분석한 결과 서열번호 1(CPKQRFWPC), 서열번호 2(CKRVTGRPC), 서열번호 3(CKNMRSSAC), 서열번호 4(CLPMRAKCC), 서열번호 5(CSRSRNKTC), 서열번호 6(CSRSKATHC) 및 서열번호 7(CARVKGTHC)의 아미노산 서열을 가지는 펩타이드가 주로 선별됨을 알 수 있었다. 아울러, 이들과 유사한 서열로, 서열번호 2에 대해서는 서열번호 8(CSRVTGRPC), 서열번호 9(CKRVTGRAC), 서열번호 10(CKRVIGRNC), 서열번호 3 및 서열번호 4에 대해서는 서열번호 11(CQNMRSSAC), 서열번호 12(CQNMRSKAC), 서열번호 5 내지 서열번호 7에 대해서는 서열번호 13(CGSRSNRAC), 서열번호 14(CRVKATH), 서열번호 15(CVRAKATHC) 및 서열번호 16(CSRSKSVHC)이 선별됨을 알 수 있었다.More specifically, in one embodiment of the present invention using a commercially available T7 phage library, each of the phages that specifically bind to the membrane of the type 2 moisture passage protein cultivated. As a result, each of the phages that specifically bind to the membrane was screened through screening. As a result of analyzing the phages, SEQ ID NO: 1 (CPKQRFWPC), SEQ ID NO: 2 (CKRVTGRPC), SEQ ID NO: 3 (CKNMRSSAC), and sequence Peptides having amino acid sequences of SEQ ID NO: 4 (CLPMRAKCC), SEQ ID NO: 5 (CSRSRNKTC), SEQ ID NO: 6 (CSRSKATHC), and SEQ ID NO: 7 (CARVKGTHC) were mainly selected. In addition, similar sequences to these include SEQ ID NO: 8 (CSRVTGRPC) for SEQ ID NO: 2, SEQ ID NO: 9 (CKRVTGRAC), SEQ ID NO: 10 (CKRVIGRNC), SEQ ID NO: 11 (CQNMRSSAC) for SEQ ID NO: 4, As for SEQ ID NO: 12 (CQNMRSKAC), SEQ ID NO: 5 to SEQ ID NO: 7, SEQ ID NO: 13 (CGSRSNRAC), SEQ ID NO: 14 (CRVKATH), SEQ ID NO: 15 (CVRAKATHC) and SEQ ID NO: 16 (CSRSKSVHC) were selected.
본 발명의 다른 실시예에서는 선별된 상기 펩타이드에 FITC를 결합하고, 이를 항이뇨호르몬(dDAVP)의 처리에 따른 속수질 집합관(IMCD) 세포에 결합여부를 확인하였다. 그 결과, 대조군 펩타이드에 비해서 본 발명의 펩타이드가 속수질 집합관의 세포막 또는 세포질내의 막에 잘 결합함을 알 수 있었다.In another embodiment of the present invention, FITC was bound to the selected peptides, and it was confirmed whether they were bound to the stromal collecting duct (IMCD) cells according to the treatment of antidiuretic hormone (dDAVP). As a result, it was found that the peptide of the present invention binds to the membrane of the stromal collecting duct or the membrane of the cytoplasm better than the control peptide.
결론적으로, 본 발명의 펩타이드가 제2형 수분통로단백이 포배된 막과 특이적으로 결합하여 생체 내에서 제2형 수분통로단백이 포배된 막의 인식 및 표적을 할 수 있음을 알 수 있었다. In conclusion, it can be seen that the peptide of the present invention specifically binds to the membrane cultured with the type 2 moisture channel protein and can recognize and target the type 2 membrane with the type 2 moisture channel protein in vivo.
참고로, 상기에서 언급한 뉴클레오타이드 및 단백질 작업에는 다음의 문헌을 참조할 수 있다(Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.(1982); Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory Press(1989); Deutscher, M., Guide to Protein Purification Methods Enzymology, vol. 182. Academic Press. Inc., San Diego, CA(1990)). For reference, reference may be made to the above-mentioned nucleotide and protein operations (Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982); Sambrook et al. ., Molecular Cloning: A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory Press (1989); Deutscher, M., Guide to Protein Purification Methods Enzymology, vol. 182. Academic Press. Inc., San Diego, CA (1990). )).
따라서, 본 발명은 서열번호 1 내지 서열번호 16으로 이루어진 군에서 선택 된 아미노산 서열을 가지는 본 발명의 펩타이드를 유효성분으로 포함하는 제2형 수분통로단백이 포배된 막 검출용 조성물을 제공한다.Accordingly, the present invention provides a composition for detecting a membrane, wherein the moisture passage protein of the second type comprising the peptide of the present invention having an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 16 as an active ingredient.
본 발명의 펩타이드의 결합 여부의 확인, 검출 및 정량을 용이하게 하기 위하여, 본 발명의 펩타이드는 표지된 상태로 제공될 수 있다. 즉, 검출가능한 표지에 링크(예: 공유 결합 또는 가교)되어 제공될 수 있다. 상기 검출가능한 표지는 발색효소(예: 퍼옥시다제, 알칼라인 포스파타제), 방사성 동위원소(예: 18F, 124I, 125I, 32P, 35S), 크로모포어(chromophore), 발광물질 또는 형광물질(예: FITC, RITC, 형광단백질(GFP(Green Fluorescent Protein); EGFP(Enhanced Green Fluorescent Protein), RFP(Red Fluorescent Protein); DsRed(Discosoma sp. red fluorescent protein); CFP(Cyan Fluorescent Protein), CGFP(Cyan Green Fluorescent Protein), YFP(Yellow Fluorescent Protein), Cy3, Cy5 및 Cy7.5), 상자성입자(super paramagnetic particles) 또는 초상자성입자(ultrasuper paramagnetic particles)일 수 있다.In order to facilitate identification, detection and quantification of the binding of the peptide of the present invention, the peptide of the present invention may be provided in a labeled state. In other words, the detectable label may be provided in a link (eg, covalently bonded or crosslinked). The detectable label may be a colorase (e.g. peroxidase, alkaline phosphatase), radioisotope (e.g. 18 F, 124 I, 125 I, 32 P, 35 S), chromophore, luminescent or Fluorescent materials (e.g., FITC, RITC, fluorescent protein (Green Fluorescent Protein); EGFP (Enhanced Green Fluorescent Protein), RFP (Red Fluorescent Protein); DsRed (Discosoma sp. Red fluorescent protein); CFP (Cyan Fluorescent Protein) , CGFP (Cyan Green Fluorescent Protein), YFP (Yellow Fluorescent Protein), Cy3, Cy5 and Cy7.5), may be super paramagnetic particles (super paramagnetic particles) or ultra super paramagnetic particles (ultrasuper paramagnetic particles).
표지에 따른 검출 방법은 당업계에 널리 알려져 있으나, 예를 들어 다음과 같은 방법에 의해 수행될 수 있다. 만약 검출가능한 표지로 형광물질을 이용하는 경우에는 면역형광염색법을 이용할 수 있다. 예컨대, 형광물질로 표지된 본 발명의 펩타이드를 시료와 반응시키고 미결합 또는 비특이적인 결합 산물을 제거한 다음 형광현미경 하에서 펩타이드에 의한 형광을 관찰할 수 있다. 또한 검출가능한 표지로 효소를 이용하는 경우에는 효소반응을 통한 기질의 발색반응에 의해 흡광도를 측정하고, 방사선 물질인 경우에는 방사선 방출량을 측정함으로써 수행할 수 있다. 아울러, 검출된 결과는 검출표지에 따른 공지된 영상화 방법에 따라 영상화될 수도 있다.Detection methods according to labels are well known in the art, but can be performed, for example, by the following method. If fluorescent material is used as a detectable label, immunofluorescence staining may be used. For example, the peptide of the present invention, labeled with a fluorescent material, may be reacted with a sample, remove unbound or nonspecific binding products, and then observe fluorescence by the peptide under a fluorescence microscope. In addition, in the case of using the enzyme as a detectable label, the absorbance may be measured by the color reaction of the substrate through the enzymatic reaction, and in the case of the radioactive substance, the radiation emission may be measured. In addition, the detected result may be imaged according to a known imaging method according to a detection label.
또한, 본 발명은 (a) 본 발명의 폴리펩타이드를 시료와 혼합하는 단계; (b) 미결합되거나 비특이적으로 결합된 상기 폴리펩타이드를 제거하는 단계; 및 (c) 상기 폴리펩타이드의 결합 여부 및 위치를 확인하는 단계를 포함하는 제2형 수분통로단백이 포배된 막의 검출 방법을 제공한다. 이 때, 본 발명의 폴리펩타이드 및 결합된 본 발명의 폴리펩타이드의 검출 방법은 상기에서 기재한 바 또는 공지된 방법에 따라 수행할 수 있다.In addition, the present invention comprises the steps of (a) mixing the polypeptide of the present invention with a sample; (b) removing the unbound or nonspecifically bound polypeptide; And (c) provides a method for detecting the membrane cultivated type 2 moisture passage protein comprising the step of identifying the binding and position of the polypeptide. At this time, the detection method of the polypeptide of the present invention and the polypeptide of the present invention to which the present invention is bound may be performed according to the above-described methods or known methods.
또한 본 발명의 펩타이드는 제2형 수분통로단백이 포배된 막에 특이적으로 결합할 수 있으므로 제2형 수분통로단백이 포배된 막을 가지는 세포(예를 들어, 속수질 집합관, 부고환 상피세포, 내이의 내림프낭 세포 (endolymphatic sac of the inner ear))에 대해서 약물을 선택적으로 전달하는 지능형 약물 전달체로서 사용할 수 있다. 따라서, 본 발명의 펩타이드를 유효성분으로 포함하는 약물 전달용 조성물을 제공한다.In addition, since the peptide of the present invention can specifically bind to a membrane in which the type 2 moisture channel protein is cultivated, a cell having a type 2 membrane of the water channel protein (for example, medulla collecting tube, epididymal epithelial cell, inner ear) It can be used as an intelligent drug carrier that selectively delivers drugs to the endolymphatic sac of the inner ear. Therefore, it provides a drug delivery composition comprising the peptide of the present invention as an active ingredient.
상기 약물 전달용 조성물이 적용되는 질환은 수분 대사 질환일 수 있다. 수분 대사 질환은 바소프레신에 의해 매개되는 소변의 농축과 희석 기전에 장애가 발생한 경우로서 농축 장애인 요붕증(diabetes insipidus, DI) 및 희석 장애인 항이뇨호르몬 과다분비 증후군(syndrome of inappropriate antidiuretic hormone secretion, SIADH)이 이에 포함될 수 있다. The disease to which the drug delivery composition is applied may be a water metabolic disease. Water metabolism is a disorder of vasopressin-mediated urine enrichment and dilution, including diabetes insipidus (DI) and a syndrome of inappropriate antidiuretic hormone secretion (SIADH). May be included.
요붕증은 다량의 희석된 소변을 배출하는 심각한 질환이다. 이는 원인에 따라 중추성 요붕증 또는 신성 요붕증으로 나뉜다. 그 중, 중추성 요붕증(central DI, CDI)은 시상하부와 뇌하수체에서 바소프레신의 생산 및 분비장애로 인해 수질 집합관 뿐만 아니라 피질 집합관에서 제 2형 수분통로를 통한 수분 재흡수 장애가 초래되며, 소변은 혈액의 삼투질 농도와 동일한 등장뇨보다 더 희석된 다량의 희석뇨를 보게 되는 심각한 질환이다. 또한 신성 요붕증 (nephrogenic DI, NDI)은 시상하부와 뇌하수체에서 바소프레신의 생산과 분비는 정상이나, 콩팥 집합관의 V2 수용체나 제2형 수분통로에 이상이 발생한 경우이다. 원인은 유전적으로 V2 수용체 유전자(Xq28)나 수분 통로 AQP2 유전자(12q13)에 기능상실 돌연변이가 일어난 경우와 후천적으로 만성 세뇨관-간질성 신염(tubulointerstitial nephritis), 다양한 약제 및 체내 전해질 이상 등에 동반되어 이차적으로 발생하는 경우가 있다. 이 또한 피질 및 수질 집합관에서 제2형 수분통로를 통한 수분 재흡수 장애가 초래되며 다량의 희석뇨를 보게 된다. Diabetes insipidus is a serious disease that produces large amounts of diluted urine. It is divided into central diabetes insipidus or nephrogenic diabetes insipidus, depending on the cause. Among them, central diabetes insipidus (central DI, CDI) is caused by disorders of vasopressin production and secretion in the hypothalamus and pituitary gland, resulting in impaired resorption of water through type 2 water channels in the cortex as well as in the medulla. This is a serious condition in which a large amount of dilute urine is diluted more than isotonic urine equal to osmolality. In addition, nephrogenic DI (NDI) is normal in the production and secretion of vasopressin in the hypothalamus and pituitary gland, but abnormality occurs in the V2 receptor or type 2 water passage in the kidney kidney tube. The cause is secondary to genetic dysfunction in the V2 receptor gene (Xq28) or the water pathway AQP2 gene (12q13), as well as acquired chronic tubulointerstitial nephritis, various medications and electrolyte abnormalities. It may occur. This also leads to impaired resorption of water through type 2 water passages in the cortex and medulla and results in large amounts of dilute urine.
항이뇨 호르몬 과다분비 증후군은 바소프레신의 과다분비시 수분의 과다섭취시 발생하며, 필요 이상의 물이 우리 몸에 투여되었을 때 소변을 희석시켜서 용질은 그대로 두고 물만 내보내야 하는데 SIADH 때는 소변이 희석되지 않으므로 결국 체액이 희석되어 저나트륨혈증을 보이게 된다.Antidiuretic hormone hypersecretion syndrome occurs when excessive intake of vasopressin causes excessive intake of water, and when more water is needed in the body, the urine should be diluted and the solute is left without water. The fluid is diluted, resulting in hyponatremia.
본 발명의 약물 전달용 조성물에 포함되는 본 발명의 펩타이드를 종래 수분 대사 질환제제와 연결하여 치료에 이용한다면 본 발명의 펩타이드에 의해 상기 제제가 신장 내 수분 대사 조절 세포인 집합관 주세포에 선택적으로 전달되기 때문에 약의 효력을 증가시킬 수 있고 동시에 정상조직에 미치는 부작용을 현저히 줄일 수 있다.When the peptide of the present invention contained in the composition for drug delivery of the present invention is used for treatment in connection with a conventional water metabolic disease agent, the agent is selectively delivered to the collecting duct main cells which are water metabolism regulating cells in the kidney by the peptide of the present invention. This can increase the efficacy of the drug and at the same time significantly reduce the adverse effects on normal tissues.
본 발명의 펩타이드에 연결될 수 있는 수분 대사 질환 제제는 집합관에서 작용할 수 있는 것이면 사용될 수 있다. 예를 들면, 프로스타글란딘; 인도메타신(indomethacin)을 포함하는 프로스타글란딘 억제제; 포스포다이에스테라제 길항제 (phosphodiesterase inhibitior), 안지오텐신 II 수용체 길항제 (angiotensin II receptor blocker), 알도스테론 수용체 길항제 (aldosterone receptor blocker), 칼슘 채널 억제제 (calcium channel blocker), 아밀로리드(amiloride) 등이 있다. 상기 제제와 본 발명의 펩타이드의 결합은 보다 선택적으로 이들 약물들이 집합관 주세포에 작용할 수 있도록 할 것이다. 상기 제제와 본 발명의 펩타이드의 연결은 당업계에 공지된 방법, 예컨대, 공유 결합, 가교 등을 통해 수행될 수 있다. 이를 위해 본 발명의 펩타이드는 필요하다면 그 활성이 소실되지 않는 범위에서 화학적으로 수식(modification)될 수 있다. 본 발명의 조성물에 포함되는 본 발명의 펩타이드의 양은 결합되는 제제의 종류 및 양에 따라 달라질 수 있다.Water metabolism disease agents that can be linked to the peptides of the invention can be used as long as they can function in the collecting duct. For example, prostaglandins; Prostaglandin inhibitors, including indomethacin; Phosphodiesterase inhibitors, angiotensin II receptor blockers, aldosterone receptor blockers, calcium channel blockers, amylolides, and the like. The combination of the agent with the peptide of the invention will more selectively allow these drugs to act on the collecting duct stem cells. Linking of the agent and the peptide of the present invention can be carried out through methods known in the art, such as covalent bonds, crosslinking and the like. To this end, the peptide of the present invention may be chemically modified if necessary so long as its activity is not lost. The amount of the peptide of the present invention included in the composition of the present invention may vary depending on the type and amount of the agent to be bound.
한편, 본 발명은 본 발명의 펩타이드 및 이와 결합된 수분 대사 질환 제제를 유효성분으로 수분 대사 질환의 예방 및 치료용 약학적 조성물을 제공한다. On the other hand, the present invention provides a pharmaceutical composition for the prevention and treatment of water metabolic diseases as an active ingredient the peptide of the present invention and a water metabolic disease agent coupled thereto.
이 때, 상기 약학적 조성물에서 수분 질환 제제, 결합 방법 및 수분 대사 질환에 대해서는 상기에서 기재한 바와 같다.At this time, the water composition, the binding method and the water metabolic disease in the pharmaceutical composition is as described above.
한편, 본 발명에 따른 약학적 조성물은 상기 펩타이드의 순수한 형태 또는 약학적으로 허용되는 담체와 함께 적합한 형태로 제형화함으로써 제공될 수 있다. '약학적으로 허용되는'이란 생리학적으로 허용되고 인간에게 투여될 때, 통상적으로 위장 장애, 현기증 등과 같은 알레르기 반응 또는 이와 유사한 반응을 일으키지 않는 비독성의 조성물을 말한다. 상기 담체로는 모든 종류의 용매, 분산매질, 수중유 또는 유중수 에멀젼, 수성 조성물, 리포좀, 마이크로비드 및 마이크로좀, 생분해성 나노입자 등이 포함된다. On the other hand, the pharmaceutical composition according to the present invention can be provided by formulating in a suitable form with a pure form of the peptide or a pharmaceutically acceptable carrier. 'Pharmaceutically acceptable' refers to a nontoxic composition which, when administered to humans, is physiologically acceptable and typically does not cause allergic or similar reactions such as gastrointestinal disorders, dizziness and the like. Such carriers include all kinds of solvents, dispersion media, oil-in-water or water-in-oil emulsions, aqueous compositions, liposomes, microbeads and microsomes, biodegradable nanoparticles and the like.
한편, 본 발명에 따른 약학적 조성물은 투여 경로에 따라 적합한 담체와 함께 제형화될 수 있다. 상기 본 발명에 따른 약학적 조성물의 투여 경로로는 이에 한정되지는 않으나 경구적 또는 비경구적으로 투여될 수 있다. 비경구적 투여 경로로는 예를 들면, 경피, 비강, 복강, 근육, 피하 또는 정맥 등의 여러 경로가 포함된다.On the other hand, the pharmaceutical composition according to the present invention can be formulated with a suitable carrier depending on the route of administration. The route of administration of the pharmaceutical composition according to the present invention is not limited thereto, but may be administered orally or parenterally. Parenteral routes of administration include, for example, several routes such as transdermal, nasal, abdominal, muscle, subcutaneous or intravenous.
본 발명의 약학적 조성물을 경구 투여하는 경우 본 발명의 약학적 조성물은 적합한 경구 투여용 담체와 함께 당 업계에 공지된 방법에 따라 분말, 과립, 정제, 환제, 당의정제, 캡슐제, 액제, 겔제, 시럽제, 현탁액, 웨이퍼 등의 형태로 제형화될 수 있다. 적합한 담체의 예로는 락토즈, 덱스트로즈, 수크로즈, 솔비톨, 만니톨, 자일리톨, 에리스리톨 및 말티톨 등을 포함하는 당류와 옥수수 전분, 밀 전분, 쌀 전분 및 감자 전분 등을 포함하는 전분류, 셀룰로즈, 메틸 셀룰로즈, 나트륨 카르복시메틸셀룰로오즈 및 하이드록시프로필메틸-셀룰로즈 등을 포함하는 셀룰로즈류, 젤라틴, 폴리비닐피롤리돈 등과 같은 충전제가 포함될 수 있다. 또한, 경우에 따라 가교결합 폴리비닐피롤리돈, 한천, 알긴산 또는 나트륨 알기네이트 등을 붕해제로 첨가할 수 있다. 나아가, 상기 약학적 조성물은 항응집제, 윤활제, 습윤제, 향료, 유화제 및 방부제 등을 추가로 포함할 수 있다.In the case of oral administration of the pharmaceutical composition of the present invention, the pharmaceutical composition of the present invention is prepared in powder, granule, tablet, pill, dragee, capsule, liquid, gel according to a method known in the art together with a suitable oral carrier. , Syrups, suspensions, wafers and the like. Examples of suitable carriers include sugars, including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol and maltitol and starch, cellulose, starch including corn starch, wheat starch, rice starch and potato starch, and the like. Fillers such as cellulose, gelatin, polyvinylpyrrolidone, and the like, including methyl cellulose, sodium carboxymethylcellulose, hydroxypropylmethyl-cellulose, and the like. In addition, crosslinked polyvinylpyrrolidone, agar, alginic acid or sodium alginate and the like may optionally be added as a disintegrant. Furthermore, the pharmaceutical composition may further include an anticoagulant, a lubricant, a humectant, a perfume, an emulsifier, and a preservative.
또한, 비경구적으로 투여하는 경우 본 발명의 약학적 조성물은 적합한 비경구용 담체와 함께 주사제, 경피 투여제 및 비강 흡입제의 형태로 당 업계에 공지된 방법에 따라 제형화될 수 있다. 상기 주사제의 경우에는 반드시 멸균되어야 하며 박테리아 및 진균과 같은 미생물의 오염으로부터 보호되어야 한다. 주사제의 경우 적합한 담체의 예로는 이에 한정되지는 않으나, 물, 에탄올, 폴리올(예를 들어, 글리세롤, 프로필렌 글리콜 및 액체 폴리에틸렌 글리콜 등), 이들의 혼합물 및/또는 식물유를 포함하는 용매 또는 분산매질일 수 있다. 보다 바람직하게는, 적합한 담체로는 행크스 용액, 링거 용액, 트리에탄올 아민이 함유된 PBS(phosphate buffered saline) 또는 주사용 멸균수, 10% 에탄올, 40% 프로필렌 글리콜 및 5% 덱스트로즈와 같은 등장 용액 등을 사용할 수 있다. 상기 주사제를 미생물 오염으로부터 보호하기 위해서는 파라벤, 클로로부탄올, 페놀, 소르빈산, 티메로살 등과 같은 다양한 항균제 및 항진균제를 추가로 포함할 수 있다. 또한, 상기 주사제는 대부분의 경우 당 또는 나트륨 클로라이드와 같은 등장화제를 추가로 포함할 수 있다. 이들 제형은 제약 화학에 일반적으로 공지된 처방서인 문헌(Remington's Pharmaceutical Science, 15th Edition, 1975, Mack Publishing Company, Easton, Pennsylvania)에 기술되어 있다.In addition, when administered parenterally, the pharmaceutical compositions of the present invention may be formulated according to methods known in the art in the form of injections, transdermal and nasal inhalants together with suitable parenteral carriers. Such injections must be sterile and protected from contamination of microorganisms such as bacteria and fungi. Examples of suitable carriers for injections include, but are not limited to, solvents or dispersion media comprising water, ethanol, polyols (e.g., glycerol, propylene glycol and liquid polyethylene glycols, etc.), mixtures thereof and / or vegetable oils Can be. More preferably, suitable carriers include Hanks' solution, Ringer's solution, phosphate buffered saline (PBS) containing triethanol amine or sterile water for injection, 10% ethanol, 40% propylene glycol and 5% dextrose Etc. can be used. In order to protect the injection from microbial contamination, various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like may be further included. In addition, the injection may in most cases further comprise an isotonic agent such as sugar or sodium chloride. These formulations are described in Remington's Pharmaceutical Science , 15th Edition, 1975, Mack Publishing Company, Easton, Pennsylvania, a prescription generally known in pharmaceutical chemistry.
흡입 투여제의 경우, 본 발명에 따라 사용되는 화합물은 적합한 추진제, 예를 들면, 디클로로플루오로메탄, 트리클로로플루오로메탄, 디클로로테트라플루오로에탄, 이산화탄소 또는 다른 적합한 기체를 사용하여, 가압 팩 또는 연무기로부터 에어로졸 스프레이 형태로 편리하게 전달 할 수 있다. 가압 에어로졸의 경우, 투약 단위는 계량된 양을 전달하는 밸브를 제공하여 결정할 수 있다. 예를 들면, 흡입기 또는 취입기에 사용되는 젤라틴 캡슐 및 카트리지는 화합물, 및 락토즈 또는 전분과 같은 적합한 분말 기제의 분말 혼합물을 함유하도록 제형화할 수 있다. In the case of inhaled dosages, the compounds used according to the invention may be pressurized packs or by means of suitable propellants, for example dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. It can be delivered conveniently from the nebulizer in the form of an aerosol spray. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount. For example, gelatin capsules and cartridges for use in inhalers or blowers can be formulated to contain a mixture of the compound and a suitable powder base such as lactose or starch.
그 밖의 약학적으로 허용되는 담체로는 다음의 문헌에 기재되어 있는 것을 참고로 할 수 있다(Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995). Other pharmaceutically acceptable carriers may be referred to those described in the following documents (Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995).
또한, 본 발명에 따른 약학적 조성물은 하나 이상의 완충제(예를 들어, 식염수 또는 PBS), 카보하이트레이트(예를 들어, 글루코스, 만노즈, 슈크로즈 또는 덱스트란), 안정화제(아황산수소나트륨, 아황산나트륨 또는 아스코르브산) 항산화제, 정균제, 킬레이트화제(예를 들어, EDTA 또는 글루타치온), 아쥬반트(예를 들어, 알루미늄 하이드록사이드), 현탁제, 농후제 및/또는 보존제(벤즈알코늄 클로라이드, 메틸- 또는 프로필-파라벤 및 클로로부탄올)를 추가로 포함할 수 있다.In addition, the pharmaceutical compositions according to the invention may contain one or more buffers (eg saline or PBS), carbohydrates (eg glucose, mannose, sucrose or dextran), stabilizers (sodium bisulfite, Sodium sulfite or ascorbic acid) antioxidants, bacteriostatic agents, chelating agents (eg EDTA or glutathione), adjuvants (eg aluminum hydroxide), suspending agents, thickening agents and / or preservatives (benzalkonium chloride , Methyl- or propyl-paraben and chlorobutanol).
또한, 본 발명의 약학적 조성물은 포유동물에 투여된 후 활성 성분의 신속, 지속 또는 지연된 방출을 제공할 수 있도록 당업계에 공지된 방법을 사용하여 제형화될 수 있다.In addition, the pharmaceutical compositions of the present invention may be formulated using methods known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal.
상기와 같은 방법으로 제형화된 약학적 조성물은 유효량으로 경구, 경피, 피하, 정맥 또는 근육을 포함한 여러 경로를 통해 투여될 수 있다. 상기에서 '유효량' 이란 환자에게 투여하였을 때, 진단 또는 치료 효과의 추적을 가능하게 하는 화합물 또는 추출물의 양을 말한다. 본 발명에 따른 약학적 조성물의 투여량은 투 여 경로, 투여 대상, 대상 질환 및 이의 중증정도, 연령, 성별 체중, 개인차 및 질병 상태에 따라 적절히 선택할 수 있다. 바람직하게는, 본 발명의 펩타이드를 포함하는 약학적 조성물은 질환의 정도에 따라 유효성분의 함량을 달리할 수 있으나, 통상적으로 성인을 기준으로 할 때 1회 투여시 10 ㎍ 내지 10 ㎎의 유효용량으로 하루에 수 차례 반복 투여될 수 있다. Pharmaceutical compositions formulated in such a manner can be administered in a effective amount via several routes, including oral, transdermal, subcutaneous, intravenous or intramuscular. As used herein, an 'effective amount' refers to an amount of a compound or extract that, when administered to a patient, enables the tracking of a diagnostic or therapeutic effect. The dosage of the pharmaceutical composition according to the present invention may be appropriately selected according to the route of administration, the subject to be administered, the target disease and its severity, age, sex weight, individual difference, and disease state. Preferably, the pharmaceutical composition comprising the peptide of the present invention may vary the content of the active ingredient according to the extent of the disease, but usually an effective dose of 10 ㎍ to 10 mg at a single dose based on an adult May be repeated several times a day.
아울러, 본 발명의 펩타이드는 제2형 수분통로단백이 포배된 막과 특이적으로 결합하므로 수분 대사 질환의 발병 부위의 영상화 및 진단에도 유용하게 사용될 수 있다. 따라서, 본 발명은 상기 펩타이드를 유효성분으로 포함하는 수분 대사 질환의 영상화 및 진단용 조성물을 제공한다. 이 때, 수분 대사 질환의 영상화 및 진단은, 이에 한정되지는 않으나, 질환의 초진 목적 뿐만아니라, 진행 경과, 치료에 대한 치료 경과, 치료제에 대한 반응 모니터링 등을 포괄하여 사용할 수 있다. 상기 펩타이드는 결합 여부의 확인, 검출 및 정량을 용이하게 하기 위하여, 표지된 상태로 제공될 수 있으며, 이에 대해서는 상기에서 기술한 바와 같다.In addition, since the peptide of the present invention specifically binds to the membrane in which the type 2 moisture passage protein is cultivated, it may be usefully used for imaging and diagnosing the site of the water metabolic disease. Accordingly, the present invention provides a composition for imaging and diagnosis of water metabolic diseases comprising the peptide as an active ingredient. At this time, the imaging and diagnosis of the water metabolic disease is not limited to this, but may be used to include not only the initial purpose of the disease but also the progress, treatment progress for treatment, response monitoring for the therapeutic agent, and the like. The peptides may be provided in a labeled state to facilitate identification, detection and quantification of binding, as described above.
본 발명에서의 도면은 다음과 같은 기재를 참조할 수 있다.The drawings in the present invention may refer to the following description.
도 1은 AQP2 면역블럿 (A) AQP2, AQP1, H+-ATPase 면역블럿 (B)에 관한 것이다. A) 실험쥐 신장의 세포막 (plasma membrane, PM) 또는 세포내 소포체 (intracellular vesicle, ICV)의 비면역분리 집적시료 (non-immunoisolated, WK), AQP2항체로 면역분리한 시료 (AQP2), 그리고 전면역 토끼 IgG로 면역분리한 시료 (Preimmune IgG)를 이용하였다. 쿠마시 블루 염색액으로 염색한 젤은 각 레인의 단백량이 일정함을 보여주었다. 면역블럿결과 AQP2는 실험쥐의 신장에서 면역분리 하지 않은 시료(WK)와 전면역 IgG 풀다운 시료와 비교하여 AQP2 항체로 면역분리한 시료(세포막과 세포내 소포체)에서 현저하게 그 발현량이 증가되었다. B) 실험쥐 신장에서 세포막 (PM) 또는 세포내 소포체의 비면역분리 집적시료(WK), AQP2항체 또는 H+-ATPase (B1-subunit)항체로 각각 면역분리한 시료를 이용하였다. 쿠마시 블루 염색액으로 염색한 젤을 통해 각 레인의 단백량이 일정함을 보여주었다. AQP2 항체로 면역분리한 시료에서 AQP2의 발현은 현저한 반면 AQP1 또는 H+-ATPase (B1-subunit) 발현량은 아주 적었으며, 이 결과는 AQP2를 발현하는 막 시료의 분리가 매우 선택적으로 이루어 졌음을 나타낸다.1 relates to AQP2 immunoblots (A) AQP2, AQP1, H + -ATPase immunoblots (B). A) Non-immunoisolated (WK), AQP2 antibody, and AQP2 antibody, and whole cells of plasma membrane (PM) or intracellular vesicles (ICV) of rat kidney A sample (Preimmune IgG) immunized with immune rabbit IgG was used. Gels stained with Coomassie blue stain showed that the protein in each lane was constant. Immunoblot results showed that AQP2 was significantly increased in the samples isolated from the AQP2 antibody (cell membrane and intracellular vesicles) compared with the non-immunized samples (WK) and the full- IgG IgG pull-down samples in the kidneys of mice. B) Immunosorbed samples of cell membrane (PM) or intracellular endoplasmic reticulum (WK), AQP2 antibody or H + -ATPase (B1-subunit) antibody were used in mouse kidney. Gels stained with Coomassie blue stains showed constant protein levels in each lane. The expression of AQP2 was remarkable in the samples immunized with AQP2 antibody, but the expression level of AQP1 or H + -ATPase (B1-subunit) was very low, and the result was very selective separation of membrane samples expressing AQP2. Indicates.
도 2는 T7 파지의 바이오패닝 및 플라크 에세이의 결과이다. (A-B) 마그네틱 비드, AQP2 항체, 세포막 또는 세포내 소포체막 직접시료의 복합체에 1 × 1011 pfu의 T7 파지 라이브러리를 반응시킴. (C-E) 비 선택적으로 결합한 파지들을 제거하고, (D) 단백 시료에 결합된 파지들을 플라크 분석을 통해 그 수를 측정함 (E). (F) 이러한 파지들은 로그 증폭(log-phase) 상태의 대장균에 감염시켜 증폭시키고, 증폭된 파지들은 새로이 준비한 마그네틱 비드, AQP2 항체, 세포막 또는 세포내소포체 집적시료의 복합체에 똑같은 방법으로 반응시켰다. 세번에 걸쳐서 이와 똑같 이 파지들을 선별하였다.2 is the result of biopanning and plaque assay of T7 phage. (AB) React 1 × 10 11 pfu of T7 phage library to a complex of magnetic beads, AQP2 antibody, cell membrane or intracellular vesicle membrane direct sample. (CE) Remove non-selectively bound phages, and (D) determine the number of phages bound to the protein sample by plaque analysis (E). (F) These phages were amplified by infecting E. coli in a log-phase state, and the amplified phages were reacted in the same manner with a newly prepared magnetic bead, AQP2 antibody, cell membrane or intracellular vesicle complex. The same phage was selected three times.
도 3은 선별한 파지 클론들을 AQP2를 발현하는 세포막과 세포내 소포체막 집적시료에 다시 반응시켜 선택적 결합력을 확인한 것이다. A) 세번에 걸친 파지 선별 후, 세포막 집적시료에서 80 플라크, 그리고 세포내 소포체막 집적시료에서 80 플라크, 총 160개의 플라크를 LB plates에서 임의로 골라내었다. B) 각각의 파지 클론들의 시퀀스를 PCR을 통해 알아내고, 이 중 높은 빈도로 동일한 시퀀스 혹은 유사한 시퀀스를 나타내는 일곱개의 파지 클론들을 선택하였다. C) 이 일곱개의 파지 클론과 파지 입자의 표면에 펩타이드 리간드를 표현하지 않는 T7 미삽입 파지 클론을 항AQP2항체로 면역분리한 세포막과 세포내 소포체막 시료에 각각 반응시키고, 그 결합력을 측정하였다. (D-E) 복합체에 결합된 파지들은 떼어 내어 (D) 플라크 에세이를 통해 파지 클론의 숫자를 헤아렸다 (E). 따라서 많은 수의 플라크를 보이는 파지 클론은 AQP2항체로 면역분리한 막 시료에 높은 결합력을 가짐을 시사한다.Figure 3 shows the selective binding of the selected phage clones by reacting again with the AQP2-expressing cell membrane and intracellular vesicle membrane integrated sample. A) After three phage screening, a total of 160 plaques were randomly selected from LB plates for 80 plaques in cell membrane samples and 80 plaques in intracellular vesicle membrane samples. B) Sequences of each phage clones were determined by PCR and seven phage clones representing the same or similar sequences were selected at high frequency. C) The seven phage clones and T7 uninserted phage clones that do not express peptide ligands on the surface of the phage particles were reacted with the cell membranes and intracellular vesicle membrane samples immunized with anti-AQP2 antibody, respectively, and the binding force was measured. Phages bound to the (D-E) complex were removed and (D) counted phage clones through plaque assays (E). Thus, phage clones showing a large number of plaques have a high binding capacity to membrane samples immunosorbed with AQP2 antibody.
도 4는 일차배양된 속수질 집합관(IMCD)세포에 FITC가 결합된 펩타이드를 염색한 것이다. A 및 C) 항이뇨호르몬 (dDAVP) 자극을 주지 않은 IMCD세포에 FITC-SRSRNKT 혹은 FITC-KNMRSSA로 염색하였을 때 세포질내 부분 혹은 세포막에서 염색되는 것을 관찰하였다. B 및 D) 단시간 dDAVP (10-8M, 20 분)를 처리한 경우, FITC- SRSRNKT와 FITC-KNMRSSA의 염색 양상이 변화하지 않았다. E 및 F) FITC-PKQRFWP로 염색하였을때 세포질내 부분과 세포막 모두에서 진하게 염색되었으며 단시간의 dDAVP 처리에 의한 변화는 관찰되지 않았다. G 및 H) FITC가 결합된 대조군 펩타이드 (FITC-NSSVDK)는 일차배양된 IMCD 세포에서 단시간의 dDAVP (10-8M, 20 분)를 처리하지 않거나 (G), 혹은 처리한 경우 (H) 염색되지 않거나 매우 약하게 염색되는 것을 관찰하였다. Figure 4 is a stained peptide FITC bound to primary cultured medullary collecting tube (IMCD) cells. A and C) When stained with FITC-SRSRNKT or FITC-KNMRSSA, IMCD cells that did not stimulate antidiuretic hormone (dDAVP) were observed to stain in the cytoplasm or in the cell membrane. B and D) Short treatment of dDAVP (10 −8 M, 20 minutes) did not change the staining pattern of FITC-SRSRNKT and FITC-KNMRSSA. E and F) When stained with FITC-PKQRFWP, the cells were stained deeply in both the cytoplasm and the cell membrane, and no change was observed with the short dDAVP treatment. G and H) FITC conjugated control peptide (FITC-NSSVDK) was treated with (G) or (H) if not treated with short dDAVP (10 -8 M, 20 min) in primary cultured IMCD cells No or very weak staining was observed.
이상 살펴본 바와 같이, 본 발명의 서열번호 1 내지 서열번호 16으로 표시되는 아미노산 서열을 가지는 펩타이드는 제2형 수분통로단백이 포배된 막과 특이적으로 결합할 수 있다. 따라서, 본 발명의 펩타이드는 제2형 수분통로단백이 포배된 막의 검출, 더 나아가 수분 대사 질환의 진단, 영상화 및 표적 약물전달 등에 다양하게 사용될 수 있다.As described above, the peptide having the amino acid sequence represented by SEQ ID NO: 1 to SEQ ID NO: 16 of the present invention can specifically bind to the membrane of the type 2 moisture passage protein cultivated. Therefore, the peptide of the present invention can be used for various purposes such as detection of membranes in which the type 2 moisture channel protein is cultivated, furthermore, diagnosis of water metabolism disorders, imaging, and targeted drug delivery.
이하. 본 발명을 실시예에 의해 상세히 설명한다.Below. The present invention will be described in detail by way of examples.
단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예에 한정되는 것은 아니다.However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.
<실험 방법>Experimental Method
1. 실험쥐 신장의 세포막 혹은 1. Cell membranes of mouse kidneys 세포내Intracellular 소포체막이Endoplasmic reticulum 집적된 시료 준비 Integrated Sample Preparation
Charles River사 (Orient Bio, 성남, 한국)로부터 무균상태의 Sprague-Dawley 숫쥐 (200 g-250 g)를 구입하였다. 실험쥐는 엔플루란(light enflurane) 흡입마취하여, 빠르게 양쪽 신장을 적출하고, 신장은 얼음 위에서 잘라 10ml 해부용 버퍼(dissecting buffer; 0.3 M sucrose, 25 mM imidazole, and 1 mM EDTA, pH 7.2, containing protease inhibitors: 8.5 μM leupeptin and 1 mM phenylmethylsulfonyl fluoride)에 넣고 균질화 시켰다 (Eppendorf 5810R, Hamburg, Germany). 이 균질액을 4°C에서 4,000g로 15 분간 원심분리 (Beckman OptimaTM L-100XP with Ti-90 rotor)하여 핵, 미토콘드리아, 그리고 세포내 큰 잔여물질 등을 제거하고(Kwon TH et al., Am J Physiol Renal Physiol 285: F152-F165, 2003.; Kwon TH et al., Am J Physiol Renal Physiol 288: F673-F684, 2005.; Marples D et al., Am J Physiol 269: C655-64, 1995.), 그 상층액을 수집하여 저속(Low speed: LS) 세포막 집적시료와 고속 (High speed: HS) 세포내 소포체 집적시료를 분리하였다(Marples D et al., Am J Physiol 269: C655-64, 1995.). LS 펠렛(pellet, 17,000 x g, 30분)은 세포막(Plasma membrane: PM)부분이 대다수 포함된 시료를 말하며, HS 펠렛 (200,000 x g, 1시간)은 세포내 소포체(intracellular vesicle: ICV)가 대다수 포함된 시료를 말한다(Marples D et al., Am J Physiol 269: C655-64, 1995.; Terris J et al., Am J Physiol 269: F775-F785, 1995.). 이러한 펠렛들은 해부용 버퍼에 용해시켰다.Aseptic Sprague-Dawley males (200 g-250 g) were purchased from Charles River (Orient Bio, Seongnam, Korea). Rats were anesthetized with light enflurane, and rapidly excised both kidneys, and the kidneys were cut on ice and 10 ml dissecting buffer (0.3 M sucrose, 25 mM imidazole, and 1 mM EDTA, pH 7.2, containing). Protease inhibitors were added to 8.5 μM leupeptin and 1 mM phenylmethylsulfonyl fluoride) and homogenized (Eppendorf 5810R, Hamburg, Germany). Centrifuge this homogenate at 4,000 g at 4 ° C for 15 minutes (Beckman OptimaTM L-100XP with Ti-90 rotor) to remove nuclei, mitochondria, and large intracellular residues (Kwon TH et al., Am J Physiol Renal Physiol 285: F152-F165, 2003 .; Kwon TH et al., Am J Physiol Renal Physiol 288: F673-F684, 2005 .; Marples D et al., Am J Physiol 269: C655-64, 1995.), the supernatants were collected to separate low speed (LS) membrane membrane samples and high speed (HS) intracellular vesicle accumulation samples (Marples D et al., Am J Physiol 269: C655-64, 1995.). LS pellet (17,000 xg, 30 minutes) refers to the sample containing the majority of the plasma membrane (PM), HS HS (200,000 xg, 1 hour) contains the majority of intracellular vesicles (ICV) Refers to the sample (Marples D et al., Am J Physiol 269: C655-64, 1995 .; Terris J et al., Am J Physiol 269: F775-F785, 1995.). These pellets were dissolved in dissection buffer.
2. 실험쥐 신장에서 2. In the rat kidney AQP2AQP2 가 선택적으로 발현되는 세포막 (Is selectively expressed in the cell membrane ( PMPM ) 집적시료 또는 ) Integrated sample or AQP2AQP2 가 선택적으로 발현되는 Is selectively expressed 세포내Intracellular 소포체막Vesicle membrane ( ( ICVICV ) 집적시료의 면역분리Immune Separation of Integrated Samples
위에서 기술한 바와 같이 실험쥐 신장으로부터 분리한 세포막 집적시료 (LS) 또는 세포막 소포체 집적시료 (HS) 모두를 마그네틱 비드(magnetic beads, Dynal M-280; Dynal Biotech ASA, Oslo, Norway, precoated with anti-rabbit IgG) 와 정제된 AQP2 항체 (~ 2 μg/107 beads)(Marples D et al., Am J Physiol 274: F384-F394, 1998.; Nielsen J et al., Am J Physiol Renal Physiol 290: F438-F449, 2006.)를 넣어 4°C 에서 하루밤 동안 일정한 교반상태에서 반응시켰다. 1) AQP2 면역블럿 분석(immunoblotting)을 위해 시료를 0.1% BSA(bovine serum albumin)를 포함하는 PBS에 10분간 3번씩 조심스럽게 씻어내고, 마그네틱 비드, AQP2 항체, 세포막 또는 소포체 집적시료의 복합체를 자석으로 분리한다. 자석으로 분리한 시료는 램리 샘플 버퍼(Laemmli sample buffer; 10 mM Tris, pH 6.8, 1.5% SDS, 6% glycerol)를 넣고, 60°C 에서 15분간 열처리 하여 단백질들을 용해시킨다. 이후 비드를 자석으로 당겨 제거하고, 면역분리한 시료는 면역블럿 분석 (그림 1) 한다. 2) AQP2를 포함하는 막 (세포막과 소포체막) 분리의 특이성을 알아보기 위한 대조군 실험(control experiment)으로 전면역 토끼 IgG 풀 다운 분석(preimmune rabbit IgG pulldown) 시료 (세포막과 소포체막)를 준비하여 AQP2 변역블럿(그림 1A)을 함께 시행하였다. 또한 면역분리한 AQP2를 발현하는 막 시료(세포막과 소포체막)와 함께, H+-ATPase (B1-subunit)를 발현하는 막 시료(세포막과 소포체막)을 따로 분리하여 AQP2, AQP1, H+-ATPase 면역블럿을 각각 실시하였다 (그림 1B). 3) 파지디스플레이 시행을 위해, 시료를 0.1% BSA in PBS에 10분간 3번씩 결합하지 않은 단백들을 조심스럽게 씻어내었고, 마그네틱 비드, AQP2 항체, 세포막 또는 소포체 집적시료의 복합체를 자석으로 당겨 얻었다 (아래 T7 파지 바이오패닝 부분에서 상세하게 기술됨).As described above, both membrane-integrated samples (LS) or membrane-vesicle-integrated samples (HS) isolated from mouse kidneys were prepared using magnetic beads, Dynal M-280; Dynal Biotech ASA, Oslo, Norway, precoated with anti- rabbit IgG) and purified AQP2 antibody (~ 2 μg / 10 7 beads) (Marples D et al., Am J Physiol 274: F384-F394, 1998 .; Nielsen J et al., Am J Physiol Renal Physiol 290: F438-F449, 2006.) was added and reacted at 4 ° C overnight under constant stirring. 1) Rinse samples carefully in PBS containing 0.1% bovine serum albumin (BSA) three times for 10 min for AQP2 immunoblotting, and prepare a complex of magnetic beads, AQP2 antibody, cell membrane or vesicle integrated sample. To separate. Magnetically separated samples were added to Laemmli sample buffer (10 mM Tris, pH 6.8, 1.5% SDS, 6% glycerol) and heat-treated at 60 ° C. for 15 minutes to dissolve the proteins. The beads are then pulled out with a magnet and the immunoblotted samples are subjected to immunoblot analysis (Figure 1). 2) Prepare a full rabbit rabbit IgG pulldown sample (cell membrane and endoplasmic reticulum membrane) as a control experiment to determine the specificity of separation of membranes (cell membrane and endoplasmic reticulum membrane) containing AQP2. AQP2 translation blots (Figure 1A) were also performed. In addition, membrane samples expressing H + -ATPase (B1-subunit) (cell membrane and endoplasmic reticulum membrane) are separately isolated along with membrane samples expressing AQP2 (Immunosorbent) and AQP2, AQP1, H + -. Each ATPase immunoblot was performed (Fig. 1B). 3) For phage display, the samples were carefully washed with 0.1% BSA in PBS that had not been bound three times for 10 minutes, and the magnetic beads, AQP2 antibody, cell membrane or vesicle complexes were pulled into the magnet ( Described in detail in the T7 phage biopanning section below).
3. 3. AQP2AQP2 , H, H ++ -- ATPaseATPase , , AQP1AQP1 의 면역 Immunity 블럿Blot
각각의 레인(lane)에 동등한 양의 단백을 투여(load)하기 위해 젤을 쿠마시 블루 염색액(Coomassie Blue dye, GelCode Blue Stain Reagent, Pierce)로 염색하고 쿠마시 블루 염색액으로 염색된 젤의 각 레인의 밴드 농도(band density)에 따라 투여량(loading volume)을 조정하였다(Kwon TH et al., Am J Physiol Renal Physiol 285: F152-F165, 2003.; Kwon TH et al., Am J Physiol Renal Physiol 288: F673-F684, 2005.; Nielsen J et al., Am J Physiol Renal Physiol 290: F438-F449, 2006.). SDS-PAGE는 12% 폴리아크릴아미드 젤(polyacrylamide gels)에서 시행하였다. 하나의 젤은 쿠마시 블루 염색액으로 다시 염색하여 각 레인들에서 투여(loading)가 균일하게 이루어졌음을 확인하고, 다른 젤은 면역블럿을 하였다. 일렉트로일루션(Electroelution)을 통해 니트로셀룰로스 막(nitrocellulose membrane)으로 젤상의 단백질을 옮긴 후에, 블럿(blots)은 PBST 내의 5% 탈지분 유(milk in PBST (80 mM Na2HPO4, 20 mM NaH2PO4, 100 mM NaCl, 0.1% Tween 20, pH 7.5)) 용액에서 한 시간 동안 차단시키고, AQP2 항체 (1:500)(Nielsen J et al., Am J Physiol Renal Physiol 290: F438-F449, 2006.), H+-ATPase 항체 (1:1,000)(Kim YH et al., Am J Physiol Renal Physiol 285: F1244-F1257, 2003.), 그리고 AQP1 항체 (1:1,000)(O'Neill H et al., Nephrol Dial Transplant 23: 1546-1555, 2008.)와 반응시켰다. AQP2 항체는 쥐 AQP2(Fushimi K et al.,, Nature 361: 549-552, 1993.)의 카르복시 말단(carboxyl terminus) 부분 (250- 271)에 KLH 부착(KLH conjugation)을 위한 NH2-말단 시스테인(NH2-terminal cysteine; NH2-CEVRRRQSVELHSPQSLPRGSKA-COOH, amino acids 250-271, 서열번호 17)을 합성하고 이를 토끼에 주입하여 생산하였다. 표지(Labeling)는 호오스래디시 퍼옥시다아제 부착(horseradish peroxidase (HRP)-conjugated) 이차항체 (P448, 1:3,000; DAKO, Glostrup, Denmark)와 ECL(enhanced chemiluminescence) 시스템으로 시각화 하여, 사진용 필름(photographic film; Hyperfilm ECL, RPN3103K, Amersham Pharmacia Biotech, Little Chalfont, UK)에 현상하였다.Gels were stained with Coomassie Blue dye (Coomassie Blue dye, GelCode Blue Stain Reagent, Pierce) and loaded with Coomassie blue stain to load the same amount of protein in each lane. The loading volume was adjusted according to the band density of each lane (Kwon TH et al., Am J Physiol Renal Physiol 285: F152-F165, 2003 .; Kwon TH et al., Am J Physiol Renal Physiol 288: F673-F684, 2005 .; Nielsen J et al., Am J Physiol Renal Physiol 290: F438-F449, 2006.). SDS-PAGE was performed on 12% polyacrylamide gels. One gel was stained again with Coomassie blue staining solution to confirm that the loading was uniform in each lane, and the other gel was immunoblotted. After transferring the gelled protein to the nitrocellulose membrane via electroroelution, the blots were milked in 5% milk in PBST (80 mM Na 2 HPO 4 , 20 mM NaH 2). PO 4 , 100 mM NaCl, 0.1
4. 4. T7T7 파지의 Phage 바이오패닝Bio Panning
T7 415-1b 파지 벡터의 파지 펩타이드 라이브러리를 사용하였으며, 이 파지는 CX7C(두개의 Cysteine사이에 임의로 배열된 일곱개의 아미노산 잔기를 포함)가 발현하도록 설계되었다 (Novagen, Madison, WI)(Lee SM et al., Mol Cancer Res 5: 11-19, 2007.). 이 라이브러리는 ~5 × 108 PFU(plaque forming unit)의 다양성을 가졌다. 파지라이브러리의 바이오패닝을 간단히 설명하면 (그림 2, 그림 3)(Hoffman JA et al., edited by Clackson T and Lowman HB. New York: Oxford University Press, 2004, p. 171-192.; Lee SM et al., Mol Cancer Res 5: 11-19, 2007.), 1 × 1011pfu의 T7 파지 라이브러리 (그림 2A)를 마그네틱 비드, AQP2 항체, 세포막 또는 소포체막 집적시료의 복합체에 4°C에서 밤새도록 일정한 교반상태에서(그림 2B) 결합시킨다. 이것을 시행하기에 앞서 T7 파지 라이브러리를 항-토끼 IgG(anti-rabbit IgG) 항체가 이미 코팅된 마그네틱 비드(Dynal M-280; Dynal Biotech ASA, Oslo, Norway)에 먼저 결합시키는데, 여기에 결합하지 않는 파지 라이브러리만 선택하여 마그네틱 비드, AQP2 항체, 세포막 또는 소포체막 집적시료의 복합체에 4°C에서 밤새도록 결합시킨다. 다음날, 1 ml 의 M9/LB 배지(10 g bactotryptone, 5 g yeast extract, 10 g NaCl, 1 g NH4Cl, 3 g KH2PO4, 3 g NaHPO4.7H2O/L containing 0.4% glucose and 1 mM MgSO4)을 이용하여 10번 씻어내어, 비 선택적으로 결합한 파지들를 제거한다 (그림 2C). 복합체에 결합한 파지는 대장균 감염을 통해 떼어내며 (그림 2D), 플라크 분석(plaque assay)을 통해 결합한 파지클론의 수를 헤아린다 (그림 2E). 또한 이 파지는 대장균(E. coli)에 감염시켜 증폭시키고 (그림 2F), 이것은 다시 새로 준비한 마그네틱 비드, AQP2 항체, 세포막 또는 소포체 집적시료의 복합체에 같은 방법으로 반응시킨다(그림 2). 세번 에 걸쳐 같은 방법을 실시하여, 세포막시료에서 80 플라크(plaques), 그리고 소포체막 시료에서 80 플라크를 LB 플레이트(plates)에서 임의로 골라 (그림 3A), 10 μl TBS (Tris 3.0285 g, NaCl 4.3838 g per 500 ml, pH 7.5)에 용해시키고, 이것을 시퀀싱한다 (그림 3B). 시퀀싱 결과 중에 동일한 시퀀스 혹은 유사한 시퀀스가 빈번히 나타난 일곱개의 파지클론을 선별하였다(표 1). 이 일곱개의 파지 클론을 다시 AQP2-면역분리 시료 (세포막과 소포체막 시료 모두)에 결합시키고, 파지입자의 표면에 펩타이드 리간드가 표현되지 않는 T7 미삽입(insertless) 파지클론의 결합능과 비교하여 선택적 결합능을 검증하였다 (그림 3C-E). 또한, 일곱개의 선별된 파지클론이 AQP2를 발현하는 세포막과 소포체막에 선택적 결합력을 가지는지를 다시 알아보기 위해 실험쥐 신장 집합관 사이세포에만 특이적인 H+-ATPase (B1-subunit)가 발현되는 세포막과 소포체막 시료를 이용하여 그 결합력을 다시 측정하였다.A phage peptide library of T7 415-1b phage vector was used, which was designed to express CX 7 C (containing seven amino acid residues randomly arranged between two Cysteine) (Novagen, Madison, WI) (Lee SM et al., Mol Cancer Res 5: 11-19, 2007.). This library had a diversity of ˜5 × 10 8 plaque forming units (PFUs). A brief description of phage library biopanning (Figure 2, Figure 3) (Hoffman JA et al., Edited by Clackson T and Lowman HB. New York: Oxford University Press, 2004, p. 171-192 .; Lee SM et al., Mol Cancer Res 5: 11-19, 2007.), Tx phage library of 1 × 10 11 pfu (Fig. 2A) was added to a complex of magnetic beads, AQP2 antibody, cell membrane or endoplasmic reticulum sample at constant stirring overnight at 4 ° C. Figure 2B) Combine. Prior to this, the T7 phage library is first bound to magnetic beads (Dynal M-280; Dynal Biotech ASA, Oslo, Norway) already coated with an anti-rabbit IgG (anti-rabbit IgG) antibody. Only phage libraries are selected and bound overnight at 4 ° C. to complexes of magnetic beads, AQP2 antibodies, cell membranes or vesicle membrane integrated samples. The next day, 1 ml of M9 / LB medium (10 g bactotryptone, 5 g yeast extract, 10 g NaCl, 1 g NH 4 Cl, 3 g KH 2 PO 4 , 3 g NaHPO 4 .7H 2 O / L containing 0.4% glucose and 1 mM MgSO 4 ) to rinse 10 times to remove non-selectively bound phages (Figure 2C). Phages bound to the complex are removed through Escherichia coli infection (Figure 2D), and the number of bound phage clones is counted by plaque assay (Figure 2E). The phage also infects E. coli Amplification (Figure 2F), which in turn reacts to the newly prepared magnetic beads, AQP2 antibody, cell membrane or vesicle complexes in the same manner (Figure 2). The same procedure was performed three times, with 80 plaques in the cell membrane sample and 80 plaques in the vesicle membrane sample randomly selected from the LB plates (Figure 3A), 10 μl TBS (Tris 3.0285 g, NaCl 4.3838 g per 500 ml, pH 7.5) and sequence it (Figure 3B). Seven phage clones with the same or similar sequence frequently selected during the sequencing results were selected (Table 1). These seven phage clones are again bound to AQP2-immunoassay samples (both cell and vesicle membrane samples) and selective binding capacity compared to the binding capacity of T7 insertless phage clones without peptide ligands expressed on the surface of phage particles. (Fig. 3C-E). In addition, to examine whether the seven selected phageclones have selective binding ability to AQP2-expressing cell membranes and vesicle membranes, cell membranes expressing H + -ATPase (B1-subunit) specific to the mouse kidney collecting stromal cells and The binding force was measured again using the vesicle membrane sample.
5. 플라크의 5. Plaque PCRPCR 증폭 및 라이브러리 분석 Amplification and Library Analysis
TBS에 용해된 파지클론들은 PCR 반응에서 프라이머(서열번호 18, 5-AGCGGACCAGATTATCGCTA-3 및 서열번호 19, 5-AACCCCTCAAGACCCGTTTA-3) (Genotech, Korea)와 함께 주형(template)으로 작용한다. PCR은 Top-TaqTM PreMix (2X) Ver 3.0 (Corebio, Korea)를 이용하여 95°C에서 3분 처리한 후 35 싸이클(cycles; 94°C 에서 50 초, 50°C 에서 1분, 72°C에서 1분)후 72°C에서 6분 동안 마지막 연 장반응(extension)을 시켰다. 4 μl의 반응물을 2% 아가로스 젤(agarose gel)에 내려서 PCR 증폭이 잘 되었는지 확인하였다. 파지 DNA를 PCR로 증폭시킨후, 이 DNA를 정제하고, T7 파지 표면에 표지되어 있던 아미노산 시퀀스를 Koma Biotech Co.(대전, 한국)를 통해 확인하였다.Phage clones dissolved in TBS act as a template with primers (SEQ ID NO: 18, 5-AGCGGACCAGATTATCGCTA-3 and SEQ ID NO: 19, 5-AACCCCTCAAGACCCGTTTA-3) (Genotech, Korea) in a PCR reaction. PCR was performed using Top-TaqTM PreMix (2X) Ver 3.0 (Corebio, Korea) for 3 minutes at 95 ° C, followed by 35 cycles; 50 seconds at 94 ° C, 1 minute at 50 ° C, 72 ° C. 1 minute) followed by a final extension for 6 minutes at 72 ° C. 4 μl of the reaction was lowered to 2% agarose gel (agarose gel) to confirm that the PCR amplification was well. After amplifying the phage DNA by PCR, the DNA was purified and the amino acid sequence labeled on the surface of the T7 phage was confirmed by Koma Biotech Co. (Daejeon, Korea).
라이브러리 분석을 통해 펩타이드 리간드 시퀀스와 상동성을 가지는 쥐의 단백질들(표 4-6)을 National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi)에서 이용할 수 있는 NCBI BLAST 프로그램의 SWISSPROT 데이터베이스를 사용하여 검색하였다. 각각의 펩타이드 시퀀스의 데이터베이스를 검색할 때 Expect (E)-값(random background noise)는 표 4-6에 같이 기술하였다. Library analysis was performed to determine the proteins of the mouse (Table 4-6) homologous to the peptide ligand sequence at the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi). The search was made using the SWISSPROT database of the available NCBI BLAST program. Expect (E) -value (random background noise) when searching the database of each peptide sequence is described in Table 4-6.
6. 실험쥐의 신장 속수질 6. Renal Medulla in Rats 집합관Assembly tube 세포의 일차배양과 Primary culture of cells 펩타이드Peptide 리간드에On the ligand 형광물질을 결합시킨 Fluorescence 펩타이드Peptide 염색 dyeing
신장 속수질 집합관세포 (IMCD 세포)가 집적된 일차배양은 무균상태의 Sprague-Dawley 숫쥐 (200 - 250g, Charles River)에서 신장을 적출하여 시행하였다(Chou CL et al., J Biol Chem 275: 36839-36846, 2000.; Lee YJ et al., Am J Physiol Renal Physiol 292: F340-F350, 2007.). IMCD세포는 인간 피브로넥틴(human fibronectin)이 코팅된 슬라이드 (Lab-Tek™ Chamber Slides™ System, Cat. No.177402, NUNC, Roskilde, Denmark)에서 키웠으며, 매 24 시간마다 신선한 배지로 갈아주었다. 이 세포들은 10% 우태아혈청(fetal bovine serum)이 첨가된 고삼투질 농도 세포배양액 (hypertonic culture medium, 640 mOsm/KgH2O)에서 자라며, 37°C의 5% CO2, 95% 대기에서 3일간 키우고, 우태아혈청이 없는 배지에서 하루를 더 키운다. 5일째, IMCD세포는 무처리(vehicle) 또는 dDAVP (10-8M)를 20 분간 처리한 후 2.5% 파라포름알데히드(paraformaldehyde; pH 7.4)로 20 분간 실온에서 고정하고, PBS에 두번 씻어 낸 후 0.3% 트리톤 X-100 함유 PBS(0.3% Triton X-100 in PBS)로 실온에서 15분간 처리하여 세포막의 투과도를 증가시켰다. 이후 세포들을 씻어내고 1% BSA 함유 0.01M PBS(1% BSA(bovine serum albumin) in 0.01 M PBS)로 30분간 차단한 다음 형광물질을 결합시킨 펩타이드 (10 μM)로 4°C에서 밤새도록 반응시킨다. 인공적으로 합성한 펩타이드(PKQRFWP, KNMRSSA, and SRSRNKT)들은 형광물질인 FITC와 결합시켰고 (Peptron, Inc., Daejeon, Korea), 또한 FITC 가 결합된 대조군 펩티드(NSSVDK)는 대조군 실험에 사용되었다. 이후 세포들을 씻어내고 안티페이딩 시약(antifading reagent)이 포함된 친수성 마운트액 (cat. no. P36930, Molecular Probes Inc, Eugene, OR)로 마운트하였다. 형광현미경은 CoolSNAP HQ camera (Photometrics, Tucson, AZ)를 갖춘 Leica DM IRB inverted microscope (Leica Microsystems, Wetzlar, Germany)를 사용하였다.Primary cultures in which kidney medullary ductal cells (IMCD cells) were accumulated were performed by assembling kidneys from aseptic Sprague-Dawley males (200-250 g, Charles River) (Chou CL et al., J Biol). Chem 275: 36839-36846, 2000 .; Lee YJ et al., Am J Physiol Renal Physiol 292: F340-F350, 2007.). IMCD cells were grown on human fibronectin coated slides (Lab-Tek ™ Chamber Slides ™ System, Cat. No. 177402, NUNC, Roskilde, Denmark) and changed to fresh medium every 24 hours. These cells grow in hypertonic culture medium (640 mOsm / KgH 2 O) with 10% fetal bovine serum and in 37% 5% CO 2 , 95% atmosphere. Raise for three days, and grow one more day in medium without fetal bovine serum. On day 5, IMCD cells were treated with vehicle or dDAVP (10 -8 M) for 20 minutes, fixed with 2.5% paraformaldehyde (pH 7.4) at room temperature for 20 minutes, washed twice in PBS Permeability of the cell membrane was increased by treatment with 0.3% Triton X-100 containing PBS (0.3% Triton X-100 in PBS) for 15 minutes at room temperature. Cells were then washed, blocked with 1% BSA containing 0.01 M PBS (1% BSA (bovine serum albumin) in 0.01 M PBS) for 30 minutes and reacted overnight at 4 ° C with a peptide-bound peptide (10 μM). Let's do it. Artificially synthesized peptides (PKQRFWP, KNMRSSA, and SRSRNKT) were combined with the fluorescent material FITC (Peptron, Inc., Daejeon, Korea), and FITC-bound control peptide (NSSVDK) was used for the control experiment. Cells were then washed and mounted with a hydrophilic mount solution (cat. No. P36930, Molecular Probes Inc, Eugene, OR) containing an antifading reagent. Fluorescence microscopy was used with a Leica DM IRB inverted microscope (Leica Microsystems, Wetzlar, Germany) equipped with a CoolSNAP HQ camera (Photometrics, Tucson, AZ).
<실험 결과><Experiment Result>
1. One. AQP2AQP2 를 발현하는 세포막과 Cell membranes that express 세포내Intracellular 소포체막의Endoplasmic reticulum 면역 분리 Immune isolation
실험쥐의 신장에서 세포막 또는 세포내 소포체막이 집적된 막 시료를 AQP2 항체, H+-ATPase (B1-subunit) 항체, 그리고 전면역 토끼 IgG를 이용하여 면역분리하였다. 면역블럿 결과, AQP2는 흰쥐 신장의 비면역분리 시료와 전면역(preimmune) IgG로 면역분리한 시료에 비하여 AQP2 항체로 면역분리된 세포막과 소포체막 시료에서 현저하게 많이 발현되었다 (그림 1A). 또한, 실험쥐 신장에서 비면역분리시료, AQP2항체로 면역분리한 시료, H+-ATPase항체로 면역분리한 시료 중 오직 AQP2항체로 면역분리한 시료에서만 AQP2 발현이 관찰되는 것을 확인하였다 (그림 1B). AQP1은 AQP2항체로 면역분리한 시료와 H+-ATPase항체로 면역분리한 시료에 비하여, 비면역분리 시료에서 우세적으로 발현되었다 (그림 1B). 또한 이와 유사하게, H+-ATPase의 발현은 비면역분리 시료와 AQP2항체로 면역분리한 시료에 비해 H+-ATPase항체로 면역분리한 시료에서 현저히 발현되었다 (그림 1B). 이러한 결과는 AQP2와 H+-ATPase를 발현하는 세포막 및 소포체막 시료 분리의 특이성을 시사한다. Membrane samples in which cell membranes or intracellular endoplasmic reticulum membranes were integrated in the kidneys of mice were immunized with AQP2 antibody, H + -ATPase (B1-subunit) antibody, and full-length rabbit IgG. As a result of the immunoblot, AQP2 was expressed more significantly in cell membranes and vesicle membranes immunized with AQP2 antibody than in the non-immunized samples of rat kidney and those immunized with preimmune IgG (Fig. 1A). In addition, AQP2 expression was observed only in samples immunized with AQP2 antibody in non-immunized samples, AQP2 antibody-immunized samples, and H + -ATPase antibody-immunized samples in mouse kidneys (Fig. 1B). ). AQP1 was predominantly expressed in non-immunized samples compared to the samples immunized with AQP2 and H + -ATPase antibody (Figure 1B). Also similarly, the H + -ATPase expression was significantly expressed in the sample separated by the immune H + -ATPase antibody than the sample, separated by a non-immune Immune separate sample and AQP2 antibody (Figure 1B). These results suggest specificity of the separation of cell membrane and vesicle membrane samples expressing AQP2 and H + -ATPase.
2. 실험쥐 신장에서 2. In the rat kidney AQP2AQP2 항체로 면역분리한 세포막과 Cell membranes immunized with antibodies 세포내Intracellular 소포체막에On the vesicle membrane 선택적으로 높은 Optionally high 결합능을Binding capacity 가진 have 파지클론Phageclone 선별 Selection
CX7C 무작위 펩타이드 시퀀스를 발현하는 T7 파지 라이브러리에서 선택적으로 실험쥐 신장에서 AQP2가 발현되는 세포막과 소포체막에 높은 결합능을 가지는 파지들을 집적시켰다. 세번에 걸친 선별작업을 통해, 각 단계에서 세포막과 소포체막 시료 모두에서 파지클론들이 유의하게 집적되는 것을 확인하였다(결과미도시). AQP2가 발현하는 세포막 또는 소포체막 시료로부터 각각 80개씩 임의로 파지클론들을 선택하였으며, 이 파지 클론들은 모두 PCR을 통해 T7 파지 캡시드(phage capsid)에 발현하고 있는 아미노산 시퀀스(sequence)에 상응하는 DNA를 증폭하여 시퀀싱(sequencing)했다. 이렇게 AQP2를 발현하는 세포막 또는 소포체막에 선택적인 파지 라이브러리 중 무작위로 추출한 160개의 파지 클론에서 높은 빈도로 동일한 시퀀스 또는 유사한 시퀀스를 보이는 7개의 파지클론을 최종 선택하였다 (표 1). 이들 중, CPKQRFWPC (7 identical sequences/160 phage clones: 4.4%), CKRVTGRPC(5/160: 3.1%), CKNMRSSAC (5/160: 3.1%)를 발현하는 파지클론이 전체 무작위로 선택한 파지클론 160개 중 11%를 차지하였다. 또한 이들 7개 파지 클론들은 다시 AQP2항체로 면역분리한 시료(세포막 또는 소포체막)에 플라크 분석을 통해 그 결합이 선택적인 결합임을 확인하였으며, 각각의 결합은 파지입자 (capsid)의 표면에 펩타이드 리간드를 발현하지 않는 T7 미삽입(insertless) 파지클론의 결합능과 비교하였다 (그림 3, 표 2). 일곱개의 파지클론들은 AQP2를 발현하는 세포막 또는 소포체막에서 T7 미삽입 파지클론들에 비해 약 50배에서 5,600배 높은 결합능을 보였다 (표 2). 또한, AQP2를 발현하는 세포막 또는 소포체막에 이들 파지클론들이 선택적으로 결합하는지를 다시 확인하기 위해, 또 다른 결합 분석을 실시하였다. 즉, 선택된 일곱개의 파지 클론을 실험쥐의 신장에서 준비한 사이세포(집합관에서 AQP2가 발현하고 있는 주세포가 아닌 다른 종류의 세포)에서만 발현하는 H+-ATPase (B1-subunit)를 포함하는 세포막 또는 소포체막 시료에 결합시켜 보았다 (표 3). 선택된 7개의 파지 클론들은 H+-ATPase를 포함하는 막 시료에서 AQP2를 발현하는 막시료에서 나타난 결과와는 달리 0.4배에서 3.5배의 낮은 결합능을 보였다(표 3). 따라서 이상의 결과는 본 연구에서 선택한 일곱개의 파지클론이 AQP2를 발현하는 세포막 및 소포체막 시료에서 높은 선택성을 가지고 있음을 나타낸다. In the T7 phage library expressing the CX 7 C random peptide sequence, phages with high binding capacity were integrated with the cell membrane and vesicle membrane expressing AQP2 in mouse kidney. Through three screening operations, it was confirmed that phageclone was significantly accumulated in both cell membrane and vesicle membrane samples at each step (results not shown). A total of 80 phage clones were randomly selected from AQP2 expressing cell membrane or endoplasmic reticulum samples. All of these phage clones amplified DNA corresponding to the amino acid sequence expressed in T7 phage capsid by PCR. By sequencing. Thus, 7 phage clones showing the same or similar sequence at high frequency were randomly selected from 160 phage clones randomly selected from cell libraries or vesicle membranes expressing AQP2 (Table 1). Of these, 160 phage clones totally randomly selected phage clones expressing CPKQRFWPC (7 identical sequences / 160 phage clones: 4.4%), CKRVTGRPC (5/160: 3.1%), and CKNMRSSAC (5/160: 3.1%) Accounted for 11% of the total. In addition, these seven phage clones were confirmed to be selective binding by a plaque analysis to the sample (cell membrane or vesicle membrane) that was immunized with AQP2 antibody again, each binding peptide peptide on the surface of the phage particles (capsid) It was compared with the binding capacity of T7 insertless phage clones that did not express (Figure 3, Table 2). Seven phage clones showed about 50- to 5,600-fold binding capacity to AQP2 expressing cell membranes or endoplasmic reticulum compared to T7 uninserted phage clones (Table 2). In addition, another binding assay was performed to reconfirm whether these phageclones selectively bind to cell or vesicle membranes expressing AQP2. That is, a cell membrane or endoplasmic reticulum containing H + -ATPase (B1-subunit) expressing only the selected phage clones in the liver cells prepared from the rat kidney (cells other than the main cells expressing AQP2 in the collection tube). The membrane samples were combined (Table 3). The seven phage clones selected showed 0.4- to 3.5-fold lower binding capacity, unlike the results of membrane samples expressing AQP2 in membrane samples containing H + -ATPase (Table 3). Therefore, the above results indicate that the seven phageclones selected in this study have high selectivity in AQP2 expressing cell membrane and vesicle membrane samples.
상기 표 1에서는 AQP2를 발현하는 세포막 (PM)과 세포내 소포체막 (ICV)에서 집적된 파지들 중 임의로 160개의 파지클론들을 선택하였으며 이 중 동일한 아미노산 잔기들이 많이 발견되거나, 유사한 시퀀스의 아미노산 잔기들이 많이 발견되는 일곱개의 파지클론들을 선별하였다.In Table 1, randomly selected 160 phage clones among phages accumulated in the cell membrane (PM) and intracellular vesicle membrane (ICV) expressing AQP2 are selected. Seven phage clones that were found are selected.
상기 표 2에서는 일곱개의 선별된 파지 클론들과 파지입자 (capsid)의 표면에 펩타이드 리간드를 발현하지 않는 T7 insertless 파지클론의 AQP2를 발현하는 세포막 또는 소포체막에 대한 결합능 비교하였다. 일곱개의 파지클론들은 AQP2를 발현하는 세포막 또는 소포체막에서 T7 insertless 파지클론들에 비해 약 50배에서 5,600배 높은 결합능을 보였다.Table 2 compares the binding capacity of the seven selected phage clones and cell membranes or vesicle membranes expressing AQP2 of T7 insertless phage clones that do not express peptide ligands on the surface of phage particles (capsid). Seven phage clones showed about 50 to 5,600 times higher binding capacity than T7 insertless phage clones in cell membranes or endoplasmic reticulum membranes expressing AQP2.
상기 표 3에서는 일곱개의 선별된 파지 클론들과 파지입자 (capsid)의 표면에 펩타이드 리간드를 발현하지 않는 T7 미삽입 파지클론의 H+-ATPase (B1-subunit)를 발현하는 세포막 또는 소포체막에 대한 결합능 비교하였다. 일곱개의 파지클론들은 H+-ATPase (B1-subunit)를 발현하는 세포막 또는 소포체막에서 T7 insertless 파지클론들에 비해 AQP2를 발현하는 막시료에서 나타난 결과와는 달리 0.4배에서 3.5배의 낮은 결합능을 보였다.Table 3 shows seven selected phage clones and cell membranes or vesicle membranes expressing H + -ATPase (B1-subunit) of T7 non-inserted phage clones that do not express peptide ligands on the surface of phage particles (capsid). Binding capacity was compared. The seven phageclones showed 0.4- to 3.5-fold lower binding capacity, compared with those found in AQP2-expressing membrane samples compared to T7 insertless phageclones in cell membranes or endoplasmic reticulum membranes expressing H + -ATPase (B1-subunit). Seemed.
3. 선택된 3. Selected 파지클론의Phageclone 펩타이드Peptide 시퀀스와Sequence and 상동성을Homology 가지는 단백질 선별 Eggplant Protein Selection
AQP2가 발현하는 세포막 또는 세포내 소포체막 시료에서 각각 80개씩 임의의 파지클론들을 선택하였으며, 총 160개의 파지클론들은 PCR을 통해 T7 파지표면에 발현하고 있는 아미노산 시퀀스에 상응하는 DNA를 증폭하여 시퀀싱했다. 이렇게 AQP2를 발현하는 세포막 또는 소포체막에 선택적인 파지 라이브러리 중에 무작위로 추출한 160개의 파지클론에서 높은 빈도로 동일한 시퀀스 또는 유사한 시퀀스를 보이는 7개의 파지클론을 선택하였다 (표 1). 이중 3개의 펩타이드 시퀀스는 소포체막 시료에서, 1개의 펩타이드 시퀀스는 세포막 시료에서, 그리고 나머지 3개의 시퀀스는 세포막과 소포체막 모두에서 확인되었다. 각각의 펩타이드 시퀀스와 상동성을 가진 실험쥐의 단백질들을 The National Center for Biotechnology Information에서 이용할 수 있는 BLAST program을 이용하여 검색하였다 (표 4 내지 6). A total of 80 phage clones were selected from AQP2 expressing cell membranes or intracellular vesicle membrane samples, and a total of 160 phage clones were sequenced by amplifying DNA corresponding to amino acid sequences expressed on T7 phage surface by PCR. . Thus, 7 phage clones showing the same or similar sequence with high frequency were selected from 160 phage clones randomly extracted from the phage library selective to AQP2 expressing cell membrane or endoplasmic reticulum membrane (Table 1). Three peptide sequences were identified in the endoplasmic reticulum membrane sample, one peptide sequence in the cell membrane sample, and the remaining three sequences in both the cell membrane and the endoplasmic reticulum membrane. Proteins of mice having homology with each peptide sequence were searched using the BLAST program available from The National Center for Biotechnology Information (Tables 4-6).
상기 표 4에서는 세포내 소포체막에서 집적된 파지클론 (높은 빈도를 보인 선택된 파지클론) 및 각각의 파지클론이 디스플레이하는 펩타이드와 시퀀스 상동성을 가지는 단백질을 나타낸다.Table 4 shows the proteins having sequence homology with the phage clones (highly selected selected phage clones) accumulated in the intracellular vesicle membrane and the peptides displayed by each phage clone.
상기 표 5에서는 세포막에서 집적된 파지클론 (높은 빈도를 보인 선택된 파지클론) 및 각각의 파지클론이 디스플레이하는 펩타이드와 시퀀스 상동성을 가지는 단백질을 나타낸다.Table 5 shows the proteins having sequence homology with the phage clones (highly selected phage clones) accumulated in the cell membrane and the peptides displayed by each phage clone.
상기 표 6에서는 세포막과 세포내 소포체막에서 동시에 집적된 파지클론 (높은 빈도를 보인 선택된 파지클론) 및 각각의 파지클론이 디스플레이하는 펩타이드와 시퀀스 상동성을 가지는 단백질을 나타낸다.Table 6 shows the proteins having sequence homology with the phageclones (highly selected selected phageclones) and phage clones displayed at the same time in the cell membrane and the intracellular vesicle membrane.
4. 일차배양된 속수질 4. Primary Cultured Water Quality 집합관Assembly tube 세포에서 In the cell FITCFITC 가 end 결합된Combined 펩타이드Peptide 염색 dyeing
본 발명자들은 일차배양된 속수질 집합관 세포>에서 항이뇨호르몬 V2-receptor 자극제인 항이뇨호르몬 (dDAVP)를 처리 (10-11M, 15분)함으로서 AQP2의 세포막으로의 이동이 유의하게 촉진됨을 이전의 연구에서 확인하였다(Lee YJ et al., Am J Physiol Renal Physiol 292: F340-F350, 2007.). 본 연구에서는 선택된 펩타이드 리간드 중에 특히 AQP2를 발현하는 소포체막에서 집적시킨 파지클론 (SRSRNKT와 KNMRSSA)의 세포 내 위치와 짧은 시간동안의 dDAVP 처리에 따른 펩타이드 리간드의 세포 내 위치이동 변화를 확인하고자 하였다. FITC가 결합된 펩타이드들을 일차배양된 속수질 집합관 세포에서 dDAVP (10-8 M)를 20분간 처리한 군과 그렇지 않은 군에서 각각 세포 염색을 실시하였다 (그림 4). dDAVP처리를 하지 않았을 때, FITC-SRSRNKT 염색은 세포질에서 진하게 발현되는 것을 확인하였고 (그림 4A), FITC-KNMRSSA의 염색은 세포질 내 구조물 (그림 4C)과 세포막 (그림 4C, 화살표로 표시된 부분)에서 모두 발현되는 것을 확인하였다. 또한 짧은 시간동안 dDAVP를 처리하였을 때 (10-8M, 20 분), FITC-SRSRNKT, FITC-NMRSSA로 염색된 세포 모두에서 세포질 내 염색패턴의 변화를 볼 수 없었다(그림 4B and D). 대조적으로 FITC 와 결합된 대조군 펩티드(FITC-NSSVDK)는 일차배양된 집합관 속수질 세포에서 염색이 되지 않거나 매우 약하게 발현하는 것을 관찰하였다 (그림 4G, H). 추가적으로 AQP2 를 발현하는 세포막과 소포체막 시료 모두에서 찾아낸 FITC-PKQRFWP로 염색해 본 결과 세포질 부분과 세포막 부분 모두에서 염색되는 것을 관찰하였다 (그림 4E). 이것은 이 파지클론이 세포막과 소포체막 모두에서 선택적 결합력을 보인 점과 일관된 결과였다 (표 2). 그러나 짧은 시간의 dDAVP 처리에 의한 염색패턴의 변화는 관찰되지 않았다 (그림 4F).The present inventors have shown that the treatment of antidiuretic hormone (dDAVP), an antidiuretic hormone V2-receptor stimulant (dDAVP), in primary cultured medullary collecting tube cells> (10 -11 M, 15 minutes) significantly promoted the migration of AQP2 to the cell membrane. (Lee YJ et al., Am J Physiol Renal Physiol 292: F340-F350, 2007.). In this study, we examined the intracellular location of phageclone (SRSRNKT and KNMRSSA) integrated in the vesicle membrane expressing AQP2 among the selected peptide ligands, and the intracellular shift of peptide ligands by dDAVP treatment for a short time. Peptides bound to FITC were stained in groups treated with dDAVP (10 -8 M) for 20 minutes in primary stromal collection duct cells (Figure 4). In the absence of dDAVP treatment, FITC-SRSRNKT staining was strongly expressed in the cytoplasm (Fig. 4A), and FITC-KNMRSSA staining was observed in the cytoplasmic structures (Fig. 4C) and in the cell membrane (Fig. 4C, arrows). It was confirmed that all are expressed. In addition, when dDAVP was treated for a short period of time (10 -8 M, 20 minutes), no change in the cytoplasmic staining pattern was observed in the cells stained with FITC-SRSRNKT and FITC-NMRSSA (Fig. 4B and D). In contrast, FITC-coupled control peptides (FITC-NSSVDK) were observed to be unstained or very weakly expressed in primary cultured stromal stromal stromal cells (Figure 4G, H). In addition, staining with FITC-PKQRFWP found in both AQP2-expressing cell and vesicle membrane samples showed that the cells were stained at both the cytoplasmic and cellular membranes (Figure 4E). This was consistent with the fact that the phageclone showed selective binding on both cell and vesicle membranes (Table 2). However, no change in staining pattern was observed by dDAVP treatment for a short time (Fig. 4F).
이상 살펴본 바와 같이, 본 발명의 서열번호 1 내지 서열번호 16으로 표시되는 아미노산 서열을 가지는 펩타이드는 신장 집합관 세포에 주로 발현되는 제2형 수분통로단백이 포배된 막과 특이적으로 결합할 수 있다. 따라서, 본 발명의 펩타이드는 제2형 수분통로단백이 포배된 막의 검출, 더 나아가 수분 대사 질환의 진단, 영상화 및 표적 약물전달 등에 다양하게 사용될 수 있다.As described above, the peptide having the amino acid sequence represented by SEQ ID NO: 1 to SEQ ID NO: 16 of the present invention can specifically bind to the membrane cultivated the type 2 water passage protein mainly expressed in renal duct cells. Therefore, the peptide of the present invention can be used for various purposes such as detection of membranes in which the type 2 moisture channel protein is cultivated, furthermore, diagnosis of water metabolism disorders, imaging, and targeted drug delivery.
도 1은 AQP2에 대한 면역블럿(A) 및 AQP2, AQP1, H+-ATPase에 대한 면역블럿(B) 결과를 나타낸 것이다.Figure 1 shows the immunoblot (A) for AQP2 and immunoblot (B) results for AQP2, AQP1, H + -ATPase.
도 2는 T7 파지의 바이오패닝 및 플라크 에세이를 모식적으로 나타낸 것이다.Figure 2 schematically shows biopanning and plaque assays of T7 phage.
도 3은 선별한 파지 클론들을 선택적 결합 확인 과정을 모식적으로 나타낸 것이다.3 schematically illustrates a process for selective binding of selected phage clones.
도 4는 속수질 집합관(IMCD)세포에 FITC가 결합된 본 발명의 펩타이드의 특이적 결합을 확인한 것이다.Figure 4 confirms the specific binding of the peptide of the present invention in which FITC is bound to the medulla collection tube (IMCD) cells.
<110> Kyungpook national university industry-academic cooperation foundation <120> Peptides for binding specifically to membrane embedded with aquaporin-2 and uses thereof <130> NP08-0049 <160> 19 <170> KopatentIn 1.71 <210> 1 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> peptide 1 <400> 1 Cys Pro Lys Gln Arg Phe Trp Pro Cys 1 5 <210> 2 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> peptide 2 <400> 2 Cys Lys Arg Val Thr Gly Arg Pro Cys 1 5 <210> 3 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> peptide 3 <400> 3 Cys Lys Asn Met Arg Ser Ser Ala Cys 1 5 <210> 4 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> peptide 4 <400> 4 Cys Leu Pro Met Arg Ala Lys Cys Cys 1 5 <210> 5 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> peptide 5 <400> 5 Cys Ser Arg Ser Arg Asn Lys Thr Cys 1 5 <210> 6 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> peptide 6 <400> 6 Cys Ser Arg Ser Lys Ala Thr His Cys 1 5 <210> 7 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> peptide 7 <400> 7 Cys Ala Arg Val Lys Gly Thr His Cys 1 5 <210> 8 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> peptide 8 <400> 8 Cys Ser Arg Val Thr Gly Arg Pro Cys 1 5 <210> 9 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> peptide 9 <400> 9 Cys Lys Arg Val Thr Gly Arg Ala Cys 1 5 <210> 10 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> peptide 10 <400> 10 Cys Lys Arg Val Ile Gly Arg Asn Cys 1 5 <210> 11 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> peptide 11 <400> 11 Cys Gln Asn Met Arg Ser Ser Ala Cys 1 5 <210> 12 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> peptide 12 <400> 12 Cys Gln Asn Met Arg Ser Lys Ala Cys 1 5 <210> 13 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> peptide 13 <400> 13 Cys Gly Ser Arg Ser Asn Arg Ala Cys 1 5 <210> 14 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> peptide 14 <400> 14 Cys Arg Val Lys Ala Thr His 1 5 <210> 15 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> peptide 15 <400> 15 Cys Val Arg Ala Lys Ala Thr His Cys 1 5 <210> 16 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> peptide 16 <400> 16 Cys Ser Arg Ser Lys Ser Val His Cys 1 5 <210> 17 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> peptide for KLH conjugation <400> 17 Cys Glu Val Arg Arg Arg Gln Ser Val Glu Leu His Ser Pro Gln Ser 1 5 10 15 Leu Pro Arg Gly Ser Lys Ala 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 18 agcggaccag attatcgcta 20 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 19 aacccctcaa gacccgttta 20 <110> Kyungpook national university industry-academic cooperation foundation <120> Peptides for binding specifically to membrane embedded with aquaporin-2 and uses approximately <130> NP08-0049 <160> 19 <170> KopatentIn 1.71 <210> 1 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> peptide 1 <400> 1 Cys Pro Lys Gln Arg Phe Trp Pro Cys 1 5 <210> 2 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> peptide 2 <400> 2 Cys Lys Arg Val Thr Gly Arg Pro Cys 1 5 <210> 3 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> peptide 3 <400> 3 Cys Lys Asn Met Arg Ser Ser Ala Cys 1 5 <210> 4 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> peptide 4 <400> 4 Cys Leu Pro Met Arg Ala Lys Cys Cys 1 5 <210> 5 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> peptide 5 <400> 5 Cys Ser Arg Ser Arg Asn Lys Thr Cys 1 5 <210> 6 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> peptide 6 <400> 6 Cys Ser Arg Ser Lys Ala Thr His Cys 1 5 <210> 7 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> peptide 7 <400> 7 Cys Ala Arg Val Lys Gly Thr His Cys 1 5 <210> 8 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> peptide 8 <400> 8 Cys Ser Arg Val Thr Gly Arg Pro Cys 1 5 <210> 9 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> peptide 9 <400> 9 Cys Lys Arg Val Thr Gly Arg Ala Cys 1 5 <210> 10 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> peptide 10 <400> 10 Cys Lys Arg Val Ile Gly Arg Asn Cys 1 5 <210> 11 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> peptide 11 <400> 11 Cys Gln Asn Met Arg Ser Ser Ala Cys 1 5 <210> 12 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> peptide 12 <400> 12 Cys Gln Asn Met Arg Ser Lys Ala Cys 1 5 <210> 13 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> peptide 13 <400> 13 Cys Gly Ser Arg Ser Asn Arg Ala Cys 1 5 <210> 14 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> peptide 14 <400> 14 Cys Arg Val Lys Ala Thr His 1 5 <210> 15 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> peptide 15 <400> 15 Cys Val Arg Ala Lys Ala Thr His Cys 1 5 <210> 16 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> peptide 16 <400> 16 Cys Ser Arg Ser Lys Ser Val His Cys 1 5 <210> 17 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> peptide for KLH conjugation <400> 17 Cys Glu Val Arg Arg Arg Gln Ser Val Glu Leu His Ser Pro Gln Ser 1 5 10 15 Leu Pro Arg Gly Ser Lys Ala 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 18 agcggaccag attatcgcta 20 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 19 aacccctcaa gacccgttta 20
Claims (14)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020080063831A KR100981531B1 (en) | 2008-07-02 | 2008-07-02 | Peptides that specifically bind to the membrane of the type 2 moisture channel protein and its use |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020080063831A KR100981531B1 (en) | 2008-07-02 | 2008-07-02 | Peptides that specifically bind to the membrane of the type 2 moisture channel protein and its use |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| KR20100003823A true KR20100003823A (en) | 2010-01-12 |
| KR100981531B1 KR100981531B1 (en) | 2010-09-10 |
Family
ID=41813524
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| KR1020080063831A KR100981531B1 (en) | 2008-07-02 | 2008-07-02 | Peptides that specifically bind to the membrane of the type 2 moisture channel protein and its use |
Country Status (1)
| Country | Link |
|---|---|
| KR (1) | KR100981531B1 (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104258155A (en) * | 2014-10-14 | 2015-01-07 | 谢显孝 | Pharmaceutical composition for treating diabetes insipidus |
| CN107607511A (en) * | 2017-10-21 | 2018-01-19 | 云南中烟工业有限责任公司 | The detection method that a kind of cigarette smoke influences on the Subcellular Localization of aquaporin 5 |
-
2008
- 2008-07-02 KR KR1020080063831A patent/KR100981531B1/en active IP Right Grant
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104258155A (en) * | 2014-10-14 | 2015-01-07 | 谢显孝 | Pharmaceutical composition for treating diabetes insipidus |
| CN107607511A (en) * | 2017-10-21 | 2018-01-19 | 云南中烟工业有限责任公司 | The detection method that a kind of cigarette smoke influences on the Subcellular Localization of aquaporin 5 |
Also Published As
| Publication number | Publication date |
|---|---|
| KR100981531B1 (en) | 2010-09-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP6676589B2 (en) | Compstatin analogs with improved pharmacokinetic properties | |
| US8907053B2 (en) | Immunosuppression modulating compounds | |
| US8877716B2 (en) | Peptide derivatives, preparation and uses thereof | |
| JP5462193B2 (en) | Aprotinin polypeptides for transporting compounds across the blood brain barrier | |
| US9539341B2 (en) | Methods and compositions for targeting adipose cells in mammals | |
| US9504731B2 (en) | Therapeutic agent, treatment method and inspection method for diseases caused by activation of neutrophils | |
| CN102282159A (en) | Peptide derivatives and use thereof as carriers for molecules in the form of conjugates | |
| Baranyai et al. | Cellular internalization and inhibition capacity of new anti-glioma peptide conjugates: physicochemical characterization and evaluation on various monolayer-and 3D-spheroid-based in vitro platforms | |
| KR20120125455A (en) | Intracelluar targeting bipodal peptide binder | |
| BG107214A (en) | Thrombopoietin receptor modulating peptide | |
| US9890197B2 (en) | RHAMM binding peptides | |
| EP2280024B1 (en) | Polypeptide specifically binding to phosphatidylserine and use thereof | |
| EP2908837B1 (en) | Compositions for drug delivery | |
| CN114621325B (en) | A fibronectin targeting polypeptide and its application in promoting tumor anoikis and chemotherapy sensitization | |
| KR100981531B1 (en) | Peptides that specifically bind to the membrane of the type 2 moisture channel protein and its use | |
| US20030232762A1 (en) | Breast homing peptides and methods of identifying same using aminopeptidase P | |
| US20230077821A1 (en) | BCL-W Polypeptides and Mimetics for Treating or Preventing Chemotherapy-Induced Peripheral Neuropathy and Hearing Loss | |
| JP7436710B2 (en) | Novel nucleolin-binding peptides and their uses | |
| CN113597318A (en) | Therapeutic nanoconjugates and uses thereof | |
| KR20240032207A (en) | Peptides that selectively bind to Trop2 and use thereof | |
| CN117838877A (en) | A polypeptide P12-drug conjugate and its application in preparing a drug for treating pancreatic cancer | |
| WO2002099379A2 (en) | Breast homing peptides and methods of identifying same using aminopeptidase p |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A201 | Request for examination | ||
| PA0109 | Patent application |
Patent event code: PA01091R01D Comment text: Patent Application Patent event date: 20080702 |
|
| PA0201 | Request for examination | ||
| PG1501 | Laying open of application | ||
| E701 | Decision to grant or registration of patent right | ||
| PE0701 | Decision of registration |
Patent event code: PE07011S01D Comment text: Decision to Grant Registration Patent event date: 20100820 |
|
| GRNT | Written decision to grant | ||
| PR0701 | Registration of establishment |
Comment text: Registration of Establishment Patent event date: 20100903 Patent event code: PR07011E01D |
|
| PR1002 | Payment of registration fee |
Payment date: 20100903 End annual number: 3 Start annual number: 1 |
|
| PG1601 | Publication of registration | ||
| FPAY | Annual fee payment |
Payment date: 20130828 Year of fee payment: 4 |
|
| PR1001 | Payment of annual fee |
Payment date: 20130828 Start annual number: 4 End annual number: 4 |
|
| FPAY | Annual fee payment |
Payment date: 20140822 Year of fee payment: 5 |
|
| PR1001 | Payment of annual fee |
Payment date: 20140822 Start annual number: 5 End annual number: 5 |
|
| FPAY | Annual fee payment |
Payment date: 20150826 Year of fee payment: 6 |
|
| PR1001 | Payment of annual fee |
Payment date: 20150826 Start annual number: 6 End annual number: 6 |
|
| FPAY | Annual fee payment |
Payment date: 20160818 Year of fee payment: 7 |
|
| PR1001 | Payment of annual fee |
Payment date: 20160818 Start annual number: 7 End annual number: 7 |
|
| FPAY | Annual fee payment |
Payment date: 20170817 Year of fee payment: 8 |
|
| PR1001 | Payment of annual fee |
Payment date: 20170817 Start annual number: 8 End annual number: 8 |
|
| FPAY | Annual fee payment |
Payment date: 20180822 Year of fee payment: 9 |
|
| PR1001 | Payment of annual fee |
Payment date: 20180822 Start annual number: 9 End annual number: 9 |
|
| FPAY | Annual fee payment |
Payment date: 20190827 Year of fee payment: 10 |
|
| PR1001 | Payment of annual fee |
Payment date: 20190827 Start annual number: 10 End annual number: 10 |
|
| PR1001 | Payment of annual fee |
Payment date: 20200831 Start annual number: 11 End annual number: 11 |
|
| PC1903 | Unpaid annual fee |
Termination category: Default of registration fee Termination date: 20220614 |