WO2024128526A1 - Antimicrobial protein cdl2200 having lytic activity to clostridioides difficile - Google Patents
Antimicrobial protein cdl2200 having lytic activity to clostridioides difficile Download PDFInfo
- Publication number
- WO2024128526A1 WO2024128526A1 PCT/KR2023/016633 KR2023016633W WO2024128526A1 WO 2024128526 A1 WO2024128526 A1 WO 2024128526A1 KR 2023016633 W KR2023016633 W KR 2023016633W WO 2024128526 A1 WO2024128526 A1 WO 2024128526A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- cdl2200
- antibacterial protein
- clostridioides difficile
- seq
- antibacterial
- Prior art date
Links
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 171
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 159
- 241000193163 Clostridioides difficile Species 0.000 title claims abstract description 87
- 230000000845 anti-microbial effect Effects 0.000 title abstract 3
- 230000002101 lytic effect Effects 0.000 title description 15
- 230000000844 anti-bacterial effect Effects 0.000 claims abstract description 173
- 239000008194 pharmaceutical composition Substances 0.000 claims abstract description 32
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 19
- 239000004480 active ingredient Substances 0.000 claims abstract description 11
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract 5
- 150000001413 amino acids Chemical class 0.000 claims description 54
- 239000003242 anti bacterial agent Substances 0.000 claims description 16
- 241000588724 Escherichia coli Species 0.000 claims description 13
- 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 claims description 12
- 239000013612 plasmid Substances 0.000 claims description 12
- 230000001131 transforming effect Effects 0.000 claims description 12
- 208000037384 Clostridium Infections Diseases 0.000 claims description 10
- 230000003115 biocidal effect Effects 0.000 claims description 10
- 241000894006 Bacteria Species 0.000 claims description 9
- 238000011282 treatment Methods 0.000 claims description 9
- 239000000645 desinfectant Substances 0.000 claims description 6
- 230000000968 intestinal effect Effects 0.000 claims description 4
- 206010009657 Clostridium difficile colitis Diseases 0.000 claims description 3
- 206010012735 Diarrhoea Diseases 0.000 claims description 3
- 206010061218 Inflammation Diseases 0.000 claims description 3
- 208000003100 Pseudomembranous Enterocolitis Diseases 0.000 claims description 3
- 206010037128 Pseudomembranous colitis Diseases 0.000 claims description 3
- 206010040047 Sepsis Diseases 0.000 claims description 3
- 239000003814 drug Substances 0.000 claims description 3
- 230000004054 inflammatory process Effects 0.000 claims description 3
- 230000017074 necrotic cell death Effects 0.000 claims description 3
- 229940124597 therapeutic agent Drugs 0.000 claims description 3
- 201000002516 toxic megacolon Diseases 0.000 claims description 3
- 206010018001 Gastrointestinal perforation Diseases 0.000 claims description 2
- 206010028851 Necrosis Diseases 0.000 claims description 2
- 208000035473 Communicable disease Diseases 0.000 claims 6
- 208000015181 infectious disease Diseases 0.000 abstract description 23
- 238000000034 method Methods 0.000 abstract description 21
- 201000010099 disease Diseases 0.000 abstract description 17
- 241000064585 Clostridioides Species 0.000 abstract description 2
- 239000004599 antimicrobial Substances 0.000 abstract 1
- 238000004519 manufacturing process Methods 0.000 description 23
- 229940088710 antibiotic agent Drugs 0.000 description 12
- 239000000203 mixture Substances 0.000 description 10
- 241001465754 Metazoa Species 0.000 description 9
- 239000000872 buffer Substances 0.000 description 9
- 239000008057 potassium phosphate buffer Substances 0.000 description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 230000009467 reduction Effects 0.000 description 5
- 238000011160 research Methods 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 241000700159 Rattus Species 0.000 description 4
- 125000000539 amino acid group Chemical group 0.000 description 4
- 238000012937 correction Methods 0.000 description 4
- 230000008520 organization Effects 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 208000035143 Bacterial infection Diseases 0.000 description 3
- 241000672609 Escherichia coli BL21 Species 0.000 description 3
- 241000233866 Fungi Species 0.000 description 3
- 108010059993 Vancomycin Proteins 0.000 description 3
- 239000002775 capsule Substances 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 239000003651 drinking water Substances 0.000 description 3
- 235000020188 drinking water Nutrition 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 230000002496 gastric effect Effects 0.000 description 3
- 229930027917 kanamycin Natural products 0.000 description 3
- 229960000318 kanamycin Drugs 0.000 description 3
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 3
- 229930182823 kanamycin A Natural products 0.000 description 3
- 230000002934 lysing effect Effects 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 244000052769 pathogen Species 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 241000894007 species Species 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 230000001225 therapeutic effect Effects 0.000 description 3
- 229960003165 vancomycin Drugs 0.000 description 3
- MYPYJXKWCTUITO-LYRMYLQWSA-N vancomycin Chemical compound O([C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=C2C=C3C=C1OC1=CC=C(C=C1Cl)[C@@H](O)[C@H](C(N[C@@H](CC(N)=O)C(=O)N[C@H]3C(=O)N[C@H]1C(=O)N[C@H](C(N[C@@H](C3=CC(O)=CC(O)=C3C=3C(O)=CC=C1C=3)C(O)=O)=O)[C@H](O)C1=CC=C(C(=C1)Cl)O2)=O)NC(=O)[C@@H](CC(C)C)NC)[C@H]1C[C@](C)(N)[C@H](O)[C@H](C)O1 MYPYJXKWCTUITO-LYRMYLQWSA-N 0.000 description 3
- MYPYJXKWCTUITO-UHFFFAOYSA-N vancomycin Natural products O1C(C(=C2)Cl)=CC=C2C(O)C(C(NC(C2=CC(O)=CC(O)=C2C=2C(O)=CC=C3C=2)C(O)=O)=O)NC(=O)C3NC(=O)C2NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(CC(C)C)NC)C(O)C(C=C3Cl)=CC=C3OC3=CC2=CC1=C3OC1OC(CO)C(O)C(O)C1OC1CC(C)(N)C(O)C(C)O1 MYPYJXKWCTUITO-UHFFFAOYSA-N 0.000 description 3
- 241000648967 Clostridioides difficile ATCC 43255 Species 0.000 description 2
- 101150056637 Hrh2 gene Proteins 0.000 description 2
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 2
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 2
- 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 2
- 229930006000 Sucrose Natural products 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 208000022362 bacterial infectious disease Diseases 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 229940041514 candida albicans extract Drugs 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 229960002227 clindamycin Drugs 0.000 description 2
- KDLRVYVGXIQJDK-AWPVFWJPSA-N clindamycin Chemical compound CN1C[C@H](CCC)C[C@H]1C(=O)N[C@H]([C@H](C)Cl)[C@@H]1[C@H](O)[C@H](O)[C@@H](O)[C@@H](SC)O1 KDLRVYVGXIQJDK-AWPVFWJPSA-N 0.000 description 2
- 230000034994 death Effects 0.000 description 2
- 231100000517 death Toxicity 0.000 description 2
- 239000003937 drug carrier Substances 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 239000003112 inhibitor Substances 0.000 description 2
- 210000000936 intestine Anatomy 0.000 description 2
- 238000011835 investigation Methods 0.000 description 2
- HQKMJHAJHXVSDF-UHFFFAOYSA-L magnesium stearate Chemical compound [Mg+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O HQKMJHAJHXVSDF-UHFFFAOYSA-L 0.000 description 2
- 229960000282 metronidazole Drugs 0.000 description 2
- VAOCPAMSLUNLGC-UHFFFAOYSA-N metronidazole Chemical compound CC1=NC=C([N+]([O-])=O)N1CCO VAOCPAMSLUNLGC-UHFFFAOYSA-N 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 238000007911 parenteral administration Methods 0.000 description 2
- 239000000546 pharmaceutical excipient Substances 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 230000002265 prevention Effects 0.000 description 2
- 239000012460 protein solution Substances 0.000 description 2
- 239000013049 sediment Substances 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 238000005507 spraying Methods 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 239000012138 yeast extract Substances 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
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- FHVDTGUDJYJELY-UHFFFAOYSA-N 6-{[2-carboxy-4,5-dihydroxy-6-(phosphanyloxy)oxan-3-yl]oxy}-4,5-dihydroxy-3-phosphanyloxane-2-carboxylic acid Chemical compound O1C(C(O)=O)C(P)C(O)C(O)C1OC1C(C(O)=O)OC(OP)C(O)C1O FHVDTGUDJYJELY-UHFFFAOYSA-N 0.000 description 1
- 244000215068 Acacia senegal Species 0.000 description 1
- 235000006491 Acacia senegal Nutrition 0.000 description 1
- 241000589291 Acinetobacter Species 0.000 description 1
- 229920001817 Agar Polymers 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
- 229930186147 Cephalosporin Natural products 0.000 description 1
- 108010078777 Colistin Proteins 0.000 description 1
- 101710112752 Cytotoxin Proteins 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
- 241000588921 Enterobacteriaceae Species 0.000 description 1
- 241000194032 Enterococcus faecalis Species 0.000 description 1
- 241000194031 Enterococcus faecium Species 0.000 description 1
- 101710146739 Enterotoxin Proteins 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 229930182566 Gentamicin Natural products 0.000 description 1
- CEAZRRDELHUEMR-URQXQFDESA-N Gentamicin Chemical compound O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N CEAZRRDELHUEMR-URQXQFDESA-N 0.000 description 1
- 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 1
- 229920000084 Gum arabic Polymers 0.000 description 1
- 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 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 229920000168 Microcrystalline cellulose Polymers 0.000 description 1
- 206010029803 Nosocomial infection Diseases 0.000 description 1
- 208000001388 Opportunistic Infections Diseases 0.000 description 1
- 206010034133 Pathogen resistance Diseases 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- 241000607142 Salmonella Species 0.000 description 1
- 101710084578 Short neurotoxin 1 Proteins 0.000 description 1
- 241000191940 Staphylococcus Species 0.000 description 1
- 241000191967 Staphylococcus aureus Species 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 101710182223 Toxin B Proteins 0.000 description 1
- 101710182532 Toxin a Proteins 0.000 description 1
- UFUVLHLTWXBHGZ-MGZQPHGTSA-N [(2r,3r,4s,5r,6r)-6-[(1s,2s)-2-chloro-1-[[(2s,4r)-1-methyl-4-propylpyrrolidine-2-carbonyl]amino]propyl]-4,5-dihydroxy-2-methylsulfanyloxan-3-yl] dihydrogen phosphate Chemical compound CN1C[C@H](CCC)C[C@H]1C(=O)N[C@H]([C@H](C)Cl)[C@@H]1[C@H](O)[C@H](O)[C@@H](OP(O)(O)=O)[C@@H](SC)O1 UFUVLHLTWXBHGZ-MGZQPHGTSA-N 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 235000010489 acacia gum Nutrition 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 229940072056 alginate Drugs 0.000 description 1
- 235000010443 alginic acid Nutrition 0.000 description 1
- 229920000615 alginic acid Polymers 0.000 description 1
- 229940024606 amino acid Drugs 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000012736 aqueous medium Substances 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 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 1
- 230000037396 body weight Effects 0.000 description 1
- 210000004556 brain 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
- 239000000378 calcium silicate Substances 0.000 description 1
- 229910052918 calcium silicate Inorganic materials 0.000 description 1
- 235000012241 calcium silicate Nutrition 0.000 description 1
- OYACROKNLOSFPA-UHFFFAOYSA-N calcium;dioxido(oxo)silane Chemical compound [Ca+2].[O-][Si]([O-])=O OYACROKNLOSFPA-UHFFFAOYSA-N 0.000 description 1
- 229940041011 carbapenems Drugs 0.000 description 1
- 238000005277 cation exchange chromatography Methods 0.000 description 1
- 239000003729 cation exchange resin Substances 0.000 description 1
- 239000012930 cell culture fluid Substances 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 235000010980 cellulose Nutrition 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 229940124587 cephalosporin Drugs 0.000 description 1
- 150000001780 cephalosporins Chemical class 0.000 description 1
- 229940024674 clindamycin injection Drugs 0.000 description 1
- 229960003346 colistin Drugs 0.000 description 1
- 231100000599 cytotoxic agent Toxicity 0.000 description 1
- 239000002619 cytotoxin Substances 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 239000008121 dextrose Substances 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 239000002270 dispersing agent Substances 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 239000003995 emulsifying agent Substances 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 229940032049 enterococcus faecalis Drugs 0.000 description 1
- 239000000147 enterotoxin Substances 0.000 description 1
- 231100000655 enterotoxin Toxicity 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 230000029142 excretion Effects 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 229940124307 fluoroquinolone Drugs 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 235000013355 food flavoring agent Nutrition 0.000 description 1
- 235000003599 food sweetener Nutrition 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 229960002518 gentamicin Drugs 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 229940090044 injection Drugs 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 229940042040 innovative drug Drugs 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 238000007912 intraperitoneal administration Methods 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 239000000314 lubricant Substances 0.000 description 1
- 235000019359 magnesium stearate Nutrition 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
- 229920000609 methyl cellulose Polymers 0.000 description 1
- 239000001923 methylcellulose Substances 0.000 description 1
- 235000010981 methylcellulose Nutrition 0.000 description 1
- LXCFILQKKLGQFO-UHFFFAOYSA-N methylparaben Chemical compound COC(=O)C1=CC=C(O)C=C1 LXCFILQKKLGQFO-UHFFFAOYSA-N 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 239000008108 microcrystalline cellulose Substances 0.000 description 1
- 229940016286 microcrystalline cellulose Drugs 0.000 description 1
- 235000019813 microcrystalline cellulose Nutrition 0.000 description 1
- 235000010446 mineral oil Nutrition 0.000 description 1
- 239000002480 mineral oil Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- JORAUNFTUVJTNG-BSTBCYLQSA-N n-[(2s)-4-amino-1-[[(2s,3r)-1-[[(2s)-4-amino-1-oxo-1-[[(3s,6s,9s,12s,15r,18s,21s)-6,9,18-tris(2-aminoethyl)-3-[(1r)-1-hydroxyethyl]-12,15-bis(2-methylpropyl)-2,5,8,11,14,17,20-heptaoxo-1,4,7,10,13,16,19-heptazacyclotricos-21-yl]amino]butan-2-yl]amino]-3-h Chemical compound CC(C)CCCCC(=O)N[C@@H](CCN)C(=O)N[C@H]([C@@H](C)O)CN[C@@H](CCN)C(=O)N[C@H]1CCNC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCN)NC(=O)[C@H](CCN)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](CC(C)C)NC(=O)[C@H](CCN)NC1=O.CCC(C)CCCCC(=O)N[C@@H](CCN)C(=O)N[C@H]([C@@H](C)O)CN[C@@H](CCN)C(=O)N[C@H]1CCNC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCN)NC(=O)[C@H](CCN)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](CC(C)C)NC(=O)[C@H](CCN)NC1=O JORAUNFTUVJTNG-BSTBCYLQSA-N 0.000 description 1
- 239000003921 oil Substances 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 150000002960 penicillins Chemical class 0.000 description 1
- 239000008177 pharmaceutical agent Substances 0.000 description 1
- XDJYMJULXQKGMM-UHFFFAOYSA-N polymyxin E1 Natural products CCC(C)CCCCC(=O)NC(CCN)C(=O)NC(C(C)O)C(=O)NC(CCN)C(=O)NC1CCNC(=O)C(C(C)O)NC(=O)C(CCN)NC(=O)C(CCN)NC(=O)C(CC(C)C)NC(=O)C(CC(C)C)NC(=O)C(CCN)NC1=O XDJYMJULXQKGMM-UHFFFAOYSA-N 0.000 description 1
- KNIWPHSUTGNZST-UHFFFAOYSA-N polymyxin E2 Natural products CC(C)CCCCC(=O)NC(CCN)C(=O)NC(C(C)O)C(=O)NC(CCN)C(=O)NC1CCNC(=O)C(C(C)O)NC(=O)C(CCN)NC(=O)C(CCN)NC(=O)C(CC(C)C)NC(=O)C(CC(C)C)NC(=O)C(CCN)NC1=O KNIWPHSUTGNZST-UHFFFAOYSA-N 0.000 description 1
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 1
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 1
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- LJPYJRMMPVFEKR-UHFFFAOYSA-N prop-2-ynylurea Chemical compound NC(=O)NCC#C LJPYJRMMPVFEKR-UHFFFAOYSA-N 0.000 description 1
- QELSKZZBTMNZEB-UHFFFAOYSA-N propylparaben Chemical compound CCCOC(=O)C1=CC=C(O)C=C1 QELSKZZBTMNZEB-UHFFFAOYSA-N 0.000 description 1
- 229960003415 propylparaben Drugs 0.000 description 1
- 238000011519 second-line treatment Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 235000010356 sorbitol Nutrition 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000000375 suspending agent Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 239000003765 sweetening agent Substances 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
- 239000000454 talc Substances 0.000 description 1
- 229910052623 talc Inorganic materials 0.000 description 1
- 235000012222 talc Nutrition 0.000 description 1
- 229930101283 tetracycline Natural products 0.000 description 1
- OFVLGDICTFRJMM-WESIUVDSSA-N tetracycline Chemical compound C1=CC=C2[C@](O)(C)[C@H]3C[C@H]4[C@H](N(C)C)C(O)=C(C(N)=O)C(=O)[C@@]4(O)C(O)=C3C(=O)C2=C1O OFVLGDICTFRJMM-WESIUVDSSA-N 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
- 239000012137 tryptone Substances 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 239000000080 wetting agent Substances 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
Definitions
- Clostridioides difficile relates to an antibacterial protein that has lytic activity against bacteria, and more specifically, a sequence that has the ability to specifically lyse Clostridioides difficile bacteria, which can infect animals, including humans, and cause disease.
- Antibacterial protein CDL2200-1 having an amino acid sequence represented by number 1
- antibacterial protein CDL2200-2 having an amino acid sequence represented by SEQ ID NO: 2
- antibacterial protein CDL2200-3 having an amino acid sequence represented by SEQ ID NO: 3, or SEQ ID NO.
- the Clostridioides difficile-specific antibacterial protein CDL2200-1, antibacterial protein CDL2200-2, antibacterial protein CDL2200-3, or antibacterial protein CDL2200-4 It relates to a pharmaceutical composition for treating infections and diseases caused by Clostridioides difficile bacteria, comprising one or more active ingredients.
- Clostridioides difficile difficile is a type of superbug, and it is estimated that 500,000 infections occur in the United States every year, and that the number of deaths within 30 days of diagnosis is approximately 29,000.
- Clostridioides difficile is a Gram-positive spore-forming anaerobic bacterium that exists as a common bacterium in the intestinal tract of humans and animals, but is known to be a pathogen that causes disease through opportunistic infection.
- Clostridioides difficile is MRSA (Methicillin-Resistant Staphylococcus) aureus ) and VRE (Vancomycin-Resistant Enterococci), it is one of the most common pathogens causing hospital-acquired infections.
- Clostridioides difficile bacteria When the balance of the normal intestinal microbial flora is disrupted due to antibiotic treatment, antibiotic-resistant Clostridioides difficile bacteria become dominant in the intestine and secrete toxin A, an enterotoxin, and toxin B, a cytotoxin. It causes inflammation and necrosis of the intestines. In addition, Clostridioides difficile causes diarrhea and pseudomembranous colitis, and if not treated properly, the condition can worsen into sepsis or toxic megacolon, and in severe cases, it can even lead to death.
- Clostridioides difficile infections Almost all antibiotics have been associated with Clostridioides difficile infections, but especially those such as fluoroquinolones, clindamycin, carbapenems, cephalosporins, and penicillins. It occurs frequently in patients taking H2 receptor inhibitors, and there have recently been reports that people taking H2 receptor inhibitors not only have a high rate of Clostridioides difficile infection, but also have a high risk of hospitalization with antibiotic prescriptions. As such, the risk of infection with Clostridioides difficile bacteria is gradually increasing.
- Antibiotics have been widely used to treat infections caused by Clostridioides difficile. It is known that oral metronidazole is mainly used as a first-line treatment and vancomycin is used as a second-line treatment. However, it is known that 8% of all Clostridioides difficile infections are caused by vancomycin-resistant strains. In particular, referring to recent reports, the frequency of emergence of resistant bacteria is steadily increasing, and there is a need for the development of new antibacterial substances that are effective against resistant bacteria. In particular, there is a great need for the development of pharmaceutical agents that have biolytic activity and can quickly remove infectious bacteria from the body, thereby providing rapid therapeutic effects.
- the present inventors provided an antibacterial protein capable of specifically lysing Clostridioides difficile. and further provides a pharmaceutical composition that can be used to treat infection with Clostridioides difficile bacteria containing the same as an active ingredient, and further provides a pharmaceutical composition that can be used to treat infection with Clostridioides difficile bacteria using the pharmaceutical composition.
- the goal is to provide a method for effectively treating infections or diseases.
- the object of the present invention is the antibacterial protein CDL2200-1, which has antibacterial activity capable of specifically lysing Clostridioides difficile bacteria and has an amino acid sequence represented by SEQ ID NO: 1, and an amino acid sequence represented by SEQ ID NO: 2.
- an antibacterial protein CDL2200-2 an antibacterial protein CDL2200-3 having an amino acid sequence shown in SEQ ID NO: 3, and an antibacterial protein CDL2200-4 having an amino acid sequence shown in SEQ ID NO: 4.
- Another object of the present invention is the antibacterial protein CDL2200-1, which has antibacterial activity capable of specifically lysing the Clostridioides difficile bacterium and has an amino acid sequence represented by SEQ ID NO: 1, and an amino acid sequence represented by SEQ ID NO: 2.
- Another object of the present invention is to provide an antibacterial protein CDL2200-1 having an amino acid sequence shown in SEQ ID NO: 1, which can specifically lyse the Clostridioides difficile bacterium, and an antibacterial protein having an amino acid sequence shown in SEQ ID NO: 2.
- another object of the present invention is to provide antibacterial protein CDL2200-1 having an amino acid sequence shown in SEQ ID NO: 1, antibacterial protein CDL2200-2 having an amino acid sequence shown in SEQ ID NO: 2, and an amino acid sequence shown in SEQ ID NO: 3.
- Infection or disease caused by Clostridioides difficile using a pharmaceutical composition containing at least one of the antibacterial protein CDL2200-3 or the antibacterial protein CDL2200-4 with the amino acid sequence shown in SEQ ID NO: 4 as an active ingredient. It provides a method of treatment.
- the inventors of the present invention utilized information on various antibacterial proteins known to have antibacterial activity against various bacterial species, prepared various antibacterial protein candidates in the form of recombinant proteins, and administered these to Clostridioides difficile bacteria.
- Antibacterial proteins with excellent lytic activity were selected by examining their lytic activity, and a method to manufacture them efficiently was developed. Finally, this was used as an active ingredient to treat infections caused by Clostridioides difficile bacteria.
- the present invention was completed by developing a pharmaceutical composition that can be used.
- the present invention provides antibacterial protein CDL2200-1, antibacterial protein CDL2200-2, antibacterial protein CDL2200-3, and antibacterial protein CDL2200 that can specifically lyse Clostridioides difficile bacteria.
- the amino acid sequence of -4 is provided.
- antibacterial protein CDL2200-1 it corresponds to the amino acid sequence of SEQ ID NO: 1
- antibacterial protein CDL2200-2 it corresponds to the amino acid sequence of SEQ ID NO: 2
- SEQ ID NO: 3 in the case of antibacterial protein CDL2200-3, it corresponds to SEQ ID NO: 3.
- the antibacterial protein CDL2200-1 which can specifically lyse Clostridioides difficile bacteria, consists of 178 amino acid residues and has a molecular weight of approximately 20.3 kDa
- the antibacterial protein CDL2200-2 consists of 191 amino acid residues and has a molecular weight of It is about 21.6 kDa
- the antibacterial protein CDL2200-3 is composed of 187 amino acid residues and has a molecular weight of about 21.0 kDa
- the antibacterial protein CDL2200-4 is composed of 185 amino acid residues and has a molecular weight of about 20.7 kDa.
- amino acid sequences of SEQ ID NO: 1 to SEQ ID NO: 4 can be partially modified by those skilled in the art using known techniques.
- the modifications include partial substitution of the amino acid sequence, partial addition of the amino acid sequence, and partial deletion of the amino acid sequence.
- the present invention relates to the production strain Escherichia coli BL21 pASK_CDL2200-1, which can be used for the production of the antibacterial protein CDL2200-1 having the amino acid sequence of SEQ ID NO: 1, and the antibacterial protein CDL2200-2 having the amino acid sequence of SEQ ID NO: 2.
- the production strain Escherichia coli BL21 pASK_CDL2200-2 which can be used for the production of the antibacterial protein CDL2200-3 having the amino acid sequence of SEQ ID NO: 3, and the antibacterial protein having the amino acid sequence of SEQ ID NO: 4
- the production strain Escherichia coli BL21 pASK_CDL2200-4 which can be used for the production of CDL2200-4, is provided.
- the production strain BL21 pASK_CDL2200-1 is a strain created to produce the antibacterial protein CDL2200-1 by transforming E.
- the production strain BL21 pASK_CDL2200-2 is a strain created to produce the antibacterial protein CDL2200-2 by transforming E. coli using a plasmid having the gene sequence of SEQ ID NO: 6 produced by the present inventors
- the production strain BL21 pASK_CDL2200-3 is a sequence produced by the present inventors. It is a strain created to produce the antibacterial protein CDL2200-3 by transforming E. coli using a plasmid having the gene sequence number 7, and the production strain BL21 pASK_CDL2200-4 has the gene sequence number 8 produced by the present inventors.
- This strain was created to produce the antibacterial protein CDL2200-4 by transforming E. coli using a plasmid.
- the production strain BL21 pASK_CDL2200-1, production strain BL21 pASK_CDL2200-2, production strain BL21 pASK_CDL2200-3, and production strain BL21 pASK_CDL2200-4 produced in this way were provided by the present inventors as biological resources of the Korea Research Institute of Bioscience and Biotechnology as of November 29, 2022. deposited at the center.
- accession number is KCTC 15213BP for production strain BL21 pASK_CDL2200-2, KCTC 15214BP for production strain BL21 pASK_CDL2200-3, and KCTC 15215BP for production strain BL21 pASK_CDL2200-4. It is 15216 BP.
- the present invention provides an antibacterial protein CDL2200-1, which is characterized by the amino acid sequence of SEQ ID NO: 1 and has specific lytic activity against Clostridioides difficile bacteria, and has an amino acid sequence of SEQ ID NO: 2.
- CDL2200-2 an antibacterial protein that is characterized and has specific lytic activity against Clostridioides difficile bacteria, is characterized by the amino acid sequence of SEQ ID NO: 3 and has specific lytic activity against Clostridioides difficile bacteria.
- Protein CDL2200-3 Clostridioides difficile containing as an active ingredient one or more of the antibacterial protein CDL2200-4, which is characterized by the amino acid sequence of SEQ ID NO: 4 and has specific lytic activity against Clostridioides difficile bacteria.
- a pharmaceutical composition that can be effectively used to treat bacterial infections or diseases is provided.
- Antibacterial protein CDL2200-1 characterized by the amino acid sequence of SEQ ID NO: 1 according to the present invention and having specific lytic activity against Clostridioides difficile bacteria, included in the pharmaceutical composition of the present invention, amino acid of SEQ ID NO: 2 CDL2200-2, an antibacterial protein characterized by sequence and having specific lytic activity against Clostridioides difficile bacteria, is characterized by the amino acid sequence of SEQ ID NO: 3 and has specific lytic activity against Clostridioides difficile bacteria.
- the antibacterial protein CDL2200-3, or the antibacterial protein CDL2200-4, which is characterized by the amino acid sequence of SEQ ID NO: 4 and has a specific lytic activity against Clostridioides difficile bacteria is, as described above, Clostridioides difficile bacteria. Since it specifically lyses, it is effective in treating various diseases caused by Clostridioides difficile bacteria. Therefore, the pharmaceutical composition of the present invention can be used to treat diseases caused by Clostridioides difficile bacteria. In addition, the pharmaceutical composition of the present invention can be implemented as an antibiotic, disinfectant, disinfectant, therapeutic agent, etc. and used to treat various diseases caused by Clostridioides difficile bacteria.
- the composition containing the antibacterial protein of the present invention is administered to an individual with Clostridioides difficile infection to treat various diseases caused by Clostridioides difficile bacteria. Provides methods for treating them.
- Disease caused by Clostridioides difficile bacteria refers to a disease caused by infection with Clostridioides difficile bacteria, including intestinal inflammation and necrosis, diarrhea, pseudomembranous colitis, gastrointestinal perforation, sepsis, It is a general term for symptoms accompanying toxic megacolon, but is not limited to this.
- “Clostridioides difficile bacteria” may be sensitive to existing antibiotics or resistant to existing antibiotics. In other words, it does not matter whether resistance to existing antibiotics is acquired or not.
- treatment refers to suppressing an infection or disease caused by Clostridioides difficile bacteria and alleviating the pathological condition of a disease caused by Clostridioides difficile bacteria. It means all actions that are ordered.
- antibacterial substances that can provide antibacterial activity against Clostridioides difficile or other bacterial species may be added to the pharmaceutical composition of the present invention.
- Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are those commonly used in preparation, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, Includes, but is limited to, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. It doesn't work.
- the pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc.
- the pharmaceutical composition of the present invention may be administered through oral or parenteral administration.
- parenteral administration it may be administered through intravenous administration, intraperitoneal administration, intramuscular administration, subcutaneous administration, or local administration.
- intravenous administration intraperitoneal administration
- intramuscular administration intramuscular administration
- subcutaneous administration or local administration.
- It can also be used as a method of applying or spraying to the diseased area, but is not limited to this.
- the pharmaceutical composition of the present invention is prepared in unit dosage form by being formulated using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily performed by a person skilled in the art to which the present invention pertains. Alternatively, it may be manufactured by placing it in a multi-capacity container. At this time, the formulation may be in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, granule, tablet, or capsule, and may additionally contain a dispersant or stabilizer.
- suitable application, spraying and dosage of the pharmaceutical composition will depend on factors such as formulation method, administration mode, age, body weight, sex, severity of disease symptoms, food, administration time, administration route, excretion rate and reaction sensitivity. It varies depending on the dose, and usually a skilled doctor or veterinarian can easily determine and prescribe an effective dosage for the desired treatment.
- the pharmaceutical composition of the present invention can be implemented as an antibiotic, disinfectant, disinfectant, therapeutic agent, etc.
- antibacterial protein CDL2200-1 having an amino acid sequence shown in SEQ ID NO: 1
- antibacterial protein CDL2200-2 having an amino acid sequence shown in SEQ ID NO: 2
- antibacterial protein CDL2200- having an amino acid sequence shown in SEQ ID NO: 3.
- a method of treating Clostridioides difficile infection using a pharmaceutical composition containing at least one of the antibacterial protein CDL2200-4 having the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4 as an active ingredient is a general Clostridioides difficile infection. It can be effective not only on Sili bacteria but also on Clostridioides difficile bacteria that have acquired resistance to existing antibiotics or antibacterial substances.
- Clostridioides difficile-specific antibacterial protein CDL2200-1, antibacterial protein CDL2200-2, antibacterial protein CDL2200-3, and antibacterial protein CDL2200-4 of the present invention exist in bodies other than Clostridioides difficile bacteria.
- Pharmaceutical compositions containing antibacterial protein CDL2200-1, antibacterial protein CDL2200-2, antibacterial protein CDL2200-3, or antibacterial protein CDL2200-4 as active ingredients do not affect normal flora, so they do not cause side effects due to use. It can be minimized compared to antibiotics.
- Existing antibiotics had the disadvantage of adversely affecting beneficial bacteria existing in the body, resulting in various side effects following use.
- Figure 1 shows antibacterial protein CDL2200-1 having an amino acid sequence shown in SEQ ID NO: 1, antibacterial protein CDL2200-2 having an amino acid sequence shown in SEQ ID NO: 2, and antibacterial protein CDL2200-3 having an amino acid sequence shown in SEQ ID NO: 3.
- an electrophoresis photograph showing the results of producing the antibacterial protein CDL2200-4 having the amino acid sequence represented by SEQ ID NO: 4 in the form of a recombinant protein, where lane M is a protein size marker, lane 1 is the antibacterial protein CDL2200-1, Lane 2 is the antibacterial protein CDL2200-2, lane 3 is the antibacterial protein CDL2200-3, and lane 4 is the antibacterial protein CDL2200-4.
- Figure 2 shows a cell count reduction assay conducted on Clostridioides difficile bacteria using antibacterial protein CDL2200-1, antibacterial protein CDL2200-2, antibacterial protein CDL2200-3, and antibacterial protein CDL2200-4. This result shows the results of the experiment.
- the vertical axis is the number of cells per unit volume (CFU/ml).
- the negative control group (Control) is the result of an experiment using the buffer itself without antibacterial protein, and the smaller the number of cells counted after adding antibacterial protein, the better the antibacterial activity.
- Example One Clostridioides difficile fungus-specific of antibacterial protein manufacturing
- Clostridioides difficile specific antibacterial protein CDL2200-1 antibacterial protein CDL2200-2, antibacterial protein CDL2200-3, and antibacterial protein CDL2200-4 will be described below.
- BL21 pASK_CDL2200-1 a strain produced by transforming E. coli using a plasmid having the gene sequence of SEQ ID NO: 5 produced by the present inventors, was used as a producing strain of the antibacterial protein CDL2200-1, and the present inventors used BL21 pASK_CDL2200-2, a strain produced by transforming E. coli using the plasmid having the gene sequence of SEQ ID NO: 6, was used as a production strain for the antibacterial protein CDL2200-2, and the gene of SEQ ID NO: 7 produced by the present inventors BL21 pASK_CDL2200-3, a strain produced by transforming E.
- coli using a plasmid having the sequence was used as a production strain for the antibacterial protein CDL2200-3, and a plasmid with the gene sequence of SEQ ID NO: 8 produced by the present inventors was used.
- the cell culture fluid was taken and centrifuged at 4,000 rpm for 15 minutes at 4°C to recover the cell sediment.
- the recovered cell sediment was suspended in 40 ml of buffer solution (60 mM Potassium phosphate buffer, pH 7.5).
- the ultrasonic disruption method was applied to the cell suspension prepared in this way to disrupt the cells.
- the ultrasonic disruption method was performed by applying ultrasound for 3 seconds to break the cells, stopping for 5 seconds, and repeating this process for a total of 20 minutes. This process was performed on ice. This was conducted in an ice bath.
- the cell disruption fluid was centrifuged at 13,000 rpm for 20 minutes at 4°C to obtain a supernatant. This obtained supernatant was subjected to a typical cation-exchange chromatography purification process to obtain the desired antibacterial protein. Purification was performed.
- FIG 1 shows the results of electrophoresis analysis of the finally obtained purified antibacterial proteins.
- Clostridioides difficile-specific antibacterial protein CDL2200-1, antibacterial protein CDL2200-2, antibacterial protein CDL2200-3, and antibacterial protein CDL2200-4 prepared according to Example 1 to Clostridioides difficile bacteria Antibacterial activity was investigated.
- Clostridioides difficile bacteria used to investigate antibacterial activity included 9 antibiotic-resistant Clostridioides difficile strains, the details of which are shown in Table 1.
- fungus strain Securing agency Clostridioides difficile ATCC 43255 American Type Culture Collection CCARM 0184 Antibiotic-resistant strain bank CCARM 0185 CCARM 0186 CCARM 0187 CCARM 0188 CCARM 0189 CCARM 0190 CCARM 0348
- Antibacterial activity was tested using cell count reduction assay.
- the experimental method of the cell count reduction method is as follows. Using buffer (20mM Tris-HCl, pH 7.5), prepare 1 ml of reaction solution so that the final concentration of the antibacterial protein is 10 ⁇ g/ml and the final concentration of the strain to be tested is 1 ⁇ 10 6 CFU/ml. did. It was left standing at 37°C for 1 or 2 hours. After standing, 100 ⁇ l was spread on a BHIS plate (Brain heart infusion 37 g/L, Yeast extract 5 g/L, 0.05% L-cysteine hydrochloride monohydrate, Agar 15 g/L), and then incubated overnight at 37°C under anaerobic conditions. The number of colonies was confirmed.
- BHIS plate Brain heart infusion 37 g/L, Yeast extract 5 g/L, 0.05% L-cysteine hydrochloride monohydrate, Agar 15 g/L
- the antibacterial activity of the antibacterial protein CDL2200-1, antibacterial protein CDL2200-2, antibacterial protein CDL2200-3, and antibacterial protein CDL2200-4 of the present invention is very specific to Clostridioides difficile bacteria. there was.
- the pharmaceutical composition containing one or more of the Clostridioides difficile-specific antibacterial protein CDL2200-1, antibacterial protein CDL2200-2, antibacterial protein CDL2200-3, or antibacterial protein CDL2200-4 of the present invention is clostri. It was found that it can be used in the same way as conventional antibiotics for the purpose of treating Dioides difficile infection.
- Example 3 Clostridioides difficile fungus-specific of antibacterial protein Clostridioides difficile Investigation of therapeutic effects on bacterial infections
- Clostridioides difficile-specific antibacterial protein CDL2200-1, antibacterial protein CDL2200-2, antibacterial protein CDL2200-3, and antibacterial protein CDL2200-4 was investigated through model experiments.
- 6-week-old SD rats [Specific pathogen-free (SPF) grade] were used as test animals.
- a total of 30 rats were provided with drinking water containing antibiotics (kanamycin 0.4 mg/ml, gentamicin 0.035 mg/ml, colistin 850 U/ml, metronidazole 0.215 mg/ml, and vancomycin 0.045 mg/ml) for 5 days. .
- antibiotics kanamycin 0.4 mg/ml, gentamicin 0.035 mg/ml, colistin 850 U/ml, metronidazole 0.215 mg/ml, and vancomycin 0.045 mg/ml
- Clindamycin (20 mg/kg) was administered intraperitoneally on the last day of provision of sterile drinking water (one day before Clostridioides difficile infection). Forced infection with Clostridioides difficile was performed the day after clindamycin injection.
- Forcible infection with Clostridioides difficile is performed by preparing Clostridioides difficile in spore form and suspending it in sterile saline solution, and then using a sonde with 0.2 ml (equivalent to approximately 10 7 CFU/ml) of the solution. This was performed by forceful injection into the stomach tube. 48 hours after forced infection, the rats were divided into 5 groups, and group 1 (6 rats) was administered 1 ml of buffer (20 mM potassium phosphate buffer, pH 7.5) (control group), and group 2 was administered 1 ml of buffer (20 mM potassium phosphate buffer, pH 7.5).
- a composition containing an antibacterial protein was prepared by mixing a freeze-dried product prepared by freeze-drying an antibacterial protein solution and an excipient (sucrose), and placing the powder in a regular enteric capsule. It was prepared to contain 2 mg of antibacterial protein per capsule. The survival rate of animals in each group was investigated until the 7th day of administration of the composition containing antibacterial protein. The results are presented in Table 2.
- the antibacterial protein CDL2200-1, antibacterial protein CDL2200-2, antibacterial protein CDL2200-3, and antibacterial protein CDL2200-4 of the present invention are effective in treating infection with Clostridioides difficile bacteria. These characteristics are such that the pharmaceutical composition containing one or more of the antibacterial protein CDL2200-1, antibacterial protein CDL2200-2, antibacterial protein CDL2200-3, or antibacterial protein CDL2200-4 of the present invention as an active ingredient is effective against Clostridioides difficile bacteria. It shows that it can be used for infection treatment purposes.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Biotechnology (AREA)
- Medicinal Chemistry (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Public Health (AREA)
- Biomedical Technology (AREA)
- Veterinary Medicine (AREA)
- Biochemistry (AREA)
- Communicable Diseases (AREA)
- Oncology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Microbiology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Plant Pathology (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Gastroenterology & Hepatology (AREA)
- Immunology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Epidemiology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
The present invention relates to an antimicrobial protein that exhibits antimicrobial activity specific for Clostridioides <i /> difficile and, more specifically, to a pharmaceutical composition comprising at least one selected from antibacterial protein CDL2200-1, antibacterial protein CDL2200-2, antibacterial protein CDL2200-3, or antibacterial protein CDL2200-4 as an active ingredient specific for Clostridioides difficile, and a method for treating infections or diseases induced by Clostridioides difficile using the pharmaceutical composition, the antibacterial proteins having the ability to specifically lyse Clostridioides difficile and having amino acid sequence represented by SEQ ID NO: 1.
Description
본 발명은 클로스트리디오이데스 디피실리 (Clostridioides difficile) 균에 대하여 용균활성을 갖는 항균단백질에 관한 것으로, 더욱 상세하게는 인간을 포함한 동물에 감염하여 질환을 일으킬 수 있는 클로스트리디오이데스 디피실리 균을 특이적으로 용균시킬 수 있는 능력을 갖고 서열번호 1로 표시되는 아미노산 서열을 가지는 항균단백질 CDL2200-1, 서열번호 2로 표시되는 아미노산 서열을 가지는 항균단백질 CDL2200-2, 서열번호 3으로 표시되는 아미노산 서열을 가지는 항균단백질 CDL2200-3, 또는 서열번호 4로 표시되는 아미노산 서열을 가지는 항균단백질 CDL2200-4, 및 상기 클로스트리디오이데스 디피실리 균 특이적인 항균단백질 CDL2200-1, 항균단백질 CDL2200-2, 항균단백질 CDL2200-3, 또는 항균단백질 CDL2200-4 중에서 하나 이상을 유효성분으로 포함하는 클로스트리디오이데스 디피실리 균에 의해 유발되는 감염 및 질환 처치용 약학적 조성물에 관한 것이다.The present invention relates to Clostridioides difficile ( Clostridioides difficile ) relates to an antibacterial protein that has lytic activity against bacteria, and more specifically, a sequence that has the ability to specifically lyse Clostridioides difficile bacteria, which can infect animals, including humans, and cause disease. Antibacterial protein CDL2200-1 having an amino acid sequence represented by number 1, antibacterial protein CDL2200-2 having an amino acid sequence represented by SEQ ID NO: 2, antibacterial protein CDL2200-3 having an amino acid sequence represented by SEQ ID NO: 3, or SEQ ID NO. Among the antibacterial protein CDL2200-4 having the amino acid sequence indicated by 4, the Clostridioides difficile-specific antibacterial protein CDL2200-1, antibacterial protein CDL2200-2, antibacterial protein CDL2200-3, or antibacterial protein CDL2200-4 It relates to a pharmaceutical composition for treating infections and diseases caused by Clostridioides difficile bacteria, comprising one or more active ingredients.
미국 질병통제예방센터 (Center for Disease Control & Prevention; CDC)의 자료에 따르면 클로스트리디오이데스 디피실리 (Clostridioides difficile) 균은 슈퍼버그의 일종으로 매년 미국에서 50만 건의 감염증이 발생하고 있으며, 감염진단 후 30일 이내에 사망자 수가 29,000여명에 달하는 것으로 추정하고 있다.According to data from the U.S. Centers for Disease Control & Prevention (CDC), Clostridioides difficile difficile ) is a type of superbug, and it is estimated that 500,000 infections occur in the United States every year, and that the number of deaths within 30 days of diagnosis is approximately 29,000.
클로스트리디오이데스 디피실리 균은 그람 양성 포자 형성 혐기성 균으로 사람과 동물의 장관에 상재균으로 존재하나 기회 감염으로 질환을 유발하는 병원균으로 알려져 있다. 클로스트리디오이데스 디피실리 균은 MRSA (Methicillin-Resistant Staphylococcus aureus), VRE (Vancomycin-Resistant Enterococci)와 더불어 병원 감염균의 대표적 원인균종 중 하나이다. 항생제 처치로 인해 정상적인 장내 미생물 군총의 균형이 무너졌을 때, 항생제 내성 클로스트리디오이데스 디피실리 균이 장내에 우점하게 되고 엔테로톡신 (Enterotoxin)인 독소 A 및 세포독소 (Cytotoxin)인 독소 B를 분비하여 장의 염증과 괴사를 유발시킨다. 또한 클로스트리디오이데스 디피실리 균은 설사, 위막성 대장염을 일으키며 적절하게 치료가 되지 않았을 경우 패혈증이나 중독성 거대결장증 (Toxic megacolon)으로 병증이 악화되게 되고 심한 경우 사망에까지 이르는 경우가 있다.Clostridioides difficile is a Gram-positive spore-forming anaerobic bacterium that exists as a common bacterium in the intestinal tract of humans and animals, but is known to be a pathogen that causes disease through opportunistic infection. Clostridioides difficile is MRSA (Methicillin-Resistant Staphylococcus) aureus ) and VRE (Vancomycin-Resistant Enterococci), it is one of the most common pathogens causing hospital-acquired infections. When the balance of the normal intestinal microbial flora is disrupted due to antibiotic treatment, antibiotic-resistant Clostridioides difficile bacteria become dominant in the intestine and secrete toxin A, an enterotoxin, and toxin B, a cytotoxin. It causes inflammation and necrosis of the intestines. In addition, Clostridioides difficile causes diarrhea and pseudomembranous colitis, and if not treated properly, the condition can worsen into sepsis or toxic megacolon, and in severe cases, it can even lead to death.
거의 모든 항생제가 클로스트리디오이데스 디피실리 균 감염과 관련이 있지만, 특히 플루오로퀴놀론 (Fluoroquinolone), 클린다마이신 (Clindamycin), 카바페넴 (Carbapenem), 세팔로스포린 (Cephalosporine), 페니실린 (Penicillin)과 같은 항생제를 투여하는 환자에게서 자주 발생하며, 최근에는 H2 수용체 억제제를 복용한 사람에게도 클로스트리디오이데스 디피실리 균 감염율이 높을 뿐만 아니라 항생제 처방과 함께 입원할 위험성이 높다는 보고도 있다. 이와같이 점차 클로스트리디오이데스 디피실리 균의 감염 위험성이 높아지고 있는 실정이다. Almost all antibiotics have been associated with Clostridioides difficile infections, but especially those such as fluoroquinolones, clindamycin, carbapenems, cephalosporins, and penicillins. It occurs frequently in patients taking H2 receptor inhibitors, and there have recently been reports that people taking H2 receptor inhibitors not only have a high rate of Clostridioides difficile infection, but also have a high risk of hospitalization with antibiotic prescriptions. As such, the risk of infection with Clostridioides difficile bacteria is gradually increasing.
한편, 근래에는 병원 내에서만이 아니라 병원 내 감염자가 지역 사회로 나와서 감염이 재발하는 경우, 또는 다른 사람에게 병원균을 전파하여 감염을 유발하는 등의 지역사회를 통한 감염 역시 문제가 되고 있다. 최근 미국 질병통제예방센터가 작성한 보고서에서는 세균들 중에 항생제 내성을 나타내는 아시네토박터 (Acinetobacter), 클로스트리디오이데스 디피실리 및 장내세균 (Enterobacteriaceae)을 가장 중대한 위협으로 판단하고 있다.Meanwhile, in recent years, infection not only within the hospital but also through the community, such as when infected people in the hospital come out into the community and the infection recurs or spread pathogens to other people and cause infection, has also become a problem. A recent report prepared by the U.S. Centers for Disease Control and Prevention judges that antibiotic-resistant Acinetobacter, Clostridioides difficile, and Enterobacteriaceae are the most serious threats.
이러한 클로스트리디오이데스 디피실리 균의 감염 치료에는 항생제가 널리 이용되었는데, 주로 1차 치료제로 경구용 메트로니다졸 (Metronidazole)을 사용하고, 2차 치료제로 반코마이신 (Vancomycin)을 사용하는 것으로 알려져 있다. 그런데, 전체 클로스트리디오이데스 디피실리 균 감염증의 8%는 반코마이신 내성균주의 감염으로 알려져 있다. 특히, 최근 보고를 참조하면 내성균주의 출현 빈도가 꾸준히 증가하고 있어 내성균에도 효과적인 새로운 항균물질의 개발이 요구되고 있다. 특히, 용균활성 (Biolytic activity)이 있어 체내로부터의 빠른 감염균 제거가 가능하여 신속한 치료 효과를 제공해 줄 수 있는 약학적 제제의 개발이 매우 절실한 형편이다. Antibiotics have been widely used to treat infections caused by Clostridioides difficile. It is known that oral metronidazole is mainly used as a first-line treatment and vancomycin is used as a second-line treatment. However, it is known that 8% of all Clostridioides difficile infections are caused by vancomycin-resistant strains. In particular, referring to recent reports, the frequency of emergence of resistant bacteria is steadily increasing, and there is a need for the development of new antibacterial substances that are effective against resistant bacteria. In particular, there is a great need for the development of pharmaceutical agents that have biolytic activity and can quickly remove infectious bacteria from the body, thereby providing rapid therapeutic effects.
또한 기존 항생제들은 장내 정상 세균총의 균형을 깨뜨려 제2의 문제를 초래할 수도 있기 때문에, 이러한 기존 항생제 치료의 문제점을 해결할 수 있는 혁신적 약물의 개발이 요구되고 있다.In addition, because existing antibiotics can disrupt the balance of normal intestinal flora and cause secondary problems, there is a need for the development of innovative drugs that can solve the problems of existing antibiotic treatment.
이에, 유해 병원성 세균인 클로스트리디오이데스 디피실리 균에 대한 기존 항생제 사용에 있어 수반되던 문제점의 해결 방안으로서, 본 발명자들은 클로스트리디오이데스 디피실리 균을 특이적으로 용균시킬 수 있는 항균단백질을 제공하고, 나아가 이를 유효성분으로 포함하는 클로스트리디오이데스 디피실리 균의 감염을 처치하는 데에 활용될 수 있는 약학적 조성물을 제공하며, 더 나아가 상기 약학적 조성물을 활용한 클로스트리디오이데스 디피실리 균의 감염 또는 질환을 효과적으로 처치하는 방법을 제공하고자 한다.Accordingly, as a solution to the problems accompanying the use of existing antibiotics against Clostridioides difficile, a harmful pathogenic bacterium, the present inventors provided an antibacterial protein capable of specifically lysing Clostridioides difficile. and further provides a pharmaceutical composition that can be used to treat infection with Clostridioides difficile bacteria containing the same as an active ingredient, and further provides a pharmaceutical composition that can be used to treat infection with Clostridioides difficile bacteria using the pharmaceutical composition. The goal is to provide a method for effectively treating infections or diseases.
따라서, 본 발명의 목적은 클로스트리디오이데스 디피실리 균을 특이적으로 용균시킬 수 있는 항균활성을 갖고 서열번호 1로 표시되는 아미노산 서열을 가지는 항균단백질 CDL2200-1, 서열번호 2로 표시되는 아미노산 서열을 가지는 항균단백질 CDL2200-2, 서열번호 3으로 표시되는 아미노산 서열을 가지는 항균단백질 CDL2200-3, 및 서열번호 4로 표시되는 아미노산 서열을 가지는 항균단백질 CDL2200-4를 제공하는 것이다.Therefore, the object of the present invention is the antibacterial protein CDL2200-1, which has antibacterial activity capable of specifically lysing Clostridioides difficile bacteria and has an amino acid sequence represented by SEQ ID NO: 1, and an amino acid sequence represented by SEQ ID NO: 2. To provide an antibacterial protein CDL2200-2, an antibacterial protein CDL2200-3 having an amino acid sequence shown in SEQ ID NO: 3, and an antibacterial protein CDL2200-4 having an amino acid sequence shown in SEQ ID NO: 4.
본 발명의 다른 목적은 상기 클로스트리디오이데스 디피실리 균을 특이적으로 용균시킬 수 있는 항균활성을 갖고 서열번호 1로 표시되는 아미노산 서열을 가지는 항균단백질 CDL2200-1, 서열번호 2로 표시되는 아미노산 서열을 가지는 항균단백질 CDL2200-2, 서열번호 3으로 표시되는 아미노산 서열을 가지는 항균단백질 CDL2200-3, 및 서열번호 4로 표시되는 아미노산 서열을 가지는 항균단백질 CDL2200-4를 효율적으로 제조할 수 있는 방법을 제공하는 것이다.Another object of the present invention is the antibacterial protein CDL2200-1, which has antibacterial activity capable of specifically lysing the Clostridioides difficile bacterium and has an amino acid sequence represented by SEQ ID NO: 1, and an amino acid sequence represented by SEQ ID NO: 2. Provides a method for efficiently producing the antibacterial protein CDL2200-2, the antibacterial protein CDL2200-3, which has the amino acid sequence shown in SEQ ID NO: 3, and the antibacterial protein CDL2200-4, which has the amino acid sequence shown in SEQ ID NO: 4. It is done.
본 발명의 또 다른 목적은 상기 클로스트리디오이데스 디피실리 균을 특이적으로 용균시킬 수 있는 서열번호 1로 표시되는 아미노산 서열을 가지는 항균단백질 CDL2200-1, 서열번호 2로 표시되는 아미노산 서열을 가지는 항균단백질 CDL2200-2, 서열번호 3으로 표시되는 아미노산 서열을 가지는 항균단백질 CDL2200-3, 또는 서열번호 4로 표시되는 아미노산 서열을 가지는 항균단백질 CDL2200-4 중에서 하나 이상을 유효성분으로 포함하는 클로스트리디오이데스 디피실리 균 감염 처치 목적의 약학적 조성물을 제공하는 것이다.Another object of the present invention is to provide an antibacterial protein CDL2200-1 having an amino acid sequence shown in SEQ ID NO: 1, which can specifically lyse the Clostridioides difficile bacterium, and an antibacterial protein having an amino acid sequence shown in SEQ ID NO: 2. Clostridioides containing at least one of protein CDL2200-2, antibacterial protein CDL2200-3 having the amino acid sequence shown in SEQ ID NO: 3, or antibacterial protein CDL2200-4 having the amino acid sequence shown in SEQ ID NO: 4 as an active ingredient. To provide a pharmaceutical composition for the purpose of treating difficile infection.
또한, 본 발명의 또 다른 목적은 서열번호 1로 표시되는 아미노산 서열을 가지는 항균단백질 CDL2200-1, 서열번호 2로 표시되는 아미노산 서열을 가지는 항균단백질 CDL2200-2, 서열번호 3으로 표시되는 아미노산 서열을 가지는 항균단백질 CDL2200-3, 또는 서열번호 4로 표시되는 아미노산 서열을 가지는 항균단백질 CDL2200-4 중에서 하나 이상을 유효성분으로 포함하는 약학적 조성물을 이용하여 클로스트리디오이데스 디피실리 균의 감염 또는 질환을 치료하는 방법을 제공하는 것이다.In addition, another object of the present invention is to provide antibacterial protein CDL2200-1 having an amino acid sequence shown in SEQ ID NO: 1, antibacterial protein CDL2200-2 having an amino acid sequence shown in SEQ ID NO: 2, and an amino acid sequence shown in SEQ ID NO: 3. Infection or disease caused by Clostridioides difficile using a pharmaceutical composition containing at least one of the antibacterial protein CDL2200-3 or the antibacterial protein CDL2200-4 with the amino acid sequence shown in SEQ ID NO: 4 as an active ingredient. It provides a method of treatment.
상기 목적들을 달성하고자, 본 발명의 발명자들은 여러 균종들에 대하여 항균활성을 가진다고 알려진 다양한 항균단백질 정보를 활용하여, 다양한 항균단백질 후보들을 재조합 단백질 형태로 제조하고 이들의 클로스트리디오이데스 디피실리 균에 대한 용균활성을 조사하여 우수한 용균활성을 갖는 항균단백질을 선별하였고, 이를 효율적으로 제조할 수 있는 방법까지 개발한 다음에, 마지막으로 이를 유효성분으로 하는 클로스트리디오이데스 디피실리 균 감염 처치 목적으로 활용될 수 있는 약학적 조성물을 개발함으로써 본 발명을 완성하였다.In order to achieve the above objectives, the inventors of the present invention utilized information on various antibacterial proteins known to have antibacterial activity against various bacterial species, prepared various antibacterial protein candidates in the form of recombinant proteins, and administered these to Clostridioides difficile bacteria. Antibacterial proteins with excellent lytic activity were selected by examining their lytic activity, and a method to manufacture them efficiently was developed. Finally, this was used as an active ingredient to treat infections caused by Clostridioides difficile bacteria. The present invention was completed by developing a pharmaceutical composition that can be used.
따라서, 본 발명의 일 양태에 따르면, 본 발명은 클로스트리디오이데스 디피실리 균을 특이적으로 용균시킬 수 있는 항균단백질 CDL2200-1, 항균단백질 CDL2200-2, 항균단백질 CDL2200-3, 및 항균단백질 CDL2200-4의 아미노산 서열을 제공한다. 구체적으로는 항균단백질 CDL2200-1의 경우에는 서열번호 1의 아미노산 서열이 해당하고, 항균단백질 CDL2200-2의 경우에는 서열번호 2의 아미노산 서열이 해당하고, 항균단백질 CDL2200-3의 경우에는 서열번호 3의 아미노산 서열이 해당하고, 항균단백질 CDL2200-4의 경우에는 서열번호 4의 아미노산 서열이 해당한다. 클로스트리디오이데스 디피실리 균을 특이적으로 용균시킬 수 있는 항균단백질 CDL2200-1은 178개의 아미노산 잔기로 구성되며 분자량은 약 20.3 kDa이며, 항균단백질 CDL2200-2는 191개의 아미노산 잔기로 구성되며 분자량은 약 21.6 kDa이며, 항균단백질 CDL2200-3은 187개의 아미노산 잔기로 구성되며 분자량은 약 21.0 kDa이며, 항균단백질 CDL2200-4는 185개의 아미노산 잔기로 구성되며 분자량은 약 20.7 kDa이다. Therefore, according to one aspect of the present invention, the present invention provides antibacterial protein CDL2200-1, antibacterial protein CDL2200-2, antibacterial protein CDL2200-3, and antibacterial protein CDL2200 that can specifically lyse Clostridioides difficile bacteria. The amino acid sequence of -4 is provided. Specifically, in the case of antibacterial protein CDL2200-1, it corresponds to the amino acid sequence of SEQ ID NO: 1, in the case of antibacterial protein CDL2200-2, it corresponds to the amino acid sequence of SEQ ID NO: 2, and in the case of antibacterial protein CDL2200-3, it corresponds to SEQ ID NO: 3. corresponds to the amino acid sequence, and in the case of the antibacterial protein CDL2200-4, it corresponds to the amino acid sequence of SEQ ID NO: 4. The antibacterial protein CDL2200-1, which can specifically lyse Clostridioides difficile bacteria, consists of 178 amino acid residues and has a molecular weight of approximately 20.3 kDa, and the antibacterial protein CDL2200-2 consists of 191 amino acid residues and has a molecular weight of It is about 21.6 kDa, and the antibacterial protein CDL2200-3 is composed of 187 amino acid residues and has a molecular weight of about 21.0 kDa, and the antibacterial protein CDL2200-4 is composed of 185 amino acid residues and has a molecular weight of about 20.7 kDa.
서열번호 1 내지 서열번호 4의 아미노산 서열은 당업자에 의해 공지의 기술을 이용하여 일부 변형될 수 있음이 자명하다. 상기 변형은 아미노산 서열의 일부 치환, 아미노산 서열의 일부 첨가, 및 아미노산 서열의 일부 결실을 포함한다. 그렇지만 본 발명에서 개시하고 있는 서열번호 1 내지 서열번호 4의 아미노산 서열을 준용하는 것이 가장 바람직하다. It is obvious that the amino acid sequences of SEQ ID NO: 1 to SEQ ID NO: 4 can be partially modified by those skilled in the art using known techniques. The modifications include partial substitution of the amino acid sequence, partial addition of the amino acid sequence, and partial deletion of the amino acid sequence. However, it is most preferable to apply the amino acid sequences of SEQ ID NO: 1 to SEQ ID NO: 4 disclosed in the present invention.
또한, 본 발명은 서열번호 1의 아미노산 서열을 갖는 항균단백질 CDL2200-1의 생산에 이용될 수 있는 생산균주 대장균 BL21 pASK_CDL2200-1, 서열번호 2의 아미노산 서열을 갖는 항균단백질 CDL2200-2의 생산에 이용될 수 있는 생산균주 대장균 BL21 pASK_CDL2200-2, 서열번호 3의 아미노산 서열을 갖는 항균단백질 CDL2200-3의 생산에 이용될 수 있는 생산균주 대장균 BL21 pASK_CDL2200-3, 및 서열번호 4의 아미노산 서열을 갖는 항균단백질 CDL2200-4의 생산에 이용될 수 있는 생산균주 대장균 BL21 pASK_CDL2200-4를 제공한다. 상기 생산균주 BL21 pASK_CDL2200-1은 본 발명자들이 제작한 서열번호 5의 유전자 서열을 갖는 플라스미드를 사용하여 대장균을 형질전환시켜 항균단백질 CDL2200-1을 생산하도록 제작된 균주이고, 상기 생산균주 BL21 pASK_CDL2200-2는 본 발명자들이 제작한 서열번호 6의 유전자 서열을 갖는 플라스미드를 사용하여 대장균을 형질전환시켜 항균단백질 CDL2200-2를 생산하도록 제작된 균주이고, 상기 생산균주 BL21 pASK_CDL2200-3은 본 발명자들이 제작한 서열번호 7의 유전자 서열을 갖는 플라스미드를 사용하여 대장균을 형질전환시켜 항균단백질 CDL2200-3을 생산하도록 제작된 균주이고, 상기 생산균주 BL21 pASK_CDL2200-4는 본 발명자들이 제작한 서열번호 8의 유전자 서열을 갖는 플라스미드를 사용하여 대장균을 형질전환시켜 항균단백질 CDL2200-4를 생산하도록 제작된 균주이다. 이렇게 제작된 생산균주 BL21 pASK_CDL2200-1, 생산균주 BL21 pASK_CDL2200-2, 생산균주 BL21 pASK_CDL2200-3, 및 생산균주 BL21 pASK_CDL2200-4는 본 발명자들에 의해 2022년 11월 29일자로 한국생명공학연구원 생물자원센터에 기탁되었다. 수탁번호는 생산균주 BL21 pASK_CDL2200-1이 KCTC 15213BP이고, 생산균주 BL21 pASK_CDL2200-2가 KCTC 15214BP이고, 생산균주 BL21 pASK_CDL2200-3이 KCTC 15215BP이고, 생산균주 BL21 pASK_CDL2200-4가 KCTC 15216BP이다. In addition, the present invention relates to the production strain Escherichia coli BL21 pASK_CDL2200-1, which can be used for the production of the antibacterial protein CDL2200-1 having the amino acid sequence of SEQ ID NO: 1, and the antibacterial protein CDL2200-2 having the amino acid sequence of SEQ ID NO: 2. The production strain Escherichia coli BL21 pASK_CDL2200-2, which can be used for the production of the antibacterial protein CDL2200-3 having the amino acid sequence of SEQ ID NO: 3, and the antibacterial protein having the amino acid sequence of SEQ ID NO: 4 The production strain Escherichia coli BL21 pASK_CDL2200-4, which can be used for the production of CDL2200-4, is provided. The production strain BL21 pASK_CDL2200-1 is a strain created to produce the antibacterial protein CDL2200-1 by transforming E. coli using a plasmid having the gene sequence of SEQ ID NO: 5 produced by the present inventors, and the production strain BL21 pASK_CDL2200-2 is a strain created to produce the antibacterial protein CDL2200-2 by transforming E. coli using a plasmid having the gene sequence of SEQ ID NO: 6 produced by the present inventors, and the production strain BL21 pASK_CDL2200-3 is a sequence produced by the present inventors. It is a strain created to produce the antibacterial protein CDL2200-3 by transforming E. coli using a plasmid having the gene sequence number 7, and the production strain BL21 pASK_CDL2200-4 has the gene sequence number 8 produced by the present inventors. This strain was created to produce the antibacterial protein CDL2200-4 by transforming E. coli using a plasmid. The production strain BL21 pASK_CDL2200-1, production strain BL21 pASK_CDL2200-2, production strain BL21 pASK_CDL2200-3, and production strain BL21 pASK_CDL2200-4 produced in this way were provided by the present inventors as biological resources of the Korea Research Institute of Bioscience and Biotechnology as of November 29, 2022. deposited at the center. The accession number is KCTC 15213BP for production strain BL21 pASK_CDL2200-2, KCTC 15214BP for production strain BL21 pASK_CDL2200-3, and KCTC 15215BP for production strain BL21 pASK_CDL2200-4. It is 15216 BP.
본 발명의 다른 일 양태에 따르면, 본 발명은 서열번호 1의 아미노산 서열로 특징지어지고 클로스트리디오이데스 디피실리 균에 대하여 특이적 용균능을 갖는 항균단백질 CDL2200-1, 서열번호 2의 아미노산 서열로 특징지어지고 클로스트리디오이데스 디피실리 균에 대하여 특이적 용균능을 갖는 항균단백질 CDL2200-2, 서열번호 3의 아미노산 서열로 특징지어지고 클로스트리디오이데스 디피실리 균에 대하여 특이적 용균능을 갖는 항균단백질 CDL2200-3, 서열번호 4의 아미노산 서열로 특징지어지고 클로스트리디오이데스 디피실리 균에 대하여 특이적 용균능을 갖는 항균단백질 CDL2200-4 중에서 하나 이상을 유효성분으로 포함하는 클로스트리디오이데스 디피실리 균의 감염 또는 질환에 대한 치료에 효과적으로 활용될 수 있는 약학적 조성물을 제공한다. According to another aspect of the present invention, the present invention provides an antibacterial protein CDL2200-1, which is characterized by the amino acid sequence of SEQ ID NO: 1 and has specific lytic activity against Clostridioides difficile bacteria, and has an amino acid sequence of SEQ ID NO: 2. CDL2200-2, an antibacterial protein that is characterized and has specific lytic activity against Clostridioides difficile bacteria, is characterized by the amino acid sequence of SEQ ID NO: 3 and has specific lytic activity against Clostridioides difficile bacteria. Protein CDL2200-3, Clostridioides difficile containing as an active ingredient one or more of the antibacterial protein CDL2200-4, which is characterized by the amino acid sequence of SEQ ID NO: 4 and has specific lytic activity against Clostridioides difficile bacteria. A pharmaceutical composition that can be effectively used to treat bacterial infections or diseases is provided.
본 발명의 약학적 조성물에 포함되는, 본 발명에 따른 서열번호 1의 아미노산 서열로 특징지어지고 클로스트리디오이데스 디피실리 균에 대하여 특이적 용균능을 갖는 항균단백질 CDL2200-1, 서열번호 2의 아미노산 서열로 특징지어지고 클로스트리디오이데스 디피실리 균에 대하여 특이적 용균능을 갖는 항균단백질 CDL2200-2, 서열번호 3의 아미노산 서열로 특징지어지고 클로스트리디오이데스 디피실리 균에 대하여 특이적 용균능을 갖는 항균단백질 CDL2200-3, 또는 서열번호 4의 아미노산 서열로 특징지어지고 클로스트리디오이데스 디피실리 균에 대하여 특이적 용균능을 갖는 항균단백질 CDL2200-4는 상술한 바와 같이 클로스트리디오이데스 디피실리 균을 특이적으로 용균시키므로, 클로스트리디오이데스 디피실리 균에 의해 유발되는 다양한 질환의 처치에 있어 효과를 나타낸다. 따라서 본 발명의 약학적 조성물은 클로스트리디오이데스 디피실리 균에 의해 유발되는 질환의 처치에 활용될 수 있다. 또한, 본 발명의 약학적 조성물은 항생제, 소독제, 살균제, 치료제 등으로 구현되어 클로스트리디오이데스 디피실리 균에 의해 유발되는 다양한 질환들의 처치에 활용될 수 있다. Antibacterial protein CDL2200-1, characterized by the amino acid sequence of SEQ ID NO: 1 according to the present invention and having specific lytic activity against Clostridioides difficile bacteria, included in the pharmaceutical composition of the present invention, amino acid of SEQ ID NO: 2 CDL2200-2, an antibacterial protein characterized by sequence and having specific lytic activity against Clostridioides difficile bacteria, is characterized by the amino acid sequence of SEQ ID NO: 3 and has specific lytic activity against Clostridioides difficile bacteria. The antibacterial protein CDL2200-3, or the antibacterial protein CDL2200-4, which is characterized by the amino acid sequence of SEQ ID NO: 4 and has a specific lytic activity against Clostridioides difficile bacteria, is, as described above, Clostridioides difficile bacteria. Since it specifically lyses, it is effective in treating various diseases caused by Clostridioides difficile bacteria. Therefore, the pharmaceutical composition of the present invention can be used to treat diseases caused by Clostridioides difficile bacteria. In addition, the pharmaceutical composition of the present invention can be implemented as an antibiotic, disinfectant, disinfectant, therapeutic agent, etc. and used to treat various diseases caused by Clostridioides difficile bacteria.
따라서, 본 발명의 다른 일 양태에 따르면, 본 발명은 본 발명의 항균단백질을 포함한 조성물을 클로스트리디오이데스 디피실리 균 감염을 갖는 개체에 투여하여 클로스트리디오이데스 디피실리 균에 의해 유발되는 다양한 질환들을 치료하는 방법을 제공한다.Therefore, according to another aspect of the present invention, the composition containing the antibacterial protein of the present invention is administered to an individual with Clostridioides difficile infection to treat various diseases caused by Clostridioides difficile bacteria. Provides methods for treating them.
본 명세서에서 "클로스트리디오이데스 디피실리 균에 의해 유발되는 질환"이란 클로스트리디오이데스 디피실리 균 감염을 원인으로 하여 유발되는 질환으로서 장의 염증과 괴사, 설사, 위막성 대장염, 위장관 천공, 패혈증, 중독성 거대결장증 등을 수반하는 증상을 총칭하지만, 이에 제한되지는 않는다.In this specification, "disease caused by Clostridioides difficile bacteria" refers to a disease caused by infection with Clostridioides difficile bacteria, including intestinal inflammation and necrosis, diarrhea, pseudomembranous colitis, gastrointestinal perforation, sepsis, It is a general term for symptoms accompanying toxic megacolon, but is not limited to this.
본 명세서에서의 "클로스트리디오이데스 디피실리 균"은 기존 항생제에 대하여 민감하든지 또는 기존 항생제에 대하여 내성을 가진 내성균이든지 상관이 없다. 즉, 기존 항생제에 대한 내성 획득 여부는 상관이 없다.As used herein, “Clostridioides difficile bacteria” may be sensitive to existing antibiotics or resistant to existing antibiotics. In other words, it does not matter whether resistance to existing antibiotics is acquired or not.
본 명세서에서 사용된 "처치" 또는 "치료"라는 용어는 클로스트리디오이데스 디피실리 균에 의해 유발된 감염 또는 질환의 억제, 및 클로스트리디오이데스 디피실리 균에 의해 유발된 질환의 병적 상태를 경감시키는 모든 행위를 의미한다.As used herein, the term "treatment" or "treatment" refers to suppressing an infection or disease caused by Clostridioides difficile bacteria and alleviating the pathological condition of a disease caused by Clostridioides difficile bacteria. It means all actions that are ordered.
이러한 활용 목적에서의 효율성을 높이기 위하여 클로스트리디오이데스 디피실리 균 또는 다른 세균 종에 대하여 항균활성을 제공할 수 있는 항균물질들이 본 발명의 약학적 조성물에 추가될 수 있다. In order to increase the efficiency for this purpose, antibacterial substances that can provide antibacterial activity against Clostridioides difficile or other bacterial species may be added to the pharmaceutical composition of the present invention.
본 발명의 약학적 조성물에 포함되는 약제학적으로 허용되는 담체는 제제 시에 통상적으로 이용되는 것으로서, 락토스, 덱스트로스, 수크로스, 솔비톨, 만니톨, 전분, 아카시아 고무, 인산칼슘, 알기네이트, 젤라틴, 규산칼슘, 미세결정성 셀룰로오스, 폴리비닐피롤리돈, 셀룰로오스, 물, 시럽, 메틸 셀룰로스, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 활석, 스테아르산 마그네슘 및 미네랄 오일 등을 포함하나, 이에 한정되는 것은 아니다. Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are those commonly used in preparation, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, Includes, but is limited to, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. It doesn't work.
본 발명의 약제학적 조성물은 상기 성분들 이외에 윤활제, 습윤제, 감미제, 향미제, 유화제, 현탁제, 보존제 등을 추가로 포함할 수 있다.In addition to the above ingredients, the pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc.
본 발명의 약학적 조성물은 경구 투여 또는 비경구 투여를 통해 투여할 수도 있으며, 비경구 투여의 경우에는 정맥 내 투여, 복강 내 투여, 근육 내 투여, 피하 투여 또는 국부 투여를 이용하여 투여할 수도 있으며, 그 밖에 질환 부위에의 도포 또는 분무하는 방법으로도 이용될 수 있으나 이에 제한되지는 않는다.The pharmaceutical composition of the present invention may be administered through oral or parenteral administration. In the case of parenteral administration, it may be administered through intravenous administration, intraperitoneal administration, intramuscular administration, subcutaneous administration, or local administration. , It can also be used as a method of applying or spraying to the diseased area, but is not limited to this.
본 발명의 약학적 조성물은 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자가 용이하게 실시할 수 있는 방법에 따라, 약학적으로 허용되는 담체 및/또는 부형제를 이용하여 제제화 됨으로써 단위 용량 형태로 제조되거나, 또는 다용량 용기 내에 내입시켜 제조될 수도 있다. 이때 제형은 오일 또는 수성 매질 중의 용액, 현탁액 또는 유화액 형태이거나 엑스제, 분말제, 과립제, 정제 또는 캡슐제 형태일 수도 있으며, 분산제 또는 안정화제를 추가적으로 포함할 수도 있다.The pharmaceutical composition of the present invention is prepared in unit dosage form by being formulated using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily performed by a person skilled in the art to which the present invention pertains. Alternatively, it may be manufactured by placing it in a multi-capacity container. At this time, the formulation may be in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, granule, tablet, or capsule, and may additionally contain a dispersant or stabilizer.
또한, 상기 약학적 조성물의 적합한 도포, 분무 및 투여량은 제제화 방법, 투여 방식, 연령, 체중, 성, 질병 증상의 정도, 음식, 투여 시간, 투여 경로, 배설 속도 및 반응 감응성과 같은 요인들에 의해 다양하며, 보통으로 숙련된 의사나 수의사는 소망하는 치료에 효과적인 투여량을 용이하게 결정 및 처방할 수 있다.In addition, suitable application, spraying and dosage of the pharmaceutical composition will depend on factors such as formulation method, administration mode, age, body weight, sex, severity of disease symptoms, food, administration time, administration route, excretion rate and reaction sensitivity. It varies depending on the dose, and usually a skilled doctor or veterinarian can easily determine and prescribe an effective dosage for the desired treatment.
본 발명의 약학적 조성물은 항생제, 소독제, 살균제, 치료제 등으로 구현될 수 있다.The pharmaceutical composition of the present invention can be implemented as an antibiotic, disinfectant, disinfectant, therapeutic agent, etc.
본 발명에 따른 서열번호 1로 표시되는 아미노산 서열을 가지는 항균단백질 CDL2200-1, 서열번호 2로 표시되는 아미노산 서열을 가지는 항균단백질 CDL2200-2, 서열번호 3으로 표시되는 아미노산 서열을 가지는 항균단백질 CDL2200-3, 또는 서열번호 4로 표시되는 아미노산 서열을 가지는 항균단백질 CDL2200-4 중에서 하나 이상을 유효성분으로 포함하는 약학적 조성물을 이용한 클로스트리디오이데스 디피실리 균 감염의 치료 방법은 일반적인 클로스트리디오이데스 디피실리 균뿐만 아니라 기존 항생제들이나 항균물질들에 대하여 내성을 획득한 클로스트리디오이데스 디피실리 균에도 효과적으로 작용할 수 있다. 한편, 본 발명의 클로스트리디오이데스 디피실리 균 특이 항균단백질 CDL2200-1, 항균단백질 CDL2200-2, 항균단백질 CDL2200-3, 및 항균단백질 CDL2200-4는 클로스트리디오이데스 디피실리 균 외의 다른 체내에 존재하는 정상 상재균에는 영향을 주지 않아 항균단백질 CDL2200-1, 항균단백질 CDL2200-2, 항균단백질 CDL2200-3, 또는 항균단백질 CDL2200-4를 유효성분으로 포함하고 있는 약학적 조성물은 사용에 따른 부작용을 기존 항생제들에 비교하여 최소화 할 수 있다. 기존 항생제들의 경우에는 체내에 존재하는 유익균들에도 악영향을 초래하여 사용에 따른 다양한 부작용을 초래하는 단점이 있었다.According to the present invention, antibacterial protein CDL2200-1 having an amino acid sequence shown in SEQ ID NO: 1, antibacterial protein CDL2200-2 having an amino acid sequence shown in SEQ ID NO: 2, and antibacterial protein CDL2200- having an amino acid sequence shown in SEQ ID NO: 3. A method of treating Clostridioides difficile infection using a pharmaceutical composition containing at least one of the antibacterial protein CDL2200-4 having the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4 as an active ingredient is a general Clostridioides difficile infection. It can be effective not only on Sili bacteria but also on Clostridioides difficile bacteria that have acquired resistance to existing antibiotics or antibacterial substances. Meanwhile, the Clostridioides difficile-specific antibacterial protein CDL2200-1, antibacterial protein CDL2200-2, antibacterial protein CDL2200-3, and antibacterial protein CDL2200-4 of the present invention exist in bodies other than Clostridioides difficile bacteria. Pharmaceutical compositions containing antibacterial protein CDL2200-1, antibacterial protein CDL2200-2, antibacterial protein CDL2200-3, or antibacterial protein CDL2200-4 as active ingredients do not affect normal flora, so they do not cause side effects due to use. It can be minimized compared to antibiotics. Existing antibiotics had the disadvantage of adversely affecting beneficial bacteria existing in the body, resulting in various side effects following use.
도 1은 서열번호 1로 표시되는 아미노산 서열을 가지는 항균단백질 CDL2200-1, 서열번호 2로 표시되는 아미노산 서열을 가지는 항균단백질 CDL2200-2, 서열번호 3으로 표시되는 아미노산 서열을 가지는 항균단백질 CDL2200-3, 및 서열번호 4로 표시되는 아미노산 서열을 가지는 항균단백질 CDL2200-4를 재조합 단백질 형태로 생산한 결과를 보여주는 전기영동 사진으로서, 레인 M은 단백질 크기 마커이고, 레인 1은 항균단백질 CDL2200-1이고, 레인 2는 항균단백질 CDL2200-2이고, 레인 3은 항균단백질 CDL2200-3이고, 레인 4는 항균단백질 CDL2200-4이다. Figure 1 shows antibacterial protein CDL2200-1 having an amino acid sequence shown in SEQ ID NO: 1, antibacterial protein CDL2200-2 having an amino acid sequence shown in SEQ ID NO: 2, and antibacterial protein CDL2200-3 having an amino acid sequence shown in SEQ ID NO: 3. , and an electrophoresis photograph showing the results of producing the antibacterial protein CDL2200-4 having the amino acid sequence represented by SEQ ID NO: 4 in the form of a recombinant protein, where lane M is a protein size marker, lane 1 is the antibacterial protein CDL2200-1, Lane 2 is the antibacterial protein CDL2200-2, lane 3 is the antibacterial protein CDL2200-3, and lane 4 is the antibacterial protein CDL2200-4.
도 2는 항균단백질 CDL2200-1, 항균단백질 CDL2200-2, 항균단백질 CDL2200-3, 및 항균단백질 CDL2200-4의 클로스트리디오이데스 디피실리 균을 대상으로 실시한 세포수감소조사법 (Cell count reduction assay)의 실험 결과를 보여주는 결과이다. 세로축은 단위 부피당의 세포수 (CFU/ml)이다. 음성대조군 (Control)은 항균단백질을 포함하지 않은 완충액 자체를 사용한 실험 결과이며, 항균단백질 첨가 후의 계수된 세포수가 작을수록 항균활성이 우수함을 나타낸다.Figure 2 shows a cell count reduction assay conducted on Clostridioides difficile bacteria using antibacterial protein CDL2200-1, antibacterial protein CDL2200-2, antibacterial protein CDL2200-3, and antibacterial protein CDL2200-4. This result shows the results of the experiment. The vertical axis is the number of cells per unit volume (CFU/ml). The negative control group (Control) is the result of an experiment using the buffer itself without antibacterial protein, and the smaller the number of cells counted after adding antibacterial protein, the better the antibacterial activity.
이하, 실시예에 의거하여 본 발명을 보다 구체적으로 설명하지만, 이들 실시예는 본 발명의 예시일 뿐이며 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail based on examples, but these examples are only illustrative of the present invention and the scope of the present invention is not limited to these examples.
실시예Example
1: One:
클로스트리디오이데스Clostridioides
디피실리difficile
균 특이적 fungus-specific
항균단백질의of antibacterial protein
제조 manufacturing
클로스트리디오이데스 디피실리 균 특이적 항균단백질 CDL2200-1, 항균단백질 CDL2200-2, 항균단백질 CDL2200-3, 및 항균단백질 CDL2200-4의 제조에 대하여 이하에 설명한다. The preparation of Clostridioides difficile specific antibacterial protein CDL2200-1, antibacterial protein CDL2200-2, antibacterial protein CDL2200-3, and antibacterial protein CDL2200-4 will be described below.
본 실시예에서는 본 발명자들이 제작한 서열번호 5의 유전자 서열을 갖는 플라스미드를 사용하여 대장균을 형질전환시켜 제작한 균주인 BL21 pASK_CDL2200-1을 항균단백질 CDL2200-1의 생산균주로 사용하였고, 본 발명자들이 제작한 서열번호 6의 유전자 서열을 갖는 플라스미드를 사용하여 대장균을 형질전환시켜 제작한 균주인 BL21 pASK_CDL2200-2를 항균단백질 CDL2200-2의 생산균주로 사용하였고, 본 발명자들이 제작한 서열번호 7의 유전자 서열을 갖는 플라스미드를 사용하여 대장균을 형질전환시켜 제작한 균주인 BL21 pASK_CDL2200-3을 항균단백질 CDL2200-3의 생산균주로 사용하였고, 본 발명자들이 제작한 서열번호 8의 유전자 서열을 갖는 플라스미드를 사용하여 대장균을 형질전환시켜 제작한 균주인 BL21 pASK_CDL2200-4를 항균단백질 CDL2200-4의 생산균주로 사용하였다. In this example, BL21 pASK_CDL2200-1, a strain produced by transforming E. coli using a plasmid having the gene sequence of SEQ ID NO: 5 produced by the present inventors, was used as a producing strain of the antibacterial protein CDL2200-1, and the present inventors used BL21 pASK_CDL2200-2, a strain produced by transforming E. coli using the plasmid having the gene sequence of SEQ ID NO: 6, was used as a production strain for the antibacterial protein CDL2200-2, and the gene of SEQ ID NO: 7 produced by the present inventors BL21 pASK_CDL2200-3, a strain produced by transforming E. coli using a plasmid having the sequence, was used as a production strain for the antibacterial protein CDL2200-3, and a plasmid with the gene sequence of SEQ ID NO: 8 produced by the present inventors was used. BL21 pASK_CDL2200-4, a strain produced by transforming E. coli, was used as a production strain for the antibacterial protein CDL2200-4.
50 μg/ml 카나마이신이 포함된 LB배지 (Tryptone 10 g/L, Yeast extract 5 g/L, NaCl 5 g/L) 20 ml에 생산균주를 20 μl 첨가하여 접종한 다음 37℃에서 한밤 동안 진탕 배양하였다. 다음날, 50 μg/ml 카나마이신이 포함된 LB배지 1 L에 한밤 배양한 배양액을 1/100 부피비로 첨가한 다음에, 200 rpm의 교반속도로, 37℃ 온도 조건에서 배양을 실시하였다. 세포 농도가 600 nm에서의 흡광도 기준으로 0.5가 되었을 때, 최종 농도가 0.2 μg/ml이 되도록 언하이드로테트라사이클린 (Anhydrotetracycline)을 첨가하여 항균단백질의 발현을 유도하였으며, 발현 유도 후에 4시간 동안 배양을 추가 실시하였다. Inoculate 20 μl of the production strain into 20 ml of LB medium (Tryptone 10 g/L, Yeast extract 5 g/L, NaCl 5 g/L) containing 50 μg/ml kanamycin, then culture with shaking at 37°C overnight. did. The next day, the overnight culture was added to 1 L of LB medium containing 50 μg/ml kanamycin at a volume ratio of 1/100, and culture was performed at a temperature of 37°C at a stirring speed of 200 rpm. When the cell concentration reached 0.5 based on absorbance at 600 nm, anhydrotetracycline was added to achieve a final concentration of 0.2 μg/ml to induce expression of the antibacterial protein. After induction of expression, the cells were cultured for 4 hours. Additional implementation was carried out.
배양 종료 후, 세포 배양액을 취하여 4,000 rpm에서 15분간 4℃에서 원심분리하여 세포 침전물을 회수하였고, 회수한 세포 침전물은 40 ml의 완충액 (60 mM Potassium phosphate buffer, pH 7.5)에 부유시켰다. 이렇게 준비된 세포 부유액에 대하여 초음파 분쇄법을 적용하여 세포를 파쇄하였고, 상기 초음파 분쇄법은 3초간 초음파를 가하여 세포를 깨고, 5초간 멈추는 조건을 총 20분간 반복하는 방식으로 실시하였으며, 이 과정은 얼음조 (Ice bath)에서 실시하였다.After completion of the culture, the cell culture fluid was taken and centrifuged at 4,000 rpm for 15 minutes at 4°C to recover the cell sediment. The recovered cell sediment was suspended in 40 ml of buffer solution (60 mM Potassium phosphate buffer, pH 7.5). The ultrasonic disruption method was applied to the cell suspension prepared in this way to disrupt the cells. The ultrasonic disruption method was performed by applying ultrasound for 3 seconds to break the cells, stopping for 5 seconds, and repeating this process for a total of 20 minutes. This process was performed on ice. This was conducted in an ice bath.
세포 파쇄 후에 세포 파쇄액을 13,000 rpm에서 20분간 4℃에서 원심분리하여 얻어진 상등액을 얻었고, 이 얻어진 상등액은 통상의 양이온-교환 크로마토그래피 (Cation-exchange chromatography) 정제 공정에 적용하여 목적하는 항균단백질의 정제를 실시하였다.After cell disruption, the cell disruption fluid was centrifuged at 13,000 rpm for 20 minutes at 4°C to obtain a supernatant. This obtained supernatant was subjected to a typical cation-exchange chromatography purification process to obtain the desired antibacterial protein. Purification was performed.
정제 공정을 간단히 설명하면 다음과 같다. 본 실시예에서는 양이온-교환수지 (Cation-exchange resin)로 5 ml의 HiTrapTM SP FF (GE Healthcare)를 사용하였다. 크로마토그래피는 칼럼을 Buffer A (60 mM Potassium phosphate buffer, pH 7.5, 20 CV)로 미리 평형화시킨 후에 실시하였고, 시료를 칼럼에 적하 (Loading)한 다음에는 5 ml/min의 유속 (Flow rate)으로 상기 buffer A를 6 CV (Column Volume) 흘려주어 세척 (Washing)을 실시하였다. 세척 후에는 5 ml/분의 유속으로 buffer A에서 buffer B (60 mM Potassium phosphate buffer, 1 M NaCl, pH 7.5)로의 농도 기울기 (Gradient)가 40%에서 60%가 되게 하는 조건으로 크로마토그래피를 수행하였다. 이 과정에서 목적하는 항균단백질의 용출이 달성되었다. 확보된 정제 분획 중에 항균단백질을 고농도로 포함하고 있는 분획들을 모았고, 이를 완충액 (20 mM Potassium phosphate buffer, pH 7.5)에 대하여 투석을 실시하여 매질 교환을 수행하였다. 이러한 과정을 통하여 90% 이상의 순도를 갖는 항균단백질 용액을 확보할 수 있었다.The purification process is briefly explained as follows. In this example, 5 ml of HiTrap TM SP FF (GE Healthcare) was used as a cation-exchange resin. Chromatography was performed after pre-equilibrating the column with Buffer A (60 mM Potassium phosphate buffer, pH 7.5, 20 CV), and after loading the sample onto the column, the column was loaded at a flow rate of 5 ml/min. Washing was performed by flowing 6 CV (Column Volume) of buffer A. After washing, chromatography was performed under conditions such that the concentration gradient from buffer A to buffer B (60 mM potassium phosphate buffer, 1 M NaCl, pH 7.5) was from 40% to 60% at a flow rate of 5 ml/min. did. In this process, elution of the desired antibacterial protein was achieved. Among the obtained purified fractions, fractions containing high concentrations of antibacterial proteins were collected, and medium exchange was performed by dialyzing them against a buffer solution (20 mM potassium phosphate buffer, pH 7.5). Through this process, it was possible to secure an antibacterial protein solution with a purity of over 90%.
도 1은 최종 확보한 정제 항균단백질들에 대한 전기영동 분석 결과를 보여준다.Figure 1 shows the results of electrophoresis analysis of the finally obtained purified antibacterial proteins.
실시예Example
2: 2:
세포수감소조사법을Cell count reduction method
통한 through
항균단백질의of antibacterial protein
항균활성 조사 Antibacterial activity investigation
실시예 1에 따라 제조된 클로스트리디오이데스 디피실리 균 특이 항균단백질 CDL2200-1, 항균단백질 CDL2200-2, 항균단백질 CDL2200-3, 및 항균단백질 CDL2200-4의 항생제 내성 클로스트리디오이데스 디피실리 균에 대한 항균활성을 조사하였다. 항균활성 조사에 사용된 클로스트리디오이데스 디피실리 균으로는 항생제 내성 클로스트리디오이데스 디피실리 균주 9종을 대상으로 하였으며, 그 상세 내역은 표 1과 같다.Antibiotic resistance of Clostridioides difficile-specific antibacterial protein CDL2200-1, antibacterial protein CDL2200-2, antibacterial protein CDL2200-3, and antibacterial protein CDL2200-4 prepared according to Example 1 to Clostridioides difficile bacteria Antibacterial activity was investigated. Clostridioides difficile bacteria used to investigate antibacterial activity included 9 antibiotic-resistant Clostridioides difficile strains, the details of which are shown in Table 1.
| 균종fungus | 균주strain | 확보기관Securing agency |
| ClostridioidesClostridioides difficile difficile | ATCC 43255ATCC 43255 | American Type Culture CollectionAmerican Type Culture Collection |
| CCARM 0184CCARM 0184 | 항생제내성균주은행Antibiotic-resistant strain bank | |
| CCARM 0185CCARM 0185 | ||
| CCARM 0186CCARM 0186 | ||
| CCARM 0187CCARM 0187 | ||
| CCARM 0188CCARM 0188 | ||
| CCARM 0189CCARM 0189 | ||
| CCARM 0190CCARM 0190 | ||
| CCARM 0348CCARM 0348 |
항균활성 조사는 세포수감소조사법 (Cell count reduction assay)을 이용하여 실시하였다. 세포수감소조사법의 실험방법은 다음과 같다. 완충액 (20 mM Tris-HCl, pH 7.5)을 이용하여 항균단백질은 최종농도가 10 μg/ml이 되게 하고 시험대상균주의 최종농도는 1× 106 CFU/ml가 되게 1 ml의 반응액을 준비하였다. 이를 37℃에서 1 시간 또는 2 시간 동안 정치하였다. 정치 후 100 μl를 BHIS plate (Brain heart infusion 37 g/L, Yeast extract 5 g/L, 0.05% L-cysteine hydrochloride monohydrate, Agar 15 g/L)에 도말한 다음에 37℃ 혐기조건에서 한밤 배양하여 콜로니 수를 확인하였다. 이때 음성대조군으로는 항균단백질이 첨가되지 않게 준비한 반응액 실험을 사용하였다.한편, 클로스트리디오이데스 디피실리 균 외의 균종에 대한 항균활성 평가도 상기와 동일 방식의 세포수감소조사법을 통하여 실시하였는데, 엔테로코쿠스 패칼리스 (Enterococcus faecalis) 2주, 엔테로코쿠스 패시움 (Enterococcus faecium) 2주, 황색포도상구균 (Staphylococcus aureus) 2주, 살모넬라 (Salmonella) 2주, 및 대장균 (Escherichia coli) 2주에 대해 실시하였다. Antibacterial activity was tested using cell count reduction assay. The experimental method of the cell count reduction method is as follows. Using buffer (20mM Tris-HCl, pH 7.5), prepare 1 ml of reaction solution so that the final concentration of the antibacterial protein is 10 μg/ml and the final concentration of the strain to be tested is 1×10 6 CFU/ml. did. It was left standing at 37°C for 1 or 2 hours. After standing, 100 μl was spread on a BHIS plate (Brain heart infusion 37 g/L, Yeast extract 5 g/L, 0.05% L-cysteine hydrochloride monohydrate, Agar 15 g/L), and then incubated overnight at 37°C under anaerobic conditions. The number of colonies was confirmed. At this time, as a negative control, a reaction solution experiment prepared without the addition of antibacterial protein was used. Meanwhile, evaluation of antibacterial activity against species other than Clostridioides difficile was also conducted using the same cell count reduction method as above. Enterococcus faecalis faecalis ) 2 weeks, Enterococcus faecium faecium ) 2 weeks, Staphylococcus aureus 2 weeks, Salmonella 2 weeks, and Escherichia coli ) was conducted for 2 weeks.
그 결과, 항균단백질 CDL2200-1, 항균단백질 CDL2200-2, 항균단백질 CDL2200-3, 및 항균단백질 CDL2200-4는 실험 대상 클로스트리디오이데스 디피실리 균 모두에 대해서 용균활성을 보였고, 다른 시험대상균주들에 대해서는 용균활성을 나타내지 않았다. 클로스트리디오이데스 디피실리 균에 대한 용균활성을 보여주는 결과로 클로스트리디오이데스 디피실리 균 ATCC 43255에 대한 결과를 도 2에 제시하였다.As a result, antibacterial protein CDL2200-1, antibacterial protein CDL2200-2, antibacterial protein CDL2200-3, and antibacterial protein CDL2200-4 showed lytic activity against all Clostridioides difficile bacteria tested and other test strains. did not show lytic activity. As a result showing the lytic activity against Clostridioides difficile bacteria, the results for Clostridioides difficile ATCC 43255 are presented in Figure 2.
이러한 결과로부터 본 발명의 항균단백질 CDL2200-1, 항균단백질 CDL2200-2, 항균단백질 CDL2200-3, 및 항균단백질 CDL2200-4의 항균활성이 클로스트리디오이데스 디피실리 균에 대하여 매우 특이적이라는 것을 확인할 수 있었다. From these results, it can be confirmed that the antibacterial activity of the antibacterial protein CDL2200-1, antibacterial protein CDL2200-2, antibacterial protein CDL2200-3, and antibacterial protein CDL2200-4 of the present invention is very specific to Clostridioides difficile bacteria. there was.
따라서, 본 발명의 클로스트리디오이데스 디피실리 균 특이적 항균단백질 CDL2200-1, 항균단백질 CDL2200-2, 항균단백질 CDL2200-3, 또는 항균단백질 CDL2200-4 중에서 하나 이상을 포함하는 약학적 조성물이 클로스트리디오이데스 디피실리 균 감염 처치 목적으로 통상의 항생제와 같은 방식으로 활용될 수 있음을 알 수 있었다.Therefore, the pharmaceutical composition containing one or more of the Clostridioides difficile-specific antibacterial protein CDL2200-1, antibacterial protein CDL2200-2, antibacterial protein CDL2200-3, or antibacterial protein CDL2200-4 of the present invention is clostri. It was found that it can be used in the same way as conventional antibiotics for the purpose of treating Dioides difficile infection.
실시예Example
3: 3:
클로스트리디오이데스Clostridioides
디피실리difficile
균 특이적 fungus-specific
항균단백질의of antibacterial protein
클로스트리디오이데스Clostridioides
디피실리difficile
균 감염에 대한 치료적 효과 조사 Investigation of therapeutic effects on bacterial infections
클로스트리디오이데스 디피실리 균 특이적 항균단백질 CDL2200-1, 항균단백질 CDL2200-2, 항균단백질 CDL2200-3, 및 항균단백질 CDL2200-4의 클로스트리디오이데스 디피실리 균 감염에 대한 치료적 효과를 감염 동물 모델 실험을 통해 조사하였다.Therapeutic effects of Clostridioides difficile-specific antibacterial protein CDL2200-1, antibacterial protein CDL2200-2, antibacterial protein CDL2200-3, and antibacterial protein CDL2200-4 on Clostridioides difficile infection in infected animals This was investigated through model experiments.
구체적으로, 6주령 SD 랫드 [Specific pathogen-free (SPF) grade]를 시험동물로 사용하였다. 총 30마리의 랫드에 항생제를 포함한 식수 (카나마이신 0.4 mg/ml, 젠타마이신 0.035 mg/ml, 콜리스틴 850 U/ml, 메트로니다졸 0.215 mg/ml, 그리고 반코마이신 0.045 mg/ml)를 5일 동안 제공하였다. 그 후 멸균된 식수를 2일 동안 제공하였다. 멸균된 식수 제공 마지막 날 (클로스트리디오이데스 디피실리 균 감염 하루 전)에 클린다마이신 (20 mg/kg)을 복강 내 주사하였다. 클린다마이신 주사 다음 날에 클로스트리디오이데스 디피실리 균 강제감염을 실시하였다. 클로스트리디오이데스 디피실리 균의 강제감염은 클로스트리디오이데스 디피실리 균을 포자 상태로 제조하여 멸균 생리식염수로 부유시켜 준비한 액 0.2 ml (약 107 CFU/ml에 해당)을 존데 (Sonde)를 이용하여 위관 내 강제로 주입하는 방식으로 실시하였다. 강제감염 실시 후, 48시간이 지난 시점에 랫드를 5그룹으로 나눈 다음에 그룹 1 (6마리)은 완충액 (Buffer; 20 mM potassium phosphate buffer, pH 7.5)을 1 ml 투여하였고 (대조군), 그룹 2 (6마리)는 1일 3회 항균단백질 CDL2200-1을 포함한 조성물을 존데를 이용하여 위관 내 강제 주입하였고, 그룹 3 (6마리)은 1일 3회 항균단백질 CDL2200-2를 포함한 조성물을 존데를 이용하여 위관 내 강제 주입하였고, 그룹 4 (6마리)는 1일 3회 항균단백질 CDL2200-3을 포함한 조성물을 존데를 이용하여 위관 내 강제 주입하였고, 그룹 5 (6마리)는 1일 3회 항균단백질 CDL2200-4를 포함한 조성물을 존데를 이용하여 위관 내 강제 주입하였다. 항균단백질을 포함한 조성물 투여는 7일 동안 지속하였다. 항균단백질을 포함한 조성물은 항균단백질 용액을 동결건조시켜 제조한 동결건조체와 부형제 (슈크로즈)를 혼합하여 제조한 분말을 통상의 장용캡슐에 넣어 제조하였다. 1캡슐 당 항균단백질이 2 mg 씩 포함되도록 제조하였다. 항균단백질을 포함한 조성물 투여 7일째까지 각 그룹 동물들의 생존률을 조사하였다. 그 결과가 표 2에 제시되어 있다. Specifically, 6-week-old SD rats [Specific pathogen-free (SPF) grade] were used as test animals. A total of 30 rats were provided with drinking water containing antibiotics (kanamycin 0.4 mg/ml, gentamicin 0.035 mg/ml, colistin 850 U/ml, metronidazole 0.215 mg/ml, and vancomycin 0.045 mg/ml) for 5 days. . Afterwards, sterilized drinking water was provided for 2 days. Clindamycin (20 mg/kg) was administered intraperitoneally on the last day of provision of sterile drinking water (one day before Clostridioides difficile infection). Forced infection with Clostridioides difficile was performed the day after clindamycin injection. Forcible infection with Clostridioides difficile is performed by preparing Clostridioides difficile in spore form and suspending it in sterile saline solution, and then using a sonde with 0.2 ml (equivalent to approximately 10 7 CFU/ml) of the solution. This was performed by forceful injection into the stomach tube. 48 hours after forced infection, the rats were divided into 5 groups, and group 1 (6 rats) was administered 1 ml of buffer (20 mM potassium phosphate buffer, pH 7.5) (control group), and group 2 was administered 1 ml of buffer (20 mM potassium phosphate buffer, pH 7.5). (6 animals) were forcibly injected into the gastric tube using a sonde with a composition containing the antibacterial protein CDL2200-1 three times a day, and Group 3 (6 animals) was injected with a composition containing the antibacterial protein CDL2200-2 three times a day using a sonde. Group 4 (6 animals) was forcibly injected into the gastric tube using a sonde with a composition containing the antibacterial protein CDL2200-3 three times a day, and Group 5 (6 animals) was injected with an antibacterial agent three times a day. A composition containing the protein CDL2200-4 was forcefully injected into the gastric tube using a sonde. Administration of the composition containing antibacterial proteins continued for 7 days. A composition containing an antibacterial protein was prepared by mixing a freeze-dried product prepared by freeze-drying an antibacterial protein solution and an excipient (sucrose), and placing the powder in a regular enteric capsule. It was prepared to contain 2 mg of antibacterial protein per capsule. The survival rate of animals in each group was investigated until the 7th day of administration of the composition containing antibacterial protein. The results are presented in Table 2.
| 생존률(%) Survival rate (%) | |||||||
| D1D1 | D2D2 | D3D3 | D4D4 | D5D5 |
D6 | D7D7 | |
| 그룹1 (완충액)Group 1 (buffer) |
100100 | 83.383.3 | 66.766.7 | 33.333.3 | 16.716.7 | 00 | 00 |
|
그룹2 (CDL2200-1) (CDL2200-1) |
100100 | 100100 | 100100 | 100100 | 100100 | 100100 | 100100 |
|
그룹3 (CDL2200-2) (CDL2200-2) |
100100 | 100100 | 100100 | 100100 | 100100 | 100100 | 100100 |
|
그룹4 (CDL2200-3) (CDL2200-3) |
100100 | 100100 | 100100 | 100100 | 100100 | 100100 | 100100 |
|
그룹5 (CDL2200-4)Group 5 (CDL2200-4) |
100100 | 100100 | 100100 | 100100 | 100100 | 100100 | 100100 |
이상의 결과로 본 발명의 항균단백질 CDL2200-1, 항균단백질 CDL2200-2, 항균단백질 CDL2200-3, 및 항균단백질 CDL2200-4가 클로스트리디오이데스 디피실리 균의 감염 치료에 효과적임을 확인할 수 있었다. 이러한 특성은 본 발명의 항균단백질 CDL2200-1, 항균단백질 CDL2200-2, 항균단백질 CDL2200-3, 또는 항균단백질 CDL2200-4 중에서 하나 이상을 유효성분으로 포함하는 약학적 조성물이 클로스트리디오이데스 디피실리 균 감염 치료 목적으로 활용될 수 있음을 보여 준다. From the above results, it was confirmed that the antibacterial protein CDL2200-1, antibacterial protein CDL2200-2, antibacterial protein CDL2200-3, and antibacterial protein CDL2200-4 of the present invention are effective in treating infection with Clostridioides difficile bacteria. These characteristics are such that the pharmaceutical composition containing one or more of the antibacterial protein CDL2200-1, antibacterial protein CDL2200-2, antibacterial protein CDL2200-3, or antibacterial protein CDL2200-4 of the present invention as an active ingredient is effective against Clostridioides difficile bacteria. It shows that it can be used for infection treatment purposes.
이상으로 본 발명의 특정한 부분을 상세히 기술하였는바, 당업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적인 기술은 단지 바람직한 구현 예일 뿐이며, 이에 본 발명의 범위가 제한되는 것이 아닌 점은 명백하다. 따라서 본 발명의 실질적인 범위는 첨부된 청구항과 그의 등가물에 의하여 정의된다고 할 것이다.As the specific parts of the present invention have been described in detail above, it is clear to those skilled in the art that these specific techniques are merely preferred implementation examples and do not limit the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.
[수탁번호][Accession number]
기탁기관명 : 한국생명공학연구원 생물자원센터(KCTC)Name of depository organization: Korea Research Institute of Bioscience and Biotechnology Biological Resources Center (KCTC)
수탁번호 : KCTC15213BPAccession number: KCTC15213BP
수탁일자 : 20221129Trust date: 20221129
기탁기관명 : 한국생명공학연구원 생물자원센터(KCTC)Name of depository organization: Korea Research Institute of Bioscience and Biotechnology Biological Resources Center (KCTC)
수탁번호 : KCTC15214BPAccession number: KCTC15214BP
수탁일자 : 20221129Trust date: 20221129
기탁기관명 : 한국생명공학연구원 생물자원센터(KCTC)Name of depository organization: Korea Research Institute of Bioscience and Biotechnology Biological Resources Center (KCTC)
수탁번호 : KCTC15215BPAccession number: KCTC15215BP
수탁일자 : 20221129Trust date: 20221129
기탁기관명 : 한국생명공학연구원 생물자원센터(KCTC)Name of depository organization: Korea Research Institute of Bioscience and Biotechnology Biological Resources Center (KCTC)
수탁번호 : KCTC15216BPAccession number: KCTC15216BP
수탁일자 : 20221129Trust date: 20221129
[규칙 제91조에 의한 정정 23.11.2023]
[Correction pursuant to Rule 91 23.11.2023]
[규칙 제91조에 의한 정정 23.11.2023]
[Correction pursuant to Rule 91 23.11.2023]
[규칙 제91조에 의한 정정 23.11.2023]
[Correction pursuant to Rule 91 23.11.2023]
[규칙 제91조에 의한 정정 23.11.2023]
[Correction pursuant to Rule 91 23.11.2023]
Claims (11)
- 클로스트리디오이데스 디피실리 (Clostridioides difficile) 균 특이적 항균활성을 갖는 서열번호 1로 표시되는 아미노산 서열을 갖는 항균단백질 CDL2200-1, 클로스트리디오이데스 디피실리 균 특이적 항균활성을 갖는 서열번호 2로 표시되는 아미노산 서열을 갖는 항균단백질 CDL2200-2, 클로스트리디오이데스 디피실리 균 특이적 항균활성을 갖는 서열번호 3으로 표시되는 아미노산 서열을 갖는 항균단백질 CDL2200-3, 또는 클로스트리디오이데스 디피실리 균 특이적 항균활성을 갖는 서열번호 4로 표시되는 아미노산 서열을 갖는 항균단백질 CDL2200-4 중에서 하나 이상을 유효성분으로 포함하는 클로스트리디오이데스 디피실리 균 감염 질환 처치용 약학적 조성물.Clostridioides difficile ( Clostridioides difficile ) CDL2200-1, an antibacterial protein having an amino acid sequence shown in SEQ ID NO: 1 and having bacteria-specific antibacterial activity, an antibacterial protein having an amino acid sequence shown in SEQ ID NO: 2 having specific antibacterial activity against Clostridioides difficile bacteria CDL2200-2, an antibacterial protein CDL2200-3 having the amino acid sequence shown in SEQ ID NO: 3, which has Clostridioides difficile-specific antibacterial activity, or SEQ ID NO: 4, which has Clostridioides difficile-specific antibacterial activity A pharmaceutical composition for treating Clostridioides difficile infectious diseases, comprising as an active ingredient one or more of the antibacterial protein CDL2200-4 having an amino acid sequence represented by .
- 제1항에 있어서, 상기 항균단백질 CDL2200-1은 178개의 아미노산으로 구성되며 분자량이 20.3 kDa인 것을 특징으로 하는 클로스트리디오이데스 디피실리 균 감염 질환 처치용 약학적 조성물.The pharmaceutical composition for treating Clostridioides difficile infectious diseases according to claim 1, wherein the antibacterial protein CDL2200-1 consists of 178 amino acids and has a molecular weight of 20.3 kDa.
- 제1항에 있어서, 상기 항균단백질 CDL2200-2는 191개의 아미노산으로 구성되며 분자량이 21.6 kDa인 것을 특징으로 하는 클로스트리디오이데스 디피실리 균 감염 질환 처치용 약학적 조성물.The pharmaceutical composition for treating Clostridioides difficile infectious diseases according to claim 1, wherein the antibacterial protein CDL2200-2 consists of 191 amino acids and has a molecular weight of 21.6 kDa.
- 제1항에 있어서, 상기 항균단백질 CDL2200-3은 187개의 아미노산으로 구성되며 분자량이 21.0 kDa인 것을 특징으로 하는 클로스트리디오이데스 디피실리 균 감염 질환 처치용 약학적 조성물.The pharmaceutical composition for treating Clostridioides difficile infectious diseases according to claim 1, wherein the antibacterial protein CDL2200-3 consists of 187 amino acids and has a molecular weight of 21.0 kDa.
- 제1항에 있어서, 상기 항균단백질 CDL2200-4는 185개의 아미노산으로 구성되며 분자량이 20.7 kDa인 것을 특징으로 하는 클로스트리디오이데스 디피실리 균 감염 질환 처치용 약학적 조성물.The pharmaceutical composition for treating Clostridioides difficile infectious diseases according to claim 1, wherein the antibacterial protein CDL2200-4 consists of 185 amino acids and has a molecular weight of 20.7 kDa.
- 제1항에 있어서, 상기 클로스트리디오이데스 디피실리 균 감염 질환은 장의 염증과 괴사, 설사, 위막성 대장염, 위장관 천공, 패혈증, 중독성 거대결장증인 것을 특징으로 하는 클로스트리디오이데스 디피실리 균 감염 질환 처치용 약학적 조성물.The Clostridioides difficile infection disease according to claim 1, wherein the Clostridioides difficile infection disease is intestinal inflammation and necrosis, diarrhea, pseudomembranous colitis, gastrointestinal perforation, sepsis, and toxic megacolon. Pharmaceutical composition for treatment.
- 제1항에 있어서, 상기 약학적 조성물은 항생제, 소독제, 살균제 또는 치료제 용도로 사용되는 것을 특징으로 하는 클로스트리디오이데스 디피실리 균 감염 질환 처치용 약학적 조성물.The pharmaceutical composition for treating Clostridioides difficile infectious diseases according to claim 1, wherein the pharmaceutical composition is used as an antibiotic, disinfectant, disinfectant, or therapeutic agent.
- 서열번호 5의 유전자 서열로 표시되는 플라스미드를 사용하여 대장균을 형질전환시켜 제작한 균주 BL21 pASK_CDL2200-1 (수탁번호 KCTC 15213BP)을 이용하여 클로스트리디오이데스 디피실리 균 특이적 항균단백질 CDL2200-1을 제조하는 방법.Clostridioides difficile-specific antibacterial protein CDL2200-1 was prepared using strain BL21 pASK_CDL2200-1 (accession number KCTC 15213BP), which was produced by transforming E. coli using the plasmid represented by the gene sequence of SEQ ID NO: 5. How to.
- 서열번호 6의 유전자 서열로 표시되는 플라스미드를 사용하여 대장균을 형질전환시켜 제작한 균주 BL21 pASK_CDL2200-2 (수탁번호 KCTC 15214BP)를 이용하여 클로스트리디오이데스 디피실리 균 특이적 항균단백질 CDL2200-2를 제조하는 방법.Clostridioides difficile-specific antibacterial protein CDL2200-2 was prepared using strain BL21 pASK_CDL2200-2 (accession number KCTC 15214BP) prepared by transforming E. coli using the plasmid represented by the gene sequence of SEQ ID NO: 6. How to.
- 서열번호 7의 유전자 서열로 표시되는 플라스미드를 사용하여 대장균을 형질전환시켜 제작한 균주 BL21 pASK_CDL2200-3 (수탁번호 KCTC 15215BP)을 이용하여 클로스트리디오이데스 디피실리 균 특이적 항균단백질 CDL2200-3을 제조하는 방법.Clostridioides difficile-specific antibacterial protein CDL2200-3 was prepared using strain BL21 pASK_CDL2200-3 (accession number KCTC 15215BP), which was produced by transforming E. coli using the plasmid represented by the gene sequence of SEQ ID NO. 7. How to.
- 서열번호 8의 유전자 서열로 표시되는 플라스미드를 사용하여 대장균을 형질전환시켜 제작한 균주 BL21 pASK_CDL2200-4 (수탁번호 KCTC 15216BP)를 이용하여 클로스트리디오이데스 디피실리 균 특이적 항균단백질 CDL2200-4를 제조하는 방법.Clostridioides difficile-specific antibacterial protein CDL2200-4 was prepared using strain BL21 pASK_CDL2200-4 (accession number KCTC 15216BP), which was produced by transforming E. coli using the plasmid represented by the gene sequence of SEQ ID NO. 8. How to.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR10-2022-0175429 | 2022-12-15 | ||
| KR1020220175429A KR20240097985A (en) | 2022-12-15 | 2022-12-15 | Antibacterial Protein CDL2200 Having Lytic Activity to Clostridioides difficile |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2024128526A1 true WO2024128526A1 (en) | 2024-06-20 |
Family
ID=91485114
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/KR2023/016633 WO2024128526A1 (en) | 2022-12-15 | 2023-10-25 | Antimicrobial protein cdl2200 having lytic activity to clostridioides difficile |
Country Status (2)
| Country | Link |
|---|---|
| KR (1) | KR20240097985A (en) |
| WO (1) | WO2024128526A1 (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2020057742A1 (en) * | 2018-09-19 | 2020-03-26 | Technische Universität Braunschweig | Vaccine and antibody against clostidioides difficile toxin |
| KR20200135145A (en) * | 2019-05-24 | 2020-12-02 | 한국식품연구원 | Lactobacillus paracasei wikim0110 having antibacterial activity against clostridioides difficile and composition comprising the same |
| KR20210130581A (en) * | 2020-04-22 | 2021-11-01 | 주식회사 인트론바이오테크놀로지 | Antibacterial Protein CDL200 Having Lytic Activity to Clostridioides difficile |
| KR20210149377A (en) * | 2020-06-02 | 2021-12-09 | 주식회사 인트론바이오테크놀로지 | Antibacterial Protein CEL200 Having Lytic Activity to Clostridioides difficile |
| KR102374480B1 (en) * | 2021-09-13 | 2022-03-15 | 동아제약 주식회사 | Lactobacillus paracasei EPS DA-BACS promoting the growth of Bifidobacterium and inhibiting Clostridium difficile and polysaccharide therefrom |
-
2022
- 2022-12-15 KR KR1020220175429A patent/KR20240097985A/en unknown
-
2023
- 2023-10-25 WO PCT/KR2023/016633 patent/WO2024128526A1/en unknown
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2020057742A1 (en) * | 2018-09-19 | 2020-03-26 | Technische Universität Braunschweig | Vaccine and antibody against clostidioides difficile toxin |
| KR20200135145A (en) * | 2019-05-24 | 2020-12-02 | 한국식품연구원 | Lactobacillus paracasei wikim0110 having antibacterial activity against clostridioides difficile and composition comprising the same |
| KR20210130581A (en) * | 2020-04-22 | 2021-11-01 | 주식회사 인트론바이오테크놀로지 | Antibacterial Protein CDL200 Having Lytic Activity to Clostridioides difficile |
| KR20210149377A (en) * | 2020-06-02 | 2021-12-09 | 주식회사 인트론바이오테크놀로지 | Antibacterial Protein CEL200 Having Lytic Activity to Clostridioides difficile |
| KR102374480B1 (en) * | 2021-09-13 | 2022-03-15 | 동아제약 주식회사 | Lactobacillus paracasei EPS DA-BACS promoting the growth of Bifidobacterium and inhibiting Clostridium difficile and polysaccharide therefrom |
Non-Patent Citations (1)
| Title |
|---|
| DATABASE Protein 8 June 2021 (2021-06-08), ANONYMOUS: "TPA: N-acetylmuramoyl-L-alanine amidase [Clostridioides difficile]", XP093182047, retrieved from NCBI Database accession no. HBF9175164.1 * |
Also Published As
| Publication number | Publication date |
|---|---|
| KR20240097985A (en) | 2024-06-27 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| WO2016108536A1 (en) | Novel clostridium perfringens bacteriophage clo-pep-1 and use thereof for inhibiting proliferation of clostridium perfringens | |
| WO2016108540A1 (en) | Novel enteropathogenic e. coli bacteriophage esc-chp-2 and use thereof for inhibiting proliferation of enteropathogenic e. coli | |
| WO2016108538A1 (en) | Novel enterohemorrhagic e. coli bacteriophage esc-chp-1 and use thereof for inhibiting proliferation of enterohemorrhagic e. coli | |
| WO2017111306A1 (en) | Novel pasteurella multocida bacteriophage pas-mup-1 and use thereof for inhibiting proliferation of pasteurella multocida | |
| WO2016108541A1 (en) | Novel shigatoxin-producing f18 type e. coli bacteriophage esc-cop-1 and use thereof for inhibiting proliferation of shigatoxin-producing f18 type e. coli | |
| KR20010071416A (en) | Lactic acid bacterium-containing compositions, drugs and foods | |
| WO2018155812A1 (en) | Novel enterococcus faecium bacteriophage ent-fap-4 and use for inhibiting enterococcus faecium proliferation of same | |
| WO2016108542A1 (en) | Novel enteroinvasive e. coli bacteriophage esc-cop-4 and use thereof for inhibiting proliferation of enteroinvasive e. coli | |
| WO2018155813A2 (en) | Novel antibacterial protein efal-2 having bacteriolytic ability with respect to enterococcus faecium | |
| WO2018101594A1 (en) | Escherichia coli bacteriophage esc-cop-7, and use thereof for suppressing proliferation of pathogenic escherichia coli | |
| WO2020013451A1 (en) | E. coli bacteriophage esc-cop-14 and use thereof in inhibiting growth of pathogenic e. coli | |
| WO2016126009A1 (en) | Novel edwardsiella tarda bacteriophage edw-tap-1 and use thereof for inhibiting proliferation of edwardsiella tarda | |
| WO2018155814A1 (en) | Novel clostridium perfringens bacteriophage clo-pep-2 and use for inhibiting clostridium perfringens proliferation of same | |
| WO2017217726A1 (en) | Novel vibrio parahaemolyticus bacteriophage vib-pap-5 and use thereof for suppressing proliferation of vibrio parahaemolyticus bacteria | |
| WO2018151417A1 (en) | Novel pseudomonas aeruginosa bacteriophage pse-aep-4 and use thereof for inhibiting proliferation of pseudomonas aeruginosa | |
| WO2018151416A1 (en) | Novel pseudomonas aeruginosa bacteriophage pse-aep-3 and use thereof for inhibiting proliferation of pseudomonas aeruginosa | |
| EP3402506A1 (en) | An antibacterial composition and a method of treating staphylococcal infections with the antibacterial composition | |
| WO2018208001A1 (en) | Novel vibrio parahaemolyticus bacteriophage vib-pap-7 and use of same for inhibiting vibrio parahaemolyticus bacteria proliferation | |
| WO2019235781A1 (en) | Novel streptococcus suis bacteriophage str-sup-1 and use thereof in inhibiting streptococcus suis bacterium proliferation | |
| WO2019235782A1 (en) | Novel streptococcus suis bacteriophage str-sup-2, and use thereof for inhibiting proliferation of streptococcus suis strains | |
| WO2021246670A1 (en) | Antibacterial protein having lytic activity against clostridioides difficile | |
| WO2021215687A1 (en) | Antibacterial protein cdl200 having lytic activity against clostridioides difficile | |
| WO2011132943A2 (en) | Composition for treatment of acute pseudomembranous colitis | |
| WO2024128526A1 (en) | Antimicrobial protein cdl2200 having lytic activity to clostridioides difficile | |
| WO2019235783A1 (en) | Novel streptococcus suis bacteriophage str-sup-3, and use thereof for inhibiting proliferation of streptococcus suis strains |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| 121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 23903730 Country of ref document: EP Kind code of ref document: A1 |