CN112574991A - Oligonucleotide, carrier, preparation method and application - Google Patents
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Abstract
The invention provides an oligonucleotide, a vector, a preparation method and application thereof, wherein the nucleotide sequence of a sense strand of the oligonucleotide is shown as SEQ ID NO.3, and the nucleotide sequence of an antisense strand is shown as SEQ ID NO. 4. The invention successfully constructs the oligonucleotide for down-regulating the Postn expression quantity through design and research. The oligonucleotide molecule is used for repairing myogenic disorder of myotonic dystrophy type I for the first time, and the oligonucleotide molecule achieves the aim of repairing myogenic dysfunction by down regulating the expression quantity of Postn protein in a DM1 disease cell model.
Description
Technical Field
The invention relates to the technical field of gene therapy, in particular to oligonucleotide, a vector, a preparation method and application.
Background
Myotonic dystrophy type I (DM1) is an autosomal dominant genetic disease characterized mainly by progressive muscle weakness, muscular atrophy and myotonia, and is also the most common muscular dystrophy disease in skeletal muscle development of adults, with incidence rates ranging from 1/8000 to 1/7000. DM1 was caused by an over-amplified trinucleotide (CTG) repeat sequence located in the 3 'untranslated region (3' UTR) of the DMPK gene. Transcription of over-amplified trinucleotide (CTG) repeats generates "toxic RNA" containing over-amplified CUG repeats, resulting in a reduction of free MBNL1 and upregulation of the level of Celf 1. Changes in the levels of MBNL1 and Celf1 resulted in changes in the variable cleavage pattern of the associated genes within the cell, leading to the development of symptoms of DM 1. The failure of the myogenic process in DM1 is one of the major causes of the skeletal muscle disorder in DM1, mainly manifested by a significant loss of myoblast differentiation capacity and a significant decrease in myoblast fusion capacity. At present, the mechanism of myogenic dysfunction in DM1 has been studied, but is controversial. Accordingly, few specific therapeutic targets and methods for DM1 myogenic dysfunction have been reported.
At present, no method for radically treating DM1 exists clinically, and only a few treatment hypotheses are proposed based on a DM1 pathogenesis, and the treatment hypotheses mainly comprise the following three methods: the first was by restoring the levels of MBNL1 and Celf1 in DM1 to correct the wrong gene splicing pattern in DM 1; secondly, degrading toxic RNA by using molecules such as polypeptide or chemical micromolecular organic matters; the third is to use gene editing means to cut out the segment encoding toxic RNA on the genome. The evaluation of the effects and clinical tests of these three treatment hypotheses is still in progress and the safety is unknown. And none of the above treatment hypotheses was specifically proposed for myogenic dysfunction in DM1, so finding a therapeutic target and therapeutic strategy for specific DM1 myogenic dysfunction was essential for the restoration of skeletal muscle dysfunction in DM 1.
Disclosure of Invention
The invention aims to solve the technical problems in the prior art. Therefore, the invention provides an oligonucleotide, a vector, a preparation method and application thereof, and aims to down-regulate the expression level of Postn protein in a DM1 disease cell model so as to achieve the purpose of repairing myogenic dysfunction.
Based on the above purpose, the invention provides an oligonucleotide, wherein the nucleotide sequence of a sense strand of the oligonucleotide is shown as SEQ ID NO.3, and the nucleotide sequence of an antisense strand is shown as SEQ ID NO. 4.
The preparation method of the oligonucleotide comprises the following steps:
step one, selecting siRNA molecules acting on 2063-2085 region on Postn mRNA to obtain sense strand sequence and antisense strand sequence of siRNA;
placing the sense strand sequence and the antisense strand sequence of the siRNA obtained in the step one in an oligonucleotide template to obtain a sense strand nucleotide sequence and an antisense strand nucleotide sequence of the oligonucleotide;
and step three, annealing and fusing the nucleotide sequence of the sense strand and the nucleotide sequence of the antisense strand of the oligonucleotide into a double strand, incubating for the first time, adding 10mM ATP and 10U/uL T4 Kinase, and continuing incubating for the second time to obtain the oligonucleotide.
In the first step, the sequence of the sense strand of the siRNA is shown as SEQ ID NO.1, and the sequence of the antisense strand is shown as SEQ ID NO. 2.
The sense strand sequence of the siRNA is 5'-GGCAGTCTTCAGCCTATTA-3',
the antisense strand sequence is 5'-TAATAGGCTGAAGACTGCC-3'.
And the first incubation in the third step is to place the mixed system fused into the double chains at 94 ℃ for incubation for 4 minutes, then place the mixed system in a water bath with the constant temperature of 70 ℃ for incubation for 10 minutes, and then close the heating of the water bath to gradually and slowly cool the water bath to the room temperature.
The second incubation in step three was first incubated at 37 ℃ for 60 minutes, followed by incubation at 65 ℃ for 10 minutes.
The invention selects siRNA molecules acting on 2063-2085 region of Postn mRNA, and the thermodynamic energy value of the combination of the siRNA molecules and the Postn mRNA is the lowest and is-6.8.
The method for constructing the oligonucleotide molecule for down-regulating the expression level of Postn is as follows:
placing the obtained sense and antisense strand sequences in an oligonucleotide template specially designed to obtain a forward sequence (sense strand nucleotide sequence) and a reverse sequence (antisense strand nucleotide sequence) of the oligonucleotide, which are respectively marked as shPostn-F and shPostn-R;
the sequence of the shPostn-F is as follows:
5’-TGGCAGTCTTCAGCCTATTATTCAAGAGATAATAGGCTGAAGACTGCCTTTTTTC-3’；
the sequence of the shPostn-R is as follows:
5’-TCGAGAAAAAAGGCAGTCTTCAGCCTATTATCTCTTGAATAATAGGCTGAAGACTGCCA-3’；
the above sequences were synthesized and diluted to 10ug/uL, respectively, and mixed in the following reaction system (10ug/uL shPostn-F5 uL, 10ug/uL shPostn-R5 uL, 5X T4 kinase forward buffer 20uL, ddH2O 45 uL). And (3) incubating the reaction system at 94 ℃ for 4 minutes, then incubating the reaction system in a water bath with the constant temperature of 70 ℃ for 10 minutes, and then closing heating of the water bath to gradually and slowly cool the water bath, so that the reaction system is slowly cooled to the room temperature. Thereafter, 8uL of the reaction mixture was removed from the reaction system, added to 1uL of 10mM ATP and 1uL of 10U/uL of T4 Kinase, and incubated at 37 ℃ for 60 minutes, followed by incubation at 65 ℃ for 10 minutes. The reaction product was diluted 50-fold, which was the oligonucleotide molecule used for the ligation reaction.
The invention also provides a carrier containing the oligonucleotide.
The invention also provides a preparation method of the carrier, which comprises the following steps:
step one, cutting a PLL4.0 plasmid by using restriction endonuclease, scaling, performing agarose gel electrophoresis, cutting a 7228bp band, and recovering gel to obtain a linearized PLL4.0 vector;
step two, connecting the oligonucleotide with a linearized PLL4.0 carrier;
and step three, transforming the ligation product obtained in the step two into DH5 alpha escherichia coli competence, coating a plate with ampicillin resistance for colony culture, selecting a single colony to extract plasmids, and sequencing to obtain the recombinant DNA.
Sequencing ensured the correctness of the construction of the shRNA plasmid ligated into pLL4.0, and the correct shRNA plasmid was designated as pLL4.0-shPostn and used for transfection of DM1 cell model.
Preferably, the restriction enzymes in the first step include HpaI and XhoI restriction enzymes.
Preferably, the descaling in the first step is a terminal descaling using FastAP phosphatase for 2 hours.
The invention also provides application of the oligonucleotide in preparing a medicament for down-regulating the Postn protein expression level in the cells of the type I disease of the ankylosing spondylitis.
The invention has the beneficial effects that:
1. the invention firstly utilizes oligonucleotide molecules to repair myogenic disorder of myotonic dystrophy type I, and the oligonucleotide molecules achieve the aim of repairing myogenic dysfunction by down regulating the expression quantity of Postn protein in a DM1 disease cell model.
2. The invention successfully constructs the oligonucleotide for down-regulating the Postn expression quantity through design and research.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a schematic diagram of the placement of sense strand sequence and antisense strand sequence of siRNA of the present invention in designed oligonucleotide template;
FIG. 2 is a graph comparing the myoblast differentiation ability of DM1 myoblast of the present invention with that of normal mouse myoblast;
FIG. 3 is a graph comparing the skeletal muscle formation area of the DM1 myoblasts of the invention with that of normal mouse myoblasts;
FIG. 4 is a graph comparing the fusion index of DM1 myoblasts of the invention with normal mouse myoblasts;
FIG. 5 is a graph of immunoblot assay detection of down-regulated Postn in DM1 disease cells of the invention versus a control group;
FIG. 6 is a graph comparing the ability of the present invention to down-regulate Postn versus control constitutive muscle in a DM1 disease cell model;
FIG. 7 is a graph comparing the area of skeletal muscle formation of the present invention down-regulated Postn in a DM1 disease cell model with a control group;
FIG. 8 is a graph comparing the skeletal muscle cell fusion index of Postn and a control group down-regulated in a DM1 disease cell model according to the invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to specific embodiments and the accompanying drawings.
It is to be noted that technical terms or scientific terms used in the embodiments of the present invention should have the ordinary meanings as understood by those having ordinary skill in the art to which the present disclosure belongs, unless otherwise defined.
Sources of the reagents used in the present invention: molecular cloning reagents such as restriction enzymes, modification enzymes and ligase, as well as culture media and other additives for cell culture media were purchased from Thermo Fisher Scientific, and other inorganic and organic reagents were purchased from Sigma Aldrich.
Example 1
1. Design of siRNA against Postn mRNA
Postn mRNA sequence (ID: ENSMUST00000081564.12) is obtained from a database, the sequence is placed in a Whitehead design platform (http:// siRNA. wi. mit. edu), and a 'NAN 19N' mode is selected to obtain a plurality of siRNA sequences. The sense strand was selected among these sequences to begin with "G" and bind the thermodynamically lowest energy siRNA to the mRNA molecule of Postn. Finally, siRNA molecules were selected that acted on the 2063-2085 region of Postn mRNA with the lowest thermodynamic energy value for binding to Postn mRNA at-6.8.
The sense strand sequence of the siRNA is 5'-GGCAGTCTTCAGCCTATTA-3',
the antisense strand sequence is 5'-TAATAGGCTGAAGACTGCC-3'.
2. Construction of oligonucleotide molecules for Down-regulating expression of Postn
The sense and antisense strand sequences obtained in step 1 were placed in a specially designed oligonucleotide template (FIG. 1) to obtain the forward (sense strand sequence) and reverse (antisense strand sequence) sequences of the oligonucleotide, which were designated as shPostn-F and shPostn-R, respectively.
The sequence of the shPostn-F is as follows:
5’-TGGCAGTCTTCAGCCTATTATTCAAGAGATAATAGGCTGAAGACTGCCTTTTTTC-3’；
the sequence of the shPostn-R is as follows:
5’-TCGAGAAAAAAGGCAGTCTTCAGCCTATTATCTCTTGAATAATAGGCTGAAGACTGCCA-3’。
the above sequences were synthesized and diluted to 10ug/uL, respectively, and mixed with the following reaction system (10ug/uL shPostn-F5 uL, 10ug/uL shPostn-R5 uL, 5X T4 kinase forward buffer 20uL, ddH2O45 uL). And (3) incubating the reaction system at 94 ℃ for 4 minutes, then incubating the reaction system in a water bath with the constant temperature of 70 ℃ for 10 minutes, and then closing heating of the water bath to gradually and slowly cool the water bath, so that the reaction system is slowly cooled to the room temperature. Thereafter, 8uL of the reaction mixture was removed from the reaction system, added to 1uL of 10mM ATP and 1uL of 10U/uL of T4 Kinase, and incubated at 37 ℃ for 60 minutes, followed by incubation at 65 ℃ for 10 minutes. The reaction product was diluted 50-fold, which was the oligonucleotide molecule used for the ligation reaction.
3. Oligonucleotide molecules for down-regulating Postn expression level are connected into plasmid vector
2ug of pLL4.0 plasmid was cut with HpaI and XhoI restriction enzymes while end-dephosphorizing with FastAP phosphatase for 2 hours in the following reaction system: pLL4.02ug, HpaI1uL, XhoI 1uL, FastAP 1uL, 10 XFastdigest buffer 2uL, plus ddH2O to a total volume of 20 uL. After the reaction, the reaction system was subjected to 1% agaroseGel electrophoresis, cutting a 7228bp band, and performing gel recovery by using a gel recovery kit to obtain the DNA which is a linearized pLL4.0 vector. The linearized pLL4.0 vector and the oligonucleotide molecule obtained in step 2 are ligated under the action of T4 ligase, and the reaction system is as follows: linearized pLL4.0 vector 2uL, oligonucleotide 6uL, 10X T4 ligase buffer 2uL, PEG 40002 uL, T4 ligase 1uL, plus ddH2O to a total volume of 20uL, and ligation was carried out at 22 ℃ for 4 hours. Thereafter, DH 5. alpha. E.coli competent was transformed with the ligation product and plated with ampicillin resistant plates for colony culture. And (3) picking a single colony to extract plasmids, and sequencing to ensure the correctness of the shRNA plasmid construction connected with pLL4.0. The correct shRNA plasmid was designated pLL4.0-shPostn and used for transfection of the DM1 cell model.
4. A mouse myoblast model of myotonic dystrophy type I (DM1) was constructed and verified for myoblast differentiation dysfunction.
The expression vectors of 200 copies and 5 copies of CUG repetitive sequences in the 3' UTR region are respectively transfected into mouse myoblast C2C12 cells, and after G418 drug screening and stabilization, a DM1 and normal mouse myoblast model are obtained. The two cell models were subjected to in vitro myogenic differentiation, and their myogenic differentiation ability was examined. Immunofluorescent staining for structural protein MF20 of skeletal muscle fibers at the end of myoblast differentiation (day six) revealed that myoblast differentiation capacity of DM1 myoblasts was significantly reduced compared to normal mouse myoblasts (fig. 2), as evidenced by statistics of skeletal muscle Area (Myotube Area) (fig. 3). Skeletal muscle cell nuclear Fusion Index (Fusion Index) measurements showed that the Fusion capacity of DM1 myoblasts was also significantly reduced compared to the normal group (fig. 4). The above phenomena are consistent with the phenotype in DM1 patients, indicating successful cell modeling.
5. Cell lines constructed for control and Down-regulated Postn in DM1 disease cell model
And (3) transferring the pLL4.0-shPostn plasmid obtained in the step (3) into the DM1 disease cell model constructed in the step (4), and screening by using puromycin until cells stably grow to obtain a DM1 disease model cell line for down-regulating Postn by using oligonucleotide. Control groups were modeled by transfecting DM1 disease cells with the pLL4.0-shScamble plasmid that had been constructed in the laboratory. The expression level of the Postn protein in the pLL4.0-shPostn group was significantly reduced by immunoblotting experiments, indicating that the cell line was successfully constructed (FIG. 5).
6. Down-regulation of Postn in the DM1 disease cell model could significantly repair the defect in myoblast differentiation in DM1
The control and Postn-down-regulated DM1 disease model cell lines obtained in step 4 were subjected to myogenic differentiation experiments and immunofluorescent staining for skeletal muscle fiber structural protein MF20 at the end of differentiation (day six), and it was found that down-regulation of Postn in the DM1 disease cell model significantly improved myogenic capacity of DM1 disease myoblasts (fig. 6). Skeletal muscle formation area measurements also showed that Postn down-regulation significantly improved myogenic capacity of DM1 disease cells (fig. 7). Skeletal muscle cell nuclear fusion index assay showed that down-regulation of Postn could repair the defect of low myoblast fusion in DM1 (figure 8). Taken together, the expression of Postn in DM1 is obviously increased, indicating that Postn is possibly related to the pathogenic mechanism of DM 1; downregulation of Postn in the DM1 myoblast model significantly repaired DM1 myogenic dysfunction, suggesting that downregulation of Postn may be useful in the treatment of skeletal myogenic dysfunction in DM 1.
Those of ordinary skill in the art will understand that: the discussion of any embodiment above is meant to be exemplary only, and is not intended to intimate that the scope of the disclosure, including the claims, is limited to these examples; within the idea of the invention, also features in the above embodiments or in different embodiments may be combined, steps may be implemented in any order, and there are many other variations of the different aspects of the invention as described above, which are not provided in detail for the sake of brevity.
The embodiments of the invention are intended to embrace all such alternatives, modifications and variances that fall within the broad scope of the appended claims. Therefore, any omissions, modifications, substitutions, improvements and the like that may be made without departing from the spirit and principles of the invention are intended to be included within the scope of the invention.
Sequence listing
<110> university of teacher's university in Anhui
<120> oligonucleotide, carrier, preparation method and application
<130> 1
<141> 2020-12-17
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<170> SIPOSequenceListing 1.0
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tcgagaaaaa aggcagtctt cagcctatta tctcttgaat aataggctga agactgcca 59
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gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg 60
atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt 120
gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc 180
tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac 240
attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat 300
atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg 360
acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt 420
tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag 480
tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc 540
attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag 600
tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt 660
ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc 720
accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg 780
gcggtaggcg tgtacggtgg gaggtctata taagcagcgc gttttgcctg tactgggtct 840
ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt 900
aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac 960
tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc 1020
gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc 1080
ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa 1140
ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg 1200
ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata 1260
aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc 1320
tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga 1380
caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc 1440
aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca 1500
aaagtaagac caccgcacag caagcggccg gccgcgctga tcttcagacc tggaggagga 1560
gatatgaggg acaattggag aagtgaatta tataaatata aagtagtaaa aattgaacca 1620
ttaggagtag cacccaccaa ggcaaagaga agagtggtgc agagagaaaa aagagcagtg 1680
ggaataggag ctttgttcct tgggttcttg ggagcagcag gaagcactat gggcgcagcg 1740
tcaatgacgc tgacggtaca ggccagacaa ttattgtctg gtatagtgca gcagcagaac 1800
aatttgctga gggctattga ggcgcaacag catctgttgc aactcacagt ctggggcatc 1860
aagcagctcc aggcaagaat cctggctgtg gaaagatacc taaaggatca acagctcctg 1920
gggatttggg gttgctctgg aaaactcatt tgcaccactg ctgtgccttg gaatgctagt 1980
tggagtaata aatctctgga acagatttgg aatcacacga cctggatgga gtgggacaga 2040
gaaattaaca attacacaag cttaatacac tccttaattg aagaatcgca aaaccagcaa 2100
gaaaagaatg aacaagaatt attggaatta gataaatggg caagtttgtg gaattggttt 2160
aacataacaa attggctgtg gtatataaaa ttattcataa tgatagtagg aggcttggta 2220
ggtttaagaa tagtttttgc tgtactttct atagtgaata gagttaggca gggatattca 2280
ccattatcgt ttcagaccca cctcccaacc ccgaggggac ccgacaggcc cgaaggaata 2340
gaagaagaag gtggagagag agacagagac agatccattc gattagtgaa cggatcggca 2400
ctgcgtgcgc caattctgca gacaaatggc agtattcatc cacaatttta aaagaaaagg 2460
ggggattggg gggtacagtg caggggaaag aatagtagac ataatagcaa cagacataca 2520
aactaaagaa ttacaaaaac aaattacaaa aattcaaaat tttcgggttt attacaggga 2580
cagcagagat ccagtttggt tagtaccggg cccgctctag agatccgacg ccgccatctc 2640
taggcccgcg ccggccccct cgcacagact tgtgggagaa gctcggctac tcccctgccc 2700
cggttaattt gcatataata tttcctagta actatagagg cttaatgtgc gataaaagac 2760
agataatctg ttctttttaa tactagctac attttacatg ataggcttgg atttctataa 2820
gagatacaaa tactaaatta ttattttaaa aaacagcaca aaaggaaact caccctaact 2880
gtaaagtaat tgtgtgtttt gagactataa atatcccttg gagaaaagcc ttgttaacgc 2940
gcggtgaccc tcgagatccg cggccgcgcc ggctctagat cgcgaacgcg tgaattctac 3000
cgggtagggg aggcgctttt cccaaggcag tctggagcat gcgctttagc agccccgctg 3060
ggcacttggc gctacacaag tggcctctgg cctcgcacac attccacatc caccggtagg 3120
cgccaaccgg ctccgttctt tggtggcccc ttcgcgccac cttctactcc tcccctagtc 3180
aggaagttcc cccccgcccc gcagctcgcg tcgtgcagga cgtgacaaat ggaagtagca 3240
cgtctcacta gtctcgtgca gatggacagc accgctgagc aatggaagcg ggtaggcctt 3300
tggggcagcg gccaatagca gctttgctcc ttcgctttct gggctcagag gctgggaagg 3360
ggtgggtccg ggggcgggct caggggcggg ctcaggggcg gggcgggcgc ccgaaggtcc 3420
tccggaggcc cggcattctg cacgcttcaa aagcgcacgt ctgccgcgct gttctcctct 3480
tcctcatctc cgggcctttc gacctgcagc ccaagcttac catgaccgag tacaagccca 3540
cggtgcgcct cgccacccgc gacgacgtcc ccagggccgt acgcaccctc gccgccgcgt 3600
tcgccgacta ccccgccacg cgccacaccg tcgatccgga ccgccacatc gagcgggtca 3660
ccgagctgca agaactcttc ctcacgcgcg tcgggctcga catcggcaag gtgtgggtcg 3720
cggacgacgg cgccgcggtg gcggtctgga ccacgccgga gagcgtcgaa gcgggggcgg 3780
tgttcgccga gatcggcccg cgcatggccg agttgagcgg ttcccggctg gccgcgcagc 3840
aacagatgga aggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg ttcctggcca 3900
ccgtcggcgt ctcgcccgac caccagggca agggtctggg cagcgccgtc gtgctccccg 3960
gagtggaggc ggccgagcgc gccggggtgc ccgccttcct ggagacctcc gcgccccgca 4020
acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag gtgcccgaag 4080
gaccgcgcac ctggtgcatg acccgcaagc ccggtgcctg acgggcgcgt ctggaacaat 4140
caacctctgg attacaaaat ttgtgaaaga ttgactggta ttcttaacta tgttgctcct 4200
tttacgctat gtggatacgc tgctttaatg cctttgtatc atgctattgc ttcccgtatg 4260
gctttcattt tctcctcctt gtataaatcc tggttgctgt ctctttatga ggagttgtgg 4320
cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt 4380
tggggcattg ccaccacctg tcagctcctt tccgggactt tcgctttccc cctccctatt 4440
gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc tcggctgttg 4500
ggcactgaca attccgtggt gttgtcgggg aagctgacgt cctttccatg gctgctcgcc 4560
tgtgttgcca cctggattct gcgcgggacg tccttctgct acgtcccttc ggccctcaat 4620
ccagcggacc ttccttcccg cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc 4680
cttcgccctc agacgagtcg gatctccctt tgggccgcct ccccgcctgg aattaattct 4740
gcagtcgaga cctagaaaaa catggagcaa tcacaagtag caatacagca gctaccaatg 4800
ctgattgtgc ctggctagaa gcacaagagg aggaggaggt gggttttcca gtcacacctc 4860
agctttaaga ccaatgactt acaaggcagc tgtagatctt agccactttt taaaagaaaa 4920
ggggggactg gaagggctaa ttcactccca acgaagacaa gatatccttg atctgtggat 4980
ctaccacaca caaggctact tccctgattg gcagaactac acaccagggc cagggatcag 5040
atatccactg acctttggat ggtgctacaa gctagtacca gttgagcaag agaaggtaga 5100
agaagccaat gaaggagaga acacccgctt gttacaccct gtgagcctgc atgggatgga 5160
tgacccggag agagaagtat tagagtggag gtttgacagc cgcctagcat ttcatcacat 5220
ggcccgagag ctgcatccgg actgtactgg gtctctctgg ttagaccaga tctgagcctg 5280
ggagctctct ggctaactag ggaacccact gcttaagcct caataaagct tgccttgagt 5340
gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt aactagagat ccctcagacc 5400
cttttagtca gtgtggaaaa tctctagcag catgtgagca aaaggccagc aaaaggccag 5460
gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca 5520
tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca 5580
ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg 5640
atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag 5700
gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt 5760
tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca 5820
cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg 5880
cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt 5940
tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc 6000
cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg 6060
cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg 6120
gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta 6180
gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg 6240
gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg 6300
ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc 6360
atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc 6420
agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc 6480
ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag 6540
tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat 6600
ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg 6660
caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt 6720
gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag 6780
atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg 6840
accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt 6900
aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct 6960
gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac 7020
tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat 7080
aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat 7140
ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca 7200
aataggggtt ccgcgcacat ttccccgaaa agtgccacct gac 7243
<210> 6
<211> 7283
<212> DNA
<213> pLL4.0-scrambling (pLL4.0-scrambles)
<400> 6
gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg 60
atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt 120
gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc 180
tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac 240
attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat 300
atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg 360
acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt 420
tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag 480
tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc 540
attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag 600
tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt 660
ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc 720
accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg 780
gcggtaggcg tgtacggtgg gaggtctata taagcagcgc gttttgcctg tactgggtct 840
ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt 900
aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac 960
tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc 1020
gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc 1080
ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa 1140
ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg 1200
ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata 1260
aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc 1320
tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga 1380
caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc 1440
aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca 1500
aaagtaagac caccgcacag caagcggccg gccgcgctga tcttcagacc tggaggagga 1560
gatatgaggg acaattggag aagtgaatta tataaatata aagtagtaaa aattgaacca 1620
ttaggagtag cacccaccaa ggcaaagaga agagtggtgc agagagaaaa aagagcagtg 1680
ggaataggag ctttgttcct tgggttcttg ggagcagcag gaagcactat gggcgcagcg 1740
tcaatgacgc tgacggtaca ggccagacaa ttattgtctg gtatagtgca gcagcagaac 1800
aatttgctga gggctattga ggcgcaacag catctgttgc aactcacagt ctggggcatc 1860
aagcagctcc aggcaagaat cctggctgtg gaaagatacc taaaggatca acagctcctg 1920
gggatttggg gttgctctgg aaaactcatt tgcaccactg ctgtgccttg gaatgctagt 1980
tggagtaata aatctctgga acagatttgg aatcacacga cctggatgga gtgggacaga 2040
gaaattaaca attacacaag cttaatacac tccttaattg aagaatcgca aaaccagcaa 2100
gaaaagaatg aacaagaatt attggaatta gataaatggg caagtttgtg gaattggttt 2160
aacataacaa attggctgtg gtatataaaa ttattcataa tgatagtagg aggcttggta 2220
ggtttaagaa tagtttttgc tgtactttct atagtgaata gagttaggca gggatattca 2280
ccattatcgt ttcagaccca cctcccaacc ccgaggggac ccgacaggcc cgaaggaata 2340
gaagaagaag gtggagagag agacagagac agatccattc gattagtgaa cggatcggca 2400
ctgcgtgcgc caattctgca gacaaatggc agtattcatc cacaatttta aaagaaaagg 2460
ggggattggg gggtacagtg caggggaaag aatagtagac ataatagcaa cagacataca 2520
aactaaagaa ttacaaaaac aaattacaaa aattcaaaat tttcgggttt attacaggga 2580
cagcagagat ccagtttggt tagtaccggg cccgctctag agatccgacg ccgccatctc 2640
taggcccgcg ccggccccct cgcacagact tgtgggagaa gctcggctac tcccctgccc 2700
cggttaattt gcatataata tttcctagta actatagagg cttaatgtgc gataaaagac 2760
agataatctg ttctttttaa tactagctac attttacatg ataggcttgg atttctataa 2820
gagatacaaa tactaaatta ttattttaaa aaacagcaca aaaggaaact caccctaact 2880
gtaaagtaat tgtgtgtttt gagactataa atatcccttg gagaaaagcc ttgtttgggt 2940
gaactcacgt cagaattcaa gagattctga cgtgagttca cccttttttc tcgagatccg 3000
cggccgcgcc ggctctagat cgcgaacgcg tgaattctac cgggtagggg aggcgctttt 3060
cccaaggcag tctggagcat gcgctttagc agccccgctg ggcacttggc gctacacaag 3120
tggcctctgg cctcgcacac attccacatc caccggtagg cgccaaccgg ctccgttctt 3180
tggtggcccc ttcgcgccac cttctactcc tcccctagtc aggaagttcc cccccgcccc 3240
gcagctcgcg tcgtgcagga cgtgacaaat ggaagtagca cgtctcacta gtctcgtgca 3300
gatggacagc accgctgagc aatggaagcg ggtaggcctt tggggcagcg gccaatagca 3360
gctttgctcc ttcgctttct gggctcagag gctgggaagg ggtgggtccg ggggcgggct 3420
caggggcggg ctcaggggcg gggcgggcgc ccgaaggtcc tccggaggcc cggcattctg 3480
cacgcttcaa aagcgcacgt ctgccgcgct gttctcctct tcctcatctc cgggcctttc 3540
gacctgcagc ccaagcttac catgaccgag tacaagccca cggtgcgcct cgccacccgc 3600
gacgacgtcc ccagggccgt acgcaccctc gccgccgcgt tcgccgacta ccccgccacg 3660
cgccacaccg tcgatccgga ccgccacatc gagcgggtca ccgagctgca agaactcttc 3720
ctcacgcgcg tcgggctcga catcggcaag gtgtgggtcg cggacgacgg cgccgcggtg 3780
gcggtctgga ccacgccgga gagcgtcgaa gcgggggcgg tgttcgccga gatcggcccg 3840
cgcatggccg agttgagcgg ttcccggctg gccgcgcagc aacagatgga aggcctcctg 3900
gcgccgcacc ggcccaagga gcccgcgtgg ttcctggcca ccgtcggcgt ctcgcccgac 3960
caccagggca agggtctggg cagcgccgtc gtgctccccg gagtggaggc ggccgagcgc 4020
gccggggtgc ccgccttcct ggagacctcc gcgccccgca acctcccctt ctacgagcgg 4080
ctcggcttca ccgtcaccgc cgacgtcgag gtgcccgaag gaccgcgcac ctggtgcatg 4140
acccgcaagc ccggtgcctg acgggcgcgt ctggaacaat caacctctgg attacaaaat 4200
ttgtgaaaga ttgactggta ttcttaacta tgttgctcct tttacgctat gtggatacgc 4260
tgctttaatg cctttgtatc atgctattgc ttcccgtatg gctttcattt tctcctcctt 4320
gtataaatcc tggttgctgt ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg 4380
cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt tggggcattg ccaccacctg 4440
tcagctcctt tccgggactt tcgctttccc cctccctatt gccacggcgg aactcatcgc 4500
cgcctgcctt gcccgctgct ggacaggggc tcggctgttg ggcactgaca attccgtggt 4560
gttgtcgggg aagctgacgt cctttccatg gctgctcgcc tgtgttgcca cctggattct 4620
gcgcgggacg tccttctgct acgtcccttc ggccctcaat ccagcggacc ttccttcccg 4680
cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg 4740
gatctccctt tgggccgcct ccccgcctgg aattaattct gcagtcgaga cctagaaaaa 4800
catggagcaa tcacaagtag caatacagca gctaccaatg ctgattgtgc ctggctagaa 4860
gcacaagagg aggaggaggt gggttttcca gtcacacctc agctttaaga ccaatgactt 4920
acaaggcagc tgtagatctt agccactttt taaaagaaaa ggggggactg gaagggctaa 4980
ttcactccca acgaagacaa gatatccttg atctgtggat ctaccacaca caaggctact 5040
tccctgattg gcagaactac acaccagggc cagggatcag atatccactg acctttggat 5100
ggtgctacaa gctagtacca gttgagcaag agaaggtaga agaagccaat gaaggagaga 5160
acacccgctt gttacaccct gtgagcctgc atgggatgga tgacccggag agagaagtat 5220
tagagtggag gtttgacagc cgcctagcat ttcatcacat ggcccgagag ctgcatccgg 5280
actgtactgg gtctctctgg ttagaccaga tctgagcctg ggagctctct ggctaactag 5340
ggaacccact gcttaagcct caataaagct tgccttgagt gcttcaagta gtgtgtgccc 5400
gtctgttgtg tgactctggt aactagagat ccctcagacc cttttagtca gtgtggaaaa 5460
tctctagcag catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt 5520
tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa 5580
gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct 5640
ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc 5700
cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg 5760
tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct 5820
tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag 5880
cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga 5940
agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga 6000
agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg 6060
gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag 6120
aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag 6180
ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat 6240
gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct 6300
taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac 6360
tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa 6420
tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg 6480
gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt 6540
gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca 6600
ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt 6660
cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct 6720
tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg 6780
cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg 6840
agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg 6900
cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa 6960
aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt 7020
aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt 7080
gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt 7140
gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca 7200
tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat 7260
ttccccgaaa agtgccacct gac 7283
Claims (7)
1. An oligonucleotide, characterized in that the nucleotide sequence of a sense strand of the oligonucleotide is shown as SEQ ID NO.3, and the nucleotide sequence of an antisense strand is shown as SEQ ID NO. 4.
2. A vector comprising the oligonucleotide of claim 1.
3. The method for preparing the oligonucleotide according to claim 1, comprising the steps of:
step one, selecting siRNA molecules acting on 2063-2085 region on Postn mRNA to obtain sense strand sequence and antisense strand sequence of siRNA;
placing the sense strand sequence and the antisense strand sequence of the siRNA obtained in the step one in an oligonucleotide template to obtain a sense strand nucleotide sequence and an antisense strand nucleotide sequence of the oligonucleotide;
and step three, annealing and fusing the nucleotide sequence of the sense strand and the nucleotide sequence of the antisense strand of the oligonucleotide into a double strand, incubating for the first time, adding 10mM ATP and 10U/uL T4 Kinase, and continuing incubating for the second time to obtain the oligonucleotide.
4. The method for preparing the oligonucleotide according to claim 3, wherein the sense strand sequence of the siRNA in the first step is shown as SEQ ID No.1, and the antisense strand sequence is shown as SEQ ID No. 2.
5. The method for preparing the oligonucleotide according to claim 3, wherein the one-time incubation in the third step is to incubate the mixed system fused into the double strand at 94 ℃ for 4 minutes, incubate the mixed system in a water bath at a constant temperature of 70 ℃ for 10 minutes, and then turn off the heating of the water bath to gradually and slowly cool the water bath to room temperature.
6. The method for preparing the oligonucleotide of claim 3, wherein the second incubation in step three is an incubation at 37 ℃ for 60 minutes, followed by an incubation at 65 ℃ for 10 minutes.
7. Use of the oligonucleotide of claim 1 in the preparation of a medicament for down-regulating the amount of Postn protein expression in a cell of a type I disorder of myotonic dystrophy.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117467664A (en) * | 2023-11-07 | 2024-01-30 | 江苏省家禽科学研究所 | Antisense oligonucleotide targeting chicken MBNL1 gene and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030211141A1 (en) * | 2001-12-11 | 2003-11-13 | Lebaron Richard G. | Genetic and protein manipulation of betaIG-H3 for the treatment and cure of muscular dystrophies |
| CN1905894A (en) * | 2003-11-25 | 2007-01-31 | 诺瓦提斯公司 | Biomarkers for the efficacy of calcitonin and parathyroid hormone treatment |
| CN102666587A (en) * | 2009-07-02 | 2012-09-12 | 菲布罗根有限公司 | Methods for treatment of muscular dystrophy |
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2020
- 2020-12-17 CN CN202011492355.9A patent/CN112574991B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030211141A1 (en) * | 2001-12-11 | 2003-11-13 | Lebaron Richard G. | Genetic and protein manipulation of betaIG-H3 for the treatment and cure of muscular dystrophies |
| CN1905894A (en) * | 2003-11-25 | 2007-01-31 | 诺瓦提斯公司 | Biomarkers for the efficacy of calcitonin and parathyroid hormone treatment |
| CN102666587A (en) * | 2009-07-02 | 2012-09-12 | 菲布罗根有限公司 | Methods for treatment of muscular dystrophy |
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| Title |
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| ANGELA LORTS ET AL.: ""Deletion of periostin reduces muscular dystrophy and fibrosis in mice by modulating the transforming growth factor-β pathway"", 《PNAS》 * |
| XIAOPENG SHEN ET AL.: ""Inhibition of Postn Rescues Myogenesis Defects in Myotonic Dystrophy Type 1 Myoblast Model"", 《FRONTIERS IN CELL AND DEVELOPMENTAL BIOLOGY》 * |
| 方肇勤: "《分子生物学技术在中医药研究中的应用》", 30 April 2018, 上海科学技术出版社 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117467664A (en) * | 2023-11-07 | 2024-01-30 | 江苏省家禽科学研究所 | Antisense oligonucleotide targeting chicken MBNL1 gene and application thereof |
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| CN112574991B (en) | 2023-03-17 |
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