CN118121701A - Use of OGDHL in preparing medicine for treating chronic heart failure - Google Patents
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- CN118121701A CN118121701A CN202410202268.7A CN202410202268A CN118121701A CN 118121701 A CN118121701 A CN 118121701A CN 202410202268 A CN202410202268 A CN 202410202268A CN 118121701 A CN118121701 A CN 118121701A
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- ogdhl
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- heart failure
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Classifications
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- 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
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
- C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- 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
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
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- C12N2310/53—Physical structure partially self-complementary or closed
- C12N2310/531—Stem-loop; Hairpin
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- C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
- C12N2750/00011—Details
- C12N2750/14011—Parvoviridae
- C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
- C12N2750/14141—Use of virus, viral particle or viral elements as a vector
- C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
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- Bioinformatics & Cheminformatics (AREA)
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- Pharmacology & Pharmacy (AREA)
- Veterinary Medicine (AREA)
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- Cardiology (AREA)
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- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Heart & Thoracic Surgery (AREA)
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Abstract
The invention discloses an application of OGDHL in preparing a medicine for treating chronic heart failure, and belongs to the technical field of medical treatment. The invention provides an application of OGDHL serving as a target in screening medicines for treating chronic heart failure and an application of OGDHL inhibitors in preparing medicines for treating chronic heart failure. The invention also provides recombinant adeno-associated virus rAAV9-cTNT-mOGDHL-shRNA for inhibiting OGDHL, which can target to knock down OGDHL on myocardial cells, reduce inflammatory cell infiltration of myocardial tissue, relieve myocardial fibrosis, reduce myocardial apoptosis and further improve heart failure symptoms. The invention has the potential of being converted into a target drug for clinically treating chronic heart failure.
Description
Technical Field
The invention belongs to the technical field of medical treatment, and particularly relates to an application of OGDHL in preparing a medicament for treating chronic heart failure.
Background
OGDHL is a kind of oxyglutarate dehydrogenase (oxoglutarate dehydrogenase-like), is a subunit of alpha-ketoglutarate dehydrogenase complex, participates in TCA cycle to catalyze alpha-ketoglutarate to succinyl coenzyme A, and is a patent application of TCA cycle with publication No. CN114660284A of speed-limiting enzyme (Deng Y,Xie M,Li Q,et al.Targeting Mitochondria-Inflammation Circuit byβ-Hydroxybutyrate Mitigates HFpEF.Circulation research.2021;128(2):232-45;Mao M,Huang RZ,Zheng J,et al.OGDHL closely associates with tumor microenvironment and can serve as a prognostic biomarker for papillary thyroid cancer.Cancer medicine.2021;10(2):728-36.)., which finds that OGDHL gene deletion can increase stability of HIF-1 alpha protein of liver cancer cells under normal oxygen environment, thereby promoting epithelial-mesenchymal transition of liver cancer cells, further leading to invasion and metastasis of liver cancer cells, and proposes OGDHL can be used as a biomarker for predicting liver cancer metastasis and prognosis, and can effectively inhibit invasion and metastasis of liver cancer by improving OGDHL expression level of cancer cells. The detection of OGDHL protein concentration has been found in the patent application published as CN113311165A to be useful in aiding the diagnosis of fetal down syndrome.
Chronic heart failure refers to the progressive appearance and persistence of symptoms and signs of heart failure based on the underlying chronic heart disease. OGDHL is not reported at present in relation to chronic heart failure.
Disclosure of Invention
According to the invention, OGDHL is found in animal experiments to increase expression in myocardial tissue of a chronic heart failure mouse, and the suppression of OGDHL expression can reduce inflammatory cell infiltration of myocardial tissue, reduce myocardial fibrosis and reduce myocardial apoptosis, so that heart functions and heart failure symptoms are improved. Based on the findings of the present invention, it is an object of the present invention to provide the use of OGDHL in the manufacture of a medicament for the treatment of chronic heart failure.
The aim of the invention is achieved by the following technical scheme:
use of OGDHL as a target in screening for a medicament for the treatment of chronic heart failure, said screening being for an inhibitor of OGDHL.
Use of OGDHL inhibitors for the manufacture of a medicament for the treatment of chronic heart failure.
The OGDHL inhibitor includes a substance inhibiting OGDHL expression, a substance inhibiting OGDHL activity, and the like. Further, the OGDHL inhibitor is a substance which specifically inhibits OGDHL in myocardial cells.
A OGDHL-suppressed shRNA having the nucleotide sequence 5'-GCACCTACTGCCAGCATATTG-3'.
A recombinant vector for inhibiting OGDHL, which comprises the above shRNA. The recombinant vector preferably contains a myocardial cell expression promoter, and can enable the shRNA to be specifically expressed in myocardial cells. The cardiomyocyte expression promoter is preferably cTnT. Furthermore, the recombinant vector takes pAAV-cTnT-master-miRNA as a skeleton vector.
A recombinant adeno-associated virus inhibiting OGDHL is prepared by using the recombinant vector inhibiting OGDHL. Furthermore, the recombinant adeno-associated virus inhibiting OGDHL can target to cardiac muscle cells and knock down rAAV9-cTNT-mOGDHL-shRNA of OGDHL, and the preparation method comprises the following steps:
(1) Designing mOGDHL-shRNA DNA primer, and synthesizing mOGDHL-shRNA fragment by using annealing buffer; the mOGDHL-shRNA DNA primer has the following sequence:
An upstream primer: 5'-CCGGGCACCTACTGCCAGCATATTGCTCGAGCAATATGCTGGCAGTAG GTGCTTTTTG-3' the process of the preparation of the pharmaceutical composition,
A downstream primer: 5'-GATCCAAAAAGCACCTACTGCCAGCATATTGCTCGAGCAATATGCTGG CAGTAGGTGC-3';
(2) The vector pAAV-cTnT-precursor-miRNA is digested by HindIII+XhoI double-cutting enzyme, so that pAAV-cTnT precursor-MIRNA HINDIII +XhoI is obtained for digestion and recovery of large fragments;
(3) Inserting mOGDHL-shRNA fragments into pAAV-cTnT-precursor-miRNA enzyme digestion and recovery large fragments through ligation reaction;
(4) Converting the connection product, and sequencing and verifying the result to obtain a recombinant plasmid pAAV-cTnT-mOGDHL-shRNA;
(5) The recombinant plasmid pAAV-cTnT-mOGDHL-shRNA containing the target fragment, the adeno-associated virus packaging plasmid pAAV2/9 and the helper plasmid pHelper are introduced into 293AAV cells, and the transfected cells are cultured, separated and purified to obtain the rAAV9-cTNT-mOGDHL-shRNA virus with high titer and target genes.
The mOGDHL-shRNA fragment synthesized in the step (1) is specifically as follows:
each primer was dissolved sufficiently in mOGDHL DNA. Mu.L of annealing buffer to synthesize a DNA fragment. Then, mOGDHL-shRNA fragments were synthesized according to the following steps:
1) Annealing buffer volume 16 μl;
2) mOGDHL-shRNA DNA 5' -end primer fragment (100. Mu.M) was 2. Mu.L in volume;
3) mOGDHL-shRNA DNA 3' -end primer fragment (100. Mu.M) was 2. Mu.L in volume;
4) Fully and uniformly mixing the components, putting the EP pipe into a water bath kettle with the temperature of 100 ℃, closing the power supply of the water bath kettle, and naturally cooling the EP pipe;
5) The annealed product was diluted 100-fold with DEPC water.
The plasmid pAAV-cTnT promoter-MIRNAHINDIII +XhoI restriction enzyme fragment obtained in the step (2) is specifically:
the pAAV-cTnT promoter-miRNA vector is sheared by HindIII+XhoI double-cutting enzyme, the reaction temperature of the enzyme cutting reaction in a water bath kettle is 37 ℃ and the time is 4 hours, and the enzyme cutting system of the vector is as follows:
1) Plasmid vector pAAV-cTnT master-miRNA (1.0. Mu.g/. Mu.L) was 2. Mu.L in volume;
2) 10X Buffer CutSmart volumes of 2. Mu.L;
3) The restriction enzyme HindIII volume was 1. Mu.L;
4) The restriction enzyme XhoI volume was 1. Mu.L;
5) ddH 2 O was 14. Mu.L in volume.
The obtained product was subjected to DNA gel electrophoresis, a 4258bp pAAV-cTnT promoter-MIRNA HINDIII +XhoI enzyme fragment was cut, and the resultant was centrifuged to obtain a supernatant (containing the pAAV-cTnT promoter-MIRNAHINDIII +XhoI enzyme fragment).
The recombinant plasmid pAAV-cTnT-mOGDHL-shRNA obtained in the step (3) is specifically:
and (3) connecting the annealing product in the step (1) with pAAV-cTnT master-miRNA in the step (2) for enzyme digestion and recovery of large fragments, and preparing a connecting reaction solution as follows:
1) mOGDHL-shRNA annealed product volume 6. Mu.L;
2) pAAV-cTnT-master-miRNA is subjected to enzyme digestion to obtain a large fragment volume of 2 mu L;
3) 10 XLigase Buffer volume 1. Mu.L;
4) T 4 DNA ligase volume 1. Mu.L;
the 4 components are fully mixed according to the proportion, and the connection reaction is carried out for 4 hours at 22 ℃.
Converting the connection product in the step (4) and verifying specifically as follows: adding 10 mu L of the ligation product obtained in the step (3) into 100 mu L of JM109 competent bacteria, uniformly mixing, placing on ice for 30min, thermally shocking at 43 ℃ for 50s, and placing on ice for 5min again. The bacteria were added to 400. Mu.L of LB medium and incubated at 37℃with shaking on a shaking table for 1h, centrifuged at 3000rpm
2Min, the supernatant was discarded. A volume of 100. Mu.L of LB medium was added, and after the homogenized bacteria were blown with a pipette, the liquid was spread on a 100ug/ML AMPICILLIN resistant LB plate. The medium was inverted and incubated overnight at 37℃in a thermostated bacterial incubator. The next day, 3 colonies with better growth were picked from the medium and inoculated into 5mL of LB medium with 100. Mu.g/ML AMPICILLIN resistance, and shaken overnight at 37 ℃. The following day, the cultured bacterial liquid was sent to the Invitrogen company for sequencing.
The rAAV9-cTNT-mOGDHL-shRNA virus with high titer and containing the target gene obtained in the step (5) is specifically as follows:
1. 293AAV cells were seeded into 15cm cell culture dishes, each containing 1.5X10 7 cells, a total of 24 dishes, cell incubator (37 ℃,5% CO 2) overnight;
2. after 24h, 2h before transfection, each dish of cells was plated with high glucose dmem+p/S containing 10% FBS;
3. plasmid transfection (the following amounts were used per 15cm dish):
(1) Taking 2 EP pipes, which are respectively marked as ① pipes and ② pipes;
(2) 1mL of CPT Buffer A was added to ① tubes;
(3) The following reagents were added sequentially to ② tubes: pAAV-cTnT-mOGDHL-shRNA 20. Mu.g, pHelper 20. Mu.g, pAAV-2/9 20. Mu.g, ddH 2 O850. Mu.L, CPT Buffer B100. Mu.L; the total volume of the mixture is 1mL, and the mixture is gently blown and evenly mixed;
4. The ② tubes of liquid are added into the ① tubes drop by drop, and are repeatedly blown and evenly mixed for about 15 times;
5. Standing at room temperature for 30min, dropwise adding the mixture into a culture dish, and then culturing in a cell incubator (37deg.C, 5% CO 2)
Culturing for 6 hours;
6. blowing cells in the culture dishes by using a pipette so that the cells fall off, and collecting all the cells and the culture medium in 24 culture dishes into 1 new 50mL centrifuge tube;
7. taking a proper amount of liquid nitrogen, immersing the centrifuge tube, putting the centrifuge tube into a water bath kettle with the temperature of 37 ℃ for thawing, repeating for 3 times, so that cells are thoroughly broken, and releasing viruses in the cells;
8. adding 50U/mL DNase and 10U/mL RNase into the obtained solution, and standing in a 37 ℃ incubator for 30min;
9. Adding 0.5% deoxycholate sodium into the obtained solution, and standing for 30min in a 37 ℃ incubator;
10. Cell debris was removed with a 0.45 μm PVDF filter;
11. PEG8000 and 2.5N NaCl are added into the solution to make the final concentration of the solution be 8 percent and 0.5N respectively, the solution is kept stand for 1 to 2 hours at the temperature of 4 ℃, and 3000g is centrifugated for 20 minutes;
12. Discarding the supernatant, re-suspending 10mL of HEPES, adding 10mL of chloroform, mixing for 5min with the mixture turned upside down vigorously, and centrifuging for 10min with 380 g;
13. heating and evaporating chloroform for 20min, and sucking supernatant;
14. Adding PEG8000 and ammonium sulfate into the supernatant to make the concentration of the supernatant be 10% and 13% respectively, and mixing the mixture for 5min with the mixture turned upside down vigorously, and standing the mixture at room temperature for 30-40 min;
15. centrifuging 3500g for 10min, wherein the content is divided into 3 layers, the protein is salted out and precipitated at the bottom or between 2 layers of liquid, and the virus is mainly at the top layer;
16. Gently sucking the uppermost solution, adding 6-12 volumes of virus preservation solution to dilute PEG8000, sucking the mixed solution into a ultrafilter tube, centrifuging 8000g for 10min, and repeating for 3-5 times to ensure complete removal of PEG8000;
17. The virus preservation solution is added into the concentrated virus solution, the total volume is 1mL, and the virus preservation solution is subpackaged and preserved in liquid nitrogen.
Application of shRNA, recombinant vector or recombinant adeno-associated virus for inhibiting OGDHL in preparation of medicines for treating chronic heart failure.
Compared with the prior art, the invention has the advantages that:
The invention uses recombinant adeno-associated virus (rAAV) as a vector to achieve the aim of treating chronic heart failure. rAAV has great prospect as gene therapy and has the following advantages as an expression vector: 1. has quite good safety and does not cause any human diseases; 2. the immunogenicity is low, the rejection reaction of the organism is small, and the safety is further proved to be good; 3. can stably express and achieve long-term curative effect by single treatment; 4. has multiple serotypes and can target body organs for treatment; 5. has tissue specific inhibition effect, i.e. can not influence the expression of OGDHL in other tissues and organs, and maximally reduces the side effect of viruses. By combining mOGDHL-shRNA with the vector and injecting the vector into a chronic heart failure mouse body through a rat tail vein, the aim of treating heart failure by targeting the heart is achieved, and animal experiment results prove that rAAV9-cTNT-mOGDHL-shRNA can reduce inflammatory cell infiltration of myocardial tissue, lighten myocardial fibrosis and reduce myocardial cell apoptosis, thereby improving heart failure symptoms. Thus, the invention has the potential to be converted into targeted drugs for clinical treatment of chronic heart failure.
Drawings
FIG. 1 is a diagram of pAAV-cTnT-master-miRNA vector.
FIG. 2 is a screen identification graph of 6 mOGDHL-shRNAs and a comparison graph of the inhibitory effect of an inhibitor on rAAV 9-cTNT-mOGDHL-shRNA.
FIG. 3 is an identification of rAAV9-cTNT-mOGDHL-shRNA targeted inhibition of myocardial tissue OGDHL gene and protein expression.
FIG. 4 is a graph showing the effect of rAAV9-cTNT-mOGDHL-shRNA on improving cardiac function in mice with chronic heart failure with hypertrophic cardiomyopathy.
FIG. 5 is a histopathological diagram of rAAV9-cTNT-mOGDHL-shRNA treatment of hypertrophic cardiomyopathy chronic heart failure mice in improving myocardial histopathological conditions.
Detailed Description
The following examples serve to further illustrate the invention but are not to be construed as limiting the invention. The technical means used in the examples are conventional means well known to those skilled in the art unless otherwise indicated.
Explanation of materials or abbreviations involved in the following examples:
pAAV-cTnT-master-miRNA: AAV eukaryotic expression plasmid vector, as shown in figure 1, contains mouse myocardial cell expression promoter cTnT, and has no fluorescence, and is purchased from Wuhan Dian Jun Biotechnology Co.
PHelper: adeno-associated viral helper plasmids, purchased from Thermo Fisher, inc. of America.
PAAV2/9: the coat protein expression plasmid of AAV virus was purchased from Thermo Fisher, inc. of America.
PAAV-cTnT-mOGDHL-shRNA: the eukaryotic expression plasmid of the adeno-associated virus carries a mouse myocardial cell expression promoter cTnT, and is self-made.
RAAV-cTnT-mOGDHL-shRNA: recombinant adeno-associated virus carrying mouse myocardial cell expression promoter cTnT.
RAAV-cTnT-Zsgreen: the empty recombinant adeno-associated virus carries a mouse myocardial cell expression promoter cTnT, has green fluorescence and is purchased from Wuhan classical Jun biotechnology Co.
EXAMPLE 1 construction of recombinant adeno-associated Virus rAAV9-cTNT-mOGDHL-shRNA
1. MOGDHL-shRNADNA primers were designed and synthesized and mOGDHL-shRNA fragments were synthesized using annealing buffer
The following 6 mOGDHL-shRNA sequences were designed:
(1)mOGDHL-1:5’-GACCATTATCGACAAGTCTAG-3’;
(2)mOGDHL-2:5’-GCCAAATATGACCGTATCTGT-3’;
(3)mOGDHL-3:5’-GGCTATTACGACTACATCAGT-3’;
(4)mOGDHL-4:5’-GCACCTACTGCCAGCATATTG-3’;
(5)mOGDHL-5:5’-GCCAGTGGATCAGACAGAAAT-3’;
(6)mOGDHL-6:5’-GCTCTGGTGATGTCAAATACC-3’。
the primers corresponding to mOGDHL-shRNA described above are shown in Table 1 below.
The designed mOGDHL-shRNA primer is designed to be added with a restriction enzyme HindIII cleavage point at the 5 'end of the primer and an XhoI cleavage point at the 3' end of the primer for subsequent cloning except for the part which is completely identical with the OGDHL gene sequence. Primer design synthesis was completed at Invitrogen corporation.
Preparation of annealing buffer stock (1M Tris-HCl,5M NaCl,pH 8.0): taking a 1L beaker, and placing the beaker into 121.1gTris; mixing 800mL of deionized water with Tris; adding hydrochloric acid to adjust the pH of the solution to 8.0, and finally adjusting the pH to 8.0 when the cooling temperature of the solution is stabilized at room temperature; 292g of NaCl solid is weighed and added into the solution to be evenly mixed; adding deionized water to fix the volume to 1L; sterilizing in high pressure steam pot.
Preparation of annealing Buffer (10 mM Tris-HCl,50mM NaCl,pH 8.0): 1 part by volume of annealing buffer stock solution, 99 parts by volume of DEPC water and pH 8.0 were adjusted with HCl.
The mOGDHL-shRNADNA primer and annealing buffer were thoroughly mixed in 200. Mu.L EP tube according to the following composition in Table 2, placed in a water bath at 100deg.C for about 1min, the water bath was turned off to allow it to return to room temperature, and the annealed product was diluted 100-fold with DEPC water.
TABLE 2
2. The vector pAAV-cTnT-precursor-miRNA is digested with HindIII+XhoI double cleavage to obtain pAAV-cTnT-precursor-MIRNAHINDIII +XhoI large fragment
PAAV-cTnT-master-miRNA vector was added to 200. Mu.L of EP tube together with HindIII+XhoI double-cutter, 10X Buffer CutSmart, ddH 2 O, and the EP tube was placed in a 37℃water bath to react for 4 hours, and the components of the vector cleavage reaction were shown in Table 3.
TABLE 3 Table 3
The cleavage reaction product was analyzed by 1% agarose gel electrophoresis, and a 4258 bp-sized pAAV-cTnT-master-MIRNA HINDIII +XhoI fragment was excised and retrieved.
3. The mOGDHL-shRNA fragment is inserted into the plasmid pAAV-cTnT-precursor-MIRNAHINDIII +XhoI enzyme-cut large fragment, and the recombinant plasmid pAAV-cTnT-mOGDHL-shRNA is obtained through ligation reaction
The mOGDHL-shRNA fragment, pAAV-cTnT-master-MIRNAHINDIII +XhoI large fragment, 10×Ligase Buffer, and T 4 DNA Ligase obtained by the annealing were added to a 200. Mu.L EP tube, vortex flash-off, and reacted in a thermocycler at 22℃for 4 hours, and the specific reaction system is shown in Table 4 below.
TABLE 4 Table 4
4. Transforming the ligation product and sequencing the result
Mu.L of the above ligation product was added to 100. Mu.L of competent bacteria of E.coli JM109, and the mixture was placed on ice for 30min, then heat-shocked at 43℃for 50s, and placed on ice again for 5min. Bacteria were added to 400. Mu.L of LB medium, incubated at 37℃with shaking for 1h, centrifuged at 3000rpm for 2min, and the supernatant was discarded. A volume of 100. Mu.L of LB medium was added, the bacteria were homogenized by pipetting, and the liquid was spread on a 100. Mu.g/ML AMPICILLIN-resistant LB plate,
The plates were inverted and incubated overnight at 37℃in a thermostated bacterial incubator. The next day, 3 colonies with better growth were picked from the medium and inoculated into 5mL of LB medium with 100. Mu.g/ML AMPICILLIN resistance, and shaken overnight at 37 ℃. After 24 hours, the cultured bacterial liquid is sent to the invitrogen for sequencing, and the result shows that mOGDHL-shRNA sequence is consistent with the design, which shows that the recombinant plasmid pAAV-cTnT-mOGDHL-shRNA is successfully constructed.
5. Introducing mOGDHL shRNA fragment-containing recombinant plasmid pAAV-cTnT-mOGDHL-shRNA, adeno-associated virus packaging plasmid pAAV2/9 and helper plasmid pHelper into 293AAV cells, and separating and purifying the cells to obtain high-titer rAAV9-cTNT-mOGDHL-shRNA virus containing target genes
S51: 293AAV cells were seeded into 15cm cell culture dishes, each containing 1.5X10 7 cells, a total of 24 dishes, cell incubator (37 ℃,5% CO 2) overnight;
S52: after 24h, 2h before transfection, each dish of cells was plated with high glucose dmem+p/S containing 10% FBS; high sugar DMEM component: contains 4500mg/L D-glucose, 584mg/L L-glutamine, 3700mg/L sodium bicarbonate.
S53: plasmid transfection (amount of 15cm dish per plate):
1) Taking 2 EP pipes, which are respectively marked as ① pipes and ② pipes;
2) 1mL of CPT Buffer A was added to ① tubes;
3) pAAV-cTnT-mOGDHL-shRNA plasmid, pHelper, pAAV-2/9, ddH 2 O, CPT Buffer B and the like were sequentially added into ② tubes, and gently stirred and mixed, the specific amounts are shown in Table 5.
TABLE 5
4) The ② tubes of liquid are added into the ① tubes drop by drop, and are repeatedly blown and evenly mixed for about 15 times;
S54: standing at room temperature for 30min, dropwise adding the mixed solution into a culture dish, and culturing in a cell incubator (37 ℃ C., 5% CO 2) for 6 hr;
S55: repeatedly blowing cells in the culture dishes by using a pipette so that the cells fall off, and collecting all the cells and culture mediums in 24 culture dishes into a new 50mL centrifuge tube;
s56: taking a proper amount of liquid nitrogen, immersing the liquid in the centrifuge tube, putting the centrifuge tube into a water bath kettle with the temperature of 37 ℃ for thawing completely, repeating for 3 times, so that cells are thoroughly broken, and releasing viruses in the cells;
S57: adding 50U/mL DNase and 10U/mL RNase into the obtained solution, and standing in a 37 ℃ incubator for 30min;
S58: adding 0.5% deoxycholate sodium into the obtained solution, and standing for 30min in a 37 ℃ incubator;
s59: cell debris was removed with a 0.45 μm PVDF filter;
S510: PEG 8000 and 2.5N NaCl were added to the solution to final concentrations of 8% and 0.5N, respectively, and the mixture was allowed to stand at 4℃for 1 to 2 hours, and 3000g was centrifuged for 20 minutes. Discarding the supernatant, re-suspending 10mL of HEPES, adding 10mL of chloroform, mixing for 5min with the mixture turned upside down vigorously, and centrifuging for 10min with 380 g;
s511: heating and evaporating chloroform for 20min, and sucking supernatant;
s512: adding PEG8000 and ammonium sulfate into the supernatant to make the concentration of the supernatant be 10% and 13% respectively, and mixing the mixture for 5min with the mixture turned upside down vigorously, and standing the mixture at room temperature for 30-40 min;
s513: centrifuging 3500g for 10min, wherein the content is divided into 3 layers, the protein is salted out and precipitated at the bottom or between 2 layers of liquid, and the virus is mainly at the top layer;
S514: gently sucking the uppermost solution, adding 6-12 volumes of virus preservation solution to dilute PEG8000, sucking the mixed solution into a ultrafilter tube, centrifuging 8000g for 10min, and repeating for 3-5 times to ensure complete removal of PEG8000;
s515: the virus preservation solution is added into the concentrated virus solution, the total volume is 1mL, and the virus preservation solution is packaged and preserved in a liquid nitrogen tank.
Example 2 experimental identification of rAAV9-cTNT-mOGDHL-shRNA silencing myocardial tissue OGDHL
RAAV9-cTNT-mOGDHL-shRNA virus is injected into 8-week-old male C57BL/6J mice by a rat tail intravenous injection method, so that OGDHL inhibition in myocardial tissues is realized. In order to screen out the sequence with the best inhibitory effect on OGDHL expression in mouse myocardial tissue, the wild type mice were randomly divided into 7 groups in this section: no-load nonsensical adenovirus rAAV-cTNT-Zsgreen group (Empty, n=5), and 6 rAAV9-cTNT-mOGDHL-shRNA virus groups (shOGDHL, n=5 for each group), with an injection of 100. Mu.L of virus solution with a titer of 1x 10 x 12 PFU/mL. Mice were sacrificed 3 weeks after injection of recombinant adenovirus and their heart tissue was taken for subsequent PCR and protein electrophoresis experiments.
FIG. 2A is a statistical plot of the results of mice myocardial tissue OGDHL mRNA from each group, showing that mOGDHL-4 sequence has the best inhibitory effect on OGDHL gene (P < 0.0001) compared to the Empty group; FIG. 2B is a statistical chart of the results of the electrophoresis of OGDHL proteins in the myocardial tissue of each group of mice, which is consistent with the results of FIG. 2A, and the mOGDHL-4 sequence has the best silencing effect on OGDHL protein expression (P < 0.0001). Therefore, mOGDHL-4 (5'-GCACCTACTGCCAGCATATTG-3') is selected as the optimal sequence of the inhibition effect according to the result, and the rAAV9-cTNT-mOGDHL-shRNA recombinant adenovirus corresponding to mOGDHL-4 is selected in the following examples.
Example 3 comparative experimental identification of rAAV9-cTNT-mOGDHL-shRNA with the inhibitor carbocarboxyethyl succinyl phosphonate (carboxyethyl ester of succinyl phosphonate, CESP) to silence myocardial tissue OGDHL
The control group, rAAV9-cTNT-mOGDHL-shRNA (SEQ ID NO: 4) was administered in the same manner as in example 2; the inhibitor groups were given a single injection of CESP (20 mg/kg/mouse, saline, sigma) and 40 mice per group. After the mice were modeled, cardiac tissue was sacrificed from week 1, OGDHL mRNA content was detected, and 5 mice were detected weekly from 7 weeks to 8 weeks continuously.
FIG. 2C is a statistical plot of the results of PCR assay OGDHL mRNA for the heart tissue rows of 3 mice, as shown: the inhibitor group started to show a OGDHL mRNA decrease in content at week 1 compared to the Empty group, reached the lowest at week 2, inhibited by 50-60% compared to the control group, showed an increase at week 3, stabilized at normal levels from week 4 to week 8; as with the inhibitor group, the rAAV9-cTNT-mOGDHL-shRNA group showed a decrease from week 1, but continued to decrease by weeks 2, 3, reaching the lowest week 3, with OGDHL mRNA expression silencing of 70-80% compared to the control group, and stabilized at the lowest level from week 4 to week 8, with no increase. In summary, intravenous injection of rAAV9-cTNT-mOGDHL-shRNA in a mode capable of inhibiting OGDHL expression at present is proved to be the method with the best effect, longest duration and most stable inhibition on OGDHL expression.
EXAMPLE 4 determination of expression of rAAV9-cTNT-mOGDHL-shRNA in organs of C57BL/6J mice
The rAAV9-cTNT-mOGDHL-shRNA virus is injected into mice by a rat tail intravenous injection method to complete animal experiments. The experiment takes 8-week-old male C57BL/6J mice as an experimental object. To examine the effect of rAAV9-cTNT-mOGDHL-shRNA virus on expression of OGDHL in various organs and tissues of mice, the wild-type mice were randomly divided into 2 groups in this section of experiment: no-load nonsensical adenovirus rAAV-cTNT-Zsgreen group (Empty, n=5), rAAV9-cTNT-mOGDHL-shRNA virus group (shOGDHL, n=5), and 100. Mu.L of virus solution with 1 x 10 x 12PFU/mL titer was injected. Mice were sacrificed 3 weeks after injection of recombinant adenovirus and their hearts, livers, spleens, lungs and kidneys were taken for subsequent experiments.
FIGS. 3A-E are statistical graphs of results of PCR experimental tests OGDHL mRNA on heart, kidney, liver, lung and spleen tissues, respectively, showing that the OGDHL mRNA content in heart tissues of shOGDHL mice is obviously reduced (P < 0.0001) compared with that of the Empty group, and no obvious change is caused in kidney, liver, lung and spleen tissues (P values are all more than 0.05); FIGS. 3F-G are Western blot bands and statistical graphs of cardiac tissue proteins, and the results indicate that OGDHL protein expression is also obviously reduced (P < 0.0001). Taken together, the results prove that the rAAV9-cTNT-mOGDHL-shRNA virus can successfully and specifically inhibit OGDHL gene and protein expression in heart tissues, and does not influence OGDHL expression in other organ tissues.
Example 5 animal experiments
1. Cardiac functional protection experiment of rAAV9-cTNT-mOGDHL-shRNA in hypertrophic cardiomyopathy chronic heart failure mice
The experiment constructs a mouse model of myocardial hypertrophy type chronic heart failure through aortic arch constriction surgery (TRANSVERSE AORTIC CONSTRICTION, TAC), which is the most commonly used means at present for establishing hypertrophic cardiomyopathy. The experiment adopts 28G (aortic constriction) to constrict the aortic arch, thereby increasing left ventricular afterload, leading to left heart failure and finally total heart failure, and the model is successfully constructed 4-8 weeks after operation. The sham operation is to open the chest of the mouse by the same procedure, but without constricting the aorta.
FIG. 4A is a schematic diagram showing construction of a mouse experimental model, in which recombinant adeno-associated virus including 2 kinds of empty nonsensical recombinant adenoviruses rAAV-cTNT-Zsgreen and rAAV9-cTNT-mOGDHL-shRNA virus were injected intravenously via the rat tail at 4 th week after TAC surgery, the virus titer was 1X 10 11 vg/mL, 100. Mu.L each was injected, and a blank control group was injected with 100. Mu.L of sterile physiological saline in the same manner. And 8, carrying out cardiac ultrasonic real-time detection and exercise tolerance experiments on mice on the 8 th week after operation, and after detection, killing the mice and taking out cardiac tissues and peripheral serum for subsequent identification.
This part of animal experiments randomly divided wild type mice into 4 groups: the sham surgery group was a control group (Ctrl), a hypertrophic cardiomyopathy chronic heart failure group (TAC), a TAC surgery and injection of Empty recombinant adenovirus rAAV-cTNT-Zsgreen group (tac+empty), a TAC surgery and injection of rAAV9-cTNT-mOGDHL-shRNA virus group (tac+ shOGDHL).
Fig. 4B is a cardiac echocardiography representation of four groups (n=5) of mice. FIGS. 4C-4G are statistics of left ventricular ejection fraction (left ventricular ejection fraction, LVEF), left ventricular short axis shortening (fraction shorting, FS), left ventricular end-diastole volume (left ventricular end diastolic volume, LVEDV), left ventricular back wall thickness (left ventricular posterior WALL THICKNESS, LVPW), cardiac weight/body weight (HEART WEIGHT/body weight, HW/BW), TAC mice significantly decreased (P values < 0.0001) relative to control mice left ventricular ejection fraction, left ventricular short axis shortening, left ventricular end-diastole volume, while left ventricular back wall thickness and cardiac weight significantly increased, suggesting that TAC mice were reduced in full cardiac function, reduced cardiac chamber volume, increased chamber wall thickness, increased heart relative to control mice model construction was successful for hypertrophic cardiomyopathy chronic heart failure mice; compared with the TAC group, the mice in the TAC+empty group have no obvious difference in the indexes (the P values are all more than 0.05), which indicates that the injection of the Empty adenovirus has no obvious influence on the heart function, the heart chamber capacity, the chamber wall thickness and the heart size of the mice, and indicates that the Empty adenovirus has no obvious influence on the heart function and the heart size of the mice; compared with the TAC+empty group, the above indexes of mice in the TAC+ shOGDHL group are obviously improved (P values are all less than 0.0001), which indicates that rAAV9-cTNT-mOGDHL-shRNA can obviously enhance the myocardial contractility of heart failure mice, enlarge the narrowed heart chamber, and reduce the thickness of the chamber wall and the heart volume.
Exercise tolerance of heart failure mice represents the severity of symptoms of total body heart failure in heart failure mice, and heart failure of mice was detected by the mouse exercise tolerance test (n=10). Mice were trained for 3 days of adaptive exercise on the treadmill prior to the initiation of the formal experiment. In the formal experiment, the mouse is heated for 3 minutes, the speed of the running machine is set to be 5 m/min, then the running machine is increased to be 14 m/min, after the running machine lasts for 2 minutes, the speed is increased by 2 m/min, the experiment is stopped until the running distance and the running time of the mouse are recorded until the power grid can not be restored due to physical exhaustion of the mouse. The results of each group of mice are shown in fig. 4H, and compared with the control group, the running distance of the mice in the TAC group is obviously reduced (P < 0.0001), which indicates that the exercise endurance of the mice in the TAC group is reduced and the mice have heart failure symptoms; compared with the TAC group, the mice in the TAC+empty group have no obvious difference in the indexes (P= 0.0646), which shows that the injection of Empty adenovirus has no obvious effect on the exercise endurance and heart failure symptoms of the mice; compared with the TAC+empty group, the indexes of mice in the TAC+ shOGDHL group are obviously increased (P values are all less than 0.0001), which proves that the rAAV9-cTNT-mOGDHL-shRNA can obviously improve exercise endurance and heart failure symptoms of heart failure mice.
Figures 4I-J show the results of the peripheral serum ELISA assay of 4 groups (n=10) of mice for BNP and NT-proBNP levels, both of which are the most clinically used peripheral serum heart failure markers, with positive correlation between magnitude and heart failure severity. Mouse NPPB/BNP ELISA Kit A Kit was purchased from Thermo Fisher, USA and the Mouse NT-proBNP ELISA Kit was purchased from Shanghai Elabscience. BNP and NT-proBNP levels in the peripheral serum of the mice were determined according to the procedure of the instructions of the manufacturer, and the results showed that: compared with the control group, the BNP and NT-proBNP content in the peripheral serum of the TAC group mice are obviously increased (P values are less than 0.0001), which indicates that the TAC mice have heart failure; compared with the TAC group, the mice in the TAC+empty group have no obvious difference (P values are all more than 0.05), which indicates that the injection of Empty adenovirus has no obvious influence on the heart failure degree of the mice; compared with the TAC+empty group, the numerical values of the BNP and NT-proBNP of the mice in the TAC+ shOGDHL group are obviously reduced (the P values are less than 0.0001), which indicates that the rAAV9-cTNT-mOGDHL-shRNA can obviously improve the heart failure degree of heart failure mice.
2. Heart histopathological detection of rAAV9-cTNT-mOGDHL-shRNA in hypertrophic cardiomyopathy chronic heart failure mice
This part of the experiment was consistent with the above mouse grouping (n=5).
Fig. 5A is a diagram showing the overall view of the heart (scale=5 mm), HE staining (scale=50 μm), masson staining (scale=50 μm), wheat germ agglutinin (WHEAT GERM agglutinin, WGA) staining (scale=20 μm), and fig. 5B to D are statistical diagrams corresponding to the three stains, respectively.
The HE staining pattern in fig. 5A and the pathology scoring results in fig. 5B show: compared with a control group, the TAC group mice have increased inflammatory cell infiltration (P is less than 0.0001), which indicates that the myocardial tissue inflammation of hypertrophic cardiomyopathy mice is aggravated; compared with the TAC group, the number of inflammatory cells infiltrated by the mice in the TAC+empty group is not obviously different (P= 0.9855), which indicates that the injection of Empty adenovirus has no obvious influence on myocardial inflammatory infiltration of the mice; compared with the TAC+empty group, the inflammatory cell infiltration of the mice in the TAC+ shOGDHL group is obviously reduced (P is less than 0.0001), which proves that the rAAV9-cTNT-mOGDHL-shRNA obviously improves the inflammatory infiltration of heart failure.
The Masson staining pattern in fig. 5A versus the area of fibrosis ratio in fig. 5C shows that: compared with the control group, the TAC group mice have increased fibrosis areas (P is less than 0.0001), which indicates that the hypertrophic cardiomyopathy mice have increased myocardial tissue fibrosis; compared with the TAC group, the TAC+empty group mice have no obvious difference in fibrosis area (P is more than 0.9999), and the injection of Empty adenovirus has no obvious influence on the myocardial fibrosis degree of the mice; compared with the TAC+empty group, the fibrosis area of the mice in the TAC+ shOGDHL group is obviously reduced (P is less than 0.0001), which indicates that rAAV9-cTNT-mOGDHL-shRNA can obviously improve heart failure myocardial fibrosis.
The WGA staining pattern in fig. 5A and the cell cross-sectional area (cross sectional area, CSA) results in fig. 5D show that: compared with the control group, the cross-sectional area of the myocardial cells of the mice in the TAC group is increased (P is less than 0.0001), which indicates that the myocardial cells of the mice in the hypertrophic cardiomyopathy heart failure are hypertrophic; compared with the TAC group, the cross-sectional area of mice in the tac+empty group has no obvious difference (p= 0.6781), which suggests that injection of Empty adenovirus has no obvious effect on the hypertrophy of myocardial cells of the mice; compared with the TAC+empty group, the cross-sectional area of the myocardial cells of the mice in the TAC+ shOG DHL group is obviously reduced (P is less than 0.0001), and the rAAV9-cTNT-mOGDHL-shRNA is proved to be capable of improving the hypertrophy of the myocardial cells of the mice with hypertrophic cardiomyopathy and heart failure.
Fig. 5E is representative myocardial tissue immunofluorescence staining images (scale bar=50 μm) of four groups (n=5) of mice. Immunofluorescence included nuclear DAPI staining (blue fluorescence), cardiomyocyte-representative molecule TNI (red fluorescence), OGDHL molecules (green fluorescence) trichromatic co-staining. Immunofluorescence image results show that: compared with a control group, the green fluorescence of OGDHL in the myocardial cells of the mice in the TAC group is enhanced, which indicates that the OGDHL expression in the myocardial cells of the mice with hypertrophic heart failure constructed by TAC operation is increased; compared with the TAC group, the green fluorescence of OGDHL in the myocardial tissue of the mice in the TAC+empty group is not obviously changed, which indicates that the injection of Empty adenovirus has no influence on the OGDHL expression in the myocardial cells of the mice; compared with the TAC+empty group, the OGDHL green fluorescence in the mice cardiomyocytes in the TAC+ shOGD HL group is obviously reduced, and the rAAV9-cTNT-mOGDHL-shRNA is proved to be capable of effectively inhibiting the OGDHL expression in the mice cardiomyocytes in the hypertrophic cardiomyopathy heart failure.
Fig. 5F is a representative immunofluorescence image of 4 groups (n=6) of mice myocardial tissue nuclei (DAPI, blue), cardiomyocytes (TNI, red), apoptotic bodies (Tunel, green) co-stained (scale bar=20 μm); FIG. 5G is a statistical plot of the number of apoptotic body positive cells, and the number of Tunel positive cells was counted under a microscope in 5 different fields of view per mouse by fluorescent staining of myocardial tissue, and the average of 5 times was analyzed statistically. The results show that: compared with the control group, tunel positive cells in the myocardial cells of the mice in the TAC group are obviously increased (P is less than 0.0001), which indicates that the apoptosis of the myocardial cells of the mice with hypertrophic cardiomyopathy is increased; compared with the TAC group, the Tunel positive cells in the myocardial tissue of the mice in the tac+empty group have no obvious change (p= 0.9988), which suggests that the Empty adenovirus has no influence on the apoptosis of the myocardial cells of the mice; compared with the TAC+empty group, the TAC+ shOGDHL group of mice has obviously reduced Tunel positive cells (P is less than 0.0001), and the rAAV9-cTNT-mOGDHL-shRNA is proved to be capable of reducing myocardial apoptosis of mice with myocardial hypertrophy type heart failure.
To sum up: 1. the invention successfully develops rAAV9-cT NT-mOGDHL-shRNA virus which can reach the heart by intravenous injection and target the expression of the rAAV9-cT NT-mOGDHL-shRNA virus to myocardial cells. 2. The rAAV9-cTNT-mOGDHL-shRNA virus prepared by the invention can provide cardiac function and histological protection for hypertrophic cardiomyopathy chronic heart failure mice.
The above examples are preferred embodiments of the present invention, and any modifications (e.g., improvements, substitutions, etc.) made within the spirit and scope of the method of the present invention are within the scope of the present invention.
Claims (10)
- The application of OGDIL as a target in screening medicines for treating chronic heart failure is characterized in that: the screening is screening OGDHL of inhibitors.
- Use of an inhibitor of ogdhl in the preparation of a medicament for the treatment of chronic heart failure.
- 3. Use according to claim 1 or 2, characterized in that: the OGDHL inhibitor includes substances inhibiting OGDHL expression and substances inhibiting OGDHL activity.
- 4. Use according to claim 1 or 2, characterized in that: the OGDHL inhibitor is a substance which specifically inhibits OGDHL in myocardial cells.
- 5. A shRNA that inhibits OGDHL, characterized by: the nucleotide sequence of the shRNA is 5'-GCACCTACTGCCAGCATATTG-3'.
- 6. A recombinant vector for inhibiting OGDHL, characterized in that: a shRNA of claim 5.
- 7. The recombinant vector according to claim 6, wherein: contains a promoter for expressing myocardial cells.
- 8. A recombinant adeno-associated virus that inhibits OGDHL, characterized by: preparation using the recombinant vector of claim 6 or 7.
- 9. The recombinant adeno-associated virus of claim 8, wherein: in order to target the myocardial cells and further knock down OGDHL rAAV9-cTNT-mOGDHL-shRNA, the preparation method comprises the following steps:(1) Designing and synthesizing mOGDHL-shRNA DNA primer, and synthesizing mOGDHL-shRNA fragment by annealing; the mOGDHL-shRNA DNA primer has the following sequence:An upstream primer: 5'-CCGGGCACCTACTGCCAGCATATTGCTCGAGCAATATGCTGGCAGTAGGTGCTTTTTG-3' the process of the preparation of the pharmaceutical composition,A downstream primer: 5'-GATCCAAAAAGCACCTACTGCCAGCATATTGCTCGAGCAATATGCTGGCAGTAGGTGC-3';(2) The vector pAAV-cTnT-master-miRNA is digested with HindIII+XhoI, so that pAAV-cTnTpromoter-MIRNA HINDIII +XhoI is obtained for digestion and recovery of large fragments;(3) Inserting mOGDHL-shRNA fragments into pAAV-cTnT-precursor-miRNA enzyme digestion and recovery large fragments through ligation reaction;(4) Converting the connection product to obtain a recombinant plasmid pAAV-cTnT-mOGDHL-shRNA;(5) The recombinant plasmid pAAV-cTnT-mOGDHL-shRNA containing the target fragment, the adeno-associated virus packaging plasmid pAAV2/9 and the helper plasmid pHelper are introduced into 293AAV cells, and the transfected cells are cultured, separated and purified to obtain the rAAV9-cTNT-mOGDHL-shRNA.
- 10. Use of the shRNA of claim 5, the recombinant vector of claim 6 or 7 or the recombinant adeno-associated virus of claim 8 or 9 in the manufacture of a medicament for the treatment of chronic heart failure.
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