Application of CES1 in preparation of medicine for treating chronic heart failure, protein, gene, transformant, medicine and preparation method of CES1

Abstract

The invention discloses application of CES1 in preparation of a medicine for treating chronic heart failure, and a protein, a gene, a transformant, a medicine and a preparation method thereof, and belongs to the field of biological medicine. The invention provides application of CES1 in preparation of a medicine for treating chronic heart failure, and provides the medicine for treating the chronic heart failure and a preparation method based on the new application of the CES1. Animal experiments prove that the anti-heart-failure medicine provided by the invention can obviously improve the heart function of animals with chronic heart failure and has an effective anti-chronic heart failure effect.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, specifically to the use of CES1 in the preparation of drugs for the treatment of chronic heart failure, as well as its protein, gene, transformant, drug and preparation method. Background Art

[0002] Cardiovascular diseases have become the leading cause of death among Chinese people. Heart failure is a clinical syndrome that occurs at the end stage of different cardiovascular diseases. Although some new drugs have been applied clinically in recent years, about 50% of heart failure patients still die within 5 years after diagnosis. Heart failure has a poor prognosis and a high mortality rate, and is one of the main causes threatening human health and increasing the medical burden.

[0003] Heart failure is divided into acute heart failure and chronic heart failure. Most patients with acute heart failure have partial relief of symptoms after hospitalization and then enter the state of chronic heart failure.

[0004] Currently, adenovirus and lentivirus are mostly used as expression vectors for gene therapy. However, lentivirus is mostly modified from leukemia virus or HIV, and the adenovirus vector has a short expression time and is immunogenic to the body, both of which are not suitable for future clinical applications. The recombinant adeno-associated virus vector (rAAV) overcomes the disadvantages that are difficult to overcome by other gene expression vectors, has no immunogenicity, and can drive the target gene to be expressed in the body for a long time, thus becoming the most promising vector for gene therapy.

[0005] The expression abundance of CES1 is extremely low during fetal development, while it is highly expressed in adult myocardium. CES1 is a metabolic regulation gene, and its homologous gene in mice is Ces1d, which encodes the protein carboxylesterase 1 (CES1). Previous studies have shown that CES1 can hydrolyze ester bonds, amide bonds and thioester bonds, and is involved in the metabolism of various drugs in the liver. In addition, CES1 can also hydrolyze triglycerides. For example, CES1 can regulate lipid metabolism by hydrolyzing triglycerides and affect the sensitivity of liver cancer cells to chemotherapeutic drugs. However, the role of CES1 in the heart has not been clarified, and there is no report on the treatment of heart diseases with CES1. Summary of the Invention

[0006] In view of the above-mentioned deficiencies and blanks objectively existing in the prior art in this field, the inventors provide

[0007] The above technical object of the present invention is achieved by the following technical solutions:

[0008] The use of CES1 in the preparation of drugs for the treatment of chronic heart failure.

[0009] The action target of the drug for the treatment of chronic heart failure is CES1;

[0010] Preferably, the CES1 is selected from: human CES1, rat CES1, mouse CES1.

[0011] A gene for treating chronic heart failure, comprising: a CES1 gene sequence.

[0012] The gene for treating chronic heart failure further comprises: a myocardial-specific promoter;

[0013] Preferably, the myocardial-specific promoter is selected from: tnt, α-MHC, MLC-2v, Desmin;

[0014] Preferably, the CES1 is selected from: human CES1, rat CES1, mouse CES1;

[0015] Preferably, the CES1 gene sequence is as shown in SEQ ID NO.1.

[0016] A recombinant expression vector for treating chronic heart failure, which is an expression vector ligated with the sequence of the gene for treating chronic heart failure.

[0017] A transformant for treating chronic heart failure, which is a host cell transformed with the recombinant expression vector for treating chronic heart failure.

[0018] Use of the gene for treating chronic heart failure, and / or the recombinant expression vector for treating chronic heart failure, and / or the transformant for treating chronic heart failure in the preparation of a drug for treating chronic heart failure.

[0019] A drug for treating chronic heart failure, comprising a pharmaceutically active ingredient, characterized in that the pharmaceutically active ingredient comprises: the gene for treating chronic heart failure, and / or the recombinant expression vector for treating chronic heart failure, and / or the transformant for treating chronic heart failure.

[0020] A method for preparing a drug for treating chronic heart failure, characterized by expressing CES1 protein.

[0021] Ligate the gene sequence of CES1 with an expression vector to obtain a recombinant expression vector;

[0022] Preferably, ligate a myocardial-specific promoter and the gene sequence of CES1 with an expression vector in sequence to obtain a recombinant expression vector;

[0023] Preferably, transform a host cell with the recombinant expression vector to obtain a transformant;

[0024] Preferably, use the calcium phosphate co-transfection method to transform a host cell with the recombinant expression vector to obtain a transformant;

[0025] Preferably, the CES1 is selected from: human CES1, rat CES1, mouse CES1;

[0026] Preferably, the expression vector is the adeno-associated virus expression vector pAAV-D(+);

[0027] Preferably, the myocardial-specific promoter is selected from: tnt, α-MHC, MLC-2v, Desmin;

[0028] Preferably, the recombinant expression vector is selected from: the recombinant adeno-associated virus expression vector pAAV-D(+)-CES1 ligated with the CES1 gene sequence, or, the recombinant adeno-associated virus expression vector pAAV-D(+)-tnt-CES1 ligated successively with the myocardial-specific promoter tnt and the CES1 gene sequence;

[0029] Preferably, the transformant is selected from: the recombinant adeno-associated virus rAAV-CES1 transformed with the recombinant adeno-associated virus expression vector pAAV-D(+)-CES1, or, the recombinant adeno-associated virus rAAV-tnt-CES1 transformed with the recombinant adeno-associated virus expression vector pAAV-D(+)-tnt-CES1;

[0030] Preferably, the CES1 gene sequence is as shown in SEQ ID NO.1.

[0031] On the premise permitted by the patent laws of some countries or regions, the present invention also claims the protection of the substance with high expression of CES1, or, the recombinant adeno-associated virus expression vector pAAV-D(+)-CES1 ligated with the CES1 gene sequence, or, the recombinant adeno-associated virus expression vector pAAV-D(+)-tnt-CES12 ligated successively with the myocardial-specific promoter tnt and the CES1 gene sequence, or, the recombinant adeno-associated virus rAAV-CES1 transformed with the recombinant adeno-associated virus expression vector pAAV-D(+)-CES1, or, the use of the recombinant adeno-associated virus rAAV-tnt-CES1 transformed with the recombinant adeno-associated virus expression vector pAAV-D(+)-tnt-CES1 in the treatment of chronic heart failure.

[0032] The present invention provides a drug for treating chronic heart failure, characterized in that the active ingredient of the drug includes the CES1 drug target and the substance capable of increasing the expression of CES1.

[0033] The active ingredient of the drug includes the substance with high expression of CES1.

[0034] The drug further includes pharmaceutically acceptable excipients, and / or, reagents for buffering, synthesizing, and / or purifying the sequence fragment.

[0035] A preparation method of a drug for treating chronic heart failure, characterized in that it includes: using the substance that can highly express CES1 as the active ingredient of the drug for treating chronic heart failure.

[0036] Insert the sequence fragment capable of expressing CES1 into an expression vector to obtain a recombinant plasmid capable of stably expressing CES1. The sequence fragment of CES1 is shown as SEQ ID NO.1.

[0037] An object of the present invention is to provide a drug for treating chronic heart failure, and the active ingredient of the drug includes using CES1 as a drug target, and the substance that highly expresses CES1 plays a therapeutic effect on chronic heart failure.

[0038] Furthermore, the active ingredient of the drug includes the substance that highly expresses CES1.

[0039] Furthermore, the substance that highly expresses CES1 contains a CES1 sequence fragment shown as SEQ ID NO.1.

[0040] Furthermore, the drug also includes pharmaceutically acceptable excipients, and / or reagents for buffering, synthesizing, and / or purifying the sequence fragment. Those skilled in the art can add various pharmaceutically acceptable adjuvants / excipients to the anti-chronic heart failure drug of the present invention according to objective needs to make various dosage forms for easy sales or promotion.

[0041] Another object of the present invention is to provide a preparation method of a drug for treating chronic heart failure, including: using the substance that highly expresses CES1 as the active ingredient of the drug for treating chronic heart failure.

[0042] In a further embodiment, the preparation method includes: inserting the sequence fragment of CES1 into an expression vector to obtain a recombinant plasmid capable of stably expressing CES1.

[0043] In a specific embodiment, the expression vector of the sequence fragment of CES1 is the adeno-associated virus expression vector pAAV-D(+).

[0044] In order to achieve the purpose of gene therapy for chronic heart failure, the present invention recombines CES1 with a recombinant adeno-associated virus vector containing a myocardial-specific promoter tnt, and after detection, a high titer meeting the treatment requirements is obtained. It is confirmed in animal experiments that it can effectively improve the cardiac function of chronic heart failure mice. Therefore, based on the above findings and results, the present invention provides a drug for treating chronic heart failure with CES1 as a therapeutic target.

[0045] The present invention relates to a drug for treating chronic heart failure. The anti-chronic heart failure drug involves the construction and preparation method of a recombinant adeno-associated virus recombinant of CES1 (rAAV-CES1), and the rAAV-CES1 with high expression of CES1 exerts the efficacy of treating chronic heart failure. The present invention constructs a pAAV-D(+)-tnt-CES1 expression plasmid by chemical synthesis method, and packages and prepares a recombinant adeno-associated virus containing the target fragment by calcium phosphate co-transfection method of three plasmids and purifies it. Animal experiments confirm that the anti-chronic heart failure drug provided by the present invention can significantly improve the cardiac function of heart failure animals and play an effective role in anti-chronic heart failure.

[0046] When the present invention conducts high-throughput detection on the hearts of heart failure mice, the gene with the most significant decrease during heart failure is Ces1. Based on this, an anti-heart failure drug targeting CES1 is developed, and its effect is verified on an animal model. It is found that high expression of CES1 can significantly improve the cardiac function of mice and treat chronic heart failure. Based on the CES1 base sequence, the present invention synthesizes the sequence of CES1 and successfully inserts it into the eukaryotic expression vector pAAV-D(+) to form a recombinant plasmid pAAV-D(+)-CES1. Then, the following three plasmids: 1) pXX9 plasmid, 2) phelper plasmid, 3) pAAV-D(+)-CES1 plasmid are transfected into 293T cells by calcium phosphate co-transfection method respectively to package and prepare a recombinant adeno-associated virus (rAAV9) capable of expressing CES1. After purification, the titer is measured by real-time PCR method. Next, the prepared recombinant adeno-associated virus of the same serotype (rAAV-CES1) is injected into a chronic heart failure mouse model through the tail vein. The ultrasound results show that the expression of CES1 mediated by the recombinant adeno-associated virus can significantly improve the cardiac function of heart failure mice induced by pressure load of transverse aortic constriction (TAC). Brief Description of the Drawings

[0047] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0048] Figure 1 It is a schematic structural diagram of the plasmid pAAV-D(+)-CES1 prepared in Experimental Example 1 of the present invention; wherein, CES1 represents the gene sequence encoding the protein carboxylesterase 1, cTNT promoter represents a cardiomyocyte-specific promoter, ITR represents an inverted repeat sequence, and poly A represents a polyadenylate tail.

[0049] Figure 2 In Experimental Example 2 of the present invention, the content of CES1 in the hearts of heart failure mice decreased significantly; among them, A is the western blot result diagram of detecting the CES1 content in the hearts of control mice and TAC-induced heart failure mice; B is the bar chart of the CES1 content of control mice and TAC-induced heart failure mice; the meanings of the labels in the figure are as follows: GAPDH represents the internal reference protein; sham represents the sham operation control group; TAC represents the mice in the heart failure model group caused by TAC.

[0050] Figure 3 It is the effect of rAAV-tnt-CES1 treatment on the heart function of TAC pressure load-induced heart failure mice detected by cardiac ultrasound in Experimental Example 3, where: A is the bar chart of ejection fraction, B is the bar chart of shortening fraction, C is the representative diagram of cardiomyocyte area; D is the western blot result diagram of successful CES1 expression; the results show that rAAV-CES1 can significantly increase the cardiac systolic function and diastolic function of TAC mice, and rAAV-CES1 can significantly reduce myocardial hypertrophy in TAC mice; the meanings of each label in the figure are listed as follows: rAAV-tnt-Ctrl represents the mice injected with the negative control recombinant adeno-associated virus containing the recombinant expression vector linked with the GFP sequence (Ctrl is the GFP sequence), rAAV-tnt-CES1 represents the mice injected with the recombinant adeno-associated virus containing the recombinant expression vector linked with the CES1 sequence specifically targeting cardiomyocytes, GAPDH represents; sham represents the mice in the sham operation control group, and TAC represents the mice in the heart failure model group. Detailed implementation manners

[0051] The technical solutions of the present invention will be clearly and completely described below in conjunction with specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without making creative efforts belong to the scope of protection of the present invention.

[0052] The sources of the instruments, reagents, consumables, and biological materials involved in the following embodiments or experimental examples are as follows:

[0053] I. Instruments

[0054] ND-1000 nucleic acid analyzer, ABI 9700 PCR instrument, ABI 7900HT fluorescence real-time quantitative PCR instrument, Beckman X-15R low-temperature high-speed centrifuge;

[0055] II. Reagents and Consumables

[0056] RNasey Mini Kit (purchased from Qiagen), TRIZOL (purchased from Invitrogen), TaKaRa Agarose Gel DNA Purification Kit Ver.2.0 (agarose gel DNA recovery kit, purchased from TaKaRa), Endo-Free Plasmid Maxi Kit (plasmid extraction kit, purchased from OMEGA), EasyPure Plasmid MiniPrep Kit (plasmid extraction kit, purchased from TransGen Biotech Co., Ltd.); The eukaryotic expression vector pAAV-D(+) was constructed and gifted by Professor Xiao Xiao of the cooperation.

[0057] III. Sources of Biological Materials

[0058] (I) The 293T cells used in Experimental Example 1 of the present invention were from the American Type Culture Collection (ATCC), and DH5α competent cells were commercially available;

[0059] (II) The C57 background mice used in Experimental Example 2 and Experimental Example 3 of the present invention were purchased from Beijing Huafukang Bioscience Co., Ltd.

[0060] Group 1 of Examples, Pharmaceutical Use of CES1

[0061] This group of examples provides the use of CES1 in the preparation of drugs for the treatment of chronic heart failure.

[0062] In a specific example, the action target of the drug for the treatment of chronic heart failure is CES1;

[0063] Preferably, the CES1 is selected from: human CES1, rat CES1, mouse CES1.

[0064] Group 2 of Examples, Gene for Treating Chronic Heart Failure of the Present Invention

[0065] This group of examples provides a gene for the treatment of chronic heart failure. All examples in this group have the following common characteristics: the gene for the treatment of chronic heart failure contains: CES1 gene sequence.

[0066] In a further example, the gene for the treatment of chronic heart failure further contains: a myocardial-specific promoter;

[0067] Preferably, the myocardial-specific promoter is selected from: tnt, α-MHC, MLC-2v, Desmin;

[0068] TNT is the TNT described in the article "Nuclear miR-320 Mediates Diabetes-Induced Cardiac Dysfunction by Activating Transcription of Fatty Acid Metabolic Genes to Cause Lipotoxicity in the Heart";

[0069] α-MHC is the α-MHC described in the article "Deletion of BCATm increases insulin-stimulated glucose oxidation in the heart";

[0070] MLC-2v is the MLC-2v described in the article "Molecular In Vivo Imaging Using a Noninvasive Cardiac-Specific MLC-2v Promoter Driven Dual-Gene Recombinant Lentivirus Monitoring System";

[0071] Desmin is the Desmin described in the article "Muscle-Specific Promoters for Gene Therapy".

[0072] Preferably, the CES1 is selected from: human CES1, rat CES1, mouse CES1;

[0073] Preferably, the CES1 gene sequence is as shown in SEQ ID NO.1.

[0074] Group 3 Examples, Recombinant Expression Vectors for Treating Chronic Heart Failure of the Present Invention

[0075] This group of examples provides a recombinant expression vector for treating chronic heart failure. All examples in this group have the following common features: the recombinant expression vector for treating chronic heart failure is an expression vector ligated with the sequence of any one of the genes for treating chronic heart failure in the second group of examples.

[0076] In specific examples, the expression vector is selected from: the adeno-associated virus expression vector pAAV-D(+);

[0077] Preferably, the recombinant expression vector having the effect of treating chronic heart failure is selected from: the recombinant adeno-associated virus expression vector pAAV-D(+)-CES1 ligated with the CES1 gene sequence, or the recombinant adeno-associated virus expression vector pAAV-D(+)-tnt-CES1 ligated successively with the myocardial-specific promoter tnt and the CES1 gene sequence.

[0078] Group 4 Examples, the transformants for treating chronic heart failure of the present invention

[0079] This group of examples provides a transformant for treating chronic heart failure. All examples in this group have the following common characteristics: The transformant for treating chronic heart failure is a host cell transformed with any one of the recombinant expression vectors for treating chronic heart failure in Group 3 Examples.

[0080] In some examples, the host cell is selected from: 293 cells;

[0081] Preferably, the transformant having the effect of treating chronic heart failure is selected from: the recombinant adeno-associated virus rAAV-CES1 transformed with the recombinant adeno-associated virus expression vector pAAV-D(+)-CES1, or the recombinant adeno-associated virus rAAV-tnt-CES1 transformed with the recombinant adeno-associated virus expression vector pAAV-D(+)-tnt-CES1.

[0082] Group 5 Examples, the pharmaceutical uses of the gene, recombinant expression vector and transformant of the present invention

[0083] This group of examples provides the application of any one of the genes for treating chronic heart failure in Group 2 Examples, and / or any one of the recombinant expression vectors for treating chronic heart failure in Group 3 Examples, and / or any one of the transformants for treating chronic heart failure in Group 4 Examples in the preparation of drugs for treating chronic heart failure.

[0084] Group 6 Examples, the drugs for treating chronic heart failure of the present invention

[0085] This group of examples provides a drug for treating chronic heart failure. All examples in this group have the following common characteristics: The drug for treating chronic heart failure includes a pharmaceutically active ingredient, and the pharmaceutically active ingredient includes: any one of the genes for treating chronic heart failure in Group 2 Examples, and / or any one of the recombinant expression vectors for treating chronic heart failure in Group 3 Examples, and / or any one of the transformants for treating chronic heart failure in Group 4 Examples.

[0086] This group of embodiments provides a drug for treating chronic heart failure. The pharmacodynamic components of the drug for treating diabetic cardiomyopathy target CES1, and the treatment effect of chronic heart failure is achieved by highly expressing CES1. The present invention discovers that the content of CES1 in the hearts of chronic heart failure mice is significantly reduced ( Figure 1 ), which indicates that CES1 may play a regulatory role in the pathophysiological process of chronic heart failure. Further mouse experiments are carried out, and by increasing the content of CES1, the heart function of mice can be significantly improved ( Figure 3 ), indicating that CES1 has a therapeutic effect on chronic heart failure.

[0087] In some alternative embodiments of the present invention, some sequence fragments of CES1 of other species, such as human CES1, rat CES1 and other sequences, are highly homologous to the mouse CES1 gene sequence. The recombinant plasmids containing CES1 of these species have anti-chronic heart failure effects similar to or equivalent to those in Experimental Example 2 below. Due to limited space, they will not be elaborated one by one herein.

[0088] In some alternative embodiments of the present invention, some other promoters of the recombinant adeno-associated virus plasmid pAAV-D(+), such as α-MHC, MLC-2v, Desmin and other sequences, can achieve the effect of specifically targeting the myocardium by the recombinant adeno-associated virus vector like tnt. The recombinant plasmid pAAV-D(+)-CES1 containing these promoter sequences has anti-chronic heart failure effects similar to or equivalent to those in Experimental Example 2 below. Due to limited space, they will not be elaborated one by one herein.

[0089] In the most specific embodiment of the present invention, the pharmacodynamic component of the drug is the recombinant adeno-associated virus plasmid pAAV-D(+)-tnt-CES1 expressing CES1; the recombinant adeno-associated virus vector (rAAV) adopted in the present invention overcomes the disadvantages that are difficult to overcome by other gene expression vectors. It can carry the target gene to transfect dividing and non-dividing cells (i.e., has a wide range of transgene scope), has no side effects (no immunogenicity), high infection efficiency, can drive the target gene to express in vivo for a long time, and successfully solves the problem of large-scale replication in vitro without adenovirus contamination, thus becoming the most promising vector for gene therapy.

[0090] More specifically, the CES1 sequence is inserted into the adenovirus expression vector pAAV-D(+) as shown in SEQ ID NO.1 to construct the pAAV-D(+)-tnt-CES1 plasmid for expression. The above double-stranded nucleotide sequence was synthesized by Wuhan Keruister Biotech Co., Ltd. and inserted into the vector pAAV-D(+).

[0091] Furthermore, the drug further comprises a pharmaceutically acceptable excipient, and / or a reagent for buffering, culturing, and / or propagating the recombinant adeno-associated virus plasmid pAAV-D(+)-CES1; those skilled in the art can, according to objective needs, add various pharmaceutically acceptable adjuvants / excipients to the anti-diabetic cardiomyopathy and chronic heart failure drug of the present invention to prepare various dosage forms for easy sales or promotion.

[0092] Group 7 Examples, Preparation Method of the Drug for Treating Chronic Heart Failure of the Present Invention

[0093] This group of examples provides a preparation method of a drug for treating chronic heart failure. All examples in this group have the following common feature: expressing CES1 protein.

[0094] In a specific example, the gene sequence of CES1 is ligated to an expression vector to obtain a recombinant expression vector;

[0095] Preferably, a myocardial-specific promoter and the gene sequence of CES1 are successively ligated to an expression vector to obtain a recombinant expression vector;

[0096] Preferably, the recombinant expression vector is transformed into a host cell to obtain a transformant;

[0097] Preferably, the calcium phosphate co-transfection method is used to transform the recombinant expression vector into a host cell to obtain a transformant;

[0098] Preferably, the CES1 is selected from: human-derived CES1, rat-derived CES1, mouse-derived CES1;

[0099] Preferably, the expression vector is the adeno-associated virus expression vector pAAV-D(+);

[0100] Preferably, the myocardial-specific promoter is selected from: tnt, α-MHC, MLC-2v, Desmin;

[0101] Preferably, the recombinant expression vector is selected from: the recombinant adeno-associated virus expression vector pAAV-D(+)-CES1 ligated with the CES1 gene sequence, or, the recombinant adeno-associated virus expression vector pAAV-D(+)-tnt-CES1 successively ligated with the myocardial-specific promoter tnt and the CES1 gene sequence;

[0102] Preferably, the transformant is selected from: the recombinant adeno-associated virus rAAV-CES1 transformed with the recombinant adeno-associated virus expression vector pAAV-D(+)-CES1, or, the recombinant adeno-associated virus rAAV-tnt-CES1 transformed with the recombinant adeno-associated virus expression vector pAAV-D(+)-tnt-CES1;

[0103] Preferably, the CES1 gene sequence is as shown in SEQ ID NO.1.

[0104] In some specific embodiments of this group, the recombinant adeno-associated virus plasmid pAAV-D(+)-CES1 with a sequence fragment capable of highly expressing CES1 is used as the active ingredient of the anti-chronic heart failure drug.

[0105] Furthermore, the CES1 double-stranded nucleotide is synthesized by the whole gene synthesis technology and cloned onto the pAAV-D(+) vector, thereby preparing a CES1 recombinant plasmid capable of stable expression. In a more specific scheme, the CES1 sequence fragment is as shown in SEQ ID NO.1; the expression vector is the adeno-associated virus expression vector pAAV-D(+).

[0106] The specific experimental operation steps of this group of examples can be referred to in Experimental Example 1.

[0107] The technical effects of the present invention will be further verified and illustrated through Experimental Examples below.

[0108] Experimental Example 1: Construction of Recombinant Adeno-Associated Virus

[0109] 1. Construction of pAAV-D(+) vector

[0110] The CES1 double-stranded nucleotide is synthesized by the whole gene synthesis technology and cloned onto the pAAV-D(+) vector ( Figure 1 ), and the double-stranded nucleotide is synthesized by Wuhan Keruister Biotechnology Co., Ltd. The CES1 sequence is as shown in SEQ ID NO.1.

[0111] 2. Plasmid transformation

[0112] The pAAV-D(+)-CES1 plasmid is added to 100 μl of DH5α competent cells and placed on ice for 30 min; after heating at 42 °C for 45 sec, it is then placed on ice for 1 min; 500 μl of antibiotic-free LB medium is added, and it is cultured with shaking at 37 °C and 100 rpm for 60 min; it is cultured on an Amp+ LB plate medium, and white monoclonal colonies are selected for identification.

[0113] 3. Small-scale plasmid extraction

[0114] Pick a single colony and add it to 3 ml of LB liquid medium containing Amp+. Incubate overnight at 37°C with shaking at 280 rpm. Extract the plasmid using the EasyPure Plasmid MiniPrep Kit from TransGen Biotech Co., Ltd. The specific operation steps are as follows: 1. Take 1.5 ml of the overnight culture bacteria and centrifuge at 10,000 g for 1 min, and aspirate as much supernatant as possible; 2. Add 250 μl of colorless solution RB (containing RNase A) and vortex to suspend the bacterial pellet; 3. Add 250 μl of blue solution LB and gently invert the mixture up and down 4 - 6 times to fully lyse the bacteria, forming a blue and transparent solution; 4. Add 350 μl of yellow solution NB and gently mix 5 - 6 times until a tight yellow agglomerate is formed, and let it stand at room temperature for 2 min; 5. Centrifuge at 15,000 g for 5 min, carefully aspirate the supernatant and add it to the adsorption column; 6. Centrifuge at 15,000 g for 1 min and discard the effluent; 7. Add 650 μl of solution WB, centrifuge at 15,000 g for 1 min and discard the effluent; 8. Centrifuge at 15,000 g for 2 min to completely remove the residual WB; 9. Place the adsorption column in a new Ep tube, add 20 μl of EB preheated at 70°C in the center of the column, and let it stand at room temperature for 1 min; 10. Centrifuge at 10,000 g for 1 min to elute the DNA, and store the eluted DNA at -20°C.

[0115] 4. Large-scale plasmid extraction

[0116] Prepare a 1 L sterile conical flask, add 300 ml of sterile LB medium, and add ampicillin solution to a final concentration of 100 μg / ml. Add 50 μl of the required plasmids (pXX9, phelper, pAAV-D(+)-Ctrl, pAAV-D(+)-CES1) respectively, and incubate overnight at 37°C with shaking at 280 rpm. Extract the plasmid according to the instructions of the Endo-Free Plasmid Maxi Kit from OMEGA Company. The specific steps are as follows: 1. Centrifuge at 5000 g for 10 min at room temperature to collect the bacteria; 2. Discard the medium, add 10 ml of Solution I / RNase A mixture, and vortex to resuspend completely; 3. Add 10 ml of Solution II to the resuspended mixture, gently invert and mix 10 - 15 times, and then let it stand at room temperature

[0117] 5. rAAV-mediated virus packaging

[0118] The 293T cells are grown to 90%. 1 - 2 hours before calcium phosphate transfection, the fresh medium (containing serum), 12 - 15 ml, is changed for each culture dish. First, calcium chloride (CaCl2) is added into a 50 ml centrifuge tube, and then the plasmid is added to form a Ca-DNA mixture, which is thoroughly mixed. 2×HEBS BUFFER is slowly added dropwise to the Ca-DNA mixture to form a Ca-DNA-P mixture. While adding 2X HEBS, the centrifuge tube is shaken to mix thoroughly to form calcium phosphate particles. After 8 - 12 hours, 18 - 20 ml of serum-free medium is changed. After 72 hours, the medium is aspirated and the cells are washed 3 times with PBS. 1 ml of Tris + NaCl (pH 8.5) is added to each culture dish, and the cells are scraped with a spatula and collected in a clean centrifuge tube and stored at -80°C.

[0119] 6. Virus purification

[0120] The cells stored at -80°C are taken out, thawed and dissolved at 37°C, and frozen and thawed 4 times repeatedly. Then, they are centrifuged at 8,000g for 15 minutes. The supernatant is transferred to a clean centrifuge tube, and the cell pellet is discarded. The pre-cooled absolute ethanol at -20°C and rAAV are thoroughly mixed at a volume ratio of 3:1. After being placed in a -20°C refrigerator for 2 hours, they are centrifuged at 4°C and 13,000 rpm for 15 minutes, and the supernatant is discarded. After the ethanol has evaporated, an appropriate volume of Tris + NaCl (pH 8.5) is added to dissolve the precipitate. It is filtered through a Millipore small filter (0.22 μm).

[0121] 7. Virus titer determination

[0122] Sample treatment:

[0123] 40 μl of rAAV virus solution

[0124] 5 μl of Proteinase K (20 mg / ml)

[0125] React at 55°C for 1 hour;

[0126] 45 μl of phenol:chloroform:isoamyl alcohol

[0127] Centrifuge at 4°C and 12,000g for 5 minutes to recover the aqueous phase;

[0128] 45 μl of chloroform

[0129] Centrifuge at 4°C and 12,000g for 5 minutes to recover the aqueous phase.

[0130] Real-time PCR:

[0131] 0.4 μl of Primer 1 (10 μm)

[0132] 0.4 μl of Primer 2 (10 μm)

[0133] SYBR Green I Mix 10μl

[0134] ddH2O 8.2μl

[0135] Template 1μl

[0136] 95℃ 30 sec---(95℃ 5 sec---60℃ 5 sec---72℃ 20 sec) × 40 cycles---Melting Curve

[0137] Experimental Example 2. Expression difference of CES1 in heart failure mice

[0138] 8-week-old C57 mice were used. Transverse Aortic Constriction (TAC) surgery was performed as the heart failure model group caused by TAC (TAC causes a chronic heart failure model). At the same time, a sham operation control group (sham group) was set up (sham means the aortic arch was not ligated, and the remaining thoracotomy and suture steps were the same as TAC). At the end of the experiment (8 weeks after TAC surgery), it was detected by western blot that the content of CES1 in the hearts of chronic heart failure mice was significantly reduced ( Figure 2 ).

[0139] Experimental Example 3. Taking the recombinant adeno-associated virus expressing CES1 of rAAV9 type as an example to detect its therapeutic effect on chronic heart failure

[0140] 8-week-old C57 mice were used. rAAV-tnt-CES1 virus and rAAV-tnt-Ctrl negative control virus were respectively injected through the tail vein, and the virus titer was 1×10 11PFU / mouse. After 2 weeks, transverse aortic constriction (TAC) surgery was performed to establish a heart failure model caused by TAC (TAC-induced chronic heart failure model). Meanwhile, a sham operation control group (sham group) was set up (in the sham group, the aortic arch was not ligated, and the other thoracotomy and suture steps were the same as those in TAC). At the end of the experiment (8 weeks after TAC surgery), echocardiography was used to detect the cardiac function of TAC mice. The method was as follows: The instrument used was an ultrasound machine equipped with a 30 MHz high-frequency probe. After anesthetizing the mice with isoflurane, the mice were placed supine on the detection platform, and two-dimensional images of the left ventricle were collected along the short-axis and long-axis sections at the level of the left ventricular papillary muscle beside the sternum of the mice. Meanwhile, under the guidance of the two-dimensional images, more than 5 consecutive M-mode echocardiogram images were obtained respectively. According to the collected images, the results were analyzed using software to obtain the cardiac hemodynamic indexes detected by echocardiography. After analysis by relevant software, the following indexes were calculated: including heart rate (HR), left ventricular internal dimension at diastole (LVIDd), left ventricular internal dimension at systole (LVIDs), left ventricular posterior wall thickness at diastole (LVPWd), left ventricular posterior wall thickness at systole (LVPWs), interventricular septal thickness at diastole (IVSd), interventricular septal thickness at systole (IVSs), ejection fraction (EF), and fractional shortening (FS), etc. The results showed that rAAV-tnt-CES1 treatment could significantly improve the cardiac function of TAC mice ( Figure 3 ).

Claims

1. Use of CES1 in the preparation of a drug for treating chronic heart failure.

2. Use of the CES1 according to claim 1 in the preparation of a medicament for treating chronic heart failure, characterized in that, The drug for treating chronic heart failure targets CES1; and / or, the CES1 is selected from: human CES1, rat CES1, mouse CES1.

3. A gene for treating chronic heart failure, characterized in that, It contains: the CES1 gene sequence.

4. A gene for treating chronic heart failure according to claim 3, characterized in that, It also contains: a myocardial-specific promoter; and / or, the myocardial-specific promoter is selected from: tnt, α-MHC, MLC-2v, Desmin; and / or, the CES1 is selected from: human CES1, rat CES1, mouse CES1; and / or, the CES1 gene sequence is as shown in SEQ ID NO.

1.

5. A recombinant expression vector for treating chronic heart failure, characterized in that, It is an expression vector linked to the sequence of a gene for treating chronic heart failure described in claim 3 or 4.

6. A transformant for treating chronic heart failure, characterized in that, A host cell transformed with a recombinant expression vector for treating chronic heart failure described in claim 5 or 6.

7. Use of a gene for treating chronic heart failure described in claim 3 or 4, and / or, a recombinant expression vector for treating chronic heart failure described in claim 5, and / or, a transformant for treating chronic heart failure described in claim 6 in the preparation of a drug for treating chronic heart failure.

8. A drug for treating chronic heart failure, comprising a pharmacologically active ingredient, characterized in that, The pharmaceutically active ingredient includes: a gene for treating chronic heart failure described in claim 3 or 4, and / or, a recombinant expression vector for treating chronic heart failure described in claim 5, and / or, a transformant for treating chronic heart failure described in claim 6.

9. A preparation method of a drug for treating chronic heart failure, characterized in that, It expresses the CES1 protein.

10. The preparation method of a drug for treating chronic heart failure according to claim 9, characterized in that, Link the gene sequence of CES1 to an expression vector to obtain a recombinant expression vector; and / or, link a myocardial-specific promoter and the gene sequence of CES1 to an expression vector in sequence to obtain a recombinant expression vector; and / or, transform a host cell with the recombinant expression vector to obtain a transformant; and / or, use the calcium phosphate co-transfection method to transform a host cell with the recombinant expression vector to obtain a transformant; and / or, the CES1 is selected from: human CES1, rat CES1, mouse CES1; and / or, the expression vector is the adeno-associated virus expression vector pAAV-D(+); and / or, the myocardial-specific promoter is selected from: tnt, α-MHC, MLC-2v, Desmin; and / or, the recombinant expression vector is selected from: the recombinant adeno-associated virus expression vector pAAV-D(+)-CES1 linked to the CES1 gene sequence, or, the recombinant adeno-associated virus expression vector pAAV-D(+)-tnt-CES1 linked in sequence with the myocardial-specific promoter tnt and the CES1 gene sequence; and / or, the transformant is selected from: the recombinant adeno-associated virus rAAV-CES1 transformed with the recombinant adeno-associated virus expression vector pAAV-D(+)-CES1, or, the recombinant adeno-associated virus rAAV-tnt-CES1 transformed with the recombinant adeno-associated virus expression vector pAAV-D(+)-tnt-CES1; and / or, the CES1 gene sequence is as shown in SEQ ID NO.1.

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