CN101648996B - Anti-myocardial remodelling polypeptide, preparation method thereof, preparations and application thereof in preparation of anti-myocardial remodelling medicament - Google Patents

Abstract

The present invention relates to a G protein competitive inhibitory peptide, starting from a 55 peptide sequence, starting from the first amino acid residue at the amino terminal, deleting any number and at least one amino acid residue at any point, but retaining at least 12 amino acid residues at the carboxyl terminal amino acid residues to obtain a series of derived polypeptides, shortening the length of the polypeptides up to 78.2%, and the polypeptides have obvious anti-myocardial remodeling effect; the present invention also relates to the preparation method of the series of derived polypeptides, and utilizes advanced peptides Synthetic technology, successfully synthesized the target polypeptide with a purity of 99.2%. The invention also relates to the preparation containing the polypeptide and its application in the preparation of drugs for treating myocardial remodeling.

Description

Anti-myocardial remodeling polypeptide, its preparation method, preparation and application in the preparation of anti-myocardial remodeling drugs

technical field

The present invention relates to a polypeptide, more specifically, the present invention relates to a competitive inhibitory peptide of Gq protein α; the present invention also relates to the preparation method of the polypeptide, the preparation containing the polypeptide and the preparation of the polypeptide against myocardial remodeling application in medicine.

Background technique

Myocardial remodeling (i.e., the usual cardiac hypertrophy) refers to the increase in the volume of cardiomyocytes while the number remains unchanged. It is the common response of cardiomyocytes to various pathological stimuli. Changes in blood rheology such as disease and increased exercise-induced pressure load, and stimulation by humoral endocrine substances such as endothelin, angiotensin II, catecholamines, transforming growth factor β, and interleukin-1 can all lead to myocardial hypertrophy, so the incidence rate is extremely high ^[1] . Only in terms of hypertension, its incidence rate has reached 15% to 20% in the West. Although China is slightly lower than this, the number of patients has exceeded 150 million. In the early stage of the disease, myocardial hypertrophy has a certain compensatory effect, but as the disease progresses, the disorder of muscle fiber remodeling and myocardial systolic dysfunction caused by myocardial hypertrophy lead to a decline in cardiac function, which further develops into heart failure. Heart failure, death from heart failure, is one of the main causes of death in the above-mentioned clinical patients. Therefore, exploring specific and effective drugs for the treatment and control of myocardial hypertrophy is not only a topic and research hotspot faced by scientists in the world today, but also one of the important health problems to be solved in the global medical field.

Due to the numerous pathogenic factors and complex mechanisms of myocardial hypertrophy, so far, there is no clinical treatment drug specifically for myocardial hypertrophy in this field. Existing studies have shown that whether it is stretch stimulation caused by changes in blood rheology or stimulation of body fluid endocrine substances (reciprocal causation in disease), almost all of them are induced by corresponding receptors and their post-receptor signal transduction process. Pathological reactions such as myocardial hypertrophy and interstitial fibrosis ^[2] . Based on this study, people aimed at a certain factor of its pathogenesis, such as trying endothelin antagonists, angiotensin-converting enzyme inhibitors, antihypertensive drugs, etc., and found that it has a certain effect. However, there are many factors and receptors, and the above-mentioned antagonists/inhibitors can cause upregulation of autoreceptors and increased compensatory secretion of other related receptor ligands while inhibiting the function of a certain signaling molecule, so their effects are very limited ^{[ 3-6]} . Therefore, it is necessary to develop specific prevention and treatment drugs from a more fundamental way.

G protein is a heterotrimeric GTP-binding protein composed of three subunits of α, β and γ, and plays a key role in transducing extracellular stimulation signals to the intracellular process. Norepinephrine (norepinephrine, NE), endothelin (endotheline, ET), angiotensin II (Ang II), etc. stimulate α ₁ -AR, AT ₁ receptors, endothelin receptors, respectively, through the G protein The Gq family activates the effector enzyme phospholipase C-β (phospholipase C, PLC-β), acts on PIP ₂ , produces DAG and IP ₃ ; jointly follow DAG-PKC-Ras-MAPK and IP ₃ -Ca ²⁺ -CaN/ CaMPK II-NFAT3/GATA-4 signaling induces intracellular embryonic gene expression leading to myocardial remodeling. In addition to activating Raf1 through integrin, stretch stimulation can also stimulate the secretion of Ang II, NE, and ET ₁ , so it is also closely related to Gq. In addition, the experiment also observed: ① During the process of pathological myocardial remodeling, the Gq signal is significantly hyperactive, and its level is significantly higher than the physiological Gq signal in normal tissue; when the expression of Gqα is increased by 2 times (or below) There is no significant change in cardiac function and shape, and cardiac hypertrophy and cardiac systolic dysfunction appear when the increase is 4 times, and heart failure occurs when the increase is 8 times; Myocardial hypertrophy and fatal heart failure; ③ Knocking out the expression of Gqα in the heart can significantly reduce the hypertrophic response of the heart to pressure load ^[7-9] . It can be seen that Gqα plays a pivotal role in the occurrence and development of myocardial remodeling, is a common target of multiple signaling pathways, and is a key signaling component that mediates multiple factors leading to myocardial remodeling/hypertrophy. Therefore, regulating Gqα is expected to become a new strategy and successful way to resist myocardial remodeling/hypertrophy.

However, transgenic animals refer to a type of animal that introduces exogenous genes by experimental methods, stably integrates them in the chromosome genome, and can be passed on to offspring; the principle is to inject target genes/fragments that have been manipulated by molecular biology with different genetic methods. The fertilized eggs/pre-implantation embryo cells of experimental animals, and then implant the fertilized eggs/pre-implantation embryo cells into the oviduct or uterus of recipient animals to develop into transgenic animals carrying foreign genes, and pass Analyze the integration status of exogenous genes in transgenic animals and the phenotype of transgenic animals to explain the function of exogenous genes, and on this basis, breed excellent genetically engineered animals through conventional genetic breeding methods. Therefore, as far as the current existing technology is concerned, it is impossible and unreasonable to use transgenic technology for the treatment of human myocardial remodeling/hypertrophy with onset in adulthood. In addition, because Gqα also has important physiological functions, knocking out the expression of Gqα in the heart will cause serious toxic side effects, so the above two strategies and methods have no practical application value in the prevention and treatment of clinical myocardial remodeling/hypertrophy.

To this end, we have prepared a series of peptides with significant anti-myocardial remodeling/hypertrophy activities by comprehensively utilizing various technologies such as molecular design, optimization, genetic engineering, peptide preparation, and in vitro and in vivo activity screening.

Contents of the invention

Based on the defects in the prior art, an object of the present invention is to provide a series of polypeptides, which not only have therapeutic activity against cardiac hypertrophy, but also have a simple production method, low cost, and are easy to be industrialized and marketed.

Another object of the present invention is to provide a production method of this series of polypeptides, which is simple and economical, and can produce a series of polypeptide products with high purity and good anti-cardiac hypertrophy activity.

Another object of the present invention is to provide preparation products containing the polypeptide and its application in the preparation of anti-cardiac hypertrophy drugs.

To achieve the purpose of the present invention, the present invention firstly provides a polypeptide according to the Gqα polypeptide sequence, which has the amino acid sequence shown in SEQ ID NO: 1 (55 peptide sequence), and it has therapeutic activity against cardiac hypertrophy.

In a preferred technical solution, the polypeptide provided by the present invention starts from the first amino acid residue at the amino terminal of SEQ ID NO: 1, deletes any number and at least one amino acid residue at any point, but retains at least the carboxyl group The polypeptide obtained from the terminal 12 amino acid residues.

In a preferred technical solution, the polypeptide provided by the present invention is deleted from the 1st to 10th amino acid residues at the amino terminal of SEQ ID NO: 1, and the resulting peptide is shown in SEQ ID NO: 2 (45 peptide sequence) of polypeptides.

In a preferred technical solution, the polypeptide provided by the present invention is deleted from the 1st to 20th amino acid residues at the amino terminal of SEQ ID NO: 1, and the resulting peptide is shown in SEQ ID NO: 3 (35 peptide sequence) of polypeptides.

In a preferred technical solution, the polypeptide provided by the present invention is deleted from the 1st to 25th amino acid residues at the amino terminal of SEQ ID NO: 1, and the resulting peptide is shown in SEQ ID NO: 4 (30 peptide sequence) of polypeptides.

In a preferred technical solution, the polypeptide provided by the present invention is deleted from the 1-28 amino acid residues at the amino terminal of SEQ ID NO: 1, and the resulting peptide is shown in SEQ ID NO: 5 (27 peptide sequence) of polypeptides.

In a preferred technical solution, the polypeptide provided by the present invention is deleted from the 1st to 30th amino acid residues at the amino terminal of SEQ ID NO: 1, and the resulting peptide is shown in SEQ ID NO: 6 (25 peptide sequence) of polypeptides.

In a preferred technical solution, the polypeptide provided by the present invention is deleted from the 1st to 35th amino acid residues at the amino terminal of SEQ ID NO: 1, and the resulting polypeptide is shown in SEQ ID NO: 7 (20 peptide sequences) of polypeptides.

In a preferred technical solution, the polypeptide provided by the present invention is deleted from the 1-38 amino acid residues at the amino terminal of SEQ ID NO: 1, and the resulting peptide is shown in SEQ ID NO: 8 (17 peptide sequence) of polypeptides.

In a preferred technical solution, the polypeptide provided by the present invention is deleted from the 1st to 40th amino acid residues at the amino terminal of SEQ ID NO: 1, and the resulting peptide is shown in SEQ ID NO: 9 (15 peptide sequences) of polypeptides.

In a preferred technical solution, the polypeptide provided by the present invention is deleted from the 1st to 43rd amino acid residues at the amino terminal of SEQ ID NO: 1, and the resulting peptide is shown in SEQ ID NO: 10 (12 peptide sequences) of polypeptides.

The polypeptide provided by the present invention can also be a polypeptide having the same or similar anti-myocardial remodeling function after substitution, deletion or addition of one or more amino acids in any of the above-mentioned polypeptide sequences.

The polypeptide provided by the present invention can also be a polypeptide containing any sequence of the above-mentioned polypeptides and having the function of anti-myocardial remodeling.

The present invention also provides nucleotide sequences encoding the above polypeptides, which are respectively:

A nucleotide sequence as shown in SEQ ID NO: 11, which encodes the polypeptide shown in SEQ ID NO: 1.

A nucleotide sequence as shown in SEQ ID NO: 12, which encodes the polypeptide shown in SEQ ID NO: 2.

A nucleotide sequence as shown in SEQ ID NO: 13, which encodes the polypeptide shown in SEQ ID NO: 3.

A nucleotide sequence as shown in SEQ ID NO: 14, which encodes the polypeptide shown in SEQ ID NO: 4.

A nucleotide sequence as shown in SEQ ID NO: 15, which encodes the polypeptide shown in SEQ ID NO: 5.

A nucleotide sequence as shown in SEQ ID NO: 16, which encodes the polypeptide shown in SEQ ID NO: 6.

A nucleotide sequence as shown in SEQ ID NO: 17, which encodes the polypeptide shown in SEQ ID NO: 7.

A nucleotide sequence as shown in SEQ ID NO: 18, which encodes the polypeptide shown in SEQ ID NO: 8.

A nucleotide sequence as shown in SEQ ID NO: 19, which encodes the polypeptide shown in SEQ ID NO: 9.

A nucleotide sequence as shown in SEQ ID NO: 20, which encodes the polypeptide shown in SEQ ID NO: 10.

The present invention also provides a recombinant vector containing any of the above nucleotide sequences.

In another preferred technical solution, the recombinant vector contains a T7 promoter.

In a preferred technical scheme, said recombinant vector contains said nucleotide sequence and pIVEX2.3MCS plasmid.

The present invention also provides a preparation containing the above polypeptide and pharmaceutically acceptable auxiliary materials.

In a preferred technical scheme, the preparation is a parenteral injection preparation.

Another aspect of the present invention provides the application of the above-mentioned polypeptide in the preparation of a drug for treating cardiac hypertrophy.

In a preferred technical solution, the polypeptide is used as an active ingredient of a medicine, and then formulated with pharmaceutically acceptable excipients.

In another aspect, the present invention provides a method for preparing the above-mentioned polypeptide, which includes the following steps:

Polypeptide synthesis was carried out on a peptide synthesizer according to the above amino acid sequence.

The present invention also provides another preparation method of the above-mentioned polypeptide, which comprises the following steps:

Forming the corresponding nucleotide sequence and vector into a recombinant vector;

Transforming the recombinant vector into a host cell;

inducing the host cell to express the polypeptide;

The polypeptide is isolated.

In a preferred technical solution of the preparation method, the vector contains a T7 promoter.

In a preferred technical solution of the preparation method, the vector is a plasmid, and the host cell is Escherichia coli.

In a preferred technical scheme of the preparation method, the plasmid is pIVEX2.3MCS, and the Escherichia coli is BL21.

Through the above technical scheme, the present invention starts from the amino acid sequence shown in SEQ ID NO: 1, starts from the first amino acid residue at the amino terminal, and deletes any number of at least one amino acid residue at any point toward the carboxyl terminal , gradually reduce its amino acid residues until 43 residues are deleted, and only 12 amino acid residues from the carboxyl terminal are retained; On the basis of improving its activity, the length of its peptide was successfully shortened by 78.2%, and the target peptide was successfully synthesized by using advanced peptide synthesis technology, with a purity of 99.2%, and its key parameters for industrialization have been met.

The present invention also successfully produces the target polypeptide through the method of genetic engineering. Because the full length of the 55-peptide gene is only 165bp, and the enzyme cutting site is about 180bp, considering factors such as economy, convenient operation, and reliability, we chose to synthesize it in two sections (4 sections), and then cloned into the expression carrier method. The pIVEX2.3MCS2-55 peptide expression plasmid was constructed, which contains the complete 55 peptide gene and is under the control of the T7 promoter. It can successfully express the 55 peptide under the prokaryotic expression system using T7 promoter.

The present invention uses technologies such as light microscopy, electron microscopy, direct weighing, and color B-ultrasound to carry out systematic pharmacodynamic evaluation on the target polypeptide, and has remarkable curative effect. Studies have confirmed that the target polypeptide has a good preventive effect on the in vitro cultured cardiomyocyte hypertrophy model caused by various factors such as angiotensin II and NE; it has a good inhibitory effect on the changes in MAPK activity stimulated by angiotensin II and other factors; It has a good therapeutic effect on the myocardial hypertrophy of the in vivo reverse aortic coarctation model (TAC) in normal mice, and the myocardial hypertrophy caused by volume overload (AVO) in normal rats; it has a good therapeutic effect on spontaneously hypertensive rats (SHR ) also has obvious anti-myocardial remodeling, anti-vascular hypertrophy and antihypertensive effects.

Preliminary safety evaluation shows that the target peptide drug is very safe. ① Cytotoxicity test, no cytotoxicity (negative); ② Genotoxicity test, no genetic toxicity (negative); ③ Mutagenicity test (Ames test): no mutagenic effect (negative). The mouse tolerance dose of the target polypeptide is at least greater than 50 mg/kg, and the ratio of the effective dose to the effective dose is at least greater than 500; while the commonly used antihypertensive drugs captopril, losartan and nifedipine with anti-cardiac hypertrophy LD50 The ratios of /common dosage (converted clinical dosage to mouse dosage) were 388, 349, and 157 respectively, indicating that the safety of the target polypeptide is significantly better than the above-mentioned drugs. In addition, no other toxic and side effects were observed during the test.

Description of drawings

Figure 1. Morphological changes of myocardial tissue in the SHR model group;

Figure 2. Changes in myocardial histomorphology in the Losartan group;

Figure 3.27 Changes in myocardial tissue morphology in the peptide group;

Figure 4. Changes in the ultrastructure of cardiomyocytes in the SHR model group;

Fig. 5. The changes of the ultrastructure of cardiomyocytes in the losartan group;

Figure 6.27 Changes in the ultrastructure of cardiomyocytes in the peptide group.

Detailed ways

The present invention will be described in more detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings.

Embodiment 1: the solid-phase synthesis of polypeptide

1. Synthesis and purification process of 27 peptide

In the following steps, 25 g of resin (with a substitution constant of 0.6 mmol/g) was charged according to the pilot scale. The production scale is 1kg resin, and the feeding amount is increased proportionally to prolong the reaction time.

(1) Synthesis process of 27 peptide

1. Accurately weigh 25g of Fmoc-Val-Wang resin, place it in a 1000ml reactor, add 500ml of DCM, shake and soak for 30min, wash twice with 500ml each of DCM, MeOH, and DMF, and remove the solvent by suction filtration.

2. Add 500ml 20% Piperidin/DMF, shake at room temperature, react for 30min, and remove the N-terminal Fmoc protecting group. After the solvent was removed by suction filtration, the resin was washed twice with 500 ml each of DMF, MeOH and DCM, and the solvent was removed by suction filtration.

3. Weigh 21.2g Fmoc-Leu-OH, 22.8g HBTU and dissolve in 500ml DMF, add 40ml DIEA, stir and react at room temperature for 30min, add to the reactor, shake at room temperature, react for 2h. After the reaction solution was removed by suction filtration, the resin was washed twice with 500 ml each of DMF, MeOH and DCM respectively, and the solvent was removed by suction filtration.

4. Repeat steps 2 and 3, except that the amino acid added in step 3 is 35.8g Fmoc-Asn(Trt)-OH, and the reaction time is 3h, other conditions remain unchanged, and the steps are the same.

5. Repeat steps 2 and 3, except that the amino acid added in step 3 is 27.6gFmoc-Tyr(tBu)-OH, other conditions remain unchanged, and the steps are the same.

6. Repeat steps 2 and 3, except that the amino acid added in step 3 is 25.5g Fmoc-Glu(OtBu)-OH, other conditions remain unchanged, and the steps are the same.

7. Repeat steps 2 and 3, except that the amino acid added in step 3 is 28.1 g Fmoc-Lys(Boc)-OH, other conditions remain unchanged, and the steps are the same.

8. Repeat steps 2 and 3, except that the amino acid added in step 3 is 21.2g Fmoc-Leu-OH, other conditions remain unchanged, and the steps are the same.

9. Repeat steps 2 and 3, except that the amino acid added in step 3 is 35.8gFmoc-Asn(Trt)-OH, other conditions remain unchanged, and the steps are the same.

10. Repeat steps 2 and 3, except that the amino acid added in step 3 is 21.2g Fmoc-Leu-OH, other conditions remain unchanged, and the steps are the same.

11. Repeat steps 2 and 3, except that the amino acid added in step 3 is 36.7gFmoc-Gln(Trt)-OH, other conditions remain unchanged, and the steps are the same.

12. Repeat steps 2 and 3, except that the amino acid added in step 3 is 21.2g Fmoc-Leu-OH, other conditions remain unchanged, and the steps are the same.

13. Repeat steps 2 and 3, except that the amino acid added in step 3 is 21.2g Fmoc-Ile-OH, other conditions remain unchanged, and the steps are the same.

14. Repeat steps 2 and 3, except that the amino acid added in step 3 is 23.9gFmoc-Thr(tBu)-OH, other conditions remain unchanged, and the steps are the same.

15. Repeat steps 2 and 3, except that the amino acid added in step 3 is 24.7gFmoc-Asp(OtBu)-OH, other conditions remain unchanged, and the steps are the same.

16. Repeat steps 2 and 3, except that the amino acid added in step 3 is 28.1 g Fmoc-Lys(Boc)-OH, other conditions remain unchanged, and the steps are the same.

17. Repeat steps 2 and 3, except that the amino acid added in step 3 is 20.4g Fmoc-Val-OH, other conditions remain unchanged, and the steps are the same.

18. Repeat steps 2 and 3, except that the amino acid added in step 3 is 19.8g Fmoc-Ala-OH, other conditions remain unchanged, and the steps are the same.

19. Repeat steps 2 and 3, except that the amino acid added in step 3 is 19.8g Fmoc-Ala-OH, other conditions remain unchanged, and the steps are the same.

20. Repeat steps 2 and 3, except that the amino acid added in step 3 is 23.2Fmoc-Phe-OH, other conditions remain unchanged, and the steps are the same.

21. Repeat steps 2 and 3, except that the amino acid added in step 3 is 20.4g Fmoc-Val-OH, other conditions remain unchanged, and the steps are the same.

22. Repeat steps 2 and 3, except that the amino acid added in step 3 is 23.2g Fmoc-Phe-OH, other conditions remain unchanged, and the steps are the same.

23. Repeat steps 2 and 3, except that the amino acid added in step 3 is 39.0gFmoc-Arg(pbf)-OH, other conditions remain unchanged, and the steps are the same.

24. Repeat steps 2 and 3, except that the amino acid added in step 3 is 21.2g Fmoc-Ile-OH, other conditions remain unchanged, and the steps are the same.

25. Repeat steps 2 and 3, except that the amino acid added in step 3 is 35.8g Fmoc-Asn(Trt)-OH, other conditions remain unchanged, and the steps are the same.

26. Repeat steps 2 and 3, except that the amino acid added in step 3 is 25.5 g Fmoc-Glu(OtBu)-OH, other conditions remain unchanged, and the steps are the same.

27. Repeat steps 2 and 3, except that the amino acid added in step 3 is 23.9g Fmoc-Thr(tBu)-OH, other conditions remain unchanged, and the steps are the same.

28. Repeat steps 2 and 3, except that the amino acid added in step 3 is 24.7g Fmoc-Asp(OtBu)-OH, other conditions remain unchanged, and the steps are the same.

29. Add 500ml 20% Piperidine/DMF, shake at room temperature, react for 30min, and remove the N-terminal Fmoc protecting group. After the solvent was removed by suction filtration, the resin was washed twice with 500 ml each of DMF, MeOH and DCM, and the solvent was removed by suction filtration. Vacuum dry the resin supernatant.

30. Weigh the dried resin, the total weight is 68g, and the weight gain is 43g. Transfer the resin to a 250ml round bottom flask, add 150ml (TFA/TA/EDT/TIS/H2O/phenol 7:1:1:0.1:0.35/0.5), and stir at room temperature for 4h. The resin was separated from the filtrate by suction filtration, 2000ml of diethyl ether at 0°C was added to the filtrate, the precipitate was separated from the diethyl ether by centrifugation, and the crude product of 27 peptide was 40g after drying.

(2) 27 peptide purification process:

1. The lyophilized 27-peptide crude peptide sample was dissolved in DMSO, separated by gradient elution with a reversed-phase high-performance liquid chromatography system, and the 27-peptide main peak fraction was collected, combined and freeze-dried to obtain a purified 27-peptide raw material. The lyophilized product after the primary purification was dissolved in 15% acetonitrile, and the reversed-phase high performance liquid chromatography system was used for secondary purification to collect the fractions of the main peak, remove impurities near the main peak, and obtain the refined 27 peptide after merging and freeze-drying.

2. The chromatographic conditions are as follows:

Chromatograph: Varian liquid chromatograph prepstar and its operation analysis software;

Chromatographic column: use Load & Lock to pack C ₁₈ chromatographic column (250x50mm);

Mobile phase: A: 0.05% TFA/2% acetonitrile/water; B: 90% acetonitrile/water;

Elution gradient: primary purification: 8-8-32-57% B mobile phase for 70 minutes, flat gradient for 5 minutes;

Secondary purification: 0-0-34-55% B mobile phase for 70 minutes, flat gradient for 5 minutes;

Flow rate: 50ml/min;

UV detection wavelength: 275nm;

2. Synthesis and purification process of 55 peptide

In the following steps, 25 g of resin (with a substitution constant of 0.6 mmol/g) was charged according to the pilot scale. The production scale is 1kg resin, and the feeding amount is increased proportionally to prolong the reaction time.

1. Accurately weigh 25g of Fmoc-Val-Wang resin, place it in a 2000ml reactor, add 500ml of DCM, shake and soak for 30min, wash twice with 500ml each of DCM, MeOH, and DMF, and remove the solvent by suction filtration.

2. Add 500ml 20% Piperidin/DMF, shake at room temperature, react for 30min, and remove the N-terminal Fmoc protecting group. After the solvent was removed by suction filtration, the resin was washed twice with 500 ml each of DMF, MeOH and DCM, and the solvent was removed by suction filtration.

3. Weigh 21.2g Fmoc-Leu-OH, 22.8g HBTU and dissolve in 500ml DMF, add 40ml DIEA, stir and react at room temperature for 30min, add to the reactor, shake at room temperature, react for 2h. After the reaction solution was removed by suction filtration, the resin was washed twice with 500 ml each of DMF, MeOH and DCM respectively, and the solvent was removed by suction filtration.

4. Repeat steps 2 and 3 for washing and deprotection, and add amino acids in sequence until the reaction reaches the end of the last amino acid;

5. Add 750ml 20% Piperidine/DMF, shake at room temperature, react for 30min, and remove the N-terminal Fmoc protecting group. After the solvent was removed by suction filtration, the resin was washed twice with 500 ml each of DMF, MeOH and DCM, and the solvent was removed by suction filtration. Vacuum dry the resin supernatant.

6. Weigh the dried resin, the total weight is 113g, and the weight gain is 88g. Transfer the resin to a 250ml round bottom flask, add 250ml (TFA/TA/EDT/TIS/H2O/phenol 7:1:1:0.1:0.35/0.5), and stir at room temperature for 4h. The resin was separated from the filtrate by suction filtration, 3000ml of diethyl ether at 0°C was added to the filtrate, the precipitate was separated from the diethyl ether by centrifugation, and the crude product of 55 peptide was 85g after drying.

7. The refining process and chromatographic conditions refer to 27 peptides.

3. Synthesis and purification process of 12 peptides

1-4. Same as above 55 peptide part

5. Add 500ml 20% Piperidine/DMF, shake at room temperature, react for 30min, and remove the N-terminal Fmoc protecting group. After the solvent was removed by suction filtration, the resin was washed twice with 500 ml each of DMF, MeOH and DCM, and the solvent was removed by suction filtration. Vacuum dry the resin supernatant.

6. Weigh the dried resin, the total weight is 46g, and the weight gain is 21g. Transfer the resin to a 250ml round bottom flask, add 100ml (TFA/TA/EDT/TIS/H2O 7:1:1:0.1:0.35), and stir at room temperature for 3h. The resin was separated from the filtrate by suction filtration, 1500ml of diethyl ether at 0°C was added to the filtrate, the precipitate was separated from the diethyl ether by centrifugation, and the crude product of 12-peptide was 19g after drying.

7. The refining process and chromatographic conditions refer to 27 peptides.

Example 2: Genetic engineering expression and purification of 55 peptide and 27 peptide

According to the gene sequences of 55 peptides and 27 peptides, the corresponding oligonucleotide sequences were designed for the construction of their expression vectors.

Synthesize four oligonucleotide single strands for 55 peptides:

55-1: 60bp

5'tcgagctccatgggtcgagaattcattctgaagatgttcgtcgactaaacgttctctgca 3'

55-2: 52bp

5'gagaacgtttagtcgacgaacatcttcagaatgaattctcgacccatggagc 3'

55-3: 85bp

5'gaggtcgacctgaacccagacagtgacaaaattatctactcccacttcacgtgtgccacagacaccg

agaatatccgctttgtct 3’

55-4: 85bp

5'tagcccggggaccagattgtactccttcaggttcagctggaggatggtgtccttgacggctgcaaag

acaaagcggatattctcg 3’

Synthesize oligonucleotide single strands for 27 peptides:

27-1:

5'catggacacccgagaatatccgctttgtctttgcagccgtcaaggacaccacctccagctgaacctga

aggagtacaatctggtctaaccc 3’

27-2:

5'ggggttagaccagattgtactccttcaggttcagctggaggatggtgtccttgacggctgcaaagacaa

agcggatattctcggtgtc 3’

The double-stranded DNA fragment with cohesive ends formed after the first and second oligonucleotides synthesized by 55 peptides were annealed was directionally cloned into the XhoI site and PstI site of the pEGFP-N1 plasmid (provided by the Genetics Teaching and Research Office of the Third Military Medical University) Between the points, the constructed plasmid was called pEGFP-A. The third and fourth oligonucleotides synthesized have 20 bases complementary to each other at the 3′ end of the single strand, and are extended into a double-stranded DNA fragment B under the action of Taq enzyme. The amplified fragment is located at 150 bp by 2.5% agarose gel electrophoresis . Fragment B was double-digested with SalI and SmaI, and cloned into pEGFP-A between the SalI and SmaI sites. The constructed plasmid was called pEGFP-55, which contained the complete 55 peptide gene. After the 55 peptide gene was excised with XhoI and SmaI double enzymes, it was inserted between the XhoI site and the SmaI site of the pIVEX2.3-MCS plasmid, and the prokaryotic expression vector constructed was called pIVEX2.3MCS-55. Its screening can be performed by XhoI and SmaI double digestion, 2.5% agarose gel electrophoresis, if there is a band around 180bp, it is a positive clone.

The first and second single-stranded oligonucleotides synthesized by 27 peptides can directly form double-stranded DNA with NcoI and SmaI sticky ends after annealing. After the pIVEX2.3 plasmid was digested with NcoI and SmaI, the plasmid fragment with cohesive ends was recovered with a gel recovery kit after electrophoresis. The 27-peptide gene was directional cloned into the pIVEX2.3 plasmid (purchased from Roche, Switzerland) with T4 DNA ligase. The positive clone contains the 27 peptide gene, called pIVEX2.3-27. Pick 6 colonies of ligated recombinant plasmid products on a plate containing ampicillin, culture them in 5ml LB medium overnight, extract the plasmids and carry out electrophoresis identification after double enzyme digestion with NcoI and SmaI; the positive clones identified by electrophoresis are then identified by sequencing , it was determined that pIVEX2.3MCS-55 and pIVEX2.3-27 were constructed successfully. Transform pIVEX2.3MCS-55 and pIVEX2.3-27 into competent BL21(DE3)pLysE bacteria (purchased from Shanghai Sangon Bioengineering Technology Co., Ltd.) Take a single colony and culture it in 2ml LB medium containing ampicillin (100μg/ml) at 37°C and 180rpm/min with shaking for 10 hours. Add 200μl of the above bacterial solution to 250ml of ampicillin-containing LB medium, shake at 37°C and 180 rpm for 10 hours, add IPTG to make the final concentration 1mmol/L, continue to cultivate at 37°C for 4-6 hours and at 30°C The incubation was continued for 12 hours. Bacteria were collected by centrifugation and stored at -70°C for later use. 1 g of collected bacteria was resuspended in 5 ml of binding buffer, ultrasonically disrupted (conditions: pulse 6 seconds, amplitude 15-20, broken on ice for 8-10 minutes), centrifuged at 12000 rpm/min for 10 minutes, and aspirate the supernatant for purification. Purification was performed by nickel chelate affinity chromatography under denaturing conditions: first equilibrate the nickel column with 5 ml of denaturing binding buffer. The sonication supernatant was passed through the column, the flow rate was controlled at no more than 10ml per hour, and the permeate was collected. Wash the column with 5ml of denaturing wash buffer A, and collect the permeate. The proportion of non-denaturing wash buffer B and denaturing wash buffer B is 0:1, 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8 : 2, 9: 1, and 1: 0 each 1ml, wash the column with the above liquids in turn, and then wash the column with 2ml of non-denaturing washing buffer B. The whole process is long and should not be less than 2 to 3 hours. Permeate. 3ml of elution buffer A was passed through the column to elute the protein, and the permeate was collected. 2×3ml elution buffer B was passed through the column to elute the protein, and the permeate was collected. And identify its purity by SDS-PAGE, the result shows that BL21 (DE3) bacterium expresses target polypeptide, and expression amount accounts for about 10% of total protein of thalline, and through nickel column purification and renaturation, output is that 250ml bacterium liquid purifies about 1.5 mg target peptide. When 500mmol/L imidazole was eluted, most of the target polypeptides were washed out, and the SDS-PAGE band was single, and its purity reached more than 98% by image analysis.

Example 3: The series of polypeptides of the present invention can significantly reduce the protein content in the rat cardiomyocyte hypertrophy model induced by norepinephrine

1. Wistar rats (purchased from the Experimental Animal Center of Third Military Medical University) 1-3 days after birth were taken out, killed by cervical dislocation, and fixed on the dissection table. Disinfect the ventral skin with 2% tincture of iodine and 75% ethanol. Take out the heart, cut it into fragments about ^1-3 mm in size, digest it repeatedly with the digestion solution containing 0.08% trypsin, 0.02% EDTA, and 0.05% collagenase II, collect the cells in DMEM containing 10% fetal bovine serum, Culture in a 37°C, 5% CO ₂ incubator.

2. After culturing the cardiomyocytes for 48 hours, replace them with serum-free DMEM medium, continue to cultivate for 24 hours, and add drugs according to the following experimental groups:

Normal control group: add PBS 10μl

Norepinephrine group: add norepinephrine (norepinephrine, NE, Serva Company, USA) 1 μmol/L

Peptide administration group: while adding NE, add the corresponding polypeptide drug 10nmol/L.

3. Continue culturing for 24 hours after adding the drug, discard the culture medium, wash with PBS, add 0.5ml of 5% trichloroacetic acid to each well, place at 4°C for 1h, dissolve the precipitate in 1ml of 0.1mol/L NaOH, and use the Lowry method Determination of protein content.

4. Results: 10nmol/L of 55 peptide, 45 peptide, 35 peptide, 30 peptide, 27 peptide, 25 peptide, 20 peptide, 17 peptide, 15 peptide can significantly reduce the protein content in hypertrophic cardiomyocytes induced by NE, and 12 peptide No obvious effect (as shown in Table 1).

Table 1. The series of polypeptides of the present invention are effective in culturing rat cardiomyocytes

Effect of protein content (n=6, x±s)

^** P＜0.01vs control group; ^# P＜0.05, ^## P＜0.01vs NE group.

Example 4. The series of polypeptides of the present invention can significantly reduce the protein content in the rat cardiomyocyte hypertrophy model induced by angiotensin II (Ang II)

1. Cardiomyocyte culture is the same as above, after 48 hours of culture, change to serum-free DMEM culture medium, continue to culture for 24 hours and add drugs according to the following experimental groups: normal control group: add PBS 10μl; Ang II group: add AngII 1μmol/L; series of peptides Administration group: while adding Ang II, 12 peptides, 15 peptides, 27 peptides, and 55 peptides were added at 10nmol/L.

2. Continue culturing for 24 hours after adding the drug, discard the culture medium, wash with PBS, add 0.5ml of 5% trichloroacetic acid to each well, place at 4°C for 1h, dissolve the precipitate in 1ml of 0.1mol/L NaOH, and use the Lowry method Determination of protein content.

3. Results: Compared with the normal control group, adding Ang II to the cell culture medium can significantly increase the protein content of cardiomyocytes (μg: 143.2±5.49vs 113.9±7.48, p<0.01). Compared with the AngII group, peptide 15, peptide 27, and peptide 55 could reduce the protein content in cardiomyocytes to varying degrees, while peptide 12 had no obvious effect (see Table 2).

Table 2. Effects of a series of polypeptides on the protein content of hypertrophic cardiomyocytes induced by angiotensin II (n=6, x±s)

Note: ^* p<.05, ^** p<0.01vs control group; ^# p<0.05, ^## p<0.01vs Ang II group

Example 5. The series of polypeptides of the present invention can significantly inhibit norepinephrine-induced myocardial hypertrophy in mice

50 Kunming mice (purchased from the Experimental Animal Center of the Third Military Medical University) were used in the test, divided into 5 groups, 10 in each group, the control group was given 0.1% ascorbic acid-3mg% potassium sodium tartrate-normal saline, and the model group was given 0.1 % ascorbic acid-6mg% norepinephrine bitartrate-physiological saline (equivalent to NE 1.5mg/kg); 3 groups were given drugs, while giving 0.1% ascorbic acid-6mg% norepinephrine bitartrate-normal saline, Peptide 15, peptide 27, and peptide 55 were administered at 30 μg/kg, intraperitoneally injected (ip), twice a day (bid), for 15 consecutive days. The administration volume of each group was 50ml/kg. At that time, the animal was killed by cervical dislocation, the heart was removed by thoracotomy, the blood was washed away with ice-cold saline, blotted dry with filter paper, weighed with a balance, the atrium and right ventricle were carefully removed (the interventricular septum was preserved), and the left ventricle was weighed.

The results showed that the series of peptides could significantly prevent the occurrence of myocardial hypertrophy in mice. Except for the heart weight of the 15-peptide group (reducing 9.9%, but p>0.05), the indicators of myocardial hypertrophy in other groups were significantly improved (see Table 3).

Table 3. Effects of series of peptides on myocardial hypertrophy in mice (x±s)

Note: BW-body weight; HW-heart weight; LVW-left ventricular weight; HI-heart index; LVI-left ventricular index or cardiac hypertrophy index

^* p<0.05, ^** p<0.01vs control group; ^# p<0.05, ^## p<0.01vs NE group

Example 6: A series of polypeptides of the present invention inhibit norepinephrine-induced cardiac hypertrophy in rats

Select 30 Wistar rats, 5 groups, 6 in each group, the control group is given 0.1% ascorbic acid-3mg% potassium sodium tartrate-normal saline, the model group is given 0.1% ascorbic acid-6mg% norepinephrine bitartrate-normal saline, Three groups of drugs were administered, while norepinephrine was given, 15-peptide, 27-peptide, and 55-peptide were given 15 μg/kg, and the volume of each group was 33ml/kg, ip, bid, for 20 consecutive days. At that time, the heart and left ventricle were weighed.

The results showed (see Table 4): 15 peptides can significantly reduce the left ventricular index, 27 peptides and 55 peptides can significantly reduce the left ventricular weight, cardiac index and left ventricular index.

Table 4 Effects of series of peptides on norepinephrine-induced myocardial hypertrophy in rats (n=6, x±s)

Note: BW-body weight; HW-heart weight; LVW-left ventricular weight; HI-heart index; LVI-left ventricular index or cardiac hypertrophy index

^* p<0.05, ^** p<0.01vs control group; ^# p<0.05, ^## p<0.01vs NE group

Example 7: The series of polypeptides of the present invention can prevent and treat myocardial hypertrophy in rats induced by abdominal aortic constriction

Healthy male Wistar rats (purchased from the Experimental Animal Center of Third Military Medical University) were randomly divided into: sham-operation group (sham-operation group), operation model group (SRS), and polypeptide administration group. Animals were fasted overnight before the operation, free to drink water, anesthetized with pentobarbital sodium, opened the abdomen, separated the abdominal aorta, tied a No. 8 injection needle (outer diameter 0.80 mm) with silk thread above the right renal artery and along the abdominal aorta Afterwards, the needle was quickly removed, and the abdomen was sutured. The sham operation group did not perform silk ligation, and other treatments were the same. From 1 day after the operation, the sham operation control group and the operation model group were given normal saline 7.5ml/kg, ip, once a day; the drugs were given 15 μg/kg of peptide 15, 27 and 55, ip, once a day. /day. After 3 weeks of routine feeding, the rats were sacrificed, and the heart was removed by opening the chest. The blood was washed away with ice-cold saline, blotted dry with filter paper, and the heart and left ventricle were weighed with a balance.

The results showed that compared with the operation model group, the series of polypeptides could significantly prevent the occurrence of myocardial hypertrophy in rats, and HW, LVW, HI and LVI were all significantly reduced (see Table 5).

The effect of table 5 series peptides on myocardial hypertrophy in rats with coarctation of the abdominal aorta (x ± SEM)

^* P<0.05, ^** P<0.01vs sham-operation group; ^#P <0.05, ^## P<0.01vsSRSgroup.

Example 8 The series of polypeptides of the present invention can prevent and treat myocardial remodeling in spontaneously hypertensive rats

1. Select 30 13-week-old spontaneously hypertensive rats (SHR, purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.), and randomly divide them into 5 groups, 6 in each group:

SHR model group (Vehicle): 0.9% normal saline, intraperitoneally injected at 5 ml/kg, twice a day.

Positive control group (Losartan): losartan potassium 6mg/kg, orally administered once a day.

15-peptide group: intraperitoneal injection of 15-peptide at 30 μg/kg, twice a day.

27-peptide group: intraperitoneal injection of 27-peptide at 30 μg/kg, twice a day.

55-peptide group: intraperitoneal injection of 55-peptide at 30 μg/kg, twice a day.

In addition, 6 WKY (Wistar-Kyoto) rats (purchased from the Experimental Animal Center of Third Military Medical University) were selected as normal controls.

2. Continuous drug intervention for 8 weeks (from the 14th week to the 21st week). The body weight of all experimental rats was measured every two weeks, and the dosage was adjusted according to the body weight.

3. The result display

(1) The series of polypeptides have a certain antihypertensive effect and can significantly reduce the arterial systolic blood pressure of SHR (see Table 6)

Table 6 The effect of series of polypeptides of the present invention on SHR systolic blood pressure (x±s n=6)

##p＜0.01vs WKY; ^** p＜0.01vs Vehicle

(2) The series of polypeptides of the present invention can significantly improve the myocardial remodeling of SHR, and can significantly reduce the HW, LVW, HI and LVI (Table 7) of rats and the end-diastolic left ventricular posterior wall thickness (left ventricular posterior wall thickness, PWT) of SHR ), interventricular septum thickness (IVST) (Table 8).

Table 7: Effects of a series of peptides on myocardial hypertrophy in rats (x±s n=6)

Note: ^** p＜0.01vs Vehicle; ^## p＜0.01vs WKY

Table 8 The influence of series of polypeptides of the present invention on SHR echocardiographic parameters (x±s n=6)

Note: ##: p<0.01vs WKY; ^* : p<0.05, ^** : p<0.01vs Vehicle

LAD: left atrium sinistrum diameter (left atrium diameter); LVEDD: left ventricular end diastolic diameter (left ventricular end diastolic diameter), EF: ejectionfraction (ejection fraction); FS: shortening fraction (short axis shortening rate), SV: strokevolume ( stroke volume)

(3) Effects of the series of polypeptides of the present invention on the morphology of SHR myocardial tissue:

Morphological analysis showed that the myocardial cell diameter (TDM) and cross-sectional area (CSA) of rats in the model group were significantly increased compared with those in the WKY group (p<0.01), while the CSA of the rats in the series of polypeptide medication group and the losartan group was significantly higher than that in the model group. Significantly decreased (p<0.01) (see Table 9).

Table 9. Morphological analysis of rat cardiomyocytes (x±s n=6)

##p<0.01vs WKY; ^** p<0.01, ^** p<0.01vs vehicle. It can be seen under the light microscope that the nucleus of cardiomyocytes is blue, the cytoplasm is pink, and the collagen is not colored.

SHR model group (Figure 1): Myocardial fibers became thicker, swollen, unclear, fractured, fused, or arranged in disorder, and some of them had obvious large-area myocardial watery degeneration; myocardial cells were obviously hypertrophic, turbid and swollen, and vacuolar changes ; Nucleus hypertrophy or pyknosis; cell contents become granular, fractured and fused, or even necrotic; punctate necrosis and focal necrosis can be seen in a few fields of view; blood vessel wall thickening; smooth muscle cell hyperplasia and hypertrophy; individual fields of view myocardium Interstitial fibrosis. But in general: the degree of fibrosis in the model group was mild; the inflammatory reaction was severe; necrotic foci could be seen in a few visual fields.

Losartan group (Figure 2): The lesions were improved, and the pathological changes were mainly infiltration of inflammatory cells. Cardiomyocytes showed obvious inflammatory changes such as swelling and hypertrophy, and necrotic lesions could still be seen in some visual fields.

Compared with the SHR model group, the 27-peptide group (Figure 3) showed that the pathological damage of the above-mentioned cardiomyocytes was significantly reduced, and the improvement of the lesions was basically close to that of the normal control group. Some inflammatory changes such as mild swelling of cardiomyocytes were occasionally seen in some visual fields, and no necrotic focus was formed. , myocardial fibers and vessel walls were normal in shape.

(4) Effect of 27 peptide on the ultrastructure of SHR cardiomyocytes (PHILIPS-TECNI10 transmission electron microscope, Netherlands):

SHR model group (Figure 4): cardiomyocyte volume increased, nuclear membrane was incomplete, nuclear hypertrophy, deformity, dissolution and other irregular changes; sarcoplasmic reticulum expansion; ) swelling, with vacuoles forming inside; the myofilament arrangement is basically normal, and some myocyte myofilaments have focal dissolution; striations are not clear in some areas, the Z line is basically normal, and individual arrangements are disordered; interstitial collagen fibers have no obvious hyperplasia.

Losartan group (Fig. 5): The shape of the nuclei was basically normal with a little irregularity; myofilaments under the lipid membrane were focally dissolved; the mitochondria were mildly swollen; the sarcoplasmic reticulum was dilated and slightly swollen;

27 peptide group (Figure 6): Compared with the SHR model group, the myocardial ultrastructure was significantly improved, the structure of myocardial cells was basically normal, the arrangement of cardiomyocyte sarcomeres and myofilaments was basically normal, the striations were clear, and the interstitial collagen fibers had no obvious hyperplasia. The Z-lines were arranged neatly, the mitochondria had no obvious hyperplasia, a small number of mitochondria were slightly swollen, myofilaments were slightly dissolved, and the capillary endothelial cells were normal.

Although the above embodiments are disclosed herein, the technical solutions of the present invention are not limited to the above embodiments; without departing from the concept of the present invention, any changes made to the technical solutions of the present invention will fall into the rights of the present invention. within the scope of the requirements.

references:

1. Cooper G 4th. Basic determinants of myocardial hypertrophy: a review of molecular mechanisms. Annu Rev Med, 1997, 48: 13-23.

2. Aoki H, Sadoshima J, Izumo S. Myosin light chain kinase mediates arcomere organization during cardiac hypertrophy in vitro. Nat Med2000, 6(2): 183-188.

3. McKinsey TA, Kass DA. Small-molecule therapies for cardiohypertrophy: moving beneath the cell surface. Nat Rev Drug Discov. 2007; 6(8): 617-635.

4. Mitchell JA, Ventura HO, Mehra MR. Early recognition and treatment of hypertensive heart disease. Curr Opin Cardiol. 2005; 20(4): 282-289.

5. Jalili T, Carlstrom J, Kim S, Freeman D, Jin H, Wu TC, Litwin SE, DavidSymons J. Quercetin-supplemented diets lower blood pressure and attenuate cardiac hypertrophy in rats with aortic constriction. J Cardiovasc Pharmacol. 2006; 47( ): 531-541.

6. Carlstrom J, Symons JD, Wu TC, Bruno RS, Litwin SE, Jalili T. Aquercetin supplemented diet does not prevent cardiovascular complications in spontaneously hypertensive rats. J Nutr. 2007; 137(3): 628-633.

7. Niizeki T, Takeishi Y, Kitahara T, et al. Diacylglycerol Kinase zetaRescues Galphaq-Induced Heart Failure in Transgenic Mice. Circ J. 2008; 72(2): 309-317.

8. Dorn GW 2nd, Tepe NM, Lorenz JN, Koch WJ, Liggett SB. Low-and high-level transgenic expression of β2-adrenergic receptors differentially affect cardiac hypertrophy and function in Gαq-overexpressing mice. ProcNatl 9 Acad 9 A US ( 11): 6400-6405.

9. Berenji K, Drazner MH, Rothermel BA, Hill JA. Does load-induced ventricular hypertrophy progress to systolic heart failure? Am J Physiol Heart Circ Physiol.2005;289(1):H8-H16.

sequence listing

<110> Li Xiaohui

 School of Pharmacy, Third Military Medical University of the Chinese People's Liberation Army

  Chongqing Zhaokang Lihui Pharmaceutical Technology Co., Ltd.

  Chongqing Qingyang Pharmaceutical Co., Ltd.

<120> Anti-myocardial remodeling polypeptide, its preparation method, preparation and application in the preparation of anti-myocardial remodeling drug

<130>DP1F080358ZX/CNSJY/YL

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<170>PatentIn version 3.3

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Claims (26)

1. the polypeptide shown in a SEQ ID NO:2.

2. the polypeptide shown in a SEQ ID NO:3.

3. the polypeptide shown in a SEQ ID NO:4.

4. the polypeptide shown in a SEQ ID NO:5.

5. the polypeptide shown in a SEQ ID NO:6.

6. the polypeptide shown in a SEQ ID NO:7.

7. the polypeptide shown in a SEQ ID NO:8.

8. the nucleotide sequence shown in the SEQ ID NO:12 of coding claim 1 described polypeptide.

9. the nucleotide sequence shown in the SEQ ID NO:13 of coding claim 2 described polypeptide.

10. the nucleotide sequence shown in the SEQ ID NO:14 of coding claim 3 described polypeptide.

11. the nucleotide sequence shown in the SEQ ID NO:15 of the described polypeptide of coding claim 4.

12. the nucleotide sequence shown in the SEQ ID NO:16 of the described polypeptide of coding claim 5.

13. the nucleotide sequence shown in the SEQ ID NO:17 of the described polypeptide of coding claim 6.

14. the nucleotide sequence shown in the SEQ ID NO:18 of the described polypeptide of coding claim 7.

15. recombinant vectors that contains the described nucleotide sequence of any one in claim 8-14.

16. recombinant vectors according to claim 15, is characterized in that, described recombinant vectors contains the T7 promotor.

17. recombinant vectors according to claim 16, is characterized in that, described recombinant vectors is to build and obtain by described nucleotide sequence being inserted in the pIVEX2.3MCS plasmid.

18. one kind contains in claim 1-7 the preparation of acceptable auxiliary material on the described polypeptide of arbitrary claim and pharmacopedics.

19. preparation according to claim 18, is characterized in that, described preparation is non-enteron aisle injection formulations.

20. the application of the described polypeptide of arbitrary claim in preparation treatment myocardial hypertrophy medicine in claim 1-7.

21. application according to claim 20, wherein said polypeptide be as active constituents of medicine, then be equipped with acceptable auxiliary material on pharmacopedics.

22. the preparation method of the described polypeptide of arbitrary claim in a claim 1-7, it comprises the following steps:

Carrying out polypeptide according to the described amino acid whose sequence of arbitrary claim in claim 1-7 on Peptide synthesizer synthesizes.

23. the preparation method of the described polypeptide of arbitrary claim in a claim 1-7, it comprises the following steps:

Corresponding nucleotide sequence and carrier are formed recombinant vectors;

Described recombinant vectors is transformed in host cell;

Induce the described polypeptide of described host cell expression;

Separate and obtain described polypeptide.

24. method according to claim 23, described carrier contains the T7 promotor.

25. method according to claim 24, described carrier are plasmid, described host cell is intestinal bacteria.

26. method according to claim 25, described plasmid are pIVEX2.3MCS, described intestinal bacteria are BL21.

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