Novel polypeptide and application thereof

Abstract

The invention provides a polypeptide. The amino acid sequence of the polypeptide is shown as SEQ ID NO. 1. Specifically, the sequence of the polypeptide is as follows: MTSRQEDASGKKTTEGAKFQGS. The invention also provides a DNA (deoxyribonucleic acid) molecule for coding the polypeptide according to the claim 1. The invention also provides a recombinant vector containing the DNA molecule. The invention also provides application of the polypeptide in preparation of medicines for treating myocardial fibrosis. The invention further provides a pharmaceutical composition, and the effective component of the pharmaceutical composition is the polypeptide. Experiments prove that the traditional Chinese medicine can relieve myocardial fibrosis.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to a polypeptide, specifically a novel polypeptide and its use. Background Art

[0002] Heart failure (HF), the terminal stage of various heart diseases, not only severely impacts patients' quality of life but can also lead to malignant arrhythmias and even sudden death. Its prevalence is increasing due to the aging of the global population, the improved effectiveness of treatment, and the survival rates of HF patients. Recent projections indicate that the prevalence of HF will increase by approximately 46% from 2012 to 2030, making it a serious global public health issue. Current clinical treatments for HF primarily include medication, interventional therapy, and heart transplantation. However, these approaches all have varying degrees of limitations, resulting in a high five-year mortality rate of approximately 50% for patients with chronic HF. Therefore, identifying safer and more effective strategies for the prevention and treatment of HF is of great significance.

[0003] Myocardial fibrosis leads to systolic and diastolic dysfunction in many cardiac pathophysiological conditions. Previous studies have shown that: (1) Alamandine alleviates cardiac dysfunction and reduces cardiac fibrosis by inhibiting the increase in collagen I, α-SMA, and TGF-β in neonatal rat cardiac fibroblasts induced by glucose deprivation (OGD). (2) HIMF induces myocardial fibrosis by activating the MAPK and CaMKII-STAT3 pathways, mediating paracrine effects from cardiomyocytes to fibroblasts. (3) AMFR catalyzes the ubiquitination of K27- and K33-linked FAM134B, enhancing endoplasmic reticulum phagocytic flux and thereby inhibiting the phosphorylation of mTORC1 downstream targets such as S6K1 and 4E-BP, thereby suppressing myocardial fibrosis.

[0004] The beneficial effects of long-term, regular exercise on the cardiovascular system have become a consensus in the industry. In recent years, with the development of relevant clinical research, the beneficial effects of exercise on the prevention and treatment of heart failure have received increasing attention. Studies have shown that regular and moderate exercise can reduce myocardial oxidative stress, activate VEGFR, increase myocardial angiogenesis, prevent cardiac remodeling after myocardial infarction in vivo, and improve myocardial diastolic and systolic function. It not only effectively reduces the risk of heart failure, but also reduces all-cause mortality and hospitalization rates in heart failure patients and significantly improves patient prognosis. Currently, an increasing number of studies have found that exercise, as a safe physiological stimulus, has a beneficial preventive and therapeutic effect on heart failure.

[0005] Studies have shown that peptides, due to their unique biological properties, are of great value in regulating human physiological functions and in clinical diagnosis and treatment, and have now become one of the hottest areas in biopharmaceutical R&D and clinical research. As an ideal candidate molecule for drug development, peptides have significant advantages such as minimal toxicity and side effects, weak immunogenicity, high safety, strong specificity, and environmentally friendly production processes. Currently, peptide drugs have demonstrated good clinical efficacy in the treatment of multiple diseases. Based on the above background, through systematic design and screening, we have successfully identified a peptide with a significant inhibitory effect on myocardial fibrosis. This invention provides a new intervention strategy for the prevention and treatment of myocardial fibrosis. Summary of the Invention

[0006] In response to the above technical problems in the prior art, the present invention provides a novel polypeptide and its use, which aims to solve the technical problem that the drugs in the prior art are not effective in treating myocardial fibrosis.

[0007] The present invention provides a polypeptide, the amino acid sequence of which is shown as SEQ ID NO.1.

[0008] Specifically, the sequence of the polypeptide is: MTSRQEDASGKKTTEGAKFQGS.

[0009] The present invention also provides a DNA molecule encoding the polypeptide according to claim 1.

[0010] The present invention also provides a recombinant vector containing the above DNA molecule.

[0011] The present invention also provides a host cell comprising the above-mentioned recombinant vector.

[0012] The present invention also provides an expression system, which contains the above-mentioned recombinant vector, or a genome in which the above-mentioned exogenous DNA molecule is integrated.

[0013] The present invention also provides use of the above polypeptide in preparing a drug for treating myocardial fibrosis.

[0014] The present invention also provides a pharmaceutical composition, characterized in that its active ingredient is the above-mentioned polypeptide.

[0015] Furthermore, the pharmaceutical composition also contains pharmaceutical excipients.

[0016] Furthermore, the pharmaceutical composition is in the form of tablets, capsules or injections.

[0017] Compared to existing polypeptides for treating myocardial fibrosis, the polypeptide of the present invention is secreted in large quantities under physiological conditions following exercise. Therefore, the polypeptide has no significant adverse effects on the organism. In normal organisms and those in the early stages of the disease, myocardial fibrosis can be alleviated by increasing the amount of exercise. In the middle and late stages of the disease, myocardial fibrosis can be alleviated by in vitro administration of the polypeptide, especially under conditions of restricted exercise. Experiments have demonstrated that the present invention can alleviate myocardial fibrosis.

[0018] Compared with existing technologies, the present invention has a positive and significant technical effect. Compared with existing drugs for treating myocardial fibrosis, the polypeptide of the present invention has fewer side effects, lower immunogenicity, and lower energy consumption for production. Moreover, the polypeptide of the present invention can cover patients with multiple risk factors, enabling better management and treatment of patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The control changes after drug treatment are shown: A. left ventricular ejection fraction of mouse heart; B. fractional shortening of mouse heart; C. left ventricular end-diastolic diameter of mouse heart; D. left ventricular end-systolic diameter of mouse heart; E. left ventricular end-diastolic volume of mouse heart; F. left ventricular end-systolic volume of mouse heart; G. left ventricular mass of mouse heart; H. systolic blood pressure level of mouse; I. diastolic blood pressure level of mouse.

[0020] Figure 2 The homology of the polypeptide of the present invention in vertebrates is shown. DETAILED DESCRIPTION

[0021] The reagents or instruments used in the examples of the present invention without indicating the manufacturer are all conventional reagent products that can be obtained through commercial purchase.

[0022] Example 1 Screening of polypeptides

[0023] We constructed an endurance exercise mouse model using endurance exercise characterized by low-intensity, long-term training. Eight-week-old C57BL / 6 male mice were used as research subjects to construct the endurance exercise model. The mice first underwent adaptive exercise training by running at a speed of 10 meters / minute for 5 minutes every day for 2 days. Then, the mice started running exercise training from an initial speed of 10 meters / minute every day, and the speed was increased by 1 meter / minute every 20 minutes until exhaustion. The exercise training lasted for 14 days.

[0024] Normally fed, non-exercised mice served as the control group.

[0025] After modeling, heart tissues of mice in each group were collected for peptide bioinformatics analysis. We screened the sequencing results based on Log2 (fold change) > 1 and P < 0.05 and found a peptide (Mus_musculus / 1-22) whose expression was significantly increased after exercise. The function and mechanism of this peptide have not been reported yet. The peptide encoded by the amino acid sequence has high homology in multiple vertebrates such as humans, mice, and rats ( Figure 2 ), showing evolutionary conservation, suggesting that it may have important biological functions.

[0026] The sequence of the polypeptide is: MTSRQEDASGKKTTEGAKFQGS.

[0027] Example 2 Preparation of polypeptide

[0028] The polypeptide described in Example 1 was prepared using conventional methods, specifically as follows:

[0029] Peptides are synthesized from the C-terminus to the N-terminus.

[0030] 1. Weigh 3 g of RINK resin (degree of substitution 0.3 mmol / g) into a 150 ml reactor and soak it with 50 ml of dichloromethane (DCM).

[0031] After 2.2 hours, the resin was washed with nitrogen-dimethylformamide (DMF) 3 times the volume of the resin, and then dried. This was repeated four times. The resin was dried and set aside for use.

[0032] 3. Add a certain amount of 20% piperidine (piperidine / DMF) to the reactor and shake on a decolorization shaker for 20 minutes to remove the Fmoc protecting group from the resin. After deprotection, wash the resin four times with DMF (3 times the resin volume) and then drain.

[0033] 4. Take a small amount of resin and test it with the ninhydrin (nine-well hydrated ninhydrin) method (two drops each of test A and test B, react at 100℃ for 1 minute). If the resin has color, it means that the deprotection is successful.

[0034] 5. Weigh an appropriate amount of the first amino acid at the C-terminus and an appropriate amount of 1-hydroxybenzotriazole (HOBT) into a 50ml centrifuge tube, add 20ml of DMF to dissolve them, then add 3ml of N,N-diisopropylcarbodiimide (DIC) and shake for 1min. After the solution is clarified, add it to the reactor, and then place the reactor in a shaker at 30℃ for reaction.

[0035] After 6.2 hours, cap the resin with a certain amount of acetic anhydride (acetic anhydride: DIEA: DCM = 1:1:2) for half an hour, then wash it four times with DMF 3 times the volume of the resin and drain it for later use.

[0036] 7. Add a certain amount of 20% piperidine (piperidine / DMF = 1:4) to the reactor and shake on a decolorization shaker for 20 minutes to remove the Fmoc protecting group from the resin. After deprotection, wash the resin four times with DMF and then drain.

[0037] 8. Take a small amount of resin and test it using the ninhydrin (nine-well hydrated ninhydrin) method (two drops each of test A and test B, react at 100℃ for 1 minute). If the resin has color, it means that the deprotection is successful.

[0038] 9. Weigh an appropriate amount of the second amino acid and HOBT into a 50ml centrifuge tube, add 25ml of DMF to dissolve them, then add 2.5ml of DIC and shake for 1min. After the solution is clarified, add it to the reactor, and then place the reactor in a shaker at 30℃ for reaction.

[0039] After 10.1 hours, take a small amount of resin for testing using the ninhydrin method (two drops each of Test A and Test B, react at 100°C for 1 minute). If the resin is colorless, the reaction is complete; if the resin has color, the condensation is incomplete and the reaction should be continued.

[0040] 11. After the reaction is complete, wash the resin four times with DMF and drain. Add a certain amount of 20% piperidine (piperidine / DMF = 1:4) to the reactor and shake on a decolorization shaker for 20 minutes to remove the Fmoc protecting group from the resin. After deprotection, wash the resin four times with DMF and drain to check for protection.

[0041] 12. Follow steps 9-11 to connect the following amino acids.

[0042] 13. After the last amino acid is attached, remove the protection and wash the resin four times with DMF. Drain the resin with methanol. Cleave the peptide from the resin using a 95% cleavage buffer (trifluoroacetic acid: 1,2-ethanedithiol: 3, isopropylsilane: water = 95:2:2:1) (10 ml of cleavage buffer per gram of resin). Centrifuge four times with icy ether (cleavage buffer: ether = 1:9). Finally, purify the peptide using HPLC and lyophilize to obtain a desired purity.

[0043] 14. Purification conditions are as follows:

[0044] Stationary phase: C18 column (commercially available);

[0045] Mobile phase configuration:

[0046] PumpA: V(tfa) / V(water)=1 / 1000;

[0047] PumpB: V(TFA) / v(acetonitrile)=1 / 1000;

[0048] Flow rate: 10 ml / min;

[0049] Retention time: between 20-30 minutes.

[0050] Example 3 Application of polypeptides

[0051] (1) Experimental animals and groups:

[0052] Eight-week-old C57BL / 6 male mice were randomly divided into four groups (n=3):

[0053] Control group: conventional feeding without intervention;

[0054] Peptide group: peptide was injected into tail vein;

[0055] Ang II model group: Ang II modeling;

[0056] Ang II + peptide group: Ang II modeling + tail vein injection of peptide.

[0057] (2) Model building:

[0058] Myocardial fibrosis model was established by subcutaneously implanting 1.5 mg / kg / d Ang II mini-osmotic pump in the back of the neck. Peptide group and Ang II+peptide group were injected with peptide-associated virus through tail vein.

[0059] (3) Cardiac ultrasound examination:

[0060] After 4 weeks of intervention, cardiac function was assessed using a high-resolution small animal ultrasound imaging system (Fujifilm VisualSonics Vevo3100, probe frequency 30 MHz). Anesthesia was maintained with 1-2% isoflurane at a flow rate of 0.6-1 L / min. Electrocardiograms were monitored simultaneously (heart rate maintained at 450 ± 25 bpm). Left ventricular mass (LVMass, mg) and end-systolic / diastolic diameters (D; s, D; d, mm) were measured. Left ventricular end-systolic / diastolic volumes (V; s, V; d, μl), ejection fraction (EF%), and fractional shortening (FS%) were calculated.

[0061] (4) Blood pressure measurement:

[0062] Hemodynamic parameters: Systolic blood pressure (SBP, mmHg) and diastolic blood pressure (DBP, mmHg) were recorded using a non-invasive tail artery blood pressure measurement system (Softron Biotechnology).

[0063] (5) Results:

[0064] Compared with the control group, the cardiac function of the AngII model group mice showed typical pathological compensation characteristics. In terms of cardiac contractile function, the left ventricular ejection fraction (EF%) and fractional shortening (FS%) of the hearts of the AngII model group mice were significantly reduced ( Figure 1 A. Figure 1 B), The end-systolic diameter (D;s) and end-diastolic diameter (D;d) increased, accompanied by the expansion of the end-diastolic volume (V;d) and end-systolic volume (V;s) ( Figure 1 C. Figure 1 D. Figure 1 E. Figure 1 F), indicating decreased left ventricular pumping efficiency and impaired myocardial contractility; in terms of structural remodeling, the left ventricular mass (LVMass) was significantly increased ( Figure 1 G); In terms of arterial blood pressure, systolic blood pressure (SBP) and diastolic blood pressure (DBP) increased significantly ( Figure 1 H. Figure 1 I).

[0065] Compared with the AngII model group, the combined intervention group (Ang II + peptide) significantly improved the cardiac pathological process. The ejection fraction (EF%) and short axis shortening rate (FS%) of the combined intervention group (Ang II + peptide) were significantly improved compared with the AngII model group ( Figure 1 A. Figure 1 B), the end-systolic diameter (D;s) and end-diastolic diameter (D;d), end-diastolic volume (V;d) and end-systolic volume (V;s) were smaller than those in the AngII group ( Figure 1 C. Figure 1 D. Figure 1 E. Figure 1 F), indicating that left ventricular systolic function improved; in terms of structural remodeling, the left ventricular mass (LVMass) of the combined intervention group (Ang II + peptide) was significantly reduced ( Figure 1 G), in terms of arterial blood pressure, the systolic blood pressure (SBP) and diastolic blood pressure (DBP) of the combined intervention group (Ang II + peptide) were lower than those of the Ang II group ( Figure 1 H. Figure 1 I), all of the above indicate that peptides can effectively inhibit ventricular dilatation and pathological hypertrophy, lower blood pressure, and effectively reduce the risk of heart failure ( Figure 1 ).

Claims

1. A polypeptide, characterized in that Its amino acid sequence is shown in SEQ ID NO.

1.

2. A DNA molecule encoding the polypeptide of claim 1.

3. A recombinant vector, characterized in that It contains the DNA molecule according to claim 2.

4. A host cell, characterized in that Comprising the recombinant vector according to claim 3.

5. An expression system, characterized in that The expression system contains the recombinant vector according to claim 3, or the exogenous DNA molecule according to claim 2 integrated into the genome.

6. Use of the polypeptide according to claim 1 in the preparation of a medicament for treating myocardial fibrosis.

7. A pharmaceutical composition, characterized in that The active ingredient is the polypeptide according to claim 1.

8. A pharmaceutical composition according to claim 7, characterized in that The pharmaceutical composition also contains pharmaceutical excipients.

9. A pharmaceutical composition according to claim 7, characterized in that The dosage form of the pharmaceutical composition is tablet, capsule or injection.

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