Selenium modification-based myocardial protection polypeptide as well as preparation method and application thereof

Abstract

The invention belongs to the field of active peptides, and particularly relates to selenium modification-based myocardial protection polypeptide as well as a preparation method and application thereof, and the selenium modification-based myocardial protection polypeptide is prepared by chelating Elabela-Apelin-TAT fusion protein as shown in SEQ ID NO: 6 and sodium selenite (Na2SeO3); the selenium peptide chelate has the effects of remarkably relieving myocardial injury, reducing the area of a myocardial infarction region, improving the oxidation resistance, inhibiting inflammatory response and resisting myocardial fibrosis for a long time, and can be used for treating and / or preventing acute myocardial infarction diseases.

Description

Technical Field

[0001] The present invention belongs to the field of bioactive peptides, and particularly relates to a selenium-modified myocardial protection polypeptide, a preparation method thereof, and an application thereof. Background Art

[0002] Cardiovascular diseases, especially acute myocardial infarction (AMI), are one of the main causes of sudden death in patients worldwide. The occurrence of myocardial infarction is mainly caused by coronary artery obstruction, leading to ischemia and hypoxia of cardiomyocytes, and then triggering apoptosis, inflammatory response, and fibrosis. At present, the treatment methods for AMI mainly include thrombolytic therapy, interventional therapy, and drug therapy. However, while these treatment methods restore myocardial blood supply, they may cause ischemia-reperfusion injury, exacerbate oxidative stress and inflammatory responses, and ultimately affect myocardial repair and functional recovery. Therefore, developing new treatment strategies that can effectively protect the myocardium and reduce ischemia-reperfusion injury has important clinical value.

[0003] Bioactive peptides have shown good application prospects in the treatment of cardiovascular diseases. Among them, Elabela and Apelin, as endogenous peptides, have been proven to play important roles in regulating cardiovascular function, protecting cardiomyocytes, and reducing myocardial fibrosis. In addition, the TAT (trans-activator of transcription) peptide segment can improve the cell penetration ability of peptides, enhance their stability and efficiency in vivo. However, there are still certain limitations in using these peptides alone, such as fast metabolic rate, poor in vivo stability, and not significant enough activity. Therefore, how to improve their biological activity and clinical application value through chemical modification or structural optimization is one of the current research focuses.

[0004] Selenium (Se) is an essential trace element with strong antioxidant and anti-inflammatory capabilities. It can reduce oxidative stress injury by regulating the activities of antioxidant enzymes such as glutathione peroxidase (GPx) and thioredoxin reductase (TrxR). In addition, selenium can also play a myocardial protection role by inhibiting the release of pro-inflammatory factors and reducing the inflammatory response. In recent years, selenium modification technology has been widely used in the functional optimization of proteins and peptides to enhance their biological activity and stability. Therefore, introducing selenium into the structure of myocardial protection peptides is expected to synergistically play myocardial protection roles such as antioxidant, anti-inflammatory, and anti-fibrotic effects, further improving their therapeutic effects.

[0005] Based on the above background, the present invention provides a selenium-modified myocardial protective polypeptide, its preparation method and application. This polypeptide is prepared by chelating Elabela-Apelin-TAT fusion protein with sodium selenite (Na2SeO3), combining the synergistic effects of Elabela and Apelin in cardiovascular protection, and enhancing its antioxidant and anti-inflammatory properties by using selenium modification technology. The selenium-peptide chelate of the present invention can significantly reduce myocardial injury, decrease the area of myocardial infarction, and improve antioxidant capacity, providing an innovative strategy for the adjuvant treatment and prevention of acute myocardial infarction. Summary of the Invention

[0006] To solve the above problems, the present invention first provides a selenium-modified myocardial protective polypeptide, which is prepared by chelating Elabela-Apelin-TAT fusion protein with inorganic selenium. This polypeptide has a significant myocardial protective effect, can reduce myocardial injury, decrease the area of myocardial infarction, and improve antioxidant capacity and inhibit inflammatory response.

[0007] In some embodiments, the Elabela-Apelin-TAT fusion protein is selected from the polypeptide shown in SEQ ID NO:6. The inorganic selenium is preferably sodium selenite (Na2SeO3), and the selenium element is combined with the Elabela-Apelin-TAT fusion protein through a chelation reaction to obtain a selenium-modified myocardial protective polypeptide chelate.

[0008] The present invention also provides a preparation method, which comprises the following steps:

[0009] 1) Provide the Elabela-Apelin-TAT fusion protein shown in SEQ ID NO:6;

[0010] 2) Mix the Elabela-Apelin-TAT fusion protein with sodium selenite (Na2SeO3);

[0011] 3) Under specific reaction conditions, make the Elabela-Apelin-TAT fusion protein and sodium selenite undergo a chelation reaction to prepare a selenium-modified myocardial protective polypeptide chelate.

[0012] Finally, the present invention provides an application, which is that the selenium-modified myocardial protective polypeptide can be used to prepare a drug for treating and / or preventing acute myocardial infarction diseases. By regulating antioxidant stress and inflammatory response, this selenium-modified polypeptide can effectively improve myocardial injury and decrease the area of myocardial infarction, and has a good myocardial protective effect.

[0013] In some embodiments, the selenium-modified cardioprotective polypeptide can be used as a single pharmaceutical preparation or in combination with other drugs to further enhance the therapeutic effect.

[0014] Compared with the prior art, the present invention has at least the following beneficial effects:

[0015] Significantly improve myocardial injury: The selenium-modified cardioprotective polypeptide can significantly reduce myocardial injury caused by acute myocardial infarction.

[0016] Reduce the area of myocardial infarction: The polypeptide can effectively reduce the area of myocardial infarction and improve cardiac function.

[0017] Improve antioxidant capacity: By introducing selenium, the polypeptide chelate can improve antioxidant capacity, thereby reducing the damage of oxidative stress to cardiomyocytes.

[0018] Inhibit inflammatory response: The polypeptide of the present invention can significantly inhibit the inflammatory response triggered during myocardial infarction and further protect myocardial tissue.

[0019] Anti-myocardial fibrosis: The polypeptide of the present invention can significantly reduce myocardial collagen deposition, inhibit the excessive production of type I collagen, and improve myocardial fibrosis.

[0020] Safety and stability: The selenium-modified polypeptide has good biocompatibility and stability, and is suitable for clinical application.

[0021] The present invention first chelates the Elabela-Apelin-TAT fusion protein with selenium to prepare a polypeptide with cardioprotective function, providing a new and effective adjuvant treatment method for the treatment and prevention of acute myocardial infarction. Brief Description of the Drawings

[0022] Figure 1 Ni-NTA column peak diagram and SDS-PAGE detection of the Elabela-Apelin-TAT fusion protein.

[0023] Figure 2 Potential analysis of the Elabela-Apelin-TAT fusion protein and its selenium chelate.

[0024] Figure 3 Cardiac function evaluation of the Elabela-Apelin-TAT fusion protein and its selenium chelate in the MI / RI mouse model.

[0025] Figure 4 Determination and analysis of myocardial infarction area of the Elabela-Apelin-TAT fusion protein and its selenium chelate in the MI / RI mouse model.

[0026] Figure 5 Analysis of the antioxidant and anti-inflammatory abilities of Elabela-Apelin-TAT fusion protein and its selenium chelate in a mouse model of MI / RI. Detailed implementation manners

[0027] To make the technical problems, technical solutions and advantages to be solved by the present invention clearer, the following will be described in detail with reference to the accompanying drawings and specific embodiments.

[0028] Example 1 Preparation of Elabela-Apelin-TAT fusion protein

[0029] According to the sequence recorded in NCBI for ELABELA [Homo sapiens] (GenBank: AHW47894.1): MRFQQFLFAFFIFIMSLLLISGQRPVNLTMRRKLRKHNCLQRRCMPLHSRVPFP (SEQ ID NO: 1);

[0030] According to the sequence recorded in NCBI for apelin [Homo sapiens] (GenBank: AAF25815.1): MNLRLCVQALLLLWLSLTAVCGGSLMPLPDGNGLEDGNVRHLVQPRGSRNGPGPWQGGRRKFRRQRPRLSHKGPMPF (SEQ ID NO: 2);

[0031] TAT cell-penetrating peptide: YGRKKRRQRRR (SEQ ID NO: 3);

[0032] Design the Elabela-Apelin-TAT fusion protein: construct the Elabela-linker-Apelin-linker-TAT module, where the TAT cell-penetrating peptide: YGRKKRRQRRR (SEQ ID NO: 4); linker: GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 5), and its amino acid sequence is as follows:

[0033] MRFQQFLFAFFIFIMSLLLISGQRPVNLTMRRKLRKHNCLQRRCMPLHSRVPFPGGGGSGGGGSGGGGSGGGGSMNLRLCVQALLLLWLSLTAVCGGSLMPLPDGNGLEDGNVRHLVQPRGSRNGPGPWQGGRRKFRRQRPRLSHKGPMPFGGGGSGGGGSGGGGSGGGGSYGRKKRRQRRR (SEQ ID NO: 6).

[0034] GenScript Biotech Corporation synthesized the recombinant expression plasmid pET-28a(+)-Elabela-Apelin-TAT, prepared competent Escherichia coli BL21(DE3), and mixed it with the recombinant expression plasmid pET-28a(+)-Elabela-Apelin-TAT. Transformation was carried out by heat shock method. After transformation, the bacteria were inoculated into LB medium containing ampicillin and cultured overnight with shaking at 37°C. Escherichia coli colonies successfully transformed were selected by ampicillin. Positive clones were picked and cultured on a small scale, and plasmids were extracted using a plasmid extraction kit for verification. The selected positive clones were inoculated into LB medium containing ampicillin (50 μg / mL) and cultured with shaking at 37°C until the optical density (OD600) of the culture broth reached 0.6 - 0.8, which is the optimal induction time for protein expression; when the culture broth reached the appropriate OD600 value, 1 mM isopropyl-β-D-thiogalactoside (IPTG) was added to induce the expression of the fusion protein, and the culture was continued for 2 - 6 hours; after induction, the culture was continued at 37°C, and attention was paid to keeping the rotation speed of the shaker within a reasonable range to ensure uniform growth of the bacteria. After induction, the bacteria expressing the protein were harvested. The specific operation was as follows: The bacterial liquid was centrifuged at a speed of 4000×g for 10 minutes. The supernatant was discarded, and the cell pellet was retained. The cell pellet was resuspended with lysis buffer containing PBS buffer (pH 7.4). The cells were ultrasonically lysed using an ultrasonic cell disruptor, and the power and time of the ultrasonic wave needed to be adjusted according to the amount of bacteria, usually carried out at 0°C - 4°C to reduce protein degradation. After lysis was completed, cell debris was removed by centrifugation, and the supernatant was taken for subsequent purification. Based on the fact that the fusion protein carried a 6×His tag, affinity chromatography was used for purification, and the operation was as follows: Ni-NTA column was used for affinity chromatography. First, the column was washed with PBS buffer (pH 7.4) to remove impurities. It was pre-washed with PBS buffer containing 20 mM imidazole to remove unbound miscellaneous proteins. The cell lysate containing the fusion protein was loaded onto the Ni-NTA affinity column and incubated for 30 - 60 minutes to allow the His tag to bind to the Ni-NTA resin. Non-specifically bound miscellaneous proteins were removed by draining and elution. Gradient elution was carried out with PBS buffer containing 20 mM, 50 mM, 100 mM, and 250 mM imidazole. Each elution fraction was collected and analyzed by SDS-PAGE to confirm the amount of protein in each fraction. The Elabela-Apelin-TAT fusion protein in the fraction was further separated and purified by an AKTA purifier to meet the requirements of subsequent experiments, as shown in Figure 1 .

[0035] Figure 1The results showed that the molecular weight of the Elabela-Apelin-TAT fusion protein was about 19.24 kDa, which met the expectations, and the protein purity was high enough for subsequent bioactivity experiments.

[0036] Example 2 Preparation and Identification of Selenium Chelate of Elabela-Apelin-TAT Fusion Protein

[0037] The purified Elabela-Apelin-TAT fusion protein was dissolved in PBS buffer solution at a concentration of 6 mg / mL, ensuring that the pH value was 7.0 to guarantee the stability of the protein. Then, an appropriate amount of sodium selenite (Na2SeO3) was taken and prepared into a sodium selenite solution with a concentration of 2 mM using deionized water, and the pH was adjusted to 7.0 to improve the solubility and reactivity of sodium selenite. The sodium selenite solution was slowly added dropwise to the Elabela-Apelin-TAT fusion protein solution while maintaining gentle stirring to make the sodium selenite evenly distributed and react with the protein. The reaction was carried out at a temperature of 20 - 25 °C for 24 hours. After the reaction, the mixed solution was transferred to a dialysis bag (800 Da molecular weight cut-off) and placed in a container containing buffer for dialysis to remove the unreacted sodium selenite. The dialysis process lasted for 24 hours, and the dialysis solution was changed every 4 hours using deionized water. After dialysis, the obtained protein selenium complex was subjected to freeze-drying treatment, and finally, the Elabela-Apelin-TAT fusion protein selenium chelate powder was obtained and stored at -20 °C.

[0038] The selenium content was determined by the 3,3′-diaminobenzidine colorimetric method: The chelate sample was mixed with 3,3′-diaminobenzidine (DAB) solution and reacted in an acidic environment. Selenium reacts with DAB under acidic conditions to form a dark blue complex, which has an obvious absorption peak at a specific wavelength. By measuring the absorbance of this absorption peak, the selenium content in the sample can be calculated according to the standard curve. The selenium chelation rate was calculated according to formula (1):

[0039] Selenium chelation rate (%) = (A1 / A2) × 100% (1)

[0040] Where: A1 is the total amount of selenium in the Elabela-Apelin-TAT fusion protein selenium chelate in grams; A2 is the total amount of selenium in the reaction system in grams; The results showed that the selenium chelation rate was 73 + 0.32%.

[0041] Potential analysis:

[0042] The potential changes of the Elabela-Apelin-TAT fusion protein and its selenium chelate were measured by a Nano-2sZEN 3600 nanoparticle size potentiometer to evaluate whether the chelation reaction was successful. 1.0 mg of the Elabela-Apelin-TAT fusion protein and the selenium chelate of the Elabela-Apelin-TAT fusion protein were separately dissolved in 1 mL of deionized water at a concentration of 1.0 mg / mL. After reacting at 37 °C for 10 minutes, the potential was measured using a potentiometer. Each sample was measured 3 times and the average value was taken to compare the potential differences. If the potential changed significantly, it indicated that selenium was successfully chelated with the Elabela-Apelin-TAT fusion protein; if the change was small, the chelation reaction was not completed, see Figure 2 .

[0043] Figure 2 The results showed that the zeta potential value of the selenium chelate of the Elabela-Apelin-TAT fusion protein was higher than that of the Elabela-Apelin-TAT fusion protein, indicating that after chelation, the negative charge on the surface of the fusion protein was significantly reduced. That is, the potential measurement results showed a significant difference in the potential value between the selenium chelate and the Elabela-Apelin-TAT fusion protein solution, indicating that selenium was successfully chelated with the Elabela-Apelin-TAT fusion protein.

[0044] Example 3 Experiment of the Elabela-Apelin-TAT fusion protein and its selenium chelate in a mouse model of acute myocardial infarction reperfusion injury (MI / RI)

[0045] Forty 8-week-old male C57 mice with a body weight of 18.5 ± 1.5 g were purchased from Xi'an Zhongkai Experimental Animal Co., Ltd. (Xi'an, China). Normal mouse food was purchased from Jiangsu Xietong Biotechnology Co., Ltd. The animals were housed in the SPF-class experimental animal room of Hubei University of Medicine. This experiment was recognized and approved by the Animal Ethics Committee of Hubei University of Medicine. To prevent the influence of selenium from drinking water on the experiment, all mice drank ddH2O (deionized water) during the experiment;

[0046] Animal grouping: The mice that had been adaptively fed for 1 week were randomly divided into 4 groups and allowed to drink water freely, denoted as the Sham group, the MI / RI model group, the fusion protein group, and the protein selenium chelate group;

[0047] Sham group: Four weeks before modeling, normal mouse chow at 300 mg / kg per day; 30 minutes after the construction of the MI / RI model, samples were taken for detection 24 hours after myocardial ischemia-reperfusion injury. After respiration stabilized, the heart was exposed. A 6-0 silk thread was passed under the junction of the pulmonary artery conus and the left atrial appendage without ligating the left anterior descending coronary artery to exclude the influence of surgical operations (such as thoracotomy, heart exposure, threading, etc.) on the experimental results itself, ensuring that the observed effects originated from ischemia-reperfusion injury or drug intervention, rather than surgical trauma.

[0048] MI / RI model group: Four weeks before modeling, normal mouse chow at 300 mg / kg per day; 30 minutes after the construction of the MI / RI model, samples were taken for detection 24 hours after myocardial ischemia-reperfusion injury. After respiration stabilized, the heart was exposed. The left anterior descending coronary artery was ligated with a 6-0 silk thread under the junction of the pulmonary artery conus and the left atrial appendage for 30 minutes, and then the ligature was loosened to restore blood flow.

[0049] Fusion protein group: Four weeks before modeling, normal mouse chow at 300 mg / kg per day + Elabela-Apelin-TAT fusion protein at 5 mg / kg (intraperitoneal injection); 30 minutes after the construction of the MI / RI model, samples were taken for detection 24 hours after myocardial ischemia-reperfusion injury. The modeling method and evaluation criteria were the same as those in the MI / RI model group.

[0050] Protein selenium chelate group: Starting four weeks before modeling, normal mouse chow at 300 mg / kg per day + Elabela-Apelin-TAT fusion protein selenium chelate at 5 mg / kg (intraperitoneal injection); 30 minutes after the construction of the MI / RI model, samples were taken for detection 24 hours after myocardial ischemia-reperfusion injury. The modeling method and evaluation criteria were the same as those in the MI / RI model group.

[0051] Construction of the acute myocardial infarction ischemia-reperfusion injury (MI / RI) model: C57 mice were anesthetized with isoflurane gas, fixed, shaved, and disinfected. A longitudinal incision was made in the neck, and a small animal ventilator (SA430) was connected to adjust the respiratory rate to 80 breaths per minute with a respiratory ratio of 1:1. After respiration stabilized, a 1-cm longitudinal incision was made at the obvious pulsation point on the left margin of the sternum to expose the heart layer by layer. The left anterior descending coronary artery was quickly ligated with a 6-0 silk thread under the junction of the pulmonary artery conus and the left atrial appendage. When the local myocardium turned white, its activity weakened and the ECG showed an ST-segment elevation greater than 0.1 mv or the appearance of a pathological Q wave, indicating the successful establishment of the acute myocardial infarction (AMI) model; the heart was returned to the chest cavity, and the ligature was loosened 30 minutes later. When the white myocardium turned rosy, it indicated the restoration of myocardial blood supply. Suture layer by layer and pay attention to maintaining negative thoracic pressure.

[0052] Cardiac function assessment: Six hours after the mice in each group woke up after the operation, color Doppler echocardiography was performed. The echocardiogram was carried out using a Vivid7 echocardiography PRO system equipped with a water-medium linear array probe of 13-14 MHz. The biplane Simpson method (biplane bi-ventricular volume method) was used to calculate the LVEF by measuring the left ventricular end-diastolic volume (LVEDV) and end-systolic volume (LVESV).

[0053]

[0054] Images of the short-axis section of the heart were obtained through the chest wall probe, and the left ventricular end-diastolic (LVIDd) and end-systolic internal diameters (LVIDs) were measured to calculate the LVFS.

[0055]

[0056] The results of the left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) in each group are shown in Figure 3 .

[0057] Figure 3 The results showed that compared with the Sham group, the left ventricular ejection fraction (LVEF) (P<0.01) and left ventricular fractional shortening (LVFS) in the MI / RI model group decreased more significantly (P<0.05); compared with the MI / RI model group, the left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) in the fusion protein group and the protein selenium chelate group were significantly improved; and the improvement effect of the left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) in the protein selenium chelate group was better than that in the fusion protein group (P<0.01), indicating that the Elabela-Apelin-TAT fusion protein selenium chelate could significantly reduce myocardial injury.

[0058] Measurement of myocardial infarction area: After the modeling was completed 24 hours after reperfusion in each group of mice, the hearts were removed, washed with PBS, taken out after being frozen in an -80°C refrigerator for 30 minutes, continuously cut into slices about 2 mm thick from the apex to the base of the heart, and the slices were placed in a 2% TTC solution and preheated at 37°C; placed in a centrifuge tube containing 10% formaldehyde overnight, and the heart slices were photographed in sequence. The ImageJ software was used to analyze the myocardial infarction area. The red area represents the area of non-infarcted myocardial tissue, and the white area represents the infarcted myocardium. Myocardial infarction area (%) = (infarcted myocardial area / left ventricular area) × 100%, see Figure 4 ;

[0059] Figure 4The results showed that compared with the Sham group, the infarct area in the MI / RI model group was larger; compared with the MI / RI model group, the infarct area in the fusion protein group and the protein selenium chelate group was significantly reduced, and the infarct area in the protein selenium chelate group was reduced most significantly (P<0.01).

[0060] Biochemical index detection: The oxidative damage and antioxidant capacity of cardiomyocytes were evaluated by detecting the oxidative stress indexes SOD and GSH in serum through qPCR; at the same time, the cytokine level indexes TNF-α and IL-6 were detected to reflect the ability to inhibit the inflammatory response of the heart, as shown in Figure 5 .

[0061] Figure 5 The results showed that compared with the Sham group, the expression levels of SOD (P<0.01) and GSH (P<0.05) in the MI / RI model group were significantly reduced; compared with the MI / RI model group, the expression levels of SOD and GSH in the fusion protein group and the protein selenium chelate group were significantly increased, and the antioxidant capacity of the protein selenium chelate group was the strongest; compared with the Sham group, the expression levels of TNF-α and IL-6 in the MI / RI model group were significantly increased, and compared with the MI / RI model group, the expression levels of TNF-α and IL-6 in the fusion protein group and the protein selenium chelate group were significantly reduced, and the protein selenium chelate group had a significant ability to inhibit the inflammatory response of the heart.

[0062] Long-term anti-myocardial fibrosis detection: Based on histopathological staining technology, Masson trichrome staining was performed on the myocardial infarction area and surrounding tissues of mice in each group at 8 weeks after surgery. Collagen fibers (blue), cardiomyocytes (red), and cell nuclei (black) were distinguished by staining, and the proportion of collagen fibers in myocardial tissue was quantified. At least 5 non-overlapping fields of view (per sample) in the infarct border area were collected under a microscope (10× or 20× objective lens). Images were saved using a high-resolution color CCD camera (to avoid overexposure), and finally quantitative analysis was performed (using ImageJ software). Open the image → Image → Color → Split Channels to separate the blue channel (collagen fibers); Process → Binary → Make Binary to binarize the image (collagen is black and the background is white); Analyze → Measure to calculate the percentage of the blue area in the total field of view area (i.e., CVF), as shown in Table 1.

[0063] Formula:

[0064]

[0065] Table 1 Results of long-term anti-myocardial fibrosis detection in mice of each group

[0066] Group Collagen volume fraction (CVF, %) Sham group 2.1±0.3 MI / RI model group 38.5±4.2** Fusion protein group <![CDATA[25.7±3.1 # > Protein selenium chelate group <![CDATA[16.4±2.6 ## >

[0067] Note: **p < 0.01 vs Sham group; #p < 0.05, ##p < 0.01 vs MI / RI model group.

[0068] Table 1 results showed that both the fusion protein group and the protein-selenium chelate group could significantly reduce the collagen volume fraction in the acute myocardial infarction reperfusion injury (MI / RI) model of mice, and the long-term anti-myocardial fibrosis effect of the protein-selenium chelate group was better than that of the fusion protein group.

[0069] The above is the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle described in the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A selenium-modified myocardial protective polypeptide, characterized in that: The polypeptide is prepared by chelating Elabela-Apelin-TAT fusion protein with inorganic selenium.

2. The polypeptide according to claim 1, characterized in that The Elabela-Apelin-TAT fusion protein is selected from the polypeptide shown in SEQ ID NO:

6.

3. The polypeptide according to claim 1 or 2, characterized in that The inorganic selenium is selected from sodium selenite (Na2SeO3).

4. A method for preparing a selenium-modified myocardial protective polypeptide, characterized in that: The Elabela-Apelin-TAT fusion protein as shown in SEQ ID NO: 6 is subjected to a chelation reaction with selenium, wherein sodium selenite (Na2SeO3) is used as a selenium source in the chelation reaction.

5. Use of the polypeptide according to any one of claims 1 to 3 in the preparation of a medicament for treating and / or preventing acute myocardial infarction.

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