Targeted selenium conjugated anti-inflammatory fusion protein and its application in repairing reperfusion injury of acute coronary syndrome

Abstract

The application discloses a kind of targeted selenium conjugated anti-inflammatory fusion protein and its application in acute coronary syndrome reperfusion injury repair, belong to biomedical technology field.The GPIb-Annexin A1 fusion protein is first constructed: human GPIb active fragment is used as N-terminal targeting element, human Annexin A1 active fragment is used as C-terminal anti-inflammatory element, is connected in series by (Gly4Ser) 3 flexible linker, and its fusion protein sequence is shown as SEQ ID NO:3, is obtained by gene synthesis, vector construction, prokaryotic expression and Ni-NTA purification;Then selenium methionine is used as selenium source, and is chelated with fusion protein in pH7.5 phosphate buffer at mass ratio 3:1, and selenium chelate is obtained after dialysis freeze-drying.Experiments prove that the selenium chelate can significantly improve the left ventricular ejection fraction, left ventricular fractional shortening of MI / RI mouse, reduce myocardial infarction area, inhibit the expression of TNF-alpha, IL-1beta, IL-6, and reduce long-term myocardial collagen volume fraction, and can be used for preparing acute coronary syndrome reperfusion injury repair drug.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the design and preparation method of a targeted selenium-coupled anti-inflammatory fusion protein, and the application of this fusion protein in the repair of acute coronary syndrome (ACS), especially acute myocardial infarction / reperfusion injury (MI / RI); more specifically, it relates to a fusion protein with platelet glycoprotein Ib (GPIb) active fragment as the targeting element and annexin A1 active fragment as the anti-inflammatory element, and its chelate with selenium, and the application of this chelate in improving cardiac function after myocardial reperfusion, reducing infarct size, inhibiting inflammatory response, and anti-myocardial fibrosis. Background Technology

[0002] Acute coronary syndrome (ACS) is one of the leading causes of cardiovascular death worldwide, with an annual incidence of approximately 150 per 100,000, according to the World Health Organization. Acute myocardial infarction (AMI) accounts for over 60% of these cases. Currently, the core clinical strategy for treating ACS is reperfusion therapy, including percutaneous coronary intervention (PCI) and intravenous thrombolysis, which can rapidly restore blood flow to the infarct-related vessels. However, clinical data shows that 30%-50% of patients undergoing reperfusion therapy develop myocardial reperfusion injury (MI / RI). Oxidative stress, neutrophil infiltration, and the massive release of pro-inflammatory cytokines (such as tumor necrosis factor-α, interleukin-1β, and interleukin-6) during reperfusion lead to further cardiomyocyte necrosis, irreversible decline in cardiac function, and ultimately heart failure, severely reducing long-term survival rates. Therefore, developing interventions that can target and repair MI / RI is a key clinical need in the cardiovascular field.

[0003] Existing interventions for myocardial infarction (MI) and respiratory inflammatory disease (RI) have significant limitations: traditional anti-inflammatory drugs (such as nonsteroidal anti-inflammatory drugs and glucocorticoids) can inhibit inflammatory responses, but they lack myocardial targeting, easily causing side effects such as gastrointestinal damage and blood glucose fluctuations, and cannot protect myocardial cells; single-target biologics (such as recombinant Annexin A1 and IL-1β antagonists) have clear anti-inflammatory activities, but Annexin A1 is difficult to accumulate in damaged myocardial areas, and insufficient targeting leads to limited efficacy, while IL-1β antagonists only target a single inflammatory factor and cannot block the multi-pathway damage mechanism of MI / RI; in addition, some studies have attempted to use platelet-targeting molecules (such as GPIb) to deliver drugs, but GPIb only has the ability to target damaged vascular endothelium and platelets, and has no anti-inflammatory activity itself. When used alone, it cannot achieve the synergistic effect of "targeted delivery + anti-inflammatory protection", which is difficult to meet the complex repair needs of MI / RI.

[0004] Selenium is an essential trace element for the human body. As a core cofactor of glutathione peroxidase, it exerts a cardioprotective effect by scavenging free radicals and inhibiting lipid peroxidation. However, existing selenium supplements (such as sodium selenite, selenium yeast, and selenomethionine) suffer from low bioavailability (only 10%-30% is absorbed after oral administration) and poor targeting, meaning that free selenium cannot specifically accumulate in damaged myocardium, and excessive intake can easily lead to selenium poisoning (such as hair loss and nerve damage). Therefore, how to integrate the three functions of "targeted delivery," "anti-inflammation," and "selenium-mediated antioxidant" to construct a MI / RI repair agent with high targeting, synergistic protective effects, and high safety is a gap that has not yet been filled in the existing technology, and it is also the core technical bottleneck that this invention aims to overcome. Summary of the Invention

[0005] To address the shortcomings of existing interventions for acute coronary syndrome (ACS) reperfusion injury (especially acute myocardial infarction reperfusion injury MI / RI), such as insufficient targeting, poor anti-inflammatory effects, and low selenium bioavailability, this invention provides the following technical solution: This invention first provides a GPIb-Annexin A1 fusion protein, designed based on the synergistic concept of "targeted delivery + anti-inflammatory protection": The active fragment of human platelet glycoprotein Ib (GPIb) (SEQ ID NO:1) is selected as the N-terminal targeting element. GPIb can specifically bind to adhesion molecules on the surface of damaged myocardial vascular endothelium, achieving targeted enrichment of the fusion protein in the MI / RI region. The active fragment of human annexin A1 (Annexin A1) (SEQ ID NO:2) is selected as the C-terminal anti-inflammatory element. Annexin A1 can block the inflammatory damage pathway of MI / RI by inhibiting neutrophil infiltration and downregulating the expression of pro-inflammatory factors (TNF-α, IL-1β). To avoid the two active fragments affecting function due to steric hindrance, a flexible linker (Gly4Ser) 3 is used to tandem the two elements, ultimately forming a fusion protein with the amino acid sequence shown in SEQ ID NO:3.

[0006] The present invention also provides a preparation method, which includes the following steps: firstly, synthesizing the fusion protein gene and cloning it into the pUC57 vector, digesting it with NcoⅠ / XhoⅠ and then ligating it into the pET-28a (+) vector to construct a recombinant expression vector; transforming the vector into BL21 (DE3) competent cells, and obtaining positive recombinant bacteria by kanamycin screening and sequencing verification; after inducing expression by IPTG, purifying the cells using Ni-NTA affinity chromatography.

[0007] Finally, this invention provides the application of the above-mentioned selenium-coupled anti-inflammatory fusion protein in the preparation of drugs for repairing acute coronary syndrome reperfusion injury.

[0008] Compared with the prior art, the present invention has at least the following beneficial effects: Highly targeted: The active fragment of GPIb enables the specific enrichment of fusion proteins into the MI / RI region, solving the problem of "systemic distribution and low local concentration" of traditional drugs; Significant synergistic protective effect: The anti-inflammatory activity of the fusion protein and the antioxidant activity of selenium work synergistically to simultaneously block the inflammatory damage and oxidative stress pathways of MI / RI, and the protective effect is better than that of the fusion protein or selenium alone; The preparation process is mature: it adopts prokaryotic expression and affinity chromatography purification, which is low in cost, high in yield, and easy to scale up industrially. Attached Figure Description

[0009] Figure 1 SDS-PAGE electrophoresis image of the GPIb-Annexin A1 fusion protein.

[0010] Figure 2A bar chart comparing the postoperative left ventricular ejection fraction (LVEF) and left ventricular shortening fraction (LVFS) in mice from different groups.

[0011] Figure 3 A bar chart comparing the myocardial infarction area of ​​mice in each group.

[0012] Figure 4 A bar chart comparing the qPCR detection levels of TNF-α expression in the serum of mice in each group.

[0013] Figure 5 A bar chart comparing the qPCR detection levels of IL-1β expression in the serum of mice in each group.

[0014] Figure 6 A bar chart comparing the qPCR detection levels of IL-6 expression in the serum of mice in each group.

[0015] Figure 7 Comparison of Masson trichrome staining and collagen volume fraction (CVF) of myocardial tissue in mice from different groups 8 weeks after surgery. Detailed Implementation

[0016] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with the accompanying drawings and specific embodiments.

[0017] Example 1: Preparation of GPIb-Annexin A1 fusion protein According to NCBI records, for platelet glycoprotein Ib beta chain precursor [Homosapiens] (NCBI Reference Sequence: NP_000398.1), the appropriate active fragment should be selected by trunculating the signal peptide: CPAPCSCAGTLVDCGRRGLTWASLPTAFPVDTTELVLTGNNLTALPPGLLDALPALRTAHLGANPWRCDCRLVPLRAWLAGRPERAPYRDLRCVAPPALRGRLLPYLAEDELRAACAPGPLCWGALAAQLALLGLGLLHALLLVLLLCRLRRLRARARAAARLSLTDPLVAERAGTDES (SEQ ID NO: 1); According to NCBI records, annexin A1 [Homo sapiens] (NCBI Reference Sequence: NP_000691.1) truncates the signal peptide to select an appropriate active fragment: VQTVKSSKGGPGSAVSPYPTFNPSSDVAALHKAIMVKGVDEATIIDILTKRNNAQRQQIKAAYLQETGKPLDETLKKALTGHLEEVVLALLKTPAQFDADELRAAMKGLGTDEDTLIEILASRTNKEIRDINRVYREELKRDLAKDITSDTSGDFRNALLSLAKGDRSEDFGVNEDLADSDARALYEAGERRKGTDVNVFNTILTTRSYPQLRRVFQKYTKYSKHDMNKVLDLELKGDIEKCLTAIVKCATSKPAFFAEKLHQAMKGVGTRHKALIRIMVSRSEIDMNDIKAFYQKMYGISLCQAILDETKGDYEKILVALCGGN (SEQ ID NO:2); Design of GPIb - Annexin A1 fusion protein: CPAPCSCAGTLVDCGRRGLTWASLPTAFPVDTTELVLTGNNLTALPPGLLDALPALRTAHLGANPWRCDCRLVPLRAWLAGRPERAPYRDLRCVAPPALRGRLLPYLAEDELRAACAPGPLCWGALAAQLALLGLGLLHALLLVLLLCRLRRLRARARARAAARLSLTDPLVAERAGTDESGGGGSGGGGSGGGGSVQTVKSSKGGPGSAVSPYPTFNPSSDVAALHKAIMVKGVDEATIIDILTKRNNAQRQQIKAAYLQETGKPLDETLKKALTGHLEEVVLALLKTPAQFDADELRAAMKGLGTDEDTLIEILASRTNKEIRDINRVYREELKRDLAKDITSDTSGDFRNALLSLAKGDRSEDFGVNEDLADSDARALYEAGERRKGTDVNVFNTILTTRSYPQLRRVFQKYTKYSKHDMNKVLDLELKGDIEKCLTAIVKCATSKPAFFAEKLHQAMKGVGTRHKALIRIMVSRSEIDMNDIKAFYQKMYGISLCQAILDETKGDYEKILVALCGGN (SEQID NO:3); Using the GPIb active fragment as the N-terminal element and the Annexin A1 active fragment as the C-terminal element, the two elements were tandemly linked by a flexible linker (Gly4Ser)3 to avoid steric hindrance affecting protein activity. The GPIb was chemically synthesized by Nanjing Genscript Biotech Co., Ltd., and then cloned into the pUC57 vector to obtain the recombinant cloning plasmid pUC57-GPIb-AnxA1. 10 μg of the synthesized recombinant cloning plasmid pUC57-GPIb-AnxA1-His and 10 μg of the HIS-tagged expression vector pET-28a (+) were added to each, along with Nco, XhoⅠ, and the corresponding buffer (5 μL). The mixture was then brought to a final volume of 50 μL with enzyme-free water and incubated at 37°C for 4 h to obtain GPIb-Annexin. The target gene fragment and the digested vector fragment of the A1 fusion protein were separated by agarose gel electrophoresis (1% agarose). The gel containing the target gene fragment and the digested vector fragment was excised under UV light. The fragments were recovered using a gel recovery kit (purchased from Omega) according to the instructions. After the concentration was determined, the gel was stored at -20℃ for later use. Take the enzyme-digested vector fragment (1 μg) and the GPIb-Annexin A1 fusion protein target gene fragment (3 μg), add T4 DNA ligase and ligation buffer (5 μL), and add enzyme-free water to make up to 50 μL. Ligate overnight at 16℃ to obtain the recombinant expression vector pET-28a-GPIb-AnxA1. Add the recombinant expression vector (10 μL) to BL21 (DE3) competent cells (100 μL), incubate on ice for 30 min, heat shock at 42℃ for 90 s, immediately incubate on ice for 2 min, add antibiotic-free LB medium (800 μL), and culture at 37℃ with shaking at 200 rpm for 1 h. Take 200 μL of culture and spread it on LB solid medium containing kanamycin (50 μg / mL), and incubate upside down at 37℃ for 12 h. Single colonies were picked and inoculated into LB liquid medium containing kanamycin (50 μg / mL), and cultured at 37℃ and 200 rpm for 8 h. After plasmid extraction, NcoⅠ / XhoⅠ double enzyme digestion was performed for verification, and sequencing verification was entrusted to Nanjing Genscript Biotech Co., Ltd. to ensure that the fusion protein gene sequence was correct. Positive recombinant bacteria BL21 / pET-28a-GPIb-AnxA1 were obtained.A single colony of the positive recombinant bacterium BL21 / pET-28a-GPIb-AnxA1 was inoculated into 10 mL of LB medium containing 50 μg / mL kanamycin and cultured at 37°C and 200 rpm for 12 h to obtain a seed culture. The culture was then expanded to 1 L LB medium (containing kanamycin) at a 1:100 ratio. When the OD600 reached 0.6-0.8, IPTG was added to a final concentration of 0.5 mM, and the culture was induced at 37°C and 180 rpm for 4 h. The cells were harvested by centrifugation at 4°C and 8000 rpm for 10 min, and washed twice with pre-cooled PBS. The cells were resuspended in 50 mL of lysis buffer (20 mM Tris-HCl, 500 mM NaCl, 20 mM imidazole, pH 8.0) and sonicated on ice (300 W, 3 s working time / 5 s interval, 30 min). The supernatant was collected by centrifugation at 4°C and 12000 rpm for 20 min and purified by 0.22 μm... After filtration through a filter membrane, the sample is loaded onto an equilibrated Ni-NTA column; elution is performed using a 50-300 mM imidazole gradient, and the elution peaks are collected and analyzed by SDS-PAGE.

[0018] Figure 1 The results showed that the theoretical relative molecular mass of the GPIb-Annexin A1 fusion protein was consistent with the band detected by SDS-PAGE (56.36 kDa), indicating that the purified protein was the target fusion protein with a purity greater than 98%.

[0019] Example 2 Preparation of GPIb-Annexin A1 fusion protein selenium chelate The recombinant expression vector pET-28a-GPIb-AnxA1 prepared in Example 1 was transformed into BL21 (DE3). Escherichia coli BL21 (DE3) was pre-cultured in methionine-free medium (containing 20 g / L glucose, 10 g / L yeast extract, and 5 g / L ammonium chloride). Single colonies were then picked and inoculated into 50 mL LB medium, and cultured at 37°C with shaking until OD600 = 0.6. The cells were collected by centrifugation, washed twice with methionine-free medium, resuspended in 1 L of methionine-free medium, and L-selenomethionine stock solution (final concentration 50 μg / mL) was added. Pre-incubation was performed at 37°C for 30 min. IPTG (final concentration 0.5 mmol / L) was added, and expression was induced at 25°C for 16 h, with the rotation speed controlled at 180 r / min to avoid excessive oxygen consumption by the cells leading to selenomethionine degradation. Following the purification method described in Example 1, the bacterial cells were collected by centrifugation, sonicated, and captured and eluted using a nickel affinity chromatography column via the histidine tag carried by the fusion protein to obtain a high-purity selenomethionine-labeled GPIb-Annexin A1 fusion protein.

[0020] The selenium content in the chelate products was determined by inductively coupled plasma mass spectrometry (ICP-MS). Sample pretreatment: Take 100 μL of LGPIb-Annexin A1 fusion protein chelate product (protein concentration 1 mg / mL), add 5 mL of nitric acid-hydrogen peroxide (volume ratio 3:1) mixture, place it in a microwave digester, set the temperature program (5℃ / min to 180℃, hold for 30 min) to ensure complete protein digestion; after digestion, remove acid at 120℃ to near dryness, make up to 10 mL with 2% nitric acid, and filter through a 0.22 μm organic filter membrane to avoid matrix interference.

[0021] Instrument parameter settings: Collision reaction cell mode (He gas flow rate 4.5 mL / min) was used to eliminate multi-atom interference; RF power 1550 W, carrier gas flow rate 1.05 L / min, sampling depth 8 mm; 10 μg / L germanium (Ge) was used as an internal standard to correct for signal drift and matrix effects.

[0022] Detection and calculation: The sample was measured in parallel three times, and the selenium response value was recorded. Based on the fixed volume and the sample amount, the selenium chelation efficiency was calculated to be 85%, which indicates that the GPIb-Annexin A1 fusion protein selenium chelate was successfully prepared.

[0023] Example 3: Experiment on GPIb-Annexin A1 fusion protein and its selenium chelate in a mouse model of acute myocardial infarction / reperfusion injury (MI / RI). Forty male 8-week-old C57 mice, weighing 18.5±1.5g, were purchased from Shulaibao (Wuhan) Biotechnology Co., Ltd. (Wuhan, China). Normal mouse food was purchased from Jiangsu Xietong Biotechnology Co., Ltd. The animals were housed at the SPF-grade Experimental Animal Center of Hubei University of Medicine. This experiment was approved and implemented by the Animal Ethics Committee of Hubei University of Medicine -- Hubei University of Medicine (Fu) No. 2023-Shi 151. To prevent the selenium source from drinking water from affecting the experiment, all mice drank ddH2O (deionized water) during the experiment. Mice that had been acclimatized for one week were randomly divided into four groups with free access to water: the Sham group, the MI / RI model group, the fusion protein group, and the protein selenium chelate group. Sham group: For 4 weeks before modeling, each mouse was fed 5g of normal rat food daily; MI / RI model was established 30 minutes ago, and myocardial ischemia-reperfusion injury was detected 24 hours later. After respiratory stabilization, the heart was exposed, and a 6-0 silk suture was threaded below the junction of the pulmonary conus and the left atrial appendage without ligating the left anterior descending coronary artery. This was to exclude the influence of surgical procedures (such as thoracotomy, heart exposure, suture threading, etc.) on the experimental results, and to ensure that the observed effect was due to ischemia-reperfusion injury or drug intervention, rather than surgical trauma.

[0024] MI / RI model group: 5g of normal rat food per animal per day for 4 weeks before modeling; MI / RI model was constructed 30 minutes ago, and myocardial ischemia-reperfusion injury was detected 24 hours later. After respiratory stabilization, the heart was exposed, and the left anterior descending coronary artery was ligated with 6-0 silk suture below the junction of the pulmonary artery conus and the left atrial appendage for 30 minutes before the ligation was released to restore blood flow. Fusion protein group: For 4 weeks before modeling, each mouse was given 5g of normal diet + 5mg / kg (intraperitoneal injection) of GPIb-Annexin A1 fusion protein prepared in Example 1 daily. The MI / RI model was constructed for 30 minutes, and samples were taken for testing 24 hours after myocardial ischemia-reperfusion injury. The modeling method and evaluation criteria were the same as those of the MI / RI model group. Protein selenium chelate group: Starting 4 weeks before modeling, each mouse was given 5g of normal diet + 5mg / kg (intraperitoneal injection) of GPIb-Annexin A1 fusion protein selenium chelate prepared in Example 2 daily. The MI / RI model was constructed 30 minutes ago, and samples were taken for testing 24 hours after myocardial ischemia-reperfusion injury. The modeling method and evaluation criteria were the same as those of the MI / RI model group.

[0025] Establishment of an acute myocardial infarction-ischemia-reperfusion injury (MI / RI) model: C57 mice were anesthetized with isoflurane gas, fixed, prepared, and disinfected. A longitudinal incision was made in the neck, and a small animal ventilator (SA430) was connected and the respiratory rate was adjusted to 80 breaths / min, with a respiratory ratio of 1:1. After respiratory stabilization, a 1cm longitudinal incision was made at the left sternal border where pulsation was obvious, exposing the heart layer by layer. The left anterior descending coronary artery was quickly ligated with 6-0 silk suture below the junction of the pulmonary conus and the left atrial appendage. When the local myocardium turned white, its activity decreased, and ECG showed ST segment elevation greater than 0.1mv or the appearance of pathological Q waves, it indicated that the acute myocardial infarction (AMI) model was successful. The heart was returned to the pleural cavity, and the ligation was released after 30 minutes. When the white myocardium turned pink, it indicated that myocardial blood supply had been restored. The incision was sutured layer by layer, taking care to maintain negative pressure in the pleural cavity.

[0026] Cardiac function assessment: Six hours after surgery, mice in each group underwent color Doppler echocardiography. Echocardiography was performed using a Vivid7 Echocardiography PRO system equipped with a water-dielectric I13 L14-MHz linear array probe. The LVEF was calculated by measuring the left ventricular end-diastolic volume (LVEDV) and end-systolic volume (LVESV).

[0027] Short-axis images of the heart are obtained using a chest wall probe. The left ventricular end-diastolic diameter (LVIDd) and end-systolic diameter (LVIDs) are measured, and LVFS is calculated.

[0028] The results of left ventricular ejection fraction (LVEF) and left ventricular shortening fraction (LVFS) for each group are shown in the figure. Figure 2 .

[0029] Figure 2 The results showed that, compared with the Sham group, the MI / RI model group had a more significant decrease in left ventricular ejection fraction (LVEF) (P<0.05) and left ventricular shortening fraction (LVFS) (P<0.05); compared with the MI / RI model group, the fusion protein group and the protein selenium chelate group showed significant improvement in left ventricular ejection fraction (LVEF) and left ventricular shortening fraction (LVFS); and the improvement effect of the protein selenium chelate group in left ventricular ejection fraction (LVEF) and left ventricular shortening fraction (LVFS) was better than that in the fusion protein group (P<0.05), indicating that the GPIb-Annexin A1 fusion protein selenium chelate can significantly reduce myocardial injury.

[0030] Myocardial infarction area determination: After reperfusion for 24 hours, the hearts of mice in each group were removed, washed with PBS, frozen at -80℃ for 30 minutes, and then cut into thin slices of approximately 2 mm from the apex to the base of the heart. The slices were placed in 2% TTC solution and preheated at 37℃; then placed in centrifuge tubes containing 10% formaldehyde overnight. The heart slices were photographed sequentially. The myocardial infarction area was analyzed using ImageJ software. 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 [reference missing]. Figure 3 ;

[0031] Figure 3 The results showed that the infarct area was larger in the MI / RI model group than in the Sham group (P<0.05); compared with the MI / RI model group, the infarct area was significantly reduced in both the fusion protein group and the protein selenium chelate group, with the protein selenium chelate group showing the most significant reduction in infarct area (P<0.05).

[0032] Anti-inflammatory marker detection: Serum cytokine levels of TNF-α, IL-1β, and IL-6, measured by qPCR, reflect the ability to inhibit cardiac inflammatory responses. (See attached image.) Figure 4-5 .

[0033] Figure 4-6 The results showed that, compared with the Sham group, the expression levels of TNF-α (P<0.01), IL-1β (P<0.01), and IL-6 (P<0.01) in the MI / RI model group were significantly increased. Compared with the MI / RI model group, the expression levels of TNF-α (P<0.05), IL-1β (P<0.05), and IL-6 (P<0.01) in the fusion protein group and the protein selenium chelate group were significantly decreased. Among them, the protein selenium chelate group had a significant ability to inhibit cardiac inflammatory response.

[0034] Long-term anti-myocardial fibrosis detection: Masson's trichrome staining was performed on the myocardial infarction area and surrounding tissues of mice in each group 8 weeks post-surgery, based on histopathological staining techniques. Staining differentiated collagen fibers (blue), cardiomyocytes (red), and cell nuclei (black), quantifying the proportion of collagen fibers in the myocardial tissue. At least five non-overlapping fields of view (per sample) were acquired from the infarct margin area using a microscope (10× or 20× objective). Images were saved using a high-resolution color CCD camera (avoiding overexposure) and finally quantitatively analyzed (ImageJ software). Open Image → Image → Color → Split Channels to separate the blue channel (collagen fibers); Process → Binary → Make Binary to binarize the image (collagen is black, background is white); Analyze → Measure to calculate the percentage of the blue area to the total field of view (i.e., CVF), see [link to image analysis]. Figure 7 .

[0035]

[0036] Figure 7 The results showed that both the fusion protein group (P<0.05) and the protein selenium chelate group (P<0.01) could significantly reduce the collagen volume fraction in the mouse acute myocardial infarction-reperfusion injury (MI / RI) model, and the long-term anti-myocardial fibrosis effect of the protein selenium chelate group was better than that of the fusion protein group.

[0037] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A targeted selenium anti-inflammatory fusion protein, characterized in that, The amino acid sequence of the fusion protein is shown in SEQ ID NO:3; wherein the methionine in the fusion protein shown in SEQ ID NO:3 is selenomethionine.

2. A method for preparing the targeted selenium anti-inflammatory fusion protein as described in claim 1, characterized in that, The selenium source of the targeted selenium anti-inflammatory fusion protein is selenomethionine (SeMet).

3. The use of the fusion protein according to claim 1 in the preparation of a medicament for treating acute coronary syndrome reperfusion injury.

Images