Application of STC-1 recombinant protein in preparation of medicine for treating myocardial infarction

Abstract

The invention belongs to the field of medicine, and particularly relates to application of STC-1 recombinant protein to preparation of medicine for treating myocardial infarction. The STC-1 recombinant protein is used for preparing medicines for treating myocardial infarction and / or medicines for improving myocardial fibrosis. The STC-1 (Stanniocalcin-1) recombinant protein has a good effect on treating a series of mouse symptoms induced by myocardial infarction, a brand-new view angle and possibility are provided for follow-up clinical development of novel medicines for treating myocardial infarction, meanwhile, when the STC-1 recombinant protein is used for preparing the medicines for treating myocardial infarction, compared with a traditional treatment method, the STC-1 recombinant protein has the advantages that the STC-1 recombinant protein has a good application prospect. The STC-1 recombinant protein has potential advantages, and can be used for directly protecting myocardial cells and reducing cardiac remodeling, so that a better treatment effect can be possibly realized. In addition, as an endogenous protein, the STC-1 is possibly higher in safety and tolerance, and the risk of side effects of drugs is reduced.

Description

Technical Field

[0001] The present application belongs to the field of medicine, and specifically relates to the application of STC-1 recombinant protein in the preparation of drugs for treating myocardial infarction. Background Art

[0002] Myocardial infarction is a common clinical heart disease caused by acute occlusion of coronary arteries, which leads to persistent and severe ischemia in the corresponding myocardial area. This ischemia is usually accompanied by tissue necrosis and infiltration of inflammatory cells. Its pathological process involves a variety of inflammatory mediators and biochemical reactions, including but not limited to ischemic injury, oxidative stress injury, activation of the complement system, release of interleukins, and aggregation / infiltration of neutrophils.

[0003] At present, the treatments for myocardial infarction mainly include interventional surgery (such as coronary stent implantation), drug therapy and cardiac rehabilitation. Interventional surgery can quickly restore coronary blood flow in a short period of time, but this method cannot completely replace drug therapy, nor can it repair damaged myocardial tissue. Drug therapy plays an important role in alleviating symptoms and reducing complications, but it also cannot achieve comprehensive repair of damaged myocardial tissue. Cardiac rehabilitation uses a series of comprehensive measures to help patients gradually restore heart function and improve their quality of life, but its effect is also limited and cannot completely cure myocardial infarction.

[0004] Stanniocalcin (STC) is produced by the Stanniocalcin body near the kidney of a teleost fish. It is mainly involved in the regulation of calcium and phosphorus in the fish and is a conserved glycoprotein. In 1995, Wagner et al. found that fish-like STC proteins also exist in the human body. According to the different homology of protein sequences, STC is divided into STC-1 and STC-2. STC-1 is a protein composed of 247 amino acids, and its amino acid sequence has about 60% homology with fish STC. The STC-1 gene is widely expressed in many tissues, but STC-1 is usually not detected in serum, indicating that STC-1 may be more inclined to act as a paracrine / autocrine factor rather than an endocrine hormone. The localization of STC-1 in the spleen and thymus indicates that it may also play a role in immune and inflammatory responses. STC-1 itself has a variety of beneficial biological effects, such as inhibiting superoxide production, inflammation, and apoptosis in mammals.

[0005] However, there are currently no records or reports on the research of STC-1 recombinant protein in treating myocardial infarction or improving myocardial fibrosis, which provides a new strategy for the subsequent clinical development of new drugs for the treatment of myocardial infarction. Summary of the invention

[0006] In view of the fact that there are no records or reports in the prior art on the research of STC-1 recombinant protein in treating myocardial infarction or improving myocardial fibrosis, the present application provides an application of STC-1 recombinant protein in the preparation of a drug for treating myocardial infarction.

[0007] The technical solution of this application is as follows:

[0008] The use of STC-1 recombinant protein in the preparation of drugs for treating myocardial infarction is used to prepare drugs for treating myocardial infarction and / or to prepare drugs for improving myocardial fibrosis.

[0009] Preferably, the method for preparing the STC-1 recombinant protein comprises the following steps:

[0010] Step S1. Heat shock method to transform plasmid:

[0011] After the competent host cells are melted in an ice bath, pure plasmid encoding the STC-1 gene or the product of connecting the STC-1 gene is added, pipetting is performed evenly, and the ice bath is continued for 30 minutes to obtain a bacterial solution, and the bacterial solution is heat-shocked and cultured on a 2×YT-AG medium to amplify the OD600 value to 0.6 to obtain a cultured bacterial solution;

[0012] Step S2. IPTG induced protein expression:

[0013] Take 1 ml of culture solution as the sample before induction, centrifuge the remaining culture solution, remove the supernatant, wash twice with 50 mM / pH 7.4 PBS, resuspend in 2×YT-AI medium without glucose, and add IPTG with a final concentration of 1 mmol / L to induce the expression of the target gene to obtain the induced solution;

[0014] Step S3. Extraction and purification of recombinant protein

[0015] The induced bacterial liquid was initially centrifuged to remove the supernatant, and the bacteria were resuspended in PBS, then ultrasonically disrupted and centrifuged again. The supernatant after disruption was taken for electrophoresis, and the precipitate was further resuspended in 50mM / pH 7.4 PBS and then electrophoresed to confirm the distribution of the recombinant protein in the supernatant and the precipitate. If the recombinant protein was in the supernatant, the protein in the supernatant was purified based on Ni+ affinity chromatography. If the recombinant protein was in the precipitate, the protein in the precipitate was purified based on Ni+ affinity chromatography to obtain the STC-1 recombinant protein.

[0016] Preferably, the competent host cell in step S1 is HB2151 Escherichia coli.

[0017] Preferably, in step S1, the ratio of μl of the competent host cells to μl of the pure plasmid encoding the STC-1 gene or the product ligated with the STC-1 gene is 100:1.

[0018] Preferably, the conditions for heat shocking the bacterial liquid in step S1 are to first place the bacterial liquid in a 42° C. water bath for 90 seconds and then place it in an ice bath for 2 minutes.

[0019] Preferably, in step S1, the culture temperature is 37°C.

[0020] Preferably, in step S2, the centrifugation conditions are: temperature at 4°C, rotation speed at 3000 rpm, and time for 10 min; and the conditions for inducing gene expression are: temperature at 37°C, and time for 15 h.

[0021] Preferably, in step S3,

[0022] The conditions for the initial centrifugation were: temperature 4°C, speed 8000 rpm, and time 15 min;

[0023] Ultrasonication conditions: amplitude 10 μm, time 1 min;

[0024] The conditions for the second centrifugation were as follows: temperature at 4°C, rotation speed at 12000 rpm, and time for 10 min.

[0025] The beneficial effects of this application are as follows:

[0026] The STC-1 (Stanniocalcin-1) recombinant protein of the present application has a good effect in treating a series of symptoms in mice induced by myocardial infarction, and provides a new perspective and possibility for the subsequent clinical development of new drugs for the treatment of myocardial infarction. Myocardial infarction is a serious disease caused by insufficient coronary blood supply leading to myocardial necrosis. After myocardial infarction, myocardial cells undergo a rapid death process, accompanied by a series of adverse cardiac remodeling, which will seriously affect the function of the heart. Current treatment methods, such as drug intervention, interventional surgery and coronary artery bypass surgery, although they can improve clinical symptoms, often cannot fundamentally repair necrotic myocardial tissue. As a multifunctional effector protein, STC-1 plays an important role in a variety of biological processes. In mammals, STC-1 is expressed in a variety of organs, including the small intestine, ileum, kidneys, placenta, etc., and exhibits a variety of physiological effects. STC-1 can protect myocardial cells from oxidative stress damage and reduce cell apoptosis, thereby maintaining the vitality and function of myocardial cells. At the same time, STC-1 may also affect the contraction and relaxation function of myocardial cells by regulating the calcium ion concentration in heart cells, further improving the overall performance of the heart. As a drug for the preparation of myocardial infarction, STC-1 recombinant protein has potential advantages over traditional treatment methods. It directly protects myocardial cells and reduces the occurrence of cardiac remodeling, which may achieve better therapeutic effects. In addition, as an endogenous protein, STC-1 may also have higher safety and tolerance, reducing the risk of drug side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solution of the embodiments of the present application, the drawings of the embodiments are briefly introduced below.

[0028] The drawings described below only relate to some embodiments of the present application and are not intended to limit the present application.

[0029] In the attached picture:

[0030] Attached Figure 1 This is a Coomassie Brilliant Blue staining image of the purified STC-1 recombinant protein of this application;

[0031] Attached Figure 2 This is the result of cardiac function in myocardial infarction model mice after STC-1 recombinant protein treatment in this application;

[0032] Attached Figure 3 This is the result of Sirius red staining of the heart of myocardial infarction model mice after treatment with STC-1 recombinant protein in this application;

[0033] Attached Figure 4 This is the result of Masson staining of the heart of mice with myocardial infarction model after treatment with STC-1 recombinant protein in this application. DETAILED DESCRIPTION

[0034] In order to further explain the technical means and effects adopted by the present application to achieve the predetermined invention purpose, the specific implementation method, structure, characteristics and effects of the present application are described in detail below in combination with the preferred embodiments.

[0035] Example 1

[0036] This example provides a method for preparing STC-1 recombinant protein. It should be noted that this example uses STC-1 coding to clone human STC-1 by PCR method, with a molecular weight of 26.9 KDa, and its protein sequence is:

[0037] MLQNSAVLLVLVISASATHEAEQNDSVSPRKSRVAAQNSAEVVRCLNSALQVGCGAFACLENSTCDTDGMYDICKSFLYSAAKFDTQGKAFVKESLKCIANGVTSKVFLAIRRCSTFQRMIAE VQEECYSKLNVCSIAKRNPEAITEVVQLPNHFSNRYYNRLVRSLLECDEDTVSTIRDSLMEKIGPNMASLFHILQTDHCAQTHPRADFNRRRTNEPQKLKVLLRNLRGEEDSPSHIKRTSHESA

[0038] The details are as follows:

[0039] Step S1. Heat shock method to transform plasmid:

[0040] Take 100 μl of competent host cells and melt them on an ice bath; add 1 μl of pure plasmid encoding STC-1 gene or product connected to STC-1 gene, gently blow and mix, continue ice bath for 30 minutes to obtain bacterial solution; put the bacterial solution into a 42°C water bath for heat shock for 90 seconds, and immediately put it into an ice bath for 2 minutes. Heat shock can temporarily change the permeability of the cell membrane, making it easier for exogenous DNA molecules (such as plasmids) to enter the competent cells, and obtain recombinant plasmids to transform HB2151, add 100 μl of 2×YT to the bacterial solution, and shake the bacteria at 150 rpm on a 37°C constant temperature shaker for 1 hour; evenly spread the bacterial solution on the surface of a 2×YT-AG plate (100 μg / ml ampicillin + 2% glucose), invert and culture at 37°C overnight, and pick a single colony from the 2×YT-AG plate on the next day and place it in 5 ml 2×YT-AG (100 μg / ml ampicillin + 2% glucose) liquid medium, cultured overnight at 37°C, 250 rpm; the culture solution was transferred to 100 ml 2×YT liquid medium at a volume ratio of 1:10, cultured at 37°C, 250 rpm, and shaken to OD600 of 0.6;

[0041] Step S2. IPTG induced protein expression:

[0042] Take 1 ml of culture solution as the pre-induction sample, centrifuge the remaining culture solution at 3000 rpm, 4°C for 10 min, carefully discard the supernatant, wash twice with pre-cooled 50 mM / pH 7.4 PBS (passed through a 0.22 μm filter membrane), resuspend with 100 ml of 2×YT-AI (without glucose) containing a final concentration of 1 mmol / L IPTG, and continue to culture at 37°C, 250 rpm for 15 hr to induce the expression of the target gene to obtain the induced bacterial solution.

[0043] Step S3. Extraction and purification of recombinant protein

[0044] Extract protein from bacteria: Pour the induced bacterial solution carefully into a 500ml centrifuge bucket, centrifuge at 4℃, 8000rpm for 15min, remove the supernatant, then suspend the bacteria with 50mM / pH 7.4 PBS, ultrasonically disrupt (amplitude 10μm, 1min) and centrifuge again, centrifuge at 4℃, 12000rpm for 10min, aspirate the supernatant after disruption, and resuspend the precipitate with 50mM / pH 7.4 PBS (add PMSF, a protease inhibitor, to both PBS and supernatant to a final concentration of 1mM, add TritonX-100 to a final concentration of 1% based on the protein solubility, and mix at room temperature for 30min to improve the concentrability of the fusion protein); run electrophoresis on the supernatant and precipitate after disruption at the same time to observe the distribution of protein in the supernatant and precipitate. After preliminary observation, it was found that the content of recombinant protein in the supernatant after disruption was much higher than that in the precipitate, so the recombinant protein in the supernatant after disruption was purified.

[0045] Re-purify the protein in the supernatant: Pour the induced bacterial solution carefully into a 500ml centrifuge bucket, centrifuge at 4℃, 8000rpm for 15min, remove the supernatant, then suspend the bacteria with 50mM / pH 7.4 PBS, ultrasonically disrupt (amplitude 10μm, 1min), centrifuge again, 4℃, 12000rpm for 10min, and absorb the supernatant after disruption (add protease inhibitor PMSF to the supernatant to a final concentration of 1mM, add TritonX-100 to a final concentration of 1% according to the protein solubility properties, and mix at room temperature for 30min to improve the concentrability of the fusion protein);

[0046] The supernatant liquid of the crushed tissue was concentrated to 1 / 3 of the original volume by ultrafiltration tube, NaCl was added to make the final concentration of NaCl 500mmpl, and the pH was adjusted to 7.8 to obtain the treated supernatant liquid of the crushed tissue; the sample was loaded onto a pre-equilibrated Ni+ resin column, and the binding buffer on the resin was drained to the top of the column; the treated supernatant liquid of the crushed tissue was loaded onto the column, and the column was washed with 6 column volumes of binding buffer (pH7.8), and the column was washed with 4 column volumes of washing buffer (PH 6.0) until the A280 of the liquid flowing through was less than 0.01, and the bound protein was eluted with 6 column volumes of 10mmol imidazole elution buffer, and 1ml of each portion was collected step by step, and the A280 was measured, and the bound protein was eluted with an elution buffer with a higher imidazole concentration (50mmol, 100mmol, 150mmol), and 1ml of each portion was collected, and its OD280 was measured; 20μl of protein was aliquoted and subjected to 10% SDS polyacrylamide gel electrophoresis to analyze the distribution of histidine tag protein.

[0047] Coomassie Brilliant Blue Analysis: Add 5×SDS sample buffer to the purified recombinant protein, boil the sample for 10 minutes, centrifuge at 12000g for 5 minutes, and take the supernatant; electrophoresis separation: load 15μl-20μl onto SDS-PAGE gel (10cm×10cm) for electrophoresis; load the sample according to the protein concentration after BCA quantification; configure the gel plate according to the manufacturer's instructions (Bio-rad Min-PROTEAN 3Cell gel system); configure the separation gel of a specific concentration in proportion, fill it into the gel plate, cover it with dd H2O or isopropanol to isolate the air, and place it at room temperature for 40 minutes; prepare the stacking gel, insert the comb, and place it at room temperature for 30 minutes; after loading the sample, perform electrophoresis at a voltage of 80V, and after bromophenol blue enters the separation gel, increase the voltage to 120V until the electrophoresis ends. Place the PAGE gel in the dye solution and heat it at 100℃ for 3 minutes, cool it naturally for 15 minutes, and heat it with a decolorizing solution until the protein bands are obvious. The results are as shown in the attached figure. Figure 1 As shown, STC-1 recombinant protein was successfully obtained.

[0048] Example 2

[0049] This example provides an effect on myocardial infarction model mice. It should be noted that the selection of animal strains and numbers follows the 3R principle, which not only meets the scientific needs of research objectives and model establishment, but also complies with current scientific standards and guidelines. The specifics of this example are as follows:

[0050] Construction of mouse myocardial infarction model: Adult male mice were randomly divided into 2 groups (Group A and Group B), with 5 male mice in each group, of which Group A was the myocardial infarction control group and Group B was the myocardial infarction group treated with STC-1 recombinant protein. The adult C57B6J male mice in Group A and Group B were depilated on the chest, and the mice were anesthetized by intraperitoneal injection of 1% chloral hydrate saline solution. After the mice were tested for no response by toe clipping, the ventilator was turned on (parameters: respiratory ratio 2:1, tidal volume 6-8mL, frequency 70 times / min), and the endotracheal tube was inserted into the mouse trachea along the glottis, and the breathing status of the mouse was observed. The intubation was successful when the chest rise and fall was consistent with the ventilator frequency, and the myocardial infarction surgery could be performed. The mice were placed in the right lateral decubitus position, the local skin was disinfected, the intercostal skin was cut, the third and fourth intercostal muscles were bluntly separated, the pericardium was opened, the left anterior descending branch of the coronary artery was exposed, and the left anterior descending branch of the coronary artery was quickly ligated with 6-0# sterile silk thread to cause myocardial ischemia of the anterior wall of the left ventricle. The chest cavity was quickly closed with a purse-string suture method, and the chest was squeezed to prevent pneumothorax.

[0051] Five days after myocardial infarction modeling, mice were anesthetized by injection of 1% chloral hydrate saline solution. After the mice were unresponsive by toe clipping, the ventilator was turned on (parameters: respiratory ratio 2:1, tidal volume 6-8mL, frequency 70 times / min), and the endotracheal tube was inserted into the mouse trachea along the glottis. The mouse's breathing condition was observed, and the intubation was successful when the chest rise and fall was consistent with the ventilator frequency. Ultrasonic media was applied to the mouse's chest and abdomen, and the mouse was subjected to ultrasonic cardiography under isoflurane anesthesia.

[0052] As attached Figure 2 As shown, the cardiac function of mice in the STC-1 recombinant protein treatment group was significantly improved. The above experimental results suggest that STC-1 recombinant protein treatment can significantly improve the reduction of cardiac function caused by myocardial infarction.

[0053] Example 3

[0054] After the myocardial infarction model was established based on Example 2, this example provides a method for treating and improving cardiac fibrosis caused by myocardial infarction in mice using a STC-1 recombinant protein, as follows:

[0055] Five days after myocardial infarction modeling, mice were killed by cervical dislocation under isoflurane anesthesia, and the hearts were removed while ensuring that the left and right ventricles were intact. The residual blood was squeezed out and the hearts were rinsed in sterile saline. The heart tissues of the same part of the mice in each group were cut along the long axis of the heart, fixed with 10% neutral formalin for 24 hours, and stained with Sirius red ( Figure 3 ) and Masson staining ( Figure 4 ) to observe the collagen structure of pathological tissue.

[0056] like Figure 3 and Figure 4As shown, after the left anterior descending branch of the coronary artery stenosis surgery, the area of ​​myocardial fibrosis in the mouse heart was significantly increased, and the STC-1 recombinant protein treatment alleviated the myocardial fibrosis caused by myocardial infarction. The above experimental results suggest that the STC-1 recombinant protein treatment can significantly improve the myocardial fibrosis caused by myocardial infarction in mice. The above is only a preferred embodiment of the present application, and does not limit the present application in any form. Although the present application has been disclosed as a preferred embodiment, it is not used to limit the present application. Any technician in this field can make some changes or modify the equivalent embodiments of equivalent changes by using the above disclosed technical content without departing from the scope of the technical solution of the present application. However, any modification, equivalent change and modification made to the above embodiments according to the technical essence of the present application without departing from the content of the technical solution of the present application still belongs to the scope of the technical solution of the present application.

Claims

1. The use of STC-1 recombinant protein in the preparation of a drug for treating myocardial infarction, characterized in that: Used for preparing drugs for treating myocardial infarction and / or for preparing drugs for improving myocardial fibrosis.

2. The use of a STC-1 recombinant protein according to claim 1 in the preparation of a drug for treating myocardial infarction, characterized in that: The method for preparing the STC-1 recombinant protein comprises the following steps: Step S1. Heat shock method to transform plasmid: After the competent host cells are melted in an ice bath, pure plasmid encoding the STC-1 gene or the product of connecting the STC-1 gene is added, pipetting is performed evenly, and the ice bath is continued for 30 minutes to obtain a bacterial solution, and the bacterial solution is heat-shocked and cultured on a 2×YT-AG medium to amplify the OD600 value to 0.6 to obtain a cultured bacterial solution; Step S2. IPTG induced protein expression: Take 1 ml of culture solution as the sample before induction, centrifuge the remaining culture solution, remove the supernatant, wash twice with 50 mM / pH 7.4 PBS, resuspend in 2×YT-AI medium without glucose, and add IPTG with a final concentration of 1 mmol / L to induce the expression of the target gene to obtain the induced solution; Step S3. Extraction and purification of recombinant protein: The induced bacterial liquid was initially centrifuged to remove the supernatant, and the bacteria were resuspended in PBS, then ultrasonically disrupted and centrifuged again. The supernatant after disruption was taken for electrophoresis, and the precipitate was further resuspended in 50mM / pH 7.4 PBS and then electrophoresed to confirm the distribution of the recombinant protein in the supernatant and the precipitate. If the recombinant protein was in the supernatant, the protein in the supernatant was purified based on Ni+ affinity chromatography. If the recombinant protein was in the precipitate, the protein in the precipitate was purified based on Ni+ affinity chromatography to obtain the STC-1 recombinant protein.

3. Use of a STC-1 recombinant protein according to claim 2 in the preparation of a drug for treating myocardial infarction, characterized in that: The competent host cell in step S1 is HB2151 Escherichia coli.

4. The use of a STC-1 recombinant protein according to claim 2 in the preparation of a drug for treating myocardial infarction, characterized in that: In the step S1, the ratio of μl of the competent host cells to μl of the pure plasmid encoding the STC-1 gene or the product ligated with the STC-1 gene is 100:

1.

5. Use of a STC-1 recombinant protein according to claim 2 in preparing a drug for treating myocardial infarction, characterized in that: The conditions for heat shocking the bacterial liquid in step S1 are to first place the bacterial liquid in a 42° C. water bath for 90 seconds and then place it in an ice bath for 2 minutes.

6. Use of a STC-1 recombinant protein according to claim 2 in the preparation of a drug for treating myocardial infarction, characterized in that: In the step S1, the culture temperature is 37°C.

7. Use of a STC-1 recombinant protein according to claim 2 in the preparation of a drug for treating myocardial infarction, characterized in that: In step S2, the centrifugation conditions are: temperature at 4° C., rotation speed at 3000 rpm, and time for 10 min; and the conditions for inducing gene expression are: temperature at 37° C., and time for 15 h.

8. Use of a STC-1 recombinant protein according to claim 2 in the preparation of a drug for treating myocardial infarction, characterized in that: In the step S3, The conditions for the initial centrifugation were: temperature at 4°C, speed at 8000 rpm, and time for 15 min; Ultrasonication conditions: amplitude 10 μm, time 1 min; The conditions for the second centrifugation were as follows: temperature at 4°C, rotation speed at 12000 rpm, and time for 10 min.

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