Recombinant human SGK3 protein kinase hydrogel as well as preparation method and application thereof in promoting myocardial regeneration

A protein kinase and cardiomyocyte technology, applied in the field of biomedicine, can solve problems such as the proliferation of cardiomyocytes that have not been discovered by SGK3 protein kinase, and achieve the effects of convenience, safety, promotion of cardiomyocyte proliferation, and promotion of myocardial regeneration and repair

Pending Publication Date: 2020-04-10
王连生
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Through an extensive search of domestic and foreign literature, no literature has been found that SGK3 protein kinase can promote the proliferation of cardiomyocytes or improve heart function

Method used

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  • Recombinant human SGK3 protein kinase hydrogel as well as preparation method and application thereof in promoting myocardial regeneration
  • Recombinant human SGK3 protein kinase hydrogel as well as preparation method and application thereof in promoting myocardial regeneration
  • Recombinant human SGK3 protein kinase hydrogel as well as preparation method and application thereof in promoting myocardial regeneration

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Embodiment 1 Preparation of recombinant human SGK3 protein kinase with biological activity

[0031] 1. TAT-SGK3 (nucleotide sequence) is shown in SEQ NO.3.

[0032] 2. Construction of pFastBacHTA-SGK3 insect cell vector

[0033]The Bac-to-BacHTVectorKit is designed for expression and purification of histidine-tagged recombinant proteins in Sf9, Sf21 or HighFive cells after baculovirus vector generation in Escherichia coli (E.coli). The pFastBacHT vector has the following features: (1) strong polyhedrin promoter for protein expression; (2) three reading frames for simplified cloning; (3) N-terminal 6xHis tag for easy purification of recombinant fusion proteins (4) TEV protease cleavage site for removal of his-tag after protein purification.

[0034] Construction process of recombinant vector

[0035] (1) Plasmid design:

[0036] pUCori, F1ori terminator: SV40poly(A) signal; restriction site 5'BamHI, 3'HindIII; TAT-SGK3 primer sequence:

[0037] F: GAGGAGGCGGCTCTATGC...

Embodiment 2

[0101] Embodiment 2 Preparation of recombinant human SGK3 protein kinase hydrogel

[0102] (1) Weigh 20 mg of colloidal precursor molecular powder and fully dissolve it with 800 μL of double distilled water at room temperature. Filter and sterilize the solution with a 0.22 μm bacteria filter, adjust the pH to 7.4 with sterile sodium bicarbonate powder, and make up the total volume of the solution to 1000 ul (hereinafter referred to as solution A).

[0103] (2) Take out the prepared recombinant human SGK3 protein kinase solution of known concentration from the -80°C refrigerator and dissolve it on ice, take a solution containing 10 mg of protein kinase, and use sterile PBS to make up the total volume of the protein solution to 1000ul ( Hereinafter referred to as liquid B)

[0104] (3) Mix 1000 ul of liquid A and liquid B at room temperature, pipette slowly to mix well, and obtain the small molecule hydrogel-recombinant human SGK3 protein kinase mixture, which must be used imme...

Embodiment 3

[0105] Example 3 Application of recombinant human SGK3 protein kinase hydrogel in promoting myocardial regeneration and repair

[0106] 1. Preparation and administration of mouse model of acute myocardial infarction

[0107] Fifty 8-week-old (P56) male mice were anesthetized by intraperitoneal injection ( / kg) of 1.2070 Avertin, and artificially ventilated with a small animal ventilator after tracheal intubation. Use ophthalmic scissors to cut the skin along the left fourth intercostal space. Ophthalmic forceps bluntly separate the intercostal muscles and enter the chest cavity to expose the left atrial appendage. Use a 6-0 needle from the lower edge of the left atrial appendage, and the anterior wall of the ventricle becomes pale. , The needle is taken out from the junction of the conus pulmonary artery and the left atrial appendage, and the LAD suture is ligated. Observing the left side indicates that the ligation is correct. The operator lifts up the two ends of the ligatur...

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Abstract

The invention provides recombinant human SGK3 protein kinase hydrogel as well as a preparation method and an application thereof in promoting myocardial regeneration, which relate to the technical field of biomedicine. The injectable hydrogel is prepared by mixing a gelling precursor molecule prepolymer solution and recombinant human SGK3 protein kinase according to a certain ratio. The recombinant human SGK3 protein kinase is formed by connecting an SGK3 protein kinase sequence and a cell-penetrating peptide sequence through a connecting peptide, and after local administration, the SGK3 protein kinase can regulate the cell cycle progress by activating a key pathway of cell proliferation, promote myocardial regeneration and repair after myocardial infarction, reduce the scar area and improve the cardiac function. Besides, after solid-phase conversion of the liquid-phase hydrogel at body temperature is generated, slow release of the recombinant human SGK3 protein kinase is facilitated to continuously play a role, and support is provided for regenerated myocardial cells.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and specifically relates to a recombinant human SGK3 protein kinase product with hydrogel as a carrier and an application for promoting myocardial regeneration. Background technique [0002] Acute myocardial infarction is one of the leading causes of death worldwide and greatly increases the social burden. With the advancement of medical technology, early reperfusion methods such as drug thrombolysis, percutaneous coronary intervention and coronary artery bypass grafting can quickly restore the blood flow in the ischemic area, save dying myocardial cells, and improve the survival of patients. However, reperfusion cannot restore the viability of necrotic myocardium. In patients who survive MI, the dead heart muscle tissue is replaced by scar tissue, which inevitably leads to ventricular remodeling, heart failure and even death. Although drug treatment can improve cardiac function and delay t...

Claims

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Application Information

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IPC IPC(8): C12N9/12C07K19/00A61K38/45A61K9/06A61K9/00A61K47/65A61K47/62A61K47/42A61P9/10A61P9/06
CPCC12N9/1205A61K9/06A61K9/0024A61K47/65A61K47/62A61K47/42A61P9/10A61P9/06C12Y207/11001C07K2319/10A61K38/00
Inventor 王连生范燚杜冲李亚飞郭雪江程毅伟陈秉瑞王昊沙家豪
Owner 王连生
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