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Recombinant human CHK1 protein kinase hydrogel for promoting myocardial regeneration as well as preparation method and application thereof

A protein kinase, CHK1 technology, applied in the field of biomedicine, can solve the problem of heart function recovery without the discovery of CHK1 protein kinase, achieve the effect of feasible implementation conditions, promote myocardial regeneration and repair, and inhibit ventricular remodeling

Inactive Publication Date: 2022-05-13
王连生
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] By searching domestic and foreign literature, there is no literature report that CHK1 protein kinase can promote the proliferation of cardiomyocytes or improve the recovery of cardiac function

Method used

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  • Recombinant human CHK1 protein kinase hydrogel for promoting myocardial regeneration as well as preparation method and application thereof
  • Recombinant human CHK1 protein kinase hydrogel for promoting myocardial regeneration as well as preparation method and application thereof
  • Recombinant human CHK1 protein kinase hydrogel for promoting myocardial regeneration as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Preparation of biologically active recombinant human CHK1 protein kinase

[0033] 1.> TAT-CHEK1 (nucleotide sequence) is shown in SEQ NO.3.

[0034] 2. Construction of pFastBacHTA-CHEK1 insect cell vector

[0035] 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.

[0036] Construction process of recombinant vector

[0037] (1) Plasmid design:

[0038]pUCori, F1ori terminator: SV40poly(A) signal; restriction site 5'BamHI, 3'HindIII; TAT-CHEK1 primer sequence:

[0039] F: CTGTATTTTTCAGGGCGCCATG...

Embodiment 2

[0105] Example 2 Preparation of recombinant human CHK1 protein kinase hydrogel

[0106] (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).

[0107] (2) Take out the prepared recombinant human CHK1 protein kinase solution of known concentration from the -80°C refrigerator and dissolve it on ice, take the solution containing 10 mg of protein kinase, and fill the total volume of the protein solution to 1000ul with sterile PBS ( Hereinafter referred to as liquid B)

[0108] (3) Mix 1000 ul of liquid A and liquid B at room temperature, and gently blow and mix with a pipette to obtain the small molecule hydrogel-recombinant human CHK1 protein kinase mixture, which must be used ...

Embodiment 3

[0109] Example 3 Application of recombinant human CHK1 protein kinase hydrogel in promoting myocardial regeneration and repair

[0110] 1. Preparation and administration of mouse model of acute myocardial infarction

[0111] 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 discloses recombinant human CHK1 protein kinase hydrogel for promoting myocardial regeneration as well as a preparation method and application of the recombinant human CHK1 protein kinase hydrogel, and belongs to the technical field of biomedicine. The recombinant human CHK1 protein kinase comprises a CHK1 protein kinase sequence and a cell-penetrating peptide sequence, after injection administration, the processes of myocardial cell DNA synthesis, cell cycle, mitosis and the like can be influenced by activating a key pathway for myocardial regeneration, myocardial regeneration and repair after acute myocardial infarction are promoted, the myocardial infarction area and fibrosis are reduced, and cardiac function recovery is improved. Meanwhile, a three-dimensional network structure formed by liquid solidification of the micromolecular hydrogel can provide mechanical supporting force for the ventricle, and ventricular remodeling is delayed; the porous structure of the small-molecule hydrogel facilitates the growth of regenerated myocardial cells so as to promote myocardial repair.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a recombinant human CHK1 protein kinase product with hydrogel as a carrier and its application. Background technique [0002] With the continuous development of clinical medical technology, although the mortality rate of patients with acute myocardial infarction has decreased, it is still a serious disease that threatens human life. After acute myocardial infarction, opening the infarction-related coronary arteries as soon as possible, adequately, and continuously, saving ischemic dying myocardium, and protecting cardiac function are the main strategies for the treatment of acute myocardial infarction. However, at present, reperfusion methods such as drug thrombolysis, percutaneous coronary intervention or coronary artery bypass grafting can only improve the blood supply of the myocardium, but cannot increase the number of myocardial cells or make the necrotic cells Myocardi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/45A61K47/64A61K9/06A61P9/10A61P9/06C12N9/12C12N15/62C12N15/866
CPCC12N9/1205A61K9/06A61P9/10A61P9/06C07K2319/10A61K38/45A61K47/645A61K47/64C12Y207/01037A61K9/0024C12N15/86C12N2710/14043
Inventor 王连生范燚王昊郭雪江陈秉瑞韦天文李亚飞王子牧程毅伟沙家豪
Owner 王连生
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