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Fusion protein of MNK2 protein kinase and cell-penetrating peptide, hydrogel of fusion protein, and application of fusion protein in promoting myocardial regeneration

A fusion protein and protein kinase technology, applied in the field of biomedicine, can solve the problem that there is no MNK2 protein kinase to promote myocardial regeneration or treat cardiomyopathy literature reports, etc., and achieve the effect of feasible implementation conditions, simple operation, and delaying ventricular remodeling.

Pending Publication Date: 2020-05-22
JIANGSU PROVINCE HOSPITAL THE FIRST AFFILIATED HOSPITAL WITH NANJING MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] By searching the existing technologies at home and abroad, there is no literature report on MNK2 protein kinase promoting myocardial regeneration or treating cardiomyopathy

Method used

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  • Fusion protein of MNK2 protein kinase and cell-penetrating peptide, hydrogel of fusion protein, and application of fusion protein in promoting myocardial regeneration
  • Fusion protein of MNK2 protein kinase and cell-penetrating peptide, hydrogel of fusion protein, and application of fusion protein in promoting myocardial regeneration
  • Fusion protein of MNK2 protein kinase and cell-penetrating peptide, hydrogel of fusion protein, and application of fusion protein in promoting myocardial regeneration

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Preparation of biologically active recombinant human MNK2 protein kinase

[0033] 1.> TAT-MNK2 (nucleotide sequence) is shown in SEQ NO.3.

[0034] 2. Construction of pFastBacHTA-MNK2 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-MNK2 primer design requirements: GC value between 40-55 is m...

Embodiment 2

[0128] Embodiment 2 Preparation of recombinant human MNK2 protein kinase hydrogel

[0129] (1) Weigh 20mg of colloidal precursor molecular powder and fully dissolve it with 800uL double distilled water at room temperature. The solution was sterilized by filtration with a 0.22um bacteria filter, the pH was adjusted to 7.4 with sterile sodium bicarbonate powder, and the total volume of the solution was added to 1000ul (hereinafter referred to as solution A).

[0130] (2) Take out the prepared recombinant human MNK2 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 use sterile PBS to make up the total volume of the protein solution to 1000ul ( Hereinafter referred to as liquid B)

[0131] (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 MNK2 protein kinase mixture, wh...

Embodiment 3

[0132] Example 3 Application of recombinant human MNK2 protein kinase hydrogel in promoting myocardial regeneration and repair

[0133] 1. Preparation and administration of mouse model of acute myocardial infarction

[0134] 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 a fusion protein of MNK2 protein kinase and a cell-penetrating peptide, a hydrogel of the fusion protein, and an application of the fusion protein in promoting myocardial regeneration, and belongs to the technical field of biomedicine. The fusion protein includes an MNK2 protein kinase sequence and a cell-penetrating peptide sequence, after injection, the fusion protein canaffect the process of cardiomyocyte cell cycle, mitosis and DNA synthesis by activating key pathways of the myocardial regeneration, reduce myocardial infarction area and fibrosis, promote myocardialregeneration repair after acute myocardial infarction, and improve recovery of a cardiac function; at the same time, a three-dimensional network structure formed by liquid coagulation of the small-molecular hydrogel can provide mechanical support force for a ventricle and delay ventricular remodeling; and the porous structure of the small-molecular hydrogel facilitates growth of regenerated cardiomyocytes, thereby promoting myocardial repair.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a recombinant human MNK2 protein kinase product with a hydrogel as a carrier, a preparation method thereof and an application for promoting myocardial regeneration. Background technique [0002] Acute myocardial infarction, as a disease that seriously endangers human life, is one of the main causes of death worldwide, so it has always received extensive attention. After the occurrence of acute myocardial infarction, the infarction-related coronary arteries should be opened as soon as possible, fully and continuously, and the main strategy for the treatment of acute myocardial infarction is to save ischemic dying myocardium and protect cardiac function. With the advancement of medical technology, especially the application of myocardial infarction reperfusion methods such as coronary artery bypass grafting or percutaneous coronary intervention, blood flow in the ischemic area ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/12C07K19/00C12N15/62A61K38/45A61K47/64A61K9/06A61P9/10A61P9/06
CPCC12N9/12C12Y207/11001A61K47/64A61K9/06A61K9/0024A61P9/10A61P9/06C07K2319/10A61K38/00
Inventor 王连生王昊陈秉瑞单天凯范燚杜冲李亚飞郭雪江程毅伟沙家豪
Owner JIANGSU PROVINCE HOSPITAL THE FIRST AFFILIATED HOSPITAL WITH NANJING MEDICAL UNIV
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