Recombinant human CDC5L fusion protein hydrogel, preparation method and application

A technology of fusion protein and hydrogel, which is applied in the field of biomedicine to achieve the effect of promoting myocardial cell proliferation, reducing myocardial ischemia infarct size and scar formation, and inhibiting cardiac pathology.

Active Publication Date: 2022-05-13
SUZHOU MUNICIPAL HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0014] Aiming at the problems existing in the existing myocardial regeneration and repair therapy after acute myocardial infarction, the present invention provides a recombinant human CDC5L fusion protein hydrogel, its preparation method and application, especially relates to a linking penetrating peptide that promotes myocardial ischemia damage Recombinant human CDC5L fusion protein hydrogel product, preparation method and functional application of promoting cardiac function recovery and myocardial regeneration

Method used

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  • Recombinant human CDC5L fusion protein hydrogel, preparation method and application
  • Recombinant human CDC5L fusion protein hydrogel, preparation method and application
  • Recombinant human CDC5L fusion protein hydrogel, preparation method and application

Examples

Experimental program
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Effect test

Embodiment 1

[0053] Example 1: Preparation and biological activity detection of recombinant human CDC5L protein kinase targeting the heart

[0054] 1. PEP1-CDC5L-EGFP (nucleotide sequence) is shown in SEQ ID NO.3.

[0055] 2. Construction of pFastBacHTA-PEP1-CDC5L-EGFP insect cell vector

[0056] The invention designs and utilizes the Bac-to-Bac baculovirus expression system to express and purify the recombinant protein with the added penetrating peptide and green fluorescence and the histidine tag in Sf9 insect cells. In addition, the pFastBacHT vector also has a strong polyhedrin promoter for protein expression, three reading frames for simplified cloning, and a TEV protease cleavage site for removing histidine tags after purification.

[0057] Construction process of recombinant vector

[0058] (1) Plasmid design:

[0059] pUCori, F1ori terminator: SV40poly(A) signal; Restriction site 5'BamHI, 3'HindIII; PEP-CDC5L-EGFP primer sequence (see SEQ ID NO.4):

[0060] F:AAAGAAACCTGGTGGGAA...

Embodiment 2

[0103] Embodiment 2: Preparation of recombinant human CDC5L protein kinase hydrogel

[0104] Weigh 20 mg of the gel-forming precursor molecular powder, and fully dissolve it with 800 ul of double-distilled water at room temperature. Filter the solution with a 0.22um filter for sterilization, adjust the pH to 7.4 with sterile sodium bicarbonate powder, and make up the total volume of the solution to 1000ul (hereinafter referred to as solution A).

[0105] Take out the prepared recombinant human CDC5L solution of known concentration from the -80°C refrigerator and dissolve it on ice, take a solution containing 10mg of protein, and make up the total volume of the protein solution to 1000ul with sterile PBS (hereinafter referred to as solution B).

[0106] Mix 1000 ul of liquid A and liquid B at room temperature, and gently blow and mix with a pipette to obtain a small molecule hydrogel-recombinant human CDC5L protein mixture, which must be used immediately.

[0107] The third as...

Embodiment 3

[0108] Example 3: Application of recombinant human CHK1 protein kinase hydrogel in promoting myocardial regeneration and repair

[0109] 1. Establishment of an adult mouse acute myocardial infarction model combined with intramyocardial microinjection

[0110] Fifty 8-week-old (P56) male ICR mice of SPF grade with similar body weight were selected and randomly divided into CDC5L gel group and control group, with 25 mice in each group. Echocardiography was performed one day before operation to evaluate cardiac function. Firstly, a mouse model of acute myocardial infarction was constructed: anesthetized by intraperitoneal injection of 1.20% Avertin (30 mL / kg), and then connected to a small animal ventilator for artificial ventilation after tracheal intubation. Use ophthalmic scissors to cut the skin along the left fourth costal intercostal space. Ophthalmic forceps bluntly separate the intercostal muscles and enter the chest cavity to expose the left atrial appendage. Use a 6-0 ...

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Abstract

The invention belongs to the technical field of biomedicine, and discloses recombinant human CDC5L fusion protein hydrogel and a preparation method and application thereof.The fusion protein comprises a CDC5L protein sequence and a cell-penetrating peptide sequence, and the CDC5L protein sequence and the cell-penetrating peptide sequence are connected through a connecting peptide; the amino acid sequence of the CDC5L protein is SEQ ID NO: 1, the amino acid sequence of the fusion protein is SEQ ID NO: 2, and the nucleotide sequence of the fusion protein is SEQ ID NO: 3. After myocardial ischemia injury, through local injection administration in myocardial of an ischemic marginal area, biological activities such as myocardial cell cycle progress, DNA synthesis and mitosis can be promoted by activating a key pathway for myocardial proliferation and regeneration, myocardial cell proliferation is promoted, and the purposes of reducing myocardial ischemia infarction area and scar formation and reducing myocardial ischemia injury are achieved. The treatment effects of reducing heart injury after myocardial infarction and promoting heart function recovery are achieved.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a recombinant human CDC5L fusion protein hydrogel, a preparation method and an application. Background technique [0002] Currently, ischemic heart disease is one of the main causes of death among Chinese residents. Among them, myocardial infarction is its most serious manifestation. As adult cardiomyocytes withdraw from the cell cycle and lose their ability to proliferate, ischemic necrotic myocardium cannot be replaced and repaired by newborn normal cardiomyocytes, so the tissue in the infarct area can only be replaced by fibrous scar. This pathological process can lead to cardiac remodeling, heart failure, and eventually death. [0003] At present, related treatment after myocardial infarction mainly focuses on ischemia-reperfusion therapy such as percutaneous coronary intervention, coronary artery bypass grafting, and drug thrombolysis. However, this can on...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K47/64A61K38/45A61K9/06A61P9/00A61P9/10C07K19/00C12N9/12
CPCA61K47/64A61K38/45A61K9/06A61K9/0024A61P9/00A61P9/10C12N9/1205C12Y207/01037C07K2319/03Y02A50/30
Inventor 李亚飞韦天文陈嘉文单天凯陈宇琼王建康张俊张海锋孙康云
Owner SUZHOU MUNICIPAL HOSPITAL
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