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Experimental technique for recombinant fusion protein (VEGF121-FC) of human vascularendothelial growth factor (VEGF) 121 and immunoglobulin Fc fragment

A technology of vegf121-fc and protein, applied in the direction of recombinant DNA technology, hybrid peptide, peptide preparation method, etc., can solve the problems of low level of correct folding of protein products, activity and toxin content difficult to meet clinical diagnosis and treatment, etc. , to achieve high expression level, improve product yield and quality, and reduce costs

Inactive Publication Date: 2012-03-07
ABZYME BIOTECH
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Problems solved by technology

[0015] At present, the VEGF121-Fc recombinant protein produced in the world is basically obtained by the E. coli expression system. The correct folding level of the protein product is not high, and it has not undergone post-translational modification. The activity and toxin content are difficult to meet the purpose of clinical diagnosis and treatment.

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  • Experimental technique for recombinant fusion protein (VEGF121-FC) of human vascularendothelial growth factor (VEGF) 121 and immunoglobulin Fc fragment
  • Experimental technique for recombinant fusion protein (VEGF121-FC) of human vascularendothelial growth factor (VEGF) 121 and immunoglobulin Fc fragment
  • Experimental technique for recombinant fusion protein (VEGF121-FC) of human vascularendothelial growth factor (VEGF) 121 and immunoglobulin Fc fragment

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Embodiment Construction

[0024] 1. Preparation of Plasmids for Transfection

[0025] The expression plasmid pR-VEGF121-Fc linked with the VEGF121-Fc gene required for transfection was prepared with a plasmid purification kit for removing endotoxin (purchased from Qiagen). See the instruction manual for the method. Dissolve the plasmid with 200 μl TE buffer, and measure its concentration with a UV spectrophotometer.

[0026] 2. Preparation of PEI for transfection

[0027] 1) Pour 450ml of ultrapure water prepared by Milli-Q into a 500ml beaker;

[0028] 2) Weigh 500 mg linear PEI (purchased from Poly Science Company) on the balance, add it to the beaker, and stir it continuously with a magnetic stirrer;

[0029]3) Add pre-prepared 12M concentrated HCl dropwise to make the pH<2.0 (about 800 μl is required). Stir the PEI solution for about 2-3 hours;

[0030] 4) Add pre-prepared 10M NaOH solution to adjust the pH to 7.0 (about 500 μl is required). Dilute the volume of PEI solution to 500ml with Mil...

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Abstract

The invention relates to a technique for transferring a human recombinant fusion gene (VEGF-FC) of a human vascularendothelial growth factor (VEGF) 121 and the Fc fragment of human immunoglobulin IgG1 into a mammalian cell for mass production of its protein products. The invention aims to establish a method for mass production of a biologically active VEGF121-FC protein with a mammalian cell. The invention can satisfy the general requirements of reducing production cost, simplifying technical process, as well as improving product output and quality during industrial production. With the technique of the invention, a lot of high purity and highly biologically active VEGF121-FC proteins can be obtained through ordinary large scale cell culture. Application of the technique provided in the invention to large scale production of the VEGF121-FC protein can substantially reduce lost, simplify technical process, as well as improve product output and quality.

Description

technical field [0001] The invention relates to a technology for transferring human recombinant human vascular endothelial growth factor 121 and the Fc region fusion gene (VEGF-Fc) of human immunoglobulin IgG1 into mammalian cells to produce its protein product on a large scale. Background technique [0002] 1. The biological function and application prospect of VEGF121 [0003] Vascular endothelial growth factor (vascular endothelial growth factor, VEGF), also known as vascular permeability factor (vascular permeability factor, VPF), is a kind of purified from bovine pituitary follicular stellate cell culture medium by Ferrara N et al in 1989 glycoprotein. VEGF is encoded by a single gene, which can form at least five different homodimeric glycoprotein products after alternative splicing, which are VEGF121, VEGF145, VEGF165, EGF189 and VEGF206. VEGF is a specific mitogen of endothelial cells. It binds to its receptors (mainly VEGFR2) to activate intracellular tyrosine kin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C07K19/00C07K1/36C07K1/22C07K1/16
Inventor 柴笑梅朱月珍杨宣武周慧芳卢守英吴咪佳
Owner ABZYME BIOTECH
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