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Experimental technology for Fc segment fusion gene (VEGF165-Fc) of recombinant human vascular endothelial growth factor 165 and immune globulin

A technology of v165-fc and protein, which is applied in the experimental technical field of recombinant human vascular endothelial growth factor 165 and immunoglobulin Fc fragment fusion protein (VEGF165-Fc), can solve the problem that the activity and toxin content are difficult to meet clinical diagnosis and treatment, Problems such as the low degree of glycosylation of protein products, to achieve high expression levels, improve product yield and quality, and reduce costs

Inactive Publication Date: 2012-02-08
ABZYME BIOTECH
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Problems solved by technology

[0015] At present, the VEGF165-Fc recombinant protein produced in the world is basically obtained by Escherichia coli or yeast expression system. The protein product is not glycosylated or the degree of glycosylation is not high, and the activity and toxin content are difficult to meet the purpose of clinical diagnosis and treatment.

Method used

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  • Experimental technology for Fc segment fusion gene (VEGF165-Fc) of recombinant human vascular endothelial growth factor 165 and immune globulin
  • Experimental technology for Fc segment fusion gene (VEGF165-Fc) of recombinant human vascular endothelial growth factor 165 and immune globulin
  • Experimental technology for Fc segment fusion gene (VEGF165-Fc) of recombinant human vascular endothelial growth factor 165 and immune globulin

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Embodiment Construction

[0024] 1. Preparation of Plasmids for Transfection

[0025] The expression plasmid pR-VEGF165-Fc linked with the VEGF165-Fc gene required for transfection was prepared with an endotoxin-removing plasmid purification kit (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 HCI dropwise to make the pH<2.0 (about 800 μl is needed). 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 Milli-Q ...

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Abstract

The invention relates to a technology for transferring an Fc segment fusion gene (VEGF165-Fc) of a human recombinant human vascular endothelial growth factor 165 and a human immune globulin IgG1 into mammalian cells so as to produce the protein products in a large scale. The invention aims to establish a method for producing VEGF165-Fc proteins with biological activity in a large scale by using the mammalian cells. The overall requirements of lowering production cost, simplifying process flow and improving product yield and quality are met in industrial production. By the method, a great number of VEGF165-Fc proteins with high purity and high biological activity are obtained by using common large-scale cell culture. The technology is applied to the large-scale production of the VEGF165-Fc proteins. By the technology, the cost can be obviously lowered, a process flow is simplified, and the yield and the quality of the products are improved.

Description

technical field [0001] The invention relates to a technique for transferring human recombinant human vascular endothelial growth factor 165 and the Fc segment fusion gene (VEGF165-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 VEGF [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江永海
Owner ABZYME BIOTECH
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