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Simple and convenient chemical industrial technology for prokaryotic expression and purification of humanized anti-vascular endothelial growth factor (Anti-VEGF) monoclonal recombinant antibody

An endothelial growth factor and recombinant antibody technology, applied in recombinant DNA technology, anti-growth factor immunoglobulin, genetic engineering, etc., can solve the problems of cumbersome purification steps, reduced economic significance, and low purity.

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

Traditional production of anti-VEGF antibody preparations requires cumbersome purification steps and low purity, which greatly reduces its economic significance

Method used

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  • Simple and convenient chemical industrial technology for prokaryotic expression and purification of humanized anti-vascular endothelial growth factor (Anti-VEGF) monoclonal recombinant antibody
  • Simple and convenient chemical industrial technology for prokaryotic expression and purification of humanized anti-vascular endothelial growth factor (Anti-VEGF) monoclonal recombinant antibody

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Embodiment Construction

[0008] 1. Eukaryotic expression of humanized anti-vascular endothelial growth factor monoclonal recombinant antibody

[0009] The encoding gene encoding humanized anti-vascular endothelial growth factor monoclonal recombinant antibody heavy chain and light chain is connected with expression vector pCDNA3.1 through EcoRI and XhoI restriction enzyme cutting sites. The plasmid was subsequently transfected into the CHO-K1 cell line.

[0010] 48 hours before transfection, 10ml of the viability is greater than 95%, and the cell density is 1.5*10 5 CHO-K1 cells / ml were plated on 10cm cell culture dish with DMEM complete medium (without anti-antibody, containing 10% fetal calf serum), and cultured in 37°C, 5% CO2 incubator. Transfection was initiated when cells reached 70-80% confluence. Add 1 μg each of the expression vectors containing heavy chain and light chain genes of anti-vascular endothelial growth factor monoclonal antibody to 200 μl 37°C preheated PBS, mix well, add 4 μl 1...

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Abstract

The invention relates to a simple and convenient chemical industrial technology for prokaryotic expression and purification of a humanized anti-vascular endothelial growth factor (Anti-VEGF) monoclonal recombinant antibody, and aims to realize expression of a large amount of humanized anti-VEGF monoclonal recombinant antibody in prokaryotic cells and obtain the antibody with higher purity through simple purification steps. The technology meets the overall requirements of short production period, low production cost and high yield in industrial production.

Description

technical field [0001] The invention relates to a simplified method for eukaryotic expression and purification of humanized anti-vascular endothelial growth factor monoclonal recombinant antibody (Anti-VEGF). Background technique [0002] Vascular endothelial growth factor (VEGF) is a disulfide-linked dimer glycoprotein with a molecular weight of 35-40 kDa. VEGF is a family of cytokines, including VEGF-A, -B, -C, -D, -E and placental growth factor. Alternative splicing of the VEGF-A gene can produce at least five protein forms, including secreted soluble proteins VEGF121, VEGF145, and VEGF165. Vascular endothelial growth factor specifically binds to three vascular endothelial growth factor receptors, initiates downstream cell signaling cascades, stimulates endothelial cell proliferation, promotes cell migration, and inhibits cell apoptosis. Vascular endothelial growth factor is mainly expressed in vascular endothelial cells, tumor cells, some inflammatory cells and interst...

Claims

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Application Information

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IPC IPC(8): C12N15/13C12N15/85C07K16/22C07K1/22
Inventor 柴笑梅朱月珍杨宣武江永海
Owner ABZYME BIOTECH
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