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Variable region coding gene of human vascular endothelial growth factor (VEGF)-resistant humanized antibody and use thereof

A technology encoding genes and variable regions, which is applied in the field of anti-human vascular endothelial growth factor humanized antibody variable region encoding genes, can solve the problems of restricting the application of antibodies and low antibody production capacity

Inactive Publication Date: 2015-01-14
BEIJING MABWORKS BIOTECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the production capacity of antibodies in the world is far lower than the demand, and the expression level of antibodies seriously restricts the application of antibodies

Method used

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  • Variable region coding gene of human vascular endothelial growth factor (VEGF)-resistant humanized antibody and use thereof
  • Variable region coding gene of human vascular endothelial growth factor (VEGF)-resistant humanized antibody and use thereof
  • Variable region coding gene of human vascular endothelial growth factor (VEGF)-resistant humanized antibody and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Rational design and gene synthesis of anti-VEGF antibody MIL60

[0038] 1. Materials:

[0039] Primer design software is biosun software, primers are synthesized by Shanghai Yingjun Biotechnology Co., Ltd.; Pyrobest DNA polymerase is a product of TAKARA company; dNTP is a product of TAKARA company; pGEM-T Easy vector system is a product of Invitrogen company; T4DNA ligase is a product of NEB company ; Gene sequencing was completed by Beijing Nuosai Genome Research Center Co., Ltd.; other related reagents were purchased from the market.

[0040] 2. Method results

[0041] 1. Gene acquisition of antibody MIL60

[0042] According to the amino acid sequence of the Avastin antibody, codons with high frequency in mammalian cells were selected, reverse-translated, rationally optimized by bioinformatics technology, and the stability of the gene and its expression level in mammalian cells were increased to obtain The antibody light chain variable region gene sequenc...

Embodiment 2

[0069] Example 2 Expression, identification and biological function identification of anti-VEGF antibody MIL60

[0070] 1. Materials

[0071] Antibody eukaryotic expression vector pTGS-FRT-DHFR was constructed by our unit and applied for national patent (patent authorization number: ZL200510064335.0); primers were designed by biosun software and synthesized by Shanghai Yingjun Biotechnology Co., Ltd.; liposomes and MTT were Invitrogen The company's products, goat anti-human IgG, and horseradish-labeled goat anti-human IgG and human IgG were prepared by our company; endonucleases were products of NEB; SKV03, MCF7, SKBR3 were purchased from ATCC; DELFIA EuTDA cytotoxicity kit It is a product of PE Company; other reagents are commercial products.

[0072] 1. Method

[0073] 1. Antibody expression

[0074] 1.1 Construction of antibody eukaryotic expression vector

[0075] The expression vector pTGS-FRT-DHFR (patent authorization number: ZL200510064335.0) obtained by our compan...

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Abstract

The invention discloses an antibody variable region coding gene. Based on the coding gene, a targeting antibody expression level is substantially improved. An external biological function evaluation result shows that a targeting antibody obtained based on the antibody variable region coding gene can specifically recognize a human vascular endothelial growth factor (VEGF) antigen and can substantially inhibit tumor tissue neovascularization thereby inhibiting tumor growth. The invention also discloses a preparation method of the above targeting antibody.

Description

technical field [0001] The invention relates to an anti-human vascular endothelial growth factor humanized antibody variable region coding gene and application thereof. By optimizing codons, a gene encoding the variable region of an anti-human vascular endothelial growth factor humanized antibody was obtained. The gene was fully synthesized using molecular biology techniques; after expression, purification, immunology and cell biology experiments were used to evaluate the expression level and biological function of the target antibody. Background technique [0002] Human vascular endothelial growth factor (VEGF) is an important regulator of normal and abnormal angiogenesis, and plays an important role in physiological and pathological angiogenesis. Its main mechanism of action is as an endothelial mitogen, promoting vascular endothelial cell division, proliferation and angiogenesis. VEGF is involved in the whole process of tumorigenesis and development. The rapid growth of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/13C07K16/22
Inventor 杨静吕明李新颖耿树生郎小玲冯健男谢波叶培黎燕沈倍奋
Owner BEIJING MABWORKS BIOTECH
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