Anti-vascular endothelial growth factor monoclonal antibody and application thereof

A technology of monoclonal antibodies and nucleic acid molecules, applied in the fields of antibody engineering and medicine, can solve the problems of differences in tolerance, speed and toxicity, affecting drug efficacy, etc.

Inactive Publication Date: 2011-10-12
BEIJING INST OF GENOMICS CHINESE ACAD OF SCI CHINA NAT CENT FOR BIOINFORMATION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The immunogenicity of different CDR sequences is different. Humanized antibodies prepared by CDR transplantat...

Method used

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  • Anti-vascular endothelial growth factor monoclonal antibody and application thereof
  • Anti-vascular endothelial growth factor monoclonal antibody and application thereof
  • Anti-vascular endothelial growth factor monoclonal antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

specific Embodiment 1

[0018] Preparation and Screening of Specific Example 1 Mouse Monoclonal Antibody

[0019] (1) Antigen preparation

[0020] human vascular endothelial growth factor VEGF 165 (Human Vascular endothelial growth factor, hVEGF 165 ) gene was amplified from the human gene transcriptome by PCR, the upstream primer was: 5'-CGCGGATCCGCACCCATGGCAGAAGGAGG-3'; the downstream primer was: 5'-CTCCTCGAGTCACCGCCTCGGCTTGTCACATC-3'. Using BamH I and Xho I double enzyme digestion, the gene fragment was connected into the prokaryotic expression vector pET32a (+), PCR and double enzyme digestion identified positive clones and then sequenced. After the sequence was determined correctly, the plasmid was extracted. Prokaryotic expression vector pET32a-VEGF 165 Transform Rosetta strain in 100ml LB medium, OD 600 When reaching about 0.5, 1mM IPTG induced expression for 4h, VEGF 165 Proteins form inclusion bodies. After sonication, the inclusion bodies were dissolved with 1×SDS loading buffer, and ...

specific Embodiment 2

[0030] Western-blot and immunofluorescence detection of specific embodiment 2 mouse monoclonal antibody

[0031] VEGF 165 The gene is connected into the expression vector pLenti7.3 / V5-TOPO by TOPO cloning method, and the eukaryotic expression vector pLenti7.3 / V5-VEGF 165Lipofectamine 2000 (Invitrogen) was used to mediated transient transfection of 293T cells, serum was diluted 1:100, and antibodies were identified by immunofluorescence. The determination showed that the antibody specificity of the 18-5 cell line was the best, and it could recognize the antigen expressed endogenously in the cell. Take the cultured cells or cell supernatant, add 3×Loading buffer, boil for 5 minutes, load the sample, and detect by westem-blot. The results showed that the molecular weight of the positive protein band was related to that of VEGF 165 unanimous.

[0032]

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PUM

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Abstract

The invention provides a specific monoclonal antibody capable of combining a human vascular endothelial growth factor (VEGF). The monoclonal antibody has the speciality of specially combining the human VEGF. The invention also provides the application of the monoclonal antibody in the aspects of scientific research and immunological test.

Description

technical field [0001] The invention relates to the fields of antibody engineering and medicine. More specifically, the present invention relates to obtaining murine monoclonal antibodies that recognize epitopes of human vascular endothelial growth factor (hVEGF). Background technique [0002] Cancer is a major disease affecting human health with a high mortality rate. In 1971, Folkman first observed that when the tumor volume exceeds 1-2mm 3 , need to form new blood vessels to continue to grow, thus putting forward the hypothesis that tumor growth is controlled by regulating angiogenesis. Current studies have shown that blocking tumor angiogenesis growth factor signaling pathways can inhibit neovascularization in the development of solid tumors, and can specifically intervene in the development of tumors. The process of neovascularization requires the participation of various stimulating factors, including vascular endothelial growth factor VEGF (vascular endothelia grow...

Claims

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Application Information

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IPC IPC(8): C07K16/22C12N15/13G01N33/577
Inventor 冯杰高静邵长君
Owner BEIJING INST OF GENOMICS CHINESE ACAD OF SCI CHINA NAT CENT FOR BIOINFORMATION
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