Variable region gene of high affinity monoclonal antibody of tumor necrosis factor and its preparation

A technology of monoclonal antibody and necrosis factor, which is applied in the field of biomedicine, can solve the problems that easily cause the immune response of the human body, and achieve the effect of great application value

Active Publication Date: 2004-11-10
FOURTH MILITARY MEDICAL UNIVERSITY
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Problems solved by technology

However, when these murine mAbs are used in humans, due to their immunogenicity, they are prone to induce immune responses in humans, which can cause clearance of antibodies and immune complex-mediated hypersensitivity reactions

Method used

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  • Variable region gene of high affinity monoclonal antibody of tumor necrosis factor and its preparation
  • Variable region gene of high affinity monoclonal antibody of tumor necrosis factor and its preparation
  • Variable region gene of high affinity monoclonal antibody of tumor necrosis factor and its preparation

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Embodiment Construction

[0060] The present invention will be further described in detail below in conjunction with the accompanying drawings.

[0061] The complete nucleotide sequence of the variable region gene, the nucleotide sequence of its functional fragment (such as CDR, etc.) and the amino acid sequence of the antibody variable region are the basis for the construction of chimeric antibodies and humanized antibodies. To this end, the applicant immunized BALB / c mice with recombinant human TNF and prepared a group of mouse anti-human TNF monoclonal antibodies. The indirect ELISA method was used to screen high-affinity F6 mAb, and the monoclonal antibody was used to block TNF-induced apoptosis of L929 cells, the monoclonal antibody blocked the TNF-induced ICAM-1 expression in ECV304 cells, and the monoclonal antibody blocked the TNF-induced NF- κB nuclear translocation experiments, etc., identified F6 mAb as having high neutralizing activity. This strain of hybridoma cells has the ability to stably s...

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Abstract

The invention discloses a process for preparing variable zone genes of high affinity tumor necrosis factor (TNF) resistant monoclonal antibody (F6 mAb) comprising, using recombinated human TNF immune BALB / c mouse to prepare a plurality of mouse Anti-TNF monoclonal antibody, screening high affinity F6 mAb using indirect ELISA method. By cloning the monoclonal antibody light chain and heavy chain variable zone genes, the monoclonal antibody light chain and heavy chain variable zone gene sequence and amino acid sequence can be obtained, and the unicity of the gene sequence and protein sequence can be confirmed.

Description

Technical field [0001] The invention belongs to the field of biomedicine technology, and relates to a monoclonal antibody gene, in particular to a method for preparing a variable region gene of a high-affinity anti-tumor necrosis factor monoclonal antibody. Background technique [0002] Tumor Necrosis Factor (TNF) is a homotrimeric soluble protein composed of 3 subunits with a molecular weight of 17kDa, mainly produced by monocytes and macrophages. There is also a membrane-bound form of TNF with a molecular weight of 26 kDa in the body. TNF increases the adhesion of neutrophils and lymphocytes by inducing the expression of adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1) on the surface of vascular endothelial cells, and stimulates the secretion of macrophages, neutrophils and vascular endothelial cells A variety of cytokines and mediators cause inflammation and cause damage to tissues such as bone and cartilage. It is currently believed that the diffuse intra...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07H21/04C07K14/525C07K16/18C12P19/34C12P21/08
Inventor 金伯泉刘雪松朱参胜杨琨
Owner FOURTH MILITARY MEDICAL UNIVERSITY
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