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Macrophagocyte transfer inhibition factor monoclonal antibody and method for making same

A technology of transfer inhibitors and monoclonal antibodies, which is applied in botany equipment and methods, biochemical equipment and methods, antibodies, etc., can solve the problems of short half-life and inability to effectively activate human effector systems, and achieve the effect of increasing production

Inactive Publication Date: 2007-09-26
胡川闽 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0012] Murine monoclonal antibody is easily rejected by the human immune system, has a short half-life, and its Fc segment cannot effectively activate the human effector system, so currently only the mouse-derived antibody, Muromonab-CD3, is still in clinical use

Method used

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  • Macrophagocyte transfer inhibition factor monoclonal antibody and method for making same
  • Macrophagocyte transfer inhibition factor monoclonal antibody and method for making same
  • Macrophagocyte transfer inhibition factor monoclonal antibody and method for making same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0043] 1. RT-PCR method to obtain hMIF gene

[0044] (1) Primer design and synthesis

[0045] 根据GenBank中已发表的hMIF cDNA序列:CCGATGTTCATCGTAAACACCAACGTGCCCCGCGCCTCCGTGCCGGACGGGTTCCTCTCCGAGCTCACCCAGCAGCTGGCGCAGGCCACCGGCAAGCCCCCCCAGTACATCGCGGTGCACGTGGTCCCGGACCAGCTCATGGCCTTCGGCGGCTCCAGCGAGCCGTGCGCGCTCTGCAGCCTGCACAGCATCGGCAAGATCGGCGGCGCGCAGAACCGCTCCTACAGCAAGCTGCTGTGCGGCCTGCTGGCCGAGCGCCTGCGCATCAGCCCGGACAGGGTCTACATCAACTATTACGACATGAACGCGGCCAATGTGGGCTGGAACAACTCCACCTTCGCCTAAGAGCCGCA,设计了一对特异引物。 The upstream primer is 5'-CGCGGATCCTGCGGCTCTTAGGCGAAGG-3' (containing the BamhI site); the downstream primer is 5'-CGCCATATGCCGATGTTCATCGTAAACAC-3' (containing the NdeI site), and the 5' ends of the upstream and downstream primers all have protective bases. Entrust Shanghai Sangon Bioengineering Co., Ltd. to carry out the synthesis.

[0046] (2) Extraction of total RNA from MDA-MB453 cells

[0047] Take out the breast cancer MDA-MB453 cells cryopreserved in liquid nitrogen, quickly put them into a 3...

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Abstract

The invention discloses a preparing method of monoclonal antibody (hMIF) to against human macrophage transferring inhibitor and getting method of amino acid sequence in biological medicine engineering technical domain, which comprises the following steps: augmenting out of hMIF gene from human breast cancer cell strain MDA-MB453 and human breast cancer tissue through reverse transcript polyose chain type reaction; constructing pET-11b / hMIF restructuring plasmid; expressing and purifying on bacillus coli BL21(DE3); immunizing Balb / c mouse; getting splenocyte of mouse and anti-hMIF monoclonal antibody 2D10 merged with SP2 / 0 cell through hybridomas technology; proceeding antibody subclass appraise and specificity test of antibody; making the secreting monoclonal antibody from 2D10 belong to IgG; setting the light chain as K type; setting the amino acid sequence of variable area of light chain as CRASGRNNAWYKGADGWYKGKAKRYAASSSGVSRSGSG SGTTTSSDATYYCHNDAVYSYGTKVKR; making the amino acid sequence of variable area as VWSGGSTVKG GRSCGYYASGTSTYSMGMNWVRAGKGWVSSSTSVDTYYYADSVKGRTSRDNAKNSYDY DVSRADTAVYYCARDGASSGWGVDATGSRVSCYWGGTVSSWMSAT. This anti-hMIF monoclonal antibody provides effective criterion for further transformation of human source, which can lay the groundwork for treating whole body inflammation in clinical therapeutics.

Description

technical field [0001] The invention relates to the technical field of biomedical engineering, and includes a method for preparing anti-macrophage metastasis inhibitory factor (MIF) monoclonal antibody and obtaining its amino acid sequence. Background technique [0002] The treatment of traumatic shock is an important clinical problem to be solved. Most previous studies attributed traumatic shock to hypovolemia, but many patients with traumatic shock died of multiple organ dysfunction syndrome (MODS) a few days after injury, with normal blood volume at the time of death . It is currently believed that severe trauma can cause severe systemic inflammatory response syndrome (systemic inflammatory response syndrome, SIRS), and SIRS is the central link in the pathogenesis of MODS, preventing and correcting the occurrence of SIRS will greatly improve the survival rate of traumatic shock, See Baue AE, Shock. 1995, 4(1):39-40; Lee CC et al., Ann Emerg Med, 2001, 38(2):170-176. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/13C12N15/31C12N1/21C07K16/18G01N33/53A61K39/395A61P29/00
Inventor 胡川闽黄洪涛王建易维京李鹏高利宏
Owner 胡川闽
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