Monoclonal antibody of human myxovirus resistance A (A-hMxA), preparation and application thereof

A monoclonal antibody and protein technology, applied in the direction of anti-enzyme immunoglobulin, biochemical equipment and methods, instruments, etc., can solve the problems of limited application range, preparation process and main technical parameters that have not been disclosed, so as to improve the detection efficiency , high affinity, high affinity effect

Inactive Publication Date: 2009-11-04
SUZHOU UNIV +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

According to the existing academic reports, the antibody is mainly used in the detection of hMxA protein by western-blot and immunocytochemistry / immunocytofluorescence (immunocytochemistry / immunocytofluorescence), and its preparation process and main technical parameters are unknown Public; moreover, their antibody did not involve immunoprecipitation (immuno-precipitation) test, which limited the scope of application of the M143 monoclonal antibody to a certain extent

Method used

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  • Monoclonal antibody of human myxovirus resistance A (A-hMxA), preparation and application thereof
  • Monoclonal antibody of human myxovirus resistance A (A-hMxA), preparation and application thereof
  • Monoclonal antibody of human myxovirus resistance A (A-hMxA), preparation and application thereof

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Experimental program
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Effect test

Embodiment 1

[0045] Embodiment 1, preparation and purification of anti-human hMxA protein monoclonal antibody

[0046] The strategy of using the prokaryotic expression product of hMxA gene as the immunogen to immunize Balb / C mice to obtain the B cells sensitized by the immunogen, the specific operation method:

[0047] 1. Preparation of Monoclonal Antibody Immunogen

[0048] 1. Amplification of the complete open reading frame fragment of 1hMxA gene

[0049] It is very difficult to obtain purified hMxA protein directly from tissues and cells to prepare antibodies. Therefore, it is an effective method to prepare antibodies through genetic engineering expression products. Obtaining genetic engineering expression products and transferring target genes is a prerequisite.

[0050] Using type I interferon to induce human cell lines in vitro for 6-12 hours can produce a large amount of hMxA gene transcripts, and then extract the total RNA, RT-PCR amplification and construct the recombinant transf...

Embodiment 2

[0129] Main technical indicators of hybridoma cell-3C2 and its secreted antibody

[0130] 1. The main technical indicators achieved by the hybridoma cell line-3C2

[0131] 1.1 Growth indicators of hybridoma cells:

[0132] The hybridoma cell growth, growth conditions and passage conditions were comprehensively analyzed, and it was found that the hybridoma cell line and the myeloma cell line-SP2 / 0 could not only survive in 10% fetal bovine serum (provided by GIBCO and hyclone companies) product) grows well, and can also grow well in the medium of 10% calf serum provided by the above two companies after being adapted to culture;

[0133] The above-mentioned 10% calf serum medium was used to carry out the first, fifth and tenth generation growth curve analysis of the above-mentioned 3C2 cell line. The doubling time of the cells was 3-4 hours, and the growth rate of each generation was basically the same.

[0134] For cell growth curves, see figure 1 The above results show that...

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Abstract

The invention discloses a monoclonal antibody against human MxA. The monoclonal antibody against the human MxA is generated by a hybridoma cell line CCTCC NO: C200916, and the monoclonal antibody is of IgG1 subtype and kappa type; the monoclonal antibody has good specificity and high affinity, can identify a prokaryotic expression product of the human MxA, an eukaryotic expression product of the human MxA, interferon or human MxA gene expression products induced by other factors, simultaneously can identify denatured protein and non-denatured protein of MxA expression protein, and well reflects the existence form of a polymer of the protein; therefore, the monoclonal antibody can simply, conveniently, quickly and accurately detect the expression level of the human MxA protein.

Description

technical field [0001] The present invention relates to the preparation and application of a human anti-myxovirus protein A-hMxA monoclonal antibody, in particular to the human anti-myxovirus protein A-hMxA monoclonal antibody and a hybridoma cell strain that secretes the antibody extracellularly; The invention also relates to various immunological methods for detecting the expression level of human hMxA gene protein by using the monoclonal antibody. Background technique [0002] Human antimyxovirus protein A-hMxA (human myxovirus resistance A) is a guanosine triphosphate hydrolase protein that is specifically induced by human type I interferon (IFNα / β) and has anti-virus activity. It can be expressed efficiently through the induction of type I interferon such as IFNα / β in various cells or tissues. At the same time, in the case of some virus infection or injection of type I interferon, the human body can directly pass human peripheral blood mononuclear cells PBMCs ( Periphe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/40G01N33/577
Inventor 吴康刘海燕刘鹏
Owner SUZHOU UNIV
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