Method for producing all-pig-source monoclonal antibody with single B cell PCR technology

A monoclonal antibody and B cell technology, applied in the biological field, to achieve the effect of good gene diversity and high efficiency

Active Publication Date: 2016-11-09
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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  • Method for producing all-pig-source monoclonal antibody with single B cell PCR technology
  • Method for producing all-pig-source monoclonal antibody with single B cell PCR technology
  • Method for producing all-pig-source monoclonal antibody with single B cell PCR technology

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Embodiment 1

[0063] Example 1 Establishment of a method for isolating whole-pig-derived anti-PEDV monoclonal antibodies using a single B cell PCR monoclonal antibody technique

[0064] 1. Animal immunity

[0065] Select 3 healthy Landrace piglets about the age of 35 days, numbering is respectively #241, #242, #243, uses the PEDV CV777 strain of 2ml respectively (10 4 TCID 50 / ml) Houhai acupoint injection (first immunization); at intervals of 2 weeks, use 2 times the dose of the first immunization to inactivate the virus before injecting Houhai acupoint (second immunization); after 2 weeks, use the same dose as the second immunization to isolate it in our laboratory The virulent PEDV strain LNCT2 strain was boosted. Monitor ELISA antibody and neutralizing antibody titers. Necropsy, anticoagulated blood and mesenteric lymph nodes were collected.

[0066] 2. Isolation and screening of single B lymphocytes:

[0067] PEDV antigen-specific porcine memory lymphocytes were selected by double...

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Abstract

The invention discloses a method for producing an all-pig-source monoclonal antibody with a single B cell PCR technology and belongs to the technical field of biology. An existing pig antibody gene library is used, multiple types of primers are designed, an antibody variable region gene is amplified from a single B lymphocyte with a nested PCR (polymerase chain reaction), and the all-pig-source monoclonal antibody with neutralizing antibody activity is screened. The method for producing the all-pig-source monoclonal antibody with a high-throughput single B cell technology is firstly established; compared with conventional antibody preparation technologies, the method has the advantages of being high in efficiency and good in gene diversity, covering all pig sources and the like. A new technological means is provided for research of antibody immune response and separation of a therapeutic antibody after proposal of the method.

Description

technical field [0001] The invention relates to a method for preparing a monoclonal antibody, in particular to a method for producing a whole porcine monoclonal antibody by using a single B cell PCR antibody technology, and the invention belongs to the field of biotechnology. Background technique [0002] Monoclonal antibody is an important tool in modern life medical science, widely used in diagnosis, treatment, research and other fields. High-throughput single B cell PCR antibody technology is currently the most advanced antibody technology in the world. The main principle is: the B cells in the target population are sorted into a 96-well plate by flow cytometry, and then the antibody gene of a single B cell is cloned into an expression vector by PCR technology, and the antibody is expressed in a mammalian cell protein expression system. This method retains the natural pairing of the light and heavy chain variable regions, and has good gene diversity, high efficiency, and...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12N15/13C12N15/10C12N15/87
CPCC07K16/10C12N15/10C12N15/11C12N15/87
Inventor 刘平黄符芳薛美冯力
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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