Expression and biological activity analysis of human-mouse chimeric antibody against anthrax protective antigen
10.3760/cma.j.issn.0254-5101.2009.12.004
- VernacularTitle:抗炭疽保护性抗原人-鼠嵌合抗体在CHO细胞中的表达和活性研究
- Author:
Bing LI
;
Jianmin LI
;
Jun ZHANG
;
Junjie XU
;
Shuling LIU
;
Jun REN
;
Jinlong ZHANG
;
Ling FU
;
Lihua HOU
;
Wei CHEN
- Publication Type:Journal Article
- Keywords:
Anthrax;
Human-mouse chimeric antibody;
CHO cells
- From:
Chinese Journal of Microbiology and Immunology
2009;29(12):1069-1074
- CountryChina
- Language:Chinese
-
Abstract:
Objective To express human-mouse chimeric antibody against anthrax protective anti-gen and to analyze its biological activities. Methods A new mammalian bipromoter expression vector was constructed with dihydrofolate reduetase(DHFR) gene as the selection and complication marker. First, the light and heavy chain variable region gene of the monoclonal antibody 5E1 were cloned by RT-PCR, at the same time the human IgG1 heavy chain constant region gene and kappa type constant region gene were cloned. Next, the human-mouse chimeric antibody genes were synthesized by fusion PCR. Then, the hu-man-mouse chimeric antibody gene were inserted into MCS of pSecTag and B1 to construct pSecTag-5E1L and B1-5E1H, respectively. Finally, heavy chain expression cassette excised from the B1-5E1H with Bgl Ⅱ/BamH Ⅰ was further cloned into the Bgl Ⅱ site of the pSecTag-5E1L to construct pSecTag-5E1. Plasmid pSecTag-5E1 was transfected into CHO(dhfr) engineering cells and high production cell clones that were screened by enhancing MTX concentration. After collecting medium and purifying chimeric antibody with af-finity chromatogram, purified chimeric antibody was analyzed by SDS-PAGE, Western blot. Results A sta-ble and high production cell line was acquired at MTX concentration 5×10~(-8) mol/L. Conclusion The hu-man-mouse chimeric antibodies were successfully expressed in CHO cells.
