Clone, prokaryotic expression and preparation of polyclonal antibody of hCD59
10.3321/j.issn:1007-8738.2009.10.018
- VernacularTitle:人CD59基因克隆、原核表达及其多克隆抗体的制备
- Author:
Jie TANG
;
Ping SUN
;
Xiaolian ZHANG
- Publication Type:Journal Article
- Keywords:
human CD59;
cloning;
polyclonal antibody
- From:
Journal of Cellular and Molecular Immunology
2009;25(10):924-926
- CountryChina
- Language:Chinese
-
Abstract:
AIM: To construct full length hCD59 eukaryotic and extracellular domain of hCD59 (hsCD59) prokaryotic expression vectors and prepare polyclonal antibody of hCD59. METHODS: cDNA fragments encoding hCD59 and hsCD59 were amplified from human PBMCs by RT-PCR and cloned into the eukaryotic vector pVAX-1 and prokaryotic vector pGEX-KG, respectively. The recombinant fusion protein GST-hsCD59 was expressed in E. coil BL21 induced by IPTG. Then the fusion protein was purified and identified. Polyclonal antibody against hCD59 was prepared by immunizing rabbit with pVAX-1-hCD59 and boosting with GSThsCD59 fusion protein, and the titer was identified. RESULTS: The recombinant eukaryotic vector pVAX-1-hCD59 and prokaryotic vector pGEX-KG-hsCD59 were successfully constructed. The GST-hsCD59 fusion protein was over-expressed in E. coli BL21 and the relative molecular mass (M~r) of the expression product was identical with predicted size. The titer of the anti-hCD59 serum was 1:3 200. CONCLUSION: We got the recombinant eukaryotic vector pVAX-1-hCD59, prokaryotic vector pGEX-KG-hsCD59 and rabbit anti-hCD59 polyclonal antibody successfully. These work would be helpful for the further study of the biological function of human CD59.
