[Clone, prokaryotic expression and preparation of polyclonal antibody of hCD59]

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 GST-hsCD59 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.