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Updated: Jun 19, 2026
Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood
Published on: February 6, 2018
[Clone, prokaryotic expression and preparation of polyclonal antibody of hCD59]
Jie Tang1, Ping Sun, Xiao-Lian Zhang
1Department of Immunology, State Key Laboratory of Virology, Wuhan University School of Medicine, Wuhan 430071, China. tjandbetty@sina.com
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.
