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


CONCLUSIONS
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.
Tang
Jie
J
Department of Immunology, State Key Laboratory of Virology, Wuhan University School of Medicine, Wuhan 430071, China. tjandbetty@sina.com
Sun
Ping
P
Zhang
Xiao-Lian
XL
D000818:Animals; D000906:Antibodies / Q000276:immunology*; D018957:CD59 Antigens / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics*; D006801:Humans; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification
D000906:Antibodies; D018957:CD59 Antigens; D011993:Recombinant Fusion Proteins