Preparation of rabbit-anti human ERA serum / 细胞与分子免疫学杂志

ABSTRACT

Aim To express h-ERA and h-ERA C-terminal domain in E.coli and prepare antiserum against human ERA. Methods Human era(h-era) cDNA and h-ERA C-terminal domain gene were amplified by PCR and ligated with prokaryotic expression vector pRSET-C and pDH respectively. E.coli TAP106 transformed with the recombinant plasmid pDH-h-era-C was induced at 42℃ to express h-ERA C-terminal domain protein. Antiserum against h-ERA was prepared by immunizing rabbit with h-ERA C-terminal domain protein purified by SDS-PAGE. E.coli BL21(DE3) transformed with the recombinant plasmid pRSET-C-h-era was induced with IPTG to express (His)6-h-ERA fusion protein for Western-blot analysis.Results The expressed h-ERA C-terminal domain and (His)6-h-ERA fusion protein occupied about 40％ and 80％ of total bacterial protein respectively. The(His)6-h-ERA fusion protein expressed in E.coli can be detected with the rabbit antiserum. Conclusion The h-ERA C-terminal domain protein and (His)6-h-ERA fusion protein were expressed with high efficiency in E.coli, and the rabbit antiserum against h-ERA was prepared successfully.