1.High level expression of 6His-hEra
Zongling JI ; Sumin CHEN ; Jizhong LIU ; Al ET
Chinese Journal of Immunology 2000;0(11):-
Objective:To study the expression of human Era.Methods:The encoding region of human Era gene was inserted into pUC19 plasmid and sequenced,the gene fragment was then cloned into the prokaryotic fusion expression vector pRSET B to contruct recombinant expression plasmid pRSET B hEra.The recombinant protein was expressed in E.coli BL21DE3(plys S).Results:6His hEra was expressed after induced by IPTG and took 51.2% of the total cell lysate and mainly existed in the inclusion body.Conclusion:High level of human Era was established and this result may shed light on the further study on function of human Era.
2.Preparation of rabbit-anti human ERA serum
Junjie ZHANG ; Yuanming WU ; Huiping LIU ; Zongling JI ; Nanchun CHEN ; Sumin CHEN
Journal of Cellular and Molecular Immunology 2001;17(1):73-75
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.