Prokaryotic expression of human scavenger receptor A extra-cellular domain

Objective To express the extracellular domain of human scavenger receptor A(SRA) in E.coli.Methods The gene fragment of the SRA was amplified by PCR from the human PBMC cDNA,and then inserted into the prokaryotic expression vector pET41a(+) and identified by PCR,restriction mapping,and sequencing.The recombinant expression vector was transformed into E.coli strain BL21(DE3) and induced to express the recombinant protein by IPTG.The fusion protein was purified by Ni-NTA gel affinity chromatography from denatured and renatured products of inclusion bodies and analyzed by SDS-PAGE and western blotting.ELISA and FCM determine the effect of expressed protein on ConA-induced PBMC proliferation and IL-2 production.Results A gene fragment of about 1 100 bp was amplified by PCR and the recombinant expression vector pET41a-SRAECD was constructed,and was confirmed by restriction maping and sequencing.The expressed products in BL21(DE3) were existed as inclusion body,from which the His-SRAECD fusion protein of Mr 75 000 were obtained by Ni-NTA gel affinity chromatography.The purified human SRAECD protein could react with anti-SRA monoclonal antibody and inhibit ConA activated T cell proliferation and the secretion of cytokines IL-2.Conclusion The recombinant human His-SRAECD protein with bioactivities is obtained,which provides the necessary material for studying the functions of SRA.