Construction and expression of two kinds of HE4 fusion protein / 中国免疫学杂志

ABSTRACT

Objective:To express human epididymis protein 4 (HE4) in prokaryotic cells,purify the expressed product and de-termine its activity by immunoassay kit.Methods: The gene encoding HE 4 was cloned using RT-PCR technique from total RNA of ovarian carcinoma cells ES-2,the amplified HE4 gene was cloned into prokaryotic expression vector pGEX-4T-1 and PET26b respective-ly.The recombinant plasmid pGEX-4T-1-HE4 and PET26b-HE4 were constructed and transformed into E.coli BL21 cells respectively, and protein ( GST-HE4 and His-HE4 ) expressions were induced by IPTG and identified by SDS-PAGE and commercial ELISA kit.Results:Restriction analysis and sequencing proved that recombinant plasmid pGEX -4T-1-HE4 and PET26b-HE4 were constructed correctly.The expressed recombinant proteins ,with the relative molecular mass of about 38 000 and 12 000 ,showed specific binding to monoclonal antibody against HE 4.Conclusion:Two kinds of recombinant HE 4 protein are successfully expressed in prokaryotic cells , which laid a foundation of preparation of immunoassay reagents.