Construction of a recombinant plasmid pGEX-Sj26GST of Schistosoma japonicum and expression in Escherichia coli BL21 (DE3) / 中国地方病学杂志

ABSTRACT

Objective To construct and express a recombinant plasmid pGEX-Sj26GST of Schistosoma japonicum(Sj) in Escherichia coli(E.coli) BL21 (DE3).Methods Total RNA was extracted from Sj adult worms by RNeasy Mini kit,26 kilodalton glutathione-S-transferases of Schistosomajaponicum (Sj26GST) antigen gene was amplified by real-time PCR(RT-PCR) from the total RNA,then cloned into a prokaryotic expression plasmid pGEX1λT and transformed into E.coli BL21 (DE3) to construct pGEX-Sj26GST; BL21 (pGEX-Sj26GST) was induced with isopropyl-beta-D-thiogalactopyranoside (IPTG),and the expressed products were analyzed and identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE) and Western blotting.Results The 676 bp Sj26GST gene was successfully amplified by RT-PCR and restriction enzyme double-digestion technique confirmed that Sj26GST antigen gene was successfully cloned into pGEX-1λT vector,the relative molecular mass of the expressed recombinant protein was approximately 52 × 103 by SDS-PAGE,and the amount of expressed protein was 20％ of the total bacterial proteins; the fusion protein could be recognized by sera from rabbits infected with Sj by Western blotting.Conclusions The recombinant plasmid pGEX-Sj26GST is successfully constructed and highly expressed in E.coli and the expressed fusion protein shows specific antigenicity.