Construction and expression of a recombinant plasmid pGEX-Sj14-3-3-Sj32 of Schistosoma japonicum in Escherichia coli BL21(DE3) / 中华地方病学杂志

ABSTRACT

Objective To construct and express a recombinant plasmid pGEX-Sj 14-3-3-Sj32 of Schistosoma japonicum in Escherichia coli (E.Coli) BL21 (DE3).Methods Sj14-3-3 and Sj32 antigen genes were amplified by PCR from template of plasmids pGEX-Sj14-3-3 and pET28α-Sj32 which were extracted from recombinant bacteria BL21 (pET28α-Sj32) and BL21 (pGEX-Sj14-3-3) stored in Institute of Infectious and Parasitic Disease of the First Affiliated Hospital of Chongqing Medical University.Sj14-3-3-Sj32 fusion gene obtained with gene SOEing was cloned into the vector pGEX-1λT to construct pGEX-Sj14-3-3-Sj32 which was identified by double digestion.The recombinant plasmid pGEX-Sj 14-3-3-Sj32 was transformed into E.Coli BL21 (DE3).The recombinant strains were induced by isopropyl-β-d-thiogalactoside (IPTG),and the expressed products were analyzed and identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting.Results The 1 750 bp Sj14-3-3-Sj32 fusion gene was successfully amplified by gene SOEing and cloned into the vector pGEX-1 λT verified by restriction analysis,the recombinant plasmid pGEX-Sj 14-3-3-Sj32 was successfully constructed.The molecular mass of the expressed recombinant protein was proximately 73 × 103 as detected by SDS-PAGE.Western blotting confirmed that the expressed protein could be recognized by the immune sera from rabbit infected with Schistosomajaponicum.Conclusion The recombinant plasmid pGEX-Sj14-3-3-Sj32 is successfully constructed and could be highly expressed in E.coli and the expressed recombinant protein has specific antigenicity.