Construction and expression of a recombinant plasmid pGEX-Sj14-3-3-Sj32 of Schistosoma japonicum in Escherichia coli BL21 (DE3)

Abstract

Objective To construct and express a recombinant plasmid pGEX-Sj14-3-3-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-Sj14-3-3-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-1λT verified by restriction analysis, the recombinant plasmid pGEX-Sj14-3-3-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 Schistosoma japonicum. 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. Key words: Schistosoma japonicum; Plasmids; Escherichia coli; Gene expression