Construction and expression of the recombinant plasmid pGEX-Eg95-EgA31 of Echinococcus granulosus in Escherichia coli BL21(DE3).

Objective To construct and express the recombinant plasmid pGEX-Eg95-EgA31 of Echinococcus granulosus in Escherichia coli BL21(DE3).Methods Total RNA was extracted from hydatid cysts by ultrasound-breaking,and the Eg95 and EgA31 antigen genes were amplified by RT-PCR of the total RNA.The Eg95-EgA31 fusion gene obtained with gene SOEing was cloned into the prokaryotic expression plasmid pGEX-1λT and transformed into E.coli BL2(DE3) to construct pGEX-Eg95-EgA31.BL21(pGEX-Eg95-EgA31) was induced with isopropyl-β-D-thiogalactopyranosid(IPTG),and the expressed products were analyzed and identified by SDS-PAGE and Western blot.Results The 1 016 bp Eg95-EgA31 fusion gene was successfully amplified by gene SOEing and cloned into pGEX-1λT by restriction analysis,the recombinant plasmid pGEX-Eg95-EgA31 was successfully constructed.The relative molecular mass of the expressed recombinant protein was approximately 62.5 ku by SDS-PAGE,and the amount of the expressed protein was 18% of the total bacterial proteins.The fusion protein could be recognized by sera from mice infected with E.granulosus by Western blot.Conclusion The recombinant plasmid pGEX-Eg95-EgA31 of E.granulosus is successfully constructed and highly expressed in E.coli in the fused form with GST,and the expressed fusion protein shows specific antigenicity.