Construction and Expression of the Recombinant Plasmid pGEX-Eg95 in Escherichia coli BL21(DE3)

Objective To construct and express the recombinant plasmid pGEX-Eg95 in Escherichia coli BL21(DE3).Methods Total RNA was extracted from hydatid cyst of Echinococcus granulosus by ultrasonication method.Eg95 gene,amplified by RT-PCR from the total RNA,was cloned into prokaryotic expression plasmid pGEX-1λT and transformed into E.coli BL2(DE3) to form pGEX-Eg95.BL21(pGEX-Eg95) was then induced with isopropyl-β-D-thiogalactopyranosid(IPTG),and the expressed products were analyzed and identified by SDS-PAGE and Western blot.Results Eg95 gene with the size of 471 bp was successfully amplified by RT-PCR and cloned into pGEX-1λT,and the recombinant plasmid pGEX-Eg95 was successfully constructed.The molecular mass of the expressed recombinant protein was approximately 42 500 as determined by SDS-PAGE.Amount of the expressed protein was 21% of the total bacterial proteins.Western blot analysis showed that tThe expressed fusion protein could be recognized by the immune sera from mice infected with E.granulosus.Conclusion The plasmid pGEX-Eg95 was successfully constructed and the fusion protein was highly expressed in E.coli.The expressed recombinant protein was also immunogenic.