Construction and expression of the recombinant plasmid pET28α-Sj32 of Schistosoma japonicum in Escherichia coli BL21(DE3)

Objective To construct and express the recombinant plasmid pQE30-Sj26GST-Sj32 of Schistosoma japonicum(Sj) in Escherichia coli BL21(DE3).Methods The total RNA was extracted from Sj adult worms by ultrasound-breaking,Sj26GST and Sj32 antigen gene was respectively amplified by RT-PCR from the total RNA;Sj26GST-Sj32 fusion gene was obtained with gene SOEing;the fusion gene was cloned into prokaryotic expression plasmid pQE30 to construct pQE30-Sj26GST-Sj32;the recombinant plasmid was transformed into E.coli BL2(DE3);the rBL21(pQE30-Sj26GST-Sj32) was induced with isopropyl-β-D-thiogalactopyranosid(IPTG),the expressed products were analyzed and identified by SDS-PAGE and Western blot.Results The 1 991 bp Sj26GST-Sj32 fusion gene was successfully amplified by gene SOEing and cloned into pQE30 by restriction analysis and PCR identification,the recombinant plasmid pQE30-Sj26GST-Sj32 was successfully constructed;the relative molecular mass of the expressed recombinant protein was approximately 61 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 rabbits infected with Sj by Western blot.Conclusion The recombinant plasmid pQE30-Sj26GST-Sj32 is successfully constructed and highly expressed in E.coli in fused form with His-tag,and the expressed fusion protein shows specific antigenicity.