Construction and expression of the recombinant plasmid pET32α-Sj26GST-Sj32 of Schistosoma japonicum in Escherichia coli BL21 (DE3).

Objective To construct and express the recombinant plasmid pET32α-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 were respectively amplified by RT-PCR from the total RNA. Sj26GST-Sj32 fusion gene obtained with gene SOEing was cloned into prokaryotic expression plasmid pET32α(+) and transformed into E.coli BL2(DE3) to construct pET32α-Sj26GST-Sj32. The transformed BL21 with pET32α-Sj26GST-Sj32 was induced with isopropyl-β-D-thiogalactopyranosid (IPTG),and the expressed products were analyzed and identified by SDS-PAGE and Western-blot, respectively. Results The 1991 bp Sj26GST-Sj32 fusion gene was successfully amplified by gene SOEing and cloned into pET32α (+) and confirmed by restriction analysis and PCR identification.The molecular mass of the expressed recombinant protein was approximately 82000 Mr by SDS-PAGE, and the amount of the expressed protein was 22% of the total bacterial proteins. The fusion protein could be recognized by sera from rabbits infected with Sj in Western-blot. Conclusion The recombinant plasmid pET32α-Sj26GST-Sj32 was successfully constructed and could be highly expressed in E.coli producing Sj26GST-Sj32 fused form with Trx-tag and His-tag, with specific antigenicity.