Constuction And Expression Of The Recombinant Bb(pGEX-Sj14-3-3-Sj32)Vaccine Of Schistosoma Japonicum

Objective Schistosomiasis is a worldwide spread and serious health-threatening parasitic zoonoses for human bings. Schistosoma japonicum, which is the most popular species in China, is still a serious public health problem.Currently, the main treatment on schistosomisis in China is praziquantel chemotherapy and snail control which are effective but can not achieve the goal of complete elimination of schistosomiasis. However the research of vaccine against schistomiasis may brings hopes to it. Due to various deficiencies, there is no safe and effective vaccines used in clinical for schistosomiasis up to now. Our study intends to amplify the Sj14-3-3 and Sj32 genes and transform it into Bifidobacterium bifidum(Bb) as the carrier to construct the recombinant Bb(pGEX-Sj14-3-3-Sj32) vaccine of Schistosoma japonicum and analyze the expression efficiency of the fusion gene Sj14-3-3-Sj32 in r Bb, to lay the foundation for the research of schistosomiasis vaccine.Methods1.Sj14-3-3 and Sj32 antigen encoding gene were amplified by PCR from template of plasmid pGEX-Sj14-3-3 and p ET28α-Sj32 respectively,Sj14-3-3-Sj32 fusion gene obtained with gene SOEing was cloned into the vector pGEX-1λT to construct pGEX-Sj14-3-3-Sj32.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 SDS-PAGE and Western blot.2.The recombinant plasmid pGEX-Sj14-3-3-Sj32 of Schistosoma japonicum was electroporated into Bifidobacteria bifidum to construct recombinant Bb(pGEX-Sj14-3-3-Sj32) vaccine. After induction with IPTG,the expression of the recombinant protein was identified by SDS-PAGE and Western blot assay.Results1.The 1750 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 73 000 Dolton as detected by SDS-PAGE. Western blot confirmed that the expressed protein could be recognized by the immune sera from rabbit infected with Schistosoma japonicum.2.Restriction analysis and PCR confirmed that the recombinant plasmid pGEX-Sj14-3-3-Sj32 was successfully transformed into Bb. SDS-PAGE analysis showed that the relative molecular weight(Mr) of the expressed recombinant protein was approximately 73 k Da, and the amount of the expressed protein was 8.2% of the total bacterial protein. Western blot confirmed that the expressed protein could be recognized by the immune sera from rabbit infected with Schistosoma japonicum.Conclusion1.The fusion gene Sj14-3-3-Sj32 was successfully synthesized and connected with vector to successfully get the recombinant plasmid pGEX-Sj-14-3-3-Sj32.2.The recombinant plasmid pGEX-Sj14-3-3-Sj32 could be highly expressed in E.coli and the expressed recombinant protein possessed specific antigenicity.3.The recombinant Bb(pGEX-Sj14-3-3-Sj32) vaccine of Schistosoma japonicum is successfully constructed. The fusion gene Sj14-3-3-Sj32 can be expressed in r Bb and the expressed target protein shows specific antigenicity.