Construction And Expression Of The Recombinant Bb(pGEX-Sj26GST-Sjl4-3-3)Vaccine Of Schistosoma Japonicum

Objective Schistosomiasis japonicum,mainly prevalent in Indonesia,China,Philippines and other Asian countries and regions,is a chronic parasite caused by Schistosoma japonicum(Sj)and becomes one of the major public health problems in our country.Over many years,the comprehensive measures such as drugs,snail control,health education and environmental renovation have been adopted in our country and made remarkable achievements.However,these measures can't completely eradicate this disease.Thus,the vaccine has become an important direction of prevention and control of the disease.Our study intends to amplify two antigen encoding genes of Sj26 GST and Sj14-3-3,to transform it into Bifidobacterium bifidum(Bb)to construct the recombinant Bb(p GEX-Sj26GST-Sj14-3-3)vaccine and analyze the expression efficiency of the fusion gene Sj26GST-Sj14-3-3 in Bb,in order to lay the foundation for further study on mechanism of vaccine immunity.Methods Sj26GST and Sj14-3-3 genewere amplified by PCR from template of recombinantplasmid p ET28?-Sj26 GST and p ET28?-Sj14-3-3 stored by our laboratory respectively.Sj26GST-Sj14-3-3was obtained by the fusion of two single genes with gene SOEing.Then the fusion gene was cloned into Escherichia coli-Bifidobacterium shuttle plasmidp GEX-1?T to constructp GEX-Sj26GST-Sj14-3-3.Therecombinant plasmid was transformed into E.coli BL21.BL21(p GEX-Sj26GST-Sj14-3-3)was induced by isopropyl-?-D-thiogalactoside(IPTG)and the expressed products were analyzed and identified by SDS-PAGE and Western blot.The recombinant plasmid p GEX-Sj26GST-Sj14-3-3 was electroporated into Bifidobacterium bifidum to construct recombinant Bb(p GEXSj26GST-Sj14-3-3)vaccine.The plasmid p GEX-Sj26GST-Sj14-3-3 from recombinant Bb was extracted and identified by PCR and restriction analysis.Thenthe vaccine was induced by IPTG and the expressed products were analyzed and identified by SDS-PAGE and Western blot.Result The 1120 bp Sj26GST-Sj14-3-3 fusion gene was successfully amplifiedand cloned into the vector p GEX-1?T,the recombinant plasmid p GEX-Sj26GST-Sj14-3-3was constructed.Therelative molecular mass(Mr)of the expressed recombinant protein was proximately 67 k Da as detected by SDS-PAGE analysis and highly expressed in 5~7h after induction.The amount of the expressed protein was 19% of the total bacterial protein.Thefusion protein could be recognized by the immune sera from rabbits infected with Schistosoma japonicum by Western blot.The recombinant plasmid p GEX-Sj26GST-Sj14-3-3 was successfullytransformed into Bb by restriction analysis and PCR identification.Therelative molecular mass of the expressed recombinant protein was approximately 67 k Da as detected by SDS-PAGE analysis and highly expressed in 3~4d after induction.The amount of the expressed protein was 7.9% of the total bacterial protein.The fusion protein could be recognized by the immune sera from rabbits infected with Schistosomajaponicum by Western blot.Conclusion 1.The recombinant plasmid p GEX-Sj26GST-Sj14-3-3 is successfully constructed and highly expressed in E.coli and the expressed fusion protein shows specific antigenicity.2.The recombinant Bb(p GEX-Sj26GST-Sj14-3-3)vaccine of Schistosoma japonicum is successfully constructed and the fusion gene Sj26GST-Sj14-3-3 can be expressed in r Bb and shows specific antigenicity.