幽门螺杆菌ureB及ureB-hspA融合基因在减毒鼠伤寒沙门菌中的表达及小鼠的免疫应答

目的　构建H .pyloriureB单基因及ureB和hspA融合基因的减毒鼠伤寒沙门菌疫苗候选株 ,并进行初步的免疫原性分析。方法　将H .pyloriureB单基因及ureB和hspA融合基因分别克隆入pTrc99A asd质粒的多克隆位点之内。挑取单菌落质粒鉴定后 ,分别将其电击经中间宿主X3730转入最终宿主X4 0 72 ,SDS PAGE和Westernblot检测蛋白表达。将疫苗候选株经口或鼻免疫BALB c小鼠。结果　疫苗候选株能够分别表达出相对分子质量 (Mr)为 6 4× 10 3的UreB蛋白及 77× 10 3的UreB HspA融合蛋白。在免疫BALB c小鼠的肠液和血清中可以分别检测到针对H .pylori的特异性分泌型IgA和IgG抗体。结论　构建了能够表达幽门螺杆菌UreB及UreB HspA融合蛋白的活菌疫苗候选株 ,为探索制备H .pylori口服活菌疫苗奠定了基础。

Construction of live recombinant attenuated Salmoella typhimurium vaccine candidate strains expressing ureB and ureB-hspA from Helicobacter pylori

Objective To construct live recombinant attenuated Salmoella typhimurium vaccine strains expressing ureB and ureB-hspA from H.pylori. Methods ureB gene and ureB-hspA fusion gene were amplified by PCR and cloned into a prokaryotic expression plasmid pTrc99A-asd. The recombinant plasmid was identified and then used to transform the attenuated S.typhimurium X3730 and S.typhimurium X4072. The recombinant vaccine strain was analyzed by SDS-PAGE and Western blot. Immunize mice with the recombinant bacteria either orogastrically (o.g) or intranasally (i.n). Results The ureB and ureB-uspA were expressed in the recombinant vaccine strain X4072 as proteins of 64kD and 77kD. Western blot analysis of UreB and UreB-HspA recombinant proteins confirmed that those specially recognized by serum from H.pylori-infected patients. Antibodies against H.pylori whole-cell sonication were detected in the sera of mice immunized with recombinant bacteria either o.g or i.n; simultaneously sIgA against H.pylori also detected in the intestine. Conclusion Recombinant live attenuated S.typhimurium vaccine strains expressing H.pylori UreB and UreB-HspA will help to develop an oral recombinant live vaccine for the control of H.pylori-related diseases in humans.