论文部分内容阅读
目的 构建表达幽门螺杆菌 (Helicobacterpylori,Hp)尿素酶B亚单位 (ureB)基因的重组减毒鼠伤寒沙门疫苗菌。方法 用PCR扩增ureB基因 ,经适当酶切 连接反应将其克隆入高效原核表达质粒 pTrc99A ,进行基因测序 ,重组质粒鉴定后再导入减毒鼠伤寒沙门菌SL32 6 1,提取重组疫苗菌质粒 ,PCR和酶切鉴定 ,筛选阳性克隆。重组ureB能在宿主菌中表达 ,用十二烷基硫酸钠 聚丙烯酰胺凝胶电泳 (SDS PAGE)和薄层扫描进行蛋白分析。重组菌C5 7BL/ 6小鼠喂灌 ,分批 2d和 10d后处死小鼠 ,取脾和末段回肠进行细菌培养 ,挑菌落提质粒鉴定。结果 构建携带ureB的重组原核表达质粒pTrc99A ureB ,并将后者成功转化入减毒鼠伤寒沙门菌SL32 6 1,阳性克隆经PCR和酶切证实。SDS PAGE电泳和薄层扫描分析表明 ,重组菌SL32 6 1(pTrc99A ureB)表达相对分子质量为6 6× 10 3的ureB ,约占菌体蛋白总量的 2 6 %。小鼠重组菌喂灌 2d或 10d后 ,脾和末段回肠均发现携目的基因的菌落。结论 构建表达HpureB的减毒鼠伤寒沙门疫苗菌 ,为探索制备Hp口服活疫苗奠定了基础。
Objective To construct a recombinant attenuated Salmonella typhimurium vaccine strain expressing the ureB gene of Helicobacter pylori (Hp). Methods UreB gene was amplified by PCR and cloned into the highly efficient prokaryotic expression plasmid pTrc99A by restriction enzyme digestion. The recombinant plasmids were identified by PCR and then introduced into attenuated Salmonella typhimurium SL32 6 1. The recombinant plasmids were extracted, PCR and restriction enzyme digestion, screening positive clones. Recombinant ureB can be expressed in host bacteria and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) and thin layer scanning. Recombinant bacteria C5 7BL / 6 mice were fed, mice were sacrificed 2d and 10d after batches, and the spleen and the distal ileum were taken for bacterial culture. Results The recombinant plasmid pTrc99A ureB carrying ureB was constructed and successfully transformed into attenuated Salmonella typhimurium SL32 6 1. The positive clones were confirmed by PCR and restriction enzyme digestion. SDS PAGE electrophoresis and thin layer scanning analysis showed that the recombinant strain SL32 6 1 (pTrc99A ureB) expressed a relative molecular weight of 6 6 × 10 3 ureB, accounting for 26% of the total bacterial protein. Mouse recombinant bacteria 2d or 10d after feeding, the spleen and the terminal ileum were found to carry the target gene colonies. Conclusion The construction of an attenuated Salmonella typhimurium vaccine expressing HpureB laid the foundation for the development of an oral Hp vaccine.