表达幽门螺杆菌尿素酶B亚单位基因减毒鼠伤寒沙门疫苗菌的构建和鉴定

Construction and identification of recombinant attenuated Salmonella typhimurium vaccine strain expressing Helicobacter pylori urease subunit B

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 live attenuated Salmonella typhimurium vaccine strain expressing Helicobacter pylori urease subunit B(ureB). Methods By genetic engineering methods, ureB gene was amplified by PCR and cloned into a prokaryotic expression plasmid pTrc99A, and the identified recombinant plasmid was then used to transform an attenuated Salmonella typhimurium vaccine strain SL3261, and the positive clones were screened by PCR and restriction enzyme digestion. The ureB expressed in the recombinant vaccine strain was analyzed by SDS PAGE and optical density scanning. Two and 10 days after recombinant strain intragastric immunization, the C57BL/6 mice were sacrificed, and the spleens and terminal ileums were cultured for recombinant strain. Results The ureB gene could be amplified from the recombinant prokaryotic expression plasmid pTrc99A ureB and the plasmids extracted from transformed SL3261 strain by PCR, and also the ureB gene flagment could be produced from these plasmids after restriction enzyme digestion. SDS PAGE and optical density scanning indicated that ureB was expressed in the recombinant vaccine strain SL3261 (pTrc99A ureB) as a protein with 66 kD of molecular weight and was 26% of the total bacterial protein. Recombinant strain was found in both spleen and terminal ileum of each mouse two and ten days after intragastric immunization. Conclusions A recombinant liver attenuated Salmonella typhimurium vaccine strain expressing Helicobacter pylori ureB was constructed and identified, and this study will help to develop an oral recombinant live vaccine against Helicobacter pylori infection.