表达幽门螺杆菌hpaA重组减毒鼠伤寒沙门氏菌的构建和鉴定

目的：构建表达幽门螺杆菌（H.pylori)hpaA的重组减毒鼠伤寒沙门低 疫苗菌。方法：用基因工程的方法将hpaA基因克隆入原核表达质粒pTrc99A,并进行了基因测序。重组质粒经鉴定后再导入减毒鼠伤寒沙门氏菌SL3261，提取重组疫苗菌质粒，PCR和酶切鉴定，筛选阳性克隆。用SDS－PAGE电泳和Western blot进行HpaA表达和鉴定。结果：经PCR和酶切证实，构建了含hpaA的重组原核表达质粒pTrc99A-hpaA，并将后者成功转化了减毒鼠伤寒沙门氏菌。HpaA能在疫苗菌中以二聚体形式表达，HpaA量约占全菌体蛋白量的17％，Westem blot证实其有免疫原性。结论：构建了表达H.pylori hpaA的重组减毒鼠伤寒沙门氏疫苗菌，为探索制备H.pylori口服活疫苗尊定了基础。

Construction andidentification of a recombinant attenuated Salmonella typhimurium vaccine strainexpressing Helicobacter pylori hpaA gene

Objective:To construct a recombinant live attenuated Salmonella typhimurium vaccine strain expressing Helicobacter pylori hpaA gene.Methods:By genetic engineering methods,hpaA gene was cloned into a procaryotic 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. HpaA expression was analyzed by SDS PAGE and Western blot.Results:Confirmed by PCR and restriction enzyme digestion ,a recombinant procaryotic expression plasmid pTrc99A hpaA was constructed,and then introduced into an attenuated Salmonella typhimurium vaccine strain SL3261 successfully. HpaA was expressed in the recombinant strains in the form of a dimer,and also its immunogenicity was confirmed by Western blot. Conclusion:A recombinant live attenuated Salmonella typhimurium vaccine strain expressing Helicobacter pylori hpaA gene was constructed and identified,and this work will help to develop an oral recombinant live vaccine against Helicobacter pylori.