携带幽门螺杆菌ureB和IL-2质粒DNA的减毒鼠伤寒沙门菌疫苗构建

目的　构建编码幽门螺杆菌 (Helicobacterpylori,H .pylori)尿素酶B亚单位 (ureB)基因和小鼠IL 2基因以减毒鼠伤寒沙门菌为载体的核酸疫苗 ,体外转染Cos 7细胞 ,鉴定其表达蛋白的免疫性。方法　应用聚合酶链式反应 (PCR)技术从H .pylori标准菌株CCUG1 7874基因组DNA扩增ureB基因 ,从重组质粒 pCIneo IL 2扩增小鼠IL 2基因 ,通过T A克隆分别插入 pUCmT载体 ,检测ureB及IL 2的核苷酸序列 ,通过酶切、连接反应分别克隆入真核表达载体 pIRES ,PCR和酶切反应进行鉴定 ;重组载体 pIRES ureB和 pIRES ureB IL 2转入减毒鼠伤寒沙门菌LB50 0 0 ,抽提质粒 ,再次转入SL72 0 7,反复传代 ,鉴定核酸疫苗载体菌的稳定性。通过脂质体法将重组载体 pIRES ureB和pIRES ureB IL 2转染Cos 7细胞 ,SDS PAGE及Westernblot法检测表达蛋白的免疫性。结果　扩增出长约 1 70 0bp的ureB基因和 51 0bpIL 2 ,测序结果表明 ,扩增出的ureB基因与基因库H .pyloriureB序列一致 ,IL 2序列和小鼠的IL 2序列一致 ,PCR和酶切鉴定结果证实ureB和IL 2基因克隆入真核表达载体 pIRES ,并成功构建了稳定的幽门螺杆菌ureB和IL 2基因以减毒鼠伤寒沙门菌为载体的核酸疫苗 ,并且以Westernblot检测到特异性的相对分子质量 (Mr)为 6 6× 1 0 3的UreB蛋白

The development of an attenuated Salmonella typhimurium vaccine carrying plamids encoding Helicobacter pylori ureB and IL-2

Objective To construct a recombinant live attenuated Salmonella typhimurium nucleic acid vaccine encoding H.pylori ureB gene and murine interleukin 2 and to identify the immunogenicity of the proteins expressed. Methods ureB gene fragment was amplified with polymerase chain reaction (PCR) from the genomic DNA of standard H.pylori strain 17874 and murine IL 2 gene was amplified from pCIneo IL 2. ureB and IL 2 were cloned into pUCmT vector respectively. Both the genes of ureB and IL 2 were cloned into the eukaryotic expression vector pIRES through a serials of enzyme digestion and ligation reactions, the recombinant plasmids were screened by PCR reaction and restriction enzyme digestion. Then, the recombinant pIRES ureB and pIRES ureB IL 2 were transformed into LB5000 and the recombinant plasmids from LB5000 were then transformed into final host SL7207. The strains were grown in LB medium repeatedly. Recombinant pIRES ureB IL 2 and pIRES ureB were transfected into Cos 7 cells using LipofectAMINE TM 2000, the immunogenicity of UreB and IL 2 protein expressed was detected with SDS PAGE and Western blot. Results The 1700 base pair ureB gene fragment and 510 bp IL 2 were amplified and they consistent with the sequence of the H.pylori ureB and murine IL 2 respectively. It was confirmed with PCR and restriction enzyme digestion that H.pylori ureB gene and murine IL 2 were inserted into the eukaryotic expression vector pIRES and a stable recombinant live attenuated Salmonella typhimurium nucleic acid vaccine encoding H.pylori ureB and murine IL 2 was successfully constructed and specific strip of 66kD ureB and 14kD IL 2 was detected through Western blot. Conclusion The recombinant attenuated Salmonella typhimurium nucleic acid vaccine strain encoding H.pylori ureB and immune adjuvant IL 2 with immunogenicity in vitro was constructed, it may be helpful for further investigation about the immune function of the nucleic acid vaccine in vivo .