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目的 构建含幽门螺杆菌(Hp)尿素酶B亚单位(ureB)基因重组减毒鼠伤寒沙门菌核酸疫苗。方法 抽提Hp标准菌株CCUG17874基因组DNA,应用聚合酶链反应(PCR)技术从基因组DNA扩增ureB基因,克隆入pUCmT载体,检测ureB基因序列,经过酶切、连接反应将其克隆入真核表达载体pIRES,转入感受态大肠杆菌DH5α,筛选阳性克隆,通过PCR和酶切反应进行鉴定。重组载体pIRES ureB转入减毒鼠伤寒沙门菌LB5000,抽提质粒,再次转入SL7207,反复传代,鉴定重组核酸疫苗菌的稳定性。通过脂质体法将构建好的重组载体pIRES ureB转染COS 7细胞,SDS PAGE Western 印迹法检测pIRES ureB表达蛋白的免疫原性。结果 扩增出长约1700 bp的ureB基因,测序结果表明扩增出的ureB基因与基因库Hp ureB序列一致,PCR和酶切鉴定结果证实ureB基因克隆入真核表达载体pIRES,并成功构建了Hp ureB基因的减毒鼠伤寒沙门菌核酸疫苗,重组核酸疫苗稳定,并且Western印迹法检测到特异性的蛋白条带。结论 构建了具有免疫反应性的Hp UreB减毒鼠伤寒沙门菌核酸疫苗,为进一步探索其体内的免疫作用奠定了基础。
Objective To construct a recombinant attenuated Salmonella typhimurium DNA vaccine containing the ureB gene of Helicobacter pylori (Hp). Methods Genomic DNA of Hp standard strain CCUG17874 was extracted. The ureB gene was amplified from genomic DNA by polymerase chain reaction (PCR) and cloned into pUCmT vector. The ureB gene sequence was detected and cloned into eukaryotic expression vector The vector pIRES was transformed into competent E. coli DH5α and positive clones were screened and identified by PCR and restriction enzyme digestion. The recombinant vector pIRES ureB was transferred into attenuated Salmonella typhimurium LB5000, plasmid was extracted, transferred to SL7207 again, and repeatedly passaged to identify the stability of the recombinant nucleic acid vaccine. The constructed recombinant vector pIRES ureB was transfected into COS 7 cells by liposome method, and the immunogenicity of pIRES ureB protein was detected by SDS PAGE Western blotting. Results A ureB gene of about 1700 bp in length was amplified. Sequencing results showed that the amplified ureB gene was consistent with the Hp ureB gene sequence. PCR and restriction enzyme digestion confirmed that the ureB gene was cloned into the eukaryotic expression vector pIRES and successfully constructed Attenuated Salmonella typhimurium nucleic acid vaccine of Hp ureB gene, the recombinant nucleic acid vaccine was stable, and the specific protein band was detected by Western blotting. Conclusion An immunoreactive Hp UreB attenuated Salmonella typhimurium nucleic acid vaccine was constructed, which lays the foundation for further exploration of its immune function in vivo.