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目的 :构建含幽门螺杆菌 (H elicobacter pylori,Hp)尿素酶 B亚单位 (Ure B)基因的核酸疫苗。方法 :抽提 Hp标准菌株 CCUG1 7874基因组 DNA,应用 PCR技术从基因组 DNA扩增 U re B基因 ,克隆入 PUCm T载体 ,检测 U re B基因序列。经过一系列酶切、连接反应将其克隆入真核表达载体 p IRES,转入感受态大肠杆菌 DH5 α,筛选阳性克隆 ,通过 PCR和酶切反应进行鉴定。通过脂质体法将构建好的重组载体 p IRES- Ure B转染 COS- 7细胞 ,Western印迹分析检测 p IRES- U re B表达 Ure B蛋白的免疫原性。 结果 :扩增出长约 1 70 0 bp的 U re B基因 ,与基因库 Hp U re B序列一致 ,PCR和酶切鉴定结果证实成功构建了含 U re B基因的 Hp核酸疫苗 p IRES- U re B,并且 Western印迹分析检测到特异性的蛋白条带。结论 :构建了具有免疫反应性 Ure B基因的 Hp核酸疫苗 ,为进一步探索其免疫作用奠定了基础
Objective: To construct a nucleic acid vaccine containing Ure B gene of H elicobacter pylori (Hp). Methods: Genomic DNA of Hp standard strain CCUG1 7874 was extracted. U re B gene was amplified from genomic DNA by PCR and cloned into PUCm T vector to detect U re B gene sequence. After a series of enzymatic digestion and ligation reaction, it was cloned into the eukaryotic expression vector pIRES and transformed into competent E. coli DH5α. The positive clones were screened and identified by PCR and restriction enzyme digestion. The constructed recombinant vector pIRES-Ure B was transfected into COS-7 cells by liposome method, and the immunogenicity of pIRES-U re B expressing Ure B protein was detected by Western blot analysis. Results: Ure B gene was amplified with a length of about 1 70 bp and was consistent with the Hp U re B sequence of the gene pool. PCR and restriction enzyme digestion confirmed that the Hp DNA vaccine p IRES-U containing the U re B gene was successfully constructed re B, and Western blot analysis detected specific protein bands. Conclusion: The Hp nucleic acid vaccine with immunoreactive Ure B gene was constructed, which laid the foundation for further exploring its immunological function