摘要
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 。
Objective:To construct nucleic acid vaccine of Helicobacter pylori (Hp) urease B subunit gene(UreB).Methods: The genomic DNA of the standard Hp strain 17 874 was isolated.UreB gene fragm ent was amplified by polymerase chain reaction (PCR) and cloned into PUCmT vector; DNA sequence of the amplified UreB gene was assayed and cloned into the eukaryotic expression vector pIRES.The recombinant plasmid was used to transfor m competent Escherichia coli DH5α,and the postive clones were screened by PCR reaction and restriction enzyme digestion.Then the recombinant pIRES UreB was transfected to COS 7 cells with Lipofectamine TM 2000; the immunogenicity of UreB protein expressed was detected with Western blot.Results: A 1 700 bp UreB gene fragment was amplified from the genomic DNA and was consi stent with the sequence analysis of the Hp UreB.PCR and restriction enzyme diges tion comfirmed that Hp UreB gene was inserted into the eukaryotic expression vec tor pIRES and the nucleic acid vaccine pIRES UreB was successfully constructed;the specific strip of UreB expressed by pIRES UreB was detected by Western blot.Conclusion: The nucleic acid vaccine pIRES UreB with immu nogenicity has been constructed,and it is helpful for further investigation on t he immune action of the nucleic acid vaccine.
出处
《第二军医大学学报》
CAS
CSCD
北大核心
2004年第1期51-54,共4页
Academic Journal of Second Military Medical University
基金
国家自然科学基金 ( 3 0 170 42 7)
