摘要
目的构建幽门螺杆菌(H.pylori)尿素通道蛋白编码基因(ureI)的真核表达重组载体,并在HeLa细胞中表达,为核酸疫苗的研制奠定基础。方法以H.pyloriSS1株基因组为模板,PCR扩增ureI目的基因片段,定向插入真核表达载体pcDNA3.1(+)多克隆酶切位点中,构建重组载体pcDNA3.1(+)/ureI;通过酶切、PCR及测序鉴定,筛选阳性重组载体;脂质体法转染重组质粒入HeLa细胞,Western blot检测其在HeLa细胞中的表达。结果以H.pyloriSS1株基因组DNA为模板扩增出特异的ureI基因片段,大小约585 bp;双酶切及测序鉴定证明成功构建了H.pylori真核表达重组载体pcDNA3.1(+)/ureI;该重组质粒能够在HeLa细胞中表达目的蛋白。结论成功构建了真核重组载体pcDNA3.1(+)/ureI,并能在HeLa细胞中表达。
Objective To construct the recombinant plasmid containing the ureI gene of Helicobacter pylori （ H. pflori）, and to detect its expression in HeLa cells, and to lay a foundation for furture studying the H. pylori DNA vaccine. Methods The ureI gene of H. pylori SS1 was amplified by polymerase chain reaction （ PCR）. The PCR product was subcloned into appropriate site of pcDNA3.1 （ ＋ ） eukaryotic expression vector by restriction enzyme digestion and linking reactions. The positive recombinant was identified by restriction endonuclease digestion, PCR amplification and sequencing. The recombinant plasmid was transfected into HeLa cells with Liposome. Western blot was applied to detect the expression of ureI gene in HeLa cells. Results A 585 bp ureI specific gene fragment was amplified. Restriction enzyme analysis and sequencing showed that the eukaryotic expression recombinant pcDNA3.1 （ ＋ ） - ureI was successfully constructed. The target recombinant protein could be expressed effectively in HeLa cells. Conclusion The recombinant pcDNA3.1 （ ＋ ）/ ureI plasmid was successfully constructed and could be expressed in Hela cells.
出处
《南华大学学报（医学版）》
2010年第2期155-158,共4页
Journal of Nanhua University(Medical Edition)
基金
湖南省自然科学基金(06JJ2093)
湖南省教育厅青年基金(08B067)项目
关键词
幽门螺杆菌
尿素通道蛋白
表达
Helieobacter pylori
urea channel protein （UreI）
express
