摘要
目的构建含人幽门螺杆菌(Helicobacter pylori,H.pylori)尿素通道蛋白编码基因(UreI)的真核表达的重组载体,并在COS-7细胞中表达,为核酸疫苗的开发奠定基础。方法以原核表达质粒pET32a(+)/UreI为模板,扩增UreI编码基因片段,将目的基因与同样进行酶切、纯化的载体pEGFP-N1进行连接,而后转化并筛选含有目的基因的重组载体pEG-FP-N1/UreI,并在COS-7细胞中表达,以荧光蛋白和Western blot法检测其表达产物。结果经酶切、测序证实插入的基因片段为H.pyloriUreI蛋白编码基因;荧光显微镜下和Western blot法等检测显示,该重组质粒能够在COS-7细胞中表达目的蛋白,同时能够被H.pylori阳性患者血清所识别。结论成功地构建了真核重组载体pEGFP-N1/UreI,并在COS-7细胞中表达。
The recombinant eukaryotic vector for gene encoding the urea channel protein （Urel） of Helicobacter pylori was constructed and expressed in COS-7 cells for the sake of providing a foundation to develop the nucleic acid vaccine. Under this subject, the eukaryotic expression plasmid pET32a（＋）/UreI was used as template and the target gene fragment encoding the UreI protein was amplified from this plasmid , digested with Hind Ⅲ and Kpn Ⅰ. In the same way, plasmid pEGFP-N1 was also digested with these two endonucleases simultaneously. Then, the objective gene and plasmid pEGFP-N1 were purified with gel kit and connected by T4 ligase at 4 ： 1 molar rate. The recombinant vector pEGFP N1/UraI was used to select, transform and meantime express in COS-7 cells. Meanwhile, the expression of this eukaryotic vector in Cos-7 cells was investigated using fluorescence microscopy and Western blotting. As demonstrated by restriction endonuclease analysis, and sequencing, the size of the inserted target gene fragment was found to be 585 bps. And this gene fragment was proved to be the gene encoding the UraI protein. As compared with the gene of H. pylori AM417609 reported by GenBank, the cloned ureI gene sequence was completely coincidental. The expression of recombinant vector pEGFP-N1/UreI in COS-7 could be detected by means of fluorescence microscopy and recognized by the anti H. pylori antibody of patients infected with this organism, sug gesting that this protein had good biological activities. It is evident that the recombinant eukaryotie vector p EGFP/UreI has been constructed and successively expressed in CO8-7 cells.
出处
《中国人兽共患病学报》
CAS
CSCD
北大核心
2009年第4期313-317,共5页
Chinese Journal of Zoonoses
基金
国家自然科学基金〔No.30371318〕
渝科发〔2002〕18-86
渝教科〔2002〕18-6联合资助
关键词
幽门螺杆菌
尿素通道蛋白蛋白
核酸疫苗
真核表达载体
Helicobacter pylori
urea channel protein （Urd）
nucleinic acid vaccine
eukaryotic expression vector
