人乳头瘤病毒16型筋基因克隆、原核表达及产物鉴定  

Cloning, Expression and Identification of Human Papillomavirus Type 16 E5 Gene in E. coli

作　　者：商庆龙[1] 王燕[1] 陈思佳[1] 李承刚[1] 魏兰兰[1] 郝美丽[1] 李立群[1] 谷鸿喜[1] 

机构地区：[1]哈尔滨医科大学微生物学教研室,150081

出　　处：《国际遗传学杂志》2008年第4期254-257,共4页International Journal of Genetics

基　　金：黑龙江省科技攻关项目（CB02C111）;黑龙江省教育厅资助项目（10553046）

摘　　要：目的克隆HPV16E5基因，构建原核重组工程菌并诱导表达，对表达产物HPV16E5蛋白进行鉴定。方法提取宫颈癌组织DNA作为模板，用PCR方法扩增HPV16E5基因，经BamHI和HindⅢ酶切后，插入相同酶切的pET21b载体质粒，转化DH5α，筛选阳性克隆。经酶切和测序鉴定后转化大肠埃希菌B121（DE3），建立重组工程菌株pET21b-HPV16ES／BL21（DE3）。经IPTG诱导表达，SDS．PAGE和Western印迹检测表达产物。结果HPV16E5基因扩增片段0．27kb。测定序列与HPV16原型株痨基因比较，出现4处核苷酸变异，分别为3979、4042、4077和4089位，引起144L和165V氨基酸改变。重组质粒经酶切和序列测定证实构建正确。SDS—PAGE分析重组菌在16kDa处出现蛋白条带。该蛋白条带可被组氨酸标签单克隆抗体特异性识别。结论成功克隆HPV16E5基因，并构建原核表达质粒，E5蛋白在B121（DE3）中表达。本实验为进一步研究E5生物学活性、转化活性和肿瘤杀伤免疫作用奠定了实验基础。Objective To construct recombinant pET21b-HPV16E5 plasmid and to express human papillomavirus （HPV） type 16 E5 protein in E. coli. Methods HPV16 E5 gene was amplified from cervical carcinoma biopsy by PCR and inserted into the plasmid pErlE1b after digestion by BamH I and Hind Ⅲ. pET21b-HPV16E5 was transformed into E. coli DH5α. The positive clones were verified by restriction enzyme digestion and sequencing. Subsequently, pET21b-HPV16E5 was transformed into BL21 （DE3） strain. After IPTG induction, the expression of His6-HPV16E5 was confirmed by SDS-PAGE and Western blot. Resuits The amplified HPV16 E5 gene is 0.27 kb. The constuction of pET21b-HPV16E5 was confirmed by restricted digestion. Several nucloetide changes were found. SDS-PAGE showed a 16 kDa band in recombi- nant strain. Western blot validated target protein with the His-tag monoclonal antibody. Conclusion HPV16 E5 gene is cloned and expressed in bacteria successfully, h lays foundation for HPV16 E5 study of biological character, tranforming activity and tumor cytotoxicity.

关 键 词：人乳头瘤病毒16型 E5蛋白 原核表达 克隆 

分 类 号：R737[医药卫生—肿瘤]