人乳头瘤病毒16型 E5基因克隆、原核表达及产物鉴定

目的 克隆HPVl6 E5基因,构建原核重组工程菌并诱导表达,对表达产物HPVl6E5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HPVl6 E5基因,经BamH I和HindⅢ双酶切后,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BL21(DE3),建屯重组工程菌株pET21b-HPVl6E5/BL21(DE3).经IPTG诱导表达,SDS-PAGE和Western印迹检测表达产物.结果 HPVl6 E5基因扩增片段0.27kb.测定序列与HPVl6原型株E5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16 kDa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HPVl6E5基因,并构建原核表达质粒,E5 蛋白在BL21(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.

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

Objective To construct recombinant pET21 b-HPVl6E5 plasmid and to express human papillomavims(HPV) type 16 E5 protein in E coli.Methods HPVl6 E5 gene was amplified from cervical carcinoma biopsy by PCR and inserted into the plasmid pET21 b after digestion by BamH I and Hind Ⅲ pET21b-HPVl6E5 was transformed into E.coli DH5α.The positive clones were vetiSed by restriction en-zyme digestion and sequencing.Subsequently,pET21 b-HPVl6E5 was transformed into BL21(DE3)strain.After IPTG indnction,the expression of His6-HPVl6E5 was confirmed by SDS-PAGE and Western blot.Re-sults The amplified tipVl6 E5 gene is 0.27 kb.The constuction of pET21 b-HPVl6E5 was confirmed by restrieted 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 HPVl6E5 gene is cloned and expressed in bacteria successfully.It lays foundation for HPVl6 E5 study of biological character,tranforming activity and tumor cytotoxieity.