HPV16-E7在Pichia pastoris酵母中的分泌表达

The Secreted Expression of HPV16-E7 in Pichia Pastoris

本研究利用酵母Pichia pastoris真核表达系统表达人乳头瘤病毒16(HPV16)E7蛋白,试图解决新疆维吾尔妇女高发宫颈癌的诊断、疫苗的研究中抗原蛋白需要的问题.依据新疆维吾尔妇女宫颈癌组织克隆获得的HPV16-E7基因序列设计引物,并分别在5’引物和3’引物中引入了EcoR Ⅰ和Xba Ⅰ酶切位点,经PCR扩增后连接到pMD18-T载体上,再将HPV16-E7克隆至穿梭质粒pGAPZαA上,获得的重组穿梭质粒pGAPZαA-E7经线性化后.采用LiCl法将重组穿梭质粒转入酵母细胞内,Zeocin+筛选阳性克隆,经小瓶发酵后,取上清作SDS-PAGE,并做Western印迹检测表达的蛋白质,结果表明HPV16-E7成功地在酵母真核表达系统获得表达,表达产物的分子量分别是22KDa和88KDa,为深入开展HPV16-E7蛋白功能和应用的研究奠定了理论基础.

In order to establish foundation for diagnose and DNA vaccine of high-risk cervical carcinoma in Xinjinag Uygur women, the experiments that protein of HPV16-E7 expressed in pichia pastoris eukaryote expression system have been performed. In this study, the 5' primer and 3' primer were designed according to HPV16-E7 gene sequence cloned from the cervical carcinoma of Xinjinag Uygur women, two restriction endonucleases' site (EcoR I & Xba I) were added to those two primers respectively. After amplified with PCR, HPV16-E7 gene was cloned into pMD18-T vector, then the identified HPV16-E7 gene was cloned into shuttle plasmid pGAPZαA. The recombinant plasmid were linearized and transformed into pichia pastoris. Before fermentation, the positive clone was screened with zeocin +. At last, the supernatant of medium was analyzed by SDS-PAGE and western blotting after fermentation. The results indicate that the HPV16-E7 protein has been expressed successfully in pichia pastoris eukaryote expression system, and the MW of HPV16-E7 protein is about 22 KDa and 88 KDa. This could lead to further investigation about the function of HPV16-E7 protein development.