HPV18 E7致癌蛋白的高效表达、纯化和鉴定

目的为进一步研究人乳头状瘤病毒18（Human papillomavirus18，HPV18）E7蛋白的结构与功能。方法构建HPV18 E7的谷胱甘肽S-转移酶融合蛋白质粒pGEX-6P-1-GST-HPV18 E7，重组质粒转入大肠埃希菌BL21进行可溶性融合蛋白的高效表达。结果柱上切除法去除GST标签，表达产物经glutathione Sepharose 4B亲和层析纯化，获得了SDS-PAGE和HPLC-ESI-MS纯度的HPV18 E7均质蛋白，非变性PAGE和凝胶过滤表明HPV18 E7以稳定的单体形式存在于水溶液中。高压液相色谱-电喷雾质谱（HPLC-ESI-MS）分析得到HPV18 E7精确分子量为12865．0 Da，与其理论值吻合。纯化蛋白经HPLC-ESI-MS／MS鉴定为目的产物，鉴定出的9个匹配肽段覆盖率为HPV18 E7整个氨基酸序列的96．5％。结论本文所建立的技术可以有效地大量制备HPV18 E7，为进一步研究其结构与功能和致癌机制奠定了重要的物质基础。

Expression, purification and identification of human papillomavirus 18 E7 oncoprotein

Objective Human papillomavirus 18 E7 oncoprotein plays a critical role in the tumorgenesis of cervical cancer. Methods To facilitate the functional and structural studies of HPV18 E7,a recombinant plasmid (pGEX-6P-1-GST-HPV18 E7) was successfully constructed by inserting HPV18 E7 gene fused with GST tag into the pGEX-6P-1 vector. Results Then, the plasmid was transferred into BL21 (DE3) and the fusion HPV18 E7 protein was overexpressed in E.coli with the induction of IPTG. GST tag was removed by using PreScission protease in column on line and the recombinant HPV18 E7 was purified to homogeneity,determined by SDS-PAGE and HPLC-ESI-MS,through two glutathione Sepharose 4B columns in tandem.The purified protein possesses an accurate molecular mass of 12865.0 Da that was pretty close to the theoretical molecular mass of HPV18 E7.Protein identification of purified HPV18 E7 was carried out by HPLC-ESI-MS/MS analysis.9 peptides,generated by the tryptic and Asp-N digestions, which occupied 96.5% of whole amino acid sequence coverage of HPV18 E7 were identified.The results from native PAGE and gel filtration chromatography indicated that the HPV18 E7 exists as a stable monomeric molecule in aqueous solution.Conclusions The results obtained from this paper provide a technique for preparation of HPV18 E7 in a large scale and provide critical basis for the further study on the structure,function and carcinogenesis of this oncoprotein.