人乳头瘤病毒18型L1蛋白的包涵体和可溶性表达及纯化  

Expression and Purification of Human Papillomavirus Type 18 L1Inclusion Body and Soluble Protein

作　　者：麻粉莲[1] 张骞[1] 郑丽舒[1] 

机构地区：[1]中国疾病预防控制中心病毒病预防控制所,北京100052

出　　处：《生物技术通讯》2014年第6期800-803,共4页Letters in Biotechnology

摘　　要：目的:原核表达系统表达人乳头瘤病毒18型(HPV18)L1蛋白,建立包涵体和可溶性表达的L1蛋白的纯化方法。方法:构建重组表达质粒p GEX-4T-1-HPV18 L1,在大肠杆菌BL21中以包涵体和可溶性方式表达HPV18 L1蛋白。通过超声波破碎菌体、洗涤包涵体、碱变性、透析复性和谷胱甘肽(GST)琼脂糖凝胶4B亲和层析纯化包涵体蛋白;在菌体中加入三磷酸腺苷(ATP)和3.5 mol/L尿素孵育后,GST 4B亲和层析纯化可溶性蛋白,凝血酶酶切。SDS-PAGE和Western印迹鉴定表达和纯化产物。结果:SDS-PAGE结果表明,HPV18 L1蛋白以包涵体和可溶性方式在大肠杆菌BL21内高效表达,均产生相对分子质量约为86 000的HPV18 L1-GST融合蛋白。Western印迹结果显示,包涵体纯化后获得的融合蛋白降解条带较多;而可溶性蛋白纯化后获得的融合蛋白未降解,凝血酶酶切后得到HPV18 L1蛋白,可与HPV18 L1蛋白单克隆抗体结合。结论:采用原核系统表达了HPV18 L1-GST融合蛋白,分别建立了包涵体和可溶性蛋白的纯化方法,获得HPV18 L1蛋白,为其进一步应用奠定了基础。Objective： To express human papillomavirus type 18（HPV18） L1 protein in a prokaryotic system, and establish the purification methods of HPV18 L1 inclusion body and soluble protein. Methods： The prokaryotic expression vector pGEX-4T-1-HPV18 L1 was constructed and transformed into E.coli BL21 cell and induced by IPTG to express HPV18 L1 protein in inclusion body and soluble forms. Inclusion body purification was conduct？ed in the following order： sonic disruption, washing, alkaline denaturation, dialysis renaturation and glutathione Sep？harose 4B（GST 4B） affinity chromatography. When the soluble protein purified, adding ATP and 3.5 mol/L urea to the lysate, and then the GST 4B affinity chromatography and thrombin cleavage was conducted. The expressed and purified products were identified by SDS-PAGE and Western blot. Results： SDS-PAGE results showed that HPV18 L1 protein was expressed efficiently in E.coli BL21 in inclusion body and soluble forms, the fusion pro？tein of Mr 86 000 was successfully induced. Western blot results showed that HPV18 L1-GST fusion protein of in？clusion body purification degenerated; the fusion protein of soluble protein purification was intact, and then HPV18 L1 protein was obtained by thrombin cleavage which has specific reaction with HPV18 L1 monoclonal anti？body. Conclusion： The fusion protein of HPV18L1-GST was prokaryotic expressed successfully; the study estab？lished the purification methods of inclusion body and soluble proteins and obtained HPV18 L1 protein which lay the foundation for application of HPV18 L1 protein further.

关 键 词：人乳头瘤病毒18型L1蛋白 包涵体 可溶性蛋白 纯化 

分 类 号：Q78[生物学—分子生物学]