人乳头瘤病毒16L1-E5表达质粒的构建及其蛋白表达

目的构建人乳头瘤病毒16L1-E5的原核表达质粒，并进行表达融合蛋白，为下一步探讨该蛋白是否能作为疫苗的研究作准备。方法以本室已构建好的阳性质粒pET32（＋）／HPV16E5为模板，PCR获得E5基因，经SalI和NotI双酶切插入已构建好的pGEX4T-1／HPV16L1，通过筛选获得正确的阳性克隆pGEX4T-1／HPV16L1-E5。pGEX4T-1／HPV16L1-E5转化入BL21感受态细胞，经IPTG诱导进行目的蛋白的表达，通过测定不同时间、不同IPTG浓度和不同温度时目的蛋白表达情况，获得蛋白表达的最佳条件，SDS-PAGE和Westernblot检测蛋白表达。结果成功构建了质粒pGEX4T-1／HPV16L1-E5，在1mmol／L IPTG、34℃、4h诱导获得最大量的目的蛋白表达。结论成功构建的pGEX4T-1／HPV16L1-E5质粒能表达出目的蛋白，为进一步研究目的蛋白功能打下了坚实的基础。

Construction of prokaryotic plasmid of HPV16 L1-E5 and its expression

Objective To construct prokaryotie expression vector PGEX4T-1/HPV16L1-E5 to observe the expression of human papillomavirus（HPV） 16 L1 and E5 fusion protein. Methods HPV16 E5 gene was amplified by PCR from PET32（＋）/HPV16E5 and inserted into the plasmid PGEX4T-1/HPV16L1 followed by Sal Ⅰ and Not Ⅰ . The recombinant plasmid pGEX4T-1 /HPV16L1-E5 was transformed into E. coli JM109 and selected with ampieillin. The positive clones were verified by restriction endonucleases Sal Ⅰ and Not Ⅰ and sequence analysis. The expression of HPV16L1-E5 protein in E coli BL21 （DE3） was identified by SDS-PAGE and Western-blotting. Results The prokaryotic plasmid pGEX4T-1 /HPV16L1-E5 was successfully constructed. E coli BL21 （DE3） transformed by the recombinant plasmid pGEX4T-1 /HPV16L1-E5 expressed HPV16L1-E5 fusion protein, efficiently. Conclusions HPV16L1-E5 fusion protein is successfully expressed, which may facilitate subsequent research of the biological function of HPV16 L1-E5 protein.