重组人乳头瘤病毒16型L1蛋白在昆虫细胞中的表达及条件优化研究

目的 利用昆虫细胞-杆状病毒表达系统表达人乳头瘤病毒16型(human papillomavirus 16,HPV16)L1蛋白,并确定含编码HPV16 LI蛋白基因杆状病毒的扩增及其相应目的 蛋白表达的最佳条件.方法 在不同感染复数(MOI)、不同时间条件下扩增病毒,以蚀斑试验检测病毒滴度,研究获得最高滴度病毒的最适条件;通过对相应蛋白进行表达条件的优化,以蛋白印迹法比较蛋白表达量,确定目的 蛋白表达的最佳条件.结果 确定了扩增含编码HPV16 L1蛋白基因杆状病毒及表达相应蛋白的最佳条件,即在sf9细胞系中,以MOI值为0.10接种病毒,扩增48 h后收获病毒;以细胞密度为(2-3)×106/ml,MOI值为10.0接种病毒,悬浮表达72 h收获目的 蛋白.蛋白印迹法检测结果证实,表达的目的 条带可与HPV16 LI蛋白的单克隆抗体发生反应.结论 建立了在昆虫细胞中表达HPV16 L1蛋白的最适条件,为下一步的纯化及免疫原性研究奠定了基础.

Optimization of condition for expressing reccmbinant HPV16 L1 protein in insect cells

Objective To determine the optimum conditions for amplification of a baculovirus encoding human papillomavirus 16(HPV16)L1 and expression of the protein.Methods The baculovirus encoding HPV16 L1 was amplified at different multiplicities of infection(MOIs)and time.The virus titers were detected by a plaque assay and the optimum condition was defined.The recombinant protein was expressed under different conditions and was identified and determined by Western Blot.Results The optimum conditions for amplifying the baculovirus encoding HPV16 L1 and expressing the protein were determined.The highest titer virus was harvested in sf 9 cells when the virus was inoculated at the MOI of 0.10 and amplified within 48 h,and the protein Was expressed by way of suspension culture within 72 h when the virus was inoculated at the MOI of 10.0 and the cell density of(2-3)×106/ml.The expressed protein showed a specific reaction with anti-HPV16 L1 monoclonal antibody.Conclusions The recombinant HPV16 L1 protein can be highly expressed in baculovirus-insect cell expression system,which lays the foundation for further purification and study on immunogenicity.