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  

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

作　　者：孙丽媛[1] 刘君[1] 张夕燕[1] 丁军颖[1] 张振龙[1] 许雪梅[2] 

机构地区：[1]北京生物制品研究所中试一室,100024 [2]中国医学科学院基础医学研究所,北京100730

出　　处：《国际生物制品学杂志》2009年第2期64-67,共4页International Journal of Biologicals

基　　金：国家高技术研究发展计划（2007AA02Z475）

摘　　要：目的利用昆虫细胞一杆状病毒表达系统表达人乳头瘤病毒16型（human papillomavirus16，HPV16）L1蛋白，并确定含编码HPV16L1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HPV16L1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48h后收获病毒；以细胞密度为（2—3）×10^6／ml，MOI值为10．0接种病毒，悬浮表达72h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HPV16L1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HPV16L1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。Objective To determine the optimum conditions for amplification of a baeulovirus encoding human papillomavirus 16 （HPV16） L1 and expression of the protein. Methods The baculovirus encoding HPVI6 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 sf9 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） x 10^6/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.

关 键 词：人乳头瘤病毒16 L1蛋白 昆虫细胞 表达 

分 类 号：R3[医药卫生—基础医学]