含编码人乳头瘤病毒18型L1蛋白基因的杆状病毒扩增及相应蛋白表达的条件优化

摘要

目的 确定以昆虫细胞sf9扩增含编码人乳头瘤病毒(human papillomavirus,HPV)18型L1蛋白(HPV18 L1)基因的杆状病毒以及相应蛋白表达的最佳条件.方法 以对数生长期的sf9细胞在不同感染复数(MOI)和不同时间条件下扩增病毒,以蚀斑试验检测病毒滴度,选择获得最高滴度病毒的最佳条件;再以sf9细胞在不同细胞密度、不同MOI值和不同时间条件下表达相应蛋白,以蛋白印迹法鉴定并比较不同条件下的蛋白表达量,选择获得最大量目的 蛋白的最佳条件.结果 对数生长期的sf9细胞在MOI值为0.50、扩增时问为48 h时,所扩增的病毒滴度最高,可达3×108 pfu/ml;在细胞密度为3×106/ml、MOI值为2.0、表达时间为72 h时,所表达的蛋白量最大,约5 mg/L.结论 确定了含编码HPV18 L1基因的杆状病毒扩增及相应蛋白表达的最佳条件.%Objective To select the optimum parameters for amplification of the baculovirus coding for human papillomavirus 18(HPV18)L1 and expression of the corresponding protein.Methods Baculovirus coding for HPVl8 L1 was amplified at different multiplicities of infection(MOls)and time.The virus titers were detected by a viral plaque assay and the optimum parameters were determined.The corresponding protein was expressed at different cell densities,MOIs and time,and was detected and quantified by Western Blot,and the optimum parameters were determined,too.Results The parameters for amplifying the baculovirus and expressing the protein were at the MOI of 0.50 for 48 h and at the cell density of 3×106/ml and MOI of 2.0 for 72 h.Conclusion The optimum parameters for amplifying the baculovirus coding for HPV18 L1 and expressing the corresponding protein are determined.