重组人乳头瘤病毒58型L1蛋白在Sf9细胞中的表达条件优化

摘要

Objective To select the optimum conditions for amplification and expression of the recombinant human papillomavirus 58 (HPV58) LI gene using baculovirus-insect cell expression system. Methods The Sf9 insect cells at logarithmic phase was used to amplify virus at different multiplicities of infection (MOI). Titers of the virus were determined by plaque assay. The expression of recombinant protein was identified by indirect immunofluorescence assay, and the expression of recombinant protein at different MOI and time was determined by Western blot. Results Recombinant baculovirus containing HPV58 LI gene were amplified at MOI of 0.5 for 48 h when the Sf9 insect cells were at logarithmic phase, and the highest titers of the virus reached 5×108 pfu/ml. The optimum conditions for expressing the recombinant protein were that the recombinant baculovirus was inoculated at MOI of 5.0 and the protein was expressed within 72 h. Conclusion The optimum conditions for amplifying the virus and expressing the recombinant protein are determined.%目的 采用昆虫细胞-杆状病毒表达系统扩增人乳头瘤病毒(human papillomavirus,HPV)58型L1蛋白基因,确定L1蛋白表达的最佳条件.方法 取处于埘数生长期的S19细胞以不同的感染复数(MOI)值扩增病毒,用噬斑试验检测病毒滴度,确定扩增重组病毒的最适条件;用间接免疫荧光法判断目的蛋白表达情况;通过蛋白印迹法测定不同的表达时间和MOI值条件下目的蛋白表达量,确定目的蛋白的最佳表达条件.结果 处于对数生长期的细胞以MOI值为0.5表达48 h条件下扩增含HPV58 LI基因的重组杆状病毒,滴度最高,可达5×108pfu/ml;以MOI值为5.0表达72 h获得最佳的目的蛋白表达量.结论 确定了扩增病毒和表达相应目的蛋白的最佳条件.