Epstein-Barr病毒膜抗原在昆虫细胞中的表达

目的　探索影响EpsteinBarr病毒膜抗原(EBVMA)在昆虫细胞中表达的因素。方法　携有截去穿膜区序列的EBVMA基因的重组昆虫杆状病毒感染不同生长阶段及生长条件的昆虫细胞,应用免疫打点法及MasterImagineVDS影像摄录分析系统测定目的蛋白的表达量。结果　重组病毒感染不同生长阶段的细胞,MA表达量不同。处于对数生长早期的细胞表达量明显高于处于对数生长晚期及静止期的细胞。将不同生长阶段的细胞补充新鲜培养液,MA的表达量在原基础上有所提高。进一步研究表明,在感染重组病毒之前,更换新鲜培养液,同时在感染过程中补充葡萄糖,L谷氨酰胺等营养物质,会大大提高MA表达量。结论　细胞培养液中营养物质的消耗和代谢产物的蓄积是影响EBVMA在昆虫细胞中表达的主要因素。

Optimization of productivity of Epstein-Barr virus membrane antigen in baculovirus infected insect cells

Objective To probe factors influencing the expression level of in insect cells.Methods Insect cells at different growth phases and in different culture media were infected with recombinant baculovirus carrying EBV MA gene, where the membrane anchor sequence was deleted. The expression level of EBV MA was detected by immunodots assay and scanner analysis.Results Cells at the early exponential phase yielded more expressed recombinant EBV MA than those at the late exponential and stationary phases. Infected cells supplemented with fresh medium at various phases of growth resulted in improved productivity of EBV MA. Further experiments revealed that replacing fresh medium prior to infection and supplementing a mixture of glucose and glutamine after infection, the productivity could be improved dramatically. Comparing the productivity of EBV MA expressed in sf9 cells with that in sf21 cells, no obvious difference was found.Conclusion Consumption of nutrients and accumulation of metabolites in cell culture media were the important factors influencing the productivity of EBV MA in insect cells.