悬浮培养昆虫细胞SF9瞬时表达GFP的条件优化.docVIP

 悬浮培养昆虫细胞 SF9 瞬时表达 GFP 的条 件优化 郭欣泳，汪才坤，袁凤媚，谢秋玲** 5 10 15 20 25 30 35 40 （暨南大学生命科学技术学院，广东省基因工程药物重点实验室，基因工程药物国家工程研 究中心，广州 510632） 摘要：动物细胞表达重组蛋白的方式包括瞬时基因表达（Transient Gene Expression）和 稳定基因表达（Stable Gene Expression），与耗时耗力的稳定基因表达相比，瞬时基因表 达省去了细胞筛选，是一种在短时间内获得大量重组蛋白的最为合适的方法，尤其适用于药 物蛋白评价和蛋白质组学研究。利用 CHO、HEK 等细胞进行瞬时表达的研究在国外已有很成 功的报道，然而昆虫细胞作为一种广泛使用的蛋白表达系统，瞬时基因表达的研究仍然较少， 在国内更是空白。本文进行了以昆虫 SF9 细胞在悬浮培养条件下进行瞬时表达 GFP 的条件 优化，以期为外源蛋白在昆虫细胞中瞬时表达打下基础。经优化后，获得在 0.5×106 cells/ml 细胞接种密度下，DNA 浓度 1 μg/ml，PEI:DNA=3：1，聚合时间 5 分钟的条件下， 能够达到最佳表达水平，转染效率达到 37%。 关键词：SF9 细胞；瞬时基因表达；聚乙烯亚胺 中图分类号：Q785 Optimization of transient gene expression of GFP in insect cell SF9 in suspension Guo Xinyong, Wang Caikun, Yuan Fengmei, Xie Qiuling (Gene engineering pharmacy national research center, Gene engineering pharmacy Guangdong key laboratory, Jinan University, GuangZhou 510632) Abstract: There are two methods for production of recombinant protein in mammalian cells: Transient Gene Expression (TGE) and Stable Gene Expression (SGE). In comparison with stable gene expression which is a costly and time-consuming process, TGE is a fast approach for r-protein production in the absence of selection of high-expression cell line. TGE has been used as a suitable method for evaluating candidate proteins rapidly and proteomic research. In fact, there are some successful reports about transient gene expression in CHO cell and HEK cell. However, there few reports about TGE in insect cell although insect cell line SF9 is a wildly used gene expression system. In this study, the factors influencing transfection efficiency, including amount of DNA, ratio of DNA and PEI, time of DNA/PEI complex formation, and cell density were optimized to express GFP in insect cell SF9 in suspension. Under the optimized conditions, 37 % of the transfection efficiency was obtained when 1 μg/ml DNA mixed with 3 μg/mL PEI to form complex for 5 min, followed by transfection in cells with cell density of 0.5×106 cells/mL. Keywords: insect cell; tran