可诱导性表达呼吸道合胞病毒蛋白细胞系的快速构建方法研究

Research of rapid construction of stable cell lines with inducible expression of RSV proteins

目的研发用以快速构建多种可诱导性表达呼吸道合胞病毒（RSV）蛋白的细胞系创新性的技术路线。方法将不同的RSV病毒蛋白基因分别连接到含有PiggyBac转座子位点的Cumate诱导表达载体上，与PiggyBac转座酶质粒共转染HEK293细胞，利用嘌呤霉素进行筛选，并用Cumate诱导病毒蛋白表达。结果流式细胞仪及Westernblot检测结果均表明，加入诱导剂Cumate后，筛选的细胞系中开始表达相应的病毒蛋白，提示细胞系构建成功。结论利用PiggyBac转座子可以高效率的构建诱导性表达RSV病毒蛋白的细胞系。

Objective An new technique was developed to construct rapidly various cell lines that could be induced to express RSV proteins. Methods The multiple RSV protein genes were cloned into the cumate-induced expression system which was positioned between two the PiggyBac transposon sites, and was co-transfected with the PiggyBae transposase plasmids into the HEK293 cell line. After screened with puromycin, and viral proteins were induced with cumate and were assayed by western blot assay and flow cytometry. Results The results of flow cytometry and western blot showed that the viral proteins were expressed in most cells in the corresponding lines after adding cumate. Conclusions Cell lines with inducible expression of RSV proteins can be efficiently constructed by using the PiggyBac transposon system.