瞬时基因表达可溶性的VEGFR2I-IV.PDFVIP

生 物 工 程 学 报 Chin J Biotech 2008, May 25; 24(5): 810-816 journals.im.ac.cn Chinese Journal of Biotechnology ISSN 1000-3061 cjb@im.ac.cn © 2008 Institute of Microbiology, CAS CSM, All rights reserved 研究报告 瞬时基因表达可溶性的VEGFR2: I-IV 李军, 易小萍, 张元兴, 孙祥明 华东理工大学生物反应器工程国家重点实验室, 上海 200237 摘 要: 通过 RT-PCR 的方法从三个月的流产绒毛组织中克隆目的基因 VEGFR2 (Vascular endothelial growth factor receptor 2, 血管内皮细胞生长因子受体2) 胞外I-IV 区, 连接到真核表达载体上构建了重组表达载体。首先在无血清悬 浮培养的HEK293 细胞中, 使用报告基因GFP(Green fluorescence protein, 绿色荧光蛋白)优化转染条件, 发现在转染时 DNA: PEI=1:2 ( W/ W) 、1.5 μg DNA/106 cells 及开始转染4 h 内使用无血清、摇床(120 r/min)时可以达到最佳的转染效率 和细胞数量。在确定转染条件之后, 将构建的表达载体分别在 HEK293 细胞、COS-7 细胞和CHO-K1 细胞中进行瞬时 转染表达, 结果发现仅在CHO-K1 细胞的培养上清中检测到目的蛋白的表达。瞬时转染CHO-K1 细胞至总体积约为1.5 L, 由于目的蛋白的羧基端有8-His 标签, 通过Ni2+-IDA 柱纯化得到5 mg 左右的目的蛋白。 关键词: VEGFR2, 瞬时基因表达, HEK293, PEI Transient Gene Expression of Soluble VEGFR2: I-IV Jun Li, Xiaoping Yi, Yuanxing Zhang, and Xiangming Sun State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China Abstract: The extracelluar domain I-IV of target gene VEGFR2 (Vascular endothelial growth factor receptor 2) was cloned from villus of trimester abortion by RT-PCR, and linked to the expression vectors. Then, the transfection conditions were optimized in serum-free suspension culture HEK293 using GFP (Green fluorescence protein) as the report gene. The results showed that the optimal transfection efficiency and cell number were obtained when the ratio of foreign DNA: PEI= 1:2 ( W/ W), DNA=1.5 g /106 cells and shaking speed (120 r/min) in serum free medium in the beginning 4 hours