构建含有成纤维细胞生长因子受体蛋白2(FGFR2) 基因序列的重组慢病毒表达载体，与慢病毒包装质粒共转染包装细胞293T，获得过表达FGFR2 的重组病毒颗粒；用重组病毒颗粒感染人胚肾细胞(HEK)293 细胞，并用嘌呤霉素(20 µg/ml) 进行筛选，最终获得过表达FGFR2 的重组细胞株。蛋白质免疫印迹(Western Blot) 试验结果显示，该重组细胞株与空白对照HEK293 细胞相比能高表达FGFR2，实时荧光定量核苷酸扩增试验(QPCR) 和酶联免疫反应(ELISA)检测结果表明FGFR2 蛋白下游相关通路的uPA 等分子的转录和分泌水平上调，克隆形成试验也证实重组HEK293 细胞促增殖能力增强。结果表明，该重组过表达FGFR2 的细胞模型可用于下一步的功能研究以及靶向FGFR2 药物的筛选。
Abstract
A recombinant FGFR2 expression vector was constructed and co-transfected with lentiviral packaging vectors into 293T to get recombinant lentiviral particles. The recombinant human embryonic kidney 293(HEK293) cells over-expressing FGFR2 were obtained after infection with the lentiviral particles and screening with 20 µg/ml puromycin. Western Blot assay verified that the expression level of FGFR2 in recombinant cells was higher compared with the blank control HEK293 cells. The results of QPCR and ELISA assays showed that the transcription and secretory expression of uPA and other molecules in FGFR2 downstream pathways were up-regulated in mRNA and protein level. Clones formation detection also suggested that the recombinant cells had higher proliferation ability. The results proved that the recombinant FGFR2 cell model could be conducted in the followed function studies and FGFR2-targeted drug screening.
关键词
成纤维细胞生长因子受体2 /
慢病毒包装 /
过表达 /
重组细胞株
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Key words
fibroblast growth factor receptor 2 /
lentiviral packaging /
over-expression /
recombinant cell line
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参考文献
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脚注
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基金
国家自然科学基金(81472337)、广东省科技计划项目(2016A020217012)和广州市科技计划项目(2016201604030039)
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