过表达FGFR2重组HEK293细胞株的构建和鉴定

发布时间：2018-05-20 03:37

本文选题：成纤维细胞生长因子受体 + 慢病毒包装　；参考：《中国医药工业杂志》2017年01期

【摘要】：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?g/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u PA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]:Recombinant lentivirus expression vector containing fibroblast growth factor receptor protein 2 (FGFR2) gene sequence was constructed and co-transfected with lentivirus packaging plasmid into packaging cells 293T to obtain recombinant viral particles that overexpressed FGFR2. Recombinant virus particles were used to infect human embryonic kidney cells (HEC) and purified by purine mycin 20 g / ml. Finally, recombinant cell lines expressing FGFR2 were obtained. Western blot assay showed that, Compared with the control HEK293 cell line, the recombinant cell line could express FGFR2.The results of real-time fluorescence quantitative nucleotide amplification assay and Elisa showed that the transcription and secretion level of UPA and other molecules related to downstream pathway of FGFR2 protein were up-regulated. Clone formation assay also confirmed that the proliferation of recombinant HEK293 cells was enhanced. The results showed that the recombinant FGFR2 overexpression cell model could be used for further functional studies and screening of targeted FGFR2 drugs.
【作者单位】：暨南大学生物医药研究院暨南大学细胞生物学系基因工程药物国家工程研究中心广东省生物工程药物重点实验室;
【基金】：国家自然科学基金(81472337) 广东省科技计划项目(2016A020217012) 广州市科技计划项目(2016201604030039)
【分类号】：R96

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