可诱导表达hFGFR1α稳定细胞系的建立与表达优化

Establishment of a stable cell line with inducible expression of hFGFR1α and optimization of its expression

目的:构建hFGFR1α(human fibroblast growth factor receptor 1 alpha, hFGFRα)稳定细胞系,优化获得最适诱导表达条件,实现可诱导的、高效的稳定表达。方法:将hFGFR1α基因编码区全长克隆入pcDNA5/FRT/TO/2 × Flag表达载体,连同pOG44重组酶质粒共转染人宿主细胞Flp-InTM-T-RexTM-293细胞系(HEK293H),潮霉素B筛选获得阳性克隆细胞系HEK293H/hFGFR1ɑ。用不同浓度强力霉素(doxycline, Dox)或者不同作用时间对HEK293H/hFGFR1ɑ细胞系进行诱导表达,Western Blot鉴定hFGFR1ɑ蛋白的表达情况。结果:构建了pcDNA5-FRT/TO/2 × Flag/hFGFR1ɑ重组表达载体,获得了可被Dox诱导表达的HEK293H/hFGFR1ɑ稳定细胞系;0.001μg/mL Dox即可诱导hFGFR1ɑ蛋白表达,0.01μg/mL Dox即可诱导表达量达峰值;Dox对该细胞系作用2 h即诱导hFGFR1ɑ蛋白表达,对其作用24 h后该细胞系的表达明显增强。结论:成功构建了可诱导的、高效的稳定表达细胞系HEK293H/hFGFR1ɑ;Dox诱导该细胞系表达具有剂量差异性和时间依赖性。

Objective: To establish a stable cell line with expression of human fibroblast growth factor receptor 1 alpha (hFGFR1α), and to optimize the condition to achieve optimal inducible and highly efficient stable expression. Methods: The full-length coding region of the hFGFR1α gene was cloned into the pcDNA5/FRT/TO/2×Flag expression vector and transfected into the human host cell line Flp-InTM-T-RexTM-293 (HEK293H) together with pOG44 recombinase plasmid. The transfected cells were screened for positive clonal HEK293H/hFGFR1α cell line by hygromycin B. The expression of HEK293H/hFGFR1α cell line was induced by different concentrations of doxycline (Dox) for different time periods and then was determined by Western blot. Results: The recombinant expression vector pcDNA5-FRT/TO/2×Flag/hFGFR1α was successfully established, and a stable cell line with Dox-inducible expression of HEK293H/hFGFR1α was obtained. The expression of hFGFR1α protein was induced by 0.001 μg/ml of Dox, and a peaked expression was induced by 0.01 μg/ml of Dox;the expression of hFGFR1α protein was induced after treatment for 2 hours, and its expression was significantly increased after treatment for 24 hours. Conclusion: A stable cell line with inducible and highly efficient expression of HEK293H/hFGFR1α has been successfully established. Dox-inducible expression of the cell line is dose-dependent and time-dependent.