Tet-off诱导表达Egr-1HEK293稳定细胞株的建立和鉴定

Establishment and detection of a Tet-off stable cell line expressing Egr-1 protein regulated by doxycycline

目的:利用大肠埃希菌Tet-off诱导表达调控系统构建多西环素调控的稳定细胞株。方法:以pEGFPEgr-1质粒为模板,扩增含早期生长反应蛋白(early growth response-1,Egr-1)完整开放阅读框的DNA序列,经酶切后插入含四环素反应元件(tetracycline responsive element,TRE)序列的表达载体pTRE2hyg,获得重组质粒pTRE2hyg-Egr-1,将其转染293Tet-off细胞株,并用G418和多西环素筛选稳定表达Egr-1的细胞株。蛋白质印迹法检测多西环素诱导表达Egr-1情况;流式细胞术检测细胞增殖能力。结果:蛋白质印迹结果显示,外源性Egr-1蛋白表达受细胞培养液中多西环素调控,当从细胞培养液中去除多西环素后,Egr-1表达量明显升高。流式细胞检测结果显示Egr-1高表达细胞的增殖能力明显高于Egr-1低表达细胞。结论:利用Tet-off体系成功构建Egr-1诱导表达的细胞株,细胞的增殖能力受Egr-1表达水平的影响。

Objective: To construct the responsive plasmid of pTRE2hyg-Egr-1 Tet-off gene expression system and examine its expression. Methods:PCR was performed to obtain the cDNA of early growth response-1(Egr-1) which was inserted into the responsive plasmid PTRE2hyg. DNA sequencing was performed after the recombinant of responsive plasmid pTRE2hyg-Egr-1 was identified by sequencing. This recombinant vector was transfected into 293Tet-offcells by means of liposome and its expression was examined by Western blotting under the control of deoxycycline. Results:The responsive plasmid pTRE2hygEgr-1 verified by sequencing,was capable of expression in 293Tet-offcould be controlled by deoxycycline concentration. Conclusion: The responsive plasmid pTRE2hyg-Egr-1 of Tet-off expression system was constructed successfully,and it can express under the regulation of deoxycycline in the 293Tet-offcells.