Endostatin、FHIT和EGFP串联表达载体的构建

Construction of endostatin,FHIT and EGFP expression vector

本研究将内皮抑素基因插入到包含人脆性组氨酸三联体基因的pIRES2-EGFP-FHIT中构建真核多基因表达载体pEF1-FHIT-EGFP-ES,脂质体法转染Hela细胞后,用G418筛选出稳定转染的细胞。RT-PCR、RT-PCR southern blot分析,结果证明单顺反子和三顺反子在RNA水平都得到表达。在蛋白表达水平上,荧光观察结果显示EGFP在Hela细胞中得到表达。本研究为基因药物和肿瘤多基因治疗奠定了基础。

Endostatin cDNA was obtained by RT--PCR. Then the fragment of endostatin was cut and ligated into eukaryotic expression vector pIRES2--EGFP--FHIT to construct the expression vector pEFI--FHIT--EGFP--ES. This recombinant expression vector was transfected into Hela cells, Total RNA was extracted from those stably transfected Hela cells, and then was checked by RT--PCR, Southern blotting. The results indicated that the moncistrons of endostatin, FHIT and EGFP as well as the tricistron all had been transcribed successfully in the transfected Hela cells. These results provides some basic information for further research on multigene therapy strategies in tumor treatment.