hTERT启动子调控的TRAIL基因载体的构建及表达

Construction and expression of TNF related apoptosis inducing ligand (TRAIL) gene driven by hTERT promoter

目的:构建hTERT启动子调控的TRAIL基因载体,探讨hTERT启动子调控的转基因表达。方法:PCR法扩增膜结合型TRAIL基因,回收产物与带有hTERT启动子的载体连接,构建重组载体ph-TERT-TRAIL;瞬时转染乳腺癌细胞系MCF-7/ADR;采用RT-PCR法检测外源基因TRAILmRNA水平的表达,流式细胞术(FCM)检测蛋白水平的表达。结果:重组载体phTERT-TRAIL经PCR、酶切出现相应长度的片段;测序结果连入TRAIL基因;目的基因的序列分析结果与Genebank中的数据高度同源;RT-PCR显示细胞中外源基因TRAIL在mRNA水平明显表达;FCM显示转染了重组载体phTERT-TRAIL的细胞较未转染带有TRAIL基因载体的细胞TRAIL蛋白表达水平上调。结论:成功构建重组载体ph-TERT-TRAIL,hTERT启动子调控的TRAIL基因在乳腺癌细胞系MCF-7/ADR中明显表达。

Objective: To construct the TRAIL gene vector driven by hTERT promoter and explore its transgene expression from the hTERT promoter. Methods: The membrane-bound TRAIL gene was amplified by PCR. The products recycled were cloned into the vector carrying hTERT promoter and the recombinant phTERT-TRAIL was then constructed. The plasmids were transfected into breast cancer cell line MCF-7/ADR in vitro and the levels on mRNA and protein of foreign gene expression were detected by RT-PCR and FCM. Results: The PCR and enzyme digestion results of the recombinant plasmid phTERT-TRAIL showed the correct fragments,and the sequence analysis of targeted gene confirmed that it was highly conserved with that of that of the Genebank. The FCM results showed that the TRAIL protein expression level in cells transfected with phTERT-vector was higher than that non-transfectants or transfectants without TRAIL gene. Conclusion: The recombinant vector phTERT-TRAIL is constructed successfully. TRAIL gene driven by hTERT promoter can be obviously expressed in breast cancer cell line MCF-7/ADR.