人端粒酶逆转录酶核心启动子调控的人TRAIL蛋白在人卵巢癌细胞中的表达

Expression of TRAIL gene eukaryotic expression vector modulated by the human telomerase reserse transcriptase gene core promoter in ovarian cancer cell line SKOV3

目的:构建人端粒酶逆转录酶基因核心启动子(hTERT)调控的肿瘤坏死因子相关凋亡诱导配体真核表达载体,并检测TRAIL在转染的卵巢癌细胞SKOV3中的表达情况。方法:利用基因重组方法将TRAIL基因克隆入带有hTERT基因核心启动子pIRES2-EGFP真核表达载体中。获得由hTERT基因核心启动子调控的绿色荧光蛋白和带有效应型TRAIL基因的真核表达载体hTERTpromoter-pIRES2-EGFP-TRAIL。采用脂质体介导的方法将hTERT调控的TRAIL基因真核表达载体转染人卵巢癌SKOV3细胞,采用G418筛选获得阳性克隆;应用免疫细胞化学法、蛋白质印迹分析、流式细胞术(FCM)结合间接免疫荧光、RT-PCR法检测转染前后卵巢癌细胞中外源基因的表达。结果:经酶切鉴定及测序结果证实所构建载体正确。转染细胞中的TRAIL表达明显高于对照组细胞。结论:成功地构建了hTRET基因核心启动子调控的TRAIL基因真核表达载体,并在人卵巢癌细胞系SKOV3中得到稳定表达,为进一步研究其对卵巢癌细胞生物学行为的影响奠定了实验基础。

AIM： To construct the TRAIL gene eukaryotic expression vector modulated by the human telomerase reserse transcription gene core promoter and to evaluate the expression of the TRAIL gene in ovarian cancer cell line SKOV3 in vitro. METHODS. The amplified TRAIL gene fragment was subsequently cloned into hTERT promoter pIRES2-EGFP vector and CMVpromoter-plRES2-EGFP vector. The hTERT promoter-plRES2-EGFP-TRAIL and CMVpromoter-plRES2-EGFP-TRAIL eukaryotic expression vectors were obtained, respectively. The plasmids were transfected into human ovadan carcinoma SKOV3 cell line by lipofeclin mediation and the positive clones were screened by G418. The expression of TRAIL gene was examined by RTPCR, Westem blot, immunocytochemistry and flow cytometry. RESULTS： All the constructed vectors were verified by enzyme digestion. The TRAIL gene of transfected cells was increased significantly （ P 〈 0.01 ）. CONCLUSION： An eukaryotic expression vector of the TRAIL gene modulated by the human telomerase reserse transcription gene core promoter has been constructed successfully and its steady expression in human ovarian cancer cell line SKOV3, which will be beneficial to further research into its role in regulatling the biological behavior of ovarian cancer cells.