【摘要】 背景:外源性端粒酶反转录酶的表达可重建端粒酶活性,诱导细胞永生化。目的:采用慢病毒载体作为基因转移载体,克隆人端粒酶反转录酶的编码区全序列,同时选用绿色荧光蛋白作为目的基因表达标志物,构建人端粒酶反转录酶慢病毒表达载体。设计、时间及地点:开放性实验,单一样本观察,于2007-06/2008-03在暨南大学生物工程研究所及暨南大学附属第一医院中心实验室完成。材料:pBABE-puro-hTERT质粒由Robert Weinberg教授惠赠,质粒pDONR221,pLenti6/V5-DEST,ccdBSurvival,Stbl3及293FT细胞由暨南大学生物工程研究所提供。方法:以pBABE-puro-hTERT质粒为模板聚合酶链反应法获取目的基因人端粒酶反转录酶(包含酶切位点BglⅡ、HindⅢ),通过BP反应克隆至pDONR221,以质粒pEGFP-N1为模板,以聚合酶链反应法获取目的基因增强型绿色荧光蛋白(包含酶切位点HindⅢ,BgⅢ),通过酶切及连接反应形成pDONR221-hTERT-EGFP。采用LR重组酶将pDONR221-hTERT-EGFP和pLenti6/V5-DEST进行重组反应,形成pLenti6/V5-DEST-hTERT-EGFP。将pLenti6/V5-DEST-hTERT-EGFP与包装质粒混合,利用脂质体共转染293FT细胞。主要观察指标:通过酶切、聚合酶链式反应及测序验证重组质粒pDONR221-hTERT-EGFP和pLenti6/V5-DEST-hTERT-EGFP是否构建成功。包装重组慢病毒,应用荧光显微镜观察报告基因绿色荧光蛋白在293FT细胞中的表达情况。结果:测序发现慢病毒入门载体pDONR221包含目的基因hTERT1547位点存在点突变(原碱基A变成G),通过定点突变技术成功诱变并鉴定慢病毒表达载体pLenti6/V5-DEST-hTERT-EGFP成功构建。转染后的293FT细胞在荧光显微镜下观察可见强绿色荧光。结论:实验成功构建了人端粒酶反转录酶和绿色荧光蛋白融合基因的慢病毒表达载体,为人端粒酶反转录酶稳定转染提供快速、简洁的方法。更多还原

【Abstract】 BACKGROUND:Exogenous telomerase reverse transcriptase(TERT) expression can reconstruct telomerase activation and induce cell immortalization.OBJECTIVE:To construct lentiviral expression vector with human TERT(hTERT) and green fluorescent protein(GFP) recombinant gene for effective reconstitution of human telomerase activity.DESIGN,TIME AND SETTING:Open trial and single sample observation was performed at the Bioengineering Institute of Jinan University and Central Laboratory of First Hospital,Jinan University between June 2007 and March 2008.MATERIALS:pBABE-puro-Htert plasmid was graciously provided by Professor Robert Weinberg;plasmids pDONR221,pLenti6/V5-DEST,ccdB Survival,Stbl3 and 293FT cells were provided by Bioengineering Institute of Jinan University.METHODS:hTERT gene with BglII and HindIII sites was amplified by PCR from pBABE-puro-hTERT plasmid and was subcloned into pDONR221 by BP reaction;EGFP gene with BglII and HindIII sites was amplified by PCR from pEGFP-N1 plasmid.Then,pDONR221-hTERT-EGFP was constructed through series of enzyme digestion and ligation reaction.PLenti6/V5-DEST-hTERT–EGFP was obtained by LR reaction between pDONR221-hTERT-EGFP and pLenti6/V5-DEST.The recombinant vector was transfected 293FT cell with lentiviral packaging system through lipofectamine.MAIN OUTCOME MEASURES:pDONR221-hTERT-EGFP and pLenti6/V5-DEST-hTERT-EGFP were detected through enzyme digestion and sequencing.The expression of green fluorescent protein was observed under fluorescent microscope after packaging of recombinant lentivirus.RESULTS:Dot mutation was found at 1 547 site(from A to G) in hTERT gene by DNA sequencing.The mutation was successfully changed by site-directed mutagenesis technique and the plenti6/V5-DEST-hTERT-EGFP was successfully constructed.The green fluorescence was observed in the 293FT cells under fluorescence microscope after transfection.CONCLUSION:The lentiviral vector with hTERT and GFP recombinant gene was successfully constructed,which provides an effective and simple method for evaluating stable transfection of hTERT.更多还原