携带有Survivin启动子的重组腺病毒载体的构建及鉴定

目的构建携带有Survivin启动子的重组腺病毒载体,检测其在人膀胱癌细胞株BIU87和人正常膀胱上皮细胞SV-HUC-1中的表达。方法用PCR扩增Survivin基因的启动子片段,构建分别携带Survivin启动子和CMV启动子的真核表达载体pSurp-EGFP和pCMV-EGFP,体外连接法制备重组腺病毒Adeno-SP-EGFP和Adeno-CMV-EGFP,分别转染BIU87及SV-HUC-1,荧光显微镜下观察EGFP的表达。结果成功克隆440 bp的Survivin基因启动子,并构建了携带有Survivin基因启动子的重组腺病毒Adeno-SP-EGFP,其转染BIU87及SV-HUC-1后,在荧光显微镜下可以观察到BIU87细胞呈现出较强绿色荧光,而SV-HUC-1细胞中仅有散在少量绿色荧光。结论成功制备的携带有Survivin启动子的重组腺病毒Adeno-SP-EGFP在BIU87细胞中表现出较高的肿瘤特异性活性,为下一步进行肿瘤特异性的基因治疗研究奠定了基础。

Construction and identification of a recombinant adenoviral vector with Survivin promoter

Objective Toconstruct a recombinant adenoviral vector with Survivin promoter,and detect its specific expression in bladder cancer cell line BIU87 and normal bladder cell line SV-HUC-1.Methods The fragment of Survivin promoter was acquired by PCR amplification,and the eukaryotic expression vector pSurp-EGFP(with Survivin promoter) and pCMV-EGFP(with CMV promoter) were constructed.The recombinant adenoviruses Ad-SPEGFP and Ad-CMV-EGFP were harvested by ligation in vitro.The two adenoviruses were transfected into BIU87 cells and SV-HUC-1 cells,respectively.The expressions of EGFP were detected by fluorescent microscope.Results A 440 bp gene fragment was amplified by PCR method from BIU87 cell genomic DNA and recombinant adenovirus Ad-SP-EGFP was constructed successfully.After transfection into BIU87 cells and SV-HUC-1 cells,a large number of green fluorescence was observed in BIU87 cells while scattered green fluorescence in SV-HUC-1 cells.Conclusions The recombinant adenovirus Ad-SP-EGFP with Survivin promoter has been constructed successfully and it has high specific activity in BIU87 cells,which might be potential in the gene therapy of bladder cancer.