腺病毒载体左侧反向末端重复区对下游基因表达特异性的影响

摘要

Aim : To study the expression of specific gene downstream of the left inverted terminal repeat ( ITR) in ad-enovectors. Methods:Construct a series of recombinant adenovirus vectors that contains green fluoresent protein (GFP) and hTERT promoter and recombinant adenovirus vectors containing the same transgene but in different orientations. The virus control group that contains CMV promoter and the other one which contains non-promotor. The recombinant adenovirus plasmids were digested with Pac I and transferred into 293 A cells mediated by Liopfectamine2000 to package recombinant adenovirus particles and then titrated using 50% tissue culture infective dose (TCID50) assay. All of the virus infected four tumor cell whith the same MOI and the green fluorescence was observed by fluorescent microscopy. Results: By statistical analysis, the fluorescent in each group does not exist significant difference and The faint fluorescent can be observed in the cell in which adenovirus absence of promoter. Conclusion: The enhancer elements of the left ITR in recombinant ade-novirus effect the expression of the upstream gene and downstream gene but the influence does not exist significant difference. Studies by this experiment have also demonstrated that the left ITR contains cryptic transcription start sites.%目的：研究腺病毒载体左侧反向末端重复区(ITR)对下游基因表达特异性的影响。方法：采用分子生物学方法及Gateway技术构建正反2个方向含有人端粒酶逆转录酶(hTERT)启动子及GFP报告基因的重组腺病毒载体。以含CMV启动子及GFP报告基因的重组腺病毒载体和不含任何启动子只含GFP报告基因的重组腺病毒载体作对照。重组腺病毒质粒经Pac Ⅰ线性化后用脂质体转染法转染293A细胞，包装并扩增出重组腺病毒颗粒，TCID50测得滴度后分别以相同的感染复数感染4种肿瘤细胞，观察荧光表达情况。结果：携带正向和反向hTERT启动子的重组腺病毒载体在各细胞中荧光表达量无差异，无启动子的重组载体在腺病毒增殖比较强的细胞中有微弱荧光表达。结论：重组腺病毒左侧ITR能够影响上下游特异启动子及其调控基因表达，但对上下游基因的影响程度无明显差异，并且左侧ITR具有类启动子功能。