Survivin启动子驱动的LRIG1基因重组腺病毒载体的构建及鉴定

Construction and identification of a recombinant adenoviral vector carrying LRIG1 gene driven by Survivin promoter

目的构建含Survivin启动子和LRIG1基因的重组复制缺陷型腺病毒并进行鉴定,为肿瘤基因治疗的研究奠定基础。方法应用分子克隆技术将Survivin启动子连接至pLRIGl-GFP上构建出pEGFP-Surp-LRIGl,将其与含有5型腺病毒右臂的质粒Pbhge3通过Lipofectamine2000共转染至人胚肾293细胞,经同源重组产生重组腺病毒Ad-Surp-LRIGl。Ad-Surp-LRIGl在人胚肾293细胞中大量扩增并通过氯化铯密度梯度离心法纯化,测其滴度。结果 pEGFP-Surp-LRIGl经酶切鉴定正确,扩增得到的Ad-Surp-LRIGl经PCR扩增证实为Survivin启动子驱动的LRIG1重组腺病毒,滴度为2.3×1010pfu/ml。结论成功构建出含有Sur-vivin启动子和LRIG1基因的重组复制缺陷型腺病毒并鉴定正确,可用于下一步膀胱癌基因治疗的研究中。

Objective To construct replication defective adenovirus that was recombinant with Survivin promoter and LRIG1 gene,and lay the foundation for following cancer gene therapy experiments.MethodsThe Survivin promoter was linked to pLRIGl-GFP and pEGFP-Surp-LRIGl was constructed,and the pEGFP-Surp-LRIGl recombinant plasmid was transfected into 293 cell with Pbhge3 containing adenovirus type 5 that included Right Arm,which produced Ad-Surp-LRIGl by the homologous recombination method.Ad-Surp-LRIGl was propagated in 293 cells and purified by cesium chloride gradient centrifugation.ResultsIdentified by restriction endonuclease analysis and PCR,recombinant adenovirus with Survivin promoter and LRIG1 gene was constructed successfully,with a viral titer of 2.3×10pfu/ml.ConclusionsThe recombinant adenovirus carrying LRIG1 gene driven by Survivin promoter has been constructed successfully,which can be used to the research of gene therapy for bladder cancer.