新型血友病B基因治疗的腺病毒-整合酶嵌合系统的构建及其体外表达鉴定

Construction and Identification of A Novel Adeno-Integrase Hybrid System for Hemophilia B

本研究构建一种既可高效感染靶细胞又能实现安全定点整合入宿主基因组的腺病毒嵌合载体。通过一系列DNA操作构建腺病毒-整合酶嵌合系统,该系统包含两个腺病毒载体:一个携带转基因表达框,表达框内有hFⅨ,红色荧光蛋白编码序列以及attB(phiC31识别位点),表达框两侧各有1个loxP(Cre识别位点);另一个腺病毒载体携带Cre和phiC31基因。同时构建只表达Cre和只表达phiC31的载体作为对照载体。在体外分别用脂质体转染293A细胞,用荧光显微镜观察荧光蛋白的表达,用RT-PCR鉴定各个基因的表达。结果表明,该腺病毒-整合酶嵌合系统构建成功。体外分别用脂质体转染293A细胞后,可见绿色荧光蛋白和红色荧光蛋白表达。RT-PCR鉴定表明,该系统能够成功表达各种目的蛋白。结论:成功构建了腺病毒-整合酶嵌合系统,为进一步研究其治疗作用提供良好基础。

This study was aimed to construct an adenovirus hybrid system with high transduction efficiency and site-specific integration.By a series of DNA manipulation,a hybrid system of two adenovirus vectors was constructed.One vector contains loxP-flanked transgene expression cassette,in which there are hFⅨ and DsRed coding sequences and attB for phiC31 recognization.The other vector carries Cre and phiC31 gene.Vectors only expressing Cre or phiC31 were used as controls.293A cells were constructed and transfected with the adenoviral vectors by Lipofectamine 2000,and the expression of target genes was identified by fluorescence microscopy and RT-PCR.The results showed that after being identified by PCR,restriction analysis and sequencing,an adeno-integrase hybrid system was successfully constructed.The system expressed RFP,GFP,hFⅨ,Cre and phiC31 in 293A cells in vitro.It is concluded that the adeno-integrase hybrid system is successfully constructed,which lays a good foundation for further investigation of its therapeutic application.