CARex和PTDtat基因重组5型腺病毒的包装及鉴定

Package of recombinant adenovirus serotype 5 carrying with CARex and PTDtat gene

目的分别构建和包装携带CARex基因、PTDtat基因、CARex和PTDtat融合基因的重组腺病毒。方法采用基因重组技术将CARex基因、PTDtat基因、CARex和PTDtat融合基因克隆在腺病毒穿梭质粒pAdtrack-CMV,经过Pme I线性化后转化BJ5183感受态细胞,与腺病毒骨架质粒pAdEasy-1同源重组,构建重组腺病毒质粒pAd-CARex、pAd-PTDtat、pAd-CARex-PTDtat,然后转化DH5α感受态细胞,大量制备重组腺病毒质粒,经过Pac I线性化后回收,在脂质体介导下转染293A细胞,通过倒置荧光显微镜观察绿色荧光蛋白(GFP)的表达来确定病毒在细胞内复制,当细胞发生病变时证明病毒包装成功。结果在293A包装细胞系中成功包装携带CARex、PTDtat、CARex和PTDtat基因的重组5型腺病毒,倒置荧光显微镜观察到明显的GFP表达,PCR检测表明包装病毒含有目的基因,半数培养组织感染量(TICD50)显示重组腺病毒具有较高的滴度。结论成功包装了pAd-CARex、pAd-PTDtat、pAd-CARex-PTDtat重组腺病毒,为进一步探讨CARex和PTDtat基因对腺病毒感染肿瘤细胞效率的影响奠定了实验基础。

Objective To construct and pack the recombinant adenovirus serotype 5 carrying CARex, PTDtat, CARex-PTDtat gene respectively. Methods The recombinant shuttle plasmid pAdtraek-CMV-CARex, pAdtrack-CMV-PTDtat, pAdtrack-CMV-CARex-PTDtat were constructed successfully by vitro ligation, the recombi- nant shuttle plasmid were linearized by Pme I and then transformed to BJ5183 cell containing the pAdEasy-1 boneplasmid and the conformed plasmid were tansformed to DH5α cells for larger scale preparation of this plasmid, pu- rifing recombinant adenovirus plasmid digested with Pac I, then transfected 293A cells using liposome. Results Recombinant adenovirus serotype 5 carrying CARex, PTDtat, CARex-PTDtat gene were packaged in 293A cells, and identified successfully by inverted fluorescence microscope and PCR. Conclusion Construction and package of the recombinant adenovirus pAd-CARex, pAd-PTDtat, pAd-CARex-PTDtat for preparation of infection the tumor ceils and testing the function of CARex and PTDtat gene in the adenovirus infections.