CARex和PTDtat基因重组5型腺病毒的包装及鉴定

发布时间：2018-06-02 09:08

本文选题：CARex + PTDtat　；参考：《安徽医科大学学报》2013年07期

【摘要】：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。
[Abstract]:Objective to construct and package the recombinant adenovirus carrying CARex gene pPTDtat gene pCARex and PTDtat fusion gene respectively. Methods CARex gene pAdtrack-CMV was cloned into adenovirus shuttle plasmid pAdtrack-CMV by gene recombination technique. After linearized with Pme I, it was transformed into BJ5183 receptive cells and was homologous recombined with adenovirus skeleton plasmid pAdEasy-1. The recombinant adenovirus plasmid pAd-CARexanpAd-PTDtatpAd-CARex-PTDtata was constructed, and then transformed into DH5 伪 competent cells. The recombinant adenovirus plasmid was linearized by Pac I and was recovered and transfected into 293A cells by liposome. The expression of green fluorescent protein (GFP) was observed by inverted fluorescence microscope to determine the replication of the virus in the cell, and it was proved that the virus was packaged successfully when the cell changed. Results Recombinant adenovirus type 5 was successfully packaged in 293A packaging cell line carrying CARexexia PTDtatCARex and PTDtat genes. Reverse fluorescence microscopy showed that the packaging virus contained the target gene. TICD 50) showed high titer of recombinant adenovirus. Conclusion the recombinant adenovirus pAd-CARex-PTDtat was successfully packaged, which laid an experimental foundation for further study of the effect of CARex and PTDtat genes on the efficiency of adenovirus infected tumor cells.
【作者单位】：安徽医科大学病原生物学教研室;安徽医科大学生物化学教研室;
【基金】：国家自然科学基金(编号:30771882、81271815)
【分类号】：R346

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