腺病毒-tk基因同源重组系统构建的研究

Study on Construction of the homologous recombinant adenovirus tk gene

目的研制能快速高效地构建携带外源目的基因tk的重组腺病毒细菌内同源重组系统。方法将pBluescriptⅡKDR-tk、ptrack与ptrackCMV质粒转化感受态细菌后扩增,通过PmeI酶线性化穿梭载体ptrackCMV-tk,然后将线性化的穿梭载体ptrackCMV-tk与pAdeasy-1进行重组构建成重组腺病毒质粒;利用整合入重组病毒质粒中的绿色荧光蛋白分别筛选重组后的腺病毒质粒。结果PCR和RT-PCR分析外源基因的表达证实tk基因在DNA和mRNA水平上均能有效表达;荧光显微镜下观察计数GFP阳性细胞数,计算病毒滴度为1.1×1011pfu/L。结论细菌内同源重组系统能快速高效地构建携带外源目的基因tk的重组腺病毒;绿色荧光蛋白方便早期证实腺病毒的产生。

Objective To explore the possibility of a high titer of the Recombinant Adenovirus Adeasy System induced Hsv-tk gene. Methods The KDR-tk fragment were taken from pBluescript II KDR-tk plasmid, then the fragment of shuttle vector ptrack and ptrack CMV in Adeasy system and tk fragment were ligated according to the instruction of Takara DNA Ligation Kit. The green fluorescence protein （GFP） positive stained cells was counted under fluorescent microscopy. Results The PCR and the RT-PCR results both indicated that the virus induced tk gene was combined in the gene of recombined adenovirus gene. It suggested that tk gene could be expressed effectively in DNA and mRNA. The GFP positive stained cells were counted, and the titer of the virus came to be 1.1 x 10H pfu/L. Conclusion The pAdeasy System may be used to produce a high titer of the recombinant adenovirus induced tk gene. The GFP expressing kit may be used directly to observe the transfection efficacy under fluorescent microscopy.