【摘要】 目的:构建携带耐药相关基因HA117的高滴度重组腺病毒,转染K562细胞,检测其在K562细胞中的表达情况。方法:用基因工程技术将ATRA相关耐药新基因HA117的cDNA亚克隆至穿梭质粒pAdTrack-CMV上,然后在细菌内与pAdeasy同源重组,后将重组腺病毒Adeasy-HA117经脂质体转染包装细胞HEK293,并收获原代的重组腺病毒Ad5easy-HA117,经乒乓交互感染的方法扩增原代腺病毒,应用RT-PCR、荧光显微镜及流式细胞仪鉴定和检测重组腺病毒感染K562细胞后HA117的表达情况。结果:酶切鉴定及PCR结果证明ATRA相关耐药新基因HA117重组腺病毒载体构建成功,携带新基因HA117重组腺病毒载体的效价达8.3×1011pfu/mL。RT-PCR检测到转染后K562细胞内HA117的表达。结论:成功获得携带新基因HA117的重组腺病毒,并能使其在K562细胞中高表达。更多还原

【Abstract】 Aim:To obtain the high titer recombinant adenovirus expressing human HA117 gene.Methods:The cDNA of HA117 gene was subcloned into the shuttle plasmid pAdTrack-CMV and then clone the homologous recombinant adenovims genomie plasmid pAdeasy in bacteria.We tranfected the DNA of identified recombinant plasmid into 293 ceils via liposome,and then did the package and amplification of adenovims, determined by fluorescence microscope.The expression of HA117 was monitored by RT-PCR,fluorescence and flow eytometry in infected K562 cells.Results:High titer recombined adnovirus carring HA117 gene was successfully obtained.The titer of the recombinant adenovims was 8.3×1011pfu/mL,and The expression of HA117 gene was observed. The specific expression of HA117 gene was identified by RT-PCR in K562 cell after infection with Ad-HA117.Conclusion:High titer recombined adnovirus carring HA117 gene was successfully obtained.Infected K562 cell by recombinant adenovims highly expressed HA117 gene.更多还原