携带EGFP与rtTA双基因的逆转录病毒载体的构建及其在PA317细胞中的表达

目的 构建表达增强型绿色荧光蛋白(EGFP)、rtTA双基因的重组逆转录病毒载体pLXSN-EGFP-rtTA,并在PA317细胞中表达.方法 以质粒pEGFP-C1为模板进行PCR反应获得EGFP基因片段,定向插入逆转录病毒载体pLXSN,筛选出正确的重组逆转录病毒载体pLXSN-EGFP.PCR方法从pTet-on质粒中扩增出rtTA基因,酶切纯化后克隆于逆转 录病毒载体pLXSN-EGFP中,限制性内切酶酶切分析及PCR鉴定筛选出正确的重组载体pLXSN-EGFP-rtTA,脂质体介导转染PA317细胞,PCR方法检测PA317细胞内EGFP、rtTA基因,RT-PCR方法检测PA317细胞内rtTA基因的mRNA表达,荧光显微镜观察EGFP基因的表达情况.以NIH3T3细胞为靶细胞测定重组逆转录病毒滴度.结果 成功构建了表达EGFP、rtTA双基因的重组逆转录病毒载体,转染PA317细胞后,RT-PCR方法可检测到rtTA基因的mRNA表达,荧光显微镜下可观察到EGFP基因表达所产生的绿色荧光,说明连接有EGFP和rtTA双基因的逆转录病毒载体在PA317细胞中可正常表达,转染pLXSN-EGFP-rtTA载体的PA317细胞可产生4.8x 104 CFU/ml病毒滴度的重组病毒.结论 成功构建表达EGFP、rtTA双基因的重组逆转录病毒载体,该载体可在PA317细胞内正常表达,获得较高滴度的病毒上清.

Construction of retroviral expression vector containing both EGFP and rtTA genes and their expression in PA317 cells

Objective To construct the retroviral vector expressing enhanced green fluorescent protein （EGFP） and rtTA genes, and their expression in the PA317 cells. Methods EGFP gene was amplified by PCR and directionally inserted into the multiple cloning site of retroviral vector pLXSN. Positive clone was identified by enzyme digesting and PCR. The rtTA gene was amplified by PCR from plasmid pTet-on and cloned into the retroviral vector pLXSN-EGFP. The correct recombinant retroviral vector pLXSN-EGFP-rtTA was identified by restriction enzymes and PCR. The pLXSN-EGFP-rtTA plasmid was transfected into PA317 cells by lipofectin. The EGFP and rtTA genes in PA317 cells were identified by PCR and the expression of EGFP was detected by fluorescence microscopy. The mRNA expression of rtTA gene in PA317 cells was detected by RT-PCR. The titer of viral supernatant produced by packaging cells PA317 was determined with NIH3T3 cells. Results Retroviral vector expressing EGFP and rtTA genes was constructed successfully and transferred into packaging cells PA317. rtTA specific mRNA expression of the PA317-GFP-rtTA was detected by RT-PCR and the EGFP was observed by fluorescence microscope. The high titer, 4.8×104 CFU/ml, viral supernatant was obtained. Conclusion Recombinant retroviral pLXSN-EGFP-rtTA plasmid was constructed, which can be expressed in PA317 cells and produced high titer viral supernatant.