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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4.8×104CFU/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。
Objective To construct recombinant retroviral vector pLXSN-EGFP-rtTA expressing enhanced green fluorescent protein (EGFP) and rtTA double gene and express in PA317 cells. Methods The plasmid pEGFP-C1 was used as template to obtain the EGFP gene fragment. The recombinant plasmid was inserted into retrovirus vector pLXSN and the correct recombinant retrovirus vector pLXSN-EGFP was screened out. PCR method was used to amplify the rtTA gene from pTet-on plasmid. After digestion and purification, the recombinant plasmid was cloned into the retroviral vector pLXSN-EGFP. Restriction endonuclease analysis and PCR analysis identified the correct recombinant vector pLXSN-EGFP- rtTA was transfected into PA317 cells by lipofectamine. The expression of EGFP and rtTA genes in PA317 cells was detected by PCR. The mRNA expression of rtTA in PA317 cells was detected by RT-PCR. The expression of EGFP gene was observed by fluorescence microscopy. Recombinant retroviral titers were determined using NIH3T3 cells as target cells. Results Recombinant retroviral vector expressing EGFP and rtTA double genes was constructed successfully. After transfection with PA317 cells, the mRNA expression of rtTA gene was detected by RT-PCR. The green fluorescence of EGFP gene was observed under fluorescence microscope , Indicating that the retroviral vector with the EGFP and rtTA double genes was normally expressed in PA317 cells and the PA317 cells transfected with pLXSN-EGFP-rtTA vector produced a recombinant virus with a viral titer of 4.8 × 10 4 CFU / ml. Conclusion Recombinant retroviral vector expressing EGFP and rtTA double genes was constructed successfully. The vector can be expressed normally in PA317 cells and a higher titer of virus supernatant can be obtained.