AQP5基因重组慢病毒载体的构建与鉴定

摘要

目的 构建人水通道蛋白5(AQP5)基因慢病毒载体.方法 体外扩增出AQP5基因全长cDNA,将慢病毒载体pWPTS-GFP与扩增出的AQP5用Mlu I和Sal I进行双酶切,将AQP5全长序列克隆人pWPTS-GFP,转化大肠杆菌DH5α感受态细胞,对长出的克隆用菌落PCR鉴定,再对阳性克隆进行测序和比对分析.重组病毒质粒与另外两种辅助包装原件载体质粒通过CaCl2共转染293T细胞(人胚肾细胞),培养48 h后收集细胞培养上清液,将病毒浓缩后在293T细胞中测定病毒滴度,并检测慢病毒载体在工具细胞SGC7901细胞(胃癌细胞)的转染效率.Western blot检测SGC7901细胞中AQP5蛋白.结果 测序结果和Westem blot检测均证明AQP5重组慢病毒载体构建正确,并能在细胞中正确表达.与辅助质粒共转包装细胞获得慢病毒颗粒,并成功感染SGC7901细胞.包装慢病毒,浓缩病毒悬液的滴度为1×108 TU/ml.结论 成功构建了稳定表达AQP5基因的重组慢病毒载体.%Objective To construct the recombinant lentiviral vector of AQP5 gene. Methods AQP5 gene sequence was amplified as target gene in vitro. The cDNA was inserted into plasmid pWPTS-GFP which was linearized by restriction endonucleases Mlu I and Sal I . The recombinant plasmid was transformed into competent DH5a celJs. The grown colonies were identified by colony PCR and DNA sequencing. Recombinant lentivector plasmids and the other helping plasmids were co-transfected into 293T cells by CaCl2, and cell culture supernatant was collected after 48 h. The virus supernatant was concentrated and titered in 293T cells. The infection efficiency of the constructed virus was determined in SGC790I cells. AQP5 expression in SGC7901 cells was determined by Western blot. Results DNA sequencing and Western blot demon strated that the lentivirus vector was constructed successfully. The constructed virus were obtained and infected SGC7901 cells. The titer of concentrated virus was 1×108 TU/ml. Conclusion Recombinant lentiu iral vector of AQP5 gene is suc cessfully constructed.