葡萄糖转运蛋白-2 逆转录病毒表达载体构建包装及鉴定.docVIP

葡萄糖转运蛋白-2逆转录病毒表达载体构建/包装及其鉴定 方 芳，凌贤龙（400037 重庆，第三军医大学新桥医院消化科） [摘要]目的 构建葡萄糖转运蛋白-2（Glucose Transporter-2，GLUT-2）基因逆转录病毒表达载体及其稳定产毒细胞系，为新型“人工胰岛β细胞”的构建奠定基础。方法 质粒pCB7/GLUT2经Eco RⅠ、Bam HⅠ双酶切，克隆至逆转录病毒载体PLXSN，构建成重组逆转录病毒表达载体pL-GLUT2-SN，经酶切及测序鉴定；转pL-GLUT2-SN至包装细胞系PA317，G418抗性筛选，检测上清病毒滴度，挑选滴度较高的稳定产毒细胞克隆行PCR及RT-PCR鉴定。结果 建立GLUT-2基因逆转录病毒表达载体pL-GLUT2-SN，经酶切及测序证实目的基因插入位点及读码框架正确；所建稳定产毒细胞克隆PA317/GLUT2，其最高病毒滴度达7.1×105 CFU/mL，PCR及RT-PCR证实GLUT-2基因整合入其中并稳定表达。结论 成功构建携GLUT-2基因的逆转录病毒表达载体及其稳定产毒细胞系。 [关键词]葡萄糖转运蛋白-2；逆转录病毒载体 Construction and identification of a retroviral vector containing GLUT-2 gene Fang Fang， Xian-Long LinDepartment of Gastroenterology， Xinqiao Hospital， Third Military Medical University of Chinese PLA， Chongqing 400037， ChinaAbstract] Objective To establish a retroviral vector containing GLUT-2 gene and a stable vector production cell line， for the construction of a newartificial beta cell”. Methods The plasmid pCB7/GLUT2 was digested by Eco RⅠ/Bam HⅠ， then cloned to retroviral vector PLXSN， constructed the recombinant plasmid PL-GLUT2-SN. The plasmid PL-GLUT2-SN was evaluated by enzyme-digestion and sequencing. The plasmid PL-GLUT2-SN was transfect to packaging cell line PA317.The titer of the recombinant virus was detected. The high- titer cell lines were chosen for culture， and then evaluated by PCR and RT-PCR. Results The retroviral vector containing GLUT-2 gene was established successfully， which was confirmed by enzyme digestion and sequence analysis. The vector production cell lines PA317/GLUT2 was established. The highest titer of the recombinant virus was 7.1×105 CFU/m. GLUT-2 gene integration and expression were detected by PCR and RT-PCR. Conclusion A retroviral vector containing GLUT-2 gene and a high-titer vector production cell line PA317/GLUT2 are successfully constructed. [Key word]Glucose Transporter-2； retroviral vectorCorresponding author：name？Tel：86- - ？；E-mail？ 【通信作者】姓名，电话：（ ） ；E-mail： 糖尿病以胰岛素绝对或相对缺乏引致各种代谢紊乱为主要特征，其病因尚未完全阐明。传统的体外胰岛素注射治疗