小鼠survivin基因慢病毒表达载体的构建及鉴定.docVIP

小鼠survivin基因慢病毒表达载体的构建及鉴定 杨 平，苟 欣* ，周青松，何卫阳，余昆 (重庆医科大学 附属第一医院 泌尿外科, 中国重庆 400016) 摘 要：应用pLV.Des3d.P/hygro构建小鼠survivin基因的慢病毒表达载体.采用Oligo核酸软件对Genbank上所发表的小鼠survivin mRNA序列进行分析，设计一对survivin基因上下游引物，利用Gateway 克隆方法，分别在其两端添加attB重组位点，运用PCR扩增mSurvivin和IRES/EmGFP，将PCR产物进行BP反应然后转化到Stbl3感受态菌，通过卡那霉素抗性筛选、PCR鉴定，选取鉴定正确的pDown-mSurvivin和pTail-IRES/EmGFP克隆测序.将pDown-mSurvivin和pTail-IRES/EmGFP与pLV.Des3d.P/hygro混合进行LR反应，构建慢病毒载体pLV.EX3d.P/hygro-EF1AmSurvivinIRES/EmGFP，再次进行PCR、测序的鉴定.结果显示基因序列克隆正确，成功构建了含有小鼠survivin基因的重组慢病毒表达载体.慢病毒表达载体pLV.EX3d.P/hygro-EF1AmSurvivinIRES/EmGFP构建成功，为下一步研究survivin在间充质干细胞中抗凋亡作用奠定基础. 关键词：骨髓间充质干细胞;慢病毒;pLV.Des3d.P/hygro;Survivin 中图分类号: Q784 文献标识码: A Construction and Identification of lentiviral vector Encoding Survivin Gene of Mice YANG Ping, GOU Xin* , ZHOU Qing-song, HE Wei-Yang，YU Kun (Department of Urology, the first Affiliated hospital, Chongqing Medical University, Chongqing 400016, China) Abstract: To Construct and Identify the lentiviral vector encoding survivin gene of mice with clone vector pLV.Des3d.P/hygro. By using Oligo nucleic acid software, we analyze the survivin gene sequence of mice which is publicated on Genbank. With gateway clone method, designed a couple of primer containing attB recombination site. mSurvivin and IRES/EmGFP were obtained by PCR, the PCR product after BP reaction was transfered to Stb13, and the correct pDown-mSurvivin and pTail-IRES/EmGFP were selected by Kanamycin resistance and PCR. The lentiviral vector pLV.EX3d.P/hygro-EF1AmSurvivinIRES/EmGFP was obtained by pDown-mSurvivin, pTail-IRES/EmGFP and pLV.Des3d.P/hygro with LR reaction. Through PCR and sequencing, the lentiviral vector Encoding Survivin Gene of Mice was constructed successfully and which lays foundation for the further study of survivin anti-apoptosis in mesenchymal stem cells. Key words: mesenchymal stem cells; lentiviral vector; pLV.Des3d.P/hygro;survivin Survivin基因是近年来发现的凋亡抑制蛋白(inhibitor of apoptosis protein, IAP)家族最小成员, 能编码产生一个由142 个氨基酸组