小鼠survivin基因慢病毒表达载体的构建及鉴定

Construction and identification of lentiviral vector encoding survivin gene of mice

背景:间充质干细胞体内有限的存活能力严重制约其用于体内治疗方面的应用,因此寻找一种能延长存活能力的实验方法是间充质干细胞进行体内实验的重要前提。目的:应用pLV.Des3d.P/hygro构建小鼠survivin基因的慢病毒表达载体。方法:采用Oligo核酸软件对Genbank上所发表的小鼠survivinmRNA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-EF1A>mSurvivin>IRES/EmGFP,再次进行PCR、测序的鉴定。结果与结论: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。说明慢病毒表达载体pLV.EX3d.P/hygro-EF1A>mSurvivin>IRES/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。

BACKGROUND：The limited viability of mesenchymal stem cells restricts its application in treatment in vivo.Therefore,it is very important to find an experiment method to prolong the viability of mesenchymal stem cells so as to lay foundation for experiment in vivo.OBJECTIVE：To construct the lentiviral vector encoding survivin gene of mice with clone vector pLV.Des3d.P/hygro.METHODS：Oligo nucleic acid software was used to design a couple of primers according to survivin gene sequence of mice which is publicated on Genbank.The attB recombination site was added to the two ends of the couple of primers,and mSurvivin and IRES/EmGFP were amplified by PCR.The PCR product after BP reaction was transferred to Stb13,and the correct pDown-mSurvivin and pTail-IRES/EmGFP were selected and sequenced.The mixture of pDown-mSurvivin,pTail-IRES/EmGFP and pLV.Des3d.P/hygro was used to do LR reaction.The lentiviral vector pLV.EX3d.P/hygro-EF1A〉mSurvivin〉IRES/EmGFP was amplified and sequenced again.RESULTS AND CONCLUSION：The results of PCR and sequencing showed that the lentiviral vector encoding survivin gene of mice was constructed successfully,which lays foundation for the further study of survivin anti-apoptosis in mesenchymal stem cells.