携带人Sox2和EGFP基因慢病毒表达载体构建

目的构建携带人Sox2和EGFP基因的慢病毒表达载体pFUSGW。方法XhoⅠ线性化pLenti-EF1a-Sox2-IRES-EGFP,回收片段并补平XhoⅠ切口,接着BamHⅠ酶切该片段,回收2.356kb片段而获连接用Sox2-IRES-EGFP;EcoRⅠ线性化pFUGW,回收并补平EcoRⅠ切口,然后BamHⅠ酶切该片段,回收9.174kb片段获连接用载体片段,最后使用DNA连接试剂盒（TaKaRa）中的SolutionⅠ将其与连接用Sox2-IRES-EGFP连接,连接产物转化,次日挑选单菌落,提取质粒并行酶切鉴定。所构建载体命名为pFUSGW。获pFUSGW后,按Invitrogen公司推荐的标准程序进行慢病毒包装和确认慢病毒是否成功生产;携带Sox2和EGFP基因的慢病毒感染小鼠胚胎成纤维细胞（MEFs）、人胚肺成纤维细胞（HLFs）和人神经胶质瘤细胞（U87）以建立相应病毒感染体系。结果酶切证实成功构建了pFUSGW,按标准程序生产的携带Sox2和EGFP基因的慢病毒上清高效率感染MEFs、HLFs及U87。结论成功构建携带人Sox2和EGFP基因的慢病毒表达载体pFUSGW,为相关后续的研究打下了良好的基础。

Construction of A Lentiviral Expression Vector Harboring Human Sox2 and EGFP Genes

Objective To generate lentiviral expression vector harboring human Sox2 gene and EGFP gene.Methods Sox2-IRES-EGFP was released from pLenti-EF1a-Sox2-IRES-EGFP by digestion with XhoⅠ,blunted at XhoⅠ site,and then by digestion with BamHⅠ,and subsequently directionally cloned into the restriction sites EcoR Ⅰ（blunted at EcoRⅠ site） and BamHⅠ of the lentiviral vector pFUGW after EGFP gene was released from pFUGW by digestion with EcoRⅠ,blunted at EcoRⅠ site,and then by digestion with BamHⅠ,designated pFUSGW,as verified by enzyme digestion.According to the standard protocol from Invitrogen,recombinant lentiviruses were produced and confirmed using EGFP assay as suggested.Lentiviruses harboring Sox2 and EGFP genes were used to infect the mouse embryonic fibroblasts（MEFs）,human embryonic lung fibroblasts（HLFs） and human neuroglioma cells（U87）.Results Results from the enzyme digestion experiment showed that pFUSGW was successfully generated.Lentivirus harboring Sox2 and EGFP genes can efficiently infect MEFs,HLFs and U87 cells.Conclusion The lentiviral expression vector harboring human Sox2 and EGFP gene was successfully constructed.