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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。
Objective To construct a lentiviral vector pFUSGW carrying human Sox2 and EGFP genes. Methods Xho Ⅰ linearization of pLenti-EF1a-Sox2-IRES-EGFP, recovery of fragments and fill level Xho Ⅰ incision, followed by BamH Ⅰ digestion of this fragment recovered 2.356kb fragment was connected with Sox2-IRES-EGFP; EcoRI linearized pFUGW, recovered and Then, the fragment was ligated with BamHI to recover the fragment of 9.174kb and ligated with the vector fragment. Finally, the product was ligated with Sox2-IRES-EGFP for ligation using Solution I in DNA Ligation Kit (TaKaRa) The next day pick single colony, plasmid extraction and parallel digestion identification. The constructed vector was named pFUSGW. After pFUSGW was obtained, the lentivirus was packaged according to the standard procedure recommended by Invitrogen and confirmed whether the lentivirus was successfully produced. The lentivirus carrying Sox2 and EGFP genes infect mouse embryonic fibroblasts (MEFs), human embryonic lung fibroblasts (HLFs) ) And human glioma cells (U87) to establish the corresponding viral infection system. Results Enzymatic digestion confirmed the successful construction of pFUSGW. MEFs, HLFs and U87 were efficiently infected by lentiviral supernatants carrying Sox2 and EGFP genes by standard procedures. Conclusion The successful construction of lentiviral vector pFUSGW carrying human Sox2 and EGFP gene has laid a good foundation for the subsequent research.