携带人c-Myc和EGFP基因慢病毒表达载体的构建

Construction of a lentiviral expression vector harboring human c-Myc and EGFP genes

目的构建携带人c-Myc和EGFP基因的慢病毒表达载体pFUMGW。方法 XhoI线性化pLenti-EF1a-c-Myc-IRES-EGFP,回收片段并补平XhoI,接着BamHI酶切该片段,回收2722bp片段而获连接用c-Myc-IRES-EGFP;EcoRI线性化pFUGW,回收并补平EcoRI,然后BamHI酶切该片段,回收9174bp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c-Myc-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pFUMGW。获pFUMGW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c-Myc和EGFP基因的慢病毒感染人胚肾细胞株293、肿瘤细胞株CNE1和C666以建立相应的病毒感染体系。结果酶切证实成功构建了pFUMGW,按标准程序生产的携带c-Myc和EGFP基因的慢病毒上清高效率感染293、CNE1和C666。结论成功构建携带人c-Myc和EGFP基因的慢病毒表达载体pFUMGW,为相关后续研究打下了良好基础。

Objective To generate lentiviral expression vector harboring human c-Myc gene and EGFP gene.Methods c-Myc-IRES-EGFP was released from pLenti-EF1a-c-Myc-IRES-EGFP by digestion with Xho I,blunted at Xho I site,and then by digestion with BamH I,and subsequently directionally cloned into the restriction sites EcoR I（blunted at EcoR I site）and BamH I of the lentiviral vector pFUGW after EGFP gene was released from pFUGW by digestion with EcoR I,blunted at EcoR I site,and then by digestion with BamH I,designated pFUMGW,as verified by enzyme digestion.According to the standard protocol from invitrogen,lentiviruses were produced,followed by confirming that lentiviruses were successfully made through EGFP assay under fluorescent microscope after infecting 293FT cells.Lentiviruses harboring c-Myc and EGFP genes were employed to infect mammalian cells（i.e.,CNE1,C666）.Results The results from enzyme digestion showed that pFUMGW was successfully generated.Lentivirus supernatant harboring c-Myc and EGFP genes efficiently infected CNE1 and C666.Conclusion The lentiviral expression vector harboring human c-Myc and EGFP gene was successfully constructed,which will lay a solid foundation for further research.