携带人miR302和EGFP基因慢病毒表达载体构建

Construction of A Lentiviral Expression Vector Harboring Human miR302 and EGFP Genes

目的构建携带人miR302和增强型绿色荧光蛋白(EGFP)基因的慢病毒表达载体pFUM3GW。方法NheⅠ和NotⅠ双酶切pmiR302ApE以释放miR302,接着补平酶切位点而获连接用miR302;BamHⅠ酶切携带EGFP的慢病毒载体pFUGW,接着补平酶切产物,并去磷酸化而获连接用载体片段,最后使用DNA连接试剂盒(TaKaRa)中的SolutionI将其与连接用miR302连接,连接产物转化,次日挑选单菌落,PCR筛选正向阳性克隆,随后将选定的含有阳性克隆的单菌落摇菌,提取质粒并行酶切鉴定及对插入的miR302测序。所构建载体命名为pFUM3GW。获pFUM3GW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miR302和EGFP基因的慢病毒感染鼻咽癌细胞株C666-1、CNE1和5-8F以建立相应病毒感染体系。结果PCR、酶切和测序证实成功构建了pFUM3GW,按标准程序生产的携带miR302和EGFP基因的慢病毒上清高效率感染小鼠胚胎成纤维细胞(MEFs)及鼻咽癌细胞株C666-1、CNE1和5-8F。结论成功构建携带人miR302和EGFP基因的慢病毒表达载体pFUM3GW,为相关后续研究打下了良好基础。

Objective To generate lentiviral expression vector harboring human miR302 and EGFP genes. Methods Human miR302 was obtained from pmiR302ApE by digestion with Nhe Ⅰ and Not Ⅰ , blunted at Nhe Ⅰ and Not Ⅰ sites, and subsequently cloned into the lentiviral vector pFUGW containing EGFP gene at the blunted and dephosphorylated BamH Ⅰ site. The insertion was verified by enzyme digestion and the resultant vector was designated as pFUM3GW. The orientation of miR302 fragment inserted in the frame was identified by PCR. The complete sequence of the miR302 was further verified by sequencing. 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 mouse embryonic fibroblasts （MEFs）. Lentiviruses harboring miR302 and EGFP genes were employed to infect human nasopharyngeal carcinoma cells （i.e., C666-1,CNE1 and 5-8F）. Results Results from PCR, enzyme digestion and sequencing showed that pFUM3GW was successfully generated. Lentivirus harboring miR302 and EGFP genes can efficiently infect MEFs, C666-1, CNE1 and 5-8F. Conclusion The lentiviral expression vector harboring human miR302 and EGFP genes was successfully constructed. Our work will lay a solid foundation for further research.