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目的构建携带人Oct4和EGFP基因的慢病毒表达载体pFUOGW。方法SacⅡ线性化pLenti-EF1a-Oct4-IRES-EGFP,回收片段并补平SacⅡ切口,接着BamHⅠ酶切该片段,回收2.565kb片段而获连接用Oct4-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Oct4-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pFUOGW。获pFUOGW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Oct4和EGFP基因的慢病毒感染293FT细胞和鼻咽癌细胞株C666-1、CNE1和6-10B以建立相应病毒感染体系。结果酶切鉴定证实成功构建了pFUOGW,按标准程序生产的携带Oct4和EGFP基因的慢病毒上清高效率感染鼻咽癌细胞株C666-1、CNE1和6-10B。结论成功构建携带人Oct4和EGFP基因的慢病毒表达载体pFUOGW,为相关后续的研究打下良好的基础。
Objective To construct a lentiviral vector pFUOGW carrying human Oct4 and EGFP genes. Methods The linearized pLenti-EF1a-Oct4-IRES-EGFP was digested with SacⅡ and ligated into SacⅡ. The fragment was digested with BamHⅠ. The 2.565kb fragment was recovered and ligated with Oct4-IRES-EGFP. EcoRⅠ linearized pFUGW, 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 Oct4-IRES-EGFP 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 pFUOGW. After obtaining pFUOGW, lentivirus packaging was performed according to the standard procedure recommended by Invitrogen and whether the lentivirus was successfully produced; 293FT cells and nasopharyngeal carcinoma cell lines C666-1, CNE1 and 6-10B were infected with lentivirus carrying Oct4 and EGFP genes Establish a corresponding virus infection system. Results The restriction enzyme digestion confirmed that pFUOGW was successfully constructed and the nasopharyngeal carcinoma cell lines C666-1, CNE1 and 6-10B were highly efficiently transfected with the lentivirus supernatants carrying Oct4 and EGFP gene by standard procedures. Conclusion The successful construction of lentiviral vector pFUOGW carrying human Oct4 and EGFP gene lays a good foundation for the follow-up research.