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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 construct a lentiviral vector pFUMGW carrying c-Myc and EGFP genes. METHODS: The fragment of pLenti-EF1a-c-Myc-IRES-EGFP was linearized by XhoI, the fragment was recovered and the XhoI was complemented. The fragment was digested with BamHI and the fragment of 2722bp was recovered and ligated with c-Myc-IRES- The EcoRI was recovered and supplemented, then the fragment was digested with BamHI, the 9174 bp fragment was recovered and ligated with the vector fragment, and finally ligation with c-Myc-IRES-EGFP using the DNA ligation kit (TaKaRa) Transformation, picked the next day single colonies, plasmid extraction and parallel digestion identification. The constructed vector was named pFUMGW. After obtaining pFUMGW, lentivirus packaging was performed according to the standard procedure recommended by Invitrogen and whether the lentivirus was successfully produced; lentivirus carrying the c-Myc and EGFP genes was used to infect human embryonic kidney cell line 293 and tumor cell lines CNE1 and C666 to establish corresponding Virus infection system. Results Enzymatic digestion confirmed that pFUMGW was successfully constructed and 293, CNE1 and C666 cells were efficiently infected with lentiviral supernatants carrying c-Myc and EGFP genes by standard procedures. Conclusion The successful construction of lentiviral vector pFUMGW carrying human c-Myc and EGFP genes lays a good foundation for further research.