摘要
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 generate lentiviral expression vectors harboring human Oct4 gene and EGFP gene.Methods Oct4-IRES-EGFP was obtained from pLenti-EF1a-Oct4-IRES-EGFP by digestion with SacⅡ,blunted at SacⅡ site,and then by digestion with BamHⅠ,and subsequently directionally cloned into the restriction sites EcoR Ⅰ（blunted at EcoRⅠ site） and BamHⅠ of the lentiviral vector pFUGW after EGFP gene was released from pFUGW by digestion with EcoRⅠ,blunted at EcoRⅠ site,and then by digestion with BamHⅠ,designated pFUOGW,as verified by enzyme digestion.According to the standard protocol from Invitrogen,lentiviruses were produced,followed by confirmation using EGFP assay under fluorescent microscope after infecting 293FT cells.Lentiviruses harboring Oct4 and EGFP genes were employed to infect human nasopharyngeal carcinoma cells（i.e.,C666-1,CNE-1 and 6-10B）.Results pFUOGW was successfully generated.Lentivirus supernatant harboring Oct4 and EGFP genes efficiently infected C666-1,CNE-1 and 6-10B.Conclusion The lentiviral expression vector harboring human Oct4 gene and EGFP gene was successfully constructed.
出处
《热带医学杂志》
CAS
2010年第3期238-240,F0004,共4页
Journal of Tropical Medicine
基金
国家自然科学基金委员会-广东省联合基金重点项目(No.u0732006)
国家自然科学基金(No.30271177)
广东省自然科学基金(No.021903
No.9151063101000015)
广东省卫生厅基金(No.A2007359)
南方医科大学优秀中青年科技人才库科研资助金
广州地区科学仪器协作共用网专用基金(No.2006176)
