携带人Kif4和EGFP基因慢病毒表达载体构建  

Construction of a Lentiviral Expression Vector Harboring Human Kif4 and EGFP Genes

作　　者：闫春江[1] 李国炜[1] 姚志芳[2] 肖高芳[2] 肖东[2] 

机构地区：[1]广东省中医院,邮政编码广州510120 [2]南方医科大学肿瘤研究所

出　　处：《微循环学杂志》2011年第2期8-9,12,I0001,F0003,共5页Chinese Journal of Microcirculation

摘　　要：目的:构建携带人Kruppel-like factor4(Kif4)和增强型绿色荧光蛋白(EGFP)基因的慢病毒表达载体pFUKGW。方法:Xho I线性化pLenti-EF1a-Kif4-IRES-EGFP,回收片段并补平XhoI酶切位点,接着BamH I酶切该片段,回收2.842kb片段而获连接用Kif4-IRES-EGFP;EcoR I线性化pFUGW,回收并补平EcoR I酶切位点,然后BamH I酶切该片段,回收9.174kb片段获连接用载体片段,最后使用DNA连接试剂盒中的Solution I将其与连接用Kif4-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pFUKGW。获pFUKGW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;将携带Kif4和EGFP基因的慢病毒感染人肿瘤细胞株CNE1、C666和SW620以建立相应病毒感染体系。结果:酶切证实成功构建了pFUKGW,按标准程序生产的携带Kif4和EGFP基因的慢病毒上清高效率感染CNE1、C666和SW620。结论:本实验成功构建携带人Kif4和EGFP基因的慢病毒表达载体pFUKGW,为开展肿瘤细胞重编程研究打下了良好基础。Objective:To generate lentiviral expression vector harboring human Kif4gene and EGFP gene.Method:Kif4-IRES-EGFP was released from pLenti-EF1a-Kif4-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 pFUKGW,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 Kif4 and EGFP genes were employed to infect mammalian cells(i.e.,CNE1,C666 and SW620).Results:The results from enzyme digestion showed that pFUKGW was successfully generated.Lentivirus supernatant harboring Kif4 and EGFP genes efficiently infected CNE1,C666 and SW620.Conclusion:The lentiviral expression vector harboring human Kif4 and EGFP gene was successfully constructed,which will lay a solid foundation for the study of cancer cell reprogramming.

关 键 词：Kif4 EGFP 慢病毒载体 

分 类 号：R34[医药卫生—基础医学]