人NSPc1慢病毒过表达系统的建立与鉴定  

Construction and identification of lentiviral over-expression system of human NSPc1 gene

作　　者：李辉[1] 龚燕华[2,3] 胡光宇[4,3] 阴彬[4,3] 

机构地区：[1]武警医学院组织胚胎学教研室,北京100005 [2]武警医学院生物化学教研室,天津300162 [3]中国医学科学院基础医学研究所医学分子生物学国家重点实验室,北京100005 [4]武警医学院

出　　处：《基础医学与临床》2011年第4期440-444,共5页Basic and Clinical Medicine

基　　金：国家自然科学基金(30971614;31070929);医学分子生物学国家重点实验室外部课题(2010)

摘　　要：目的建立并鉴定人NSPc1慢病毒过表达系统,以便应用过表达技术进一步研究NSPc1功能。方法设计针对人NSPc1cDNA序列的带酶切位点引物,PCR扩增获得双链DNA后,与酶切后的pLenti6-TO-EGFP-TRIP载体片段进行连接、转化,酶切及DNA测序鉴定重组克隆。提取阳性克隆质粒,转染293T细胞并用Western blot检测质粒过表达效果。用293T细胞制备慢病毒颗粒,感染293T细胞后收集细胞全蛋白,用Western blot检测慢病毒系统过表达效果。结果证实人NSPc1正确插入慢病毒载体,人NSPc1慢病毒过表达质粒及相应病毒颗粒能有效过表达NSPc1。结论人NSPc1慢病毒表达系统的成功建立,为应用过表达技术研究人NSPc1功能打下了基础。Objective To construct and identify lentiviral over-expression system to study the function of NSPc1 using over-expression technique.Methods The cDNA sequence of human NSPc1 was chosen to design the primer,and restricted enzyme site was added to primer sequences.The DNA purified from PCR was digested with double restriction enzymes and then linked with linearized pLenti6-TO-EGFP-TRIP.The recombinants were identified by restriction digestion and DNA sequencing.The plasmids of positive clones were transfected into 293T cells,and Western blot was used to identify the over-expression efficiency of the lentiviral vector.Then 293T cells were used to package the lentiviral particles and infected by obtained lentiviral particles.The over-expression efficiency of NSPc1 lentiviral system was examined by Western blot.Results The human NSPc1 sequences was successfully inserted into pLenti6-TO-EGFP-TRIP vector,which was identified by double restriction digestion and DNA sequencing.Western blot validated that both the lentiviral vector and lentiviral particle can effectively over-express human NSPc1 gene in 293T cells.Conclusion The construction of over-expression lentiviral system of human NSPc1 gene may support the further study of function of NSPc1 gene as a potential over-expression technique.

关 键 词：过表达 NSPC1 慢病毒载体 多梳家族 

分 类 号：R373[医药卫生—病原生物学]