NSPc1-shRNA慢病毒表达载体的构建与鉴定  

Construction and identification of lentiviral vectors expressing shRNA of NSPc1 gene

作　　者：李辉[1] 龚燕华[2,3] 胡光宇[2] 阴彬[2] 

机构地区：[1]武警医学院组织胚胎学教研室,天津300162 [2]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 [3]武警医学院生物化学教研室

出　　处：《山西医科大学学报》2010年第9期775-779,共5页Journal of Shanxi Medical University

基　　金：国家自然科学基金面上项目(30971614;31070929);医学分子生物学国家重点实验室开放课题

摘　　要：目的构建并鉴定NSPc1-shRNA慢病毒表达载体,以便应用RNAi技术以及慢病毒感染系统进一步研究NSPc1的功能。方法设计针对NSPc1mRNA靶序列的小发夹状RNA,化学合成含茎环结构的正义链与反义链,退火形成带内切酶粘/平末端的双链后,与酶切后的pLentiLox3.7载体片段进行连接、转化。用双酶切及DNA测序鉴定重组克隆。提取阳性克隆质粒并转染293T细胞,收集细胞全蛋白用Western检测RNAi效果。结果重组克隆经酶切证实shRNA正确插入慢病毒载体,DNA测序证实插入的序列正确,Western检测证实设计的三条RNA干扰序列有效敲低了293T细胞中的内源性NSPc1。结论 3个NSPc1-shRNA慢病毒表达载体的成功构建为应用基于慢病毒系统的RNAi技术来研究NSPc1基因的功能打下了基础。Objective To construct and identify three lentiviral vectors for studying the function of NSPc1 using RNAi technique and lentivirus system. Methods Three mRNA sequences of NSPe 1 were chosen , and the small hairpin RNA sequences were designed.The shRNA sequences were annealed and linked with linearized pLentiLox3.7. The recombinants were identified by double restriction digestion and DNA sequencing. The plasmids of positive clones were transfected into 293T cells, and then the RNAi efficiency of the lentiviral vectors was determined by Western blot. Results The small hairpin RNA sequences were successfully inserted into pLenti- Lox3.7 vector, and the sequences were identified by DNA sequencing. Further, Western blot results validated that the three small hairpin RNAs effectively knockdowned the expression of endogenous NSPc1 in 293T cells. Conclusion The construction of small hairpin RNA expression vectors of NSPc1 gene makes a further study of function of NSPc1 gene using lentivirus system-based RNAi technique.

关 键 词：RNA干扰 NSPC1 小发夹状RNA 慢病毒载体 多梳家族 

分 类 号：Q782[生物学—分子生物学]