小鼠XBP1基因RNA干扰慢病毒载体的构建及筛选  

Construction and screening of lentiviral vector of RNA interference of mouse XBP1 gene

作　　者：钟河江[1] 蒋建新[1] 王海燕[1] 杨策[1] 张冲[1] 严军[1] 刘庆[1] 黄苏娜[1] 

机构地区：[1]第三军医大学大坪医院野战外科研究所第四研究室全军交通医学研究所,全军交通伤重点实验室,创伤,烧伤与复合伤国家重点实验室,重庆400042

出　　处：《现代生物医学进展》2009年第8期1401-1404,共4页Progress in Modern Biomedicine

基　　金：国家重点基础研究发展规划项目("973"项目)(2005CB522602);全军医学科研"十一五"科技攻关项目(06G080)

摘　　要：目的:构建小鼠XBP1基因RNA干扰(RNA interference,RNAi)慢病毒载体,筛选具有较好干扰效率的小鼠XBP1 siRNA靶序列。方法:针对小鼠XBP1基因特异性序列,设计4个RNAi靶序列及1个阴性对照序列,合成含有正义和反义OligoDNA的互补DNA序列,退火形成双链DNA,并克隆到经AgeⅠ和EcoRⅠ酶切后的pGCL-GFP载体连接产生短发卡RNA(shRNA)慢病毒载体,PCR筛选阳性克隆,DNA测序鉴定。由病毒包装系统进行包装,经滴度测定后感染NIH3T3细胞,应用Real-timePCR鉴定干扰效率。结果:PCR鉴定与DNA测序证实合成的寡核苷酸链插入正确,293T细胞测定病毒滴度为1×108TU/ml。Real-timePCR证实XBP1-siRNA-3靶点的干扰效率最高,其干扰效率达到95%以上。结论:成功构建并筛选了小鼠XBP1基因RNAi慢病毒载体,为研究XBP1在巨噬细胞免疫功能调控中的作用奠定了基础。Objective： To construct lentiviral vector of RNA interference（RNAi） of mouse X-box binding protein 1 （XBP1） gene, and screen the effective sequence of siRNA targeting XBP1 gene. Methods： According to the GenBank information of mouse XBP1 gene, four interfering sequences and a negative sequence were designed. The complementary DNA containing both sense and antisense Oligo DNA of the targeting sequence was synthesized, and then were formed into double-stranded DNA by annealing, The obtained products were cloned into the pGCL-GFP vector digested by the restriction enzyme of Age Ⅰ and EcoR Ⅰ to construct lentiviral vector which expressed short hairpin RNA （shRNA）, and it was identified by PCR and DNA sequencing. The successful construct vector was packaged by packaging plasmid mix. The titer of virus was tested. NIH3T3 cells were infected by lentivirus, and the interfering efficiency was determined by Real-time PCR. Results： PCR identification and DNA sequencing demonstrated that insertion of oligonucleotide was correct. The titer of virus tested according to the expression level of GFP was 1 × 10^8 TU/ml. Real-time PCR anaylsis confirmed XBP1-siRNA-3 had the highest interfering efficiency, and the interfering efficiency of lentivirus was more 95%. Conclusion： The lentivirus RNAi vector of mouse XBP1 was constructed and screened successfully, which lays a foundation for further study of the role of XBP1 in the regulation ofmacrophage immune function.

关 键 词：RNA干扰 XBP1 慢病毒 

分 类 号：Q953-3[生物学—动物学] Q75