含鼠LINGO-1 shRNA重组腺病毒载体的构建和鉴定  

Construction and identification of replication recombinent adenovirus vector of mice LINGO-1 shRNA

作　　者：高峰[1] 孙红辉[1] 杨有庚[1] 

机构地区：[1]吉林大学第二附属医院骨科,吉林长春130041

出　　处：《中国实验诊断学》2009年第11期1491-1493,共3页Chinese Journal of Laboratory Diagnosis

摘　　要：目的构建含鼠LINGO-1短发夹RNA(short hairpin RNA,shRNA)的复制缺陷型腺病毒载体。方法通过PCR法将带有LINGO-1 shRNA序列构建至U6启动子(U6sh LINGO-1)下游,与T载体连接,将质粒转化入大肠杆菌。将U6sh LINGO-1连到穿梭质粒pDC316上,与腺病毒基因组质粒pBHGlox△E1、3Cre共转染293T细胞,得到重组腺病毒载体(rAd LINGO-1)。经PCR鉴定正确后,进行扩增、纯化、滴度测定及感染性鉴定。结果PCR法得到U6sh LINGO-1,rAd LINGO-1具有良好的感染性,腺病毒滴度可达109TCID50/ml。双引物PCR法鉴定Ad LINGO-1可扩增出Ad及特异性片段U6sh LINGO-1。结论成功构建了能表达LINGO-1 shRNA的重组腺病毒载体rAd LINGO-1,可能为临床应用RNA干涉技术促进脊髓损伤的恢复提供新的方法。Objective To construct the recombinant adenovirus vector with mice gene LINGO-1 （shRNA, short hairpin RNA）by Cre/lox P system. Methods U6 promoters following LINGO-1 shRNA （U6sh LINGO-1 ）were obtained with PCR. Then the promoters were ligated to T-vector. After plasnfids pT-U6sfi LINGO-1 being formed, these plasmids were transforuled into E. coli JMI09. The target gene! fragment was subcloned into shuttle plasmid pDC316 to construct recombinant shuttle plasmid pDC316- LINGO-1. Then the combinant shuttle plasmid and adenovirus ＇genomic plasmid pBHGlox△E1,3Cre were contransfected into 293T cell to construct recombinant adenovirus Ad LINGO-l, and it was identified by infection test and PCR amplification. After purification and concentra- tion, the titer of Ad LINGO- 1 reached 109TCIDso/ml. Ad LINGO- 1 could transfect 293T cell. Results U6sh LINGO- 1 was got by PCR method, rAd LINGO-1 has a good infection, adenovirus titers up to 109TCID50/ml. Double-PCR method to identify Ad LINGO-1 can be amplified and Ad-speeific fragment U6sh LINGO-1. Conclusion Can be successfully constructed the expression of LINGO-1 shRNA adenoviral vector tAd LINGO- 1, may be the clinical application of RNA interference technology for the recovery of spinal cord injury to provide a new method.

关 键 词：LINGO-1 腺病毒载体 RNA干扰 脊髓损伤 基因治疗 

分 类 号：R346[医药卫生—基础医学]