LRIG2基因短发夹RNA表达载体的构建、鉴定和稳定株的筛选  

Construction of LRIG2 Specific Short-hairpin RNA Expressing Vector and Screening of Stably Transfected Cell Clone

作　　者：王宝峰[1] 蔡明俊[1] 陈如东[1] 韩林[1] 郭东生[1] 雷霆[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院神经外科,武汉430030

出　　处：《华中科技大学学报（医学版）》2009年第3期351-354,共4页Acta Medicinae Universitatis Scientiae et Technologiae Huazhong

基　　金：国家自然科学基金资助项目(No.30500521)

摘　　要：目的构建针对富含亮氨酸重复序列免疫球蛋白样蛋白2(LRIG2)基因的短发夹RNA(shRNA)表达载体,建立稳定转染的细胞株,观察其对目的基因表达的影响。方法根据GenBank中LRIG2基因序列设计2条RNA干扰序列,命名LRIG2-shRNA1、LRIG2-shRNA2,同时设计1条非特异性序列作为阴性对照。据此设计合成各自的寡核苷酸链,退火后连接入pGenesil2载体,转化扩增后进行序列测定。用不同浓度的G418作用于GL15细胞,得到G418对于GL15细胞的筛选浓度。3种重组表达载体转染胶质瘤细胞系GL15细胞,用G418筛选后挑单克隆后扩增获得稳定株。逆转录RT-PCR和Western印迹法分别在mRNA和蛋白水平上检测LRIG2的表达。结果3种重组表达载体(pGenesil 2-LRIG2-shRNA1、pGenesil 2-LRIG2-shRNA2和pGenesil 2-negative shRNA)经限制性酶切及DNA测序分析证明序列插入正确。G418对于GL15细胞的筛选浓度为600μg/ml,筛选出稳定转染三种质粒的GL15细胞,转染pGenesil2-LRIG2-shRNA2组细胞LRIG2 mRNA和蛋白表达明显低于转染pGenesil 2-LRIG2-shRNA1、pGenesil 2-negative shRNA组。结论成功构建了针对LRIG2基因的shRNA表达载体(pGenesil2-LRIG2-shRNA2),转染细胞后可抑制LRIG2基因表达,为研究LRIG2基因的功能提供了基础。Objective To construct effective short-hairpin RNA （shRNA） expression vector encoding shRNA targeting LRIG2 gene, and to screen the stably transfected cell clone. Methods Two shRNAs sequences based on the sequence of LRIG2 mRNA in the GenBank were designed and synthesized, and one scrambled shRNA sequences served as negative control. The synthesized sequences were cloned into shRNA expression vector pGenesil2. Accordingly the fatal dose of G418 to GL15 cell, and the selection concentration of G418 to GL15 cell were determined. The three shRNA vectors were transfeeted into GL15 by Metafectene respectively. The stably transfected cell clones were obtained after being screened with G418. Reverse transcription-polymerase chain reaction （RT-PCR） and Western blot were performed to examine the inhibitory effect at the RNA level and protein level. Results The recombinant plasmids containing shRNA were analyzed by restriction endonuclease analysis and DNA sequencing. The screening concentration of G418 to GL15 cells was 600 ng/ml. The LRIG2 expression was significantly down-regulated by siRNA as validated by RT-PCR and Western blot. Conclusion RNA interference （RNAi） mediated by the shRNA expression vector could significantly down-regulate the expression of LRIG2 in glioma cell line GL15. The stably transfected cell clone was obtained for further study.

关 键 词：富含亮氨酸重复序列免疫球蛋白样蛋白2 RNA干扰 短发夹RNA 

分 类 号：Q7[生物学—分子生物学]