LRIG1基因特异性RNA干扰表达载体的构建、鉴定和稳定株的筛选

Construction of LRIG1 Specific RNAi Expressing Vector and Screening of Stably Transfected Cell Clone

目的构建针对LRIG1LRIG1(leucine-rich repeats andi mmunoglobulin-like domains1,LRIG1)基因的特异性RNA干扰质粒,稳定转染人胶质瘤GL15细胞系,观察其对目的基因LRIG1表达的影响,为探讨LRIG1基因沉默对人脑胶质瘤细胞的生物学行为调控奠定基础。方法根据GenBank提供的LRIG1基因序列设计2条RNA干扰序列,命名为LRIG1-shRNA1、LRIG1-shRNA2,并设计1条非特异性序列作为阴性对照,命名为pGenesil2-negativesh RNA。合成各自的寡核苷酸链,退火后与pGenesil2质粒载体连接,转化扩增后测定序列。用不同浓度的G418作用于GL15细胞确定G418对GL15的筛选浓度。将3种重组表达载体转染GL15细胞,G418筛选后挑单克隆并扩增获得稳定株。Western印迹法在蛋白水平上检测LRIG1的表达。结果重组pGenesil2-LRIG1-sh RNA质粒经限制性酶切及DNA测序分析证明序列插入正确。G418对GL15细胞的筛选浓度为600mg/L,筛选出稳定转染三种质粒的GL15细胞,转染pGenesil2-LRIG1-sh RNA1组细胞LRIG1蛋白表达明显低于转染pGenesil2-negativesh RNA组。结论成功构建了针对LRIG1基因的特异性shRNA表达载体(pGenesil2-LRIG1-sh RNA1),转染细胞后可抑制LRIG1基因表达,为下一步研究其功能奠定基础。

Objective To construct eukaryotic expression vector encoding RNA interference sequences specific for LRIG1 gene, to screen the stably transfected cell clone, and to observe its effect on the expression of LRIG1. Methods Two shRNAs sequences based on the sequence of LRIG1 mRNA in the Gen- Bank were designed and one scrambled shRNA sequence was regarded as negative control. The synthesized sequences were inserted into shRNA expression vector pGenesil2 and sequenced. The shRNA vectors were transfected into GL15 by Metafectene. The stably transfected cell clones were obtained after being screened with G418. Western Blotting was performed to examine the inhibitory effect at the protein level. Results The recombinant plasmidS containing shRNA were analyzed by restriction endonuclease analysis and DNA sequencing. The screening concentration of G418 to GL15 cell was 600 mg/L. LRIGI expression was significantly down-regulated by siRNA as validated by Western Bloting. Conclusion RNA interfering （RNAi） mediated by the shRNA expression vector could significantly down-regulate the expression of LRIG1 in glioma cell line GL15. The stably transfected cell clone was obtained for further study.