摘要
目的探讨构建人类富含亮氨酸重复和免疫球蛋白样结构域1(LRIG1)基因过表达慢病毒表达载体并转染胶质瘤细胞系U87细胞的技术方法,为研究LRIG1的功能提供帮助。方法用EcoRⅠ及BamHⅠ双酶切LRIG1基因和plvxDsRed-monomer-n1慢病毒载体,琼脂糖凝电泳回收LRIG1片段和载体片段;通过T4连接酶将LRIG1基因连接至慢病毒载体上;按Lenti-XHT慢病毒包装试剂盒说明包装慢病毒;用EcoRⅠ及BamHⅠ双酶切法鉴定重组慢病毒载体;然后用plvxDsRed-monomer-n1和plvxDsRed-monomer-n1-3×flagLRIG1分别转染293T细胞和胶质瘤细胞系U87细胞,荧光定量PCR和western blot检测LRIG1 m RNA和蛋白表达。结果 U87细胞感染病毒载体后经嘌呤霉素筛选显示细胞红色荧光较空载体感染细胞减弱;实时PCR结果显示LRIG1 m RNA过表达组较对照明显升高;提取感染后细胞蛋白,Western blot鉴定flag标签蛋白表达成功。结论 LRIG1基因慢病毒表达载体能感染胶质瘤细胞系U87细胞,可使外源基因获得稳定表达。
Objectives To construct a lentiviral vector over-expressing leucine-rich repeats and immunoglobulin bulin-like domain protein 1 （LRIG1） gene, and to examine the LRIG1 gene expressed by the transfected cells in order to provide the basis for stundy of LRIG1 gene function. Methods The full length of the LRIG1 gene was cut from plasmid Zeroblunt-topo-LRIG1 by BamH Ⅰ and Ecor Ⅰ , subjected to electrophoresis by agarose gel and then extracted with DNA Gel Extraction Kit. The lentiviral vector PlvxDsRed-monomer-n1 was cut by BamH I and Ecor I, and then was inserted into the full length of the LRIG1 by T4 DNA Ligase. Each vector and the Lenti-X HTX Packaging mixture were cotransfected into 293T cells. Human glioma cell line U87 cells were infected by the lentivirus. The LRIG1 gene expression was determined by quantitative PCR and western blot. Results The results of gel electrophoresis showed that the LRIG1 gene was cloned into the lentiviral vector. PCR and western blot showed that LRIG1 gene was expressed. Conclusion The lentiviral vector over-expressing LRIG1 gene was successfully constructed, and lentivirus can transfect cells and exnous gene was expressed stably.
出处
《中国临床神经外科杂志》
2011年第10期605-608,共4页
Chinese Journal of Clinical Neurosurgery
基金
国家自然科学基金(NO.30872653)
