小鼠Cxcl10基因shRNA慢病毒表达载体构建与基因沉默效果鉴定  

Construction and Identification of Lentivector Encoding Chemokine CxcllO shRNAs

作　　者：卜慧莲[1] 舒斌[1] 王伟[1] 高峰[1] 田玉科[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院麻醉学教研室,武汉市430030

出　　处：《医学分子生物学杂志》2013年第5期254-258,共5页Journal of Medical Molecular Biology

基　　金：国家自然科学基金（No.81171259,81070890）

摘　　要：目的构建携带趋化因子一Cxcl10shRNA的慢病毒表达载体，并鉴定其在BV一2细胞中的沉默效果。方法将含有针对小鼠Cxcl10的ShRNA序列克隆到pGC—LV载体中，包装浓缩后用293T细胞测定病毒滴度。将Cxcl10ShRNA慢病毒载体分别以感染复数MOI为1，10和100的条件感染BV一2细胞，72h后采用real．timePCR技术和Western印迹检测shRNA对Cxcl10基因的沉默效果。结果Cxcl10ShRNA被成功构建入慢病毒表达载体pGC—LV，病毒滴度为5×10。TU／ml。用感染复数（MOI）为1或10的shRNA慢病毒感染BV．2细胞，可沉默约35％的Cxcl10基因表达，当MOI为100时，Cxcl10基因的沉默效果为65％，并可有效下调CXCLl0蛋白的表达。结论成功构建了携带Cxcl10shRNA的慢病毒载体．为研究趋化因子CXCLl0在疼痛、吗啡镇痛中的作用提供了有效的研究工具。Objective To construct a lentivector that can encode short hairpin RNAs （shRNAs） targeting the gene, Cxcll0, and to examine its gene-silencing efficiency. Methods The special ShRNA sequences targeting mouse Cxcl10 were cloned into pGC-LV vector. The lentivirus particles were packaged and their titer was detected in 293T cells. The gene-silencing efficiency of the Cxcll0 shRNA lentiviral vector in BV-2 cells was measured by real-time PCR and western blotting when MOI was at series of 1, 10, and 100. Results Cxcl10 ShRNAs were successfully cloned into pGC-LV. The titer of the concentrated lentivirus was 5 ×10s TU/ml. The gene-silencing efficiency of the constructed virus in BV-2 cells was about 35% when the MOI was 1 or 10, and the Cxcll0 mRNA expression was down-regulated by 65% when the MOI was 100. CXCL10 protein was also effectively down-regulated by Cxcll0 shRNA. Conclusions Lentivectors encoding Cxcl10 shRNA were successfully constructed, which provide an effective tool for researching the role of Cxcll0 gene in the regulation of pain and morphine analgesia.

关 键 词：慢病毒载体 趋化因子 基因沉默 短发卡RNA 

分 类 号：R349.6[医药卫生—基础医学]