CD47-shRNA慢病毒载体构建及其介导Hep-2细胞CD47基因沉默效应鉴定  

Construction of targeted lentiviral vector shRNA of CD47 gene and its effect of CD47 gene silence in Hep-2 cells

作　　者：高树峰[1] 张少容[1] 徐莲[1] 李黎[1] 杨春平[1] 刘建国[1] 汪美群[1] 刘红兵[1] 刘月辉[1] 

机构地区：[1]南昌大学第二附属医院耳鼻咽喉头颈外科,江西南昌330006

出　　处：《中国现代医学杂志》2014年第9期1-6,共6页China Journal of Modern Medicine

基　　金：江西省自然科学基金资助项目(No:20114BAB205069);江西省卫生厅科技计划资助项目(No:20133067)

摘　　要：目的构建CD47靶向shRNA慢病毒干扰载体,并观察其对Hep-2细胞CD47基因的抑制效应。方法设计CD47靶点特异性的寡核苷酸序列,利用酶切反应合成含特异性系列线性化的pGCSIL-GFP载体,包装293T细胞产生慢病毒并行滴度测定,再转染人喉癌Hep-2细胞,通过实时荧光定量PCR及Western Blotting检测重组慢病毒对CD47靶基因在mRNA和蛋白水平上的沉默抑制情况。结果阳性克隆PCR及测序证实针对CD47基因shRNA慢病毒干扰载体构建成功,RT-PCR及Western Blotting检测载体在mRNA和蛋白水平上可抑制喉癌Hep-2细胞的CD47表达。结论成功构建针对CD47基因的慢病毒干扰载体,为进一步行喉癌CD47基因功能研究提供了较好的实验工具。[ Objective ] To construct short hairpin RNA interference lentiviral vector of CD47 gene and to evaluate their inhibitory effect in Hep-2 cells. [ Methods ] The CD47 target specific oligonucleotide sequence was designed, synthesized and connected into the pGCSIL-GFP vector digested, 293T cells were packaged to produce the lentivirus, and then transfected with Hep-2 cells. The inhibitory effect on CD47 gene was tested by the real-time quantitative PCR and Western Blotting. [Results] The PCR and DNA sequencing of positive clones demonstrated that the lentivirus shRNA vector of CD47 gent was constructed successfully. The real-time quantitative PCR and Western Blotting confirmed that the vector could suppress the expression level of CD47 at mRNA and protein levels. [ Conclusions ] CD47-shRNA lentivirus vector has been constructed successfully, and our recombined lentivirus vector will be a good model for further study and utilization on gene function research about CD47.

关 键 词：CD47 RNA干扰 慢病毒载体 构建鉴定 HEP-2细胞 

分 类 号：R346[医药卫生—基础医学]