CX43腺病毒表达载体的构建及其在人胃癌BGC-823细胞系的表达  

CX43 adenovirus vector and its expression in human gastric cancer BGC-823 cell line

作　　者：吴瑾[1] 刘文涛[1] 李永庆 周红凤[1] 刘丹[1] 岳晓龙[1] 孙喜文[3] 石光跃[1] 赵艳滨[1] 王亚丽[1] 仇鑫[1] 

机构地区：[1]哈尔滨医科大学附属第三医院肿瘤内科,黑龙江哈尔滨150081 [2]美国Fred Hutchinson癌症研究中心,美国西雅图98109 [3]哈尔滨医科大学肿瘤研究所,黑龙江哈尔滨150081

出　　处：《现代肿瘤医学》2012年第11期2231-2236,共6页Journal of Modern Oncology

基　　金：黑龙江省教育厅海外学人科研资助项目(编号:1154h06)

摘　　要：目的:构建并鉴定CX43-IRES2-EGFP的重组腺病毒小质粒表达载体,观察其在人胃癌BGC-823细胞系中的表达。方法:将目的基因CX43片段与IRES2-EGFP进行PCR拼接,重组到腺病毒穿梭载体pAd/CMV/V5-DEST上,构建成腺病毒表达载体pAd-CX43-IRES2-EGFP;经酶切后转染293A细胞进行包装,测定滴度;重组病毒转染人胃癌BGC-823细胞后,通过RT-PCR检测耐药基因MDR的变化,Westernblot检测CX43超表达。结果:经测序验证,腺病毒载体pAd-CX43-IRES2-EGFP构建成功。pAd-CX43-IRES2-EGFP病毒的滴度为:2.8×106ifu/ml。转染人胃癌BGC-823细胞后经Western blot检测到CX43蛋白超表达,耐药基因MDR受到抑制。结论:成功构建腺病毒载体pAd-CX43-IRES2-EGFP高效体外转染表达体系,检测其对人胃癌BGC-823细胞系具有超表达CX43和抑制耐药基因MDR的作用,为CX43基因转染的研究及基于CX43为靶标的基因药物筛选平台的奠定了基础。Objective：To construct and identify the CX43-IRES2-EGFP recombinant adenovirus small plasmid expression vector,and observed expression in human gastric cancer cell line BGC-823.Methods：CX43 gene fragments for PCR with IRES2-EGFP splicing,recombinant adenovirus shuttle vector pAd/CMV/V5-DEST to the constructed adenovirus expression vector pAd-CX43-IRES2-EGFP;after transfection by digestion 293A packaging cells,titer was determined;recombinant virus-transfected human gastric cancer BGC-823 cells was tested by RT-PCR,changes in drug resistance gene MDR,western blot to detect CX43 expression.Results：After sequencing,adenoviral vector pAd-CX43-IRES2-EGFP was successfully constructed.pAd-CX43-IRES2-EGFP virus titer：2.8 × 10^6 ifu / ml.Transfection of human gastric cancer BGC-823 cells by western blot to detect CX43 protein overexpression,resistance gene MDR inhibition.Conclusion：We successfully constructed adenovirus pAd-CX43-IRES2-EGFP transfection efficiency in vitro expression system,to detect the human gastric cancer BGC-823 cells,with overexpression of Cx43 and inhibition of drug resistance gene MDR role for the CX43 gene research and CX43-targeting gene-based drug discovery platform to establish the foundation.

关 键 词：连接蛋白43 重组 腺病毒 载体 

分 类 号：R730[医药卫生—肿瘤] R735.2[医药卫生—临床医学]