【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。更多还原

【Abstract】 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 × 106 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.更多还原