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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 CX43-IRES2-EGFP recombinant adenovirus small plasmid expression vector and observe its expression in human gastric cancer BGC-823 cell line. Methods: CX43 fragment of target gene and IRES2-EGFP were spliced by PCR and recombined into adenovirus shuttle vector pAd / CMV / V5-DEST to construct adenovirus expression vector pAd-CX43-IRES2-EGFP. 293A cells were packaged for determination of titer. The recombinant adenovirus was transfected into human gastric cancer cell line BGC-823 and the expression of CX43 was detected by RT-PCR. The expression of CX43 was detected by Western blot. Results: The adenovirus vector pAd-CX43-IRES2-EGFP was successfully constructed. The titer of pAd-CX43-IRES2-EGFP virus was 2.8 × 10 6 cells / ml. After transfection with human gastric cancer cell line BGC-823, CX43 protein was overexpressed by Western blot, and the MDR resistance gene was inhibited. CONCLUSION: The adenovirus vector pAd-CX43-IRES2-EGFP is successfully constructed and transfected into human gastric cancer cell line BGC-823 with overexpression of CX43 and MDR. Research and based on the CX43 target gene drug screening platform laid the foundation.