重组腺病毒pCMV-NM23-IRES-EGFP载体的构建及鉴定

Construction and identification of recombinant adenovirus pCMV-NM23-IRES-EGFP

目的构建携带有NM23的重组腺病毒载体并观察其对人胃癌细胞株BGC-823凋亡的影响。方法以质粒NM23-PIRES2-EGFP为模版,采用PCR扩增NM23基因,克隆至腺病毒载体p CMV-MCS-IRES-EGFP上,形成重组腺病毒载体p CMV-NM23-IRES-EGFP并酶切鉴定及基因测序。将人胃癌细胞株BGC-823分为实验组(NM23-OE组,感染p CMV-NM23-IRES-EGFP)、空白组(Ctrl组),阴性对照组(NC组,感染p CMV-MCS-IRES-EGFP);Real Time PCR和Western Blot分别检测3组细胞中NM23mRNA和蛋白表达;流式细胞仪检测3组细胞凋亡率。结果经酶切鉴定和基因测序证实成功构建重组腺病毒p CMV-NM23-IRES-EGFP;经扩增后,重组腺病毒的滴度为4.0×1010ifu/m L;RTPCR及Western blot结果显示,阴性对照组与空白组相比差异无统计学意义(P>0.05),而实验组的NM23的mRNA和蛋白表达显著高于其他两组(P<0.05);实验组BGC-823细胞的凋亡率也显著高于其他两组(P<0.05)。结论成功构建了过表达NM23的重组腺病毒p CMV-NM23-IRES-EGFP载体,通过上调NM23基因的表达,可观察到胃癌细胞株BGC-823的凋亡水平有明显升高,为后续研究NM23基因对人胃腺癌细胞生物学功能的影响提供实验基础。

Objective To construct the re-combinant adenovirus vector carrying with gene NM23 and observe the influence on the apoptosis of human carcinoma of stomach cell line BGC-823.Methods The NM23 gene was amplified by PCR using plasmid NM23-PIRES2-EGFP as templateand cloned into adenovirus vector pCMV-MCS-IRES-EGFP.The recombinant adenovirus vector pCMV-NM23-IRES-EGFP was identified by restriction enzyme digestion and gene sequencing.The human gastric cancer cell line BGC-823 was divided into experimental group （NM23-OE group,infected pCMV-NM23-IRES-EGFP）,blank group （Ctrl group）,negative control group （NC group,infected pCMV-MCS-IRES-EGFP）.The mRNA and protein expressions of NM23 gene were detected by real Time PCR and western blot assay.The apoptosis was detected by flow cytometry.Results The recombinant adenovirus pCMV-NM23-IRES-EGFP was supported by restriction enzyme digestion and gene sequencing.After amplification,the titer of re-constructed adenovirus was 4.0×10^10 ifu/ml.Compared with the NC group,there was no significant difference between the Ctrl group and the NC group （P〉0.05）.The mRNA and protein expressions of NM23 gene in the NM23-OE group were higher than those in the other two groups（P〈0.05）.The apoptosis rate of human gastric cancer cell line BGC-823 in the NM23-OE group was also higher than that in the other two groups （P〈0.05）.Conclusion The recombinant adenovirus pCMV-NM23-IRES-EGFP vector is successfully constructed.The expression of NM23 gene is significantly increased by up-regulating the expression of NM23 gene BGC-823.These data could provide an experimental basis for the subsequent study on the effect of NM23 gene on the biological function of human gastric adenocarcinoma cells.