鸡MDV L-meq基因与该病毒感染的鸡胚成纤维细胞中p53基因的相关性

Relationship between Chicken Marek Disease Virus(MDV) L-meq Gene and p53 Gene in MDV-infected Chicken Embryo Fibroblasts

目的探讨鸡马立克病病毒(Marek disease virus,MDV)L-meq基因与MDV感染的鸡胚成纤维细胞(Chicken em-bryo fibroblast,CEF)中p53基因的相关性。方法双酶切质粒pMD18-T-L-meq,获得L-meq基因片段,克隆入载体pcDNA3.1(+),构建重组真核表达质粒pcDNA-L-meq,转染CEF细胞,利用实时定量RT-PCR检测转染细胞中p53基因mRNA的表达量,TRAP法检测转染细胞中端粒酶的活性,流式细胞术检测转染细胞细胞周期的变化。结果重组真核表达质粒pcDNA-L-meq经双酶切鉴定构建正确。转染L-meq基因后48 h,CEF细胞中p53基因mRNA的表达量略有升高,72 h时下降。转染L-meq基因后48、72 h,CEF细胞的端粒酶活性比未转染的CEF细胞分别高16、1个活力单位。转染L-meq基因后48 h,CEF细胞G0/G1期比例较转染空载体pcDNA3.1(+)的细胞(对照)显著增加(P<0.01);转染后72 h,实验组G0/G1及G2/M期细胞比例较对照组显著增加(P<0.01);转染后48和72 h,S期细胞比例较对照组均显著减少(P<0.01)。结论 L-meq基因对MDV的增殖具有一定程度的抑制作用,在病毒的增殖过程中可能为一种转录抑制因子。

Objective To investigate the relationship between chicken Marek disease virus（MDV） L-meq gene and p53 gene in MDV-infected chicken embryo fibroblasts（CEFs）.Methods Recombinant plasmid pMD18-T-L-meq was digested with NcoⅠ and XhoⅠ,and the obtained L-meq gene fragment was cloned into vector pcDNA3.1（＋）.CEFs were transfected with the constructed recombinant plasmid pcDNA-L-meq,and determined for expression level of p53 mRNA by real-time quantitative RT-PCR,for telomerase activity by TRAP method,and for cell cycle by flow cytometry.Results Restriction analysis proved that recombinant plasmid pcDNA-L-meq was constructed correctly.The expression level of p53 mRNA in CEFs increased slightly 48 h,while decreased 72 h after transfection with L-meq gene.The telomerase activity of CEFs 48 and 72 h after transfection with L-meq gene were 16 and 1 unit higher than those of untransfected CEFs respectively.The percentage of CEFs at G0 / G1 phase 48 h after transfection with L-meq gene was significantly higher that with empty vector pcDNA3.1（＋）（P 0.01）.The percentages of CEFs at G0 / G1 and G2 / M phases 72 h after transfection were significantly higher in test group than in control group（P 0.01）.However,the percentage of CEFs at S phage 48 and 72 h after transfection was significantly lower in test group than in control group（P 0.01）.Conclusion L-meq gene showed a certain inhibitory effect on proliferation of MDV,which might be served as a transcription inhibitor during virus proliferation.