鸡马立克病病毒Meq蛋白抑制p53的转录激活作用

Meq protein of Marek's disease virus inhibits the transactivity of p53

目的构建鸡马立克病病毒野生型Meq基因(Meq-wt)和p53结合区域缺失的突变型Meq基因(Meq-mut)表达质粒,研究Meq蛋白对p53转录激活功能的影响,为进一步了解其生物学功能奠定基础。方法采用聚合酶链反应(PCR)方法克隆鸡马立克病病毒Meq-wt基因,并采用突变PCR方法缺失Meq与p53结合区域,分别构建了Meq-wt和Meq-mut基因表达质粒。转染鸡胚成纤维细胞(CEF)后,用Western印迹法检测Meq蛋白的表达,利用荧光素酶报告基因系统证实Meq蛋白对p53转录激活的抑制作用。同时借助免疫荧光技术,采用荧光显微镜观察Meq蛋白与p53蛋白在细胞中的定位。结果DNA序列测序表明克隆的Meq-wt、Meq-mut基因插入位点和核苷酸序列完全正确。荧光素酶报告基因系统证实Meq蛋白对p53转录激活具有明显的抑制作用。间接免疫荧光试验证实野生型Meq蛋白主要在胞质中表达,而缺失与p53结合区域的突变型Meq在胞质/胞核中均有表达。野生型Meq蛋白与内源性p53蛋白在胞核内能够很好地重合。结论Meq蛋白对p53的转录激活具有抑制作用,可能是通过直接与p53相互结合,来抑制对p53的转录活性。

Objective To construct recombinant plasmids expressing wild type Meq （Meq-wt） and mutant Meq （Meq-mut） from the Marek＇ s disease virus （MDV）, and to use the plasmids in order to examine the inhibitory activity of the Meq gene on the transactivity of p53.Methods Meq-wt was cloned by PCR, and Meq-mut, which has the p53 binding domain deleted, was generated using PCR-based site- directed mutagenesis. The expression of Meq-wt and -mut in primary chicken embryo fibroblasts （CEF） was detected by Western blot. The p53-Luc reporter plasmid was co-tranfected with the Meq-wt and -mut plasmids into CEF cells to evaluate the inhibitory activity of the Meq gene on the transactivity of p53. The sub-cellular localizations of Meq proteins and p53 were also examined by immunofluorescent staining. Results DNA sequencing analysis showed that Meq-wt and -mut were obtained and properly inserted into the expression vector. The p53-Luc reporter gene assay showed that Meq inhibited the transactivity of p53. The Meq-wt protein was mostly localized in the nucleus of transfected cells and co-localized with p53, whereas the Meq-mut protein was distributed throughout the cytoplasm and the nucleus. Conclusion Meq protein of MDV inhibits the transactivity of p53 and this inhibitory activity of Meq protein is exerted probably through direct binding to p53.