早期蛋白p52对人巨细胞病毒基因复制作用的实验研究

目的 :探讨HCMVDNA聚合酶辅助蛋白p5 2在病毒基因复制过程中的作用。方法 :采用体外实验方法建立HCMV感染细胞模型 ,在感染后不同时间段 ,FQ PCR法测定细胞内HCMVDNA拷贝数 ,免疫组化法检测病毒蛋白p5 2 ,计算机图像分析系统处理结果。同时光镜观察致细胞病变作用 ,电镜检查其超微结构的改变。结果 :感染后各组细胞内均能检测到HCMVDNA及p5 2表达 ,随感染时间延长 ,二者水平均升高 ,p5 2定位于感染细胞核内。与感染后 12h相比 ,此后各时段p5 2表达量均明显增高 (P <0 0 1) ;而至感染后 48h ,细胞内HCMVDNA量方明显高于感染后 12h(P <0 0 5 ) ,持续到感染晚期 ;同时细胞开始出现病变 ,并随感染时间延长、细胞内p5 2表达水平升高及HCMVDNA量增多而逐渐加重。结论 :早期蛋白p5 2在HCMVDNA复制过程中发挥重要作用 ,可有效促进病毒基因复制。

Experimental study of human cytomegalovirus (HCMV) DNA polymerase accessory protein p52 on HCMV DNA replication

Objective To explore the effect of h um an cytomegalovirus (HCMV) DNA polymerase accesory protein p52 on HCMV DNA replic a ton. Methods HCMV infected-cell model was established in vit r o by coincubating passage cultured HEL and HCMV AD 169 with titer of 100TCID 50 . Then intracellular HCMV genome copies were evaluated by FQ-PCR and the ex p ression of p52 was detected by immunohistochemical test and semi-quantitated by computer image analysis. Meanmwhile, the cytopathologic effects (CPE) was obs erved by microscope and ultrasturctural changes were studied by transmisson elec tron microscope (TEM). Results Both HCMV DNA replication and p52 expression were detected 12 h post-infection and their levels were elevate d subsequently. p52 was localized in the nuclei after infection. Compared with th at of 12 h post-infection, p52 expression was signficantly elevated at 24 h, 48 h and 96 h after infection (P<0.01); while until 48 h post-infection, int r acellular HCMV genome copies showed a significant increase (P<0.05) and mo re significant since 96 h post-infection (P<0.01). Cytopathogenesis was o b served at 48 h post-infection, and aggravated from then on with the increase of p52 expression and HCMV genome copies in infected cells. Conclusion p52 appears to accelerate effectively HCMV DNA repilication. It suggests that p52 plays important role in HCMV DNA replication.