丙型肝炎病毒NS4B对YAP转录、表达、磷酸化及细胞内分布的影响  

Effect of the hepatitis C virus non-structural protein 4B on the levels of YAP transcription,expression,phosphorylation and intracellular distribution

作　　者：黎允诗[1] 张舒婷 周少虎[1] 张战锋[2] LI Yun-shi;ZHANG Shu-ting;ZHOU Shao-hu;ZHANG Zhan-feng(Department of Reproductive Medicine,Guangzhou Guangdong 510405,China;Laboratory Medicine,The First Affiliated Hospital of Guangzhou University of Chinese Medicine,Guangzhou Guangdong 510405,China)

机构地区：[1]广州中医药大学第一附属医院生殖医学科,广东广州510405 [2]广州中医药大学第一附属医院检验科,广东广州510405

出　　处：《蚌埠医学院学报》2022年第5期566-569,575,共5页Journal of Bengbu Medical College

基　　金：广东省中医药局科研项目(20222053);广州中医药大学第一附属医院创新强院青年科研人才培优项目(2015QN08);广州中医药大学学科研究重点项目及特色培育项目(XKP2019001)。

摘　　要：目的:构建丙型肝炎病毒(HCV)非结构蛋白4B(NS4B)细胞内荧光表达质粒,检测NS4B对Yes相关蛋白(YAP)转录、表达、磷酸化及细胞内分布影响。方法:根据现有含有HCV 1b型病毒NS4B完整基因的质粒设计NS4B的特异性扩增引物,扩增NS4B完整序列并构建NS4B荧光质粒,质粒转染进入HEPG2和293T细胞,通过Real-time PCR检测YAP基因转录水平;通过Western blotting检测YAP蛋白表达及磷酸化水平;通过激光共聚焦和核质分离实验检测YAP细胞内分布。结果:成功扩增出NS4B完整序列并构建其荧光质粒;Real-time PCR和Western blotting结果显示,NS4B并不影响YAP的转录和表达水平,但是能够调节其磷酸化水平;激光共聚焦和核质分离试验结果提示NS4B能够调节YAP在细胞质和细胞核内的比例。结论:HCV NS4B可能通过对YAP的影响发挥生物学功能。Objective:To construct the intracellular fluorescent expression plasmid of non-structural protein 4B(NS4B)of hepatitis C virus(HCV),and detect the effects of NS4B on the levels of transcription,expression,phosphorylation and intracellular distribution of Yes-associated protein(YAP).Methods:The specific amplification primers for NS4B were designed according to the existing plasmid containing HCV 1b virus NS4B complete gene.The complete sequence of NS4B was amplified to construct the NS4B fluorescent plasmid,and the plasmid was transfected into HEPG2 and 293T cells.The transcription level of YAP gene,and expression and phosphorylation levels of YAP protein were detected using Real-time PCR and Western blotting,respectively.The intracellular distribution of YAP was detected by confocal laser and nucleo-cytoplasmic separation assay.Results:The complete NS4B sequence was successfully amplified,and its fluorescent plasmid was constructed.The results of Real-time PCR and Western blotting showed that the NS4B did not affect the transcription and expression levels of YAP,but could regulate its phosphorylation level.The results of laser confocal and nucleo-cytoplasmic separation results suggested that the NS4B could regulate the ratio of YAP in cytoplasm and nucleus.Conclusions:The HCV NS4B may play a biological role through its effects on YAP.

关 键 词：丙型肝炎病毒 非结构蛋白4B Yes相关蛋白 细胞增殖 

分 类 号：R373.2[医药卫生—病原生物学]