丝裂原激活的蛋白激酶激酶2对猪瘟病毒复制的调控作用

发布时间：2018-06-16 17:03

  本文选题：猪瘟病毒 + 丝裂原激活的蛋白激酶激酶2　； 参考：《吉林农业大学》2015年硕士论文

【摘要】：猪瘟(Classical swine fever, CSF)是严重危害世界和我国养猪业的一种急性、热性、高度接触性和致死性传染病,以高热稽留、广泛性出血和免疫抑制为主要特征。猪瘟的病原是猪瘟病毒(Classical swine fever virus, CSFV)。CSFV是黄病毒科瘟病毒属的重要成员,其长约12.3 kb的单股正链RNA基因组仅含有一个大的开放阅读框(Open reading frame, ORF),编码一个分子量约438kDa的多聚蛋白,该多聚蛋白被病毒和宿主蛋白酶水解为8种非结构蛋白(NPro、p7、NS2、NS3、NS4A、NS4B、NS5A和NS5B)和4种结构蛋白(C、Erns、E1和E2)。丝裂原激活的蛋白激酶激酶2(Mitogen-activated protein kinase kinase 2/extracellular regulated kinase, MEK2)是细胞外信号调节激酶(Extracellular signaling-regulation kinase, ERK)信号通路的重要激酶。此外,MEK2还参与丙型肝炎、登革热等病毒的复制,而且MEK2与CSFV之间存在相互作用。CSFV感染PK-15细胞后能够激活宿主的ERK通路并促进MEK2的表达,应用特异性抑制剂阻断ERK信号通路能明显抑制CSFV复制。为了进一步研究MEK2对CSFV复制的影响,本研究利用慢病毒载体系统分别构建MEK2上调表达的PK-15稳定细胞系(PK-EGFP-MEK2)和MEK2下调表达的PK-15细胞系(PK-shMEK2),通过流式细胞术筛选EGFP阳性细胞,免疫荧光试验和Western blotting试验证实MEK2上调和下调表达的细胞系构建成功。细胞增殖试验表明MEK2上调和下调后均不影响细胞的活力及增殖水平。将所构建的细胞系进行CSFV感染试验,分别在病毒感染后的48 h和72 h,收集病毒感染样品,进行荧光定量RT-PCR和病毒效价的测定,并对感染后的细胞蛋白进行Western blotting检测以及荧光素酶报告系统检测等试验明确MEK2对CSFV复制的影响。结果显示,过表达MEK2能够明显提高病毒RNA拷贝数和病毒滴度,细胞裂解物中病毒Npro蛋白的表达量增加,表明上调表达MEK2可以促进CSFV的复制。相反,下调表达MEK2的细胞培养上清中病毒RNA拷贝数和病毒滴度明显低于对照组,细胞裂解物中Npro蛋白的表达量也显著减少,表明下调表达MEK2能够明显抑制CSFV的复制。总之,本研究发现宿主蛋白MEK2能够正调控猪瘟病毒的复制。
[Abstract]:Classical swine fever (CSF) is an acute, hot, highly contagious and fatal infectious disease which seriously endangers the world and the pig industry in our country. It is mainly characterized by high fever, extensive bleeding and immunosuppression. The pathogen of swine fever is that the swine fever virus (Classical swine fever virus, CSFV).CSFV is the weight of the yellow virus family. Members, its 12.3 KB single strand RNA genome contains only a large open reading frame (Open reading frame, ORF), encoding a polyprotein with a molecular weight of about 438kDa, which is hydrolyzed to 8 non structural proteins by the virus and the host protease (NPro, P7, NS2, NS3, NS4A, NS4A, NS4A, etc.) and 4 structural proteins And E2). The mitogen activated protein kinase kinase 2 (Mitogen-activated protein kinase kinase 2/extracellular regulated kinase, MEK2) is an important kinase in the signal transduction pathway of extracellular signal regulated kinase (Extracellular signaling-regulation kinase). Furthermore, it is also involved in the replication of hepatitis C, dengue and other viruses. The interaction between K2 and CSFV can activate the ERK pathway of the host and promote the expression of MEK2, and the application of specific inhibitors to blocking the ERK signaling pathway can significantly inhibit the replication of CSFV. In order to further study the effect of MEK2 on CSFV replication, this study uses the slow virus vector system to construct MEK2 up and up expression PK. -15 stable cell line (PK-EGFP-MEK2) and MEK2 down regulated PK-15 cell line (PK-shMEK2), EGFP positive cells were screened by flow cytometry. Immunofluorescence test and Western blotting test confirmed that the cell lines of MEK2 up and down expression were successfully constructed. Cell proliferation test showed that the activity of MEK2 was not affected by the up and down regulation of MEK2. The proliferation level of the cell lines was tested by CSFV infection test, 48 h and 72 h after the virus infection, the samples of the virus infection were collected, the fluorescence quantitative RT-PCR and the virus titer were measured, and the Western blotting detection and the fluorescein report system test of the infected cell protein were made to clarify the MEK2 to CSFV replication. The results showed that overexpression of MEK2 could significantly increase the number of viral RNA copies and virus titers, and the expression of Npro protein in cell lysates increased, indicating that up regulation of MEK2 could promote the replication of CSFV. On the contrary, the number of viral RNA copies and virus titers in cell culture supernatant were significantly lower than those of the control group. The expression of Npro protein in the lysates also decreased significantly, indicating that down regulation of MEK2 could significantly inhibit the replication of CSFV. In conclusion, this study found that the host protein MEK2 was able to regulate the replication of the swine fever virus.
【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.651
，

本文编号：2027454