本文選題：豬瘟病毒 + 絲裂原激活的蛋白激酶激酶2��； 參考：《吉林農(nóng)業(yè)大學(xué)》2015年碩士論文

【摘要】：豬瘟(Classical swine fever, CSF)是嚴(yán)重危害世界和我國(guó)養(yǎng)豬業(yè)的一種急性、熱性、高度接觸性和致死性傳染病,以高熱稽留、廣泛性出血和免疫抑制為主要特征。豬瘟的病原是豬瘟病毒(Classical swine fever virus, CSFV)。CSFV是黃病毒科瘟病毒屬的重要成員,其長(zhǎng)約12.3 kb的單股正鏈RNA基因組僅含有一個(gè)大的開(kāi)放閱讀框(Open reading frame, ORF),編碼一個(gè)分子量約438kDa的多聚蛋白,該多聚蛋白被病毒和宿主蛋白酶水解為8種非結(jié)構(gòu)蛋白(NPro、p7、NS2、NS3、NS4A、NS4B、NS5A和NS5B)和4種結(jié)構(gòu)蛋白(C、Erns、E1和E2)。絲裂原激活的蛋白激酶激酶2(Mitogen-activated protein kinase kinase 2/extracellular regulated kinase, MEK2)是細(xì)胞外信號(hào)調(diào)節(jié)激酶(Extracellular signaling-regulation kinase, ERK)信號(hào)通路的重要激酶。此外,MEK2還參與丙型肝炎、登革熱等病毒的復(fù)制,而且MEK2與CSFV之間存在相互作用。CSFV感染PK-15細(xì)胞后能夠激活宿主的ERK通路并促進(jìn)MEK2的表達(dá),應(yīng)用特異性抑制劑阻斷ERK信號(hào)通路能明顯抑制CSFV復(fù)制。為了進(jìn)一步研究MEK2對(duì)CSFV復(fù)制的影響,本研究利用慢病毒載體系統(tǒng)分別構(gòu)建MEK2上調(diào)表達(dá)的PK-15穩(wěn)定細(xì)胞系(PK-EGFP-MEK2)和MEK2下調(diào)表達(dá)的PK-15細(xì)胞系(PK-shMEK2),通過(guò)流式細(xì)胞術(shù)篩選EGFP陽(yáng)性細(xì)胞,免疫熒光試驗(yàn)和Western blotting試驗(yàn)證實(shí)MEK2上調(diào)和下調(diào)表達(dá)的細(xì)胞系構(gòu)建成功。細(xì)胞增殖試驗(yàn)表明MEK2上調(diào)和下調(diào)后均不影響細(xì)胞的活力及增殖水平。將所構(gòu)建的細(xì)胞系進(jìn)行CSFV感染試驗(yàn),分別在病毒感染后的48 h和72 h,收集病毒感染樣品,進(jìn)行熒光定量RT-PCR和病毒效價(jià)的測(cè)定,并對(duì)感染后的細(xì)胞蛋白進(jìn)行Western blotting檢測(cè)以及熒光素酶報(bào)告系統(tǒng)檢測(cè)等試驗(yàn)明確MEK2對(duì)CSFV復(fù)制的影響。結(jié)果顯示,過(guò)表達(dá)MEK2能夠明顯提高病毒RNA拷貝數(shù)和病毒滴度,細(xì)胞裂解物中病毒Npro蛋白的表達(dá)量增加,表明上調(diào)表達(dá)MEK2可以促進(jìn)CSFV的復(fù)制。相反,下調(diào)表達(dá)MEK2的細(xì)胞培養(yǎng)上清中病毒RNA拷貝數(shù)和病毒滴度明顯低于對(duì)照組,細(xì)胞裂解物中Npro蛋白的表達(dá)量也顯著減少,表明下調(diào)表達(dá)MEK2能夠明顯抑制CSFV的復(fù)制�？傊�,本研究發(fā)現(xiàn)宿主蛋白MEK2能夠正調(diào)控豬瘟病毒的復(fù)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.
【學(xué)位授予單位】：吉林農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S852.651
，

本文編號(hào)：2027454