本文關(guān)鍵詞： 馬立克氏病病毒 pp38蛋白 Toll樣受體 免疫應(yīng)答 細(xì)胞因子　出處：《揚(yáng)州大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：馬立克氏病(MD)是一種由馬立克氏病病毒(Marek's Diseas Virus,MDV)感染禽類引起的具有高度細(xì)胞結(jié)合特性的傳染性腫瘤病,馬立克氏病病毒(MDV)是一種致T細(xì)胞瘤的皰疹病毒,然而病毒的致腫瘤機(jī)制仍不十分清楚。所有致腫瘤的MDV均屬于血清I型,而與I型MDV致腫瘤相關(guān)的3個(gè)基因:132-bPr、pp38、meq基因中,pp38基因編碼的38 000磷蛋白(pp38)在腫瘤和細(xì)胞系中的表達(dá)存在差異,而I型特異性的pp38復(fù)合體是迄今為止在MDV誘發(fā)的腫瘤及非生產(chǎn)性腫瘤細(xì)胞系中唯一能檢出的MDV特異性抗原,pp38則被證實(shí)可抑制機(jī)體的免疫應(yīng)答。CVI988株屬于MDVI型疫苗株,RB1B株屬于MDV I型強(qiáng)毒株。本研究通過PCR法,從感染MDV強(qiáng)毒株RBIB與疫苗株CVI988的CEF細(xì)胞總RNA中擴(kuò)增pp38基因序列。實(shí)驗(yàn)結(jié)果表明,疫苗株與強(qiáng)毒株均可擴(kuò)增出約870bp的pp38基因片段。序列分析比對(duì)結(jié)果顯示,除了在第107和109位氨基酸發(fā)生變異外,其余部分完全相同。為了進(jìn)一步了解pp38在細(xì)胞感染MDV后免疫應(yīng)答中的作用機(jī)理,將擴(kuò)增出的基因片段用限制性內(nèi)切酶HindⅢ、XhoⅠ酶切消化,然后插入經(jīng)HindⅢ及XhoⅠ酶切好的真核表達(dá)載體pCMV-C-Flag中,經(jīng)酶切鑒后構(gòu)建成pCMV-pp38-Flag質(zhì)粒。構(gòu)建的表達(dá)質(zhì)粒轉(zhuǎn)染CEF細(xì)胞,通過間接免疫熒光試驗(yàn)(IFA)和western-blot實(shí)驗(yàn)驗(yàn)證pp38磷蛋白的表達(dá)。真核表達(dá)載體pCMV-CVI988-pp38和pCMV-RBlB-pp38的正確構(gòu)建為進(jìn)一步研究MDV I型強(qiáng)毒株與致弱株間pp38基因?qū)λ拗骷?xì)胞免疫應(yīng)答抑制機(jī)理的差異提供了試驗(yàn)材料。為研究馬立克氏病病毒pp38蛋白對(duì)宿主細(xì)胞天然免疫的影響,本研究將構(gòu)建的真核表達(dá)載體 pCMV-CVI988-pp38 和 pCMV-RBlB-pp38 轉(zhuǎn)染至 CEF 細(xì)胞中,經(jīng) poly(I:C)刺激,分別在6,12,24小時(shí)收集細(xì)胞RNA,利用熒光定量的方法檢測(cè)各個(gè)采樣時(shí)間點(diǎn)TLR基因家族,包括TLR3、TLR7、TLR15、TLR21和TLR2、TLR4,以及TLR3信號(hào)通路相關(guān)基因的相對(duì)表達(dá)量。結(jié)果表明pp38對(duì)TLR3-TRIF通路有一定的抑制作用,并影響通路下游炎性因子的表達(dá),且不同致病力的毒株pp38在分子水平的差異明顯。同時(shí)也檢測(cè)了MyD88通路下游基因IRAK4、TRAF6、IFNA、IFNB等基因以及其他一些細(xì)胞因子的相對(duì)表達(dá)水平。實(shí)驗(yàn)結(jié)果顯示pp38對(duì)這些因子表達(dá)量的影響與MDV的毒力有關(guān)。本研究發(fā)現(xiàn)MDV疫苗株與強(qiáng)毒株pp38蛋白對(duì)TLR3-TIRF途徑有抑制作用,且這種抑制作用在不同毒株之間均存在,而pp38對(duì)TLR2、7、15以及一些下游細(xì)胞因子表達(dá)量的影響與毒力有關(guān)。這些結(jié)果為研究不同MDV毒株的pp38蛋白在體內(nèi)外對(duì)細(xì)胞天然免疫的反應(yīng)提供了依據(jù),也為研究pp38免疫抑制作用在不同毒力的毒株間的差異性奠定了基礎(chǔ),將更有利于全面了解pp38在MDV致腫瘤中的作用機(jī)制。
[Abstract]:Marek's disease (MDD) is caused by Marek's disease virus (Marekhos Diseas Virus). Marek's disease virus (MDV) is a herpesvirus that causes T-cell tumor. However, the tumorigenic mechanism of the virus is still unclear. All the tumor-causing MDV belong to serotype I, while three genes: 1 132-b Prnp 38 are associated with type I MDV. There were differences in the expression of 38000 phosphoprotein in meq gene between tumor and cell lines. So far, type I specific pp38 complex is the only MDV specific antigen detected in MDV induced tumor and unproductive tumor cell lines. Pp38 was confirmed to inhibit the immune response. CVI988 strain belongs to MDVI vaccine strain RB1B strain belongs to MDVI type virulent strain. PCR method was used in this study. The sequence of pp38 gene was amplified from the total RNA of CEF cells infected with MDV virulent strain RBIB and vaccine strain CVI988. About 870bp pp38 gene fragment was amplified from the vaccine strain and virulent strain. The sequence analysis showed that the amino acids at the 107th and 109th sites were mutated except for the 107th and 109th amino acids. The other parts were identical. In order to further understand the role of pp38 in the immune response after MDV infection, the amplified gene fragment was amplified by restriction endonuclease Hind 鈪，

本文編號(hào)：1450418