本文關鍵詞： 馬立克氏病病毒 pp38蛋白 Toll樣受體 免疫應答 細胞因子　出處：《揚州大學》2017年碩士論文　論文類型：學位論文

【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胞結合特性的傳染性腫瘤病,馬立克氏病病毒(MDV)是一種致T細胞瘤的皰疹病毒,然而病毒的致腫瘤機制仍不十分清楚。所有致腫瘤的MDV均屬于血清I型,而與I型MDV致腫瘤相關的3個基因:132-bPr、pp38、meq基因中,pp38基因編碼的38 000磷蛋白(pp38)在腫瘤和細胞系中的表達存在差異,而I型特異性的pp38復合體是迄今為止在MDV誘發(fā)的腫瘤及非生產性腫瘤細胞系中唯一能檢出的MDV特異性抗原,pp38則被證實可抑制機體的免疫應答。CVI988株屬于MDVI型疫苗株,RB1B株屬于MDV I型強毒株。本研究通過PCR法,從感染MDV強毒株RBIB與疫苗株CVI988的CEF細胞總RNA中擴增pp38基因序列。實驗結果表明,疫苗株與強毒株均可擴增出約870bp的pp38基因片段。序列分析比對結果顯示,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。為了進一步了解pp38在細胞感染MDV后免疫應答中的作用機理,將擴增出的基因片段用限制性內切酶HindⅢ、XhoⅠ酶切消化,然后插入經HindⅢ及XhoⅠ酶切好的真核表達載體pCMV-C-Flag中,經酶切鑒后構建成pCMV-pp38-Flag質粒。構建的表達質粒轉染CEF細胞,通過間接免疫熒光試驗(IFA)和western-blot實驗驗證pp38磷蛋白的表達。真核表達載體pCMV-CVI988-pp38和pCMV-RBlB-pp38的正確構建為進一步研究MDV I型強毒株與致弱株間pp38基因對宿主細胞免疫應答抑制機理的差異提供了試驗材料。為研究馬立克氏病病毒pp38蛋白對宿主細胞天然免疫的影響,本研究將構建的真核表達載體 pCMV-CVI988-pp38 和 pCMV-RBlB-pp38 轉染至 CEF 細胞中,經 poly(I:C)刺激,分別在6,12,24小時收集細胞RNA,利用熒光定量的方法檢測各個采樣時間點TLR基因家族,包括TLR3、TLR7、TLR15、TLR21和TLR2、TLR4,以及TLR3信號通路相關基因的相對表達量。結果表明pp38對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,且不同致病力的毒株pp38在分子水平的差異明顯。同時也檢測了MyD88通路下游基因IRAK4、TRAF6、IFNA、IFNB等基因以及其他一些細胞因子的相對表達水平。實驗結果顯示pp38對這些因子表達量的影響與MDV的毒力有關。本研究發(fā)現(xiàn)MDV疫苗株與強毒株pp38蛋白對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TLR2、7、15以及一些下游細胞因子表達量的影響與毒力有關。這些結果為研究不同MDV毒株的pp38蛋白在體內外對細胞天然免疫的反應提供了依據(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,將更有利于全面了解pp38在MDV致腫瘤中的作用機制。
[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：1450418