马立克氏病毒pp38基因对Toll样受体通路的影响

发布时间：2018-01-21 04:07

  本文关键词： 马立克氏病病毒 pp38蛋白 Toll样受体 免疫应答 细胞因子　出处：《扬州大学》2017年硕士论文　论文类型：学位论文

【摘要】：马立克氏病(MD)是一种由马立克氏病病毒(Marek's Diseas Virus,MDV)感染禽类引起的具有高度细胞结合特性的传染性肿瘤病,马立克氏病病毒(MDV)是一种致T细胞瘤的疱疹病毒,然而病毒的致肿瘤机制仍不十分清楚。所有致肿瘤的MDV均属于血清I型,而与I型MDV致肿瘤相关的3个基因:132-bPr、pp38、meq基因中,pp38基因编码的38 000磷蛋白(pp38)在肿瘤和细胞系中的表达存在差异,而I型特异性的pp38复合体是迄今为止在MDV诱发的肿瘤及非生产性肿瘤细胞系中唯一能检出的MDV特异性抗原,pp38则被证实可抑制机体的免疫应答。CVI988株属于MDVI型疫苗株,RB1B株属于MDV I型强毒株。本研究通过PCR法,从感染MDV强毒株RBIB与疫苗株CVI988的CEF细胞总RNA中扩增pp38基因序列。实验结果表明,疫苗株与强毒株均可扩增出约870bp的pp38基因片段。序列分析比对结果显示,除了在第107和109位氨基酸发生变异外,其余部分完全相同。为了进一步了解pp38在细胞感染MDV后免疫应答中的作用机理,将扩增出的基因片段用限制性内切酶HindⅢ、XhoⅠ酶切消化,然后插入经HindⅢ及XhoⅠ酶切好的真核表达载体pCMV-C-Flag中,经酶切鉴后构建成pCMV-pp38-Flag质粒。构建的表达质粒转染CEF细胞,通过间接免疫荧光试验(IFA)和western-blot实验验证pp38磷蛋白的表达。真核表达载体pCMV-CVI988-pp38和pCMV-RBlB-pp38的正确构建为进一步研究MDV I型强毒株与致弱株间pp38基因对宿主细胞免疫应答抑制机理的差异提供了试验材料。为研究马立克氏病病毒pp38蛋白对宿主细胞天然免疫的影响,本研究将构建的真核表达载体 pCMV-CVI988-pp38 和 pCMV-RBlB-pp38 转染至 CEF 细胞中,经 poly(I:C)刺激,分别在6,12,24小时收集细胞RNA,利用荧光定量的方法检测各个采样时间点TLR基因家族,包括TLR3、TLR7、TLR15、TLR21和TLR2、TLR4,以及TLR3信号通路相关基因的相对表达量。结果表明pp38对TLR3-TRIF通路有一定的抑制作用,并影响通路下游炎性因子的表达,且不同致病力的毒株pp38在分子水平的差异明显。同时也检测了MyD88通路下游基因IRAK4、TRAF6、IFNA、IFNB等基因以及其他一些细胞因子的相对表达水平。实验结果显示pp38对这些因子表达量的影响与MDV的毒力有关。本研究发现MDV疫苗株与强毒株pp38蛋白对TLR3-TIRF途径有抑制作用,且这种抑制作用在不同毒株之间均存在,而pp38对TLR2、7、15以及一些下游细胞因子表达量的影响与毒力有关。这些结果为研究不同MDV毒株的pp38蛋白在体内外对细胞天然免疫的反应提供了依据,也为研究pp38免疫抑制作用在不同毒力的毒株间的差异性奠定了基础,将更有利于全面了解pp38在MDV致肿瘤中的作用机制。
[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，

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