替换LaSota株V蛋白基因的重组新城疫病毒拯救及对干扰素的影响

发布时间：2018-01-18 11:44

  本文关键词：替换LaSota株V蛋白基因的重组新城疫病毒拯救及对干扰素的影响　出处：《吉林大学》2017年硕士论文　论文类型：学位论文

  更多相关文章： 新城疫病毒 V蛋白 反向遗传 拮抗干扰素 毒力

【摘要】：新城疫由新城疫病毒(Newcastle disease virus,NDV)引起,为高度接触性传染病。NDV属副粘病毒科(Paramyxoviridae)禽腮腺炎病毒属(Avulavirus),是单股负链、有囊膜、不分节段的RNA病毒。新城疫病毒P基因通过RNA编辑(RNA editing)机制,转录时在484位碱基处(AAAAAGGG)额外插入1到2个G产生V基因(1个G)和W基因(2个G)m RNA。V基因编码的V蛋白以多种方式拮抗宿主干扰素,使病毒完成免疫逃避,影响病毒的致病性以及宿主嗜性。目前,不同毒力型V蛋白在感染过程中对病毒毒力的影响及拮抗干扰素作用强弱尚不明确。为阐明不同毒力型V蛋白在病毒感染过程中对毒力的影响及拮抗干扰素作用的强弱,本文以La Sota弱毒株为病毒骨架,拯救表达弱、中、强毒株V蛋白的重组新城疫病毒,V蛋白终止及补偿表达弱、中、强V蛋白的重组新城疫病毒。通过测定重组病毒毒力、在不同细胞的生长曲线、感染后干扰素下游信号通路m RNA表达水平,感染后细胞上清干扰素水平来比较不同毒力型V蛋白在感染过程中的作用强弱。研究分为以下几个部分:1.表达新城疫弱、中、强毒株V蛋白和V蛋白终止及补偿表达弱、中、强毒株V蛋白的全长c DNA构建:利用p BRN-FL La Sota质粒的Pme I酶切位点,分别构建插入表达基因Ⅳ型弱毒疫苗株La Sota(La)、II型中毒株Beaudette C(BC)、VII型强毒株河北株(HB)V蛋白的全长c DNA,L-La,L-BC和L-HB;利用Kfl I及Pme I酶切位点构建V蛋白终止并插入红光蛋白RFP标签的重组病毒Lvs-R,及补偿表达弱、中、强毒V蛋白的全长c DNA,Lvs-La,Lvs-BC和Lvs-HB。2.重组病毒拯救及毒力鉴定:重组质粒和辅助质粒转染BSR细胞,5天后接种9-11日龄SPF鸡胚,成功拯救5株重组病毒。测定重组病毒毒力:MDT、IVPI与母本重组病毒一致,ICPI测定中L-BC及L-HB分别为0.4、0.2,其余为0。结果表明中毒、强毒V蛋白对毒力有提升,仍保持在弱毒范畴。3.重组病毒的细胞致病能力及对I型IFN释放的影响:测定重组病毒在VERO和DF1细胞的生长曲线、观察感染后细胞病变、q RT-PCR测定感染后I型IFN信号通路相关因子MX和PKR的m RNA水平、ELISA测定感染后上清IFN-β含量,VSV干扰素滴定实验来比较感染后病毒对细胞致病能力及宿主干扰素产生水平。结果表明,重组病毒感染过程中,不同毒力型V蛋白的插入及替换会影响宿主细胞干扰素产生水平,毒力弱的NDV毒株V蛋白,呈现出干扰素拮抗系统能力强的趋势;毒力强的NDV毒株V蛋白,呈现出细胞破坏能力强的趋势。经研究发现,V蛋白的插入和替换会影响La Sota株的干扰素拮抗能力,与V蛋白所属毒力型呈现出负相关趋势;而细胞致病能力与V蛋白所属毒力型呈正相关趋势。
[Abstract]:Newcastle disease is caused by Newcastle disease virus (NDV). NDV belongs to Paramyxoviridae.Avulavirus.NDV belongs to Avulavirus.NDV is a single-stranded negative chain with envelope. Newcastle disease virus P gene was edited by RNA. Insert additional 1 to 2 G to produce V gene (1 GG) and W gene (2 GG) at 484 base site. The V protein encoded by m RNA.V gene antagonizes the host interferon in many ways. Make the virus complete immune escape, affect the pathogenicity of the virus as well as host tropism. At present. The effect of different virulence V protein on virus virulence and the antagonistic effect of interferon on virus virulence in the course of infection is not clear. In order to elucidate the virulence of different virulence V protein in the course of virus infection and the antagonistic effect of interferon. Is strong or weak. In this paper, the virus skeleton of La Sota attenuated strain was used as the virus skeleton, and the expression of the V protein of the virulent strain V was weak, while that of the V protein of the virulent strain V was weak and the expression of the V protein of the recombinant Newcastle disease virus was weak. The virulence of recombinant Newcastle disease virus (NDV), the expression level of m RNA in different cell growth curves and downstream signal pathway of interferon after infection, was determined by detecting the virulence of recombinant virus. The level of interferon in supernatant of infected cells was used to compare the effect of different virulence V protein in the process of infection. The study was divided into the following parts: 1. Expression of Newcastle disease weak, moderate. The full-length c DNA of V protein of virulent strain V was constructed by using the Pme I site of p BRN-FL La Sota plasmid. The attenuated vaccine strain La Sotae Lajiao II inserted the expressed gene type 鈪，

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