本文關(guān)鍵詞：替換LaSota株V蛋白基因的重組新城疫病毒拯救及對干擾素的影響　出處：《吉林大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 新城疫病毒 V蛋白 反向遺傳 拮抗干擾素 毒力

【摘要】：新城疫由新城疫病毒(Newcastle disease virus,NDV)引起,為高度接觸性傳染病。NDV屬副粘病毒科(Paramyxoviridae)禽腮腺炎病毒屬(Avulavirus),是單股負(fù)鏈、有囊膜、不分節(jié)段的RNA病毒。新城疫病毒P基因通過RNA編輯(RNA editing)機制,轉(zhuǎn)錄時在484位堿基處(AAAAAGGG)額外插入1到2個G產(chǎn)生V基因(1個G)和W基因(2個G)m RNA。V基因編碼的V蛋白以多種方式拮抗宿主干擾素,使病毒完成免疫逃避,影響病毒的致病性以及宿主嗜性。目前,不同毒力型V蛋白在感染過程中對病毒毒力的影響及拮抗干擾素作用強弱尚不明確。為闡明不同毒力型V蛋白在病毒感染過程中對毒力的影響及拮抗干擾素作用的強弱,本文以La Sota弱毒株為病毒骨架,拯救表達弱、中、強毒株V蛋白的重組新城疫病毒,V蛋白終止及補償表達弱、中、強V蛋白的重組新城疫病毒。通過測定重組病毒毒力、在不同細(xì)胞的生長曲線、感染后干擾素下游信號通路m RNA表達水平,感染后細(xì)胞上清干擾素水平來比較不同毒力型V蛋白在感染過程中的作用強弱。研究分為以下幾個部分:1.表達新城疫弱、中、強毒株V蛋白和V蛋白終止及補償表達弱、中、強毒株V蛋白的全長c DNA構(gòu)建:利用p BRN-FL La Sota質(zhì)粒的Pme I酶切位點,分別構(gòu)建插入表達基因Ⅳ型弱毒疫苗株La Sota(La)、II型中毒株Beaudette C(BC)、VII型強毒株河北株(HB)V蛋白的全長c DNA,L-La,L-BC和L-HB;利用Kfl I及Pme I酶切位點構(gòu)建V蛋白終止并插入紅光蛋白RFP標(biāo)簽的重組病毒Lvs-R,及補償表達弱、中、強毒V蛋白的全長c DNA,Lvs-La,Lvs-BC和Lvs-HB。2.重組病毒拯救及毒力鑒定:重組質(zhì)粒和輔助質(zhì)粒轉(zhuǎn)染BSR細(xì)胞,5天后接種9-11日齡SPF雞胚,成功拯救5株重組病毒。測定重組病毒毒力:MDT、IVPI與母本重組病毒一致,ICPI測定中L-BC及L-HB分別為0.4、0.2,其余為0。結(jié)果表明中毒、強毒V蛋白對毒力有提升,仍保持在弱毒范疇。3.重組病毒的細(xì)胞致病能力及對I型IFN釋放的影響:測定重組病毒在VERO和DF1細(xì)胞的生長曲線、觀察感染后細(xì)胞病變、q RT-PCR測定感染后I型IFN信號通路相關(guān)因子MX和PKR的m RNA水平、ELISA測定感染后上清IFN-β含量,VSV干擾素滴定實驗來比較感染后病毒對細(xì)胞致病能力及宿主干擾素產(chǎn)生水平。結(jié)果表明,重組病毒感染過程中,不同毒力型V蛋白的插入及替換會影響宿主細(xì)胞干擾素產(chǎn)生水平,毒力弱的NDV毒株V蛋白,呈現(xiàn)出干擾素拮抗系統(tǒng)能力強的趨勢;毒力強的NDV毒株V蛋白,呈現(xiàn)出細(xì)胞破壞能力強的趨勢。經(jīng)研究發(fā)現(xiàn),V蛋白的插入和替換會影響La Sota株的干擾素拮抗能力,與V蛋白所屬毒力型呈現(xiàn)出負(fù)相關(guān)趨勢;而細(xì)胞致病能力與V蛋白所屬毒力型呈正相關(guān)趨勢。
[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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