【摘要】：新城疫是由新城疫病毒引起的一種急性高度接觸性傳染病,對全世界的養(yǎng)禽業(yè)造成了巨大的經(jīng)濟(jì)損失。新城疫病毒(Newcastle disease virus,NDV)屬于副黏病毒科、禽副黏病毒屬。為單股負(fù)鏈RNA病毒,全基因組編碼6種主要病毒蛋白:核衣殼蛋白(NP)、磷蛋白(P)、基質(zhì)蛋白(M)、融合蛋白(F)、血凝素-神經(jīng)氨酸酶(HN)、大蛋白(L)。另外,在P基因RNA編輯過程中,產(chǎn)生兩種非結(jié)構(gòu)蛋白V和W。P、V和W蛋白的N端氨基酸序列相同,而C端氨基酸序列不同。V蛋白具有拮抗宿主I型干擾素(IFNα和β)的作用。目前,NDV V蛋白拮抗I型IFN信號通路的機(jī)制仍不清楚。有研究發(fā)現(xiàn)V蛋白能夠通過降解STAT1和抑制STAT1的磷酸化來阻斷I型IFN信號通路。為了進(jìn)一步研究V蛋白拮抗I型IFN信號通路的具體機(jī)制,本文通過NDV感染細(xì)胞,以及NDV陜西榆林(Yulin)株V蛋白轉(zhuǎn)染細(xì)胞等方法,檢測Ⅰ型IFN信號通路中相關(guān)因子的mRNA表達(dá)水平。同時(shí),構(gòu)建NDV Yulin株全長cDNA克隆,為成功拯救出Yulin株后研究V蛋白的功能奠定基礎(chǔ)。主要結(jié)果如下:1.NDV V蛋白拮抗Ⅰ型干擾素(IFN)的能力為了檢測NDV V蛋白對Ⅰ型IFN信號通路的作用,本研究采用雙熒光素酶報(bào)告基因檢測干擾素刺激反應(yīng)元件(IFN-stimulated response element,ISRE)啟動(dòng)子的活性。結(jié)果顯示NDV V蛋白抑制Ⅰ型IFN信號通路的ISRE啟動(dòng)子的活性,強(qiáng)毒株Yulin株抑制ISRE啟動(dòng)子的活性強(qiáng)于弱毒株La Sota株。2.NDV V蛋白影響Ⅰ型IFN信號通路中相關(guān)因子mRNA表達(dá)水平NDV強(qiáng)毒株Yulin、疫苗弱毒株La Sota感染Hep-2細(xì)胞,采用熒光定量PCR檢測Ⅰ型IFN信號通路中相關(guān)因子的mRNA的表達(dá)水平,結(jié)果顯示:Ⅰ型IFN信號通路相關(guān)因子ISG56、ISG15、STAT1的mRNA表達(dá)水平有明顯的增加。Yulin、La Sota株的V基因轉(zhuǎn)染細(xì)胞后,采用熒光定量PCR檢測Ⅰ型信號通路中相關(guān)因子的mRNA的表達(dá)水平。結(jié)果顯示:轉(zhuǎn)染NDV V蛋白之后,Ⅰ型IFN信號通路相關(guān)因子ISG15、STAT1的mRNA表達(dá)水平有明顯的降低。3.Yulin株全長cDNA的構(gòu)建為了獲得NDV Yulin株全長cDNA,本研究通過針對Yulin株全基因組序列采用分段(共8個(gè)片段)克隆全長cDNA的構(gòu)建策略。通過RT-PCR擴(kuò)增出每個(gè)片段(F1至F8),并在F1片段的上游引入T7 RNA聚合酶的啟動(dòng)子,在F8片段的下游引入T7 RNA聚合酶的終止子和丁型肝炎病毒核酶序列。通過酶切連接到經(jīng)過改造的pBR322載體的方法成功構(gòu)建了一株NDV的全長cDNA。經(jīng)序列比對,全長有4個(gè)核苷酸的改變,其中有3處不引起氨基酸的改變,分析表明另外一處氨基酸改變,但在NDV的其它毒株中也同樣存在,不會(huì)影響到后續(xù)病毒的拯救。Yulin株全長cDNA的構(gòu)建為下一步拯救出重組病毒及運(yùn)用反向遺傳系統(tǒng)研究新城疫V蛋白拮抗干擾素奠定基礎(chǔ)。
[Abstract]:Newcastle disease (NDV) is an acute highly contagious disease caused by Newcastle disease virus (NDV), which has caused enormous economic losses to poultry industry all over the world. Newcastle disease virus (Newcastle disease virusNDV) belongs to paramyxovirus family, avian paramyxovirus genus. The whole genome encodes six major viral proteins: nucleocapsid protein (NP), phosphorous protein (P), matrix protein (M), fusion protein (F), hemagglutinin neuraminidase (HN), large protein (L). In addition, the N-terminal amino acid sequences of two kinds of nonstructural proteins V and W.PnV and W protein were the same during the RNA editing of the P gene, while the C-terminal amino acid sequences of different .V proteins could antagonize the host type I interferon (IFN 偽 and 尾). At present, the mechanism of IFN protein antagonizing type I IFN signaling pathway is still unclear. It has been found that V protein can block type I IFN signaling pathway by degrading STAT1 and inhibiting STAT1 phosphorylation. In order to further study the specific mechanism of V protein antagonizing type I IFN signaling pathway, the mRNA expression level of related factors in type I IFN signaling pathway was detected by means of NDV infection and V protein transfection of NDV Shaanxi Yulin (Yulin) strain. At the same time, the full-length cDNA clone of NDV Yulin strain was constructed, which laid a foundation for studying the function of V protein after successfully saving Yulin strain. The main results are as follows: 1. The ability of NDV protein to antagonize interferon I (IFN). In order to detect the effect of NDV V protein on type I IFN signaling pathway, the activity of the promoter of interferon stimulating element (IFN-stimulated response element-ISRE) was detected by double luciferase reporter gene. The results showed that NDV V protein inhibited the activity of ISRE promoter in type I IFN signaling pathway. The inhibitory activity of virulent strain Yulin against ISRE promoter was stronger than that of attenuated strain La Sota. 2. NDV protein affected the expression of mRNA in type I IFN signaling pathway. NDV virulent strain Yulin was infected with attenuated virus strain La Sota. Fluorescence quantitative PCR was used to detect the level of mRNA expression of related factors in type 鈪，

本文編號：2148677