新城疫病毒Yulin株V蛋白对Ⅰ型IFN信号通路相关因子的影响及Yulin株全长cDNA的构建
发布时间：2018-07-27 17:52
【摘要】：新城疫是由新城疫病毒引起的一种急性高度接触性传染病,对全世界的养禽业造成了巨大的经济损失。新城疫病毒(Newcastle disease virus,NDV)属于副黏病毒科、禽副黏病毒属。为单股负链RNA病毒,全基因组编码6种主要病毒蛋白:核衣壳蛋白(NP)、磷蛋白(P)、基质蛋白(M)、融合蛋白(F)、血凝素-神经氨酸酶(HN)、大蛋白(L)。另外,在P基因RNA编辑过程中,产生两种非结构蛋白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型干扰素(IFNα和β)的作用。目前,NDV V蛋白拮抗I型IFN信号通路的机制仍不清楚。有研究发现V蛋白能够通过降解STAT1和抑制STAT1的磷酸化来阻断I型IFN信号通路。为了进一步研究V蛋白拮抗I型IFN信号通路的具体机制,本文通过NDV感染细胞,以及NDV陕西榆林(Yulin)株V蛋白转染细胞等方法,检测Ⅰ型IFN信号通路中相关因子的mRNA表达水平。同时,构建NDV Yulin株全长cDNA克隆,为成功拯救出Yulin株后研究V蛋白的功能奠定基础。主要结果如下:1.NDV V蛋白拮抗Ⅰ型干扰素(IFN)的能力为了检测NDV V蛋白对Ⅰ型IFN信号通路的作用,本研究采用双荧光素酶报告基因检测干扰素刺激反应元件(IFN-stimulated response element,ISRE)启动子的活性。结果显示NDV V蛋白抑制Ⅰ型IFN信号通路的ISRE启动子的活性,强毒株Yulin株抑制ISRE启动子的活性强于弱毒株La Sota株。2.NDV V蛋白影响Ⅰ型IFN信号通路中相关因子mRNA表达水平NDV强毒株Yulin、疫苗弱毒株La Sota感染Hep-2细胞,采用荧光定量PCR检测Ⅰ型IFN信号通路中相关因子的mRNA的表达水平,结果显示:Ⅰ型IFN信号通路相关因子ISG56、ISG15、STAT1的mRNA表达水平有明显的增加。Yulin、La Sota株的V基因转染细胞后,采用荧光定量PCR检测Ⅰ型信号通路中相关因子的mRNA的表达水平。结果显示:转染NDV V蛋白之后,Ⅰ型IFN信号通路相关因子ISG15、STAT1的mRNA表达水平有明显的降低。3.Yulin株全长cDNA的构建为了获得NDV Yulin株全长cDNA,本研究通过针对Yulin株全基因组序列采用分段(共8个片段)克隆全长cDNA的构建策略。通过RT-PCR扩增出每个片段(F1至F8),并在F1片段的上游引入T7 RNA聚合酶的启动子,在F8片段的下游引入T7 RNA聚合酶的终止子和丁型肝炎病毒核酶序列。通过酶切连接到经过改造的pBR322载体的方法成功构建了一株NDV的全长cDNA。经序列比对,全长有4个核苷酸的改变,其中有3处不引起氨基酸的改变,分析表明另外一处氨基酸改变,但在NDV的其它毒株中也同样存在,不会影响到后续病毒的拯救。Yulin株全长cDNA的构建为下一步拯救出重组病毒及运用反向遗传系统研究新城疫V蛋白拮抗干扰素奠定基础。
[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 伪 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，
本文编号：2148677
[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 伪 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，
本文编号：2148677
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