鸭源呼肠孤病毒的表型和分子特征分析及基因2型毒株编码的新蛋白的鉴定

发布时间：2018-01-14 01:21

  本文关键词：鸭源呼肠孤病毒的表型和分子特征分析及基因2型毒株编码的新蛋白的鉴定　出处：《中国农业大学》2016年博士论文　论文类型：学位论文

  更多相关文章： 鸭源呼肠孤病毒 表型 分子特征 p10蛋白 p18蛋白

【摘要】：根据宿主和病变,鸭呼肠孤病毒病分为3种主要类型,即,番鸭“白点病”、番鸭“新肝病”和北京鸭“脾坏死病”,其病原分别为番鸭呼肠孤病毒(Muscovy duck reovirus, MDRV)、新致病型番鸭呼肠孤病毒(New pathotype of Muscovy duck reovirus, N-MDRV)和鸭呼肠孤病毒(Duck reovirus, DRV)。MDRV于1972年首次发现于法国,目前被归为禽呼肠孤病毒(Avian orthoreovirus,ARV)的番鸭源分离株(ARV-Md), N-MDRV和DRV是我国研究者分别于2009年和2011年报道的新毒株。迄今为止,不同鸭源呼肠孤病毒毒株之间的准确分类关系还有待于阐明。以ARV-Md/815-12、N-MDRV/J18和DRV/091为材料,对不同鸭源呼肠孤病毒的表型特征进行比较。结果显示,N-MDRV和DRV在实验室感染宿主范围、在细胞质内排列方式、致细胞融合活性、沉淀反应抗原、对北京鸭的致病性和组织嗜性等表型特征上表现出相似性。与N-MDRV和DRV相比,ARV-Md在表型特征上存在明显差异。3株病毒均对番鸭有致病性,但所引起的病变和组织嗜性有所不同。为明确三类鸭源呼肠孤病毒的基因组结构特点及其与其它水禽源毒株和鸡源ARV的遗传演化关系,用SDS-PAGE分析了3株病毒的核酸电泳型,用RT-PCR和5RACE测定了ARV-Md/815-12的基因组序列以及N-MDRV/J18和DRV/091的S1基因节段序列,由此获得了3株鸭源呼肠孤病毒的基因组序列。SDS-PAGE结果显示,N-MDRV和DRV具有相同的基因组核酸电泳型。与N-MDRV和DRV相比,ARV-Md S组的核酸电泳型表现出显著的差异。序列分析结果显示,所测3株病毒均具有ARV的基因组结构特点,但在多顺反子基因节段的长度和基因结构上,N-MDRV和DRV等新毒株与ARV-Md存在显著差异,即,J-MDRV和DRV的多顺反子节段位于S1,含3个ORF,分别编码p10、p18和σC蛋白；而ARV-Md的多顺反子基因节段位于S4,仅含2个ORF,分别编码p10和σC蛋白。N-MDRV和DRV的p10蛋白与ARV-Md的p10蛋白缺乏序列同源性。进化分析结果显示,水禽源和鸡源毒株的9个基因节段(除M2)均按宿主聚为两个明显不同的分支；在M2节段,相对于ARV-Md, N-MDRV和DRV等水禽源新毒株与鸡源毒株具有更近的遗传演化关系。综合分析,可将水禽源毒株鉴定为ARV的成员,但现有的序列同源性分类标准需要调整。根据外衣壳蛋白序列的进化分析结果,可将水禽源呼肠孤病毒区分为2个不同的基因型,ARV-Md属于基因1型,N-MDRV和DRV属于基因2型。相对于基因1型毒株,N-MDRV和DRV等基因2型毒株最显著的基因组结构特点是其多顺反子节段含有3个ORF,但在复制过程中,预测的ORF1和ORF2是否会表达成熟的p10和p18蛋白,尚不清楚。为回答这一问题,开展了下述研究。用Western Blot进行检测,结果显示,DRV/091的免疫血清可与p18重组蛋白发生反应,而p18重组蛋白的免疫血清亦可从DRV/091感染的Vero细胞中识别出18 kDa的蛋白；用MALDI-TOF/TOF法进行分析,从DRV/091感染的Vero细胞中鉴定出p18蛋白的氨基酸序列：由此可见,p18是DRV S1基因节段编码的一种新蛋白。序列分析显示,在p18蛋白的118-135位可能存在一个核定位信号(Nuclear localization signal, NLS)序列(118KRRR121-X10-132KRRR135),提示该蛋白可能具有入核活性。为验证这一观点,用激光共聚焦显微镜观察了p18蛋白的亚细胞定位,结果显示,在感染细胞和转染细胞的细胞核内,见有p18蛋白聚集。突变分析显示,p18蛋白的入核活性由NLS决定,而位于NLS N-端的’18KRRR121序列是引导p18蛋白入核的关键基序。经检测,p18蛋白在Vero细胞中过表达能够引起细胞凋亡。将DRV/091感染Vero细胞,用p10蛋白的单克隆抗体进行间接免疫荧光试验,从感染细胞内检测到荧光信号,以此证实p10蛋白是病毒复制所产生的成熟蛋白。N-MDRV和DRV的p10蛋白含有融合蛋白基序,提示该蛋白具有致细胞融合活性。为验证这一观点,用表达p10蛋白的真核表达质粒转染Vero细胞,在转染后24 h可见细胞融合现象。然而,用表达病毒其它蛋白的真核表达质粒转染Vero细胞,均未见细胞融合现象。由此可见,p10蛋白是决定DRV等基因2型毒株致细胞融合活性的关键因素。上述研究证实,DRV等基因2型毒株的S1基因节段为三顺反子。为初步了解该基因节段的转录和翻译策略,用DRV/091感染、Aero细胞,在感染后不同时间提取细胞内总RNA,并用识别S1基因节段的核酸探针进行Northern Blot检测,在感染后2-8 h可检测到单-mRNA条带,长度与S1基因节段长度(1568 bp)相符。由此可见,在DRV的复制过程中,其S1基因节段可转录出全长mRNA。结果提示,ORF2和ORF3可能是从多顺反子mRNA的中途起始翻译的。
[Abstract]:According to the host and disease, duck reovirus disease is divided into 3 main types, namely, "white spot disease", Muscovy duck and Beijing duck "new" disease "spleen necrosis disease", the pathogens were muscovy duck reovirus (Muscovy duck, reovirus, MDRV), a new pathotype of Muscovy duck reovirus virus (New pathotype of Muscovy duck reovirus, N-MDRV) and duck reovirus (Duck reovirus, DRV.MDRV) was first discovered in 1972 in France, is currently classified as avian reovirus (Avian orthoreovirus, ARV) of the Muscovy Duck isolates (ARV-Md), N-MDRV and DRV are new researchers in China strain respectively in 2009 and 2011 reported. So far, different accurate classification of duck avian reovirus strain relationship remains to be elucidated. In ARV-Md/815-12, N-MDRV/J18 and DRV/091 as materials, comparing the phenotypic characteristics of duck reovirus call on different source. The results showed that N-M DRV and DRV in the laboratory host range, arranged in the cytoplasm, cell fusion activity, precipitation reaction of Beijing duck antigen, virulence and tissue tropism and other phenotypic characteristics showed similarity. Compared with N-MDRV and DRV, ARV-Md in phenotypic characteristics there were significantly differences in.3 strains of virus pathogenicity the Muscovy duck, but caused by the lesions and tissue tropism. Different genetic relationship between genome structure of reovirus is clearly three duck source and strain and other waterfowl source and chicken ARV, were analyzed by SDS-PAGE 3 strains of virus nucleic acid electrophoresis, ARV-Md/815-12 genome sequence and N-MDRV/J18 and DRV/091 S1 gene segment sequences were determined by RT-PCR and 5RACE,.SDS-PAGE genome sequence results thus obtained 3 strains of duck reovirus showed that N-MDRV and DRV have the same type of genomic DNA electrophoresis. Compared to N-MDRV and DRV, ARV-Md type nucleic acid electrophoresis S group showed significant difference. The results of sequence analysis showed that the genomic structural characteristics of 3 strains of the virus are ARV, but in many along the length and gene structure gene segment, N-MDRV and DRV new strain and ARV-Md are significant difference that is, J-MDRV, and DRV polycistron segment is located in S1, 3 ORF, respectively, encoding P10, P18 and sigma C protein; ARV-Md polycistron gene segment is located in S4, with only 2 ORF, respectively. P10 protein and ARV-Md encoding P10 and C proteins.N-MDRV and DRV. The lack of p10 protein sequence homology. Phylogenetic analysis showed that 9 gene segments and avian strains of waterfowl origin (except M2) according to the host clustered into two distinct branches; in the M2 segment, compared to ARV-Md, N-MDRV and DRV and other waterfowl new strains of chicken origin and source strains have evolved the more recent genetic relationship. Comprehensive Analysis can be identified from waterfowl is a member of ARV, but the existing sequence classification standards need to be adjusted according to the coat protein sequence evolution analysis results, the waterfowl reovirus is divided into 2 different genotypes, ARV-Md belonged to genotype 1, N-MDRV and DRV belong to genotype 2 compared with genotype 1 strains, N-MDRV and DRV gene type 2 strain genome structure of the most notable feature is the Multicistron segment containing 3 ORF, but in the process of replication, ORF1 and ORF2 can predict whether the expression of mature P10 and P18 proteins, is not clear. To answer this the problem, carried out the following research. Were detected with Western Blot showed that DRV/091 immune serum can react with recombinant P18 protein, recombinant P18 protein and immune serum from DRV/091 infected Vero cells can be identified in 18 kDa protein; using MALDI-TOF/ TOF method Analysis and identification of amino acid sequence of P18 protein from DRV/091 infected Vero cells: thus, P18 is a kind of new protein DRV S1 gene segment encoding. Sequence analysis showed that 118-135 P18 protein may be in the presence of a nuclear localization signal (Nuclear localization, signal, NLS), suggesting that the sequence (118KRRR121-X10-132KRRR135) protein may have nuclear activity. In order to verify this view, with laser confocal microscopy. The subcellular localization of P18 protein, the results showed that in infected cells and transfected into cell nucleus, see P18 protein aggregation. Mutation analysis showed that P18 protein into the nucleus was determined by NLS, and is located in NLS N- '18KRRR121 sequence is to guide the key motif of P18 protein into the nucleus. After testing, the overexpression of P18 protein can induce apoptosis in Vero cells. DRV/091 infection of Vero cells with monoclonal p10 protein Antibodies by indirect immunofluorescence assay, detected from infected cells, which confirmed that the p10 protein is a mature protein of.N-MDRV and replication of DRV produced by p10 protein containing the fusion protein motif, suggesting that the protein can cause the cell fusion activity. Based on this point of view, with the eukaryotic expression plasmid was transfected into Vero cells the expression of p10 protein in H 24 cell confluence after transfection. However, with the eukaryotic expression plasmid was transfected into Vero cells for virus expression of other proteins, no cell fusion phenomenon. Thus, p10 protein is a key factor in determining DRV gene type 2 strain induced cell fusion activity. The study confirmed that the S1 gene segment section DRV genotype 2 strains for three cistron. As a preliminary understanding of the gene segments of the transcription and translation strategies, with DRV/091 infection, Aero cells in different time after infection with total RNA extracted from cells, and Northern Blot detection of nucleic acid probe recognition S1 gene segment, after infection 2-8 h can detect single -mRNA bands, and the length of S1 gene segment length (1568 BP). Therefore, in the process of DRV replication, the S1 gene segment can be transcribed from the full-length mRNA. results suggest that ORF2 and ORF3 may be translated from polycistron mRNA starting midway.

【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S852.65
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本文编号：1421409