本文關鍵詞：鴨源呼腸孤病毒的表型和分子特征分析及基因2型毒株編碼的新蛋白的鑒定　出處：《中國農業(yè)大學》2016年博士論文　論文類型：學位論文

  更多相關文章： 鴨源呼腸孤病毒 表型 分子特征 p10蛋白 p18蛋白

【摘要】：根據(jù)宿主和病變,鴨呼腸孤病毒病分為3種主要類型,即,番鴨“白點病”、番鴨“新肝病”和北京鴨“脾壞死病”,其病原分別為番鴨呼腸孤病毒(Muscovy duck reovirus, MDRV)、新致病型番鴨呼腸孤病毒(New pathotype of Muscovy duck reovirus, N-MDRV)和鴨呼腸孤病毒(Duck reovirus, DRV)。MDRV于1972年首次發(fā)現(xiàn)于法國,目前被歸為禽呼腸孤病毒(Avian orthoreovirus,ARV)的番鴨源分離株(ARV-Md), N-MDRV和DRV是我國研究者分別于2009年和2011年報道的新毒株。迄今為止,不同鴨源呼腸孤病毒毒株之間的準確分類關系還有待于闡明。以ARV-Md/815-12、N-MDRV/J18和DRV/091為材料,對不同鴨源呼腸孤病毒的表型特征進行比較。結果顯示,N-MDRV和DRV在實驗室感染宿主范圍、在細胞質內排列方式、致細胞融合活性、沉淀反應抗原、對北京鴨的致病性和組織嗜性等表型特征上表現(xiàn)出相似性。與N-MDRV和DRV相比,ARV-Md在表型特征上存在明顯差異。3株病毒均對番鴨有致病性,但所引起的病變和組織嗜性有所不同。為明確三類鴨源呼腸孤病毒的基因組結構特點及其與其它水禽源毒株和雞源ARV的遺傳演化關系,用SDS-PAGE分析了3株病毒的核酸電泳型,用RT-PCR和5RACE測定了ARV-Md/815-12的基因組序列以及N-MDRV/J18和DRV/091的S1基因節(jié)段序列,由此獲得了3株鴨源呼腸孤病毒的基因組序列。SDS-PAGE結果顯示,N-MDRV和DRV具有相同的基因組核酸電泳型。與N-MDRV和DRV相比,ARV-Md S組的核酸電泳型表現(xiàn)出顯著的差異。序列分析結果顯示,所測3株病毒均具有ARV的基因組結構特點,但在多順反子基因節(jié)段的長度和基因結構上,N-MDRV和DRV等新毒株與ARV-Md存在顯著差異,即,J-MDRV和DRV的多順反子節(jié)段位于S1,含3個ORF,分別編碼p10、p18和σC蛋白；而ARV-Md的多順反子基因節(jié)段位于S4,僅含2個ORF,分別編碼p10和σC蛋白。N-MDRV和DRV的p10蛋白與ARV-Md的p10蛋白缺乏序列同源性。進化分析結果顯示,水禽源和雞源毒株的9個基因節(jié)段(除M2)均按宿主聚為兩個明顯不同的分支；在M2節(jié)段,相對于ARV-Md, N-MDRV和DRV等水禽源新毒株與雞源毒株具有更近的遺傳演化關系。綜合分析,可將水禽源毒株鑒定為ARV的成員,但現(xiàn)有的序列同源性分類標準需要調整。根據(jù)外衣殼蛋白序列的進化分析結果,可將水禽源呼腸孤病毒區(qū)分為2個不同的基因型,ARV-Md屬于基因1型,N-MDRV和DRV屬于基因2型。相對于基因1型毒株,N-MDRV和DRV等基因2型毒株最顯著的基因組結構特點是其多順反子節(jié)段含有3個ORF,但在復制過程中,預測的ORF1和ORF2是否會表達成熟的p10和p18蛋白,尚不清楚。為回答這一問題,開展了下述研究。用Western Blot進行檢測,結果顯示,DRV/091的免疫血清可與p18重組蛋白發(fā)生反應,而p18重組蛋白的免疫血清亦可從DRV/091感染的Vero細胞中識別出18 kDa的蛋白；用MALDI-TOF/TOF法進行分析,從DRV/091感染的Vero細胞中鑒定出p18蛋白的氨基酸序列：由此可見,p18是DRV S1基因節(jié)段編碼的一種新蛋白。序列分析顯示,在p18蛋白的118-135位可能存在一個核定位信號(Nuclear localization signal, NLS)序列(118KRRR121-X10-132KRRR135),提示該蛋白可能具有入核活性。為驗證這一觀點,用激光共聚焦顯微鏡觀察了p18蛋白的亞細胞定位,結果顯示,在感染細胞和轉染細胞的細胞核內,見有p18蛋白聚集。突變分析顯示,p18蛋白的入核活性由NLS決定,而位于NLS N-端的’18KRRR121序列是引導p18蛋白入核的關鍵基序。經(jīng)檢測,p18蛋白在Vero細胞中過表達能夠引起細胞凋亡。將DRV/091感染Vero細胞,用p10蛋白的單克隆抗體進行間接免疫熒光試驗,從感染細胞內檢測到熒光信號,以此證實p10蛋白是病毒復制所產生的成熟蛋白。N-MDRV和DRV的p10蛋白含有融合蛋白基序,提示該蛋白具有致細胞融合活性。為驗證這一觀點,用表達p10蛋白的真核表達質粒轉染Vero細胞,在轉染后24 h可見細胞融合現(xiàn)象。然而,用表達病毒其它蛋白的真核表達質粒轉染Vero細胞,均未見細胞融合現(xiàn)象。由此可見,p10蛋白是決定DRV等基因2型毒株致細胞融合活性的關鍵因素。上述研究證實,DRV等基因2型毒株的S1基因節(jié)段為三順反子。為初步了解該基因節(jié)段的轉錄和翻譯策略,用DRV/091感染、Aero細胞,在感染后不同時間提取細胞內總RNA,并用識別S1基因節(jié)段的核酸探針進行Northern Blot檢測,在感染后2-8 h可檢測到單-mRNA條帶,長度與S1基因節(jié)段長度(1568 bp)相符。由此可見,在DRV的復制過程中,其S1基因節(jié)段可轉錄出全長mRNA。結果提示,ORF2和ORF3可能是從多順反子mRNA的中途起始翻譯的。
[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.

【學位授予單位】：中國農業(yè)大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：S852.65
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本文編號：1421409