【作者】 于天飞；

【导师】 王君伟；

【作者基本信息】 东北农业大学 ， 预防兽医学， 2007， 博士

【摘要】 鹅细小病毒病(Gosling pavovirusis,GPVS)是雏鹅的烈性传染病,又称小鹅瘟或Derzsy’s病。多发生于4日龄至20日龄以内的雏鹅。发病率和死亡率可高达90%～100%。该病病原为鹅细小病毒(Goose parvovirus,GPV),能感染各种品种的雏鹅,也可以感染雏番鸭。目前,该病仍为养鹅业最主要的传染病之一,造成巨大的经济损失。由于本病长期困扰着养鹅业的发展,所以需要一种,准确、简便的诊断方法来对该病作出早期诊断;也需要通过分析病毒的抗原成分筛选合适的肽段作为亚单位疫苗用于本病的免疫防治。但这种诊断试剂和亚单位疫苗的研制,必须建立在对病毒蛋白抗原性的研究之上。细小病毒都具有基因组短小,衣壳结构简单的特点,所以更容易将它们无关的抗原成分筛除,在制造亚单位疫苗和重组诊断抗原上具有很多优势。本研究首先构建了含有GPV H1株非结构蛋白(NS1)和结构蛋白(VP1)基因的重组克隆载体pMD18-T-NS1和pMD18-T-VP1。经测序鉴定正确后,分别作为截短表达片段PCR扩增的模板。为了对NS1蛋白(627aa)进行抗原表位作图,设计了一套覆盖整个NS1蛋白的短肽,这些短肽长为50-60个氨基酸,有10-15个氨基酸重叠。为了表达这些短肽,共设计合成了15对引物。将这些亚克隆的基因片段插入至原核表达载体pGEX-6P-1中,经IPTG诱导获得了表达。利用切胶纯化的方法对表达蛋白进行了纯化,纯化蛋白经抗GST单克隆抗体鉴定。应用GPV H1株活疫苗毒免疫的10周龄鹅血清对表达的15个重组蛋白的抗原性进行了检测,Western blot结果表明,表达的融合蛋白NS(453-514)、NS(485-542)、NS(533-598)和NS(575-627)能被GPV免疫的鹅血清所识别。根据以上试验结果,进一步设计表达了覆盖NS1蛋白C末端453-627aa的7个截短短肽,这些短肽长为30个氨基酸左右,有10～15个氨基酸重叠。Western blot结果表明,表达的融合蛋白NS(485-514)、NS(498-532)、NS(523-556)、NS(543-573)、NS(564-598)和NS(599-627)能被GPV免疫的鹅血清所识别。为了对VP1蛋白(732aa)进行抗原表位作图,设计并表达了19个覆盖整个VP1蛋白的短肽,这些短肽长为50～60个氨基酸,有10～15个氨基酸重叠。Western blot结果表明,表达的融合蛋白VP(35-100)、VP(81-136)、VP(124-161)、VP(146-198)、VP(423-491)、VP(531-595)、VP(616-669)和VP(678-732)能被GPV免疫的鹅血清所识别。根据以上试验结果,进一步设计表达了16个覆盖VP1蛋白抗原性片段的短肽,这些短肽长为30个氨基酸左右,有10～15个氨基酸重叠。Western blot结果表明,表达的融合蛋白VP(35-63),VP(50-81),VP(111-145),VP(136-161),VP(423-453),VP(462-491),VP(531-560),VP(548-577),VP(616-647),VP(634-669),VP(678-706)和VP(697-732)能被GPV免疫的鹅血清所识别。综合上述试验结果,GPV H1株NS1蛋白的B细胞线性抗原表位区位于NS1蛋白的羧基末端485-627aa;GPV H1株VP1蛋白的B细胞线性抗原表位区位于35-81aa、111～198aa、423～453aa、462～491aa、531～577aa、616～669aa和678～732aa。总之,对以体液免疫反应为主的GPV来说,鉴定其B细胞抗原表位可以揭示GPV体液免疫的本质,填补GPV抗原表位研究上的空白,对深入了解GPV的抗原分子特性,病毒变异对免疫原性及致病性的影响,建立基于抗原表位水平的特异性诊断方法以及设计新型的抗GPV疫苗等具有重要意义。更多还原

【Abstract】 Goose parvovirus(GPV) infection,also known as goose plaque or Derzsy’s disease,causes high mortality in domestic goslings(Anser anser vardom) and Muscovy ducklings(Cairina moschata).It caused 90 to 100%mortality in susceptible goslings under the age of 4 days to 20 days.GPV infection has been reported from many goose-producing countries,and caused huge economic loss.Owing to the hindrance to development of goose cultural industry because of this desease,there needs accurate,convenient diagnostic method for early diagnosis avoiding the interference of maternal antibodies.Meanwhile,it needs monitor the level of maternal antibodies. Furthermore,it needs analyze the viral antigen to select the suitable peptides be as subunits vaccine for desease prevention.Notwithstanding many veterinary researchers paid close attention to GPV,the study about the antigenicity of viral protein fell behind other aspects.The genome of the members of parvovirinae is small and the capsid structure is simple,so it is easy to eliminate independent antigenic component.Thus,it has superiority in prepareing subunit vaccine and recombinant diagnostic antigen.Except for this,GPV not only has the high mortality feature of autonomously replicating parvoviruses,but also has the genetic feature of dependovirus.Therefore,we can study the GPV collateing the research finding of other parvovirus.In this study,two recombinant plasmids harboring non-structural and structural protein gene named pMD18-T-NS1 and pMD18-T-NS1 were constructed and sequenced.The two recombinant plasmids were used as the templates for PCR in the next procedure.To map the antigenic epitopes of non-structural protein,a set of partially overlapping fragments of 50-60aa spanning the protein were designed.The totals of 15 pairs of primers were synthesized. All the forward primers contained a BamHI restriction site,and reverse primers contained a termination codon and an XhoI restriction site.PCR products were cloned into expression vector, pGEX-6P-1(Amersham Pharmacia Biotech,Sweden) and the map of these fragments confirmed by sequencing.Each fragment was expressed as glutathione S-transferase(GST) fusion proteins in Escherichia coli Rosetta(DE3)pLysS and purified by elution from sodium dodecyl sulphate(SDS) polyacrylamide gels.Anti-GST tag MAb was used to identify the purified protein.Then western blot reactivity of these short peptide fused protein to viral infected sera were surveyed.Linear immunodominant B-cell epitopes were primarily found in four fragments:NS(453-514), NS(485-542),NS(533-598),NS(575-627).The further 7 overlapping fragments of the NS1 protein from 453 to 627aa were also expressed in E.coli.The further results of mapping NS1 protein immunodominant epitopes are fragments NS(498-532) which showed strongly positive,fragments NS(485-514),NS(523-556),NS(543-573),NS(564-598) and NS(599-627) which were weakly positive.To map the antigenic epitopes of structural protein,a set of partially overlapping fragments of 50-60aa spanning the protein were designed.The totals of 19 pairs of primers were synthesized. Western blot reactivity of these short peptide fused protein to viral infected sera were surveyed. Linear immunodominant B-cell epitopes were primarily found in fragments:VP(35-100), VP(81-136),VP(124-161),VP(146-198),VP(423-491),VP(531-595),VP(616-669) and VP(678-732).The further 16 overlapping fragments of the VP1 antigenic protein were also expressed in E.coli.The further results of mapping VP1 antigenic protein immunodominant epitopes are fragments:VP(35-63),VP(50-81),VP(111-145),VP(136-161),VP(423-453), VP(462-491),VP(531-560),VP(548-577),VP(616-647),VP(634-669),VP(678-706) and VP(697-732).Thus,the non-structural protein linear B-cell epitopes are located on the C-terminal, (485-627aa).The structural protein linear B-cell epitopes are located on the 35-81aa,111-198aa, 423-453aa,462-491aa,531-577aa,616-669aa and 678-732aa.After goose parvovirus infection,the host principal immune reaction is humoral immune reaction.Identification of B-cell epitopes can reveal the essence of humoral immune reaction and supply the blank in GPV research.Meanwhile,because of GPV supposed to be the antecedent of autonomous parvovirus and dependovirus,the study about GPV non-structural and structural protein antigenicity can complete the knowledge of interaction between parvovirus and their hosts. In a word,identification of antigenic epitopes of the non-structural and structural protein of GPV may be helpful in understanding molecular properties of non-structural and structural protein of GPV,as well as relationship between immunogenicity or pathogenesis and gene variations of the virus.Therefore,the results from the study may also be useful for diagnosis of GPV infection based on antigenic epitope or developing a new strategy for vaccine design.更多还原