【作者】 赵丽荣；

【导师】 相文华；

【作者基本信息】 东北农业大学 ， 预防兽医学， 2006， 博士

【摘要】 鹅细小病毒(goose parvovirus GPV)是细小病毒科(Parvoviridate)、细小病毒属(Parvovirus Genus)的成员。主要感染1月龄内雏鹅和雏番鸭以急性肠炎及肝、肾、心实质脏器炎症为特征的烈性传染病。该病危害程度与日龄密切相关,10日龄内雏鹅发病率和死亡率可达100%,在易感群中的致死率高达70℅,以后发病随日龄增大而逐渐减少。但近几年有报道称该病的发病日龄有增大趋势,超过30日龄的占14.3℅,这可能与鹅细小病毒毒力增强和基因变异有关。自1956年我国学者方定一首次发现并分离到病毒至今,国内外一直有分离到该病毒的报道,说明该病已经呈世界范围流行,对养鹅业发展威胁很大。该病毒可造成大鹅呈一过性隐性感染,不表现发病症状,但可带毒、排毒造成病毒的水平或垂直传播。目前本病的预防主要是采用疫苗进行免疫接种。小鹅除采用疫苗接种外,还要结合小鹅瘟高免血清进行紧急预防。因此寻找该病毒的毒力基因及抗原性基因遗传演化规律,筛选合适的疫苗进行有效防控;同时研制安全无毒有效的基因工程疫苗替代活苗免疫,将是逐步控制、消除本病的大趋势。本研究首先分离鉴定了黑龙江省鹅细小病毒发病地区的一株病毒,命名为GPVHG\21,该毒株的ELD50值显示其为一强毒株。以GPVHG\21株和两致弱疫苗株GPVHg1株、GPVHg2株为研究对象,分别克隆、测序三个病毒株的结构蛋白基因片段,共得到九个克隆片段,即GPVHG\21VP1、GPV HG\21VP2、GPVHG\21VP3片段;GPVHg1VP1、GPVHg1VP2、GPVHg1VP3片段和GPVHg2VP1、GPVHg2VP2、GPVHg2VP3片段。之后采用DNAStar分子生物学分析软件,对上述三个毒株的共九个克隆片段进行比较分析。通过系统演化分析,揭示了GPV的遗传演化与地域因素的相关性规律;通过对GPVHG\21株结构蛋白三个完整基因片段的抗原性预测图谱比较,发现在三段基因片段的非重叠区,也存在抗原性位点的信息;通过比较还发现GPVHg2疫苗株与GPVHG\21分离株抗原性图谱更为接近,即采用以GPVHg2疫苗株去防治以GPVHG\21毒株引发的鹅细小病毒病将更为合理有效。另外通过对强毒株GPVHG\21和弱毒株GPVHg1、GPVHg2结构蛋白基因VP1序列的比较研究,发现了与GPV毒力强弱有关的基因位点,即VP1基因的第1611位和第2127位的核苷酸差异与毒株毒力强弱有关。此外以pcDNA5/TO为载体,以GPV HG\21株的三个结构蛋白基因片段为研究对象,分别构建了三个结构蛋白基因片段的真核表达载体,近而搭建了进行结构蛋白基因免疫研究的平台,通过对三个真核表达质粒体外转染鹅胚成纤维细胞的体外表达检测和免疫小鼠、大鹅等动物的体内表达检测结果,说明三个真核基因表达质粒都可以在体外细胞中成功表达,并能诱发动物体内的体液免疫和细胞免疫两种形式的机体免疫反应。进一步为鹅细小病毒核酸疫苗的研究提供了理论依据。更多还原

【Abstract】 Goose parvovirus (GPV), belonging to Parvoviridate Parvovirus Genus,mainly infects one–month geese and ducks. The virus causes acute inflammation in intestines,liver ,kidney and heart.The degree of damage is closely relevant to born days. Mortality to 10 days old was up to 100%.GPV was first found in 1956 by fangdingyi and was reported in the all world. The threat to geese was great.The virus can infect geese recessively , has no symptom and can be transmitted to the gosling from the infected mother and other geese by repiratory system and digestive system.. Presently vaccine was used to prevent the disease and serum was for urgency prevention. It is necessary to explore genes relative to pathogenesis and study its evolution rule,furthermore, screening suitable vaccine for GPV. At the same time, gene engineering vaccine was explored to substitute for active vaccine for controling and annihilating the disease.The study isolated and identified GPV HG\21 happened in Heilongjiang province. The virulence of the strain was not super strong known by ELD50. Structural protein genes VP1 ,VP2, VP3 of GPV HG\21 and attenuated vaccine strain Hg1、Hg2 were cloned and sequenced. DNAStar software was used to analyze their evolution, antigen forecast analysis, molecular comparation research on strong and attenuated strains.By comparing antigen forecast analysis profile of GPV VP1、VP2和VP3,we find that antigen determinant could exist in non-overlap position of three genes;Furthermore,1611 and 2127 site of VP1 gene was determined to be possively relative to virulence of viruses by analyzing sequences of different pathogenesis. Besides, vaccine screening principle was derived ,that was , serum type should be near to gene type GPVHg2 can be more efficient to prevent GPV disease caused by HG\21. Eukaryotic expressing plasmids of structural protein genes VP1,VP2, VP3 of GPV HG\21 was constructed and transfected geese eggs fibroblast cells.Gene expression in mice and geese was assayed.The results showed that three eukaryotic expressing plasmids can expressed in vitro cell,also, induce physical and cell immunity response.The results provide theoretic foundation for DNA vaccination of GPV.更多还原