摘要
禽脑脊髓炎病毒(Avian Encephalomyelitis Virus,AEV)是一种小RNA病毒。侵害鸡的中枢神经系统和其它实质性器官,该病毒通过口-粪途径传播,具有水平和垂直传播的能力。由于它极大的稳定性,被污染的区域可能长期保持传染性。鸡胚适应株Van Roekel是高度嗜神经的,并且在鸡的肠道内不能有效的生长,而野毒株却是嗜肠道型的。尽管这样,野毒株和Van Roekel株的病毒多肽具有一致的抗原性。幼鸡感染该病毒后,引起麻痹、头颈震颤甚至共济失调,而成鸡常呈亚临床感染或导致产蛋量和孵化率下降。病毒的感染性不受氯仿、低pH、胃蛋白酶、胰酶和脱氧核糖核酸酶的影响,镁离子可增强病毒对热的稳定性,病毒的浮密度为1.31g/ml,直径为22-30nm,该病毒主要在鸡胚中增殖,在大多数细胞培养物中不生长。
1932年Jones首次报道了在1930年美国新英格兰地区幼鸡群爆发的禽脑脊髓炎。随后十年中,在欧洲、加拿大、日本和澳大利亚都有此病的报道。八十年代,该病传入我国。AEV一直被认为是最重要的禽病病原之一,尽管它很重要,但有关其基础生物学,发病机理的了解却较少。本项研究,旨在利用分子生物学的技术手段,搞清楚AEV基因组的序列和结构,从而探讨基因结构与功能的关系。
本研究共分四个部分:第一部分为AEV的增殖,纯化和电镜观察,用1:5倍稀释的AEV-NH937株和阴性对照PBS分别经卵黄囊接种于6dSPF鸡胚,继续孵化10d后,收集尿囊液。比较接种组和健康对照组鸡胚的大小,结果显示,健康对照组鸡胚明显大于接种组。分离、提纯AEV,把纯化的病毒在电镜下观察,证明确有大量AEV病毒粒子存在,说明AEV在鸡胚中成功扩增;第二部分是AEV-NH937基因组的序列测定工作。根据Calnek疫苗株序列设计了9对引物,利用反转录聚合酶链式反应,成功地扩增了AEV-NH937毒株的全基因。把它们分段克隆在pUCm-T载体上,经序列分析,获得了AEV-NH937毒株的基因组全序列及推导的氨基酸序列,基因组全长为7055个核苷酸,与calnek疫苗株具有94.3%的同源性,编码一个含2134个氨基酸的多聚蛋白。这是国内首次对AEV基因组全序列的分析;第三部分:AEV外壳蛋白VP_1基因在大肠杆菌中的融合表达、纯化及活性研究。将VP_1基因编码区的810bp片段插入载体pGEX-4T1构建表达重组质粒,然后转化大肠杆菌BL_(21),经IPTG诱导后,用聚丙稀酰氨凝被电泳分析。结果表明,表达产物为53KD的融合蛋白。经超声波裂解,用尿素溶解包涵体,电泳纯化后,利用ELISA检测VP_1
外壳蛋白,表明具有一定抗原性;第四部分:应用原位杂交检测 AEV i用 D i g
标记的探针与sd、10d、20d的攻毒鸡脑组织杂交,来检测那V的RNA。结果
显示,sd、10d、20d的攻毒脑组织切片均有阳性杂交信号,阴性对照却没有
杂交信号,该方法与其它检测方法具有良好的平行关系。
Avian encephalomyelitis virus (AEV) is a picornavirus with a predilection for the central nervous system and other parenchymous organs of chickens that is transmited by the oral-faecal route.The virus may be spread by the vertical and horizontalroutes, and because of its great stability,contaminated areas may remain infectious for long periods.The egg-adapted Van Roekel strain is highly neurotropic and does not grow efficiently in the enteric tract of the chicken,and the field isolates of AEV is usually enterotropic .Despite this.the virion polypeptides of both naturally-occurring strains and the Van Roekel strain are antigenically identical. In young chickens AEV induces paralysis, ataxia and muscular dystrophy, while in older chickens, infection is usually subclinical,resulting in a decline in egg production and hatchability.Infectivity was shown to remain unaffected by chloroform,low pH, pepsin, trypsin and deoxyribonuclease.Magnesium cations were shown to stabilise preparations of the virus against heat
inactivation.The buoyant density of virions are 1.31g/ml.The diameter of the virion was estimated to be 22 to 30nm.The AEV can be adapted to grow in chicken embryo.The inability of AEV to grow effeciently in most cell cultures.
In 1932,outbreaks of a disease causing neurotropic dysfunction in young chickens in the New England region of the USA in 1930 were first reported by Jones.Over the next decade,outbreaks of the disease were reported in Europe, Canadajapan and Australia.In 1980s,The disease was transmited into China. AEV has long been recognised as the aetidaogical agent of one of the most significant disease of poultry.Despite its importance,relatively little progress had been made towards an understanding of its basic biology and pathogenesis.
The purpose of this study was to learn the sequence and structure of the AEV by technology of the molecular biology.to reveal the relation between genome features and function. The research consist of four parts.The first part is multiplication,purification and electron microscope examination of the avian encephalomyelitis virus.A 1:5 dilution of isolate-NH937 of AEV and control group of PBS were inoculated to susceptible 6-day-old chickens
embryos.respectively.After incubation for 10 days,the urinay vesicle liquid was collected.Making a comparison the size of the chickens embryos between the test group and the control group,the results showed that the size of the control group is bigger than that of the test group.Purified virions were examined under the electron microscope,the result revealed that there are a lot of virions and the AEV-NH937 was multiplicated in embryos.The second part was seguence analysis of the genome of the AEV-NH937.Nine pairs of primers were designed according to published Calnek vaccine strain of AEV. RT-PCR was used to amplify the cDNA of the genome .These cDNA fragments were cloned into the plasmid pUCm-T.The result indicated that the seguence of the genome was obtained. The genome of AEV-NH937 composed of 7055 nucleotides, potentially encodes a polyprotein of 2134 amino acids. The genome of AEV-NH937 has 94.3% nucleotides identity with the Calnek vaccine strain of AEV. it was firstly reported in China.The third part
rthe capsid protein VP| gene was expressed.810 bp fragment was inserted into vector pGEX-4Tl,and then transformed into E.coli BL21. SDS-PAGE analysis showed that the expressed product is a 53KD fusion protein aftor induction with IPTG. The expressed product was lysised with supersonic wave and purified by SDS-PAGE.ELISA analysis revealed that the antigenicity of the VPi protein has been detected. The forth part-detection of AEV-NH937 strain by In Situ Hybridization(1SH)-Probe was labelled with digoxigenin(Dig).Then the probe hybrid with 5d,10d,and 20d postinfection brain tissue of chicken.The results of the ISH showed that the positive signal was found in 3 cases,while control group was negative.there has been a reasonable correlatien between this method and other detection test.
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