【作者】 刘刚；

【导师】 张彦明；

【作者基本信息】 西北农林科技大学 ， 预防兽医学， 2008， 硕士

【摘要】 犬瘟热(Canine distemper, CD)是由犬瘟热病毒(Canine distemper virus, CDV)引起的一种以犬为主的多种动物共患急性传染病,对动物的危害很大。CDV基因组编码核衣壳蛋白(N)、磷蛋白(P)、基质膜蛋白(M)、大蛋白(L)、融合蛋白(F)和血凝蛋白(H)6个蛋白,其中H蛋白是CDV感染细胞过程中侵袭宿主所必需的,也是产生中和抗体的主要抗原,同时也是变异最大的结构蛋白;分析和研究H蛋白的分子进化和变异情况能反映CDV的流行和进化趋势。虽然之前开发的犬瘟热弱毒疫苗已被广泛使用并取得很好的保护效果,但是近来犬瘟热又在全世界流行,疫苗免疫失败的现象越来越多。出现这种现象的原因一方面可能是在免疫压力下,CDV抗原表位可能发生漂移,尤其是主要结构蛋白H蛋白极易发生变异,从而使疫苗失去效用;另一方面也可能由于免疫失败或者动物机体的抗体水平下降而导致机体在受到CDV感染时不能产生有效的保护。本研究旨在探明杭州地区CDV的流行特征和变异趋势,为疫苗的选择或开发奠定基础;同时,表达H蛋白用于动物机体的免疫状况的监测,为犬瘟热的防治提供参考。为此,2007年1月至2008年4月间我们收集了44份临床疑似犬瘟热病料并进行PCR检测,克隆测序13株CDV H基因序列,用于遗传进化分析;同时截短表达H蛋白,并进行免疫印迹试验以确证该重组蛋白的反应原性。1.根据CDV N基因的保守区域设计引物,在对RT-PCR法和反转录-套式PCR扩增的N基因片段克隆测序的基础上,比较了两种检测方法的灵敏性和特异性。结果显示,PCR扩增片段和测序结果与预期一致,特异性分析表明两种检测方法对犬细小病毒阳性病料无扩增条带,具有很高的特异性,RT-PCR法和反转录-套式PCR法的阳性检出率分别为11.36%和63.64%,反转录-套式PCR法比RT-PCR法更加敏感。2.为了进一步探明杭州地区CDV的流行特征和变异趋势,克隆测序了13份CDV阳性病料的H基因。分子遗传进化分析显示,所有毒株可分为7个大的分支,且各分支间具有一定的地域相关性。亚洲国家主要存在CDVⅠ型和Ⅴ型,但在中国未发现Ⅴ型毒株的存在;杭州毒株除一株属于Ⅶ型外,其余与中国大部分毒株同处于Ⅰ型,而只有少量中国毒株属于Ⅵ型和Ⅶ型。潜在的天冬酰胺糖基化位点分析发现不同毒株之间的数目差距较大,疫苗株含4～7个糖基化位点,野毒株为7～9个;杭州地区的毒株除HZ005为8个和HZ011为6个外,其余均为9个。进一步分析了H基因变异情况,发现H基因整体变异较大,但氨基酸同义置换的概率大于非同义置换的概率,表明H基因同义突变的趋势更加明显。3.利用MDCK细胞从临床犬瘟热病犬病料中分离CDV,结果表明第3、6、9和12代感染细胞均能检测到病毒特异性条带(病毒命名为HZ026),其原代病毒和第12代病毒的H基因测序分析显示在传代过程中H基因没有发生变异。进一步截短表达H基因的两个片段,Western blot鉴定表明,表达的蛋白对于CDV阳性血清具有较强的反应原性,可用于进一步的血清学研究。更多还原

【Abstract】 Canine distemper (CD),caused by canine distemper virus (CDV), is an acute and highly contagious viral disease of dogs as well as some other animals. The virus genome consists of genes encoding N, P, M, L, F and H proteins. Many studies have shown that Hemagglutinin protein (H) of CDV is necessary during infection and a major target antigen for the host immunesystem. In addition, H protein is the most variable structural protein which can serve as the indicator in analysis of molecular evolution and epidemic study of CDV. The use of live attenuated vaccines has successfully prevented CDV infections in domestic dogs. However, in recent years, several episodes of CD in vaccinated animals have been reported throughout the world, which might be due to the frequent variation of epitopes especially in H protein and the failure of vaccination as well as the lower level of antibodies after uncorrected vaccination strategies. This study was aimed to find out the epidemic and evolutionary trend of CDV in Hangzhou, which was usefull for vaccine development. In addition, the H protein was expressed and analyzed. Thus, during January 2007 and April 2008, fourty-four blood samples or conjunctival swabs of diseased dogs suspected of canine distemper were collected. After detection for CDV, the H gene were cloned and sequenced for analysis of genetic characterization.1. The diagnosis of CD infection is usually based on clinical symptoms, which may result in misdiagnosis in cases. So a sensitive and specific method for detection of CDV infection is necessary. In this stuy, an one-step RT-PCR and a nested PCR are compared for their role in CDV detection. Both the methods reacted with the CDV vaccinal strain, but not with canine parvovirus. The expected fragment of the N gene was detected in 11.36% samples by one-step RT-PCR and 63.64% by nested PCR. So the nested PCR is more reliable than the one-step RT-PCR.2. To gain insight in epidemic and evolutionary trend of CDV in Hangzhou, phylogenetic analysis based on H gene was conducted. The results revealed that all the CDV isolates were segregated into seven groups which showed relationships with regard to geographic regions. Obviusly, most isolates of Asian country were in groupⅠand groupⅤ, but no Chinese isolates was in groupⅤ. Most isolates of our study (except one in group VII) were clustered in groupⅠt ogether with the most Chinese isolates. Prediction of potential N-glycosylation sites revealed that there were different sites in different genotypes. Compared to vaccine isolates of four to seven sites and other wildtype isolates of seven to nine sites, most of the isolates from Hangzhou contain nine potential N-glycosylation sites, except for HZ005 and HZ011 (eight and six respectively). Variability of the CDV H protein was analyzed by the differences between non-synonymous (dN) and synonymous (dS) rates and the entropy values. Majority of the positions within H were subject to negative selection. Our results suggest that recent isolates from Hangzhou are predominantly of groupⅠand they have shifted away from vaccine strains used in the area.3. To isolate CDV, canine epithelial kidney cells (MDCK) were inoculated with lung specimens from suspected dogs. Then one isolate of CDV, designated as HZ026, was found positive at three, six, nine, twelve passages by nested PCR. Then two fragments of H gene, containing 816bp and 459bp of its 3′end, were amplified and cloned into the prokaryotic expression vector pET30a for induction, which showed two bands of 36.4ku and 22.2ku respectively as analysed by SDS-PAGE. Moreover, both of the two fusion proteins could be recognized by CDV positive serum by Western blotting, indicating that the proteins had the antigenitic epitopes of H gene of CDV and could be used in future serological studies.更多还原