【作者】 梁冬莹；

【导师】 华育平； 赵广英；

【作者基本信息】 东北林业大学 ， 野生动植物保护与利用， 2007， 博士

【摘要】 水貂阿留申病(Aleutian Disease，AD)是由细小病毒亚科(Parvovirinae)阿留申水貂病毒属(Amdovirus)水貂阿留申病毒(Aleutian mink disease virus，ADV)引起的免疫系统病理性失调，是一种主要侵染水貂、病程缓慢的传染性疾病。该病以垂直和水平传播两种形式在世界各国的貂群中广泛流行，导致没有抗体的新出生幼貂的急性间质性肺炎甚至死亡，成年水貂的繁殖能力、皮张质量、健康状况下降以及死亡率增加，对水貂养殖业造成不可低估的经济损失。近年来，我国水貂养殖区阿留申病的流行情况在国内时有报道，高水平的AD感染一直是严重影响我国水貂养殖业高效、健康发展的一个非常重要的因素之一。由于目前针对该病缺少有效的防治方法，普遍采用的是通过定期检测，淘汰患病貂群，从而达到净化种群的目的。因此，在国内进行分离鉴定病毒株及进行分子生物学特性研究，建立国内适用的血清学诊断方法，将会对今后国内开展水貂阿留申病的临床诊断、有效防制及进一步的深入研究，提供更多的科学依据。在本研究中，首先通过CIEP以及本实验室建立的ADV PCR检测方法，对黑龙江某水貂养殖场发病送检貂以及辽宁某水貂养殖区疑似阿留申病貂进行检测，将经两种方法检测均为阳性结果的毒株进行PCR扩增产物的序列测定，从中抽取序列较为保守的四株病毒与标准对照毒株进行序列比对和同源性分析，与ADV-G相应片段的核酸同源率分别为92.8％、93.9％、93.2％和93.5％，与参考株ADV-Utah的同源率分别为94.5％、98％、94.6％和95.5％，确定为ADV特异性核苷酸片段。依据鉴定的结果，成功鉴定了四株病毒，分别命名为MS-1、DL-1、DL-2和DL-3。将MS-1、DL-1、DL-2和DL-3株病毒经组织病料提取总DNA，采用依据标准毒株ADV-G的DNA序列设计并合成的四对引物，经PCR扩增后分别构建重组质粒并测序，将测序结果用DNA Star软件中的SeqMan程序进行拼接，获得四株病毒的近全长DNA序列。用DNA Star软件MegAlign程序中的Jotun Hein method将实验测得的毒株与国外病毒参考株的对应核苷酸序列进行同源性比较和进化树分析。结果表明，ADV-Utah与四个实验毒株的序列同源性较高，同源率为92.9-93.4％，ADV-G、ADV-SL3与实验毒株的同源性较低；四个实验毒株和三个参考毒株被分别划归为两个组，分属于两个进化分支；实验毒株中ADV-DL1与DL2和DL3虽然同为大连分离株，却表现出较远的亲缘关系。用DNA Star软件MegAlign程序中的Clustal W method将实验测得的ADV毒株核苷酸序列、ADV参考株序列以及细小病毒其它四个属的代表毒株全序列进行进化树分析，发现14株病毒共划归为五个相对独立的进化分支，ADV与牛犬细小病毒作为细小病毒亚科中新增的属，与其它属的成员分属于不同的进化分支，分析结果验证了ICTV8公布的新分类体系的合理性。选择VP2蛋白主要抗原表位区相对保守的ADV MS-1毒株作为种毒，分别设计合成两对引物，用PCR方法扩增其VP2基因中主要抗原表位区的两个片段，分别将其克隆到原核表达载体pMAL-c2的多克隆位点中，构建重组表达质粒pMAL-VP2a和pMAL-VP2b，经PCR扩增、酶切和测序分析确证其正确插入到表达载体中，阅读框正确，成功地构建了原核表达载体。阳性重组质粒转化宿主菌TB1，用IPTG进行诱导表达，对表达产物进行SDS-PAGE检测和免疫印迹分析。结果表明两段融合蛋白均以包涵体形式获得了有效表达，表达产物分别占总菌体蛋白的32.7％和37.41％；表达产物的分子质量分别约为63kD和65kD，与理论推测的分子质量一致；在终浓度为1mM的IPTG诱导下，4h时其表达量达到高峰：Western blot分析表明表达蛋白能被兔抗MBP抗体所识别。将分离和初步纯化的表达包涵体蛋白作为抗原，对阳性血清进行CIEP检测，验证其免疫活性，并与标准抗原的CIEP检测结果相比较，二者的符合率达到94.3％。以本实验获得的重组蛋白作为诊断抗原的特异性、重复性、敏感性均较好，初步建立了临床诊断方法。总之，本研究对ADV国内分离毒株进行分离鉴定和全序列测定，为研究国内ADV病毒株的进化特点和规律提供基础性资料；在国内首次利用原核表达载体成功地对VP2主要抗原表位区进行表达，并以其为抗原，初步建立了ADV抗体的CIEP检测方法，将为国内养貂场有效开展阿留申病血清学调查提供有效的技术手段，在貂群的净化中发挥巨大的作用。更多还原

【Abstract】 Aleutian Disease (AD), which caused by Aleutian mink disease virus belonged to Parvovirinae Amdovirus, is a major chronic infectious disease of mink. AD would cause immunologic system patho-disharmony. And it was wide spread all over the world in the mink with both vertical and horizontal transmission. The disease caused acute interstitial pneumonia even death of the new birth young mink without antibody and the descent of reproductive capacity, fur quality and health status of the adult minks. AD resulted in increase of death rate and enormous economic loss of mink cultivation. For the past few years, the popular information was reported in Chinese mink cultivation. High level AD infection is one of the all-important effect factors for developing Chinese mink cultivation healthy. Now, regular detection and condemn infected minks to clean the population was used widely. So the clinical diagnosis of AD, prevention and cure methods and more scientific information would be provided by identifying the AD strain, studying the virus molecular characters and founding the fit sero-diagnosis methods.In the study, some Hei Long Jiang and Liao Ning province mink nursery were detected by CIEP and ADV PCR method founded by our laboratory. The PCR productions of positive strains in both methods were sequenced. And four conservative strains were draw-off to sequence compare and homology analyze with the standard contrast strain. The result showed that the nucleate homology with the corresponding ADV-G region was 92.8%, 93.9%, 93.2% and 93.5% respectively. And the homology with the collate strain ADV-Utah was 94.5%, 98%, 94.6% and 95.5% respectively. So the virus strain was determined ADV virus specific nuclear fragment. According to the result, the four viruses were named as MS-1, DL-1, DL-2 and DL-3 respectively.The total DNA of MS-1, DL-1, DL-2 and DL-3 were extracted from infective organizations. Four pairs of primers based on the standard virus strain ADV-G were designed. The recombinant plasmids were constructed with PCR productions and sequenced. The four proper complete DNA sequences were obtained by splicing the sequenced results with the software SeqMan of the DNA Star. The homology compare of the corresponding nuclear sequences and the cladogram analyze between the obtained virus strain and abroad virus collate strain were measured by Jotun Hein method in software DNA Star. The results suggested that there is high homology, from 92.9% to 93.4%, between the ADV-Utah and the experimental virus strain. And ADV-G and ADV-SL3 had the low homology with the experimental virus strain. The four experimental virus strains and the three collate virus strains were divided into two groups, and belonged two evolutionary branches. Although ADV-DL1, DL2 and DL3 were all belonged to the Da Lian separated strains, they had the ulterior genetic relationship. The nuclear sequences of the obtained experimental ADV virus strains, ADV virus collate virus strains and typify virus strains of other four Parvovirus generic were cladogram analyzed with the Clustal W method of software DNA Star. The results show that fourteen virus were divided into five independent evolutionary branches. ADV and Bovine parvovirus, as the new genus of the parvovirus subfamily, had the different evolutionary branches with the other genus viruses. The result validated the rationality of the new classify system reported by ICTV8.ADV MS-1 strain was selected with a relative conserved main antigenic region of VP2 protein. According to the two pairs of primers designed, two main antigen domains of VP2 gene of ADV strain were amplified by PCR. Then the amplified DNA products were cloned into the multiple cloning sites of prokaryotic expression vector pMAL-c2, respectively, through which recombinant vectors pMAL-VP2a and pMAL-VP2b were constructed. The insert position, the size and the reading frame were affirmed all right by restriction digestion, PCR and the sequence analysis which showed that the prokaryotic expression vectors pMAL-VPa and pMAL-VPb were constructed successfully. Then the positive recombinant vectors were transformed into recipient germs TB1 for expression by IPTG inducing. Through SDS-PAGE and Western blot, the two proteins were detected to be expressed successfully in the form of cytoryctes. The molecular weights of the expressed protein were 63KDa and 65 KD respectively which was identical with the theoretically presumed. Induced at a concentration of 1 mmol/L IPTG, it took 4 hours for the quantity of expressed protein to amount to peak value. Western blot indicated that the expressed antigen proteins could be recognized by the rabbit anti-MBP antiserum. To detect the immunoreactivity, the protein was isolated and purified as the antigen and CIEP was performed on the positive serum. The result was showed to be 94.3% similar to the CIEP with control antigen. It was believed that the expressed recombinant protein in this study can be used as the antigen in the clinical diagnosis with better specificity, reproducibility and sensitivity.In a word, this study provided lots of foundational information for the separating, identifying and complete sequencing of the ADV separated strains in China. And the results would be useful for the evolutionary characters and regularities of the ADV virus strains. VP2 major antigens epitope were expressed by the prokaryotic expression vector first time in China. Using the expression vector as the antigens, the CIEP detection was set up initially. All of these would be useful in AD sero-survey of the mink cultivation.更多还原