【作者】 宗春苗；

【导师】 王全凯； 苏凤艳；

【作者基本信息】 吉林农业大学 ， 野生动植物保护与利用， 2007， 硕士

【摘要】 犬瘟热是由犬瘟热病毒(canine distemper virus，CDV)引起的一种急性、传染性极强的烈性传染病。CDV属于副粘病毒科麻疹病毒属，该病毒只有一个血清型不同毒株拥有共同的可溶性抗原，各毒株在抗原性上没有区别。该病毒呈世界性分布，自然条件下感染肉食目中犬科、鼬科、浣熊科等多种动物，尤其对毛皮经济动物危害严重。特种经济动物因为其本身的经济价值很高，一旦发生传染病损失严重，因此有必要对患传染病的动物进行治疗和紧急预防，将损失降到最小。但目前犬瘟热没有治疗的良方，只能通过疫苗来控制。因此犬瘟热疫苗的研究一直是世界各地学者研究的热点。本实验采用Vero细胞从山东某貂场的疑似CD的貂肝脏内分离出1株病毒，对其培养特性、细胞毒力、生物学特性、免疫学和分子生物学鉴定。经鉴定该病毒为犬瘟热病毒(CDV)，命名为CDVSJ。根据GeneBank中所发表的CDV Onderstepoort株的H基因设计了1对特异性引物，对CDVSJ H基因进行RT-PCR扩增，扩增产物与pMD18-T simple vector连接，转化到大肠杆菌JM109感受态细胞中进行克隆。提取质粒进行PCR和双酶切鉴定，鉴定为阳性的送去测序。去掉两端酶切位点测出长1596bp的目的基因条带与预计的完全相符。并将测序结果与Genebank中发表的15个CDV株的核苷酸序列和推导的氨基酸序列进行了比较分析。分析的结果表明：所有的基因分为2组，一组由弱毒株组成，另一组由野生型组成一组，野生型又可分为不同的基因型。本实验室分离的CDVSJ在基因进化树上与弱毒为一组，它与国内的低毒疫苗株strain CDV3的核苷酸序列和推导的氨基酸序列的同源性均最高分别为99.1％和98.1％，而与野生型Hamamatsu核苷酸和推导的氨基酸序列的同源性均最低分别为90.9％和90.4％。编码氨基酸比较结果，所有毒株都含有12个半胱氨酸残基，其中只有strain CDV3有一处不同在414位(其它在304位)，在CDV毒株中半胱氨酸残基是很保守的。CDVSJ此段H基因含有5个潜在的天冬氨酸糖基化位点；此段疫苗株仅含3个；strain CDV3和Convac都含有6个与分离株不同的在530aa～532aa；Hamatsu有8个，其它3处在236Aa～238Aa、511aa～513aa和530aa～532aa；Danish dog有7处，其它在236Aa～238Aa和530aa～532aa。在511aa～513aa是Hamamatsu强毒株所特有的而弱毒株没有的。本实验通过对该分离株的研究表明，该分离株可能是一株弱毒株，为弱毒疫苗提供了后备毒株；同时对该分离株的H基因的序列及编码氨基酸的比较表明它与其它的CDV毒株有一定的差异，这些差异为研究犬瘟热免疫失败提供分子生物学依据。为犬瘟热的预防提供理论依据。更多还原

【Abstract】 The canine distemper was one kind acute and the infectious greatly strengthened ardent infectionwhich caused by the canine distemper virus. CDV belonged Paramyxoviridae Morbillivirus genus, thisvirus has only one serotype and has the common soluble antigen in different strain, which has notdistinguished in the antigenicity. This virus assumed the worldwide basis distribution, under the naturalcondition infects the carnivorous dog branch, the weasel branch, the raccoon branch, and so on many kindsof animals, is especially serious to the fur economical animal harm. The special economical animal wasvery high because of itself economic value, once has the infectious disease to lose seriously, therefore hasthe necessity to the animal which contracts the infectious disease to carry on the treatment and the urgentprevention, will lose falls slightly to. But the present canine distemper has no treatment effectiveprescription, only has been able to control through the vaccine. Therefore the canine distemper vaccineresearch always is a hot spot which the world each place scholar studies.Canine distemper virus strain was isolated from the liver of mink from Shandong using Vero cell. Itwas demonstrated to be a virulent strain of CDV by a series of systematic identification such asmorphology, cell virulence test, biological property, immunoassay and molecular virology test. The strainwas named CDVSJ. One pare of primers was designed and synthesized based on the Haemagglutinin(H)protein gene sequence the Onderstepoort strain of canine distemper virus(CDV).The length H genefragment was amplified by polymerase chain reaction Amplification product was linked topMD18-Tsimple vector and transformed in JM109 of coli bacillus competent cell. Plasmid was made toidentify by PCR and emzyme cut. The correct positive recombinant was used for sequencing. The sequencelength was 1596 bp as expected. The nucleic acid and deduced amino acid sequence of the H protein of theCDVSJ strain were compared with those of other CDV strain extracted from the Genebank database. Allthe strains were divided to two groups. One is virulent strain group; the other is vaccine strain group.CDVSJ strain was part of vaccine group. The nucleic acid and amino acid sequence homology the highestand the lowest of CDVSJ was Chinese strain CDV3 and Hamamatsu, leave each other 99.1％, 98.1％and 90.9％, 90.4％. There are 12 cysteine residue site in all strains, but strain CDV3 has a different site which isat 414(other 304), cysteine residue in all CDV is conservative. The strain CDVSJ owned 5 potentialN-glycosylation sites but 3 potential sites for Onderstepoort strain; there were 6 potential N-glycosylationsites in strain CDV3 and Convac, among which there was differentce of CDVSJ in 530aa～532aa;Hamamatsu owned 8 sites, other sites located 236Aa～238Aa、511aa～513aa and 530aa～532aa; aDanish dog owned 7sites, other sites located 236Aa～238Aa and 530aa～532aa. A potentialN-glycosylation site which located in the position of 511～513 amino acid was unique to yield isolate.This study indicated that this separation research was a low virulent strain, which may become avaccine strain. Meanwhile to the CDVSJ H gene sequence and the code amino acid comparison indicated ithas certain difference with other CDV strain, these differences were to study offer the molecular biologybasis for the distemper research immunity defeat and the theory basis for the canine distemper prevention.更多还原