【作者】 简中友；

【导师】 王全凯；

【作者基本信息】 吉林农业大学 ， 预防兽医学， 2008， 博士

【摘要】 犬瘟热（Canine distemper CD）系由副粘病毒科麻疹病毒属的犬瘟热病毒引起犬科、鼬科等多种食肉动物的一种急性、热性、高度接触性的传染病，分布范围广，易感动物种类多，发病率和死亡率高。自发现以来，犬瘟热一直未停止对动物健康的威胁。截至目前，世界各国凡是有犬科动物生存的地区都有本病发生，即使美、英、法、澳大利亚、新西兰、日本等动物卫生水平较高的发达国也不例外，我国自1968年首次在黑龙江一野生动物饲养场发现犬瘟热，现已蔓延到吉林、辽宁、江苏、四川、山东、广西等二十几个省区。犬瘟热的流行与传播严重地影响了经济动物养殖、野生动物保护和动物贸易的健康发展。随着CDV对动物流行因素的不断适应，其自然感染宿主范围在不断扩大，加之CDV变异株的出现，犬瘟热的发病率和致死率仍居高不下，流行趋势由散发向群发发展，临床症状越来越复杂，其危害也越来越大。特别是国宝大熊猫等珍稀动物及猕猴等灵长类动物的CDV感染，使CDV的致病地位日益突出。此外，最新研究证实CDV在体外可感染人的前体破骨细胞，在破骨细胞内增殖，表明CDV的自然宿主可能已扩展到了人类，犬瘟热可能是犬传染给人的第二个病毒性传染病。鉴于上述因素，国际动物卫生组织（OIE）始终将其作为重点关注的疫病之一，我国也在CD的诊断、防治等领域进行大量的研究。本研究由国家质检总局资助，在口岸动物检疫特种经济动物重点试验完成，旨在收集关于CD疫情信息、跟踪研究进展、开发诊断技术、分析流行趋势，为进一步研究防治措施、完善标准体系奠定基础，主要包括以下内容：1．狐源犬瘟热病毒的分离与鉴定本研究用MDCK细胞从具有疑似犬瘟热症状的病狐血液中分离到一株病毒，该病毒在MDCK细胞上生长良好，并能形成典型而规律的CPE。经电镜观察、理化特性、滴度测定、动物回归试验、RT-PCR和荧光定量RT-PCR鉴定，证实分离到的病毒为一株犬瘟热病毒强毒株，并命名为CDV-FOX-TA。该病毒的分离与鉴定的成功，为进一步开展狐狸犬瘟热的综合性防制和诊断技术研究奠定了基础，具有重要的理论价值和实践意义。2．根据Genbank上发表的犬瘟热病毒（CDV）N基因核酸序列，设计、合成一对CDV N基因特异性引物，并在上下游引物分别引入酶切位点Kpn I／XhoI，采用RT-PCR扩增出狐源CDV泰安分离株（CDV-FOX-TA）的N基因片段，纯化扩增的N基因后，将其克隆入pMD18-T载体。进行测序分析，证实CDV-FOX-TA N基因含有1个ORF，全长1572bp，共编码523个氨基酸，N蛋白含有高度保守Tyr-Pro-Ala-Leu-Gly-Leu-His-Glu-Phe九肽序列，是T细胞表位，可致敏靶细胞。CDV-FOX-TA N基因与CDV疫苗株Ondersteport、Convac的N基因的同源性分别为96.0％和95.9％，与CDV野毒株N基因的同源性在98.4％-98.9％之间。对CDV N基因进行系统发生分析，结果发现CDV-FOX-TA与CDV强毒株的亲缘关系较为密切。3．根据GenBank发表的犬瘟热病毒N基因全序列，设计合成了1对特异扩增CDV N基因的引物。以山东泰安分离的CDV-FOX-TA株细胞毒中提取病毒RNA来制模板，利用RT-PCR扩增出了1.6kb的N基因，将其克隆到pIREShyg载体上，构建了pIRES-N真核表达载体。然后通过脂质体转染CHO-K1细胞，用潮霉素筛选得到阳性克隆。间接免疫荧光实验（IFA）鉴定N基因在CHO细胞中的表达，RT-PCR方法则从转录水平证实N基因在CHO-K1细胞中的表达。CHO／CDV-N细胞株的成功构建，为进一步研究犬瘟热血清学检测技术和基因疫苗奠定了基础。4．RT-PCR技术扩增出犬瘟热病毒（CDV）核衣壳（N）蛋白基因的高度保守序列后，将其TA克隆至pMD18-T载体中，再将测序正确的N基因的目的片段亚克隆到原核表达载体pET24b中6×His Tag编码基因的上游，并将该重组质粒转化大肠杆菌Rosetta 2（DE3）株，经IPTG诱导，N基因融合蛋白获得了高效表达。SDS-PAGE分析和Western blot分析的结果显示，表达产物的分子质量为15 kD，与CDV标准阳性血清呈阳性反应，间接ELISA结果也表明重组表达产物具有良好的抗原性，能够有效区分CDV标准阳性与阴性血清，表明大肠杆菌表达的CDV N蛋白在免疫原性上具有与天然N蛋白一定的相似性，可作为诊断用抗原，为下一步建立检测CDV的间接ELISA试剂盒奠定了基础。5将构建好的含重组质粒且能正确表达N蛋白的Rosetta 2（DE3）菌株中，在最佳诱导条件下获得重组N蛋白。随后用镍亲和层析方法对表达产物进行纯化，对纯化效果及纯化产物的特异性分别用SDS-PAGE电泳及Western-blot试验检测。在此基础上，以纯化的重组N蛋白作为包被抗原，对各种条件进行优化（如抗原的包被，作用时间），确定了判定标准，建立了检测CDV抗体的间接ELISA方法。用此方法检测了270份血清样品，并与法国Synbiotics公司ELISA试剂盒检测结果相比较，符合率达92.4％。6．实验选择CDV的M基因保守序列作为引物和探针并对CDV RT-PCR和Taq Man荧光定量RT-PCR的反应体系和反应条件进行了优化，建立了CDVTaq Man荧光定量RT-PCR检测cdv技术。利用检测系列稀释的标准阳性样品建立标准曲线，可对待检样品中病毒含量定量，精确度可达0.01 TCID₅₀／ml，敏感性比常规的RT-PCR的方法高100倍；对Rabies virus、CPV、CAV-1和CAV-2进行检测不产生扩增，具有高度特异性。CDV TaqMan荧光定量RT-PCR方法在检测CDV整个过程中从RNA提取到出现检测结果只需3小时，检测时间大大缩短。本法既可对血液、扁桃体等体内样品中检测病毒，又可对尿液、结膜拭子、粪拭子等体外样品进行检测，样品收集不受限制，微量样品也可进行检测，而且本法一次可检测多个样品，检测通量高。应用结果表明，TaqMan荧光定量RT-PCR与RT-PCR和电镜观察的符合率高，具有准确定量、灵敏度高和特异性强、降低污染以及省时省力等优点，且提取RNA后完全是自动的，大大减少手工操作、降低实验室污染机会，实用性较强。更多还原

【Abstract】 Canine distemper(CD) is a contagious, incurable, often fatal, multi-systemic viraldisease that affects the respiratory, gastrointestinal, and central nervous systems ofvarious carnivorous animals such as Canidae and Mustelidae. Canine distemper iscaused by the canine distemper virus (CDV), a kind of morbillivirus, of theparamyxovirus family.Canine distemper was treated animal since it was discovered. It occursworldwide even in countries with high animal health level including the USA, theEU, Australia, New Zealand and Japan. It was first described in a wildlife farm in HeLongjiang province, China in 1968 and is now widespread in more than 20 provincesand autonomous regions such as Jilin, Liaoning, Jiangsu, Sichuan, Shandong,Guangxi provinces etc.Canine distemper once was the leading cause of death in unvaccinated puppiesand impacted on the development of animal trade, wildlife protection and Economicanimal farming. With the varying CDV and widowing host spectrum, CD now tendsto present with more complex symptom and more influence on animal health.Especially it can affect endangered species such as panda and Primates. More andmore attention is being paid to CD.Moreover most recent research showed that CDV could infect the humanprecursor osteoclast and propagate in it. It implies that CDV nature host maybe haveincluded human being, and men possibly be the second human virus disease derivedfrom canine.As mentioned above, CD was put on the list of notifiable disease and muchresearch on CD in diagnostic and prevention field has been done in China. Thisproject, funded by AQSIQ, was finished in the key laboratory of Economic AnimalDisease investigation, targeted collecting information, following up the advance,developing testing methods, analysis epidemic trend of CD, and laid the foundationfor further investigation on prevention measures and integraled standard system. Thisstudy includes the followings:1. Isolation and Identification of Canine Distemper Virus from FoxA virus strain, named CDV-FOX-TA, was isolated from the blood of fox, thisvirus can grow well and produce stabilization cytopathic effects in MDCK cells. Itwas demonstrated to be canine distemper viruses by a series of systematic identification such as electron microscope observe morphology physicochemically,animals infection test, RT-PCR and real time RT-PCR.2. Cloning and Sequence Analysis of the N Gene of Canine Distemper VirusTai’an Isolate in a FoxOne pair of primers was designed according to the N genes of canine distempervirus strains reported in Genebank. The N protein gene of CDV-FOX-TA wasamplified with the primers by RT-PCR. The PCR product was cloned into pMD 18-T.The positive recombinant was identified, sequenced and analyzed. As a result, thelarge open reading frame of the N gene of CDV-FOX-TA included 1572bp, whichencoded 523 amino acids. The nucleotide homology of the N genes between CDV-FOX-TA and CDV Ondertepoort strain was 96.0%, and it was 95.9% between CDV-FOX-TA and CDV Convac strain. However, the nucleotide homologies of the Ngenes of CDV-FOX-TA and the CDV wild strains, was from 98.4% to 98.9%. A ninepeptidessequence in the N protein of CDV-FOX-TA was Tyr-Pro-Ala-Leu-Gly-Leu-His-Glu-Phe, which was T lymphocyte defined antigen and senstized target cells. Onthe basis of phylogenetic analysis, it implied that CDV-FOX-TA and the CDV wildstrains had the same ancestor.3. Eukaryotic Expression and Identification of Nucleocapsifl Protein N Geneof Canine Distemper VirusA pair of primers, based on the published sequence of N gene of CDV, wasdesigned, synthesized and can amplify CDV N gene specifically. The N gene’s cDNAfragment of about 1.6kb obtained by RT-PCR from FOX TA strain of CDV wascloned into pIREShyg Vector, and then the constructed Eukaryotic Expression Vectornamed as pIRES-N was transfected into CHO-K1 cells with liposome. Positive cellswere selected with hygromycine. The N gene protein expressed in the CHO cells wasidentified by IFA. RT-PCR also showed that N gene was expressed in CHO cells attranscription stage. The success construction of CHO/CDV-N cell strain providedevidence for developing CDV serum detection methods and gene vaccine.4. Prokaryotic Expression and Identification of Nucleocapsid Gene ofCanine Distemper Virus CDV-FOX-TA strain from FoxThe high conservative sequence of Canine Distemper Virus (CDV) N gene wasamplified with RT-PCR, cloned into pMD18-T vector, and then constructed intoupstream site of His Tag coding sequence in prokaryotic expression vector pET24b.The N fusion protein was highly expressed when the recombinant plasmid was transformed E coli Rosetta 2(DE3) strain and induced with IPTG. SDS-PAGE andWestern blot showed the 15kD expressed products reacted with standard positiveserum against CDV. An indirect ELISA demonstrated recombinant expressed proteinhad good antigencity and can distinguish effectively anti-CDV positive serum andnegative serum. The results showed expressed CDV N protein had similarity ofantigencity with natural N protein, and can be used as diagnosis antigen. It providedevidence for establishment of an indirect ELISA method for CDV detection.5 Establishment and Application of Recombinant N Protein Based ELISAfor Detection of Antibody against CDVThe recombinant plasmid pET-N was transformed into E. coli Rosetta 2 (DE3)host cell and the expression products-recombinant nucleocapsid protein of CDV wasobtained under the optimized condition of host cell cultivation and IPTG induction.Subsequently, the expression product was purified by the means of his-bind resinprotein purification procedure. Following SDS-PAGE and Western-blot were used todetect the purification effect and the specificity of purified recombinant nucleocapsidprotein of CDV. On this basis, the purified N protein was used to be coated on thewell of 96-well plate, each following step was optimized, such as coatingconcentration of recombinant nucleocapsid protein, sample diluent, chromogen (TMB)and stop solution and its concentration. As a result, confirmed the determinantstandard and an indirect ELISA were constructed to detect antibody against CDV.About 270 serum samples were detected by this method and Synbiotics ELISA kit,respectively. The agreement ratio between the two methods is 92.4%6. Establishment and Preliminary Application of a TaqMan-based Real-timeRT-PCR method for Detection of Canine Distemper VirusTo establish a TaqMan-based real-time RT-PCR method for detection of caninedistemper virus, the pair of palmers and the TaqMan-based probe was designedaccording to the M protein genes of canine distemper virus strains. The reactiveconditions were optimized to improve the sensitivity and specificity of the method.The specificity and sensitivity of the method were high. Canine distemper virus in thethirty-eight samples had been tested by the TaqMan-based real-time RT-PCR, thecommon RT-PCR and electron microscopy. As a result, the sensitivity of theTaqMan-based real-time RT-PCR was higher than the common RT-PCR and electronmicroscopy. It implied that the method may be used in clinical diagnosis, epizooticstudy and laboratory research.更多还原