毛皮动物犬瘟热病毒分离鉴定及分子生物学特性研究

发布时间：2018-03-30 17:33

本文选题：CDV　切入点：分子流行病学调查　出处：《山东农业大学》2017年硕士论文

【摘要】：2015年6月至2016年6月,对山东省中东部地区送检的118份病死水貂、貉子和狐狸肺脏病料检测,水貂83份、貉子17份、狐狸18份。根据Genbank已发表犬瘟热病毒(Canine distemper virus,CDV)N基因高度保守区域设计引物,建立了其RT-PCR检测方法,阳性结果为71份、13份与13份,阳性率为85.54%、76.47%与72.22%。选取部分阳性病料经MDCK细胞分离,貂源分离获得4株CDV,貉源分离1株,狐源没有分离得到,5株分离株分别命名为CDV103、CDV114、CDV122、CDVDH2与CDV LC2株。接种细胞后部分样品呈现明显CPE亦有部分样品无明显CPE,经RT-PCR检测分析发现部分无CPE细胞冻融液检测结果呈阳性,表明CDV对MDCK细胞的适应性有所不同。根据Genbank已发表的CDV F、H与N全基因序列分别设计扩增F、H与N全基因的特异性引物,研究CDV分子生物学特性。采用RT-PCR扩增及琼脂凝胶回收、克隆,对克隆纯化获得的相关片段测序与分析。经DNAstar软件对分离株核苷酸与氨基酸序列分析,根据Genbank中已发表的犬瘟热毒株作参考株,构建遗传进化树分析。H基因核苷酸序列分析结果:CDV103、C DV114、CDV122、CDVDH2与CDVLC2株彼此同源性为90.7%-100.0%,CDVLC2与CDVDH2株的同源性最高100.0%,CDVLC2、CDVDH2株与CDV103、CDV114、CD V122株彼此间有相对较高同源性为90.7%-91.1%。H蛋白氨基酸序列潜在的N-联糖基化位点分析结果:CDV103、CDV114与CDV122株糖基化位点均为9个;第309-311位糖基化位点为野毒株所独有得,Asia-1型中部分毒株具有9个潜在N-联糖基化位点,CDVDH2与CDVLC2毒株糖基化位点位7个,这与Convac弱毒疫苗株H蛋白氨基酸序列的糖基化位点一致。H基因遗传进化树分析结果:CDV103、CDV114与CDV122株属于Asia-1型,与JN(08)(2009ChinaFJ810213fox)株有较近亲缘关系;CD VDH2与CDVLC2分离株属于America-1型,与Convac、Onderstepoort疫苗株关系较近。F基因核苷酸序列分析结果:CDV103、CDV114、CDV122、CDVDH2与CDVLC2株核苷酸序列同源性为90.3%-99.0%,CDV103、CDV114与CDV122株同Onderstepoort疫苗株同源性为90.4%-90.7%,CDVDH2、CDVLC2株与Onderstepoort疫苗株同源性最高为99.0%。Fsp推导氨基酸序列潜在N-联糖基化位点分析结果:CDV103、CDV114与CDV122株均含有2个潜在的N-联糖基化位点,位于第62位和108位,CDV103、CDV114与CDV122株糖基化位与Asia-1型毒株一致。F基因构建遗传进化树分析结果:5株CDV划分于2个基因型,CDV103、CDV114与CDV122株属于Asia-1型,与ZYL(2016ChinaKX499865fox)株存在较近亲缘关系;CDVDH2与CDVLC2株属于America-1型,同Onderstepoort疫苗株关系较近。N基因核苷酸序列分析结果:CDV103、CDV114、CDV122、CDVDH2与CDVLC2株彼此间同源性均高于99.5%,为99.5%-100.0%,CDV103与CDV114株相似性最高为100.0%,CDVLC2与CDVDH2株同源性最低为99.5%。N蛋白氨基酸序列存在较高同源性,5株分离株彼此同源性为99.0%-100.0%。与Onderstepoort弱毒疫苗株相似性较高,为98.3%-98.9%;N基因遗传进化树分析结果:5株分离株与Onderstepoort疫苗株存在较近亲缘关系,划分于America-1型。本研究对山东省中东部地区毛皮动物犬瘟热病毒流行病学调查,分离鉴定,主要基因测序,构建遗传进化发生树。本研究主要克服犬瘟热病毒分离困难的问题,总结犬瘟热病毒分离过程中的经验。为做好犬瘟热病毒防制和监测等相关工作提供了重要的分子流行病学资料。为犬瘟热病毒分子生物学提供了最新的调查数据。
[Abstract]:From June 2015 to June 2016, 118 copies of mink for inspection of Shandong Province in the eastern region, and the fox raccoon lung disease material detection, 83 copies of 17 copies of mink, raccoon, fox 18. According to the published Genbank of canine distemper virus (Canine distemper virus, CDV) N gene is highly conserved region primers were designed and established the RT-PCR detection methods, positive results for 71 copies, 13 copies and 13 copies, the positive rate was 85.54%, 76.47% and 72.22%. were part of a positive disease after MDCK cells were isolated, 4 strains of CDV isolated from mink source, 1 strains of raccoon dog, fox source is not isolated, 5 strains of isolates were named as CDV103 and CDV114. CDV122, CDVDH2 and CDV LC2 strains. After inoculation samples showed no obvious CPE is also part of the sample CPE, RT-PCR analysis found no CPE cell lysate was tested positive, showed that the adaptability of CDV on MDCK cells were different. According to the Genbank CDV: F, H and N were designed to amplify the gene sequence of F, specific primers H and N genes, characteristics of molecular biology of CDV. Amplified and cloned by RT-PCR agar gel recovery, purification, sequencing and analysis of related fragments obtained by DNAstar software. Cloning of isolates of nucleotide and amino acid sequence analysis according to the published Genbank, the canine distemper virus strains were reference strains, phylogenetic tree construction analysis of nucleotide sequences of.H gene: CDV103, C DV114, CDV122, CDVDH2 and CDVLC2 strains of each homology was 90.7%-100.0%, the highest homology of 100%, CDVLC2 and CDVDH2 CDVLC2 strain, CDVDH2 strain and CDV103, CDV114, CD among the V122 strains have a relatively high homology of amino acid sequence of 90.7%-91.1%.H protein N- potential glycosylation site analysis results: CDV103, CDV114 and CDV122 strains of glycosylation sites were 9; the 309-311 glycosylation sites Unique to wild strains of type Asia-1 strains have 9 potential glycosylation sites of N-, CDVDH2 and CDVLC2 strains of glycosylation site 7, which with Convac attenuated vaccine strain H protein amino acid sequence genetic glycosylation sites consistent.H gene phylogenetic analysis results: CDV103 and CDV114. CDV122 strains belonged to Asia-1 type, and JN (08) (2009ChinaFJ810213fox) strains have a close genetic relationship; CD VDH2 and CDVLC2 isolates belong to type America-1, Convac and Onderstepoort, the closer relationship between vaccine strain.F gene nucleotide sequence analysis results: CDV103, CDV114, CDV122, CDVDH2 and CDVLC2 strains of nucleotide sequence homology of 90.3%-99.0%. CDV103, CDV114 and CDV122 strains with Onderstepoort vaccine strain homology to 90.4%-90.7%, CDVDH2, CDVLC2 strain and Onderstepoort vaccine strain had the highest homology to the deduced amino acid sequence of 99.0%.Fsp N- potential glycosylation site analysis results: CDV103, CD V114 and CDV122 strains contained 2 potential N- linked glycosylation sites, located in sixty-second and 108, CDV103, construction of phylogenetic analysis results of glycosylation and the Asia-1 strain.F gene CDV114 and CDV122 strains, 5 CDV strains into 2 genotypes, CDV103, CDV114 and CDV122 strains belong to type Asia-1, and ZYL (2016ChinaKX499865fox) strains had a close genetic relationship; CDVDH2 and CDVLC2 belonged to America-1 type, with Onderstepoort vaccine strain is closely related to the.N gene nucleotide sequence analysis results: CDV103, CDV114, CDV122, CDVDH2 and CDVLC2 strains of each homology were higher than 99.5%, 99.5%-100.0%, CDV103 and CDV114 strain the highest is 100%, CDVLC2 and CDVDH2 were the lowest homology to the amino acid sequence of 99.5%.N protein has high homology, 5 isolates each homology was 99.0%-100.0%. and Onderstepoort attenuated vaccine strain of high similarity to 98.3%-98.9%; N Phylogenetic tree analysis results: 5 isolates and Onderstepoort vaccine strain has close relationship, divided in America-1. The research on the epidemiology of canine distemper virus fur animal in middle part of Shandong province mainly investigation, isolation and identification, gene sequencing, genetic construction of a phylogenetic tree. This research is mainly to overcome the difficulty in separation of canine distemper virus isolation of canine distemper virus, summarize the experience in the process. Provide important data for the molecular epidemiology of canine distemper virus prevention and monitoring and other related work. The molecular biology of canine distemper virus provides the latest survey data.

【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.65

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