不同毒株犬瘟热病毒结构基因的通用扩增及序列测定  

Universal Amplification and Sequencing of Structural Genes of Different Strains of Canine Distemper Virus

作　　者：卜研 史一珺 薛向红[1] 赵传芳[1] 陈杰 廉士珍[1] 朱言柱[1] 胡博[1] BU Yan;SHI Yi-jun;XUE Xiang-hong;ZHAO Chuan-fang;CHEN Jie;LIAN Shi-zhen;ZHU Yan-zhu;HU Bo(Institute of Special Wild Economic Animals and Plants,Chinese Academy of Agricultural Sciences,Changchun 130112,China;Yantai Animal Disease Prevention and Control Center,Yantai 264003,China)

机构地区：[1]中国农业科学院特产研究所,吉林长春130112 [2]烟台市动物疫病预防与控制中心,山东烟台264003

出　　处：《特产研究》2020年第2期9-14,共6页Special Wild Economic Animal and Plant Research

基　　金：“十三五”国家重点研发计划(2016YFD0501001)。

摘　　要：为了更准确、快速、高通量地追溯犬瘟热病毒流行毒株来源,揭示其进化规律,本研究分析比对了GenBank中已公布的23株犬瘟热病毒全基因组序列,针对6个结构基因的保守区设计了特异性通用引物,用于扩增犬瘟热病毒野毒株和疫苗株的6个结构基因。经过优化引物对的特异性、通用性、工作浓度及退火温度等,最终确定6对特异性通用扩增引物,对本室保存的3株疫苗毒CDV-3株、OR12株、CDV/R-20/8株,及2个流行毒HBF-1株和SD(14)07株的6个结构蛋白基因进行RT-PCR通用扩增。取5μL扩增产物进行1%琼脂糖凝胶电泳,结果显示,以上述5株犬瘟热病毒的cDNA为模板,用6对通用引物扩增,分别获得6个特异性条带,大小与预期相符。选取疫苗毒CDV-3株和野毒HBF-1株N、P、M、F、H和L基因连接至p EASY-Blunt载体上,进行序列测定。测序结果显示,与GenBank中已公布的序列同源性均大于99.0%,证实筛选的6对特异性引物能够实现对犬瘟热病毒野毒株和疫苗株的不同结构基因的准确扩增,并获得精准序列。这提高了犬瘟热病毒检测鉴定的效率和特异性,有利于人们快速、准确地获取犬瘟热病毒未知毒株结构基因的序列,追溯病原来源和分析其进化规律,为疫苗研制提供思路。To track the sources of canine distemper virus strains quickly,accurately,high throughput,and reveal its evolutionary rules.In this study,23 strains of published genomic sequences of canine distemper virus were compared and analyzed,and six paired,specific and universal primers targeting to the six structural genes of canine distemper virus were designed,respectively.The working concentrations and annealing temperatures of six paired-primers were optimized and achieved the specific and universal amplification of N,P,M,F,H and L genes for two wild type strains of CDV HBF-1 and SD(14)07,and three vaccine strains of CDV-3,OR12 and CDV/R-20/8.5 L PCR amplification products were used for 1%agarose gel electrophoresis.6 specific bands of expected sizes were obtained with six pairs of universal primers for the above five CDV strains,respectively.The amplifications of N,P,M,F,H and L genes of CDV vaccine strain CDV-3 and wild-type strain HBF-1 were selected and ligated to the pEASY-Blunt vector.The recombinant plasmids were sequenced and compared to the published sequences of CDV-3 and Hebei strains.Compared with the published sequences in GenBank,the homologies was more than99.0%,which confirmed that the 6 pairs of specific primers could achieve accurate amplifications of six structural genes for different canine distemper viruses(both wild-type and vaccine strain),and obtain accurate sequences.This method improved the efficiency and specificity of detection of canine distemper virus,and was conducive to the quickly and accurately obtaining the structural protein sequence of unknown canine distemper virus strains,tracing the source of CDV and analyzing its evolutionary rules,so as to provid ideas for vaccine development.

关 键 词：通用引物 犬瘟热病毒 结构基因扩增 

分 类 号：S852.655[农业科学—基础兽医学] Q786[农业科学—兽医学]