山羊痘病毒n1l基因缺失重组毒株的构建及其生物特性研究预防兽医学专业论文.docxVIP

用，阐明GTPV的发病机理奠定基础。 用，阐明GTPV的发病机理奠定基础。 关键词：山羊痘病毒同源重组基因缺失重组病毒 万方数据 Construction Construction of N 1 L Gene Deletion Recombinant Goatpox Virus and Research on its Biological Properties AB STRACT In the research，we constructed the transfer vector plasmid pLR-EGFP-gpt expressing reporter gene EGFP and screening gene gpt with GTPV antigen as a template and N 1 L gene flanking sequences as the homologous recombinant arms．Vero cells pre-infected with GTPV were subsequently transfected with resulting plasmid．Finally,through recombination，selection culture，purification and homologous identification，recombinant GTPV without NI L gene were obtained．While we constructed recombinant transfer vector pLR-EGFP-gpt-N 1 Lrev after the N 1 gene deletion by the reverse insertion of N 1 L gene in pLR-EGFP-gpt and screened the revertant virus GTPV N1 Lrev after co-transfection with GTPV．Genetic stability and biological characteristics show that GTPV／＼N 1 L could be genetic stably at least 1 0 generations with the growth characteristics of the parental strains in cell cultures，but the copies of GTPV／k N 1 L were 30 times of parental strains in the same titer．N I L may be one of the virulence factor of GTPV GTPV N 1 L gene fragment was amplified by PCR and cloned to eiakaryotic expression vector pcDNA3．O(一)．After the the acquired plasmid transfected into Hela cells，G4 1 8 selection medium was used to screen，and the 万方数据 cell cell strains expressing N 1 protein was obtained．Identification and analysis results of the cell line by RT-PCR and Western-blotting，The results show that the Hela cell lines stably expressing GTPV N 1 protein were established successfully． The research will lay a foundation for exploring the function of N 1 protein in the process of GTPV infection host cells． Key words：Goatpox virus；homologous recombination；gene deletion； recombinant virus 万方数据 目 目 录 弓I 言 ．．1 第一章文献综述 。3 1．1羊痘病毒的研究概况 ．3 1．1．1病原的分类及形态特征 ．3 1．1．2地理分布 一3 1．1．3宿主范围 ．．4 1．1．4传播方式 。5 1．1．5临床症状