动物rna病毒反向遗传系统的研究和建立预防兽医学专业论文.docxVIP

建立RNA聚合酶I启动转录的重组质粒。然后把SVDV全长eDNA装配进该载体，在IBRS-2 建立RNA聚合酶I启动转录的重组质粒。然后把SVDV全长eDNA装配进该载体，在IBRS-2 细胞内和乳鼠体内成功拯救出了SVDV，第一次证明了聚合酶I系统能够高效拯救细胞质复制的 正链RNA病毒。并利用该拯救系统对FMDV进行了拯救，首次证明该聚合酶I系统能够转录出 至少长8．2 kb的转录本。在此基础上，构建含有外源性生物标记5819的SVDV HK／70全长cDNA 克隆。利用聚合酶I反向遗传拯救系统拯救出含有该标记的病毒。为制备含有基因标记疫苗和建 立鉴别诊断方法奠定一定的基础。该设计思路的实现，拓宽了高效病毒拯救的途径，为病毒反向 遗传学研究提供了更为高效和广泛应用的病毒拯救技术方法。 关键词：反向遗传学，病毒拯救系统，17 RNA聚合酶，RNA聚合酶I。SVDV，FMDV，CSFV Ⅱ AbstractThe Abstract The rcvelse genetics of RNA virus allows precise study of the mechanisms as antigenicity, virulence,pathogenesis,maturation and replication ofthe virus at the molecular level．In this study,加 vitro and加vivo transcription systems were developed． 1 Testing of biologicel properties,analyzing of bioinformaties of SVDV HK／70 strain and development in in vitro transcription system based on T7 RNA polymerase for the virus To develop／n vRro transcription system based on I7 RNA polymerase(T7 RNAP)for RNA virus, the swine vesicular disease virus HK／70 strain was selected硒a model virus of cytoplasmic positive-strand RNA virus in the present study．Firstly，the virus Was isolated and cultured i11 moase and IBRS-2 cells．Then，the biological properties such 50％tissue culture infecting dose(TODso)and mouse virl_Ilence,and SO on,were tested．The bioinformatics of tbe virus genome Was alSO删v∞d based complete genome sequence by some software． At last,the full·length eDNA clone ofswine vesicular disease virus HK／70 strain named pSVOKl2 Was constructed in order to stIldy the antigenicity,replication,maturation and pathogenicity of swine vesicular disease virus．／n v#ro transcription RNA from pSVOKl2 uansfected IBRS-2 cells and the recovered virus RNA was isolated and sequenced,then indirect hemagglutination test,indirect immunofluorescence assays，eleectron microscope test,50％tissue culture infecting dose(TCID蚰) assays and mouse virulence studies were used to study the antigenicity and virulence ofthe recovered virus．m resdt showed that the infectious clones has obtained and