【摘要】 为构建猫杯状病毒(FCV)2280株感染性克隆,本研究在FCV 2280株基因组5’末端和3’末端分别引入T7 RNA聚合酶启动子和丁型肝炎核酶(Hdv Rz)序列;并将FCV基因组中唯一的XbaⅠ限制性酶切位点序列进行同义突变作为遗传标记;将基因组分为3段分别进行RT-PCR扩增,通过酶切依次克隆于pOK-12载体中,构建FCV基因组全长cDNA重组质粒。以其为模板经体外转录制备其全长基因组RNA,并在5’末端加帽,转染CRFK细胞,24 h后出现典型的细胞病变。提取出现细胞病变的病毒液的总RNA制备cDNA模板,PCR扩增FCV遗传标记片段并进行XbaⅠ酶切鉴定分析,表明拯救的病毒为FCV重组病毒。而且拯救的病毒生长动力学与亲本病毒无明显差异。本实验为进一步研究FCV的生物学特性和致病机制奠定了基础。更多还原

【Abstract】 For establishment of reverse genetic system of the feline calicivirus(FCV), the genomic cDNA of the FCV 2280 strain was cloned into pOK-12 to construct infectious clone of pOK-FCV2280, in which the T7 RNA polymerase promoter and ribozyme sequence of hepatitis delta virus(Hdv Rz) were introduced into 5’ end and 3’ end of the FCV genomic cDNA,respectively, and a genetic marker was created by the synonymous mutation to disable the only one XbaⅠ cleavage site within the viral genome. Then the viral genomic RNAs with cap structure in 5’ end were prepared by in vitro transcription, and transfected into CRFK cells, which displayed typical cytopathic effect about 24 hrs post transfection. In addition, the recombinant FCV(r FCV)was rescued and identified by Xba Ⅰ digesting the viral cDNA fragment. Moreover, the replication kinetics of the r FCV was similar to the parental virus. The established FCV reverse genetic system provided a basis for further study on biological characteristics and pathogenic mechanism of FCV.更多还原