嵌合犬瘟热病毒rHBF-vacH株的构建及鉴定

Generation and Identification of a Chimeric Canine Distemper Virus rHBF-vacH Strain

为了明确我国犬瘟热病毒流行毒株的关键毒力基因及其致病机制,本研究构建了一株表达疫苗毒株H基因的嵌合犬瘟热病毒rHBF-vacH。犬瘟热病毒的囊膜蛋白H和F,决定着犬瘟热病毒宿主嗜性和致病性,也是产生免疫保护的关键抗原。因此,本研究以GenBank中公布的犬瘟热病毒疫苗毒株Onderstepoort序列(AF378705)为模板,利用Primer Premier 6.0设计特异性引物,扩增其H基因,将Onderstepoort的H基因定向克隆至pcDNA3.2-HBF-1中,筛选获得正确的全长重组质粒pcDNA3.2-HBF-vacH之后,通过病毒拯救,获得嵌合病毒rHBF-vacH,最后,通过免疫荧光、RT-PCR和电镜观察,初步鉴定嵌合病毒rHBF-vacH的基因组中携带有犬瘟热病毒疫苗毒Onderstepoort的H基因。本研究构建的嵌合病毒rHBF-vacH为国内犬瘟热病毒流行毒株H基因致病性研究提供了技术平台,有助于设计重组犬瘟热疫苗。

To clarify the key virulence gene and its pathogenic mechanism of the epidemic canine distemper virus in China,a chimeric canine distemper virus rHBF-vacH strain expressing the H gene of the vaccine strain was constructed.As known that the enveloped proteins H and F of canine distemper virus determine its host tropism and pathogenicity,and they are the key protective antigens.Therefore,the specific primers to amplify the H gene of Onderstepoort strain with the Primer Premier 6.0 soft based on the whole genome sequence as a template of Onderstepoort(AF378705)published in GenBank was designed.Then the H gene of Onderstepoort to the restriction sites in pcDNA3.2-HBF-1,a full-length infectious clone of an isolate canine distemper virus HBF-1 strain was cloned,which would obtain a correct recombinant plasmid pcDNA3.2-HBF-vacH.The chimeric virus rHBF-vacH was recovered by co-transfections of pcDNA3.2-HBF-vacH and three helper plasmids pcDNA3.2-N,pcDNA3.2-P and pcDNA3.2-L expressing N protein,P protein and L protein,respectively.The chimeric virus rHBF-vacH was preliminarily identified by immunofluorescence,RT-PCR and electron microscope observation.The results demonstrated that the chimeric virus rHBF-vacH reacted strongly with the canine distemper virus specific anti-N monoclonal antibodies,carried an H gene of Onderstepoort strain.It wasn’t its original wild type H gene,it was round-shapes with envelope membrane of about 200 nm size,the typical morphology of canine distemper virus.The construction and identification of chimeric virus rHBF-vacH in the present study provided a basis to further clarify the role of canine distemper virus H gene in pathogenicity of canine distemper virus epidemic strains in China,which would provided an evidence for the precise design of canine distemper vaccine in future.