表达鹅α干扰素基因重组禽痘病毒的构建及表达产物抑制鹅细小病毒活性的初步研究

Expression of goose interferon-α gene in recombinant fowlpox and detection for its activity against GPV

为研究表达鹅α干扰素(GoIFN-α)基因重组活载体疫苗对抑制病毒增殖活性,本实验从植物血凝素刺激的延边白鹅外周血淋巴细胞中提取总RNA,采用RT-PCR扩增gIFN-α基因,其大小为576 bp。将其与LacZ表达盒串联克隆于pSY681质粒中构建pSY681-IFN-α-LacZ转移重组质粒。采用脂质体将重组质粒转染于预感染禽痘病毒(FPV)017株的鸡胚成纤维细胞(CEF),通过蓝/白斑筛选获得重组禽痘病毒rFPV-IFN-α。采用间接免疫荧光和western blot方法对重组毒鉴定结果显示,GoIFN-α基因在CEF中获得表达,分子质量约为29 ku。表达的CoIFN-α对鹅细小病毒在鹅胚成纤维细胞中的复制具有抑制作用。本研究为进一步开展GoIFN-α基因重组FPV活载体疫苗的体内试验奠定了基础。

To construct the recombinant fowlpox virus （FPV） expressing goose interferon-α （GolFN-α） and evaluate its antiviral activity, the 576 bp GOIFN-α gene was amplified by RT-PCR from the total RNA of phytohemagglutinin （PHA） stimulated Yanbian white goose peripheral blood lymphocytes, which was ligated with LacZ cassette and clone into pSY681 vector to construct the recombinant transfer vector of pSY681-IFN-α-LacZ. The recombinant virus of rFPV-IFN-tx was generated by transfection of the transfer vector into the pre-infected CEF with FPV-017 and selected the blue virus plaque in present of X-gal. In additionally, the expression of GolFN-α from rFPV-IFN-α was identified by indirect immunofluorescent assay and western blot. Furthermore, the cytopathic effect inhibition tests showed that the GOIFN-α possessed antiviral activity evaluated by using gosling plague virus （GPV） in geese embryo fibroblast. These results laid the foundation for fiarther study of the recombinant viral vaccines expressing GolFN-α in vivo.