H3N8亚型马流感病毒M1基因在昆虫细胞中的表达及活性分析

Expression and biological characterization of M1 protein of H3N8 equine influenza virus in insect cells

为建立H3N8亚型马流感血清学诊断方法,将H3N8亚型马流感病毒A/equine/Xinjiang/3/2007(H3N8)M1基因插入到杆状病毒转移载体pFastBac Dual中,构建重组转移质粒pFastBac Dual-M1;将pFastBac Dual-M1转化到DH10Bac感受态细胞中,通过蓝白斑筛选重组穿梭质粒rBD-M1;在脂质体转染试剂介导下将rBD-M1转染对数生长期的Sf9昆虫细胞,拯救重组杆状病毒rBV-M1;rBV-M1感染Sf9细胞后,通过Western-blotting和免疫组织化学染色进行蛋白表达分析。结果表明,获得了分子质量为27ku的特异性M1蛋白,而且该蛋白具有良好的免疫活性。M1蛋白的成功表达为以M1蛋白作为诊断抗原的研究奠定了基础。

To establish a serodiagnostic method for H3N8 subtype equine influenza,the M1 gene of H3N8 subtype equine influenza virus A/equine/Xinjiang/3/2007（H3N8） was subcloned into the transfer plasmid pFastBac Dual,then the recombinant transfer plasmid pFastBac Dual-M1 was transformed into DH10Bac competent cells.The recombinant shuttle plasmid rBD-M1 was obtained by screening of white plaque and then identified by PCR.After the rBD-M1 transfected into Sf9 cells,the recombinant baculovi-rus rBV-M1 was harvested.The expressed M1 protein was analysed by Western-blot and immunohistochemistry staining.A specific protein band of 27 ku was obtained and its immunoreactivity was confirmed.The recombinant M1 protein laid a foundation for establishing a diagnostic method.