禽流感病毒GD/1/96株M基因在sf9昆虫细胞中的表达

Expression of avian influenza virus M gene of the isolate GD/1/96 in insect sf9 cells

从pUCM 1质粒中亚克隆禽流感病毒M基因至转移载体 pFastBac1质粒 ,PCR鉴定后 ,转化DH10 BAC感受态细胞。提取重组杆粒rBacM ,以M 13为通用引物作PCR分析 ,证明M基因已转入其中。用CellFectin试剂包裹rBacM杆粒转染sf9细胞 ,96h收取感染细胞。细胞表达产物裂解后作SDS PAGE蛋白电泳和Westernblot分析 ,证明M基因在昆虫细胞中成功表达且具有与天然M 1蛋白相似的活性。琼脂扩散试验结果进一步证明 ,重组M 1蛋白可作为诊断抗原。

Matrix encoding cDNA of avian influenza virus (AIV) GD/1/96 strain was subcloned into pFastBac1 vector from pUCM1plasmid. The presentation of the M1 cDNA in recombinant plasmid was confirmed by PCR analysis. Recombinant bacmid (rBacM) was obtained by transform plasmid pBMinto DH10baccells. After confirmed by PCR, the rBacM was trans infected into sf9 cells packed with CellFECTIN regent. The expression of M gene in sf9 cells was determined by SDS PAGE and Western blotting. The results showed that the expressed product has a molecular mass of approximate 27 ku, accounting for 8.9% approximately in whole cellar proteins. The AGP test showed that recombinant M1 protein has potentialof diagnosis antigen.