【摘要】 从质粒pGEM-HA中扩增H5N1亚型禽流感病毒(Avian influenza virus,AIV)血凝素(Hemagglutinin,HA)基因,构建转移载体pFastBacHT-HA并与大肠杆菌(Escherichia coli)DH10Bac中的Bacmid质粒重组,构建重组转座质粒rBacmid-HA。在脂质体介导下将rBacmid-HA转染sf9昆虫细胞获得重组杆状病毒。在sf9昆虫细胞中表达HA蛋白,通过SDS-PAGE、Western blot和血细胞吸附试验鉴定重组蛋白。结果显示,重组HA蛋白分子量约为66kD,该蛋白与AIVH5亚型鸡血清发生特异性反应,与H7和H9亚型鸡血清不反应,转染重组杆状病毒后的细胞能吸附鸡的红血球,证明HA基因在sf9昆虫细胞中获得了正确表达,不仅具有亚型特异性,而且有良好的生物反应性。更多还原

【Abstract】 The hemagglutinin(HA)gene of H5N1 Avian influenza virus(AIV)was amplified from the plasmid pGEM-HA and inserted into Baculovirus transfer plasmid pFastBacHT to construct recombinant transfer vector pFastBacHT-HA.The pFastBacHT-HA was transformed into Escherichia coli DH10Bac competent cells,transposited with Baculovirus shuttle vector(Bacmid)and constructed recombinant transposition rBacmid-HA.After the rBacmid-HA transfected into sf9 cells,the recombinant Baculovirus was harvested.The expressed HA protein was analyzed by SDS-PAGE,Western blot and hemadsorption assay.The specific protein band of 66 kD was identified,which could only react with chicken sera against H5 subtype AIV and not react with the sera against H7 and H9 subtype,respectively.Hemadsorption assay showed that sf9 cells that infected rBacmid-HA Baculovirus could absorb chicken red blood cells.These results indicated that the HA protein was accurately expressed in sf9 cells,and displayed nice specificity to H5 subtype antisera and biologic activation.更多还原