Ⅰ型鸭甲型肝炎病毒VP3基因在昆虫细胞中的表达与鉴定

摘要

为在昆虫细胞中表达Ⅰ型鸭甲型肝炎病毒(Duck hepatitis A virus type-Ⅰ,DHAV-I)SH株的主要结构蛋白VP3,首先根据DHAV-I SH株VP3基因序列设计1对引物,RT-PCR方法扩增出VP3基因,克隆至杆状病毒表达载体pFastBac1,获得重组杆状病毒转移载体pFB-VP3,将其转化到DH10Bac感受态细胞中,经抗性和蓝白斑筛选,获得重组穿梭质粒rBacmid-VP3,在脂质体介导下转染昆虫细胞Sf9,获得重组杆状病毒rBac-VP3.间接免疫荧光结果显示重组蛋白获得了正确表达,能与鸭抗全病毒阳性血清发生特异性反应.Western blot结果显示表达的重组蛋白分子量约为27000.表明,DHAV-I SH株的主要结构蛋白VP3在昆虫细胞中获得了成功表达,为VP3结构蛋白的功能研究及相关亚单位疫苗的研制奠定了基础.%In order to express the main structural protein VP3 of duck hepatitis A virus type-Ⅰ ( DHAV-I) in insect cells, one pair of specific primers were designed according to the published genome sequences of DHAV-I to amplify VP3 genes by PCR, and the amplified fragment was cloned into baculovirus expression vector pFastBac1. The recombinant vector pFB-VP3 was transformed into DH10Bac Escherichia coli, and the positive recombinant bacmid rBacmid-VP3 was selected through resistance and blue-white plague screening. The recombinant bacmid rBacmid-VP3 was then transfected into the Sf9 insect cells by liposome. Indirect immunofluorescence analysis revealed that the recombinant protein was expressed, which could be recognized by the positive anti-virus serum, and the protein was about 27000 in molecular weight. The successful expression of protein VP3 of DHAV-I in insect cells lays a foundation of function study of VP3 protein.