Ⅰ型鸭甲型肝炎病毒VP0基因在杆状病毒表达系统中的表达

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株的结构蛋白VP0,首先根据DHAV-Ⅰ SH株VP0基因序列设计一对引物,RT-PCR方法扩增出VP0基因,亚克隆至杆状病毒转移载体pFast Bac1,获得重组质粒p FB-VP0,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-VP0,在脂质体介导下转染昆虫细胞Sf9,获得重组杆状病毒rBac-VP0。Western-blot结果显示,表达的重组蛋白相对分子量约为29 k D,间接免疫荧光结果显示重组蛋白能与鸭抗全病毒阳性血清发生特异性反应。研究结果表明,DHAV-Ⅰ SH株的结构蛋白VP0在昆虫细胞中获得了成功表达,为VP0结构蛋白的功能研究和基因工程疫苗的研制奠定了基础。

Expression of VP0 genes of duck hepatitis A virus type-Ⅰ in insect cells

In order to express the structural protein VP0 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 VP0 genes by PCR,the amplified fragment was cloned into Baculovirus expression vector pFastBac1.The recombinant vector pFB-VP0 was transformed into DH10Bac E.coli,and the positive recombinant bacmid rBacmid-VP0 was screened according to the resistant and the blue-white plague screening.The recombinant bacmid rBacmid-VP0 was transfected into the Sf9 insect cells by liposome.Once the cytopathic effect was found,the rBac-VP0 was aquired.The result of Western blot showed that the molecular weight of the recombinant proteins was about 29 kD.Indirect immunofluorescence analysis showed that the recombinant proteins could be recognized by the positive anti-virus serum.These results suggest that the structural proteins VP0 of DHAV-Ⅰ have been expressed successfully in insect cells,which can lay the foundation of function study on VP0 protein.