狂犬病病毒磷蛋白在杆状病毒系统的表达及鉴定

EXPRESSION AND CHARACTERIZATION OF PHOSPHOPROTEIN OF RABIES VIRUS IN BACULOVIRUS

为了建立狂犬病毒磷蛋白(phosphoprotein,P)的体外表达系统,本研究将RT-PCR扩增的狂犬病病毒ERA株P蛋白基因克隆于杆状病毒转移载体pFastBacHTA,构建重组质粒pFastBacHTA-P,并转染昆虫细胞Sf9包装形成重组杆状病毒。SDS-PAGE分析显示重组质粒pFastBacHTA-P转染的Sf9细胞中出现了分子量约为42 kDa的蛋白条带,Western blot证实分子量约为42 kDa的蛋白能与抗His的单克隆抗体发生特异性反应,间接免疫荧光(indirect immunofluorescence assay,IFA)分析进一步显示Sf9细胞表达的P蛋白与抗P蛋白单克隆抗体能特异性结合。这些实验证明,狂犬病病毒P蛋白不仅在Sf9细胞中获得表达,而且具有良好的免疫反应性。狂犬病病毒P蛋白杆状病毒表达体系的建立,为蛋白结构的解析和诊断试剂的研制奠定了基础。

To construct an in vitro expression system of phosphoprotein of Rabies virus ERA strain,the phosphoprotein gene was amplified in RT-PCR and cloned into the baculovirus vector pFastBacHTA.The recombinant baculovirus expressing phosphoprotein was obtained by transfecting the resulting plasmid into Sf9 cells.The phosphoprotein expressed in Sf9 cells was detected to be a 42 kDa protein as determined in SDS-PAGE and Western blot using anti-His monoclonal antibody.Furthermore,the expressed phosphoprotein was recognized in immunofluorescence assay using monoclonal antibody against phosphoprotein of Rabies virus.These results confirmed that the phosphoprotein of Rabies virus was expressed in Sf9 cells and retained its immunoreactivity.The expression of phosphoprotein of Rabies virus in baculovirus expression system provided a tool for structural analysis and development of diagnostic reagent for Rabies virus.