Grass carp reovirus (GCRV) is a tentative member of the Aquareovirus genus in the family Reoviridae. The mature virion comprises 11 dsRNA genomes enclosed by two concentric icosahedral proteins shells that is comprise...Grass carp reovirus (GCRV) is a tentative member of the Aquareovirus genus in the family Reoviridae. The mature virion comprises 11 dsRNA genomes enclosed by two concentric icosahedral proteins shells that is comprised of five core proteins and two outer capsid proteins. The genome sequence and 3D structure demonstrate there is a higher level of sequence homology in structural proteins between GCRV and mammalian orthoreoviruses (MRV) compared to other members of the family. To understand the pathogenesis of GCRV infection, the outer capsid protein VP5, a homology of the μ1 protein of MRV, was expressed in E.coli. It was found that the recombinant VP5 was highly expressed, and the expressed His-tag fusion protein was involved in the formation of the inclusion body. Additionally, specific anti-VP5 serum was prepared from purified protein and western blot demonstrated that the expressed protein was able to bind immunologically to rabbit anti GCRV particle serum and the immunogenicity was determined by ELISA assay. Additional experiments in investigating the functional properties of VP5 will further elucidate the role of the GCRV outer capsid protein VP5 during entry into host cells, and its interaction among viral proteins and host cells during the infection process.展开更多
大菱鲆呼肠孤病毒(Scophthalmus maximus reovirus,SMRe V)属于呼肠孤病毒科水生呼肠孤病毒属水生呼肠孤病毒A型(AQRV-A),其病毒粒子具有双层衣壳,外衣壳由VP5和VP7蛋白构成。从SMRe V基因组中克隆出表达外衣壳蛋白VP5的全长基因vp5(2 0...大菱鲆呼肠孤病毒(Scophthalmus maximus reovirus,SMRe V)属于呼肠孤病毒科水生呼肠孤病毒属水生呼肠孤病毒A型(AQRV-A),其病毒粒子具有双层衣壳,外衣壳由VP5和VP7蛋白构成。从SMRe V基因组中克隆出表达外衣壳蛋白VP5的全长基因vp5(2 057 bp),构建包含该基因全长的原核表达质粒p ET32a-vp5,转化E.coli表达菌BL21(DE3)。经诱导获得的融合蛋白以包涵体的形式表达,大小约为88 k D。将纯化的融合蛋白免疫小鼠,制备出抗SMRe V VP5的多抗血清。经Western blot杂交分析显示,该抗体制备成功,且能识别SMRe V的VP5蛋白,大小约为69 k D。进一步经间接免疫荧光分析显示,VP5呈颗粒状分布在宿主CIK细胞质中。展开更多
基金National Basic Research Program ofChina (973 Program) (2009CB118701)National NaturalScientific Foundation of China (30671615, 30871940)+1 种基金Innovation Project of the Chinese Academy of Sciences(KSCX2-YW-N-021)Science and Technology Foundation of Zhejiang Province (2007C22052)
文摘Grass carp reovirus (GCRV) is a tentative member of the Aquareovirus genus in the family Reoviridae. The mature virion comprises 11 dsRNA genomes enclosed by two concentric icosahedral proteins shells that is comprised of five core proteins and two outer capsid proteins. The genome sequence and 3D structure demonstrate there is a higher level of sequence homology in structural proteins between GCRV and mammalian orthoreoviruses (MRV) compared to other members of the family. To understand the pathogenesis of GCRV infection, the outer capsid protein VP5, a homology of the μ1 protein of MRV, was expressed in E.coli. It was found that the recombinant VP5 was highly expressed, and the expressed His-tag fusion protein was involved in the formation of the inclusion body. Additionally, specific anti-VP5 serum was prepared from purified protein and western blot demonstrated that the expressed protein was able to bind immunologically to rabbit anti GCRV particle serum and the immunogenicity was determined by ELISA assay. Additional experiments in investigating the functional properties of VP5 will further elucidate the role of the GCRV outer capsid protein VP5 during entry into host cells, and its interaction among viral proteins and host cells during the infection process.
文摘大菱鲆呼肠孤病毒(Scophthalmus maximus reovirus,SMRe V)属于呼肠孤病毒科水生呼肠孤病毒属水生呼肠孤病毒A型(AQRV-A),其病毒粒子具有双层衣壳,外衣壳由VP5和VP7蛋白构成。从SMRe V基因组中克隆出表达外衣壳蛋白VP5的全长基因vp5(2 057 bp),构建包含该基因全长的原核表达质粒p ET32a-vp5,转化E.coli表达菌BL21(DE3)。经诱导获得的融合蛋白以包涵体的形式表达,大小约为88 k D。将纯化的融合蛋白免疫小鼠,制备出抗SMRe V VP5的多抗血清。经Western blot杂交分析显示,该抗体制备成功,且能识别SMRe V的VP5蛋白,大小约为69 k D。进一步经间接免疫荧光分析显示,VP5呈颗粒状分布在宿主CIK细胞质中。
