草鱼Ⅲ型呼肠孤病毒NS66抗体制备及应用

背景]草鱼Ⅲ型呼肠孤病毒(grass carp reovirus,GCRV genotypeⅢ) 104株可导致典型性草鱼出血病,对其编码片段的分析有望为临床免疫学检测提供依据。目的]研究GCRV104株s6基因节段编码蛋白NS66的可能功能,制备良好的GCRV104株NS66蛋白多克隆抗体并分析其特异性。方法]PCR方法扩增GCRV104株s6基因片段,并克隆至表达载体pGEX-4T-3,转化到大肠杆菌BL21后用IPTG诱导表达,其产物经SDS-PAGE鉴定分析后,通过纯化获得目的蛋白。然后用纯化的pGEX-4T-3-NS66重组蛋白免疫小鼠,获得Anti pGEX-4T-3-NS66多克隆抗体,Western blot测定抗体效价,Western blotting和间接免疫荧光试验(indirect immunofluorescence assay,IFA)鉴定抗体特异性。结果]SDS-PAGE分析显示表达的重组蛋白约66 kD,大小与预期相符,主要存在于包涵体中;Western blotting测得制备的多克隆抗体效价大于1:50 000,Western blotting和IFA结果表明,制备的多克隆抗体能特异性识别GCRV104病毒。结论]GCRV104病毒编码的非结构蛋白NS66可能参与了复制和组装过程,形成病毒包涵体,这为建立GCRV104免疫诊断方法及研究GCRV编码的NS66蛋白的功能奠定了前期基础。

Polyclonal antibody preparation and application of grass carp reovirus genotype III nonstructural protein NS66

Background] Grass carp reovirus (GCRV) 104 strain can cause typical grass carp hemorrhagic disease, the analysis of its coding fragments is helpful to provide a basis for clinical immunological detection. Objective] To study the possible function of the NS66 protein encoded by the s6 gene segment of GCRV 104 strain, the highly specific polyclonal antibody against NS66 protein was prepared, and its specificity was identified. Methods] s6 gene segment amplified by PCR was cloned into the expression vector pGEX-4T-3, transformed into E. coli BL21 and induced by IPTG. After analysis and identification by SDS-PAGE, the target protein is obtained through purification. Mice were then immunized with purified recombinant protein to obtain polyclonal antibody. Titers were determined by Western blot, and antibody specificity was identified by Western blot and IFA (indirect immunofluorescence assay). Results] The recombinant protein analysed by SDS-PAGE was about 66 kD, the same size as expected. The target protein was mainly present in inclusion bodies. The polyclonal antibody titer prepared by Western blot was more than 1:50 000. Western blot and IFA showed that the prepared polyclonal antibody recognizes the 104 strain, and the polyclonal antibody recognizes a single band with high specificity. Conclusion] The study showed that the polyclonal antibody against NS66 protein can specifically recognize GCRV104 virus, which lays a foundation for the establishment of GCRV104 immunodiagnostic method and the study of GCRV-encoded NS66 protein.