蓝舌病病毒NS2蛋白单克隆抗体的制备及其应用

本研究旨在制备针对蓝舌病病毒(bluetongue virus,BTV)非结构蛋白NS2的单克隆抗体,进而用于C-ELISA鉴别BTV自然感染动物与BTV灭活疫苗免疫动物。通过原核表达BTV NS2蛋白的高保守多肽(1~228 aa)来制备抗原,并通过免疫BALB/c小鼠制备抗BTV NS2蛋白的单克隆抗体,结果,获得1株分泌单克隆抗体的小鼠杂交瘤细胞,命名为BTV-2A4。鉴定结果显示,该单克隆抗体的亚类/亚型为IgG1/κ;其抗原表位在NS2蛋白的91~138 aa区间;能特异性地与1~24型BTV的NS2蛋白反应;可应用于Western-blot、间接免疫荧光、间接ELISA及C-ELISA技术。C-ELISA应用的试验数据表明,BTV疫苗免疫动物及未被BTV感染动物的血清中检测不到NS2抗体,而BTV感染动物的血清中大多可被检出NS2抗体。

Preparation and application of monoclonal antibody to bluetongue virus NS2 protein

This study aimed to produce one of monoclonal antibody(m Ab)to NS2 protein of bluetongue virus(BTV),and made a C-ELISA kit based on this m Ab to distinguish the BTV infected animals and the BTV vaccine immunized animals.A NS2 antigen of 1-228 aa with high homology was prepared by prokaryotic expression and purification.Subsequently,BABL/c mice were immunized by the NS2 antigen to produce m Ab.Finally,we acquired a strain of mouse hybridoma(named as BTV-2A4)stably secreting m Ab to BTV NS2.The results of series experiments showed that:The m Ab is constructed by IgG1/κpeptides;The epitope is located in the region of 91-138 aa of NS2;The m Ab can specifically recognize the BTV NS2 of 1-24 serotypes;The m Ab can be used in Western-blot,indirect immunofluorescence assay,indirect ELISA and C-ELISA.The results of C-ELISA suggested that anti-NS2 IgG cannot be detected from the sera of the animals without BTV infection or only with vaccine immunization,but can be detected from the most sera of animals infected by BTV.