Ⅲ型登革病毒NS1单克隆抗体的制备及鉴定

目的 研制Ⅲ型登革病毒(DV3)非结构蛋白1(NS1)单克隆抗体,鉴定其血清型特异性.方法 以具有良好抗原性的重组DV3-NS1蛋白与DV3交替免疫Balb/c小鼠,取其脾细胞与小鼠骨髓瘤细胞融合,间接ELISA筛选阳性的杂交瘤细胞,并结合免疫荧光(IFA)和Weatern Blot对抗体的特异性进行鉴定.结果 经交替免疫法免疫Balb/c小鼠,共获得14株抗DV3-NS1单抗,其亚类测定1株为IgG2a,余为lgG1.其中3株特异性结合DV3及DV3-NS1蛋白,4株能同时结合4型登革病毒NS1蛋白,其余7株与其他3型登革病毒NS1蛋白存在交叉反应.结论 成功获得了针对DV3-NS1的特异性单抗及交叉性单抗,将为进一步研究登革病毒NS1蛋白的结构与功能及临床诊断试剂的研发奠定基础.

Preparation and characterization of monoclonai antibody against DV3 nonstructural 1 protein

Objective To prepare monoclonal antibodies against dengue serotype 3 virus(DV3) nonstructural 1 protein and to identify the specificity of antibodies.Methods Eight Balb/c mice were immunized by mixed immunity:recombinant DV3 nonstructural 1 protein of high antigenicity and inactive DV3 virus.Splenocytes of immunized mice were fused with myeloma cells NS-1 to produce hybridoma cell line,secreting anti-DV nonstructural 1 protein antibodies.Enzyme-linked immunosorbent assay(ELISA),immunofluorescence assay(IFA) and Western blot analysis were applied to identify specificity of antibodies.Results Fourteen strains of hybridoma cell lines steadily secreting antibodies of nonstructural 1 protein were obtained.Among them,one strain was identified as IgG2a isotype and others were all IgG1.Three out of fourteen strains were specific to DV3 and DV3-NS1 protein.Others had cross-reactivity with other three serotypes of DV-NS1 protein.Conclusion Monoclonal antibodies specific to DV3 nonstructural 1 protein with high activity and cross-reactive with other serotypes of DV-NS1 protein had been successfully established.These results can provide a potential value for structural and functional studies of DV-NS1 and early diagnosis of dengue virus infection.