Ⅲ型登革病毒NS1单克隆抗体的制备及鉴定

Preparation and characterization of monoclonal antibody against DV3 nonstructural 1 protein

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IgG1。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。

Objective To prepare monoclonal antibodies against dengue serotype 3 virus （DV3） nonstruetural 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. Enzymelinked immunosorbent assay （ELISA）, immunofluoreseence assay （IFA） and Western blot analysis were ap- plied 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.