Ⅱ型登革病毒非结构蛋白NS1血清型特异性单克隆抗体的制备与鉴定

Preparation and characterization of monoclonal antibodies specific to the non-structure 1 protein of dengue virus serotype 2

目的制备并鉴定Ⅱ型登革病毒非结构蛋白NS1(DV2-NS1)的血清型特异性单克隆抗体。方法以具有良好抗原性的重组DV2-NS1蛋白与灭活的Ⅱ型登革病毒(DV2)混合免疫Balb/c小鼠,取其脾细胞与小鼠骨髓瘤细胞融合,杂交瘤细胞经间接ELISA法、IFA法筛选,ELISA、IFA及WesternBlot对mAbs的类型及亚类、交叉实验及特异性等进行鉴定。结果免疫的Balb/c小鼠经多次融合筛选,共获得14株血清型特异性抗DV2-NS1蛋白的mAbs,其亚类测定两株为IgG2b,余均为IgG1。ELISA和免疫印迹显示这些mAbs与重组DV2-NS1蛋白和DV2抗原均特异性结合,IFA结果显示这14株单抗特异结合Ⅱ型登革病毒,与其它3型登革病毒无交叉。结论成功获得了特异性针对DV2-NS1蛋白的mAb,为进一步研究NS1蛋白的结构和功能以及研制早期诊断试剂奠定基础。

To prepare and characterize monoclonal antibodies specific to the non-structure 1 protein of dengue virus serotype 2, Balb/c mice were immunized by recombinant dengue virus non-structure 1 protein with high immunogenicity and inactive dengue virus serotype 2. Splenocytes of immunized mice were fused with myeloma cells NS1 to produce hybridoma cell lines Secreting anti-non-structure 1 protein antibodies of dengue virus serotype 2. Enzyme-linked immunosorbent assay （ELISA）, immnofluorescence assay （IFA） and Western blot analysis were applied to identify specificity of antibodies. Fourteen strains of hybridoma cell lines steadily secreting antibodies against non-structure 1 protein of dengue virus serotype 2 were obtained. Two of those antibodies were IgG2b in subtype, the others were IgG1. These antibodies had characteristics of specific binding to dengue virus serotype 2 and recombinant non-structure 1 protein of dengue virus serotype 2. All of those strains were specific to non-structure 1 protein of dengue virus serotype 2 without detectable cross-reactivity with other three serotypes of dengue virus with IFA. It is evident that monoclonal antibodies specific to non-structure 1 protein of dengue virus serotype 2 with high activity and specificity had been successfully established. These results established a foundation for early diagnosis of dengue virus infection.