DEN2重组E蛋白单克隆抗体的制备及其生物学活性

Preparation and Biological Activations of Monoclonal Antibodies Against DEN Envelope Proteins

[目的]研制登革病毒E蛋白特异性单克隆抗体,鉴定其各种生物学特性.[方法]用纯化的Pichia pastoris酵母表达的DEN2重组E蛋白作为抗原,免疫Balb/c小鼠,采用传统的细胞融合、有限稀释法克隆化杂交瘤细胞,制备稳定分泌McAbs的细胞株.采用硫酸铵沉淀法纯化腹水中的McAbs,用ELISA、IFA、Western Blot和空斑减数中和实验等鉴定McAbs的各种生物学活性.[结果]用ELISA、IFA检测证实5株杂交瘤细胞产生的McAbs与DEN2重组E蛋白和DEN全病毒均有较高的亲和力;Western Blot分析发现其中3株杂交瘤细胞(3G3,6G11,4E3)分泌的McAbs能与其相对分子质量Mr=(66～69)×103的DEN2重组E蛋白结合.空斑减数中和实验证实所获得的单克隆抗体具有中和登革病毒感染C6/36细胞的作用.[结论]成功地制备了5株抗登革病毒E蛋白特异性的McAbs,为进一步研究E蛋白的结构和功能及临床诊断试剂盒研发打下了基础.

To obtain monoclonal antibodies (McAbs) against DEN envelope proteins. In the first place, Balb/c mice were immunized using the antigens of purified recombinant E protein form DEN2 virus expressed in yeast Pichia pastoris. Then, the splenocytes of one of the immunized mice were fused with myeloma cells SP2/0 to produce hybridoma cell line, which could secrete anti-DEN envelope protein antibodies. Enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay (IFA), and Western blot analysis were applied to identify specificity of antibodies. Plaque reduction neutralization test (PRNT) were performed to study the protective effect of McAbs. Five established strains of hybridoma cell lines (3G3, 4E3, 6G11, 6G12, 9D9) could steadily secrete antibodies of E protein. These antibodies had characteristics of specific binding to DEN and E protein. PRNT indicated that the McAbs could reduce the plaque of DEN infecting C6/36 cells. [Conclusion] The monoclonal antibodies against DEN Envelope protein with high activity and specificity had been established successfully. These results could provide a potential value for vaccine researches and diagnosis of DEN.