小鼠肝炎病毒N蛋白单克隆抗体的制备及鉴定

[目的]制备并鉴定小鼠肝炎病毒(MHV)N蛋白的单克隆抗体(mAb).[方法]以Bac-to-Bac杆状病毒表达系统表达的重组N蛋白为免疫原,免疫BALB/c小鼠,应用常规杂交瘤技术将免疫小鼠脾细胞与Sp2/0骨髓瘤进行融合,用间接ELISA方法筛选分泌N蛋白mAb的杂交瘤细胞系.并用Western blot和IFA方法对所获得的mAb进行鉴定.[结果]共获得4株稳定分泌抗N蛋白mAb的杂交瘤细胞系,其亚类鉴定2株为IgG2a,1株为IgG2b,1株为IgG1,轻链均为k型.经鉴定4株mAb均能与重组N蛋白发生特异性反应.[结论]成功获得了特异性抗MHV N蛋白的mAb,为进一步研究N蛋白的结构功能及建立诊断学方法奠定了基础.

Preparation and identification of monoclonal antibodies against recombinant N protein of mouse hepatitis virus

AIM: To prepare and characterize the monoclonal antibodies(mAbs) against N protein of mouse hepatitis virus(MHV).METHODS: BALB/c mice were immunostimulated with the purified recombinant N protein expressed by Bac-to-Bac baculovirus expression system.Then the spleen cells of the immunized mice were fused with Sp2/0 myeloma cells by conventional hybridoma techniques.An indirect ELISA using recombinant N protein as antigen was established to screen antibody-producing hybridoma cell lines.Western blotting and IFA were applied to characterize the mAbs.RESULTS: We obtained four hybridoma cell lines secreting mAbs against recombinant N protein.The subclasses of two mAbs were IgG2a,one IgG2b and the other IgG1,and light chains were both Kappa.Western blotting showed that the mAbs specifically recognized certain antigenic epitope of N protein of MHV.CONCLUSION: mAbs against N protein of MHV with high activity and specificity were prepared successfully,which provides a potential basis for further researches on N protein function and diagnostic methods.