犬瘟热病毒F蛋白单克隆抗体的制备及鉴定

[目的]获得犬瘟热病毒F蛋白特异性单克隆抗体。[方法]用纯化的大肠杆菌BL21表达的犬瘟热病毒重组F蛋白作为抗原,免疫BALB/c小鼠,采用传统的杂交瘤技术制备单克隆抗体。用ELISAI、FA、Western-blot、细胞中和试验鉴定各株单抗的生物学特性。[结果]获得7株稳定分泌犬瘟热F蛋白单克隆抗体的杂交瘤细胞株(2D6,3E10,3A2,5B7,5F6,6H8,9B6);单抗亚类鉴定结果分别为IgG2a,IgG1,IgG2b,IgG1,IgG1,IgG2a和IgG1;细胞中和试验初步证实2株单抗(5B7,9B6)具有中和犬瘟热病毒感染Vero细胞的作用,中和效价分别为1∶320和1∶640;经ELISA相加试验证实7株单抗的作用位点不同,其中2D6,5F6的作用位点非常相近。[结论]该研究成功制备了7株抗犬瘟热F蛋白特异性单抗,为进一步研究犬瘟热病毒F蛋白和临床诊断打下良好的基础。

Preparation and Identification of Monoclonal Antibodies against Canine Distemper F Protein

[Objective] The study aimed to obtain the special monoclonal antibodies against canine distemper F protein.[Method] BALB/c mice were immunized by using the immunogen of purified recombinant fusion protein of canine distemper virus(CDV) expressed in Escherichia coli BL21.The monoclonal antibodies were produced by using the traditional hybridoma technique,then the biology characteristic of each monoclonal antibody was identified by indirect enzyme-linked immunosorbent assay(ELISA),western-blot analysis and Immunofluorescence(IFA),westernblot and cell neutralization test.[Result] Seven cell strains of hybridoma(2D6,3E10,3A2,5B7,5F6,6H8 and 9B6) which secreted the anti-CDV F McAbs were obtained.The subtypes of monoclonal antibodies were identified as IgG2a,IgG1,IgG2b,IgG1,IgG1,IgG2a and IgG1.Neutralization test indicated that 5B7 and 9B6 had certain neutralizing ability to CDV infecting Vero cells,with the neutralizing titres of 1∶320 and 1∶640.The results of epitope analysis with addition ELISA revealed that 7 McAbs could recognize the different epitopes,in which,the epitopes of 2D6 and 5F6 were very close.[Conclusion] The CDV fusion protein McAbs were successfully prepared,which provide a good foundation for further research on the fusion protein and clinical diagnose.