抗牛传染性鼻气管炎病毒gD蛋白单克隆抗体的制备及鉴定

Preparation and identification of monoclonal antibody against gD protein of bovine infectious rhinotracheitis virus

目的制备牛传染性鼻气管炎病毒(infectious bovine rhinotracheities virus,IBRV)gD蛋白单克隆抗体,并鉴定其生物学特性。方法采用超速离心纯化的IBRV全病毒免疫BALB/c小鼠,取免疫后血清抗体呈阳性的小鼠脾细胞,通过融合剂PEG与骨髓瘤细胞SP2/O融合,采用间接免疫荧光法(IFA)筛选杂交瘤细胞,并制备腹水,分析单抗的生物学特性。结果筛选出1株稳定分泌抗IBRVgD蛋白单抗的杂交瘤细胞,命名为3C1。单抗的重链为IgG1亚类,轻链为kappa链;上清和腹水效价分别为1∶40和1∶12800;单抗可与IBRV及gD重组蛋白分别在相对分子质量71000和96000处发生特异性反应,可与感染IBRV的MDBK细胞发生特异性结合,产生荧光。结论成功制备了抗IBRVgD蛋白的单克隆抗体,为深入研究gD蛋白功能及IBR的临床诊断方法筛选等提供了数据材料。

Objective To prepare the monoclonal antibody(MAb)against gD protein of infectious bovine rhinotracheities virus(IBRV)and identify its biological characteristics. Methods BALB / c mice were immunized with the complete IBRV purified by ultracentrifugation. The spleen cells of immunized mice positive for serum antibody were fused with myeloma SP2 / 0 cells by using PEG. Hybridoma cells were screened by indirect immunofluorescence(IFA),with which the ascites was prepared,and the biological characteristics of MAb was analyzed. Results A hybridoma cell line stably secreting MAb against IBRV gD protein was screened and named as 3C1. The heavy chain of the MAb was of IgG1 subclass while the light chain was kappa chain. The titers of MAb in supernatant and ascite were 1 ∶ 40 and 1 ∶ 12 800 respectively. The MAb showed specific reaction bands,with relative molecular masses of 71 000 and 96 000,with IBRV and gD protein, respectively,as well as the specific binding to the MDBK cells infected with IBRV. Conclusion The MAb against IBRV gD protein was prepared successfully,which provided data for further study on the function of gD protein and clinical diagnosis of IBR.