牛传染性鼻气管炎病毒gC蛋白单克隆抗体的制备与初步应用

Development and Preliminary Application of Monoclonal Antibody against IBRV gC and Identification of Its Biological Characteristic

为制备牛传染性鼻气管炎病毒(IBRV)gC蛋白的特异性单克隆抗体,并分析其免疫学特性,以原核表达并纯化的重组gC蛋白为免疫原注射免疫BALB/c小鼠,取其脾细胞与骨髓瘤细胞融合。建立间接ELISA方法筛选分泌gC蛋白单克隆抗体的杂交瘤细胞,获得1株能稳定分泌gC蛋白单克隆抗体的杂交瘤细胞株,命名为3D6。3D6的亚类为IgG1型,轻链为κ链;上清、腹水抗体效价分别为6.4×103、1.28×105;Western blot和间接免疫荧光试验表明该单克隆抗体能识别牛gC蛋白。利用3D6细胞株分泌的单克隆抗体建立了检测IBRV的双抗体夹心ELISA方法,结果证明,该方法特异性和敏感性良好,为快速诊断IBRV和研究gC蛋白功能奠定了基础。

The objective of this study was to prepare monoclonal antibody against glycoprotein C （gC） protein of infectious bovine rhinotracheitis virus （IBRV） and to analyze its biological char- acteristic. BALB/c mice were immunized with purified recombinant gC protein expressed by E. coli and the spleen cells of immunized mouse were fused with SP2/0 myeloma cells. An indirect ELISA using the purified gC protein was developed to screen positive antibody-producing cells. A hybridoma stably secreting MAb designated as 3D6 was obtained against gC protein. The mono- clonal antibody belongs to IgG1, and the light chain was κ. The titers of supernatant and ascites were 6.4× 10^3 and 1.28× 10^4 as detected by ELISA, respectively. The result of western blot as- say and IFA suggested that the MAb could recognize IBRV. A sandwich ELISA was established by using 3D6 secreted monoclonal antibody. The results indicated that the ELISA possessed good specificity and higher sensitivity. The monoclonal antibody could be used to diagnose IBRV and {urther research gC protein function.