摘要
为制备针对重组牛γ-干扰素(rBoIFN-γ)的单克隆抗体(mAb),以纯化的原核表达融合蛋白rHis-BoIFN-γ为免疫原,用纯化的rGST-BoIFN-γ为筛选抗原,应用淋巴细胞杂交瘤技术,研制抗rBoIFN-γ的mAb。结果获得13株稳定分泌抗BoIFN-γmAb的细胞株,命名为1C12、1F7、1G5、3E6、4D5、5E11、5G4、6F8、6G6、7E9、8D3、8F8和9G11。腹水的效价除单抗9G11外,其余效价都在1∶80 000以上;除5G4mAb亚类为IgG2b外,其余均为IgG1。Dot-ELISA结果表明,所得单抗只与融合蛋白rHis-BoIFN-γ和rGST-BoIFN-γ反应,而不与其它重组细胞因子和对照细菌反应,显示其良好的特异性。Western blot显示所获单抗能与相应的融合蛋白发生反应,出现特异性条带。同时所获单抗均能与牛IFN-γ标准品反应。13株单抗均为针对rBoIFN-γ的特异性mAb,为进一步研究rBoIFN-γmAb在牛的免疫调控、牛疫病的致病机制及其诊断中的应用奠定基础。
Seven week-old BALB/c mice were immunized with purified fusion protein rHis-BoIFN-γ that was obtained from a prokaryotic expression system.Splenocytes from immunized mice were fused with Sp2/0-Ag-14 myeloma cells.The supernatants of hybridoma clones were screened by indirect ELISA using purified rGST-BoIFN-γ.Thirteen hybridoma cell lines secreting MAbs against BoIFN-γ were obtained and assigned as 1C12,1F7,1G5,3E6,4D5,5E11,5G4,6F8,6G6,7E9,8D3,8F8 and 9G11.The immunoglobulin subclass of MAb 5G4 was IgG2b and all others were IgG1.The ascitic titers of these MAbs were 640 000,160 000,640 000,320 000,160 000,320 000,2 560 000,2 560 000,320 000,640 000,80 000,2 560 000 and 10 000,respectively.In Dot-ELISA,all MAbs specifically reacted with the immunogen and the screening antigen.Western blot analysis confirmed that all MAbs reacted with the corresponding recombinant proteins.The MAbs also reacted with the standard recombinant bovine IFN-γ with biological activity.The availability of these MAbs provides a basis for further research and clinical applications of BoIFN-γ in pathogenesis and diagnosis of bovine diseases.
出处
《中国动物传染病学报》
CAS
2009年第4期15-19,共5页
Chinese Journal of Animal Infectious Diseases
基金
973计划课题(2006CB504404)
国家科技重大专项(2008ZX10003-010)
江苏省科技攻关计划(BE2007340)
国家公益性行业科研专项(200903027)
关键词
重组牛γ-干扰素
原核表达
单克隆抗体
Recombinant bovine interferon-gamma
prokaryotic expression
monoclonal antibody
