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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。
To prepare monoclonal antibodies (mAbs) against recombinant bovine IFN-γ (rBoIFN-γ), the purified prokaryotic expression vector rHis-BoIFN-γ was used as the immunogen and purified rGST-BoIFN-γ as the screening antigen. The anti-rBoIFN-γ mAb was developed by using lymphocyte hybridoma technology. As a result, 13 cell lines stably secreting anti-BoIFN-γ mAb were obtained and named as 1C12, 1F7, 1G5, 3E6, 4D5, 5E11, 5G4, 6F8, 6G6, 7E9, 8D3, 8F8 and 9G11. The potency of ascites except McAb 9G11, the remaining potency in 1: 80 000 above; except 5G4 mAb subclass IgG2b, the rest are IgG1. The results of Dot-ELISA showed that the McAbs reacted only with the fusion proteins rHis-BoIFN-γ and rGST-BoIFN-γ, but did not react with other recombinant cytokines and control bacteria and showed good specificity. Western blot showed that the McAb reacted with the corresponding fusion protein, showing a specific band. At the same time the McAb can react with the bovine IFN-γ standard. All the 13 mAbs were specific mAb against rBoIFN-γ, which laid the foundation for further study on rBoIFN-γmAb in the immune regulation of cattle, pathogenic mechanism of bovine disease and its diagnosis.