抗鸡γ-干扰素单克隆抗体的研制及初步鉴定

目的:制备抗鸡γ-干扰素单克隆抗体(mAb)。方法:应用淋巴细胞杂交瘤技术,以重组菌BL21(DE3)(pET-ChIFN-γ)表达产物的包涵体作为抗原免疫BALB/c鼠,以纯化的GST-ChIFN-γ作为检测抗原,制备抗鸡γ-干扰素mAb;采用ELISA、Dot-ELISA和Westernblot鉴定mAb的特异性。结果:获得2株可稳定分泌抗鸡γ-干扰素mAb的杂交瘤细胞株1G5、5E3,其Ig亚类均为IgG2a,腹水mAb1G5、5E3的ELISA效价分别为1∶1600000,1∶120000。在Dot-ELISA试验中,这2株mAb均只与表达His-ChIFN-γ及GST-ChIFN-γ的重组大肠杆菌反应,与未表达这2种IFN-γ的其他8种菌株均不发生反应。在蛋白质印迹试验中,2株mAb均能与融合蛋白GST-ChIFN-γ、His-ChIFN-γ发生反应,出现特异性条带。结果表明,mAb1G5、5E3是针对鸡γ-干扰素的特异性mAb。结论:成功地制备抗鸡γ-干扰素的mAb,它们在免疫检测、免疫细胞功能分析和免疫调节研究等方面有应用价值。

Preparation and characterization of monoclonal antibodies against chicken interferon-γ

AIM: To prepare monoclonal antibodies (mAb) against chicken interferon-gamma (ChIFN-gamma). METHODS: By using lymphocyte hybridoma technique, the inclusion body of the recombined bacteria, BL21(DE3) (pET-ChIFN-gamma), was harvested and used to immunize BALB/c mice. With the purified GST-ChIFN-gamma as detecting antigen, mAbs against ChIFN-gamma were prepared, and positive hybridoma clones were screened by indirect ELISA. The specificity of the mAb was characterized by indirect ELISA, Dot-ELISA and Western blot. RESULTS: Two hybridoma cell lines secreting mAbs against ChIFN-gamma named 1G5, 5E3 were obtained. The immunoglobulin subclasses of both 2 mAbs were IgG2a, and the ELISA titers of 2 mAbs ascitic fluids were 1:160,000, 1:12,000 respectively. In Dot-ELISA test, the 2 mAbs could only react with BL21 (DE3) (pET-ChIFN-gamma), BL21 (pGEX-6P-1-ChIFN-gamma), which expressed His-ChIFN-gamma, GST-ChIFN-gamma, respectively. Western blot analysis confirmed that the 2 mAbs could only react with GST-ChIFN-gamma and His-ChIFN-gamma proteins. CONCLUSION: Two mAbs specific to the protein of chicken interferon gamma are obtained, which may have important application value in further studies on immune detection, the functions of immune cells and immune regulation.