鸡毒支原体丙酮酸脱氢酶E1β亚基单克隆抗体的制备和鉴定

Preparation and identification of monoclonal antibodies against pyruvate dehydrogenase subunit E1β from Mycoplasma gallisepticum

为制备鸡毒支原体丙酮酸脱氢酶E1β亚基(PDHB)的单克隆抗体,将鸡毒支原体Rlow株的pdhb基因进行克隆和原核表达。表达产物纯化后作为免疫原,按常规方法免疫BALB/c小鼠。取免疫小鼠的脾细胞与SP2/0骨髓瘤细胞融合,经过克隆和筛选,获得3株能稳定分泌抗鸡毒支原体PDHB抗体的杂交瘤细胞株,分别被命名为2D11、3B5和4D9。ELISA检测结果表明,3株杂交瘤细胞培养上清的抗体效价为1∶1 024~1∶2 048,小鼠腹水的抗体效价为1∶12 800~1∶25 600。单克隆抗体特异性试验显示,获得的腹水单克隆抗体均能与鸡毒支原体各菌株发生反应,而不与其他禽类病原发生反应。3株单克隆抗体的抗体亚型均为IgG1,轻链均为κ链。稳定性鉴定结果表明,获得的3株杂交瘤细胞均能稳定分泌单克隆抗体。鸡毒支原体PDHB单克隆抗体的成功制备,为研究丙酮酸脱氢酶的生物学功能和进一步建立检测方法奠定了基础。

To prepare a monoclonal antibody(McAb)against pyruvate dehydrogenase subunit E1β(PDHB)of Mycoplasma gallisepticum,the PDHB was expressed in a prokaryotic system,purified and used as antigen to immunize BALB/c mice.The spleen cells from the immunized mice were fused with mouse myeloma SP2/0cells.Three positive hybridoma cell lines secreting McAbs against PDHB were selected by indirect enzyme-linked immunosorbent assay(ELISA).The antibody titers in the cell culture supernatant and ascites were from 1∶1 024to 1∶2 048and from 1∶12 800to 1∶25 600,respectively.Western-blot and ELISA showed that McAbs reacted with the recombinant PDHB protein and M.gallisepticumtotal protein specifically,and had no cross reactions with other avian pathogens.The immunoglobulin subtypes of McAbs were both IgG1,and light chains wereκ.Three hybridoma cell lines could secret McAbs against PDHB protein stably in vitro.These McAbs were successfully prepared,providing a tool for establishing detection method for M.gallisepticumand further studying biological functions of the PDHB protein.