鸡白细胞介素18成熟蛋白的表达、纯化及其单克隆抗体的制备与特性鉴定

Preparation and identification of monoclonal antibodies against mature chicken interleukin-18

将鸡白细胞介素18成熟蛋白(mature chicken interleukin 18,mChIL-18)基因亚克隆至原核表达载体pET-28a(+),并在大肠杆菌中高效表达,采用Ni-NTA亲和层析方法纯化重组蛋白,免疫BALB/c小鼠,经融合、筛选制备特异性单克隆抗体(mAb),并通过间接ELISA、间接免疫荧光(IFA)和Western blot等方法对mAb做初步鉴定。结果表明,试验成功表达了mChIL-18蛋白,并获得2株持续且稳定分泌抗体的杂交瘤细胞(1G9、2E6),其腹水ELISA效价分别为1:5.12×105和1:3.2×104,亚类鉴定结果均为IgM。Western blot结果显示,2株单抗均能特异性识别原核表达的mChIL-18蛋白,IFA分析表明它们均能与真核表达的mChIL-18发生特异性反应,ELISA叠加试验证实1G9与2E6针对2个不同的抗原表位。本研究所获单抗可以用于建立鸡IL-18的检测方法,为进一步开展其生物学功能研究奠定了基础。

Mature chicken interleukin-18 （mChlL-18） gene was cloned into a pET-28a（＋） vector, the protein was expressed in E. coli and purified. Two hybridoma cell lines （1G9 and 2E6） stably secreting monoclonal antibody （mAb） specific to mChlL-18 were established and characterized by immunizing BALB/c mice with purified recombinant mChlL-18 as an antigen. The mAbs had an ascites titers of 1 ： 5.12× 10^5 and 1 ： 3.2× 10^4 ,respectively and both were subtype IgM. Westernblot analysis confirmed that these mAbs specifically recognized mChIL-18 only. IFA assay revealed that both mAbs could react to 293T cells transfected by pcDNA3. 1-mChlL18. ELISA showed that 1G9 and 2E6 were for the different epitope. Therefore the mAb developed in this work lays a foundation for further studying and detection of ChlL-18.