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Position: Home > Articles > 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 Chinese Journal of Veterinary Science 2013,33 (5) 684-688

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

作  者:
苏倩倩;王肖祎;李恩扩;彭志成;胡敬东
单  位:
山东农业大学动物科技学院
关键词:
鸡白细胞介素18;单克隆抗体;特异性鉴定
摘  要:
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.
单  位:
College of Veterinary Medicine,Shandong Agricultural University%Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention,Taian,Shandong 271018,China
关键词:
chicken interleukin-18%monoclonal antibody%specificity identification
摘  要:
Mature chicken interleukin-18(mChIL-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 mChIL-18 were established and characterized by immunizing BALB/c mice with purified recombinant mChIL-18 as an antigen.The mAbs had an ascites titers of 1:5.12*105 and 1:3.2*104,respectively and both were subtype IgM.Western-blot 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-mChIL18.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 ChIL-18.

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