抗人中性粒细胞明胶酶相关脂质运载蛋白单克隆抗体识别的抗原表位区域鉴定

Identification of antigenic epitope regions recognized by monoclonal antibodies against human neutrophil gelatinase-associated lipoprotein

目的对抗人中性粒细胞明胶酶相关脂质运载蛋白(NGAL)单克隆抗体H5识别的抗原表位区域进行鉴定。方法设计4个NGAL截短体,通过PCR扩增截短体DNA片段并克隆到PET42a质粒中表达GST-NGAL截短体融合短肽。以此为抗原鉴定H5识别的抗原表位区域。随后根据生物信息学预测结果,化学合成2段潜在B细胞表位肽,用ELISA和Western blot方法进一步鉴定。结果 H5能与NGAL蛋白分子第20～36位氨基酸结合。结论单克隆抗体H5的抗原表位结合位点存在于成熟NGAL蛋白分子氨基末端第20～36位氨基酸处,其深入研究为建立高效灵敏的NGAL免疫学检测体系和进一步研究NGAL分子抗原表位结构奠定基础。

Objective To identify the epitope region of monoclonal antibody H5 against human neutrophil gelatinase-associated lipoprotein(NGAL).Methods Four NGAL truncated DNA fragments were designed and amplified by PCR and cloned into PET42a plasmid to express GST-NGAL truncated fusion peptide.The antigen epitope region recognized by H5 was identified as antigen.Two potential B cell epitope peptides were synthesized and identified by ELISA and Western blot.Results H5 could bind to the amino acid of the NGAL protein at 20-36 position.Conclusion The antigen epitope binding site of monoclonal antibody H5 exists at at the amino terminal 20-36 amino acid of mature NGAL protein molecule,which lays a foundation for the establishment of a highly sensitive immunoassay system for NGAL and the further study of the epitope structure of NGAL molecule.