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目的:为了揭示循环血液中存在的软骨寡聚基质蛋白(Cartilage Oligomeric Matrix Protein,COMP)片段,探索COMP逃避自身免疫耐受的途径及机制,需制备高效、专一的识别COMP不同结构域的单克隆抗体。方法:利用真核表达系统对人COMP进行了一系列的表达、并用亲和层析法结合离子交换层析法进行纯化。纯化的COMP为抗原免疫BALB/c小鼠,采用杂交瘤技术结合有限稀释传代法筛选得到稳定分泌抗人COMP单克隆抗体的杂交瘤细胞株,用COMP单结构域片段结合生物信息学方法分析鉴定单克隆抗体结合表位。用不同种属来源的COMP分析单克隆抗体结合表位的保守性。结果:COMP及单结构域片段为分泌到细胞外的可溶性分子利于蛋白的纯化,筛选获得4株稳定分泌抗人COMP单克隆抗体细胞株,4株单克隆抗体的亚型为IgG1,经COMP单结构域片段检测证明4株单克隆抗体识别不同的表位,表位预测软件初步确定4株单克隆抗体识别表位所在区域,间接ELISA及免疫组化证实其中3株单克隆抗体与不同种属来源的COMP有较好的结合特性。结论:成功制备了4株识别不同表位的抗COMP单克隆抗体,为揭示血液中存在的COMP片段和进一步研究COMP逃避自身免疫耐受机理提供了研究工具。
OBJECTIVE: To reveal the pathways and mechanisms by which COMP suppresses cartilage Oligomeric Matrix Protein (COMP), and to find a way to identify the different domains of COMP Clone antibody. METHODS: Human COMP was expressed in a series of eukaryotic expression systems and purified by affinity chromatography using ion exchange chromatography. Purified COMP was used to immunize BALB / c mice against the antigen. The hybridoma cell line secreting anti-human COMP monoclonal antibody was screened by hybridoma technique combined with limited dilution passage. The hybridoma cells were identified by COMP single domain fragment and bioinformatics analysis Monoclonal antibodies bind to epitopes. Analysis of the conservation of monoclonal antibody binding epitopes using COMP from different species sources. RESULTS: The COMP and single domain fragments were soluble in extracellular molecules, which was beneficial to the purification of the protein. Four stable cell lines secreting anti-human COMP monoclonal antibody were screened. The subtype of four monoclonal antibodies was IgG1. The detection of the domain fragment showed that the four monoclonal antibodies recognized different epitopes. The epitope prediction software initially identified the region where the four monoclonal antibodies recognized the epitopes. Indirect ELISA and immunohistochemistry confirmed that three of the monoclonal antibodies were related to different species The source of COMP has better binding characteristics. CONCLUSIONS: Four monoclonal anti-COMP monoclonal antibodies recognizing different epitopes were successfully prepared, providing a research tool for revealing the COMP fragments present in blood and further studying the mechanism by which COMP escape autoimmune tolerance.