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目的:制备鼠抗人CMTM7(CKLF-like MARVEL transmembrane domain containing 7)蛋白的单克隆抗体,探讨CMTM7蛋白的分子结构和生物学功能。方法:通过生物信息学对CMTM7的蛋白序列进行分析,选取了3段序列合成多肽并与钥孔戚血蓝素(keyhole limpet hemocyanin,KLH)偶联,混合后免疫Balb/c小鼠。取免疫小鼠的脾细胞和同系小鼠的骨髓瘤细胞SP2/0进行常规融合,间接ELISA方法进行筛选和有限稀释克隆化,获得鼠抗CMTM7蛋白单克隆抗体的杂交瘤细胞株,用Western blot、免疫荧光以及免疫细胞化学等方法对其特性进行鉴定。结果:成功地建立了2株稳定分泌抗CMTM7蛋白的单克隆抗体杂交瘤细胞株,分别命名为MC9和2C9,其免疫球蛋白亚类均为IgG1。MC9单抗识别的抗原表位位于CMTM7氨基酸残基163~175区域(CMTM7163-175),可用于Western blot、免疫荧光和免疫细胞化学分析(immunocytochemistry,ICC)。2C9单抗识别的抗原表位位于CMTM7氨基酸残基19~44区域(CMTM719-44),该单抗只能用于Western blot分析。初步的功能研究提示,CMTM7蛋白在植物血凝素(phytohemagglutinin,PHA)活化早期的外周血淋巴细胞中表达上调。结论:获得了特异性好、能够识别不同抗原表位以及不同用途的鼠抗人CMTM7蛋白的单克隆抗体,为研究CMTM7蛋白的分子结构以及功能特性奠定了基础。
OBJECTIVE: To prepare monoclonal antibodies against murine CMTM7 (CKLF-like MARVEL transmembrane domain containing 7) protein and to investigate the molecular structure and biological function of CMTM7 protein. Methods: The protein sequence of CMTM7 was analyzed by bioinformatics. The 3-mer peptide was synthesized and conjugated with keyhole limpet hemocyanin (KLH). Balb / c mice were immunized after mixing. The spleen cells of immunized mice and myeloma cells SP2 / 0 of homologous mice were subjected to conventional fusion, screening by indirect ELISA and limiting dilution cloning to obtain the hybridoma cell strain of mouse monoclonal antibody against CMTM7 protein. Western blot , Immunofluorescence and immunocytochemistry and other methods to identify its characteristics. RESULTS: Two hybridoma cell lines stably secreting anti-CMTM7 protein were successfully established and named as MC9 and 2C9, respectively. The immunoglobulin subclasses were both IgG1. The epitope recognized by MC9 McAb is located in the region of 163-175 of CMTM7 amino acid residues (CMTM7163-175) and can be used for Western blot, immunofluorescence and immunocytochemistry (ICC). The epitopes recognized by 2C9 McAb are located in the region 19-44 of CMTM7 amino acid residues (CMTM719-44) and this monoclonal antibody can only be used for Western blot analysis. Preliminary functional studies suggest that CMTM7 protein is upregulated in peripheral blood lymphocytes activated by phytohemagglutinin (PHA). Conclusion: The monoclonal antibodies against murine CMTM7, which have good specificity, can recognize different epitopes and can be used for different purposes, have laid a foundation for studying the molecular structure and functional properties of CMTM7 protein.