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目的:制备重组锌指蛋白KOX12(肿瘤相关蛋白),并制备其特异性的单克隆抗体,用免疫组化观察它在肿瘤组织中的表达。方法:通过逆转录-酶链聚合反应(RT-PCR)得到KOX12 cDNA片段,构建真核表达质粒和原核表达质粒,在大肠杆菌M15和293T细胞中进行表达。以KOX12蛋白为抗原,免疫BALB/c小鼠,用聚乙二醇(PEG)融合法进行细胞融合制备产生抗KOX12单克隆抗体的杂交瘤细胞株;该抗体用免疫组化法对结肠癌、肺癌组织进行染色。结果:构建了含KOX12的真核细胞和原核细胞的表达质粒,并表达及纯化了KOX12重组蛋白,建立了产生抗KOX12单克隆抗体的杂交瘤细胞株6C8,酶联免疫吸附试验(ELISA)和蛋白质免疫印迹试验(Western blot)显示6C8对KOX12有高度的特异性,免疫组化染色结果提示在结肠癌组织和肺癌组织切片中有少数细胞呈阳性反应。结论:6C8单克隆抗体是锌指蛋白KOX12特异的单克隆抗体,该抗体为IgG1型、k链亚类免疫球蛋白。在肺癌组织及对肠癌组织中有阳性细胞可见。该文为KOX12蛋白的研究工作创造了一个平台,为肿瘤的防治提供了一条新的可能途径。
OBJECTIVE: To prepare the recombinant human zinc finger protein KOX12 (tumor associated protein) and to prepare its specific monoclonal antibody, and to observe its expression in tumor tissue by immunohistochemistry. Methods: KOX12 cDNA fragment was obtained by reverse transcription-polymerase chain reaction (RT-PCR). The eukaryotic expression plasmid and prokaryotic expression plasmid were constructed and expressed in E. coli M15 and 293T cells. BALB / c mice were immunized with KOX12 protein as antigen, and the hybridoma cell lines producing monoclonal antibodies against KOX12 were prepared by cell fusion with polyethylene glycol (PEG) fusion method. The antibodies against colon cancer, Lung cancer tissue staining. Results: KOX12-containing eukaryotic and prokaryotic expression plasmids were constructed, and KOX12 recombinant protein was expressed and purified. The hybridoma cell line 6C8, enzyme-linked immunosorbent assay (ELISA) Western blot analysis showed that 6C8 was highly specific to KOX12, and immunohistochemical staining showed that a few cells were positive in colon cancer and lung cancer tissue sections. Conclusion: 6C8 monoclonal antibody is a specific monoclonal antibody against zinc finger protein KOX12, which is an IgG1 type, k chain immunoglobulin. In lung cancer and colon cancer positive cells can be seen. This article provides a platform for the research of KOX12 protein and provides a new possible approach for the prevention and treatment of cancer.