锌指蛋白KOX12单克隆抗体的制备及在肿瘤组织中的表达

Preparation of Monoclonal Antibody Against zinc Finger Protein KOX12 and Identification of KOX12 in Tumor Tissues

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 specific monoclonal antibody against zinc finger protein KOX12 （tumor associated protein） and study the identification of KOX12 in tumor tissues. Methods The cDNA sequence of KOX12 was cloned with RT-PCR. The eukaryotic expression plasmid and prokaryotic expression plasmid were constructed with DNA recombinant technique,and then transfected into 293 cells and E.coli M15 respectively for ampli fication. BALB/c mice were immunized with repeated injections of KOX12. The spleen lymphocytes and NS-1 cells were hybridized using PEG as fusing agent. Monoclonal antibodies against KOX12 were obtained from the secretion of the hybridoma. The mAbs were used to detect expression of KOX12 in colon cancer and lung cancer tissues with immunohistochemistry （IHC）. Results The eukaryotic expression plasmid and prokaryotic expression plasmid of KOX12 were success fully constructed. The recombinant KOX12 proteins were expressed and purified. Monoclonal antibody against KOX12 was obtained from hybridoma cell line 6C8. ELISA and western blot technique confirmed that the mAbs reacted specifically with KOX12. Positive cells were found to be expressed in colon cancer and lung cancer tissues with IHC in present study. Conclusion MAb 6C8 is a specific monoclonal antibody against the recombinant zinc finger protein KOX12 the MAb is of IgG1 subclass,k-chain immunoglobulin. Some positive cells with KOX12 expression are found in colon cancer and lung cancer tissues. This experiment creates a platform for studying KOX12,which might prove to be useful for prevention and treatment of tumors.