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目的:制备抗人肌糖蛋白C(tenescin-C,TN-C)单克隆抗体并鉴定。方法:根据人TN-C蛋白序列,用软件预测B细胞表位;化学合成优势表位蛋白肽,分别与钥孔戚血蓝素(KLH)和兔血清白蛋白(RSA)交联;取KLH-TN-C常规免疫BALB/c小鼠,取免疫小鼠脾细胞与Sp2/0细胞融合,用分别预包被KLH-TN-C或者RSA-TN-C融合蛋白的ELISA板筛选高亲和力单克隆抗体;取骨肉瘤组织进行免疫组化染色。结果:获得蛋白优势表位数据;成功将TN-C肽交联至KLH和RSA;获得3株抗人TN-C单克隆抗体,分别为IgG1和IgG2a亚型,腹水效价为10-6左右,免疫组化有阳性着染。结论:成功获得抗人TN-C单克隆抗体。
Objective: To prepare and identify monoclonal antibodies against tenescin-C (TN-C). Methods: B cell epitopes were predicted by software based on the human TN-C protein sequence. The dominant epitope peptide was chemically synthesized and cross-linked with keyhole limpet hemocyanin (KLH) and rabbit serum albumin (RSA) respectively. KLH The BALB / c mice were immunized with TNT-CT and the splenocytes of the immunized mice were fused with Sp2 / 0 cells. The high affinity monofilament was screened with ELISA plates pre-coated with KLH-TN-C or RSA-TN-C fusion protein Cloned antibody; osteosarcoma tissue immunohistochemical staining. Results: The dominant protein epitope data were obtained. The TN-C peptide was successfully cross-linked to KLH and RSA. Three anti-human TN-C monoclonal antibodies were obtained, which were IgG1 and IgG2a subtypes, respectively. The ascites titer was about 10-6 , Immunohistochemistry positive staining. Conclusion: The anti-human TN-C monoclonal antibody was successfully obtained.