抗日本血吸虫重组信号蛋白14-3-3单克隆抗体的制备和鉴定

Preparation and identification of monoclonal antibodies against recombinant signal protein 14-3-3 of Schistosoma japonicum

目的制备抗日本血吸虫重组信号蛋白14-3-3单克隆抗体,并鉴定抗体性质。方法以重组表达纯化的日本血吸虫信号蛋白14-3-3为抗原免疫BALB/c小鼠,采用杂交瘤技术制备单克隆抗体,采用酶联免疫吸附试验及免疫印迹法对获得的单克隆抗体进行类和亚类、效价、浓度、纯度、亲和常数、特异性和检测灵敏度的测定与鉴定。结果共获得6株针对日本血吸虫信号蛋白14-3-3的单克隆抗体,分别为5G9、3F1、3F7、5C6、5D1和1G6,抗体类型分别为IgG1、IgG1、IgG2a、IgG2b、IgG1和IgG1。对其中的单克隆抗体5G9分析显示,制备的腹水抗体效价为1∶1.28×105,亲和层析纯化后的浓度为5.3 mg/ml,纯度>95%,亲和常数为5.1×107mol/L。免疫印迹试验结果表明,该单克隆抗体可与体外重组表达纯化的日本血吸虫14-3-3蛋白特异性结合,同时也能与日本血吸虫可溶性虫卵抗原、成虫排泄分泌抗原和成虫抗原特异性反应,而与大肠埃希菌、健康人血清和华支睾吸虫抗原均无明显交叉反应。HRP标记的单克隆抗体5G9检测14-3-3蛋白的灵敏度为31.25 ng/ml。结论本研究获得了6株能稳定分泌抗日本血吸虫14-3-3蛋白的单克隆抗体杂交瘤细胞株,为建立新的日本血吸虫活动性感染检测方法奠定了良好的基础。

Objective To generate and analyze monoclonal antibodies against recombinant signal protein 14-3-3 of Schistosoma japonicum.MethodsThe recombinant 14-3-3 protein obtained previously was used to immunize BALB/c mice.Hybridoma technique was applied to prepare and screen the hybridoma cell lines,which could secret the target antibodies.The subclass,titer,affinity constant,specificity and detection limit of the monoclonal antibodies were identified by ELISA and Western blot.ResultsSix hybridoma cell lines were obtained and named as 5G9,3F1,3F7,5C6,5D1 and 1G6.Their subclasses were IgG1,IgG1,IgG2a,IgG2b,IgG1 and IgG1.The analyzed data of monoclonal antibody 5G9 showed that its antibody titer in ascites was 1∶1.28×105,its concentration after purification was 5.3 mg/ml,its purity was 95% and its affinity constant was 5.1×107 mol/L.The monoclonal antibody 5G9 could recognize the recombinant signal protein 14-3-3 of Schistosoma japonicum and the natural signal protein 14-3-3 in SEA,ESA and AWA without cross reactions with the protein of E.coli,healthy human serum and Clonorchis sinensis in Western blot.The detection limit of HRP labeled monoclonal antibody 5G9 was 31.25 ng/ml.ConclusionsSix hybridoma cell lines that could stably secrete the target antibodies are raised,which will lay a solid foundation for developing a new diagnosis method to the active infection of Schistosoma japonicum.