混合抗原免疫结合蛋白质芯片通量化制备单克隆抗体体系的初步建立

发布时间：2019-05-03 20:12

【摘要】： 单克隆抗体具有高度的特异性及均一性，应用广泛，目前制备单克隆抗体的方法成本高，产量低，程序繁杂，周期长。近年来蛋白质组学研究的迅速发展对高亲和力的单克隆抗体的需求迅速增加，对传统的杂交瘤技术提出了挑战。蛋白质芯片是将大量蛋白质分子按预先设置的排列固定于一种载体表面行成微阵列，根据蛋白质分子间特异性结合的原理以实现对生物分子的准却、快速、大信息量的检测。将单克隆抗体的特异性同蛋白质芯片的高通量相结合是未来高通量制备单克隆抗体的趋势。本实验的目的在于寻找高通量制备单克隆抗体的方法。首先，用基因工程的方法分别在两个表达载体构建了4个蛋白的融合载体，并在大肠杆菌中进行表达，不同的载体蛋白带有不同的标签，分别用来免疫及筛选，解决了单抗筛选中抗标签抗体的干扰；传统方法在抗原免疫时采用一只小鼠一次免疫一种抗原，本实验将4个纯化后的蛋白同时免疫一只小鼠后用传统方法制备单克隆抗体；对单抗进行Western blot及间接免疫荧光的验证，同时进行了蛋白质芯片的检测，比较常规的ELISA方法与蛋白质芯片的吻合性。结果显示，8个融合蛋白在宿主菌中以包涵体的形式得到高表达；混合免疫小鼠后，通过一次细胞融合，用杂交瘤技术筛选得到了针对4个蛋白的24株单克隆抗体；并对24株抗体进行了Western blot及间接免疫荧光验证，其中的7株能用于Western blot实验，且具有良好的特异性，5株能用于间接免疫荧光实验，识别胞浆内蛋白；同时对24株单克隆抗体进行了蛋白质芯片的检测，共检测了88个样品，传统的ELISA方法和蛋白质芯片检测结果的复合率为76％，，显示两者具有良好的一致性。本实验通过混合免疫和以抗原包板的芯片进行筛选的方法，尝试建立通量制备鼠单克隆抗体的新方法。
[Abstract]:Monoclonal antibodies are highly specific and homogeneous, and are widely used. At present, the preparation of monoclonal antibodies has the advantages of high cost, low yield, complicated program and long period. With the rapid development of proteomics in recent years, the demand for monoclonal antibodies with high affinity has increased rapidly, which challenges the traditional hybridoma technique. Protein chip is a kind of microarray formed by fixing a large number of protein molecules on the surface of a carrier in a predetermined arrangement. According to the principle of specific binding between protein molecules, the detection of biomolecules can be realized quickly and with a large amount of information. Binding the specificity of monoclonal antibody to high throughput of protein chip is the trend of producing monoclonal antibody in the future. The aim of this experiment is to find a high-throughput method for the preparation of monoclonal antibodies. Firstly, the fusion vectors of four proteins were constructed in two expression vectors by genetic engineering, and expressed in E. coli. Different vector proteins were labeled with different tags, and were used for immunization and screening, respectively. The interference of anti-label antibody in the screening of monoclonal antibody was solved. In the traditional method, one mouse was used to immunize one antigen at a time. In this experiment, four purified proteins were simultaneously immunized into one mouse and then monoclonal antibodies were prepared by traditional methods. The monoclonal antibody was tested by Western blot and indirect immunofluorescence, and the protein chip was detected. The coincidence of the conventional ELISA method with the protein chip was compared. The results showed that eight fusion proteins were highly expressed in the form of inclusion bodies in host bacteria, and 24 monoclonal antibodies against four proteins were obtained by hybridoma technique after the mice were immunized by mixed immunization with one cell fusion, and 24 strains of monoclonal antibodies against 4 proteins were obtained by hybridoma technique, and 24 monoclonal antibodies against 4 proteins were obtained by hybridoma technique. The results of Western blot and indirect immunofluorescence showed that 7 of the 24 antibodies could be used for Western blot test and had good specificity. 5 strains could be used for indirect immunofluorescence assay to recognize cytoplasmic proteins. At the same time, 24 monoclonal antibodies were detected by protein chip, and 88 samples were detected. The combination rate of traditional ELISA method and protein chip detection was 76%, which showed good consistency between the two methods. In this experiment, we tried to establish a new method of producing mouse monoclonal antibody by mixed immunization and screening with antigen-coated chip.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392

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