SOX4单克隆抗体的制备及其在肿瘤细胞表达分析中的应用

Preparation of the monoclonal antibody against SOX4 protein and detection of SOX4 expression level in different tumor cell lines

摘要:

应用分子生物学技术, 构建了含SOX4编码序列的原核表达载体, 在大肠杆菌 DH5a中获得了GST-SOX4融合蛋白的可溶性表达。应用谷胱甘肽-Sepharose 4B对重组蛋白进行了纯化, 利用纯化的融合蛋白免疫小鼠, 制备了可特异性识别SOX4的单克隆抗体。通过间接 ELISA 法鉴定了抗体的效价为1 × 10-5, Western blotting 分析证实了抗体的特异性。结果显示, 该抗体可识别细胞内外源性过表达及内源性的SOX4蛋白。在培养细胞系、小鼠不同组织中, SOX4蛋白的表达存在显著的差异。本研究制备的SOX4单克隆抗体具有良好的特异性, 为进一步研究SOX4在肿瘤发生中的作用提供了重要的工具。

Abstract:

In the present study, we constructed a prokaryotic expression vector containing SOX4 protein encoding sequences. The GST-SOX4 soluble protein was expressed in Escherichia coli DH5a and purified by glutathione sepharose-4B. The purified recombinant protein was used to immunize Balb/C mice and the monoclonal antibody against SOX4 was prepared by using hybridoma technique. The titer of the antibody was determined as 1×10-5 by indirect ELISA. The specificity of the antibody was verified by Western blotting analysis. The monoclonal antibody specifically recognized the overexpressed exogenous SOX4 protein as well as endogenous SOX4 protein. The expression level of SOX4 protein in different cell lines and mouse tissues was detected by using the antibody. Differential expression of the protein was demonstrated by Western blotting. The data indicated that the antibody was specific. The antibody can be used as an important tool for further exploration of the role of SOX4 in tumorigenesis.