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目的制备针对HAUSP蛋白的单克隆抗体并进行鉴定。方法扩增人HAUSP基因片段,构建含HAUSP编码序列的原核表达载体,在大肠杆菌Rosetta中表达融合蛋白。应用Ni-NTA agarose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HAUSP的单克隆抗体。通过间接ELISA法鉴定了抗体的效价,Western blotting和Immunoprecipitation(IP)实验检测抗体的特异性。结果 HAUSP基因长539 bp,表达融合蛋白在其2～180位氨基酸,相对分子质量44 800,纯化后蛋白纯度≥85%,制备的HAUSP单克隆抗体可识别Jurkat和Hela细胞内的HAUSP蛋白,并且具有良好的特异性。结论本研究制备了效价高、特异性好的HAUSP的单克隆抗体,为进一步揭示HAUSP在肿瘤发生中的功能提供了有效工具。
Objective To prepare monoclonal antibodies against HAUSP protein and identify them. Methods The human HAUSP gene fragment was amplified and a prokaryotic expression vector containing the HAUSP coding sequence was constructed. The fusion protein was expressed in Escherichia coli Rosetta. The recombinant protein was purified by Ni-NTA agarose. The purified fusion protein was used to immunize mice to prepare a monoclonal antibody that can specifically recognize HAUSP. The antibody titer was identified by indirect ELISA. The specificity of the antibody was detected by Western blotting and Immunoprecipitation (IP) assays. Results The HAUSP gene was 539 bp in length. The HAUSP gene was expressed at amino acids 2 ~ 180, with a relative molecular mass of 44 800. After purified, the purity of HAUSP gene was over 85%. The prepared HAUSP monoclonal antibody recognized the HAUSP protein in Jurkat and Hela cells Has good specificity. Conclusions The monoclonal antibody against HAUSP with high potency and specificity was prepared in this study, which provides an effective tool for further revealing the function of HAUSP in tumorigenesis.