疱疹病毒相关性泛素特异蛋白酶(HAUSP)单克隆抗体的制备及鉴定  

Preparation and identification of the monoclonal antibody against HAUSP protein

作　　者：张娜[1] 刘学芳[1] 

机构地区：[1]河南中医学院科研实验中心,郑州450046

出　　处：《免疫学杂志》2013年第11期957-961,共5页Immunological Journal

基　　金：河南中医学院"博士科研基金"(BSJJ2010-33)

摘　　要：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。To pre pare and indentify the monoclonal antibody against HAUSP protein, we amplified the herpesvirus-associated ubiquitin specific protease （HAUSP） DNA and constructed a prokaryotie expression vector containing HAUSP protein encoding sequences. The fusion protein was expressed in Escherichia coli Rosetta and purified by Ni-NTA agarose. And then the purified recombinant protein was used to immunize Balb/c mice and the monoclonal antibody against HAUSP was prepared by hybridoma technique. The titer of the antibody was determined by indirect ELISA, while the specificity of the antibody was verified by Western blotting and Immuno- preipitation （IP） analysis. Result showed the total length of HAUSP gene was 539 bp, and the fusion protein （2-180 aa） was 44.8 kD, whose purity was up to 95% after purification. And the monoclonal antibody could specifically recognize the HAUSP protein in Jurkat and Hela cell lines. In conclusion, this study has prepared the monoclonal antibody against HAUSP protein, whieh was specific and of high titer. This antibody can be used as an important tool for further exploration of the role of HAUSP in tumorigenesis.

关 键 词：HAUSP 单克隆抗体 鉴定 

分 类 号：R392.1[医药卫生—免疫学]