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目的制备兔抗人同源异形盒基因HOXB9的多克隆抗体,为后续深入研究HOXB9蛋白的功能提供工具。方法采用聚合酶链反应(PCR)和DNA重组技术,构建HOXB9基因5’端7-458碱基片段到原核表达载体pGEX-4T-1和pMal-C2x中,热休克法转化及异丙基-β-D-硫代吡喃半乳糖苷诱导表达谷胱甘肽转移酶标签的HOXB9 N端融合蛋白和麦芽糖结合蛋白标签的HOXB9 N端融合蛋白。纯化GST-HOXB9 N端融合蛋白免疫新西兰白兔,ELISA鉴定效价后,颈动脉放血,收集血清,纯化抗体。免疫印迹法及免疫沉淀法鉴定抗体有效性及特异性。结果本实验制备的抗体能够特异识别目标分子的单一条带,并且具有较强的免疫沉淀天然构象的HOXB9蛋白的能力;采用所制备的抗体检测发现,HOXB9蛋白在小鼠免疫器官、胰脏、结肠、肺、小脑、雌性生殖系统及睾丸中表达较高,其他器官表达较低或无表达。结论成功制备了特异而有效的兔抗人HOXB9蛋白多克隆抗体,此多克隆抗体可用于免疫印迹法分析及免疫沉淀实验。
Objective To prepare a polyclonal antibody against HOXB9, a rabbit antihuman homeobox gene, to provide a tool for further study on the function of HOXB9 protein. Methods The 7-458-base 5 ’end of HOXB9 gene was cloned into prokaryotic expression vector pGEX-4T-1 and pMal-C2x by polymerase chain reaction (PCR) and DNA recombination technology. The heat shock method and isopropyl- β-D-thiogalactopyranoside induces a glutathione transferase-tagged HOXB9 N-terminal fusion protein and a maltose-binding protein tagged HOXB9 N-terminal fusion protein. New Zealand white rabbits were immunized with GST-HOXB9 N-terminal fusion protein. After the titer was identified by ELISA, the carotid arteries were exsanguinated, serum was collected and antibodies were purified. Immunoblotting and immunoprecipitation were used to identify the effectiveness and specificity of the antibody. Results The antibody prepared in this experiment could specifically recognize a single band of target molecule and had a strong ability of immunoprecipitation of HOXB9 protein in natural conformation. Using the prepared antibody, it was found that HOXB9 protein had a strong immunogenicity in mouse immune organs, pancreas, Colon, lung, cerebellum, female reproductive system and testes higher expression of other organs were low or no expression. Conclusion The specific and effective rabbit polyclonal anti-human HOXB9 antibody was successfully prepared. The polyclonal antibody can be used in western blot and immunoprecipitation assays.