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目的制备人Ⅱ型高尔基体膜蛋白73(Golgi protein 73,GP73)的多克隆抗体,为进一步研究GP73的功能奠定基础。方法利用重组原核表达载体PET-32a-GP73在大肠杆菌BL21中诱导表达,利用切胶方法回收纯化蛋白,并免疫新西兰兔制备多克隆抗体。制备的抗体经硫酸铵沉淀纯化后,用ELISA法和Western blot对多克隆抗体进行效价和特异性检测,用免疫组织化学法(IHC)检测纯化抗体在不同肝组织中识别GP73的能力。结果 GP73蛋白表达成功,并成功获得纯化的GP73蛋白及兔抗GP73多克隆抗体;ELISA法测定多克隆抗体效价>16 000;Western blot证明多克隆抗体的特异性良好;IHC显示GP73蛋白在正常肝组织中仅表达于胆管上皮细胞,而在肝癌组织中表达于肝癌细胞和胆管上皮细胞。结论成功获得了纯化的GP73蛋白及高效价的多克隆抗体,肝脏组织的IHC验证了其良好的特异性。
Objective To prepare polyclonal antibody of human type Ⅱ Golgi protein 73 (GP73), which lays the foundation for further study on the function of GP73. Methods The recombinant prokaryotic expression vector PET-32a-GP73 was induced in E. coli BL21. The purified protein was recovered by gel-cutting and immunized New Zealand rabbit to prepare polyclonal antibody. The purified antibody was precipitated with ammonium sulfate, and the titer and specificity of polyclonal antibody were detected by ELISA and Western blot. The ability of purified antibody to recognize GP73 in different liver tissues was detected by immunohistochemistry (IHC). Results The GP73 protein was expressed successfully and the purified GP73 protein and rabbit anti-GP73 polyclonal antibody were successfully obtained. The polyclonal antibody titer was> 16 000 by ELISA. The specificity of polyclonal antibody was confirmed by Western blot. Liver tissue is expressed only in biliary epithelial cells, and in liver cancer tissue expressed in hepatocellular carcinoma cells and bile duct epithelial cells. Conclusion The purified GP73 protein and high titer polyclonal antibody were successfully obtained. The IHC of liver tissue proved its good specificity.