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目的:制备兔抗人羧酸酯酶1(CES1)多克隆抗体(pAb),并对抗体进行特性鉴定。方法:以人正常肝cDNA文库为模板,构建重组表达质粒pGEX-4T-1-CES1(即CES1-GST)和pET-32a-CES1(即CES1-His)。CES1-GST融合蛋白作为免疫原制备兔pAb,辛酸-硫酸铵法纯化抗体,采用Western blot和免疫组化鉴定抗体的特异性。结果:CES1-GST和CES1-His融合蛋白均在大肠杆菌中以包涵体形式大量表达。兔抗人CES1pAb效价为1∶64000。Western blot鉴定表明,该抗体可识别重组人CES1蛋白,也可特异地识别人肝微粒体中的CES1亚基。免疫组织化学鉴定显示该抗体可特异性识别正常肝组织中的CES1蛋白。结论:成功地制备了兔抗人CES1pAb,该抗体可应用于ELISA、Western blot、免疫组化等实验,为进一步研究CES1的功能提供了特异性检测工具。
Objective: To prepare rabbit anti-human carboxylesterase 1 (CES1) polyclonal antibody (pAb) and characterize the antibody. Methods: The recombinant plasmid pGEX-4T-1-CES1 (CES1-GST) and pET-32a-CES1 (CES1-His) were constructed using human normal liver cDNA library as a template. CES1-GST fusion protein was used as immunogen to prepare rabbit pAb. The protein was purified by caprylic acid-ammonium sulfate method. The specificity of the antibody was identified by Western blot and immunohistochemistry. Results: Both CES1-GST and CES1-His fusion proteins were highly expressed in inclusion bodies in E. coli. Rabbit anti-human CESlpAb titer was 1: 64000. Western blot analysis showed that this antibody can recognize recombinant human CES1 protein and specifically recognize CES1 subunit in human liver microsomes. Immunohistochemistry showed that the antibody could specifically recognize CES1 protein in normal liver tissue. CONCLUSION: Rabbit anti-human CES1pAb was successfully prepared and could be used in ELISA, Western blot, immunohistochemistry and other experiments to provide a specific test tool for further study on the function of CES1.