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目的:构建人羧酸酯酶-Ⅱ(hCE-Ⅱ)原核表达质粒并在大肠杆菌中表达,制备多克隆抗体(pAb),并对其特性进行初步鉴定。方法:以人正常肝cDNA文库为模板,构建重组表达质粒pGEX-4T-1-hCE-Ⅱ(即hCE-Ⅱ-GST)和pET-32a-hCE-Ⅱ(即hCE-Ⅱ-His)。在宿主菌BL21内进行诱导表达,hCE-Ⅱ-GST融合蛋白作为免疫原免疫大耳白兔,用辛酸-硫酸铵分步沉淀法初步纯化抗血清,并用间接ELISA、West-ernblot及免疫组化检测抗体的灵敏度和特异性。结果:成功制备了抗hCE-Ⅱ的多克隆抗体。Westernblot鉴定表明该抗体可特异性识别重组蛋白以及天然的人肝微粒体蛋白。免疫组织化学鉴定显示该抗体可特异性结合肝包浆中的hCE-Ⅱ蛋白,而不与血管平滑肌细胞结合。结论:制备的pAb有较高的效价和较好的特异性,为肝癌诊断试剂盒的研究奠定了实验基础。
OBJECTIVE: To construct a prokaryotic expression plasmid of human carboxylesterase-II (hCE-Ⅱ) and express it in E. coli. The polyclonal antibody (pAb) was prepared and its characteristics were preliminarily identified. Methods: The recombinant plasmid pGEX-4T-1-hCE-Ⅱ (hCE-Ⅱ-GST) and pET-32a-hCE-Ⅱ (hCE-Ⅱ-His) were constructed using human normal liver cDNA library as a template. The recombinant protein was induced in host strain BL21. The hCE-Ⅱ-GST fusion protein was used as immunogen to immunize the rabbits. The antisera were purified by octanoic acid-ammonium sulfate fractionation and identified by indirect ELISA, West-ernblot and immunohistochemistry Detection of antibody sensitivity and specificity. Results: Polyclonal antibodies against hCE-Ⅱ were successfully prepared. Westernblot identification showed that the antibody specifically recognizes the recombinant protein as well as native human liver microsomal protein. Immunohistochemistry showed that the antibody could specifically bind hCE-Ⅱprotein in hepatic plasma without binding to vascular smooth muscle cells. Conclusion: The prepared pAb has high titer and good specificity, which lays the foundation for the research of liver cancer diagnostic kit.