抗人羧酸酯酶-Ⅱ多克隆抗体的制备、鉴定及应用  

Preparation,characterization and application of polyclonal antibody against human carboxylesterases-Ⅱ

作　　者：吴春铃[1] 包红霞 唐晓波[1] 朱大岭[1] 

机构地区：[1]哈尔滨医科大学药学院生物制药学教研室,黑龙江省生物医药工程重点实验室-省部共建国家重点实验室培育基地,药物与内源性物质代谢省高校重点实验室,黑龙江哈尔滨150081

出　　处：《细胞与分子免疫学杂志》2009年第2期158-160,163,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：黑龙江省卫生厅科研课题(2007-471);黑龙江省博士后启动金(LRB04-196);黑龙江省教育厅振兴老工业基地重大科技项目(1151zgd11);哈尔滨市科技创新人才研究专项基金项目(RC2006XK003010);黑龙江省生物医药工程重点实验室研究基金(2004-001);哈尔滨医科大学医学基础学科青年科学基金项目(060043);黑龙江省卫生厅科研课题(2007-471)

摘　　要：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。AIM： To construct a prokaryotic plasmid expressing human carboxyles-terases- Ⅱ （ hCE- 11 ）, purify the recombinant protein and investigate the rabbit polyclonal antibody against hCE-Ⅱ. METHODS： A recombinant plasmid expressing pGEX-4T-I-hCE- Ⅱ （ hCE- Ⅱ-GST） and pET- 32a- hCE-Ⅱ （hCE-Ⅱ-His） was constructed and then it was expressed in E. coli BL21 induced by IPTG. The polyclonal antibody was prepared by immunizing the rabbits with the purified recombinant protein. The sensitivity and specificity of the antibody was detected using enzyme -linked immunosorbent assay, immunohistochem ical staining and Western blot analysis. RESULTS： The polyclonal antibody against hCE-Ⅱ was successfully prepared. Western blot analysis showed that the antibody specifically reacted with the recombinant protein and natural human liver microsomal proteins. Immunohistochemis result demonstrated that the pAb could combine with liver cytoplasm protein but not vas- cular smooth muscle cell protein. CONCLUSION： The successful preparation of the pAb against hCE- Ⅱ with high titer and specificity lays a foundation for the investigation of diagnosis kit of liver cancer.

关 键 词：hCE-Ⅱ 原核表达 融合蛋白 多克隆抗体 

分 类 号：R392.11[医药卫生—免疫学]