【作者】 武正华； 唐晓波； 王志刚； 纪宏宇； 于磊； 刘慧文； 朱大岭；

【Author】 WU Zheng-hua1, TANG Xiao-bo1,4, WANG Zhi-gang1, JI Hong-yu2, YU Lei1, LIU Hui-wen5 , ZHU Da-ling * 1,2,3,(4Harbin Medical University: 1Biopharmacy Department of Pharmacy College; 2Pharmacy Department of the Second Affiliated Hospital; 3Key Laboratory of Biopharmaceutical Engineering of Heilongjiang province; 4Key Laboratory of Drug and Endogenous Substance Metabolism of Province Higher Learning Institution; 5Histology and Embryology Department of Basic Medicine College, Harbin # Heilongjiang 150081, China)

【机构】 哈尔滨医科大学药学院生物制药教研室； 哈尔滨医科大学附属二院药学部； 哈尔滨医科大学基础医学院组胚教研室；

【摘要】 目的:制备鼠抗人羧酸酯酶-Ⅱ(human carboxylesterases-Ⅱ,hCE-Ⅱ)单克隆抗体(mAb)并对其特性进行鉴定。方法:以含hCE-Ⅱ的人肝脏微粒体蛋白混合抗原免疫BALB/c小鼠,采用杂交瘤技术制备鼠抗hCE-Ⅱ的mAb,并用Protein-G亲和层析法纯化mAb。用间接ELISA法测定mAb效价、Western blot鉴定抗体特异性、免疫组化染色法对抗体相应的抗原进行组织定位。用免疫沉淀及基质辅助激光解吸电离飞行时间质谱(matrix-assisted laser desorption/ionization time-of-flight mass spectrometry,MALDI-TOF-MS)得到相应的肽质量指纹谱(peptide mass fingerprint,PMF),搜索数据库鉴定mAb对应的抗原。结果:获得1株可持续、稳定分泌抗hCE-Ⅱ的mAb的杂交瘤细胞系。杂交瘤细胞分泌的mAbIg亚类(型)为IgG1(κ),腹水mAb的效价达1×10-7。Western blot分析显示,在Mr为62000处有1条清晰的条带。免疫组化染色显示,该mAb可与肝细胞的浆蛋白结合(hCE-Ⅱ存在于肝细胞浆内微粒体),而不与血管平滑肌细胞内蛋白结合。该mAb免疫沉淀物的Mr为62000,与hCE-Ⅱ的Mr相符。对免疫沉淀的蛋白质进行质谱鉴定证实,该mAb对应的抗原为hCE-Ⅱ。结论:制备出的鼠抗hCE-Ⅱ的mAb效价高、特异性良好,为进一步研究hCE-Ⅱ的功能及其在肝癌等癌症的诊断和治疗中的作用提供了工具。更多还原

【Abstract】 OBJECTIVE To prepare monoclonal antibody (mAb) against human carboxylesterases-Ⅱ (hCE-Ⅱ) and characterize its properties. METHODS BALB/c mice were immunized with human liver microsome proteins, and mAb was prepared by hybridoma technique. The crude mAb was purified by protein-G affinity chromatography. The titer of mAb was detected by ELISA, specificity of mAb by Western blot analysis and antigen tissue localization by immunohistochemical staining. Antigen was appraised by peptide mass fingerprint (PMF) that was obtained by immunoprecipitation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). RESULTS One clone of hybridoma secreting specific mAb against hCE-Ⅱ was obtained. The Ig subclass of mAb was of IgG1(κ). The titer of mAb was 1×10-7. Western blot analysis demonstrated 1 clear belt in the Mr 62,000; Immunohistochemistry demonstrated that mAb had specificity suffusion combination with the liver cytoplasm protein, but did not have specificity combination with the vascular smooth muscle cell protein. Immunoprecipitation obtained 1 clear belt in the Mr 62,000 (conformity with the Mr of hCE-Ⅱ). The protein of immunoprecipitation was analyzed with mass spectrometry to identify antigen picked up by mAb, which was confirmed to be hCE- II. CONCLUSION The mAb with high titer and specificity against hCE-Ⅱhas been obtained, which lays the foundation for investigation of functions of hCE-Ⅱ and diagnosis and treatment of liver cancer and so on.更多还原