血红蛋白A2单克隆抗体的制备与鉴定_医学论文.docVIP

血红蛋白A2单克隆抗体的制备与鉴定_医学论文 血红蛋白A2单克隆抗体的制备与鉴定_医学论文 作者：陈志成，杨金菊，刘蓉，曲海霞，王婉，刘莉，柳晓兰，陈勇 刘莹，高建恩，孙启鸿【摘要】 本研究目的是制备鼠抗人血红蛋白A2（HBA2）单克隆抗体（McAb）并进行初步鉴定。将正常人胎肝组织匀浆离心并分离出人胎肝核总蛋白, 用人胎肝核总蛋白免疫BALB/c 小鼠, 采用杂交瘤技术制备单克隆抗体, 并通过间接ELISA法、Western blot及免疫组织化学的方法对单克隆抗体进行特异性鉴定, 通过免疫沉淀和Uni表达文库筛选鉴定抗原。结果表明： 通过间接ELISA筛选获得分泌AEE091抗体的杂交瘤细胞1株，其分泌的单克隆抗体Ig亚类（型）为IgG1 (κ)； Western blot 结果显示，该抗体识别分子量为15 kD的蛋白； Western blot识别AEE091免疫沉淀获得的分子量为15 kD的抗原；同时应用单克隆抗体AEE091对人肝脏cDNA表达文库（Uni）进行筛选，筛选所获得的阳性噬菌斑测序结果显示两个阳性克隆插入序列均为HBA2。结论: 经筛选获得了分泌AEE091抗体的杂交瘤细胞株。AEE091抗体能特异识别HBA2抗原，因而其在HBA2的功能研究和珠蛋白生成障碍性贫血筛查等方面具有应用价值。 【关键词】 地中海贫血 Preparation and Identification of Monoclonal Antibody against Homo Sapiens Hemoglobin alpha 2 (HBA2) Abstract This study was purposed to prepare and identify monoclonal antibodies (McAbs) against Homo sapiens hemoglobin alpha 2 (HBA2). Normal human fetal liver tissues were homogenized, and human liver nuclear proteins were isolated by centrifugation. The total human fetal liver nuclear proteins were used to immunize BALB/c mice for preparing McAbs by hybridoma technique. The McAbs specificity was identified by ELISA, Western blot, and immunohistochemistry. The antigen was identified by UniZAP expression library screening. The results showed that one hybridoma cell line, AEE091, secreting specific McAb against HBA2 was established. The Ig subclass of this McAb was IgG1 (κ). Data from immunohistochemistry assay showed that AEE091 could recognize human liver nuclear protein. Using AEE091 McAb, isolation of the protein antigen by IP revealed that AEE091 McAb could recognize 15 kD protein. Screening the UniZAP XR premade liver cDNA library with AEE091 hybridoma cell supernatants demonstrated that AEE091 McAb specially reacted with HBA2. It is concluded that a hybridoma cell line stably secreting specific McAb against HBA2 is established. The specific McAb against HBA2 would be very useful for studying HBA2 function and screening thalassanemia. Key words Homo sapiens hemoglobin alpha 2 McAb cDNA expr