抗尿苷二磷酸葡萄糖焦磷酸化酶2单克隆抗体制备及鉴定.docVIP

抗尿苷二磷酸葡萄糖焦磷酸化酶2单克隆抗体制备及鉴定 　 作者：王婉，高媛，杨金菊，柳晓兰，鞠艳芳，刘莉，陈志成 刘蓉， 毕业论文 ; 迟俊，邢维贤，高建恩，安立国，孙启鸿 毕业论文 【摘要】; 　　本研究制备抗人尿苷二磷酸葡萄糖焦磷酸化酶2（UDP?glucose pyrophosphorylase 2）的单克隆抗体（mAb）并进行初步鉴定。将正常成人肝组织匀浆离心并分离肝脏胞浆总蛋白，用肝脏胞浆总蛋白免疫BALB/c小鼠，采用杂交瘤技术制备单克隆抗体，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性鉴定，通过Uni?ZAP XR肝脏cDNA表达文库筛选鉴定抗体特异性。结果表明：通过间接ELISA筛选获得1株可稳定分泌抗人UGP2 mAb 的杂交瘤细胞系BAD062，其分泌的单克隆抗体的Ig亚类(型)为IgG2b (κ)，Western blot 结果显示该抗体可以特异地识别分子量为56 kD的蛋白；应用单克隆抗体BAD062对人肝脏cDNA表达文库（Uni?ZAP XR）进行筛选，阳性噬菌斑测序结果显示2个阳性克隆插入序列均为UGP2。结论：单克隆抗体BAD062特异地识别抗原UGP2，该抗体可用于ELISA检测、Western blot、免疫组织化学实验，为UGP2的研究提供了有力的工具。 毕业论文 【关键词】; 尿苷二磷酸葡萄糖焦磷酸化酶2 单克隆抗体 cDNA表达文库 毕业论文 　　Preparation and Identification of Monoclonal Antibody Against 毕业论文 Abstract ; The study was aimed to generate monoclonal antibodies (mAbs) against homo sapiens UDP?glucose pyrophosphorylase 2 (UGP2).; Normal human liver tissues; homogenized, and cytosolic proteins; isolated by centrifugation were used to immunize BALB/c mice to generate mAbs by hybridoma technique. The mAbs were identified by ELISA, Western blot, and immunohistochemistry; assay. The antibody specificity was confirmed by Uni?ZAP expression library screening. The results indicated that; one hybridoma BAD062 secreting specific mAb against UGP2 was established. The Ig subclass of this mAb was IgG2b (κ), and it could be used in ELISA, Western blot, immunohistochemistry assay. The antigen recognized by BAD062 mAb was localized in the hepatocyte cytoplasm, with molecular weight of 56 kD in the cytosolic proteins of human liver tissue. The BAD062 mAb was further confirmed by immunoscreening of Uni?ZAP XR liver cDNA expression library. It is concluded that a hybridoma cell line stably secretes specific mAb against UGP2. This mAb reacted with UGP2 in ELISA, Western blot, immunohistochemistry assay, and would be very useful for the UGP2 studies. 毕业论文 ; Key words ; UGP2; mAb; cDNA expression library 毕业论文 ; 尿苷二磷酸葡萄糖焦磷酸化酶2（UDP?glucose pyrophosphorylase2, UGP2）是糖原生物合成过程中的一种酶