抗丝瓜籽核糖体失活蛋白luffin b单克隆抗体的制备及应用  

Preparation and Use of a Monoclonal Antibody against Luffin b

作　　者：严明[1] 杨欣秀[1] 张茹平 平蓓芳[1] 李臻[1] 张祖传[1] 

机构地区：[1]中国科学院上海生命科学研究院生物化学与细胞生物学研究所,上海200031

出　　处：《生物化学与生物物理学报》2001年第1期93-98,共6页

基　　金：中国科学院"九五"重大项目!No .KJ .95 1 B1 6 0 6&&

摘　　要：丝瓜籽核糖体失活蛋白luffinb是至今已分离到的毒性最强的单链植物核糖体失活蛋白之一 ,它已被成功地用于免疫毒素的制备[1] 。用高纯度的luffinb免疫Balb/c小鼠 ,采用细胞融合方法筛选到两株能够稳定分泌高专一性的抗luffinb单克隆抗体的杂交瘤细胞株 1E5和 2E1,它们的亲和常数分别为 1.1× 10 9mol-1·L和 7.5×10 8mol-1·L ,腹水ELISA效价均达到 2× 10 7。用单克隆抗体 1E5制备的亲和凝胶 ,能够快速、高效地纯化luffinb。此外 ,单抗Luffin b is one of the most toxic single chain plant ribosome inactivating proteins. It has been successfully used to prepare an immunotoxin against human melanoma cells. Two strains of hybridomas (1E5 and 2E1) were screened out using cell fusion technique which steadily secreted monoclonal antibodies against luffin b. These antibodies specifically reacted with luffin b. The affinity constants of 1E5 and 2E1 monoclonal antibodies were determined to be 1.1×10 9 mol -1 ·L and 7.5×10 8 mol -1 ·L, by RIA, respectively. An immunoaffinity gel composed with the 1E5 monoclonal antibody and Sepharose 4B was prepared. The luffin b was successfully purified by one step immunoaffinity chromatography from the crude extract of Luffa cylindrica seeds. An immunoconjugate 1E5 HRP was also prepared and it was successfully used in Western blotting for detection of recombinant luffin b from E.coli total proteins.

关 键 词：丝瓜籽核糖体失活蛋白luffinb 抗luffinb单克隆抗体 亲和层析 免疫印迹 植物毒蛋白 

分 类 号：Q51[生物学—生物化学]