隔离、局部特征和分子克隆人类结肠腺癌的细胞表面糖蛋白的识别C215鼠单克隆抗体。
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比约克P,琼森U,斯维德贝格H,拉尔森K,林德P, Dillner J,荷得拉得G, T Dohlsten M, Kalland说
隔离、局部特征和分子克隆人类结肠腺癌的细胞表面糖蛋白的识别C215鼠单克隆抗体。
生物化学杂志。1993年11月15日;268 (32):24232 - 41。
- PubMed ID
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7693697 (在PubMed]
- 文摘
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单克隆抗体C215 (IgG2a)获得的免疫BALB / c小鼠与人类结肠恶性腺瘤细胞系科罗拉多州205年和用于大肠癌癌的目标。C215目标分子的局部特征和净化从205年科罗拉多州可溶性膜表明它是不可或缺的膜糖蛋白non-mucin类型。变性抗原作为主要40-kDa形式出现在西方的屁股后SDS-polyacrylamide凝胶电泳和迁移作为一个单体的36-kDa物种分子内二硫还原裂解后的桥梁。使用五步过程,抗原纯化4300倍从205年科罗拉多州肿瘤生长在裸小鼠评估通过毛细管电泳时95%的同质性。由肽清除N-linked碳水化合物:N-glycosidase治疗并不影响的可视化纯化抗原在免疫印迹,但导致更快的SDS凝胶迁移。氨基酸序列的部分决定。17连续NH2-terminal氨基酸被确定和恰好与残留GA733-2蛋白克隆的82 - 98年Szala et al。(Szala, S。Froehlich, M。Scollon, M。开赛,Y。、Steplewske Z。Koprowski, H。Linnenback, a . j . (1990) Proc。国家的。学会科学。 U.S.A. 87, 3542-3546). Therefore, the predicted amino acid sequence of this protein was used to prepare overlapping synthetic peptides that cover the entire extracellular domain in order to identify the C215 epitope. A likely epitope, close to the NH2 terminus and corresponding to the first distinct hydrophilic stretch after the putative signal sequence, was identified in a peptide enzyme-linked immunosorbent assay. Moreover, GA733-2 cDNA was used for the cloning of the C215 protein from COLO 205 cells and the subsequent transfection to K36.16 mouse T cell leukemia cells. The transfected cells were C215 reactive in fluorescence-activated cell sorter analysis, and a 42 kDa band was visualized in Western blots under both non-reducing and reducing conditions. Our findings indicate a close relationship between the C215 antigen and other members of the GA-733 family, some of which are currently being used as targets in clinical trials with monoclonal antibodies. The mammalian expression system described here will enable further studies into the biological role of this protein and the construction of animal models in order to develop optimal therapeutic strategies.