MC3是我们自主建立的一株结肠癌特异的单抗，前期研究利用蛋白质组学技术鉴定出其识别的抗原为Txl-2,并发现Txl-2能够显著促进结肠癌转移。酵母双杂和免疫共沉淀发现Txl-2的第2个亚型Txl-2b与小G蛋白家族成员Ran相互作用，而Txl-2其它2个亚型与Ran没有相互作用；Ran近年来被报道在多种肿瘤中高表达且促进肿瘤进展，但机制不清。本课题拟采用激光共聚焦、配对免疫组化和截短突变免疫共沉淀等进一步证实Ran与Txl-2b亚型特异的相互作用；慢病毒共感染、体内体外功能实验观察两者相互作用对结肠癌细胞转移能力的影响;Pull Down实验检测二者相互作用后Ran GTP活化形式的表达水平；位点突变明确Txl-2b酶学催化位点对其与Ran相互作用的影响；芯片筛选、抑制剂干预进一步寻找下游信号通路；本项目将深入阐明蛋白相互作用对肿瘤恶性表型影响的机制，为开发肿瘤新的治疗策略提供新思路。

MC3 is a monoclonal antibody previously prepared in our laboratory, which could detect colon cancer with high sensitivity and specificity. Our previous work identified thioredoxin-like protein 2 (Txl-2) as the target of MC3 and indicated that overexpression of Txl-2 in colon cancer cells significantly promoted cancer cell motility, and the invasive and metastatic abilities. Furthermore, isoform-specific interaction was identified between Txl-2b and Ran, a Ras-related protein, by yeast two-hybrid assay and co-immunoprecipitation. Recently, emerging evidence has been reported on Ran associating with various cancer malignant phenotypes. However, the underlying mechanism is still poorly understood. In the present study, confocal study, paired immunostaining,deleting mutation and co-IP will be employed to further confirm the interaction between Txl-2b and Ran and detect the detailed binding sequences. Lentiviral co-transfection of Txl-2 and Ran will be done, followed by the observation of changes in cell invasive and metastatic potential in vitro and in vivo. Ran GTP level will be monitored before or after Txl-2b and Ran interaction. Mutant variants will be generated to determine whether the enzyme active sites of Txl-2b may influence the interaction between Txl-2b and Ran. Gene arrays, pathway inhibitors, qPCR and western blot analysis will be used to further explore the involved downstream events and pathways of Txl-2b-Ran mediated colon cancer metastasis. The current study may provide a deep insight into how protein interaction functions in cancer metastasis and open an avenue to a novel therapeutic strategy against colon cancer.