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目的 为深入研究源于噬菌体抗体库特定单链抗体 (scFv)的功能和其识别抗原的分子特征 ,将识别KG1a细胞的单链抗体 5C1scFv高效表达、纯化 ;用scFv测定未知抗原的相对分子质量 (Mr) ;并研究 5C1scFv对KG1a部分细胞生物学特性的影响。方法 基因重组构建表达 5C1scFv的载体pSTE 5C1,在大肠杆菌中诱导表达 ,金属离子螯和亲和层析法纯化 ,获得高纯度的活性 5C1scFv ;借助生物素 链亲和素的高亲和力和高灵敏度的显色系统 ,用Westernblot分析其识别的KG1a细胞膜蛋白的Mr;用聚集实验分析 5C1scFv对KG1a细胞同型聚集的影响。结果 5C1scFv在大肠杆菌中获高效表达 ,纯化后活性蛋白产量可达每升培养物 5 0～ 6 0mg ,纯度大于 95 % ;成功地测定了其识别抗原的Mr 为 (85 .0 /12 4.5 )× 10 3;并确认 5C1scFv以浓度依赖方式抑制KG1a细胞的同型聚集。结论 高效表达纯化的 5C1scFv特异性识别Mr 为 (85 .0 /12 4.5 )× 10 3 的KG1a细胞膜表面分子 ,此分子可能是一种在KG1a细胞表面表达的参与细胞同型聚集的分子或受体。这些结果为进一步深入研究抗原本质及克隆抗原分子基因奠定了良好的基础
OBJECTIVE: To further investigate the function of specific scFv derived from phage antibody library and the molecular characteristics of its recognition antigen, 5C1scFv, a single chain antibody recognizing KG1a cells, was efficiently expressed and purified. The relative molecular mass of unknown antigen Mr); and 5C1scFv on KG1a part of the biological characteristics of cells. Methods The recombinant plasmid pSTE 5C1 expressing 5C1scFv was constructed and expressed in Escherichia coli. The purified 5C1 scFv was purified by metal ion chelation and affinity chromatography. With the high affinity and high sensitivity of biotinylated streptavidin Color system, Western blot analysis of KG1a cell membrane protein recognition Mr; aggregation experiment analysis of 5C1scFv KG1a cells with the type of aggregation. Results 5C1scFv was highly expressed in E.coli. The purified protein could reach 50 ~ 600mg per liter of culture with a purity of more than 95%. The McAb was successfully assayed for the antigen (85 .0 / 12 4.5) × 10 3. It was also confirmed that 5C1scFv inhibited the islet aggregation of KG1a cells in a concentration-dependent manner. CONCLUSION: The highly expressed and purified 5C1scFv specifically recognizes Mr1 (85.0 / 12 4.5) × 10 3 cell surface molecules of KG1a, which may be a molecule or receptor involved in the homolocation of cells expressed on the surface of KG1a cells. These results lay a good foundation for further study of antigenicity and molecular cloning of antigen