摘要
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。
Objective To express and purify a single chain antibody (scFv)5C1 which specifically reacted to acute myelogenous leukemia cell line KG1a cell. To determine the apparent molecular weight ( M r) of 5C1 scFv′s corresponding antigen and study the effect of 5C1 scFv on the homoaggregation of KG1a cells. Methods An expression vector pSTE 5C1 was constructed by DNA recombination. Expression of the 5C1 scFv was induced by IPTG in E.coli , and the expressed product was purified with immobilized metal affinity chromatography (IMAC) from periplasmic inclusion bodies. With the purified scFv, the apparent molecular weight of 5C1 antigen was determined by an improved Western blotting assay. The homoaggregation effect of 5C1 on KG1a cells were investigated by a modified semiquantitative method. Results The 5C1 scFv was expressed highly efficiently in E.coli , the yield of purified protein was up to 50 60mg/L cultures. Two protein bands with apparent M r of 85.0×10 3 and 124.5×10 3 were visible in cell lysates of KG1a in Western blotting using the 5C1 scFv. The 5C1 scFv could markedly block homoaggregate formation of KG1a cells in a dose dependent manner. Conclusions The single chain antibody 5C1 was specifically reactive to a KG1a cell surface molecule with apparent M r of (85.0/ 124.5 )×10 3, which may be a cell surface molecule or receptor involved in the homoaggregation of KG1a cells. This result is important for further study of molecular characterization and cloning cDNA of 5C1 antigen.
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2001年第4期437-441,共5页
Chinese Journal of Microbiology and Immunology
