Furuta M, Uchikawa M, Ueda Y, Yabe T, Taima T, Tsumoto K, Kojima S, Juji T, Kumagai I
Japanese Red Cross Central Blood Center, Hiroo, Tokyo.
Protein Eng. 1998 Mar;11(3):233-41. doi: 10.1093/protein/11.3.233.
An expression system for mono- and bivalent single-chain Fv fragments (scFv) of a human antibody against D antigen in the Rh blood group system was established in Escherichia coli. The cDNA encoding the Fv fragment of the anti-D monoclonal antibody D10 was cloned using the polymerase chain reaction and expressed in E.coli by fusing with a peptide tag link in the C-terminus of the light chain variable region. The scFv fragment expressed by the bacteria produced specific agglutination of human D positive red cells in the presence of an anti-peptide tag antibody. Flow cytometric analysis clearly indicated that the bacterially prepared scFv showed high specificity and affinity for D antigen, which was identical with that of the parental IgG. In order to construct bivalent D10 scFv for use in direct cell agglutination, the scFv was fused with a dimeric protein, bacterial alkaline phosphatase (BAP). The fusion protein produced significant agglutination of human red blood cells with D antigen, confirming that the bacterially expressed fusion protein is a functional bivalent antibody fragment. Specific agglutination of D positive red cells by D10 scFv-BAP was enhanced in the presence of anti-BAP antibody, suggesting that further multimerization of scFv led to highly efficient cell agglutination. By grafting BAP enzymatic activity into the scFv fragment (enzyme-linked scFv), blood typing could conveniently be performed. These results indicate that bacterially expressed scFv and scFv-BAP would be of practical use in blood typing. The system reported here could also be applied to the examination of other cell surface antigens and cell agglutination.
在大肠杆菌中建立了一种表达系统，用于表达针对Rh血型系统中D抗原的人源抗体的单价和双价单链Fv片段（scFv）。利用聚合酶链反应克隆编码抗D单克隆抗体D10的Fv片段的cDNA，并通过与轻链可变区C末端的肽标签连接体融合，在大肠杆菌中表达。在抗肽标签抗体存在的情况下，细菌表达的scFv片段能使人D阳性红细胞产生特异性凝集。流式细胞术分析清楚地表明，细菌制备的scFv对D抗原具有高特异性和亲和力，这与亲本IgG相同。为了构建用于直接细胞凝集的双价D10 scFv，将scFv与二聚体蛋白细菌碱性磷酸酶（BAP）融合。融合蛋白能使人D抗原红细胞产生明显凝集，证实细菌表达的融合蛋白是一种有功能的双价抗体片段。在抗BAP抗体存在的情况下，D10 scFv-BAP对D阳性红细胞的特异性凝集增强，表明scFv的进一步多聚化导致高效细胞凝集。通过将BAP酶活性嫁接到scFv片段（酶联scFv）中，可以方便地进行血型鉴定。这些结果表明，细菌表达的scFv和scFv-BAP在血型鉴定中具有实际应用价值。本文报道的系统也可应用于其他细胞表面抗原的检测和细胞凝集。
