应用噬菌体抗体库技术制备特异抗人纤维蛋白鼠单链抗体

Making Specific Against Human Fibrin Mouse Single-Chain Fv Fragment (ScFv) by Phage Antibody Library

应用Pharmacia公司的重组噬菌体抗体系统,从经过人交联纤维蛋白特异抗原D二聚体(DD)免疫过的鼠脾细胞mRNA中构建出组合单链抗体(ScFv)cDNA文库。文库cDNA克隆到噬菌粒载体pCANTAB5E,转化大肠杆菌TG1,得到2.5×10~5个氨苄抗性菌落。通过噬菌体表面呈现,用DD对表达的重组噬菌体单链抗体文库进行三轮亲和富集获得一株特异抗DD的噬菌体单链抗体(ScFvA11)。经Phage-ELISA鉴定,呈现在噬菌体表面的ScFvA11与DD结合的ELISA阳性滴度小于10~7tfu/ml,而与人纤维蛋白原结合的ELISA滴度大于10^(10)tfu/ml,两者相差1000倍以上。表明ScFvA11具有较好的DD结合特异性。经序列分析,ScFvA11cDNA全长729bp,其中Vh基因354bp,编码118个氨基酸;Vl基因327bp,编码108个氨基酸;Vh与Vl之间为(Gly_4Ser)_315个氨基酸连接肽。

We used the recombinant phage antibody system to construct a combinatory single-chain Fv fragment (ScFv) cDNA library from a human cross-linked fibrin specific antigen D -dimer(DD) immunized mouse splenic mRNA. The library cDNA was cloned into a phagemid vector pCANTABSE and introduced into E. Coli TGI. 2. 5 × 105 ampicillin resistant clones were obtained. By phage displaying.expressed ScFvs were displayed on the surface of phage particle. Panning against DD coated on the solidate surface by three rounds,a number of DD binding phage-ScFvs were collected. From these ellected clones, one phage-ScFv .ScFv A11, was found to be more specific for binding DD than for binding human fibrinogen. The titer of Phage-ELISA of ScFvAllfor binding DD is lower than 107tfu/ml .while for binding human fibrinogen is higher than 1010tfu/ml. The differance is more than 1000-fold. By sequencing, the entire ScFvAll cDNA is 729bp.consisting of Vh gene 354bp .encoding 118 animo acids; Vl gene 327bp, encoding 109 animo acids. Between Vh and Vl is a 45bp long linker DNA,encoding a (Gly4Ser)3 linker pep-tid.