【摘要】 目的　构建大容量噬菌体单链抗体库 ,从中筛选人源单链抗体 (ScFv)。方法　从正常成人外周血和新生儿脐血分离淋巴细胞 ,用RT PCR扩增轻链可变区基因 (VL)和重链可变区基因(VH) ,通过重叠PCR法将VH 和VL 拼接形成ScFv基因 ,并克隆入噬菌体表达载体PDF ,得到ScFv初级噬菌体抗体库。以高MOI超感染cre+ 菌株BS136 5 ,通过loxp cre定位重组系统 ,介导轻重链的组合配对 ,得到大容量抗体库 ,用多种抗原对抗体库进行生物淘筛 ,鉴定抗体库的性能。结果　获得了 6×10 10 的大容量单链噬菌体抗体库。分别用卵清蛋白、胃蛋白酶、铁蛋白、人角蛋白、人TNF α、地高辛等6种抗原进行筛选 ,均得到多样性的特异性噬菌体抗体。结论　经loxp cre定位重组系统在单细胞内重组成功地构建了大容量单链噬菌体抗体库 ,初步尝试对 6种抗原进行筛选均获成功 ,提示该抗体库可用于制备具有应用前景的人源抗体更多还原

【Abstract】 Objective To construct a large single chain phage antibody library for human single chain antibody cloning. Methods Diverse V L and V H genes were amplified by RT PCR from lymphocytes collected from adult peripheral blood and new born cord blood. The V genes were spliced to form ScFv by overlap PCR and cloned into vector PDF, obtaining a primary library in which the V H genes were flanked by two non homologous loxp sites. The V H and V L genes in this primary library were shuffled by infecting the phagemid into cre expressing bacteria at high multiplicity of infection(MOI) to create a very large phage antibody library. The library was tested by selecting human antibodies against various antigens through bio panning. Results A large phage antibody of 6×10 10 in size was constructed. Diverse specific human ScFvs against all the 6 tested antigens were obtained from the library by biopanning. Conclusion The loxp cre mediated recombination in single bacteria cell is efficient in constructing very large phage antibody library. The success in cloning various human antibodies suggests that the constructed phage antibody library can be used in human antibody preparation.更多还原