【摘要】 目的:构建大容量易于改造成Diabody的噬菌体单链抗体库,筛选人源性抗体。方法:从正常成人外周血和新生儿脐血中分离淋巴细胞提取RNA,经RT-PCR扩增轻重链可变区基因(VL和VH),通过重叠PCR(over-lapPCR)将VL和VH拼接成单链抗体(ScFv)基因(其中VH两侧是两个非同源的loxp基因),并克隆入噬菌体表达载体PDF,得到初级噬菌体抗体库。将初级抗体库以高MOI超感染Cre+菌株BS1365,通过loxp-cre定位重组系统,介导轻重链在菌内重组配对,然后低感染XL1-Blue菌,得到大容量的次级抗体库。以多种不同抗原对库进行筛选,得到多样性较好的特异性抗体。结果:经超感染重组,得到1·2×1010的大容量抗体库。经三种蛋白抗原筛选,均得到多株特异性较好的噬菌体抗体,并成功构建为活性较好的Diabody。结论:经Loxp-Cre定位重组系统在单细胞内重组,能够高效地构建大容量噬菌体单链抗体库。更多还原

【Abstract】 Objective:To create a large human phage antibody library from which easy to get diabody and select human antibody clones.Methods:VL and VH genes were amplified by RT-PCR from RNA which came from normal adult peripheral blood and new-born cord blood lymphocytes. VL and VH genes were reamplified to add a region of overlap in the ScFv linker to form ScFv genes in which VH genes were flanked by two non-homologous loxp sites. The ScFv genes were cloned into PDF to obtain a primary library. This primary library was used to infect bacteria Bs1365(expressing cre recombinase) with high multiplicity of infection(MOI) 200∶1. This procedure resulted in a very large phage antibody library through VL and VH recombined by the cre recombinase. Following recombination, phagemide were derived from these bacteria and used to infect bacteria not expressing cre(XL1-Blue) at MOI<1 to couple phenotype and genetype, then the library was tested by selection on a number of different protein antigens.Results:A 1.2×10~ 10 phage antibody library was created by cre-loxp mediated recombination. Antibodies were obtained against all three different protein antigens and specifically recognized their antigens. A functional diabody was constructed successfully.Conclusion:It is efficient way in creating very large phage ScFv library using loxp-cre mediated recombination in single bacteria cell.更多还原