Santi E, Capone S, Mennuni C, Lahm A, Tramontano A, Luzzago A, Nicosia A
Istituto di Ricerche di Biologia Molecolare P. Angeletti, Via Pontina Km 30.600;, Roma, 00040 Pomezia, Italy.
J Mol Biol. 2000 Feb 18;296(2):497-508. doi: 10.1006/jmbi.1999.3471.
We describe the construction and characterization of two lambda surface displayed cDNA expression libraries derived from human brain and mouse embryo. cDNA inserts were obtained by tagged random-priming elongation of commercially available cDNA libraries and cloned into a novel lambda vector at the 3' end of the D capsid protein gene, which produced highly complex repertoires (1x10(8) and 2x10(7) phage). These libraries were affinity selected with a monoclonal antibody against the neural specific factor GAP-43 and with polyclonal antibodies that recognize the EMX1 and EMX2 homeoproteins. In both cases rapid identification of specific clones was achieved, which demonstrates the great potential of the lambda display system for generating affinity selectable cDNA libraries from complex genomes.
我们描述了从人脑和小鼠胚胎中构建并鉴定的两个λ噬菌体表面展示的cDNA表达文库。通过对市售cDNA文库进行标记随机引物延伸获得cDNA插入片段，并将其克隆到新型λ载体中D衣壳蛋白基因的3'端，产生了高度复杂的文库（分别为1×10⁸和2×10⁷个噬菌体）。这些文库用抗神经特异性因子GAP - 43的单克隆抗体以及识别EMX1和EMX2同源结构域蛋白的多克隆抗体进行亲和筛选。在这两种情况下都实现了对特定克隆的快速鉴定，这证明了λ噬菌体展示系统在从复杂基因组中生成亲和选择的cDNA文库方面具有巨大潜力。
