Dübel S, Breitling F, Fuchs P, Braunagel M, Klewinghaus I, Little M
Division of Diagnosis and Experimental Therapy, German Cancer Research Center, Heidelberg.
Gene. 1993 Jun 15;128(1):97-101. doi: 10.1016/0378-1119(93)90159-z.
Expression vectors for surface display and production of single-chain (Fv) antibodies (scAb) have been constructed based on the phagemid pSEX, which expresses DNA encoding a scAb fused to the gene III product of filamentous phage [Breitling et al., Gene 104 (1991) 147-153]. A smaller version of this phagemid, pSEX20, was made by removing an unnecessary cat. To produce a vector for the surface display of other proteins and peptides, the scAb of pSEX20 was substituted by a polycloning site (MCS) to give pSEX40. For the presentation of Ab on the surface of Escherichia coli, phagemid pAP10 was derived from pSEX20 by substituting gene III with a gene encoding the peptidoglycan-associated lipoprotein (PAL). Vectors for producing scAb that can be purified by antibody and metal affinity chromatography were constructed by substituting gene III in the vector pSEX20 with DNA encoding a peptide with a C-terminal epitope recognised by a monoclonal antibody (phagemid pOPE40) or with five C-terminal histidines (pOPE 90).
基于噬菌粒pSEX构建了用于单链(Fv)抗体(scAb)表面展示和生产的表达载体,该噬菌粒表达编码与丝状噬菌体基因III产物融合的scAb的DNA[Breitling等人,《基因》104(1991)147 - 153]。通过去除不必要的cat构建了该噬菌粒的较小版本pSEX20。为了构建用于其他蛋白质和肽表面展示的载体,将pSEX20的scAb替换为多克隆位点(MCS),得到pSEX40。为了在大肠杆菌表面展示抗体,噬菌粒pAP10由pSEX20衍生而来,通过将基因III替换为编码肽聚糖相关脂蛋白(PAL)的基因。通过将载体pSEX20中的基因III替换为编码具有单克隆抗体识别的C端表位的肽的DNA(噬菌粒pOPE40)或五个C端组氨酸(pOPE 90),构建了可通过抗体和金属亲和色谱纯化的scAb生产载体。