Houimel M, Corthesy-Theulaz I, Fisch I, Wong C, Corthesy B, Mach J, Finnern R
Institute of Biochemistry, University of Lausanne, Epalinges, Switzerland.
Tumour Biol. 2001 Jan-Feb;22(1):36-44. doi: 10.1159/000030153.
Single chain Fv antibody fragments (scFv) binding to purified Helicobacter pylori urease were selected from a nonimmune human antibody repertoire displayed on filamentous phage. After three rounds of screening on solid phase urease, 44 clones were found to bind the enzyme and four distinct scFv were identified by sequencing their heavy and light chain variable region genes (V(H) and V(L)). Two of the selected human scFv (scFv B4 and scFv D9) inhibited the activity of H. pylori urease with inhibitory constants (K(i)) of 7 and 2 microM, respectively. Their affinity (K(d)) for H. pylori urease as determined by surface plasmon resonance ranged from 17 to 42 nM. Both scFv were able to bind to urease present on the surface of living H. pylori organisms as demonstrated by flow cytometry analysis. The binding sites of scFv B4 and D9 were mapped by the use of two random hexapeptide libraries (X6 and CX6C) displayed on filamentous bacteriophage. The selected peptide sequences were shown to inhibit scFv binding to H. pylori urease and thus could be used in a vaccination strategy as epitopes mimicking (mimotopes) the region of urease recognized by these human scFv antibody fragments.
从展示在丝状噬菌体上的非免疫人抗体库中筛选出与纯化的幽门螺杆菌脲酶结合的单链Fv抗体片段（scFv）。在对固相脲酶进行三轮筛选后，发现44个克隆与该酶结合，并通过对其重链和轻链可变区基因（V(H)和V(L)）进行测序鉴定出4种不同的scFv。所选择的两种人scFv（scFv B4和scFv D9）抑制幽门螺杆菌脲酶的活性，抑制常数（K(i)）分别为7和2 microM。通过表面等离子体共振测定，它们对幽门螺杆菌脲酶的亲和力（K(d)）在17至42 nM之间。流式细胞术分析表明，这两种scFv都能够与活的幽门螺杆菌表面存在的脲酶结合。通过使用展示在丝状噬菌体上的两个随机六肽库（X6和CX6C）对scFv B4和D9的结合位点进行了定位。所选择的肽序列显示出抑制scFv与幽门螺杆菌脲酶的结合，因此可以作为模拟这些人scFv抗体片段识别的脲酶区域的表位（模拟表位）用于疫苗接种策略。
