短肽库中血管内皮细胞生长因子受体Flt-1拮抗剂筛选条件的改进

目的:改进噬菌体肽库的筛选条件,提高筛选阳性率及阳性克隆重复性.使血管内皮细胞生长因子受体Flt-1拮抗剂的筛选更为有效.方法:通过对血管内皮细胞生长因子受体Flt-1的原核表达产物的变性复性处理,获得相对纯化的可溶性Flt-1受体.将该可溶性受体固相化,对噬菌体表达12肽库进行4 ～ 5轮Bio-panning后,挑取单个噬菌斑制备高滴度噬菌体上清.用ELISA法初步鉴定各噬菌体克隆与sFlt-1的特异性结合并测定阳性克隆的DN A 顺序.在Bio-panning过程中增加非特异性蛋白的预吸附,并减少输入噬菌体数量, 再次筛选短肽库.结果:经过第一次筛选,获得了一系列具有一定重复性与同源性的噬菌体克隆. 改进筛选条件后,筛选阳性率由2%提高到8.3%,阳性克隆的重复性也得到显著提高.该高重复阳性克隆的氨基酸顺序与前一次筛选获得的阳性克隆有较高同源性.结论:Bio-panning 条件的改进提高了筛选效率,为不纯蛋白筛选短肽库积累了经验,并为血管内皮细胞生长因子受体拮抗剂的研制奠定了基础.

The improvement in screening for vascular endothe lial growth factor receptor Flt-1 antagonists in a phage display library

Objective: To increase the positive rate of screening for VEGF (vascular endothelial growth factor) receptor antagonist and obtain more desirable positive clones. Methods: Soluble VEGF receptor Flt 1 was first prepared through denaturation and refolding, then used to screen a phage display 12 mers peptide library for obtaining the clones which could mimic the binding of VEGF to receptor Flt 1. After 4-5 rounds of bio panning, single positive clones were further determined by phage ELISA. The amino acid sequence of the positive peptides was obtained through DNA sequencing. To obtain more specific and higher affinity peptide clones binding to Flt 1, the peptide library was pre absorbed with non specific protein before bio panning and more less eluted phages were added after second round bio panning for next round screening in the modified procedure. Results: After pre absorption and decreasing the input phages, the positive clone rate was increased from 2% to 8.3% and repetition of the positive clones increased remarkably. Conclusion: The modified bio panning could increase the efficiency of peptide library screening greatly.