噬菌体展示法筛选与EphB2结合的短肽  

Identification of the Peptides that Bind to Tyrosine Kinase Receptor EphB2 by Phage Display

作　　者：张晓光[1] 药立波[1] 刘新平[1] 韩炯[1] 韩景田[2] 聂晓燕[1] 苏成芝[1] 

机构地区：[1]第四军医大学生物化学与分子生物学教研室,西安710032 [2]武警医学院微生物教研室,天津300162

出　　处：《生物化学与生物物理学报》2000年第5期475-479,共5页

基　　金：国家杰出青年科学基金资助项目!No .3 982 5 113;国家自然科学基金资助项目!No .3 980 0 0 2 7&&

摘　　要：酪氨酸蛋白激酶受体EphB2的Ig类似区 ,克隆到融合表达质粒载体 pET2 8a中 ,阳性克隆经IPTG诱导 ,表达出了氨基端带 6个连续组氨酸残基的融合蛋白。利用Ni NTA金属螯合亲和层析法在变性条件下对表达的蛋白进行纯化 ,纯度大于 94%。以此纯化蛋白为靶 ,将其包被于ELISA板上 ,经过三轮亲和筛选 ,从噬菌体随机 7肽库中筛选到 19个具有结合EphB2活性的重组噬菌体克隆 ,对阳性噬菌体克隆的短肽序列进行了分析。The gene encoding the Ig-like domain of tyrosine protein kinase rece ptor EphB2 was cloned into the expressing vector pET28a. Under induction with IP TG, the positive strain expressed the fusion protein with a hexahistidine tail o n the N-terminal. The protein was purified under denaturing conditions using me tal chelate chromatography. The purity was up to 94%. The purified-protein-coa ted ELISA plate was used as target to screen recombinant phages able to bind onto i t; and after three rounds of affinity screening, 19 phages that could bind spe ci fically with EphB2 were isolated from a random phage-displayed seven-peptide l ibrary. The peptide sequences of the positive phage clones were analyzed.

关 键 词：EPHB2 金属螯合层析 噬菌休展示 随机短肽库 

分 类 号：Q786[生物学—分子生物学]