摘要
目的应用噬菌体随机七肽库生物淘选人角质细胞生长因子(keratinocyte growth factor,KGF)噬菌体模拟肽。方法以人KGF单克隆抗体为靶,对噬菌体随机七肽库进行4轮生物淘选,测定噬菌体滴度。随机挑取单克隆噬菌体行ELISA检测其结合力,对结合活性较好的单克隆噬菌体提取噬菌体DNA,测序,并应用Basic BLAST系统行序列相似性和同源性分析。结果经过4轮生物淘选获得的噬菌体滴度逐渐升高,特异性噬菌体模拟肽获得了富集,行ELISA检测的单克隆噬菌体滴度均可达到2.0×1014 pfu/mL。取吸光度值显示与特异性抗体具有较好结合性的单克隆噬菌体进行测序,共获得26个与促进分裂、生长有关的碱基序列,其中有2个序列与人KGF相似;同源序列分析显示26个碱基序列的共同序列与人KGF的部分序列相似。结论从噬菌体随机七肽库中可淘选到与人KGF DNA序列相似或相关的噬菌体模拟肽,有望改善人KGF性能,促进创面愈合和改善组织工程皮肤替代物的性能。
Objective To harvest human keratinocyte growth factor （KGF） mimic peptides with Ph.D.-7^TM phage display peptide library. Methods Ph.D.-7^TM phage display peptide library was biopanned for 4 rounds to harvest monoclonal anti-body human KGF and then phage tilter was detected. ELISA detection was performed to detect the binding force of random-selected monoclonal phages, thereafter DNA extracted from phages with better binding activity was sequenced and the Basic BLAST system was applied to conduct the sequence similarity and homology analysis. Results After 4 rounds of biopanning, the titer of phages was increased gradually and the enrichment of specific phage mimic peptides was obtained. The titers of monoclonal phages were up to 2.0 × 10^14 pfu/mL according to ELISA detection. According to the absorbance value, the monoclonal phages with better binding activities to certain specific antibodies were sequenced, and 26 base sequences related to the promotion of division and growth were verified, 2 of which were similar to human KGF. Homology sequence analysis revealed that the common sequence of those 26 base sequences was similar to the partial sequences of human KGF. Conclusion The phage mimic peptides resembling or related to human KGF DNA can be harvested from Ph.D.-7TM phage display peptide 1 ibrary, which may be conducive to improve tissue engineered skin substitutes.
出处
《中国修复重建外科杂志》
CAS
CSCD
北大核心
2009年第2期183-187,共5页
Chinese Journal of Reparative and Reconstructive Surgery
基金
国家自然科学基金资助项目(30670571
30772258)
山东省教育厅科研发展计划资助项目(J07WD03)~~
关键词
噬菌体随机七肽库
人角质细胞生长因子模拟肽
生物淘选
测序
Sequencing Ph.D.-7TM phage display peptide library human KGF performance, wound healing and the quality of Human keratinocyte growth factor mimic peptide Biopanning
