天花粉蛋白结合肽的筛选及潜在靶点蛋白的同源分析  

Binding Peptide Screening and Potential Target Protein Identification of Trichosanthin

作　　者：赵峻[1] 时磊[1] 张芳[2] ZHAO Jun;SHI Lei;ZHANG Fang(ThE-Fifth Affiliated Hospital of Zhengzhou University,Zhengzhou,450052,China;School of Pharmacy,Nanjing University of ChinesE-Medicine,Nanjing,210023,China)

机构地区：[1]郑州大学第五附属医院,河南郑州450052 [2]南京中医药大学药学院,江苏南京210023

出　　处：《南京中医药大学学报》2020年第1期73-77,共5页Journal of Nanjing University of Traditional Chinese Medicine

基　　金：国家自然科学基金青年基金(81102899,81773837)

摘　　要：目的通过噬菌体展示技术,筛选天花粉蛋白(TCS)的结合多肽,并通过蛋白质同源分析,研究TCS在体内可能直接作用的靶点蛋白。方法用构建的十五肽库和购买的十二肽库分别对TCS进行4轮固相亲和筛选,并通过噬菌体ELISA检验阳性克隆与靶蛋白的亲和力,对阳性克隆进行测序,并将多肽序列与已登记蛋白序列进行同源性分析。结果每轮筛选的回收率,及多克隆噬菌体ELISA结果显示筛选有效。通过单克隆噬菌体ELISA结果挑取阳性克隆,测序分析得到若干噬菌体展示多肽序列。经蛋白质同源性分析,TCS特异性结合多肽与蛋白激酶C(PKC)的磷酸化位点具有同源序列。结论噬菌体展示技术是筛选中药成分靶点蛋白的有效手段,PKC可能为TCS潜在的靶点蛋白。OBJECTIVE To screen binding peptides and identify potential target protein of trichosanthin(TCS)by homology analysis.METHODS TCS was used to pan against the fifteen peptide library and a random twelve peptide library by solid-phase screening,respectively.A series of positive clones that bind specifically to TCS were obtained after 4 rounds biopanning process,and the monoclonal phage ELISA was used to test the affinity between positive clones and TCS.The amino acid sequence of positive peptides was sequenced and homology analysis was performed to identify the potential target protein of TCS.RESULTS The yield and polyclonal phage ELISA showed that the panning was effective.Positive clones were picked by monoclonal ELISA and the displayed peptide sequence was identified by sequencing.Some proteins that had homology with registered proteins in PROSITE and BLAST were found.The potential target protein of TCS probably was protein kinase C.CONCLUSION Phage display random peptide library technology is a powerful method for exploring the target protein of traditional Chinese medicine ingredients.Protein kinase C probably is the target protein of TCS.

关 键 词：噬菌体展示 天花粉蛋白 筛选 同源分析 

分 类 号：R285.5[医药卫生—中药学]