人源血清白蛋白结合二价肽的设计

发布时间：2018-04-29 07:19

本文选题：人血白蛋白 + 噬菌体展示库　；参考：《湖北工业大学》2017年硕士论文

【摘要】：人血清白蛋白(Human Serum Albumin,HSA)是一种药物递送的天然载体。近年来,HSA已广泛用于延长多肽药物的半衰期,取得了较大的进展。本实验室前期研究,利用Ph.D.-12噬菌体展示肽库技术淘洗出一种与HSA结合力较高的小肽(称ABD6肽,LPHSHRAHSLPP,Kd值1.67μmol/L),并成功地运用于介导GLP-1药物缓释。缓释多肽药物的途径是在HSA与多肽间引入一段高亲和HSA的结合肽,结合肽-多肽融合物与HSA可逆性结合,这种新型多肽缓释方法能延长多肽药物半衰期。本研究基于多价分子热力学原理,即△G=△G_1+△G_2→lnK_(d1)+lnK_(d2)→K_(d1)×K_(d2),探索设计一种含ABD6肽序列的HSA亲和二价肽,在邻近ABD6肽结合HSA位点处再延伸一段HSA亲和肽,课题采用两种策略来延长ABD6肽序列以获得特异性结合HSA的二价肽。其一,利用M13噬菌体展示技术在ABD6肽序列的N端延伸5个随机氨基酸,构建噬菌体展示二价肽库,用于筛选HSA亲和二价肽。其二,利用结构生物学方法,制备ABD6肽与HSA复合物晶体,通过解析复合物晶体的三维结构,研究ABD6肽与HSA的结合机制,为合理延伸ABD6肽序列提供结构学依据。噬菌体展示二价肽文库的构建分为两个部分,一是,在噬菌体质粒M13KE中先插入ABD6肽序列,再引入5个随机氨基酸;二是,直接将ABD6肽与5个随机氨基酸的融合肽插入到M13KE中。目前已将ABD6肽序列正确插入到M13KE中。另一方面,复合物晶体制备实验,利用悬滴和座滴两种结晶方法,均获得了大量的HSA单晶体,结晶条件为150~200 mg HSA,28%~35%PEG3350和pH7.5,50 mmol/L磷酸钾溶液。HSA晶体浸泡在9 mmol ABD6肽溶液中很稳定,利用20%甘油与结晶液用做冷冻保护剂,该方法为进一步制备复合物晶体提供基础。本课题致力于获得特异性结合HSA的二价肽,多肽药物以非共价键与HSA结合的载药方式对开发多肽药物有促进作用,二价肽还可作为亲和配体与HSA结合,用于大规模初步纯化HSA。
[Abstract]:Human Serum albumin (HSA) is a natural carrier for drug delivery. In recent years, HSA has been widely used to prolong the half-life of polypeptide drugs, and has made great progress. In our previous study, a small peptide (ABD6 peptide) with high binding ability to HSA was obtained by using Ph.D.-12 phage display peptide library technique. The KD value of LPHSHRAHSLPPN was 1.67 渭 mol / L, and was successfully used to mediate the sustained release of GLP-1. The way of sustained-release polypeptide drug is to introduce a high affinity HSA binding peptide between HSA and polypeptide, and combine peptide fusion with HSA reversibility. This new method can prolong the half life of polypeptide drug. This study is based on the multivalent molecular thermodynamics principle, that is, G = G1G / T _ 2 / lnK / T _ 1) lnK / D _ 2) 脳 K _ T _ d _ 2). We explore the design of a HSA affinity divalent peptide containing ABD6 peptide sequence, and extend a HSA affinity peptide near the ABD6 peptide binding HSA site. Two strategies were used to extend the sequence of ABD6 peptides to obtain bivalent peptides specifically bound to HSA. Firstly, using M13 phage display technique to extend 5 random amino acids in the N-terminal of ABD6 peptide sequence, a phage display bivalent peptide library was constructed to screen HSA affinity bivalent peptide. Secondly, the crystal of ABD6 peptide and HSA complex was prepared by structural biology method. By analyzing the three-dimensional structure of the complex crystal, the binding mechanism of ABD6 peptide and HSA was studied, which provided the structural basis for the rational extension of ABD6 peptide sequence. The construction of phage display bivalent peptide library is divided into two parts: first, insert ABD6 peptide sequence into phage display plasmid M13KE, then introduce 5 random amino acids; second, insert ABD6 peptide and 5 random amino acid fusion peptides into M13KE directly. The ABD6 peptide sequence has been inserted into M13KE correctly. On the other hand, in the preparation of complex crystals, a large number of HSA single crystals were obtained by means of both suspension and drop crystallization methods. The crystallization conditions were 150 ~ 200mg HSA-28g ~ (28) PEG3350 and pH7.550 mmol/L potassium phosphate solution .HSA crystal was stable in 9 mmol ABD6 peptide solution. Using 20% glycerol and crystallization solution as freezing protectant, this method provides a basis for further preparation of complex crystals. The aim of this study is to obtain bivalent peptides specifically bound to HSA. Polypeptide drugs can promote the development of polypeptide drugs in the form of non-covalent binding with HSA. Divalent peptides can also be used as affinity ligands to bind to HSA, and can be used to purify HSA on a large scale.
【学位授予单位】：湖北工业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：TQ460.1

【参考文献】