本文選題：人血白蛋白 + 噬菌體展示庫(kù)��； 參考：《湖北工業(yè)大學(xué)》2017年碩士論文

【摘要】：人血清白蛋白(Human Serum Albumin,HSA)是一種藥物遞送的天然載體。近年來(lái),HSA已廣泛用于延長(zhǎng)多肽藥物的半衰期,取得了較大的進(jìn)展。本實(shí)驗(yàn)室前期研究,利用Ph.D.-12噬菌體展示肽庫(kù)技術(shù)淘洗出一種與HSA結(jié)合力較高的小肽(稱(chēng)ABD6肽,LPHSHRAHSLPP,Kd值1.67μmol/L),并成功地運(yùn)用于介導(dǎo)GLP-1藥物緩釋。緩釋多肽藥物的途徑是在HSA與多肽間引入一段高親和HSA的結(jié)合肽,結(jié)合肽-多肽融合物與HSA可逆性結(jié)合,這種新型多肽緩釋方法能延長(zhǎng)多肽藥物半衰期。本研究基于多價(jià)分子熱力學(xué)原理,即△G=△G_1+△G_2→lnK_(d1)+lnK_(d2)→K_(d1)×K_(d2),探索設(shè)計(jì)一種含ABD6肽序列的HSA親和二價(jià)肽,在鄰近ABD6肽結(jié)合HSA位點(diǎn)處再延伸一段HSA親和肽,課題采用兩種策略來(lái)延長(zhǎng)ABD6肽序列以獲得特異性結(jié)合HSA的二價(jià)肽。其一,利用M13噬菌體展示技術(shù)在ABD6肽序列的N端延伸5個(gè)隨機(jī)氨基酸,構(gòu)建噬菌體展示二價(jià)肽庫(kù),用于篩選HSA親和二價(jià)肽。其二,利用結(jié)構(gòu)生物學(xué)方法,制備ABD6肽與HSA復(fù)合物晶體,通過(guò)解析復(fù)合物晶體的三維結(jié)構(gòu),研究ABD6肽與HSA的結(jié)合機(jī)制,為合理延伸ABD6肽序列提供結(jié)構(gòu)學(xué)依據(jù)。噬菌體展示二價(jià)肽文庫(kù)的構(gòu)建分為兩個(gè)部分,一是,在噬菌體質(zhì)粒M13KE中先插入ABD6肽序列,再引入5個(gè)隨機(jī)氨基酸;二是,直接將ABD6肽與5個(gè)隨機(jī)氨基酸的融合肽插入到M13KE中。目前已將ABD6肽序列正確插入到M13KE中。另一方面,復(fù)合物晶體制備實(shí)驗(yàn),利用懸滴和座滴兩種結(jié)晶方法,均獲得了大量的HSA單晶體,結(jié)晶條件為150~200 mg HSA,28%~35%PEG3350和pH7.5,50 mmol/L磷酸鉀溶液。HSA晶體浸泡在9 mmol ABD6肽溶液中很穩(wěn)定,利用20%甘油與結(jié)晶液用做冷凍保護(hù)劑,該方法為進(jìn)一步制備復(fù)合物晶體提供基礎(chǔ)。本課題致力于獲得特異性結(jié)合HSA的二價(jià)肽,多肽藥物以非共價(jià)鍵與HSA結(jié)合的載藥方式對(duì)開(kāi)發(fā)多肽藥物有促進(jìn)作用,二價(jià)肽還可作為親和配體與HSA結(jié)合,用于大規(guī)模初步純化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.
【學(xué)位授予單位】：湖北工業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：TQ460.1

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