新型人血清白蛋白特异结合肽ML的筛选及鉴定

doi:10.13523/j.cb.20150511

中国生物工程杂志

2015, Vol. 35

Issue (5): 74-80 DOI: 10.13523/j.cb.20150511

技术与方法

新型人血清白蛋白特异结合肽ML的筛选及鉴定

马义, 赵绍军, 洪岸

暨南大学细胞生物学系暨南大学生物医药研究院广东省生物工程药物重点实验室基因工程药物国家工程研究中心广州 510632

Screening and Identification of the New Human Serum Albumin-specific Binding Peptides

MA Yi, ZHAO Shao-jun, HONG An

Department of Cell Biology of Jinan University, Institute of Biological Medicine of Jinan University, Guangdong Provincial Key Laboratory of Bioengineering Medicine, National Engineering Research Center of Genetic Medicine, Guangzhou 510632, China

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摘要：

利用噬菌体展示技术筛选和鉴定新型人血清白蛋白(HSA)特异结合7肽,分析与垂体腺苷酸环化酶激活肽27(PACAP27)构成的融合多肽 ML-PACAP27与HSA的亲和结合力,以确定筛选的7肽 (ML1 和 ML2)在与其他药物多肽或蛋白融合状态下仍具有较高的HSA结合活力。利用噬菌体展示7肽库,经四轮筛选、筛选克隆的DNA测序、酶联免疫吸附技术分析(ELISA)初步鉴定筛选克隆与HSA的亲和作用,并利用等离子共振技术(SPR)定量测定合成的ML-PACAP27融合多肽与人血清白蛋白的亲和力常数。实验结果表明:经筛选获得2个与人血清白蛋白特异性结合的7肽序列,其氨基酸序列分别为:ML1:LKSCKPL和ML2:SLKSHAL; ELISA分析结果显示,含有ML1和ML2的噬菌体均可亲和结合HSA,而且ML2的亲和结合作用高于ML1;SPR分析结果显示,ML1-PACAP27和ML2-PACAP27与HSA的解离常数(K_D)分别为:8.1×10^-6mol/L和 3.7×10^-6mol/L,ML1-PACAP27与HSA的结合力高于ML1-PACAP27。筛选和鉴定了两个可与HSA特异性结合的7肽序列,该7肽序列可用于特异偶联HSA的长效分子药物的重组融合表达或设计,可为延长分子药物的半衰期及新型药物研发提供新工具。

关键词： 人血清白蛋白; 噬菌体展示; 特异性结合; 药物半衰期

Abstract:

New human serum albumin (HSA) specific binding 7-amino-acid peptides were screened and identificated by using phage display technology. The affinity of the fusion polypeptide, comprising the screened 7-amino-acid peptides and pituitary adenylate cyclase activating peptide 27 (ML-PACAP27), were quantitatively determined,so that it was determine that the screened 7 peptides (ML1 or ML2) in fusion proteins including other peptide or protein drugs still remain the high binding activity for HSA. Using M13 phage-displayed 7-mer peptide library, the affinity effect of ML1 or ML2 and HSA were preliminarily identified through enzyme-linked immunosorbent assay (ELISA) after four screening rounds and DNA sequencing of the screened clones. Affinity constants of ML1-PACAP27 or ML2-PACAP27 and HSA were assayed by surface plasmon resonance (SPR). The experimental results showed two new 7-amino-acid peptides ML1 and ML2 were obtained, and their amino acid sequence is LKSCKPL (ML1) and SLKSHAL (ML2). The ELISA results showed the screened clones containing ML1 and ML2 can bind to HSA, and the affinity effect of ML2 for HSA is higher than that of ML1. The SPR results showed the dissociation constants of ML1 and ML2 for HSA is respectively 8.1×10^-6and 3.7×10^-6mol/L,and the affinity of ML2 and HSA is higher than that of ML1. In the study, two new HSA-specifically binding 7-amino-acid peptides that were screened and identificated may be used for the fusion expression or design of HSA-conjugated molecule drugs with long-acting effect, which can provide the new tools for extending the half-life of molecule drugs and the development of the novel drugs.

Key words: Human serum albumin(HSA) Phage-display Specific binding Drug half-life

收稿日期: 2015-03-02 出版日期: 2015-05-25

ZTFLH:

Q816

基金资助:

国家自然科学基金面上项目(81373314),广东省自然科学基金(S2012010008756),高等学校博士学科点专项科研基金(20124401120012),广东省教育部产学研结合项目(2010B090400544, 2013B090500105)资助项目

通讯作者: 马义 E-mail: tmayi@jnu.edu.cn

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引用本文:

马义, 赵绍军, 洪岸. 新型人血清白蛋白特异结合肽ML的筛选及鉴定[J]. 中国生物工程杂志, 2015, 35(5): 74-80.

MA Yi, ZHAO Shao-jun, HONG An. Screening and Identification of the New Human Serum Albumin-specific Binding Peptides. China Biotechnology, 2015, 35(5): 74-80.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20150511 或 https://manu60.magtech.com.cn/biotech/CN/Y2015/V35/I5/74

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