HβD-3在大肠杆菌中的融合表达与纯化

中国生物工程杂志

2013, Vol. 33

Issue (11): 68-74

技术与方法

HβD-3在大肠杆菌中的融合表达与纯化

朱钧萍¹, 李改瑞¹, 杜启科³, 郭中敏², 陆家海¹

1 中山大学公共卫生学院热带病防治研究教育部重点实验室广东省重大传染病预防和控制技术研究中心广州 510080;
2 中山大学实验动物中心广州 510080;
3 山东省银香伟业集团山东省动物肽生物工程中心曹县 274400

Fusion Expression and Purification of Human Beta Defensin-3 in E.coli

ZHU Jun-ping¹, LI Gai-rui¹, DU Qi-ke³, GUO Zhong-min², LU Jia-hai¹

1. School of Public Health, Sun Yat-sen University, Key Laboratory of Tropical Disease Control(Sun Yat-sen University), Ministry of Education, Guangdong Provincial Research Center for Severe Infectious Disease Prevention and Control Technology, Guangzhou 510080, China;
2. Experiment Animal Center, Sun Yat-sen University, Guangzhou 510080, China;
3. Good Earth Group, Animal Peptide Bio-Engineering Center of Shandong Province, Caoxian 274400, China

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摘要： 抗菌肽是生物体内合成的一种具有抗菌活性的多肽类物质，对多种病原体乃至耐药性菌株具有明显的杀伤作用，是固有免疫及适应性免疫的重要参与物质。HβD-3是人体内一类内源性多肽，与其它β-防御素相比，耐高盐，具有灭活多种耐药性菌株的能力。针对如何高效表达和快速纯化这个问题，研究选用pET-EI质粒载体在E. coli BLR（DE3）表达原始序列HβD-3及密码子优化序列 HβD-3i。经密码子改造后的重组抗菌肽HβD-3i的表达量是原始序列HβD-3表达量的约12倍之多。对融合表达产物进行 “ITC法”纯化蛋白，纯化后的HβD-3i具有与天然HβD-3相似的生物学活性，对金黄色葡萄球菌的活性强于伤寒沙门菌。表明利用pET-EI原核表达系统表达HβD-3是可行的，且因该纯化方法经济简便，为实现HβD-3大规模生产奠定基础。

关键词： HβD-3; HβD-3i; pET-EI表达系统

Abstract: Antimicrobial peptides (AMPs) which are produced by most living organisms have shown robust activity against a wide variety of pathogens, including drug-resistant bacteria. They are important components of the innate and adaptive system. Human beta-defensin-3 is an endogenous, cysteine-rich antimicrobial peptide that resistants to high salt and contributes more powerful to host defense against multi-resistant bacteria compared with other beta-defensins. There is a great need for a less expensive and increasing efficiency peptide-production platform.This research aims at expressing human beta-defensin-3 and a novel sequence human beta-defensin-3i based on E. coli codon preference in E.coli BLR(DE3) via pET-EI plasmid vector. HβD-3i were 12 times higher yields than HβD-3. All the peptides, expressed as fusion protein, were isolated from the protein debris by the method called Inverse Transition Cycling(ITC).Fully reduced peptides that were purified exhibited expected antimicrobial activity that towarded to Staphylococus aureus was stronger than Salmonella.It is an attractive way to express human beta-defensin-3 via pET-EI prokaryotic plasmid vector. This approach described here is a low-cost, convenient and potential way for large-scale human beta-defensin-3 production.

Key words: HβD-3 HβD-3i pET-EI expression vector

收稿日期: 2013-05-09 出版日期: 2013-11-25

ZTFLH:

Q812

基金资助: 省部产学研合作项目（2007A090302042）、广东省科技计划项目社会发展（2010B031600076）、广州市医药卫生科技重大项目（201102A211003）资助项目

通讯作者: 郭中敏, 陆家海 E-mail: jiahailu@aliyun.com.cn;zhongminguo@aliyun.com.cn

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引用本文:

朱钧萍, 李改瑞, 杜启科, 郭中敏, 陆家海. HβD-3在大肠杆菌中的融合表达与纯化[J]. 中国生物工程杂志, 2013, 33(11): 68-74.

ZHU Jun-ping, LI Gai-rui, DU Qi-ke, GUO Zhong-min, LU Jia-hai. Fusion Expression and Purification of Human Beta Defensin-3 in E.coli. China Biotechnology, 2013, 33(11): 68-74.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2013/V33/I11/68

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