生物技术通报 ›› 2017, Vol. 33 ›› Issue (12): 144-150.doi: 10.13560/j.cnki.biotech.bull.1985.2017-0373

• 研究报告 • 上一篇    下一篇

片球菌素pedA基因的原核表达、纯化及生物信息学分析

黄宇良1, 汪立平1,2, 陆克文3, 邵会娟3, 王正全2   

  1. 1. 上海海洋大学食品学院 食品热加工工程技术研究中心，上海 201306;
    2. 上海海洋大学食品学院 上海水产品加工及贮藏工程技术研究中心，上海 201306;
    3. 上海邦成生物工程有限公司，上海 201506
  • 收稿日期:2017-05-09 出版日期:2017-12-25 发布日期:2017-12-21
  • 作者简介:黄宇良，男，硕士研究生，研究方向：食品生物技术；E-mail：791313564@qq.com
  • 基金资助:
    国家自然科学基金青年基金项目（31401486），上海市科学技术委员会工程中心建设项目（11DZ2280300）

Prokaryotic Expression，Purification and Bioinformatics Analysis of Pediocin pedA Gene

HUANG Yu-liang1, WANG Li-ping1, 2, LU Ke-wen3, SHAO Hui-juan3, WANG Zheng-quan2   

  1. 1. College of Food Science and Technology，Shanghai Ocean University，Engineering Research Center of Food Thermal-processing Technology，Shanghai 201306;
    2. College of Food Science and Technology，Shanghai Ocean University，Shanghai Engineering Research Center of Aquatic-Product Processing & Preservation，Shanghai 201306;
    3. Shanghai Bangcheng Biotechnology Co. Ltd.，Shanghai 201506
  • Received:2017-05-09 Published:2017-12-25 Online:2017-12-21

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摘要： 旨在构建片球菌素PA-1结构基因pedA的原核表达载体，在大肠杆菌体系中实现PA-1的外源表达，运用生物信息学手段分析重组蛋白的理化性质及分子结构。以戊糖片球菌C-2-1的DNA为模板，扩增pedA基因，扩增产物克隆入pET28a（+）原核表达载体，构建pET28a-pedA重组质粒，转入大肠杆菌BL21（DE3）中；30℃，以1 mmol/L IPTG 诱导表达5 h；采用Ni-NTA 树脂亲和层析纯化重组蛋白，检测纯化后重组蛋白的抑菌活性；利用生物信息学手段探究重组蛋白的理化性质及分子结构信息。结果显示，pET28a-pedA重组质粒构建成功，带有His标签的PA-1重组片球菌素在大肠杆菌中实现了可溶性表达，利用Ni-NTA亲和层析获得纯化的重组蛋白，Tricine-SDS-PAGE电泳分析表明纯化后的重组蛋白分子量约为7.8 kD，与理论值相符。琼脂扩散法抑菌活性实验抑菌圈效果明显，表明纯化的重组片球菌素具有抑菌活性。生物信息学方法对比分析其蛋白结构发现，获得的重组蛋白含有信号肽，α螺旋占23.53%，疏水性提高，可能促进了重组蛋白的可溶性表达。该实验成功构建了片球菌素PA-1的原核表达载体，实现了在大肠杆菌中的可溶性表达，该表达可能与其含有信号肽及信号肽疏水性有关，纯化后的重组蛋白抑菌活性明显。

关键词: pedA基因, 片球菌素PA-1, 原核表达, 抑菌活性, 生物信息学

Abstract: The aim of this study is to construct a prokaryotic expression vector for pedA gene of pediocin PA-1 to realize the exogenous expression of PA-1，and to analyze the physicochemical properties and molecular structure of recombinant protein by bioinformatics. The pedA gene was amplified by DNA of Pediococcus pentosaceus C-2-1 as template，and the PCR product was cloned into prokaryotic expression vector pET28a（+）. The recombinant plasmid pET28a-pedA was thus constructed and transformed into Escherichia coli BL21（DE3）competent cells. Next，the transformant was induced to express protein by IPTG（1 mmol/L）at 30℃ for 5 h. Then，the recombinant protein was purified by Ni-NTA resin affinity chromatography，and the antibacterial activity of the recombinant protein was detected. Finally，the physicochemical properties and molecular structure of recombinant protein was explored by bioinformatics method. The results showed that the recombinant plasmid pET28a-pedA was successfully constructed，and recombinant pediocin of PA-1 with His-tag was expressed in E. coli. The molecular size of the purified recombinant protein by Ni-NTA affinity chromatography was determined to be about 7.8 kD via Tricine-SDS-PAGE，which was consistent with the theoretical value. Agar diffusion method demonstrated that antibacterial colony was remarkable，indicating that the purified recombinant pediocin had antibacterial activity. Comparative analysis of the protein structure and characteristics by bioinformatics method showed that the recombinant protein contained the signal peptide and consisted of 23.53% alpha helix，meaning that its hydrophobicity increased，thus which might enhance the soluble expression of the recombinant protein. Conclusively，in this experiment，the prokaryotic expression vector of pediocin PA-1 was successfully constructed，and the recombinant protein was at soluble expression in E. coli. The soluble expression may be related to signal peptide and the hydrophobicity of the signal peptide，and the antibacterial activity of the purified recombinant protein was significant.

Key words: pedA gene, pediocin PA-1, prokaryotic expression, antibacterial activity, bioinformatics

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