生物学杂志

生物学杂志 ›› 2023, Vol. 40 ›› Issue (5): 16-.doi: 10.3969/j.issn.2095-1736.2023.05.016

• 研究报告 • 上一篇    下一篇

人干扰素ε的基因合成、原核表达及质谱表征

杨银河1， 狄　斌2， 杨大松1   

  1. 1. 大理大学 云南省昆虫生物医药研发重点实验室， 大理 671000；
    2. 中国药科大学 药学院， 南京 210009
  • 出版日期:2023-10-18 发布日期:2023-10-17
  • 通讯作者: 杨大松，博士，教授，研究方向为多肽药物，E-mail:yangds@cpu.edu.cn；狄斌，博士，教授，研究方向为多肽药物，E-mail:dibin@cpu.edu.cn；杨大松和狄斌为共同通信作者
  • 作者简介:杨银河，博士，副教授，研究方向为微生物药学，E-mail:yyh8612@126.com
  • 基金资助:
    国家自然科学基金项目（81903924, 81903926）； 云南省中青年学术和技术带头人后备人才项目（202105AC160062）； 云南省重大科技专项计划项目（202002AA100007）

Gene synthesis, prokaryotic expression of human interferon ε and its characterization by mass spectrometry

YANG Yinhe1, DI Bin2, YANG Dasong1   

  1. 1. Yunnan Provincial Key Laboratory of Entomological Biopharmaceutical R&D, Dali University, Dali 671000, China;
    2. School of Pharmacy, China Pharmaceutical University, Nanjing 210009, China
  • Online:2023-10-18 Published:2023-10-17

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摘要： 首先人工合成了人源干扰素ε的基因，经密码子优化后分别构建表达载体Ifn-ε/pET-21a (+)和Ifn-ε/pET-22b (+)。将表达载体转化入大肠埃希菌中表达，优化表达条件筛选出两株优势表达菌Ifn-ε/pET-21a/BL21和Ifn-ε/pET-22b/BL21。通过发酵、提取、纯化，成功地从Ifn-ε/pET-22b/BL21表达菌中得到纯度较高的IFN-ε，通过质谱对其结构进行确证。研究建立了人干扰素ε高效表达体系，为获取大量蛋白进行结构和作用机制研究奠定基础。此外通过基因合成建立的表达体系为后期以表达的方式对蛋白的氨基酸序列进行突变进而研究构效关系奠定基础。

关键词: 干扰素ε, 基因合成, 密码子优化, 原核表达, 质谱

Abstract: First of all, the gene of human Ifn-ε was synthesized, the expression vectors of Ifn-ε/pET-21a(+) and Ifn-ε/pET-22b(+) were constructed respectively after codon optimization. The expression vector was transformed into Escherichia coli and the expression conditions were further optimized. Two dominant strains Ifn-ε/pET-21a/BL21A and Ifn-ε/pET-22b/BL21A were obtained. At last, IFN-ε with high purity was isolated from Ifn-ε/pET-22b/BL21A throgh fermentation, extraction, purification, and its structure was confirmed by mass spectrometry. An effective expression system of human IFN-ε has been established to provide large quantity of protein for the study of its structure and mechanism. Moreover, the establishment of expression system through gene synthesis laid the foundation for the study of structure-activity relationship by amino acid mutation.

Key words: interferon epsilon, gene synthesis, codon optimization, prokaryotic expression, mass spectrometry

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