生物技术通报 ›› 2019, Vol. 35 ›› Issue (2): 53-57.doi: 10.13560/j.cnki.biotech.bull.1985.2018-0702

• 研究报告 • 上一篇    下一篇

人参thionins的克隆、原核表达和活性研究

张鹏飞, 边帅, 赵月, 赵雨, 王佳雯   

  1. 长春中医药大学 吉林省人参科学研究院,长春 130117
  • 收稿日期:2018-08-02 出版日期:2019-02-26 发布日期:2019-03-07
  • 作者简介:张鹏飞,男,硕士研究生,研究方向:中药化学;E-mail:269412461@qq.com
  • 基金资助:
    吉林省教育厅“十三五”科学技术项目(JJKH20181265KJ)

Cloning,Prokaryotic Expression and Activity Research of Panax ginseng Thionins

ZHANG Peng-fei, BIAN Shuai, ZHAO Yue, ZHAO Yu, WANG Jia-wen   

  1. Jilin Ginseng Academy,Changchun University of Traditional Chinese Medicine,Changchun 130117
  • Received:2018-08-02 Published:2019-02-26 Online:2019-03-07

摘要: 抗菌肽在人参的生长过程中发挥了抑菌抗逆等重要作用。通过研究人参硫堇蛋白(Thionins)的抗菌活性,为未来进行大规模生产和生物农药研究奠定基础。分析人参thionins序列,构建原核表达质粒,诱导蛋白表达,对包涵体进行亲和纯化,使用纸片扩散法和质膜通透性实验检测纯化蛋白的抗菌活性。人参thionins长度为228 bp,编码75个氨基酸,选取其成熟肽区域(29-75 aa)进行后续的表达和纯化。成功构建了原核表达质粒pGEX-4T1-thionins,在大肠杆菌BL21中获得目的蛋白的高效表达,通过包涵体纯化获得目的蛋白,并进行复性和浓缩。人参硫堇蛋白在3和9 μmol/L的浓度下,对黑斑病菌丝的生长抑制率分别为36.0%和51.1%,对黑斑孢子的半数抑制浓度为0.87 μmol/L。人参硫堇蛋白具有显著抑制人参黑斑病真菌的活性的作用。

关键词: 人参, 硫堇, 原核表达, 蛋白纯化, 抑真菌活性

Abstract: Antibacterial peptides play an important role in bacteriostasis and resistance to stress in the growth process of ginseng. Studying the antibacterial activity of ginseng thionins may lay a foundation for large-scale production and biological pesticide research in the future. First,the gene sequence of ginseng thionins was analyzed and prokaryotic expression plasmid was constructed to induce the expression of the protein.. Then the affinity and purification of inclusion bodies was performed,the anti-fungal activity of the purified protein was detected by paper tablet diffusion method and membrane permeability assay. The ginseng thionins was 228 bp in length and encoded 75 amino acids,and its mature peptide region(29-75 aa)was selected for subsequent expression and purification. The prokaryotic expression plasmid pGEX-4T1-thionins was successfully constructed,the efficient expression of the target protein was obtained in Escherichia coli BL21,and the target protein was purified by inclusion body and then renatured and concentrated. The growth inhibition rate of ginseng thionins on Alternaria panax was 36.0% and 51.1% at 3 μmol/L and 9μmol/L,respectively. The half-inhibitory concentration of the fungal spores was 0.87 μmol/L. In conclusion,the ginseng thionins has a significant effect of inhibiting the fungus A. panax.

Key words: Panax ginseng, thionins, prokaryotic expression, protein purification, antifungal activity