人参thionins的克隆、原核表达和活性研究

doi:10.13560/j.cnki.biotech.bull.1985.2018-0702

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

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

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

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/Abstract

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

张鹏飞, 边帅, 赵月, 赵雨, 王佳雯. 人参thionins的克隆、原核表达和活性研究[J]. 生物技术通报, 2019, 35(2): 53-57.

ZHANG Peng-fei, BIAN Shuai, ZHAO Yue, ZHAO Yu, WANG Jia-wen. Cloning,Prokaryotic Expression and Activity Research of Panax ginseng Thionins[J]. Biotechnology Bulletin, 2019, 35(2): 53-57.

参考文献

[1] 崔巍, 赵洪艳, 王燕嬉. 人参皂苷抗衰老的研究进展[J]. 中国老年学杂志, 2006, 26(11):1578-1581.
[2] 胡圣望, 胡勇, 王平. 人参皂苷Rg1对慢性应激大鼠空间学习能力的影响[J]. 四川中医, 2004, 22(3):14-15.
[3] Wesnes KA, Ward T, et al.The memory enhancing effects of a Ginkgo balboa/Panax ginseng combination in health middle aged volunteers[J]. Pschopharmacology, 2000, 152(4):353-361.
[4] 田建明, 李浩, 叶金梅, 等. 人参皂苷Rg2对大鼠化学性心肌缺血的影响[J]. 中国中药杂志, 2003, 28(12):1191-1192.
[5] 张秋梅, 张喆, 于德民, 等. 人参糖肽治疗2型糖尿病的临床观察[J]. 中国现代医学杂志, 2003, 13(6):59.
[6] Shergis JL, Di YM, et al.Therapeutic potential of Panax ginseng and ginsenosides in the treatment of chronic obstructive pulmonary disease[J]. Complement Ther Med, 2014, 22(5):944-953.
[7] Cai K, Wang J, Wang M, et al.Molecular cloning, recombinant expression, and antifungal functional characterization of the lipid transfer protein from Panax ginseng[J]. Biotechnology Letters, 2016, 38(7):1229-1235.
[8] Zhang H, Wang J, Li S, et al.Molecular cloning, expression, purifcation and functional characterization of an antifungal cyclophilin protein from Panax ginseng[J]. Biomedical Reports, 2017, 7(6):527-531.
[9] Tam JP, Wang S, Wong KH, et al.Antimicrobial peptides from plants[J]. Pharmaceuticals, 2015, 8(4):711-757.
[10] Ghosh M, Antifungal properties of haem peroxidase from Acorus calamus[J]. Annals of Botany, 2006, 98(6):1145-1153.
[11] Guida V, Cantarella M, Chambery A, et al.Purification and characterization of novel cationic peroxidases from Asparagus acutifolius L. with biotechnological applications[J]. Molecular Biotechnology, 2014, 56(8):738-746.
[12] Ajesh K, Sreejith K.A novel antifungal protein with lysozyme-like activity from seeds of Clitoria ternatea[J]. Applied Biochemistry and Biotechnology, 2014, 173(3):682-693.
[13] Candido Ede S, Pinto MF, Pelegrini PB, et al.Plant storage proteins with antimicrobial activity:novel insights into plant defense mechanisms[J]. FASEB Journal, 2011, 25(10):3290-3305.
[14] Wong JH, Ng TB, et al.Proteins with antifungal properties and other medicinal applications from plants and mushrooms[J]. Appl Microbiol Biotechnol, 2010, 87(4):1221-1235.
[15] Kulko AB, Kisl OV, Sadykova VS, et al.investigation of thionins from black cumin(nigella sativa L.)seeds showing cytotoxic, regulatory and antifungal activity[J]. Antibiol Khimioter, 2016, 61(9/10):8-16.
[16] Ji H, Gheysen G, et al.The role of thionins in rice defence against root pathogens[J]. Mol Plant Pathol, 2015, 16(8):870-881.
[17] Lee OR, Kim Y, Balusamy SRD, et al.Ginseng g-thionin is localized to cell wall-bound extracellular spaces and responsive to biotic and abiotic stresses[J]. Physiol Mol Plant Pathol, 2011, 76(2):82-89.
[18] Harper S, Speicher DW.Purification of proteins fused to glutathione S-transferase[J]. Methods Mol Biol, 2011, 681:259-280.
[19] Yu H, Li H, Gao D, et al.Secretory production of antimicrobial peptides in Escherichia coli using the catalytic domain of a cellulase as fusion partner[J]. J Biotechnol, 2015, 214:77-82.
[20] Barbosa PP, Del Sart RP, Silva ON, et al.Antibacterial peptides from plants:what they are and how they probably work[J]. Biochem Res Int, 2011, 2011(5):250349.

人参thionins的克隆、原核表达和活性研究

Cloning,Prokaryotic Expression and Activity Research of Panax ginseng Thionins

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics

本文评价

[1]	梅欢, 李玥, 刘可蒙, 刘吉华. 小檗碱桥酶高效原核表达及生物合成l-SLR的研究[J]. 生物技术通报, 2023, 39(7): 277-287.
[2]	索青青, 吴楠, 杨慧, 李莉, 王锡锋. 水稻咖啡酰辅酶A-O-甲基转移酶基因的原核表达、抗体制备和应用[J]. 生物技术通报, 2022, 38(8): 135-141.
[3]	覃雪晶, 王雨涵, 曹一博, 张凌云. 青杄PwHAP5基因原核表达及多克隆抗体制备[J]. 生物技术通报, 2022, 38(8): 142-149.
[4]	王光丽, 范婵, 王辉, 卢惠芳, 夏灵尹, 黄健, 闵迅. 霍乱弧菌溶血素HlyA的原核表达、纯化及多克隆抗体制备与鉴定[J]. 生物技术通报, 2022, 38(7): 269-277.
[5]	汪巧菊, 胡雨萌, 温亚亚, 宋丽, 孟闯, 潘志明, 焦新安. 新型冠状病毒S1蛋白的表达及活性鉴定[J]. 生物技术通报, 2022, 38(3): 157-163.
[6]	沈俊强, 张莉萍, 于瑞明, 王永录, 潘丽, 刘霞, 刘新生. 猪嵴病毒结构蛋白VP0与VP1原核表达及间接ELISA方法的建立[J]. 生物技术通报, 2022, 38(10): 243-253.
[7]	山草梅, 叶蕾, 张连虎, 况卫刚, 孙晓棠, 马建, 崔汝强. 水稻抗潜根线虫基因OsRAI1的克隆及功能分析[J]. 生物技术通报, 2021, 37(7): 146-155.
[8]	曾福源, 苏泽辉, 周诗慧, 谢妙, 庞欢瑛. 溶藻弧菌PEPCK蛋白原核表达及其乙酰化、琥珀酰化修饰的鉴定[J]. 生物技术通报, 2021, 37(5): 84-91.
[9]	张西西, 张怡青, 李玉林, 韩笑, 王国强, 王晓军, 王旭东, 王云龙. 新型冠状病毒（SARS-CoV-2）N蛋白C端重组蛋白的原核表达、纯化及应用[J]. 生物技术通报, 2021, 37(5): 92-97.
[10]	白福美, 李至敏, 王小琴, 胡紫微, 鲍玲玲, 李志敏. 集胞藻PCC6803中N-乙酰鸟氨酸转氨酶的生化表征及结构分析[J]. 生物技术通报, 2021, 37(5): 98-107.
[11]	瞿欢, 李成, 陈汭, 廖艺杰, 曹三杰, 文翼平, 颜其贵, 黄小波. 猪δ冠状病毒S1-CTD的截短表达及间接ELISA抗体方法的建立[J]. 生物技术通报, 2021, 37(5): 273-280.
[12]	彭利忠, 张鹏, 周雯雯, 曾旭辉, 张小宁. 精子特异性蛋白Cabs1多克隆抗体的制备及多用途验证[J]. 生物技术通报, 2021, 37(3): 261-270.
[13]	贺扬, 余巧玲, 王均, 覃川杰, 李华涛. 罗非鱼原核表达基因研究进展[J]. 生物技术通报, 2021, 37(2): 195-202.
[14]	唐禄, 董丽平, 尹茉莉, 刘磊, 董媛, 王会岩. 成纤维细胞生长因子20单克隆抗体的制备及鉴定[J]. 生物技术通报, 2021, 37(10): 179-185.
[15]	段应策, 胡姿仪, 杨帆, 李金涛, 邬向丽, 张瑞颖. 香菇草酰乙酸水解酶基因LeOAH1克隆及表达分析[J]. 生物技术通报, 2020, 36(9): 227-234.