麻疯树核糖体失活蛋白Curcin2的原核可溶性表达及其抗真菌活性研究

中国生物工程杂志

2013, Vol. 33

Issue (7): 43-49

研究报告

麻疯树核糖体失活蛋白Curcin2的原核可溶性表达及其抗真菌活性研究

邵子静, 蒋楠, 晏华立, 詹诚, 徐莺, 陈放

四川大学生命科学学院生物资源与生态环境教育部重点实验室成都 610064

The Soluble Prokaryotic Expressed Ribosome-inactivating Protein Curcin2 with Anti-fungal Activity from Jatropha curcas L.

SHAO Zi-jing, JIANG Nan, YAN Hua-li, ZHAN Cheng, XU Ying, CHEN Fang

Key Laboratory of Bio-resources and Eco-environment of Ministry of Education, College of Life Sciences, Sichuan University, Chengdu, 610064, China

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摘要： Curcin2是麻疯树幼苗在真菌侵染、干旱及高低温胁迫下诱导产生的一种核糖体失活蛋白。采用分子克隆的方法,从麻疯树基因组中扩增到 Curcin2 成熟肽编码基因,分别连接到质粒载体pGEX-6p-1、pMAL-c5E上,转化大肠杆菌最终获得重组菌PGC(pGEX-6p-1-curcin2)和PMC(pMAL-c5E-curcin2)。在不同温度、IPTG浓度、时间诱导下,Curcin2在重组菌PGC中均以包涵体形式表达,在重组菌PMC中主要以可溶性融合蛋白形式表达,且蛋白质的表达量与诱导条件相关。重组菌PMC表达可溶性curcin2的优化条件为:温度28℃,IPTG 0.3mmol/L,时间8h,此条件下目的蛋白占总蛋白表达量的30.6%, 1L培养物中可获得19.74mg电泳纯的重组蛋白。重组蛋白可被MBP Trap^TMHP柱亲和纯化并与curcin抗体发生抗原抗体反应。体外抗真菌活性实验表明,纯化后的curcin2融合蛋白有抑制真菌生长作用,且对小麦赤霉、油菜菌核的抑制作用强于curcin。此蛋白的获得为其相关功能的研究奠定了基础。

关键词： 麻疯树; 核糖体失活蛋白; 原核表达; 抗真菌活性

Abstract: The open reading frame (ORF) encoding mature curcin2 protein, a ribosomal inactivating protein induced by several kinds of stress, was amplified from genomic DNA of Jatropha curcas by PCR method.The sequence of curcin2 gene was synthesized in pMD-19-T. The gene was cloned in pGEX-6p-1 and pMAL-c5E prokaryotic expression vectors and expressed in E.coli BL21 strain. Curcin 2 protein could be expressed under different IPTG concentration, temperature and induction time but the soluble recombinant curcin2 could only be obtained in the PMAL expression system.The MBP-curcin2 fusion protein was purified through MBP Trap^TMHP column and could be recognized by curcin antibody. Its antibacterial activity was tested in vitro and a better inhibition effections were found towards Gibberelle zeae (Schw.) Petch and Sclerotinia sclerotiorum (Lib) de Bary compared with those of curcin. These results may provide valuable clues and foundation to study the functions of curcin2.

Key words: Jatropha curcas L. Ribosome-inactivating protein Prokaryotic expression Antifungal activity

收稿日期: 2013-04-02 出版日期: 2013-07-25

ZTFLH:

Q344

基金资助: 国家"十二五"科技支撑项目(2011DFB22B08)

通讯作者: 徐莺 E-mail: xuying@scu.edu.cn

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引用本文:

邵子静, 蒋楠, 晏华立, 詹诚, 徐莺, 陈放. 麻疯树核糖体失活蛋白Curcin2的原核可溶性表达及其抗真菌活性研究[J]. 中国生物工程杂志, 2013, 33(7): 43-49.

SHAO Zi-jing, JIANG Nan, YAN Hua-li, ZHAN Cheng, XU Ying, CHEN Fang. The Soluble Prokaryotic Expressed Ribosome-inactivating Protein Curcin2 with Anti-fungal Activity from Jatropha curcas L.. China Biotechnology, 2013, 33(7): 43-49.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2013/V33/I7/43

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