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2025年4月3日 星期四
  2015, Vol. 23 Issue (3): 380-387    
  研究论文与报告 本期目录 | 过刊浏览 | 高级检索 |
斑点叉尾鮰铁调素成熟肽在毕赤酵母中的表达及其抑菌活性
宋长丰1,陶妍1,赵冬梅2,沈彦萍3
1. 上海海洋大学 食品学院
2.
3. 上海沈李科工贸有限公司
Expression of Channel Catfish (Ictalurus punctatus) Hepcidin Mature Peptide in Pichia pastoris and Its Antibacterial Activity
1, 1, 1
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摘要 铁调素(hepcidin)是一种在动物肝脏中特异表达的碱性小分子抗菌肽,在机体免疫系统中发挥重要作用,被认为是抗生素的理想替代品。本研究根据毕赤酵母(Pichia pastoris)偏爱性密码子人工合成斑点叉尾鮰(Ictalurus punctatus) hepcidin成熟肽基因(mCH)。通过PCR方法在mCH的5'端和3'端分别引入EcoRⅠ和NotⅠ酶切位点,扩增到的目的片段与表达载体pPIC9K连接构建重组表达载体pPIC9K-mCH后,转化至毕赤酵母GS115细胞;以不同浓度梯度的G418筛选高拷贝转化子,1.0%甲醇、30 ℃、pH 6.0诱导表达72 h,获得重组体mCH。结果显示,斑点叉尾鮰hepcidin成熟肽区域含25个残基,8个保守的半胱氨酸残基位于成熟肽的羧基端,表明该区域对hepcidin的抗菌活性具有重要作用。Tricine-SDS-PAGE分析显示,分泌表达的重组体mCH的分子量约为3 800 D,通过阳离子交换层析获得纯化的mCH。抑菌实验表明,重组体抗菌肽mCH对金黄色葡萄球菌(Staphylococcus aureus)和大肠杆菌(Escherichia coli)有抑菌活性。本研究首次实现了斑点叉尾鮰hepcidin成熟肽在毕赤酵母中的重组DNA表达,为其产业化制备提供了重要的基础资料。
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宋长丰
陶妍
赵冬梅
沈彦萍
关键词 斑点叉尾鮰铁调素成熟肽(mCH)毕赤酵母重组表达抗菌活性    
Abstract:Hepcidin is a kind of small cysteine-rich cationic antimicrobial peptide which mainly expressed in livers of animals, and play an important role in immune response against microbial invasion. Thus, it is considered to be good substitutes for traditional antibiotics. In the present study, the gene encoding channel catfish (Ictalurus punctatus) hepcidin mature peptide (mCH) was synthesized with reference to its native DNA sequence according to preference of Pichia pastoris. EcoRⅠand NotⅠrestriction sites were added to 5' and 3' ends of the mCH by PCR, respectively. The expected fragment was ligated with pPIC9K vector to construct a recombinant expression vector pPIC9K-mCH which then was transformed into competent Pichia pastoris GS115 cells. The transformants (His+Mut+) containing multicopy gene insertion were screened with increasing concentration of antibiotic G418. Recombinant mCH was induced with 1.0% (V/V) methanol at 30 ℃, pH 6.0 for different times. The results showed that channel catfish mCH consisted of 25 residues, and 8 conserved cysteine residues were located at its C-terminal, suggesting that this region was responsible for its antibacterial activity. The total protein in the supernatant culture reached a peak concentration at 72 h during the induction period, which indicated that 72 h was an optimal expression time. Furthermore, the recombinant mCH was purified by SP Sepharose Fast Flow column with 20 mmol/L PB (pH 6.5) containing 500 mmol/L NaCl. Tricine-SDS-PAGE analysis demonstrated that molecular mass of the purified recombinant mCH was 3 800 D. According to the concentration of purified mCH, expression yield of the recombinant mCH was calculated to be 51 mg/L. Antibacterial assays showed that the fermentation supernatant concentrated for 10 and 15 times containing the recombinant mCH had bioactivities against Staphylococcus aureus and Escherichia coli. The present study first realized the recombinant DNA expression of channel catfish hepcidin mature peptide in Pichia pastoris, which provides the important initial value for industrial production of hepcidin antimicrobial peptide.
Key wordsChannel catfish    Hepcidin mature peptide (mCH)    Pichia pastoris    Recombinant expression    Antibacterial activity
收稿日期: 2014-09-01      出版日期: 2015-02-02
通讯作者: 陶妍     E-mail: ytao@shou.edu.cn
引用本文:   
宋长丰 陶妍 赵冬梅 沈彦萍. 斑点叉尾鮰铁调素成熟肽在毕赤酵母中的表达及其抑菌活性[J]. , 2015, 23(3): 380-387.
链接本文:  
http://journal05.magtech.org.cn/Jwk_ny/CN/     或     http://journal05.magtech.org.cn/Jwk_ny/CN/Y2015/V23/I3/380
 
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