文章摘要
孙立人,陶妍,高北.斑点叉尾(鱼回)LEAP2成熟肽在毕赤酵母中的表达及其抑菌活性[J].上海海洋大学学报,2017,26(1):23-30.
SUN Liren,TAO Yan,GAO Bei.Expression of channel catfish (Ictalurus punctatus) LEAP2 mature peptide in Pichia pastoris and its antibacterial activity[J].Journal of Shanghai Ocean University,2017,26(1):23-30.
斑点叉尾(鱼回)LEAP2成熟肽在毕赤酵母中的表达及其抑菌活性
Expression of channel catfish (Ictalurus punctatus) LEAP2 mature peptide in Pichia pastoris and its antibacterial activity
投稿时间:2016-04-28  修订日期:2016-05-12
DOI:10.12024/jsou.20160401759
中文关键词: 斑点叉尾(鱼回)  LEAP2成熟肽  毕赤酵母  重组DNA表达
英文关键词: channel catfish  LEAP2 mature peptide  Pichia pastoris  recombinant DNA expression
基金项目:上海市教育委员会产学研项目(15CXY30);农业部都市农业(南方)重点实验室开放基金项目(UA201307)
作者单位E-mail
孙立人 上海海洋大学 食品学院, 上海 201306
上海水产品加工及贮藏工程技术研究中心, 上海 201306 
 
陶妍 上海海洋大学 食品学院, 上海 201306
上海水产品加工及贮藏工程技术研究中心, 上海 201306 
ytao@shou.edu.cn 
高北 上海海洋大学 食品学院, 上海 201306
上海水产品加工及贮藏工程技术研究中心, 上海 201306 
 
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中文摘要:
      LEAP2(Liver expressed antimicrobial peptide-2)抗菌肽是一类主要在动物肝脏中表达的小分子肽,它们在动物的免疫系统中发挥了重要作用。斑点叉尾(鱼回)(Ictalurus punctatus)LEAP2的一级结构由信号肽和原前体肽组成;原前体肽由66个氨基酸残基组成,近羧基端的41个残基组成了其成熟肽区域(mLEAP2),该区域决定了LEAP2的生物学活性。本文以斑点叉尾(鱼回)mLEAP2为研究对象,通过建立毕赤酵母表达系统,实现了mLEAP2在毕赤酵母中的重组DNA表达,优化的表达条件为:29℃、pH 6、250 r/min、0.5%甲醇、120 h。Tricine-SDS-PAGE分析表明:表达产物经固化金属离子亲和层析(IMAC)纯化后,获得了高纯度的目的蛋白。经Folin-酚法测定,纯化产物的浓度为0.22 mg/mL,推算至表达量为2.2 mg/L。进一步通过MALDI-TOF/TOF质谱分析对纯化产物的结构进行鉴定,结果证明:获得的纯化产物为预期的重组体mLEAP2(rmLEAP2)。此外,抑菌实验结果显示rmLEAP2具有明显的抑制枯草芽孢杆菌(Bacillus subtilis)的活性。
英文摘要:
      LEAP2(Liver expressed antimicrobial peptide-2) antibacterial peptide is a kind of small molecular peptide expressed mainly in the liver of animals, and they play important roles in the immune systems of animals. The primary structure of LEAP2 from channel catfish(Ictalurus punctatus) is composed of signal peptide and prepeptide which is composed of 66 amino acid residues. The 41 residues near carboxyl terminal of the prepeptide form mature peptide region(mLEAP2), which is responsible for biological activity of the LEAP2 antibacterial peptide. The present study focused on the mLEAP2 for channel catfish. Recombinant DNA expression of the mLEAP2 in Pichia pastoris was realized by constructing P. pastoris expression system. The optimized expression conditions were as follows:29℃, pH 6, 250 r/min, 0.5% methanol and 120 h. Tricine-SDS-PAGE analysis indicated that the expressed product was highly purified by immobilized metal affinity chromatography(IMAC). The concentration of the purified product was 0.22 mg/mL, thus the expression yield of recombinant protein was calculated to be about 2.2 mg/L. Structure of the purified product was further identified by MALDI-TOF/TOF analysis, and the result demonstrated that the purified product was the recombinant mLEAP2(rmLEAP2) expected. In addition, antibacterial assay showed that the fermentation supernatant containing the rmLEAP2 had bacteriostatic activity against Bacillus subtilis.
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