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2025年8月12日 星期二
农业生物技术学报  2019, Vol. 27 Issue (11): 1912-1922    DOI: 10.3969/j.issn.1674-7968.2019.11.002
  研究论文与报告 本期目录 | 过刊浏览 | 高级检索 |
鲤鱼c型溶菌酶在毕赤酵母中的重组表达及其抑菌活性
颜倩倩, 陶妍*, 李雯, 谢晶, 钱韻芳
上海海洋大学 食品学院/上海水产品加工及贮藏工程技术研究中心,上海 201306
Recombinant Expression of Common Carp (Cyprinus carpio) c-type Lysozyme in Pichia pastoris and Its Antibacterial Activity
YAN Qian-Qian, TAO Yan*, LI Wen, XIE Jing, QIAN Yun-Fang
College of Food Science and Technology/Shanghai Engineering Research Center of Aquatic-Product Processing & Preservation, Shanghai Ocean University, Shanghai 201306, China
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摘要 c型溶菌酶是鱼类在先天性免疫机制中抵抗细菌感染的关键性免疫蛋白。鲤鱼(Cyprinus carpio) c型溶菌酶具有鱼类c型溶菌酶的基本特征:N端含有信号肽;C端成熟肽中的Glu35和Asp51构成酶的活性位点,并含有8个保守的半胱氨酸残基。本研究通过反转录PCR (reverse transcription PCR, RT-PCR)获得鲤鱼c型溶菌酶成熟肽的cDNA,该序列全长381 bp,编码由127个氨基酸残基组成的成熟肽;通过PCR在其5'端添加限制性内切酶XhoⅠ位点和6×His标签、3'端添加XbaⅠ位点;获得的目的基因,即鲤鱼c型溶菌酶基因(common carp c-type lysozyme, CLYc),与表达载体pPICZαA连接后电转至毕赤酵母(Pichia pastoris) X-33,通过高浓度博来霉素筛选高拷贝酵母转化子;在29 ℃、250 r/min条件下,使用1.5%甲醇诱导表达120 h,表达上清液经固化金属离子亲和层析(immobilized metal affinity chromatography, IMAC)纯化,获得分子量为15.4 kD的重组蛋白,表达量约为40 mg/L;Western blot分析和MALDI-TOF/TOF质谱鉴定表明该重组蛋白为预期的目的重组蛋白CLYc。抑菌试验证明,含重组CLYc的培养上清液对革兰氏阳性的单增李斯特菌(Listeria monocytogenes)和金黄色葡萄球菌(Staphylococcus aureus)以及革兰氏阴性的大肠杆菌(Escherichia coli)和副溶血性弧菌(Vibrio parahemolyticus)具有明显的抑菌活性;纯化的重组CLYc对单增李斯特菌、枯草芽胞杆菌(Bacillus subtilis)、大肠杆菌和副溶血性弧菌显示了相同的最小抑菌浓度。本研究结果为鱼类来源的天然抗菌剂的开发和生产提供了技术途径。
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颜倩倩
陶妍
李雯
谢晶
钱韻芳
关键词 鲤鱼c型溶菌酶成熟肽毕赤酵母重组表达抗菌活性    
Abstract:The c-type lysozyme is a key immune protein to resist bacterial infection for the fish with innate immune system. Common carp (Cyprinus carpio) c-type lysozyme has some characteristics that are common in fish c-type lysozyme: Its N-terminal is a signal peptide sequence, and its C-terminal region is a mature peptide sequence. Glu35 and Asp51 are 2 amino acid residues located in the mature peptide and form the active site of the c-type lysozyme. In addition, 8 conserved cysteine residues exist in the mature peptide and form 4 pairs of disulfide bonds in spatial structure which are related to the activity and stability of the c-type lysozyme. In the present study, the cDNA encoding common carp c-type lysozyme mature peptide was cloned by reverse transcription PCR (RT-PCR), and the 381 bp cDNA encoded the mature peptide consisting of 127 residues. PCR were performed to add XhoⅠ restriction site and 6×His-tag to its 5' end, and XbaⅠ site to its 3' end. This target gene was named CLYc (common carp c-type lysozyme), and ligated to pPICZαA vector and electronically transformed into competent Pichia pastoris X-33. The yeast transformants containing multi-copy gene insertions were screened using high-concentration zeocin. The target protein was induced for 120 h with 1.5% methanol at 29 ℃, 250 r/min. The recombinant protein with molecular weight of 15.4 kD was purified by immobilized metal ion affinity chromatography (IMAC); Western blot and MALDI-TOF/TOF mass spectrometry analysis proved that this recombinant protein was the expected recombinant CLYc, and its expression yield was about 40 mg/L. The antimicrobial activity of the culture medium supernatant containing recombinant CLYc was tested on Gram-positive (including Listeria monocytogenes and Staphylococcus aureus) and Gram-negative (including Escherichia coli and Vibrio parahemolyticus) bacteria using micro liquid-bacterial colony notation, demonstrating that the crude culture medium supernatant had notable antimicrobial activity against these bacteria, implying the potential use of cell-free P. pastoris medium in fish feed without the need for further peptide purification. Furthermore, the minimum inhibitory concentration (MIC) of purified recombinant CLYc was determined on these bacteria and Bacillus subtilis, indicating that the recombinant CLYc had the same MIC for L. monocytogenes, B. subtilis, E. coli and V. parahemolyticus. The results of this study provide a technical approach for the development and production of natural antibacterial agents from fish.
Key wordsCommon carp    c-type lysozyme    Mature peptide    Pichia pastoris    Recombinant expression    Antibacterial activity
收稿日期: 2019-02-24     
ZTFLH:  S188  
基金资助:国家十三五重点研发计划(No. 2016YFD0400106)
通讯作者: ytao@shou.edu.cn   
引用本文:   
颜倩倩, 陶妍, 李雯, 谢晶, 钱韻芳. 鲤鱼c型溶菌酶在毕赤酵母中的重组表达及其抑菌活性[J]. 农业生物技术学报, 2019, 27(11): 1912-1922.
YAN Qian-Qian, TAO Yan, LI Wen, XIE Jing, QIAN Yun-Fang. Recombinant Expression of Common Carp (Cyprinus carpio) c-type Lysozyme in Pichia pastoris and Its Antibacterial Activity. 农业生物技术学报, 2019, 27(11): 1912-1922.
链接本文:  
http://journal05.magtech.org.cn/Jwk_ny/CN/10.3969/j.issn.1674-7968.2019.11.002     或     http://journal05.magtech.org.cn/Jwk_ny/CN/Y2019/V27/I11/1912
 
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