CN115536737B - 一种眼镜王蛇抗菌肽oh-cath30在制备细菌生长抑制剂中的应用 - Google Patents

一种眼镜王蛇抗菌肽oh-cath30在制备细菌生长抑制剂中的应用 Download PDF

Info

Publication number
CN115536737B
CN115536737B CN202210381217.6A CN202210381217A CN115536737B CN 115536737 B CN115536737 B CN 115536737B CN 202210381217 A CN202210381217 A CN 202210381217A CN 115536737 B CN115536737 B CN 115536737B
Authority
CN
China
Prior art keywords
cath30
cobra
antibacterial peptide
application
antibacterial
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202210381217.6A
Other languages
English (en)
Other versions
CN115536737A (zh
Inventor
沈李元
朱新鹏
陈义培
刘杰
涂翰卿
卢玉平
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Jiangsu Kangjun Biotechnology Co ltd
Original Assignee
Jiangsu Kangjun Biotechnology Co ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Jiangsu Kangjun Biotechnology Co ltd filed Critical Jiangsu Kangjun Biotechnology Co ltd
Priority to CN202210381217.6A priority Critical patent/CN115536737B/zh
Publication of CN115536737A publication Critical patent/CN115536737A/zh
Application granted granted Critical
Publication of CN115536737B publication Critical patent/CN115536737B/zh
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Wood Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Mycology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Plant Pathology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Microbiology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Communicable Diseases (AREA)
  • Oncology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Toxicology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

本发明涉及眼镜王蛇抗菌肽OH‑CATH30在抗水产动物致病菌中的应用，属于生物医药技术领域。眼镜王蛇抗菌肽OH‑CATH30分子量为4693.71Da，等电点为12.08，其氨基酸序列为KRFKKFFKKLKNSVKKRAKKFFKKPRVIGVSIPFHHHHHH；眼镜王蛇抗菌肽OH‑CATH30在水产养殖中的应用。本发明的效果在于：眼镜王蛇抗菌肽OH‑CATH30在抗水产动物致病菌中的应用，能对嗜水气单胞菌、蜡状芽孢杆菌、铜绿假单胞菌等多种菌种有明显抑菌效果。

Description

一种眼镜王蛇抗菌肽OH-CATH30在制备细菌生长抑制剂中的 应用
技术领域
本发明属于生物医学技术领域，具体涉及一种眼镜王蛇抗菌肽OH-CATH30在制备细菌生长抑制剂中的应用。
背景技术
目前，抗生素的滥用问题极其严重，抗生素的残留以及耐药性的问题非常突出，养殖业也同样是抗生素滥用的重灾区。水产养殖行业滥用抗生素，不仅会给动物性食品的安全带来潜在隐患，也会对人类的健康及生存环境造成危害。因此，寻找高效广谱且不易产生耐药性的抗细菌和病毒的药物替代抗生素在水产养殖方面应用成为一项紧迫的任务。
抗菌肽是存在于生物体内能抵抗外界微生物侵害的一类小分子多肽，它是先天免疫系统的重要组成部分，具有抗微生物活性的作用，对革兰氏阳性和革兰氏阴性细菌、真菌和病毒都有抑杀作用。与抗生素相比，抗菌肽不易产生耐药性，在农业、医疗、畜牧、食品及保健品等领域的开发研究与应用方面均有广泛的前景和价值。
眼镜王蛇抗菌肽OH-CATH30由34个氨基酸序列组成，目前研究发现对大肠杆菌、金黄色葡萄球菌等人体病原菌具有抑菌效果。
水产养殖十大病原菌之一的嗜水气单胞菌是为革兰氏阴性短杆菌，广泛分布于自然界的各种水体，是多种水生动物的原发性致病菌，具传染性，可以产生毒性很强的外毒素。由嗜水气单胞菌感染的疾病一般病势较猛，多为恶性传染病，死亡率很高。
本发明以眼镜王蛇抗菌肽OH-CATH30为研究对象，研究其对嗜水气单胞菌、蜡状芽孢杆菌、铜绿假单胞菌等多种细菌的抑菌活性，明确了眼镜王蛇抗菌肽OH-CATH30在抗水产动物致病菌中的应用。
发明内容
本发明的目的在于提供一种眼镜王蛇抗菌肽OH-CATH30。
本发明的另一个目的在于提供眼镜王蛇抗菌肽OH-CATH30在抗水产动物致病菌中的应用。
本发明是通过以下技术方案实现的：眼镜王蛇抗菌肽OH-CATH30，所述眼镜王蛇抗菌肽OH-CATH30由34个氨基酸序列组成；其分子量为4693.71Da，等电点为12.08，氨基酸序列为KRFKKFFKKLKNSVKKRAKKFFKKPRVIGVSIPFHHHHHH。
进一步的，所述眼镜王蛇抗菌肽OH-CATH30采用毕赤酵母发酵的方法获得。
本发明提供眼镜王蛇抗菌肽OH-CATH30在抗水产动物致病菌中的应用。
本发明的优点：制得眼镜王蛇抗菌肽OH-CATH30基因工程表达产物，对其抑菌活性进行鉴定，实验表明，眼镜王蛇抗菌肽OH-CATH30发挥抑制/杀灭致病菌作用，对嗜水气单胞菌、蜡状芽孢杆菌、铜绿假单胞菌等多种细菌具有显著的抑菌活性，明确在制备细菌生长抑制剂中的应用。眼镜王蛇抗菌肽OH-CATH30对水产致病菌如嗜水气单胞菌具有显著的抑菌活性，明确在抗水产动物致病菌中的应用。
具体实施方式
1.眼镜王蛇抗菌肽OH-CATH30，来自眼镜王蛇(Ophiophagus hannah)的毒腺。
2.载体及宿主细胞：pPIC9K质粒和毕赤酵母GS115菌株均购自于淼灵质粒平台。
3.细菌菌株购自中国普通微生物菌种保藏管理中心。
除非特别指明，本发明以下实施例所用的试剂均为分析纯试剂，且可从常规渠道商购获得。
下面对本发明做进一步的描述，本发明的保护范围不局限于以下所述：
实施例1：眼镜王蛇抗菌肽OH-CATH30的制备
S01.眼镜王蛇抗菌肽OH-CATH30表达载体pPIC9K-OH-CATH30的构建，具体操作如下：PCR扩增眼镜王蛇抗菌肽OH-CATH30基因，获得抗菌肽OH-CATH30的目的基因片段，将OH-CATH30基因片段与pPIC9K载体连接，得到pPIC9K-OH-CATH30重组载体；
S02.将步骤S01获得的重组载体转化入宿主细胞毕赤酵母GS115，并对宿主细胞进行诱导表达，获得表达产物；
S03.对步骤S02获得的表达产物进行分离纯化，具体操作为：先对表达产物进行离心，收集上清液，进行透析后，最后进行亲和层析法纯化，获得重组融合多肽OH-CATH30。
实施例2：眼镜王蛇抗菌肽OH-CATH30的抑菌试验，具体如下：
(1)将由按实施例1中的方法所获得的眼镜王蛇抗菌肽OH-CATH30多肽样品过滤除菌后，以眼镜王蛇抗菌肽OH-CATH30的合成肽为标准品应用绘制蛋白浓度标准曲线，根据公式计算出OH-CATH30重组多肽的浓度。
(2)将多肽溶液倍比稀释至1-32μg/mL。
(3)进行MIC(Minimum Inhibitory Concentration，最小抑菌浓度)的测定，所述MIC测定在96孔细胞培养板上进行。具体操作如下：
①革兰氏阳性菌如金黄色葡萄球菌、蜡状芽孢杆菌，革兰氏阴性菌如铜绿假单胞菌、嗜水气单胞菌四种菌悬液的准备：取被测细菌划线于MH平板，培养12个小时；
②调整菌浓度至6×105CFU/ml备用。
③每种被测菌按照以下操作设置空白对照组、阳性对照组和待测样品实验组，每组设置2个平行样，每种菌重复3次：
Ⅰ阳性对照组：加入50μl磷酸钠盐缓冲液和50μl菌悬液；
Ⅱ空白对照组：加入50μl待测蛋白样品和50μl磷酸钠盐缓冲液；
Ⅲ样品实验组：加入50μl待测蛋白样品和50μl菌悬液；
④细菌培养24～48h后，观察MIC结果。
抑菌实验结果显示，眼镜王蛇抗菌肽OH-CATH30具有广谱的抑菌活性，能够有效的抑制革兰氏阳性菌如金黄色葡萄球菌(MIC 2～4μg/mL)和蜡状芽孢杆菌(MIC 2～4μg/mL)，革兰氏阴性菌铜绿假单胞菌(MIC 2～4μg/mL)和嗜水气单胞菌(MIC 4～8μg/mL)的生长。结果如表1
表1眼镜王蛇抗菌肽OH-CATH30的抑菌活性
CGMCC No.：中国微生物菌种保藏编号；MIC：最小抑菌浓度，用(a)-(b)表示，(a)表示肉眼可见菌体生长的最高浓度，(b)表示不见菌体生长的最小浓度。
从表1数据可知，重组表达获得的眼镜王蛇抗菌肽OH-CATH30对革兰氏阳性菌金黄色葡萄球菌、蜡状芽孢杆菌和革兰氏阴性菌铜绿假单胞菌、嗜水气单胞菌均具有较好的抑菌效果。
序列表
<110> 江苏亢钧生物科技有限公司
<120> 一种眼镜王蛇抗菌肽OH-CATH30在制备细菌生长抑制剂中的应用
<130> 20220411
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 105
<212> DNA
<213> 眼镜王蛇(Ophiophagus hannah)
<400> 1
aagagattca agaaattttt caagaagctg aagaacagcg tgaagaaacg tgccaagaaa 60
ttcttcaaga agccgagggt catcggagtc tccatcccct tctaa 105
<210> 2
<211> 40
<212> PRT
<213> 眼镜王蛇(Ophiophagus hannah)
<400> 2
Lys Arg Phe Lys Lys Phe Phe Lys Lys Leu Lys Asn Ser Val Lys Lys
1 5 10 15
Arg Ala Lys Lys Phe Phe Lys Lys Pro Arg Val Ile Gly Val Ser Ile
20 25 30
Pro Phe His His His His His His
35 40

Claims (1)

1.一种眼镜王蛇抗菌肽OH-CATH30在制备细菌生长抑制剂中的应用，其特征在于，所述细菌为嗜水气单胞菌、蜡状芽孢杆菌，所述眼镜王蛇抗菌肽OH-CATH30的氨基酸序列为KRFKKFFKKLKNSVKKRAKKFFKKPRVIGVSIPFHHHHHH。
CN202210381217.6A 2022-04-13 2022-04-13 一种眼镜王蛇抗菌肽oh-cath30在制备细菌生长抑制剂中的应用 Active CN115536737B (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202210381217.6A CN115536737B (zh) 2022-04-13 2022-04-13 一种眼镜王蛇抗菌肽oh-cath30在制备细菌生长抑制剂中的应用

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202210381217.6A CN115536737B (zh) 2022-04-13 2022-04-13 一种眼镜王蛇抗菌肽oh-cath30在制备细菌生长抑制剂中的应用

Publications (2)

Publication Number Publication Date
CN115536737A CN115536737A (zh) 2022-12-30
CN115536737B true CN115536737B (zh) 2023-10-03

Family

ID=84724214

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202210381217.6A Active CN115536737B (zh) 2022-04-13 2022-04-13 一种眼镜王蛇抗菌肽oh-cath30在制备细菌生长抑制剂中的应用

Country Status (1)

Country Link
CN (1) CN115536737B (zh)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115558613B (zh) * 2022-08-17 2024-04-09 江苏亢钧生物科技有限公司 一种提高诱导眼镜王蛇抗菌肽oh-cath30表达效率的培养基及其配制方法
CN116396372B (zh) * 2023-02-18 2024-05-24 青岛农业大学 一种载肽纳米颗粒及在宠物眼部感染治疗上的应用
CN117016448A (zh) * 2023-08-29 2023-11-10 江苏亢钧生物科技有限公司 一种利用眼镜蛇抗菌肽提高大口黑鲈抗病能力的方法

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020060401A2 (en) * 2018-09-17 2020-03-26 Universiteit Leiden Bioactive peptides derived from snakes
WO2020061553A1 (en) * 2018-09-21 2020-03-26 Emory University Using multiple heteroresistance to guide combination antibiotic regimens
CN113980112A (zh) * 2021-11-25 2022-01-28 中国科学院昆明动物研究所 一种眼镜王蛇抗菌肽oh-cath30的表达载体和表达产物及其构建制备方法
CN115536738A (zh) * 2022-04-13 2022-12-30 江苏亢钧生物科技有限公司 眼镜王蛇抗菌肽oh-cath30在治疗鱼类肠炎中的应用
CN115558019A (zh) * 2022-04-13 2023-01-03 江苏亢钧生物科技有限公司 一种眼镜王蛇抗菌肽oh-cath30的基因工程制备方法

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3621632A4 (en) * 2017-05-11 2021-04-28 Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center ANTIMICROBIAL PEPTIDES WITH ALPHA CORE HELICES

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020060401A2 (en) * 2018-09-17 2020-03-26 Universiteit Leiden Bioactive peptides derived from snakes
WO2020061553A1 (en) * 2018-09-21 2020-03-26 Emory University Using multiple heteroresistance to guide combination antibiotic regimens
CN113980112A (zh) * 2021-11-25 2022-01-28 中国科学院昆明动物研究所 一种眼镜王蛇抗菌肽oh-cath30的表达载体和表达产物及其构建制备方法
CN115536738A (zh) * 2022-04-13 2022-12-30 江苏亢钧生物科技有限公司 眼镜王蛇抗菌肽oh-cath30在治疗鱼类肠炎中的应用
CN115558019A (zh) * 2022-04-13 2023-01-03 江苏亢钧生物科技有限公司 一种眼镜王蛇抗菌肽oh-cath30的基因工程制备方法

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
"Antimicrobial peptides (AMPs): Ancient compounds that represent novel weapons in the fight against bacteria";J M Ageitos 等;《Biochem Pharmacol》;第133卷;第117-138页 *
"Antimicrobial peptides, conventional antibiotics, and their synergistic utility for the treatment of drug-resistant infections";Yiyun Zhu 等;《medicinal research reviews》;第42卷(第4期);第1377-1422页 *
"Cathelicidin antimicrobial peptide from Alligator mississippiensis has antibacterial activity against multi-drug resistant Acinetobacter baumanii and Klebsiella pneumoniae";Stephanie M Barksdale 等;《Dev Comp Immunol》;第70卷;第135-144页 *
"Hitchhiking with Nature: Snake Venom Peptides to Fight Cancer and Superbugs";Clara Pérez-Peinado 等;《Toxins 2020》;第12卷(第4期);doi.org/10.3390/toxins12040255 *
"Identification and characterization of novel reptile cathelicidins from elapid snakes";Hui Zhao 等;《Peptides》;第29卷(第10期);第1685-1691页 *
"Structure–function relationship of king cobra cathelicidin";Yong Zhang 等;《Peptides》;第31卷(第8期);表1、2 *
"Venom peptides cathelicidin and lycotoxin cause strong inhibition of Escherichia coli ATP synthase";Sofiya Azim 等;《Int J Biol Macromol》;第87卷;第246-251页 *
"细鳞鱼Cathelicidins的鉴定及宿主防御肽CATHBRALE的结构与功能分析";厉政;《中国优秀硕士学位论文全文数据库 (农业科技辑)》(第8期);D052-23 *

Also Published As

Publication number Publication date
CN115536737A (zh) 2022-12-30

Similar Documents

Publication Publication Date Title
CN115536737B (zh) 一种眼镜王蛇抗菌肽oh-cath30在制备细菌生长抑制剂中的应用
CN104151415B (zh) 一种天然抗菌肽Alligatorin4及其应用
CN111892646B (zh) 抗菌肽衍生物及其在制备抗细菌感染药物中的应用
EP2938352B1 (en) Cyclic cationic peptides with antibmicrobial activity
WO2022084978A1 (en) A process for the preparation of an antimicrobial peptide
CN107252475B (zh) 天然宿主防御肽Alligatorin4的应用
Yin et al. A specific antimicrobial protein CAP-1 from Pseudomonas sp. isolated from the jellyfish Cyanea capillata
CN104177485B (zh) 一种扬子鳄抗菌肽Alligatorin6及其应用
CN116270973A (zh) 克氏原螯虾甘露糖结合蛋白在抑制细菌中的应用
CN112321697B (zh) 一种黑水虻抗菌肽Cecropin-α及其应用
CN113321708A (zh) 一种人工设计抗菌肽的制备及其在水产上的应用
CN105104712B (zh) 一种微生物饲料添加剂及其制备方法
CN116063440B (zh) 一种可口革囊星虫抗菌肽及其应用
Zare-Zardini et al. Antimicrobial peptides of innate immune system as a suitable compound for cancer treatment and reduction of its related infectious disease
CN113599497B (zh) 斜带石斑鱼piscidin1及其合成多肽在制备抗病毒或抗细菌药物中的应用
CN113603748B (zh) 一种β-折叠抗菌肽HINGE-RV及其制备方法和应用
CN109627286A (zh) 一种新型广谱抗菌肽samp1-a4及其制备方法
CN105777875B (zh) 一种抗菌肽cstc24及其应用
CN114516899A (zh) 抗菌肽yhx-5及其应用
CN110317253B (zh) 一种鸡源抗菌肽及其制备方法
CN107261113B (zh) 天然宿主防御肽Alligatorin5的应用
CN115974996A (zh) 杂交鲈鱼抗菌肽wb-moronecidin在抗水产动物致病菌中的应用
CN113234137B (zh) 分离自草鱼的CXCL20a蛋白在作为抗菌肽中的应用
CN115925872B (zh) 一种靶向变形假单胞菌的抗菌肽skl17-2及其应用
CN117069819B (zh) 一种黑腹狼蛛抗菌肽lc-amp-i1及其应用

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant