WO2021068531A1 - 一种黑鲷抗菌肽AS-hepc3 (48-56)及其应用 - Google Patents

一种黑鲷抗菌肽AS-hepc3 (48-56)及其应用 Download PDF

Info

Publication number
WO2021068531A1
WO2021068531A1 PCT/CN2020/093727 CN2020093727W WO2021068531A1 WO 2021068531 A1 WO2021068531 A1 WO 2021068531A1 CN 2020093727 W CN2020093727 W CN 2020093727W WO 2021068531 A1 WO2021068531 A1 WO 2021068531A1
Authority
WO
WIPO (PCT)
Prior art keywords
hepc3
antibacterial
drug
sea bream
black sea
Prior art date
Application number
PCT/CN2020/093727
Other languages
English (en)
French (fr)
Inventor
王克坚
朱德鹏
彭会
陈芳奕
陈慧芸
Original Assignee
厦门大学
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 厦门大学 filed Critical 厦门大学
Publication of WO2021068531A1 publication Critical patent/WO2021068531A1/zh
Priority to US17/373,042 priority Critical patent/US11299514B2/en

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • 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
    • 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
    • C07K14/461—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from fish
    • 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

Definitions

  • the invention belongs to the technical field of marine molecular biology, and specifically relates to a black sea bream antibacterial peptide and its application.
  • antibiotics have a single site of action, and bacteria can develop drug resistance through inherent drug resistance, adaptive drug resistance, and acquired drug resistance, while antimicrobial peptides have diverse sites of action and are not easy to develop drug resistance.
  • antimicrobial peptides have diverse sites of action and are not easy to develop drug resistance.
  • Antimicrobial Peptide also known as host defense peptide, is a kind of amphiphilic, mostly positively charged, short peptide with antibacterial function. It is widely present in animals, plants, microorganisms and other organisms, and it is the innate immune system. An important part of the antibacterial activity is strong, the antibacterial spectrum is broad, and it is not easy to produce drug resistance. Antimicrobial peptides have the characteristics of simple structure, convenient and fast chemical synthesis, etc. They have good application prospects in production and application. At present, a variety of peptide drugs have been used in clinical treatment, providing new ideas and ideas for reducing the occurrence of drug resistance. Strategy.
  • the purpose of the present invention is to overcome the shortcomings of the prior art and provide a black sea bream antibacterial peptide AS-hepc3 (48-56) and its application, which solves the above-mentioned problems in the background art.
  • One of the technical solutions adopted by the present invention to solve its technical problems is to provide a black sea bream antimicrobial peptide AS-hepc3 (48-56) , its molecular formula is C 48 H 86 N 24 O 10 S 3 , and its amino acid sequence is SEQ ID NO: 01:
  • the molecular weight of the black sea bream antimicrobial peptide is 1255.567 Daltons, including 5 positively charged amino acid residues and 3 cysteine residues.
  • the second technical solution adopted by the present invention to solve its technical problems is to provide an application of black sea bream antibacterial peptide AS-hepc3 (48-56) in the preparation of antibacterial drugs.
  • the third technical solution adopted by the present invention to solve its technical problems is to provide an antibacterial drug, the effective ingredients of which include black sea bream antimicrobial peptide AS-hepc3 (48-56) , and the black sea bream antimicrobial peptide AS-hepc3 (48 The amino acid sequence of -56) is SEQ ID NO:01.
  • the active ingredient of the antibacterial drug is black sea bream antimicrobial peptide AS-hepc3 (48-56)
  • the amino acid sequence of the black sea bream antimicrobial peptide AS-hepc3 (48-56) is SEQ ID NO: 01.
  • the antibacterial drug is used to inhibit and/or kill Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli.
  • the black sea bream antimicrobial peptide AS-hepc3 (48-56) of the present invention is composed of 9 amino acids, the molecular formula is C 48 H 86 N 24 O 10 S 3 , the molecular weight is 1255.567 Daltons, and 5 of them are positively charged.
  • the antimicrobial peptide is predicted to have an isoelectric point of 11.40 and an average coefficient of hydrophilicity of -1.356. It has good water solubility and is a
  • the positively charged cationic polypeptide has the characteristics of short length, easy synthesis, broad antibacterial spectrum, high antibacterial activity and stability, and has good application prospects in drug research.
  • the black sea bream antibacterial polypeptide AS-hepc3 (48-56) of the present invention has good antibacterial properties against clinically isolated drug-resistant Pseudomonas aeruginosa, drug-resistant Staphylococcus aureus and drug-resistant Escherichia coli Active and not easy to produce drug resistance;
  • AS-hepc3 (48-56) has no cytotoxicity to normal mouse liver cells AML12 and human kidney epithelial cells 293T.
  • Figure 1 shows the comparison of black sea bream antibacterial polypeptide AS-hepc3 (48-56) and antibiotic meropenem against Pseudomonas aeruginosa PAO1 antimicrobial resistance; where the abscissa is time (day), and the ordinate is the change in MIC concentration and The initial antibacterial concentration ratio.
  • Figure 2 is the MTS-PMS method to detect the cytotoxicity of the black sea bream antibacterial polypeptide AS-hepc3 (48-56) ; where a is AML12 cells, b is 293T cells, and the abscissa is the protein concentration of AS-hepc3 (48-56) ( ⁇ M), the ordinate is the cell proliferation rate (%).
  • amino acid sequence of the black sea bream antibacterial polypeptide AS-hepc3 (48-56) in this example is:
  • the black sea bream antibacterial polypeptide AS-hepc3 (48-56) was entrusted to Jill Biochemical (Shanghai) Co., Ltd. to synthesize by solid-phase synthesis method, and the black sea bream antibacterial polypeptide AS-hepc3 (48-56) with a purity of more than 95% was obtained. And provide peptide molecular weight, HPLC and other detection information, related physical and chemical parameters are shown in Table 1.
  • the black sea bream antibacterial polypeptide AS-hepc3 (48-56) of this example has a small molecular weight, good stability, and strong water solubility, and is a positively charged cationic polypeptide.
  • Example 2 Determination of Minimum Inhibition Concentration (MIC: Minimum Inhibition Concentration) and Minimum Bactericidal Concentration (MBC: Minimum Bactericidal Concentration):
  • the strains involved are: Pseudomonas aeruginosa PAO1, clinical isolation of drug-resistant Pseudomonas aeruginosa QZ19121, QZ19122, QZ19123, QZ19124, QZ19125, clinical isolation of drug-resistant Acinetobacter baumannii ) QZ18050, QZ18055, clinical isolation of drug-resistant Staphylococcus aureus QZ18090, QZ18091, clinical isolation of drug-resistant Klebsiella pneumoniae QZ18106, clinical isolation of drug-resistant Escherichia coli QZ18109, QZ18110.
  • PAO1 was purchased from the Culture Collection Center of the Institute of Microbiology, Chinese Academy of Sciences, and the clinically isolated strains were from the Laboratory Department of the Second affiliated Hospital of Fujian Medical University.
  • Blank control group 50 ⁇ L protein sample to be tested and 50 ⁇ L culture medium;
  • Negative control group 50 ⁇ L sterile Milli-Q water and 50 ⁇ L bacterial suspension
  • MIC minimum inhibitory concentration ( ⁇ M), expressed as a-b; a: the highest protein concentration that can be seen by the naked eye; b: the lowest protein concentration where no bacterial growth is seen by the naked eye;
  • MBC The minimum bactericidal concentration ( ⁇ M), the concentration that kills 99.9% of bacteria.
  • Dissolve antibiotic meropenem powder in sterile Milli-Q water configure 5mg/mL stock solution, filter with 0.22 ⁇ m membrane, and dilute to different working concentrations, 0.0625 ⁇ g/mL, 0.125 ⁇ g/mL, 0.25 ⁇ g/mL, 0.5 ⁇ g/mL, 1 ⁇ g/mL, 2 ⁇ g/mL, 4 ⁇ g/mL, 8 ⁇ g/mL, 16 ⁇ g/mL, 32 ⁇ g/mL, 48 ⁇ g/mL, 64 ⁇ g/mL, 96 ⁇ g/mL, 128 ⁇ g/mL, 192 ⁇ g /mL, 256 ⁇ g/mL, 512 ⁇ g/mL;
  • a blank control group, a negative control group and an experimental group to be tested are set for each type of bacteria to be tested, and three parallel groups are set for each group:
  • Blank control group 50 ⁇ L protein sample to be tested and 50 ⁇ L culture medium;
  • Negative control group 50 ⁇ L sterile Milli-Q water and 50 ⁇ L bacterial suspension
  • the antimicrobial peptide AS-hepc3 (48-56) was incubated with AML12 cells ( Figure 2-a) and 293T cells ( Figure 2-b) for 24 hours. Compared with the experimental group, the cell survival rate of the control group was above 95%, indicating that the antimicrobial peptide AS-hepc3 (48-56) had no cytotoxicity.
  • the invention discloses a black sea bream antimicrobial peptide AS-hepc3 (48-56) and its application.
  • the molecular formula of the black sea bream antimicrobial peptide AS-hepc3 (48-56) is C 48 H 86 N 24 O 10 S 3 , which The amino acid sequence is SEQ ID NO: 01.
  • the antibacterial peptide AS-hepc3 (48-56) of the present invention is used as an effective ingredient of antibacterial drugs. It has the characteristics of short length, high antibacterial activity, resistance to resistance, and resistance to a variety of clinical drug-resistant bacteria. The research and development of drug-resistant bacteria has broad prospects and has industrial applicability.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Zoology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Animal Behavior & Ethology (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Communicable Diseases (AREA)
  • Oncology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Biophysics (AREA)
  • Toxicology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)

Abstract

本发明公开了一种黑鲷抗菌肽AS-hepc3 (48-56)及其应用，该黑鲷抗菌肽AS-hepc3 (48-56)的分子式为C 48H 86N 24O 10S 3，其氨基酸序列为SEQ ID NO：01。本发明的抗菌肽AS-hepc3 (48-56)，作为抗菌药物的有效成分应用，具有长度短、抗菌活性高且不易产生耐药性，以及能够抗多种临床耐药细菌等特点，在抗耐药菌药物研发方面具有广阔前景。

Description

一种黑鲷抗菌肽AS-hepc3 (48-56)及其应用 技术领域
本发明属于海洋分子生物学技术领域，具体涉及一种黑鲷抗菌肽及其应用。
背景技术
1928年英国科学家Fleming发现了第一种抗生素——青霉素。从此，抗生素不断被发现、改进并用于现代医疗。随着抗生素的广泛应用，抗生素耐药性也不断被发现，世界卫生组织2014年报告中提出，抗生素耐药性细菌已在全球范围蔓延，我们已经迈入了后抗生素时代。目前，全球范围均有抗生素耐药性的报道，成为全球亟待解决的重要问题之一。WHO指出每年至少有70万人死于耐药性疾病，如不采取有效措施这一数目在2050年将达到1000万。
在临床分离耐药细菌中，不仅有单一耐药菌株，还发现多重耐药性、极高耐药性和完全耐药性细菌，给临床治疗带来巨大挑战。2017年WHO提出需要优先研发新药治疗耐碳青霉烯类铜绿假单胞菌、耐碳青霉烯类鲍曼不动杆菌、耐碳青霉烯类和耐三代头孢霉素的肠杆菌科细菌、耐万古霉素肠球菌、耐甲氧西林和万古霉素金黄色葡萄球菌等十二类耐药性细菌。目前认为抗生素因其作用位点单一，细菌可通过固有耐药、适应性耐药和获得性耐药等方式产生耐药性，而抗菌肽作用位点多样，不易产生耐药性，被认为是替代抗生素的选择之一。
抗菌肽(Antimicrobial Peptide，AMP)又称为宿主防御肽，是一种多为正电荷的两亲性具有抗菌功能的短肽，广泛存在于动物、植物、微生物等生物体中，为先天免疫系统的重要组成部分，具有抗菌活性强，抗菌谱广，不易产生耐药性等特点。抗菌短肽具有结构简单，化学合成方便快捷等特点在生产应用中具有很好的应用前景，目前已有多种多肽类药物用于临床治疗，为降低药物耐药性的发生提供新的思路和策略。
发明内容
本发明的目的在于克服现有技术的不足之处，提供了一种黑鲷抗菌肽AS-hepc3 (48-56)及其应用，解决了上述背景技术中的问题。
本发明解决其技术问题所采用的技术方案之一是：提供了一种黑鲷抗菌肽AS-hepc3 (48-56)，其分子式为C 48H 86N 24O 10S 3，其氨基酸序列为SEQ ID NO：01：
Arg-Arg-Arg–Arg–Cys-Arg-Phe–Cys-Cys(SEQ ID NO：01)。
所述黑鲷抗菌肽的分子量为1255.567道尔顿，包括5个带正电的氨基酸残基和3个半胱氨酸残基。
本发明解决其技术问题所采用的技术方案之二是：提供了一种黑鲷抗菌肽AS-hepc3 (48-56)在制备抗菌药物中的应用。
本发明解决其技术问题所采用的技术方案之三是：提供了一种抗菌药物，其有效成分包括黑鲷抗菌肽AS-hepc3 (48-56)，所述黑鲷抗菌肽AS-hepc3 (48-56)的氨基酸序列为SEQ ID NO：01。
在本发明一较佳实施例中，所述抗菌药物的有效成分为黑鲷抗菌肽AS-hepc3 (48-56)，所述黑鲷抗菌肽AS-hepc3 (48-56)的氨基酸序列为SEQ ID NO：01。
在本发明一较佳实施例中，所述抗菌药物用于抑制和/或杀灭铜绿假单胞菌、金黄色葡萄球菌、大肠埃希氏菌。
本技术方案与背景技术相比，它具有如下优点：
1.本发明的黑鲷抗菌肽AS-hepc3 (48-56)由9个氨基酸组成，分子式为C 48H 86N 24O 10S 3，分子量为1255.567道尔顿，其中含有5个带正电的氨基酸残基和3个半胱氨酸残基，根据氨基酸残基电荷预测该抗菌肽等电点为11.40，亲水性平均系数为﹣1.356，具有很好的水溶性，是一种带有正电荷的阳离子多肽，具有长度短、易合成、 抗菌谱广、抗菌活性高且稳定等特点，在药物研究方面具有很好应用前景。
2.本发明的黑鲷抗菌多肽AS-hepc3 (48-56)对临床分离的耐药性铜绿假单胞菌、耐药性金黄色葡萄球菌和耐药性大肠埃希氏菌均具有良好抗菌活性且不易产生耐药性；此外，AS-hepc3 (48-56)对正常小鼠肝细胞AML12和人肾上皮细胞293T无细胞毒性。
附图说明
图1为黑鲷抗菌多肽AS-hepc3 (48-56)与抗生素美罗培南对铜绿假单胞菌PAO1抗菌耐药性产生比较；其中，横坐标为时间(day)，纵坐标为MIC浓度变化与初始抗菌浓度比值。
图2为MTS-PMS法检测黑鲷抗菌多肽AS-hepc3 (48-56)细胞毒性实验图；其中，a为AML12细胞，b为293T细胞，横坐标为AS-hepc3 (48-56)蛋白浓度(μM)，纵坐标为细胞增殖率(％)。
具体实施方式
实施例1
本实施例的一种黑鲷抗菌多肽AS-hepc3 (48-56)的氨基酸序列为：
Arg-Arg-Arg–Arg–Cys-Arg-Phe–Cys-Cys(SEQ ID NO：01)
本实施例黑鲷抗菌多肽AS-hepc3 (48-56)委托吉尔生化(上海)有限公司以固相合成方法合成，获得纯度达95％以上的黑鲷抗菌多肽AS-hepc3 (48-56)，并提供多肽分子量、HPLC等检测信息，相关理化参数如表1所示。
表1抗菌多肽AS-hepc3 (48-56)理化参数
Figure PCTCN2020093727-appb-000001
Figure PCTCN2020093727-appb-000002
由表1可知本实施例的黑鲷抗菌多肽AS-hepc3 (48-56)分子量小、稳定性较好，具有很强的水溶性，是一种带有正电荷的阳离子多肽。
实施例2：最小抑菌浓度(MIC：Minimum Inhibition Concentration)和最小杀菌浓度(MBC：Minimum Bactericidal Concentration)的测定：
1、涉及到的菌株有：铜绿假单胞菌(Pseudomonas aeruginosa)PAO1，临床分离耐药铜绿假单胞菌QZ19121、QZ19122、QZ19123、QZ19124、QZ19125，临床分离耐药鲍曼不动杆菌(Acinetobacter baumannii)QZ18050，QZ18055，临床分离耐药金黄色葡萄球菌(Staphylococcus aureus)QZ18090、QZ18091，临床分离耐药肺炎克雷伯氏菌(Klebsiella pneumoniae)QZ18106，临床分离耐药大肠埃希氏菌(Escherichia coli)QZ18109、QZ18110。PAO1购自中国科学院微生物研究所菌种保藏中心，临床分离菌株来源于福建医科大学附属第二医院检验科。
2、具体方法如下：
(1)将保种菌株划线于MH平板，于37℃条件下倒置培养过夜；
(2)挑取单克隆于MH液体培养基中，37℃，200rpm培养至对数期；
(3)5000g，2min收集细菌，用10mM磷酸钠缓冲液(pH＝7.4)将细菌重悬，最后用MH液体培养基稀释细菌，使得菌体的最终浓度为5×10 5cfu/mL；
(4)将已合成的AS-hepc3 (48-56)粉末溶于无菌Milli-Q水，倍比稀释蛋白浓度至2 μM、4μM、8μM、16μM、32μM、64μM；
(5)在96孔聚丙烯无菌培养板上，每种待测菌设置空白对照组、阴性对照组和待测实验组，每组设置三个平行：
a空白对照组：50μL待测蛋白样品和50μL培养基；
b阴性对照组：50μL无菌Milli-Q水和50μL菌悬液；
c待测实验组：50μL待测蛋白样品和50μL菌悬液；
将96孔培养板置于37℃培养箱中，培养18-24h，观察待测实验组中MIC结果；将待测实验组吹打混匀后，吸取适量的菌液涂布于MH平板上，于37℃倒置培养过夜，观察MBC结果。
3、黑鲷抗菌肽AS-hepc3 (48-56)MIC、MBC观察结果如表2所示。
表2黑鲷抗菌肽AS-hepc3 (48-56)的抗菌活性
Figure PCTCN2020093727-appb-000003
Figure PCTCN2020093727-appb-000004
注：MIC：最小抑菌浓度(μM)，用a-b表示；a：肉眼可见菌体生长的最高蛋白浓度；b：肉眼未见菌体生长的最低蛋白浓度；
MBC：最小杀菌浓度(μM)，杀死99.9％细菌浓度。
实施例3：、耐药性产生对比实验：
1、黑鲷抗菌多肽AS-hepc3 (48-56)与抗生素美罗培南抗铜绿假单胞菌PAO1耐药性产生比较，其中铜绿假单胞菌(Pseudomonas Aeruginosa)PAO1，购自中国科学院微生物研究所菌种保藏中心。
2、具体方法如下：
(1)将保种铜绿假单胞菌划线于MH平板，于37℃条件下倒置培养过夜；
(2)挑取单克隆于MH液体培养基中，37℃，200rpm培养至对数期；
(3)5000g，2min收集细菌，用10mM磷酸钠缓冲液(pH＝7.4)将细菌重悬，最后用10mM磷酸钠缓冲液与MH液体培养基混合液稀释细菌，使得菌体的最终浓度为5×10 5cfu/mL；
(4)将已合成的AS-hepc3 (48-56)粉末溶于无菌Milli-Q水，倍比稀释蛋白浓度至8μM、12μM、16μM、24μM、32μM、48μM、64μM、96μM；
(5)将抗生素美罗培南粉末溶于无菌Milli-Q水，配置5mg/mL储液，用0.22μm滤膜过滤，倍比稀释至不同工作浓度，0.0625μg/mL、0.125μg/mL、0.25μg/mL、0.5μg/mL、1μg/mL、2μg/mL、4μg/mL、8μg/mL、16μg/mL、32μg/mL、48μg/mL、64μg/mL、96μg/mL、128μg/mL、192μg/mL、256μg/mL、512μg/mL；
(6)在96孔细胞培养板上，每种待测菌设置空白对照组、阴性对照组和待测实验组，每组设置三个平行：
a空白对照组：50μL待测蛋白样品和50μL培养基；
b阴性对照组：50μL无菌Milli-Q水和50μL菌悬液；
c待测实验组：50μL待测蛋白样品和50μL菌悬液；
将96孔细胞培养板置于37℃培养箱中，培养18-24h，观察待测实验组中MIC结果；取细菌可生长最高浓度抗菌物质组细菌稀释1000倍取50μL用于下一代抗菌实验，如此往复150代。
3、结果如图1。在使用抗生素美罗培南作用铜绿假单胞菌时，3天后其抗铜绿假单胞菌MIC值升高至初始MIC值的4倍，连续使用10天后，其抗铜绿假单胞菌MIC值升高至初始MIC值16倍并不断升高，在作用90天后则可达到1024倍，表明铜绿假单胞菌对抗生素美罗培南产生较强的耐药性；而使用抗菌肽AS-hepc3 (48-56)对铜绿假单胞菌连续作用150天后，其抗铜绿假单胞菌MIC值仅为初始MIC值的2倍，无明显变化，说明铜绿假单胞菌对抗菌肽AS-hepc3 (48-56)并没有产生明显耐药性。
实施例4：、细胞毒性测定
1、选取小鼠肝细胞(AML12)和人肾上皮细胞(293T)，对黑鲷抗菌肽AS-hepc3 (48-56)细胞毒性进行测定。
2、具体方法如下：
(6)收集生长状态良好的小鼠肝细胞(AML12)和人肾上皮细胞(293T)，调整细胞浓度为10 3-10 4个/mL，将细胞均匀吹散，在96孔细胞培养板中每孔加入100μL 细胞悬液，置于37℃，0.5％CO 2条件下静置培养，50％以上细胞贴壁。
(7)小心吸出培养基，加入含有不同浓度(0μM、40μM、80μM)AS-hepc3 (48-56)的相应培养基，置于37℃，0.5％CO 2条件下静置培养24h。
(8)加入20μL MTS-PMS溶液后避光孵育3h后，使用酶标仪测得OD 492值，评价AS-hepc3 (48-56)的细胞毒性。
3、结果如图2。
在5倍和10倍MIC浓度(40μM和80μM)条件下，抗菌肽AS-hepc3 (48-56)与AML12细胞(图2-a)和293T细胞(图2-b)共孵24h后，与对照组相比实验组细胞存活率均在95％以上，说明抗菌肽AS-hepc3 (48-56)无细胞毒性。
以上所述，仅为本发明较佳实施例而已，故不能依此限定本发明实施的范围，即依本发明专利范围及说明书内容所作的等效变化与修饰，皆应仍属本发明涵盖的范围内。
工业实用性
本发明公开了一种黑鲷抗菌肽AS-hepc3 (48-56)及其应用，该黑鲷抗菌肽AS-hepc3 (48-56)的分子式为C 48H 86N 24O 10S 3，其氨基酸序列为SEQ ID NO：01。本发明的抗菌肽AS-hepc3 (48-56)，作为抗菌药物的有效成分应用，具有长度短、抗菌活性高且不易产生耐药性，以及能够抗多种临床耐药细菌等特点，在抗耐药菌药物研发方面具有广阔前景,具有工业实用性。

Claims (7)

  1. 一种黑鲷抗菌肽AS-hepc3 (48-56)，其特征在于：其分子式为C 48H 86N 24O 10S 3，其氨基酸序列为SEQ ID NO：01。
  2. 根据权利要求1所述的一种黑鲷抗菌肽AS-hepc3 (48-56)，其特征在于：所述黑鲷抗菌肽的分子量为1255.567道尔顿。
  3. 根据权利要求1所述的一种黑鲷抗菌肽AS-hepc3 (48-56)，其特征在于：包括5个带正电的氨基酸残基和3个半胱氨酸残基。
  4. 如权利要求1～3中任一项所述的一种黑鲷抗菌肽AS-hepc3 (48-56)在制备抗菌药物中的应用。
  5. 一种抗菌药物，其特征在于：其包括黑鲷抗菌肽AS-hepc3 (48-56)，所述黑鲷抗菌肽AS-hepc3 (48-56)的氨基酸序列为SEQ ID NO：01。
  6. 根据权利要求5所述的一种抗菌药物，其特征在于：其有效成分为黑鲷抗菌肽AS-hepc3 (48-56)。
  7. 根据权利要求5或6任一项所述的一种抗菌药物，其特征在于：所述抗菌药物用于抑制和/或杀灭铜绿假单胞菌、金黄色葡萄球菌、大肠埃希氏菌。
PCT/CN2020/093727 2019-10-09 2020-06-01 一种黑鲷抗菌肽AS-hepc3 (48-56)及其应用 WO2021068531A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US17/373,042 US11299514B2 (en) 2019-10-09 2021-07-12 Antimicrobial peptide AS-hepc3(48-56) of Acanthopagrus schlegelii and method thereof

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201910953094.7A CN110776560B (zh) 2019-10-09 2019-10-09 一种黑鲷抗菌肽AS-hepc3(48-56)及其应用
CN201910953094.7 2019-10-09

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US17/373,042 Continuation US11299514B2 (en) 2019-10-09 2021-07-12 Antimicrobial peptide AS-hepc3(48-56) of Acanthopagrus schlegelii and method thereof

Publications (1)

Publication Number Publication Date
WO2021068531A1 true WO2021068531A1 (zh) 2021-04-15

Family

ID=69384864

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/093727 WO2021068531A1 (zh) 2019-10-09 2020-06-01 一种黑鲷抗菌肽AS-hepc3 (48-56)及其应用

Country Status (3)

Country Link
US (1) US11299514B2 (zh)
CN (1) CN110776560B (zh)
WO (1) WO2021068531A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116143883A (zh) * 2022-10-19 2023-05-23 厦门大学 一种广谱抗微生物肽Scymicrosin7-26及其应用

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110776560B (zh) * 2019-10-09 2021-02-02 厦门大学 一种黑鲷抗菌肽AS-hepc3(48-56)及其应用
CN113121666B (zh) * 2021-03-10 2023-09-12 厦门大学 抗菌肽Scybaumancin105-127及其应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1641025A (zh) * 2004-11-29 2005-07-20 中国水产科学研究院黄海水产研究所 真鲷抗菌肽基因和重组酵母表达载体及其制备方法
CN110170045A (zh) * 2019-06-28 2019-08-27 福州大学 抗菌肽Lchamp2-3在抗肿瘤细胞增殖药物中的应用
CN110776560A (zh) * 2019-10-09 2020-02-11 厦门大学 一种黑鲷抗菌肽AS-hepc3(48-56)及其应用

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7723063B2 (en) * 2004-04-28 2010-05-25 Intrinsic Lifesciences Methods for measuring levels of bioactive human hepcidin
CN101063145B (zh) * 2007-04-20 2010-04-14 厦门大学 黑鲷抗菌肽Hepcidin的表达载体和表达产物及其构建制备方法
CN101906165B (zh) * 2010-07-09 2012-11-14 厦门大学 两种鱼类抗菌肽基因的串联表达产物及其表达方法
CN102517293A (zh) * 2011-11-28 2012-06-27 厦门大学 黑鲷抗菌肽表达载体与构建方法及表达产物与制备方法
CN104812241A (zh) * 2012-06-26 2015-07-29 生物原子价有限公司 快速的无特定病原动物
CN105732790B (zh) * 2016-05-06 2017-04-05 青岛农业大学 一种鱼源抗菌肽hepcidin突变体及其应用

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1641025A (zh) * 2004-11-29 2005-07-20 中国水产科学研究院黄海水产研究所 真鲷抗菌肽基因和重组酵母表达载体及其制备方法
CN110170045A (zh) * 2019-06-28 2019-08-27 福州大学 抗菌肽Lchamp2-3在抗肿瘤细胞增殖药物中的应用
CN110776560A (zh) * 2019-10-09 2020-02-11 厦门大学 一种黑鲷抗菌肽AS-hepc3(48-56)及其应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
YANG, M. WANG, K.J. CHEN, J.H. QU, H.D. LI, S.J.: "Genomic organization and tissue-specific expression analysis of hepcidin-like genes from black porgy (Acanthopagrus schlegelii B.)", FISH & SHELLFISH IMMUNOL, ACADEMIC PRESS, LONDON,, GB, vol. 23, no. 5, 10 September 2007 (2007-09-10), GB, pages 1060 - 1071, XP022240675, ISSN: 1050-4648, DOI: 10.1016/j.fsi.2007.04.011 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116143883A (zh) * 2022-10-19 2023-05-23 厦门大学 一种广谱抗微生物肽Scymicrosin7-26及其应用

Also Published As

Publication number Publication date
CN110776560A (zh) 2020-02-11
US11299514B2 (en) 2022-04-12
CN110776560B (zh) 2021-02-02
US20210340179A1 (en) 2021-11-04

Similar Documents

Publication Publication Date Title
WO2021068531A1 (zh) 一种黑鲷抗菌肽AS-hepc3 (48-56)及其应用
WO2022268115A1 (zh) 拟穴青蟹抗菌多肽Spampcin 56-86及其应用
Steinberg et al. Protegrin-1: a broad-spectrum, rapidly microbicidal peptide with in vivo activity
JP4484941B2 (ja) 生物活性を有する短ペプチド及び該ペプチドの使用方法
CN102702333B (zh) 抗耐药性病原菌感染多肽及其用途
CN113121666B (zh) 抗菌肽Scybaumancin105-127及其应用
CN110903347A (zh) 抗菌肽l7及其应用
CN112341522B (zh) 一种抗菌肽及其应用
WO2023274024A1 (zh) 一种抗菌肽Scyampcin 44-63及其应用
CN106146643A (zh) 人α‑防御素5改造体及其应用
CN114516899B (zh) 抗菌肽yhx-5及其应用
CN114702598A (zh) 一种重组抗菌肽及其应用
CN114014910B (zh) 一种拟穴青蟹抗真菌多肽Spamptin72-91及其应用
CN116870132B (zh) 一种抗菌肽rh-16及其在制备抗耐药抑菌药物中的应用
CN117069819B (zh) 一种黑腹狼蛛抗菌肽lc-amp-i1及其应用
CN116813713B (zh) 一种改造体抗菌肽ri-18及其应用
CN114539378B (zh) 一种抗菌肽及其在防治多重耐药菌中的应用
CN108373510B (zh) 一种PhaP-Tac的微生物合成及其对疏水材料PHBV的修饰方法
Ng Study of Kinetic Killing of Antibacterial Peptide Pam-5 on Escherichia Coli and Pseudomonas Aeruginosa
CN109535238B (zh) 一种经结构改造的抗菌肽Cec4或其盐及应用
US20110269669A1 (en) Self-decontaminating coatings containing antimicrobial peptides
Farrag Synthesis and Biological Activity of Newly designed Cyclic Peptides
TW202229313A (zh) 抗微生物胜肽及其用於預防或治療感染的方法
CN116789770A (zh) 一种抗生物膜肽及其应用
CN117106025A (zh) 一种拟穴青蟹抗菌肽Scyovacin19-30及其应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20874344

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 20874344

Country of ref document: EP

Kind code of ref document: A1