WO2020103511A1 - 一种拟穴青蟹抗菌肽Scyreprocin及其应用 - Google Patents

一种拟穴青蟹抗菌肽Scyreprocin及其应用

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WO2020103511A1
WO2020103511A1 PCT/CN2019/103237 CN2019103237W WO2020103511A1 WO 2020103511 A1 WO2020103511 A1 WO 2020103511A1 CN 2019103237 W CN2019103237 W CN 2019103237W WO 2020103511 A1 WO2020103511 A1 WO 2020103511A1
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scyreprocin
peptide
antibacterial
pseudo
blue crab
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French (fr)
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王克坚
杨盈
陈芳奕
陈炎超
陈慧芸
彭会
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Xiamen University
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Xiamen University
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    • 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
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43509—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from crustaceans
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/142—Amino acids; Derivatives thereof
    • A23K20/147—Polymeric derivatives, e.g. peptides or proteins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/195—Antibiotics
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/10—Feeding-stuffs specially adapted for particular animals for ruminants
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/70—Feeding-stuffs specially adapted for particular animals for birds
    • A23K50/75—Feeding-stuffs specially adapted for particular animals for birds for poultry
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • 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
    • 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
    • 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
    • A61P31/10—Antimycotics
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2250/00—Food ingredients
    • A23V2250/54—Proteins
    • A23V2250/55—Peptide, protein hydrolysate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag

Definitions

  • the invention belongs to the technical field of crustacean genetic engineering, and in particular relates to an anti-peptide blue crab antibacterial peptide Scyreprocin and its application.
  • Antimicrobial peptide also known as host defense peptide (host defense peptide, HDP), is a small molecule peptide substance with antibacterial activity or defense function produced by biological organisms. It is an important component of the animal's innate immune defense system. section. In 1972, Swedish scientist Boman et al. First discovered antimicrobial peptides and their immune function in fruit flies. In 1981 Boman et al. Successfully isolated two antimicrobial peptides-Cecropin A and B from Hyalophora Cecropin for the first time. Since then, antibacterial peptides have been found in plants, insects, amphibians, aquatic species, mammals and even some bacteria. To date, more than 2,800 antimicrobial peptides have been isolated, and more than 100 have been found in various vertebrates. After more than 20 years of research, people have a deeper understanding of the structure, function and mechanism of antimicrobial peptides.
  • Natural antimicrobial peptides are usually composed of 12 to 100 amino acid residues, and most of them have strong alkalinity, thermal stability and broad-spectrum antibacterial characteristics. They not only kill bacteria, some fungi, protozoa, and drug-resistant bacteria. It can also selectively kill tumor cells and inhibit the replication of hepatitis B virus.
  • antimicrobial peptides can exert their active functions through a variety of different mechanisms of action, including the following six pathways: 1 Most antimicrobial peptides (such as Cecropin, cecropin isolated from pig small intestine, etc.) It destroys the plasma membrane structure of microorganisms or cancer cells through the attack of the cell membrane, causing a large amount of content to leak out, which eventually leads to the death of tumor cells and microorganisms; 2Some antimicrobial peptides (such as fusion antimicrobial peptide DP1, antimicrobial peptide RGD tachyplesin, etc.) pass induced cells Apoptosis to kill tumor cells; 3Human salivary antimicrobial peptide MUC7 and antimicrobial peptide Thanatin exert their bactericidal function by attacking microbial mitochondria, inhibiting cell respiration; 4the antibacterial peptide dermaseptinS4 is transformed to obtain a new antibacterial peptide PVS4 (13) wearable The cell membrane directly acts on the
  • antimicrobial peptides found above not only have a strong inhibitory effect on bacteria. With the deepening of research, the antimicrobial peptides' ability to kill fungi, protozoa, viruses and cancer cells has attracted widespread attention. Among them, Cecropin A and Magainin , Nisin and other antimicrobial peptides have been used in medicine, food and agriculture. As a new type of antibacterial substance, antibacterial peptides have the characteristics of broad antibacterial spectrum and stable biological activity. Their physical adsorption and destruction of the microbial membrane mechanism are not easy to produce drug resistance, making them a powerful alternative to antibiotics.
  • the purpose of the present invention is to provide an anti-bacterial peptide Scyreprocin of pseudo-blue crab.
  • Another object of the present invention is to provide the application of the anti-bacterial peptide Scyreprocin.
  • An anti-pseudo-blue crab antibacterial peptide Scyreprocin whose amino acid sequence includes the sequence shown in SEQ ID NO 01. It is composed of 84 amino acids and is derived from the pseudo-blue crab, which is a polypeptide with antibacterial function.
  • the 84 amino acids of the antimicrobial peptide contains 15 positively charged amino acid residues and 7 negatively charged amino acid residues. According to the charge of the amino acid residues, the antimicrobial peptide is predicted to have an isoelectric point of 9.61 at pH 7.0 7 positive charges.
  • the antibacterial peptide has an average coefficient of hydrophilicity of -0.968, has strong water solubility, and is a positively charged cationic antibacterial peptide.
  • the nucleotide sequence of its open reading frame includes the sequence shown in SEQ ID NO 02.
  • its molecular weight is 9107.258 Daltons and its molecular formula is C 396 H 636 N 106 O 127 S 4 .
  • a person of ordinary skill in the art can easily adopt known methods, such as directed evolution and point mutation methods, to mutate the nucleotide sequence shown in the encoding SEQ ID NO 02 of the present invention.
  • the sequence functions shown in ID NO 01 are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention.
  • identity refers to sequence similarity to a natural nucleic acid sequence. “Identity” includes: the nucleotide sequence (SEQ ID NO 02) of a protein composed of the amino acid sequence shown in SEQ ID NO 01 in the present invention is 70% or higher, or 85% or higher, or 90% Or higher, or 95% or higher identity nucleotide sequence. Identity can be evaluated with the naked eye or computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to assess the identity between related sequences.
  • An antibacterial composition the effective ingredients of which include the above-mentioned antibacterial peptide Scyreprocin.
  • a feed composition whose effective ingredients include the above-mentioned anti-bacterial peptide Scyreprocin.
  • An anti-mildew and anti-corrosive composition the effective ingredients of which include the above-mentioned antibacterial peptide Scyreprocin.
  • the antimicrobial peptide Scyreprocin of the pseudocrane blue crab of the present invention has a significant antibacterial effect on a variety of Gram-negative bacteria, Gram-positive bacteria, fungi and molds.
  • the minimum inhibitory concentration against Vibrio fluvialis is 1-2 ⁇ M
  • the minimum inhibitory concentration against important pathogenic bacteria Pseudomonas stutzeri is 0.5-1 ⁇ M
  • the minimum inhibitory concentration of Staphylococcus aureus is ⁇ 0.5 ⁇ M
  • the minimum bactericidal concentration of various Gram-negative bacteria and Gram-positive bacteria is 2-4 ⁇ M.
  • the minimum inhibitory concentration for the important clinical pathogenic streptococcus albicans is 2-4 ⁇ M
  • the minimum inhibitory concentration for Cryptococcus neoformans is 1-2 ⁇ M and the minimum bactericidal concentration is 8-16 ⁇ M
  • the minimum inhibitory concentration for Aspergillus niger 4-8 ⁇ M is 8-16 ⁇ M. Therefore, compared with many known marine animal antibacterial peptides, it has a wide antibacterial spectrum, good antibacterial effect, and fast sterilization rate, which shows great application value and has good application in the preparation of antibacterial agents.
  • the anti-microbial peptide Scyreprocin of the pseudo-blue crab of the present invention is non-cytotoxic to mouse liver parenchymal cells AML12, human normal liver cells L02 and the like, and can be safely used for drug therapy or as a feed additive.
  • FIG. 1 is a genetic engineering expression and purification diagram of the antimicrobial peptide Scyreprocin of the pseudo-blue crab in Example 1 of the present invention.
  • the left picture shows the results of SDS-PAGE electrophoresis of the Scyreprocin protein obtained after purification and concentration of the strain: M, protein Marker26616; 1, total protein of strain before induction; 2, total protein of strain after induction of 24h; 3, ultrasound of strain Crush the cell supernatant; 4. Purify the obtained Scyreprocin protein.
  • the figure on the right is the peak of protein purification, and the arrow indicates the peak of Scyreprocin protein purification.
  • Fig. 2 is an experimental diagram of the detection of the antimicrobial peptide Scyreprocin of the pseudo-crypt blue crab to inhibit the spore germination of Aspergillus niger.
  • Figure 3 is the bactericidal kinetics curve diagram of the antibacterial peptide Scyreprocin of the pseudo-crypt blue crab against Cryptococcus neoformans (top left), Candida albicans (bottom left), Pseudomonas stutzeri (top right), and Micrococcus lyticus (bottom right) .
  • the abscissa is time (min)
  • the ordinate is the percentage of the number of viable colonies relative to the number of initial colonies.
  • FIG. 4 is an experimental diagram of MTS method for detecting the effect of anti-peptide blue crab antibacterial peptide Scyreprocin on proliferation of non-cancer cell line mouse parenchymal cells (AML12) and human normal liver cells (L02).
  • Escherichia coli Escherichia coli BL21 (Escherichia coli BL21), Staphylococcus aureus (Staphylococcus aureus), Vibrio fluvialis (Pseudomonas vibrio) stutzeri), Micrococcus lysoleikticus, Candida albicans, Candida krusei, Candida tropicalalis, Cryptococcus neoformans , Aspergillus niger (Aspergillus niger), etc., were purchased from the Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences.
  • Escherichia coli MC1061 Esscherichia coli MC1061
  • Pichia pastoris GS115 was purchased from Invitrogen.
  • the full-length cDNA sequence of Scyreprocin gene was obtained and verified by RACE technology.
  • the open reading frame of the Scyreprocin gene is 255bp (including the stop codon TAA), GenBank accession number: MH488960.
  • the reaction system is as follows:
  • the 3'UTR amplification method of the Scyreprocin gene is similar to the 5'UTR amplification method.
  • the resulting amplified products are sequenced and spliced to obtain the full length of the Scyreprocin gene cDNA.
  • the appropriate primers are designed.
  • the full-length Scyreprocin gene was verified, and the PCR reaction system was as follows:
  • the Scyreprocin gene is derived from the pseudo-blue crab, and its amino acid sequence is:
  • Scyreprocin has a molecular formula of C 396 H 636 N 106 O 127 S 4 and a molecular weight of 9107.258 Daltons. It is predicted by SignalP4.1 software that Scyreprocin has no signal peptide sequence, and the total length of 84 amino acids contains 15 positively charged amino acid residues and 7 negatively charged amino acid residues.
  • the antimicrobial peptide is predicted based on the amino acid residue charge The electric point is 9.61, with 7 positive charges when the pH is 7.0.
  • the antibacterial peptide has an average coefficient of hydrophilicity of -0.968, has strong water solubility, and is a positively charged cationic antibacterial peptide.
  • the genetically engineered expression technology method can be used to obtain the anti-bacterial peptide Scyreprocin of the pseudo-blue crab with a purity of more than 85%.
  • the antimicrobial peptide Scyreprocin of the pseudo-blue crab was obtained by our laboratory through genetic engineering technology (the results are shown in Figure 1).
  • Negative control group add 50 ⁇ L of sterilized ultrapure water and 50 ⁇ L of bacterial suspension;
  • Blank control group add 50 ⁇ L of protein sample to be tested and 50 ⁇ L phosphate buffer;
  • Sample experiment group add 50 ⁇ L of protein sample to be tested and 50 ⁇ L of bacterial suspension.
  • MIC minimum inhibitory concentration ( ⁇ M), expressed as a-b.
  • a The highest protein concentration in which the bacterial growth can be seen with the naked eye;
  • b The lowest protein concentration in which the bacterial growth cannot be seen with the naked eye
  • MBC minimum bactericidal concentration ( ⁇ M), the lowest protein concentration capable of killing 99.9% of bacteria
  • Example 4 Determination of the antimicrobial peptide Scyreprocin of blue crab crabs inhibiting the germination of Aspergillus niger spores
  • Positive control group add 50 ⁇ L of spore suspension and 50 ⁇ L of 10 mM phosphate buffer.
  • Experimental group to be tested add 50 ⁇ L of spore suspension and 50 ⁇ L of protein sample to be tested.
  • the 96-well cell culture plate was placed in a 28 ° C incubator for 24 hours, and the germination results of Aspergillus niger spores were observed with an optical microscope. As shown in Figure 2, the results show that at a concentration of 4 ⁇ M, the antimicrobial peptide Scyreprocin of the pseudocrane blue crab can significantly inhibit the germination of Aspergillus niger spores.
  • a Gram-negative bacterium (Pseudomonas stutzeri), a Gram-positive bacterium (Micrococcus lyticus) and two fungi (Candida albicans, Cryptococcus neoformans) were selected as the test.
  • Positive control group add 100 ⁇ L of bacterial suspension and 100 ⁇ L of 10 mM phosphate buffer.
  • Experimental group to be tested add 100 ⁇ L of bacterial suspension and 100 ⁇ L of protein sample to be tested.
  • human normal hepatocytes L02 and mouse liver parenchymal cells AML12 were selected as the test cell strains, and the effects of Scyreprocin on the proliferation of different cells were tested.
  • the results show that the anti-bacterial peptide Scyreprocin of the pseudo-blue crab has no effect on inhibiting the growth of mouse liver parenchymal cells AML12 and human normal liver cells L02.
  • the present invention provides an antibacterial peptide Scyreprocin of a pseudo-blue crab and its application.
  • the antibacterial peptide Scyreprocin of a pseudo-blue crab of the present invention has a significant antibacterial effect against various Gram-negative bacteria, Gram-positive bacteria, fungi and mold .
  • the minimum inhibitory concentration against Vibrio fluvialis is 1-2 ⁇ M
  • the minimum inhibitory concentration against important pathogenic bacteria Pseudomonas stutzeri is 0.5-1 ⁇ M
  • the minimum inhibitory concentration of Staphylococcus aureus is ⁇ 0.5 ⁇ M
  • the minimum bactericidal concentration of various Gram-negative bacteria and Gram-positive bacteria is 2-4 ⁇ M.
  • the minimum inhibitory concentration for the important clinical pathogenic streptococcus albicans is 2-4 ⁇ M
  • the minimum inhibitory concentration for Cryptococcus neoformans is 1-2 ⁇ M and the minimum bactericidal concentration is 8-16 ⁇ M
  • the minimum inhibitory concentration for Aspergillus niger 4-8 ⁇ M is 8-16 ⁇ M. Therefore, compared with many known marine animal antibacterial peptides, it has a wide antibacterial spectrum, good antibacterial effect, and fast sterilization rate, which shows great application value and has good application in the preparation of antibacterial agents.
  • the anti-bacterial peptide Scyreprocin of the pseudo-blue crab of the present invention is non-cytotoxic to mouse liver parenchymal cells AML12, human normal liver cells L02 and the like, can be safely used for drug treatment or used as a feed additive, and has good industrial practicality.

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Abstract

提供了一种拟穴青蟹抗菌肽Scyreprocin及其应用，其氨基酸序列包括如SEQ ID NO 01所示的序列，采用基因工程技术表达、纯化获得。该抗菌肽的抗菌谱广、抗菌效果好、杀菌速率快，显示出极大的应用价值，在制备抗菌剂方面有良好的应用。该抗菌肽对小鼠肝实质细胞AML12、人正常肝细胞L02等无细胞毒性，可安全用于药物治疗或者作为饲料组合物使用。

Description

一种拟穴青蟹抗菌肽Scyreprocin及其应用 技术领域
本发明属于甲壳动物基因工程技术领域，具体涉及一种拟穴青蟹抗菌肽Scyreprocin及其应用。
背景技术
为满足世界人口快速增长伴随对食品需求的增长，近年来畜禽、水产养殖业快速发展，大量抗生素被应用于产业以抵抗各类病原菌。抗生素的应用提高了饲料利用效率及产业经济效益，而抗生素的滥用也使病原微生物产生耐药性及二重感染，残留于养殖土壤及水体、畜禽水产产品中的抗生素使产品质量及安全性受到严重影响，威胁人类健康。因此，寻找高效、绿色抗生素替代品迫在眉睫，成为近年来的研究热点。安全高效、不产生抗药性、无残留和可体内降解的抗菌肽被认为是抗生素的理想替代品之一。
抗菌肽(antimicrobial peptide,AMP)又称宿主防御肽(host defense peptide,HDP)，是由生物有机体产生具有抗菌活性或防御功能的小分子肽类物质，是动物体先天性免疫防御系统的重要组成部分。1972年，瑞典科学家Boman等首先在果蝇中发现抗菌肽及其免疫功能。1981年Boman等首次从美国天蚕(Hyalophora cecropin)中成功地分离到两种抗菌肽——天蚕素(Cecropin)A和B。此后，人们在植物、昆虫、两栖类、水产类以及哺乳类动物甚至一些细菌中都发现了抗菌肽类物质。迄今为止，大约有2800多种抗菌肽被分离出来，各种脊椎动物中已发现100多种。经过20多年的研究，人们对抗菌肽结构、功能及作用机制有了较深的认识。
天然抗菌肽通常由12至100个氨基酸残残基组成，多具有强碱性、热稳定性及广谱抗菌等特点，不仅对细菌、部分真菌、原生动物、耐药性细菌具有杀灭作用，亦可选择性杀伤肿瘤细胞，抑制乙型肝炎病毒等的复制。迄今为止，研究者们发现抗菌肽可通过多种不同的作用机制发挥其活性功能，主要包括以下六个途径：①大多数抗菌肽(如天蚕素Cecropin，猪小肠分离得到的抗菌肽Cecp等)通过胞膜攻击作用破坏微生物或癌细胞质膜结构，引起内容物大量渗出，最终导致肿瘤细胞和微生物的死亡；②某些抗菌肽(如融合抗菌肽DP1，抗菌肽RGD tachyplesin等)通过诱导细胞凋亡以杀灭肿瘤细胞；③人唾液抗菌肽MUC7及抗菌肽Thanatin通过攻击微生物线 粒体，抑制细胞呼吸作用发挥其杀菌功能；④对抗菌肽dermaseptinS4进行改造后获得新型抗菌肽PVS4 (13)可穿过细胞膜直接作用于肿瘤细胞染色质发挥杀伤作用；⑤天蚕素B、B1和B3不仅使癌细胞膜发生穿孔，还可以进一步破坏细胞微管的正常功能进而引起细胞骨架的完整性，阻止纺锤丝形成和游戏分裂的过程，破坏细胞器，最终杀伤肿瘤细胞；⑥人源抗菌肽β2defensin3通过抑制细菌蛋白质合成及细胞壁的形成，使细菌无法维持正常的形态、生长受阻，最终导致细菌细胞壁穿孔，内容物外泄，达到其杀菌的效果。
以上已发现的大多数抗菌肽不仅对细菌有较强的抑杀效果，随着研究的深入开展，抗菌肽对真菌、原虫、病毒及癌细胞的杀伤能力受到广泛的关注，其中Cecropin A、Magainin、Nisin等抗菌肽已先后在医药、食品和农业上得到了应用。抗菌肽作为新型抗菌物质，具有抗菌谱广、生物活性稳定等特点，其物理吸附、破坏微生物膜作用机制不易产生耐药性使其成为强有力的抗生素替代品。随着对抗菌肽结构及其作用机理的不断深入研究，更多新型、高效、广谱且低毒的抗菌肽及其改造产品将会在临床医学、饲料添加剂和畜禽水产养殖领域具有良好的应用前景。
发明内容
本发明的目的在于提供一种拟穴青蟹抗菌肽Scyreprocin。
本发明的另一目的在于提供上述拟穴青蟹抗菌肽Scyreprocin的应用。
本发明的技术方案如下：
一种拟穴青蟹抗菌肽Scyreprocin，其氨基酸序列包括如SEQ ID NO 01所示的序列。其由84个氨基酸组成，来源于拟穴青蟹，是具有抗菌功能的多肽。该抗菌肽84个氨基酸中包含15个带正电荷的氨基酸残基和7个带负电荷的氨基酸残基，根据氨基酸残基电荷预测该抗菌肽等电点为9.61，在pH等于7.0时带有7个正电荷。该抗菌肽的亲水性平均系数为-0.968，具有较强的水溶性，是一种带有正电荷的阳离子抗菌肽。
在本发明的一个优选实施方案中，其开放阅读框的核苷酸序列包括如SEQ ID NO 02所示的序列。
在本发明的一个优选实施方案中，其分子量为9107.258道尔顿，分子式为C 396H 636N 106O 127S 4。
本领域普通技术人员可以很容易地采用已知的方法，例如定向进化和点突变的方法，对本发明的编码SEQ ID NO 02所示的核苷酸序列进行突变。那些经过人工修饰的，具有与本发明分离得到的SEQ ID NO 02所示的核苷酸序列70％或者更高同一 性的核苷酸，只要编码SEQ ID NO 01所示的序列、且具有SEQ ID NO 01所示的序列功能，均是衍生于本发明的核苷酸序列并且等同于本发明的序列。
这里使用的术语“同一性”指与天然核酸序列的序列相似性。“同一性”包括：与本发明中SEQ ID NO 01所示的氨基酸序列构成的蛋白质的核苷酸序列(SEQ ID NO 02)具有70％或更高，或85％或更高，或90％或更高，或95％或更高同一性的核苷酸序列。同一性可以用肉眼或计算机软件进行评价。使用计算机软件，两个或多个序列之间的同一性可以用百分比(％)表示，其可以用来评价相关序列之间的同一性。
本发明的技术方案之二：
一种抗菌组合物，其有效成分包括上述拟穴青蟹抗菌肽Scyreprocin。
本发明的技术方案之三：
一种饲料组合物，其有效成分包括上述拟穴青蟹抗菌肽Scyreprocin。
本发明的技术方案之四：
一种防霉防腐组合物，其有效成分包括上述拟穴青蟹抗菌肽Scyreprocin。
本发明的技术方案之五：
上述拟穴青蟹抗菌肽Scyreprocin在制备抗菌组合物中的应用。
本发明的技术方案之六：
上述拟穴青蟹抗菌肽Scyreprocin在制备饲料组合物中的应用。
本发明的技术方案之七：
上述拟穴青蟹抗菌肽Scyreprocin在制备防霉防腐组合物中的应用。
本发明的有益效果是：
1、本发明的拟穴青蟹抗菌肽Scyreprocin对多种革兰氏阴性菌、革兰氏阳性菌、真菌及霉菌有显著的抗菌效果。其中，对河流弧菌的最小抑菌浓度为1-2μM，对重要致病菌施氏假单胞菌最小抑菌浓度为0.5-1μM、金黄色葡萄球菌最小抑菌浓度为<0.5μM；对多种革兰氏阴性菌、革兰氏阳性菌的最小杀菌浓度为2-4μM。此外，对重要临床致病菌白色链球菌的最小抑菌浓度为2-4μM，新型隐球菌的最小抑菌浓度为1-2μM且最小杀菌浓度为8-16μM；对黑曲霉的最小抑菌浓度为4-8μM。因此与许多已知的海洋动物抗菌肽比较，其抗菌谱广、抗菌效果好、杀菌速率快，显示出极大的应用价值，在制备抗菌剂方面有良好的应用。
2、本发明的拟穴青蟹抗菌肽Scyreprocin对小鼠肝实质细胞AML12、人正常肝细 胞L02等无细胞毒性，可安全用于药物治疗或者作为饲料添加剂使用。
附图说明
图1为本发明实施例1中拟穴青蟹抗菌肽Scyreprocin的基因工程表达纯化图。在图1中，左图为菌株表达纯化、浓缩后获得的Scyreprocin蛋白SDS-PAGE电泳结果：M，蛋白Marker26616；1，诱导前菌株总蛋白；2，诱导24h后菌株总蛋白；3，菌株超声破碎细胞上清；4，纯化获得的Scyreprocin蛋白。右图为蛋白纯化峰图，箭头所指为Scyreprocin蛋白纯化峰。
图2为拟穴青蟹抗菌肽Scyreprocin抑制黑曲霉孢子萌发检测的实验图。
图3为拟穴青蟹抗菌肽Scyreprocin对新型隐球菌(左上)、白假丝酵母(左下)、施氏假单胞菌(右上)、溶壁微球菌(右下)的杀菌动力学曲线图。在图3中，横坐标为时间(min)，纵坐标为存活菌落数相对于起始菌落数的百分比。
图4为MTS法检测拟穴青蟹抗菌肽Scyreprocin对非癌细胞系小鼠肝实质细胞(AML12)、人正常肝细胞(L02)增殖影响的实验图。
具体实施方式
以下通过具体实施方式对本发明的技术方案进行进一步的说明和描述。
下述实施例涉及的菌种有：大肠杆菌(Escherichia coli)、大肠杆菌BL21(Escherichia coli BL21)、金黄色葡萄球菌(Staphylococcus aureus)、河流弧菌(Vibrio fluvialis)施氏假单胞菌(Pseudomonas stutzeri)、溶壁微球菌(Micrococcus lysoleikticus)、白假丝酵母菌(Candida albicans)、克鲁假丝酵母菌(Candida krusei)、热带假丝酵母菌(Candida tropicalis)、新型隐球菌(Cryptococcus neoformans)、黑曲霉(Aspergillus niger)等，均购自中国科学院微生物研究所菌种保藏中心。大肠杆菌MC1061(Escherichia coli MC1061)购自Gene Power公司，毕赤酵母(Pichia pastoris GS115)购自Invitrogen公司。
实施例1拟穴青蟹抗菌肽Scyreprocin的制备
所述拟穴青蟹抗菌肽Scyreprocin的开放阅读框序列为：
Figure PCTCN2019103237-appb-000001
Figure PCTCN2019103237-appb-000002
通过RACE技术获得并验证了Scyreprocin基因全长cDNA序列。Scyreprocin基因的开放阅读框为255bp(含终止密码子TAA)，GenBank登录号：MH488960。
根据Scyreprocin基因的cDNA序列，设计基因特异性引物通过RACE技术进行重复验证(表1)。
表1 Scyreprocin序列扩增引物列表
Figure PCTCN2019103237-appb-000003
通过RACE技术扩增青蟹Scyreprocin基因非编码序列(以5'UTR扩增为例)：
以申请人前期制备的RACE cDNA为PCR模板，具体反应体系如下：
①Outer PCR反应：
Figure PCTCN2019103237-appb-000004
Figure PCTCN2019103237-appb-000005
反应程序如下所述：
1)95℃预变性3min；
2)95℃变性30s，60℃退火30s，72℃延伸2min，20个循环；
3)72℃延伸10min；
4)4℃停止。
将上述第一轮PCR产物稀释50-100倍，作为模板进行巢式PCR扩增，反应体系如下：
②Inner PCR反应：
Figure PCTCN2019103237-appb-000006
混合均匀，进行PCR反应。反应程序如下所述：
1)95℃预变性5min；
2)95℃变性30s，60℃退火30s，72℃延伸2min，30个循环；
3)72℃延伸10min；
4)4℃停止。
Scyreprocin基因的3'UTR扩增方法同5'UTR扩增方法类似，所得扩增产物测序后拼接获得Scyreprocin基因cDNA全长，设计合适的引物，以拟穴青蟹cDNA为模 板，对拼接获得的Scyreprocin基因全长进行验证，PCR反应体系如下：
Figure PCTCN2019103237-appb-000007
混合均匀，进行PCR反应。反应程序如下所述：
1)95℃预变性5min；
2)95℃变性30s，55℃退火30s，72℃延伸1min，30个循环；
3)72℃延伸5min；
4)4℃停止。
所述Scyreprocin基因来源于拟穴青蟹，其氨基酸序列为：
MKEDSNILDKTAKMTKQNKALLFTAGGAAAFMAGYYYYHCNYRNPAPKKSGSTTSQDKTDAQAVQSIPSPSGNKGKESKDPKVK(SEQ ID NO 01)
Scyreprocin的分子式为C 396H 636N 106O 127S 4，分子量为9107.258道尔顿。经SignalP4.1软件预测，Scyreprocin无信号肽序列，全长84个氨基酸中包含15个带正电荷的氨基酸残基和7个带负电荷的氨基酸残基，根据氨基酸残基电荷预测该抗菌肽等电点为9.61，在pH等于7.0时带有7个正电荷。该抗菌肽的亲水性平均系数为-0.968，具有较强的水溶性，是一种带有正电荷的阳离子抗菌肽。
采用基因工程表达技术方法即可获得纯度达85％以上的拟穴青蟹抗菌肽Scyreprocin。本实施例中拟穴青蟹抗菌肽Scyreprocin由本实验室通过基因工程技术表达纯化获得(结果如图1所示)。
实施例2拟穴青蟹抗菌肽Scyreprocin基因工程表达产物的获得
(a)从拟穴青蟹cDNA模板中克隆获得拟穴青蟹抗菌肽Scyreprocin开放阅读框序列，克隆重组到载体pET28a，通过碱基测序确定读码正确的重组载体，转入表达菌株大肠杆菌BL21(Escherichia coli BL21)，获得可表达带融合His标签的拟穴青蟹 抗菌肽Scyreprocin的重组表达菌株。
(b)挑取表达菌株单克隆于液体LB培养基中，于37℃下以180rpm摇瓶培养10-14h后，以1：1000转接到含0.5％葡萄糖的液体LB培养基中继续培养至OD 600＝0.3，加入IPTG使其终浓度为0.5mM以诱导表达，于16℃以160rpm转速继续培养表达菌株，24h后离心收集菌体，用50mM磷酸盐缓冲液(pH8.0)以1：10体积比重悬菌体。
(c)获得重悬拟穴青蟹抗菌肽Scyreprocin表达后菌体中加入PMSF以抑制蛋白酶活性，利用超声破碎仪破碎菌体，12000rpm高速离心30min以获得含有拟穴青蟹抗菌肽Scyreprocin的菌体裂解上清液。菌体裂解上清液经过0.22μm滤膜过滤除去杂质后，通过镍柱亲和层析方法获得融合His标签的拟穴青蟹抗菌肽Scyreprocin。
(d)获得的融合His标签拟穴青蟹抗菌肽Scyreprocin于透析液(50mM Tris-HCl，50mM NaCl，pH8.0)中透析16-18h，以去除蛋白纯化液中的咪唑；再转移到MilliQ中继续透析10-12h，以去除蛋白纯化液中的盐离子。
(e)将透析后的重组表达拟穴青蟹抗菌肽Scyreprocin通过超滤管进行超滤浓缩，并以Bradford法测定浓缩后蛋白浓度，获得的浓缩后拟穴青蟹抗菌肽Scyreprocin分装保存于-80℃待用。获得的纯蛋白通过变性凝胶电泳及质谱分析，确定表达产物为拟穴青蟹抗菌肽Scyreprocin。
实施例3拟穴青蟹抗菌肽最小抑菌浓度(MIC：minimum inhibition concentration)的测定
(a)将保种的大肠杆菌、大肠杆菌MC1061、施氏假单胞菌、嗜水汽假单胞菌、金黄色葡萄球菌、溶壁微球菌等在营养肉汤平板上划线，河流弧菌在2216E海水肉汤平板上划线，在相应培养最适温度倒置培养12-16h；白假丝酵母菌、毕赤酵母、克鲁假丝酵母菌、热带假丝酵母菌、新型隐球菌在YPG平板上划线，于28℃培养1-2周；黑曲霉孢子涂布于PDA平板上，于28℃倒置培养3-7d。从各平板上挑取菌落接种于相应培养基斜面上，细菌继续培养10-16h，真菌继续培养1-3d，霉菌继续培养3-7d后，用10mM磷酸盐缓冲液(pH7.4)冲洗斜面，细菌用MH液体培养基：10mM磷酸缓冲液＝2：3的混合培养基调整稀释至OD 600＝0.003；弧菌用海水培养基调整稀释至OD 600＝0.0006；酵母用YPD液体培养基：10mM磷酸缓冲液＝2：3的混合培养基调整稀释至OD 600＝0.00067；霉菌用PDA液体培养基：10mM磷酸缓冲液＝1：1的 混合培养基调整稀释孢子浓度至5×10 4个/mL(具体操作见实施例4)；弧菌用海水培养基2216E调整稀释至OD 600＝0.0006。准备好的菌液须在20min内使用。
(b)将抗菌肽Scyreprocin经0.22μm滤膜过滤后，利用Bradford法测定蛋白浓度，倍比稀释蛋白浓度为0.5、1、2、4、8、16、32μM，4℃保存备用。
(c)在96孔细胞培养板上进行最小抑菌浓度检测实验，每种被测菌按照以下操作设置空白对照组、阴性对照组和待测样品实验组，每组设置3个平行样：
阴性对照组：加50μL灭菌超纯水和50μL菌悬液；
空白对照组：加50μL待测蛋白样品和50μL磷酸盐缓冲液；
样品实验组：加50μL待测蛋白样品和50μL菌悬液。
对于细菌，将培养板置于各菌种相对应的最适培养温度下，培养18-24h，观察结果；
对于真菌、弧菌和霉菌，将培养板置于28℃培养1-2d，观察结果。
将96孔细胞培养板待测实验组中肉眼观察未有菌生长的孔中培养液混匀后，吸取2μL接种于相应固体培养基平板上，置于各最适生长温度继续培养24-48h，观察MBC(minimum bactericidal concentration)结果。
拟穴青蟹抗菌肽Scyreprocin MIC、MBC观察结果如表2所示：
表2拟穴青蟹抗菌肽Scyreprocin抗菌活性测定
Figure PCTCN2019103237-appb-000008
Figure PCTCN2019103237-appb-000009
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)，能够杀死99.9％菌体的最低蛋白浓度
实施例4拟穴青蟹抗菌肽Scyreprocin抑制黑曲霉孢子萌发的测定
(a)将保种的黑曲霉涂布于PDA平板上，于28℃倒置培养3-7d；
(b)从平板上挑取菌丝体接种于PDA斜面上，于28℃继续培养3-7d。用10mM磷酸盐缓冲液(pH7.4)冲洗斜面，用细胞筛(孔径＝100nm)过滤除去菌丝，收集霉菌孢子。用PDA液体培养基：10mM磷酸盐缓冲液＝1：1的混合培养基调整孢子浓度为5×10 4个/mL。
(c)将Scyreprocin蛋白表达产物经0.22μm滤膜过滤后，倍比稀释蛋白至0.5、1、2、4、8、16、32μM，置于冰上待用。
(d)在96孔细胞培养板上，如下设置阳性对照组、待测实验组，每组设置三个平行样：
阳性对照组：加50μL孢子悬液和50μL 10mM磷酸盐缓冲液。
待测实验组：加50μL孢子悬液和50μL待测蛋白样品。
将96孔细胞培养板置于28℃培养箱中培养24h，用光学显微镜观察黑曲霉孢子的萌发结果。如图2所示，结果表明：在4μM浓度下，拟穴青蟹抗菌肽Scyreprocin可显著抑制黑曲霉孢子的萌发。
实施例5拟穴青蟹抗菌肽Scyreprocin杀菌动力学曲线
本实施例选取一种革兰氏阴性菌(施氏假单胞菌)、一种革兰氏阳性菌(溶壁微球菌)及两种真菌(白假丝酵母、新型隐球菌)作为被测菌进行拟穴青蟹抗菌肽Scyreprocin杀菌动力学曲线的测定。
(a)将保种的施氏假单胞菌及溶壁微球菌涂布于营养肉汤平板上，将保种的白假丝酵母及新型隐球菌涂布于YPD平板上，在最适培养温度倒置培养。
(b)从平板上挑取菌落接种于斜面上继续培养，用10mM磷酸盐缓冲液(pH7.4)冲洗斜面，用MH液体培养基(或YPD液体培养基)：10mM磷酸缓冲液＝2：3的混合培养基调整菌悬液浓度至OD 600＝3.3×10 4cfu/mL。
(c)将Scyreprocin蛋白表达产物经过0.22μm滤膜过滤后，倍比稀释蛋白至0.5、1、2、4、8、16、32μM，置于冰上待用。
(d)在96孔细胞培养板上，设置阳性对照组、待测实验组，每组设置三个平行样：
阳性对照组：加100μL菌悬液和100μL 10mM磷酸缓冲液。
待测实验组：加100μL菌悬液和100μL待测蛋白样品。
将96孔细胞培养板置于28℃培养箱中继续培养，于0、15、30、60、90、120、180、240min后取适量培养液稀释后涂布营养肉汤平板或YPD平板，平板于菌种最适温度继续培养至单克隆生长，计数每个平板上克隆数目，统计分析并作杀菌动力学曲线(如图3所示)。结果表明：2倍MBC浓度可在作用100min内杀死50％的新型隐球菌及白假丝酵母，在500min后杀菌指数达到100％；2倍MBC浓度的拟穴青蟹抗菌肽Scyreprocin与溶壁微球菌作用60min后，杀菌指数达到100％；2倍MBC浓度的拟穴青蟹抗菌肽Scyreprocin与施氏假单胞菌作用180min后，杀菌指数达到100％。
实施例6拟穴青蟹抗菌肽Scyreprocin对动物细胞系增殖的影响
本实施例选取人正常肝细胞L02、小鼠肝脏实质细胞AML12作为受试细胞株，进行拟穴青蟹Scyreprocin对不同细胞增殖影响的检测。
(a)将AML12细胞、L02细胞在对应细胞培养基中培养至细胞接合度80-90％后，用含EDTA的胰酶消化并用相应细胞培养基重悬，以相应细胞培养基调整细胞密度，在96孔细胞培养板中每孔接种2×10 5个细胞，置于37℃细胞培养箱在5％CO 2条件下过夜培养。
(b)待每孔细胞生长到接合度约60-70％时，吸取细胞培养基，用相应细胞培养基倍比稀释Scyreprocin蛋白至0.5、1、2、4、8、16μM，以正常细胞培养基为对照，每组设置3个平行样。
将96孔细胞培养板放入37℃、5％CO 2条件下继续培养48h后，每孔加入20μL MTS试剂，放入培养环境反应2h后，用酶标仪测定在波长为490nm时的吸光值。如图4所示，结果表明：拟穴青蟹抗菌肽Scyreprocin对小鼠肝脏实质细胞AML12、人正常肝细胞L02无抑制其生长的作用。
以上所述，仅为本发明的较佳实施例而已，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
本发明提供了一种拟穴青蟹抗菌肽Scyreprocin及其应用，本发明的拟穴青蟹抗菌肽Scyreprocin对多种革兰氏阴性菌、革兰氏阳性菌、真菌及霉菌有显著的抗菌效果。其中，对河流弧菌的最小抑菌浓度为1-2μM，对重要致病菌施氏假单胞菌最小抑菌浓度为0.5-1μM、金黄色葡萄球菌最小抑菌浓度为<0.5μM；对多种革兰氏阴性菌、革兰氏阳性菌的最小杀菌浓度为2-4μM。此外，对重要临床致病菌白色链球菌的最小抑菌浓度为2-4μM，新型隐球菌的最小抑菌浓度为1-2μM且最小杀菌浓度为8-16μM；对黑曲霉的最小抑菌浓度为4-8μM。因此与许多已知的海洋动物抗菌肽比较，其抗菌谱广、抗菌效果好、杀菌速率快，显示出极大的应用价值，在制备抗菌剂方面有良好的应用。本发明的拟穴青蟹抗菌肽Scyreprocin对小鼠肝实质细胞AML12、人正常肝细胞L02等无细胞毒性，可安全用于药物治疗或者作为饲料添加剂使用,具有良好的工业实用性。

Claims (12)

  1. 一种拟穴青蟹抗菌肽Scyreprocin，其特征在于：其氨基酸序列包括SEQ ID NO01所示的序列。
  2. 一种拟穴青蟹抗菌肽Scyreprocin，其特征在于：其开放阅读框的核苷酸序列包括SEQ ID NO 02所示的序列。
  3. 编码权利要求1或2所述拟穴青蟹抗菌肽Scyreprocin及其核酸分子。
  4. 下述1)-4)中的任一种生物材料：
    1)含有权利要求3所述核酸分子的表达盒；
    2)含有权利要求3所述核酸分子的重组载体；
    3)含有所述重组载体及氨基酸的纳米颗粒；
    4)含有权利要求3所述Scyreprocin的抗菌涂料。
  5. 一种抗菌组合物，其特征在于：其有效成分包括权利要求1至2中任一权利要求所述的拟穴青蟹抗菌肽Scyreprocin。
  6. 一种饲料组合物，其特征在于：其有效成分包括权利要求1至2中任一权利要求所述的拟穴青蟹抗菌肽Scyreprocin。
  7. 一种防霉防腐组合物，其特征在于：其有效成分包括权利要求1至2中任一权利要求所述的拟穴青蟹抗菌肽Scyreprocin。
  8. 一种抗肿瘤组合物，其特征在于：其有效成分包括权利要求1至2中任一权利要求所述的拟穴青蟹抗菌肽Scyreprocin。
  9. 权利要求1至2中任一权利要求所述的拟穴青蟹抗菌肽Scyreprocin在制备抗菌组合物中的应用。
  10. 权利要求1至2中任一权利要求所述的拟穴青蟹抗菌肽Scyreprocin在制备饲料组合物中的应用。
  11. 权利要求1至2中任一权利要求所述的拟穴青蟹抗菌肽Scyreprocin在制备防霉防腐剂中的应用。
  12. 权利要求1至2中任一权利要求所述的拟穴青蟹抗菌肽Scyreprocin在制备 抗肿瘤制剂及相关与其他抗肿瘤药物联合使用方案中的应用。
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