CN114149486A - 一种抗菌肽mamp-01及其应用 - Google Patents
一种抗菌肽mamp-01及其应用 Download PDFInfo
- Publication number
- CN114149486A CN114149486A CN202110591731.8A CN202110591731A CN114149486A CN 114149486 A CN114149486 A CN 114149486A CN 202110591731 A CN202110591731 A CN 202110591731A CN 114149486 A CN114149486 A CN 114149486A
- Authority
- CN
- China
- Prior art keywords
- gram
- mamp
- polypeptide
- positive
- negative bacteria
- 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.)
- Granted
Links
- 239000003910 polypeptide antibiotic agent Substances 0.000 title claims description 17
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 39
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 33
- 229920001184 polypeptide Polymers 0.000 claims abstract description 26
- 241000894006 Bacteria Species 0.000 claims abstract description 15
- 241000605862 Porphyromonas gingivalis Species 0.000 claims abstract description 14
- 241000194019 Streptococcus mutans Species 0.000 claims abstract description 14
- 241000191967 Staphylococcus aureus Species 0.000 claims abstract description 13
- 241000588724 Escherichia coli Species 0.000 claims abstract description 12
- 239000003814 drug Substances 0.000 claims abstract description 12
- 102000014171 Milk Proteins Human genes 0.000 claims abstract description 11
- 108010011756 Milk Proteins Proteins 0.000 claims abstract description 11
- 235000021239 milk protein Nutrition 0.000 claims abstract description 11
- 241000192125 Firmicutes Species 0.000 claims abstract description 8
- 244000005700 microbiome Species 0.000 claims abstract description 8
- 230000000813 microbial effect Effects 0.000 claims abstract description 6
- 235000013305 food Nutrition 0.000 claims abstract description 5
- 239000004480 active ingredient Substances 0.000 claims abstract description 3
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract 3
- 239000003966 growth inhibitor Substances 0.000 claims description 6
- 102000044503 Antimicrobial Peptides Human genes 0.000 claims description 4
- 108700042778 Antimicrobial Peptides Proteins 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 4
- 208000025157 Oral disease Diseases 0.000 claims description 3
- 208000030194 mouth disease Diseases 0.000 claims description 3
- 208000017520 skin disease Diseases 0.000 claims description 3
- 230000002924 anti-infective effect Effects 0.000 claims description 2
- 239000000969 carrier Substances 0.000 claims description 2
- 208000015181 infectious disease Diseases 0.000 claims description 2
- 239000000463 material Substances 0.000 claims description 2
- 239000002537 cosmetic Substances 0.000 claims 5
- 239000013588 oral product Substances 0.000 claims 4
- 229940023486 oral product Drugs 0.000 claims 3
- 229960005475 antiinfective agent Drugs 0.000 claims 1
- 239000004599 antimicrobial Substances 0.000 claims 1
- 230000002401 inhibitory effect Effects 0.000 abstract description 9
- 229940079593 drug Drugs 0.000 abstract description 5
- 150000001875 compounds Chemical class 0.000 abstract description 3
- 239000002994 raw material Substances 0.000 abstract 1
- 238000002474 experimental method Methods 0.000 description 12
- 150000001413 amino acids Chemical class 0.000 description 11
- 230000005764 inhibitory process Effects 0.000 description 11
- 230000000844 anti-bacterial effect Effects 0.000 description 10
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 9
- 230000000694 effects Effects 0.000 description 8
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 8
- 235000018102 proteins Nutrition 0.000 description 8
- 102000004169 proteins and genes Human genes 0.000 description 8
- 108090000623 proteins and genes Proteins 0.000 description 8
- 239000000523 sample Substances 0.000 description 8
- 239000000243 solution Substances 0.000 description 8
- 239000001963 growth medium Substances 0.000 description 7
- 230000003385 bacteriostatic effect Effects 0.000 description 6
- 239000002609 medium Substances 0.000 description 5
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 4
- 229920001817 Agar Polymers 0.000 description 4
- 102000007079 Peptide Fragments Human genes 0.000 description 4
- 108010033276 Peptide Fragments Proteins 0.000 description 4
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 4
- 239000008272 agar Substances 0.000 description 4
- 230000001580 bacterial effect Effects 0.000 description 4
- 238000012258 culturing Methods 0.000 description 4
- 235000019253 formic acid Nutrition 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 208000035473 Communicable disease Diseases 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 108091005804 Peptidases Proteins 0.000 description 3
- 239000004365 Protease Substances 0.000 description 3
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 3
- 239000003242 anti bacterial agent Substances 0.000 description 3
- 229940088710 antibiotic agent Drugs 0.000 description 3
- 239000007864 aqueous solution Substances 0.000 description 3
- 150000002500 ions Chemical class 0.000 description 3
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 description 3
- 235000013336 milk Nutrition 0.000 description 3
- 239000008267 milk Substances 0.000 description 3
- 210000004080 milk Anatomy 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- XXMFJKNOJSDQBM-UHFFFAOYSA-N 2,2,2-trifluoroacetic acid;hydrate Chemical compound [OH3+].[O-]C(=O)C(F)(F)F XXMFJKNOJSDQBM-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- 241000233866 Fungi Species 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 229940124350 antibacterial drug Drugs 0.000 description 2
- 230000003115 biocidal effect Effects 0.000 description 2
- 239000007382 columbia agar Substances 0.000 description 2
- 230000007717 exclusion Effects 0.000 description 2
- 238000000034 method Methods 0.000 description 2
- 244000000010 microbial pathogen Species 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- 239000002243 precursor Substances 0.000 description 2
- 239000008223 sterile water Substances 0.000 description 2
- 101001008231 Bos taurus Beta-lactoglobulin Proteins 0.000 description 1
- GHXZTYHSJHQHIJ-UHFFFAOYSA-N Chlorhexidine Chemical compound C=1C=C(Cl)C=CC=1NC(N)=NC(N)=NCCCCCCN=C(N)N=C(N)NC1=CC=C(Cl)C=C1 GHXZTYHSJHQHIJ-UHFFFAOYSA-N 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241001646716 Escherichia coli K-12 Species 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 206010022678 Intestinal infections Diseases 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- 102000008192 Lactoglobulins Human genes 0.000 description 1
- 108010060630 Lactoglobulins Proteins 0.000 description 1
- 239000006137 Luria-Bertani broth Substances 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 206010034133 Pathogen resistance Diseases 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 230000021736 acetylation Effects 0.000 description 1
- 238000006640 acetylation reaction Methods 0.000 description 1
- -1 aliphatic amino acid Chemical class 0.000 description 1
- 229940124599 anti-inflammatory drug Drugs 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 239000005018 casein Substances 0.000 description 1
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 1
- 235000021240 caseins Nutrition 0.000 description 1
- 125000002091 cationic group Chemical group 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 229960003260 chlorhexidine Drugs 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 238000013480 data collection Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000010494 dissociation reaction Methods 0.000 description 1
- 230000005593 dissociations Effects 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 230000009036 growth inhibition Effects 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 230000005596 ionic collisions Effects 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 238000005556 structure-activity relationship Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000004885 tandem mass spectrometry Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
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
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/18—Peptides; Protein hydrolysates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
-
- 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
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
-
- 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
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- 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)
- General Health & Medical Sciences (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Organic Chemistry (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- General Chemical & Material Sciences (AREA)
- Mycology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Communicable Diseases (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Genetics & Genomics (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Oncology (AREA)
- Nutrition Science (AREA)
- Engineering & Computer Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Birds (AREA)
- Epidemiology (AREA)
- Dermatology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Cosmetics (AREA)
- Peptides Or Proteins (AREA)
Abstract
本发明涉及一种来自水解牛奶蛋白的具有微生物抑制活性的多肽化合物MAMP‑01，其氨基酸序列为Ala‑Ala‑Ser‑Asp‑Ile‑Ser‑Leu‑Leu(AASDISLL)。多肽MAMP‑01对革兰氏阳性菌和革兰氏阴性菌具有广泛的抑制活性，包括大肠杆菌、金黄色葡萄球菌、牙龈卟啉单胞菌和变异链球菌等。具有原料天然安全和生产成本低的优点，可作为抑制微生物生长的活性成分应用于药物、食品、日用品中。
Description
技术领域
本发明涉及一种来源于水解牛奶蛋白的抗菌肽MAMP-01(AASDISLL)及其应用。
背景技术
抗生素是能抵抗致病微生物的药物，是抗菌消炎药中最大的一类。抗生素是由细菌、真菌或其他微生物在生活过程中所产生的物质，具有抑制或杀灭细菌、真菌等致病微生物的作用，因此作为抗感染药物广泛地应用于各种感染性疾病。抗生素在治疗感染中的显著效果也导致了细菌耐药性，使部分感染性疾病的治疗变得愈加困难。抗菌肽作为一种新型的抗生素，具有广谱的抗菌活性，不会轻易使微生物产生耐药性，具有广阔的应用前景。
抗菌肽在自然界中广泛分布，作为被广泛关注的一种新型抗生素，越来越多的抗菌肽在微生物、动植物体内被发现。目前解析的抗菌肽构效关系中有两个较清晰的特征，分别是“带正电荷的阳离子”和“两亲性序列结构”。然而抗菌肽作为抗菌药物仍然面临许多待解决的问题：部分阳离子抗菌肽可以对哺乳动物的细胞产生较大的毒性作用；两亲性结构则要求氨基酸序列中有一定数量的碱性氨基酸，而碱性氨基酸易被蛋白酶水解的特性使得其作为药物进入人体后发挥效果大大受限。因此，开发安全、稳定、低细胞毒性的抗菌肽具有重要意义。
牛奶作为优质的天然食物，是丰富的蛋白质和多肽资源，兼顾抗菌肽的优势及优质安全的蛋白来源两个特质。牛奶中蛋白含量丰富，在蛋白酶的水解作用下，形成稳定存在的抗菌肽，作为微生物生长抑制剂具有良好的潜质。因此，从水解牛奶蛋白肽出发进一步寻找具有抑菌活性的肽会为健康安全的微生物生长抑制剂的制备提供新的方向。
发明内容
本发明的目的是提供一种来源于水解牛奶蛋白的抗菌肽MAMP-01，在制备预防和/或降低由微生物引起的肠道感染疾病、口腔疾病、皮肤疾病的药物、日用品或者微生物生长抑制剂中的应用。
为实现上述目的，本发明以所述多肽MAMP-01为抑制微生物生长活性的有效成份。
其具有序列表SEQ ID NO：1中氨基酸序列；多肽MAMP-01为抗革兰氏阴性菌和革兰氏阳性菌药物、食品、日用品的活性成份，其中可添加药学、食品、日用品上可接受的载体或辅料。
具有对革兰氏阴性和革兰氏阳性菌抑制活性的多肽化合物MAMP-01的氨基酸序列为AASDISLL，Ala-Ala-Ser-Asp-Ile-Ser-Leu-Leu。分子量788.90Da，白色粉末状，易溶于水，对大肠杆菌、金黄色葡萄球菌、牙龈卟啉单胞菌、变异链球菌的生长存在较强的抑制作用。
本发明与现有技术相比，具有如下有益效果：
本发明从水解牛奶蛋白中获得并确定了上述多肽的结构，首次验证了上述多肽具有较好的抑菌活性，因此作为预防和/或降低由大肠杆菌、金黄色葡萄球菌、牙龈卟啉单胞菌、变异链球菌引起的感染疾病的药物和/或保健品和/或日用品或者微生物生长抑制剂具有良好的应用前景。
附图说明
图1本发明多肽对大肠杆菌、金黄葡萄球菌、牙龈卟啉单胞菌和变异链球菌的抑菌结果，其中：图1A为多肽MAMP-01抑制大肠杆菌的实验结果；图1B为多肽MAMP-01抑制金黄色葡萄球菌的实验结果；图1C为多肽MAMP-01抑制牙龈卟啉单胞菌的实验结果图；图1D为多肽MAMP-01抑制变异链球菌的实验结果。
具体实施方式
实施例1
水解牛奶蛋白肽的制备及鉴定。
(1)样品制备
取1g牛奶酪蛋白用10ml水溶解后加入20mg胰蛋白酶于37℃反应2小时，酶解过程结束后在14000×g、4℃条件下离心20min，上清液为水解牛奶蛋白肽样品。
(2)LC-MS/MS分析
水解牛奶蛋白肽溶液样品按如下步骤以C18柱(Waters Oasis HLB SPE柱，1cc/30mg，30um)除盐：C18柱用1.5mL甲醇活化后加入1.5mL 0.1％(V/V)TFA(三氟乙酸)-H2O溶液平衡，将样品以1ml 0.1％(V/V)TFA-H2O溶液复溶后加入C18柱中，以1.5mL 80％(V/V)ACN(乙腈)/0.1％(V/V)TFA-H2O溶液进行洗脱，收集洗脱液分装冻干于-80℃保存。脱盐后的样品以0.1％(V/V)FA(甲酸)-H2O溶解并制备成浓度为0.2g/L的溶液，上样量为8μL，加载到15cm毛细管分析柱(内径为180μm)进行质谱分析。LC-MS/MS系统由Agilent 1100HPLC系统和LTQ-Orbitrap Velos质谱仪组成。流动相A为含0.1％(V/V)甲酸的水溶液，流动相B为含0.1％甲酸(V/V)和98％乙腈(V/V)的水溶液。梯度洗脱程序如下：0～80min，5～25％B(V/V，下同)；80～95min，25％～35％B；95～97min，35％～90％B；97～107min，90％B；107～109min，90％～0％B；109～126min，0％B；流速70μL/min。在Orbitrap质量分析仪中以60000的分辨率获得了完整的质谱扫描图(m/z 400～2000)。对其中最强的15个离子进行离子碰撞解离(CID)，再进行MS/MS扫描，扫描范围在m/z 400～2000。动态排除功能设置如下：重复2；持续时间30s；排除持续时间为60s。系统控制和数据收集由Xcalibur软件进行。每个样品进行三次质谱分析。
(3)数据检索
Xcalibur采集的*.RAW文件用MaxQuant软件在蛋白数据库(实验室在http://www.uniprot.org/下载蛋白质信息，建立牛奶蛋白数据库，蛋白质数目为7)中进行检索。搜库参数如下：不设置酶切、最大漏切数和固定修饰，可变修饰设置为甲硫氨酸的氧化(+15.9949Da)，乙酰化修饰(+42.011Da，磷酸化修饰(+79.966Da)。母离子的质量容忍偏差为20ppm，碎片离子为0.8Da。导出肽段时控制假阳性率(FDR)<1％，对导出结果Score>20的肽段视为有效数据进行分析。
(4)LC-MS/MS得到目标肽段信息
在水解牛奶蛋白肽样品中鉴定到五百条以上的肽段信息，其中约10％以上来源于β-乳球蛋白。其中多肽AASDISLL蛋白质前体为牛β-乳球蛋白(f41-48)，肽段等电点为3.80，分子质量为788.90Da，具有8个氨基酸，较短的序列及较低的相对分子质量，结合质谱鉴定结果表明该序列可以稳定存在不易继续被蛋白酶酶解。通过在线工具获取SEQ ID NO：1所示氨基酸序列的生物学信息表示，该肽段的不稳定系数为8.75，脂肪族氨基酸指数和亲水性分别为171.25和1.325，表现为亲水性，具有抗菌肽的特征。
SEQ ID No.1的信息
(a)序列特征
*长度：8氨基酸
*类型：氨基酸
*链型：单链
(b)分子类型：蛋白
序列描述：
SEQ ID No.1
AASDISLL
实施例2
多肽的抗菌活性。
于上海杰肽科技有限公司定制化学合成多肽MAMP-01，纯度为97.7％。以革兰氏阴性菌中的大肠杆菌和牙龈卟啉单胞菌、革兰氏阳性菌中的金黄色葡萄球菌和变异链球菌分别为靶标菌种，考察多肽MAMP-01的抗菌活性。
(1)菌株及复苏
大肠杆菌(Escherichia coli K12)、金黄色葡萄球菌(Staphylococcus aureus)、牙龈卟啉单胞菌(Porphyromonas gingivalis，P.g.,ATCC33277)和变异链球菌(Streptococcus mutans，M.s.,ATCC25175)均购自中国菌种资源库保藏中心，以甘油保藏法保存于-80℃。实验前将大肠杆菌接种于LB肉汤培养基(北京索莱宝科技有限公司)中，将金黄色葡萄球菌接种于TSB肉汤培养基(北京索莱宝科技有限公司)中，将牙龈卟啉单胞菌和变异链球菌分别接种于BHI肉汤培养基(北京索莱宝科技有限公司)中，靶标菌种均以1％体积量接种。于37℃条件下培养12h，将菌液以1％体积量再次接种至新鲜培养基中培养复苏。重复接种复苏操作2次，使菌株恢复活力。
(2)培养基
实验使用边长为90mm的方形培养皿。大肠杆菌和金黄色葡萄球菌的抑菌实验使用双层培养基，下层以20mL 2％(m/V，g/mL)的琼脂水溶液做支撑，上层分别加入8mL含0.7％(m/V，g/mL)琼脂、菌浓度为1×10^5CFU/mL的LB培养基和TSB培养基，分别制成LB平板和TSB平板。将制备好的两种平板培养基密封后于4℃保存。牙龈卟啉单胞菌的抑菌实验使用哥伦比亚琼脂培养基(南京全隆生物技术有限公司)，15mL的培养基制成平板，平板上加入200μL、菌浓度为1×10^5CFU/mL的菌液，利用滚珠涂布均匀。变异链球菌的抑菌实验使用含1.5％(m/V，g/mL)琼脂、菌浓度为1×10^5CFU/mL的BHI培养基，每个培养皿中加入含菌的培养基15mL制成平板。
(3)抗菌实验
在LB平板和TSB平板上层制作出内径2mm的孔、BHI平板中制作内径8mm的孔、哥伦比亚琼脂平板中加入内径8mm牛津杯分别用于抗菌实验。
将抗菌肽样品用无菌水溶解配制成100mg/ml的溶液，分别加到琼脂平板孔和牛津杯中20μL。大肠杆菌和金黄色葡萄球菌以无菌水作为阴性对照，牙龈卟啉单胞菌和变异链球菌以0.3mg/mL氯己定作阳性对照。阴性对照和阳性对照的加样体积与抗菌肽样品溶液相同。
加样后的平板于4℃冰箱中静置3h，待孔中样液完全吸收扩散后取出，大肠杆菌和金黄色葡萄球菌在37℃恒温培养箱中倒置培养16h，牙龈卟啉单胞菌和变异链球菌在80％氮气(V/V)、10％氢气(V/V)、10％二氧化碳(V/V)的厌氧培养箱37℃条件下倒置培养16h。培养结束后观察是否有抑菌圈出现，测量抑菌圈大小并记录。
(4)实验结果
对四种菌的抑菌结果见表1。结果表明多肽MAMP-01对革兰氏阴性菌和革兰氏阳性菌均有生长抑制活性。
表1多肽MAMP-01对大肠杆菌和金黄色葡萄球菌的抑菌结果(抑菌圈，mm)
1,数字表示抑菌圈直径(mm)，如5.0*5.0表示抑菌圈直径5.0(mm)；
表2多肽MAMP-01对牙龈卟啉单胞菌和变异链球菌的抑菌结果(抑菌圈，mm)
1,数字表示抑菌圈直径(mm)，如13.0*13.0表示抑菌圈直径13.0(mm)。
Claims (8)
1.一种抗菌肽MAMP-01，其特征在于：所述抗菌肽为多肽AASDISLL，具有序列表SEQ IDNO：1中氨基酸序列；该多肽的氨基酸序列具体为：
Ala-Ala-Ser-Asp-Ile-Ser-Leu-Leu。
2.按照权利要求1所述的抗菌肽，其特征在于：为来源于水解牛奶蛋白的抗菌肽，命名为MAMP-01。
3.一种权利要求1或2所述抗菌肽在制备微生物生长抑制剂、抗感染药物、口腔用品或化妆品中的应用。
4.按照权利要求3所述的应用，其特征在于：所述微生物生长抑制剂、抗感染药物、口腔用品或化妆品是以多肽MAMP-01为活性成份，其中可添加药物学、食品、口腔用品、化妆品中可接受的载体或辅料中的一种或两种以上。
5.按照权利要求3或4所述的应用，其特征在于：所述微生物为革兰氏阴性或革兰氏阳性菌中的一种或二种。
6.按照权利要求3或4所述的应用，其特征在于：所述感染为由大肠杆菌、金黄色葡萄球菌、牙龈卟啉单胞菌、变异链球菌中的一种或两种以上菌引起感染或菌群失衡。
7.按照权利要求3或4所述的应用，其特征在于：
所述药物为预防和/或降低或治疗由革兰氏阳性和革兰氏阴性菌引起的口腔疾病的药品；
所述口腔用品为预防和/或降低或治疗由革兰氏阳性和革兰氏阴性菌引起的口腔疾病的口腔用品。
8.按照权利要求3或4所述的应用，其特征在于：
所述药物为预防和/或降低或治疗由革兰氏阳性和革兰氏阴性菌引起的皮肤疾病的药品；
所述化妆品为预防和/或降低或治疗由革兰氏阳性和革兰氏阴性菌引起的皮肤疾病的化妆品。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110591731.8A CN114149486B (zh) | 2021-05-28 | 2021-05-28 | 一种抗菌肽mamp-01及其应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110591731.8A CN114149486B (zh) | 2021-05-28 | 2021-05-28 | 一种抗菌肽mamp-01及其应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114149486A true CN114149486A (zh) | 2022-03-08 |
| CN114149486B CN114149486B (zh) | 2023-05-12 |
Family
ID=80460955
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110591731.8A Active CN114149486B (zh) | 2021-05-28 | 2021-05-28 | 一种抗菌肽mamp-01及其应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114149486B (zh) |
Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU5334296A (en) * | 1996-03-25 | 1997-10-17 | Wolf-Georg Forssmann | Antibiotic peptides from bovine milk |
| US20030022299A1 (en) * | 2001-06-26 | 2003-01-30 | Pe Corporation (Ny) | Isolated human secreted proteins, nucleic acid molecules encoding human secreted proteins, and uses thereof |
| US20040071714A1 (en) * | 2000-09-25 | 2004-04-15 | Germond Jacques Edouard | Lactic acid bateria capable of reducing an individual's tendency to develop allergic reactions |
| CN102876764A (zh) * | 2012-10-05 | 2013-01-16 | 甘肃华羚生物技术研究中心 | 牦牛乳酪蛋白抑菌肽的制备方法 |
| US20140148378A1 (en) * | 2012-11-27 | 2014-05-29 | The Regents Of The University Of California | Antibacterial peptides |
| CN105237626A (zh) * | 2015-10-19 | 2016-01-13 | 河南科技学院 | 一种抗菌肽hjh-3及其应用 |
| CN107427553A (zh) * | 2015-03-18 | 2017-12-01 | N·V·努特里奇亚 | 通过β‑乳球蛋白衍生肽结合益生菌的给药来诱导口服耐受性的方法 |
| US20200157142A1 (en) * | 2017-11-13 | 2020-05-21 | Jiangsu University | Ultrasound-assisted simulated digestion method of milk protein active peptide and application thereof in health foods |
| WO2021091369A1 (en) * | 2019-11-07 | 2021-05-14 | N.V. Nutricia | Extensive whey protein hydrolysate with tolerogenic peptides |
| CN112824430A (zh) * | 2019-11-21 | 2021-05-21 | 中国科学院大连化学物理研究所 | 一种人乳内源性的抗菌肽及其应用 |
| CN112824429A (zh) * | 2019-11-21 | 2021-05-21 | 中国科学院大连化学物理研究所 | 一种人乳内源性抗菌多肽及其在在制备抗炎药物中的应用 |
-
2021
- 2021-05-28 CN CN202110591731.8A patent/CN114149486B/zh active Active
Patent Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU5334296A (en) * | 1996-03-25 | 1997-10-17 | Wolf-Georg Forssmann | Antibiotic peptides from bovine milk |
| US20040071714A1 (en) * | 2000-09-25 | 2004-04-15 | Germond Jacques Edouard | Lactic acid bateria capable of reducing an individual's tendency to develop allergic reactions |
| US20030022299A1 (en) * | 2001-06-26 | 2003-01-30 | Pe Corporation (Ny) | Isolated human secreted proteins, nucleic acid molecules encoding human secreted proteins, and uses thereof |
| CN102876764A (zh) * | 2012-10-05 | 2013-01-16 | 甘肃华羚生物技术研究中心 | 牦牛乳酪蛋白抑菌肽的制备方法 |
| US20140148378A1 (en) * | 2012-11-27 | 2014-05-29 | The Regents Of The University Of California | Antibacterial peptides |
| CN107427553A (zh) * | 2015-03-18 | 2017-12-01 | N·V·努特里奇亚 | 通过β‑乳球蛋白衍生肽结合益生菌的给药来诱导口服耐受性的方法 |
| CN105237626A (zh) * | 2015-10-19 | 2016-01-13 | 河南科技学院 | 一种抗菌肽hjh-3及其应用 |
| US20200157142A1 (en) * | 2017-11-13 | 2020-05-21 | Jiangsu University | Ultrasound-assisted simulated digestion method of milk protein active peptide and application thereof in health foods |
| WO2021091369A1 (en) * | 2019-11-07 | 2021-05-14 | N.V. Nutricia | Extensive whey protein hydrolysate with tolerogenic peptides |
| CN112824430A (zh) * | 2019-11-21 | 2021-05-21 | 中国科学院大连化学物理研究所 | 一种人乳内源性的抗菌肽及其应用 |
| CN112824429A (zh) * | 2019-11-21 | 2021-05-21 | 中国科学院大连化学物理研究所 | 一种人乳内源性抗菌多肽及其在在制备抗炎药物中的应用 |
Non-Patent Citations (9)
| Title |
|---|
| DANIEL MARTÍNEZ-MAQUEDA等: "Effect of β-lactoglobulin hydrolysate and β-lactorphin on intestinal mucin secretion and gene expression in human goblet cells" * |
| GENBANK: "KAF3007304.1: hypothetical protein E8E14_004018 [Neopestalotiopsis sp. 37M]", 《GENBANK》 * |
| LAURA G. GÓMEZ-MASCARAQUE等: "Microencapsulation of a whey protein hydrolysate within micro-hydrogels: Impact on gastrointestinal stability and potential for functional yoghurt development", 《JOURNAL OF FUNCTIONAL FOODS》 * |
| MARLOES SCHURINK等: "Novel peptides with tyrosinase inhibitory activity", 《PEPTIDES》 * |
| SØREN DRUD NIELSEN等: "Milk bioactive peptide database: A comprehensive database of milk protein-derived bioactive peptides and novel visualization" * |
| 于洋、祁艳霞、靳艳: "乳源生物活性肽研究进展", 《食品与发酵工业》 * |
| 翟青新、张源淑、哈惠馨: "牛乳酪蛋白源抗菌肽的研究进展", 《生物技术通讯》 * |
| 胡玉伟等: "抗菌肽在农业和食品工业上的应用" * |
| 陈娅娇: "酶法制备乳源抗菌肽的抗菌性研究" * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114149486B (zh) | 2023-05-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Xuan et al. | Antimicrobial peptides for combating drug-resistant bacterial infections | |
| CN112824429B (zh) | 一种人乳内源性抗菌多肽及其在制备抗炎药物中的应用 | |
| CN112707961B (zh) | 一种贝类抗菌肽p-amp153及其应用 | |
| CN107298706B (zh) | 一种荷斯坦奶牛脾脏源抗菌肽及其制备方法与应用 | |
| CN1683399A (zh) | 胡蜂抗菌肽及其制备方法和应用 | |
| CN114149486B (zh) | 一种抗菌肽mamp-01及其应用 | |
| CN114149485B (zh) | 一种抗菌肽mamp-02及其应用 | |
| CN108093637A (zh) | 新型双环脂羊毛硫肽、制备及作为抗微生物剂的用途 | |
| CN114149484B (zh) | 一种抗菌肽mamp-03及其应用 | |
| Kim et al. | Antimicrobial activity of antimicrobial peptide LPcin-YK3 derived from bovine lactophoricin | |
| CN112824430B (zh) | 一种人乳内源性的抗菌肽及其应用 | |
| CN105801669A (zh) | 一种杂合式抗生物型抗菌肽及制备方法和应用 | |
| CN114149483B (zh) | 一种抗菌肽组合物及其应用 | |
| CN118063544A (zh) | 一种抗菌肽或组合物及其应用 | |
| CN114763377B (zh) | 一种人乳内源性的抗菌肽及其应用 | |
| CN112851799B (zh) | 人乳内源性抗菌多肽及其在制备抗炎药物中的应用 | |
| CN112961210B (zh) | 一种人乳内源性抗菌多肽及其制备抗炎药物中的应用 | |
| CN112851762B (zh) | 一种人乳内源性抗菌多肽及其在制备抗炎药物中的应用 | |
| CN112851798B (zh) | 一种人乳内源性抗菌多肽和其在制备抗炎药物中的应用 | |
| CN112851752B (zh) | 人乳内源性抗菌多肽和其在制备抗炎药物中的应用 | |
| Babich et al. | Structure and properties of antimicrobial peptides produced by antagonist microorganisms isolated from Siberian natural objects. | |
| CN117886913A (zh) | 乳源抗菌多肽及其药物和应用 | |
| CN116693607A (zh) | 抗菌多肽及应用和细菌生长抑制剂、抗感染药物或保健品 | |
| CN116693620A (zh) | 抗菌多肽和应用以及细菌生长抑制剂、抗感染药物或保健品 | |
| CN116694599A (zh) | 抗菌多肽和应用及抑菌剂、抗感染药物或保健品 |
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 |