CN114773435B - 一种蚯蚓抗菌肽及其在广谱抗菌方面的应用 - Google Patents
一种蚯蚓抗菌肽及其在广谱抗菌方面的应用 Download PDFInfo
- Publication number
- CN114773435B CN114773435B CN202210356514.5A CN202210356514A CN114773435B CN 114773435 B CN114773435 B CN 114773435B CN 202210356514 A CN202210356514 A CN 202210356514A CN 114773435 B CN114773435 B CN 114773435B
- Authority
- CN
- China
- Prior art keywords
- antibacterial
- peptide
- earthworm
- ewamp
- antibacterial peptide
- 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
Links
- 239000003910 polypeptide antibiotic agent Substances 0.000 title claims abstract description 44
- 241000361919 Metaphire sieboldi Species 0.000 title claims abstract description 37
- 230000000844 anti-bacterial effect Effects 0.000 title abstract description 28
- 241000894006 Bacteria Species 0.000 claims abstract description 11
- 241000191967 Staphylococcus aureus Species 0.000 claims abstract description 8
- 241000588724 Escherichia coli Species 0.000 claims abstract description 6
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 6
- 229940124350 antibacterial drug Drugs 0.000 claims abstract description 6
- 241000192125 Firmicutes Species 0.000 claims abstract description 5
- 239000000654 additive Substances 0.000 claims abstract description 5
- 239000003755 preservative agent Substances 0.000 claims abstract description 5
- 108700042778 Antimicrobial Peptides Proteins 0.000 claims description 17
- 102000044503 Antimicrobial Peptides Human genes 0.000 claims description 17
- 230000000845 anti-microbial effect Effects 0.000 claims description 7
- 230000000996 additive effect Effects 0.000 claims description 2
- 230000002335 preservative effect Effects 0.000 claims description 2
- 125000000539 amino acid group Chemical group 0.000 abstract description 4
- 238000003766 bioinformatics method Methods 0.000 abstract description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 22
- 230000001580 bacterial effect Effects 0.000 description 12
- 230000002401 inhibitory effect Effects 0.000 description 12
- 102000004196 processed proteins & peptides Human genes 0.000 description 12
- 108010045269 tryptophyltryptophan Proteins 0.000 description 9
- UJGDFQRPYGJBEH-AAEUAGOBSA-N Trp-Ser-Gly Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CO)C(=O)NCC(=O)O)N UJGDFQRPYGJBEH-AAEUAGOBSA-N 0.000 description 8
- 239000007788 liquid Substances 0.000 description 8
- 108010094001 arginyl-tryptophyl-arginine Proteins 0.000 description 7
- 238000000034 method Methods 0.000 description 7
- QUBKBPZGMZWOKQ-SZMVWBNQSA-N Arg-Trp-Arg Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@H](CCCN=C(N)N)N)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O)=CNC2=C1 QUBKBPZGMZWOKQ-SZMVWBNQSA-N 0.000 description 6
- LKJCZEPXHOIAIW-HOTGVXAUSA-N Gly-Trp-Lys Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)CN LKJCZEPXHOIAIW-HOTGVXAUSA-N 0.000 description 6
- GFWLIJDQILOEPP-HSCHXYMDSA-N Lys-Ile-Trp Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)O)NC(=O)[C@H](CCCCN)N GFWLIJDQILOEPP-HSCHXYMDSA-N 0.000 description 6
- OSYOKZZRVGUDMO-HSCHXYMDSA-N Trp-Lys-Ile Chemical compound [H]N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O OSYOKZZRVGUDMO-HSCHXYMDSA-N 0.000 description 6
- UPADCCSMVOQAGF-LBPRGKRZSA-N Gly-Gly-Trp Chemical compound C1=CC=C2C(C[C@H](NC(=O)CNC(=O)CN)C(O)=O)=CNC2=C1 UPADCCSMVOQAGF-LBPRGKRZSA-N 0.000 description 4
- NLOZZWJNIKKYSC-WDSOQIARSA-N Lys-Arg-Trp Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@@H](N)CCCCN)C(O)=O)=CNC2=C1 NLOZZWJNIKKYSC-WDSOQIARSA-N 0.000 description 4
- YCEHCFIOIYNQTR-NYVOZVTQSA-N Trp-Trp-Ser Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CC3=CNC4=CC=CC=C43)C(=O)N[C@@H](CO)C(=O)O)N YCEHCFIOIYNQTR-NYVOZVTQSA-N 0.000 description 4
- 210000000170 cell membrane Anatomy 0.000 description 4
- 229920001184 polypeptide Polymers 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- KIAOPHMUNPPGEN-PEXQALLHSA-N Ile-Gly-His Chemical compound CC[C@H](C)[C@@H](C(=O)NCC(=O)N[C@@H](CC1=CN=CN1)C(=O)O)N KIAOPHMUNPPGEN-PEXQALLHSA-N 0.000 description 3
- YOZCKMXHBYKOMQ-IHRRRGAJSA-N Leu-Arg-Lys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCCCN)C(=O)O)N YOZCKMXHBYKOMQ-IHRRRGAJSA-N 0.000 description 3
- XXROXFHCMVXETG-UWVGGRQHSA-N Val-Gly-Val Chemical compound CC(C)[C@H](N)C(=O)NCC(=O)N[C@@H](C(C)C)C(O)=O XXROXFHCMVXETG-UWVGGRQHSA-N 0.000 description 3
- 125000000637 arginyl group Chemical group N[C@@H](CCCNC(N)=N)C(=O)* 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 241001233061 earthworms Species 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 238000010532 solid phase synthesis reaction Methods 0.000 description 3
- 239000008223 sterile water Substances 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- CFGHCPUPFHWMCM-FDARSICLSA-N Arg-Ile-Trp Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)O)NC(=O)[C@H](CCCN=C(N)N)N CFGHCPUPFHWMCM-FDARSICLSA-N 0.000 description 2
- 238000009631 Broth culture Methods 0.000 description 2
- 241001360526 Escherichia coli ATCC 25922 Species 0.000 description 2
- FXTUGWXZTFMTIV-GJZGRUSLSA-N Gly-Trp-Arg Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)NC(=O)CN FXTUGWXZTFMTIV-GJZGRUSLSA-N 0.000 description 2
- RVKIPWVMZANZLI-UHFFFAOYSA-N H-Lys-Trp-OH Natural products C1=CC=C2C(CC(NC(=O)C(N)CCCCN)C(O)=O)=CNC2=C1 RVKIPWVMZANZLI-UHFFFAOYSA-N 0.000 description 2
- 108010087066 N2-tryptophyllysine Proteins 0.000 description 2
- 108700026244 Open Reading Frames Proteins 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 108010068380 arginylarginine Proteins 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 125000003588 lysine group Chemical group [H]N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 2
- 108010054155 lysyllysine Proteins 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- JBQORRNSZGTLCV-WDSOQIARSA-N Arg-Trp-Lys Chemical compound C1=CC=C2C(C[C@@H](C(=O)N[C@@H](CCCCN)C(O)=O)NC(=O)[C@@H](N)CCCN=C(N)N)=CNC2=C1 JBQORRNSZGTLCV-WDSOQIARSA-N 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- 208000035143 Bacterial infection Diseases 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 241001535558 Ewingia Species 0.000 description 1
- 102000002812 Heat-Shock Proteins Human genes 0.000 description 1
- 108010004889 Heat-Shock Proteins Proteins 0.000 description 1
- XDVKZSJODLMNLJ-GGQYPGDFSA-N Ile-Trp-Trp Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@H](CC=3C4=CC=CC=C4NC=3)NC(=O)[C@@H](N)[C@@H](C)CC)C(O)=O)=CNC2=C1 XDVKZSJODLMNLJ-GGQYPGDFSA-N 0.000 description 1
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 1
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 1
- RYOLKFYZBHMYFW-WDSOQIARSA-N Lys-Trp-Arg Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@@H](N)CCCCN)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O)=CNC2=C1 RYOLKFYZBHMYFW-WDSOQIARSA-N 0.000 description 1
- YUTZYVTZDVZBJJ-IHPCNDPISA-N Lys-Trp-Lys Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@@H](N)CCCCN)C(=O)N[C@@H](CCCCN)C(O)=O)=CNC2=C1 YUTZYVTZDVZBJJ-IHPCNDPISA-N 0.000 description 1
- 102100037611 Lysophospholipase Human genes 0.000 description 1
- AJHCSUXXECOXOY-UHFFFAOYSA-N N-glycyl-L-tryptophan Natural products C1=CC=C2C(CC(NC(=O)CN)C(O)=O)=CNC2=C1 AJHCSUXXECOXOY-UHFFFAOYSA-N 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 108010058864 Phospholipases A2 Proteins 0.000 description 1
- 108010040201 Polymyxins Proteins 0.000 description 1
- YMTLKLXDFCSCNX-BYPYZUCNSA-N Ser-Gly-Gly Chemical compound OC[C@H](N)C(=O)NCC(=O)NCC(O)=O YMTLKLXDFCSCNX-BYPYZUCNSA-N 0.000 description 1
- CDPXXGFRDZVVGF-OYDLWJJNSA-N Trp-Arg-Trp Chemical compound [H]N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(O)=O CDPXXGFRDZVVGF-OYDLWJJNSA-N 0.000 description 1
- ZHZLQVLQBDBQCQ-WDSOQIARSA-N Trp-Lys-Arg Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)N ZHZLQVLQBDBQCQ-WDSOQIARSA-N 0.000 description 1
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- -1 antibacterial drugs Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 208000022362 bacterial infectious disease Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000007123 defense Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 108010084389 glycyltryptophan Proteins 0.000 description 1
- 230000002519 immonomodulatory effect Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 125000001500 prolyl group Chemical group [H]N1C([H])(C(=O)[*])C([H])([H])C([H])([H])C1([H])[H] 0.000 description 1
- 125000000430 tryptophan group Chemical group [H]N([H])C(C(=O)O*)C([H])([H])C1=C([H])N([H])C2=C([H])C([H])=C([H])C([H])=C12 0.000 description 1
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
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
- A01N37/44—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a nitrogen atom attached to the same carbon skeleton by a single or double bond, this nitrogen atom not being a member of a derivative or of a thio analogue of a carboxylic group, e.g. amino-carboxylic acids
- A01N37/46—N-acyl derivatives
-
- 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
- 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
- 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
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicinal Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Communicable Diseases (AREA)
- Oncology (AREA)
- Plant Pathology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pest Control & Pesticides (AREA)
- Pharmacology & Pharmacy (AREA)
- Dentistry (AREA)
- Public Health (AREA)
- Environmental Sciences (AREA)
- Wood Science & Technology (AREA)
- Agronomy & Crop Science (AREA)
- Mycology (AREA)
- Nutrition Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
本发明提供了一种新型蚯蚓抗菌肽及其在广谱抗菌方面的应用，所述抗菌肽的氨基酸序列如SEQ ID NO.14所示，具有13个氨基酸残基，其等电点为12.48。本发明主要是采用生物信息学的方法寻找新的蚯蚓抗菌肽，再进行氨基酸序列优化，最终得到抗菌效果较好的抗菌肽。所得抗菌肽对以大肠杆菌为代表的革兰氏阴性菌和以金黄色葡萄球菌为代表的革兰氏阳性菌具有较好的抗菌活性，可应用于制备抗菌类产品，包括抗细菌药物、添加剂和防腐剂等。
Description
技术领域
本发明涉及生物技术领域，具体地，涉及一种蚯蚓抗菌肽及其在广谱抗菌方面的应用，该新型蚯蚓抗菌肽对革兰氏阴性/阳性细菌均具有良好的抗菌活性。
背景技术
目前被称为细菌感染最后一道防线的糖肽类抗生素和环脂肽多粘菌素类抗生素也出现了耐药细菌。因此，开发新型抗菌药物已经迫在眉睫。大量研究证明，抗菌肽具有成为新型抗菌药物的潜力。按其二级结构划分，抗菌肽主要分为α螺旋肽、β折叠肽和其他类型肽。就抗菌机理而言，大部分α螺旋肽可以破坏细菌细胞膜，少数可以与核酸相互作用；部分β折叠肽可以在细菌细胞膜上形成环形孔，部分β折叠肽还可以影响DNA和蛋白质相互作用、改变细菌细胞膜电位或抑制生物大分子合成等作用；其他类型肽大部分含有较多的色氨酸、精氨酸或脯氨酸残基，可以与热休克蛋白等靶蛋白结合，抑制或杀灭细菌。某些抗菌肽还具有免疫调节作用，例如激活磷脂酶A2等。
目前在许多生物中都发现了抗菌肽的存在，蚯蚓也如此。目前已有较多蚯蚓抗菌肽被发现，但是在蚯蚓的基因组中仍然可能具有许多可以转录翻译为抗菌肽的ORFs(开放性阅读框)。为了进一步利用蚯蚓生物资源，可以继续在蚯蚓中寻找抗菌肽作为抗菌药物的备选。
发明内容
针对现有技术中的缺陷，本发明的目的是提供一种新型蚯蚓抗菌肽及其在广谱抗菌方面的应用。
本发明的目的是通过以下方案实现的：
<第一方面>
本发明提供一种蚯蚓抗菌肽EWAMP-R，所述蚯蚓抗菌肽EWAMP-R是由蚯蚓抗菌肽EWAMP.15序列优化获得。
所述蚯蚓抗菌肽EWAMP.15的氨基酸序列如SEQ ID NO.1所示。
所述新蚯蚓抗菌肽EWAMP-R具有13个氨基酸残基，其等电点为12.48
优选的，所述蚯蚓抗菌肽EWAMP-R包括5个色氨酸残基和4个精氨酸残基，等电点为12.48。
所述蚯蚓抗菌肽EWAMP-R的氨基酸序列如SEQ ID NO.14所示。
<第二方面>
所述的新型蚯蚓抗菌肽EWAMP-R在广谱抗菌方面的应用也属于本发明的保护范围。
所述蚯蚓抗菌肽EWAMP-R应用于对革兰氏阴性菌和革兰氏阳性菌的抗菌。
所述革兰氏阴性菌为大肠杆菌，所述革兰氏阳性菌为金黄色葡萄球菌。
所述蚯蚓抗菌肽EWAMP-R对大肠杆菌的最小抑菌浓度为8μg/mL。
所述蚯蚓抗菌肽EWAMP-R对金黄色葡萄球菌的最小抑菌浓度为16μg/mL。
<第三方面>
本发明还提供一种包含所述的蚯蚓抗菌肽EWAMP-R的抗菌药物、添加剂、或防腐剂等衍生产品。
本发明的新型蚯蚓抗菌肽具有13个氨基酸残基，具有两亲性，其等电点为12.48，具有阳离子特性，其通过破坏细菌细胞膜或者与细胞内的分子相互作用而起抑菌作用。
与现有技术相比，本发明具有如下的有益效果：
1、本发明对蚯蚓抗菌肽EWAMP.15序列拆分成多个多肽序列，通过比较研究，筛选出具有抗菌活性的EWAMP.15 12-24肽段；
2、本发明进一步对EWAMP.15 12-24肽段进行优化，在EWAMP.15 12-24的基础上，设计了精氨酸残基和赖氨酸残基相互取代的衍生肽，并通过筛选，得到所述蚯蚓抗菌肽EWAMP-R；所得EWAMP-R对以大肠杆菌(E.coil)为代表的革兰氏阴性菌和以金黄色葡萄球菌(S.aureus)为代表的革兰氏阳性菌具有抗菌活性；EWAMP-R最小抑菌浓度为MICE.coil＝8μg/mL、MICS.aureus＝16μg/mL，该抗菌肽具有良好的抗菌作用，可应用于制备抗细菌类产品，包括抗菌药物、添加剂和防腐剂等。
具体实施方式
下面结合具体实施例对本发明进行详细说明。以下实施例将有助于本领域的技术人员进一步理解本发明，但不以任何形式限制本发明。应当指出的是，对本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变化和改进。这些都属于本发明的保护范围。
实施例1、蚯蚓抗菌肽EWAMP.15的抗菌活性的测定
利用生物信息学方法在安德爱胜蚓全基因组DNA序列GWHACBE00000000中发掘了一个新的蚯蚓抗菌肽EWAMP.15，其氨基酸序列如SEQ ID NO.1所示，具体为MLRKVGVIGHWKIWWSGGWKRWRWR。委托吉尔生化(上海)有限公司利用固相合成法人工合成，纯度大于95％。将上一步人工合成的肽用最小抑菌浓度法测定其抗菌活性。具体步骤如下：
(1)在MH琼脂培养基中平板划线分别培养大肠杆菌ATCC25922和金黄色葡萄球菌ATCC29213，37℃培养过夜；
(2)分别挑取大肠杆菌ATCC25922和金黄色葡萄球菌ATCC29213的单菌落，接种于MH肉汤培养基中，37℃180rpm培养过夜；
(3)取50μL步骤(2)过夜培养的菌液，加入5mL的MH肉汤培养基中，37℃180rpm培养至OD600＝0.5左右；
(4)将10μL步骤(3)所得的菌液加入10mL的MH肉汤培养基中，得到测定所需菌液；
(5)将本发明所述的蚯蚓抗菌肽EWAMP.15分别用无菌水稀释到10240、5120、2560、1280、640、320、160、80、40、20、10、5μg/mL；
(6)在无菌96孔板中加入90μL步骤(4)得到的测定所需菌液，然后分别加入10μL步骤(5)中稀释后的各种浓度的蚯蚓抗菌肽溶液EWAMP.15，作为实验组。阳性对照组为：加入90μL步骤(4)得到的测定所需菌液和10μL无菌水；阴性对照组为：加入90μL无菌MH肉汤培养基和10μL无菌水；37℃培养18-24h；
(7)观察各孔中菌液的浑浊情况和是否有细菌沉淀，菌液澄清无沉淀的最低浓度作为最小抑菌浓度。表1为合成的EWAMP.15序列及其最小抑菌浓度试验结果。
表1EWAMP.15序列及最小抑菌浓度
实施例2、蚯蚓抗菌肽EWAMP.15的抗菌活性区域的筛选
根据抗菌肽设计原则，将EWAMP.15序列拆分成多个多肽序列，委托吉尔生化(上海)有限公司利用固相合成法人工合成，纯度大于95％。将上一步人工合成的肽用最小抑菌浓度法测定其抗菌活性。表2为拆分出的多肽序列及其最小抑菌浓度试验结果。
表2、拆分出的多肽序列及最小抑菌浓度
由上表可知EWAMP.15 12-24，序列为KIWWSGGWKRWRW(SEQ ID NO.8)，抗菌活性较好，最小抑菌浓度为MICE.coil＝16μg/mL、MICS.aureus＝16μg/mL。因此12-24位的氨基酸残基属于是EWAMP.15的抗菌活性区域。
实施例3、蚯蚓抗菌肽EWAMP.15 12-24的序列优化
在EWAMP.15 12-24的基础上，设计了精氨酸残基和赖氨酸残基相互取代的衍生肽，委托吉尔生化(上海)有限公司利用固相合成法人工合成，纯度大于95％。将上一步人工合成的肽用最小抑菌浓度法测定其抗菌活性。表3为衍生肽序列及其最小抑菌浓度试验结果。
表3、衍生肽序列及最小抑菌浓度
由上表可知EWAMP.15 12-24.01 09R，序列为RIWWSGGWRRWRW(SEQ ID NO.14)，抗菌活性较好，最小抑菌浓度为MICE.coil＝8μg/mL、MICS.aureus＝16μg/mL，将其重命名为EWAMP-R。
由试验结果可知，本发明所获得的蚯蚓抗菌肽EWAMP-R对大肠杆菌和金黄色葡萄球菌均具有较好的抗菌活性，可用于抗菌药物、添加剂和防腐剂等的开发。
以上对本发明的具体实施例进行了描述。需要理解的是，本发明并不局限于上述特定实施方式，本领域技术人员可以在权利要求的范围内做出各种变化或修改，这并不影响本发明的实质内容。在不冲突的情况下，本申请的实施例和实施例中的特征可以任意相互组合。
序列表
<110> 上海交通大学
<120> 一种蚯蚓抗菌肽及其在广谱抗菌方面的应用
<130> KAG48337
<160> 15
<170> SIPOSequenceListing 1.0
<210> 1
<211> 25
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Met Leu Arg Lys Val Gly Val Ile Gly His Trp Lys Ile Trp Trp Ser
1 5 10 15
Gly Gly Trp Lys Arg Trp Arg Trp Arg
20 25
<210> 2
<211> 14
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Leu Arg Lys Val Gly Val Ile Gly His Trp Lys Ile Trp Trp
1 5 10
<210> 3
<211> 13
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 3
Leu Arg Lys Val Gly Val Ile Gly His Trp Lys Ile Trp
1 5 10
<210> 4
<211> 14
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
Trp Lys Ile Trp Trp Ser Gly Gly Trp Lys Arg Trp Arg Trp
1 5 10
<210> 5
<211> 13
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 5
Trp Lys Ile Trp Trp Ser Gly Gly Trp Lys Arg Trp Arg
1 5 10
<210> 6
<211> 12
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 6
Trp Lys Ile Trp Trp Ser Gly Gly Trp Lys Arg Trp
1 5 10
<210> 7
<211> 13
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 7
Lys Ile Trp Trp Ser Gly Gly Trp Lys Arg Trp Arg Trp
1 5 10
<210> 8
<211> 12
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 8
Ile Trp Trp Ser Gly Gly Trp Lys Arg Trp Arg Trp
1 5 10
<210> 9
<211> 12
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 9
Lys Ile Trp Trp Ser Gly Gly Trp Lys Arg Trp Arg
1 5 10
<210> 10
<211> 13
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 10
Arg Ile Trp Trp Ser Gly Gly Trp Lys Arg Trp Arg Trp
1 5 10
<210> 11
<211> 13
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 11
Lys Ile Trp Trp Ser Gly Gly Trp Arg Arg Trp Arg Trp
1 5 10
<210> 12
<211> 13
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 12
Lys Ile Trp Trp Ser Gly Gly Trp Lys Lys Trp Arg Trp
1 5 10
<210> 13
<211> 13
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 13
Lys Ile Trp Trp Ser Gly Gly Trp Lys Arg Trp Lys Trp
1 5 10
<210> 14
<211> 13
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 14
Arg Ile Trp Trp Ser Gly Gly Trp Arg Arg Trp Arg Trp
1 5 10
<210> 15
<211> 13
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 15
Lys Ile Trp Trp Ser Gly Gly Trp Lys Lys Trp Lys Trp
1 5 10
Claims (5)
1.一种蚯蚓抗菌肽EWAMP-R，其特征在于，所述蚯蚓抗菌肽EWAMP-R的氨基酸序列如SEQID NO .14所示。
2.如权利要求1所述的蚯蚓抗菌肽EWAMP-R在广谱抗菌方面的应用。
3.根据权利要求2所述的应用，其特征在于，所述蚯蚓抗菌肽EWAMP-R应用于对革兰氏阴性菌和革兰氏阳性菌的抗菌。
4.根据权利要求2所述的应用，其特征在于，所述革兰氏阴性菌为大肠杆菌，所述革兰氏阳性菌为金黄色葡萄球菌。
5.一种包含权利要求1所述的蚯蚓抗菌肽EWAMP-R的抗菌药物、添加剂、或防腐剂。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210356514.5A CN114773435B (zh) | 2022-04-06 | 2022-04-06 | 一种蚯蚓抗菌肽及其在广谱抗菌方面的应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210356514.5A CN114773435B (zh) | 2022-04-06 | 2022-04-06 | 一种蚯蚓抗菌肽及其在广谱抗菌方面的应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114773435A CN114773435A (zh) | 2022-07-22 |
| CN114773435B true CN114773435B (zh) | 2023-10-31 |
Family
ID=82426371
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210356514.5A Active CN114773435B (zh) | 2022-04-06 | 2022-04-06 | 一种蚯蚓抗菌肽及其在广谱抗菌方面的应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114773435B (zh) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115433258B (zh) * | 2022-09-06 | 2023-09-26 | 北京林业大学 | 一种抗菌多肽及其应用 |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101319007A (zh) * | 2007-06-06 | 2008-12-10 | 中国农业科学院饲料研究所 | 具有高抑菌活性的新抗菌肽设计与合成 |
| CN106496305A (zh) * | 2016-11-09 | 2017-03-15 | 湖南科技学院 | 一组广谱高效的抗菌肽及其应用 |
| CN111892646A (zh) * | 2020-08-13 | 2020-11-06 | 中国人民解放军军事科学院军事医学研究院 | 高效低毒的抗菌肽衍生物及其在制备抗细菌感染药物中的应用 |
-
2022
- 2022-04-06 CN CN202210356514.5A patent/CN114773435B/zh active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101319007A (zh) * | 2007-06-06 | 2008-12-10 | 中国农业科学院饲料研究所 | 具有高抑菌活性的新抗菌肽设计与合成 |
| CN106496305A (zh) * | 2016-11-09 | 2017-03-15 | 湖南科技学院 | 一组广谱高效的抗菌肽及其应用 |
| CN111892646A (zh) * | 2020-08-13 | 2020-11-06 | 中国人民解放军军事科学院军事医学研究院 | 高效低毒的抗菌肽衍生物及其在制备抗细菌感染药物中的应用 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114773435A (zh) | 2022-07-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Wang | Unifying the classification of antimicrobial peptides in the antimicrobial peptide database | |
| KR101983668B1 (ko) | 그람 음성 박테리아에 대한 강한 항균 활성을 갖는 벌거숭이두더지쥐 유래의 항균성 폴리펩티드 및 이를 포함하는 약학 조성물 | |
| CN111057134B (zh) | 一类具有广谱抗菌活性、低毒性且无诱导耐药性的抗菌肽及其应用 | |
| CN114773435B (zh) | 一种蚯蚓抗菌肽及其在广谱抗菌方面的应用 | |
| Grafskaia et al. | Discovery of novel antimicrobial peptides: A transcriptomic study of the sea anemone Cnidopus japonicus | |
| Santana et al. | Alpha-helical domain from IL-8 of salmonids: Mechanism of action and identification of a novel antimicrobial function | |
| CN112094833B (zh) | 一种抑菌蛋白及其编码基因、用途和菌株 | |
| Jaradat et al. | Solid-phase synthesis and antibacterial activity of an artificial cyclic peptide containing two disulfide bridges | |
| CN110938112A (zh) | 脂肪酸修饰的超短序列抗菌肽类似物及其应用 | |
| CN112457413B (zh) | 一种利用sumo融合表达抗菌肽的方法 | |
| CN105622763A (zh) | 抗菌肽融合蛋白及其制备方法和应用 | |
| CN116813712B (zh) | 一种富含Trp的α-螺旋结构抗菌肽W33及其制备方法和应用 | |
| CN116987150A (zh) | 一种抗菌多肽、多肽衍生物及其制备方法与应用 | |
| Kong et al. | Insights into the antibacterial activity of cottonseed protein-derived peptide against Escherichia coli | |
| CN105837675B (zh) | 一组阳离子抗菌肽及其制备方法 | |
| CN115819544A (zh) | 一种抗菌肽及其制备方法和应用 | |
| CN107298707B (zh) | 一种类Bac5抗菌肽及其应用 | |
| CN113185577B (zh) | 具有不同电荷排列模式及不同电荷种类的低毒广谱抗菌肽及其应用 | |
| CN113603748B (zh) | 一种β-折叠抗菌肽HINGE-RV及其制备方法和应用 | |
| CN111253474B (zh) | 一种抗菌肽rg-27及其应用 | |
| CN113929760B (zh) | 一种防御素样免疫调节十四肽rv14及其制备方法和应用 | |
| Tamaki et al. | Antimicrobially active cycloundecapeptides related to gramicidin S having a novel turn structure with cisd-Phe-Pro peptide bond | |
| Sharma et al. | In vitro antibacterial and antimalarial activity of dehydrophenylalanine-containing undecapeptides alone and in combination with drugs | |
| CN112480230A (zh) | 一种具有更好抗热功能的抗菌肽及其应用 | |
| Mello et al. | Membrane selectivity of antimicrobial peptides |
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 |