CN107383175B - A kind of antibacterial peptide VK-21 and its application - Google Patents
A kind of antibacterial peptide VK-21 and its application Download PDFInfo
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- CN107383175B CN107383175B CN201710779621.8A CN201710779621A CN107383175B CN 107383175 B CN107383175 B CN 107383175B CN 201710779621 A CN201710779621 A CN 201710779621A CN 107383175 B CN107383175 B CN 107383175B
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- 239000003910 polypeptide antibiotic agent Substances 0.000 title claims abstract description 68
- 230000000844 anti-bacterial effect Effects 0.000 claims abstract description 11
- 230000003115 biocidal effect Effects 0.000 claims abstract description 8
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 6
- 238000002360 preparation method Methods 0.000 claims description 5
- 239000003795 chemical substances by application Substances 0.000 claims description 4
- 241000192125 Firmicutes Species 0.000 claims description 3
- 239000002537 cosmetic Substances 0.000 claims description 3
- 208000035143 Bacterial infection Diseases 0.000 claims description 2
- 239000003755 preservative agent Substances 0.000 claims description 2
- 230000002335 preservative effect Effects 0.000 claims description 2
- 239000000654 additive Substances 0.000 claims 1
- 230000000996 additive effect Effects 0.000 claims 1
- 241000191967 Staphylococcus aureus Species 0.000 abstract description 14
- 230000001408 fungistatic effect Effects 0.000 abstract description 13
- 241000607598 Vibrio Species 0.000 abstract description 9
- 230000002401 inhibitory effect Effects 0.000 abstract description 9
- 241000588724 Escherichia coli Species 0.000 abstract description 6
- 241000607618 Vibrio harveyi Species 0.000 abstract description 6
- RJQXTJLFIWVMTO-TYNCELHUSA-N Methicillin Chemical compound COC1=CC=CC(OC)=C1C(=O)N[C@@H]1C(=O)N2[C@@H](C(O)=O)C(C)(C)S[C@@H]21 RJQXTJLFIWVMTO-TYNCELHUSA-N 0.000 abstract description 5
- 229960003085 meticillin Drugs 0.000 abstract description 5
- 241000589517 Pseudomonas aeruginosa Species 0.000 abstract description 4
- 230000002949 hemolytic effect Effects 0.000 abstract description 4
- 108090000765 processed proteins & peptides Proteins 0.000 description 16
- 239000000047 product Substances 0.000 description 16
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 15
- 241000894006 Bacteria Species 0.000 description 13
- 229920005989 resin Polymers 0.000 description 12
- 239000011347 resin Substances 0.000 description 12
- 102000004196 processed proteins & peptides Human genes 0.000 description 11
- 239000000243 solution Substances 0.000 description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 10
- 210000003743 erythrocyte Anatomy 0.000 description 8
- 125000003345 AMP group Chemical group 0.000 description 7
- 150000001413 amino acids Chemical class 0.000 description 7
- 229920006227 ethylene-grafted-maleic anhydride Polymers 0.000 description 7
- 229920001184 polypeptide Polymers 0.000 description 7
- 238000012913 prioritisation Methods 0.000 description 7
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- 244000052616 bacterial pathogen Species 0.000 description 6
- 239000007788 liquid Substances 0.000 description 5
- 238000000034 method Methods 0.000 description 5
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 4
- 241001465754 Metazoa Species 0.000 description 4
- 238000009360 aquaculture Methods 0.000 description 4
- 244000144974 aquaculture Species 0.000 description 4
- 230000001580 bacterial effect Effects 0.000 description 4
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- 238000006243 chemical reaction Methods 0.000 description 4
- 239000003814 drug Substances 0.000 description 4
- 238000004128 high performance liquid chromatography Methods 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- UMRUUWFGLGNQLI-QFIPXVFZSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-6-[(2-methylpropan-2-yl)oxycarbonylamino]hexanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CCCCNC(=O)OC(C)(C)C)C(O)=O)C3=CC=CC=C3C2=C1 UMRUUWFGLGNQLI-QFIPXVFZSA-N 0.000 description 3
- 241000196324 Embryophyta Species 0.000 description 3
- 206010018910 Haemolysis Diseases 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 3
- 239000012043 crude product Substances 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 238000010438 heat treatment Methods 0.000 description 3
- 230000008588 hemolysis Effects 0.000 description 3
- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 description 3
- 230000001717 pathogenic effect Effects 0.000 description 3
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 3
- 238000012795 verification Methods 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 description 2
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- 125000000539 amino acid group Chemical group 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 230000002924 anti-infective effect Effects 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
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- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 description 2
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- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 description 1
- JFLSOKIMYBSASW-UHFFFAOYSA-N 1-chloro-2-[chloro(diphenyl)methyl]benzene Chemical compound ClC1=CC=CC=C1C(Cl)(C=1C=CC=CC=1)C1=CC=CC=C1 JFLSOKIMYBSASW-UHFFFAOYSA-N 0.000 description 1
- 102000044503 Antimicrobial Peptides Human genes 0.000 description 1
- 108700042778 Antimicrobial Peptides Proteins 0.000 description 1
- 241000255789 Bombyx mori Species 0.000 description 1
- 241000195493 Cryptophyta Species 0.000 description 1
- 206010013654 Drug abuse Diseases 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 241000276427 Poecilia reticulata Species 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
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- 230000000843 anti-fungal effect Effects 0.000 description 1
- 229940121375 antifungal agent Drugs 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 208000022362 bacterial infectious disease Diseases 0.000 description 1
- 239000003899 bactericide agent Substances 0.000 description 1
- 239000000022 bacteriostatic agent Substances 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
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- 238000009395 breeding Methods 0.000 description 1
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- NWFNSTOSIVLCJA-UHFFFAOYSA-L copper;diacetate;hydrate Chemical compound O.[Cu+2].CC([O-])=O.CC([O-])=O NWFNSTOSIVLCJA-UHFFFAOYSA-L 0.000 description 1
- 238000010511 deprotection reaction Methods 0.000 description 1
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- 239000002778 food additive Substances 0.000 description 1
- 235000013373 food additive Nutrition 0.000 description 1
- 230000000855 fungicidal effect Effects 0.000 description 1
- 239000000417 fungicide Substances 0.000 description 1
- 239000002955 immunomodulating agent Substances 0.000 description 1
- 229940121354 immunomodulator Drugs 0.000 description 1
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- JEIPFZHSYJVQDO-UHFFFAOYSA-N iron(III) oxide Inorganic materials O=[Fe]O[Fe]=O JEIPFZHSYJVQDO-UHFFFAOYSA-N 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
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- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000012452 mother liquor Substances 0.000 description 1
- CMWYAOXYQATXSI-UHFFFAOYSA-N n,n-dimethylformamide;piperidine Chemical compound CN(C)C=O.C1CCNCC1 CMWYAOXYQATXSI-UHFFFAOYSA-N 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 150000002825 nitriles Chemical class 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 230000010355 oscillation Effects 0.000 description 1
- 150000003053 piperidines Chemical class 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 230000028706 ribosome biogenesis Effects 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 230000011664 signaling Effects 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
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- 231100000419 toxicity Toxicity 0.000 description 1
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
-
- 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
- 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
- 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
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Polymers & Plastics (AREA)
- Health & Medical Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Food Science & Technology (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- General Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Gastroenterology & Hepatology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Animal Husbandry (AREA)
- Medicinal Chemistry (AREA)
- Biophysics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Mycology (AREA)
- Nutrition Science (AREA)
- Cosmetics (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
This application discloses a kind of antibacterial peptide VK-21 of field of biotechnology, the amino acid sequences of the antibacterial peptide are as follows: Val-Lys-Arg-Lys-Lys-Lys-Pro-Gln-Ser-Trp-Lys-Thr-Trp-Trp- Thr-Lys-Trp-Trp-Thr-Lys-Lys.Antibacterial peptide of the invention not only has apparent inhibiting effect to Escherichia coli, pseudomonas aeruginosa, staphylococcus aureus and four kinds of methicillin-resistant staphylococcus aureus, and also there is good fungistatic effect to vibrio parahaemolytious ever-present in aquatic products and vibrio harveyi, there is very low hemolytic activity simultaneously, stability is good, antibacterial activity is strong, with high-efficiency broad spectrum bacteriostasis, Substitutes For Antibiotic use can be used as.
Description
Technical field
The present invention relates to a kind of small peptides of field of biotechnology, and in particular to a kind of antibacterial peptide VK-21 and its application.
Background technique
Aquatic products are the animal produced in ocean, rivers and lake or the general designation of algae, due to life movable in water body
Species are more, and water is also the medium of germ easy to breed, so also often inevitably contain pathogenic bacteria in aquatic products, example
It is pathogenic bacteria common in aquatic products such as vibrio parahaemolytious and vibrio harveyi, it is pathogenic or even extensive dead frequently results in aquatic products
It dies, causes huge economic losses to culture fishery.Currently used bacteriostatic agent is substantially chemical bactericide, but uses chemistry
Fungicide is inhibited, then can be polluted to ecological environment, also can inevitably be remained in aquatic products, and the safety of such people and animals is all
It will receive threat.
Generation when antibacterial peptide (Antimicrobial peptides, AMPs) is organism defence external world's pathogen invasion
Encoded by gene, the small molecule active peptides of Ribosome biogenesis are the important sets of innate defence system in organism
At ingredient.AMPs generally has antibacterium, fungi, virus, protozoon isoreactivity as a kind of bioactive small molecule.In addition,
AMPs is also used as drug carrier system, antitumor agent, immunomodulator and signaling molecule etc..
Antibiotic is as a kind of important anti-infection drug, extensively by application and clinical, animal husbandry and aquaculture
Equal fields, but in recent years, since the problems such as drug abuse medicament residue and bacterial drug resistance, gets worse, more and more countries
It is sought for Substitutes For Antibiotic, and AMPs is because of its unique bioactivity and different from the special effect of conventional antibiotic
Mechanism, it has also become one of most potential Substitutes For Antibiotic, and meanwhile it is anti-in novel foodstuff, drug, skin care item and cosmetics
The application of rotten agent and feed addictive etc. is also more and more extensive, has good development prospect.
The correlative study of AMPs can trace back to 1975 earliest, and Sweden scientist G.Bomam etc. was cherishing guppy day at that time
Escherichia coli are injected in silkworm chrysalis, have found a kind of basic polypeptide class object with antibacterial activity in its blood lymphocytes later
Matter, i.e. antibacterial peptide Ceropins.By research in forties years, had found from animal, plant, bacterium and virus at present super
Cross 2500 kinds of antibacterial peptides.
Although natural A MPs has the advantages that universal, there is also certain clearly disadvantageous.Quite a few is natural
Antibacterial peptide bacteriostatic activity is lower, stability is poor, toxicity is higher, or causes eukaryocyte that haemolysis etc. occurs；In addition, part
AMPs is poor to the inhibitory effect of drug-fast bacteria, is not able to satisfy the requirement of practical application；And by the way that natural A MPs is transformed or is led to
Design and rational is crossed, completely new artificial AMPs is synthesized, certain of the above even all disadvantages can be improved, largely to adapt to
Different application demand.
Summary of the invention
The present invention is intended to provide the inhibiting effect of a kind of pair of drug-fast bacteria is strong, it can effectively inhibit vibrio parahaemolytious and Kazakhstan arc
The antibacterial peptide VK-21 that bacterium breeds.
In order to achieve the above object, the present invention provides following basic technology scheme: a kind of antibacterial peptide VK-21, the antibacterial
The amino acid sequence of peptide VK-21 are as follows:
Val-Lys-Arg-Lys-Lys-Lys-Pro-Gln-Ser-Trp-Lys-Thr-Trp-Trp-Thr-Lys-Trp-
Trp-Thr-Lys-Lys。
In antibacterial peptide VK-21 of the invention, V and K are the abbreviations of small peptide the first two amino acid, and 21 be amino acid number, for this
The conventional naming method in field.
Antibacterial peptide of the invention is not only to Escherichia coli, pseudomonas aeruginosa, staphylococcus aureus and four kinds of resistance to methoxies
XiLin staphylococcus aureus has apparent inhibiting effect, and to vibrio parahaemolytious ever-present in aquatic products and Kazakhstan arc
Bacterium also has good fungistatic effect, while having very low hemolytic activity, and stability is good, and antibacterial activity is strong, has high-efficiency broad spectrum
Bacteriostasis can be used as Substitutes For Antibiotic use.
It is the optimization to basic technology scheme below:
Prioritization scheme one: the antibacterial peptide VK-21 is α spiral straight-chain polypeptide, contains 21 amino acid residues, and molecular weight is big
Small is 2845.46Da, isoelectric point 11.51.
Prioritization scheme two, be based on base case or prioritization scheme one: the antibacterial peptide VK-21 is in preparing antibacterial agent
Using.Because antibacterial peptide VK-21 provided by the invention has stronger fungistatic effect, and to common Escherichia coli, verdigris
Pseudomonad, staphylococcus aureus and four kinds of methicillin-resistant staphylococcus aureus have apparent inhibiting effect, so its
It can be used for preparing antibacterial agent, in order to use.
Prioritization scheme three, be based on base case or prioritization scheme one: the antibacterial peptide VK-21 is preparing feed addictive
In application.Because antibacterial peptide VK-21 provided by the invention also there is stronger inhibition to make vibrio parahaemolytious and vibrio harveyi
With, and both germs be commonly present in aquatic products, it is possible to antibacterial peptide VK-21 is added in aquatic feeds, feeding is passed through
Process the growth of vibrio parahaemolytious and vibrio harveyi breeding is effectively inhibited, prevent aquatic products from infecting or even illness
It is dead.
Prioritization scheme four, is based on base case or prioritization scheme one: the antibacterial peptide VK-21 in food additives or
Application in cosmetics preservative.
Specific embodiment
Below with reference to embodiment, technical scheme of the present invention is further explained:
Comparative example: on October 12nd, 2016 announce application No. is a kind of 2016106321452 Chinese patents " antibacterial peptides
SE37 and its application " as a comparison, the amino acid sequence of the antibacterial peptide of the patent are as follows:
Ser-Glu-Thr-Arg-Pro-Val-Leu-Asn-Arg-Leu-Phe-Asp-Lys-Ile-Arg-Gln-Val-
Ile-Arg-Lys-Phe-Glu-Lys-Gly-Ile-Lys-Glu-Lys-Ser-Lys-Arg-Phe-Phe-Asp-Gly-Leu-
Leu。
Antibacterial peptide SE37 be α spiral straight-chain polypeptide, contain 37 amino acid residues, molecular size range 4504.40Da, etc.
Electricity point is 11.34.
Patent antibacterial peptide SE37 can also be to Escherichia coli, pseudomonas aeruginosa, staphylococcus aureus, four kinds of resistance to methoxies
XiLin staphylococcus aureus has apparent inhibiting effect.
Embodiment: antibacterial peptide VK-21 product of the present invention is the amino acid sequence in SEQ ID NO. 1, and sequence information is
Val-Lys-Arg-Lys-Lys-Lys-Pro-Gln-Ser-Trp-Lys-Thr-Trp-Trp-Thr-Lys-Trp-Trp-Thr-
Lys-Lys 。
Sequence signature: length 21, type are amino acid sequence, and chain is straight chain, artificial synthesized.
The present embodiment antibacterial peptide VK-21 product is using automatic Peptide synthesizer according to conventional polypeptide synthesis in solid state, finally
Obtained antibacterial peptide VK-21 is through efficient liquid phase chromatographic analysis, purity >=98%.Its specific preparation step is as follows:
1) resin swelling: the 0.6g 2-Chlorotrityl Chloride Resin that degree of substitution is 0.4mmol/g is weighed
Resin is put into reaction tube by resin, adds DCM(15mL/g), vibrate 30min.
2) it connects first amino acid: solvent being leached out by husky core, the Fmoc-L-Lys of 3 times of molar excess is added
(Boc)-OH amino acid, HBTU three times are excessive, add the DIEA of 10 times of molar excess, are eventually adding a small amount of DMF dissolution, oscillation
1h.With DMF and DCM alternately cleaning 6 times.
3) it detects: taking a small amount of resin, ninhydrin, pyridine, each 3 drop of phenol solution, 2 drops, 2 drops, 110 degree of heating are added
3min, color of resin is transparent, illustrates that Fmoc-L-Lys (Boc)-OH reaction is upper.
4) it is deprotected: adding 20% Piperidine/DMF solution of 15mL (15mL/g), 5min removes and adds 20% piperidines DMF of 15mL again
Solution (15mL/g), 15min.Piperidine solution is taken out, 6 times is washed with DMF and drains.
6) it detects: taking a small amount of resin, ninhydrin, pyridine, each 3 drop of phenol solution, 2 drops, 2 drops, 110 degree of heating are added
3min, resin deepen blue to have removed Fmoc.
6) be condensed: protected amino acid Fmoc-L-Lys (Boc)-OH three times are excessive, and HBTU three times are excessive, and use is few as far as possible
DMF dissolution, is added reaction tube, is added immediately ten times of excess of DIEA, reacts 30 min.With DMF and DCM alternately cleaning 6 times.
7) it detects: taking a small amount of resin, ninhydrin, pyridine, each 3 drop of phenol solution, 2 drops, 2 drops, 110 degree of heating are added
3min, color of resin is transparent, illustrates that Fmoc-L-Lys (Boc)-OH reaction is upper.
8) operation of two to seven steps, the amino acid being sequentially connected in sequence from right to left are repeated.
9) after the connection of the last one amino acid, deprotection washes resin: DMF(10mL/g in following manner) twice, DCM
(10mL/g) twice, methanol (10mL/g) is twice.Drain 10min.
10) polypeptide is cut from resin: preparing cutting liquid (10mL/g) TFA 95%；2.5% TIS 2.5% of water, will set
Rouge is fitted into flask or centrifuge tube, and resin and cutting liquid proportional are according to 10mL/g, isothermal vibration, time: 120min.
11) drying washing: lysate being dried up as far as possible with nitrogen, is separated out with ether layer, then washes six times with ether, so
Room temperature volatilizes afterwards.Up to crude product peptide sequence.
12) HPLC purified polypeptide is used:
(1) crude product peptide 200mg is taken to be put into vessel, it, can be slightly ultrasonic with the acetonitrile solution dissolved clarification of 2-5mL 50%
2min。
(2) with 0.45 μm of membrane filtration lysate.
(3) it analyzes: 3 μ L being taken to analyze crude product with analysis level HPLC.Mobile phase is water and acetonitrile, time 30min, and gradient is washed
It is de-, HPLC start gradient is first balanced into 5min then sample introduction, start gradient water 95%, acetonitrile 5% terminates ratio water 5%, acetonitrile
95%
(4) prepare: the sample that will have been dissolved does sample introduction preparation.It prepares HPLC and balances 10min, start gradient water 95%, second
Nitrile 5% terminates gradient water 25%, 75% gradient timetable 40min of acetonitrile.Collect the sample come out from detector.
(5) identify: the sample that will be collected, sampling carry out the identification of purity and MS.
13) solution after purification is lyophilized, obtains finished product.
14) by the polypeptide of white powder, sealed package, -20 degree preservations.
Verification test one:
The minimal inhibitory concentration (MIC) of antibacterial peptide VK-21 and antibacterial peptide SE37 are measured:
Respectively by Escherichia coli (ATCC8739), pseudomonas aeruginosa (CMCC10104), staphylococcus aureus
(ATCC6538) logarithmic phase is cultivated, is diluted to 2 × 10 with 2 × liquid MHB culture medium5CFU/mL.It is sequentially added in 96 orifice plates
It is diluted to the 50 μ L of antibacterial peptide mother liquor of gradient, the 50 μ L of bacterium solution diluted is added into each hole, is trained after mixing in 37 DEG C of standings
It supports 16 hours, the absorbance value at 600nm is measured after concussion, does positive control with 100 μ g/mL ampicillins.Result judgement:
Take the hole that can't detect bacterial growth as minimal inhibitory concentration.The results are shown in Table 1.
The fungistatic effect of table 1 antibacterial peptide VK-21 and antibacterial peptide SE37
As can be seen from the above table, VK-21 and antibacterial peptide SE37 have fungistatic effect to Gram-negative and positive bacteria,
Show that inhibitory effect is best to the antibacterial peptide SE37 of staphylococcus aureus, still, the antibacterial peptide of embodiment group known to comparison
The MIC value of VK-21 is significantly less than the antibacterial peptide SE37 of comparative example group, illustrates that the fungistatic effect of antibacterial peptide VK-21 compares antibacterial peptide
The fungistatic effect of SE37 is more significant, it is seen that antibacterial peptide VK-21 of the present invention has preferable research and development value.
Verification test two:
The hemolytic activity of product of the present invention antibacterial peptide VK-21 detects:
1) fresh mouse blood is acquired, to stratification, upper serum is removed, physiological saline is added, gently with suction pipe
The red blood cell of tube bottom is dispelled, 1000 rpm are centrifuged 5 min, upper layer physiological saline is carefully drawn with suction pipe and is discarded, until supernatant
Liquid is without red.
2) red blood cell 2 for taking bottom to be compacted drips, and red blood cell is resuspended in the isotonic PBS that 2.0mL is added, and it is blood red thin to be configured to 4%
Born of the same parents' suspension.
3) experimental group: being added 50 μ L various concentrations, then the antibacterial peptide dissolved with isotonic PBS is added 50 μ L and configures
4% red blood cell suspension.
4) positive control: Triton configured 4% erythrocyte of X-100,50 μ L that 50 μ L2% are added in each hole is outstanding
Liquid.Negative control: the isotonic PBS of 50 μ L, configured 4% red blood cell suspension of 50 μ L is added in each hole.
5) after 37 DEG C of 1 h of incubation, after 1 000 g are centrifuged 96 orifice plate, 5 min, 50 μ L supernatants to 96 orifice plates are drawn from each hole
In, 415 nm wavelength measure OD value, calculate percent hemolysis=[(experimental port OD value-negative hole OD value)/(positive hole OD value-yin
Property hole OD value)] × 100.
The result shows that antibacterial peptide VK-21 is about 2.46% to the hemolysis rate of red blood cell when concentration reaches 352 μM；Explanation
Antibacterial peptide VK-21 of the present invention influences less the brittleness of seeing through of red blood cell, and safety is very high.
Confirmatory experiment three:
The heat stability test of product of the present invention antibacterial peptide VK-21:
1) it is real to carry out thermal stability for a kind of selection antibacterial peptide bacterium (staphylococcus aureus) best to its fungistatic effect
It tests.
2) picking bacterium single bacterium is fallen in the LB culture medium of 5 mL, 37 DEG C of 200 12 h of rpm shaking table culture.
3) concentration is higher than the antibacterial peptide of MIC value certain multiple respectively at 100 DEG C of 0 min of water bath processing, 30 min, 60
Min and 90 min takes out the MIC value for measuring sample after being cooled to room temperature respectively.
The results show that VK21 is through 100 DEG C of water bath processing 60min, bacteriostatic activity is unaffected, and thermal stability is fabulous.
Confirmatory experiment four:
Respectively with antibacterial peptide VK-21 and antibacterial peptide SE37 to the Antibacterial Activity for being clinically separated antibody-resistant bacterium:
Using front MIC value measuring method, antibacterial peptide VK-21 and antibacterial peptide SE37 is measured respectively, four plants of clinical examinations are obtained
The bacteriostatic activity of the methicillin-resistant staphylococcus aureus bacterial strain arrived, the results are shown in Table 2.
Table 2 antibacterial peptide VK-21 and antibacterial peptide SE37 is to methicillin-resistant staphylococcus aureus fungistatic effect
Antibacterial peptide VK-21 is than antibacterial peptide SE37 to 4 plants of methicillin-resistant staphylococcus aureus bacterium as can be seen from the above table
The fungistatic effect of strain is more preferable, has good Substitutes For Antibiotic drug development research value.
Verification test five:
Antibacterial Activity of the product of the present invention antibacterial peptide VK-21 to culture fishery common pathogen:
Using front MIC value measuring method, VK-21 is measured respectively to aquaculture pathogenic bacteria vibrio parahaemolytious and Kazakhstan arc
Bacteriostatic activity of the bacteria strain in the case where salinity is respectively 0.5% and 1% condition of culture, the results are shown in Table 3.
Fungistatic effect of the 3 antibacterial peptide VK-21 of table to vibrio parahaemolytious and vibrio harveyi
As can be seen from the above table, antibacterial peptide VK-21 to aquaculture pathogenic bacteria vibrio parahaemolytious and vibrio harveyi bacterial strain all
There is fabulous fungistatic effect, even if fungistatic effect is still fine when salinity reaches 1%, there is the addition of good aquatic feeds
Agent Development volue.
In conclusion antibacterial peptide product cell hemolytic of the present invention is low, has a broad antifungal spectrum, to gram-positive bacteria and gram
Negative bacterium all has good antibacterial action.So product of the present invention antibacterial peptide VK-21 is preparing anti-infective gram-positive bacteria
Or/and it can preferably be applied in Gram negative bacterial disease drug, while in aquaculture pathogenic bacteria prevention and treatment preparation
It can obtain good application.
The foregoing is only a preferred embodiment of the present invention, is not intended to restrict the invention, although referring to aforementioned reality
Applying example, invention is explained in detail, for those skilled in the art, still can be to aforementioned each implementation
Technical solution documented by example is modified or equivalent replacement of some of the technical features.It is all in essence of the invention
Within mind and principle, any modification, equivalent substitution, improvement and etc. done be should all be included in the protection scope of the present invention.
Claims (5)
1. a kind of antibacterial peptide VK-21, which is characterized in that the amino acid sequence of the antibacterial peptide VK-21 are as follows:
Val-Lys-Arg-Lys-Lys-Lys-Pro-Gln-Ser-Trp-Lys-Thr-Trp-Trp-Thr-Lys-Trp-Trp-
Thr-Lys-Lys。
2. the application of antibacterial peptide VK-21 as described in claim 1, it is characterised in that: the antibacterial peptide VK-21 is treated in preparation
Application in the extensive pedigree antibiotic of gram-positive bacteria or gram positive bacterial infection.
3. the application of antibacterial peptide VK-21 as described in claim 1, it is characterised in that: the antibacterial peptide VK-21 is in preparation antibacterial
Application in agent.
4. the application of antibacterial peptide VK-21 as described in claim 1, it is characterised in that: the antibacterial peptide VK-21 is preparing feed
Application in additive.
5. the application of antibacterial peptide VK-21 as described in claim 1, it is characterised in that: the antibacterial peptide VK-21 is added in food
Application in agent or cosmetics preservative.
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| CN110317248B (en) * | 2019-06-05 | 2020-12-29 | 遵义医科大学珠海校区 | Artificially synthesized antibacterial peptide and design method and application thereof |
| CN111253474B (en) * | 2019-10-10 | 2023-07-07 | 应连心 | Antibacterial peptide RG-27 and application thereof |
| CN113201057B (en) * | 2021-03-16 | 2022-10-21 | 广东佰欧斐丝细胞科研中心有限公司 | Deep-sea top clam antibacterial protein and application thereof |
| CN115246878B (en) * | 2021-03-31 | 2024-02-13 | 禾美生物科技(浙江)有限公司 | Antibacterial peptide and application thereof in cosmetics |
| CN113307850B (en) * | 2021-06-09 | 2022-03-29 | 温州大学 | Antibacterial peptide, composition containing same and application |
| CN113880933B (en) * | 2021-11-12 | 2023-10-20 | 青岛农业大学 | Antibacterial peptide SsNKL27 and application thereof |
| CN116023431B (en) * | 2022-07-26 | 2024-03-15 | 青岛大学 | Antibacterial short peptide and application thereof |
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| CN1609121A (en) * | 2003-10-17 | 2005-04-27 | 上海高科联合生物技术研发有限公司 | Antibiotic peptides and their prepn process and application |
| CN103333225A (en) * | 2013-04-11 | 2013-10-02 | 浙江大学 | Antibacterial peptide, preparation method and applications thereof |
| CN105294838A (en) * | 2015-09-22 | 2016-02-03 | 徐州市玛泰生物科技有限公司 | Antibacterial peptide and application thereof |
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| CN1609121A (en) * | 2003-10-17 | 2005-04-27 | 上海高科联合生物技术研发有限公司 | Antibiotic peptides and their prepn process and application |
| CN103333225A (en) * | 2013-04-11 | 2013-10-02 | 浙江大学 | Antibacterial peptide, preparation method and applications thereof |
| CN105294838A (en) * | 2015-09-22 | 2016-02-03 | 徐州市玛泰生物科技有限公司 | Antibacterial peptide and application thereof |
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