CN107383175A - 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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- CN107383175A CN107383175A CN201710779621.8A CN201710779621A CN107383175A CN 107383175 A CN107383175 A CN 107383175A CN 201710779621 A CN201710779621 A CN 201710779621A CN 107383175 A CN107383175 A CN 107383175A
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- antibacterial peptide
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- 239000003910 polypeptide antibiotic agent Substances 0.000 title claims abstract description 72
- 230000003115 biocidal effect Effects 0.000 claims abstract description 8
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 6
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 15
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 10
- 229920001184 polypeptide Polymers 0.000 claims description 8
- 241000192125 Firmicutes Species 0.000 claims description 3
- 125000000539 amino acid group Chemical group 0.000 claims description 3
- 230000002421 anti-septic effect Effects 0.000 claims description 3
- 239000002537 cosmetic Substances 0.000 claims description 3
- 208000035143 Bacterial infection Diseases 0.000 claims description 2
- 239000000654 additive Substances 0.000 claims description 2
- 230000000996 additive effect Effects 0.000 claims description 2
- 239000003755 preservative agent Substances 0.000 claims description 2
- 230000002335 preservative effect Effects 0.000 claims description 2
- 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
- 230000000844 anti-bacterial effect Effects 0.000 abstract description 9
- 241000607618 Vibrio harveyi Species 0.000 abstract description 7
- 230000002401 inhibitory effect Effects 0.000 abstract description 7
- 241000588724 Escherichia coli 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
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 18
- 239000000047 product Substances 0.000 description 16
- 241000894006 Bacteria Species 0.000 description 12
- 229920005989 resin Polymers 0.000 description 11
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- 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
- 238000012913 prioritisation Methods 0.000 description 7
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 6
- 244000052616 bacterial pathogen Species 0.000 description 6
- 241001465754 Metazoa Species 0.000 description 5
- 230000001580 bacterial effect Effects 0.000 description 5
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 4
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 description 4
- 238000009360 aquaculture Methods 0.000 description 4
- 244000144974 aquaculture Species 0.000 description 4
- 230000003385 bacteriostatic effect Effects 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 239000003814 drug Substances 0.000 description 4
- 238000004128 high performance liquid chromatography Methods 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 238000002360 preparation 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
- 238000002474 experimental method Methods 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
- 238000006467 substitution reaction Methods 0.000 description 3
- 238000012360 testing method 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
- 241000700605 Viruses Species 0.000 description 2
- 230000002924 anti-infective effect Effects 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 239000003899 bactericide agent Substances 0.000 description 2
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- 230000005764 inhibitory process Effects 0.000 description 2
- 239000006166 lysate Substances 0.000 description 2
- 238000000034 method Methods 0.000 description 2
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 description 2
- 244000052769 pathogen Species 0.000 description 2
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- 150000003384 small molecules Chemical class 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 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
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 1
- 241000276427 Poecilia reticulata Species 0.000 description 1
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- 208000022362 bacterial infectious disease Diseases 0.000 description 1
- 239000000022 bacteriostatic agent Substances 0.000 description 1
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- 230000005494 condensation Effects 0.000 description 1
- NWFNSTOSIVLCJA-UHFFFAOYSA-L copper;diacetate;hydrate Chemical compound O.[Cu+2].CC([O-])=O.CC([O-])=O NWFNSTOSIVLCJA-UHFFFAOYSA-L 0.000 description 1
- 238000013461 design Methods 0.000 description 1
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- 239000002955 immunomodulating agent Substances 0.000 description 1
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- 230000002584 immunomodulator Effects 0.000 description 1
- 230000009545 invasion Effects 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
- 239000013642 negative control Substances 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 150000003053 piperidines Chemical class 0.000 description 1
- 239000000843 powder Substances 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
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- 230000028706 ribosome biogenesis Effects 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 biological technical field, the amino acid sequence of the antibacterial peptide is：Val‑Lys‑Arg‑Lys‑Lys‑Lys‑Pro‑Gln‑Ser‑Trp‑Lys‑Thr‑Trp‑Trp‑Thr‑Lys‑Trp‑Trp‑Thr‑Lys‑Lys.The antibacterial peptide of the present invention not only has obvious inhibitory action to Escherichia coli, pseudomonas aeruginosa, staphylococcus aureus and four kinds of methicillin-resistant staphylococcus aureus, and also there is good fungistatic effect to ever-present vibrio parahaemolytious 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, can be used as Substitutes For Antibiotic.
Description
Technical field
The present invention relates to a kind of small peptide of biological technical field, and in particular to a kind of antibacterial peptide VK-21 and its application.
Background technology
Aquatic products are the general designations for the animal or algae produced in ocean, rivers and lake, due to life movable in water body
Species are more, and water is also the medium of easy growth germ, 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
Die, huge economic losses are caused to culture fishery.Currently used bacteriostatic agent is substantially chemical bactericide, but uses chemistry
Bactericide is suppressed, then ecological environment can be polluted, and can also be remained unavoidably in aquatic products, and the safety of such people and animals is all
It can be on the hazard.
Antibacterial peptide (Antimicrobial peptides, AMPs) is that organism is defendd to produce during extraneous pathogen invasion
By gene code, the small molecule active peptides of Ribosome biogenesis, be innate defence system in organism important set
Into composition.AMPs is as a kind of bioactive small molecule, generally with antibacterium, fungi, virus, protozoon isoreactivity.In addition,
AMPs is also used as drug carrier system, antitumor agent, immunomodulator and signaling molecule etc..
Antibiotic is employed and clinical, animal husbandry and aquaculture extensively as a kind of important anti-infection drug
Deng field, but in recent years, getting worse the problems such as due to drug abuse medicament residue and bacterial drug resistance, increasing country
Substitutes For Antibiotic is sought for, and AMPs is because of its unique bioactivity and the special effect different from conventional antibiotic
Mechanism, it has also become one of most potential Substitutes For Antibiotic, while prevent in novel foodstuff, medicine, skin care item and cosmetics
The application of rotten agent and feed addictive etc. is also more and more extensive, has good development prospect.
AMPs correlative study 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, are found that a kind of basic polypeptide class thing with antibacterial activity in its blood lymphocytes afterwards
Matter, i.e. antibacterial peptide Ceropins.By the research of forties years, it has been found that from animal, plant, bacterium and virus at present super
Cross 2500 kinds of antibacterial peptides.
Although natural A MPs has the advantages of universal, there is also some clearly disadvantageous.Quite a few is natural
Antibacterial peptide bacteriostatic activity is relatively low, less stable, toxicity are higher, or causes eukaryotic that haemolysis etc. occurs；In addition, part
AMPs is poor to the inhibition of drug-fast bacteria, it is impossible to meets the requirement of practical application；And by the way that natural A MPs is transformed or led to
Design and rational is crossed, synthesizes brand-new artificial AMPs, can largely improve certain of the above even all shortcomings, to adapt to
Different application demand.
The content of the invention
The present invention is intended to provide a kind of inhibitory action to drug-fast bacteria is strong, it can effectively suppress vibrio parahaemolytious and Kazakhstan arc
The antibacterial peptide VK-21 that bacterium grows.
In order to achieve the above object, the present invention provides following basic technology scheme：A kind of antibacterial peptide VK-21, the antibacterial
Peptide VK-21 amino acid sequence is：
Val-Lys-Arg-Lys-Lys-Lys-Pro-Gln-Ser-Trp-Lys-Thr-Trp-Trp-Thr-Lys-Trp-Trp-
Thr-Lys-Lys。
In the antibacterial peptide VK-21 of the present 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.
The antibacterial peptide of the present invention is not only to Escherichia coli, pseudomonas aeruginosa, staphylococcus aureus and four kinds of resistance to methoxies
XiLin staphylococcus aureus has obvious inhibitory action, and to ever-present vibrio parahaemolytious in aquatic products and Kazakhstan arc
Bacterium also has good fungistatic effect, while has very low hemolytic activity, and stability is good, and antibacterial activity is strong, has high-efficiency broad spectrum
Bacteriostasis, it can be used as Substitutes For Antibiotic.
It is the optimization to basic technology scheme below：
Prioritization scheme one：The antibacterial peptide VK-21 is α spiral straight-chain polypeptides, and containing 21 amino acid residues, molecular size range is
2845.46Da, isoelectric point 11.51.
Prioritization scheme two, based on base case or prioritization scheme one：The antibacterial peptide VK-21 is in antiseptic is prepared
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 obvious inhibitory action, so its
It can be used for preparing antiseptic, in order to use.
Prioritization scheme three, 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 suppression to make vibrio parahaemolytious and vibrio harveyi
With, and both germs be commonly present with aquatic products, it is possible to antibacterial peptide VK-21 is added in aquatic feeds, passes through feeding
Process to vibrio parahaemolytious and vibrio harveyi growth breeding effectively suppressed, prevent aquatic products from infecting, in addition illness
It is dead.
Prioritization scheme four, based on base case or prioritization scheme one：The antibacterial peptide VK-21 in food additives or
Application in cosmetics preservative.
Embodiment
Technical solution of the present invention is described further with reference to embodiment：
Comparative example：The Chinese patent of Application No. 2016106321452 announced with October 12nd, 2016《A kind of antibacterial peptide SE37
And its application》As a comparison, the amino acid sequence of the antibacterial peptide of the patent is：
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 is α spiral straight-chain polypeptides, containing 37 amino acid residues, molecular size range 4504.40Da, etc.
Electricity point is 11.34.
Patent antibacterial peptide SE37 also can be to Escherichia coli, pseudomonas aeruginosa, staphylococcus aureus, four kinds of resistance to methoxies
XiLin staphylococcus aureus has obvious inhibitory action.
Embodiment：Antibacterial peptide VK-21 products of the present invention are the amino acid sequence in SEQ ID NO. 1, and its 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 is 21, and type is amino acid sequence, and chain is straight chain, artificial synthesized.
The present embodiment antibacterial peptide VK-21 products are according to conventional polypeptide synthesis in solid state, finally using automatic Peptide synthesizer
Obtained antibacterial peptide VK-21 is through efficient liquid phase chromatographic analysis, its purity >=98%.Its specific preparation process is as follows：
1）Resin swelling：Weigh the 0.6g 2-Chlorotrityl Chloride Resin trees that substitution value is 0.4mmol/g
Fat, resin is put into reaction tube, adds DCM（15mL/g）, vibrate 30min.
2）Connect first amino acid：Solvent is leached out by husky core, adds the Fmoc-L-Lys of 3 times of molar excess
(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 dissolvings, vibration
1h.With DMF and DCM alternately cleaning 6 times.
3）Detection：A small amount of resin is taken, adds ninhydrin, pyridine, each 3 drop of phenol solution, 2 drops, 2 drops, 110 degree of heating
3min, color of resin is transparent, illustrates that Fmoc-L-Lys (Boc)-OH reactions are upper.
4）Deprotection：Add the Piperidine/DMF solutions of 15mL 20%（15mL/g）, 5min, remove and add the piperidines DMF of 15mL 20% again
Solution（15mL/g）, 15min.Piperidine solution is taken out, 6 times is washed with DMF and drains.
6）Detection：A small amount of resin is taken, adds ninhydrin, pyridine, each 3 drop of phenol solution, 2 drops, 2 drops, 110 degree of heating
3min, resin become navy blue to have removed Fmoc.
6）Condensation：Protected amino acid Fmoc-L-Lys (Boc)-OH three times are excessive, and HBTU three times are excessive, with as far as possible few
DMF dissolves, and adds reaction tube, is added immediately ten times of excess of DIEA, reacts 30 min.With DMF and DCM alternately cleaning 6 times.
7）Detection：A small amount of resin is taken, adds ninhydrin, pyridine, each 3 drop of phenol solution, 2 drops, 2 drops, 110 degree of heating
3min, color of resin is transparent, illustrates that Fmoc-L-Lys (Boc)-OH reactions are upper.
8）Repeat the operation of two to seven steps, the amino acid being sequentially connected from right to left in sequence.
9）After the connection of last amino acid, deprotection, resin is washed in following manner：DMF（10mL/g）Twice, DCM
（10mL/g）Twice, methanol（10mL/g）Twice.Drain 10min.
10）Polypeptide is cut from resin：Prepare cutting liquid（10mL/g） TFA 95%；The TIS 2.5% of water 2.5%, will set
Fat 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 is dried up as far as possible with nitrogen, separated out with ether layer, then six times are washed with ether, so
Normal temperature volatilizes afterwards.Produce crude product peptide sequence.
12）With HPLC purified polypeptides:
(1) crude product peptide 200mg is taken to be put into vessel, can somewhat ultrasonic 2min with 2-5mL 50% acetonitrile solution dissolved clarification.
(2) with 0.45 μm of membrane filtration lysate.
(3) analyze：3 μ L are 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 is first balanced into 5min and then sample introduction with start gradient, start gradient water 95%, acetonitrile 5%, terminates ratio water 5%, acetonitrile
95%
(4) prepare：The sample that will have been dissolved, do sample introduction preparation.Preparation HPLC balance 10min, start gradient water 95%, acetonitrile 5%,
Terminate gradient water 25%, the gradient timetable 40min of acetonitrile 75%.Collect the sample come out from detector.
(5) identify：The sample that will be collected, sampling carry out purity and MS identification.
13）Solution after purification is freezed, obtains finished product.
14）By the polypeptide of white powder, pack, -20 degree preserve.
Checking test one：
To antibacterial peptide VK-21 and antibacterial peptide SE37 minimal inhibitory concentration（MIC）It is measured：
Respectively by Escherichia coli（ATCC8739）, pseudomonas aeruginosa (CMCC10104), staphylococcus aureus (ATCC6538)
Logarithmic phase is cultivated, 2 × 10 are diluted to 2 × liquid MHB culture mediums5CFU/mL.Sequentially added in 96 orifice plates and be diluted to gradient
The μ L of antibacterial peptide mother liquor 50, the μ L of bacterium solution 50 diluted are added into each hole, are mixed after 37 DEG C of quiescent cultures 16 hours, shake
The absorbance value at 600nm is determined after swinging, positive control is done with 100 μ g/mL ampicillins.Result judgement：Take and can't detect
The hole of bacterial growth is as minimal inhibitory concentration.As a result it is as shown in table 1.
The antibacterial peptide VK-21 of table 1 and antibacterial peptide SE37 fungistatic effect
As can be seen from the above table, VK-21 and antibacterial peptide SE37 have fungistatic effect to Gram-negative and positive bacteria, show
In the antibacterial peptide SE37 to staphylococcus aureus, inhibition is best, and still, contrast understands the antibacterial peptide VK- of embodiment group
21 MIC value is significantly less than the antibacterial peptide SE37 of comparative example group, illustrates antibacterial peptide VK-21 fungistatic effect than antibacterial peptide SE37
Fungistatic effect it is more notable, it is seen that antibacterial peptide VK-21 of the present invention have preferably research and development be worth.
Checking test two：
Product antibacterial peptide VK-21 of the present invention hemolytic activity detection：
1）Fresh mouse blood is gathered, treats stratification, removes upper serum, physiological saline is added, is gently dispelled with suction pipe
The red blood cell of ttom of pipe, 1000 rpm centrifuge 5 min, upper strata physiological saline are carefully drawn with suction pipe and is discarded, until supernatant does not have
There is red.
2）The red blood cell 2 for taking bottom to be compacted drips, and red blood cell is resuspended in the isotonic PBS for adding 2.0mL, and it is blood red thin to be configured to 4%
Born of the same parents' suspension.
3）Experimental group：50 μ L various concentrations are added, the antibacterial peptide dissolved with isotonic PBS, 50 μ L is then added and configures
4% red blood cell suspension.
4）Positive control：Each hole adds 50 μ L2% Triton X-100, and 4% erythrocyte that 50 μ L have been configured hangs
Liquid.Negative control：Each hole adds the isotonic PBS of 50 μ L, 4% red blood cell suspension that 50 μ L have been configured.
5）After 37 DEG C are incubated 1 h, after 1 000 g centrifugation 96 orifice plates 5 min, 50 μ L of supernatant to 96 orifice plates are drawn from each hole
In, 415 nm wavelength measure OD values, calculating percent hemolysis=[（Experimental port OD values-negative hole OD values）/（Positive hole OD values-the moon
Property hole OD values）]×100.
As a result show, when concentration reaches 352 μM, antibacterial peptide VK-21 is about 2.46% to the hemolysis rate of red blood cell；Explanation
Antibacterial peptide VK-21 of the present invention influences less on the fragility of seeing through of red blood cell, and security is very high.
Confirmatory experiment three：
Product antibacterial peptide VK-21 of the present invention heat stability test：
1）Select a kind of antibacterial peptide bacterium best to its fungistatic effect（Staphylococcus aureus）Carry out heat endurance experiment.
2）Picking bacterium single bacterium is fallen within 5 mL LB culture mediums, 37 DEG C of 200 h of rpm shaking table cultures 12.
3）By antibacterial peptide of the concentration higher than MIC value certain multiple respectively at 100 DEG C of min of water bath processing 0,30 min, 60
Min and 90 min, take out and be cooled to after room temperature the MIC value of determination sample respectively.
As a result show, VK21 is through 100 DEG C of water bath processing 60min, and its bacteriostatic activity is unaffected, and heat endurance is fabulous.
Confirmatory experiment four：
Respectively with antibacterial peptide VK-21 and antibacterial peptide SE37 to being clinically separated the Antibacterial Activity of antibody-resistant bacterium：
Using above MIC value assay method, determine what antibacterial peptide VK-21 and antibacterial peptide SE37 obtained to four plants of clinical examinations respectively
The bacteriostatic activity of methicillin-resistant staphylococcus aureus bacterial strain, the results are shown in Table 2.
The antibacterial peptide VK-21 of table 2 and antibacterial peptide SE37 are to methicillin-resistant staphylococcus aureus fungistatic effect
As can be seen from the above table antibacterial peptide VK-21 than antibacterial peptide SE37 to 4 plants of methicillin-resistant staphylococcus aureus bacterial strains
Fungistatic effect is more preferable, and there is good Substitutes For Antibiotic drug development research to be worth.
Checking test five：
Antibacterial Activities of the product antibacterial peptide VK-21 of the present invention to culture fishery common pathogen：
Using above MIC value assay method, VK-21 is determined respectively to aquaculture pathogenic bacteria vibrio parahaemolytious and vibrio harveyi bacterium
Bacteriostatic activity of the strain in the case where salinity is respectively 0.5% and 1% condition of culture, the results are shown in Table 3.
The antibacterial peptide VK-21 of table 3 is to vibrio parahaemolytious and the fungistatic effect of vibrio harveyi
As can be seen from the above table, antibacterial peptide VK-21 has pole to aquaculture pathogenic bacteria vibrio parahaemolytious and vibrio harveyi bacterial strain
Good fungistatic effect, even if fungistatic effect is still fine when salinity reaches 1%, there is good feeding additive aquatic animal to open
Make an offer value.
In summary, 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 antibacterial peptide VK-21 of the present invention is preparing anti-infective gram-positive bacteria
Or/and can preferably be applied in Gram negative bacterial disease medicine, while in aquaculture pathogenic bacteria prevent and treat preparation
Good application can be obtained.
The preferred embodiments of the present invention are the foregoing is only, are not intended to limit the invention, although with reference to foregoing reality
Apply example the present invention is described in detail, for those skilled in the art, it still can be to foregoing each implementation
Technical scheme described in example is modified, or carries out equivalent substitution to which part technical characteristic.All essences in the present invention
God any modification, equivalent substitution and improvements done etc., should be included in the scope of the protection with principle.
Claims (6)
1. a kind of antibacterial peptide VK-21, it is characterised in that the amino acid sequence of the antibacterial peptide VK-21 is：
Val-Lys-Arg-Lys-Lys-Lys-Pro-Gln-Ser-Trp-Lys-Thr-Trp-Trp-Thr-Lys-Trp-Trp-
Thr-Lys-Lys。
2. antibacterial peptide VK-21 as claimed in claim 1, it is characterised in that：The antibacterial peptide VK-21 is α spiral straight-chain polypeptides,
Contain 21 amino acid residues, molecular size range 2845.46Da, isoelectric point 11.51.
3. antibacterial peptide VK-21 as claimed in claim 1 or 2 application, it is characterised in that：It is prepared by the antibacterial peptide VK-21
Treat the application in the extensive pedigree antibiotic of gram-positive bacteria or gram positive bacterial infection.
4. antibacterial peptide VK-21 as claimed in claim 1 or 2 application, it is characterised in that：It is prepared by the antibacterial peptide VK-21
Application in antiseptic.
5. antibacterial peptide VK-21 as claimed in claim 1 or 2 application, it is characterised in that：It is prepared by the antibacterial peptide VK-21
Application in feed addictive.
6. antibacterial peptide VK-21 as claimed in claim 1 or 2 application, it is characterised in that：The antibacterial peptide VK-21 is in food
Application in additive or cosmetics preservative.
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| CN110317248A (en) * | 2019-06-05 | 2019-10-11 | 遵义医科大学珠海校区 | A kind of artificial synthetic antimicrobial peptide and its design method and application |
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| CN113201057A (en) * | 2021-03-16 | 2021-08-03 | 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 |
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| CN112480228A (en) * | 2019-06-05 | 2021-03-12 | 遵义医科大学珠海校区 | Artificially synthesized antibacterial peptide and application thereof |
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| CN111253474A (en) * | 2019-10-10 | 2020-06-09 | 应连心 | Antibacterial peptide RG-27 and application thereof |
| CN111253474B (en) * | 2019-10-10 | 2023-07-07 | 应连心 | Antibacterial peptide RG-27 and application thereof |
| CN113201057A (en) * | 2021-03-16 | 2021-08-03 | 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 |
| CN115246878A (en) * | 2021-03-31 | 2022-10-28 | 禾美生物科技(浙江)有限公司 | Antibacterial peptide and application thereof in cosmetics |
| 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 |
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| CN113307850A (en) * | 2021-06-09 | 2021-08-27 | 温州大学 | Antibacterial peptide, composition containing same and application |
| CN113880933A (en) * | 2021-11-12 | 2022-01-04 | 青岛农业大学 | Antibacterial peptide SssNKL 27 and application thereof |
| CN113880933B (en) * | 2021-11-12 | 2023-10-20 | 青岛农业大学 | Antibacterial peptide SsNKL27 and application thereof |
| CN116023431A (en) * | 2022-07-26 | 2023-04-28 | 青岛大学 | Antibacterial short peptide 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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