CN106432426B - It can inhibit and kill the antibacterial peptide of gram-positive bacteria and Gram-negative bacteria - Google Patents
It can inhibit and kill the antibacterial peptide of gram-positive bacteria and Gram-negative bacteria Download PDFInfo
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- CN106432426B CN106432426B CN201610984016.XA CN201610984016A CN106432426B CN 106432426 B CN106432426 B CN 106432426B CN 201610984016 A CN201610984016 A CN 201610984016A CN 106432426 B CN106432426 B CN 106432426B
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- 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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- 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
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Abstract
A kind of antibacterial peptide that can inhibit and kill gram-positive bacteria and Gram-negative bacteria disclosed by the invention, amino acid sequence is as shown in SEQ ID NO.1-3.The antibacterial peptide is for killing or inhibiting using methicillin-resistant staphylococcus aureus, Listeria as the gram-positive bacteria of representative or using Escherichia coli as the significant effect of the Gram-negative bacteria of representative.
Description
Technical field
The present invention relates to antibacterial peptides, can specifically inhibit and kill the antibacterial of gram-positive bacteria and Gram-negative bacteria
Peptide.
Background technique
Abuse of antibiotics causes pathogenic microorganism to generate high-level drug resistance and multi-drug resistance (multidrug
Resistance, MDR) have become global problem.In recent years, " superbug " with MDR was in phase all over the world
After outburst, the life and health safety of the mankind is seriously threatened.A large number of studies show that pathogenic bacteria pass through the horizontal transfer etc. of gene
Mechanism can get MDR, and resist the antibiotic that one line of various clinical uses." super drug-resistant bacteria " is come from increasingly sternly to cope with
It is anti-more to place hope on excavation other than carrying out modifying improvement to existing antibiotic and excavating new antibiotic by high challenge, people
The substitute of raw element is to alleviate world health security crisis caused by " super drug-resistant bacteria ".
Summary of the invention
An object of the present invention, which is to provide, a kind of can inhibit and kill the anti-of gram-positive bacteria and Gram-negative bacteria
Bacterium peptide, to solve the problems, such as above-mentioned background technique.
The antibacterial peptide of the present invention that can inhibit and kill gram-positive bacteria and Gram-negative bacteria, it is specifically a kind of
It can inhibit or kill using methicillin-resistant staphylococcus aureus, Listeria as the gram-positive bacteria of representative or with large intestine
Bacillus is the novel antimicrobial peptide of the Gram-negative bacteria of representative.
The antibacterial peptide that can inhibit and kill gram-positive bacteria and Gram-negative bacteria of the invention, amino acid sequence is such as
Shown in SEQ ID NO.1-3.
That is named corresponding to above-mentioned SEQ ID NO.1-3 is encoded to XH-14, and sequence is as follows:
XH-14A:Phe-Ile-Lys-Arg-Ile-Ala-Arg-Leu-Leu-Arg-Lys-Ile-P he-Arg(SEQ ID
NO.1)；
XH-14B:Phe-Ile-Lys-Arg-Ile-Ala-Arg-Leu-Leu-Arg-Lys-Ile-L ys-Arg(SEQ ID
NO.2)；
XH-14C:Phe-Ile-Lys-Arg-Ile-Ala-Arg-Leu-Leu-Arg-Lys-Ile-T rp-Arg(SEQ ID
NO.3).
The preparation method of above-mentioned antibacterial peptide is prepared using the BOC method in existing solid-state chemical reaction method method.
It specifically includes:
One, resins synthesis
1) Peptide acid Merrifield Resin and PAM Resin
2) Peptidecarboxamide MBHA Resin
Two, peptide chain synthesizes
1) coupling of amino acid；
2) excision of the end N- Boc group；
The end N- Boc blocking group must be removed with TFA before HF cutting.The end hand cut N- Boc group methodologies are to use
Solution 15 min of washing reaction at room temperature that TFA/DCM ratio is 1:1.
Three, it cuts
1) resin before cutting prepares: all resins must completely clean before cutting and drying.
Treatment on special problems:
A) the cutting of the Dnp blocking group of His: with the DMF swellable resins of minimum volume；It is handled with the benzenethiol of 20 mol
1-2 h；It transfers a resin into and coheres in glass funnel, with DMF, methanol and cold ether frequency before with HF liquid or TFMSA processing
Numerous flushing.
B) the deformylase of the polypeptide resin containing Trp: being put into round-bottomed flask with volume ratio for 1:10 piperidines/DMF liquid, and
It is cooling in ice bath；It is added polypeptide resin (1 g/10 mL), 2 h is stirred to react at 0 DEG C；With DMF(5 times of volume) it washes twice,
Twice, MeOH is twice by DCM；The above-mentioned gained resin of high vacuum dry at least 4 h before being cut with HF.
2) HF is cut
Standard HF patterning method (0.2 mmol)
A) polypeptide resin, polyfluortetraethylene pipe and purification agent composition are added in reaction vessel.Polypeptide containing Cys is used
HF/ methyl phenyl ethers anisole/DMS/p- benzene methylthio phenol (10:1:1:0.2), and HF/DMS/ methyl phenyl ethers anisole (10:1:1) is used without Cys.
B) lid is screwed, preceding at least 5 min cooling in the pure methanol of ice of cutting.
C the HF of 10mL) is distilled under the explanation referring to manufacturer into bottle.Because the polypeptide cutting containing Arg (Tos) can be held
Continuous 2 h or more.
D) reaction is finally, pass through nitrogen flow evaporator HF and DMS.
E the polypeptide under cutting off) is drawn out from resin with TFA.
F) resin is removed in filtering under negative pressure, washes resin twice with TFA.8-10 times of cold ether is added in filtering screening.Sometimes
Need to evaporate most of TFA to obtain the sediment of crude product.
3) post-processing is cut:
A it) precipitates, under vacuum with the peptide of hardened filter paper filtering precipitating in Hirsch funnel.Sediment, In are rinsed with cold ether
It dissolves in suitable buffer solution, is then lyophilized.
B it) is centrifuged, the tert-butyl ether of a small amount of volume is added in residue, grind, until obtaining suspended matter, by suspended matter
It is transferred in a clean centrifuge tube, closed centrifugation, automatic centrifuge is necessary during this.Then by ether from pipe
Careful pours out, and is washed repeatedly with ether, then the dissolution residual substance in suitable buffer solution is lyophilized.
C) water-soluble peptide after precipitating, adds water into residue, then mixture is transferred in separatory funnel, Ke Yijia
Enter a small amount of ethyl alcohol hydrotropy.
D the funnel of blocking, dispersed mixture) are sufficiently rocked, static separation separates subnatant (water).
E) plus a small amount of water is into funnel, and repetition is above-mentioned to rock-static-separation three times, removes upper liquid, then places
In clean flask, mixed liquor is transferred in separatory funnel.
F) plus it is a small amount of newly match Anaesthetie Ether, repeat above-mentioned to rock-static-separation two to three times, every time ether
Layer is removed, and water layer is put into separatory funnel, collects water layer to clean flask, freeze-drying.
It is a further object to provide the purposes of above-mentioned antibacterial peptide.
The purposes is: in preparation treatment using methicillin-resistant staphylococcus aureus, Listeria as Gram-negative
Bacterium or using Escherichia coli as the application in the gram positive bacterial infection drug of representative especially treats or prevents in preparation for animals
Or the application in the drug of people, to kill and inhibit methicillin-resistant staphylococcus aureus, Listeria, large intestine bar
Bacterium.
The drug may include one of antibacterial peptide defined in aforementioned present invention or a variety of compositions；
The drug can also include one or more kinds of pharmaceutically acceptable carriers or additive, such as in order to
Realize the organized enzyme for promoting enzymatic hydrolysis, or the activating agent etc. of enhancing dispersibility.
The purposes further relates to the application in terms of the feed and/or feed addictive of preparation livestock and poultry.
The dosage and use condition of antibacterial peptide of the invention in above-mentioned application, can be according to the known formula of this field
Method determines.
The present invention our experiments show that, be with methicillin-resistant staphylococcus aureus, Listeria for inhibiting and killing
The gram-positive bacteria that represents has significant effect by the Gram-negative bacteria of representative of Escherichia coli.
Detailed description of the invention
Fig. 1 is Cecropin X C-16A: killing methicillin-resistant staphylococcus aureus, Listeria, the effect of Escherichia coli
Fruit figure；
Fig. 2 is Cecropin X C-16B: killing methicillin-resistant staphylococcus aureus, Listeria, the effect of Escherichia coli
Fruit figure；
Fig. 3 is Cecropin X C-16C: killing methicillin-resistant staphylococcus aureus, Listeria, the effect of Escherichia coli
Fruit figure；
Specific embodiment:
Embodiment 1
Minimal inhibitory concentration (MIC) assessment, using micro broth dilution method.
A, antibacterials and culture medium preparation
Antibacterials and culture medium preparation, with constant broth dilution method.
Drug sensitive test antibacterials concentration range: according to NCCLS sensitivity testing to antibacterials operation standard, drug
Concentration range should include drug resistance, intermediary and sensitive boundary point value.
Culture medium: NCCLS is recommended to use Mueller-Hinton(MH) meat soup, pH7.2 ~ 7.4.Aerobic bacteria and facultative detest
Oxygen bacterium well-grown in this culture medium.The preparation of inoculum, connects by similar bacterium colony 3 ~ 5 to be checked with oese picking form
Kind is in caseinhydrolysate (MH) meat soup of 4 ~ 5mL, 35 DEG C of 2 ~ 6h of incubation.Logarithmic growth phase bacterium solution physiological saline after increasing bacterium
Or MH meat soup corrected concentrations to 0.5 Maxwell than turbid standard, containing about 1 ~ 2 × 108CFU/mL。
B, prepared by MIC plate
The preparation of MIC plate: the antibacterials solution of various concentration after doubling dilution is added separately to the 96 of sterilizing by sterile working
In hole polystyrene plate, the 1st to the 11st hole adds medical fluid, every hole 10W μ L, and the 12nd hole not dosing is used as growth control, and frost is dry
Sealing after dry, -20 DEG C or less save backup.
C, prepared by inoculum
It will be equivalent to bacteria suspension of 0.5 Maxwell than turbid standard with the concentration of growth method or the preparation of direct bacteria suspension method, through MH
It after meat soup 1:1000 dilution, to every 100 μ L of Kong Zhongjia, seals in 35 DEG C of normal air incubators of postposition, is incubated for 16 ~ 20h judgement
As a result.At this point, the 1st hole to the 11st hole drug concentration is respectively 128,64,32,16,8,4,2,1,0.5,0.25,0.125 μ g/
mL。
D, result judges
As a result judge: to completely inhibit the lowest concentration of drug of bacterial growth in aperture as MIC.Work as Positive control wells
(being free of antibiotic) interior obvious growth test of bacterium is just significant.When there is single jump hole in micro broth dilution method,
The highest drug concentration for inhibiting bacterial growth should be recorded.
As a result it see the table below
| Polypeptide number | Methicillin-resistant staphylococcus aureus | Listeria monocytogenes | Escherichia coli |
| XH-14A | 8μg/ml | 4 μg/ml | 16 μg/ml |
| XH-14B | 32μg/ml | 16 μg/ml | 32μg/ml |
| XH-14C | 16μg/ml | 8 μg/ml | 32 μg/ml |
It is used in this experiment: Escherichia coli (Escherich) ATCC25922, Listeria (Monocute
proliferation) ATCC19115。
Embodiment 2
Minimum bactericidal concentration tests (MBC)
Minimum bactericidal concentration (minimum bactericidal concentration, MBC): killing 99.9%(reduces by 3
It is a
The order of magnitude) for examination Institute of Micro-biology need lowest concentration of drug.
Minimum bactericidal concentration is surveyed in doubling dilution: drug concentration successively exactly being halved to (doubling dilution), such as in 96 orifice plates
Certain row/column be MBC, 10 μ L medical fluids are added in each hole, and first hole liquor strength is 100 μ g/mL, and second hole is 50 μ
G/mL, 25 μ g/mL ... of third.Operating is exactly that such as first hole adds 20 μ L medical fluids, draws 10 μ L from first hole
Medical fluid adds 10 μ L of culture solution to the second hole, after mixing, therefrom draws 10 μ L and adds in third hole, then plus 10 μ L of culture solution it is mixed
It is even ....Most down to which concentration, there is no bacterium living, which is MBC.
The result is shown in Figure 1 ~ 3.
Wherein, HR--- hours, μ g/ml--- concentration, CFU reduction %--- bacterium colony reduced percentage.
It is used in this experiment: Escherichia coli (Escherich) ATCC25922, Listeria (Monocute
proliferation) ATCC19115。
XH-14A:Phe-Ile-Lys-Arg-Ile-Ala-Arg-Leu-Leu-Arg-Lys-Ile-P he-Arg(SEQ ID
NO.1)
XH-14B:Phe-Ile-Lys-Arg-Ile-Ala-Arg-Leu-Leu-Arg-Lys-Ile-L ys-Arg(SEQ ID
NO.2)
XH-14C:Phe-Ile-Lys-Arg-Ile-Ala-Arg-Leu-Leu-Arg-Lys-Ile-T rp-Arg(SEQ ID
NO.3)
Claims (4)
1. can inhibit and kill gram-positive bacteria/Gram-negative bacteria antibacterial peptide, it is characterised in that: its amino acid sequence is such as
Shown in SEQ ID NO.3.
2. a kind of application of antibacterial peptide as described in claim 1, it is characterised in that: treat methicillin-resistant staphylococcus Portugal in preparation
Grape coccus, Listeria, the application in coli-infection drug.
3. application according to claim 2, it is characterised in that: be used to prepare people with or livestock and poultry drug.
4. antibacterial peptide described in claim 1 is preparing the application in additive for farm animal feed.
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| CN201610984016.XA CN106432426B (en) | 2016-11-09 | 2016-11-09 | It can inhibit and kill the antibacterial peptide of gram-positive bacteria and Gram-negative bacteria |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101063102A (en) * | 2007-04-26 | 2007-10-31 | 武汉大学 | East-Asia scorpion antibiotic peptide gene and preparation method and application |
| CN104292298A (en) * | 2013-07-17 | 2015-01-21 | 武汉摩尔生物科技有限公司 | Polypeptide, DNA molecule for coding polypeptide, vector, and preparation method and applications of polypeptide |
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2016
- 2016-11-09 CN CN201610984016.XA patent/CN106432426B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101063102A (en) * | 2007-04-26 | 2007-10-31 | 武汉大学 | East-Asia scorpion antibiotic peptide gene and preparation method and application |
| CN104292298A (en) * | 2013-07-17 | 2015-01-21 | 武汉摩尔生物科技有限公司 | Polypeptide, DNA molecule for coding polypeptide, vector, and preparation method and applications of polypeptide |
Non-Patent Citations (1)
| Title |
|---|
| Identification and functional characterizationofnovelscorpionvenom peptides with no disulfide bridgefromButhusmartensii Karsch;Zeng X C et al;《Peptides》;20041231(第25期);143-150 * |
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