CN106632611A - A novel polypeptide capable of inhibiting and killing a plurality of drug-resistant bacteria - Google Patents
A novel polypeptide capable of inhibiting and killing a plurality of drug-resistant bacteria Download PDFInfo
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- CN106632611A CN106632611A CN201610983977.9A CN201610983977A CN106632611A CN 106632611 A CN106632611 A CN 106632611A CN 201610983977 A CN201610983977 A CN 201610983977A CN 106632611 A CN106632611 A CN 106632611A
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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
-
- 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
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
A novel polypeptide capable of inhibiting and killing a plurality of drug-resistant bacteria is disclosed. The amino acid sequence of the polypeptide is shown as SEQ ID NO.1-2. The antibacterial peptide has significant effects when the antibacterial peptide is used for inhibiting and killing Gram-positive bacteria represented by methicillin-resistant staphylococcus aureus and listeria, or Gram-negative bacteria represented by escherichia coli.
Description
Technical field
The present invention relates to antibacterial peptide, can particularly suppress and kill the polypeptide of bacterium, specifically one kind can suppress and kill
The novel polypeptide of various drug tolerant bacterias.
Background technology
Abuse of antibiotics causes pathogenic microorganism to produce the high-level resistance to the action of a drug and multi-drug resistance (multidrug
Resistance, MDR) have become a global difficult 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 threaten.Numerous studies show that pathogenic bacteria pass through horizontal transfer of gene etc.
Mechanism can obtain MDR, and resist the antibiotic that the line of various clinical one is used.It is increasingly tight from " super drug-resistant bacteria " in order to tackle
High challenge, people more place hope on excavation anti-in addition to modifying improvement is carried out to existing antibiotic and new antibiotic is excavated
The substitute of element is given birth to alleviate the world health security crisis caused by " super drug-resistant bacteria ".
The content of the invention
It is an object of the invention to provide a kind of novel polypeptide that can suppress and kill various drug tolerant bacterias, to solve the above-mentioned back of the body
Problem present in scape technology.
The novel polypeptide that can suppress and kill various drug tolerant bacterias of the present invention, it is particularly a kind of to suppress or kill
The gram-positive bacteria gone out with methicillin-resistant staphylococcus aureus, Listeria as representative, or with Escherichia coli as representative
Gram-negative bacteria novel polypeptide.
The novel polypeptide that can suppress and kill various drug tolerant bacterias of the present invention, its amino acid sequence such as SEQ ID NO.1-
Shown in 2.
Corresponding to above-mentioned SEQ ID NO.1-2 named be encoded to XC-16, its sequence is as follows：
XC-16A：Lys-Leu-Leu-Asp-Ile-Val-Lys-Lys-Arg-Val-Arg-Ala-Phe-Trp-Ser-Leu
（SEQ ID NO.1）；
XC-16B：Lys-Leu-Leu-Asp-Ile-Val-Lys-Lys-Val-Val-Arg-Ala-Phe-Arg-Ser-Leu
（SEQ ID NO.2）；
The preparation method of above-mentioned antibacterial peptide, is prepared using the BOC methods in existing solid-state chemical reaction method method.
Specifically include：
First, resins synthesis
1) Peptide acid Merrifield Resin and PAM Resin
2) Peptidecarboxamide MBHA Resin
2nd, peptide chain synthesis
1) coupling of amino acid；
2) excision of N- ends Boc groups；
N- ends Boc blocking group TFA must be removed before HF cuttings.Hand cut N- end Boc group methodologies are to use TFA/
DCM ratios are 1：The 1 solution min of washing reaction 15 at ambient temperature.
3rd, cut
1）Resin before cutting prepares：All resins must completely be cleaned before cutting and are dried.
Treatment on special problems：
A）The cutting of the Dnp blocking groups of His：With the DMF swellable resins of minimum volume；Processed with the benzenethiol of 20 mol
1-2 h；Transfer a resin into and cohere in glass funnel, with HF liquid or TFMSA before processings with DMF, methyl alcohol and cold diethyl ether frequency
Numerous flushing.
B）The deformylase of the polypeptide resin containing Trp：With volume ratio as 1：10 piperidines/DMF liquid is put into round-bottomed flask,
And cool down in ice bath；Add polypeptide resin（1 g/10 mL）, the h of stirring reaction 2 at 0 DEG C；Use DMF（5 times of volumes）Wash
Twice, twice, MeOH is twice for DCM；With high vacuum dry above-mentioned gained resin at least 4 h before HF cuttings.
2）HF cuts
Standard HF patterning method（0.2 mmol）
A）By polypeptide resin, in polyfluortetraethylene pipe and purification agent composition addition reaction vessel.Polypeptide containing Cys HF/ benzene
Methyl ether/DMS/p- benzene methylthio phenols（10：1：1：0.2）, and do not contain Cys use HF/DMS/ methyl phenyl ethers anisoles（10：1：1）.
B）Lid is screwed, at least 5 min are cooled down in the pure methyl alcohol of ice before cutting.
C）The HF of 10 mL is distilled under the explanation with reference to manufacturer in bottle.Because the cutting meeting of the polypeptide containing Arg (Tos)
Continue 2 more than h.
D）Reaction is last, by nitrogen flow evaporator HF and DMS.
E）The polypeptide under excision is drawn out from resin with TFA.
F）Filter under negative pressure and remove resin, resin is washed twice with TFA.Filtering screening, adds 8-10 times of cold diethyl ether.Sometimes
Need to evaporate sediments of most of TFA to obtain crude product.
3）The cutting later stage is processed：
A）Precipitation, filters the peptide of precipitation in Hirsch funnels with hardened filter paper under vacuo.Sediment is rinsed with cold ether, suitable
Cushioning liquid in dissolve, then freeze.
B）Centrifugation, adds the tert-butyl ether of a small amount of volume in residue, grinds, until obtaining suspension, by suspension
In being transferred to a clean centrifuge tube, closed centrifugation, automatic centrifuge is necessary during this.Then by ether from pipe
Careful pours out, and uses ether repeated washing, and then the dissolution residual substance in suitable cushioning liquid freezes.
C）Water-soluble peptide, after precipitation, adds water in residue, then mixture is transferred in separatory funnel, Ke Yijia
Enter a small amount of ethanol hydrotropy.
D）The funnel of blocking is fully rocked, dispersed mixture, static separation separates subnatant（Water）.
E）Plus a small amount of water is in funnel, repetition it is above-mentioned rock-it is static-separate three times, remove upper liquid, then place
In clean flask, mixed liquor is transferred in separatory funnel.
F）Plus it is a small amount of newly match somebody with somebody Anaesthetie Ether, repetition it is above-mentioned rock-it is static-separate two to three times, every time ether
Layer is removed, and water layer is put into separatory funnel, collects water layer to clean flask, is freezed.
It is a further object to provide the purposes of above-mentioned novel polypeptide.
The purposes is：Treatment is being prepared with methicillin-resistant staphylococcus aureus, Listeria as Gram-negative
Application in bacterium or the gram positive bacterial infection medicine with Escherichia coli as representative；
It is further the application in preparing treatment or preventing for animals or people medicine, to kill and suppress methicillin-resistant
Staphylococcus aureus, Listeria, Escherichia coli.
Described medicine can include the composition of one or more in antibacterial peptide defined in the invention described above；
Described medicine can also include one or more pharmaceutically acceptable carriers or additive, such as in order to realize
The organized enzyme of promotion enzymolysis, or strengthen the activating agent of dispersibility etc.；
The purposes further relates to the application in terms of the feed and/or feed addictive that prepare livestock and poultry.
Dosage and use condition of the antibacterial peptide of the present invention in above-mentioned application, can be according to the known formula of this area
Method is determining.
The present invention our experiments show that, be with methicillin-resistant staphylococcus aureus, Listeria for suppressing and killing
The gram-positive bacteria of representative or the Gram-negative bacteria with Escherichia coli as representative have significant effect.
Description of the drawings
Fig. 1 is Cecropin X C-16A：Kill methicillin-resistant staphylococcus aureus（A）, Listeria（B）, large intestine bar
Bacterium（C）Design sketch；
Fig. 2 is Cecropin X C-16B：Kill methicillin-resistant staphylococcus aureus（A）, Listeria（B）, Escherichia coli（C）
Design sketch
Specific embodiment：
Embodiment 1
Minimal inhibitory concentration（MIC）Assessment, using micro broth dilution method.
It is prepared by A, antibacterials and culture medium
Prepared by antibacterials and culture medium, with constant broth dilution method.
Drug sensitive test antibacterials concentration range：According to NCCLS sensitivity testing to antibacterials operation standards, medicine
Concentration range should be comprising resistance, intermediary and sensitive boundary point value.
Culture medium：NCCLS recommends Mueller-Hinton（MH）Meat soup, pH 7.2 ~ 7.4.Aerobic bacteria and facultative detest
Oxygen bacterium well-grown in this culture medium.The preparation of inoculum, with oese picking plesiomorphism bacterium colony to be checked 3 ~ 5, connects
Plant in the caseinhydrolysate of 4 ~ 5 mL（MH）In meat soup, 35 DEG C of 2 ~ 6 h of incubation.Increase the exponential phase bacterium solution physiology salt after bacterium
Water or MH meat soups corrected concentrations to 0.5 Maxwell than turbid standard, containing about 1 ~ 2 × 108 CFU/mL。
It is prepared by B, MIC plate
It is prepared by MIC plates：Sterile working, 96 holes that the antibacterials solution of variable concentrations after doubling dilution is added separately to sterilize
In XPS, the 1st to the 11st hole adds liquid, and per the W μ L of hole 10, the 12nd hole not dosing is used as growth control, and frost is dry
Sealing after dry, less than -20 DEG C save backup.
It is prepared by C, inoculum
By the concentration prepared with growth method or direct bacteria suspension method equivalent to 0.5 Maxwell than turbid standard bacteria suspension, Jing MH meat
Soup 1：After 1000 dilutions, to the μ L of every Kong Zhongjia 100, in sealing rearmounted 35 DEG C of normal air incubators, 16 ~ 20 h of incubation judge
As a result.Now, the 1st hole is respectively 128,64,32,16,8,4,2,1,0.5,0.25,0.125 μ to the 11st hole drug concentration
g/mL。
D, result judge
As a result judge：Lowest concentration of drug with the complete inhibition bacterial growth in aperture is as MIC.Work as Positive control wells（I.e. not
Containing antibiotic）The obvious growth test of interior bacterium is just meaningful.When there is single jump hole in micro broth dilution method, should record
The highest drug concentration of bacteria growing inhibiting.
As a result see the table below
| Polypeptide is numbered | Methicillin-resistant staphylococcus aureus | Listeria monocytogenes | Escherichia coli |
| XC-16A | 16 μg/ml | 16 μg/ml | 64 μg/ml |
| XC-16B | 16 μg/ml | 8 μg/ml | 32 μg/ml |
Used in this experiment：Escherichia coli (Escherich) ATCC 25922, Listeria (Monocute
proliferation) ATCC 19115。
Embodiment 2
Minimum bactericidal concentration is tested（MBC）
MBC（Minimum bactericidal concentration, MBC）：Kill 99.9%（Reduce by 3
The order of magnitude）For examination Institute of Micro-biology need lowest concentration of drug.
MBC is surveyed in doubling dilution：Exactly drug concentration is halved successively（Doubling dilution）, such as in 96 orifice plates
Certain row/column be MBC, each hole adds 10 μ L liquids, and first hole liquor strength is 100 μ g/mL, and second hole is
50 μ g/mL, the 3rd 25 μ g/mL ....It is exactly such as first hole plus 20 μ L liquids to operate, and is inhaled from first hole
10 μ L liquids to the second hole, plus the μ L of nutrient solution 10 are taken, after mixing, 10 μ L is therefrom drawn and is added in the 3rd hole, then add culture
The μ L of liquid 10 are mixed ....Most as little as which concentration, lived bacterium, and the concentration is MBC.
As a result Fig. 1 ~ 2 are seen.
Wherein, HR--- hours, μ g/ml--- concentration, CFU reduction %--- bacterium colonies reduce percentage.
Used in this experiment：Escherichia coli (Escherich) ATCC 25922, Listeria (Monocute
proliferation) ATCC 19115。
XC-16A：Lys-Leu-Leu-Asp-Ile-Val-Lys-Lys-Arg-Val-Arg-Ala-Phe-Trp-Ser-Leu
（SEQ ID NO.1）
XC-16B：Lys-Leu-Leu-Asp-Ile-Val-Lys-Lys-Val-Val-Arg-Ala-Phe-Arg-Ser-Leu
（SEQ ID NO.2）
Claims (6)
1. a kind of energy suppresses and kills the polypeptide of various drug tolerant bacterias, it is characterised in that：Its amino acid sequence such as SEQ ID
Shown in NO.1-2.
2. a kind of application of polypeptide as claimed in claim 1, it is characterised in that：Described polypeptide is preparing treatment Gram-positive
Apply in the medicine of bacterium or gram positive bacterial infection.
3. polypeptide according to claim 2, it is characterised in that：Use or livestock and poultry treatment or prevention methicillin-resistant people is prepared
Application in staphylococcus aureus, Listeria, coli-infection medicine.
4. the application according to Claims 2 or 3, it is characterised in that：The medicine is included and resisted as defined in claim 1
The composition of one or more of bacterium peptide.
5. the application according to one of claim 2-4, it is characterised in that：One or more are mixed with the medicine
Pharmaceutically acceptable carrier or additive.
6. a kind of application of the antibacterial peptide as described in claim 1, it is characterised in that：Prepare livestock and poultry treatment or prevent leather blue
Application in family name's positive bacteria or gram positive bacterial infection feed.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610983977.9A CN106632611B (en) | 2016-11-09 | 2016-11-09 | Polypeptide capable of inhibiting and killing multiple drug-resistant bacteria |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610983977.9A CN106632611B (en) | 2016-11-09 | 2016-11-09 | Polypeptide capable of inhibiting and killing multiple drug-resistant bacteria |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106632611A true CN106632611A (en) | 2017-05-10 |
| CN106632611B CN106632611B (en) | 2020-03-27 |
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| CN201610983977.9A Active CN106632611B (en) | 2016-11-09 | 2016-11-09 | Polypeptide capable of inhibiting and killing multiple drug-resistant bacteria |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113633600A (en) * | 2020-08-07 | 2021-11-12 | 湖南科技学院 | Alcohol-free water-washing-free disinfection hand sanitizer and preparation method thereof |
| CN113717261A (en) * | 2021-10-13 | 2021-11-30 | 西北农林科技大学 | Polypeptide compound with broad-spectrum bactericidal activity on gram-positive bacteria |
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| CN106496305B (en) * | 2016-11-09 | 2019-10-01 | 湖南科技学院 | The antibacterial peptide of one group of broad-spectrum high efficacy and its application |
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| CN104072583A (en) * | 2014-05-10 | 2014-10-01 | 冯小荣 | Polypeptide capable of inhibiting and killing multiple medicine-resisting bacteria |
| CN106496305B (en) * | 2016-11-09 | 2019-10-01 | 湖南科技学院 | The antibacterial peptide of one group of broad-spectrum high efficacy and its application |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113633600A (en) * | 2020-08-07 | 2021-11-12 | 湖南科技学院 | Alcohol-free water-washing-free disinfection hand sanitizer and preparation method thereof |
| CN113633600B (en) * | 2020-08-07 | 2024-02-13 | 湖南科技学院 | Alcohol-free washing-free disinfection hand sanitizer and preparation method thereof |
| CN113717261A (en) * | 2021-10-13 | 2021-11-30 | 西北农林科技大学 | Polypeptide compound with broad-spectrum bactericidal activity on gram-positive bacteria |
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| CN106632611B (en) | 2020-03-27 |
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