CN108467424A - Linear antibacterial oligopeptides SLAP-S25 and its application - Google Patents
Linear antibacterial oligopeptides SLAP-S25 and its application Download PDFInfo
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- CN108467424A CN108467424A CN201810282987.9A CN201810282987A CN108467424A CN 108467424 A CN108467424 A CN 108467424A CN 201810282987 A CN201810282987 A CN 201810282987A CN 108467424 A CN108467424 A CN 108467424A
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Abstract
The invention discloses linear antibacterial oligopeptides SLAP S25 and its applications.Linear antibacterial oligopeptides SLAP S25 are that the nitrogen end of oligopeptides shown in sequence in sequence table 1 is carried out acetylation modification, carbon teminal progress amidation modification, obtained oligopeptides.Experiments have shown that, linear antibacterial oligopeptides SLAP S25 have certain bacteriostasis to a variety of gram-positive bacterias and Gram-negative bacteria, good synergistic effect is all had with antibacterials (such as tetracycline, vancomycin, Ofloxacin, rifampin), it can restore sensibility of the Gram-negative bacteria to polymyxins of resistance to polymyxins, enhance sensibility of the Friedlander's bacillus to a variety of antibacterials, without hemolytic, has preferable histocompatbility, it not only can effectively inhibit polymyxins drug-fast bacteria, but also bacteremia can be treated.Linear antibacterial oligopeptides SLAP S25 molecular weight is small, simple in structure, convenient for extensive synthesis.The present invention has great application value.
Description
Technical field
The present invention relates to biotechnologies, and in particular to linear antibacterial oligopeptides SLAP-S25 and its application.
Background technology
Antibacterials play vital effect in terms for the treatment of and controlling microorganism infection disease, have ensured the mankind
With the health of animal.But the extensive and lack of standardization of antibacterials uses caused bacterial resistance sex chromosome mosaicism getting worse, causes
Make the effect of a variety of antibacterials are to bacterium infection reduction or invalid.South Asia in 2011 finds that the gram-negative of NDM-1 can be generated
Property the bacterium and pathogenic bacteria Shigella and comma bacillus that have not been reported；The same year North America is found and the relevant methicillin-resistant in community
Staphylococcus aureus (CA-MRSA)；China finds plasmid-mediated polymyxins drug resistance (MCR-1) Escherichia coli within 2015,
" last line of defense " for treating resistant Gram negative bacterium is precarious.As unreasonable control and solution bacterial drug resistance are asked
Topic, it will grave danger is caused to human health, causes the fear of society.
To ensure the sustainable development of human health and animal husbandry, effective therapeutic scheme or searching novel antibacterial drug are developed
And its synergist is imperative.First, by antibacterials use in conjunction or antibacterials synergist, antibacterials can be improved
Bioavilability and service life, reduce existing Antibiogics usage amount, effectively prevent and control drug-resistant bacteria development.As most
Antibacterials synergist-the methoxybenzyl aminopyrimidine early found, itself has weaker antibacterial action, when itself and sulfonamides
After object shares, the antibacterial efficacy of sulfa drugs can be improved decades of times；Researches show that aspergillomarasmine A can restore pan belong to β-it is interior
Sensibility (Nature, 2014,510,503-506) of amidase (NDM-1) Gram-negative bacteria to Meropenem.In addition, line
Property antibacterial oligopeptides (short linear antibacterial peptide, SLAP) have it is simple in structure, production cost is low, peace
The advantages that property is high entirely, application prospect is good.Antibacterials synergist of the novel linear oligopeptides as wide spectrum is developed, restore or is improved
The antibacterial activity of existing antibacterials is one of the important means of control bacterial drug resistance.To sum up, antibacterials synergist and line
Property antibacterial oligopeptides drug-fast bacteria development can provide a favorable security in order to control, there is higher R and D value.
Invention content
The object of the present invention is to provide a kind of Antibacterials.
Present invention firstly provides a kind of oligopeptides, can be a1) or a2) or a3) a4) or a5) or a6)：
A1) oligopeptides shown in sequence 1 in sequence table；
A2) by a1) nitrogen end of the oligopeptides carries out acetylation modification, carbon teminal carries out amidation modification, obtained oligopeptides；
A3) structural formula oligopeptides as shown in formula (I)；
A4) change a3) shown in oligopeptides at least one amino acid chirality, obtained oligopeptides；
A5) by a1) or a2) or a3) or a4) shown in oligopeptides join end to end, obtained cyclic oligopeptides；
A6) by a1) or a2) or a3) a4) or a5) shown in oligopeptides amino acid sequence pass through one or several amino acid
The substitution of residue and/or the oligopeptides with the same function lacked and ored add.
Above-mentioned a6) in, the substitution by one or several amino acid residues and/or to lack and or add can be not
More than 4 amino acid residues substitution and/or lack and or add.
Above-mentioned a6) in, the amino acid can be rare amino acid and/or natural amino acid.
Above-mentioned a6) described in oligopeptides concretely sequence 2 in sequence table, sequence 3, sequence 4, sequence 5, sequence 6, sequence 7,
Sequence 8, sequence 9, sequence 10, sequence 11, sequence 12, sequence 13, sequence 14, sequence 15,18 institute of sequence 16, sequence 17 or sequence
Show oligopeptides (be corresponding in turn to SLAPS25-1, SLAPS25-2 in embodiment table 7, SLAPS25-3, SLAPS25-4,
SLAPS25-5、SLAPS25-6、SLAPS25-7、SLAPS25-8、SLAPS25-9、SLAPS25-10、SLAPS25-11、
SLAPS25-12, SLAPS25-13, SLAPS25-14, SLAPS25-15, SLAPS25-16 and SLAPS25-17).SLAPS25-1
To SLAPS25-14 be by structural formula the oligopeptides as shown in formula (I) pass through no more than 4 amino acid residues substitution obtain.
SLAPS25-15 to SLAPS25-17 is that the oligopeptides as shown in formula (I) passes through and is no more than lacking for 4 amino acid residues by structural formula
What mistake obtained.
Above-mentioned a4) described in oligopeptides concretely in embodiment 7 SLAPS25-18, SLAPS25-19 of table 7 or
SLAPS25-20 (is corresponding in turn to sequence 19 in sequence table, sequence 20 and sequence 21).
The amino acid of all oligopeptides shown in table 7 in embodiment 7, nitrogen end carries out acetylation modification, the amino acid of carbon teminal
Carry out amidation modification.
The application of the oligopeptides also belongs to protection scope of the present invention；The application of the oligopeptides can be b1) or b2) or b3)
Or b4) b5) or b6) b7) or b8)：B1) inhibit bacterium；B2 Antibacterial) is prepared；B3 caused by) preventing bacterium infection
Disease；B4 the product of disease caused by) preparing for preventing bacterium infection；B5 disease caused by bacterium infection) is treated；B6 it) makes
It is ready for use on the product of disease caused by treatment bacterium infection；B7 food spoilage) is prevented；B8 it) prepares for preventing food spoilage
Product.
The application of the oligopeptides joint antibacterials also belongs to protection scope of the present invention；The oligopeptides combines antibacterials
Application can be c1) c2) or c3) c4) or c5) or c6)：C1) inhibit bacterium；C2 Antibacterial) is prepared；C3) prevent
Disease caused by bacterium infection；C4 the product of disease caused by) preparing for preventing bacterium infection；C5) treatment bacterium infection is drawn
The disease risen；C6 the product of disease caused by) preparing for treating bacterium infection.The antibacterials can be Tetracyclines medicine
Object, Beta-lactam medicine, sugared peptide medicament, quinolone drugs, rifamycin drug or cyclic peptide drug.
The concretely bacteremia of disease caused by any of the above-described bacterium infection.
Any of the above-described product can be drug or vaccine.
The present invention also protects the oligopeptides in sensibility of the Gram-negative bacteria to polymyxins for restoring resistance to polymyxins
In application.
In above application, the Gram-negative bacteria can be the Gram-negative bacteria of non-Friedlander's bacillus.It is described resistance to
The Gram-negative bacteria (non-Friedlander's bacillus) of the Gram-negative bacteria of the polymyxins concretely drug resistant gene containing mcr.
The Gram-negative bacteria of the drug resistant gene containing the mcr concretely Escherichia coli of the drug resistant gene containing mcr-1, drug resistance containing mcr-1
The solution ornithine of the Acinetobacter baumannii of gene, the Citrobacter freundii of the drug resistant gene containing mcr-1, the drug resistant gene containing mcr-1
Ural bacterium, the serratia marcescens of the drug resistant gene containing mcr-1, the salmonella of the drug resistant gene containing mcr-1, drug resistance containing mcr-3
The plant life crow of the Aeromonas veronii of gene, the production alkali Providence of the drug resistant gene containing mcr-1, the drug resistant gene containing mcr-1
La Er bacterium or the enterobacter cloacae of the drug resistant gene containing mcr-1.The Escherichia coli of the drug resistant gene containing mcr-1 concretely large intestine
Bacillus (Escherichiacoli) 1-1 (mcr-1), Escherichia coli (Escherichiacoli) 1-5 (mcr-1), Escherichia coli
(Escherichiacoli) 1-6 (mcr-1), Escherichia coli (Escherichiacoli) 2-1 (mcr-1), Escherichia coli
(Escherichiacoli) 2-6 (mcr-1), Escherichia coli (Escherichiacoli) 2-8 (mcr-1) or Escherichia coli
(Escherichia coli)B2(NDM-5+mcr-1).The Acinetobacter baumannii of the drug resistant gene containing mcr-1 concretely Bao
Family name's acinetobacter calcoaceticus (Acinetobacterbaumannii) 2-38 (mcr-1).The Freund citric acid of the drug resistant gene containing mcr-1
Bacillus concretely Citrobacter freundii (Citrobacterfreumdii) 2-4 (mcr-1).The drug resistant gene containing mcr-1
Solution ornithine Ural bacterium concretely solve ornithine Ural bacterium (Raoultellaornithinolytica) 16-68
(mcr-1).The serratia marcescens of the drug resistant gene containing mcr-1 concretely serratia marcescens
(Serratiamarcescens)16-99(mcr-1).The salmonella of the drug resistant gene containing mcr-1 concretely Salmonella
Bacterium (Salmonellaenterica) SH30 (mcr-1), salmonella (Salmonellaenterica) SH170 (mcr-1), sand
Door Salmonella (Salmonellaenterica) SH1-3 (mcr-1), salmonella (Salmonellaenterica) SH2-8 (mcr-
Or salmonella (Salmonellaenterica) 8H-3 (mcr-1) 1).The Aeromonas veronii of the drug resistant gene containing mcr-3
Concretely Aeromonas veronii (Aeromonas veronii) 172 (mcr-3).The production alkali of the drug resistant gene containing mcr-1 is general
Luo Weidengsi bacterium concretely produce alkali Providence (Providenciaalcalifaciens) 16-1 (mcr-1).It is described
The plant life Ural bacterium of the drug resistant gene containing mcr-1 concretely plants raw Ural bacterium (Raoultellaplanticola) 16-15
(mcr-1).The enterobacter cloacae of the drug resistant gene containing mcr-1 concretely enterobacter cloacae
(Enterobactercloacae)16-15(mcr-1)。
The present invention also protects the oligopeptides to combine antibacterials in resistance to polymyxins or intolerant to the kerekou pneumonia of polymyxins
The application of the synergistic effect of primary Salmonella.The antibacterials can be rifamycin drug, quinolone drugs, Tetracyclines medicine
Object or beta-lactam.
In above application, the Friedlander's bacillus of the resistance to polymyxins can be the kerekou pneumonia of the drug resistant gene containing mcr
Primary Salmonella.The Friedlander's bacillus intolerant to polymyxins can be the Friedlander's bacillus without mcr drug resistant genes.Institute
State the Friedlander's bacillus of the drug resistant gene containing mcr concretely Friedlander's bacillus (Klebsiella pneumoniae)
16-63 (mcr-1), Friedlander's bacillus (Klebsiella pneumoniae) 4-20 (mcr-6) or Friedlander's bacillus
(Klebsiella pneumoniae)91(mcr-6).The Friedlander's bacillus intolerant to polymyxins can be kerekou pneumonia
Primary Salmonella (Klebsiella pneumoniae) 2.
The present invention also protects application of the oligopeptides joint antibacterials in the synergistic effect of bacterium.The bacterium can be
Gram-positive bacteria or Gram-negative bacteria.When the oligopeptides combines antibacterials in the synergistic effect of gram-positive bacteria
In application, antibacterials can be Beta-lactam medicine.When oligopeptides joint antibacterials are in the collaboration of Gram-negative bacteria
It is active in application, antibacterials can be Beta-lactam medicine, tetracycline medication, sugared peptide medicament, Du-6859a
Object, rifamycin drug or cyclic peptide drug.
The present invention also protects a kind of Antibacterial, contains the oligopeptides.
The Antibacterial can also contain antibacterials.The antibacterials can be tetracycline medication, beta-lactam
Class drug, sugared peptide medicament, quinolone drugs, rifamycin drug or cyclic peptide drug.
Any of the above-described tetracycline medication concretely tetracycline.Any of the above-described sugared peptide medicament is concretely
Vancomycin.Any of the above-described quinolone drugs concretely Ofloxacin.Any of the above-described rifamycin drug
Concretely rifampin.Any of the above-described cyclic peptide drug concretely polymyxins.Any of the above-described beta-lactam
Drug can be ampicillin or Imipenem.
Any of the above-described bacterium can be gram-positive bacteria or Gram-negative bacteria.The gram-positive bacteria can be gold
Staphylococcus aureus (Staphylococcusaureus) or enterococcus or bacillus subtilis (Bacillus subtilis) or
Other gram-positive bacteriums.The staphylococcus aureus (Staphylococcusaureus) concretely golden yellow grape
Coccus (Staphylococcusaureus) ATCC No.29213, staphylococcus aureus (Staphylococcusaureus)
215 (LZD of MRSA T144 or staphylococcus aureus (Staphylococcusaureus)R+cfr).The enterococcus can be excrement
Enterococcus (Enterococcus faecalis).The enterococcus faecalis (Enterococcus faecalis) concretely excrement intestines
Coccus (Enterococcus faecalis) VRE A4.The bacillus subtilis (Bacillus subtilis) can be withered grass
Bacillus (Bacillus subtilis) ATCC No.6051.The Gram-negative bacteria can be Escherichia coli
(Escherichiacoli), salmonella (Salmonellaenterica), Friedlander's bacillus
(Klebsiellapneumoniae), Pseudomonas aeruginosa (Pseudomonasaeruginosa), Acinetobacter baumannii
(Acinetobacterbaumannii), Aeromonas veronii (Aeromonas veronii), Citrobacter freundii
(Citrobacterfreumdii), solution ornithine Ural bacterium (Raoultellaornithinolytica), Serratia
Bacterium (Serratiamarcescens), production alkali Providence (Providenciaalcalifaciens) plant raw Ural
Bacterium (Raoultellaplanticola) or enterobacter cloacae (Enterobactercloacae).The Escherichia coli
(Escherichiacoli) it can be Escherichia coli (Escherichiacoli) ATCC No.25922, Escherichia coli
(Escherichiacoli) C3 (NDM-1), Escherichia coli (Escherichiacoli) G6 (NDM-5), Escherichia coli
(Escherichiacoli)3CZ20E(mcr-1+FLOR), Escherichia coli (Escherichia coli) B2 (NDM-5+mcr-
1), Escherichia coli (Escherichia coli) I1 (CRE+mcr-1), Escherichia coli (Escherichiacoli) L14 (ESBL+
Mcr-1), Escherichia coli (Escherichiacoli) F9 (ESBL+mcr-1), Escherichia coli (Escherichiacoli) 1-1
(mcr-1), Escherichia coli (Escherichiacoli) 1-5 (mcr-1), Escherichia coli (Escherichiacoli) 1-6 (mcr-
1), Escherichia coli (Escherichiacoli) 2-1 (mcr-1), Escherichia coli (Escherichiacoli) 2-6 (mcr-1) or
Escherichia coli (Escherichiacoli) 2-8 (mcr-1).
The salmonella (Salmonellaenterica) can be salmonella (Salmonellaenterica) ATCC
No.13076, salmonella (Salmonellaenterica) SH30 (mcr-1), salmonella (Salmonellaenterica)
SH170 (mcr-1), salmonella (Salmonellaenterica) SH1-3 (mcr-1), salmonella
(Salmonellaenterica) SH2-8 (mcr-1), salmonella (Salmonellaenterica) 8H-3 (mcr-1), sramana
Salmonella (Salmonellaenterica) 4CZ2 (mcr-1) or salmonella (Salmonellaenterica) 4M (NDM-1).Institute
It can be Friedlander's bacillus to state Friedlander's bacillus (Klebsiellapneumoniae)
(Klebsiellapneumoniae) 1, Friedlander's bacillus (Klebsiellapneumoniae) 2, Friedlander's bacillus
(Klebsiellapneumoniae) 3, Friedlander's bacillus (Klebsiellapneumoniae) 4, Friedlander's bacillus
(Klebsiellapneumoniae) 16-80 (mcr-1), Friedlander's bacillus (Klebsiellapneumoniae) 16-87
(mcr-1), Friedlander's bacillus (Klebsiellapneumoniae) 16-63 (mcr-1), Friedlander's bacillus
(Klebsiellapneumoniae) 4-20 (mcr-6) or Friedlander's bacillus (Klebsiellapneumoniae) 91
(mcr-6).The Pseudomonas aeruginosa (Pseudomonasaeruginosa) can be Pseudomonas aeruginosa
(Pseudomonasaeruginosa) PAO1 ATCC No.47085 or Pseudomonas aeruginosa (Pseudomonasaeruginosa)
PA14.The Acinetobacter baumannii (Acinetobacterbaumannii) can be Acinetobacter baumannii
(Acinetobacterbaumannii)2-38(mcr-1).The Aeromonas veronii (Acinetobacterbaumannii)
It can be Aeromonas veronii (Aeromonas veronii) 172 (mcr-3).The Citrobacter freundii
(Citrobacterfreumdii) it can be Citrobacter freundii (Citrobacterfreumdii) 2-4 (mcr-1).The solution
Ornithine Ural bacterium (Raoultellaornithinolytica) can be solution ornithine Ural bacterium
(Raoultellaornithinolytica)16-68(mcr-1).The serratia marcescens (Serratiamarcescens)
Can be serratia marcescens (Serratiamarcescens) 16-99 (mcr-1).The production alkali Providence
(Providenciaalcalifaciens) it can be production alkali Providence (Providenciaalcalifaciens) 16-1
(mcr-1).The raw Ural bacterium (Raoultellaplanticola) of the plant can be to plant raw Ural bacterium
(Raoultellaplanticola)16-15(mcr-1).The enterobacter cloacae (Enterobactercloacae) can be the moon
Enterobacter cloacae (Enterobactercloacae) 16-15 (mcr-1).
It is demonstrated experimentally that the oligopeptides all has certain antibacterial work to a variety of gram-positive bacterias and Gram-negative bacteria
With the minimal inhibitory concentration to gram-positive bacteria (containing drug-fast bacteria) is 8-64 μ g/mL, to Gram-negative bacteria (containing drug-fast bacteria)
Minimal inhibitory concentration be 0.5-32 μ g/mL；Oligopeptides and antibacterials (such as tetracycline, vancomycin, Ofloxacin, rifampin,
Polymyxins, ampicillin, Imipenem) all have good synergistic effect；Oligopeptides can restore the gram of resistance to polymyxins
Sensibility of the negative bacterium to polymyxins；Oligopeptides does not have hemolytic, has preferable histocompatbility；Oligopeptides can not only have
The inhibition polymyxins drug-fast bacteria of effect, and bacteremia can be treated.In addition, oligopeptides molecular weight is small, and it is simple in structure, convenient for big rule
Mould synthesizes.The present invention has great application value.
Description of the drawings
The hemolytic that Fig. 1 is linear antibacterial oligopeptides SLAP-S25 is tested.
Fig. 2 is that linear antibacterial oligopeptides SLAP-S25 treats mouse bacteria infection；Wherein PBS is PBS groups, S25 SLAP-
S25 treatment groups, polymyxins are polymyxins treatment group, and polymyxins+S25 is synergistic treatment group.
Fig. 3 is that linear antibacterial oligopeptides SLAP-S25 treats mouse bacteremia；Wherein PBS is PBS groups, S25 SLAP-S25
Treatment group, polymyxins are polymyxins treatment group, and polymyxins+S25 is synergistic treatment group.
Fig. 4 is that linear antibacterial oligopeptides SLAP-S25 treats greater wax moth larva bacterium infection；Wherein PBS is PBS groups, and S25 is
SLAP-S25 treatment groups, polymyxins are polymyxins treatment group, and polymyxins+S25 is synergistic treatment group.
Specific implementation mode
Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method is unless otherwise specified conventional method.Experiment material as used in the following examples is unless otherwise specified certainly
What routine biochemistry reagent shop was commercially available.Quantitative experiment in following embodiment is respectively provided with three repeated experiments, as a result makes even
Mean value.
BALB/c female mices are Beijing Vital River Experimental Animals Technology Co., Ltd.'s product.
MHB broth bouillons are 1.5g/L containing powdered beef, soluble starch 1.5g/L and acid hydrolyzed casein 17.5g/L
Aqueous solution.
Embodiment 1, the discovery of linear antibacterial oligopeptides SLAP-S25 and artificial synthesized
One, the discovery of linear antibacterial oligopeptides SLAP-S25
Pass through bioinformatic analysis, optimization peptide chain structure, the linear oligopeptides of structure to a variety of linear antibacterial oligopeptide sequences
Library, evaluation obtain linear oligopeptides antibacterial effect and its with a variety of antibacterials (such as beta-lactam, Tetracyclines, sugar
Peptides, quinolones, cyclic peptide and rifomycins) synergetic antibacterial effect, the present inventor obtains linear few line
Property antibacterial oligopeptides SLAP-S25.(nitrogen end 2,4-diamino-butanoic shown in the structural formula such as formula (I) of linear antibacterial oligopeptides SLAP-S25
Acetylation modification is carried out, carbon teminal alanine carries out amidation modification), molecular weight 1229.10.
Two, linear antibacterial oligopeptides SLAP-S25's is artificial synthesized
Linear antibacterial oligopeptides SLAP-S25 shown in artificial synthesized formula (I).
In the amino acid sequence such as sequence table of linear antibacterial oligopeptides SLAP-S25 shown in sequence 1.
Linear antibacterial oligopeptides SLAP-S25 hereinafter abbreviation S25.
The measurement of embodiment 2, linear antibacterial oligopeptides SLAP-S25 bacteriostatic activities
Strain to be tested refers to table 1.
Table 1
Note：A, staphylococcus aureus (Staphylococcusaureus) ATCC No.29213, bacillus subtilis
(Bacillus subtilis) ATCC No.6051, Escherichia coli (Escherichiacoli) ATCC No.25922, Salmonella
Bacterium (Salmonellaenterica) ATCC No.13076 and Pseudomonas aeruginosa (Pseudomonasaeruginosa) PAO1 ATCC
No.47085 is preserved in American Type Culture collection warehousing (abbreviation ATCC, address：American Type Culture
20110 USA of Collection (ATCC) 10801University Boulevard Manassas, VA), the public can be from the U.S.
Type culture collection warehousing obtains.
B, 215 (LZD of staphylococcus aureus (Staphylococcusaureus)R+ cfr) carry drug resistant gene cfr
And to Linezolid drug resistance.Enterococcus faecalis (Enterococcus faecalis) VRE A4 are the antibody-resistant bacterium of vancomycin.Gold
Staphylococcus aureus (Staphylococcusaureus) MRSA T144 are methicillin-resistant S staphylococcus.Name
The bacterial strain for containing " ESBL " in title is can the ESBLs-producing bacteria mediation drug resistant bacterial strain of beta-lactam antibiotic.Name
The bacterial strain containing " NDM " is that production New Delhi metallo-β-lactamase mediates the drug resistant bacterial strain of beta-lactam antibiotic in title.
The bacterial strain containing " CRE " is carbapenem antibiotic antibody-resistant bacterium in title.Contain FLO in titleRBacterial strain indicate fluorobenzene
Buddhist nun examines drug resistant bacterial strain.The bacterial strain containing " mcr " is the bacterial strain for carrying polymyxins drug resistant gene mcr in title.
C, document 1 is Liu Y, Ding S, Dietrich R,E, Zhu K.A biosurfactant
inspired heptapeptide with improved specificity to kill MRSA[J].Angewandte
Chemie International Edition, 2017,56 (6), 1486-1490. enterococcus faecalis (Enterococcus
Faecalis) VRE A4, staphylococcus aureus (Staphylococcus aureus) MRSA T144, staphylococcus aureus
(Staphylococcusaureus)215(LZDR+ cfr) and Pseudomonas aeruginosa (Pseudomonasaeruginosa) PA14 in text
Offer the title in 1 be followed successively by Enterococcus faecalis VRE A4, MRSA T144,
Staphylococcusaureus215(LZDR+ cfr) and PseudomonasaeruginosaPA14.
D, document 2 is Yang Z Q, Huang Y L, Zhou H W, et al.Persistent carbapenem-
resistant Klebsiella pneumoniae：A Trojan horse [J] .Lancet Infectious Diseases,
2017,18 (1):22. Friedlander's bacillus (Klebsiellapneumoniae) 1, Friedlander's bacillus
(Klebsiellapneumoniae) 2, Friedlander's bacillus (Klebsiellapneumoniae) 3 and Friedlander's bacillus
(Klebsiellapneumoniae) 4 title in document 2 is followed successively by CRKP1, CRKP2, CRKP3 and CRKP4.
E, document 3 is Ling Z, Yin W, Li H, et al.Chromosome-mediated mcr-3variants in
Aeromonas veronii from chicken meat [J] .Antimicrobial agents and chemotherapy,
2017,61 (11)：E01272-17. name of the Aeromonas veronii (Aeromonas veronii) 172 (mcr-3) in document 3
Referred to as Aeromonas veronii 172.
F, the separation process of the bacterial strain in source voluntarily to detach is as follows：(1) provinces such as Shandong, Henan, Sichuan are acquired respectively
Part distance different slaughterhouses farther out, hog snout swab in farm, be sold chicken, pork etc. in supermarket, be impregnated in BHI
5min in fluid nutrient medium, and filtered, filtrate is stored in spare in ESwab pipes；(2) different filtrates are dipped with oese to apply
It is distributed on the Ke Majia urethra CHROMagar orientation mediums of the polymyxins containing 2 μ g/mL, 37 DEG C of culture 18h select red (large intestine
Bacillus), blue (Klebsiella Pneumoniae category, citrobacter category, Enterobacter) or cream-colored translucent (pseudomonas)
Monoclonal bacterial strain be transferred on BHI tablets, 37 DEG C culture 18h.Appropriate bacterium transfer is scraped from tablet with super thick filter paper
Into 2ml sterile test tubes, it is placed in -20 DEG C of refrigerators and saves backup；(3) monoclonal inoculation is selected in BHI fluid nutrient mediums
Middle increasing bacterium, then extracts genomic DNA；(4) using the genomic DNA of step (3) extraction as template, using standard PCR amplification
16SrRNA and the method for sequencing further confirm Pseudomonas；Meanwhile expanding mcr genetic tests.The primer of amplification 16S rRNA is
5 ＇-AGAGTTTGATCCTGGCTCAG-3 ＇ and 5 ＇-TACGGCTACCTTGTTACGACTT-3 '；Reacting operating parameter is：94℃
10min；94 DEG C of 30s, 55 DEG C of 40s, 72 DEG C, 2min, 30cycles；72 DEG C, 10min.The primer for expanding mcr-1 is 5 '-
CGGTCAGTCCGTTTGTTC-3 ' and 5 '-CTTGGTCGGTCTGTAGGG-3 '；Reacting operating parameter is：94℃10min；94℃
30s, 55 DEG C of 30s, 72 DEG C, 50s, 36cycles；72 DEG C, 5min.The primer for expanding mcr-3 is 5 '-
TTGGCACTGTATTTTGCATTT-3 ' and 5 '-TTAACGAAATTGGCTGGAACA-3 '；Reacting operating parameter is：95 DEG C of 30s,
50 DEG C of 30s, 72 DEG C, 45s, 30cycles；72 DEG C, 7min.The primer for expanding mcr-6 is 5 '-AACCGCCAGAGCACAGAATT-
3 ' and 5 '-AACCGCCAGAGCACAGAATT-3 '；Reacting operating parameter is：94℃10min；94 DEG C of 30s, 60 DEG C of 30s, 72 DEG C,
50s, 36cycles；72 DEG C, 5min.
Using broth dilution method (Clinical and Laboratory Standards Institute, CLSI, 2017)
Detect minimal inhibitory concentrations (MIC) of the linear antibacterial oligopeptides SLAP-S25 to strain to be tested.Detect linear antibacterial oligopeptides SLAP-
The step of S25 is to the minimal inhibitory concentration of each strain to be tested is as follows：
1, with MHB broth bouillon suspension strains to be tested, it is 1 × 10 to obtain bacteria concentration8The bacteria suspension of CFU/mL.
2, line taking antibacterial oligopeptides SLAP-S25 dilutes with water dissolution and with MHB broth bouillons, obtains a concentration of 512 μ
The linear antibacterial oligopeptides SLAP-S25 solution of g/mL.
3,96 orifice plates are taken, MHB broth bouillons (per 100 μ L of hole) are added, the linear of step 2 preparation is added per hole for first row
Antibacterial oligopeptides SLAP-S25 solution (per 100 μ L of hole), doubling dilution to the tenth hole, the tenth hole discards 100 μ L liquid, later per hole
Be added the bacteria suspension of 100 μ L steps 1 preparation, final linear antibacterial oligopeptides SLAP-S25 in hole a concentration of 0.25,0.5,
1.0,2.0,4.0,8.0,16.0,32.0 μ g/mL, 64.0 μ g/mL or 128.0 μ g/mL, 37 DEG C of stationary culture 16h-20h, observation
It is MIC to inhibit the minimum concentration of bacterial growth in hole.Negative control hole and Positive control wells are set in 96 orifice plates.It is each cloudy
Property control wells be added 200 μ LMHB broth bouillons.100 μ LMHB broth bouillons and 100 μ L steps are added in each Positive control wells
Rapid 1 bacteria suspension prepared.
Experimental result is shown in Table 2.The result shows that linear antibacterial oligopeptides SLAP-S25 is to gram-positive bacteria (such as golden yellow Portugal
Grape coccus, enterococcus faecalis, bacillus subtilis) and Gram-negative bacteria (Escherichia coli, salmonella, Friedlander's bacillus,
Pseudomonas aeruginosa, Acinetobacter baumannii, Aeromonas veronii, Citrobacter freundii, solution ornithine Ural bacterium, Serratia
Bacterium, plants raw Ural bacterium and enterobacter cloacae at production alkali Providence) good antibacterial activity is all had, to gram sun
Property bacterium (contain drug-fast bacteria) minimal inhibitory concentration be 8-64 μ g/mL, it is antibacterial dense to the minimum of Gram-negative bacteria (containing drug-fast bacteria)
Degree is 0.5-32 μ g/mL.
Table 2
The synergistic effect of embodiment 3, linear antibacterial oligopeptides SLAP-S25 and antibacterials
Tetracycline belongs to tetracycline medication.Vancomycin belongs to sugared peptide medicament.Ofloxacin belongs to Du-6859a
Object.Rifampin belongs to rifamycin drug.Polymyxins belongs to cyclic peptide drug.It is interior that ampicillin and Imipenem belong to β-
Amides drug.
One, the synergistic effect of linear antibacterial oligopeptides SLAP-S25 and antibacterials
1, the synergistic effect of linear antibacterial oligopeptides SLAP-S25 and antibacterials to Gram-negative bacteria
Antibacterials are tetracycline, vancomycin, Ofloxacin, rifampin, polymyxins or Imipenem.
The synergistic effect of linear antibacterial oligopeptides SLAP-S25 and different antibacterials are detected using chessboard analytic approach, test bacterium
Strain is Escherichia coli (Escherichia coli) B2 (NDM-5+mcr-1), and chessboard analytic approach is as follows：
1) MHB broth bouillon suspension test strains are used, it is 1 × 10 to obtain bacteria concentration8The bacteria suspension of CFU/mL.
2) line taking antibacterial oligopeptides SLAP-S25 dilutes with water dissolution and with MHB broth bouillons, obtains a concentration of 256 μ
The linear antibacterial oligopeptides SLAP-S25 solution of g/mL.
3) antibacterials are taken, is diluted with water dissolution and with MHB broth bouillons, obtains the antimicrobial of a concentration of 128 μ g/mL
Object solution.
4) 96 orifice plates are taken, 100 μ LMHB broth bouillons are added per hole, last column is added 100 μ L steps 2) per hole and prepares
Linear antibacterial oligopeptides SLAP-S25 solution (a concentration of 512 μ g/mL in the first hole), from the 8th row doubling dilution to the second row；The
Antibacterials solution (per 100 μ L of hole) prepared by step 3) is added per hole for one row, and doubling dilution is to the tenth row, later per hole
Be added 100 μ L steps 1) preparation bacteria suspension, 37 DEG C of stationary culture 16h-20h, observe linear antibacterial oligopeptides SLAP-S25 with not
Minimum concentration both when inhibition bacterial growth is used in combination with antibacterials combines.Positive control wells are set, it is each positive right
The bacteria suspension of 100 μ LMHB broth bouillons and the preparation of 100 μ L steps 1) is added according to hole.
Classification Mlc FIC indexes are calculated according to following formula：
FIC=MIC (A use in conjunction)/MIC (A is applied alone)+MIC (B use in conjunction)/MIC (B is applied alone)
Experimental result is shown in Table 3.The result shows that linear antibacterial oligopeptides SLAP-S25 can be by antibacterials to Escherichia coli
The MIC of (Escherichia coli) B2 (NDM-5+mcr-1) reduces 4-128 times.Linear antibacterial oligopeptides SLAP-S25 and antibacterial
The FIC indexes of drug (Imipenem, tetracycline, vancomycin, Ofloxacin, rifampin or polymyxins) are below 0.5, because
This linear antibacterial oligopeptides SLAP-S25 and above-mentioned six kinds of antibacterials all have good synergistic effect.
Table 3
| FICAntibacterials | FICS25 | FIC indexes | |
| Tetracycline | 0.031 | 0.250 | 0.281 |
| Vancomycin | 0.031 | 0.063 | 0.094 |
| Ofloxacin | 0.031 | 0.063 | 0.094 |
| Rifampin | 0.031 | 0.001 | 0.032 |
| Polymyxins | 0.016 | 0.031 | 0.047 |
| Imipenem | 0.0625 | 0.125 | 0.187 |
2, the synergistic effect of linear antibacterial oligopeptides SLAP-S25 and antibacterials to gram-positive bacteria
Antibacterials are tetracycline, vancomycin, Ofloxacin, ampicillin, rifampin or polymyxins.
Collaboration using the linear antibacterial oligopeptides SLAP-S25 of chessboard assay and antibacterials to gram-positive bacteria
Effect, for specific experiment method with step 1, test strain is staphylococcus aureus (Staphylococcus aureus) ATCC
29213, staphylococcus aureus (Staphylococcus aureus) MRSA T144, bacillus subtilis (Bacillus
subtilis)ATCC6051。
Experimental result is shown in Table 4.The result shows that gram-positive bacteria, linear antibacterial oligopeptides SLAP-S25 and ampicillin
Play stronger synergistic effect.To three kinds of gram-positive bacterias, the FIC indexes of linear antibacterial oligopeptides SLAP-S25 and ampicillin
It is below 0.5, therefore, linear antibacterial oligopeptides SLAP-S25 and ampicillin make gram-positive bacteria with good collaboration
With.
Table 4
Two, linear antibacterial oligopeptides SLAP-S25 restores sensitivity of the drug resistant Gram-negative bacteria of polymyxins to polymyxins
Property
The Gram-negative bacteria of the drug resistant gene containing mcr refers to the row of table 5 the 1st, is the bacterial strain of the mcr drug resistant gene positives.
Using the linear antibacterial oligopeptides SLAP-S25 of chessboard assay and antibacterials to mcr positive Gram-negative bacterias
Synergistic effect, specific experiment method is the same as in step 11.
Experimental result is shown in Table 5.The result shows that linear antibacterial oligopeptides SLAP-S25 and the FIC indexes of polymyxins are below
0.375, therefore linear antibacterial oligopeptides SLAP-S25 can restore the Gram-negative bacteria of resistance to polymyxins (except Friedlander's bacillus
Outside) to the sensibility of polymyxins.
Table 5
Three, the synergistic effect of linear antibacterial oligopeptides SLAP-S25 and antibacterials to Friedlander's bacillus
Antibacterials are tetracycline, vancomycin, Ofloxacin, ampicillin, Imipenem, rifampin or more Acarasiales
Element.
Measure linear antibacterial oligopeptides SLAP-S25 and antibacterials to polymyxins drug resistance and not drug resistant kerekou pneumonia primary
The synergistic effect of Salmonella, wherein polymyxins drug resistance Friedlander's bacillus are Friedlander's bacillus (Klebsiella
Pneumoniae) 16-63 (mcr-1), Friedlander's bacillus (Klebsiella pneumoniae) 4-20 (mcr-6) or lung
Scorching Klebsiella (Klebsiella pneumoniae) 91 (mcr-6), drug resistance Friedlander's bacillus is not lung to polymyxins
Scorching Klebsiella (Klebsiella pneumoniae) 2.
The same step of synergistic effect specific method of the linear antibacterial oligopeptides SLAP-S25 of chessboard assay and antibacterials
1 in one.
Experimental result is shown in Table 6：For polymyxins drug resistance and non-drug resistance Friedlander's bacillus, linear antibacterial oligopeptides
The FIC indexes of SLAP-S25 and rifampin are below 0.065, the FIC indexes of linear antibacterial oligopeptides SLAP-S25 and Ofloxacin
It is below 0.127, the FIC indexes of linear antibacterial oligopeptides SLAP-S25 and tetracycline are below 0.129；For polymyxins intolerant to
The FIC indexes of medicine Friedlander's bacillus, linear antibacterial oligopeptides SLAP-S25 and Imipenem are less than 0.187.The result shows that line
Property antibacterial oligopeptides SLAP-S25 can reduce antibacterials rifampin, Ofloxacin, tetracycline and Imipenem to kerekou pneumonia
The minimal inhibitory concentration of primary Salmonella.To Friedlander's bacillus, linear antibacterial oligopeptides SLAP-S25 and antibacterials rifampin, oxygen
Flucloxacillin, tetracycline and Imipenem can play stronger synergistic effect.
Table 6-1
Table 6-2
Table 6-3
The hemolytic experiment of embodiment 4, linear antibacterial oligopeptides SLAP-S25
PBS buffer solution is the PBS buffer solution of pH7.4,0.01M.
The preparation method of 8% red blood cell suspension is as follows：(1) de- fiber sheep blood 10mL, 3000g is taken to centrifuge 10min, abandon
Clearly, it is precipitated；(2) precipitation for taking step (1) to obtain, is cleaned twice with PBS buffer solution, obtains 100% red blood cell suspension；
(3) 100% red blood cell suspensions of 8mL and 92mL PBS buffer solution are mixed, obtains 8% red blood cell suspension.
Then line taking antibacterial oligopeptides SLAP-S25 is diluted with water dissolution with PBS buffer solution, obtain a concentration of 1024 μ g/
The linear antibacterial oligopeptides SLAP-S25 solution of mL.
1,96 orifice plates are taken, 100 μ L PBS buffer solution are added per hole.
2, after completing step 1, take 96 orifice plate, the first hole that the linear antibacterial oligopeptides SLAP-S25 solution of 100 μ L is added, times
Than being diluted to octal；In second hole to octal, the concentration of linear antibacterial oligopeptides SLAP-S25 is followed successively by 512 μ g/mL, 256 μ
G/mL, 128 μ g/mL, 64 μ g/mL, 32 μ g/mL, 16 μ g/mL and 8 μ g/mL (three multiple holes of each concentration).
3, after completing step 2,96 orifice plate is taken, 100 μ L, 8% red blood cell suspensions are added per hole, 37 DEG C are incubated 1h,
3000g centrifuges 10min, draws 100 μ L supernatants.
4, the OD for the supernatant that measuring process 3 obtains576nmValue, i.e., linear antibacterial oligopeptides SLAP-S25 solution Os D576nmValue.
5, step 2 is replaced with into step a, other steps are constant, obtain negative control OD576nmValue.Step a is：Take institute
96 orifice plates are stated, 100 μ L PBS buffer solution are added per hole.
6, step 2 is replaced with into step b, other steps are constant, obtain positive control OD576nmValue.Step b is：Take institute
96 orifice plates are stated, 100 μ L Triton X-100 are added per hole.
Hemolysis rate is calculated according to following formula：
Hemolysis rate=(linear antibacterial oligopeptides SLAP-S25 solution Os D576nmValue-negative control OD576nmValue)/(positive control
OD576nmValue-negative control OD576nmValue) × 100%.
Part of test results is shown in Fig. 1.The result shows that when a concentration of 8-256 μ g/mL of linear antibacterial oligopeptides SLAP-S25,
Do not have hemolytic.
Embodiment 5, linear antibacterial oligopeptides SLAP-S25 are in treatment mouse bacteria infection and bacteremia
One, linear antibacterial oligopeptides SLAP-S25 treats mouse bacteria infection
PBS buffer solution is the PBS buffer solution of pH7.4,0.01M.
E. coli suspension：Escherichia coli (Escherichia coli) B2 (NDM-5+mcr- are resuspended with PBS buffer solution
1) E. coli suspension, is obtained；In the E. coli suspension, Escherichia coli (Escherichia coli) B2 (NDM-5+
Mcr-1) a concentration of 1.0 × 109CFUs/mL。
Polymyxins solution：800mg polymyxins is dissolved in 1L sterile waters to obtain.
SLAP-S25 solution：The linear antibacterial oligopeptides SLAP-S25 of 800mg are dissolved in 1L sterile waters to obtain.
Mixed solution：800mg polymyxins and the linear antibacterial oligopeptides SLAP-S25 of 800mg are dissolved in 1L sterile waters and obtained.
1, mice group is handled
It is the BALB/c female mices of 18-22g to take 24 weight, is randomly divided into PBS groups, SLAP-S25 treatment groups, glues more
Rhzomorph treatment group and synergistic treatment group (every group 6), are handled as follows respectively：
PBS groups：Right side of mice leg muscle injects the E. coli suspension of 0.1mL；1h pneumoretroperitoneums inject 0.2mL PBS
Buffer solution；
SLAP-S25 treatment groups：Right side of mice leg muscle injects the E. coli suspension of 0.1mL；1h pneumoretroperitoneums are injected
0.2mLSLAP-S25 solution；
Polymyxins treatment group：Right side of mice leg muscle injects the E. coli suspension of 0.1mL；1h pneumoretroperitoneums are injected
0.2mL polymyxins solution；
Synergistic treatment group：Right side of mice leg muscle injects the E. coli suspension of 0.1mL；1h pneumoretroperitoneums are injected
0.2mL mixed solutions.
2, the clump count of mouse thigh infection is measured
By the mouse euthanasia of 48h after completion step 1, right lateral thigh muscle is taken, then meat piece homogenate, mixing take
It is coated on TSA culture mediums in right amount, measures clump count.
Experimental result is shown in Fig. 2.The result shows that compared with polymyxins treatment group, synergistic treatment group can effectively reduce small
The clump count of mouse thigh infection.
Two, linear antibacterial oligopeptides SLAP-S25 treats mouse bacteremia
PBS buffer solution is the PBS buffer solution of pH7.4,0.01M.
E. coli suspension：Escherichia coli (Escherichia coli) B2 (NDM-5+mcr- are resuspended with PBS buffer solution
1) E. coli suspension, is obtained；In the E. coli suspension, Escherichia coli (Escherichia coli) B2 (NDM-5+
Mcr-1) a concentration of 1.5 × 1010CFUs/mL。
Polymyxins solution：800mg polymyxins is dissolved in 1L sterile waters to obtain.
SLAP-S25 solution：The linear antibacterial oligopeptides SLAP-S25 of 800mg are dissolved in 1L sterile waters to obtain.
Mixed solution：800mg polymyxins and the linear antibacterial oligopeptides SLAP-S25 of 800mg are dissolved in 1L sterile waters and obtained.
1, mice group is handled
It is the BALB/c female mices of 18-22g to take 24 weight, is randomly divided into PBS groups, SLAP-S25 treatment groups, glues more
Rhzomorph treatment group and synergistic treatment group (every group 6), are handled as follows respectively：
PBS groups：Mouse peritoneal injects the E. coli suspension of 0.2mL；1h pneumoretroperitoneums inject 0.2mL PBS buffer solution；
SLAP-S25 treatment groups：Mouse peritoneal injects the E. coli suspension of 0.2mL；1h pneumoretroperitoneums are injected
0.2mLSLAP-S25 solution；
Polymyxins treatment group：Mouse peritoneal injects the E. coli suspension of 0.2mL；It is more that 1h pneumoretroperitoneums inject 0.2mL
Colistin solution；
Synergistic treatment group：Mouse peritoneal injects the E. coli suspension of 0.2mL；It is molten that 1h pneumoretroperitoneums inject 0.2mL mixing
Liquid.
2, survival rate is counted
To the mouse of 48h after completion step 1, survival rate is counted.
Experimental result is shown in A in Fig. 3.The result shows that compared with polymyxins treatment group, the mouse survival rate of synergistic treatment group
It significantly improves.
3, the clump count in mouse organs is measured
The mouse of 48h after step 1 is taken into, separation organ (such as heart, liver, spleen, lung and kidney) then measures each organ
Interior clump count.
Experimental result is shown in B in Fig. 3.The result shows that compared with polymyxins treatment group, the mouse core of synergistic treatment group, liver,
Clump count in spleen, lung or kidney is significantly reduced.
The above results show that linear antibacterial oligopeptides SLAP-S25 can be used for treating mouse bacteria infection and bacteremia.
Embodiment 6, linear antibacterial oligopeptides SLAP-S25 treat greater wax moth larva bacterium infection
PBS buffer solution is the PBS buffer solution of pH7.4,0.01M.
E. coli suspension：Escherichia coli (Escherichia coli) B2 (NDM-5+mcr- are resuspended with PBS buffer solution
1) E. coli suspension, is obtained；In the E. coli suspension, Escherichia coli (Escherichia coli) B2 (NDM-5+
Mcr-1) a concentration of 1.0 × 107CFUs/mL。
Polymyxins solution：160mg polymyxins is dissolved in 1L sterile waters to obtain.
SLAP-S25 solution：The linear antibacterial oligopeptides SLAP-S25 of 160mg are dissolved in 1L sterile waters to obtain.
Mixed solution：160mg polymyxins and the linear antibacterial oligopeptides SLAP-S25 of 160mg are dissolved in 1L sterile waters and obtained.
1, greater wax moth larva packet transaction
It is the greater wax moth larva of 190-210mg to take 32 weight, is randomly divided into PBS groups, SLAP-S25 treatment groups, glues more
Rhzomorph treatment group and synergistic treatment group (every group 8), are handled as follows respectively：
PBS groups：The second abdominal foot of greater wax moth larva lower-left injects 0.01mL E. coli suspensions；Second abdominal foot under 1h rear rights
Inject 0.01mL PBS buffer solution；
SLAP-S25 treatment groups：The second abdominal foot of greater wax moth larva lower-left injects 0.01mL E. coli suspensions；1h rear rights
Lower second abdominal foot injects 0.01mL SLAP-S25 solution；
Polymyxins treatment group：The second abdominal foot of greater wax moth larva lower-left injects 0.01mL E. coli suspensions；1h rear rights
Lower second abdominal foot injects 0.01mL polymyxins solution；
Synergistic treatment group：The second abdominal foot of greater wax moth larva lower-left injects 0.01mL E. coli suspensions；Under 1h rear rights
Two abdominal foots inject 0.01mL mixed solutions.
2, survival rate is counted
The 1st day, the 2nd day, the 3rd day, the 4th day and the 5th day after completing step 1, the survival of greater wax moth larva is counted
Rate.
Experimental result is shown in Fig. 4.The result shows that compared with polymyxins treatment group, the greater wax moth larva of synergistic treatment group is deposited
Motility rate significantly improves.
Embodiment 7, the derivative of linear antibacterial oligopeptides SLAP-S25 and its application
1, substitution that the amino acid sequence of linear antibacterial oligopeptides SLAP-S25 is passed through to four or less amino acid residues, obtains
(substituted amino acid letter overstriking shows that the amino acid of all oligopeptides nitrogen ends carries out second to oligopeptides shown in the 2nd to 15 row in table 7
The amino acid of acylated modification, carbon teminal carries out amidation modification).The amino acid sequence of linear antibacterial oligopeptides SLAP-S25 is passed through four
The missing of a following amino acid residue obtains oligopeptides shown in the 16th to 18 row in table 7.Change linear antibacterial oligopeptides SLAP-S25
In at least one amino acid chirality, obtaining oligopeptides shown in the 19th to 21 row in table 7, (Val changes into L by D, and Phe is changed by D
For L).
Each oligopeptides shown in artificial synthesized table 7.
According to the method for embodiment 2, the bacteriostatic activity of oligopeptides shown in table 7 is measured.Strain to be tested is Escherichia coli
(Escherichia coli) ATCC No.25922, Escherichia coli (Escherichia coli) B2 (NDM-5+mcr-1) or gold
Staphylococcus aureus (Staphylococcusaureus) ATCC 29213.
4th row during experimental result is shown in Table 7.
Table 7
2, the synergistic effect of oligopeptides and antibacterials to Gram-negative bacteria shown in table 7
Test strain is Escherichia coli (Escherichia coli) B2 (NDM-5+mcr-1).
The synergistic effect of each oligopeptides and polymyxins shown in chessboard assay table 7, specific method is the same as embodiment 3
1 in step 1.
Part of test results is shown in Table 8.The result shows that the FIC indexes of each oligopeptides and polymyxins shown in table 7 are below
0.5.Therefore each oligopeptides and polymyxins shown in table 7 all have good synergistic effect.
Table 8
| Oligopeptides title | FIC |
| SLAPS25-1 | 0.375 |
| SLAPS25-2 | ≤0.15625 |
| SLAPS25-3 | 0.0468 |
| SLAPS25-4 | 0.375 |
| SLAPS25-5 | ≤0.3125 |
| SLAPS25-6 | 0.0468 |
| SLAPS25-7 | 0.078 |
| SLAPS25-8 | 0.078 |
| SLAPS25-9 | 0.093 |
| SLAPS25-10 | 0.09375 |
| SLAPS25-11 | 0.031 |
| SLAPS25-13 | 0.125 |
| SLAPS25-15 | 0.094 |
| SLAPS25-16 | 0.078 |
| SLAPS25-17 | 0.156 |
| SLAPS25-18 | 0.25 |
| SLAPS25-19 | 0.25 |
| SLAPS25-20 | 0.156 |
<110>China Agricultural University
<120>Linear antibacterial oligopeptides SLAP-S25 and its application
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<170> PatentIn version 3.5
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Claims (10)
- Be a1 1. a kind of oligopeptides) or a2) or a3) a4) or a5) or a6)：A1) oligopeptides shown in sequence 1 in sequence table；A2) by a1) nitrogen end of the oligopeptides carries out acetylation modification, carbon teminal carries out amidation modification, obtained oligopeptides；A3) structural formula oligopeptides as shown in formula (I)；A4) change a1) or a2) or a3) shown in oligopeptides at least one amino acid chirality, obtained oligopeptides；A5) by a1) or a2) or a3) or a4) shown in oligopeptides join end to end, obtained cyclic oligopeptides；A6) by a1) or a2) or a3) a4) or a5) shown in oligopeptides amino acid sequence pass through one or several amino acid residues Substitution and/or the oligopeptides with the same function that lacks and ors add.
- Be b1 2. the application of oligopeptides described in claim 1) or b2) or b3) b4) or b5) b6) or b7) or b8)：B1) inhibit bacterium；B2 Antibacterial) is prepared；B3) prevent disease caused by bacterium infection；B4 it) prepares thin for preventing The product of disease caused by bacterium infection；B5 disease caused by bacterium infection) is treated；B6 it) prepares and causes for treating bacterium infection Disease product；B7 food spoilage) is prevented；B8 the product for preventing food spoilage) is prepared.
- 3. the application of oligopeptides joint antibacterials, is c1 described in claim 1) or c2) or c3) c4) or c5) or c6)：C1) inhibit bacterium；C2 Antibacterial) is prepared；C3) prevent disease caused by bacterium infection；C4 it) prepares thin for preventing The product of disease caused by bacterium infection；C5 disease caused by bacterium infection) is treated；C6 it) prepares and causes for treating bacterium infection Disease product.
- 4. application as claimed in claim 3, it is characterised in that：The antibacterials are tetracycline medication, beta-lactam Drug, sugared peptide medicament, quinolone drugs, rifamycin drug or cyclic peptide drug.
- 5. oligopeptides described in claim 1 is restoring the Gram-negative bacteria of resistance to polymyxins to answering in the sensibility of polymyxins With.
- 6. oligopeptides described in claim 1 combines antibacterials in resistance to polymyxins or intolerant to the Friedlander's bacillus of polymyxins Synergistic effect application.
- 7. application of the oligopeptides joint antibacterials in the synergistic effect of bacterium described in claim 1.
- 8. a kind of Antibacterial contains oligopeptides described in claim 1.
- 9. Antibacterial as claimed in claim 8, it is characterised in that：The Antibacterial also contains antibacterials.
- 10. the application or the Antibacterial as described in claim 8 or 9, feature as described in claim 2,3,4 or 7 exist In：The bacterium is gram-positive bacteria or Gram-negative bacteria.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113999282A (en) * | 2021-01-21 | 2022-02-01 | 扬州大学 | Antibacterial peptide LI7, repeated polypeptides LI14 and LI21 thereof, and derivatives and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010029196A1 (en) * | 2008-09-10 | 2010-03-18 | Universidad De Barcelona | Antibacterial peptide compounds |
| CN106632606A (en) * | 2016-12-26 | 2017-05-10 | 中国农业大学 | Antimicrobial lipopeptide bacaucin derivatives and application thereof in bacterial infection inhibition |
| CN106674331A (en) * | 2016-12-26 | 2017-05-17 | 中国农业大学 | Antibacterial lipopeptide bacaucin and preparation method and application thereof |
| CN107188944A (en) * | 2017-06-08 | 2017-09-22 | 中国农业科学院饲料研究所 | Extra large earthworm antimicrobial peptide NZ17074 derived peptides N6 and its application |
-
2018
- 2018-04-02 CN CN201810282987.9A patent/CN108467424B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010029196A1 (en) * | 2008-09-10 | 2010-03-18 | Universidad De Barcelona | Antibacterial peptide compounds |
| CN106632606A (en) * | 2016-12-26 | 2017-05-10 | 中国农业大学 | Antimicrobial lipopeptide bacaucin derivatives and application thereof in bacterial infection inhibition |
| CN106674331A (en) * | 2016-12-26 | 2017-05-17 | 中国农业大学 | Antibacterial lipopeptide bacaucin and preparation method and application thereof |
| CN107188944A (en) * | 2017-06-08 | 2017-09-22 | 中国农业科学院饲料研究所 | Extra large earthworm antimicrobial peptide NZ17074 derived peptides N6 and its application |
Non-Patent Citations (4)
| Title |
|---|
| YUAN LIU: "Nonribosomal antibacterial peptides that target multidrug-resistant bacteria", 《NATURAL PRODUCT REPORTS》 * |
| YUAN LIU等: "Discovery of Linear Low-Cationic Peptides to Target Methicillin-Resistant Staphylococcus aureus in Vivo", 《ACS INFECT. DIS.》 * |
| 刘源: "新型非核糖体肤类衍生物的发掘及其抗菌机制研究新型非核糖体肤类衍生物的发掘及其抗菌机制研究", 《中国博士学位论文全文数据库（电子期刊）农业科技辑》 * |
| 杨志强等: "非核糖体肽SLAP-25联合头孢他啶对耐碳青霉烯肺炎克雷伯菌的体外抗菌作用研究", 《中国畜牧兽医学会兽医药理毒理学分会第十五次学术讨论会论文集》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113999282A (en) * | 2021-01-21 | 2022-02-01 | 扬州大学 | Antibacterial peptide LI7, repeated polypeptides LI14 and LI21 thereof, and derivatives and application thereof |
| CN113999282B (en) * | 2021-01-21 | 2023-08-22 | 扬州大学 | Antibacterial peptide LI7, repeated polypeptides LI14 and LI21 thereof, derivatives and application thereof |
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