CN108467424B - Linear antibacterial oligopeptide SLAP-S25 and application thereof - Google Patents
Linear antibacterial oligopeptide SLAP-S25 and application thereof Download PDFInfo
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Abstract
The invention discloses a linear antibacterial oligopeptide SLAP-S25 and application thereof. The linear antibacterial oligopeptide SLAP-S25 is obtained by performing acetylation modification on the nitrogen end and performing amidation modification on the carbon end of an oligopeptide shown in a sequence 1 in a sequence table. Experiments prove that the linear antibacterial oligopeptide SLAP-S25 has certain antibacterial action on various gram-positive bacteria and gram-negative bacteria, has good synergistic action with antibacterial drugs (such as tetracycline, vancomycin, ofloxacin and rifampicin), can restore the sensitivity of polymyxin-resistant gram-negative bacteria to polymyxin, enhances the sensitivity of klebsiella pneumoniae to various antibacterial drugs, has no hemolytic property and good histocompatibility, can effectively inhibit polymyxin-resistant bacteria and can treat bacteremia. The linear antibacterial oligopeptide SLAP-S25 has small molecular weight and simple structure, and is convenient for large-scale synthesis. The invention has great application value.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a linear antibacterial oligopeptide SLAP-S25 and application thereof.
Background
The antibacterial drug plays a vital role in treating and controlling microbial infection diseases, and the health of human and animals is guaranteed. However, the problem of bacterial resistance due to the large-scale and non-regulated use of antibacterial agents is becoming more serious, rendering many antibacterial agents less effective or ineffective against bacterial infections. 2011 south Asia finds gram-negative bacteria capable of producing NDM-1 and has not reported pathogenic bacteria Shigella and Vibrio cholerae; the discovery of community-associated methicillin-resistant staphylococcus aureus (CA-MRSA) in north america of the same year; in 2015, China discovered that plasmid-mediated polymyxin-resistant (MCR-1) escherichia coli, and a 'last line of defense' for treating drug-resistant gram-negative bacteria was at a risk. If the problem of the drug resistance of bacteria is unreasonably controlled and solved, the health of human beings is threatened greatly, and the social panic is caused.
In order to ensure the health of human beings and the continuous development of animal husbandry, the development of effective treatment schemes or the search for novel antibacterial drugs and synergists thereof is imperative. Firstly, the bioavailability and the service life of the antibacterial can be improved, the use amount of the existing antibacterial is reduced, and the development of drug-resistant bacteria is effectively prevented and controlled by the combined application of the antibacterial or the antibacterial synergist. For example, the earliest discovered antibacterial drug synergist, trimethoprim, has weak antibacterial action, and when the synergist is used with sulfonamides, the antibacterial effect of the sulfonamides can be improved by tens of times; studies have shown that Aspergillus A restores the sensitivity of metal-producing beta-lactamase (NDM-1) gram-negative bacteria to meropenem (Nature, 2014, 510, 503-. In addition, the linear antibacterial oligopeptide (SLAP) has the advantages of simple structure, low production cost, high safety and the like, and has a good application prospect. The development of a novel linear oligopeptide as a broad-spectrum antibacterial drug synergist to recover or improve the antibacterial activity of the existing antibacterial drugs is one of important means for controlling the drug resistance of bacteria. In conclusion, the antibacterial drug synergist and the linear antibacterial oligopeptide can provide a favorable guarantee for controlling the development of the drug-resistant bacteria, and have higher development and research values.
Disclosure of Invention
The invention aims to provide an antibacterial preparation.
The invention firstly provides an oligopeptide which can be a1) or a2) or a3) or a4) or a5) or a 6):
a1) oligopeptides shown in a sequence 1 in a sequence table;
a2) performing acetylation modification on the nitrogen end and amidation modification on the carbon end of the oligopeptide of a1) to obtain the oligopeptide;
a3) oligopeptide with a structural formula shown as a formula (I);
a4) changing the chirality of at least one amino acid in the oligopeptide shown in a3) to obtain an oligopeptide;
a5) cyclic oligopeptides are obtained by connecting the oligopeptides a1) or a2) or a3) or a4) end to end;
a6) the oligopeptide with the same function is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence of the oligopeptide shown in a1) or a2) or a3) or a4) or a 5).
In the above a6), the substitution and/or deletion and/or addition of one or more amino acid residues may be a substitution and/or deletion and/or addition of not more than 4 amino acid residues.
In the above a6), the amino acid may be a rare amino acid and/or a natural amino acid.
The oligopeptide described in a6) may be specifically oligopeptide represented by SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 16, SEQ ID NO 17 or SEQ ID NO 18 (which correspond to SLAPS25-1, SLAPS25-2, SLAPS25-3, SLAPS25-4, SLAPS25-5, SLAPS25-6, SLAPS25-7, SLAPS25-8, SLAPS25-9, SLAPS25-10, SLAPS25-11, SLAPS 3985-12, SLAPS 38-13, SLAPS 48-14, SLAPS25-15, SLAPS25-16 and SLAPS25-17 in Table 7 in the above). SLAPS25-1 to SLAPS25-14 are obtained by substituting oligopeptide with a structural formula shown in formula (I) by not more than 4 amino acid residues. SLAPS25-15 to SLAPS25-17 are obtained by deleting not more than 4 amino acid residues from oligopeptide with a structural formula shown in formula (I).
The oligopeptide in a4) can be specifically SLAPS25-18, SLAPS25-19 or SLAPS25-20 (which correspond to sequence 19, sequence 20 and sequence 21 in the sequence table in sequence) in Table 7 in example 7.
In all of the oligopeptides shown in Table 7 in example 7, the nitrogen-terminal amino acid was modified by acetylation, and the carbon-terminal amino acid was modified by amidation.
The application of the oligopeptide also belongs to the protection scope of the invention; the application of the oligopeptide can be b1) or b2) or b3) or b4) or b5) or b6) or b7) or b 8): b1) inhibiting bacteria; b2) preparing an antibacterial preparation; b3) preventing diseases caused by bacterial infection; b4) preparing a product for preventing diseases caused by bacterial infection; b5) treating diseases caused by bacterial infection; b6) preparing a product for treating diseases caused by bacterial infection; b7) preventing food spoilage; b8) preparing a product for preventing food spoilage.
The application of the oligopeptide in combination with antibacterial drugs also belongs to the protection scope of the invention; the application of the oligopeptide combined with antibacterial drugs can be c1), c2), c3), c4), c5) or c 6): c1) inhibiting bacteria; c2) preparing an antibacterial preparation; c3) preventing diseases caused by bacterial infection; c4) preparing a product for preventing diseases caused by bacterial infection; c5) treating diseases caused by bacterial infection; c6) preparing the product for treating diseases caused by bacterial infection. The antibacterial drug can be tetracycline drugs, beta-lactam drugs, glycopeptide drugs, quinolone drugs, rifamycin drugs or cyclic peptide drugs.
The disease caused by any of the above bacterial infections may specifically be bacteremia.
Any of the above products may be a medicament or a vaccine.
The invention also protects the use of said oligopeptides in restoring the sensitivity of polymyxin-resistant gram-negative bacteria to polymyxin.
In the above application, the gram-negative bacterium may be a gram-negative bacterium other than klebsiella pneumoniae. The polymyxin-resistant gram-negative bacterium can be a gram-negative bacterium (non-Klebsiella pneumoniae) containing mcr drug resistance gene. The gram-negative bacteria containing the mcr drug-resistant gene can be escherichia coli containing the mcr-1 drug-resistant gene, acinetobacter baumannii containing the mcr-1 drug-resistant gene, citrobacter freundii containing the mcr-1 drug-resistant gene, uracilus ornithinolyticus containing the mcr-1 drug-resistant gene, serratia marcescens containing the mcr-1 drug-resistant gene, salmonella containing the mcr-1 drug-resistant gene, aeromonas veronii containing the mcr-3 drug-resistant gene, providencia alcaligenes containing the mcr-1 drug-resistant gene, uracilus planticola containing the mcr-1 drug-resistant gene or enterobacter cloacae containing the mcr-1 drug-resistant gene. The Escherichia coli containing the mcr-1 drug-resistant gene can be Escherichia coli (Escherichia coli)1-1(mcr-1), Escherichia coli (Escherichia coli)1-5(mcr-1), Escherichia coli (Escherichia coli)1-6(mcr-1), Escherichia coli (Escherichia coli)2-1(mcr-1), Escherichia coli (Escherichia coli)2-6(mcr-1), Escherichia coli (Escherichia coli)2-8(mcr-1) or Escherichia coli (Escherichia coli) B2(NDM-5+ mcr-1). The Acinetobacter baumannii containing mcr-1 drug-resistant gene can be Acinetobacter baumannii (Acinetobacter baumannii)2-38 (mcr-1). The Citrobacter freundii containing mcr-1 drug-resistant gene can be specifically Citrobacter freundii (Citrobacterfreumdii)2-4 (mcr-1). The Uralensis ornithinolyticus containing mcr-1 drug-resistant gene can be specifically Uralensis ornithinolyticus (Raoultella ornithinolytica)16-68 (mcr-1). The Serratia marcescens containing mcr-1 drug-resistant gene can be specifically Serratia marcescens (Serratiamarcescens)16-99 (mcr-1). The salmonella containing the mcr-1 drug resistance gene can be salmonella (Salmonella enterica) SH30(mcr-1), salmonella (Salmonella enterica) SH170(mcr-1), salmonella (Salmonella enterica) SH1-3(mcr-1), salmonella (Salmonella enterica) SH2-8(mcr-1) or salmonella (Salmonella enterica)8H-3 (mcr-1). The Aeromonas veronii containing mcr-3 drug-resistant gene can be specifically Aeromonas veronii 172 (mcr-3). The alkali-producing providencia strain containing mcr-1 drug-resistant gene can be specifically alkali-producing providencia strain (providencia calixafaciens) 16-1 (mcr-1). The Uralensis planticola containing mcr-1 drug-resistant gene can be specifically Uralensis planticola 16-15 (mcr-1). The enterobacter cloacae containing mcr-1 drug-resistant gene can be enterobacter cloacae (Enterobacteracterchloroaceae) 16-15 (mcr-1).
The invention also protects the application of the oligopeptide and antibacterial drug in the synergistic action of polymyxin-resistant or polymyxin-intolerant Klebsiella pneumoniae. The antibacterial drug can be rifamycin drugs, quinolone drugs, tetracycline drugs or beta-lactams.
In the above application, the polymyxin-resistant klebsiella pneumoniae can be klebsiella pneumoniae containing mcr drug-resistant genes. The polymyxin-intolerant klebsiella pneumoniae can be klebsiella pneumoniae that does not contain a mcr resistance gene. The Klebsiella pneumoniae containing mcr drug resistance gene can be Klebsiella pneumoniae (Klebsiella pneumoniae)16-63(mcr-1), Klebsiella pneumoniae (Klebsiella pneumoniae)4-20(mcr-6) or Klebsiella pneumoniae (Klebsiella pneumoniae)91 (mcr-6). The polymyxin-intolerant Klebsiella pneumoniae can be Klebsiella pneumoniae (Klebsiella pneumoniae) 2.
The invention also protects the application of the oligopeptide and antibacterial drug in the synergistic action of bacteria. The bacteria may be gram positive or gram negative. When the oligopeptide is combined with an antibacterial drug to be applied to the synergistic effect of gram-positive bacteria, the antibacterial drug can be beta-lactam drugs. When the oligopeptide is combined with an antibacterial drug to be applied to the synergistic effect of gram-negative bacteria, the antibacterial drug can be beta-lactam drugs, tetracycline drugs, glycopeptide drugs, quinolone drugs, rifamycin drugs or cyclic peptide drugs.
The invention also provides an antibacterial preparation containing the oligopeptide.
The antibacterial preparation may further contain an antibacterial agent. The antibacterial drug can be tetracycline drugs, beta-lactam drugs, glycopeptide drugs, quinolone drugs, rifamycin drugs or cyclic peptide drugs.
Any one of the tetracycline drugs can be tetracycline. Any one of the glycopeptide drugs may specifically be vancomycin. The quinolone drug may be ofloxacin. Any one of the rifamycin class drugs described above may specifically be rifampicin. Any one of the cyclic peptide drugs can be specifically polymyxin. Any one of the beta-lactam drugs may be ampicillin or imipenem.
Any of the above bacteria may be gram-positive bacteriaOr a gram negative bacterium. The gram positive bacteria may be staphylococcus aureus (staphylococcus aureus) or enterococcus or Bacillus subtilis (Bacillus subtilis) or other gram positive bacteria. The Staphylococcus aureus (Staphylococcus aureus) can be Staphylococcus aureus (ATCC No. 29213), Staphylococcus aureus (Staphylococcus aureus) MRSA T144 or Staphylococcus aureus (Staphylococcus aureus)215 (LZD)R+ cfr). The Enterococcus may be Enterococcus faecalis (Enterococcus faecalis). The Enterococcus faecalis (Enterococcus faecalis) may specifically be Enterococcus faecalis (Enterococcus faecalis) VRE a 4. The Bacillus subtilis can be Bacillus subtilis ATCC No. 6051. The gram-negative bacterium may be Escherichia coli (Escherichia coli), Salmonella (Salmonella enterica), Klebsiella pneumoniae (Klebsiella pneumoniae), Pseudomonas aeruginosa (Pseudomonas aeruginosa), Acinetobacter baumannii (Acinetobacter baumannii), Aeromonas veronii (Aeromonas veronii), Citrobacter freundii (Citrobacter freundii), Uralensis ornithinovarum (Raoultellaria), Serratia marcescens (Serratia marcescens), providencia alcaliensis (Providence caligenes), Uralensis planticola (Raoultellaria), or Enterobacter cloacae (Enterobacter cloacae). The Escherichia coli (Escherichia coli) can be Escherichia coli (Escherichia coli) ATCC No.25922, Escherichia coli (Escherichia coli) C3(NDM-1), Escherichia coli (Escherichia coli) G6(NDM-5), Escherichia coli (Escherichia coli)3CZ20E (mcr-1+ FLO)R) Escherichia coli (Escherichia coli) B2(NDM-5+ mcr-1), Escherichia coli (Escherichia coli) I1(CRE + mcr-1), Escherichia coli (Escherichia coli) L14(ESBL + mcr-1), Escherichia coli (Escherichia coli) F9(ESBL + mcr-1), Escherichia coli (Escherichia coli)1-1(mcr-1), Escherichia coli (Escherichia coli)1-5(mcr-1), Escherichia coli (Escherichia coli)1-6(mcr-1), Escherichia coli (Escherichia coli)2-1(mcr-1), Escherichia coli (Escherichia coli)2-6(mcr-1), Escherichia coli (Escherichia coli)2-8 (mcr-1).
The Salmonella (Salmonella enterica) may be Salmonella (Salmonella enterica) ATCC No.13076, Salmonella (Salmonella enterica) SH30(mcr-1), Salmonella (Salmonella enterica) SH170(mcr-1), Salmonella (Salmonella enterica) SH1-3(mcr-1), Salmonella (Salmonella enterica) SH2-8(mcr-1), Salmonella (Salmonella enterica)8H-3(mcr-1), Salmonella (Salmonella enterica)4CZ2(mcr-1) or Salmonella (Salmonella enterica)4M (NDM-1). The Klebsiella pneumoniae (Klebsiella pneumoniae) can be Klebsiella pneumoniae (Klebsiella pneumoniae)1, Klebsiella pneumoniae (Klebsiella pneumoniae)2, Klebsiella pneumoniae (Klebsiella pneumoniae)3, Klebsiella pneumoniae (Klebsiella pneumoniae)4, Klebsiella pneumoniae (Klebsiella pneumoniae)16-80(mcr-1), Klebsiella pneumoniae (Klebsiella pneumoniae)16-87(mcr-1), Klebsiella pneumoniae (Klebsiella pneumoniae)16-63(mcr-1), Klebsiella pneumoniae (Klebsiella pneumoniae)4-20(mcr-6) or Klebsiella pneumoniae (Klebsiella pneumoniae) 91-6. The pseudomonas aeruginosa (pseudomonas aeruginosa) may be pseudomonas aeruginosa (pseudomonas aeruginosa) PAO1 ATCC No.47085 or pseudomonas aeruginosa (pseudomonas aeruginosa) PA 14. The Acinetobacter baumannii (Acinetobacter baumannii) can be Acinetobacter baumannii (Acinetobacter baumannii)2-38 (mcr-1). The Aeromonas veronii (Acinetobacter erbaumannii) can be Aeromonas veronii (Aeromonas veronii)172 (mcr-3). The Citrobacter freundii may be Citrobacter freundii 2-4 (mcr-1). The Uralensis ornitholitica (Raoultella ornitholinolytica) can be Uralensis ornitholitica (Raoultella ornitholinolytica) 16-68 (mcr-1). The Serratia marcescens (Serratiamarcescens) can be Serratia marcescens (Serratiamarcescens)16-99 (mcr-1). The alkali-producing providencia bacterium (providencialcaliforiens) can be 16-1(mcr-1) of alkali-producing providencia bacterium (providencialcaliforiens). The Uralensis planticola (Raoultella lanicola) can be Uralensis planticola (Raoultella lanicola) 16-15 (mcr-1). The Enterobacter cloacae (Enterobactererchloroacae) can be Enterobacter cloacae (Enterobactererchloroacae) 16-15 (mcr-1).
Experiments prove that the oligopeptide has certain bacteriostatic action on various gram-positive bacteria and gram-negative bacteria, the minimum bacteriostatic concentration on the gram-positive bacteria (containing drug-resistant bacteria) is 8-64 mu g/mL, and the minimum bacteriostatic concentration on the gram-negative bacteria (containing drug-resistant bacteria) is 0.5-32 mu g/mL; the oligopeptide and antibacterial drugs (such as tetracycline, vancomycin, ofloxacin, rifampin, polymyxin, ampicillin and imipenem) have good synergistic effect; the oligopeptide can restore the sensitivity of polymyxin-resistant gram-negative bacteria to polymyxin; the oligopeptide has no hemolytic property and good histocompatibility; the oligopeptide can effectively inhibit polymyxin drug-resistant bacteria and can treat bacteremia. In addition, the oligopeptide has small molecular weight and simple structure, and is convenient for large-scale synthesis. The invention has great application value.
Drawings
FIG. 1 shows the hemolytic assay for the linear antimicrobial oligopeptide SLAP-S25.
FIG. 2 is a graph of the linear antimicrobial oligopeptide SLAP-S25 for the treatment of bacterial infection in mice; wherein PBS is PBS group, S25 is SLAP-S25 treatment group, polymyxin is polymyxin treatment group, and polymyxin + S25 is synergistic treatment group.
FIG. 3 is a graph of the linear antimicrobial oligopeptide SLAP-S25 for the treatment of bacteremia in mice; wherein PBS is PBS group, S25 is SLAP-S25 treatment group, polymyxin is polymyxin treatment group, and polymyxin + S25 is synergistic treatment group.
FIG. 4 is a graph of the linear antimicrobial oligopeptide SLAP-S25 for the treatment of bacterial infection in the larvae of wax moth; wherein PBS is PBS group, S25 is SLAP-S25 treatment group, polymyxin is polymyxin treatment group, and polymyxin + S25 is synergistic treatment group.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The experimental materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. In the quantitative experiments in the following examples, three replicates were set up and the results averaged.
The BALB/c female mouse is a product of experimental animal technology Limited of Beijing Wittingerli.
MHB broth medium is an aqueous solution containing 1.5g/L of beef powder, 1.5g/L of soluble starch and 17.5g/L of acid hydrolyzed casein.
Example 1 discovery and Artificial Synthesis of a Linear antibacterial oligopeptide SLAP-S25
Discovery of linear antibacterial oligopeptide SLAP-S25
By bioinformatics analysis of various linear antibacterial oligopeptide sequences, optimization of peptide chain structures, construction of linear oligopeptide libraries and evaluation of the antibacterial effect of the obtained linear oligopeptides and the synergistic antibacterial effect of the linear oligopeptides and various antibacterial drugs (such as beta-lactams, tetracyclines, glycopeptides, quinolones, cyclic peptides and rifamycins), the inventor of the invention obtains linear oligo linear antibacterial oligopeptides SLAP-S25. The structural formula of the linear antibacterial oligopeptide SLAP-S25 is shown in the formula (I) (acetylation modification is carried out on nitrogen-end 2, 4-diaminobutyric acid, amidation modification is carried out on carbon-end alanine), and the molecular weight is 1229.10.
Second, artificial synthesis of linear antibacterial oligopeptide SLAP-S25
Synthesizing the linear antibacterial oligopeptide SLAP-S25 shown in the formula (I) artificially.
The amino acid sequence of the linear antibacterial oligopeptide SLAP-S25 is shown as a sequence 1 in a sequence table.
The linear antibacterial oligopeptide SLAP-S25 is hereinafter abbreviated as S25.
Example 2 determination of the bacteriostatic Activity of the Linear antibacterial oligopeptide SLAP-S25
The strains to be tested are detailed in Table 1.
TABLE 1
Note: a. staphylococcus aureus (Staphylococcus aureus) ATCC No.29213, Bacillus subtilis (Bacillus subtilis) ATCC No.6051, Escherichia coli (Escherichia coli) ATCC No.25922, Salmonella (Salmonella enterica) ATCC No.13076, and Pseudomonas aeruginosa (Pseudomonas aeruginosa) PAO1 ATCC No.47085 are all deposited in American Type Culture Collection (ATCC for short, address: American Type Culture Collection (ATCC)10801University Boulevans, Va 20110 USA), and publicly available from the American Type Culture Collection.
b. Staphylococcus aureus (Staphyloccocusareus) 215 (LZD)R+ cfr) carries a drug resistance gene cfr and is resistant to linezolid. Enterococcus faecalis (Enterococcus faecalis) VRE a4 is a drug-resistant strain of vancomycin. Staphylococcus aureus (Staphyloccocusareus) MRSA T144 is methicillin resistant Staphylococcus aureus. The strains containing the ESBL are all strains capable of generating the drug resistance of the extended-spectrum beta-lactamase mediated beta-lactam antibiotics. The bacterial strains containing the NDM are all bacterial strains which generate new Delhi metallo beta-lactamase mediated beta-lactam antibiotics drug resistance. The strains containing "CRE" in the name are carbapenem antibiotic resistant strains. Name containing FLORThe strain of (a) represents a florfenicol-resistant strain. The strains with the name of "mcr" are all strains carrying polymyxin drug resistance gene mcr.
c. Reference 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-R+ cfr) and Pseudomonas aeruginosa (Pseudomonas aeruginosa) PA14 in document 1 are sequentially named Enterococcus faecalis VRE A4, MRSA T144, Staphyloccocusareureus 215 (LZD)R+ cfr) and pseudomonas aeruginosa pa 14.
d. Document 2 is Yang Z Q, Huang Y L, Zhou H W, et al, personal carbapenem-resistant Klebsiella pneumoniae: a Trojan horse [ J ]. Lancet Infectious Diseases, 2017, 18(1):22. Klebsiella pneumoniae (Klebsiella pneumoniae)1, Klebsiella pneumoniae (Klebsiella pneumoniae)2, Klebsiella pneumoniae (Klebsiella pneumoniae)3 and Klebsiella pneumoniae (Klebsiella pneumoniae)4 are named as CRKP1, CRKP2, CRKP3 and CRKP4 in sequence in the document 2.
e. Literature 3 is link Z, Yin W, Li H, et al, chromosome-mediated mcr-3variants in Aeromonas veronii from chip mean [ J ]. Antimicrobial agents and chemitherapy, 2017, 61 (11): e01272-17 Aeromonas veronii (Aeromonas veronii)172(mcr-3) is named Aeromonas veronii 172 in reference 3.
f. The isolation of the strains from which they were self-isolated was as follows: (1) collecting pig nose swabs in slaughterhouses and breeding farms with long distance in Shandong, Henan, Sichuan provinces, and the like, retail chicken, pork, and the like in supermarkets, soaking in BHI liquid culture medium for 5min, filtering, and storing the filtrate in ESwab tubes for later use; (2) different filtrates are dipped by an inoculating loop and spread on a Kemajia urethra positioning and developing culture medium containing polymyxin of 2 mu g/mL, the mixture is cultured for 18h at 37 ℃, monoclonal strains with red (escherichia coli), blue (Klebsiella pneumoniae, Citrobacter and Enterobacter) or semitransparent (Pseudomonas) cream color are selected and transferred to a BHI plate, and the mixture is cultured for 18h at 37 ℃. Scraping appropriate bacteria from the flat plate by using an ultra-thick filter paper sheet, transferring the bacteria into a 2ml sterile test tube, and storing the sterile test tube in a refrigerator at the temperature of minus 20 ℃ for later use; (3) selecting a monoclonal strain, inoculating the monoclonal strain into a BHI liquid culture medium for enrichment, and then extracting genome DNA; (4) taking the genomic DNA extracted in the step (3) as a template, and further confirming the genus by adopting a conventional PCR (polymerase chain reaction) 16SrRNA amplification and sequencing method; and meanwhile, detecting the amplified mcr gene. The primers for amplifying 16S rRNA are 5 '-AGAGAGTTTGATCCTGGCTCAG-3' and 5 '-TACGGCTACCTTGTTACGACTT-3'; the reaction operating parameters were: 10min at 94 ℃; 30cycles at 94 ℃ for 30s, 55 ℃ for 40s, 72 ℃ for 2 min; 72 ℃ for 10 min. Primers for amplifying mcr-1 are 5'-CGGTCAGTCCGTTTGTTC-3' and 5'-CTTGGTCGGTCTGTAGGG-3'; the reaction operating parameters were: 10min at 94 ℃; 94 ℃ for 30s, 55 ℃ for 30s, 72 ℃, 50s, 36 cycles; 72 ℃ for 5 min. Primers for amplifying mcr-3 are 5'-TTGGCACTGTATTTTGCATTT-3' and 5'-TTAACGAAATTGGCTGGAACA-3'; the reaction operating parameters were: 30cycles at 95 ℃ for 30s, 50 ℃ for 30s, 72 ℃ for 45 s; 72 ℃ for 7 min. Primers for amplifying mcr-6 are 5'-AACCGCCAGAGCACAGAATT-3' and 5'-AACCGCCAGAGCACAGAATT-3'; the reaction operating parameters were: 10min at 94 ℃; 94 ℃ for 30s, 60 ℃ for 30s, 72 ℃, 50s, 36 cycles; 72 ℃ for 5 min.
The Minimum Inhibitory Concentration (MIC) of the linear antimicrobial oligopeptide SLAP-S25 against the strain to be tested was determined by broth dilution (Clinical and Laboratory Standards Institute, CLSI, 2017). The steps for detecting the minimum inhibitory concentration of the linear antibacterial oligopeptide SLAP-S25 on each strain to be detected are as follows:
1. suspending the test strain in MHB broth to obtain a concentration of 1X 108CFU/mL of bacterial suspension.
2. The linear antimicrobial oligopeptide SLAP-S25 was dissolved in water and diluted with MHB broth to give a solution of 512. mu.g/mL linear antimicrobial oligopeptide SLAP-S25.
3. Adding MHB broth culture medium (100 mu L per well) into a 96-well plate, adding the linear antibacterial oligopeptide SLAP-S25 solution (100 mu L per well) prepared in the step 2 into each well of the first row, diluting the solution in a multiple mode to a tenth well, discarding 100 mu L of liquid from the tenth well, then adding 100 mu L of the bacterial suspension prepared in the step 1 into each well, and finally, enabling the concentration of the linear antibacterial oligopeptide SLAP-S25 in the wells to be 0.25, 0.5, 1.0, 2.0, 4.0, 8.0, 16.0, 32.0 mu g/mL, 64.0 mu g/mL or 128.0 mu g/mL, and standing the wells at 37 ℃ for 16h-20h, wherein the lowest concentration for inhibiting the bacterial growth in the wells is MIC. Negative control wells and positive control wells were set in a 96-well plate. 200 μ LMHB broth was added to each negative control well. Each positive control well was added 100. mu.L of LMHB broth and 100. mu.L of the bacterial suspension prepared in step 1.
The results are shown in Table 2. The results show that the linear antibacterial oligopeptide SLAP-S25 has good antibacterial activity on gram-positive bacteria (such as staphylococcus aureus, enterococcus faecalis and bacillus subtilis) and gram-negative bacteria (such as escherichia coli, salmonella, klebsiella pneumoniae, pseudomonas aeruginosa, acinetobacter baumannii, aeromonas veronii, citrobacter freundii, uracillus ornithii, serratia marcescens, providencia alcaligenes, uracillus planticus and enterobacter cloacae), the minimum inhibitory concentration on the gram-positive bacteria (containing drug-resistant bacteria) is 8-64 mu g/mL, and the minimum inhibitory concentration on the gram-negative bacteria (containing drug-resistant bacteria) is 0.5-32 mu g/mL.
TABLE 2
Example 3 synergistic Effect of Linear antimicrobial oligopeptide SLAP-S25 and antimicrobial drug
Tetracyclines belong to the class of tetracyclines. Vancomycin belongs to glycopeptide drugs. Ofloxacin belongs to quinolone drugs. Rifampicin belongs to the class of rifamycins. Polymyxins belong to cyclic peptide drugs. Ampicillin and imipenem belong to the group of beta-lactams.
First, the synergistic effect of the linear antibacterial oligopeptide SLAP-S25 and the antibacterial drug
1. Synergistic effect of linear antibacterial oligopeptide SLAP-S25 and antibacterial drug on gram-negative bacteria
The antibacterial agent is tetracycline, vancomycin, ofloxacin, rifampin, polymyxin or imipenem.
The synergistic effect of the linear antibacterial oligopeptide SLAP-S25 and different antibacterial drugs is detected by adopting a chessboard analysis method, a test strain is Escherichia coli (Escherichia coli) B2(NDM-5+ mcr-1), and the chessboard analysis method comprises the following specific steps:
1) test strains were suspended in MHB broth to a concentration of 1X 108CFU/mL of bacterial suspension.
2) The linear antimicrobial oligopeptide SLAP-S25 was dissolved in water and diluted with MHB broth to give a solution of 256 μ g/mL linear antimicrobial oligopeptide SLAP-S25.
3) The antibacterial agent was taken, dissolved with water and diluted with MHB broth to give an antibacterial agent solution having a concentration of 128. mu.g/mL.
4) Taking a 96-well plate, adding 100 mu of LMHB broth culture medium into each well, adding 100 mu of the linear antibacterial oligopeptide SLAP-S25 solution prepared in the step 2) into each well in the last row (the concentration of the first well is 512 mu g/mL), and diluting to the second row from the eighth row in a multiple ratio; adding 100 mu L of the antibacterial agent solution prepared in the step 3) into each well of the first row, diluting the mixture to the tenth row in a multiple ratio, adding 100 mu L of the bacterial suspension prepared in the step 1) into each well, standing and culturing the mixture for 16-20 h at 37 ℃, and observing the lowest concentration combination of the linear antibacterial oligopeptide SLAP-S25 and different antibacterial agents when the linear antibacterial oligopeptide and the different antibacterial agents are combined for inhibiting the growth of bacteria. Positive control wells were set, and 100. mu.L of LMHB broth and 100. mu.L of the bacterial suspension prepared in step 1) were added to each positive control well.
The fractional antibacterial concentration FIC index is calculated according to the following formula:
FIC ═ MIC (A combination)/MIC (A alone) + MIC (B combination)/MIC (B alone)
The results are shown in Table 3. The results show that the linear antibacterial oligopeptide SLAP-S25 can reduce the MIC of antibacterial drugs to Escherichia coli B2(NDM-5+ mcr-1) by 4-128 times. The FIC indexes of the linear antibacterial oligopeptide SLAP-S25 and antibacterial drugs (imipenem, tetracycline, vancomycin, ofloxacin, rifampicin or polymyxin) are all lower than 0.5, so the linear antibacterial oligopeptide SLAP-S25 and the six antibacterial drugs have good synergistic effect.
TABLE 3
| FICAntibacterial agent | FICS25 | FIC index | |
| Tetracycline derivatives | 0.031 | 0.250 | 0.281 |
| Vancomycin | 0.031 | 0.063 | 0.094 |
| Ofloxacin | 0.031 | 0.063 | 0.094 |
| Rifampicin | 0.031 | 0.001 | 0.032 |
| Polymyxin | 0.016 | 0.031 | 0.047 |
| Imipenem | 0.0625 | 0.125 | 0.187 |
2. Synergistic effect of linear antibacterial oligopeptide SLAP-S25 and antibacterial drug on gram-positive bacteria
The antibacterial agent is tetracycline, vancomycin, ofloxacin, ampicillin, rifampicin or polymyxin.
A chessboard analysis method is adopted to determine the synergistic effect of the linear antibacterial oligopeptide SLAP-S25 and the antibacterial drug on gram-positive bacteria, the specific experimental method is the same as the step 1, and the test strains are Staphylococcus aureus (Staphylococcus aureus) ATCC 29213, Staphylococcus aureus (Staphylococcus aureus) MRSA T144 and Bacillus subtilis ATCC 6051.
The results are shown in Table 4. The result shows that the linear antibacterial oligopeptide SLAP-S25 has strong synergistic effect on gram-positive bacteria and ampicillin. The FIC indexes of the linear antibacterial oligopeptides SLAP-S25 and ampicillin for three gram-positive bacteria are all lower than 0.5, so the linear antibacterial oligopeptides SLAP-S25 and ampicillin have good synergistic effect on the gram-positive bacteria.
TABLE 4
Second, the Linear antibacterial oligopeptide SLAP-S25 restores the sensitivity of polymyxin-resistant gram-negative bacteria to polymyxin
The details of gram-negative bacteria containing mcr drug-resistant gene are shown in the 1 st column of table 5, and all the bacteria are mcr drug-resistant gene positive strains.
And (3) determining the synergistic effect of the linear antibacterial oligopeptide SLAP-S25 and the antibacterial agent on mcr positive gram-negative bacteria by adopting a chessboard analysis method, wherein the specific experimental method is the same as the step 1 in the first step.
The results are shown in Table 5. The results show that both the linear antibacterial oligopeptide SLAP-S25 and polymyxin have FIC indices below 0.375, and thus the linear antibacterial oligopeptide SLAP-S25 restores the sensitivity of polymyxin-resistant gram-negative bacteria (except Klebsiella pneumoniae) to polymyxin.
TABLE 5
Thirdly, the synergistic effect of the linear antibacterial oligopeptide SLAP-S25 and antibacterial drugs on the Klebsiella pneumoniae
The antibacterial agent is tetracycline, vancomycin, ofloxacin, ampicillin, imipenem, rifampin or polymyxin.
The synergistic effect of the linear antibacterial oligopeptide SLAP-S25 and the antibacterial drug on polymyxin resistant and non-resistant Klebsiella pneumoniae is determined, wherein the polymyxin resistant Klebsiella pneumoniae is Klebsiella pneumoniae (Klebsiella pneumoniae)16-63(mcr-1), Klebsiella pneumoniae (Klebsiella pneumoniae)4-20(mcr-6) or Klebsiella pneumoniae (Klebsiella pneumoniae)91(mcr-6), and the polymyxin non-resistant Klebsiella pneumoniae is Klebsiella pneumoniae (Klebsiella pneumoniae) 2.
The specific method for determining the synergistic effect of the linear antibacterial oligopeptide SLAP-S25 and the antibacterial drugs by the chessboard analysis method is the same as the step 1 in the first step.
The results are shown in Table 6: aiming at polymyxin drug-resistant and drug-intolerant Klebsiella pneumoniae, FIC indexes of the linear antibacterial oligopeptide SLAP-S25 and rifampicin are both lower than 0.065, FIC indexes of the linear antibacterial oligopeptide SLAP-S25 and ofloxacin are both lower than 0.127, and FIC indexes of the linear antibacterial oligopeptide SLAP-S25 and tetracycline are both lower than 0.129; the FIC index of the linear antibacterial oligopeptides SLAP-S25 and imipenem is lower than 0.187 aiming at polymyxin-intolerant Klebsiella pneumoniae. The results show that the linear antibacterial oligopeptide SLAP-S25 can reduce the minimum inhibitory concentration of antibacterial drugs rifampicin, ofloxacin, tetracycline and imipenem to Klebsiella pneumoniae. Has strong synergistic effect on Klebsiella pneumoniae, linear antibacterial oligopeptide SLAP-S25 and antibacterial drugs rifampin, ofloxacin, tetracycline and imipenem.
TABLE 6-1
TABLE 6-2
Tables 6 to 3
Example 4 hemolytic test of Linear antimicrobial oligopeptide SLAP-S25
The PBS buffer was 0.01M PBS buffer, pH 7.4.
The preparation method of the 8% erythrocyte suspension comprises the following steps: (1) centrifuging 10mL of defibrinated sheep blood at 3000g for 10min, and removing supernatant to obtain precipitate; (2) taking the precipitate obtained in the step (1), and washing twice by using a PBS (phosphate buffer solution) to obtain a 100% erythrocyte suspension; (3) 8mL of 100% erythrocyte suspension and 92mL of PBS buffer were mixed to obtain 8% erythrocyte suspension.
Dissolving the linear antibacterial oligopeptide SLAP-S25 in water, and then diluting the solution with PBS buffer solution to obtain a solution of the linear antibacterial oligopeptide SLAP-S25 with the concentration of 1024 mu g/mL.
1. A96-well plate was prepared, and 100. mu.L of PBS buffer was added to each well.
2. After the step 1 is finished, taking the 96-pore plate, adding 100 mu L of linear antibacterial oligopeptide SLAP-S25 solution into the first pore, and diluting the solution in multiple proportions to the eighth pore; the concentrations of the linear antibacterial oligopeptide SLAP-S25 in the second to eighth wells were 512. mu.g/mL, 256. mu.g/mL, 128. mu.g/mL, 64. mu.g/mL, 32. mu.g/mL, 16. mu.g/mL and 8. mu.g/mL in this order (three duplicate wells per concentration).
3. After completing step 2, the 96-well plate was taken, 100. mu.L of 8% erythrocyte suspension was added to each well, incubated at 37 ℃ for 1h, centrifuged at 3000g for 10min, and 100. mu.L of supernatant was aspirated.
4. Measuring OD of the supernatant obtained in step 3576nmValue, i.e. OD of solution of linear antibacterial oligopeptide SLAP-S25576nmThe value is obtained.
5. Replacing step 2 with step a, and obtaining negative control OD without changing other steps576nmThe value is obtained. The step a is as follows: the 96-well plate was taken and 100. mu.L of PBS buffer was added to each well.
6. Will be provided withStep 2 is replaced by step b, other steps are not changed, and positive control OD is obtained576nmThe value is obtained. The step b is as follows: the 96-well plate was taken and 100. mu.L Triton X-100 was added to each well.
The hemolysis rate was calculated according to the following formula:
hemolysis rate (linear antibacterial oligopeptide SLAP-S25 solution OD576nmValue-negative control OD576nmvalue)/(Positive control OD576nmValue-negative control OD576nmValue) × 100%.
The results of some of the experiments are shown in FIG. 1. The results show that the linear antibacterial oligopeptide SLAP-S25 has no hemolytic property when the concentration is 8-256 mu g/mL.
Example 5 use of the Linear antibacterial oligopeptide SLAP-S25 in the treatment of bacterial infection and bacteremia in mice
Linear antibacterial oligopeptide SLAP-S25 for treating mouse bacterial infection
The PBS buffer was 0.01M PBS buffer, pH 7.4.
E.coli suspension: resuspending E.coli (Escherichia coli) B2(NDM-5+ mcr-1) in PBS buffer to obtain E.coli suspension; the E.coli (Escherichia coli) B2(NDM-5+ mcr-1) concentration in the E.coli suspension was 1.0X 109CFUs/mL。
Polymyxin solution: 800mg of polymyxin was dissolved in 1L of sterile water.
SLAP-S25 solution: obtained by dissolving 800mg of linear antibacterial oligopeptide SLAP-S25 in 1L of sterile water.
Mixing the solution: obtained by dissolving 800mg polymyxin and 800mg linear antibacterial oligopeptide SLAP-S25 in 1L sterile water.
1. Mouse grouping treatment
24 BALB/c female mice weighing 18-22 g were randomly divided into PBS group, SLAP-S25 treatment group, polymyxin treatment group and co-treatment group (6 per group), and treated as follows:
PBS group: injecting 0.1mL of Escherichia coli suspension into right thigh muscle of the mouse; injecting 0.2mL PBS buffer solution into the abdominal cavity after 1 h;
SLAP-S25 treatment group: injecting 0.1mL of Escherichia coli suspension into right thigh muscle of the mouse; injecting 0.2mLSLAP-S25 solution into abdominal cavity after 1 h;
polymyxin treatment group: injecting 0.1mL of Escherichia coli suspension into right thigh muscle of the mouse; injecting 0.2mL polymyxin solution into the abdominal cavity after 1 h;
the co-treatment group: injecting 0.1mL of Escherichia coli suspension into right thigh muscle of the mouse; the mixed solution was injected 0.2mL into the abdominal cavity after 1 h.
2. Determination of the number of colonies infected by the thigh of mice
And (3) euthanizing the mice 48h after the step 1, taking right thigh muscle, homogenizing and uniformly mixing the meat mass, then coating a proper amount of the meat mass on a TSA culture medium, and determining the colony number.
The results of the experiment are shown in FIG. 2. The results show that the synergistic treatment group was effective in reducing the number of colonies infected in the thigh of mice compared to the polymyxin treatment group.
Second, linear antibacterial oligopeptide SLAP-S25 for treating bacteremia of mice
The PBS buffer was 0.01M PBS buffer, pH 7.4.
E.coli suspension: resuspending E.coli (Escherichia coli) B2(NDM-5+ mcr-1) in PBS buffer to obtain E.coli suspension; the E.coli (Escherichia coli) B2(NDM-5+ mcr-1) concentration in the E.coli suspension was 1.5X 1010CFUs/mL。
Polymyxin solution: 800mg of polymyxin was dissolved in 1L of sterile water.
SLAP-S25 solution: obtained by dissolving 800mg of linear antibacterial oligopeptide SLAP-S25 in 1L of sterile water.
Mixing the solution: obtained by dissolving 800mg polymyxin and 800mg linear antibacterial oligopeptide SLAP-S25 in 1L sterile water.
1. Mouse grouping treatment
24 BALB/c female mice weighing 18-22 g were randomly divided into PBS group, SLAP-S25 treatment group, polymyxin treatment group and co-treatment group (6 per group), and treated as follows:
PBS group: injecting 0.2mL of escherichia coli suspension into the abdominal cavity of the mouse; injecting 0.2mL PBS buffer solution into the abdominal cavity after 1 h;
SLAP-S25 treatment group: injecting 0.2mL of escherichia coli suspension into the abdominal cavity of the mouse; injecting 0.2mLSLAP-S25 solution into abdominal cavity after 1 h;
polymyxin treatment group: injecting 0.2mL of escherichia coli suspension into the abdominal cavity of the mouse; injecting 0.2mL polymyxin solution into the abdominal cavity after 1 h;
the co-treatment group: injecting 0.2mL of escherichia coli suspension into the abdominal cavity of the mouse; the mixed solution was injected 0.2mL into the abdominal cavity after 1 h.
2. Statistic survival rate
For the mice at 48h after completion of step 1, the survival rate was counted.
The results are shown in FIG. 3, panel A. The results show that the survival rate of mice in the co-treated group was significantly improved compared to the polymyxin-treated group.
3. Determination of colony counts in mouse organs
The mice at 48h after completion of step 1 were taken, organs (e.g., heart, liver, spleen, lung and kidney) were isolated, and then the number of colonies in each organ was determined.
The results are shown in FIG. 3B. The results show that the colony counts in heart, liver, spleen, lung or kidney were also significantly reduced in the mice of the co-treated group compared to the polymyxin-treated group.
The above results indicate that the linear antibacterial oligopeptide SLAP-S25 can be used for treating bacterial infection and bacteremia in mice.
Example 6 treatment of bacterial infection of Chilo suppressalis larvae with the Linear antibacterial oligopeptide SLAP-S25
The PBS buffer was 0.01M PBS buffer, pH 7.4.
E.coli suspension: resuspending E.coli (Escherichia coli) B2(NDM-5+ mcr-1) in PBS buffer to obtain E.coli suspension; the E.coli (Escherichia coli) B2(NDM-5+ mcr-1) concentration in the E.coli suspension was 1.0X 107CFUs/mL。
Polymyxin solution: 160mg of polymyxin was dissolved in 1L of sterile water.
SLAP-S25 solution: 160mg of the linear antibacterial oligopeptide SLAP-S25 was dissolved in 1L of sterile water.
Mixing the solution: 160mg polymyxin and 160mg linear antibacterial oligopeptide SLAP-S25 were dissolved in 1L sterile water.
1. Grouping treatment of galleria mellonella larvae
32 larvae of galleria mellonella, which weigh 190-210 mg, were randomly divided into a PBS group, a SLAP-S25 treatment group, a polymyxin treatment group and a co-treatment group (8 in each group), and the following treatments were performed, respectively:
PBS group: injecting 0.01mL of escherichia coli suspension into the lower left second gastropod of the galleria mellonella larvae; injecting 0.01mL PBS buffer solution into the lower right second gastropod after 1 h;
SLAP-S25 treatment group: injecting 0.01mL of escherichia coli suspension into the lower left second gastropod of the galleria mellonella larvae; after 1h, injecting 0.01mL of SLAP-S25 solution into the lower right second gastropod;
polymyxin treatment group: injecting 0.01mL of escherichia coli suspension into the lower left second gastropod of the galleria mellonella larvae; injecting 0.01mL polymyxin solution into the second lower right gastropod after 1 h;
the co-treatment group: injecting 0.01mL of escherichia coli suspension into the lower left second gastropod of the galleria mellonella larvae; the second gastropod was injected 0.01mL of the mixed solution after 1 h.
2. Statistic survival rate
And (4) counting the survival rate of the larvae of the galleria mellonella on the 1 st day, the 2 nd day, the 3 rd day, the 4 th day and the 5 th day after the step 1 is finished.
The results of the experiment are shown in FIG. 4. The results show that the survival rate of the larvae of the galleria mellonella of the synergistic treatment group is obviously improved compared with the polymyxin treatment group.
Example 7 derivatives of Linear antibacterial oligopeptide SLAP-S25 and uses thereof
1. The amino acid sequence of the linear antibacterial oligopeptide SLAP-S25 was substituted with four or less amino acid residues to obtain oligopeptides shown in rows 2 to 15 in Table 7 (the substituted amino acid letters are bold, and all the amino acids at the nitrogen terminal of the oligopeptide are acetylated and the amino acids at the carbon terminal are amidated). The amino acid sequence of the linear antimicrobial oligopeptide SLAP-S25 was subjected to deletions of four or less amino acid residues to yield the oligopeptides shown in rows 16 to 18 of Table 7. The chirality of at least one amino acid of the linear antibacterial oligopeptide SLAP-S25 was altered to give the oligopeptides shown in Table 7 at lines 19 to 21 (Val was changed from D to L, Phe was changed from D to L).
Each of the oligopeptides shown in Table 7 was synthesized manually.
The antibacterial activity of the oligopeptides shown in Table 7 was determined according to the method of example 2. The tested strain is Escherichia coli (Escherichia coli) ATCC No.25922, Escherichia coli (Escherichia coli) B2(NDM-5+ mcr-1) or Staphylococcus aureus (Staphylococcus aureus) ATCC 29213.
The results are shown in column 4 of Table 7.
TABLE 7
2. Synergistic effect of oligopeptides and antibacterial agents on gram-negative bacteria as shown in Table 7
The test strain was E.coli (Escherichia coli) B2(NDM-5+ mcr-1).
The synergy of each oligopeptide shown in table 7 and polymyxin was determined by the checkerboard assay, which was the same as in step 1 of example 3.
Some experimental results are shown in table 8. The results showed that the FIC index was less than 0.5 for each of the oligopeptides and polymyxins shown in Table 7. Thus, the respective oligopeptides and polymyxins shown in Table 7 have good synergistic effects.
TABLE 8
| Name of oligopeptide | 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> university of agriculture in China
<120> linear antibacterial oligopeptide SLAP-S25 and application thereof
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Claims (10)
1. An oligopeptide of a1) or a2) or a3) or a 4):
a1) performing acetylation modification on the nitrogen end and amidation modification on the carbon end of the oligopeptide shown in the sequence 1 in the sequence table to obtain the oligopeptide;
a2) oligopeptide with a structural formula shown as a formula (I);
a3) changing the chirality of Phe or/and Val in the oligopeptide shown in a1) or a2) to obtain an oligopeptide;
a4) and (b) carrying out substitution and/or deletion of not more than 4 amino acid residues on the amino acid sequence of the oligopeptide shown in a1) or a2) or a3) to obtain the oligopeptide with the same function, wherein the amino acid sequence of the oligopeptide with the same function is not shown as sequence 7, sequence 13 or sequence 15 in the sequence table.
2. The oligopeptide of claim 1, which is b1) or b2) or b3) or b4) or b5) or b 6):
b1) inhibiting bacteria; b2) preparing an antibacterial preparation; b3) preparing a product for preventing diseases caused by bacterial infection; b4) preparing a product for treating diseases caused by bacterial infection; b5) preventing food spoilage; b6) preparing a product for preventing food spoilage;
the bacteria are gram-negative bacteria;
the use is a non-disease diagnostic or/and therapeutic use.
3. The use of the oligopeptide according to claim 1 in combination with an antibacterial agent, c1) or c2) or c3) or c 4):
c1) inhibiting bacteria; c2) preparing an antibacterial preparation; c3) preparing a product for preventing diseases caused by bacterial infection; c4) preparing a product for treating diseases caused by bacterial infection;
the bacteria are gram-negative bacteria;
the use is a non-disease diagnostic or/and therapeutic use.
4. Use according to claim 3, characterized in that: the antibacterial drugs are tetracycline drugs, beta-lactam drugs, glycopeptide drugs, quinolone drugs, rifamycin drugs or cyclic peptide drugs.
5. Use of an oligopeptide according to claim 1 for restoring the sensitivity of a polymyxin-resistant gram-negative bacterium to polymyxin; the use is a non-disease diagnostic or/and therapeutic use.
6. Use of the oligopeptide of claim 1 in combination with an antibacterial agent for synergistic effect against polymyxin-resistant or polymyxin-intolerant klebsiella pneumoniae; the use is a non-disease diagnostic or/and therapeutic use.
7. Use of the oligopeptide of claim 1 in combination with an antibacterial agent for synergistic effect on bacteria;
the bacteria are gram-negative bacteria;
the use is a non-disease diagnostic or/and therapeutic use.
8. An antibacterial preparation comprising the oligopeptide of claim 1.
9. The antibacterial formulation of claim 8, wherein: the antibacterial preparation also contains antibacterial drugs.
10. The antibacterial formulation of claim 8 or 9, characterized in that: the bacteria are gram negative bacteria.
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