CN116036231A - Application of antibacterial peptide - Google Patents
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- CN116036231A CN116036231A CN202211656479.5A CN202211656479A CN116036231A CN 116036231 A CN116036231 A CN 116036231A CN 202211656479 A CN202211656479 A CN 202211656479A CN 116036231 A CN116036231 A CN 116036231A
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
- A61K38/08—Peptides having 5 to 11 amino acids
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- A61P31/04—Antibacterial agents
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Abstract
The invention provides an application of an antibacterial peptide in preparation of an antibacterial agent, wherein the antibacterial peptide comprises any one or more of polypeptides with a sequence of RCASSCGAK, RCASSCGAKS, TGMRCASSCGAK, TGMRCASSCGAKS, and the polypeptide sequence comprises the following modifications: cysteine is modified to an oxazolone-thioamide group; wherein the content of the polypeptide with the sequence of RCASSCGAK is higher than 60 percent. The antibacterial peptide has excellent antibacterial activity, has antibacterial effects on various bacteria such as escherichia coli, staphylococcus aureus, pseudomonas aeruginosa, klebsiella pneumoniae, acinetobacter baumannii and the like, is particularly excellent in antibacterial activity on multi-drug-resistant acinetobacter baumannii, and has good application prospects in the fields of antibacterial anti-infective drugs, preservatives and the like.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to application of antibacterial peptide.
Background
In recent years, due to the evolution of bacteria and the abuse of antibiotics, multi-drug resistant bacteria are growing, so that the difficulty of clinical anti-infection treatment is increased, more and more patients die continuously because proper antibacterial agents cannot be found, and the patients become the second leading cause of death worldwide, and great trouble and challenges are caused to first-line clinicians. Existing antibiotic therapies have failed to meet the clinical needs of treatment, and thus there is an urgent need to find new alternative drugs or therapeutic strategies.
Antibacterial peptide (AMP) is a low molecular weight protein with broad spectrum antibacterial and immunomodulatory activity against infectious bacterial (gram positive and gram negative), viral and fungal infections. At the same time, microorganisms are difficult to develop resistance to most antimicrobial peptides, which makes them a long-term effective product. In addition, the antibacterial peptide also has the characteristics of high specificity, low toxicity, biodiversity and the like. There is great interest in the hope of overcoming the increasingly serious problem of antibiotic resistance.
The methanoxybacteria (Mdns) is a small molecule peptide which is secreted by methanoxybacteria and used for capturing copper, and the structure and the composition of the methanoxybacteria produced by different methanoxybacteria are different, but the polypeptides are all peptides with post-translational modification groups. Early structural characterization of methanotrophic bacteria was difficult to perform because of their extreme susceptibility to degradation, and Kim et al were first studied and developed in 2004 as a result of the structure of methanotrophic bacteria secreted by the bacterial strain of campylobacter sporangium (Methylosinus trichosporium) OB3 b:
the LCGSCYPCSCM sequence is taken as a precursor peptide sequence, and the second cysteine and the eighth cysteine are modified into oxazolone-thioamide groups. Several other strains have been subsequently secreted with Mbn structures, all with typical modification groups, which generally include oxazolone-thioamides, 2, 6-dihydroxypyrazine-thioamides, etc., but to date, the number of structurally defined Mbn is still small (Dassama LM, kenney GE, rosenzweig AC. Methanobactins: from genome to function. Metallics.2017 Jan 25;9 (1): 7-20.Doi:10.1039/c6mt00208k.PMID: 27905610; PMCID: PMC 5269455.).
At present, researchers have carried out many researches and reports on biological activities of Mns including antioxidant activity, metal chelating property and the like, and found that some Mns also exhibit a certain antibacterial activity, and have great prospects as antibacterial peptides for use in anti-infective drugs (Zhou Qiqiong, xin Jiaying, zhang Yingxin, dong Jing, song, xia Chungu. Biological activity research progress of methane-oxidizing bacteria [ J ]. Biotechnology communication, 2009,20 (05): 723-725.).
However, since there are few Mbs with definite structures, the preparation difficulty is also high, and the activity change of Mbs with different structures is difficult to be accurately expected. Therefore, the development of new methane-oxidizing bacteria antibiotic peptide with excellent antibacterial activity has important significance.
Disclosure of Invention
The invention aims to provide an application of novel methane-oxidizing bacteria antibiotic peptide as an antibacterial agent with excellent antibacterial effect.
The invention provides application of an antibacterial peptide in preparation of an antibacterial agent, wherein the antibacterial peptide comprises any one or a mixture of more of the following polypeptides:
(a) A polypeptide with a sequence shown as SEQ ID NO. 1;
(b) A polypeptide with a sequence shown as SEQ ID NO. 2;
(c) A polypeptide with a sequence shown as SEQ ID NO. 3;
(d) A polypeptide with a sequence shown as SEQ ID NO. 4;
and the polypeptide sequence comprises the following modifications: cysteine is modified to an oxazolone-thioamide group;
wherein the polypeptide with the sequence shown as SEQ ID NO.1 accounts for more than 60 percent of the total peptide fragments.
Further, the antibacterial agent is a drug against any one or more of Acinetobacter baumannii, escherichia coli, staphylococcus aureus, pseudomonas aeruginosa and klebsiella pneumoniae.
Further, the antibacterial agent is a drug against multiple drug-resistant acinetobacter baumannii.
Further, the antibacterial peptide is prepared by the following method:
(1) Preparing recombinant bacteria containing RrmbnA genes, rrmbnB genes and RrmbnC genes;
(2) Inducing and culturing the recombinant bacteria in the step (1) to obtain bacterial liquid containing antibacterial peptide;
(3) Purifying the bacterial liquid obtained in the step (2);
the RrmbnA gene sequence is shown in SEQ ID NO. 5; the RrmbnB gene sequence is shown as SEQ ID NO. 6; the RrmbnC gene sequence is shown in SEQ ID NO. 7.
Furthermore, the preparation method of the recombinant bacterium in the step (1) comprises the following steps:
1) Respectively constructing a recombinant plasmid A containing nucleotide fragments with the sequences shown as SEQ ID NO.5 and a recombinant plasmid B containing nucleotide fragments with the sequences shown as SEQ ID NO.6 and SEQ ID NO. 7;
2) And (3) co-converting the recombinant plasmids A and B prepared in the step (1) into engineering bacteria to obtain positive clones.
Furthermore, in the step 1) of the preparation method of the recombinant bacterium, the recombinant plasmid A contains a nucleotide fragment P4 formed by fusing an RBS region with SEQ ID NO. 5; recombinant plasmid B contains a nucleotide fragment of sequence SEQ ID NO.7 with a protein tag, preferably a 6 XHis tag.
Further, the nucleotide fragment P4 formed by fusing the RBS region and SEQ ID NO.5 consists of: f1 and R1, F2 and R2, and F3 and R3 are templates and primers, and are obtained through PCR amplification; the nucleotide sequence of F1 is shown as SEQ ID NO. 8; the nucleotide sequence of R1 is shown as SEQ ID NO. 9; the F2 nucleotide sequence is shown as SEQ ID NO. 10; the nucleotide sequence of R2 is shown as SEQ ID NO. 11; the F3 nucleotide sequence is shown as SEQ ID NO. 12; the nucleotide sequence of R3 is shown as SEQ ID NO. 13.
Further, the PCR amplification is as follows: respectively taking F1/R1, F2/R2 and F3/R3 as templates and primers, and performing PCR amplification to obtain three short fragments P1, P2 and P3 forming RrmbnA genes; and (3) using P1, P2 and P3 as templates and F1/R3 as primers, and performing overlap extension PCR amplification to obtain a fusion fragment P4 of the RBS region and the whole length of the RrmbnA gene.
Further, the inducer used in the induction culture in the step (2) is IPTG, and the recombinant bacterium is escherichia coli BL21DE3; the bacterial liquid containing the antibacterial peptide is obtained by adding protease inhibitor into recombinant bacterial cells after induction culture and crushing; preferably, the protease inhibitor is PMSF.
Further, the purification in the step (3) includes the following steps:
1') crude separation: passing the bacterial liquid through a nickel column affinity chromatographic column, and eluting to obtain a compound;
2') ultrafiltration separation: centrifuging the compound obtained in the step 1') in an ultrafiltration tube, and collecting the filtrate of the ultrafiltration tube;
3') buffer replacement: purifying the filtrate obtained in the step 2') by macroporous adsorption resin, washing with ultrapure water to remove buffer solution, eluting target components by using 50% -70% acetonitrile or ethanol, and freeze-drying;
4') molecular sieve repurification: resuspension the lyophilized powder obtained in step 3'), separating by Superdex 75/300 GL gel chromatographic column, collecting eluting peak, and lyophilizing to obtain purified antibacterial peptide.
The invention has the beneficial effects that: the methanoxybacteria antibacterial peptide with the novel structure provided by the invention has excellent antibacterial activity, has antibacterial effects on various bacteria such as escherichia coli, staphylococcus aureus, pseudomonas aeruginosa, klebsiella pneumoniae, acinetobacter baumannii and the like, is particularly excellent in antibacterial activity on multi-drug-resistant acinetobacter baumannii, and has good application prospects in the fields of antibacterial anti-infective drugs, preservatives and the like.
The term of the invention: the structure of the oxazolone-thioamide group is as follows:
the cysteine of the invention is modified into oxazolone-thioamide group, namely the cysteine part structure in the polypeptide sequence:
become->
"RBS region" refers to the ribosome binding site and is a purine-rich untranslated region upstream of the start codon.
It should be apparent that, in light of the foregoing, various modifications, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
The above-described aspects of the present invention will be described in further detail below with reference to specific embodiments in the form of examples. It should not be understood that the scope of the above subject matter of the present invention is limited to the following examples only. All techniques implemented based on the above description of the invention are within the scope of the invention.
Drawings
FIG. 1 is an SDS-PAGE identification of a co-expression complex by nickel column affinity chromatography, wherein M represents a protein Marker, C represents a whole cell sample, S represents a supernatant sample separated after disruption, F represents a flow-through sample of nickel column affinity chromatography, and E represents a sample eluted by a nickel column;
FIG. 2 shows the antibacterial peptide RrMbina OXA The grey dotted line, the black solid line and the grey solid line respectively represent peak types corresponding to the UV detectors of 270nm, 280nm and 335nm, the mature peptide form of RrMnA has characteristic absorption peaks at 270nm and 335nm, and the peak types in the graph are single and have no impurity protein pollution;
FIG. 3 shows the antibacterial peptide RrMbina OXA Ultraviolet-visible absorption spectrum (UV-Vis) identification patterns of (C), black solid line, gray dashed line represent RrMbnBC control, rrMbinaBC co-expression product complex, purified RrMbina, respectively OXA UV-Vis absorption spectrum of (c). Purified RrMbina OXA Has characteristic ultraviolet absorption of methane-oxidizing mycotins, namely absorption peaks around 270nm and 335nm as shown in figure 3.
FIG. 4 is the primary and secondary mass spectra results of RCASSCGAK fragments.
FIG. 5 is the primary and secondary mass spectrum results of RCASSCGAKS fragments.
FIG. 6 is the primary and secondary mass spectra results of TGMRCASSCGAK fragments.
FIG. 7 is the primary and secondary mass spectra results of TGMRCASSCGAKS fragments.
FIG. 8 shows the antibacterial peptide RrMbina OXA Antibacterial activity against E.coli;
FIG. 9 shows the antibacterial peptide RrMbina OXA Antibacterial activity against staphylococcus aureus;
FIG. 10 shows the antibacterial peptide RrMbina OXA Antibacterial activity against pseudomonas aeruginosa;
FIG. 11 shows the antibacterial peptide RrMbina OXA Antibacterial activity against klebsiella pneumoniae;
FIG. 12 shows the antibacterial peptide RrMbina OXA Antibacterial activity against a standard strain of acinetobacter baumanii;
FIG. 13 results of multiple drug resistance experiments of different antibiotics against clinical isolates of Acinetobacter baumannii;
FIG. 14 antibacterial peptide RrMbina of the present invention OXA Antibacterial results on multi-drug resistant acinetobacter baumannii;
FIG. 15 antibacterial peptide RrMbina of the present invention OXA Real-time monitoring results of growth inhibition of Acinetobacter baumannii clinical isolates;
FIG. 16 shows the antibacterial activity of the antibacterial peptide against Acinetobacter baumannii AB9 at different concentrations.
FIG. 17 shows the antibacterial activity of the antibacterial peptide against Acinetobacter baumannii AB13 at different concentrations.
FIG. 18 shows the antibacterial activity of the antibacterial peptide against Acinetobacter baumannii AB14 at different concentrations.
FIG. 19 shows the antibacterial activity of the antibacterial peptide against Acinetobacter baumannii AB15 at various concentrations.
FIG. 20 shows the antibacterial activity of the antibacterial peptide against Acinetobacter baumannii AB9 at different pH values.
FIG. 21 shows the antibacterial activity of the antibacterial peptide against Acinetobacter baumannii AB13 at different pH values.
FIG. 22 shows the antibacterial activity of the antibacterial peptide against Acinetobacter baumannii AB14 at different pH values.
FIG. 23 shows the antibacterial activity of the antibacterial peptide against Acinetobacter baumannii AB15 at different pH values.
FIG. 24 shows the antibacterial activity of the antibacterial peptide against Acinetobacter baumannii AB9 at different temperatures.
FIG. 25 shows the antibacterial activity of the antibacterial peptide against Acinetobacter baumannii AB13 at different temperatures.
FIG. 26 shows antibacterial activity of antibacterial peptides against Acinetobacter baumannii AB14 at different temperatures.
FIG. 27 shows antibacterial activity of antibacterial peptides against Acinetobacter baumannii AB15 at different temperatures.
Detailed Description
The raw materials and equipment used in the invention are all known products and are obtained by purchasing commercial products.
EXAMPLE 1 antimicrobial peptide RrMbina of the invention OXA Is prepared from
(1) Precursor peptide RrMnA expression vector construction: first, 3 pairs of primers are designed:
f1 (sequence shown in SEQ ID NO. 8):
5’-CGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACC-3’；
r1 (sequence shown in SEQ ID NO. 9):
5’-CTGAATCTCCACTTTTTTAACGATCACGATTTTCATGGTATATCTCCTTCTTA-3’；
f2 (sequence shown in SEQ ID NO. 10):
5'-AAAAAGTG GAGATTCAGGTTGCAGGCCGTACCGGCATGCGT-3'; r2 (sequence shown in SEQ ID NO. 11):
5’-GCTTTTTGCACCACAAGAAGAAGCGCAACGCATGCCGGTACG-3’；
f3 (sequence shown in SEQ ID NO. 12):
5’-TGTGGTGCAAAAAGCTAAGAGAACCTGTACTTCCAAGGGCATCATCATCATCA-3’；
r3 (sequence shown in SEQ ID NO. 13):
5’-TGGTGGTGGTGGTGCTCGAGTCAGTGATGATGATGATGATGCC-3’；
underlined is a fragment homologous to the vector; respectively taking F1/R1, F2/R2 and F3/R3 as templates and primers, and performing PCR amplification to obtain three short fragments P1, P2 and P3 forming RrmbnA gene (the N end carries an RBS region); p1, P2 and P3 are used as templates, F1/R3 is used as a primer, and fusion fragment P4 of the RBS region and the whole length of RrmbnA gene is obtained through overlapping extension PCR amplification,
the sequence of the RrmbnA gene is (SEQ ID NO. 5):
ATGAAAATCGTGATCGTTAAAAAAGTGGAGATTCAGGTTGCAGGCCGTACCGGCATGCGTTGCGCTTCTTCTTGTGGTGCAAAAAGCTAA
the sequence of the fusion fragment P4 of the RBS region and the full length of the RrmbnA gene is (SEQ ID NO. 14):
CGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACCATGAAAATCGTGATCGTTAAAAAAGTGGAGATTCAGGTTGCAGGCCGTACCGGCATGCGTTGCGCTTCTTCTTGTGGTGCAAAAAGCTAAGAGAACCTGTACTTCCAAGGGCATCATCATCATCATCACTGACTCGAGCACCACCACCACCA
the vector pET28b is subjected to double-enzyme tangentially-cutting by XbaI and XhoI, homologous recombination is carried out on the vector and the fragment P4, so that an expression vector pET28b-RrmbnA is formed, DH5 alpha competence is transformed, and sequencing identification is carried out.
(2) Construction of an expression vector of the biological synthesis holoenzyme RrMbnBC: the codon optimized RrmbnB (SEQ ID NO. 6) and RrmbnC gene sequences (SEQ ID NO. 7) were synthesized by Jin Weizhi Biotechnology Inc. and constructed onto pETDuet-1 vector, where RrmbnC was constructed to the first MCS region with a 6 XHis tag and RrmbnB was constructed to the second MCS region without tag, the recombinant plasmid was designated pETDuet-1-RrmbnBC.
SEQ ID NO.6：
ATGCGCATTGGCTTTAACTTTACCCTGGGCGAAACCCTGCCGCTGGTGCGTCAGTTAGCGCAAGAAGGTGCGATTGATTATTGCGAACTGCTGATTGATAACTTTATGCAAGTGCCGCCGCAAGAACTGGCGGAAGCGTTTGATGTGCCGGTGGGCTTTCATATTATGTTTAGCCGCTTTATTGAAAGCGATGAAGAACAGCTGCGCGATTTTGCGGCGCGCCTGCGCCCGTATATTGAAGCGCTGCGCCCGCTGTATGTGAGCGATCATATTGCGTATTTTAGCCATCAAGGCCGCGCGCTGTATCATCTGGGCGAAATTGATTATGCGGCGGATTATGAACGCGTGCGCGCGCGCGCGGCGCTGTGGCAGAGCCTGCTGGGTCAGACCATTCATTTTGAAAACTATCCGAGCATTGTGGATGGCGGCCATGCGGCGCCGGCGTTTTTTCAGCGCCTGGCGCGCGATACCGGCGCGGGCGTGCTGTTTGATGTTAGCAACGCGGTGTGCGCGTGGCGCAACGATGGCCCGGAGGTTGCGGCCTGGCGCGGCGTGATGGCGGGCGCGAGCCATTTTCATGTGGGCGGCTATGCGGGCGCGTTTATTGATGAAGGCGTGACCGTGGATACCCATGATCGCGCGCTGGCGCAAGATACCCTGGATAGCCTGCGCCGCCATCGCGATGTGCTGGATAAACCGGGCGCGACCATTACCTATGAACGCGATGAAAACATTGACATTGACGGTGTGCGCGCGGATCTGCTGGCGCTGCGCGCCATCTTTCCGCGTGGCCAAGCCCGTCAGCCGGCGCCGGCGGAAGCGGTGGCGAGCTAA
SEQ ID NO.7：
ATGAACGCGCCGACCACCGCGGCCGCGGGCGCGGCGCCGGGCCGCCAAGTGAAAGATAGCGAACTGCTGGCGCGCCTGGCCGATCCGGCGGCGCGCGGCGATTTTCCGCCGGGCTGCCGCGCGCATGTGCGCATTGATATTAGCATTCGCGCGTATTGGCATACCCTGTTTGATATTTGCCCGGGCCTGCTGGATATTGCCGATCCGGATGGCATGGCGATTTTTGCGCCGTTTATGGATTGGGCGCGCCGCGAAAACCTGACCATGGGCTGGAGCTTTTATATTTGGGTGGGCCGCTGGCTGGCGCAGAGCCCGTGGCGCGAACGCCTGGATGAAGAACTGACCCAAGCGCTGTTAAGCGCGAGCGCGGCGCGCTGGGCGGTGTTAGATCGTAGCGCCGATGTGGGCGTGGTTCTGGGCCGCCGCGGCAGCGATGATTGGATTATCGGCTGGAAACCGAACACCCTGGCGGCGGGCCGCCGCGTGGAACTGGTGAGCTTAGATGGTCAGCTGCCGCGCCCGGCGGAAGATGTGGGCGTGTTTCATCTGGCGGGCTATGAACTGGATAGCTTTCCGGGCTGGTTAGCGCTGCCGCGCTAA
(3) Co-transformation of double plasmids: the plasmid pET28b-RrmbnA with kanamycin resistance and the plasmid pETDuet-1-RrmbnBC with ampicillin resistance are co-transformed into competent cells of the expression bacterium BL21DE3, and the competent cells are coated on a double-resistance plate added with ampicillin and kanamycin, and positive clones coexisting with double plasmids are selected.
(4) Induction of expression: selecting 5 positive clones obtained in the step (3) to 100mL of LB liquid medium, and culturing for 3 hours at 220rpm at the temperature of 37 ℃ on a shaking table; according to 1:50 volume ratio was transferred to 15 1L LB medium and cultured at 220rpm to OD at shaker 37℃ 600 Cooling the shaking table to 16 ℃ at a speed of 180rpm for 30min;the inducer IPTG was added to a final concentration of 100mM per 1L of the culture, and the culture was continued for 16 hours.
(5) And (3) bacterial harvesting and crude extraction: centrifuging 15L of the bacterial liquid in the step (4) at 3800rpm for 12min, discarding the supernatant, and resuspending bacterial precipitate with a buffer solution containing 10mM Hepes (pH 8.0); adding a protease inhibitor PMSF with the final concentration of 1mM, and crushing thalli by a high-pressure cell crusher; separating supernatant and precipitate by centrifuging at 15000rpm for 30min; loading the supernatant onto nickel column affinity chromatography column, eluting enzyme and RrMbNA OXA SDS-PAGE identification of co-expression complexes by nickel column affinity chromatography is shown in figure 1, wherein M represents a protein Marker, C represents a whole-cell sample, S represents a supernatant sample separated after disruption, F represents a flow-through sample of nickel column affinity chromatography, and E represents a sample eluted by a nickel column.
(6) Ultrafiltration separation: centrifuging the eluted complex in step (5) in 10kDa ultrafilter tube at 3800rpm for 30min, collecting ultrafilter tube filtrate, and collecting main component RrMbnA with smaller molecular weight OXA 。
(7) Buffer replacement: purifying the filtrate collected in (6) by macroporous adsorption resin HP-20, washing with ultrapure water to remove the buffer component, eluting the target component with 200mL of 60% acetonitrile finally, and freeze-drying overnight.
(8) And (3) purifying the molecular sieve: resuspending the lyophilized powder obtained in step (7) with 2mL of ultrapure water, separating with Superdex 75/300 GL gel chromatography column, collecting eluting peak, performing absorption spectrum analysis, and lyophilizing again to obtain final product of antibacterial peptide RrMbina as shown in figures 2 and 3 OXA 。
EXAMPLE 2 antimicrobial peptide RrMbina of the invention OXA Structural confirmation of (a)
(1) The method comprises the following steps: the antibacterial peptide RrMbina prepared in example 1 was taken OXA Liquid chromatography tandem mass spectrometry (HPLC-MS/MS) assays were performed on a Orbitrap Fusion Lumos mass spectrometer (ThermoFisher Scientific, san jose, california) equipped with a nano ESI ion source that provided HCD, CID and ETD fragmentation analysis. The peptide fragments were separated by analytical capillary column packed with 3 μm spherical C18 reversed phase material (Dr. Masch GmbH, germany). Using EASY-nLC 1200 (Sieimer's Feier family USA)The following HPLC gradient was generated: 0% -65% b in 50 min, 50% -80% b in 5min, 80% b in 2min (a=0.1% formic acid, 99.9% h 2 O; b=80% acetonitrile, 20% H 2 O, 0.1% formic acid). The mass spectrometer was operated in a data dependent mode with one MS scan at the highest speed for 3 seconds, followed by HCD (high energy collisional dissociation) MS/MS scan and ETHCD (electron transfer/high energy collisional dissociation). Raw data were analyzed using Thermo Xcalibur QualBrowser and Thermo Proteome Discoverer 2.5.0.400 to identify and confirm peptide fragments therein.
(2) Results: as shown in fig. 4-7, the mass of the primary mass spectrum matches the expected modification of the two sites of the peptide fragment, and the secondary mass spectrum further identifies the composition of the peptide fragment and the specific site at which the modification occurs; wherein the peptide segment corresponding to the peak with the mass-to-charge ratio of 870.32727 is RCASSCGAK after modification, and the content of the peptide segment is the highest and accounts for more than 60% of the total peptide segment; the modified RCASSCGAKS, TGMRCASSCGAK, TGMRCASSCGAKS fragments corresponding to parent ions with mass-to-charge ratios of 961.35809, 556.24329 and 625.73944, respectively, indicate that the antimicrobial peptide RrMnA OXA Contains polypeptides with fragments RCASSCGAK (SEQ ID NO. 1), RCASSCGAKS (SEQ ID NO. 2), TGMRCASSCGAK (SEQ ID NO. 3) and TGMRCASSCGAKS (SEQ ID NO. 4). The modification process involves multiple reaction steps of oxygenation, hydrogen abstraction, dehydration, hydrogenation and the like, and finally the mass of the single cysteine is reduced by-4 Da when the modification is finished. The results of the secondary mass spectrum showed a mass change of-4 Da at each cysteine, indicating that oxazolone-thioamide modification occurred for both cysteines in the polypeptide. Since HPLC separation is performed in a strong acid environment, partial polypeptides undergo acid hydrolysis, -30Da is derived from hydrolysis of-4 Da, which is equivalent to-4 Da modification.
(3) Conclusion: as shown in Table 1, the antibacterial peptide RrMbnA of the present invention OXA Contains fragment structure. The lower case letter c of the sequences in the table, i.e. the position of cysteine at which a mass change of-4 Da occurs on the mass spectrum, i.e. oxazolone-thioamide modification occurs, -30Da is the acid hydrolysis formation of the-4 Da modification during HPLC loading, and is equivalent to the-4 Da modification before hydrolysis. Antibacterial peptide RrMbina OXA The fragments in (a) are mainly RcASSCGAK, and the cleavage between the last two amino acids K and S is random, and each purification is performedThere will be some variation in the outgoing ratio.
TABLE 1
| Sequence annotated after mass spectral library search | Corresponding modification |
| [AM].RcASScGAK.[S] | C2(-4)；C6(-4) |
| [AM].RcASScGAKS.[-] | C2(-4)；C6(-4) |
| [AR].TGMRcASScGAKS.[-] | C5(-4)；C9(-4) |
| [AR].TGMRcASScGAK.[-] | C5(-30)；C9(-30) |
The following experiments prove the beneficial effects of the antibacterial peptide.
Experimental example 1 broad-spectrum antibacterial Activity of the antibacterial peptide of the present invention
In this example, the polypeptide RrMbina of the present invention was verified by a double-layer plate method OXA The broad-spectrum antibacterial activity of (2) comprises the following specific steps:
1) Preparation of the Strain (E.coli DH 5. Alpha., staphylococcus aureus Newman, pseudomonas aeruginosa PAO1, klebsiella pneumoniae ATCC11296, acinetobacter baumannii ATCC 19606)
-80 ℃ frozen bacterial liquid or 4 ℃ preserved bacterial liquid 1:100 was transferred to fresh LB medium, and shaken to mid-log growth at 220rpm/37℃for about 3 hours.
2) Preparation of double-layer agar
The lower glue is prepared in advance: about 10mL of LB (containing 1.5% agar powder) after high pressure is spread on a 10cm dish, uncapped and air-dried for about ten minutes, and then collected for standby.
Preparation of the upper layer adhesive: after high pressure LB containing 0.3% of agar powder, the mixture is placed in a 50 ℃ oven for standby.
3) Sub-packaging of upper glue
Prepare 10mL centrifuge tubes, add 4mL of supersize gum to each tube, and place rapidly in a 42 ℃ water bath for use.
4) Diluting the bacterial liquid, and mixing with upper layer glue
The bacterial liquid in the mid-log growth stage is used in fresh medium 1:5, adding a 10mL centrifuge tube containing the upper layer glue after dilution, uniformly mixing the materials for about 10 times in an upside-down way, rapidly pouring the materials on the lower layer agar prepared in advance, uniformly dispersing the materials, covering the materials, and standing the materials in a biosafety cabinet for 10 minutes until the materials solidify.
5) Antibacterial peptide RrMbina OXA Is added dropwise to
Three concentration gradients were set, 23mg/mL, 2.3mg/mL, and 0.23mg/mL. Dripping the set groups on the double-layer agar respectively, wherein the concentration is 1.5 mu L/drop; the same volume of ultrapure water was simultaneously added dropwise as a control.
6) Placing in a incubator at 37 ℃ for overnight incubation
Standing on the surface of the biosafety cabinet for a plurality of minutes, sucking the liquid drop, carefully placing the liquid drop into a incubator, and culturing the liquid drop in an inverted mode after half an hour.
7) Observing whether or not there is clear antibacterial spot
Observing whether antibacterial spots exist at the drop adding position on a photoresist camera, and photographing.
The results are shown in fig. 8, 9, 10, 11 and 12: wherein FIG. 8 is an antibacterial peptide RrMbina OXA Antibacterial activity against E.coli; FIG. 9 shows the antibacterial peptide RrMbina OXA Antibacterial activity against staphylococcus aureus; FIG. 10 shows the antibacterial peptide RrMbina OXA Antibacterial activity against pseudomonas aeruginosa; FIG. 11 is a Klebsiella pneumoniae RrMbina OXA Antibacterial activity against E.coli; FIG. 12 shows the antibacterial peptide RrMbina OXA Antibacterial activity against a standard strain of acinetobacter baumanii;
antibacterial peptide RrMbina OXA The antibacterial agent has broad-spectrum antibacterial activity on escherichia coli, staphylococcus aureus, pseudomonas aeruginosa, klebsiella pneumoniae and acinetobacter baumannii standard strains, and has high-efficiency inhibition effect on 5 bacterial plates. Antibacterial peptide RrMbina OXA Obvious inhibition zones appear around the liquid drops, and concentration dependence appears, while no inhibition zone appears in the control part.
Experimental example 2 determination of multiple drug resistance of Acinetobacter baumannii clinical isolate by double-layer plate method and inhibition of the antibacterial peptide of the present invention on the multiple drug resistant Acinetobacter baumannii clinical isolate
1) Preparation of strains
4 Acinetobacter baumannii clinical isolates were selected and labeled as AB9, AB13, AB14 and AB15, respectively. -80 ℃ frozen bacterial liquid or 4 ℃ preserved bacterial liquid 1:100 was transferred to fresh LB medium, and shaken to mid-log growth at 220rpm/37℃for about 3 hours.
2) Preparation of double-layer agar
The lower glue is prepared in advance: about 10mL of LB (containing 1.5% agar powder) after high pressure is spread on a 10cm dish, and the dish is opened and air-dried for about ten minutes, and then stored for standby.
Preparation of the upper layer adhesive: after high pressure LB containing 0.3% of agar powder, the mixture is placed in a 50 ℃ oven for standby.
3) Sub-packaging of upper glue
Preparing 10mL centrifuge tubes, adding 4mL of upper layer adhesive to each tube, and rapidly placing in a water bath kettle at 42 DEG C
4) Diluting the bacterial liquid, and mixing with upper layer glue
The bacterial liquid in the mid-log growth stage is used in fresh medium 1: and 5, respectively adding 10mL centrifuge tubes containing the upper layer glue after dilution, uniformly mixing the materials for about 10 times in an upside-down way, rapidly pouring the materials on the lower layer agar prepared in advance, uniformly dispersing the materials, covering the materials, and standing the materials in a biosafety cabinet for 10 minutes until the materials solidify. Two double-layered agar plates were prepared for each strain.
5) Experimental treatment of double-layer Flat plate
(1) Determination of multiple drug resistance of Acinetobacter baumannii clinical isolates
The double-layer agar plate of each strain is divided into 5 areas, and different drug sensitive tablets (20 antibiotics are respectively placed in different areas of 4 plates) are placed in the center of each area, so that the two areas are fully adhered with the culture medium, and the culture medium is placed for 15 minutes at room temperature.
(2) Determination of the antibacterial peptide RrMbina OXA Inhibition of multiple drug resistant Acinetobacter baumannii clinical isolates
Antibacterial peptide RrMbina OXA Three concentration gradients of 23mg/mL, 2.3mg/mL and 0.23mg/mL were set, and were added dropwise to each of the bacteria on double-layered agar plates, 1.5. Mu.L/drop, respectively; the same volume of sterile water was added dropwise at the same time as a control. Left at room temperature for 15 minutes.
6) After the plate is turned over, the plate is placed in a 37 ℃ incubator for overnight incubation;
judging the drug sensitivity of Acinetobacter baumannii according to the existence and the size of the inhibition zone.
The results are shown in FIG. 13:
FIG. 13 shows that the 4 Acinetobacter baumannii strains (AB 9, AB13, AB14 and AB15 are all clinical isolates from Huaxi Hospital of Sichuan university) used in the invention all show multiple drug resistance, and the antibacterial circles with different sizes appear around different antibiotic filter paper sheets, so that the antibacterial circles show different sensitivities; table 1 shows the antibiotic resistance of 4 Acinetobacter baumannii strains.
TABLE 1 Acinetobacter baumannii resistant to antibiotics
Meanwhile, the antibacterial peptide RrMbnA OXA Has remarkable inhibiting effect on multiple drug-resistant Acinetobacter baumannii clinical isolates, and as shown in figure 14, the antibacterial peptides RrMbina of 4 clinical isolates OXA A clear zone of inhibition appears around and concentration dependence appears.
Experimental example 3, growth curve method, verifies the polypeptide RrMbnA of the invention OXA Antibacterial activity against 4 multiple drug-resistant Acinetobacter baumannii
1) Treatment of bacterial liquid
-80 ℃ frozen bacterial liquid or 4 ℃ preserved bacterial liquid 1:100 is transferred to fresh culture medium, and is rocked to logarithmic growth medium at 220rpm/37 ℃ for about 3 hours; fresh medium 1 was then used: after 50-fold dilution, the bacterial liquid was dispensed into 96-well cell culture plates with a row gun at 100. Mu.L/well.
(2) Antibacterial peptide RrMbina OXA Is processed by (a)
Setting antibacterial peptide RrMbnA OXA The final concentration was 75. Mu.g/mL, and the mixture was applied to the wells of the above 96-well plate, 3 parallel wells/concentration, and 2. Mu.L/well. Control groups were also established: without addition of RrMbnA OXA Filling with sterile ultrapure water.
(3) Real-time measuring bacterial liquid growth condition by enzyme-labeled instrument
After the 96-well plate is capped, the plate is sealed by a sealing tape and is added into an enzyme-labeled instrument for measurement. Parameter setting: the detection temperature is 37 ℃, the detection wavelength is 600nm, and the detection interval is 15min.
(4) Data processing
The software used was Graphpad Prism 8.3.
Results:
as shown in FIG. 15, the polypeptide RrMbina of the present invention OXA Can effectively inhibit the growth of the Acinetobacter baumannii clinical isolate.
Experimental example 4 determination of the minimum effective bacteriostatic concentration
The antibacterial peptide RrMbnA is diluted by the doubling ratio OXA Then, the antimicrobial peptides RrMbina with different concentration gradients are determined by a growth curve method OXA (300. Mu.g/mL, 150. Mu.g/mL, 75. Mu.g/mL, 38. Mu.g/mL, 19. Mu.g/mL, 9.5. Mu.g/mL, 4.7. Mu.g/mL, 2.3. Mu.g/mL, and 1.2. Mu.g/mL). The test was repeated 3 times. Due to the antimicrobial peptide RrMbina in this patent OXA Is effective bacteriostatic activity, and cannot thoroughly kill bacteria, so the minimum bacteriostatic concentration (MIC) is defined as: the lowest concentration of antimicrobial peptide effective to inhibit bacterial growth compared to the control growth curve.
Results:
the Minimum Inhibitory Concentration (MIC) of the antibacterial peptide on Acinetobacter baumannii was determined by a double dilution method, and the results are shown in FIGS. 16-19, wherein FIG. 16 shows the antibacterial activity of the antibacterial peptide on Acinetobacter baumannii AB9 at different concentrations; FIG. 17 shows different concentrationsAntibacterial activity of the lower antibacterial peptide on Acinetobacter baumannii AB 13; FIG. 18 shows the antibacterial activity of the antibacterial peptide against Acinetobacter baumannii AB14 at different concentrations; FIG. 19 shows the antibacterial activity of the antibacterial peptide against Acinetobacter baumannii AB15 at various concentrations. From the figure, it can be seen that the antibacterial peptide RrMbnA OXA MIC was similar for the 4 Acinetobacter baumannii strains measured and was 38. Mu.g/mL. The results show that the antibacterial peptide RrMbnA OXA Has good antibacterial activity.
Experimental example 5 influence of different pH values on antibacterial peptide bacteriostatic effect
The antibacterial peptide RrMbina OXA Mixing with aqueous solutions (pH is adjusted by NaOH and HCl) of pH4, 5, 6, 7, 8, 9 and 10, respectively, and allowing to act at 37deg.C for 30min, and determining antibacterial activity of the antibacterial peptide by growth curve method. The test was repeated 3 times.
Results:
the antibacterial peptide RrMbina OXA Diluting with water solutions with different pH values, performing action for 30min, and determining antibacterial activity on 4 Acinetobacter baumannii AB9, AB13, AB14 and AB15, wherein the results are shown in figures 20-23; wherein, figure 20 shows the antibacterial activity of the antibacterial peptide on acinetobacter baumannii AB 9; FIG. 21 shows the antibacterial activity of the antibacterial peptide against Acinetobacter baumannii AB 13; FIG. 22 shows the antibacterial activity of the antibacterial peptide against Acinetobacter baumannii AB 14; FIG. 23 shows the antibacterial activity of the antibacterial peptide against Acinetobacter baumannii AB 15. From the figure, it can be seen that the antibacterial peptide RrMbnA OXA The antibacterial activity is good at the pH of 4-10, and no obvious difference exists between different pH values. Indicating that the antibacterial peptide RrMbnA OXA Has better acid-base stability.
Experimental example 6 influence of different temperatures on antibacterial peptide bacteriostatic Effect
The antibacterial peptide RrMbina OXA Respectively placing at-80deg.C, 0deg.C, 25deg.C, 50deg.C, 65deg.C, 75deg.C and 100deg.C for 30min, and measuring antibacterial activity of the antibacterial peptide by growth curve method. The test was repeated 3 times.
Results:
the antibacterial peptide RrMbina OXA The antibacterial activity against 4 Acinetobacter baumannii AB9, AB13, AB14 and AB15 was measured after 30min of action at different temperatures, and the results are shown in FIGS. 24-27, respectively. Wherein FIG. 24 shows the temperature at different temperaturesAntibacterial activity of the antibacterial peptide on Acinetobacter baumannii AB 9; FIG. 25 shows antibacterial activity of antibacterial peptides against Acinetobacter baumannii AB13 at different temperatures; FIG. 26 shows antibacterial activity of antibacterial peptides against Acinetobacter baumannii AB14 at different temperatures; FIG. 27 shows antibacterial activity of antibacterial peptides against Acinetobacter baumannii AB15 at different temperatures. As is clear from the figure, the antibacterial peptide RrMbnA was found to have a weak decrease in antibacterial activity in addition to a 30min action at 100 ℃ OXA The antibacterial activity of the composition has no obvious fluctuation along with the temperature change, and has good antibacterial activity. Indicating that the antibacterial peptide RrMbnA OXA Has better heat stability.
In summary, the invention provides an application of an antibacterial peptide in preparation of an antibacterial agent. The antibacterial peptide has excellent antibacterial activity, has antibacterial effects on various bacteria such as escherichia coli, staphylococcus aureus, pseudomonas aeruginosa, klebsiella pneumoniae, acinetobacter baumannii and the like, is particularly excellent in antibacterial activity on multi-drug-resistant acinetobacter baumannii, and has good application prospects in the fields of antibacterial anti-infective drugs, preservatives and the like.
Claims (10)
1. Use of an antimicrobial peptide in the preparation of an antimicrobial agent, wherein the antimicrobial peptide comprises a mixture of any one or more of the following polypeptides:
(a) A polypeptide with a sequence shown as SEQ ID NO. 1;
(b) A polypeptide with a sequence shown as SEQ ID NO. 2;
(c) A polypeptide with a sequence shown as SEQ ID NO. 3;
(d) A polypeptide with a sequence shown as SEQ ID NO. 4;
and the polypeptide sequence comprises the following modifications: cysteine is modified to an oxazolone-thioamide group;
wherein the polypeptide with the sequence shown as SEQ ID NO.1 accounts for more than 60 percent of the total peptide fragments.
2. The use according to claim 1, wherein the antibacterial agent is a medicament against any one or more of acinetobacter baumannii, escherichia coli, staphylococcus aureus, pseudomonas aeruginosa, klebsiella pneumoniae.
3. The use according to claim 2, wherein the antibacterial agent is an anti-multidrug-resistant acinetobacter baumannii drug.
4. Use of an antimicrobial peptide according to any one of claims 1 to 3, wherein the antimicrobial peptide is prepared by the following method:
(1) Preparing recombinant bacteria containing RrmbnA genes, rrmbnB genes and RrmbnC genes;
(2) Inducing and culturing the recombinant bacteria in the step (1) to obtain bacterial liquid containing antibacterial peptide;
(3) Purifying the bacterial liquid obtained in the step (2);
the RrmbnA gene sequence is shown in SEQ ID NO. 5; the RrmbnB gene sequence is shown in SEQ ID NO. 6; the RrmbnC gene sequence is shown in SEQ ID NO. 7.
5. The use according to claim 4, wherein the recombinant bacterium of step (1) is prepared by the following method:
1) Respectively constructing a recombinant plasmid A containing nucleotide fragments with the sequences shown as SEQ ID NO.5 and a recombinant plasmid B containing nucleotide fragments with the sequences shown as SEQ ID NO.6 and SEQ ID NO. 7;
2) And (3) co-converting the recombinant plasmids A and B prepared in the step (1) into engineering bacteria to obtain positive clones.
6. The use according to claim 5, wherein in step 1) of the method for preparing the recombinant bacterium, the recombinant plasmid A comprises a nucleotide fragment P4 fused by RBS region and SEQ ID NO. 5; recombinant plasmid B contains a nucleotide fragment of sequence SEQ ID NO.7 with a protein tag, preferably a 6 XHis tag.
7. The use according to claim 6, wherein the nucleotide fragment P4 fused to the RBS region of SEQ ID No.5 consists of: f1 and R1, F2 and R2, and F3 and R3 are templates and primers, and are obtained through PCR amplification; the nucleotide sequence of F1 is shown as SEQ ID NO. 8; the nucleotide sequence of R1 is shown as SEQ ID NO. 9; the F2 nucleotide sequence is shown as SEQ ID NO. 10; the nucleotide sequence of R2 is shown as SEQ ID NO. 11; the F3 nucleotide sequence is shown in SEQ ID NO. 12; the nucleotide sequence of R3 is shown as SEQ ID NO. 13.
8. The use of claim 7, wherein the PCR amplification is: respectively taking F1/R1, F2/R2 and F3/R3 as templates and primers, and performing PCR amplification to obtain three short fragments P1, P2 and P3 forming RrmbnA genes; and (3) using P1, P2 and P3 as templates and F1/R3 as primers, and performing overlap extension PCR amplification to obtain a fusion fragment P4 of the RBS region and the whole length of the RrmbnA gene.
9. The use according to claim 4, wherein the inducer used in the induction culture of step (2) is IPTG and the recombinant bacterium is e.coli BL21DE3; the bacterial liquid containing the antibacterial peptide is obtained by adding protease inhibitor into recombinant bacterial cells after induction culture and crushing; preferably, the protease inhibitor is PMSF.
10. The use according to claim 4, wherein the purification of step (3) comprises the steps of:
1') crude separation: passing the bacterial liquid through a nickel column affinity chromatographic column, and eluting to obtain a compound;
2') ultrafiltration separation: centrifuging the compound obtained in the step 1') in an ultrafiltration tube, and collecting the filtrate of the ultrafiltration tube;
3') buffer replacement: purifying the filtrate obtained in the step 2') by macroporous adsorption resin, washing with ultrapure water to remove buffer solution, eluting target components by using 50% -70% acetonitrile or ethanol, and freeze-drying;
4') molecular sieve repurification: resuspension the lyophilized powder obtained in step 3'), separating by Superdex 75/300 GL gel chromatographic column, collecting eluting peak, and lyophilizing to obtain purified antibacterial peptide.
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| CN116675740A (en) * | 2023-07-31 | 2023-09-01 | 中国医学科学院医学生物学研究所 | Antibacterial peptide TC-LAR-18 and application thereof in preparation of antibacterial infection medicines |
| CN118324849A (en) * | 2022-12-22 | 2024-07-12 | 四川大学华西医院 | Antibacterial peptide and preparation method and application thereof |
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| WO2005078116A1 (en) * | 2004-01-16 | 2005-08-25 | Qiuyun Liu | A method of isolating antibacterial peptides and the isolated peptides thereof |
| CN114057835A (en) * | 2021-11-24 | 2022-02-18 | 辽宁师范大学 | Antibacterial peptide analogue and preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2005078116A1 (en) * | 2004-01-16 | 2005-08-25 | Qiuyun Liu | A method of isolating antibacterial peptides and the isolated peptides thereof |
| CN114057835A (en) * | 2021-11-24 | 2022-02-18 | 辽宁师范大学 | Antibacterial peptide analogue and preparation method and application thereof |
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| CN118324849A (en) * | 2022-12-22 | 2024-07-12 | 四川大学华西医院 | Antibacterial peptide and preparation method and application thereof |
| CN116675740A (en) * | 2023-07-31 | 2023-09-01 | 中国医学科学院医学生物学研究所 | Antibacterial peptide TC-LAR-18 and application thereof in preparation of antibacterial infection medicines |
| CN116675740B (en) * | 2023-07-31 | 2023-09-29 | 中国医学科学院医学生物学研究所 | Antibacterial peptide TC-LAR-18 and application thereof in preparation of antibacterial infection medicines |
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