CN117487029B - A bifunctional antibacterial peptide and its synthesis method and application - Google Patents

Abstract

The invention belongs to the technical field of biology, and particularly relates to a difunctional antibacterial peptide, a synthesis method and application thereof. The bifunctional antibacterial peptide consists of 34 amino acids, and the amino acid sequence of the bifunctional antibacterial peptide is shown as SEQ ID NO in a sequence table: 1. The invention provides a novel bifunctional antibacterial peptide, which has the characteristics of small molecular weight, simple synthesis, low hemolytic activity and good stability; experimental study shows that the novel bifunctional antibacterial peptide designed by the invention can damage the membrane integrity of bacteria, can increase the concentration of antibacterial peptide in bacterial cells, inhibit protein translation, exert double inhibition effects, reduce the generation of bacterial drug resistance and reduce toxic and side effects. In addition, the bifunctional antibacterial peptide can be applied to the fields of medicine, cosmetics, food preservation and cultivation industry.

Description

A bifunctional antibacterial peptide and its synthesis method and application

Technical field

The invention belongs to the field of biotechnology, and specifically relates to a bifunctional antibacterial peptide and its synthesis method and application.

Background technique

Gram-negative bacteria often cause intra-abdominal infections, urinary tract infections, bacteremia, and induce systemic inflammatory reactions. The emergence of drug-resistant bacteria increases the difficulty of treating infectious diseases and seriously increases the economic burden on patients.

Antibacterial peptide (AMP), as part of the host's natural defense system, is a small molecule peptide consisting of 10-100 amino acid residues and has certain antibacterial activity. Due to its natural antibacterial function and low probability of drug resistance, it is considered an effective alternative to antibiotics. According to its antibacterial mechanism, it is mainly divided into two categories: targeting bacterial membranes and intracellular biological macromolecules, but the two categories have single targets. Natural antimicrobial peptides have shortcomings such as high toxic side effects and poor permeability. Bacterial membranes and ribosomes are important targets for clinical antibiotics, and studies have shown that two antibiotics or antimicrobial peptides have synergistic effects in vitro and in vivo. Peptide drug molecules have many advantages such as high safety and target affinity, easy synthesis and transformation, and low immunogenicity. However, many antimicrobial peptides currently have low antibacterial activity and low efficiency, and are highly toxic to cells or have a series of other side effects. Therefore, a safe and effective drug that can specifically act on Gram-negative bacteria can not only destroy the membrane integrity of bacteria, but also increase the concentration of intracellular antimicrobial peptides in bacteria, inhibit protein translation, exert a dual inhibitory effect, and reduce bacterial infection. The development of drug resistance is a technical difficulty and demand for antibacterial research.

Contents of the invention

The purpose of the present invention is to solve the deficiencies of the existing technology and provide a bifunctional antibacterial peptide and its preparation method and application. Specifically, the following technical solutions are adopted:

In the first aspect of the present invention, a bifunctional antibacterial peptide is provided. The antibacterial peptide consists of 34 amino acids, and its amino acid sequence is:

Val Asp Lys Pro Pro Tyr Leu Pro Arg Pro Arg Pro Gly Gly Gly Gly SerPhe Trp Lys Lys Ile Lys Asn Ser Val Lys Lys Arg Ala Lys Lys Phe Phe, all amino acids are in the L-form.

The abbreviated amino acid sequence of the above-mentioned bifunctional antibacterial peptide is shown in SEQ ID No. 1: VDKPPYLPRPRPGGGGSFWKKIKNSVKKRAKKFF.

The molecular weight of the above-mentioned bifunctional antimicrobial peptide is 3915.62, and the isoelectric point is 11.7.

Another object of the present invention is to provide a method for preparing bifunctional antimicrobial peptides, as follows: by combining the snake Cathelicidin family FP-CATH (whose amino acid sequence is shown in SEQ ID No. 2: KRFKKFWKKIKNSVKKRAKKFFRKPRVIAVSIPF) and the proline antimicrobial peptide family Oncocin (whose amino acid sequence is shown in SEQ ID No. 3: VDKPPYLPRPRPPRRIYNR) was analyzed comparatively. By truncating the polypeptide and testing the antibacterial activity of the truncated peptide and the affinity of lipopolysaccharide and ribosomal protein, two types of peptide pharmacophores that can bind lipopolysaccharide and ribosomal protein were initially screened. By combining the two types of pharmacophores The peptide segments were arranged and combined in different ways, and the antibacterial activity was tested and screened. The N-terminal (Oncocin12) was designed using the VDKPPYLPRPRP amino acid sequence (tested to have no antibacterial activity), and the C-terminal (FP-CATH17) was designed using the FWKKIKNSVKKRAKKFF amino acid sequence, using GGGGS. Connect them with the amino acid linker sequence, and the final sequence is shown as SEQ ID No. 1, named FPON. The designed polypeptide is synthesized through artificial solid phase and desalted and purified by HPLC-C18 reversed phase column chromatography. After measuring its molecular weight by LC-MS/MS method and identifying its purity by high-performance liquid chromatography (HPLC), the preparation of the antibacterial peptide is completed.

This invention innovatively introduces peptide pharmacophore that inhibits bacterial protein translation based on the structure of membrane antimicrobial peptide FP-CATH, and designs and synthesizes a bifunctional peptide FPON. In vitro experiments show that FPON destroys the integrity of bacterial membranes and inhibits bacterial protein translation, indicating that It has a bifunctional antibacterial effect, has low hemolytic activity and exhibits good antibacterial activity against Gram-negative bacteria. The novel bifunctional antimicrobial peptide designed by the present invention can not only destroy the membrane integrity of bacteria, but also increase the concentration of intracellular antimicrobial peptides in bacteria, inhibit protein translation, exert a dual inhibitory effect, reduce the occurrence of bacterial resistance, and reduce toxicity. side effect.

In a second aspect of the present invention, a method for preparing the above-mentioned bifunctional antibacterial peptide is also provided, comprising the following steps:

First, we conduct a comparative analysis of the snake Cathelicidin (cathelicidin) family and the proline antimicrobial peptide family; then we truncate the peptides, detect the antibacterial activity of the truncated peptides, and the affinity of lipopolysaccharide and ribosomal proteins, and initially screen out the ones that can bind The amino acid sequences of the two types of peptide pharmacophores of lipopolysaccharide and ribosomal protein were finally combined with the complete sequences using an automatic peptide synthesizer and desalted and purified by reverse-phase column chromatography using high-performance liquid chromatography.

In addition to the above steps, it also includes: using high-performance liquid chromatography HPLC method to identify its purity, using LC-MS/MS to identify the sequence, isoelectric focusing electrophoresis to determine the isoelectric point, circular dichroism spectroscopy to determine the secondary structure, and SWISS software to predict amino acids. The tertiary structure of the sequence.

In a third aspect of the present invention, a pharmaceutical composition for inhibiting bacterial growth is also provided, comprising the above-mentioned bifunctional antibacterial peptide and a pharmaceutically acceptable carrier; wherein the above-mentioned bacteria include Escherichia coli, At least one of Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa.

In the fourth aspect of the present invention, the use of a bifunctional antibacterial peptide in preparing a preservative is also provided.

In the fifth aspect of the present invention, the use of a bifunctional antimicrobial peptide in preparing animal feed additives is also provided.

In the sixth aspect of the present invention, the use of a bifunctional antibacterial peptide in the preparation of cosmetic additives is also provided.

In the seventh aspect of the present invention, the use of a bifunctional antibacterial peptide in preparing an antibacterial agent is also provided. In addition, the bifunctional antimicrobial peptide provided by the present invention can be used in antibiotic adjuvants, and the bifunctional antimicrobial peptide can be used in combination with any antibiotic.

The beneficial effects of the present invention are:

The bifunctional antibacterial peptide FPON proposed by the present invention can be obtained through chemical synthesis. Experimental results show that the peptide is effective against clinically multi-drug-resistant Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa. Monocystis and other Gram-negative bacteria have relatively strong antibacterial activity. In addition, the antimicrobial peptide has the characteristics of small molecular weight, simple synthesis, low hemolytic activity and good stability, and can be used in medicine, cosmetics, food preservation and aquaculture.

Description of the drawings

Figure 1 shows the mass spectrometry identification chart of bifunctional antimicrobial peptides;

Figure 2 shows a transmission electron microscope image of Gram-negative bacterial membrane damaged by bifunctional antimicrobial peptides;

Figure 3 shows a diagram of bifunctional antimicrobial peptides inhibiting bacterial protein translation in vitro;

Figure 4 shows a diagram of the hemolytic activity of bifunctional antimicrobial peptides on red blood cells;

Figure 5 shows the toxicity of bifunctional antimicrobial peptides to Raw264.7 cells.

Detailed ways

In order to facilitate understanding of the present application, the present application will be described more fully below with reference to the relevant drawings. Several embodiments of the present application are shown in the drawings. However, the present application may be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the technical field to which this application belongs. The terminology used herein in the description of the application is for the purpose of describing specific embodiments only and is not intended to limit the application. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

In this article, unless otherwise stated, the term "%" refers to "mass %"; the term "μg/mL" refers to: micrograms per milliliter. As used herein, unless otherwise stated, the term "%" refers to the total weight of the composition of the present application.

As used herein, all ranges defined are meant to include each specific range within a given range and every combination of subranges between the given ranges. For example, the range of 1 to 5 specifically includes 1, 2, 3, 4 and 5, and also includes sub-ranges such as 2 to 5, 3 to 5, 2 to 3, 2 to 4, 1 to 4, etc.

Example 1

FP-CATH was truncated to FP-CATH9 (its amino acid sequence is shown in SEQ ID No. 4: KRFKKFWKK), FP-CATH16 (its amino acid sequence is shown in SEQ ID No. 5: KKFFRKPRVIAVSIPF) and FP-CATH17 (its amino acid sequence is shown in SEQ ID No. 5: KKFFRKPRVIAVSIPF). The amino acid sequence is shown in SEQ ID No. 6: FWKKIKNSVKKRAKKFF); truncated Oncocin is Oncocin7 (the amino acid sequence is shown in SEQ ID No. 7: VDKPPYL), Oncocin10 (the amino acid sequence is shown in SEQ ID No. 8: PRPPRRIYNR ) and Oncocin12 (whose amino acid sequence is shown in SEQ ID No. 9: VDKPPYLPRPRP); detect the antibacterial activity of the truncated peptide, the affinity of lipopolysaccharide and ribosomal protein, and initially screen out the compounds that can bind lipopolysaccharide (FP-CATH17) and Two types of peptide pharmacophores of ribosomal protein (Oncocin12), but the antibacterial activity results show that Oncocin12 does not have antibacterial activity (Table 1). By arranging and combining the N-terminal to C-terminal of the two types of pharmacophore peptides in different ways, the antibacterial activity, lipopolysaccharide and ribosomal protein affinity were again tested. The screening found that FPON has the best antibacterial effect. Further testing was carried out to determine that FPON is a bifunctional Antimicrobial peptide consists of 34 amino acids, and its amino acid sequence is as follows:

Val Asp Lys Pro Pro Pro Tyr Leu Pro Arg Pro Arg Pro Gly Gly Gly Gly SerPhe Trp Lys Lys Ile Lys Asn Ser Val Lys Lys Arg Ala Lys Lys Phe Phe, the amino acid abbreviation is VDKPPYLPRPRPGGGGSFWKKIKNSVKKRAKKFF, all amino acids are L-form.

Example 2

A method for synthesizing bifunctional antibacterial peptides, which specifically includes the following steps:

Step 1: First, synthesize the FPON polypeptide through artificial solid-phase synthesis, and then desalt and purify it through HPLC-C18 reversed-phase column chromatography;

Step 2: Determine its molecular weight using LC-MS/MS method. The results are shown in Figure 1;

Step 3: Use high-performance liquid chromatography (HPLC) to identify the purified FPON to have a purity >95%, a molecular weight of 3915.62, and an isoelectric point of 11.7 measured by isoelectric focusing electrophoresis.

Example 3

This example is a bifunctional antibacterial peptide FPON antibacterial activity detection experiment. The minimum inhibitory concentration (MIC) was determined using the double dilution method. E.coli BW25113, K. pneumoniae _KPC-2 MDR, and K. pneumoniae _NDM-1 MDR were selected. P. aeruginosa ATCC 27853, A. baumannii ATCC 19606, S. aureus and S. aureus MRSA strains were tested and the MIC determination experiment was carried out.

Pick the monoclonal test strain (above), inoculate it into fresh MH liquid culture medium (purchased from Shanghai Guandao Bioengineering Co., Ltd.), culture it with constant temperature shaking at 37°C for about 6 hours (logarithmic growth phase), and use fresh MH liquid The culture medium was diluted to 1.5×10 ⁸ cfu/mL for later use; 10 μL MH liquid culture medium was first added to each well of the sterile 96-well plate, and then 10 μL of the peptide sample (FPON, FP-CATH17, and Oncocin 12) solution was pipetted ( Accurately weigh 8 mg of the polypeptide, dissolve it in 1 mL of physiological saline, filter it through a 0.22 μm pore filter), and dilute it one by one in order. The last hole is the negative control hole; dilute the above bacterial liquid 200 times, take 90 μL of the bacterial liquid and add it to each wells, and then place the 96-well plate in a 37°C constant-temperature incubator for static culture for 18 hours, and measure the light absorption at OD ₆₀₀ ; the minimum inhibitory concentration is the lowest sample concentration at which bacterial growth cannot be seen, and the results are shown in Table 1;

In this experiment, the experimental steps for MIC determination of K. pneumoniae _KPC-2 MDR, K. pneumoniae _NDM-1 MDR, P. aeruginosa ATCC27853, A. baumannii ATCC 19606, S. aureus and S. aureus MRSA were the same as the above steps.

Table 1 Different peptides have antibacterial activity against Gram-negative bacteria in various bacteria

As can be seen from the above table, Oncocin 12 does not have antibacterial activity; the effective MIC of FPON is 4 to 8 times lower than FP-CATH17, and the effective MIC of FPON is 64 to 128 times lower than Oncocin 12; the antibacterial activity of FPON is better than FP-CATH17 and Oncocin 12, and is specific against Gram-negative bacteria.

Example 4

This example is an experiment to detect bacterial membrane damage caused by bifunctional antibacterial peptide FPON:

The test bacteria E.coli BW25113 (-80℃) were inoculated into sterilized Luria-Bertani (LB) solid medium plates using the three-zone marking method, and cultured upside down in a 37℃ constant temperature incubator for 12 hours; then Pick single colonies from the inoculation loop and transfer them to sterilized liquid LB culture medium, and culture them with shaking at 37°C and 150 rpm until the logarithmic growth phase. The OD ₆₀₀ value is between 0.6-0.8; then wash with physiological saline. 3 times, resuspend at OD ₆₀₀ to 0.8, and incubate the mixture of bacteria and peptide (2× MIC final concentration) at 37°C for 60 min; use 3% (w/v) glutaraldehyde and 0.075% (w/v) ruthenium. Red was fixed in 0.1 M PBS for 1 hour in the dark, then fixed with 0.075% (w/v) ruthenium red and 1% (w/v) osmium tetroxide for 2 hours in the dark; samples were dehydrated through graded ethanol and transferred to pure Spurr In the resin, after the resin solidified and hardened, it was sectioned with an ultrathin microtome, double stained with uranyl acetate-lead citrate, and observed under a transmission electron microscope. As shown in Figure 2, the cell surface and cytoplasmic contents were released, and the cell membrane was obviously irregular. The bacterial outer membrane has gaps; bacterial cells treated with the peptide showed more aggregation and clumping than control cells.

Example 5

This experiment is an in vitro protein translation inhibition detection experiment:

The inhibitory effect of peptides on cell-free GFP synthesis was determined by a transcription-translation coupling method based on Escherichia coli lysates. According to the instructions of myTXTL Sigma70 Master Mix Kit, which is based on E.coli lysis solution, mix Ready-to-Use master mix (4 μL) and deGFP plasmid (1 μL), add reaction buffer (4 μL), and add FPON/polymyxa B (1 μL, concentration 100 μg/mL), so that the total trace reaction system is 10 μL, and the final concentration of peptide/drug is 10 μg/mL. Examine the effects on in vitro transcription-translation in the presence or absence of peptides. Quantification was performed by detecting GFP protein expression within 30 min (λ _excitation = 460 nm, λ _emission = 525 nm), and the fluorescence intensity was calculated from the fluorescence signal at the 30 min time point. All measurements were performed in triplicate; the results are shown in Figure 3, Polymyxa Phin B (PB) has no in vitro translation inhibitory effect, and FPON can inhibit the protein translation of Escherichia coli GFP in vitro.

Example 6

This example is a cytotoxicity detection experiment of bifunctional antibacterial peptide FPON:

(1) Determination of hemolytic activity

Take the standard human blood cell reagent (Changchun Boxun Biotechnology Co., Ltd.), wash it three times with physiological saline, and prepare a red blood cell suspension of 10 ⁷ cells/mL. Mix the red blood cell suspension with different concentrations of bifunctional antimicrobial peptides dissolved in physiological saline, incubate at 37°C for 1 hour, and then centrifuge at 1000 rpm, 4°C for 10 minutes; draw 100 μL of the supernatant and measure at OD ₅₄₀ Absorbance values. The use of physiological saline as a negative control is represented by A _{negative control} , 1% TritonX-100 is used as a positive control and is represented by A _{positive control} , and the addition of polypeptide is represented by A _sample ; this example also tested the hemolytic activity of polymyxin B. The experimental process is the same as that of the bifunctional antimicrobial peptide. The results are shown in Figure 4. From Figure 4, it can be seen that the hemolytic activity of the bifunctional antimicrobial peptide of the present invention is lower than that of polymyxin B.

The percentage of hemolysis rate is calculated according to the following formula: Hemolysis rate %=A _sample ^- A _{negative control} /A _{positive control} ×100%.

(2) Determination of cytotoxicity

The CCK8 method was used to determine the cytotoxicity of bifunctional peptides on mouse Raw264.7 cells. Dissolve the bifunctional peptide in serum-free RPMI 1640 medium and dilute it into different concentration gradients (256 μg/mL, 128 μg/mL, 64 μg/mL, 32 μg/mL, 16 μg/mL, 8 μg/mL, 4 μg/mL, 2 μg /mL, 1μg/mL), and then added to a 96-well plate containing mouse Raw264.7 cells (2×10 ⁴ cells/well). Three duplicate wells were set up for each concentration of peptide, and the cells without peptide were added to the 96-well plate. Serum RPMI 1640 culture medium was used as a blank control; after 24 hours of constant temperature incubation at 37°C, 10 μL of CCK8 reagent was added to each well; after 4 hours of incubation, the absorbance at OD ₄₅₀ was measured; the results of three independent experiments were averaged. And the cell viability was calculated as %=[A administration-blank]/[A0 administration-blank]×100%. Among them, blank represents the absorbance of the wells containing culture medium and CCK8 solution without cells; A administration represents the absorbance of the wells of CCK8 solution containing peptide, culture medium and cells; A0 administration represents no peptide, containing culture medium and cells. The absorbance of the wells of the CCK8 solution of cells; the results are shown in Figure 5.

Figure 4 shows a diagram of the hemolytic activity of bifunctional antimicrobial peptides on red blood cells; it can be seen from Figure 4 that the bifunctional antimicrobial peptides of the present invention have low hemolytic activity, and when the concentration is 256 μg/mL, the hemolytic activity is 7.2±1.2 About %, its concentration far exceeds its effective bactericidal concentration, indicating that the bifunctional antibacterial peptide has low hemolytic toxicity.

Figure 5 shows the toxicity chart of the bifunctional antimicrobial peptide to Raw264.7 cells; it can be seen from Figure 5 that the bifunctional antimicrobial peptide has low cytotoxicity, and when the concentration is 256 μg/mL, the cell survival rate is 92.3± About 3.5%.

In summary, the bifunctional antimicrobial peptide FPON proposed by the present invention can be obtained through chemical synthesis. This bifunctional antimicrobial peptide is clinically effective against multi-drug-resistant Escherichia coli, Klebsiella pneumoniae, and Acinetobacter baumannii. Bacillus, Pseudomonas aeruginosa and other Gram-negative bacteria, all have relatively strong antibacterial activity. Secondly, the antimicrobial peptide has the characteristics of small molecular weight, simple synthesis, low hemolytic activity and good stability, and can be used in the fields of medicine, cosmetics, food preservation and aquaculture.

Although the present invention has been described in considerable detail and in particular to several of the described embodiments, it is not intended to be limited to any such details or embodiments or to any particular embodiment, but rather is to be considered by reference The appended claims are intended to provide the broadest possible interpretation of these claims, taking into account the prior art, to effectively cover the intended scope of the invention. In addition, the above description of the present invention is based on embodiments foreseeable by the inventor for the purpose of providing a useful description, and those non-substantive changes to the present invention that are not yet foreseen can still represent equivalent changes of the present invention.

Claims (10)

1. A bifunctional antibacterial peptide, characterized in that the antibacterial peptide consists of 34 amino acids, and its amino acid sequence is: Val Asp Lys Pro Pro Tyr Leu Pro Arg Pro Arg Pro Gly Gly Gly Gly Ser PheTrp Lys Lys Ile Lys Asn Ser Val Lys Lys Arg Ala Lys Lys Phe Phe, all amino acids are in the L-form. 2. The bifunctional antimicrobial peptide according to claim 1, characterized in that the molecular weight of the bifunctional antimicrobial peptide is 3915.62 and the isoelectric point is 11.7. 3. A synthesis method of the bifunctional antimicrobial peptide according to any one of claims 1-2, characterized in that the synthesis method includes: First, a comparative analysis was conducted on the cathelicidin family and the proline antimicrobial peptide family; then the polypeptide was truncated, the antibacterial activity of the truncated peptide segment, and the affinity of lipopolysaccharide and ribosomal protein were detected, and the peptides that could bind lipopolysaccharide and ribosomal protein were initially screened. The amino acid sequences of the two types of peptide pharmacophores of ribosomal proteins were finally combined with their complete sequences using an automatic peptide synthesizer, and then purified by high-performance liquid chromatography, reverse-phase column chromatography, desalting and purification. 4. A pharmaceutical composition for inhibiting bacterial growth, characterized by comprising the bifunctional antimicrobial peptide according to any one of claims 1-2. 5. The pharmaceutical composition according to claim 4, further comprising a pharmaceutically acceptable carrier. 6. The pharmaceutical composition according to claim 4, wherein the bacteria include at least one of Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa. 7. Application of the bifunctional antimicrobial peptide according to any one of claims 1-2 in the preparation of preservatives. 8. Application of the bifunctional antimicrobial peptide according to any one of claims 1-2 in the preparation of animal feed additives. 9. Application of the bifunctional antimicrobial peptide according to any one of claims 1-2 in the preparation of cosmetic additives. 10. Application of the bifunctional antibacterial peptide according to any one of claims 1-2 in the preparation of antibacterial agents.