Antibacterial polypeptide and application thereof

Abstract

The invention relates to the technical field of biology, and particularly discloses an antibacterial polypeptide and application thereof. The amino acid sequence of the polypeptide disclosed by the invention is as shown in SEQ ID NO. 1; the molecular marker is represented by SEQ ID NO. 1: C14-FFFKKKKK-NH2. The molecular marker is represented by SEQ ID NO. The polypeptide provided by the invention has broad-spectrum antibacterial activity, can inhibit the activity of gram-negative bacteria and / or gram-positive bacteria, is low in cytotoxicity, has good biocompatibility, and has clinical application potential.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to an antibacterial polypeptide and application thereof. Background Art

[0002] In recent years, the frequent and irregular use of antibiotics has led to increased mutations and evolution in pathogenic microorganisms, resulting in increasing drug resistance and the emergence of superbugs. Currently, the frequent and irregular use of antibiotics in the livestock industry is rapidly losing their effectiveness, and the problem of drug resistance caused by antibiotic misuse is becoming increasingly serious. In addition to the resistance caused by antibiotics, bacterial metabolites themselves can also confer resistance to certain conventional antimicrobial agents.

[0003] Antimicrobial peptides possess broad-spectrum antimicrobial activity and are less susceptible to developing drug resistance. These small peptides can penetrate bacterial membranes and eliminate infected bacteria, while exhibiting low cytotoxicity. Therefore, designing antimicrobial peptides with bactericidal activity is a strategy for eliminating drug-resistant bacteria. Summary of the Invention

[0004] One of the purposes of the present invention is to provide a novel polypeptide having broad-spectrum antibacterial activity and good biocompatibility.

[0005] The present invention provides a polypeptide (3FK), the amino acid sequence of which is shown in SEQ ID NO.1; SEQ ID NO. 1: C14-FFFFKKKKKK-NH2.

[0006] Through specialized polypeptide chain design, this invention provides a novel polypeptide with broad-spectrum antibacterial properties. The use of a C14 alkyl chain as a hydrophobic group enables the polypeptide to effectively penetrate bacterial membranes and exert its antibacterial efficacy. A specific number of overlapping aromatic amino acid (phenylalanine) segments is employed to balance membrane surface affinity. K6 peptide is incorporated to enhance biocompatibility, increase the polypeptide's positive charge, and balance its hydrophilicity. C-terminal amino acid ligation further enhances the polypeptide's stability. Antibacterial activity testing revealed that the resulting polypeptide, 3FK, exhibits broad-spectrum antibacterial activity, including clearance and inhibition of Escherichia coli and Staphylococcus aureus. Furthermore, the peptide exhibits low cytotoxicity and good biocompatibility, suggesting potential for clinical application.

[0007] The polypeptides of the present invention can be prepared by methods known in the art, such as solid-phase chemical synthesis or inducing host cell expression (introducing a gene encoding the polypeptide into a host cell to achieve expression of the polypeptide).

[0008] The present invention also provides a DNA molecule encoding the above polypeptide.

[0009] Those skilled in the art can design genes with different nucleotide sequences encoding the above polypeptides according to the needs for polypeptide expression, based on the principle of codon degeneracy and the codon usage preferences of different species.

[0010] The present invention also provides a biological material comprising the above DNA molecule, wherein the biological material is an expression cassette, a recombinant vector or a host cell.

[0011] The present invention also provides the use of the above polypeptide or DNA molecule or biological material in the preparation of antibacterial products.

[0012] In the application of the present invention, the antibacterial product can inhibit Gram-negative bacteria and / or Gram-positive bacteria.

[0013] In the application of the present invention, the antibacterial product can inhibit Escherichia coli and / or Staphylococcus aureus.

[0014] The present invention also provides the use of the above polypeptide or DNA molecule or biological material in the preparation of an antiseptic and / or antibacterial composition.

[0015] The present invention also provides an antibacterial agent, which comprises the above polypeptide.

[0016] The antibacterial agent of the present invention may contain the above-mentioned polypeptide as the sole active ingredient, or may contain other active ingredients with similar or different functions.

[0017] The present invention also provides application of the polypeptide in livestock and poultry breeding.

[0018] The present invention also provides an animal feed or feed additive, which comprises the above polypeptide.

[0019] The beneficial effects of the present invention are at least: The polypeptide 3FK provided by the present invention has broad-spectrum antibacterial activity and has antibacterial effects on both Gram-negative bacteria such as Staphylococcus aureus and Gram-positive bacteria, and has very good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the mass spectrum of the polypeptide 3FK in Example 1 of the present invention.

[0021] Figure 2 This is a liquid chromatogram of the polypeptide 3FK in Example 1 of the present invention.

[0022] Figure 3 This is the result of the hemolytic activity test of the polypeptide in Example 3 of the present invention.

[0023] Figure 4This is the result of the polypeptide cytotoxicity test in Example 4 of the present invention. DETAILED DESCRIPTION

[0024] The preferred embodiments of the present invention will be described in detail below with reference to the examples. It should be understood that the following examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0025] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods. Unless otherwise specified, the materials, reagents, etc. used in the following examples are all commercially available or prepared according to conventional methods in the art.

[0026] Example 1 Synthesis of antimicrobial peptide 3FK by solid phase chemical synthesis In this example, the antimicrobial peptide 3FK was synthesized by solid phase chemical synthesis. The amino acid sequence of the antimicrobial peptide is shown in SEQ ID NO. 1. The liquid chromatography and mass spectrometry of the peptide are shown in Figure 1 and Figure 2 As shown, the specific synthesis method is as follows: The preparation of antimicrobial peptides is carried out one by one from the C-terminus to the N-terminus using a peptide synthesizer. First, Fmoc-X (X is the first amino acid at the C-terminus of the antimicrobial peptide) is attached to Wang resin, and then the Fmoc group is removed to obtain X-Wang resin. Next, Fmoc-Y-Trt-OH (9-fluorenylmethoxycarboxyl-trimethyl-Y is removed from the protecting group, and Y is the second amino acid at the C-terminus of the antimicrobial peptide) is added. Following this procedure, the synthesis is continued from the C-terminus to the N-terminus until the synthesis is complete, resulting in a resin with the side chain protection removed from the Fmoc group. A cleavage reagent was added to the peptide resin obtained above, and the mixture was reacted at 20°C in the dark for 2 hours, and filtered; the precipitate was washed with TFA (trifluoroacetic acid), and the washing liquid was mixed with the above filtrate, concentrated on a rotary evaporator, and then about 10 times the volume of pre-cooled anhydrous ether was added. The mixture was precipitated at -20°C for 3 hours to precipitate a white powder. The mixture was centrifuged at 2500g for 10 minutes, the precipitate was collected, and the precipitate was washed with anhydrous ether and vacuum dried to obtain a polypeptide. The cleavage reagent was prepared by mixing TFA, water, and TIS (triisopropylsilyl chloride) in a mass ratio of 95:2.5:2.5.

[0027] The column was equilibrated with 0.2 mol / L sodium sulfate (adjusted to pH 7.5 with phosphoric acid) for 30 min, the polypeptide was dissolved with 90% acetonitrile aqueous solution, filtered, and eluted with a C18 reverse-phase atmospheric pressure column using gradient elution (eluent: methanol and sodium sulfate aqueous solution mixed in a volume ratio of 30:70 to 70:30) at a flow rate of 1 mL / min and a detection wavelength of 220 nm. The main peak was collected and lyophilized. The peptide was further purified using a reverse-phase C18 column with eluent A being 0.1% TFA / water solution and eluent B being 0.1% TFA / acetonitrile solution with an elution concentration of 25% B to 40% B, an elution time of 12 min, and a flow rate of 1 mL / min. The main peak was collected and lyophilized as above.

[0028] Identification of antimicrobial peptides: The antimicrobial peptides obtained above were analyzed by electrospray mass spectrometry. Figure 1 ) is basically consistent with the theoretical molecular weight of 1621.11; the purity of the antimicrobial peptide is greater than 95% ( Figure 2 The liquid chromatography column was Kromasil C18-5 (4.6×250 mm, 220 nm, 10 μL), and a nonlinear gradient of water / acetonitrile (containing 0.1% trifluoroacetic acid) was used at a flow rate of 1.0 mL / min).

[0029] Example 2 Determination of antimicrobial activity of antimicrobial peptide 3FK The minimum inhibitory concentration of the peptide was determined using the microdilution method. A diluent containing 0.01% acetic acid and 0.2% fetal bovine serum albumin was added to a 96-well plate, and a series of gradient peptide solutions were prepared using the doubling dilution method so that the volume of the solution in each well was 50 μL. Then 50 μL of the test bacterial solution (about 10 5 CFU / mL) were placed in each well in MHB culture medium. The cells were incubated at 37°C for 18-24 hours. The optical density at 492 nm was then measured using a microplate reader to determine the minimum inhibitory concentration (MIC) of peptide 3FK against the bacteria. A value less than 0.1 was considered inhibitory. Each test was performed in duplicate and repeated three times. The results are shown in Table 1.

[0030] Table 1 Antibacterial activity of antimicrobial peptide 3FK against bacteria The results in Table 1 show that the antimicrobial peptide 3FK has a broad-spectrum antimicrobial activity against Escherichia coli and Staphylococcus aureus.

[0031] Example 3 Determination of hemolytic activity of antimicrobial peptide 3FK The hemolytic activity of the antimicrobial peptide prepared in Example 1 was determined as follows: Fresh porcine red blood cells (RBCs) were used to measure the hemolytic activity of antimicrobial peptides. Red blood cells (RBCs) were washed with PBS (pH 7.4) until the supernatant was clear. RBCs were resuspended in PBS to prepare a 10% (v / v) suspension, and serially diluted antimicrobial peptides were added to the suspension (1 mL). After incubation at 37°C for 2 hours, RBCs were centrifuged at 6000 rpm for 10 minutes, and the absorbance of the supernatant was measured at 570 nm. A PBS-treated group served as a negative control, and a 0.2% Triton X-100-treated group served as a positive control.

[0032] Hemolysis rate (%) = [(OD570 sample absorbance - OD570 negative control absorbance) / (OD570 positive control absorbance - OD570 negative control absorbance)] × 100%.

[0033] The results of the peptide hemolytic activity test are shown in Figure 3 The results showed that when pig red blood cells were treated with the antimicrobial peptide 3FK at a concentration of 128 μM, the hemolysis rate was about 1.7%, indicating that the red blood cell hemolytic activity of 3FK was relatively small and it has certain clinical application potential.

[0034] Example 4 Determination of Cytotoxicity of Antimicrobial Peptide 3FK The cytotoxicity of the antimicrobial peptide prepared in Example 1 was determined as follows: The toxicity of antimicrobial peptides to eukaryotic cells was determined using the MTT colorimetric assay. Human embryonic kidney cell line 293T cells, frozen in liquid nitrogen, were revived and inoculated into a culture medium containing 10% fetal bovine serum. The cells were subcultured at 37°C and 5% CO2. After the cells grew to 80% confluence in the culture dish, 2 mL of 0.25% trypsin was added to digest the cells. The cell concentration was adjusted with culture medium to a final concentration of approximately 1 × 10 5 cells / well. After the cells are completely attached, 50 μL of cell suspension is mixed with 50 μL of serially diluted antimicrobial peptide in a 96-well plate and incubated at 37°C and 5% CO2 for 6 hours. Then, 25 μL of MTT (5 mg / mL) is added to each well and incubated for another 2 hours. After the incubation, the supernatant is discarded. The crystals at the bottom of the well are dissolved with 150 μL of DMSO, and the absorbance value is measured at 570 nm using an enzyme reader. Positive controls (containing cells but not antimicrobial peptides) and negative controls (containing only culture medium) are set up respectively. The experiment is set up in two parallels and repeated three times. See the results. Figure 4 The cell viability was calculated according to the following formula: Cell viability = [(test well OD570 - negative control OD570) / (positive control OD570 - negative control OD570)] × 100%.

[0035] The results showed that when human embryonic kidney cells 293T were treated with the antimicrobial peptide 3FK at a concentration of 128 μM, the cell survival rate was 93.9%, indicating that 3FK has low cytotoxicity and has certain clinical application potential.

[0036] The above results show that the antimicrobial peptide 3FK has broad-spectrum antimicrobial activity, low cytotoxicity, and has wide application potential.

[0037] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. A polypeptide, characterized in that The amino acid sequence is shown in SEQ ID NO.1; SEQ ID NO. 1: C14-FFFFKKKKKK-NH2.

2. A DNA molecule, characterized in that Encodes the polypeptide according to claim 1.

3. A biomaterial, characterized in that Comprising the DNA molecule according to claim 2, the biological material is an expression cassette, a recombinant vector or a host cell.

4. Use of the polypeptide according to claim 1, the DNA molecule according to claim 2, or the biomaterial according to claim 3 in the preparation of an antibacterial product.

5. The use according to claim 4, characterized in that The antibacterial product can inhibit Gram-negative bacteria and / or Gram-positive bacteria.

6. The use according to claim 4, characterized in that The antibacterial product can inhibit Escherichia coli and / or Staphylococcus aureus.

7. Use of the polypeptide according to claim 1, the DNA molecule according to claim 2, or the biomaterial according to claim 3 in the preparation of an antiseptic and / or antibacterial composition.

8. An antibacterial agent, characterized in that Comprising the polypeptide of claim 1.

9. Use of the polypeptide according to claim 1 in livestock and poultry breeding.

10. An animal feed or feed additive, characterized in that Comprising the polypeptide of claim 1.

Images