Targeted antibacterial peptide SWP aiming at escherichia coli as well as preparation method and application thereof

Abstract

The invention discloses a targeted antibacterial peptide SWP aiming at escherichia coli and a preparation method and application thereof, and belongs to the technical field of biology, and the amino acid sequence of the targeted antibacterial peptide SWP is shown as SEQ ID No.1. A peptide fragment SGNLTKY which is obtained by screening through a phage display peptide library and has escherichia coli affinity is used as a targeting region of the targeted antibacterial peptide, an active peptide WKKIWKPGIKKWIK is used as a functional region, the peptide fragment and the active peptide are connected to construct the novel antibacterial peptide SWP with the targeted escherichia coli, the novel antibacterial peptide SWP shows a good targeting effect, and under the condition that the influence on other bacteria is small, the antibacterial peptide SGNLTKY and the active peptide WKKIPGIKKWIK can be used for preparing the antibacterial peptide SWP with the targeted escherichia coli affinity. The compound has efficient antibacterial activity on escherichia coli. In addition, the antibacterial peptide SWP has relatively low hemolytic activity and cytotoxicity.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to an Escherichia coli targeted antimicrobial peptide SWP and a preparation method and application thereof. Background Art

[0002] Escherichia coli is a common intestinal pathogen that causes a large number of diarrheal diseases in animals and humans every year and causes significant economic losses. Currently, most known antimicrobial peptides have broad-spectrum antimicrobial properties, but often lead to damage to beneficial microbiota. This broad antimicrobial effect may disrupt the balance of intestinal microorganisms, leading to a series of negative consequences, including aggravation of the disease and prolonged treatment cycles. Therefore, it is particularly important to develop highly selective "intelligent" targeted antimicrobial peptides. Such antimicrobial peptides can effectively kill pathogenic bacteria while minimizing the impact on the microbial environment, thereby helping to restore the balance of the microecology and improve the health of the body. Therefore, there is an urgent need for a targeted antimicrobial peptide against Escherichia coli that can effectively combat the infection of this pathogen and reduce its threat to host health. Summary of the invention

[0003] Based on the above shortcomings, the purpose of the present invention is to provide a targeted antimicrobial peptide SWP for Escherichia coli, which has the ability to selectively kill enterotoxigenic Escherichia coli.

[0004] The object of the present invention is achieved in this way: a targeted antimicrobial peptide SWP against Escherichia coli, the sequence of which is shown in SEQ ID No.1.

[0005] Another object of the present invention is to provide a method for preparing an Escherichia coli targeted antimicrobial peptide SWP, which is as follows: by extracting Escherichia coli proteins and co-incubating the Escherichia coli proteins with a phage display peptide library, a targeted short peptide with Escherichia coli affinity is screened out, and its sequence is shown in SEQ ID No.2; and an α-helical peptide WP against Gram-negative bacteria is designed, and its sequence is shown in SEQ ID No.3, and finally the short peptide is connected with the α-helical peptide WP to obtain a polypeptide SWP, and its sequence is shown in SEQ ID No.1; the polypeptide is prepared by solid phase chemical synthesis, and after reversed-phase high performance liquid chromatography purification and mass spectrometry identification, the polypeptide is finally named as the antimicrobial peptide SWP after the antibacterial activity, hemolytic activity and eukaryotic cytotoxicity are determined.

[0006] Another object of the present invention is to provide the use of the antimicrobial peptide SWP as described above in the preparation of a drug for treating Escherichia coli infectious diseases.

[0007] Furthermore, the application is application as a feed additive in animal husbandry production to treat Escherichia coli infectious diseases.

[0008] Another object of the present invention is to provide a drug suitable for treating Escherichia coli infection, wherein the drug contains the antimicrobial peptide SWP as described above.

[0009] The beneficial effects and advantages of the present invention are as follows: The method of the present invention obtains a short peptide with affinity for Escherichia coli by screening a phage display peptide library, and the sequence is as follows: SGNLTKY, which has the function of binding to Escherichia coli, but does not have bactericidal activity. On the basis of this affinity sequence, combined with the study of the relationship between structure and function, an α-helical peptide WKKIWKPGIKKWIK is added to increase the positive charge, thereby improving its mutual attraction with the negatively charged bacterial cell membrane, and enhancing its permeability to the bacterial cell membrane through hydrophobic amino acids. The obtained antimicrobial peptide SWP was tested for antibacterial and biosafety, and the results showed that the antimicrobial peptide SWP had extremely strong antibacterial activity against Escherichia coli. In addition, the antimicrobial peptide SWP showed extremely weak or no antibacterial activity against Staphylococcus aureus, Staphylococcus epidermidis, Lactobacillus rhamnosus and Lactococcus lactis. It is worth noting that the hemolysis rate of red blood cells caused by the antimicrobial peptide SWP at a concentration of 128 μM is only 3.5%, which does not reach the hemolysis threshold of 10%. At the same concentration, the survival rate of porcine intestinal epithelial cells IPEC-J2 reached 95.6%. In summary, the antimicrobial peptide SWP is a typical targeted antimicrobial peptide with strong targeting against Escherichia coli and high application value, showing the potential to become a substitute for feed antibiotics. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 The high performance liquid chromatogram of the antimicrobial peptide WP in the embodiment

[0011] Figure 2 It is the high performance liquid chromatogram of the antimicrobial peptide SWP in the example.

[0012] Figure 3 It is the mass spectrum of the antimicrobial peptide WP in the embodiment.

[0013] Figure 4 The mass spectrum of the antimicrobial peptide SWP in the examples.

[0014] Figure 5 It is a graph showing the hemolytic activity of the antimicrobial peptides WP, SWP and melittin in the examples.

[0015] Figure 6 The cytotoxicity graphs of the antimicrobial peptides WP, SWP and melittin in the examples are shown. DETAILED DESCRIPTION

[0016] The present invention is further described in detail below with reference to the accompanying drawings. The experimental methods used in the following examples are conventional methods unless otherwise specified, and the materials, reagents, etc. used are all commercially available unless otherwise specified.

[0017] Example 1

[0018] Design of antimicrobial peptides

[0019] In this embodiment, a bacterial protein extraction kit is used to extract Escherichia coli protein, and the extracted protein is incubated with a phage display peptide library. After three rounds of panning, a targeted short peptide S with affinity for Escherichia coli is screened out, and its amino acid sequence is: SGNLTKY; and an α-helical peptide WP against Gram-negative bacteria is designed, and its amino acid sequence is: WKKIWKPGIKKWIK; the targeted short peptide and the α-helical peptide are connected to design a new peptide SWP with strong targeted antibacterial activity against Escherichia coli, and the above antimicrobial peptide is synthesized by solid phase synthesis using a peptide synthesizer. The amino acid sequence of the antimicrobial peptide SWP is: SGNLTKYWKKIWKPGIKKWIK. The sequence of the antimicrobial peptide is shown in Table 1.

[0020] Table 1 Amino acid sequences of antimicrobial peptides.

[0021]

[0022] Example 2

[0023] Synthesis of antimicrobial peptides WP and SWP by solid phase chemical synthesis

[0024] 1. The preparation of antimicrobial peptides is carried out one by one from the C-terminus to the N-terminus, and is completed by a peptide synthesizer. First, Fmoc-X (X is the first amino acid at the C-terminus of each antimicrobial peptide) is connected to the Wang resin, and then the Fmoc group is removed to obtain the X-Wang resin: then Fmoc-Y-Trt-OH (9-fluorenylmethoxycarboxyl-trimethyl-Y, Y is the second amino acid at the C-terminus of each antimicrobial peptide): according to this procedure, it is synthesized from the C-terminus to the N-terminus in sequence until the synthesis is completed, and the side chain protection resin with the Fmoc group removed is obtained;

[0025] 2. Add a cleavage agent to the peptide resin obtained above, react at 20°C in the dark for 2 hours, filter; wash with precipitated TFA (trifluoroacetic acid), mix the washing liquid with the above filtrate, concentrate on a rotary evaporator, add about 10 times the volume of pre-cooled anhydrous ether, precipitate at -20°C for 3 hours, precipitate a white powder, centrifuge at 2500g for 10 minutes, collect the precipitate, wash the precipitate with anhydrous ether, and vacuum dry to obtain a polypeptide, wherein the cleavage agent is a mixture of TFA, water and TIS (triisopropylsilyl chloride) in a mass ratio of 95:2.5:2.5;

[0026] 3. Use 0.2 mol / L sodium sulfate (adjusted to pH 7.5 with phosphoric acid) to balance the column for 30 min, dissolve the polypeptide with 90% acetonitrile aqueous solution, filter, C18 reverse phase atmospheric pressure column, use gradient elution (eluent is methanol and sodium sulfate aqueous solution mixed in a volume ratio of 30:70 to 70:30), flow rate is 1 mL / min, detection wave is 220 nm, collect the main peak, and freeze-dry; further purify using reverse phase C18 column, eluent A is 0.1% TFA / water solution: eluent B is 0.1% TFA / acetonitrile solution, elution concentration is 25% B to 40% B, elution time is 12 min, flow rate is 1 mL / min, collect the main peak as above, and freeze-dry;

[0027] 4. Identification of antimicrobial peptides: The antimicrobial peptides obtained above were analyzed by electrospray ionization mass spectrometry. The molecular weights (e.g. Figure 1-4 The molecular weight of the antimicrobial peptide was basically consistent with the theoretical molecular weight in Table 1, and the purity of the antimicrobial peptide was greater than 95%.

[0028] Example 3

[0029] Determination of biological activity of antimicrobial peptides

[0030] 1. Determination of antibacterial activity: The minimum inhibitory concentration of several antimicrobial peptides was determined by the microbroth dilution method. 0.01% acetic acid (containing 0.2% BSA) was used as the diluent, and a series of gradient antimicrobial peptide solutions were prepared in sequence using the doubling dilution method. 100 μL of the above solution was placed in a 96-well cell culture plate, and then an equal volume of the test bacterial solution (~10 5 pc / mL) in each well. Set up positive control (containing bacterial solution but not antimicrobial peptide) and negative control (containing neither bacterial solution nor peptide) respectively. Incubate at 37℃ for 14-18h and read the sample at 492nm (OD 492nm ) to determine the minimum inhibitory concentration. The test results are shown in Table 2.

[0031] Table 2 Antibacterial activity of antimicrobial peptides WP and SWP

[0032]

[0033] 2. Determination of hemolytic activity: Collect 1 mL of fresh human blood, dissolve it in 2 mL of PBS solution after anticoagulation with heparin, centrifuge at 3000 rpm for 10 min, collect red blood cells; wash with PBS 3 times, and resuspend with 10 mL PBS; take 50 μL of red blood cell suspension and 50 μL of antimicrobial peptide solution of different concentrations dissolved in PBS, mix them evenly, and incubate them in a 37°C incubator for 1 hour; take out after the incubation, centrifuge at 4°C and 3000 rpm for 10 min; take out the supernatant and measure the absorbance at 570 nm with an enzyme marker. 50 μL of red blood cells plus 50 μL of PBS is used as a negative control; 50 μL of red blood cells plus 50 μL of 0.1% Tritonx-100 is used as a positive control. The minimum hemolytic concentration is the antimicrobial peptide concentration when the antimicrobial peptide causes a hemolysis rate of 10%. The test results are shown in Figure 5 .pass Figure 5 It can be seen that the antimicrobial peptide SWP at a concentration of 128 μM only caused 3.5% hemolysis of erythrocytes and failed to cause 10% hemolysis of erythrocytes, which was significantly different from the control group melittin.

[0034] Table 3 Minimum hemolytic concentration MHC (μM), mean minimum inhibitory concentration GM (μM), therapeutic index and targeting index of antimicrobial peptides.

[0035]

[0036] a Pathogenic bacteria: pathogens other than E. coli. b Therapeutic index: calculated as MHC / GM (E. coli). Larger values ​​indicate greater cell selectivity. c Targeting index represents the ability of antimicrobial peptides to specifically target. Targeting index is calculated as GM (E. coli) / GM (other pathogenic strains) and GM (E. coli) / GM (probiotics). Smaller values ​​indicate stronger specificity. It can be seen from Table 3 that the antimicrobial peptide SWP has significantly improved targeted antibacterial activity against E. coli compared with WP.

[0037] 3. Determination of eukaryotic cytotoxicity: The MTT method was used to detect the cytotoxicity of porcine small intestinal epithelial cells IPEC-J2.

[0038] (1) Preparation of culture medium and cell culture: DMEM (culture medium) and fetal bovine serum were mixed at a ratio of 9:1 to prepare complete culture medium, and porcine intestinal epithelial cells IPEC-J2 were resuscitated in liquid nitrogen until the cells covered 80%-90% of the bottom of the flask.

[0039] (2) Treatment of cells to be tested: Wash and resuspend the cells three times with sterile PBS, digest the cells with 0.25% trypsin solution to make them fall off the bottom of the bottle, rinse with complete culture medium to obtain a single cell suspension, and fill a 96-well plate with a final concentration of about 2×10 450 μL of cell suspension.

[0040] (3) Antimicrobial peptide treatment: 10 μL of antimicrobial peptide was added to the first well of a 96-well plate and diluted in multiples. 50 μL of the diluted cell suspension was added to wells 1-10 of the 96-well plate. 50 μL of complete culture medium was added to well 11 and 100 μL of complete culture medium was added to well 12. The cells were cultured at a constant temperature for 4 h.

[0041] (4) Toxicity detection: 50 μL of 5 mg / mL MTT solution was added to a 96-well plate. After culturing for 3-4 h, 150 μL of DMSO was added and the OD value was measured by a microplate reader. 570nm The absorbance was measured at Figure 4 .pass Figure 6 It can be seen that the antimicrobial peptide SWP did not show toxicity to porcine small intestinal epithelial cells IPEC-J2 within the detection range, and showed significant differences from the control group melittin.

Claims

1. A targeted antimicrobial peptide SWP for Escherichia coli, characterized in that: Its sequence is shown as SEQ ID No.

1.

2. The method for preparing a targeted antimicrobial peptide SWP against Escherichia coli according to claim 1, characterized in that: The method is as follows: by extracting Escherichia coli protein and co-incubating the Escherichia coli protein with a phage display peptide library, a targeted short peptide with Escherichia coli affinity is screened out, and its sequence is shown in SEQ ID No. 2; and an α-helical peptide WP against Gram-negative bacteria is designed, and its sequence is shown in SEQ ID No. 3; finally, the short peptide is connected with the α-helical peptide WP to obtain a polypeptide SWP, and its sequence is shown in SEQ ID No. 1; The polypeptide was prepared by solid phase chemical synthesis, and after reverse phase high performance liquid chromatography purification and mass spectrometry identification, and then after the determination of antibacterial activity, hemolytic activity and eukaryotic cytotoxicity, it was finally named antimicrobial peptide SWP.

3. Use of the Escherichia coli targeted antimicrobial peptide SWP according to claim 1 in the preparation of a drug for treating Escherichia coli infectious diseases.

4. The use according to claim 3, characterized in that: The application is application as a feed additive in animal husbandry production to treat Escherichia coli infectious diseases.

5. A drug suitable for treating Escherichia coli infection, comprising the antimicrobial peptide SWP according to claim 1.

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