Targeted antibacterial peptide SWP against Escherichia coli, its preparation method and application

Abstract

The present invention discloses a targeted antibacterial peptide SWP against Escherichia coli, its preparation method and application, belonging to the field of biotechnology, and its amino acid sequence is shown as SEQ ID No.1. The peptide segment SGNLTKY with Escherichia coli affinity obtained by screening a phage display peptide library in the present invention is used as the targeting region of the targeted antibacterial peptide, and the active peptide WKKIWKPGIKKWIK is used as the functional region. The two are connected to construct a novel antibacterial peptide SWP targeting Escherichia coli, which shows good targeting effect and has high antibacterial activity against Escherichia coli with less influence on other bacteria. In addition, the antibacterial peptide SWP has low hemolytic activity and cytotoxicity.

Description

Technical Field

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

[0002] Escherichia coli is a common intestinal pathogenic bacterium that causes a large number of animal and human diarrheas every year, resulting in significant economic losses. Currently, most known antibacterial peptides have broad-spectrum antibacterial properties, but often cause damage to beneficial microbiota. This broad antibacterial effect may disrupt the intestinal microbial balance, thereby triggering a series of negative consequences, including disease exacerbation and extended treatment cycles. Therefore, it is particularly important to develop highly selective "intelligent" targeted antibacterial peptides. Such antibacterial peptides can effectively kill pathogenic bacteria while minimizing the impact on the microbiota environment, thereby helping to restore the microecological balance and improve the health level of the body. Therefore, there is an urgent need for an Escherichia coli-targeted antibacterial peptide that can effectively combat the infection of this pathogenic bacterium and reduce its threat to host health. Summary of the Invention

[0003] Based on the above deficiencies, the purpose of the present invention is to provide an Escherichia coli-targeted antibacterial peptide SWP, which has the ability to target and selectively kill enterotoxigenic Escherichia coli.

[0004] The purpose of the present invention is achieved as follows: An Escherichia coli-targeted antibacterial peptide SWP, whose sequence is shown in SEQ ID No.1.

[0005] Another purpose of the present invention is to provide a preparation method of an Escherichia coli-targeted antibacterial peptide SWP 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. Finally, the short peptide is linked with the α-helical peptide WP to obtain a polypeptide SWP, and its sequence is shown in SEQ ID No.1; The preparation of the polypeptide is completed by using solid-phase chemical synthesis, followed by purification by reverse-phase high-performance liquid chromatography and mass spectrometry identification, and then through the determination of antibacterial activity, hemolytic activity, and eukaryotic cell cytotoxicity, and finally named antibacterial peptide SWP.

[0006] Another purpose of the present invention is to provide the application of the above-mentioned antibacterial peptide SWP in the preparation of drugs for treating Escherichia coli infectious diseases.

[0007] Furthermore, the above application is the application in drugs for treating Escherichia coli infectious diseases as a feed additive in livestock production.

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

[0009] The beneficial effects and advantages of the present invention are as follows: The method of the present invention screens a short peptide with Escherichia coli affinity through a phage display peptide library, and the sequence is as follows: SGNLTKY. This sequence has the function of binding to Escherichia coli, but does not have bactericidal activity. On the basis of this affinity sequence, combined with the research on the relationship between structure and function, an α-helical peptide WKKIWKPGIKKWIK is added to increase the positive charge, thereby enhancing its mutual attraction with the negatively charged bacterial cell membrane, and enhancing its penetration of the bacterial cell membrane through hydrophobic amino acids. Antibacterial and biosafety tests were carried out on the obtained antibacterial peptide SWP. The results showed that the antibacterial peptide SWP had extremely strong antibacterial activity against Escherichia coli. In addition, the antibacterial 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 antibacterial peptide SWP at a concentration of 128 μM was only 3.5%, which did not reach the hemolysis threshold of 10%. At the same concentration, the survival rate of porcine intestinal epithelial cells IPEC-J2 reached 95.6%. To sum up, the antibacterial peptide SWP is a typical targeted antibacterial peptide, with strong targeting against Escherichia coli and high application value, showing the potential to become a substitute for feed antibiotics. Description of the Drawings

[0010] Figure 1 High performance liquid chromatography of antibacterial peptide WP in the examples

[0011] Figure 2 High performance liquid chromatography of antibacterial peptide SWP in the examples.

[0012] Figure 3 Mass spectrum of antibacterial peptide WP in the examples

[0013] Figure 4 Mass spectrum of antibacterial peptide SWP in the examples

[0014] Figure 5 Hemolytic activity diagram of antibacterial peptides WP, SWP and Melittin in the examples

[0015] Figure 6 Cytotoxicity diagram of antibacterial peptides WP, SWP and Melittin in the examples Detailed Description of the Invention

[0016] The present invention will be further described in detail below with reference to the accompanying drawings of the specification. Unless otherwise specified, the experimental methods used in the following examples are all conventional methods, and the materials, reagents, etc. used can be obtained from commercial sources unless otherwise specified.

[0017] Example 1

[0018] Design of Antibacterial Peptide

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

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

[0021]

[0022] Example 2

[0023] Synthesis of Antibacterial Peptides WP and SWP by Solid-Phase Chemical Synthesis Method

[0024] 1. The preparation of the antibacterial peptide is carried out one by one from the C-terminus to the N-terminus and completed by a peptide synthesizer. First, Fmoc-X (X is the first amino acid at the C-terminus of each antibacterial peptide) is attached to Wang resin, and then the Fmoc group is removed to obtain X-Wang resin; then Fmoc-Y-Trt-OH (9-fluorenylmethoxycarbonyl-trimethyl-Y, Y is the second amino acid at the C-terminus of each antibacterial peptide): The synthesis is carried out from the C-terminus to the N-terminus in this program in turn until the synthesis is completed to obtain the resin with side-chain protection after removing the Fmoc group;

[0025] 2. In the peptide resin obtained above, a cleavage reagent is added, and the reaction is carried out at 20 °C in the dark for 2 h, and then filtered; the precipitate is washed with TFA (trifluoroacetic acid), the washing solution is mixed with the above filtrate, concentrated by a rotary evaporator, and then about 10 times the volume of precooled anhydrous ether is added, and precipitated at -20 °C for 3 h to precipitate a white powdery substance, centrifuged at 2500 g for 10 min, the precipitate is collected, and the precipitate is washed with anhydrous ether and dried in vacuo to obtain the polypeptide, wherein the cleavage reagent is composed of TFA, water and TIS (triisopropylchlorosilane) mixed in a mass ratio of 95:2.5:2.5;

[0026] 3. Equilibrate the column with 0.2 mol / L sodium sulfate (adjusted to pH 7.5 with phosphoric acid) for 30 min. Dissolve the polypeptide in 90% aqueous acetonitrile solution, filter it, and use a C18 reversed-phase normal-pressure column. Perform gradient elution (the eluent is a mixture of methanol and sodium sulfate aqueous solution in a volume ratio of 30:70 to 70:30), with a flow rate of 1 mL / min, a detection wavelength of 220 nm, collect the main peak, and lyophilize it; then further purify it using a reversed-phase C18 column. Eluent A is 0.1% TFA / aqueous solution, and eluent B is 0.1% TFA / acetonitrile solution. The elution concentration is 25% B to 40% B, the elution time is 12 min, the flow rate is 1 mL / min, collect the main peak as above, and lyophilize it;

[0027] 4. Identification of the antimicrobial peptide: Analyze the obtained antimicrobial peptide by electrospray mass spectrometry. The molecular weight shown in the mass spectrum (as Figures 1-4 shown) is basically consistent with the theoretical molecular weight in Table 1, and the purity of the antimicrobial peptide is greater than 95%.

[0028] Example 3

[0029] Determination of the biological activity of the antimicrobial peptide

[0030] 1. Determination of antibacterial activity: Use the microbroth dilution method to determine the minimum inhibitory concentration of several antimicrobial peptides. Use 0.01% acetic acid (containing 0.2% BSA) as the diluent, and use the serial dilution method to prepare a series of gradient antimicrobial peptide solutions in turn. Take 100 μL of the above solution and place it in a 96-well cell culture plate, and then add an equal volume of the test bacterial solution (~10 5 CFU / mL) to each well. Set positive control (containing bacterial solution but no antimicrobial peptide) and negative control (neither containing bacterial solution nor peptide) respectively. Incubate at 37°C for 14 - 18 h, and measure the absorbance at 492 nm (OD 492nm ) with an enzyme-linked immunosorbent assay reader to determine the minimum inhibitory concentration. The test results are shown in Table 2.

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

[0032]

[0033] 2. Determination of hemolytic activity: 1 mL of fresh human blood was collected, anticoagulated with heparin and dissolved in 2 mL of PBS solution. After centrifugation at 3000 rpm for 10 min, the red blood cells were collected; washed 3 times with PBS and then resuspended in 10 mL of PBS; 50 μL of the red blood cell suspension was mixed evenly with 50 μL of antibacterial peptide solutions with different concentrations dissolved in PBS, and incubated at a constant temperature in an incubator at 37 °C for 1 h; after incubation, it was taken out and centrifuged at 4 °C and 3000 rpm for 10 min; the supernatant was taken out and the light absorption value was measured at 570 nm with an enzyme-linked immunosorbent assay (ELISA) reader. Among them, 50 μL of red blood cells plus 50 μL of PBS was used as a negative control; 50 μL of red blood cells plus 50 μL of 0.1% Tritonx-100 was used as a positive control. The minimum hemolytic concentration is the concentration of the antibacterial peptide when it causes a 10% hemolysis rate. The test results are shown in Figure 5 . Through Figure 5 It can be seen that the antibacterial peptide SWP caused only 3.5% hemolysis of red blood cells at a concentration of 128 μM and did not cause 10% hemolysis of red blood cells. There was a significant difference compared with the control group melittin.

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

[0035]

[0036] a Pathogenic bacteria: Pathogenic bacteria other than Escherichia coli. b Therapeutic index: Calculated as MHC / GM (E. coli). A larger value indicates greater cell selectivity. c The targeting index represents the ability of the antibacterial peptide to specifically target. The targeting index is calculated as GM (E. coli) / GM (other pathogenic strains) and GM (E. coli) / GM (probiotics). A smaller value indicates stronger specificity. It can be seen from Table 3 that compared with WP, the antibacterial peptide SWP has significantly improved antibacterial activity against Escherichia coli.

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

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

[0039] (2) Treatment of the cells to be tested: The cells were washed and resuspended 3 times with sterile PBS, and digested with 0.25% trypsin solution to make them detach from the bottom of the flask, and rinsed with the complete culture medium to obtain a single cell suspension. At the same time, the final concentration of about 2×10 450 μL of cell suspension.

[0040] (3) Antimicrobial peptide treatment: Add 10 μL of antimicrobial peptide to the first well of a 96-well plate and perform serial dilutions. Add 50 μL of the diluted cell suspension to wells 1-10 of the 96-well plate, add 50 μL of complete medium to well 11, and add 100 μL of complete medium to well 12. Incubate at a constant temperature for 4 h.

[0041] (4) Toxicity detection: Add 50 μL of 5 mg / mL MTT solution to the 96-well plate. After continued incubation for 3-4 h, add 150 μL of DMSO. Measure the absorbance at OD 570nm . The test results are shown in Figure 4 . It can be seen through Figure 6 that the antimicrobial peptide SWP did not show toxicity to porcine intestinal epithelial cells IPEC-J2 within the detection range, showing a significant difference from the control group melittin.

Claims

1. A targeted antimicrobial peptide SWP for Escherichia coli, characterized in that: Its sequence is shown in 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, 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 targeted antimicrobial peptide SWP against Escherichia coli 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 the application as a feed additive in animal husbandry production to prepare medicines for treating 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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