Targeted antibacterial peptide HI-WK against Escherichia coli K88, its preparation method and application

Abstract

The present invention provides a targeted antibacterial peptide HI-WK targeting Escherichia coli K88, its preparation method and applications, belonging to the field of biotechnology, and its sequence is as shown in SEQ ID No.1. The peptide segment HI with affinity for Escherichia coli was screened from a phage display heptapeptide library. On the basis of this affinity sequence, an active peptide WK was added, and its sequence is as shown in SEQ ID No.3, to improve the penetration of the Escherichia coli cell membrane, resulting in the antibacterial peptide HI-WK. Biological safety detection found that the minimum inhibitory concentration of the antibacterial peptide HI-WK against Escherichia coli K88 was increased by 2 times compared with the active peptide WK, while the geometric mean of the minimum inhibitory concentrations against other Escherichia coli decreased by 1.78 times. In addition, the antibacterial peptide HI-WK did not show hemolytic activity at a concentration of 128 μM and has high application value.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a targeted antibacterial peptide HI-WK against Escherichia coli K88, a preparation method thereof, and applications thereof. Background Art

[0002] Enterotoxigenic Escherichia coli is the most common pathogenic Escherichia coli causing diarrhea in humans and young livestock, and can be lethal in severe cases. It can be transmitted through various routes such as food, water sources, feces, etc., increasing the risk of Escherichia coli outbreaks in young livestock, young poultry, and humans. It not only causes serious economic losses to the breeding industry, but also seriously threatens human health. In addition, Escherichia coli is also one of the most common drug-resistant strains isolated clinically.

[0003] Previously, adding antibiotics to livestock and poultry feed was the main measure to prevent and control diseases such as diarrhea caused by Escherichia coli infection. However, in the context of prohibiting the abuse of antibiotics, seeking effective alternative solutions to prevent and control Escherichia coli diseases in livestock and poultry has become a key link and an urgent task for ensuring the sustainable development of the breeding industry. Antibacterial peptides are a class of natural small-molecule polypeptides with a wide range of sources and high safety. They are widely present in animals, plants, and microorganisms and are not easily resistant due to their unique cell membrane action mechanism. Antibacterial peptides not only have antibacterial activity, but also have killing effects on viruses, parasites, tumors, etc. Therefore, antibacterial peptides have become a research hotspot in the biological field, especially having broad market application prospects in the field of animal husbandry and veterinary medicine.

[0004] However, most of the existing antibacterial peptides are still mainly broad-spectrum antibacterial, killing pathogenic bacteria while also killing other beneficial bacteria indiscriminately, breaking the flora balance, reducing intestinal homeostasis, and thus reducing the resistance of livestock and poultry. Therefore, it is of great significance to design a targeted peptide that can selectively kill Escherichia coli in the body of livestock and poultry while having no effect or little effect on normal bacteria. Summary of the Invention

[0005] Based on the above deficiencies, the purpose of the present invention is to provide a targeted antibacterial peptide HI-WK against Escherichia coli K88, which has targeting to Escherichia coli K88.

[0006] The purpose of the present invention is achieved as follows: The sequence of a targeted antibacterial peptide HI-WK against Escherichia coli K88 is as shown in SEQ ID No.1.

[0007] Another purpose of the present invention is to provide a preparation method of a targeted antibacterial peptide HI-WK against Escherichia coli K88 as described above, as follows: Through a phage display heptapeptide library (Ph.D. TM.-7Phage display peptide library) The short peptide HI with affinity for Escherichia coli obtained by screening has the sequence shown in SEQ ID No. 2. This short peptide has the function of binding to Escherichia coli but does not have bactericidal activity; on the basis of the short peptide HI, an active peptide WK is added, and its sequence is shown in SEQ ID No. 3. Finally, the short peptide HI and the active peptide WK are linked to obtain the polypeptide HI-WK, and its sequence is shown in SEQ ID No. 1; using the solid-phase chemical synthesis method, after reverse-phase high-performance liquid chromatography purification and mass spectrometry identification, the preparation of the polypeptide is completed, and then the antibacterial activity, hemolytic activity and eukaryotic cell toxicity are measured, and finally it is named antibacterial peptide HI-WK.

[0008] Another object of the present invention is to provide the application of a targeted antibacterial peptide HI-WK against Escherichia coli K88 as described above in the preparation of a drug for treating infectious diseases caused by Escherichia coli K88.

[0009] Furthermore, the above application is the application in a drug for treating infectious diseases caused by Escherichia coli K88 as a feed additive in livestock production.

[0010] Another object of the present invention is to provide a drug suitable for treating Escherichia coli K88 infection as described above, and the drug contains the antibacterial peptide HI-WK as described above.

[0011] The beneficial effects and advantages of the present invention are as follows: The short peptide HI with Escherichia coli affinity obtained by screening a phage display peptide library in the method of the present invention has the sequence: HIMPIQA, and 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 study of structure-activity relationship, an active peptide WK with the sequence: WKWPKPKPKPKWKW is added on the basis of this sequence to improve the penetration of the Escherichia coli cell membrane. The antibacterial and hemolytic activities of the obtained antibacterial peptide were detected, and it was found that compared with the active peptide WK, the antibacterial peptide HI-WK had improved antibacterial activity against Escherichia coli K88, while the activity against other bacteria decreased. The minimum inhibitory concentration of the antibacterial peptide HI-WK against Escherichia coli K88 was increased by 2 times compared with the active peptide WK, while the geometric mean of the minimum inhibitory concentrations against other Escherichia coli decreased by 1.78 times. Moreover, the targeting peptide has very weak or no antibacterial activity against Staphylococcus aureus, Salmonella typhimurium, Staphylococcus epidermidis, Enterococcus faecalis, etc. In addition, the antibacterial peptide HI-WK did not show hemolytic activity at a concentration of 128 μM. And it has low hemolytic activity and eukaryotic cell toxicity. The antibacterial peptide HI-WK caused 2.3% erythrocyte hemolysis at a concentration of 128 μM and did not cause 10% erythrocyte hemolysis. The survival rate of porcine intestinal epithelial cells IPEC-J2 reached 90.28% at a concentration of 128 μM. To sum up, the antibacterial peptide HI-WK is a typical targeted antibacterial peptide, has strong targeting to Escherichia coli K88, has high application value, and already has the development potential to become a substitute for feed antibiotics. Description of the Drawings

[0012] Figure 1 It is the high performance liquid chromatography diagram of the active peptide WK in the embodiment;

[0013] Figure 2 It is the high performance liquid chromatography diagram of the antibacterial peptide HI-WK in the embodiment;

[0014] Figure 3 It is the mass spectrometry diagram of the active peptide WK in the embodiment;

[0015] Figure 4 It is the mass spectrometry diagram of the antibacterial peptide HI-WK in the embodiment;

[0016] Figure 5 It is the hemolytic activity diagram of the active peptide WK, antibacterial peptide HI-WK and melittin ME in the embodiment.

[0017] Figure 6 It is the cytotoxicity diagram of the active peptide WK, antibacterial peptide HI-WK and melittin ME in the embodiment. Detailed Embodiments

[0018] 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.

[0019] Example 1

[0020] Screening of targeting peptide segments

[0021] Escherichia coli K88 in the logarithmic phase was collected, and the outer membrane proteins of Escherichia coli were extracted according to the bacterial outer membrane extraction kit. A phage display heptapeptide library (Ph.D.-7TM Phage display peptide library) was used to screen out the polypeptides that specifically bind to the bacterial outer membrane proteins. According to the instructions, 10 μL of the phage display heptapeptide library containing 2.0×10 11 PFU was added to the purified bacteria and incubated at room temperature for 10 min. Unbound phages were extensively washed away with Tris buffer (TBST) containing 0.1% Tween 20, and the phages bound to the bacteria were eluted with glycine-HCl solution. The phage titer was determined by serial dilution, and the phage DNA was extracted according to the operating steps of the M13 phage single-stranded DNA extraction kit (BioLabo, China). The extracted DNA was subjected to amplicon sequencing, and the targeting peptide segment HI with the amino acid sequence: HIMPIQA was obtained by comparison and screening.

[0022] Example 2

[0023] Design of antibacterial peptides

[0024] In this example, we designed an active antibacterial peptide WK with the amino acid sequence: WKWPKPKPKPKWKW; HI and the active peptide WK were connected to design a new polypeptide HI-WK with strong targeting antibacterial activity against Escherichia coli. Using a peptide synthesizer, the above polypeptide was synthesized by solid-phase synthesis method, and the amino acid sequence was: HIMPIQAWKWPKPKPKPKWKW, and the sequence is shown in Table 1.

[0025] Table 1 Amino acid sequences of active peptide WK and antibacterial peptide HI-WK.

[0026]

[0027] Example 3

[0028] Synthesis of active peptide WK and antibacterial peptide HI-WK by solid-phase chemical synthesis method

[0029] 1. The preparation of the antimicrobial 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 antimicrobial peptide) is connected 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 antimicrobial peptide): According to this procedure, it is synthesized from the C-terminus to the N-terminus in turn until the synthesis is completed, and the resin with side-chain protection after removing the Fmoc group is obtained;

[0030] 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 powder, 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;

[0031] 3. Column equilibration is carried out for 30 min with 0.2 mol / L sodium sulfate (adjusted to pH 7.5 with phosphoric acid), the polypeptide is dissolved in a 90% acetonitrile aqueous solution, filtered, and a C18 reversed-phase normal pressure column is used. Gradient elution is adopted (the eluent is a mixture of methanol and sodium sulfate aqueous solution in a volume ratio of 30:70 to 70:30), the flow rate is 1 mL / min, the detection wavelength is 220 nm, the main peak is collected and freeze-dried; further purification is carried out using a reversed-phase C18 column, the eluent A is 0.1% TFA / aqueous solution: the 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, and the main peak is collected and freeze-dried as above;

[0032] 4. Identification of the antimicrobial peptide: The antimicrobial peptide obtained above is analyzed by electrospray mass spectrometry. The molecular weight shown in the mass spectrum (as Figure 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%.

[0033] Example 4

[0034] Determination of the biological activity of the antimicrobial peptide

[0035] 1. Determination of antibacterial activity: The minimum inhibitory concentration of several antimicrobial peptides is determined by the microbroth dilution method. Using 0.01% acetic acid (containing 0.2% BSA) as the diluent, a series of gradient antimicrobial peptide solutions are prepared in turn using the serial dilution method. 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(in CFU / mL) into each well. Positive control (containing bacterial solution but no antimicrobial peptide) and negative control (containing neither bacterial solution nor peptide) were set respectively. Incubate at a constant temperature of 37 °C for 14 - 18 h, and measure the absorbance at 492 nm (OD 492nm ) with an enzyme-linked immunosorbent assay (ELISA) reader to determine the minimum inhibitory concentration. The test results are shown in Table 2.

[0036] Table 2 Antibacterial activity of antimicrobial peptides

[0037]

[0038]

[0039] As can be seen from Table 3, compared with the active peptide WK, the target antibacterial activity of the antimicrobial peptide HI-WK against Escherichia coli K88 was significantly improved, while the activity against other Gram-negative bacteria such as other Escherichia coli decreased.

[0040] Table 3 Minimum hemolytic concentration MHC (μM), average minimum inhibitory concentration GM (μM), therapeutic index, and target index of antimicrobial peptides.

[0041]

[0042] a Escherichia coli: Escherichia coli other than Escherichia coli K88; b Gram-negative bacteria: Gram-negative bacteria other than Escherichia coli

[0043] 2. Determination of hemolytic activity: Collect 1 mL of fresh human blood, anticoagulate with heparin and dissolve it in 2 mL of PBS solution, centrifuge at 3000 rpm for 10 min, and collect red blood cells; wash 3 times with PBS and then resuspend with 10 mL of PBS; take 50 μL of the red blood cell suspension and mix it evenly with 50 μL of antimicrobial peptide solutions with different concentrations dissolved in PBS, and incubate at a constant temperature in an incubator at 37 °C for 1 h; after incubation, take it out and centrifuge at 4 °C and 3000 rpm for 10 min; take out the supernatant and measure the absorbance at 570 nm with an enzyme-linked immunosorbent assay (ELISA) reader. Among them, 50 μL of red blood cells plus 50 μL of PBS is used as the negative control; 50 μL of red blood cells plus 50 μL of 0.1% Tritonx-100 is used as the positive control. The minimum hemolytic concentration is the antimicrobial peptide concentration when the antimicrobial peptide causes a 10% hemolysis rate. The test results are shown in Figure 5 .

[0044] 3. Determination of eukaryotic cell toxicity: The MTT method was used to detect the cytotoxicity of porcine intestinal epithelial cells IPEC-J2.

[0045] (1) Preparation of culture medium and cell culture: Mix DMEM (culture medium) and fetal bovine serum at a ratio of 9:1 to prepare a complete culture medium, and resuscitate porcine intestinal epithelial cells IPEC-J2 in liquid nitrogen. It is appropriate when the cells cover 80% - 90% of the bottom of the flask.

[0046] (2) Treatment of cells to be tested: Wash and resuspend the cells 3 times with sterile PBS, and digest the cells with 0.25% trypsin solution to make them detach from the bottom of the flask. Rinse with complete medium to obtain a single-cell suspension. At the same time, add 50 μL of cell suspension with a final concentration of approximately 2×10 4 to the 96-well plate.

[0047] (3) Treatment with antimicrobial peptide: Add 10 μL of antimicrobial peptide to the first well of the 96-well plate and perform serial dilution. 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;

[0048] (4) Toxicity detection: Add 50 μL of 5 mg / mL MTT solution to the 96-well plate. After continuing to culture for 3-4 h, add 150 μL of DMSO. Measure the absorbance at OD 570nm . The test results are shown in Figure 6 . It can be seen from this figure that the antimicrobial peptide HI-WK did not show toxicity to porcine intestinal epithelial cells IPEC-J2 within the test range, showing a significant difference from the control group melittin ME.

Claims

1. A targeted antimicrobial peptide HI-WK against Escherichia coli K88, characterized in that: Its sequence is shown in SEQ ID No.

1.

2. The method for preparing a targeted antimicrobial peptide HI-WK against Escherichia coli K88 according to claim 1, characterized in that: The method is as follows: a short peptide HI with Escherichia coli affinity is obtained by screening a phage display seven-peptide library, the sequence of which is shown in SEQ ID No. 2, and an active peptide WK is connected to the C-terminus of the short peptide HI, the sequence of which is shown in SEQ ID No. 3, to obtain a polypeptide HI-WK, the sequence of which is shown in SEQ ID No. 1; The polypeptide was prepared by solid phase chemical synthesis, followed by reverse phase high performance liquid chromatography purification and mass spectrometry identification, and then tested for antimicrobial activity, hemolytic activity and eukaryotic cell toxicity, and was finally named antimicrobial peptide HI-WK.

3. Use of the targeted antimicrobial peptide HI-WK against Escherichia coli K88 according to claim 1 in the preparation of a drug for treating Escherichia coli K88 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 K88 infectious diseases.

5. A drug suitable for treating Escherichia coli K88 infection, comprising the antimicrobial peptide HI-WK as claimed in claim 1.

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