Targeted antibacterial peptide HI-WK aiming at escherichia coli K88 as well as preparation method and application of targeted antibacterial peptide HI-WK

Abstract

The invention provides a targeting antibacterial peptide HI-WK targeting escherichia coli K88 as well as a preparation method and application of the targeting antibacterial peptide HI-WK, and belongs to the technical field of biology, and the sequence of the targeting antibacterial peptide HI-WK is shown as SEQ ID No.1. A peptide fragment HI with escherichia coli affinity is obtained through screening of a phage display heptapeptide library, an active peptide WK is added on the basis of an affinity sequence, the sequence of the active peptide WK is shown as SEQ ID No.3, and the active peptide WK is used for improving penetrability to escherichia coli cell membranes, so that the antibacterial peptide HI-WK is obtained. Biological safety detection shows that the minimum inhibitory concentration of the antibacterial peptide HI-WK on escherichia coli K88 is increased by 2 times compared with that of the active peptide WK, and the geometric average of the minimum inhibitory concentration of the antibacterial peptide HI-WK on other escherichia coli is reduced by 1.78 times. In addition, the antibacterial peptide HI-WK does not show hemolytic activity when the concentration is 128 [mu] M, and the antibacterial peptide HI-WK has high application value.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a targeted antimicrobial peptide HI-WK for Escherichia coli K88, a preparation method and an application thereof. Background Art

[0002] Enterotoxigenic E. coli is the most common pathogenic E. coli that causes diarrhea in humans and young animals, and can be fatal in severe cases. It can be transmitted through food, water, feces, and other channels, increasing the risk of E. coli outbreaks in young animals, poultry, and humans, not only causing serious economic losses to the breeding industry, but also posing a serious threat to human health. In addition, E. coli is also one of the most common drug-resistant strains isolated clinically.

[0003] In the past, adding antibiotics to livestock and poultry feed was the main measure to prevent and treat diseases such as diarrhea caused by E. coli infection. However, in the context of prohibiting the abuse of antibiotics, seeking effective alternatives to prevent and treat E. coli disease in livestock and poultry has become a key link and urgent task to ensure the sustainable development of the breeding industry. Antimicrobial 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. Due to their unique cell membrane action mechanism, they are not easy to develop drug resistance. Antimicrobial peptides not only have antibacterial activity, but also have killing effects on viruses, parasites and tumors. Therefore, antimicrobial peptides have become a research hotspot in the biological field, especially in the field of animal husbandry and veterinary medicine, with broad market application prospects.

[0004] However, most of the existing antimicrobial peptides are still broad-spectrum antibacterial, killing pathogenic bacteria while also indiscriminately killing other beneficial bacteria, disrupting the balance of the flora, 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 livestock and poultry, while having no effect or little effect on normal bacteria. Summary of the invention

[0005] Based on the above shortcomings, the purpose of the present invention is to provide a targeted antimicrobial peptide HI-WK for Escherichia coli K88, which is targeted to Escherichia coli K88.

[0006] The object of the present invention is achieved in this way: the sequence of a targeted antimicrobial peptide HI-WK for Escherichia coli K88 is shown as SEQ ID No.1.

[0007] Another object of the present invention is to provide a method for preparing the targeted antimicrobial peptide HI-WK of Escherichia coli K88 as described above, as follows: by displaying a seven-peptide library (Ph.D. TM.-7Phage display peptide library) screened the short peptide HI with E. coli affinity, and its sequence is shown in SEQ ID No.2. The short peptide has the function of binding to E. 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 connected to obtain the polypeptide HI-WK, and its sequence is shown in SEQ ID No.1; the preparation of the polypeptide is completed by solid phase chemical synthesis, and then after reversed phase high performance liquid chromatography purification and mass spectrometry identification, the antibacterial activity, hemolytic activity and eukaryotic cell toxicity are determined, and finally named antimicrobial peptide HI-WK.

[0008] Another object of the present invention is to provide the use of the above-mentioned targeted antimicrobial peptide HI-WK against Escherichia coli K88 in the preparation of drugs for treating diseases infected by Escherichia coli K88.

[0009] Furthermore, the application is the application as a feed additive in animal husbandry production to treat Escherichia coli K88 infectious diseases.

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

[0011] The beneficial effects and advantages of the present invention are as follows: the method of the present invention obtains a short peptide HI with affinity for Escherichia coli by screening a phage display peptide library, and its sequence is: HIMPIQA, which has the function of binding to Escherichia coli but does not have bactericidal activity; on the basis of the affinity sequence, an active peptide WK is added to the sequence in combination with the structure-activity relationship study, and its sequence is: WKWPKPKPKPKWKW to improve the permeability to the cell membrane of Escherichia coli. The obtained antimicrobial peptides were tested for antibacterial and hemolytic activity, and it was found that the antibacterial activity of the antimicrobial peptide HI-WK against Escherichia coli K88 was improved compared with the active peptide WK, while the activity against other bacteria was reduced. The minimum inhibitory concentration of the antimicrobial 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 concentration for 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 antimicrobial peptide HI-WK showed no hemolytic activity at a concentration of 128 μM. It also had low hemolytic activity and eukaryotic cytotoxicity. The antimicrobial peptide HI-WK caused 2.3% hemolysis of erythrocytes at a concentration of 128 μM, but failed to cause 10% hemolysis of erythrocytes. The survival rate of porcine intestinal epithelial cells IPEC-J2 reached 90.28% at a concentration of 128 μM. In summary, the antimicrobial peptide HI-WK is a typical targeted antimicrobial peptide with strong targeting to Escherichia coli K88 and high application value. It has the potential to become a substitute for feed antibiotics. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 is the HPLC chromatogram of the active peptide WK in the examples;

[0013] Figure 2 is a high performance liquid chromatogram of the antimicrobial peptide HI-WK in the examples;

[0014] Figure 3 is the mass spectrum of the active peptide WK in the examples;

[0015] Figure 4 is the mass spectrum of the antimicrobial peptide HI-WK in the embodiment;

[0016] Figure 5 It is a graph showing the hemolytic activity of the active peptide WK, antimicrobial peptide HI-WK and melittin ME in the examples.

[0017] Figure 6 The cytotoxicity graphs of the active peptide WK, antimicrobial peptide HI-WK and melittin ME in the examples are shown. DETAILED DESCRIPTION

[0018] 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.

[0019] Example 1

[0020] Screening of targeting peptides

[0021] E. coli K88 in the logarithmic phase was collected, and the outer membrane protein of E. coli was extracted according to the bacterial outer membrane extraction kit. The peptides that specifically bind to bacterial outer membrane protein were screened using the Ph.D.-7TM Phage display peptide library. According to the instructions, 10 μL of 2.0×10 11 The phage display heptapeptide library of PFU was added to the purified bacteria and incubated at room temperature for 10 min. Unbound phages were extensively washed with Tris buffer containing 0.1% Tween 20 (TBST), and phages bound to bacteria were eluted with glycine-HCl solution. The phage titer was determined by the doubling dilution method, and phage DNA was extracted according to the M13 phage single-stranded DNA extraction kit (BioLab, China). The extracted DNA was sequenced for amplicon sequencing, and the targeted peptide HI was obtained by alignment screening, and its amino acid sequence was: HIMPIQA.

[0022] Example 2

[0023] Design of antimicrobial peptides

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

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

[0026]

[0027] Example 3

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

[0029] 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;

[0030] 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;

[0031] 3. Use 0.2 mol / L sodium sulfate (phosphoric acid adjusted to pH 7.5) to balance the column for 30 minutes, 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;

[0032] 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%.

[0033] Example 4

[0034] Determination of biological activity of antimicrobial peptides

[0035] 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. 100uL of the above solution was placed in a 96-well cell culture plate, and then an equal volume of the test bacterial solution (~10 5pc / 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.

[0036] Table 2 Antibacterial activity of antimicrobial peptides

[0037]

[0038]

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

[0040] Table 3 Minimum hemolytic concentration MHC (μM), mean minimum inhibitory concentration GM (μM), therapeutic index and targeting 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, 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 .

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

[0045] (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.

[0046] (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 4 50 μL of cell suspension.

[0047] (3) Antimicrobial peptide treatment: Add 10 μL of antimicrobial peptide to the first well of a 96-well plate and dilute it in multiples. Add 50 μL of the diluted cell suspension to wells 1-10 of the 96-well plate. Add 50 μL of complete culture medium to well 11 and 100 μL of complete culture medium to well 12. Incubate at constant temperature for 4 h.

[0048] (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 6 ,It can be seen from the figure that the antimicrobial peptide HI-WK did not show toxicity to porcine small intestinal epithelial cells IPEC-J2 within the detection range, and showed significant differences compared with the control group bee venom ME.

Claims

1. A targeted antimicrobial peptide HI-WK against Escherichia coli K88, characterized in that: Its sequence is shown as 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 application as a feed additive in animal husbandry production to treat 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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