Artificially synthesized antibacterial peptide, derivative thereof and application of artificially synthesized antibacterial peptide in escherichia coli resistance

Abstract

The invention belongs to the technical field of biological medicines, and discloses an artificially synthesized antibacterial peptide, a derivative thereof and an application of the artificially synthesized antibacterial peptide in resisting escherichia coli. The antibacterial peptide provided by the invention has a strong inhibition effect on escherichia coli and comprises multiple drug-resistant strains. Experiments show that the antibacterial peptide has high in-vitro antibacterial activity, excellent thermal stability, acid-base stability and no hemolytic activity, has no obvious toxicity to mammalian cells, and shows a remarkable treatment effect in a chicken escherichia coli infection model. The invention provides a novel antibacterial preparation for coping with escherichia coli, especially infection of drug-resistant strains.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a synthetically produced antimicrobial peptide, its derivatives, and their application in combating Escherichia coli. Background Technology

[0002] Bacterial infections, especially those caused by Gram-negative bacteria, pose a significant challenge to clinical and public health fields. Escherichia coli, one of the most common Gram-negative bacteria, can cause a variety of infectious diseases. With the widespread use of antibiotics, multidrug-resistant strains of E. coli are constantly emerging, significantly reducing the effectiveness of existing antibiotics and making the development of novel antimicrobial drugs particularly urgent.

[0003] Antimicrobial peptides (AMPs), as an important component of the organism's innate immune system, possess advantages such as broad-spectrum antibacterial activity and low likelihood of inducing drug resistance, and are considered potential antibiotic alternatives. However, natural antimicrobial peptides often suffer from limitations such as poor stability, high cytotoxicity, and limited mechanisms of action. Therefore, developing novel antimicrobial peptides with high efficiency, stability, and safety is of great significance. Summary of the Invention

[0004] The purpose of this invention is to provide a synthetically produced antimicrobial peptide, the amino acid sequence of which is shown in SEQ ID NO.11.

[0005] Another object of the present invention is to provide a synthetically produced derivative of an antimicrobial peptide, wherein the amino acid sequence of the derivative of the antimicrobial peptide is any one of the sequences described in SEQ ID NO. 1-6.

[0006] The final object of this invention is to provide an application of a synthetically produced antimicrobial peptide and / or its derivatives.

[0007] To achieve the above objectives, the present invention adopts the following technical measures:

[0008] A synthetically produced antimicrobial peptide, wherein the antimicrobial peptide is any one of the proteins shown in SEQ ID NO. 1-6 or SEQ ID NO. 11.

[0009] The fusion protein obtained by fusing the above-mentioned antimicrobial peptide protein with a protein tag.

[0010] The genes encoding the aforementioned antimicrobial peptides or fusion proteins.

[0011] Expression cassettes, recombinant vectors, recombinant microorganisms, or in vitro recombinant cells containing the above-mentioned coding genes.

[0012] The application of the above-mentioned antimicrobial peptides, fusion proteins, genes encoding antimicrobial peptides or fusion proteins, or expression cassettes containing the above-mentioned encoding genes, recombinant vectors, recombinant microorganisms, or ex vivo recombinant cells in the preparation of antimicrobial agents for Escherichia coli.

[0013] The use of the above-mentioned antimicrobial peptides, fusion proteins, genes encoding antimicrobial peptides or fusion proteins, or expression cassettes containing the above-mentioned encoding genes, recombinant vectors, recombinant microorganisms, or ex vivo recombinant cells in the preparation of drugs for the prevention or treatment of Escherichia coli infection.

[0014] The application of the above-mentioned antimicrobial peptides, fusion proteins, genes encoding antimicrobial peptides or fusion proteins, or expression cassettes having the above-mentioned encoding genes, recombinant vectors, recombinant microorganisms, or in vitro recombinant cells in the preparation of additives for livestock and poultry feed or drinking water.

[0015] A drug for treating Escherichia coli infection, said drug comprising one or more antimicrobial peptides shown in SEQ ID NO. 1-6 and / or SEQ ID NO. 11.

[0016] In the above-described applications, preferably, the *Escherichia coli* includes multidrug-resistant *Escherichia coli*.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] (1) Novel sequence and high activity: The antimicrobial peptide provided by the present invention has strong inhibitory activity against standard strains of Escherichia coli and multidrug-resistant clinical isolates. Its minimum inhibitory concentration (MIC) value is in the microgram per milliliter (μg / mL) level, showing good antimicrobial potential.

[0019] (2) Excellent stability: The antimicrobial peptide can still maintain more than 90% of its antimicrobial activity in acidic and alkaline environments of pH 2.0-10.0 and after high-temperature treatment at 100℃, and has excellent environmental and process stability.

[0020] (3) High safety: Preliminary safety evaluation shows that the antimicrobial peptide has no hemolytic activity at effective antimicrobial concentrations and low toxicity to mammalian cells, and has the potential for further development.

[0021] (4) Significant in vivo therapeutic effect: In the Escherichia coli infection model of chicks, the antimicrobial peptide can significantly reduce the mortality rate, showing good in vivo therapeutic potential.

[0022] (5) Provides multiple implementation methods: This invention not only provides the antimicrobial peptide itself, but also its encoding gene, expression system and preparation method, providing multiple feasible technical routes for large-scale production and application. Attached Figure Description

[0023] Figure 1 The graph shows the test results of acid-base stability (A) and thermal stability (B) of the antimicrobial peptide AIP-1 in Example 2 of the present invention.

[0024] Figure 2 This is a bar chart showing the results of the minimum inhibitory concentration (MIC) determination of the antimicrobial peptide AIP-1 in Example 3 of the present invention. Detailed Implementation

[0025] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments, but the scope of protection of the present invention is not limited thereto. Experimental methods in the following embodiments that do not specify specific conditions are generally performed according to conventional conditions in the art or conditions recommended by the reagent manufacturer.

[0026] Example 1:

[0027] Antimicrobial peptide design and synthesis:

[0028] Eleven peptides were artificially synthesized according to Table 1.

[0029] Table 1. List of antimicrobial peptide sequences designed in this invention

[0030]

[0031] Example 2:

[0032] Table 1 shows the stability tests of the antimicrobial peptides.

[0033] 1. Acid-base stability: AIP-1 (or the antimicrobial peptide shown in SEQ ID NO.2-11) was dissolved in buffer solutions with different pH values ​​(2.0, 4.0, 6.0, 8.0, 10.0), incubated at 37°C for 2 hours, and then the pH was adjusted to neutral with a neutralizing solution. The minimum inhibitory concentration (MIC) against the standard strain of *Escherichia coli* ATCC 25922 was determined, and the activity retention rate was calculated. Specific results for AIP-1 are as follows... Figure 1 As shown in Figure A, the antibacterial activity retention rate of AIP-1 is higher than 90% in the pH range of 2.0-10.0, indicating that it has good acid and alkali stability.

[0034] Activity retention rate = (control MIC / treated MIC) × 100%, or 1 / (MIC fold change) × 100%.

[0035] Results for other peptides: Except for sequence AIP-1 (SEQ ID NO.1), the other antimicrobial peptides described in this invention (SEQ ID NO.2 to SEQ ID NO.11) also exhibited excellent acid-base stability. Processed and tested using the same method as AIP-1 in Example 2, the results showed that the acid-base stability of AIP2-6 and 11 was not significantly different from that of AIP1. The remaining peptides (SEQ ID NO.7 to SEQ ID NO.10) retained more than 85% of their antimicrobial activity against the Escherichia coli standard strain ATCC 25922 after incubation over a wide pH range of 2.0 to 10.0.

[0036] Table 2. Activity retention rate of antimicrobial peptide AIP-1 after treatment under different pH conditions

[0037]

[0038] 2. Thermal stability: AIP-1 solution (0.5 mg / mL) was placed in water baths at 4℃, 50℃, 70℃, 90℃, and 100℃ for 30 minutes, respectively, and then rapidly cooled to room temperature. The MIC against *Escherichia coli* ATCC 25922 was then determined. Results are as follows: Figure 1 As shown in Table B and Table 3, even after being treated at 100°C for 30 minutes, its antibacterial activity retention rate still exceeds 90%, proving that it has excellent thermal stability.

[0039] Results for the remaining peptides: Following the same testing methods as AIP-1 in Example 2, the results showed that the thermal stability of AIP2-6 and 11 was not significantly different from that of AIP1. The remaining peptides (SEQ ID NO.7 to SEQ ID NO.10) maintained an antibacterial activity retention rate of over 85% against the Escherichia coli standard strain ATCC 25922 after water bath treatment at different temperatures.

[0040] Table 3. Activity retention rate of antimicrobial peptide AIP-1 after treatment at different temperatures.

[0041]

[0042] Example 3:

[0043] Table 1 shows the in vitro antimicrobial activity assay (MIC assay) of the antimicrobial peptides:

[0044] The antimicrobial activity of the antimicrobial peptide AIP-1 against Escherichia coli was determined using the microbroth dilution method recommended by the Clinical Laboratory Standards Institute (CLSI).

[0045] Test strains: Standard Escherichia coli strain ATCC 25922, and three clinically isolated multidrug-resistant Escherichia coli strains (MDR-Eco 1, MDR-Eco 2, and MDR-Eco 3). All three clinical isolates exhibited high levels of resistance to multiple common antibiotics, including ampicillin (a penicillin), ceftriaxone (a third-generation cephalosporin), and ciprofloxacin (a quinolone).

[0046] Method Summary: AIP-1 (or the antimicrobial peptide shown in SEQ ID NO. 2-11) was serially diluted with Mueller-Hinton (MH) broth and added to 96-well plates. The prepared bacterial culture (final concentration approximately 5 × 10^5 CFU / mL) was added to each well. A growth control without the drug and a blank control without bacteria were also included. The 96-well plates were incubated at 37°C for 16–20 hours. The lowest drug concentration at which no bacterial growth was observed visually was defined as the minimum inhibitory concentration (MIC).

[0047] Result: As Figure 2 As shown, the MIC of the antimicrobial peptide AIP-1 against the standard Escherichia coli strain ATCC 25922 was 2 μg / mL. The MIC values ​​against three clinically multidrug-resistant strains were 4.0 μg / mL (MDR-Eco 1), 8.0 μg / mL (MDR-Eco 2), and 8.0 μg / mL (MDR-Eco 3), indicating that AIP-1 also has inhibitory activity against drug-resistant strains. The positive control drug ampicillin had MICs greater than 64 μg / mL against all three drug-resistant strains, and was therefore ineffective.

[0048] Except for sequence AIP-1 (SEQ ID NO.1), the in vitro antibacterial activity of other antimicrobial peptides (SEQ ID NO.2 to SEQ ID NO.11) described in this invention was also systematically determined according to the CLSI microbroth dilution method described above.

[0049] As shown in Table 4, the antimicrobial peptides (SEQ ID NO.2 to SEQ ID NO.6) and the core sequence SEQ ID NO.11 all exhibited clear inhibitory activity against the Escherichia coli standard strain ATCC 25922, with minimum inhibitory concentrations (MICs) ranging from 4.0 μg / mL to 32 μg / mL. Specifically, SEQ ID NO.2 and SEQ ID NO.6 both had an MIC of 4.0 μg / mL against the standard strain, showing potent activity similar to the preferred sequence AIP-1; sequences SEQ ID NO.3, 5, and 11 showed moderate activity; while SEQ ID NO.7 to SEQ ID NO.10 showed relatively weak activity.

[0050] Meanwhile, SEQ ID NO.2 and SEQ ID NO.6 also show good anti-drug resistance potential against clinically multidrug-resistant Escherichia coli strains (MDR-Eco1, 2, 3).

[0051] Table 4. Minimum inhibitory concentrations (MICs) of antimicrobial peptides AIP-1 to AIP-11 against Escherichia coli strains.

[0052]

[0053] Example 4:

[0054] Table 1 shows the safety evaluation of the antimicrobial peptides:

[0055] 1. Hemolysis test

[0056] Fresh rabbit anticoagulated blood was collected, centrifuged, and washed to obtain rabbit red blood cells. A 2% (v / v) red blood cell suspension was prepared using phosphate-buffered saline (PBS). Different concentrations of AIP-1 solution (0, 32, 64, 128 μg / mL) (or the antimicrobial peptide shown in SEQ ID NO.2-11) were mixed with an equal volume of the red blood cell suspension and incubated at 37°C for 1 hour. PBS was used as a negative control (0% hemolysis), and 1% Triton X-100 was used as a positive control (100% hemolysis). After incubation, the cells were centrifuged, and the absorbance of the supernatant was measured at 540 nm. The hemolysis rate at each concentration was calculated.

[0057] The results showed that even at a high concentration of 128 μg / mL (far exceeding its MIC value), the hemolysis rate induced by AIP-1 was still less than 1%, indicating that it had virtually no hemolytic toxicity.

[0058] According to the same method described above, the antimicrobial peptides of this invention (SEQ ID NO.2 to SEQ ID NO.11) induced rabbit erythrocyte hemolysis rates of less than 2.0% at test concentrations as high as 128 μg / mL (far exceeding their respective effective antibacterial concentrations). This result indicates that the series of antimicrobial peptides of this invention all possess excellent blood compatibility and exhibit virtually no hemolytic toxicity at effective antibacterial concentrations, providing important assurance for their safety when administered intravenously or systemically.

[0059] 2. Cytotoxicity assay

[0060] The toxicity of AIP-1 to porcine small intestinal epithelial cells IPEC-J2 and HEK293 was detected by the CCK-8 assay.

[0061] IPEC-J2 or HEK293 cells were seeded into 96-well plates and cultured until adherent. The medium was then replaced with fresh medium containing different concentrations of AIP-1 (0, 32, 64, 128, 256 μg / mL) (or the antimicrobial peptide shown in SEQ ID NO.2-11), with multiple replicates for each concentration. After culturing for another 24 hours, CCK-8 reagent was added to each well, and after incubation for a certain period, the absorbance at 450 nm was measured using a microplate reader. Cell viability was calculated, and the half-maximal inhibitory concentration (IC50) was calculated using software.

[0062] The results, as shown in Table 5, indicate that AIP-1 exhibited weak growth inhibition against IPEC-J2 and HEK293 cells within the tested concentration range, with an IC50 value greater than 256 μg / mL, demonstrating good cellular safety. The therapeutic index was calculated as: TI = IC50. 50 / MIC.

[0063] The antimicrobial peptides described in SEQ ID NO.2 to SEQ ID NO.11 were evaluated using the CCK-8 assay and showed extremely low cytotoxicity against porcine small intestinal epithelial cells IPEC-J2 and human embryonic kidney cells HEK293. At concentrations up to 256 μg / mL, cell viability in each peptide treatment group was significantly higher than 80%, and the calculated half-maximal inhibitory concentration (IC50) was significantly higher than the target value. 50 All were greater than 256 μg / mL.

[0064] Table 5. In vitro safety evaluation of antimicrobial peptide AIP-1

[0065]

[0066] Based on combined hemolysis and cytotoxicity data, all the claimed antimicrobial peptide sequences (SEQ ID NO.1 to SEQ ID NO.6) and the core sequence AIP-11 (SEQ ID NO.11) of this invention exhibit a high therapeutic index. The MIC values ​​(e.g., 8 μg / mL) and IC50 values ​​against multidrug-resistant strains... 50 The therapeutic index (TI = IC50) is calculated based on the value (>256 μg / mL). 50 The concentration ratio (MIC) is greater than 32. This demonstrates that the antimicrobial peptide series of the present invention has a wide safety window between effective antimicrobial concentration and potential toxic concentration, and possesses good drug development potential. Therefore, it was further investigated in animal experiments as the peptide to be protected.

[0067] Example 5:

[0068] In vivo efficacy validation (chick infection model)

[0069] A chick Escherichia coli infection model was established to evaluate the in vivo protective effect of the antimicrobial peptide AIP-1 (or the antimicrobial peptide shown in SEQ ID NO.2-6, 11).

[0070] One-day-old SPF chicks were randomly divided into nine groups (n=20 per group): a blank control group (saline), an infection model group (saline + infection), an AIP-1 treatment group (SEQ ID NO.1, 5 mg / kg / d + infection), and AIP-2~6 and AIP-11 treatment groups (corresponding to SEQ ID NO.2, 3, 4, 5, 6, 11, respectively, all at a dose of 5 mg / kg / d + infection). Each treatment group received an oral dose of the corresponding antimicrobial peptide solution for 7 consecutive days before infection, while the blank and model groups received an equal volume of saline. On day 8, except for the blank control group, all other groups received an intraperitoneal injection of a lethal dose of ESBL-producing clinical isolates of *Escherichia coli* (1×10^8 CFU / chick). The mortality rate of chicks in each group was continuously observed and recorded for 14 days, and the mortality rate, survival rate, and relative protection rate were calculated. The relative protection rate (%) was used to quantify the therapeutic effect, calculated using the following formula:

[0071] Relative protection rate (%) = [(Model group mortality rate - Treatment group mortality rate) / Model group mortality rate] × 100%

[0072] The results showed that the cumulative mortality rate in the infection model group was as high as 65% on day 14. In contrast, the mortality rate in the AIP-1 treatment group was significantly reduced to 15%, close to the survival rate of the blank control group (0%). These results indicate that treatment with the antimicrobial peptides of this invention significantly reduced mortality in all groups, with the antimicrobial peptide AIP-1 showing the best effect. It effectively prevents and treats infections caused by *E. coli* in vivo, significantly improving the survival rate of infected animals (Table 6).

[0073] Table 6. Protective effects of antimicrobial peptides AIP-1~6 and AIP-11 in a chick Escherichia coli infection model.

[0074]

[0075] Example 6: Preparation of the pharmaceutical composition

[0076] Mix 1% AIP-1 peptide powder with 90% corn starch and 9% β-cyclodextrin excipients to prepare an oral powder or add it to feed as a feed additive.

[0077] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A synthetically produced antimicrobial peptide, wherein the antimicrobial peptide is any one of the proteins shown in SEQ ID NO. 1-6 or SEQ ID NO.

11.

2. The fusion protein obtained by fusing the antimicrobial peptide protein of claim 1 with a protein tag.

3. The gene encoding the antimicrobial peptide protein of claim 1 or the fusion protein of claim 2.

4. An expression cassette, recombinant vector, recombinant microorganism, or ex vivo recombinant cell having the gene encoding as described in claim 3.

5. The use of the antimicrobial peptide protein of claim 1, the fusion protein of claim 2, the gene of claim 3 or an expression cassette having the encoding gene of claim 3, a recombinant vector, a recombinant microorganism or an ex vivo recombinant cell in the preparation of an antibacterial agent for Escherichia coli.

6. The use of the antimicrobial peptide protein of claim 1, the fusion protein of claim 2, the gene of claim 3 or an expression cassette having the encoding gene of claim 3, a recombinant vector, a recombinant microorganism or an ex vivo recombinant cell in the preparation of a drug for the prevention or treatment of Escherichia coli infection.

7. The use of the antimicrobial peptide protein of claim 1, the fusion protein of claim 2, the gene of claim 3 or an expression cassette having the encoding gene of claim 3, a recombinant vector, a recombinant microorganism or an ex vivo recombinant cell in the preparation of additives for livestock and poultry feed or drinking water.

8. A medicament for treating Escherichia coli infection, said medicament comprising one or more antimicrobial peptides shown in SEQ ID NO. 1-6 and / or SEQ ID NO.

11.

9. The application according to claims 5-6, or the drug according to claim 7, wherein the *Escherichia coli* is a multidrug-resistant *Escherichia coli*.

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