A derivative peptide KR-23 and a preparation method and application thereof

Abstract

The application provides a derivative peptide KR-23 and a preparation method and application thereof, and the sequence of the derivative peptide KR-23 is shown in the sequence table SEQ ID No. 1. The application takes a high-toxicity frog-derived antibacterial peptide AR-23 as raw material, replaces hydrophobic amino acids by alkaline amino acids, destroys the amphiphilic structure of the alpha-helix of the original peptide, keeps the length of the original peptide unchanged, and the derivative peptide KR-23 significantly reduces the cytotoxicity and slightly improves the antibacterial activity, and the therapeutic index is 83 times of that of AR-23. The cytotoxicity of the peptide is obviously improved through the method, a new source can be provided for the selection of replacement products, and the healthy development of the animal husbandry is facilitated.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a derived peptide KR-23, its preparation method, and its application. Background Technology

[0002] Antimicrobial peptides and antibiotics have different antibacterial mechanisms. Antimicrobial peptides interfere with the function of multiple biological processes in a "mild" way, rather than acting on highly specific target sites to render them ineffective, while antibiotics tend to act on a single target within the bacterial cell. Antimicrobial peptides leave no residue in animals, cause no environmental pollution, meet the needs of safe livestock production, are suitable for use in feed production, and have the potential to become a new generation of green feed additives. Currently, the development and application of antimicrobial peptides are widely carried out in animal husbandry and the medical and health fields.

[0003] However, compared with traditional antibiotics, there are still gaps in the production and application of natural antimicrobial peptides, and the problem of high cytotoxicity cannot be ignored. Therefore, artificially designing antimicrobial peptides or molecularly modifying existing natural antimicrobial peptides is one of the effective ways to develop highly effective antimicrobial agents. Summary of the Invention

[0004] The purpose of this invention is to provide a derived peptide KR-23, its preparation method, and its application; this derived peptide KR-23 has a high therapeutic index.

[0005] The objective of this invention is achieved through the following technology: a derived peptide KR-23, the sequence of which is shown in SEQ ID No. 1 of the sequence listing.

[0006] The preparation method of the derived peptide KR-23 as described above is as follows: (1) For the highly hemolytic frog-derived antimicrobial peptide AR-23 (i.e. AR-23), the derivative peptide KR-23 was generated by replacing Ala1, Ala8 and Ile17 with Lys respectively, and by exchanging the positions of Ile2 and Lys11 and C-terminal amidation. The sequence is shown in SEQ ID No.1 of the sequence listing. (2) Peptide resin was obtained by solid-phase chemical synthesis using a peptide synthesizer. The obtained peptide resin was then cleaved by TFA to obtain the derived peptide KR-23. (3) After purification by reversed-phase high-performance liquid chromatography, the preparation of the derived peptide KR-23 is completed.

[0007] The application of the derivative peptide KR-23 as described above in the preparation of drugs for treating infectious diseases caused by Gram-positive or Gram-negative bacteria.

[0008] The experimental technique for preparing the derived peptide KR-23 using this method is simple. Antibacterial and hemolytic activity tests on the obtained derived peptide KR-23 revealed that it possesses broad-spectrum antibacterial activity and extremely low hemolytic activity. In conclusion, KR-23 is an antimicrobial peptide with high application value. Attached Figure Description

[0009] Figure 1 The diagram shows the helical wheel of the AR-23 and KR-23.

[0010] Figure 2 The amino acid sequence is KR-23.

[0011] Figure 3 The antibacterial activity of AR-23 and KR-23.

[0012] Figure 4 The hemolytic activity and therapeutic index of AR-23 and KR-23. Detailed Implementation

[0013] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0014] Example 1: A derived peptide KR-23, the sequence of which is shown in SEQ ID No. 1 of the sequence listing.

[0015] The design of the derived peptide KR-23: For the highly hemolytic frog-derived antimicrobial peptide AR-23, Lys was used to replace Ala1, Ala8, and Ile17 in the AR-23 sequence to increase the proportion of positively charged amino acids and reduce the peptide's hydrophobicity; Ile2 and Lys11 were interchanged in the sequence to disrupt the peptide's amphiphilic α-helix structure, as shown in the appendix. Figure 1 As shown; C-terminal amidation increases peptide stability to generate the derived peptide KR-23, the sequence of which is attached. Figure 2 As shown.

[0016] Example 2: A method for preparing the derivative peptide KR-23, wherein the derivative peptide KR-23 is synthesized by solid-phase chemical synthesis.

[0017] 1. The preparation of the derived peptide KR-23 was carried out sequentially from the C-terminus to the N-terminus using a peptide synthesizer. First, Fmoc-X (X is the first amino acid at the C-terminus of each antimicrobial peptide) was inoculated into Wang resin, and then the Fmoc group was removed to obtain X-Wang resin; then Fmoc-Y-Trt-OH (9-fluorenylmethoxycarboxylic acid-trimethyl-Y, where Y is the second amino acid at the C-terminus of each antimicrobial peptide) was added; following this procedure, the peptides were synthesized sequentially from the C-terminus to the N-terminus until the synthesis was complete, resulting in a resin with the side chain protection removed by the Fmoc group. 2. Add the cleavage reagent to the peptide resin obtained above, react at 20°C in the dark for 2 hours, and filter. Wash the precipitate with TFA (trifluoroacetic acid), mix the washings with the above filtrate, concentrate by rotary evaporator, add about 10 times the volume of pre-cooled anhydrous ether, precipitate at -20°C for 3 hours, and a white powder will precipitate. Centrifuge at 2500g for 10 minutes, collect the precipitate, wash the precipitate with anhydrous ether, and vacuum dry to obtain the peptide. The cleavage reagent is composed of TFA, water and TIS (triisopropylchlorosilane) in a mass ratio of 95:2.5:2.5. 3. Equilibrate the column for 30 min using 0.2 mol / L sodium sulfate (adjusted to pH 7.5 with phosphoric acid). Dissolve the peptide in 90% acetonitrile aqueous solution, filter, and elute using a C18 reversed-phase atmospheric pressure column with gradient elution (eluting agent is a mixture of methanol and sodium sulfate aqueous solution in a volume ratio of 30:70~70:30), a flow rate of 1 mL / min, and a detection wavelength of 220 nm. Collect the main peak and freeze-dry. Further purify using a reversed-phase C18 column with elution buffer A being 0.1% TFA / acetonitrile solution and elution buffer B being 0.1% TFA / aqueous solution, with an elution concentration of 50%~90%, an elution time of 30 min, and a flow rate of 1 mL / min. Collect the main peak again and freeze-dry to obtain the purified derived peptide KR-23. 4. Identification of the derived peptide KR-23: The derived peptide KR-23 obtained above was analyzed by electrospray mass spectrometry. The theoretical molecular weight was basically consistent with the measured molecular weight, and the purity of the derived peptide KR-23 was greater than 95%.

[0018] Example 3: Application of a derived peptide KR-23 in the preparation of drugs for treating infectious diseases caused by Gram-positive or Gram-negative bacteria.

[0019] Determination of the bioactivity of the derived peptide KR-23 in applications.

[0020] 1. Determination of antibacterial activity: A 2.56 mM stock solution of the derived peptide KR-23 was prepared for use. The minimum inhibitory concentrations (MICs) of KR-23 and AR-23 were determined using the microbroth dilution method. A series of KR-23 and AR-23 solutions were prepared sequentially using a two-fold dilution method with 0.01% acetic acid (containing 0.2% BSA) as the diluent. 100 μL of each solution was placed in a 96-well cell culture plate, and then an equal volume of the test bacterial culture (~10 μL) was added. 5 The bacterial culture was injected into each well (number of bacteria per mL). A positive control (containing bacterial culture but not KR-23 or AR-23) and a negative control (containing neither bacterial culture nor KR-23 or AR-23) were set up. The cultures were incubated at 37°C for 20 hours. The minimum inhibitory concentration (MIC) was determined by the absence of visible turbidity at the bottom of the well.

[0021] The results are attached. Figure 3 and attached Figure 4 As shown, KR-23 exhibited varying degrees of antibacterial activity against Gram-negative and Gram-positive bacteria. Compared to AR-23, KR-23 showed slightly increased antibacterial activity against Gram-negative bacteria, while its activity against Gram-positive bacteria decreased slightly. Geometric mean analysis of the minimum inhibitory concentration (MIC) indicated that KR-23 demonstrated improved antibacterial efficacy, suggesting that the molecular design preserved the broad-spectrum antibacterial activity of the original peptide.

[0022] 2. Determination of hemolytic activity: 1 mL of fresh human blood was collected, anticoagulated with heparin, and dissolved in 2 mL of PBS solution. The mixture was centrifuged at 1000g for 5 min, and red blood cells were collected. The red blood cell suspension was washed three times with PBS and resuspended in 10 mL of PBS. 50 µL of the red blood cell suspension was mixed thoroughly with 50 µL of KR-23 and AR-23 solutions of different concentrations dissolved in PBS, and incubated at 37℃ for 1 h. After incubation, the mixture was centrifuged at 4℃ for 5 min. The supernatant was then measured at 570 nm using a microplate reader. The average value of each group was taken and compared. 50 µL of red blood cells with 50 µL of PBS served as a negative control; 50 µL of red blood cells with 50 µL of 0.1% Tritonx-100 served as a positive control. The minimum hemolytic concentration was the concentration at which KR-23 and AR-23 caused a 5% hemolysis rate.

[0023] As attached Figure 4 As shown, KR-23 did not exhibit hemolytic activity within the detection range, while the control AR-23 showed high hemolytic activity at a concentration of 4 μM. This indicates that the design of KR-23 significantly reduced cytotoxicity.

[0024] The results in summary show that KR-23 exhibits slightly enhanced antibacterial activity and lower hemolytic activity compared to AR-23. A comprehensive analysis of the antibacterial and hemolytic activities of antimicrobial peptides can be performed using the therapeutic index (the ratio of hemolytic concentration to antibacterial concentration) to more fully evaluate the biological activity of each antimicrobial peptide, as shown in the appendix. Figure 4 As shown, KR-23 has a significantly higher therapeutic index of 41.5, an improvement of 83 times. Therefore, the designed KR-23 antimicrobial peptide has high potential for development as an alternative antibiotic.

Claims

1. A derived peptide KR-23, characterized in that: Its amino acid sequence is LysLysGlySerIleLeuGlyLysLeuAlaIleGlyLeuProThrLeuLysSerTrpIleLysAsnArg-NH2.

2. The method for preparing the derived peptide KR-23 as described in claim 1, characterized in that: The preparation method is as follows: For the highly hemolytic frog-derived antimicrobial peptide AR-23, the derived peptide KR-23 as described in claim 1 was generated by replacing Ala1, Ala8 and Ile17 with Lys respectively, and by exchanging the positions of Ile2 and Lys11 and performing C-terminal amidation. Peptide resin was obtained by solid-phase chemical synthesis using a peptide synthesizer. The obtained peptide resin was then cleaved by TFA to obtain the derived peptide KR-23. After purification by reversed-phase high-performance liquid chromatography, the preparation of the derived peptide KR-23 is completed.

3. The use of the derivative peptide KR-23 as described in claim 1 in the preparation of an antibacterial drug, wherein the antibacterial activity is the inhibition of any one or more of the following bacteria: Escherichia coli, Salmonella Pullorum, Salmonella Typhimurium, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, and Bacillus subtilis.

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