Double-branched-chain antibacterial peptide as well as preparation method and application thereof

Abstract

The invention discloses a double-branched-chain antibacterial peptide as well as a preparation method and application thereof, and belongs to the field of bioengineering, and the sequence molecular formula of the double-branched-chain antibacterial peptide is as shown in formula (I). The invention also provides an application of the double-branched-chain antibacterial peptide VOHyp in preparation of a medicine for treating diseases infected by gram-positive bacteria or / and gram-negative bacteria. The double-branched-chain antibacterial peptide disclosed by the invention has high-efficiency antibacterial activity on various pathogenic microorganisms and has no toxicity on human red blood cells, so that the double-branched-chain antibacterial peptide has the potential of becoming a feed antibiotic substitute.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering, specifically relating to a bibranched antimicrobial peptide VOHyp, its preparation method, and its application. Background Technology

[0002] Natural antimicrobial peptides (AMPs) consist of approximately 10–30 amino acid residues and can achieve broad-spectrum bactericidal activity by directly disrupting bacterial cell membranes, without relying on blocking metabolic pathways. Therefore, they are less likely to induce drug resistance and are considered ideal candidates for feed antibiotics. However, many currently available AMPs still have limitations such as insufficient activity, narrow antimicrobial spectrum, or potentially high toxicity, which restrict their application in animal husbandry.

[0003] By precisely adjusting the physicochemical characteristics such as amino acid sequence, hydrophobicity, and amphiphilicity, the selectivity and bactericidal efficiency against pathogenic bacteria can be significantly improved. Recent research shows that peptides with multi-branched chains often exhibit stronger antibacterial activity compared to conventional linear structures. Therefore, it is necessary to add bibranched chains to linear antimicrobial peptides to develop a novel broad-spectrum antimicrobial peptide, providing a theoretical basis and technical support for the development of feed-grade antimicrobial peptides. Summary of the Invention

[0004] The purpose of this invention is to disclose a bibranched antimicrobial peptide, VOHyp, which provides a broad-spectrum antimicrobial peptide with the potential to become a substitute for feed antibiotics.

[0005] The objective of this invention is achieved as follows: the molecular formula of a bibranched antimicrobial peptide VOHyp is shown in formula (I):

[0006] Formula (I).

[0007] This invention also provides a method for preparing the bibranched antimicrobial peptide VOHyp as described above, as follows: It comprises four short peptide chains, one hydroxyproline, and two ornithine residues. The sequence of short peptide chain a is N-VKVVKK-C, the sequence of short peptide chain b is N-VKVVKKV-C, the sequence of short peptide chain c is N-VKVVKK-C, and the sequence of short peptide chain d is N-VKKVVKV-C. The C-terminus of short peptide chain a is connected to the N-terminus of the first ornithine residue, the N-terminus of short peptide chain b is connected to the C-terminus of the first ornithine residue, the amino group of the first ornithine side chain is connected to the C-terminus of the hydroxyproline, and the C-terminus of the second ornithine residue is connected to the N-terminus of the hydroxyproline. The amino group of the second ornithine side chain is linked to the C-terminus of short peptide chain c, and the N-terminus of the second ornithine is linked to the C-terminus of short peptide chain d. The N-terminus of short peptide chains a, c, and d are acetylated, and the C-terminus of short peptide chain b is amidated. The molecular formula of the constructed peptide is shown in formula (I). The peptide is synthesized by solid-phase chemical synthesis, purified by reversed-phase high-performance liquid chromatography and identified by mass spectrometry, and then subjected to antibacterial activity and hemolytic activity determination. Finally, it is named antimicrobial peptide VOHyp.

[0008] The present invention also provides the use of the bibranched antimicrobial peptide VOHyp as described above in the preparation of a medicament for treating infectious diseases caused by Gram-positive bacteria and / or Gram-negative bacteria.

[0009] Furthermore, in the application described above, the Gram-positive bacteria are Staphylococcus aureus, Enterococcus faecalis, or Staphylococcus epidermidis.

[0010] Furthermore, in the application described above, the Gram-negative bacteria are Escherichia coli or Salmonella typhimurium.

[0011] Furthermore, as described above, the application of the drug in the context of feed additives is its use as a medicated feed additive.

[0012] The present invention also provides a medicament for treating and / or preventing infections caused by Gram-positive and / or Gram-negative bacteria, as described above, the medicament containing a bibranched antimicrobial peptide VOHyp as described above.

[0013] The beneficial effects and advantages of the present invention are as follows: Biological activity tests on the antimicrobial peptide of the present invention revealed that it has high antibacterial activity against Gram-negative bacteria such as Escherichia coli and Salmonella typhimurium, as well as Gram-positive bacteria such as Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, and Enterococcus faecalis. The antimicrobial peptide caused only 6.13% hemolysis of erythrocytes at a concentration of 128 μM, and it has the potential to become a substitute for feed antibiotics. Attached Figure Description

[0014] Figure 1This is a high-performance liquid chromatogram of the bibranched antimicrobial peptide VOHyp of the present invention.

[0015] Figure 2 This is a matrix-assisted laser desorption / ionization time-of-flight mass spectrum of the bibranched antimicrobial peptide VOHyp of the present invention.

[0016] Figure 3 This is a diagram showing the hemolytic activity of the bibranched antimicrobial peptide VOHyp and bee venom ME of the present invention. Detailed Implementation

[0017] The invention will be further described below with reference to the accompanying drawings:

[0018] Example 1

[0019] This embodiment provides a bibranched antimicrobial peptide VOHyp, comprising four short peptide chains, one hydroxyproline, and two ornithine residues. The sequence of short peptide chain a is N-VKVVKK-C, the sequence of short peptide chain b is N-VKVVKKV-C, the sequence of short peptide chain c is N-VKVVKK-C, and the sequence of short peptide chain d is N-VKKVVKV-C. The C-terminus of short peptide chain a is connected to the N-terminus of the first ornithine residue, and the N-terminus of short peptide chain b is connected to the first ornithine residue. The C-terminus of the first ornithine side chain is linked to the C-terminus of hydroxyproline, the C-terminus of the second ornithine is linked to the N-terminus of hydroxyproline, the amino group of the second ornithine side chain is linked to the C-terminus of short peptide chain c, and the N-terminus of the second ornithine is linked to the C-terminus of short peptide chain d. The N-termini of short peptide chains a, c, and d are acetylated, and the C-terminus of short peptide chain b is amidated. The resulting bibranched antimicrobial peptide VOHyp has the structural formula shown in formula (I).

[0020]

[0021] Formula (I)

[0022] Its amino acid sequence is as follows:

[0023]

[0024] The antimicrobial peptide VOHyp has a net charge of +11 and a peptide chain length of 29. VOHyp not only exhibits highly effective antimicrobial activity against a variety of pathogenic microorganisms, but also shows no toxicity to human erythrocytes, making it a potential alternative to animal feed antibiotics.

[0025] Example 2

[0026] Solid-phase chemical synthesis of bibranched antimicrobial peptides

[0027] 1. The preparation of bibranched antimicrobial peptides proceeds sequentially from the C-terminus to the N-terminus using a peptide synthesizer: First, Fmoc-X (where X is the first amino acid at the C-terminus of each antimicrobial peptide) is inscribed into Wang resin, and then the Fmoc group is removed to obtain X-Wang resin; next, Fmoc-Y-Trt-OH (9-fluorenylmethoxycarboxylic acid-trimethyl-Y, where Y is the second amino acid at the C-terminus of each antimicrobial peptide, and its side chain may be protected with groups such as Trt as needed) is mixed with coupling reagents and activators, and then reacts with the free amino groups on the resin to form peptide bonds. After the reaction is complete, the resin is thoroughly washed to remove excess reagents; this procedure is repeated sequentially from the C-terminus to the N-terminus until the synthesis is complete, yielding resin with the side chain protection removed by the Fmoc group;

[0028] 2. Add the cleavage reagent to the peptide resin obtained above, react at 20 ℃ in the dark for 2 h, 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 diethyl ether, precipitate at -20 ℃ for 3 h, and a white powder will precipitate. Centrifuge at 2500 g for 10 min, collect the precipitate, wash the precipitate with anhydrous diethyl 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.

[0029] 3. Equilibrate the column for 30 min using 0.2 M 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 to 70:30), at a flow rate of 1 mL / min and a detection wavelength of 220 nm. Collect the main peak and lyophilize it. Further purify using a reversed-phase C18 column with elution buffer A being 0.1% TFA / aqueous solution and elution buffer B being 0.1% TFA / acetonitrile solution, with an elution concentration of 25% B to 40% B, an elution time of 12 min, and a flow rate of 1 mL / min. Collect the main peak again and lyophilize it.

[0030] 4. Identification of antimicrobial peptides: The antimicrobial peptides obtained above were analyzed by electrospray ionization mass spectrometry. The molecular weight (e.g., ...) shown in the mass spectrum is determined by the electrospray ionization mass spectrometry method. Figure 1 , 2 The molecular weight (as shown in Table 1) is basically consistent with the theoretical molecular weight, and the purity of the antimicrobial peptide is greater than 95%.

[0031] Example 3

[0032] 1. Determination of antibacterial activity: The minimum inhibitory concentration (MIC) of the bibranched antimicrobial peptide VOHyp was determined after treatment with a serial dilution method. A series of antimicrobial peptide solutions were prepared using a 2-fold dilution method with 2 mg / ml BSA (containing 0.01% acetic acid) 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 to each well. 5 (Cells / mL) were added to each well. A positive control (containing bacterial culture but no antimicrobial peptides) and a negative control (containing neither bacterial culture nor antimicrobial peptides) were also included. After incubation at 37 ℃ for 14-18 h, the OD values ​​were... 492nm The minimum inhibitory concentration (MIC) is the concentration of the antimicrobial peptide that inhibits the growth of 99.9% of microorganisms when absorbance is measured. The test results are shown in Table 1.

[0033] Table 1. Minimum inhibitory activity of the bibranched antimicrobial peptide VOHyp against pathogenic microorganisms.

[0034]

[0035] As shown in Table 1, the antimicrobial peptide VOHyp exhibits high antibacterial activity against both Gram-negative bacteria (E. coli 25922, E. coli K88, E. coli UB1005, and S. typhimurium 14028) and Gram-positive bacteria (S. aureus 29213, S. aureus 25923, S. epidermidis ATCC12228, S. aureus 43300, and E. faecalis 29212).

[0036] 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 3000 rpm for 10 min, and red blood cells were collected. The red blood cell suspension was washed three times with PBS solution and resuspended in 10 mL of PBS solution. 50 µL of the red blood cell suspension was mixed with 50 µL of solutions containing different concentrations of the antimicrobial peptide VOHyp and incubated at 37 ℃ for 1 h. Subsequently, the mixture was centrifuged at 3000 rpm for 10 min at 4 ℃. The supernatant was collected and the absorbance was measured at 570 nm using a microplate reader. 50 µL of red blood cells with 50 µL of PBS solution served as a negative control, and 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 of the antimicrobial peptide that caused a 10% hemolysis rate. The test results are shown in […]. Figure 3 .pass Figure 3It can be seen that the antimicrobial peptide VOHyp did not exhibit hemolytic activity within the detection range. At a concentration of 128 µM, it caused 6.13% hemolysis of erythrocytes, but failed to cause 10% hemolysis of erythrocytes. This was significantly different from the control group bee venom, indicating that the antimicrobial peptide VOHyp has the potential to be developed into a feed-grade antimicrobial peptide.

Claims

1. A bibranched antimicrobial peptide, VOHyp, characterized in that, Its molecular formula is shown in formula (I): Formula (I).

2. The method for preparing the bibranched antimicrobial peptide VOHyp according to claim 1, characterized in that, The method is as follows: It includes four short peptide chains, one hydroxyproline, and two ornithine residues. The sequence of short peptide chain a is N-VKVVKK-C, the sequence of short peptide chain b is N-VKVVKKV-C, the sequence of short peptide chain c is N-VKVVKK-C, and the sequence of short peptide chain d is N-VKKVVKV-C. The C-terminus of short peptide chain a is linked to the N-terminus of the first ornithine residue, the N-terminus of short peptide chain b is linked to the C-terminus of the first ornithine residue, the amino group of the first ornithine side chain is linked to the C-terminus of the hydroxyproline, and the C-terminus of the second ornithine residue is linked to the hydroxyproline side chain. The N-terminus of the second ornithine is linked to the C-terminus of short peptide chain c, and the N-terminus of the second ornithine is linked to the C-terminus of short peptide chain d. The N-terminus of short peptide chains a, c, and d are acetylated, and the C-terminus of short peptide chain b is amidated. The molecular formula of the constructed peptide is shown in formula (I). The peptide is synthesized by solid-phase chemical synthesis, purified by reversed-phase high-performance liquid chromatography and identified by mass spectrometry, and then subjected to antibacterial activity and hemolytic activity determination. Finally, it is named antimicrobial peptide VOHyp.

3. The use of the bibranched antimicrobial peptide VOHyp according to claim 1 in the preparation of a medicament for treating infectious diseases caused by Gram-positive bacteria and / or Gram-negative bacteria.

4. The application according to claim 3, characterized in that: The Gram-positive bacteria mentioned are Staphylococcus aureus, Enterococcus faecalis, or Staphylococcus epidermidis.

5. The application according to claim 3, characterized in that: The Gram-negative bacteria mentioned are Escherichia coli or Salmonella typhimurium.

6. The application according to claim 3, characterized in that: The application of the drug is as a medicated feed additive.

7. A drug suitable for treating and / or preventing infections caused by Gram-positive and / or Gram-negative bacteria, characterized in that, The drug contains a bibranched antimicrobial peptide VOHyp as described in claim 1.

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