Heterozygosis alpha spiral swine antibacterial peptide, and preparation method and application thereof

Abstract

The invention provides a heterozygosis alpha spiral swine antibacterial peptide, and a preparation method and application thereof. The heterozygosis alpha spiral swine antibacterial peptide has the sequence shown as SEQ No.1 in a sequence table. According to the method, firstly, 16 amino acid residues before N- tail end in the swine antibacterial peptide PMAP-36 are modified, and are then subjected to heterozygosis with the front 15 amino acid of Fowlicidin-2 to obtain the antibacterial peptide PR-FO; the tail end of the carboxyl of the PR-FO is amidated so as to increase the positive charge and to improve the peptide stability; the sequence is shown as SEQ No. 1; then, a polypeptide synthesis instrument is used for synthesizing the antibacterial peptide PR-FO by a solid-phase synthesis method. The antibacterial peptide provided by the invention has higher antibacterial activity and low cell toxicity; the cell selectivity is greatly improved. The method can lay the feasible theoretical and practice foundation for making the antibacterial peptide into antibiotic substitutes.

Description

technical field

[0001] The invention belongs to the field of agricultural, animal husbandry and veterinary applications, and specifically relates to a hybrid α-helical pig-derived antimicrobial peptide and its preparation method and application. Background technique

[0002] Antibiotics have always been a powerful weapon for humans to treat diseases caused by pathogenic microbial infections. However, with the extensive use of traditional antibiotics, the drug resistance of pathogenic bacteria has gradually increased, and the era of antibiotics is gradually coming to an end. The best antibiotics are also gradually losing their effectiveness, so the search for new antibacterial drugs has become the focus of much attention. Especially the recent reports on superbugs have sounded the alarm for more people. Finding a new green feed additive to replace antibiotics has become an urgent task.

[0003] There are many kinds of antimicrobial peptides, which are widely distributed in ...

Examples

Embodiment 1

[0016] Example 1 Design and synthesis of hybrid α-helical porcine antimicrobial peptide sequence

[0017] The antimicrobial peptide RI16 was obtained by intercepting the first 16 amino acid residues at the N-terminal of the porcine antimicrobial peptide PMAP-36, and the antimicrobial peptide PRW4 was obtained by replacing the 7th and 11th amino acids with tryptophan, and then PRW4 was combined with α-helix The first 15 amino acids of the type antibacterial peptide Fowlicidin-2 were hybridized to obtain the antibacterial peptide PR-FO, and the carboxyl terminal of PR-FO was amidated to increase a positive charge and increase the stability of the peptide. The amino acid sequence of the antimicrobial peptide is shown in Table 1 Show.

[0018] Amino acid sequence and molecular weight of table 1 antimicrobial peptide

[0019]

[0020] Using a peptide synthesizer, the above-mentioned antimicrobial peptides were synthesized by solid-phase synthesis.

Embodiment 2

[0021] Destruction of the bacterial cell membrane of embodiment 2

[0022] 1 Destruction of the outer membrane by antimicrobial peptides

[0023] Escherichia coli was cultured to the logarithmic growth phase, centrifuged and collected, washed with 5 mM HEPES buffer solution (5 mM glucose, pH 7.4) and suspended in the buffer solution. Take 2mL of bacterial solution and put it in a quartz cuvette, and add 1mM NPN solution to mix with it, so that the final concentration of NPN is 10μM. Use F-4500 fluorescence spectrophotometer to detect the fluorescence intensity (excitation 350nm emission 420nm), add different concentrations of antimicrobial peptides, and detect the change of fluorescence intensity. The result is as figure 1 shown. Depend on figure 1 It can be seen that both PRW4 and PR-FO have a certain degree of permeabilization of the outer membrane, but the permeation intensity of PR-FO is stronger, indicating that the destruction effect of antimicrobial peptides on the ...

Embodiment 3

[0026] Bacteriostatic activity and hemolytic activity of embodiment 3 antimicrobial peptides

[0027] 1 Bacteriostatic activity of antimicrobial peptides

[0028] The antimicrobial peptides PRW4, FO and PR-FO were prepared as stock solution with a certain concentration for use. The minimum inhibitory concentrations of several antimicrobial peptides were determined by the broth microdilution method. Using 0.01% acetic acid (containing 0.2% BSA) as the diluent, a series of gradient antimicrobial peptide solutions were sequentially prepared using the double dilution method. Take 100 μL of the above solution and place it in a 96-well cell culture plate, then add an equal volume of the bacteria solution to be tested (~10 5 individual / mL) in each well. Positive controls (containing bacterial fluid but not antimicrobial peptides) and negative controls (neither bacterial fluid nor peptides) were set up. Incubate at a constant temperature of 37°C for 20 hours, and the minimum inhib...

technical field

[0001] The invention belongs to the field of agricultural, animal husbandry and veterinary applications, and specifically relates to a hybrid α-helical pig-derived antimicrobial peptide and its preparation method and application. Background technique

[0002] Antibiotics have always been a powerful weapon for humans to treat diseases caused by pathogenic microbial infections. However, with the extensive use of traditional antibiotics, the drug resistance of pathogenic bacteria has gradually increased, and the era of antibiotics is gradually coming to an end. The best antibiotics are also gradually losing their effectiveness, so the search for new antibacterial drugs has become the focus of much attention. Especially the recent reports on superbugs have sounded the alarm for more people. Finding a new green feed additive to replace antibiotics has become an urgent task.

[0003] There are many kinds of antimicrobial peptides, which are widely distributed in ...

Examples

Embodiment 1

[0016] Example 1 Design and synthesis of hybrid α-helical porcine antimicrobial peptide sequence

[0017] The antimicrobial peptide RI16 was obtained by intercepting the first 16 amino acid residues at the N-terminal of the porcine antimicrobial peptide PMAP-36, and the antimicrobial peptide PRW4 was obtained by replacing the 7th and 11th amino acids with tryptophan, and then PRW4 was combined with α-helix The first 15 amino acids of the type antibacterial peptide Fowlicidin-2 were hybridized to obtain the antibacterial peptide PR-FO, and the carboxyl terminal of PR-FO was amidated to increase a positive charge and increase the stability of the peptide. The amino acid sequence of the antimicrobial peptide is shown in Table 1 Show.

[0018] Amino acid sequence and molecular weight of table 1 antimicrobial peptide

[0019]

[0020] Using a peptide synthesizer, the above-mentioned antimicrobial peptides were synthesized by solid-phase synthesis.

Embodiment 2

[0021] Destruction of the bacterial cell membrane of embodiment 2

[0022] 1 Destruction of the outer membrane by antimicrobial peptides

[0023] Escherichia coli was cultured to the logarithmic growth phase, centrifuged and collected, washed with 5 mM HEPES buffer solution (5 mM glucose, pH 7.4) and suspended in the buffer solution. Take 2mL of bacterial solution and put it in a quartz cuvette, and add 1mM NPN solution to mix with it, so that the final concentration of NPN is 10μM. Use F-4500 fluorescence spectrophotometer to detect the fluorescence intensity (excitation 350nm emission 420nm), add different concentrations of antimicrobial peptides, and detect the change of fluorescence intensity. The result is as figure 1 shown. Depend on figure 1 It can be seen that both PRW4 and PR-FO have a certain degree of permeabilization of the outer membrane, but the permeation intensity of PR-FO is stronger, indicating that the destruction effect of antimicrobial peptides on the ...

Embodiment 3

[0026] Bacteriostatic activity and hemolytic activity of embodiment 3 antimicrobial peptides

[0027] 1 Bacteriostatic activity of antimicrobial peptides

[0028] The antimicrobial peptides PRW4, FO and PR-FO were prepared as stock solution with a certain concentration for use. The minimum inhibitory concentrations of several antimicrobial peptides were determined by the broth microdilution method. Using 0.01% acetic acid (containing 0.2% BSA) as the diluent, a series of gradient antimicrobial peptide solutions were sequentially prepared using the double dilution method. Take 100 μL of the above solution and place it in a 96-well cell culture plate, then add an equal volume of the bacteria solution to be tested (~10 5 individual / mL) in each well. Positive controls (containing bacterial fluid but not antimicrobial peptides) and negative controls (neither bacterial fluid nor peptides) were set up. Incubate at a constant temperature of 37°C for 20 hours, and the minimum inhib...

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