CN105777889A - Heterozygosis alpha spiral swine antibacterial peptide, and preparation method and application thereof - Google Patents

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

A kind of heterozygosis α screw type pig derived antimicrobial peptide and its preparation method and application

Technical field

This invention belongs to agricultural animal and veterinary application, is specifically related to a kind of heterozygosis α screw type pig source antibacterial Peptide and its preparation method and application.

Background technology

The powerful mean of the disease that antibiotic always human treatment causes because of cause pathogeny imcrobe infection, but with A large amount of uses of conventional antibiotic, the drug resistance of pathogenic bacterium gradually strengthens, and the antibiotic epoch are the most gradually gone to the greatest extent Head.The most best antibiotic the most gradually loses effect, has become receive much concern so finding new antibacterials Focus.Especially recently, about the report of superbacteria, more people have beaten alarm bell.Find a kind of novel Green feed additive, with substitute antibiotics, has become as a urgent task.

Antibacterial peptide is of a great variety, is distributed extremely wide in nature, but is really applied to medicine and herding neck The example in territory is little, and the factor limiting antibacterial peptide application is a lot, wherein antibacterial work for some antibacterial peptide Property low be limit its application major influence factors.Therefore, ensureing normal cell is not produced the premise of toxicity Under, a kind of method finding effective raising antibacterial peptide antibacterial activity is imperative.Hybrid design is a kind of fine Strategy be used for optimize these antibacterial peptide molecules.This method can be good at retaining lives the biology of required fragment Property, and do not affected by the change of a certain parameter.Additionally, due to only retentive activity center, the most this side Method can be significantly shorter peptide chain length, reduces chemosynthesis cost.

Summary of the invention

It is an object of the invention to provide a kind of heterozygosis α screw type pig derived antimicrobial peptide and its preparation method and application. The pig derived antimicrobial peptide transformed is carried out heterozygosis with alpha-helix type antibacterial peptide Fowlicidin-2 by the present invention, thus obtains To brand-new antibacterial peptide, to improve its antibacterial effect.

The technology that the present invention uses is as follows: a kind of heterozygosis α screw type pig derived antimicrobial peptide, its sequence such as sequence table Shown in SEQ No.1.

Invention also has a following technical characteristic: 1, a kind of heterozygosis α screw type pig derived antimicrobial peptide as above Preparation method is as follows:

(1) intercept front 16 amino acid residues of N-end in former peptide PMAP-36 and obtain antibacterial peptide RI16, The aminoacid of the wherein the 7th and the 11st is replaced to tryptophan and obtains antibacterial peptide PRW4, such as sequence table SEQ Shown in No.2；

(2) front 15 aminoacid heterozygosis of PRW4 and alpha-helix type antibacterial peptide Fowlicidin-2 are resisted Bacterium peptide PR-FO, and by the terminal-carboxy amidation of PR-FO to improve a positive charge and to increase stablizing of peptide Property；

(3) use solid-state chemical reaction method method, synthesize above-mentioned antibacterial peptide by Peptide synthesizer.

2, a kind of heterozygosis α screw type pig derived antimicrobial peptide as above is in preparation treatment gram positive bacteria or leather orchid Application in family name's negative microbial infections disease medicament.

The antibacterial peptide of the present invention has higher antibacterial activity, low cytotoxicity, substantially increases cell selective. The method is that antibacterial peptide becomes antibiotic substitute and established feasible theory and practice basis.

Accompanying drawing explanation

Fig. 1 is the antibacterial peptide permeabilization figure to bacterial cell adventitia of the present invention.

Fig. 2 is the antibacterial peptide permeabilization figure to bacterial cell inner membrance of the present invention.

Fig. 3 is the hemolytic activity figure of the antibacterial peptide of the present invention.

Detailed description of the invention

Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention It is not limited to this.

The design of embodiment 1 heterozygosis α screw type pig derived antimicrobial peptide sequence and synthesis

Intercept front 16 amino acid residues of N-end in pig derived antimicrobial peptide PMAP-36 and obtain antibacterial peptide RI16, will Wherein the aminoacid of the 7th and the 11st replaces to tryptophan and obtains antibacterial peptide PRW4, then by PRW4 Yu α-spiral shell Front 15 aminoacid heterozygosis of rotation type antibacterial peptide Fowlicidin-2 obtain antibacterial peptide PR-FO, by the carboxyl of PR-FO Terminus amidated to improve a positive charge and to increase the stability of peptide, the aminoacid sequence of antibacterial peptide such as table 1 institute Show.

The aminoacid sequence of table 1 antibacterial peptide and molecular weight

Use Peptide synthesizer, utilize solid-phase synthesis to synthesize above-mentioned antibacterial peptide.

The destruction of embodiment 2 bacterial cell membrane

The destruction of 1 antibacterial peptide external membrane

Escherichia coli bacterium was cultivated to the logarithm growth stage, is centrifuged and collects thalline, molten by 5mM HEPES buffering Liquid (5mM glucose, pH 7.4) washs and is suspended in this buffer solution.The bacterial solution taking 2mL is put In quartz cuvette, and add 1mM NPN solution and be mixed with so that final concentration of 10 μMs of NPN. Use F-4500 spectrofluorophotometer fluorescence intensity (exciting 350nm to launch 420nm), add difference The antibacterial peptide of concentration, the change of fluorescence intensity.Result is as shown in Figure 1.As seen from Figure 1, PRW4 Produce certain permeabilization with PR-FO all external membranes, but the saturatingization intensity of PR-FO is higher, shows through color ammonia After acid substitutes, the destruction of bacterial cell adventitia is strengthened by antibacterial peptide.

The destruction to inner membrance of 2 antibacterial peptides

Escherichia coli bacterium was cultivated to the logarithm growth stage, is centrifuged and collects thalline, add 5mM HEPES buffering Liquid (20mM glucose, pH 7.4), bacterial sediment is resuspended, add in the bacterial suspension configured The DiSC3 (5) of final concentration of 0.4 μM stores liquid, and 1-1.5h is hatched in the concussion of room temperature lucifuge；Add final concentration KCl for 4M stores liquid, and 15-30min is hatched in the concussion of room temperature lucifuge.The bacterial suspension taking 2mL joins In the quartz cuvette of F-4500 spectrofluorophotometer, add antibacterial peptide to be measured, 622nm excitation wavelength, 670nm launches fluorescence intensity under wavelength, and slit width is 10nm, and fluorescence burst size is 360s.Result As shown in Figure 2.As seen from Figure 2, the fluorescence intensity that antibacterial peptide PR-FO after heterozygosis produces compared with former peptide It is remarkably reinforced, shows that PR-FO is higher to the unpolarizing of bacterial cell plasma membrane, and presentative time concentration depends on The relation of relying.

The bacteriostatic activity of embodiment 3 antibacterial peptide and hemolytic activity

The bacteriostatic activity of 1 antibacterial peptide

Antibacterial peptide PRW4, FO and PR-FO are configured as finite concentration and store liquid in case using.Utilize micro- The minimal inhibitory concentration of amount several antibacterial peptide of broth dilution method determination.With 0.01% acetic acid (containing 0.2%BSA) As diluent, doubling dilution is used to configure the antibacterial peptide solution of graded series successively.Take above-mentioned solution 100 μ L is placed in 96 porocyte culture plates, adds isopyknic bacterium solution to be measured (～10 the most respectively⁵Individual/mL) in In each hole.It is respectively provided with positive control (not containing antibacterial peptide containing bacterium solution) and negative control (both without bacterium Liquid is also without peptide).37 DEG C of constant temperature culture 20h, have no bottom hole that with naked eyes the minimum that is having research of chaotic phenomenon presses down Bacteria concentration.Result is as shown in table 2.From Table 2, it can be seen that the minimal inhibitory concentration of PR-FO is 0.5-2 μM, Demonstrate the strongest suppression Gram-negative and the activity of positive bacteria growth.

The minimal inhibitory concentration (MIC) of the former peptide of table 2 and T1249

Minimal inhibitory concentration (MIC): the Cmin of antibacterial peptide bacteria growing inhibiting, in this concentration perusal less than the growth of antibacterial.

The hemolytic activity of 2 antibacterial peptides

Gathering the fresh blood 1mL of people, be dissolved in 2mL PBS solution after anticoagulant heparin, 1000g is centrifuged 5 Min, collects erythrocyte；Wash 3 times with PBS, more resuspended with 10mLPBS；Take 100 μ L red cell suspensions The antibacterial peptide solution of the variable concentrations dissolved with 100 μ L PBS is mixed homogeneously, and in 37 DEG C of incubators, constant temperature is incubated Taking out after educating 1h, 4 DEG C, 1000g is centrifuged 5min；Take out the light-metering at 540nm of supernatant microplate reader to absorb Value；Often group is averaged, and comparative analysis.Wherein 100 μ LPBS are as negative control；100μL 0.2%Tritonx-100 is as positive control.Result is as shown in Figure 3.

In conjunction with table 2 with Fig. 3 it can be seen that compared with PRW4 the antibacterial activity of PR-FO be remarkably reinforced, with this with Time keep again relatively low hemolytic activity.

The minimum hemolytic concentration that definition is antibacterial peptide of therapeutic index and the geometric mean of minimal bactericidal concentration Ratio.The highest cell selective of therapeutic index is the strongest, and PR-FO has higher cell selective.

Claims (3)

1. a heterozygosis α screw type pig derived antimicrobial peptide PR-FO, it is characterised in that its sequence such as sequence table SEQ Shown in No.1.

A kind of heterozygosis α screw type pig derived antimicrobial peptide PR-FO the most according to claim 1, its feature exists In, preparation method is as follows:

(1) intercept front 16 amino acid residues of N-end in former peptide PMAP-36 and obtain antibacterial peptide RI16, The aminoacid of the wherein the 7th and the 11st is replaced to tryptophan and obtains antibacterial peptide PRW4, such as sequence table SEQ Shown in No.2；

(2) front 15 aminoacid heterozygosis of PRW4 with α screw type antibacterial peptide Fowlicidin-2 are resisted Bacterium peptide PR-FO, and by the terminal-carboxy amidation of PR-FO to improve a positive charge and to increase stablizing of peptide Property；

(3) use solid-state chemical reaction method method, synthesize above-mentioned antibacterial peptide by Peptide synthesizer.

A kind of heterozygosis α screw type pig derived antimicrobial peptide PR-FO the most according to claim 1 is at preparation treatment leather Application in Lan Shi positive bacteria or gram positive bacterial infection disease medicament.