CN106589135A - Targeting antibacterial peptide and preparation method and application thereof - Google Patents

Abstract

The invention discloses a targeting antimicrobial peptide and a preparation method and application thereof. A sequence of the targeting antimicrobial peptide cCF10-C4 is shown in a sequence table SEQ ID No.1. The preparation method comprises the steps that enterococcus faecalis serves as a target bacterium to obtain the targeting antimicrobial peptide cCF10-C4 through design, a series of multi-structural-domain antimicrobial peptide molecules having selectivity to the enterococcus faecalis are established in a heterozygous mode. The design of a series of antimicrobial peptide molecules mainly includes two independent functional areas, namely a sterilization area and an identification area respectively. The targeting antimicrobial peptide has targeting selection capability to the target bacterium. The targeting antimicrobial peptide cCF10-C4 is conductive to reestablishment of microecological balance after treatment.

Description

A kind of targeting antibacterial peptide and its preparation method and application

Technical field

The invention belongs to biological technical field, and in particular to a kind of targeting antibacterial peptide and its preparation method and application.

Background technology

Antibacterial peptide is the small molecule polypeptide being present in organism natural immune defence system, participates in the immunity of body Defense function, is that body resists first barrier that pathogenic microorganism is attacked.Antibacterial peptide is generally by 12-50 amino acid residue group Into molecular weight is less than 10kDa, and its common feature is containing positively charged aminoacid and hydrophobic amino acid, with amphipathic And cationic, there is killing or inhibiting effect to various bacteria, funguses, virus, parasite even cancerous cell.

But antibacterial peptide is identical with conventional antibiotic mostly to be extensive pedigree antibiotic, not only microorganism resistance mutation is produced Great selection pressure.Importantly, extensive pedigree antibiotic kill pathogen while also kill it is beneficial just Often flora, serious destruction is caused to microecological balance, cause the antibiotic such as clinically common secondary infection it is related and Send out disease, result in the state of an illness further deteriorate, the serious negative consequences such as extended treatment cycle.Therefore, a class is especially needed at present " intelligent " targeting antibacterials selective to pathogen, can be while pathogenic bacterium be killed, to microecological environment Cause minimally to destroy, this is beneficial to rebuild microecological balance, and for body digital preservation is provided.

The content of the invention

Based on above weak point, it is an object of the invention to provide a kind of targeting antibacterial peptide cCF10-C4 and its preparation side Method and application, the antibacterial peptide has targeting selective power to object bacteria.

The purpose of the present invention is realized by following technology：A kind of targeting antibacterial peptide cCF10-C4, its sequence such as sequence table SEQ Shown in ID No.1.

The present invention also has following technical characteristic：

1st, a kind of preparation method of targeting antibacterial peptide cCF10-C4 is as follows：

(1) using broad spectrum antimicrobial peptide C6 as female peptide, from the pheromone that there is specific recognition to act on to enterococcus faecalis CCF10 connects the two functional areas using GGG as connector as identification region, and design has been obtained to be had to object bacteria Selective antibacterial peptide cCF10-C6；

(2) replace the K in the 16th, cCF10-C6 active center with negatively charged E to reduce net positive charge number, design is obtained There is the targeting antibacterial peptide cCF10-C4 of specificity to object bacteria；

(3) cCF10-C4 peptide resins are obtained by Peptide synthesizer using solid-state chemical reaction method method, by the peptide resin for obtaining After TFA cuttings, a polypeptide is obtained；

(4) after reversed-phase high-performance liquid chromatography purification and Mass Spectrometric Identification, that is, the preparation of polypeptide is completed.

2nd, a kind of targeting antibacterial peptide cCF10-C4 as described above, the application in targeting antibacterials are prepared.

The experimental technique of the antibacterial peptide prepared by this method is simple, and the antibacterial peptide to obtaining carries out antibacterial and hemolytic activity Detection, find cCF10-C4 can specific killing enterococcus faecalis, to staphylococcus aureuses, staphylococcus epidermidiss, escherichia coli, S. pullonum, Salmonella typhimurium do not have inhibitory action, show accurately targeting specific, and with very low Hemolytic activity.In sum, cCF10-C4 is a kind of targeting antibacterial peptide with higher using value, that is, prove the method pair In the accurate targeting antibacterial peptide effect is significant of design.

Description of the drawings

Fig. 1 is the hemolytic activity result figure of antibacterial peptide.

Specific embodiment

With reference to embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited In this.

Embodiment 1

Two methods of the present embodiment combining information element labelling technique and optimization positive changes be optimal to object bacteria The effect of specificity, antibacterial peptide cCF10- is obtained using enterococcus faecalis (Enterococcus faecalis) as object bacteria design C4, is constructed a series of to the selective Multidomain antibacterial peptide molecule of enterococcus faecalis by way of heterozygosis.The series The MOLECULE DESIGN of antibacterial peptide mainly includes two independent functional areas, and respectively sterilize region and identification region.

The design of antibacterial peptide

Using broad spectrum antimicrobial peptide C6 as female peptide, from the pheromone cCF10 that there is specific recognition to act on to enterococcus faecalis As identification region, the two functional areas are connected using GGG as connector, design has obtained having selectivity to object bacteria Antibacterial peptide cCF10-C6, replace the K in the 16th, cCF10-C6 active center with negatively charged E on this basis reduce it is net just Charge number, so as to further improve specificity of the targeting antibacterial peptide to object bacteria, design obtains having specificity to object bacteria Targeting antibacterial peptide cCF10-C4.The aminoacid sequence of antibacterial peptide is as shown in table 1.

The aminoacid sequence of the antibacterial peptide of table 1

Embodiment 2

Solid-state chemical reaction method method composite signal element labelling antibacterial peptide

1st, the preparation of antibacterial peptide is carried out one by one from C-terminal to N-terminal, is completed by Peptide synthesizer.First by Fmoc-X (X It is first aminoacid of C-terminal of each antibacterial peptide) Wang resins are linked into, then slough and X-Wang trees are obtained after Fmoc groups Fat；Again by Fmoc-Y-Trt-OH (9- fluorenes methoxy carboxyl-trimethyl-Y, Y is second aminoacid of each antibacterial peptide C-terminal)；According to This program is synthesized to N-terminal from C-terminal successively, until synthesis is finished, the resin of the side chain protected for obtaining sloughing Fmoc groups；

2nd, in peptide resin obtained above, cutting reagent is added, under 20 DEG C of lucifuges 2h is reacted, filtered；Precipitation TFA (three Fluoroethanoic acid) washing, washing liquid is mixed with above-mentioned filtrate, Rotary Evaporators concentration, the pre-cooling for adding 10 times or so volumes is anhydrous Ether, -20 DEG C of precipitation 3h, separates out white powder thing, and 10min is centrifuged with 2500g, collects precipitation, then washs heavy with absolute ether Form sediment, vacuum drying obtains polypeptide, wherein cutting reagent by TFA, water and TIS (tri isopropyl chlorosilane) according to mass ratio 95: 2.5:2.5 mix；

3rd, column equilibration 30min is carried out using 0.2mol/L sodium sulfate (phosphoric acid is adjusted to pH7.5), uses 90% acetonitrile solution Dissolving polypeptide, filters, the anti-phase normal pressure posts of C18, and (eluant is methanol and aqueous sodium persulfate solution according to volume ratio to adopt gradient elution For 30:70～70:30 mixing), flow velocity is 1mL/min, and detection ripple is 220nm, collects main peak, lyophilizing；Recycle anti-phase C18 posts It is further purified, eluent A is 0.1%TFA/ aqueous solutions；Eluent B is 0.1%TFA/ acetonitrile solutions, and wash-out concentration is 25% B～40%B, elution time is 12min, and flow velocity is 1mL/min, then ibid collects main peak, lyophilizing；

4th, the identification of antibacterial peptide：Antibacterial peptide obtained above is analyzed through electron spray mass spectrometry, the purity of antibacterial peptide is big In 95%.

Embodiment 3

The measure of antibacterial peptide activity

1st, the measure of antibacterial activity：Antibacterial peptide is configured as into certain storing liquid in case using.Using micro broth dilution Method determines the minimal inhibitory concentration of several antibacterial peptides.It is dilute using two times using 0.01% acetic acid (containing 0.2%BSA) as diluent Interpretation of the law configures successively the antibacterial peptide solution of graded series.Take the μ L of above-mentioned solution 100 to be placed in 96 porocyte culture plates, then distinguish Add isopyknic bacterium solution to be measured (～10⁵Individual/mL) in each hole.It is respectively provided with positive control (not containing containing bacterium solution anti- Bacterium peptide) and negative control (both without bacterium solution or without peptide).37 DEG C of constant temperature culture 20h, have no that bottom hole portion has muddy existing with naked eyes The as minimal inhibitory concentration of elephant.As a result it is as shown in table 2.

The bacteriostatic activity of the antibacterial peptide of table 2

It can be seen from Table 2 that, pheromone cCF10 does not have bacteriostatic activity, and antibacterial peptide C6 has broad spectrum antibiotic activity, even Meeting cCF10 can improve bacteriostatic activity of the antibacterial peptide to enterococcus faecalis (E.faecalis), and to the antibacterial work of other non-target bacterium Property show substantially reduction；When positive changes are reduced to 4 (cCF10-C4), lose to the antibacterial of all non-targeted bacterial strains Ability, but the bacteriostatic activity to E.faecalis does not have significant change, shows accurately targeting specific.

2nd, the measure of hemolytic activity：The fresh blood 1mL of collection people, during 2mLPBS solution is dissolved into after anticoagulant heparin, 1000g is centrifuged 5min, collects erythrocyte；Washed with PBS 3 times, then it is resuspended with 10mL PBS；Take 50 μ L red cell suspensions and 50 μ L The antibacterial peptide solution mix homogeneously of the variable concentrations dissolved with PBS, the constant-temperature incubation 1h in 37 DEG C of incubators；Take out after l h, 4 DEG C, 1000g centrifugation 5min；Take out supernatant microplate reader light-metering absorption value at 570nm；Average per group, and compare point Analysis.Wherein 50 μ L erythrocyte add 50 μ LPBS as negative control；50 μ L erythrocyte add 50 μ L0.1%Tritonx-100 as sun Property control.Minimum hemolytic concentration is antibacterial peptide concentration when antibacterial peptide causes 10% hemolysis rate.As a result it is as shown in Figure 1.As figure can To find out, the antibacterial peptide hemolytic activity compared with female peptide after link information element cCF10 is slightly improved, but cCF10-C4 is in maximum survey The hemolysis rate for determining to cause under 256 μM of concentration is still below 15%, illustrates that the antibacterial peptide has relatively low cytotoxicity.

Result above shows, the targeting antibacterial peptide cCF10-C4 that present invention design is obtained can specific killing enterococcus faecalis, To escherichia coli, staphylococcuses etc. without effect.Simultaneously relatively low cytotoxicity is shown, with developing into the anti-enterococcus faecalis of targeting The potentiality of medicine.

The ＞ Northeast Agricultural Universities of ＜ 110

A kind of targeting antibacterial peptides of the ＞ of ＜ 120 and its preparation method and application

The ＞ 1 of ＜ 160

The ＞ 1 of ＜ 210

The ＞ 24 of ＜ 211

The ＞ PRT of ＜ 212

The ＞ artificial sequences of ＜ 213

The ＞ 1 of ＜ 400

Leu Val Thr Leu Val Phe Val Gly Gly Gly Trp Lys Trp Lys Trp Glu Asn Gly Lys Trp

1 5 10 15 20

Lys Trp Lys Trp-NH2

21 24

Claims (3)

1. a kind of targeting antibacterial peptide cCF10-C4, it is characterised in that its sequence is as shown in sequence table SEQ ID No.1.

2. a kind of preparation method of targeting antibacterial peptide cCF10-C4, it is characterised in that method is as follows：

(1) using broad spectrum antimicrobial peptide C6 as female peptide, made from the pheromone cCF10 that there is specific recognition to act on to enterococcus faecalis For identification region, the two functional areas are connected using GGG as connector, design has obtained selective to object bacteria Antibacterial peptide cCF10-C6；

(2) replace the K in the 16th, cCF10-C6 active center with negatively charged E to reduce net positive charge number, design is obtained to mesh Mark bacterium has the targeting antibacterial peptide cCF10-C4 of specificity；

(3) cCF10-C4 peptide resins are obtained by Peptide synthesizer using solid-state chemical reaction method method, the peptide resin for obtaining is passed through After TFA cuttings, a polypeptide is obtained, sequence is as shown in sequence table SEQ ID No.1；

(4) after reversed-phase high-performance liquid chromatography purification and Mass Spectrometric Identification, that is, the preparation of polypeptide is completed.

3. applications of a kind of targeting antibacterial peptide cCF10-C4 in targeting antibacterials are prepared according to claim 1.