CN106749531A - Tryptophan slide fastener β hair fastener antibacterial peptides and preparation method thereof and with application - Google Patents

Abstract

The present invention provides a kind of tryptophan slide fastener β hair fastener antibacterial peptides and preparation method and application.Its sequence is as shown in sequence table SEQ ID No.1, preparation method is with tryptophan zipper as template, with reference to the amino acid composition and design feature of antibacterial peptide, interacted as the Stability Analysis of Structures factor by the use of the W W across chain, and charge residue lysine and hydrophobic amino acid tryptophan are arranged in the two-arm of β hair fasteners as repetition binary sequence unit symmetrical cycle, antibacterial peptide WK3 is obtained, with bacteriostatic activity and cell selective higher, therapeutic index is up to 161.27.The antibacterial peptide designed by the method does not need disulfide bond to bind but has high structural stability, improve selectivity of the antibacterial peptide between bacterial cell and mammalian cell, possesses the application potential quality as antibiotic substitute, the antibacterial peptide activity is higher and cytotoxicity is relatively low.

Description

Tryptophan slide fastener β-hair fastener antibacterial peptide and preparation method thereof and with application

Technical field

The invention belongs to biological technical field, and in particular to a kind of tryptophan slide fastener β-hair fastener antibacterial peptide and preparation method thereof And with application.

Background technology

The fast development of feed industry be unable to do without important function and the contribution that antibioticses feed addictive is played.But it is long The excessive use even abuse of antibiotics of phase result in medicament residue and antibody-resistant bacterium worldwide generally to be occurred, to public Health and Environmental security cause directly threat.The features such as antibacterial peptide has antibacterial activity high, noresidue, easily produces, and Its antimicrobial mechanism for being different from conventional antibiotic makes bacterium be not likely to produce drug resistance, therefore possesses and be developed into antimicrobial of new generation The potential quality of thing.Wherein, β-hairpin structure antibacterial peptide because of it there is antibacterial activity higher and cell selective to receive people's Extensive concern.But the research to β-hairpin structure antibacterial peptide still has some deficits at present, its main cause is that small peptide is hardly formed stabilization β-hairpin structure, it usually needs more than one pair of cysteine formed intramolecule disulfide bond be cyclized antibacterial peptide molecule so as to Stablize its secondary structure, be easily contained within by organism this not only adds the synthesis difficulty and production cost of peptide, and disulfide bond The material of mercapto alcohol radical is cracked causes the decline of its BA even to be lost.Therefore, exploitation has structural stability higher Linear β-hair fastener model peptide system for β-hair fastener antibacterial peptide research with application have great importance.

The content of the invention

It is an object of the invention to provide a kind of tryptophan slide fastener β-hair fastener antibacterial peptide and preparation method thereof and with application；Should Antibacterial peptide does not need disulfide bond binding but has the β-hairpin structure of stabilization；The antibacterial peptide activity is higher and cytotoxicity is relative It is relatively low.

The purpose of the present invention is realized by following technology：A kind of tryptophan slide fastener β-hair fastener type antibacterial peptide WK3, sequence such as sequence Shown in list SEQ ID No.1.

The present invention also has following technical characteristic：

1st, a kind of preparation method of tryptophan slide fastener β-hair fastener type antibacterial peptide WK3, comprises the following steps：

(1) with tryptophan zipper as template, with reference to the amino acid composition and design feature of antibacterial peptide, by water transport property Tryptophan W paired being arranged in is allowed to be formed and is interacted so as to steady across the W-W of chain on the non-hydrogen key of hair fastener two-arm formation site Determine β-hairpin structure, the lysine with positive charge is arranged in hydrogen bond and is formed makes antibacterial peptide in cationic, design on site Linear tryptophan slide fastener β-hair fastener antibacterial peptide is obtained：(WK)_n ^DPG(KW)_n-NH₂；As n=3, antibacterial peptide is named as WK3, Sequence is as shown in sequence table SEQ ID No.1；

(2) peptide resin is obtained by Peptide synthesizer using solid-state chemical reaction method method, the peptide resin that will be obtained is cut by TFA After cutting, antibacterial peptide WK3 is obtained；

(3) by after RPLC purifying and Mass Spectrometric Identification, that is, completing the preparation of antibacterial peptide WK3.

2nd, a kind of tryptophan slide fastener β-hair fastener type antibacterial peptide WK3 as described above, prepare treatment gram-positive bacteria or Application in gram positive bacterial infection disease medicament.

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, finds WK3 not only to Escherichia coli, staphylococcus aureus, MRSE, salmonella typhimurium, white diarrhea Five kinds of strains of salmonella have obvious inhibitory action, and with very low hemolytic activity.In sum, WK3 is a kind of tool There is the antibacterial peptide of application value higher.

Brief description of the drawings

Fig. 1 is the molecular structure of antibacterial peptide WK3.

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

The MOLECULE DESIGN of antibacterial peptide

According to the molecular structure of Fig. 1, the paired non-hydrogen keys for being arranged in hair fastener two-arm of the tryptophan W of water transport property are formed into position It is allowed to be formed on point and is interacted so as to stablize β-hairpin structure across the W-W of chain, the lysine K from positive charge is arranged in hydrogen bond Being formed makes antibacterial peptide in cationic on site, so extend the arm lengths of hair fastener two in couples with WK and KW as recurring unit, if Meter has obtained WK series antibacterial peptides：(WK)_n ^DPG(KW)_n-NH₂, n=2,3,4；As n=2, antibacterial peptide is named as WK2；Work as n= When 3, antibacterial peptide is named as WK3, and as n=4, antibacterial peptide is named as WK4.Its amino acid sequence is as shown in table 1.

The amino acid sequence of the derived peptide of table 1

By tri- terminal-carboxy amidations of peptide of WK2, WK3, WK4 improving a positive charge and increase the stability of peptide.

Embodiment 2

Solid-state chemical reaction method method synthesizes tri- antibacterial peptides of WK2, WK3 and WK4

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 amino acid of C-terminal of each antibacterial peptide) Wang resins are linked into, obtain X-Wang trees after then sloughing Fmoc groups Fat；Again by Fmoc-Y-Trt-OH (9- fluorenes methoxy carboxyl-trimethyl-Y, Y is second amino acid 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, 2h is reacted under 20 DEG C of lucifuges, filtered；Precipitation TFA (three Fluoroacetic acid) washing, washing lotion is mixed with above-mentioned filtrate, Rotary Evaporators concentration, the precooling 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 wash 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 sulphate (phosphoric acid is adjusted to pH7.5), uses 90% acetonitrile solution Dissolving polypeptide, filtering, the anti-phase normal pressure posts of C18, (eluant, eluent is methyl alcohol and aqueous sodium persulfate solution according to volume ratio to use gradient elution It is 30:70~70:30 mixing), flow velocity is 1mL/min, and detection ripple is 220nm, collects main peak, is freezed；Recycle anti-phase C18 posts It is further purified, eluent A is the 0.1%TFA/ aqueous solution；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, is freezed；

4th, the identification of antibacterial peptide：Antibacterial peptide obtained above is analyzed by electron spray mass spectrometry, molecular weight and table 1 is surveyed In theoretical molecular it is basically identical, the purity of antibacterial peptide is more than 95%.

Embodiment 3

The measure of antibacterial peptide antibacterial activity

1st, the measure of antibacterial activity：Peptide is configured as certain storing liquid in case using.Surveyed using micro broth dilution method The minimal inhibitory concentration of fixed several antibacterial peptides.Doubling dilution, as dilution, is used using 0.01% acetic acid (containing 0.2%BSA) The antibacterial peptide solution of graded series is configured successively.Take the μ L of above-mentioned solution 100 to be placed in 96 porocyte culture plates, then add respectively Isometric bacterium solution to be measured (~10⁵Individual/mL) in each hole.It is respectively provided with positive control (antibacterial peptide is not contained containing bacterium solution) With negative control (both without bacterium solution or without peptide).37 DEG C of incubated 20h, with naked eyes have no bottom hole portion have research of chaotic phenomenon i.e. It is minimal inhibitory concentration.Testing result is as shown in table 2.

The bacteriostatic activity of the antibacterial peptide of table 2

It can be seen from Table 2 that, WK2, WK3 and WK4 show the suppression of different depths for Gram-negative and positive bacteria Bacterium activity, and WK3 and WK4 bacteriostatic activities are significantly higher than WK2, and illustrating the bacteriostatic activity of antibacterial peptide increasing with cycle-index and carrying It is high.

2nd, the measure of hemolytic activity：The new blood 1mL of people is gathered, is dissolved into after anticoagulant heparin in 2mLPBS solution, 1000g is centrifuged 5min, collects red blood cell；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 of the various concentrations dissolved with PBS is well mixed, the constant-temperature incubation 1h in 37 DEG C of incubators；Taken out after l h, 4 DEG C, 1000g centrifugation 5min；Take out supernatant ELIASA light-metering absorption value at 570nm；Every group is averaged, and is compared point Analysis.Wherein 50 μ L red blood cells add 50 μ LPBS as negative control；50 μ L red blood cells add 50 μ L0.1%Tritonx-100 as sun Property control.Minimum hemolytic concentration is antibacterial peptide concentration when antibacterial peptide causes 5% hemolysis rate.Testing result is as shown in table 3.

The measure of the antibacterial peptide hemolytic activity of table 3

It can be seen from Table 3 that, WK2 and WK3 does not show hemolytic activity in detection range, and WK4 is in 8 μM of concentration The hemolysis rate for causing alreadys exceed 5%, illustrates to be significantly improved with the increase hemolytic activity of peptide chain length.

Result above shows that, as WK or KW increases symmetrical tip length in antibacterial peptide, its antibacterial activity and haemolysis are equal There is different degrees of rising.The antibacterial and hemolytic activity of comprehensive analysis antibacterial peptide, can be by therapeutic index (hemolytic concentration and suppression The ratio of bacteria concentration) more fully evaluate the BA of each antibacterial peptide.As can be seen from Table 3, WK3 has highest Therapeutic index, shows that designing the WK3 antibacterial peptides for obtaining has the development potentiality of substitute antibiotics higher.

<110>Northeast Agricultural University

<120>Tryptophan slide fastener β-hair fastener antibacterial peptide and preparation method thereof and with application

<160> 3

<210> 1

<211> 14

<212> PRT

<213>Artificial sequence

<400> 1

Trp Lys Trp Lys Trp Lys Pro Gly Trp Lys Trp Lys Trp Lys-NH2

1 5 10 14

<210> 2

<211> 10

<212> PRT

<213>Artificial sequence

<400> 2

Trp Lys Trp Lys Pro Gly Trp Lys Trp Lys-NH2

1 5 10

<210> 3

<211> 18

<212> PRT

<213>Artificial sequence

<400> 3

Trp Lys Trp Lys Trp Lys Trp Lys Pro Gly Trp Lys Trp Lys Trp Lys Trp Lys-NH2

1 5 10 15

Claims (3)

1. a kind of tryptophan slide fastener β-hair fastener type antibacterial peptide WK3, it is characterised in that sequence is as shown in sequence table SEQ ID No.1.

2. the preparation method of a kind of tryptophan slide fastener β-hair fastener type antibacterial peptide WK3, it is characterised in that comprise the following steps：

(1) with tryptophan zipper as template, with reference to the amino acid composition and design feature of antibacterial peptide, by the color ammonia of water transport property Sour W it is paired be arranged in the non-hydrogen key of hair fastener two-arm formed be allowed to be formed on site across the W-W of chain interact so as to stablize β- Hairpin structure, the lysine with positive charge is arranged in hydrogen bond and is formed makes antibacterial peptide in cationic on site, design is obtained Linear tryptophan slide fastener β-hair fastener antibacterial peptide：(WK)_n ^DPG(KW)_n-NH₂；As n=3, antibacterial peptide is named as WK3, sequence As shown in sequence table SEQ ID No.1；

(2) peptide resin is obtained by Peptide synthesizer using solid-state chemical reaction method method, the peptide resin that will be obtained cuts by TFA Afterwards, antibacterial peptide WK3 is obtained；

(3) by after RPLC purifying and Mass Spectrometric Identification, that is, completing the preparation of antibacterial peptide WK3.

3. a kind of tryptophan slide fastener β according to claim 1-hair fastener type antibacterial peptide WK3, it is characterised in that controlled in preparation Treat the application in gram-positive bacteria or gram positive bacterial infection disease medicament.