CN102827255A

CN102827255A - Antibacterial peptide GW13 and its preparation method and use

Info

Publication number: CN102827255A
Application number: CN2012102967585A
Authority: CN
Inventors: 单安山; 董娜; 马清泉; 胡婉宁
Original assignee: Northeast Agricultural University
Current assignee: Northeast Agricultural University
Priority date: 2012-08-09
Filing date: 2012-08-09
Publication date: 2012-12-19
Anticipated expiration: 2032-08-09
Also published as: CN102827255B

Abstract

The invention relates to an antibacterial peptide GW13 and its preparation method and use. The antibacterial peptide GW13 has an amino acid sequence of GlyLeuArgProLysTyrSer(ArgTrpLeu)2-NH2. The preparation method comprises the following steps of 1, cutting out 13 amino acid fragments from a linear chicken beta-defensin 4 carboxyl end by a fixed-point amino acid fragment cutting method, removing disulfide bonds to obtain a GW10 (GlyLeuArgProLysTyrSerArgTrpLeu-NH2) fragment, and repeating connecting characteristic three-residue complexes ArgTrpLeu to obtain the antibacterial peptide GW13, and 2, synthesizing a peptide resin in a polypeptide synthesizer by a solid-phase synthesis method, carrying out TFA cutting to obtain a polypeptide, and carrying out reversed-phase high-performance liquid chromatography purification. The antibacterial peptide GW13 obtained by the preparation method has strong bacteriostatic activity, low hemolytic activity, the highest therapeutic index and a large development potential.

Description

A kind of antibacterial peptide GW13

Technical field

The present invention relates to a kind of antibacterial peptide, be specifically related to a kind of antibacterial peptide GW13.

Background technology

Antibacterial peptide is extensively to have a kind of active polypeptide with anti-microbial effect in the organism, is the immunne response product of biological nonspecific defense system, has broad-spectrum antimicrobial and antiviral, antimycotic, parasiticide and biological activity such as antitumor.The antifungal mechanism of antibacterial peptide is different with microbiotic, it be through the physics osmosis in bacterial cell membrane, destroy phospholipid bilayer on the film, thereby make material outflow in the film, cause bacterial death, therefore, antibacterial peptide does not produce resistance.Antibacterial peptide itself is a kind of polypeptides matter, does not have the residual problem, so the antibacterial peptide optimal surrogate that is microbiotic has very wide market application prospect.At present, thousands of kinds of antibacterial peptides extract from various animal and plant bodies, but the antibacterial peptide that is used for medical field as the microbiotic substitute seldom.The reason that the restriction antibacterial peptide becomes antibiotic substitute has following two aspects: be that antibacterial peptide is for normal cell on the one hand; Also cause hemolytic action such as red corpuscle etc.; Just cell selective is low, and so, it is imperative to seek a kind of method that improves the antibacterial peptide cell selective.Cell selective is the different choice property effect of antibacterial peptide to different cells, just harmful microorganism is had and suppresses or lethal effect, and normal cell is not had toxic action.The therapeutic index of antibacterial peptide is to estimate the critical index of its cell selective.The therapeutic index of antibacterial peptide is big more, and the cell selective of antibacterial peptide is just high more, and it is big more to explain that this antibacterial peptide becomes the feasibility of microbiotic substitute.Second aspect that influences the antibacterial peptide application is the production cost problem.For the structure function research of antibacterial peptide, mainly the method through chemosynthesis obtains antibacterial peptide at present, and amino acid number is many more, and synthetic cost is high more, so the antibiotic little peptide that cell selective is high becomes the focus of people's research.Antibacterial peptide of a great variety, therefore complex structure is difficult to find unified model to represent all antibacterial peptides to study.In addition, the engineered method of people's use is blindly expressed antibacterial peptide, and the 26S Proteasome Structure and Function and the in vitro tests of antibacterial peptide is not comprehensively groped, and it is not high to cause expressing the most activity of the antibacterial peptide that obtains, and perhaps has hemolytic activity and cytotoxicity.So far, there have been a variety of methods to study the relation between the 26S Proteasome Structure and Function of antibacterial peptide.Such as method screening antibacterial peptide, or utilize Method for minimization only to be contained the methods such as brand-new antibacterial peptide of several seed amino acids through structure antibacterial peptide storehouse.One section amino-acid residue fragment of the natural antibacterial peptide that the present invention exists through intercepting naturally, and its C-terminal characteristic three residue amino acid complexes circulation connected once, design obtains brand-new antibacterial peptide.This three residues amino-acid residue comprises Arg (l-arginine; R), Trp (tryptophane; W), Leu (leucine; L).Arg is a kind of basic aminoacids, the positive charge that provides can with the negative charge generation electrostatic attraction effect of bacterium surface, and this sucking action is stronger than other two kinds of basic aminoacidss (Lys and His).Trp and Leu all are hydrophobic amino acids, and the phosphatide generation hydrophobicity effect that can show with bacterium destroys the immobilized artificial membrane of bacterium, thereby produce BA.Discover that the antibacterial potentiality that are rich in Trp and Leu antibacterial peptide are better than other hydrophobic amino acids.The little peptide of also not seeing the C-terminal fragment intercepting that utilizes natural antibacterial peptide both at home and abroad connects report and the research that the antibacterial peptide structure-function relationship is studied in multiple (RWL) constitutional features unit.

Summary of the invention

The present invention is in order to obtain higher and the antibacterial peptide that cytotoxicity is relatively low of anti-microbial activity, thereby has designed antibacterial peptide GW13 of a kind of high cell selective and preparation method thereof.

The aminoacid sequence of antibacterial peptide GW13 is: GlyLeuArgProLysTyrSer (ArgTrpLeu) ₂-NH ₂

The preparation method of antibacterial peptide GW13 is following:

1) according to pinpoint amino acid fragment intercept method, 13 amino acid fragments of linear chicken beta-alexin 4 C-terminals of intercepting, and remove disulfide linkage, and obtain the GW10 antibacterial peptide, typical segments ArgTrpLeu is repeated to connect, obtain the GW13 antibacterial peptide.

2) adopt the solid state chemistry synthesis method to obtain peptide resin through Peptide synthesizer; After the peptide resin process TFA cutting that obtains, obtain two polypeptide; Through after RPLC purifying and the mass spectrum evaluation, promptly accomplish the preparation of polypeptide then;

3) minimal inhibitory concentration and the hemolytic activity to antibacterial peptide carried out analyzing and testing, and its therapeutic index is calculated, and the result shows through the ArgTrpLeu additivity and connects, and has improved the antibacterial peptide activity and the therapeutic index of peptide; GW13 has higher cell selective, has the development potentiality that becomes antibiotic substitute.

The experimental technique of the antibacterial peptide through present method preparation is simple; Carry out antibiotic and the hemolytic activity detection to the antibacterial peptide that obtains; Find that GW13 is not only to intestinal bacteria, Pseudomonas aeruginosa, streptococcus aureus, staphylococcus epidermidis, Salmonella typhimurium, Salmonella gallinarum; Subtilis, eight kinds of bacterial classifications of streptococcus faecium have the obvious suppression effect, and have very low hemolytic activity.Thereby in general, GW13 is a kind of antibacterial peptide with higher using value.

Description of drawings

Fig. 1 is the mass spectrum of antibacterial peptide GW10;

Fig. 2 is the mass spectrum of antibacterial peptide GW13.

Embodiment

Embodiment 1:

The aminoacid sequence of linear chicken beta-alexin 4 is:

Arg Tyr His Met Gln Cys Gly Tyr Arg Gly Thr Phe Cys Thr Pro

1 5 10 15

Gly Lys Cys Pro Tyr Gly Asn Ala Tyr Leu Gly Leu Cys Arg Pro ；

20 25 30

Lys Tyr Ser Cys Cys Arg Trp Leu-NH2

35 38

13 amino acid fragments of linear chicken beta-alexin 4 C-terminals of fixed point intercepting, and remove disulfide linkage, obtain GW10 (GlyLeuArgProLysTyrSerArgTrpLeu-NH ₂) fragment.

Typical segments ArgTrpLeu is repeated to connect, obtain GW13 (GlyLeuArgProLysTyrSer (ArgTrpLeu) ₂-NH ₂) antibacterial peptide.GW10 and GW13 are 10 and 13 little peptide for amino acid quantity.

Embodiment 2:

Use Peptide synthesizer to synthesize above-mentioned two antibacterial peptides, method is the solid state chemistry synthesis method, and concrete steps are:

1) preparation of antibacterial peptide holds the N end to carry out one by one from C, accomplishes through Peptide synthesizer.At first Fmoc-X (X is first amino acid of C end of each antibacterial peptide) is linked into the Wang resin, obtains the X-Wang resin after sloughing the Fmoc group then; Again with Fmoc-Y-Trt-OH (9-fluorenes methoxy carboxyl-trimethylammonium-Y, Y are second amino acid of each antibacterial peptide C end); Be synthesized to the N end from C end successively according to this program, finish, obtain sloughing the resin of the side chain protected of Fmoc group until synthetic;

In the above-mentioned peptide resin that obtains, add cutting reagent, reaction is 2 hours under 20 ℃ of lucifuges, filters; Deposition TFA (trifluoroacetic acid) washing mixes washing lotion with above-mentioned filtrating, Rotary Evaporators concentrates; The precooling anhydrous diethyl ether that adds 10 times of left and right sides volumes again ,-20 ℃ of deposition 3h separate out the white powder thing; With the centrifugal 10min of 2500g, collecting precipitation is used the anhydrous diethyl ether washing precipitation again; Vacuum-drying obtains polypeptide, and wherein cutting reagent is mixed according to mass ratio by TFA, water and TIS (tri isopropyl chlorosilane) at 95: 2.5: 2.5;

Use 0.2mol/L sodium sulfate (phosphoric acid is adjusted to pH7.5) to carry out column equilibration 30min,, filter with 90% acetonitrile solution dissolving polypeptide; C18 anti-phase normal pressure post; Adopt gradient elution (eluent is that methyl alcohol and aqueous sodium persulfate solution are mixing in 30: 70～70: 30 according to volume ratio), flow velocity is 1ml/min, and the detection ripple is 220nm; Collect main peak, freeze-drying; Utilize anti-phase C18 post to be further purified again, elutriant A is the 0.1%TFA/ aqueous solution; Elutriant B is the 0.1%TFA/ acetonitrile solution, and wash-out concentration is 25%B～40%B, and elution time is 12min, and flow velocity is 1ml/min, the same again collection main peak, freeze-drying;

The evaluation of antibacterial peptide: with the above-mentioned antibacterial peptide process electron spray mass spectrometry analysis that obtains, the theoretical molecular basically identical in molecular weight that shows in the mass spectrum (seeing accompanying drawing 1,2) and the table one, the purity of antibacterial peptide is greater than 95%.

The design of table 1 antibacterial peptide

Embodiment 3:

Compare detection to designing and synthesizing the antibacterial peptide that obtains through external antibacterial the test with hemolytic activity;

The mensuration of anti-microbial activity: peptide is configured as certain storage liquid in order to using.Utilize the minimal inhibitory concentration of several kinds of antibacterial peptides of micro-broth dilution method.As diluent, use doubling dilution to dispose the antibacterial peptide solution of serial gradient successively with 0.01% acetate (containing 0.2%BSA).Get above-mentioned solution 100 μ l and place 96 porocyte culture plates, add isopyknic bacterium liquid (～10 to be measured then respectively ⁵Individual/ml) in each hole.Positive control (contain bacterium liquid and do not contain antibacterial peptide) and negative control (neither contain bacterium liquid and also do not contain peptide) are set respectively.37 ℃ of constant temperature culture 20h do not see that with naked eyes the minimal inhibitory concentration that is of muddy phenomenon is arranged at the bottom, hole.

Detected result is seen table 2.Can find out through table 2; GW13 shows stronger bacteriostatic activity for Gram-negative and positive bacteria; And GW10 does not almost detect bacteriostatic activity in sensing range, explains through 3 amino-acid residue additivities of C-terminal to connect the bacteriostatic activity that has improved antibacterial peptide greatly.

Antibacterial and the hemolytic activity of table 2 antibacterial peptide

The mensuration of hemolytic activity: gather people's fresh blood 1mL, be dissolved into behind the anticoagulant heparin in the 2mlPBS solution, the centrifugal 5min of 1000g collects red corpuscle; With PBS washing 3 times, use 10ml PBS resuspended again; Get 50 μ L red cell suspensions and 50 μ L and mix with the antibacterial peptide solution of PBS dissolved different concns, constant temperature is hatched 1h in 37 ℃ of incubators; Take out 4 ℃, the centrifugal 5min of 1000g behind the lh; Take out supernatant with ELIASA in 570nm place photometry absorption value; Average for every group, and comparative analysis.Wherein 50 μ L red corpuscle add 50 μ lPBS as negative control; 50 μ L red corpuscle add 50 μ l 0.1%Tritonx-100 as positive control.Minimum hemolytic concentration is the antibacterial peptide concentration of antibacterial peptide when causing 10% hemolysis rate.Detected result is seen table 2.Hemolytic concentration is big more, shows that hemolytic activity is more little; Can find out that through table 2 GW10 and GW13 all do not have hemolytic activity in sensing range.

Above result shows that along with the binary stack of ArgTrpLeu elementary cell, anti-microbial activity significantly improves, and hemolytic activity does not change.Antibacterial and the hemolytic activity of analysis-by-synthesis antibacterial peptide can more comprehensively estimate the cell selective of each antibacterial peptide through therapeutic index (ratio of hemolytic concentration and Mlc).Can find out that by table 2 GW13 has higher therapeutic index, show that the GW13 antibacterial peptide that design obtains has higher alternative development of antibiotics potentiality.

Claims

1. an antibacterial peptide GW13 is characterized in that, aminoacid sequence is:

GlyLeuArgProLysTyrSer(ArgTrpLeu) ₂-NH ₂。

2. one kind prepares the preparation method of antibacterial peptide GW13 according to claim 1, it is characterized in that method is following:

1) according to pinpoint amino acid fragment intercept method; 13 amino acid fragments of linear chicken beta-alexin 4 C-terminals of intercepting; And removal disulfide linkage; Obtain the GW10:GlyLeuArgProLysTyrSerArgTrpLeu fragment, typical segments ArgTrpLeu is repeated to connect, obtain GW13:GlyLeuArgProLysTyrSer (ArgTrpLeu) ₂-NH ₂: antibacterial peptide;

2) adopt the solid state chemistry synthesis method to obtain peptide resin through Peptide synthesizer; After the peptide resin process TFA cutting that obtains, obtain two polypeptide; Through after RPLC purifying and the mass spectrum evaluation, promptly accomplish the preparation of polypeptide then.

3. the application of an a kind of antibacterial peptide GW13 as claimed in claim 1 is characterized in that: use in the medicine of treatment gram-positive microorganism or gram positive bacterial infection.