CN102827255B

CN102827255B - Antibacterial peptide GW13 and its preparation method and use

Info

Publication number: CN102827255B
Application number: CN201210296758.5A
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: 2014-04-16
Anticipated expiration: 2032-08-09
Also published as: CN102827255A

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 and its preparation method and application

Technical field

The present invention relates to a kind of antibacterial peptide, be specifically related to a kind of antibacterial peptide GW13 and its preparation method and application.

Background technology

Antibacterial peptide is in organism, extensively to have a kind of active polypeptide with anti-microbial effect, is the immunne response product of biological nonspecific defense system, has broad-spectrum antimicrobial and antiviral, antimycotic, parasiticide and the biological activity such as antitumor.The antifungal mechanism of antibacterial peptide is different from microbiotic, it be by physics osmosis in bacterial cell membrane, destroy phospholipid bilayer on film, thereby make material outflow in film, cause bacterium dead, therefore, antibacterial peptide does not produce resistance.Antibacterial peptide itself is a kind of polypeptides matter, does not have residual problem, so the antibacterial peptide optimal surrogate that is microbiotic has very wide market application foreground.At present, thousands of kinds of antibacterial peptides extract from various animal and plant bodies, but as Substitutes For Antibiotic for the antibacterial peptide of medical field seldom.The reason that restriction antibacterial peptide becomes antibiotic substitute has following two aspects: be that antibacterial peptide is for normal cell on the one hand, such as red corpuscle etc. also causes hemolytic action, namely cell selective is low, so, finds a kind of method that improves antibacterial peptide cell selective imperative.Cell selective is the different choice effect of antibacterial peptide to different cells, namely harmful microorganism is had and is suppressed or lethal effect, and normal cell is not had to toxic action.The therapeutic index of antibacterial peptide is to evaluate the critical index of its cell selective.The therapeutic index of antibacterial peptide is larger, and the cell selective of antibacterial peptide is just higher, illustrates that this antibacterial peptide becomes the feasibility of Substitutes For Antibiotic larger.Second aspect that affects antibacterial peptide application be, production cost problem.For the structure function research of antibacterial peptide, mainly the method by chemosynthesis obtains antibacterial peptide at present, and amino acid number is more, and synthetic cost is higher, so the antibacterial little peptide that cell selective is high becomes the focus of people's research.Antibacterial peptide of a great variety, complex structure, is therefore difficult to find unified model to represent that all antibacterial peptides study.In addition, the engineered method of people's use is blindly expressed antibacterial peptide, and the structure and function of antibacterial peptide and in vitro tests is not comprehensively groped, and causes expressing the most activity of antibacterial peptide obtaining not high, or has hemolytic activity and cytotoxicity.So far, there have been a variety of methods to study the relation between the structure and function of antibacterial peptide.Such as screening antibacterial peptide by building the method in antibacterial peptide storehouse, or utilize Method for minimization to obtain only containing the methods such as several amino acid whose brand-new antibacterial peptides.One section of amino-acid residue fragment of the natural antibacterial peptide that the present invention exists by intercepting nature, and its C-terminal characteristic three residue amino acid complex circulations are connected once, design obtains brand-new antibacterial peptide.This three residues amino-acid residue comprises, Arg (arginine; R), Trp (tryptophane; W), Leu (leucine; L).Arg is a kind of basic aminoacids, the positive charge providing 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 are all hydrophobic amino acids, and the phosphatide generation hydrophobicity effect that can show with bacterium destroys the immobilized artificial membrane of bacterium, thereby produce biologic activity.Research finds that the antibacterial potentiality that are rich in Trp and Leu antibacterial peptide are better than other hydrophobic amino acids.The little peptide that yet there are no the C-terminal fragment intercepting that utilizes natural antibacterial peptide both at home and abroad connects report and the research that antibacterial peptide structure-function relationship is studied in (RWL) constitutional features unit repeating.

Summary of the invention

The present invention is in order to obtain anti-microbial activity compared with antibacterial peptide high and that cytotoxicity is relatively low, 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 as follows:

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

2) adopt solid state chemistry synthesis method to obtain peptide resin by Peptide synthesizer; The peptide resin obtaining, after TFA cutting, is obtained to two polypeptide; Then after RPLC purifying and Mass Spectrometric Identification, complete the preparation of polypeptide;

3) minimal inhibitory concentration of antibacterial peptide and hemolytic activity have been carried out to analyzing and testing, and its therapeutic index is calculated, result shows to connect by ArgTrpLeu additivity, has improved 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 of preparing by present method is simple, the antibacterial peptide obtaining is carried out to antibacterial and hemolytic activity detection, 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 obvious restraining effect, and have very low hemolytic activity.Thereby in general, GW13 is a kind of antibacterial peptide with higher using value.

Accompanying drawing explanation

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 β-defensin 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 fixed point linear chicken β-defensin 4 C-terminal of 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 that amino acid quantity is 10 and 13 little peptide.

Embodiment 2:

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

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

In peptide resin obtained above, add cutting reagent, under 20 ℃ of lucifuges, react 2 hours, filter; Precipitation TFA (trifluoroacetic acid) washing, washing lotion is mixed with above-mentioned filtrate, and Rotary Evaporators is concentrated, then adds the precooling anhydrous diethyl ether of 10 times of left and right volumes,-20 ℃ of precipitation 3h, separate out white powder thing, with the centrifugal 10min of 2500g, collecting precipitation, use again anhydrous diethyl ether washing precipitation, 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, with 90% acetonitrile solution, dissolve polypeptide, filter, the anti-phase normal pressure post of C18, adopt gradient elution (eluent is that methyl alcohol and aqueous sodium persulfate solution are to mix for 30: 70～70: 30 according to volume ratio), flow velocity is 1ml/min, and detection ripple is 220nm, collect main peak, freeze-drying; Recycle anti-phase C18 post and be further purified, elutriant A is the 0.1%TFA/ aqueous solution; Elutriant B is 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, more the same collection main peak, freeze-drying;

The evaluation of antibacterial peptide: through electron spray mass spectrometry analysis, the molecular weight showing in mass spectrum (seeing accompanying drawing 1,2) is basically identical with the theoretical molecular in table one by antibacterial peptide obtained above, and the purity of antibacterial peptide is greater than 95%.

The design of table 1 antibacterial peptide

Embodiment 3:

To designing and synthesizing the antibacterial peptide obtaining, by In Vitro Bacteriostasis and hemolytic activity test, compare detection;

The mensuration of anti-microbial activity: peptide is configured as to certain storage liquid in order to using.Utilize the minimal inhibitory concentration of several antibacterial peptides of micro-broth dilution method.Using 0.01% acetic acid (containing 0.2%BSA) as diluent, use doubling dilution to configure successively the antibacterial peptide solution of serial gradient.Get above-mentioned solution 100 μ l and be placed in 96 porocyte culture plates, then add respectively isopyknic bacterium liquid (～10 to be measured ⁵individual/ml) in each hole.Positive control (contain bacterium liquid and do not contain antibacterial peptide) and negative control (neither containing bacterium liquid also containing peptide) are set respectively.37 ℃ of constant temperature culture 20h, have no with naked eyes the minimal inhibitory concentration that is that research of chaotic phenomenon is arranged at bottom, hole.

Detected result is in Table 2.By table 2, can find out, GW13 shows stronger bacteriostatic activity for Gram-negative and positive bacteria, and GW10 does not almost detect bacteriostatic activity in sensing range, illustrate by 3 amino-acid residue additivities of C-terminal and connect the bacteriostatic activity that has greatly improved antibacterial peptide.

Antibacterial and the hemolytic activity of table 2 antibacterial peptide

The mensuration of hemolytic activity: gather people's fresh blood 1mL, be dissolved in 2mlPBS solution after anticoagulant heparin, the centrifugal 5min of 1000g, collects red corpuscle; With PBS washing 3 times, then use 10ml PBS resuspended; Get 50 μ L red cell suspensions and mix with the antibacterial peptide solution of the different concns of PBS dissolving with 50 μ L, constant-temperature incubation 1h in 37 ℃ of incubators; After lh, take out 4 ℃, the centrifugal 5min of 1000g; Taking out supernatant liquor uses microplate reader 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 while causing 10% hemolysis rate.Detected result is in Table 2.Hemolytic concentration is larger, shows that hemolytic activity is less; By table 2, can find out, GW10 and GW13 all do not have hemolytic activity in sensing range.

Above result demonstration, along with the binary stack of ArgTrpLeu elementary cell, anti-microbial activity significantly improves, and hemolytic activity does not change.The comprehensive antibacterial and hemolytic activity of analyzing antibacterial peptide, can more fully evaluate by therapeutic index (ratio of hemolytic concentration and Mlc) cell selective of each antibacterial peptide.As can be seen from Table 2, GW13 has higher therapeutic index, shows to design the development potentiality that the GW13 antibacterial peptide obtaining has higher substitute antibiotics.

Claims

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

GlyLeuArgProLysTyrSer(ArgTrpLeu) ₂-NH ₂。

2. prepare a preparation method of antibacterial peptide GW13 as claimed in claim 1, it is characterized in that, method is as follows:

1) according to pinpoint amino acid fragment intercept method, intercept 13 amino acid fragments of linear chicken β-defensin 4 C-terminal, and remove disulfide linkage, obtain GW10:GlyLeuArgProLysTyrSerArgTrpLeu fragment, typical segments ArgTrpLeu is repeated to connect, obtain GW13:GlyLeuArgProLysTyrSer (ArgTrpLeu) ₂-NH ₂: antibacterial peptide;

2) adopt solid state chemistry synthesis method to obtain peptide resin by Peptide synthesizer; The peptide resin obtaining, after TFA cutting, is obtained to two polypeptide; Then after RPLC purifying and Mass Spectrometric Identification, complete the preparation of polypeptide.

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