CN106432513A

CN106432513A - Efficient hybrid antibacterial peptide LI and preparation method and application thereof

Info

Publication number: CN106432513A
Application number: CN201611075909.9A
Authority: CN
Inventors: 董娜; 刘春玉; 李欣然
Original assignee: Northeast Agricultural University
Current assignee: Northeast Agricultural University
Priority date: 2016-11-29
Filing date: 2016-11-29
Publication date: 2017-02-22
Anticipated expiration: 2036-11-29
Also published as: CN106432513B

Abstract

The invention relates to efficient hybrid antibacterial peptide LI and a preparation method and application thereof. A sequence of the efficient hybrid antibacterial peptide LI is shown as SEQ ID No.1 of a sequence table. The preparation method includes following steps: according to a fixed-point amino acid fragment intercepting method, intercepting an amino acid residue fragment from the 14th locus to the 21st locus of human-derived antibacterial peptide LL37 and an amino acid residue fragment from the 5th locus to the 13th locus of bovine-derived antibacterial Indolicidin, and sequentially connecting the two fragments to obtain the antibacterial peptide LI; 2), using a polypeptide synthesizer for solid-phase synthesis to synthesize peptide resin; obtaining polypeptide after TFA cutting, and adopting reverse-phase high-performance liquid chromatography for purifying. The antibacterial peptide LI obtained by the method has high antibacterial activity, weak hemolytic activity, high treatment index and huge development potential.

Description

A kind of efficiently hybridization antibacterial peptide LI and its preparation method and application

Technical field

The present invention relates to a kind of efficiently hybridize antibacterial peptide LI and its preparation method and application.

Background technology

Antibacterial peptide is the first line of defence that animal resists foreign pathogens.It not only has the function of killing pathogen, also Can be with biological functions such as antifungal, virus, parasite and inhibiting tumor cell.Antibacterial peptide has the bactericidal mechanism of uniqueness so as to become For the most promising antibiotic substitute.Antibacterial peptide molecule has positively charged aminoacid, such as arginine, lysine and group ammonia Acid and there is the hydrophobic amino acid of hydrophobic residue, such as leucine, isoleucine, L-Valine, tryptophan, Phenylalanine, Alanine etc..The mutual electrostatic attraction with negatively charged bacterial cell membrane of antibacterial peptide with quiet positive charge, antibacterial peptide is near antibacterial Cell, hydrophobic residue probes into the lipid bilayer of somatic cells, makes somatic cells film form micropore or cavernous structure, causes Intracellular matter outflows, thus leading to bacterial death.Antibacterial peptide is hindered to be applied to medical science, cosmetics, food and herding etc. at present , there are following two aspects the reason industry：First, the toxicity of antibacterial peptide.Natural antibacterial peptide has higher cytotoxicity.Find one The method for designing planting raising antibacterial peptide cell selective is imperative.The cell selective of antibacterial peptide refers to that antibacterial peptide is thin to cause of disease Bacterium has killing function and does not have toxic and side effects to normal animal somatic cell, shows the selectively killing effect to cell.Anti- The therapeutic index of bacterium peptide is the critical index evaluating its cell selective.2nd, the production cost of antibacterial peptide.Current antibacterial peptide Preparation is mainly two methods of chemical method and genetic engineering.Chemical method produces antibacterial peptide symthesis precisely, but yield is few, cost High.Genetic engineering produces the method for antibacterial peptide just in process of the test, the condition of the screening of engineering bacteria and gene expression process Control to be always and restrict the principal element that the method produces antibacterial peptide.Blindness using engineered method express antibacterial peptide, The 26S Proteasome Structure and Function and in vitro tests of antibacterial peptide is not comprehensively groped, lead to express the antibacterial peptide obtaining and mostly live Property is not high, or has the cytotoxicities such as haemolysis.

Antibacterial peptide molecular weight is minimum, and species is various, complex structure.At present, multiple methods have been had to study antibacterial peptide Relation between structure and function, for example, is transformed to natural antibacterial peptide and the research method such as brand-new design antibacterial peptide.

Content of the invention

Based on above weak point, the present invention provides a kind of efficiently hybridization antibacterial peptide LI and its preparation method and application, should Antibacterial activity is high and cytotoxicity is low.

The technology used in the present invention is as follows：A kind of efficiently hybridization antibacterial peptide LI, its sequence such as sequence table SEQ ID No.1 Shown.

The present invention also has following technical characteristic：

1st, a kind of preparation method of efficient hybridization antibacterial peptide LI comprises the steps as above：

1) according to pinpoint amino acid fragment intercept method, intercept people's derived antimicrobial peptide LL37 14-21 amino acids residue and Cattle derived antimicrobial peptide Indolicidin 5-13 amino acid residue segment, above-mentioned two fragment is sequentially connected, and obtains LI antibacterial Peptide；

2) peptide resin is obtained by Peptide synthesizer using solid-state chemical reaction method method；The peptide resin obtaining is cut through TFA After cutting, tentatively obtain polypeptide products；After being then passed through reversed-phase high-performance liquid chromatography purification and Mass Spectrometric Identification, that is, complete this antibacterial peptide Preparation.

2nd, one kind efficiently hybridizes antibacterial peptide LI as above, in preparation treatment gram positive bacteria or gram negative bacteria Application in infectious disease medicament.

The present invention passes through to intercept natural antibacterial peptide LL37 14-21 amino acids residue (GlyLysGluPheLysArgIleVal) and Indolicidin (In13) characteristic amino acid residue segment (LysTrpProTrpTrpProTrpArgArg), hybrid design obtains brand-new antibacterial peptide LI (GlyLysGluPheLysArgIleValLysTrpProTrpTrpProTrpArgArg-NH₂).Antibacterial peptide LL37 is by people source The protein derived α spiral antibacterial peptide of hCAP18, has the biological activitys such as the antibacterium of wide spectrum, funguses, virus.Indolicidin It is derived from a kind of small peptide rich in tryptophan and 13 aminoacid of proline of cattle neutrality grain born of the same parents.Tryptophan is hydrophobicity Aminoacid, there is hydrophobicity effect in the phospholipid that can show with antibacterial, destroy the immobilized artificial membrane of antibacterial, thus producing biologic activity. The two is respectively provided with certain hemolytic cytotoxicity.Yet there are no both at home and abroad and hybridize to study antibacterial peptide structure using the two characteristic fragment With emic report.The experimental technique of the antibacterial peptide prepared by this method is simple, carries out antibacterial to the antibacterial peptide obtaining With hemolytic activity detection, this antibacterial activity is high and cytotoxicity is low, finds LI not only to escherichia coli, bacillus pyocyaneus, chicken sramana Salmonella, Salmonella typhimurium, staphylococcus aureuses, staphylococcus epidermidiss, streptococcus faecalis, bacillus subtilises, eight kinds of bacterium Planting has obvious inhibitory action, and has very low hemolytic activity.Thus, in general, LI is that one kind has higher application The antibacterial peptide being worth.

Brief description

Fig. 1 is the three-dimensional prediction figure of the antibacterial peptide that design obtains；

Fig. 2 is the mass spectrum of the antibacterial peptide that design obtains.

Specific embodiment

Below according to Figure of description citing, the present invention will be further described：

Embodiment 1

The aminoacid sequence of people derived antimicrobial peptide LL37 is：

The aminoacid sequence of cattle derived antimicrobial peptide In13 is：

Ile Leu Pro Trp Lys Trp Pro Trp Trp Pro Trp Arg Arg-NH2

1 5 10 13

According to pinpoint amino acid fragment intercept method, intercept people's derived antimicrobial peptide LL37 14-21 amino acids residue And cattle derived antimicrobial peptide In13 5-13 amino acid residue segment (GlyLysGluPheLysArgIleVal) (LysTrpProTrpTrpProTrpArgArg).

Above-mentioned two fragment is sequentially connected, obtains LI (GlyLysGluPheLysArgIleValLysTrpProTrpTrp ProTrpArgArg-NH₂) antibacterial peptide.

Embodiment 2

Above-mentioned two antibacterial peptide is synthesized using Peptide synthesizer, method is solid-state chemical reaction method method, concrete steps For：

1) preparation of antibacterial peptide is carried out from C-terminal to N-terminal one by one, is completed by Peptide synthesizer.First by Fmoc-X (X It is first aminoacid of C-terminal of each antibacterial peptide) it is linked into Wang resin, obtain X-Wang tree after then sloughing Fmoc group 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 finishes, obtains sloughing the resin of the side chain protected of Fmoc group；

In peptide resin obtained above, add cutting reagent, react 2 hours under 20 DEG C of lucifuges, filter；Precipitation TFA (three Fluoroethanoic acid) washing, washing liquid is mixed with above-mentioned filtrate, Rotary Evaporators concentrate, the pre-cooling adding 10 times about volumes is anhydrous Ether, -20 DEG C precipitation 3h, separate out white powder thing, with 2500g be centrifuged 10min, collect precipitation, then wash with absolute ether sink Form sediment, vacuum drying, obtain polypeptide, wherein cutting reagent by TFA, water and TIS (tri isopropyl chlorosilane) according to mass ratio 95: 2.5:2.5 mixing；

Carry out column equilibration 30min using 0.2mol/L sodium sulfate (phosphoric acid is adjusted to pH7.5), molten with 90% acetonitrile solution Solution polypeptide, filters, C18 anti-phase normal pressure post, and using gradient elution, (eluant is methanol and aqueous sodium persulfate solution according to volume ratio is 30:70～70:30 mixing), flow velocity is 1ml/min, and detection ripple is 220nm, collects main peak, lyophilizing；Anti-phase C18 post is recycled to enter One step purification, eluent A is 0.1%TFA/ aqueous solution；Eluent B is 0.1%TFA/ acetonitrile solution, and wash-out concentration is 25%B ～40%B, elution time is 12min, and flow velocity is 1ml/min, more ibid collects main peak, lyophilizing；

The identification of antibacterial peptide：Antibacterial peptide obtained above is analyzed through electron spray mass spectrometry, in mass spectrum, display divides Son amount (see photo) is basically identical with the theoretical molecular in table one, and the purity of antibacterial peptide is more than 95%.

The design of table 1 antibacterial peptide

Embodiment 3

It is compared detection to designing and synthesizing the antibacterial peptide obtaining by In Vitro Bacteriostasis and Hemolytic activity assays；

The mensure of antibacterial activity：Peptide is configured as certain storing liquid in case using.Using micro-broth dilution method The minimal inhibitory concentration of several antibacterial peptides.Using 0.01% acetic acid (containing 0.2%BSA) as diluent, using doubling dilution according to The antibacterial peptide solution of secondary configuration graded series.Above-mentioned solution 100 μ l is taken to be placed in 96 porocyte culture plates, then add respectively etc. The bacterium solution to be measured (～10 of volume⁵Individual/ml) in each hole.Be respectively provided with positive control (not containing antibacterial peptide containing bacterium solution) and Negative control (had not both contained bacterium solution and had not contained peptide).With naked eyes, 37 DEG C of constant temperature culture 20h, have no that there is being of research of chaotic phenomenon in bottom hole portion Minimal inhibitory concentration.

Testing result is shown in Table 2.It can be seen from Table 2 that, LI shows stronger suppression for Gram-negative and positive bacteria Bacterium activity, its bacteriostatic activity is more than LL37 and In13., illustrates to be carried by the characteristic amino acid residue segment of heterozygosis LL37 and In13 The high bacteriostatic activity of antibacterial peptide.

The antibacterial and hemolytic activity of table 2 antibacterial peptide

The mensure of hemolytic activity：The fresh blood 1mL of collection people, is dissolved into after anticoagulant heparin in 2mlPBS solution, 1000g Centrifugation 5min, collects erythrocyte；Washed with PBS 3 times, more resuspended with 10ml PBS；Take 50 μ L red cell suspensions and 50 μ L PBS The antibacterial peptide solution mix homogeneously of the variable concentrations of dissolving, constant-temperature incubation 1h in 37 DEG C of incubators；Take out after 1h, 4 DEG C, 1000g is centrifuged 5min；Take out supernatant microplate reader light-metering absorption value at 570nm；Average for every group, and comparative analysiss. Wherein 50 μ L erythrocyte add 50 μ l PBS as negative control；50 μ L erythrocyte add 50 μ l 0.1%Tritonx-100 as sun Property comparison.Minimum hemolytic concentration is that antibacterial peptide causes antibacterial peptide concentration during 10% hemolysis rate.

Testing result is shown in Table 2.Hemolytic concentration is bigger, shows that hemolytic activity is less；It can be seen from Table 2 that, LI is in detection In the range of all there is no hemolytic activity, and LL37 and In13 all shows certain hemolytic activity.

Result above shows, intercepting people derived antimicrobial peptide LL37 and two characteristic fragments of cattle derived antimicrobial peptide Indolicidin are simultaneously It is sequentially connected obtained antibacterial peptide LI and there is higher antibacterial activity, and do not have toxicity to hemocyte.Comprehensive analysis antibacterial The antibacterial and hemolytic activity of peptide, more fully can be evaluated each by therapeutic index (ratio of hemolytic concentration and Mlc) The cell selective of individual antibacterial peptide.As can be seen from Table 2, LI has higher therapeutic index, shows to design the LI antibacterial obtaining Peptide has the development potentiality of higher substitute antibiotics.

<110>Northeast Agricultural University

<120>A kind of efficiently hybridization antibacterial peptide LI and its preparation method and application

<160> 1

<210> 1

<211> 17

<212> PRT

<213>Artificial sequence

<400> 1

Gly Lys Glu Phe Lys Arg Ile Val Lys Trp

1 5 10

Pro Trp Trp Pro Trp Arg Arg-NH2

11 15 17

Claims

1. a kind of antibacterial peptide LI that efficiently hybridizes is it is characterised in that its sequence is as shown in sequence table SEQ ID No.1.

2. according to claim require described in 1 a kind of efficiently hybridize antibacterial peptide LI it is characterised in that preparation method include as follows Step：

1) according to pinpoint amino acid fragment intercept method, people's derived antimicrobial peptide LL37 14-21 amino acids residue and Niu Yuan are intercepted Antibacterial peptide Indolicidin 5-13 amino acid residue segment, above-mentioned two fragment is sequentially connected, and obtains LI antibacterial peptide；

2) peptide resin is obtained by Peptide synthesizer using solid-state chemical reaction method method；By the peptide resin obtaining after TFA cutting, Tentatively obtain polypeptide products；After being then passed through reversed-phase high-performance liquid chromatography purification and Mass Spectrometric Identification, that is, complete the system of this antibacterial peptide Standby.

3. a kind of efficiently hybridization antibacterial peptide LI according to claim 1 is it is characterised in that treat Gram-positive in preparation Application in bacterium or gram positive bacterial infection disease medicament.