CN106589135B - A kind of targeting antibacterial peptide and its preparation method and application - Google Patents

Abstract

The present invention discloses a kind of targeting antibacterial peptide and its preparation method and application, and the sequence of targeting antibacterial peptide cCF10 C4 is as shown in sequence table SEQ ID No.1.Preparation method designs to obtain targeting antibacterial peptide cCF10 C4 using enterococcus faecalis as object bacteria, is constructed by way of heterozygosis a series of to the selective Multidomain antibacterial peptide molecule of enterococcus faecalis.The MOLECULE DESIGN of the series antibacterial peptide includes mainly two independent functional areas, respectively sterilizes region and identification region.The antibacterial peptide has targeting selective power to object bacteria.Targeting antibacterial peptide cCF10 C4 rebuild microecological balance after being conducive to treatment.

Description

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

Technical field

The invention belongs to biotechnologies, 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 immune of body Defense function is first of barrier that body resists pathogenic microorganism invasion.Antibacterial peptide is usually by 12-50 amino acid residue group At molecular weight is less than 10kDa, and common feature is that have amphipathic containing positively charged amino acid and hydrophobic amino acid And cationic, there is killing or inhibiting effect to various bacteria, fungi, virus, parasite even cancer cell.

But it is mostly extensive pedigree antibiotic that antibacterial peptide is identical as conventional antibiotic, is not only generated to microorganism resistance mutation Great selection pressure.Importantly, extensive pedigree antibiotic also killed while killing pathogen it is beneficial just Normal flora causes serious destruction to microecological balance, it is relevant simultaneously to cause the antibiotic such as clinically common secondary infection Send out disease, result in the state of an illness further deteriorate, the serious negative consequences such as extended treatment period.Therefore, one kind is especially needed at present " intelligent " targeting antibacterials selective to pathogen, can be while killing pathogenic bacteria, to micro-ecological environment It causes minimally to destroy, this is beneficial to rebuild microecological balance, and digital preservation is provided for body.

Invention content

Based on the above shortcoming, the purpose of the present invention is to provide a kind of targeting antibacterial peptide cCF10-C4 and its preparation sides Method and application, the antibacterial peptide have 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, sequence such as sequence table SEQ Shown in ID No.1.

The present invention also has following technical characteristic：

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

(1) using broad spectrum antimicrobial peptide C6 as female peptide, the pheromones that there is specific recognition effect to enterococcus faecalis are selected CCF10 connects the two functional areas using GGG as connector as identification region, and design has obtained having object bacteria The antibacterial peptide cCF10-C6 of selectivity；

(2) K in the 16th, the activated centres cCF10-C6 is replaced to reduce net positive charge number with negatively charged E, design obtains 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, the peptide resin that will be obtained After TFA is cut, a polypeptide is obtained；

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

2, a kind of targeting antibacterial peptide cCF10-C4 as described above is preparing the application in targeting antibacterials.

Experimental technique by the antibacterial peptide of this method preparation is simple, and antibacterial and hemolytic activity are carried out to obtained antibacterial peptide Detection, find cCF10-C4 can specific killing enterococcus faecalis, to staphylococcus aureus, staphylococcus epidermis, Escherichia coli, S. pullonum, salmonella typhimurium do not have inhibiting effect, show accurately targeting specific, and with very low Hemolytic activity.In conclusion cCF10-C4 is a kind of targeting antibacterial peptide with higher application value, that is, prove this method pair Antibacterial peptide significant effect is precisely targeted in design.

Description of the drawings

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

Specific implementation mode

Present invention will now be described in further detail with reference to the embodiments and the accompanying drawings, but embodiments of the present invention are unlimited 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, designs to obtain antibacterial peptide cCF10- using enterococcus faecalis (Enterococcus faecalis) as object bacteria 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 includes mainly two independent functional areas, respectively sterilizes region and identification region.

The design of antibacterial peptide

Using broad spectrum antimicrobial peptide C6 as female peptide, the pheromones cCF10 that there is specific recognition effect to enterococcus faecalis is selected 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 activated centres with negatively charged E on this basis reduce it is net just Charge number, to further increase specificity of the targeting antibacterial peptide to object bacteria, design obtains having specificity to object bacteria Target antibacterial peptide cCF10-C4.The amino acid sequence of antibacterial peptide is as shown in table 1.

The amino acid sequence of 1 antibacterial peptide of table

Embodiment 2

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

1, the preparation of antibacterial peptide carries 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 finishes, obtains the resin for sloughing the side chain protection of Fmoc groups；

2, in peptide resin obtained above, cutting reagent is added, 20 DEG C are protected from light lower reaction 2h, filtering；Precipitate 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 precipitate 3h, and white powder object is precipitated, and 10min is centrifuged with 2500g, collect precipitation, then washed and sunk with anhydrous 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 mixing；

3, column equilibration 30min is carried out using 0.2mol/L sodium sulphate (phosphoric acid is adjusted to pH7.5), with 90% acetonitrile solution Polypeptide is dissolved, filtering, C18 reverse phase normal pressure columns, using gradient elution, (eluant, eluent is methanol and aqueous sodium persulfate solution according to volume ratio It is 30:70~70:30 mixing), flow velocity 1mL/min, detection wave is 220nm, collects main peak, freeze-drying；Recycle reverse phase C18 columns It is further purified, eluent A is 0.1%TFA/ aqueous solutions；Eluent B is 0.1%TFA/ acetonitrile solutions, wash-out concentration 25% B~40%B, elution time 12min, flow velocity 1mL/min, then main peak is ibid collected, freeze-drying；

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

Embodiment 3

The active measurement of antibacterial peptide

1, the measurement of antibacterial activity：Antibacterial peptide is configured as certain storing liquid in case using.Utilize micro broth dilution Method measures the minimal inhibitory concentration of several antibacterial peptides.It is dilute using two times using 0.01% acetic acid (containing 0.2%BSA) as dilution Interpretation of the law configures in order the antibacterial peptide solution of graded series.It takes 100 μ L of above-mentioned solution to be placed in 96 porocyte culture plates, then distinguishes The isometric bacterium solution to be measured (~10 of addition⁵A/mL) in each hole.Positive control is respectively set (containing bacterium solution without containing anti- Bacterium peptide) and negative control (be both free of bacterium solution or be free of peptide).37 DEG C of constant temperature incubation 20h, visually to have no that it is muddy existing that hole bottom has Elephant is minimal inhibitory concentration.The results are shown in Table 2.

The bacteriostatic activity of 2 antibacterial peptide of table

It can be seen from Table 2 that pheromones cCF10 does not have bacteriostatic activity, and antibacterial peptide C6 has broad spectrum antibiotic activity, even The bacteriostatic activity that antibacterial peptide can be improved to enterococcus faecalis (E.faecalis) in cCF10 is connect, and to the antibacterial work of other non-target bacterium Property shows to be substantially reduced；When positive changes are reduced to 4 (cCF10-C4), lose to the antibacterial of all non-targeted bacterial strains Ability, but there is no significant change to the bacteriostatic activity of E.faecalis, show accurately targeting specific.

2, the measurement of hemolytic activity：The new blood 1mL of people is acquired, is dissolved into 2mLPBS solution after anticoagulant heparin, 1000g centrifuges 5min, collects red blood cell；It is washed 3 times with PBS, then is resuspended with 10mL PBS；Take 50 μ L red cell suspensions and 50 μ L It is uniformly mixed with the antibacterial peptide solution of the various concentration of PBS dissolvings, the constant-temperature incubation 1h in 37 DEG C of incubators；It is taken out after l h, 4 DEG C, 1000g centrifuge 5min；It takes out supernatant microplate reader and surveys absorbance value at 570nm；Every group is averaged, and compares 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 10% hemolysis rate.The results are shown in Figure 1.As figure can To find out, the antibacterial peptide hemolytic activity compared with female peptide after link information element cCF10 slightly improves, but cCF10-C4 is surveyed in maximum Determine caused hemolysis rate under 256 μM of concentration and be still below 15%, illustrates that the antibacterial peptide has lower cytotoxicity.

The above result shows that the targeting antibacterial peptide cCF10-C4 that designs of the present invention can specific killing enterococcus faecalis, To Escherichia coli, staphylococcus etc. without effect.Lower cytotoxicity is shown simultaneously, has exploitation at the anti-enterococcus faecalis of targeting The potentiality of drug.

110 ＞ Northeast Agricultural Universities of ＜

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

160 ＞ 1 of ＜

210 ＞ 1 of ＜

211 ＞ 24 of ＜

212 ＞ PRT of ＜

213 ＞ artificial sequences of ＜

400 ＞ 1 of ＜

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, which is characterized 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, which is characterized in that method is as follows：

(1) it using broad spectrum antimicrobial peptide C6 as female peptide, selects and the pheromones cCF10 that enterococcus faecalis has specific recognition effect is made 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) K in the 16th, the activated centres cCF10-C6 is replaced with negatively charged E to reduce net positive charge number, design is obtained to mesh Marking 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, obtained peptide resin 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 purifying and Mass Spectrometric Identification, that is, the preparation of polypeptide is completed.

3. a kind of targeting antibacterial peptide cCF10-C4 is preparing answering in targeting anti-enterococcus faecalis drug according to claim 1 With.