CN109705195A

CN109705195A - A kind of Escherichia coli targeting antibacterial peptide KI-QK and preparation method and application

Info

Publication number: CN109705195A
Application number: CN201910095960.3A
Authority: CN
Inventors: 单安山; 谭鹏; 邵长轩; 来振衡; 朱永杰
Original assignee: Northeast Agricultural University
Current assignee: Northeast Agricultural University
Priority date: 2019-01-31
Filing date: 2019-01-31
Publication date: 2019-05-03
Anticipated expiration: 2039-01-31
Also published as: CN109705195B

Abstract

The present invention provides the design method of Escherichia coli targeting antibacterial peptide KI-QK a kind of, and sequence is as shown in sequence table SEQ ID No.1.Using the peptide KI of a α-helixstructure, in its C-terminal link peptide QK, meanwhile, it has used three flexible amino acid glycine as connector between KI and QK, this peptide is named as KI-QK.In biological activity test, we have found that KI-QK has very strong bacteriostatic activity to Escherichia coli, the geometric mean to Escherichia coli minimal inhibitory concentration is 3.667, improves 4.2 times compared to former peptide KI, therapeutic index is 69.812, improves 4.7 times compared to former peptide.In conclusion KI-QK is a kind of typical targeting antibacterial peptide, there is strong targeting, application value with higher to Escherichia coli.

Description

A kind of Escherichia coli targeting antibacterial peptide KI-QK and preparation method and application

Technical field

The invention belongs to field of biotechnology, and in particular to a kind of Escherichia coli targeting antibacterial peptide KI-QK and preparation method And application.

Background technique

Currently, the excessive use of antibiotic, causes pathogenic microorganism to generate drug resistance, or even cause the medicine in animal products Object residual, seriously affects the health status, product quality and its safety of livestock and poultry, to threaten the health of the mankind.Antibacterial Peptide (Antimicrobial peptides, AMPs), the important component of organism natural immune defence system constitutes people The immune the first line of defence of body.The biological characteristics of antibacterial peptide are wherein with broad spectrum antibacterial.Most antibacterial peptides can be with Kill the various bacteria including Gram-negative bacteria and gram-positive bacteria.However, division bacteria beneficial bacterium and pathogenic bacteria, Intracorporal beneficial bacterium is such as: Bacillus acidi lactici, middenstead coccus.The normal flora on human mucosa surface is to nutrient absorption and protection body It resists external microorganism and plays key effect, and current antibacterial peptide is because have broad-spectrum bactericidal action, it is with understanding non-selectivity right Beneficial bacterium and pathogenic bacteria in mucomembranous surface flora have bactericidal effect.The destruction of normal flora can cause two double infection of mucomembranous surface The appearance of dye and patience bacterial strain, this just needs to design a kind of targeting antibacterial peptide, can selectively kill normal mucosa surface flora In pathogenic bacteria and to normal bacteria without effect or effect it is smaller, to keep the ecological balance of normal flora.

Summary of the invention

It is an object of the invention to disclose a kind of Escherichia coli targeting antibacterial peptide KI-QK and preparation method and application, to big Enterobacteria has strong targeting, application value with higher.

The object of the present invention is achieved like this: a kind of Escherichia coli targeting antibacterial peptide KI-QK, sequence such as sequence table Shown in SEQ IDNo.1.

The present invention also has following technical characteristic

1, the preparation method of a kind of Escherichia coli targeting antibacterial peptide KI-QK as described above is as follows:

Using the peptide KI of a α-helixstructure, sequence connects as shown in sequence table SEQ ID No.2 in its C-terminal Meet peptide QK, sequence as shown in sequence table SEQ ID No.3, while used between peptide KI and peptide QK three glycine as Connector；Peptide KI-QK is obtained using solid-state chemical reaction method method, sequence is as shown in sequence table SEQ ID No.1, by reverse phase height After effect liquid phase chromatogram purifying and Mass Spectrometric Identification, the preparation of polypeptide is completed.

2, a kind of Escherichia coli targeting antibacterial peptide KI-QK as described above is in preparation treatment coli-infection disease Application in drug.

The invention has the following advantages and beneficial effects: it is simple by the experimental technique of the antibacterial peptide of this method preparation, it is right Obtained antibacterial peptide carries out antibacterial and hemolytic activity detection, and discovery KI-QK has very strong bacteriostatic activity to Escherichia coli, KI-QK does not show hemolytic activity when concentration is 128 μM, and is better than former peptide KI to the biological activity of Escherichia coli, right The geometric mean of Escherichia coli minimal inhibitory concentration is 3.667, improves 4.2 times compared to former peptide KI, therapeutic index is 69.812,4.7 times are improved compared to former peptide.To Pseudomonas aeruginosa, staphylococcus aureus, Streptococcusagalactiae, mouse typhus sramana Salmonella, staphylococcus epidermis and probiotics lactobacillus plantarum, Lactobacillus rhamnosus have very weak or without bacteriostatic activity.To sum up Described, KI-QK is a kind of typical targeting antibacterial peptide, has strong targeting to Escherichia coli, with higher to apply valence Value.

Detailed description of the invention

Fig. 1 is the mass spectrogram of antibacterial peptide KI；

Fig. 2 is the mass spectrogram of antibacterial peptide QK；

Fig. 3 is the mass spectrogram of antibacterial peptide KI-QK.

Specific embodiment

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

The design of antibacterial peptide

In the present embodiment, we used screened known to one using display technique of bacteriophage and Escherichia coli Peptide QK (103590116 A of CN) with strong affinity, has the α-helixstructure peptide KI of Escherichia coli antibacterial activity with one It is connected, to design a completely new peptide KI-QK to Escherichia coli with strong targeting antibacterial activity.In the KI of former peptide C-terminal connect QK antibacterial peptide, three glycine are then added between KI and QK as connector, using Peptide synthesizer, Above-mentioned antibacterial peptide is synthesized using solid-phase synthesis.The amino acid sequence of antibacterial peptide are as follows:

The sequence of antibacterial peptide is as shown in table 1.

The amino acid sequence of 1 peptide of table

The charge number of KI-QK is+10, hydrophobic value 0.244.The connector that two sections of peptide chains are formed with flexible amino acid GlyGlyGly is connected, to guarantee the biological activity brought into normal play.This method makes peptide have the high antibacterial work of targeting to Escherichia coli Property while have lower hemolytic activity, be able to maintain that microbial balance in body, have as antibiotic substitute hair Open up potentiality.

Embodiment 2

Solid-state chemical reaction method method synthesizes KI-QK

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) it is linked into Wang resin, X-Wang tree is obtained after then sloughing Fmoc group Rouge；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 successively synthesized to N-terminal from C-terminal, until synthesis finishes, obtains the resin for sloughing the side chain protection of Fmoc group；

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 pre-cooling for adding 10 times or so volumes is anhydrous White powder object is precipitated in ether, -20 DEG C of precipitating 3h, is centrifuged 10min with 2500g, collects precipitating, then washed and sunk with anhydrous ether It forms sediment, vacuum drying obtains polypeptide, wherein cutting reagent is 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 column, using gradient elution, (eluant, eluent is methanol and aqueous sodium persulfate solution according to volume ratio For 30:70~70:30 mixing), flow velocity 1mL/min, detection wave is 220nm, collects main peak, freeze-drying；Recycle reverse phase C18 column It is further purified, eluent A is 0.1%TFA/ aqueous solution；Eluent B is 0.1%TFA/ acetonitrile solution, 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, is shown in mass spectrogram Molecular weight (as shown in Figure 1, 2) and the theoretical molecular weight in table 1 are almost the same, and the purity of antibacterial peptide is greater than 95%.

Embodiment 3: the measurement of antibacterial peptide biological activity

1, the measurement of antibacterial activity: the minimal inhibitory concentration of several antibacterial peptides of micro-broth dilution method is utilized.With 0.01% acetic acid (containing 0.2%BSA) is used as dilution, and the antibacterial peptide solution of graded series is configured in order using coubling dilution. It takes above-mentioned 100 μ L of solution to be placed in 96 porocyte culture plates, then adds isometric bacterium solution to be measured (~10 respectively⁶A/mL) In each hole.Positive control (containing bacterium solution without containing antibacterial peptide) is respectively set and negative control (had both been free of bacterium solution or had been free of Peptide).37 DEG C of constant temperature incubation 24-25h measure absorbance value with microplate reader at 492nm (OD492nm), determine minimum antibacterial dense Degree.Testing result is shown in Table 2.

The bacteriostatic activity of 2 antibacterial peptide of table

It can be seen from Table 2 that KI-QK compared with former peptide KI, significantly improves the targeting antibacterial activity of Escherichia coli.

2, the measurement of hemolytic activity: acquiring the new blood 1mL of people, be dissolved into 2mLPBS solution after anticoagulant heparin, 1000g is centrifuged 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 dissolution, the constant-temperature incubation 1h in 37 DEG C of incubators；It is taken out after lh, 4 DEG C, 1000g be centrifuged 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 5% hemolysis rate.Testing result is shown in Table 3.

The measurement of 3 antibacterial peptide hemolytic activity of table

It can be seen from Table 3 that KI-QK does not show hemolytic activity, and the life to Escherichia coli when concentration is 128 μM Object activity is better than former peptide KI, and therapeutic index 69.812 improves 4.7 times than former peptide KI.

The above results show that connecting Phage Display Peptide in targeting antibacterial peptide end, Targeting Effect can be significantly improved, together When, by analyzing the antibacterial and hemolytic activity of antibacterial peptide, therapeutic index (hemolytic concentration and Mlc geometric average can be passed through Several ratio) more fully evaluate the biological activity of each antibacterial peptide, as can be seen from Table 3, KI-QK is with higher to be controlled Index is treated, to sum up shows that antibacterial peptide KI-QK has the potentiality of substitute antibiotics as a kind of targeting antibacterial peptide of Escherichia coli.

Sequence table

<110>Northeast Agricultural University

<120>a kind of Escherichia coli targeting antibacterial peptide KI-QK and preparation method and application

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 27

<212> PRT

<213> Artificial sequence

<400> 1

Trp Lys Lys Ile Trp Lys Pro Gly Ile Lys Lys Trp Ile Lys Gly Gly

1 5 10 15

Gly Gln Lys Arg Pro Arg Val Arg Leu Ser Ala

20 25

<210> 2

<211> 14

<212> PRT

<213> Artificial sequence

<400> 2

Trp Lys Lys Ile Trp Lys Pro Gly Ile Lys Lys Trp Ile Lys

1 5 10

<210> 3

<211> 10

<212> PRT

<213> Artificial sequence

<400> 3

Gln Lys Arg Pro Arg Val Arg Leu Ser Ala

1 5 10

Claims

1. a kind of Escherichia coli target antibacterial peptide KI-QK, which is characterized in that its sequence is as shown in sequence table SEQ ID No.1.

2. the preparation method of Escherichia coli targeting antibacterial peptide KI-QK according to claim 1 a kind of, which is characterized in that side Method is as follows: using the peptide KI of a α-helixstructure, peptide KI sequence is connected as shown in sequence table SEQ ID No.2 in its C-terminal Peptide QK, peptide QK sequence as shown in sequence table SEQ ID No.3, while used between peptide KI and peptide QK three glycine as Connector；Peptide KI-QK is obtained using solid-state chemical reaction method method, sequence is as shown in sequence table SEQ ID No.1, by reverse phase height After effect liquid phase chromatogram purifying and Mass Spectrometric Identification, the preparation of polypeptide is completed.

3. a kind of Escherichia coli targeting antibacterial peptide KI-QK according to claim 1 treats coli-infection in preparation Application in the drug of disease.