CN109705195B

CN109705195B - Escherichia coli targeted antibacterial peptide KI-QK and preparation method and application thereof

Info

Publication number: CN109705195B
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: 2021-12-14
Anticipated expiration: 2039-01-31
Also published as: CN109705195A

Abstract

The invention provides a design method of escherichia coli targeted antibacterial peptide KI-QK, wherein the sequence of the escherichia coli targeted antibacterial peptide KI-QK is shown in a sequence table SEQ ID No. 1. A peptide KI with an alpha-helix structure is adopted, the C terminal of the peptide KI is connected with a peptide QK, and meanwhile, three flexible amino acid glycines are used as connecting bodies between the KI and the QK, and the peptide is named as KI-QK. In a biological activity test, the KI-QK has very strong antibacterial activity on escherichia coli, the geometric mean of the minimum antibacterial concentration of the KI-QK on the escherichia coli is 3.667, the KI is improved by 4.2 times compared with the original peptide KI, and the treatment index is 69.812, and the KI-QK is improved by 4.7 times compared with the original peptide. In conclusion, the KI-QK is a typical targeted antibacterial peptide, has strong targeting property on escherichia coli, and has high application value.

Description

Escherichia coli targeted antibacterial peptide KI-QK and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to escherichia coli targeted antibacterial peptide KI-QK and a preparation method and application thereof.

Background

At present, the overuse of antibiotics causes drug resistance of pathogenic microorganisms, even causes drug residues in livestock and poultry products, seriously affects the health condition, the product quality and the safety of livestock and poultry, thereby threatening the health of human beings. Antimicrobial peptides (AMPs), an important component of the natural immune defense system of organisms, constitute the first line of defense in human immunity. One of the biological properties of antimicrobial peptides is that they have broad antimicrobial activity. Most antimicrobial peptides can kill a variety of bacteria including gram-negative and gram-positive bacteria. However, bacteria are classified into beneficial and pathogenic bacteria, beneficial bacteria in the body such as: lactobacillus, faecalis, and the like. The normal flora on the surface of human mucosa plays a key role in absorbing nutrition and protecting organisms against foreign microorganisms, and the existing antibacterial peptide has a broad-spectrum bactericidal effect and can indiscriminately sterilize beneficial bacteria and pathogenic bacteria in the flora on the surface of the mucosa. The destruction of normal flora can cause the occurrence of double infection and resistant strains on the mucosal surface, so that a target antibacterial peptide is required to be designed, pathogenic bacteria in the normal flora on the mucosal surface can be selectively killed, and the target antibacterial peptide has no or little effect on the normal bacteria, so that the ecological balance of the normal flora is maintained.

Disclosure of Invention

The invention aims to disclose an escherichia coli targeted antibacterial peptide KI-QK as well as a preparation method and application thereof, which have strong targeting property on escherichia coli and higher application value.

The purpose of the invention is realized as follows: an escherichia coli targeted antibacterial peptide KI-QK, the sequence of which is shown in a sequence table SEQ ID No. 1.

The invention also has the following technical characteristics

1. The preparation method of the escherichia coli targeted antibacterial peptide KI-QK comprises the following steps:

the peptide KI adopts an alpha-helix structure, the sequence of the peptide KI is shown in a sequence table SEQ ID No.2, the C terminal of the peptide KI is connected with the peptide QK, the sequence of the peptide QK is shown in a sequence table SEQ ID No.3, and three glycines are used as a connecting body between the peptide KI and the peptide QK; the peptide KI-QK is obtained by adopting a solid phase chemical synthesis method, the sequence of the peptide KI-QK is shown in a sequence table SEQ ID No.1, and the preparation of the polypeptide is completed after reversed phase high performance liquid chromatography purification and mass spectrum identification.

2. The application of the escherichia coli targeted antibacterial peptide KI-QK in preparing the medicines for treating the escherichia coli infectious diseases is described.

The invention has the following advantages and beneficial effects: the antibacterial peptide prepared by the method has simple experimental technology, and antibacterial and hemolytic activity detection is carried out on the obtained antibacterial peptide, so that the KI-QK has very strong antibacterial activity on escherichia coli, the KI-QK does not show hemolytic activity when the concentration is 128 mu M, the biological activity on the escherichia coli is stronger than that of the original peptide KI, the geometric mean of the minimum antibacterial concentration on the escherichia coli is 3.667, the minimum antibacterial concentration on the escherichia coli is improved by 4.2 times compared with the original peptide KI, and the treatment index is 69.812, and the minimum antibacterial activity on the escherichia coli is improved by 4.7 times compared with the original peptide KI. Has very weak or no bacteriostatic activity on pseudomonas aeruginosa, staphylococcus aureus, streptococcus agalactiae, salmonella typhimurium, staphylococcus epidermidis, probiotics lactobacillus plantarum and lactobacillus rhamnosus. In conclusion, the KI-QK is a typical targeted antibacterial peptide, has strong targeting property on escherichia coli, and has high application value.

Drawings

FIG. 1 is a mass spectrum of an antimicrobial peptide KI;

FIG. 2 is a mass spectrum of antimicrobial peptide QK;

FIG. 3 is a mass spectrum of antimicrobial peptide KI-QK.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.

Example 1

Design of antimicrobial peptides

In this example, a peptide QK (CN 103590116A) having strong affinity with Escherichia coli, which is selected by phage display technology, was linked to a peptide KI having an alpha-helix structure with Escherichia coli antibacterial activity, thereby designing a novel peptide KI-QK having strong target antibacterial activity against Escherichia coli. The antibacterial peptide was synthesized by connecting QK antibacterial peptide to the C-terminal of KI of the propeptide, adding three glycines between KI and QK as linkers, and using a peptide synthesizer to synthesize the antibacterial peptide by a solid phase synthesis method. The amino acid sequence of the antibacterial peptide is as follows:

the sequences of the antimicrobial peptides are shown in table 1.

TABLE 1 amino acid sequence of the peptides

The charge number of KI-QK was +10 and the hydrophobicity was 0.244. The two peptide chains are connected by a connector GlyGlyGly consisting of flexible amino acid to ensure the normal biological activity. The method has the advantages that the peptide has high targeted antibacterial activity on escherichia coli and lower hemolytic activity, can maintain the microbial balance in an organism, and has the development potential of becoming an antibiotic substitute.

Example 2

KI-QK synthesized by solid phase chemical synthesis method

1. The preparation of the antibacterial peptide is carried out one by one from the C end to the N end and is completed by a polypeptide synthesizer. Firstly, Fmoc-X (X is the first amino acid of the C end of each antibacterial peptide) is grafted to Wang resin, and then an Fmoc group is removed to obtain X-Wang resin; then Fmoc-Y-Trt-OH (9-fluorenylmethoxycarbonyl-trimethyl-Y, Y is the second amino acid at the C end of each antibacterial peptide); synthesizing the resin from the C end to the N end in sequence according to the procedure until the synthesis is finished to obtain the resin with the side chain protection of the Fmoc group removed;

2. adding a cutting reagent into the obtained peptide resin, reacting for 2 hours at 20 ℃ in a dark place, and filtering; washing precipitate TFA (trifluoroacetic acid), mixing washing liquor with the filtrate, concentrating by a rotary evaporator, adding precooled anhydrous ether with the volume about 10 times of that of the filtrate, precipitating for 3 hours at the temperature of-20 ℃, separating out white powder, centrifuging for 10min by 2500g, collecting precipitate, washing the precipitate by the anhydrous ether, and drying in vacuum to obtain polypeptide, wherein a cutting reagent is prepared by mixing TFA, water and TIS (triisopropylchlorosilane) according to the mass ratio of 95:2.5: 2.5;

3. performing column equilibrium with 0.2mol/L sodium sulfate (pH is adjusted to 7.5 by phosphoric acid) for 30min, dissolving polypeptide with 90% acetonitrile water solution, filtering, performing C18 reversed-phase normal pressure column, performing gradient elution (eluent is methanol and sodium sulfate water solution are mixed according to a volume ratio of 30: 70-70: 30), the flow rate is 1mL/min, the detection wave is 220nm, collecting main peak, and freeze-drying; further purifying with reverse phase C18 column, wherein eluent A is 0.1% TFA/water solution; eluent B is 0.1% TFA/acetonitrile solution, the elution concentration is 25% B-40% B, the elution time is 12min, the flow rate is 1mL/min, and then the main peak is collected and freeze-dried as above;

4. identification of antibacterial peptides: the obtained antibacterial peptide is analyzed by electrospray mass spectrometry, the molecular weight (shown in figures 1 and 2) shown in a mass spectrogram is basically consistent with the theoretical molecular weight in table 1, and the purity of the antibacterial peptide is more than 95%.

Example 3: determination of biological Activity of antimicrobial peptides

1. Determination of antibacterial Activity: the minimum inhibitory concentrations of several antimicrobial peptides were determined using the broth dilution method. Serial gradients of antimicrobial peptide solutions were prepared in order using a double dilution method with 0.01% acetic acid (containing 0.2% BSA) as the diluent. Taking 100 mu L of the solution, placing the solution into a 96-hole cell culture plate, and then respectively adding the bacterial liquid to be detected (10-10) with the same volume⁶one/mL) in each well. Positive controls (containing the bacterial solution but not the antimicrobial peptide) and negative controls (containing neither the bacterial solution nor the peptide) were set separately. Culturing at 37 deg.C for 24-25h, measuring light absorption value at 492nm (OD492nm) with microplate reader, and determining minimum inhibitory concentration. The results are shown in Table 2.

TABLE 2 bacteriostatic Activity of antimicrobial peptides

As can be seen from Table 2, KI-QK has a significantly improved targeted antibacterial activity against Escherichia coli as compared with the propeptide KI.

2. Determination of hemolytic Activity: collecting 1mL of fresh human blood, dissolving heparin in a 2mLPBS solution after anticoagulation, centrifuging for 5min at 1000g, and collecting erythrocytes; washed 3 times with PBS and resuspended in 10mL PBS; uniformly mixing 50 mu L of erythrocyte suspension with 50 mu L of antibacterial peptide solution dissolved by PBS and having different concentrations, and incubating for 1h at constant temperature in an incubator at 37 ℃; taking out after lh, centrifuging at 4 ℃ for 5min at 1000 g; taking out the supernatant, and measuring the light absorption value at 570nm by using an enzyme-labeling instrument; the average value of each group is taken and compared and analyzed. Wherein 50 μ L red blood cells plus 50 μ LPBS served as negative controls; 50 μ L of red blood cells plus 50 μ L of 0.1% Tritonx-100 served as a positive control. The minimum hemolytic concentration is the concentration of antimicrobial peptide at which antimicrobial peptide causes a 5% hemolytic rate. The results are shown in Table 3.

TABLE 3 determination of the hemolytic Activity of the antimicrobial peptides

As can be seen from Table 3, KI-QK showed no hemolytic activity at a concentration of 128. mu.M, and the biological activity against E.coli was stronger than that of the pro-peptide KI, with a therapeutic index of 69.812, which was 4.7-fold higher than that of the pro-peptide KI.

The results show that the phage display peptide is connected to the tail end of the targeted antibacterial peptide, so that the targeted effect can be remarkably improved, meanwhile, the biological activity of each antibacterial peptide can be comprehensively evaluated through the therapeutic index (the ratio of the hemolytic concentration to the geometric mean of the antibacterial concentration) by analyzing the antibacterial activity and the hemolytic activity of the antibacterial peptide, and the KI-QK has a high therapeutic index as can be seen from the table 3.

Sequence listing

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Claims

1. An escherichia coli targeted antibacterial peptide KI-QK is characterized in that the sequence is shown in a sequence table SEQ ID No. 1.

2. The preparation method of the targeted antibacterial peptide KI-QK for escherichia coli as claimed in claim 1, wherein the method comprises the following steps: the peptide KI is of an alpha-helix structure, the sequence of the peptide KI is shown in a sequence table SEQ ID No.2, the C terminal of the peptide KI is connected with the peptide QK, the sequence of the peptide QK is shown in a sequence table SEQ ID No.3, and three glycines are used as connecting bodies between the peptide KI and the peptide QK; the peptide KI-QK is obtained by adopting a solid phase chemical synthesis method, the sequence of the peptide KI-QK is shown in a sequence table SEQ ID No.1, and the preparation of the polypeptide is completed after reversed phase high performance liquid chromatography purification and mass spectrum identification.

3. The use of the targeted antimicrobial peptide KI-QK for Escherichia coli according to claim 1 in the preparation of a medicament for treating infectious diseases of Escherichia coli.