CN109485701B

CN109485701B - Antibacterial peptide, antibacterial drug and preparation method

Info

Publication number: CN109485701B
Application number: CN201811431760.2A
Authority: CN
Inventors: 刘袆帆; 王琴; 李素芬
Original assignee: Zhongkai University of Agriculture and Engineering
Current assignee: Zhongkai University of Agriculture and Engineering
Priority date: 2018-11-27
Filing date: 2018-11-27
Publication date: 2020-06-16
Anticipated expiration: 2038-11-27
Also published as: CN109485701A

Abstract

The invention discloses an antibacterial peptide, an antibacterial drug and a preparation method thereof, and relates to the technical field of antibacterial peptides. The amino acid sequence of the antibacterial peptide disclosed by the invention is shown in SEQ ID NO.1 or SEQ ID NO. 2. The antibacterial peptide has antibacterial effect on Pseudomonas aeruginosa, Escherichia coli and Candida albicans. The compound can be used for preparing antibacterial drugs, such as drugs for inhibiting pseudomonas aeruginosa, escherichia coli or candida albicans, and has wide application prospects.

Description

Antibacterial peptide, antibacterial drug and preparation method

Technical Field

The invention relates to the technical field of antibacterial peptides, and particularly relates to an antibacterial peptide, an antibacterial drug and preparation methods thereof.

Background

Drug resistance is an urgent threat to the effective prevention and treatment of bacterial infections, necessitating the adoption of alternative antibiotic strategies. Antimicrobial peptides (AMPs) may be promising alternatives to current antibiotics or as adjuvants to improve antibiotic efficacy.

Antimicrobial peptides are basic polypeptides with antimicrobial activity in the body and are an important component of the natural defense mechanism of most organisms against pathogen invasion. The antibacterial peptide has wide inhibiting effect and good application prospect in the fields of agriculture, medicine and the like.

Disclosure of Invention

The invention aims to provide an antibacterial peptide which has an antibacterial effect on pseudomonas aeruginosa, escherichia coli and candida albicans.

The invention also aims to provide an antibacterial drug which takes the antibacterial peptide as an active ingredient and has bacteriostatic effects on pseudomonas aeruginosa, escherichia coli and candida albicans.

The invention also aims to provide a method for preparing the antibacterial peptide, which can prepare the antibacterial peptide and has bacteriostatic effect on pseudomonas aeruginosa, escherichia coli and candida albicans.

The invention is realized by the following steps:

in one aspect, the invention provides an antibacterial peptide, the amino acid sequence of which is shown as SEQ ID NO.1 or SEQ ID NO. 2.

The invention separates, purifies and sequences two peptides from a product fermented by taking dendrobium aphyllum as a raw material and lactobacillus as a strain, and the amino acid sequences of the two peptides are shown as SEQ ID NO.1 and SEQ ID NO. 2. Experiments prove that the two peptides have good bacteriostatic effects on pseudomonas aeruginosa, escherichia coli and candida albicans.

Wherein, the Minimum Inhibitory Concentration (MIC) of the peptide shown in SEQ ID NO.1 to pseudomonas aeruginosa, escherichia coli and candida albicans is respectively as follows: 18.08, 2.26, 1.13 mg/mL.

The peptide shown in SEQ ID NO.2 has the Minimum Inhibitory Concentration (MIC) on pseudomonas aeruginosa, escherichia coli and candida albicans respectively as follows: 4.44, 8.87, 2.22 mg/mL.

The two antibacterial peptides can be used for preparing antibacterial drugs, such as drugs for inhibiting pseudomonas aeruginosa, escherichia coli or candida albicans, and have wide application prospects.

On the other hand, the invention provides the application of the antibacterial peptide in preparing antibacterial drugs.

For example, the antibacterial peptide can be used for preparing medicines for inhibiting pseudomonas aeruginosa, escherichia coli or candida albicans based on the bacteriostatic effect of the antibacterial peptide on pseudomonas aeruginosa, escherichia coli and candida albicans. Can provide wider selection for raw materials for preparing antibacterial drugs.

In another aspect, the present invention provides an antibacterial agent comprising the above antibacterial peptide, and a pharmaceutically acceptable carrier.

Based on the bacteriostatic effect of the antibacterial peptide on pseudomonas aeruginosa, escherichia coli and candida albicans, the antibacterial drug also has the same bacteriostatic effect on pseudomonas aeruginosa, escherichia coli and candida albicans.

In another aspect, the present invention provides a method for preparing the above antimicrobial peptide, comprising: and (2) sequentially adding the following raw materials into a reactor filled with resin to synthesize the polypeptide shown in SEQ ID NO. 1: Fmoc-Tyr (otbu) -OH, Fmoc-Asp (otbu) -OH and Fmoc-Asp (otbu) -OH;

or, adding the following raw materials into a reactor filled with resin in sequence to synthesize the polypeptide shown in SEQ ID NO. 2: Fmoc-Asp (otbu) -OH, Fmoc-Tyr (otbu) -OH and Fmoc-Asp (otbu) -OH.

Further, in some embodiments of the invention, after each addition of starting material, the following operations are performed:

step (a): placing the reactor in a shaking table for reaction;

step (b): piperidine solution was added to the reactor to remove the Fmoc protecting group and washed with DMF.

Further, in some embodiments of the present invention, in the step (a), the temperature of the shaker is controlled to be 29 to 31 ℃ and the reaction time is controlled to be 90 to 150 min.

Further, in some embodiments of the invention, in step (b), the piperidine solution comprises piperidine and DMF.

Further, in some embodiments of the invention, the volume ratio of piperidine to DMF is 1: 4.

Further, in some embodiments of the invention, at the time of the 1 st addition of the feedstock to the reactor, DCM and DIEA are added to the reactor with the added feedstock;

when the raw material was added to the reactor at any of the 2 nd to 4 th times, HOBT and DIC were added to the reactor together with the raw material added at that time.

Further, in some embodiments of the invention, after the 1 st feed to the reactor, the following is performed: adding a methanol solution into the reactor for sealing.

The invention provides a preparation method for preparing the antibacterial peptide by adopting a polypeptide synthesis method, and the preparation method has the characteristics of simple operation, high purity, high recovery rate and the like.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 shows the HPLC detection results of a lyophilized powder sample of DDDY polypeptide synthesized in example 2;

FIG. 2 shows the MS detection results of lyophilized powder samples of DDDY polypeptide synthesized in example 2;

FIG. 3 is an HPLC assay of a lyophilized powder sample of the DYDD polypeptide synthesized in example 3;

fig. 4 is a MS measurement of a lyophilized powder sample of the DYDD polypeptide synthesized in example 3.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The features and properties of the present invention are described in further detail below with reference to examples.

Example 1

Method for preparing antibacterial peptide by lactobacillus fermentation

1. Sample preparation

Cutting leaves and roots of the new stem of the dendrobium aphyllum, and drying the stem to constant weight by using a dryer to obtain the dry stem of the dendrobium aphyllum. Pulverizing into powder with a pulverizer, and sieving with 100 mesh sieve to obtain herba Dendrobii powder.

2. Microbial solid state fermentation

Through the research, Lactobacillus amylolyticus 6 is selected as the microbial solid fermentation strain. MRS broth was prepared at a concentration of 54g/L and sterilized in a sterile kettle (121 ℃, 15 min). The strain stored at-40 deg.C was recovered, 50. mu.l of the thawed cryobacteria was dropped into 25ml of MRS broth, sealed with a sealing strip to avoid contamination, and cultured in a 37 deg.C incubator for 48 hours. And (5) cleaning the recovered lactobacillus by using sterile water. Centrifuging the cultured lactobacillus for 2 times (3000r/min, 10min, normal temperature), pouring out supernatant each time, adding 25ml of sterile water for cleaning, pouring out supernatant, adding 10ml of sterile water, and collecting suspension for later use. 20g of dried Dendrobium aphyllum powder was placed in 4 petri dishes, moistened with distilled water to maintain a water content of 50%, and sterilized in a sterilization pot (121 ℃, 15 min). Then inoculating a sterile sample (10 percent of inoculation amount), uniformly spreading the lactobacillus suspension on the moistened dendrobium aphyllum sample powder, sealing the powder by using a sealing strip to avoid pollution, and placing the powder in an incubator at 37 ℃ for 48 hours.

3. Extraction of dendrobium aphyllum crude polypeptide

After fermentation was complete, the samples were dried in an oven at 60 ℃ and immediately stored in a refrigerator at-20 ℃. Then, the ratio of 1: dissolving 10 of the crude extract in distilled water, stirring for 30min by using a magnetic stirrer, after complete dissolution, centrifuging (3500r/min, 10min) for 3 times by using a centrifugal machine, taking supernatant liquid each time, adding water again for centrifugation, storing at-40 ℃, and freeze-drying by using a freeze-drying machine to obtain a crude dendrobium aphyllum polypeptide sample, and immediately storing in a refrigerator at-20 ℃.

DEAE Sepharose-FF anion exchange chromatography

The ion column can separate the polypeptides according to different charges in the elution process. The crude sample obtained by freeze-drying was dissolved in distilled water at a concentration of 10mg/ml, and the sample was eluted with pure water and 0.05mol/L and 0.15mol/L sodium chloride solutions, respectively. First, 15ml of the sample solution was gradually dropped along the tube wall into the column, and then collection was started, with the number of pump revolutions kept at 26.8, and 60 tubes were collected and absorbance was measured at 220nm using a spectrophotometer. After each group of samples is eluted, the ion column 100 tube is washed by 4mol/L sodium chloride, and then the ion column 100 tube is washed by distilled water. The obtained dendrobium aphyllum polypeptide is named as DPP.

HPLC-MS/MS analysis

High resolution ultra high pressure liquid chromatography was used to characterize the amino acid sequences in the DPP fraction. Taking a proper amount of DPP component, preparing 1mg/ml solution by ultrapure water, and passing through a 0.22um microporous filter membrane. 20ul of the solution was injected into a column (Agilent SB-C18RRHD, 1.8um, 2.1X 50 mm). Gradient elution was carried out using acetonitrile (mobile phase A) and 0.1% formic acid (mobile phase B) as mobile phases. The elution gradient was: 0-2-5-7-8-9-10min, 90-75-75-15-15-90-90% of A. The detection wavelength is 220nm, and the flow rate is 0.2 ml/min. The eluted peptide fragments were sprayed through an electrospray interface to a tandem mass spectrometer (Bruker Daltonik GmbH, Germany). In terms of acquisition parameters, the source type was ESI, positive ion scan was used, and the dry heater was set to 180 ℃. The scanning range is 50-1000 (m/z). Peptide sequences were analyzed using the Proteomic Toolkit software in conjunction with manual calculations.

6. Analysis results

2 peptides were identified, the amino acid sequences of which were: DDDY (SEQ ID NO.1) and DYDD (SEQ ID NO. 2).

Example 2

Synthesizing the polypeptide DDDY according to the sequence of the polypeptide.

1. The weight of each material was calculated from the weight of the target polypeptide.

2. 5g of resin was placed in a 150ml reactor and 50ml DCM was added and soaked for 2 hours

3. The resin was washed with DMF and then drained, and this was repeated four times and the resin was drained.

4. 0.02mol of Fmoc-Tyr (tBu) -OH (the first amino acid at the C-terminus) +10ml of DCM +5ml of DIEA was weighed into a reactor, and the reactor was placed in a shaker at 30 ℃ for 2 hours.

5. Blocked with 50ml of methanol solution (methanol: DIEA: DCM ═ 1: 1: 2) for half an hour, then washed four times with DMF and drained.

6. The Fmoc protecting group was removed by adding 50ml of 20% piperidine solution (piperidine/DMF ═ 1:4) to the reactor. After deprotection was washed four times with DMF and then drained.

7. And detecting a small amount of resin by an indetrione method, wherein the resin has color, which indicates that the deprotection is successful.

8. 0.06ml of Fmoc-Asp (otbu) -OH (the second amino acid at the C-terminus) +20ml of HOBT +10ml of DIC were weighed into a reactor, and the reactor was placed in a shaker at 30 ℃ for 1 hour.

9. And (3) detecting a small amount of resin by using an indantrione method, and if the resin has a color, indicating that condensation is incomplete, and continuing to react. If the resin is colorless, the reaction is complete; after the reaction was complete, the resin was washed four times with DMF and then drained.

10. The Fmoc protecting group on the resin was removed by adding a quantity of 20% piperidine (piperidine/DMF ═ 1:4) to the reactor and shaking on a decolourizing shaker for 20 min. After the protection is removed, washing with DMF for four times, and then draining to detect whether the protection is removed.

11. And detecting a small amount of resin by an indetrione method, wherein the resin has color, which indicates that the deprotection is successful.

12. The amino acids were attached by sequential addition of Fmoc-Asp (otbu) -OH + HOBT + DIC and Fmoc-Asp (otbu) -OH + HOBT + DIC starting materials according to steps 8-11.

13. The polypeptide protecting groups are cleaved off completely with a cleavage reagent and cleaved from the resin for purification.

14. Separating the target peptide fragment from impurities by a High Performance Liquid Chromatography (HPLC), freeze-drying 500mg of the target peptide fragment DDDY into powder, and carrying out QC quality inspection. The results are shown in FIGS. 1 and 2. FIG. 1 shows the HPLC detection results of the lyophilized powder sample of DDDY polypeptide, and FIG. 2 shows the MS detection results of the lyophilized powder sample of DDDY polypeptide.

Example 3

Synthesizing polypeptide DYDD according to the sequence of the polypeptide.

4. 0.02mol of Fmoc-Asp (otbu) -OH (the first amino acid at the C-terminus) +10ml of DCM +5ml of DIEA was weighed into a reactor, and the reactor was placed in a shaker at 30 ℃ for 2 hours.

8. 0.06mol of Fmoc-Asp (otbu) -OH (the second amino acid at the C-terminus) +20ml of HOBT +10ml of DIC were weighed into a reactor, and the reactor was placed in a shaker at 30 ℃ for 1 hour.

12. According to the steps 8-11, 0.06mol of Fmoc-Tyr (otbu) -OH +20ml of HOBT +10ml of DIC and 0.06mol of Fmoc-Asp (otbu) -OH +20ml of HOBT +10ml of DIC as raw materials were added in sequence to link the amino acids.

14. Separating the target peptide segment from impurities by a High Performance Liquid Chromatography (HPLC), freeze-drying the target peptide segment DYDD into powder, and sending the powder for QC quality inspection. The results are shown in FIGS. 3 and 4. Fig. 3 shows HPLC assay results for lyophilized powder samples of DYDD polypeptide, and fig. 4 shows MS assay results for lyophilized powder samples of DYDD.

Examples of the experiments

The antibacterial effect of the antibacterial peptides DDDY and DYDD is reflected by measuring the minimum inhibitory concentration of the antibacterial peptides DDDYDD and DYDD, wherein the minimum inhibitory concentration is the lowest sample concentration at which the growth of bacteria cannot be detected. A double dilution method is adopted, and the specific method is as follows:

1. cultivation of bacterial species

1.1 bacteria

(1) Activating strains: the slant-preserved bacteria (Pseudomonas aeruginosa or Escherichia coli) were inoculated into 200mL of TSB broth and cultured at 37 ℃ for 24 hours on a shaker at 180 rmp.

(2) Obtaining a single colony: the activated bacteria were inoculated into TSA medium (plate) and cultured in an incubator at 37 ℃ for 24 hours.

(3) Culturing a single colony: single colonies were picked up in 4ml of TSB broth and cultured for 14h at 37 ℃ on a 180rmp shaker.

(4) Diluting the bacterial count: 1ml of the single colonies cultured were inoculated into TSB broth diluted 100-fold and cultured for 6 hours at 37 ℃ on a shaker at 180 rmp.

1.2 Yeast (Candida albicans)

(1) Activating strains: the slant-preserved bacteria were inoculated into 200mL of SDB liquid medium and cultured at 35 ℃ for 24 hours on a 180rmp shaker.

(2) Obtaining a single colony: the activated bacteria were inoculated into SDA medium (plate) and cultured in a 35 ℃ incubator for 24 hours.

(3) Culturing a single colony: single colonies were picked up in 4ml SDB broth and incubated for 14h at 35 ℃ on a 180rmp shaker.

(4) Diluting the bacterial count: 1ml of the cultured single colony was inoculated into SDB liquid medium diluted 100-fold and cultured for 6 hours at 35 ℃ on a shaker at 180 rmp.

2. Preparation of samples to be tested

Dissolving a frozen sample, namely the polypeptide prepared in the embodiment, into distilled water to a certain concentration, performing primary filtration by using a 0.22-micron pinhole filter membrane and a pinhole, adding 1ml of the filtered sample into a first centrifuge tube and a second centrifuge tube, adding 1ml of TSB liquid culture medium into the second centrifuge tube, uniformly mixing, sucking 1ml of the filtered sample into the second centrifuge tube, adding into a third centrifuge tube, adding 1ml of TSB liquid culture medium into the third centrifuge tube, uniformly mixing, and so on.

3. Drug sensitivity test

(1) Preparation of inoculumPreparing: bacteria and yeast were diluted to 1x10 with TSB and SDB, respectively⁶cfu/ml (OD600 of 0.1, concentration of fungi of 1.5X 10)⁸cfu/ml)。

(2) ① pouring the prepared solid culture medium into a plate, after the culture medium is solidified, sucking 0.5mL of the bacterial liquid diluted by the culture medium by using a pipette gun, uniformly coating the bacterial liquid on the surface of the solidified solid culture medium by using an applicator, standing for 10 minutes, after the bacterial liquid is completely absorbed by the solid culture medium, putting 3 Oxford cups into the plate by using tweezers, standing for 3-5 minutes, preventing the Oxford cups from sliding when the plate is moved, and adding 100 microliters of sample liquid into each Oxford cup by using the pipette gun.

② setting negative control, adding 100 microliters of TSB culture medium into each oxford cup on the bacteria culture dish, and adding 100 microliters of SDB culture medium into each oxford cup on the yeast culture dish

③ blank control is set, wherein no bacterial liquid is added into the solid culture medium, and the solid culture medium is directly placed in an Oxford cup

(3) Culturing: culturing the culture dish added with the bacterial liquid at 37 ℃ for 12 h; the petri dish with the yeast solution added thereto was incubated at 35 ℃ for 12 h.

(4) And (3) determination: the diameter of the transparent antibacterial ring is accurately measured by a vernier caliper.

Table 1: minimum antibacterial concentration table of antibacterial peptide DDDY to each bacterium

Test bacterial body	DDDY MIC(mg/mL)
		Pseudomonas aeruginosa	18.08
Escherichia coli	2.26
		Candida albicans	1.13

Table 2: minimum inhibitory concentration table of antibacterial peptide DYDD to each bacterium

Test bacterial body	DYDD MIC(mg/mL)
		Pseudomonas aeruginosa	4.44
Escherichia coli	8.87
		Candida albicans	2.22

As can be seen from the above table, the Minimum Inhibitory Concentrations (MIC) of the polypeptide DDDY to Pseudomonas aeruginosa, Escherichia coli and Candida albicans are respectively: 18.08, 2.26, 1.13 mg/mL.

The Minimum Inhibitory Concentrations (MIC) of the polypeptide DYDD on pseudomonas aeruginosa, escherichia coli and candida albicans are respectively as follows: 4.44, 8.87, 2.22 mg/mL.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

SEQUENCE LISTING

<110> agriculture engineering college of Durch Caesar

<120> antibacterial peptide, antibacterial drug and preparation method

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<170>PatentIn version 3.5

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<213> Artificial sequence

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Asp Asp Asp Tyr

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<213> Artificial sequence

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Asp Tyr Asp Asp

1

Claims

1. The application of the antibacterial peptide in preparing the antibacterial drug is characterized in that the amino acid sequence of the antibacterial peptide is shown as SEQ ID No.1 or SEQ ID No.2, and the antibacterial drug is a drug for resisting pseudomonas aeruginosa, escherichia coli or candida albicans.