CN114106197B

CN114106197B - Narrow-spectrum antibacterial peptide and application thereof

Info

Publication number: CN114106197B
Application number: CN202111298440.6A
Authority: CN
Inventors: 王菊芳; 王蒙; 马毅
Original assignee: South China University of Technology SCUT
Current assignee: South China University of Technology SCUT
Priority date: 2021-11-04
Filing date: 2021-11-04
Publication date: 2023-06-16
Anticipated expiration: 2041-11-04
Also published as: CN114106197A

Abstract

The invention belongs to the technical fields of protein engineering and biopharmaceuticals, and discloses a narrow-spectrum antibacterial peptide and application thereof, wherein the amino acid sequence of the narrow-spectrum antibacterial peptide (G2) is shown as SEQ ID No: 1. The application of the narrow-spectrum antibacterial peptide in preparing an antimicrobial infection medicament is that microorganisms are mainly intestinal tracts and food-borne pathogenic bacteria. The results show that the G2 antibacterial peptide has a narrow antibacterial spectrum, has stronger antibacterial activity on clostridium difficile (Clostridium difficile), salmonella typhimurium (Salmonella typhimurium) and Escherichia coli (Escherichia coli) O157, and has good temperature, pH and serum stability; biosafety experiments show that the G2 antibacterial peptide has no obvious hemolytic activity and cytotoxicity.

Description

Narrow-spectrum antibacterial peptide and application thereof

Technical Field

The invention belongs to the technical field of protein engineering and biopharmaceuticals, and particularly relates to an antibacterial peptide and design and application thereof, wherein the antibacterial peptide has a narrow antibacterial spectrum and obvious antibacterial effects on pathogenic bacteria such as clostridium difficile, escherichia coli O157 and salmonella typhimurium.

Background

In recent years, with the advent of multi-drug resistant pathogenic bacteria, most pathogenic bacteria have developed serious drug resistance to antibiotics, resulting in a greatly reduced therapeutic effect of antibiotics, which seriously threatens the life health of human beings. Antibacterial peptide (Antimicrobial peptide, AMP) is a polypeptide secreted by natural organisms and has important immune protection function, has small molecular weight and is generally composed of 10-100 amino acids. Because of the characteristics of unique antibacterial mechanism, difficult generation of drug resistance and the like, the novel antibacterial agent is closely paid attention to by scientists in recent years. To date, scientists have successfully found about 3180 natural antimicrobial peptides from nature (including bacteria, archaea, fungi, protozoa, plants and animals), most of which are extracted from animal endosomes, which exhibit good broad-spectrum antimicrobial activity against g+ and G-bacteria, fungi, viruses and tumor cells, etc. However, natural antibacterial peptides have certain drawbacks such as large differences in antibacterial activity, low stability, toxicity to mammalian cells, and the like, which seriously hamper further studies of antibacterial peptides. In recent years, scientists have adopted various means such as amino acid mutation, from the head design and other strategies to directionally design novel antibacterial peptides with high antibacterial activity, good stability and high biological safety. For example, zhu et al designed the antimicrobial peptide by selecting a series of amino acid sequences that avoid recognition by trypsin and pepsin, and finally screened the obtained II-I4-II antimicrobial peptide not only has good thermal stability, but also shows good stability in protease, salt ion, serum and acid-base solution (Rational Avoidance of Protease Cleavage Sites and Symmetrical End-Tagging Significantly Enhances the Stability and Therapeutic Potential of Antimicrobial peptides. J Med Chem 2020,63 (17): 9421-9435); feng et al studied the effect of tryptophan mutation on cytotoxicity and antibacterial activity of the antibacterial peptide dCATH extracted from duck meat by amino acid mutation, and finally successfully screened to obtain dCATH derivative peptides dCATH (1-16) and dCATH (5-20) with high antibacterial activity and biosafety (The Critical Role of Tryptophan in the Antimicrobial Activity and Cell Toxicity of the Duck Antimicrobial Peptide DCATH.front microbiol.2020 May 28; 11:1146.). However, most of the antibacterial peptides researched and designed at present are broad-spectrum antibacterial peptides, and the antibacterial peptides can simultaneously show good antibacterial activity on various pathogenic bacteria commonly infected in a hospital, such as pseudomonas aeruginosa, staphylococcus aureus, multi-drug resistant acinetobacter baumannii or pneumococcus and the like, but have obvious inhibition effect on most probiotics, and have few reports on narrow-spectrum antibacterial peptides with high specific antibacterial activity on single or few pathogenic bacteria. In recent years, with the increase of serious resistance of most pathogenic bacteria to traditional antibiotics, the population dying from pathogenic bacteria infection tends to rise year by year, especially the pathogenic bacteria such as clostridium difficile (Clostridium difficile, c.difficile), pseudomonas aeruginosa (Pseudomonas aeruginosa, p.aeromonas), salmonella (salmonella), escherichia coli (O157) and the like, which are infected with serious pathogenic bacteria at present. Therefore, the narrow-spectrum antibacterial peptide with high design specificity and capability of correctly identifying normal flora and pathogenic bacteria has important significance for controlling the infection of special pathogenic bacteria.

Disclosure of Invention

The primary aim of the invention is to overcome the defects in the prior art, and design and screen a narrow-spectrum antibacterial peptide, which comprises the following steps: the escherichia coli membrane channel protein CsgG is used as a design template, and the amino acid composition and the tertiary structure characteristics of a CsgG spiral transmembrane region are analyzed, and the amino acid sequence of the PEW300 antibacterial peptide is combined to design the antibacterial peptide G2. And the G2 antibacterial peptide and the self-aggregation peptide ELK16 are subjected to fusion expression through a fusion strategy, so that the high-efficiency expression and purification of the G2 in escherichia coli are realized, and the antibacterial activity, stability and biosafety of the prepared G2 antibacterial peptide are tested. The antibacterial experiment result shows that the antibacterial peptide has remarkable antibacterial activity on clostridium difficile, escherichia coli O157 and salmonella typhimurium; has no remarkable antibacterial activity on other bacteria such as bifidobacterium, staphylococcus aureus, vibrio parahaemolyticus, pseudomonas aeruginosa, bacillus cereus, lactobacillus rhamnosus and lactobacillus.

The invention further aims to provide the application of the antibacterial peptide G2 based on the antibacterial peptide G2 designed as above, which is expressed with high efficiency and is characterized in antibacterial performance.

The aim of the invention is achieved by the following technical scheme:

a narrow-spectrum antibacterial peptide (G2), the amino acid sequence of which is shown in SEQ ID No: 1.

The nucleotide sequence of the gene for encoding the narrow-spectrum antibacterial peptide is shown as SEQ ID No: 2.

The application of the narrow-spectrum antibacterial peptide in preparing the antimicrobial infection medicines.

Preferably, the microorganism is an intestinal tract or a food-borne pathogenic bacterium.

Preferably, the microorganism is clostridium difficile (Clostridium difficile), salmonella typhimurium (Salmonella typhimurium) and Escherichia coli (Escherichia coli) O157.

A preparation method of a narrow-spectrum antibacterial peptide G2 comprises the following steps:

(1) The amino acid sequence and physicochemical property characteristics of the coliform membrane channel protein CsgG and PEW300 antibacterial peptide (the antibacterial peptide is mutant peptide of cecropin A antibacterial peptide) are analyzed. According to the analysis of charge characteristics and hydrophobic characteristics, designing and obtaining an antibacterial peptide G2, and determining the gene sequence according to the amino acid sequence of the G2;

(2) Connecting the antibacterial peptide gene in the step (1) to an expression vector to obtain a recombinant expression vector;

(3) Converting the recombinant expression vector obtained in the step (2) into escherichia coli expression host bacteria to obtain engineering bacteria;

(4) Adding an expression Inducer (IPTG) into the engineering bacteria obtained in the step (3) for induced expression, centrifugally collecting the bacteria, crushing the bacteria, centrifugally obtaining sediment (namely fusion protein active aggregate), adding a cutting inducer (DTT) into the sediment for induced cutting, centrifugally collecting the sediment, adding glacial acetic acid into the sediment for treatment, and centrifugally collecting the supernatant to obtain the G2 antibacterial peptide.

Preferably, the recombinant expression vector of step (2) comprises a G2 antimicrobial peptide fusion protein consisting of: g2 antibacterial peptide-self-cleaving short peptide-connecting peptide-self-aggregating short peptide.

Preferably, the self-shearing short peptide is Mxe gyrA, and the nucleotide sequence of the self-shearing short peptide is shown as SEQ ID No: 3.

Preferably, the self-aggregation short peptide is ELK16, and the nucleotide sequence of the self-aggregation short peptide is shown in SEQ ID No: 4.

Preferably, the connecting peptide is PT linker, and the nucleotide sequence of the connecting peptide is shown as SEQ ID No: shown at 5.

Preferably, the concentration of IPTG in step (4) is 0.1mM; the concentration of DTT was 40mM; the temperature of the induced expression is 16 ℃, and the time of the induced expression is 24 hours; the temperature of the induced cutting is 4 ℃, and the cutting time is 12h.

Preferably, the glacial acetic acid concentration in step (4) is 3% (v/v).

Compared with the prior art, the invention has the following advantages and beneficial effects:

(1) The G2 antibacterial peptide has a narrow antibacterial spectrum, shows remarkable antibacterial activity on clostridium difficile, salmonella typhimurium and escherichia coli O157, and has weak resistance on other indicator bacteria.

(2) The G2 antibacterial peptide has good pH stability, heat stability and serum stability.

(3) The G2 antibacterial peptide has good biological safety: the addition of 250 ng/. Mu.L of G2 antibacterial peptide and the co-incubation of the mouse erythrocytes show no obvious hemolytic activity; different concentrations (10-240 ng/. Mu.L) of the G2 antibacterial peptide were incubated with HEK293 cells, and the results showed that HEK293 cells grew well, indicating that the G2 antibacterial peptide had no significant cytotoxicity.

Drawings

FIG. 1 is a diagram showing structural information of G2 antimicrobial peptides. A: g2 antimicrobial peptide modeling results graph; b: a graph of the results of the Helical wheel of G2 antimicrobial peptides; wherein LWMFIV is a hydrophobic amino acid, K is a positively charged amino acid, AG is a non-polar amino acid, and T and P are uncharged amino acids.

FIG. 2 shows plasmid information of pET30-gme and colony PCR identification result. A: pET30-gme plasmid information map; b: pET30-gme colony PCR identification results, lanes 1-8: 8 single clones selected; m: DNA marker.

FIG. 3 shows graphs of the results of the expression of small amounts of fusion protein G2-ELK 16; wherein: w is the crushed whole liquid sample; s is a crushed supernatant sample; p is the sediment sample after crushing. No is the uninduced group, IPTG is the induced group to which different concentrations of IPTG were added, respectively.

FIG. 4 is a graph showing the results of protein quantification of G2 antimicrobial peptides; s is S ₁ -S ₅ : standard protein (aprotinin, available from Shanghai Ind Chemicals) M at 0.05, 0.1, 0.2, 0.3, 0.4. Mu.g/well, respectively ₁ And M ₂ Is a protein standard molecular weight marker; g2 is the purified G2 antibacterial peptide.

FIG. 5 is a graph showing the results of measurement of the stability of G2 antibacterial peptide. A: a pH stability measurement result graph; b: a temperature stability measurement result graph; c: serum stability assay results.

FIG. 6 G2 biosafety assay results. A: hemolytic activity measurement result diagram, trition X-100 is used as positive control; b: HEK293 cell activity detection result diagram; c: cell observations plot, NC: PBS as negative control, PC: trition X-100 treatment was used as a positive control.

Detailed Description

The present invention will be described in further detail with reference to examples, but embodiments of the present invention are not limited thereto.

The G2 antibacterial peptide designed in the invention is rationally designed based on the amino acid sequence of the escherichia coli membrane channel protein CsgG and PEW300 antibacterial peptide. Wherein the ID of the amino acid sequence of the escherichia coli membrane channel protein CsgG in the NCBI database is CAD6011129; the amino acid sequence of PEW300 antimicrobial peptide was identified in the NCBI database as AZU96665, and the following examples were conducted under conditions described in conventional molecular gram Long Shouce with no specific reference to the experimental method.

Example 1: rational design of antibacterial peptide and screening of G2 antibacterial peptide

(1) Analyzing the amino acid sequence and structural characteristics of the escherichia coli membrane channel protein CsgG, selecting and collecting a transmembrane helix region of the escherichia coli membrane channel protein CsgG, and performing antibacterial performance prediction analysis (comprising charge number, hydrophobicity, isoelectric point, stability and amphipathy) on the peptide segment of the selected transmembrane region by using online analysis software; meanwhile, according to the secondary structure of cecropin mutant PEW300 antibacterial peptide, the charge and the hydrophobicity of the cecropin mutant PEW300 antibacterial peptide are analyzed, and different truncated fragments are selected for antibacterial performance analysis.

(2) According to the analysis result of the antibacterial property of the peptide segment, the antibacterial peptide with high antibacterial potential is designed and constructed by combining fusion, mutation and other technologies.

(3) The antibacterial peptide designed above is subjected to antibacterial activity analysis, and finally G2 is screened to perform antibacterial performance test, so that the antibacterial peptide has a narrow spectrum and excellent antibacterial activity on clostridium difficile, salmonella typhimurium and escherichia coli O157. The antibacterial peptide consists of 79-88 th peptide of escherichia coli CsgG protein and 1-10 th peptide of PEW300 antibacterial peptide, and the middle is connected by GP. The information of the G2 antibacterial peptide is shown in fig. 1 and table 1, the result shows that the G2 belongs to the alpha helix antibacterial peptide, the relative molecular weight of the G2 is about 2.5kDa, the G2 has good hydrophobicity and amphipathy, and carries 6 positive charges, and the predicted result shows that the G2 has good antibacterial potential.

Table 1: physicochemical properties of G2 antibacterial peptide

Wherein GRAVY represents the amphipathic nature of the antimicrobial peptide and μH represents the hydrophobic nature of the antimicrobial peptide.

Example 2: construction of recombinant expression vector pET30-gme

(1) Plasmid information of recombinant expression vector pET30-gme is shown in FIG. 2A. Based on the gene sequence of the G2 antibacterial peptide, the DNA sequences G2 to pUC57 vector were synthesized at gene synthesis company (Shanghai Biotechnology). The following RF clone (Restriction Free Clone) primers (RF-g 2-F/RF-g 2-R) were designed for the RF cloning reaction:

(2) The preparation of the G2 linear fragment was carried out using the synthesized plasmid containing the G2 antibacterial peptide gene (pUC 57-G2) as a template and the above-mentioned RF cloning primers, and the specific steps were as follows:

PCR reaction System (50. Mu.L):

PCR amplification reaction procedure:

(3) After the PCR reaction was completed, 2.5% (w/v) agarose gel electrophoresis was performed to identify, thereby obtaining a gene fragment containing G2 having a size of about 116bp, and the PCR product was purified and recovered and the recovery concentration of the gene fragment containing G2 antibacterial peptide was measured by a DNA concentration meter.

(4) The recombinant expression vector pET30-PEW300 (the plasmid carries PEW300-Mxe-ELK16 fusion protein, see literature: design, expression, and characterization of a novel cecropin A-derived peptide with high antibacterial activity, appl Microbiol Biotechnol,103 (2019) 1765-1775.) is used as an RF cloning (Restriction Free Clone) template, and the PCR product containing the G2 antibacterial peptide gene recovered by purification is used as a linear amplification reaction primer for linear amplification reaction, wherein the specific reaction system is as follows:

PCR reaction System (50. Mu.L):

PCR amplification reaction procedure:

(5) After the PCR reaction is finished, the PCR product obtained in the step (4) is subjected to Dpn I digestion, and the specific operation is as follows:

cleavage reaction System (20. Mu.L):

the mixture is placed in a constant temperature PCR instrument at 37 ℃ for enzyme digestion reaction for 1h.

(6) The digested product was transformed into E.coli DH 5. Alpha. Competent cells by chemical transformation, and the bacterial solution was spread on LB solid plates (10 g/L sodium chloride, 10g/L peptone, 5g/L yeast extract, 1.5g/L agar powder) containing kanamycin (final concentration: 50. Mu.g/mL), and incubated overnight in an incubator at 37 ℃.

(7) Colony PCR was performed on the monoclonal antibodies grown in the plates, and the results are shown in FIG. 2B. Positive clones No. 1 and No. 3 were randomly picked for sequencing identification. Screening recombinant expression vector pET30-gme carrying G2 antibiotic peptide gene correctly.

Example 3: expression and condition optimization of G2-Mxe-ELK16 fusion protein

(1) The recombinant expression vector pET30-gme constructed in the example 2 is transferred into competent cells of escherichia coli BL21 (DE 3) by a chemical conversion method to obtain BL21 (DE 3) engineering bacteria containing the recombinant expression vector pET30-gme. The engineering bacteria solution was then spread on LB solid plates containing 50. Mu.g/mL kanamycin and incubated overnight at 37 ℃. Wherein BL21 (DE 3) competent cells were purchased from Piano Biotech Inc.

(2) The single clone in step (1) was selected and inoculated into LB liquid medium (10 g/L sodium chloride, 10g/L peptone, 5g/L yeast extract) containing 50. Mu.g/mL kanamycin, and cultured overnight at 37℃and 220rpm as a seed solution. Seed solution was inoculated into 4 test tubes containing fresh LB liquid medium (containing 50. Mu.g/mL kanamycin) at an inoculum size (v/v) of 1%, placed in a 37℃incubator, shake-cultured at 220rpm, and the test tubes were subjected to

numbers

1, 2, 3, 4. When OD is ₆₀₀ When=0.6 to 0.8, inducer (IPTG) was added at different concentrations (0.1, 0.5, 1 mM) to test tubes numbered 2, 3, and 4, respectively, for induction expression. Wherein, the test tube No. 1 is not added with an inducer as a control, and 4 test tubes are placed in a shaking table at 16 ℃ to induce expression for 24 hours at low temperature. After the expression is finished, OD is measured on the expressed bacterial liquid ₆₀₀ And (5) value and bacterial collection.

(3) The cells collected above were resuspended at a ratio of 1mL PBS buffer per 10OD, and the cells were disrupted by sonication. After the thallus is crushed, 80 mu L of a whole liquid sample after crushing is sucked, and then 12000rpm is used for centrifugation for 2min to obtain a crushed supernatant and a sediment, and 80 mu L of the whole liquid sample after crushing is sucked to prepare the crushed supernatant and the sediment sample respectively. After the sample preparation, SDS-PAGE gel electrophoresis is carried out to analyze the expression level of the fusion protein and determine the optimal expression condition. As a result, as shown in FIG. 3, the fusion protein was successfully expressed after IPTG induction, and the vast majority was present in E.coli cells in a "precipitated" form. According to the IPTG optimization result, the expression level of the fusion protein reaches equilibrium when the addition concentration of the inducer IPTG is 0.1mM. Thus, the optimal addition amount of IPTG was selected to be 0.1mM.

Example 4: purification and preparation of G2 antibacterial peptide

(1) The G2-Mxe-ELK16 fusion protein was expressed in large amounts (1L) according to the optimal conditions optimized in example 3. After the completion of the expression, the cells were collected by centrifugation at 8000rpm for 10 min. After the cells were sonicated, the cells were centrifuged at 12000rpm for 30min, and the crushed precipitate was collected. To the pellet, PBS buffer was added to suspend the pellet in a proportion of 1mL PBS buffer per 10 OD. To the suspended precipitate, 40mM of an induced cleavage agent (DTT) was added, and the mixture was placed in a constant temperature refrigerator at 4℃for cleavage for 12 hours, thereby inducing self-cleavage of the self-cleaving peptide Mxe gyrA in the fusion protein.

(2) After the completion of the cleavage reaction, the mixture was centrifuged at 12000rpm for 30 minutes, and the precipitate was collected. The pellet was washed twice with PBS, and glacial acetic acid at a concentration of 3% (v/v) was added to the pellet after cleavage, the pellet was suspended, incubated at room temperature for 2min, and then centrifuged at 12000rpm for 5min to collect the supernatant. Acetic acid was then removed with a 1kDa ultrafiltration tube, and the G2 antimicrobial peptide was finally successfully obtained. The concentration of the G2 antibacterial peptide was obtained by gray scale scanning calculation using commercial aprotinin (Shanghai) as a standard protein for the quantification of the antibacterial peptide. The quantitative result of the G2 antibacterial peptide is shown in FIG. 4, and the concentration of the G2 antibacterial peptide obtained after purification is 142. Mu.g/mL.

Example 5: g2 antibacterial peptide antibacterial spectrum determination and application

The MIC (minimum inhibitory concentration) of G2 antimicrobial peptides against 12 indicator bacteria, including major intestinal probiotics and pathogenic bacteria (clostridium difficile, bacillus cereus, escherichia coli, lactobacillus acidophilus, lactobacillus rhamnosus and bifidobacterium) and other common pathogenic bacteria (salmonella typhimurium, salmonella enteritidis, pseudomonas aeruginosa, staphylococcus aureus, vibrio parahaemolyticus) was determined by adding a series of G2 antimicrobial peptide solutions (264, 132, 66, 33, 16.5, 8.25, 4.1, 2.05, 1.03, 0.51, 0.25ng/μl) to 96-well plates according to a two-fold serial dilution method, and determining the antimicrobial profile of the G2 antimicrobial peptides. Taking aerobic escherichia coli and anaerobic clostridium difficile as examples, the specific operation method is as follows:

(1) Preparation of indicator suspensions

E.coli is selected and inoculated into MHB liquid culture medium for culturing overnight at 37 ℃; diluting the bacterial liquid to OD with fresh MHB liquid culture medium ₆₂₅ 0.08 to 0.13, and then continuously diluting 100 times as indicator bacteria suspension for standby;

clostridium difficile is selected and monoclonal into BHI liquid culture medium, and is placed into an anaerobic incubator at 37 ℃ for culturing for 18h. Diluting the bacterial liquid to OD by using fresh BHI liquid culture medium ₆₂₅ Is 0.08 to 0.13, and then is continuously diluted by 100 times to be used as indicator bacteriaThe suspension was ready for use.

(2) Bacteriostasis experiment

a. Colibacillus bacteriostasis experiment

Sterile 96-well plates were taken and numbered 1-12 in columns from left to right, with 3 multiplex wells per column. 50. Mu.L of sterilized MHB liquid medium was added to each of the three wells of columns 2 to 11, and 100. Mu.L of MHB liquid medium was added to the 12 th well as a positive control. 100 μl 264 ng/. Mu.l of the G2 antibacterial peptide solution was added to each of the three wells of column 1, then 50 μl was aspirated from each of the three wells of column 1 to the three wells of column 2, 50 μl was taken from each of the three wells of column 2 to the three wells of column 3, and so on to the three wells of

column

10, and 50 μl was aspirated from each of the three wells of column 10 and discarded. Finally, 50 mu L of the indicator bacteria liquid prepared in the step (1) is respectively added into the compound holes of the rows 2 to 11, and the mixture is placed at 37 ℃ for culture for 16 to 20 hours.

b. Clostridium difficile bacteriostasis experiment

The specific operation method is the same as that of the step (2) a. Except that the medium used was fresh BHI broth and was run in an anaerobic bench.

(3) Determination of MIC values by observation

The bacteriostasis is observed with naked eyes, the lowest concentration of the antimicrobial peptide which can not grow in the indicator bacteria is the MIC value of the antimicrobial peptide for the indicator bacteria, and the result is shown in Table 2: the G2 antibacterial peptide has remarkable inhibition effect on clostridium difficile, escherichia coli O157 and salmonella typhimurium ATCC14028 (the strains can be purchased from proprietary microorganism strain preservation institutions such as the culture collection of the Guangdong province microorganism research institute, and the like), and has weaker antibacterial activity on other indicator bacteria, so that the G2 antibacterial peptide has narrower antibacterial spectrum.

Table 2 characterization of the antimicrobial activity of the G2 antimicrobial peptides

Example 6: stability and biosafety assay for G2 antimicrobial peptides

The stability and biosafety of the high purity G2 antibacterial peptide prepared in example 5 were measured using clostridium difficile as an indicator. Stability includes: pH, temperature and serum stability; biosafety includes: hemolytic activity, serum stability and cytotoxicity experiments.

(1) Detection of pH stability

Buffer solutions with different pH values are respectively prepared, and the following specific steps are as follows:

100mM glycine-HCL buffer at ph=2.0, 100mM sodium acetate buffer at ph=4.0, 100mM sodium phosphate buffer at ph=6.0, 100mM Tris-HCL buffer at ph=8.0, 100mM glycine-NaOH buffer at ph=10.0. The purified 126.3 ng/. Mu.L of G2 antibacterial peptide was adjusted to the corresponding pH buffer and left at 37℃for 4 hours. The change in antibacterial activity of the G2 antibacterial peptide under different pH conditions was then determined. The results are shown in FIG. 5A: the G2 antibacterial peptide has good pH stability, and the activity of the G2 antibacterial peptide is slightly influenced in an acidic environment.

(2) Thermal stability detection

The antibacterial activity change of the G2 antibacterial peptide under different temperature conditions was measured after the antibacterial peptide having a concentration of 135 ng/. Mu. L G2 was reacted at 4℃and 20℃and 40℃and 60℃and 80℃and 100℃for 1 hour, respectively, as shown in FIG. 5B: the G2 antibacterial peptide has strong thermal stability and can tolerate 80-100 ℃.

(3) Serum stability assay

The G2 antimicrobial peptide solution at 86.2 ng/. Mu.l was incubated with fresh bovine serum (50% by volume) at 37 ℃ for the following incubation times: 0h,6h,12h,24h,48h. The antibacterial activity of the incubated antibacterial peptides was then verified, with sterile PBS and bovine serum as controls, respectively. The results are shown in fig. 5C: serum has no effect on the activity of the G2 antibacterial peptide.

(4) Haemolytic property detection

The tail venous blood of the mice was taken, treated with citric acid to stop clotting, then centrifuged at 3000g for 5min to collect the erythrocytes, washed three times with 0.9% physiological saline, resuspended in 10mM PBS (ph=7.4), and treated with antimicrobial peptide for 1h. PBS and 1% Triton X-100 were used as controls. After 1h, centrifugation was performed to measure the OD value (amount of hemoglobin released) at 570nm of the supernatant. The results are shown in FIG. 6A: the G2 antibacterial peptide has no obvious hemolytic activity.

(5) Cytotoxicity detection

Cytotoxicity of the G2 antibacterial peptide was characterized by detecting cell viability of HEK 293. The method comprises the following steps: to an initial concentration of 1X 10 ⁵ Fresh DMEM medium (100 mg/mL streptomycin, 100U/mL penicillin and 10% bovine serum) was added to HEK293 cells in cells/mL and placed at 37℃with 5% CO ₂ Is cultured in an incubator of (a). The following day the cells were counted according to 1X 10 ⁴ cell/well ratio HEK293 cells were sub-packed in 96 well plates and labeled; g2 antibacterial peptide solution with concentration of 10-240 ng/. Mu.L is added into labeled 96-well plates respectively, and placed at 37 ℃ and 5% CO ₂ For 48h in an incubator, three replicates were set for each set of experiments. The cell morphology was observed with a microscope and the cell activity of HEK293 was detected with MTT assay kit. The results are shown in fig. 6B and 6C: the addition of different concentrations of G2 antibacterial peptide has no influence on the growth of HEK293 cells, and the cell activity is good.

The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

Sequence listing

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Claims

1. A narrow-spectrum antibacterial peptide, which is characterized in that the amino acid sequence is shown as SEQ ID No: 1.

2. A gene encoding the narrow spectrum antibacterial peptide of claim 1, wherein the nucleotide sequence is as set forth in SEQ ID No: 2. as shown.

3. The use of a narrow spectrum antibacterial peptide according to claim 1 for the manufacture of a medicament for the treatment of microbial infections, wherein the microorganism is clostridium difficile @ aClostridium difficile) Salmonella typhimurium (Salmonella typhimurium)Salmonella typhimurium) Coli @ andEscherichia coli）O157。