Cyclic bacteriocin Flexusin A and application thereof

Abstract

The invention provides a cyclic bacteriocin FlexusinA with antibacterial activity, the amino acid sequence of the cyclic bacteriocin FlexusinA is an amino acid sequence which is obtained by substitution and / or deletion and / or addition of one or more amino acid residues on the amino acid sequence as shown in SEQ ID NO: 1 and has the same function, and the sequence similarity of the cyclic bacteriocin FlexusinA is greater than or equal to 70%; a nucleotide sequence for synthesizing the cyclic bacteriocin FlexusinA is shown as a sequence table SEQ ID NO: 2, and the cyclic bacteriocin FlexusinA is composed of 201 nucleotides in total; the propeptide amino acid sequence of the cyclic bacteriocin Flexusin A before cyclization is as shown in SEQ ID NO: 3 in a sequence table, and the number of amino acids is 66. The cyclic bacteriocin Flexusin A has bacteriostatic activity on a part of gram-positive pathogenic bacteria such as staphylococcus aureus, methicillin-resistant staphylococcus aureus, listeria monocytogenes, enterococcus faecalis, streptococcus agalactiae and bacillus cereus, and particularly has inhibitory activity on drug-resistant bacteria.

Description

Technical Field

[0001] The invention belongs to the development of antimicrobial peptides in the field of microbial engineering, and specifically relates to a cyclic bacteriocin with antimicrobial activity and a use thereof. Background Art

[0002] Bacteriocins are a class of peptide substances with antibacterial activity that are synthesized by ribosomes, activated by post-translational modification, and transported to the extracellular space through a specific transport system to exert their effects. In the classification scheme of bacteriocins, cyclic bacteriocins are considered to belong to class II unmodified peptides and are usually classified as subclass IIc.

[0003] Cyclic bacteriocins are first synthesized as linear propeptides containing a signal peptide sequence of variable size, which is cleaved during maturation and then covalently linked to the C-terminus to form a structurally conserved cyclic peptide backbone. Mature cyclic bacteriocins are usually composed of 58 to 70 amino acid residues and have a molecular weight ranging from 5.6 to 7.2 kDa. To date, the cyclic bacteriocins identified are produced by Gram-positive bacteria of the Firmicutes phylum, mainly lactic acid bacteria, but also a few from Bacillus and Clostridium genera.

[0004] WHO has announced six types of drug-resistant bacteria "ESKAPE" that pose a serious threat to human health and need to be conquered first. They are Enterococcus faecalis (E), Staphylococcus aureus (S), Klebsiella pneumoniae (K), Acinetobacter baumannii (A), Pseudomonas aeruginosa (P) and Enterobacter species (E). Finding and discovering substances that are effective against Gram-positive pathogens and some drug-resistant pathogens in "ESKAPE" is of practical significance and urgency in the face of the increasingly serious situation of drug resistance. Summary of the invention

[0005] The present invention provides a cyclic bacteriocin Flexusin A with high antibacterial activity, which has good application and development prospects as an antibacterial agent and / or antibacterial drug in the fields of medical treatment, food, breeding, etc. Its primary structure consists of 60 amino acid sequences covalently linked via the N-terminus and the C-terminus to form a structurally stable cyclic peptide skeleton.

[0006] Furthermore, the amino acid sequence of the cyclic bacteriocin Flexusin A is:

[0007] 1) the amino acid sequence shown in SEQ ID NO: 1; or,

[0008] 2) An amino acid sequence with the same function as the amino acid sequence shown in SEQ ID NO: 1 after one or more amino acid residues are substituted and / or deleted and / or added, and the sequence similarity is ≥ 70%.

[0009] Furthermore, the preparation method of the bacteriocin Flexusin A is as follows:

[0010] 1) Synthesis of structural gene fleh1 and signal peptidase gene lepB;

[0011] 2) Construction of co-expression vector of structural gene flel and signal peptidase gene lepB;

[0012] 3) Transformation of expression strains;

[0013] 4) Inducible expression of expression strains.

[0014] Furthermore, the expression strain is Escherichia coli.

[0015] Another aspect of the present invention provides a propeptide of a synthetic cyclic bacteriocin Flexusin A, wherein the amino acid sequence of the propeptide is shown in SEQ ID NO:3.

[0016] In a third aspect, the present invention provides an expression vector containing a gene encoding a synthetic cyclic bacteriocin Flexusin A, wherein the gene encoding the gene contains a nucleotide sequence as shown in SEQ ID NO:2.

[0017] The fourth aspect of the present invention provides the use of the cyclic bacteriocin Flexusin A in the preparation of an antibacterial preparation.

[0018] Furthermore, the cyclic bacteriocin Flexusin A can inhibit the growth of Gram-positive bacteria.

[0019] Furthermore, the Gram-positive bacteria are Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Listeria monocytogenes, Enterococcus faecalis, Streptococcus agalactiae, and Bacillus cereus.

[0020] Furthermore, the inhibition spectrum experiment found that Flexusin A has a narrow spectrum of inhibition. + Bacterial activity: the MIC value of the Flexusin A against S. aureus ATCC6538 is 1 μg / mL, and the Flexusin A exhibits an antibacterial activity superior to that of ampicillin against E. faecalis CICC21605 and clinically isolated drug-resistant strain S. aureus 07. The Flexusin A has the use of inhibiting drug-resistant bacteria.

[0021] Further, the use is that Flexusin A at a concentration of 1×MIC can cause intracellular K + and protein efflux, or inhibiting the formation of cell biofilm.

[0022] The last aspect of the present invention provides a Gram-positive bacteria inhibitor, which comprises one or more pharmaceutically acceptable carriers, excipients and / or diluents of the cyclic bacteriocin Flexusin A.

[0023] Beneficial effects of the present invention:

[0024] 1) The present invention provides a cyclic bacteriocin Flexusin A with high antibacterial activity, which is a new strain. The strain can be obtained by inducing Escherichia coli and has a significant inhibitory effect on Gram-positive bacteria.

[0025] 2) The cyclic bacteriocin Flexusin A provided by the present invention has a significant use in inhibiting drug-resistant bacteria. Flexusin A has a narrow spectrum inhibition part G + Bacterial activity: the MIC value of the drug against S. aureus ATCC6538 was 1 μg / mL, and it showed better antibacterial activity than ampicillin against E. faecalis CICC21605 and clinically isolated drug-resistant strain S. aureus07.

[0026] 3) The present invention evaluated the low hemolytic activity (hemolytic rate was 1.79% at a concentration of 1×MIC (1 μg / mL)) and weak cytotoxicity (the survival rate was 99.80% when acting on mouse L929 fibroblasts at a concentration of 1×MIC (1 μg / mL) for 24 hours) of Flexusin A through hemolytic and cytotoxic experiments.

[0027] 4)Flexusin A has no antibacterial activity against beneficial probiotics. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Flexusin A biosynthetic gene cluster prediction;

[0029] Figure 2 pCDFDuet-1 plasmid vector map and multiple cloning site information;

[0030] Figure 3 Gel images of recombinant plasmids after restriction digestion (A: plasmid pCD-1; B: plasmid pCD-2. Lane 1: empty plasmid; Lane 2: plasmid after restriction digestion; Lane 3: Marker);

[0031] Figure 4 PCR verification of colonies containing recombinant plasmids (A: single colony containing pCD-2 plasmid, primers are 5155-1_1 and 5155-1_14; B: single colony containing pCD-2 plasmid, primers are 5155-2_1 and 5155-2_6. Lane 1: Marker; Lanes 2-9: 8 randomly selected colonies);

[0032] Figure 5 Monoclonal induced expression WB results;

[0033] Figure 6 Amino acid sequence analysis of the primary structure of Flexusin A (AD are the four resolved fragments of the secondary mass spectrum of Flexusin A);

[0034] Figure 7 Hemolysis rate of Flexusin A on mouse erythrocytes (A: hemolysis rate; B: hemolysis graph);

[0035] Figure 8 Flexusin A is toxic to mouse L929 fibroblasts;

[0036] Fig. 9 Intracellular potassium ion release curve after treatment with different concentrations of Flexusin A;

[0037] Fig.10 Intracellular protein release curve after treatment with different concentrations of Flexusin A;

[0038] Fig.11 Effects of different concentrations of Flexusin A on biofilm formation;

[0039] Fig.12 TEM ultrathin section observation of S. aureus after Flexusin A treatment (A, C, E are 6k times, 10k times, and 20k times electron micrographs after Flexusin A treatment, B, D, and F are 6k times, 10k times, and 20k times electron micrographs under negative control, respectively). DETAILED DESCRIPTION

[0040] The present invention is further described below in conjunction with the examples, which are used to illustrate the present invention but are not intended to limit the scope of the present invention. The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

[0041] Example 1 Biosynthesis of Flexusin A

[0042] The whole genome of the cyclic bacteriocin-producing bacteria was sequenced to obtain the chromosome sequence and 5 plasmid sequences of the strain. The obtained chromosome and plasmid sequences were uploaded to the antiSMASH and BAGEL4 databases for prediction of related metabolite gene clusters, thereby obtaining the biosynthetic gene cluster of the cyclic bacteriocin Flexusin A (such as Figure 1 ), which contains the propeptide structural gene fleh and signal peptidase gene lepB necessary for the synthesis of Flexusin A.

[0043] The target sequence 5155-1 for expressing the signal peptidase gene lepB and the target sequence 5155-2 for expressing the Flexusin A propeptide structural gene fl1 were synthesized by two rounds of PCR reaction. The 5155-1 sequence was inserted into the linear plasmid pCDFDuet-1 after restriction digestion to obtain the pCD-1 plasmid, and the 5155-2 sequence was inserted into the linear plasmid pCD-1 after restriction digestion to obtain the pCD-2 plasmid. The plasmid map and multiple cloning site information are shown in Figure 2 To check the accuracy of the synthesized target sequence and its insertion into the plasmid, three recombinant plasmids were randomly digested and the fragment lengths of the digested products were analyzed by nucleic acid electrophoresis. The results are shown in Figure 3 As shown. Among them, the target sequence insertion site of plasmid pCD-1 is MCS1_BamHI / EcoRI, and the theoretical length of the fragment obtained after digestion with BamHI-XhoI should be 3527bp and 855bp; the target sequence insertion site of plasmid pCD-2 is MCS2_NdeI / BglII, and the theoretical length of the fragment obtained after digestion with BamHI-XhoI should be 3520bp and 1086bp. Figure 3 It can be seen that the actual length of the fragments of the enzyme cleavage products is consistent with the theoretical length, which shows that the insertion of the target sequence in each plasmid is correct.

[0044] After the verified recombinant plasmid pCD-2 was transformed into E. coli Rosetta competent cells, the monoclonal colonies with the transferred plasmid were obtained by Streptomycin resistance plate screening. Eight colonies were randomly selected, and colony PCR was performed using 5155-1_1 / 5155-1_14 and 5155-2_1 / 5155-2_6 as primers, respectively. Positive clones were identified by electrophoresis, and the results are shown in Figure 4.

[0045] Pick a successfully transformed single clone and culture it in liquid until the bacterial OD 600 When the concentration of IPTG was 0.6-0.8, 1 mM IPTG was added to induce expression at 28°C, and the bacterial solution was collected after 16 hours.

[0046] The bacterial solution collected after induction of expression was used for Western Blot detection, and the bacterial solution sample without IPTG induction expression was used as a negative control. The Western Blot detection steps are as follows:

[0047] 1) Take 200 μL of bacterial solution, collect the bacterial precipitate by centrifugation, add 50 μL of distilled water to resuspend the bacteria, then add 5× loading buffer and cook at 100°C for 10 min;

[0048] 2) Wet transfer was used for membrane transfer, voltage 300V, current 90mA, and membrane transfer time 2h;

[0049] 3) After transfer, wash the membrane with TBST for 5 min, and then block it with blocking solution containing 5% skim milk powder in TBST for 1 h;

[0050] 4) After blocking, pour off the blocking solution, wash with TBST for 15 minutes, repeat three times, and then add the primary antibody (Mouse Anti-His Tag (1:1000 dilution)) and incubate at 4°C for 3 hours;

[0051] 5) After the primary antibody incubation, the membrane was washed with TBST for 15 min, repeated three times, and then the secondary antibody (Goat Anti-Mouse IgG, HRP (1:10000 dilution)) was added and incubated at 4°C for 0.5 h;

[0052] 6) After incubation with the secondary antibody, wash the membrane with TBST for 15 minutes, repeat three times, and then use the color developing solution to develop the image. The expression results are as follows Figure 5 shown.

[0053] Example 2 Analysis of the amino acid sequence of the primary structure of Flexusin A

[0054] For the Flexusin A component that has been purified by Tricine-SDS-PAGE to obtain a single band, it was sequenced by protein shotgun analysis after gel recovery, and the protein library obtained after whole genome sequencing was used to assist the secondary mass spectrometry analysis to provide a mutually verifiable method for the analysis of cyclic bacteriocins. The Flexusin A component after gel recovery was enzymatically digested by chymotrypsin to obtain 4 groups of peptides of different sizes. After secondary mass spectrometry analysis, the sequences of these 4 peptides were: IAGALGISEY, ANVIVTAIEAGSTVL, ALISMFASF, GLTSALILTAKSL, see for details. Figure 6 Based on the obtained 4 amino acid sequences, the protein database was searched and it was found that the 4 peptide sequences of Flexusin A determined by secondary mass spectrometry were highly consistent with the propeptide sequence encoded by the cyclic bacteriocin gene cluster, which also confirmed Figure 1 The predicted cyclic bacteriocin gene cluster is the biosynthetic gene cluster of Flexusin A. By comparing with the known cyclic bacteriocin propeptide sequence, the first 6 amino acids MEVVAY at the amino terminal were determined to be the leader peptide sequence.

[0055] The linear peptide sequence of Flexusin A without the leader peptide was uploaded to the TMHMM-2.0 database for transmembrane region prediction. There are two obvious transmembrane regions in the amino acids 1-25 and 29-51 in the primary sequence of Flexusin A, that is, 80% of the amino acid sequence is in the transmembrane region. Therefore, Flexusin A exhibits hydrophobic characteristics as a whole. Example 3 MIC determination of Flexusin A

[0056] Six pathogenic bacteria, including Enterococcus faecalis CICC21605, E. faecium CICC10004, Listeria monocytogenes ATCC 19115, Staphylococcus aureus ATCC6538, S. aureus 07 and MRSA01, and five probiotic bacteria, including Clostridium butyricum CB03, Lactobacillus plantarum RS-1, Lactobacillus lactis RS-16, Bacillus coagulans 1.3220 and Bacillus subtilis Y-1, were selected as indicator bacteria. Single colonies were picked and cultured in liquid culture medium until the logarithmic growth phase, and then diluted with fresh sterile culture medium to a final cell concentration of 10 6 CFU / mL for later use. Dissolve the purified and freeze-dried Flexusin A sample in water to prepare a stock solution with a concentration of 2560μg / mL, and dilute it 2-fold with sterile water to the 12th tube. Prepare another positive control with an initial concentration of 320μg / mL ampicillin, and dilute it 2-fold to the 12th tube.

[0057] Take a sterile 96-well plate and add the above 11 indicator bacteria solutions (the final concentration of indicator bacteria cells is about 10 6CFU / mL), and 90 μL of bacterial solution was added to each well. Subsequently, 10 μL of Flexusin A sample or ampicillin dilution was added to the wells where the bacterial solution was added, and 2 groups were parallel for each dilution, and the water-added group was used as a negative control. After the addition of the sample, the 96-well plate was sealed and placed at 37°C and 80r / min for 12 hours. The culture medium without bacterial solution was adjusted to zero, and the absorbance of each group was detected at a wavelength of 600nm by an enzyme marker. The lowest sample concentration and the lowest ampicillin concentration at which the indicator bacteria in the 96-well plate were completely inhibited were taken as the MIC values ​​of the samples and drugs for each indicator bacteria, and the results are shown in Table 1. It is worth mentioning that one of the strains, S. aureus 07, was a drug-resistant bacteria isolated from the blood samples of ICU patients. It survived the clinical combination of antibiotics such as amoxicillin, cephalexin and azithromycin, and has broken through the defense line of existing antimicrobial drugs. In addition, the test results showed that Flexusin A showed almost no antibacterial activity against five probiotics (Clostridium butyricum CB03, Lactobacillus plantarum RS-1, Lactobacillus lactis RS-16, Bacillus coagulans1.3220 and Bacillus subtilis Y-1).

[0058] Table 1 MIC determination of Flexusin A and Amp

[0059]

[0060] Example 4 Hemolysis experiment of Flexusin A

[0061] The hemolytic test was performed by obtaining mouse heart blood using the mouse heart blood sampling method, and collecting red blood cells after treatment with physiological saline. The experiment set up 11 groups, and each group took 450 μL of red blood cell suspension into a 1.5mL EP tube, and added 50 μL of Flexusin A sample solutions of different concentrations in sequence, so that the final concentrations were 1, 2, 4, 8, 16, 32, 64, 128, and 256 μg / mL, respectively. Physiological saline was used as a negative control, and 2% Triton-100 was used as a positive control. The above group samples were incubated at 37°C for 1 hour, and then transferred to a 96-well plate to detect its absorbance at a wavelength of 540nm. Three parallels were set for each treatment group, and then the hemolysis rate (%) of each group of samples was calculated according to Formula 1.

[0062] Formula 1:

[0063] Hemolysis rate % = (A 样品 -A 0 ) / (A 100 -A 0)×100%·············1

[0064] Among them, A 样品 is the absorbance value of each treatment group, A 0 is the absorbance value of the saline treatment group, A 100 is the absorbance value of the Triton-100 treated group.

[0065] The results are as follows Figure 7 As shown, Flexusin A showed slight hemolysis. The hemolysis rate was 1.79% at a concentration of 1 μg / mL, which was significantly different from the control group (P<0.05).

[0066] Example 5 Cytotoxicity Experiment of Flexusin A

[0067] Mouse L929 fibroblasts were selected for cytotoxicity experiments and tested using the Cell Counting Kit-8 (CKK-8) method. L929 fibroblasts were inoculated into DMEM high-glucose medium (containing 10% fetal bovine serum) and cultured to the logarithmic growth phase. After rinsing and digestion, the cell concentration was adjusted to about 40,000 cells / mL and the cells were collected. The diluted cell suspension was pipetted into a 96-well plate, 100 μL per well, and incubated at 37°C, 5% CO 2 The cells were cultured under the conditions for about 24 hours until the cells attached to the wall. The culture medium was removed, and Flexusin A samples of different concentrations pre-diluted with fresh culture medium were added to the final concentrations of 1, 2, 4, 8, 16, 32, and 64 μg / mL. Fresh culture medium without sample was used as the negative control, and cultured at 37°C for 24 hours. Three parallel groups were set for each treatment. After the culture was completed, 10 μL of CCK-8 solution was added to each well and returned to the cell culture incubator for continued incubation for about 4 hours until an obvious color reaction was observed with the naked eye. The absorbance was detected at a wavelength of 450 nm by an enzyme reader, and the cell survival rate (%) of each group of samples was calculated according to Formula 2.

[0068] Formula 2:

[0069] Survival rate % = (A 样品 -A 0 ) / (A CK -A 0 )×100%·············2

[0070] Among them, A 样品 : absorbance value of each treatment group; A 0 : Culture medium containing CCK-8 but without cells and Flexusin A samples; A CK : Culture medium containing CCK-8 and cells, without Flexusin A sample.

[0071] The results are as follows Figure 8 As shown, Flexusin A has weak cytotoxicity. At a concentration of 1×MIC (1 μg / mL), the survival rate of mouse L929 fibroblasts was 99.80% after acting on them for 24 hours.

[0072] Example 6 Study on the antibacterial mechanism of Flexusin A

[0073] The study of the antibacterial mechanism examined the effect of Flexusin A on the intracellular K + and protein concentration. First, prepare and collect the concentration of about 10 6 CFU / mL of S. aureus cells, take 5mL of bacterial suspension into 5 50mL triangular flasks, add Flexusin A samples of different final concentrations to 1×MIC, 2×MIC, and 4×MIC in 3 of them, add Amp solution to the final concentration of 2×MIC in the other 2 bottles as positive control, and add normal saline as negative control. The above groups were shaken at 80r / min, and samples were taken at 0, 5, 10, 30, and 60min in turn. The obtained samples were centrifuged at 5000r / min for 10min, the bacteria were discarded, and the supernatant was sterilized with a 0.22μm membrane to obtain a sterile supernatant sample. The K in the supernatant was determined by atomic absorption flame retardant and BCA trace protein concentration determination kit. + and protein concentration. + and protein standard curve to obtain the regression equation.

[0074] The results are as follows Fig. 9 , 10 As shown in Figure 2, 1×MIC Flexusin A can rapidly induce intracellular K + The outflow of proteins indicated that Flexusin A acted on S. aureus cells and changed the permeability of cell membrane.

[0075] Example 7 Study on the effect of Flexusin A on biofilm formation

[0076] To investigate the inhibition of biofilm formation by Flexusin A, we first prepared a 10 6CFU / mL of S.aureus ATCC6538 was added to a 96-well plate (90 μL per well), and then Flexusin A samples were added to the final concentrations of 4×MIC, 2×MIC and 1×MIC, with sterile water as the negative control and 20% sodium lactate as the positive control. After culturing at 37°C for 48 hours, staining and decolorization were performed: 1) The culture medium in the well was aspirated, washed twice with PBS, and then 100 μL of 0.1% crystal violet solution was added for staining for 20 minutes; 2) The crystal violet stain was removed and washed with PBS until the supernatant was colorless; 3) 100 μL of 95% ethanol was added to each well and decolorized for 30 minutes; 4) Finally, the absorbance of each group of samples was measured at a wavelength of 630 nm using an ELISA reader.

[0077] The inhibition rate (%) of Flexusin A sample on S. aureus ATCC6538 biofilm formation was calculated according to Formula 3:

[0078]

[0079] The results are as follows Fig.11 As shown, Flexusin A exhibited a good inhibitory effect on the formation of S. aureus biofilm, and the inhibitory efficiency of 4×MIC on the formation of S. aureus biofilm reached 71.24%, which was significantly better than the positive control sodium lactate (66.26%).

[0080] Example 8 TEM ultrathin section observation of S. aureus cell integrity after Flexusin A treatment

[0081] TEM ultrathin sections can be used to visually observe the integrity of cells after the action of Flexusin A. The cell concentration is about 10 6 CFU / mL of S. aureus ATCC6538 bacterial solution, add Flexusin A sample to a final concentration of 1×MIC, and add sterile water as a negative control. After incubation at 37℃ for 1 hour, collect the bacterial cells, rinse and add fresh 2.5% glutaraldehyde to fix overnight, and observe the cell morphology under TEM.

[0082] Electron microscopy results Fig.12 As shown in the figure, after the action of 1×MIC Flexusin A, holes appeared on the cell membrane surface of S. aureus A bacteria, and the release of the contents was clearly observed ( Fig.12 In addition, it was observed that some of the cell contents were completely leaked out, the inside of the cell was completely transparent, and the cell shrank and collapsed as a whole ( Fig.12C), in contrast, the bacterial edges of the control group were complete and smooth, with clear boundaries, and the internal substances of the bacterial cells were uniformly dense (12B, D, E). Under the electron microscope, representative fields of view were selected at 6k, 10k, and 20k magnifications for observation and photography. The number of identifiable cells in the field of view of the treatment group and the control group at 6k and 10k magnifications also showed that after the action of Flexusin A, a large number of cells disintegrated, making it difficult to distinguish the outline of the cells in the field of view.

[0083] The above embodiments only illustrate the technical solutions of the present invention, rather than limiting it. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for a person skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to replace some of the technical features therein by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions claimed to be protected by the present invention.

Claims

1. A cyclic bacteriocin FlexusinA, characterized in that The amino acid sequence of the bacteriocin is selected from: 1) the amino acid sequence shown in SEQ ID NO: 1; or, 2) An amino acid sequence with the same function as the amino acid sequence shown in SEQ ID NO: 1 after one or more amino acid residues are substituted and / or deleted and / or added, and the sequence similarity is ≥ 70%.

2. The cyclic bacteriocin FlexusinA according to claim 1, characterized in that The preparation method of the bacteriocin is as follows: 1) Synthesis of structural gene fleh1 and signal peptidase gene lepB; 2) Construction of co-expression vector of structural gene flel and signal peptidase gene lepB; 3) Transformation of expression strains; 4) Induced expression of expression strains.

3. The cyclic bacteriocin FlexusinA according to claim 2, characterized in that The expression strain is Escherichia coli.

4. A propeptide of a synthetic cyclic bacteriocin FlexusinA, characterized in that: The amino acid sequence of the propeptide is shown in SEQ ID NO:

3.

5. An expression vector containing a gene encoding a synthetic cyclic bacteriocin FlexusinA, characterized in that: The coding gene contains the nucleotide sequence shown in SEQ ID NO:

2.

6. Use of the cyclic bacteriocin Flexusin A in the preparation of antibacterial preparations.

7. The use according to claim 6, characterized in that The bacteria are Gram-positive bacteria. Preferably, the Gram-positive bacteria are Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Listeria monocytogenes, Enterococcus faecalis, Streptococcus agalactiae, and Bacillus cereus.

8. The use according to claim 6, characterized in that The bacteria are drug-resistant bacteria.

9. The use according to claim 6, characterized in that Described Use: 1) FlexusinA at 1×MIC concentration can lead to intracellular K + and protein outflow, resulting in the disintegration of target bacteria, or 2) Inhibit the formation of cell biofilm.

10. A Gram-positive bacteria inhibitor, characterized in that: One or more pharmaceutically acceptable carriers, excipients and / or diluents comprising the cyclic bacteriocin Flexusin A according to claim 1.

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