Use of a polypeptide in inhibiting bacteria or in the manufacture of an agent for inhibiting bacteria

Abstract

The application discloses application of a polypeptide in inhibiting bacteria or preparing a bacterium-inhibiting agent and belongs to the technical field of biotechnology. In the application, a polypeptide coding gene of unknown function from myxobacteria is designed according to the preferred codon of bacillus subtilis, and a nucleotide sequence is obtained. Then, the gene engineering technology is used to make the polypeptide coding gene recombinantly expressed in bacillus subtilis. The fermentation liquor of the recombinant bacillus subtilis shows good antibacterial activity on gram-positive bacteria represented by staphylococcus aureus, gram-negative bacteria represented by pseudomonas aeruginosa, salmonella, escherichia coli and vibrio parahaemolyticus. The application first discovers the antibacterial function of the polypeptide in myxobacteria, that is, a novel antibacterial peptide is provided. Compared with traditional antibiotics, the antibacterial peptide has equivalent or higher antibacterial activity, a wide antibacterial spectrum and low preparation cost, and has wide application prospects in the fields of antibacterial preparations, food preservatives and the like.

Description

Technical Field

[0001] The present application belongs to the field of biotechnology, and specifically relates to the use of a polypeptide in inhibiting bacteria or preparing an agent for inhibiting bacteria. Background Art

[0002] Antimicrobial peptides are small, biologically active polypeptides produced during microbial secondary metabolism, typically consisting of 10 to 50 amino acid residues. They can inhibit and kill bacteria, fungi, and viruses. Compared with traditional antibiotics, antimicrobial peptides offer the advantages of low resistance risk and rapid bactericidal activity, and are therefore considered promising candidates for addressing the antibiotic resistance crisis. However, natural antimicrobial peptides often suffer from narrow antimicrobial spectra and insufficient stress resistance, making the development of novel, highly effective, broad-spectrum antimicrobial peptides crucial. Furthermore, genomic data analysis indicates that currently known peptide compounds represent only the tip of the iceberg in nature, with a vast number of unknown peptide compounds remaining to be discovered and elucidated.

[0003] Myxobacteria, one of the richest sources of natural products, produce a variety of secondary metabolites. Sequencing of the myxobacterial genome has revealed that it may also encode a polypeptide with the amino acid sequence shown in SEQ ID NO. 1. However, the function of this polypeptide has not yet been annotated or reported, and further research is needed. Summary of the Invention

[0004] 1. Problem to be solved

[0005] In view of the fact that existing antimicrobial peptides generally have a narrow antimicrobial spectrum, while potential antimicrobial peptide compounds may exist in the secondary metabolites produced by myxobacteria, this application aims to obtain more efficient and broad-spectrum new antimicrobial peptides. The polypeptide encoding genes mined from the myxobacterial genome are designed according to the codon preference of Bacillus subtilis, and recombinant expression is achieved in Bacillus subtilis. The obtained recombinant Bacillus subtilis fermentation broth is tested for antibacterial activity and found to have good antibacterial activity against both Gram-positive and Gram-negative bacteria, thereby providing a new efficient and broad-spectrum antimicrobial peptide derived from myxobacteria.

[0006] 2. Technical Solution

[0007] In order to solve the above problems, the technical solutions adopted in this application are as follows:

[0008] The present application provides the use of a polypeptide in inhibiting bacteria or preparing an agent for inhibiting bacteria. The amino acid sequence of the polypeptide is shown in SEQ ID NO. 1. The polypeptide in the present application is derived from myxobacteria, and its function is not yet known. By designing a gene encoding the polypeptide and then using genetic engineering technology to recombinantly express it in Bacillus subtilis, the fermentation broth of the recombinant Bacillus subtilis showed active inhibition against bacteria, thereby discovering the antibacterial function of the polypeptide.

[0009] Furthermore, the above-mentioned bacteria include Gram-positive bacteria and / or Gram-negative bacteria.

[0010] Furthermore, the above-mentioned bacteria include Gram-positive bacteria.

[0011] Furthermore, the above-mentioned bacteria include Gram-negative bacteria.

[0012] Furthermore, the above-mentioned bacteria include Gram-positive bacteria and Gram-negative bacteria.

[0013] Furthermore, the above-mentioned Gram-positive bacteria include Staphylococcus aureus ( Staphylococcus aureus ).

[0014] Furthermore, the above-mentioned Gram-negative bacteria include Pseudomonas aeruginosa ( Pseudomonas aeruginosa ),salmonella( salmonella ), Escherichia coli ( Escherichia coli ) and / or Vibrio parahaemolyticus ( Vibrio Parahemolyticus ).

[0015] Furthermore, the above-mentioned Gram-negative bacteria include Vibrio parahaemolyticus.

[0016] Furthermore, the antibacterial agent is a fermentation broth containing the polypeptide.

[0017] Furthermore, the fermentation broth is that of recombinant Bacillus subtilis, which includes a nucleic acid encoding the polypeptide. This application selects Bacillus subtilis as the host bacteria to express the polypeptide because of its advantages: 1) strong natural secretion ability, which simplifies the downstream process; 2) high safety and application compatibility. It is certified as GRAS (Generally Recognized as Safe) by the FDA and is suitable for food and pharmaceutical grade production without worrying about endotoxin contamination; 3) mature genetic manipulation tools and mature gene editing systems (such as CRISPR-Cas9 and homologous recombination), which facilitate the knockout of protease genes to reduce antimicrobial peptide degradation; 4) natural tolerance to the toxicity of antimicrobial peptides. Its cell membrane contains a high proportion of negatively charged phosphatidylglycerol (PG) and cardiolipin (CL), which has natural resistance to positively charged antimicrobial peptides (such as cationic peptides).

[0018] Furthermore, the nucleotide sequence of the above nucleic acid is shown in SEQ ID NO.2. In this application, the above polypeptide encoding gene was designed based on the codon preference of Bacillus subtilis to obtain the nucleotide sequence, the purpose of which is to improve the expression of the polypeptide in recombinant Bacillus subtilis.

[0019] Furthermore, the recombinant Bacillus subtilis includes a recombinant plasmid containing the nucleic acid.

[0020] Furthermore, the plasmid is selected from any one of pWB980, pTTB1, pHT01, pHT253, and pHY-p43.

[0021] Furthermore, the above-mentioned plasmid is selected from pWB980.

[0022] Furthermore, the above-mentioned Bacillus subtilis is selected from Bacillus subtilis 168 、 Bacillus subtilis WB800N 、 Bacillus subtilis Bsn5 、 Bacillus subtilis ATCC6051a 、 Bacillus subtilis var.natto 、 Bacillus subtilis RIK1285 Any one of .

[0023] Furthermore, the above-mentioned Bacillus subtilis is selected from Bacillus subtilis WB800N .

[0024] The present application also provides a reagent for inhibiting bacteria, which comprises the above-mentioned polypeptide, and the amino acid sequence of the polypeptide is shown in SEQ ID NO.1.

[0025] Furthermore, the above reagent is a fermentation broth containing the above polypeptide, and the fermentation broth is a fermentation broth of recombinant Bacillus subtilis, and the recombinant Bacillus subtilis includes a nucleic acid encoding the above polypeptide.

[0026] The present application also provides a method for inhibiting bacteria, which comprises: adding the above-mentioned polypeptide or the above-mentioned agent for inhibiting bacteria to the living environment of the bacteria to be inhibited.

[0027] 3. Beneficial effects

[0028] Compared with the prior art, the present application has the following advantages:

[0029] (1) This application provides a polypeptide for use in inhibiting bacteria or preparing an agent for inhibiting bacteria. This application designs a polypeptide encoding a gene (SEQ ID NO. 2) for an unknown function of a polypeptide with an amino acid sequence as shown in SEQ ID NO. 1, which was mined from the myxobacterium genome, in combination with the codons preferred by Bacillus subtilis. Using genetic engineering techniques, the polypeptide is recombinantly expressed in Bacillus subtilis. Antibacterial activity tests show that the polypeptide has antibacterial function, thereby providing a novel antimicrobial peptide derived from myxobacteria.

[0030] (2) The present application provides a polypeptide for use in inhibiting bacteria or preparing an agent for inhibiting bacteria. The present application tests the antibacterial activity of the polypeptide-containing fermentation broth against representative Gram-positive bacteria, Staphylococcus aureus, and representative Gram-negative bacteria, such as Pseudomonas aeruginosa, Salmonella, Escherichia coli, and Vibrio parahaemolyticus. It was found that the polypeptide exhibits antibacterial activity against both Gram-positive and Gram-negative bacteria. Among them, the inhibitory effect against Staphylococcus aureus and Vibrio parahaemolyticus is more significant. The polypeptide has broad-spectrum and highly effective antibacterial properties and has good application prospects in agriculture, medicine, and other fields.

[0031] (3) The present application provides a polypeptide for use in inhibiting bacteria or preparing an agent for inhibiting bacteria. Bacillus subtilis is selected to heterologously express the novel broad-spectrum antimicrobial peptide. The peptide has a strong ability to secrete natural proteins, and the protein product is not easy to form inclusion bodies. Compared with other host bacteria, Bacillus subtilis can effectively reduce the production cost of antimicrobial peptides. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a graph showing the antibacterial activity test results of the polypeptide provided in this application against Staphylococcus aureus.

[0033] Figure 2 This is a graph showing the antibacterial activity test results of the polypeptide provided in this application against Pseudomonas aeruginosa.

[0034] Figure 3 This is a graph showing the antibacterial activity test results of the polypeptide provided in this application against Escherichia coli.

[0035] Figure 4 This is a graph showing the antibacterial activity test results of the polypeptide provided in this application against Salmonella.

[0036] Figure 5 This is a graph showing the antibacterial activity test results of the polypeptide provided in this application against Vibrio parahaemolyticus. DETAILED DESCRIPTION

[0037] The present application is further described below with reference to specific examples. Unless otherwise specified, the materials, reagents, etc. used in the following examples can be obtained from commercial sources.

[0038] The culture medium formula involved in the embodiment is as follows:

[0039] LB liquid medium: peptone 10 g / L; yeast extract 5 g / L; NaCl 10 g / L.

[0040] LB solid medium: Add 2% agarose to the above LB liquid medium formula.

[0041] Example 1

[0042] This example provides the construction of polypeptide genetically engineered bacteria.

[0043] (1) Synthetic polypeptide gene sequence

[0044] The polypeptide used in this example is derived from myxobacteria ( Myxococcus fulvus ), whose amino acid sequence is shown in SEQ ID NO. 1 (NCBI Reference Sequence: WP_046714561.1). This application first designed a polypeptide-encoding gene based on the codon preference of Bacillus subtilis and obtained the nucleotide sequence (SEQ ID NO. 2) through codon optimization. Then, to construct a recombinant expression vector, a target gene fragment containing PstI and HindIII restriction endonuclease sites was synthesized using whole-gene synthesis technology:

[0045] SEQ ID NO.1 (polypeptide amino acid sequence):

[0046] MSNKPTHVDVELNDAQLDQVVGGQEVLSLQMMDVGQAEVPNCVSSVSCNSSASCESSASAVVSAAT;

[0047] SEQ ID NO.2 (nucleotide sequence encoding polypeptide):

[0048] ATGTCAAATAAACCGACACATGTTGATGTTGAACTGAATGATGCACAACTGGATCAAGTTGTTGGCGGCCAAGAAGTTTCTGTCACTGCAAATGATGGATGTTGGCCAAGCAGAAGTTCCGAATTGCGTTTCATCAGTTTCATGCAATAGCTCAGCATCATGCGAATCATCAGCAAGCGCAGTTGTTTCAGCAGCAACA.

[0049] (2) Construction of recombinant expression vector

[0050] The vector used in this example was plasmid pWB980. The synthesized target gene (encoding a polypeptide nucleotide) and pWB980 were double-digested with PstI and HindIII restriction endonucleases, respectively, followed by agarose gel electrophoresis. The digested target gene fragment and pWB980 were recovered from the gel and ligated with T4 DNA ligase. The ligation product was transformed into competent DH5α cells, and positive clones were selected using 50 μg / mg kanamycin, indicating that the recombinant expression vector was successfully constructed.

[0051] (3) Construction of genetically engineered bacteria

[0052] The recombinant expression vector pWB980-MF was transformed into Bacillus subtilis WB800N by electroporation. The following steps were performed: WB800N competent cells were removed from a -80°C freezer and placed on ice. 500 ng of the recombinant expression vector was mixed with 80 μL of the competent cells and incubated on ice for 2 min. The cells were then transferred to a pre-chilled 1 mm electroporation cuvette using the following electroporation settings: 2.0 kV, 1 mm, 1 shock. After the shock, the cuvette was removed and immediately filled with 1 ml of LB liquid medium (containing 1 M sorbitol, 50% by volume). The cells were incubated at 37°C, 200 rpm, and allowed to recover for 2 h. The cells were then plated onto LB solid medium containing 50 μg / mL kanamycin and incubated in an inverted position at 37°C overnight. A single clone was picked from the plate and inoculated into LB liquid medium for expansion culture. The recombinant engineered bacterial solution was preserved with 25% final concentration of glycerol at -80°C for future use. Part of it was sent to Anshengda Biotechnology Co., Ltd. for testing. The sequence analysis verified that it was correct, indicating that the polypeptide recombinant engineered bacteria was successfully constructed, named WB800N-MF in this application.

[0053] Example 2

[0054] In this example, WB800N-MF was fermented and cultured.

[0055] (1) Strain activation

[0056] The WB800N-MF in Example 1 was inoculated into LB liquid culture medium containing 50 μg / ml kanamycin and cultured overnight at 37° C. and 220 rpm.

[0057] (2) Expand training

[0058] Transfer the above culture into 50 mL of fresh LB liquid medium at a ratio of 1:100 and ferment at 37°C for 24 hours. Measure the optical density at 600 nm (OD600) to approximately 8. Centrifuge at 12,000 rpm and 4°C for 10 minutes, remove the supernatant, and store the fermentation broth containing the polypeptide at -20°C until needed.

[0059] Example 3

[0060] This example provides a test of the antibacterial activity of the polypeptide against Staphylococcus aureus.

[0061] (1) Antibacterial activity experiment

[0062] In this example, Staphylococcus aureus was used as a representative of Gram-positive bacteria, and the double-layer Oxford cup inhibition zone method was used to determine the antibacterial activity of the polypeptide against it. The specific operation was as follows:

[0063] Pour 10 mL of melted LB solid medium onto a sterile plate. After solidification, place an Oxford cup vertically on the surface of the medium and set aside. Pick a single colony of Staphylococcus aureus and transfer it to LB liquid medium. Culture the suspension at 37°C and 180 rpm for 12 hours. At this point, the bacterial suspension should have an OD600 of approximately 6-8. Dilute the suspension 1:10,000 with sterile water. Then, pipette the suspension into LB solid medium cooled to 50°C, shake well, and pour 20 mL onto the surface of the plate with the Oxford cup. After solidification, remove the Oxford cup and pipette 200 μL of the peptide-containing fermentation broth into the wells. A control well should receive antibiotics (100 μg / mL neomycin sulfate). Incubate the wells at 37°C for 16-20 hours. Observe the inhibitory activity and measure the diameter of the inhibition zone.

[0064] (2) Result analysis:

[0065] The antibacterial activity of the polypeptide in this application against Staphylococcus aureus is as follows Figure 1 As shown in the figure, the diameter of the inhibition zone of the control group was 20 mm, and the diameter of the inhibition zone of the experimental group was 22 mm, indicating that the polypeptide fermentation broth in the present application can inhibit the growth of Staphylococcus aureus, and the inhibitory effect is better than that of 100 μg / ml neomycin sulfate.

[0066] Example 4

[0067] This example provides a test of the antibacterial activity of the polypeptide against Pseudomonas aeruginosa.

[0068] (1) Antibacterial activity experiment

[0069] In this example, Pseudomonas aeruginosa was used as a representative of Gram-negative bacteria, and the double-layer Oxford cup inhibition zone method was used as in Example 3 to determine the antibacterial activity of the polypeptide against Pseudomonas aeruginosa.

[0070] The specific operation is the same as that of Example 3, except that Staphylococcus aureus is replaced by Pseudomonas aeruginosa.

[0071] (2) Results analysis

[0072] The antibacterial activity of the polypeptides in this application against Pseudomonas aeruginosa is as follows Figure 2 As shown, the diameter of the inhibition zone of the control group was 22 mm, and the diameter of the inhibition zone of the experimental group was 19 mm, indicating that the polypeptide fermentation broth in the present application can inhibit the growth of Pseudomonas aeruginosa, and its antibacterial effect is equivalent to that of 100 μg / ml neomycin sulfate.

[0073] Example 5

[0074] This example provides a test of the antibacterial activity of the polypeptide against Escherichia coli.

[0075] (1) Antibacterial activity experiment

[0076] In this example, Escherichia coli was used as a representative of Gram-negative bacteria, and the double-layer Oxford cup inhibition zone method was used as in Example 3 to determine the antibacterial activity of the polypeptide against Escherichia coli.

[0077] The specific operation is the same as that of Example 3, except that Staphylococcus aureus is replaced by Escherichia coli.

[0078] (2) Results analysis

[0079] The antibacterial activity of the polypeptides in this application against Escherichia coli is as follows Figure 3 As shown, after measurement, the diameter of the inhibition zone of the control group was 22 mm, and the diameter of the inhibition zone of the experimental group was 21.1 mm, indicating that the polypeptide fermentation broth in the present application can inhibit the growth of Escherichia coli, and its antibacterial effect is equivalent to that of 100 μg / ml neomycin sulfate.

[0080] Example 6

[0081] This example provides a test of the antibacterial activity of the polypeptide against Salmonella.

[0082] (1) Antibacterial activity experiment

[0083] In this example, Salmonella was used as a representative of Gram-negative bacteria, and the double-layer Oxford cup inhibition zone method was used as in Example 3 to determine the antibacterial activity of the polypeptide.

[0084] The specific operation is the same as that of Example 3, except that Staphylococcus aureus is replaced by Salmonella.

[0085] (2) Results analysis

[0086] The antibacterial activity of the polypeptides in this application against Salmonella is as follows Figure 4 As shown, the diameter of the inhibition zone of the control group was 22 mm, and the diameter of the inhibition zone of the experimental group was 22.3 mm, indicating that the polypeptide fermentation broth in the present application can inhibit the growth of Salmonella, and the inhibitory effect is equivalent to that of 100 μg / ml neomycin sulfate.

[0087] Example 7

[0088] This example provides a test of the antibacterial activity of the polypeptide against Vibrio parahaemolyticus.

[0089] (1) Antibacterial activity experiment

[0090] In this example, Vibrio parahaemolyticus was used as a representative of Gram-negative bacteria, and the double-layer Oxford cup inhibition zone method was used as in Example 3 to determine the antibacterial activity of the polypeptide against the bacteria.

[0091] The specific operation is the same as that of Example 3, except that Staphylococcus aureus is replaced by Vibrio parahaemolyticus.

[0092] (2) Results analysis

[0093] The antibacterial activity of the polypeptides in this application against Vibrio parahaemolyticus is as follows Figure 5 As shown in the results, the diameter of the inhibition zone of the control group was 22 mm, and the diameter of the inhibition zone of the experimental group was 25 mm, indicating that the polypeptide fermentation broth in the present application can inhibit the growth of Vibrio parahaemolyticus, and the inhibitory effect is more significant than that of 100 μg / ml neomycin sulfate.

[0094] In summary, the polypeptide described in this application is a novel antimicrobial peptide that exhibits broad-spectrum antibacterial activity against Gram-positive bacteria, represented by Staphylococcus aureus, as well as Gram-negative bacteria, such as Pseudomonas aeruginosa, Salmonella, Escherichia coli, and Vibrio parahaemolyticus. The inhibitory activity against Staphylococcus aureus and Vibrio parahaemolyticus is particularly significant. Its use as an alternative to antibiotics is of great significance in addressing the antibiotic resistance crisis.

Claims

1. Use of a polypeptide in the preparation of an agent for inhibiting bacteria, characterized in that: The amino acid sequence of the polypeptide is shown in SEQ ID NO.1; the bacteria are Gram-positive bacteria and / or Gram-negative bacteria; the Gram-positive bacteria are Staphylococcus aureus ( Staphylococcus aureus ); The Gram-negative bacteria is Pseudomonas aeruginosa ( Pseudomonas aeruginosa ),salmonella( salmonella ), Escherichia coli ( Escherichia coli ) and / or Vibrio parahaemolyticus ( Vibrio Parahemolyticus ).

2. The use according to claim 1, characterized in that The agent for inhibiting bacteria is a fermentation broth containing the polypeptide.

3. The use according to claim 2, characterized in that The fermentation broth is a fermentation broth of recombinant Bacillus subtilis, and the recombinant Bacillus subtilis comprises a nucleic acid encoding the polypeptide.

4. The use according to claim 3, characterized in that The nucleotide sequence of the nucleic acid is shown in SEQ ID NO.

2.

5. The use according to claim 3 or 4, characterized in that The recombinant Bacillus subtilis comprises a recombinant plasmid containing the nucleic acid.

6. The use according to claim 5, characterized in that The plasmid is selected from any one of pWB980, pTTB1, pHT01, pHT253, and pHY-p43; and / or the Bacillus subtilis is selected from Bacillus subtilis 168 、 Bacillus subtilis WB800N 、 Bacillus subtilis Bsn5 、 Bacillus subtilis ATCC6051a 、 Bacillus subtilis var.natto 、 Bacillus subtilis RIK1285 Any one of .

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