A strain of Bacillus licheniformis, antibacterial agents, methods for preparing antibacterial active substances and their applications

Abstract

This invention belongs to the field of biotechnology, specifically relating to a strain of Bacillus licheniformis, an antibacterial agent, a method for preparing antibacterial active substances, and their applications. The Bacillus licheniformis DCGGN-121 provided by this invention has the preservation number CCTCC NO: M 2024878. The bacteriocins prepared from this strain are effective against African swine fever virus (ASFV), Seneca virus (SVA), and enterovirus 71 (EV71). The results of the examples show that the bacteriocins prepared from Bacillus licheniformis DCGGN-121 can significantly reduce the replication levels of ASFV, SVA, and EV71, as well as reduce the expression of ASFV-p72, SVA-VP2, and EV71-VP1 proteins. Bacillus licheniformis DCGGN-121 can be used to prepare natural microecological preparations, suitable for livestock farming.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a strain of Bacillus licheniformis, an antibacterial agent, a method for preparing antibacterial active substances, and their applications. Background Technology

[0002] Bacteriocins are small cationic peptides produced by bacterial ribosomes that have antibacterial effects. They have advantages such as strong antibacterial activity, safety without side effects, no inhibition by antibacterial agents, no impact on the intestinal microbiota, and low likelihood of developing widespread drug resistance. They are at the forefront of research on novel microecological preparations in recent years.

[0003] Bacteriocins are antimicrobial peptides synthesized by many bacteria via ribosomes during their primary growth stages, possessing antimicrobial, anticancer, and antibiofilm effects. Studies have found that probiotics produce various bacteriocins, protein metabolites with antiviral effects. Bacteriocins can block host cell receptor sites to prevent viral invasion, induce cytopathic effects while reducing viral release, and also resist viruses by regulating the mucosal system and systemic immune system in humans and animals. For example, bacteriocins secreted by some Enterococci can fight influenza and polioviruses; bacteriocins produced by *Lactobacillus mucosa* have activity against African swine fever virus and pseudorabies virus; and plantaricin BN and plantaricin W can combat SARS-CoV-2 infection by blocking the cell receptor-binding domain of the SARS-CoV-2 S protein or the interaction between the S protein and the host cell angiotensin-converting enzyme 2 (ACE2) receptor protein. These findings demonstrate the potential of probiotic-produced bacteriocins in the prevention and treatment of viral diseases.

[0004] Existing technologies have reported that Bacillus licheniformis can effectively reduce and inhibit white spot syndrome virus (see CN104388333A A Bacillus licheniformis and its application), but its antiviral effect is limited. Therefore, the search for novel broad-spectrum Bacillus licheniformis and the preparation of bacteriocins that inhibit broad-spectrum viruses are of great significance. Summary of the Invention

[0005] The purpose of this invention is to provide a strain of Bacillus licheniformis, an antibacterial agent, a method for preparing antibacterial active substances, and their applications. The bacteriocins prepared from Bacillus licheniformis DCGGN-121 provided by this invention can resist African swine fever virus, Seneca virus, and EV71 virus.

[0006] To address the aforementioned technical problems, the present invention provides the following technical solution:

[0007] This invention provides a strain of Bacillus licheniformis DCGGN-121, with accession number CCTCC NO: M 2024878.

[0008] This invention provides the application of Bacillus licheniformis DCGGN-121 described in the above technical solution in the preparation of antibacterial active substances.

[0009] Preferably, the antibacterial active substance includes bacteriocins; the amino acid sequence of the bacteriocins is shown in SEQ ID NO.1.

[0010] This invention provides a method for preparing antibacterial active substances using Bacillus licheniformis DCGGN-121 as described in the above technical solution, comprising:

[0011] The Bacillus licheniformis DCGGN-121 was inoculated into a culture medium and cultured to obtain a cell-free fermentation broth.

[0012] The cell-free fermentation broth was mixed with ammonium sulfate and allowed to stand to obtain crude active substances.

[0013] The crude active substance was subjected to ultrafiltration and purification to obtain an antibacterial active substance.

[0014] The present invention provides an antibacterial agent comprising the metabolites of Bacillus licheniformis DCGGN-121 described in the above technical solution.

[0015] Preferably, the metabolite includes cell-free fermentation broth or antibacterial active substances prepared by the method described above.

[0016] This invention provides the application of the antibacterial active substance or the antibacterial agent prepared by the above-described Bacillus licheniformis DCGGN-121 or by the above-described preparation method in the preparation of antiviral drugs.

[0017] Preferably, the antiviral agent includes one or more of the following: anti-African swine fever virus, anti-Seneca virus, and anti-EV71 virus.

[0018] This invention provides the application of the antibacterial active substance prepared by the above-described Bacillus licheniformis DCGGN-121 or the preparation method described in the above-described technical solution, or the antibacterial agent described in the above-described technical solution, in the preparation of microecological preparations.

[0019] Preferably, the product form of the microecological preparation includes feed additives.

[0020] The beneficial effects of this invention: This invention provides a strain of Bacillus licheniformis DCGGN-121, with the preservation number CCTCCNO: M 2024878. The bacteriocin prepared from Bacillus licheniformis DCGGN-121 provided by this invention is effective against African swine fever virus (ASFV), Seneca virus (SVA), and enterovirus 71 (EV71). The results of the examples show that the bacteriocin prepared from Bacillus licheniformis DCGGN-121 can significantly reduce the replication levels of ASFV, SVA, and EV-71, and reduce the expression of ASFV-p72, SVA-VP2, and EV71-VP1 proteins, exhibiting significant in vitro anti-ASFV, SVA, and EV71 virus activity. Bacillus licheniformis DCGGN-121 can be used to prepare a safe, efficient, and green natural microecological preparation, suitable for livestock farming, protecting public health, and other fields. Attached Figure Description

[0021] Figure 1 Colony morphology and Gram staining microscopic examination (1000×) of Bacillus licheniformis DCGGN-121. The left side shows the colony morphology, and the right side shows the microscopic examination results.

[0022] Figure 2 Phylogenetic analysis diagram of Bacillus licheniformis DCGGN-121;

[0023] Figure 3 Image showing the anti-ASFV activity of Bacillus licheniformis DCGGN-121 in pigs.

[0024] Figure 4 SDS-PAGE electrophoresis image of bacteriocin produced by Bacillus licheniformis DCGGN-121;

[0025] Figure 5 Mass spectrometry analysis of bacteriocins produced by Bacillus licheniformis DCGGN-121;

[0026] Figure 6 The figure shows the effect of bacteriocins produced by Bacillus licheniformis DCGGN-121 on RNA replication and protein expression of ASFV, SVA and EV71. In the figure, A represents RNA replication and protein expression of EV71, B represents RNA replication and protein expression of SVA and C represents RNA replication and protein expression of ASFV.

[0027] Biological Preservation Instructions

[0028] Bacillus licheniformis DCGGN-121 was deposited on May 8, 2024, at the China Center for Type Culture Collection (CCTCC), with accession number CCTCC NO: M2024878, located at Wuhan University, Wuhan, China. Detailed Implementation

[0029] This invention provides a strain of Bacillus licheniformis DCGGN-121, with accession number CCTCC NO: M 2024878. The 16S rRNA gene sequence of Bacillus licheniformis DCGGN-121 described in this invention is shown in SEQ ID NO.25.

[0030]

[0031] This invention uses fresh pig gastrointestinal contents as raw material to isolate Bacillus licheniformis DCGGN-121, which has broad-spectrum antiviral activity.

[0032] The Bacillus licheniformis DCGGN-121 described in this invention has a colony diameter of 2-3 mm on LB agar plates. It is a white colony with irregular edges, an uneven surface, and wrinkles.

[0033] This invention utilizes the 16S rRNA sequence of Bacillus licheniformis DCGGN-121 for BLAST homology sequence search in NCBI's GenBank, constructs a phylogenetic tree using MEGA8 software, and identifies strain DCGGN-121 as Bacillus licheniformis based on morphological characteristics. The Bacillus licheniformis DCGGN-121 provided by this invention is acid-resistant, bile-resistant, and heat-resistant, exhibiting significant antiviral activity against African swine fever virus (ASFV), Seneca virus (SVA), and enterovirus 71 (EV71). Furthermore, safe, efficient, and green natural antiviral drugs can be prepared using Bacillus licheniformis DCGGN-121, applicable to livestock farming and public health.

[0034] This invention provides the application of Bacillus licheniformis DCGGN-121 described in the above technical solution in the preparation of antibacterial active substances.

[0035] In this invention, as one embodiment, the antibacterial active substance includes bacteriocins; the amino acid sequence of the bacteriocins is shown in SEQ ID NO.1. The prepared bacteriocins are effective against African swine fever virus (ASFV), Seneca virus (SVA), and enterovirus 71 (EV71).

[0036] This invention provides a method for preparing antibacterial active substances from Bacillus licheniformis DCGGN-121 as described in the above technical solution, comprising:

[0037] The Bacillus licheniformis DCGGN-121 described in the above technical solution was inoculated into a culture medium and cultured to obtain a cell-free fermentation broth;

[0038] The cell-free fermentation broth was mixed with ammonium sulfate and allowed to stand to obtain crude active substances.

[0039] The crude active substance was subjected to ultrafiltration and purification to obtain an antibacterial active substance.

[0040] In one implementation, the present invention inoculates the *Bacillus licheniformis* DCGGN-121 described in the above-mentioned technical solution into a culture medium for cultivation to obtain a cell-free fermentation broth. In another embodiment, the present invention inoculates the seed culture of *Bacillus licheniformis* DCGGN-121 described in the above-mentioned technical solution into a culture medium for cultivation, centrifugation, and filtration to obtain a cell-free fermentation broth.

[0041] As one embodiment, the method for preparing the seed culture of Bacillus licheniformis DCGGN-121 according to the present invention can be as follows: a single colony of Bacillus licheniformis DCGGN-121 is inoculated onto an LB solid medium plate and cultured to obtain the DCGGN-121 seed culture. The present invention does not impose any special limitations on the culture parameters; conventional methods can be used.

[0042] In one embodiment, the inoculation amount of the seed culture in this invention can be 1% to 3% of the culture medium volume. In specific embodiments of this invention, the inoculation amount can be 1%, 2%, or 3% of the culture medium volume. The culture temperature in this invention can be 35℃ to 40℃; in specific embodiments of this invention, the culture temperature can be 35, 36, 37, 38, 39, or 40℃; the culture time can be 22 to 26 hours; in specific embodiments of this invention, the culture time can be 22 hours, 23 hours, 24 hours, 25 hours, or 26 hours. The culture medium in this invention can be LB liquid medium, and the preferred composition of the LB liquid medium is: 10g peptone, 5g yeast extract, 10g sodium chloride, and 1000mL distilled water.

[0043] As one implementation method, after obtaining the culture medium, the present invention can centrifuge the culture medium at a speed of 8000 rpm for 30 min at a temperature of 4°C.

[0044] In one implementation method, after centrifugation, a supernatant is obtained. This supernatant is then filtered using a 0.22 μm diameter filter membrane. The resulting filtration yields a cell-free fermentation broth. The purpose of filtration is to remove antibacterial substances from the metabolites. The cell-free fermentation broth of this invention contains only the metabolites of Bacillus licheniformis DCGGN-121 and does not contain Bacillus licheniformis DCGGN-121 cells.

[0045] This invention involves mixing and allowing the cell-free fermentation broth to stand, thereby obtaining a crude extract of active substances. As one embodiment, ammonium sulfate can be added to the cell-free fermentation broth until the ammonium sulfate saturation in the broth reaches 80%. The ammonium sulfate can be added in powder form. The mixing method may include a first stirring and a second stirring; the addition may accompany the first stirring, and the addition should be slow; the first stirring should stop after the ammonium sulfate addition is complete; the first stirring can be performed using a stirring rod; the purpose of the first stirring is to promote the dissolution of ammonium sulfate. As one embodiment, the second stirring and standing are performed after the ammonium sulfate saturation in the cell fermentation broth reaches 80%, i.e., after the ammonium sulfate addition is complete. The second stirring can be performed using a magnetic stirrer, and the second stirring time can be 1 hour. The standing temperature can be 4°C, and the standing time can be 12–24 hours, or overnight. As one embodiment, after standing, the obtained product is centrifuged to obtain a precipitate, which is the crude extract of active substances. The centrifugation speed can be 10,000 rpm, and the time can be 30 minutes. Ammonium sulfate can precipitate and concentrate the protein components in the cell-free fermentation broth.

[0046] In one embodiment, the crude active substance is subjected to ultrafiltration and purification to obtain an antibacterial active substance. In another embodiment, the crude active substance is dialyzed and then subjected to ultrafiltration and purification to obtain an antibacterial active substance. Before dialysis, the crude active substance is mixed with phosphate-buffered saline (PBS) and then dialysis is performed. The dialysis bag used has a molecular weight cutoff of 3.5 kDa, and the dialysis solution used is PBS buffer. The purpose of dialysis is to remove ammonium sulfate.

[0047] In one embodiment, ultrafiltration can be performed using ultrafiltration centrifuge tubes. The molecular weight cutoff of the ultrafiltration centrifuge tubes can be 3 kDa or 30 kDa, and proteins with molecular weights between 3 kDa and 30 kDa are obtained after ultrafiltration centrifugation. In another embodiment, the proteins retained by the ultrafiltration centrifuge tubes are reconstituted with PBS and filtered through a filter membrane for further purification. Conventional parameters are sufficient for PBS reconstitution. The diameter of the filter membrane can be 0.22 μm. The purification method can be an ion exchange column. During the purification process, African swine fever virus (ASFV) strain CN / GS / 2018 can be used to monitor antiviral activity to ensure successful separation of the antibacterial active substance produced by *Bacillus licheniformis* DCGGN-121. The antibacterial active substance prepared by this invention is the protein component in the cell-free fermentation broth.

[0048] The present invention provides an antibacterial agent comprising metabolites of Bacillus licheniformis DCGGN-121 as described in the above technical solution.

[0049] In this invention, as one implementation, the metabolites of Bacillus licheniformis DCGGN-121 in the antibacterial agent may include cell-free fermentation broth or antibacterial active substances prepared by the method described above. The preparation method of the cell-free fermentation broth has been described above and will not be discussed further here.

[0050] This invention provides the application of the antibacterial active substance or the antibacterial agent prepared by the above-described Bacillus licheniformis DCGGN-121 or the method described in the above-described technical solution in the preparation of antiviral drugs.

[0051] As one implementation method, the virus may include one or more of African swine fever virus, Seneca virus, and EV71 virus.

[0052] This invention provides the application of Bacillus licheniformis DCGGN-121 as described in the above technical solution, or the antibacterial active substance or antibacterial agent as described in the above technical solution, in microecological preparations.

[0053] As one implementation method, the product form of the microecological preparation may include feed additives.

[0054] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0055] Example 1: Isolation and identification of Bacillus licheniformis DCGGN-121:

[0056] 1. Materials, strains, and cell sources: Strains were screened using fresh porcine gastrointestinal contents. Samples were sourced from Diebu County, Gannan Prefecture, Gansu Province, and transported to the laboratory under sterile plastic bags at low temperatures. Porcine alveolar macrophages (PAMs), hamster kidney fibroblasts (BHK-21), and bovine kidney cells (MDBK) were preserved in our laboratory.

[0057] 2. The indicator strains used in the antiviral experiments were African swine fever virus (ASFV) CN / GS / 2018 strain, bovine viral diarrhea virus (BVDV) Oregon C24V strain, and foot-and-mouth disease virus (FMDV) O-MYA98 strain preserved in our laboratory.

[0058] 3. Culture medium and other biochemical reagents

[0059] LB medium: Dissolve 10g peptone, 5g yeast extract, and 10g sodium chloride in 1000mL distilled water by heating to obtain LB liquid medium; LB solid medium is obtained by adding 1.5% agar to LB liquid medium. Autoclave LB medium at 121℃ for 15min.

[0060] Nutrient broth culture medium: 10g peptone, 3g beef extract powder, 5g sodium chloride, dissolved in 1000mL distilled water by heating, and then autoclaved at 121℃ for 15min.

[0061] Glucose-tryptone medium: 10g tryptone, 5g glucose, 0.04g bromocresol purple, and 15g agar are dissolved in 1000mL distilled water by heating to adjust the pH to 6.9±0.2. The mixture is then autoclaved at 121℃ for 15min and poured into plates for later use.

[0062] DNA Ligation Kit (Mighty Mix), Taq DNA Polymerase, Molecular Marker, ChamQ Universal qPCR Master Mix was purchased from Nanjing Novizan Biotechnology Co., Ltd.; MEM / DMEM cell culture medium, PBS solution, 0.25% EDTA trypsin and newborn calf serum (FBS) were all purchased from BI; Trizol reagent was purchased from Baoriyi Biotechnology (Beijing) Co., Ltd.; enhanced chemiluminescence (ECL) substrate was purchased from Advansta Biotechnology Co., Ltd.; pre-stained protein markers were purchased from Yaxin Biotechnology Co., Ltd.; self-made rabbit anti-ASFV-p72, FMDV-VP0, and BVDV-Core antibodies were stored in our laboratory; HRP goat anti-mouse IgG and HRP goat anti-rabbit IgG were purchased from Beijing Bio-Long Immunotherapy Co., Ltd.

[0063] Hydrochloric acid and sodium chloride were domestically produced analytical grade reagents. The bacterial DNA extraction kit was purchased from Beijing Tiangen Biotech Co., Ltd., and the Gram staining kit was purchased from Sigma-Aldrich (USA).

[0064] 4. Experimental Methods

[0065] (1) Isolation of Bacillus licheniformis DCGGN-121: In a clean bench, fresh pig gastrointestinal contents were collected, labeled, and approximately 1g of each was dispensed into sterile test tubes. 5mL of nutrient broth was added to each tube. The tubes were incubated at 37℃ in a shaker for 2 hours, then in a water bath at 80℃ for 15 minutes to kill non-spore-forming bacteria. After cooling, 100μL was spread onto LB agar plates and incubated upside down at 37℃ for 24 hours, resulting in white colonies with a diameter of 2–3mm, irregular edges, a rough surface, and wrinkles. Single colonies were picked and cultured in LB liquid for further incubation. The streaking process was repeated until single colonies were obtained.

[0066] (2) Preliminary screening of strains: Resuscitated strains: The strains obtained in step (1) were inoculated into LB liquid medium and cultured in a shaker at 37°C until OD600 The concentration was set to 0.8, and bacterial solutions of each strain were obtained.

[0067] A: Acid tolerance screening: Prepare LB liquid culture medium with pH values ​​of 2.0, 2.5, 3.0, 3.5, 4.0 and 6.8 respectively.

[0068] The bacterial cultures of each strain were transferred to 5 mL of culture medium with different pH values ​​at a ratio of 5% of the culture medium volume, and normal LB liquid medium with pH 6.8 was used as a blank control.

[0069] After transfer, each culture medium was placed in a 37℃ incubator for 2 hours. Then, 100 μL of the resulting culture solution was plated on glucose tryptone medium and incubated upside down for 24 hours. The plates were counted and the survival rate of each strain was calculated. The strains with strong acid resistance that could survive for 2 hours in LB liquid medium at pH 2.0 and had a survival rate of more than 35% were preliminarily screened.

[0070] B: Bile salt tolerance screening: Prepare LB liquid medium containing 0.1%, 0.2%, 0.3%, 0.4%, and 0.5% ox bile salts. Transfer the bacterial suspension of each acid-tolerant strain to 5 mL of LB liquid medium with different bile salt concentrations at a ratio of 1% (v / v). Incubate at 37°C for 12 h. Spread 100 μL of the resulting culture onto LB agar plates and incubate for 24 h. Count the plates and calculate the survival rate of the strains. Preliminary screening is conducted for strains with a survival rate of over 50% after 12 h of treatment in 0.3% bile salt medium, demonstrating strong bile salt tolerance.

[0071] C: High-temperature resistance screening: The obtained acid-resistant and bile-salt-resistant strains were inoculated into LB liquid medium and cultured overnight at 37°C to obtain bacterial suspensions. The obtained bacterial suspensions were then treated in metal baths at 70°C, 75°C, 80°C, 85°C, 90°C, 95°C, and 100°C for 10 min, respectively. After cooling, 100 μL of the bacterial suspension was plated. The bacterial suspensions without high-temperature treatment served as blank controls. After culturing both the high-temperature treatment group and the control group for 24 h, plate counting was performed to preliminarily screen strains with a survival rate of over 50% after treatment at 85°C for 10 min.

[0072] After preliminary screening, a strain that is resistant to acid, bile salts and heat was obtained and named DCGGN-121. The colony morphology on LB medium is as follows: white colonies with a diameter of 2-3 mm, irregular edges, rough surface and wrinkles. Figure 1 Microscopic examination of Bacillus licheniformis colonies and Gram staining (1000×) revealed that strain DCGGN-121 is a Gram-positive short bacillus.

[0073] (3) Activation identification and preservation of strain: single colonies of strain DCGGN-121 were transferred to 5 mL of LB liquid culture medium and cultured at 37℃ for 48 h. Then, the culture was subcultured twice with an inoculation amount of 1% (v / v) and stored after activation.

[0074] The strain DCGGN-121 was amplified by PCR using Bli-man-1F (5'-AGCCATGGATATTAAATA AC-3', SEQ ID NO:21); Bli-man-1R (5'-TATTCCCTTACAATAAGACG-3', SEQ ID NO:22) and 16S rRNA primers (27F-AGAGTTTGATCMTGGCTCAG, SEQ ID NO:23, 1492R-TACGGTYACCTTGTTGTTACGACTT, SEQ ID NO:24). The resulting products were sequenced, and the sequences were uploaded to NCBIblast for alignment. A phylogenetic tree was constructed using MEGA8 software. Figure 2 As shown. Figure 2 Phylogenetic analysis diagram of Bacillus licheniformis DCGGN-121 Figure 2 WBL 009 in the text represents DCGGN-121. According to... Figure 2 It is known that strain DCGGN-121 (i.e. WBL 009) is Bacillus licheniformis, which has a high similarity to Bacillus licheniformis strain SR5 in the GenBank database and is on the same evolutionary branch.

[0075] (4) Culture of indicator virus: PAMs cells were seeded in 12-well plates. When the cell density reached 70%–80%, ASFV cells were infected with a multiplicity of infection (MOI) of 1. ASFV cell samples were collected 24 hours after infection and recorded as the first-generation seed virus. The fourth-generation ASFV seed virus was obtained by propagation with PAMs cells and the titer was 10. 6 HAD 50 / 100μL, dispensed and stored at 80℃ for later use.

[0076] Using the same method of culturing ASFV seed virus, FMDV was propagated in BHK-21 cells to obtain the 4th generation FMDV seed virus with a titer of 10. 6 TCID 50 / mL, aliquot and store at 80℃ for later use.

[0077] Using the same method of culturing ASFV seed virus, BVDV was propagated in MDBK cells to obtain the 4th generation BVDV seed virus with a titer of 10. 5 TCID 50 / mL, aliquot and store at 80℃ for later use.

[0078] Antiviral activity identification of Bacillus licheniformis DCGGN-121: A single colony of Bacillus licheniformis DCGGN-121 was transferred to 5 mL of LB liquid medium and incubated at 37 °C for 48 h. The culture was then inoculated into 1 L of LB liquid medium at 1% (v / v). The cultured culture for 24 h was centrifuged at 8000 rpm and 4 °C for 30 min. The supernatant was filtered through a 0.22 μm filter membrane to obtain cell-free fermentation broth.

[0079] PAMs cells, BHK-21 cells, and MDBK cells were seeded in 12-well plates. When the cell density reached 70%–80%, the cells were infected with a multiplicity of infection (MOI) of 1 (PAMs cells were infected with 4th generation ASFV, BHK-21 cells with 4th generation FMDV, and MDBK cells with 4th generation BVDV). At the same time, 200 μL of the cell-free fermentation broth was added to the cell culture medium. Cell samples were collected 24 h after virus infection. The effect on the replication of the virus was analyzed by quantitative real-time PCR (primers are shown in Table 1) and Western blotting. Bacillus licheniformis DCGGN-121 showed anti-ASFV activity.

[0080] Example 2: Isolation, identification, and antiviral study of bacteriocins produced by Bacillus licheniformis DCGGN-121:

[0081] 1. Materials, strains, and cell sources: The cell-free fermentation broth isolated in Example 1 was used, and the indicator bacteria for the antibacterial activity assays in the following experiments were all African swine fever virus (ASFV) strain CN / GS / 2018. Antiviral activity monitoring was performed after each purification step to ensure the successful isolation of bacteriocins produced by Bacillus licheniformis DCGGN-121. PAMs cells, BHK-21 cells, MDBK cells, and HEK293T cells were preserved in our laboratory.

[0082] The indicator strains used in the antiviral experiments were African swine fever virus (ASFV) CN / GS / 2018 strain, bovine viral diarrhea virus (BVDV) Oregon C24V strain, foot-and-mouth disease virus (FMDV) O-MYA98 strain, enteritis virus type 71 (EV-71), and Seneca virus (SVA) CH-FJ-2017 strain preserved in our laboratory.

[0083] 2. Preparation of culture media, other biochemical reagents and related liquids

[0084] DMEM cell culture medium, PBS solution, 0.25% trypsin-EDTA, and newborn calf serum (FBS) were all purchased from BI; Trizol reagent was purchased from Baori Medical (Beijing) Co., Ltd.; ChamQ Universal... qPCR Master Mix was purchased from Novizan Biotechnology Co., Ltd.; enhanced chemiluminescence (ECL) substrate was purchased from Advansta Biotechnology Co., Ltd.; and pre-stained protein markers were purchased from Yageo Biotechnology Co., Ltd.

[0085] HiTrap Capto S pre-loaded columns were purchased from Cytiva, Sweden; ultrafiltration tubing was purchased from Merck Millipore.

[0086] Ethanol, ammonium sulfate, hydrochloric acid, and sodium chloride were all domestically produced analytical grade. Dimethyl sulfoxide (DMSO) was purchased from AMRESCO. Mouse anti-β-actin monoclonal antibody (ARM2001) was purchased from Shanghai Nolai Biotechnology. Self-made rabbit anti-ASFV-p72, FMDV-VP0, BVDV-Core, SVA-VP2, and EV71-VP1 antibodies were stored in our laboratory. HRP goat anti-mouse IgG and HRP goat anti-rabbit IgG were purchased from Beijing Bio-Long Immunotherapy Co., Ltd.

[0087] The reagents involved in the examples were prepared as follows:

[0088] Staining solution: Weigh 1g of R-250, add 250mL of isopropanol, 100mL of glacial acetic acid, and 600mL of double-distilled water. Stir to dissolve each step, and finally filter through filter paper to remove particles before use.

[0089] Decolorizing solution: Add 100mL of glacial acetic acid, 50mL of ethanol and 850mL of double-distilled water in sequence.

[0090] Buffer A: 50mM sodium acetate (4.1g sodium acetate added to 1L buffer A), pH adjusted to 5.0 with concentrated hydrochloric acid, used for filtration through a 0.22μm filter membrane.

[0091] Buffer B: 50mM sodium acetate, 1M NaCl (add 58.5g NaCl and 4.1g sodium acetate to 1L of buffer B), adjust the pH to 5.0 with concentrated hydrochloric acid, and filter through a 0.22μm filter membrane.

[0092] 3. Experimental Methods

[0093] (1) Preparation of cell-free fermentation broth of Bacillus licheniformis DCGGN-121: The DCGGN-121 strain preserved in glycerol tubes at -80℃ was activated by streaking on LB solid medium plates. After activation and culture at 37℃ for 24h, a single colony was picked and transferred to 5mL of LB liquid medium and cultured at 37℃ for 48h. The culture was then inoculated into 1L of LB liquid medium at 1% (v / v) and cultured for 24h. The resulting bacterial culture was centrifuged at 8000rpm and 4℃ for 30min. The supernatant was filtered through a 0.22μm filter membrane to obtain cell-free fermentation broth.

[0094] (2) Preparation of crude antiviral extract of Bacillus licheniformis DCGGN-121: The cell-free fermentation broth obtained in step (1) was slowly added to the ground ammonium sulfate powder while stirring, so that the saturation of ammonium sulfate reached 80%. After stirring slowly on a magnetic stirrer for 1 hour, it was placed in a refrigerator at 4°C overnight. The next day, it was taken out and centrifuged at 10,000 rpm for 30 minutes to obtain a precipitate, which is the crude active substance. 2 mL of 0.01 M phosphate buffer (PBS) was added to the above crude active substance to dissolve the precipitate. The precipitate was placed in a dialysis bag with a molecular weight cutoff of 3.5 kDa. The dialysis bag was then placed in 2 L of PBS buffer for overnight dialysis to remove ammonium sulfate and obtain the crude antiviral extract.

[0095] (3) Purification of the crude antiviral extract of Bacillus licheniformis DCGGN-121: The crude antiviral extract obtained in step (2) was centrifuged sequentially using ultrafiltration tubes with molecular weight cutoffs of 3 kDa and 30 kDa. The cutoff protein was reconstituted with PBS and filtered through a 0.22 microporous membrane to obtain the protein. The obtained protein was used for antiviral activity (ASFV) monitoring, using the same method as in "Identification of Antiviral Activity of Bacillus licheniformis DCGGN-121" in Example 1, except that 200 μL of cell-free fermentation broth added to the infected cells was replaced with the protein. The results showed that the protein could resist ASFV virus.

[0096] (4) Select protein samples with antiviral activity from step (3) for ion exchange column purification: First, equilibrate the pre-packed column with buffer A, load the sample, rinse with buffer A and collect the flow-through, collect flow-through 1; then perform gradient elution with buffer A and buffer B, the molar concentration of NaCl in the eluent during gradient elution is 20mM, 40mM, 80mM, 100mM and 200mM respectively (when eluting with 20mM, collect elution 1 and elution 2 in segments, when eluting with 40mM, collect elution 1 and elution 2 in segments with buffer B, when eluting with 80mM, when eluting with 100mM, collect elution 1 and elution 2 in segments with buffer B, and when eluting with 200mM, collect elution 2 in segments with buffer B). The results show that when the molar concentration of NaCl in the eluent is 100mM, the collected protein sample has anti-ASFV activity, and the target protein eluted with 100mM NaCl and buffer B is bacteriocin.

[0097] (5) PAMs cells were seeded in 12-well plates. When the cell density reached 70%–80%, the cells were infected with ASFV at a multiplicity of infection (MOI) of 1. Different concentrations of bacteriocin (0, 25, 100, and 500 μg / mL) were added simultaneously. Cell samples were collected 24 hours after infection. The effect of bacteriocin on viral replication was analyzed by quantitative real-time PCR (primers are shown in Table 1) and Western blotting. The results are shown in [Table 1]. Figure 3 It can be seen that bacteriocins have anti-ASFV activity.

[0098] (6) Identification of bacteriocin produced by Bacillus licheniformis DCGGN-121: The collected protein samples were subjected to SDS-PAGE electrophoresis. The target band was found to be approximately 14 kDa. The results are shown in […]. Figure 4 . Figure 4 SDS-PAGE electrophoresis analysis of bacteriocin produced by Bacillus licheniformis DCGGN-121 (1: loaded sample; M: Marker; 2: flow-through 1; 3: elution with 20mM B solution 1; 4: elution with 20mM B solution 2; 5: elution peak 1 with 40mM B solution; 6: elution peak 2 with 40mM B solution; 7: elution with 80mM B solution; 8: elution with 100mM B solution 1; 9: elution with 100mM B solution 2; 10: elution with 200mM B solution). The loaded sample was the protein obtained in step (3).

[0099] according to Figure 4 It can be seen that the target protein (bacteriocin) can be eluted with 100mM NaCl B buffer, and its size is about 14Kd.

[0100] (7) The protein concentration of bacteriocins was determined by BCA and sent to Shanghai Baipu Biotechnology Co., Ltd. for LC-MS / MS determination of the amino acid sequence of the purified bacteriocins, see SEQ ID NO:1. The molecular weight of the purified bacteriocins is about 14.2 kDa and the N-terminal sequence is MKFEESGIEGLTADLNR (SEQ ID NO:20). The amino acid sequence of the bacteriocins produced by Bacillus licheniformis DCGGN-121 encodes a total of 119 amino acids. Figure 5 Mass spectrometry analysis of bacteriocin production by Bacillus licheniformis DCGGN-121. Figure 5 This indicates that the corresponding bacteriocins can be identified by the unique peptide segment corresponding to the N-terminal sequence.

[0101] SEQ ID NO: 1: MKFEESGIEGLTADLNRLDDIMENEGFVRAGQWDYERV TYDRKFDMVEGRFYLRVFGYAIEGDVGARRAVIKLMTPLLGKYYYPHGVEYGEDEHFPPQLLKTSKTVLNSIKEKVSALEV.

[0102] (8) Identification of antiviral activity of bacteriocin produced by Bacillus licheniformis DCGGN-121: PAMs cells, PK15 cells, BHK-21 cells, MDBK cells, and HEK293T cells were seeded in 12-well plates. When the cell density reached 70%–80%, they were infected with viruses at a multiplicity of infection (MOI) of 1. PAMs cells were infected with ASFV, PK15 cells with SVA, BHK-21 cells with FMDV, MDBK cells with BVDV, and HEK293T cells with EV-71. Different concentrations of bacteriocin (0, 25, 100, and 500 μg / mL) were added to each infected cell type. Cell samples were collected 24 h after virus infection. The effect of bacteriocin on the replication of the above viruses was analyzed by quantitative real-time PCR (primers are shown in Table 1) and Western blotting. Figure 6 The study investigated the effects of bacteriocin produced by Bacillus licheniformis DCGGN-121 on RNA replication and protein expression of different viruses. A represents EV71, B represents SVA, and C represents ASFV. The results showed that the bacteriocin significantly reduced the replication levels of EV-71, ASFV, and SVA, as well as the expression of EV71-VP1, ASFV-p72, and SVA-VP2 proteins, demonstrating significant in vitro antiviral activity against ASFV, SVA, and EV71. However, it had no inhibitory effect on FMDV and BVDV.

[0103] Table 1 Primers for Real-Time Quantitative PCR (RT-qPCR)

[0104] Primer name Primer sequence ASFV-qPCR-F GATACCACAAGATCAGCCGT(SEQ ID NO:2) ASFV-qPCR-R CTGCTCATGGTATCAATCTATCGA(SEQ ID NO:3) EV-qPCR-F GAGTGGCAGATGTGATTGA(SEQ ID NO:4) EV-qPCR-R TCCAGTGTCTAAGCGATGA(SEQ ID NO:5) SVA-qPCR-F GAATTTGGAAGCCATGCTC(SEQ ID NO:6) SVA-qPCR-R AGCCAACATAGARACCAGATTGC(SEQ ID NO:7) FMDV-qPCR-F CACTGGTGACAGGCTAAGG(SEQ ID NO:8) FMDV-qPCR-R CCCTTCTCAGATTCCGAGT(SEQ ID NO:9) BVDV-qPCR-F GCCATGCCCTTAGTAGGACT(SEQ ID NO:10) BVDV-qPCR-R CACCCTATCAGGCTGTRTYC(SEQ ID NO:11) Pig-GAPDH-qPCR-F ACATGGCCTCCAAGGAGTAAGA(SEQ ID NO:12) Pig-GAPDH-qPCR-R GATCGAGTTGGGGCTGTGACT(SEQ ID NO:13) Human-GAPDH-qPCR-F GATTCCACCCATGGCAAATTC(SEQ ID NO:14) Human-GAPDH-qPCR-R CTGGAAGATGGTGATGGGATT(SEQ ID NO:15) Mouse-GAPDH-qPCR-F GTGGCAAAGTGGAGATTGTTG(SEQ ID NO:16) Mouse-GAPDH-qPCR-R CGTTGAATTTGCCGTGAGTG(SEQ ID NO:17) Cattle-GAPDH-qPCR-F AGCGAGATCCTGCCAACATCAAG(SEQ ID NO:18) Cattle-GAPDH-qPCR-R GCAGGAGGCATTGCTGACAATCT(SEQ ID NO:19)

[0105] In summary, the *Bacillus licheniformis* DCGGN-121 provided by this invention can produce bacteriocins, with a molecular weight of 14280 Da. These bacteriocins exhibit broad-spectrum antiviral activity, possessing antiviral activity against African swine fever virus (ASFV), Seneca virus (SVA), and EV71 virus. Therefore, *Bacillus licheniformis* DCGGN-121 can be used to prepare a safe, efficient, and green natural microecological preparation suitable for livestock farming and public health protection.

[0106] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A strain of Bacillus licheniformis ( bacillus licheniformis DCGGN-121, accession number CCTCC NO: M2024878.

2. The use of Bacillus licheniformis DCGGN-121 as described in claim 1 in the preparation of bacteriocins; The amino acid sequence of the bacteriocin is shown in SEQ ID NO.

1.

3. A method for preparing bacteriocins using Bacillus licheniformis DCGGN-121 as described in claim 1, characterized in that, include: The Bacillus licheniformis DCGGN-121 was inoculated into a culture medium and cultured to obtain a cell-free fermentation broth. The cell-free fermentation broth was mixed with ammonium sulfate and allowed to stand to obtain crude active substances. The crude active substance was subjected to ultrafiltration and purification to obtain bacteriocin.

4. An antibacterial agent, characterized in that, Includes metabolites of Bacillus licheniformis DCGGN-121 as described in claim 1; The metabolite is a cell-free fermentation broth or a bacteriocin prepared using the method described in claim 3.

5. The use of the Bacillus licheniformis DCGGN-121 of claim 1, or the bacteriocin prepared by the preparation method of claim 3, or the antibacterial agent of claim 4, in the preparation of antiviral drugs; The antiviral agents include one or more of the following: anti-African swine fever virus, anti-Seneca virus, and anti-EV71 virus.

6. The use of the Bacillus licheniformis DCGGN-121 of claim 1, the bacteriocin prepared by the preparation method of claim 3, or the antibacterial agent of claim 4 in the preparation of microecological preparations.

7. The application according to claim 6, characterized in that, The product form of the microecological preparation includes feed additives.

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