Lactobacillus mucosa with porcine virus resisting activity and application thereof

Abstract

The invention discloses lactobacillus mucosa with porcine virus resisting activity and application of the lactobacillus mucosa, and relates to the technical field of microorganism application and virus prevention and control. It is found for the first time that the lactobacillus mucosa has broad-spectrum antiviral activity, not only has a remarkable inhibition effect on porcine reproductive and respiratory syndrome virus type 2 (PRRSV2) strains, but also can further resist porcine epidemic diarrhea virus (PEDV) and porcine rotavirus (PoRV); according to the present invention, the Lactobacillus mucosa is adopted to culture the supernatant, such that the supernatant does not have toxicity on Marc-145 cells; according to the PRRSV2 strain treated by using the supernatant, the expression level of virus RNA and protein and the filial generation virus titer are remarkably reduced, so that the infection ability is weakened; the culture supernatant directly acts with virus particles to block infection and replication of the virus particles; the product disclosed by the invention is derived from natural probiotics, is high in safety, environment-friendly and not easy to resist drugs, can be used for preparing a broad-spectrum anti-porcine virus biological preparation, provides a new strategy for preventing and controlling porcine virus diseases, and has a good industrial application prospect.

Description

Technical Field

[0001] This invention relates to the field of microbial application and virus control technology, specifically to a mucosal lactobacillus with anti-swine virus activity and its application. Background Technology

[0002] Porcine reproductive and respiratory syndrome (PRRS) is a highly contagious viral disease caused by Porcine reproductive and respiratory syndrome virus (PRRSV). Initially identified in the late 1980s, it has rapidly spread globally and is one of the most damaging diseases in the swine industry. PRRSV2 belongs to the Arteriviridae family and is a positive-sense single-stranded RNA virus. Pigs are its only natural host, and porcine alveolar macrophages are its primary target cells. Porcine reproductive and respiratory syndrome virus (PRRSV) is the main target cell in vivo; in vitro, PRRSV2 can replicate and spread in the monkey kidney cell line MARC-145; PRRSV2 mainly affects the lungs and respiratory tract, causing symptoms such as fever and fatigue; when infected in pregnant sows, the virus can cause reproductive disorders, manifested as late abortion, fetal mummification, and congenitally infected weak piglets; it causes severe economic losses to the pig industry worldwide and puts enormous pressure on disease control; although inactivated and attenuated vaccines have been developed, these interventions have significant limitations, and there is currently no specific drug against PRRSV2. The lack of cost-effective and effective drugs in clinical use remains a challenge, so there is an urgent need to explore safe and effective therapeutics to combat PRRS.

[0003] Porcine epidemic diarrhea virus (PEDV) and porcine rotavirus (PoRV) are the main pathogens causing viral diarrhea in pigs. PEDV is an alpha coronavirus whose S protein mediates viral invasion. PoRV belongs to the Reoviridae family, with group A being the main pathogenic serotype. Both target the epithelial cells of the small intestinal villi and are transmitted via the fecal-oral route, leading to villi atrophy, malabsorption, and severe watery diarrhea and dehydration. PEDV can cause up to 100% mortality in newborn piglets, while PoRV infection often causes disease in piglets aged 2-8 weeks. In recent years, mixed infections of the two have become increasingly serious, synergistically causing disease through mechanisms such as metabolic reprogramming, exacerbating intestinal damage. Existing vaccines have limited protective efficacy against variant strains, and there is an urgent need to develop novel antiviral strategies that target the intestinal mucosa and block viral invasion. Summary of the Invention

[0004] To address the problems existing in related technologies, one of the objectives of this invention is to provide a mucosal lactobacillus with anti-swine virus activity (…). Lactobacillus mucosae The *Lactobacillus mucosa* was deposited on March 16, 2026, at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The proposed classification name is *Lactobacillus mucosa*. Lactobacillus mucosae The accession number is CGMCC No. 37932.

[0005] The second objective of this invention is to propose a mucosal lactobacillus with anti-swine virus activity. Lactobacillus mucosae The preparation method of culture supernatant is as follows: Lactobacillus mucosa ( Lactobacillus mucosae The bacteria were inoculated onto MRS medium (selective isolation and counting medium for lactic acid bacteria) for fermentation culture to obtain fermentation broth; the fermentation broth was centrifuged, the supernatant was collected, the pH was adjusted, and the broth was filtered to obtain the culture supernatant of Lactobacillus mucosa.

[0006] The third objective of this invention is to propose a mucosal lactobacillus with anti-swine virus activity. Lactobacillus mucosae The supernatant has the following two applications: (1) The mucosal lactobacillus ( Lactobacillus mucosae Application of ) in suppressing swine viruses.

[0007] (2) The mucosal lactobacillus ( Lactobacillus mucosae Application of ) in the preparation of anti-swine virus biological agents.

[0008] Preferably, the porcine virus of the present invention is one or more of porcine reproductive and respiratory syndrome virus type 2 (PRRSV2), porcine epidemic diarrhea virus (PEDV), or porcine rotavirus (PoRV).

[0009] The beneficial effects of this invention are: (1) This invention is the first to discover a mucosal lactobacillus with broad-spectrum anti-swine virus biological activity. Lactobacillus mucosae It not only has a highly effective inhibitory effect on porcine reproductive and respiratory syndrome virus type 2 (PRRSV2), but can also further resist infection by porcine epidemic diarrhea virus (PEDV) and porcine rotavirus (PoRV), and has broad-spectrum antiviral potential across viral families (arteritisviridae, coronavirusidae, and reoviridae).

[0010] (2) After co-incubation with porcine virus, the culture supernatant of Lactobacillus mucosa can effectively inhibit / kill the virus strain, or reduce its relative expression level of viral RNA, expression level of viral protein, progeny virus titer and degree of cytopathic effect (CPE) caused by the virus, significantly weakening its virulence. This indicates that the culture supernatant of Lactobacillus mucosa can directly act on the virus particles and effectively block its infection and replication process.

[0011] (3) This invention is the first to demonstrate that mucosal lactobacilli can effectively antagonize PRRSV2 infection through a direct action mechanism, and preliminarily reveals its inhibitory potential against PEDV and PoRV, laying a theoretical foundation for the in-depth development and application of the biological functions of mucosal lactobacilli.

[0012] (2) Compared with traditional antiviral methods, the culture supernatant of *Lactobacillus mucosa* described in this invention is entirely derived from natural probiotics, with high safety, good environmental compatibility, and low susceptibility to inducing viral drug resistance. *Lactobacillus mucosa* and its culture supernatant are expected to be developed into a safe, efficient, broad-spectrum, eco-friendly anti-swine virus biological agent, pioneering a new path for broad-spectrum anti-swine virus biological control with probiotic products as active ingredients. This provides a new strategy for the prevention and control of various important swine viral diseases such as porcine reproductive and respiratory syndrome, porcine epidemic diarrhea, and porcine rotavirus disease, and has important scientific research value and industrial application prospects. Attached Figure Description

[0013] Figure 1 This is a diagram showing the pathological effect of Marc-145 cells in Example 2 of the present invention.

[0014] Figure 2 This is a diagram showing the pathological effect of Vero cells in Example 3 of the present invention. Figure 2 (a) is the negative control group. Figure 2 (b) is the PEDV virus group. Figure 2 (c) PEDV group after treatment with supernatant of Lactobacillus mucosa culture.

[0015] Figure 3 This is a diagram showing the pathological effect of MA104 cells in Example 4 of the present invention. Figure 3 (a) is the negative control group. Figure 3 (b) represents the PoRV virus group. Figure 3 (c) PoRV group after treatment with supernatant of Lactobacillus mucosa culture.

[0016] Figure 4 This refers to the inhibitory effect of RT-qPCR on the relative expression level of porcine reproductive and respiratory syndrome virus (PRRSV) RNA in the supernatant of Lactobacillus mucosa culture in Example 5 of this invention.

[0017] Figure 5This refers to the inhibitory effect of the supernatant of Lactobacillus mucosa culture on the expression level of N protein of porcine reproductive and respiratory syndrome virus (PRRSV) detected by immunoblotting in Example 6 of the present invention.

[0018] Figure 6 This is the inhibitory effect of the supernatant of Lactobacillus mucosa culture on the activity of porcine reproductive and respiratory syndrome virus detected by the indirect immunofluorescence method in Example 7 of the present invention. Detailed Implementation

[0019] The present invention will be further described in detail below with reference to specific embodiments and accompanying drawings, but the scope of protection of the present invention is not limited to the contents described therein; unless otherwise specified, the instruments and equipment used in the present invention are all conventional instruments and equipment, the reagents used are all commercially available analytical grade reagents, and the experimental methods used are all conventional methods.

[0020] The cells, viruses, and main reagents involved in this invention are as follows: (1) African green monkey kidney epithelial cells Marc-145 (overexpressing CD163 receptor), African green monkey kidney cells Vero, African green monkey embryonic kidney epithelial cells MA104, porcine reproductive and respiratory syndrome virus type 2 (PRRSV2), PEDV, PoRV strain and PRRSV-N protein monoclonal antibody were all obtained from this experiment. Among them, Marc-145 overexpressed CD163 receptor in order to make PRRSV2 better inoculated on Marc-145. Vero cells, MA104 cells, PRRSV2, PEDV, PoRV strain and PRRSV-N protein monoclonal antibody can be purchased through conventional commercial channels.

[0021] (2) SweScript RT II First Strand cDNA Synthesis Kit (Wuhan Swewell Biotechnology Co., Ltd., catalog number: G3333-50); 2×Fast SYBR Green qPCR Master Mix (Wuhan Swewell Biotechnology Co., Ltd., catalog number: G3325-15); 5×SDS-PAGE protein loading buffer (Wuhan Swewell Biotechnology Co., Ltd., catalog number: G2075-1ML); HRP-labeled goat anti-mouse IgG (Wuhan Swewell Biotechnology Co., Ltd., catalog number: GB23301); ultrasensitive ECL chemiluminescence kit (Wuhan Swewell Biotechnology Co., Ltd., catalog number: G2020-50ML); Alexa Fluor 488-labeled goat anti-mouse IgG (Wuhan Swewell Biotechnology Co., Ltd., catalog number: GB25301).

[0022] Example 1 Isolation and Identification of Mucosal Lactobacillus Lung tissue samples were taken from healthy pigs, disinfected, and transferred to a laminar flow hood. Using sterile forceps and surgical scissors, the lung tissue was cut into soybean-sized pieces and placed in a sterile grinder. 10% PBS buffer was added to the lung tissue for further grinding. The ground tissue solution was then diluted 10... 5 The culture medium was evenly spread onto MRS agar plates (selective isolation and counting medium for lactic acid bacteria) and placed in an anaerobic workstation at 37°C for constant temperature anaerobic incubation until single colonies grew on the plates. Well-grown, well-defined, milky-white, round, raised single colonies were picked from the plates and further cultured in 8 mL of MRS broth. After amplification, bacterial genomic DNA was extracted from the bacterial culture. PCR amplification was performed using universal primers 27F (see SEQ ID NO:1) and 1492R (see SEQ ID NO:2) for the bacterial 16S rRNA gene. Positive PCR products were bidirectionally sequenced. The obtained bidirectional sequences were spliced ​​using SeqMan software to obtain the 16S rRNA gene sequence (see SEQ ID NO:3), which was then uploaded to the NCBI database for homology comparison analysis using the BLAST program. The results showed that the obtained spliced ​​sequence was consistent with the standard strain of *Lactobacillus mucosa*. Lactobacillus mucosae The sequence homology of (GenBank: MT545152.1) reached over 99.9%, confirming it as *Lactobacillus mucosa*.

[0023] Example 2 Inhibitory effect of mucosal lactobacillus culture supernatant on PRRSV2 infection-induced cytopathic effect.

[0024] (1) Marc-145 cells were cultured at a concentration of 1.5 × 10⁻⁶. 5 The cells were seeded at a density of 1 cell / mL into 12-well cell culture plates. 1 mL of DMEM complete medium (containing 100 U / mL penicillin and 100 μg / mL streptomycin) was added to each well. The plates were then incubated at 37°C and 5% CO2 for 48 h until the cells reached 80% monolayer density.

[0025] (2) Incorporate Lactobacillus mucosa ( Lactobacillus mucosae Inoculate into MRS medium for fermentation culture until OD... 600 =1.6, to obtain the fermentation broth; after centrifuging the fermentation broth, take the supernatant, adjust the pH to neutral, and then filter it through a 0.22μm hydrophilic filter membrane to obtain the culture supernatant of Lactobacillus mucosa.

[0026] (3) Take different lineages of porcine reproductive and respiratory syndrome virus type 2 (PRRSV2-L1, PRRSV2-L3, PRRSV2-L5 and PRRSV2-L8) at 500 TCID50. 50The titer of / well was added to serum-free DMEM medium, and the supernatant of Lactobacillus mucosa culture was added at the same time to make the volume percentage of supernatant in DMEM medium 12.5%. After mixing, it was co-incubated at 37°C for 1 h to obtain the virus mixture.

[0027] (4) The virus mixture treated with the supernatant of Lactobacillus mucosa culture was inoculated into Marc-145 cell monolayers at a rate of 1 mL / well as the experimental group. The cell layer without virus was used as the negative control, and the cell layer with untreated virus (PRRSV2-L1) was used as the PRRSV2-L1 group. The cells were incubated at 37°C for 1.5 h. After incubation, the culture medium was discarded, and the cells were gently washed three times with PBS buffer. Then, DMEM maintenance medium containing 2% fetal bovine serum was added to each well. Three parallel replicates were set up for each experimental group. At the same time, in order to further rule out the possibility that MRS medium inhibits PRRSV2, the cell layer with MRS medium added to the PRRSV2 group was used as the PRRSV2-L1-MRS group. The cell culture plates were placed in a 37°C, 5% CO2 incubator and cultured for another 60 h (corresponding to the logarithmic replication phase of the virus).

[0028] (5) After the culture was completed, the cytopathic effect (CPE) of Marc-145 cells infected by PRRSV2-L1 was observed using a regular optical microscope.

[0029] The results are as follows Figure 1 As shown, this embodiment uses African green monkey kidney epithelial cells (Marc-145) as an in vitro infection model to systematically evaluate the antiviral effect of *Lactobacillus mucosa*. Compared with the PRRSV2-L1 group and the PRRSV2-L1-MRS group, PRRSV2-L1 (PRRSV2-L1-*Lactobacillus mucosa*), PRRSV2-L3 (PRRSV2-L3-*Lactobacillus mucosa*), PRRSV2-L5 (PRRSV2-L5-*Lactobacillus mucosa*), and PRRSV2-L8 (PRRSV2-L8-*Lactobacillus mucosa*) treated with *Lactobacillus mucosa* culture supernatant did not induce typical cytopathic effects (CPE) such as shrinkage, deformation, and aggregation in Marc-145 cells. This indicates that the supernatant can effectively inhibit the in vitro infection ability of different lineages of PRRSV2, and the inhibitory ability on PRRSV2 is independent of the MRS culture medium. Compared with the negative control group, the culture supernatant did not show significant cytotoxic effects on Marc-145 cells, demonstrating good in vitro biocompatibility.

[0030] Example 3 Inhibitory effect of mucosal lactobacillus culture supernatant on PEDV-induced cytopathic effect.

[0031] (1) Vero cells were subjected to a concentration of 1.5 × 10⁻⁶ 5The cells were seeded at a density of 1 cell / mL into 12-well cell culture plates, with 1 mL of DMEM complete medium (containing 100 U / mL penicillin and 100 μg / mL streptomycin) added to each well. The plates were then incubated in a constant temperature incubator at 37°C and 5% CO2 until the cells reached 80% monolayer density.

[0032] (2) Take porcine epidemic diarrhea virus (PEDV) at 50 TCID50 50 The titer of each well was diluted in serum-free DMEM medium, and the culture supernatant of *Lactobacillus mucosa* prepared in step (2) of Example 2 was added. The volume percentage of the culture supernatant of *Lactobacillus mucosa* in the medium was 12.5%. After mixing, the mixture was incubated at 37°C for 1 h.

[0033] (3) The above-treated virus mixture was seeded into Vero cell monolayer at a rate of 1 mL / well and incubated at 37°C for 1.5 h. After incubation, the virus mixture was discarded, and the cells were gently washed three times with PBS buffer. Then, DMEM maintenance medium containing 2% fetal bovine serum was added to each well. Three parallel replicates were set up for each experimental group. The cell culture plate was placed in a 37°C, 5% CO2 incubator and cultured for another 48 h (corresponding to the logarithmic replication phase of PEDV).

[0034] (4) After the culture was completed, the cytopathic effect (CPE) of Vero cells caused by PEDV infection was observed using a regular optical microscope.

[0035] The results are as follows Figure 2 As shown, compared with the untreated virus control group ( Figure 2 (b) Compared to the PEDV-infected group treated with the supernatant of Lactobacillus mucosa culture ( Figure 2 (c) Vero cells did not show typical PEDV-characteristic CPE such as syncytial bodies or cell shedding, indicating that the culture supernatant also had a significant inhibitory effect on PEDV infection.

[0036] Example 4 Inhibitory effect of mucosal lactobacillus culture supernatant on cytopathic effects induced by PoRV infection.

[0037] (1) MA104 cells were subjected to a concentration of 1.5 × 10⁻⁶ cells. 5 The cells were seeded at a density of 1 cell / mL into 12-well cell culture plates, with 1 mL of DMEM complete medium (containing 100 U / mL penicillin and 100 μg / mL streptomycin) added to each well. The plates were then incubated in a constant temperature incubator at 37°C and 5% CO2 until the cells reached 80% monolayer density.

[0038] (2) Take porcine rotavirus (PoRV) at 100 TCID50. 50The titer of / well was diluted in serum-free DMEM medium, and the culture supernatant of Lactobacillus mucosa prepared in step (2) of Example 2 was added at the same time. The volume percentage of Lactobacillus mucosa culture supernatant in the culture medium was 12.5%. After mixing, it was co-incubated at 37°C for 1 h.

[0039] (3) The above-treated virus mixture was inoculated into a monolayer of MA104 cells at a rate of 1 mL / well and incubated at 37°C for 1.5 h. After incubation, the virus mixture was discarded, and the cells were gently washed three times with PBS buffer. Then, DMEM maintenance medium containing 2% fetal bovine serum (containing 10% trypsin to activate porcine rotavirus) was added to each well. Three parallel replicates were set up for each experimental group. The cell culture plate was placed in a 37°C, 5% CO2 incubator and cultured for another 48 h (corresponding to the logarithmic replication phase of PoRV).

[0040] (4) After the culture was completed, the cytopathic effect (CPE) of MA104 cells infected with PoRV was observed using a regular optical microscope.

[0041] The results are as follows Figure 3 As shown, compared with the untreated virus group ( Figure 3 (b) Compared to the PoRV infection group treated with the supernatant of Lactobacillus mucosa culture ( Figure 3 (c) MA104 cells did not show typical cytopathic effects, indicating that the culture supernatant also had an inhibitory effect on PoRV infection.

[0042] Example 5 The inhibitory effect of the above-mentioned Lactobacillus mucosa culture supernatant on RNA expression in PRRSV2 was detected by RT-qPCR.

[0043] (1) Marc-145 cells were cultured at a concentration of 1.5 × 10⁻⁶. 5 Cells were seeded at a density of 1 cell / mL in 12-well cell culture plates, with 1 mL of DMEM complete medium (containing 100 U / mL penicillin and 100 μg / mL streptomycin) added to each well. The plates were then incubated at 37°C in a 5% CO2 incubator for 48 h. Cells were then used after reaching 80% confluence monolayer.

[0044] (2) Take PRRSV2-L1 with 500 TCID 50The titer of the virus mixture was diluted in serum-free DMEM medium, and the supernatant of the *Lactobacillus mucosa* culture was added simultaneously. The volume percentage of the *Lactobacillus mucosa* culture supernatant in the medium was 12.5%. After mixing, the mixture was incubated at 37°C for 1 h. The above-treated virus mixture was seeded into a monolayer of Marc-145 cells at 1 mL / well and incubated at 37°C for 1.5 h. The virus solution was discarded, and the cells were gently washed three times with PBS buffer. DMEM maintenance medium containing 2% fetal bovine serum was added to each well, and the cells were cultured for another 48 h. The PRRSV2-L1 infection group without the addition of *Lactobacillus mucosa* culture supernatant was used as a positive control, and normal cells without virus infection were used as a negative control. Three replicates were set up for each experimental group.

[0045] (3) After culture, discard the cell culture supernatant, wash three times with PBS, add 700 μL of TRIzol lysis buffer to each well to extract total RNA, and use the SweScript RT II First Strand cDNA Synthesis Kit to reverse transcribe and synthesize cDNA as a template for reverse transcription real-time quantitative PCR (RT-qPCR); use GAPDH as an internal reference gene, and employ 2 -ΔΔCT The relative expression level of the PRRSV-N protein gene in PRRSV2-L1 was detected to assess the relative content of viral RNA in infected cells.

[0046] The upstream primer sequence of PRRSV-N is detailed in SEQ ID NO:4, and the downstream primer sequence of PRRSV-N is detailed in SEQ ID NO:5.

[0047] Add 10 μL of 2×Fast SYBR Green qPCR Master Mix, 0.5 μL of upstream primer, 0.5 μL of downstream primer, and 2 μL of cDNA template to a PCR tube in an ice bath, and bring the volume to 20 μL with RNase-Free H2O; centrifuge the PCR tube briefly to remove air bubbles.

[0048] The RT-qPCR reaction procedure is as follows: Table 1 The results are as follows Figure 4 As shown, compared with the positive control group without the addition of Lactobacillus mucosa culture supernatant, the relative expression level of PRRSV-N gene RNA in the PRRSV2-L1 infection group treated with Lactobacillus mucosa culture supernatant was significantly reduced (P<0.01), indicating that Lactobacillus mucosa culture supernatant can effectively inhibit the transcription and replication of PRRSV2-L1 in host cells.

[0049] Example 6 Western blotting was used to further verify the anti-PRRSV2 activity of Lactobacillus mucosa culture supernatant and to detect the expression level of PRRSV-N protein in virus-infected cells. The specific steps are as follows: (1) Marc-145 cells were cultured at a concentration of 1.5 × 10⁻⁶. 5 The cells were seeded at a density of 1 cell / mL in 12-well cell culture plates, and 1 mL of DMEM complete medium (containing 100 U / mL penicillin and 100 μg / mL streptomycin) was added to each well. The plates were then incubated at 37°C and 5% CO2 for 48 h until the cells reached 80% confluence and were ready for use.

[0050] (2) Take PRRSV2-L1 with 500 TCID 50 The titer of each well was diluted in serum-free DMEM medium, and the supernatant of the *Lactobacillus mucosa* culture was added simultaneously. The volume percentage of the *Lactobacillus mucosa* culture supernatant in the medium was 12.5%. After mixing, the mixture was incubated at 37°C for 1 hour.

[0051] (3) The above-treated virus mixture was seeded into a monolayer of Marc-145 cells at 1 mL / well and adsorbed at 37°C for 1.5 h. The virus mixture was discarded, and the cells were gently washed three times with PBS buffer. DMEM maintenance medium containing 2% fetal bovine serum was added to each well and cultured for another 48 h. The PRRSV2-L1 infection group without the addition of Lactobacillus mucosa culture supernatant was used as the positive control (PRRSV2-L1 group, PRRSV2-L1-MRS group), and normal cells without virus infection were used as the negative control. Three parallel replicates were set up for each experimental group.

[0052] (4) After the culture is completed, discard the cell culture supernatant, wash three times with PBS, add RIPA lysis buffer (weak) to each well and lyse at room temperature for 5 min, and collect the cell lysis buffer; take 100 μL of cell lysis suspension into a 1.5 mL EP tube, add 25 μL of SDS-PAGE loading buffer, mix well and boil in a water bath for 10 min to prepare protein samples.

[0053] (5) The expression of PRRSV-N protein in PRRSV2-L1 was detected by Western Blot: 10 μL of the protein sample obtained in step (4) was added to the sample wells in sequence. After separation by SDS-PAGE electrophoresis, the sample was transferred to a PVDF membrane. The primary antibody was PRRSV-N protein antibody and GAPDH internal reference antibody (dilution ratio of 1:1000), and the membrane was incubated at room temperature for 2 h. The secondary antibody was HRP-labeled goat anti-mouse IgG (dilution ratio of 1:5000), and the membrane was incubated at room temperature for 2 h. Finally, the membrane was developed according to the instructions of the ultrasensitive ECL chemiluminescence kit to detect the expression of PRRSV-N protein in the cells.

[0054] The results are as follows Figure 5 As shown, with GAPDH as an internal control to ensure consistent loading, and based on the same cell number, compared with the positive control group without the addition of Lactobacillus mucosa culture supernatant, the expression level of PRRSV-N protein in the Lactobacillus mucosa culture supernatant treatment group was significantly reduced, indicating that Lactobacillus mucosa culture supernatant can effectively inhibit the expression of PRRSV-N protein.

[0055] Example 7 The anti-PRRSV2 activity of Lactobacillus mucosa culture supernatant was detected using indirect immunofluorescence assay. The specific steps are as follows: (1) Marc-145 cells were cultured at a concentration of 1.5 × 10⁻⁶. 5 The cells were seeded at a density of 1 cell / mL in 12-well cell culture plates, and 1 mL of DMEM complete medium (containing antibiotics) was added to each well. The cells were cultured for 48 h until 80% confluence monolayer was achieved and ready for use.

[0056] (2) Take PRRSV2-L1 strain at 500 TCID 50 The titer of each well was diluted in serum-free DMEM medium, and the supernatant of the *Lactobacillus mucosa* culture was added simultaneously. The volume percentage of the *Lactobacillus mucosa* culture supernatant in the medium was 12.5%. After mixing, the mixture was incubated at 37°C for 1 hour.

[0057] (3) The virus mixture was inoculated into a monolayer of Marc-145 cells, adsorbed at 37°C for 1.5 h and then discarded. The cells were washed three times with PBS, and DMEM maintenance medium containing 2% fetal bovine serum was added. The cells were cultured for another 48 h. The PRRSV2 infection group without the addition of Lactobacillus mucosa culture supernatant was used as the positive control (PRRSV2-L1 group, PRRSV2-L1-MRS group), and normal cells were used as the negative control.

[0058] (4) After the culture was completed, the supernatant was discarded, and the cells were washed three times with PBS. 500 μL of 4% paraformaldehyde was added to each well for 30 min at room temperature. After washing three times with PBS, 500 μL of 0.1% Triton X-100 (immunostaining permeabilization solution) was added to each well for 20 min at room temperature. After washing three times with PBS, 1 mL of 2% BSA (bovine serum albumin) was added for 1.5 h at room temperature. PRRSV-N protein monoclonal antibody (1:300 dilution) was added and incubated overnight at 4°C. After washing three times with TBST (Tris salt buffer + Tween-20), Alexa Fluor 488-labeled goat anti-mouse IgG (1:500 dilution) was added and incubated at room temperature in the dark for 1.5 h. After washing three times with TBST, the cells were observed and photographed under a fluorescence microscope. The cell nuclei showed blue fluorescence after DAPI counterstaining, and the PRRSV2-L1 positive signal showed green fluorescence.

[0059] The results are as follows Figure 6As shown, compared with the positive control group without the addition of Lactobacillus mucosa culture supernatant, the PRRSV2-L1 specific green fluorescence signal basically disappeared in the group treated with Lactobacillus mucosa culture supernatant, indicating that Lactobacillus mucosa culture supernatant can significantly inhibit the infection and replication of PRRSV2-L1 in host cells.

[0060] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A mucosal lactobacillus with anti-swine virus activity ( Lactobacillus mucosae ), characterized in that, The preservation number of the Lactobacillus mucosa is: CGMCC No.37932.

2. The *Lactobacillus mucosa* with anti-swine virus activity according to claim 1 (… Lactobacillus mucosae Application of ) in suppressing swine viruses.

3. A mucosal lactobacillus with anti-swine virus activity as described in claim 1 ( Lactobacillus mucosae Application of ) in the preparation of anti-swine virus biological agents.

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