Monocytogenes bacteriophage lysing enzyme lysP70 and application thereof

Abstract

The present application relates to the technical field of biology, in particular to a kind of monocyte producing Lee's bacillus bacteriophage lytic enzyme LysP70 and application, the amino acid sequence of lytic enzyme LysP70 is as shown in SEQ ID No.1;Can cleave monocyte producing Lee's bacillus, with good lytic activity and stability, sterilization speed is very fast;Lytic enzyme LysP70 has lytic activity to milk and lettuce;Lytic enzyme LysP70 effectively inhibits the formation of biofilm by destroying biofilm structure, reducing the amount of biofilm state bacteria and damaging bacterial morphology, while it can also effectively destroy and remove the early biofilm and mature biofilm formed in advance, has significant inhibitory effect on bacterial metabolic activity in monocyte producing Lee's bacillus biofilm, can inhibit the production of monocyte producing Lee's bacillus extracellular polysaccharide, and with the concentration increasing, inhibitory effect enhances;Can inhibit the production of monocyte producing Lee's bacillus extracellular protein.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a Listeria monocytogenes phage lysin LysP70 and its applications. Background Technology

[0002] Listeria monocytogenes is a zoonotic pathogen, a Gram-positive short rod-shaped bacterium that is non-spore-forming, facultatively anaerobic, flagellated, and resistant to low temperatures (growing at 4°C), salt (10% NaCl), and bile. It can cause listeriosis, posing a serious threat to pregnant women, newborns, and adults. Currently, antibiotics are the most common treatment. However, this bacterium exhibits strong environmental adaptability and can form biofilms, showing some resistance to environmental disinfectants and antibiotics. Statistics show that timely treatment of pregnant women can reduce fetal risk, but the neonatal mortality rate remains 20-30%. The mortality rate for meningitis / septicemia is approximately 20-30%, and survivors may suffer from neurological sequelae.

[0003] To effectively kill this bacterium, it is first necessary to disrupt the biofilm of Listeria monocytogenes, and further damage its cell wall. Lyases are a crucial class of enzymes in the process of bacteriophage infection of bacteria; their binding and catalytic domains can bind to and degrade the bacterial cell wall. Phage lyases are structurally and functionally similar to bacterial autolysins and are closely related to the mammalian peptidoglycan recognition protein family. For Gram-positive bacteriophages, most lyases consist of two domains: an N-terminal enzymatically active domain (EAD) and a C-terminal cell wall binding domain (CBD), linked by a short polypeptide chain. The EAD facilitates the cleavage of different bonds in peptidoglycan, while the CBD recognizes and specifically binds to bacterial cell wall receptor proteins. Although Listeria monocytogenes exhibits some resistance to lyases, modifying lyases or combining them with other antibacterial agents can enhance their bactericidal effect. In other bacterial studies, antibacterial agents based on lyases have been developed and widely used in the treatment of bacterial infection-related diseases, with no observed bacterial resistance. Meanwhile, since natural lyases are difficult to extract from bacteriophages, a prokaryotic expression system is used to express the lyase in a soluble manner to ensure that it has lyase activity.

[0004] Based on the aforementioned technical problems, the inventors discovered a lyase LysP7 in Listeria monocytogenes phage P70 during the research process. The inventors expressed the lyase in prokaryotes and systematically studied its enzymatic properties and antibacterial activity, including its effect on bacterial biofilms. They further explored the effects of the lyase LysP70 in the preliminary application of food (milk, lettuce). Summary of the Invention

[0005] The first aspect of the present invention is to provide a Listeria monocytogenes phage lysin LysP70, the amino acid sequence of which is shown in SEQ ID No. 1.

[0006] A second aspect of the present invention is to provide a gene encoding the lysin LysP70, the nucleotide sequence of which is shown in SEQ ID No. 2.

[0007] A third aspect of the present invention is to provide a recombinant expression vector for expressing the lyase LysP70, said expression vector comprising the nucleotide sequence of said lyase LysP70.

[0008] A fourth aspect of the present invention is to provide a recombinant engineered bacterium expressing the lysin LysP70, wherein the recombinant engineered bacterium is formed by transforming the recombinant expression vector into a host bacterium.

[0009] A fifth aspect of the invention is the use of the lysin LysP70, the recombinant expression vector, or the recombinant engineered bacteria in the preparation of reagents for lysing Listeria monocytogenes.

[0010] A sixth aspect of the present invention is to provide a reagent for lysing Listeria monocytogenes, the reagent comprising the lysin LysP70; the amino acid sequence of the lysin LysP70 is shown in SEQ ID No. 1.

[0011] A seventh aspect of the present invention is to provide an antibacterial agent, the main active ingredient of which is the lyase LysP70, the amino acid sequence of which is shown in SEQ ID No. 1.

[0012] An eighth aspect of the present invention is to provide an antibacterial composition comprising the aforementioned antibacterial agent and an antibiotic.

[0013] The beneficial effects of this invention are:

[0014] (1) The present invention provides a Listeria monocytogenes phage lysin LysP70, the amino acid sequence of which is shown in SEQ ID No.1 and the nucleotide sequence of which is shown in SEQ ID No.2.

[0015] (2) The lyase LysP70 described above can lyse Listeria monocytogenes, exhibiting good lysing activity, rapid bactericidal speed, and a wide lysing range against Listeria, but it has no lysing ability against Staphylococcus and Salmonella. The activity of this lyase is relatively stable within a certain range of pH, temperature, metal ion concentration, storage temperature, and time.

[0016] (3) The bacteria treated with the lyase LysP70 showed increased sensitivity to ampicillin (AMP), doxycycline (DO) and piperacillin (PIP), as evidenced by an increase in the diameter of the inhibition zone; however, they still exhibited strong resistance to ceftazidime (CAZ) and polymyxin B (PB), and no inhibition zone was produced.

[0017] (4) The lyase LysP70 described above exhibits lysing activity against both milk and lettuce. LysP70 effectively inhibits biofilm formation by disrupting biofilm structure, reducing the amount of biofilm-bearing bacteria, and damaging bacterial morphology. It can effectively destroy and remove pre-formed early and mature biofilms. It has a significant inhibitory effect on the cellular metabolic activity of Listeria monocytogenes biofilms, with the highest inhibition rate of 50% at 400 μg / mL. The inhibitory effect weakens as the concentration decreases.

[0018] (5) LysP70 can inhibit the production of extracellular polysaccharides in Listeria monocytogenes, and the inhibitory effect is enhanced as the concentration increases; it can also inhibit the production of extracellular proteins in Listeria monocytogenes. Attached Figure Description

[0019] Figure 1 (A) SDS-PAGE and (B) Western blot validation results of recombinant protein LysP70

[0020] Note: M: Marker; 1: Uninduced pET28a; 2: Induced pET28a; 3: Uninduced pET28a-LysP70; 4: Induced pET28a-LysP70; 5: Total protein before lysis; 6: Total protein after lysis; 7: Supernatant; 8: Precipitate; 9: Purified protein; 10: Washing buffer

[0021] Figure 2 Protein standard curve

[0022] Figure 3 Lysis zone of lyase LysP70

[0023] Figure 4 Lysogenic activity of lyase LysP70 in liquid environment. Note: 1: Negative control; 2: 400 μg / mL; 3: 200 μg / mL; 4: 100 μg / mL; 5: 50 μg / mL; 6: Blank control.

[0024] Figure 5 In vitro bactericidal activity of lyase LysP70

[0025] Figure 6 Effect of pH on the cleavage activity of LysP70 lyase

[0026] Figure 7 Effect of temperature on the cleavage activity of lysin LysP70

[0027] Figure 8 Effects of different metal ions on the cleavage activity of the lysin LysP70 (Note: (A) Na) + Effect on the cleavage activity of the lysin LysP70; (B)K + Effect of Ca on the cleavage activity of the lysin LysP70; (C)Ca 2+ Effect of (D)Zn on the cleavage activity of lysin LysP70; 2+ Effect on the cleavage activity of the lysin LysP70 Figure 9 Effect of storage conditions on the cleavage activity of lysin LysP70

[0028] Note: (A) Plate drop method; (B) Plate counting method

[0029] Figure 10 Lysase LysP70 cleavage activity in milk. Note: (A) Lysase LysP70 cleavage activity in milk at 4℃; (B) Lysase LysP70 cleavage activity in milk at 30℃.

[0030] Figure 11 Lysogenic activity of lyase LysP70 in lettuce. Note: (A) Lysogenic activity of lyase LysP70 in lettuce at 4℃; (B) Lysogenic activity of lyase LysP70 in lettuce at 30℃.

[0031] Figure 12 Determination of bacterial biofilm formation ability

[0032] Figure 13 Inhibitory effect of lyase LysP70 on the biofilm of Listeria monocytogenes: (A) Crystal violet staining; (B) Fluorescent staining; (C) Scanning electron microscopy.

[0033] Figure 14 The scavenging effect of the lyase LysP70 on the biofilm of Listeria monocytogenes: (A) Crystal violet staining; (B) Fluorescent staining; (C) Scanning electron microscopy.

[0034] Figure 15 Effects of the lyase LysP70 on the metabolic activity of Listeria monocytogenes biofilm cells

[0035] Figure 16 Effects of lyase LysP70 on the self-aggregation and hydrophobicity of Listeria monocytogenes

[0036] Note: (A) Self-aggregation; (B) Hydrophobicity

[0037] Figure 17Effect of lyase LysP70 on extracellular polysaccharides of Listeria monocytogenes biofilm

[0038] Note: (A) Standard curve of glucose content; (B) Effect of lyase LysP70 on extracellular polysaccharides.

[0039] Figure 18 Effects of the lyase LysP70 on extracellular proteins of the biofilm of Listeria monocytogenes

[0040] Note: (A) Protein standard curve; (B) Effect of lyase LysP70 on extracellular proteins. Detailed Implementation

[0041] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0042] It should be noted that, unless otherwise specified, the methods described in the following embodiments are all conventional methods, and the reagents described are all commercially available.

[0043] In the following examples, GraphPad Prism 8.0 and Excel 2016 software were used for chart creation and data analysis. The t-test and one-way ANOVA were used for significance testing. Where no as or ns indicates no significant difference (p>0.05), * indicates a significant difference (p<0.05), and ** and *** both indicate extremely significant differences (p<0.01 and p<0.001, respectively).

[0044] Example 1: Bioinformatics Analysis, Expression, and Purification of LysP70 Lysis Enzyme

[0045] 1 Experimental Methods

[0046] 1.1 Obtaining the lysin LysP70

[0047] Download the lyase coding region sequence (gene lookup number NC_018831.1) and amino acid sequence (protein ID: YP_006905871.1) of Listeria monocytogenes phage P70 from the NCBI (National Center for Biotechnology Information) database.

[0048] Sequence analysis revealed the sequences of multiple lyases. One of the lyase sequences was identified and named LysP70, with its amino acid sequence shown in SEQ ID No. 1 and its nucleotide sequence shown in SEQ ID No. 2.

[0049] 1.2 Construction of the pET28a-LysP70 vector for lysin

[0050] The target fragment LysP70 gene sequence was synthesized by Sangon Biotech (Shanghai) Co., Ltd. to prepare the pET28a-LysP70 recombinant plasmid.

[0051] 1.3 Plasmid Extraction

[0052] The Top10 plasmids containing recombinant plasmids returned by the company were streaked and inoculated into the Kan-containing plasmids. + Incubate overnight at 37°C on LB agar plates containing 50 μg / mL of Kanamycin. The next day, pick single colonies and inoculate them onto plates containing Kanamycin. + The plasmid was cultured in LB liquid medium at 37°C and 180 rpm on a shaker until the logarithmic growth phase, and the plasmid was extracted according to the instructions of the plasmid DNA mini-extraction kit.

[0053] 1.4 Transformation of Recombinant Plasmids

[0054] Remove E. coli competent cells BL21(DE3) from a -80℃ freezer and place them on ice to thaw. In a laminar flow hood, add 50 μL of competent cells and 5 μL of recombinant plasmid to a 1.5 mL centrifuge tube, mix gently, incubate on ice for 30 min, heat shock at 42℃ for 90 s, and immediately place on ice for 3 min. In a laminar flow hood, add 600 μL of antibiotic-free LB liquid medium to the centrifuge tube, mix gently, and incubate at 37℃ and 180 rpm for 1 h. After incubation, centrifuge at 4000 rpm for 5 min, discard 500 μL of supernatant, resuspend the bacterial pellet, and spread evenly with a spreader onto a plate containing Kansas. + Incubate on LB solid culture plates at 37°C in an inverted incubator for 14 hours.

[0055] 1.5 Identification of Recombinant Plasmids

[0056] Four monoclonal colonies were randomly selected from the plate and inoculated into a solution containing Kans. + The culture was carried out in LB liquid medium at 37℃ and 180 r / min for 12 h, followed by bacterial PCR verification. Universal primers T7 and T7ter were used for identification, with primer sequences T75′-TAATACGACTCACTATAGGG-3′ and T7ter 5′-GCTAGTTATTGCTCAGCGG-3′. The PCR amplification system is shown in Table 1.

[0057] Table 1 PCR amplification system

[0058]

[0059] Reaction conditions: 94℃ pre-denaturation for 5 min, 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 2 min, for a total of 30 cycles; 72℃ extension for 10 min.

[0060] The amplified PCR samples were subjected to 1% agarose gel electrophoresis. The bands were observed and photographed in a gel imaging system to check for correctness.

[0061] 1.6 strains cryopreservation

[0062] The PCR-identified strain was placed in a Kan + Expand the culture in LB liquid medium. Add 40% glycerol and bacterial solution (autoclaved) to the cryovial in a 1:1 ratio, mix well, and store at -80°C.

[0063] 1.7 Expression and purification of recombinant proteins

[0064] 1.7.1 Induced Expression

[0065] Verify that the correct strain is streaked onto the sample containing Kan + Incubate overnight at 37°C on LB agar plates. Pick a single colony and add a solution containing Kansei culture medium. + After incubating the culture in 4 mL of LB liquid medium at 37°C with shaking for 6 h, the culture was expanded at a ratio of 1:100, and the culture was then transferred to a medium containing Kansas. + In 600 mL LB liquid medium, cultured at 37°C with shaking until OD 600 The concentration was 0.6–0.8. IPTG was added to the bacterial culture to a final concentration of 0.5 mmol / L, and the culture was incubated overnight at 16°C and 200 rpm for 14 h. After induction, the bacterial culture was centrifuged at 4°C and 8000 rpm for 15 min, the culture medium was discarded, and the bacterial pellet was collected. The pellet was washed three times with PBS and resuspended in Binding Buffer. PMSF was added to a final concentration of 1 mmol / L, and the cells were sonicated at 220 W with a 3-second sonication cycle until the bacterial suspension was clear. The suspension was then centrifuged at 4°C and 8000 rpm for 20 min, and the supernatant was collected.

[0066] 1.7.2 Protein Purification

[0067] Recombinant proteins were purified using a Ni-NTA affinity chromatography column. The column protection buffer (20% ethanol) was discarded. The column was washed five times with 5 column volumes of ultrapure water, followed by equilibration five times with 5 column volumes of binding buffer. The collected supernatant was filtered through a 0.22 μm filter and mixed with the equilibrated resin. The column was then incubated on a horizontal shaker at 4°C for 4 hours. After the binding buffer adhered to the column, the eluent was collected and re-adsorbed to improve the binding rate. Impurities were eluted with wash buffer five times, followed by elution buffer to elute the target protein. The eluent was collected. After elution, the nickel column was washed, and 20% ethanol solution was added. The column was then stored at 4°C. The collected samples were prepared and subjected to SDS-PAGE electrophoresis.

[0068] The purified target protein was added to a 30 kDa ultrafiltration centrifuge tube and centrifuged at 5000 rpm for 20 min at 4 °C. 1 mL of PBS was added after each centrifugation to replace the buffer, and this process was repeated 8 times to remove imidazole and concentrate the protein. Finally, the concentrated protein in the ultrafiltration tube was gently pipetted to mix, collected in a centrifuge tube, and filtered through a 0.22 μm filter membrane for subsequent experiments.

[0069] 1.8 SDS-PAGE and Western blot validation

[0070] 1.8.1 SDS-PAGE electrophoresis detection

[0071] (1) Preparation of SDS-PAGE protein electrophoresis gel: Install the gel-casting glass plates on the gel-casting rack, fill the gaps between the glass plates with distilled water to check for leaks; prepare the lower layer of 10% separating gel (6 mL / plate) according to Table 2-4, mix well and immediately inject it between the installed glass plates with a pipette, add anhydrous ethanol to press the gel to prevent the generation of air bubbles, and let it stand for 40 min until the separating gel is completely solidified; discard the upper layer of anhydrous ethanol, absorb the residual ethanol with filter paper, prepare the lower layer of 5% stacking gel (2.5 mL / plate) according to Table 2-4, mix well by blowing and quickly inject it into the glass plates, immediately insert the comb (to avoid air bubbles in the gel wells), and let it stand until it solidifies before electrophoresis.

[0072] (2) Sample preparation: Add 5×SDS-PAGE loading buffer and protein at a ratio of 1:4, boil in a boiling water bath for 15 min, centrifuge and take the supernatant for loading.

[0073] (3) Electrophoresis: Place the prepared electrophoresis gel in the electrophoresis tank, add 1×SDS-PAGE electrophoresis buffer until it covers the comb, pull the comb vertically, add 10μL of protein sample to each well, and add protein marker at the same time. Set the constant voltage to 80V. When the sample is observed to move to the edge of the stacking gel in the lane, adjust the voltage to 120V. Turn off the power when the sample electrophoresis reaches about 1 / 10 of the distance from the bottom of the glass plate.

[0074] (4) Staining: After electrophoresis, gently pry open the glass plate, rinse the gel into the petri dish, add Coomassie brilliant blue staining solution, and stain at room temperature for 2 hours on a horizontal shaker.

[0075] (6) Destaining: Discard the staining solution, wash several times with distilled water, and then destain with the destaining solution. Destain on a horizontal shaker for 30 minutes until the solution turns blue. Replace the staining solution and destain until the background is transparent and the protein bands are clear. Take pictures of the protein gel and analyze the results.

[0076] The protein gel preparation system is shown in Table 2.

[0077] Table 2 SDS-PAGE electrophoresis system

[0078]

[0079]

[0080] 1.8.2 Western blot validation

[0081] (1) Electrophoresis: Same as 1.8.1.

[0082] (2) Transfer: The PVDF membrane was activated by immersing it in anhydrous methanol for 10 seconds. The filter paper and screen used for transfer were immersed in the transfer solution. The separating gel was cut off and placed on the electrophoresis clamp in the following order: screen, filter paper, gel, PVDF membrane, filter paper, screen. The gel was placed on the side closer to the negative electrode, and the membrane was placed on the side closer to the positive electrode. Air bubbles were removed during the placement of each layer. The clamped electrophoresis clamp was placed in the electrophoresis tank and the transfer solution was poured in. Transfer was performed under ice bath conditions, with a constant current of 300 mA for 50 minutes.

[0083] (3) Blocking: After taking out the PVDF membrane and washing it in TBST buffer, put it into the incubation box, pour in the blocking solution, and place it on a horizontal shaker at room temperature for 4 hours.

[0084] (4) Primary antibody incubation: Discard the blocking solution, pour in TBST buffer, place on a shaker to wash the membrane for 10 min each time, and wash 3 times; select His-tag antibody as the primary antibody, dilute the antibody with TBST buffer (1:5000), add the diluted primary antibody to the membrane, place on a horizontal shaker, and incubate overnight at 4°C.

[0085] (5) Secondary antibody incubation: Remove the PVDF membrane, recover the primary antibody, wash the membrane with TBST buffer, and follow the same steps as above; add diluted goat anti-mouse IgG-HRP secondary antibody (1:5000) and incubate at room temperature on a horizontal shaker for 2 hours.

[0086] (6) Color development: Remove the PVDF membrane, recover the secondary antibody, and wash the membrane as above. Prepare color development solution A and solution B in a 1:1 ratio, add them dropwise to the membrane, expose it on a chemiluminescence analyzer for a few minutes to form an image, observe the target band and take a picture to record.

[0087] 1.9 Determination of protein concentration

[0088] The concentration of recombinant LysP70 protein lysin was determined using a BCA (Bicinchoninic Acid) protein quantification kit. A 25 mg / mL bovine serum albumin standard solution was diluted to a concentration of 0.5 mg / mL. In a 96-well plate, 0, 1, 2, 4, 8, 12, 16, and 20 μL of protein standard were added to each well, respectively. Standard diluent was added to bring the total volume to 20 μL, resulting in standard concentrations of 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mg / mL. 20 μL of the protein sample to be tested was added to each well. 200 μL of BCA working solution (A:B = 50:1) was then added to each well, and the plate was incubated at 37°C for 30 min. The absorbance at 562 nm was measured using a microplate reader. A standard curve was plotted with protein concentration on the x-axis and absorbance at 562 nm on the y-axis. The protein concentration of the sample was then calculated based on the standard curve.

[0089] 2. Results Analysis

[0090] 2.1 Validation using SDS-PAGE and Western blot

[0091] After successfully transforming the recombinant plasmid pET28a-LysP70 into BL21(DE3), expression was induced by IPTG, and the expression product was purified. SDS-PAGE results showed a protein band around 35 kDa, and the protein molecular weight was similar to the predicted size. Figure 1 A). Western blot analysis also revealed clear bands, indicating successful expression of the LysP70 protein. Figure 1 B).

[0092] 2.6 Determination of protein concentration

[0093] Protein concentration was determined using the BCA method for subsequent experiments. After ultrafiltration and concentration of the purified LysP70 lysin, the protein concentration was calculated using the established protein standard curve, with the formula: y = 0.0012x + 0.1052, R0. 2 =0.9956, and the calculated concentration of the lysin LysP70 protein is 1097 μg / mL. The protein standard curve is shown in [reference needed]. Figure 2 As shown.

[0094] Example 2: Determination of the lysing activity of the lysin LysP70

[0095] 1. Experimental Methods

[0096] 1.1 Lytic activity of lyase LysP70 on plates

[0097] Listeria monocytogenes strains stored at -80℃ were retrieved and revived by streak plating. Single colonies from the plates were inoculated into BHI liquid medium and cultured overnight. The culture was then transferred at a 1:100 ratio and incubated at 37℃ with constant temperature shaking at 200 rpm until OD500. 600 The value is approximately 0.6. Take 100 μL of bacterial suspension and mix it in 1.2% BHI semi-solid medium at a suitable temperature. Pour it onto a 0.7% water agar plate. After the upper layer solidifies, take 8 μL of lysin LysP70 and add it to the surface of the medium. The control group uses PBS instead of lysin LysP70. Place the plate in an inverted incubator at 37℃ for 8 hours and observe whether transparent or translucent lysis zones appear on the plate.

[0098] 1.2 Lysogenic activity of lyase LysP70 in liquid environment

[0099] strain Li4 cultured to OD 600 =0.6, centrifuge the bacterial suspension at 8000 r / min for 5 min, discard the supernatant, wash the bacterial pellet three times with sterile PBS and resuspend to prepare a bacterial suspension. Add 2 mL of bacterial suspension and different concentrations (400 μg / mL, 200 μg / mL, 100 μg / mL and 50 μg / mL) of the lysin LysP70 to four sterile test tubes, and set up a negative control group treated with PBS solution, a blank control with an equal volume of PBS, and protein control groups with different concentrations. Incubate all test tubes at 37℃ with shaking for 2 h, observe whether the test tubes become turbid and measure the OD using an ELISA reader. 600 The value is used to determine the bactericidal effect of the lysin LysP70.

[0100] 1.3 In vitro bactericidal activity of lysin LysP70

[0101] The bacterial suspension preparation is the same as in 1.2. Take 100 μL of the prepared bacterial suspension and add an equal volume of lysin LysP70 to a 1.5 mL centrifuge tube, resulting in LysP70 concentrations of 400 μg / mL, 200 μg / mL, 100 μg / mL, and 50 μg / mL. Mix thoroughly. Incubate the mixture in a 37°C shaker for 2 hours. Every 20 minutes, take 100 μL and perform a 10-fold serial dilution, then plate it onto BHI agar plates and incubate at 37°C for 24 hours. Calculate the viable cell count using Logarithmic count. 10 Observe the order of magnitude of the decrease. Viable bacteria count (CFU / mL) = colony count × 10 × dilution factor.

[0102] 1.4 Determination of the cleavage profile of the lysin LysP70

[0103] The lysis profile of the lyase LysP70 was detected using the plate drop method, as described in 1.1. The strains selected for the experiment included 23 mononuclear Listeria monocytogenes strains, 1 Listeria oryzae strain, 1 Listeria wiltii strain, 1 harmless Listeria monocytogenes strain, 2 Staphylococcus strains, and 6 Salmonella strains.

[0104] 2. Results Analysis

[0105] 2.1 Lytic activity of lyase LysP70 on plates

[0106] The cleavage activity of LysP70 was detected by plate cleavage assay, and the results are as follows: Figure 3 As shown, compared with the control group, the experimental group with added lyase showed a clear lysis zone, indicating that the purified lyase LysP70 can lyse Listeria monocytogenes and has good lysis activity.

[0107] 2.2 Lysogenic activity of lyase LysP70 in liquid environment

[0108] After incubation of the lysin LysP70 and the bacterial suspension resuspended in PBS at 37°C for 2 hours on a shaker, the results are as follows: Figure 4 As shown in A and 9B, when the lysin concentration was between 50 μg / mL and 400 μg / mL, the bacterial culture became clearer and more transparent compared to the control group, with a higher OD value. 600nm The value was significantly reduced (P<0.001); the turbidimetric assay showed that the lyase LysP70 had good lysis activity.

[0109] 2.3 In vitro bactericidal activity of lysin LysP70

[0110] Results of in vitro bactericidal activity test as follows Figure 5 As shown, compared with the control group, a lysin concentration of 400 μg / mL reduced the viable bacterial count by approximately 3.4 Log. 10 CFU / mL (P<0.001); when the lyase concentration was 50 μg / mL, the viable bacterial count decreased by approximately 2.7 Log. 10 The concentration of CFU / mL (P<0.001) indicates that the lyase LysP70 has a strong bactericidal effect. Furthermore, it can be seen that after incubating the bacterial culture with the lyase LysP70 for 20 minutes, the number of viable bacteria decreased significantly, indicating that the lyase LysP70 has a rapid bactericidal rate.

[0111] 2.4 Determination of the cleavage profile of the lysin LysP70

[0112] The lysis range of the lysin LysP70 was determined by observing the formation of lysis zones. The results are shown in Table 2. Lysin LysP70 could lyse 23 strains of Listeria monocytogenes, 1 strain of Listeria elodea, 1 strain of Listeria wiltii, and 1 harmless Listeria strain, but could not lyse Staphylococcus or Salmonella. The experimental results indicate that LysP70 has a broad lysis range against Listeria species, but lacks lysis ability against Staphylococcus and Salmonella.

[0113] Table 3 Results of LysP70 lysis profile determination.

[0114]

[0115]

[0116] Example 3: Determination of the stability of the lysin LysP70

[0117] 1. Experimental Methods

[0118] 1.1 Effect of pH on the cleavage activity of the lysin LysP70

[0119] strain Li4 was shaken to the logarithmic growth phase (OD). 600 =0.6~0.8), centrifuge the bacterial solution at 8000 r / min for 5 min, discard the supernatant, wash the bacterial pellet three times with sterile PBS, and resuspend the bacterial pellet with PBS buffer of different pH values. The experimental group was 100 μL of bacterial solution with LysP70 lyase added to a final concentration of 400 μg / mL, and the control group was 100 μL of bacterial solution with an equal volume of PBS. Incubate at 37℃ for 2 h. Take out 100 μL and perform 10-fold serial dilutions and spread it on plates. Incubate upside down at 37℃ for 24 h and then count the colonies.

[0120] 1.2 Effect of temperature on the cleavage activity of the lysin LysP70

[0121] Strain Li4 was cultured to the logarithmic phase, centrifuged for 5 min, the supernatant was discarded, and the precipitate was washed with PBS and resuspended. The protein was incubated in a water bath at different temperatures (4℃, 20℃, 30℃, 37℃, 40℃, 50℃, 60℃, 70℃, 80℃, 90℃) for 30 min, and then mixed with 100 μL of bacterial culture after returning to room temperature. The final concentration of the lysin LysP70 was 400 μg / mL. An equal volume of PBS was added to the control group. The mixture was incubated at 37℃ for 2 h. 100 μL of the mixture was then serially diluted 10-fold and plated. The plates were incubated upside down at 37℃ for 24 h before colony counting.

[0122] 1.3 Effects of different metal ions on the cleavage activity of the lysin LysP70

[0123] The bacterial suspension was prepared as in 2.3.2. The purified lysin LysP70 was added to 100 μL of the prepared bacterial suspension. Different concentrations of Na+ were added to the mixture. + K + Ca 2+ and Zn 2+ The control group used PBS instead of LysP70 lysin, and was cultured at 37°C with shaking for 2 hours. 100 μL of the solution was diluted and spread on BHI plates, and colony counts were performed after incubation at 37°C for 24 hours.

[0124] 1.4 Effects of different storage temperatures and times on the lytic activity of the lysin LysP70

[0125] After storing the protein at different temperatures (RT, 4℃, and -80℃) for 1, 3, 5, 7, 10, 14, 20, 25, and 30 days, its lytic activity was determined using the plate drop method and plate count method. The plate drop method is the same as in 2.1.1, and the plate count method is as follows:

[0126] strain Li4 cultured to the logarithmic growth phase (OD50) 600 =0.6~0.8), centrifuge the bacterial suspension at 8000 r / min for 5 min, discard the supernatant, wash the bacterial pellet three times with sterile PBS and resuspend it to prepare a bacterial suspension, take 100 μL of bacterial suspension and mix with an equal volume of lysin LysP70, add PBS to the control group, place in a shaker at 37℃ and culture for 2 h, take 100 μL for serial dilution and spread on plates, incubate at 37℃ for 24 h, and count the colonies.

[0127] 2. Results Analysis

[0128] 2.1 Effect of pH on the cleavage activity of lysin LysP70

[0129] In the pH stability test, the results are as follows: Figure 6 As shown, the lysin LysP70 exhibits high enzyme activity within the pH range of 3–11, indicating its acid and alkali resistance; at pH 7, it shows relatively high lysogenic activity, with a decrease in viable cell count of approximately 3.3 Log compared to the control group. 10 The CFU / mL (p<0.001) indicates that pH 7 is the optimal pH for the lysin LysP70.

[0130] 2.2 Effect of temperature on the cleavage activity of the lysin LysP70

[0131] Temperature stability test results are as follows Figure 7As shown, the lyase LysP70 exhibits high activity in the temperature range of 20℃ to 50℃. Its activity is significantly reduced after heat treatment at 60℃ to 90℃, but it still retains activity, indicating that it can withstand high temperatures. 37℃ is the optimal temperature for the lyase LysP70.

[0132] 2.3 Effects of different metal ions on the cleavage activity of the lysin LysP70

[0133] Na + In stability tests, such as Figure 8 As shown in A, when Na + At a concentration of 0 mM, the viable bacterial count decreased by approximately 3.6 Log. 10 CFU / mL; when Na + At a concentration of 300 mM, the viable bacterial count decreased by approximately 2.5 Log. 10 CFU / mL indicates Na + It can inhibit the enzyme activity of lyase LysP70, but compared with the control group LysP70 still has enzyme activity (p<0.001); at 50mM to 300mM, the inhibitory effect increases with increasing ion concentration; at 300mM to 600mM, the inhibitory effect decreases with increasing ion concentration.

[0134] K + and Ca 2+ In stability tests, such as Figure 8 As shown in B and 8C, when K + and Ca 2+ At a concentration of 0 mM, the viable bacterial count decreased by approximately 2.8 Log compared to the control group. 10 CFU / mL and 2.9 Log 10 CFU / mL; when K + and Ca 2+ At a concentration of 200 mM, the number of viable bacteria decreased by approximately 2.1 Log compared to the control group. 10 CFU / mL and 1.3 Log 10 CFU / mL indicates K + and Ca 2+ It can inhibit the enzyme activity of the lyase LysP70, and the inhibitory effect is enhanced with increasing ion concentration.

[0135] Zn 2+ In stability tests, such as Figure 8 As shown in D, when Zn 2+ At a concentration of 0 mM, the viable bacterial count decreased by approximately 4.1 Log. 10 CFU / mL; when Zn 2+ At a concentration of 0.5 mM, the viable bacterial count decreased by approximately 5.3 Log. 10 CFU / mL indicates Zn2+ Enzyme activity that can promote the lysin LysP70; 0.1mM~10mM Zn 2+ The addition of [a specific ingredient] promoted the enzyme activity of LysP70 lyase, but the changes were irregular.

[0136] 2.4 Effects of different storage temperatures and times on the lytic activity of the lysin LysP70

[0137] The effect of storage conditions on the lytic activity of the lysin LysP70 enzyme was determined by plate drop and flat counting methods. Figure 9 As shown in Figure A, clear lysis zones were observed under different storage conditions, indicating that the lyase LysP70 still exhibits good lysis activity after 30 days of storage at different temperatures. Figure 9 As shown in Figure B, after co-incubating the lyase LysP70 with bacteria under different storage conditions for 2 hours, the viable bacterial count in the experimental group decreased by approximately 1.35–3.51 Log10 CFU / mL compared to the control group, with a significant difference (p<0.05). In conclusion, the lyase LysP70 can be stored at RT, 4℃, and -80℃ for approximately 30 days without losing its lysing activity. Example 4: Effect of LysP70 on Bacterial Drug Resistance

[0138] 1. Experimental Methods

[0139] The Kirby-Bauer disk diffusion method was used to test the antibiotic susceptibility of Listeria monocytogenes. Strains Li4 were treated with LysP70 (experimental group) and PBS (control group), then plated and incubated at 37°C for 24 h. Single colonies from the plates were picked and cultured in BHI liquid medium at 37°C with shaking until OD... 600 The value was 0.4. 100 μL of bacterial suspension was spread on BHI plates. After a few minutes, antibiotic susceptibility test strips were attached to the surface of the culture medium. The plates were incubated at 37°C for 24 hours. The diameter of the inhibition zone was measured with vernier calipers, and the results of sensitivity, intermediate and resistance of each antibiotic were determined. The judgment criteria were in accordance with the Staphylococcus spp. criteria in the CLSI standard. The specific judgment criteria are shown in Table 4.

[0140] Table 4. Types of antibiotics and judgment criteria for drug susceptibility testing.

[0141]

[0142]

[0143] 2. Results Analysis

[0144] The effects of lyase LysP70 on drug resistance in Listeria monocytogenes are shown in Table 5. Compared with the control group, the bacteria treated with lyase LysP70 showed increased sensitivity to ampicillin (AMP), doxycycline (DO), and piperacillin (PIP), as evidenced by an increase in the diameter of the inhibition zone. However, they still exhibited strong resistance to ceftazidime (CAZ) and polymyxin B (PB), with no inhibition zone produced.

[0145] Table 5. Results of antimicrobial susceptibility testing of bacteria under different treatment conditions.

[0146]

[0147] Example 5: Lysase LysP70 cleavage activity in milk

[0148] 1. Experimental Methods

[0149] Single colonies of Listeria monocytogenes Li4 were picked from PALCAM agar plates, cultured to the logarithmic growth phase, washed with PBS, and resuspended. The bacterial suspension was then inoculated into milk at a ratio of 1:100 to achieve a bacterial concentration of 10. 6 CFU / mL. Lysin was added to milk samples at concentrations of 400 μg / mL and 200 μg / mL. A control group with an equal volume of PBS was also included. Samples were incubated statically at 4℃ and 30℃ for 0, 2, 4, 6, 8, 12, 24, and 48 hours. Samples were then serially diluted, plated on BHI plates, and incubated at 37℃ for 24 hours before colony counting.

[0150] 2. Experimental Results

[0151] The cleavage activity of the lyase LysP70 in milk at 4℃ and 30℃ was studied using the plate count method. The results are as follows: Figure 15 As shown, under 4℃ conditions, the viable bacterial count in the experimental group decreased by approximately 0.3-0.5 Log compared to the control group after 2-12 hours using the lysin LysP70. 10 CFU / mL. At 24h and 48h, the viable bacterial count in the experimental group was basically the same as that in the control group, with no significant difference between the two. Figure 10 A). Under 30℃ conditions, the number of viable bacteria in the control group increased continuously over 12 hours, from an initial count of 6.3 Log. 10 CFU / mL increased to 8.3 Log 10 CFU / mL. Incubation with 400 μg / mL and 200 μg / mL LysP70 lysin for 4 h resulted in a 1.9 Log decrease in viable bacterial count compared to the control group. 10The optimal lytic activity was achieved at 1.3 Log10 CFU / mL and 1.3 Log10 CFU / mL. At 24 h and 48 h of incubation, the viable cell counts in the experimental group were essentially the same as those in the control group, with no significant difference between the two. Figure 10 B). In summary, the lysin LysP70 exhibits stronger cleavage activity at 30°C than at 4°C.

[0152] Example 6: Lysogenic activity of lyase LysP70 in lettuce

[0153] 1. Experimental Methods

[0154] Rinse the fresh lettuce thoroughly with distilled water and cut it into squares (1×1cm). 2 Place the sample in a sterile petri dish and irradiate with UV light for 30 minutes to ensure sterility. Inoculate the lettuce slices with 100 μL of Listeria monocytogenes Li4 suspension (10 μL). 8 CFU / cm 2 The samples were dried in a biosafety cabinet for 15–20 min. 100 μL of the lysin LysP70 was evenly spread on the surface of lettuce leaves. An equal volume of PBS buffer was added to the lettuce leaves of the control group, and the samples were incubated at 4°C and 30°C. At 0, 2, 4, 6, 8, 12, 24, and 48 h, the lettuce leaves were removed and placed in sterile tubes containing 1 mL of PBS, and homogenized. 100 μL of the homogenate was taken, serially diluted 10-fold with sterile PBS, spread on BHI plates, and incubated at 37°C for 24 h. The bacterial count was then determined.

[0155] 2. Results Analysis

[0156] like Figure 11 As shown, under 4℃ conditions, the number of viable bacteria in the experimental group decreased by approximately 0.53-1.23 Log during a 24-hour incubation period with the lysin LysP70 compared to the control group. 10 CFU / mL Figure 11 A). At 30°C, after incubation with the lysin LysP70 for 4 hours, the number of viable bacteria decreased by 1.29 Log₂O₅ compared to the control group. 10 CFU / mL and 1.33 Log 10 CFU / mL, the lysis activity reached its optimal level. Figure 11 B). When incubated at 4℃ and 30℃ for 48 hours, there was no significant difference in the number of viable bacteria between the experimental group and the control group.

[0157] Example 7: Determination of the ability of the lysin LysP70 to lyse the biofilm of Listeria monocytogenes.

[0158] 1. Experimental Methods

[0159] 1.1 Determination of biofilm formation ability

[0160] Three strains of Listeria monocytogenes were selected, and single colonies were inoculated into BHI liquid medium and cultured at 37°C with constant temperature shaking at 200 rpm until OD500. 600nm The value was approximately 0.6. 200 μL of a 100-fold dilution of BHI culture medium was added to a 96-well plate, with BHI liquid medium as a negative control. The plates were incubated at 37°C for 12 h, 24 h, 36 h, 48 h, 60 h, 72 h, and 96 h, respectively. After incubation, the medium containing airborne bacteria was gently aspirated with a pipette, and the plates were washed three times with 200 μL of sterile PBS and air-dried at room temperature. 200 μL of methanol was added to each well for fixation for 15 min, the methanol was removed, and the plates were air-dried for 15 min. 200 μL of 0.1% crystal violet staining solution was added to each well and stained for 30 min. The staining solution was discarded, the plates were washed six times with PBS, and the water was patted dry. 200 μL of 95% ethanol solution was added to each well and the plates were gently shaken for 15 min to ensure complete destaining. The OD was measured using a microplate reader. 595nm Each sample was tested in triplicate, and the experiment was repeated three times.

[0161] 1.2 Inhibitory effect of lyase LysP70 on the biofilm of Listeria monocytogenes

[0162] 1.2.1 Crystal Violet Staining Method

[0163] Listeria monocytogenes was cultured at 37°C with shaking until OD. 600 The value was approximately 0.6. The bacterial culture was diluted 100-fold with BHI medium. 100 μL of the bacterial culture was added to a 96-well plate, and 100 μL of different concentrations of lyase LysP70 were added to each well. The plates were mixed thoroughly to achieve concentrations of lyase of 400 μg / mL, 200 μg / mL, 100 μg / mL, and 50 μg / mL, respectively. An equal volume of PBS was added to the control group. The plates were incubated at 37°C for 24 h and 48 h, respectively. The subsequent procedures were the same as in section 2.1.

[0164] 1.2.2 Fluorescent staining method

[0165] The bacterial culture was prepared as above. Cell smears were placed in 12-well plates, and 1 mL of bacterial culture and an equal volume of LysP70 lyase were added to each well. The mixture was thoroughly mixed. PBS was added to the control group. The cells were incubated at 37°C for 24 h and 72 h. The supernatant was discarded, and the cells were washed three times with sterile PBS and air-dried at room temperature. The cells were fixed with 2.5% glutaraldehyde for 20 min, and then stained with fluorescent staining solution (STYO9 / PI was added to sterile water at a 1:1 ratio) for 20 min in the dark. The staining solution was discarded, and the cells were washed three times with PBS and air-dried. The cells were mounted with a fluorescence quenching mounting medium and observed under a fluorescence microscope.

[0166] 1.2.3 Scanning Electron Microscopy

[0167] Sample preparation was the same as that for fluorescence staining. The prepared slides were fixed in 2.5% glutaraldehyde fixative, and the fixed samples were washed three times with PBS for 15 min each time. They were then fixed with 1% osmium tetroxide at room temperature in the dark for 2 h, washed three times with PBS, and dehydrated with ethanol of different concentrations (30%, 50%, 70%, 80%, 90%, 95%, and 100%). The samples were dried in a critical point desiccator, then sputter-coated with gold, and observed under a scanning electron microscope.

[0168] 1.3 The effect of lysin LysP70 on the removal of biofilm from Listeria monocytogenes

[0169] 1.3.1 Crystal Violet Staining Method

[0170] Listeria monocytogenes was cultured at 37°C with shaking until OD. 600 The value was approximately 0.6. The bacterial culture was diluted 100-fold with BHI medium, and 200 μL of the bacterial culture was added to each well of a 96-well plate. The plates were incubated at 37°C for 24 h and 48 h, respectively. After incubation, the culture medium containing airborne bacteria was gently aspirated with a pipette and washed three times with 200 μL of sterile PBS. 200 μL of different concentrations of lyase LysP70 were added to the medium, with concentrations of lyase being 400 μg / mL, 200 μg / mL, 100 μg / mL, and 50 μg / mL, respectively. An equal volume of PBS was added to the control group. The plates were incubated at 37°C for 2 h, the supernatant was discarded, and the plates were washed twice with PBS. The plates were then air-dried at room temperature. Subsequent procedures were the same as in 2.1.

[0171] 1.3.2 Fluorescent staining method

[0172] Cell smears were placed in 12-well plates, and 2 mL of bacterial culture was added to each well. The plates were incubated at 37°C for 24 h and 72 h. The supernatant was discarded, and the cells were washed three times with sterile PBS. 1 mL of LysP70 lyase was added. The control group was incubated with PBS and incubated at 37°C for 2 h. The supernatant was discarded, and the cells were washed and dried. The staining method was the same as in 2.2.2.

[0173] 1.3.3 Scanning Electron Microscopy

[0174] Cell smears were placed in 12-well plates, and 2 mL of bacterial culture was added to each well. The plates were incubated at 37°C for 24 h and 72 h. 1 mL of LysP70 lyase was added to the experimental group, and PBS was added to the control group to act on the cultured biofilm. Scanning electron microscopy samples were prepared, and the sample processing method was the same as in 2.2.3.

[0175] 1.4 Effects of the lysin LysP70 on cellular metabolic activity

[0176] Listeria monocytogenes was cultured at 37°C with shaking until OD. 600 The value is approximately 0.6. A 1:100 transfer ratio results in a bacterial concentration of 10. 6CFU / mL. 100 μL of bacterial culture was added to each well of a 96-well plate. The experimental groups received 100 μL of different concentrations (400, 200, 100, 50, 25 μg / mL) of the lysin LysP70, while the control group received 100 μL of PBS. After incubating the 96-well plates at 37°C for 72 h, the plates were washed three times with PBS, air-dried at room temperature for 30 min, and then incubated at 37°C for 4 h with 100 μL of PBS and 10 μL of CCK-8 reagent. OD was then measured. 450nm Value. Record the OD value of the control group. 450nm OD0, experimental group OD 450nm The formula for calculating the metabolic activity inhibition rate is: Metabolic activity inhibition rate % = (OD0 - OD1) / OD0 × 100%.

[0177] 1.5 Effects of the lyase LysP70 on the self-aggregation and cell surface hydrophobicity of Listeria monocytogenes

[0178] Listeria monocytogenes, after activation, was inoculated into BHI medium containing different concentrations of the lysin LysP70 (400, 200, 50, and 0 μg / mL) and cultured at 37°C for 24 h. The cells were then centrifuged at 12000 rpm for 10 min at 4°C, and resuspended twice in sterile PBS. The OD of the bacterial suspension at this point was... 600nm The reading is recorded as H0 or A0. Divide the bacterial suspension into two parts. After mixing one part by shaking, incubate it in a 37°C water bath for 2 hours, and then measure the OD of the bacterial suspension. 600nm , denoted as A t And according to the formula, self-polymerization rate / % = A0 - A t / A0×100%, calculate its autoaggregation property.

[0179] Another portion was mixed with xylene by shaking, and then incubated in a 37°C water bath for 15 minutes. The aqueous phase was then pipetted to determine the OD. 600nm And denoted as H t The hydrophobicity of the cell is calculated according to the formula: hydrophobicity / % = H0 - Ht / H0 × 100%.

[0180] 1.6 Effect of the lyase LysP70 on the extracellular polysaccharides of Listeria monocytogenes

[0181] Extracellular polysaccharides were determined using the phenol-sulfuric acid method. 1 mL of the lyase LysP70 (final concentration 400 μg / mL) was added to each well of a 12-well plate. PBS was used as the control group. Listeria monocytogenes cells were inoculated into the wells (final concentration 10). 6(CFU / mL) was added to sterile cell smears, and the mixture was incubated at 37°C for 72 h. After washing twice with PBS, the cell smears were removed with forceps and placed in a 50 mL centrifuge tube. 3 mL of PBS was added for elution, followed by sonication for 3 min and centrifugation at 8000 rpm for 30 min. The supernatant was collected by filtration. The extracellular polysaccharide content was determined using a total polysaccharide assay kit. 200 μL of the supernatant was placed in a centrifuge tube, 200 μL of 6% phenol was added, and after mixing, 500 μL of concentrated sulfuric acid was added. The mixture was incubated at 90°C for 20 min, and after cooling, 200 μL was used to determine the OD. 490nm The sample concentration (mg / mL) was calculated based on the standard curve.

[0182] 1.7 Effect of lyase LysP70 on extracellular proteins of Listeria monocytogenes

[0183] The preparation of the supernatant was the same as above. Extracellular protein concentration was determined using a BCA kit. 20 μL of the prepared supernatant was added to a 96-well plate, followed by 200 μL of BCA working solution. The plate was incubated at 37°C for 30 min, and the OD was measured. 562nm The concentration of extracellular proteins was calculated based on the standard curve.

[0184] 1.8 Data Statistical Analysis

[0185] GraphPad Prism 8.0 and Excel 2016 software were used for chart creation and data analysis. t-tests and one-way ANOVA were used for significance testing. No asterisk (*) or ns indicates no significant difference (p>0.05), * indicates a significant difference (p<0.05), and ** and *** indicate extremely significant differences (p<0.01 and p<0.001, respectively).

[0186] 2. Results Analysis

[0187] 2.1 Determination of biofilm formation ability

[0188] The ability of Listeria monocytogenes to form a biofilm was determined by crystal violet staining, and the results are as follows: Figure 12 As shown, the biofilm amount of the three bacterial strains increased over time within 36 hours; when cultured for 72 hours, the biofilm amount of Li4 ​​and D40 reached its maximum, and then began to decrease. The strength of biofilm formation varied among the different strains, with Li4 exhibiting a stronger biofilm-forming ability, forming an early biofilm at 24 hours and a mature biofilm at 72 hours. Therefore, the early biofilm formed at 24 hours and the mature biofilm formed at 72 hours were selected for the experiment.

[0189] 2.2 Inhibitory effect of lyase LysP70 on the biofilm of Listeria monocytogenes

[0190] Three methods were used to investigate the inhibitory effect of the lyase LysP70 on biofilms. Crystal violet staining showed that the OD values ​​of different concentrations of LysP70 treatment groups increased. 595 The OD value was significantly lower than that of the control group (p<0.05), and the OD value increased with increasing concentration. 595 The smaller the value, the more significantly the lysin LysP70 can inhibit biofilm formation after co-incubation with bacteria for 24h and 72h. Figure 13 A). Fluorescent staining showed that after 24 h and 72 h of treatment, the number of viable bacteria in the LysP70 lyase-treated group was less than that in the control group, and the structure became looser. Figure 13 B). Scanning electron microscopy showed that after 24 h and 72 h of treatment, the control group cells remained intact with regular edges, while the cells treated with the lysin P70 enzyme exhibited morphological damage and deformation, losing their typical bacillus morphology. Figure 13 C). These results indicate that the lyase LysP70 effectively inhibits biofilm formation by disrupting biofilm structure, reducing biomass, and impairing bacterial morphology.

[0191] 2.3 The scavenging effect of the lysin LysP70 on the biofilm of Listeria monocytogenes

[0192] To investigate the ability of the lyase LysP70 to remove pre-formed biofilms, biofilms were cultured for 24 h and 72 h, and then treated with LysP70 for 2 h to evaluate its removal effect on existing biofilms. Crystal violet staining showed that the OD values ​​of different concentrations of LysP70 treatment groups increased. 595 The value was significantly lower than that of the control group (p<0.001), indicating that the lysin LysP70 can remove existing biofilms, and the higher the concentration, the better the removal effect. Figure 14 A). Fluorescent staining showed that the biofilm in the control group exhibited a dense network structure with a large number of viable bacteria, while the biofilm treated with the lyase LysP70 became more dispersed, and the number of bacteria decreased. Figure 14 B). Scanning electron microscopy showed that, compared with the control group, the morphology of bacteria treated with the lysin P70 was disrupted and deformed. Figure 14 C). These findings indicate that the lyase LysP70 can effectively disrupt and remove pre-formed early and mature biofilms.

[0193] 2.4 Effects of the lysin LysP70 on cellular metabolic activity

[0194] The metabolic activity of Listeria monocytogenes after treatment with the lysin LysP70 was determined by the CCK-8 assay, and the results are as follows: Figure 15As shown, the lyase LysP70 significantly inhibited the metabolic activity of Listeria monocytogenes biofilms, with the highest inhibition rate of 50% at 400 μg / mL. The inhibitory effect weakened as the concentration decreased.

[0195] 2.5 Effects of the lyase LysP70 on the self-aggregation and cell surface hydrophobicity of Listeria monocytogenes

[0196] The effects of the lyase LysP70 on the self-aggregation and cell surface hydrophobicity of Listeria monocytogenes, such as Figure 16 As shown in the figure, the self-aggregation and cell surface hydrophobicity of Listeria monocytogenes significantly decreased after treatment with different concentrations of the lyase LysP70. After treatment with 400 μg / mL of the lyase LysP70, the cell self-aggregation rate decreased from 39.15% to 14.42%, and the cell surface hydrophobicity decreased from 40.24% to 23.73%. Furthermore, with increasing concentration, both the self-aggregation rate and cell surface hydrophobicity decreased.

[0197] 2.6 Effect of the lyase LysP70 on the extracellular polysaccharides of Listeria monocytogenes biofilm

[0198] The extracellular polysaccharide content of each group can be calculated using the standard curve in Figure 17A. Figure 17 As shown in B, the extracellular polysaccharide content of Listeria monocytogenes was significantly reduced after treatment with the lyase LysP70 (p<0.01), indicating that the lyase LysP70 can inhibit the production of extracellular polysaccharides in Listeria monocytogenes, and the inhibitory effect is enhanced with increasing concentration.

[0199] 2.7 Effect of the lyase LysP70 on extracellular proteins of the biofilm of Listeria monocytogenes

[0200] The extracellular protein content of each group can be calculated using the standard curve in Figure 18A. Figure 18 As shown in B, when Listeria monocytogenes was treated with different concentrations of the lyase LysP70, the extracellular protein concentration decreased by 53 μg / mL, 42 μg / mL, and 28 μg / mL, respectively, compared with the control group. This indicates that the lyase LysP70 can inhibit the production of extracellular proteins in Listeria monocytogenes.

[0201] In summary, this invention provides a Listeria monocytogenes phage lysin LysP70, the amino acid sequence of which is shown in SEQ ID No. 1 and the nucleotide sequence in SEQ ID No. 2. The LysP70 lysin can lyse Listeria monocytogenes, exhibiting good lytic activity, rapid bactericidal speed, and a broad lytic range against Listeria species, but lacks lytic ability against Staphylococcus and Salmonella. The activity of this lysin is relatively stable within a certain range of pH, temperature, metal ion concentration, storage temperature, and time. Bacteria treated with LysP70 show increased sensitivity to ampicillin (AMP), doxycycline (DO), and piperacillin (PIP), as evidenced by an increased diameter of the inhibition zone; however, they still exhibit strong resistance to ceftazidime (CAZ) and polymyxin B (PB), with no inhibition zone produced. The LysP70 lyses milk and lettuce. LysP70 effectively inhibits biofilm formation by disrupting biofilm structure, reducing the amount of biofilm-covered bacteria, and damaging bacterial morphology. It can effectively destroy and remove pre-formed early and mature biofilms. It significantly inhibits cellular metabolic activity in Listeria monocytogenes biofilms, with the highest inhibition rate of 50% at 400 μg / mL; the inhibitory effect weakens as the concentration decreases. LysP70 also inhibits the production of extracellular polysaccharides in Listeria monocytogenes, with the inhibitory effect increasing with increasing concentration; it can also inhibit the production of extracellular proteins in Listeria monocytogenes.

Claims

1. A Listeria monocytogenes phage lysin LysP70, characterized in that, The amino acid sequence of the lysin LysP70 is shown in SEQ ID No.

1.

2. The gene encoding the lyase LysP70 according to claim 1, characterized in that, The nucleotide sequence of the encoding gene is shown in SEQ ID No.

2.

3. A recombinant expression vector for expressing the lysin LysP70, characterized in that, The expression vector comprises the nucleotide sequence of the lyase LysP70 as described in claim 2 and the vector.

4. A recombinant engineered bacterium expressing the lysin LysP70, characterized in that, The recombinant engineered bacteria are generated by transforming the recombinant expression vector described in claim 3 into the host bacteria.

5. The use of the lysin LysP70 as described in claim 1, the recombinant expression vector as described in claim 3, or the recombinant engineered bacteria as described in claim 4 in the preparation of reagents for lysing Listeria monocytogenes.

6. A reagent for lysing Listeria monocytogenes, characterized in that, The reagent includes the lyase LysP70 of claim 1, the amino acid sequence of which is shown in SEQ ID No.

1.

7. An antibacterial agent, characterized in that, Its main active ingredient is at least one of the following: LysP70 lyase, a recombinant expression vector containing LysP70 lyase expression element, an expression cassette containing LysP70 lyase expression element, or a recombinant engineered bacterium containing LysP70 lyase expression element, wherein the amino acid sequence of LysP70 lyase is shown in SEQ ID No.

1.

8. An antibacterial composition, characterized in that, Includes the antibacterial agent and antibiotic as described in claim 7.

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