Streptococcus suis bacteriophage lyase with wide degradation spectrum as well as coding gene and application thereof

Abstract

The invention discloses a streptococcus suis bacteriophage lyase with a wide degradation spectrum as well as a coding gene and application thereof. The amino acid sequence of the streptococcus suis bacteriophage lyase provided by the invention is as shown in SEQ ID No.1, and the nucleotide sequence of the coding gene is as shown in SEQ ID No.2. Tests prove that the streptococcus suis bacteriophage lyase is high in environmental tolerance, and can keep remarkable lysis activity in the pH value range of 3.0-11.0, the temperature range of 4-55 DEG C and the salt concentration range of 0-600mM; the broad-spectrum splitting capacity is achieved, and the good inhibition effect on 32 different types of streptococcus suis, 3 streptococcus agalactiae and 1 staphylococcus aureus can be achieved; the piglet feed additive has the advantages of simple preparation process, high degradation resistance, high safety and no side effect, and can still play a good bacteriostatic role in a piglet serum medium within 24 hours. The invention has application prospects in preparation of feed additives and medicines for resisting streptococcus suis.

Description

Technical Field

[0001] The present invention relates to a phage lytic enzyme and its application field, in particular to a Streptococcus suis phage lytic enzyme and its encoding gene and its application in preparing a drug for inhibiting bacteria or as an animal feed additive, belonging to the field of Streptococcus suis phage lytic enzyme and its application field. Background Art

[0002] Streptococcus suis is a major zoonotic pathogen, widespread in the natural environment and posing a serious threat to the swine industry and public health. Multiple pathogenic strains of S. suis can cause diseases such as meningitis and sepsis in pigs, and can also infect humans, leading to serious health problems. Currently, antibiotics are the mainstay of treatment for S. suis infections. However, the widespread use of antibiotics has led to the increasing prevalence of bacterial resistance and a decline in treatment effectiveness.

[0003] Phage lytic enzymes are hydrolases encoded by bacteriophage genomes that specifically cleave bacterial cell walls. They offer broad-spectrum antimicrobial activity, are less susceptible to drug resistance, and exhibit no adverse effects on animal cells. Therefore, phage lytic enzymes hold great promise as a new class of antimicrobial agents. However, existing phage lytic enzymes still have limitations in terms of environmental tolerance, resistance to degradation, and lytic activity, failing to meet practical application requirements. Summary of the Invention

[0004] One of the purposes of the present invention is to provide a Streptococcus suis phage lytic enzyme Ply900 with a broad degradation spectrum.

[0005] The second purpose of the present invention is to provide the coding gene of the Streptococcus suis phage lytic enzyme Ply900.

[0006] The third object of the present invention is to provide an expression vector or recombinant host cell containing the encoding gene.

[0007] The fourth object of the present invention is to apply the Streptococcus suis phage lytic enzyme Ply900, the coding gene of the Streptococcus suis phage lytic enzyme Ply900, or the expression vector or recombinant host cell containing the coding gene to the preparation of antibacterial drugs or as animal feed additives.

[0008] In order to achieve the above objectives, the main technical solutions adopted by the present invention include:

[0009] On one hand, the present invention provides a Streptococcus suis phage lytic enzyme Ply900 with a broad degradation spectrum.

[0010] In a preferred embodiment of the present invention, the amino acid sequence of the Streptococcus suis phage lytic enzyme Ply900 is selected from any one of the following amino acid sequences (a) to (c):

[0011] (a) the amino acid sequence shown in SEQ ID No. 1;

[0012] (b) deleting, inserting, or / and replacing one or more amino acids in the amino acid sequence shown in SEQ ID No. 1 to obtain a mutant amino acid sequence, wherein the mutant amino acid sequence still has the activity of inhibiting Streptococcus suis;

[0013] (c) an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID No. 1, and that still has the activity of inhibiting Streptococcus suis.

[0014] Another aspect of the present invention is to provide the coding gene of the Streptococcus suis phage lytic enzyme Ply900.

[0015] In a preferred embodiment of the present invention, the gene encoding the Streptococcus suis phage lytic enzyme Ply900 is selected from any one of the nucleotide sequences described in (a) to (e) below:

[0016] (a) the polynucleotide sequence shown in SEQ ID No. 2;

[0017] (b) a polynucleotide sequence encoding the amino acid sequence shown in SEQ ID No. 1;

[0018] (c) a polynucleotide sequence capable of hybridizing with the polynucleotide sequence described in (a) or (b) under stringent hybridization conditions, wherein the protein encoded by the polynucleotide sequence still has the function of inhibiting the activity of Streptococcus suis;

[0019] (d) a polynucleotide sequence that is at least 95% identical to the polynucleotide sequence shown in any one of (a) to (c), wherein the protein encoded by the polynucleotide sequence still has the function of inhibiting the activity of Streptococcus suis;

[0020] (e) A polynucleotide sequence that is complementary to the polynucleotide sequence described in any one of (a) to (d), wherein the protein encoded by the polynucleotide sequence still has the function of inhibiting the activity of Streptococcus suis.

[0021] Another aspect of the present invention is to provide an expression vector or a recombinant host cell containing the encoding gene.

[0022] The Streptococcus suis phage lytic enzyme Ply900 of the present invention has strong environmental tolerance and can maintain significant lytic activity within the pH range of 3.0-11.0, the temperature range of 4°C-55°C, and the salt concentration range of 0-600mM. The Streptococcus suis phage lytic enzyme Ply900 has strong lytic activity, with a minimum inhibitory concentration (MIC) and a minimum bactericidal concentration (MBC) of 20μg / mL. Bactericidal kinetic tests show that bacterial turbidity can be reduced by approximately 70% within 15 minutes. The Streptococcus suis phage lytic enzyme Ply900 has a broad-spectrum lytic ability and can effectively inhibit 32 strains of Streptococcus suis of different types, 3 strains of Streptococcus agalactiae, and 1 strain of Staphylococcus aureus. The Streptococcus suis phage lytic enzyme Ply900 has strong anti-degradation ability and can still exert a good antibacterial effect in a piglet serum medium for 24 hours.

[0023] Another aspect of the present invention is to use the Streptococcus suis phage lytic enzyme Ply900, the coding gene of the Streptococcus suis phage lytic enzyme Ply900, or the expression vector or recombinant host cell containing the coding gene to prepare drugs for inhibiting bacteria or as animal feed additives.

[0024] In a preferred embodiment of the present invention, the Streptococcus suis phage lytic enzyme Ply900, the coding gene of the Streptococcus suis phage lytic enzyme Ply900, or the expression vector or recombinant host cell containing the coding gene is used to prepare a drug for inhibiting bacteria.

[0025] In a preferred embodiment of the present invention, the bacteria is Streptococcus suis, Streptococcus agalactiae or Staphylococcus aureus.

[0026] In a preferred embodiment of the present invention, the Streptococcus suis is type 1 Streptococcus suis, type 2 Streptococcus suis, type 3 Streptococcus suis, type 7 Streptococcus suis, type 8 Streptococcus suis, type 9 Streptococcus suis, type 12 Streptococcus suis, type 18 Streptococcus suis, type 20 Streptococcus suis or untyped Streptococcus suis.

[0027] For reference, the present invention also provides a pharmaceutical composition for inhibiting Streptococcus suis, comprising an active ingredient for inhibiting Streptococcus suis and a carrier or excipient acceptable for pharmaceutical preparations, wherein the active ingredient for inhibiting Streptococcus suis is the Streptococcus suis phage lytic enzyme Ply900.

[0028] In a preferred embodiment of the present invention, the pharmaceutically acceptable carrier is one or more of xylitol, mannitol, lactose, fructose, dextran, glucose, polyvinyl pyrrolidone, low molecular weight dextran, sodium chloride, calcium gluconate or calcium phosphate; the excipient can be an antioxidant chelating agent, a filler, a skeleton material, etc.

[0029] In a preferred embodiment of the present invention, the pharmaceutical composition is in the form of a spray, a water-soluble powder, a granule, a tablet or an injection.

[0030] In a preferred embodiment of the present invention, the Streptococcus suis phage lytic enzyme Ply900, the coding gene of the Streptococcus suis phage lytic enzyme Ply900, or the expression vector or recombinant host cell containing the coding gene is used as an animal feed additive.

[0031] In a preferred embodiment of the present invention, the animal feed additive contains the Streptococcus suis phage lytic enzyme Ply900.

[0032] The invention obtains the Streptococcus suis phage lytic enzyme Ply900 through expression, purification and Western blot detection. Experiments have shown that the Streptococcus suis phage lytic enzyme Ply900 has strong environmental tolerance and can maintain significant lytic activity within the pH range of 3.0-11.0, the temperature range of 4°C-55°C, and the salt concentration range of 0-600mM. The Streptococcus suis phage lytic enzyme Ply900 has strong lytic activity, with its minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) both being 20μg / mL. Bactericidal kinetic tests showed that bacterial turbidity could be reduced by approximately 70% within 15 minutes. The Streptococcus suis phage lytic enzyme Ply900 has broad-spectrum lytic ability and can effectively inhibit 32 strains of Streptococcus suis of different types, 3 strains of Streptococcus agalactiae, and 1 strain of Staphylococcus aureus. The Streptococcus suis phage lytic enzyme Ply900 also has strong anti-degradation ability and can still exert a good antibacterial effect in piglet serum medium for 24 hours. The Streptococcus suis phage lytic enzyme Ply900 provided by the present invention can be used alone as a medicine or an animal feed additive, has no toxic side effects and is highly safe. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is the SDS-PAGE result of the expression and purification of Streptococcus suis phage lytic enzyme Ply900; where M: Marker; 1: uninduced whole bacteria; 2: induced whole bacteria; 3: induced supernatant; 4: purified supernatant.

[0034] Figure 2 The figure shows the Western blot results of Streptococcus suis phage lytic enzyme Ply900; wherein, M: Marker; 1: purified Streptococcus suis phage lytic enzyme Ply900.

[0035] Figure 3 This is the experimental result of the effect of temperature on the activity of Streptococcus suis phage lytic enzyme Ply900.

[0036] Figure 4 This is the experimental result of the effect of pH value on the activity of Streptococcus suis phage lytic enzyme Ply900.

[0037] Figure 5 This is a graph showing the effect of salt ion concentration on the activity of Streptococcus suis phage lytic enzyme Ply900.

[0038] Figure 6 This is a graph showing the bactericidal kinetic test results of Streptococcus suis phage lytic enzyme Ply900.

[0039] Figure 7 This is the lysis spectrum of Streptococcus suis phage lytic enzyme Ply900 against some strains.

[0040] Figure 8 The figure shows the bactericidal activity of Streptococcus suis phage lytic enzyme Ply900 in piglet serum. The negative control is healthy piglet serum with Streptococcus suis type 2 added but without Streptococcus suis phage lytic enzyme Ply900. The positive control is an equal volume of PBS buffer with Streptococcus suis type 2 and Streptococcus suis phage lytic enzyme Ply900 added. DETAILED DESCRIPTION

[0041] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, it should be understood that the embodiments are merely exemplary and do not limit the scope of the present invention in any way. It should be understood by those skilled in the art that the details and forms of the technical solutions of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but such modifications or replacements fall within the scope of protection of the present invention.

[0042] Reagents, culture media, strains

[0043] Amp-resistant LB plates, Amp-resistant LB liquid culture medium, LB liquid culture medium, and BL21 / Pcold-his monoclonal strain were all purchased from Sangon Biotech (Shanghai) Co., Ltd.; Ni agarose purification resin was purchased from Merck Life Sciences Sigma-Aldrich.

[0044] PBS buffer: final concentration is 10 mM tris (tris), final concentration is 100 mM NaCl, pH is 7.5.

[0045] The Streptococcus suis used in the present invention are all standard strains of Streptococcus suis type 2, which can be purchased through conventional commercial channels.

[0046] Example 1 Expression and purification of Streptococcus suis phage lytic enzyme Ply900

[0047] Pcold-Ply900 monoclonal strains were picked and activated overnight on ampicillin (Amp, 50 μg / mL)-resistant LB solid medium. The Pcold-Ply900 activated bacterial suspension was transferred to 500 mL of LB liquid medium at a 1% inoculum volume (5 mL). Amp was added at a final concentration of 50 μg / mL and cultured at 37°C, 180 rpm until the OD 600 to between 0.5 and 0.8; after an ice bath for 15 minutes, add IPTG with a final concentration of 0.5 mM, shake evenly, and induce overexpression at 16°C and 210 rpm for 24 hours; collect the bacteria by centrifugation at 4000 rpm and 4°C for 10 minutes, resuspend the bacteria in 50 mL of PBS buffer and wash twice; resuspend the washed bacteria in 50 mL of PBS buffer, fix in an ice box and ultrasonically disrupt (30 W, work 3 seconds, rest 3 seconds, 45 minutes), set the alarm temperature to 28°C, and cycle 3 times; after the ultrasonication, centrifuge at 12000 rpm and 4°C for 10 minutes to separate the supernatant and precipitate, and take the precipitate for SDS-PAGE electrophoresis (120 V, 80 minutes) to identify the expression of the protein. Subsequently, the purified Ply900 lytic enzyme (protein) was obtained according to the instruction manual of Ni agarose purification resin and stored at -80°C for future use. After purification, the concentration of Streptococcus suis phage lytic enzyme Ply900 was measured by BCA protein concentration determination kit (purchased from Sangon Biotech (Shanghai) Co., Ltd.) to be 2.2 mg / mL. The results of SDS-PAGE detection are as follows. Figure 1 As shown, the target protein is mostly located in the supernatant and is a soluble protein with a size of approximately 25.4 kDa.

[0048] The amino acid sequence of Streptococcus suis phage lytic enzyme Ply900 is shown in SEQ ID No. 1:

[0049]

[0050] The nucleotide sequence of the gene encoding the Streptococcus suis phage lytic enzyme Ply900 is shown in SEQ ID No. 2:

[0051]

[0052] Example 2 Western blot detection of Streptococcus suis phage lytic enzyme Ply900

[0053] The proteins expressed in Example 1 were subjected to SDS-PAGE gel electrophoresis. At the end of the electrophoresis, a PVDF membrane of appropriate size was cut and immersed in anhydrous methanol for 10 seconds for activation. The soaked PVDF membrane, two pieces of filter paper, and SDS-PAGE gel were placed in transfer solution and immersed for several minutes. The filter paper, PVDF membrane, SDS-PAGE gel, and sponge were placed in a pyramid shape on the transfer apparatus in order from bottom to top to remove bubbles. Adjust the voltage to 30V and the time to 15min. After the transfer is completed, take out the PVDF membrane and wash it in PBST buffer for 1min. Immerse the PVDF membrane in blocking solution (5% skim milk powder) and incubate it slowly at room temperature for 2h. Take out the PVDF membrane from the blocking solution and wash it in PBST buffer 3 times, 10min each time. Dilute the mouse anti-His tag antibody with the primary antibody diluent at a ratio of 1:1000. Take out the PVDF membrane and place it in a container containing the primary antibody and incubate it at 4℃ overnight. Take out the PVDF membrane from the primary antibody solution and wash it in PBST buffer 3 times, 10min each time. Dilute the HRP-labeled goat anti-mouse secondary antibody with PBST at a ratio of 1:1000. Take out the washed PVDF membrane and place it in a container containing the secondary antibody and incubate it at room temperature for 1h. Take out the PVDF membrane from the secondary antibody solution and wash it in PBST 3 times, 10min each time. Then use the chemiluminescence kit for color development and use the color development instrument to develop and take pictures. The results are as follows Figure 2 As shown, a specific band appeared at the position of 25.4 kDa, proving that the Streptococcus suis phage lytic enzyme Ply900 was successfully expressed.

[0054] Experimental Example 1 Effect of Temperature on the Activity of Streptococcus suis Phage Lytic Enzyme Ply900

[0055] 1 Test method

[0056] Type 2 Streptococcus suis was used as the test strain to prepare a resuspended bacterial solution. The prepared bacterial suspension and a 20 μg / mL Streptococcus suis phage lytic enzyme Ply900 solution were pre-incubated at 4°C, 16°C, 22°C, 26°C, 37°C, 42°C, 50°C, 55°C, and 60°C for 60 minutes. Then, the Streptococcus suis phage lytic enzyme Ply900 was pre-mixed with the bacterial suspension. The final concentration of the Streptococcus suis phage lytic enzyme Ply900 was 10 μg / mL. The solution was incubated at 4°C, 16°C, 22°C, 26°C, 37°C, 42°C, 50°C, 55°C, and 60°C for 60 minutes. The same volume of PBS buffer was added to the control group. After 2 hours, the OD difference between the treatment group and the control group was calculated. 600 The effect of different temperatures on the activity of Streptococcus suis phage lytic enzyme Ply900 was determined by the ratio of .All reactions were repeated three times independently.

[0057] 2 Test results

[0058] The test results are as follows Figure 3 As shown in the results, the Streptococcus suis phage lytic enzyme Ply900 has good lytic ability within the range of 4°C-55°C. The lytic activity of the Streptococcus suis phage lytic enzyme Ply900 is highest at 37°C. Above 37°C, its activity gradually decreases with increasing temperature, and the lytic activity drops significantly at 60°C. This indicates that the Streptococcus suis phage lytic enzyme Ply900 has good temperature tolerance.

[0059] Experimental Example 2 Effect of pH on the Activity of Streptococcus suis Phage Lytic Enzyme Ply900

[0060] 1 Test method

[0061] Prepare a resuspended bacterial solution of type 2 Streptococcus suis. Use hydrochloric acid and sodium hydroxide solutions to adjust the pH value of PBS buffer to 2.0-12.0, respectively. Resuspend the bacteria with PBS buffer of different pH values. Pre-incubate 20 μg / mL Streptococcus suis phage lytic enzyme Ply900 in a solution with the same pH value as the bacteria for 10 minutes. Then, premix the Streptococcus suis phage lytic enzyme Ply900 with the bacterial suspension to a final concentration of 10 μg / mL. Incubate at 37°C for 2 hours. Add PBS buffer of the same pH value to the control group. After incubation for 2 hours, the OD value between the treated group and the control group is calculated. 600 The effect of different pH values ​​on the activity of Streptococcus suis phage lytic enzyme Ply900 was determined by the ratio of pH to pH. All reactions were repeated three times independently.

[0062] 2 Test results

[0063] The test results are as follows Figure 4 As shown in the data, at pH 7, the lytic activity of Streptococcus suis phage lytic enzyme Ply900 was the highest; at pH 2.0 and pH 12.0, the activity of Streptococcus suis phage lytic enzyme Ply900 dropped sharply, that is, in the pH range of 3.0-11.0, the lytic activity of Streptococcus suis phage lytic enzyme Ply900 was higher, indicating that Streptococcus suis phage lytic enzyme Ply900 has a wide pH tolerance and can function in environments and samples with different pH values.

[0064] Experimental Example 3 Effect of Salt Ion Concentration on the Activity of Streptococcus suis Phage Lytic Enzyme Ply900

[0065] 1 Test method

[0066] Usually, phage lytic enzymes need to have lytic activity under a certain salt concentration, but the lytic activity of Streptococcus suis phage lytic enzyme Ply900 is not affected by the salt ion concentration. Prepare PBS buffer containing 1M sodium chloride concentration for use, and prepare type 2 Streptococcus suis resuspended bacteria. Add 100μL of bacterial suspension to a 96-well plate, and use PBS buffer containing 1M sodium chloride to make the final salt ion concentration of the reaction system 0, 50, 100, 150, 300, 400, 500, 750mM and 1M, respectively. Add Streptococcus suis phage lytic enzyme Ply900 to a final concentration of 20μg / mL. Add the same volume of PBS buffer to the control group and incubate at 37℃ for 60min. The OD value between the treatment group and the control group is 0. 600 The effect of different salt ion concentrations on the Streptococcus suis phage lytic enzyme Ply900 was determined by the ratio of 1:1 to 2:1. All reactions were repeated three times independently.

[0067] 2 Test results

[0068] The test results are as follows Figure 5 As shown in the figure, the lytic activity of Streptococcus suis phage lytic enzyme Ply900 is almost unaffected by salt ion concentration, which indicates that Streptococcus suis phage lytic enzyme Ply900 has a wider range of applications.

[0069] Test Example 4 Bactericidal Kinetics Detection Test of Streptococcus suis Phage Lytic Enzyme Ply900

[0070] 1 Test method

[0071] Prepare a resuspended bacterial suspension of type 2 Streptococcus suis, add the prepared bacterial suspension to a 96-well plate, and add Streptococcus suis phage lytic enzyme Ply900 to the bacterial suspension to a final concentration of 40, 20, 15, 10, 5, and 1 μg / mL, respectively. Place the plate in a microplate reader and incubate at 37°C. Shake for 2 seconds with an amplitude of 2 mm before measurement, and read the OD every 15 minutes. 600 Record the OD value within 60 minutes 600 All reactions were repeated three times independently.

[0072] 2 Test results

[0073] The test results are as follows Figure 6 As shown in the figure, as the concentration decreases, the lysis efficiency continues to decrease, and the initial lysis rate of Streptococcus suis phage lytic enzyme Ply900 decreases more significantly, but the lysis endpoint can be reached within 60 minutes.

[0074] Test Example 5 Bactericidal Kinetics Detection Test of Streptococcus suis Phage Lytic Enzyme Ply900

[0075] 1 Test method

[0076] 35 test bacteria strains stored in the laboratory were used to prepare a resuspended bacterial solution. The 35 test bacteria strains included 32 strains of Streptococcus suis, 3 strains of Streptococcus agalactiae, and 1 strain of Staphylococcus aureus. The serotypes of the 35 test bacteria strains are shown in Table 1. The prepared resuspended bacterial solution was mixed with Streptococcus suis phage lytic enzyme Ply900 to a final concentration of 20 μg / mL. After incubation at 37°C for 1 hour, the OD value was measured using a microplate reader. 600 Determination of.

[0077] Table 1 Names and serotypes of 35 test strains

[0078]

[0079]

[0080] Note: NT stands for unclassified.

[0081] 2 Test results

[0082] The test results are as follows Figure 7 As shown, the Streptococcus suis phage lytic enzyme Ply900 showed high lytic activity against 32 strains of Streptococcus suis, 3 strains of Streptococcus agalactiae and 1 strain of Staphylococcus aureus.

[0083] Test Example 6 Bactericidal Detection Test of Streptococcus suis Phage Lysing Enzyme Ply900 in Healthy Piglet Serum

[0084] 1 Test method

[0085] Prepare a resuspended bacterial solution of type 2 Streptococcus suis and add 100 μL of the bacterial suspension to a 96-well plate. Mix healthy piglet serum with Streptococcus suis phage lytic enzyme Ply900 to a final concentration of 20 μg / mL and place in a 37°C incubator for co-culture. Take 100 μL of the co-culture solution and add it to the prepared resuspended bacterial solution of type 2 Streptococcus suis at 0.5h, 1h, 2h, 6h, 12h, 18h, 24h, 36h, and 48h, incubate at 37°C for 1h, and measure the OD value with a microplate reader. 600 The OD values ​​were calculated between the treatment group and the control group. 600 The effect of the incubation time with Streptococcus suis on the lytic enzyme Ply900 was determined by the ratio of 1:1 to 2:1. All reactions were repeated three times independently.

[0086] 2 Test results

[0087] The test results are as follows Figure 8 As shown in the figure, with the increase of the co-incubation time at 37°C, the lysis efficiency continued to decrease, and the lysis efficiency decreased significantly after 36 hours, while a good lysis effect was still retained within 0-24 hours.

[0088] Test Example 7: Experiment on killing Streptococcus suis in the environment by Streptococcus suis phage lytic enzyme Ply900

[0089] 1mL of a concentration of 10 5 A single smear of Streptococcus suis (CFU / mL) was evenly applied to a blank bacterial culture dish to simulate the floor of a piglet barn. The dish was then sprayed with 40 ng / mL of the Streptococcus suis phage lytic enzyme Ply900. After 40 minutes, the number of Streptococcus suis on the blank bacterial culture dish dropped to 100 CFU / mL, as detected by plate colony counts. After 60 minutes, no Streptococcus suis could be detected. This demonstrates that the Streptococcus suis phage lytic enzyme Ply900 can effectively kill Streptococcus suis in the environment.

[0090] Test Example 8 Toxicity Detection Test of Streptococcus suis Phage Lytic Enzyme Ply900

[0091] Mice (4-6 weeks old) were intraperitoneally injected with 2 mg / mL of Streptococcus suis phage lytic enzyme Ply900 (400 μL) every 12 hours for 48 hours. Over the course of 14 days, the mice displayed normal activity levels, including cage locomotion, environmental exploration, social behavior, and feeding. Food and water consumption was consistent with expectations, and no abnormal behaviors were observed during the observation period. This demonstrates the safety of Streptococcus suis phage lytic enzyme Ply900 and its potential use in the prevention and treatment of Streptococcus suis infections.

Claims

1. A Streptococcus suis phage lytic enzyme Ply900, characterized in that The amino acid sequence is selected from any one of the following amino acid sequences (a) to (c): (a) the amino acid sequence shown in SEQ ID No. 1; (b) deleting, inserting, or / and replacing one or more amino acids in the amino acid sequence shown in SEQ ID No. 1 to obtain a mutant amino acid sequence, wherein the mutant amino acid sequence still has the activity of inhibiting Streptococcus suis; (c) an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID No. 1, and that still has the activity of inhibiting Streptococcus suis.

2. The gene encoding the Streptococcus suis phage lytic enzyme Ply900 according to claim 1.

3. The coding gene according to claim 2, characterized in that The nucleotide sequence is selected from any one of the nucleotide sequences described in (a) to (e) below: (a) the polynucleotide sequence shown in SEQ ID No. 2; (b) a polynucleotide sequence encoding the amino acid sequence shown in SEQ ID No. 1; (c) a polynucleotide sequence capable of hybridizing with the polynucleotide sequence described in (a) or (b) under stringent hybridization conditions, wherein the protein encoded by the polynucleotide sequence still has the function of inhibiting the activity of Streptococcus suis; (d) a polynucleotide sequence that is at least 95% identical to the polynucleotide sequence shown in any one of (a) to (c), wherein the protein encoded by the polynucleotide sequence still has the function of inhibiting the activity of Streptococcus suis; (e) A polynucleotide sequence that is complementary to the polynucleotide sequence described in any one of (a) to (d), wherein the protein encoded by the polynucleotide sequence still has the function of inhibiting the activity of Streptococcus suis.

4. An expression vector, characterized in that Contains the coding gene according to claim 2 or 3.

5. A recombinant host cell, characterized in that Contains the expression vector according to claim 4.

6. Use of the Streptococcus suis phage lytic enzyme Ply900 according to claim 1, the encoding gene according to claim 2 or 3, the expression vector according to claim 4 or the recombinant host cell according to claim 5 in the preparation of a drug for inhibiting bacteria; preferably, the bacteria is Streptococcus suis, Streptococcus agalactiae or Staphylococcus aureus.

7. The use according to claim 6, characterized in that The Streptococcus suis is selected from type 1 Streptococcus suis, type 2 Streptococcus suis, type 3 Streptococcus suis, type 7 Streptococcus suis, type 8 Streptococcus suis, type 9 Streptococcus suis, type 12 Streptococcus suis, type 18 Streptococcus suis, type 20 Streptococcus suis or untyped Streptococcus suis.

8. A pharmaceutical composition for inhibiting bacteria, comprising an active ingredient and a carrier acceptable for pharmaceutical preparations, characterized in that: The active ingredient is the Streptococcus suis phage lytic enzyme Ply900 according to claim 1; preferably, the Streptococcus suis, Streptococcus agalactiae or Staphylococcus aureus; more preferably, the Streptococcus suis is selected from type 1 Streptococcus suis, type 2 Streptococcus suis, type 3 Streptococcus suis, type 7 Streptococcus suis, type 8 Streptococcus suis, type 9 Streptococcus suis, type 12 Streptococcus suis, type 18 Streptococcus suis, type 20 Streptococcus suis or untyped Streptococcus suis.

9. The preparation prepared from the antibacterial pharmaceutical composition according to claim 8, preferably, the preparation comprises a spray, a water-soluble powder, a granule, a tablet or an injection.

10. An animal feed additive, characterized in that Contains the Streptococcus suis phage lytic enzyme Ply900 according to claim 1.

Images