Lyase preparation aiming at various pathogenic bacteria of cow mastitis source as well as preparation method and application of lyase preparation

Abstract

The invention provides a lyase preparation for various pathogenic bacteria of a cow mastitis source as well as a preparation method and application of the lyase preparation, and the lyase preparation comprises lyase LysJPP30, lyase LysECP2 and lyase Art-17; wherein the amino acid sequence of the lyase LysJPP30 is as shown in SEQ ID NO. 1, the amino acid sequence of the lyase LysECP2 is as shown in SEQ ID NO. 2, and the amino acid sequence of the lyase Art-17 is as shown in SEQ ID NO. 3. The lyase preparation can efficiently lyse staphylococcus aureus, streptococcus agalactiae, streptococcus dysgalactiae, escherichia coli and klebsiella pneumoniae, has the advantages of high lysis rate, high stability and excellent antibacterial effect, reduces the use of antibiotics, improves the treatment effect, and is suitable for various administration modes such as breast perfusion and breast soaking.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a lysin preparation targeting multiple pathogens causing bovine mastitis, its preparation method, and its application. Background Technology

[0002] Bovine mastitis is one of the most common diseases in the global dairy industry, seriously affecting cow health, milk yield, and dairy product quality, leading to significant economic losses. Statistics show that the global incidence of bovine mastitis ranges from 15% to 40%, with bacterial infections being the most prevalent. Common pathogens include Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, Escherichia coli, and Klebsiella pneumoniae. These pathogens have different pathogenic mechanisms and antibiotic resistance, resulting in limited effectiveness of traditional antibiotic treatments. Furthermore, long-term antibiotic use may lead to the spread of drug-resistant strains and dairy product residue problems, threatening food safety and public health.

[0003] Existing non-antibiotic treatments, such as vaccines, immune enhancers, and probiotics, are inconsistent in their efficacy against specific pathogens and struggle to address mixed infections caused by multiple pathogens simultaneously. Lysins, as enzymes capable of specifically degrading bacterial cell walls, possess broad-spectrum antibacterial potential. However, current lysin preparations for bovine mastitis primarily target single pathogens (such as Staphylococcus aureus), lacking multi-pathogen lysin preparations, and their stability and application methods have not yet been optimized.

[0004] Therefore, there is an urgent need to develop a highly efficient, broad-spectrum, and safe lysin preparation that can simultaneously target multiple major pathogens of bovine mastitis, reduce antibiotic use, and improve treatment efficacy. Summary of the Invention

[0005] In view of this, the present invention proposes a lysin preparation for multiple pathogens causing mastitis in dairy cows, its preparation method and application. The lysin preparation of the present invention can efficiently lyse Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, Escherichia coli and Klebsiella pneumoniae, with high lysis rate, high stability, and excellent antibacterial effect, reducing the use of antibiotics, improving the therapeutic effect, and is suitable for various administration methods such as mammary infusion and mammary soaking.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] In a first aspect, the present invention provides a lysin preparation targeting multiple pathogens causing mastitis in dairy cows, including lysin LysJPP30, lysin LysECP2 and lysin Art-17.

[0008] The amino acid sequence of the lysin LysJPP30 is shown in SEQ ID NO.1: MKSQQQAKDWIYKHEGVGVDFDGAYGFQCMDLAVAYIYYITDGKVRMWGNAKDAINNDFKGLATVYENTPSFKPQLGDVAVYTNSQYGHIQCVTSGNLDYYTCLEQNWLGGGFDGWEKATIRTHYYDGVTHFIRPKFSASNSNELETSKVNTFGNWKQNQYGTYYRNENATFTCGFLPIFARVGSPKLSEPNGYWFQPNGYTPYDEVCLSDGYVWIGYNWQGTRYYLPVRQWNGKTGNSYSVGIPWGVFS;

[0009] The amino acid sequence of the lysin LysECP2 is shown in SEQ ID NO.2:

[0010] MKICFPARKANGEQYATLDDMMQSLCQEPHGSWLAGTNNMWHGGIHITRKSAPGSVLINETADTAVPLQFMAGGEVVAWRVNQDYLTSKYINNPLQYSSTLVLVKSTCTPDPEKQDNSLDFYSLYIGLAPLSAFPEHTLYQVTDKGDGLSQREYTGKEKDGDKAPVAKNKLKAGDCVIVLREITFDLKGQKQTFGLARMLNSKSEITGGAFWVSLNPQYITPAGKQRAYLPAWMQQAVAQGTFDTVVKPATKMEVAAGDAVGYLAEDIAPDSMGAVEKSAFAHIEVLSTDSRMVDFLRNKAQVKSGQKYVYIHPESFIYSRSGDSFTQTKGKVKKDIHKIMPQDKCHPFKDSSDKRWFDIGDGAWVSEDDADADIGQYDLDKLGFKAFEEQSTSDMTKSLREGWIKDGFTRMAEWVRPERGIQESQVSDYYKALLRKMDSDNSGDLSGEELRHAVNYAELDVRDIAARMVVKHDSEWFGGSGHHRWRTFLKQLDPLCISYVRQWFDDMEWMSKVDGFSSGEPVWHMHPVIFLNALSDGEEMITYEQLKAMLPADKEKYVQMYLEPLNETMKLFEINTPLRKVHFMAQILHETGFFKYTEETASGKAYEGRSDLGNNKAGDGPLFKGRGLLQIYGRDNYTKCQQYLRTKLNDNSFDITSTTAKASHLSNNPRYATLASGYFWRYIKPKLNSSADKDDIYWVSVYVNGWAVQKKPYYPDKAKEPNHMDDRVAKLSITKEAFGLE;

[0011] The amino acid sequence of the lyase Art-17 is shown in SEQ ID NO.3:

[0012] RGLRRLGRKIAHGVKKYGPTVLRIIRIAGGGGGSGGGGSGGGGSMLKGIDVSEHQGRIDWERVKGNIDFAILRAGYGRNNIDKQFIRNIEECNRLCIPVGIYWFSYAWNEEMAKNEARYVLEAIKGYRVDYPISYDLEYDTLNYASKNGVTIGKRLATDMVKAFCDEINRNGYRAMNYTNQDFLLNKFYM NELTNYPLWYAWYNSKLNRDCAIWQYSENGQVPGIPGSSVDMNYCYEDFLKKDFTLENATTCNVDTELNIRAKGTTGATIVGSIPAGDRFRIKWVDSDYLGWYYIEYQGITGYVSQDYVEKLQMATTCNVDSVLNVRAEGNTSSNIVATINPGEVFRIDWVDSDFIGWYRITTANGFVKSDFVKKL.

[0013] Based on the above technical solution, the amino acid sequence of the lyase LysJPP30 further includes the amino acid sequence of a protein that has at least 95% homology with the amino acid sequence of the lyase LysJPP30.

[0014] The amino acid sequence of the lysin LysECP2 also includes the amino acid sequence of a protein that has at least 95% homology with the amino acid sequence of the lysin LysECP2.

[0015] The amino acid sequence of the lyase Art-17 also includes the amino acid sequence of a protein that has at least 95% homology with the amino acid sequence of the lyase Art-17.

[0016] Based on the above technical solution, the amino acid sequence of the lyase Art-17 further includes an antimicrobial peptide, the amino acid sequence of which is shown in SEQ ID NO.4: RGLRRLGRKIAHGVKKYGPTVLRIIRIAG.

[0017] Secondly, the present invention provides a method for preparing the above-mentioned lysin preparation targeting multiple pathogens causing bovine mastitis, comprising the following steps:

[0018] S1. Mix the lyase LysJPP30, LysECP2 and Art-17 thoroughly.

[0019] S2. Add the buffer system, stabilizer and preservative and mix well to obtain the lysin preparation.

[0020] The preservative is sodium benzoate, which prevents microbial contamination and extends the shelf life of the preparation.

[0021] The surfactant is Tween-20, which can improve the dispersibility and uniformity of the lysin preparation.

[0022] Sterilization and dispensing of lyase preparations: The prepared lyase preparations are sterilized by filtration through a 0.22μm filter membrane, dispensed into sterile glass or plastic bottles, sealed, and stored at 2-8℃.

[0023] Quality control: The purity and molecular weight of the lysin were determined by SDS-PAGE; the bactericidal efficiency of the formulation against Staphylococcus aureus, Escherichia coli, Streptococcus agalactiae, Streptococcus dysgalactiae, and Klebsiella pneumoniae were determined by in vitro antibacterial experiments; the pH value (6.5-7.5), clarity, and storage stability of the formulation were determined (activity remained ≥90% after 6 months of storage at 4℃).

[0024] Based on the above technical solution, the lyase LysJPP30, lyase LysECP2 and lyase Art-17 are further mixed in a mass ratio of 1:(0.2-1.2):(0.2-1.2) to make the total concentration of lyase 3 mg / mL.

[0025] Based on the above technical solutions, the stabilizer further includes glycerol, sorbitol and trehalose.

[0026] The stabilizer includes glycerol to enhance the freeze resistance and solution stability of the lysin preparation under storage conditions of 2-8°C.

[0027] Sorbitol protects the secondary and tertiary structures of lysins through hydrogen bonding, preventing protein aggregation.

[0028] Trehalose is used to form a glassy matrix, thereby improving the stability of the lysin preparation during long-term storage or freeze-drying.

[0029] Based on the above technical solutions, the buffer solution further includes Tris-HCl buffer solution.

[0030] The buffer system is a Tris-HCl buffer solution with pH 7.0 to maintain the acid-base stability of the formulation.

[0031] Thirdly, the present invention provides the use of the above-mentioned lysin preparation in the preparation of a drug for preventing and treating mastitis in dairy cows caused by one or more of Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, Escherichia coli, and / or Klebsiella pneumoniae.

[0032] Based on the above technical solutions, the lysin preparation is further applicable to the prevention and treatment of mastitis in dairy cows caused by one or more of Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, Escherichia coli, and / or Klebsiella pneumoniae.

[0033] When used to prevent and treat mastitis in dairy cows caused by one or more of Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, Escherichia coli, and / or Klebsiella pneumoniae, the administration methods include the following two:

[0034] (1) Breast soaking: The lysin preparation is administered through a breast soaking cup twice daily for 5 consecutive days;

[0035] (2) Mammary infusion: The lysin preparation is infused into the infected mammary area through the nipple tube, 10 mL per mammary area, twice a day for 5 consecutive days.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] The lysin preparation of this invention can efficiently lyse Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, Escherichia coli, and Klebsiella pneumoniae, exhibiting high lysis rate, high stability, and excellent antibacterial effect. It can reduce antibiotic use, improve therapeutic efficacy, and is suitable for various administration methods such as breast infusion and breast soaking. Attached Figure Description

[0038] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0039] Figure 1 The lysis zone of the lysin preparation against the pathogen causing mastitis in dairy cows;

[0040] Figure 2 The in vitro bactericidal effect curve of the lysin preparation against the pathogen causing mastitis in dairy cows is shown. Detailed Implementation

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

[0042] LysJPP30 is a Staphylococcus aureus phage lysin with broad-spectrum lytic activity.

[0043] LysECP2 is a bacteriophage lysin of Escherichia coli with broad-spectrum lytic activity.

[0044] Art-17 is an engineered lyase that binds an antimicrobial peptide to Clostridium perfringens phage. Its structural formula is SMAP-29-linker-LysCPP5, and the amino acid sequence of LysCPP5 is shown in SEQ ID. Shown in NO.5: MLKGIDVSEHQGRIDWERVKGNIDFAILRAGYGRNNIDKQFIRNIEECNRLCIPVGIYWFSYAWNEEMAKNEARYVLEAIKGYRVDYPISYDLEYDTLNYASKNGVTIGKRLATDMVKAFCDEINRNGYRAMNYTNQDFLLNKFYMNELTNYPLWYAWYNSKLN RDCAIWQYSENGQVPGIPGSSVDMNYCYEDFLKKDFTLENATTCNVDTELNIRAKGTTGATIVGSIPAGDRFRIKWVDSDYLGWYYIEYQGITGYVSQDYVEKLQMATTCNVDSVLNVRAEGNTSSNIVATINPGEVFRIDWVDSDFIGWYRITTANGANGFVKSDFVKKL.

[0045] The Staphylococcus aureus (GRNSTA015), Streptococcus agalactiae (GRNSag002), Streptococcus dysgalactiae (GRNSdy010), Escherichia coli (GRNEC008), and Klebsiella pneumoniae (GRNKp004) used in this invention were isolated, identified, and preserved by our company.

[0046] In the following specific implementation, the Tris-HCl buffer solution was purchased from Biosharp, catalog number BL1979A.

[0047] Example 1: Construction of the lyase gene expression vector

[0048] 1. Amplify gene fragments encoding lyases LysJPP30, LysECP2, and Art-17, respectively;

[0049] The amino acid sequence of the lyase LysJPP30 is shown in SEQ ID NO.1; the amino acid sequence of the lyase LysECP2 is shown in SEQ ID NO.2; and the amino acid sequence of the lyase Art-17 is shown in SEQ ID NO.3.

[0050] 2. The gene fragments encoding the lyases LysJPP30, LysECP2, and Art-17 obtained by amplification were cloned into the BamHI / SalI site of the expression vector pET28a(+), and an N-terminal His tag was added to obtain recombinant plasmids pET28a-LysJPP30, pET28a-LysECP2, and pET28a-Art-17.

[0051] 3. After sequencing verification, the recombinant plasmids pET28a-LysJPP30, pET28a-LysECP2, and pET28a-Art-17 were transformed into Escherichia coli BL21(DE3) host bacteria to facilitate subsequent expression and purification experiments.

[0052] Example 2: Purification and Expression of Lysase

[0053] Positive clones were picked from LB agar plates containing 50 mg / L kanamycin and inoculated onto LB agar (containing 50 mg / L kanamycin) and cultured until OD. 600 Once the concentration reached 0.7, IPTG was added to a final concentration of 0.1 mM for induction expression, and the mixture was continuously induced at 15°C for 12 hours to improve protein solubility.

[0054] After cultivation, the bacterial cells were collected and lysed by sonication, followed by Ni... 2 + Affinity chromatography column for His-tagged purification.

[0055] The eluted proteins were dialyzed to remove impurities, and the purity and molecular weight of the proteins were analyzed by SDS-PAGE. At the same time, the expression level of the proteins was verified by Nanodrop instrument. Finally, LysJPP30, LysECP2 and Art-17 lyases with a purity of 96% were obtained.

[0056] Example 3: Preparation of lysin preparation

[0057] LysJPP30, LysECP2, and Art-17 were mixed in a 1:1:1 mass ratio. Tris-HCl (pH 7.0) was added to a final concentration of 20 mM, along with 10% (w / v) glycerol, 10% (w / v) sorbitol, 10% (w / v) trehalose, 0.1% (w / v) Tween-20, and 0.1% (w / v) sodium benzoate to prepare a lysin preparation with a final concentration of 3 mg / mL. The mixture was filtered sterilized, dispensed into 10 mL aliquots into sterile bottles, and stored at 4°C.

[0058] Example 4: Preparation of lysin preparation

[0059] The difference between this embodiment and Example 3 is that the mass ratio of lyase LysJPP30, lyase LysECP2 and lyase Art-17 is 1:0.2:1.2, and a final concentration of lyase 3 mg / mL is prepared.

[0060] Example 5: Preparation of lysin preparation

[0061] The difference between this embodiment and Example 3 is that the mass ratio of lyase LysJPP30, lyase LysECP2 and lyase Art-17 is 1:1.2:0.2, and a final concentration of lyase 3 mg / mL is prepared.

[0062] Example 6: Preparation of lysin preparation

[0063] The difference between this embodiment and Example 3 is that the mass ratio of lyase LysJPP30, lyase LysECP2 and lyase Art-17 is 1:0.2:0.2, and a final concentration of lyase 3 mg / mL is prepared.

[0064] Example 7: Preparation of lysin preparation

[0065] The difference between this embodiment and Example 3 is that the mass ratio of lyase LysJPP30, lyase LysECP2 and lyase Art-17 is 1:1.2:1.2, and the final concentration of lyase is 3 mg / mL.

[0066] Comparative Example 1

[0067] The difference between this comparative example and Example 3 is that it contains only the lyase LysJPP30 and is prepared into a formulation with a final concentration of lyase of 3 mg / mL.

[0068] Comparative Example 2

[0069] The difference between this comparative example and Example 3 is that it contains only the lyase LysECP2 and is prepared into a formulation with a final concentration of lyase of 3 mg / mL.

[0070] Comparative Example 3

[0071] The difference between this comparative example and Example 3 is that it contains only the lyase Art-17 and is prepared into a formulation with a final concentration of lyase of 3 mg / mL.

[0072] Comparative Example 4

[0073] The difference between this comparative example and Example 3 is that the mass ratio of lyase LysJPP30, lyase LysECP2 and lyase Art-17 is 1:0.05:0.05.

[0074] Comparative Example 5

[0075] The difference between this comparative example and Example 3 is that the mass ratio of lyase LysJPP30, lyase LysECP2 and lyase Art-17 is 1:0.05:1.

[0076] Comparative Example 6

[0077] The difference between this comparative example and Example 3 is that the mass ratio of lyase LysJPP30, lyase LysECP2 and lyase Art-17 is 1:1:0.05.

[0078] Comparative Example 7

[0079] The difference between this comparative example and Example 3 is that the lyase Art-17 is not modified with antimicrobial peptides, and is instead the lyase LysCPP5, whose amino acid sequence is shown in SEQ ID NO.5.

[0080] Experimental Example 1: Determination of the lysing ability of lysin preparations

[0081] The lysis effect of the lysin preparation on Staphylococcus aureus (GRNSTA015), Streptococcus agalactiae (GRNSag002), Streptococcus dysgalactiae (GRNSdy010), Escherichia coli (GRNEC008), and Klebsiella pneumoniae (GRNKp004) was determined by the double-layer agar plate method.

[0082] 10 μL of the lyase preparation prepared in Example 3 was dropped onto an agar plate containing bacterial growth. After incubation at 37°C for 18 hours, the lysis zone was observed to evaluate the lysing activity of the lyase preparation.

[0083] The results are as follows Figure 1 As shown, by Figure 1 It can be seen that the lysin preparation has a significant lysing effect on all five pathogens causing mastitis in dairy cows.

[0084] Experimental Example 2: In vitro bactericidal ability test of lysin preparations against five pathogenic bacteria causing mastitis in dairy cows

[0085] Staphylococcus aureus (GRNSTA015), Streptococcus agalactiae (GRNSag002), Streptococcus dysgalactiae (GRNSdy010), Escherichia coli (GRNEC008), and Klebsiella pneumoniae (GRNKp004) were cultured separately in 200 mL of TSB serum liquid medium and diluted to OD. 600 The concentration of the lyase was 0.05 mg / L, and the lyase preparation prepared in Example 3 was added to make the concentration of the lyase 50 mg / L. The blank control group was given an equal volume of Normal saline. The cultures were incubated at 37°C and 200 rpm, and the OD of the culture medium was measured at 1-hour intervals. 600 value.

[0086] This experiment used a two-well parallel experiment and calculated the average value, and plotted the growth curve of the strain.

[0087] The results are as follows Figure 2 As shown, the lysin preparation can effectively inhibit the growth of five bovine mastitis pathogens, demonstrating excellent bactericidal ability.

[0088] Experimental Example 3: The therapeutic effect of lysin preparations on mastitis in dairy cows

[0089] To investigate the efficacy of this bacteriophage in treating subclinical mastitis in dairy cows, a feeding experiment was conducted. The specific methods and results are as follows: Bacterial isolation was performed on dairy cows with subclinical mastitis, similar parity, similar milk production, and in late lactation. 300 dairy cows with consistent health status and mixed infections of two or more of the following bacteria were selected and randomly divided into 10 groups of 30 cows each. Each group was fed the same brand, specification, and batch of feed (blank feed) and under the same feeding conditions.

[0090] The blank group received no treatment, while the control group received 500,000 units of kanamycin injected into the nipple tube after milking, twice daily.

[0091] Examples 3-7: Each group was injected with 10 mL of the 3 mg / mL lysin preparation prepared in Examples 3-7 through the teat tube after milking, twice a day.

[0092] Comparative Examples 1-7: Each group was injected with 10 mL of the lysin preparation prepared in Comparative Examples 1-7 at a concentration of 3 mg / mL through the teat tube after milking, twice daily.

[0093] The cure rate for each group was calculated on the 7th, 14th and 21st days of treatment, and the results are shown in Table 1.

[0094] Table 1. Statistics on the treatment effects of mastitis in dairy cows

[0095]

[0096] As shown in Table 1, the group treated with the lysin preparation of the present invention had a higher cure rate. This significant efficacy may be due to the complementary effects among the three lysins: LysJPP30, LysECP2, and Art-17 each have good lysing effects against different pathogens. LysJPP30 is effective against Staphylococcus aureus, Streptococcus agalactiae, and Streptococcus dysgalactiae; LysECP2 has excellent inhibitory effects against Escherichia coli; and Art-17, as an engineered lysin fused with antimicrobial peptides, not only retains lysing activity but also enhances membrane penetration, showing good efficacy against Klebsiella pneumoniae. The group treated with the lysin preparation in Example 3 had the highest cure rate. The 1:1:1 ratio of each lysin established the optimal synergistic relationship among the components, which can broadly cover multiple pathogens and enhance their respective effects, thus exhibiting the strongest therapeutic ability in the mixed infection model.

[0097] A comparison of Comparative Examples 1-3 with Example 3 shows that a single component is insufficient to meet the treatment needs in complex infection models. This is because the bactericidal spectrum of lysins is usually limited, and a single enzyme preparation cannot simultaneously cover all pathogenic species. Therefore, single-component treatment is unlikely to achieve ideal therapeutic effects.

[0098] A comparison of Comparative Examples 4-6 and Example 3 shows that decreasing or increasing the proportion of any of the three lysins significantly reduced the cure rate, indicating that dosage imbalance severely affected the therapeutic effect. This phenomenon occurs because insufficient dosage of a particular component reduces its ability to target and clear pathogens, thus hindering the overall therapeutic efficacy of the combination. Furthermore, low proportions of components may not reach effective concentrations, failing to fully participate in the synergistic bactericidal process, disrupting the synergistic enhancement effect among the three components, and thus limiting the overall therapeutic effect.

[0099] Comparison of Comparative Example 7 and Example 3 revealed that the unmodified version (LysCPP5) showed a certain gap in therapeutic efficacy compared to the antimicrobial peptide-modified Art-17. Art-17 without antimicrobial peptide modification lacks the ability to penetrate the outer membrane or biofilm of Gram-negative bacteria, making it difficult to exert its intended lytic effect. Lacking this enhancement mechanism, LysCPP5 also cannot fully exert its synergistic effect when used in combination with LysJPP30 and LysECP2, thus limiting the overall therapeutic effect.

[0100] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A lysin preparation targeting multiple pathogens causing mastitis in dairy cows, characterized in that, Including lyases LysJPP30, LysECP2, and Art-17; The amino acid sequence of the lyase LysJPP30 is shown in SEQ ID NO.1, the amino acid sequence of the lyase LysECP2 is shown in SEQ ID NO.2, and the amino acid sequence of the lyase Art-17 is shown in SEQ ID NO.

3.

2. The method for preparing a lysin preparation targeting multiple pathogens causing bovine mastitis as described in claim 1, characterized in that, The amino acid sequence of the lyase Art-17 contains an antimicrobial peptide, the amino acid sequence of which is shown in SEQ ID NO.

4.

3. A method for preparing a lysin preparation targeting multiple pathogens causing bovine mastitis as described in any one of claims 1 to 2, characterized in that, Includes the following steps: S1. Mix the lyase LysJPP30, LysECP2 and Art-17 thoroughly. S2. Add the buffer system, stabilizer and preservative and mix well to obtain the lysin preparation.

4. The method for preparing a lysin preparation targeting multiple pathogens causing bovine mastitis as described in claim 3, characterized in that, The lyases LysJPP30, LysECP2, and Art-17 were mixed at a mass ratio of 1:(0.2-1.2):(0.2-1.2) to make the total concentration of lyases 3 mg / mL.

5. The method for preparing a lysin preparation targeting multiple pathogens causing bovine mastitis as described in claim 3, characterized in that, The stabilizers include glycerol, sorbitol, and trehalose.

6. The method for preparing a lysin preparation targeting multiple pathogens causing bovine mastitis as described in claim 3, characterized in that, The buffer solution includes Tris-HCl buffer.

7. The use of the lysin preparation according to any one of claims 1 to 2 in the preparation of a medicament for the prevention and treatment of mastitis in dairy cows caused by one or more of Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, Escherichia coli, and / or Klebsiella pneumoniae.

Images