Streptococcus equi subspecies equi prophage lytic enzyme and its preparation method and application

Abstract

The present invention discloses a prophage lytic enzyme of Streptococcus equi subspecies equi, and its preparation method and application, which belong to the technical field of veterinary medicine. The amino acid sequence of the lytic enzyme is shown in SEQ ID NO.29. The polynucleotide encoding the prophage lytic enzyme of Streptococcus equi subspecies equi, the expression vector containing the polynucleotide, and the host bacteria containing the expression vector are also within the protection scope of the present invention. Among them, preferably, the sequence of the polynucleotide is shown in SEQ ID NO.30. The present invention compares the prophage lytic enzyme genes in the genome sequence of Streptococcus equi subspecies equi, designs specific primers, amplifies and clones the lytic enzyme genes in the Streptococcus equi strains preserved in the laboratory, performs recombinant expression, and evaluates the antibacterial effects of different lytic enzymes, and finally screens out the lytic enzyme with the best bactericidal effect, thereby providing a new technical means for the prevention and treatment of equine strep.

Description

Technical Field

[0001] The present invention relates to a prophage lytic enzyme of Streptococcus equi subspecies equi, and also relates to a preparation method of the lytic enzyme and application in the treatment of equine striae. The present invention belongs to the technical field of veterinary medicine. Background Art

[0002] Streptococcus equi ( Streptococcus equi ) belongs to the Streptococcus family ( Streptococcaceae ), Streptococcus ( Streptococcus ), is a Gram-positive bacterium belonging to the Lancefield C group. Initially, the bacterium was considered a separate species, but through 16S rRNA gene sequence, whole genome comparison, and virulence genotype analysis, it was found that the bacterium can be further divided into two subspecies, namely Streptococcus equi subspecies equi ( Streptococcus equi subsp. equi , S.equi ) and Streptococcus equi subsp. zooepidemicus ( Streptococcus equi subsp. Zooepidemicus , S.zoo ). S.equi It is the main pathogen that causes strangles in horses, donkeys and other equine animals. It has high host specificity and pathogenicity. Clinically, the typical symptoms of the affected animals are fever, inflammation of the upper respiratory tract mucosa, swelling and suppuration of lymph nodes in the mandible, neck and other parts, and mucopurulent nasal discharge.

[0003] Bacteriophages, a general term for viruses that infect microorganisms such as bacteria, fungi, algae, actinomycetes, or spirochetes, are the most abundant biological entities on Earth. The bacteriophage life cycle is primarily divided into two phases: the lytic phase and the lysogenic phase. Endolysins, a class of hydrolases encoded by the phage or prophage genome, hydrolyze the bacterial cell wall during the late lytic phase of the phage lysis cycle, or when the prophage is activated and enters the late lytic phase, releasing progeny phage into the external environment. Because endolysins efficiently cleave the peptidoglycan structure of the bacterial cell wall and are highly specific for host bacteria, they are considered potential alternatives to antimicrobial drugs. Compared to traditional antibiotics, endolysins offer advantages such as a unique mechanism of action, a low risk of drug resistance, high bactericidal efficiency, and minimal impact on the host's normal flora.

[0004] Currently targeting S.equi There has been no large-scale or systematic research on the lytic enzymes of Streptococcus aureus, which may be due to the lack of application research and development in this field and the relatively high cost of studying horses as large animal models. A small number of in vitro studies have shown that some streptococcal lytic enzymes have significant bactericidal activity against a variety of pathogenic streptococci, suggesting that they are effective against S.equi Potentially universal. However, S.equiThe specific toxicity characteristics of the lyases are used to screen or optimize suitable lyases, and safety and efficacy tests are conducted in horses or alternative animal models. In theory, lyases can play a role in the following aspects: ① Preventive application: In horse farms, racecourses or equestrian clubs, lyase preparations are sprayed regularly to reduce the risk of infection. S.equi ② Early treatment: In the early stages of equine glandular fever (such as when mild upper respiratory tract infection symptoms appear), lytic enzymes are directly delivered through aerosol inhalation or nasal spray to inhibit the colonization of pathogens and reduce the spread of infection. ③ Adjuvant treatment: For cases where abscesses have formed, lytic enzymes can be applied topically after abscess incision and drainage to kill residual bacteria. S.equi ④ Combined immunization: Combined with vaccination, lytic enzymes can be used to control clinical symptoms, allowing the immune system to better respond to infection, while reducing the horse's carrier status and reducing the continued spread of the disease.

[0005] The present invention is through S.equi By comparing prophage lytic enzyme genes in genomic sequences, specific primers were designed to amplify and clone the lytic enzyme gene from a laboratory-conserved strain of Streptococcus equi. Recombinant expression was performed, and the antibacterial effects of different lytic enzymes were evaluated. Ultimately, the lytic enzyme with the best bactericidal effect was screened, providing a new technical approach for the prevention and treatment of equine strep. Summary of the Invention

[0006] The purpose of the present invention is to provide a Streptococcus equi subspecies equi prophage lytic enzyme and a preparation method and application thereof.

[0007] In order to achieve the above object, the present invention adopts the following technical means:

[0008] The present invention provides a Streptococcus equi subspecies equi prophage lytic enzyme, and the amino acid sequence of the lytic enzyme is shown in SEQ ID NO.29.

[0009] The polynucleotide encoding the Streptococcus equi subspecies equi prophage lytic enzyme, the expression vector containing the polynucleotide, and the host bacteria containing the expression vector are also within the protection scope of the present invention.

[0010] Preferably, the sequence of the polynucleotide is shown as SEQ ID NO.30.

[0011] Among them, preferably, the expression vector is a pET-28a vector containing the polynucleotide.

[0012] Among them, preferably, the host bacteria is E. coli BL21(DE3).

[0013] Furthermore, the present invention also proposes the use of the Streptococcus equi subspecies equi prophage lytic enzyme in the preparation of reagents or drugs against Streptococcus equi subspecies equi and Streptococcus equi subspecies zooepidemicus, as well as the use of the Streptococcus equi subspecies equi prophage lytic enzyme in the preparation of drugs for treating equine glanders.

[0014] Furthermore, the present invention also provides a biological antibacterial agent for preventing and treating equine glandular disease, wherein the antibacterial agent contains the prophage lytic enzyme of Streptococcus equi subspecies equi.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] The present invention is through S.equi The prophage lytic enzyme genes in the genome were compared and the antimicrobial efficacy of different lytic enzymes was evaluated. Ultimately, the lytic enzyme with the best bactericidal activity was identified and named LysHLJ1-9. In vitro experiments demonstrated that LysHLJ1-9 exhibited the broadest spectrum of lytic activity, lysing 20 strains of Streptococcus equi and 2 strains of Streptococcus dysgalactiae subsp. equisimilis, while showing no activity against Escherichia coli, demonstrating good targeting. Within one hour, LysHLJ1-9 reduced the number of SD2018 strains by approximately 1 log, with a clear dose-dependent bactericidal activity. No drug-resistant strains were observed after 12 consecutive passages of SD2018 strains treated with LysHLJ1-9. Biochemical characterization revealed that LysHLJ1-9 maintained stable activity at temperatures ranging from 4°C to 42°C and pH values ​​from 3 to 9, with an optimal pH of 7, demonstrating excellent environmental stability. Compared with penicillin, LysHLJ1-9 has a stronger ability to destroy bacterial biofilms. In vivo experiments (in a mouse model of acute bacteremia) demonstrated that LysHLJ1-9, administered at a dose of 1500 μg per mouse, achieved a 100% survival rate. Treatment significantly reduced bacterial loads in the blood and organs, including the liver, spleen, lungs, and kidneys (with a maximum reduction of 3 log). Pathological tissue sections revealed that LysHLJ1-9 effectively alleviated inflammation and tissue damage. No toxic side effects were observed after a single injection of 2000 μg of the lyase, demonstrating good biosafety.

[0017] In summary, the present invention proposes a method based on S.equi The prophage lytic enzyme LysHLJ1-9 has a good antibacterial spectrum, environmental stability and in vivo protective efficacy. The invention provides a new technical means for the prevention and treatment of equine strep. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 for the amplification of prophage lytic enzymes;

[0019] Among them, AH are electrophoresis diagrams of amplification using primers Lys1–Lys8, respectively; lane 1 is a no-template control (H2O), lanes 2–11 are the amplification results of Streptococcus equi subsp. equi strains HLJ2018, SD2018, SE-1, SE-2, SE-3, SE-4, SE-5, SE-6, SE-7, and SE-8, respectively; M is a DNA molecular weight standard;

[0020] Figure 2 is a heat map of similarity of different lytic enzyme protein sequences;

[0021] Figure 3 For pET-28a and amplification of the lytic enzyme gene;

[0022] Among them, M: DNA marker; 1: linear pET-28a; 2: LysHLJ1-9; 3: LysHLJ1-18; 4: LysHLJ3-11; 5: LysHLJ4-11; 6: LysHLJ5-13; 7: LysHLJ6-11;

[0023] Figure 4 SDS-PAGE analysis of the lytic enzyme expression product;

[0024] Among them, A–F are the protein expression products of recombinant plasmids pET-28a-LysHLJ1-9, pET-28a-LysHLJ1-18, pET-28a-LysHLJ3-11, pET-28a-LysHLJ4-11, pET-28a-LysHLJ5-13, and pET-28a-LysHLJ6-11, respectively; M: protein marker; 1-5 are pET-28a empty vector, bacterial solution before induction, whole bacterial solution after induction, precipitate, and supernatant, respectively;

[0025] Figure 5 This is the SDS-PAGE analysis of the purified product of the lytic enzyme;

[0026] Wherein, M: protein marker; 1: purified LysHLJ1-9; 2: purified LysHLJ1-18; 3: purified LysHLJ3-11; 4: purified LysHLJ4-11; 5: purified LysHLJ5-13; 6: purified LysHLJ6-11;

[0027] Figure 6 is the lytic activity of different lytic enzymes in liquid environment;

[0028] Wherein, A: cleavage activity of lyase LysHLJ1-9; B: cleavage activity of lyase LysHLJ1-18; C: cleavage activity of lyase LysHLJ3-11; D: cleavage activity of lyase LysHLJ4-11; E: cleavage activity of lyase LysHLJ5-13; F: cleavage activity of lyase LysHLJ6-11;

[0029] Figure 7 is the OD of the bactericidal effect of different lytic enzymes on SD2018 600nm Determination;

[0030] Figure 8 The in vitro bactericidal activity of lyase LysHLJ1-9 against SD2018;

[0031] Figure 9 is the resistance of SD2018 to LysHLJ1-9;

[0032] Figure 10 The biochemical characteristics of LysHLJ1-9;

[0033] Among them, A: Effect of temperature on the activity of lyase LysHLJ1-9; B: Effect of pH value on the activity of lyase LysHLJ1-9;

[0034] Figure 11 is the LysHLJ1-9 cleavage spectrum;

[0035] Figure 12 The effect of LysHLJ1-9 on bacterial biofilm;

[0036] Ns: no significant difference; ****: p < 0.0001;

[0037] Figure 13 LysHLJ1-9 was used to treat SD2018-induced bacteremia in mice;

[0038] A: Determination of the minimum lethal dose of SD2018; B: Determination of the minimum therapeutic dose of LysHLJ1-9; C: Safety assessment of LysHLJ1-9; D: Bacterial load in mouse blood; E: Bacterial load in mouse viscera, including (a) liver, (b) lung, (c) kidney, and (d) spleen; ***: p < 0.001;

[0039] Figure 14 For histopathological analysis of mouse lung and liver;

[0040] Figure 15 For histopathological analysis of mouse spleen and kidney. DETAILED DESCRIPTION

[0041] The present invention is further described below by way of examples, which are intended only to provide a better understanding of the present invention and are not intended to limit the scope of the present invention. The following examples, except for special examples, are all conventional experimental methods and operating procedures in the art.

[0042] Example 1 Screening and expression of lytic enzymes

[0043] 1 Experimental Materials

[0044] 1.1 Bacterial strains and plasmids

[0045] The 20 strains of Streptococcus equi used in this experiment (including 10 strains of S. equi subsp. equi and 10 strains of S. equi subsp. zooepidemicus) were isolated and maintained in our laboratory (Table 1). The prokaryotic expression vector pET-28a was also maintained in our laboratory, and competent E. coli DH5α and BL21 (DE3) strains were purchased from Qingke Biotechnology.

[0046] ;

[0047] 1.2 Main Reagents

[0048] ;

[0049] 1.3 Solution preparation

[0050] Todd-Hewitt broth (THB): Place 800 mL of distilled water in a beaker, add 36.4 g of THB, stir well, and dilute to 1 L. Autoclave at 115°C for 30 min and store at 4°C in the dark.

[0051] The 5× SDS-Page Sample Loading Buffer, LB medium, and LB solid medium containing kanamycin used in this experiment were all prepared by our laboratory.

[0052] 2 Experimental methods

[0053] 2.1 Acquisition and screening of prophage lytic enzymes

[0054] This study used bioinformatics methods to obtain the " Streptococcus equi subsp equi "The complete genome sequence of the gene encoding the lytic enzyme was screened by consulting the genome annotation information. The specific method is as follows: First, search the NCBI genome database with the keyword " Streptococcus equi subsp equi AND complete genome" was searched to download annotated bacterial genome sequences (GenBank format, .gbff). The genome sequence files were opened using SnapGene software (GSL Biotech, USA). Genes annotated as containing "lysin," "CHAP domain," or "amidase domain" were searched within the genome annotation information for preliminary screening of candidate lytic enzyme genes. Based on the results of functional annotation and domain analysis, gene sequences containing clear lytic functional domains (such as CHAP domain or Amidase-2 domain) were selected and their corresponding nucleotide sequences (FASTA format) were saved as candidate lytic enzyme genes for subsequent primer design and experimental verification.

[0055] Functional domain analysis of the candidate lytic enzyme protein sequences was performed using the UniProt database (https: / / www.uniprot.org / ) to ensure that the target gene contained a typical cell wall hydrolysis domain. Specific primers were then designed using the Primer-BLAST tool (https: / / www.ncbi.nlm.nih.gov / tools / primer-blast / ) for PCR amplification. The specific primer sequences are shown in Table 3.

[0056] ;

[0057] 2.2 Extraction of genomic DNA from Streptococcus equi subspecies equi

[0058] Laboratory-stored Streptococcus equi subsp. equi strains were taken from a -80°C glycerol stock, revived, and inoculated onto Columbia blood agar plates. They were then incubated inverted at 37°C for 18 to 24 hours. A single colony was inoculated into 5 mL of THB medium and incubated at 37°C and 200 rpm until the logarithmic growth phase (OD 600nm (Approximately 0.6 to 0.8). Take 1 to 2 mL of the above culture medium and centrifuge at 12,000 rpm for 2 minutes at 4°C. Discard the supernatant and collect the bacterial pellet. Use the TIANGEN Bacterial Genomic DNA Extraction Kit according to the manufacturer's instructions to extract DNA. Determine the concentration and purity of the resulting genomic DNA using a visible light spectrophotometer and store at -20°C until needed.

[0059] 2.3 PCR amplification of the lytic enzyme gene

[0060] Using genomic DNA extracted from Streptococcus equi subsp. equi as a template, specific primers designed and synthesized based on previous bioinformatics analysis were used to amplify the target lytic enzyme gene. Primers were synthesized by Ruiboxingke (Harbin) Biotechnology Co., Ltd.; the primer sequences are shown in Table 3.

[0061] High-fidelity KOD high-fidelity enzyme was used for amplification reaction. The PCR reaction system was 50 μL and the components were as follows:

[0062] ;

[0063] ;

[0064] PCR products were run on a 1% agarose gel. The amplified product band sizes were observed and recorded using a gel imaging system to verify amplification specificity. Gels with specific bands were excised, and the target fragments were purified using the FastPure Gel DNA Extraction Mini Kit according to the kit's instructions. The recovered products were sequenced by Jilin Kumei Biotechnology Co., Ltd.

[0065] 2.4 Recombinant expression of lytic enzymes

[0066] 2.4.1 Plasmid construction

[0067] Based on the pET-28a vector sequence and the target cleavage enzyme gene sequence, specific primers for homologous recombination were designed. Fragments homologous to the vector sequence (typically 15-20 bp in length) were introduced at the 5' ends of the primers to achieve efficient homologous recombination. Primers were synthesized by Ruibo Xingke (Harbin) Biotechnology Co., Ltd. The primer sequences are shown in Table 6, with the underlined portions representing the homology arms to the pET-28a vector. The PCR reaction system and protocol were the same as those in Tables 4 and 5.

[0068] ;

[0069] Add 1 μL of methylation-sensitive restriction enzyme to the amplified linearized vector. DpnⅠ Incubate in a 37°C water bath for 45 minutes to remove the template plasmid. Run the PCR product on a 1% agarose gel. Observe the size of the amplified product band using a gel imaging system to verify amplification specificity. Excise the gel containing the specific band and purify the target fragment using the FastPure Gel DNA Extraction Mini Kit according to the manufacturer's instructions.

[0070] The cleavage enzyme gene fragment, amplified and purified by PCR, was homologously recombined with the PCR-linearized expression vector pET-28a using MonClone™ Single Assembly Cloning Mix. The recombination reaction system (10 μL) was as follows: linearized pET-28a vector (50 ng): 1 μL; cleavage enzyme gene fragment (PCR product, 100 ng): 1 μL; MonClone™ Single Assembly Cloning Mix: 5 μL; ddH2O: make up to 10 μL. Reaction conditions: 50°C for 30 min, briefly placed on ice, and then immediately transformed.

[0071] 2.4.2 Transformation and identification of recombinant plasmids

[0072] The recombinant reaction product was added to 50 μL of E. coli DH5α competent cells, incubated on ice for 15 minutes, heat-shocked at 42°C for 60 seconds, and immediately incubated on ice for 2 minutes. 900 μL of sterile LB liquid medium was added to the competent cells, and the cells were incubated at 37°C and 180 rpm with shaking for 1 hour. After centrifugation at 6000 rpm for 1 minute, the cells were harvested and resuspended in 30 μL of LB liquid medium. The bacterial suspension was spread onto LB solid medium containing kanamycin and incubated inverted at 37°C for 12–16 hours. A single colony was selected and added to LB medium containing kanamycin and incubated at 37°C and 180 rpm with shaking for 5 hours. Plasmids were extracted from the bacterial suspension using an Invitrogen plasmid extraction kit according to the manufacturer's instructions and then sent to Jilin Kumei Biological Co., Ltd. for sequencing.

[0073] 2.4.3 Expression and purification of recombinant proteins

[0074] The recombinant plasmids confirmed by sequencing were transformed into E. coli BL21 (DE3) was used for protein expression. A single positive colony was selected and inoculated into LB liquid medium containing kanamycin. The culture was shaken at 37°C and 180 rpm for 12 h to serve as seed culture. The next day, the seed culture was diluted 1:100 into fresh LB medium and cultured at 37°C and 180 rpm until the OD value reached 0. 600nm Reach 0.6 ~ 0.8.

[0075] When the bacteria reached logarithmic growth phase, IPTG was added to induce expression at a final concentration of 0.5 mM. The culture was then incubated at 16°C and 130 rpm for 12 h. Following induction, the cells were harvested by centrifugation at 8000 rpm for 10 min at 4°C and resuspended in PBS (pH 7.4). The resuspended cells were sonicated on ice (power 39%, 5-second intervals, 6-second rest intervals, for a total of 10 minutes) and centrifuged at 12000 rpm for 10 min. The supernatant and precipitate were collected and used for subsequent SDS-PAGE analysis.

[0076] Protein purification was performed using His-tag affinity chromatography, and the specific operations were carried out according to the instructions of the Beyotime His-tag protein purification kit.

[0077] 2.4.4 Dialysis of purified protein and determination of protein concentration

[0078] The purified lytic enzyme protein was dialyzed to remove impurities and small molecule interfering agents in the buffer. The purified protein solution was placed in a dialysis bag (molecular weight cutoff, 10 kDa) and dialyzed against sterile PBS buffer (pH 7) at 4°C. The initial dialysate volume was 200 times the sample volume, and the dialysate was replaced every 2 hours for three consecutive dialysis cycles. After dialysis, the protein solution was collected and centrifuged at 12,000 rpm for 10 minutes at 4°C. The supernatant was used for subsequent experiments. The purified protein samples were aliquoted into sterile EP tubes and stored at -80°C for subsequent antimicrobial activity testing.

[0079] Protein concentration was determined using the BCA assay using the Pierce™ BCA Protein Assay Kit according to the manufacturer's instructions. All samples were assayed in triplicate.

[0080] 3 Experimental Results

[0081] 3.1 Screening and Acquisition of Prophage Lytic Enzymes

[0082] This study was based on 26 publicly available genomes of Streptococcus equi subspecies in the NCBI database. A total of 30 protein sequences with lytic enzyme potential were screened and amplified by PCR using 8 pairs of primers. Primers Lys1, Lys3, Lys4, Lys5, and Lys6 all amplified specific bands, while primers Lys2, Lys7, and Lys8 did not. The amplification results are shown in Figure 2. Figure 1By comparing the sequencing results of specific bands, a total of six different lyase gene sequences were obtained. These six lyases were named LysHLJ1-9, LysHLJ1-18, LysHLJ3-11, LysHLJ4-11, LysHLJ5-13, and LysHLJ6-11, respectively. The lyase source strains / primers and their lengths are shown in Table 7.

[0083] ;

[0084] The obtained lysases (LysHLJ1-9, LysHLJ1-18, LysHLJ3-11, LysHLJ4-11, LysHLJ5-13 and LysHLJ6-11) were compared by NCBI BLASTP and similarity analysis was performed with published streptococcal lysases (including Ply30, Ply5218, Ply7917, PlyS2, PlyS59, LysFL1 and Ply1228) using Jalview 2.11.4.1 software. The Python programming language was used for visualization analysis, and the seaborn and matplotlib libraries were used to draw similarity heat maps to intuitively display the sequence identity levels between different lysases. The NCBI BLASTP results showed that LysHLJ1-9, LysHLJ1-18, LysHLJ3-11, LysHLJ4-11 and lysin ( Streptococcus phageJavan158) has 88% homology, but Streptococcus There is no literature report on the function of lysin of phage Javan158; BLASTP analysis of LysHLJ5-13 and LysHLJ6-11 revealed that these two lysins did not show significant homology with known lysin proteins in public databases. S.equi A coding region with certain sequence identity was retrieved from the partial complete genome sequence of the genome of the human.

[0085] Similarity heatmap such as Figure 2As shown, LysHLJ1-9, LysHLJ1-18, and LysHLJ3-11 showed extremely high sequence identities, at 98.77%, 95.06%, and 94.81%, respectively. LysHLJ4-11 shared approximately 88% similarity with LysHLJ1-9, LysHLJ1-18, and LysHLJ3-11, while LysHLJ5-13 shared 89.6% similarity with LysHLJ6-11. The similarities with the first four lyases were relatively low, ranging from 15% to 17.2%. The sequence identities of the six amplified lyases with previously published streptococcal lyases were generally low, ranging from 12% to 67%. These data indicate that six new lyases were discovered in this study.

[0086] 3.2 Construction of recombinant expression plasmid for lytic enzyme

[0087] To further verify the biological activity of the lyases, the six lyase genes screened in this study were constructed into the prokaryotic expression vector pET-28a. Using homologous recombination technology, the His-tagged lyase coding sequence was successfully inserted into the expression vector. Using primers Lys1, Lys3, Lys4, Lys5, and Lys6, fragments LysHLJ1-9, LysHLJ1-18, LysHLJ3-11, LysHLJ4-11, LysHLJ5-13, and LysHLJ6-11 were successfully amplified ( Figure 3 Sequencing identification showed that the pET-28a-LysHLJ1-9, pET-28a-LysHLJ1-18, pET-28a-LysHLJ3-11, pET-28a-LysHLJ4-11, pET-28a-LysHLJ5-13, and pET-28a-LysHLJ6-11 vectors were successfully constructed.

[0088] 3.3 Recombinant expression and purification of lytic enzyme

[0089] The recombinant expression plasmids pET-28a-LysHLJ1-9, pET-28a-LysHLJ1-18, pET-28a-LysHLJ3-11, pET-28a-LysHLJ4-11, pET-28a-LysHLJ5-13 and pET-28a-LysHLJ6-11 successfully transformed into BL21 (DE3) were induced for expression, and the bacterial liquid before and after induction, the supernatant after ultrasonic disruption and the precipitate were collected for SDS-PAGE electrophoresis. The results are shown in the figure. Figure 4Specific bands appeared at approximately 44.4 kDa, 44.4 kDa, 44.4 kDa, 44.4 kDa, 47.9 kDa, and 45.1 kDa in the supernatant of LysHLJ1-9, LysHLJ1-18, LysHLJ3-11, LysHLJ4-11, pET-28a-LysHLJ5-13, and pET-28a-LysHLJ6-11 after induction, respectively, indicating successful expression of the recombinant proteins.

[0090] To further verify the expression and purification effect of the recombinant protein, the supernatant of the expression strain after ultrasonic disruption was purified and the purified product was analyzed by SDS-PAGE. Figure 5 The results showed that the recombinant proteins of each lysing enzyme had few impurity bands, indicating that each recombinant protein could be successfully expressed and purified through His tag affinity purification to obtain highly pure soluble proteins. Among them, the amino acid sequence of LysHLJ1-9 is shown in SEQ ID NO.29, and its encoding nucleotide sequence is shown in SEQ ID NO.30.

[0091] Example 2 Study on the bactericidal activity of lytic enzyme in vitro

[0092] 1 Experimental Materials

[0093] 1.1 Bacterial strains

[0094] 2 strains of Streptococcus dysgalactiae subsp. equisimilis ( Streptococcus dysgalactiae subspecies equisimilis , SDSE) were isolated and preserved by our laboratory, and the other bacterial strains required for the experiment were the same as those in Example 1.

[0095] 1.2 Preparation of main reagents and solutions

[0096] BHI culture medium (Oxoid) and other main reagents were the same as those in Example 1.

[0097] Todd-Hewitt broth (THB): Place 800 mL of distilled water in a beaker, add 36.4 g of THB, stir well, and dilute to 1 L. Autoclave at 115°C for 30 min and store at 4°C in the dark.

[0098] Sterile PBS buffer: Weigh 8.0 g of NaCl, 0.2 g of KCl, 2.9 g of Na₂HPO₄·12H₂O, and 0.24 g of KH₂PO₄ and dissolve them in 800 mL of ultrapure water. Stir thoroughly, adjust the pH to 7.4 with 1 M NaOH or 1 M HCl, and finally bring the volume to 1000 mL. Autoclave the solution at 121°C for 15 min and store at 4°C until needed.

[0099] BHI solid medium: Weigh 37 g of BHI medium powder and 15 g of agar into 1 L of ultrapure water, stir to dissolve completely, and adjust the pH to 7.4. Make up to 1 L of the solution and autoclave at 121°C for 15 minutes. After sterilization, cool the medium to approximately 50°C. Aseptically pour the medium into a sterile Petri dish and allow it to solidify. Store at 4°C until ready for use.

[0100] 2 Experimental methods

[0101] 2.1 Comparison of the antimicrobial spectra of different lytic enzymes

[0102] Twenty strains of Streptococcus equi (including 10 strains of subspecies equi and 10 strains of subspecies zooepidemicus) were selected to determine the cleavage spectrum of the lytic enzyme. The above strains were cultured to the logarithmic growth phase (OD 600nm = 0.6 ~ 0.8), 200 μL of bacterial liquid was evenly spread on BHI solid culture medium, 10 μL of purified lytic enzyme protein (200 μg) was added dropwise to the plate surface, and after the liquid was dried, the plate was inverted and incubated in a 37°C incubator for 12 h. The target host range of the lytic enzyme was determined by observing whether an inhibition ring was formed, and the lytic enzyme with the broadest antibacterial spectrum was screened.

[0103] 2.2 Lytic activity of different lyases in liquid environments

[0104] Streptococcus equi subspecies SD2018 (Isolation and identification of the pathogen of donkey plague in large-scale donkey farms and its infectivity to mice, Wang Ning et al., Heilongjiang Animal Husbandry and Veterinary Medicine, 2020(22):67-70,76,166) was used as an indicator bacteria. A single colony of SD2018 was picked and inoculated into THB medium and cultured at 37°C and 180 rpm overnight. The fully recovered bacterial solution was inoculated into fresh THB medium at a ratio of 1:100 and cultured at 37°C and 180 rpm until the logarithmic phase of growth (OD 600nm = 0.6 ~ 0.8). Centrifuge at 4°C, 6000 rpm for 2 min, discard the supernatant, and wash the cells three times with sterile PBS solution and resuspend to OD 600nm≈ 1.0. Add 1 mL of resuspended bacterial solution to each of four sterile test tubes. Dilute the purified lytic enzyme protein with sterile PBS buffer and add it to each of the four test tubes, achieving final concentrations of 50 µg / mL, 100 µg / mL, 150 µg / mL, and 200 µg / mL, respectively. Add an equal volume of sterile PBS buffer to the control group. Incubate the five test tubes at 37°C and 180 rpm with shaking. Observe the bactericidal effect of the lytic enzyme protein on the bacterial solution for 1 hour to identify the lytic enzyme with the best lytic activity.

[0105] 2.3 Bactericidal activity of lyase LysHLJ1-9 against SD2018

[0106] Streptococcus equi subspecies equi SD2018 was cultured to the logarithmic growth phase (OD 600nm = 0.6 to 0.8), the cells were washed three times with sterile PBS buffer and divided into five groups. The most effective lytic enzyme proteins from 2.1 and 2.2 were added to the four experimental groups to final concentrations of 50 µg / mL, 100 µg / mL, 150 µg / mL, and 200 µg / mL, respectively; an equal volume of sterile PBS buffer was added to the control group. All samples were incubated in a 37°C water bath for 1 hour, with samples collected every 15 minutes. Colonies were then counted using the serial dilution-plating method: the cells were serially diluted 10-fold, and the dilutions were plated on Columbia agar plates. The dilutions were incubated at 37°C for 24 hours before counting. This evaluated the in vitro bactericidal activity of the lytic enzyme proteins.

[0107] 2.4 Analysis of SD2018 resistance to lyase LysHLJ1-9

[0108] The clinical isolate of Streptococcus equi subspecies equi SD2018 was used as an indicator bacterium and cultured to the logarithmic growth phase (OD 600nm = 0.6 to 0.8), spread 200 µL of the bacterial solution evenly on Columbia blood agar plates. After the solution dried naturally, add 10 µL of 200 µg / mL lytic enzyme protein to the plate surface. Incubate the plate in a 37°C incubator for 12 hours. Pick a single colony at the edge of the inhibition zone and inoculate it into THB medium, incubating at 37°C overnight. Assay the lytic activity of the lytic enzyme protein according to step 2.3. This process was repeated for seven consecutive passages, followed by five additional passages. The lytic activity of the lytic enzyme protein against each passage of the SD2018 strain was assayed.

[0109] 2.5 Antimicrobial spectrum determination of LysHLJ1-9

[0110] Twenty strains of Streptococcus equi were cultured as indicator bacteria according to step 2.2 until the logarithmic growth phase. The cells were washed three times with sterile PBS buffer and resuspended, and the initial colony count was determined. Subsequently, the lytic enzyme LysHLJ1-9 was added to the bacterial suspension to a final concentration of 200 µg / mL. An equal volume of sterile PBS buffer was added to the control group. All samples were incubated in a 37°C water bath for 1 hour, followed by colony count. The difference between the initial and post-incubation colony counts was used to assess the lytic activity of the lytic enzyme against the different strains.

[0111] 2.6 Determination of biochemical characteristics of lysase LysHLJ1-9

[0112] 2.6.1 Effect of temperature on the activity of LysHLJ1-9

[0113] Strain SD2018 was cultured according to the method described in 2.2. The culture medium was washed with sterile PBS buffer and resuspended. The resuspended culture was mixed with lytic enzyme to a final concentration of 200 µg / mL. An equal volume of sterile PBS buffer was added to the control group. Samples were incubated at 4°C, 16°C, 25°C, 37°C, and 42°C for 1 h. Colonies were then counted using the serial dilution-plating method. The effect of different temperature conditions on lytic enzyme activity was evaluated by calculating the difference in colony counts before and after incubation.

[0114] 2.6.2 Effect of pH on the activity of lyase LysHLJ1-9

[0115] The SD2018 strain was cultured to OD 600nm = 0.6 to 0.8, followed by centrifugation at 6000 rpm for 2 min at 4°C and washing three times with sterile PBS buffer. Seven pH gradients were established, with the pH of the sterile PBS buffer adjusted to 3, 4, 5, 6, 7, 8, and 9 using 1 M HCl and 1 M NaOH solutions, respectively. Lysase was diluted in sterile PBS buffer of the corresponding pH value to a final concentration of 400 µg / mL. Simultaneously, washed cells were resuspended in sterile PBS buffer of the above-mentioned different pH values, and colony counts were performed. Lysase solutions of different pH values ​​were mixed with the resuspended bacterial solution of the corresponding pH value in a 1:1 volume ratio. An equal volume of sterile PBS buffer of the same pH value was added to the control group. All samples were incubated in a 37°C water bath for 1 h, followed by colony counts. The effect of different pH conditions on lysase activity was evaluated by calculating the change in colony count before and after incubation.

[0116] 2.7 Determination of the lytic effect of LysHLJ1-9 on clinical isolates of Streptococcus equi

[0117] Twenty strains of Streptococcus equi (including 10 strains of equi subspecies and 10 strains of zooepidemicus subspecies) and two strains of Streptococcus dysgalactiae subspecies equisimilis were selected to determine the cleavage spectrum of the lytic enzyme. The above strains were cultured to the logarithmic growth phase (OD 600nm = 0.6 to 0.8), centrifuged 1 mL of bacterial suspension at 6000 rpm at 4°C for 5 minutes, discarded the supernatant, washed three times with sterile PBS (pH 7.4), and resuspended. Lysase was added to a final concentration of 100 μg / mL, and incubated at 37°C for 1 hour. A gradient dilution-plating method was then used to determine the changes in colony counts before and after treatment to analyze the bactericidal efficacy of lysase against different strains.

[0118] 2.8 Effects of LysHLJ1-9 on Bacterial Biofilms

[0119] The SD2018 strain was cultured at 37°C and 180 rpm for 12 hours. The next day, the bacterial suspension was diluted 1:100 and inoculated into a 96-well plate. 200 μL of the diluted suspension was added to each well and incubated at 37°C for 24 hours to allow biofilm formation. After incubation, the culture medium was discarded, and each well was gently washed three times with sterile PBS buffer to remove unadhered bacteria. A 200 μL lytic enzyme solution (final concentration: 200 μg / mL) was added to the experimental group, while an equal volume of sterile PBS buffer was added to the control group. A penicillin solution (final concentration: 200 μg / mL) was added to the penicillin group for comparison. Three replicate wells were set up in each group, and the samples were incubated at 37°C for 1 hour to test the effects of lytic enzyme and penicillin on biofilm formation.

[0120] After incubation, the solution in the wells was discarded, each well was washed three times with PBS buffer, and 200 μL of 0.1% crystal violet solution was added for staining for 15 minutes. After staining, the staining solution was discarded, and the wells were washed three times with PBS. 200 μL of 95% ethanol was added to each well and allowed to stand at room temperature for 30 minutes to dissolve the crystal violet in the membrane. The absorbance value (OD) was measured at 570 nm. 570nm ) to reflect the formation of biofilm.

[0121] 2.9 Data Analysis

[0122] All data were analyzed using GraphPad Prism 8.0 software (GraphPad Software Inc., San Diego, CA, USA) using one-way analysis of variance (ANOVA). P values ​​< 0.05 were considered statistically significant. Error bars represent the standard error of the mean.

[0123] 3 Experimental Results

[0124] 3.1 Antimicrobial spectrum of different lytic enzymes

[0125] The lysis range of different lyases (200 μg) against 20 strains of Streptococcus equi (including 10 strains of S. equi and 10 strains of S. zooepidemicus) and 2 strains of Streptococcus dysgalactiae subsp. equisimilis was determined by observing the formation of inhibition zones on BHI solid plates. LysHLJ1-9 and LysHLJ1-18 were positive (+) against all tested strains; LysHLJ3-11 had lytic activity against all tested S. equi (including S. equi and S. zooepidemicus), but was ineffective against S. dysgalactiae subsp. equisimilis and Escherichia coli; LysHLJ4-11 had lytic activity against 17 strains of S. equi, excluding SE-4, SE-5, and SZ-4; LysHLJ5-13 was effective against 19 strains of S. equi, excluding SE-7. LysHLJ6-11 had the narrowest range of activity compared to the other five lyases, lysing only four strains. S.equi and 5 strains S.zoo Effective. LysHLJ3-11, LysHLJ4-11, LysHLJ5-13, and LysHLJ6-11 were ineffective against the tested Streptococcus dysgalactiae subsp. equisimilis. All lytic enzymes had no lytic effect on Escherichia coli DH5α and E. coli BL21 (DE3). Lysis results for different strains are shown in Table 8.

[0126] ;

[0127] Note: 1: Streptococcus equi subsp. equi; 2: Streptococcus equi subsp. zooepidemicus; 3: Streptococcus dysgalactiae subsp. equisimilis; 4: Escherichia coli; +: plaque formation; -: no plaque formation

[0128] 3.2 Lytic activity of different lyases in liquid environments

[0129] To further evaluate the lytic ability of each lytic enzyme, this study used SD2018 as an indicator bacterium to detect the effects of six lytic enzymes at different concentrations (50 μg / mL, 100 μg / mL, 150 μg / mL, and 200 μg / mL) on the transparency of the bacterial solution. The results are shown in Figure 2. Figure 6 and Figure 7 As shown, the degree of clarification of the bacterial solution and OD 600nm The value reflects the degree of bacterial lysis.

[0130] In all control groups, the bacterial solution remained turbid, LysHLJ1-9 ( Figure 6 A) At 50 μg / mL, it showed obvious lysis effect. As the concentration increased, the bacterial solution gradually became clear, and it was almost completely clear at 200 μg / mL. In contrast, LysHLJ1-18 ( Figure 6B) Although it can cause a certain degree of clarification at higher concentrations, the cleavage intensity is significantly weaker than that of LysHLJ1-9. LysHLJ3-11 and LysHLJ4-11 ( Figure 6 C, D) The clarification effect on bacterial suspension was weak at all concentrations, with only a slight change observed at 200 μg / mL, suggesting limited lytic activity.

[0131] LysHLJ5-13 ( Figure 6 E) There was almost no significant lysis effect on the bacterial solution at each concentration, and the difference was not significant compared with the control group. Figure 6 F) There was essentially no effect at a concentration of 50 μg / mL. At a concentration of 200 μg / mL, the bacterial solution became somewhat transparent, but the overall lysis efficiency was low.

[0132] To evaluate the bactericidal effects of the six recombinant lytic enzymes against SD2018, the bacterial cultures were treated with different concentrations (50 μg / mL, 100 μg / mL, 150 μg / mL, and 200 μg / mL), and the OD values ​​were measured. 600nm The value reflects the bacterial growth. Figure 7 As shown in the figure, LysHLJ1-9 and LysHLJ1-18 treated groups showed concentration-dependent bactericidal effects at different concentrations. 600nm The value dropped to 0.259 at 200 μg / mL; the OD of the LysHLJ1-18 treated group 600nm The value drops to about 0.452.

[0133] OD of LysHLJ3-11, LysHLJ4-11, LysHLJ5-10 and LysHLJ6-11 treated groups 600nm The value changes were small and maintained at a high level overall. At 200 μg / mL, OD 600nm The values ​​remained above 0.7. These results indicate that LysHLJ1-9 has stronger bactericidal activity than the other five lyases.

[0134] 3.3 In vitro bactericidal activity of LysHLJ1-9 against SD2018

[0135] To further evaluate the in vitro bactericidal ability of LysHLJ1-9 and its relationship with time and concentration, this study measured the changes in the number of surviving bacteria after the action of the lyase at different concentrations (50, 100, 150, and 200 μg / mL) and at different time points (0 min, 15 min, 30 min, 45 min, and 60 min). Figure 8As shown, LysHLJ1-9 exhibited moderate bactericidal activity at various concentrations in a dose-dependent manner. The bactericidal effect significantly increased with increasing lyase concentration. Within 60 minutes, LysHLJ1-9 at concentrations of 50 μg / mL, 100 μg / mL, and 150 μg / mL reduced the colony count by approximately 0.35 lg, 0.54 lg, and 0.78 lg, respectively. At 200 μg / mL, the bacterial count decreased from approximately 8.6 lg (CFU / mL) to approximately 7.6 lg (CFU / mL) within 60 minutes, achieving a bactericidal effect of approximately 1 lg.

[0136] 3.4 Analysis of SD2018 resistance to lyase LysHLJ1-9

[0137] To verify whether the bacteria would develop resistance to the lytic enzyme, the strain SD2018 was serially passaged for 12 generations, and the resistance of SD2018 to the lytic enzyme LysHLJ1-9 was analyzed in each generation. Figure 9 As shown in the results, the bactericidal effect of LysHLJ1-9 on SD2018 did not change significantly among generations, indicating that the strain did not develop resistance to LysHLJ1-9 within 12 generations.

[0138] 3.5 Determination of biochemical characteristics of lysase LysHLJ1-9

[0139] 3.5.1 Effect of temperature on the activity of lyase LysHLJ1-9

[0140] The lyase LysHLJ1-9 was exposed to temperatures of 4°C, 16°C, 25°C, 37°C, and 42°C for 1 h to determine the effects of different temperatures on the activity of LysHLJ1-9. Figure 10 As shown in Figure A, the activity of the lyase remained stable within the 4°C to 42°C range, and different temperatures had little effect on its bactericidal efficacy. Compared with 4°C, 16°C, 25°C, and 37°C, the lyase activity decreased slightly at 42°C. At 42°C, LysHLJ1-9 reduced the colony count by approximately 0.96 log within 1 hour.

[0141] 3.5.2 Effect of pH on LysHLJ1-9 Activity

[0142] A total of 7 pH buffer gradients were set up. After acting under different pH conditions, the activity changes of the lyase LysHLJ1-9 were detected by gradient dilution-plate coating method. Figure 10As shown in Figure B, LysHLJ1-9 maintains relatively stable bactericidal activity within a pH range of 3-9. Maximum activity is achieved at pH 7, reducing the SD2018 colony count by approximately 1 log. While relatively low in acidic environments with a pH of 3-6, the lyase still maintains a moderate bactericidal effect. At alkaline pH 9, the lyase's activity approaches that of a neutral environment.

[0143] 3.6 Determination of the lytic effect of LysHLJ1-9 on clinical isolates of Streptococcus

[0144] By selecting clinical isolates of Streptococcus (including 10 strains S.equi, 10 strains S.zoo and 2 strains of SDSE) as Figure 11 , lyase LysHLJ1-9 S.equi The overall lysis effect of the strain was good, and LysHLJ1-9 had a good S.equi The strains (HLJ2018, SD2018, SE-1 to SE-8) all had strong lysis ability, with bacterial reduction rates ranging from 55% to 95%. S. zoo Among the strains (SZ-1 to SZ-10), some (such as SZ-3, SZ-9, and SZ-10) showed high sensitivity, with lysis rates exceeding 80%, while others (such as SZ-1 and SZ-6) had lower lysis rates of only around 40%. The two SDSE strains (SDSE-1 and SDSE-2) also showed high lysis rates, both exceeding 70%. The viable cell reduction rate at 200 μg / mL of protein ranged from 30% to 95%.

[0145] 3.7 Effects of LysHLJ1-9 on Bacterial Biofilms

[0146] The inhibitory effects of LysHLJ1-9 and penicillin on bacterial biofilm were detected by crystal violet staining, and the OD value at 570 nm was measured to evaluate the residual amount of biofilm. Figure 12 As shown, OD of PBS-treated group 570nm The highest value was about 0.56; after treatment with 200 μg / mL penicillin, OD 570nm The value remained at about 0.51, and the OD of the LysHLJ1-9 treated group was 570nm The OD values ​​were significantly lower than those in the PBS group. 570nm The value decreased by about 0.4.

[0147] Example 3 In vivo antibacterial effect and safety evaluation of lyase LysHLJ1-9

[0148] 1 Experimental Materials

[0149] 1.1 Bacterial strains and experimental animals

[0150] The experimental animals were SPF female BALB / c mice, 6-8 weeks old (18-20 g), purchased from Liaoning Changsheng Biotechnology Co., Ltd. The bacterial strain required for the experiment was Streptococcus equi subspecies equi SD2018 preserved in the laboratory.

[0151] 1.2 Preparation of bacterial solution

[0152] Laboratory-stored SD2018 glycerol stock was streaked onto Columbia blood agar plates and incubated at 37°C for 12 hours. A single colony was then selected and inoculated into THB liquid medium and incubated at 37°C with shaking at 180 rpm for 8 hours. The culture was then transferred to fresh THB medium at a 1:100 volume ratio and incubated under the same conditions until the bacteria reached the logarithmic phase. Viable cells were counted in the culture medium at this stage and used as the infection medium in subsequent experiments.

[0153] 2 Experimental methods

[0154] 2.1 Establishment of the SD2018 strain infection mouse model

[0155] Prepare fresh SD2018 bacterial suspension in the logarithmic growth phase according to the method in 1.3, and measure its concentration by spreading on blood agar plates after dilution. According to the measured colony count, prepare different concentrations of infection bacterial suspension, set as 1×10³, 1×10 4 , 1×10 5 , 1×10 6 and 1×10 7 CFU / 0.2 mL. Balb / C mice of the same sex and similar health conditions were randomly divided into 6 groups, with 8 mice in each group. One group was the control group, which was injected intraperitoneally with only 0.2 mL of THB medium. The other five groups were injected intraperitoneally with different concentrations (10 3 ~ 10 7 CFU / 0.2 mL). Mice were housed under uniform conditions, and their survival was observed and recorded daily. The survival rate of each group was calculated to assess the minimum lethal dose (MLD) of the SD2018 strain in mice.

[0156] 2.2 Evaluation of the therapeutic effect of LysHLJ1-9 cleavage in mice

[0157] 2.2.1 Determination of the therapeutic dose of LysHLJ1-9 in mice

[0158] Mice were randomly divided into 4 groups, with 8 mice in each group. SD2018 bacterial solution at a dose of 2×MLD was injected into the peritoneal cavity unilaterally. Blood samples were collected from the tail vein of the mice every 1 hour within 7 hours after infection, and the number of colonies in the blood was detected by gradient dilution method. When the number of colonies reached 1×10 4 When the number of CFU reached 1,000 mice, systemic infection was considered, and treatment was initiated. The experimental groups received intraperitoneal injections of 500, 1000, and 1500 μg of the lytic enzyme LysHLJ1-9, respectively, while the control group received an equal volume of sterile PBS as a control. Treatment was performed by intraperitoneal injection into the contralateral side of the abdomen, and the survival of the mice was observed and recorded daily for 7 days. The lowest dose that achieved 100% survival was determined as the minimum therapeutic dose of LysHLJ1-9.

[0159] 2.2.2 Safety assessment of LysHLJ1-9 in mice

[0160] Mice were randomly divided into two groups, with six mice in each group. The experimental group received an intraperitoneal injection of 2000 μg of LysHLJ1-9, while the control group received an equal volume of sterile PBS. The mice were observed for 7 consecutive days, and their health status was recorded and assessed using a 5-0 scoring system: good health (5 points); matted hair and decreased activity (4 points); lethargy and hunched posture (3 points); periocular discharge (2 points); impending death (1 point); and death (0 points). The health score of each mouse was recorded and used for subsequent analysis.

[0161] 2.2.3 Determination of bacterial load in mouse blood

[0162] Mice were randomly divided into three groups: the PBS-treated group was inoculated with a 2×MLD dose of SD2018 and then injected with an equal volume of sterile PBS 1 hour after infection; the LysHLJ1-9-treated group was inoculated with a 2×MLD dose of SD2018 and then injected intraperitoneally with 1500 μg of LysHLJ1-9 1 hour later; and the control group was injected with an equal volume of sterile PBS. Blood was collected from three mice randomly selected from each group at 1, 2, 3, 4, 5, 6, 7, 12, 24, 36, and 48 hours after infection. Blood samples were diluted with sterile PBS and plated on Columbia blood agar plates, which were then incubated inverted at 37°C. Colonies formed on the plates were counted to calculate blood bacterial loads. The trends in colony counts between the treated and control groups at each time point were further analyzed.

[0163] 2.2.4 Determination of bacterial load in major mouse organs

[0164] At 1, 12, and 24 hours after infection, three mice were randomly selected from each group for dissection and isolation of liver, spleen, lung, and kidney tissues. Each tissue was placed in 1 mL of sterile PBS, thoroughly homogenized, and serially diluted with sterile PBS. Subsequently, the diluted samples were plated onto Columbia blood agar plates and incubated inverted at 37°C. Colony counts were then counted to assess changes in bacterial load in various organs at different time points, thereby determining the in vivo bactericidal efficacy of LysHLJ1-9.

[0165] 2.2.5 Examination of histopathological changes in important organs

[0166] At 24 and 48 hours after infection, three mice were randomly selected from each group. Liver, spleen, lung, and kidney tissues were removed after dissection and fixed in 4% PFA. Paraffin sections were prepared from the fixed organs and stained with HE to observe pathological changes.

[0167] 2.3 Data Analysis

[0168] All data were statistically analyzed using GraphPad Prism 8.0 software (GraphPad Software Inc., San Diego, CA, USA). Differences between groups were tested using one-way analysis of variance (ANOVA), and P values ​​less than 0.05 were considered statistically significant. Error bars in the figures represent the standard error of the mean.

[0169] 3 Experimental Results

[0170] 3.1 Determination of the minimum lethal dose

[0171] To establish the infection model of Streptococcus equi subspecies SD2018, different doses of bacterial solution (1×10 3 CFU / unit, 1×10 4 CFU / cell, 1×10 5 CFU / unit, 1×10 6 CFU / cell, 1×10 7 CFU / mouse) to evaluate its lethality. The results showed that 1×10 3 The survival rate of mice in the CFU group was 50% within 7 days, and 1×10 4 All the CFU group died within 7 days, while 1×10 5 All mice in the CFU group died within 4 days. 6 CFU and above dose groups showed 100% mortality within 3 days ( Figure 13 A). All mice in the control group survived without any abnormal symptoms. Therefore, the MLD of the SD2018 strain was determined to be 1.0×106 CFU / piece.

[0172] 3.2 Determination of the therapeutic dose of LysHLJ1-9 in mice

[0173] To verify the protective effect of lytic enzymes on the mouse infection model, mice were intraperitoneally injected with 2×MLD (2×10 6 CFU / mouse) to establish an acute bacteremia model. All mice that did not receive any treatment died within 48 hours. 1 hour after infection, different doses of lytic enzyme were given for treatment. The results are shown in the figure. Figure 13 Figure B shows that intraperitoneal injection of 1500 μg of lytic enzyme resulted in 100% survival in mice, while 1000 μg of lytic enzyme resulted in a survival rate of 62.5%. At a dose of 500 μg, the survival rate dropped to 37.5%. Therefore, the minimum effective therapeutic dose of lytic enzyme is 1500 μg per mouse, which effectively improves the resistance of mice to bacteremia.

[0174] 3.3 Safety assessment of LysHLJ1-9 in mice

[0175] To evaluate the potential toxic side effects of LysHLJ1-9, mice were intraperitoneally injected with 2000 μg of LysHLJ1-9, and an equal amount of sterile PBS control group was set up. Figure 13 As shown in Figure C, within 7 days, all mice treated with 2000 μg of LysHLJ1-9 survived and remained in good condition, with normal food and water intake, showing no significant abnormalities compared to the normal control group. This result demonstrates that LysHLJ1-9 has a good safety profile, and a single intraperitoneal injection of 2000 μg does not significantly affect the health of the mice.

[0176] 3.4 Determination of bacterial load in mouse blood

[0177] The mouse infection model was established by intraperitoneal injection of Streptococcus equi subspecies SD2018. At 1 h after infection, the bacterial load in the blood reached approximately 10 4 CFU / mL. The infected mice were then treated with LysHLJ1-9 and PBS, and the changes in the bacterial load in the blood of the mice were monitored at different time points. The results are as follows: Figure 13 D. From 2 h after infection, the number of colonies in the blood of mice in the PBS group increased significantly to about 10 5 CFU / mL and maintained a high level until the end of the experiment, indicating that the mice were in a state of persistent bacteremia. In contrast, the bacterial load in the LysHLJ1-9 treatment group decreased rapidly after treatment and maintained a steady downward trend, dropping to 10 CFU / mL at 48 h. 3The level of HLJ1-9 in the LysHLJ1-9-treated group was significantly lower than that in the PBS group starting at 2 h (p < 0.001). LysHLJ1-9 effectively reduced the bacterial load in the peripheral blood of mice infected with SD2018, demonstrating strong in vivo antibacterial activity.

[0178] 3.5 Determination of bacterial load in important organs of mice

[0179] At 1 h, 12 h, and 24 h after infection, liver, lung, kidney, and spleen tissues were collected and the number of colonies was determined. Figure 13 E. At 1 hour, bacterial loads in organs were similar across all groups, with liver loads at 4.92 ± 0.21 lg CFU / g and lung loads at 5.01 ± 0.15 lg CFU / g, suggesting no significant differences in the initial infection phase. Twelve hours later, liver loads in the PBS group rose to 6.69 ± 0.18 lg CFU / g, while those in the LysHLJ1-9 group decreased to 4.51 ± 0.09 lg CFU / g. Lung loads were significantly different (p < 0.01), with 5.67 ± 0.20 lg CFU / g in the PBS group and 4.54 ± 0.12 lg CFU / g in the LysHLJ1-9 group. By 24 hours, the bacterial load in the liver, lung, kidney, and spleen of the treatment group decreased to 3.75 ± 0.11, 3.73 ± 0.09, 4.11 ± 0.10, and 4.52 ± 0.12 lg CFU / g, respectively, compared with 6.73 ± 0.14, 6.63 ± 0.13, 5.58 ± 0.10, and 6.65 ± 0.15 lg CFU / g in the PBS group, representing decreases of 3, 2.9, 1.4, and 2.1 lg CFU / g, respectively. These data indicate that LysHLJ1-9 significantly inhibits bacterial colonization and spread in multiple organs.

[0180] 3.6 Histopathological changes of important organs

[0181] At 24 hours and 48 hours after SD2018 infection, the lungs, livers, spleens, and kidneys of mice were collected and pathological tissue sections were prepared to observe the pathological changes of these organs in the blank group, PBS treatment group, and LysHLJ1-9 treatment group. LysHLJ1-9 significantly improved the pathological damage of the liver, spleen, lungs, and kidneys of mice infected with SD2018. According to the observation results of the tissue pathological sections (such as Figure 14 and 15 ), pathological changes in the liver, spleen, lungs and kidneys are as follows:

[0182] Lungs: Mice in the PBS-treated group exhibited significant pathological changes in the lungs, including marked damage to alveolar structure, thickened alveolar septa accompanied by pronounced inflammatory cell infiltration, and extensive erythrocyte exudate, suggesting significant hemorrhage and inflammation. However, the pathological changes in the lungs of mice in the LysHLJ1-9-treated groups (24 and 48 hours) were significantly improved, with clearer alveolar structure and significantly reduced inflammatory cell infiltration and erythrocyte exudate. The 48-hour group showed even more pronounced recovery, approaching the levels of the normal control group.

[0183] Spleen: Mice in the PBS-treated group showed significant lesions in the spleen, with blurred white and red pulp structure, extensive lymphocytic necrosis, and severe congestion and hemorrhage. In contrast, the LysHLJ1-9-treated group (24 and 48 hours) showed significant relief of splenic inflammation and necrosis, with gradual restoration of tissue structure. At 48 hours, the white and red pulp structure became clear, lymphocytes increased significantly, and histopathological changes were significantly improved.

[0184] Liver: The liver tissue of mice in the PBS group showed significant pathological changes, including marked hepatocyte edema, localized cytolysis and necrosis, and large, elongated eosinophilic necrotic foci within the liver parenchyma. In the LysHLJ1-9 treatment group (24 and 48 hours), liver tissue structure improved significantly, with reduced hepatocyte swelling and cell necrosis. By 48 hours, liver tissue had essentially returned to normal.

[0185] Kidneys: Mice in the PBS group also exhibited significant pathological changes in renal tissue, including tubular epithelial cell edema and necrosis and interstitial vascular congestion. However, in the LysHLJ1-9-treated group (24 and 48 hours), renal tissue structure improved significantly, interstitial vascular congestion was significantly reduced, and the degree of tubular lesions decreased significantly, approaching the level of the normal group by 48 hours.

Claims

1. A Streptococcus equi subspecies equi ( Streptococcus equi subsp. equi , S.equi ) a prophage lytic enzyme, characterized in that The amino acid sequence of the lyase is shown in SEQ ID NO.

29.

2. A polynucleotide encoding the Streptococcus equi subsp. equi prophage lytic enzyme according to claim 1.

3. The polynucleotide according to claim 2, wherein The sequence of the polynucleotide is shown as SEQ ID NO.

30.

4. An expression vector, characterized in that The expression vector contains the polynucleotide according to claim 2 or 3.

5. The expression vector according to claim 4, wherein The expression vector is a pET-28a vector containing the polynucleotide according to claim 2 or 3.

6. A host bacterium, characterized in that The host bacteria contains the expression vector according to claim 4 or 5.

7. The host bacteria according to claim 6, characterized in that The host bacteria is E. coli BL21(DE3).

8. The Streptococcus equi subspecies equi prophage lytic enzyme according to claim 1 is used in the preparation of anti-Streptococcus equi subspecies equi and Streptococcus equi subspecies zooepidemicus ( Streptococcus equi subsp. Zooepidemicus , S.zoo ) reagents or drugs.

9. Use of the Streptococcus equi subspecies equi prophage lytic enzyme according to claim 1 in the preparation of a drug for preventing and treating equine strangles.

10. A biological antimicrobial agent for preventing and treating equine strep, characterized in that: The biological antibacterial agent contains the prophage lytic enzyme of Streptococcus equi subspecies equi described in claim 1.

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