Streptococcus equi subsp. Equi probacteriophage lyase as well as preparation method and application thereof

Abstract

The invention discloses streptococcus equi subsp. Equi probacteriophage lyase as well as a preparation method and application thereof, and belongs to the technical field of veterinary drugs. The amino acid sequence of the lyase is as shown in SEQ ID NO. 29. Polynucleotide for coding the streptococcus equi subsp. Equi probacteriophage lyase, an expression vector containing the polynucleotide and host bacteria containing the expression vector are also within the protection range of the invention. Wherein, preferably, the sequence of the polynucleotide is as shown in SEQ ID NO. 30. According to the invention, prophage lyase genes in a streptococcus equi subsp. Equi genome sequence are compared, specific primers are designed, lyase genes in streptococcus equi strains stored in a laboratory are amplified and cloned, recombinant expression is carried out, and antibacterial effects of different lyase are evaluated. Finally, the lyase with the optimal sterilization effect is screened out, and a new technical means is provided for prevention and treatment of equine adenitis.

Description

Technical Field

[0001] The present invention relates to a Streptococcus equi subsp. equi prophage lyase, and also to a preparation method of the lyase and its application in the treatment of strangles. The present invention belongs to the technical field of veterinary drugs. Background Art

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

[0003] Phages are a general term for viruses that infect microorganisms such as bacteria, fungi, algae, actinomycetes or spirochetes, and are the most abundant biological entities on Earth. The life cycle of phages is mainly divided into two types: the lytic cycle and the lysogenic cycle. Endolysin is a class of hydrolases encoded by phage or prophage genomes, which can hydrolyze the bacterial cell wall in the later stage of the phage lytic cycle or when the prophage is activated and enters the later stage of the lytic cycle, so that the progeny phages are released into the external environment. Since endolysin can efficiently lyse the peptidoglycan structure of the bacterial cell wall and has high specificity for host bacteria, it is considered a potential alternative to antibacterial drugs. Compared with traditional antibiotics, endolysin has advantages such as a unique mechanism of action, being less likely to induce drug resistance, highly efficient bactericidal activity, and having less impact on the normal flora of the host.

[0004] Currently, there has been no large-scale or systematic report on the research of endolysins against S.equi . The possible reasons are that there is less application research in this field, and it is also related to the relatively high research cost of using horses as large animal models. The results of a small number of in vitro studies show that some streptococcal endolysins have significant bactericidal activity against a variety of pathogenic streptococci, suggesting their potential universality against S.equi . However, further research is still needed for S.equiScreen for specific virulence characteristics to optimize suitable lytic enzymes and conduct safety and efficacy tests in horses or alternative animal models. Theoretically, lytic enzymes can play a role in the following aspects: ① Preventive application: Regularly spray lytic enzyme preparations in the environment of horse farms, racetracks, or equestrian clubs to reduce S.equi the contamination level and reduce the risk of environmental transmission. ② Early treatment: Directly deliver lytic enzymes through nebulization or nasal spray in the early stage of strangles (such as when mild upper respiratory tract infection symptoms appear) to inhibit pathogen colonization and reduce the spread of infection. ③ Adjuvant treatment: For cases with abscesses already formed, lytic enzymes can be applied locally after incision and drainage of the abscess to kill the remaining S.equi , reduce the risk of recurrence, and reduce the use of antibiotics. ④ Combined immunization: Combined with vaccination, lytic enzymes can be used to control clinical symptoms, enable the immune system to better respond to infections, and at the same time reduce the carrier state of horses and reduce the continuous spread of the disease.

[0005] In the present invention, by aligning the prophage lytic enzyme gene in the S.equi genomic sequence, designing specific primers, amplifying and cloning the lytic enzyme gene in the Streptococcus equi strain preserved in the laboratory, performing recombinant expression, and evaluating the antibacterial effects of different lytic enzymes. Finally, the lytic enzyme with the best bactericidal effect was screened out, providing a new technical means for the prevention and treatment of strangles. Summary of the Invention

[0006] The object of the present invention is to provide a prophage lytic enzyme of Streptococcus equi subsp. equi, its preparation method and application.

[0007] In order to achieve the above object, the present invention adopts the following technical means: A prophage lytic enzyme of Streptococcus equi subsp. equi according to the present invention, the amino acid sequence of the lytic enzyme is as shown in SEQ ID NO.29.

[0008] The polynucleotide encoding the prophage lytic enzyme of Streptococcus equi subsp. equi, the expression vector containing the polynucleotide, and the host bacterium containing the expression vector are also within the protection scope of the present invention.

[0009] Among them, preferably, the sequence of the polynucleotide is as shown in SEQ ID NO.30.

[0010] Among them, preferably, the expression vector is the pET-28a vector containing the polynucleotide.

[0011] Among them, preferably, the host bacterium is E. coli BL21(DE3).

[0012] Furthermore, the present invention also provides the use of the Streptococcus equi subsp. equi prophage lyase in the preparation of reagents or drugs against Streptococcus equi subsp. equi and Streptococcus equi subsp. zooepidemicus, as well as the use of the Streptococcus equi subsp. equi prophage lyase in the preparation of drugs for the treatment of strangles.

[0013] Moreover, the present invention also provides a biological antibacterial agent for the prevention and treatment of strangles, and the antibacterial agent contains the Streptococcus equi subsp. equi prophage lyase.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention compares the prophage lyase genes in the S.equi genomic sequences and evaluates the antibacterial effects of different lyases. Finally, a lyase with the best bactericidal effect is screened out and named LysHLJ1-9. In vitro experiments show that LysHLJ1-9 has the broadest lytic activity, can lyse 20 strains of Streptococcus equi and 2 strains of Streptococcus dysgalactiae subsp. equisimilis, has no effect on Escherichia coli, and has good targeting. LysHLJ1-9 can reduce the number of SD2018 strains by about 1 lg within 1 hour, and its bactericidal activity has an obvious dose-dependence. By subculturing the remaining bacteria after treating the SD2018 strain with LysHLJ1-9 for 12 consecutive generations, no drug-resistant strains were observed. Biochemical property analysis shows that LysHLJ1-9 can maintain stable activity at 4 °C to 42 °C and pH 3 to 9, with the optimal pH of 7, showing good environmental stability. Compared with penicillin, LysHLJ1-9 has a stronger ability to destroy bacterial biofilms. In vivo experiments (mouse acute bacteremia model) show that LysHLJ1-9 can achieve 100% survival rate when injected at a dose of 1500 μg / animal. The treatment can significantly reduce the bacterial load in the blood and organs such as the liver, spleen, lung, and kidney (the maximum reduction is up to 3 lg). Pathological tissue sections show that LysHLJ1-9 can effectively relieve the inflammatory response and tissue damage. No toxic and side effects were observed after a single injection of 2000 μg of the lyase, showing good biological safety.

[0015] In summary, the present invention provides a lyase LysHLJ1-9 derived from S.equi a prophage. The lyase LysHLJ1-9 has a good antibacterial spectrum, environmental stability, and in vivo protective efficacy. The present invention provides a new technical means for the prevention and treatment of strangles. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Amplification of the prophage lyase; Among them, A-H are the electrophoresis maps amplified using primers Lys1–Lys8; lane 1 is the template-free control (H2O), and 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 the DNA molecular weight standard; Figure 2 is the heat map of the similarity of different lyase protein sequences; Figure 3 is the amplification of pET-28a and the lyase gene; 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; Figure 4 is the SDS-PAGE analysis of the lyase expression product; 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 the pET-28a empty vector, the bacterial liquid before induction, the whole bacteria after induction, the precipitate, and the supernatant in sequence; Figure 5 is the SDS-PAGE analysis of the purified lyase product; Among them, 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; Figure 6 is the lysis activity of different lyases in a liquid environment; Among them, A: lysis activity of lyase LysHLJ1-9; B: lysis activity of lyase LysHLJ1-18; C: lysis activity of lyase LysHLJ3-11; D: lysis activity of lyase LysHLJ4-11; E: lysis activity of lyase LysHLJ5-13; F: lysis activity of lyase LysHLJ6-11; Figure 7 is the OD of the bactericidal effect of different lyases on SD2018 600nm measurement; Figure 8 For the in vitro bactericidal activity of lyase LysHLJ1-9 against SD2018; Figure 9 For the resistance of SD2018 to LysHLJ1-9; Figure 10 For the biochemical characteristics of LysHLJ1-9; 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; Figure 11 For the lysis spectrum of LysHLJ1-9; Figure 12 For the effect of LysHLJ1-9 on bacterial biofilms; Ns: No significant difference; ****: p < 0.0001; Figure 13 For LysHLJ1-9 to treat murine bacteremia caused by SD2018; Among them, A: Determination of the minimum lethal dose of SD2018; B: Determination of the minimum therapeutic dose of lyase LysHLJ1-9; C: Safety determination of lyase LysHLJ1-9; D: Bacterial load in murine blood; E: Bacterial load in murine internal organs, where (a) liver, (b) lung, (c) kidney, (d) spleen; ***: p < 0.001; Figure 14 For histopathological analysis of murine lung and liver tissues; Figure 15 For histopathological analysis of murine spleen and kidney tissues. Specific embodiments

[0017] The present invention is further illustrated by the following examples, and its purpose is only to better understand the research content of the present invention rather than to limit the protection scope of the present invention. Unless otherwise specified in the following examples, all are conventional experimental methods and operation steps in the art.

[0018] Example 1 Screening and expression of lyase 1 Experimental materials 1.1 Bacterial strains and plasmids Twenty Streptococcus equi strains used in this experiment (including 10 Streptococcus equi subsp. equi strains and 10 Streptococcus equi subsp. zooepidemicus strains) were isolated and preserved by our laboratory (Table 1). The prokaryotic expression vector pET-28a was preserved by our laboratory, and the competent cells of Escherichia coli DH5α and Escherichia coli BL21(DE3) were all purchased from Tsingke Biotechnology Co., Ltd.

[0019] ; 1.2 Main reagents ; 1.3 Solution preparation Todd-Hewitt broth medium (Todd-Hewitt broth, THB): Take 800 mL of distilled water and place it in a beaker. Add 36.4 g of THB medium, stir evenly, make up the volume to 1 L, then autoclave at 115 °C for 30 min, and store it in the dark at 4 °C; The 5×SDS-Page Sample Loading Buffer, LB medium, and LB solid medium containing kanamycin used in this experiment were all prepared in this laboratory.

[0020] 2 Experimental methods 2.1 Acquisition and screening of prophage lyase In this study, bioinformatics methods were used to obtain the complete genome sequence of " Streptococcus equi subsp equi " from the NCBI GenBank database (https: / / www.ncbi.nlm.nih.gov / genbank / ). By referring to the genome annotation information, gene sequences that might encode lyase were screened. The specific method is as follows: First, search in the NCBI genome database with the keyword " Streptococcus equi subsp equi AND completegenome", and download the annotated bacterial genome sequences (GenBank format,.gbff). Use SnapGene software (GSL Biotech, USA) to open the genome sequence file, and search for genes annotated as containing "lysin", "CHAP domain", or "amidase domain" in the genome annotation information as candidate lyase genes for preliminary screening. According to the results of functional annotation and domain analysis, select gene sequences containing clear lytic functional domains (such as CHAP domain or Amidase-2 domain), and save their corresponding nucleotide sequences (FASTA format) as candidate lyase genes for subsequent primer design and experimental verification.

[0021] The functional domain analysis of the obtained candidate lyase protein sequences was performed using the UniProt database (https: / / www.uniprot.org / ) to ensure that the target gene contains a typical cell wall hydrolysis domain. Subsequently, the Primer-BLAST tool (https: / / www.ncbi.nlm.nih.gov / tools / primer-blast / ) was used to design specific primers for PCR amplification, and the specific primer sequences are shown in Table 3.

[0022] ; 2.2 Extraction of genomic DNA of Streptococcus equi subsp. equi For the Streptococcus equi subsp. equi strain preserved in the laboratory, after taking it out from the -80 °C glycerol strain bank, it was revived and inoculated onto a Columbia blood agar plate, and cultured in an inverted position at 37 °C for 18 - 24 h. The single colony after culture was inoculated into 5 mL of THB medium and cultured at 37 °C and 200 r / m until the logarithmic growth phase (OD 600nm was approximately 0.6 - 0.8). Take 1 - 2 mL of the above-mentioned cultured bacterial liquid, centrifuge at 12000 r / m for 2 min at 4 °C, discard the supernatant, and collect the bacterial cell precipitate. The genomic DNA was extracted using the TIANGEN bacterial genomic DNA extraction kit according to the instructions. The concentration and purity of the obtained genomic DNA were measured using a visible light spectrophotometer and stored at -20 °C for later use.

[0023] 2.3 PCR amplification of the lyase gene Using the genomic DNA of the Streptococcus equi subsp. equi strain extracted as a template, the specific primers designed and synthesized by previous bioinformatics analysis were used to amplify the target lyase gene. The primers were synthesized by Ruibo Xingke (Harbin) Biotechnology Co., Ltd., and the primer sequences are the same as those in Table 3.

[0024] The high-fidelity KOD high-fidelity enzyme was used for the amplification reaction. The PCR reaction system was 50 μL, and the components were as follows: ; ; The PCR products were subjected to 1% agarose gel electrophoresis. The size of the amplified product bands was observed and recorded under a gel imaging system to verify the amplification specificity. The gel with specific bands was cut, and the target fragment was purified and recovered using the FastPure Gel DNA Extraction Mini Kit gel recovery kit according to the steps of the kit instructions. The recovered product was sent to Jilin Kumei Biotechnology Co., Ltd. for sequencing.

[0025] 2.4 Recombinant expression of the lyase 2.4.1 Construction of Plasmid Specific primers for homologous recombination were designed according to the pET-28a vector sequence and the target lyase gene sequence. Fragments homologous to the vector sequence (usually 15 - 20 bp in length) were introduced at the 5' ends of the primers to achieve efficient homologous recombination. The primers were synthesized by Ruibo Xingke (Harbin) Biotechnology Co., Ltd. The primer sequences are shown in Table 6, and the underlined parts are the homologous arms of the vector pET-28a. The PCR reaction system and reaction program are the same as those in Tables 4 and 5.

[0026] ; 1 μL of methylation-sensitive restriction endonuclease was added to the amplified linearized vector DpnⅠ , and incubated in a water bath at 37 °C for 45 min to remove the template plasmid. The PCR products were subjected to 1% agarose gel electrophoresis, and the size of the amplified product bands was observed under a gel imaging system to verify the amplification specificity. The gel with specific bands was cut, and the target fragment was purified and recovered using the FastPure Gel DNA Extraction Mini Kit according to the instructions.

[0027] The lyase gene fragment amplified by PCR and purified was subjected to homologous recombination 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; lyase gene fragment (PCR product, 100 ng): 1 μL; MonClone™ Single Assembly Cloning Mix: 5 μL; ddH2O: made up to 10 μL. Reaction conditions: react at 50 °C for 30 min, and immediately transform after a brief placement on ice.

[0028] 2.4.2 Transformation and Identification of Recombinant Plasmid The above recombinant reaction product was added to 50 μL of Escherichia coli DH5α competent cells, incubated on ice for 15 min, heat-shocked at 42 °C for 60 s, and immediately incubated on ice for 2 min. 900 μL of sterile LB liquid medium was added to the competent cells, and the mixture was shaken at 37 °C and 180 r / m for 1 h. Then, it was centrifuged at 6000 r / m for 1 min to collect the bacteria, which were resuspended in 30 μL of LB liquid medium. The bacterial suspension was spread on LB solid medium containing kanamycin and incubated inverted at 37 °C for 12 - 16 h. Single colonies were selected and added to LB medium containing kanamycin, and shaken at 37 °C and 180 r / m for 5 h. The plasmid was extracted from the bacterial suspension using the Invitrogen plasmid extraction kit according to the instructions and then sent to Jilin Kumei Biotechnology Co., Ltd. for sequencing.

[0029] 2.4.3 Expression and purification of recombinant protein The recombinant plasmid confirmed to be correct by sequencing was transformed into E. coli BL21(DE3) for protein expression. A positive single colony was selected and inoculated into LB liquid medium containing kanamycin, and shaken at 37 °C and 180 r / m for 12 h as a seed solution for standby. The next day, the seed solution was diluted 1:100 and inoculated into fresh LB medium, and cultured continuously at 37 °C and 180 r / m until the OD 600nm reached 0.6 - 0.8.

[0030] When the bacterial suspension reached the logarithmic growth phase, IPTG was added for induction at a final concentration of 0.5 mM, and the culture system was transferred to 16 °C and 130 r / m for induction for 12 h. After the induction, the bacteria were collected by centrifugation at 8000 r / m for 10 min at 4 °C and resuspended in PBS with pH 7.4. The resuspended bacteria were sonicated under ice bath conditions (power 39%, sonication for 5 s, intermittent for 6 s, cumulative sonication for 10 min), and centrifuged at 12000 r / m for 10 min. The supernatant and precipitate were collected and used for subsequent SDS-PAGE analysis of the expression.

[0031] Protein purification was carried out by His-tag affinity chromatography, and the specific operation was carried out according to the instructions of the Beyotime His-tag protein purification kit.

[0032] 2.4.4 Dialysis of purified protein and determination of protein concentration The purified lysozyme protein was dialyzed to remove impurities and small molecule interferents in the buffer. The purified protein solution was placed in a dialysis bag (molecular weight cut-off 10 kDa), and sterile PBS buffer (pH = 7) was used as the dialysis solution. Dialysis was carried out at 4 °C. The initial volume of the dialysis solution was 200 times that of the sample volume, and the dialysis solution was changed every 2 h for 3 consecutive times. After dialysis, the protein solution was collected and centrifuged at 12,000 r / m for 10 min at 4 °C, and the supernatant was taken for subsequent experiments. The purified protein samples were aliquoted into sterile EP tubes and stored at -80 °C for subsequent antibacterial activity detection experiments.

[0033] The protein concentration was determined by the BCA method. The Pierce™ BCA Protein Assay Kit was used according to the instructions, and all samples were set with 3 replicates.

[0034] 3 Experimental results 3.1 Screening and acquisition of prophage lysozymes Based on 26 publicly available genomes of Streptococcus equi subsp. equi in the NCBI database, the whole genome-encoded proteins were preliminarily annotated and screened in this study. A total of 30 protein sequences with lysozyme potential were screened. The genome of Streptococcus equi subsp. equi was amplified by PCR using 8 pairs of designed primers. Specific bands could be amplified with primers Lys1, Lys3, Lys4, Lys5, and Lys6, while no specific bands were amplified with primers Lys2, Lys7, and Lys8. The amplification results were as Figure 1 . By comparing the sequencing results of the specific bands, a total of 6 different lysozyme gene sequences were obtained. These 6 lysozymes were named LysHLJ1-9, LysHLJ1-18, LysHLJ3-11, LysHLJ4-11, LysHLJ5-13, and LysHLJ6-11, respectively. The lysozyme source strains / primers and their lengths are shown in Table 7.

[0035] ; The obtained lyases (LysHLJ1-9, LysHLJ1-18, LysHLJ3-11, LysHLJ4-11, LysHLJ5-13 and LysHLJ6-11) were aligned by NCBI BLASTP, and similarity analysis was performed with the published streptococcal lyases (including Ply30, Ply5218, Ply7917, PlyS2, PlyS59, LysFL1 and Ply1228) using Jalview 2.11.4.1 software. Visual analysis was carried out using the Python programming language, and seaborn and matplotlib libraries were used to draw a similarity heatmap to intuitively show the sequence identity levels among different lyases. The NCBI BLASTP results showed that LysHLJ1-9, LysHLJ1-18, LysHLJ3-11, and LysHLJ4-11 had 88% homology with lysin ( Streptococcus phageJavan158), but Streptococcus the function of the lysin of phage Javan158 has not been reported in the relevant literature; through BLASTP analysis of LysHLJ5-13 and LysHLJ6-11, it was found that neither of these two lyases showed significant homology with known lyase proteins in the public database, and only coding regions with a certain sequence identity were retrieved in the partial complete genomic sequences of S.equi .

[0036] The similarity heatmap is as Figure 2 shown. The sequence identities of LysHLJ1-9, LysHLJ1-18 and LysHLJ3-11 are extremely high, being 98.77%, 95.06% and 94.81% respectively. The similarity between LysHLJ4-11 and LysHLJ1-9, LysHLJ1-18, LysHLJ3-11 is about 88%, while the similarity between LysHLJ5-13 and LysHLJ6-11 is 89.6%, and their similarities with the first four lyases are all relatively low, being 15% - 17.2%. The sequence identities between the six amplified lyases and the published streptococcal lyases are generally low, ranging from 12% to 67%. The above information indicates that six new lyases were discovered in this study.

[0037] 3.2 Construction of recombinant expression plasmids of lyases To further verify the biological activity of the lyase, in this study, the six screened lyase genes were constructed into the prokaryotic expression vector pET-28a. Using homologous recombination technology, the lyase coding sequence with a His tag was successfully inserted into the expression vector. Fragments LysHLJ1-9, LysHLJ1-18, LysHLJ3-11, LysHLJ4-11, LysHLJ5-13, and LysHLJ6-11 were successfully amplified using primers Lys1, Lys3, Lys4, Lys5, and Lys6 ( Figure 3 ). Sequencing identification showed that the vectors 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 were successfully constructed.

[0038] 3.3 Recombinant expression and purification of lyase 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, which were successfully transformed into BL21(DE3), were induced for expression. The bacterial cultures before and after induction, the supernatant and precipitate after ultrasonic disruption were collected and subjected to SDS-PAGE electrophoresis. The results are shown in Figure 4 . Specific 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 after induction of LysHLJ1-9, LysHLJ1-18, LysHLJ3-11, LysHLJ4-11, pET-28a-LysHLJ5-13, and pET-28a-LysHLJ6-11, indicating successful expression of the recombinant proteins.

[0039] To further verify the expression and purification effects of the recombinant proteins, the supernatant of the sonicated expression strain was purified, and the purified products were analyzed by SDS-PAGE. The results are shown in Figure 5 . The results showed that there were few impurity bands in each recombinant lyase protein, indicating that each recombinant protein could be successfully expressed and highly pure soluble proteins could be obtained by His-tag affinity purification. 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.

[0040] Example 2 Study on the in vitro bactericidal activity of lyase 1 Experimental materials 1.1 Bacterial strains Two strains of Streptococcus dysgalactiae subsp. equisimilis ( Streptococcus dysgalactiae subspecies equisimilis , SDSE) were isolated and preserved by this laboratory. The other bacterial strains required for the experiments were the same as those in Example 1.

[0041] 1.2 Preparation of main reagents and solutions BHI medium (Oxoid). The other main reagents were the same as those in Example 1.

[0042] Todd-Hewitt broth medium (Todd-Hewitt broth, THB): Place 800 mL of distilled water in a beaker, add 36.4 g of THB medium, stir evenly, make up the volume to 1 L, then autoclave at 115 °C for 30 min, and store in the dark at 4 °C; 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, dissolve them in 800 mL of ultrapure water, stir well, adjust the pH to 7.4 with 1 M NaOH or 1 M HCl, and finally make up the volume to 1000 mL. The solution was autoclaved at 121 °C for 15 min and stored at 4 °C for later use.

[0043] BHI solid medium: Weigh 37 g of BHI medium powder and 15 g of agar, add them to 1 L of ultrapure water, stir to dissolve completely, and adjust the pH to 7.4. Make up the volume of the solution to 1 L and autoclave at 121 °C for 15 min. After sterilization, wait for the medium to cool to about 50 °C, pour it into sterile petri dishes under aseptic conditions, let it stand until solidified, and store at 4 °C for later use.

[0044] 2 Experimental methods 2.1 Comparison of antibacterial spectra of different lytic enzymes Twenty strains of Streptococcus equi (including 10 strains of subsp. equi and 10 strains of subsp. zooepidemicus) were selected to determine the lysis spectra of lytic enzymes. The above strains were cultured to the logarithmic growth phase (OD 600nm = 0.6 - 0.8), 200 μL of the bacterial solution was evenly spread on the BHI solid medium, 10 μL of the purified lytic enzyme protein (200 μg) was added dropwise to the surface of the plate. After the liquid dried, the plate was inverted and cultured in an incubator at 37 °C for 12 h. By observing whether an inhibition zone was formed, the target host range of the lytic enzyme was determined, and the lytic enzyme with the broadest antibacterial spectrum was screened out.

[0045] 2.2 Lytic activities of different lytic enzymes in liquid environment Using Streptococcus equi subsp. zooepidemicus SD2018 (Isolation and Identification of the Pathogen of Donkey Glanders 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) as the indicator bacterium, pick a single colony of SD2018 and inoculate it into THB medium, and culture it overnight at 37 °C and 180 r / m. Inoculate the fully resuscitated bacterial solution into fresh THB medium at a ratio of 1:100, and culture it at 37 °C and 180 r / m until the logarithmic growth phase (OD 600nm = 0.6 - 0.8). Centrifuge at 4 °C and 6000 r / m for 2 min, discard the supernatant, wash the bacterial cells three times with sterile PBS solution and then resuspend them to OD 600nm ≈ 1.0. Add 1 mL of the resuspended bacterial solution into 4 sterile test tubes respectively. After diluting the purified lyase protein with sterile PBS buffer, add it into the 4 test tubes respectively, so that the final concentrations of the lyase protein are 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. Place the 5 test tubes in a shaker at 37 °C and 180 r / m, and observe the bactericidal effect of the lyase protein on the bacterial solution within 1 h to screen out the lyase with the best lysis activity.

[0046] 2.3 Bactericidal Activity of Lyase LysHLJ1-9 against SD2018 After culturing Streptococcus equi subsp. zooepidemicus SD2018 to the logarithmic growth phase (OD 600nm = 0.6 - 0.8), wash it three times with sterile PBS buffer, and divide the bacterial solution into 5 groups. Add the lyase protein with the best effect in 2.1 and 2.2 into 4 groups of bacterial solutions respectively in the experimental groups, so that their final concentrations are 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 all samples in a water bath at 37 °C for 1 h, and take samples every 15 min. Subsequently, use the gradient dilution - plate spreading method for colony counting: Dilute the bacterial solution in a 10-fold serial gradient, take the diluted solution and spread it on Columbia agar plates, and count after culturing at 37 °C for 24 h. Evaluate the in vitro bactericidal activity of the lyase protein.

[0047] 2.4 Resistance Analysis of SD2018 to Lyase LysHLJ1-9 Using the clinical isolate Streptococcus equi subsp. zooepidemicus SD2018 as the indicator bacterium, culture it to the logarithmic growth phase (OD 600nmAfter (OD = 0.6 - 0.8), 200 μL of the bacterial solution was evenly spread on a Columbia blood agar plate. After the bacterial solution dried naturally, 10 μL of the lysozyme protein with a concentration of 200 μg / mL was added dropwise onto the surface of the plate. The plate was incubated in an incubator at 37 °C for 12 h. Single colonies were picked from the edge of the inhibition zone and inoculated into THB medium, and cultured overnight at 37 °C. The lysis effect of the lysozyme protein was detected according to the steps in 2.3. This process was subcultured 7 times continuously, and then an additional 5 subcultures were carried out, and the lysis activity of the lysozyme protein against each generation of SD2018 strains was detected.

[0048] 2.5 Determination of the antibacterial spectrum of lysozyme LysHLJ1-9 Twenty Streptococcus equi strains were used as indicator bacteria, cultured to the logarithmic growth phase according to the steps in 2.2, washed three times with sterile PBS buffer and resuspended, and the initial colony count was determined. Subsequently, lysozyme LysHLJ1-9 was added to the bacterial solution to make its final concentration reach 200 μg / mL, and an equal volume of sterile PBS buffer solution was added to the control group. All samples were incubated in a water bath at 37 °C for 1 h, and then colony counting was carried out. The difference between the colony count after incubation and the initial colony count was used to evaluate the lysis activity of the lysozyme against different strains.

[0049] 2.6 Determination of the biochemical properties of lysozyme LysHLJ1-9 2.6.1 Effect of temperature on the activity of lysozyme LysHLJ1-9 The SD2018 strain was cultured according to the method in 2.2, the medium was washed with sterile PBS buffer and resuspended, the resuspended bacterial solution was mixed with the lysozyme to make the final concentration of the lysozyme 200 μg / mL, and an equal volume of sterile PBS buffer was added to the control group. The samples were incubated at 4 °C, 16 °C, 25 °C, 37 °C and 42 °C for 1 h respectively, and then the gradient dilution-plate spreading method was used for colony counting. By calculating the difference in colony counts before and after incubation, the effect of different temperature conditions on the lysozyme activity was evaluated.

[0050] 2.6.2 Effect of pH on the activity of lysozyme LysHLJ1-9 The SD2018 strain was cultured until OD 600nm= 0.6 - 0.8, and then centrifuge at 4 °C and 6000 r / m for 2 min, and wash three times with sterile PBS buffer. Set 7 pH gradients, and use 1 M HCl solution and 1 M NaOH solution to adjust the pH value of sterile PBS buffer to 3, 4, 5, 6, 7, 8, and 9 respectively. Dilute the lysozyme with sterile PBS buffer of the corresponding pH value to make its final concentration reach 400 µg / mL. At the same time, resuspend the washed bacterial cells in sterile PBS buffer of the above different pH values, and perform colony counting. Mix the lysozyme solutions of different pH values with the resuspended bacterial solutions of the corresponding pH according to a volume ratio of 1:1, and add an equal volume of sterile PBS buffer of the same pH to the control group. Incubate all samples in a 37 °C water bath for 1 h, and then perform colony counting. By calculating the change in the number of colonies before and after incubation, evaluate the effect of different pH conditions on the activity of lysozyme.

[0051] 2.7 Determination of the lysis effect of lysozyme LysHLJ1-9 on clinical isolates of Streptococcus equi Select 20 strains of Streptococcus equi (including 10 strains of S. equi subsp. equi and 10 strains of S. equi subsp. zooepidemicus) and 2 strains of S. dysgalactiae subsp. equisimilis to determine the lysis spectrum of the lysozyme. Culture the above strains to the logarithmic growth phase (OD 600nm = 0.6 - 0.8), take 1 mL of the bacterial solution, centrifuge at 4 °C and 6000 r / m for 5 min, discard the supernatant, wash 3 times with sterile PBS (pH = 7.4) buffer, and resuspend the bacterial cells. Add lysozyme to make its final concentration reach 100 μg / mL, and incubate at 37 °C for 1 h. Then, use the gradient dilution-plate coating method to determine the change in the number of colonies before and after treatment, and analyze the bactericidal effect of the lysozyme on different strains.

[0052] 2.8 Effect of lysozyme LysHLJ1-9 on bacterial biofilms Culture the SD2018 strain at 37 °C and 180 r / m for 12 h. The next day, dilute the bacterial solution 1:100 and inoculate it into a 96-well plate. Add 200 μL of the diluted bacterial solution to each well, and statically culture at 37 °C for 24 h to form a biofilm. After the culture is completed, discard the culture solution in the wells, gently wash each well 3 times with sterile PBS buffer to remove unadhered bacteria. Add 200 μL of a lysozyme solution with a final concentration of 200 μg / mL to the experimental group, and add an equal volume of sterile PBS buffer to the control group. Add a penicillin solution with a final concentration of 200 μg / mL to the penicillin group as a control group for comparison. Set 3 replicate wells for each group, and incubate the samples at 37 °C for 1 h to detect the effect of lysozyme and penicillin on biofilms.

[0053] After incubation, discard the solution in the wells, wash each well 3 times with PBS buffer, and add 200 μL of 0.1% crystal violet solution to stain for 15 min. After staining, discard the staining solution, wash 3 times with PBS, add 200 μL of 95% ethanol to each well, and let stand at room temperature for 30 min to dissolve the crystal violet in the biofilm, and measure the absorbance at 570 nm (OD 570nm ), to reflect the formation of biofilm.

[0054] 2.9 Data analysis All statistical analyses of the data were performed using GraphPad Prism 8.0 software (GraphPad Software Inc., San Diego, CA, USA), and the data were analyzed by one-way analysis of variance (ANOVA). A P value < 0.05 was considered statistically significant. Error bars represent the standard error of the mean.

[0055] 3 Experimental results 3.1 Antibacterial spectra of different lytic enzymes The lysis range of lytic enzymes against 20 Streptococcus equi strains (including 10 S. equi subsp. equi and 10 S. equi subsp. zooepidemicus) and 2 S. dysgalactiae subsp. equisimilis strains was determined by observing the formation of inhibition zones of different lytic enzymes (200 μg) on BHI solid plates. LysHLJ1-9 and LysHLJ1-18 were positive (+) against all tested strains; LysHLJ3-11 had lytic activity against all tested Streptococcus equi strains (including S. equi subsp. equi and S. equi subsp. zooepidemicus), but was ineffective against the tested S. dysgalactiae subsp. equisimilis and Escherichia coli strains; LysHLJ4-11 had lytic effects on 17 Streptococcus equi strains except SE-4, SE-5, and SZ-4; LysHLJ5-13 was effective against 19 Streptococcus equi strains except SE-7, and the activity range of LysHLJ6-11 was the narrowest among the other 5 lytic enzymes, only effective against 4 S.equi and 5 S.zoo strains. LysHLJ3-11, LysHLJ4-11, LysHLJ5-13, and LysHLJ6-11 were all ineffective against the tested S. dysgalactiae subsp. equisimilis, and all lytic enzymes had no lytic effect on Escherichia coli DH5α and Escherichia coli BL21 (DE3). The lysis of different strains is shown in Table 8.

[0056] ; Note: 1: S. equi subsp. equi; 2: S. equi subsp. zooepidemicus; 3: S. dysgalactiae subsp. equisimilis; 4: Escherichia coli; +: plaque formation; -: no plaque formation 3.2 Lytic activity of different lytic enzymes in liquid environment To further evaluate the lytic ability of each lyase, in this study, SD2018 was used as the indicator bacterium to detect the effects of 6 lyases at different concentrations (50 μg / mL, 100 μg / mL, 150 μg / mL, 200 μg / mL) on the transparency of the bacterial solution. The results are as Figure 6 and Figure 7 shown. The degree of bacterial lysis was reflected by the degree of clarification of the bacterial solution and the OD 600nm value.

[0057] In all control groups, the bacterial solution remained turbid. LysHLJ1-9 ( Figure 6 A) showed obvious lysis at 50 μg / mL. As the concentration increased, the bacterial solution gradually clarified and was basically completely clarified at 200 μg / mL. In contrast, LysHLJ1-18 ( Figure 6 B) could cause a certain degree of clarification at higher concentrations, but the lysis intensity was significantly weaker than that of LysHLJ1-9. LysHLJ3-11 and LysHLJ4-11 ( Figure 6 C, D) had weak clarification effects on the bacterial solution at all concentrations, and only slight changes were observed at 200 μg / mL, indicating limited lysis activity.

[0058] LysHLJ5-13 ( Figure 6 E) had almost no obvious lysis effect on the bacterial solution at each concentration, and there was no significant difference from the control group. LysHLJ6-11 ( Figure 6 F) had basically no effect at 50 μg / mL concentration, and a certain degree of transparency of the bacterial solution appeared at 200 μg / mL, but the overall lysis efficiency was low.

[0059] To evaluate the bactericidal effects of 6 recombinant lyases on SD2018, the bacterial solution was treated with different concentrations (50 μg / mL, 100 μg / mL, 150 μg / mL and 200 μg / mL), and the OD 600nm value was measured to reflect the bacterial growth. As Figure 7 shown, the LysHLJ1-9 and LysHLJ1-18 treatment groups showed concentration-dependent bactericidal effects at different concentrations. After treatment with LysHLJ1-9, the OD 600nm value decreased to 0.259 at 200 μg / mL; the OD 600nm value of the LysHLJ1-18 treatment group decreased to about 0.452.

[0060] The OD 600nm value changes of the LysHLJ3-11, LysHLJ4-11, LysHLJ5-10 and LysHLJ6-11 treatment groups were small, and overall remained at a relatively high level. At 200 μg / mL, the OD600nm The value still remained above 0.7. These results indicated that LysHLJ1-9 had stronger bactericidal activity compared with the other five lyases.

[0061] 3.3 Bactericidal activity of lyase LysHLJ1-9 against SD2018 in vitro To further evaluate the in vitro bactericidal ability of LysHLJ1-9 and its relationship with action time and concentration, in this study, the changes in the number of surviving bacteria after the action of the lyase were measured at different concentrations (50, 100, 150, and 200 μg / mL) and different time points (0 min, 15 min, 30 min, 45 min, and 60 min). As Figure 8 shown, LysHLJ1-9 showed certain bactericidal activity at different concentrations, and it was significantly dose-dependent. With the increase in the concentration of the lyase, the bactericidal effect was significantly enhanced. LysHLJ1-9 at 50 μg / mL, 100 μg / mL, and 150 μg / mL could reduce the colony count by approximately 0.35 lg, 0.54 lg, and 0.78 lg respectively within 60 min. LysHLJ1-9 at 200 μg / mL decreased the bacterial count from approximately 8.6 lg (CFU / mL) to approximately 7.6 lg (CFU / mL) within 60 min, and the bactericidal amplitude was approximately 1 lg.

[0062] 3.4 Resistance analysis of SD2018 to lyase LysHLJ1-9 To verify whether bacteria would develop resistance to the lyase, the strain SD2018 was passaged continuously for 12 generations, and whether SD2018 developed resistance to the lyase LysHLJ1-9 was analyzed in each generation. The results were as Figure 9 shown. There was no obvious change in the bactericidal effect of the lyase LysHLJ1-9 against SD2018 among different generations, indicating that within 12 generations, this strain did not develop resistance to LysHLJ1-9.

[0063] 3.5 Determination of biochemical characteristics of lyase LysHLJ1-9 3.5.1 Effect of temperature on the activity of lyase LysHLJ1-9 The lyase LysHLJ1-9 was incubated at 4 °C, 16 °C, 25 °C, 37 °C, and 42 °C for 1 h respectively, and the effect of different temperatures on the enzyme activity of LysHLJ1-9 was measured. The results were as Figure 10As shown in A, within the range of 4 °C to 42 °C, the activity of the lyase remained stable, and different temperatures had little effect on its bactericidal effect. Compared with 4 °C, 16 °C, 25 °C, and 37 °C, the lysis activity at 42 °C decreased slightly. Under the condition of 42 °C, LysHLJ1-9 could reduce the colony count by about 0.96 lg within 1 h.

[0064] 3.5.2 Effect of pH on the activity of lyase LysHLJ1-9 A total of 7 gradients of pH buffer were set. After acting under different pH conditions, the activity changes of lyase LysHLJ1-9 were detected by gradient dilution-plate coating method. The results are as Figure 10 shown in B. Within the pH range of 3-9, LysHLJ1-9 could maintain relatively stable bactericidal activity. The enzyme activity was the highest at pH 7, and the colony count of SD2018 decreased by about 1 lg. In the acidic environment with pH values of 3-6, the activity of the lyase was relatively low, but it could still maintain a certain bactericidal effect. Under the alkaline condition with pH value of 9, the activity of the lyase was similar to that in the neutral environment.

[0065] 3.6 Determination of the lysis effect of lyase LysHLJ1-9 on clinical isolates of streptococcus By selecting clinical isolates of streptococcus (including 10 strains S.equi, 10 strains S.zoo and 2 strains of SDSE) as Figure 11 , the overall lysis effect of lyase LysHLJ1-9 on S.equi strains was relatively good. LysHLJ1-9 had strong lysis ability on S.equi strains (HLJ2018, SD2018, SE-1 to SE-8), and the bacterial reduction rate was between 55% and 95%. Among S. zoo strains (SZ-1 to SZ-10), some strains (such as SZ-3, SZ-9, SZ-10) showed relatively high sensitivity, and the lysis rate reached more than 80%. While for other strains (such as SZ-1, SZ-6), the lysis rate was relatively low, only about 40%. For the two strains of SDSE (SDSE-1, SDSE-2), the lysis rate was also relatively high, both above 70%. The viable bacteria reduction ratio of 200 μg / mL protein was between 30% and 95%.

[0066] 3.7 Effect of lyase LysHLJ1-9 on bacterial biofilms The inhibitory effects of lyase LysHLJ1-9 and penicillin on bacterial biofilms were detected by crystal violet staining method, and the OD value was measured at 570 nm to evaluate the residual amount of biofilms. The results are as Figure 12 shown. The OD of the PBS treatment group570nm had the highest value, approximately 0.56; after treatment with 200 μg / mL penicillin, the OD 570nm value remained at approximately 0.51, and the OD value of the lyase LysHLJ1-9 treatment group decreased significantly. Compared with the PBS group, its OD 570nm value decreased by approximately 0.4. 570nm

[0067] Example 3 Evaluation of the in vivo antibacterial effect and safety of lyase LysHLJ1-9 1 Experimental materials 1.1 Bacterial strains and experimental animals The experimental animals were SPF-grade 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 subsp. zooepidemicus SD2018 preserved in the laboratory.

[0068] 1.2 Preparation of bacterial suspension The SD2018 glycerol bacteria preserved in the laboratory were streaked on Columbia blood agar plates and cultured at 37 °C for 12 h. Then, single colonies were picked and inoculated into THB liquid medium, and cultured with shaking at 37 °C and 180 r / min for 8 h. Subsequently, the culture solution was transferred to fresh THB medium at a volume ratio of 1:100 and continued to be cultured under the same conditions until the bacteria grew to the logarithmic phase. The viable bacteria count of the bacterial suspension at this stage was performed, and it was used as the infectious bacterial suspension for the subsequent experiment.

[0069] 2 Experimental methods 2.1 Establishment of a mouse model infected with SD2018 strain According to the method in 1.3, fresh SD2018 bacterial suspension in the logarithmic growth phase was prepared, and its concentration was measured by the method of spreading and coating on blood agar plates after dilution. According to the measured colony counts, different concentrations of infectious bacterial suspensions were prepared, set as five gradients of 1×10³, 1×10 4 、1×10 5 、1×10 6 and 1×10 7 CFU / 0.2 mL. Balb / C mice with the same sex and similar health status were selected and randomly divided into 6 groups, with 8 mice in each group. Except for one group as the control group, which was intraperitoneally injected with 0.2 mL of THB medium only, the other five groups were intraperitoneally injected with different concentrations (10 3 ~ 10 7 CFU / 0.2 mL) of the bacterial suspension. The mice were raised under unified feeding conditions, and their survival conditions were observed and recorded daily. The survival rate of each group was calculated to evaluate the minimum lethal dose (MLD) of the SD2018 strain to mice. ​

[0070] 2.2 Evaluation of the Therapeutic Effect of LysHLJ1-9 on Mice 2.2.1 Determination of the Therapeutic Dose of Lytic Enzyme LysHLJ1-9 for Mice The mice were randomly divided into 4 groups, with 8 mice in each group. A single intraperitoneal injection of SD2018 bacterial solution at a dose of 2×MLD was given. Within 7 hours after infection, mouse tail vein blood samples were collected every 1 hour, and the number of colonies in the blood was detected by the serial dilution method. When the number of detected colonies reached 1×10 4 CFU, it was considered that the mice had developed systemic infection, and then the treatment measures were initiated. The experimental groups were respectively given an intraperitoneal injection of 500 μg / mouse, 1000 μg / mouse, and 1500 μg / mouse of the lytic enzyme LysHLJ1-9, while the control group was injected with an equal volume of sterile PBS as a control. The treatment method was to inject on the opposite side of the peritoneum, and the survival of the mice was observed and recorded every day for 7 days. The lowest dose that could achieve 100% survival rate was determined as the minimum therapeutic dose of LysHLJ1-9.

[0071] 2.2.2 Determination of the Safety of Lytic Enzyme LysHLJ1-9 for Mice The mice were randomly divided into two groups, with 6 mice in each group. The experimental group of mice was given an intraperitoneal injection of 2000 μg LysHLJ1-9, and the control group was injected with an equal volume of sterile PBS. They were continuously observed for 7 days, and the health status of the mice was recorded and evaluated using a 5-0 scoring standard: a good health status was recorded as 5 points; disheveled hair and reduced activity were recorded as 4 points; lethargy and hunchback were recorded as 3 points; if there were secretions around the eyes, it was recorded as 2 points; a dying state was recorded as 1 point; death was recorded as 0 points. The health scores of each mouse were recorded and used for subsequent analysis.

[0072] 2.2.3 Determination of Bacterial Load in Mouse Blood The experimental mice were randomly divided into three groups: the PBS treatment group was inoculated with 2×MLD dose of SD2018 and injected with an equal volume of sterile PBS as treatment 1 hour after infection; the LysHLJ1-9 treatment group was inoculated with 2×MLD dose of SD2018 and treated with an intraperitoneal injection of 1500 μg of LysHLJ1-9 on the other side 1 h later; the control group of mice was injected with an equal volume of sterile PBS. At 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 12 h, 24 h, 36 h, and 48 h after infection, 3 mice were randomly selected from each group for tail vein blood collection. The collected blood samples were diluted with sterile PBS and then inoculated onto Columbia blood agar plates, and incubated upside down at 37 °C. By counting the colonies formed on the plates, the bacterial load in the blood was calculated, and the changing trends of the colony numbers in the treatment groups and the control group at each time point were further compared and analyzed.

[0073] 2.2.4 Determination of Bacterial Load in Major Organs of Mice At 1 h, 12 h, and 24 h after infection, three mice were randomly selected from each group, dissected, and liver, spleen, lung, and kidney tissues were isolated. Each tissue was placed in 1 mL of sterile PBS, ground thoroughly to make a homogenate, and serially diluted with sterile PBS. Subsequently, the diluted samples were spread on Columbia blood agar plates and incubated upside down at 37 °C. The number of colonies was counted to evaluate the changes in bacterial load in each organ at different time points, thereby determining the bactericidal effect of LysHLJ1-9 in vivo.

[0074] 2.2.5 Examination of Histopathological Changes in Vital Organs At 24 h and 48 h after mouse infection, three mice were randomly selected from each group. After dissection, liver, spleen, lung, and kidney tissues were taken out and fixed in 4% PFA fixative. Paraffin sections of each fixed organ were made, and pathological changes of each organ were observed after HE staining.

[0075] 2.3 Data Analysis All data were statistically analyzed using GraphPad Prism 8.0 software (GraphPad Software Inc., San Diego, CA, USA). The differences between groups were tested by one-way analysis of variance (ANOVA), and a P value less than 0.05 was considered statistically significant. The error bars in the figures represent the standard error of the mean.

[0076] 3 Experimental Results 3.1 Determination of the Minimum Lethal Dose To establish a Streptococcus equi subsp. zooepidemicus SD2018 infection model, different doses of bacterial suspension (1×10 3 CFU / mouse, 1×10 4 CFU / mouse, 1×10 5 CFU / mouse, 1×10 6 CFU / mouse, 1×10 7 CFU / mouse) were injected intraperitoneally to evaluate their lethality. The results showed that the survival rate of mice in the 1×10 3 CFU group was 50% within 7 days, all mice in the 1×10 4 CFU group died within 7 days, and all mice in the 1×10 5 CFU group died within 4 days. All mice in the 1×10 6 CFU and higher dose groups showed 100% death within 3 days ( Figure 13 A). All mice in the control group survived without any abnormal manifestations. Therefore, the MLD of the SD2018 strain in mice was determined to be 1.0×10 6 CFU / mouse.

[0077] 3.2 Determination of the therapeutic dose of lyase LysHLJ1-9 in mice To verify the protective effect of the lyase on the mouse infection model, 2×MLD (2×10 6 CFU / mouse) of the strain was injected intraperitoneally into mice to establish an acute bacteremia model. All mice without any treatment died within 48 h. At 1 h after infection, different doses of the lyase were given for treatment, and the results are shown in Figure 13 Figure B. Intraperitoneal injection of 1500 μg of the lyase could make 100% of the mice survive, while 1000 μg of the lyase made the survival rate of the mice 62.5%, and the survival rate decreased to 37.5% at a dose of 500 μg. Therefore, the minimum effective therapeutic dose of the lyase is 1500 μg / mouse, which can effectively improve the resistance of mice to bacteremia.

[0078] 3.3 Determination of the safety of lyase LysHLJ1-9 in mice To evaluate the potential toxic and side effects of lyase LysHLJ1-9, 2000 μg of lyase LysHLJ1-9 was injected intraperitoneally into mice once, and an equal amount of sterile PBS control group was set. The results are shown in Figure 13 Figure C. Within 7 days, all mice treated with 2000 μg of lyase LysHLJ1-9 injection survived, were in good condition, had normal food intake and drinking water, and there was no obvious abnormality compared with the normal group. This result indicates that lyase LysHLJ1-9 has good safety, and a single intraperitoneal injection of 2000 μg will not cause significant impact on the health of mice.

[0079] 3.4 Determination of bacterial load in mouse blood A mouse infection model was established by intraperitoneal injection of Streptococcus equi subsp. zooepidemicus SD2018. At 1 h after infection, the bacterial load in the blood reached about 10 4 CFU / mL. Subsequently, the infected mice were treated with lyase LysHLJ1-9 and PBS respectively, and the changes in the bacterial load in the blood of the mice at different time points were monitored. The results are shown in Figure 13 Figure D. From 2 h after infection, the number of colonies in the blood of the mice in the PBS group increased significantly to about 10 5 CFU / mL and remained at 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 stable downward trend, and decreased to 10 3Less than CFU / mL. Starting from 2 h, there was a highly significant difference between the LysHLJ1-9 treatment group and the PBS group (p < 0.001). The lyase LysHLJ1-9 could effectively reduce the peripheral blood bacterial load in the mouse SD2018 infection model, showing strong in vivo antibacterial activity.

[0080] 3.5 Determination of bacterial loads in important organs of mice At 1 h, 12 h, and 24 h after infection, liver, lung, kidney, and spleen tissues were collected respectively to detect the number of colonies. The results were as Figure 13 E. At 1 h, the bacterial loads in the organs of each group were similar. For example, in the liver, it was 4.92 ± 0.21 lg CFU / g, and in the lung tissue, it was 5.01 ± 0.15 lg CFU / g, indicating that there were no significant differences at the initial stage of infection. After 12 h, the bacterial load in the liver of the PBS group increased to 6.69 ± 0.18 lg CFU / g, while that in the LysHLJ1-9 group decreased to 4.51 ± 0.09 lg CFU / g; the bacterial load in the lungs of the PBS group was 5.67 ± 0.20 lg CFU / g, and that in the lyase group was 4.54 ± 0.12 lg CFU / g, with a significant difference (p < 0.01). By 24 h, the bacterial loads 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 the PBS group's 6.73 ± 0.14, 6.63 ± 0.13, 5.58 ± 0.10, and 6.65 ± 0.15 lg CFU / g, the decreases were 3, 2.9, 1.4, and 2.1 lg CFU / g respectively. The data showed that LysHLJ1-9 significantly inhibited the colonization and spread of bacteria in multiple organs.

[0081] 3.6 Histopathological changes in important organs At 24 h and 48 h after infection of SD2018-infected mice, the lungs, livers, spleens, and kidneys of the mice were collected respectively to make pathological tissue sections, and the pathological changes of the above-mentioned organs in the blank group, PBS treatment group, and LysHLJ1-9 treatment group were observed. The lyase LysHLJ1-9 had obvious improvement effects on the pathological damage of the liver, spleen, lung, and kidney of SD2018-infected mice. According to the observation results of the tissue pathological sections (such as Figure 14 and 15 ), the pathological changes of the liver, spleen, lung, and kidney were as follows: Lungs: In the PBS treatment group, obvious pathological changes were observed in the lungs of mice. The alveolar structure was significantly damaged, the alveolar septum was thickened with obvious inflammatory cell infiltration, and a large amount of red blood cells exudation was visible, indicating obvious hemorrhage and inflammatory reaction. In contrast, in the LysHLJ1-9 treatment groups (24 h and 48 h), the pathological changes in the lung tissues of mice were significantly improved. The alveolar structure gradually became clear, the infiltration of inflammatory cells and the exudation of red blood cells were significantly reduced, and the recovery in the 48 h group was more obvious, approaching the level of the normal group.

[0082] Spleen: In the PBS treatment group, obvious pathological manifestations appeared in the spleens of mice. The structure of white pulp and red pulp was blurred, extensive lymphocyte necrosis occurred, accompanied by severe congestion and hemorrhage. In contrast, in the LysHLJ1-9 treatment groups (24 h and 48 h), the inflammatory and necrotic lesions in the spleen tissues were significantly alleviated, the tissue structure gradually recovered. Especially at 48 h, the structure of white pulp and red pulp was clear, the number of lymphocytes increased significantly, and the histopathological state was significantly improved.

[0083] Liver: In the PBS group, significant pathological changes were shown in the liver tissues of mice. Obvious hepatocyte edema occurred, local cell lysis and necrosis were observed, and large and long eosinophilic necrosis foci were present in the liver parenchyma. In the LysHLJ1-9 treatment groups (24 h and 48 h), the liver tissue structure was significantly improved, the swelling of hepatocytes was reduced, and cell necrosis was significantly decreased. By 48 h, the liver tissue had basically returned to the normal state.

[0084] Kidney: In the PBS group, obvious pathological changes also occurred in the renal tissues of mice, including tubular epithelial cell edema, necrosis and interstitial vascular congestion. In the LysHLJ1-9 treatment groups (24 h and 48 h), the renal tissue structure was significantly improved, the interstitial vascular congestion was significantly reduced, and the degree of tubular lesions was significantly decreased, approaching the level of the normal group at 48 h.

Claims

1. A Streptococcus equi subsp. equi Streptococcus equi subsp. equi , S.equi ) prophage lyase, 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 lyase according to claim 1.

3. The polynucleotide according to claim 2, characterized in that The sequence of the polynucleotide is shown in 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, characterized in that The expression vector is the pET-28a vector containing the polynucleotide according to claim 2 or 3.

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

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

8. Use of the Streptococcus equi subsp. equi prophage lyase according to claim 1 in the preparation of a reagent or drug against Streptococcus equi subsp. equi and Streptococcus zooepidemicus Streptococcus equi subsp. Zooepidemicus , S.zoo ).

9. Use of the Streptococcus equi subsp. equi prophage lyase according to claim 1 in the preparation of a drug for preventing and treating strangles.

10. A biological antibacterial agent for preventing and treating strangles, characterized in that The biological antibacterial agent contains the Streptococcus equi subsp. equi prophage lyase according to claim 1.

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