Production and application of antibacterial protein targeting enterococcus casseliflavus

Abstract

The application discloses production and application of antibacterial proteins targeting Enterococcus casseliflavus. Specifically, the antibacterial proteins comprise an amino acid sequence as shown in any one of SEQ ID NO. 15-28 or an active fragment or an analog thereof. The application finds potential antibacterial proteins from the genome of bacteria or bacteriophages, which can target and inhibit Enterococcus casseliflavus. The antibacterial proteins with biological activity can be obtained through heterologous expression, which can target and eliminate Enterococcus casseliflavus, and further relieve and treat diseases caused by Enterococcus casseliflavus. The antibacterial proteins of the application can target and kill bacteria without destroying normal human symbiotic flora, and can avoid side effects caused by traditional antibiotic treatment.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and particularly relates to production and application of antibacterial protein targeting Enterococcus casseliflavus. BACKGROUND

[0002] Enterococcus casseliflavus is a gram-positive, facultative anaerobic bacteria, belonging to Enterococcus. Enterococcus casseliflavus is a common opportunistic pathogen in immunocompromised or chronically ill patients, and the most common form of infection is sepsis, usually associated with biliary tract and liver. The second most common form of Enterococcus casseliflavus infection is endophthalmitis, usually associated with trauma, especially in active young and middle-aged patients. Other infections of Enterococcus casseliflavus include infective endocarditis, meningitis, peritonitis and pyothorax, etc. Infectious diseases associated with Enterococcus casseliflavus: 1. Enterococcus casseliflavus can be transmitted through blood, causing bacteremia, especially in intensive care patients or immunocompromised individuals (such as cancer and organ transplant patients); 2. The enteric origin of Enterococcus casseliflavus makes it easy to cause peritonitis and other abdominal infections after gastrointestinal surgery or trauma; 3. There are occasional reports of heart valve infections, and infective endocarditis caused by Enterococcus casseliflavus usually involves native or prosthetic valves; 4. Enterococcus casseliflavus often causes urinary tract infections in the elderly and long-term catheter users, and these infections are often resistant to multiple antibiotics, increasing the complexity of treatment. In addition, Enterococcus casseliflavus is also a bacterial symbiont in widely distributed insects, which can degrade chlorantraniliprole insecticides by breaking amide bonds and dehalogenation, enhance the resistance of host insects to chlorantraniliprole insecticides, and thus make chlorantraniliprole insecticides ineffective.

[0003] Currently, the main treatment for Enterococcus casseliflavus infection is broad-spectrum antibiotics, but these methods have some obvious problems. The main problems of existing treatment technology: 1. Antibiotic resistance, Enterococcus casseliflavus gradually develops resistance to many commonly used antibiotics, especially vancomycin. This makes the effectiveness of broad-spectrum antibiotic therapy decrease, and is easy to lead to further spread of drug-resistant strains. During treatment, doctors often face the problem of limited choice of antibiotic species. 2. Intestinal microecological destruction, the use of antibiotics not only kills Enterococcus casseliflavus, but also affects the beneficial flora in the intestine, leading to intestinal microecological imbalance. The destruction of intestinal flora may trigger more serious secondary infections, such as Clostridium difficile-related infections, and may even exacerbate the patient's condition. 3. Non-specific effect, the current antibiotic therapy lacks specificity and often acts on the entire bacterial population. This non-targeted treatment can harm normal flora, especially in the case of long-term medication, increasing the risk of opportunistic infections by other pathogens (such as fungi and drug-resistant bacteria). 4. Immune-related side effects, for immunosuppressed patients, the treatment of Enterococcus casseliflavus infection is particularly difficult. Antibiotics themselves may not be effective in controlling infection in cases of low immune function, and further suppression of the immune system by the drug may exacerbate the condition.

[0004] In summary, antibiotic therapy for Enterococcus casseliflavus faces problems such as drug resistance, microecological destruction, and non-specific side effects, and the problem of drug resistance exacerbates the difficulty of treatment. This study focuses on developing new antibacterial strategies to address the pathogenic effects of Enterococcus casseliflavus in the human body and the problem of chlorantraniliprole pesticide failure caused by Enterococcus casseliflavus in crop pest control. SUMMARY

[0005] The purpose of the present application is to provide a method for preventing or treating infections caused by Enterococcus casseliflavus or diseases related to Enterococcus casseliflavus. The present application seeks potential antibacterial proteins from the genomes of bacteria or bacteriophages that can target and inhibit Enterococcus casseliflavus. By means of heterologous expression, biologically active antibacterial proteins can be obtained, which can target and eliminate Enterococcus casseliflavus, thereby alleviating and treating related infectious diseases caused by Enterococcus casseliflavus. In addition, the present application also aims to achieve targeted bactericidal without destroying normal human symbiotic flora to avoid the side effects of traditional antibiotics.

[0006] Based on this, the first aspect of the present application provides an antibacterial protein or its chimeric targeting Enterococcus casseliflavus, wherein the antibacterial protein comprises an amino acid sequence as shown in any one of SEQ ID NO. 15-28 or an active fragment, analog thereof, the active fragment, analog thereof can inhibit the growth of Enterococcus casseliflavus; the active fragment, analog thereof includes one or more of the following:

[0007] (a) has at least 85% sequence identity to the amino acid sequence shown in any one of SEQ ID NO. 15-28 or an alternative amino acid sequence having the same functional group;

[0008] (b) a derivative sequence of the amino acid sequence shown in any one of SEQ ID NO. 15-28 with one or several amino acids substituted, added and / or deleted and having the same function as the amino acid sequence shown in any one of SEQ ID NO. 15-28.

[0009] In some embodiments, the active fragment, analog comprises a catalytic domain and / or a cell wall binding domain.

[0010] In some embodiments, the catalytic domain comprises one or more of Amidase_2, Amidase_3, Amidase_5, CHAP, Glyco_hydro_25, Peptidase_M23, Phage_lysozyme and Glucosaminidase.

[0011] In some embodiments, the cell wall binding domain comprises one or more of SH3, CW_7, PG_binding_1, the amino acid fragment shown in SEQ ID NO. 15 from position 161 to 231, the amino acid fragment shown in SEQ ID NO. 17 from position 165 to 307, the amino acid fragment shown in SEQ ID NO. 20 from position 202 to 272, the amino acid fragment shown in SEQ ID NO. 21 from position 204 to 275, the amino acid fragment shown in SEQ ID NO. 24 from position 169 to 341 and the amino acid fragment shown in SEQ ID NO. 27 from position 198 to 259.

[0012] In some embodiments, the chimeric protein comprises two or more of the amino acid sequences of the complete antibacterial protein, the catalytic domain sequence, the cell wall binding domain sequence as described above in combination.

[0013] In some embodiments, the antibacterial protein or chimeric protein thereof is capable of lysing Enterococcus casseliflavus.

[0014] The second aspect of the present application provides a nucleic acid molecule encoding the antibacterial protein or chimeric protein thereof of the first aspect of the present application.

[0015] In some embodiments, the nucleic acid molecule comprises the nucleic acid sequence shown in any one of SEQ ID NO. 1-14 or a synonymous codon sequence thereof.

[0016] The third aspect of the application provides a vector comprising the nucleic acid molecule of the second aspect of the application.

[0017] The fourth aspect of the application provides a host cell comprising the nucleic acid molecule of the second aspect of the application or the vector of the third aspect of the application.

[0018] The fifth aspect of the application provides a method of producing the antibacterial protein of the first aspect of the application or a chimera thereof, comprising:

[0019] The antibacterial protein of the first aspect of the application or a chimera thereof is produced in vitro by transcription using the nucleic acid molecule of the second aspect of the application or the vector of the third aspect of the application or the host cell of the fourth aspect of the application.

[0020] The sixth aspect of the application provides a pharmaceutical preparation for preventing or treating an infection caused by Enterococcus casseliflavus or a disease associated with Enterococcus casseliflavus, the pharmaceutical preparation comprising the antibacterial protein of the first aspect of the application or a chimera thereof.

[0021] The pharmaceutical preparation of the application can be administered in any manner, including but not limited to, orally, parenterally, transdermally, transmucosally, topically, by inhalation, by oral or intranasal or intravesical administration, or a combination thereof.

[0022] In some embodiments, the disease associated with Enterococcus casseliflavus includes but is not limited to septicemia, endophthalmitis, infective endocarditis, meningitis, peritonitis, empyema, bacteremia, or urinary tract infection.

[0023] The seventh aspect of the application provides use of the antibacterial protein of the first aspect of the application or a chimera thereof, the nucleic acid molecule of the second aspect of the application, the vector of the third aspect of the application, the host cell of the fourth aspect of the application, or the pharmaceutical preparation of the sixth aspect of the application in the manufacture of a medicament or medical device for preventing or treating an infection caused by Enterococcus casseliflavus or a disease associated with Enterococcus casseliflavus, or an Enterococcus casseliflavus inhibitor.

[0024] In some embodiments, the Enterococcus casseliflavus inhibitor includes a disinfectant, a bactericide, a pharmaceutical preparation, or a pesticide adjuvant.

[0025] In some embodiments, the Enterococcus casseliflavus inhibitor further includes a physiologically acceptable carrier, diluent, or excipient.

[0026] In some embodiments, the medicament further includes a pharmaceutically acceptable carrier and / or adjuvant.

[0027] In the present application, the pharmaceutically acceptable carriers and / or excipients include but are not limited to diluents, binders, surface active agents, wetting agents, adsorptive carriers, lubricants, fillers, disintegrants.

[0028] In some embodiments, the dosage form of the drug includes tablets, capsules, injectable solutions, eye washes, mouthwashes, sprays, aerosols, creams or ointments.

[0029] In some embodiments, the disease associated with Enterococcus casseliflavus includes septicemia, endophthalmitis, infective endocarditis, meningitis, peritonitis, keratitis, polycystic intrahepatic infection, empyema, bacteremia or urinary tract infection.

[0030] The colonization of Enterococcus casseliflavus in pests can lead to the failure of chlorantraniliprole insecticides. The antibacterial protein described in the present application can be used as an insecticide adjuvant to enhance the insecticidal effect of chlorantraniliprole insecticides.

[0031] In some embodiments, the insecticide adjuvant described in the present application can enhance the insecticidal effect of chlorantraniliprole insecticides.

[0032] The eighth aspect of the present application provides a method for inhibiting the growth of Enterococcus casseliflavus, which comprises adding the antibacterial protein or its chimera of the first aspect of the present application to a system in need thereof.

[0033] The ninth aspect of the present application provides a method for preventing or treating infection caused by Enterococcus casseliflavus or a disease associated with Enterococcus casseliflavus, which comprises administering the antibacterial protein or its chimera of the first aspect of the present application to a subject.

[0034] In some embodiments, the disease associated with Enterococcus casseliflavus includes but is not limited to septicemia, endophthalmitis, infective endocarditis, meningitis, peritonitis, keratitis, polycystic intrahepatic infection, empyema, bacteremia or urinary tract infection.

[0035] Beneficial effects:

[0036] Through simulation, experiment and verification, the present application obtains a plurality of bacteriophage lytic enzymes of Enterococcus casseliflavus, and successfully prepares high-purity lytic enzyme protein through genetic engineering means. Experiments prove that the bacteriophage lytic enzyme protein can effectively kill Enterococcus casseliflavus, has good targeting, does not affect other common microorganisms in the intestinal tract, and can effectively kill drug-resistant Enterococcus casseliflavus of different sources.

[0037] Compared with the treatment of Enterococcus casseliflavus with antibiotics, the bacteriophage lytic enzyme has high specificity and does not produce drug resistance, etc., which can be roughly divided into the following points:

[0038] 1. Specificity: Phage lysins have high specificity against specific bacterial species or strains, meaning they can precisely kill Enterococcus casseliflavus without affecting other beneficial gut flora.

[0039] 2. Reduced resistance: Unlike antibiotics, the use of phage lysins has a relatively low chance of developing resistant strains. The overuse of antibiotics has led to many bacteria developing resistance to commonly used antibiotics, but phage lysins act on bacteria through different mechanisms, so it is unlikely to cause this situation.

[0040] 3. Safety: Due to the specificity of phage lysins, they are generally harmless to human cells, with better safety and fewer side effects.

[0041] The phage lysins or chimeras provided by the present application can specifically and effectively kill Enterococcus casseliflavus while keeping other symbiotic bacteria intact, providing a safe and effective solution for Enterococcus casseliflavus-related infections and diseases, suitable for long-term use without the risk of microecological imbalance and microbial resistance.

[0042] The advantages of phage lysin-based targeted therapy: 1. High specificity of bactericidal activity, phage lysins can recognize and lyse the cell wall of bacteria, and have high specificity for pathogenic bacteria, usually only targeting specific bacterial species or strains. This high specificity means that lysins can target and kill Enterococcus casseliflavus without damaging beneficial bacteria in the human body, avoiding the broad-spectrum damage of antibiotics. 2. Resistance, phage lysins achieve bactericidal effect by binding to specific molecules in the bacterial cell wall, making it difficult for bacteria to resist lysin through traditional drug resistance mechanisms such as changing metabolic pathways or producing drug-resistant enzymes. Therefore, drug resistance develops slowly and can be an effective alternative to deal with drug-resistant strains. 3. Rapid and effective action, lysins act quickly and can kill target bacteria within a few minutes to a few hours. This is particularly effective for treating acute infections or controlling the spread of infection in critically ill patients. In addition, the use of lysins does not depend on the function of the host immune system, so it is also effective in immunosuppressed patients. 4. Reduced side effects, as lysins are targeted, they do not cause imbalance of the gut flora like antibiotics, greatly reducing the risk of secondary infection. This makes phage lysins safer and more tolerable in long-term treatment or repeated infections. 5. Flexible engineering, phage lysins can be optimized for their bactericidal properties through genetic engineering techniques, such as enhancing their ability to lyse specific strains or increasing their stability. This flexibility makes lysin therapy have broad application potential, and even allows for customized therapy based on the specific characteristics of the pathogenic bacteria. BRIEF DESCRIPTION OF DRAWINGS

[0043] Figure 1is the three-dimensional structure of LysEC06 in Example 1 and other lytic enzymes with the same cell wall binding domain, wherein the gray part is the catalytic domain and the black part is the cell wall binding domain;

[0044] Figure 2 is the plasmid map of the lytic enzyme LysEC06 expression vector in Example 2;

[0045] Figure 3 is the electrophoresis map of the lytic enzyme LysEC06 protein heterologous expression identification in Example 2;

[0046] Figure 4 is the protein electrophoresis map of the lytic enzyme LysEC06 after purification in Example 3;

[0047] Figure 5 is the diagram of the lytic enzyme LysEC06 high-efficiency killing of Enterococcus casseliflavus in Example 4;

[0048] Figure 6 is the diagram of the lytic activity of the lytic enzyme LysEC06 on different intestinal symbiotic bacteria in Example 5;

[0049] Figure 7 is the lytic activity of the lytic enzyme LysEC06 and lysozyme (chicken egg white source, CAS: 12650-88-3) on different intestinal symbiotic bacteria. DETAILED DESCRIPTION

[0050] Before further description of the specific embodiments of the present application, it is to be understood that the application is not limited to the particular specific embodiments described below; it is also to be understood that the terminology used in the present application is for the purpose of describing the particular specific embodiments only and is not intended to limit the scope of the present application.

[0051] When the examples give numerical ranges, it is to be understood that, unless otherwise specified by the present application, both endpoints of each numerical range and any number between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present application have the same meaning as commonly understood by one skilled in the art. In addition to the specific methods, devices, materials used in the examples, any method, device and material of the prior art similar or equivalent to those described in the examples of the present application can also be used to implement the present application according to the mastery of the prior art by those skilled in the art and the description of the present application.

[0052] Unless otherwise specified, the experimental methods, detection methods, preparation methods not described in detail in the present application use conventional techniques in the art.

[0053] Example 1 Identification and protein sequence analysis of lytic enzyme gene targeting Enterococcus casseliflavus

[0054] The phage and prophage genomic sequences targeting Enterococcus casseliflavus were collected, and the lytic enzyme genes were identified by sequence alignment and domain alignment using BLAST and Interproscan. More than 300 potential phage lytic enzyme gene sequences were found. The identified lytic enzymes were clustered according to sequence similarity, and classified according to the set threshold. According to the threshold that the sequence similarity and sequence coverage are greater than or equal to 80%, the identified phage-derived lytic enzyme genes were divided into 14 categories. A representative lytic enzyme sequence (LysEC01-LysEC14) was selected from each category, and a representative sequence was selected for expression vector construction. The preferred representative sequence is named LysEC06.

[0055] The nucleic acid sequences of the 14 categories of lytic enzymes after codon optimization for Escherichia coli are as follows:

[0056] The nucleic acid sequence of LysEC01 is SEQ ID NO. 1; the nucleic acid sequence of LysEC02 is SEQ ID NO. 2; the nucleic acid sequence of LysEC03 is SEQ ID NO. 3; the nucleic acid sequence of LysEC04 is SEQ ID NO. 4; the nucleic acid sequence of LysEC05 is SEQ ID NO. 5; the nucleic acid sequence of LysEC06 is SEQ ID NO. 6; the nucleic acid sequence of LysEC07 is SEQ ID NO. 7; the nucleic acid sequence of LysEC08 is SEQ ID NO. 8; the nucleic acid sequence of LysEC09 is SEQ ID NO. 9; the nucleic acid sequence of LysEC10 is SEQ ID NO. 10; the nucleic acid sequence of LysEC11 is SEQ ID NO. 11; the nucleic acid sequence of LysEC12 is SEQ ID NO. 12; the nucleic acid sequence of LysEC13 is SEQ ID NO. 13; and the nucleic acid sequence of LysEC14 is SEQ ID NO. 14.

[0057] The protein sequences of the 14 categories of lytic enzymes are as follows:

[0058] LysEC01 protein sequence: SEQ ID NO. 15; LysEC02 protein sequence: SEQ ID NO. 16; LysEC03 protein sequence: SEQ ID NO. 17; LysEC04 protein sequence: SEQ ID NO. 18; LysEC05 protein sequence: SEQ ID NO. 19; LysEC06 protein sequence: SEQ ID NO. 20; LysEC07 protein sequence: SEQ ID NO. 21; LysEC08 protein sequence: SEQ ID NO. 22; LysEC09 protein sequence: SEQ ID NO. 23; LysEC10 protein sequence: SEQ ID NO. 24; LysEC11 protein sequence: SEQ ID NO. 25; LysEC12 protein sequence: SEQ ID NO. 26; LysEC13 protein sequence: SEQ ID NO. 27; LysEC14 protein sequence: SEQ ID NO. 28.

[0059] The protein formed by the amino acid sequence shown in any one of SEQ ID NO: 15 to SEQ ID NO: 28 of the present application contains catalytic domain and cell wall binding domain and the position information thereof as shown in Table 1, wherein the catalytic domain includes Amidase_2, Amidase_3, Amidase_5, CHAP, Glyco_hydro_25, Peptidase_M23, Phage_lysozyme and Glucosaminidase. The cell wall binding domain includes SH3, CW_7 and PG_binding_1, etc. As can be seen from Table 1, the lytic enzyme LysEC06 has the same catalytic domain Amidase_3 as LysEC14, and the cell wall binding domain of the plurality of lytic enzymes including LysEC06 is in unknown state. Through structure prediction, the present application found that the lytic enzyme LysEC06 has the same cell wall binding domain as LysEC01, LysEC07 and LysEC13, as shown in the black part in Table 1. Figure 1

[0060] Table 1: Catalytic domain and cell wall binding domain of antibacterial protein LysEC01 to LysEC14

[0061]

[0062] Example 2 Heterologous expression of lytic enzymes targeting Enterococcus casseliflavus

[0063] ​The identified lyase gene was codon-optimized for E. coli, and then the corresponding gene was cloned into pET28a(+) or pCold II vector with 6x-His tag at the C- or N-terminus, and the gene was controlled by lactose operon. Figure 2 is the LysEC06 expression vector plasmid map of the constructed lyase. The expression plasmid includes replicon, antibiotic resistance gene, lactose operon and other elements, and the lyase gene N-terminus is fused with 6x-His tag.

[0064] The constructed plasmid containing lyase LysEC06 gene was transformed into BL21(DE3) competent cells, and then uniformly coated on LB plate (containing 50 μg / mL kanamycin sulfate), and then inverted in 37°C incubator overnight. Single colonies were selected from the transformed plate, inoculated into 1L of LB medium (containing 50 μg / mL kanamycin sulfate), and when the culture reached OD600 of 0.6-0.8, IPTG was added to the test tube culture to a final concentration of 0.1-1 mM, and then placed at 16-37°C, 100 rpm, 18h to induce protein expression.

[0065] The induced culture was centrifuged at 12000 rpm for 5 min, the medium was removed, PBS was added to resuspend the strain, and finally SDS-PAGE loading buffer was added to heat the sample at 100°C for 30 min, and the supernatant was electrophoresed after centrifugation. 10 min before electrophoresis, 100-150V constant voltage electrophoresis, when the bromophenol blue indicator entered the separation gel, 200V constant voltage electrophoresis until the bromophenol blue band migrated to the bottom of the gel 1 cm, the gel was stained with coomassie brilliant blue staining solution, and then transferred to the decolorizing solution, and decolorized until the background was clear.

[0066] The whole bacteria were lysed by ultrasonic or high pressure homogenization in PBS buffer, and the Ni-IDA affinity chromatography column was equilibrated with PBS buffer, at least three times, and then the target protein was eluted with different concentrations of imidazole equilibration buffer, and each elution fraction was collected for SDS-PAGE analysis. The SDS results are shown in Figure 3 (Figure M is SDS-PAGE Protein marker, 1 is whole cell before induction, 2 is whole cell after induction, 3 is supernatant after induction, 4 is induction expression precipitate).

[0067] Figure 3 The electrophoresis map for identification of LysEC06 heterologous expression in E. coli. Among them, lane 1 is before induction, lane 2 is after induction, lane 3 is supernatant after cell disruption, and lane 4 is precipitate after cell disruption.

[0068] As Figure 3 It is shown that after crushing and separation, the lyase LysEC06 protein is expressed in soluble form in E. coli.

[0069] Example 3 Purification of Lysing Enzyme Targeting Enterococcus casseliflavus

[0070] The cultured bacteria were centrifuged at 4000 rpm for 10 min, and the bacteria were collected. Then the bacteria were resuspended with Lysis buffer and broken. The supernatant was collected by centrifugation at 18000 rpm for 15 min at 4°C. The supernatant was incubated with Ni resin at 4°C for 2 h. After the supernatant was flowed out, the column was washed with different concentrations of imidazole in Wash buffer until the Bradford reagent was no longer blue. Then 10 ml of Elute buffer was added for elution, and SDS-PAGE was used for detection. The protein was concentrated to 1 ml, and the impurities were removed by low-temperature centrifugation. Then the protein was collected by molecular sieve, and SDS-PAGE was used for detection. The protein was concentrated, and the concentration was determined and stored. Figure 4 The protein electrophoretogram after purification of LysEC06 shows that high-purity LysEC06 can be obtained through multiple purification steps.

[0071] Example 4 Determination of the Lysing Activity of Lysing Enzyme Protein on Enterococcus casseliflavus

[0072] Enterococcus casseliflavus was inoculated into anaerobic BHI liquid medium, and the Enterococcus casseliflavus was cultured to OD600=0.6. The bacteria were centrifuged from the culture medium at 4000 rpm for 5 min, washed twice with buffer (50 mM Tris-HCl [pH 7.0], 100 mM NaCl), and resuspended with the buffer after washing. The buffer containing the bacteria was added to a 96-well plate, and LysEC06 lysing enzyme protein was added to a final concentration of 5 micromoles, and the final volume was 200 microliters. After 3 h of culture, the experimental data were arranged, and the lysis activity was calculated. The lysis rate calculation formula is: [test ΔOD600 (add lysing enzyme)-ΔOD600 control (only buffer)] / initial OD600. According to the specific embodiments of the present application, the Enterococcus casseliflavus is isolated from human intestine, and the Latin name is Enterococcus casseliflavus, and the strain name is DO99 (the strain is provided by Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, and has been publicly preserved in the General Microbiological Center of China Microorganism Culture Collection Management Committee for public sharing). The genome of the strain contains the vancomycin-resistant gene VanC4XY, and it is a vancomycin-resistant strain. Enterococcus casseliflavus can be transmitted through blood, causing bacteremia; causing peritonitis and other abdominal infections after gastrointestinal surgery or trauma; occasionally reported in heart valve infections, and Enterococcus casseliflavus-induced infective endocarditis usually involves native valves or artificial valves; Enterococcus casseliflavus often causes urinary tract infections in the elderly and long-term catheter users.

[0073] Figure 5The results show that LysEC06 can efficiently lyse Enterococcus casseliflavus. After adding the lysing enzyme LysEC06, the OD of the culture solution rapidly decreases, indicating that LysEC06 can effectively lyse Enterococcus casseliflavus. After adding the LysEC06 lysing enzyme, the absorbance OD600 has decreased by more than 50% in about 60 min, and after 200 min, Enterococcus casseliflavus has been almost completely lysed, indicating that the lysing enzyme LysEC06 has strong lytic activity on Enterococcus casseliflavus.

[0074] Example 5 Determination of the targeting of the lysing enzyme protein

[0075] Using the purified protein LysEC06, the lytic activity of the protein on different symbiotic intestinal bacteria was determined, and the determination method of the lytic activity was the same as that in Example 4. Briefly, different symbiotic intestinal bacteria were inoculated into anaerobic BHI liquid medium, and Enterococcus casseliflavus was cultured to OD600 = 0.6. The bacterial solution was centrifuged at 4000 rpm for 5 min, washed twice with buffer (50 mM Tris-HCl [pH 7.0], 100 mM NaCl), and then resuspended in the buffer after washing. The buffer containing the bacterial solution was added to a 96-well plate, and LysEC06 lysing enzyme protein was added to a final concentration of 5 micromoles, and the final volume of each well was 200 microliters. The 96-well plate was placed in an enzyme marker and the change in absorbance was continuously determined. A control group without lysing enzyme was also set up. After 2 h, the change in absorbance was calculated. The lytic activity was calculated.

[0076] Figure 6 The lytic activity of the lysing enzyme LysEC06 on different Enterococcus bacteria. The lysis rate is the proportion of cells lysed in the total cells within a specified time, and the closer the ratio is to 1, the higher the lytic activity. As can be seen from Table 1, Figure 6 It can be seen from Table 1 that LysEC06 exhibits high lytic activity on Enterococcus casseliflavus, with a lysis rate close to 0.9. LysEC06 also has certain lytic activity on Enterococcus gallinarum, with a lysis rate of about 0.28, but does not exhibit obvious lytic activity on other tested Enterococcus bacteria, such as Enterococcus hirae, Enterococcus faecalis, and Enterococcus faecium. This indicates that LysEC06 has high targeting and can target and remove Enterococcus casseliflavus and Enterococcus gallinarum in the intestine, but has no lytic activity on other Enterococcus bacteria.

[0077] Figure 7 The lytic activity of the lysing enzyme LysEC06 and lysozyme (chicken egg white source, CAS: 12650-88-3) on different intestinal symbiotic bacteria. As Figure 7As shown in Table A, LysEC06 exhibited high lytic activity against E. casseliflavus; but did not exhibit lytic activity against the other 11 tested intestinal symbiotic bacteria (information and taxonomic status of the tested strains are shown in Table 2). These tested intestinal symbiotic bacteria were from 5 common intestinal symbiotic bacterial phyla, including Bacteroidetes, Firmicutes, Actinobacteria, Proteobacteria and Fusobacteria. This indicates that the lytic enzyme LysEC06 has high targeting ability, and can target and eliminate E. casseliflavus and E. gallinarum in the intestine, without affecting other intestinal symbiotic bacteria. Lysozyme from chicken egg white has certain lytic activity against E. casseliflavus, but the lytic ability is much lower than LysEC06. However, lysozyme from chicken egg white also causes the lysis of many intestinal symbiotic bacteria, and has no obvious targeting ability. Figure 7 Table B.

[0078] Table 2: Information of intestinal symbiotic bacteria used for testing the targeting ability of lytic enzyme LysEC06

[0079] Bacterial species name Bacterial strain name Bacterial classification Bacteroides uniformis ATCC 8492 Bacteroidetes Parabacteroides distasonis ATCC 8503 Bacteroidetes Blautia obeum DA69 Firmicutes Dorea longicatena DA136 Firmicutes Clostridium symbiosum DA229 Firmicutes Anaerostipes hadrus DA538 Firmicutes Faecalibacterium prausnitzii DA726 Firmicutes Bifidobacterium adolescentis DA06 Actinobacteria Collinsella aerofaciens DA394 Actinobacteria Escherichia coli DH5a Proteobacteria Fusobacterium varium DA690 Fusobacteria

[0080] The above description of the embodiments is to facilitate those of ordinary skill in the art to understand and use the present application. Those skilled in the art can easily make various modifications to these embodiments, and apply the general principles described herein to other embodiments without having to go through creative labor. Therefore, the present application is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present application without departing from the scope of the present application should be within the scope of protection of the present application.

Claims

1. The use of an antimicrobial protein, a nucleic acid molecule encoding the antimicrobial protein, a carrier containing the nucleic acid molecule, a host cell containing the nucleic acid molecule or the carrier, or a pharmaceutical preparation containing the antimicrobial protein in the preparation of a medicament for treating infections caused by Enterococcus faecalis; wherein the amino acid sequence of the antimicrobial protein is shown in SEQ ID NO.

20.

2. The application according to claim 1, wherein, The drug also includes pharmaceutically acceptable carriers and / or excipients.

3. The application according to claim 1, wherein, The dosage forms of the drug include tablets, capsules, injectable solutions, eye wash, mouthwash, sprays, aerosols, creams, or ointments.

4. The use of an antimicrobial protein, a nucleic acid molecule encoding the antimicrobial protein, a carrier containing the nucleic acid molecule, a host cell containing the nucleic acid molecule or the carrier, or a pharmaceutical preparation containing the antimicrobial protein in the preparation of an inhibitor of Enterococcus faecalis; wherein the amino acid sequence of the antimicrobial protein is shown in SEQ ID NO.

20.

5. The application according to claim 4, wherein, The aforementioned Enterococcus faecalis inhibitors include disinfectants or bactericides.

6. The application according to claim 4, wherein, The aforementioned Enterococcus faecalis inhibitors also include physiologically acceptable carriers, diluents, or excipients.

7. A method for inhibiting the growth of Enterococcus pyogenes for non-therapeutic purposes, the method comprising adding an antimicrobial protein to a system where necessary; the amino acid sequence of said antimicrobial protein is shown in SEQ ID NO.20.

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