Production and application of antibacterial protein targeting enterococcus pluvialis

Abstract

The invention discloses production and application of an antibacterial protein targeting enterococcus pluvialis. Specifically, the antibacterial protein comprises an amino acid sequence as shown in any one of SEQ ID NO.15-28 or an active fragment and an analogue of the amino acid sequence. According to the present invention, the potential antibacterial protein is found from the genome of the bacteria or the bacteriophage, and the antibacterial protein can inhibit the enterococcus pluvialis in the targeting manner; the antibacterial protein with biological activity can be obtained in a heterologous expression mode and can remove the enterococcus pluvialis in a targeted mode, and then related diseases caused by the enterococcus pluvialis are relieved and treated. The antibacterial protein provided by the invention can perform targeted sterilization without damaging normal human symbiotic flora, and can avoid side effects caused by traditional antibiotic treatment.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and specifically relates to the production and application of an antibacterial protein targeting Enterococcus pyogenes. Background Art

[0002] Enterococcus pyogenes is a gram-positive, facultative anaerobic bacterium belonging to the genus Enterococcus. It is a common opportunistic pathogen in immunocompromised or chronically ill patients. The most common form of infection with Enterococcus pyogenes is sepsis, often associated with the biliary tract and liver. The second most common form of infection with Enterococcus pyogenes is endophthalmitis, often associated with trauma, especially in active young and middle-aged patients. Other infections with Enterococcus pyogenes include infective endocarditis, meningitis, peritonitis, and empyema. Infectious diseases associated with Enterococcus pyogenes: 1. Enterococcus pyogenes can be transmitted through the blood and cause bacteremia, which is especially common in patients in intensive care or those with compromised immune systems (such as cancer and organ transplant patients); 2. The intestinal origin of Enterococcus pyogenes 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 pyogenes usually involves native valves or artificial valves; 4. Enterococcus pyogenes often causes urinary tract infections in the elderly and long-term catheter users. These infections are often resistant to multiple antibiotics, increasing the complexity of treatment. In addition, Enterococcus pyogenes is also a widely distributed bacterial symbiont in insects. It can degrade chlorantraniliprole insecticides by destroying amide bonds and dehalogenation, enhancing the host insects' resistance to chlorantraniliprole insecticides, thereby rendering chlorantraniliprole insecticides ineffective.

[0003] At present, the main means of treating Enterococcus aureus infection is broad-spectrum antibiotics, but these methods have some obvious problems. The main problems of existing treatment technologies: 1. Antibiotic resistance. Enterococcus aureus gradually develops resistance to a variety of commonly used antibiotics, especially vancomycin. This reduces the effectiveness of treatment with broad-spectrum antibiotics and easily leads to the further spread of resistant strains. During treatment, doctors often face the difficulty of choosing a limited number of antibiotics. 2. Intestinal microecological destruction. The use of antibiotics will not only kill Enterococcus aureus, but also affect the beneficial flora in the intestine, leading to intestinal microecological imbalance. The destruction of intestinal flora may cause more serious secondary infections, such as Clostridium difficile-related infections, and may even aggravate the patient's condition. 3. Non-specific effects. Current antibiotic therapies lack specificity and often work on the entire bacterial community. This non-targeted treatment can cause damage to 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 aureus infection is particularly difficult. Antibiotics alone may not be effective in controlling infection in immunocompromised individuals, and further suppression of the immune system by medications may exacerbate the condition.

[0004] In summary, antibiotic treatment of E. piloflavus faces problems such as drug resistance, microecological damage and nonspecific side effects, which exacerbate the difficulty of treatment. This study focuses on developing new antimicrobial strategies to address the pathogenic effects of E. piloflavus in the human body and the failure of chlorfenapyr pesticides caused by E. piloflavus in the field of crop pest control. Summary of the invention

[0005] The purpose of the present invention is to provide a method for preventing or treating infections caused by Enterococcus casseliflavus or diseases related to Enterococcus casseliflavus. The present invention searches for potential antimicrobial proteins from the genome of bacteria or bacteriophages, and such antimicrobial proteins can target and inhibit Enterococcus casseliflavus. Antimicrobial proteins with biological activity can be obtained by heterologous expression, and they can target and eliminate Enterococcus casseliflavus, thereby alleviating and treating related infectious diseases caused by Enterococcus casseliflavus. In addition, the present invention also aims to achieve targeted sterilization without destroying the normal human symbiotic flora, so as to avoid the side effects of traditional antibiotics.

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

[0007] (a) having at least 85% sequence identity with the amino acid sequence shown in any one of SEQ ID NOs. 15-28 or an alternative amino acid sequence having the same functional groups;

[0008] (b) A derivative sequence of the amino acid sequence shown in any one of SEQ ID NOs. 15-28, wherein one or more amino acids are replaced, added and / or deleted and has the same function as the amino acid sequence shown in any one of SEQ ID NOs. 15-28.

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

[0010] In some embodiments, the catalytic domain includes 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 includes SH3, CW_7, PG_binding_1, the amino acid fragment shown at positions 161 to 231 in SEQ ID NO.15, the amino acid fragment shown at positions 165 to 307 in SEQ ID NO.17, the amino acid fragment shown at positions 202 to 272 in SEQ ID NO.20, the amino acid fragment shown at positions 204 to 275 in SEQ ID NO.21, the amino acid fragment shown at positions 169 to 341 in SEQ ID NO.24 and one or more of the amino acid fragment shown at positions 198 to 259 in SEQ ID NO.27.

[0012] In some embodiments, the chimera comprises a chimeric protein formed by combining two or more of the amino acid sequence of the complete antibacterial protein described above, the catalytic domain sequence, and the cell wall binding domain sequence.

[0013] In some embodiments, the antimicrobial protein or chimera thereof is capable of lysing Enterococcus pyogenes.

[0014] The second aspect of the present invention provides a nucleic acid molecule encoding the antibacterial protein or its chimera according to the first aspect of the present invention.

[0015] In some embodiments, the nucleic acid molecule comprises a 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 present invention provides a vector comprising the nucleic acid molecule described in the second aspect of the present invention.

[0017] The fourth aspect of the present invention provides a host cell, which comprises the nucleic acid molecule described in the second aspect of the present invention or the vector described in the third aspect of the present invention.

[0018] The fifth aspect of the present invention provides a method for preparing the antimicrobial protein or its chimera according to the first aspect of the present invention, comprising:

[0019] The antimicrobial protein or its chimera described in the first aspect of the present invention is prepared by in vitro transcription using the nucleic acid molecule described in the second aspect of the present invention, the vector described in the third aspect of the present invention, or the host cell described in the fourth aspect of the present invention.

[0020] The sixth aspect of the present invention provides a pharmaceutical preparation for preventing or treating infections caused by Enterococcus pyogenes or diseases associated with Enterococcus pyogenes, wherein the pharmaceutical preparation comprises the antimicrobial protein or its chimera according to the first aspect of the present invention.

[0021] The pharmaceutical formulations of the present invention may 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 diseases associated with Enterococcus fusiformis include, but are not limited to, sepsis, endophthalmitis, infective endocarditis, meningitis, peritonitis, empyema, bacteremia, or urinary tract infection.

[0023] The seventh aspect of the present invention provides the use of the antibacterial protein or its chimera described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect of the present invention, the vector described in the third aspect of the present invention, the host cell described in the fourth aspect of the present invention, or the pharmaceutical preparation described in the sixth aspect of the present invention in the preparation of drugs or medical devices for preventing or treating infections caused by Enterococcus flavus or diseases related to Enterococcus flavus, or Enterococcus flavus inhibitors.

[0024] In some embodiments, the inhibitor of Enterococcus leucoderma comprises a disinfectant, a fungicide, a pharmaceutical preparation, or an insecticide adjuvant.

[0025] In some embodiments, the inhibitor of Enterococcus leucoderma further comprises a physiologically acceptable carrier, diluent or excipient.

[0026] In some embodiments, the drug further comprises a pharmaceutically acceptable carrier and / or excipient.

[0027] In the present invention, the pharmaceutically acceptable carriers and / or excipients include, but are not limited to: diluents, adhesives, surfactants, wetting agents, adsorption carriers, lubricants, fillers, and 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 fusiformis comprises sepsis, endophthalmitis, infective endocarditis, meningitis, peritonitis, keratitis, polycystic intrahepatic infection, empyema, bacteremia, or urinary tract infection.

[0030] The colonization of Enterococcus leucoderma in pests can lead to the ineffectiveness of chlorantraniliprole insecticides. The antimicrobial 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 invention can enhance the insecticidal effect of chlorantraniliprole insecticides.

[0032] The eighth aspect of the present invention provides a method for inhibiting the growth of Enterococcus pyogenes, wherein the method comprises adding the antimicrobial protein or its chimera described in the first aspect of the present invention to a system in need thereof.

[0033] The ninth aspect of the present invention provides a method for preventing or treating infection caused by Enterococcus pyogenes or diseases associated with Enterococcus pyogenes, the method comprising administering the antimicrobial protein or its chimera described in the first aspect of the present invention to a subject.

[0034] In some embodiments, the diseases associated with Enterococcus fusiformis include, but are not limited to, sepsis, 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 invention obtains phage lytic enzymes of various Enterococcus pyogenes, and successfully prepares high-purity lytic enzyme proteins through genetic engineering. Experiments have shown that phage lytic enzyme proteins can effectively kill Enterococcus pyogenes, have good targeting, will not affect other common intestinal microorganisms, and can effectively kill drug-resistant Enterococcus pyogenes from different sources.

[0037] Compared with the use of antibiotics to treat Enterococcus pyogenes, phage lytic enzymes have the characteristics of high specificity and no drug resistance, which can be roughly divided into the following points:

[0038] 1. Specificity: Phage lytic enzymes are highly specific to specific bacterial species or strains, which means that they can precisely kill Enterococcus ulmoides without affecting other beneficial flora in the intestines.

[0039] 2. Reduced drug resistance: Unlike antibiotics, the use of phage lytic enzymes is less likely to produce resistant strains. The overuse of antibiotics has caused many bacteria to become resistant to commonly used antibiotics, but phage lytic enzymes act on bacteria through different mechanisms and may not cause this.

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

[0041] The phage lytic enzyme or its chimera provided by the present invention can specifically and effectively kill Enterococcus pyogenes while keeping other symbiotic bacteria intact, and can provide a safe and effective solution to Enterococcus pyogenes-related infections and diseases, which is suitable for long-term use without the risk of dysbiosis and microbial resistance.

[0042] Advantages of targeted therapy based on phage lytic enzymes: 1. Highly specific bactericidal, phage lytic enzymes can recognize and lyse the cell walls of bacteria, are highly specific to pathogens, and are usually only targeted at specific bacterial species or strains. This high specificity means that the lytic enzyme can target and kill Enterococcus pyogenes without destroying the beneficial flora in the human body, avoiding the broad-spectrum destructive effects of antibiotics. 2. Anti-resistance, phage lytic enzymes achieve bactericidal effects by binding to specific molecules in the bacterial cell wall, and it is difficult for bacteria to resist the action of lytic enzymes through traditional resistance mechanisms (such as changing metabolic pathways or producing resistant enzymes). Therefore, drug resistance develops slowly and can become an effective alternative to deal with resistant strains. 3. Fast and effective, lytic enzymes act quickly and can kill target bacteria within minutes to hours. This is particularly effective for treating acute infections or controlling the spread of infection in critically ill patients. In addition, the use of lytic enzymes does not depend on the function of the host immune system, so it is also effective in immunosuppressed patients. 4. Reduced side effects. Since lytic enzymes are targeted, they will not cause an imbalance in the intestinal flora like antibiotics, and the risk of secondary infection is greatly reduced. This makes phage lytic enzymes safer and more tolerable in long-term treatment or repeated infections. 5. Flexible engineering. Phage lytic enzymes can be optimized for their bactericidal properties through genetic engineering technology, such as enhancing their ability to lyse specific strains or increasing their stability. This flexibility gives lytic enzyme therapy a wide range of application potential, and even allows for customized therapies based on the specific characteristics of pathogens. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1The three-dimensional structure of LysEC06 in Example 1 and other lysing 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 expression vector of lyase LysEC06 in Example 2;

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

[0046] Figure 4 is the protein electrophoresis diagram of the purified lyase LysEC06 in Example 3;

[0047] Figure 5 This is a diagram showing that the lytic enzyme LysEC06 in Example 4 efficiently kills Enterococcus pyogenes;

[0048] Figure 6 is a graph showing the lytic activity of the lytic enzyme LysEC06 against different intestinal commensal bacteria in Example 5;

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

[0050] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing the specific embodiments rather than for limiting the scope of protection of the present invention.

[0051] When the embodiments give numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the grasp of the prior art by those skilled in the art and the record of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention can also be used to realize the present invention.

[0052] Unless otherwise specified, experimental methods, detection methods, and preparation methods not described in detail in the present invention all adopt conventional techniques in the technical field.

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

[0054] Phage and prophage genome sequences targeting Enterococcus pyogenes 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, and the identified lytic enzymes were clustered according to sequence similarity, and classified according to the set threshold. According to the threshold of sequence similarity and sequence coverage greater than or equal to 80%, the identified phage-derived lytic enzyme genes were divided into 14 major categories, and a representative lytic enzyme sequence (LysEC01-LysEC14) was selected from each major category, and one representative sequence was selected for expression vector construction, and the preferred representative sequence was named LysEC06.

[0055] The nucleic acid sequences of 14 major types of lytic enzymes after codon optimization for E. coli are as follows:

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

[0057] The protein sequences of the 14 major classes of lytic enzymes are shown below:

[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: SEQID 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 IDNO.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 invention contains a catalytic domain and a cell wall binding domain and their position information 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 lyase LysEC06 and LysEC14 have the same catalytic domain Amidase_3, while the cell wall binding domains of multiple lyases including the lyase LysEC06 are in an unknown state. Through structural prediction, the present invention found that the lyase LysEC06 has the same cell wall binding domain as LysEC01, LysEC07 and LysEC13, such as Figure 1 Shown in black.

[0060] Table 1: Catalytic domains and cell wall binding domains of antimicrobial proteins LysEC01 to LysEC14

[0061]

[0062] Example 2 Heterologous expression of lytic enzyme targeting Enterococcus pyogenes

[0063] The identified lytic enzyme genes were codon-optimized for E. coli, and then the corresponding genes were cloned into pET28a(+) or pCold II vectors, with a 6x-His tag added at the C-terminus or N-terminus, and the genes were controlled by the lactose operon. Figure 2 This is the constructed LysEC06 expression vector plasmid map of the lyase. The expression plasmid includes multiple elements such as replicon, antibiotic resistance gene, lactose operon, etc. The N-terminus of the lyase gene is fused with a 6x-His tag.

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

[0065] Take the induced culture solution and centrifuge it at 12000rpm for 5min, remove the culture medium, add PBS solution to resuspend and wash the strain, and finally add SDS-PAGE loading buffer to heat the sample at 100℃ for 30min, centrifuge and take the supernatant for electrophoresis. 10min before electrophoresis, 100-150V constant voltage electrophoresis, after the bromophenol blue indicator enters the separation gel, 200V constant voltage electrophoresis until the bromophenol blue band migrates to 1cm from the bottom of the gel, take out the gel and stain it with Coomassie brilliant blue staining solution, then transfer it to the decolorizing solution and decolorize it until the background is clear.

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

[0067] Figure 3 This is an electrophoresis diagram for identification of heterologous expression of LysEC06 in E. coli, wherein lane 1 is before induction, lane 2 is after induction, lane 3 is the supernatant after cell lysis, and lane 4 is the precipitate after cell lysis.

[0068] like Figure 3 It was shown that the lytic enzyme LysEC06 protein was expressed in a soluble form in Escherichia coli after fragmentation and separation.

[0069] Example 3 Purification of the Lytic Enzyme Targeting Enterococcus pyogenes

[0070] The cultured bacteria were centrifuged at 4000rpm for 10min to collect the bacteria. Then the bacteria were resuspended with Lysis buffer and broken. Then centrifuged at 18000rpm at 4℃ for 15min to collect the supernatant; the supernatant obtained above was incubated with Ni resin at 4℃ for 2h; after the supernatant flowed through, the column was washed with Wash buffer with different concentration gradients of imidazole until the effluent did not turn blue; 10ml Elute buffer was added for elution and SDS-PSGE detection was performed; the column was concentrated to 1ml, centrifuged at low temperature to remove impurities and then passed through molecular sieves, the protein was collected and tested by SDS-PAGE gel; the protein was concentrated, the concentration was determined and frozen. Figure 4 The electrophoresis diagram of the purified LysEC06 protein shows that highly pure lytic enzyme protein LysEC06 can be obtained through multiple purification steps.

[0071] Example 4 Determination of the lytic activity of lytic enzyme protein against Enterococcus pyogenes

[0072] Enterococcus casseliflavus was inoculated into anaerobic BHI liquid culture medium and cultured to OD600 = 0.6. The bacterial solution was centrifuged at 4000rpm for 5min from the culture medium, washed twice with buffer (50mM Tris-HCl [pH7.0], 100mM NaCl), and the bacteria were resuspended with the buffer after washing. The buffer containing the bacteria was added to a 96-well plate, and LysEC06 lyase protein was added to a final concentration of 5 micromolar, and the final volume was 200 microliters. After 3h of culture, the experimental data was sorted and the lysis activity was calculated. The lysis rate calculation formula is: [tested ΔOD600 (lyase added)-ΔOD600 control (buffer only)] / initial OD600. According to a specific embodiment of the present invention, the Enterococcus casseliflavus was isolated from the human intestine, the Latin name is Enterococcuscasseliflavus, and the strain name is DO99 (the strain was provided by the Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, and has been publicly preserved and shared for public welfare at the General Microbiological Center of the China Microbiological Culture Collection Administration). The genome of this strain contains the vancomycin resistance gene VanC4XY, which is a vancomycin-resistant strain. Enterococcus pyogenes can be transmitted through blood and cause bacteremia; it can cause peritonitis and other abdominal infections after gastrointestinal surgery or trauma; it is occasionally reported in heart valve infections, and infective endocarditis caused by Enterococcus pyogenes usually involves native valves or artificial valves; Enterococcus pyogenes is more likely to cause urinary tract infections in the elderly and long-term catheter users.

[0073] Figure 5This indicates that LysEC06 can efficiently lyse Enterococcus pyogenes. After adding the lytic enzyme LysEC06, the OD of the culture solution decreased rapidly, proving that LysEC06 can effectively lyse Enterococcus pyogenes. After adding LysEC06 lytic enzyme, the absorbance OD600 had dropped by more than 50% in about 60 minutes, and after 200 minutes, Enterococcus pyogenes was basically completely lysed, indicating that the lytic enzyme LysEC06 has a strong lysis activity against Enterococcus pyogenes.

[0074] Example 5 Targeting determination of lytic enzyme protein

[0075] The purified protein LysEC06 was used to determine its lytic activity against different symbiotic intestinal bacteria, and the method for determining the lytic activity was the same as in Example 4. In short: Different symbiotic intestinal bacteria were inoculated into anaerobic BHI liquid culture medium, and Enterococcus pyogenes was cultured to OD600 = 0.6. The bacterial solution was centrifuged at 4000rpm for 5min from the culture medium, washed twice with buffer (50mMTris-HCl [pH7.0], 100mM NaCl), and the bacteria were resuspended with the buffer after washing. The buffer containing the bacteria was added to a 96-well plate, and LysEC06 lytic enzyme protein was added to a final concentration of 5 micromolar, and the final volume of each well was 200 microliters. It was placed in a 96-well plate and placed in an ELISA instrument to continuously measure the absorbance changes. At the same time, a control group without lytic enzyme was set up. After 2h, the absorbance changes were calculated. Calculate the lytic activity.

[0076] Figure 6 is the lytic activity of the lytic enzyme LysEC06 against different Enterococcus bacteria. The lysis rate is the proportion of cells lysed to the total cells within a specified time. The closer the ratio is to 1, the higher the lytic activity. Figure 6 It can be seen that LysEC06 exhibits efficient lysis activity against Enterococcus foetida, with a lysis rate of nearly 0.9. It also has a certain lysis activity against Enterococcus gallinarum, with a lysis rate of about 0.28. However, LysEC06 does not show obvious lysis activity against other Enterococcus bacteria tested, such as Enterococcus hirae, Enterococcus faecalis, and Enterococcus faecium. This shows that LysEC06 has a high targeting ability and can eliminate Enterococcus foetida and Enterococcus gallinarum in the intestine, but has no lysis activity against other Enterococcus bacteria.

[0077] Figure 7 The lytic activity of LysEC06 and lysozyme (from chicken egg white, CAS: 12650-88-3) on different intestinal symbiotic bacteria. Figure 7As shown in A, LysEC06 exhibited efficient lytic activity against Enterococcus foetida; but for the other 11 intestinal symbiotic bacteria tested (the information and taxonomic status of the test strains are shown in Table 2), LysEC06 did not show lytic activity. These intestinal symbiotic bacteria tested came from five common intestinal symbiotic bacterial phyla, including Bacteroidetes, Firmicutes, Actinobacteria, Proteobacteria, and Fusobacterium. This indicates that the lytic enzyme LysEC06 has a high degree of targeting and can target and eliminate Enterococcus foetida and Enterococcus gallinarum in the intestine without affecting other intestinal symbiotic bacteria. Lysozyme derived from egg white has certain lytic activity against Enterococcus foetida, and its lytic ability is much lower than that of LysEC06, but lysozyme derived from egg white will also cause the lysis of multiple intestinal symbiotic bacteria at the same time, and there is no obvious targeting ( Figure 7 (middle B).

[0078] Table 2: Information on intestinal commensal bacteria used for targeting test of LysEC06

[0079] Bacterial species name Bacterial strain name Bacterial classification Bacteroides uniformis ATCC8492 Bacteroidetes Parabacteroides distasonis ATCC8503 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 DH5α Proteobacteria Fusobacterium varium DA690 Fusobacteria

[0080] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention 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 invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. An antimicrobial protein or a chimera thereof targeting Enterococcus pyogenes, wherein: The antibacterial protein comprises an amino acid sequence as shown in any one of SEQ ID NO.15-28 or an active fragment or analog thereof, and the active fragment or analog can inhibit the growth of Enterococcus fusca; The active fragments and analogs include one or more of the following: (a) having at least 85% sequence identity with the amino acid sequence shown in any one of SEQ ID NOs. 15-28 or an alternative amino acid sequence having the same functional groups; (b) A derivative sequence of the amino acid sequence shown in any one of SEQ ID NOs. 15-28, wherein one or more amino acids are replaced, added and / or deleted and has the same function as the amino acid sequence shown in any one of SEQ ID NOs. 15-28.

2. The antimicrobial protein or chimera thereof according to claim 1, wherein: The active fragments and analogs contain a catalytic domain and / or a cell wall binding domain; Preferably, the catalytic domain comprises one or more of Amidase_2, Amidase_3, Amidase_5, CHAP, Glyco_hydro_25, Peptidase_M23, Phage_lysozyme and Glucosaminidase; Preferably, the cell wall binding domain comprises one or more of SH3, CW_7, PG_binding_1, the amino acid fragment shown at positions 161 to 231 in SEQ ID NO.15, the amino acid fragment shown at positions 202 to 272 in SEQ ID NO.20, the amino acid fragment shown at positions 204 to 275 in SEQ ID NO.21, and the amino acid fragment shown at positions 198 to 259 in SEQ ID NO.27; Preferably, the chimera comprises a chimeric protein formed by combining two or more sequences of the complete amino acid sequence of the antibacterial protein in claim 1, the catalytic domain sequence, and the cell wall binding domain sequence.

3. A nucleic acid molecule encoding the antibacterial protein or its chimera according to claim 1 or 2; Preferably, the nucleic acid molecule comprises a nucleic acid sequence shown in any one of SEQ ID NO.1-14 or a synonymous codon sequence thereof. A vector comprising the nucleic acid molecule according to claim 3 . A host cell comprising the nucleic acid molecule according to claim 3 or the vector according to claim 4.

6. A method for preparing the antimicrobial protein or its chimera according to claim 1 or 2, comprising: The antibacterial protein or its chimera according to claim 1 or 2 is prepared by in vitro transcription using the nucleic acid molecule according to claim 3, the vector according to claim 4, or the host cell according to claim 5.

7. A pharmaceutical preparation for preventing or treating infections caused by Enterococcus pyogenes or diseases associated with Enterococcus pyogenes, the pharmaceutical preparation comprising the antimicrobial protein or a chimera thereof according to claim 1 or 2.

8. Use of the antimicrobial protein or chimera thereof according to claim 1 or 2, the nucleic acid molecule according to claim 3, the vector according to claim 4, the host cell according to claim 5, or the pharmaceutical preparation according to claim 7 in the preparation of a drug or medical device for preventing or treating infections caused by Enterococcus pyogenes or diseases associated with Enterococcus pyogenes, or an Enterococcus pyogenes inhibitor.

9. The use according to claim 8, wherein: The inhibitor of Enterococcus pyogenes includes a disinfectant, a bactericide, a pharmaceutical preparation or an insecticide adjuvant. Preferably, the inhibitor of Enterococcus pyogenes also includes a physiologically acceptable carrier, a diluent or an excipient; Preferably, the drug further comprises a pharmaceutically acceptable carrier and / or excipient, and preferably, the dosage form of the drug comprises tablets, capsules, injectable solutions, eye washes, mouthwashes, sprays, aerosols, creams or ointments; Preferably, the diseases associated with Enterococcus leucoderma include sepsis, endophthalmitis, infective endocarditis, meningitis, peritonitis, keratitis, polycystic intrahepatic infection, empyema, bacteremia or urinary tract infection; Preferably, the insecticide adjuvant can enhance the insecticidal effect of chlorantraniliprole insecticides.

10. A method for inhibiting the growth of Enterococcus pyogenes, the method comprising adding the antimicrobial protein or its chimera according to claim 1 or 2 to a system in need thereof.

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