Peptidoglycan hydrolase lysph1 having broad-spectrum lytic activity, and mutant and use thereof

Abstract

A peptidoglycan hydrolase LysPH1 having a broad-spectrum lytic activity, and a mutant and the use thereof, which belong to the technical field of biological formulations. An antibacterial peptide LysPH1-CP, a peptidoglycan hydrolase LysPH1 containing the antibacterial peptide LysPH1-CP and a peptidoglycan hydrolase mutant LysPH1-K containing the peptidoglycan hydrolase LysPH1 all have good thermal stability, and can broadly lyse both Gram-negative bacteria and Gram-positive bacteria. Moreover, the peptidoglycan hydrolase LysPH1 and the peptidoglycan hydrolase mutant LysPH1-K can be solubly expressed in Escherichia coli, and have high enzymatic activity. Therefore, the antibacterial peptide LysPH1-CP, the peptidoglycan hydrolase LysPH1 and the peptidoglycan hydrolase mutant LysPH1-K have good application prospects in the preparation of an anti-infective drug and the research and development of feed additives.

Description

Peptidoglycan hydrolase LysPH1 with broad-spectrum lytic activity, mutants and applications thereof TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological agents, and particularly relates to an antibacterial peptide LysPH1-CP, a peptidoglycan hydrolase LysPH1 and a mutant LysPH1-K thereof, and applications thereof. BACKGROUND

[0002] Antibiotics were once considered the most powerful weapon for treating bacterial infectious diseases. However, with the increasing abuse of antibiotics, the problem of bacterial drug resistance is becoming increasingly serious, and the speed of new antibiotic development is much lower than that of drug-resistant bacteria. There are very few new antibiotics that can be used as alternatives, and they are expensive, resulting in a large number of drug-resistant bacteria infections in the clinic dying due to lack of available drugs. Under the influence of such a severe antibiotic resistance form, bacteriophages, which can replace antibiotics as new antibacterial agents, have attracted the attention of scholars at home and abroad.

[0003] Bacteriophages are a class of viruses that use microorganisms (bacteria, fungi, actinomycetes or spirochetes) as hosts. They have no cell structure and are composed of only capsid proteins and internal genetic material, and must rely on the host for replication and proliferation. As natural killers of bacteria, bacteriophages are widely present in nature and are the most diverse organisms on Earth, with a number about 10 times that of bacteria. For more than a century, the safety and effectiveness of bacteriophage therapy have been confirmed in a large number of animal experiments. After infecting host bacterial cells, lytic bacteriophages immediately initiate the expression of their early genes, and through these early gene expression products, the host DNA is degraded, thus terminating the host's gene expression and taking over the host's DNA replication machinery to synthesize a large amount of nucleic acid. When enough bacteriophages are produced, these nucleic acids are used as templates to produce a large number of bacteriophage structural proteins, which process and package the bacteriophage genome to produce the next generation of bacteriophage particles. Once the bacteriophage regulatory proteins accumulate to a certain level, the bacteriophage holin is expressed in large quantities, which forms a hole in the host cell, causing the bacteriophage lysozyme to pass through the inner membrane and approach the cell wall, thus achieving the purpose of lysing the bacteria.

[0004] In addition to the complete phage particles, the phage-encoded peptidoglycan hydrolase-lysin is also a new type of antibacterial molecule with great development potential. Lysin is a kind of hydrolytic enzyme encoded by double-stranded DNA phage in the late stage of infection of host, which can hydrolyze the peptidoglycan of bacterial cell wall, leading to the lysis of bacteria and the release of progeny phage. The lysis of Gram-positive bacteria has a relatively systematic and mature research, and a large number of natural lysins and chimeric lysins have been proved to be safe and effective in animal infection models, among which multiple lysins against drug-resistant Staphylococcus aureus infection have entered the clinical trial stage. The advantages of lysin, such as high efficiency, specificity, low drug resistance and synergistic effect with existing antibiotics, make it a new and promising antibacterial drug.

[0005] At present, the application of phage lysin is mainly to lyse Gram-positive bacteria, such as Staphylococcus aureus and Listeria, and the research on phage lysin with lysis activity to Gram-negative bacteria is less. Therefore, it is necessary to develop new phage lysin with high lysis activity to Gram-negative bacteria. SUMMARY

[0006] The purpose of the present application is to provide a peptidoglycan hydrolase LysPH1 with broad-spectrum lysis activity and its mutants and applications. The antibacterial peptide LysPH1-CP, the peptidoglycan hydrolase LysPH1 and the peptidoglycan hydrolase mutant LysPH1-K in the present application have broad-spectrum lysis activity and can lyse Gram-negative bacteria and Gram-positive bacteria.

[0007] In a first aspect, the present application provides an antibacterial peptide LysPH1-CP with broad-spectrum lysis activity, and the amino acid sequence of the antibacterial peptide LysPH1-CP is shown in SEQ ID NO: 1.

[0008] In a second aspect, the present application provides a peptidoglycan hydrolase LysPH1 with broad-spectrum lysis activity, the peptidoglycan hydrolase LysPH1 comprises the above antibacterial peptide LysPH1-CP, and the peptidoglycan hydrolase LysPH1 is selected from any one of the following proteins: A1) a protein with an amino acid sequence shown in SEQ ID NO: 2; A2) a protein with the same function obtained by changing one or more amino acid residues in the amino acid sequence shown in SEQ ID NO: 2, wherein the change is selected from at least one of substitution, deletion and addition; A3) a protein with more than 90% (for example, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to the amino acid sequence defined in any one of A1) to A2) and having the same function.

[0009] In a third aspect, the present application provides a peptidoglycan hydrolase mutant LysPH1-K having broad-spectrum lytic activity, the peptidoglycan hydrolase mutant LysPH1-K comprising the above-mentioned antibacterial peptide LysPH1-CP or the above-mentioned peptidoglycan hydrolase LysPH1; and the peptidoglycan hydrolase mutant LysPH1-K is selected from any one of the following proteins: A1) a protein having an amino acid sequence as set forth in SEQ ID NO: 3; A2) a protein having the same function as the amino acid sequence as set forth in SEQ ID NO: 3, which is obtained by changing one or more amino acid residues, wherein the change is selected from at least one of substitution, deletion, and addition; A3) a protein having 90% or more (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to the amino acid sequence as defined in any one of A1) to A2) and having the same function.

[0010] The above-mentioned antibacterial peptide LysPH1-CP, the above-mentioned peptidoglycan hydrolase LysPH1, or the above-mentioned peptidoglycan hydrolase mutant LysPH1-K provided by the present application can be a natural, recombinant, or synthetic active polypeptide, which can be a naturally purified product, a chemically synthesized product, or a product produced using a recombinant technique from a prokaryotic host (e.g., Escherichia coli) or a eukaryotic host (e.g., yeast, higher plants).

[0011] In some embodiments, the above-mentioned peptidoglycan hydrolase LysPH1 or the above-mentioned peptidoglycan hydrolase mutant LysPH1-K is obtained by introducing a recombinant vector containing a gene encoding the same into an expression host (e.g., Escherichia coli BL21 (DE3)) to obtain a recombinant genetically engineered strain, then culturing the recombinant genetically engineered strain, and inducing expression to obtain the peptidoglycan hydrolase LysPH1 or the peptidoglycan hydrolase mutant LysPH1-K.

[0012] In a fourth aspect, the present application provides a nucleic acid molecule encoding the above antibacterial peptide LysPH1-CP or the above peptidoglycan hydrolase LysPH1 or the above peptidoglycan hydrolase mutant LysPH1-K, the nucleic acid molecule being selected from any one of the following nucleic acid molecules: B1) a nucleic acid molecule having a nucleotide sequence as set forth in SEQ ID NO: 4, as set forth in SEQ ID NO: 5, or as set forth in SEQ ID NO: 6; B2) a nucleic acid molecule hybridizing with the nucleic acid molecule defined in B1) and encoding the above antibacterial peptide LysPH1-CP, the above peptidoglycan hydrolase LysPH1, or the above peptidoglycan hydrolase mutant LysPH1-K; B3) a nucleic acid molecule having 90% or more (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity with the nucleic acid molecule defined in B1) or B2) and encoding the above antibacterial peptide LysPH1-CP, the above peptidoglycan hydrolase LysPH1, or the above peptidoglycan hydrolase mutant LysPH1-K.

[0013] The above nucleic acid molecule provided by the present application can be generally obtained by a method of PCR amplification or artificial synthesis.

[0014] As used herein, the term "hybridizes under stringent conditions" means that two nucleic acid molecule fragments hybridize to each other under standard hybridization conditions as described in the section "Expression of cloned genes in E. coli" of Sambrook et al. Molecular Cloning: A Laboratory Manual (1989) (Cold Spring Harbor Laboratory Press, New York, USA). Such conditions are, for example, hybridization in 6.0 x SSC at 45°C followed by a washing step in 2 x SSC at 50°C. In order to select the stringency, the salt concentration in the washing step can be chosen, for example, between 2.0 x SSC at 50°C for low stringency and 2.0 x SSC at 50°C for high stringency. In addition, the temperature in the washing step can vary between about room temperature of about 22°C for low stringency and 65°C for high stringency.

[0015] As used herein, the term "sequence identity" can be assessed by eye or by computer software, such as the software programs described in Ausubel et al. eds. (2007) in Current Protocols in Molecular Biology. When a position in the compared sequences is occupied by the same base or amino acid, then the molecules are identical at that position. A degree of identity between two or more sequences can be expressed as a percentage (%) which can be used to assess the identity between related sequences. A polynucleotide sequence or an amino acid sequence has a certain percentage (e.g., 90%, 95%, 98%, or 99%) of "sequence identity" to another sequence if the percentage of bases or amino acids in the two sequences that are identical when the sequences are aligned.

[0016] In a fifth aspect, the present application provides an expression cassette comprising the above-mentioned nucleic acid molecule.

[0017] The expression cassette in the present application can further comprise an enhancer sequence or other regulatory elements.

[0018] In a sixth aspect, the present application provides a recombinant vector comprising the above-mentioned nucleic acid molecule or the above-mentioned expression cassette.

[0019] The recombinant vector in the present application comprises a cloning vector for replicating relevant sequences and an expression vector for expressing relevant genes, wherein the vector used in constructing the expression vector can be a pET28b vector.

[0020] In a seventh aspect, the present application provides a recombinant cell comprising the above-mentioned nucleic acid molecule, the above-mentioned expression cassette or the above-mentioned recombinant vector.

[0021] In some embodiments, the method for preparing the recombinant cell comprises the step of transforming the above-mentioned recombinant vector into an expression host cell.

[0022] In an eighth aspect, the present application provides a method for preparing the above-mentioned peptidoglycan hydrolase LysPH1 or the above-mentioned peptidoglycan hydrolase mutant LysPH1-K, characterized in that it comprises the following steps: culturing the above-mentioned recombinant cell, inducing expression to obtain a culture; and isolating the peptidoglycan hydrolase LysPH1 or the peptidoglycan hydrolase mutant LysPH1-K from the culture.

[0023] In the present application, the culture method and culture conditions are not particularly required, as long as the recombinant cell can grow normally. The methods for isolating the above-mentioned peptidoglycan hydrolase LysPH1 or the above-mentioned peptidoglycan hydrolase mutant LysPH1-K from the culture are all conventional methods in the art.

[0024] In some embodiments, the culture medium used in the method for preparing the above-mentioned peptidoglycan hydrolase LysPH1 or the above-mentioned peptidoglycan hydrolase mutant LysPH1-K is a culture medium that can express proteins in the art, preferably an LB culture medium.

[0025] In a ninth aspect, the present application provides the use of the above-mentioned antibacterial peptide LysPH1-CP, the above-mentioned peptidoglycan hydrolase LysPH1 or the above-mentioned peptidoglycan hydrolase mutant LysPH1-K in any one of the following: C1) lysing gram-negative bacteria and / or gram-positive bacteria; C2) preparing a drug for resisting gram-negative bacterial and / or gram-positive bacterial infection; C3) preparing a feed additive for resisting gram-negative bacterial and / or gram-positive bacterial infection.

[0026] In some embodiments, the gram-negative bacteria include one or more of Acinetobacter baumannii, Escherichia coli, Salmonella, Pseudomonas aeruginosa, and Klebsiella pneumoniae; the gram-positive bacteria include one or more of Staphylococcus aureus, Enterococcus faecalis, Streptococcus suis, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus pyogenes, and Streptococcus pneumoniae.

[0027] The present application has the following beneficial effects: Different from the prior art, the antibacterial peptide LysPH1-CP, the peptidoglycan hydrolase LysPH1 containing the antibacterial peptide LysPH1-CP, or the peptidoglycan hydrolase mutant LysPH1-K containing the peptidoglycan hydrolase LysPH1 all have good thermal stability, and have good lysis effect on Acinetobacter baumannii, Escherichia coli, Salmonella, Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus aureus, Enterococcus faecalis, Streptococcus suis, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus pyogenes, and Streptococcus pneumoniae in vitro, and can lyse gram-negative bacteria and gram-positive bacteria in a broad spectrum; meanwhile, the peptidoglycan hydrolase LysPH1 and the peptidoglycan hydrolase mutant LysPH1-K can be expressed in Escherichia coli in a soluble manner, and have high enzyme activity. Therefore, the antibacterial peptide LysPH1-CP, the peptidoglycan hydrolase LysPH1, and the peptidoglycan hydrolase mutant LysPH1-K have good application prospect in the research and development of anti-infection drugs and feed additives. BRIEF DESCRIPTION OF DRAWINGS

[0028] FIG. 1 is a chromatographic purity diagram of the antibacterial peptide LysPH1-CP in the embodiment 1 of the present application;

[0029] FIG. 2 is an SDS-PAGE gel diagram of the peptidoglycan hydrolase LysPH1 and the peptidoglycan hydrolase mutant LysPH1-K after purification in the embodiment 2 of the present application;

[0030] FIG. 3 is a result diagram of the influence of time on the lysis of A. baumannii 3437 by the peptidoglycan hydrolase LysPH1 in the embodiment 3 of the present application;

[0031] FIG. 4 is a result diagram of the activity of the peptidoglycan hydrolase LysPH1 in lysis of A. baumannii 3437 in different growth periods in the embodiment 4 of the present application;

[0032] FIG. 5 is a result diagram of the activity of the peptidoglycan hydrolase LysPH1 in lysis of A. baumannii 3437 after water bath heating at different temperatures for 1 h and cooling at room temperature for 1 h in the embodiment 5 of the present application;

[0033] FIG. 6 is a result diagram of the broad spectrum of the peptidoglycan hydrolase LysPH1 in lysis of different gram-negative bacteria and positive bacteria strains in vitro in the embodiment 6 of the present application;

[0034] Figure 7 is a graph showing the activity of the antibacterial peptide LysPH1-CP against strains at different growth stages according to the embodiment 7 of the present application;

[0035] Figure 8 is a graph showing the activity of the antibacterial peptide LysPH1-CP against A. baumannii 3437 after being heated at 100℃ for 1h and then cooled at room temperature for 1h according to the embodiment 8 of the present application;

[0036] Figure 9 is a graph showing the results of the peptidoglycan hydrolase LysPH1 in treating drug-resistant bacteria infection in vivo according to the embodiment 9 of the present application;

[0037] Figure 10 is a graph showing the bactericidal activity of the peptidoglycan hydrolase mutant LysPH1-K in vitro according to the embodiment 10 of the present application. DETAILED DESCRIPTION

[0038] The technical solutions in the embodiments of the present application will be described below in a clear and complete manner with reference to the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the scope of the present application.

[0039] The experimental methods not specified in the embodiments are generally performed according to the conventional experimental methods in the field of molecular biology, including but not limited to the experimental methods described in Molecular Cloning: A Laboratory Manual by M. R. Green, Molecular Biology by Robert F. Weaver, or the experimental methods suggested by the manufacturers of reagent kits and instrument equipment. The reagents and biological materials used in the embodiments can be obtained from commercial channels unless otherwise specified.

[0040] The primers and sequencing work used in the present application were completed by Wuhan Jin Kai Rui Biological Engineering Co., Ltd.; the antibacterial peptide LysPH1-CP was synthesized by Wuhan Dai'an Biological Technology Co., Ltd.

[0041] Example 1 Synthesis of antibacterial peptide LysPH1-CP

[0042] The inventors found a sequence with accession number WP_038349544.1 by constructing a phylogenetic tree through Blastp homology search on the NCBI (https: / / www.ncbi.nlm.nih.gov / ) website using P307 as a template, which was named as peptidoglycan hydrolase LysPH1. The inventors found an antibacterial peptide LysPH1-CP from the sequence of LysPH1, and the amino acid sequence of the antibacterial peptide LysPH1-CP is shown as SEQ ID NO. 1. The antibacterial peptide LysPH1-CP was synthesized by Wuhan Dai'an Biotechnology Co., Ltd., and the purity of the synthesized antibacterial peptide LysPH1-CP was 99.16%, and the molecular weight was 4550.31. The purity of the antibacterial peptide LysPH1-CP was determined by high performance liquid chromatography, and the chromatographic purity graph of the antibacterial peptide LysPH1-CP is shown in Figure 1, and the analysis results are shown in Table 1.

[0043] Amino acid sequence of antibacterial peptide LysPH1-CP (SEQ ID NO: 1)

[0044] Nucleotide sequence encoding antibacterial peptide LysPH1-CP (SEQ ID NO: 4)

[0045] Table 1 Composition table of antibacterial peptide LysPH1-CP detected by liquid chromatography

[0046] The liquid chromatography conditions of high performance liquid chromatography are as follows:

[0047] Chromatographic column: Kromasil 100-5-C18 (4.6 mm x 150 mm, 5 μm); mobile phase: mobile phase A is 0.1% TFA acetonitrile solution, mobile phase B is 0.1% TFA aqueous solution, gradient elution is carried out, and the gradient program elution is shown in Table 2; flow rate 1.0 mL / min, column temperature 25℃, injection volume 10 μL, detection wavelength 220 nm.

[0048] Table 2 Gradient program elution table

[0049] Example 2 Expression and purification of peptidoglycan hydrolase LysPH1 and peptidoglycan hydrolase mutant LysPH1-K

[0050] 2.1 Obtaining of sequences of peptidoglycan hydrolase LysPH1 and peptidoglycan hydrolase mutant LysPH1-K

[0051] The inventors found a sequence with accession number WP_038349544.1 by Blastp homology search on the NCBI (https: / / www.ncbi.nlm.nih.gov / ) website and constructed a phylogenetic tree, and named the sequence as peptidoglycan hydrolase LysPH1. The amino acid sequence of the peptidoglycan hydrolase LysPH1 is shown as SEQ ID NO. 2, and the nucleotide sequence encoding the peptidoglycan hydrolase LysPH1 is shown as SEQ ID NO. 5.

[0052] The amino acid sequence of the peptidoglycan hydrolase LysPH1 (SEQ ID NO: 2)

[0053] The nucleotide sequence encoding the peptidoglycan hydrolase LysPH1 (SEQ ID NO: 5)

[0054] Further, a sequence was inserted into the amino acid sequence shown as SEQ ID NO. 2 to obtain a peptidoglycan hydrolase mutant LysPH1-K. The amino acid sequence of the peptidoglycan hydrolase mutant LysPH1-K is shown as SEQ ID NO. 3, and the nucleotide sequence encoding the peptidoglycan hydrolase mutant LysPH1-K is shown as SEQ ID NO. 6.

[0055] The amino acid sequence of the peptidoglycan hydrolase mutant LysPH1-K (SEQ ID NO: 3)

[0056] The nucleotide sequence encoding the peptidoglycan hydrolase mutant LysPH1-K (SEQ ID NO: 6)

[0057] 2.2 Construction of a recombinant expression vector

[0058] The 5' end and 3' end of the gene fragment encoding the peptidoglycan hydrolase LysPH1 and the gene fragment encoding the peptidoglycan hydrolase mutant LysPH1-K were added with NcoI restriction site sequence and XhoI restriction site sequence, respectively, and then synthesized by a biological company. The above synthesized gene fragment encoding the peptidoglycan hydrolase LysPH1 and the gene fragment encoding the peptidoglycan hydrolase mutant LysPH1-K were subjected to enzyme digestion with NcoI restriction enzyme and XhoI restriction enzyme, and then connected with the vector pET28b subjected to enzyme digestion with the same NcoI restriction enzyme and XhoI restriction enzyme, to obtain recombinant expression vectors pET28b-LysPH1 and pET28b-LysPH1-K, respectively, and then the recombinant expression vectors pET28b-LysPH1 and pET28b-LysPH1-K were respectively transformed into E. coli BL21 (DE3), positive clones were selected, and sequencing verification was performed, to obtain E. coli BL21 (DE3)-pET28b-LysPH1 recombinant bacteria and E. coli BL21 (DE3)-pET28b-LysPH1-K recombinant bacteria, respectively.

[0059] 2.3 Expression and purification of peptidoglycan hydrolase LysPH1 and peptidoglycan hydrolase mutant LysPH1-K

[0060] The above prepared E. coli BL21 (DE3)-pET28b-LysPH1 recombinant bacteria and E. coli BL21 (DE3)-pET28b-LysPH1-K recombinant bacteria were cultured in 500 mL LB medium containing 50 μg / mL kanamycin to OD 600nm 0.5-0.6, and then induced with 0.5 mM isopropyl β-D-thiogalactoside (Thermo Scientific) for 16 h; then the cells were collected by centrifugation at 4°C, 8000 rpm for 10 min, washed once with 20 mM imidazole, and resuspended in 20 mM imidazole; the cells were broken by a cell disrupter on ice, and the supernatant was filtered through a 0.22 μm filter membrane and subjected to affinity chromatography by passing through a nickel column, and the fragments eluted with 250 mM imidazole were collected and dialyzed in 20 mM Tris buffer (pH 6.8) at 4°C overnight, to obtain the peptidoglycan hydrolase LysPH1 and the peptidoglycan hydrolase mutant LysPH1-K. The SDS-PAGE gel map of the purified peptidoglycan hydrolase LysPH1 and the peptidoglycan hydrolase mutant LysPH1-K is shown in FIG. 2.

[0061] As can be seen from FIG. 2, the size of the peptidoglycan hydrolase LysPH1 is about 16.5 kDa, and the size of the peptidoglycan hydrolase mutant LysPH1-K is about 19.5 kDa.

[0062] Effect of time on the activity of the peptidoglycan hydrolase LysPH1 in cleaving A. baumannii 3437

[0063] A. baumannii 3437 was cultured to the logarithmic phase (OD 600nm = 0.4-0.6), and the precipitate was collected by low-temperature centrifugation, washed once with a Tris-HCl buffer, and then dissolved in the above buffer to obtain a bacterial solution of A. baumannii 3437. The peptidoglycan hydrolase LysPH1 prepared in Example 2 was mixed with the above bacterial solution so that the final concentration of the peptidoglycan hydrolase LysPH1 was 50 μg / ml, and an equal amount of a mixture of the buffer and the above bacterial solution was used as a negative control. Incubation was performed at 37°C, and sampling was performed at different time points for plate counting, and the results are shown in FIG. 3.

[0064] As can be seen from FIG. 3, the number of A. baumannii 3437 bacteria was significantly reduced after 1 h of incubation, and the number of A. baumannii 3437 bacteria decreased most rapidly within 15 min. The above results show that the peptidoglycan hydrolase LysPH1 has good cleavage activity on A. baumannii 3437.

[0065] Example 4: Activity test of the peptidoglycan hydrolase LysPH1 in cleaving A. baumannii 3437 at different growth stages

[0066] A. baumannii 3437 was cultured to the logarithmic phase (OD 600nm = 0.4-0.6) and the stationary phase (OD 600nm = 1.2-1.4), and the precipitate was collected by low-temperature centrifugation, washed once with a Tris-HCl buffer, and then dissolved in the above buffer to obtain a bacterial solution of A. baumannii 3437. The peptidoglycan hydrolase LysPH1 prepared in Example 2 was mixed with the above bacterial solution so that the final concentration of the peptidoglycan hydrolase LysPH1 was 50 μg / ml, and an equal amount of a mixture of the buffer and the above bacterial solution was used as a negative control. Incubation was performed at 37°C for 1 h, and plate counting was performed, and the results are shown in FIG. 4.

[0067] As can be seen from FIG. 4, the peptidoglycan hydrolase LysPH1 has higher cleavage activity on A. baumannii 3437 in the logarithmic phase.

[0068] Example 5: Effect of different temperatures on the activity of the peptidoglycan hydrolase LysPH1 in cleaving A. baumannii 3437

[0069] A.baumannii 3437 was cultured to logarithmic phase (OD 600nm = 0.4-0.6), and the precipitate was collected by low temperature centrifugation, washed once with Tris-HCl buffer, and then resuspended in the above buffer to obtain A.baumannii 3437 bacterial solution. The peptidoglycan hydrolase LysPH1 prepared in Example 2 was diluted with the above buffer to a final concentration of 50 μg / ml, and then heated in a water bath at 4, 25, 37, 45, 55, 65, 75, 85, 100°C for 1 h, and cooled at room temperature for 1 h. The mixture of the above bacterial solution and the peptidoglycan hydrolase LysPH1 was incubated at 37°C for 1 h, and then plated for counting. The negative control was a mixture of equal amounts of buffer and the above bacterial solution. The results are shown in Figure 5.

[0070] As can be seen from Figure 5, the lysis activity of the peptidoglycan hydrolase LysPH1 on A.baumannii 3437 was slightly reduced as the temperature increased to 45°C, and the lysis activity of the peptidoglycan hydrolase LysPH1 on A.baumannii 3437 was higher as the temperature continued to rise, indicating that the peptidoglycan hydrolase LysPH1 has good thermal stability.

[0071] Example 6: Test of the broad-spectrum lysis of the peptidoglycan hydrolase LysPH1 on different strains of gram-negative and positive bacteria in vitro

[0072] A.baumannii, E.coli, Salmonella, P.aeruginosa, and K.pneumoniae strains were cultured to logarithmic phase (OD 600nm = 0.4-0.6), and S.aureus, E.faecalis, S.suis, S.agalactiae, S.dysgalactiae, S.pyogenes, and S.pneumoniae strains were cultured to logarithmic phase (OD 600nm = 0.4-0.6) and stationary phase (OD 600nm=1.2~1.4), after collecting the precipitate by low-temperature centrifugation, wash it once with Tris-HCl buffer, and then dissolve the washed precipitate in the above buffer to obtain various bacterial solutions. Take the peptidoglycan hydrolase LysPH1 prepared in Example 2 and mix it with the above bacterial solutions to make the final concentration of peptidoglycan hydrolase LysPH1 50 μg / ml, of which the final concentration of peptidoglycan hydrolase LysPH1 mixed with Enterococcus faecalis is 10 μg / ml. At the same time, use an equal volume of buffer and the above bacterial solution as a negative control. After incubation at 37℃ for 1 h, perform TLC counting. The results are shown in Figure 6.

[0073] As shown in Figure 6, peptidoglycan hydrolase LysPH1 exhibits good lytic effects on a variety of Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus aureus, Enterococcus faecalis, Streptococcus suis, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus pyogenes, and Streptococcus pneumoniae. The results indicate that peptidoglycan hydrolase LysPH1 has a broad spectrum of lytic activity against both Gram-negative and Gram-positive bacteria.

[0074] Example 7: Effect of antimicrobial peptide LysPH1-CP on the activity of strains at different growth stages

[0075] Acinetobacter baumannii 3437, Pseudomonas aeruginosa 2555, Staphylococcus aureus N315, and Enterococcus faecalis EF493 were cultured to the logarithmic developmental stage (OD200). 600nm =0.4~0.6) and the stable period (OD) 600nm =1.2~1.4), after collecting the precipitate by low-temperature centrifugation, wash it once with Tris-HCl buffer, and then dissolve the washed precipitate in the above buffer to obtain various bacterial solutions. Take the antimicrobial peptide LysPH1-CP synthesized in Example 1 and mix it with the above bacterial solutions to make the final concentration of antimicrobial peptide LysPH1-CP 50 μg / ml. Use an equal volume of buffer and the above bacterial solution as a negative control. Incubate at 37°C for 1 h and perform TLC counting. The results are shown in Figure 7.

[0076] As can be seen from Figure 7, the antimicrobial peptide LysPH1-CP has a higher bactericidal effect on bacteria in the logarithmic phase than on those in the stationary phase.

[0077] Example 8: Activity test of antimicrobial peptide LysPH1-CP after heating in a 100°C water bath for 1 hour and cooling at room temperature for 1 hour followed by cleavage of A. baumannii 3437.

[0078] Acinetobacter baumannii 3437 was cultured to the logarithmic growth phase (OD2). 600nm= 0.4-0.6), and after the precipitate was collected by low temperature centrifugation, the precipitate was washed once with Tris-HCl buffer, and then the washed precipitate was dissolved in the above buffer to obtain A. baumannii 3437 bacterial solution. The antibacterial peptide LysPH1-CP synthesized in Example 1 was mixed with the above bacterial solution so that the final concentration of the antibacterial peptide LysPH1-CP was 50 μg / ml. An equal amount of buffer and the above bacterial solution mixture was used as a negative control. The mixtures were incubated at 37°C for 1 h, and then the plate count was performed. The results are shown in Fig. 8.

[0079] As can be seen from Fig. 8, the bactericidal activity of the antibacterial peptide LysPH1-CP remained essentially unchanged after the antibacterial peptide LysPH1-CP was heated at 100°C for 1 h and then cooled at room temperature for 1 h. The results show that the antibacterial peptide LysPH1-CP has good thermal stability.

[0080] Example 9 In vivo test of peptidoglycan hydrolase LysPH1 and peptidoglycan hydrolase mutant LysPH1-K for treatment of drug-resistant bacteria infection

[0081] On the first day, BALB / c mice were injected intraperitoneally with anesthetic isoflurane, and then the hair on the back was cut and removed with depilatory cream to form a 2 cm 2 hairless area on the back. On the second day, the back of the mouse was disinfected with 75% alcohol, and then wiped with sterile water to remove the disinfectant. Then A. baumannii 3437 (10 8 CFU / mL / mouse, 25 μl / mouse) was injected on the right side of the back. After the bacteria were colonized for 24 h, the mice were randomly divided into 3 groups for drug treatment, in which Tris-HCl was used as a negative control, antibiotic minocycline (4 μg) was used as a positive control, and the experimental groups were injected with peptidoglycan hydrolase LysPH1 or peptidoglycan hydrolase mutant LysPH1-K (10 μg). The body weight of the mice was monitored every 24 hours. After 5 days of treatment, the mice were killed by cervical dislocation, and the number of bacteria in the infected skin on the back was detected. The results are shown in Fig. 9.

[0082] As can be seen from Fig. 9, the peptidoglycan hydrolase LysPH1 and the peptidoglycan hydrolase mutant LysPH1-K can effectively reduce the number of pathogenic bacteria in the infected skin tissue, and their effects are better than that of the antibiotic. Moreover, the peptidoglycan hydrolase mutant LysPH1-K has a better bactericidal effect than the wild-type peptidoglycan hydrolase LysPH1.

[0083] Example 10 In vitro test of bactericidal activity of peptidoglycan hydrolase LysPH1 and peptidoglycan hydrolase mutant LysPH1-K

[0084] A.baumannii 3437, A.baumannii 140, A.baumannii 143, A.baumannii 147, P. aeruginosa 2555, P. aeruginosa 2, P. aeruginosa 53, P. aeruginosa 2965, P. aeruginosa 3763, E. coli 091, E. coli 097, E. coli 0149, E. coli MG1655, E. coli O157:H7, K. pneumoniae 23, K. pneumoniae 27, S. aureus N315, S. mutans UA159, S. agalactiae C001 and S. pyogenes 12344 were cultured to logarithmic phase (OD 600nm =0.4~0.6), and the precipitate was collected by low temperature centrifugation, washed once with PBS buffer, and then the washed precipitate was dissolved in the above buffer to obtain a bacterial suspension. The peptide glycan hydrolase LysPH1 and the peptide glycan hydrolase mutant LysPH1-K prepared in Example 2 were mixed with the above bacterial solution so that the final concentration of the peptide glycan hydrolase LysPH1 and the peptide glycan hydrolase mutant LysPH1-K was 25 μg / ml, and an equal amount of buffer and the above bacterial solution were mixed as a negative control. After incubation at 37°C for 1 h, spot plate counting was performed, and the results are shown in FIG. 10.

[0085] As can be seen from FIG. 10, the peptide glycan hydrolase mutant LysPH1-K has higher bactericidal activity than the wild-type peptide glycan hydrolase LysPH1 in vitro against A. baumannii, P. aeruginosa, E. coli, and K. pneumoniae, and the results show that the peptide glycan hydrolase mutant LysPH1-K having higher bactericidal activity is further inserted into the wild-type peptide glycan hydrolase LysPH1.

[0086] In summary, the antibacterial peptide LysPH1-CP, the peptide glycan hydrolase LysPH1 containing the antibacterial peptide LysPH1-CP, and the peptide glycan hydrolase mutant LysPH1-K containing the peptide glycan hydrolase LysPH1 all have good thermal stability, and have good lysis effect in vitro on A. baumannii, E. coli, Salmonella, P. aeruginosa, K. pneumoniae, S. aureus, E. faecalis, S. suis, S. agalactiae, S. dysgalactiae, S. pyogenes and S. pneumoniae, and can lyse gram-negative bacteria and gram-positive bacteria in a broad spectrum.

[0087] It should be noted that the above embodiments all belong to the same inventive concept, and the description of each embodiment has its own emphasis. If the description is not exhaustive in an individual embodiment, the description in other embodiments can be referred to.

[0088] The above-described embodiments only express the implementation of the present application, and the description is more specific and detailed, but it should not be understood as a limitation on the scope of the patent. It should be noted that for ordinary skilled persons in the art, without departing from the inventive concept, a number of modifications and improvements can be made, which are within the scope of the present application. Therefore, the protection scope of the present application patent should be subject to the appended claims.

Claims

1. An antibacterial peptide LysPH1-CP having broad spectrum lytic activity, characterized in that, The amino acid sequence of the antibacterial peptide LysPH1-CP is shown in SEQ ID NO:

1.

2. A peptidoglycan-hydrolysing enzyme LysPH1 having broad-spectrum lytic activity, characterized in that, The peptidoglycan hydrolase LysPH1 comprises the antibacterial peptide LysPH1-CP according to claim 1, and the peptidoglycan hydrolase LysPH1 is selected from any one of the following proteins: A1) a protein having an amino acid sequence shown in SEQ ID NO: 2; A2) a protein having the same function as the amino acid sequence shown in SEQ ID NO: 2, which is obtained by changing one or several amino acid residues, wherein the change is selected from at least one of substitution, deletion, addition; A3) a protein having 90% or more sequence identity to the amino acid sequence defined in any one of A1) to A2) and having the same function.

3. A mutant of a peptidoglycan hydrolase, LysPHl-K, having broad-spectrum lytic activity, characterized in that, The peptidoglycan hydrolase mutant LysPH1-K comprises the antibacterial peptide LysPH1-CP according to claim 1 or the peptidoglycan hydrolase LysPH1 according to claim 2; and the peptidoglycan hydrolase mutant LysPH1-K is selected from any one of the following proteins: A1) a protein having an amino acid sequence shown in SEQ ID NO: 3; A2) a protein having the same function as the amino acid sequence shown in SEQ ID NO: 3, which is obtained by changing one or several amino acid residues, wherein the change is selected from at least one of substitution, deletion, addition; A3) a protein having 90% or more sequence identity to the amino acid sequence defined in any one of A1) to A2) and having the same function.

4. A nucleic acid molecule encoding the antibacterial peptide LysPHl-CP of claim 1 or the peptidoglycan-hydrolysing enzyme LysPHl of claim 2 or the peptidoglycan-hydrolysing enzyme mutant LysPHl-K of claim 3, characterized in that, The nucleic acid molecule is selected from any one of the following nucleic acid molecules: B1) a nucleic acid molecule having a nucleotide sequence shown in SEQ ID NO: 4, shown in SEQ ID NO: 5 or shown in SEQ ID NO: 6; B2) a nucleic acid molecule hybridizing to the nucleic acid molecule defined in B1) and encoding the antibacterial peptide LysPH1-CP according to claim 1, the peptidoglycan hydrolase LysPH1 according to claim 2 or the peptidoglycan hydrolase mutant LysPH1-K according to claim 3; B3) a nucleic acid molecule having 90% or more sequence identity to the nucleic acid molecule defined in B1) or B2) and encoding the antibacterial peptide LysPH1-CP according to claim 1, the peptidoglycan hydrolase LysPH1 according to claim 2 or the peptidoglycan hydrolase mutant LysPH1-K according to claim 3.

5. An expression cassette comprising, comprising the nucleic acid molecule according to claim 4.

6. A recombinant vector, characterized in that, comprising the nucleic acid molecule according to claim 4 or the expression cassette according to claim 5.

7. A recombinant cell, characterized in that, comprising the nucleic acid molecule according to claim 4, the expression cassette according to claim 5 or the recombinant vector according to claim 6.

8. A method for the production of the peptidoglycan-hydrolysing enzyme LysPHl of claim 2 or the peptidoglycan-hydrolysing enzyme mutant LysPHl-K of claim 3, characterized in that, comprising the following steps: culturing the recombinant cell according to claim 7, inducing expression to obtain a culture; isolating the peptidoglycan hydrolase LysPH1 or the peptidoglycan hydrolase mutant LysPH1-K from the culture.

9. Use of the antibacterial peptide LysPHl-CP of claim 1, the peptidoglycan hydrolase LysPHl of claim 2 or the peptidoglycan hydrolase mutant LysPHl-K of claim 3 in any one of the following: C1) for lysing Gram-negative and / or Gram-positive bacteria; C2) for preparing a medicament against Gram-negative and / or Gram-positive bacterial infection; C3) for preparing a feed additive against Gram-negative and / or Gram-positive bacterial infection.

10. Use according to claim 9, characterized in that, The Gram-negative bacteria include one or more of Acinetobacter baumannii, Escherichia coli, Salmonella, Pseudomonas aeruginosa and Klebsiella pneumoniae; the Gram-positive bacteria include one or more of Staphylococcus aureus, Enterococcus faecalis, Streptococcus suis, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus pyogenes and Streptococcus pneumoniae.

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