Antimicrobial peptides P104 and lysine LysP53 with broad lytic activity and their applications

Abstract

The present invention provides an antimicrobial peptide P104 and lysine LysP53 having broad lytic activity, and their applications. The amino acid sequence of the antimicrobial peptide P104 is shown in SEQ ID NO.1, and the gene sequence of the antimicrobial peptide P104 is shown in SEQ ID NO.3. The amino acid sequence of the antimicrobial peptide LysP53 is shown in SEQ ID NO.2, and the gene sequence of the antimicrobial peptide LysP53 is shown in SEQ ID NO.4. The antimicrobial peptide P104 and the lysine LysP53 have good lytic effect against Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Escherichia coli in vitro, and can lyse a broad range of Gram-negative bacteria. In addition, the lysine LysP53 can be soluble expressed in Escherichia coli and has high enzymatic activity. Therefore, the antimicrobial peptide P104 and the lysine LysP53 have excellent applicability in the research and development of anti-infective drugs.

Description

[Technical field]

[0001] The present invention relates to the field of biopharmaceuticals, and more particularly to an antimicrobial peptide P104 and lysine LysP53 having broad lytic activity, and their applications. [Background technology]

[0002] In the past, antibiotics were considered the most powerful weapon for treating bacterial infectious diseases, but as the abuse of antibiotics becomes more serious day by day, the problem of bacterial drug resistance is also becoming more serious. However, the research and development of new antibiotics is lagging far behind the rate at which drug-resistant bacteria emerge. In addition, the number of alternative new antibiotics is extremely small and their costs are rising, so many people infected with drug-resistant bacteria die helplessly in clinical practice. Under the severe effects of antibiotic resistance, bacteriophages, which can replace antibiotics as a new type of antibacterial agent, have attracted the attention of scholars at home and abroad.

[0003] Bacteriophages are a type of virus that use microorganisms (bacteria, fungi, actinomycetes, or spirochetes) as hosts. Bacteriophages have no cellular structure, and are composed only of nucleocapsid proteins and their internal genetic material. They must depend on the host for replication and growth. Bacteriophages are widespread in nature as natural killers of bacteria, and are considered to be the most abundant and diverse organisms on Earth. There are approximately 10 times as many bacteriophages as there are bacteria. For almost a century, the safety and efficacy of bacteriophage therapy has been demonstrated in many animal experiments. Upon infecting a host bacterial cell, lytic phages immediately begin to express their own early genes, and the expression products of these early genes degrade the host's DNA. This stops the host's gene expression, and they use the host's DNA replication apparatus to synthesize large amounts of their own nucleic acids. After sufficient bacteriophages are generated, these nucleic acids serve as templates to produce large amounts of bacteriophage structural proteins. These structural proteins process and encapsulate the bacteriophage genome to produce the next generation of bacteriophage particles. When the bacteriophage regulatory proteins accumulate to a certain level, the bacteriophage perforin is expressed in large amounts. Then, perforin forms a pore in the host cell, allowing the bacteriophage lysin to penetrate the inner membrane and access the cell wall, thereby achieving the goal of lysing the bacteria.

[0004] In addition to the completed bacteriophage particles being potential antibacterial agents, the peptidoglycan hydrolase (lysin) encoded by the bacteriophage is also a new type of antibacterial molecule with excellent development potential. Lysin is a type of hydrolase encoded by double-stranded DNA bacteriophages at the late stage of host infection, which hydrolyzes the peptidoglycan in the bacterial cell wall, lysing the bacteria and releasing progeny bacteriophages. There are comparative and mature studies on lysin against Gram-positive bacteria, and the safety and efficacy of many natural and chimeric lysins have been demonstrated in animal infection models. Among them, several lysins against drug-resistant Staphylococcus aureus infections have already progressed to the clinical trial stage. Lysin is a new antibacterial drug with excellent future potential due to its advantages such as high efficiency, specificity, low drug resistance, and synergistic effects with conventional antibiotics.

[0005] At present, bacteriophage lysin is mainly applied to lyse Gram-positive bacteria, such as Staphylococcus aureus, Listeria monocytogenes, and Enterococcus faecalis, and there are few studies on bacteriophage lysin with lytic activity against Gram-negative bacteria. Therefore, there is a need to develop a new bacteriophage lysin with highly efficient lytic activity against Gram-negative bacteria. Summary of the Invention [Problem to be solved by the invention]

[0006] The object of the present invention is to provide an antimicrobial peptide P104 and lysine LysP53 having broad-spectrum lytic activity and their applications, which can overcome the drawbacks of the prior art. The antimicrobial peptide P104 and the lysine LysP53 have good lytic effects against Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae and Escherichia coli in vitro, and can lyse a broad range of Gram-negative bacteria. [Means for solving the problem]

[0007] One of the objects of the present invention is to provide an antimicrobial peptide P104 that has broad spectrum lytic activity.

[0008] In the antimicrobial peptide P104 having broad spectrum lytic activity, the amino acid sequence of the antimicrobial peptide P104 is as shown in SEQ ID NO.1.

[0009] Furthermore, the gene sequence of the antimicrobial peptide P104 is shown in SEQ ID NO.3.

[0010] Another object of the present invention is to provide a lysine, LysP53, which has broad spectrum lytic activity.

[0011] LysP53 has broad spectrum lytic activity, and contains the antimicrobial peptide P104. The amino acid sequence of LysP53 is as shown in SEQ ID NO.2.

[0012] Furthermore, the gene sequence of said lysine LysP53 is as shown in SEQ ID NO.4.

[0013] Furthermore, the sequences of the primers for amplifying the nucleotide sequence of said lysine LysP53 are as shown in SEQ ID NO.5 and SEQ ID NO.6.

[0014] The present invention further provides a recombinant expression vector comprising the gene for lysine LysP53.

[0015] In the recombinant expression vector containing the gene for lysine LysP53, the recombinant expression vector is a prokaryotic expression vector pET28a-LysP53.

[0016] The present invention further provides a host cell comprising the recombinant expression vector.

[0017] In the host cell containing the recombinant expression vector, the host cell is E. coli BL21(DE3).

[0018] Furthermore, the method for preparing lysine LysP53 comprises the steps of:

[0019] S1: Amplify the lysine LysP53 gene from the genome of bacteriophage P53.

[0020] S2: Constructing a recombinant expression vector pET28a-LysP53 that expresses the gene for lysine LysP53.

[0021] S3: Transform the recombinant expression vector pET28a-LysP53 into competent cells of E. coli BL21 (DE3), and screen for engineered strains expressing the lysine LysP53 gene.

[0022] S4: Expression is induced using IPTG to obtain the expression product of the recombinant gene.

[0023] S5: The expression product of the recombinant gene is purified and separated using a nickel column to obtain the lysine LysP53.

[0024] Finally, the present invention provides the application of the antimicrobial peptide P104 with broad lytic activity and / or the lysine LysP53 with broad lytic activity in the lysis of Gram-negative bacteria.

[0025] Further, the Gram-negative bacteria include some of Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Escherichia coli. Effect of the Invention

[0026] Compared with the prior art, the present invention has the following advantages:

[0027] The present invention provides an antimicrobial peptide P104 having a broad lytic activity and a lysine LysP53 having a broad lytic activity. The antimicrobial peptide P104 and the lysine LysP53 have good lytic effects against Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae and Escherichia coli in vitro, and can lyse a broad range of Gram-negative bacteria. The lysine LysP53 can be soluble expressed in Escherichia coli and has high enzymatic activity. Therefore, the antimicrobial peptide P104 and the lysine LysP53 have excellent applicability in the research and development of anti-infective drugs.

[0028] In order to more clearly describe the technical solutions of the embodiments of the present invention, the drawings that need to be used in the description of the embodiments are briefly described below. It should be noted that the drawings described below are only some of the embodiments of the present invention, and those skilled in the art can obtain other drawings from these drawings without requiring creative labor. [Brief description of the drawings]

[0029] [Figure 1] FIG. 1 is a graph showing the purity of the antimicrobial peptide P104 in Example 1 of the present invention when it was subjected to chromatography. [Diagram 2] FIG. 2 is an image of an SDS-PAGE gel after purification of lysine LysP53 in Example 2 of the present invention. [Diagram 3] FIG. 3 is a graph showing the results of time-dependent changes when A. baumannii WHG40137 was dissolved with lysine LysP53 in Example 3 of the present invention. [Figure 4] FIG. 4 is a graph showing the activity results when lysine LysP53 in Example 4 of the present invention lyses A. baumannii WHG40137 at different growth stages. [Diagram 5] FIG. 5 is a graph showing the activity results when A. baumannii WHG40137 was lysed at different pH values ​​by lysine LysP53 in Example 5 of the present invention. [Figure 6]FIG. 6 is a diagram showing the results of broad spectrum lysis of different Gram-negative bacterial strains in vitro by lysine LysP53 in Example 6 of the present invention. [Figure 7] FIG. 7 is a diagram showing the activity results when A. baumannii WHG40137 was killed by lysine LysP53 at different EDTA concentrations in Example 7 of the present invention. [Figure 8] FIG. 8 is a graph showing the activity results when lysine LysP53 in Example 8 of the present invention killed A. baumannii WHG40137 on the skin surface of a mouse. [Figure 9] FIG. 9 is a diagram showing the results of broad spectrum lysis of different Gram-negative bacterial strains in vitro by the antimicrobial peptide P104 and lysine LysP53 in Example 8 of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] In the following, the technical solutions of the embodiments of the present invention will be clearly and concisely described in combination with the drawings in the embodiments of the present invention. It should be noted that the described embodiments are only some of the embodiments of the present invention, and are not all of the embodiments. All other embodiments that a person skilled in the art can obtain based on the embodiments of the present invention without creative labor are all within the scope of protection of the present invention.

[0031] Unless otherwise specified, all of the reagents, gram-negative bacterial strains and equipment used in the present invention are commercially available.

[0032] Unless otherwise stated, the experimental methods used in the present invention are all common experimental methods. In addition, the primers used and the sequencing operations were completed by Shanghai Biotechnology Co., Ltd., and the antibacterial peptide P104 was synthesized by Shanghai Qiangyao Biotechnology Co., Ltd.

[0033] The gene sequence of the antimicrobial peptide P104 and the gene sequence of the lysine LysP53 in the present invention were obtained from the genome of bacteriophage P53, which has been uploaded to the NCBI (https: / / www.ncbi.nlm.nih.gov / ) website under the accession number MW590698.

[0034] Example 1 Synthesis of antimicrobial peptide P104

[0035] The inventors have found the antimicrobial peptide P104 from the genome of bacteriophage P53 through a large amount of experiments and analysis. The amino acid sequence of the antimicrobial peptide P104 is as shown in SEQ ID NO.1, and the gene sequence of the antimicrobial peptide P104 is as shown in SEQ ID NO.3. The antimicrobial peptide P104 was synthesized by Shanghai Qianyao Biotechnology Co., Ltd. The synthesized antimicrobial peptide P104 had a purity of 98.58% and a molecular weight of 3885.57. The purity of the antimicrobial peptide P104 was measured by high performance liquid chromatography. See FIG. 1 for a diagram of the purity of the antimicrobial peptide P104 when it was subjected to chromatography. The analysis results are shown in Table 1 below.

[0036] [Table 1]

[0037] The liquid phase conditions for the high performance liquid chromatography were as follows:

[0038] Chromatography column: Kromasil 100-5-C18 (4.6 mm x 150 mm, 5 μm).

[0039] Mobile phase: Gradient elution was performed using a 0.1% TFA acetonitrile solution as mobile phase A and a 0.1% TFA aqueous solution as mobile phase B. For gradient elution, see Table 2. The flow rate was 1.0 mL / min, the column temperature was 25° C., the injection volume was 10 μL, and the detection wavelength was 220 nm.

[0040] [Table 2]

[0041] Example 2 Expression and purification of lysine LysP53

[0042] Through extensive experiments and analysis, the inventors have found lysine LysP53 in the genome of bacteriophage P53, the amino acid sequence of said lysine LysP53 being as shown in SEQ ID NO.2, and the gene sequence of said lysine LysP53 being as shown in SEQ ID NO.4.

[0043] 2.1 Construction of recombinant expression vectors

[0044] Based on the gene sequence of lysine LysP53, the following primer sequences were designed using a primer design software commonly used in the prior art.

[0045] 53P37-F: ctttaagaaggagatataccatggATGACGATGACAACAAAACGTA (as shown in SEQ ID NO.5, with NcoI enzyme cleavage site).

[0046] 53P37-R: tggtggtggtggtggtgctcgagCCCCGCCAATTCAAAGTGTGGGCT (as shown in SEQ ID NO.6, with XhoI enzyme cleavage site).

[0047] The target gene fragment may be synthesized by a biotechnology company, or the genome of bacteriophage P53 may be used as a template to amplify the target gene. In the present invention, the genome of bacteriophage P53 was used as a template to amplify the target gene. The amplification system was a 50 μL reaction system, and specifically, it contained 5 μL of 10×buffer (Mg2+ concentration is 20 mmol / L), 4 μL of dNTPs (2.5 mmol / L), 2 μL of 53P37-F (10 μmol / L), 2 μL of 53P37-R (10 μmol / L), 0.5 μL of template DNA, 0.2 μL of Taq DNA polymerase (5 U / μL), and 36.3 μL of ddH20 as components.

[0048] PCR reaction conditions: 30 cycles of pre-deformation at 98°C for 5 min, denaturation at 98°C for 10 s, annealing at 55°C for 15 s, and extension at 72°C for 50 s, followed by extension at 72°C for 5 min. After the reaction was completed, the PCR amplified product was subjected to agarose gel electrophoresis detection, purification, and sequence verification, and then enzymatic cleavage using NcoI and XhoI restriction enzymes. The recombinant expression vector pET28a-LysP53 was obtained by combining with the vector pET28a that had been enzymatically cleaved with the same NcoI and XhoI restriction enzymes. The recombinant expression vector pET28a-LysP53 was then introduced into E. coli BL21 (DE3), and a positive clone was selected and sequence verification was performed.

[0049] 2.2 Expression and purification of lysine LysP53

[0050] E. coli BL21(DE3) containing the correct sequence and pET28a-LysP53 was cultured in 500 mL of LB medium containing 50 μg / mL kanamycin until OD600 reached 0.5-0.6, and then induced with 0.5 mM isopropyl-β-D-thiogalactopyranoside (Thermo Scientific) for 16 h. The cells were then collected by centrifugation at 4°C and 8000 rpm for 10 min, washed once with 20 mM imidazole, and resuspended in 20 mM imidazole. The cells were disrupted on ice using a cell disrupter and centrifuged at 4°C and 8000 rpm for 20 min. The supernatant was then filtered using a 0.22 μm membrane filter, and the fragments eluted with 250 mM imidazole were collected by affinity chromatography through a nickel column. The lysine LysP53 was obtained by placing it in 20 mM Tris buffer (pH 6.8) at 4° C. and dialyzing it overnight. See FIG. 2 for an SDS-PAGE gel image of the purified lysine LysP53.

[0051] As is clear from FIG. 2, the size of the lysine LysP53 was approximately 24 kDa.

[0052] Example 3 Effect of time on the lytic activity of lysine LysP53 against A. baumannii WHG40137

[0053] Acinetobacter baumannii A. baumannii WHG40137 was cultured until the logarithmic phase (OD600nm=0.4-0.6), centrifuged at low temperature to collect the precipitate, and then washed once with Tris-HCl buffer. The washed precipitate was dissolved in the above buffer to obtain a bacterial solution of A. baumannii WHG40137. Next, lysine LysP53 prepared in Example 2 was taken and mixed with the above bacterial solution so that the final concentration of lysine LysP53 was 100μg / ml. In addition, a mixture of an equal amount of buffer and the above bacterial solution was used as a negative control. These were cultured under 37°C conditions, and samples were taken at different times, and then counted using a cell count plate. The results obtained above are shown in FIG. 3.

[0054] As can be seen from the results in Figure 3, after 15 min of incubation, the concentration of Acinetobacter baumannii WHG40137 in the experimental group was significantly reduced, and after 1 h of incubation, the concentration of Acinetobacter baumannii WHG40137 was reduced to 10 cfu / ml. The results showed that lysine LysP53 had good lytic activity against Acinetobacter baumannii WHG40137.

[0055] Example 4 Results of lytic activity of lysine LysP53 against A. baumannii WHG40137 at different growth stages

[0056] Acinetobacter baumannii A. baumannii WHG40137 was cultured to the logarithmic phase (OD600nm = 0.4-0.6) and stationary phase (OD600nm = 1.2-1.4), centrifuged at low temperature to collect the precipitate, and then washed once with Tris-HCl buffer. The washed precipitate was dissolved in the above buffer to obtain a bacterial solution of A. baumannii WHG40137. Next, lysine LysP53 prepared in Example 2 was taken and mixed with the above bacterial solution so that the final concentration of lysine LysP53 was 100 μg / ml. In addition, a mixture of an equal amount of buffer and the above bacterial solution was used as a negative control. These were cultured at 37 ° C for 1 h, and then counted using a cell count plate. The results obtained above are shown in FIG. 4.

[0057] As can be seen from the results in Figure 4, the lytic activity of lysin LysP53 against logarithmic-phase Acinetobacter baumannii A. baumannii WHG40137 was higher.

[0058] Example 5 Effect of Different pH on the Lytic Activity of Lysine LysP53 against A. baumannii WHG40137

[0059] Acinetobacter baumannii A. baumannii WHG40137 was cultured until the logarithmic phase (OD600nm = 0.4-0.6), centrifuged at low temperature to collect the precipitate, and then washed once with Tris-HCl buffer. The washed precipitate was then resuspended in the above buffers (pH 5-8) with different pH values ​​to obtain a bacterial solution of A. baumannii WHG40137. Next, the lysine LysP53 prepared in Example 2 was taken and mixed with the above bacterial solution so that the final concentration of lysine LysP53 was 100 μg / ml. A mixture of an equal amount of the buffer and the above bacterial solution was used as a negative control. These were cultured at 37 °C for 1 h, and then counted using a cell count plate. The results obtained above are shown in Figure 5.

[0060] As can be seen from the results in Figure 5, the lytic activity of lysine LysP53 against Acinetobacter baumannii WHG40137 was higher when the pH was between 5.0 and 6.5, and the bacterial concentration was reduced to 10 cfu / ml. In addition, as the pH of the solution increased (7.0 to 8.0), the lytic activity of lysine LysP53 against Acinetobacter baumannii WHG40137 decreased. The results showed that lysine LysP53 has higher lytic activity against Acinetobacter baumannii WHG40137 under acidic conditions.

[0061] Example 6. Broad spectrum results of in vitro lysis of different Gram-negative bacterial strains by lysine LysP53

[0062] Several strains of Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae and Escherichia coli were cultured until they reached stationary phase (OD600nm=1.2-1.4), centrifuged at low temperature to collect the precipitate, and then washed once with Tris-HCl buffer. The washed precipitate was dissolved in the above buffer to obtain a bacterial solution of A. baumannii WHG40137. Next, the lysine LysP53 prepared in Example 2 was taken and mixed with the above bacterial solution so that the final concentration of lysine LysP53 was 100μg / ml. In addition, a mixture of an equal amount of the buffer and the above bacterial solution was used as a negative control. These were cultured at 37℃ for 1h, and then counted using a cell count plate. The results obtained above are shown in FIG. 6.

[0063] As is clear from the results in Figure 6, lysine LysP53 had a good lysis effect against multiple types of Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Escherichia coli. The results showed that lysine LysP53 has a broad spectrum of activity in lysing Gram-negative bacteria.

[0064] Example 7 Effect of Different EDTA Concentrations on the Lytic Activity of Lysin LysP53 against A. baumannii WHG40137

[0065] Acinetobacter baumannii A. baumannii WHG40137 was cultured until stationary phase (OD600nm=1.2-1.4), centrifuged at low temperature to collect the precipitate, and then washed once with Tris-HCl buffer. The washed precipitate was dissolved in the above buffer to obtain a bacterial solution of A. baumannii WHG40137. Next, lysine LysP53 prepared in Example 2 was taken and mixed with the above bacterial solution so that the final concentration of lysine LysP53 was 100 μg / ml, and EDTA was added until the final concentrations were 0 μM, 62.5 μM, 125 μM, 250 μM, and 500 μM, respectively. In addition, a mixture of an equal amount of buffer and the above bacterial solution was used as a negative control. These were cultured under 37°C conditions, and samples were taken at different times, and then counted using a cell count plate. The results obtained above are shown in FIG. 7.

[0066] As is clear from the results in Figure 7, the lytic activity of lysine LysP53 against Acinetobacter baumannii WHG40137 increased with increasing EDTA concentration. The results showed that EDTA had a significant enhancing effect on the lysis of Acinetobacter baumannii WHG40137 by lysine LysP53.

[0067] Example 8 Lysine LysP53 activity in lysing A. baumannii WHG40137 on mouse skin surface

[0068] Six to eight week old female BALB / c mice were anesthetized with 40mg / kg pentobarbital intraperitoneally, and 2cm2 of the back was shaved with an electric razor. The exposed skin was then partially burned by exposing it to 65°C water for 12s. Next, 10μL of Acinetobacter baumannii WHG40137 at a concentration of 108CFU / ml was inoculated into the burn site, and the mice were allowed to settle for 24h before being randomly divided into three groups. The first group consisted of 12 mice, and each mouse received 14μg of lysine LysP53 at the burn site. The second group consisted of 12 mice, and each mouse received 4μg of minocycline at the burn site. The third group consisted of 9 mice, and each mouse received an equal volume of buffer at the burn site. Four hours after treatment, the mice were sacrificed by cervical dislocation, and the skin was harvested. The infected skin was excised and infiltrated with 1 ml of PBST containing 0.1% Triton X-100 for 2 minutes, and then the tissue was homogenized using a tissue cell homogenizer (NewZongKe, Wuhan). The tissue was then diluted and plated on LB plates containing 4 μg / ml gentamicin and 2 μg / ml meropenem, incubated overnight, and counted. The results obtained above are shown in FIG. 8.

[0069] As is clear from the results in Figure 8, the lytic activity of lysine LysP53 against Acinetobacter baumannii WHG40137 was higher than that of minocycline, and there was a significant difference between the two.

[0070] Example 9. Broad spectrum results of the in vitro lysis of different Gram-negative bacterial strains by the antimicrobial peptide P104 and lysine LysP53

[0071] A number of strains of Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae and Escherichia coli were cultured until they reached stationary phase (OD600nm=1.2-1.4), centrifuged at low temperature to collect the precipitate, and then washed once with Tris-HCl buffer. The washed precipitate was dissolved in the above buffer to obtain a bacterial solution of A. baumannii WHG40137. Next, lysine LysP53 prepared in Example 2 and antimicrobial peptide P104 prepared in Example 1 were taken and mixed with the above bacterial solution so that the final concentrations of lysine LysP53 and antimicrobial peptide P104 were 100 μg / ml, respectively. In addition, a mixture of an equal amount of buffer and the above bacterial solution was used as a negative control. These were cultured at 37°C for 1 h, and then counted using a cell count plate. The results obtained above are shown in FIG. 9.

[0072] As is clear from the results in Figure 9, lysine LysP53 and antimicrobial peptide P104 had stronger lytic activity against Acinetobacter baumannii, and had a certain lytic effect against Pseudomonas aeruginosa, Klebsiella pneumoniae, and Escherichia coli. The results showed that lysine LysP53 and antimicrobial peptide P104 have a broad spectrum of activity in lysing Gram-negative bacteria.

[0073] The above examples are merely specific embodiments of the present invention, and are intended to illustrate the technical solutions of the present invention, but are not limiting. The scope of protection of the present invention is not limited thereto, and although the present invention has been described in detail with reference to the above examples, those skilled in the art should understand the following: That is, those skilled in the art can modify the technical solutions described in the above examples within the technical scope disclosed by the present invention. Or, they can easily come up with modifications or make equivalent replacements for some technical features. However, these modifications, modifications or replacements do not deviate from the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention, and all should be included in the scope of protection of the present invention.

Claims

1. In the antimicrobial peptide P104, The amino acid sequence of the antimicrobial peptide P104 is as shown in SEQ ID NO.

1.

2. The antimicrobial peptide P104 according to claim 1, characterized in that the gene sequence of the antimicrobial peptide P104 is as shown in SEQ ID NO.

3.

3. In lysine LysP53, The lysine LysP53 comprises the antimicrobial peptide P104 of claim 1, and the amino acid sequence of the lysine LysP53 is as shown in SEQ ID NO.

4. The lysine LysP53 according to claim 3, characterized in that the gene sequence of the lysine LysP53 is as shown in SEQ ID NO.

5. Lysine LysP53 according to claim 3 or 4, characterized in that the sequences of the primers for amplifying the nucleotide sequence of said lysine LysP53 are as shown in SEQ ID NO. 5 and SEQ ID NO.

6.

6. A recombinant expression vector comprising the gene for lysine LysP53 according to claim 4, The recombinant expression vector is a prokaryotic expression vector pET28a-LysP53.

7. A host cell comprising the recombinant expression vector of claim 6, The host cell is Escherichia coli BL21(DE3).

8. The method for preparing lysine LysP53 comprises the steps of: S1: Amplification of the lysine LysP53 gene from the genome of bacteriophage P53; S2: Constructing a recombinant expression vector pET28a-LysP53 that expresses the lysine LysP53 gene; S3: Transform the recombinant expression vector pET28a-LysP53 into competent cells of E. coli BL21 (DE3) and screen for engineered strains expressing the lysine LysP53 gene; S4: Induce expression using IPTG to obtain the expression product of the recombinant gene; S5: The recombinant gene expression product is purified and separated using a nickel column to obtain the lysine LysP53; 5. Lysine LysP53 according to claim 3 or 4, characterized in that it comprises the steps of:

9. A method for lysing Gram-negative bacteria in vitro using the antibacterial peptide P104 described in claim 1.

10. The method for lysing Gram-negative bacteria in vitro according to claim 9, characterized in that the Gram-negative bacteria include some of Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae and Escherichia coli.

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