Heat-resistant and acid-alkali-resistant bacteriophage lytic enzyme ply152 and application thereof

Abstract

The application discloses a heat-resistant and acid-alkali-resistant bacteriophage lytic enzyme Ply152 and an application thereof, and belongs to the technical field of enzyme preparations.The amino acid sequence of the lytic enzyme Ply152 is shown as SEQ ID NO.2.The lytic enzyme Ply152 has good thermal stability and excellent pH tolerance, can retain a high enzyme activity in the range of 4-80 DEG C and pH 3.0-12.0, shows a significant lysis effect on various salmonellas and enterotoxigenic escherichia coli, has broad-spectrum antibacterial activity, and can effectively kill pathogenic bacteria in cooperation with citric acid.The lytic enzyme Ply152 has potential application value in preparation of a novel antibacterial agent and prevention and control of bacterial infections caused by salmonellas and pathogenic escherichia coli.

Description

Technical Field

[0001] This invention relates to the field of enzyme preparation technology, and in particular to a heat- and acid- and alkali-resistant bacteriophage lysin Ply152 and its applications. Background Technology

[0002] Piglets are highly susceptible to diarrhea and death during weaning due to stress, an immature digestive system, and unstable gut microbiota, resulting in significant economic losses. Enterotoxigenic Escherichia coli is one of the main pathogens causing this disease, with widespread transmission routes (feed, environment, vertical transmission) and a high rate of asymptomatic carriers, making control difficult. In addition, Salmonella and Shigella are also common pathogens. The long-standing overuse of antibiotics has not only led to drug residue problems but also exacerbated the drug resistance problem of pathogens such as Salmonella, with multidrug-resistant strains constantly emerging and traditional drug treatments becoming less effective, posing a dual threat to the livestock industry and human public health. Piglet diarrhea is one of the most challenging diseases in the pig industry, causing enormous economic losses to the global pig farming sector.

[0003] Bacteriophage lysins are peptidoglycan hydrolases expressed by bacteriophages in the later stages of host infection and are the most crucial lysing components for bacteriophages to kill pathogens. Compared to bacteriophage viruses, which are prone to mutation, lysins, as proteins, have better biosafety and therefore have great research and application value as novel and highly effective antibacterial agents. Existing research has confirmed that phage-encoded lysins play a key role in the lysis of host bacteria. However, most lysins have strong antibacterial activity against Gram-positive bacteria, while there are few reports on lysins targeting Gram-negative bacteria. This is mainly because the outer membrane of Gram-negative bacteria, which is mainly composed of anionic lipopolysaccharides, prevents lysins from entering the peptidoglycan layer to exert their bactericidal function. Salmonella and Escherichia coli are Gram-negative bacteria, and research on their lysins is still relatively limited. Due to the limitations of the acidity and alkalinity of the animal gastrointestinal tract and the destruction of feed enzymes during high-temperature feed processing, the development and application of enzyme preparations urgently need to be acid and alkali resistant and heat resistant, with a wide acid-base range and temperature range. Summary of the Invention

[0004] The purpose of this invention is to provide a heat- and acid / alkali-resistant phage lyase, Ply152, and its applications to address the problems existing in the prior art. This invention yields a phage lyase, Ply152, which possesses peptidoglycan hydrolysis capabilities and, when used in conjunction with citric acid, exhibits highly efficient lysis of Salmonella and enterotoxigenic Escherichia coli.

[0005] To achieve the above objectives, the present invention provides the following solution: The present invention provides a heat- and acid- and alkali-resistant phage lysin Ply152, the amino acid sequence of which is shown in SEQ ID NO.2.

[0006] The present invention also provides a gene encoding the above-mentioned phage lysin Ply152, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0007] The present invention also provides a recombinant vector containing the above-mentioned genes.

[0008] The present invention also provides a recombinant microorganism comprising the above-described recombinant vector.

[0009] The present invention also provides the use of the above-mentioned phage lysin Ply152, the above-mentioned gene, the above-mentioned recombinant vector, or the above-mentioned recombinant microorganism in the preparation of bacterial lysing agents, wherein the bacteria include Escherichia coli and Salmonella.

[0010] The present invention also provides a bacterial lysis agent comprising the above-mentioned phage lysis enzyme Ply152.

[0011] The present invention also provides the use of citric acid in combination with the above-mentioned phage lysin Ply152 or the above-mentioned bacterial lysing agent in the preparation of products for preventing and treating diseases caused by bacteria, including Escherichia coli and Salmonella.

[0012] The present invention also provides a product for preventing and treating diseases caused by bacteria, the product comprising a composition of citric acid and the above-mentioned phage lysin Ply152, or a composition of citric acid and the above-mentioned bacterial lysin.

[0013] The present invention discloses the following technical effects: This invention provides a lyase Ply152 derived from Enterobacter bacteriophage. This lyase has a molecular weight of approximately 15.46 kDa and an isoelectric point of 6.91. Its active site is composed of three key amino acid residues: His66, Asp73, and His133. The optimal reaction temperature for this lyase Ply152 is 30°C, and it exhibits good thermostability within a temperature range of 4°C to 80°C. The optimal reaction pH is 8.0, and it demonstrates excellent pH tolerance and stability over a wide pH range of 3.0 to 12.0. Verification has shown that the lyase Ply152 of this invention exhibits significant lytic effects against various Salmonella strains and enterotoxigenic Escherichia coli, possessing broad-spectrum antibacterial activity. When used in synergy with citric acid, it can effectively kill pathogenic bacteria. Furthermore, the lyase Ply152 of this invention can be efficiently prepared using a prokaryotic expression system, and the purified protein has high purity. It has potential application value in the preparation of novel antibacterial agents and the prevention and control of bacterial infections caused by Salmonella and pathogenic Escherichia coli. Attached Figure Description

[0014] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0015] Figure 1 A comparative diagram of the amino acid sequence of the lyase Ply152; Figure 2 This represents the tertiary structure of the lyase Ply152; Figure 3 SDS-PAGE electrophoresis image of the lyase Ply152; where 1 is the supernatant enzyme solution of the recombinant expression of the blank vector pET-29a; 2 is the supernatant enzyme solution of the recombinant expression of the vector pET-29a-ply152; 3 is the lyase protein eluted with 200 mM imidazole. Figure 4 The optimal temperature for the lysin Ply152; Figure 5 Temperature stability of the lyase Ply152; Figure 6 The optimal pH for the lysin Ply152; Figure 7 pH stability of the lysin Ply152; Figure 8 This study investigated the antibacterial activity of the lyase Ply152 against Salmonella and Escherichia coli. Detailed Implementation

[0016] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0017] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0018] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0019] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0020] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0021] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the instruments and equipment used in the following examples are all conventional laboratory instruments and equipment; unless otherwise specified, the experimental materials used in the following examples were all purchased from conventional biochemical reagent stores.

[0022] Example 1: Obtaining the phage lysin Ply152 For those from Escherichia phage Genome-wide bioinformatics analysis of the EC148 phage yielded an E. coli phage lyase gene, named Ply152. The nucleotide sequence of the phage lyase gene Ply152 is shown in SEQ ID NO.1, and the amino acid sequence of the phage lyase Ply152 it encodes is shown in SEQ ID NO.2.

[0023] SEQ ID NO.1: ATGAGTTTTAAATTTAGTCAAAGAAGTTTAGATAGATTAAAAGGTGTTCACCCCGACTTAGTAAAAGTAACGCATAGGGCTTTAGAATTAAGCCCTTATGATTTTACTATTACAGAAGGACTTCGTTCCCTAGAACAAAGTGCTAAGAACATTGCTAATGGTACATCTTTCTTAAAAGATCCTAGCAAATCTAAGCATGTTCAAGGC AGGCGGTAGATTTTGCTCCGCTTAAGGATGGAAAGATTGACTGGAATGATCTAGAATCCTTTAAGTTAGTGGCAGATGCCTTCTTCAAAGCAGCCGAAGAGCTAGGAGTGAAGATTAGATGGGGTGGTGATTGGAACCAAAATGGAAGTTATAAGGATGAAATCCAACGCGGTACTTTTGACGGACCACACATAGAATTGATTTAA.

[0024] SEQ ID NO.2: MSFKFSQRSLDRLKGVHPDLVKVTHRALELSPYDFTITEGLRSLEQSAKNIANGTSFLKDPSKSKHVQGRAVDFAPLKDGKIDWNDLESFKLVADAFFKAAEELGVKIRWGGDWNQNGSYKDEIQRGTFDGPHIELI.

[0025] The isoelectric point and molecular weight of the phage lyase Ply152 were predicted using the online website Expasy (https: / / web.expasy.org / compute_pi / ). The results showed that the molecular weight of phage lyase Ply152 was 15.46 kDa and the isoelectric point was 6.91. The amino acid sequence of Ply152 was submitted to NCBI, and sequences with high similarity were downloaded from Uniprot and Protein Data Bank databases for alignment. The amino acid sequence alignment websites used were ESPript 3.2 and Multiple Sequence Alignment - CLUSTALW. The alignment results are shown below. Figure 1 As shown, ★ indicates the predicted active site. The three-dimensional structure of the phage lysin Ply152 was obtained using the AlphaFold Server online prediction method, as shown below. Figure 2As shown, His66, Asp73, and His133 in Ply152 are active site residues.

[0026] Example 2: Obtaining the full-length gene fragment of the phage lysin Ply152 Primers were designed based on the predicted restriction sites of the nucleotide sequence of the phage lysin Ply152. Using phage genomic DNA as a template, the full-length Ply152 gene was amplified by PCR to obtain the full-length sequence of the Ply152 gene (SEQ ID NO.1). The primers used for PCR amplification were F and R, and their nucleotide sequences are shown in SEQ ID NO.3 and SEQ ID NO.4.

[0027] F:TCCATGGCTGATATCGGATCCATGAGTTTTAAATTTAGTCAAAGAAGTTTAGA( Bam HI), SEQ ID NO.3; R: GTGGTGGTGGTGGTGCTCGAGAATCAATTCTATGTGTGGTCCGTC (XhoI), SEQ ID NO. 4.

[0028] In addition to obtaining the full-length gene fragment of the lyase Ply152 of this invention by PCR amplification, it can also be obtained by other techniques commonly used in the field, such as in vitro chemical synthesis techniques.

[0029] Example 3: Construction and Validation of Recombinant Clones and Expression Vector pET-29a-ply152 The pET-29a vector was used separately. Bam Double digestion with HI and XhoI was performed using the following digestion system: 5 μL 10×swift buffer and 2.5 μL swift cut. Bam HI, 2.5 μL swift-cut XhoI, 15 μL pET-29a(+), and ddH2O were added to a final volume of 50 μL. The mixture was recovered by agarose gel electrophoresis, and purified PCR product (prepared in Example 2) and 5 μL 5×CE MixV3 were added. Ligation was carried out at 50°C for 0.25 h. The ligation product was then used to transform *E. coli* DH5α, and the mixture was plated onto a culture dish containing 50 μg / mL Kanamycin and incubated at 37°C for 10–15 h.

[0030] Multiple single colonies were extracted from transformation plates, and plasmids were extracted using Novizan's plasmid mini-extraction kit. The obtained plasmids were verified by double enzyme digestion, and the recombinant plasmids were sent to Youkang Biotechnology Co., Ltd. for sequencing. Sequencing results showed that the cloned target fragment (414 bp in length) was inserted into the pET-29a(+) vector, thus yielding the recombinant expression vector pET-29a(+)-ply152.

[0031] Example 4: Recombinant Expression and Purification of Phage Lysis Enzyme Ply152 The recombinant expression vector pET-29a-ply152 (prepared in Example 3) and the blank vector pET-29a were transformed into the host bacteria by heat shock. E. coli BL21(DE3) was used to obtain recombinant bacteria containing the recombinant plasmid. A single positive colony was inoculated into 4 mL of Luria-Bertani broth (LB) medium containing 50 μg / mL kanamycin and cultured at 37°C with shaking at 220 rpm for 12 h. The plasmid was extracted using a Novizan plasmid mini-extraction kit for verification. After confirmation, the bacterial culture was inoculated into a 250 mL shake flask containing 50 mL of medium and cultured at 37°C with shaking at 220 rpm for 8 h. Then, it was transferred to a 1000 mL shake flask containing 400 mL of medium and cultured at 37°C with shaking at 220 rpm for 3.5 h. When OD... 600 When the concentration reached 0.4-0.6, IPTG was added to a final concentration of 1 mmol / L, and expression was induced at 16℃ and 180 rpm for 18-20 h. The culture medium was centrifuged at 5000 rpm for 0.5 h, and the cells were collected. The cells were resuspended and washed with 20 mmol / L pH 8.0 Tris-HCl buffer, centrifuged at 5000 rpm for 0.5 h, and the cells were recovered. Then, the cells were disrupted with 20 mmol / L pH 8.0 Tris-HCl buffer in an ice-water bath using an ultrasonic cell disruptor, and centrifuged at 12000 rpm for 10 min at 4℃ to obtain a crude extract containing the lyase Ply152.

[0032] The crude extract was purified using a Ni-NTA affinity chromatography column (method as described in the instruction manual for the Ni NTA Beads 6FF gravity column from Changzhou Tiandi Renhe Biotechnology Co., Ltd.). The specific procedures are as follows: (1) Fix the Ni NTA Beads 6FF gravity column on the iron stand, remove the lower end plug and the upper end plug in turn, and drain the protective liquid from the gravity column.

[0033] (2) Add 5 column volumes of Lysis Bufer to the column tube to equilibrate, so that the packing material is in the same buffer system as the target protein.

[0034] (3) Add the sample to the balanced gravity column and retain it for at least 2 minutes to ensure sufficient contact between the target protein and the medium, thereby improving the recovery rate of the target protein. Collect the eluent for SDS-PAGE analysis of protein binding.

[0035] (4) Wash with 10-15 column volumes of Wash Bufer to remove non-specifically adsorbed proteins and collect the washing solution.

[0036] (5) Elute sequentially with Eluton Bufer solution containing 10 mM, 100 mM, and 200 mM imidazole, with an elution volume of 5-10 times the column volume, and collect in fractions.

[0037] (6) Use 3 column volumes of Lysis Bufer and 5 column volumes of deionized water balance packing material in sequence. Store the gravity column in an equal volume of 20% ethanol at 2-8℃ to prevent bacterial contamination of the packing material.

[0038] The purity and molecular weight of the obtained recombinant protein were determined by SDS-PAGE, and the results are shown in the figure. Figure 3 The results showed that the purified Ply152 protein eluted with 200 mM imidazole had high purity and a molecular weight of approximately 15.4 kDa.

[0039] Example 5: Optimal reaction temperature and temperature stability of phage lysin Ply152 1. Optimal reaction temperature of phage lysin Ply152 Will Salmonella CICC 21484 was cultured in LB medium to the logarithmic phase. The cells were collected by centrifugation at 8000 rpm for 5 min at 4 °C. The cells were resuspended in pre-cooled Tris-HCl buffer (pH 8.0) and centrifuged again. The cells were then treated with 50 mM citrate buffer (pH 3.0) at room temperature for 0.25 h. The cells were then rapidly neutralized with an equal or twice the volume of 1 M Tris-HCl (pH 8.0), centrifuged at 8000 rpm for 5 min, and washed three times with 20 mM Tris-HCl (pH 8.0).

[0040] The bacterial cells were resuspended in 20 mM Tris-HCl solution at pH 8.0, and the OD was adjusted. 600 To achieve an OD value of 1.0, 1 mL of bacterial culture was mixed with 10 μL of Ply152 enzyme solution (9 μg / mL). The control group was treated with an equal volume of Tris-HCl buffer instead of Ply152. Both groups of bacterial cultures were incubated at different temperatures: 4℃, 20℃, 30℃, 37℃, 40℃, 50℃, 60℃, and 70℃. OD was measured after 10 min. 600The value of ΔOD is calculated. 600 Values, with 3 repetitions per group. ΔOD 600 The reaction temperature at which the value is maximized is the optimum reaction temperature. The maximum ΔOD of the phage lysin is defined as... 600 The value is relative enzyme activity of 100%. Relative enzyme activity is expressed as a percentage of maximum enzyme activity: Relative enzyme activity = ΔOD 600 / Maximum ΔOD 600 ×100%.

[0041] The optimal temperature for phage lysin Ply152 is as follows: Figure 4 As shown, the optimal temperature for phage lysin Ply152 is 30℃. Its relative enzyme activity remains above 70% between 4℃ and 60℃. Even when the temperature is above 60℃, the relative enzyme activity of phage lysin Ply152 still retains 65%, indicating that phage lysin Ply152 has good heat resistance.

[0042] 2. Temperature stability of phage lysin Ply152 The phage lysin Ply152 (9 μg / mL) was incubated at 4℃, 20℃, 30℃, 40℃, 50℃, 60℃, 70℃, and 80℃ for different times. After incubation, 1 mL of the above solution was taken... Salmonella CICC 21484 bacterial culture was mixed with 10 μL of phage lysin Ply152 incubated at different temperatures. An equal volume of Tris-HCl buffer was added to the control group instead of Ply152. The mixtures were then incubated statically at the optimal reaction temperature. OD was measured after 10 min. 600 The values ​​are set to 3 repetitions per group, and ΔOD is calculated. 600 The value is then used to calculate the relative enzyme activity using the method described above.

[0043] The temperature stability results of phage lysin Ply152 are as follows: Figure 5 As shown, the ΔOD decrease of lyase Ply152 after incubation at 4°C for 0 min is expressed as... 600 The enzyme activity was 100%, and the Ply152 lyase remained stable after treatment at 4℃-80℃ for 10 min. After treatment at 50℃ for 90 min, the enzyme activity was 92.77%. After treatment at 60℃, 70℃, and 80℃ for 45 min, the residual enzyme activities were 83.60%, 70.45%, and 67.08%, respectively. Therefore, the Ply152 lyase exhibits good thermostability.

[0044] Example 6: Optimal pH and pH stability of phage lysin Ply152 1. Optimal pH of phage lysin Ply152 Prepare buffer solutions with different pH values ​​(pH 6.0-8.0 Na2HPO4-NaH2PO4; pH 7.0-9.0 Tris-HCl; pH 9.0-10.0 Glycine-NaOH). Salmonella CICC 21484 was cultured in LB medium to the logarithmic growth phase. Cells were collected by centrifugation at 8000 rpm for 5 min at 4°C. The cells were resuspended in pre-chilled Tris-HCl buffer (pH 8.0) and centrifuged again. The resulting cells were treated with 50 mM citrate buffer (pH 3.0) at room temperature for 0.25 h, followed by rapid neutralization with an equal or twice-volume volume of 1 M Tris-HCl (pH 8.0). The cells were centrifuged at 8000 rpm for 5 min and washed three times with 20 mM Tris-HCl (pH 8.0). The cells were then resuspended in buffers of different pH values ​​to adjust the OD. 600 To determine the OD value, 1.0 mL of bacterial culture was mixed with 10 μL of Ply152 (9 μg / mL). For the control group, an equal volume of Tris-HCl buffer was added instead of the phage lysin Ply152. Both mixtures were incubated statically at 30°C. OD was measured after 10 min. 600 The value of ΔOD is calculated. 600 Values, with 3 repetitions per group. ΔOD 600 The pH at which the value is highest is the optimal reaction pH, and is counted as 100% of the relative enzyme activity. The relative enzyme activity is calculated according to the method in Example 5.

[0045] The optimal pH for phage lysin Ply152 is as follows: Figure 6 As shown, the results indicate that the optimal pH for the phage lysin Ply152 reaction is 8.0. Under neutral and weakly alkaline conditions (pH 7.0-8.0 Na₂HPO₄-NaH₂PO₄; pH 7.0-9.0 Tris-HCl), the enzyme activity remains above 60%; at pH 6.0 (Na₂HPO₄-NaH₂PO₄), the relative enzyme activity is 37.76%; and at pH 10, the relative enzyme activity of phage lysin Ply152 is 42.60%.

[0046] 2. pH stability of phage lysin Ply152 Phage lysin Ply152 was mixed with buffers of different pH values ​​(pH 3-6 citrate-sodium citrate; pH 6-8 Na2HPO4-NaH2PO4; pH 7-9 Tris-HCl; pH 9-10 Glycine-NaOH; pH 11-12 Na2CO3-NaOH) and incubated on ice for 24 h. Then, 10 μL of the enzyme solution (9 μg / mL) treated with different pH buffers was mixed with 1 mL of bacterial culture and incubated statically at the optimal pH and reaction temperature. ΔOD was measured and calculated after 10 min. 600 Values, with 3 repetitions per group, ΔOD 600 The maximum value represents 100% of the relative enzyme activity. The relative enzyme activity was calculated according to the method in Example 5.

[0047] The pH stability of the lyase Ply152 is as follows: Figure 7 As shown, the results indicate that after treating the phage lysin Ply152 in a buffer system with a pH of 3.0-12.0 for 24 h, the relative enzyme activity was higher than 80% under the optimal pH reaction conditions, indicating that the lysin Ply152 can maintain good stability under pH conditions of 3.0-12.0.

[0048] Example 7: Inhibitory effect of phage lysin Ply152 on different Salmonella and Escherichia coli strains. Will Salmonella CICC 10437 Salmonella CICC 21483 Salmonella CICC 21484 Salmonella CICC 21487 Salmonella CICC 21488 Salmonella CICC 21493 Salmonella CICC21496 Salmonella CICC 21497 Salmonella CICC 21498 Salmonella CICC 21527 Salmonella CICC 21534 Salmonella CICC 21535 Salmonella CICC 21680, ETEC O8:K87, ETEC O147:K89, EHEC O157:H7, and EPEC O26:K60 were inoculated into LB liquid medium at a volume ratio of 1:100 and cultured at 37°C with shaking until the logarithmic growth phase (OD200). 600= 0.6). Take 100 mL of bacterial culture, centrifuge at 8000 rpm for 5 min at 4℃ to collect bacterial cells, resuspend in pre-cooled Tris-HCl buffer (pH 8.0) and centrifuge again. Treat the obtained bacterial cells with 50 mM citrate buffer (pH 3.0) at room temperature for 0.25 h, then rapidly neutralize with an equal volume or twice the volume of 1 M Tris-HCl (pH 8.0), centrifuge at 8000 rpm for 5 min, wash 3 times with 20 mM Tris-HCl (pH 8.0), and adjust to OD. 600 =0.7. Mix 1 mL of bacterial culture with 10 μL of phage lysin Ply152 (9 μg / mL), and add an equal volume of Tris-HCl buffer to the control group. Incubate the mixture at 30℃ for 10 min, and measure and calculate ΔOD after 10 min. 600 Values, with 3 repetitions per group.

[0049] like Figure 8 As shown, the phage lysin Ply152 exhibits lytic activity against a variety of bacteria, demonstrating broad-spectrum lytic performance. Furthermore, phage lysin Ply152 can... Salmonella CICC 21535, ETEC O8:K87 OD 600 The value decreased by 0.5 (i.e., ΔOD) 600 An increase of 0.5 or more indicates a strong pyrolysis effect.

[0050] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A heat and acid and alkali resistant bacteriophage lytic enzyme Ply152, characterized in that, The amino acid sequence of the phage lysin Ply152 is shown in SEQ ID NO.

2.

2. A gene encoding the phage lysin Ply152 of claim 1, characterized in that, The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

3. A recombinant vector comprising the gene of claim 2.

4. A recombinant microorganism comprising the gene of claim 2.

5. The use of the phage lysin Ply152 of claim 1, the gene of claim 2, the recombinant vector of claim 3, or the recombinant microorganism of claim 4 in the preparation of bacterial lysing agents, characterized in that, The bacteria include Escherichia coli and Salmonella.

6. A bacterial lysis agent, characterized in that, The bacterial lysing agent contains the phage lysin Ply152 as described in claim 1.

7. The use of citric acid in combination with the phage lysin Ply152 of claim 1 or the bacterial lysing agent of claim 6 in the preparation of products for the prevention and treatment of bacterial diseases, characterized in that, The bacteria include Escherichia coli and Salmonella.

8. A product for preventing and treating diseases caused by bacteria, characterized in that, The product comprises a composition of citric acid and the phage lysin Ply152 of claim 1, or a composition of citric acid and the bacterial lysin of claim 6.

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