Phage lytic enzymes and uses thereof

Abstract

A bacteriophage lytic enzyme and its application. The name of the bacteriophage lytic enzyme is PlyNJ3; the gene fragment encoding the lytic enzyme PlyNJ3 is amplified from streptococcus suis by PCR, and a prokaryotic expression vector plasmid pET32a-PlyNJ3 containing the PlyNJ3 gene is constructed, the recombinant plasmid is obtained by transforming DH5 alpha competent cells, and the protein is massively expressed in an escherichia coli expression system. Then, the lytic enzyme PlyNJ3 is purified by affinity purification medium, the high-efficiency expression of the lytic enzyme PlyNJ3 in vitro is realized, and the lytic effect of the lytic enzyme PlyNJ3 on different serotypes of streptococcus suis, streptococcus agalactiae and other common streptococci is verified in vitro. The bacteriophage lytic enzyme PlyNJ3 prepared by the application can selectively lyse target pathogenic bacteria, and has a good application prospect in the development of new bacteriophage drugs.

Description

Technical Field

[0001] This invention belongs to the field of bacteriophage and its lysin technology application, and in particular relates to a bacteriophage lysin and its application. Background Technology

[0002] Streptococcus suis is an important zoonotic pathogen. Clinical symptoms of Streptococcus suis infection mainly include meningitis, arthritis, and endocarditis. In humans, Streptococcus suis infection can also cause systemic infections such as meningitis and arthritis, posing a serious threat to livestock and public health. Besides pigs and humans, it can also infect other mammals and rodents such as cattle, sheep, horses, dogs, and cats. Based on bacterial capsular polysaccharide antigens, it can be classified into 33 serotypes (1–31, 33, and 1 / 2) and untyped strains. In recent years, with the expansion of pig farming, Streptococcus suis has become a major pathogen seriously endangering the pig industry. Streptococcus suis not only causes serious economic losses to the pig industry but also threatens public health and food safety, and even human health, thus attracting widespread attention. Currently, treatment for Streptococcus suis infection still relies on antibiotics and other antimicrobial drugs. However, with the extensive use of antibiotics in clinical practice, drug resistance has emerged and is becoming increasingly serious. In recent years, the resistance of Streptococcus suis isolated from laboratory clinics to commonly used antibiotics has been increasing year by year, leading to unprecedented challenges for antibiotics in the field of antibacterial infection. At present, the development of new bacteriophage preparations that can lyse pathogens has become a major focus.

[0003] Bacteriophages are a general term for viruses that can infect microorganisms such as bacteria, fungi, actinomycetes, or spirochetes. Referring to the International Commission on Viruses (ICTV) and other classification criteria, bacteriophages can be subdivided based on their protein structure, morphological characteristics, infecting structures, and nucleic acid composition. Based on their different mechanisms of bacterial infection, bacteriophages can be divided into virulent bacteriophages and temperate bacteriophages (lysogenic bacteriophages). Common virulent bacteriophage infection of bacteria involves five stages: adsorption, invasion, synthesis, assembly, and release. The bacteriophage particle specifically adsorbs onto receptors on the surface of the bacterial cell wall, injecting its own genetic material into the bacterial cell. Using materials within the bacterial cell, it synthesizes and assembles progeny bacteriophages, ultimately lysing the bacterial cell wall and releasing a large number of its own bacteriophages. The mechanism of bacterial infection by temperate bacteriophages differs from that of virulent bacteriophages. After a temperate bacteriophage infects a bacterium, it integrates its genomic DNA into the bacterial genome and replicates along with the bacterial DNA. Under specific conditions (such as ultraviolet light or mitomycin C induction), the bacteriophage DNA cleaves from the bacterial DNA, enters a lysis cycle, and uses its own genetic material as a template to synthesize progeny bacteriophages using raw materials within the bacteria, thus lysing the bacteria. A prophage is a bacteriophage in an integrated state that has been infected by some temperate bacteriophages and whose nucleic acid has integrated into the host bacterial chromosome.

[0004] Bacteriophage lysins offer several key advantages over antibiotics, making them an attractive alternative. First, phage lysins exhibit high host specificity, reducing the likelihood of secondary infections, and to date, they have not shown any significant toxic side effects on mammalian cells. Second, due to their different mechanisms of action compared to antibiotics, phages can also treat multidrug-resistant bacteria. Furthermore, phage lysin therapy is an alternative treatment option for patients allergic to antibiotics. Currently, most lysins reported domestically and internationally are derived from virulent phages, which have relatively narrow lysis spectra. Many pathogens are easily infected by virulent phages during their growth and development, leading to cell wall rupture and bacterial death. This not only results in slow growth but also makes it difficult to isolate virulent phages from bacteria, thus posing challenges to the development of lysin-related formulations. Research has found that most bacterial genomes contain prophage sequences, which generally contain genes encoding lysins, providing new strategies and pathways for lysin development. This invention involves performing whole-genome sequencing on a clinically isolated Streptococcus suis strain, analyzing its sequence, and then recombinantly expressing a lysin encoded by a prophage, which is then applied to the treatment of Streptococcus suis, Streptococcus agalactiae, and other streptococcal infections. Summary of the Invention

[0005] Technical problem solved: In view of the above-mentioned technical problem, the present invention provides a phage lysin and its application in the preparation of products for treating drug-resistant streptococci.

[0006] Technical solution: A phage lysin PlyNJ3, the amino acid sequence of which is shown in SEQ ID NO.1.

[0007] The nucleotides encoding the phage lysin PlyNJ3 described above, and the DNA sequence are shown in SEQ ID NO.2.

[0008] Plasmids containing the above amino acid sequence.

[0009] Vectors containing the above-mentioned plasmids.

[0010] The application of the above-mentioned phage lysin in the preparation of products for treating drug-resistant streptococci.

[0011] The concentration of phage lysin in the above products is not less than 10 μg / mL.

[0012] The aforementioned drug-resistant streptococci are either Streptococcus suis or Streptococcus agalactiae.

[0013] This invention provides a method for expressing and applying a lysin encoded by the ΦNJ3 prephage, comprising the following steps:

[0014] (1) Discovery of phage lysin genes: By performing whole-genome sequencing on Streptococcus suis NJ3 and analyzing and comparing the genes of the ΦNJ3 prephage in the genome, gene sequences expressing lysins were screened out.

[0015] (2) Construction of recombinant plasmid: Design primers to amplify the gene encoding the lyase PlyNJ3 and its promoter and terminator, and add restriction endonuclease digestion sites at both ends. The amplified product and the plasmid stored in the laboratory are double-digested with restriction endonuclease, and the digestion products are recombined with DNA ligase to construct the recombinant plasmid.

[0016] (3) Transformation into Escherichia coli expression system: The vector constructed in step (2) is transformed into competent Escherichia coli cells DH5α and BL21(DE3) so that the recombinant plasmid can be expressed in large quantities in the Escherichia coli expression system.

[0017] (4) Expression and purification of lyase PlyNJ3: Escherichia coli BL21(DE3) containing recombinant plasmid was transferred to broth containing Kan resistance (50 μg / mL) for large-scale expression, and lyase PlyNJ3 was isolated and purified from the culture medium.

[0018] The expression of the lyase PlyNJ3 in step (4) is induced expression.

[0019] The plasmid is an expression vector for Escherichia coli, specifically pET32a(+).

[0020] The method used in step (4) to separate and purify the lysin PlyNJ3 is Ni-NTA agarose affinity chromatography.

[0021] The specific steps are as follows: Two primers, upper and lower, were designed based on the gene sequence encoding the lyase PlyNJ3 in the ΦNJ3 prephage. A BamHI restriction endonuclease site was added to the 5' end of each primer, and a SalI restriction endonuclease site was added to the 3' end. Using *Streptococcus suis* NJ3 containing the ΦNJ3 prephage as a template, the fragment was amplified. It was then double-digested with pET32a using BamHI and SalI restriction endonucleases, respectively. After recovering the correct gene fragment from the gel, the target fragment was ligated to the linearized vector plasmid using T4 DNA ligase. The recombinant product was transformed into competent *E. coli* DH5α cells and plated on LB agar (containing 50 μg / mL ampicillin). After single colonies were observed to be visible to the naked eye, PCR identification was performed, and *E. coli* DH5α cells containing the recombinant plasmid pET32a-PlyNJ3 were preserved.

[0022] The recombinant plasmid pET32a-PlyNJ3 was transformed into BL21(DE3) competent cells. After PCR identification, pET32a-PlyNJ3-BL21 cells containing the correct recombinant plasmid were preserved. The bacterial culture was transferred 1:100 to 500 mL of LB broth (containing 50 μg / mL ampicillin) and cultured at 37°C and 180 rpm in a shaker. When the absorbance of the bacterial culture at 600 nm was 0.4-0.6, IPTG was added to a final concentration of 1 mM, and the culture was transferred to a shaker at 16°C and 180 rpm for overnight culture. After centrifugation at 4°C and 10,000 rpm for 10 minutes, the supernatant was discarded, and the bacterial pellet was used for subsequent purification.

[0023] The purification method for the lyase PlyNJ3 is as follows: Bacterial protein lysis buffer (50 mM Tris-HCl, 0.2 mM PMSF, pH 7.0-9.2) was added to the bacterial cell pellet and resuspended. The pellet was then sonicated on ice for 30 minutes. After lysis, the pellet was centrifuged, and the supernatant was collected and filtered through a 0.45 μm cell filter. The filtered supernatant was then passed through a Ni-NTA agarose gravity chromatography column. The column was washed with 10 column volumes of non-denaturing wash buffer, and the flow-through was collected in fractions with non-denaturing eluent. Once the band sizes were verified by SDS-PAGE, the purified lyase PlyNJ3 was obtained.

[0024] The lysis effect of purified lysin PlyNJ3 on different pathogens was determined using a turbidity assay. Streptococci and Escherichia coli isolated from laboratory clinical samples were transferred to broth and cultured overnight. After washing with sterile 1X PBS, the absorbance at 600 nm was adjusted to 1.0. The purified lysin PlyNJ3 was then added and incubated in a microplate reader, and the change in absorbance at 600 nm was measured.

[0025] This invention utilizes gene recombination technology to amplify and construct a recombinant plasmid by integrating a fragment encoding a phage lyase from the ΦNJ3 family prophage gene integrated into Streptococcus suis. This plasmid is then transformed into an Escherichia coli expression system, and protein expression is induced by the addition of IPTG, resulting in high-level expression in the supernatant of the E. coli expression system. The purified lyase PlyNJ3 is obtained by Ni-NTA agarose affinity chromatography for further experiments to test its lysis effect on different bacterial species and to determine the optimal reaction conditions.

[0026] Beneficial effects: This invention uses purified lysin to co-incubate pathogens isolated from laboratory clinical samples at different temperatures, resulting in a final concentration of lysin of 500 μg / mL and a final concentration of pathogens of 1 × 10⁻⁶. 5 CFU / mL. Observe the change in absorbance of the mixture at 600 nm. Tests showed that for most Streptococcus suis, the absorbance at 600 nm decreased by more than 50% within half an hour of adding the lysin. Compared to Streptococcus suis, the lysin also showed a lytic effect on some agalactiae, but had a poor or no inhibitory effect on Escherichia coli and Staphylococcus aureus. After measuring the change in absorbance of the mixture at 600 nm in vitro, the co-incubated product was diluted 10-fold and dripped into solid culture medium. Comparison showed a significant reduction in the number of viable bacteria, proving that the lysin does indeed have a certain inhibitory effect on Streptococcus suis. Attached Figure Description

[0027] Figure 1 Electrophoresis image (1467 bp) showing the amplification of the PlyNJ3 gene encoding the lyase PlyNJ3 from ΦNJ3 family prophages. Lane M represents the DL5000 DNA molecular weight standard, and lane 1 represents the PlyNJ3 lyase gene.

[0028] Figure 2SDS-PAGE analysis of the expression of the lyase PlyNJ3 in the recombinant vector. Lane M represents protein molecular weight standards (10-180 kDa), lane 1 represents uninduced pET32a-PlyNJ3-BL21 bacterial culture, lane 2 represents pET32a-PlyNJ3-BL21 bacterial culture induced overnight at 1mM IPTG at 16℃, lane 3 represents uninduced supernatant protein, lane 4 represents supernatant protein induced overnight at 1mM IPTG at 16℃, lane 5 represents uninduced inclusion body protein, and lane 6 represents inclusion body protein induced overnight at 1mM IPTG at 16℃.

[0029] Figure 3 SDS-PAGE analysis of purified lyase PlyNJ3. Lane M represents protein molecular weight standards (10-180 kDa), lane 1 represents supernatant protein after overnight induction with 1 mM IPTG at 16°C, lane 2 represents the flow-through buffer after supernatant flow through a Ni-NTA column, lane 3 represents the first wash with non-denaturing wash buffer, lane 4 represents the second wash with non-denaturing wash buffer, lane 5 represents the first elution with non-denaturing elution buffer, lane 6 represents the second elution with non-denaturing elution buffer, lane 7 represents the third elution with non-denaturing elution buffer, lane 8 represents the fourth elution with non-denaturing elution buffer, and lane 9 represents the fifth elution with non-denaturing elution buffer.

[0030] Figure 4 The lysis profiles of different Streptococcus suis strains were determined by the lysin PlyNJ3 lysing enzyme.

[0031] Figure 5 The graph shows the relationship between the lysis effect of the lysin PlyNJ3 on Streptococcus suis ML3-12 and different concentrations.

[0032] Figure 6 This is a comparison of viable cell counts after co-incubation of the lysin PlyNJ3 with different Streptococcus suis strains. Specific implementation methods

[0033] This invention provides a method for the expression and application of a lysin encoded by the ΦNJ3 prephage. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention is further described in detail below. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention. Those skilled in the art can make various modifications or alterations to this invention, but these equivalent forms also fall within the scope defined by the appended claims.

[0034] Example 1. Obtaining the gene encoding the phage lysin PlyNJ3

[0035] 1. Using the whole genome sequenced Streptococcus suis NJ3 as a template, the gene encoding the phage lysin PlyNJ3 was amplified. The full-length coding sequence of this gene is 1467 bp.

[0036] (1) The primers used for amplification include SEQ ID NO.3 and SEQ ID NO.4. SEQ ID NO.3 is the forward primer: 5'-TCGGTACCCTCGAG GGATCC GGAAAACATCTAGTCATTTGTG-3'; SEQ ID NO.4 is the reverse primer: 5'-TCTAGACTGCAG GTCGAC TTATGATATTCTAAACCAACCTACAAC-3'. The underlined portions of the forward and reverse primers represent the restriction endonuclease sites BamHI and SalI, respectively.

[0037] 2. Polymerase chain amplification (PCR) reaction and product recovery

[0038] The gene was amplified by PCR using the following mixture: 12.5 μL of 2×KeyPo Master Mix (Dye Plus), 1 μL of upstream primer, 1 μL of downstream primer, 1 μL of DNA template (NJ3 DNA), and 9.5 μL of sterile water. The amplification conditions were: 98℃ for 10 seconds, 59℃ for 5 seconds, and 72℃ for 30 seconds, for 32 cycles. After the reaction, 5 μL was added to a 1.5% agarose gel for electrophoresis analysis. Figure 1 Based on the electrophoresis results, select the correctly amplified fragments for PCR product recovery.

[0039] Example 2. Construction of recombinant plasmid pET32a-PlyNJ3

[0040] To ligate the PlyNJ3 gene fragment to the expression vector plasmid pET32a, PlyNJ3 and pET32a with sticky ends need to be constructed using restriction endonucleases BamHI and SalI, and the recombinant expression plasmid pET32a-PlyNJ3 is obtained through ligation transformation.

[0041] 1. Preparation of PlyNJ3 with viscous ends

[0042] PlyNJ3 with sticky ends was prepared using a double enzyme digestion method. A PCR system containing 1 μL BamHI, 2 μL 10× Loading Buffer, 1 μL PlyNJ3 DNA, and 15 μL sterile water was used for digestion at 30°C for 1 hour. After digestion, 1 μL SalI was added, and the digestion continued at 37°C for another hour.

[0043] 2. Preparation of pET32a with viscous ends

[0044] pET32a(+)-DH5α Escherichia coli preserved in our laboratory was transferred to Kan-resistant LB agar plates using a disposable sterile inoculation loop. After overnight culture, a single colony was picked and inoculated into 5 mL of Kan-resistant (50 μg / mL) LB broth. After overnight culture, the circular plasmid pET32a was extracted using a plasmid extraction kit.

[0045] (1) Centrifuge at 10000rcf for 1 minute at room temperature, discard the supernatant, add 250μL Solution I / RNase A mixture and vortex; add 250μL Solution II and invert to mix 4-6 times; add 350μL Solution III and gently invert to mix.

[0046] (2) Centrifuge at 13000 rcf for 10 minutes, transfer the supernatant to a DNA binding column fitted with a collection tube, centrifuge at 12000 rcf for 1 minute at room temperature, and discard the filtrate; add 500 μL HBC Buffer, centrifuge at 12000 rcf for 1 minute, and discard the filtrate; add 700 μL DNA Wash Buffer, centrifuge at 12000 rcf for 1 minute, and discard the filtrate; after centrifuging the empty column at 12000 rcf for 2 minutes, transfer the DNA binding column to a 1.5 mL centrifuge tube, add 40 μL sterile water, let stand for 1 minute, and then centrifuge at 13000 rcf for 1 minute to elute the plasmid. The concentration of the eluted plasmid was determined using a NanoDrop microspectrophotometer.

[0047] pET32a with sticky ends was prepared by double enzyme digestion. The enzyme digestion system was prepared by 1 μL BamHI, 2 μL 10× Loading Buffer, 1 μL pET32a and 15 μL sterile water in a PCR instrument at 30℃ for 1 hour. After digestion, 1 μL SalI was added and the enzyme digestion was continued at 37℃ for 1 hour.

[0048] 3. Fragment ligation of recombinant plasmids

[0049] Using 3 μL each of PlyNJ3 and pET32a (digested with sticky ends after double enzyme digestion) as templates and 6 μL of DNA Ligation Kit Mighty Mix, the mixture was incubated at 16 °C for 30 minutes. The resulting solution was transformed into competent E. coli DH5α cells and plated onto Kan-resistant (50 μg / mL) LB plates. Correctly ligated pET32a-PlyNJ3 clones were picked and stored at -20 °C.

[0050] Example 3. Large-scale expression of recombinant PlyNJ3 lysin protein in a prokaryotic expression vector

[0051] The pET32a-PlyNJ3 recombinant plasmid stored in DH5α was extracted using a plasmid extraction kit and transformed into BL21(DE3) competent E. coli cells. After overnight culture, the BL21 cells containing the recombinant plasmid were transferred 1:100 to 500 mL of Kan-resistant (50 μg / mL) LB broth and cultured until the absorbance at 600 nm was 0.4-0.6. IPTG was then added to a final concentration of 1 mM, and the cells were transferred to a shaker at 16°C and 180 rpm for further overnight culture. After centrifugation at 4°C and 10,000 rpm for 10 minutes, the supernatant was discarded, and the bacterial pellet was resuspended in 10 mL of bacterial protein lysis buffer containing 50 mM Tris, 500 mM NaCl, and pH 7.5. The pellet was then sonicated for 30 minutes, with sonication intervals of 2 seconds and 2-second intervals. After sonication, centrifuge at 10,000 rpm for 30 minutes at 4°C. Transfer the supernatant to a sterile disposable syringe and filter using a 0.45 μm cell filter. Retain 40 μL of each fraction, mix with 10 μL of 10× Loading Buffer, and load onto the sample in sequence for SDS-PAGE electrophoresis. The estimated size of the lysin PlyNJ3 protein is 70.9 kDa, therefore a 12% separating gel was selected for electrophoresis. Run at 80V for 45 minutes, then at 120V for 1.5 hours. After electrophoresis, transfer the gel to a glass dish, wash with water, stain with Coomassie Brilliant Blue for 30 minutes, and destain overnight with deionized water. See the electrophoresis results image below. Figure 2 .

[0052] Example 4. Purification of the lyase PlyNJ3

[0053] The filtered supernatant obtained in Example 3 was purified on ice after SDS-PAGE electrophoresis confirmed its accuracy. Since both ends contain His tags, a Ni-NTA-agarose purification resin pre-packed column was used to purify the lyase PlyNJ3. After allowing the 20% ethanol preservation solution in the pre-packed column to flow out naturally, the column was equilibrated with 10 mL of Binding / Wash Buffer, and the flow rate was adjusted to 0.5-1 mL / min to allow the buffer to flow out slowly. The supernatant was mixed with Binding / Wash Buffer at a 1:1 ratio to prepare a sample solution with a total volume of 10 mL. This solution was added to the pre-packed column to allow flowthrough, and the flowthrough was collected and repeated three times. The column was washed with 10 mL of Binding / Wash Buffer, repeated five times. The tagged protein, i.e., the lyase PlyNJ3, on the column was eluted with 1 mL of Elution Buffer. This process was repeated five times, and each eluent was stored at -80°C. The concentration of the purified lyase PlyNJ3 was subsequently determined using a Bradford protein assay kit.

[0054] Example 5. Determination of the cleavage spectrum of the lysin PlyNJ3

[0055] The lysis efficiency of the purified lysin PlyNJ3 obtained in Example 4 was determined by co-incubation with clinically isolated Streptococcus suis preserved in the laboratory. The specific determination methods and results are as follows:

[0056] Fourteen strains of Streptococcus suis isolated clinically in the laboratory were selected and transferred to 50 mL of THB broth, incubated overnight at 37°C, centrifuged at 4000 rpm for 3 minutes, washed three times with sterile 1×PBS, and resuspended in sterile 1×PBS. The absorbance at 600 nm was adjusted to 1.0, and 100 μL of each strain was added to a 96-well plate. 100 μL of lysin PlyNJ3 (200 mg / mL) was added to each well, and the mixture was thoroughly mixed by pipette. The plates were incubated at 37°C using a microplate reader, and the absorbance at 600 nm was measured every 10 minutes for 1 hour. Each group was tested in duplicate. The negative control group received 100 μL of sterile 1×PBS in each well, except for the bacterial resuspension; all other treatment conditions remained unchanged. The results of the lysis profile determination for different Streptococcus suis strains are shown below. Figure 4 .

[0057] Example 6. Evaluation of the antibacterial effect of the lysin PlyNJ3

[0058] The antibacterial effect of the lysin PlyNJ3 enzyme was determined by co-incubating the effective Streptococcus suis strain from Example 5 with the purified lysin and then dripping it onto a solid culture medium. The number of surviving bacteria was recorded. The specific measurement method and results are as follows:

[0059] Streptococcus suis strains exhibiting a more than 50% decrease in absorbance at 600 nm within 1 hour, as described in Example 5, were selected and transferred to 1 mL of THB broth. After overnight incubation at 37°C, the cultures were centrifuged at 4000 rpm for 3 minutes, washed three times with sterile 1×PBS, and resuspended in 200 μL of sterile 1×PBS. 100 μL of each culture was added to a 96-well plate, with 100 μL of lyase PlyNJ3 (200 mg / mL) added to one well and mixed thoroughly by pipetting. The other well was treated with 100 μL of sterile 1×PBS as a negative control. All wells were incubated at 37°C for 1 hour. After 1 hour, 100 μL of the co-incubated product was added to 900 μL of sterile 1×PBS, vortexed, and the above steps were repeated for a total of 6 dilutions, bringing the dilution to 10 times the original product. -6 Multiple, select 10 -4 10 -5 10 -6For each of the three dilutions, 10 μL of the mixture was dropped into a square THA medium, allowing the droplets to fall naturally. Two replicates were prepared for each group. The negative control group was treated using the same method. The medium was incubated overnight at 37°C. Once single colonies were visible to the naked eye, dilutions with colony counts between 30 and 300 were selected, and the colonies were counted and compared between the control and treatment groups.

[0060] Example 7. Determination of the optimal reaction concentration of lyase PlyNJ3

[0061] ML3-12 of *Streptococcus suis*, which showed good performance in lysis spectrum assays, was selected for determining the optimal reaction concentration. ML3-12 was transferred to 50 mL of THB broth and incubated overnight at 37°C. After centrifugation at 4000 rpm for 3 minutes, the bacteria were washed three times with sterile 1×PBS and resuspended in sterile 1×PBS. The absorbance at 600 nm was adjusted to 1.0. The lysis reaction was measured using a flat-bottomed 96-well plate. 100 μL of the resuspended bacterial solution was added to each well, and the lysin was diluted to 150 μg / mL, 100 μg / mL, 40 μg / mL, and 20 μg / mL, resulting in final concentrations of 75 μg / mL, 50 μg / mL, 20 μg / mL, and 10 μg / mL, respectively. Mix thoroughly by pipetting, preheat the microplate reader to 37°C, and incubate the 96-well plate in the reader. Measure the absorbance of the mixture at 600 nm every 10 minutes for 1 hour, with two replicates per group. For the negative control group, add 100 μL of sterile 1×PBS to each group except for the bacterial resuspension; all other treatment conditions remain unchanged. Results are as follows: Figure 5 As shown.

Claims

1. A phage lysin PlyNJ3, characterized in that, The amino acid sequence is shown in SEQ ID NO.

1.

2. The nucleotide encoding the phage lysin PlyNJ3 of claim 1, characterized in that, The DNA sequence is shown in SEQ ID NO.

2.

3. A plasmid containing the amino acid sequence of claim 1.

4. A vector containing the plasmid of claim 3.

5. The use of the phage lysin of claim 1 in the preparation of a product for treating streptococcal infection in pigs.

6. The application according to claim 5, characterized in that, The concentration of phage lysin in the product is not less than 10 μg / mL.

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