CN116640755B - Streptococcus prophage lyase lys1519 and application thereof - Google Patents

Abstract

The invention discloses a streptococcus prophage lyase lys1519 and application thereof. Belongs to the technical field of bioengineering. The streptococcus prophage lyase lys1519 is cloned from the streptococcus uberis SD-7 genome, and the soluble lyase is induced and expressed by adopting a prokaryotic expression mode. The streptococcus prophage lyase lys1519 has broad-spectrum antibacterial effect on streptococcus agalactiae, streptococcus dysgalactiae, streptococcus uberis and the like which can cause cow mastitis in streptococcus, and lays a foundation for preventing and treating diseases of bacterial infection.

Description

Streptococcus prophage lyase lys1519 and application thereof

Technical Field

The invention relates to the technical field of bioengineering, in particular to streptococcus prophage lyase lys1519 and application thereof.

Background

Streptococcus (streptococci) bacteria belong to the gram-positive group, and are widely distributed in the nature, in human and animal faeces and in the nasopharynx of healthy people, among which are a wide variety of pathogenic bacteria. The streptococcus agalactiae, streptococcus agalactiae and streptococcus uberis infected with cattle can cause the symptoms of cow mastitis and the like, and cause great loss for livestock breeding industry.

The presence of antibiotics allows for a certain control of streptococcal infection. However, with the irregular use of antibiotics in the livestock breeding industry, the drug resistance of the antibiotics becomes stronger gradually, and the emerging multi-drug resistant bacteria are not treated by the available antibiotics. Therefore, there is an urgent need to develop a new class of antibacterial drugs or agents for the prevention and control of streptococcus infection associated with bovine mastitis.

Phage is a virus capable of infecting a prokaryotic microorganism such as bacteria, archaea, etc., which can rapidly lyse bacteria through a "perforin-lyase" binary system, so that phage and its derived lyase can be used as an alternative antimicrobial agent for antibiotics.

Phage lysis spectra are generally narrow and limited in their use because bacteria can rapidly evolve resistance to surface receptors. The lyase derived from phage has a relatively wide cleavage spectrum, can be used as a high-efficiency and safe natural antibacterial agent for preventing and controlling drug-resistant pathogenic bacteria.

The use of phage lytic enzymes as antibacterial agents has the following advantages:

(1) Can specifically and rapidly kill host bacteria;

(2) The peptidoglycan acted on the bacterial surface by the lyase has high intra-species conservation because the peptidoglycan is a key component of the cell wall, so the lyase shows a broader spectrum of lytic activity and also determines that the bacteria are more difficult to generate resistance to the lyase;

(3) When the lyase is used for treating animals infected by pathogenic bacteria, the host animal will produce antibodies against the lyase, but the active center of the lyase is not generally an epitope exposed on the surface, so that the antibodies against the lyase do not impair its lytic bactericidal effect;

(4) The combination of the lyase and other antibacterial agents such as antibiotics has synergistic antibacterial effect, so that the cleavage spectrum can be widened, the antibacterial activity can be effectively exerted, and the probability of bacteria resistance generation is reduced.

Aiming at the diseases caused by various streptococcus infections related to cow mastitis, a broad-spectrum effective streptococcus phage lyase is not developed at present, and is applied to prevention and control of the streptococcus infections. Because of the greater difficulty in isolating streptococcal virulent phages, there are fewer reports of phage and phage-derived lytic enzymes involved.

In summary, how to provide a broad-spectrum efficient streptococcal phage lyase is a problem that needs to be solved by those skilled in the art.

Disclosure of Invention

In view of this, the present invention provides a streptococcus prophage lyase lys1519 and uses thereof.

The invention discovers that more prophages exist in the genome of streptococcus agalactiae, streptococcus dysgalactiae and streptococcus uberis which can cause cow mastitis, and phage lyase in the prophage genome is a potential antibacterial protein which is not mined yet.

In order to achieve the above purpose, the present invention adopts the following technical scheme:

a streptococcus prophage lyase lys1519 with the amino acid sequence shown in SEQ ID NO. 1.

MTDSIQEMRRLQSIPVRYDMGDRYGNDADRDGRIEMDCSSAVSKALGISMTNNTETLQQALPEIGYGKIHDAVDGIFDMKAYDVIIWAPRDGSSSLGSFGHVLIATSPTTAIHCNYGSDGITENDYNYIWDLNGRPREIVFRKGSSTTVQSAPQSDFDKELAVEARLEKSNQPYYEATLSEDYFVEAGPTITAKDKEFLRAGTRVRVYEKLNGWSRINHPESAQWVEDSYLIDATDM，SEQ ID NO.1。

Further, the nucleotide sequence of the gene encoding the streptococcus prophage lyase lys1519 is shown in SEQ ID NO. 2.

atgacagacagtattcaagaaatgagacgattacaatctattccggtccgctatgatatgggcgaccgttacggaaatgatgctgacagagatggcaggattgagatggactgctcatctgcggtctctaaagctcttggcataagtatgacaaacaatactgagacattgcaacaggccttgccagagattggatatggtaaaatccatgatgcggttgatggtatatttgatatgaaagcttatgatgttatcatttgggcgcctcgagacggctcaagctctcttggctcattcggtcacgtacttattgcgacaagtccaacaacggcaatccattgcaattatggatcggacggaataactgaaaatgactataattatatttgggatttaaacggtcggcctcgtgaaattgtctttaggaaaggttcttctactactgttcaatcagcacctcagtctgattttgataaagagctagccgttgaggctcgactggagaaatctaatcaaccttactacgaagctacgctatctgaagattactttgtagaggcaggtcctacaattactgcaaaagacaaagaattcttgcgagcaggaactcgtgtaagagtgtacgaaaaacttaacggttggtcacgaattaatcatccagagtctgcacaatgggttgaagatagttatcttattgacgcaacagatatg，SEQ ID NO.2。

Further, primers for amplifying the genes are as follows:

lys1519-F:aggagatataccatgggatccatgacagacagtattcaaga，SEQ ID NO.3；

lys1519-R:aaaatacaggttttcggtacccatatctgttgcgtcaataa，SEQ ID NO.4。

the preparation method of the streptococcus prophage lyase lys1519 comprises the following specific steps:

(1) Amplifying the nucleotide sequence of streptococcus pre-phage lyase lys1519 by taking the streptococcus uberis SD-7 genome as a template;

(2) Cloning the nucleotide sequence obtained in the step (1) into an expression vector to obtain a recombinant expression vector;

(3) Transforming the recombinant expression vector obtained in the step (2) into competent cells of escherichia coli to obtain recombinant engineering bacteria;

(4) Inducing and expressing streptococcus prophage lyase lys1519 by using recombinant engineering bacteria;

(5) Extracting and purifying the streptococcus prophage lyase lys1519 obtained in the step (4).

The beneficial effects are that: the method can obtain a large number of products with definite enzyme activity and physicochemical properties, has low production cost and strong product activity, and can meet the production requirements of popularization and application.

Furthermore, bamHI and Kpn I double digestion is adopted in the cloning process of the nucleotide sequence in the step (2).

Further, the expression vector in the step (2) is pEC, has a 6xHis tag at the C terminal, has a T7 lac promoter, and can adopt Ni ⁺ And (5) purifying by affinity chromatography. The expression vector may also be selected from other vectors suitable for expression of exogenous genes in E.coliThe vector used is such as pET series, pGEX series, pMAL series or pBAD series.

Furthermore, the engineering bacteria may be E.coli BL21 (DE 3) or E.coli Rosseta or other expression host bacteria.

Further, the inducer used for inducing expression in the step (4) is IPTG, the induction concentration is 0.5mmol/L, and the induction time is 16 h.

Further, the culture temperature of the engineering bacteria is 37 ℃ and the induction expression temperature is 25 ℃.

Use of the streptococcus prophage lyase lys1519 described above for:

1) The application of the streptococcus prophage lyase lys1519 in specific streptococcus lysis;

2) The application of the streptococcus prophage lyase lys1519 in preparing biological agents for cracking streptococcus;

3) The application of the streptococcus prophage lyase lys1519 in preparing medicaments for preventing or treating dairy cow mastitis.

Further, the streptococcus is streptococcus agalactiae, streptococcus dysgalactiae and streptococcus uberis.

Compared with the prior art, the invention has the beneficial effects that: the streptococcus prophage lyase lys1519 is cloned from the streptococcus uberis SD-7 genome, and the soluble lyase is induced and expressed by adopting a prokaryotic expression mode. The streptococcus prophage lyase lys1519 has broad-spectrum antibacterial effect on streptococcus agalactiae, streptococcus dysgalactiae, streptococcus uberis and the like which can cause cow mastitis in streptococcus, and lays a foundation for preventing and treating diseases of bacterial infection.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only embodiments of the present invention, and that other drawings can be obtained according to the provided drawings without inventive effort for a person skilled in the art.

FIG. 1 is a diagram showing SDS-PAGE results of prokaryotic expression and purification of Streptococcus prophage lyase lys1519 in example 3 of the present invention,

wherein M is a standard protein molecular weight marker;

lane 1 is the whole protein of the expression engineering strain transferred into the empty plasmid;

lane 2 is recombinant engineered strain whole protein;

lanes 3 and 4 are the purification results of streptococcal prophage lyase lys1519;

FIG. 2 shows the domain of Streptococcus prophage lyase lys1519 in example 4 of the present invention,

wherein Amidase-5 is a cleavage domain and SH3-3 is a binding domain;

amidase-5 starts at value 29 and ends at value 147 in the protein sequence;

SH3-3 begins at value 163 and ends at value 226 in the protein sequence;

FIG. 3 is a three-dimensional structure representation of the alpha fold predicted streptococcal prophage lyase lys1519;

FIG. 4 is a graph showing the in vitro bacteriostatic effect of Streptococcus dysgalactiae SD24 by Streptococcus prophage lyase lys1519 in example 5 of the present invention.

FIG. 5 is a graph showing the effect of the pre-Streptococcus phage lyase lys1519 of example 5 on Streptococcus dysgalactiae SD for 24 hours,

wherein, the left side is bacterial liquid without adding streptococcus prophage lyase lys1519,

the right side is the bacterial liquid state after adding the streptococcus prophage lyase lys1519 with the final concentration of 300 mug/mL for 1 hour.

Detailed Description

The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

The required medicament is a conventional experimental medicament and is purchased from a commercial channel;

the test methods not mentioned are conventional test methods and will not be described in detail herein.

Example 1 PCR amplification and recovery

The nucleotide sequence shown as SEQ ID NO.2 was amplified using the primer pair lys 1519-F/R.

Wherein the sequence of lys1519-F is shown as SEQ ID NO.3, and the sequence of lys1519-R is shown as SEQ ID NO. 4.

The primer sequence contains a homology arm fragment at the ligation site of the expression vector.

atgacagacagtattcaagaaatgagacgattacaatctattccggtccgctatgatatgggcgaccgttacggaaatgatgctgacagagatggcaggattgagatggactgctcatctgcggtctctaaagctcttggcataagtatgacaaacaatactgagacattgcaacaggccttgccagagattggatatggtaaaatccatgatgcggttgatggtatatttgatatgaaagcttatgatgttatcatttgggcgcctcgagacggctcaagctctcttggctcattcggtcacgtacttattgcgacaagtccaacaacggcaatccattgcaattatggatcggacggaataactgaaaatgactataattatatttgggatttaaacggtcggcctcgtgaaattgtctttaggaaaggttcttctactactgttcaatcagcacctcagtctgattttgataaagagctagccgttgaggctcgactggagaaatctaatcaaccttactacgaagctacgctatctgaagattactttgtagaggcaggtcctacaattactgcaaaagacaaagaattcttgcgagcaggaactcgtgtaagagtgtacgaaaaacttaacggttggtcacgaattaatcatccagagtctgcacaatgggttgaagatagttatcttattgacgcaacagatatg，SEQ ID NO.2。

aggagatataccatgggatccatgacagacagtattcaaga，SEQ ID NO.3。

aaaatacaggttttcggtacccatatctgttgcgtcaataa,SEQ ID NO.4。

The reaction system: 2X Phanta Mix Master Buffer. Mu.L, 1. Mu.L of template DNA, 2. Mu.L of each of the upstream and downstream primers, ddH ₂ O 20μL。

Reaction conditions: pre-denaturation at 95 ℃ for 3min, starting the cycle: 95 denaturation 15s, annealing at 60℃15s, extension at 72℃50s,30 cycles, and final extension at 72℃for 10min.

Detecting PCR amplified products by 1% agarose gel electrophoresis, and recovering target fragments amplified by PCR by using a Norpraise rubber cutting recovery kit.

EXAMPLE 2 ligation and transformation of recombinant expression plasmids competent cells

Carrying out double enzyme digestion on the expression vector, and carrying out enzyme digestion system: pEC plasmid 10. Mu.L, bamHI 1. Mu.L, kpnI 1. Mu.L, 10 XM Buffer 2. Mu.L, ddH ₂ O6. Mu.L. And (3) carrying out water bath for 4 hours at 37 ℃, verifying the enzyme digestion system by 1% agarose gel electrophoresis, and recovering the gel to obtain the carrier with the sticky end.

According to the target gene PCR gel recovery product and plasmid concentration after enzyme digestion, 20 mu L of a one-step cloning connection system is determined: 2 mu L of target gene, 2 mu L of plasmid enzyme digestion product, 4 mu L of 5 XCE II Buffer and ddH ₂ O12. Mu.L. And the connection is carried out for 30min at 37 ℃.

mu.L of the ligation product was added to 100. Mu.L of E.coli DH 5. Alpha. Competent cells, gently mixed and left on ice for 30min. Then rapidly placed on ice for 2min after heat shock at 42 ℃ for 90 s. 890. Mu.L of LB medium was added thereto, and the culture was continued at 37℃for 1 hour with shaking. After centrifugation at 5000rpm for 5min, 900. Mu.L of the supernatant was discarded, and the remaining 100. Mu.L of the resuspended broth was used and spread evenly on a solid LB medium containing Kan (final concentration 50. Mu.g/mL) and incubated in an incubator at 37℃overnight with inversion.

And (3) selecting a single colony on a Kan plate, inoculating the single colony into an LB liquid culture medium containing Kan for culture, identifying positive bacterial liquid through PCR after bacterial suspension is obtained, simultaneously sending a PCR identification system to Shanghai biological stock company for sequencing, and comparing a sequencing result with a target gene sequence.

After overnight incubation of the properly sequenced recombinant DH 5. Alpha. Strain, the recombinant plasmid was extracted using the Norfluzamide plasmid extraction kit.

10. Mu.L of the recombinant plasmid was added to 100. Mu.L of competent cells of E.coli BL21, and the mixture was left on ice for 30min after mixing. Then rapidly placed on ice for 2min after heat shock at 42 ℃ for 90 s. 890. Mu.L of LB medium was added thereto, and the culture was continued at 37℃for 1 hour with shaking. 100. Mu.L of the bacterial liquid was uniformly spread on a solid LB medium containing Kan (final concentration: 50. Mu.g/mL), and cultured in an incubator at 37℃overnight with inversion.

EXAMPLE 3 prokaryotic expression and purification of Streptococcus prophage lyase lys1519

And (3) picking a single colony on a Kan plate, inoculating the single colony into an LB liquid culture medium containing Kan for culture, and identifying positive bacterial liquid through PCR after bacterial suspension is obtained. Transferring positive bacterial liquid into LB liquid culture medium, shake culturing at 37deg.C to OD ₆₀₀ After about 0.6-0.8, IPTG with a final concentration of 0.5mmol/L is added, and the mixture is oscillated overnight at 25 ℃ to induce expression. An expression engineering bacterium transformed into an empty plasmid was used as a negative control. The recombinant engineering bacteria are subjected to expansion culture and then induced expression, and then the thallus is cracked by ultrasonic crushing. After centrifugation at 12000rpm at 4℃for 10min, the supernatant was removed and subjected to SDS-PAGE analysis. As a result, as shown in FIG. 1, the recombinant engineering bacteria showed a thicker band at 38kDa (lane 2) than the expression engineering bacteria (lane 1) into which the empty plasmid had been transferred.

The cells were resuspended in 50mM imidazole wash, sonicated and lysed, and the supernatant was taken using Ni ⁺ The affinity chromatography column is used for purifying the lyase protein. Balancing the chromatographic column for 2-3 times by using a balancing liquid, and then loading the supernatant onto the column for multiple times to enable the target protein to be compatible with the affinity chromatography Ni ⁺ The filler was fully bound and washed 6 times with 50mM imidazole to wash off the contaminating proteins. The target protein was then eluted using 200mM imidazole and the results of protein purification are shown in lanes 3 and 4 of FIG. 1.

EXAMPLE 4 physicochemical Property analysis of Streptococcus prophage lyase lys1519

Amino acid number: 237.

Relative molecular mass: 26590.43.

theoretical pI: 4.68.

Amino acid composition:

amino acid number ratio

Ala (A) 18 7.6%

Arg (R) 15 6.3%

Asn (N) 10 4.2%

Asp (D) 22 9.3%

Cys (C) 2 0.8%

Gln (Q) 8 3.4%

Glu (E) 16 6.8%

Gly (G) 17 7.2%

His (H) 4 1.7%

Ile (I) 17 7.2%

Leu (L) 13 5.5%

lys (K) 9 3.8%

Met (M) 7 3.0%

Phe (F) 6 2.5%

Pro (P) 9 3.8%

Ser (S) 21 8.9%

Thr (T) 15 6.3%

Trp (W) 4 1.7%

Tyr (Y) 12 5.1%

Val (V) 12 5.1%

Pyl (O) 0 0.0%

Sec (U) 0 0.0%

(B) 0 0.0%

(Z) 0 0.0%

(X) 0 0.0%

Total number of negatively charged residues (asp+glu): 38.

Total number of positively charged residues (arg+lys): 24.

Atomic composition:

Carbon C 1159

Hydrogen H 1791

Nitrogen N 321

Oxygen O 380

Sulfur S 9

the molecular formula: c (C) ₁₁₅₉ H ₁₇₉₁ N ₃₂₁ O ₃₈₀ S ₉

Total atomic number: 3660.

Extinction coefficient:

extinction coefficient at M ^-1 cm ^-1 In units, measured in 280nm water.

The extinction coefficient was 40005.

The absorbance was 0.1% (=1 g/l) at 1.504, assuming all pairs of Cys residues form cystines.

Estimated half-life:

the N-terminal of the protein sequence is methionine (Met).

The estimated half-life is 30 hours (mammalian reticulocytes, in vitro);

>20 hours (yeast, in vivo)；

>10 hours (Escherichia coli, in vivo)。

instability index:

the Instability Index (II) was calculated as 37.02.

This indicates that the protein classification is stable.

The Aliphatic index (aliphatics index) was 71.65.

The overall average hydrophilicity (GRAVY) was-0.571.

Domain

Submitting the amino acid sequence of the lyase to a Pfam server) The prediction of the domain shows that the domain of the streptococcal prophage lyase lys1519 comprises an N-terminal Amidase-5 cleavage domain and a C-terminal SH3-3 binding domain.

Further, the N-terminal Amidase-5 cleavage domain is from position 29 to 147 of the amino acid sequence shown in SEQ ID NO.1, and the SH3-3 binding domain at the C-terminal is from position 163 to position 226 of the amino acid sequence shown in SEQ ID NO.1 (FIG. 2).

MTDSIQEMRRLQSIPVRYDMGDRYGNDADRDGRIEMDCSSAVSKALGISMTNNTETLQQALPEIGYGKIHDAVDGIFDMKAYDVIIWAPRDGSSSLGSFGHVLIATSPTTAIHCNYGSDGITENDYNYIWDLNGRPREIVFRKGSSTTVQSAPQSDFDKELAVEARLEKSNQPYYEATLSEDYFVEAGPTITAKDKEFLRAGTRVRVYEKLNGWSRINHPESAQWVEDSYLIDATDM，SEQ ID NO.1

Three-dimensional structure prediction of the pre-streptococcal phage lyase lys1519 was performed using the AlphaFold Colab website (https:// Colab. Research. Google. Com/gitub/sokrypton/ColabFold/blob/main/AlphaFold 2.Ipynb #scrollto=azikiidicacan), submitting its amino acid sequence (SEQ ID No. 1), and mapping its domain using pymol software after downloading the prediction results, as shown in fig. 3.

Example 5 in vitro antibacterial Activity assay and cleavage Spectrum assay of Streptococcus prophage lyase lys1519

Streptococcus dysgalactiae SD24 was cultured in BHI medium to logarithmic phase, the bacterial liquid was centrifuged, and the pellet was washed 2 times with Tris-HCI (pH 7.5), then the bacterial cells were resuspended with Tris-HCI and OD was adjusted ₆₀₀ About 0.6, and then concentrated to 1/2 of the original volume. 100. Mu.L of the concentrated bacterial solution was mixed with 100. Mu.L of Streptococcus prophage lyase lysase 1519 at various concentrations (0, 400, 600, 800, 1000. Mu.g/mL) in 96-well plates to give an OD of the mixture ₆₀₀ The final concentration of the streptococcus prophage lyase lys1519 was about 0.6, and was 0, 200, 300, 400, 500. Mu.g/mL, respectively. Taking 100 μl of concentrated bacterial liquid and 100 μl of Tris-HCl mixed solution as control, placing 3 groups of the bacterial liquid in parallel, standing at 37deg.C in a multifunctional enzyme labeling instrument for culturing, and measuring OD every 10min ₆₀₀ Numerical value, OD is plotted ₆₀₀ A time-dependent curve.

As a result, as shown in FIG. 4, the OD of the Streptococcus prophage lyase lys 1519-treated group was different in concentration ₆₀₀ The values were gradually decreasing, the bacterial solution was gradually cleared (FIG. 5), and the OD of the Streptococcus prophage lyase lys1519 was not added ₆₀₀ The numerical values are not changed basically. Wherein, the descending trend of the 200 mug/mL lyase group is slightly worse; OD when the concentration of the lyase is more than 300. Mu.g/mL ₆₀₀ The decrease of the values tends to be consistent, which indicates that the application concentration of 300 mug/mL can achieve the best antibacterial effect.

The total 14 strains of streptococcus uberis, streptococcus dysgalactiae, streptococcus agalactiae, staphylococcus aureus, listeria monocytogenes, escherichia coli and pseudomonas aeruginosa are respectively taken for the determination of the lysis spectrum of the streptococcus prophage lyase lys1519. Culturing the strain to be tested at 37 ℃ to logarithmic growth medium phase, centrifuging the bacterial liquid, washing precipitate with Tris-HCI (pH 7.5) for 2 times, and then re-suspending the bacterial cells with Tris-HCI and adjusting OD ₆₀₀ About 0.6, and then concentrated to 1/2 of the original volume. mu.L of the concentrated bacterial solution was mixed with 600. Mu.g/mL of 100. Mu.L of Streptococcus prophage lyase lysase lys1519 in a 96-well plate to give an OD ₆₀₀ The value is about 0.6, and the streptococcus prophage lyase lys1519 is the endThe concentration is 300 mug/mL, 100 mug of concentrated bacterial liquid and 100 mug of Tris-HCl mixed liquid are used as control, 3 groups of bacteria are parallel, and the bacteria are placed in a multifunctional enzyme labeling instrument for static culture at 37 ℃. OD was measured at 0h and 1h ₆₀₀ Numerical value, calculate OD ₆₀₀ Ratio of decrease in value, in OD ₆₀₀ A value decrease ratio > 20% indicates that the lysis was possible to determine the lysis profile of the streptococcal prophage lyase lys1519.

The results of the lytic spectrum are shown in Table 1, and the pre-streptococcus phage lytic enzyme lys1519 has a lytic effect on all of Streptococcus agalactiae, streptococcus dysgalactiae and Streptococcus mammitis in 14 strains of Streptococcus tested, and has no lytic effect on other gram-positive bacteria tested, such as Staphylococcus aureus, listeria monocytogenes and gram-negative bacteria, such as Escherichia coli, pseudomonas aeruginosa and the like. The results show that the streptococcus prophage lyase lys1519 has broad-spectrum antibacterial capacity on the dairy cow mastitis-related streptococcus.

TABLE 1 results of cleavage spectrum measurement of lyase lys1519

In the present specification, each embodiment is described in a progressive manner, and each embodiment is mainly described in a different point from other embodiments, and identical and similar parts between the embodiments are all enough to refer to each other.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims (4)

1. A streptococcus prophage lyase lys1519 is characterized in that the amino acid sequence is shown in SEQ ID NO. 1.

2. A streptococcus prophage lyase lys1519 according to claim 1, wherein the nucleotide sequence of the gene encoding said streptococcus prophage lyase lys1519 is shown in SEQ ID No. 2.

3. A streptococcus prophage lyase lys1519 according to claim 2, wherein the primers for amplifying the gene are as follows:

lys1519-F:aggagatataccatgggatccatgacagacagtattcaaga，SEQ ID NO.3；

lys1519-R:aaaatacaggttttcggtacccatatctgttgcgtcaataa，SEQ ID NO.4。

4. use of the streptococcus prophage lyase lys1519 according to claim 1 for:

1) The application of the streptococcus prophage lyase lys1519 in specific streptococcus lysis;

2) The application of the streptococcus prophage lyase lys1519 in preparing biological agents for cracking streptococcus;

the streptococcus is streptococcus agalactiae, streptococcus agalactiae and streptococcus uberis.