CN114990098A

CN114990098A - Preparation method and application of lyase, coding gene, composition and bacteriostatic agent

Info

Publication number: CN114990098A
Application number: CN202210642388.XA
Authority: CN
Inventors: 潘强; 任慧英; 于凌云; 孙虎芝; 田延军
Original assignee: Beijing Nuoan Parkway Pharmaceutical Technology Co ltd
Current assignee: Beijing Nuoan Parkway Pharmaceutical Technology Co ltd
Priority date: 2022-06-08
Filing date: 2022-06-08
Publication date: 2022-09-02
Anticipated expiration: 2042-06-08
Also published as: CN114990098B

Abstract

The application belongs to the technical field of molecular biology, and particularly relates to a preparation method and application of lyase, a coding gene, a composition, a bacteriostatic agent. A staphylococcal bacteriophage lyase PB50 has an amino acid sequence shown in SEQ ID NO. 1. A gene encoding the staphylococcal lyase PB50 of claim 1, the gene sequence being represented by SEQ ID No. 2. The lyase can rapidly cleave staphylococcus, has strong heterologous expression solubility and less inclusion bodies.

Description

Preparation method and application of lyase, coding gene, composition and bacteriostatic agent

Technical Field

The application belongs to the technical field of molecular biology, and particularly relates to a preparation method and application of lyase, a coding gene, a composition and a bacteriostatic agent.

Background

Since the discovery of penicillin by Fleming, antibiotics are widely and rapidly applied to medical treatment, agriculture, animal husbandry, breeding industry and the like due to the advantages of broad spectrum and high efficiency, pathogenic bacteria with natural drug resistance or acquired drug resistance are propagated in large quantities, and the drug resistance of the bacteria is continuously enhanced. Thus causing bacterial resistance to become a global problem. The development rate of antibiotics is far from the evolution rate of drug-resistant bacteria, and with the increase of drug resistance, bacterial infection finally becomes untreatable. It has been reported that antibiotics cause adverse reactions including anaphylaxis, renal toxicity, cardiac toxicity, hepatic toxicity and neurotoxicity, and thus, the development of safer and more efficient substitute antibiotic products is required.

At present, more developed anti-substitution products comprise plant extracts, acidifiers, microecologics, phages, lyases thereof and the like. Phage lytic enzymes are cell wall hydrolases that are expressed by double-stranded DNA phages at a later stage of infection of a host cell. Lyase has the following advantages in "killing" bacteria: firstly, compared with the phage from which the lyase is derived, the lyase has a wider lysis spectrum and can act on more pathogenic bacteria; secondly, the cracking rate of the lyase is high, and the cracking process can be completed within minutes; thirdly, the combination effect of the antibacterial agent and other antibacterial agents such as antibiotics, lysozyme and the like is better, the side effect of a single medicine can be reduced, and the generation of drug resistance is reduced; fourth, no reports relating to drug resistance were found. Under the large background of resistance reduction and resistance replacement, the lyase has a plurality of advantages, so that the lyase has high exploitable value in the fields of agriculture, cultivation, food safety, medical treatment and the like.

The staphylococcus is a group of gram-positive cocci, can cause a plurality of serious infections of human and animals, and has extremely high detection rate in human diseases, livestock and poultry diseases, various environmental microorganisms and food-source microorganisms. The spread of methicillin-resistant staphylococcus aureus (MRSA) presents a significant challenge to clinical treatment. The lyase capable of dealing with staphylococcus infection is expected to be found, more novel and broad-spectrum lyases are excavated from the phage genome, and more possibilities are opened up for the anti-substitution industry reduction in the fields of agriculture, breeding, food safety, medical treatment and the like. The efficient broad-spectrum phage lyase has great industrial value in the aspects of treatment and disinfection of staphylococcus infection. However, phage lytic enzymes are often poorly soluble and have a number of inclusion bodies when expressed heterologously. It is necessary to obtain a high efficiency, broad spectrum of phage lytic enzymes.

Disclosure of Invention

In order to obtain the staphylococcus phage lyase with high efficiency and broad spectrum, the application provides a preparation method and application of the lyase, a coding gene, a composition and a bacteriostatic agent.

In a first aspect, the present application provides a staphylococcal bacteriophage lytic enzyme PB50, having an amino acid sequence as set forth in SEQ ID No. 1.

In a second aspect, the present application provides a gene encoding the staphylococcal lyase PB50 of claim 1, the gene sequence being represented by SEQ ID No. 2.

In a third aspect, the present application provides the use of a gene encoding staphylococcal phage lyase PB50 for recombinant expression to produce a staphylococcal lytic enzyme according to claim 1.

In a fourth aspect, the present application provides a bacteriostatic composition comprising the staphylococcal phage lyase PB50 described above.

In a fifth aspect, the application provides a bacteriostatic agent, the main active ingredient of the bacteriostatic agent is at least one of staphylococcal phage lyase PB50, a vector containing a PB50 expression element, an expression cassette containing a PB50 expression element or a host cell containing a PB50 expression element, and the amino acid sequence of the lyase PB50 is shown in SEQ ID No.1 or the gene sequence coding for staphylococcal lyase PB50 is shown in SEQ ID No. 2.

Preferably, the bacterial inhibition spectrum is staphylococcus of human origin and staphylococcus of animal origin.

In a sixth aspect, the present application provides a PCR primer set comprising primer pairs pET22b-PB50-F 'and pET22b-PB 50-R', pET25b-PB50-F 'and pET25b-PB 50-R', pET28a-PB50-F 'and pET28a-PB 50-R' or pET32a-PB50-F 'and pET32a-PB 50-R' for specifically amplifying the PB50 gene of staphylococcal bacteriophage,

the nucleotide sequences of the primers are respectively as follows:

pET22b-PB 50-F': GGAATTCCATATGAAAACAAAACTCAAGCTCTCTTGnde I cleavage site

pET22b-PB50-R’：CCGCTCGAGACTAAATGTACCCCATGCAGCAC

Xhol I cleavage site

pET25b-PB 50-F': GGAATTCCATATGAAAACAAAACTCAAGCTCTCTTGnde I cleavage site

pET25b-PB50-R’：CCGCTCGAGACTAAATGTACCCCATGCAGCAC

Xhol I cleavage site

pET28a-PB50-F’：CGGGATCCATGAAAACAAAAACTCAAGCTCTTG

BamH I cleavage site

pET28a-PB50-R’：CCGCTCGAGTTAACTAAATG TACCCCATGC AGCACCA

Xhol I cleavage site

pET32a-PB50-F’：CGGGATCCATGAAAACAAAAACTCAAGCTCTTG

BamH I cleavage site

pET32a-PB50-R’：CCGCTCGAGTTAACTAAATG TACCCCATGC AGCACCA

The restriction enzyme site of Xhol I,

the size of the target fragment for amplification of the primer pair is 798 bp.

In a seventh aspect, the present application provides a method for preparing a staphylococcal bacteriophage lytic enzyme PB50, comprising the steps of:

the method comprises the steps of taking a staphylococcus phage genome as a template, adding a primer, carrying out PCR amplification, recovering a coding gene of staphylococcus phage lyase PB50, connecting the coding gene with an expression vector skeleton after the same enzyme digestion, transforming an expression host BL21(DE3) by a verified positive recombinant expression vector pET-PB50, culturing by a liquid culture medium, carrying out induced expression, collecting thalli, extracting, and purifying, wherein the staphylococcus phage lyase PB50 has an amino acid sequence shown as SEQ ID No. 1.

Preferably, the enzyme is double-digested by Nde I/Xho I or BamH I/Xho I.

Preferably, the skeleton of the expression vector is pET22b, pET25b, pET28a or pET32 a.

In summary, the present application has at least the following advantageous effects.

1. The lyase can rapidly cleave staphylococcus, has strong heterologous expression solubility and less inclusion bodies, and solves the technical problems of poor solubility and more inclusion bodies of the conventional phage lyase in heterologous expression.

2. Compared with the phage from which the lyase is derived, the lyase has a wider lysis spectrum and can act on more pathogenic bacteria.

3. The lyase has fast cracking rate.

4. The antibacterial agent has better effect when being combined with other antibacterial agents such as antibiotics, lysozyme and the like, can reduce the side effect of a single medicament, and reduces the generation of drug resistance.

5. Under the large background of resistance reduction and resistance substitution, the lyase has a plurality of advantages, so that the lyase has higher development value in the fields of agriculture, cultivation, food safety, medical treatment and the like.

Drawings

FIG. 1: electrophoresis chart (one) of expression of PB50 in different expression strains.

FIG. 2 is a schematic diagram: electrophoresis chart (II) of PB50 expression in different expression strains.

FIG. 3: PB50 crude enzyme solution bacteriostatic effect diagram.

FIG. 4: PB50 protein purification electrophoretogram.

FIG. 5: lysis of 5ml of the cell suspension with 100ul of the enzyme solution.

In FIG. 1:

M：marker。

disruption of the supernatant in pET22b-PB50/BL21 induction group.

pET22b-PB50/BL21 induced disruption of the pellet.

The control, broken supernatant, pET22b-PB50/BL 21.

pET25b-PB50/BL21 induced group disruption supernatant.

pET25b-PB50/BL21 induced disruption of the pellet.

In FIG. 2:

M：marker。

disruption of the supernatant in the pET25b-PB50/BL21 induction group.

pET25b-PB50/BL21 induced disruption of the pellet.

Disruption of the supernatant in the pET32a-PB50/BL21 induction group.

pET32a-PB50/BL21 induced disruption of the pellet.

Disruption of the supernatant in the pET28a-PB50/BL 21-induced group.

pET28a-PB50/BL21 induced disruption of the pellet.

Disruption of the supernatant in the pET28a-PB50/BL 21-induced group.

pET28a-PB50/BL21 induced disruption of the pellet.

In fig. 4:

M：Marker。

CL: crude enzyme solution;

FT: (ii) a transudate;

W1-W4: a heteroprotein wash solution;

E1-E7: and (3) eluting the target protein.

Detailed Description

Strains, plasmids, culture media and reagents

Large intestine competence BL21(DE 3): purchased from Beijing Tiangen Biochemical technology Ltd.

Expression vector: purchased from Novengen corporation.

NB medium: purchased from BD company. 10g of tryptone, 5g of yeast extract and 10g of sodium chloride, and supplementing water to 1L. Solid NB medium: 15g of agar powder was added to the NB medium. Autoclaving at 121 deg.C for 20 min.

1. Isolation of phages

In 2021, 12 months, staphylococcus phage was isolated from the lung of mink in the garden enterochirurgia urban farm, comprising the following specific steps: a biological sample (5 g) was taken and soaked in 10mL phage buffer (phase buffer) for 30 min. The phage were allowed to well enter the buffer, centrifuged at 4500g for 10min, the supernatant carefully aspirated, and filter sterilized with a 0.22 μm filter.

2. Whole genome sequencing

The phage is purified and then subjected to whole genome sequencing. Through RAST online prediction, 297 open reading frames are obtained, and a hypothetical protein sequence is obtained and named as a gene PB 50. The total length of the PB50 gene is 798bp, and 265 amino acids are coded. The protein sequence has CHAP and SH3 structural domains.

3. Construction of recombinant expression vectors

Four recombinant plasmids, namely pET22b-PB50, pET25b-PB50, pET28a-PB50 and pET32a-PB50, were constructed. To carry a histidine purification tag or other amino acid modification to facilitate expression, in this example a histidine purification tag. Using the phage genome as a template, primers were designed as follows:

pET22b-PB50-F’：GGAATTCCATATGAAAACAAAAACTCAAGCTCTTG

nde I cleavage site

pET22b-PB50-R’：CCGCTCGAGACTAAATGTACCCCATGCAGCAC

Xhol I cleavage site

pET25b-PB50-F’：GGAATTCCATATGAAAACAAAAACTCAAGCTCTTG

Nde I cleavage site

pET25b-PB50-R’：CCGCTCGAGACTAAATGTACCCCATGCAGCAC

Xhol I cleavage site

pET28a-PB50-F’：CGGGATCCATGAAAACAAAAACTCAAGCTCTTG

BamH I cleavage site

pET28a-PB50-R’：CCGCTCGAGTTAACTAAATG TACCCCATGC AGCACCA

Xhol I cleavage site

pET32a-PB50-F’：CGGGATCCATGAAAACAAAAACTCAAGCTCTTG

BamH I cleavage site

pET32a-PB50-R’：CCGCTCGAGTTAACTAAATG TACCCCATGC AGCACCA

Xhol I cleavage site

2ul genome is taken as a template, 1ul primer is added respectively, and the Prime STAR Max is adopted to carry out PCR amplification on the target gene. The PCR procedure was as follows: (1) at 98 ℃ for 2 min; (2) 30 cycles of 98 ℃, 10s, 58 ℃, 10s, 72 ℃, 5 s; (3)72 ℃ for 10 min. And (5) recovering the PCR product by gel electrophoresis, and determining that the size of the band is in accordance with the expectation. The PB50 gene was ligated to 4 plasmids by double digestion with T4 ligase at 16 ℃ overnight to construct different recombinant plasmids, which were transformed into expression strain BL21 using conventional heat shock method (DE 3). The expression of PB50 protein was induced by 0.1mM IPTG, after 24h induction at 20 ℃, the cells were washed 2 times with PBS (50mM, pH7.0), resuspended in 1/10 volume of PBS, and disrupted by sonication to release intracellular protein, under sonication conditions of 200W, 3s sonication, 5s pause, and 70 cycles. The supernatant and the precipitate of the disruption solution were collected by centrifugation, and the protein solubility was analyzed by SDS-PAGE.

With reference to FIG. 1, PB50 was soluble expressed in pET22b-SA210/BL21 and pET25b-SA210/BL21 strains, and with reference to FIG. 2, PB50 was soluble expressed in pET25b-SA210/BL21, pET28a-SA210/BL21 and pET32a-SA210/BL21 strains. When the protein is expressed in pET28a-SA210/BL21 strain, the inclusion bodies are more, and are about 2 times of soluble protein; when the expression is carried out in pET32a-SA210/BL21 strain, the expression is almost soluble; when expressed in pET22b-SA210/BL21 and pET25b-SA210/BL21 strains, the soluble protein was 2-3 times that of inclusion bodies. In conclusion, PB50 can be expressed in different expression strains in a soluble manner, and the selection of plasmids and enzyme cutting sites influences the relative specific gravity of soluble protein and inclusion bodies. When the phage lyase is expressed in a heterologous way, the solubility is poor and the number of inclusion bodies is large, but when the plasmid and the enzyme cutting site are adopted, the solubility of the lyase is strong in the heterologous expression, and the number of the inclusion bodies is small.

pET22b-PB50/BL21(DE3) strain was selected for the following steps:

referring to FIG. 3, the supernatant of the disruption solution is spotted on a solid plate of a strain of Staphylococcus hominis, obvious plaques are visible, and PB50 crude enzyme solution is proved to have the cracking activity.

4. Purification of phage lytic enzymes

The constructed expression strain is induced by 0.1mM IPTG for 24 hours at low temperature, and the supernatant of the crushing liquid is collected. Purifying by using a Biyunyan protein purification kit (purchased from Biyunyan, product number P2226). 1ml of the uniformly mixed 50% Beyogold His-tag Purification Resin is taken, centrifuged at 4 ℃ and the liquid is discarded; adding non-denaturing lysate equilibrium gel into the centrifuge tube twice; 4ml of the supernatant of the disruption solution was added thereto, and the mixture was slowly shaken overnight at 4 ℃. Transferring the broken solution and the mixture to an affinity chromatography column tube, and collecting the flow-through solution. The column was washed 6 times, and 0.5ml of non-denaturing wash solution was added each time to remove contaminating proteins. Eluting the target protein for 10 times, and adding 0.5ml of non-denatured eluent each time until the eluate is free of protein. All the transudates were collected and subjected to SDS-PAGE analysis to determine the purification conditions.

Referring to FIG. 4, the crude enzyme solution was shaken overnight at 4 ℃ and was completely bound to the gel, and the transudate contained no target protein. The chromatographic column can be completely removed by washing for four times, namely W1-W4 in figure 4, so that the influence of the foreign protein is avoided. The purified target protein was completely eluted by 7-10 elution, e.g., E1-E7 in FIG. 4, and the active transudate was dialyzed overnight at 4 ℃ against PBS buffer (50mM, pH7.0) to obtain a purified enzyme solution. The purity of the protein obtained by analysis by using Biovision software and matching with a Quantum CX5 Edge 18.02a imaging system can reach more than 90%.

5. Detection of antibacterial rate and antibacterial spectrum of PB50 protein antibacterial active agent

(1) 100ul of pure enzyme solution is added into 5ml of bacterial solution and mixed evenly, and the bacterial solution can be changed from turbid to obvious clear after about 10 min. Referring to FIG. 5, the left test tube in FIG. 5 is a control of 5ml of bacterial liquid; the right test tube is 5ml of bacterial liquid, 100ul of pure enzyme liquid is added and mixed evenly, and a state diagram after 10min shows that the test tube is clear obviously, which indicates that the cracking rate of the lyase is high.

(2) 200 mul of the tested staphylococcus proliferation liquid is mixed with the solid NB culture medium melted to about 60 ℃, and the plate is quickly poured. After solidification, a puncher with the diameter of 6mm is used for punching a culture medium, 50 mu l of purified PB50 enzyme liquid is sucked to a sample hole, the sample hole is kept stand for about 2 hours at 4 ℃, after the enzyme liquid is completely absorbed, the sample hole is kept stand and cultured overnight at 37 ℃, and the diameter of a bacteriostatic circle is measured. The results of the specific cleavage spectra are shown in Table 1.

TABLE 1 cleavage spectra of PB50 lyase

All test strains were from the unit, with standard strains awarded by the university of Qingdao agriculture.

As can be seen from Table 1, lyase PB50 has a good cracking effect on staphylococci from different sources such as human source, pet source, pig source and cattle source, and has a total cracking rate of 89% and high cracking activity.

Based on phage genome analysis, we obtained a protein with high lytic activity against staphylococci. The lyase PB50 has broad-spectrum and high-efficiency bactericidal activity in various infections caused by staphylococcus in the fields of human medicine, pet diseases and livestock breeding, and has great significance in developing novel anti-staphylococcus drugs and controlling staphylococcus infection in vitro.

6. Staphylococcal phage antibiogram detection

Similarly, 200. mu.l of the test Staphylococcus proliferation solution was mixed with the solid NB medium thawed to about 60 ℃ and quickly transferred to a plate. After coagulation, fresh staphylococcal phage multiplication fluid (titer 10) was used ⁸ PFU/ml), 5ul of the solution was pipetted onto the plate, left to stand at 4 ℃ for about 0.5 hour, and after complete absorption, left to stand at 37 ℃ for overnight culture, and the presence or absence of plaque formation was recorded. The results of the specific cleavage spectra are shown in Table 2.

TABLE 2 lysis profiles of staphylococcal bacteriophages

All test strains were from the unit, with standard strains awarded by the university of Qingdao agriculture. "+"

Plaque is indicated and "-" indicates no plaque.

As can be seen from Table 1, lyase PB50 has a good cracking effect on staphylococci from different sources such as human source, pet source, pig source and cattle source, the total cracking rate reaches 89%, and the cracking activity is high. Under the same conditions, as shown in Table 2, the staphylococcus phage obtained from the strain 92 showed only 39% of lysis rate for the test bacteria.

6. Antibacterial composition of staphylococcus phage lyase PB50

PB50 has good lysis effect on human staphylococcus, and the lysis rate reaches 91.3%, so that the product can be used for various infections caused by staphylococcus on facial skin. Adding 1% PB50 pure enzyme solution into a skin-moistening emulsion mixture containing 0.5% of PEG-20 methyl glucose sesquistearate, 0.5% of glycerol stearate, 0.5% of hydrogenated lecithin, 0.8% of cetearyl alcohol, 2.0% of polydimethylsiloxane, 2% of hydrogenated rice bran oil, 5% of caprylic triglyceride, 4% of propylene glycol, 5% of glycerol, 0.05% of sodium hyaluronate, 0.2% of hydroxyethyl acrylate/sodium acryloyldimethyl taurate copolymer and 54% of deionized water, uniformly mixing, and measuring the bacteriostatic effect by a plate coating method, wherein the emulsion mixture added with PB50 has the cracking activity and can form cracking spots on a bacterial membrane.

The above embodiments are preferred embodiments of the present application, and the protection scope of the present application is not limited by the above embodiments, so: all equivalent changes made according to the structure, structure and principle of the present application shall be covered by the protection scope of the present application.

Sequence listing

<110> Beijing Nuo' an Baihui pharmaceutical science and technology Co., Ltd

<120> preparation methods and applications of lyase, coding gene, composition and bacteriostatic agent

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 265

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Met Lys Thr Lys Thr Gln Ala Leu Asp Trp Val Asn Ser Arg Ile Gly

1 5 10 15

Arg Arg Leu Asp Phe Asp Gly Trp Tyr Gly Ala Gln Cys Met Asp Leu

20 25 30

Thr Ile Gly Tyr Cys Asn Tyr Ile Ser Gly Gly Ser Phe Arg Pro Trp

35 40 45

Gly Asn Ala Ile Asn Leu Lys Asp Asn Thr Met Pro Ala Gly Trp Lys

50 55 60

Leu Ile Lys Asn Thr Pro Ser Phe Leu Pro Gln Pro Gly Asp Ile Ala

65 70 75 80

Ile Trp Ala Tyr Ala Pro Tyr Asp Val Tyr Gly His Thr Gly Ile Ile

85 90 95

Thr Ser Ala Asn Leu Asn Asn Phe Tyr Ser Val Asp Gln Asn Trp Phe

100 105 110

Asn Ala Gly Ser Asn Gly Ser Pro Ala Ala Lys Val Phe His Asp Tyr

115 120 125

Thr Gly Phe Trp Gly Val Ile Arg Pro Ala Phe Gly Ser Thr Ser Thr

130 135 140

Lys Lys Ala Thr Pro Lys Lys Ala Ala Pro Lys Lys Lys Val Val Lys

145 150 155 160

Lys Ala Ala Thr Lys Lys Ala Ala Thr Thr Ala Thr Trp Lys Arg Asn

165 170 175

Ser Ala Gly Ile Leu Trp Lys Thr Glu Lys Ala Lys Phe Thr Cys Asn

180 185 190

Val Ser Ser Gly Ile Ile Thr Arg Lys Asn Gly Pro Trp Thr Gly Trp

195 200 205

Ala Gln Gly Pro Phe Met Lys Lys Gly Asp Thr Ile Lys Tyr Asp Glu

210 215 220

Ile Gln Asp Phe Asp Gly His Ile Trp Val Ser Gly Asn Phe Lys Gly

225 230 235 240

Gln Tyr Val Tyr Val Pro Ile Gly Lys Ser Asn Gly Lys Gly Gln Arg

245 250 255

Ile Gly Ala Ala Trp Gly Thr Phe Ser

260 265

<210> 2

<211> 798

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

atgaaaacaa aaactcaagc tcttgattgg gttaatagtc gtattggtcg tagactagat 60

tttgatggtt ggtatggagc tcagtgtatg gatttaacta taggttactg taattatatc 120

tcaggtggtt ctttccgtcc ttggggtaat gcaattaatc ttaaagacaa cacaatgcca 180

gctggatgga aattaattaa aaatactcca tcattcttac ctcaacctgg tgatattgct 240

atttgggctt atgcacctta tgatgtttat ggtcatacag gtattattac ttcagctaac 300

ttaaataact tctattcagt tgaccaaaac tggtttaatg caggaagtaa cggttcaccg 360

gcagctaaag tatttcatga ttatacaggt ttctggggag taattcgtcc agcttttggt 420

agtacatcta ctaagaaagc aactcctaag aaagcggctc ctaagaaaaa agtagttaaa 480

aaagcagcta ctaagaaagc agctacaact gctacttgga aacgtaattc tgcaggaatt 540

ctatggaaaa ctgaaaaagc taaattcaca tgtaatgttt cttcaggaat tattactcgt 600

aaaaatggtc catggactgg atgggctcaa ggtccattta tgaagaaagg tgatactatt 660

aaatatgatg aaattcaaga tttcgatgga catatttggg tatcaggtaa ctttaaaggt 720

caatatgttt atgtaccaat tggtaaatca aatggtaaag gacaacgtat tggtgctgca 780

tggggtacat ttagttaa 798

Claims

1. A staphylococcal phage lyase PB50 characterized by: the amino acid sequence is shown in SEQ ID NO. 1.

2. A gene encoding the staphylococcal lyase PB50 of claim 1 characterized by: the gene sequence is shown in SEQ ID NO. 2.

3. Use according to claim 2 of the gene encoding the staphylococcal phage lyase PB50 of claim 1 wherein: when in use, the gene is used for recombinant expression to produce the enzyme which can crack staphylococcus according to claim 1.

4. A bacteriostatic composition, characterized in that: comprising the staphylococcal phage lyase PB50 of claim 1 or 2.

5. A bacteriostatic agent is characterized in that: the main active ingredients of the staphylococcus phage lyase are at least one of staphylococcus phage lyase PB50, a vector containing a PB50 expression element, an expression cassette containing a PB50 expression element or a host cell containing a PB50 expression element, and the amino acid sequence of the lyase PB50 is shown in SEQ ID No.1 or the gene sequence of the coded staphylococcus lyase PB50 is shown in SEQ ID No. 2.

6. The bacteriostatic agent according to claim 5, characterized in that: the antibacterial spectrum is human staphylococcus and animal staphylococcus.

7. A PCR primer set, comprising: primer pairs pET22b-PB50-F 'and pET22b-PB 50-R', pET25b-PB50-F 'and pET25b-PB 50-R', pET28a-PB50-F 'and pET28a-PB 50-R' or pET32a-PB50-F 'and pET32a-PB 50-R' comprising specific amplification of the staphylococcal bacteriophage PB50 gene,

the nucleotide sequences of the primers are respectively as follows:

pET22b-PB50-F’：GGAATTCCATATGAAAACAAAAACTCAAGCTCTTG

nde I cleavage site

pET22b-PB50-R’：CCGCTCGAGACTAAATGTACCCCATGCAGCAC

Xhol I cleavage site

pET25b-PB50-F’：GGAATTCCATATGAAAACAAAAACTCAAGCTCTTG

Nde I cleavage site

pET25b-PB50-R’：CCGCTCGAGACTAAATGTACCCCATGCAGCAC

Xhol I cleavage site

pET28a-PB50-F’：CGGGATCCATGAAAACAAAAACTCAAGCTCTTG

BamH I cleavage site

pET28a-PB50-R’：CCGCTCGAGTTAACTAAATGTACCCCATGCAGCACCA

Xhol I cleavage site

pET32a-PB50-F’：CGGGATCCATGAAAACAAAAACTCAAGCTCTTG

BamH I cleavage site

pET32a-PB50-R’：CCGCTCGAGTTAACTAAATGTACCCCATGCAGCACCA

The restriction enzyme site of Xhol I,

the size of the target fragment for amplification of the primer pair is 798 bp.

8. The preparation method of the staphylococcus phage lyase PB50 is characterized by comprising the following steps: the method comprises the steps of taking a staphylococcus phage genome as a template, adding a primer, carrying out PCR amplification, recovering a coding gene of staphylococcus phage lyase PB50, connecting the coding gene with an expression vector skeleton after the same enzyme digestion, transforming an expression host BL21(DE3) by a verified positive recombinant expression vector pET-PB50, culturing by a liquid culture medium, carrying out induction expression, collecting thalli, extracting, and purifying, wherein the staphylococcus phage lyase PB50 has an amino acid sequence shown as SEQ ID No. 1.

9. The method for producing staphylococcal phage lyase PB50 of claim 8, wherein: the enzyme digestion adopts Nde I/Xho I or BamH I/Xho I double enzyme digestion.

10. The method for preparing staphylococcal phage lyase PB50 according to claim 8 or 9, wherein the expression vector backbone is pET22b, pET25b, pET28a, or pET32 a.