CN116179525A - Phage lyase and application thereof - Google Patents

Abstract

A phage lyase and its application are provided. The name of the phage lyase is PlyNJ3; the gene fragment encoding the lyase PlyNJ3 is amplified from streptococcus suis by utilizing PCR, a prokaryotic expression vector plasmid pET32a-PlyNJ3 containing the gene encoding the PlyNJ3 is constructed, a recombinant plasmid is obtained by transforming DH5 alpha competent cells, and the recombinant plasmid is introduced into an escherichia coli expression system for large-scale expression of proteins. And then purifying by an affinity purification medium to realize the efficient expression of the lyase PlyNJ3 in vitro, and verifying that the lyase PlyNJ3 has better cracking effect on common streptococcus such as streptococcus suis, streptococcus agalactiae and the like with different serotypes in vitro. The phage lyase PlyNJ3 prepared by the invention can selectively lyse target pathogenic bacteria, and has good application prospect in the development of novel phage drugs.

Description

Phage lyase and application thereof

Technical Field

The invention belongs to the technical application field of phage and lyase thereof, and particularly relates to phage lyase and application thereof.

Background

Streptococcus suis (Streptococcus suis) is an important zoonotic primordium. Clinical symptoms of streptococcus suis infection are mainly represented by meningitis, arthritis, endocarditis and the like, and meanwhile, the streptococcus suis infected people can also cause systemic infection such as meningitis, arthritis and the like, which causes serious harm to animal husbandry and public health safety. In addition to pigs and humans, mammals such as cattle, sheep, horses, dogs, cats, and rodents can also be infected. Bacterial capsular polysaccharide antigens can be classified into 33 serotypes (1-31, 33 and 1/2) and strains that are not committed. In recent years, with the expansion of the pig raising scale, streptococcus suis has become an important pathogen that seriously jeopardizes the pig raising industry. Streptococcus suis not only causes serious economic loss to the pig industry, but also threatens public health and food safety, even threatens human health, and therefore, is receiving a great deal of attention. At present, the streptococcus suis infection treatment still depends on antibiotics and other antibacterial drugs, but with the use of a large amount of antibacterial drugs in clinic, the problem of drug resistance is generated and is increasingly serious. In recent years, the clinical isolation of streptococcus suis from laboratories has increased in resistance to common antibiotics year by year, resulting in unprecedented challenges in the field of antibacterial infections, and the development of novel phage preparations capable of lysing pathogenic bacteria has been a major hotspot.

Phage (phage) is a generic 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 bases, phages can be subdivided according to their protein structure, morphological characteristics, the structure of the infecting phage, and the nucleic acid composition constituting the phage. Phages can be classified into virulent phages and temperate phages (lysogenic phages) according to the mechanism by which they infect bacteria. The common virulent phage-infected bacteria are divided into five phases, respectively: adsorption, invasion, synthesis, assembly and release. The phage particles specifically adsorb receptors on the cell wall surfaces of bacteria, self genetic materials are injected into the bacteria, raw materials in the bacteria are utilized to synthesize and assemble progeny phage in the bacteria, and finally the bacterial cell wall is cracked, so that a large number of self-carried phages are released. The mechanism by which temperate phages infect bacteria is different from virulent phages. After the temperate phage infects bacteria, the genome DNA thereof is integrated on the genome of the bacteria and replicated along with the replication of the bacterial DNA, under specific conditions (such as ultraviolet and mitomycin C induction), the phage DNA is separated from the bacterial DNA, enters a lysis cycle, takes self genetic material as a template, synthesizes progeny phage by using raw materials in the bacteria body, and lyses the bacteria. Prophages are phages whose nucleic acid integrates into the chromosome of the host bacterium after infection of the bacterium with certain temperate phages, and are called prophages.

Phage lytic enzymes have several key advantages over antibiotics, making them attractive alternatives. Firstly, phage lyase has high specificity to a host, is not easy to generate secondary infection, and does not show any obvious toxic or side effect to mammalian cells by the current phage; secondly, because the phage has different action mechanisms compared with antibiotics, the phage can also treat multi-drug resistant bacteria; furthermore, phage lyase therapy is also an alternative treatment option for treating patients allergic to antibiotics. Most of the lytic enzymes reported at home and abroad at present are derived from virulent phage, the lytic spectrum is relatively narrow, and more pathogenic bacteria are easy to be infected by phage in the growing and developing process due to the characteristic of the virulent phage, so that cell walls are broken to cause bacterial death, the growth is slow, and the virulent phage is difficult to separate from thalli, so that a certain difficulty is brought to the research and development of a preparation related to the lytic enzymes. Research has found that the presence of Prophage (Prophage) sequences in the genome of most bacteria, and that they typically contain genes encoding lytic enzymes, provides a new strategy and path for developing lytic enzymes. The invention is to carry out whole genome sequencing on a streptococcus suis strain separated clinically and analyze the sequence of the streptococcus suis strain, and then to recombinant express a prophage-encoded lyase, and the protease is applied to the treatment of streptococcus suis infection, streptococcus agalactiae infection and the like.

Disclosure of Invention

The technical problems to be solved are as follows: aiming at the technical problems, the invention provides phage lyase and application thereof in preparing a product for treating drug-resistant streptococcus.

The technical scheme is as follows: a phage lyase PlyNJ3 has an amino acid sequence shown in SEQ ID NO. 1.

The nucleotide for encoding the phage lyase PlyNJ3 has a DNA sequence shown in SEQ ID NO. 2.

A plasmid containing the above amino acid sequence.

A vector containing the above plasmid.

The application of the phage lyase in preparing a product for treating drug-resistant streptococcus.

The concentration of phage lyase in the above product is not less than 10. Mu.g/mL.

The drug-resistant streptococcus is streptococcus suis or streptococcus agalactiae.

The invention provides an expression method and application of lyase based on phi NJ3 prophage encoding, comprising the following steps:

(1) Digging phage lyase genes: the gene sequence of the prophage of phi NJ3 in the genome of the gene sequence is analyzed and compared by carrying out whole genome sequencing on the streptococcus suis NJ3, and the gene sequence for expressing the lyase is screened.

(2) Construction of recombinant plasmids: designing a primer to amplify a gene encoding the lyase PlyNJ3 and a promoter and a terminator thereof, adding restriction endonuclease cleavage sites at two ends, respectively carrying out double cleavage on an amplified product and plasmids stored in a laboratory by using restriction endonucleases, and recombining the cleavage products by using DNA ligase to construct a recombinant plasmid.

(3) Transformation of E.coli expression systems: and (3) transforming the vector constructed in the step (2) into competent cells DH5 alpha and BL21 (DE 3) of the escherichia coli, so that the recombinant plasmid can be expressed in a large amount in an escherichia coli expression system.

(4) Expression and purification of the lyase PlyNJ 3: coli BL21 (DE 3) containing the recombinant plasmid was transferred into a broth containing Kan resistance (50. Mu.g/mL) for mass expression, and the lyase PlyNJ3 was isolated and purified from the culture broth.

Wherein the expression of the lyase PlyNJ3 of step (4) is induced expression.

The plasmid is a vector for expressing escherichia coli and pET32a (+).

The method for separating and purifying the lyase PlyNJ3 adopted in the step (4) is Ni-NTA agarose affinity column chromatography.

The specific steps are that two primers are respectively designed according to the gene sequence of the encoding lyase PlyNJ3 in the phi NJ3 prophage, a restriction endonuclease BamHI enzyme cutting site is added at the 5 'end, and a restriction endonuclease SalI enzyme cutting site is added at the 3' end. The streptococcus suis NJ3 integrated with the phi NJ3 prophage is used as a template for fragment amplification, restriction endonucleases BamHI and SalI are respectively used for double enzyme digestion with pET32a, after correct gene fragments are recovered by gel cutting, the target fragments are connected with a linearization vector plasmid by using T4DNA ligase, the recombinant product is transformed into escherichia coli competent DH5 alpha, the escherichia coli competent DH5 alpha is coated on LB agar (containing 50 mug/mL ampicillin) plates, and after macroscopic single bacterial colonies grow out, PCR identification is carried out, and escherichia coli DH5 alpha containing the recombinant plasmid pET32a-PlyNJ3 is saved.

The recombinant plasmid pET32a-PlyNJ3 is transformed into BL21 (DE 3) competent cells, and the pET32a-PlyNJ3-BL21 containing the correct recombinant plasmid is preserved after PCR identification. Bacterial liquid 1:100 was transferred to 500mL of LB broth (containing 50. Mu.g/mL ampicillin) and cultured in a shaker at 37℃and 180rpm until the absorbance of the broth at 600nm was 0.4-0.6, and IPTG was added to a final concentration of 1mM and transferred to 16℃and 180rpm shaking overnight. After the overnight cultured bacterial liquid was centrifuged at 10000rpm at 4℃for 10 minutes, the supernatant was discarded, and the bacterial pellet was used for the subsequent purification.

The purification method of the lyase PlyNJ3 is as follows: bacterial proteins were added to the bacterial pellet to prepare lysates (50 mM Tris-HCl,0.2mM PMSF,pH7.0-9.2), resuspended, and sonicated on ice for 30 min. After disruption, the supernatant was collected by centrifugation, and the supernatant was filtered through a 0.45 μm cell filter. Passing the filtered supernatant through a Ni-NTA agarose gravity chromatographic column, washing the column with 10 times of column volume non-denaturing washing liquid, adding non-denaturing washing liquid in batches to collect the flow-through liquid, and verifying the size of a strip through SDS-PAGE to obtain purified lyase PlyNJ3.

The purified lyase PlyNJ3 adopts a turbidity test method to determine the cleavage effect of the purified lyase on different pathogenic bacteria. Bacteria such as streptococcus and escherichia coli which are clinically separated in a laboratory are selected to be transferred into broth for overnight culture, the bacteria are washed by sterile 1 XPBS and then adjusted to have a light absorption value of 1.0 at a wavelength of 600nm, purified lyase PlyNJ3 is added, and the bacteria are placed in a microplate reader for co-incubation, so that the change of the light absorption value at the wavelength of 600nm is measured.

The invention uses gene recombination technology to amplify the fragment which can code phage lyase in the phi NJ3 family prophage gene integrated in streptococcus suis and constructs recombinant plasmid, transforms the recombinant plasmid into an escherichia coli expression system, induces protein expression by adding IPTG, makes the recombinant plasmid express in a large amount in the escherichia coli expression system supernatant, and obtains purified lyase PlyNJ3 by Ni-NTA agarose affinity chromatography technology, so as to carry out the next test and test the determination of the lysis effect and the optimal reaction condition of bacteria of different species.

The beneficial effects are that: the invention uses purified lyase to incubate with pathogenic bacteria clinically separated in laboratory at different temperatures, so that the final concentration of the lyase is 500 mug/mL, and the final concentration of the pathogenic bacteria is 1 multiplied by 10 ⁵ CFU/mL. The change in absorbance of the mixture at 600nm was observed. Through tests, the absorbance of most streptococcus suis at 600nm can be reduced by more than 50% within half an hour of adding lyase; compared with streptococcus suis, the lyase also has a lysis effect on some streptococcus agalactiae, and has a poor or no inhibition effect on escherichia coli and staphylococcus aureus. After the change of the absorbance value of the mixture at 600nm is measured in vitro, the co-incubated product is diluted by 10 times and is dripped into a solid culture medium, and the number of the living bacteria of the co-incubated product can be obviously reduced through comparison, so that the lyase is proved to have a certain antibacterial effect on streptococcus suis.

Drawings

FIG. 1 is an electrophoretogram (1467 bp) of the plynJ3 gene of the prophage encoding lyase of the ΦNJ3 family. Wherein lane M is the DL5000 DNA molecular weight standard and lane 1 is the lyase PlyNJ3 gene.

FIG. 2 is a SDS-PAGE analysis for examining the expression of the lyase PlyNJ3 in the recombinant vector. Lane M is the protein molecular weight standard (10-180 kDa), lane 1 is the uninduced pET32a-PlyNJ3-BL21 broth, lane 2 is the pET32a-PlyNJ3-BL21 broth after overnight induction at 1mM IPTG16 ℃, lane 3 is the uninduced supernatant, lane 4 is the 1mM IPTG16 ℃ overnight induced supernatant, lane 5 is the uninduced inclusion body protein, and Lane 6 is the inclusion body protein after overnight induction at 1mM IPTG16 ℃.

FIG. 3 is a SDS-PAGE analysis of purified lyase PlyNJ3. Lane M is the protein molecular weight standard (10-180 kDa), lane 1 is 1mM IPTG over night after induction at 16℃and is the flow-through of the supernatant through the Ni-NTA column, lane 2 is the first wash of the non-denaturing wash, lane 4 is the second wash of the non-denaturing wash, lane 5 is the first wash of the non-denaturing eluate, lane 6 is the second wash of the non-denaturing eluate, lane 7 is the third wash of the non-denaturing eluate, lane 8 is the fourth wash of the non-denaturing eluate, and Lane 9 is the fifth wash of the non-denaturing eluate.

FIG. 4 is a cleavage spectrum assay for the efficient cleavage of different Streptococcus suis by the cleavage enzyme PlyNJ3.

FIG. 5 is a graph showing the effect of the lyase PlyNJ3 on the cleavage of Streptococcus suis ML3-12 in relation to the various concentrations.

FIG. 6 is a graph comparing viable counts after incubation of the lyase PlyNJ3 with different Streptococcus suis.

Detailed description of the preferred embodiments

The invention provides an expression and application method of a lyase based on a phi NJ3 prophage code. The present invention will be described in further detail below in order to make the objects, technical solutions and effects of the present invention clearer and more specific. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention. Various changes and modifications to the present invention may be made by one skilled in the art, and such equivalents are intended to fall within the scope of the invention as defined in the claims appended hereto.

Example 1 acquisition of the gene encoding phage lyase PlyNJ3

1. The gene encoding phage lyase PlyNJ3 is amplified by taking the whole genome sequenced streptococcus suis NJ3 as a template, and the full length of the coding sequence of the gene is 1467bp.

(1) The primers used for amplification include SEQ ID NO.3 and SEQ ID NO.4.SEQ ID NO.3 is a forward primer: 5' -TCGGTACCCTCGAGGGATCCGGAAAACATCTAGTCATTTGTG-3'; SEQ ID NO.4 shows the reverse primer: 5' -TCTAGACTGCAGGTCGACTTATGATATTCTAAACCAACCTACAAC-3'. Wherein the forward primer and reverse primer underlined sections represent the restriction endonuclease BamHI and SalI cleavage sites, respectively.

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

The genes were PCR amplified by the following system: 2 XKeyPo Master Mix (Dye Plus) 12.5. Mu.L, upstream primer 1. Mu.L, downstream primer 1. Mu. L, DNA template (NJ 3 DNA) 1. Mu.L, sterile water 9.5. Mu.L. The amplification conditions were: 98℃for 10 seconds, 59℃for 5 seconds, 72℃for 30 seconds, 32 cycles. After completion of the reaction, 5. Mu.L was added to 1.5% agarose gel for electrophoresis analysis (FIG. 1). And selecting the correctly amplified fragments according to the electrophoresis result to recycle the PCR products.

EXAMPLE 2 construction of recombinant plasmid pET32a-PlyNJ3

In order to ligate the PlyNJ3 gene fragment to the expression vector plasmid pET32a, it was necessary to construct PlyNJ3 and pET32a with cohesive ends using restriction endonucleases BamHI and SalI, and to obtain recombinant expression plasmids pET32a-PlyNJ3 by ligation transformation.

1. Preparation of PlyNJ3 with cohesive ends

The PlyNJ3 with sticky ends at both ends was prepared by double cleavage. The mixture was digested with BamHI 1. Mu.L, 10×loading Buffer 2. Mu. L, plyNJ3 DNA 1. Mu.L and sterilized water 15. Mu.L at 30℃for 1 hour in a PCR instrument, and after completion of the digestion, salI 1. Mu.L was added and the digestion was continued at 37℃for 1 hour.

2. Preparation of pET32a with cohesive ends

The pET32a (+) -DH 5. Alpha. E.coli stored in the laboratory was transferred to a Kan-resistant LB plate using a disposable sterile inoculating loop, and after overnight culture, single colonies were picked up and inoculated into 5mL Kan-resistant (50. Mu.g/mL) LB broth, and after overnight culture, circular plasmid pET32a was extracted using a plasmid extraction kit.

(1) Centrifuging at 10000rcf for 1 min at room temperature, discarding the supernatant, adding 250 μl of Solution I/RNase A mixture, and shaking by vortex; adding 250 μl of Solution II, and mixing for 4-6 times; add 350. Mu.L Solution III and gently mix by inversion.

(2) 13000rcf for 10 min, transferring supernatant to DNA binding column sleeved with collecting tube, centrifuging 12000rcf for 1 min at room temperature, and discarding filtrate; adding 500 mu L of HBC Buffer, centrifuging for 1 min at 12000rcf, and discarding the filtrate; adding 700 mu L DNA Wash Buffer and 12000rcf, centrifuging for 1 minute, and discarding the filtrate; after centrifugation of the empty column 12000rcf for 2 minutes, the DNA binding column was placed in a 1.5mL centrifuge tube, 40. Mu.L of sterilized water was added, and after standing for 1 minute, the plasmid was eluted by centrifugation of 13000rcf for 1 minute. The concentration of the eluted plasmid was measured using a NanoDrop micro-spectrophotometer.

pET32a with sticky ends is prepared by a double enzyme digestion method, 1 mu L of BamHI, 1 mu L of 10×Loading Buffer2 mu L, pET a and 15 mu L of sterilized water are used for enzyme digestion for 1 hour at 30 ℃ in a PCR instrument, after the completion of which 1 mu L of SalI is added, and enzyme digestion is continued for 1 hour at 37 ℃.

3. Fragment ligation of recombinant plasmids

The system containing 3 mu L of PlyNJ3 with sticky ends and pET32a as templates after double enzyme digestion is reacted for 30 minutes at 16 ℃, transformed E.coli competent DH5 alpha is coated on LB plates with Kan resistance (50 mu g/mL), and pET32a-PlyNJ3 clones with correct connection are picked and stored in a refrigerator at-20 ℃.

EXAMPLE 3 Large expression of the lyase PlyNJ3 recombinant protein in prokaryotic expression vectors

pET32a-PlyNJ3 recombinant plasmid stored in DH5 alpha is extracted by using a plasmid extraction kit, transformed into BL21 (DE 3) escherichia coli competence, BL21 containing the recombinant plasmid is subjected to night culture, 1:100 is transferred into 500mL of LB broth with Kan resistance (50 mug/mL), when the absorbance at 600nm is 0.4-0.6, IPTG is added to a final concentration of 1mM, and the mixture is transferred to a shaking table at 16 ℃ and 180rpm for continuous overnight culture. After the overnight cultured bacterial liquid was centrifuged at 10000rpm at 4℃for 10 minutes, the supernatant was discarded, and the bacterial pellet was resuspended in 10mL of a lysate prepared from bacterial protein containing 50mM Tris, 500mM NaCl, pH 7.5, sonicated for 30 minutes, sonicated for 2 seconds, and the interval was 2 seconds. After sonication was completed, the supernatant was transferred to a disposable sterile syringe by centrifugation at 10000rpm for 30 minutes at 4℃and filtered with a 0.45 μm cell filter. After each fraction was kept at 40. Mu.L and mixed with 10. Mu.L of 10×loading Buffer, SDS-PAGE was performed sequentially, and the predicted size of the cleavage enzyme PlyNJ3 protein was 70.9kDa, so that 12% of the separation gel was selected for electrophoresis. After 45 minutes of 80V operation, 120V operation was performed for 1.5 hours. After electrophoresis, the gel was transferred to a glass plate, washed with water, stained with coomassie brilliant blue for 30 minutes, and destained overnight with deionized water. The result of electrophoresis is shown in FIG. 2.

EXAMPLE 4 purification of the lyase PlyNJ3

The filtered supernatant obtained in example 3 was subjected to SDS-PAGE electrophoresis, and then purified on ice. Since both ends contained His tag, the cleavage enzyme PlyNJ3 was purified using a Ni-NTA-agarose purification resin pre-cartridge. Naturally flowing out 20% ethanol preservation solution in the pre-packed column, balancing the column by using 10mL Binding/Wash Buffer, and regulating the flow rate to 0.5-1 mL/min to enable Buffer solution to slowly flow out of the column; mixing the supernatant with Binding/Wash Buffer 1:1 to prepare a sample solution, enabling the total volume to be 10mL, adding a pre-packed column to flow through the sample solution, and collecting the flow-through solution to repeatedly lift the sample solution for 3 times; the column was washed with 10mL Binding/Wash Buffer and repeated 5 times; the tag protein on the column, the lyase PlyNJ3, was eluted with 1mL Elution Buffer. The procedure was repeated 5 times, each time the eluate was stored at-80℃and the concentration of the purified cleavage enzyme PlyNJ3 was subsequently determined using the protein concentration determination kit according to the Bradford method.

EXAMPLE 5 determination of the schizolysis spectrum of the lyase PlyNJ3

The cleavage effect of purified lyase PlyNJ3 obtained in example 4 was determined by co-incubation with clinically isolated Streptococcus suis stored in the laboratory. The specific measurement method and the results are as follows:

a total of 14 strains of streptococcus suis clinically separated in a laboratory are selected, respectively transferred into 50mL of THB broth, cultured overnight at 37 ℃, centrifuged at 4000rpm for 3 minutes, sterilized by 1 XPBS for 3 times, resuspended by sterilized by 1 XPBS, adjusted to a light absorption value of 1.0 at a wavelength of 600nm, 100 mu L of the solution is absorbed and added into a flat-bottomed 96-well plate, 100 mu L of lyase PlyNJ3 (200 mg/mL) is added into each well, the solution is blown and mixed uniformly by a pipette, and the mixture is incubated in a 37 ℃ enzyme-labeled instrument, the light absorption value of the mixture at a wavelength of 600nm is measured once every 10 minutes, and the measurement is carried out continuously for 1 hour, and two groups are arranged in parallel. Negative control groups 100. Mu.L of sterile 1 XPBS was added to each group except for the bacterial heavy suspension, the remaining treatment conditions were unchanged. The results of the cleavage spectrum assays for the different Streptococcus suis are shown in FIG. 4.

EXAMPLE 6 evaluation of antibacterial Effect of lyase PlyNJ3

The bacteriostatic effect of the lyase PlyNJ3 was determined by selecting the effective streptococcus suis of example 5, co-incubating with purified lyase and dropping into solid medium, and recording the number of surviving bacteria. The specific measurement method and the results are as follows:

streptococcus suis with a 50% decrease in absorbance at 600nm wavelength over 1 hour in example 5 was selected, each was transferred to 1mL THB broth, incubated overnight at 37℃and centrifuged at 4000rpm for 3 minutes, sterilized 1 XPBS was used to wash 3 times, 200. Mu.L of sterilized 1 XPBS was used to resuspend, 100. Mu.L of each was added to a flat bottom 96-well plate, 100. Mu.L of the lyase PlyNJ3 (200 mg/mL) was added to one well, and the other was mixed by pipetting with a pipetting gun, 100. Mu.L of sterilized 1 XPBS was added as a negative control and incubated in a 37℃incubator for 1 hour. After 1 hour, 100. Mu.L of the co-incubated product was taken up into 900. Mu.L of sterile 1 XPBS, vortexed and mixed well, and the above procedure was repeated for a total of 6 dilutions, i.e., 10% of the original product ^-6 Multiple of 10 is selected ^-4 、10 ^-5 、10 ^-6 Three mixed solutions with dilution factors are sucked into a square THA culture medium by 10 mu L of the mixed solutions, so that the liquid drops naturally drop, and each group is provided with two parallel liquid drops. The negative control group was plated using the same method. The culture medium was placed in an incubator at 37 ℃ overnight for culture, and when macroscopic single colonies were formed, dilution factors of 30-300 colonies were selected, counted and compared for differences between the control group and the treatment group.

Example 7 determination of optimal reaction concentration of the lyase PlyNJ3

And (3) selecting streptococcus suis ML3-12 with better effect in the schizobacteria spectrum measurement to perform the optimal reaction concentration measurement. ML3-12 was transferred to 50mL THB broth, incubated overnight at 37℃and centrifuged at 4000rpm for 3 minutes, sterilized 3 times with sterile 1 XPBS and resuspended in sterile 1 XPBS to adjust the absorbance at 600nm to 1.0. The cleavage reaction was assayed using a flat bottom 96-well plate. Each well was added with 100. Mu.L of the resuspended bacterial liquid, and the resulting solution was diluted to a final concentration of 75. Mu.g/mL, 50. Mu.g/mL, 20. Mu.g/mL, 10. Mu.g/mL, and the resulting solution was diluted to 150. Mu.g/mL, 100. Mu.g/mL, 40. Mu.g/mL, and 20. Mu.g/mL with the lyase. The mixture is blown and evenly mixed by a pipetting gun, an enzyme-labeled instrument is preheated to 37 ℃, a 96-well plate is put into the enzyme-labeled instrument for co-incubation, the absorbance value of the mixture at the wavelength of 600nm is measured every 10 minutes, the continuous measurement is carried out for 1 hour, and two groups are arranged in parallel. Negative control groups 100. Mu.L of sterile 1 XPBS was added to each group except for the bacterial heavy suspension, the remaining treatment conditions were unchanged. The results are shown in FIG. 5.

Claims (7)

1. A phage lyase PlyNJ3 is characterized in that the amino acid sequence is shown in SEQ ID NO. 1.

2. A nucleotide encoding the phage lyase PlyNJ3 of claim 1, wherein the DNA sequence is shown in SEQ ID No. 2.

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

4. A vector comprising the plasmid of claim 3.

5. Use of a phage lyase according to claim 1 for the preparation of a product for the treatment of drug-resistant streptococcus.

6. The use according to claim 5, wherein the concentration of phage lyase in the product is not less than 10 μg/mL.

7. The use according to claim 5, wherein the drug-resistant streptococcus is streptococcus suis or streptococcus agalactiae.

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