CN111808837A

CN111808837A - Staphylococcus aureus bacteriophage lyase and preparation method and application thereof

Info

Publication number: CN111808837A
Application number: CN202010691524.5A
Authority: CN
Inventors: 潘强; 任慧英; 王佳; 孙虎芝
Original assignee: Qingdao No Antibiotics Biotechnology Co ltd
Current assignee: Qingdao No Antibiotics Biotechnology Co ltd
Priority date: 2020-07-17
Filing date: 2020-07-17
Publication date: 2020-10-23

Abstract

The invention discloses a staphylococcus aureus bacteriophage lyase and a preparation method and application thereof, and relates to the technical field of biological engineering. The invention provides a staphylococcus aureus bacteriophage lyase LysSA2, the amino acid sequence of which is Seq ID No. 1; the invention provides a coding gene of a staphylococcus aureus bacteriophage lyase LysSA2, and the nucleotide sequence of the coding gene is Seq ID No. 2. The invention clones the phage lyase LysSA2 from staphylococcus aureus phage SA2, and adopts a prokaryotic expression mode to induce and express soluble lyase; the phage lyase LysSA2 has strong bacteriostatic action on staphylococcus, has broad-spectrum bacteriostatic effect on staphylococcus from different sources, and lays a foundation for treating and inhibiting staphylococcus diseases.

Description

Staphylococcus aureus bacteriophage lyase and preparation method and application thereof

Technical Field

The invention relates to the technical field of bioengineering, and particularly relates to a staphylococcus aureus bacteriophage lyase as well as a preparation method and an application thereof.

Background

Staphylococcus aureus is a gram-positive bacterium, mostly a pathogenic bacterium, which can cause local pyogenic infection. The pathogenicity of staphylococcus aureus is strongly dependent on the toxins and aggressive enzymes it produces, for example: hemolytic toxins, enterotoxins, deoxyribonuclease, plasma coagulase, and the like.

Staphylococcus aureus is widely found in nature and is highly pathogenic, thus being a great hazard to animal husbandry. Various poultry are sensitive to staphylococcus aureus, umbilical cord infection of chicks is common, and staphylococcus arthritis, septicemia and the like frequently occur in the breeding stage of adult chickens and broiler breeders. Staphylococcus aureus infection can cause acute, subacute or chronic mastitis, necrotizing staphylococcus dermatitis and impetigo of breast of livestock, and if the treatment is not timely, septicemia or arthritis is further caused, which causes large-area death of livestock and brings huge economic loss to the breeding industry. Exudative dermatitis of pigs, also known as piglet oily skin disease, is the most common cause of staphylococcus aureus infection.

Bacteriophages are a class of viruses that specifically recognize and infect bacteria, which are widely found in nature, and find their corresponding bacteriophages in nature no matter how serious the pathogenic bacteria are. Compared with the traditional antibiotics for treating bacterial diseases of livestock, the bacteriophage has unique advantages: the wide drug resistance can not be generated; in addition, because the phage is propagated by the host, a small dose of a single dose can kill the bacteria. However, the phage is a live virus, and the genome may have integrase gene, so that the absolute safety of the phage cannot be guaranteed in practical application. This is also a big problem that seriously affects the popularization and application of the phage preparation.

Lytic enzymes derived from bacteriophages, which are essentially proteases, are capable of cleaving and destroying the bacterial cell wall, thereby having the ability to kill bacteria. As a novel bacteriostatic agent with higher safety, the phage lyase has the application advantage of being wider than antibiotics and phage. However, no effective staphylococcus aureus phage lyase has been isolated, purified or developed and used to inhibit and kill staphylococcus aureus. How to solve the technical problems is a technical problem to be solved in the technical field of bioengineering at present.

Disclosure of Invention

In order to solve the problems in the prior art, the invention aims to provide a staphylococcus aureus bacteriophage lyase and a preparation method and application thereof.

The technical scheme adopted by the invention is as follows:

the invention provides a staphylococcus aureus bacteriophage lytic enzyme LysSA2, wherein the amino acid sequence of the staphylococcus aureus bacteriophage lytic enzyme LysSA2 has a sequence shown as Seq ID No. 1. That is, the staphylococcus aureus phage lyase LysSA2 of the present invention may be a protease formed by the amino acid sequence shown in Seq ID No.1 itself, a protease variant formed by adding a cleavage site or other marker fragment to both ends of the amino acid sequence shown in Seq ID No.1, or a protease formed by further modifying the amino acid sequence shown in Seq ID No. 1. The modification comprises intramolecular cross-linking for improving enzyme stability, modification of side chain groups, or addition of purification tags at two ends of a sequence and the like.

Secondly, the staphylococcus aureus bacteriophage lytic enzyme LysSA2 has at least 95% homology with the amino acid sequence shown in Seq ID No.1 and has an amino acid sequence with basically the same enzyme activity. Since the sequences of microorganisms (such as phages or other expression strains) are highly susceptible to mutation during replication under natural conditions, it is preferred that at least 95% homologous mutants of the aforementioned staphylococcus aureus phage lyase LysSA2 (which retain their enzymatic activity) are also within the scope of the present application. Enzyme variants of this kind are within the scope of the present invention, since they have a very small number of amino acid mutations in non-critical positions and influence on the lyase activity. The mutant can be point mutation, deletion mutation or addition mutation, and for those skilled in the art, it is not necessary to creatively work to select a mutant with homology of 95% similar to the character of the lyase sequence provided by the invention. More preferably, the lyase mutants have 96%, 97%, 98% and 99% identity to the natural amino acid sequence as shown in Seq ID No. 1.

The invention provides a coding gene of a staphylococcus aureus bacteriophage lyase LysSA2, which has a nucleotide sequence shown in Seq ID No.2 or a nucleotide sequence of lyase which has at least 95% of homology with the sequence shown in Seq ID No.2 and can express basically the same enzyme activity.

Specifically, the domain of the staphylococcus aureus bacteriophage lytic enzyme LysSA2 comprises an N-terminal CHAP hydrolysis domain, an intermediate Amidase _2 hydrolysis domain and a C-terminal SH3_5 binding domain. Alternatively, the N-terminal CHAP hydrolysis domain is from position 5 to position 114 of the amino acid sequence as shown in Seq ID No.1, the intermediate Amidase _2 hydrolysis domain is from position 193 to position 323 of said amino acid sequence, and the C-terminal SH3_5 binding domain is from position 394 to position 459 of said amino acid sequence.

The invention provides a recombinant expression vector, which is formed by recombining an expression vector and the coding gene of the staphylococcus aureus phage lyase LysSA 2.

The invention provides a recombinant engineering bacterium, which is formed by transforming the recombinant expression vector into host bacteria.

The invention provides a preparation method of the staphylococcus aureus phage lyase LysSA2, which comprises the following specific steps:

(1) cloning the lyase LysSA2 nucleotide sequence into an expression vector to obtain a recombinant expression vector;

(2) transferring the recombinant expression vector obtained in the step 1 into escherichia coli competent cells to obtain recombinant engineering bacteria;

(3) inducing and expressing lyase LysSA2 by using recombinant engineering bacteria;

(4) and extracting and purifying the lyase LysSA2 obtained in the step 3.

Specifically, the cleavage method adopted in the cloning process of the lyase LysSA2 nucleotide sequence is SacI and XhoI double-cleavage.

Preferably, the expression vector is pCold TF carrying a fusion protein capable of promoting soluble expression.

Specifically, the escherichia coli competent cell is escherichia coli DH5 α or escherichia coli BL21, and may also be escherichia coli Top10, escherichia coli JM109, escherichia coli Rosseta, or the like. Because the escherichia coli is a model strain, the high-density fermentation condition is mature, the enzyme production period is short, and the escherichia coli is widely applied to the field of microbial fermentation industry.

In particular, the purification method employed is an affinity chromatography method. By the affinity chromatography method, the yield and purity of the target lyase can be improved, and the quality of the enzyme product can be improved.

The invention also provides application of the staphylococcus aureus bacteriophage lyase LysSA2 in preparing a medicament or a bacteriostatic agent for preventing or treating staphylococcal diseases; preferably, the staphylococcal disease is a disease caused by infection with s. The staphylococcus includes poultry-derived, animal-derived and human-derived staphylococcus, such as swine-derived, chicken-derived, rabbit-derived, cattle-derived and human-derived staphylococcus, and the staphylococcus aureus phage lyase LysSA2 has a lysis effect on the staphylococcus aureus of different sources, has a broad-spectrum lysis effect and is wide in application range.

The invention provides a detection kit, which comprises the staphylococcus aureus phage lyase LysSA 2. The above-described Staphylococcus aureus phage lyase LysSA2 can be used by those skilled in the art to prepare test kits for detecting specifically lysed Staphylococcus aureus thereof, or for identifying and controlling diseases caused by infection with Staphylococcus aureus, the host strain thereof, based on the present disclosure and general knowledge in the art.

The invention also provides a pair of specific primers for amplifying the coding gene of the staphylococcus aureus phage lyase LysSA2, wherein the primers are respectively the primer LysSA2-F described in the sequence table Seq ID No.3 and the primer LysSA2-R described in the sequence table Seq ID No. 4. The pair of primers can be used for detecting the encoding gene of the lyase LysSA2 in the recombinant engineering bacteria or the expression vector.

The invention has the beneficial effects that:

the invention clones the phage lyase LysSA2 from staphylococcus aureus phage SA2, and adopts a prokaryotic expression mode to induce and express soluble lyase; the phage lyase LysSA2 has strong bacteriostatic action on staphylococcus, has broad-spectrum bacteriostatic effect on staphylococcus from different sources, and lays a foundation for treating and inhibiting staphylococcus diseases. In addition, the phage lyase LysSA2 has the advantages of safe use and environmental protection, and can meet the production requirements of popularization and application by carrying out mass production through recombinant expression vectors.

Drawings

FIG. 1 is a schematic diagram showing the structure of the lysSA2 protein sequence,

wherein CHAP is a hydrolysis domain, Amidase _2 is a hydrolysis domain, and SH3_5 is a binding domain;

CHAP is in the protein sequence, starting at value 5 and ending at value 114;

amidase _2 in the protein sequence, starting at value 193 and ending at value 323;

SH3_5 is in the protein sequence, starting at value 394 and ending at value 459;

FIG. 2 is a diagram showing the result of PCR amplification of the gene of the lyase LysSA2,

wherein M is a standard DNA molecular weight marker; 1 is the full-length gene of the lyase LysSA2 with the total length of 1443 bps; 2 is lyase LysSA2-2 fragment gene, contains CHAP and Amidase _2 hydrolysis structural domain, total 969 bps; 3 is a lyase LysSA2-1 fragment gene which contains a CHAP structural domain and has 342 bps;

FIG. 3 is a diagram showing the results of a double digestion experiment on recombinant plasmids,

wherein M is a standard DNA molecular weight marker; 1 is the result of plasmid double digestion by the lyase LysSA 2; 2 is the result of double enzyme digestion of the lysSA2-1 fragment plasmid; 3 is the result of double enzyme digestion of the lysSA2-2 fragment plasmid;

FIG. 4 is a graph showing the MegAlign alignment of the lyase LysSA2 with a standard sequence;

FIG. 5 is a diagram showing the result of SDS-PAGE in which a lyase is expressed from a prokaryotic cell,

wherein, a is the expression of the full-length protein of the lyase LysSA 2; b is lyase LysSA2-1 fragment protein expression; c is lyase LysSA2-2 fragment protein expression;

m is the standard protein molecular weight; 1 is a thallus lysis supernatant; 2, cracking and precipitating thalli; 3 is empty plasmid induced expression;

FIG. 6 is a graph showing the in vitro inhibitory effects of the lytic enzymes LysSA2, LysSA2-1 and LysSA2-2 on Staphylococcus aureus.

FIG. 7 shows the results of pH stability assay of the lyase LysSA 2;

FIG. 8 shows the results of temperature stability of the lyase LysSA 2.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

EXAMPLE 1 Staphylococcus aureus phage lyase Gene cloning and vector construction

1.1 strains and vectors

The staphylococcus aureus bacteriophage SA2 is preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms, the preservation date is 7 months and 27 days in 2017, and the preservation code is CGMCC No. 14331.

Coli DH 5. alpha. competent cells and E.coli BL21 competent cells were purchased from Takara Bio Inc.

Expression vector pCold TF was purchased from Takara Bio engineering (Dalian) Ltd.

1.2 primer design

Determining the gene sequence of lyase LysSA2 of the phage SA2 according to the whole genome sequence of the phage SA2, and cloning the coding sequences of lyase LysSA2, lyase LysSA2-1 and lyase LysSA2-2 according to the domain characteristics of the lyase LysSA 2; wherein, the encoding gene of the LysSA2-1 only reserves the CHAP structural domain at the N end of the lyase LysSA2 gene sequence (such as Seq ID NO. 2); the encoding gene of LysSA2-2 retains the N-terminal CHAP and the intermediate Amidase Amidase _2 domain. According to the sequence of a target gene and the sequence of an expression vector pCold TF, primers of lyase LysSA2, lyase LysSA2-1 and lyase LysSA2-2 are respectively designed by using Primer Premier 5.0 software, SacI enzyme cutting sites and XhoI enzyme cutting sites are respectively added at two ends of the primers, and the primers are synthesized by Shanghai bioengineering GmbH.

LysSA 2-F: gaaggtaggcatatgGAGCTCATGACAGCAAATTTAACGAAGAAAGA (see Seq ID NO.3)

LysSA 2-R: cttgaattcggatccCTCGAGTTATTTAATTGTACCCCATAAATCACC (see Seq ID NO.4)

LysSA2-1-F：gaaggtaggcatatgGAGCTCATGACAGCAAATTTAACGAAGAAAGA

LysSA2-1-R：

cttgaattcggatccCTCGAGTTAGTTTTGCTCTGCTACAATAATATAATCA

LysSA2-2-F：gaaggtaggcatatgGAGCTCATGACAGCAAATTTAACGAAGAAAGA

LysSA2-2-R：cttgaattcggatccCTCGAGTTATCTGTGAGGACAAGCTGTAGGA

1.3 PCR amplification and recovery

And (3) respectively establishing 25 mu L of PCR reaction systems by taking the bacteriophage SA2 proliferation solution as a template: phage amplification solution SA 21 μ L, upstream and downstream primers 1 μ L, DNA Polymerase 0.5 μ L, dNTP Mix 0.5 μ L, 2 xmix buffer 12.5 μ L, ddH2O 8.5.5 μ L. Reaction conditions are as follows: pre-denaturation at 94 ℃ for 5min, start cycle: denaturation at 94 ℃ for 1min, annealing at 61.4 ℃ for 1min, extension at 72 ℃ for 2min, 30 cycles, and final total extension at 72 ℃ for 10 min. The PCR amplification products were examined by 1% agarose gel electrophoresis to obtain bands of interest corresponding to expected sizes of 1443bp, 969bp, and 342bp, as shown in FIGS. 1 and 2.

The target fragment amplified by PCR was recovered using a SanPrep column type DNA gel recovery kit.

1.4 ligation of recombinant expression plasmids and transformation of competent cells

Carrying out double enzyme digestion reaction on the expression vector pCold TF plasmid, wherein an enzyme digestion system comprises: pCold TF plasmid 10. mu.L, Sac 11. mu.L, Xho 11. mu.L, 10 XM Buffer 2. mu.L, ddH2O 6. mu.L. And (3) carrying out water bath for 4h at 37 ℃, verifying the enzyme digestion system through 1% agarose gel electrophoresis, and recovering the gel to obtain the vector with the viscous tail end.

According to the concentrations of the target gene PCR gel recovery product and the pCold TF plasmid double enzyme digestion recovery product, a 10 mu L connection system is determined: mu.L of gel recovery product, 1. mu.L of plasmid double-restriction enzyme product, 1. mu.L of 10 Xligation Buffer, and 1. mu.L of T4 DNAligase. Ligation was performed at 50 ℃ for 10 min.

mu.L of the ligation product was added to 100. mu.L of E.coli DH 5. alpha. competent cells, mixed gently and ice-cooled for 30 min. Heating in 42 deg.C water bath for 90s, and rapidly placing on ice for 2-3 min. 890. mu.L of LB broth was added and cultured at 37 ℃ for 90 min. Centrifugation is carried out for 5min at the speed of 12,000 Xg, 800 mu L of supernatant is discarded, and then the bacterial liquid is re-suspended. And (3) coating 100 mu L of bacterial liquid on a nutrient agar plate containing Amp, and after the liquid is completely absorbed, carrying out inversion culture at constant temperature of 37 ℃ overnight.

1.5 double digestion and sequencing identification of recombinant expression plasmids

Randomly picking a single bacterial colony on an Amp plate, inoculating the single bacterial colony into an LB broth culture medium containing Amp for culture to obtain a bacterial suspension, and identifying a positive bacterial liquid through PCR; PCR positive plasmid was extracted for SacI and XhoI double restriction enzyme identification, and the cleavage products were checked by 1% agarose gel electrophoresis, see FIG. 3.

Meanwhile, the PCR positive bacteria liquid is sent to Shanghai Senno biotechnology and technology limited for sequencing, and a MegAlign tool in a Lasergene software package is used for comparing with a target gene sequence according to a sequencing result, so that the result shows that the gene inserted into the recombinant plasmid is consistent with the target gene sequence, and the figure is shown in figure 4.

Example 2 inducible expression and purification of recombinant expression plasmid bacteria

2.1 transfer of recombinant expression plasmid into BL21 competent cell

Adding 1 μ L of recombinant plasmid into 100 μ L of Escherichia coli BL21 competent cells, mixing, and ice-cooling for 30 min; heating in water bath at 42 deg.C for 90s, rapidly placing on ice for 3min, adding 900 μ L LB broth, and shake culturing at 37 deg.C for 90 min; 100 mu L of the bacterial liquid is taken and spread on a nutrient agar plate containing Amp, and the culture is carried out at the constant temperature of 37 ℃ overnight.

2.2 inducible expression of recombinant expression plasmid bacteria

Single colonies transformed into BL21 competent cells were randomly picked, inoculated into LB liquid broth containing Amp, cultured with shaking at 37 ℃ until OD600 became about 0.6-0.8, added with IPTG at a final concentration of 0.1mM, and expressed with shaking overnight at 16 ℃. The recombinant proteins were named lysssa 2, LysSA2-1 and LysSA2-2, respectively, and expressed-inducing pCold TF empty plasmid was used as a control. After the recombinant bacteria are subjected to amplification culture, induction expression is carried out, intermittent ultrasonic bacteria are carried out in ice bath, after the bacteria are cracked, centrifugation is carried out at 4 ℃, and supernatant and sediment are respectively taken for SDS-PAGE electrophoretic analysis, which is shown in figure 5.

The results showed that the lyase LysSA2, LysSA2-1 and LysSA2-2 recombinant proteins were soluble expression.

2.3 purification of the recombinant proteins LysSA2, LysSA2-1 and LysSA2-2

And centrifuging the recombinant protein subjected to ultrasonic cracking at 4 ℃, taking the supernatant, and purifying the protein by using a His-tagged protein purification kit. And (2) balancing the chromatographic column with non-denatured lysate for 2-3 times, loading the recombinant protein on the column for multiple times to ensure that the protein is fully combined with the affinity chromatography Ni column filler, washing the protein with 2mM imidazole for 5 times respectively, and finally eluting the protein with 50mM imidazole for 6 times respectively to obtain the purified recombinant protein with His labels at different concentrations. The obtained eluate was subjected to SDS-PAGE electrophoresis, and the electrophorogram is shown in FIG. 5, and the sizes of the recombinant proteins LysSA2, LysSA2-1 and LysSA2-2 were 105kDa, 65kDa and 88kDa, respectively. The dialysis bags were boiled with NaHCO3 to a final concentration of 2% and EDTA to a final concentration of 1mM, and the purified proteins were placed in the dialysis bags, respectively, and dialyzed against PBS at 4 ℃ for 24 hours by changing PBS every 6 hours to obtain the finally purified LysSA2, LysSA2-1 and LysSA2-2 proteins.

Example 3 determination of the in vitro inhibitory Activity and detection of the inhibitory spectra of the lytic enzymes LysSA2, LysSA2-1 and LysSA2-2

3.1 in vitro assays for the bacteriostatic Activity of the lysssa 2, LysSA2-1 and LysSA2-2 lyssenzymes

The staphylococcus aureus is cultured in an LB liquid culture medium to a logarithmic phase, the bacterial liquid is centrifuged, washed and precipitated for 2 times by PBS, then the bacterial is used for resuspending the bacterial and the absorbance OD600 is adjusted to be about 0.65. mu.L of the resuspended suspension was mixed with 100. mu.L of each of the recombinant proteins LysSA2, LysSA2-1 and LysSA2-2 at a concentration of 500. mu.g/mL in a 96-well plate, and 3 of each group were incubated at 37 ℃ in an incubator with control of the expression-inducing empty plasmid pCold TF protein. OD600 values were measured at 30min intervals after the start of the culture, and OD600 was plotted as a function of time.

The results showed that the OD600 values of the groups LysSA2, LysSA2-1 and LysSA2-2 gradually decreased, the turbidity of the suspension gradually cleared, and the OD600 value of the empty plasmid pCold TF protein increased. The value of the turbidity OD600 of the full-length proteome bacterial liquid of the lyase LysSA2 is reduced fastest, which shows that the lyase has the most obvious effect of cracking staphylococcus aureus, the LysSA2-2 protein is inferior, and in 6h, 3 recombinant proteins have significant difference, and the figure is 6.

3.2 detection of the lytic enzyme LysSA2 bacterial inhibition Profile

In order to further verify the bacteriostasis spectra of the staphylococcus aureus bacteriophage lytic enzymes LysSA2, LysSA2-1 and LysSA2-2, staphylococcus aureus, staphylococcus epidermidis, staphylococcus saprophyticus, escherichia coli and salmonella which are stored in a laboratory are respectively selected, 64 strains in total are taken as detection bacteria, and the bacteriostasis spectra of the lytic enzymes are researched. The test strains were cultured overnight at 37 ℃ and entered logarithmic growth phase. mu.L of the resuspended suspension was mixed with 100. mu.L of each of the recombinant proteins LysSA2, LysSA2-1 and LysSA2-2 at a concentration of 500. mu.g/mL in a 96-well plate, 3 of each group were set up in parallel with Staphylococcus aureus phage SA2 as a control, and incubated at 37 ℃ in an incubator. Adding 100 mu LPBS buffer solution into the control group, and measuring the OD600 value in each well in 0h and 6h respectively; calculating the reduction ratio of the OD600 value; complete lysis is indicated by a > 50% decrease in OD. Meanwhile, the bacteriostatic effect of the lyase on staphylococcus aureus phage SA2 is compared.

The result shows that the lyase LysSA2 has obvious bacteriostatic action on 52 strains in 61 staphyloccocus with different sources, and the cracking rate is 85%; the lyase LysSA2-1 has obvious bacteriostatic action on 25 strains of 61 different staphylococci, and the cleavage rate is 41%; the lyase LysSA2-2 has obvious bacteriostatic action on 36 strains of 61 different staphylococci, and the cleavage rate is 59%; the staphylococcus aureus bacteriophage SA2 has obvious bacteriostasis on 15 strains of 61 different staphylococcus strains, and the cracking rate is only 25%.

Thus, the cleavage spectra of the lysssa 2, LysSA2-1 and LysSA2-2 were all broader than that of staphylococcus aureus phage SA 2. Among the three lysssa 2, the LysSA2 has the widest cleavage spectrum and the highest cleavage activity; but the enzyme has no bacteriostatic action on escherichia coli, salmonella and pseudomonas aeruginosa and has strong specificity. The results of the bacteriostatic spectra are shown in Table 1.

TABLE 1 antibacterial spectrum detection experiment results

Example 4 determination of pH stability and temperature stability of the lyase LysSA2

1. The experimental method comprises the following steps:

staphylococcus aureus was cultured to log phase, the cells were collected by centrifugation, washed three times with PBS and resuspended to an OD600 of about 1.0. The average was dispensed into 2mL centrifuge tubes. The same volume of 300. mu.g/mL LysSA2 protein was added and incubated at different temperatures for 1h, followed by determination of OD600, in triplicate.

The bacteria were harvested as described above, reselected with PBS buffer at different pH (2-12), added with equal amounts of purified LysSA2, incubated at optimal temperature for 1h, and then measured for OD600, and repeated three times. 2. Experimental results and analysis:

as shown in FIG. 7, LysSA2 was stable at pH 6-9, showed good lytic activity, and maintained some lytic activity under conditions of pH4 and pH 10. As shown in the results of FIG. 8, LysSA2 was adapted to a wide temperature range, and exhibited good cleavage activity at 30-37 ℃, but the cleavage activity was the strongest at a temperature of about 37 ℃ and began to decrease at a temperature exceeding 37 ℃.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Sequence listing

<110> Qingdao Nonbert Biotechnology Ltd

<120> staphylococcus aureus phage lyase and preparation method and application thereof

<160>4

<170>SIPOSequenceListing 1.0

<210>1

<211>480

<212>PRT

<213> LysSA2 Wild type phage lyase (Wild type phase lyase LysSA2)

<400>1

Met Thr Ala Asn Leu Thr Lys Lys Glu Phe Val Lys Trp Ile Lys Gln

1 5 1015

Ser Ile Gly Lys Gln Tyr Asn Phe Asp Gly Trp Phe Gly Phe Gln Cys

20 25 30

Phe Asp Ser Ala Asn Glu Gly Trp Ser Gln Leu Phe Pro Gly Glu Arg

35 40 45

Leu Lys Gly Phe Ser Ala Val Asn Ile Pro Asn Ala Asn Asp Phe Lys

50 55 60

Gly Lys Ala Lys Val Tyr Asn Asn Thr Glu Ser Phe Lys Ala Glu Pro

65 70 75 80

Gly Asp Met Val Val Phe Asn Asn Ser Tyr Gly Ala Gly His Gly His

85 90 95

Val Ala Trp Val Leu Glu Ala Thr Leu Asp Tyr Ile Ile Val Ala Glu

100 105 110

Gln Asn Trp Leu Gly Gln Gly Trp Thr Asp Gly Ile Asp Gln Pro Gly

115 120 125

Trp Gly Pro Glu Thr Val Thr Lys Arg Lys His Ala Tyr Asp Phe Pro

130 135 140

Met Trp Phe Ile Arg Pro Glu Phe Lys Ser Glu Asn Pro Ser Lys Val

145 150 155 160

Lys Pro Glu Thr Ala Lys Lys Pro Ala Lys Lys Pro Ala Lys Lys Pro

165 170 175

Gln Lys Leu Thr Val Ser Lys Asn Thr Ile Asn Tyr Asn Met Ala Asn

180 185 190

Arg Gly Tyr Lys Pro Lys Gly Ile Val Val His Asn Asp Ala Gly Gly

195 200 205

Ser Ser Ala Gln Gln Tyr Glu Asn Ser Leu Ala Asn Ala Gly Tyr Asn

210 215 220

Arg Phe Val Asn Gly Ile Ala His Ala Tyr Thr Ser Lys Gly Tyr Val

225 230 235 240

Trp Glu Ala Ile Pro Asp Gly Lys Val Ala Trp His Thr Gly Asp Gly

245 250 255

Thr Gly Lys Gly Thr Gly Asn His Glu Tyr Tyr Gly Glu Glu Val Cys

260 265 270

Gln Ser Met Ser Ala Ser Asp Ala Glu Phe Leu Lys Asn Glu Gln Ala

275 280 285

Val Phe Gln His Ala Ala Gln Lys Leu Lys Glu Trp Gly Leu Gln Ala

290 295 300

Asn Arg Asn Thr Val Arg Leu His Met Glu Phe Val Pro Thr Ala Cys

305 310 315 320

Pro His Arg Ser Met Ala Leu His Thr Gly Trp Asp Pro Val Lys Lys

325 330 335

Gly Arg Pro Ser Glu Ala Thr Met Val Lys Leu Lys Asp Tyr Phe Ile

340 345 350

Lys Gln Ile Arg Ala Tyr Met Asp Gly Lys Val Pro Thr Ala Thr Val

355 360 365

Ser Lys Asp Ser Pro Ala Ser Ser Asn Thr Ala Lys Pro Val Ala Gly

370 375 380

Lys Trp Arg Arg Asn Glu Tyr Gly Thr Tyr Tyr Met Ser Glu Ser Ala

385 390 395 400

Arg Phe Thr Asn Gly Asn Gln Pro Ile Val Ala Arg Thr Val Gly Pro

405 410 415

Phe Arg Ser Cys Pro Phe Ala Tyr Asn Phe Gln Pro Gly Gly Tyr Cys

420 425 430

Asp Tyr Asp Thr Val Met Leu Gln Asp Gly His Val Trp Ile Gly Tyr

435 440 445

Asp Trp Gln Gly Gln Arg Tyr Tyr Leu Pro Ile Arg Pro Trp Asn Gly

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Val Ala Pro Pro Asn Gln Gly Leu Gly Asp Leu Trp Gly Thr Ile Lys

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atgacagcaa atttaacgaa gaaagaattt gttaaatgga taaaacagtc aatagggaag 60

caatataatt ttgatggttg gttcggattt caatgttttg atagcgccaa tgaaggatgg 120

tctcagctgt ttccaggaga aagacttaaa ggattttcag cagtaaatat tcctaatgca 180

aacgacttta aaggtaaagc taaggtatat aataacactg aatcattcaa agctgaacca 240

ggagatatgg tagtgtttaa caactcttat ggagctgggc acggacatgt cgcttgggta 300

ttagaagcta ctcttgatta tattattgta gcagagcaaa actggttggg tcaaggatgg 360

acagacggta tcgaccaacc aggatggggt cctgaaacag taacgaaacg taaacacgct 420

tatgatttcc caatgtggtt catacgtcca gagtttaaat cagaaaatcc tagcaaagta 480

aaacctgaaa cagctaaaaa accagctaag aaaccagcta agaaacctca aaaattaaca 540

gtatcaaaaa atactattaa ttataacatg gctaatcgtg gatataaacc caagggtatt 600

gtagttcata atgatgcggg tggttctagt gcccagcaat acgaaaacag tttagctaat 660

gcaggttaca acagatttgt caatggtatt gctcatgctt atacaagtaa aggttatgta 720

tgggaagcta ttccagacgg aaaagttgca tggcacacag gtgatggaac aggtaaagga 780

actggaaacc atgaatatta tggtgaagaa gtttgtcaat caatgagcgc aagtgatgcc 840

gagttcctta aaaacgaaca agcagttttc caacacgctg cacaaaaact aaaagagtgg 900

ggacttcaag ccaacagaaa tacagttaga cttcatatgg aatttgttcc tacagcttgt 960

cctcacagaa gtatggcact tcatactggt tgggaccctg ttaaaaaagg tagaccttct 1020

gaagccacaa tggttaaatt gaaagactac ttcattaaac aaatccgtgc ctacatggat 1080

ggtaaagttc caacagctac agtatctaaa gattctcctg cttctagtaa cacggctaaa 1140

cctgtggcag gtaaatggcg tagaaatgag tatggtactt actatatgtc agaaagcgca 1200

cgtttcacta atggtaatca accaatagta gctagaacag taggaccatt tagaagctgt 1260

ccatttgcat ataacttcca acctggtggt tattgtgatt acgataccgt aatgctacaa 1320

gatggtcacg tatggattgg ttatgattgg caaggacaac gttactacct tccaatcaga 1380

ccatggaatg gtgtcgctcc accaaatcaa ggacttggtg atttatgggg tacaattaaa 1440

taa 1443

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<211>48

<212>DNA

<213> artificially synthesized sequence LysSA2-R (Synthetic sequence LysSA2-R)

<400>4

cttgaattcg gatccctcga gttatttaat tgtaccccat aaatcacc 48

Claims

1. A staphylococcus aureus bacteriophage lytic enzyme LysSA2, having an amino acid sequence as set forth in Seq ID No. 1; or an amino acid sequence which has at least 95% homology with the amino acid sequence shown in Seq ID No.1 and has substantially the same enzyme activity.

2. The staphylococcus aureus phage lyase LysSA2 according to claim 1, wherein the lyase LysSA2 is derived from a staphylococcus aureus phage.

3. The staphylococcus aureus phage LysSA2 according to claim 1 or 2, wherein the domain of the staphylococcus aureus phage LysSA2 comprises an N-terminal CHAP hydrolysis domain, an intermediate Amidase _2 hydrolysis domain and a C-terminal SH3_5 binding domain.

4. The gene encoding the bacteriophage LysSA2 of claim 1, which has the nucleotide sequence shown in Seq ID No.2 or a nucleotide sequence having at least 95% homology to the sequence shown in Seq ID No.2 and expressing a lyase having substantially the same enzymatic activity.

5. A recombinant expression vector, which is obtained by recombining an expression vector with the gene encoding the Staphylococcus aureus phage lyase LysSA2 according to claim 4.

6. A recombinant engineering bacterium, which is obtained by transforming the recombinant expression vector of claim 5 into a host bacterium.

7. The method for preparing the staphylococcus aureus phage lyase LysSA2 according to claim 1, comprising the following steps:

(1) cloning the nucleotide sequence of the lyase LysSA2 of claim 3 into an expression vector to obtain a recombinant expression vector;

(4) and extracting and purifying the lyase LysSA2 obtained in the step 3.

8. The method of claim 7, wherein the expression vector is pCold TF.

9. The method of claim 7, wherein the competent cell of Escherichia coli is Escherichia coli DH5 a or Escherichia coli BL 21.

10. Use of the staphylococcus aureus bacteriophage lytic enzyme LysSA2 of claim 1 in the manufacture of a medicament or bacteriostatic agent for preventing or treating staphylococcal disease; preferably, the staphylococcal disease is a disease caused by infection with s.

11. A test kit comprising the staphylococcus aureus phage lyase LysSA2 of claim 1 or 2.

12. A pair of primers specific for amplifying the gene coding for the bacteriophage lysiSA 2 Staphylococcus aureus according to claim 1 or 2, wherein the primers LysSA2-F and LysSA2-R are described in sequence Listing, Seq ID No.3 and Seq ID No. 4.