CN114507653B

CN114507653B - Phosphodiesterase BSP, biological agent and application thereof

Info

Publication number: CN114507653B
Application number: CN202210139703.7A
Authority: CN
Inventors: 陈红歌; 王明道; 夏国庆; 林晖; 楚梦晓; 朱华楠
Original assignee: Henan Agricultural University
Current assignee: Henan Agricultural University
Priority date: 2022-02-16
Filing date: 2022-02-16
Publication date: 2023-06-30
Anticipated expiration: 2042-02-16
Also published as: CN114507653A

Abstract

The invention provides phosphodiesterase BSP, a biological agent and application thereof, and relates to the technical field of genetic engineering. The invention provides a novel phosphodiesterase BSP which has phosphodiesterase activity and phosphomonoesterase activity, and experiments show that the bacteriostasis rate of the phosphodiesterase BSP for treating staphylococcus aureus reaches 67.63 percent and the bacteriostasis rate for treating methicillin-resistant staphylococcus aureus reaches 53.20 percent. Meanwhile, the phosphodiesterase BSP is combined with zinc chloride and erythromycin, so that the antibacterial rate of the methicillin-resistant staphylococcus aureus reaches 95.2%, and the antibacterial and bactericidal functions of the methicillin-resistant staphylococcus aureus are remarkable.

Description

Phosphodiesterase BSP, biological agent and application thereof

Technical Field

The invention relates to the technical field of genetic engineering, in particular to phosphodiesterase BSP, a biological agent and application thereof.

Background

Staphylococcus aureus (Staphylococcus aureus) is a common gram-positive pathogen that can cause a variety of diseases in humans and animals. In livestock breeding, staphylococcus aureus infection can cause animal dermatitis, pneumonia, mammitis, systemic bacteremia and the like, resulting in great loss of breeding yield and benefit. The prevention and treatment of staphylococcus aureus infections is more troublesome than the massive emergence of staphylococcus aureus drug-resistant strains. After the first isolation of methicillin-resistant staphylococcus aureus (MRSA) from jemons, a uk scholars 1961, MRSA began to be widely found worldwide. MRSA has multiple drug resistance, not only resists beta-lactam antibiotics, but also has stronger drug resistance to aminoglycosides, macrolides, tetracyclines, fluoroquinolones and sulfonamide antibiotics, becomes super bacteria, and causes quite high morbidity and mortality of people and animals.

On the one hand, new antibiotics aiming at MRSA are continuously discovered for solving MRSA infection, on the other hand, the drug sensitivity of the MRSA is restored through the use of antibiotic auxiliary agents, such as D-tyrosine (Yao Zeming, etc. 2017), clove oil (Min, etc. 2019), sanguisorba officinalis ethanol extract (Chen, et al 2015) and the like, which can be combined with beta-lactam antibiotics to generate a certain inhibition effect on the MRSA. However, there is still a great lack of effective chemical or biological agents to potentiate antibiotic action to control MRSA.

Teichoic acid is a characteristic component of the cell wall of gram-positive bacteria, is a linear polymer formed by taking glycerophosphate or ribitol phosphate with substituent groups as monomers and connecting the monomers through phosphodiester bonds, and accounts for 50% of the dry weight of the cell wall. Teichoic acid plays an important role in host colonization by gram-positive pathogenic bacteria, peptidoglycan synthesis, and cell division (Atilano, et al 2010; campbell, et al 2011). Recent studies have found that teichoic acid is also associated with resistance of MRSA strains to antibiotics. While some phosphodiester enzymes can act on phosphodiester linkages in the phosphowall acid backbone of gram-positive bacteria to produce free phosphoric acid, glycerol, or ribitol, the backbone structure of the phosphowall acid is broken, and enzymes with such an effect are also known as phosphowall acid enzymes. In 2015, the first phosphowall enzyme gene was identified (Myers, et al 2015), which is the GP12 gene in the genome of bacillus subtilis phage phi 29, and GP12 protein was an accessory protein of phage phi 29, and was demonstrated to degrade teichoic acid. However, little information is currently available about the enzyme proteins and genes capable of degrading teichoic acid, impeding the progress of bacteriological research, and lacking biological agents for the treatment of traumatic infections and the prevention of eating disorders caused by gram-positive bacteria.

Disclosure of Invention

In view of the above, the present invention aims to provide a phosphodiesterase BSP having phosphodiesterase and phosphodiesterase activities and having an obvious inhibitory effect on the growth of Staphylococcus aureus.

In order to solve the technical problems, the invention provides the following technical scheme:

the invention provides a phosphodiesterase BSP, the amino acid sequence of which is shown as SEQ ID NO. 1.

Preferably, the phosphodiesterase BSP is derived from a strain of Bacillus subtilis BGSC3A 28.

The invention provides a gene for encoding the phosphodiesterase BSP, and the nucleotide sequence of the gene is shown as SEQ ID NO. 2.

The invention also provides application of the phosphodiesterase BSP in inhibiting staphylococcus aureus.

Preferably, the staphylococcus aureus includes drug resistant staphylococcus aureus and non-drug resistant staphylococcus aureus.

More preferably, the use includes a feed additive for the treatment of an animal's epidermic infection caused by staphylococcus aureus and for the inhibition of an intestinal infection by staphylococcus aureus.

The invention also provides a biological agent with an antibacterial effect, which comprises the phosphodiesterase BSP or a combined reagent.

Preferably, the combined reagent comprises zinc chloride and/or an antibiotic.

More preferably, the antibiotic includes any one of ampicillin, neomycin and erythromycin.

The invention also provides application of the biological agent in inhibiting or killing staphylococcus aureus.

The invention provides a novel phosphodiesterase BSP which has phosphodiesterase activity and phosphomonoesterase activity, can inhibit bacterial growth by hydrolyzing a teichoic acid structure in a staphylococcus aureus cell wall, and has no mechanism for generating drug resistance to the BSP. Experiments show that the antibacterial rate of the phosphodiesterase BSP for treating staphylococcus aureus reaches 67.63%, the antibacterial rate for treating methicillin-resistant staphylococcus aureus reaches 53.20%, and the phosphodiesterase BSP is suitable for treating animal epidermic infection caused by staphylococcus aureus and drug-resistant staphylococcus aureus or can be used for inhibiting staphylococcus aureus intestinal infection by a feed additive. Meanwhile, the phosphodiesterase BSP disclosed by the invention is combined with zinc chloride or erythromycin, so that the antibacterial rate of the methicillin-resistant staphylococcus aureus reaches 95.2%, the methicillin-resistant staphylococcus aureus has remarkable antibacterial and bactericidal functions, and a new solution is provided for health threat caused by methicillin-resistant staphylococcus aureus infection.

Drawings

FIG. 1 shows the expression of BSP protein in E.coli; wherein M is a protein marker,1 is a whole cell component, 2 is a supernatant after cell disruption, and 3 is a precipitate after cell disruption.

FIG. 2 shows the self-cleavage site of BSP protein.

FIG. 3 is a purified BSP protein; wherein M is a protein marker, and 1 is a purified BSP.

FIG. 4 is a graph showing the determination of BSP optimum action temperature and optimum action pH; wherein a) the temperature of BSP optimum action is measured, b) the pH of BSP optimum action is measured.

FIG. 5 is a graph showing the effect of metal ions on the catalytic activity of BSP.

Fig. 6 is the effect of BSP on staphylococcus aureus (Sa) growth.

FIG. 7 is an effect of BSP on methicillin-resistant Staphylococcus aureus (MRSA).

FIG. 8 is an OD of Staphylococcus aureus under the action of BSP at different concentrations ₆₀₀ Values.

FIG. 9 is an OD of treating Staphylococcus aureus with different metal ions and BSP ₆₀₀ Values.

FIG. 10 is an OD of BSP protein in combination with ampicillin after various times of MRSA treatment ₆₀₀ Values.

FIG. 11 shows OD after various times of treatment of MRSA with BSP protein and neomycin ₆₀₀ Values.

FIG. 12 is an OD of BSP protein and erythromycin combined treatment of MRSA at various times ₆₀₀ Values.

FIG. 13 shows the change in MRSA culture color upon treatment with zinc chloride and erythromycin in combination with BSP; wherein, the 1, 2 and 3 tubes are culture solution without BSP addition culture for 24 hours; 4.

tubes

5 and 6 are added with culture solution for 24 hours for BSP.

Detailed Description

In the invention, the nucleotide sequence of the encoding phosphodiesterase BSP gene is shown as SEQ ID NO. 2. The phosphodiesterase BSP is derived from bacillus subtilis BGSC3A28 strain (Bacillus subtilisBGSC A28) and has phosphodiesterase activity and phosphomonoesterase activity. The BSP has no obvious homology with the known phosphodiesterase, has the homology with GP12 protein of only 39.29 percent, and is a novel phosphodiesterase. The phosphodiesterase BSP of the present invention is expressed in a soluble form, and the BSP protein cleaves between the positions 674-675. The mature BSP protein contains 674 amino acid residues and the molecular weight of the protein is 71.4kDa.

In the present invention, the staphylococcus aureus includes drug-resistant staphylococcus aureus and non-drug-resistant staphylococcus aureus. In a specific embodiment of the invention, the resistant staphylococcus aureus is preferably Methicillin-resistant staphylococcus aureus (Methicillin-resistant Staphylococcus aureus, denoted MRSA) and the non-resistant staphylococcus aureus is preferably staphylococcus aureus (Staphylococcus aureus, denoted Sa). In the present invention, the use includes a feed additive for the epidermic infection of animals caused by staphylococcus aureus and for inhibiting intestinal tract infection of staphylococcus aureus. The specific application method is not particularly limited, and the application method conventional in the art can be selected according to different application objects.

In the present invention, the combined reagent is preferably zinc chloride and/or an antibiotic; the antibiotic is preferably any one of ampicillin, neomycin and erythromycin, more preferably erythromycin.

The present invention will be described in detail below with reference to examples for the purpose of making the objects, technical solutions and advantages of the present invention more apparent, but they should not be construed as limiting the scope of the present invention.

In the following examples, conventional methods are used unless otherwise specified.

Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

EXAMPLE 1 preparation of BSP protein

And searching a gene sequence of the BSP protein WP-032721605.1 in NCBI (NCBI accession number is NZ-FODG 01000008.1), and obtaining a gene sequence of the encoding BSP protein after codon optimization, wherein the gene sequence is shown in SEQ ID NO.2, and enzyme cutting sites of NdeI and XhoI are respectively added at the 5 'end and the 3' end of the encoding gene. The amino acid sequence of the BSP protein obtained by encoding the gene is shown as SEQ ID NO. 1. The SEQ ID NO.2 gene sequence was submitted to gene synthesis by Suzhou Jin Weizhi and constructed into pET-21a (+) vector. And (3) transforming the recombinant pET-21a (+) vector obtained by construction into competent cells of the escherichia coli BL21 (DE 3) to obtain the escherichia coli transformant with correct sequencing.

Recombinant E.coli was inoculated into 100mL of LB medium and cultured at 37℃and 250rpm for 2.5h to OD ₆₀₀ When the value reaches 0.8-0.9, 0.1mM IPTG is added, and the induction is performed at 16 ℃ and 180rpm for 20 hours. The cells were collected by centrifugation, washed 2 times with 20mL of equilibration buffer (NaCl 8.766g, imidazole 0.340g, tris) 1.21g in 400mL of deionized water, pH adjusted to 7.4 with hydrochloric acid, and constant volume to 500 mL), resuspended in 50mL of centrifuge tube with 10mL of lysis buffer, and sonicated under ice water bath conditions for 20min (6 mm horn, power 200W, 3s working, 5s suspension). After sonication, the mixture was centrifuged at 12000 Xg for 10min, and the supernatant was taken as a soluble protein component, and the expression of BSP protein was detected by SDS-PAGE, and the results are shown in FIG. 1.

It can be seen that BSP is expressed in soluble form, but the molecular weight of the expressed protein is significantly lower than the theoretical molecular weight of 92.0kDa for BSP, about 70kDa. As can be seen from an analysis of the amino acid sequence of BSP, the BSP contains a self-cleaving domain (peptidase_G2 (pfam 11962), and amino acid residues 618-847 may exert self-cleaving function.

Purifying the N-terminal part and the C-terminal part of the BSP with (His) 6 tag sequence by using a Ni-NTA column, loading the supernatant after ultrasonic disruption and centrifugation to the Ni-NTA column, flushing the column by using a balance buffer, dissolving 1.21g of Tris (Tris) in 400mL of deionized water by using an elution buffer (NaCl 8.766g, imidazole 10.212g, hydrochloric acid to adjust the pH to 7.4 and the volume to 500 mL), eluting the BSP protein with (His) 6 tag at a flow rate of 0.5mL/min, judging the purity according to SDS-PAGE, and carrying out mass spectrometry on the BSP protein which is uniform and pure by SDS-PAGE to the self-cleavage site of the BSP. The results are shown in FIG. 2. The purified BSP protein was subjected to electrophoresis, and the results were shown in FIG. 3.

As can be seen from FIGS. 2 and 3, cleavage of the BSP protein did occur between positions 674-675, the mature BSP protein contained 1-674 amino acid residues, the theoretical molecular weight was 71.4kDa, and the size of the purified BSP protein was consistent with the theoretical value of the mature BSP protein.

EXAMPLE 2 phosphatase Activity of BSP

1. And respectively taking bis-p-nitrophenyl-sodium phosphate (bis-pNPP) and p-nitrophenyl-disodium phosphate (pNPP) as substrates to detect whether the BSP has phosphodiesterase activity and phosphomonoesterase activity.

The reaction system for measuring the enzyme activity is as follows: in a final reaction system of 100. Mu.L, 40mM Tris-HCl, 1mM CaCl was contained ₂ 10mM bis-pNPP (or pNPP), 1.8. Mu.M BSP protein (control tube in Na addition ₂ CO ₃ The same amount of BSP was added after the termination of the reaction, the reaction tube and the control tube were allowed to stand at 37℃for 30 minutes, and 200. Mu.L of 2M Na was then added ₂ CO ₃ The reaction was stopped and after 2.9mL deionized water was added, the OD was measured ₄₀₀ The value of the amount of released p-nitrophenol was converted (extinction coefficient of p-nitrophenol: 1.7X10) ⁴ mol ^-1 ·cm ^-1 ·L)。

Definition of enzyme activity unit: under the above conditions, the amount of enzyme catalyzing bis-pNPP (or pNPP) to produce μmol p-nitrophenol within 1min is 1 enzyme activity unit (IU).

As a result of the measurement, whether bis-pNPP is used as a substrate or pNPP is used as a substrate, the BSP generates p-nitrophenol, and the p-nitrophenol has phosphodiesterase activity and phosphomonoesterase activity corresponding to the BSP respectively. The phosphodiesterase activity of BSP was 1.40IU/mg and the phosphodiesterase activity was 0.92IU/mg.

2. Taking bis-pNPP as a substrate, and determining the optimal action temperature of BSP under the condition that the pH of a reaction system is 7.2 and the reaction time is 15 min. Meanwhile, under the condition that the temperature of a reaction system is 37 ℃ and the reaction time is 15min, the optimal action pH of BSP is measured. The results are shown in FIG. 4.

It can be seen that the novel BSP enzymes of the present invention have phosphodiesterase activity and phosphomonoesterase activity. Taking bis-pNPP as a substrate, the optimal catalytic temperature for catalyzing the hydrolysis of bis-pNPP by BSP is 85 ℃, and the optimal catalytic pH is 8.5.

3. To determine the effect of metal ions on BSP activity, the effect of different metal ions on BSP activity was determined as follows: in the reaction system described in 1 (bis-pNPP was used as a substrate), EDTA and various metal ion chlorides were added at a final concentration of 1mM, and the phosphodiesterase activity of BSP was measured in the same manner as in 1, and the relative enzyme activities of BSP in the presence of EDTA and various metal ions were calculated with the enzyme activity of BSP without EDTA or metal ions being 100%, and the results of the measurement are shown in FIG. 5.

It can be seen that BSP remains hydrolytically active in the presence of EDTA. Most divalent ions have a promoting effect on the catalytic activity of BSP, wherein Mn ²⁺ And Co ²⁺ The promotion effect of (2) is most obvious, and is respectively improved by 414 percent and 331 percent, and Fe ³ ⁺ The presence of (2) significantly reduces the catalytic activity of the BSP.

The kinetics of BSP action on bis-pNPP was further determined using the Eadie-Hofstee mapping method: in the reaction system described in 1, the concentration of substrate bis-pNPP was set to 0.5, 1.0, 2.0, 5.0, 10, 30mM, respectively, and the reaction tube and the control tube were placed at 37℃for 10 minutes of incubation, and the reaction system described in 1 was used for the remainder to determine the initial reaction rate at different substrate concentrations. The ratio of the initial reaction rate to the substrate concentration (v/[ S ]]) V is calculated from the curve of (1), i.e. the Eadie-Hofstee curve _max And K _m K is as follows _cat . The results are shown in Table 1 below.

TABLE 1 kinetic parameters of BSP

It can be seen that BSP catalyzes the conversion k of bis-pNPP hydrolysis _cat Is 6.99min ^-1 。

Example 3

Inhibition of BSP on growth of Staphylococcus aureus (Sa) and methicillin-resistant Staphylococcus aureus (MRSA)

3mL of LB liquid medium was taken, purified BSP was added in an amount of 50ug/mL final concentration (3.5 mL final volume of culture medium), 100uL of pre-activated Staphylococcus aureus (Sa) and methicillin-resistant Staphylococcus aureus (MRSA) species were added, respectively, and the mixture was supplemented to 3.5mL with sterilized 50mM Tris buffer pH 7.5. The treatment group and the control group were each set with three replicates with the addition of equal amounts of inactivated BSP as control. Each culture tube was cultured in a shaking incubator at a constant temperature of 37℃at 220rpm, 100uL of the bacterial liquid was taken out at 24 hours and 48 hours, and the bacterial liquid was applied to a solid LB plate for viable count after gradient dilution, and the results are shown in FIG. 6 and FIG. 7.

The results show that for Sa, the bacteriostasis rates of the BSP treatment for 24h and 48h are 53.33% and 67.63% respectively; for MRSA, the antibacterial rates of BSP treatment for 24h and 48h are 53.20% and 49.08% respectively, which shows that BSP has obvious capability of inhibiting the growth of staphylococcus aureus and drug-resistant staphylococcus aureus.

Minimum effective concentration of BSP against Staphylococcus aureus

The BSP primordial enzyme solution purified by affinity chromatography in example 1 was subjected to 1:1, and sequentially carrying out 1 on diluted enzyme solution: 1 to obtain 4 BSP concentrations, respectively adding BSP original enzyme solution and diluted enzyme solution according to the culture system in example 2 to make the final concentration of BSP in the culture solution be respectively 50ug/mL, 25ug/mL, 12.5ug/mL and 6.25ug/mL, placing in a shaking table at 37 deg.C, culturing at 220rpm for 24 hr, quickly determining OD of the culture solution ₆₀₀ Values, results are shown in fig. 8.

According to OD ₆₀₀ As can be seen from the value analysis of the inhibition of BSP on the growth of staphylococcus aureus, the inhibition on the growth of staphylococcus aureus is gradually reduced along with the reduction of the concentration of BSP, although the BSP still has a certain antibacterial effect at the concentrations of 12.5ug/mL and 6.25ug/mL, the optimal antibacterial effect is considered under the condition of the lowest enzyme consumption, so that the concentration of BSP of 25ug/mL is selected as the optimal addition concentration.

EXAMPLE 4 action of biological Agents

Divalent Metal ion combination of BSP

3 metal ions which have a promoting effect on the BSP enzyme activity and are colorless per se are selected:calcium, manganese and zinc are directly added into the culture solution in the form of chloride solution, so that the final concentration of the calcium, manganese and zinc is 1mM, the final concentration of BSP is 25ug/mL, and whether 3 metal ions can promote the BSP to generate larger inhibition effect on the growth of staphylococcus aureus is examined. In order to eliminate the influence of metal ions on bacterial growth, a single metal ion treatment group is also arranged in the embodiment. OD of the culture medium was measured by sampling at different incubation times ₆₀₀ Values, results are shown in fig. 9. In fig. 9, column 1 is a control group of BSP enzyme-treated staphylococcus aureus, and each 2 columns are a group of BSP enzyme-treated staphylococcus aureus, wherein the metal ions are added singly and enzyme-added respectively, and the sequence of the metal ions is calcium, manganese and zinc.

As can be seen from FIG. 9, in the case of single metal ion, i.e., 2, 4, 6 columns of each group, calcium ion and manganese ion do not affect the growth of Staphylococcus aureus much like zinc ion, and both do not greatly promote the growth of Staphylococcus aureus (3, 5 columns of each group) under the condition of enzyme-linked use, but zinc ion can act together with BSP protein on the basis of itself inhibiting the growth of Staphylococcus aureus (6 th column of each group), so that the effect of inhibiting the growth of Staphylococcus aureus is most obvious (7 th column of each group). Therefore, zinc ions can be used as a metal ion combination agent of BSP proteins to enhance the effect of BSP on inhibiting the growth of staphylococcus aureus.

Effect of bsp-combined antibiotics on restoring drug sensitivity to MRSA strains

With MRSA strain ATCC43300 as a test subject, 4 antibiotics of different action types were selected according to the resistance profile of MRSA strain ATCC 43300: ampicillin (beta-lactam, standard working concentration 100 ug/mL), tetracycline hydrochloride (tetracyclines, standard working concentration 5 ug/mL), neomycin (aminoglycosides, standard working concentration 20 ug/mL) and erythromycin (macrolides, standard working concentration 100 ug/mL) were added to MRSA strain culture broth at 2X standard working concentration, 1X standard working concentration, 0.5X standard working concentration, respectively, and the antibiotic-free MRSA strain culture broth was used as a control, and OD of the culture broth was measured after 24 hours of culture ₆₀₀ Values.

The results indicated that MRSA strain ATCC43300 was completely resistant to ampicillin, erythromycin and neomycin and sensitive to tetracycline hydrochloride. Therefore, ampicillin, neomycin and erythromycin were selected for subsequent BSP antibiotic combination tests to examine whether BSP antibiotic combination had a drug susceptibility restoring effect on MRSA.

3. Taking MRSA ATCC43300 as a test strain, respectively adding ampicillin 100ug/mL, neomycin 20ug/mL and erythromycin 100ug/mL as a control, adding BSP protein with the final concentration of 25ug/mL, culturing with a culture medium of pH7.0, taking PBS buffer solution as a blank control of BSP protein, culturing at 37 ℃ with a shaker at 220rpm for different time sampling to determine OD ₆₀₀ Values, results are shown in FIGS. 10-12.

It can be seen that when the combination of the 3 antibiotics is cultivated for 24 hours, the drug sensitivity of MRSA resistant bacteria is recovered by the combination of the BSP, and compared with a control group without the BSP, the concentration of bacterial liquid is reduced in a test group, which proves that the combination of the BSP and the antibiotics can inhibit MRSA strains with resistance to the antibiotics, wherein the inhibition amplitude is the largest and the effect is the most remarkable.

EXAMPLE 5 inhibition of MRSA growth by BSP in combination with Zinc chloride and erythromycin

The results of the analysis by live bacteria count method after 24 hours of incubation with MRSA as the test strain, pH7.0 of the medium, 1mM of zinc chloride added, 100ug/mL of erythromycin, 25ug/mL of BSP added, and no BSP protein added (replaced with equal volume of PBS buffer) were shown in FIG. 13.

It can be seen that the color of MRSA culture solution is obviously changed under the condition of combining BSP with zinc chloride and erythromycin, and the turbidimetry method for measuring the concentration of bacteria is not applicable. Therefore, the viable count analysis was continued for 3 control tubes and 3 test tubes, and the viable count of the control tubes was (191.9.+ -. 17.7). Times.10 ⁹ cfu/mL, the viable count of the test tube is (9.2+/-1.8) multiplied by 10 ⁹ The inhibition rate of the combined treatment of the zinc chloride and the erythromycin on the MRSA reaches 95.2 percent by cfu/mL, which proves that the combined treatment of the zinc chloride and the erythromycin on the BSP has obvious inhibition effect on the drug-resistant MRSA strain. Analysis of the cause, in addition to the direct effect of BSP on Staphylococcus aureus, there is also the possibility that BSP causes changes in bacterial cell walls and overall physiological statusThe bacterial drug sensitivity is restored, so that the antibiotics can effectively act on bacteria, and the bacteria are generally represented by the inhibiting and killing effects of BSP combined with zinc chloride and erythromycin on drug-resistant bacteria MRSA.

The foregoing description is only illustrative of the present invention and is not intended to limit the scope of the invention, and all equivalent structures or equivalent processes or direct or indirect application in other related technical fields are included in the scope of the present invention.

Sequence listing

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

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Ile Lys Gln Thr Phe Glu Asp Ile Leu Lys Gln Val Ser Ser Val Ser

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Ser Val Ile Lys Leu His Lys Asp Ala Pro Ala Tyr Ser His Val Ile

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

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Val His Ala Lys Asp Ala Gly Leu His Gly Phe Asp Val Thr Ser Pro

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Ser Tyr Asn Ser Leu Thr Asp Thr Glu Asp Val Tyr Gln Pro Gly Gly

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Ser Lys Tyr Val Trp Ile Asn Asn Cys Thr Ala Thr Asn Phe Gly Asp

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Asp Gly Phe Thr Thr His Phe Ser Glu Tyr Val Phe Ile Thr Asn Cys

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Tyr Ser Tyr Asp Gly Asn Gly Ser Ala His Thr Ser Gly Ala Ser Asn

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Thr Asn Gly Phe Glu Ile Asp Asp Gly Ser Met Lys Val Trp Leu Ile

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Asn Cys Val Ser Lys Asn Asn Cys Arg Gly Phe Glu Ala Lys Ala His

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Glu His Ala Pro Ala Ala Gln Asn Val Thr Phe Leu Asn Cys Val Ser

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Glu Asn Asp Ile Arg Ser Phe Asp Phe Arg His Ile Gly Phe His Lys

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Phe Val Pro Lys Asn Ile Lys Gly Gly Thr Arg Ile Ala Thr Thr Ser

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Gly Tyr Ala Ala Lys Asn Thr Ser Leu Val Ala Ala Ser Ser Gly Gly

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Gly Gln Ala Thr Gly Thr Ala Ser Ala Val Ile Ala Thr Thr Gly Gly

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Ser Lys Ala Asp Gly Pro Arg Asn Val Val Ile Ala Ser Ser Gly Gly

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Ser Lys Thr Thr Ser Glu Gly Ser Arg Ser Met Val Ala Ala Ser Asn

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Asn Ser Ser Ile Glu Gly Thr Gly Ser Ser Arg Met Val Ile Ala Ser

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Gln Gly Val Ala Asn Lys Thr Gly Tyr Thr Val Ala Leu Gly Tyr Ala

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

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Lys Asn Gly Asn Ile Asn Leu Thr Gly Gln Val Lys Gly Ala Ser Thr

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ggcaccggca aactgagtgt gattaaactg cataaagatg cccctgccta tagccacgtg 480

atcaccaccg tgcagaatag taagtatatc atttttgaaa acctgctgtt agattggaat 540

ctgcagaaaa caaacaacag catcagcagc ggcccgaata gcagttgcct gaatatcacc 600

aatagccagt ttgtgtgggt gaacaacgtg catgcaaaag acgcaggcct gcatggtttc 660

gacgtgacaa gccctagtta caacagtctg accgacaccg aggatgtgta tcagccgggc 720

ggcagcaaat acgtttggat caacaactgc accgccacca acttcggcga cgatggcttt 780

accacccatt ttagtgagta tgtgtttatt acaaattgtt atagttatga tggtaatggt 840

agtgcacata ccagcggcgc cagcaataca aacggcttcg agatcgacga cggtagcatg 900

aaagtttggc tgattaactg tgtgagcaaa aacaattgtc gtggcttcga ggccaaagca 960

cacgaacacg ccccggcagc ccagaacgtt accttcctga attgcgtgag cgagaacgac 1020

atccgcagct ttgacttccg tcatattggc tttcataaag ccagcgaccc ggagagcaaa 1080

acagcccgca atatcatggc cagcaactgc acagccatca aaccgatctt caatgaccgt 1140

ctgtacgcag gcatgacccc gcgtgccctg gttattagcg catataaaaa tgttaacatt 1200

agcaacttca cagcaatcgg cgacccgagc tacgattaca aaggcaatcc tgccatcgca 1260

acccagtaca aaagccgtaa tattaccttt aacaatgtta gcagtagcgg cttcaagacc 1320

gccggcgcag atatctacat ttacggcggc agccagaaga gtgatttcgt tagcctgagt 1380

aatatcaacg tgctggagag cgccctgatt ggcattcgta ttggcagcgc catcaaaaac 1440

gccagtgtga accaggcaag cctgattggt tacagtaaag agggcagtat cggcttatac 1500

tgtacaaata gccaggttga tattaatgca gtgaattgtg acaagtacgc catcccgagt 1560

aagatcgccg gcaaggcata taccagcttt gttccgaaga acatcaaggg cggcacacgc 1620

attgccacca caagcggtta tgcagccaaa aataccagtc tggttgccgc cagtagcggt 1680

ggtggtcaag ccacaggtac agccagcgca gtgattgcaa ccaccggtgg cagcaaagca 1740

gacggcccgc gtaacgtggt gatcgccagt agcggcggca gtaaaaccac aagtgaaggc 1800

agtcgcagca tggtggcagc cagcaacaac agcagtattg aaggcaccgg cagtagccgc 1860

atggtgatcg caagccaggg cgtggcaaac aagacaggtt acacagttgc cttaggctac 1920

gccgcaaccg gtgcaccgag caccgccaac acaaagatcc agctggatgc aaagaacggc 1980

aatattaatc tgaccggtca ggttaaaggc gccagcacct tcagcgatta cgccgagtac 2040

ttcgaaagca ttgacggcaa ggcaattccg agcggctact ttgttacctt agagggcgac 2100

aagatccgca aagcaaacgc cggtgataaa gtgctgggcg tgattagcga gacagccggc 2160

gttgttctgg gtgaagcagc attcaattgg cagggccgct atctgaagaa tgaatttggc 2220

ggtttaatct atgaggacat cgatgttacc gttaccaacg aagatggcac ccaacgcatt 2280

gaaaccaaga ccgtgccgaa agagaatccg tattacgagc cgagtgagga ctacatcgca 2340

cgtagcgatc gtcctgagtg gaatattgtg ggcatgttcg gccagatttt tgttcgtatt 2400

gacggcaccg ttgcagcagg tgatcgcatc atccctaaag ccggtaaagg cagcaaaagc 2460

gaagacggta gtggctatta tgtgatgcgc atcacaaccc cgtatagcca ggaacgcggc 2520

tatggcgtgg cactgtgcct gattaccccg acaattctcg ag 2562

Claims

1. Use of phosphodiesterase BSP for the non-therapeutic purpose in inhibiting staphylococcus aureus; the amino acid sequence of the phosphodiesterase BSP is shown as SEQ ID NO. 1; the gene sequence for encoding the phosphodiesterase BSP is shown as SEQ ID NO. 2.

2. The use according to claim 1, wherein the staphylococcus aureus comprises drug resistant staphylococcus aureus and non-drug resistant staphylococcus aureus.

3. A biologic having bacteriostatic action, characterized in that it comprises the phosphodiesterase BSP of claim 1 and zinc chloride.

4. The biologic of claim 3, wherein said biologic further comprises an antibiotic.

5. The biologic of claim 4, wherein said antibiotic comprises any one of ampicillin, neomycin and erythromycin.

6. Use of a biological agent according to any one of claims 3 to 5 for the non-therapeutic purpose in inhibiting or killing staphylococcus aureus.