CN114507653A

CN114507653A - Phosphodiesterase BSP, biological agent and application thereof

Info

Publication number: CN114507653A
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: 2022-05-17
Anticipated expiration: 2042-02-16
Also published as: CN114507653B

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 to staphylococcus aureus reaches 67.63 percent and the bacteriostasis rate to methicillin-resistant staphylococcus aureus reaches 53.20 percent. Meanwhile, the phosphodiesterase BSP is combined with zinc chloride and erythromycin, so that the inhibition rate of the phosphodiesterase BSP on methicillin-resistant staphylococcus aureus reaches 95.2%, and the phosphodiesterase BSP has obvious inhibition and sterilization functions on the methicillin-resistant staphylococcus aureus.

Description

Phosphodiesterase BSP, biological agent and application thereof

Technical Field

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

Background

Staphylococcus aureus (Staphylococcus aureus) is a common gram-positive pathogen and can cause various diseases in humans and animals. In livestock breeding, staphylococcus aureus infection can cause animal pyoderma, pneumonia, mastitis, systemic bacteremia and the like, and huge loss of yield and benefit of the breeding industry is caused. The prevention and treatment of staphylococcus aureus infection diseases is more troublesome due to the occurrence of a large number of staphylococcus aureus drug-resistant strains. MRSA began to be widely discovered worldwide after the first isolation of methicillin-resistant Staphylococcus aureus (MRSA) from Jevons, a UK scholar in 1961. MRSA has multiple drug resistance, not only to β -lactam antibiotics, but also to aminoglycoside, macrolide, tetracycline, fluoroquinolone, and sulfonamide antibiotics, and becomes a "super bacterium" that causes considerable morbidity and mortality in humans and animals.

On one hand, the problem of MRSA infection is solved by continuously discovering novel antibiotics aiming at MRSA, on the other hand, the drug sensitivity of MRSA is recovered by using an antibiotic adjuvant, for example, D-tyrosine (Yaoheming, et al, 2017), clove oil (Qimin, et al, 2019), sanguisorba alcohol extract (Chen, et al, 2015) and the like are used together with beta-lactam antibiotics to generate a certain degree of inhibition effect on MRSA. However, there is still a substantial lack of effective chemical or biological agents to potentiate the antibiotic action to control MRSA.

Teichoic acid is a specific component of the cell wall of gram-positive bacteria, and is a linear polymer formed by connecting glycerol phosphate or ribitol phosphate with substituent groups as monomers through phosphodiester bonds, and the mass of the linear polymer accounts for 50% of the dry weight of the cell wall. Teichoic acid plays an important role in host colonization, peptidoglycan synthesis and cell division of gram-positive pathogenic bacteria (atliano, 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 act on the phosphodiester bond in the teichoic acid skeleton of gram-positive bacteria to produce free phosphate, glycerol or ribitol which disrupts the teichoic acid skeleton, enzymes with this action are also known as teichoic enzymes. In 2015, the first gene for teichoic acid was identified (Myers, et al 2015), which is the GP12 gene in the genome of bacillus subtilis phage phi 29, and GP12 protein, which is an accessory protein of phage phi 29, was shown to degrade teichoic acid. However, the information on the enzyme proteins and genes capable of degrading teichoic acid is still poor, which hinders the progress of bacteriological studies, and there is a lack of biological agents for the treatment of traumatic infections caused by gram-positive bacteria and the prevention of eating diseases.

Disclosure of Invention

In view of the above, the present invention aims to provide a phosphodiesterase BSP having phosphodiesterase and phosphomonoesterase activities and having a significant 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 phosphodiesterase BSP, and the amino acid sequence of the phosphodiesterase BSP is shown as SEQ ID NO. 1.

Preferably, the phosphodiesterase BSP is derived from a Bacillus subtilis BGSC3A28 strain.

The invention provides a gene for coding 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 comprises a feed additive for epidermal infection of animals caused by staphylococcus aureus and for inhibiting intestinal infection by staphylococcus aureus.

The invention also provides a biological agent with a bacteriostatic action, which comprises the phosphodiesterase BSP or a combined reagent.

Preferably, the combination 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 cause the inhibition of bacterial growth by hydrolyzing the structure of teichoic acid in the cell wall of staphylococcus aureus, and has no mechanism of generating drug resistance to BSP. Experiments show that the phosphodiesterase BSP provided by the invention has an antibacterial rate of 67.63% for treating staphylococcus aureus and an antibacterial rate of 53.20% for treating methicillin-resistant staphylococcus aureus, is suitable for animal epidermal infection caused by staphylococcus aureus and drug-resistant staphylococcus aureus, or can be used for inhibiting intestinal infection of staphylococcus aureus by using feed additives. Meanwhile, by combining the phosphodiesterase BSP with zinc chloride or erythromycin, the bacteriostasis rate of the phosphodiesterase BSP on methicillin-resistant staphylococcus aureus reaches 95.2%, the phosphodiesterase BSP has obvious bacteriostasis and sterilization functions on the methicillin-resistant staphylococcus aureus, and a new solution is provided for health threats caused by methicillin-resistant staphylococcus aureus infection.

Drawings

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

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

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

FIG. 4 is a measurement of the optimum temperature and pH for BSP; wherein, a) the determination of the optimum action temperature of BSP, b) the determination of the optimum action pH of BSP.

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

FIG. 6 is a graph showing the effect of BSP on the growth of Staphylococcus aureus (Sa).

FIG. 7 is a graph of the effect of BSP on the growth of methicillin-resistant Staphylococcus aureus (MRSA).

FIG. 8 is the OD of Staphylococcus aureus with BSP at various concentrations₆₀₀The value is obtained.

FIG. 9 shows OD of Staphylococcus aureus treated with different metal ions in combination with BSP₆₀₀The value is obtained.

FIG. 10 is OD of BSP protein after different time periods in combination with ampicillin treatment of MRSA₆₀₀The value is obtained.

FIG. 11 is OD of MRSA treated with BSP protein in combination with neomycin over different periods of time₆₀₀The value is obtained.

FIG. 12 is OD of MRSA treated with BSP protein in combination with erythromycin over different time periods₆₀₀The value is obtained.

FIG. 13 is a color change in MRSA cultures when BSP is treated with zinc chloride in combination with erythromycin; wherein,

tubes

1, 2 and 3 are culture solution without BSP addition culture for 24 h; 4.

tubes

5 and 6 are BSP added and cultured for 24 h.

Detailed Description

In the invention, the nucleotide sequence for coding the phosphodiesterase BSP gene is shown as SEQ ID NO. 2. The phosphodiesterase BSP is derived from a Bacillus subtilis BGSC3A28 strain (Bacillus subtilis BGSC3A28) 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 invention is expressed in a soluble form, and the BSP protein is cut between a 674-675 site. The mature BSP protein contains 674 amino acid residues, and the molecular weight of the protein is 71.4 kDa.

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

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

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

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

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

EXAMPLE 1 preparation of BSP protein

Searching a gene sequence of BSP protein WP _032721605.1 in NCBI (NCBI includes a number of NZ _ FODG01000008.1), and obtaining a gene sequence of the encoding BSP protein through codon optimization as shown in SEQ ID NO.2, wherein NdeI and XhoI enzyme digestion sites 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 the gene coding is shown as SEQ ID NO. 1. The SEQ ID NO.2 gene sequence was subjected to gene synthesis by Jinzhi corporation, Suzhou, and constructed into a pET-21a (+) vector. And (3) transforming the constructed recombinant pET-21a (+) vector into an escherichia coli BL21(DE3) competent cell to obtain an escherichia coli transformant with correct sequencing.

The recombinant Escherichia coli was inoculated into 100mL of LB medium and cultured at 37 ℃ and 250rpm for 2.5h to OD₆₀₀When the value reached 0.8-0.9, 0.1mM IPTG was added and the mixture was induced at 16 ℃ and 180rpm for 20 hours. The thalli is collected by centrifugation, 20mL of balance buffer solution (NaCl 8.766g, 0.340g of imidazole, 1.21g of Tris (hydroxymethyl) aminomethane (Tris) is dissolved in 400mL of deionized water, the pH value is adjusted to 7.4 by hydrochloric acid, the volume is constant to 500mL) is used for washing the thalli for 2 times, the washed thalli is suspended in a 50mL centrifuge tube by 10mL of lysis buffer solution, and the thalli is lysed by ultrasonic treatment for 20min (6mm variable-amplitude rod, power 200W, working time 3s and pause time 5s) under the condition of ice water bath. After the sonication, the mixture was centrifuged at 12000 Xg for 10min at 4 ℃ and the supernatant was taken as a soluble protein fraction, and the expression of BSP protein was detected by SDS-PAGE, the results of which 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 BSP, 92.0kDa, which is about 70 kDa. Analysis of the amino acid sequence of BSP revealed that BSP contains an auto-cleavage domain (Peptidase _ G2(pfam 11962)), and that amino acid residues 618-847 may exert the auto-cleavage function.

Purifying the N-terminal part of BSP with a (His)6 tag sequence and the C-terminal part of the BSP by using a Ni-NTA column, loading the supernatant obtained by the centrifugation after the ultrasonication to the Ni-NTA column, washing the column by using an equilibrium buffer solution, eluting the BSP protein with the (His)6 tag by using an elution buffer solution (NaCl 8.766g, imidazole 10.212g and Tris (Tris)1.21g in 400mL of deionized water, adjusting the pH to 7.4 by using hydrochloric acid, and fixing the volume to 500mL) at a flow rate of 0.5mL/min, judging the purity according to SDS-PAGE, and feeding the BSP protein uniformly pure by SDS-PAGE to a Shanghai pharmaceutical company to analyze the self-cleavage site of the BSP by mass spectrometry. The results are shown in FIG. 2. The purified BSP protein was electrophoresed, and the results are shown in fig. 3.

As can be seen from FIGS. 2 and 3, the cleavage did occur between the 674-675 site in the BSP protein, the mature BSP protein contains 1-674 amino acid residues and has a theoretical molecular weight of 71.4kDa, and the size of the purified BSP protein matches the theoretical value of the mature BSP protein.

EXAMPLE 2 phosphatase Activity of BSP

1. Respectively using bis-p-nitrophenol-sodium phosphate (bis-pNPP) and p-nitrophenol-disodium phosphate (pNPP) as substrates to detect whether BSP has phosphodiesterase activity and phosphomonoesterase activity.

The reaction system for measuring the enzyme activity was as follows: in a final reaction system of 100. mu.L, 40mM Tris-HCl and 1mM CaCl₂10mM bis-pNPP (or pNPP), 1.8. mu.M BSP protein (control tubes in addition of Na)₂CO₃Adding the same amount of BSP after the solution is stopped reaction), placing the reaction tube and the control tube at 37 ℃ for heat preservation reaction for 30min, and then adding 200 mu L of 2M Na₂CO₃The solution was quenched, 2.9mL of deionized water was added, and OD was measured₄₀₀Value, converted to the amount of p-nitrophenol released (extinction coefficient of p-nitrophenol: 1.74X 10)⁴mol^-1·cm^-1·L)。

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

The measurement result shows that no matter bis-pNPP is used as the substrate or pNPP is used as the substrate, BSP generates p-nitrophenol, and the BSP has phosphodiesterase activity and phosphomonoesterase activity respectively corresponding to the BSP. The phosphodiester activity of BSP was 1.40IU/mg and the phosphomonoester activity was 0.92 IU/mg.

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

It can be seen that the novel BSP enzyme of the present invention has phosphodiesterase activity and phosphomonoesterase activity. bis-pNPP is used as a substrate, the optimum catalysis temperature of BSP for catalyzing hydrolysis of the bis-pNPP is 85 ℃, and the optimum catalysis pH is 8.5.

3. In order to determine the influence of metal ions on the BSP activity and determine the influence of different metal ions on the BSP activity, the specific determination method comprises the following steps: in the reaction system described in 1 (with bis-pNPP as a substrate), EDTA and various metal ion chlorides were added to the reaction system in a final concentration of 1mM, respectively, and the phosphodiesterase activity of BSP was measured in the same manner as in 1, and the relative enzyme activity of BSP in the presence of EDTA and various metal ions was calculated with the enzyme activity of BSP without EDTA or metal ions as 100%, and the results of the detection are shown in FIG. 5.

It can be seen that BSP is still hydrolytically active in the presence of EDTA. Most of the divalent ions have a promoting effect on the catalytic activity of BSP, among which Mn²⁺And Co²⁺The promotion effect is most obvious, the promotion effect is respectively improved by 414 percent and 331 percent, and Fe³ ⁺The presence of (b) significantly reduces the catalytic activity of BSP.

Further determining kinetic parameters of BSP acting on bis-pNPP by adopting an Eadie-Hofstee mapping method: in the reaction system described in 1, the concentrations of the substrates bis-pNPP were set to 0.5, 1.0, 2.0, 5.0, 10, and 30mM, respectively, the reaction tube and the control tube were incubated at 37 ℃ for 10min, and the initial reaction rates at different substrate concentrations were measured in the same manner as in the reaction system described in 1. Plotting the initial reaction rate versus the ratio of initial reaction rate to substrate concentration (v/[ S ]]) I.e. Eadie-Hofstee curve, calculating V_maxAnd K_mAnd K_cat. The results are shown in table 1 below.

TABLE 1 kinetic parameters of BSP

It can be seen that the turnover number k of BSP catalyzing the hydrolysis of bis-pNPP_catIs 6.99min^-1。

Example 3

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

Taking 3mL of LB liquid culture medium, adding purified BSP according to the amount of 50ug/mL (the final volume of the culture solution is 3.5mL) of the final concentration, respectively adding 100uL of previously activated staphylococcus aureus (Sa) and methicillin-resistant staphylococcus aureus (MRSA) strains, and then supplementing to 3.5mL by a sterilized 50mM Tris buffer solution with the pH value of 7.5. Three replicates of each of the treated and control groups were run against an equivalent amount of inactivated BSP. The culture tubes were cultured in a shaking incubator at constant temperature of 37 ℃ at 220rpm, 100uL of the bacterial solution was taken at 24 hours and 48 hours, respectively, and after gradient dilution, the bacterial solution was applied to a solid LB plate to count viable bacteria, and the results are shown in FIGS. 6 and 7.

The result shows that for Sa, the bacteriostasis rates of the BSP are 53.33% and 67.63% respectively when the BSP is treated for 24 hours and 48 hours; for MRSA, the bacteriostasis rates of the MRSA are 53.20 percent and 49.08 percent respectively when the MRSA is treated by BSP for 24 hours and 48 hours, and the BSP has obvious capacity of inhibiting the growth of staphylococcus aureus and drug-resistant staphylococcus aureus.

Minimum effect concentration of BSP on Staphylococcus aureus bacteriostasis

The BSP stock solution purified by affinity chromatography in example 1 was subjected to 1: 1, sequentially diluting the diluted enzyme solution by the following steps of 1: 1 to finally obtain 4 BSP concentrations, adding BSP proenzyme solution and the diluted enzyme solution respectively according to the culture system in the example 2 to ensure that the final concentrations of the BSP in the culture solution are respectively 50ug/mL, 25ug/mL, 12.5ug/mL and 6.25ug/mL, placing the mixture in a shaking table at 37 ℃, and rapidly determining the OD of the culture solution when culturing for 24 hours at 220rpm₆₀₀The results are shown in FIG. 8.

According to OD₆₀₀The value analysis shows that the inhibition of BSP on the growth of staphylococcus aureus is gradually reduced along with the reduction of the concentration of BSP, although the BSP still has certain inhibition at the concentrations of 12.5ug/mL and 6.25ug/mL, the best inhibition effect is considered under the lowest enzyme dosage, and therefore the BSP concentration of 25ug/mL is selected as the optimal addition concentrationAnd (4) degree.

EXAMPLE 4 Effect of biological Agents

Divalent Metal ion Co-agent of BSP

3 metal ions which have a promoting effect on the BSP enzyme activity and are colorless per se were 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, the manganese and the zinc is 1mM, the final concentration of BSP is 25ug/mL, and whether 3 metal ions can promote BSP to generate larger inhibition effect on the growth of staphylococcus aureus is examined. In order to eliminate the influence of the metal ions on the growth of bacteria, the embodiment is also provided with a single metal ion treatment group. Sampling at different culture times to determine OD of culture solution₆₀₀The results are shown in FIG. 9. In fig. 9, the 1 st column in each group is a control group of BSP enzyme-treated staphylococcus aureus, and each subsequent 2 columns are a group of single metal ion and enzyme metal ion, respectively, and the metal ions are calcium, manganese and zinc in this order.

As can be seen from FIG. 9, in the case of adding metal ions alone, i.e., 2, 4, 6 columns of each group, calcium ions and manganese ions do not affect the growth of Staphylococcus aureus as much as zinc ions, and the calcium ions and manganese ions do not greatly promote the growth of Staphylococcus aureus under the condition of being combined with enzyme (3, 5 columns of each group), while zinc ions can act together with BSP protein on the basis of inhibiting the growth of Staphylococcus aureus by themselves (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, the zinc ions can be used as a metal ion combination agent of BSP protein to enhance the effect of BSP in inhibiting the growth of staphylococcus aureus.

Effect of BSP in combination with antibiotics on recovery of drug sensitivity of MRSA strains

Taking the MRSA strain ATCC43300 as a test object, selecting 4 antibiotics with different action types according to the drug resistance spectrum of the MRSA strain ATCC 43300: ampicillin (beta-lactam, standard working concentration 100ug/mL), tetracycline hydrochloride (tetracycline, standard working concentration 5ug/mL), neomycin (aminoglycoside, standard working concentration 20ug/mL) and erythromycin (macrolide, standard working concentration 100ug/mL) were added at 2 × standard working concentration, 1 × standard working concentration, and 0.5 × standard working concentration, respectivelyAdding into MRSA strain culture solution, and determining OD of the culture solution after culturing for 24 hr with MRSA strain culture solution without antibiotic as control₆₀₀The value is obtained.

The results show that MRSA strain ATCC43300 is completely resistant to ampicillin, erythromycin and neomycin, and is sensitive to tetracycline hydrochloride. Therefore, ampicillin, neomycin and erythromycin are selected in the subsequent BSP combined antibiotic test to investigate whether the BSP combined antibiotic has the effect of restoring drug sensitivity to MRSA.

3. MRSA ATCC43300 was used as a test strain, 100ug/mL of ampicillin, 20ug/mL of neomycin, and 100ug/mL of erythromycin were added as controls, BSP protein was added based on the control, the final concentration of BSP protein was 25ug/mL, the pH of the medium was 7.0, PBS buffer was used as a blank control of BSP protein, shaking table at 37 ℃ and cultivation at 220rpm were carried out for different times, and OD was measured by sampling₆₀₀The results are shown in FIGS. 10-12.

It can be seen that, after 24h of culture, the drug sensitivity of the MRSA drug-resistant bacteria is recovered by combining the 3 kinds of antibiotics with BSP, and the concentration of the bacterial liquid is reduced in the test group compared with the control group without BSP, which indicates that the MRSA strain having resistance to the antibiotics can be inhibited by combining the BSP with the antibiotics, wherein the maximum inhibition range and the most significant effect are erythromycin.

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

MRSA was used as a test strain, the pH of the medium was 7.0, the final concentration of zinc chloride was 1mM, the final concentration of erythromycin was 100ug/mL, and the final concentration of BSP was 25ug/mL, and the viable cell count method was used for analysis after 24h of culture with the control of a culture solution without BSP protein (replaced with an equal volume of PBS buffer), as shown in FIG. 13.

As can be seen, under the condition of combining BSP with zinc chloride and erythromycin, the color of the MRSA culture solution is obviously changed, and the turbidimetry measurement of the bacteria concentration is not applicable. Therefore, the viable cell count of the control tube was (191.9. + -. 17.7). times.10 as seen by continuing the viable cell count analysis of the 3 control tubes and the 3 test tubes⁹cfu/mL, the number of viable bacteria in the test tube is (9.2 +/-1.8) multiplied by 10⁹Inhibition of MRSA by cfu/mL, BSP in combination with Zinc chloride and erythromycin treatmentThe bacteria rate reaches 95.2%, and the remarkable inhibition effect of BSP combined with zinc chloride and erythromycin on the drug-resistant MRSA strain is proved. The analysis reason is that besides the direct influence of BSP on staphylococcus aureus, the change of bacterial cell wall and whole physiological state caused by BSP can cause the recovery of drug sensitivity of bacteria, so that the antibiotic can effectively act on the bacteria, and the general expression shows that BSP combined with zinc chloride and erythromycin can inhibit and kill drug-resistant bacteria MRSA.

The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes performed by the present specification and drawings, or directly or indirectly applied to 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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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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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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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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aaccagatcg gcaacatcga tgacttccat atcaccggta ccaacctggt ggagaagatc 240

ctgaacgcac tgatccaacg tcgtgtgagt gtgaccgatt tcggtgcaaa gggtgacggc 300

gttaccgacg ataccgcagc ctttaataaa gccttcgaaa tgggcaatgc cgaggtgttt 360

gtgccggccg gtacatacat ggttaagggc ctgaaggtgc ctagctatac ccgtctgtac 420

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. A phosphodiesterase BSP is characterized in that the amino acid sequence of the phosphodiesterase BSP is shown as SEQ ID NO. 1.

2. The phosphodiesterase BSP according to claim 1, wherein the phosphodiesterase BSP is derived from a Bacillus subtilis BGSC3A28 strain.

3. A gene encoding the phosphodiesterase BSP according to claim 1, wherein the nucleotide sequence of the gene is shown in SEQ ID NO. 2.

4. Use of the phosphodiesterase BSP according to claim 1 or 2 for the inhibition of Staphylococcus aureus.

5. The use of claim 4, wherein said Staphylococcus aureus comprises drug-resistant Staphylococcus aureus and non-drug-resistant Staphylococcus aureus.

6. The use according to claim 4, wherein the use comprises a feed additive for epidermal infection of animals caused by Staphylococcus aureus and for inhibiting intestinal infection by Staphylococcus aureus.

7. A biological agent with bacteriostatic effect, characterized in that it comprises the phosphodiesterase BSP according to claim 1 or further comprises a combined reagent.

8. The biological preparation according to claim 7, wherein the combination reagent comprises zinc chloride and/or an antibiotic.

9. The biological agent according to claim 8, wherein the antibiotic comprises any one of ampicillin, neomycin and erythromycin.

10. Use of a biological agent according to any one of claims 7 to 9 for inhibiting or killing staphylococcus aureus.