A bacteriophage 573 lytic enzyme LysZB48, fusion protein and application thereof

Abstract

The present invention relates to a bacteriophage 573 lytic enzyme LysZB48, a fusion protein and an application thereof. The present invention uses S.aureus 573 as a host bacterium to isolate S.aureus phage Sau-p573, analyzes and obtains the sequence of the S.aureus phage Sau-p573 lytic enzyme LysZB48, and successfully expresses the fusion protein in a soluble form. LysZB48 has good bactericidal activity, and has the best lysis effect at 37°C to 42°C and a pH range of 8 to 9. It has the effect of removing biofilms and is expected to be applied to the development of biological antibacterial products.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial antibacterial agents, and specifically relates to a lytic enzyme LysZB48 screened from Staphylococcus aureus phage 573, a fusion protein containing the lytic enzyme, and an application thereof in the field of preparing antibacterial agents. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Staphylococcus aureus is a widely spread pathogen in mammals. It usually attaches to the skin, mucous membranes, intestines and other tissues of mammals, and is commonly found in the nasal cavity. It can cause a series of diseases such as mastitis and sepsis in dairy cows. Staphylococcus aureus is prone to drug resistance and can spread drug-resistant genes vertically and horizontally. The biofilm it produces is also an important cause of bacterial resistance. With the abuse of antibiotics, more and more drug-resistant bacteria have begun to appear, resulting in increasingly difficult clinical treatment of dairy cow mastitis. Therefore, the development of new bactericidal biological agents has become extremely important.

[0004] Phage is a virus that uses bacteria as hosts. It can inject genetic material into the host and continue to reproduce in the host bacteria, eventually killing the host bacteria and releasing progeny phages. Oliveira et al. divided staphylococcal phages into four major families: ABCD. Most of the phages of Staphylococcus aureus belong to family B. Most of the phages in this family are temperate phages and belong to the family of Long-tailed Viruses. Lytic enzymes are a type of enzyme that hydrolyzes bacterial cell wall peptidoglycan expressed in the late stage of phage infection. They usually carry two important domains: the N-terminal catalytic domain and the C-terminal catalytic domain. According to the different action sites, they can be divided into five types: lysozyme, transglycosylase, glucosaminidase, amidase or N-acetylmuramoyl-L-alanine amidase and endopeptidase. The first three enzymes act on the β-1,4-glycosidic bond in N-acetylmuramic acid and N-acetylglucosamine in the cell wall, while the latter two have a hydrolysis effect on the amide bond between N-acetylmuramic acid and the first amino acid in the side chain connected to it. Lytic enzymes have the advantages of host specificity, trace efficiency, low resistance to drug resistance in bacteria, and relative safety. They have played a role in many fields and have great development prospects. Summary of the invention

[0005] The present invention uses S. aureus 573 as a host bacteriophage to extract a bacteriophage from a wastewater sample of a cattle farm, and the bacteriophage is named Sau-p573. Through a perspective electron microscope, it can be observed that Sau-p573 has an irretractable long tail, a head that is a typical icosahedral head of a bacteriophage, a total length of 411±3nm, and a tail length of 316±3nm. According to the latest classification method formulated by the 9th meeting of the International Committee on Taxonomy of Viruses, the bacteriophage belongs to the order Caudovirales and the family Siphoviridae.

[0006] The above-mentioned bacteriophage Sau-p573 has a lytic effect on Staphylococcus aureus. The titer of bacteriophage Sau-p573 measured by the double-layer plate method is 7×10 10 PFU / mL.

[0007] It is well known in the art that S. aureus is a conditional pathogen that attaches to the skin and mucous membranes of mammals. The abuse of antibiotics has led to the discovery of more and more drug-resistant bacteria such as MRSA and VISA, and the development of the animal husbandry industry is facing severe challenges. Bacteriophages are a type of virus that uses bacteria as hosts. Today, they are being re-emphasized in order to combat the growing drug resistance of bacteria. Compared with antibiotics, bacteriophages have the advantages of being effective only against specific host bacteria, being easy to extract, and not easily causing bacteria to develop resistance.

[0008] In the study of the ORF encoded by the above phage, ORF48 has a high similarity with the known lytic enzyme, and it has been verified to be a fragment encoding the lytic enzyme in the phage Sau-p573. The present invention expresses ORF48 through a recombinant plasmid to obtain a purified protein molecule, which is named lytic enzyme LysZB48. The present invention also investigates the physicochemical properties of lytic enzyme LysZB48, and the results show that:

[0009] (1) When the final concentration is 25 μg / mL to 200 μg / mL, the lytic enzyme LysZB48 has a certain bactericidal effect on the host bacteria. The bactericidal effect gradually becomes more significant as the concentration increases, and the bactericidal effect can be quickly exerted within 30 minutes of contact with the bacteria. In addition, the above-mentioned lytic enzyme LysZB48 can also destroy the bacterial biofilm at a lower concentration, thereby reducing the probability of bacteria adhering to the body or the surface of production equipment, and playing an auxiliary bactericidal effect;

[0010] (2) LysZB48 has a broad-spectrum bactericidal effect. The present invention uses Staphylococcus aureus and Escherichia coli as examples for verification. The lysase can exert a lysis effect on multiple strains of Staphylococcus aureus and Escherichia coli, indicating that the above-mentioned lysase is expected to be used as a broad-spectrum bactericide in the development of biocides;

[0011] (3) The lyase LysZB48 has good tolerance and can exert cleavage activity in the range of 4°C to 42°C, and has higher cleavage activity in the higher temperature range (37°C to 42°C). In addition, the lyase has certain activity at pH 6-9, and has a good cleavage effect at pH 8-9. It can tolerate a wider pH condition and is more suitable for alkaline environment.

[0012] Phages express lytic enzymes at the end of infection. Lytic enzymes kill bacteria by acting on specific bonds on the peptidoglycan of the host bacterial cell wall to break it. They are widely studied because of their specific lytic spectrum and rapid bactericidal effect. The present invention confirms that the lytic enzyme LysZB48 has good bactericidal activity and is expected to be used to combat drug-resistant bacteria, with good development prospects in medical treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0014] Figure 1 This is the plaque morphology of Sau-p573;

[0015] Figure 2 This is an electron microscopic image of bacteriophage Sau-p573 (magnified 60,000 times, scale bar 200 nm);

[0016] Figure 3 The results of comparison between bacteriophage Sau-p573 and other bacteriophage genomes;

[0017] Figure 4 is the evolutionary tree of bacteriophage Sau-p573;

[0018] Figure 5 It is a schematic diagram of the circular structure of the bacteriophage Sau-p573 genome;

[0019] Figure 6 Annotate the structure map of ORF48 domain;

[0020] Figure 7 This is the SDS-PAGE electrophoresis diagram of the recombinant LysZB48 purification;

[0021] Figure 8 The effect of different concentrations of LysZB48 on Staphylococcus aureus;

[0022] Fig. 9 The inhibitory effects of different concentrations of LysZB48 on Staphylococcus aureus;

[0023] Fig.10The effect of milk powder and bovine serum on the effect of LysZB48;

[0024] Fig.11 is the optimal reaction temperature of LysZB48;

[0025] Fig.12 The effect of incubation time at 37°C on the lytic enzyme activity described in Example 2;

[0026] Fig.13 is the optimal reaction pH of LysZB48;

[0027] Fig.14 These are the test results of the ability of LysZB48 to clear the formed biofilm. DETAILED DESCRIPTION

[0028] It should be noted that the following detailed descriptions are all illustrative and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0029] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0030] As introduced in the background technology, bacteriophages and lytic enzymes have good development prospects in combating drug-resistant bacteria. The present invention screened a Staphylococcus aureus phage Sau-p573, and isolated a lytic enzyme LysZB48 with good bactericidal effect from the phage, which is expected to be applied to the development of pharmaceutical products against drug-resistant bacteria.

[0031] In a first aspect, the present invention provides a lyase LysZB48, wherein the lyase has the sequence shown below:

[0032] MLMTRNQAEKWFDNSLGKQFNPDGWYGFQCYDYANMFFMLATGERLQGLYAYNIPFDNKAKIEKYGQIIKNYDSFLPQKLDIVVFPSKYGGGAGHVEIVESANLNTFISFGQNWNGKGWTNG VAQPGWGPETVTRHVHYYDNPMYFIRLNFPDSISVKDKAKGIIKQATAKKEAVIKPKRIMLVAGHGYNDPGAVGNGTNERDFIRKYITPNIAKYLRHAGHEVALYGGSSQSQDMYQDTAYGV NVGNKKDYGLYWVKSQGYDIVLEIHLDAAGESASGGHVIISSQFNADTIDKSIQDVIKNNLGQIRGITPRNDLLNVNVSAEININYRLSELGFITNKNDMDWIKKNYDLYSKLIAGAIHGKP IGGLVASEVKAPVKNEKNPPVPAGYTLDSKGVPYKKEQGNYTVANVKGNNVRDGYSTNSRITGVLPNNTTITYDGAYCINGYRWITYIANSGQRRYIATGEVDKAGNRISSFGKFSTI (such as SEQ ID NO.1).

[0033] The second aspect of the present invention provides a fusion protein, which includes the lytic enzyme LysZB48 described in the first aspect, and also includes other active fragments, wherein the other active fragments are fragments that enhance the activity of the lytic enzyme LysZB48 or have a binding effect on effector cells.

[0034] Preferably, the other active fragments include but are not limited to membrane-penetrating peptides, serum albumin, polyethylene glycol or other active proteins, and the other active fragments can be connected to the lyase LysZB48 by chemical or genetic modification;

[0035] Further, in order to obtain better solubility and purification effect, the other active fragment is maltose binding protein (MBP), which is connected to the N-terminus or C-terminus of the lyase LysZB48. In a specific embodiment of this preferred technical solution, the fusion protein also includes a target tissue site recognition sequence, a linker and a membrane-penetrating peptide, and the specific sequence of the fusion protein is as follows:

[0036]

[0037]

[0038] (shown in SEQ ID NO.2).

[0039] In the above fusion protein sequence,

[0040] "MAEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDN AGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQT(SEQID NO.3)" is the MBP-tagged protein, which can improve the soluble expression of the protein and will be removed later.

[0041] "DDDDK (shown in SEQ ID NO.4)" is an enterokinase site, which can be cut by enterokinase to obtain the target recombinant protein.

[0042] "RRRRRRRRRR (shown in SEQ ID NO.5)" is a membrane-penetrating peptide that can help proteins enter cells and exert their effects.

[0043] "GGGGS (shown in SEQ ID NO.6)" is a linker that connects the membrane-penetrating peptide and the lytic enzyme.

[0044] “HHHHHH (shown in SEQ ID NO. 7)” is a his tag used for protein purification.

[0045] The third aspect of the present invention provides a nucleic acid material encoding the lytic enzyme LysZB48 described in the first aspect or the fusion protein described in the second aspect.

[0046] The nucleic acid material includes a coding nucleic acid that can be translated to obtain the above-mentioned lytic enzyme LysZB48 due to codon degeneracy, including cDNA, genomic DNA or artificially synthesized DNA, which can be double-stranded or single-stranded, and can include a coding strand or a non-coding strand. The method for isolating the nucleic acid material can be prepared by automatic DNA synthesis and / or recombinant DNA technology, or it can be separated from the above-mentioned bacteriophage.

[0047] In a specific embodiment, the nucleic acid sequence encoding the fusion protein is shown in SEQ ID NO:8.

[0048] According to a fourth aspect of the present invention, an expression vector is provided, wherein the expression vector comprises the nucleic acid substance according to the third aspect.

[0049] Preferably, the expression vector is a bacterial plasmid or a yeast plasmid, including pET series vectors, pMAL series vectors, pGEX series vectors, pBAD series vectors, pMBP series vectors or pUC series vectors.

[0050] According to a fifth aspect of the present invention, a host cell is provided, wherein the host cell comprises the nucleic acid substance according to the third aspect or the expression vector according to the fourth aspect.

[0051] The host cell is an engineered bacterium used to express the lytic enzyme LysZB48 described in the first aspect or the fusion protein described in the second aspect, and its starting strain is often Escherichia coli or yeast; in a specific embodiment of the present invention, the starting strain is Escherichia coli.

[0052] The sixth aspect of the present invention provides the use of the lytic enzyme LysZB48 described in the first aspect and the fusion protein described in the second aspect in the field of preparing antibacterial agents.

[0053] Preferably, the antibacterial agent is a broad-spectrum antibacterial agent, which can be used to inhibit common pathogenic bacteria such as Escherichia coli, Staphylococcus aureus, and Salmonella; further, the antibacterial agent is used to prevent or treat Escherichia coli or Staphylococcus aureus infection.

[0054] Preferably, the antibacterial agent is used in fields including but not limited to food, beverages, livestock and poultry farming, etc.; further, the antibacterial agent can be used to sterilize dairy products, or to prevent and treat diseases related to bovine mastitis; in a specific example, the dairy product is skim milk, low-fat milk or milk powder.

[0055] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in combination with specific embodiments and comparative examples.

[0056] Example 1

[0057] 1 Materials and methods

[0058] 1.1 Materials

[0059] 1.1.1 Plasmids and strains

[0060] Plasmid pMBP2 was purchased from Jinan Shangbo Biotechnology Co., Ltd., and 39 strains of Staphylococcus aureus and 7 strains of Escherichia coli were preserved in our laboratory.

[0061] 1.1.2 Main reagents and instruments

[0062] DNA extraction kit, Aikerui Biotechnology Co., Ltd.; PBS buffer, Meilun Biotechnology Co., Ltd.; BCA kit, Dextrin Beads 6FF, Dextrin beads, Beijing Solebow Technology Co., Ltd.

[0063] Cell ultrasonic disruptor, Ningbo Xinzhi Biotechnology Co., Ltd.; Horiba LAQUAtwin pH test pen, HORIBA Manufacturing, Japan; Mini Protean II vertical plate electrophoresis system, BIO-RAD, USA.

[0064] 1.2 Methods

[0065] 1.2.1 Isolation and purification of bacteriophage

[0066] Mixed sewage was obtained from a cattle farm in Shandong. The supernatant was taken after centrifugation and filtered through a 0.22μm filter membrane. 50mL of the preliminarily treated sewage was taken, and 2mL of the host bacteria S.aureus 573 was added and shaken at 37℃ overnight. The culture solution was centrifuged at 4℃ and 12,000rpm for 10min, and the supernatant was filtered through a 0.22μm filter membrane. The resulting liquid was the phage stock solution. The phage was isolated using the double-layer plate method, and after multiple purifications, the phage Sau-p573 was obtained.

[0067] 1.2.2 Electron microscopy and sequencing of bacteriophages

[0068] Measure the titer of amplified phage and reach 10 9 The above is enough. 10 μL of sample was dropped onto the copper grid and placed for about 10 to 15 minutes, excess liquid was removed, treated with 2% phosphotungstic acid for 10 seconds, and its morphological structure was observed with a transmission electron microscope after drying. Phage sequencing was performed by Shanghai Lingen Biotechnology Co., Ltd. using the Illumina novaseq6000 sequencing platform for whole genome sequencing, and the optimized sequence was spliced ​​with multiple Kmer parameters using ABySS splicing software to obtain the optimal assembly result.

[0069] 1.2.3 Phage whole genome sequencing analysis and prediction of lytic enzymes

[0070] The open reading frames (ORFs) of bacteriophages were predicted by BLAST and GeneMark. The sequences of each ORF were compared and analyzed using NCBI and phaster to analyze their possible functions. The protein sequences of the predicted genes were compared with NR, Swiss-Prot, eggNOG, KEGG, GO and other databases to obtain the annotation information of the predicted genes and find out the existing lytic enzyme gene sequences.

[0071] 1.2.4 Expression and purification of lyase LysZB48

[0072] In order to make the lysase LysZB48 soluble, an MBP tag was added to the N-terminus of the lysase LysZB48 sequence and a his tag was added to the C-terminus, and then constructed into the pMBP2 vector. The MBP tag can promote the efficient and soluble expression of the target protein. The lysase expression vector pMBP2-LysZB48 was constructed and synthesized by Jinan Boshang Biotechnology Co., Ltd.

[0073] The successfully constructed expression bacteria were added to 200 mL of liquid culture medium containing Amp resistance and cultured on a shaker at 37°C, 180 rpm until the logarithmic growth phase. IPTG was added to a final concentration of 1 mM and induced expression at 37°C for 3.5 hours. The mixture was centrifuged at 4°C, 4,000 rpm for 15 minutes, and the supernatant was discarded. The precipitated bacteria were resuspended and washed with PBS, and then 30 ml of PBST was added to suspend the bacteria. PMSF was finally added to a final concentration of 1 mM, and the mixture was broken by ultrasonic 200W for 6 minutes under ice bath conditions. After incubation at 4°C on a shaker for 1 hour, the mixture was centrifuged at 8,000 rpm, 4°C for 15 minutes, and the supernatant was taken and added to a 1 ml Dextrin column for binding overnight at 4°C. The Dextrin column was collected (centrifuged at 2,000 rpm for 5 minutes), and the beads were washed with 1 mL of PBS washing solution to remove impurities. The mixture was washed three times in total. PBS eluent containing 10 mM maltose was added, and the eluent was allowed to fully bind to the beads for 1 hour, and the supernatant was collected by centrifugation. Add 300uL of elution buffer to the beads again, elute for 1h, collect the supernatant by centrifugation, and combine the two elutions into one tube. SDS-PAGE was used to identify the protein molecular weight, and the BCA kit was used to measure the concentration of the expressed protein. 1.2.5 Lysis effect of lyase LysZB48 at different concentrations

[0074] The lysis activity test was designed based on the principle that the turbidity of the bacterial suspension decreases after bacterial lysis and death. S. aureus was cultured to the logarithmic growth phase, centrifuged at 3,000 rpm for 10 min, the supernatant was discarded, and the precipitate was resuspended in PBS solution to the required OD 630nm The lyase LysZB48 was diluted to an appropriate concentration with PBS solution, and added to the 96-well plate in sequence with the bacterial solution. The OD was measured at 37°C. 630nmThe value was measured once every minute for a total of 60 minutes, and there were three repeated groups in the experiment.

[0075] To verify the actual bactericidal effect of the lysing enzyme LysZB48, 100 μL of the diluted lysing enzyme with a concentration of 400 μg / mL, 200 μg / mL, 100 μg / mL, and 50 μg / mL was resuspended in PBS solution to the appropriate OD 630nm 100 μL of S. aureus solution with the same value was added into a 96-well plate. After incubation for 1 hour, 100 μL of the mixture was evenly spread on a solid LB plate. Three replicate groups were set up in the experiment.

[0076] 1.2.6 Determination of the cleavage spectrum of the lyase LysZB48

[0077] S. aureus was cultured to the logarithmic growth phase, centrifuged at 3,000 rpm for 10 min, the supernatant was discarded, and the precipitate was resuspended in PBS solution to the desired OD 630nm 100 μL of 100 μg / mL LysZB48 and 100 μL of bacterial solution were added to a 96-well plate and the OD was measured within 60 min. 630nm The change of the value is used to verify whether it has a lysis effect on the bacteria.

[0078] 1.2.7 Effects of interfering substances on the effect of lysase LysZB48

[0079] S. aureus cultured to an appropriate concentration was dispensed into 2 mL centrifuge tubes, centrifuged at 3,000 rpm for 10 min, the supernatant was discarded, the precipitates were resuspended with PBS, 4%, 8%, 12% milk powder solution and bovine serum, 100 μL was taken and added to a 96-well plate together with 100 μL of 200 μg / mL lytic enzyme LysZB48, a control group was set up, and after culturing in a 37°C incubator for 1 hour, 100 μL of the culture solution was evenly spread on a solid LB plate and incubated inverted at 37°C overnight. The experiment had three replicate groups.

[0080] 1.2.8 Effect of temperature on the effect of lyase LysZB48

[0081] The experiment is similar to 1.2.5, and the OD per minute is measured at 4℃, 16℃, 20℃, 37℃, and 42℃ respectively. 630nm The changes in the values ​​were measured for 1 h, and three repeated groups were set up in the experiment.

[0082] 1.2.9 Effect of storage time at 37°C on the activity of LysZB48

[0083] Incubate LysZB48 at a concentration of 100 μg / mL in a 37°C incubator for 5 h. Take out 100 μL of the solution every h and add it to a 96-well plate together with 100 μL of PBS resuspended bacterial solution to mix well. Test the OD within 30 min. 630nm The change of the value was used to determine whether LysZB48 had cleavage activity. The experiment was repeated in three groups.

[0084] 1.2.10 Effect of pH on the effect of lyase LysZB48

[0085] The pH of the PBS solution was adjusted to 3, 5, 6, 7, 8, 9, 11, and 13 with H2PO4 and NaOH. After the bacteria were cultured to the appropriate concentration, the supernatant was discarded by centrifugation, and the precipitate was resuspended with PBS solutions of different pH. Lysase LysZB48 was diluted to 200 μg / mL with PBS solutions of different pH. 100 μL LysZB48 and 100 μL bacterial solution were added to a 96-well plate, and the OD per minute was measured. 630nm The changes in the values ​​were measured for 1 h, and three repeated groups were set up in the experiment.

[0086] 1.2.11 Effect of LysZB48 on biofilm removal

[0087] Add 200 μL of S. aureus cultured to an appropriate concentration to a 96-well plate, culture in an incubator at 37°C for 24 hours, then remove the bacterial solution with a pipette and wash three times with PBS solution. Divide the treated wells into 5 groups, add 100 μL of 200 μg / mL, 100 μg / mL, 50 μg / mL, 25 μg / mL of lytic enzyme LysZB48 and PBS solution, culture at 37°C for 2 hours, aspirate the liquid, wash three times with PBS solution, and bake in an oven until the surface is dry. Add 100 μL of crystal violet solution and place at room temperature for 10 minutes, then wash 5 to 6 times with PBS solution, pat dry, add 200 μL of 95% ethanol, incubate at room temperature for 15 minutes, and measure OD 630nm The experiment was set up with three repeated groups.

[0088] 2 Results and analysis

[0089] 2.1 Isolation and purification of bacteriophage

[0090] In this example, the double-layer plate method was used to successfully isolate and extract the bacteriophage Staphylococcus aureus phage 573 (Sau-p573) from the wastewater sample of the cattle farm through the host bacteria S. aureus 573. After multiple purifications, the plaques obtained were relatively uniform in shape and size, as shown in FIG. Figure 1 The titer of bacteriophage Sau-p573 was 7×10 10 PFU / mL.

[0091] 2.2 Electron microscopic observation of bacteriophage

[0092] like Figure 2 It can be clearly observed that the bacteriophage Sau-p573 has a non-retractable long tail. It belongs to the order Caudovirales, family Caudoviridae. The head is a typical icosahedral head of the phage, with a total length of 411±3nm and a tail length of 316±3nm.

[0093] 2.3 Complete genome results and analysis of bacteriophage Sau-p573

[0094] Bacteriophage Sau-p573 is a double-stranded circular DNA with a total genome size of 45,637 bp, of which the base composition is A (37.44%), G (19.54%), T (28.70%), C (14.33%), and the CG content is 33.87%. The results of the complete genome sequence of bacteriophage Sau-p573 were used to align the nucleic acid sequence using the Basic Local Alignment Search Tool in NCBI. The results are as follows Figure 3 The results showed that phage Sau-p573 had a certain degree of similarity with several phages: Staphylococcus phage phiSa2wa_st121mssa (Query coverage 75%, Identity 98.78%), Staphylococcus phage phi 12 (Query coverage 77%, Identity 97.87%), Staphylococcus phage phiSa2wa_st1 (Query coverage 78%, Identity 98.17%), Staphylococcus phage tp310-2 (Query coverage 77%, Identity 97.49%). In addition, phage Sau-p573 also had some unique sequences, such as about 5,000-5,500 bp and about 41,000-42,000 bp, which were not found in similar phages.

[0095] The evolutionary tree was drawn using the software MEGA7. The results are as follows Figure 4As shown, bacteriophage Sau-p573 is in the same large evolutionary tree branch with Staphylococcus phage phiSa2wa_st121mssa, Staphylococcus phage phi 12, Staphylococcus phage phiSa2wa_st1, and Staphylococcus phage tp310-2, and the average credibility of the evolutionary tree branch is 89.25. It can be judged that bacteriophage Sau-p573 is closely related to other Staphylococcus phage involved in the construction of the evolutionary tree, and they have a certain degree of genetic and evolutionary relationship.

[0096] Through BLAST and GeneMark prediction, it can be known that bacteriophage Sau-p573 encodes 67 ORFs, the largest of which is ORF38, with a gene size of 6,225 bp and a protein size of 499.72 kDa, and the smallest is ORF63, with a gene size of 111 bp and a protein size of 9.63 kDa.

[0097] Among all ORFs of LysZB48, the NR database and CDD database analysis revealed that ( Figure 6 ) ORF48 is a protein containing CHAP domain, amidase and SH3b binding domain, and is adjacent to perforin (ORF47). Software comparison analysis revealed that ORF48 has a high degree of similarity with known lyase sequences, so it is inferred that ORF48 is a fragment of LysZB48 encoding lyase.

[0098] 2.4 Expression and purification of LysZB48

[0099] The recombinant plasmid pMBP2-LysZB48 successfully transformed into BL21 (DE3) was induced by IPTG and then ultrasonically lysed. After centrifugation, it was passed through a Dextrin column and the protein solution eluted with PBS containing 10 mM maltose was collected. SDS-PAGE analysis showed that the molecular weight of the LysZB48 protein connected with the mbp tag was similar to the predicted size of 113.14 kDa.

[0100] 2.5 Bactericidal effect of lyase LysZB48

[0101] like Figure 8 As shown, from OD 630nm The change in the value shows that LysZB48 has a certain bactericidal effect when the final concentration is 25μg / mL~200μg / mL, and the bactericidal effect of LysZB48 is more significant when the final concentration is 100μg / mL~200μg / mL. At a concentration of 200μg / mL, it can be seen that the OD630nm The change range of OD value was the largest within 30-60min, and the change range was not obvious. With the decrease of LysZB48 concentration, OD 630nm The change in value becomes more gradual.

[0102] from Fig. 9 It can be seen that the host bacteria concentration is 3×10 8 200μg / mL of LysZB48 can reduce S.aureus573 by 2.1lg CFU / mL in 1h, and 100μg / mL of LysZB48 can reduce S.aureus 573 by 1.9lg CFU / mL.

[0103] 2.6 Determination of the cleavage spectrum of the lyase LysZB48

[0104] This example tests whether the lytic enzyme LysZB48 has a lytic effect on 39 strains of S. aureus and 7 strains of E. coli stored in the laboratory. The results are shown in Table 1. LysZB48 has a lytic effect on 26 of the 39 strains of S. aureus and 4 of the 7 strains of E. coli.

[0105] Table 1 LysZB48 fragmentation spectrum determination results

[0106]

[0107]

[0108] 2.7 Effects of interfering substances on the activity of LysZB48

[0109] The effect of different concentrations of milk powder and bovine serum on the bactericidal effect of LysZB48 shows the possibility of LysZB48 application in treating mastitis in dairy cows. Fig.10 Bovine serum and milk powder had a certain effect on the bactericidal effect of LysZB48. In bovine serum, LysZB48 could reduce the concentration of host bacteria by 1.74 lg CFU / mL. LysZB48 containing 4% milk powder could reduce the concentration of host bacteria by 1.7 lg CFU / mL. The bactericidal effect gradually decreased with the increase of concentration.

[0110] 2.8 Effect of temperature on the effect of lyase LysZB48

[0111] Generally speaking, the increase of temperature within a certain range can accelerate the movement of molecules and thus accelerate the enzymatic reaction. The cleavage effect of LysZB48 at different temperatures is as follows: Fig.11 It can be seen that the cleavage effect of LysZB48 accelerates with the increase of temperature in the range of 4℃ to 42℃, and the best effect is in the range of 37℃ to 42℃.

[0112] 2.9 Effect of storage time at 37°C on the activity of LysZB48

[0113] like Fig.12 LysZB48 lost its cleavage activity after incubation at 37°C for 3 h, and the cleavage activity gradually decreased with the incubation time within 3 h.

[0114] 2.10 Effect of pH on the effect of lyase LysZB48

[0115] like Fig.13 The lytic enzyme LysZB48 has certain activity at pH 6-9, and has a good lytic effect at pH 8-9. The effect of LysZB48 gradually disappears with the increase and decrease of pH.

[0116] 2.11 Effect of LysZB48 on the Removal of Biofilm

[0117] According to laboratory data, S. aureus573 can produce biofilm after incubation at 37°C for 24 h. The biofilm removal effect of the lytic enzyme LysZB48 is as follows: Fig.14 As shown in the figure, all four concentrations of LysZB48 can clear biofilms, and can also play a role at lower concentrations.

[0118] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A fusion protein, characterized in that The sequence of the fusion protein is shown in SEQ ID NO.

2.

2. A nucleic acid substance encoding the fusion protein according to claim 1.

3. The nucleic acid material encoding the fusion protein according to claim 2, characterized in that: The nucleic acid sequence encoding the fusion protein is shown in SEQ ID NO:

8.

4. An expression vector, characterized in that: The expression vector comprises the nucleic acid substance according to claim 2 or 3.

5. The expression vector according to claim 4, characterized in that The expression vector is a bacterial plasmid or a yeast plasmid, including pET series vectors, pMAL series vectors, pGEX series vectors, pBAD series vectors, pMBP series vectors or pUC series vectors.

6. A host cell, characterized in that The host cell comprises the nucleic acid substance of claim 2 or 3 or the expression vector of claim 4 or 5; The host cell is an engineered bacterium used to express the fusion protein of claim 1, and its starting strain is Escherichia coli or yeast.

7. The host cell according to claim 6, characterized in that The starting strain is Escherichia coli.

8. Use of the fusion protein according to claim 1 in the preparation of an antibacterial agent, wherein the antibacterial agent is used to inhibit S. aureus ATCC25923.

9. Use of the fusion protein according to claim 8 in the preparation of antibacterial agents, characterized in that: The antibacterial agent is used for preventing or treating S. aureus ATCC25923 infection.

10. Use of the fusion protein according to claim 8 in the preparation of antibacterial agents, characterized in that: The antibacterial agent is used in the fields including but not limited to food, beverage, and livestock and poultry breeding.

11. Use of the fusion protein according to claim 10 in the field of preparing antibacterial agents, characterized in that: The antibacterial agent is used for sterilizing dairy products, or for preventing and treating diseases related to mastitis in dairy cows.

12. Use of the fusion protein according to claim 11 in the field of preparing antibacterial agents, characterized in that: The dairy product is skimmed milk, low-fat milk or milk powder.

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