Application of bacteriophage lyase in preparation of antibacterial agent

Abstract

The invention discloses an application of bacteriophage lyase in preparation of an antibacterial agent. The bacteriophage lyase has a remarkable splitting effect on gram-negative bacteria, so that the bacteriophage lyase can be used as a broad-spectrum antibacterial protein. For example, the antibacterial effect on gram-negative bacteria such as escherichia coli, pseudomonas aeruginosa and klebsiella pneumoniae is remarkable. The antibacterial protein has relatively high temperature tolerance and pH tolerance, can replace antibiotics, and is applied to prevention and treatment of bacterial diseases as an external medicine or an internal medicine and sterilization of medical instruments and medical places; and the product can also be used as an additive to be applied to the fields of feed processing, food industry, animal husbandry, beer industry, aquaculture, cosmetic production, fruit preservation and the like.

Description

Technical Field

[0001] The invention relates to application of bacteriophage lytic enzyme in preparing antibacterial agent, belonging to the field of bacteriophage lytic enzyme. Background Art

[0002] With the widespread and long-term use of antibiotics, the emergence of more and more drug-resistant strains has become a difficult medical problem, and super bacteria (bacteria resistant to almost all antibiotics) have emerged. The terrible thing about this kind of bacteria is not its lethality to humans, but its resistance to ordinary antibiotics. For this kind of bacteria, people have almost no medicine to use. The widespread use of antibiotics in food processing, animal husbandry, agricultural production, ecological prevention and control and other fields has also led to a more serious problem of drug resistance. Globally, it is a general trend to vigorously control the abuse of antibiotics. Therefore, it is urgent to find new antibacterial drugs to deal with the diseases, environment, agricultural production and food safety problems caused by drug resistance. Phage lytic enzymes are highly specific bacterial cell wall hydrolases encoded by potent phage genes. They can effectively recognize and degrade peptidoglycan, the main component of bacterial cell walls, thereby specifically killing bacteria. Many studies have shown that phage lytic enzymes have high antibacterial activity both in vitro and in vivo. In the context of multidrug-resistant pathogens, exogenous sterilization of lytic enzymes is undoubtedly a new type of antibacterial development. Due to its high antibacterial activity, broad antibacterial spectrum, many types, and wide range of options, it is considered to be an antibiotic alternative with the best development prospects and application potential. There are a large number of bacteriophages in the natural environment, which have become a potential source of highly effective antibacterial proteins. It is a class of microbial antibacterial agents with great application potential. Summary of the invention

[0003] Purpose of the invention: The technical problem to be solved by the present invention is to provide an application of bacteriophage lytic enzyme in the preparation of antibacterial agents.

[0004] Technical solution: To solve the above technical problems, the present invention provides a phage lytic enzyme having an amino acid sequence as shown in SEQ ID NO.1 or an active fragment or analog thereof for use in the preparation of an antibacterial agent, wherein the active fragment or analog has a sequence identity of at least 85% with the amino acid sequence shown in SEQ ID NO.1 or has an alternative amino acid sequence with the same functional group.

[0005] Preferably, the bacteriophage lytic enzyme or its binding domain is used in combination with a signal molecule.

[0006] Preferably, the bacteriophage lytic enzyme or its binding domain is fused with the signal molecule to form a fusion through gene fusion or chemical coupling.

[0007] The replacement amino acid sequence is obtained by conservatively replacing amino acids in the same amino acid group, wherein the amino acid group includes aliphatic amino acids, hydroxyl or sulfur / selenium-containing amino acids, cyclic amino acids, aromatic amino acids or basic acidic amino acids and their amides.

[0008] Among them, the aliphatic amino acids include glycine, alanine, valine, leucine or isoleucine; the hydroxyl or sulfur / selenium-containing amino acids include serine, cysteine, threonine or methionine; the cyclic amino acids include proline; the aromatic amino acids include phenylalanine, tyrosine or tryptophan; the basic amino acids include histidine, lysine or arginine; the acidic amino acids and their amides include aspartic acid, glutamic acid, asparagine or glutamine.

[0009] Wherein, the antibacterial agent is used to inhibit and / or kill Gram-negative bacteria.

[0010] Wherein, the Gram-negative bacteria include Escherichia coli, Klebsiella pneumoniae or Pseudomonas aeruginosa.

[0011] Among them, the phage lytic enzyme is Klebsiella pneumoniae phage lytic enzyme, which is a bacterial cell wall hydrolase.

[0012] The present invention also provides an antibacterial agent for inhibiting and / or killing Gram-negative bacteria, wherein the antibacterial agent contains an antibacterial protein with an amino acid sequence as shown in SEQ ID NO. 1. The amino acid sequence of the antibacterial protein is as shown in SEQ ID NO. 1: MDIFGMLRIDEGYDSKIYKDTEGYWTIGIGHLLTKDPSKSLAISNLDKLVGRS TGGQITQAEAEVIFAKDVEKAIKGIVANATLNPVYNVLDDVRRAALINMVFQMGVSG VAGFPASMRLLLAKKWEAAAKELANSRWYRQTPNRARRVIETMRTGTWSAYQGK.

[0013] The nucleotide encoding the antibacterial protein comprises the sequence shown in SEQ ID NO.2, or its synonymous codon sequence. The nucleotide sequence of the antibacterial protein is shown in SEQ ID NO.2: ATGGATATTTTTGGC ATGTTGCGTATCGATGAAGGATATGACAGCAAAATTTATAAAGATACCGAAGGGTATTGGACCATTGGTATTGGCCACTTACTGACTAAAGACCCGTCAAAATCTTTGGCTATTTCTAATCTGGACAAACTGGTAGGTCGTTCTACTGGTGGTCAAATTACTCAGGCTGAGGCAGAAGTAATTTTTGCCAAAGATGTTGAGAAGGCAATTAAAGGTATTGTTGCTAATGCTACATTAAA CCCGGTATAATGTATTAGATGATGTTCGTAGAGCTGCTCTGATTAACATGGTATTCCAAATGGGTGTGTCTGGTGTAGCCGGGTTCCCAGCTTCAATGAGGTTATTACTCGCTAAAAAGTGGGAAGCTGCTGCCAAGGAACTCACGTTGGTATCGTCAGACACCTAATCGTGCTCGTCGTGTAATTGAAACAATGCGGACCGGAACTTGGTCTGCTTATCAAGGAAAA.

[0014] Among them, the phage lytic enzyme has a strong inhibitory effect on Gram-negative bacteria, has a broad-spectrum antibacterial property, and has good thermal stability. It can effectively replace antibiotics and is widely used in the feed industry, medicine, food industry, animal husbandry, beer industry, aquaculture and other fields.

[0015] Among them, the products include external or internal medicines, feed or food additives, disinfectants, beauty products or medical devices.

[0016] Furthermore, the Gram-negative bacteria include Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae and the like.

[0017] Wherein, the concentration of the bacteriophage lytic enzyme is 0.1-1 mg / mL.

[0018] Wherein, the antibacterial agent also contains EDTA.

[0019] Wherein, the concentration of EDTA is 0.1-10 mM.

[0020] Furthermore, the concentration of the EDTA is 0.5-10 mM.

[0021] Furthermore, the concentration of the EDTA is 1-10 mM.

[0022] Furthermore, the concentration of the EDTA is 5-10 mM.

[0023] Wherein, the applicable temperature of the antibacterial agent is 4-70°C.

[0024] Furthermore, the applicable temperature of the antibacterial agent is 4 to 50°C.

[0025] Wherein, the applicable pH of the antibacterial agent is 3-11.

[0026] Furthermore, the applicable pH of the antibacterial agent is 3-10.

[0027] The present invention also provides a method for preparing the bacteriophage lytic enzyme, comprising the following steps: centrifugation after culturing, collecting the supernatant and separating and purifying the bacteriophage lytic enzyme with a Ni-TED column, then performing a dialysis replacement step, and vacuum concentrating and freeze-drying to obtain the bacteriophage lytic enzyme; the molecular weight of the bacteriophage lytic enzyme is 18.9 kDa, and the isoelectric point is 9.58.

[0028] Preferably, the culture conditions are: 16-30°C, 180-220rpm, 12-18h.

[0029] Specifically, the preparation method of the bacteriophage lytic enzyme is:

[0030] S1: obtain culture solution at 30°C and 180rpm;

[0031] S2: The culture fluid is centrifuged to collect the precipitate, the bacteria are broken and centrifuged to obtain the supernatant, which is separated and purified by Ni-TED column, and then subjected to a dialysis replacement step, vacuum concentrated and freeze-dried to obtain the phage lytic enzyme.

[0032] Wherein, during the induced expression of Klebsiella pneumoniae phage lytic enzyme ATCCa, the final concentration of IPTG was 0.2-1 mM and the temperature was 16-37°C.

[0033] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0034] (1) A novel antibacterial protein isolated and obtained by the present invention has a strong inhibitory effect on Gram-negative bacteria, especially on pathogenic Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, etc., and has the characteristics of broad-spectrum antibacterial property and good stability. The antibacterial protein can replace antibiotics and can be applied to the prevention and treatment of bacterial diseases, as well as the sterilization of medical devices and medical places; it can also be used as an additive in feed processing, food industry, animal husbandry, beer industry, aquaculture cosmetics production, fruit preservation and other fields.

[0035] (2) The antibacterial protein disclosed in the present invention is a soluble protein, and the natural antibacterial protein is produced from Escherichia coli, has the characteristics of rapid reproduction, strong vitality, safety and non-toxicity, etc., which is convenient for large-scale production of the natural antibacterial protein and has huge economic value. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is a schematic diagram of the plasmid expressing vector of Klebsiella pneumoniae phage lytic enzyme;

[0037] Figure 2 To optimize the concentration of isopropyl-BD-thiogalactoside (IPTG) inducing conditions during the expression of lytic enzymes;

[0038] Figure 3 To optimize the induction temperature during the expression of lytic enzymes;

[0039] Figure 4 The SDS PAGE electrophoresis diagram of the lytic enzyme engineered bacteria after expression, fragmentation and purification; wherein: 1 is the marker, 2 is the total bacterial cells after fragmentation, 3 is the precipitate after fragmentation, and 4 is the supernatant after fragmentation;

[0040] Figure 5 Analysis of the optimal inhibitory concentration of lytic enzyme;

[0041] Figure 6 Analysis of optimal EDTA concentration for lysing enzyme;

[0042] Figure 7 For the lytic enzyme host spectrum;

[0043] Figure 8 To test the temperature tolerance of lyase;

[0044] Fig. 9 This is a test for pH tolerance of lysing enzymes. DETAILED DESCRIPTION

[0045] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0046] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be obtained commercially.

[0047] Experimental Materials:

[0048] Enzymes and reagents: The enzymes used in the molecular biology operations in the examples were purchased from TakaRa, and the corresponding operation steps were carried out in full accordance with the relevant product instructions.

[0049] The Klebsiella pneumoniae liquid culture medium and solid culture medium were purchased from Solebao Biotechnology, and the corresponding preparation methods were carried out in full accordance with the relevant product instructions. The Klebsiella pneumoniae standard strain ATCC 13883 was purchased from Beijing Baosai Biotechnology Co., Ltd. The primer design and sequencing of the Klebsiella pneumoniae phage lytic enzyme and expression vector involved in the embodiment were completed by Sangon Biotechnology (Shanghai) Co., Ltd. Other raw materials and auxiliary materials without indicating the source are all commercially available products.

[0050] Example 1 Construction of Klebsiella pneumoniae phage lytic enzyme ATCCa expression vector

[0051] Using the DNA of Klebsiella pneumoniae phage pATCCa obtained by screening sludge from the Xiqing District sewage treatment plant in Tianjin with ATCC13883 as the host bacteria as a template, a primer pair ATCCa-F (5'-GGAATTCCATATGatggatatttttggcatgttgcgta-3') and ATCCa-R (5'-CCGCTCGAGttttccttgataagcagaccaagtt-3') were designed to amplify the lytic enzyme gene.

[0052] Table 1 PCR system

[0053]

[0054] Table 2 PCR reaction program

[0055]

[0056] The amplified lytic enzyme gene and vector pET30a were digested with restriction endonucleases NdeI and XhoI, respectively, and then ligated with T4 ligase. Figure 1 As shown, the recombinant plasmid pET30a-ATCCa was successfully constructed, and then transformed into Escherichia coli BL21 (DE3) to construct recombinant Escherichia coli BL21 / pET30a-ATCCa.

[0057] Example 2 Optimization of expression and purification conditions of Klebsiella pneumoniae phage lytic enzyme ATCCa

[0058] 1. Optimization of IPTG concentration-induced expression conditions: Use a sterile pipette tip to randomly pick a single colony containing the recombinant plasmid, inoculate it into liquid LB containing 50μg / ml Kanamycin, and culture it in a 37°C shaker overnight; 1% was transferred to 50ml of liquid LB containing 50μg / ml Kanamycin, and cultured in a 37°C, 220rpm constant temperature shaker until the logarithmic phase, and IPTG was added to the final concentrations of 0.2mM, 0.4mM, 0.6mM, 0.8mM, and 1mM, respectively. Another bottle without IPTG was used as an uninduced control; cultured overnight in a constant temperature shaker at 25°C and 180rpm, and the bacteria were collected by centrifugation at 4°C; ultrasonic disruption was performed (ultrasound conditions: 46% power; 8min, ultrasonic 5s, interval 5s) until the bacterial solution was transparent and clear; the total protein was taken for protein electrophoresis. The results are as follows Figure 2 As shown, the protein expression level increased significantly under the induction of IPTG. The expression level of ATCCa was slightly different under the induction of different IPTG concentrations. The optimal IPTG concentration in the following experimental expression process was 0.2 mM.

[0059] 2. Optimization of temperature-induced expression conditions: Use a sterile pipette tip to randomly pick a single colony containing the recombinant plasmid, inoculate it into liquid LB containing 50μg / ml Kanamycin, and culture it in a 37°C shaker overnight; 1% was transferred to 50ml of liquid LB containing 50μg / ml Kanamycin, and cultured in a constant temperature shaker at 37°C and 220rpm until the logarithmic phase, and the optimal IPTG obtained from the above experiment was added to the final concentration, and the temperature was set to 16°C, 20°C, 25°C, 30°C, 37°C, and 180rpm for overnight culture in a constant temperature shaker at 4°C, and the bacteria were collected by centrifugation; ultrasonic disruption was performed (ultrasound conditions: 46% power; 8min, ultrasonic 5s, interval 5s) until the bacterial solution was transparent and clear; the total protein was taken for protein electrophoresis. The results are as follows Figure 3 As shown, the protein expression levels under different temperature induction conditions are different. The protein expression level under the 30°C induction condition is the highest, and the optimal temperature induction condition is 30°C.

[0060] 3. Expression and purification of Klebsiella pneumoniae phage lytic enzyme ATCCa: Recombinant Escherichia coli was inoculated into LB liquid culture medium containing 50μg / ml Kanamycin, cultured at 37°C, 1% was transferred to 50ml liquid LB containing 50μg / ml Kanamycin, cultured at 37°C, 220rpm constant temperature shaker until exponential growth phase, IPT G was added to a final concentration of 0.2mM, cultured overnight at 30°C constant temperature shaker, and the bacteria were collected by centrifugation at 4°C; ultrasonic disruption (ultrasound conditions: 46% power; 8min, ultrasonic 5s, interval 5s) until the bacterial solution was clear and transparent; after the expressed protein was disrupted, it was purified using nickel-nitrotriacetic acid chromatography (Ni-NTA columns; Qiagen, Dusseldorf, Germany), and the imidazole elution concentration was 2ml 20mM and 2ml 30mM to elute impurities, and 4ml 100mM to elute the target protein. The protein was then dialyzed into PBS with a pH of 7. The results are shown in Figure 4 As shown, by constructing the expression strain BL21 / pET30a-ATCCa of the antimicrobial protein ATCCa, the lytic enzyme ATC Ca was soluble expressed and purified in Escherichia coli, and its lytic activity was determined. Wherein, the amino acid sequence of the antimicrobial protein ATCCa is shown in SEQ ID NO.1: MDIFGMLRIDEGYDSKIYKDTEGYWTIGIGHLLTKDPSKSLAISNLDKLVGRSTGGQITQAEAEVIFAKDVEKAIKGIVANATLNPVYNVLDDVRRAALINMVFQMGVSGVAGFPASMRLLLAKKWEAAAKELANSRWYRQTPNRARRVIETMRTGTWSAYQGK

[0061] Example 3 Exploration of the Optimal Inhibitory Concentration of Klebsiella Pneumoniae Phage Lytic Enzyme ATCCa

[0062] The bactericidal activity of lysozyme is weak when it acts alone. Many studies have shown that low concentrations of EDTA (ethylenediaminetetraacetic acid) can produce synergistic effects with lysozyme. In order to verify whether the expressed and purified lysozyme in the present invention can also produce effects with low concentrations of EDTA, low concentrations of EDTA were added while the lysozyme was treating the bacteria to observe the difference in the test results.

[0063] Randomly pick a monoclonal host bacterium and culture it in liquid LB medium to the logarithmic phase, collect the bacteria, treat it with 100mM, pH=7 EDTA at 37℃ for 10min, and then resuspend the bacteria with PBS. Set up 4 groups: one group with antimicrobial protein ATCCa and EDTA, one group with only antimicrobial protein ATCCa, one group with only EDTA, and one group as blank control. Among them, the final concentrations of antimicrobial protein ATCCa were set to 0.1, 0.5 and 1mg / ml, respectively, and the final concentration of EDTA in the group with EDTA was 10mM; each group was placed in a 37℃ constant temperature incubator for 6h, and the plates were smeared and counted. There were 3 parallel experiments for the above. The results are as follows Figure 5 As shown, when 0.5 mg / ml antimicrobial protein ATCCa is used alone, the survival rate of Klebsiella pneumoniae ATCC 13883 in the logarithmic phase can be reduced to 26.49%. When used in combination with EDTA, the ATCC survival rate is reduced to 0.09%. When used in combination with EDTA, the antibacterial activity is further enhanced.

[0064] Example 4 Exploration of the Optimal EDTA Concentration of Klebsiella Pneumoniae Phage Lytic Enzyme ATCCa

[0065] Pick a monoclonal host bacterium and incubate it in liquid LB medium until the logarithmic phase, collect the bacteria, treat it with 100mM, pH=7 EDTA at 37℃ for 10min, and resuspend the bacteria with PBS. Use the optimal protein concentration (0.5mg / ml) obtained in Example 3 to set EDTA groups with final concentrations of 0, 0.1, 0.5, 1, 5 and 10mM respectively; each group is placed in a 37℃ constant temperature incubator for 6h, and the plates are plated and counted every 2 hours. The above experiments were repeated 3 times. The results are as follows Figure 6 As shown, when the EDTA concentration was 5 mM, the concentration of TCC13883 bacteria increased from 2 × 10 8 cfu / mL decreased to 5×10 4 cfu / mL, and the optimal EDTA concentration for the protein's antibacterial effect was 5mM.

[0066] Example 5 Determination of host spectrum of Klebsiella pneumoniae phage lytic enzyme ATCCa

[0067] The present invention determines the host spectrum of different test strains by treating the antimicrobial protein ATCCa. In order to detect whether the antimicrobial protein has a wide host spectrum, 6 clinical Klebsiella pneumoniae, 1 Escherichia coli BL21 (DE3) and 1 clinical Pseudomonas aeruginosa were selected. Pick a monoclonal host bacterium in liquid LB culture medium to the logarithmic phase, collect the bacteria, treat with 100mM, pH=7EDTA37℃ for 10min, and resuspend the bacteria with PBS; select the most suitable protein concentration and EDTA concentration in Examples 3 and 4; place each group in a 37℃ constant temperature incubator for 6h; spot plate to observe the number of colonies on the plate, and the above tests are repeated 3 times.

[0068] Table 3 Determination of host spectrum of lytic enzymes

[0069] Strain name Lyase ATCCa Klebsiella pneumoniae ATCC13883 + Clinical Klebsiella pneumoniae S15 + Clinical Klebsiella pneumoniae S21 + Clinical Klebsiella pneumoniae S24 + Clinical Klebsiella pneumoniae S34 + Clinical Klebsiella pneumoniae S55 + Clinical Pseudomonas aeruginosa P8W + Escherichia coli BL21(DE3) +

[0070] The results are shown in Table 3 and Figure 7 As shown, in addition to its antibacterial effect on its own host bacteria, the protein also has a significant antibacterial effect on other Gram-negative bacteria, such as Escherichia coli, Pseudomonas aeruginosa and drug-resistant Klebsiella pneumoniae.

[0071] Example 6 Temperature tolerance test of Klebsiella pneumoniae phage lytic enzyme ATCCa

[0072] Pick a monoclonal host bacterium and incubate it in liquid LB medium until the logarithmic phase, collect the bacteria, treat it with 100mM, pH=7 EDTA at 37℃ for 10min, and then resuspend the bacteria with pH=7.4 PBS; select the most suitable protein concentration in Example 3, set different temperature gradients (4, 16, 25, 37, 50 and 70℃) for treatment; select the most suitable EDTA concentration in Example 4; place each group in a 37℃ constant temperature incubator for 6h and count the plates; the above experiments were repeated 3 times. The results are as follows Figure 8 As shown in the figure, in terms of temperature tolerance, the antimicrobial protein ATCCa can remain stable after being treated at 4, 16, 25, 37 and 50°C, and the antimicrobial activity is reduced to about 80% after being exposed to 70°C for the same duration. Therefore, the antimicrobial protein has good temperature tolerance.

[0073] Example 7 Klebsiella pneumoniae phage lytic enzyme ATCCapH tolerance test

[0074] Pick a monoclonal host bacterium and incubate it in liquid LB medium until the logarithmic phase, collect the bacteria, treat it with EDTA for 10 minutes, and then resuspend it with PBS; select the most suitable protein concentration in Example 3, set different pH gradients (3, 4, 5, 6, 7, 8, 9, 10, 11) for treatment; select the most suitable EDTA concentration in Example 4; place each group in a 37°C constant temperature incubator for 6 hours and count the plates; the above experiments were repeated 3 times. The results are as follows Fig. 9 As shown, in terms of pH tolerance, the protein activity remains basically unchanged at pH values ​​of 3 to 10, and the antibacterial activity is reduced to about 90% at pH 11. Therefore, the antibacterial protein has good pH tolerance.

Claims

1. Use of a bacteriophage lytic enzyme having an amino acid sequence as shown in SEQ ID NO.1 in the preparation of an antibacterial agent.

2. The application according to claim 1, characterized in that: The antimicrobial agent is used to inhibit and / or kill Gram-negative bacteria.

3. The application according to claim 2, characterized in that: The Gram-negative bacteria include Escherichia coli, Klebsiella pneumoniae or Pseudomonas aeruginosa.

4. The use according to claim 1, characterized in that: The method for obtaining the bacteriophage lytic enzyme comprises the following steps: using the DNA genome of the Klebsiella pneumoniae phage pATCCa as a template, amplifying by PCR, enzyme cutting and recombination to obtain a complete gene fragment, and then expressing the bacteriophage lytic enzyme ATCCa by Escherichia coli.

5. An antibacterial agent for inhibiting and / or killing Gram-negative bacteria, characterized in that: The antibacterial agent contains a bacteriophage lytic enzyme with an amino acid sequence as shown in SEQ ID NO.

1.

6. The antibacterial agent according to claim 5, characterized in that: The concentration of the antibacterial protein is 0.1-1 mg / mL.

7. The antibacterial agent according to claim 5 or 6, characterized in that: The antibacterial agent also contains EDTA.

8. The antibacterial agent according to claim 7, characterized in that: The concentration of the EDTA is 0.1-10 mM.

9. The antibacterial agent according to any one of claims 5 to 8, characterized in that The applicable temperature of the antibacterial agent is 4-70°C.

10. The antibacterial agent according to any one of claims 5 to 8, characterized in that The applicable pH of the antibacterial agent is 3-11.

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