Application of antibacterial agent and bacteriophage lyase

Abstract

The invention discloses application of an antibacterial agent and bacteriophage lyase. 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 an antimicrobial agent and bacteriophage lytic enzyme, and belongs to the field of bacteriophage lytic enzyme. Background Art

[0002] With the widespread and long-term use of antibiotics, the emergence of increasingly drug-resistant strains has become a daunting medical challenge. The widespread use of antibiotics in food processing, animal husbandry, agricultural production, and ecological protection has also led to a growing problem of drug resistance. Therefore, there is an urgent need to identify new antimicrobial agents to address the disease, environmental, agricultural, and food safety issues caused by drug resistance. Bacteriophage lytic enzymes are highly specific bacterial cell wall hydrolases, encoded by potent phage genes. They effectively recognize and degrade peptidoglycan, the main component of the bacterial cell wall, thereby specifically killing bacteria. Numerous studies have demonstrated that phage lytic enzymes possess high antimicrobial activity both in vitro and in vivo. In the context of multidrug-resistant pathogens, exogenous sterilization with lytic enzymes is undoubtedly a novel antimicrobial development. Due to their high antimicrobial activity, broad antimicrobial spectrum, diverse antimicrobial activity, and wide range of available options, they are considered to be the most promising antibiotic alternatives with the greatest development prospects and application potential. Bacteriophages are abundant in the natural environment, providing a potential source of highly effective antimicrobial proteins and representing a class of microbial antimicrobial agents with significant potential for application. 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 an antibacterial agent and a bacteriophage lytic enzyme.

[0004] Technical solution: To solve the above technical problems, the present invention provides an antimicrobial agent for inhibiting and / or killing Gram-negative bacteria, wherein the antimicrobial agent contains an antimicrobial protein with an amino acid sequence as shown in SEQ ID NO.1.As shown in Figure 1: MAIERQAVQGLAPVQSTGGPRGSATQAIQ VGTPQTQPGLGNFVDDLFNAAGSVAGLATDIMNRSVEDDKVVQYDRALRGLLPSDEATVGGTRAHMLVQLQNDVIASTAQLQEDAKRFTGDDAEWEQHVIKSRNAIQDTVIQKYPGLGADKETGKIITNAFMEQQPKVFAARQSAKLEREGAERAQSMQSRILLVTKGLSGEPLNEALHQLQQEALTMQLTKPEYEAMVADVALSKASVGDSSFIEATKSLKDKDGVSLYERNGKLMTGEISANRTWASLNQVELFEKKNAAIEAYTKGELNKDEMLQIMENHNRLSGGTAWSDGEITTLFNNVAKQHAKTAQLADLVKRGEGGSPLGLQDISDKDRKSYAEAISGVYTKLADDEILKTGATGEAAEAIRGKYERARYAKLGEQLIKDPNITDRYQALMSMSSANLKDMKAESEAMKTLMQARDAIPEDARRAVMGDKEYAFAENYERATRMGYNPGQAVEFAQAASQGDKLPSSVLKELNSDVDGVVSDVAGGSWLTRGDNMSDMGKDLMLQDAGEIARAMKVAGHNNDTIKRHLTEYLKGQYSQLSEGFFTSGVLVKGDVRGLGEALKTNQKDMPLALRQYLNDNKQALLDASGGMDEKDLYFDVDMKRGLFTIRAGSGRTPVTPAMPLSEIKAQGLLKKYYESEVKARDDGNKAFREQQMKMGSWGISPTTVKDPKDVTAKNVGKMGISSFLMSPAFADGANLPSNFEFGYKENQNMFYDYVAKAENSINAGFDPRAGTYSVYRDEGGDNIGFGHLLTEEEKRNGYVTIGDEKVPFRQGQSELTPARARQLLEQDIQAHQPSTSGWAVPFDSMHPGVQRGILDLSYNLGKQGIANAPKAYADFKAGRFTEGFINMLDTNYTKGARSPGLLRRRAEAYNMAMAASGMPKITQVRTEKDGSMYAKFASADIANFLREGLASKIGKDGWLQVNGPSKLTENSRVGTLSV。

[0005]

[0006] Among them, the phage lytic enzyme has a strong inhibitory effect on Gram-negative bacteria, has broad-spectrum antibacterial properties, 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.

[0007] The products include medicines for external or internal use, feed or food additives, disinfectants, beauty products or medical devices.

[0008] Furthermore, the Gram-negative bacteria include Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae and the like.

[0009] Wherein, the antibacterial agent also contains EDTA.

[0010] Wherein, the applicable temperature of the antibacterial agent is 4-70°C.

[0011] Furthermore, the applicable temperature of the antibacterial agent is 4 to 50°C.

[0012] Furthermore, the applicable temperature of the antibacterial agent is 4 to 37°C.

[0013] Furthermore, the applicable temperature of the antibacterial agent is 4 to 25°C.

[0014] Furthermore, the applicable temperature of the antibacterial agent is 4 to 16°C.

[0015] Wherein, the applicable pH of the antibacterial agent is 3-11.

[0016] Furthermore, the applicable pH of the antibacterial agent is 4-11.

[0017] Furthermore, the applicable pH of the antibacterial agent is 4-10.

[0018] Furthermore, the applicable pH of the antibacterial agent is 5-10.

[0019] The present invention also provides the use of a phage lytic enzyme having an amino acid sequence as shown in SEQ ID NO.1 or an active fragment or analog thereof 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 an alternative amino acid sequence having the same functional group.

[0020] Preferably, the bacteriophage lytic enzyme or its binding domain is used in combination with a signal molecule.

[0021] Preferably, the phage lytic enzyme or its binding domain is fused with the signal molecule to form a fusion through gene fusion or chemical coupling.

[0022] 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.

[0023] 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.

[0024] Wherein, the antibacterial agent is used to inhibit and / or kill Gram-negative bacteria.

[0025] Wherein, the Gram-negative bacteria include Escherichia coli, Klebsiella pneumoniae or Pseudomonas aeruginosa.

[0026] Among them, the phage lytic enzyme is Klebsiella pneumoniae phage lytic enzyme, which is a bacterial cell wall hydrolase.

[0027] Wherein, the phage lytic enzyme is used in combination with EDTA.

[0028] Wherein, the concentration of the phage lytic enzyme is 0.1-1 mg / mL.

[0029] Furthermore, the concentration of the phage lytic enzyme is 0.5-1 mg / mL.

[0030] Wherein, the concentration of the EDTA is 0.1-10 mM.

[0031] Furthermore, the concentration of the EDTA is 0.5-10 mM.

[0032] Furthermore, the concentration of the EDTA is 1-10 mM.

[0033] Furthermore, the concentration of the EDTA is 5-10 mM.

[0034] The present invention also provides a method for preparing the phage lytic enzyme, comprising the following steps: centrifugation after culture, collecting the supernatant and separating and purifying the phage lytic enzyme using a Ni-TED column, then performing a dialysis replacement step, and vacuum concentrating and freeze-drying to obtain the phage lytic enzyme.

[0035] Preferably, the culture conditions are: 16-30°C, 180-220 rpm, 12-18 hours.

[0036] Specifically, the preparation method of the phage lytic enzyme is:

[0037] S1: obtain culture solution at 30°C and 180 rpm;

[0038] S2: The culture solution is centrifuged to collect the precipitate, the bacteria are broken and centrifuged, and the supernatant is taken, 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.

[0039] During the induction expression of Klebsiella pneumoniae phage lytic enzyme S16c, the final concentration of IPTG is 0.2-1 mM and the temperature is 16-37°C.

[0040] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0041] (1) The present invention isolates a novel antimicrobial protein that has a strong inhibitory effect on Gram-negative bacteria, particularly pathogenic Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, and the like, and exhibits broad-spectrum antimicrobial activity and good stability. This antimicrobial protein can replace antibiotics and be used in the prevention and treatment of bacterial diseases, as well as in sterilization of medical devices and medical facilities. It can also be used as an additive in feed processing, the food industry, animal husbandry, the beer industry, aquaculture, cosmetics production, and fruit preservation.

[0042] (2) The antimicrobial protein of the present invention is a soluble protein, and the natural antimicrobial protein is expressed by Escherichia coli, which has the characteristics of rapid reproduction, strong vitality, safety and non-toxicity, etc., which facilitates the large-scale production of the natural antimicrobial protein and has great economic value.

[0043] (3) The antimicrobial protein of the present invention is still active after shearing. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 To optimize the concentration of isopropyl-BD-thiogalactopyranoside (IPTG) for induction during lytic enzyme expression;

[0045] Figure 2 To optimize the induction temperature during the expression of lytic enzymes;

[0046] Figure 3 The figure shows the SDSPAGE electrophoresis of the engineered bacteria expressing the lytic enzyme after fragmentation and purification; 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.

[0047] Figure 4 To analyze the optimal inhibitory concentration of lytic enzyme;

[0048] Figure 5Analysis of optimal EDTA concentration for lytic enzyme;

[0049] Figure 6 For the lytic enzyme host spectrum;

[0050] Figure 7 To test the temperature tolerance of lyase;

[0051] Figure 8 This is a test for pH tolerance of lyase. DETAILED DESCRIPTION

[0052] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0053] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in this field can be used. If the manufacturer of the reagents or instruments is not specified, they are all commercially available conventional reagents.

[0054] Experimental Materials:

[0055] 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.

[0056] Klebsiella pneumoniae liquid and solid culture media were purchased from Solebao Biotechnology, and the corresponding preparation methods were carried out in full accordance with the relevant product instructions. Primer design and sequencing of the Klebsiella pneumoniae phage lytic enzyme and expression vector involved in the examples were completed by Sangon Biotechnology (Shanghai) Co., Ltd. All other raw materials and excipients whose sources are not indicated are commercially available products.

[0057] Example 1 Construction of Klebsiella pneumoniae phage lytic enzyme S16c expression vector

[0058] Sludge was collected from the sewage treatment plant in Xiqing District, Tianjin. The obtained Klebsiella pneumoniae phage pS16c DNA (NCBI accession number PV239576) was used as a template to design primer pairs S16c-F (5'-AAAACTGCAGatggctattgagcgccaagcagtgc-3') and S16c-R (5'-CCGCTCGAGgaccgaaagtgttcccactcgggag-3'). The lytic enzyme gene was amplified according to the system and procedures in Tables 1 and 2.

[0059] Table 1 PCR system

[0060]

[0061] Table 2 PCR reaction procedure

[0062]

[0063] The amplified lytic enzyme gene and vector pET30a were digested with restriction endonucleases NdeI and XhoI, respectively, and then ligated with T4 ligase to successfully construct the recombinant plasmid pET30a-S16c. Subsequently, the recombinant plasmid pET30a-S16c was transformed into Escherichia coli BL21 (DE3) to construct the recombinant Escherichia coli BL21 / pET30a-S16c.

[0064] Example 2 Optimization of expression and purification conditions for Klebsiella pneumoniae phage lytic enzyme S16c

[0065] 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 LB liquid culture medium containing 50μg / ml Kanamycin, and culture it in a 37°C shaker overnight; transfer it to 50ml LB liquid culture medium containing 50μg / ml Kanamycin at a 1% inoculation volume, and culture it in a 37°C, 220rpm constant temperature shaker until the logarithmic phase, add IPTG to a final concentration of 0.2mM, 0.4mM, 0.6mM, 0.8mM, and 1mM, respectively. Another bottle without IPTG is used as an uninduced control; culture it in a constant temperature shaker at 25°C and 180rpm overnight, collect the bacteria by centrifugation at 4°C, perform ultrasonic disruption (ultrasound conditions: 46% power; 8min, ultrasonic 5s, interval 5s) until the bacterial solution is transparent and clear; take the total protein for protein electrophoresis. The results are as follows Figure 1 As shown in the figure, the protein expression level increased significantly under the induction of IPTG. The expression level of S16c was slightly different under the induction of different IPTG concentrations. The optimal IPTG concentration in the following experimental expression process was 0.6 mM.

[0066] 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 LB liquid culture medium containing 50μg / ml Kanamycin, and culture it in a 37°C shaker overnight; transfer it to 50ml LB liquid culture medium containing 50μg / ml Kanamycin at a 1% inoculation rate, and culture it in a 37°C, 220rpm constant temperature shaker to the logarithmic phase, add the IPTG obtained in the above experiment to a final concentration of 0.6mM, and culture it in a constant temperature shaker at 16°C, 20°C, 25°C, 30°C, 37°C, and 180rpm overnight, collect the bacteria at 4°C, and perform ultrasonic disruption (ultrasonic conditions: 46% power; 8min, ultrasonic 5s, interval 5s) until the bacterial solution is transparent and clear; take the total protein for protein electrophoresis. The results are as follows Figure 2 As shown in the figure, the protein expression levels were different under different temperature induction conditions. The protein expression level was the highest under the induction condition of 25℃, and the optimal temperature induction condition was 25℃.

[0067] 3. Expression and Purification of Klebsiella pneumoniae phage lytic enzyme S16c: A single colony of recombinant Escherichia coli was inoculated into LB liquid medium containing 50 μg / ml kanamycin and cultured at 37°C. A 1% inoculum was transferred to 50 ml of LB liquid medium containing 50 μg / ml kanamycin and cultured in a shaker at 37°C, 220 rpm until the exponential growth phase. IPTG was added to a final concentration of 0.6 mM and cultured overnight in a shaker at 25°C. The cells were harvested by centrifugation at 4°C and ultrasonicated (ultrasonication conditions: 46% power; 8 min, 5 s sonication, 5 s intervals) until the cell culture became clear. After fragmentation, the expressed protein was purified using nickel-nitrotriacetic acid chromatography (Ni-NTA columns; Qiagen, Dusseldorf, Germany) with 2 ml of 20 mM imidazole and 2 ml of 30 mM imidazole to elute contaminants, and 4 ml of 100 mM imidazole to elute the target protein. The protein was then dialyzed into PBS at pH 7. Figure 3 As shown, by constructing the expression strain BL21 / pET30a-S16c for the antimicrobial protein S16c, the lytic enzyme S16c was soluble expressed and purified in Escherichia coli, and its lytic activity was determined. The amino acid sequence of the antimicrobial protein S16c is shown in SEQ ID NO.1.

[0068] Example 3 Exploration of the Optimal Inhibitory Concentration of Klebsiella Pneumoniae Phage Lytic Enzyme S16c

[0069] Pick one 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°C for 10 minutes, and then resuspend the bacteria with PBS. Set up 4 groups: one group with antimicrobial protein S16c and EDTA, one group with only antimicrobial protein S16c, one group with only EDTA, and one group as a blank control. Among them, the final concentration of antimicrobial protein S16c in each group was set to 0.1, 0.5 and 1 mg / ml, respectively, and the final concentration of EDTA in the group with EDTA was 10mM; each group was placed in a 37°C constant temperature incubator for 6 hours, and the plates were smeared and counted. There were 3 parallel experiments for all of the above. The results are as follows Figure 4 As shown in the data, in the presence of antimicrobial protein S16c, the survival rate of Klebsiella pneumoniae S16c in the logarithmic phase was the lowest. In addition, when 0.5 mg / ml antimicrobial protein S16c was used alone, the survival rate of Klebsiella pneumoniae S16c in the logarithmic phase was reduced to 84.7%. When used in combination with EDTA, the ATCC survival rate was reduced to 5.95%. This shows that the antimicrobial activity of antimicrobial protein S16c was further enhanced when used in combination with EDTA.

[0070] Example 4 Exploration of the Optimal EDTA Concentration for Klebsiella Pneumoniae Phage Lytic Enzyme S16c

[0071] One monoclonal host bacterium was selected and cultured in liquid LB medium until the logarithmic phase. The bacteria were collected and treated with 100mM, pH=7 EDTA at 37°C for 10 minutes, and then resuspended in PBS. A group with antimicrobial protein S16c and EDTA was set up: at the optimal protein concentration (0.5mg / ml) obtained in Example 3, the final EDTA concentrations were set to 0, 0.1, 0.5, 1, 5 and 10mM respectively; each group was placed in a 37°C constant temperature incubator and cultured for 6 hours, and the plates were plated and counted every 2 hours. The above experiments were repeated 3 times. The results are shown in the figure below. Figure 5 As shown in the figure, when the EDTA concentration was 10 mM, the concentration of S16 bacteria increased from 1.77×10 9 cfu / mL decreased to 4.4×10 8 cfu / mL, and the optimal EDTA concentration for the protein's antibacterial effect was 10 mM.

[0072] Example 5 Determination of the host spectrum of Klebsiella pneumoniae phage lytic enzyme S16c

[0073] The present invention uses the antimicrobial protein S16c to treat different test strains to determine its host spectrum. To test whether the antimicrobial protein has a broad host spectrum, five clinical Klebsiella pneumoniae strains provided by Nankai University (accession numbers: S15: SAMN43028724, S21: SAMN43028728, S24: SAMN43028729, S34: SAMN43028732, S55: SAMN43028742), one Escherichia coli BL21 (DE3) strain, and one clinical Pseudomonas aeruginosa PBW strain (NZCP081477.2) were selected. A monoclonal host bacterium was selected and grown in liquid LB medium until the logarithmic phase. The cells were collected and treated with 100 mM EDTA, pH = 7, at 37°C for 10 min, and then resuspended in PBS. The optimal protein concentration and EDTA concentration determined in Examples 3 and 4 were selected. Each group was cultured in a 37°C constant temperature incubator for 6 h. The number of colonies on the plate was observed by spotting. The above experiments were repeated 3 times.

[0074] Table 3 Lysase host spectrum determination

[0075] Strain name Lyase ATCCa 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) +

[0076] The results are shown in Table 3 and Figure 6 As shown, the protein has a significant antibacterial effect on Escherichia coli, Pseudomonas aeruginosa and drug-resistant Klebsiella pneumoniae (such as S21, S24, S34 and S55), but has no antibacterial effect on drug-resistant Klebsiella pneumoniae S15.

[0077] Example 6 Temperature tolerance test of Klebsiella pneumoniae phage lytic enzyme S16c

[0078] Pick a monoclonal host bacteria and grow it in liquid LB medium until the logarithmic phase. Collect the bacteria, treat with 100mM, pH=7 EDTA at 37℃ for 10min, and then resuspend the bacteria in pH=7.4 PBS. Select the most suitable protein concentration in Example 3 and set different temperature gradients (4, 16, 25, 37, 50 and 70℃) for treatment. Select the most suitable EDTA concentration in Example 4. Each group is placed in a 37℃ constant temperature incubator for 6h and plated and counted. The above experiments are repeated 3 times. The results are as follows. Figure 7 As shown in the figure, in terms of temperature tolerance, the antimicrobial protein S16c remained stable after treatment at 4, 16, 25, 37 and 50°C. After exposure to 70°C for the same duration, the antimicrobial activity was reduced to approximately 50%. Therefore, the antimicrobial protein has good temperature tolerance.

[0079] Example 7 pH tolerance test of Klebsiella pneumoniae phage lytic enzyme S16c

[0080] Pick a monoclonal host bacteria and grow it in liquid LB medium until the logarithmic phase. Collect the bacteria, treat with EDTA for 10 minutes, and resuspend them in PBS. Select the most suitable protein concentration in Example 3 and set different pH gradients (3, 4, 5, 6, 7, 8, 9, 10, 11) for treatment. Select the most suitable EDTA concentration in Example 4. Each group is placed in a 37°C constant temperature incubator for 6 hours and the plate is plated and counted. The above experiments are repeated 3 times. The results are as follows. Figure 8 As shown in the figure, in terms of pH tolerance, the protein activity remained basically unchanged at pH 4 to 11, and the antibacterial activity was reduced to about 65% at pH 3. Therefore, the antibacterial protein has good pH tolerance.

Claims

1. 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.

2. The antibacterial agent according to claim 1, characterized in that The antibacterial agent also contains EDTA.

3. The antibacterial agent according to claim 1 or 2, characterized in that The applicable temperature of the antibacterial agent is 4 to 50°C.

4. The antibacterial agent according to any one of claims 1 to 3, characterized in that The applicable pH of the antibacterial agent is 4-11.

5. Use of a bacteriophage lytic enzyme having an amino acid sequence as shown in SEQ ID NO. 1 in inhibiting and / or killing Gram-negative bacteria.

6. The application according to claim 5, characterized in that The Gram-negative bacteria are Escherichia coli, Klebsiella pneumoniae or Pseudomonas aeruginosa.

7. The use according to claim 5, characterized in that The nucleotide sequence encoding the bacteriophage lytic enzyme is shown in SEQ ID NO.

2.

8. The application according to claim 5, characterized in that: The phage lytic enzyme is used in combination with EDTA.

9. The use according to claim 5, characterized in that: The concentration of the phage lytic enzyme is 0.1-1 mg / mL.

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

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