Phage DNA enzyme and application thereof
The phage DNA enzyme prepared through genetic engineering directly acts on Pseudomonas aeruginosa, solving the treatment problem of multidrug-resistant Pseudomonas aeruginosa infection, and achieving the effect of efficiently inhibiting bacterial growth and reducing drug resistance risks.
Patent Information
- Application Number
- CN202410956283.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-07-17
- Publication Date
- 2025-01-10
AI Technical Summary
The existing antibiotics have limited therapeutic effects on multidrug-resistant Pseudomonas aeruginosa infection, resulting in high infection severity and mortality, and increased drug resistance has become a serious threat.
A phage DNA enzyme was developed, expressed and purified in E. coli through genetic engineering, and used to prepare a novel antibacterial drug against Pseudomonas aeruginosa. It uses phage DNA enzyme to act directly on bacterial genomic DNA and inhibit its growth.
This phage DNA enzyme significantly inhibits the growth of Pseudomonas aeruginosa, is highly specific, reduces the impact on the host's normal bacterial flora, reduces adverse reactions and side effects during the treatment process, and is not easy to cause bacteria to develop drug resistance.
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Abstract
Description
Technical Field
[0001] The invention relates to the preparation and application of bacteriophage DNA enzyme, and belongs to the technical field of biochemistry, molecular biology and genetic engineering. Background Art
[0002] Deoxyribonucleases (DNases) are a heterogeneous class of enzymes that catalyze the hydrolysis of deoxyribonucleic acid (DNA). The two main types of DNases are DNase I and DNase II, both of which are endonucleases that generate 3′-oligonucleotides and 5′-oligonucleotides, respectively. DNases play an important role in the pathogenesis of various diseases and in programmed cell death (apoptosis).
[0003] Currently, bacterial infections are one of the most worrying problems worldwide. Since the first discovery of antibiotics in the 20th century, antibiotics have been the best treatment option for bacterial infections. However, due to the abuse of antibiotics, many bacteria have developed genetic conditions that are resistant to antibiotic treatment to some extent. The emergence of drug-resistant bacteria has become a key medical problem threatening human health. It is estimated that by 2050, more than 10 million people will die from these drug-resistant bacterial infections worldwide. Pseudomonas aeruginosa ( Pseudomonas aeruginosa ) is one of the three major human pathogens, which are very likely to develop multi-drug resistance to antibiotics under the selective pressure of infection and natural environment. Infection with multi-drug resistant isolates limits the choice of antibiotic treatment and increases morbidity and mortality. Therefore, Pseudomonas aeruginosa is listed by the World Health Organization as one of the key pathogens that urgently need new treatment options.
[0004] Bacteriophages are natural enemies of bacteria and are the most abundant and common organisms on earth. In the current context of increasing antibiotic resistance, alternative antibiotic therapies are being developed, and lytic phages are currently considered one of the best options for treating infections caused by multidrug-resistant bacteria. Ongoing clinical trials have also demonstrated that phage therapy has some advantages over conventional antibiotic treatment, such as low toxicity and high host specificity. Currently, the search for phage-derived antibacterial agents through the study of small molecule proteins encoded by phages is considered another potential approach for phage therapy. Summary of the invention
[0005] The purpose of the present invention is to provide a bacteriophage DNA enzyme and a preparation method and application thereof, aiming at developing a new type of antibacterial drug.
[0006] The implementation process of the present invention is as follows: A bacteriophage DNA enzyme, the amino acid sequence of the bacteriophage DNA enzyme is shown in SEQ ID NO: 1.
[0007] A gene, which encodes the above-mentioned bacteriophage DNA enzyme, and the nucleotide sequence of the gene is SEQ ID NO: 2.
[0008] A recombinant vector is provided, wherein the coding gene of the bacteriophage DNA enzyme is inserted into the plasmid pET22b.
[0009] A recombinant cell is obtained by transforming the above recombinant vector into a host cell.
[0010] The preparation method of the above-mentioned bacteriophage DNA enzyme is as follows: the above-mentioned recombinant vector is transformed into Escherichia coli, and the bacteriophage DNA enzyme is obtained by inducing expression and purification.
[0011] The bacteriophage DNA enzyme is used for preparing a drug for treating clinical drug-resistant pathogenic bacteria infection, wherein the clinical drug-resistant pathogenic bacteria is Pseudomonas aeruginosa.
[0012] Advantages of the present invention: (1) Ped1 can significantly inhibit the growth of Pseudomonas aeruginosa, quickly eliminate the number of infected Pseudomonas aeruginosa, and reduce the severity and duration of infection.
[0013] (2) Ped1 only inhibits the growth of Pseudomonas aeruginosa. This high specificity can reduce the impact on the host's normal flora and reduce adverse reactions and side effects during treatment.
[0014] (3) Ped1 inhibits the growth of P. aeruginosa by acting directly on the genomic DNA of P. aeruginosa through its DNase activity, a mechanism that is not likely to lead to bacterial resistance. This means that even with long-term use, P. aeruginosa is unlikely to adapt and develop resistance to this treatment. BRIEF DESCRIPTION OF THE DRAWINGS
[0015] Figure 1 This is the result of double restriction digestion of pET22b and pME6032 vectors; Figure 2 This is a diagram of the purification results of Ped1 protein; Figure 3 This is the result of the in vitro DNA enzyme activity verification of Ped1 protein; Figure 4 This is a diagram showing the in vitro bactericidal validation results of Ped1 protein; Figure 5 This is the result of the in vivo DNA enzyme activity verification of Ped1 protein; Figure 6 is pET22b- ped1 Gene map of the recombinant vector. DETAILED DESCRIPTION
[0016] The technical solution of the present invention is described in detail below in conjunction with the accompanying drawings.
[0017] Pseudomonas aeruginosa is a heterotrophic, motile, Gram-negative bacterium that is highly diverse in genetic background and antibiotic resistance profiles among clinically isolated strains and frequently causes nosocomial ventilator- and surgical instrument-associated infections with high mortality. Infections with this pathogen are particularly lethal in immunocompromised patients, burn patients, and patients with cystic fibrosis (CF). P. aeruginosa can cause bacteremia in burn patients, urinary tract infections in urinary tract patients, and hospital-acquired pneumonia in ventilator-treated patients. It is also a major cause of morbidity and mortality in patients with cystic fibrosis, where abnormalities in the respiratory epithelium allow P. aeruginosa to colonize the lungs for long periods of time. Infections with P. aeruginosa are generally difficult to eradicate, in large part because P. aeruginosa develops resistance to antibiotics, which ultimately leads to lung failure and death.
[0018] Pseudomonas aeruginosa phage P5 is a lytic dsDNA phage, and its host is the standard strain PAO1. P5 can effectively lyse Pseudomonas aeruginosa. Ped1 is an early gene encoded by Pseudomonas aeruginosa phage P5, which has DNase activity and can cause the breakage and degradation of Pseudomonas aeruginosa genomic DNA, thereby inhibiting the growth of Pseudomonas aeruginosa. It can be used to prepare drugs for treating Pseudomonas aeruginosa infections.
[0019] The specific technical solution is: Ped1 is composed of A protein consisting of the amino acid sequence of “MTQANWCKGVRILGGKRDGETVLFTDAHFSESMGMAGGDPELVEALPMPAALRLLVKWNKLCEQMKLDYRYKLL”; In Ped1, the mutation of S (serine) at position 32 to A (alanine) or the mutation of W (tryptophan) at position 58 to A (alanine) in its amino acid sequence will affect the DNA hydrolase function of Ped1 and its application in treating Pseudomonas aeruginosa infection. Proteins with equivalent activity obtained by replacing, deleting or inserting one or more amino acids in the amino acid sequence shown belong to the present invention.
[0020] The present invention uses genetic engineering to express and purify the bacteriophage DNA enzyme in Escherichia coli. The vector used is pET22b. The nucleotide sequence of the exogenous gene is "ATGACCCAAGCTAACTGGTGCAAAGGCGTCCGTATACTTGGCGGGAAGCGGGACGGCGAGACCGTCCTGTTCACCGACGCTCACTTCAGCGAGTCGATGGGTATGGCAGGCGGCGACCCTGAACTGGTGGAAGCTCTGCCCATGCCTGCGGCTCTTCGTTTGCTGGTCAAATGGAACAAACTGTGCGAACAAATGAAGCTTGACTACCGCTACAAACTGCTGTAG" The nucleotide sequences obtained by replacing, deleting or inserting one or several nucleotides into the nucleotide sequence shown all belong to the content of the present invention.
[0021] The invention discloses an application of the bacteriophage DNA enzyme in the preparation of a drug for assisting Pseudomonas aeruginosa infection.
[0022] The technical solution of the present invention is further described in detail below in conjunction with specific implementation methods.
[0023] Amplification of target gene fragment: PCR amplification of Ped1 gene from phage P5 genomic DNA by designing upstream and downstream primers ped1 , the primer sequences are as follows: Forward primer pET22b- ped1 -S: TATCATTGACCCAAGCTAACTGGTGCAAAGG Reverse primer pET22b- ped1 -AS:TATCTCGAGCAGCAGTTTTGTAGCGGTAGTCAAGC Use the following system and procedure to perform PCR amplification of the target gene sequence.
[0024]
[0025] After the PCR reaction product is added to 10 μL of 6 × DNA Loading Buffer and mixed, nucleic acid gel electrophoresis is performed. Under ultraviolet light, a blade is used to cut out the gel block containing DNA fragments, and then the DNA fragments in the gel block are recovered and purified. The specific operation is as follows: (1) Cut the target band under UV light and try to remove the part that does not contain the target band. The smaller the volume, the better.
[0026] (2) Place the target band into a 1.5 mL centrifuge tube and add 500 μL of Buffer GN.
[0027] (3) Incubate in a 65°C water bath for 4–6 min, mixing by inverting every 2–3 min until the gel is completely melted and the solution becomes light yellow. (4) Transfer the solution into the adsorption column, centrifuge at 12,000 × g for 1 min, discard the waste liquid, and return the adsorption column to the empty collection tube; (5) Add 600 μL of Buffer W1 to the adsorption column, centrifuge at 12,000 × g for 1 min, and discard the waste liquid; (6) Repeat step 5 once; (7) Place the adsorption column back into the empty collection tube and centrifuge at 12,000 × g for 2 min. (8) Take out the adsorption column, place it in a clean 1.5 mL centrifuge tube, and leave it uncapped at 20-25°C for 2 min. Add 35-50 μL of Elution Buffer to the middle of the adsorption membrane, leave it at 20-25°C for 2 min, and centrifuge it at 12,000 × g for 2 min. Transfer the resulting solution back into the adsorption column and centrifuge it for 2 min to obtain more DNA.
[0028] Connection of target fragment and vector: Use plasmid micro-extraction kit to recover the vector. The specific operation is as follows: (1) Pick a single clone of the strain to be tested and transfer it to 5 mL of liquid LB medium. Culture it overnight in a shaking incubator at 37°C for 12-16 hours.
[0029] (2) Take 1 to 4 mL of bacterial culture and centrifuge at 12,000 × g for 1 min to collect the cells. Remove as much supernatant as possible.
[0030] (3) Add 250 μL of Buffer P1 to resuspend the cells and shake thoroughly until a sterile block is obtained.
[0031] (4) Add 250 μL of Buffer P2 and gently invert 6 to 8 times to ensure complete bacterial lysis.
[0032] (5) Add 350 μL of Buffer P3 and gently invert 6 to 8 times to mix thoroughly. Centrifuge at 12,000 × g for 15 min.
[0033] (6) Aspirate the supernatant (do not aspirate the precipitate), transfer the supernatant to the adsorption column, centrifuge at 12,000 × g for 1 min, discard the waste liquid, and return the adsorption column to the empty collection tube.
[0034] (7) Add 600 μL of Buffer W1 to the adsorption column, centrifuge at 12,000 × g for 1 min, and discard the waste liquid.
[0035] (8) Repeat the above steps once.
[0036] (9) Place the adsorption column back into the empty collection tube and centrifuge at 12,000 × g for 2 min.
[0037] (10) Take out the adsorption column and place it in a clean 1.5 mL centrifuge tube. Let it stand for 2 min to allow the residual ethanol to evaporate. Add 35-50 μL Elution Buffer to the middle of the adsorption membrane, let it stand for 2 min, and centrifuge at 12,000 × g for 2 min. Transfer the resulting solution back into the adsorption column and centrifuge for 2 min to obtain more DNA.
[0038] After plasmid extraction, digest the vector and the recovered target fragment according to the following system. After digestion, purify and recover using a DNA recovery kit. Connect at 16°C overnight according to the following connection system.
[0039]
[0040] Verification of plasmid vector: Transform the ligation system into DH5α competent cells by heat shock transformation. The specific operation is as follows: (1) Take the above ligation product and add it to the competent cells (the competent cells need to be thawed on ice in advance, about 5 min), mix it by gently pipetting, and place it on ice for 30 min.
[0041] (2) After an ice bath, heat shock the tube at 42°C for 45 seconds, then quickly return the tube to ice and let it stand for 2 minutes (note that shaking should be minimized as it will reduce transformation efficiency).
[0042] (3) Add 500 μL of liquid LB medium or SOC medium to the centrifuge tube and incubate at 37°C at 200 rpm for 0.5-1 h for recovery.
[0043] (4) After the culture is completed, centrifuge at 8000 rpm for 5 min, retain about 100 μL of the supernatant and resuspend the bacteria. Apply the bacterial solution to solid culture medium with appropriate concentrations of antibiotics. After culturing in a 37°C incubator for 12-16 h, screen the desired single clone.
[0044] Perform PCR verification on the single clone grown overnight according to the system below. Culture the strain with the same size as the target band at 37°C overnight, then use the plasmid micro-extraction kit to extract the plasmid, and perform enzyme digestion verification on the vector according to the enzyme digestion verification system below. If the enzyme digestion produces a band with the same size as the target gene, the recombinant vector is successfully constructed ( Figure 1 as shown).
[0045]
[0046] Protein purification: Transform the correct plasmid into E. coli BL21, refer to the method above, and purify the protein as follows: In order to express and purify His-tagged recombinant proteins, the vector pET22b- ped1 Transform into Escherichia coli BL21 (DE3). Then, culture 5 mL of the bacterial solution at 37°C until the stable period, transfer to 1 L of liquid LB, culture at 37°C until OD600nm is 0.6, add 1 mL of 1 M IPTG, and then continue to culture at 16°C for 20 hours. Collect the cells by centrifugation, then suspend the cells with His Buffer A and disrupt them with ultrasound on ice. After disruption, centrifuge and take the supernatant through a His nickel column, then use the AKTA purification system to remove impurities with 10% His Buffer B, and use 100% His Buffer B to gradient elute the target protein. After SDS-PAGE electrophoresis, the collected target protein is stained with Coomassie Brilliant Blue (such as Figure 2 The concentrated protein was desalted using a HiTrap desalting column and then transferred to His Buffer C.
[0047] DNase in vitro activity assay: Ped1 protein and DNA were mixed according to the system below and incubated at 37°C for 30 min. After the reaction was completed, 0.5 μL 250 mM EDTA was added to terminate the reaction, and then 1 μL 6 × Loaging buffer was added to detect nucleic acid degradation by nucleic acid gel electrophoresis.
[0048]
[0049] like Figure 3 As shown in a, PAO1 genomic DNA was incubated with purified Ped1 in DNase I reaction buffer at 37°C for 15, 30, 45, 60, and 75 min, respectively. DNase I was used as a positive control for this reaction.
[0050] like Figure 3 b shows the reaction of PAO1 genomic DNA with purified Ped1 and Ped1 S32L / W58L 、Ped1 S32A and Ped1 W58A The cells were incubated in DNase I reaction buffer at 37°C for 30 min, and the reaction results were displayed by agarose gel electrophoresis.
[0051] In vitro bactericidal validation results of Ped1 protein: Spot plate method: Pick a single clone of the strain PAO1 to be tested and place it in 5 mL of liquid LB, shake it overnight for 12-16 hours. Then evenly spread PAO1 on the LB solid plate, spot 2 μL, 4 μL, and 6 μL of 1 mg / mL Ped1 protein on the plate, and culture it overnight at 37°C (such as Figure 4 a).
[0052] Turbidimetric method: Pick a single clone of the strain PAO1 to be tested and place it in 5 mL of liquid LB. Shake overnight for 12-16 hours. After adjusting the OD 600 nm to a consistent value, transfer it to a test tube containing 3 mL of liquid LB at a 1% inoculum volume and add Ped1 at a final concentration of 1 μg / μL. Take a sample every two hours to measure the OD 600 nm of the cells and obtain a 10-hour growth curve of Pseudomonas aeruginosa (such as Figure 4 b).
[0053] In vivo DNA enzyme activity verification experiment of Ped1 The pME6032- ped1 The expression vector was transformed into PAO1 cells by electroporation. The specific operation is as follows: Inoculate the PAO1 monoclonal clone into 5 mL of liquid LB medium and culture overnight at 37°C. Collect the cells by centrifugation at 8000 g for 3 min. Suspend the cells with 0.3 M sucrose solution, then centrifuge at 8000 g for 3 min to collect the cells. Repeat this process three times. Finally, suspend the cells with an appropriate amount of sucrose solution and dispense into 1.5 mL centrifuge tubes, 50 μL per tube. Add about 200 ng of plasmid pME6032- ped1 After mixing, transfer to an electroporation cup and use an electroporator for electroporation. After electroporation, add 500 μL of liquid LB culture medium to the electroporation cup and mix well. Place in a 37°C incubator and culture for about 1 hour. Remove and centrifuge, discard the supernatant, and retain a small amount of supernatant to suspend the bacteria. Then use a spreading rod to evenly spread it on an LB solid plate (containing tetracycline at a concentration of 100 μg / μL).
[0054] DAPI (4',6-diamidino-2-phenylindole dihydrochloride) staining of in vivo DNA: ped1The plasmid bacteria were cultured at 37°C with 1 mM IPTG concentration to the stable phase, and then 1 / 10 of the volume of the culture medium was added to the cell culture medium with 15 μg / mL DAPI solution. The cells were then cultured at 37°C for 10-20 min, and then the cells were collected by centrifugation and washed twice with PBS buffer, and then suspended with 100 μL PBS buffer. 10 μL was aspirated and spotted on a glass slide, which was then covered with a coverslip. The slide was placed under a laser confocal microscope and imaged using the DAPI channel. The results are shown below. Figure 5 As shown in a.
[0055] One-step TUNEL (TdT-mediated dUTP Nick-End Labeling) detection of bacterial DNA fragmentation: ped1 The plasmid bacteria were cultured at 37°C with 1 mM IPTG concentration to the stable phase. After centrifugation to collect the cells, they were washed once with PBS buffer and fixed with 4% paraformaldehyde for 30 min. After fixation, the bacteria were suspended with PBS containing 0.5% Triton X-100 and incubated at room temperature for 5 min. After washing the cells twice with PBS buffer, 50 μL of TUNEL solution was added and incubated at 37°C in the dark for 60 min. Then, the cells were washed twice with PBS buffer and suspended with 25 μL PBS buffer. 10 μL was aspirated and spotted on a slide, which was then covered with a coverslip. The slide was placed under a laser confocal microscope and imaged using the TUNEL-FITC channel. The results are shown in the figure. Figure 5 As shown in b.
[0056] Protein sequences and nucleotide sequences of the present invention: Protein sequence: SEQ ID NO: 1 MTQANWCKGVRILGGKRDGETVLFTDAHFSESMGMAGGDPELVEALPMPAALRLLVKWNKLCEQMKLDYRYKLL Nucleotide sequence: SEQ ID NO: 2 ATGACCCAAGCTAACTGGTGCAAAGGCGTCCGTATACTTGGCGGGAAGCGGGACGGCGAGACCGTCCTGTTCACCGACGCTCACTTCAGCGAGTCGATGGGTATGGCAGGCGGCGACCCTGAACTGGTGGAAGCTCTGCCCATGCCTGCGGCTCTTCGTTTGCTGGTCAAATGGAACAAACTGTGCGAACAAATGAAGCTTGACTACCGCTACAAACTGCTGTAG The above description is only a preferred specific implementation manner of the present invention, and the protection scope of the present invention is not limited thereto. Any simple change or equivalent substitution made by any relevant person familiar with the technical field within the technical scope introduced in the present invention to the technical solution falls within the protection scope of the present invention.
Claims
1. A bacteriophage DNA enzyme, characterized in that The amino acid sequence of the bacteriophage DNA enzyme is shown in SEQ ID NO:
1.
2. A gene, characterized in that The gene encodes the bacteriophage DNA enzyme according to claim 1.
3. The gene according to claim 2, characterized in that The nucleotide sequence of the gene is shown in SEQ ID NO:
2.
4. A recombinant vector, characterized in that: The gene encoding the bacteriophage DNA enzyme according to claim 1 is inserted into the plasmid pET22b.
5. The recombinant vector according to claim 4, characterized in that The recombinant vector carries the gene according to claim 2.
6. A recombinant cell, characterized in that: The recombinant vector according to claim 4 is transformed into a host cell to obtain the result.
7. The method for preparing the bacteriophage DNA enzyme according to claim 1, characterized in that: The recombinant vector according to claim 3 is transformed into Escherichia coli, and the phage DNA enzyme is obtained by inducing expression and purification.
8. Use of the bacteriophage DNA enzyme according to claim 1 for preparing a drug for treating clinical drug-resistant pathogenic bacterial infections.
9. The use according to claim 8, characterized in that: The clinical drug-resistant pathogenic bacteria is Pseudomonas aeruginosa.