A polypeptide for eliminating Pseudomonas aeruginosa and its application
Through the polypeptide PA0726.1 screened from the phage source, it has an efficient killing effect on Pseudomonas aeruginosa, solving the problem of limited efficacy in the treatment of Pseudomonas aeruginosa in the prior art, and is biosafe.
Patent Information
- Application Number
- CN202411172801.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-08-26
- Publication Date
- 2025-03-11
- Estimated Expiration
- 2044-08-26
AI Technical Summary
The prior art has limited therapeutic effect on Pseudomonas aeruginosa, and commonly used antibiotic treatments are prone to the generation of multiple drug resistance.
A polypeptide PA0726.1 was screened from the phage source. This peptide has a highly effective bactericidal ability to have a significant toxic effect on Pseudomonas aeruginosa, but has no effect on other common strains.
PA0726.1 shows significant killing effects on both laboratory and clinical strains of Pseudomonas aeruginosa and is non-toxic to other bacteria, providing a new and safe treatment method against Pseudomonas aeruginosa.
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Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of polypeptides, and particularly relates to a polypeptide for eliminating Pseudomonas aeruginosa and an application thereof. Background Art
[0002] Pseudomonas aeruginosa is widely distributed in many natural places, causing a variety of infectious diseases such as respiratory tract and urinary system. It is a very common conditional pathogen in hospital infection, and can produce a variety of pathogenic factors such as adhesin, polysaccharide capsule, endotoxin and exotoxin. The bacteria are extremely resistant to chemical drugs, and have strong resistance to physical and chemical factors such as disinfectants, drying, ultraviolet rays and adverse environments. It is easy to form biofilms, and it is not easy to be completely removed after forming biofilms. Existing treatment methods for Pseudomonas aeruginosa infection include organic acids, antibiotics, phage therapy, nanomaterials and antimicrobial peptides. Weak organic acids can penetrate bacterial cell membranes and have certain antibacterial ability. Boric acid, salicylic acid and ascorbic acid are commonly used to eliminate Pseudomonas aeruginosa in local wound infections. However, the current research and use of organic acids are limited to small-scale wounds, and their therapeutic effects on Pseudomonas aeruginosa are limited.
[0003] β-lactamase produced by Pseudomonas aeruginosa can hydrolyze β-lactam antibiotics. In response to this drug resistance mechanism, researchers have proposed the use of large doses of β-lactam antibiotics for long-term treatment. β-lactam antibiotics include two major categories: semi-synthetic penicillins and cephalosporins. During the treatment process, drugs can be used in combination to enhance antibacterial activity and protect antibiotics from being hydrolyzed. There are many reports of the use of broad-spectrum combination preparations composed of tazobactam and cephalosporins in existing treatments. However, improper use of antibiotics can easily lead to the emergence of multidrug-resistant Pseudomonas aeruginosa.
[0004] In response to the phenomenon of increasing drug resistance in pathogenic bacteria, many studies have focused on replacing antibiotic treatment with phage therapy. Phage therapy is a means of treating diseases caused by pathogenic bacteria by lysing host bacteria with phages. In recent years, phage therapy has made significant progress in the treatment of bacterial diseases such as Escherichia coli, Mycobacterium, Klebsiella pneumoniae, and Pseudomonas aeruginosa. The limitation of phage therapy is that the host spectrum is narrow, and a phage can often only infect certain strains of a bacterium. Today, multiple phage combination preparations and phage-antibiotic combination therapy have bright prospects in the treatment of Pseudomonas aeruginosa pneumonia.
[0005] At present, a variety of nanomaterials have been shown to significantly inhibit the quorum response of Pseudomonas aeruginosa, the production of pyocyanin and the formation of biofilm, such as metal ions such as silver, copper, iron and zinc and their oxides. However, the use of metal nanomaterials to treat human diseases may bring certain side effects and need to be used with caution.
[0006] Some antimicrobial peptides are also good means of fighting Pseudomonas aeruginosa infection, and the frequency of drug resistance they produce is lower than that of antibiotic use. For example, colistin is currently the most typical natural antimicrobial peptide for treating severe Pseudomonas aeruginosa infection, and has a broad-spectrum bactericidal effect. However, while some natural antimicrobial peptides have an antibacterial effect on the target pathogens, they are also toxic to eukaryotic cells, and their activity is significantly affected by environmental factors and is easily degraded by bacterial or host proteases. Therefore, the clinical application of natural antimicrobial peptides is limited.
[0007] Existing drugs and technologies have limited therapeutic effects on Pseudomonas aeruginosa, so there is an urgent need to explore new effective and safe anti-Pseudomonas aeruginosa treatments and drugs. Summary of the invention
[0008] The purpose of this patent is to explore a class of phage-derived peptides that can specifically eliminate Pseudomonas aeruginosa, detect their killing effects on Pseudomonas aeruginosa of model and clinical sources, and evaluate their bactericidal effects on Pseudomonas aeruginosa and other common strains, so as to achieve the effect of specific treatment of Pseudomonas aeruginosa infection.
[0009] The present invention aims to find a specific antibacterial polypeptide that is derived from a bacteriophage and has a highly effective bactericidal ability against Pseudomonas aeruginosa and has no toxic effects on other bacteria, thereby providing a new idea for treating infections caused by Pseudomonas aeruginosa.
[0010] The first object of the present invention is to provide a use of a polypeptide in the preparation of a Pseudomonas aeruginosa inhibitor, wherein the amino acid sequence of the polypeptide is shown in SEQ ID NO.3 or SEQ ID NO.4.
[0011] The second object of the present invention is to provide a polypeptide for use in preparing a drug for treating Pseudomonas aeruginosa infection, wherein the amino acid sequence of the polypeptide is shown in SEQ ID NO.3 or SEQ ID NO.4.
[0012] The third object of the present invention is to provide an agent or drug for inhibiting or eliminating Pseudomonas aeruginosa, which contains a component for expressing the polypeptide shown in SEQ ID NO.3 or SEQ ID NO.4 in Pseudomonas aeruginosa.
[0013] Preferably, the reagent or drug contains a component that integrates the sequence shown in SEQ ID NO.1 or SEQ ID NO.2 into Pseudomonas aeruginosa.
[0014] Preferably, the component for integrating the sequence shown in SEQ ID NO.1 or SEQ ID NO.2 into Pseudomonas aeruginosa is a microorganism containing a recombinant vector containing the sequence shown in SEQ ID NO.1 or SEQ ID NO.2.
[0015] Preferably, the recombinant vector containing the sequence shown by SEQ ID NO.1 or SEQ ID NO.2 is a pHERD20T plasmid containing the sequence shown by SEQ ID NO.1 or SEQ ID NO.2.
[0016] Preferably, the agent or drug increases the cell membrane permeability of Pseudomonas aeruginosa.
[0017] Preferably, the Pseudomonas aeruginosa is MPAO1 or PAO1.
[0018] Preferably, the drug further contains pharmaceutically acceptable excipients.
[0019] The fourth object of the present invention is to provide an agent or drug for treating Pseudomonas aeruginosa infection, wherein the agent or drug contains a component for expressing the polypeptide shown in SEQ ID NO.3 or SEQ ID NO.4 in Pseudomonas aeruginosa.
[0020] Advantages of the present invention:
[0021] It is a matter of concern to explore new antibacterial drugs to maintain the stability of normal human flora and control the release of endotoxins after the lysis of pathogens while treating multidrug-resistant Pseudomonas aeruginosa infections. The antibacterial polypeptide PA0726.1 found in the present invention showed significant toxicity to both standard strains and clinical strains of Pseudomonas aeruginosa, and had no effect on the growth of non-Pseudomonas aeruginosa Escherichia coli, Halomonas and Providencia stuartii, which indicates that the polypeptide has the potential to resist Pseudomonas aeruginosa and certain biosafety. Subsequent experiments can be conducted to find the most suitable drug carrier for PA0726.1. If the polypeptide PA0726.1 can be loaded on the carrier, the loss of the drug can be reduced and its therapeutic efficiency can be improved.
[0022] The polypeptide PA0726.1 found in the filamentous phage of Pseudomonas aeruginosa MPAO1 of the present invention exhibits bactericidal activity against Pseudomonas aeruginosa, which not only enhances people's understanding of the functions of accessory genes of Pseudomonas aeruginosa, but also provides new ideas for the treatment of Pseudomonas aeruginosa infection. This discovery has clinical value. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] Figure 1 The base sequence, amino acid sequence and gene locus of PA0726.1. M represents middle length (M), and S represents short length (S).
[0024] Figure 2 The full-length fragment of PA0726.1 is toxic to the laboratory standard strains of Pseudomonas aeruginosa MPAO1 and PAO1, while the truncated fragment is non-toxic.
[0025] Figure 3 Live and dead cell staining experiments proved that PA0726.1 had a significant killing effect on MPAO1 cells.
[0026] Figure 4 Pf4-PA0726.1 expresses toxicity against clinical pathogenic strains of Pseudomonas aeruginosa.
[0027] Figure 5 Pf4-PA0726.1 does not express toxicity against Escherichia coli, Providencia stuartii and Halomonas.
[0028] Figure 6 PA0726.1 is localized on the cell membrane of MPAO1.
[0029] Figure 7 PA0726.1 significantly increased the cell membrane permeability of MPAO1. DETAILED DESCRIPTION
[0030] The following examples are provided to further illustrate the present invention, rather than to limit the present invention.
[0031] Example:
[0032] The present invention focuses on discovering a phage-derived polypeptide that kills Pseudomonas aeruginosa, evaluating its toxic effects on Pseudomonas aeruginosa and other common strains, and expressing it in Pseudomonas aeruginosa to inhibit the growth and reproduction of pathogenic bacteria, thereby achieving the effect of treating Pseudomonas aeruginosa infection.
[0033] The primers used in the present invention are shown in Table 1.
[0034] The specific implementation methods include the following:
[0035] 1. After comparative analysis of the genomes of two filamentous phages Pf4 and Pf6 coexisting in Pseudomonas aeruginosa MPAO1, it was found that both encode a 102bp gene PA0726.1, named Pf4-PA0726.1 (nucleotide sequence as shown in SEQ ID NO.1) and Pf6-PA0726.1 (nucleotide sequence as shown in SEQ ID NO.2), respectively. Both genes can be translated into polypeptides consisting of 33 amino acids (nucleotide sequences as shown in SEQ ID NO.3 and SEQ ID NO.4), and both contain 3 sequences starting from methionine, each consisting of 11 amino acids. The two polypeptides have two amino acid differences ( Figure 1 ).
[0036] 2. We cloned two full-length PA0726.1 genes (Pf4-PA0726.1 and Pf6-PA0726.1) and truncated fragments of PA0726.1 (Pf4-M-PA0726.1, Pf6-M-PA0726.1, and Pf-S-PA0726.1) from the MPAO1 genome ( Figure 1 After integrating these fragments into the laboratory standard strains of Pseudomonas aeruginosa MPAO1 and PAO1, the toxicity plate experiment showed that the full-length fragment of PA0726.1 was toxic to Pseudomonas aeruginosa MPAO1 and PAO1, while the truncated clones of PA0726.1 were not toxic ( Figure 2 ). The specific operations are as follows:
[0037] (1) Inoculate MPAO1 from a -80°C refrigerator onto an LB plate, place it in a 37°C incubator and culture it overnight. Pick a single colony and transfer it to LB liquid culture medium. Cultivate it in a shaker at 37°C until the OD 600 =1.
[0038] (2) Take 1 mL of bacterial solution and centrifuge at 12,000 rpm for 1 min. Discard the supernatant and use the bacterial genome extraction kit (Cat. No.: DP302-02) from Tiangen Biochemical Technology (Beijing) Co., Ltd. to extract the MPAO1 genomic DNA in the precipitate.
[0039] (3) Using 2×Phanta Max Master Mix of Nanjing Novezan Biotechnology Co., Ltd., PCR amplification was performed with MPAO 1 genomic DNA as template using primer pairs Pf4-PA0726.1-F / R and Pf6-PA0726.1-F / R to obtain the full-length sequences Pf4-PA0726.1 and Pf6-PA0726.1 with pHERD20T homology arms; primer pairs Pf4-M-PA0726.1-F / R, Pf6-M-PA0726.1-F / R and Pf-S-PA0 PCR amplification was performed on 726.1-F / R to obtain the truncated fragments Pf4-M-PA0726.1, Pf6-M-PA0726.1 and Pf-S-PA0726.1 with pHERD20T homology arms (PCR program: 95°C, 10 min; 95°C, 30 s, 60°C, 30 s, 72°C, 20 s, 30 cycles; 72°C, 5 min, the same below), which were sent to the company for DNA sequencing verification. The PCR products with correct sequencing results were subjected to agarose gel electrophoresis and then recovered using Omega's gel recovery kit (Cat. No.: D2500-02) to obtain DNA fragments Pf4-PA0726.1, Pf6-PA0726.1, Pf4-M-PA0726.1, Pf6-M-PA0726.1, and Pf-S-PA0726.1 with pHERD20T homology arms.
[0040] (4) The vector pHERD20T was double-digested with NcoⅠ and HindⅢ, followed by agarose gel electrophoresis and gel recovery.
[0041] (5) The recovered DNA fragment and the vector fragment were connected using the One Step Multi-Fragment Cloning Kit (Cat. No.: C113-01) of Nanjing Novezan Biotechnology Co., Ltd.
[0042] (6) The ligation product was transformed into E. coli WM3064 competent cells, and after PCR verification using the pHERD20T-F / R primer pair (PCR procedure is the same as above), it was sent to the company for DNA sequencing verification.
[0043] (7) The WM3064 strain (donor bacteria) containing the recombinant plasmid and Pseudomonas aeruginosa MPAO1 and PAO1 (recipient bacteria) were cultured in LB liquid medium at 37°C in a shaker overnight. The donor bacteria and the recipient bacteria were mixed in a volume ratio of 5:1 and washed twice with LB liquid medium, centrifuged at 8,000 rpm for 1 min each time. The precipitate was suspended with 100 μL LB for the last time, dropped on an LB solid plate containing 0.3 mM diaminoacrylic acid, and placed in a 37°C incubator for 8 h. The culture was streaked on an LB solid plate containing 100 mg / L carbenicillin, and the plate was placed in a 37°C incubator for overnight culture. The recipient strain that successfully combined and transferred was selected, and after PCR verification using the pHERD20T-F / R primer pair (PCR procedure is the same as above), it was sent to the company for DNA sequencing verification.
[0044] (8) Pseudomonas aeruginosa standard strains MPAO1 and PAO1 containing different recombinant plasmids (pHERD20T-Pf4-PA0726.1, pHERD20T-Pf6-PA0726.1, pHERD20T-Pf4-M-PA0726.1, pHERD20T-Pf6-M-PA0726.1, pHERD20T-Pf-S-PA0726.1) were cultured in a shaker at 37°C overnight. The inoculation loop was burned red on an alcohol lamp, and after cooling, a loop of bacterial solution was taken and streaked on an LB solid plate containing 100 mg / L carbenicillin. After streaking, the inoculation loop was burned red on an alcohol lamp; after the inoculation loop was cooled, a loop of bacterial solution was taken and streaked on an LB solid plate containing 100 mg / L carbenicillin and 0.3% L-arabinose. The plate was placed in a 37°C incubator for overnight culture and observed and photographed.
[0045] 3. To explore the killing effect of PA0726.1 on Pseudomonas aeruginosa, we used the Live / DeadBacLight TM The MPAO1 overexpressing Pf4-PA0726.1 was stained with a bacterial viability detection kit and observed under a microscope. Through live-dead cell staining experiments, we further demonstrated that PA0726.1 can effectively kill Pseudomonas aeruginosa ( Figure 3 ). The specific operations are as follows:
[0046] (1) MPAO1 containing pHERD20T empty vector and pHERD20T-Pf4-PA0726.1 recombinant plasmid was cultured in LB liquid medium at 37°C in a shaking incubator overnight. The bacterial solution was diluted to OD 600 =0.6, add 0.3% L-arabinose, and culture in a shaker at 37°C for 8 h.
[0047] (2) Take 50 μL of bacterial solution and add 0.5 mL of SYTO 9 and PI staining working solution. Incubate at room temperature in the dark for 15 min. Prepare slides and observe under a microscope.
[0048] 4. Pf4-PA0726.1 is known to be toxic to laboratory standard strains of Pseudomonas aeruginosa. To explore whether the toxicity of Pf4-PA0726.1 to Pseudomonas aeruginosa is universal, Pf4-PA0726.1 was transformed into different hospital clinical strains of Pseudomonas aeruginosa. Through the toxicity plate experiment, it was found that PA0726.1 has a wide range of toxic effects on Pseudomonas aeruginosa, which has clinical significance ( Figure 4 ). The specific operations are as follows:
[0049] (1) The WM3064 strain containing the pHERD20T-Pf4-PA0726.1 recombinant plasmid and different clinical pathogenic strains of Pseudomonas aeruginosa (WK172, PA-16, PA-19, PA-20, and PA-22) were cultured in LB liquid medium in a shaker at 37°C overnight, and the WM3064 strain containing the pHERD20T-Pf4-PA0726.1 recombinant plasmid was mixed with other clinical pathogenic strains of Pseudomonas aeruginosa at a bacterial liquid volume ratio of 5:1 for conjugation transfer. The recipient strain that was successfully conjugated and transferred was selected, and after PCR verification using the pHERD20T-F / R primer pair (PCR procedure is the same as above), it was sent to the company for DNA sequencing verification.
[0050] (2) Different clinical pathogenic strains of Pseudomonas aeruginosa containing the pHERD20T-Pf4-PA0726.1 recombinant plasmid were cultured in a 37°C shaker overnight. The bacterial solution was dipped in turn using a sterilized inoculation loop and streaked on an LB solid plate containing 100 mg / L carbenicillin and an LB solid plate containing 100 mg / L carbenicillin and 0.3% L-arabinose, respectively. The plates were placed in a 37°C incubator for static culture overnight, and the plates were observed and photographed for record.
[0051] 5. To explore whether PA0726.1 is toxic to other species of bacteria, we transformed pHERD20T-Pf4-PA0726.1 into Escherichia coli MG1655 and Providencia stuartii, and transformed pHGECm-Pf4-PA0726.1 into Halomonas 43005. Through the toxicity plate experiment, it was found that PA0726.1 was non-toxic to Escherichia coli MG1655, Providencia stuartii, and Halomonas 43005 ( Figure 5 ), it was preliminarily determined that PA0726.1 had a specific toxic effect on Pseudomonas aeruginosa. The specific operation was as follows:
[0052] (1) Primers pHGECm-PA0726.1-EcoRI-F and pHGECm-PA0726.1-HindⅢ-R were used to amplify Pf4-PA0726.1 with homology arms of pHGECm vector (disclosed in the document "Mobile Genetic Elements Used by Competing Coral Microbial Populations Increase Genomic Plasticity") by PCR (PCR procedure was the same as above). The PCR product was subjected to agarose gel electrophoresis and gel recovery.
[0053] (2) The pHGECm vector was double-digested with EcoRI and HindIII, followed by agarose gel electrophoresis and gel recovery.
[0054] (3) The recovered DNA fragment and pHGECm vector fragment were connected using the One Step Multi-Fragment Cloning Kit (Cat. No.: C113-01) of Nanjing Novezan Biotechnology Co., Ltd.
[0055] (4) The ligation product was transformed into E. coli WM3064 competent cells, verified by PCR using the pHGECm-F / R primer pair (the PCR procedure was the same as above), and then sent to the company for DNA sequencing verification.
[0056] (5) The WM3064 strain containing the recombinant plasmid pHGECm-Pf4-PA0726.1 and Halomonas 43005 were mixed at a logarithmic bacterial liquid volume ratio of 1:1 for conjugation transfer; the WM3064 strain containing the recombinant plasmid pHERD20T-Pf4-PA0726.1 was mixed with Escherichia coli MG1655 and Providencia stuartii at a logarithmic bacterial liquid volume ratio of 5:1 for conjugation transfer. The recipient strains that were successfully conjugated and transferred were selected, and PCR verification was performed using the pHGECm-F / R and pHERD20T-F / R primer pairs (PCR procedure is the same as above), and then sent to the company for DNA sequencing verification.
[0057] (6) Halomonas 43005 containing the recombinant plasmid pHGECm-Pf4-PA0726.1 was cultured in 2216E medium in a shaking incubator at 30°C overnight. The bacterial solution was dipped into a sterilized inoculation loop and streaked on a 2216E solid plate containing 30 mg / L chloramphenicol and a 2216E solid plate containing 30 mg / L chloramphenicol and 1 μM isopropylthiogalactoside, respectively. The plates were placed in a 30°C incubator for overnight static culture and observed and photographed. Escherichia coli MG1655 and Providencia stuartii containing the recombinant plasmid pHERD20T-Pf4-PA0726.1 were cultured in LB liquid culture medium at 37°C in a shaker overnight. The bacterial liquid was dipped in turn using a sterilized inoculation loop and streaked on LB solid plates containing 100 mg / L carbenicillin and 100 mg / L carbenicillin and 0.3% L-arabinose, respectively. The plates were placed in a 37°C incubator for static culture overnight, and the plates were observed and photographed for record.
[0058] 6. The software Alphafold predicted that PA0726.1 contained a transmembrane domain. We connected the GFP gene to the PA0726.1 gene and transferred it into MPAO1 for expression. Through microscopic observation, we confirmed that PA0726.1 was located on the cell membrane of MPAO1 ( Figure 6 ). The specific operations are as follows:
[0059] (1) Using primers GFP-Pf4-PA0726.1-F and Pf4-PA0726.1-HindⅢ-R, MPAO1 genomic DNA was used as a template to PCR amplify Pf4-PA0726.1 with GFP and pHERD20T homology arms; using primers GFP-Pf6-PA0726.1-F and Pf6-PA0726.1-HindⅢ-R, MPAO1 genomic DNA was used as a template to PCR amplify Pf6-PA0726.1 with GFP and pHERD20T homology arms; using primers NcoI-GFP-F and GFP-Pf-PA0726.1-R, GFP genome (nucleotide sequence shown in SEQ ID NO.5) was used as a template to PCR amplify GFP tag (PCR procedure is the same as above). The PCR products were subjected to agarose gel electrophoresis and gel recovery.
[0060] (2) Using primer pairs NcoI-GFP-F and Pf-PA0726.1-HindⅢ-R, Pf4-PA0726.1 and Pf6-PA0726.1 were linked to GFP by PCR (PCR procedure was the same as above) to obtain GFP-Pf4-PA0726.1 and GFP-Pf6-PA0726.1. The PCR products were subjected to agarose gel electrophoresis and gel recovery.
[0061] (3) The recovered DNA fragments and the double-enzyme-digested (NcoⅠ and HindⅢ) fragments of the pHERD20T vector were connected using the One Step Multi-Fragment Cloning Kit (Cat. No.: C113-01) of Nanjing Novezan Biotechnology Co., Ltd.
[0062] (4) The ligation product was transformed into E. coli WM3064 competent cells, verified by PCR using the pHERD20T-F / R primer pair, and then sent to the company for DNA sequencing verification.
[0063] (5) The WM3064 strain containing the recombinant plasmid and Pseudomonas aeruginosa MPAO1 were mixed at a logarithmic bacterial liquid volume ratio of 5:1 for conjugation transfer. The recipient strain that successfully conjugated and transferred was selected, and after PCR verification using the pHERD20T-F / R primer pair, it was sent to the company for DNA sequencing verification.
[0064] (6) Culture the MPAO1 containing the recombinant plasmid in a shaker at 37°C overnight. Dilute the bacterial solution to OD 600 =0.6, add 0.3% L-arabinose, culture in a shaker at 37°C for 4 h, prepare slices, and observe under a microscope.
[0065] 7. To investigate the effect of PA0726.1 on the cell membrane of Pseudomonas aeruginosa, we used SYTOX TM Green dead cell dye was used to observe the cell membrane permeability of Pseudomonas aeruginosa under a microscope. Since high SYTOX green fluorescence levels represent the formation of pores in the cell membrane, fluorescence microscopy experiments showed that PA0726.1 increased the cell membrane permeability of MPAO1 ( Figure 7 ).
[0066] The specific operations are as follows:
[0067] (1) Culture the MPAO1 containing the pHERD20T empty vector and the pHERD20T-Pf4-PA0726.1 recombinant plasmid in a shaking incubator at 37°C overnight. Dilute the bacterial solution to OD 600 =0.6, add 0.3% L-arabinose, and culture in a shaker at 37°C for 1 h.
[0068] (2) Take 50 μL of bacterial solution and add 200 nM of SYTOX TM Green dead cell dye was added, incubated at room temperature in the dark for 5 min, sliced, and observed under a microscope.
[0069] Table 1 Primers used in the present invention
[0070]
[0071]
[0072] SEQ ID NO.1
[0073] ATGATCGATTGGGAATTCCTCGCCCCGGTGGCGATGGGCTGGGCGCTGCATCACTGGTGGACGGTGATGACGGCGCTAGCGGCGGTAGGGGTGCCGCCATGASEQ ID NO.2
[0074] ATGATCGATTGGGAATTCCTCGTTCCGGTGGCGATGGGCTGGGCGCTGCATCACTGGTGGTCGGTGATGACGGCGCTAGCGGCGGTAGGGGTGCCGCCATGASEQ ID NO.3
[0075] MIDWEFLAPVAMGWALHHWWTVMTALAAVGVPPSEQ ID NO.4
[0076] MIDWEFLVPVAMGWALHHWWSVMTALAAVGVPPSEQ ID NO.5
[0077] 。
Claims
1. Use of a polypeptide in the preparation of a Pseudomonas aeruginosa inhibitor, characterized in that: The amino acid sequence of the polypeptide is shown in SEQ ID NO.3 or SEQ ID NO.
4.
2. The use according to claim 1, characterized in that: The Pseudomonas aeruginosa is MPAO1 or PAO1.