Mucus type pseudomonas aeruginosa phage separation method based on recombinant alginate lyase

Abstract

The invention discloses a mucus type pseudomonas aeruginosa bacteriophage separation method based on recombinant alginate lyase, according to the separation method, mucus type pseudomonas aeruginosa bacteriophage is obtained based on recombinant alginate lyase separation, and the amino acid sequence of the recombinant alginate lyase is shown as SEQ ID NO.2. An alginate lyase gene algL in genomic DNA of a pseudomonas aeruginosa standard strain PAO1 is used as a target gene, a recombinant plasmid is constructed, induced expression is completed in escherichia coli, and the recombinant alginate lyase with high purity and high alginate degradation activity is obtained. Based on the recombinant alginate lyase, the bacteriophage which shows a high splitting effect on the mucus type pseudomonas aeruginosa is screened, the problem that the mucus type pseudomonas aeruginosa bacteriophage is difficult to separate is solved, and a technical basis is provided for development of drugs for treating infectious diseases caused by the mucus type pseudomonas aeruginosa.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a method for isolating mucinous Pseudomonas aeruginosa bacteriophages based on recombinant alginate lyase. Background Technology

[0002] Pseudomonas aeruginosa ( Pseudomonas aeruginosa , P. aeruginosa Pseudomonas aeruginosa (PA) is widely distributed in the environment and is a common Gram-negative bacterium in clinical practice. Pseudomonas aeruginosa has a mucoid form. Pseudomonas aeruginosa (mPA) and non-myxoid types. Myxoid and non-myxoid Pseudomonas aeruginosa can transform into each other. Under the stimulation of conditions such as high osmotic pressure, high sodium chloride level and low phosphate level, non-myxoid Pseudomonas aeruginosa transforms into a high alginate-producing state, which leads to the formation of myxoid Pseudomonas aeruginosa.

[0003] The development of specific drug resistance in *Pseudomonas aeruginosa* (mPA) is primarily due to the alginate-rich extracellular matrix, with abundant mucopolysaccharides enabling the formation of a thicker and denser biofilm. This biofilm formation helps bacteria evade phagocytosis by immune cells such as macrophages. Secondly, the physical barrier of the biofilm hinders or delays the penetration of antibiotics and other antimicrobial agents, making it difficult for them to directly contact and act on the bacteria. Furthermore, the reduced metabolic activity within the biofilm significantly decreases the sensitivity of bacteria to antimicrobial drugs, further enhancing their survival ability. However, as bacterial resistance increases, infections caused by *Pseudomonas aeruginosa* gradually develop into chronic, persistent, recurrent, and difficult-to-treat diseases.

[0004] Phage therapy treats pathogenic bacterial infections by lysing bacteria with lysing phages. Compared to antibiotics, phages can simultaneously disrupt the microbiome, exhibiting high host specificity, high safety, and low cost, making them significant in addressing the increasingly serious threat of drug-resistant Pseudomonas aeruginosa and the failure of antibiotics worldwide. Myxobolus aeruginosa secretes large amounts of alginate polysaccharides and other sticky substances: ① forming a slimy "barrier" on the outside of the bacteria, preventing direct contact between the phage and the host bacteria; ② limiting phage diffusion, thus affecting the phage's infection and lysis of the host bacteria, and impacting phage isolation and identification. However, the alginate-based extracellular matrix of mPA makes mPA phage isolation difficult, and even when phages are isolated, the bactericidal effect is not ideal. Therefore, there is an urgent need to explore effective mPA phage isolation strategies to obtain myxobolus aeruginosa phages with significant bactericidal effects. Summary of the Invention

[0005] In view of this, the present invention proposes a recombinant alginate lyase, and the present invention also proposes a method for isolating myxomorphic Pseudomonas aeruginosa bacteriophages based on the recombinant alginate lyase.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: The recombinant alginate lyase of this invention has the amino acid sequence shown in SEQ ID NO.2, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.1. The beneficial effects are: the recombinant alginate lyase of this invention has excellent alginate degradation activity and can be used for screening myxotrophic Pseudomonas aeruginosa phages.

[0007] This invention also proposes a method for preparing recombinant alginate lyase. The method involves first constructing a recombinant plasmid expressing alginate lyase using recombinant homology, then transforming the correctly constructed plasmid into *E. coli* DH5α for amplification, followed by transformation into BL21(DE3) for induced expression to obtain a crude enzyme solution. The crude enzyme solution is then purified by column chromatography to obtain the recombinant alginate lyase. The amino acid sequence of the recombinant alginate lyase in this invention is shown in SEQ ID NO.2, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.1.

[0008] The beneficial effects are: This invention utilizes the alginate lyase gene from the genomic DNA of the Pseudomonas aeruginosa standard strain PAO1. algL As the target gene, a recombinant plasmid was constructed and induced to express in Escherichia coli BL21(DE3), resulting in a high-purity recombinant alginate lyase, thus ensuring its alginate degradation activity.

[0009] This invention also proposes a method for isolating myxobolus aeruginosa bacteriophages based on recombinant alginate lyase, comprising the following steps: First, inoculating a myxobolus aeruginosa strain into LB liquid medium and culturing overnight to obtain a seed culture of the myxobolus aeruginosa strain; inoculating the seed culture into LB liquid medium supplemented with recombinant alginate lyase (amino acid sequence as shown in SEQ ID NO.2), adding sewage for culturing, and enriching to obtain bacteriophages; and separating and obtaining bacteriophage plaques using double-layer agar plates. The second step involves sequential plaque selection from the phage plaques isolated in the first step, followed by phage plaque formation rate determination to obtain phage P1030, which lyses *Pseudomonas aeruginosa*. This phage is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 46655. Phage P1030 of this invention possesses complete structural protein, replication regulation, and lysis system gene modules. Its genome is compact and functionally well-defined, and its genetic characteristics are highly consistent with the phenotype of *Pseudomonas aeruginosa*, which efficiently lyses *Pseudomonas aeruginosa*, providing a theoretical basis for its application in the preparation of drugs or biological agents for treating infectious diseases caused by *Pseudomonas aeruginosa*.

[0010] Preferably, in the first step, the final concentration of recombinant alginate lyase in LB liquid medium and the final concentration of recombinant alginate lyase in the upper layer of the bilayer agar plate during phage enrichment are both 80 mg / mL. More preferably, the titer of phage P1030 is 10. 7 -10 11 PFU / mL. The beneficial effects are: This invention utilizes recombinant alginate lyase to degrade the extracellular alginate matrix of mPA, significantly improving the enrichment and separation efficiency of phages, thus successfully obtaining phage P1030 and overcoming the problem of difficult isolation of existing myxotropic Pseudomonas aeruginosa phages. Secondly, the phage P1030 obtained by this invention has a high titer (at 10...). 7 -10 11 The phage P1030 of this invention exhibits high titer (at 10 PFU / mL), is genetically stable, and shows enhanced lysis against myxotrophic Pseudomonas aeruginosa with the assistance of alginate lysin. 7 -10 11 (PFU / mL), genetically stable, and exhibits enhanced lytic activity against *Pseudomonas aeruginosa* with the assistance of alginate lyase, providing a reliable technical basis for the development of drugs to treat infectious diseases caused by *Pseudomonas aeruginosa*.

[0011] Compared with the prior art, the advantages of the present invention are as follows: This invention uses the alginate lyase gene in the genomic DNA of the Pseudomonas aeruginosa standard strain PAO1. algL A recombinant plasmid was constructed as the target gene and induced to express in *Escherichia coli* BL21(DE3), yielding a high-purity and high-activity (alginate degradation activity) recombinant alginate lyase. This invention, based on the recombinant alginate lyase, screened bacteriophages exhibiting high lytic activity against *Pseudomonas aeruginosa*, overcoming the difficulty in isolating *P. aeruginosa* bacteriophages and providing a reliable technical foundation for the development of drugs to treat infectious diseases caused by *P. aeruginosa*. Attached Figure Description

[0012] Figure 1 This is in Embodiment 1 of the present invention algL Agarose gel electrophoresis image of gene fragments (Line 1 is the marker, Lines 2-5 are...) algL (Gene fragments).

[0013] Figure 2 This is an agarose gel electrophoresis image of the plasmid in Example 1 of the present invention (Line 1 is the marker, Line 2 is the undigested pET28a(+) plasmid (i.e. the original plasmid), and Lines 2-5 are the linearized plasmids).

[0014] Figure 3 This is the gene map of the recombinant plasmid in Example 1 of the present invention.

[0015] Figure 4 This is an electrophoresis diagram of the recombinant alginate lyase in Example 1 of the present invention (Line 1 is the marker, Line 2 is the purified enzyme solution, Line 3 is the enzyme solution after dialysis, Line 4 is the enzyme solution purified by column chromatography, Line 5 is the precipitate sample separated by cell lysis after induced expression, Line 6 is the supernatant separated by cell lysis after induced expression, Line 7 is the bacterial culture after induced expression, and Line 8 is the bacterial culture before induced expression).

[0016] Figure 5 This is a Western blot result of the recombinant alginate lyase in Example 1 of the present invention (Line 1 is the marker; Lines 2-5 are the target protein, approximately 44KD in size).

[0017] Figure 6 This is a graph showing the enzyme activity verification results of the recombinant alginate lyase of the present invention (the blue column represents the negative control group, and the red column represents the experimental group with added recombinant alginate lyase).

[0018] Figure 7 This is a staining diagram of the biofilm removal ability of the recombinant alginate lyase of this invention.

[0019] Figure 8 This is a graph showing the biofilm removal capability of the recombinant alginate lyase of this invention.

[0020] Figure 9 This is an enrichment diagram of bacteriophages in Example 3 of the present invention (the left image is the recombinant alginate lyase, and the right image is the control group without the addition of recombinant alginate lyase).

[0021] Figure 10 The images show the morphology of phage plaques (left) and a transmission electron microscope scan (right) of phage P1030.

[0022] Figure 11This is a circular diagram of the genome of the bacteriophage P1030 of this invention.

[0023] Figure 12 This is the developmental tree of the bacteriophage P1030 of this invention.

[0024] Figure 13 This is a diagram showing the bactericidal effect of the recombinant alginate lyase-assisted phage in Example 4 of the present invention. Detailed Implementation

[0025] The present invention will now be described in more detail with reference to specific embodiments. It should be noted that the host strain, *Pseudomonas aeruginosa* strain 240731-02, is deposited at the China General Microbiological Culture Collection Center (CGMCC), accession number: CGMCC No. 36713; deposit date: November 13, 2025; the *Pseudomonas aeruginosa* standard strain PAO1 was donated by Ocean University of China.

[0026] It should be noted that the myxotropic Pseudomonas aeruginosa phage in this invention was obtained by screening based on recombinant alginate lyase (amino acid sequence as shown in SEQ ID NO.2), named phage P1030, and deposited at the China General Microbiological Culture Collection Center (CGMCC); accession number: CGMCC No.46655; accession date: November 13, 2025.

[0027] Example 1: Expression, purification, and enzyme activity verification of the recombinant alginate lyase described in this invention. I. In this embodiment, the alginate lyase gene in the genomic DNA of the Pseudomonas aeruginosa standard strain PAO1 is used. algL The engineered bacteria used to construct and express recombinant alginate lyase (amino acid sequence as shown in SEQ ID NO.2) for the target gene are detailed below: The first step is to construct recombinant plasmids. S11, design primer pairs for amplification of the alginate lyase gene. algL Gene fragments were amplified by PCR, and the PCR amplification products were verified by agarose gel electrophoresis (results are shown in...). Figure 1 The DNA sequence of the PCR amplification product is shown in SEQ ID NO.1, and its size is 1146 bp; In this primer pair design, two restriction enzyme sites, EcoRI and XhoRI, were selected. Homologous recombination was also chosen to ligate the linearized plasmid to the target gene. Therefore, the two ends of the primers were designed as two homologous arms complementary to the restriction enzyme sites on the plasmid. After the design was completed, Beijing Ruiboxingke Biotechnology Co., Ltd. synthesized the primers. The upstream primer is shown in SEQ ID NO.4, and the downstream primer is shown in SEQ ID NO.5, as detailed below: algL F: ATGGGTCGCGGATCCGAATTCATGAAAACGTCCCACCTGATCC algL R: GTGGTGGTGGTGGTGCTCGAGTCAACTTCCCCCTTCCGG; S12, using pET28a(+) carrying the kanamycin resistance gene (with a His-tag) as the plasmid, double digestion with EcoRI and XhoI was performed to obtain a linearized plasmid, which was verified by agarose gel electrophoresis (results are shown in Figure 12). Figure 2 ); S13, the amplification product was directionally inserted into a linearized plasmid using homologous recombination to construct a recombinant plasmid, which was then sequenced for verification. The gene map of the recombinant plasmid is shown below. Figure 3 As shown; The second step involves transforming the correctly sequenced recombinant plasmid into E. coli DH5α for amplification and preservation. Then, the recombinant plasmid is extracted from E. coli DH5α and transformed into BL21(DE3) strain for IPTG (final concentration 0.4 mM) induction expression at 16℃ and 150 rpm for 24 h. After induction, the plasmid is disrupted by sonication (or high-pressure homogenization), centrifuged, and the supernatant and precipitate are obtained and electrophoresed separately. The third step involved purifying the supernatant from the second step using nickel ion affinity chromatography. The elution buffers used in the purification process are shown in Table 1. The elution buffers were collected and dialyzed. During dialysis, 5 mM reduced glutathione (GSH) and 1 mM oxidized glutathione (GSSG) were added to the dialysate to help the protein fold correctly. The purified recombinant alginate lyase was obtained after dialysis.

[0028] Table 1. Column chromatography eluent and elution buffer It should be noted that in this invention, the pET28a(+) plasmid with a His-tag is used as the plasmid during the construction of the recombinant plasmid. The target protein after induced expression is fused with the tag protein of the plasmid. This tag protein has 36 residues, specifically MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRGSEF. Therefore, the amino acid sequence of the target protein obtained by induced expression and nickel ion affinity chromatography in this invention has a total of 404 residues (as shown in SEQ ID NO.3), and its molecular weight corresponding to electrophoretic analysis is approximately 44 kDa. Therefore, this invention verifies the results by SDS-polyacrylamide gel electrophoresis on the samples processed in the above steps. Figure 4A distinct and specific protein band was observed at approximately 44 kDa, confirming the successful expression of the recombinant alginate lyase. Specifically, the expression level of the target protein (i.e., the recombinant alginate lyase) significantly increased after induction compared to before induction; subsequently, after Ni... 2+ After affinity chromatography purification, the protein bands became more uniform and the purity was higher.

[0029] II. Verification of Recombinant Alginate Lyase This invention further validates the purified recombinant alginate lyase using Western blot (WB) experiments (validation results are shown in [link to WB]). Figure 5 The results showed a clear target protein band at approximately 44 kDa, indicating that the present invention yielded the correct recombinant alginate lyase.

[0030] III. Enzymatic activity of recombinant alginate lyase 1% sodium alginate was prepared using PBS buffer as a substrate. Multiple portions of sodium alginate were divided into two groups. One group was supplemented with recombinant alginate lyase at a final concentration of 80 μg / mL, and the other group was supplemented with PBS buffer of the same volume as the enzyme as a negative control. The third group was PBS buffer as a blank control. The reaction was carried out at 40℃ for 20 min. After the reaction, 100 μL of sample solution was taken, 100 μL of 4% phenol and 500 μL of concentrated sulfuric acid were added, the mixture was vortexed for 10 s, and then 700 μL of pure water was added for dilution. The absorbance of the sample at 495 nm was measured. Figure 6 The results showed that the absorbance value of the experimental group was significantly higher than that of the negative control group (P<0.0001), indicating that the recombinant alginate lyase prepared in this invention has good alginate degradation activity.

[0031] Example 2: Biofilm removal capacity of the recombinant alginate lyase of the present invention The first step was to select a single colony of *Pseudomonas aeruginosa* strain 240731-02 (myxomorphic) and inoculate it into freshly prepared LB liquid medium. The culture was then incubated overnight at 37°C and 200 rpm. After the culture was completed, the turbidity of the bacterial culture was adjusted to 1.0 using LB liquid medium (measured using a McFarland turbidimeter). The second step involved dividing the newly prepared LB liquid medium into three groups. One group was treated with the recombinant alginate lyase purified in Example 1, serving as the experimental group. Another group was treated with an equal volume of PBS buffer, serving as the negative control group. The third group was treated with LB liquid medium, serving as the blank control group. The bacterial suspension with a turbidity of 1.0 obtained in the first step was added to the experimental and control groups, while the blank group was treated with LB medium (180 μL was added to each well using a whole-well filling method). The mixture was then incubated at 37°C for 24 hours. Third, after cultivation, discard the bacterial culture in each well, wash each well with PBS buffer (wash three times), and then add 200 μL of 1% crystal violet solution to each well. Incubate at room temperature for 30 min to stain. After staining, rinse off excess crystal violet solution with deionized water and dry the plate (dry at room temperature for 1-2 h or in a 50℃ oven for 30 min). After the wells are completely dry, add 200 μL of 30% glacial acetic acid solution to each well to dissolve the crystal violet bound to the biofilm, and shake at 100 rpm for 10 min at room temperature. Using 30% glacial acetic acid as a blank, measure the OD using a microplate reader. 550 Absorbance was used to quantify biofilms. Biofilm clearance rate = (average OD of control group biofilm - average OD of experimental group biofilm) / average OD of control group biofilm × 100%.

[0032] exist Figure 7 In the diagram, six wells in section 1 represent the staining results of the experimental group, six wells in section 2 represent the staining results of the negative control group, and six wells in section 3 represent the staining results of LB liquid medium. Figure 7 It can be seen that the color of the experimental group was significantly lighter than that of the negative control group, indicating that there was less biofilm in the experimental group. Figure 8 It can be seen that the recombinant enzyme has a biofilm removal rate of 59% against myxobolus aeruginosa, which is a significant removal effect, proving that the recombinant alginate lyase of the present invention can effectively degrade the biofilm of myxobolus aeruginosa.

[0033] Example 3: Method for isolating myxotropic Pseudomonas aeruginosa bacteriophages according to the present invention This invention uses the purified recombinant alginate lyase from Example 1 to screen bacteriophages. The enrichment and screening of bacteriophages uses *Pseudomonas aeruginosa* 240731-02 as the host cell. The purification, proliferation, and titer determination of the bacteriophages all use the *Pseudomonas aeruginosa* standard strain PAO1 as the host cell. The wastewater used in the enrichment process is from the Changping Wastewater Treatment Plant in Beijing, filtered, and then used. The bacterial solutions used in the cork plate spotting are all logarithmic-phase solutions, specifically including the following: 1. Enrichment of bacteriophages Recombinant alginate lyase was added to LB liquid medium to a final concentration of 80 mg / mL. 200 μL of *Pseudomonas aeruginosa* 240731-02 culture (cultured to the logarithmic phase, hereinafter the same) was inoculated into LB liquid medium containing recombinant alginate lyase (4 mL), and 1 mL of filtered wastewater was added. The medium was then incubated at 37°C for 6 h on a shaker to screen for bacteriophages (this group was the experimental group). In this example, *Pseudomonas aeruginosa* 240731-02 was inoculated into LB liquid medium without recombinant alginate lyase as a negative control group, and 1 mL of filtered wastewater was added. The medium was then incubated at 37°C on a shaker for 6 h. During the incubation, the liquid in the experimental group was clear, while the liquid in the negative control group without recombinant alginate lyase was turbid (see...). Figure 9 This indicates that adding the recombinant alginate lyase of the present invention helps to enrich bacteriophages.

[0034] 2. Phage screening After the phage enrichment culture was completed, the clarified liquid was filtered through a 0.22 μm filter membrane and further screened using the double-layer agar plate method (i.e., bacteria and phages were cultured in two layers of culture medium in a petri dish). In this embodiment, the lower layer of the double-layer agar plate is a solid LB plate; the upper soft plate includes 7 mL EP and 4 mL semi-solid LB medium. 200 μL of myxobolus aeruginosa bacterial suspension, 100 μL of treated clear liquid and recombinant alginate lyase with a final concentration of 80 μg / mL are added to the soft plate. The plate is mixed by inverting and poured onto the solidified solid LB plate. The plate is then left unsealed and incubated upside down at 37°C for 24 h after solidification. The negative control group for this step is as follows: The upper soft plate consists of 7 mL EP and 4 mL semi-solid LB medium. Add 200 μL of myxobolus aeruginosa bacterial culture, 100 μL of the treated clear liquid and PBS buffer (equal volume to the enzyme recombinant alginate lyase) to the soft plate, mix by inverting, pour it onto the solidified solid LB plate, open the lid and place it in the container. After solidification, invert it and incubate at 37°C for 24 h. Plate screening results of the phage enrichment clarification solution showed that clear phage plaques could be observed when recombinant alginate lyase was added to the upper plate; however, no phage plaques could be detected when PBS buffer was used instead of recombinant alginate lyase, demonstrating that recombinant alginate lyase significantly improved the isolation efficiency of mPA phage. 3. Perform continuous phage selection and EOP assay on the phages from step two to achieve phage isolation and purification. This specifically includes the following: S31, take 1 mL of physiological saline or PBS buffer, gently poke the phage plaque, insert it into the EP tube and pipette to obtain the phage stock solution; centrifuge the phage stock solution at 12000 rpm for 10 min; add physiological saline or PBS buffer to the 96-well plate, and perform serial dilutions on the centrifuged phage sample for later use; S32. The diluted phages were purified using the Pseudomonas aeruginosa standard strain PAO1. Purification was performed using the double-layer plate method, repeated 2-3 times until uniformly sized and transparent phage plaques appeared on the double-layer plates. The phages on these plates were then considered purified. Figure 10 The left image.

[0035] During the purification process, due to the different shapes and sizes of phage plaques, it is necessary to sample and purify phage plaques of different shapes separately in order to ensure that phages with good lysis effects can be obtained as much as possible in the future.

[0036] 4. Proliferation of bacteriophages The purified phage was cultured for proliferation: 200 μL of bacterial culture (PAO1 strain cultured to the logarithmic cycle) and 100 μL of phage solution were added to 4 mL of liquid LB medium and cultured in a shaker at 37℃ for 6 h.

[0037] 5. Phage titer The *Pseudomonas aeruginosa* standard strain PAO1 and the proliferated phage were subjected to double-layer agar plate speckle assays (three parallel groups) to determine the electrophoretic potency (EOP). The phage potency was calculated as: phage potency = number of plaques × dilution factor × 10. The results showed that the phages screened in this example had a propagation potency as high as 10. 10 PFU / mL.

[0038] The purified and isolated bacteriophage was named P1030 in this invention. Its transmission characterization results showed that the bacteriophage exhibited typical Myocaudae characteristics, with a head of approximately 78 nm and a tail of approximately 128 × 20 nm. See details... Figure 10 The image on the right.

[0039] 6. Sequencing analysis of the bacteriophage of this invention In this invention, the whole genome of bacterial cell P1030 was extracted using LEAGENE's λ phage genomic DNA extraction kit (PEG precipitation method) and then sequenced. The results showed that the whole genome sequence size of bacterial cell P1030 was 66,081 bp, and the GC content was 56%.

[0040] This invention uses the online software RAST (https: / / rast.nmpdr.org / ) and NCBI's BLASTp function (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) to predict and annotate open reading frames (ORFs) in bacteriophage genomes. The results show that the bacteriophage P1030 genome contains 94 ORFs, accounting for approximately 92.0% of the total genome length (66,081 bp). The maximum length of the ORF is 3108 bp, and the minimum length is 96 bp. These ORFs encode functional proteins related to bacteriophage P1030 and phage DNA replication, transcriptional regulation, structural protein assembly, host lysis, and nucleic acid metabolism, indicating that bacteriophage P1030 possesses typical genomic organization characteristics of lytic bacteriophages. Specifically, the ORFs of bacteriophage P1030 in this invention mainly include structural protein modules, replication and transcriptional regulation modules, lysis system modules, and functionally unknown protein modules. In the structural protein module, several typical phage structure-related proteins were identified and annotated, including ORF15 (terminase large subunit), ORF34 (head maturation protease), ORF36 (capsid protein), ORF30 and ORF31 (small capsid / head proteins), ORF10 (tail tape measure protein), ORF41 (tail completion / neck protein), ORF54 (substrate protein), and ORF43, ORF47, ORF48, ORF49, ORF55, and ORF57 (tail filament and substrate-related proteins). These proteins showed high sequence similarity to previously reported Pseudomonas aeruginosa phage structural proteins in the NCBI non-redundant (nr) protein database and the RefSeq protein database, indicating that phage P1030 has a complete and typical phage assembly system. In the lysis system module, ORF59 is annotated to encode endolysin, a protein that can specifically hydrolyze the peptidoglycan structure of the host cell wall. This protein is a key functional factor for lysis-type phages to complete host lysis and release progeny phages, indicating that the phage P1030 of this invention has the ability to complete its life cycle through the classical lysis pathway.In the replication and nucleic acid metabolism-related modules, multiple phage replication-related proteins were identified and annotated, including ORF66, ORF67, and ORF80 (DNA helicases), ORF68 (DNA polymerase IIIα subunit), ORF61 (DNA ligase), ORF69 (3′–5′ exonuclease), ORF85 and ORF87 (DNA primases / phage-associated primases), ORF70 (polynucleotide kinase / 3′ phosphatase), and ORF72 (thymidine nucleotide synthase ThyX), providing a complete molecular basis for phage genome replication and nucleotide metabolism. In summary, the genome of phage P1030 of this invention contains key functional modules such as structural proteins, replication regulation, and lysis systems. Its ORF composition and functional distribution characteristics are highly consistent with those of previously reported lysis phages, indicating that phage P1030 possesses typical genomic organization characteristics of lysis phages.

[0041] This invention uses the Proksee online tool (https: / / proksee.ca / ) to draw a phagocyte genome loop map, see [link to related documentation]. Figure 11 The results showed that the circular diagram clearly demonstrated the distribution of genes of phage P1030 on the positive and negative strands and its functional modularity. The overall arrangement was compact, and no obvious redundant sequences or large non-coding regions were found, which is consistent with the structural characteristics of a highly efficient lytic phage genome.

[0042] This invention performs phylogenetic analysis based on the whole genome sequence of bacteriophage P1030. NCBI's BLAST function is used to find sequences similar to the bacteriophage genome. A phylogenetic tree based on whole genome distance is constructed using the VICTOR platform. The phylogenetic tree is then beautified using the Chiplot online tool (https: / / www.chiplot.online / ). See below. Figure 12 The results showed that the bacteriophage P1030 of the present invention clustered into the same branch as several previously reported Pseudomonas aeruginosa bacteriophages, indicating a close evolutionary relationship. However, the genome sequences still showed certain differences, suggesting that the bacteriophage of the present invention is a novel bacteriophage with unique genetic characteristics.

[0043] Example 4: Application of the bacteriophage for lysing myxotropic Pseudomonas aeruginosa as described in this invention This invention uses *Pseudomonas aeruginosa* strain 240731-02 as the host strain and investigates the bacteriophage isolated in Example 3 of this invention against *Pseudomonas aeruginosa*. The specific details are as follows: Bacteriophages and host strains (Pseudomonas aeruginosa strain 240731-02) were spotted onto double-layer agar plates. The lower layer was LB fixation medium, and the upper layer was 5 mL of semi-solid LB medium and 200 μL of bacterial suspension of Pseudomonas aeruginosa strain 240731-02 (grown to the logarithmic phase). In the preparation of the upper culture medium, 1 mL of recombinant alginate lyase was added to the upper culture medium as the experimental group, and 1 mL of PBS buffer was added to the upper culture medium as the control group. The proliferated phage stock solution was diluted 10-fold sequentially, and the different concentrations of phage were spotted onto double-layer agar plates of the experimental and control groups. The plates were inverted and incubated overnight at 37°C. Results are shown below. Figure 13 ( Figure 13 In the diagram, 0 corresponds to the proliferated phage stock solution, -1 indicates that the phage stock solution has been diluted 10 times, and -2 indicates that the phage stock solution has been diluted 10 times. 2 This serial dilution was repeated until the phage stock solution was diluted to 10⁻⁶. 7 times).

[0044] Depend on Figure 13 It can be seen that only the phage stock solution in the control group showed a light-colored inhibition zone (i.e., Figure 13 (The position corresponding to 0 in the control group); after the addition of recombinant alginate lyase, the inhibition zone significantly expanded, and clear single phage plaques could be observed (see...). Figure 13 Experimental group), with phage diluted 10 -4 Plaques were still observed at higher dilutions, indicating that the phage of this invention still exhibits bactericidal effects against *Pseudomonas aeruginosa* at higher dilutions. The results show that the recombinant alginate lyase of this invention can significantly enhance the infectivity and lysis ability of the phage described in this invention against *Pseudomonas aeruginosa*.

[0045] In summary, the recombinant alginate lyase of this invention can effectively degrade the extracellular alginate matrix of *Pseudomonas aeruginosa* phages, significantly improving the enrichment and separation efficiency of the phages of this invention, thereby solving the technical problem of difficult isolation of existing *P. aeruginosa* phages. The phage P1030 obtained by screening based on the recombinant alginate lyase of this invention has high titer and good genetic stability, and exhibits significant lytic ability against *P. aeruginosa* with the assistance of alginate lyase, providing reliable technical support for the preparation of drugs (or biological agents) for treating infectious diseases caused by *P. aeruginosa*.

Claims

1. A recombinant alginate lyase, characterized in that: The amino acid sequence of the recombinant alginate lyase is shown in SEQ ID NO.2, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.

1.

2. A method for preparing the recombinant alginate lyase as described in claim 1, characterized in that: The preparation method is as follows: First, a recombinant plasmid expressing alginate lyase is constructed using recombinant homology. The correctly constructed recombinant plasmid is then transformed into E. coli DH5α for amplification, and then transformed into BL21(DE3) for induced expression to obtain crude enzyme solution. The crude enzyme solution is purified by column chromatography to obtain recombinant alginate lyase.

3. A method for isolating myxotropic Pseudomonas aeruginosa phages based on recombinant alginate lyase, characterized in that: The method is based on the recombinant alginate lyase described in claim 1 or the recombinant alginate lyase prepared in claim 2, and the specific steps are as follows: The first step was to inoculate the myxobolus aeruginosa strain into LB liquid medium and culture it overnight to obtain the seed culture of the myxobolus aeruginosa strain. The seed culture was inoculated into LB liquid medium supplemented with recombinant alginate lyase, and sewage was added to enrich the phages; phage plaques were obtained by separation using double-layer agar plates. The second step involved selecting consecutive plaques from the plaques isolated in the first step and determining the plaque formation rate of the phages to obtain the myxomorphic Pseudomonas aeruginosa phage P1030, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 46655.

4. The method for isolating myxotropic Pseudomonas aeruginosa bacteriophages according to claim 3, characterized in that: In the first step, the final concentration of recombinant alginate lyase in LB liquid medium and the final concentration of recombinant alginate lyase in the upper layer of the bilayer agar plate during phage enrichment are 80 mg / mL.

5. The method for isolating myxotropic Pseudomonas aeruginosa bacteriophages according to claim 3, characterized in that: The titer of the bacteriophage P1030 was 10. 7 -10 11 PFU / mL.

Images