Phage depolymerases Depo17 as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and discloses a bacteriophage depolymerases Depo17 and a preparation method and application thereof, the amino acid sequence of the bacteriophage depolymerases Depo17 is shown as SEQ ID No: 2, the bacteriophage depolymerases Depo17 can be used for preparing a preparation for enhancing the phagocytosis of macrophages on klebsiella pneumoniae, and a new strategy is provided for anti-infection treatment.

Description

Technical Field

[0001] This application relates to a phage depolymerase Depo17, its preparation method, and its application, belonging to the field of biomedical technology. Background Technology

[0002] Klebsiella pneumoniae is a common opportunistic pathogenic Gram-negative bacterium that can cause a variety of infectious diseases, including pneumonia, sepsis, urinary tract infections, and liver abscesses. With the widespread use of antibiotics such as carbapenems, the emergence and prevalence of multidrug-resistant Klebsiella pneumoniae strains carrying resistance genes have made clinical treatment more challenging.

[0003] Capsular polysaccharide (CPS) is a key virulence factor in Klebsiella pneumoniae, composed of repeating polysaccharide units that tightly coat the bacterial cell surface, forming a thick physical barrier. This structure plays a central role in bacterial pathogenicity and immune evasion: on the one hand, the capsule can inhibit the activation of the complement system and its deposition on the bacterial membrane surface, thereby resisting complement-mediated lysis; on the other hand, the capsule obscures pathogen-related molecular patterns, interfering with the recognition and uptake processes of phagocytes. Therefore, the capsule is not only a key determinant of the pathogenicity of Klebsiella pneumoniae, but also one of the important reasons why drug-resistant bacterial infections are difficult to eradicate.

[0004] Studies have shown that phage-derived depolymerases can effectively degrade capsules and biofilms in vitro, demonstrating the potential to weaken bacterial pathogenicity. However, current research on depolymerase function mainly focuses on in vitro degradation activity and molecular characterization. Existing research still lacks information on whether and how depolymerases affect the host immune system's clearance efficiency of bacteria after capsule degradation. This limitation prevents a comprehensive evaluation of the application value of depolymerases as an anti-infection strategy and hinders their further development towards clinical treatment. Summary of the Invention

[0005] The purpose of this application is to provide a phage depolymerase Depo17, its preparation method, and its application, so as to solve the technical problems of existing phage depolymerases in enhancing host immune clearance capabilities.

[0006] To achieve the above objectives, the technical solution adopted in this application is as follows:

[0007] In a first aspect, this application provides the use of phage depolymerase Depo17 in the preparation of a formulation for enhancing the phagocytic activity of macrophages against Klebsiella pneumoniae, wherein the amino acid sequence of phage depolymerase Depo17 is shown in SEQ ID No:2.

[0008] In conjunction with the first aspect, the phage depolymerase Depo17 is further encoded by a gene with a nucleotide sequence as shown in SEQ ID No:1.

[0009] Furthermore, the Klebsiella pneumoniae is capsular serotype KL64.

[0010] Furthermore, the formulation comprises phage depolymerase Depo17 and a pharmaceutically acceptable carrier.

[0011] Furthermore, the formulation is a pharmaceutical composition or an in vitro treatment agent.

[0012] Secondly, this application provides a method for preparing phage depolymerase Depo17, comprising the following steps: amplifying a gene encoding an amino acid sequence as shown in SEQ ID No:2; ligating the gene into an expression vector to construct a recombinant plasmid; transforming the recombinant plasmid into an expression host for induced expression; and collecting the induced bacterial culture.

[0013] The bacterial culture was purified by centrifugation to obtain the phage depolymerase Depo17.

[0014] In conjunction with the second aspect, the expression vector is pET28a, the expression host is Escherichia coli BL21(DE3), and the inducing agent for expression is IPTG.

[0015] Thirdly, this application provides a phage depolymerase Depo17, prepared according to the preparation method described in the second aspect.

[0016] Fourthly, this application provides an in vitro treatment agent for enhancing the phagocytic activity of macrophages against Klebsiella pneumoniae type KL64, comprising an effective amount of the phage depolymerase Depo17 according to the third aspect, wherein the effective amount is not less than 0.1 µg of phage depolymerase Depo17 per milliliter of the treatment agent.

[0017] Compared with the prior art, the beneficial effects achieved by this application are as follows:

[0018] This application discloses a Klebsiella pneumoniae phage P7247. The phage depolymerase Depo17 was obtained by prokaryotic expression and purification of the 17th open reading frame (ORF17) of Klebsiella pneumoniae phage P7247. This phage depolymerase Depo17 can specifically remove capsular polysaccharides and biofilms formed by Klebsiella pneumoniae type KL64, and has a significant inhibitory effect on biofilm formation. By weakening the bacterial surface defense barrier, phage depolymerase Depo17 enhances the phagocytic efficiency of host macrophages, and has good clinical application potential in the development of antibiotic alternatives. Attached Figure Description

[0019] Figure 1 Agarose gel electrophoresis image of the PCR product for identification of the recombinant expression strain BL21-pET28a-depo17 positive clone of Escherichia coli provided in the embodiments of this application;

[0020] Figure 2 This is an SDS-PAGE protein electrophoresis image of the purified phage depolymerase Depo17 provided in the embodiments of this application;

[0021] Figure 3 This is a graph showing the inhibitory effect of different concentrations of phage depolymerase Depo17 on KL64 type Klebsiella pneumoniae Kp7247 in the embodiments of this application;

[0022] Figure 4 These are transmission electron microscopy images of phage depolymerase Depo17 before and after treatment with Klebsiella pneumoniae Kp7247 in the embodiments of this application;

[0023] Figure 5 A quantitative analysis diagram showing the effect of phage depolymerase Depo17 on the capsular polysaccharide of Klebsiella pneumoniae Kp7247, based on uronic acid detection, provided in the embodiments of this application.

[0024] Figure 6 Statistical results on the effect of treatment with the phage depolymerase Depo17 provided in the embodiments of this application on the ability of RAW264.7 macrophages to phagocytose Klebsiella pneumoniae Kp7247. Detailed Implementation

[0025] The following embodiments are used to further illustrate this application, but the scope of protection of this application is not limited thereto. Unless otherwise specified, the experimental methods used in the following embodiments are conventional experimental methods; unless otherwise specified, the reagents and materials used are commercially available.

[0026] Example 1:

[0027] This embodiment describes the preparation of the phage depolymerase Depo17, and the steps are as follows:

[0028] (1) Target gene amplification and vector construction;

[0029] Based on the genome sequence of Klebsiella pneumoniae phage P7247, specific primers were designed targeting its 17th open reading frame (ORF17) to construct an expression vector.

[0030] The Klebsiella pneumoniae phage P7247 of this application has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20252697, classified as Przondovrus P7247, and deposited on November 27, 2025. The address of CCTCC is Wuhan University, Wuhan, China.

[0031] The upstream primer F (SEQ ID No:3) is:

[0032] 5'-CGGCATATGATGGACCAAGATACTAAAACAATTATCCAATACC-3';

[0033] The downstream primer R (SEQ ID No:4) is:

[0034] 5'-CGACTCGAGTTAGGCGTTTAGGTAAACACCGGTA-3'.

[0035] The PCR amplification program was as follows: pre-denaturation at 95℃ for 5 minutes; 30 cycles were performed: denaturation at 95℃ for 30 seconds, annealing at 58℃ for 30 seconds, extension at 72℃ for 2 minutes and 40 seconds; after the cycle, extension at 72℃ for 7 minutes was performed to obtain the PCR product.

[0036] The obtained PCR product was digested and purified by NdeI and XhoI restriction endonucleases, and then ligated into the pET28a plasmid vector that had been digested with the same enzymes to construct the recombinant plasmid pET28a-depo17.

[0037] (2) Construction and screening of recombinant strains;

[0038] The recombinant plasmid pET28a-depo17 was transformed into Escherichia coli BL21(DE3) competent cells. The specific steps were as follows:

[0039] Take 100 μL of competent cells, add plasmid, and incubate on ice for 30 minutes. After heat shock in a 42°C metal bath for 90 seconds, quickly place on ice and incubate for 2 minutes.

[0040] Add 900 μL of LB medium, mix well, and incubate at 37°C with shaking at 180 rpm for 1 hour. After incubation, centrifuge at 5000×g for 3 minutes, discard the supernatant, resuspend the bacterial cells in the remaining medium, and spread evenly on LB solid medium plates containing 50 μg / mL kanamycin. Incubate the plates overnight at 37°C.

[0041] Single colonies were selected and colony PCR was performed using universal T7 primers. Following this, BL21-pET28a-depo17 positive Escherichia coli clones were obtained by agarose gel electrophoresis. The agarose gel electrophoresis image is shown below. Figure 1 As shown, lane M is the DNA molecular weight standard, and lanes 1, 2, and 3 are three randomly selected single colony PCR products. The target bands of the expected size are visible, indicating that the positive recombinant strain BL21-pET28a-depo17 has been successfully obtained.

[0042] (3) Induced expression of protein;

[0043] The above-mentioned positive recombinant strain BL21-pET28a-depo17 was inoculated into 1L of LB liquid medium containing 50μg / mL kanamycin and cultured at 37℃ with shaking until the logarithmic growth phase.

[0044] Isopropyl-β-D-thiogalactoside (IPTG) was added to the culture medium to a final concentration of 0.2 mM, and the culture was induced by shaking at 16 °C and 180 rpm for 6 hours.

[0045] (4) Protein purification;

[0046] Collect the induced bacterial culture into a centrifuge tube, centrifuge at 4000 rpm for 15 minutes in a pre-cooled centrifuge at 4℃, discard the supernatant, and collect the bacterial cells.

[0047] The collected bacterial cells were resuspended in 30 mL of lysis buffer, and 300 µL of benzyl sulfonyl fluoride, 75 µL of lysozyme, 30 µL of DNase I, and 75 µL of MgCl2 were added. The mixture was stirred on a magnetic stirrer for 5 minutes. Subsequently, the bacterial cells were sonicated under ice bath conditions for 6 seconds followed by a 4-second pause, for a total sonication time of 10 minutes. The beaker was then removed and stirred on a magnetic stirrer for 5 minutes. This sonication process was repeated four times. The cells were centrifuged, and the supernatant was collected as a sample.

[0048] The supernatant sample was loaded onto a pre-equilibrated Ni-NTA affinity chromatography column. After the supernatant had fully bonded to the Ni column, the nickel strand was washed sequentially with lysis buffer, high-salt buffer, and low-imidazole wash buffer, for at least two column volumes at each step. The eluent from each wash step was collected. Finally, the target protein was eluted with two column volumes of elution buffer containing 250 mM imidazole.

[0049] The eluent was collected and subjected to ion affinity chromatography and ultrafiltration to obtain purified phage depolymerase Depo17, the nucleotide sequence of which is shown in SEQ ID No: 1; the amino acid sequence of which is shown in SEQ ID No: 2. The concentration of the purified phage depolymerase Depo17 was determined using a BCA protein quantification kit, and the concentration was 1.2 mg / mL. The purified phage depolymerase was aliquoted and stored at -80°C.

[0050] (5) Protein purity identification;

[0051] The purity and molecular weight of the purified protein were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

[0052] Prepare a 10% PAGE gel according to the instructions of the PAGE gel rapid preparation kit.

[0053] The effluent sample collected in step 4 of sample purification was boiled for 10 minutes, then loaded onto the plate and electrophoresed at 150V for 1 hour. Coomassie brilliant blue staining was performed, followed by destaining and observation. Figure 2 As shown, lane M is the protein molecular weight standard, lane 1 is the lysed whole bacterial sample, lane 2 is the supernatant of the lysed sample, lane 3 is the flow-through buffer, lane 4 is the lysis buffer used to wash the sample, lane 5 is the high-salt buffer used to wash the sample, lane 6 is the low-imidazolium buffer used to wash the sample, and lane 7 is the elution buffer. A clear main band is visible at approximately 70-100 kDa, indicating that high-purity Depo17 protein was successfully obtained.

[0054] Example 2:

[0055] This embodiment utilizes the phage depolymerase Depo17 to degrade the capsular polysaccharide of KL64 Klebsiella pneumoniae. A dot assay was used to detect the degradation activity of phage depolymerase Depo17 on the KL64 Klebsiella pneumoniae capsule, as follows:

[0056] 100 μL of 0.5% semi-solid LB medium containing logarithmic-phase bacteria was evenly spread on solid LB medium to form a bacterial lawn, and then allowed to air dry. 10 μL of protein dilutions containing different concentrations of phage depolymerase Depo17 (10 µg, 5 µg, 2 µg, 1 µg, 0.5 µg, 0.2 µg, 0.1 µg, 0.01 µg) were spotted onto the bilayer medium, with an equal volume of PBS as a negative control. The mixture was incubated overnight at 37°C. Results are shown below. Figure 3 As shown, all samples with different amounts of phage depolymerase Depo17 formed translucent spots on the bacterial colony, while the PBS control did not exhibit this phenomenon, indicating that phage depolymerase Depo17 can effectively degrade the bacterial capsule.

[0057] Kp7247 bacterial culture cultured overnight was washed with PBS. Then, 2 µg of purified phage depolymerase Depo17 was added to the treatment group, while the control group received no treatment. The mixture was incubated at 37°C for 2 hours. After washing the bacterial cells three times with PBS, 2 mL of 1.5% glutaraldehyde was added, and the mixture was incubated overnight at 4°C. Transmission electron microscopy was then performed, and the results are shown below. Figure 4 As shown, Figure 4 In Figure a, the treatment group shows that the capsule layer around the bacterial cells is significantly thinned or even disappears; in Figure b, the control group shows that the bacterial cells are surrounded by an intact and thick capsule. This result directly confirms the capsule degradation function of the phage depolymerase Depo17.

[0058] The Kp7247 bacterial culture culture incubated overnight was adjusted to OD using PBS. 600 For a concentration of 1.0, take 500 µL of the solution and add 2 µg of purified phage depolymerase Depo17 to the treatment group. Add an equal volume of PBS to the control group and incubate at 37 °C for 2 hours. After centrifugation to remove the supernatant, resuspend the cells in 500 µL of PBS, add 100 µL of 1% amphoteric detergent 3-14, mix, and incubate at 50 °C for 30 minutes. Centrifuge at 14000 rpm for 2 minutes, transfer 250 µL of the supernatant to a new 1.5 mL centrifuge tube, add 1 mL of pre-chilled anhydrous ethanol, and incubate at 4 °C for 30 minutes to form a precipitate. Centrifuge at 14000 rpm for 5 minutes, discard the supernatant, and allow the solution to dry completely. Dissolve the precipitate in 200 µL of deionized water. Then add 1.2 mL of sodium tetraborate / concentrated sulfuric acid solution to the extract and vortex to mix. Boil for 5 minutes and then cool to room temperature. Add 20 μL of 3-hydroxydiphenol and measure its absorbance at 520 nm after the reaction.

[0059] Simultaneously, solutions of glucuronic acid at different concentrations were prepared for standard curve determination. Based on the standard curves, the uronic acid content in different treatment groups was calculated. The final results are as follows: Figure 5 As shown, compared with the PBS control group, the uronic acid content of the bacterial cells treated with phage depolymerase Depo17 was significantly reduced, which statistically and quantitatively confirmed the degradation effect of phage depolymerase Depo17 on capsular polysaccharides.

[0060] Example 3:

[0061] This embodiment verifies whether treatment with the phage depolymerase Depo17 can enhance the phagocytic clearance ability of macrophages against Klebsiella pneumoniae.

[0062] (1) Cell culture

[0063] Mouse macrophage line RAW264.7 was cultured in DMEM medium containing 10% heat-inactivated fetal bovine serum, 100 U / mL penicillin and 100 μg / mL streptomycin in a 37°C, 5% CO2 incubator.

[0064] (2) Bacterial preparation

[0065] Klebsiella pneumoniae strain Kp7247 was cultured at 37°C with shaking until mid-log phase, approximately 4 hours. The bacterial concentration was adjusted to OD500 using PBS. 600 Approximately 1.0, 1 mL of bacterial culture was added to phage depolymerase Depo17 to a final concentration of 10 μg / mL, and incubated at 37°C for 2 h. The treated bacterial culture was then washed with PBS and resuspended in DMEM medium; the PBS-treated group served as a negative control.

[0066] (3) Macrophage invasion

[0067] The inoculum concentration in the six-well plate was 1 × 10⁻⁶. 6 RAW264.7 macrophages were cultured at a density of cells / mL to form a monolayer. Kp7247 bacteria, pretreated with phage depolymerase Depo17 or PBS, were added to the corresponding wells at a bacterial-to-cell ratio of 10:1 and cultured at 37°C for 2 h.

[0068] (4) Lysis of cells and colony count

[0069] After culture, the plates were washed three times with PBS. Then, DMEM medium containing 100 μg / mL apramycin was added to each well, and the plates were incubated at 37°C for 1 h to remove unphagocytosed bacteria. The plates were washed three more times with PBS, and 1 mL of PBS solution containing 0.5% Triton X-100 was added to each well to lyse the cells, with repeated pipetting to ensure complete lysis. The lysates were serially diluted tenfold and spotted onto LB agar plates. After incubation, the colony count was performed to calculate the phagocytosis rate.

[0070] The results are as follows Figure 6 As shown, the number of bacterial colonies phagocytosed by RAW264.7 macrophages after pretreatment with phage depolymerase Depo17 was significantly higher than that in the PBS-treated group, indicating that phage depolymerase Depo17 can effectively enhance the macrophages' ability to recognize, phagocytose, and clear Klebsiella pneumoniae by degrading the capsule.

[0071] The phage depolymerase Depo17 provided in this application can specifically degrade the capsular polysaccharide of Klebsiella pneumoniae type KL64 and significantly improve the phagocytic efficiency of macrophages. It makes up for the shortcomings of existing technologies with unclear action spectrum or limited effect, and provides a more targeted solution for the prevention and control of drug-resistant bacterial infections. It has outstanding innovation and application value.

[0072] The above description is only a preferred embodiment of this application. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of this application, and these improvements and modifications should also be considered within the scope of protection of this application.

Claims

1. Use of a bacteriophage depolymerase Depo 17 for the preparation of a preparation for enhancing the phagocytosis of Klebsiella pneumoniae by macrophages, characterized in that, The amino acid sequence of the phage depolymerase Depo17 is shown as SEQ ID No:

2.

2. Use according to claim 1, characterized in that, The phage depolymerase Depo17 is encoded by a gene with a nucleotide sequence shown as SEQ ID No:

1.

3. Use according to claim 1, characterized in that, The Klebsiella pneumoniae is a capsular serotype KL64.

4. Use according to claim 1, characterized in that, The preparation comprises the phage depolymerase Depo17 and a pharmaceutically acceptable carrier.

5. The use according to claim 1, characterized in that, The preparation is a pharmaceutical composition or an in vitro treatment agent.

6. A method for the production of the bacteriophage depolymerase Depo 17, characterized by, The method comprises the following steps: amplifying a gene encoding an amino acid sequence shown as SEQ ID No: 2; linking the gene to an expression vector to construct a recombinant plasmid; transferring the recombinant plasmid into an expression host for induced expression, and collecting the bacterial liquid of induced expression; centrifuging and purifying the bacterial liquid to obtain the phage depolymerase Depo17.

7. The production method according to claim 6, wherein The expression vector is pET28a, the expression host is Escherichia coli BL21(DE3), and the inducer for induced expression is IPTG.

8. A bacteriophage depolymerase Depo 17, characterized in that, The preparation method is prepared according to claim 6 or 7.

9. An in vitro treatment agent for enhancing the phagocytosis of KL64 type Klebsiella pneumoniae by macrophages, characterized by, The phage depolymerase Depo17 of claim 8 is contained in an effective amount, and the effective amount is not less than 0.1 µg of the phage depolymerase Depo17 per milliliter of the treatment agent.

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