Use of bacteriophage depolymerase dep253 in degrading bacterial capsular polysaccharides

Abstract

The present invention belongs to the field of bacteriophage technology, and specifically relates to a use of a bacteriophage depolymerase dep253 in degrading bacterial capsular polysaccharides. The amino acid sequence of the bacteriophage depolymerase dep253 is shown in SEQ ID NO.1. The bacteriophage depolymerase dep253 is prepared from a Klebsiella pneumoniae phage P253 strain; the Klebsiella pneumoniae phage P253 strain has a deposit number of: CCTCC NO: M20241683, a deposit date of: July 25, 2024, a deposit unit of: China Center for Type Culture Collection, and a deposit address of: Wuhan, China, Wuhan University. The bacteriophage depolymerase dep253 provided by the present invention is a K5 serotype-specific depolymerase, which can be used to specifically degrade K5 serotype Klebsiella pneumoniae capsular polysaccharides, and can be used to inhibit the formation of K5 serotype Klebsiella pneumoniae biofilms, thereby playing the role of preventing and treating diseases caused by Klebsiella pneumoniae.

Description

Technical Field

[0001] The invention belongs to the technical field of bacteriophages, and in particular relates to use of bacteriophage depolymerase dep253 in degrading bacterial capsular polysaccharides. Background Art

[0002] Klebsiella pneumoniae is a common zoonotic pathogen. In recent years, multidrug-resistant Klebsiella pneumoniae has become one of the most important pathogens of nosocomial infections. The prevention and control of this pathogen mainly relies on antibiotics. However, the widespread use of antibiotics has also brought some negative effects: first, long-term and large-scale use of antibiotics will lead to increased bacterial resistance, that is, some bacteria are no longer sensitive to antibiotics. Secondly, the abuse of antibiotics will also cause food safety problems. Antibiotic residues in poultry products may affect the health of consumers. Bacteria resistant to antibiotics may be transmitted to humans through the food chain, increasing health risks.

[0003] Most Klebsiella pneumoniae have the ability to synthesize and secrete capsular polysaccharides. Capsular polysaccharides, as a natural barrier for bacteria, can maintain bacterial virulence, adhesion, and block the penetration of some antibiotics. They are important virulence factors of Klebsiella pneumoniae. In addition, Klebsiella pneumoniae can form biofilms, which are membrane-like structures formed by bacterial cells wrapped in extracellular polysaccharide matrix, lipoproteins, fibrin, etc. produced by the bacteria themselves. They can significantly increase the bacteria's resistance to antibiotics and their ability to evade host immune system recognition, thereby accelerating bacterial colonization in lesions. Therefore, biofilms are one of the main pathogenic factors of persistent bacterial infections in hospitals.

[0004] Phage depolymerase degrades bacterial surface polysaccharides, thereby guiding phages to adsorb to host outer membrane proteins. The enzyme randomly attacks glycosidic bonds to release the repeating units of the polymer, thereby achieving targeted degradation of capsular polysaccharides. Many domestic and foreign studies have shown that phage depolymerase can effectively remove and inhibit the formation of biofilms, and has certain application potential in the field of controlling pathogenic infections. However, phage depolymerases from different sources, different nucleic acid sequences, and different protein structures have very different degradation performances on different types of capsular polysaccharides and biofilms. Although depolymerases for the capsule of Klebsiella pneumoniae serotype K5 have been reported, the product use of phage depolymerases in degrading bacterial capsular polysaccharides has not yet been found. Summary of the invention

[0005] In order to solve the problem that there is no product use of phage depolymerase in degrading bacterial capsular polysaccharides in the prior art, the present invention provides a use of phage depolymerase dep253 in degrading bacterial capsular polysaccharides. The present invention performs whole genome sequencing and annotation on the Klebsiella pneumoniae phage isolated from nature, and uses a prokaryotic expression platform to screen out phage depolymerases with serotype specificity from phage sources, treat diseases caused by Klebsiella pneumoniae, reduce economic losses, and protect the health of humans and animals.

[0006] To achieve the above purpose, the present invention adopts the following technical solution.

[0007] Klebsiella pneumoniae is a Gram-negative bacillus with many serotypes. Serotypes are classified based on the differences in the capsular polysaccharide antigens of bacteria. Currently, there are more than 80 known capsular serotypes of Klebsiella pneumoniae. A notable feature of this bacterium is that the bacteria are wrapped in thick capsular polysaccharides and are prone to form biofilms. Based on this, the present invention provides a use of a bacteriophage depolymerase dep253 in degrading bacterial capsular polysaccharides. A bacteriophage depolymerase dep253 is a K5 serotype-specific depolymerase, which can be used to specifically degrade K5 serotype Klebsiella pneumoniae capsular polysaccharides, inhibit the formation of K5 serotype Klebsiella pneumoniae biofilms, and thus prevent and treat diseases caused by Klebsiella pneumoniae.

[0008] A Klebsiella pneumoniae phage P253 strain, the Klebsiella pneumoniae phage P253 strain has a deposit number of CCTCC NO: M 20241683, a deposit time of July 25, 2024, a deposit unit of China Center for Type Culture Collection, and a deposit address of Wuhan University, Wuhan, China.

[0009] The phage strain P253 uses the K5 serotype Klebsiella pneumoniae strain KP181 as the host bacteria and is isolated from the medical sewage of Zhangjiagang City Hospital by the double-layer plate method. Subsequent evaluation of the lysis ability of the phage strain P253 on 46 strains of Klebsiella pneumoniae found that the phage strain P253 only lyses the K5 serotype Klebsiella pneumoniae strain. The plaques of the phage strain P253 are surrounded by a layer of turbid halo, indicating that the phage strain P253 encodes a depolymerase.

[0010] Bacteriophage is a virus that can infect bacteria and can specifically infect and kill bacteria. Bacteriophage-derived depolymerase is a class of enzymes encoded by bacteriophage that can degrade bacterial extracellular polysaccharides, which helps to enhance the antibacterial effect of antibiotics or bacteriophages.

[0011] Phage therapy has some significant advantages over antibiotics, especially when facing resistant bacteria and treating specific types of infections. Highly specific: Phages are able to infect and kill specific bacteria with high specificity, without affecting the body's normal microbiome. This is in contrast to the broad-spectrum bactericidal effect of antibiotics, which can more precisely target pathogenic bacteria and reduce the impact on the body's beneficial flora. Low toxicity and side effects: Phages act on specific bacteria and are harmless to human cells. Therefore, compared with antibiotics, phages have fewer side effects. This makes phages safer to use during treatment, especially for patients who have been unable to be treated with traditional antibiotics due to drug resistance issues. Natural selection advantage: Phages are able to naturally select their targets based on the type of bacteria infecting, which makes them more effective during treatment. In contrast, although the broad-spectrum bactericidal effect of antibiotics is powerful, it is also easy to cause disturbances in the microbiome and the emergence of resistant strains. Environmentally friendly: Phages are naturally occurring viruses, and their therapeutic use does not have a negative impact on the environment. In contrast, the widespread use of antibiotics has been shown to have potential impacts on environmental microbial communities and ecosystems. Based on this, the present invention provides a use of a bacteriophage depolymerase dep253 in degrading bacterial capsular polysaccharides, and the amino acid sequence of the bacteriophage depolymerase dep253 is shown in SEQ ID NO.1. The amino acid sequence of the bacteriophage depolymerase dep253 involved in the present invention has only about 30% homology with the amino acid sequence of the depolymerase reported previously. In addition, the previously reported K5 serotype-specific depolymerase did not mention the inhibitory effect of biofilm formation, and the bacteriophage depolymerase dep253 mentioned in the present invention can significantly inhibit the biofilm formation of K5 type Klebsiella pneumoniae strains.

[0012] Preferably, the bacteriophage depolymerase dep253 is prepared from a Klebsiella pneumoniae phage P253 strain.

[0013] The preservation number of the Klebsiella pneumoniae phage P253 strain is: CCTCC NO: M20241683, the preservation time is July 25, 2024, the preservation unit is: China Center for Type Culture Collection, and the preservation address is: Room 211, China Center for Type Culture Collection, Wuhan University, Wuchang District, Wuhan City, Hubei Province (opposite the First Affiliated Primary School of Wuhan University).

[0014] Preferably, the nucleotide sequence encoding the bacteriophage depolymerase dep253 is shown as SEQ ID NO.2.

[0015] Preferably, the bacteriophage depolymerase dep253 is the depolymerase encoded by the 17th open reading frame of the Klebsiella pneumoniae phage P253 strain.

[0016] Preferably, the method for preparing the bacteriophage depolymerase dep253 comprises the following steps:

[0017] The dep253 gene fragment of the 17th open reading frame of the Klebsiella pneumoniae phage P253 strain is obtained by PCR, double enzyme digestion, and ligation molecular cloning, and the dep253 gene fragment is connected to a plasmid vector to obtain a recombinant plasmid.

[0018] The recombinant plasmid is transferred into a host cell, and the recombinant host cell containing the recombinant plasmid is obtained by screening.

[0019] The recombinant host cell is cultured to obtain a culture fluid, and the liquid is collected after solid-liquid separation of the culture fluid to obtain the bacteriophage depolymerase dep253.

[0020] Preferably, the plasmid vector comprises pET28a. The pET-28a vector carries an N-terminal His / Thrombin / T7 protein tag and also contains an optional C-terminal His tag.

[0021] Preferably, the host cell is Escherichia coli DE3. Escherichia coli DE3 is suitable for the T7 promoter-driven recombinant protein inducible expression system, such as the common prokaryotic expression vector pET series. This strain can effectively avoid the degradation of recombinant expression proteins and is widely used in the expression of recombinant proteins.

[0022] Preferably, the bacteriophage depolymerase dep253 is used to degrade Klebsiella pneumoniae capsular polysaccharide and biofilm. The typical feature of Klebsiella pneumoniae strains is that the bacteria are wrapped in a thick capsule and are prone to form biofilm.

[0023] Preferably, the bacteriophage depolymerase dep253 is used to prepare a bacteriophage disinfection product, wherein the bacteriophage depolymerase dep253 is the only active ingredient.

[0024] Preferably, the bacteriophage disinfection product is made of the bacteriophage depolymerase dep253 and a solvent.

[0025] The concentration of the bacteriophage depolymerase dep253 is 0.002 mg / mL to 2 mg / mL.

[0026] Preferably, the solvent is PBS; the concentration of PBS is 0.01 mol / L; and the pH is 7.2 to 7.4. The full name of PBS in English is phosphate buffer saline, and its corresponding Chinese name is phosphate buffered saline.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] 1. The present invention provides the use of phage depolymerase dep253 in degrading bacterial capsular polysaccharides. The phage depolymerase dep253 provided by the present invention is a K5 serotype-specific depolymerase, which can be prepared from a Klebsiella pneumoniae phage P253 strain. The Klebsiella pneumoniae phage P253 strain has a strong specific lysis ability against K5 serotype Klebsiella pneumoniae, and can be used alone or in combination with other substances to provide a safe and non-toxic phage disinfection product for disinfecting and purifying the environment.

[0029] 2. The present invention also provides a depolymerase dep253 encoded by the Klebsiella pneumoniae phage P253 strain, which can specifically degrade the capsular polysaccharide of Klebsiella pneumoniae serotype K5, and can also significantly inhibit the formation of biofilm of K5K5 serotype Klebsiella pneumoniae, which is beneficial to the removal of K5 serotype Klebsiella pneumoniae.

[0030] 3. The present invention performs whole genome sequencing and annotation on the Klebsiella pneumoniae phage isolated from nature, and uses a prokaryotic expression platform to screen out phage-derived serotype-specific phage depolymerases to treat diseases caused by Klebsiella pneumoniae, reduce economic losses, and protect the health of humans and animals. Among them, the key technical scheme for obtaining K5 serotype-specific depolymerases through whole genome sequencing annotation and cloning expression is as follows:

[0031] (1) Genome sequencing: For various types of bacteria already existing and newly isolated in the laboratory, representative strains are selected based on pathogenicity for whole genome sequencing.

[0032] (2) Genome assembly: After obtaining the offline data, unicycler is mainly used for assembly. After writing the script by yourself, high-throughput automated assembly of the sequenced strains can be performed.

[0033] (3) Genome annotation: RAST was used to annotate and analyze the genome to obtain genes that may encode K5 depolymerase.

[0034] (4) Cloning and expression of depolymerase: The depolymerase protein was cloned and expressed through a prokaryotic expression system, and its in vitro activity was verified. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The plaque morphology of the Klebsiella pneumoniae phage P253 strain in the present invention at 24 hours, 48 ​​hours and 60 hours; wherein, Figure 1 Figure A shows the plaque morphology of Klebsiella pneumoniae phage P253 strain at 24 hours; Figure 1 Figure B shows the plaque morphology of Klebsiella pneumoniae phage P253 strain at 48 hours; Figure 1 Figure C shows the plaque morphology of Klebsiella pneumoniae phage P253 strain at 60 hours.

[0036] Figure 2 The electron microscopic morphology of the Klebsiella pneumoniae phage P253 strain in the present invention.

[0037] Figure 3 Agarose gel electrophoresis is used to screen the positive prokaryotic expression strains of bacteriophage depolymerase dep253 in the present invention.

[0038] Figure 4 The 24-hour, 48-hour and 60-hour inhibition effect diagram of the bacteriophage depolymerase dep253 of the present invention on the K5 type Klebsiella pneumoniae KP181 bacterial lawn; wherein, Figure 4 Figure A shows the inhibitory effect of bacteriophage depolymerase dep253 on K5 type Klebsiella pneumoniae KP181 bacterial lawn for 24 hours; Figure 4 Figure B shows the inhibitory effect of bacteriophage depolymerase dep253 on K5 type Klebsiella pneumoniae KP181 bacterial lawn for 48 hours; Figure 4 Figure C shows the 60-hour inhibitory effect of bacteriophage depolymerase dep253 on K5 type Klebsiella pneumoniae KP181 bacterial lawn.

[0039] Figure 5 The colony morphology of Klebsiella pneumoniae KP181 cultured overnight on a sheep blood agar plate in the present invention.

[0040] Figure 6 This is a diagram showing the effect of the bacteriophage depolymerase dep253 on K5 type Klebsiella pneumoniae KP181 in the present invention; wherein, Figure 6 Figure A shows the effect of bacteriophage depolymerase dep253 on K5 type Klebsiella pneumoniae KP 181; Figure 6 Figure B shows the effect of PBS on K5 type Klebsiella pneumoniae KP181.

[0041] Figure 7 The present invention shows that the bacteriophage depolymerase dep253 has an inhibitory effect on the biofilm formation of K5 serotype Klebsiella pneumoniae KP181. DETAILED DESCRIPTION

[0042] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0043] The materials and methods used in the following examples are as follows:

[0044] 1.1 Main reagents and instruments:

[0045] SM buffer was purchased from Regeneron Biotechnology.

[0046] The transmission electron microscope was Hitachi H-7650.

[0047] DNase and RNase were purchased from Abcam Biotechnology Co., Ltd.

[0048] PBS was purchased from Shanghai Double Helix Biotechnology Co., Ltd. with a concentration of 0.01 mol / L and a pH of 7.4.

[0049] The λ phage genomic DNA extraction kit was purchased from Abcam Biotechnology Co., Ltd.

[0050] Crystal violet was purchased from Zhuhai Beso Biotechnology Co., Ltd.

[0051] 1.2 Plasmid and bacterial samples:

[0052] The source of pET28a plasmid was Beijing Qingke Biotechnology Co., Ltd.

[0053] Klebsiella pneumoniae was derived from clinical strains collected by Jiangsu Provincial Center for Disease Control and Prevention.

[0054] Escherichia coli DE3 was obtained from Beijing Qingke Biotechnology Co., Ltd.

[0055] 1.3 Materials:

[0056] The sewage samples used in the present invention were taken from Zhangjiagang Municipal Hospital.

[0057] Klebsiella pneumoniae was derived from clinical strains collected by Jiangsu Provincial Center for Disease Control and Prevention.

[0058] Example 1: Isolation and purification of bacteriophage

[0059] A Klebsiella pneumoniae phage P253 strain, the Klebsiella pneumoniae phage P253 strain is deposited in the China Center for Type Culture Collection, the deposit number is CCTCC NO: M 20241683, and the deposit time is July 25, 2024. The 17th open reading frame of the genome of the Klebsiella pneumoniae phage P253 strain encodes a depolymerase.

[0060] The separation and purification method of bacteriophage strain P253 comprises the following specific steps:

[0061] After centrifuging 10mL of sewage sample at 5000g for 5 minutes, filter the supernatant with a 0.22μm filter membrane and collect the filtrate. Take 100μL of sewage sample filtrate and 100μL of Klebsiella pneumoniae strain KP181 to LB semi-solid and mix well, then pour into LB solid plate. After the plate solidifies, invert it and place it in a 37℃ incubator for overnight culture. Purify the phage plaques until a single plaque morphology appears on the plate. Add 5mL of SM buffer to the plate and shake it at 60rpm for 4 hours. After the shaking is completed, collect the liquid and centrifuge it at 5000×g for 5min, and collect the supernatant after centrifugation. The supernatant is filtered with a 0.22μm filter membrane, and the purified phage is stored in SM buffer at 4℃ to obtain the enriched phage strain P253.

[0062] Klebsiella pneumoniae strain KP181 was cultured in LB liquid medium at 37°C in a shaker at 180rpm overnight. 100μL of strain KP181 culture and 100μL of phage strain P253 culture were added to LB semi-solid medium, mixed thoroughly and poured into LB solid plates. After solidification, the plates were inverted and placed in a 37°C incubator for 60 hours. The plaques of phage strain P253 were observed and recorded at 24 hours, 48 ​​hours, and 60 hours. The results are shown in Figure 2. Figure 1 shown.

[0063] Depend on Figure 1 It can be seen that the plaques of the phage strain P253 are surrounded by a turbid halo, and the halo surrounding the plaques continues to grow as the culture time in the 37°C incubator increases, indicating that the phage strain P253 encodes a depolymerase.

[0064] Example 2: Transmission electron microscopy observation of bacteriophage P253

[0065] The purified phage strain P253 in Example 1 was taken for electron microscopy observation. The specific operation steps were as follows: 10 μL of phage strain P253 was dropped on a copper mesh, and it was allowed to precipitate for 15 minutes. Excess liquid was absorbed with filter paper, and the mixture was stained with 2% phosphotungstic acid for 2 minutes. After drying, the mixture was observed using a transmission electron microscope.

[0066] The English abbreviation of phosphotungstic acid is PTA.

[0067] like Figure 2 Transmission electron microscopy results showed that the phage strain P253 had a polyhedral head with a diameter of approximately 58.9±0.5nm and a tail length of approximately 83.7±0.5nm.

[0068] Example 3: Genome analysis of bacteriophage strain P253

[0069] 10 μg / mL deoxyribonuclease and ribonuclease were added to the SM buffer containing the phage strain P253, and the mixture was treated at 37°C for 1 hour. The phage strain P253 genome was extracted using the λ phage genomic DNA extraction kit, and the whole genome was sequenced at a biological company. The open reading frame of the phage strain P253 genome was analyzed using the RAST online website, and the protein encoded by the open reading frame was compared with the NCBI database to clarify its function. The pathogenic bacteria virulence factor database was used to detect the presence of virulence genes, and the comprehensive drug resistance database was used for comparative analysis to detect drug resistance genes.

[0070] The genome sequencing analysis of bacteriophage strain P253 revealed that it had no virulence factors, antibiotic resistance genes, or lysogenic genes. According to the latest classification standards of the International Committee on Taxonomy of Viruses, bacteriophage strain P253 belongs to the genus Drulisvirus of the family Autographiviridae.

[0071] The nucleotide sequence of bacteriophage strain P253 is a linear genome containing 50722 bp.

[0072] Among them, the English name of deoxyribonuclease is DNase; the English name of ribonuclease is RNase.

[0073] The English abbreviation of Pathogen Virulence Factor Database is VFDB.

[0074] The English abbreviation of the Comprehensive Antimicrobial Resistance Database is CARD.

[0075] Example 4: Determination of lysis spectrum of bacteriophage strain P253

[0076] 46 strains of Klebsiella pneumoniae were grown at 37°C overnight. 100 μL of each bacterial culture was added to LB semi-solid and 100 μL of phage strain P253 culture solution. After thorough mixing, the plates were poured into LB solid plates. After solidification, the plates were inverted and placed in a 37°C incubator for overnight culture. The lysis of 46 strains of Klebsiella pneumoniae by phage strain P253 was observed.

[0077] Among them, overnight culture refers to a culture time ≥ 12h.

[0078] The information of the above 46 strains of Klebsiella pneumoniae and the lysis spectrum results of the phage strain P253 are shown in Table 1.

[0079] Table 1 Strain information and phage lysis spectrum

[0080] Bacterial strains KL / K type Bacteriophage strain P253 Bacterial strains KL / Ktype Bacteriophage strain P253 KP1 KL25 - KP311 K30 - KP3 KL14 - KP313 KL61 - KP7 K3 - KP318 K21 - KP24 KL64 - KP319 KL47 - KP25 KL64 - KP320 KL47 - KP29 KL139 - KP324 KL47 - KP41 K3 - KP325 KL19 - KP47 K3 - KP326 KL47 - KP52 K3 - KP334 KL47 - KP72 K3 - KP341 KL112 - KP83 K2 - KP345 KL110 - KP101 K54 - KP350 KL112 - KP182 K2 - KP355 KL112 - KP186 K3 - KP364 KL19 - KP205 KL19 - KP365 K24 - KP216 K57 - KP366 KL47 - KP218 K2 - KP370 KL116 - KP225 KL61 - KP371 KL116 - KP226 KL47 - KP181 K5 + KP232 KL30 - KP435 K5 + KP234 KL21 - KP858 K5 + KP248 KL149 - KP905 K5 + KP253 KL103 - 57949 K5 +

[0081] Note: "+" means the bacteria can be lysed by phage strain P253, and "-" means the bacteria cannot be lysed by phage strain P253.

[0082] The results showed that the bacteria lysed by the phage strain P253 all belonged to the K5 serotype, and other serotypes could not be lysed by it.

[0083] Example 5: Cloning, expression and purification of the depolymerase gene of bacteriophage strain P253

[0084] Using the primers with the sequence shown in SEQ ID NO.3: 5'-CAGCAAATGGGTCGCGGATCCATGACCATTATCAAACGTGCAGAC-3' and the primers with the sequence shown in SEQ ID NO.4: 5'-CTCGAGTGCGGCCGCAAGCTTTTAAGCGGACTGCGCGGA-3', the 2475 bp depolymerase gene predicted from the Klebsiella pneumoniae phage strain P253 was amplified by PCR, and the PCR product was digested and purified by restriction endonucleases BamH I and Hind III and then connected to the pET28a plasmid. Agarose gel electrophoresis was used to identify and screen clones that met the expected size. The results are shown in FIG. Figure 3 As shown, after DNA sequencing confirmed that the recombinant plasmid was correct, it was transformed into Escherichia coli DE3 to obtain DE3 / pET28a-dep253.

[0085] DE3 / pET28a-dep253 was inoculated into 200 mL of liquid LB medium containing 50 μg / mL kanamycin and cultured at 37°C and 180 rpm until OD 600 is 0.6. Add isopropyl-β-D-thiogalactoside to the culture medium to a final concentration of 0.1mM, and then shake and culture at 16°C and 180rpm for 16h. Centrifuge the induced expression DE3-dep253 bacterial solution at 6000rpm / min for 10 minutes. After centrifugation, collect the bacteria. Wash the bacteria three times with 20mL of pre-cooled PBS, and then suspend the bacteria with 20mL of pre-cooled protein purification buffer. The suspended DE3-dep253 bacterial solution was ultrasonically broken after an ice bath, centrifuged at 10000g for 10 minutes, and the supernatant was aspirated and filtered with a 0.22μm filter membrane. Gently add the filtered supernatant sample to the balanced Ni-NTA affinity chromatography column. After the supernatant is fully combined with the Ni column, collect the effluent for subsequent analysis. The Ni column was washed with 4 column volumes of washing buffer containing 50mM imidazole, 100mM imidazole, and 250mM imidazole, respectively, to remove impurities. Then, the target protein was eluted with 4 column volumes of eluent containing 500mM imidazole. The eluent was collected and ultrafiltered through a 30KDa ultrafiltration tube to obtain purified bacteriophage depolymerase dep253. After the concentration of bacteriophage depolymerase dep253 was determined, it was aliquoted and stored in a -80°C refrigerator.

[0086] The reaction system for PCR amplification was 50 μL, including 25 μL high-fidelity enzyme 2×Phanta Flash MasterMix, 17 μL ddH2O, 2 μL upstream primer F and 2 μL downstream primer, and 4 μL bacteriophage P253 strain genome.

[0087] The reaction program for PCR amplification was 95°C for 5 minutes, followed by 30 cycles of: 95°C for 15 seconds, 55°C for 15 seconds, 72°C for 2 minutes, and 72°C for 10 minutes.

[0088] The conditions for ultrasonic crushing were 5 seconds of operation, 5 seconds of rest, 30 minutes of cumulative crushing, and an ice bath throughout the whole process.

[0089] The target protein is bacteriophage depolymerase dep253. The nucleotide sequence encoding bacteriophage depolymerase dep253 is shown in SEQ ID NO.2.

[0090]

[0091] The amino acid sequence of phage depolymerase dep253 is shown in SEQ ID NO.1:

[0092] MTIIKRADLGRPLTWDELDDNFRQVDDLRAAASAAVSSATASATAAAGSATNSLNSANSAAASALDASNSSEVAINALMNSTFEPSSFDFATGGTLDATDRNKAVYNPADNNWYSWSGTLPHVVAAGTDPTTDSNWKPRTDQLLRQELAGTDDETLGDAMIGVRQPYTGSVSRTMHDKVKESISIADFGGAPGTSASSALSSFLTSHIADAVSAAFGLRGEYLIDSASQITLTEGQSMDVDFSSAVFIQNANVSPLVISNGFTGPWTVNSITNEQYNLGDGGTNSAVSILDIPDHGLAVGDSAKIISDDICAFNENANQRRGEWFIVAAVSGNTVVTSGRLSETYSSNVKVVRPSNASVNIRGLRFKSTIADGTTAAMFTVRGFFRPKIEVSFEDLNATGLSVTGCFQAEVMVSGAYLKNRPDLSAYGYLVNDSSSQETQVVGLRCYYARHAYTTTTGSTAANDDNWYNRGRTIDSQVRGGVAHGCANAFDTHGPALRVTFIGCKAVNDYRGYSTGGSGFQIRGQKCRIVACESKGSKVGASVTGAYATEEQFADIDLVYSGEDGTTAVLCSNASSSFAQNVNVKVSAETPASVIVDARNSNVTLKDPDIRANFTANSGIVAQLQDGAKLYVKGGHVDLSRSTATSHIIVKHMDASTEAEVDGLKISGVNRMAYLAQHDAYQAKSRWDNLRLDTALPGVAFLTSVSLTNASVTYRTSASQKPLKYRALTLATGAQAVDLQYSGHDAVNLRVSAAAAGTVINSLTRGAFAGQEISIGVASNSANSIVVQNASAGLIAMPASVTVAAGKALRMYWDGSNWQSAQSA。

[0093] Example 7: Activity verification of phage depolymerase dep253

[0094] The spot assay was used to detect the activity of the bacteriophage depolymerase dep253. The specific steps are as follows:

[0095] 46 strains of Klebsiella pneumoniae were grown overnight at 37°C. 100 μL of each bacterial culture was added to LB semi-solid, mixed thoroughly and poured into LB solid plates. After natural drying, 10 μL of phage depolymerase dep253 with a concentration of 0.2 mg / mL was dropped on the double-layer plate and cultured in a 37°C incubator for 60 hours. The depolymerase action morphology was recorded at 24 hours, 48 ​​hours and 60 hours. The results are as follows Figure 4 The buffer solution was used as negative control and cultured in the same manner.

[0096] Among them, overnight refers to a time ≥12h.

[0097] The information of the above 46 strains of Klebsiella pneumoniae and the results of the activity spectrum of the bacteriophage depolymerase dep253 are shown in Table 2.

[0098] Table 2 Strain information and depolymerase action spectrum

[0099] Bacterial strains KL / K type Bacteriophage strain P253 Bacterial strains KL / K type Bacteriophage strain P253 KP1 KL25 - KP311 K30 - KP3 KL14 - KP313 KL61 - KP7 K3 - KP318 K21 - KP24 KL64 - KP319 KL47 - KP25 KL64 - KP320 KL47 - KP29 KL139 - KP324 KL47 - KP41 K3 - KP325 KL19 - KP47 K3 - KP326 KL47 - KP52 K3 - KP334 KL47 - KP72 K3 - KP341 KL112 - KP83 K2 - KP345 KL110 - KP101 K54 - KP350 KL112 - KP182 K2 - KP355 KL112 - KP186 K3 - KP364 KL19 - KP205 KL19 - KP365 K24 - KP216 K57 - KP366 KL47 - KP218 K2 - KP370 KL116 - KP225 KL61 - KP371 KL116 - KP226 KL47 - KP181 K5 + KP232 KL30 - KP435 K5 + KP234 KL21 - KP858 K5 + KP248 KL149 - KP905 K5 + KP253 KL103 - 57949 K5 +

[0100] “+” means that the bacteriophage depolymerase dep253 has depolymerization activity on the bacterial capsule, and “-” means that the bacteriophage depolymerase dep253 has no depolymerization activity on the bacterial capsule.

[0101] The phage depolymerase dep253 formed a turbid halo on the bacterial lawn of Klebsiella pneumoniae strain, indicating that the phage depolymerase dep253 can degrade the capsular polysaccharide of the strain. Conversely, the phage depolymerase dep253 could not form a turbid halo on the bacterial lawn of Klebsiella pneumoniae strain, indicating that the phage depolymerase dep253 could not degrade the capsular polysaccharide of the strain.

[0102] After Klebsiella pneumoniae strain KP181 was streaked on a sheep blood agar plate and cultured overnight at 37°C, large gray-white mucus colonies formed, indicating that Klebsiella pneumoniae strain KP181 contained a large amount of capsules, such as Figure 5 shown.

[0103] Herein, overnight culture refers to a culture time ≥ 12 h.

[0104] 50 mL of Klebsiella pneumoniae strain KP181 was cultured in LB medium until OD 600When the concentration was 1.0, centrifuge at 10,000 g for 5 minutes and wash twice with 10 mL of PBS. Resuspend the bacterial pellet with 1 mL of PBS and transfer it to a 1.5 mL EP tube and centrifuge at 10,000 g for 5 minutes. The experimental group was resuspended with 1 mL of PBS containing 5 μg of phage depolymerase dep253, and the control group was resuspended with 1 mL of PBS. After the two groups were shaken at 37°C for 12 hours, they were centrifuged at 10,000 g for 5 minutes. Figure 6 As shown, the bacterial precipitate in the group treated with phage dep253 was more compact and smaller in size, while the bacterial precipitate in the group treated with PBS was more compact and slightly larger in size.

[0105] It can be seen from the above experimental results that the phage depolymerase dep253 of the present invention can degrade the capsular polysaccharide of Klebsiella pneumoniae serotype K5.

[0106] Example 8: Inhibition of Klebsiella pneumoniae KP181 biofilm using purified bacteriophage depolymerase dep253

[0107] Put 80 μL of OD 600 The K5 type Klebsiella pneumoniae KP181 with a concentration of 1.0 was inoculated into a 96-well cell culture plate containing 100 μL LB medium in each well, and the brand of the 96-well cell culture plate was Sigma-Aldrich, USA. The purified phage depolymerase dep253 with a concentration of 2 mg / mL was diluted with PBS buffer PBS buffer to obtain phage depolymerase dep253 dilutions with concentrations of 0.2 mg / mL, 0.02 mg / mL, and 0.002 mg / mL, respectively, and 20 μL of the above-mentioned phage depolymerase dep253 dilutions of different concentrations were added to each well of the 96-well cell culture plate containing the biofilm. Among them, the contents of phage depolymerase dep253 in 20 μL of phage depolymerase dep253 dilutions of different concentrations were 4 μg, 0.4 μg, and 0.04 μg, respectively.

[0108] After culturing at 37°C for 24 hours, the cell culture plate was removed, the culture medium was discarded with a pipette, and the plate was washed twice with 200 μL of sterilized PBS buffer to remove free bacteria and enzymes. Then 200 μL of methanol was added to each well and fixed for 10 minutes. After discarding the fixative and allowing it to air dry naturally, the biofilm was stained with 200 μL of 0.1% by mass crystal violet at room temperature for 10 minutes. After staining, the staining solution was discarded and washed twice with 200 μL of PBS buffer to remove free staining solution. The cell culture plate was placed in a 50°C oven to dry, and after taking it out, 200 μL of 33% acetic acid solution was added to each well and placed on a shaker for 30 minutes to release the crystal violet in the biofilm. Finally, an enzyme reader was used to measure its absorbance at a wavelength of 595 nm. According to OD 595The value determines the amount of biofilm residue.

[0109] like Figure 7 As shown, the biofilm formation amount of the 4μg, 0.4μg and 0.04μg phage depolymerase dep253 treatment groups was significantly reduced. From the above experimental results, it can be seen that the phage depolymerase dep253 of the present invention can significantly inhibit the formation of biofilm.

[0110] It should be noted that when the present invention involves a numerical range, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes a preferred embodiment.

[0111] Although preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they understand the basic inventive concepts, and all such changes and modifications fall within the scope of the present invention.

Claims

1. Use of a bacteriophage depolymerase dep253 in the preparation of a bacteriophage disinfection product for degrading K5 serotype Klebsiella pneumoniae capsular polysaccharide, characterized in that: The amino acid sequence of the bacteriophage depolymerase dep253 is shown in SEQ ID NO.

1.

2. The use of a bacteriophage depolymerase dep253 according to claim 1 in preparing a bacteriophage disinfection product for degrading K5 serotype Klebsiella pneumoniae capsular polysaccharide, characterized in that: The nucleotide sequence encoding the bacteriophage depolymerase dep253 is shown in SEQ ID NO.

2.

3. The use of a bacteriophage depolymerase dep253 according to claim 1 in preparing a bacteriophage disinfection product for degrading K5 serotype Klebsiella pneumoniae capsular polysaccharide, characterized in that: The preparation method of the bacteriophage depolymerase dep253 comprises the following steps: The dep253 gene fragment of the 17th open reading frame of the Klebsiella pneumoniae phage P253 strain was obtained by PCR, double enzyme digestion, and ligation molecular cloning, and the dep253 gene fragment was ligated to a plasmid vector to obtain a recombinant plasmid; Transforming the recombinant plasmid into host cells, and screening to obtain recombinant host cells containing the recombinant plasmid; Cultivating the recombinant host cell to obtain a culture fluid, separating the culture fluid into solid and liquid, and collecting the liquid to obtain the bacteriophage depolymerase dep253; The preservation number of the Klebsiella pneumoniae phage P253 strain is: CCTCC NO: M 20241683, the preservation time is July 25, 2024, the preservation unit is: China Center for Type Culture Collection, and the preservation address is: Wuhan University, Wuhan, China.

4. The use of a bacteriophage depolymerase dep253 according to claim 3 in preparing a bacteriophage disinfection product for degrading K5 serotype Klebsiella pneumoniae capsular polysaccharide, characterized in that: The plasmid vector includes pET28a.

5. Use of a bacteriophage depolymerase dep253 according to claim 3 in preparing a bacteriophage disinfection product for degrading K5 serotype Klebsiella pneumoniae capsular polysaccharide, characterized in that: The host cell is Escherichia coli DE3.

6. Use of a bacteriophage depolymerase dep253 according to claim 1 in the preparation of a bacteriophage disinfection product for inhibiting the formation of K5 serotype Klebsiella pneumoniae biofilm, characterized in that: The amino acid sequence of the bacteriophage depolymerase dep253 is shown in SEQ ID NO.

1.

7. The use according to claim 1 or claim 6, characterized in that: The bacteriophage disinfecting product is made of the bacteriophage depolymerase dep253 and a solvent; The concentration of the bacteriophage depolymerase dep253 is 0.002 mg / mL to 2 mg / mL.

8. The use according to claim 7, characterized in that The solvent is PBS; The concentration of PBS is 0.01 mol / L; the pH is 7.2~7.4.

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