CN119060987A - Use of bacteriophage depolymerase dep253 in degrading bacterial capsular polysaccharides - Google Patents
Use of bacteriophage depolymerase dep253 in degrading bacterial capsular polysaccharides Download PDFInfo
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Abstract
本发明属于噬菌体技术领域，具体涉及一种噬菌体解聚酶dep253在降解细菌荚膜多糖中的用途。所述噬菌体解聚酶dep253的氨基酸序列如SEQ ID NO.1所示。所述噬菌体解聚酶dep253由一种肺炎克雷伯菌噬菌体P253菌株制备得到；所述肺炎克雷伯菌噬菌体P253菌株的保藏编号为：CCTCC NO：M20241683，保藏时间为：2024年7月25日，保藏单位为：中国典型培养物保藏中心，保藏地址为：中国.武汉.武汉大学。本发明所提供的噬菌体解聚酶dep253为一种K5血清型特异性的解聚酶，可以用于特异性地降解K5血清型肺炎克雷伯菌荚膜多糖，可以用于抑制K5血清型肺炎克雷伯菌生物被膜形成，进而起到防治肺炎克雷菌引起疾病的效果。
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 bacteriophage P253 strain; the deposit number of the Klebsiella pneumoniae bacteriophage P253 strain is: CCTCC NO: M20241683, the deposit time is: July 25, 2024, the deposit unit is: China Center for Type Culture Collection, and the deposit address is: China. Wuhan. 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
The invention belongs to the technical field of phage, and particularly relates to application of phage depolymerase dep253 in degrading bacterial capsular polysaccharide.
Background
Klebsiella pneumoniae is a common pathogen for human and livestock co-occurrence, and in recent years, multi-drug resistant Klebsiella pneumoniae has become one of the most important pathogens for iatrogenic infection. The prevention and control of the pathogenic bacteria mainly depend on antibiotics. However, the widespread use of antibiotics has also had some negative effects, firstly, the prolonged, high-volume use of antibiotics leads to increased bacterial resistance, i.e. certain bacteria are no longer susceptible to antibiotics. Second, abuse of antibiotics can also cause food safety problems and antibiotic residues in poultry products can have an impact on the health of the consumer. Bacteria resistant to antibiotics may be transmitted to humans through the food chain, increasing health risks.
Most klebsiella pneumoniae has the capability of synthesizing and secreting capsular polysaccharide, and the capsular polysaccharide serves as a natural barrier of bacteria, can maintain bacterial virulence, adhere and block permeation of antibiotics, and is an important virulence factor of the klebsiella pneumoniae. In addition, klebsiella pneumoniae can form a biological envelope, namely a membranous structure formed by bacterial cells wrapped by extracellular polysaccharide matrixes, lipoproteins, fibrin and the like generated by bacteria, and can obviously improve the drug resistance of the bacteria to antibiotics and the ability of escaping from the recognition of a host immune system, so that the colonization of the bacteria in a focus is accelerated. Thus, biofilms are one of the major causative factors of persistent infection by bacteria in hospitals.
Phage depolymerases direct phage adsorption to host outer membrane proteins by degrading bacterial surface polysaccharides. The enzyme can realize targeted degradation of capsular polysaccharide by randomly attacking glycosidic bond to release polymer repeating units, and various domestic and foreign researches show that phage depolymerizing enzyme can effectively remove and inhibit formation of biological envelope and has certain application potential in the field of pathogen infection control. However, phage depolymerases of different sources, different nucleic acid sequences and different protein structures also show a very large difference in degradation performance for different types of capsular polysaccharides and biofilms. Although depolymerases of the K5 serotype Klebsiella pneumoniae capsule have been reported at present, no product use of phage depolymerases in degrading bacterial capsular polysaccharides has been found.
Disclosure of Invention
In order to solve the problem that no phage depolymerase has been developed in the prior art for the product purpose in the aspect of degrading bacterial capsular polysaccharide, the invention provides the purpose of phage depolymerase dep253 in the aspect of degrading bacterial capsular polysaccharide. The invention carries out whole genome sequencing and annotation on the klebsiella pneumoniae bacteriophage separated in the nature, screens out phage depolymerase with serotype specificity of phage sources by utilizing a prokaryotic expression platform, treats diseases caused by klebsiella pneumoniae, reduces economic loss and protects human and animal health.
In order to achieve the above purpose, the present invention adopts the following technical scheme.
Klebsiella pneumoniae is a gram-negative bacillus of numerous serotypes. Serotypes are classified based on differences in bacterial capsular polysaccharide antigens, with more than 80 capsular serotypes of klebsiella pneumoniae currently known. The bacterium has the remarkable characteristic that the bacterium body wraps the thick capsular polysaccharide, and a biofilm is easy to form, and based on the bacterium, the invention provides the application of phage depolymerase dep253 in degrading the capsular polysaccharide of the bacterium. Phage depolymerase dep253 is a K5 serotype specific depolymerase and can be used for specifically degrading K5 serotype Klebsiella pneumoniae capsular polysaccharide and inhibiting K5 serotype Klebsiella pneumoniae biofilm formation, thereby having the effect of preventing and treating diseases caused by Klebsiella pneumoniae.
A Klebsiella pneumoniae phage P253 strain has a preservation number of CCTCC NO of M20241683, a preservation time of 2024 and 7 months and 25 days, a preservation unit of China center for type culture collection, and a preservation address of China university of Wuhan.
The phage strain P253 is separated from medical sewage of Zhang Kong municipal hospital by a double-layer flat plate method by using K5 serotype Klebsiella pneumoniae strain KP181 as a host bacterium. Subsequent evaluation of phage strain P253 on the lytic ability of 46 Klebsiella pneumoniae strains revealed that phage strain P253 only lyses K5 serotype Klebsiella pneumoniae strains. A cloudy halo was wrapped around the plaques of phage strain P253, indicating that phage strain P253 encodes a depolymerase.
Phage is a virus that can infect bacteria, and can specifically infect and kill bacteria. Phage-derived depolymerases are a class of enzymes encoded by phage that degrade bacterial extracellular polysaccharides, which are beneficial to enhancing the bacteriostatic effect of antibiotics or phage.
Phage therapy has some significant advantages over antibiotics, especially when faced with drug-resistant bacteria and treating specific infection types. Highly specific, phage are capable of highly specific infection and killing of specific bacteria without affecting the normal microbiota of the human body. This is in contrast to the broad spectrum bactericidal action of antibiotics, which can more precisely target pathogenic bacteria with reduced impact on the beneficial flora of the human body. Low toxic side effect, that is, the bacteriophage acts on specific bacteria and is harmless to human cells. Thus, the phage has fewer side effects than antibiotics. This allows the phage to be used more safely during the course of treatment, especially for patients who have been unable to be treated with conventional antibiotics because of drug resistance issues. Natural selection has the advantage that phages are able to select their targets naturally according to the type of bacteria infected, which makes them more efficient in the course of treatment. In contrast, the broad-spectrum bactericidal effect of antibiotics is strong, but also tends to cause disturbance of the microbial population and emergence of resistant strains. Environmentally friendly, phages are naturally occurring viruses whose therapeutic use does not adversely affect the environment. In contrast, the widespread use of antibiotics has been demonstrated to have a potential impact on the environmental microbiota and ecosystem. Based on the above, the invention provides an application of phage depolymerase dep253 in degrading bacterial capsular polysaccharide, wherein the amino acid sequence of phage depolymerase dep253 is shown as SEQ ID NO. 1. The amino acid sequence of the bacteriophage depolymerizing enzyme dep253 related to the invention has about 30 percent of homology with the amino acid sequence of the depolymerizing enzyme reported in the prior art. Furthermore, the previously reported K5 serotype specific depolymerase did not mention biofilm formation inhibitory effect, and the phage depolymerase dep253 mentioned in the present invention was able to significantly inhibit K5-type Klebsiella pneumoniae strain biofilm formation.
Preferably, the phage depolymerase dep253 is prepared from a klebsiella pneumoniae phage P253 strain.
The Klebsiella pneumoniae phage P253 strain has a preservation number of CCTCC NO: M20241683, a preservation time of 2024, 7 months and 25 days, a preservation unit of China center for typical culture collection, and a preservation address of 211 chambers of China center for typical culture collection in Wuhan university of Wuhan district of Wuhan City in Hubei province.
Preferably, the nucleotide sequence encoding said phage depolymerase dep253 is shown in SEQ ID NO. 2.
Preferably, said phage depolymerase dep253 is a depolymerase encoded by the open reading frame at position 17 of said klet bacteriophage P253 strain.
Preferably, the preparation method of the phage depolymerizing enzyme dep253 comprises the following steps:
Obtaining a dep253 gene fragment of the 17 th open reading frame of the Klebsiella pneumoniae bacteriophage P253 strain by means of PCR, double enzyme digestion and connecting molecule cloning, and connecting the dep253 gene fragment to a plasmid vector to obtain a recombinant plasmid.
Transferring the recombinant plasmid into a host cell, and screening to obtain the recombinant host cell containing the recombinant plasmid.
Culturing the recombinant host cell to obtain a culture solution, and collecting liquid after solid-liquid separation of the culture solution to obtain the phage depolymerizing enzyme dep253.
Preferably, the plasmid vector comprises pET28a. The pET-28a vector has an N-terminal His/Thrombin/T7 protein tag and also contains an optional C-terminal His tag.
Preferably, the host cell is E.coli DE3. Coli DE3 is suitable for T7 promoter driven recombinant protein inducible expression systems, such as the common prokaryotic expression vector pET series. The strain can effectively avoid degradation of recombinant expression proteins and is widely used for expression of the recombinant proteins.
Preferably, the phage depolymerase dep253 is used to degrade klebsiella pneumoniae capsular polysaccharide and biofilm. The klebsiella pneumoniae strain is typically characterized in that the thallus is wrapped with a thick capsule, and a biofilm is easy to form.
Preferably, the phage depolymerase dep253 is used to prepare phage decontamination products. The phage killing product takes the phage depolymerizing enzyme dep253 as the only active ingredient.
Preferably, the phage killing product is made from the phage depolymerase dep253 and a solvent.
The concentration of the phage depolymerase dep253 is 0.002 mg/mL-2 mg/mL.
Preferably, the solvent is PBS, the concentration of the PBS is 0.01mol/L, and the pH is 7.2-7.4. The English language of PBS is known as phosphate buffer saline, and the corresponding Chinese name is phosphate buffered saline.
Compared with the prior art, the invention has the following beneficial effects:
1. The invention provides the use of phage depolymerase dep253 in degrading bacterial capsular polysaccharides. The bacteriophage depolymerizing enzyme dep253 provided by the invention is a K5 serotype specific depolymerizing enzyme and can be prepared from a Klebsiella pneumoniae bacteriophage P253 strain. The Klebsiella pneumoniae bacteriophage P253 strain has strong specific cracking capacity to K5 serotype Klebsiella pneumoniae, can be used singly or in combination with other substances, and provides a safe and nontoxic bacteriophage disinfection product for disinfection and purification environment.
2. The invention also provides a depolymerizing enzyme dep253 coded by the Klebsiella pneumoniae bacteriophage P253 strain, which can specifically degrade Klebsiella pneumoniae capsular polysaccharide of the K5 serotype, can obviously inhibit the formation of a Klebsiella pneumoniae biofilm of the K5K5 serotype, and is beneficial to the removal of Klebsiella pneumoniae of the K5 serotype.
3. The invention carries out whole genome sequencing and annotation on the klebsiella pneumoniae bacteriophage separated in the nature, screens out phage depolymerase with serotype specificity of phage sources by utilizing a prokaryotic expression platform, treats diseases caused by klebsiella pneumoniae, reduces economic loss and protects human and animal health. The key technical scheme for obtaining K5 serotype specific depolymerase through whole genome sequencing annotation and clone expression is as follows:
(1) Genome sequencing, namely, carrying out whole-gene sequencing on various bacteria which are existing and newly separated in the laboratory and are representative strains selected by combining pathogenicity.
(2) Genome splicing, namely carrying out automatic splicing with high flux on the sequencing strain after obtaining the off-machine data and mainly using unicycler for splicing and writing a script by oneself.
(3) Genome annotation the genome was annotated using RAST to obtain a gene potentially encoding K5 depolymerase.
(4) The depolymerizing enzyme clone expression, clone expression of depolymerizing enzyme protein by a prokaryotic expression system and verification of the in vitro activity.
Drawings
FIG. 1 shows plaque patterns of Klebsiella pneumoniae phage P253 strain at 24 hours, 48 hours and 60 hours, wherein FIG. 1A shows plaque patterns of Klebsiella pneumoniae phage P253 strain at 24 hours, FIG. 1B shows plaque patterns of Klebsiella pneumoniae phage P253 strain at 48 hours, and FIG. 1C shows plaque patterns of Klebsiella pneumoniae phage P253 strain at 60 hours.
FIG. 2 shows the electron microscope morphology of Klebsiella pneumoniae bacteriophage P253 strain in the present invention.
FIG. 3 shows agarose gel electrophoresis of a prokaryotic expression strain positive for phage depolymerizing enzyme dep253 selected in the present invention.
FIG. 4 shows the effects of phage depolymerase dep253 on K5 type Klebsiella pneumoniae KP181 lawn for 24 hours, 48 hours and 60 hours, wherein FIG. 4 shows the effects of phage depolymerase dep253 on K5 type Klebsiella pneumoniae KP181 lawn for 24 hours, FIG. 4 shows the effects of phage depolymerase dep253 on K5 type Klebsiella pneumoniae KP181 lawn for 48 hours, and FIG. 4 shows the effects of phage depolymerase dep253 on K5 type Klebsiella pneumoniae KP181 lawn for 60 hours.
FIG. 5 shows colony morphology of Klebsiella pneumoniae KP181 of the present invention cultured overnight in sheep blood plates.
FIG. 6 shows the effect of the bacteriophage depolymerizing enzyme dep253 on K5 type Klebsiella pneumoniae KP181, wherein FIG. 6A shows the effect of the bacteriophage depolymerizing enzyme dep253 on K5 type Klebsiella pneumoniae KP181, and FIG. 6B shows the effect of PBS on K5 type Klebsiella pneumoniae KP 181.
FIG. 7 shows inhibition of K5 serotype Klebsiella pneumoniae KP181 biofilm formation by phage depolymerase dep253 of the present invention.
Detailed Description
The present invention will now be described in detail with reference to the drawings and specific examples, which should not be construed as limiting the invention. Unless otherwise indicated, 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 are commercially available unless otherwise indicated.
The materials and methods used in the examples below were as follows:
1.1 main reagents and instrumentation:
SM buffer was purchased from Lei Gen Bio Inc.
The transmission electron microscope is Hitachi H-7650.
Deoxyribonucleases and ribonucleases were purchased from ibodia corporation.
PBS was purchased from Shanghai double helix Biotechnology Co., ltd, at a concentration of 0.01mol/L and pH 7.4.
Lambda phage genomic DNA extraction kit was purchased from idebi biosystems.
Crystal violet was purchased from pearl oyster Biotechnology Co.
1.2 Plasmid and bacterial samples:
the source of the pET28a plasmid is Beijing Optimu Biotech Co.
Klebsiella pneumoniae is derived from clinical strains collected by Jiangsu province disease control and prevention control centers.
Coli DE3 was from Beijing qingke biotechnology Co.
1.3 Materials:
the sewage sample used in the present invention was taken from the Zhang Kong City hospital.
Klebsiella pneumoniae is derived from clinical strains collected by Jiangsu province disease control and prevention control centers.
EXAMPLE 1 isolation and purification of phages
The Klebsiella pneumoniae phage P253 strain is preserved in China Center for Type Culture Collection (CCTCC) No. M20241683, and the preservation time is 2024, 7 months and 25 days. The 17 th open reading frame of the genome of the klebsiella pneumoniae bacteriophage P253 strain codes for a depolymerase.
The separation and purification method of the phage strain P253 comprises the following specific steps:
After centrifugation of 10mL of the wastewater sample at 5000g for 5 minutes, the supernatant was filtered using a 0.22 μm filter membrane, and the filtrate was collected. 100. Mu.L of the sewage sample filtrate was taken and mixed with 100. Mu.L of Klebsiella pneumoniae strains KP181 to LB semisolid and poured into LB solid plates. After the plate solidified, it was placed upside down in a 37℃incubator overnight for cultivation. Phage plaques were purified until a single plaque morphology was presented on the plate. 5mL of SM buffer was added to the plate and the plate was shaken at 60rpm for 4 hours. After the completion of the shaking, the liquid was collected and centrifuged at 5000 Xg for 5min, and the supernatant after centrifugation was collected. The supernatant was filtered with a 0.22 μm filter, and the purified phage was stored in SM buffer at 4℃to give enriched phage strain P253.
Klebsiella pneumoniae strain KP181 was shake-cultured overnight at 180rpm in LB liquid medium at 37 ℃. 100. Mu.L of strain KP181 culture and 100. Mu.L of phage strain P253 culture solution were taken and put into LB semisolid, poured into LB solid plate after being fully mixed, after solidification, put into 37 ℃ incubator for cultivation for 60 hours, and plaque condition of phage strain P253 at 24 hours, 48 hours and 60 hours was observed and recorded, respectively. The results are shown in FIG. 1.
As can be seen from FIG. 1, phage strain P253 had a cloudy halo around the plaque and the halo surrounding the plaque was also growing larger with prolonged incubation time at 37℃in the incubator, indicating that phage strain P253 encodes a depolymerase.
Example 2 phage P253 Transmission Electron microscopy
Taking the phage strain P253 purified in the example 1, and carrying out electron microscope observation, wherein the specific operation steps are that 10 mu L of phage strain P253 is added to be dripped on a copper mesh, the sediment is carried out for 15 minutes, the excessive liquid is sucked by filter paper, the liquid is dyed for 2 minutes by phosphotungstic acid with the volume fraction of 2%, and the liquid is observed by using a transmission electron microscope after being dried.
The english language of phosphotungstic acid is abbreviated as PTA.
As shown in the transmission electron microscope results of FIG. 2, phage strain P253 had a polyhedral head with a diameter of about 58.9.+ -. 0.5nm and a tail length of about 83.7.+ -. 0.5nm.
EXAMPLE 3 phage Strain P253 genomic analysis
10. Mu.g/mL of deoxyribonuclease and ribonuclease were added to SM buffer containing phage strain P253, treated at 37℃for 1 hour, and phage strain P253 genome was advanced with lambda phage genomic DNA extraction kit and subjected to whole genome sequencing at Bio Inc. The open reading frame of the phage strain P253 genome is analyzed by using a RAST online website, the function of the protein coded by the open reading frame is determined by comparing the protein coded by the open reading frame with a NCBI database, the virulence factor database of pathogenic bacteria is used for detecting the virulence genes of the pathogenic bacteria, and the drug resistance genes are detected by comparing and analyzing the protein in a comprehensive drug resistance database.
After genome sequencing analysis, the phage strain P253 is found to have no virulence factor, antibiotic drug resistance gene and lysogenic gene. In combination with the latest classification standards of the International Commission on viral classification, phage strain P253 belongs to genus Drulisvirus of Autographiviridae.
The nucleotide sequence of phage strain P253 is a linear genome containing 50722 bp.
Wherein, the English name of the deoxyribonuclease is DNase, and the English name of the ribonuclease is RNase.
The English name of the pathogenic bacteria virulence factor database is VFDB.
English of the comprehensive drug resistance database is called CARD for short.
EXAMPLE 4 determination of the cleavage Spectrum of phage Strain P253
46 Klebsiella pneumoniae bacteria grow overnight at 37 ℃, 100 mu L of each bacterial culture is respectively mixed with 100 mu L of phage strain P253 culture solution to LB semisolid, the mixture is poured into LB solid plates after being fully mixed, after solidification, the mixture is placed into a 37 ℃ incubator for overnight culture, and the cracking condition of the phage strain P253 on 46 Klebsiella pneumoniae bacteria is observed.
Wherein, the overnight culture means that the culture time is more than or equal to 12 hours.
The information of the 46 Klebsiella pneumoniae bacteria and the results of the phage strain P253 lysis profile are shown in Table 1.
TABLE 1 Strain information and phage lysis spectra
| Bacterial strains | KL/K type | Phage strain P253 | Bacterial strains | KL/Ktype | Phage 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 | + |
Note that "+" indicates that the bacteria were able to be lysed by phage strain P253 and "-" indicates that the bacteria were unable to be lysed by phage strain P253.
As a result, it was found that the bacteria lysed by the phage strain P253 all belong to the K5 serotype, and none of the other serotype strains could be lysed by them.
EXAMPLE 5 expression and purification of the depolymerase Gene clone of phage Strain P253
The 2475bp depolymerizing enzyme gene predicted from Klebsiella pneumoniae phage strain P253 is amplified by PC R using a primer with the sequence shown as SEQ ID NO.3 and 5'-CAGCAAATGGGTCGCGGATCCATG ACCATTATCAAACGTGCAGAC-3' and a primer with the sequence shown as SEQ ID NO.4 and 5'-CTCGAGTGCGGCCGCAAGCTTTTAAGCGGACTGCGCGGA-3', and the PCR product is connected to pET28a plasmid after restriction enzyme digestion and purification by restriction enzymes BamHI and HindIII. Clones meeting the expected size are identified and screened by agarose gel electrophoresis. As shown in FIG. 3, the recombinant plasmid was transformed into E.coli DE3 after DNA sequencing to obtain DE3/pET28a-dep253.
DE3/pET28a-dep253 was inoculated into 200mL of liquid LB medium containing 50. Mu.g/mL kanamycin and cultured at 37℃and 180rpm to OD 600 of 0.6. isopropyl-beta-D-thiogalactoside was added to the culture broth to a final concentration of 0.1mM and further cultured with shaking at 16℃and 180rpm for 16 hours. DE3-dep253 bacterial liquid for inducing expression is centrifuged for 10 minutes under the condition of 6000 rpm/min. After centrifugation, the cells were collected. The cells were washed 3 times with 20mL of pre-chilled PBS and then suspended with 20mL of pre-chilled protein purification buffer. The suspension of DE3-dep253 bacteria was subjected to ice bath and then to ultrasonic disruption, centrifuged at 10000g for 10 minutes and the supernatant was aspirated and filtered through a 0.22 μm filter membrane. And (3) lightly adding the filtered supernatant sample into the balanced Ni-NTA affinity chromatography column, and collecting effluent for subsequent analysis after the supernatant is fully combined with the Ni column. The Ni column was washed to remove the contaminating proteins using 4 column volumes of wash buffer containing 50mM imidazole, 100mM imidazole, 250mM imidazole in sequence. The target protein was then eluted with 4 column volumes of an eluent containing 500mM imidazole. The eluent was collected and then ultrafiltered through a 30kDa ultrafiltration tube to give purified phage depolymerizing enzyme dep253. After the concentration of phage depolymerase dep253 was determined, it was dispensed and stored in a-80℃refrigerator.
Wherein the PCR amplification reaction system was 50. Mu.L, including 25. Mu.L of high-fidelity enzyme 2X PHANTA FLASH MASTER Mix, 17. Mu. LddH 2 O, 2. Mu.L of upstream primer F and 2. Mu.L of downstream primer, 4. Mu.L of phage P253 strain genome.
The reaction procedure for PCR amplification was 95℃for 5 minutes, followed by 30 cycles of 95℃for 15 seconds, 55℃for 15 seconds, 72℃for 2 minutes, and 72℃for 10 minutes.
The condition during ultrasonic crushing is 5 seconds of operation, 5 seconds of intermission, 30 minutes of accumulated crushing and the whole process of ice bath.
The protein of interest is phage depolymerase dep253. The nucleotide sequence of the encoding phage depolymerase dep253 is shown in SEQ ID NO. 2.
atgaccattatcaaacgtgcagacctgggtcgacccttaacgtgggacgaactggatgataacttccggcaagttgatgatttgagagccgccgcatcggcggctgtatcgagtgcaacggcttcagctacagccgctgcaggcagtgctacgaattcacttaatagtgcaaatagtgctgctgcctcggccttggatgcttcgaattcgtccgaagtagctatcaatgcgttgatgaattctacatttgaaccaagtagtttcgacttcgccaccggtggcactcttgatgccactgaccgcaataaagcagtgtacaacccggcggataataattggtattcatggtcgggcactctgccgcatgtggttgccgctgggacggaccctacaacggacagtaactggaagccacgcacggaccagttattgcgacaggagttggccgggacagacgatgagacgttgggtgacgcaatgataggtgttcgtcagccgtacacggggtctgtctcccggacaatgcacgataaagtgaaggaatcaatttctattgcagattttggcggcgcaccgggcacatctgcatcatctgccttgtcatctttcctcacatcgcacatcgcagatgcggtgtccgccgcgttcgggctgcgcggtgagtatttgatagactccgcctcccaaataacgctgaccgaaggccagtcaatggatgtggatttcagctctgcagtgtttatacaaaatgccaatgtcagcccattggttatatctaacggtttcactggtccgtggactgtgaactcaataactaatgaacagtataacttaggcgacggcggcaccaacagcgcggtatccattttagatataccggaccacggtctagcggtcggggatagcgcgaaaataatatctgatgatatttgcgcctttaacgaaaacgcaaaccaacggcgcggcgagtggtttattgttgctgccgtgtctgggaacactgttgttacctctggacggctttcggaaacatactccagtaatgttaaggttgtgcggccgtccaacgcctctgttaatatcaggggtttgaggttcaagagcacgatagccgacgggactaccgcagcgatgttcactgttcgcggcttcttccgcccaaaaatagaagtttcattcgaggacctgaacgcgaccggtctcagtgttactggttgtttccaggccgaagttatggtttcgggggcttatcttaaaaaccgtcctgacctgtctgcatatggttatttggtaaatgacagcagttcccaggaaactcaagttgtcgggcttcgctgttattatgcccgacacgcgtacacaaccaccactggcagtactgccgccaacgacgacaactggtacaaccgtgggcggactattgattctcaggtgcgaggtggcgtcgcccacggttgtgcaaacgcgttcgacacacacgggccagctttgcgtgtgaccttcatcgggtgtaaagccgtgaatgattacagaggttacagcaccggcggcagtggtttccagatccggggacaaaaatgccgcattgtggcttgtgagtctaaggggtctaaagttggagcatccgtaaccggggcttacgccaccgaagaacagtttgccgatatagacctcgtttactcaggggaagatggcaccaccgcggttttgtgctctaacgcaagttcttcctttgcgcaaaacgtcaatgttaaagtgagcgcggagactcctgccagcgtgatagtggatgcccgaaactctaacgtgacactgaaggacccagacatacgggccaacttcaccgccaactccgggatagtggcacaattacaggatggtgcgaaactatacgtcaaaggaggccatgtcgacctctcaaggtccacggcaacctcgcacatcattgttaaacatatggacgcttcgacagaagccgaggttgatgggttgaagatatccggcgttaatcgcatggcctatttggcacagcatgacgcttatcaggctaaatcaagatgggataaccttcgcctggacaccgcattgccgggtgtggctttcctcaccagtgtgtcgctgacgaacgcctccgtgacttatcgcacctcggcgtctcaaaagcctcttaaatatcgcgcactcactctcgcaacaggtgcgcaagcggttgacctgcaatactccggacatgatgcggtaaacctgcgtgtgtctgcagctgctgcgggtacggtgattaacagtctgacgcggggtgcttttgcgggtcaagaaatatcaataggcgtggcgtctaattccgccaacagcatcgttgtccagaacgcttctgctggtcttatcgcaatgcccgcctcggttaccgtggccgcaggtaaagctctacgcatgtattgggatggctctaactggcaatccGcgcagtccgcttaa.
The amino acid sequence of phage depolymerase dep253 is shown in SEQ ID NO. 1:
MTIIKRADLGRPLTWDELDDNFRQVDDLRAAASAAVSSATASATAAAGSATNSLNSANSAAASALDASNSSEVAINALMNSTFEPSSFDFATGGTLDATDRNKAVYNPADNNWYSWSGTLPHVVAAGTDPTTDSNWKPRTDQLLRQELAGTDDETLGDAMIGVRQPYTGSVSRTMHDKVKESISIADFGGAPGTSASSALSSFLTSHIADAVSAAFGLRGEYLIDSASQITLTEGQSMDVDFSSAVFIQNANVSPLVISNGFTGPWTVNSITNEQYNLGDGGTNSAVSILDIPDHGLAVGDSAKIISDDICAFNENANQRRGEWFIVAAVSGNTVVTSGRLSETYSSNVKVVRPSNASVNIRGLRFKSTIADGTTAAMFTVRGFFRPKIEVSFEDLNATGLSVTGCFQAEVMVSGAYLKNRPDLSAYGYLVNDSSSQETQVVGLRCYYARHAYTTTTGSTAANDDNWYNRGRTIDSQVRGGVAHGCANAFDTHGPALRVTFIGCKAVNDYRGYSTGGSGFQIRGQKCRIVACESKGSKVGASVTGAYATEEQFADIDLVYSGEDGTTAVLCSNASSSFAQNVNVKVSAETPASVIVDARNSNVTLKDPDIRANFTANSGIVAQLQDGAKLYVKGGHVDLSRSTATSHIIVKHMDASTEAEVDGLKISGVNRMAYLAQHDAYQAKSRWDNLRLDTALPGVAFLTSVSLTNASVTYRTSASQKPLKYRALTLATGAQAVDLQYSGHDAVNLRVSAAAAGTVINSLTRGAFAGQEISIGVASNSANSIVVQNASAGLIAMPASVTVAAGKALRMYWDGSNWQSAQSA.
Example 7 Activity verification of phage depolymerizing enzyme dep253
The activity of phage depolymerizing enzyme dep253 was detected by a spot assay, which was performed as follows:
46 Klebsiella pneumoniae bacteria were grown overnight at 37℃and each bacterial culture was taken 100. Mu.L of each bacterial culture to LB semisolid, poured into LB solid plates after thoroughly mixing, naturally dried, and 10. Mu.L of phage depolymerase dep253 at a concentration of 0.2mg/mL was dropped on double-layered plates and allowed to stand for 60 hours in a 37℃incubator, followed by recording the depolymerase action patterns for 24 hours, 48 hours and 60 hours. The results are shown in FIG. 4. Buffer as a negative control, cultures were performed in the same manner.
Wherein, overnight means that the time is more than or equal to 12 hours.
The information of the 46 Klebsiella pneumoniae bacteria and the results of the phage depolymerizing enzyme dep253 enzyme activity profile are shown in Table 2.
TABLE 2 Strain information and depolymerase Activity Spectrum
| Bacterial strains | KL/K type | Phage strain P253 | Bacterial strains | KL/K type | Phage 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 | + |
"+" Indicates that phage depolymerase dep253 has depolymerising activity on the bacterial capsule and "-" indicates that phage depolymerase dep253 has no depolymerising activity on the bacterial capsule.
The formation of a cloudy halo on the lawn of the klebsiella pneumoniae strain by phage depolymerase dep253 suggests that phage depolymerase dep253 is capable of degrading the capsular polysaccharide of the strain. In contrast, the inability of phage depolymerase dep253 to form a cloudy halo on the lawn of a klebsiella pneumoniae strain suggests that phage depolymerase dep253 is unable to degrade the capsular polysaccharide of that strain.
Klebsiella pneumoniae strain KP181 was streaked on sheep blood platelets and cultured overnight at 37℃to form larger gray-white mucus colonies, suggesting that Klebsiella pneumoniae strain KP181 contained a large amount of capsules, as shown in FIG. 5.
Wherein, the overnight culture refers to the culture time of more than or equal to 12 hours.
50ML of Klebsiella pneumoniae strain KP181 was incubated in LB medium to an OD 600 of 1.0, centrifuged at 10000g for 5 min, and washed twice with 10mL of PBS. The bacterial pellet was resuspended in 1mL of PBS and transferred to a 1.5mLEP tube and centrifuged at 10000g for 5 minutes. The test group was resuspended with 1 mLPBS. Mu.g of phage depolymerase dep253 and the control group was resuspended with 1mL of PBS. Both groups were centrifuged at 10000g for 5 minutes after 12 hours of shaking treatment at 37 ℃. As shown in FIG. 6, phage depolymerase dep253 treated group bacterial pellet was more compact and less bulky, while PBS treated group bacterial pellet depolymerase dep253 treated group was compact and slightly more bulky.
As shown by the experimental results, the bacteriophage depolymerizing enzyme dep253 can degrade capsular polysaccharide of K5 serotype Klebsiella pneumoniae.
Example 8 inhibition of Klebsiella pneumoniae KP181 biofilm Using purified phage depolymerizing enzyme dep253
80. Mu.L of Klebsiella pneumoniae KP181, type K5 with an OD 600 of 1.0, was inoculated into 96-well cell culture plates containing 100. Mu.L LB medium per well, 96-well cell culture plates brand Sigma-Aldrich, USA. Phage depolymerase dep253 with the concentration of 2mg/mL after purification is diluted by PBS buffer solution respectively to obtain phage depolymerase dep253 diluted solutions with the concentration of 0.2mg/mL, 0.02mg/mL and 0.002mg/mL in sequence, and 20 mu L of phage depolymerase dep253 diluted solutions with different concentrations are added into each well of a 96-well cell culture plate containing a biological film. Wherein, the content of phage depolymerase dep253 in 20 mu L of phage depolymerase dep253 dilutions at different concentrations was 4 mu g, 0.4 mu g and 0.04 mu g, respectively.
After 24 hours of incubation at 37 ℃, the cell culture plates were removed, the medium was discarded with a pipette and washed twice with 200 μl of sterilized PBS buffer to remove free bacteria and enzymes. Then 200. Mu.L of methanol was added to each well and the mixture was allowed to stand for 10 minutes. After the fixation solution was discarded and naturally dried in air, the biofilm was stained with 200. Mu.L of 0.1% by mass crystal violet at room temperature for 10 minutes. After completion of the staining, the staining solution was discarded, and the solution was washed twice with 200. Mu.L of PBS buffer to remove the free staining solution. The cell culture plate was dried in a 50℃oven, 200. Mu.L of 33% strength acetic acid solution was added to each well after removal, and the mixture was placed on a shaker for 30 minutes to release crystal violet in the biofilm. Finally, the absorbance at 595nm was measured using an enzyme-labeled instrument. The residual amount of the biofilm is determined according to the value of OD 595.
As shown in FIG. 7, biofilm formation was significantly reduced in the 4. Mu.g, 0.4. Mu.g and 0.04. Mu.g phage depolymerization enzyme dep253 treated groups. From the above experimental results, it was found that phage depolymerizing enzyme dep253 of the present invention significantly inhibited biofilm formation.
It should be noted that, when numerical ranges are referred to in the present invention, it should be understood that two endpoints of each numerical range and any numerical value between the two endpoints are optional, and in order to prevent redundancy, the present invention describes a preferred embodiment.
While the preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts, all such variations and modifications are within the scope of the invention.
Claims (10)
1. The application of the bacteriophage depolymerizing enzyme dep253 in degrading bacterial capsular polysaccharide is characterized in that the amino acid sequence of the bacteriophage depolymerizing enzyme dep253 is shown as SEQ ID NO. 1.
2. The use of a bacteriophage depolymerase dep253 according to claim 1 for degrading bacterial capsular polysaccharides, wherein said bacteriophage depolymerase dep253 is a metabolite of a klebsiella pneumoniae bacteriophage P253 strain;
The Klebsiella pneumoniae phage P253 strain has a preservation number of CCTCC NO: M20241683, a preservation time of 2024, 7 and 25 days, a preservation unit of China center for type culture collection, and a preservation address of China university of Wuhan.
3. Use of a bacteriophage depolymerizing enzyme dep253 according to claim 1 for degrading bacterial capsular polysaccharides, wherein the nucleotide sequence encoding said bacteriophage depolymerizing enzyme dep253 is shown in SEQ ID No. 2.
4. Use of a bacteriophage depolymerizing enzyme dep253 according to claim 2 for degrading bacterial capsular polysaccharides, characterized in that the preparation method of the bacteriophage depolymerizing enzyme dep253 comprises the following steps:
Obtaining a dep253 gene fragment of the 17 th open reading frame of the Klebsiella pneumoniae bacteriophage P253 strain by means of PCR, double enzyme digestion and connecting molecule cloning, and connecting the dep253 gene fragment to a plasmid vector to obtain a recombinant plasmid;
transferring the recombinant plasmid into a host cell, and screening to obtain a recombinant host cell containing the recombinant plasmid;
culturing the recombinant host cell to obtain a culture solution, and collecting liquid after solid-liquid separation of the culture solution to obtain the phage depolymerizing enzyme dep253.
5. The use of a bacteriophage depolymerizing enzyme dep253 according to claim 4 for degrading bacterial capsular polysaccharides, wherein the plasmid vector comprises pET28a.
6. The use of a bacteriophage depolymerizing enzyme dep253 according to claim 4 for degrading bacterial capsular polysaccharides, wherein the host cell is e.coli DE3.
7. Use of a bacteriophage depolymerizing enzyme dep253 for degrading bacterial capsular polysaccharides according to claim 1, wherein said bacteriophage depolymerizing enzyme dep253 is used for degrading klebsiella pneumoniae capsular polysaccharides and inhibiting biofilm formation.
8. Use of a bacteriophage depolymerizing enzyme dep253 for degrading bacterial capsular polysaccharides according to claim 1, wherein said bacteriophage depolymerizing enzyme dep253 is used for the preparation of bacteriophage sterilizing products;
The phage killing product takes the phage depolymerizing enzyme dep253 as the only active ingredient.
9. The use of a phage depolymerase dep253 in degrading bacterial capsular polysaccharides according to claim 8, wherein the phage-killing product is made of the phage depolymerase dep253 and a solvent;
the concentration of the phage depolymerase dep253 is 0.002 mg/mL-2 mg/mL.
10. Use of a bacteriophage depolymerizing enzyme dep253 according to claim 9 for degrading bacterial capsular polysaccharides, wherein the solvent is PBS;
The concentration of PBS is 0.01mol/L, and the pH is 7.2-7.4.
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| CN119410611A (en) * | 2025-01-06 | 2025-02-11 | 中国科学院青岛生物能源与过程研究所 | Phage polysaccharide degrading enzyme, nucleic acid molecule, expression vector, recombinant cell, bactericidal composition and bactericide and application |
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| CN112501135A (en) * | 2020-12-10 | 2021-03-16 | 南京农业大学 | Klebsiella pneumoniae phage strain P560, phage Depo43 and application |
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| CN112501135A (en) * | 2020-12-10 | 2021-03-16 | 南京农业大学 | Klebsiella pneumoniae phage strain P560, phage Depo43 and application |
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| CN119410611A (en) * | 2025-01-06 | 2025-02-11 | 中国科学院青岛生物能源与过程研究所 | Phage polysaccharide degrading enzyme, nucleic acid molecule, expression vector, recombinant cell, bactericidal composition and bactericide and application |
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