Recombinant bacteriophage displaying phage holins and methods of making and using the same

Abstract

The application belongs to the technical field of bacteriophages, and discloses a recombinant bacteriophage for displaying bacteriophage perforin as well as a preparation method and application of the recombinant bacteriophage. The preparation method of the recombinant bacteriophage comprises the following steps: S1, using a bacteriophage genome as a template, amplifying a bacteriophage perforin coding gene sequence by using a fusion primer to obtain the bacteriophage perforin coding gene sequence, and performing enzyme digestion and recovery on the amplification product and an expression vector; S2, connecting the bacteriophage perforin coding gene sequence with a homologous arm and the enzyme-digested expression vector skeleton, and transforming the same into Escherichia coli to obtain recombinant Escherichia coli; and S3, performing bacteriophage rescue on the recombinant Escherichia coli to obtain the recombinant bacteriophage for displaying bacteriophage perforin. The lysis spectrum of the recombinant bacteriophage prepared by the method is greatly widened, and the recombinant bacteriophage can cross-species lyse various gram-positive bacteria and mycoplasma, and the lysis efficiency is also improved, so that the recombinant bacteriophage has important significance and value in antibacterial treatment and clinical application.

Description

Technical Field

[0001] This invention relates to the field of phage technology, and in particular to a recombinant phage displaying phage perforin, its preparation method, and its application. Background Technology

[0002] With the widespread use of antibiotics in various fields, pathogens are generally exhibiting multidrug resistance, making the prevention and control of various diseases caused by these pathogens increasingly difficult. Therefore, antibiotic resistance (AMR) is a significant global public health security issue.

[0003] Escherichia coli ( Escherichia coli Escherichia coli (E. coli) is one of the nine pathogens of great concern in antibiotic resistance research. It exhibits strong acquired resistance and is commonly found in human medical and veterinary clinical infections. In the United States, approximately 50–60% of community-acquired E. coli infections are resistant to commonly used clinical antibiotics (such as amoxicillin, cefixime, and ciprofloxacin), leading to difficulties in outpatient treatment. Conservative estimates suggest that 17% of severe septicemia cases are caused by E. coli, and approximately 40,000 people died from severe septicemia caused by E. coli in 2001. The direct cost of treating E. coli is estimated to be at least $1.1-2.8 billion annually in the United States. Antibiotic-resistant E. coli has been reported in fruits and vegetables worldwide. Therefore, the development of safe and antibiotic-alternative prevention and control strategies is urgently needed.

[0004] bacteriophage ( Bacteriophages Bacteriophages are viruses capable of specifically infecting bacteria. Although the first case of using bacteriophages to treat bacterial infections occurred a century ago, the widespread use of antibiotics has hindered research into their application. However, in recent years, with the increasing prevalence of super-resistant bacteria posing a serious challenge to clinical practice, phage therapy has gradually become a research hotspot as an antibiotic alternative. Compared to traditional antibiotics, bacteriophages have several important advantages: 1) High specificity: They are specific to bacteria, even specific strains and species, do not infect human cells, and have little or no impact on the normal microbiota, making them safe to use; 2) Bacteriophages possess extremely rich diversity and multifunctionality, and there are virtually unlimited phages in the natural environment. Therefore, various treatment strategies can be designed and implemented, including conventional phage therapy, phage cocktails, phage-derived proteins, and combined therapies with antibiotics and the immune system. Studies have shown that, to date, phages and their preparations have rapidly lysed target strains without causing harm to human or animal cells, demonstrating extremely high biosafety. Furthermore, with ongoing research, more and more phages are being discovered and applied in various fields.

[0005] However, existing bacteriophages have a narrow lysis spectrum due to their specific lysis specificity against the host bacteria, which limits their application. Bacteriophage resources with a wide lysis spectrum, especially those capable of cross-species lysis, are extremely rare.

[0006] Therefore, the existing technology needs further improvement. Summary of the Invention

[0007] To address the aforementioned problems, this invention provides a recombinant phage displaying a broad lysis spectrum and high lysis efficiency, and also provides a method for preparing this recombinant phage. By displaying phage perforin on its surface, this phage significantly improves its cross-species lysis spectrum and lysis efficiency, and can be widely used in the inhibition of various pathogens and the prevention and control of related diseases.

[0008] To achieve the above objectives, this application provides the following technical solution:

[0009] In a first aspect, this application provides a method for preparing a recombinant phage displaying phage perforin, comprising the following steps:

[0010] S1. Using the phage genome as a template, the phage perforin coding gene sequence was amplified with fusion primers to obtain the phage perforin coding gene sequence with homologous arms. The amplification product was recovered. The expression vector was digested with enzymes, and the digested expression vector backbone was recovered.

[0011] S2. The phage perforin coding gene sequence with homologous arms is ligated to the enzyme-digested expression vector backbone through homologous recombination, and the ligation product is then transformed into competent Escherichia coli cells to obtain recombinant Escherichia coli.

[0012] S3. Then, use an auxiliary phage to perform phage rescue on the recombinant Escherichia coli obtained in the previous step to obtain a recombinant phage displaying phage perforin.

[0013] Perforin is a hydrophobic membrane protein with a high charge and a C-terminal domain, expressed by bacteriophages in the late transcriptional stage. Within a specific timeframe, it spontaneously assembles into oligomers, forming non-specific transmembrane channels in the cell membrane. This damages the cell membrane, allowing the release of intracellular lysins, ultimately leading to bacterial death or lysis. In vitro, perforin can cause cell death without the use of endosomalins. The lethal effect of perforin on bacterial viability is primarily due to the pores formed by perforin in the cell membrane, not cell rupture. Perforin exhibits good antibacterial activity against both Gram-positive and Gram-negative bacteria. Perforin acts like a "molecular timer," determining the end of the infection cycle and making the bacterial lysis process somewhat controllable.

[0014] Phage display technology is a molecular technique that utilizes phage DNA gene modification to express target substances on the phage surface by binding peptide, protein, or antibody fragments to the phage coat protein. This process involves introducing a foreign DNA sequence into a specific location in the phage genome nucleotide sequence. During phage infection, the phage gene begins to be expressed within the bacterial host, and the inserted peptide or antibody fragment, as a combination product encoding the coat protein and the cloning sequence, can be effectively displayed on the phage surface.

[0015] This application innovatively utilizes phage display technology to display phage perforin protein on the phage surface, enabling the recombinant phage prepared by the above method to directly act on the bacterial cell membrane, eliminating the need for the recognition-invasion-release process required by traditional phages. Furthermore, the recombinant phage displaying perforin can improve the highly specific bactericidal mechanism of phages to a certain extent. Upon adsorption onto the bacterial surface, the perforin on the surface can exert its bactericidal effect, thus significantly expanding the range of bacteria it inhibits. In addition, after the displayed phage invades the host bacteria, it can proliferate extensively within the host, thereby increasing perforin production and improving lysis efficiency.

[0016] Therefore, the recombinant phages obtained by the above-described method for preparing recombinant phages displaying phage perforin in this application greatly expand the lysis spectrum and improve lysis efficiency, showing great application potential. Experiments have demonstrated that the recombinant phages can not only lyse Gram-positive bacteria such as Bacillus subtilis and Staphylococcus aureus across species, but also exhibit excellent antibacterial activity against mycoplasma, while existing control phages lack these activities, representing a significant breakthrough in their biological lysis activity.

[0017] Furthermore, compared to traditional antibiotics, bacteria are less likely to develop resistance to the recombinant phage due to its unique mechanism of action based on bacteriophages and perforin. This method and the resulting recombinant phage are of great significance for the treatment of drug-resistant bacterial infections. Therefore, phage display of perforin provides a new approach and method for antibacterial therapy and can serve as a novel antibacterial agent, holding significant importance and value in antibacterial treatment and clinical applications.

[0018] In one optional embodiment, the phage perforin is the perforin protein of Escherichia coli phage DE7, whose amino acid sequence is shown in SEQ ID NO. 1.

[0019] Preferably, the nucleotide sequence of the gene encoding the Escherichia coli bacteriophage perforin protein is shown in SEQ ID NO. 2.

[0020] Based on the selection of the perforin protein of E. coli phage DE7 in the above method, in step S1, the fusion primers are: holDE7-F with the sequence as described in SEQ ID NO.5 and holDE7-R with the sequence as described in SEQ ID NO.6, and the primer sequences are as follows:

[0021] holDE7-F:

[0022] 5′-tgttcctttctatgcggcccagccggccATGAAGTGGCTAGAGGAGGCTTT-3′

[0023] holDE7-R:

[0024] 5′-ggcaccggcgcacctgcggccgcTTACTTTTCAGTATTCTTTGTGTTCTTTTC-3′

[0025] Alternatively, in another embodiment, the phage perforin is the perforin of Pseudomonas aeruginosa phage, the amino acid sequence of which is shown in SEQ ID NO. 3.

[0026] Preferably, the sequence of the gene encoding the Escherichia coli bacteriophage perforin protein is shown in SEQ ID NO. 4.

[0027] In other cases, the phage perforin protein may be selected from other existing phage perforin proteins, and the protein is not limited to the types listed in the embodiments of this application.

[0028] Based on the perforin protein selection of Pseudomonas aeruginosa phage holASP in the above method, in step S1, the fusion primers used are: holASP-F with the sequence as described in SEQ ID NO.7 and holASP-R with the sequence as described in SEQ ID NO.8, and the primer sequences are as follows:

[0029] holASP-F:

[0030] 5′-tgttcctttctatgcggcccagccggccatgatgattgataccgccac-3′

[0031] holASP-R:

[0032] 5′-ggcaccggcgcacctgcggccgctcacttcttgaatctccggc-3′

[0033] Optionally, in the method for preparing recombinant phages displaying phage perforin, the expression vector is pCANTAB 5E.

[0034] Optionally, the expression vector may also be an expression vector suitable for 3+3 / 8+8 phage display types, such as pComb3. The 3+3 / 8+8 type refers to a display system based on phage particles and helper phages, where a foreign gene is linked to the phage coat protein, while the wild-type gene is present on the helper phage genome. Display is achieved through the regulation of DNA replication and transcription.

[0035] Optionally, the helper phage is M13K07.

[0036] Optionally, the helper phage may also include other helper phages suitable for phage display of 3+3 / 8+8 types, such as VCSM13, but not limited thereto.

[0037] Secondly, this application also provides a recombinant phage displaying phage perforin, which is prepared using any of the methods described above.

[0038] Thirdly, this application also provides the application of the above-mentioned recombinant phage in antibacterial activity.

[0039] Optionally, the recombinant phage can be used to inhibit bacteria or fungi such as Bacillus subtilis, Staphylococcus aureus, and Streptococcus, and to prevent and control diseases caused by these pathogens.

[0040] Fourthly, this application also provides the application of the above-mentioned recombinant phage in inhibiting mycoplasma.

[0041] The mycoplasma is mycoplasma of sheep origin, mycoplasma of pig origin, or mycoplasma of chicken origin.

[0042] The present invention has the following beneficial effects:

[0043] 1. The recombinant phage provided by this invention utilizes phage display technology to display perforin protein on the phage surface, allowing it to directly act on the bacterial cell membrane without the need for the recognition-invasion-release process required by traditional phages. Furthermore, the perforin-displayed phage can improve the highly specific bactericidal mechanism of phages to a certain extent. Upon adsorption onto the bacterial surface, the perforin on the surface can exert its bactericidal effect, expanding the range of bacteria it inhibits and exhibiting a good synergistic effect. Simultaneously, after the displayed phage invades the host bacteria, it can proliferate extensively within the host, thereby increasing perforin production and improving lysis efficiency.

[0044] 2. Compared with traditional antibiotics, bacteria are less likely to develop resistance due to the unique mechanisms of action of bacteriophages and perforin, which is of great significance for the treatment of drug-resistant bacterial infections. Therefore, bacteriophage-displayed perforin provides a new approach and method for antibacterial therapy and can serve as a novel antibacterial agent with significant meaning and value in antibacterial treatment and clinical applications.

[0045] 3. The resulting recombinant phage has both the advantages of phage and the sterilization activity of perforin, thus improving the lysis spectrum and lysis efficiency.

[0046] 4. The recombinant Escherichia coli phage provided in this application has significant antibacterial activity against Gram-positive bacteria and mycoplasma, while the original phage did not have this antibacterial activity. Attached Figure Description

[0047] Figure 1 The results are shown in the electrophoresis diagram of the perforin gene; M is the marker, and 1, 2, and 3 are the amplification maps of the perforin gene of E. coli phage DE7.

[0048] Figure 2 These are the PCR electrophoresis results of single-clone colonies after proliferation; M: marker, 1, 2, 3, 4, 5, 6. PCR samples of single-clone colonies after homologous recombination;

[0049] Figure 3 Plates for measuring the DE7 titer of recombinant phage after rescue;

[0050] Figure 4 The results of the antibacterial experiment on phage DE7 are shown below; the results, in order, show the antibacterial activity of recombinant phage DE7 and control phage M13K07 against bacteria. B. subtilis WB800N, strain S.aureus ATCC 29213 and strain S. pneumoniae The antibacterial results of CICC24947;

[0051] Figure 5 The inhibitory effect of recombinant phage DE7 on mycoplasma is shown in column A1 (sheep mycoplasma experimental group), column A2 (swine mycoplasma experimental group), and column A3 (chicken mycoplasma experimental group). Columns 1-12 represent samples diluted 10, 20, 40, 80, 160, 320, 640, 1280, and 2560 times with recombinant phage DE7, the original phage control, the tiamulin control, and the culture medium control group, respectively.

[0052] Figure 6 To demonstrate the antibacterial experimental results of the recombinant holASP phage; the antibacterial results of the recombinant holASP phage and the control phage against three pathogens M13K07 are shown respectively;

[0053] Figure 7To demonstrate the inhibitory effect of recombinant holASP phage on mycoplasma, A1 represents the sheep mycoplasma experimental group, A2 represents the pig mycoplasma experimental group, and A3 represents chicken mycoplasma. Columns 1-12 represent samples with 10, 20, 40, 80, 160, 320, 640, 1280, and 2560-fold dilutions of the recombinant holASP phage, the original phage control, the tiamulin control, and the culture medium control group, respectively. Detailed Implementation

[0054] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. In the present invention, unless otherwise specified, the equipment and raw materials used can be purchased from the market or are commonly used in the art. Unless otherwise specified, the methods in the following embodiments are conventional methods in the art.

[0055] Experimental materials

[0056] 1. Strains and plasmids

[0057] Escherichia coli competent cells TG1: purchased from Beijing Tiangen Biotech Co., Ltd.

[0058] Expression vector: pCANTAB 5E plasmid was used, which was purchased from Novengen.

[0059] Phage: Helper phage M13K07 was used, purchased from New England Biolabs.

[0060] strain B. subtilis WB800N (Bacillus subtilis) was purchased from Changsha Aibiwei Biotechnology Co., Ltd.

[0061] strain S.aureus ATCC 29213 (Staphylococcus aureus), purchased from Haibo Biotechnology.

[0062] S.aureus ATCC 25923 (Staphylococcus aureus), purchased from Haibo Biotechnology.

[0063] Streptococcus pneumoniae S. pneumoniae CICC 24947 was purchased from the China Industrial Microbial Culture Collection Center.

[0064] 2. Culture medium and reagents

[0065] NB medium: purchased from BD Company. 10g tryptone, 5g yeast extract, 10g sodium chloride, add water to 1L. Solid NB medium: add 15g agar powder to NB medium. Autoclave at 121℃ for 20min.

[0066] LB medium: Tryptone 10g / L, yeast extract 5g / L, sodium chloride 10g / L, add water to 1L. Solid LB medium: Add 15g agar powder to LB medium. Autoclave at 121℃ for 15min.

[0067] LB / 2% Glu-Amp medium: Based on the above LB medium preparation, add 20 g / L glucose, and when the culture medium temperature drops to 50℃, add Amp (final concentration 0.1 mg / mL).

[0068] 2×YT / 2% Glu / Kan+Amp medium: Add the finished 2×YT medium (31g / L) to an Erlenmeyer flask, dissolve it, seal the flask, and sterilize it in an autoclave. Before use, add Amp (final concentration 0.1mg / mL) and Kan (final concentration 0.025mg / mL).

[0069] PEG6000 / NaCl solution: Add distilled water to the required culture medium volume into the conical flask. Weigh out the corresponding PEG (400g / L) and NaCl (146g / L) according to the ratio using an electronic balance and add them to the conical flask. After dissolving, seal the flask and place it in an autoclave at 121℃ for 15 minutes for sterilization.

[0070] Example 1 demonstrates the construction of a recombinant phage of DE7 perforin from Escherichia coli.

[0071] This embodiment provides a method for preparing a recombinant phage displaying Escherichia coli phage DE7 perforin.

[0072] 1. Experimental Method:

[0073] 1.1.1 Proliferation of TG1 bacterial culture

[0074] The inoculation loop was dipped into TG1 preservation solution and streaked onto an LB agar plate for activation. The plate was then incubated overnight at 37°C. A single colony was picked and inoculated into 5 mL of LB liquid medium to obtain the bacterial culture.

[0075] 2. Preparation of TG1 competent cells

[0076] (1) Incubate 5 mL of TG1 at 37℃ for about 2-3 h (to reach the logarithmic phase), then transfer to a 15 mL centrifuge tube.

[0077] (2) Ice bath for 10 min, 4℃, 4000 rpm, 10 min, discard the supernatant.

[0078] (3) Resuspend in 200 µL of Cacl2 (0.1 mol / mL) (pre-cooled), 4℃, 4000 rpm, 10 min, and discard the supernatant.

[0079] (4) Repeat (3).

[0080] (5) Resuspend the sample in 200 µL of a 15% glycerol aqueous solution containing a final concentration of 0.1 mol / mL CaCl2, mix well, and dispense 100 µL / tube at -80℃.

[0081] 3. Extraction of plasmid pCANTAB 5E

[0082] Using an inoculation loop, streak the TG1 Escherichia coli containing pCANTAB 5E onto an LB agar plate (containing 0.1% Amp) for activation. Incubate overnight at 37°C. Pick a single colony and inoculate it into 5 mL of LB liquid medium (containing 0.1% Amp). Culture to obtain bacterial culture. Extract plasmid pCANTAB 5E using a plasmid extraction kit.

[0083] 4. Amplification of M13K07 helper phage

[0084] (1) Add 20 µL of M13K07 proliferation solution to 20 ml TG1 (OD=0.5) and adsorb at 37 ℃ for 30 min. Add Kan and incubate overnight at 37 ℃ and 200 rpm.

[0085] (2) Transfer to a centrifuge tube, centrifuge at 4 ℃ and 8500 rpm for 20 min. Transfer the supernatant to a beaker and add an equal volume of saturated ammonium sulfate (add 1 mL of distilled water to the EP tube and then add solid ammonium sulfate until the ammonium sulfate no longer dissolves). Stir magnetically on ice for 30 min. Centrifuge the liquid at 4 ℃ and 6000 rpm for 20 min, and discard the supernatant.

[0086] (3) Resuspend 1 ml of finished TBS (pH=7.4) at 14000 rpm for 2 min, collect the supernatant, mix with 50% glycerol and store.

[0087] The proliferation titer of M13K07 helper phage is 1×10⁻⁶. 9 PFU / ml, the concentrated potency is 1.9×10 10 PFU / ml.

[0088] 5. Primer design and amplification of the DE7 perforin gene from bacteriophages

[0089] The primers for phage DE7 perforin amplification are as follows:

[0090] holDE7-F: 5′-tgttcctttctatgcggcccagccggccATGAAGTGGCTAGAGGAGGCTTT-3′ (as shown in SEQ ID NO.5)

[0091] holDE7-R: 5′-ggcaccggcgcacctgcggccgcTTACTTTTCAGTATTCTTTGTGTTCTTTTC-3′ (as shown in SEQ ID NO. 6)

[0092] Note: In the primers above, lowercase letters represent vector homologous fragments, and uppercase letters represent phage lysin sequences. The target gene is inserted between the Sfi I and NotI restriction sites.

[0093] Using the DE7 phage genome as a template, 2 μL of the above primers were added, and the target gene was amplified by PCR using 2×Accurate TaqMaster Mix.

[0094] The PCR program was as follows: (1) 95℃, 5min; (2) 95℃, 1min, 68℃, 30s, 72℃, 30s, 30 cycles; (3) 72℃, 10min.

[0095] The amplification products were detected by electrophoresis, and the electrophoresis results are as follows: Figure 1 As shown, the band size of the PCR product is as expected. The PCR product was recovered by gel electrophoresis and used for subsequent experiments.

[0096] 6. Enzyme digestion of pCANTAB 5E expression vector

[0097] (1) Sfi I enzyme digestion

[0098] Table 1. Sfi I enzyme digestion system

[0099]

[0100] Incubate in a water bath at 37 ℃ for 6 h. After enzyme digestion, recover the digested product using a PCR product recovery kit and determine its concentration.

[0101] (2) Not I enzyme digestion

[0102] Table 2 Not I enzyme digestion system

[0103]

[0104] Incubate in a water bath at 37 ℃ for 6 h. After enzyme digestion, recover the digested product using a PCR product recovery kit and determine its concentration.

[0105] 7. Homologous recombination transformation and positive identification

[0106] (1) The aforementioned target fragment was ligated into the enzyme-digested pCANTAB 5E expression vector using a homologous recombination kit (VazymeC113-02).

[0107] Table 3 Homologous recombination system

[0108]

[0109] After bathing in water at 37 ℃ for 10 minutes, immediately place it in ice.

[0110] (2) Transformation of Escherichia coli:

[0111] Add 10 µL of the homologous recombination product and 1 µL of the empty vector control to two tubes containing 100 µL of TG1 competent cells, respectively. Mix well by tapping, incubate on ice for 30 min, heat shock at 42 ℃ for 90 s, then incubate on ice for 120 s. Take 1 ml of LB and add it to a 1.5 ml EP tube, incubate at 37 ℃ and 220 rpm for 1 h, then incubate at 10000 rpm for 3 min. Discard the supernatant (reserve 100 µL), resuspend the precipitate, and spread it on an LB / 2% Glu-Amp plate. Incubate at 37 ℃ for 12 h.

[0112] (3) Positive identification

[0113] Using the bacterial culture after monoclonal proliferation as a template, primer PCR identification was performed, and positive clones were obtained. The system procedure was the same as described in step 5. This yielded TG1 transformed with recombinant pCANTAB-holDE7, which was then used for the next step of phage rescue.

[0114] 8. Phage rescue

[0115] (1) 200 µL of Escherichia coli TG1 carrying pCANTAB-holDE7 recombinant plasmid was added to 200 ml of 2×YT / 2% Glu-Amp and incubated at 37 °C for 225 r / min to the logarithmic phase to obtain bacterial culture.

[0116] (2) Add 30 µL of 7.8×10 to the bacterial culture in 8(1). 13 PFU / ml of helper phage M13K07 was gently mixed and then allowed to stand at 37 °C for 40 min to obtain the mixture.

[0117] (3) Centrifuge the mixture in 8(2) at 25 ℃ and 3000×g for 20 min, discard the supernatant, resuspend the precipitate in 400 ml of 2×YT / Kan+Amp and treat it at 37 ℃ and 225 rpm for 14 h.

[0118] (4) The bacterial solution obtained in the previous step was incubated at 4 ℃ and 4500× g Centrifuge for 35 min under the specified conditions, collect the supernatant (do not mix in the precipitate), divide the supernatant into two sterile 500 ml Erlenmeyer flasks, add 40 ml of PEG6000 / NaCl solution to each flask, mix well, and incubate on ice for 12-24 h.

[0119] (5) Then at 4 ℃ 6000× g Centrifuge for 1 h under the specified conditions, discard the supernatant, resuspend the precipitate in 800 µL of 1×PBS (pH 7.4), transfer to a sterile 1.5 ml EP tube, and incubate at 4 ℃, 225 rpm for 12–14 h on a shaker, followed by incubation at 4 ℃ and 13000 × 10⁻⁶ rpm. g After 20 minutes, the supernatant was collected to obtain the recombinant phage, which was stored at 4 °C in the dark for subsequent titer determination.

[0120] 9. Titer determination of recombinant phages

[0121] (1) Take 30 µL of the above recombinant phage supernatant and perform 10-fold serial dilutions using 2×YT / 2%Glu-Amp medium. Then take the 10-fold dilution. -7 ~10 -10 200 µL of recombinant phage supernatant was mixed with an equal volume of Escherichia coli TG1 culture cultured to the logarithmic phase and incubated at 37 °C for 35 min.

[0122] (2) Spread the mixed bacterial solution after standing on LB / 2%Glu-Amp plates, absorb at 4℃ for 30 min, incubate at 37℃ for 12 h, count the number of single colonies growing on the plates, and calculate the titer of recombinant phage.

[0123] The results are as follows Figure 3 As shown, the titer of the recombinant phage was determined to be 5 × 10⁻⁶. 8 pfu / mL.

[0124] Example 2 demonstrates the application of recombinant phage DE7 perforin from Escherichia coli.

[0125] 1. Plate antibacterial test

[0126] (1) Experimental methods

[0127] strains B. subtilis WB800N (Bacillus subtilis), strain S.aureus ATCC 29213 (Staphylococcus aureus) and strain S. pneumoniae CICC 24947 (Streptococcus pneumoniae) was used as the experimental strain to determine the antibacterial activity of the recombinant phage prepared in Example 1.

[0128] The method for determining antibacterial activity is as follows: Select strains B. subtilis WB800N (Bacillus subtilis), strain S.aureus ATCC29213 (Staphylococcus aureus) and strain S. pneumoniae CICC 24947 (Streptococcus pneumoniae) was inoculated into 5 mL of NB liquid medium for activation (37°C) and cultured overnight. 100 μL of the bacterial culture was mixed with 15 mL of NB solid medium and poured onto a plate. After solidification, 10 μL of the recombinant phage sample was spotted onto the plate. The plate was then placed in a 4°C refrigerator for 30 min to allow absorption of the liquid, and then transferred to a 37°C incubator for further incubation. The activity of the recombinant phage was observed.

[0129] (2) Experimental results and analysis

[0130] like Figure 4 As shown, the recombinant phage from Example 1 and the control phage M13K07 were respectively dropped onto... B. subtilis WB800N, S.aureus ATCC29213 and S. pneumoniae On CICC 24947 plates, after overnight incubation, the recombinant phage displaying DE7 formed an inhibition zone, while the control phage M13K07 showed no inhibitory effect.

[0131] The above results show that the recombinant phage can inhibit Gram-positive bacteria such as Bacillus subtilis, Staphylococcus aureus, and Streptococcus, and is no longer limited to lysing Gram-negative bacteria such as Escherichia coli. Its lysis spectrum has been greatly broadened. This indicates that the recombinant phage displaying phage perforin can greatly expand the lysis spectrum, enabling it to perform cross-species lysis and greatly improving its lysis performance.

[0132] 2. Inhibitory effect of recombinant bacteriophages on mycoplasma

[0133] This experiment used three types of mycoplasma as experimental subjects: Mycoplasma hyopneumoniae in sheep, Mycoplasma hyopneumoniae in pigs, and Mycoplasma gallisepticum.

[0134] 2.1 Drug susceptibility testing

[0135] (1) The aforementioned recombinant phage sample (titer 5 × 10⁻⁶) was cultured in mycoplasma broth medium. 8 (pfu / mL) diluted 5 times.

[0136] (2) Add 100 μL of mycoplasma broth medium to columns 2-12;

[0137] (3) Take 100 μL of the 5-fold diluted sample and add it to columns 1-2;

[0138] (4) After mixing the second column, take 100ul to the third column, mix well, and take 100ul to the fourth column. Continue this process until the tenth column, then discard 100ul.

[0139] (5) Dilute each fresh culture of mycoplasma with the corresponding culture medium at a ratio of 1:10000; the preparation method of each fresh culture of mycoplasma is as follows: add 1800ul of culture medium to a 5ml stoppered test tube, aspirate 200ul of the original mycoplasma liquid, mix well, and culture at 37℃ for about 3-4 days under microaerophilic conditions.

[0140] (6) Add 100 μL of diluted mycoplasma to each well in columns 1 to 10;

[0141] (7) Seal the cell culture plate with sealing film and observe at 37℃ for 10 days.

[0142] (8) Record the time of color change.

[0143] 2.2 Detection of the inhibitory effect of recombinant phage on mycoplasma

[0144] (1) 180 μL of culture medium per well in columns 1-12;

[0145] (2) Add 20 μL of fresh culture to column 1, mix, and then transfer 20 μL to column 2; replace the tip, mix, and then transfer 20 μL to column 3; continue mixing and transferring to column 11. Column 12 is the culture medium control.

[0146] (3) Seal the cell culture plate with sealing film, observe at 37℃ for 10 days, and record the color change.

[0147] 2.3 Experimental Results and Analysis

[0148] After the above treatment, row A1 was designated as the sheep mycoplasma experimental group, row A2 as the swine mycoplasma experimental group, and row A3 as the chicken mycoplasma experimental group. Wells in columns 1-12 contained, respectively, samples diluted 10, 20, 40, 80, 160, 320, 640, 1280, and 2560 times with recombinant phage, a prophage control, a tiamulin control, and a culture medium control group. When the solution in the well turned yellow, it indicated that the phage had no inhibitory activity against mycoplasma.

[0149] like Figure 5 As shown, the recombinant phage, after being diluted 20-40 times, still exhibited antibacterial activity against Mycoplasma sheepii, Mycoplasma suis, and Mycoplasma gallisepticum, with a relatively significant antibacterial effect.

[0150] Example 3 demonstrates the preparation and application of recombinant phage containing the perforin holASP from Pseudomonas aeruginosa.

[0151] 1. Demonstration of the preparation of recombinant phages containing the perforin holASP of Pseudomonas aeruginosa.

[0152] The method for preparing the recombinant phage containing the Pseudomonas aeruginosa phage perforin holASP is the same as in Example 1, except that the original perforin is replaced with Pseudomonas aeruginosa phage perforin, the amino acid sequence of which is shown in SEQ ID NO.3, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.4. The titer of the recombinant phage prepared after rescue is 6 × 10⁻⁶. 8 PFU / ml.

[0153] 2. Demonstrating the application of recombinant phages containing the perforin holASP from Pseudomonas aeruginosa.

[0154] (1) Plate antibacterial test

[0155] A. Experimental Methods

[0156] strains B. subtilis WB800N (Bacillus subtilis), strain S.aureus ATCC29213 (Staphylococcus aureus) and strain S.aureus ATCC 25923 (Staphylococcus aureus) was used as the experimental strain to test the antibacterial activity of the perforin holASP recombinant phage.

[0157] The method for determining antibacterial activity is as follows: Select strains B. subtilis WB800N (Bacillus subtilis), strain S.aureus ATCC29213 (Staphylococcus aureus) and strain S.aureus ATCC 25923 (Staphylococcus aureus) was inoculated into 5 mL of NB liquid medium for activation (37°C) and cultured overnight. 100 μL of the bacterial culture was mixed with 15 mL of NB solid medium and poured onto a plate. After solidification, 10 μL of the recombinant phage sample was spotted onto the plate. The plate was then placed in a 4°C refrigerator for 30 min to allow absorption of the liquid, and then transferred to a 37°C incubator for further incubation. The activity of the recombinant phage was observed.

[0158] B. Experimental Results and Analysis

[0159] Recombinant phages of perforin holASP (10) will be displayed. 8 (pfu / mL) and control phage M13K07 were respectively dropped onto... B. subtilis WB800N, S.aureus ATCC29213 and S.aureus After overnight incubation on an ATCC 25923 plate. (The text abruptly ends here.) Figure 6As shown, the treatment group with recombinant holASP perforin formed inhibition zones on the plate, while the control phage M13K07 had no inhibitory effect on the three pathogens mentioned above.

[0160] This demonstrates that phage M13K07, after exhibiting perforin holASP, has an expanded lysis spectrum, enabling it to perform cross-species lysis and exhibiting good lysis performance against Bacillus subtilis and Staphylococcus aureus.

[0161] 2. Inhibitory effect of recombinant bacteriophages on mycoplasma

[0162] A. Experimental Methods

[0163] The method is the same as in Example 2.

[0164] The settings on the well plate are as follows: Row A1 is the sheep mycoplasma experimental group, and Row A2 is the porcine mycoplasma experimental group; columns 1-12 are respectively filled with recombinant phage diluted 10, 20, 40, 80, 160, 320, 640, 1280, and 2560 times, prophage control, tiamulin control, and culture medium control (e.g., ...). Figure 7 (As shown).

[0165] B. Experimental Results and Analysis

[0166] The results are as follows Figure 7 As shown in the figure, the results indicate that the recombinant phage of this embodiment, after being diluted 10-20 times, has an antibacterial effect against Mycoplasma sheepii, Mycoplasma swineii, and Mycoplasma gallisepticum. Figure 7 If the solution turns yellow, it indicates that the bacteriophage has no antibacterial activity against mycoplasma.

[0167] It is understood that those skilled in the art can make equivalent substitutions or modifications to the technical solutions and concepts of this invention, and all such substitutions or modifications should fall within the protection scope of the appended claims.

Claims

1. The application of a recombinant phage displaying phage perforin in the preparation of antibacterial pathogen products, characterized in that, The method for preparing the recombinant phage includes the following steps: S1. Using the phage genome as a template, the phage perforin coding gene sequence is amplified using fusion primers to obtain the phage perforin coding gene sequence with homologous arms, and the amplification product is recovered; the expression vector is digested with enzymes, and the digested expression vector backbone is recovered; the fusion primers are: holDE7-F with the sequence shown in SEQ ID NO.5 and holDE7-R with the sequence shown in SEQ ID NO.6; S2. The phage perforin coding gene sequence with homologous arms was ligated to the enzyme-digested expression vector backbone by homologous recombination, and the ligation product was then transformed into competent Escherichia coli cells and recombinant Escherichia coli was obtained by screening. S3. Then, use the helper phage to perform phage rescue on the recombinant E. coli obtained in the previous step to obtain recombinant phage displaying phage perforin; The phage perforin is the perforin protein of Escherichia coli phage DE7, and its amino acid sequence is shown in SEQ ID NO.1; The expression vector is pCANTAB 5E; the helper phage is M13K07; The pathogens are Bacillus subtilis, Staphylococcus aureus, or Streptococcus.

2. The application of a recombinant phage displaying phage perforin in the preparation of a mycoplasma-inhibiting product, characterized in that, The method for preparing the recombinant phage includes the following steps: S1. Using the phage genome as a template, the phage perforin coding gene sequence is amplified using fusion primers to obtain the phage perforin coding gene sequence with homologous arms, and the amplification product is recovered; the expression vector is digested with enzymes, and the digested expression vector backbone is recovered; the fusion primers are: holDE7-F with the sequence shown in SEQ ID NO.5 and holDE7-R with the sequence shown in SEQ ID NO.6; S2. The phage perforin coding gene sequence with homologous arms was ligated to the enzyme-digested expression vector backbone by homologous recombination, and the ligation product was then transformed into competent Escherichia coli cells and recombinant Escherichia coli was obtained by screening. S3. Then, use the helper phage to perform phage rescue on the recombinant E. coli obtained in the previous step to obtain recombinant phage displaying phage perforin; The phage perforin is the perforin protein of Escherichia coli phage DE7, and its amino acid sequence is shown in SEQ ID NO.1; The expression vector is pCANTAB 5E; the helper phage is M13K07; and the mycoplasma is Mycoplasma ovis, Mycoplasma hyopneumoniae, or Mycoplasma gallisepticum.

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