Recombinant bacteriophage for displaying bacteriophage perforin as well as preparation method and application of recombinant bacteriophage

Abstract

The invention belongs to the technical field of bacteriophages, and discloses a recombinant bacteriophage for displaying bacteriophage perforin and a preparation method and application thereof.The preparation method of the recombinant bacteriophage comprises the following steps that S1, a bacteriophage genome is used as a template, a fusion primer is used for amplifying a bacteriophage perforin coding gene sequence, and a recombinant bacteriophage is obtained; obtaining a bacteriophage perforin coding gene sequence, and carrying out enzyme digestion and recovery on an amplification product and an expression vector; s2, connecting a bacteriophage perforin coding gene sequence with a homologous arm with the expression vector skeleton subjected to enzyme digestion, and transforming into Escherichia coli to obtain recombinant Escherichia coli; and S3, carrying out phage rescue on the recombinant escherichia coli to obtain the recombinant phage displaying the phage perforin. The lysis spectrum of the recombinant bacteriophage prepared by the method is greatly widened, various gram-positive bacteria and mycoplasmas can be subjected to cross-species lysis, the lysis efficiency is also improved, and the recombinant bacteriophage has important significance and value in the aspects of antibacterial treatment, clinical application and the like.

Description

Technical Field

[0001] The present invention relates to the field of bacteriophage technology, and in particular to a recombinant bacteriophage displaying bacteriophage perforin, and a preparation method and application thereof. Background Art

[0002] With the promotion and use of antibiotics in various fields, pathogens generally have multi-drug resistance problems, making the prevention and treatment of various diseases caused by pathogens more difficult. Therefore, antibiotic resistance (Antimicrobial Resistance, AMR) is an important issue of global public health security.

[0003] Escherichia coli ( Escherichia coli ) is one of the nine pathogens that have received much attention in antibiotic resistance research. It has a strong ability to acquire drug resistance and is common in human and veterinary clinical infections. In the United States, about 50–60% of community-acquired E. coli infections are resistant to commonly used clinical antibiotics (such as amoxicillin, cefixime, and ciprofloxacin), making outpatient treatment difficult. According to conservative estimates, 17% of severe sepsis cases are caused by E. coli. In 2001, about 40,000 people died from severe sepsis caused by E. coli. It is estimated that the direct cost of E. coli treatment is at least $1.1-2.8 billion per year in the United States. It is reported that antibiotic-resistant E. coli have been detected in fruits and vegetables worldwide. Therefore, it is urgent to develop safe and alternative antibiotic prevention and treatment plans.

[0004] Bacteriophage ( Bacteriophages ) is a virus that can specifically infect bacteria. Although the first case of using phages to treat bacterial infections appeared a century ago, the widespread use of antibiotics has hindered the application of phages. In recent years, as more super-resistant bacteria pose a severe challenge to clinical practice, phage therapy has gradually become a research hotspot as an alternative to antibiotics. Compared with traditional antibiotics, phages have several important advantages: 1) High specificity: They are specific to bacteria and even specific strains and species of bacteria, do not infect human cells, and have little or no effect on normal microbial flora, making them safe to use; 2) Phages have extremely rich diversity and versatility, and there are almost unlimited phages in the natural environment, so a variety of treatment strategies can be designed and implemented, including conventional phage therapy, phage cocktails, phage-derived proteins, and combined treatments with antibiotics and the immune system. Studies have shown that so far, phages and their preparations have not been found to cause harm to human or animal cells while rapidly lysing target strains, showing extremely high biosafety. At present, with the progress of research, more and more phages have been discovered and applied in various fields.

[0005] However, the existing phages have a narrow lysis spectrum due to their specific lysis specificity for host bacteria, which limits their application. There are very few phage resources with a wide lysis spectrum, especially cross-species lysis.

[0006] Therefore, the prior art needs to be further improved. Summary of the invention

[0007] In view of the above problems, the present invention provides a recombinant phage displaying phage perforin with a wide cleavage spectrum and high cleavage efficiency, and also provides a method for preparing the recombinant phage. By displaying phage perforin on the surface, the phage greatly improves its cross-species cleavage spectrum and cleavage efficiency, and can be widely used in the inhibition of various pathogens and the prevention and treatment of related diseases.

[0008] To achieve the above objectives, this application provides the following technical solutions: In a first aspect, the present application provides a method for preparing a recombinant bacteriophage displaying bacteriophage perforin, which comprises the following steps: S1. Using the bacteriophage genome as a template, amplifying the bacteriophage perforin encoding gene sequence with a fusion primer to obtain the bacteriophage perforin encoding gene sequence with homology arms, and recovering the amplified product; performing enzyme digestion on the expression vector, and recovering the expression vector skeleton after enzyme digestion; S2, connecting the bacteriophage perforin encoding gene sequence with homology arms to the expression vector backbone after enzyme digestion through homologous recombination, and then transforming the connection product into Escherichia coli competent cells to obtain recombinant Escherichia coli; S3. Reusing the helper phage to perform phage rescue on the recombinant Escherichia coli obtained in the previous step to obtain a recombinant phage displaying phage perforin.

[0009] Perforin protein is a hydrophobic membrane protein with high charge and carboxyl terminal domain that is expressed by late transcription of bacteriophage. It spontaneously assembles into oligomers within a specific time, forming nonspecific transmembrane channels in the cell membrane, damaging the cell membrane so as to release intracellular lytic enzymes, thereby causing bacterial death or even lysis. In vitro, perforin can cause cell death without passing through endolysins. The fatal cause of bacterial loss of vitality is mainly the holes formed by perforin on the cell membrane, and is not related to cell rupture. Perforin shows good antibacterial effects on both G+ and G-. Perforin is like a "molecular timer" that determines the end of the infection cycle, making the process of bacterial lysis controllable to a certain extent.

[0010] Phage display technology is a molecular technology that uses phage DNA gene modification to express the target on the phage surface by combining peptides, proteins or antibody fragments with phage coat proteins. This process involves introducing exogenous DNA sequences into specific locations of the nucleotide sequence of the phage genome. When phage infection occurs, phage genes begin to express in the bacterial host, and the inserted peptides and antibody fragments can be effectively displayed on the phage surface as a combination product of the encoded coat protein and cloned sequence.

[0011] This application innovatively uses phage display technology to display the phage perforin protein on the phage surface, so that the recombinant phage prepared by the above method can directly act on the bacterial cell membrane, without having to rely on the recognition-invasion-release process like traditional phages. In addition, the recombinant phage displaying perforin can improve the highly specific bactericidal mechanism of the phage to a certain extent. When the phage is adsorbed to the bacterial surface, the perforin on the surface can exert a bactericidal effect, thereby significantly expanding its antibacterial species. In addition, after the display phage invades the host bacteria, it can proliferate in large quantities in the host body, thereby increasing the perforin production and thus improving the lysis efficiency.

[0012] Therefore, the recombinant phage obtained by the preparation method of the recombinant phage displaying phage perforin of the present application greatly expands the lysis spectrum, improves the lysis efficiency, and has a good application prospect. Experiments have shown that the recombinant phage can not only lyse Gram-positive bacteria such as Bacillus subtilis and Staphylococcus aureus across species, but also has excellent antibacterial activity against mycoplasma, while the existing phage of the control does not have these activities, and its biological lysis activity has been greatly improved.

[0013] In addition, compared with traditional antibiotics, based on the unique mechanism of action of bacteriophage and perforin, it is difficult for bacteria to develop drug resistance to the recombinant bacteriophage, and the method and the recombinant bacteriophage obtained are of great significance for the treatment of drug-resistant bacterial infections. Therefore, phage display perforin provides a new idea and method for antibacterial treatment, and can be used as a new type of antibacterial preparation, which has important significance and value in antibacterial treatment and clinical application.

[0014] In an alternative embodiment, the bacteriophage perforin is the perforin protein of Escherichia coli bacteriophage DE7, and its amino acid sequence is shown in SEQ ID NO.1.

[0015] Preferably, the nucleotide sequence of the gene encoding the Escherichia coli bacteriophage perforin protein is as shown in SEQ ID NO.2.

[0016] Based on the selection of the perforin protein of Escherichia coli phage DE7 in the above method, in the step S1, the fusion primers are: holDE7-F having a sequence as described in SEQ ID NO.5 and holDE7-R having a sequence as described in SEQ ID NO.6, and the primer sequences are as follows: holDE7-F: 5′-tgttcctttctatgcggcccagccggccATGAAGTGGCTAGAGGAGGCTTT-3′ holDE7-R: 5′-ggcaccggcgcacctgcggccgcTTACTTTTCAGTATTCTTTGTTGTTCTTTTC-3′ Alternatively, in another embodiment, the bacteriophage perforin is the perforin of Pseudomonas aeruginosa bacteriophage, and its amino acid sequence is shown in SEQ ID NO.3.

[0017] Preferably, the sequence of the gene encoding the Escherichia coli bacteriophage perforin protein is shown in SEQ ID NO.4.

[0018] In other cases, the bacteriophage perforin protein may be selected from other existing bacteriophage perforin proteins, and the protein is not limited to the types listed in the embodiments of the present application.

[0019] Based on the perforin protein in the above method, the perforin holASP of Pseudomonas aeruginosa phage is selected. In step S1, the fusion primers used are: holASP-F with a sequence as described in SEQ ID NO.7 and holASP-R with a sequence as described in SEQ ID NO.8. The primer sequences are as follows: holASP-F: 5′-tgttcctttctatgcggcccagccggccatgatgattgataccgccac-3′ holASP-R: 5′-ggcaccggcgcacctgcggccgctcacttcttgaatctccggc-3′ Optionally, in the method for preparing a recombinant phage displaying bacteriophage perforin, the expression vector is pCANTAB 5E.

[0020] Optionally, the expression vector may also be an expression vector suitable for 3+3 type / 8+8 type phage display type, such as pComb3. The 3+3 type / 8+8 type refers to a display system based on phagemid and helper phage, in which the foreign gene is connected to the phagemid coat protein, and the wild-type gene exists on the genome of the helper phage, and the display is achieved by regulating DNA replication and transcription.

[0021] Optionally, the helper phage is M13K07.

[0022] Optionally, the helper phage also includes: other helper phages suitable for 3+3 type / 8+8 type phage display, such as VCSM13, but not limited thereto.

[0023] In a second aspect, the present application also provides a recombinant phage displaying bacteriophage perforin, which is prepared by any of the aforementioned methods.

[0024] In a third aspect, the present application also provides the use of the above-mentioned recombinant bacteriophage in antibacterial treatment.

[0025] Optionally, the recombinant bacteriophage can be used to inhibit bacteria or fungi such as Bacillus subtilis, Staphylococcus aureus, Streptococcus, etc., and prevent and control diseases caused by these pathogens.

[0026] In a fourth aspect, the present application also provides the use of the above-mentioned recombinant phage in inhibiting mycoplasma.

[0027] The mycoplasma is ovine mycoplasma, porcine mycoplasma or chicken mycoplasma.

[0028] The present invention has the following beneficial effects: 1. The recombinant phage provided by the present invention uses phage display technology to display perforin protein on the phage surface, so that it can directly act on the bacterial cell membrane, without the need for the recognition-invasion-release process like traditional phages. And the phage displaying perforin can improve the highly specific bactericidal mechanism of phage to a certain extent. When the phage is adsorbed to the bacterial surface, the perforin on the surface can exert the bactericidal effect, expand its antibacterial species, and show good synergistic effects. At the same time, after the display phage invades the host bacteria, it can proliferate in large quantities in the host body, thereby increasing the perforin production and improving the lysis efficiency.

[0029] 2. Compared with traditional antibiotics, it is difficult for bacteria to develop drug resistance to phages and perforins based on their unique mechanism of action, which is of great significance for the treatment of drug-resistant bacterial infections. Therefore, phage-displayed perforins provide new ideas and methods for antibacterial treatment and can be used as a new type of antibacterial agent, which has important significance and value in antibacterial treatment and clinical application.

[0030] 3. The obtained recombinant phage has both the advantages of phage and the bactericidal activity of perforin, which improves the lysis spectrum and lysis efficiency.

[0031] 4. The recombinant Escherichia coli phage provided in the present application has significant antibacterial activity against Gram-positive bacteria and mycoplasmas, while the phage before the transformation does not have such antibacterial activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 is the electrophoresis result of perforin gene; M is marker, 1, 2, 3 are the amplification maps of Escherichia coli phage DE7 perforin gene; Figure 2 The results of colony PCR electrophoresis after monoclonal proliferation; M: marker, 1, 2, 3, 4, 5, 6. Monoclonal colony PCR samples after homologous recombination; Figure 3 This is the plate for titer determination of recombinant phage DE7 after rescue; Figure 4 The results of the bacteriophage DE7 antibacterial experiment are as follows: B. subtilis WB800N, strain S. aureus ATCC 29213 and strains S. pneumoniae The antibacterial results of CICC24947; Figure 5 The inhibition effect of recombinant bacteriophage DE7 on mycoplasma; A1 is the experimental group of ovine mycoplasma, A2 is the experimental group of porcine mycoplasma; A3 is the experimental group of gallisepticum mycoplasma; Columns 1-12 are the samples diluted by adding recombinant bacteriophage DE7 10, 20, 40, 80, 160, 320, 640, 1280, 2560 times, original phage control, tylosin control, and culture medium control group, respectively; Figure 6 To show the antibacterial experimental results of the recombinant phage of holASP; the antibacterial results of the recombinant phage holASP and the control phage against three pathogenic bacteria M13K07; Figure 7 To demonstrate the inhibitory effect of holASP recombinant phage on mycoplasma; A1 is the Mycoplasma ovis experimental group, A2 is the Mycoplasma suis experimental group; A3 is the Mycoplasma gallisepticum; columns 1-12 are 10, 20, 40, 80, 160, 320, 640, 1280, and 2560 times diluted samples with the recombinant phage displaying holASP, the original phage control, the tylosin control, and the culture medium control group, respectively. DETAILED DESCRIPTION

[0033] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those skilled in the art without creative work 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. The methods in the following embodiments are conventional methods in the art unless otherwise specified.

[0034] Experimental Materials 1. Strains and plasmids Escherichia coli competent TG1: purchased from Beijing Tiangen Biochemical Technology Co., Ltd.

[0035] Expression vector: plasmid pCANTAB 5E was used, which was purchased from Novengen.

[0036] Phage: Helper phage M13K07 was purchased from New England Biolabs.

[0037] strain B. subtilis WB800N (Bacillus subtilis) was purchased from Changsha Abiwei Biotechnology Co., Ltd.

[0038] strain S. aureus ATCC 29213 (Staphylococcus aureus) was purchased from Hibo Biotechnology.

[0039] S. aureus ATCC 25923 (Staphylococcus aureus) was purchased from Hibo Biotechnology.

[0040] Streptococcus pneumoniae S. pneumoniae CICC 24947 was purchased from China Industrial Microbiological Culture Collection Center.

[0041] 2. Culture medium and reagents NB medium: purchased from BD. Tryptone 10g, yeast extract 5g, sodium chloride 10g, add water to 1L. Solid NB medium: add 15g agar powder to NB medium. Autoclave at 121℃ for 20min.

[0042] LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, add water to 1 L. Solid LB medium: add 15 g agar powder to the LB medium. Autoclave at 121°C for 15 min.

[0043] LB / 2% Glu-Amp medium: Based on the above LB medium configuration, add 20 g / L glucose, and when the culture temperature drops to 50°C, add Amp (final concentration 0.1 mg / mL).

[0044] 2×YT / 2% Glu / Kan+Amp medium: Add the finished 2×YT medium (31 g / L) into a conical flask, seal it after dissolving, and put it into a high pressure sterilizer for sterilization. Add Amp (final concentration 0.1 mg / mL) and Kan (final concentration 0.025 mg / mL) before use.

[0045] PEG6000 / Nacl solution: Add distilled water of the required volume of culture medium into a conical flask, use an electronic balance to weigh the corresponding PEG (400 g / L) and Nacl (146 g / L) in proportion and add them to the conical flask. After dissolution, seal the flask and place it in an autoclave at 121°C for 15 minutes for sterilization.

[0046] Example 1 Construction of a recombinant phage displaying Escherichia coli phage DE7 perforin This embodiment provides a method for preparing a recombinant phage displaying Escherichia coli phage DE7 perforin.

[0047] 1. Experimental methods: 1.1.1 Growth of TG1 bacterial culture Dip the inoculation loop into the TG1 seed solution and streak it on the LB medium plate for activation. Culture it at 37°C overnight. Pick a single colony and inoculate it into 5 mL of LB liquid medium to obtain a bacterial solution.

[0048] 2. Preparation of TG1 competent cells (1) Culture 5 mL of TG1 at 37°C for about 2-3 h (to reach the logarithmic phase) and transfer to a 15 mL centrifuge tube.

[0049] (2) Incubate on ice for 10 min, incubate at 4000 rpm at 4°C for 10 min, and discard the supernatant.

[0050] (3) Resuspend in 200 µL of CaCl2 (0.1 mol / mL) (pre-cooled), incubate at 4000 rpm at 4°C for 10 min, and discard the supernatant.

[0051] (4) Repeat (3).

[0052] (5) Resuspend the sample in 200 µL of 15% glycerol aqueous solution containing a final concentration of 0.1 mol / mL CaCl2. Mix well and dispense 100 µL / tube at -80°C.

[0053] 3. Extraction of plasmid pCANTAB 5E Use an inoculation loop to dip the seed solution of TG1 Escherichia coli carrying pCANTAB 5E and streak it on an LB medium plate (added with 0.1% Amp) for activation. Culture it overnight at 37°C, pick a single colony and inoculate it into 5 mL of LB liquid culture medium (added with 0.1% Amp), culture the bacterial solution to obtain it, and use a plasmid extraction kit to extract the plasmid pCANTAB 5E.

[0054] 4. M13K07 assists in the amplification of phage (1) Add 20 µL of M13K07 proliferation medium to 20 ml TG1 (OD = 0.5) and adsorb at 37 °C for 30 min. Add Kan and culture overnight at 37 °C and 200 rpm.

[0055] (2) Transfer to a centrifuge tube and centrifuge at 4°C, 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 does not dissolve). Stir magnetically on ice for 30 min. Centrifuge the liquid at 4°C, 6000 rpm for 20 min and discard the supernatant.

[0056] (3) Resuspend in 1 ml TBS (pH = 7.4) and centrifuge at 14,000 rpm for 2 min. Collect the supernatant and mix with 50% glycerol for storage.

[0057] The proliferation titer of M13K07 helper phage was 1×10 9 PFU / ml, the titer after concentration is 1.9×10 10 PFU / ml.

[0058] 5. Primer design and amplification of bacteriophage DE7 perforin gene The primers for amplification of bacteriophage DE7 perforin are as follows: holDE7-F: 5′-tgttcctttctatgcggcccagccggccATGAAGTGGCTAGAGGAGGCTTT-3′ (shown in SEQ ID NO.5) holDE7-R: 5′-ggcaccggcgcacctgcggccgcTTACTTTTCAGTATTCTTTGTGTTCTTTTC-3′ (as shown in SEQ ID NO. 6) Note: The lowercase letters in the above primers indicate that the part is the vector homologous fragment, and the uppercase letters indicate that the part is the phage lytic enzyme sequence. The target gene is inserted between the Sfi I and Not I restriction sites.

[0059] Using the bacteriophage DE7 genome as a template, 2ul of the above primers were added, and 2×Accurate TaqMaster Mix was used to amplify the target gene by PCR.

[0060] The PCR program was as follows: (1) 95°C, 5 min; (2) 95°C, 1 min, 68°C, 30 s, 72°C, 30 s, 30 cycles; (3) 72°C, 10 min.

[0061] The amplified product was detected by electrophoresis. The electrophoresis results were as follows: Figure 1 As shown, the band size of the PCR product was consistent with expectations, and the PCR product was recovered by gel electrophoresis for subsequent experiments.

[0062] 6. Enzyme digestion of pCANTAB 5E expression vector (1) Sfi I digestion Table 1 Sfi I restriction enzyme system

[0063] Incubate in a water bath at 37 °C for 6 h. After the digestion, use a PCR product recovery kit to recover the digestion product and determine its concentration.

[0064] (2) Not I digestion Table 2 Not I enzyme digestion system

[0065] Incubate in a water bath at 37 °C for 6 h. After the digestion, use a PCR product recovery kit to recover the digestion product and determine its concentration.

[0066] 7. Homologous recombination transformation and positive identification (1) Use the homologous recombination kit (VazymeC113-02) to connect the above-mentioned target fragment into the pCANTAB 5E expression vector after restriction digestion.

[0067] Table 3 Homologous recombination system

[0068] Incubate in a water bath at 37°C for 10 min and then immediately place on ice.

[0069] (2) Transformation of Escherichia coli: Add the above 10 µL homologous recombination product and 1 µL empty vector control to 2 tubes of 100 µL TG1 competent cells, respectively, flick to mix, incubate on ice for 30 min, heat shock at 42 ℃ for 90 s, and incubate on ice for 120 s. Take 1 ml LB and add it to a 1.5 ml EP tube, incubate at 37 ℃ 220 rpm for 1 h, then 10,000 rpm for 3 min, discard the supernatant (reserve 100 µL), resuspend the precipitate and apply it to LB / 2% Glu-Amp plate, and incubate at 37 ℃ for 12 h.

[0070] (3) Positive identification The bacterial solution after monoclonal proliferation was used as a template, and primer PCR was performed to identify the positive clones. The system procedure was the same as that described in step 5. That is, TG1 transformed with recombinant pCANTAB-holDE7 was obtained, which was used for the next step of phage rescue.

[0071] 8. Phage rescue (1) Add 200 μL of Escherichia coli TG1 carrying the pCANTAB-holDE7 recombinant plasmid to 200 ml of 2×YT / 2% Glu-Amp and incubate at 37°C and 225 rpm until the logarithmic phase to obtain a bacterial suspension.

[0072] (2) Add 30 µL of 7.8×10 13 PFU / ml of helper phage M13K07 was gently mixed and allowed to stand at 37°C for 40 min to obtain a mixed solution.

[0073] (3) Centrifuge the mixture in 8(2) at 3000×g at 25℃ for 20 min, discard the supernatant, resuspend the precipitate in 400 ml 2×YT / Kan+Amp and incubate at 37℃, 225 rpm for 14 h.

[0074] (4) The bacterial solution obtained in the previous step was heated at 4°C and 4500× g Centrifuge for 35 min under the same conditions, collect the supernatant (do not mix with the precipitate), divide the supernatant equally into two sterilized 500 ml conical flasks, add 40 ml PEG6000 / NaCl solution to each flask, mix well, and let stand on ice for 12-24 h.

[0075] (5) Then, heat at 4°C at 6000× g The pellet was resuspended in 800 µL of 1×PBS (pH 7.4) and transferred to a sterile 1.5 ml EP tube. The tube was shaken at 4°C, 225 rpm for 12-14 h at 4°C, 13000× gAfter 20 min, the supernatant was collected to obtain the recombinant phage, which was stored at 4 °C in the dark for subsequent titer determination.

[0076] 9. Titer determination of recombinant phage (1) Take 30 µL of the above recombinant phage supernatant and dilute it 10-fold using 2×YT / 2%Glu-Amp medium. -7 ~10 -10 200 μL of the recombinant phage supernatant was mixed with an equal volume of Escherichia coli TG1 cultured to the logarithmic phase and incubated at 37°C for 35 min.

[0077] (2) Spread the mixed bacterial solution after static incubation on LB / 2% Glu-Amp plates, absorb for 30 min at 4°C, and incubate in a 37°C incubator for 12 h. Count the number of monoclonal colonies growing on the plates and calculate the recombinant phage titer.

[0078] The results are as follows Figure 3 As shown, the titer of the recombinant phage was 5×10 8 pfu / mL.

[0079] Example 2 Demonstrates the use of recombinant phages of Escherichia coli phage DE7 perforin 1. Plate antibacterial experiment (1) Experimental methods Use strains B. subtilis WB800N (Bacillus subtilis), strain S. aureus ATCC 29213 (Staphylococcus aureus) and strains 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.

[0080] The method for determining the antibacterial activity is as follows: B. subtilis WB800N (Bacillus subtilis), strain S. aureus ATCC29213 (Staphylococcus aureus) and strains S. pneumoniae CICC 24947 (Streptococcus pneumoniae) was inoculated in 5 mL NB liquid medium for activation (37°C) and cultured overnight. 100 μL of bacterial solution was mixed with 15 mL NB solid medium and poured onto the plate. After solidification, 10 μL of recombinant phage sample was applied and placed in a 4°C refrigerator for 30 minutes to allow the liquid to be absorbed. The plate was then transferred to a 37°C incubator for culture and the action of the recombinant phage was observed.

[0081] (2) Experimental results and analysis like Figure 4As shown, the recombinant phage of Example 1 and the control phage M13K07 were dropped into B. subtilis WB800N, S. aureus ATCC29213 and S. pneumoniae On the CICC 24947 plate, after overnight culture, the recombinant phage displaying DE7 was observed to form an inhibition zone, while the control phage M13K07 had no inhibitory effect.

[0082] 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. The width of its lysis spectrum has undergone a major breakthrough; this shows that the recombinant phage displaying phage perforin can greatly expand the lysis spectrum, enabling it to have the ability of cross-species lysis, and the lysis performance is greatly improved.

[0083] 2. Inhibitory effect of recombinant phage on mycoplasma This experiment used three mycoplasmas as experimental objects: Mycoplasma ovipneumoniae, Mycoplasma hyopneumoniae and Mycoplasma gallisepticum.

[0084] 2.1 Drug sensitivity test (1) The above-mentioned recombinant phage sample (titer of 5×10 8 pfu / mL) was diluted 5-fold.

[0085] (2) Add 100 μl of mycoplasma broth culture medium to columns 2-12; (3) Take 100ul of the 5-fold diluted sample and add it to columns 1-2; (4) After mixing the second column, transfer 100ul to the third column. After mixing, transfer 100ul to the fourth column, and continue to the tenth column. Discard 100ul.

[0086] (5) Dilute the fresh culture of each mycoplasma at a ratio of 1:10,000 with the corresponding culture medium. The preparation method of the fresh culture of each mycoplasma is as follows: add 1800 μl of culture medium to a 5 ml stoppered test tube, aspirate 200 μl of the original mycoplasma liquid, mix well, and culture at 37°C for about 3-4 days.

[0087] (6) Add 100 μl / well of diluted mycoplasma to the wells in columns 1 to 10; (7) Seal the cell culture plate with sealing film and observe at 37°C for 10 days.

[0088] (8) Record the time of color change.

[0089] 2.2 Detection of the inhibitory effect of recombinant phage on mycoplasma (1) 180ul culture medium per well in columns 1-12; (2) Add 20ul of fresh culture medium to column 1, mix and pipette 20ul into column 2; replace the tip, mix and pipette 20ul into column 3; mix and pipette into column 11. Column 12 is the culture medium control.

[0090] (3) Seal the cell culture plate with sealing film, observe at 37°C for 10 days, and record the color change.

[0091] 2.3 Experimental Results and Analysis After the above treatment, A1 is the sheep mycoplasma experimental group, A2 is the porcine mycoplasma experimental group, and A3 is the chicken mycoplasma experimental group; 1-12 columns of wells are respectively added with 10 times, 20 times, 40 times, 80 times, 160 times, 320 times, 640 times, 1280 times, 2560 times diluted samples of recombinant phage, original phage control, tylosin control and culture medium control group. When the solution in the well turns into a yellow area, it means that the phage has no antibacterial activity against mycoplasma.

[0092] like Figure 5 As shown, the recombinant phage still has an antibacterial effect on Mycoplasma ovis, Mycoplasma suis and Mycoplasma gallisepticum after being diluted 20-40 times, and the antibacterial effect is more obvious.

[0093] Example 3 Preparation and application of recombinant phage displaying Pseudomonas aeruginosa phage perforin holASP 1. Preparation of recombinant phage displaying Pseudomonas aeruginosa phage perforin holASP The preparation method of the recombinant phage displaying the Pseudomonas aeruginosa phage perforin holASP is similar to the method of Example 1, except that the original perforin is replaced by the Pseudomonas aeruginosa phage perforin, whose amino acid sequence 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 8 PFU / ml.

[0094] 2. Application of recombinant phages displaying Pseudomonas aeruginosa phage perforin holASP (1) Plate antibacterial test A. Experimental Methods Use strains B. subtilis WB800N (Bacillus subtilis), strain S. aureus ATCC29213 (Staphylococcus aureus) and strains S. aureus ATCC 25923 (Staphylococcus aureus) was used as the experimental strain to detect the antibacterial activity of the perforin holASP recombinant phage.

[0095] The method for determining the antibacterial activity is as follows: B. subtilisWB800N (Bacillus subtilis), strain S. aureus ATCC29213 (Staphylococcus aureus) and strains S. aureus ATCC 25923 (Staphylococcus aureus) was inoculated in 5 mL NB liquid medium for activation (37°C) and cultured overnight. 100 μL of bacterial solution was mixed with 15 mL NB solid medium and poured onto the plate. After solidification, 10 μL of recombinant phage sample was applied and placed in a 4°C refrigerator for 30 minutes to allow the liquid to absorb. The plate was then transferred to a 37°C incubator for culture and the action of the recombinant phage was observed.

[0096] B. Experimental Results and Analysis The recombinant phage displaying perforin holASP (10 8 pfu / mL) and control phage M13K07 were dropped into B. subtilis WB800N, S. aureus ATCC29213 and S. aureus ATCC 25923 plates after overnight incubation. Figure 6 As shown, inhibition zones were formed on the plates of the treatment group of the recombinant phage displaying perforin holASP, while the control phage M13K07 had no inhibitory effect on the above three pathogens.

[0097] It can be seen that the phage M13K07 displaying the perforin holASP has expanded its lysis spectrum, enabling it to have the ability of cross-species lysis, and has good lysis performance against Bacillus subtilis and Staphylococcus aureus.

[0098] 2. Inhibitory effect of recombinant phage on mycoplasma A. Experimental Methods The method is the same as in Example 2.

[0099] The settings on the well plate are as follows: A1 is the Mycoplasma ovis test group, A2 is the Mycoplasma suis test group; columns 1-12 are added with 10-fold, 20-fold, 40-fold, 80-fold, 160-fold, 320-fold, 640-fold, 1280-fold, and 2560-fold dilution samples of the recombinant phage, the original phage control, the tylosin control, and the culture medium control group (such as Figure 7 as shown).

[0100] B. Experimental Results and Analysis The results are as follows Figure 7 As shown, the results show that the recombinant phage of this example has an antibacterial effect on Mycoplasma ovis, Mycoplasma suis and Mycoplasma gallisepticum after being diluted 10-20 times. Figure 7 If the solution turns yellow, it means that the phage has no antibacterial activity against mycoplasma.

[0101] It is understandable that those skilled in the art can make equivalent substitutions or changes based on the technical solution and concept of the present invention, and all these changes or substitutions should fall within the protection scope of the claims attached to the present invention.

Claims

1. A method for preparing a recombinant bacteriophage displaying bacteriophage perforin, characterized in that: The following steps are involved: S1. Using the bacteriophage genome as a template, amplifying the bacteriophage perforin encoding gene sequence with a fusion primer to obtain the bacteriophage perforin encoding gene sequence with homology arms, and recovering the amplified product; performing enzyme digestion on the expression vector, and recovering the expression vector skeleton after enzyme digestion; S2, connecting the bacteriophage perforin encoding gene sequence with homology arms to the expression vector backbone after enzyme digestion through homologous recombination, and then transforming the connection product into Escherichia coli competent cells to screen and obtain recombinant Escherichia coli; S3. Use the helper phage to perform phage rescue on the recombinant Escherichia coli obtained in the previous step to obtain a recombinant phage displaying phage perforin.

2. The method for preparing a recombinant bacteriophage displaying bacteriophage perforin according to claim 1, characterized in that: The bacteriophage perforin is the perforin protein of Escherichia coli bacteriophage DE7, and its amino acid sequence is shown in SEQ ID NO.

1.

3. The method for preparing a recombinant bacteriophage displaying bacteriophage perforin according to claim 2, characterized in that: In step S1, the fusion primers are: holDE7-F having a sequence as described in SEQ ID NO.5 and holDE7-R having a sequence as described in SEQ ID NO.

6.

4. The method for preparing a recombinant bacteriophage displaying bacteriophage perforin according to claim 1, characterized in that: The bacteriophage perforin is holASP, a perforin of Pseudomonas aeruginosa phage, and its amino acid sequence is shown in SEQ ID NO.

3.

5. The method for preparing a recombinant bacteriophage displaying bacteriophage perforin according to claim 4, characterized in that: In step S1, the fusion primers are: holASP-F with a sequence as shown in SEQ ID NO.7 and holASP-R with a sequence as shown in SEQ ID NO.

8.

6. The method for preparing a recombinant bacteriophage displaying bacteriophage perforin according to claim 1, characterized in that: The expression vector is pCANTAB 5E.

7. The method for preparing a recombinant bacteriophage displaying bacteriophage perforin according to claim 1, characterized in that: The helper phage is M13K07.

8. A recombinant bacteriophage displaying bacteriophage perforin, characterized in that: The method is prepared according to any one of claims 1 to 7.

9. Use of the recombinant bacteriophage as claimed in claim 8 in inhibiting pathogenic bacteria.

10. Use of the recombinant bacteriophage according to claim 8 in inhibiting mycoplasma.

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