M13 Filamentous Phage Double-Modified with Capsid and Tail Fiber Proteins and Enhanced Delivery Method

Abstract

The present invention discloses an M13 filamentous phage with dual capsid and fiber protein modifications and a method for enhancing delivery. The M13 filamentous phage with dual capsid and fiber protein modifications provided in the present invention is based on the M13 filamentous phage. A peptide sequence with high affinity for Pseudomonas aeruginosa exopolysaccharide is inserted into the M13 filamentous phage capsid protein. The amino acid sequence of the fiber protein of the M13 filamentous phage is edited to include a peptide sequence capable of binding to a polyvalent phage. This improves the affinity of the M13 filamentous phage for biofilms, thereby enhancing the delivery of the polyvalent phage and improving the efficiency of removing biofilms using the phage as a bactericide.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to an M13 filamentous phage with double modifications of capsid and tail fiber proteins and a method for enhancing its delivery. Background Art

[0002] Water supply systems often rely on residual chlorine to eliminate harmful microorganisms in piped water. However, due to fluctuations in source water quality, extended transportation distances, and aging pipe network equipment, pipe network microorganisms, under the stress of hydraulic scouring and residual chlorine, primarily exist in the form of biofilms. Biofilms provide a stable microenvironment for the growth of pathogenic microorganisms and the spread of drug resistance. When biofilms age and fall off, the pathogenic and drug-resistant bacteria they carry with them enter the water body, increasing the risk of microbial water quality. Biofilms protect and promote the growth of problematic bacteria such as acid-producing and iron-oxidizing bacteria, accelerating pipe network corrosion and causing water quality deterioration. The presence of biofilms accelerates the consumption of residual chlorine, reducing the efficiency of pathogen inactivation while generating disinfection byproducts with carcinogenic risks.

[0003] Given the inability of residual chlorine to fully penetrate biofilms in pipe networks and eliminate potential pathogens, bacteriophages, which can target and lyse the host without causing secondary contamination, have become a new biocide option. Bacteriophages are viruses that infect prokaryotes exclusively and lack intrinsic metabolism, relying on the metabolic machinery of host cells to support their reproduction. Virulent phages can replicate and assemble into new phage particles within infected host cells, then lyse the host cells and release new phage particles to infect neighboring cells. However, in actual water supply systems, phage infection of biofilms is often hindered by hydrodynamic conditions. This can shorten phage-biofilm contact time, reduce phage concentration, and cause phage adsorbed on biofilms to escape the target. This can result in inefficient phage delivery as a biocide, potentially stimulating biofilm growth rather than eradicating it. Therefore, enhancing phage delivery to target biofilms is crucial for biofilm removal in water supply systems. Summary of the Invention

[0004] The purpose of the present invention is to solve the problem of low administration efficiency of bacteriophage as a bactericidal agent in the prior art, and to provide an M13 filamentous phage with double modification of capsid and tail fiber protein, a peptide segment, a method and an application.

[0005] In order to achieve the above-mentioned object of the invention, the specific technical solutions adopted by the present invention are as follows:

[0006] In a first aspect, the present invention provides an M13 filamentous phage with double modifications of capsid and fiber proteins, comprising:

[0007] A first oligonucleotide corresponding to a Pseudomonas aeruginosa-specific affinity peptide is inserted into the pVIII gene of the M13 filamentous phage using gene editing technology. After expression of the first oligonucleotide, the M13 filamentous phage capsid protein has a peptide sequence that has affinity for adsorbing Pseudomonas aeruginosa. Simultaneously, a second oligonucleotide corresponding to a polyvalent phage-specific affinity peptide is inserted into the pIII gene of the M13 filamentous phage using gene editing technology. After expression of the second oligonucleotide, the M13 filamentous phage tail fiber protein has a peptide sequence that has affinity for adsorbing polyvalent phages.

[0008] As a preferred embodiment of the first aspect, the Pseudomonas aeruginosa-specific affinity peptide is screened by phage display technology, and the screening method is:

[0009] The phages in the phage display peptide library are mixed and incubated with fresh Pseudomonas aeruginosa culture medium, and the phages adsorbed on Pseudomonas aeruginosa are centrifuged and eluted from the cell aggregates using glycine-hydrochloric acid. The phages are then propagated and purified in a culture medium containing Escherichia coli, and then precipitated with polyethylene glycol (PEG) to obtain M13 phages. The precipitated M13 phages are resuspended in PBS, completing one round of Pseudomonas aeruginosa affinity screening. Multiple rounds of the Pseudomonas aeruginosa affinity screening are repeated to isolate the first phage with affinity for Pseudomonas aeruginosa from the peptide library.

[0010] The screened phages were amplified and cultured in soft agar plates to obtain dispersed plaques. PCR was performed on the phages in each plaque to amplify the DNA fragments corresponding to the specific peptides, which were then sequenced and identified to determine the Pseudomonas aeruginosa-specific affinity peptides.

[0011] As a preference of the first aspect above, the phage display peptide library is the Doctor No. 7 phage display peptide library.

[0012] As a preferred embodiment of the first aspect above, the Pseudomonas aeruginosa-specific affinity peptide sequence is YHLPVES, as shown in SEQ ID No. 1.

[0013] As a preferred embodiment of the first aspect, the multivalent phage-specific affinity peptides are screened by phage display technology, and the screening method is:

[0014] Magnetic nanoparticles capable of immobilizing polyvalent bacteriophages are used as first magnetic beads, the first magnetic beads are added to a polyvalent bacteriophage solution to immobilize the polyvalent bacteriophages on the first magnetic beads, thereby forming second magnetic beads, and the first and second magnetic beads are treated with a bovine serum albumin solution to block vacant binding sites on the magnetic beads;

[0015] First magnetic beads treated with a bovine serum albumin solution are added to a phage display peptide library for a first incubation, so that phages that can be adsorbed to the bovine serum albumin and the magnetic nanoparticles themselves are adsorbed on the first magnetic beads. The supernatant is then separated by magnetic decantation. Second magnetic beads treated with a bovine serum albumin solution are then added to the supernatant for a second incubation. After the incubation is complete, the second magnetic beads are separated and the phages bound to the second magnetic beads are eluted and enriched, completing one round of polyvalent phage affinity screening. Multiple rounds of polyvalent phage affinity screening are repeated to isolate second phages with affinity for the polyvalent phages from the peptide library.

[0016] The screened second phage is amplified and cultured in a soft agar plate to obtain dispersed plaques. The phage in each plaque is subjected to PCR to amplify the DNA fragment corresponding to the specific peptide segment, which is then sequenced and identified to determine the multivalent phage-specific affinity peptide segment.

[0017] As a preferred embodiment of the first aspect, the phage display peptide library is formed by mixing two phage display peptide libraries, namely, Dr. No. 7 and Dr. No. 12.

[0018] As a preferred embodiment of the first aspect, the Pseudomonas aeruginosa-specific affinity peptide sequence is HDYTDWYWLLSY, as shown in SEQ ID No. 2.

[0019] As a preferred embodiment of the first aspect, the method of inserting the first oligonucleotide into the pVIII gene of the M13 filamentous phage by gene editing technology is:

[0020] Phage M13SK was generated by introducing the restriction sites PstI and BamHI into the pVIII gene of the M13KE phage and deleting the previously existing PstI restriction sites in multiple cloning sites.

[0021] Complementary oligonucleotides corresponding to the Pseudomonas aeruginosa-specific affinity peptide were synthesized and annealed, and then the annealed complementary oligonucleotides were inserted into the phage M13SK genome between the PstI and BamHI restriction sites using T4 ligase to complete the insertion modification of the first oligonucleotide.

[0022] As a preferred embodiment of the first aspect, the method of inserting the second oligonucleotide into the pill gene of the M13 filamentous phage by gene editing technology is:

[0023] After synthesizing complementary oligonucleotides corresponding to the multivalent phage-specific affinity peptides and performing annealing treatment, the annealed complementary oligonucleotides are inserted into the pIII gene of the phage M13SK after modification of the first oligonucleotide using T4 ligase, and the insertion position is between the EagI and Acc65I restriction sites.

[0024] In a second aspect, the present invention provides a method for enhancing phage transport by using a double-modified phage to carry a multivalent phage, the specific method of which is as follows: the multivalent phage is mixed with the M13 filamentous phage with double modification of the capsid and tail fiber protein as described in any scheme of the first aspect above to form a conjugate of the two phages, in which the multivalent phage is adsorbed on the tail fiber protein of the M13 filamentous phage; the conjugate of the two phages is then introduced into the water environment where the target biofilm is located, so that the M13 filamentous phage carrying the multivalent phage is further affinity-adsorbed to the Pseudomonas aeruginosa in the target biofilm through the peptide sequence modified on the capsid protein, thereby enhancing the transport of the multivalent phage to the target biofilm.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The present invention has developed a method for modifying the capsid and fiber proteins of the M13 filamentous phage. Phage display technology is used to screen for peptides with high affinity for Pseudomonas aeruginosa and polyvalent phages. Then, through gene editing, oligonucleotides corresponding to the two peptides are inserted into the genome of the M13 filamentous phage to obtain a double-modified M13 filamentous phage. Furthermore, the present invention also uses the M13 filamentous phage obtained by this method to enhance the delivery of polyvalent phage to target biofilms. Specifically, using the polyvalent phage as a carrier can improve the delivery efficiency of the phage as a biofilm bactericide. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 It is a water supply network simulation device;

[0028] Figure 2 Comparison of biofilm removal efficiency of X and X+M13 conjugates;

[0029] Figure 3 Live / dead cell coverage was obtained by microscopic analysis four hours after phage treatment (the bars represent biofilm coverage, and the broken line represents the live-dead cell ratio). DETAILED DESCRIPTION

[0030] The present invention will be further described and illustrated below with reference to the accompanying drawings and specific embodiments. The technical features of each embodiment of the present invention may be combined accordingly, provided that there is no conflict between them.

[0031] In order to make the above-mentioned objects, features and advantages of the present invention more clearly understood, the specific embodiments of the present invention are described in detail below with reference to the accompanying drawings. In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways than those described herein, and those skilled in the art can make similar improvements without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below. The technical features in the various embodiments of the present invention can be combined accordingly without conflicting with each other.

[0032] In the description of the present invention, it should be understood that when an element is considered to be "connected" to another element, it can be directly connected to the other element or indirectly connected, that is, there are intermediate elements. On the contrary, when an element is said to be "directly" connected to another element, there are no intermediate elements.

[0033] In the description of the present invention, it should be understood that the terms "first" and "second" are used solely for descriptive purposes and are not to be construed as indicating or implying relative importance or implicitly specifying the number of technical features being described. Therefore, features defined as "first" or "second" may explicitly or implicitly include at least one of such features.

[0034] The present invention provides an M13 filamentous phage with dual modifications of capsid and fiber protein. The phage is based on the M13 filamentous phage, and a peptide sequence with high affinity for Pseudomonas aeruginosa exopolysaccharide (commonly present on the surface of biofilms in natural and engineered systems) is inserted into the M13 filamentous phage capsid protein. The amino acid sequence of the fiber protein of the M13 filamentous phage is edited to contain a peptide sequence that can bind to a polyvalent phage, thereby improving the affinity of the M13 filamentous phage for biofilms, thereby achieving the purpose of enhancing the delivery of polyvalent phages and improving the efficiency of removing biofilms using phages as bactericides.

[0035] In a preferred embodiment of the present invention, a capsid and tail fiber protein dual-modified M13 filamentous phage is provided, wherein a first oligonucleotide corresponding to a Pseudomonas aeruginosa-specific affinity peptide is inserted into the pVIII gene of the M13 filamentous phage by gene editing technology, and after expression of the first oligonucleotide, the M13 filamentous phage capsid protein has a peptide sequence that has affinity for adsorbing Pseudomonas aeruginosa; simultaneously, a second oligonucleotide corresponding to a polyvalent phage-specific affinity peptide is inserted into the pIII gene of the M13 filamentous phage by gene editing technology, and after expression of the second oligonucleotide, the M13 filamentous phage tail fiber protein has a peptide sequence that has affinity for adsorbing polyvalent phages.

[0036] It should be noted that the polyvalent phages herein refer to phages capable of lysing a variety of bacteria, i.e., host cells. The specific bacterial types lysed are not limited, and the phages are intended to encompass the major bacterial types in the target biofilm. Polyvalent phages can be screened using phage screening techniques, which will not be described in detail.

[0037] In the present invention, the Pseudomonas aeruginosa-specific affinity peptide is a polypeptide fragment with a strong affinity for Pseudomonas aeruginosa, and the polyvalent phage-specific affinity peptide is a polypeptide fragment with a strong affinity for polyvalent phage. The purpose of modifying these two peptides on the M13 filamentous phage is to improve the attachment efficiency of the phage on the biofilm during the process of adding the phage as a bactericide to the water body where the biofilm is located. Because Pseudomonas aeruginosa is an extremely common type of bacteria in the biofilm of both natural water bodies and engineered water bodies. When the M13 filamentous phage has a high affinity adsorption performance for Pseudomonas aeruginosa, it can be quickly and efficiently adsorbed on the Pseudomonas aeruginosa in the biofilm. The tail fiber protein of the M13 filamentous phage has a peptide sequence that has affinity for adsorbing multivalent phages. Therefore, by pre-adsorbing the multivalent phages on the tail fiber protein of the M13 filamentous phage, the multivalent phages can be efficiently adsorbed onto the biofilm using the M13 filamentous phage as a carrier, thereby improving the infection efficiency.

[0038] In addition, in the present invention, the key to modifying the M13 filamentous phage is to find Pseudomonas aeruginosa-specific affinity peptides and multivalent phage-specific affinity peptides, which can be screened by phage display technology.

[0039] As an implementation method of the embodiment of the present invention, the above-mentioned Pseudomonas aeruginosa-specific affinity peptides are screened by phage display technology, and the screening method is:

[0040] The phages in phage display peptide library A are mixed and incubated with fresh Pseudomonas aeruginosa culture medium, and the phages adsorbed on Pseudomonas aeruginosa are centrifuged and eluted from the cell aggregates using glycine-hydrochloric acid. The phages are then propagated and purified in a culture medium containing Escherichia coli, and then precipitated with polyethylene glycol (PEG) to obtain M13 phages. The precipitated M13 phages are resuspended in PBS, completing one round of Pseudomonas aeruginosa affinity screening. Multiple rounds of the Pseudomonas aeruginosa affinity screening are repeated to isolate the first phage with affinity for Pseudomonas aeruginosa from the peptide library.

[0041] The screened phages were amplified and cultured in soft agar plates to obtain dispersed plaques. PCR was performed on the phages in each plaque to amplify the DNA fragments corresponding to the specific peptides, which were then sequenced and identified to determine the Pseudomonas aeruginosa-specific affinity peptides.

[0042] During the screening process of the above-mentioned Pseudomonas aeruginosa-specific affinity peptides, the phage display peptide library A should cover as many different peptides as possible to be screened. Here, the phage display peptide library A is preferably the Doctor No. 7 phage display peptide library.

[0043] In an embodiment of the present invention, through multiple rounds of Pseudomonas aeruginosa affinity screening of the Dr. 7 phage display peptide library, a Pseudomonas aeruginosa-specific affinity peptide sequence YHLPVES was finally obtained, as shown in SEQ ID No. 1.

[0044] Similarly, as an implementation of an embodiment of the present invention, the above-mentioned multivalent phage-specific affinity peptides are screened by phage display technology, and the screening method is:

[0045] Magnetic nanoparticles capable of immobilizing polyvalent phages are used as first magnetic beads. The first magnetic beads are added to a polyvalent phage solution to immobilize the polyvalent phages on the first magnetic beads, thereby forming second magnetic beads. The first and second magnetic beads are treated with a bovine serum albumin solution (BSA) to block vacant binding sites on the magnetic beads.

[0046] First magnetic beads treated with a bovine serum albumin solution are added to a phage display peptide library B for a first incubation, so that phages capable of adsorbing to the bovine serum albumin and the magnetic nanoparticles themselves are adsorbed on the first magnetic beads. The supernatant is then separated by magnetic decantation. Second magnetic beads treated with a bovine serum albumin solution are then added to the supernatant for a second incubation. After the incubation is complete, the second magnetic beads are separated and the phages bound to the second magnetic beads are eluted and enriched, completing one round of polyvalent phage affinity screening. Multiple rounds of polyvalent phage affinity screening are repeated to isolate second phages having affinity for the polyvalent phages from the peptide library.

[0047] The screened second phage is amplified and cultured in a soft agar plate to obtain dispersed plaques. The phage in each plaque is subjected to PCR to amplify the DNA fragment corresponding to the specific peptide segment, which is then sequenced and identified to determine the multivalent phage-specific affinity peptide segment.

[0048] During the screening process of the above-mentioned multivalent phage-specific affinity peptides, the phage display peptide library B should cover as many different peptides as possible to be screened. Here, it is preferred that the phage display peptide library B is a mixed peptide library formed by mixing the two phage display peptide libraries, Dr. No. 7 and Dr. No. 12.

[0049] In an embodiment of the present invention, a polyvalent phage-specific affinity peptide sequence HDYTDWYWLLSY was finally obtained by affinity screening of the phage display peptide libraries of Dr. 7 and Dr. 12, as shown in SEQ ID No. 2.

[0050] Once the Pseudomonas aeruginosa-specific affinity peptides and the multivalent phage-specific affinity peptides are obtained, the corresponding encoding nucleotide sequences can be determined from the peptides. These encoding nucleotide sequences are then inserted into the genome of the M13 filamentous phage at the corresponding locations. Upon DNA expression, the corresponding specific affinity peptides are formed on the capsid and fiber proteins. This process is a common technique in bioengineering and can be implemented using any existing gene editing technology.

[0051] As an implementation method of the embodiment of the present invention, the method of inserting the first oligonucleotide into the pVIII gene of the M13 filamentous phage by gene editing technology is:

[0052] Phage M13SK was generated by introducing the restriction sites PstI and BamHI into the pVIII gene of the M13KE phage and deleting the previously existing PstI restriction sites in multiple cloning sites.

[0053] Complementary oligonucleotides corresponding to the Pseudomonas aeruginosa-specific affinity peptide were synthesized and annealed, and then the annealed complementary oligonucleotides were inserted into the phage M13SK genome between the PstI and BamHI restriction sites using T4 ligase to complete the insertion modification of the first oligonucleotide.

[0054] Among them, when synthesizing the complementary oligonucleotide corresponding to the Pseudomonas aeruginosa-specific affinity peptide, in order to increase the probability of successful peptide fusion, additional amino acids can be added to the 3' end of the Pseudomonas aeruginosa-specific affinity peptide, for example, amino acid P is added to the 3' end of the peptide YHLPVES to form the YHLPVESP peptide, and then the complementary oligonucleotide corresponding to the YHLPVESP peptide is synthesized and inserted through gene editing.

[0055] Furthermore, based on the above completion of the insertion and modification of the first oligonucleotide, as an implementation method of an embodiment of the present invention, a method for inserting a second oligonucleotide into the pill gene of the M13 filamentous phage by gene editing technology is as follows:

[0056] After synthesizing complementary oligonucleotides corresponding to the multivalent phage-specific affinity peptides and performing annealing treatment, the annealed complementary oligonucleotides are inserted into the pIII gene of the phage M13SK after modification of the first oligonucleotide using T4 ligase, and the insertion position is between the EagI and Acc65I restriction sites.

[0057] Once the capsid and fiber protein-dual-modified M13 filamentous phage is obtained, it can be used in practical applications. In another preferred embodiment of the present invention, a method for enhancing phage delivery using a dual-modified phage carrying a multivalent phage is provided. Specifically, the method comprises: mixing a multivalent phage capable of lysing bacteria in a target biofilm with the aforementioned capsid and fiber protein-dual-modified M13 filamentous phage to form a conjugate of the two phages, wherein the multivalent phage in the conjugate is adsorbed onto the fiber protein of the M13 filamentous phage; then, introducing the conjugate of the two phages into the water environment where the target biofilm is located, whereby the M13 filamentous phage carrying the multivalent phage further affinity-adsorbs onto Pseudomonas aeruginosa in the target biofilm via the peptide sequence modified on the capsid protein, thereby enhancing the delivery of the multivalent phage to the target biofilm.

[0058] It should be noted that the target biofilm in the present invention can be a biofilm grown in natural water bodies (such as rivers, lakes, ponds, etc.), or a biofilm grown in engineering water bodies (such as water pipes, sewage pipes, water treatment facilities, etc.), without limitation.

[0059] The following specific examples will specifically demonstrate the preparation process of the aforementioned M13 filamentous phage with double modification of capsid and fiber proteins, and the effect of using the prepared double-modified phage to enhance phage delivery.

[0060] Example

[0061] In this embodiment, the experimental process is as follows:

[0062] Step 1: Screening for high-affinity peptides

[0063] (1) 10 11 Each phage was mixed with 1 mL of a Pseudomonas aeruginosa culture medium (OD600 = 0.1) in the stationary growth phase and incubated for 1 hour. Adsorbed phage was eluted from the cell aggregates by centrifugation using glycine-HCl (pH 2.2), then propagated in ER2738 medium containing Escherichia coli. After purification, the phage was precipitated with PEG to obtain M13 phage. The precipitated M13 phage was resuspended in PBS to complete one round of affinity screening. This affinity screening process was repeated four times to isolate a phage with high affinity for Pseudomonas aeruginosa, designated phage A.

[0064] (2) Prepare magnetic nanoparticles (referred to as magnetic beads M) that can fix polyvalent phages, add magnetic beads M to the polyvalent phage solution, and fix the polyvalent phages on magnetic beads M to form magnetic beads M'. Use 4% bovine serum albumin solution BSA to treat pure magnetic beads M and phage-coated magnetic beads M' respectively to block the vacant binding sites on magnetic beads M and M'. First, add 1 mg of pure magnetic beads M treated with BSA to the mixed phage display peptide library of Doctor No. 7 and Doctor No. 12 (5*10 10 phages, 10 11 Phages were mixed in 1 mL of PBS and incubated for 1 hour. After magnetic decantation using a neodymium magnet, the supernatant was separated (the supernatant now retains phage that lacks affinity for BSA and pure magnetic beads; this step ensures that phage subsequently isolated are not screened out due to magnetic forces or BSA attraction). To this supernatant, 1 mg of magnetic beads M', whose vacant binding sites have been blocked with BSA, was added for a second incubation. Bound phage were then eluted and enriched, completing one round of affinity screening. This affinity screening process was repeated four times to obtain phage with high affinity for the multivalent phage, designated phage B.

[0065] The magnetic beads M can be any magnetic nanoparticles that can effectively immobilize multivalent bacteriophages. In this embodiment, the magnetic beads M can be prepared by the following method:

[0066] First, 1 mL of 4-(2-aminophenyl)aniline (Sigma-Aldrich, 97%) was mixed with 20 mL of distilled water, followed by the addition of 2 mL of hydrochloric acid to form a dark yellow 4-aminoaniline mixture solution. 2 g of nano-iron carbide (Fe3C) magnetic particles were dispersed in 100 mL of distilled water and ultrasonicated for 15 minutes. The prepared 4-aminoaniline mixture was then added to the suspension and ultrasonicated to form a mixed solution. 820 mg (12 mmol / L) of sodium nitrite (NaNO2, Sigma-Aldrich) in cooled distilled water (0°C) was then added dropwise to the mixed solution to generate diazonium ions. Finally, the nanoparticles were recovered and washed to obtain surface-amino-modified nanoparticles (NPs-NH2). Then 1 mg of NPs-NH2 was taken and activated with 0.4 mL of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC, 20 mg / mL) and 0.3 mL of amine-reactive N-hydroxysuccinimide (NHS, 20 mg / mL) to obtain magnetic beads M.

[0067] (3) The above-mentioned phage A and phage B each contain a corresponding specific affinity peptide segment. Therefore, the following experiments can be performed on phage A and phage B to determine the peptide segment sequence:

[0068] Phage (A or B) containing the peptide fragments were individually dissociated and amplified on soft agar plates to form dispersed plaques. The target plaque was gently removed from the soft agar plate using a sterile inoculating loop, avoiding contact with nearby plaques. The plaque was immersed in 10 μL of SM buffer in a sterile microcentrifuge tube (0.2 mL). The tube was then vortexed vigorously for 5 minutes to accelerate the transfer of phage from the plaque to the SM buffer, resulting in a phage suspension. 1 μL of the phage suspension was then added to the PCR mixture as a DNA template. The resulting PCR reaction mixture contained 10 μL of Phusion Flash High-Fidelity PCR Master Mix, 1 μL of the phage suspension, 0.5 μM primers (forward primer: 5'-ATTCACCTCGAAAGCAAGCTG-3', reverse primer: 5'-AGACAGCCCTCATAGTTAGC-3'), and DNA-free water, for a total volume of 20 μL. PCR was performed in a T100 thermal cycler (Bio-Rad, USA) with the following temperature settings: 95°C for 5 minutes (for phage lysis and PCR enzyme activation), 98°C for 20 seconds, 33 cycles of 98°C for 6 seconds, annealing at 62.5°C for 16 seconds, annealing at 72°C for 20 seconds, and a final extension at 72°C for 3 minutes. DNA fragments corresponding to specific peptides were amplified by PCR. PCR products were purified using a DNA cleanup and concentration kit (ZYMO research, USA), and DNA concentration was determined using a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA). Finally, Sanger sequencing was performed to identify the corresponding specific affinity peptides.

[0069] In this example, a possible Pseudomonas aeruginosa-specific affinity peptide sequence was ultimately identified as YHLPVES (as shown in SEQ ID No. 1) based on phage A. Five possible multivalent phage-specific affinity peptide sequences were identified based on phage B: HDYTDWYWLLSY (as shown in SEQ ID No. 2), LTSSNQS (as shown in SEQ ID No. 3), WHWYQFELPDVV (as shown in SEQ ID No. 4), SNPETPDRPFRH (as shown in SEQ ID No. 5), and QGPTQFN (as shown in SEQ ID No. 6).

[0070] Step 2: Double modification of M13 phage capsid and tail fibers

[0071] (1) The restriction sites PstI (T1372A) and BamHI (C1381G) were introduced into the pVIII gene of the M13KE phage, and the previously existing PstI restriction site (A6250T) in multiple cloning sites was deleted by whole-plasmid mutagenesis to prepare the phage M13SK. In the process of preparing phage M13SK, primers 5'-TGTCTTTCGCTGCAGAGGGTGA-3' and 5'-TCACCCTCTGCAGCGAAAGACA-3' were used to introduce the PstI site; primers 5'-AGGGTGCTGCAGCCAAAAGC-3' and 5'GCTTTTGCGGGATCCTCACCCT-3' were used to introduce the BamHI site; and primers 5'CTTGCATGCCAGCAGGTCCTC-3' and 5'-GAGGACCTGCTGGCATGCAAG-3' were used to delete the existing PstI site.

[0072] (2) An additional amino acid P was added to the 3' end of the peptide segment YHLPVES to increase the probability of successful peptide fusion. Complementary oligonucleotides corresponding to the YHLPVESP peptide segment (with overhanging nucleotides) were then synthesized, with the sequences being 5'-GAATATCATTTACCTGTTGAATCACCG-3' and 5'GATCCGGTGATTCAACAGGTAAATGATATTCTGCA-3'. The synthesized complementary oligonucleotides were heated at 95°C for 2 minutes at a final concentration of 1 μmol / mL in annealing buffer (10 mM Tris, pH 7.5, 50 mM NaCl, 1 mM EDTA), and then cooled to 25°C for 50 minutes. The annealed complementary oligonucleotides were inserted into the M13SK genome between the PstI and BamHI restriction sites using T4 ligase (pre-digested with PstI and BamHI to open the two restriction sites) to form a recombinant phage genome.

[0073] (3) The recombinant phage genome was transformed into homemade electroporation Escherichia coli ER2738 using the Gene Pulser Xcell electroporation system (Bio-Rad, USA), and then plaque detection was performed. The resulting plaques were verified by PCR and pVIII gene sequencing (forward primer: 5'-TTACCCGTTTAATGGAAACTTC-3', reverse primer: 5'-AAGGAGCCTTTAATTGTATCG-3'). After verification, the genetically recombinant M13SK phage was used to continue the modification and insertion of multivalent phage-specific peptides.

[0074] (4) Similarly, for the five possible polyvalent phage-specific affinity peptide sequences HDYTDWYWLLSY, LTSSNQS, WHWYQFELPDVV, SNPETPDRPFRH, and QGPTQFN, complementary oligonucleotides corresponding to the polyvalent phage-specific peptides were synthesized. Then, for each combination of complementary oligonucleotides synthesized in this step, based on the M13SK phage recombined in step (2), the synthesized complementary oligonucleotides were further inserted between the EagI and Acc65I restriction sites of the pill gene for modification. The gene modification method was the same as the modification method of the pVIII gene in the aforementioned step (2). Finally, a group of double-modified M13 phages were obtained for each multivalent phage-specific affinity peptide sequence, which were named double-modified M13 phage III-1, double-modified M13 phage III-2, double-modified M13 phage III-3, double-modified M13 phage III-4, and double-modified M13 phage III-5, respectively.

[0075] Step 3: Treat biofilms with dual-modified M13 phage combined with multivalent phage and characterize the treatment effects

[0076] (1) Take the exponential phase of Pseudomonas aeruginosa (PAO1) and Escherichia coli (NDM-1) and dilute them with PBS to an OD600 of 0.5. Then add 0.25 mL of the diluted Escherichia coli and 0.25 mL of the diluted Pseudomonas aeruginosa to a plastic culture dish containing 5 mL of modified M63 culture medium and mix them well. Place a hydrophobic PVDF membrane with a pore size of 0.2 μm in the mixed solution of the culture dish and incubate at 37°C with horizontal shaking at 50 rpm for 36 hours. After the incubation, wash the floating cells 3 times with PBS to obtain Pseudomonas aeruginosa and Escherichia coli. Double biofilm .

[0077] (2) A polyvalent phage (named X) that can infect both Pseudomonas aeruginosa and Escherichia coli was isolated in advance. The double-modified M13 phages III-1 to III-5 were mixed with polyvalent phage X in equal volumes in PBS, rotated at 10 rpm at 4°C overnight, and then diluted 500-fold with PBS (until the titers of both phages in the dilution solution reached 106 PFU / mL) to obtain a conjugate of the two phages.

[0078] (3) Design Figure 1 The water supply network simulation device shown;

[0079] Two polymethyl methacrylate plates (Goodfellow, USA) were used to simulate the water supply network, and a flow channel (1 cm in diameter and 10 cm in length) was engraved on the bottom plate. The two plates were fixed together using screws and silicone gaskets to prevent leakage. The PVDF membrane on which the biofilm was attached and grown was cut into appropriate sizes and embedded in the bottom of the channel to simulate the growth of the biofilm on the inner surface of the water supply network. Three groups were designed, and pure multivalent phage (106 PFU / mL in PBS, group name is X treatment), the two phage conjugate prepared in the previous step (2) (group name is X+M13 treatment), or pure PBS (group name is control group) was passed through the water channel embedded with the biofilm at different speeds (0-1 cm / s) for 30 minutes to allow phage attachment. The biofilm was then carefully removed, gently washed three times with PBS to remove free phage, and then incubated in PBS for 4 hours to allow the phage adsorbed on the biofilm to lyse the cells it infected and further disperse the phage.

[0080] (4) The residual biofilms from the different groups were separated from the PVDF membranes and analyzed using a confocal laser scanning microscope. The residual biofilms on the membranes were stained with SYTO 9 and propidium iodide (PI). The membranes were then mounted on glass slides and observed using a 20x objective lens under an Olympus IX-71 microscope. Live bacteria stained with SYTO 9 radiated green light under excitation with a 488 nm laser line, while dead bacteria stained with PI radiated red light under excitation with a 560 nm laser line.

[0081] (5) The PVDF membrane containing the residual biofilm was immersed in 0.2% PBST at 0°C, vortexed vigorously for 3 minutes, and sonicated at 40 kHz for 15 minutes in a bath sonicator (Branson, USA) at 4°C to separate the biofilm and disperse the bacteria. The viable bacteria were then counted using the plate method, and the total bacterial count was determined using quantitative PCR (qPCR) targeting the 16s rRNA gene. The biofilm removal efficiency was determined as the relative difference between the treated biofilm and the control biofilm.

[0082] The final results showed that among the double-modified M13 phage III-1 to 5, only the double-modified M13 phage III-1 modified with the multivalent phage-specific affinity peptide sequence HDYTDWYWLLSY (as shown in SEQ ID No. 2) had a true multivalent phage-specific affinity effect. Therefore, based on this group of double-modified M13 phage III-1, the specific technical effects of this embodiment are demonstrated, as shown in FIG. Figure 2 and Figure 3 shown.

[0083] like Figure 2As shown, a comparison of the biofilm removal efficiency in the X group and the X+M13 group in this example is shown. As can be seen from the results, under stagnant water conditions (i.e., flow rate = 0 cm / s, which is typical in water tanks and house plumbing systems), only the multivalent phage treatment resulted in a 16.3±12.3% reduction in viable biofilm bacteria (determined by plate count) and a 11.6±5.0% reduction in total bacteria (determined by 16s quantification). The phage conjugate further reduced the biomass, as the viable bacteria decreased by 77.4±6.8% and the total bacteria decreased by 74.8±11.3%, indicating that the biofilm removal efficiency was higher due to enhanced phage delivery and higher infection rate. At different water flow rates, the biofilm removal efficiency of the multivalent phage and double-modified M13 phage conjugate treatments was higher than that of the multivalent phage treatment alone.

[0084] like Figure 3 The figure shows the live / dead cell coverage obtained by microscopic analysis four hours after phage treatment (bars represent biofilm coverage, and broken lines represent live / dead cell ratio). Microscopic analysis revealed that PEBX treatment reduced biofilm coverage from 96.7±1.5% (no phage control) to 74.9±4.7%, and further reduced it to 13.9±3.9% with phage conjugate treatment. The remaining biofilm coverage after conjugate treatment was significantly less than that after treatment with the polyvalent phage alone, and the live / dead cell ratio was also lower than that after treatment with the polyvalent phage alone, indicating that the conjugate enhanced the efficiency of the phage as a bactericidal agent in removing biofilm.

[0085] The embodiment described above is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Persons skilled in the art may make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, any technical solution obtained by equivalent substitution or equivalent transformation falls within the scope of protection of the present invention.

Claims

1. An M13 filamentous phage with double modification of capsid and tail fiber protein, characterized in that: include: A first oligonucleotide corresponding to a specific affinity peptide segment of Pseudomonas aeruginosa is inserted into the pVIII gene of the M13 filamentous phage using gene editing technology. After expression of the first oligonucleotide, the M13 filamentous phage capsid protein has a peptide sequence that has affinity for adsorbing Pseudomonas aeruginosa. Simultaneously, a second oligonucleotide corresponding to a specific affinity peptide segment of a polyvalent phage is inserted into the pIII gene of the M13 filamentous phage using gene editing technology. After expression of the second oligonucleotide, the M13 filamentous phage tail fiber protein has a peptide sequence that has affinity for adsorbing polyvalent phages. The Pseudomonas aeruginosa-specific affinity peptide sequence is YHLPVESP, which is inserted between the PstI and BamHI restriction sites in the genome of bacteriophage M13SK; bacteriophage M13SK is obtained by introducing restriction sites PstI and BamHI into the pVIII gene of M13KE phage and deleting the PstI restriction site previously existing in multiple cloning sites; The polyvalent phage-specific affinity peptide sequence is HDYTDWYWLLSY, and is inserted between the EagI and Acc65I restriction sites in the pill gene of the phage M13SK.

2. The M13 filamentous phage with double modifications of capsid and fiber proteins according to claim 1, characterized in that: The method for inserting the first oligonucleotide into the pVIII gene of the M13 filamentous phage by gene editing technology is as follows: Phage M13SK was generated by introducing the restriction sites PstI and BamHI into the pVIII gene of the M13KE phage and deleting the previously existing PstI restriction sites in multiple cloning sites. Complementary oligonucleotides corresponding to the Pseudomonas aeruginosa-specific affinity peptide were synthesized and annealed, and then the annealed complementary oligonucleotides were inserted into the phage M13SK genome between the PstI and BamHI restriction sites using T4 ligase to complete the insertion modification of the first oligonucleotide.

3. The M13 filamentous phage with double modifications of capsid and fiber proteins according to claim 2, characterized in that: The method for inserting the second oligonucleotide into the pill gene of the M13 filamentous phage by gene editing technology is as follows: After synthesizing complementary oligonucleotides corresponding to the multivalent phage-specific affinity peptides and performing annealing treatment, the annealed complementary oligonucleotides are inserted into the pIII gene of the phage M13SK after modification of the first oligonucleotide using T4 ligase, and the insertion position is between the EagI and Acc65I restriction sites.

4. A method for enhancing phage delivery using a double-modified phage carrying a multivalent phage, characterized in that: The multivalent phage is mixed with the M13 filamentous phage with double modification of the capsid and tail fiber protein according to any one of claims 1 to 3 to form a conjugate of the two phages, wherein the multivalent phage is adsorbed on the tail fiber protein of the M13 filamentous phage; the conjugate of the two phages is then introduced into the water environment where the target biofilm is located, so that the M13 filamentous phage carrying the multivalent phage is further affinity-adsorbed to the Pseudomonas aeruginosa in the target biofilm through the peptide sequence modified on the capsid protein, thereby enhancing the delivery of the multivalent phage to the target biofilm.

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