Recombinant M13 bacteriophage, K88 targeting antibacterial preparation containing bacteriophage and application of K88 targeting antibacterial preparation

Abstract

The invention discloses a recombinant M13 bacteriophage, a K88 targeting antibacterial preparation containing the bacteriophage and application of the K88 targeting antibacterial preparation, and belongs to the field of biotechnology and veterinary medicine. The antibacterial preparation comprises a recombinant M13 bacteriophage rM13; a K88 targeting element is displayed on a tail filament pIII protein of the recombinant M13 bacteriophage, and the K88 targeting element is a nano antibody NBK88 of which the amino acid sequence is as shown in SEQ ID NO: 1. Through engineering modification, the rM13 can specifically recognize, infect and split enterotoxigenic escherichia coli expressing K88 fimbriae, so that the host range of the wild type M13 is accurately guided to pathogenic bacteria. The invention also provides application of the preparation in preparation of medicines for preventing or treating ETEC infection, and the dosage form of the preparation is preferably a freeze-dried preparation or an enteric-coated preparation capable of being released in a targeted intestinal tract. The preparation provided by the invention has the remarkable advantages of strong targeting property, self-proliferation, no damage to intestinal microecology and capability of overcoming bacterial drug resistance.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and veterinary medicine, specifically to a recombinant M13 bacteriophage, a K88-targeted antibacterial agent containing the bacteriophage, and their applications. Background Technology

[0002] Enterotoxigenic Escherichia coli (ETEC) is one of the main pathogens causing diarrhea in newborn and weaned piglets, with strains expressing K88 fimbriae being the predominant pathogenic serotype. K88 fimbriae mediate bacterial adhesion to the small intestinal epithelial cells of piglets, a prerequisite for ETEC pathogenicity. Currently, the pig industry mainly relies on antibiotics to prevent and control ETEC infection; however, long-term and widespread use has led to increasingly serious problems of bacterial resistance, drug residues, and food safety risks, while also increasing farming costs. Therefore, developing novel, highly effective, and safe antibiotic alternatives is urgently needed.

[0003] Bacteriophages are viruses capable of specifically infecting and lysing bacteria. Due to their strong host specificity, high self-replication capacity, minimal impact on beneficial bacteria, and low residue levels, they are considered potential alternatives to antibiotics. M13 bacteriophage is a filamentous bacteriophage that uses *E. coli* as its host. Its well-defined genetic structure makes it easy to manipulate genetically, making it an ideal platform for engineering modification. However, the natural M13 bacteriophage exhibits strict host receptor specificity (*E. coli* F-type fimbriae), and its natural host range has a low overlap with the pathogenic ETEC K88 strain, resulting in its inability to effectively recognize and lyse ETEC K88. This significantly limits its application in the prevention and control of ETEC. Summary of the Invention

[0004] To overcome the problem of the narrow antibacterial spectrum of M13 bacteriophage, this invention performs targeted modification of the M13 bacteriophage genome to display specific target molecules on its pIII protein, thereby reprogramming its host recognition range so that it can accurately target and eliminate specific pathogens.

[0005] This invention provides a recombinant M13 phage, wherein the recombinant M13 phage displays a K88 targeting element on its tail fibula pIII protein, and the recombinant M13 phage is named rM13 phage.

[0006] Furthermore, the K88 targeting element is displayed at the N-terminus of the pIII protein of the tail filament via gene fusion.

[0007] Furthermore, the K88 targeting element replaces the native receptor-binding domain of the pIII protein of the tail filament.

[0008] Furthermore, the K88 targeting element is a nanobody NBK88, and the amino acid sequence of the nanobody NBK88 is shown in SEQ ID NO:1.

[0009] The present invention also provides a K88-targeted antimicrobial agent comprising recombinant M13 phage and a pharmaceutically or veterinarily acceptable carrier.

[0010] The present invention also provides the use of a K88-targeted antibacterial agent in the preparation of a medicament for the prevention or treatment of enterotoxigenic Escherichia coli infection, wherein the enterotoxigenic Escherichia coli infection is an ETEC infection expressing K88 fimbriae.

[0011] Furthermore, the drug is used to prevent or treat piglet diarrhea caused by ETEC expressing K88 fimbriae.

[0012] Furthermore, the dosage form of the drug is a lyophilized preparation, a liquid preparation, or an enteric-coated preparation.

[0013] Furthermore, the enteric-coated formulation is a high-molecular-weight enteric-coated sustained-release formulation, wherein the high-molecular-weight enteric-coated sustained-release formulation comprises at least one selected from Eudragit L100, Eudragit S100, hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate succinate, cross-linked sodium alginate, and complex chitosan.

[0014] Furthermore, the enteric-coated sustained-release formulation is in tablet form, and the polymeric material is Eudragit L100, which is the main component of the tablet coating.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: I. This invention successfully recombines the K88 targeting element onto the pIII protein of the M13 phage tail filament, enabling it to accurately recognize and infect pathogenic ETEC K88 strains, thus effectively solving the technical problem of mismatch between the antibacterial spectrum of natural M13 phage and ETEC serotype.

[0016] Second, the modified phage of this invention can replicate and proliferate within target bacteria, achieving self-amplification and continuous bactericidal action. It can achieve efficient lysis at the site of infection with only a low initial dose, and is especially suitable for the intestinal environment.

[0017] Third, the recombinant phage of this invention specifically targets and expresses ETEC with K88 fimbriae, without affecting other beneficial microbial communities in the gut. It can effectively maintain intestinal microecological stability while clearing pathogens, avoiding the dysbiosis problems caused by broad-spectrum antibiotics. Furthermore, its bactericidal mechanism does not depend on the target of traditional antibiotics, thus it is also effective against multidrug-resistant ETEC K88 strains, providing a novel solution to combat bacterial resistance.

[0018] Fourth, by designing an enteric-coated formulation, this invention can ensure that bacteriophages remain active in the gastric acid environment and are precisely released at the target site in the intestine, which significantly improves the delivery efficiency and bioavailability of live bacteria and ensures the final preventive and therapeutic effects of the formulation. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 This is the result of the inhibition zone experiment of the rM13 phage of the present invention against some K88-positive Escherichia coli.

[0021] Figure 2 This is a comparison of growth curves of the strains after inoculating K88-positive Escherichia coli with the rM13 phage of this invention. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, 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, 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.

[0023] Example 1 Construction of recombinant M13 phage (rM13) 1.1 Construction of rM13 recombinant plasmid Wild-type M13 phage ( Escherichia phage M13, Inovirus M13 (GenBank: PL346599.1) was purchased from Ningbo Mingzhou Biotechnology Co., Ltd.

[0024] The amino acid sequence of the target nanobody NBK88 is shown in SEQ ID NO:1: QVQLQESGGGLVQAGGSLRLSCAASGSVSSISTMGWFRQAPGKEREFVAAITSDALTEYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCHASQWSTVVARGIDYWGQGTQVTVSS The coding gene sequence of the target Nanobody NBK88 is as SEQ ID NO:2 shows: CATATGCAAGTACAGCTACAAGAATCAGGTGGAGGGTTGGTTCAGGCGGGTGGCTCCCTGCGCCTGTCTTGTGCGGCGTCTGGCAGCGTTTCCTCGATCAGCACGATGGGCTGGTTTCGTCAAGCTCCGGGTAAAGAGCGCGAGTTCGTGGCCGCGATCACCAGCGACGCACTGACCGAATATG CGGATAGCGTGAAAGGCCGTTTCACCATTAGCCGTGACAACGCCAAGAACACCGTTTATCTGCAAATGAATAGCCTTAAGCCGGAAGATACCGCTGTGTACTACTGCCACGCAAGCCAATGGTCAACGGTCGTGGCGCGTGGTATTGACTACTGGGGTCAGGGTACTCAGGTTACCGTAAGCTCCCTCGAG The primer sequences used are as follows: pM13-1:ACTGTTGAAAGTTGTTTAGCAAAAC pM13-2: CATGTTGAAAATCTCCAAAAAAAAAGGC pNB-1: GAGATTTTCAACATGCAAGTACAGCTACAAGAATCAG pNB-2:ACAACTTTCAACAGTGAGGGAGCTTACGGTAACC The genome of wild-type M13 phage was amplified using primer pair pM13-1 / pM13-2, yielding a linearized vector fragment of approximately 6350 bp. The coding DNA sequence of NBK88 was amplified using primer pair pNB-1 / pNB-2, yielding a target gene fragment of approximately 450 bp. Both PCR products were purified by agarose gel electrophoresis.

[0025] Subsequently, using the In-Fusion Snap Assembly Master Mix (Takara, Code No. 638947) seamless cloning kit, the purified NBK88 DNA fragment was inserted into the 5' end of the linearized M13 vector PIII protein gene (GIII). The resulting recombinant plasmid can fuse the NBK88 nanobody with the N-terminus of the PIII protein for expression; the construct at this stage is recombinant plasmid DNA.

[0026] Those skilled in the art should understand that any substantially identical technical solutions that use different primer sequences, enzyme restriction sites, or ligation methods to achieve the fusion expression of NBK88 nanoantibody at the N-terminus of the PⅢ protein are all within the scope of protection of the claims of this invention.

[0027] 1.2 Preparation of competent cells and transformation of recombinant plasmids Escherichia coli TG1 strain (host bacterium) was purchased from Sangon Biotech (Shanghai) Co., Ltd. The preparation of competent cells was performed according to the heat shock method described in *Molecular Cloning: A Laboratory Manual (Third Edition)*, with the specific steps as follows: S1: Pick a small amount of bacteria from the TG1 puncture culture medium and inoculate it into 20 mL of LB liquid medium. Incubate overnight at 37°C with shaking at 180 rpm.

[0028] S2: Take 1 mL of overnight activated bacterial culture and transfer it to 100 mL of fresh LB medium. Incubate at 37°C and 180 rpm until OD500. 600 The value is approximately 0.4.

[0029] S3: Transfer the bacterial culture to a pre-cooled centrifuge tube and incubate in an ice water bath for 10 minutes, gently mixing during the process to aid cooling.

[0030] S4: Centrifuge at 4℃ and 4000×g for 10 minutes, and discard the supernatant.

[0031] S5: Add 6 mL of pre-cooled 0.1 M CaCl2 solution, gently resuspend the bacterial cells, and incubate on ice again for 10 minutes.

[0032] S6: Centrifuge at 4℃ and 4000×g for 10 minutes, and discard the supernatant.

[0033] S7: Add 5 mL of pre-cooled 0.1 M CaCl2 solution (containing 15% glycerol), gently resuspend the bacterial cells, and dispense 100 μL into each sterile centrifuge tube to obtain TG1 competent cells.

[0034] Take 100 μL of the above competent cells and add 5 μL of the recombinant plasmid ligation product obtained in step 1.1, then gently mix. After incubating on ice for 30 minutes, heat shock in a water bath at 42°C for 60 seconds, then quickly transfer to ice to cool for 2 minutes. Add 900 μL of fresh LB medium and incubate at 37°C with shaking at 180 rpm for 1 hour to recover and culture.

[0035] Those skilled in the art should understand that other host strains, competent cell preparation methods, transformation methods, or culture conditions can also be used to achieve the purpose of this invention and fall within the scope of protection of this invention.

[0036] 1.3 rM13 plaque culture and titer determination S1: Preparation of TG1 bacterial culture in logarithmic growth phase (OD) 600 ≈0.4), the method is the same as S1-S2 in 1.2.

[0037] S2: Perform continuous 10-fold serial dilutions on the transformed bacterial culture obtained in step 1.2, usually 5-10 dilutions.

[0038] S3: Take 1 mL of each gradient dilution and quickly mix it with 4 mL of melted LB semi-solid medium (containing 0.5-0.7% agar). Immediately pour the mixture onto the solidified LB solid medium plate and shake the plate to spread it evenly.

[0039] S4: After the top layer of culture medium has completely solidified, invert the plate in a 37°C incubator and incubate overnight (about 12-16 hours).

[0040] S5: Count the clear plaques formed on the plate and calculate the titer (pfu / mL) of rM13 phage in the original sample using the formula: C = X × 10 n Where C is the original sample titer (pfu / mL), X is the number of plaques on the plate suitable for counting, and n is the dilution corresponding to the plate.

[0041] 1.4 Amplification and purification of rM13 phage S1: Preparation of TG1 bacterial culture in logarithmic growth phase (OD) 600 (≈0.4), the method is the same as before.

[0042] S2: Select a single, clearly morphologically distinct plaque, grind and resuspend it in a small amount of LB medium, and inoculate it into 1 mL of the above TG1 bacterial suspension. Incubate at 37°C and 180 rpm for 1 hour. Then, transfer the entire mixed culture to 100 mL of fresh LB medium and amplify it by shaking at 37°C and 180 rpm for 16 hours.

[0043] S3: Collect the amplified bacterial culture, centrifuge at 3200×g for 30 minutes at 4℃, and collect the supernatant.

[0044] S4: Add 1 / 4 volume of PEG / NaCl solution (polyethylene glycol 6000 200 g / L, sodium chloride 146.25 g / L, diluted to 1 L with deionized water) to the supernatant, mix well, and let stand in an ice-water bath for 30 minutes to allow the phage to precipitate.

[0045] S5: Centrifuge at 3200×g for 30 minutes at 4℃, and carefully discard the supernatant.

[0046] S6: Resuspend the precipitate in 1 mL of pre-cooled PBS buffer, transfer the resuspended solution to a new 1.5 mL centrifuge tube, and centrifuge at 4°C and 20,000 × g for 1 minute to remove residual cell debris.

[0047] S7: Carefully aspirate the supernatant into a new sterile centrifuge tube. This is the purified rM13 phage suspension, which can be stably stored for several months at 4°C or room temperature. Its titer can be determined according to the method described in 1.3.

[0048] Example 2: Analysis of the in vitro antibacterial effect of rM13 bacteriophage To evaluate the targeted antibacterial activity of rM13 against K88-positive Escherichia coli, the standard strain K88 was selected. E. coli Enterotoxigenic pathogenic Escherichia coli (strain number K88+) was used as a positive control (purchased from the NTCC Type Culture Collection Center), and 10 clinical isolates of Escherichia coli isolated from the excrement of diseased piglets (identified by PCR, 5 of which were K88 positive, numbered K88-1~K88-5; 5 were K88 negative, numbered K88Y-1~K88Y-5) were used for inhibition zone experiments.

[0049] 2.1 Experimental Methods: S1: Phage Sample Preparation: Dilute the purified rM13 and wild-type M13 (wtM13) with PBS buffer to 1×10⁻⁶. 6 pfu / mL. Take a sterile circular filter paper disc (5 mm in diameter), add 100 µL of diluted phage suspension to each disc, and store in a sealed container at 4°C for later use.

[0050] S2: Indicator strain preparation: Inoculate each test strain into LB liquid medium and activate overnight at 37°C and 180 rpm. Transfer 1 mL of the overnight bacterial culture to 100 mL of fresh LB medium and incubate at 37°C with shaking until OD reaches 100%. 600 ≈0.4 (logarithmic growth phase).

[0051] S3: Spreading and Plating: Take 0.2 mL of each indicator bacterial suspension and spread it evenly on an LB agar plate using a sterile spreader. After the bacterial suspension is absorbed by the culture medium, place a phage filter paper plate prepared in step S1 in the center of the plate.

[0052] S4: Incubation and Observation: After incubating the plates upside down in a 37℃ incubator for 16 hours, measure the diameter of the inhibition zone (R, unit: mm). The width of the inhibition zone (d, unit: mm) is calculated using the formula: d = (R - 5) / 2. Each experimental group has three replicates.

[0053] 2.2 Results and Analysis: Results of inhibition zone experiments for some strains are as follows: Figure 1 As shown in Table 1.

[0054] Table 1. Diameter of inhibition zones (mm) of rM13 and wild-type M13 against different Escherichia coli.

[0055] The results showed that recombinant phage rM13 produced significant inhibition zones against all six K88-positive Escherichia coli strains, while wild-type M13 had no inhibitory effect. For K88-negative strains, the inhibition spectrum of rM13 was essentially the same as that of wild-type M13. This demonstrates that by displaying the NBK88 nanobody, rM13 successfully expanded its host recognition range and could specifically inhibit the growth of K88-positive Escherichia coli.

[0056] Example 3: Effect of rM13 bacteriophage on the growth curve of pathogenic bacteria 3.1 Experimental Methods: S1: Dilute the purified rM13 and wild-type M13 with PBS to a concentration of 1×10⁻⁶. 6 pfu / mL.

[0057] S2: After overnight activation of K88 positive strains (K88+, K88-1~K88-5), the initial OD is used. 600 The concentration of 0.01 was inoculated into 100 mL of fresh LB medium and incubated at 37°C and 180 rpm for 2 hours.

[0058] S3: Add 100 µL of rM13 or wild-type M13 suspension to each bacterial culture, and add an equal volume of LB medium to the control group. Continue to culture at 37℃ and 180 rpm with shaking. Starting from phage inoculation (0 hours), take samples every 2 hours to measure OD. 600 The value will be continuously monitored for 14 hours.

[0059] 3.2 Results and Analysis: Growth curves as follows Figure 2As shown. In the experimental group inoculated with rM13, the growth of all six K88-positive strains was significantly inhibited, and their OD... 600 The growth rate of the bacteria was significantly slowed down, and the final bacterial density was much lower than that of the control group. Wild-type M13 had no significant effect on the growth of the above strains. These results further confirm that rM13 has specific lytic ability and growth-inhibiting effect on K88-positive Escherichia coli.

[0060] Example 4: Application of rM13 in the prevention and treatment of ETEC infectious diarrhea in piglets 4.1 Experimental Design and Processing S1: Amplify and purify rM13 according to the method in Example 1, resuspend in sterile physiological saline and determine the titer, and finally dilute to 1×10⁻⁶. 12 pfu / mL available for use.

[0061] S2: Incubate K88-1, a K88-positive strain, overnight and adjust the bacterial concentration to 1 OD using physiological saline. 600 Units per 10 mL are used as the challenge bacterial solution.

[0062] S3: Fifty healthy, 24-28 day old, 6.0±0.5 kg three-way crossbred (Duroc × Landrace × Large White) weaned piglets were selected. All piglets came from farms with no ETEC infection records in the past 3 months. They were randomly divided into 5 groups of 10 piglets each. The specific grouping and treatment are shown in Table 2.

[0063] Table 2 Animal Experiment Group Design

[0064] During the experiment, all piglets were housed independently in a clean animal facility, fed a basal diet, and received no drug treatment. Diarrhea, mortality, and weight changes in each group of piglets were continuously observed and recorded from 0 to 20 days.

[0065] 4.2 Experimental Results The experimental results are recorded in Table 3.

[0066] Table 3. The preventive and therapeutic effects of rM13 on ETEC-infected piglets.

[0067] The experimental results showed that, compared with the positive control group, both the rM13 prevention and treatment groups effectively reduced the diarrhea rate and diarrhea index in piglets, and significantly improved the survival rate and daily weight gain. The protective effect of prophylactic administration was superior to that of therapeutic administration, indicating that rM13 phage can be used to effectively prevent and treat diarrhea in piglets caused by ETEC infection.

[0068] Example 5: Preparation and activity evaluation of rM13 lyophilized formulation 5.1 Preparation of rM13 lyophilized formulation To improve the stability and ease of use of rM13 formulations, it was prepared as a lyophilized powder.

[0069] rM13 phage was prepared according to the method in Example 1. The purified rM13 phage precipitate was resuspended using a 5% (w / v) skim milk solution sterilized by autoclaving as a protectant. After determining the initial phage titer, the phage suspension was diluted to 1 × 10⁻⁶ with the same protectant solution. 11 -1×10 12 pfu / mL. The diluted suspension was dispensed into lyophilization bottles and placed in a fully automatic freeze dryer. The bottles were then freeze-dried according to a preset program to obtain a loose, porous freeze-dried powder.

[0070] 5.2 Determination of activity retention rate of rM13 lyophilized formulation Accurately weigh 50 mg of lyophilized powder and place it in a sterile centrifuge tube. Add 1 mL of sterile ultrapure water and vortex to fully reconstitute the phage. Immediately determine the titer of the reconstituted phage according to the method described in Example 1.3. Compare the titer with the original titer before lyophilization and calculate the activity retention rate (%).

[0071] The activity test results of three independent batches of freeze-dried products are shown in Table 4.

[0072] Table 4 Activity retention rate of rM13 lyophilized formulation

[0073] Example 6: Preparation of rM13 enteric-coated positioning tablets This example illustrates the preparation of an enteric-coated sustained-release formulation—rM13 enteric-coated positioning tablets. The specific steps are as follows: 6.1 Tablet Core Preparation The basic components and proportions of the tablet core (by weight percentage) are shown in the table below: Table 5. Composition of Tablet Core Formulation

[0074] The preparation process of the chip core includes the following steps: S1: The rM13 lyophilized preparation, anhydrous lactose, and crospovidone were passed through an 80-mesh sieve.

[0075] S2: Place the sieved raw materials into a three-dimensional mixer and mix at 20 rpm for 30 minutes to ensure thorough and uniform mixing.

[0076] S3: Add magnesium stearate and colloidal silica to the main mixture, adjust the mixer speed to 10 rpm, and continue mixing for 5 minutes to ensure that the low-content components are evenly distributed.

[0077] S4: The final mixed powder is compressed into tablet cores using a rotary tablet press. The tablet core hardness is controlled at 6 kPa, and the tablet weight is 0.5 g ± 0.025 g. The resulting tablet cores are used for subsequent coating.

[0078] 6.2 Preparation of Enteric Coating Solution The basic components and proportions of the coating solution (by weight percentage) are shown in the table below: Table 6. Coating solution formulation composition

[0079] The preparation process of the coating solution includes the following steps: S1: Add most of the anhydrous ethanol (approximately 90% of the total prescription volume) to the mixing tank. While continuously stirring mechanically, slowly add Eudragit L100 powder and continue stirring for at least 2 hours until a clear and transparent polymer solution is formed.

[0080] S2: Add diethyl phthalate to the above polymer solution and continue stirring for 30 minutes to ensure uniform mixing.

[0081] S3: In another container, mix the remaining anhydrous ethanol with colloidal silica and disperse the mixture for 3 minutes at 5000 rpm using a high-speed shear emulsifier to prepare a uniform anti-sticking slurry.

[0082] S4: Under slow stirring, slowly add the anti-sticking agent slurry to the polymer solution containing the plasticizer. After the addition is complete, continue stirring for 1 hour to ensure uniform dispersion. The resulting coating solution is then filtered through a 100-mesh sieve for later use.

[0083] 6.3 Coating process and formulation molding A high-efficiency coating pan is used to coat the tablet cores. The coating process parameters are as follows: tablet bed temperature 30-40°C; inlet air temperature 40-50°C; coating pan rotation speed 10-20 rpm; spray gun air pressure 0.3-0.5 MPa; spray rate, initially 2-3 mL / min, gradually increased to 8-10 mL / min after the tablet cores are preheated and the coating film has initially formed.

[0084] The coating process continues until the coating layer gains 5-8% of the tablet core weight. After coating, the coated tablets are placed in a 30°C oven for curing for 6 hours to obtain the rM13 enteric-coated positioning tablets.

[0085] Enteric-coated polymers remain stable in the acidic environment of the stomach, but rapidly dissolve or swell in the neutral to weakly alkaline environment of the small intestine, thereby achieving targeted intestinal release of the drug. In this embodiment, Eudragit L100, an anionic polyacrylic acid resin, is selected. Its molecular chain contains carboxyl groups, and it does not dissociate or dissolve in the stomach. When it enters the duodenum (pH approximately 5.5-6.0), the carboxyl groups ionize, and the polymer dissolves, thus achieving precise drug release in the duodenum and upper jejunum. This region is the core site for ETEC K88 colonization and pathogenicity, effectively protecting the bacteriophage from the acidic environment of the stomach and improving its bioavailability in the lesion area. The enteric-coated tablets prepared in this embodiment maintain structural integrity in the acidic environment of the stomach and target the dissolution of the coating layer in the neutral pH environment of the duodenum, releasing the active ingredient rM13, thereby achieving targeted intestinal delivery of the drug.

[0086] In summary, the core of this invention lies in genetically engineering M13 phage to display the element nanobody NBK88, which specifically targets K88 fimbriae, on its tail fimbriae pIII protein. This "reprograms" its host range, enabling it to accurately recognize and efficiently lyse enterotoxin-producing *Escherichia coli* (ETEC) expressing K88 fimbriae. The resulting recombinant rM13 phage not only possesses high specificity and bactericidal efficiency but also can self-reproduce at the infection site, achieving a long-lasting effect. Furthermore, its highly specific mode of action avoids disruption of the normal intestinal flora, effectively maintaining microecological stability and making it suitable for addressing the growing problem of antibiotic resistance.

[0087] Furthermore, this invention innovatively develops an enteric-coated formulation suitable for rM13, ensuring the safe delivery of the active ingredients to the intestinal lesion site, effectively improving bioavailability and ultimate preventive and therapeutic effects. Experimental results show that this formulation is significantly effective in preventing and treating piglet diarrhea caused by ETEC K88, demonstrating excellent application potential and market value.

[0088] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A recombinant M13 phage, characterized in that, The recombinant M13 phage exhibits a K88 targeting element on its tail fibula pIII protein, and the recombinant M13 phage is named rM13 phage.

2. The recombinant M13 phage according to claim 1, characterized in that, The K88 targeting element is displayed at the N-terminus of the pIII protein of the tail filament via gene fusion.

3. The recombinant M13 phage according to claim 1, characterized in that, The K88 targeting element replaces the native receptor-binding domain of the pIII protein in the tail filament.

4. The recombinant M13 phage according to any one of claims 1 to 3, characterized in that, The K88 targeting element is the nanobody NBK88, and the amino acid sequence of the nanobody NBK88 is shown in SEQ ID NO:

1.

5. A K88-targeted antibacterial agent, characterized in that, The antimicrobial agent comprises the recombinant M13 phage as described in claim 4 and a pharmaceutically or veterinarily acceptable vector.

6. The use of the K88-targeted antibacterial agent as described in claim 5 in the preparation of a medicament for the prevention or treatment of enterotoxigenic Escherichia coli infection, characterized in that, The enterotoxigenic Escherichia coli infection is an ETEC infection expressing K88 fimbriae.

7. The use according to claim 6, characterized in that, The drug is used to prevent or treat diarrhea in piglets caused by ETEC expressing K88 fimbriae.

8. The use according to claim 7, characterized in that, The dosage form of the drug is a lyophilized preparation, a liquid preparation, or an enteric-coated preparation.

9. The use according to claim 8, characterized in that, The enteric-coated formulation is a high-molecular-weight enteric-coated sustained-release formulation, and the high-molecular-weight enteric-coated sustained-release formulation contains at least one of Eudragit L100, Eudragit S100, hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate succinate, cross-linked sodium alginate, and complex chitosan.

10. The use according to claim 9, characterized in that, The enteric-coated sustained-release formulation is in tablet form, and the polymeric material is Eudragit L100, which is the main component of the tablet coating.

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