A recombinant m13 bacteriophage, a k88-targeting antibacterial preparation comprising the same, and use thereof

Abstract

The application discloses a recombinant M13 bacteriophage, a K88-targeting antibacterial preparation containing the bacteriophage and application of the antibacterial preparation, and belongs to the field of biotechnology and veterinary medicine. The antibacterial preparation contains the recombinant M13 bacteriophage rM13; a K88-targeting element is displayed on the tail fiber pIII protein of the recombinant M13 bacteriophage, and the K88-targeting element is a nanobody NBK88 with an amino acid sequence shown in SEQ ID NO:1. The engineering in the application enables the rM13 to specifically recognize, invade and lyse enterotoxigenic Escherichia coli expressing K88 fimbriae, so that the host range of the wild-type M13 is accurately guided to pathogenic bacteria. The application also provides use of the preparation in preparation of a medicine for preventing or treating ETEC infection, and a dosage form of the medicine is preferably a freeze-dried preparation or an enteric preparation capable of releasing in the intestinal tract. The preparation provided by the application has the advantages of high targeting, self-proliferation, non-damage to intestinal microecology and overcoming of bacterial drug resistance.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology and veterinary technology, in particular to a recombinant M13 bacteriophage, a K88-targeting antibacterial preparation containing the bacteriophage and application thereof. BACKGROUND

[0002] Enterotoxigenic Escherichia coli (ETEC) is one of the main pathogenic bacteria causing diarrhea in newborn and weaned piglets, and the strain expressing K88 fimbriae is the main pathogenic serotype. K88 fimbriae can mediate the adhesion of bacteria to the small intestinal epithelial cells of piglets, which is a prerequisite for ETEC pathogenesis. At present, the pig industry mainly relies on antibiotics to prevent and treat ETEC infection, but long-term and extensive use has led to increasingly serious problems of bacterial resistance, drug residues and food safety hazards, and also increased the cost of breeding. Therefore, it is urgent to develop new, efficient and safe antibiotic alternatives.

[0003] Bacteriophages are viruses that can specifically infect and lyse bacteria. They are considered as potential alternatives to antibiotics due to their strong host specificity, strong self-replication ability, little impact on beneficial bacteria, and difficulty in producing residues. M13 bacteriophage is a filamentous bacteriophage with Escherichia coli as the host, and its gene structure is clear and easy to operate genetically, making it an ideal engineering platform. However, the natural M13 bacteriophage has strict host receptor specificity (Escherichia coli F pilus), and its natural host range has low coincidence rate with pathogenic ETEC K88 strains, which greatly limits its application in ETEC prevention and treatment. SUMMARY

[0004] In order to overcome the problem of narrow antibacterial spectrum of M13 bacteriophage, the present application reprograms the host recognition range of M13 bacteriophage by displaying specific targeting molecules on its pIII protein, so that it can accurately target and eliminate specific pathogenic bacteria.

[0005] The present application provides a recombinant M13 bacteriophage, which displays a K88 targeting element on its tail fiber pIII protein, and the recombinant M13 bacteriophage is named rM13 bacteriophage.

[0006] Further, the K88 targeting element is displayed on the N-terminus of the pIII protein of the tail fiber by gene fusion.

[0007] Further, the K88 targeting element replaces the natural receptor binding domain of the pIII protein of the tail fiber.

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

[0009] The present application also provides a K88-targeting antibacterial preparation, which comprises a recombinant M13 bacteriophage and a pharmaceutically or veterinarily acceptable carrier.

[0010] The present application also provides a use of a K88-targeting antibacterial preparation in the preparation of a medicament for preventing or treating enterotoxigenic Escherichia coli infection, which is an ETEC infection expressing K88 fimbriae.

[0011] Further, the medicament is used for preventing or treating diarrhea of piglets caused by ETEC expressing K88 fimbriae.

[0012] Further, the dosage form of the medicament is a lyophilized preparation, a liquid preparation or an enteric preparation.

[0013] Further, the enteric preparation is a high-molecular enteric sustained-release preparation, and the high-molecular material contained in the high-molecular enteric sustained-release preparation is at least one selected from Eudragit L100, Eudragit S100, hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate succinate, cross-linked sodium alginate and complex chitosan.

[0014] Further, the dosage form of the high-molecular enteric sustained-release preparation is a tablet, and the high-molecular material is Eudragit L100, which is used as a main component for coating the tablet.

[0015] Compared with the prior art, the present application has the following beneficial effects:

[0016] Firstly, the present application successfully recombines the host specificity of the K88 targeting element by displaying the K88 targeting element on the M13 bacteriophage tail fiber pIII protein, so that the K88 targeting element can accurately recognize and infect pathogenic ETEC K88 strains, and effectively solves the technical problem that the natural M13 bacteriophage antibacterial spectrum does not match the ETEC serotype.

[0017] Secondly, the modified bacteriophage of the present application can replicate and proliferate in the target bacteria, realize self-amplification and continuous sterilization, and only needs a low initial dose to achieve high-efficiency lysis at the infection site, and is especially suitable for the intestinal environment.

[0018] Thirdly, the recombinant bacteriophage of the application only specifically targets ETEC expressing K88 fimbriae, has no effect on other beneficial microflora in the intestinal tract, can effectively maintain intestinal microecological stability while eliminating pathogens, and avoids the problem of bacterial flora imbalance caused by broad-spectrum antibiotics. At the same time, its bactericidal mechanism does not depend on the target of traditional antibiotics, so it is also effective for multiple drug-resistant ETEC K88 strains, providing a new solution to bacterial drug resistance.

[0019] Fourthly, the application can ensure that the bacteriophage remains active in the gastric acid environment and is accurately released at the target site in the intestinal tract, significantly improving the efficiency of live bacteria delivery and bioavailability, and guaranteeing the final prophylactic and therapeutic effect of the preparation. BRIEF DESCRIPTION OF DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or the prior art description. Obviously, the drawings in the following description are some embodiments of the application, and those skilled in the art can also obtain other drawings according to these drawings without creative labor.

[0021] Figure 1 The experiment results of the bacteriostatic circle of the rM13 bacteriophage of the application on part of K88 positive E. coli.

[0022] Figure 2 The growth curve comparison chart of K88 positive E. coli after being inoculated with the rM13 bacteriophage of the application. DETAILED DESCRIPTION

[0023] In order to make the purpose, technical solutions and advantages of the embodiments of the application more clear, the following will combine the drawings in the embodiments of the application to clearly and completely describe the technical solutions in the embodiments of the application. Obviously, the described embodiments are some embodiments of the application, not all embodiments. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative labor are within the protection scope of the application.

[0024] Embodiment 1

[0025] Construction of genetically recombinant M13 bacteriophage (rM13)

[0026] 1.1 Construction of rM13 recombinant plasmid

[0027] Wild-type M13 bacteriophage (M13) Escherichia phage M13, Inovirus M13) (GenBank: PL346599.1) was purchased from Ningbo Mingzhou Biological Technology Co., Ltd.

[0028] The amino acid sequence of the target Nanobody NBK88 is shown as SEQ ID NO: 1 : QVQLQESGGGLVQAGGSLRLSCAASGSVSSISTMGWFRQAPGKEREFVAAITSDALTEYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCHASQWSTVVARGIDYWGQGTQVTVSS

[0029] The encoding gene sequence of the target Nanobody NBK88 is shown as SEQ ID NO: 2: CATATGCAAGTACAGCTACAAGAATCAGGTGGAGGGTTGGTTCAGGCGGGTGGCTCCCTGCGCCTGTCTTGTGCGGCGTCTGGCAGCGTTTCCTCGATCAGCACGATGGGCTGGTTTCGTCAAGCTCCGGGTAAAGAGCGCGAGTTCGTGGCCGCGATCACCAGCGACGCACTGACCGAATATGCGGATAGCGTGAAAGGCCGTTTCACCATTAGCCGTGACAACGCCAAGAACACCGTTTATCTGCAAATGAATAGCCTTAAGCCGGAAGATACCGCTGTGTACTACTGCCACGCAAGCCAATGGTCAACGGTCGTGGCGCGTGGTATTGACTACTGGGGTCAGGGTACTCAGGTTACCGTAAGCTCCCTCGAG

[0030] The primer sequences used are as follows:

[0031] pM13-1 : ACTGTTGAAAGTTGTTTAGCAAAAC

[0032] pM13-2: CATGTTGAAAATCTCCAAAAAAAAAGGC

[0033] pNB-1 : GAGATTTTCAACATGCAAGTACAGCTACAAGAATCAG

[0034] pNB-2: ACAACTTTCAACAGTGAGGGAGCTTACGGTAACC

[0035] The wild type M13 phage genome was amplified by primer pair pM13-1 / pM13-2 to obtain a linearized vector fragment of about 6350 bp. The coding DNA sequence of NBK88 was amplified by primer pair pNB-1 / pNB-2 to obtain a target gene fragment of about 450 bp. The two PCR products were recovered and purified by agarose gel electrophoresis.

[0036] Subsequently, the purified NBK88 DNA fragment was inserted into the 5' end of the linearized M13 vector PIII protein gene (GIII) using an In-Fusion Snap Assembly Master Mix (Takara, Code No. 638947) seamless cloning kit. The recombinant plasmid thus constructed can express the NBK88 nanobody fused to the N-terminus of the PIII protein, and the construct at this stage is a recombinant plasmid DNA.

[0037] Those skilled in the art will understand that any substantially identical technical solutions that use different primer sequences, enzyme cutting sites, or ligation methods to achieve the fusion expression of the NBK88 nanobody at the N-terminus of the PIII protein are within the scope of protection of the present application.

[0038] 1.2 Preparation of competent cells and transformation of recombinant plasmid

[0039] The E. coli TG1 strain (host bacteria) was purchased from Shengong Bioengineering (Shanghai) Co., Ltd. The preparation of competent cells was performed according to the heat shock method described in the Molecular Cloning Laboratory Guide (3rd Edition), and the specific steps are as follows:

[0040] S1: A small amount of bacteria was taken from the TG1 puncture bacteria and inoculated into 20 mL of LB liquid medium, which was incubated at 37°C and 180 rpm overnight.

[0041] S2: 1 mL of the overnight activated bacterial solution was transferred to 100 mL of fresh LB medium and incubated at 37°C and 180 rpm until the OD 600 value was about 0.4.

[0042] S3: The bacterial solution was transferred to a pre-cooled centrifuge tube and placed in an ice water bath for 10 minutes, during which it was gently mixed to aid cooling.

[0043] S4: Centrifuge at 4°C and 4000 x g for 10 minutes, and discard the supernatant.

[0044] S5: Add 6 mL of pre-cooled 0.1 M CaCl2 solution, gently resuspend the bacterial cells, and again ice bath for 10 minutes.

[0045] S6: Centrifuge at 4°C and 4000 x g for 10 minutes, and discard the supernatant.

[0046] S7: Add 5 mL of pre-cooled 0.1 M CaCl2solution (containing 15% glycerol) to the bacteria, resuspend gently, and aliquot 100 μL per sterile centrifuge tube to obtain TG1competent cells.

[0047] Take 100 μL of the above competent cells, add 5 μL of the recombinant plasmid ligation product obtained in step 1.1, and mix gently. After 30 minutes of ice bath, heat shock at 42°C for 60 seconds, then quickly transfer to ice for 2 minutes. Add 900 μL of fresh LB medium, and incubate at 37°C with 180 rpm shaking for 1 hour.

[0048] Those skilled in the art will understand that other host strains, methods of preparing competence, methods of transformation, or culture conditions can also be used to achieve the purposes of the present application and are within the scope of the present application.

[0049] 1.3 rM13 phage plaque culture and titer determination

[0050] S1: Prepare TG1bacterial solution in the logarithmic growth phase (OD 600 ≈0.4) according to the method of S1-S2 in 1.2.

[0051] S2: Perform serial 10-fold gradient dilution of the transformed bacterial solution obtained in step 1.2, usually diluting 5-10 gradients.

[0052] S3: Take 1 mL of each gradient dilution and mix rapidly with 4 mL of melted LB semi-solid medium (containing 0.5-0.7% agar), then immediately pour onto the solidified LB medium plate and shake the plate to evenly spread it.

[0053] S4: After the top layer of medium completely solidifies, invert the plate in a 37°C incubator and incubate overnight (about 12-16 hours).

[0054] S5: Count the clear phage plaques formed on the plate and calculate the titer of rM13 phage in the original sample (pfu / mL) using the formula:

[0055] C = X x 10 n

[0056] Where C is the titer of the original sample (pfu / mL), X is the number of plaques on the plate suitable for counting, and n is the dilution corresponding to the plate.

[0057] 1.4 Amplification and purification of rM13 phage

[0058] S1: Prepare TG1bacterial solution in the logarithmic growth phase (OD 600 ≈0.4) according to the method described above.

[0059] S2: Pick one single, well-isolated plaque, resuspend it in a small volume of LB medium, and inoculate 1 mL of the above TG1 culture. Incubate at 37°C, 180 rpm for 1 hour. Then, transfer the whole culture into 100 mL of fresh LB medium, and incubate at 37°C, 180 rpm for 16 hours for amplification.

[0060] S3: Collect the amplified culture, centrifuge at 4°C, 3200 x g for 30 minutes, and collect the supernatant.

[0061] S4: Add 1 / 4 volume of PEG / NaCl solution (polyethylene glycol 6000 200 g / L, sodium chloride 146.25 g / L, and deionized water to 1 L) to the supernatant, mix well, and then place in an ice water bath for 30 minutes to allow the phage to precipitate.

[0062] S5: Centrifuge at 4°C, 3200 x g for 30 minutes, and carefully discard the supernatant.

[0063] S6: Resuspend the precipitate with 1 mL of pre-cooled PBS buffer, transfer the resuspension to a new 1.5 mL centrifuge tube, centrifuge at 4°C, 20000 x g for 1 minute, and remove the residual cell debris.

[0064] S7: Carefully pipette the supernatant into a new sterile centrifuge tube, which is the purified rM13 phage suspension. The phage suspension can be stored at 4°C or room temperature for several months. Its titer can be determined according to the method described in 1.3.

[0065] Example 2: Analysis of the in vitro bacteriostatic effect of rM13 phage

[0066] To evaluate the targeted bacteriostatic ability of rM13 against K88 positive E. coli, the standard strain K88 E. coli ETEC enterotoxin-producing pathogenic E. coli (strain number K88+) as a positive control (purchased from the NTCC typical culture collection) and 10 E. coli clinical isolates isolated from the feces of sick piglets (5 of which were K88 positive, numbered K88-1~K88-5, and 5 were K88 negative, numbered K88Y-1~K88Y-5) were used for the bacteriostatic circle experiment.

[0067] 2.1 Experimental method:

[0068] S1: Preparation of phage samples: Dilute the purified rM13 and wild-type M13 (wtM13) to 1 x 10 6pfu / mL. Sterile round filter paper pieces (5 mm in diameter) were taken and each piece was added with 100 μL of the diluted phage suspension, and stored at 4 °C for later use.

[0069] S2: Indication bacteria preparation: each of the test strains was inoculated in LB liquid medium and activated at 37 °C, 180 rpm overnight. 1 mL of the overnight bacterial solution was transferred to 100 mL of fresh LB medium and cultured at 37 °C with shaking until the OD 600 ≈0.4 (logarithmic growth phase).

[0070] S3: Spreading and patching: 0.2 mL of each of the indication bacteria was taken and evenly spread on LB solid medium plates using a sterile spreading rod. After the bacterial solution was absorbed by the medium, a piece of the phage filter paper prepared in step S1 was placed in the center of the plate.

[0071] S4: Culturing and observation: after the plates were cultured in a 37 °C incubator for 16 hours, the diameter of the inhibition zone (R, unit: mm) was measured. The width of the inhibition zone (d, unit: mm) was calculated according to the formula: d = (R-5) / 2. Three parallel repeats were set for each experimental group.

[0072] 2.2 Results and analysis:

[0073] The inhibition zone experiment results of some strains are shown in Figure 1 and Table 1.

[0074] Table 1 Inhibition zone diameter (mm) of rM13 and wild-type M13 on different E. coli

[0075]

[0076] The results show that the recombinant phage rM13 produces obvious inhibition zones on all 6 K88 positive E. coli strains, while the wild-type M13 has no inhibitory effect. For K88 negative strains, the inhibition spectrum of rM13 and wild-type M13 is basically the same. This proves that by displaying NBK88 nanobodies, rM13 has successfully expanded the host recognition range and can specifically inhibit the growth of K88 positive E. coli.

[0077] Example 3: Effect of rM13 phage on the growth curve of pathogenic bacteria

[0078] 3.1 Experimental method:

[0079] S1: The purified rM13 and wild-type M13 were diluted to 1 x 10 6 pfu / mL with PBS.

[0080] S2: After the K88 positive strains (K88+, K88-1~K88-5) were activated overnight, 1 mL of the overnight bacterial solution was transferred to 100 mL of fresh LB medium and cultured at 37 °C with shaking until the OD 600The concentration of 0.01 was inoculated into 100 mL of fresh LB medium and incubated at 37°C and 180 rpm for 2 hours.

[0081] 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.

[0082] 3.2 Results and Analysis:

[0083] Growth curves as follows Figure 2 As 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.

[0084] Example 4: Application of rM13 in the prevention and treatment of ETEC infectious diarrhea in piglets

[0085] 4.1 Experimental Design and Processing

[0086] 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.

[0087] 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.

[0088] 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.

[0089] Table 2 Animal Experiment Group Design

[0090]

[0091] 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.

[0092] 4.2 Experimental results

[0093] The experimental results are recorded in Table 3.

[0094] Table 3 rM13 prophylactic effect on ETEC infected piglets

[0095]

[0096] The experimental results show that, compared with the positive control group, both the rM13 prophylactic group and the rM13 therapeutic group can effectively reduce the diarrhea rate and diarrhea index of piglets, and significantly improve the survival rate and daily weight gain. Among them, the protective effect of prophylactic administration is better than that of therapeutic administration, which shows that the rM13 bacteriophage can be used to effectively prevent and treat piglet diarrhea caused by ETEC infection.

[0097] Example 5: Preparation and activity evaluation of rM13 freeze-dried preparation

[0098] 5.1 Preparation of rM13 freeze-dried preparation

[0099] In order to improve the stability and convenience of use of rM13 preparation, it is prepared as a freeze-dried powder.

[0100] rM13 bacteriophage was prepared according to the method of Example 1. Purified rM13 bacteriophage precipitate was resuspended with sterilized 5% (w / v) skim milk solution as a protective agent. After determining the initial bacteriophage titer, the bacteriophage suspension was diluted to 1 x 10 11 -1 x 10 12 pfu / mL with the same protective agent solution. The diluted suspension was aliquoted into freeze-drying bottles, and placed in a full-automatic freeze-drying machine for freeze-drying according to the preset program, and finally a loose porous freeze-dried powder was obtained.

[0101] 5.2 Activity retention rate determination of rM13 freeze-dried preparation

[0102] Accurately weigh 50 mg of freeze-dried powder into a sterile centrifuge tube, add 1 mL of sterile ultrapure water, vortex to fully dissolve. Immediately determine the bacteriophage titer after reconstitution according to the method described in Example 1.3. Compare with the original titer before freeze-drying, and calculate the activity retention rate (%).

[0103] The activity determination results of three independent batches of freeze-dried preparations are shown in Table 4.

[0104] Table 4 Activity retention rate of rM13 freeze-dried preparation

[0105]

[0106] Example 6: Preparation of rM13 enteric positioning tablets

[0107] This example is a preparation example of enteric sustained-release preparation, preparation of rM13 enteric positioning tablets, the specific steps are as follows:

[0108] 6.1 The basic ingredients and proportions of the tablet core (in percentage by weight) are shown in the following table:

[0109] Table 5 Tablet core prescription composition

[0110]

[0111] The preparation process of the tablet core includes the following steps:

[0112] S1: rM13 freeze-dried preparation, anhydrous lactose and cross-linked povidone are respectively passed through an 80 mesh sieve.

[0113] S2: The sieved raw materials are placed in a three-dimensional mixer and mixed at a speed of 20 rpm for 30 minutes to ensure uniform mixing.

[0114] S3: Add magnesium stearate and colloidal silicon dioxide to the main mixture, adjust the mixer speed to 10 rpm, and continue mixing for 5 minutes to ensure uniform distribution of low content components.

[0115] S4: Use a rotary tablet press to compress the final mixed powder into a tablet core. Control the tablet hardness to be 6 kPa, and the tablet weight to be 0.5g±0.025 g. The obtained tablet core is used for subsequent coating.

[0116] 6.2 Preparation of enteric coating solution The basic ingredients and proportions of the coating solution (in percentage by weight) are shown in the following table:

[0117] Table 6 Coating solution prescription composition

[0118]

[0119] The preparation process of the coating solution includes the following steps:

[0120] S1: Add most of the anhydrous ethanol (about 90% of the total prescription) to the liquid preparation tank, slowly add Eudragit L100 powder under continuous mechanical stirring, and continue stirring for at least 2 hours until a clear and transparent polymer solution is formed.

[0121] S2: Add diethyl phthalate to the above polymer solution and continue stirring for 30 minutes to ensure uniform mixing.

[0122] S3: In another container, mix the remaining anhydrous ethanol with colloidal silicon dioxide, and use a high-speed shear emulsifier to disperse at a speed of 5000 rpm for 3 minutes to prepare a uniform anti-sticking agent slurry.

[0123] S4: The anti-sticking agent slurry was slowly added into the polymer solution containing plasticizer under slow stirring, and the stirring was continued for 1 hour after the addition was completed to ensure uniform dispersion. The obtained coating solution was filtered through a 100 mesh screen and was ready for use.

[0124] 6.3 Coating process and formulation molding

[0125] The tablet core was coated using a high-efficiency coating pan. The coating process parameters were as follows: tablet bed temperature 30-40°C; air inlet temperature 40-50°C; coating pan rotation speed 10-20 rpm; spray gun air pressure 0.3-0.5 MPa; spray liquid speed, 2-3 mL / min in the initial stage, and gradually increased to 8-10 mL / min after the tablet core was preheated and the coating film was initially formed.

[0126] The coating process was continued until the coating layer weight gain reached 5-8% of the weight of the tablet core. After the coating was completed, the coated tablets were placed in a 30°C oven for curing treatment for 6 hours, and the rM13 enteric positioning tablets were obtained.

[0127] The enteric polymer material remains stable in the gastric acid environment, but rapidly dissolves or swells in the small intestine neutral to weak alkaline environment, thereby achieving intestinal targeted release of the drug. Eudragit L100 selected in this embodiment is an anionic polyacrylic acid resin, and its molecular chain contains carboxyl groups, which do not dissociate and dissolve in the stomach; when entering the duodenum (pH about 5.5-6.0), the carboxyl groups ionize and the polymer dissolves, thereby achieving precise release of the drug in the duodenum and the upper part of the jejunum, which is the core site of ETEC K88 colonization and pathogenesis, and can effectively protect the phage from passing through the gastric acid environment and improve its bioavailability in the lesion area. The enteric tablets prepared by this embodiment can maintain structural integrity in the gastric acid environment, and the coating layer dissolves in the neutral pH environment of the duodenum to release the active ingredient rM13, thereby achieving intestinal positioning delivery of the drug.

[0128] In summary, the core of the present application is to genetically engineer the M13 phage to display the nanobody NBK88, which is a specific targeting element of K88 pilus, on its tail fiber pIII protein, thereby "reprogramming" its host range, enabling it to accurately recognize and efficiently lyse enterotoxigenic Escherichia coli ETEC expressing K88 pilus. The recombinant rM13 phage obtained thereby not only has the characteristics of high specificity and high bactericidal efficiency, but also can self-propagate at the infection site to achieve long-acting effect. At the same time, its highly specific mode of action can avoid damage to the normal flora of the intestinal tract, effectively maintaining the stability of the microecology, and can be used to address the growing problem of antibiotic resistance.

[0129] In addition, the application also innovatively develops an enteric preparation suitable for rM13, which can ensure that the active ingredient safely reaches the lesion site in the intestinal tract, effectively improving the bioavailability and the final prevention and treatment effect. Experimental results show that the preparation has a significant effect in preventing and treating diarrhea of piglets caused by ETEC K88, and has excellent application potential and market value.

[0130] Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present application, but not to limit them; although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing embodiments, or make equivalent replacement for part or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A recombinant M13 bacteriophage, characterized in that, The recombinant M13 phage displays a K88 targeting element on its tail fiber pIII protein, the K88 targeting element is a nanobody NBK88, the amino acid sequence of the nanobody NBK88 is shown as SEQ ID NO: 1, and the recombinant M13 phage is named as rM13 phage.

2. The recombinant M13 bacteriophage of claim 1, wherein, The K88 targeting element is displayed on the N-terminus of the pIII protein of the tail fiber by means of gene fusion.

3. The recombinant M13 bacteriophage of claim 1, wherein, The K88 targeting element replaces the natural receptor binding domain of the pIII protein of the tail fiber.

4. A K88-targeted antibacterial agent, characterized by, The antibacterial preparation comprises the recombinant M13 phage according to any one of claims 1-3 and a pharmaceutically or veterinarily acceptable carrier.

5. Use of the K88 targeting antibacterial preparation according to claim 4 for the manufacture of a medicament for the prevention or treatment of an infection with enterotoxigenic Escherichia coli, characterized in that, The enterotoxigenic Escherichia coli infection is an ETEC infection expressing K88 pili.

6. Use according to claim 5, characterized in that, The drug is used for preventing or treating piglet diarrhea caused by ETEC expressing K88 pili.

7. Use according to claim 6, characterized in that, The dosage form of the drug is a lyophilized preparation, a liquid preparation or an enteric preparation.

8. Use according to claim 7, characterized in that, The enteric preparation is a high molecular enteric sustained-release preparation, and the high molecular material contained in the high molecular enteric sustained-release preparation is at least one selected from Eudragit L100, Eudragit S100, hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate succinate, cross-linked sodium alginate and composite chitosan.

9. Use according to claim 8, characterized in that, The dosage form of the high molecular enteric sustained-release preparation is a tablet, and the high molecular material is Eudragit L100.

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