Engineered bacteriophage that simultaneously lyse both sensitive and resistant strains of salmonella and uses thereof

Abstract

The application discloses an engineered bacteriophage capable of simultaneously lysing sensitive strains and resistant strains of Salmonella and application thereof. The engineered bacteriophage is obtained by introducing a T6 tail fiber protein coding gene sequence of a second bacteriophage into a corresponding editing region in a genome of a first bacteriophage through a gene editing technology. The engineered bacteriophage is capable of lysing a sensitive host bacterium of the first bacteriophage and a resistant bacterium induced by the first bacteriophage, and maintains corresponding biological activities of the bacteriophages. The engineered bacteriophage can be used for preparing a medicine or an antibacterial preparation for treating Salmonella infection.

Description

Technical Field

[0001] This invention relates to the fields of phage engineering and antimicrobial biological agents, specifically to an engineered phage capable of lysing susceptible strains of Salmonella and its phage-resistant strains, a method for constructing the engineered phage, and its applications. Background Technology

[0002] Salmonella is a common zoonotic pathogen that can cause infection in poultry and other animals, and can impact public health through animal-derived food products. Salmonella infection can lead to decreased animal growth performance, morbidity, and death, causing economic losses to the livestock industry.

[0003] Antimicrobial drugs can be used to control Salmonella infections, but long-term or irrational use may promote the emergence and spread of drug-resistant strains. Therefore, developing antimicrobial technologies with different mechanisms of action is of great significance for reducing drug dependence and controlling drug-resistant bacteria.

[0004] Bacteriophages are a class of viruses capable of specifically infecting bacteria and causing lysis of the host bacteria under certain conditions. Vicious bacteriophages are characterized by high host specificity, the ability to proliferate in susceptible host bacteria, and relatively minimal impact on non-target bacteria, thus making them suitable for use in antimicrobial agent research. On October 13, 2023, the European Medicines Agency published the "Guidelines on the Quality, Safety and Efficacy of Veterinary Drugs Designed Specifically for Bacteriophage Therapies," which sets forth technical requirements for the quality, safety, and efficacy evaluation of bacteriophage veterinary drug products.

[0005] Phage infection typically begins with the recognition and adsorption of receptor-binding proteins onto bacterial surface receptors. Bacteria can reduce phage adsorption through receptor mutations, altered receptor expression, changes in cell surface structure, or extracellular matrix shielding, thus developing phage resistance. The emergence of phage resistance can lead to a decrease or loss of the lytic activity of a single phage, and is a significant factor limiting the sustained effectiveness of phage preparations.

[0006] Existing technologies can mitigate the impact of resistant bacteria by employing phage cocktails, combined drug therapy, or phage replacement. However, it is still necessary to screen for new recognition elements in strains that have developed phage resistance and to use them for the engineering modification of the original phage. Therefore, it is necessary to provide an engineered phage capable of simultaneously lysing both the original susceptible bacteria and its phage-resistant strains, along with a method for its construction. Summary of the Invention

[0007] The purpose of this invention is to provide an engineered bacteriophage that can simultaneously lyse both susceptible and resistant strains of Salmonella, its construction method, and its application, in order to solve the problem that the lysis ability of a single bacteriophage decreases after the host bacteria develop phage resistance.

[0008] To achieve the above objectives, the present invention provides an engineered bacteriophage.

[0009] The genome of the engineered phage contains a T6 tail filament protein coding sequence derived from the second phage, which is inserted after the T2 tail filament protein coding sequence of the first phage. The GenBank accession number for the whole genome of the first phage is ON550260.1, and the GenBank accession number for the whole genome of the second phage is OP828573.1.

[0010] The engineered bacteriophage is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC M 2026963, on May 14, 2026, and is taxonomically named *Salmonella enteritidis* bacteriophage. Salmonella enteritidis phage GCD18, Latin scientific name Salmonella enteritidis phage GCD18 。

[0011] In one specific embodiment, the nucleic acid molecule encoding the T2 tail filament protein has the sequence shown in SEQ ID NO:2, and the nucleic acid molecule encoding the T6 tail filament protein has the sequence shown in SEQ ID NO:6.

[0012] This invention also provides a method for constructing the above-mentioned engineered phage, comprising: inserting the nucleic acid molecule of the T6 tail fimbriae protein of the second phage shown in SEQ ID NO:6 into the pHCY-26D plasmid. Sal I and EcoR I. Between the restriction enzyme sites, and insert the N20 sequence shown in SEQ ID NO:15 between the gRNA and the scaffold sequence to obtain the T6-pHCY-26D plasmid; transform the pHCY-25A plasmid and the T6-pHCY-26D plasmid into Salmonella competent cells to obtain recombinant bacteria containing the two plasmids; infect the recombinant bacteria with the first phage, and obtain the engineered phage after screening.

[0013] In one specific embodiment, the Salmonella competent cells are prepared from Salmonella CVCC1806 and transformed with plasmids using a heat shock method.

[0014] In one specific embodiment, the recombinant bacteria were inoculated into a culture medium containing ampicillin and kanamycin and cultured. IPTG, L-arabinose, and the first phage were added sequentially and cultured at 30°C. After the culture was completed, the phage fluid was separated and engineered phages were screened from the phage fluid.

[0015] The present invention also provides the use of the engineered phage in the preparation of medicaments for treating Salmonella infection, as well as antimicrobial agents, pharmaceutical compositions and medicaments for treating Salmonella infection comprising the engineered phage.

[0016] This invention obtains phage-resistant bacteria through screening with a first phage, then uses these resistant bacteria as hosts to screen for a second phage, and identifies the host recognition-related tail fimbriae proteins of the first and second phages. Based on this, the T6 tail fimbriae protein coding sequence of the second phage is inserted into the genome of the first phage to obtain the engineered phage GCD18. The results of the examples show that GCD18 can lyse both the susceptible host bacteria of the first phage and the screened resistant bacteria, and maintains the corresponding phage biological activity and in vitro and in vivo antibacterial effects under the tested conditions.

[0017] This invention does not simply replace the tail filament protein of phages, but addresses the problem of decreased cytotoxicity caused by the generation of resistant bacteria during phage therapy. It achieves the construction of engineered phages targeting specific resistance mechanisms by inducing the generation of resistant bacteria, screening new phages using the resistant bacteria as hosts, identifying host recognition-related proteins, and importing the new host recognition module into the original phage genome. Attached Figure Description

[0018] Figure 1 These are the results of the phage D1 resistant bacteria screening plate from Example 1; Figure 2 The bacteriophage D1 resistant bacteria in the examples invA Gene PCR identification results; Figure 3 This is the result of phage susceptibility identification of the D1 resistant bacteria in Example 1; Figure 4 These are the results of tail filament protein induction expression and solubility identification in Example 2; Figure 5 This is the purification result of the tail filament protein in Example 2; Figure 6 The results of the adsorption inhibition test of phage D1 tail filament protein in Example 2; Figure 7 The results of the adsorption inhibition test of phage D8 tail filament protein in Example 2; Figure 8 This is a schematic diagram of the construction of the T6-pHCY-26D vector in Example 3; Figure 9 This is the identification result of the recombinant strain CVCC1806-25A-26D in Example 3; Figure 10 This is the PCR identification result of engineered bacteriophage GCD18 in Example 3; Figure 11This is the result of the optimal multiple of infection (MOI) determination for phage D1 in Example 4; Figure 12 This is the result of the optimal multiple of infection (MOI) determination for phage D8 in Example 4; Figure 13 This is the result of the optimal multiple of infection (MOI) determination for engineered phage GCD18 in Example 4; Figure 14 These are the one-step growth curve determination results of phages D1, D8, and GCD18 in Example 4; Figure 15 This is the pH stability test result of phage D1 in Example 4; Figure 16 This is the pH stability test result of phage D8 in Example 4; Figure 17 This is the pH stability test result of engineered bacteriophage GCD18 in Example 4; Figure 18 This is the result of the temperature stability test of phage D1 in Example 4; Figure 19 This is the result of the temperature stability test of phage D8 in Example 4; Figure 20 This is the result of the temperature stability test of engineered bacteriophage GCD18 in Example 4; Figure 21 These are the in vitro lysis curves of bacteriophages D1, D8, and GCD18 in Example 4; Figure 22 This refers to the results of mouse weight changes in Example 4; Figure 23 This is the result of the Salmonella load determination in the mouse intestine in Example 4. Detailed Implementation

[0019] The present invention will be further illustrated below with reference to the embodiments. These embodiments are used to illustrate the technical solutions of the present invention, and are not intended to limit the scope of protection of the present invention. Unless otherwise stated, the reagents and materials used in the embodiments are commercially available, and the molecular biology, microbiology, and biochemical operations employed are performed according to conventional methods in the art or product instructions. The experimental conditions described in each embodiment should be based on actual experimental records.

[0020] Example 1: Screening of phage-resistant bacteria and new phages 1. Screening for bacteriophage-resistant bacteria The CVCC1806 bacterial culture was inoculated into 5 mL of fresh LB liquid medium at a ratio of 1:100 and cultured at 37°C and 180 rpm until the logarithmic growth phase. The first phage (Salmonella phage PSE-D1, GenBank: ON550260.1, donated by Guangxi University, hereinafter referred to as phage D1, phage titer >10) was then added. 8 PFU / mL) and the above-mentioned logarithmic phase bacteria were mixed at a 1:1 volume ratio (110µL each), and incubated at 37℃ for 10 min. Then, 5mL of preheated semi-solid LB medium was added, mixed well, poured onto solid LB plates, and incubated overnight at 37℃. Figure 1 As shown, the single colonies that grew on the phage plaques were inoculated onto xylose-lysine-deoxycholate (XLD) agar the following day. After five consecutive passages, the colonies were analyzed using the conserved gene for Salmonella invasive antigens. invA Specific primers were used for PCR identification. The primers used are shown in Table 1, the PCR reaction system is shown in Table 2, and the PCR reaction procedure is shown in Table 3.

[0021] Table 1 Salmonella invA Gene primers Table 2 PCR reaction system Table 3 PCR reaction procedure Through the above screening, six strains that still exhibited resistance to phage D1 after continuous subculturing were obtained and named D1K1, D1K2, D1K3, D1K4, D1K5, and D1K6, respectively. (See also...) Figure 2 PCR verification confirmed that all six bacteriophage-resistant bacteria were Salmonella. Figure 2 In the diagram, M stands for DL ​​2000 Marker, 1 for D1K1, 2 for D1K2, 3 for D1K3, 4 for D1K4, 5 for D1K5, and 6 for D1K6.

[0022] 2. Phage susceptibility identification of resistant bacteria Single colonies of the preliminarily screened phage-resistant strains were streaked across four zones. Each colony was picked and inoculated into LB broth liquid medium and incubated at 37°C and 180 rpm until the logarithmic growth phase. This process was repeated five times, and the bacteria of the fifth generation were labeled F5. 200 μL of the F5 generation culture was spread onto LB solid medium and allowed to air dry. Then, 10 μL of phage suspension was added dropwise to each LB agar plate. After the drop was completely absorbed and dried, the plate was inverted and incubated at 37°C for 16–18 hours. The next day, the phage plaques formed at each dilution were observed and recorded. Strains that still exhibited phage resistance were preserved and labeled. The phage susceptibility identification results of the phage-resistant bacteria are shown below. Figure 3 As shown, D1K1~D1K6 all exhibited resistance to phage D1, and D1K1 was selected for subsequent experiments.

[0023] 3. Phage screening for lysing phage-resistant bacteria Using D1K1 as the host bacterium, a spot-dotting method was used to screen for new phages capable of lysing D1K1 from the phage library. The host bacterium was cultured to the logarithmic growth phase, and 200 μL of the bacterial culture was spread onto LB agar. After air-drying, 5 μL of phage enrichment solution was dropped onto the plate. After the phages dried, the plate was inverted and incubated overnight at 37°C. The spot-dotting results were observed the next day; a clear, circular empty spot on the plate surface indicated a resistant phage. The phage exhibiting the best performance based on the spot-dotting results was defined as the second phage. This second phage was identified as *Salmonella phage* 3384-D8 (GenBank: OP828573.1, donated by Guangxi University, hereinafter referred to as phage D8, phage titer >10). 8 (PFU / mL) was used as a phage capable of lysing D1K1 for subsequent experiments.

[0024] Example 2: Identification of phage receptor-binding proteins 1. Phage genome analysis Analysis of the genome sequences of phages D1 and D8 revealed that phage D1 has three tail filament proteins that may adsorb onto the CVCC1806 surface receptor, denoted as T1, T2, and T3, while phage D8 has three tail filament proteins that may adsorb onto the CVCC1806 surface receptor, denoted as T4, T5, and T6.

[0025] 2. Construction of a bacteriophage tail fimbriae expression vector Tail filament proteins T1, T2, T3, T4, T5, and T6 were cloned into the space between the EcoRI and HindIII restriction sites of the pET-28a(+) vector. The expression vector was synthesized by Suzhou Genewiz Biotechnology Co., Ltd., and after sequencing verification, it was introduced into BL21(DE3) for use.

[0026] 3. Expression and purification of bacteriophage tail fimbriae 3.1 Induced expression of tail fimbriae The revived BL21(DE3) strain containing the recombinant pET-28a(+) vector was inoculated into 20 mL of LB broth containing 50 μg / mL kanamycin and cultured at 37°C with shaking at 180 rpm until the logarithmic growth phase. The culture was then transferred at a 1:50 ratio to LB broth with a final concentration of 50 μg / mL kanamycin for further expansion. When the culture medium… OD 600 When the value reaches 0.6, add IPTG to a final concentration of 1 mM and induce culture overnight at 16℃ and 120 rpm.

[0027] 3.2 Ultrasonic fragmentation Collect 1 L of overnight induced bacterial culture into a large-volume centrifuge bottle, balance with ddH2O, and place in a pre-cooled 4°C high-speed floor centrifuge. Centrifuge at 5000×g for 10 min. Discard the supernatant, resuspend the bacterial pellet in 40 mL of PBS solution, wash, centrifuge at 5000×g for 10 min, discard the supernatant, resuspend the bacterial pellet in 40 mL of PBS solution again, transfer to centrifuge tubes, and sonicate on ice for 1 h. The sonication program was set as follows: power 40%, sonication for 4 seconds, interval for 4 seconds.

[0028] 3.3 SDS-PAGE Identification The ultrasonically disrupted bacterial culture was centrifuged at 8000×g for 15 min in a pre-cooled benchtop centrifuge at 4°C. After centrifugation, the supernatant was transferred to a new 50 mL centrifuge tube for storage, and the bacterial pellet was resuspended in 4 mL PBS. 40 µL of the supernatant and the resuspended pellet were placed in 1.5 mL EP tubes, and 5×SDS-PAGE loading buffer was added in the appropriate ratio. The mixture was vortexed and heated in a metal bath at 95°C for 10 min, then briefly centrifuged to collect the liquid on the tube wall for electrophoresis. A 15% polyacrylamide gel was fixed in the protein electrophoresis tank, and electrophoresis buffer was added to cover the wells. Samples were added to the wells in the following order: protein marker, heated supernatant, and pellet. Electrophoresis was performed at a constant voltage of 120 V for 55 minutes. After electrophoresis, Coomassie Brilliant Blue was added to the protein gel and incubated in a shaker at 90 rpm for 20 min for staining. The staining solution was then discarded, and the protein gel was washed with water until the background color completely disappeared. The expression of the target protein was then observed to determine whether it was successfully induced, whether the protein band size was normal, and whether the protein expression was located in the supernatant or in inclusion bodies.

[0029] The results are as follows Figure 4As shown, tail filament proteins T1, T3, T4, and T5 are expressed in inclusion bodies, while T2 and T6 are expressed in the supernatant. Purification of tail filament proteins T1, T3, T4, and T5 requires denaturation and renaturation. Figure 4 The labels and their corresponding substances are as follows: M: Protein Marker; 1: T1-pET28a-BL21 supernatant; 2: T1-pET28a-BL21 precipitate; 3: T2-pET28a-BL21 supernatant; 4: T2-pET28a-BL21 precipitate; 5: T3-pET28a-BL21 supernatant; 6: T3-pET28a-BL21 precipitate; 7: T4-pET28a-BL21 supernatant; 8: T4-pET28a-BL21 precipitate; 9: T5-pET28a-BL21 supernatant; 10: T5-pET28a-BL21 precipitate; 11: T6-pET28a-BL21 supernatant; 12: T6-pET28a-BL21 precipitate.

[0030] 3.4 Expression and purification of tail fimbriae Positive colonies containing the recombinant expression vector were expanded and inoculated into LB liquid medium containing kanamycin at a ratio of 1:100, and cultured at 37°C and 180 rpm / min until the bacterial culture was complete. OD 600 Within the range of 0.6–0.8, add IPTG to a final concentration of 0.5 mM and induce overnight at 16°C and 200 rpm / min. Collect the induced bacterial culture, centrifuge at 8000 rpm / min for 15 min at 4°C to collect the bacterial cell pellet, discard the supernatant, and resuspend in 20 mL of PBS with shaking. Place the bacterial culture on ice and sonicate for 4 s at 60 Hz, pause for 6 s, until the bacterial culture is clear and transparent. Centrifuge the sonicated lysate at 10000 rpm / min for 15 min at 4°C to separate the supernatant and pellet. Filter the protein expressed in the supernatant through a 0.45 μm filter membrane for later use; resuspend the protein expressed in the pellet in 20 mL of 8 M urea, shake for 2–4 h until the pellet dissolves, centrifuge at 10000 rpm / min for 15 min at 4°C, and filter the supernatant through a 0.45 μm filter membrane for later use. Purify the protein from the filtered bacterial lysate supernatant as follows: (1) Add Ni-NTA filler to the empty column of the gravity column, with a volume not exceeding 1 / 3; (2) Rinse with pure water for 5 CV; (3) Column balance; (4) Load the sample and let it rest overnight; (5) Wash impurities with 10mM, 30mM, and 50mM imidazole washing solutions respectively; (6) Elute with 100mM and 300mM imidazole eluents respectively.

[0031] SDS-PAGE analysis was performed on the sample flow-through, washing buffers at various imidazole concentrations, and elution buffers. Based on the electrophoresis results, elution fractions containing the target protein at 100 mmol / L and / or 300 mmol / L imidazole concentrations were collected and dialyzed in PBS containing 5% glycerol using a dialysis bag with a molecular weight cutoff of 12 kDa.

[0032] Purification of inclusion body proteins requires the preparation of imidazole with 8M urea, following the same steps as described above. After obtaining high-purity protein, refolding is necessary: ​​the protein solution is placed in a 12kDa dialysis bag and dialyzed for 8 hours in a PBS solution containing 6M urea and 5% glycerol. Subsequently, dialysis is performed in a gradient of 4M urea, 2M urea, 1M urea, and 0M urea to obtain the target protein.

[0033] After purification, the tail filament protein was subjected to SDS-PAGE electrophoresis and staining, and the results are as follows: Figure 5 As shown, the tail protein T1 band size is 51.5 kDa, tail protein T2 is 22.2 kDa, tail protein T3 is 15.1 kDa, tail protein T4 is 31.4 kDa, tail protein T5 is 129.4 kDa, and tail protein T6 is 79.0 kDa. The bands of each tail protein are of correct size and relatively uniform, indicating good purification results. Figure 5 The labels and their corresponding substances are as follows: M: Protein Marker; 1: T1 protein; 2: T2 protein; 3: T3 protein; 4: T4 protein; 5: T5 protein; 6: T6 protein.

[0034] 4. Tail filament protein adsorption inhibition test strain CVCC1806 was cultured for approximately 10... 8 CFU / mL, after centrifugation, the bacterial pellet was resuspended in LB liquid medium, and the pellet was washed three times with LB liquid to adjust the bacterial cell count. OD 600 =0.5 for later use. The purified protein is then transferred from... Remove from the 80℃ freezer and quickly place on ice to thaw. Take 200 μL of purified tail filament protein (1 mg / mL) and 200 μL of CVCC1806 ( OD 600 = 0.5) was incubated at 37°C for 30 min, with an equal volume of LB liquid as a negative control. 100 μL of phage D1 (10) was added to the incubated mixture. 5 The mixture was incubated at 37°C for 10 min and then centrifuged at 8000×g for 6 min. The adsorption rate of the phage was then measured. Each experimental group was repeated three times.

[0035] Recombinant tail filament protein can competitively bind to adsorption sites on the bacterial surface with bacteriophages. The adsorption rate of bacteriophages can indirectly reflect the adsorption activity of recombinant tail filament protein on bacteriophage receptors. Results are as follows... Figure 6 and Figure 7 As shown in the figure, compared with the negative control group, the adsorption rate of phage D1 decreased after T2 protein treatment, indicating that T2 protein can compete with D1 for adsorption sites on the CVCC1806 surface; no adsorption inhibition of the same degree was observed in the T1 and T3 protein treatment groups. Correspondingly, the adsorption rate of phage D8 for D1K1 decreased after T6 protein treatment. Based on the above results, T2 and T6 were identified as tail filament proteins related to host recognition of D1 and D8 under the conditions of this embodiment. Figure 6 Assay for the adsorption inhibition of phage D1 tail filament protein. Figure 7 For the phage D8 tail filament protein adsorption inhibition assay, ** P <0.01; *** P <0.001.

[0036] Example 3 Construction of engineered bacteriophages Using the CRISPR Cas9 system, the T6 tail filament protein gene sequence of phage D8 was inserted after the T2 tail filament protein gene sequence of phage D1. The plasmids used in this experiment were pHCY-25A and pHCY-26D. The pHCY-25A plasmid contains the SpCas9 coding gene and the λ-Red recombination system. The SpCas9 protein is responsible for cleaving the genome, and the λ-Red system mediates homologous recombination to repair the cleaved genome. The pHCY-26D plasmid expresses the N20 sequence and provides a template for homologous recombination, guiding Cas9 cleavage and achieving integration of the target fragment.

[0037] 1. N20 Design The N20 sequence was designed using the CHOPCHOP online website (https: / / chopchop.cbu.uib.no), and the result is as follows: 5'-actcacagaacttcgatacg-3' (SEQ ID NO:15).

[0038] 2. Construction of donor plasmids like Figure 8 As shown, using pHCY-26D as a vector, restriction enzyme sites were selected. Sal I. EcoR I. The designed complement fragment with homologous arms was inserted into the restriction enzyme site, and the N20 sequence was inserted between the gRNA and the scaffold feature. The constructed recombinant vector was then synthesized by Suzhou Genewise Biotechnology Co., Ltd. After the sequencing results were confirmed to be correct, it was introduced into DH5α for storage and later use, and designated as T6-pHCY-26D.

[0039] 3. Introduction of recombinant vector into host bacteria 3.1 Plasmid Extraction E. coli DH5α containing pHCY-25A plasmid was inoculated at 1% in 20 mL of LB broth containing 50 μg / mL kanamycin and cultured overnight at 37°C with shaking at 180 rpm. E. coli DH5α containing T6-pHCY-26D plasmid was inoculated at 1% in 20 mL of LB broth containing 100 μg / mL ampicillin and cultured overnight at 30°C with shaking at 180 rpm. The next day, pHCY-25A and T6-pHCY-26D plasmids were extracted according to the plasmid miniprep kit instructions, their concentrations were measured, and the samples were stored at -20°C.

[0040] 3.2 Preparation of competent bacteria Inoculate 1 mL of Salmonella CVCC1806 into 100 mL of LB and incubate at 37°C until... OD 600 =0.4~0.6 (approximately 1.5~2.5h); Aliquot the bacterial culture into two 50mL EP tubes and incubate on ice for 30min; Centrifuge at 4℃ and 4000rpm for 10min, discard the supernatant, resuspend in 10mL of pre-cooled 0.1M CaCl2 solution, incubate on ice for 30min, centrifuge at 4℃ and 4000rpm for 10min, discard the supernatant, and finally resuspend in 2mL of 0.1M CaCl2 solution and 2mL of 30% glycerol, aliquot into EP tubes, and store at -80℃ for later use.

[0041] 3.3 Thermal shock transformation of plasmids Thaw 50 μL of CVCC1806 competent cells slowly on ice, add 5 μL each of pHCY-25A and T6-pHCY-26D plasmids, incubate on ice for 30 min, heat shock in a 42°C water bath for 1 min, remove and place on ice for 30 s, add 1 mL of LB broth, and incubate at 30°C with shaking at 180 rpm for 1 h. Finally, concentrate the bacterial culture to 100 μL and spread it entirely on LB agar plates containing 100 μg / mL ampicillin, 50 μg / mL kanamycin, and 0.1% glucose, and incubate overnight at 30°C.

[0042] 3.4 Identification and Preservation Select positive bacteria and inoculate them into 2 mL LB, add 2 μL ampicillin (100 mg / mL) and 2 μL kanamycin (50 mg / mL), incubate for 8 h and then perform PCR detection. The primers are shown in Table 4. Select colonies that show bands in both primer pairs as positive colonies, and the recombinant bacteria are successfully constructed.

[0043] After PCR identification, recombinant CVCC1806 bacteria carrying the pHCY-25A and T6-pHCY-26D plasmids were successfully screened. The PCR results are as follows. Figure 9 As shown, the recombinant bacteria is designated as CVCC1806-25A-26D. Figure 9 The symbols and corresponding substances are as follows: M: DL2000 Marker; 1: CVCC1806-25A-26D; 2: CVCC1806-25A-26D; 3: pHCY-25A; 4: T6-pHCY-26D; 5: Negative control.

[0044] Table 4. Primers for plasmid introduction identification 4. Interaction between bacteriophages and recombinant bacteria Inoculate CVCC1806-25A-26D bacterial culture into 2 mL LB, add 2 μL ampicillin (100 mg / mL) and 2 μL kanamycin (50 mg / mL), and incubate at 30℃ for 2 h. OD 600 Approximately 0.2), then add 20 μL IPTG (100 mM), incubate at 30℃ for 1 h, then add 40 μL L-arabinose (1 M), add 1% phage D1, and incubate at 30℃ and 180 rpm for 3 h. After incubation, filter through a 0.22 μm filter to obtain phage fluid, and then serially dilute the phage fluid 10-fold to 10. -7 100 μL of each dilution gradient was mixed with the recombinant bacteria in an equal proportion and incubated at 37°C for 10 min. 200 μL of the mixture was then plated in a double layer (ampicillin + kanamycin + L-arabinose) and incubated overnight at 30°C. Single plaques were picked from the double-layer agar plates for continuous purification, and PCR identification was performed using the primers shown in Table 5. Figure 10 As shown, the candidate phage amplified to obtain a specific fragment of the expected size, while the parental phage D1 did not obtain an amplification product of the same size. The candidate phage that met the expected PCR identification was further purified and amplified into plaques and named GCD18. (Accession number CCTCC M 2026963, accession date 2026.05.14). Figure 10 The symbols and corresponding substances are as follows: M: DL 2000 Marker; 1: GCD18; 2: D1; 3: Negative control.

[0045] Table 5 Primers for Phage Modification and Identification Example 4: Identification of the bactericidal effect of engineered bacteriophages 1. Spot assay for determining the host spectrum of engineered bacteriophages Existing Salmonella and the phage-resistant bacteria cultured in Example 1 were inoculated and cultured to the logarithmic growth phase. 200 μL of the bacterial suspension was spread onto a standard solid culture medium and allowed to air dry. Then, 10 μL of engineered phage GCD18 suspension was vertically added to a pre-labeled LB agar plate. After the droplet was completely absorbed and dried, the plate was inverted and incubated at 37°C for 16–18 hours. The plaques formed at each dilution were observed and recorded the results the following day.

[0046] The results are shown in Table 6. Under the spot test conditions of this embodiment, bacteriophages D1, D8, and GCD18 formed lysing plaques of varying degrees on some tested Salmonella bacterial motifs. D1 showed lysis on 14 tested strains, D8 showed lysis on 15 tested strains, and GCD18 showed lysis on 21 tested strains; GCD18 also formed lysing plaques on bacterial motifs D1K1-D1K6. These results indicate that GCD18 can lyse a wider range of strains than D1 under these test conditions. Surprisingly, compared to bacteriophages D1 and D8, the engineered bacteriophage GCD18 showed a significantly improved lysis ability on some strains.

[0047] Table 6. Results of phage host profile determination Note: "+" indicates that lysis has occurred in the spotted area, and the number of "+" indicates the relative clarity of the lysis spots; "-" indicates that no obvious lysis was observed.

[0048] 2. Determination of biological characteristics of engineered bacteriophages 2.1 Optimal Multiple of Infection (MOI) After culturing the host bacteria to the logarithmic growth phase, dilute it 10-fold to a bacterial concentration of approximately 10. 7 CFU / mL, take 500μL of the diluted bacterial culture and dilute it to 10 with 500μL of the diluted culture. 3 10 4 10 5 10 6 10 7 10 8 10 9Phage solutions of PFU / mL were mixed 1:1, resulting in MOIs of 0.0001, 0.001, 0.01, 0.1, 1, 10, and 100, with three replicates for each MOI. The mixtures were incubated at 37°C with shaking at 180 rpm for 5 h. The EP tubes were then centrifuged at 8000×g for 10 min. The supernatant was collected, serially diluted to an appropriate gradient, and the titer was determined using the double-layer agar method. The ratio with the highest titer is the optimal MOI for that phage.

[0049] Bacteriophages D1, D8, and GCD18 were co-cultured with Salmonella CVCC1806 for 5 h under different multiples of infection (MOI) conditions. The results are as follows: Figures 11-13 As shown, when the MOI is 0.0001, the phage titers of both D1 and D8 reach their highest value of 10. 9 PFU / mL; and when the MOI was 0.001, the phage titer of GCD18 was the highest, also 10. 9 PFU / mL. Therefore, the optimal MOIs for phages D1, D8, and GCD18 are 0.0001, 0.0001, and 0.001, respectively.

[0050] 2.2 One-step growth curve Add 200 μL of the host bacterium CVCC1806 to 5 mL of LB broth and incubate at 37°C with shaking at 180 rpm until the growth phase is reached, resulting in a bacterial concentration of approximately 10. 8 CFU / mL. Take 200 μL of bacterial culture and 10 6 Mix PFU / mL phage solution (at the optimal MOI ratio) thoroughly, set up 3 replicates, and incubate at 37°C for 10 min to allow the phage to fully adsorb onto the bacterial surface. Then, centrifuge the mixture at 8000×g for 10 min, discard the supernatant, and resuspend the precipitate in 500 μL of LB liquid medium. Transfer the entire bacterial culture to a test tube containing 5 mL of LB liquid medium and incubate at 37°C with shaking at 180 rpm. During the first 100 min, take 200 μL of culture every 10 min, centrifuge at 8000×g for 1 min, and dilute the supernatant to an appropriate gradient. Measure the phage titer using the double-layer agar method, taking the sample every 20 min after 100 min, until the titer reaches 160 min.

[0051] The results of the one-step growth curve determination of the three bacteriophages are as follows: Figure 14As shown, the incubation period of D1 is 10 min, it takes 120 min to reach the plateau phase, and the burst rate is 105.4 PFU / cell; the incubation period of D8 is 20 min, it takes 100 min to reach the plateau phase, and the burst rate is 276.9 PFU / cell; the incubation period of GCD18 is 10 min, it takes 140 min to reach the plateau phase, and the burst rate is 81.5 PFU / cell. All three phages can reach a maximum order of magnitude of 10. 9 PFU / mL.

[0052] 2.3 Acid and alkali tolerance Concentrated hydrochloric acid or sodium hydroxide was added to SM buffer to prepare SM solutions with pH values ​​of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and 14, respectively. 100 μL of phage was mixed with 100 μL of SM buffer at different pH values ​​in a 1.5 mL EP tube, and incubated in a 37°C water bath for 1 h. The phage titer was then determined using the double-layer agar method.

[0053] The survival ability of bacteriophages D1, D8, and GCD18 at different pH values ​​was detected. Figures 15-17 When D1, D8, and GCD18 were incubated in SM solution with a pH of 3–12 for 1 hour, the phage titers did not change significantly. Under extreme acidic and alkaline conditions (pH values ​​of 1, 2, 13, and 14), no phages were detected after 1 hour of incubation, indicating that the three phage strains have strong stability and infectious activity in an environment with a pH of 3–12.

[0054] 2.4 Temperature Sensitivity Take 500 μL of phage and incubate it in a metal bath at 40℃, 50℃, 60℃, 70℃ and 80℃. Repeat the experiment three times at each temperature. Determine the phage titer using the double agar method at 30 min and 60 min respectively.

[0055] The temperature sensitivities of the three bacteriophages are as follows: Figures 18-20 As shown, D1, D8, and GCD18 maintained their activity and were relatively stable after 1 hour of treatment at 40℃ and 50℃. After 30 minutes of treatment at 60℃, the activity of all three phages decreased significantly, and after 60 minutes, none of the three phages were detected. At 70℃ and 80℃, none of the three phages were detected.

[0056] 3. Determination of the in vitro lysis efficiency of engineered bacteriophages Culture the host bacterium CVCC1806 to the logarithmic growth phase, then take 500 μL of 10 9 Add CFU / mL host bacteria to 50 mL LB broth. Adjust the titers of D1, D8, and engineered phage GCD18 to 10. 7PFU / mL, 50 μL of phage D1, D8, and GCD18 were added to the above culture system, respectively. The positive control group was added only with CVCC1806, and the negative control was liquid LB. The conical flasks of each group were placed in a shaker at 37℃ and 180r / min for 20h. 3mL of bacterial solution was taken every 2h for determination. OD 600， Continuous detection for 20 hours, plotting OD 600 The value change curve is used to determine the in vitro antibacterial effect of bacteriophage.

[0057] The results are as follows Figure 21 As shown. In the bacterial control group without phage, CVCC1806 continued to grow and entered the plateau phase; after adding D1, D8, or GCD18, the culture system showed growth in the first 8 hours. OD600 The OD600 values ​​remained at a low level for all groups, then gradually increased. The OD600 values ​​of the D8 and GCD18 groups were lower than those of the D1 group at some time points. These results indicate that GCD18 can inhibit the in vitro growth of CVCC1806 under the experimental conditions, and its antibacterial trend is no less than that of the parental phage D1.

[0058] 4. Evaluation of the protective effect of engineered bacteriophages on animals The bacterial strain CVCC1806 was cultured in LB broth for 4 hours to reach the logarithmic growth phase, washed twice with PBS, and the bacterial concentration was adjusted to 10. 9 CFU / mL, phages D1, D8 and GCD18 were cultured to 10... 10 PFU / mL was prepared for use. Twenty-five 4-week-old female BALB / c mice were randomly divided into 5 groups of 5 mice each. After pretreatment with streptomycin in drinking water (5 g / L) for 24 h, the mice were deprived of water and food for 24 h. Then, the mice were challenged by gavage with the prepared CVCC1806 bacterial solution, 200 μL / mouse. The negative control group was challenged by gavage with the same dose of PBS solution. The mice were challenged twice, with an interval of 24 h between each challenge. Five h after the second challenge, the mice were treated as follows: D1 group mice were gavage with 100 μL of phage D1 plus 100 μL of PBS, D8 group mice were gavage with 100 μL of phage D8 plus 100 μL of PBS, GCD18 group mice were gavage with 100 μL of phage GCD18 plus 100 μL of PBS, the positive control group mice were gavage with 200 μL of PBS, and the negative control group mice were gavage with 200 μL of PBS. After treatment, the mice were weighed daily, and a weight change curve was plotted. The mice's mental state, appetite, fecal morphology, and symptoms such as diarrhea and pasty anus were observed. On the seventh day, the mice were sacrificed, a small intestinal fragment was taken, weighed, and thoroughly homogenized in PBS using a tissue homogenizer. The tissue homogenate was then serially diluted 10-fold and spread on XLD agar plates to measure the Salmonella load in the mouse intestines.

[0059] Following phage infection, mice in the infected group exhibited symptoms such as disheveled fur, lethargy, decreased appetite, and softened feces. After phage treatment, the clinical condition of mice in all treatment groups gradually improved. Weight changes are shown in the figures below. Figure 22 As shown, on day 7, the average weight of mice in the phage treatment group was higher than that in the infected control group, and the average weight of mice in the GCD18 group was higher than that in the D1 group.

[0060] The results of the intestinal Salmonella load on day 7 of the experiment are as follows: Figure 23 As shown. The bacterial load in the infected control group was 10. 5 CFU / mL, bacterial load in the phage D8 and GCD18 treatment groups was 10 4 CFU / mL, the bacterial load in the GCD18 treatment group was significantly lower than that in the D1 group. Figure 23 middle,* P <0.05.

[0061] It should be understood that the above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention should be considered equivalent substitutions and are included within the protection scope of the present invention.

Claims

1. An engineered bacteriophage capable of simultaneously lysing both susceptible and resistant strains of Salmonella, characterized in that: The engineered phage was formed by inserting the T6 tail filament protein coding sequence of the second phage into the T2 tail filament protein coding sequence of the first phage using gene editing tools. The GenBank accession number of the whole genome of the first phage is ON550260.1, and the GenBank accession number of the whole genome of the second phage is OP828573.

1.

2. The engineered bacteriophage according to claim 1, characterized in that, The T2 tail filament protein is encoded by the nucleic acid sequence shown in SEQ ID NO:2, and the T6 tail filament protein is encoded by the nucleic acid sequence shown in SEQ ID NO:

6.

3. An engineered bacteriophage capable of simultaneously lysing both susceptible and resistant Salmonella strains, characterized in that: The engineered bacteriophage is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC M 2026963 and deposit date of May 14, 2026.

4. A method for constructing an engineered bacteriophage according to any one of claims 1 to 3, characterized in that, include: Insert the nucleic acid molecule shown in SEQ ID NO:6 into the pHCY-26D plasmid. Sal I and EcoR I. Between the restriction sites, the N20 sequence shown in SEQ ID NO:15 was inserted between the gRNA and the scaffold sequence to obtain the T6-pHCY-26D plasmid; The pHCY-25A plasmid and the T6-pHCY-26D plasmid were transformed into Salmonella competent cells to obtain recombinant bacteria containing the two plasmids. The recombinant bacteria were infected with the first phage with the whole genome accession number ON550260.1 in GenBank, and the engineered phage was obtained after screening.

5. The construction method according to claim 4, characterized in that: The Salmonella competent cells were prepared from Salmonella CVCC1806.

6. The construction method according to claim 4 or 5, characterized in that, The pHCY-25A plasmid and T6-pHCY-26D plasmid were used to transform the Salmonella competent cells using the heat shock method.

7. The construction method according to any one of claims 4 to 6, characterized in that, The recombinant bacteria were inoculated into a culture medium containing ampicillin and kanamycin and cultured. IPTG, L-arabinose and the first phage were added sequentially and cultured at 30°C. After the culture was completed, the phage fluid was separated and the engineered phage was screened from the phage fluid.

8. The use of the engineered bacteriophage according to any one of claims 1 to 3 in the preparation of a medicament for treating Salmonella infection.

9. A bacterial agent for inhibiting Salmonella antibiotics, characterized in that, Includes the engineered bacteriophage according to any one of claims 1 to 3.

10. A pharmaceutical composition, characterized in that, Includes the engineered phage as described in any one of claims 1 to 3 and pharmaceutically acceptable vectors.

11. A drug for treating Salmonella infection, characterized in that, The engineered phage comprising a therapeutically effective amount as claimed in any one of claims 1 to 3.

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