Salmonella engineered cell-penetrating bacteriophage, and construction method therefor and use thereof

Abstract

Provided are a Salmonella engineered cell-penetrating bacteriophage, and a construction method therefor and a use thereof. The Salmonella engineered cell-penetrating bacteriophage comprises a bacteriophage and a cell-penetrating peptide, the surface of the bacteriophage has a surface protein, the surface protein is fused with the cell-penetrating peptide, and the surface protein is a protein containing an Ig-like domain. By means of a bioinformatics method, proteins which are suitable for fusion expression with foreign proteins, namely proteins containing Ig-like domains, are screened out in a high-throughput manner from structural proteins of a Salmonella typhimurium bacteriophage, and then, fusion expression of a cell-penetrating peptide is implemented on the surface of the Salmonella typhimurium bacteriophage to obtain an engineered bacteriophage capable of efficiently killing Salmonella typhimurium in cells, thereby increasing the titer of the Salmonella typhimurium bacteriophage entering cells, and extending phage therapy to treatment of intracellular infections.

Description

A salmonella engineered membrane-penetrating phage and its construction method and application Technical Field

[0001] The present invention relates to the field of life science technology, and in particular to a Salmonella engineered transmembrane phage and a construction method and application thereof. Background Art

[0002] Salmonella typhimurium is the second most common foodborne pathogen, causing approximately 200,000 deaths each year. The pathogenic mechanism of Salmonella typhimurium mainly involves its ability to infect intestinal epithelial cells. It first forms vesicles containing Salmonella inside the cells, which then escape into the cytoplasm to multiply in large numbers, leading to potential foci of infection and causing prolonged infection. In addition, Salmonella typhimurium can not only invade epithelial cells but also infect phagocytes and even fibroblasts. Antibiotics are an effective treatment method. With their widespread use, highly resistant Salmonella typhimurium has emerged and begun to spread globally. Due to the presence of cell membranes, antibiotics are difficult to penetrate into cells to reach effective concentrations, making it extremely difficult to effectively kill intracellular bacteria. Therefore, there is an urgent need to find new and effective alternatives to treat Salmonella typhimurium infections in clinical practice.

[0003] As the natural enemy of bacteria, bacteriophages can effectively target and kill extracellular host bacteria. However, their effects on intracellular host bacteria are rarely reported. As the most abundant and diverse biological entity, bacteriophages are ubiquitous in the environment and human body. It is reported that there are up to 2×10 12 A growing number of studies have shown that phages, when administered orally or by inhalation, can be widely distributed throughout the human body, including the liver, spleen, kidneys, heart, and even brain tissue, demonstrating their potential for transcellular transport. Recent studies have shown that phages can enter mammalian cells through specific or nonspecific interactions, remaining stable for 24 hours. However, the efficiency of phage entry is related to factors such as phage morphology, concentration, size, and cell type, providing preliminary evidence that phages can enter mammalian cells.

[0004] Currently, the ability of natural phages to enter cells is very limited. Improving phage entry has great potential for treating intracellular bacteria. To improve the ability of phages to cross the plasma membrane and reach intracellular targets, people have been widely considering methods to encapsulate phages into carriers including liposomes, polymer particles, and nanoparticles. However, chemical modification has disadvantages such as being expensive, time-consuming, and labor-intensive. In recent years, with the rapid development of synthetic biology, it has become possible to use gene editing technology to obtain cell-penetrating peptides (CPPs) to display engineered phages to kill intracellular bacteria. Cell-penetrating peptides are small peptides composed of 5-30 amino acids that have the potential to carry large molecules including DNA, RNA, and proteins into cells. As a classic transport carrier, CPPs are favored in medical applications due to their efficient internalization ability and low toxicity. There are many types of CPPs, the most common of which are those that enter cells nonspecifically, such as the transactivator of transcription (TAT) of the human immunodeficiency virus (HIV), the influenza virus hemagglutinin protein (HA), the Drosophila homeotic transcription factor ANTP, and synthetic polyarginine and polylysine. Tao Pan et al. achieved efficient phage entry by expressing the transmembrane peptide TAT fused to the hoc site of the T4 head modification protein. With the rapid development of phage gene editing technology, the use of transmembrane peptides to modify phage entry holds great promise. However, current research on transmembrane peptide fusions has primarily focused on colon model phages. Some scholars have proposed that combining gene editing with phage display technology can effectively improve phage entry efficiency. In 2018, Frances and three other scientists won the Nobel Prize for phage display technology. This technology fuses nucleic acid sequences encoding random short peptides or proteins with genes encoding phage surface proteins. As the phage is propagated, the fused peptides or proteins are displayed on the phage surface, maintaining their corresponding spatial structure and biological activity. This technology plays a crucial role in molecular biology fields such as vaccine development and antibody preparation. In 1990, Scott successfully constructed the first phage-displayed random peptide library by fusing a large number of random gene sequences to the surface of filamentous phage.In recent years, phage display technology has made great progress. Phage display technology has been used to screen vascular-targeting peptides RGD, NGR, and SFITGv6 for cancer treatment. Mann used the CX7C T7 phage library to screen the connective tissue growth factor (CTGF / CCN2) targeting peptide DAG, which can be used for the diagnosis of Alzheimer's disease. The T7 phage display random peptide library was used to screen the selective tumor inhibitor K-RAS, which can be used to develop cancer-related signaling pathway inhibitors.

[0005] Phage display technology is to clone the coding gene or target gene fragment of a polypeptide or protein into the appropriate position of the phage coat protein structural gene, so that the foreign polypeptide or protein is fused with the coat protein and expressed under the condition that the reading frame is correct and the normal function of other coat proteins is not affected. The fusion protein is then displayed on the phage surface as the daughter phage is reassembled. However, the current phage display technology is mainly carried out on E. coli related model phages and is based on a clear protein structure. In nature, there are as many as 10 30 -10 31 The huge number of phages, high phage diversity, little understanding of structural proteins that can be matched and modified, and many unknown genes have greatly restricted the development of non-model phage display.

[0006] Therefore, the existing technology still needs to be improved and developed.

[0007] Summary of the Invention

[0008] In view of the above-mentioned deficiencies in the prior art, the object of the present invention is to provide a Salmonella engineered transmembrane phage and its construction method and application, aiming to solve the problem of low efficiency of existing phage entry into cells.

[0009] The technical solutions of the present invention are as follows:

[0010] In a first aspect, the present invention provides an engineered Salmonella penetrating phage, which comprises a phage and a penetrating peptide, wherein the phage has a surface protein on its surface, the surface protein is fused with the penetrating peptide, and the surface protein is a protein containing an Ig-like domain.

[0011] Optionally, the surface protein is GP94 protein.

[0012] Optionally, the cell-penetrating peptide comprises one of HA-TAT, cTAT, Intergrin, R8, R7, P8 and Transportan.

[0013] Further optionally, the cell-penetrating peptide is HA-TAT.

[0014] The present invention utilizes phage display technology, the principle of which is to recombinate a DNA fragment encoding a polypeptide with a phage capsid protein-encoding gene and express it on the surface of the phage as a fusion protein. Specifically, the present invention uses bioinformatics methods to screen the structural proteins of Salmonella typhimurium phage for high-throughput expression of proteins suitable for fusion expression with exogenous proteins, namely proteins containing Ig-like domains. Subsequently, the cell-penetrating peptide is fused and expressed on the surface of Salmonella typhimurium phage to obtain an engineered membrane-penetrating phage that can effectively kill Salmonella typhimurium in cells, thereby increasing the potency of Salmonella typhimurium phage to enter cells and expanding phage therapy to the treatment of intracellular infections.

[0015] Specifically, the present invention used the hmmscan command in the HMMER3 bioinformatics software to search and screen for proteins containing Ig-like domains. Furthermore, AlphaFold2 predicted the structure of the GP94 protein from the Salmonella typhimurium SL7207 phage, suggesting that the protein contains three domains, two of which are Ig-like domains. Therefore, the GP94 protein has the potential for phage display.

[0016] In a second aspect, the present invention provides a method for constructing a Salmonella engineered transmembrane phage according to the present invention, comprising the following steps:

[0017] Construct a recombinant pDonor plasmid containing homology arms, CPP, and sgRNA;

[0018] Electroporating the spCas9 plasmid and the recombinant pDonor plasmid into host Salmonella to obtain a host bacterium containing two plasmids;

[0019] The wild-type phage is propagated on the host bacteria containing the double plasmid to obtain the engineered transmembrane phage.

[0020] In a third aspect, the present invention provides a use of the above-mentioned engineered transmembrane phage of Salmonella in the preparation of a drug for treating intracellular infection of Salmonella.

[0021] Optionally, the Salmonella is Salmonella typhimurium.

[0022] In a fourth aspect, the present invention provides a drug for treating intracellular Salmonella infection, comprising the engineered transmembrane phage of Salmonella according to the present invention.

[0023] The present invention screens proteins suitable for fusion expression with exogenous proteins from the structural proteins of Salmonella typhimurium phage, and then allows the phage surface to express cell-penetrating peptides in fusion, thereby obtaining an engineered transmembrane phage that can effectively kill intracellular Salmonella typhimurium, providing a new approach for treating intracellular Salmonella typhimurium infections.

[0024] Beneficial effects: The present invention uses bioinformatics methods to screen phage surface proteins in high throughput, which can quickly and efficiently obtain target proteins and expand the application scope of phage display technology. Furthermore, by fusing the screened phage surface proteins with cell-penetrating peptides, the potency of phages entering mammalian cells is increased, and phage therapy is extended to the treatment of intracellular bacterial infections, providing new ideas for the treatment of intracellular Salmonella typhimurium infections. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 shows the prediction results of Alphafold 2 for the GP94 protein structure.

[0026] Figure 2 shows the average local distance difference test (LDDT) predicted by AlphaFold 2.

[0027] FIG3 is a Western blot image of HRP-labeled streptavidin on a PVDF membrane in Example 1.

[0028] Figure 4 shows the wild type selz phage (left) and selz in Example 1 GBP TEM image of bacteriophage (right) after reaction with 10 nm gold nanoparticles; scale bar, 100 nm.

[0029] FIG5 is a schematic diagram showing the difference in the efficiency of wild-type phage and various engineered transmembrane phage entering different tissue cell lines in Example 3.

[0030] Figure 6 shows the wild-type phage and the engineered transmembrane phage selz in Example 4. HA-TAT Schematic comparison of therapeutic efficacy against intracellular Salmonella Typhimurium infection.

[0031] Figure 7 shows the wild-type phage and engineered transmembrane phage selz in Example 4. HA-TAT Schematic comparison of titers in therapeutic cell infection models.

[0032] Figure 8 is a flow cytometry analysis of mCherry-SL1344 in Hela cells in wild-type phage and engineered transmembrane phage selz in Example 4 HA-TAT Schematic representation of the differences in mean fluorescence intensity after treatment. DETAILED DESCRIPTION

[0033] The present invention provides an engineered transmembrane phage for Salmonella, its construction method, and its application. To clarify the objectives, technical solutions, and effects of the present invention, the present invention is described in further detail below. It should be understood that the specific embodiments described herein are intended only to illustrate the present invention and are not intended to limit the present invention.

[0034] Phage display technology involves cloning a polypeptide or protein-encoding gene or a target gene fragment into the appropriate location of a phage coat protein structural gene. In the correct reading frame and without affecting the normal function of other coat proteins, the exogenous polypeptide or protein is fused to the coat protein and expressed. The fusion protein is then displayed on the phage surface as the progeny phage reassembles. However, existing phage display technology can only express cell-penetrating peptides by fusion on model phages. Expressing cell-penetrating peptides by fusion on non-model phage surface proteins remains a significant challenge, making phage therapy difficult to use for the treatment of intracellular bacterial infections.

[0035] The inventors analyzed the immunoglobulin-like (Ig-like) domains in the phage genome:

[0036] Proteins containing Ig-like domains can be screened from existing phage genomes. For example, the hmmscan command in HMMER3 (Bioinformatics Software) can be used to search a portion of the phage genome (with an e-value threshold set to 1e-5). The results can be filtered to identify proteins containing Ig-like domains in the Pfam.v.35 database (Table 1).

[0037] Ig-like domains are frequently located on the surface of tailed double-stranded DNA (dsDNA) phages, with nearly a quarter of these phages possessing Ig-like domains. Ig-like domains are among the most common and widespread domains in nature, composed of at least seven β-strands. Proteins with Ig-like domains are commonly found in bacteria and are most often involved in cell-cell adhesion or extracellular sugar hydrolysis. Furthermore, phages can interact with mammalian mucus via these displayed Ig-like domains, further suggesting that these Ig-like domains are exposed on the phage surface. Studies have reported that Ig-like domain proteins from coliform phages, such as Hoc (T4), pb10 (T5), and gp17 (N4), are highly associated with host adhesion and can be used for phage display. Therefore, it is hypothesized that phage proteins containing Ig-like domains could serve as potential sites for phage display.

[0038] Table 1. Information on proteins containing Ig-like domains in existing phage genomes

[0039] Based on this, the present invention provides a Salmonella engineered transmembrane phage, which includes a phage and a transmembrane peptide. The phage surface has a surface protein, the surface protein is fused with the transmembrane peptide, and the surface protein is a protein containing an Ig-like domain.

[0040] Specifically, the present invention screens out proteins suitable for fusion expression with exogenous proteins from the structural proteins of Salmonella typhimurium phage, allows the phage surface to express cell-penetrating peptides, and obtains engineered transmembrane phages that can effectively kill intracellular Salmonella typhimurium, providing new ideas for the clinical treatment of intracellular Salmonella typhimurium.

[0041] After analyzing that proteins containing Ig-like domains in phages can serve as potential sites for phage display, we further speculated that the phage selz GP94 protein could be used for phage display:

[0042] The results showed that GP94 (amino acid sequence shown in Table 2) contains an Ig-like domain (Table 1). Further prediction of the GP94 structure using AlphaFold2 revealed that the protein contains three domains, two of which are Ig-like domains (see Figure 1).

[0043] Figure 2 shows the average local distance difference test score (LDDT) predicted by AlphaFold 2. These scores are indicators that measure the amino acid positions predicted by AlphaFold2. At the same time, the structure was compared with the protein structure database (PDB) using the DALI server, showing that GP94 has a high degree of similarity with three phage structural proteins, including the hoc protein of phage RB49, the tail tube protein (pb6) of phage T5, and the tail tube protein (gpV) of phage lambda (see Table 3). In fact, the hoc protein of phage RB49 and the gpV protein of phage lambda have been reported for phage display. Therefore, it is speculated that GP94 has the potential for phage display.

[0044] Table 2. Amino acid sequence of selz phage GP94 protein

[0045] Table 3. DALI comparison analysis results of GP94 domain 2 (amino acids 92-171) and domain 3 (amino acids 172-269)

[0046] Wherein, rmsd is the root mean square deviation of carbon atoms in the least squares superposition of carbon atoms of equivalent structures; lali is the number of amino acids in equivalent structures; %id is the percentage of identical amino acids in all amino acids in all equivalent structures.

[0047] That is to say, in the engineered transmembrane phage, the surface protein on the phage surface is GP94 protein.

[0048] In some embodiments, the cell-penetrating peptide comprises one of HA-TAT, cTAT, Intergrin, R8, R7, P8, and Transportan, etc. Preferably, the cell-penetrating peptide is HA-TAT.

[0049] Specifically, the present invention uses bioinformatics methods to high-throughput screen the structural proteins of Salmonella typhimurium phage for proteins suitable for fusion expression with exogenous proteins, namely the GP94 protein containing an Ig-like domain. Subsequently, the cell-penetrating peptide is fused and expressed on the surface of the Salmonella typhimurium phage to obtain an engineered phage that can effectively kill Salmonella typhimurium in cells, thereby increasing the potency of Salmonella typhimurium phage entering cells and expanding phage therapy to the treatment of intracellular infections.

[0050] The present invention also provides a method for constructing an engineered transmembrane phage of Salmonella, which comprises the following steps:

[0051] S1: Construction of recombinant pDonor plasmid containing homology arms, CPP, G4S Linker and SgRNA;

[0052] S2: electroporating the spCas9 plasmid and the recombinant pDonor plasmid into host Salmonella to obtain a host bacterium containing two plasmids;

[0053] S3: The wild-type phage is propagated on the host bacteria containing the double plasmid for one generation, and the engineered transmembrane phage is obtained by homologous recombination.

[0054] The present invention uses bioinformatics methods to screen the phage surface proteins of Salmonella typhimurium in high throughput, which can quickly and efficiently obtain the target protein. By fusing and expressing cell-penetrating peptides with the screened phage surface proteins, the titer of phage entry into cells is increased, and phage therapy is extended to the treatment of intracellular bacterial infections.

[0055] The present invention also provides use of the engineered transmembrane phage of Salmonella according to the present invention in preparing a drug for treating intracellular infection of Salmonella.

[0056] In some embodiments, the Salmonella is Salmonella typhimurium.

[0057] The present invention also provides a drug for treating intracellular infection of Salmonella, which comprises the Salmonella engineered transmembrane phage according to the present invention.

[0058] The present invention will be further described below with reference to specific examples.

[0059] Example 1 Verification of the GP94 protein on phage selz for phage display

[0060] (1) Linearization of pDonor plasmid: Use BamHI restriction endonuclease to digest the pDonor plasmid, then use primers to perform inverse PCR to obtain high-concentration plasmid backbone DNA, and finally detect the PCR product.

[0061] The specific steps for PCR product detection are as follows: prepare an agarose gel composed of 1×TAE (nucleic acid electrophoresis buffer) + 1% agarose + 1 / 1000SYBR safe gel dye, take 5μl of PCR product and load it for electrophoresis detection; use gel recovery kit, according to Follow the instructions of the Extraction Kit to obtain a DNA solution. Measure the recovered DNA concentration on a Nano-drop instrument, label and record the solution, and store it at -20°C for a long term.

[0062] (2) Obtaining a phage gene DNA fragment containing the GP94 gene: A DNA fragment containing 500 bp homology arms upstream and downstream of the GP94 gene, a gene sequence expressing Avi-tag or gold-binding peptide (GBP) fused to the start of the GP94 gene, and a 20 bp sgRNA sequence was constructed by overlapping PCR.

[0063] Among them, PCR was performed using the bacteriophage selz genome as a template to obtain homology arms, and then overlapping PCR was performed using the high-fidelity polymerase 2xPhanta Max Master Mix. The primers used are shown in Table 4.

[0064] Table 4. PCR primers used

[0065] Obtaining the recombinant pDonor plasmid containing the DNA fragment of the GP94 gene: First, use the one-step cloning enzyme (ClonExpress II One Step Cloning Kit) to obtain the linear plasmid and the recombinant pDonor plasmid containing the DNA fragment of the GP94 gene. Then, take the PCR reaction product for sequencing verification. Then, use the mini-extraction kit (Tiangen Biochemical) to obtain the recombinant plasmid solution. Finally, use the Nano-drop instrument (microspectrophotometer) to measure the plasmid concentration, keep a record of the plasmid concentration, and store it at -20℃ for a long time.

[0066] (3) Obtaining a host bacterium containing two plasmids: The spCas9 plasmid and the recombinant pDonor plasmid prepared in step (3) were electroporated into the host Salmonella SL7207.

[0067] The specific steps are as follows: 200ng of spCas9 plasmid and 200ng of recombinant pDonor plasmid were added to the host Salmonella SL7207 competent cells (logarithmic phase bacteria, 10% glycerol treated, stored at -80°C), then added to the electroporation instrument sample cell, and the electroporation instrument sample cell was electroporated at 1.8kV, 2.5ms. Subsequently, 37°C preheated LB (lysate broth) was added, and the cells were placed in a 37°C shaker for 1 hour before being coated with the corresponding resistance plate. The next day, single clones were picked and the successful electroporation of the dual plasmids was verified by PCR product.

[0068] (4) Obtaining Salmonella engineered phage: First, wild-type phage (WT) was used to infect the host bacteria containing the double plasmid for one generation, and then primers seq 94FW and seq 94Rev were used for PCR electrophoresis and Sanger sequencing; then, the engineered phage containing Avi-tag (GLNDIFEAQKIEWHE) was obtained after repeated purification three times on a double-layer plate. Avitag and GBP (VSGSSPDS) engineered phage selz GBP .

[0069] The specific steps are as follows: the phage after subculture is gradiently diluted and first mixed with 200 μl of logarithmic phase host bacteria, then mixed with 5 ml of 0.5% LB agar, and then immediately poured into a 1.5% LB solid agar plate and dried, and cultured at 37°C overnight to form a single plaque. The next day, a single plaque was picked and dissolved in 50 μl of SM buffer and mixed. Primers seq 94 FW and seq 94 Rev were used to use 0.5 μl of the buffer as a PCR reaction template for PCR to verify whether the band size was correct. This operation was repeated 3 times, and Sanger sequencing was performed to obtain the purified engineered phage.

[0070] (5) Verification of engineered phage biotinylation: The phage was biotinylated according to the instructions of the Avi tag protein biotin labeling kit (BirA method), and then the in vitro biotinylated high-purity phage selz Avitag (after purification by CsCl density gradient ultracentrifugation) and selz were detected by western-blot.

[0071] The specific steps are as follows: first, use 4-12% high-resolution gradient precast gel to mix 5x SDS-PAGE buffer with high-purity phage (>10 11After mixing with 5 μl of marker and 20 μl of sample in a ratio of 1:4, the mixture was boiled in a metal bath at 100°C for 10 minutes and equilibrated to room temperature. 5 μl of marker and 20 μl of sample were added to the SDS-PAGE gel well and slowly injected along the well wall to avoid sample overflow and mutual contamination. The samples were then pressed into the same plane using 80V and 30min electrophoresis conditions. After the samples ran to the junction of the stacking gel and the separation gel, the electrophoresis conditions were changed to 120V and 60min to run to the bottom of the gel. After activating the PVDF membrane with anhydrous methanol for 1 minute, the cotton-PVDF membrane-precast gel-cotton soaked in the transfer liquid were placed vertically in a sandwich manner in sequence. After air bubbles were exhausted, a semi-dry transfer method was used with the constant voltage set to 15V, 1mA, and 10W, and the semi-dry transfer operation was performed for 15 minutes. The transferred PVDF membrane was then washed 3 times with PBST solution for 5 minutes each time. Then incubate with 5% BSA at 37°C for 2 hours; then incubate the PVDF membrane with 1:2000 horseradish peroxidase-labeled streptavidin at 37°C for 2 hours; finally, wash it repeatedly with PBST three times, mix the ECL developer in a 1:1 ratio in the dark, completely cover the PVDF membrane, and develop it using an imager after 3 minutes.

[0072] (6) Verification of the binding of engineered phage nanogold particles: First, the high-purity and high-concentration selz purified by CsCl density gradient ultracentrifugation was GBP The selz phages were mixed with gold nanoparticles at a ratio of 1:10 at room temperature for 5 h. The copper mesh with the thin film carbon support was then placed with the carbon side down in 20 μl of the phage mixture solution for 20 min. After washing three times in PBS, the solution was negatively stained with 20 μl of 2% PTA for 1 min. Finally, a HITACH HT7700 transmission electron microscope was used for observation.

[0073] Western-Blot detection of high concentration and high purity in vitro biotinylated selz Avitag and selz, the results showed that the engineered phage selz fused with Avi-tag Avitag The Avi-tagged protein biotin labeling kit can be successfully used for in vitro biotinylation, and the GP94 (28KD) protein band can be detected by horseradish peroxidase-labeled streptavidin reaction, as shown in Figure 3. However, there is no obvious band in the wild-type phage selz, which indirectly indicates that GP94 is not only a structural protein, but also can successfully realize the phage display of Avi tags. Further transmission electron microscopy can be used to observe that the gold nanoparticles can specifically bind to the engineered phage selz fused with GBP. GBP The reaction was observed under an electron microscope, and gold particles were mainly observed in the tail fibers of the phage, as shown in Figure 4. However, no obvious gold particles were found in the wild-type phage group. These results confirm that the GP94 protein of selz phage can be used for exogenous peptide display.

[0074] Example 2 Construction of Salmonella engineered transmembrane phage

[0075] The engineered transmembrane phage of Salmonella was achieved by fusion expression of CPP at the N-terminus of selz GP94 protein, wherein the amino acid sequences of CPP and Linker are shown in Table 5.

[0076] The method for constructing a CPP-displaying engineered transmembrane phage was the same as in Example 1: First, a recombinant pDonor plasmid containing homology arms, a CPP sequence, a G4S linker, and an sgRNA was constructed. After verifying the recombinant plasmid sequence by Sanger sequencing, the spCas9 plasmid and the recombinant pDonor plasmid were electroporated into the host Salmonella SL7207. Wild-type phage was then propagated for one generation on host Salmonella SL7207 containing the pDonor plasmid and the spCas9 plasmid carrying the target fragment. Under the pressure of the Cas9 system, homologous recombination was performed to obtain a CPP-displaying engineered phage. Finally, the presence of the recombinant phage was verified using primers seq_94_FW and seq_94Rev. The modified CPP-displaying engineered transmembrane phage was purified three times. The sequences of all primers used are detailed in Table 6.

[0077] Table 5. Amino acid sequences of CPPs and Linkers involved

[0078] Table 6. PCR primers used

[0079] Example 3 Functional assay of engineered transmembrane phage

[0080] Hela, A549, and Caco2 cell lines were selected to screen the engineered transmembrane phages constructed in Example 2. The culture medium for Hela, A549, and Caco2 cells was DMEM + 10% fetal bovine serum (FBS) + penicillin-streptomycin. The cell culture medium was changed every 2 days. When the cells grew to about 80% confluence, they were digested with 0.25% trypsin and then passaged. All cells were cultured in a 37°C incubator with 5% CO2. When the different cell types grew to 80% confluence, 1.5 x 10 9 PFU / ml of various engineered transmembrane phages; wild-type phage served as a control group. After 4 hours of co-incubation, the supernatant was aspirated and discarded. The cells were washed four times with PBS to fully remove extracellular phage. DDH2O was added to lyse the cells, and the titer of intracellular functional phage was determined. Three to four replicates were set up for each group, and repeated twice. This experiment preliminarily screened for engineered transmembrane phages with efficient transmembrane ability.

[0081] Furthermore, the phages were incubated with the cells for 4 h and then counted on a double-layer plate. HA-TAT 、selzTransportan、selz Intergrin and selz R7 The uptake of selz in A549 cells was significantly higher than that in wild-type phage (P<0.05). HA-TAT The uptake of selztransportan (P<0.0001) and selztransportan (P<0.01) was increased, and intracellular functional phage selz was also observed in Caco-2 cells. HA-TAT The concentration of the bacteriophage was significantly higher than that of the wild-type phage (P<0.001), and the specific difference is shown in Figure 5.

[0082] Notably, in all epithelial cells tested, selz HA-TAT It showed the best membrane penetration efficiency, which was 10 to 30 times higher than that of wild-type phage, especially in A549 cells. HA-TAT The cell entry efficiency was 29 times higher than that of the wild type. The results showed that it is feasible to display the GP94 protein of selz via CPP to improve the cell entry efficiency of phage.

[0083] Example 4 selz HA-TAT Verification of intracellular bactericidal activity of engineered transmembrane phages in vitro

[0084] (1) Salmonella typhimurium SL1344 (mCherry-SL1344) containing plasmid pBBR1MCS-Tac-mCherry was selected as the host bacteria and 5x10 4 The cells were cultured in 12-well plates, and within 24 hours, Salmonella typhimurium mCherry-SL1344 with an MOI of 10 was added and cultured for 12 hours. The next day, the extracellular Salmonella were repeatedly washed with high-concentration gentamicin (100 μg / ml) PBS to kill the extracellular Salmonella. Then, 1.5×10 9 PFU / ml of engineered transmembrane phage selz HA-TAT The cells were incubated with 1% FBS cell culture medium (added with low concentration of gentamicin 10 μg / ml) and wild-type phage selz for 4 hours, and the PBS group was used as a negative control group. After 4 hours of phage treatment, the cells were washed three times with PBS to fully wash off the extracellular phage and bacteria. The cells were incubated with ddH2O for 5-10 minutes and the bottom of the well plate was repeatedly scraped with a pipette tip to mix and fully lyse the cells. Then, the bacterial colony count (CFU) was counted on the plate and the phage titer (PFU) was counted on the spot plate.

[0085] As shown in Figure 6, in three different epithelial cell intracellular infection models, the engineered transmembrane phage selz HA-TAT After treatment in Hela cells, CFU results showed that the phage selz HA-TAT After treatment, the number of intracellular bacterial colonies was significantly lower than that of the wild-type treatment group and the untreated group (P<0.5) (see Figure 6A); in the A549 cell intracellular infection model, selz HA-TAT The number of intracellular bacterial colonies in the treated group was significantly lower than that in the untreated group (P<0.5), while there was a decreasing trend in the wild-type treated group with no significant difference (P>0.5) (see Figure 6B); in Caco-2 cells, phage selz HA-TAT The bacterial colony count in the treatment group showed a downward trend compared with the wild-type phage treatment group and the untreated group, but it was not statistically significant (P>0.5) (see Figure 6C); while there was no significant difference in the wild-type phage treatment in the three epithelial cell intracellular infection models compared with the untreated group (P>0.5). As shown in Figure 7, in the corresponding intracellular functional phage titer determination experiment, phage selz was detected in almost all epithelial cells. HA-TAT The levels of the treated group were significantly higher than those of the wild-type phage treated group (P<0.5).

[0086] (2) Engineered transmembrane phage selz HA-TAT The procedure for treating intracellular Salmonella typhimurium infection with wild-type phage selz in vitro was the same as (1). After 4 h of phage treatment, cells were washed three times with pre-chilled PBS containing a high concentration of gentamicin, then digested with 0.25% trypsin. The cells were then resuspended three times with 4°C pre-chilled PBS and immediately analyzed by flow cytometry. The fluorescence intensity of each group was compared, which indirectly reflects the number of intracellular bacteria.

[0087] As shown in Figure 8, it can be observed that in the engineered transmembrane phage selz HA-TAT The mean fluorescence intensity (MFI) of the cells in the treatment group was significantly lower than that in the wild-type treatment group and the untreated group (P<0.5).

[0088] The above results indicate that the engineered transmembrane phage selz constructed by the present invention HA-TAT It can significantly enter epithelial Hela cells and has the ability to control intracellular Salmonella typhimurium infection, and has the potential to be used in the treatment of intracellular pathogenic bacterial infections.

[0089] In summary, the present invention provides an engineered Salmonella transmembrane phage, its construction method, and application. By using bioinformatics methods to screen phage surface proteins for high-throughput screening, target proteins can be obtained quickly and efficiently, expanding the application scope of phage display technology. Furthermore, by fusing and expressing cell-penetrating peptides with the screened phage surface proteins, the potency of phage entering mammalian cells is increased, extending phage therapy to the treatment of intracellular bacterial infections.

[0090] It should be understood that the application of the present invention is not limited to the above examples. For those skilled in the art, improvements or changes can be made based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to the present invention.

Claims

1. A Salmonella engineered transmembrane phage, characterized in that: The method comprises a bacteriophage and a membrane-penetrating peptide. The surface of the bacteriophage has a surface protein, the surface protein is fused with the membrane-penetrating peptide, and the surface protein is a protein containing an Ig-like structural domain.

2. The engineered transmembrane-penetrating phage of Salmonella according to claim 1, characterized in that: The surface protein is GP94 protein.

3. The engineered transmembrane-penetrating phage of Salmonella according to claim 1, characterized in that: The cell-penetrating peptide includes one of HA-TAT, cTAT, Intergrin, R8, R7, P8 and Transportan.

4. The engineered transmembrane-penetrating phage of Salmonella according to claim 3, characterized in that: The cell-penetrating peptide is HA-TAT.

5. A method for constructing a Salmonella engineered transmembrane phage according to any one of claims 1 to 4, characterized in that: The steps include: Construct a recombinant pDonor plasmid containing homology arms, CPP and SgRNA; Electrotransforming the spCas9 plasmid and the recombinant pDonor plasmid into host Salmonella to obtain a host bacterium containing two plasmids; The wild-type phage is propagated on the host bacteria containing the double plasmid to obtain the engineered membrane-penetrating phage.

6. Use of the engineered transmembrane-penetrating phage of Salmonella according to any one of claims 1 to 4 in the preparation of a drug for treating intracellular infection with Salmonella.

7. The use according to claim 6, characterized in that: The salmonella is salmonella typhimurium.

8. A drug for treating intracellular infection with Salmonella, characterized in that: The invention comprises the engineered transmembrane-penetrating phage of Salmonella as described in any one of claims 1 to 4.

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