CN116555197B

CN116555197B - Salmonella engineering membrane penetrating phage and construction method and application thereof

Info

Publication number: CN116555197B
Application number: CN202310744710.4A
Authority: CN
Inventors: 马迎飞; 谭新; 赵敏
Original assignee: Shenzhen Institute of Advanced Technology of CAS
Current assignee: Shenzhen Advanced Science And Technology Cci Capital Ltd
Priority date: 2023-06-25
Filing date: 2023-06-25
Publication date: 2023-09-01
Anticipated expiration: 2043-06-25
Also published as: CN116555197A

Abstract

The invention relates to the technical field of life science, in particular to a salmonella engineering membrane penetrating phage and a construction method and application thereof. The salmonella engineering membrane penetrating phage comprises phage and membrane penetrating peptide, wherein the surface of the phage is provided with surface protein, the surface protein is fused with the membrane penetrating peptide, and the surface protein is protein containing Ig-like structural domain. The invention screens out the protein which is suitable for fusion expression with exogenous protein, namely the protein containing Ig-like structural domain, from the structural protein of salmonella typhimurium phage by a bioinformatics method, then allows the salmonella typhimurium phage surface to fuse and express cell penetrating peptide, thus obtaining engineering phage which can kill salmonella typhimurium in cells with high efficiency, improving the titer of salmonella typhimurium phage entering cells, and expanding phage therapy to the treatment of intracellular infection.

Description

Salmonella engineering membrane penetrating phage and construction method and application thereof

Technical Field

The invention relates to the technical field of life science, in particular to a salmonella engineering membrane penetrating phage and a construction method and application thereof.

Background

Salmonella typhimurium, the second largest food-borne infectious pathogen, can cause about 20 tens of thousands of deaths each year. The pathogenic mechanism of salmonella typhimurium mainly relates to the infection of intestinal epithelial cells, and vesicles containing salmonella are formed in the cells firstly, and can escape to cytoplasm for mass propagation in the later period, so that potential infection sites are caused, and the infection is not healed. In addition, salmonella typhimurium can invade epithelial cells and can infect phagocytes, even fibroblasts and the like. Antibiotics are used as effective treatment means, and with the wide use of antibiotics, salmonella typhimurium with high drug resistance appears and starts to spread globally, and due to the existence of cell membranes, antibiotics are difficult to permeate into cells to reach effective action concentration, so that great difficulty is brought to effective killing of intracellular bacteria, and therefore, a new and effective alternative mode for treating salmonella typhimurium infection is urgently needed to be found clinically.

The phage can be used as natural enemy of bacteria, and can efficiently target and kill extracellular host bacteria. However, their effect on intracellular host bacteria has been rarely reported. As the most abundant and most diverse biological entity, phage are ubiquitous in the environment and human body, reportedly having up to 2x 10 in the human gut ¹² And (3) phage particles. More and more studies have shown that phage can be widely distributed throughout various organs of the human body, including liver, spleen, kidney, heart and even brain tissue, when treated by oral or inhalation of phage, indicating the potential of phage to traverse cells. Recent and increasing studies suggest that phages can enter cells and exist stably for 24 hours through specific or non-specific interactions with mammalian cells, but that the efficiency of entry into cells is related to factors such as the morphology, concentration, size and different cell types of phages, and initially suggest that phages can enter mammalian cells.

At present, the capability of natural phage entering cells is very limited, and how to improve the capability of phage entering cells has great potential for treating intracellular bacteria. In order to enhance the ability of phage to pass through plasma membranes and intracellular targets, methods of encapsulating phage into carriers including liposomes, polymer particles and nanoparticles have been widely considered, however chemical modification has disadvantages of being expensive, time consuming and labor consuming. With the rapid development of synthetic biology in recent years, it has become possible to obtain Cell-penetrating peptide (CPP) display engineering phage through gene editing technology to kill intracellular bacteria. The membrane penetrating peptide is a small peptide consisting of 5-30 amino acids, and has the potential of carrying macromolecules including DNA, RNA, proteins and the like into cells. CPPs are favored for medical applications as a classical transport vehicle due to their efficient internalization ability and lower toxicity. CPPs are of a wide variety, most commonly non-specific into cells such as reverse transcription activator of HIV (Transactivator of transcription, TAT), influenza hemagglutinin protein (The influenza virus haemagglutinin protein, HA), drosophila homeotranscriptional factor ANTP, and artificially synthesized polyarginine and polylysine. TaoPan et al realized efficient entry of phage by fusion expression of the transmembrane peptide TAT at the T4 head-modified protein hoc site. Along with the rapid development of phage gene editing technology, the realization of phage cell entry through membrane-penetrating peptide modification has great prospect. However, the current work on transmembrane peptide fusion has focused mainly on coliform phages. The scholars propose that the use of gene editing in combination with phage display technology (Phage display technology) can effectively increase the efficiency of phage entry. Three scientists in 2018 Franes obtain Nobel prize by phage display technology, which fuses nucleic acid sequence encoding random short peptide sequence or protein with gene encoding phage surface protein, and then presents the fused polypeptide or protein on phage surface along with phage passage, and maintains corresponding space structure and biological activity. Has important effects in the fields of molecular biology such as vaccine development, antibody preparation and the like. The first random peptide library for phage display was constructed by Scott scholars in 1990 by first fusing a large number of random gene sequences to the surface of filamentous phage. In recent years, phage display technology has been greatly developed, and peptide segments RGD, NGR and SFITGv6 targeting blood vessels are screened by using phage display technology so as to facilitate cancer treatment; mann targeting connective tissue growth factor (CTGF/CCN 2) targeting peptide DAG screened using CX7C T7 phage library can be used for diagnosis of Alzheimer's disease; the T7 phage display random peptide library is used for screening a selective tumor inhibitor K-RAS, and can be used for developing cancer related signal path inhibitors and the like.

Phage display technology is to clone the encoding gene or target gene fragment of the polypeptide or protein into phage coat proteinThe proper position of the structural gene enables the fusion expression of the exogenous polypeptide or protein and the coat protein under the condition that the reading frame is correct and the normal functions of other coat proteins are not affected, and the fusion protein is displayed on the surface of phage along with the reassembly of the progeny phage. However, phage display technology is currently mainly developed in E.coli-related mode phage and is based on a well-defined protein structure basis. While the number of phage in nature is as high as 10 ³⁰ -10 ³¹ The number of phages is large, the phage diversity is high, and factors such as little understanding of structural proteins capable of being matched and modified, numerous unknown genes and the like greatly limit the development of non-model phage display work.

Accordingly, the prior art is still in need of improvement and development.

Disclosure of Invention

In view of the defects of the prior art, the invention aims to provide a salmonella engineering membrane penetrating phage and a construction method and application thereof, and aims to solve the problem of low efficiency of the existing phage entering cells.

The technical scheme of the invention is as follows:

in a first aspect, the salmonella engineering penetrating phage provided by the invention comprises phage and penetrating peptide, wherein the phage surface is provided with surface protein, the surface protein is fused with the penetrating peptide, and the surface protein is protein containing Ig-like structural domain.

Alternatively, the surface protein is GP94 protein.

Optionally, the transmembrane peptide comprises one of HA-TAT, cTAT, intergrin, R8, R7, P8 and transporter.

Further alternatively, the transmembrane peptide is HA-TAT.

The invention uses phage display technology, and the principle is that DNA fragment encoding polypeptide and phage capsid protein encoding gene are recombined and expressed on the surface of phage in the form of fusion protein. Specifically, the invention screens out the protein suitable for fusion expression with foreign protein, namely the protein containing Ig-like structural domain, from the structural protein of the salmonella typhimurium phage by a bioinformatics method, then allows the salmonella typhimurium phage surface to fuse and express cell penetrating peptide, thus obtaining engineering penetrating phage capable of efficiently killing salmonella typhimurium in cells, improving the titer of the salmonella typhimurium phage entering cells, and expanding phage therapy to the treatment of intracellular infection.

Specifically, the invention uses hmmscan commands in HMMER3 signaling software to search and screen proteins containing Ig-like domains. Further, the structure of the GP94 protein in phage of salmonella typhimurium SL7207 was predicted by AlphaFold2, suggesting that the protein includes 3 domains, 2 of which are Ig-like domains. Thus, GP94 protein has the potential for phage display.

In a second aspect, the invention provides a construction method of salmonella engineering penetrating phage, which comprises the following steps:

constructing a recombinant pDONOOR plasmid containing homology arms, CPP and SgRNA;

the spCas9 plasmid and the recombinant pDOor plasmid are electrically transferred into a host salmonella to obtain a host bacterium containing double plasmids;

and propagating wild phage on the host bacteria containing the double plasmids to obtain the engineering membrane penetrating phage.

In a third aspect, the invention provides an application of the salmonella engineering transmembrane phage in preparing a medicament for treating intracellular infection salmonella.

Optionally, the salmonella is salmonella typhimurium.

In a fourth aspect, the invention provides a medicine for treating salmonella infection in cells, which comprises the salmonella engineering penetrating bacteriophage.

According to the invention, the protein suitable for fusion expression with the exogenous protein is screened from the salmonella typhimurium phage structural protein, and then the phage surface is fused to express the cell penetrating peptide, so that the engineering penetrating phage capable of efficiently killing salmonella typhimurium in cells is obtained, and a new thought is provided for treating salmonella typhimurium infected in cells.

The beneficial effects are that: the invention screens phage surface protein with high flux by bioinformatics method, can obtain target protein rapidly and efficiently, and expands the application range of phage display technology; furthermore, through fusion expression of cell penetrating peptide on the surface protein of the screened phage, the titer of the phage entering the mammalian cells is improved, phage therapy is expanded to treatment of intracellular bacterial infection, and a new idea is provided for treating intracellular infected salmonella typhimurium.

Drawings

FIG. 1 is a graph showing the prediction results of the structure of the GP94 protein by alpha fold 2.

Fig. 2 is an AlphaFold2 predictive average local distance difference test score (LDDT).

FIG. 3 is a Western blot of PVDF membrane using HRP-labeled streptavidin in example 1.

FIG. 4 shows wild-type selz phages (left) and selz from example 1 _GBP TEM image of phage (right) reacted with 10 nm nm gold particles; wherein, the scale bar, 100 nm.

FIG. 5 is a graph showing the difference in the efficiency of the wild-type phage and the various engineered transmembrane phages entering different tissue cell lines in example 3.

FIG. 6 shows the wild type phage and engineered transmembrane phage selz of example 4 _HA-TAT A comparative schematic of the effect of treatment on salmonella typhimurium infection in cells.

FIG. 7 shows the wild type phage and engineered transmembrane phage selz of example 4 _HA-TAT Comparison of titers in the model of therapeutic cell infection is schematically shown.

FIG. 8 is a flow assay of Hela intracellular mCherry-SL1344 in example 4 on wild type phage and engineered transmembrane phage selz _HA-TAT Schematic of the difference in mean fluorescence intensity after treatment.

Detailed Description

The invention provides a salmonella engineering membrane penetrating phage and a construction method and application thereof, and the invention is further described in detail below for making the purposes, technical schemes and effects of the invention clearer and more definite. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.

The phage display technology is to clone the encoding gene or target gene fragment of polypeptide or protein into proper position of phage coat protein structure gene, and to fuse and express exogenous polypeptide or protein and coat protein under the condition of correct reading frame and no influence on normal functions of other coat proteins, and the fusion protein is displayed on phage surface along with the reassembly of progeny phage. However, the existing phage display technology only can fusion express cell penetrating peptide on a model phage, and fusion expression of cell penetrating peptide on a non-model phage surface protein still has a great challenge, so that phage therapy is difficult to be used for treating intracellular bacterial infection.

The inventors analyzed immunoglobulin-like (Ig-like) domains in phage genomes:

proteins containing Ig-like domains were screened from the existing phage genome. For example, a portion of the phage genome can be searched using hmmscan command in HMMER3 (letter software) (e-value threshold set to 1 e-5), and the results screened for proteins containing Ig-like domains in the pfam.v.35 database (table 1).

Ig-like domains are often located on the surface of a tailed double-stranded DNA (dsDNA) phage, where nearly 1/4 of the tailed double-stranded DNA phage has Ig-like domains. Ig-like is one of the most common and most widely distributed domains in nature, consisting of at least seven beta strands. Proteins with Ig-like domains are common in bacteria and are most commonly involved in cell-cell adhesion or extracellular glycolysis. In addition, phage can interact with mucus in mammalian organisms through these displayed Ig-like domains, further indicating that these Ig-like domains are exposed on phage surfaces. The present study reports that Ig-like domain proteins such as coliphage Hoc (T4), pb10 (T5), gp17 (N4) and the like are highly correlated with host adhesion and can be used for phage display. Thus, it is speculated that proteins containing Ig-like domains in phage may serve as potential sites for phage display.

TABLE 1 information about Ig-like Domain-containing proteins in the genome of existing phages

Based on the above, the invention provides a salmonella engineering membrane penetrating phage, which comprises phage and a membrane penetrating peptide, wherein the phage surface is provided with 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.

Specifically, the invention screens out the protein suitable for fusion expression with the exogenous protein in the salmonella typhimurium phage structural protein, and allows the phage surface to fuse and express cell penetrating peptide, thus obtaining engineering penetrating phage capable of effectively killing salmonella typhimurium in cells, and providing a new idea for clinical salmonella typhimurium in cells.

After analysis of phage display to find that proteins containing Ig-like domains can be used as potential sites for phage display, phage selz GP94 protein is further speculated to be useful for phage display:

phage selz of salmonella typhimurium SL7207, which shows that GP94 (amino acid sequence see table 2) contains an Ig-like domain (table 1). Further use of AlphaFold2 to predict GP94 structure shows that the protein contains 3 domains, 2 of which are Ig-like domains (see figure 1).

Fig. 2 is an AlphaFold2 predictive average local distance difference test score (LDDT). These scores are a measure of the amino acid position that alpha fold2 predicts. At the same time, comparing this structure with the protein structure database (PDB) using DALI server, it showed high similarity of GP94 with three phage structural proteins, including the hoc protein of phage RB49, the tail tube protein of phage T5 (pb 6) and the tail tube protein of phage lambda (gpV) (see table 3). In fact, the hoc protein of phage RB49 and the gpV protein of phage lambda have been reported for phage display. Thus, GP94 is presumed to have the potential for phage display.

TABLE 2 amino acid sequence of the GP94 protein of the selz phage

DALI alignment analysis results for Table 3, GP94 domain 2 (amino acids 92-171) and domain 3 (amino acids 172-269)

Wherein, rmsd: the root mean square deviation of carbon atoms in the least square superposition of carbon atoms of the equivalent structure; lali: number of equivalent structural amino acids; % id: the percentage of identical amino acids in all equivalent structures.

That is, in the engineered transmembrane phage, the surface protein of the phage surface is GP94 protein.

In some embodiments, the transmembrane peptide comprises one of HA-TAT, cTAT, intergrin, R8, R7, P8, and tranportan, among others. Preferably, the membrane penetrating peptide is HA-TAT.

Specifically, the invention screens out the protein suitable for fusion expression with foreign protein, namely GP94 protein containing Ig-like structural domain, from structural protein of salmonella typhimurium phage by bioinformatics method, then allows the salmonella typhimurium phage surface to fuse and express cell penetrating peptide, thus obtaining engineering phage capable of killing salmonella typhimurium in cells with high efficiency, improving the titer of salmonella typhimurium phage entering cells, and expanding phage therapy to treatment of intracellular infection.

The invention also provides a construction method of the salmonella engineering membrane penetrating phage, which comprises the following steps:

s1: constructing a recombinant pDONOOR plasmid containing homology arms, CPP, G4S Linker and SgRNA;

s2: the spCas9 plasmid and the recombinant pDOor plasmid are electrically transferred into a host salmonella to obtain a host bacterium containing double plasmids;

s3: propagating the wild phage on the host bacteria containing the double plasmids for the first generation, and carrying out homologous recombination to obtain the engineering membrane penetrating phage.

According to the invention, the phage surface proteins of salmonella typhimurium are screened in a high throughput manner by a bioinformatics method, target proteins can be obtained quickly and efficiently, and cell penetrating peptides are expressed by fusion of the screened phage surface proteins, so that the titer of phage entering cells is improved, and phage therapy is expanded to the treatment of intracellular bacterial infection.

The invention also provides application of the salmonella engineering membrane penetrating phage in preparing medicines for treating intracellular infection salmonella.

In some embodiments, the salmonella is salmonella typhimurium.

The invention also provides a medicine for treating salmonella infection in cells, which comprises the salmonella engineering penetrating bacteriophage.

The invention is further illustrated by the following specific examples.

Example 1 validation of GP94 protein on phage selz for phage display

pDONOR plasmid linearization: the pDONER plasmid was digested with BamHI restriction enzymes, then inverse PCR was performed using primers to obtain high concentration plasmid backbone DNA, and finally PCR product detection was performed.

The PCR product detection comprises the following specific steps: preparing agarose gel composed of 1 xTAE (nucleic acid electrophoresis buffer) +1% agarose+1/1000 SYBR safe gel dye, taking 5 μl of PCR product and loading to carry out electrophoresis detection; the gel recovery kit is adopted, and E.Z.N.A is adopted. ^® Gel Extraction Kit, obtaining DNA-containing solution, measuring the concentration of recovered DNA on a Nano-drop instrument, making a mark record, and storing at-20 ℃ for a long time.

Obtaining phage gene DNA fragments containing GP94 gene: a DNA fragment containing the gene sequence expressing Avi-tag or Gold Binding Peptide (GBP) and the SgRNA sequence of 20 bp fused at the beginning of the GP94 gene was constructed by the overlap PCR method with 500 bp homology arms on the upstream and downstream of the GP94 gene.

Wherein PCR was performed using phage selz genome as template to obtain homology arms followed by overlap PCR using high fidelity polymerase 2xPhanta Max Master Mix, the primers used are shown in table 4.

TABLE 4 PCR primers used

Obtaining recombinant pDonor plasmid containing DNA fragment of GP94 gene: firstly, a one-step cloning enzyme (ClonExpress II One Step Cloning Kit) is used for obtaining a linear plasmid and a recombinant pDONER plasmid containing a DNA fragment of the GP94 gene, then a PCR reaction product is taken for sequencing and verification, then a small extraction kit (Tiangen biochemistry) is used for obtaining a recombinant plasmid solution, finally a Nano-drop instrument (micro-spectrophotometer) is used for measuring the plasmid concentration, and a plasmid concentration record is made for long-term storage at-20 ℃.

Obtaining host bacteria containing double plasmids: the spCas9 plasmid and the recombinant pDOor plasmid prepared in the step (3) are electrotransferred into a host salmonella SL7207 through electrotransfer.

The method comprises the following specific steps: 200ng of spCas9 plasmid and 200ng of recombinant pDONOR plasmid were added to host Salmonella SL7207 competent cells (log phase bacteria, 10% glycerol treatment, -80℃for preservation), then to an electroreformer cuvette, which was shocked at 1.8 kv, 2.5 ms, then to LB (lysis broth) preheated at 37℃and, after shaking culture at 37℃for 1 h, the corresponding resistance plates were plated. The following day, monoclonal colonies were picked and the success of the double plasmid electrotransformation was verified by PCR products.

Acquisition of salmonella engineering phage: first infecting a host bacterium generation containing double plasmids by using Wild type phage (Wild-type, WT), and then performing PCR electrophoresis and Sanger sequencing by using primers seq 94 FW and seq 94 Rev; then obtaining the engineering phage selz containing Avi-tag (GLNDIFEAQKIEWHE) after repeated purification for 3 times through double-layer plates _Avitag GBP (V)SGSSPDS) engineering phage selz _GBP 。

The method comprises the following specific steps: after passage, phage were diluted in a gradient and mixed with 200. Mu.l of log-phase host bacteria, then mixed with 5 ml of 0.5% LB agar, immediately poured into 1.5% LB solid agar plates, then dried, incubated overnight at 37℃to form individual plaques, the individual plaques were picked the next day and dissolved in 50. Mu.l of SM buffer, and after mixing uniformly, 0.5. Mu.l of the buffer was used as a PCR reaction template for PCR verification of correct band size using primers seq 94 FW and seq 94 Rev, the procedure was repeated 3 times, and Sanger sequencing was performed to obtain purified engineered phage.

Engineering phage biotin assay: biotinylation of phage was performed according to Avi tag protein biotin labelling kit (BirA method) instructions, followed by in vitro biotinylation of high purity phage selz, respectively _Avitag (after purification by CsCl density gradient ultracentrifugation) and selz were subjected to western-blot detection.

The method comprises the following specific steps: the 5x SDS-PAGE buffer and high purity phage (> 10) are first prepared with 4-12% high resolution gradient pre-gel ¹¹ PFU) is mixed in a ratio of 1:4, and the metal bath is boiled for 10 min at 100 ℃ and balanced to room temperature; then 5 mu l of Marker and 20 mu l of sample are added into SDS-PAGE gel holes, and the samples are slowly injected along the hole walls, so that the overflow of the samples and the mutual pollution are avoided; then adopting electrophoresis conditions of 80V and 30 min to press the samples on the same plane, changing the electrophoresis conditions to 120V and 60 min to the bottom of the gel when the samples run to the junction of the concentrated gel and the separation gel; after the PVDF film is activated by using absolute methanol for 1 min, the cotton-PVDF film-prefabricated glue-cotton soaked by the transfer molding liquid is vertically aligned in sequence by adopting a sandwich mode, air bubbles are emptied, and parameters of constant pressure 15V, 1 mA and 10W are set by adopting a semi-dry transfer mode, so that semi-dry transfer operation is carried out for 15 min; the PVDF membrane after the transfer was then washed 3 times with PBST solution for 5min each. Then incubated with 5% BSA at 37℃for 2 h; then incubating the PVDF membrane with horseradish peroxidase labeled streptavidin at a ratio of 1:2000 at 37 ℃ for 2 h; finally, after 3 times of PBST washing, the mixture was washed in dark according to the formula 1: the ECL developer was mixed in a ratio of 1, completely covered with PVDF film, and developed using an imager after 3 min.

Engineering phage nano-gold particle binding verification: csCl density gradient ultracentrifuge is first used to purify high purity high concentration selz _GBP And selz phage with gold nanoparticles at room temperature at 1: 10, mixing reaction 5 h; then placing the carbon surface of the copper mesh with the film carbon carrier downward in 20 μl phage mixture solution for reaction for 20 min, washing 3 times in PBS, and then negatively staining with 20 μl 2% PTA for 1 min; finally, the sample was observed by using a HITACH HT7700 transmission electron microscope.

Detection of high concentration high purity in vitro biotinylated selz by Western-Blot _Avitag And selz, the results show that the Avi-tag fused engineering phage selz _Avitag The Avi tag protein biotin labeling kit can be successfully utilized for in vitro biotinylation, and protein bands with the same size as GP94 (28 KD) can be detected by horseradish peroxidase-labeled streptavidin reaction, as shown in figure 3, but no obvious bands exist on wild phage selz, so that the GP94 is not only structural protein, but also phage display of the Avi tag can be successfully realized. It was further observed by transmission electron microscopy that the gold nanoparticles could be specifically fused to the engineered phage selz fused to GBP _GBP In response, and under electron microscopy, gold particles were observed to appear predominantly at the fiber sites of phages, as shown in FIG. 4, whereas the wild-type phage group had no apparent gold particles. The GP94 protein of phage selz was validated for exogenous peptide display in combination with the above results.

EXAMPLE 2 construction of Salmonella engineered transmembrane phage

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

The CPP display engineering transmembrane phage construction method is the same as in example 1: firstly, constructing a recombinant pDONOOR plasmid containing a homology arm, a CPP sequence, a G4S Linker and SgRNA; after verifying the recombinant plasmid sequence by Sanger sequencing, the spCas9 plasmid and the recombinant pDONER plasmid are electrically transferred into a host salmonella SL 7207; and then propagating the wild phage on a host salmonella SL7207 carrying a pDOOR plasmid of the target fragment and a spCas9 plasmid for one generation, and carrying out homologous recombination under the pressure of a Cas9 system to obtain the CPP display engineering phage. Finally, the presence of recombinant phage was verified using primers seq_94_fw and seq_94 Rev. Purifying for 3 times to obtain the modified CPP display engineering membrane penetrating phage. All primer sequences used are detailed in Table 6. TABLE 5 CPP and Linker amino acid sequences involved

TABLE 6 PCR primers used

/>

Example 3 functional assay of engineered transmembrane phage

Hela, A549 and Caco2 cell lines are selected, and engineering membrane penetrating phage obtained through the construction of the example 2 is screened. Wherein, hela, a549 and Caco2 cell culture medium is dmem+10% fetal bovine serum (Fetal bovine serum, FBS) +green streptomycin. Changing the liquid of the cell culture medium every 2 days, and carrying out cell passage after the cell growth is converged to about 80%, and digestion by 0.25% pancreatin; all cells were incubated at 37℃in 5% CO ₂ Culturing; when the growth of different types of cells is converged to 80%, 1.5x10 is added respectively ⁹ PFU/ml various engineering transmembrane phages; wild-type phage was used as a control group, the supernatant was aspirated after incubation for 4 hours, extracellular phage was removed after repeated 4 times of washing with PBS, and ddH was added ₂ O lyses cells and intracellular functional phage titer assays were performed, each set of 3-4 secondary wells, repeated 2 times. Engineering membrane penetrating phage with high-efficiency membrane penetrating capacity is primarily screened out through the experiment.

Further, after incubation of phage with cells 4h, double-plate spot counting was performed. Phage selz _HA-TAT 、selz _Transportan 、selz _Intergrin And selz _R7 Uptake in a549 cells was significantly higher thanWild type phage (P)<0.05 A) is provided; at the same time, selz was observed in HeLa cells _HA-TAT (P<0.0001 And selz) _Transportan (P<0.01 Increased uptake, intracellular functional phage selz was also observed in Caco-2 cells _HA-TAT Concentration was significantly higher than wild-type phage (P<0.001 The specific differences are shown in fig. 5.

Notably, in all the tested epithelial cells, selz _HA-TAT Shows the optimal membrane penetrating efficiency which is 10-30 times higher than that of wild phage, especially in A549 cells, selz _HA-TAT The efficiency of cell entry is improved by 29 times compared with the wild type. The results indicate that CPP display of the GP94 protein of phage selz is feasible to improve phage entry efficiency.

Example 4 selz _HA-TAT In-vitro intracellular sterilization verification of engineering membrane-penetrating phage

(1) Salmonella typhimurium SL1344 (mCherry-SL 1344) containing plasmid pBBR1MCS-Tac-mCherry was selected as host bacteria for inoculation of 5X10 ⁴ Cells were cultured in 12 well plates, 24 h were incubated with Salmonella typhimurium mCherry-SL1344 having an MOI of 10 for 12 h; repeatedly washing and killing extracellular salmonella with high-concentration gentamicin (100 mug/ml) PBS for daily use, and then respectively containing 1.5x10 ⁹ PFU/ml engineering transmembrane phage selz _HA-TAT And wild-type phage selz, 1% FBS-containing cell culture medium (10 μg/ml plus low concentration gentamicin) was incubated with cells for 4h, with PBS added as negative control; after 4h of phage treatment, PBS was repeatedly washed 3 times to thoroughly wash off extracellular phage and bacteria, using ddH ₂ O is incubated for 5-10 min, and the bottom of the orifice plate is repeatedly scraped by a gun head and blown and evenly mixed to fully lyse cells, and then the bacterial colony Count (CFU) and phage titer (PFU) are counted by plate plating.

As shown in FIG. 6, in 3 different models of epithelial intracellular infection, the phage sel z was engineered to pass through the membrane _HA-TAT After treatment, in Hela cells, CFU results showed phage-induced selz _HA-TAT After treatment, intracellular bacterial colony count was significantly lower than in the wild-type treated group and untreated group (P<0.5 (see fig. 6 a); intracellular infection in A549 cellsSelz in model _HA-TAT The intracellular bacterial colony count was significantly reduced in the treated group compared to the untreated group (P<0.5 No significant difference in trend of decrease for wild type treatment group (P)>0.5 (see B in fig. 6); phage selz in Caco-2 cells _HA-TAT The treated group had a decreasing trend in bacterial colony count relative to the wild-type phage treated group and untreated group but was not statistically significant (P>0.5 (see C in fig. 6); while wild type phage were not significantly different after treatment with the 3 epithelial cell intracellular infection model than the untreated group (P)>0.5). As shown in FIG. 7, in the intracellular functional phage titer assay corresponding thereto, phage selz was found in almost all epithelial cells _HA-TAT The treatment group was significantly elevated relative to the wild-type phage treatment group (P<0.5)。

(2) Engineering membrane penetrating phage selz _HA-TAT The step of treating the salmonella typhimurium infection in vitro by the wild phage selz is the same as (1). After phage treatment for 4h, the cells were repeatedly washed 3 times with pre-chilled gentamicin-containing PBS, then digested with 0.25% pancreatin, resuspended 3 times with 4℃pre-chilled PBS, and immediately subjected to flow analysis, and the fluorescence intensity between groups was compared to indirectly reflect intracellular bacterial numbers.

As shown in FIG. 8, it can be observed that the phage selz was penetrated by a membrane in engineering _HA-TAT The mean fluorescence intensity (Mean fluorescence intensity, MFI) of the cells of the treated group was significantly lower than that of the wild-type treated group and the untreated group (P<0.5)。

The above results indicate that the engineering membrane penetrating phage selz constructed by the invention _HA-TAT Can obviously enter epithelial Hela cells, has the capability of controlling the infection of salmonella typhimurium, and has potential for treating the infection of intracellular pathogenic bacteria.

In conclusion, the salmonella engineering membrane penetrating phage and the construction method and application thereof provided by the invention can rapidly and efficiently obtain target proteins by screening phage surface proteins in a high throughput manner through a bioinformatics method, and expand the application range of phage display technology; further, phage therapy is extended to the treatment of intracellular bacterial infection by increasing the titer of phage entry into mammalian cells by fusion of cell-penetrating peptides to the screened phage surface proteins.

It is to be understood that the invention is not limited in its application to the examples described above, but is capable of modification and variation in light of the above teachings by those skilled in the art, and that all such modifications and variations are intended to be included within the scope of the appended claims.

Claims

1. The salmonella engineering membrane penetrating phage is characterized by comprising a phage and a membrane penetrating peptide, wherein the surface of the phage is provided with 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;

the surface protein is GP94 protein, and the amino acid sequence of the GP94 protein is MPTITVLVAPEVVRNKPETERNHVVTGVAKGWQKTSLNQDPDEILTECKGLDALLTKSNLQADGVTKVDPTKPIGFQVSYEIHDPNAILTTGLVITPATASGEIGQFVELLATVSPANATYQGVNWYSGDLTKAIHIGGGKFKLLQSGSVTVYGVTVEGNHTDSTVITIAGLLSLTTDLAASQDVADGADATFTIVAAGGTTPYSYAWYYSDTPGGEGVVIDAGVNPTAATASLVNHAVTAASEGEYWCVVEDADGHSVTSTRCELAVV;

the membrane penetrating peptide is HA-TAT, and the amino acid sequence of the HA-TAT is GDIMGEWGNEIFGAIAGFLGYGRKKRRQRR.

2. Use of the salmonella-engineered, transmembrane bacteriophage of claim 1 in the manufacture of a medicament for treating intracellular infection with salmonella;

the cells are Hela cells;

the salmonella is Salmonella typhimurium.

3. A medicament for treating salmonella infection in a cell, comprising the salmonella engineering transmembrane phage of claim 1;

the cells are Hela cells;

the salmonella is Salmonella typhimurium.