Recombinant salmonella strain and use thereof

Abstract

The present invention relates to a spheroplast protein y (Spy)-exogenous protein-secreting Salmonella strain, which possesses a Spy protein-coding gene and has the rpoE gene deleted. The recombinant Salmonella strain according to the present invention, while exhibiting attenuated virulence due to the Spy protein, can maintain structural stability when fused with exogenous proteins such as pathogenic viruses, therapeutic proteins, or antigenic epitopes, through the structure and chaperone functions of the Spy protein. Therefore, the present invention can serve as a live attenuated vaccine. Furthermore, the present invention can be used as a protein delivery vehicle, a drug delivery vehicle, and in antigen-antibody reaction-based techniques for applications in other areas of biotechnology.

Description

Recombinant Salmonella strain and its use

[0001] The present invention relates to a novel Salmonella strain, and more particularly, to a recombinant Salmonella strain secreting a Spy outer protein and its use.

[0002] Some bacterial species have been reported to prefer proliferation and accumulation within tumors. Furthermore, bacteria possess advantageous characteristics, including the ability to simultaneously transport and express multiple therapeutic proteins and the ability to be easily eliminated by antibiotics. This suggests that bacterial therapy could be a promising new strategy for disease treatment.

[0003] Among these, regulation of gene expression is crucial for the selective delivery of therapeutic macromolecules, such as proteins and nucleic acids, using Salmonella. Salmonella possesses a Spy protein, which is expressed only within the periplasmic space, and Spy proteins have been confirmed to function as chaperones that repair damaged proteins within cells. The recently discovered Spy structure is a small, crib-shaped dimer structure that is expected to be highly flexible and is expected to function as a chaperone or carrier protein for a wide variety of substrate proteins.

[0004] However, Spy proteins are proteins that do not exist in the cytoplasm but are located only in the periplasmic space, and the technology to induce production and secretion of Spy proteins from bacteria has not yet been discovered.

[0005] The present invention aims to provide a Salmonella strain that secretes a Spy-exoprotein, which has a Spy (Spheroplast protein y) protein coding gene and a deletion of the rpoE gene.

[0006] The present invention aims to provide a protein production method comprising a step of culturing a Salmonella strain that secretes a Spy-exoprotein.

[0007] The present invention aims to provide a drug delivery system comprising a Salmonella strain secreting a Spy outer protein.

[0008] 1. A Salmonella strain that possesses the Spy (Spheroplast protein y) protein coding gene and has a defective rpoE gene.

[0009] 2. In the above 1, a strain of the genus Salmonella, which is Salmonella typhimurium, Salmonella enterica or Salmonella enteritidis.

[0010] 3. A Salmonella strain in which a genetic construct containing an external protein coding gene has been transfected into the strain in the above 1.

[0011] 4. In the above 3, the gene construct comprises at least one tag peptide selected from the group consisting of CBP (Calmodulin binding peptide) tag, His-tag (Histidine tag), Myc-tag, FLAG-tag, SUMO-tag (small ubiquitin-like modifier tag), CYD-tag (covalent yet dissociable NorpD peptide tag), HPC-tag (heavy chain of protein C tag), GST-tag (glutathione s transferase), Intein-tag, MBP-tag (maltose binding protein), and HA-tag (hemagglutinin-tag), a Salmonella strain.

[0012] 5. A strain of the genus Salmonella, wherein in the above 3, the genetic construct comprises at least one proteolytic cleavage site selected from the group consisting of a trypsin cleavage site, a thrombin cleavage site, an enterokinase cleavage site, a Factor Xa cleavage site, a collagenase cleavage site, and a TEV protease cleavage site.

[0013] 6. In the above 3, the gene construct is a strain of Salmonella genus, linked to the N-terminus, C-terminus or both ends of the Spy protein coding gene.

[0014] 7. In the above 3, the gene construct is included in a vector or inserted into the chromosome of the strain, a strain of the genus Salmonella.

[0015] 8. A Salmonella strain in which the Spy protein or the Spy-external protein bound to the external protein in the above 3 is secreted inside and outside the strain.

[0016] 9. In the above 3, the foreign protein is at least one selected from the group consisting of an antigen, an antibody, an antibody fragment, a structural protein, a regulatory protein, a transcription factor, a toxic protein, a hormone, a hormone analog, a cytokine, an enzyme, an enzyme inhibitor, a transport protein, a receptor, a receptor fragment, a biological defense inducer, a storage protein, a movement protein, an exploitative protein, and a reporter protein, a Salmonella strain.

[0017] 10. A method for producing a protein, comprising a step of culturing a Salmonella strain of any one of 1 to 9 above.

[0018] 11. A method for producing a protein, wherein the protein in the above 10 is a Spy protein or a Spy-external protein.

[0019] 12. A drug delivery system comprising any one of the Salmonella strains 1 to 9 above.

[0020] 13. In the above 12, the drug is a drug delivery system that is a Spy-exoprotein bound to a disease-treating protein or antigen epitope that is contained within the strain or secreted by the strain.

[0021] The Spy-exoprotein secreting Salmonella strain according to the present invention has reduced virulence due to the Spy protein and maintains structural stability when fused with external proteins such as pathogenic viruses, disease-treating proteins, and antigenic epitopes through the structure and chaperone function of the Spy protein, thereby providing functions such as attenuated live vaccines.

[0022] Furthermore, it can be utilized as a protein delivery vehicle, drug delivery vehicle, and antigen-antibody reaction-based technology that can be utilized in other biotechnology fields.

[0023] Figure 1 is a schematic diagram of the production of a Chromosol Spy-FLAG recombinant Salmonella strain according to one embodiment.

[0024] FIG. 2 is a diagram showing a Chromosol Spy-FLAG recombinant Salmonella strain Spy-FLAG tag fusion sequence (SEQ ID NO: 3) according to one embodiment.

[0025] Figure 3 is a diagram showing the results of an immunoblot in which a Spy protein tagged with Spy-FLAG is detected after culturing a Chromosol Spy-FLAG recombinant Salmonella strain under SPI-1 or SPI-2 inducing conditions according to one embodiment. A: Results detected in the periplasm, B: Results detected in a cell lysate and a protein sample (Secretion Protein) obtained from a culture medium from which bacteria were removed.

[0026] Figure 4 is a schematic diagram of the production of a Chromosol Spy-FLAG recombinant Salmonella strain lacking the rpoE gene according to one embodiment.

[0027] FIG. 5 is an image of a Chromosol Spy-FLAG recombinant Salmonella strain in which the rpoE gene is deleted according to one embodiment, with 180 bp from the start codon of the rpoE gene deleted and a chlorampenicol cassette inserted in that position.

[0028] Figure 6 is a diagram showing the results of immunoblotting of Spy-FLAG fusion proteins secreted into a culture medium of a Chromosol Spy-FLAG recombinant Salmonella strain lacking the rpoE gene according to one embodiment. Top: Spy-FLAG bands of the Chromosol Spy-FLAG recombinant Salmonella strain lacking the rpoE gene (rpoEspy-FLAG) and the wild type (WT) were confirmed at a concentration ratio of 1:16 for each sample amount. Bottom: Under the exposure conditions for confirming the normal band of the Chromosol Spy-FLAG recombinant Salmonella strain lacking the rpoE gene (rpoEspy-FLAG), the Spy-Flag band of the Chromosol Spy-FLAG recombinant Salmonella strain (WT spy-FLAG) was not confirmed, so a separate experiment was conducted to confirm the presence of the band.

[0029] The present invention relates to a novel Salmonella strain, and more particularly, to a Salmonella strain that secretes a large amount of Spy protein expressed in the periplasmic space, which is a space inherent to bacteria, to the outside of the bacteria, and its use.

[0030] “Spy (Spheroplast protein y) protein” is a small chaperone protein with 138 amino acids, which exists only in the bacterial domain genome, and many gram-negative bacteria, some gram-positive bacteria including Streptococcus pneumoniae, and pathogens such as Mycobacterium tuberculosis have Spy protein. Spy protein was first identified as a protein that is not present in the cytoplasm but is expressed in the periplasm when spheroplasts are formed when the cell wall of E. coli is partially removed by various stimuli.

[0031] Spy proteins are expected to contribute to the structural stabilization of many types of substrate proteins and act as chaperones or carrier proteins because they have flexibility by forming a unique folding structure with a long hairpin-like structure of four α-helices.

[0032] The present inventors expected that the Spy protein's structure and chaperone function could be used to deliver it to the outside of bacteria while maintaining structural stability when fused with external proteins such as pathogenic viruses, disease-modifying proteins, and antigenic epitopes, or to simultaneously induce antibody production by utilizing this property. They confirmed whether it was possible to create a Salmonella strain that secretes Spy-exoprotein.

[0033] Accordingly, the present inventors successfully confirmed that the Spy-FLAG protein was detected in the cell lysate and secreted into the bacterial culture medium as a result of producing and culturing a Salmonella strain in which the terminal region of the Spy gene and the CBP-TEV-FLAG tag encoding gene were fused on the chromosome of the Salmonella strain. In addition, the present invention was completed by confirming that a large amount of the Spy-FLAG protein was secreted outside the bacteria as a result of culturing a Salmonella strain in which the rpoE gene was deleted.

[0034] The present invention relates to a Salmonella strain having a Spy (spheroplast protein y) protein coding gene and a deletion of the rpoE gene.

[0035] In the present invention, the Salmonella strain may be a strain into which a genetic construct including an external protein coding gene has been transduced.

[0036] In the present invention, “gene” should be considered in the broadest sense and is not limited to a nucleic acid molecule encoding a specific amino acid sequence (polypeptide), but is interpreted to include a nucleic acid molecule encoding a polypeptide having an amino acid sequence that exhibits substantial identity to a specific amino acid sequence. Substantial identity means an amino acid sequence that exhibits at least 60% homology, more preferably at least 80% homology, and most preferably at least 90% homology, when the amino acid sequence encoded by the gene of the present invention is aligned with any other sequence to the greatest extent possible and the aligned sequence is analyzed using an algorithm commonly used in the art. Furthermore, a polypeptide having identity includes, for example, a polypeptide having an amino acid sequence in which one or more amino acids are deleted, substituted, inserted, and / or added. Such a polypeptide is composed of an amino acid sequence in which one or more amino acid residues are deleted, substituted, inserted, and / or added, and it is preferable that the number of deletions, substitutions, insertions, and / or additions of amino acid residues is small.

[0037] Two or more of the above genes may be operably linked. "Operably linked" refers to a functional connection between a nucleic acid expression regulatory sequence and a nucleic acid sequence encoding a desired protein, allowing them to perform a general function. For example, a promoter and a nucleic acid sequence encoding a protein or RNA may be operably linked to influence the expression of the coding sequence.

[0038] In the present invention, the foreign protein may be at least one selected from the group consisting of, but is not limited to, an antigen, an antibody, an antibody fragment, a structural protein, a regulatory protein, a transcription factor, a toxin protein, a hormone, a hormone analog, a cytokine, an enzyme, an enzyme inhibitor, a transport protein, a receptor, a fragment of a receptor, a biological defense inducer, a storage protein, a movement protein, an exploitative protein, and a reporter protein.

[0039] In the present invention, the genetic construct may be a nucleic acid molecule containing only the minimum elements for protein expression in a cell, and preferably may contain the components mentioned in the present invention as the minimum essential components.

[0040] In the present invention, the gene construct may include at least one tag peptide selected from the group consisting of CBP (Calmodulin binding peptide) tag, His-tag (Histidine tag), Myc-tag, FLAG-tag, SUMO-tag (small ubiquitin-like modifier tag), CYD-tag (covalent yet dissociable NorpD peptide tag), HPC-tag (heavy chain of protein C tag), GST-tag (glutathione s transferase), Intein-tag, MBP-tag (maltose binding protein), and HA-tag (hemagglutinin-tag), but is not limited thereto.

[0041] In the present invention, the genetic construct may include at least one proteolytic cleavage site selected from the group consisting of a trypsin cleavage site, a thrombin cleavage site, an enterokinase cleavage site, a Factor Xa cleavage site, a collagenase cleavage site, and a TEV protease cleavage site, but is not limited thereto.

[0042] In the present invention, the genetic construct may be one in which external protein coding genes are operably sequentially linked.

[0043] In the present invention, the genetic construct may be a recombinant vector to which a Spy protein coding gene is operably linked, but is not limited thereto.

[0044] In the present invention, the genetic construct may be, but is not limited to, a sequential peptide affinity (SPA) tag. In one embodiment, the SPA tag used was a CBP-TEV-FLAG tag represented by the base sequence of SEQ ID NO: 2.

[0045] In the present invention, the genetic construct can bind to the N-terminus, C-terminus, or both ends of the Spy protein coding gene present in a Salmonella strain, and is preferably bound to the C-terminus, but is not limited thereto. Preferably, it can bind to the C-terminus.

[0046] In the present invention, the gene construct may be incorporated into a vector or inserted into the chromosome of the strain, and preferably, may be incorporated into the chromosome. Inclusion into the vector may be accomplished by operatively linking the gene construct using a genetic recombination technique well known in the art, and site-specific DNA cleavage and linkage may be accomplished using enzymes generally known in the art. The chromosomal insertion may be accomplished using conventional homologous recombination methods, transposon-mediated gene insertion, or the Cre-LoxP recombination system.

[0047] In one embodiment, the gene construct may comprise a dual affinity tag consisting of three modified FLAG sequences (3X FLAG) and CBP, and a sequential peptide affinity tag (SPA-tag) separated by a TEV protease cleavage site. The gene construct may be introduced into the chromosome of a Salmonella strain and linked to the C-terminus of the Spy protein encoding gene.

[0048] According to one embodiment of the present invention, when a Chromosol Spy-FLAG recombinant Salmonella strain in which SPA-FLAG (CBP-TEV-FLAG tag) was introduced at the end of the Spy gene on the wild-type Salmonella typhimurium chromosome was cultured, Spy-Flag protein was detected in cell lysate and bacterial secretion.

[0049] In the present invention, the Salmonella strain can secrete a Spy protein or a Spy-exoprotein bound to an external protein inside and outside the strain.

[0050] In the present invention, the rpoE gene is a gene encoding a sigma factor (Sigma E) that promotes the initiation of transcription of a group of genes in a Salmonella strain. It is known that a Salmonella strain lacking the rpoE gene loses virulence in experimental animals (mice).

[0051] In addition, the Spy protein secreted outside the cell by the Salmonella strain according to the present invention can exhibit a virulence reduction effect of the Salmonella strain because the original function of Spy required for the virulence of the Salmonella strain is limited due to the bound foreign protein.

[0052] According to one embodiment of the present invention, it was confirmed that a Salmonella strain lacking the rpoE gene significantly increases SPA-FLAG protein secretion, and thus, it is expected that the Salmonella strain of the present invention can be used not only as a protein delivery or attenuated live vaccine strain, but also as a technology for mass production of Spy-exoprotein.

[0053] By introducing a foreign protein such as a pathogenic virus, a disease-treating protein, or an antigen epitope into a Salmonella strain according to the present invention and fusing it with a Spy protein, the structural stability of the foreign protein is maintained through the structure and chaperone function of the Spy protein, and a protein delivery system capable of delivering the foreign protein to the outside, or an attenuated live vaccine, can be provided as a functionality.

[0054] In the present invention, the Salmonella strain may be Salmonella typhimurium, Salmonella enterica or Salmonella Enteritidis, and preferably Salmonella typhimurium.

[0055] The present invention also relates to a method for producing a protein, comprising a step of culturing the aforementioned Salmonella strain. The protein may be a Spy protein or a Spy external protein.

[0056] The present invention also relates to a drug delivery system comprising the aforementioned Salmonella strain. The drug may be a disease-treating protein or an antigenic epitope bound to a Spy-exoprotein, either endogenous to the Salmonella strain according to the present invention or secreted by the strain.

[0057] The above-mentioned foreign protein may be at least one selected from the group consisting of, but not limited to, an antigen, an antibody, an antibody fragment, a structural protein, a regulatory protein, a transcription factor, a toxic protein, a hormone, a hormone analog, a cytokine, an enzyme, an enzyme inhibitor, a transport protein, a receptor, a receptor fragment, a biological defense inducer, a storage protein, a movement protein, an exploitative protein, and a reporter protein. In addition, the above-mentioned foreign protein may be a disease-treating protein or an antigen epitope.

[0058] In the present invention, the disease-treating protein may be an anticancer protein, a growth factor, a cytokine, an antibody, a chemokine receptor, or an angiogenic factor. For example, the anticancer protein may be a protein toxin, an antibody specific for a cancer antigen or a fragment of the antibody, a tumor suppressor gene, or an antiangiogenic factor. In this case, the protein toxin may be botulinum toxin, tetanus toxin, Shiga toxin, diphtheria toxin (DT), ricin, Pseudomonas exotoxin (PE), cytolysin A (ClyA), or r-Gelonin. For example, the growth factor may be a growth factor such as hepatocyte growth factor (HGF), vascular epidermal growth factor (VEGF), nerve growth factor (NGF), brain-derived nerve growth factor (BDNF), fibroblast growth factor (FGF), brain-derived neurotrophic factor (BDNF), insulin-like growth factor (IGF), transforming growth factor (TGF), platelet-derived growth factor (PDGF), bone-derived growth factor (BMP), colony-stimulating factor (CSF), epidermal growth factor (EGF), keratinocyte growth factor (KGF). For example, the cytokine may be IL-2, IL-4, TNF, IF-γ, G-CSF, GM-CSF, etc.For example, the angiogenic factor may be VEGF (vascular endothelial growth factor), angiopoietin 1 (Ang1), angiopoietin 2 (Ang2), transforming growth factor-β (TGF-β), integrin, vascular endothelial cadherin (VE-cadherin), plasminogen activator (PA), ephrin, AC-133, platelet-derived growth factor (PDGF), monocyte chemotactic protein-1 (MCP-1), fibroblast growth factor (FGF), or placenta growth factor (PIGF). The above tumor suppressor genes are genes that suppress the development of tumors, and representative examples thereof include VHL (von Hippel Lindau), APC (Adenomatous polyposis coli), CD95 (cluster of differentiation 95), ST5 (Suppression of tumorigenicity 5), YPEL3 (Yippee like 3), ST7 (Suppression of tumorigenicity 7), and ST14 (Suppression of tumorigenicity 14). The above anti-angiogenic proteins include anti-VEGF antibodies, angiostatin, endostatin, and the Kringle V domain of apolipoprotein.

[0059] In the present invention, the antigenic epitope may be derived from a therapeutic protein selected from the group consisting of antibodies, coagulation factors, and proteins used in replacement therapy. Alternatively, it may be derived from an isolated protein, or from a synthetic peptide comprising the amino acid sequence of the protein or a fragment thereof, wherein the isolated protein may be derived from a tumor, an intracellular pathogen, an autoantigen, a biological protein, an allergen, or a chronic inflammatory agent.

[0060]

[0061] Hereinafter, the present invention will be described in detail with examples. However, these examples are presented as preferred examples of the present invention and should not be construed as limiting the present invention.

[0062] Anything not described here will be omitted as it is technically feasible for those skilled in this field to infer.

[0063]

[0064] Prepare the ingredients

[0065] The Salmonella typhimurium wild type (WT) parent strain, mutant strain, plasmid and primers used for transformation used in the present invention are summarized in Table 1.

[0066] StrainsGenotypeIB1S.Typhimurium 14028s, Wild-typeIB1026Δspy:: CMIB1894Δspy:: CM / pBAD30-spyIB1930WT / ppstS-gfpIB2183Δspy:: CM / ppstS-gfpIB2184ΔssaV:: CM / ppstS-gfpIB2190Δspy:: CMsipC:: Tn5lacZYIB2191Δspy:: CMsipA:: Tn5lacZYIB2192Δspy:: CMinvF:: Tn5lacZYIB2365spy-SPAIB2702spy-SPA ΔrpoE:: CMPlasmidpBAD30Gene expression vectorpJL148Plasmid carrying the sequential peptide affinity (SPA) tagpTP233Plasmid encoding IPTG-inducible lambda red recombinaseOligonucleotidesspylinkerF-SacI: SEQ ID NO: 6R-XbaI: SEQ ID NO: 7MCS† of pBADplasmid (flanking)F: SEQ ID NO: 8R: SEQ ID NO: 9MCS† of pFPV25plasmid (flanking)F: SEQ ID NO: 10ΔrpoE::CMF: SEQ ID NO: 11R: SEQ ID NO: 12rpoE(flanking)F: SEQ ID NO: 13R: SEQ ID NO: 14spy-SPA-TFSEQ ID NO: 15spy-SPA-TRSEQ ID NO: 16spy(flanking)F: SEQ ID NO: 17R: SEQ ID NO: 18†Multi-cloning site

[0067] Luria-Bertani (LB) nutrient medium was used for strain culture. All strains used were cultured in a shaking incubator (HST, Korea) at 37°C and 220 rpm, and antibiotics were added to the medium to be used, including ampicillin (200 μg / mL, AP) and chloramphenicol (20 μg / mL, CM).

[0068]

[0069] Example 1: Recombinant Salmonella strain

[0070] 1-1. Confirmation of external secretion of Spy protein of plasmid recombinant Salmonella strain

[0071] To observe the production of Spy protein by Salmonella strains, a Salmonella strain transformed with a Spy protein expression plasmid clone was prepared using a Spy gene knockout mutant Salmonella strain.

[0072] Specifically, to create a Spy protein expression plasmid clone, the primer pair spylinker F-SacI and spylinker R-XbaI with restriction enzyme site bases were used to amplify the gene corresponding to the Spy promoter region from the purified chromosome using Salmonella Typhimurium 14028s as a template. After PCR amplification using iPFU polymerase (iNtRON), the generated PCR product was purified, digested with SacI and XbaI, and ligated to the pBAD30 plasmid treated with the same enzymes, and transformed into E. coli DH5α. The plasmids of the transformed colonies were isolated, and after confirming the base sequence of the inserted gene, they were transformed into a Spy gene knockout mutant Salmonella strain.

[0073] The parent strain and the transformed Spy gene knockout mutant Salmonella strains were cultured under conditions that induce the expression of SPI-1 and SPI-2 genes (the culture grown overnight in LB medium was diluted 1:100 in LB containing 0.3 M NaCl (LBN) and low magnesium minimum (MgM) medium [170 mM Mes(2-N-morpholino ethanesulfonic acid), 5 mM KCl, 7.5 mM (NH4)2SO4, 0.5 mM K2SO4, 1 mM KH2PO4, 8 μM MgCl2, 38 mM glycerol, 0.1% casamino acids, pH 5.8]).

[0074] StrainAccessionsProteinmol%Δspy / pSpy-#1ACY88067.1Spy6.0911Δspy / pSpy-#1ACY88926.1STM14_2476 3.9264Δspy / pSpy-#1ACY89758.1FljB3.6921Δspy / pSpy-#1ACY88172.1SseB3.4579Δspy / pSpy-#1ACY88383. 1STM14_19122.8894Δspy / pSpy-#1ACY88044.1GapA2.4271Δspy / pSpy-#1ACY90116.1Pgk2.1272Δspy / pSpy-# 1ACY91356.1HupA2.0991Δspy / pSpy-#1ACY87983.1PagC2.0616Δspy / pSpy-#1ACY88898.1STM14_24481.9616

[0075] As shown in Table 2, the LC-MS / MS analysis results confirmed that the spy gene mutant Salmonella strain transformed with the Spy protein expression plasmid clone secreted the Spy protein outside the bacteria.

[0076]

[0077] 1-2. Confirmation of external secretion of Spy protein of chromosomally recombinant Salmonella strains

[0078] To further confirm whether the recombinant Salmonella strain according to the present invention can secrete Spy protein under natural gene regulation, an external peptide SPA (Sequential Peptide Affinity) tag consisting of three FLAG tag sequences (3X FLAG) was fused to the end of the Spy gene (SEQ ID NO: 1) on the chromosome of the wild-type Salmonella strain.

[0079] Specifically, the CBP-TEV-FLAG coding gene (SEQ ID NO: 2), consisting of CBP (Calmodulin binding peptide)-TEV site (TEV protease cleavage site)-FLAG tag, was PCR amplified using a pair of primers (spy-SPA-TF and spy-SPA-TR) and the pJL148 plasmid as a template. The generated DNA product was inserted into the chromosome of Salmonella Typhimurium (WT) 14028s according to the protocol of the bacteriophage λ-mediated recombination method, thereby constructing a Chromosol Spy-FLAG (Spy-CBP-TEV-FLAG) recombinant Salmonella strain fused to the stop codon of the Spy gene (Fig. 1). The fusion sequence of the Spy-FLAG tag construct constructed on the Salmonella Typhimurium chromosome is shown in Fig. 2.

[0080] Wild-type Salmonella strains lacking the FLAG tag and Chromosol Spy-FLAG recombinant Salmonella strains were cultured under SPI-1 or SPI-2 inducing conditions. SPI-1 or SPI-2 inducing media are known to be the primary environments in which Salmonella secrete proteins necessary for animal infection. Before immunoblotting, protein samples from the cultured recombinant Salmonella strains were separated by SDS-PAGE (12%), and Spy-FLAG was detected using an anti-FLAG monoclonal antibody. Anti-RNA polymerase β-subunit was used as an internal loading control, and β-lactoglobulin was used as a method for quantitative analysis of secreted and precipitated proteins.

[0081] Looking at Figure 3A, it was confirmed that the Spy protein with a Spy-FLAG tag attached to the C-terminus was successfully expressed and localized by being detected in the periplasm of the Salmonella typhimurium strain.

[0082] In addition, looking at B of FIG. 3, Spy proteins with Spy-FLAG tags were detected in cell lysates and protein samples (Secretion Protein) obtained from culture media from which bacteria had been removed, confirming that they were secreted and localized outside the bacteria. These results confirm that the wild-type Salmonella strain introducing the recombinant technology according to the present invention can secrete Spy proteins into the extrabacterial space.

[0083]

[0084] Example 2: Confirmation of Spy protein exocytosis in rpoE-deficient mutants

[0085] A mutant lacking the sigma factor Sigma E gene, rpoE, was prepared based on the Chromosol Spy-FLAG recombinant Salmonella strain of Example 1-2 (Fig. 4).

[0086] The gene encoding rpoE (SEQ ID NO: 4) has 576 bp of bases in the Salmonella chromosome. Using a single-step gene mutagenesis method utilizing the bacteriophage λRed recombination method, the rpoE gene was deleted (SEQ ID NO: 5). From 224 bp to 180 bp from the start codon of the rpoE gene, a chlorampenicol cassette with a size of approximately 1 kbp was inserted in its place (Fig. 5). Then, PCR was performed using an external DNA primer containing the rpoE gene. As a result, the size of the Chromosol Spy-FLAG recombinant Salmonella strain was 2509 bp, and the rpoE deletion mutant strain produced a PCR product with a size of 1696 bp, confirming that the Salmonella mutant with rpoE deletion was normally created.

[0087] The results of immunoblotting of secreted proteins by culturing wild type (WT), Chromosol Spy-FLAG recombinant Salmonella strain (WT spy-FLAG), and Chromosol Spy-FLAG recombinant Salmonella mutant strain lacking the rpoE gene (rpoEspy-FLAG) are shown in Figure 6. Since there is a limitation in simultaneously showing the Spy-FLAG protein secretion amount of the relatively small Chromosol Spy-FLAG recombinant Salmonella strain (WT spy-FLAG) compared to the Spy-FLAG protein secretion amount of the rpoE gene-deficient Chromosol Spy-FLAG recombinant Salmonella strain (rpoEspy-FLAG), a separate experiment was conducted and the immunoblotting results are shown.

[0088] As a result, no Spy-FLAG band was detected in the wild type (WT), whereas Spy-FLAG bands were detected in the Chromosol Spy-FLAG recombinant Salmonella strain (WT spy-FLAG) and the Chromosol Spy-FLAG recombinant Salmonella strain lacking the rpoE gene (rpoEspy-FLAG). In particular, in the case of the recombinant Salmonella strain lacking the rpoE gene, the amount of spy-FLAG fusion protein detected was significantly increased, confirming that it was secreted more than 500 times more.

Claims

1. A Salmonella strain that possesses the Spy (spheroplast protein y) protein coding gene and has a defective rpoE gene.

2. A strain of the genus Salmonella, which is Salmonella typhimurium, Salmonella enterica or Salmonella enteritidis, according to claim 1.

3. A Salmonella strain according to claim 1, wherein a genetic construct containing a foreign protein coding gene is transfected into the strain.

4. A strain of Salmonella genus, wherein the genetic construct comprises at least one tag peptide selected from the group consisting of a calmodulin binding peptide (CBP) affinity tag, a His-tag (Histidine tag), a Myc-tag, a FLAG-tag, a SUMO-tag (small ubiquitin-like modifier tag), a CYD-tag (covalent yet dissociable NorpD peptide tag), a HPC-tag (heavy chain of protein C tag), a GST-tag (glutathione s transferase), an Intein-tag, an MBP-tag (maltose binding protein), and an HA-tag (hemagglutinin-tag).

5. A strain of Salmonella spp., wherein the genetic construct according to claim 3 comprises at least one proteolytic cleavage site selected from the group consisting of a trypsin cleavage site, a thrombin cleavage site, an enterokinase cleavage site, a Factor Xa cleavage site, a collagenase cleavage site, and a TEV protease cleavage site.

6. In claim 3, the genetic construct is a strain of Salmonella genus, linked to the N-terminus, C-terminus or both ends of a Spy protein coding gene.

7. A strain of the genus Salmonella according to claim 3, wherein the genetic construct is included in a vector or inserted into the chromosome of the strain.

8. A Salmonella strain according to claim 3, wherein the Spy protein or the Spy-external protein bound to the external protein is secreted inside and outside the strain.

9. A strain of Salmonella genus, wherein the foreign protein according to claim 3 is at least one selected from the group consisting of an antigen, an antibody, an antibody fragment, a structural protein, a regulatory protein, a transcription factor, a toxic protein, a hormone, a hormone analog, a cytokine, an enzyme, an enzyme inhibitor, a transport protein, a receptor, a receptor fragment, a biological defense inducer, a storage protein, a movement protein, an exploitative protein, and a reporter protein.

10. A method for producing a protein, comprising a step of culturing a Salmonella strain of any one of claims 1 to 9.

11. A method for producing a protein according to claim 10, wherein the protein is a Spy protein or a Spy-external protein.

12. A drug delivery vehicle comprising a Salmonella strain according to any one of claims 1 to 9.

13. A drug delivery system according to claim 12, wherein the drug is a Spy-exoprotein bound to a disease-treating protein or antigen epitope that is inherently present in the strain or secreted by the strain.

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