Cell surface mass display technology using extracellular membrane lipoprotein PrsA display system

Abstract

The present invention relates to a carrier system for displaying a cell surface of a protein of interest comprising a lactic acid bacteria extracellular membrane protein PrsA having a function of displaying an anchoring motif on the cell surface, a sequence encoding the PrsA, and a potent promoter present on the upper part of the PrsA gene.

Description

Technical Field

[0001] The present invention relates to a vector for cell surface display, which, for the purpose of displaying a target protein on the cell surface, comprises a lipoprotein PrsA present in the outer membrane of lactic acid bacteria cells, its derivatives, a PrsA promoter for inducing a large amount of display of PrsA, and a base sequence encoding the PrsA. Background Art

[0002] A cell surface display system displays a protein or peptide on the cell surface and exposes it to the outside of the cell. This system allows the protein or peptide labeled on the cell surface to freely expose to the extracellular space. Labeling foreign proteins on the cell surface has attracted attention in the fields of bioengineering and industrial applications such as vaccine development, whole cell biocatalysis, bioabsorbants, and biosensors.

[0003] If a protein is to be displayed on the cell surface, a secretion signal that enables the protein biosynthesized intracellularly to pass through the cell membrane should be present in the primary sequence of the protein. Also, the surface display system varies depending on the type of cell on which a target protein, enzyme, peptide, etc. is to be displayed. In the case of bacteria, a protein synthesized in the cytoplasm or near the cell membrane should have a signal sequence in order to be inserted into the cell membrane or secreted to the outside. In particular, in the case of Gram-negative bacteria, it should be arranged in such a way that it can be inserted through the inner cell membrane and the intermembrane space and attached to the outer cell membrane so as to protrude outside the membrane. For this purpose, a secretion signal and a targeting signal for inserting into the cell surface are required, which are present in outer membrane proteins and enzymes or toxin proteins secreted to the outside of the cell.

[0004] There are four types of anchor proteins in the thick cell wall of Gram-positive bacteria such as lactic acid bacteria that can immobilize foreign proteins. The types are: (i) transmembrane anchor; (ii) lipoprotein anchor; (iii) LPXTG anchor; (iv) LysM-repeat anchor (Boekhorst et al. Microbiology, 2006, 152: 3175 - 3183; Michon et al. Microb Cell Fact. 2016, 15: 70).

[0005] Transmembrane anchor proteins, which belong to the common membrane protein types of Gram-positive and Gram-negative bacteria, form an anchor by inserting 20 to 30 amino acids at the N-terminus or C-terminus composed of hydrophobic amino acids into the cell membrane in an α-helix structure. When abundantly presented in the cell membrane, it has a considerable impact on the rigidity of the membrane, so there is a limitation that it cannot be abundantly presented. The second type of lipoprotein anchor protein exists on the cell surface by a mechanism in which the -SH group of the cysteine residue, where the secretion signal exists behind, is covalently linked to the carbon atom of the glycerol head of the lipid that is a cell membrane component. Such lipoprotein anchor proteins are different from the transmembrane anchor proteins described above. Instead of inserting into the cell membrane to affect the membrane rigidity, they exist in the form of hanging on the lipid bilayer, so they have the advantage of being able to be abundantly presented on the cell surface.

[0006] On the other hand, in most cases such as Gram-positive and Gram-negative bacteria or yeast, the biggest obstacle in the commercialization of vaccine development and biocatalyst development using the microbial surface display system is the difficulty in abundantly presenting the target protein on the cell surface. In particular, through molecular biology research over the past fifty-odd years, the development of an abundant display system using changes in the base sequence of promoters in the display system or derivatives (inducers) that bind to repressors, and the development of microbial hosts that can stably present the target protein in the cell through specific gene manipulation or removal of specific genes in the chromosome of a specific bacterium used as a carrier have been carried out. However, except for specific industrial strains including Escherichia coli, it can be said that there is still no artificial technology that can present more than 1% of a target protein in the total protein mass per microorganism. At the same time, a system that can abundantly present on the cell membrane rather than in the cytoplasm or abundantly present on the outer cell membrane can be said to be in a blank state. In the commercialization of the cell surface display technology of biocatalysts used in biotransformation engineering, the crucial core lies in the amount of the enzyme presented on the surface and the stability of the enzyme. Especially in vaccine development, due to side effects such as inflammation, the amount of bacteria that can be administered into the body is limited. When the total amount of antigens that can be provided is limited relative to the amount of microbial administration bacteria, the induced immune response may be insufficient in disease defense. Therefore, the amount of cell surface display of the target antigen can be said to be an important core matter.

[0007] Worldwide, lactic acid bacteria widely used in fermented foods containing lactic acid bacteria have long been recognized as safe (GRAS, Generally Recognized As Safe) microorganisms. Therefore, various studies have been carried out using GRAS microorganisms that have almost no toxic side effects and can minimize the possibility of safety problems.

[0008] On the other hand, Korean Patent Publication No. 2019-0037481 discloses a "method for cell surface display of target proteins using a cell-fixed matrix derived from coryneform bacteria", and Korean Patent Publication No. 2002-0010428 discloses a "novel cell wall attachment-mediated protein isolated from yeast, its gene, and a cell surface display system using the same" using four GPI-anchored protein (glycosyl phosphatidylinositol anchor protein) genes, namely HpSED1, HpGAS1, HpTIP1, and HpCWP1, isolated from Hansenula polymorpha, a methanol-utilizing yeast. Korean Patent Publication No. 2004-0032824 discloses a "vector for surface display of a Bacillus-derived poly-γ-glutamic acid synthesis gene and a method for microbial surface display of proteins using the same" isolated from cheonggukjang. However, there is no record of the "description of cell surface mass display using the extracellular membrane lipoprotein PrsA or its variant and the PrsA promoter as lactic acid bacteria surface proteins of the present invention". Summary of the Invention

[0009] Technical Problem

[0010] The present invention was derived from the above requirements. The inventors isolated and cultured lactic acid bacteria, which are GRAS microorganisms, from kimchi, a traditional fermented food, and screened PrsA, an extracellular membrane lipoprotein that is constantly and abundantly displayed, and its promoter from the surface of GRAS lactic acid bacteria isolated by the proteolytic surface-shaving method.

[0011] The structure of the screened PrsA protein was analyzed, and a cell surface display vector operably linked to the coding sequence of PrsA wild-type, a variant with the serine-rich domain of PrsA removed, a variant with the hinge region of PrsA removed, or a variant with both the hinge region and the serine-rich domain of PrsA removed was constructed under the regulation of the PrsA promoter. After cloning a reporter gene into the vector, it was transformed into lactic acid bacteria to analyze whether the reporter protein was displayed on the cell surface. As a result, stable display of the reporter protein on the cell surface was observed in all lactic acid bacteria transformed with the vector containing the coding sequence of PrsA protein or its variant. Through this, it was confirmed that the PrsA protein and its variant of the present invention can be used as surface display anchor motifs, thus completing the present invention.

[0012] Technical Solution

[0013] To solve the above problems, the present invention provides a recombinant vector for displaying a target protein on the cell surface, which is characterized in that a polynucleotide encoding PrsA or a variant thereof consisting of the amino acid sequence of SEQ ID NO: 2 and a gene encoding the target protein are successively linked downstream of the PrsA promoter consisting of the base sequence of SEQ ID NO: 3.

[0014] Moreover, the present invention provides a microorganism transformed with the recombinant vector.

[0015] Furthermore, the present invention provides a method for displaying a target protein on the surface of a microorganism, comprising the step of transforming a microorganism with the recombinant vector.

[0016] In addition, the present invention provides a method for preparing a microorganism having a target protein displayed on the cell surface, comprising: the step of displaying the target protein on the cell surface by culturing the microorganism transformed with the recombinant vector; and the step of recovering the microorganism having the target protein displayed on the cell surface.

[0017] Also, the present invention provides a microorganism having a target protein displayed on the cell surface prepared by the above method.

[0018] Furthermore, the present invention provides an injectable preparation comprising the microorganism having a target protein displayed on the cell surface as an active ingredient.

[0019] Moreover, the present invention provides an oral preparation comprising the microorganism having a target protein displayed on the cell surface as an active ingredient.

[0020] In addition, the present invention provides a composition for inducing immunity in non-human vertebrates comprising the microorganism as an active ingredient.

[0021] Furthermore, the present invention provides a method for preparing a protein array, comprising the step of immobilizing the microorganism having a target protein displayed on the surface prepared by the method of the present invention on the surface of a substrate.

[0022] Also, the present invention provides a method for inducing immunity in vertebrates, comprising the step of administering to a vertebrate the microorganism having an antigen displayed on the surface prepared by the method of the present invention.

[0023] Effects of the Invention

[0024] The present invention uses a protein isolated from lactic acid bacteria, which are GRAS bacteria, to provide a method for stably displaying foreign proteins on the surface of lactic acid bacteria, which can be applied to existing methods for displaying foreign proteins on the cell surface that are difficult to stably display or difficult to display in large quantities on the cell surface, thus expecting high industrial utilization.

[0025] Moreover, when using the display system of the present invention to display ligand proteins, receptor proteins, or enzyme proteins related to in-vivo signal transduction of ordinary cells including their own proteins, it can be used as a therapeutic agent for metabolic diseases. When displaying ligands or receptors of immune cells, it can be used as an immunotherapeutic agent. When displaying bacteria or bacterial antigens, it can be used as a preventive or therapeutic vaccine. Thus, the display system of the present invention can be effectively utilized in the pharmaceutical industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 MS / MS results of the sequence of amino acids at positions 255 to 264 (SEQ ID NO: 6) as a representative peptide in the tandem mass spectrometry (MS / MS) analysis of 5 Lys-C cleavage peptides to be confirmed.

[0027] Figure 2 Shows the base sequence and amino acid sequence information of the PrsA protein of Lactobacillus sakei. And the hinge region (dashed underline + bold font) and serine-rich domain (serine-rich domain) (solid underline + bold font) deleted when preparing the PrsA protein derivative are marked.

[0028] Figure 3 Plasmid map of pGOSTalpha:PrsA containing the PrsA promoter sequence and the coding sequence of the PrsA anchoring motif.

[0029] Figure 4 For use in Figure 3 Results of confirming the display of PrsA from Lactobacillus sakei in Lactobacillus paracasei transformed with the pGOSTα:PrsA vector. Part A is a CBB-stained gel photograph of total proteins. Part B is the result of Western blotting using an anti-PrsA antibody after transferring the total proteins to a polyvinylidene difluoride (PVDF) membrane. Part C is a CBB-stained gel photograph of each fraction after separating the total proteins by high-speed centrifugation to separate cytoplasmic proteins and membrane proteins. The position of the PrsA-anchored membrane protein (theoretical molecular weight, 31.4 kDa) is marked with a red arrow. Figure 4 For parts A and B, lane M: Protein size marker (ExcelBang three-color broad-range protein marker, #PM2700 Thermo), lane 1: Non-transformed Lactobacillus paracasei, lane 2: Transformed Lactobacillus paracasei. Figure 4For part C, column M: Protein size marker, lane 1: Total protein, lane 2: Cytoplasmic protein fraction, lane 3: Cell membrane protein fraction.

[0030] Figure 5 For cloning the reporter gene sfGFP in the vector of Figure 3 Vector map of pGOSTa:PrsA-sfGFP.

[0031] Figure 6 Results of analyzing the protein display level by culturing Lactobacillus paracasei transformed with recombinant vectors (GOSTa:PrsA-sfGFP, GOSTa:PrsA DS-sfGFP, GOSTa:PrsA DH-sfGFP, GOSTa:PrsA WD-sfGFP in turn) that fuse the sfGFP gene to the 3' end of the PrsA, PrsA serine-rich domain deletion variant, PrsA hinge region deletion variant, or PrsA serine-rich domain and hinge region deletion variant genes. Part A is a CBB-stained gel photo, and part B is the result of Western blotting using an anti-GFP antibody.

[0032] Figure 7 A graph showing the amount of surface-displayed sfGFP compared outside the cell membrane by analyzing sfGFP displayed on the surface of Lactobacillus cells using an anti-GFP antibody through whole-cell ELISA after culturing Lactobacillus paracasei transformed with recombinant vectors (GOSTa:PrsA-sfGFP, GOSTa:PrsA DS-sfGFP, GOSTa:PrsA DH-sfGFP, GOSTa:PrsA WD-sfGFP in turn) that fuse the sfGFP gene to the 3' end of the PrsA, PrsA serine-rich domain deletion variant, PrsA hinge region deletion variant, or PrsA serine-rich domain and hinge region deletion variant genes.

[0033] Figure 8 Shows the base sequence (part A) and amino acid sequence (part B) information of the protein that fuses the mouse B7-H1 gene to the 3' end of the PrsA gene. The base sequence and amino acid sequence of PrsA are in normal font, and the sequence of mouse B7-H1 is in bold font.

[0034] Figure 9Gel photograph (Part A) of total proteins stained with CBB after culturing Lactobacillus paracasei transformed with a vector encoding a fusion protein in which the 3'-end of the PrsA gene is fused with the mouse B7-H1 gene, and the result of Western blotting using an anti-PrsA antibody (Part B). M: Protein size marker, 1: Non-transformed Lactobacillus paracasei, 2: Control group of Lactobacillus paracasei transformed with GOSTa PrsA, 3: Lactobacillus paracasei transformed with GOSTa:PrsA-mB7H1, a: Position of PrsA protein size, b: Position of PrsA-mB7H1 fusion protein size.

[0035] Figure 10 Results of measuring the anti-B7H1 antibody level in serum 4 weeks after injecting Lactobacillus paracasei transformed with the GOSTa:PrsA-mB7H1 vector into mice intramuscularly twice at 2-week intervals. Part A shows the animal experiment stage, marking the time points of intramuscular injection administration and serum collection, and Part B shows the results of analyzing anti-B7H1 antibodies in 6 mice administered with GOSTa:PrsA-mB7H1 / Lactobacillus paracasei by enzyme-linked immunosorbent assay (ELISA). Results of t-test analysis, * indicates p < 0.05, ** indicates significance of p < 0.01. Detailed implementation mode

[0036] To achieve the object of the present invention, the present invention provides a recombinant vector for displaying a target protein on the cell surface, which is characterized in that a polynucleotide encoding PrsA or a variant thereof consisting of the amino acid sequence of SEQ ID NO: 2 and a gene encoding a target protein are successively connected downstream of the PrsA promoter consisting of the base sequence of SEQ ID NO: 3.

[0037] The PrsA protein of the present invention may consist of the amino acid sequence of SEQ ID NO: 2 derived from Lactobacillus sakei, but is not limited thereto.

[0038] The scope of PrsA of the present invention includes a protein having the amino acid sequence represented by SEQ ID NO: 2 and a functional equivalent of the protein. "Functional equivalent" refers to a protein that, as a result of addition, substitution or deletion of amino acids, has a sequence homology of more than 30%, preferably more than 40%, more preferably more than 50% with the amino acid sequence represented by SEQ ID NO: 2, and shows substantially the same physiological activity as the protein represented by SEQ ID NO: 2. "Substantially the same physiological activity" refers to the activity of displaying a target protein on the cell surface.

[0039] Moreover, in the recombinant vector of the present invention, the variant of PrsA may have a deletion of residues 162 to 166 in the amino acid sequence of SEQ ID NO: 2, or a deletion of residues 281 to 303 in the amino acid sequence of SEQ ID NO: 2, or a deletion of residues 162 to 166 and residues 281 to 303 in the amino acid sequence of SEQ ID NO: 2, but is not limited thereto.

[0040] In the present invention, residues 162 to 166 in the amino acid sequence of SEQ ID NO: 2 are the hinge region of the PrsA protein, and residues 281 to 303 in the amino acid sequence of SEQ ID NO: 2 are the serine-rich domain.

[0041] In one example of the present invention, the polynucleotide encoding PrsA consisting of the amino acid sequence of SEQ ID NO: 2 may consist of the base sequence of SEQ ID NO: 1, but is not limited thereto. Moreover, homologs of the base sequence are also included within the scope of the present invention. The "%" of sequence homology with respect to a polynucleotide can be confirmed by comparison of the comparison region between two optimally aligned sequences. A portion of the polynucleotide sequence in the comparison region may include additions or deletions (i.e., gaps) compared to the reference sequence of the optimal alignment of the two sequences (excluding additions or deletions).

[0042] The term "recombinant" in this specification refers to a cell that replicates a heterologous nucleic acid, or expresses the nucleic acid, or expresses a peptide, a heterologous peptide, or a protein encoded by a heterologous nucleic acid. A recombinant cell can express a gene or gene fragment that is not found in the natural form of the cell in either the sense or antisense form. Moreover, a recombinant cell can express a gene found in a cell in its natural form, but the gene is modified and introduced into the cell by artificial means.

[0043] Moreover, the term "vector" is used when referring to a DNA fragment or nucleic acid molecule that is transferred into a cell. A vector can replicate DNA and can reproduce independently within a host cell. The term "carrier" is often used interchangeably with "vector". The term "expression vector" refers to a recombinant DNA molecule that contains a coding sequence and the appropriate nucleic acid sequences necessary for the coding sequence to be operably linked and expressed in a specific host organism.

[0044] In the present invention, the polynucleotide encoding PrsA or its variant and the gene sequence encoding the target protein can be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to a bacterial plasmid, phage, yeast plasmid, plant cell virus, virus, mammalian cell virus or other vectors. Generally, any plasmid and vector that can replicate and be stabilized in a host cell can be used. An important characteristic of the expression vector is that it has an origin of replication, a promoter, a marker gene and translation control elements.

[0045] An expression vector containing the polynucleotide encoding PrsA or its variant, the gene sequence encoding the target protein and appropriate transcriptional / translational regulatory signals of the present invention can be constructed by methods well-known to those of ordinary skill in the art to which the present invention pertains. Such methods include in vitro recombinant DNA technology, DNA synthesis technology and in vivo recombinant technology, etc. In order to direct the synthesis of mRNA, the DNA sequence can be effectively ligated to an appropriate promoter in the expression vector. And the expression vector can contain a ribosome binding site as the translation start site and a transcription terminator.

[0046] The term "polynucleotide" in this specification is a polymer of nucleotides in which nucleotide monomers are covalently linked into a long chain, and is a DNA or RNA strand having a length above a specified length. More specifically, it can be a polynucleotide fragment encoding the variant.

[0047] Moreover, in the recombinant vector of an example of the present invention, the polynucleotide encoding PrsA or its variant can be operably linked downstream of the PrsA promoter consisting of the base sequence of SEQ ID NO: 3, but is not limited thereto.

[0048] In this specification, "operably linked" means that one nucleic acid fragment is combined with another nucleic acid fragment, and its function or expression is affected by the other nucleic acid fragment. That is, the polynucleotide encoding PrsA or its variant can be linked thereto in such a way that its expression can be regulated by the PrsA promoter consisting of the base sequence of SEQ ID NO: 3.

[0049] The term "target protein" in this specification refers to a protein that those of ordinary skill in the art to which the present invention pertains can display on the cell surface of a transformed microorganism by inserting the polynucleotide encoding the protein into a recombinant vector.

[0050] In the recombinant vector of the present invention, the target protein may be one or more selected from the group consisting of ligand proteins, receptor proteins, enzyme proteins, proteins derived from viruses and bacteria, but is not limited thereto, and proteins such as hormones, hormone derivatives, enzyme inhibitors, antibodies or fragments thereof, toxin proteins, cytokines, transcriptional regulators or coagulation factors are also included within the scope of the target protein of the present invention.

[0051] The recombinant vector of the present invention is characterized in that it can display the target protein on the cell surface and is an Escherichia coli-Lactobacillus shuttle vector.

[0052] Furthermore, the present invention provides a microorganism transformed with the recombinant vector of the present invention for displaying the target protein on the cell surface.

[0053] In the present invention, preferably, the microorganism may be a lactic acid bacterium, and more preferably, it may be a lactic acid bacterium belonging to the genus Lactobacillus, but is not limited thereto.

[0054] Moreover, the present invention also provides a method for displaying the target protein on the surface of a microorganism, including the step of transforming the microorganism with the recombinant vector of the present invention for displaying the target protein on the cell surface.

[0055] In the method for displaying the target protein on the surface of a microorganism of the present invention, the recombinant vector for cell surface display, the target protein and the microorganism are as described above.

[0056] The method for transporting the recombinant vector of the present invention into the microorganism, that is, the transformation method, can be carried out by the CaCl 2 method, Hanahan method (Hanahan, D., 1983 J. Mol. Biol. 166, 557-580), conjugation (Heinze et al. BMC microbiology 2018, 18:56) and electroporation method, etc., but is not limited thereto.

[0057] Furthermore, the present invention also provides a method for preparing a microorganism with the target protein displayed on the cell surface and a microorganism with the target protein displayed on the cell surface prepared by this method. The method includes: the step of displaying the target protein on the cell surface by culturing the microorganism transformed with the recombinant vector; and the step of recovering the microorganism with the target protein displayed on the cell surface.

[0058] In the method for preparing a microorganism with the target protein displayed on the cell surface of the present invention, the recombinant vector for cell surface display, the target protein and the microorganism are as described above.

[0059] Furthermore, the cultivation of the transformed microorganism can be achieved using well-known techniques in a culture medium suitable for the production of the target protein. Suitable culture media can be purchased commercially or prepared according to the ingredients and composition ratios described in publications such as the instructions of the American Type Culture Collection, but are not limited thereto.

[0060] In the microorganism displaying the target protein on the cell surface in an example of the present invention, the microorganism can be a lactic acid bacterium displaying B7 homolog 1 (B7-H1, also known as Programmed death-ligand 1 or cluster of differentiation 274) or a fragment thereof on the cell surface, but is not limited thereto. B7-H1 (or PD-L1) is a type of immune checkpoint inhibitor and is a factor that is the main target of immune anticancer agents used in research and development to activate immune cells to attack cancer cells.

[0061] The present invention provides an injectable preparation or an oral preparation, comprising, as an active ingredient, a microorganism that uses a bacterium belonging to the genus Lactobacillus, which is a GRAS microorganism, as an antigen carrier and displays the B7-H1 or a fragment thereof as the target protein on the cell surface.

[0062] The term "injectable preparation" used in the present invention refers to a preparation suitable for injection into humans and / or vertebrates, and the injection is intradermal, subcutaneous, intramuscular or intravenous injection. Such a preparation is sterile, free of pyrogens, and has a biologically acceptable pH. The pH of the injectable preparation is particularly related to the safety and comfort during injection, and is particularly important when the preparation is supplied as a liquid preparation. Suitable preparations may contain preservatives, for example, including sodium benzoate, methyl p-hydroxybenzoate and propyl p-hydroxybenzoate, etc., and at a temperature of 25 °C, its pH can be 6.8 - 8.0. Preferably, the pH is maintained by a buffer.

[0063] Furthermore, the oral preparation can be a powder, granule, tablet, capsule, liquid medicine, suspension, emulsion, syrup or aerosol. One or more excipients can be mixed in the solid preparation for oral administration, for example, starch, calcium carbonate, sucrose or lactose, gelatin, etc., to prepare it. And, in addition to simple excipients, lubricants such as magnesium stearate and talc can also be used.

[0064] Furthermore, the present invention also provides a composition for inducing immunity in vertebrates other than humans, comprising the microorganism as an active ingredient.

[0065] The composition for immune induction of the present invention contains, as an active ingredient, lactic acid bacteria that display an antigen as a target protein on the cell surface using the recombinant vector for displaying a target protein on the cell surface of the present invention, and an immune response can be induced by administering the lactic acid bacteria to a vertebrate other than a human. Preferably, the vertebrate may be a mammal other than a human, but is not limited thereto.

[0066] The composition for immune induction of the present invention displays an antigen on the cell surface of lactic acid bacteria whose safety as a GRAS microorganism is recognized, can successfully induce immune responses by ordinary administration methods, has effective and excellent immune induction effects, and as a result, can have a disease treatment effect through immune induction.

[0067] Moreover, the present invention provides a method for preparing a protein array, including a step of immobilizing a microorganism displaying a target protein prepared by the microorganism preparation method of the present invention on the surface of a substrate.

[0068] A protein array provides a means for analyzing the presence or absence, degree of display, etc. of a desired target protein in a specific cell by arranging various proteins, especially antibodies, such as a DNA array or a DNA chip, on a solid surface. To prepare a protein array, it is necessary to ensure the proteins to be arranged and immobilize the proteins on the solid surface. During the analysis using a protein array, in order to bind to the immobilized proteins and wash away the unbound proteins, various treatments such as changes in temperature, salt concentration, and pH are performed. Therefore, immobilization of stabilized proteins that can withstand such harsh environments is required. However, cloning the genes of up to thousands or even tens of thousands of proteins into an expression vector, expressing and separating them, and then immobilizing them on a solid surface requires repeating many operations to achieve. Therefore, it is necessary to make the operations simple and rapid.

[0069] In the preparation process of the protein array of the present invention, a preparation method commonly used in the technical field to which the present invention pertains can be adopted. The protein array prepared by the method of the present invention can be used in diagnostic kits, gene expression analysis, analysis of the interaction between proteins or between a protein and a ligand, and between an antigen and an antibody, metabolic process analysis, exploration of new enzymes, combinatorial biochemical synthesis, and biosensors, etc.

[0070] Solid substrates that can be used in the present invention are glass (for example, glass exposing functional groups), Si, Ge, GaAs, GaP, SiO, SiN 4, modified silicone nitrocotton, polyvinylidene fluoride, polystyrene, polytetrafluoroethylene, polycarbonate, nylon, fiber, or a combination thereof. To immobilize proteins on the above substrate, linker molecules can also be attached, and preferably, the remaining unspotted portions are blocked. On the other hand, the amount of the surface-displaying cells of the present invention applied to each spot (or address) is determined according to the morphology of the array. The interaction between the proteins surface-displayed by the present invention immobilized on the solid substrate and the sample can be detected by utilizing the inherent properties of the proteins (e.g., immunoreactivity), or alternatively, the signal change of the labeling substance can be detected by binding an appropriate labeling substance (e.g., fluorescent substance, luminescent substance, radioactive substance, antigenic determinant) to the proteins surface-displayed. The analysis of the final result of the protein array of the present invention can be implemented using an automated device known as a "scanner" or "reader" in the technical field to which the present invention pertains.

[0071] Furthermore, the present invention also provides a method for inducing immunity in vertebrates, comprising the step of administering to a vertebrate a microorganism surface-displaying an antigen prepared by the microorganism preparation method of the present invention.

[0072] In the method for inducing immunity in vertebrates of the present invention, preferably, the vertebrate can be a mammal other than humans, but is not limited thereto.

[0073] In this specification, when it is mentioned that a certain part "comprises" a certain structural element, unless there is a particularly opposing record, it means that other structural elements can also be included, rather than excluding other structural elements. Also, in this specification, "their combination" included in the Markush format expression refers to a mixture or combination of one or more selected from the group consisting of the structural elements described in the Markush format expression, indicating the meaning of including one or more selected from the group consisting of the said structural elements.

[0074] Hereinafter, the present invention will be described in detail through examples. However, the following examples are only used to illustrate the present invention, and the content of the present invention is not limited to the following examples.

[0075] Example 1. Isolation and Screening of Lactic Acid Bacteria from Traditional Fermented Pickles

[0076] To isolate lactic acid bacteria as GRAS microorganisms from mustard pickles and Chinese cabbage pickles prepared by traditional methods in Changwon City, Gyeongsangnam-do, Korea, in 100 mL of MRS (phosphate-yeast-peptone-sodium nitrate; 6 g / L of K 2 HPO 4 、2 g / L of KH 2 PO 4, 5 g / L of Bacto tryptone, 2.55 g / L of NaNO 3 1 mL of pickled vegetable soup was inoculated into a liquid medium (containing 5 g / L of Bacto tryptone, 2.55 g / L of NaNO, and 10 g / L of yeast extract) and subjected to enrichment culture under anaerobic conditions at 30 °C for 24 hours. After diluting the obtained enrichment cultures, they were spread on MRS agar medium and cultured under anaerobic conditions at 30 °C for 48 hours. Then, the grown colonies were screened and 16S rDNA sequencing analysis was performed on them. As a result, as shown in Table 1 below, various known microorganisms with a 16S rDNA sequence homology of 99.6% or more could be isolated from each sample.

[0077] Table 1

[0078] Results of 16S rDNA sequencing analysis of microorganisms isolated from mustard pickles

[0079]

[0080] Example 2. Screening for overexpression of cell membrane proteins in Lactobacillus sakei

[0081] The strain to be used in the experiment (sample #: FT007, Lactobacillus sakei) was cultured in MRS medium (Difco Lactobacilli MRS Broth, BD) under anaerobic conditions for 24 hours, washed 3 times with 0.85% saline, and then treated with 50 mM ammonium bicarbonate (NH TM HCO 4 HCO 3, it was suspended and treated with Lysyl Endopepticase (Lys-C, Cat#NC9223464, Wako) in a buffer solution (pH 8.0) for 2 hours. Subsequently, it was centrifuged at 4500×g for 20 minutes to remove lactic acid bacteria. After purifying the peptide fragments of Lys-C, which were decomposed from the protein source present in the cell outer membrane and contained in the buffer solution as the supernatant, using a C18 SepPak cartridge (Sep-Pak C18 1ccVac, WAT054955, Waters), the Lys-C peptide fragments recovered in an 80% acetonitrile solution were lyophilized at -80°C for 24 hours. After dissolving the dried peptide pool in 20 μl of ammonium bicarbonate buffer, peptides were separated from 5 μl of it using a nano-LC (EASY-nLC1000, Thermo Fisher) liquid chromatograph equipped with a C18 column. After electrospray ionization of the separated eluate, the sequence of each peptide was analyzed using a mass spectrometer (LTQ Orbitrap Velos LC-MS / MS, Thermo). The amino acid sequences of the peptides identified by separation with a concentration gradient from 2% to 30% acetonitrile in a C18 reversed-phase column were used to identify cell outer membrane proteins through a MASCOT database search (Matrix Science), thereby confirming the presence of the PrsA lipomembrane protein, which is abundantly present in the cell outer membrane. Figure 1 Shows the MS / MS results of the sequence of amino acids at positions 255 to 264, which is the representative peptide among the 5 Lys-C cleavage MS / MS analysis peptide sequences confirmed as the PrsA lipomembrane protein, i.e., N-WANDQTVMAK-C (SEQ ID NO: 6).

[0082] Example 3. Cloning of the genes encoding the PrsA promoter and PrsA protein from Lactobacillus sakei

[0083] Based on the strain information and the identified protein information obtained in the above-described Example 1 and Example 2, it can be speculated that the PrsA protein is a membrane protein with a high possibility of being used as an anchor protein for cell surface display, and since it is abundantly displayed on the cell outer wall, it can be speculated that the promoter inducing the display of the PrsA protein is a very strong promoter. Therefore, the PrsA gene information of Lactobacillus sakei and the information on the promoter regulating the synthesis of the mRNA of PrsA were confirmed in the KEGG database (https: / / www.kegg.jp). The base sequence of 300 bases in the 5' upstream direction starting from before the ATG start codon of the PrsA gene was regarded as the PrsA promoter, and using the primer pair (5'-end promoter of PrsA of sFT007: 5'-aa actgca gga aat caa aac aac agc tg-3' (underlined: PstI recognition epitope, SEQ ID NO: 7)) and the 3'-end of PrsA of sFT007: 5'-ttt- tct-ata -tta tta gga tcc ttt tga tga tga ttt gac-3' (underlined: XbaI recognition epitope, SEQ ID NO: 8)), the chromosomal DNA of Lactobacillus sakei isolated and purified was used as a template, and after amplifying a 1.22 kb gene by polymerase chain reaction (PCR), it was cloned using PstI and XbaI into the lactic acid bacterium - Escherichia coli shuttle vector pFT003 (Korean Patent Publication No. 10-0469800, identical to pHCE1LB:BCA) to prepare pGOSTa:PrsA( Figure 3 ).

[0084] Example 4. Inducing the display of PrsA on the cell outer membrane of Lactobacillus paracasei

[0085] After introducing the pGOSTa:PrsA prepared in Example 3 into the Lactobacillus paracasei FT003 strain shown in Table 1 by the electroporation method, the transformed lactic acid bacterium colonies were inoculated into 15 mL of MRS liquid medium and cultured under anaerobic conditions at 30 °C for 24 hours. After recovering 5 mL of the culture solution, it was washed 3 times in 0.85% physiological saline, and then 20 μg of the total protein recovered after being disrupted by a bead beater was electrophoresed in a 12.5% sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) to perform CBB (coomassie brilliant blue) staining( Figure 4Part A). Also, after transferring the protein bands separated by different sizes in SDS-PAGE to a PVDF membrane, Western blotting was performed using an anti-PrsA antibody. As a result, the exact position and size of the PrsA protein could be confirmed, and the display amount of the PrsA protein in the total protein could be confirmed ( Figure 4 Part B).

[0086] Generally, methods for stably and abundantly displaying target proteins by genetic engineering in lactic acid bacteria are not common. But as Figure 4 shown, by CBB staining and Western blotting using a PrsA-specific antibody, it was simultaneously confirmed that the PrsA promoter from Lactobacillus sakei did not compete with the PrsA promoter present in the chromosome of Lactobacillus paracasei in the Lactobacillus paracasei host cell, but could stably and abundantly display the PrsA from Lactobacillus sakei ( Figure 4 Part A and Figure 4 Part B). In particular, it could be confirmed that the anti-PrsA polyclonal antibody (KOMA#24462) prepared using the purified PrsA protein from Lactobacillus sakei did not cross-react with the PrsA from Lactobacillus paracasei (the amino acid sequence homology between the PrsA protein from Lactobacillus sakei and the PrsA protein from Lactobacillus paracasei was 54%, and the similarity was 71%). It could be confirmed that the PrsA protein from Lactobacillus sakei biosynthesized in the pGOSTa:PrsA surface display system accounted for more than 3% of the total proteins displayed in Lactobacillus paracasei.

[0087] Next, to confirm whether the PrsA protein from Lactobacillus sakei that had been confirmed to be abundantly displayed was present in the cell membrane of Lactobacillus paracasei, a total protein solution with a concentration of 1.5 mg / mL broken using a bead mill was centrifuged at 20,000×g at 4°C for 4 hours, and then the supernatant was transferred to a new test tube, and the precipitate was redissolved using 0.5 mL of PBS. After mixing 8 μl of each protein solution containing the total protein with 2 μl of 5X SDS-PAGE sample buffer and heat-treating at 97°C for 5 minutes, electrophoresis was performed in SDS-PAGE for CBB staining. As a result, as Figure 4 shown in C, the 31.4 kDa PrsA protein from Lactobacillus sakei in the total protein sample in lane 1 was not found in the cytoplasmic solution in lane 2, but was found in the cell membrane protein solution in lane 3. Therefore, it could be confirmed that most of the PrsA protein abundantly displayed in the GOSTa:PrsA vector was present in the cell membrane of Lactobacillus paracasei.

[0088] Example 5. Preparation of PrsA-sfGFP fusion protein expression vector (pGOSTa:PrsA-sfGFP)

[0089] The sfGFP gene cloned into the pQI30 vector was amplified by PCR using the forward primer sfGFP BamHI (5'-aaa aggatcc atgag caa agg aga ag-3' (underlined: BamHI recognition site, SEQ ID NO: 9)) and the reverse primer sfGFP KpnⅠ (5'-tct tgg tacc tt tgt aga gct cat cca-3' (underlined: KpnⅠ recognition site, SEQ ID NO: 10)). After treatment with BamHI and KpnI, the pGOSTa:PrsA vector prepared in Example 3 was treated with the same restriction enzymes, and then two DNA fragments were ligated to prepare pGOSTa:PrsA-sfGFP( Figure 5 ).

[0090] Example 6. Analysis of PrsA structure and construction of a fusion surface display system of PrsA modified anchor motif and sfGFP

[0091] The PrsA protein forms a structure firmly fixed to the cell membrane by covalently linking the cysteine at the 21st amino acid at the N-terminus to the carbon in the glycerol molecule, which is the main structural component of the cell membrane. The C-terminus of PrsA forms a structure of the NC domain close to the N-terminus. Moreover, the PrsA protein has a single dimer structure, and the structure of the dimer has a loop shape with the N-terminus and C-terminus approaching each other. It is also judged that the serine-rich domain present at the C-terminus of each PrsA plays an important role in the formation of the NC domain structure, and it is speculated that it interacts with the peptidoglycan layer, which is a component of the cell wall structure of Gram-positive bacteria, and plays a role in maintaining structural stability within the cell wall.

[0092] The core of surface display on lactic acid bacteria is to maximize the display of specific proteins outside the outer membrane rather than inside the peptidoglycan layer. Therefore, the present inventors judged that it is possible to remove 23 amino acid residues (DLSDILSSYGVNAKKSSAKSSSK, SEQ ID NO: 4) of the serine-rich domain of PrsA Figure 2)to more effectively transport foreign proteins to the outside of the cell wall than the PrsA wild type. To remove the serine-rich domain of the PrsA protein, using the pGOSTa:PrsA vector prepared in Example 3 as a template, the 5'-terminal promoter primer of PrsA of sFT007 (SEQ ID NO: 7) and the primer for deleting the C-terminal serine-rich domain of PrsA (delete C-term serine-rich domain of PrsA) (5'-ggg ggg tac ctt atc aggatcc TT TAT CCT TGA TTG TTA CGT CGG C-3' (underlined: BamHI recognition site, SEQ ID NO: 11)) were used for PCR, and the amplified 1.4 kb DNA fragment was recovered by treating with restriction enzymes PstI and BamHI. The DNA fragment treated with the restriction enzyme was ligated to the 7.25 kb vector DNA fragment recovered after treating pGOSTa:PrsA-sfGFP prepared in Example 5 with the same restriction enzymes PstI and BamHI to obtain pGOSTa:PrsA DS-sfGFP.

[0093] As shown in the content related to the structure of PrsA described above, it is known that PrsA has a bent structure with the N-terminus and C-terminus approaching each other. In the present invention, after confirming through structural analysis that the hinge region is located in the middle position of the PrsA protein sequence, it was judged that the "KDNST" amino acid sequence (SEQ ID NO: 5, Figure 2 ) which is speculated to play a core role in the hinge region could be removed to effectively transport a specific protein fused to PrsA to the outside of the cell wall. To remove the 5 amino acids in the hinge region, using the pGOSTa:PrsA-sfGFP vector obtained in Example 5 as a template, the hinge deletion forward primer (5'-ccc c ctcgagaag aa gta ctc aac aga t-3' (underlined: XhoI recognition site, SEQ ID NO: 12)) and the hinge deletion reverse primer (5'-ccc c ctcgag ct taa gtt cag aaataa c-3' (underlined: XhoI recognition site, SEQ ID NO: 13)) were used for PCR, and then the 8.45 kb PCR product was recovered. After treating the recovered PCR product with the restriction enzyme XhoI, pGOSTa:PrsA DH-sfGFP was obtained by self-ligation.

[0094] The PrsA WD variant that simultaneously removes the serine-rich domain and hinge region of PrsA uses the pGOST:PrsADS-sfGFP plasmid as a template and uses the hinge deletion full sequence forward primer (5'-ccc cct cga g aa gaa gta ctc aac aga t-3' (underlined: XhoI recognition site, SEQ ID NO: 14)) and the hinge deletion full sequence reverse primer (5'-ccc c ct cga g ct taa gtt cag aaa taa c-3' (underlined: XhoI recognition site, SEQ ID NO: 15)) for PCR. After treating the amplified 7.27 kb DNA fragment with the restriction enzyme XhoI, pGOST:PrsA WD-sfGFP was obtained by performing self-ligation.

[0095] Example 7. Confirmation of Lactobacillus cell surface display of sfGFP fused with PrsA modified anchoring motif

[0096] After introducing four plasmids obtained from pGOST:PrsA-sfGFP, pGOST:PrsA DS-sfGFP, pGOST:PrsA DH-sfGFP, and pGOST:PrsA WD-sfGFP into Lactobacillus paracasei isolated from kimchi by electroporation, the four transformed Lactobacillus paracasei strains were cultured anaerobically in MRS liquid medium for 24 hours. After recovering 5 ml of the four Lactobacillus paracasei cultures, they were washed three times with 0.85% saline and then disrupted using a bead mill to obtain total proteins. After quantifying the total proteins of each Lactobacillus paracasei by the bicinchoninic acid (BCA) method, 20 μg of the total proteins were electrophoresed on two pre-prepared 12.5% SDS-PAGEs to separate the proteins by size. After electrophoresis, one polyacrylamide gel (PAGE) was stained with CBB, and the bands on the other gel were transferred to a PVDF membrane and then Western blotting was performed using an anti-sfGFP antibody (Santa Cruz Biotechnology, Cat#sc-9996).

[0097] The results are as Figure 6As shown, the CBB photographs show that the proteins of the control group and the five samples containing Lactobacillus paracasei are all equally developed in SDS-PAGE, and the Western blot photographs confirm that sfGFP fused with PrsA and PrsA variants is stably displayed in Lactobacillus paracasei. Also, at the position corresponding to the theoretical molecular weight, each variant of each fusion protein shows a slight difference. Compared with the PrsA wild-type anchor protein, the anchor proteins with the PrsA hinge region removed, the PrsA hinge region and the serine-rich domain removed, and the PrsA serine-rich domain removed show slightly different levels of surface display in turn. From the above results, it was confirmed that in lactic acid bacteria, the target protein sfGFP is stably displayed from four plasmids, pGOST:PrsA-sfGFP, pGOST:PrsA DS-sfGFP, pGOST:PrsA DH-sfGFP, and pGOST:PrsAWD-sfGFP, which contain PrsA and PrsA modified anchor motifs.

[0098] Next, the whole-cell ELISA method (Bonnie L. Elder et al., J. Clin. Microbiol. 1982, 16:141-144; Albritton et al., PLOS One 2017, 12(8):e0183101) was used to compare and analyze whether sfGFP displayed by fusion with PrsA or PrsA modified anchor motifs in the four plasmids is effectively displayed on the outer surface of Lactobacillus paracasei cells.

[0099] The results are as Figure 7 shown. The levels of sfGFP displayed on the cell surface were confirmed in the order of the anchor protein with the PrsA hinge region removed (PrsA DH), the anchor protein with the PrsA hinge region and the serine-rich domain removed (PrsA WD), the anchor protein with the PrsA serine-rich domain removed (PrsA DS), and the PrsA wild-type anchor protein (PrsA) motif. From the above results, it can be seen that the PrsA wild-type and PrsA modified anchor motifs can effectively display the target protein on the cell surface of lactic acid bacteria, and the PrsA modified anchor motif shows a better effect of displaying the target protein on the cell surface than the PrsA wild-type.

[0100] Example 8. Preparation of the surface display vector pGOSTa:PrsA-mB7-H1 and surface display

[0101] Using the MouseTagged ORF clone (ORiGENE, Cat#MR203953) of PD-L1 (CD274) (NM_021893) containing the mouse B7-H1 gene as a template, and using the mouse B7-H1 forward primer (5'-AAA GGA TCC GAC TTG TAC GTG GTG GAG-3' (underlined: BamHI recognition site, SEQ ID NO: 16)) and the mouse B7-H1 reverse primer (5'-GGG TCT AGA ACT AGT GTC GAC TTA GTT GAT TTT GCGGTA TGG GGC ATT-3' (underlined: XbaI recognition site, SEQ ID NO: 17)), after amplifying the mouse B7-H3 gene by PCR, treat the restriction enzymes BamHI and XbaI to recover a DNA fragment of approximately 360 bp in size. Treat the recovered 360-bp DNA fragment and the vector prepared in Example 3 with BamHI and XbaI respectively, and ligate the recovered 360-bp DNA fragment with the 7.7-kb DNA fragment to prepare pGOSTa:PrsA-mB7-H1. The nucleotide sequence of the full gene encoding the PrsA-mB7-H1 fusion protein and the amino acid sequence of the fusion protein are respectively as Figure 8 shown.

[0102] After introducing the obtained pGOSTa:PrsA-mB7-H1 plasmid into the Lactobacillus paracasei FT003 strain isolated in Example 1 by electroporation, culture the transformed lactic acid bacteria colonies by the method described in Example 4. After recovering 5 mL of the culture solution, wash it 3 times in 0.85% physiological saline and then break it using a bead mill. Electrophorese 20 μg of the recovered total protein in 12.5% SDS-PAGE and then perform CBB staining ( Figure 9 Part A). Also, transfer the protein bands separated by size in SDS-PAGE to a PVDF membrane, and perform Western blotting using an anti-B7-H1 antibody (R&D Systems, Cat#AF1019) ( Figure 9 Part B). As a result, the large display and size of the PrsA-mB7-H1 fusion protein can be confirmed. Also, the display amount of the PrsA-mB7-H1 fusion protein in the total protein can be confirmed, and it is confirmed that the display amount of the PrsA-mB7-H1 fusion protein is most stably and abundantly displayed to the extent that it can also be confirmed as a large display in SDS-PAGE CBB staining.

[0103] Example 9. Antibody response of lactic acid bacteria with surface-displayed mouse B7-H1 protein

[0104] To investigate the antigenicity of the surface proteins of the Lactobacillus paracasei FT003 strain transformed with pGOSTa:PrsA-mB7-H1 prepared in the aforementioned Example 8 and to determine whether neutralizing antibodies are formed. After subjecting the Lactobacillus paracasei displaying the antigen on its surface to a sterilization process using ethanol, it was washed 3 times with 0.85% sodium chloride solution. Six 6-week-old male BALB / c mice that were purchased at 5 weeks of age and acclimated for 1 week were each administered 1×10 7 cells of heat-killed bacteria twice at 2-week intervals by intramuscular injection. Six mice were also used for each of the 0.85% sodium chloride group as the control group and the Lactobacillus paracasei alone experimental group. Starting from the time point of the second intramuscular injection, sera of each test group were collected 4 weeks later, and neutralizing antibodies against each antigen were measured and compared by the ELISA method. The results are as shown in Figure 10 Part B of. Compared with the 0.85% sodium chloride administration group as the control group and the group administered only with the Lactobacillus paracasei FT003 lactic acid bacterium carrier, the anti-B7-H1 antibody in the group administered with the Lactobacillus paracasei transformed with pGOSTa:PrsA-mB7-H1 was higher, and statistical significance was respectively confirmed.

[0105] From the above results, it can be seen that the pGOSTa:PrsA vector of the present invention can use PrsA as a cell membrane anchor protein to serve as a platform technology for displaying various target proteins on the surface of microbial cells. It can be confirmed that when the microbial cells transformed with "pGOSTa:PrsA-antigen" are administered to animals for the purpose of using the surface-displayed target protein as an antigen, antibodies against the antigen can be induced.

Claims

1. A recombinant vector for displaying a target protein on the cell surface, characterized in that, a polynucleotide encoding PrsA or a variant thereof consisting of the amino acid sequence of SEQ ID NO: 2 and a gene encoding a target protein are successively linked downstream of the PrsA promoter consisting of the base sequence of SEQ ID NO:

3.

2. The recombinant vector for displaying a target protein on the cell surface according to claim 1, characterized in that, the variant of PrsA lacks residues 162 to 166 in the amino acid sequence of SEQ ID NO:

2.

3. The recombinant vector for displaying a target protein on the cell surface according to claim 1, characterized in that, the variant of PrsA lacks residues 281 to 303 in the amino acid sequence of SEQ ID NO:

2.

4. The recombinant vector for displaying a target protein on the cell surface according to claim 1, characterized in that, the variant of PrsA lacks residues 162 to 166 and residues 281 to 303 in the amino acid sequence of SEQ ID NO:

2.

5. The recombinant vector for displaying a target protein on the cell surface according to claim 1, characterized in that, the target protein is any one selected from the group consisting of ligand proteins, receptor proteins, enzyme proteins, and proteins derived from viruses and bacteria.

6. A microorganism, characterized in that, it is transformed with the recombinant vector for displaying a target protein on the cell surface according to any one of claims 1 to 5.

7. The microorganism according to claim 6, characterized in that, the microorganism is lactic acid bacteria.

8. A method for displaying a target protein on the surface of a microorganism, characterized in that, it includes the step of transforming a microorganism with the recombinant vector for displaying a target protein on the cell surface according to any one of claims 1 to 5.

9. A method for preparing a microorganism with a target protein displayed on the cell surface, characterized in that, it includes: a step of displaying a target protein on the cell surface by culturing the transformed microorganism according to claim 6; and a step of recovering the microorganism with a target protein displayed on the cell surface.

10. The method for preparing a microorganism with a target protein displayed on the cell surface according to claim 9, characterized in that, the target protein is any one selected from the group consisting of ligand proteins, receptor proteins, enzyme proteins, and proteins derived from viruses and bacteria.

11. A microorganism with a target protein displayed on the cell surface, characterized in that, it is prepared by the method for preparing a microorganism with a target protein displayed on the cell surface according to claim 9.

12. The microorganism with a target protein displayed on the cell surface according to claim 11, characterized in that, the target protein is B7 homolog 1 or a fragment thereof.

13. An injectable preparation, characterized in that, it contains the microorganism with a target protein displayed on the cell surface according to claim 12 as an active ingredient.

14. An oral preparation, characterized in that, Comprising the microorganism with the target protein displayed on the cell surface as described in claim 12 as an active ingredient.

15. A method for preparing a protein array, characterized in that it includes the step of immobilizing the microorganism with the target protein displayed on the surface prepared by the method for preparing the microorganism with the target protein displayed on the cell surface as described in claim 9 on the surface of a matrix.

16. A method for inducing immunity in a vertebrate, characterized in that it includes the step of administering to the vertebrate the microorganism with the antigen displayed on the surface prepared by the method for preparing the microorganism with the target protein displayed on the cell surface as described in claim 9.

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