KR20190115262A - Nano particle comprising self-assembled ferritin structure conjugated with antigen peptide and adjuvant and use thereof - Google Patents

Abstract

The present invention relates to ferritin nanoparticles to which an antigen peptide and an immune enhancer are bound. By indirectly binding the antigen peptide or the immune enhancer to a ferritin self-assembly using an HTTcys peptide and a VL12.3cys peptide, it is possible to more easily produce fusion protein compared to a case of direct fusion of an antigen and the immune enhancer. In addition, by the same, it is possible to easily control a relative molar ratio of the antigen and the immune enhancer, thereby being able to be usefully used as a vaccine composition.

Description

Nano particle comprising self-assembled ferritin structure conjugated with antigen peptide and adjuvant and use approximately}

The present invention relates to nanoparticles in which an antigen peptide and an immune enhancer are bound to a ferritin self-assembly.

Vaccines are antigens used to actively immunize animals for the prevention of infectious diseases, or biological agents containing the antigen as an active ingredient, which was proposed by the French microbiologist L. Pasteur. In general, when a vaccine is administered, the antibody is produced in the living body, and immunization is obtained. Once the generated antibody remains in the living body for a relatively long time, even if an infection caused by the causative agent of the disease occurs, it is possible to defend against the disease. Can be.

In general, vaccine vaccines include killed vaccines used for killing bacteria, inactivated vaccines, live vaccines using live bacteria, and attenuated strains that weaken live bacteria. Attenuated vaccines, bacterial toxoids, or derivatives thereof are present.In order to develop effective vaccines, the immune response in vivo can be appropriately developed by smoothing the formation of antibodies against disease causing agents. Since it should be induced, it is desirable to develop it in a form similar to a possible disease agent. For this reason, live vaccines that use live bacteria without any treatment can have the best effect as vaccines. However, in the case of live vaccines because the disease causing agent causing disease is used alive, it is converted to a toxic strain in the future and has a risk of causing the disease, which is used only in very few infectious diseases. As an alternative to overcome these drawbacks, attenuated vaccines that have attenuated live bacteria have been developed and used.However, in this case, antigenicity is weakened compared to live vaccines, which makes them less effective as vaccines and is still safe. There is no problem. Although the bacteriophage vaccine developed due to safety in use can solve the above problems in terms of safety, the antigenicity is much lower than that of live vaccine or attenuated vaccine. Accordingly, there is a demand for the development of an ideal new vaccine formulation that is safe to use, such as live vaccine, and excellent antigenicity, such as live vaccine.

Cage proteins are proteins that can form macromolecules of tens to hundreds of times the molecular weight of a monolith by the precise self-assembly of low molecular weight monoliths. In nature, these include viral capsid proteins, ferritins, heat shock proteins, and Dps proteins, and each of the monomers that make up the cage has very regular and precise interactions with neighboring monomers. To form a structure.

Ferritin is a protein that stores iron and is widely present in prokaryotes and eukaryotes. The molecular weight of the ferritin cage is about 500,000 Da, and the same monomer of 20 kDa (a single monomer or a heterologous monomer composed of either heavy or light chain) is self-assembled to form a giant spherical tertiary structure. The outer diameter is about 12 nm and the inner diameter is about 8 nm. Ferritin disperses into monomers depending on pH conditions and forms nanoparticles with 24 monomers bound together.

In addition, influenza hemagglutinin may be genetically fused to the N-terminus of the ferritin monomer to be expressed on the outer surface of the ferritin cage (Kanekiyo, M et al., Self-assembling influenza nanoparticle vaccines elicit broadly neutralizing H1N1 antibodies , Nature 2013; 499: 102-6), especially when the sequences of the invariable regions of hemagglutinin are expressed on the surface of ferritin, can also be used to produce candidate vaccines against a wide range of influenza strains (Yassine). , HM et al., Hemagglutinin-stem nanoparticles generate heterosubtypic influenza protection, Nat Med 2015; 21: 1065-70).

The inventors have therefore found that HTT ^cys forms antigen-antibody bonds and disulfide bonds. By using a peptide and V _L 12.3 ^cys peptide to prepare nanoparticles that combine antigen peptides and immune enhancers to ferritin self-assembly, fusion proteins can be produced more easily than direct fusion of antigen and immune enhancer peptides. It was confirmed that the molar ratio of the antigen peptide to the assembly and the immune enhancer can be easily controlled. In addition, the inventors have completed the present invention by confirming that the composition comprising the nanoparticles can increase total immunoglobulin levels and enhance cellular and humoral immunity.

It is an object of the present invention to provide ferritin nanoparticles in which an antigen peptide and an immune enhancer are bound to a ferritin self-assembly.

In order to achieve the above object, the present invention provides a self-assembly of 24 fusion proteins of HTT ^cys peptide and ferritin monomer; Fusion proteins fused to a _VL 12.3 ^cys peptide and an antigen peptide; And a nanoparticle comprising a fusion molecule of a V _L 12.3 ^cys peptide and an immunoadjuvant, wherein the antigenic peptide and the immune enhancer are linked to a ferritin self-assembly by binding between the HTT ^cys peptide and the V _L 12.3 ^cys peptide. To provide nanoparticles.

The present invention also provides a vaccine composition comprising the nanoparticles.

The nanoparticles of the present invention are antigen peptides or immune enhancers coupled to ferritin self-assembly via HTT ^cys peptides and V _L 12.3 ^cys peptides, which can produce fusion proteins more easily than direct fusion of antigens and immune enhancers. Since the molar ratio of the antigen peptide and the immune enhancer can be easily controlled, it can be usefully used as a composition for vaccine production.

1 is HTT HTT ^cys can additionally introduce disulfide bonds to antigen-antibody bonds between the peptide and the V _L 12.3 peptide The sequence of the peptide and V _L 12.3 ^cys peptide is shown.
Figure 2 shows the fusion protein domains of Salmonella flagellin (FliC), Escherichia coli ferritin (Ftn) and hemagglutinin (influenza A virus (A / California / 04/2009 (H1N1), HA).
Figure 3 shows the nanoparticles bound to flagellin, an immune enhancing peptide, (A-1) is Ftn-HA-ΔFliC nanoparticles, (B-1) is Ftn-PA-ΔFliC nanoparticles and (C-1 ) Represents I3-01-HA-ΔFliC nanoparticles, (A-2) is the size exclusion chromatography profile of the nanoparticles of (A-1), (B-2) is the nanoparticles of (B-1) The size exclusion chromatography profile of (C-2) shows the size exclusion chromatography profile of the nanoparticles of (C-1), and (A-3) shows the nanoparticles purified by chromatography of (A-2). The results of SDS-PAGE analysis are shown.
Figure 4 shows the degree of TLR5 activation of HEK293 cells incubated with Ftn, Ftn-ΔFliC (N) and Ftn-FliC, respectively.
5 shows the total IgG and IgG subclass values of mouse serum immunized with Ftn-HA-ΔFliC (N) and Ftn-HA-FliC (N), respectively.
(Ftn: HA = 24:10;
HA: FliC (or ΔFliC) = 4: 1).
6 shows the degree of TLR5 activation of HEK293 cells incubated with Ftn + ΔFliC, Ftn-ΔFliC (C), Ftn-ΔFliC (I) and Ftn-ΔFliC (N), respectively.
Figure 7 (A) shows the total IgG and IgG subclass values of mouse serum immunized with HA, Ftn + ΔFliC + HA and Ftn-ΔFliC (I) + HA, respectively, (B) is Ftn-HA + ΔFliC And IgG levels of mouse serum immunized with Ftn-HA-ΔFliC (I), respectively.
(Ftn-ΔFliC = 24: 10 in Ftn-ΔFliC;
Ftn-HA to Ftn: HA = 24:10;
Ftn-HA-ΔFliC = 24: 10: 3.3) in Ftn-HA-ΔFliC (I).
FIG. 8 shows IgG levels of mouse serum immunized with Ftn-HA-ΔFliC prepared by differently controlling the molar ratios of Ftn-HA and HA and ΔFliC, wherein (A) is total IgG, (B) is IgG1, (C) shows IgG2a, (D) shows IgG2b and (E) shows IgG1 / IgG2a.
(Ftn: HA: ΔFliC = 24: 10: 10 in Ftn-HA-ΔFliC of 1: 1;
Ftn: HA: ΔFliC = 24: 10: 5 in Ftn-HA-ΔFliC at 2: 1;
Ftn: HA: ΔFliC = 24: 10: 3.3 in Ftn-HA-ΔFliC at 3: 1;
Ftn: HA: ΔFliC = 24: 10: 2.5) at 4: 1 Ftn-HA-ΔFliC.
9 is Ftn-HA; And the amounts of cytokines IFN-γ and IL-4 secreted from splenocytes of mice immunized with Ftn-HA-ΔFliC, which were prepared by differently controlling the molar ratio of HA and ΔFliC.
(Ftn: HA: ΔFliC = 24: 10: 10 in Ftn-HA-ΔFliC of 1: 1;
Ftn: HA: ΔFliC = 24: 10: 5 in Ftn-HA-ΔFliC at 2: 1;
Ftn: HA: ΔFliC = 24: 10: 2.5) at 4: 1 Ftn-HA-ΔFliC.
10 is Ftn-HA prepared by differently adjusting the molar ratio of Ftn and HA; And the total IgG levels of mouse serum immunized with Ftn-HA-ΔFliC, each of which adjusted the molar ratio of HA to ΔFliC by 3: 1, (A) for total IgG, (B) for IgG1, and (C) for IgG2a, (D) shows IgG2b and (E) shows IgG1 / IgG2a levels
(From Low's Ftn-HA, Ftn: HA = 24: 4;
Ftn-HA at High Ftn: HA = 24:24;
Ftn: HA: ΔFliC = 24: 10: 3.3) at 3: 1 Ftn-HA-ΔFliC.

Hereinafter, the present invention will be described in detail.

The present invention is HTT ^cys Autoassembles of 24 fusion proteins of peptide and ferritin monomers; Fusion proteins fused to a _VL 12.3 ^cys peptide and an antigen peptide; And a fusion molecule of a V _L 12.3 ^cys peptide and an immune adjuvant, wherein the antigenic peptide and the immune enhancer are the HTT ^cys Peptides with V _L 12.3 ^cys Nanoparticles are provided that are linked to the ferritin self-assembly by binding between peptides.

As used herein, the term "fusion protein" refers to an artificially synthesized protein in which two or more proteins or peptides are linked to each other. The fusion protein of the present invention may be prepared by chemical synthesis methods known in the art or by expressing a polynucleotide encoding the fusion protein of the present invention by cloning into an expression vector. The fusion protein may further comprise an amino acid sequence designed for the purpose of increasing the stability of the targeting sequence, tag, labeled residue, half-life or peptide, wherein some amino acids of the known amino acid sequence are added, substituted Mutantchen protein mutated by a method such as deletion or deletion may also be included in the category of fusion proteins provided by the present invention.

In the present invention, the ferritin self-assembly means that 24 ferritin monomers derived from living organisms form nanoparticles in the form of hollow cages through self-assembly.

The ferritin monomers may be homologous or heterologous to each other, but may be derived from human, Helicobacter bacteria, or E. coli , but is not limited thereto. Preferably the ferritin monomer may be derived from E. coli and more preferably may be composed of the amino acid sequence of SEQ ID NO: 1.

SEQ ID NO 1:

MLKPEMIEKLNEQMNLELYSSLLYQQMSAWCSYHTFEGAAAFLRRHAQEEMTHMQRLFDYLTDTGNLPRINTVESPFAEYSSLDELFQETYKHEQLITQKINELAHAAMTNQDYPTFNFLQWYVSEQHEEEKLFKSIIDKLSLAGKSGEGDTIFNKELSTLDT

HTT ^cys in the present invention Peptide refers to a variant of the short alpha helical huntingtin peptide (hereinafter referred to as HTT) present in the huntingtin protein. Specifically, the HTT ^cys The peptide is a mutated alanine (Alanine, A), the sixth amino acid of the HTT peptide consisting of the amino acid sequence of SEQ ID NO: 2 with cysteine (C), may be composed of the amino acid sequence of SEQ ID NO: 3.

designation SEQ ID NO: Sequence type order HTT 2 Amino acid sequence EKLMK A FESLKSFQ HTT ^cys 3 Amino acid sequence EKLMK C FESLKSFQ

The HTT ^cys The peptide may be fused to or inserted into the N-terminus or C-terminus of the ferritin monomer, preferably fused to the N-terminus. When the HTT ^cys peptide is a fusion protein fused to the N-terminus of the ferritin monomer, when the ferritin self-assembly is formed, HTT ^cys The peptide may be located on the outer surface of the ferritin autoassembly. According to a specific embodiment of the present invention, the HTT ^cys The peptide is fused to the N-terminus of the ferritin monomer and the HTT ^cys The fusion protein in which the peptide is fused to the N-terminus of the ferritin monomer may be composed of the amino acid sequence of SEQ ID NO.

The HTT ^cys In the ferritin self-assembly consisting of 24 fusion proteins of peptides and ferritin monomers, the HTT ^cys The peptide may be located on the inner or outer surface of the ferritin self-assembly, preferably may be located on the outer surface of the ferritin self-assembly.

In the present invention, the V _L 12.3 ^cys peptide refers to a variant of the V _L 12.3 peptide, which is an immunoglobulin fragment that binds to the huntingtin peptide. Specifically, the V _L 12.3 ^cys peptide is a mutated tyrosine (Y), which is the 53rd amino acid of the V _L 12.3 peptide consisting of the amino acid sequence of SEQ ID NO: 4, to cysteine (C), and the amino acid sequence of SEQ ID NO: 5 It may be configured.

designation SEQ ID NO: Sequence type order V _L 12.3 4 Amino acid sequence MGSQPVLTQSPSVSAAPRQRVTISVSGSNSNIGSNTVNWIQQLPGRAPELLM Y DDDLLAPGVSDRFSGSRSGTSASLTISGLQSEDEADYYAATWDDSLNGWVFGGGTKVTVLSA V _L 12.3 ^cys 5 Amino acid sequence MGSQPVLTQSPSVSAAPRQRVTISVSGSNSNIGSNTVNWIQQLPGRAPELLM C DDDLLAPGVSDRFSGSRSGTSASLTISGLQSEDEADYYAATWDDSLNGWVFGGGTKVTVLSA

The V _L 12.3 ^cys peptide may be fused to the N- terminus or the C- terminus of the peptide antigen or inserted therein.

The antigen peptide is hemagglutinin (HA), protective antigen (PA), prostate-specific antigen (PSA), hantavirus glycoprotein antigen, SFTS virus glycoprotein antigen, etc. However, as long as it is a peptide constituting an antigen capable of inducing an immune response, it can be used without limitation.

The adjuvant may be flagellin, monophosphoryl lipid A, MPL-A, bacterial lipoprotein, or the like, but enhances the function of an antigen peptide that induces an immune response in a direct or indirect manner. As long as it can, it can be used without limitation. Preferably the immune enhancer may be an immune enhancing peptide, more preferably a flagellin peptide.

designation SEQ ID NO: Sequence type order Hemagglutinin
(hemagglutinin, HA) 8 Amino acid sequence DTLCIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDKHNGKLCKLRGVAPLHLGKCNIAGWILGNPECESLSTASSWSYIVETPSSDNGTCYPGDFIDYEELREQLSSVSSFERFEIFPKTSSWPNHDSNKGVTAACPHAGAKSFYKNLIWLVKKGNSYPKLSKSYINDKGKEVLVLWGIHHPSTSADQQSLYQNADTYVFVGSSRYSKKFKPEIAIRPKVRDQEGRMNYYWTLVEPGDKITFEATGNLVVPRYAFAMERNAGSGIIISDTPVHDCNTTCQTPKGAINTSLPFQNIHPITIGKCPKYVKSTKLRLATGLRNIPSIQSRGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADLKSTQNAIDEITNKVNSVIEKMNTQFTAVGKEFNHLEKRIENLNKKVDDGFLDIWTYNAELLVLLENERTLDYHDSNVKNLYEKVRSQLKNNAKEIGNGCFEFYHKCDNTCMESVKNGTYDYPKYSEEAKLNREEIDGVKLESTRIYQI Battlefield Flaglin
(full-length flagellin, Flic) 9 Amino acid sequence MAQVINTNSLSLLTQNNLNKSQSALGTAIERLSSGCRINSAKDDAAGQAIANRFTANIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAEITQRLNEIDRVSGQTQFNGVKVLAQDNTLTIQVGANDGETIDIDLKQINSQTLGLDTLNVQQKYKVSDTAATVTGYADTTIALDNSTFKASATGLGGTDQKIDGDLKFDDTTGKYYAKVTVTGGTGKDGYYEVSVDKTNGEVTLAGGATSPLTGGLPATATEDVKNVQVANADLTEAKAALTAAGVTGTASVVKMSYTDNNGKTIDGGLAVKVGDDYYSATQNKDGSISINTTKYTADDGTSKTALNKLGGADGKTEVVSIGGKTYAASKAEGHNFKAQPDLAEAAATTTENPLQKIDAALAQVDTLRSDLGAVQNRFNSAITNLGNTVNNLTSARSRIECSDYATEVSNMSRAQILQQAGTSVLAQANQVPQNVLSLLR Digested flagellin
(truncated flagellin, ΔFlic) 10 Amino acid sequence FTANIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAEITQRLNEIDRVSGQTQFNGVKVLAQDNTLTIQVGANDGETIDIDLKQINSQTLGLDTLNVQQKYKVSDTAATVTGYADTTIALDNSTFKASATGLGGTDQKIDGDLKFDDTTGKYYAKVTVTGGTGKDGYYEVSVDKTNGEVTLAGGATSPLTGGLPATATEDVKNVQVANADLTEAKAALTAAGVTGTASVVKMSYTDNNGKTIDGGLAVKVGDDYYSATQNKDGSISINTTKYTADDGTSKTALNKLGGADGKTEVVSIGGKTYAASKAEGHNFKAQPDLAEAAATTTENPLQKIDAALAQVDTLRSDLGAVQNRFNSAITNLGNTVNNLTSARSRIECSDYATEVSNMSRAQILQQAGTSVLAQANQVPQNVLSLLR

V _L 12.3 ^cys The peptide may be fused to or inserted into the N-terminus or C-terminus of the antigenic peptide or immune enhancing peptide. In a specific embodiment of the present invention, the V _L 12.3 ^cys Peptides were fused to the C-terminus of the antigen peptide hemagglutinin or to the N-terminus, C-terminus, or inside of full-length flagellin, an immune enhancing peptide.

The HTT ^cys Peptides huntingtin (hungtingtin) a variant of a short alpha helix of huntingtin (HTT) peptides present in the protein, wherein the V _L 12.3 ^cys peptide is a variant of an immunoglobulin fragment of V _L 12.3 peptide coupled to the huntingtin peptide The HTT ^cys peptide and the V _L 12.3 ^cys peptide may be bound by an antigen-antibody reaction. In addition, the HTT ^cys Peptide and V _L 12.3 ^cys peptide were each HTT As a variant in which cysteine is introduced into a peptide and a V _L 12.3 peptide, the HTT ^cys Peptides and V _L 12.3 ^cys peptides may be bound by disulfide bonds. Thus, the antigen peptide and the immune enhancer fused with the _VL 12.3 ^cys peptide are the HTT ^cys It can be linked to the ferritin autoassembly by binding between the peptide and the V _L 12.3 ^cys peptide. Thus the HTT ^cys Binding between the peptide and the V _L 12.3 ^cys peptide may include antigen-antibody binding and disulfide bonds, thereby providing the HTT ^cys The bond between the peptide and the V _L 12.3 ^cys peptide may form a stronger bond than if only the antigen-antibody bond was formed.

The molar ratio of the antigen peptide and the immune enhancing peptide in the nanoparticles can be appropriately controlled. Preferably it may be 0.1-10: 1, More preferably, it may be 1-4: 1, Most preferably, it may be 1-3: 1.

The nanoparticles can be prepared by a manufacturing method comprising the following steps:

1) HTT ^cys Fusion Proteins of Peptides and Ferritin Monomers Obtaining ferritin autoassembly from 24 monomers; And

2) a fusion protein in which the V _L 12.3 ^cys peptide and the antigen peptide are fused to the ferritin self-assembly of 1); And mixing the fusion molecule to which the V _L 12.3 ^cys peptide and the immunoadjuvant are fused.

The present invention also provides a vaccine composition comprising the nanoparticles of the present invention. Nanoparticles of the present invention is easier to manufacture when fusion of the antigen and immune enhancer directly to the ferritin monomer protein and can easily control the molar ratio of the antigen peptide and immune enhancer.

The vaccine can increase total IgG levels.

In addition, the vaccine can enhance cellular immunity and humoral immunity.

The vaccine composition of the present invention may further comprise a pharmaceutically acceptable carrier, appropriate adjuvant, other conventional substances, and may be administered in an immunologically effective amount. As used herein, the term "immunologically effective amount" means an amount sufficient to exhibit an immune enhancing effect and an amount sufficient to not cause side effects or serious or excessive immune responses, and the exact dosage concentration depends on the specific immunogen to be administered. To determine the occurrence of an immune response, one skilled in the art can determine this using known methods. It may also vary depending on the dosage form and route, the age, health and weight of the recipient, the nature and extent of the symptoms, the type of current treatment, and the number of treatments.

Carriers are known in the art and may include stabilizers, diluents, buffers. Suitable stabilizers include carbohydrates such as sorbitol, lactose, mannitol, starch, sugars, dextran and glucose; Proteins such as albumin or casein and the like. Suitable diluents may include salts, Hanks balance salts, Ringer's solution and the like. Suitable buffers include alkali metal phosphates, alkali metal carbonates, alkaline earth metal carbonates, and the like. The vaccine may also include one or more immune enhancers (adjuvant) to improve or enhance the immune response. Examples of suitable immune enhancers may include aluminum hydroxide, Freud's complete or incomplete adjuvant, DEAE dextran, levamisol, PCG and poly I: C or poly A: U. Vaccine compositions of the invention can be administered via known routes of administration. Such methods may include, but are not limited to, oral, transdermal, muscle, peritoneal, intravenous, subcutaneous, nasal routes, and may be administered by any device that allows the active substance to migrate to the target cell.

According to a specific embodiment of the present invention, the present inventors are HTT ^cys The nanoparticles were stably bound to the hemagglutinin antigen peptide and the flagellin immune enhancing peptide to the ferritin self-assembly via the peptide and the V _L 12.3 ^cys peptide (see FIG. 3).

In addition, the present inventors wish to test the effect on the induction of an immune response of flagellin D0 domains that have a tendency to proteolytic degradation and polymerization, and thus, full-length flagellin comprising the D0 to D3 domains of flagellin (hereinafter, FliC). Nanoparticles bound to the ferritin self-assembly (Ftn-FliC); And the TLR5 activation ability of the nanoparticles (Ftn-ΔFliC) bound to the ferritin self-assembled flagellin (hereinafter referred to as ΔFliC) in which the D0 domain was cleaved, and the immunoglobulin levels in the mouse serum induced by the nanoparticles, It was confirmed that the D0 domain of flagellin, which may limit the production of large amounts of protein, has a slight effect on the IgG response (see FIGS. 4 and 5).

In addition, the present inventors have fused to ferritin self N- terminal, C- terminal, or internal (D3 domain) in V _L 12.3 ^cys peptide of Plastic jelrin (ΔFliC), Plastic jelrin (ΔFliC) to confirm the attachment sites suitable for assembly The TLR5 activation ability of nanoparticles (represented as Ftn-ΔFliC (N), Ftn-ΔFliC (C) and Ftn-ΔFliC (I), respectively) bound to the ferritin self-assembly was confirmed. As a result, Ftn-ΔFliC (N) and Ftn- ΔFliC (I) was found to be more effective in stimulating the TLR5 signaling pathway than Ftn-ΔFliC (C) (see FIG. 6).

In addition, the inventors confirmed that when hemagglutinin and flagellin are simultaneously bound to the ferritin self-assembly, they induce a stronger immune response than simply mixing hemagglutinin and flagellin without binding (FIG. 7). Reference).

In addition, to determine the minimum amount of flagellin required for immune enhancing activity, the present inventors have found that the immunoglobulin levels and splenocytes in mouse serum derived from nanoparticles with controlled molar ratios of hemagglutinin to flagellin bound to ferritin autoassembly. As a result of measuring the amount of cytokines, it was confirmed that the molar ratio of hemagglutinin to flagellin bound to the ferritin self-assembly can reduce the amount of flagellin up to 3: 1 without affecting IgG2a class conversion and Th1 response. In addition, the co-binding of hemagglutinin and flagellin to the ferritin self-assembly was confirmed to enhance humoral immune responses and stimulate antigen-specific T cell responses (see FIGS. 8 and 9).

The present inventors also found that nanoparticles (Ftn-HA (Low), Ftn-HA (High), respectively) in which 4- or 24-molecule hemagglutinin is bound to the ferritin self-assembly, and hemagglutinin and flagellin 3 Immunoglobulin levels in mouse serum induced with nanoparticles (Ftn-HA-ΔFliC) bound to ferritin autoassembly at a molar ratio of 1: 1 revealed that humagglutinin bound to ferritin autoassembly was a humoral immune response. Although useful for inducing γ, it was confirmed that flagellin should be bound to produce nanoparticles having autoimmune enhancing activity (see FIG. 10).

Therefore, the nanoparticles of the present invention is HTT ^cys An antigen peptide and an immune enhancer are coupled to a ferritin self-assembly via a peptide and a V _L 12.3 ^cys peptide, and the mole ratio of the antigen peptide and the immune enhancer in the nanoparticles can be easily controlled, resulting in a risk of toxicity. The content can be optimized. In addition, the administration of nanoparticles that combine antigen peptides and immune enhancers together can greatly enhance Th1 cytokine secretion and IgG2a class conversion, as well as total IgG, thus making the nanoparticles useful for vaccine compositions. Can be.

Hereinafter, the present invention will be described in detail by the following examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited by the following examples.

< Example  1> Disulfide  Amino Acid Sequence Design for Bond Formation

V _L 12.3 is a single domain antibody fragment consisting of 115 amino acids (SEQ ID NO: 4), which binds to an HTT peptide sequence (SEQ ID NO: 2) by antigen-antibody binding in a human huntingtin protein. V _L 12.3 ^cys and HTT ^cys introduced cysteine sequences into HTT peptide and V _L 12.3 respectively using disulfide-by-design web server Peptides were prepared (FIG. 1, http://cptweb.cpt.wayne.edu/DbD2/). V _L 12.3 ^cys and HTT ^cys peptides can form robust covalent bonds through additional disulfide bridge formation in addition to antigen-antibody binding (FIG. 1, Table 1 and Table 2).

< Example  2> Self-assembly  Monomer manufacturers

< Example  2-1> HTT ^cys Peptide  Fused ferritin ( ferritin ) Preparation of Monomer

In order to fuse HTT ^cys to the N-terminus of E. coli ferritin monomer (hereinafter referred to as Ftn) (FIG. 2), polynucleotides encoding the ferritin monomer fused with HTT ^cys (Table 4) were amplified by PCR. It was. Specifically, as a PCR template, HTT ^cys was used as Addgene plasmid # 23966 (Addgene, Cambridge, Mass.), And ferritin monomer (Ftn) was used as Addgene bacterial strain # 61440. PCR was performed at 30 seconds at 72 ° C. and at 30 seconds.

(HTT ^cys forward primer: ATC ATC CAT GGG CGA AAA GCT GAT GAA GTG TTT C (SEQ ID NO: 25);

HTT ^cys reverse primer: ATC ACT CTA GAA CCA CCC TGG AAG GAC TTG AG (SEQ ID NO: 26);

Ftn forward primer: TGC GGT CTA GAA TGC TGA AAC CAG AAA TGA TTG AA (SEQ ID NO: 27);

Ftn reverse primer: AGG CCA GCC ACT CGA GAC TAG TTC ATT AGT TTT GTG TGT C (SEQ ID NO: 28))

Polynucleotides encoding amplified HTT ^cys -ferritin (HTT ^cys- Ftn) were cloned into pET28a vector (Addgene, Cambridge, Mass.).

designation SEQ ID NO: Sequence type order HTT ^cys -ferritin monomer
(HTT ^cys -Ftn) 6 Amino acid sequence MGEKLMKCFESLKSFQGGSRMLKPEMIEKLNEQMNLELYSSLLYQQMSAWCSYHTFEGAAAFLRRHAQEEMTHMQRLFDYLTDTGNLPRINTVESPFAEYSSLDELFQETYKHEQLITQKINELAHAAMTNQDYPTFNFLQWYVSEQHEEEKLAKKIDIDSL 7 Sequence atgggcgaaaagctgatgaagtgtttcgagtccctcaagtccttccagggtggttctagaatgctgaaaccagaaatgattgaaaaacttaatgagcagatgaacctggaactgtactcttcactgctttatcagcaaatgagcgcctggtgcagctatcataccttcgaaggtgctgccgcgttcctgcgccgtcacgcccaggaagagatgacgcatatgcagcgtctgtttgattacctgactgataccggcaatttaccgcgtattaataccgttgaatctccgtttgctgaatattcctcacttgatgaattattccaggaaacctataaacacgaacaattaatcacccagaaaattaacgaactggctcatgctgcaatgaccaatcaggactacccaacatttaatttcctgcagtggtatgtttctgagcagcatgaagaagagaaactgttcaaatcgattattgataaattaagcctggcaggcaaaagcggcgaaggtctgtattttatcgacaaagaactctctaccctcgacacacaaaactaa

The cloned vector was transformed into E. coli BL21 (DE3) strain and when the OD ₆₀₀ value of the bacterial culture reached 0.7, 1 mM IPTG was added to induce HTT ^cys -ferritin monomer fusion protein production. After 4 hours of incubation at 37 ° C, E. coli (DE3) was harvested by centrifugation at 4000 rpm for 30 minutes at 4 ° C.

Pellets of harvested cells are resuspended in lysis buffer containing 20 mM Tris (pH 8.0), 200 mM NaCl, 10 mM β-mercaptoethanol and 0.1 mM PMSF, It was homogenized using a microfludizer (microfludizer, Microfluidics, Westwood, Mass.) And then centrifuged at 15,000 rpm for 40 minutes at 4 ° C. The supernatant was heated at 80 ° C. for 10 minutes, then centrifuged at 15,000 rpm for 30 minutes at 4 ° C., and purified by a Q-Sepharose anion exchange column to obtain a fraction containing HTT ^cys -ferritin monomer fusion protein. Concentrated.

< Example  2-2> HTT ^cys Peptide  Preparation of Fused I3-01 Monomers

HTT ^cys for attaching antigen or immune enhancing peptides to ferritin self-assembly Peptides and V _L 12.3 ^cys To confirm that the binding between peptides is also useful in self-assembly of other proteins, the HTT ^cys sequence was fused to the N-terminus of the artificial cage protein I3-01 monomer. I3-01 self-assembly is a computer designed protein that forms a 250 angstrom cage consisting of 60 monomer subunits.

Specifically, PCR templates include HTT ^cys , Addgene plasmid # 23966 (Addgene, Cambridge, MA), and I3-01 self-assembly. Baker's I3-01 cDNA was used, and PCR was performed using the following primers under conditions of 30 seconds at 95 ° C, 60 seconds at 62 ° C, and 30 seconds at 72 ° C.

(HTT ^cys- I3-01 forward primer: ATT ATC ATA TGC ATC ATC ATC ATC ATC ACG GTG GAA GCG GTG GAA GC (SEQ ID NO: 29);

HTT ^cys -I3-01 reverse primer: TGC GCC TCG AGC TAT TAT TCG GTG CAG CCA CGA ATC (SEQ ID NO: 30))

name SEQ ID NO: Sequence type order HTT ^cys -I3-01 11 Amino acid sequence MHHHHHHGGSGGSEKLMKCFESLKSFQGGSGGSMKMEELFKKHKIVAVLRANSVEEAKKKALAVFLGGVHLIEITFTVPDADTVIKELSFLKEMGAIIGAGTVTSVEQCRKAVESGAEFIVSPHLDEEISQFCKEKGVFYMPGVMTPTELVKAMKLVVGPKGPVKKVVKVVKKVVKVVKKVVKVGKVGKVGKVGKVGK 12 Sequence atgcatcatcatcatcatcacggtggaagcggtggaagcgaaaagctgatgaagtgtttcgagtccctcaagtccttccagggtggtagcggcggttctatgaagatggaagagctgttcaagaaacacaagatcgttgccgtgctgcgtgccaatagtgtggaagaagcgaaaaagaaagcgctggcggttttcctgggcggcgttcatctgattgaaattacctttaccgtgccggatgcggataccgtgattaaggaactgagctttctgaaggaaatgggcgcgattattggtgcgggcaccgtgaccagcgtggagcagtgccgtaaagcggtggaaagtggcgccgaattcattgtgagtccgcacctggacgaggaaattagccaattttgcaaggagaagggtgtgttctatatgccaggcgttatgaccccgaccgaactggtgaaagccatgaaactgggccataccatcttaaaactgtttccgggtgaggtggtgggtccgcagtttgttaaagcgatgaaaggtccgtttccgaatgtgaaatttgtgccaaccggcggtgttaatctggacaatgtgtgcgaatggttcaaagcgggcgtgctggccgtgggcgtgggcagcgcgttagtgaaaggcaccccggtggaagtggcggaaaaggccaaggcgttcgttgagaagattcgtggctgcaccgaataa

The cloned vector was transformed into E. coli BL21 (DE3) strain and when the OD ₆₀₀ value of the bacterial culture reached 0.5, 0.5 mM IPTG was added to induce HTT ^cys- I3-01 fusion protein production. After 4 hours of incubation at 37 ° C, E. coli (DE3) was harvested by centrifugation at 4000 rpm for 30 minutes at 4 ° C.

Pellets of harvested cells are resuspended in lysis buffer containing 20 mM Tris (pH 8.0), 200 mM NaCl, 10 mM β-mercaptoethanol and 0.1 mM PMSF, After homogenization using a microfludizer (Microfludizer, Microfluidics, Westwood, Mass.), It was centrifuged for 40 minutes at 4 ℃ at 15,000 rpm. The supernatant was heated at 80 ° C. for 5 minutes, centrifuged at 15,000 rpm for 30 minutes at 4 ° C., and purified by a Q-Sepharose anion exchange column to obtain a fraction containing HTT-I3-01 fusion protein. Concentrated.

< Example  3> antigen Peptide  Produce

< Example  3-1> V _L 12. 3 ^cys Peptide  And Hemagglutinin ( hemagglutinin , HA) Preparation of fusion protein fused antigen

Fragments (18D-530I) of hemagglutinin (hereinafter referred to as HA) (A / California / 04/2009 (H1N1)) of influenza A virus were used as antigen peptides (FIG. 2).

V _L 12.3 ^cys at the C-terminus of the HA antigen fragment To obtain the protein with the domain fused, polynucleotides encoding it (Table 6) were amplified by PCR. Specifically, as a PCR template, HA is a cDNA clone of Swine Flu H1N1 hemagglutinin (aa 1-530) (A / California / 04/2009), V _L 12.3 ^cys The cDNA of V _L 12.3 single domain intrabody obtained from the Massachusetts Institute of Technology (MIT) Dr Wittrup Laboratories, was used for 30 seconds at 95 ℃, 30 seconds at 58 ℃, 1 minute 30 seconds at 72 ℃ using the following primers PCR was performed.

(HA-V _L 12.3 ^cys forward primer: AGG CCT CTA GAA GCA TGG GTA GTC AAC CAG TAC TT (SEQ ID NO: 31);

HA-V _L 12.3 ^cys reverse primer: GTG ATG ATG ATG ATG ATG GCT ACT GCC TGC GGA CAG CAC CGT (SEQ ID NO: 32))

Amplified HA-V _L 12.3 ^cys encoding polynucleotides were cloned into pAcGP67A baculovirus transfer vector (BD Biosciences, San Jose, Calif.).

name SEQ ID NO: Sequence type order HA-V _L 12.3 ^cys 13 Amino acid sequence ADPDTLCIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDKHNGKLCKLRGVAPLHLGKCNIAGWILGNPECESLSTASSWSYIVETPSSDNGTCYPGDFIDYEELREQLSSVSSFERFEIFPKTSSWPNHDSNKGVTAACPHAGAKSFYKNLIWLVKKGNSYPKLSKSYINDKGKEVLVLWGIHHPSTSADQQSLYQNADTYVFVGSSRYSKKFKPEIAIRPKVRDQEGRMNYYWTLVEPGDKITFEATGNLVVPRYAFAMERNAGSGIIISDTPVHDCNTTCQTPKGAINTSLPFQNIHPITIGKCPKYVKSTKLRLATGLRNIPSIQSRGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADLKSTQNAIDEITNKVNSVIEKMNTQFTAVGKEFNHLEKRIENLNKKVDDGFLDIWTYNAELLVLLENERTLDYHDSNVKNLYEKVRSQLKNNAKEIGNGCFEFYHKCDNTCMESVKNGTYDYPKYSEEAKLNREEIDGVKLESTRIYQIGSRSMGSQPVLTQSPSVSAAPRQRVTISVSGSNSNIGSNTVNWIQQLPGRAPELLMCDDDLLAPGVSDRFSGSRSGTSASLTISGLQSEDEADYYAATWDDSLNGWVFGGGTKVTVLSAGSSHHHHHH 14 Sequence gcggatcccgacacattgtgcattgggtaccatgctaataactcaacagatacagtagatactgtgttggaaaaaaacgtaacggtaacacactcagtgaacttactagaagacaaacataatggaaagttatgtaaactaagaggggttgccccattacatctgggtaaatgcaatattgcagggtggattctaggaaacccagagtgtgagtcactatccactgcatcaagttggtcatacatcgtggaaactccaagctcagataatggcacatgttatccgggagactttatcgattatgaggaattaagggaacagttgagcagtgttagctcgttcgaaagattcgagatattccccaagacatcaagttggccaaatcatgactccaacaaaggagtaacagcggcatgccctcacgccggagctaaatcattctacaagaacctgatttggctagttaagaaaggcaattcttacccaaaactgtcaaagtcttatattaatgacaaaggtaaagaagtattggttctatgggggatacatcaccccagcacaagcgctgaccaacagtcactataccagaatgccgatacttacgtatttgtggggagtagcagatattctaaaaagttcaagccggaaattgcaattaggccgaaggtgagagaccaagaaggtagaatgaattactattggacattagtagagccgggtgataaaataacatttgaagcaactggaaatttggtggttccaagatacgcattcgctatggagagaaacgccggtagcggaatcattataagtgatacaccagtccacgactgtaacacaacctgccaaactcccaagggagcaataaacacgagcctaccgtttcaaaatattcacccaattacaatagggaaatgtccaaaatatgtaaaatcaacaaagttgagactggcaacagggctcagaaacataccctctattcagtccagaggtctatttg gggcaattgccggattcatcgaggggggatggacaggtatggtcgatggctggtatgggtaccaccatcagaacgagcaaggatctggttacgccgcgatctaaagtcaacacaaaatgcaattgatgaaatcacaaataaggtaaattcagtaatagagaagatgaatacacagttcacagcagtaggcaaagaatttaatcacctggagaaaagaatagaaaacctcaataaaaaggtagacgatggtttcttagacatttggacatacaatgcagaactcctagtactactcgaaaatgaaaggacgctggactaccatgactcaaacgttaaaaatctatatgaaaaagtaagatcacagttgaaaaataatgccaaagagataggaaatggatgctttgaattttaccataaatgtgacaatacatgtatggaatcagttaaaaatgggacttacgattatccaaagtatagcgaagaagctaagctgaatagagaagaaatagatggggtaaaactagaatcaactaggatttatcagattggctctagaagcatgggtagtcaaccagtacttacacaaagcccgtctgtgtctgccgctccacgtcagagagtcaccatctcagttagtggttctaattcaaatataggatcgaacacagtgaactggattcagcagttgcctggccgtgccccggagttattaatgtgtgatgatgatttattagcacccggagtatcagatcgcttttcaggaagccgtagtggcactagtgcgtccttaaccattagcgggttacagtctgaagacgaagcggactattacgcagctacgtgggatgatagtttaaatggctgggtttttggtggtggtactaaagttacggtgctgtccgcaggcagtagccatcatcatcatcatcactaa

The cloned vector was infected with High Five insect cells (High Five insect cells, Invitrogen, Waltham, Mass.) And cells were obtained after incubation at 28 ° C. for 2 days. The supernatant was obtained by centrifuging the cell culture at 4 ° C. at 4,000 rpm for 30 minutes. The supernatant was treated with 0.1 mM PMSF, 10 mM β-mercaptoethanol, and the supernatant was treated with Ni-NTA chelating agar. HA-V _L 12.3 ^cys fusion proteins were purified by loading onto Ross CL-6B and Q-Sepharose anion exchange columns.

< Example  3-2> V _L 12. 3 ^cys Peptide  And preparation of a fusion protein to which a protective antigen (PA) antigen is fused.

Domain 4 (596F-735G) of the protective antigen (PA) of Bacillus anthracis was used as the antigen peptide. Domain 4 of PA mediates the binding of PA to the capillary morphogenesis protein 2 (CMG2) receptor and is a recognition site for anthrax neutralizing antibody.

To obtain a protein in which the V _L 12.3 ^cys domain was fused to the C-terminus of the PA antigen fragment, polynucleotides encoding it (Table 7) were amplified by PCR. Specifically, as the PCR template, PA was used as Addgene plasmid # 11079, and V _L 12.3 ^cys was used as the cDNA of V _L 12.3 single domain intrabody obtained from MIT Dr. M. Wittrup, Massachusetts Institute of Technology (MIT). PCR was performed at 30 seconds, 58 seconds at 30 seconds, and 72 seconds at 30 seconds.

(PA-V _L 12.3 ^cys forward primer: AAG AGC CAT GGG CTT TCA TTA TGA TAG AAA TAA C (SEQ ID NO: 33);

PA-V _L 12.3 ^cys reverse primer: AGC ATC TCG AGT GCG GAC AGC ACC GTA ACT TTA (SEQ ID NO: 34))

The polynucleotide encoding the amplified PA-V _L 12.3 ^cys was cloned into the pET28a vector.

name SEQ ID NO: Sequence type order PA-V _L 12.3 ^cys 15 Amino acid sequence MGFHYDRNNIAVGADESVVKEAHREVINSSTEGLLLNIDKDIRKILSGYIVEIEDTEGLKEVINDRYDMLNISSLRQDGKTFIDFKKYNDKLPLYISNPNYKVNVYAVTKENTIINPSENGDTSTNGIKKILIFSKKGYEIGSGGSMGSQPVLTQSPSVSAAPRQRVTISVSGSNSNIGSNTVNWIQQLPGRAPELLMCDDDLLAPGVSDRFSGSRSGTSASLTISGLQSEDEADYYAATWDDSLNGWVFGGGTKVTVLSALEHHHHHH 16 Sequence atgggctttcattatgatagaaataacatagcagttggggcggatgagtcagtagttaaggaggctcatagagaagtaattaattcgtcaacagagggattattgttaaatattgataaggatataagaaaaatattatcaggttatattgtagaaattgaagatactgaagggcttaaagaagttataaatgacagatatgatatgttgaatatttctagtttacggcaagatggaaaaacatttatagattttaaaaaatataatgataaattaccgttatatataagtaatcccaattataaggtaaatgtatatgctgttactaaagaaaacactattattaatcctagtgagaatggggatactagtaccaacgggatcaagaaaattttaatcttttctaaaaaaggctatgagataggatccggtggtagcatgggtagtcaaccagtacttacacaaagcccgtctgtgtctgccgctccacgtcagagagtcaccatctcagttagtggttctaattcaaatataggatcgaacacagtgaactggattcagcagttgcctggccgtgccccggagttattaatgtgtgatgatgatttattagcacccggagtatcagatcgcttttcaggaagccgtagtggcactagtgcgtccttaaccattagcgggttacagtctgaagacgaagcggactattacgcagctacgtgggatgatagtttaaatggctgggtttttggtggtggtactaaagttacggtgctgtccgcactcgagcaccaccaccaccaccactga

The cloned vector was transformed into E. coli BL21 (DE3) strain, and when the OD ₆₀₀ value of the bacterial culture reached 0.5, 0.4 mM IPTG was added to PA-V _L 12.3 ^cys Induced fusion protein production. PA-V _L 12.3 ^cys fusion protein was incubated at 30 ° C. for 3.5 hours and then harvested E. coli (DE3) by centrifugation at 4000 rpm for 30 minutes at 4 ° C.

Cell pellets expressing PA-V _L 12.3 ^cys fusion protein were lysis buffer containing 20 mM Tris (pH 8.0), 200 mM NaCl, 10 mM β-mercaptoethanol and 0.1 mM PMSF. Resuspended and homogenized using a microfludizer (Microfludizer, Microfluidics, Westwood, Mass.). After centrifugation at 4 ° C. at 15,000 rpm for 40 minutes, the proteins were purified by Ni-NTA chelating agarose CL-6B (Incospharm, Daejeon, Korea) and Q-Sepharose (GE Healthcare, Pittsburgh, PA) anion exchange columns. It was. Fractions containing PA-V _L 12.3 ^cys fusion protein were obtained and concentrated.

< Example  4> V _L 12. 3 ^cys end  Fused Flagellin  Produce

Full-length flagellin containing the D0 domain is known to have higher activity to stimulate an immune response than flagellin, in which the D0 domain is truncated, while the flagellin D0 domain is responsible for proteolysis and polymerization. There is a tendency to increase, and production of recombinant proteins, including the D0 domain, is often difficult.

To test the effect of the D0 domain in the nanoparticle system of the present invention, a full-length flagellin (hereinafter referred to as FliC) containing all of D0 to D3 and a cleaved flagellin containing D1 to D3 (hereinafter And denoted ΔFliC. For full-length flagellin (FliC), it includes SS5 mutations that introduce disulfide bonds to reduce proteolysis. The SS5 mutation is a disulfide bond of the L36C and D456C residues of flagellin (FliC) and reduces proteolysis of flagellin. Also, to identify the most suitable fusion site with flagellin, V _L 12.3 ^cys was fused inside the N-terminal, C-terminal, or D3 domain (between 238G and 239G, I) of flagellin (FIG. 2).

As shown in Table 8, polynucleotides encoding various fusion proteins of flagellin and V _L 12.3 ^cys were amplified by PCR. Specifically, as a PCR template, FliC is Addgene plasmid # 85438, V _L 12.3 ^cys The cDNA of V _L 12.3 single domain intrabody obtained from the Massachusetts Institute of Technology (MIT) Dr Wittrup laboratory was used, 30 seconds at 95 ° C, 30 seconds at 58 ° C, 30 seconds to 1 minute at 72 ° C using the following primers: PCR was performed under the conditions.

(FliC (N) -V _L 12.3 ^cys forward primer: AGC GGC ATA TGG GTA GTC AAC CAG TAC TTA CAC AAA GCC CG (SEQ ID NO: 35);

FliC (N) -V _L 12.3 ^cys reverse primers: ATT ATG CGG CCG CTC AAC GCA GTA AAG AGA GGA CG (SEQ ID NO: 36);

ΔFliC (N) -V _L 12.3 ^cys forward primers: ACT GGT GGT ACC ATG GGT AGT CAA CCA GTA CTT (SEQ ID NO: 37);

ΔFliC (N) -V _L 12.3 ^cys reverse primers: AGT TAC TCG AGA CGG GCA GAA GTC AGG TTG TT (SEQ ID NO: 38);

ΔFliC (C) -V _L 12.3 ^cys forward primers: ATA ATC CAT GGG CTT TAC CGC GAA CAT CAA AGG T (SEQ ID NO: 39);

ΔFliC (C) -V _L 12.3 ^cys reverse primer: AGC ATC TCG AGT GCG GAC AGC ACC GTA AC (SEQ ID NO: 40);

ΔFliC (I) -V _L 12.3 ^cys forward primers: ATA ATC CAT GGG CTT TAC CGC GAA CAT CAA AGG T (SEQ ID NO: 41);

ΔFliC (I) -V _L 12.3 ^cys reverse primer: AGT TAC TCG AGA CGG GCA GAA GTC AGG TTG TT (SEQ ID NO: 42))

The amplified polynucleotides were cloned into pET28a vector (Addgene, Cambridge, MA).

name SEQ ID NO: Sequence type order FliC 9 Amino acid sequence MAQVINTNSLSLLTQNNLNKSQSALGTAIERLSSGCRINSAKDDAAGQAIANRFTANIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAEITQRLNEIDRVSGQTQFNGVKVLAQDNTLTIQVGANDGETIDIDLKQINSQTLGLDTLNVQQKYKVSDTAATVTGYADTTIALDNSTFKASATGLGGTDQKIDGDLKFDDTTGKYYAKVTVTGGTGKDGYYEVSVDKTNGEVTLAGGATSPLTGGLPATATEDVKNVQVANADLTEAKAALTAAGVTGTASVVKMSYTDNNGKTIDGGLAVKVGDDYYSATQNKDGSISINTTKYTADDGTSKTALNKLGGADGKTEVVSIGGKTYAASKAEGHNFKAQPDLAEAAATTTENPLQKIDAALAQVDTLRSDLGAVQNRFNSAITNLGNTVNNLTSARSRIECSDYATEVSNMSRAQILQQAGTSVLAQANQVPQNVLSLLR ΔFliC 10 amino acid
order FTANIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAEITQRLNEIDRVSGQTQFNGVKVLAQDNTLTIQVGANDGETIDIDLKQINSQTLGLDTLNVQQKYKVSDTAATVTGYADTTIALDNSTFKASATGLGGTDQKIDGDLKFDDTTGKYYAKVTVTGGTGKDGYYEVSVDKTNGEVTLAGGATSPLTGGLPATATEDVKNVQVANADLTEAKAALTAAGVTGTASVVKMSYTDNNGKTIDGGLAVKVGDDYYSATQNKDGSISINTTKYTADDGTSKTALNKLGGADGKTEVVSIGGKTYAASKAEGHNFKAQPDLAEAAATTTENPLQKIDAALAQVDTLRSDLGAVQNRFNSAITNLGNTVNNLTSAR FliC (N) -V _L 12.3 ^cys
17 Amino acid sequence MGSSHHHHHHSSGLVPRGSHMGSQPVLTQSPSVSAAPRQRVTISVSGSNSNIGSNTVNWIQQLPGRAPELLMCDDDLLAPGVSDRFSGSRSGTSASLTISGLQSEDEADYYAATWDDSLNGWVFGGGTKVTVLSAGGSAMAQVINTNSLSLLTQNNLNKSQSALGTAIERLSSGCRINSAKDDAAGQAIANRFTANIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAEITQRLNEIDRVSGQTQFNGVKVLAQDNTLTIQVGANDGETIDIDLKQINSQTLGLDTLNVQQKYKVSDTAATVTGYADTTIALDNSTFKASATGLGGTDQKIDGDLKFDDTTGKYYAKVTVTGGTGKDGYYEVSVDKTNGEVTLAGGATSPLTGGLPATATEDVKNVQVANADLTEAKAALTAAGVTGTASVVKMSYTDNNGKTIDGGLAVKVGDDYYSATQNKDGSISINTTKYTADDGTSKTALNKLGGADGKTEVVSIGGKTYAASKAEGHNFKAQPDLAEAAATTTENPLQKIDAALAQVDTLRSDLGAVQNRFNSAITNLGNTVNNLTSARSRIECSDYATEVSNMSRAQILQQAGTSVLAQANQVPQNVLSLLR 18 Sequence atgggtagtcaaccagtacttacacaaagcccgtctgtgtctgccgctccacgtcagagagtcaccatctcagttagtggttctaattcaaatataggatcgaacacagtgaactggattcagcagttgcctggccgtgccccggagttattaatgtgtgatgatgatttattagcacccggagtatcagatcgcttttcaggaagccgtagtggcactagtgcgtccttaaccattagcgggttacagtctgaagacgaagcggactattacgcagctacgtgggatgatagtttaaatggctgggtttttggtggtggtactaaagttacggtgctgtccgcaggtggtagcgccatggcacaagtcattaataccaacagcctgtcgctgttgacccagaataacctgaacaaatcccagtccgctctgggcaccgctatcgagcgtctgtcttcctgcctgcgtatcaacagcgcgaaagacgatgcggcaggtcaggcgattgctaaccgttttaccgcgaacatcaaaggtctgactcaggcttcccgtaacgctaacgacggtatctccattgcgcagaccactgaaggcgcgctgaacgaaatcaacaacaacctgcagcgtgtgcgtgaactggcggttcagtctgctaacagcaccaactcccagtctgacctcgactccatccaggctgaaatcacccagcgcctgaacgaaatcgaccgtgtatccggccagactcagttcaacggcgtgaaagtcctggcgcaggacaacaccctgaccatccaggttggtgccaacgacggtgaaactatcgatatcgatctgaagcagatcaactctcagaccctgggtctggatacgctgaatgtgcaacaaaaatataaggtcagcgatacggctgcaactgttacaggatatgccgatactacgattgctttagacaatagtacttttaaagcctcggctactggtcttggtggtactgacc agaaaattgatggcgatttaaaatttgatgatacgactggaaaatattacgccaaagttaccgttacggggggaactggtaaagatggctattatgaagtttccgttgataagacgaacggtgaggtgactcttgctggcggtgcgacttccccgcttacaggtggactacctgcgacagcaactgaggatgtgaaaaatgtacaagttgcaaatgctgatttgacagaggctaaagccgcattgacagcagcaggtgttaccggcacagcatctgttgttaagatgtcttatactgataataacggtaaaactattgatggtggtttagcagttaaggtaggcgatgattactattctgcaactcaaaataaagatggttccataagtattaatactacgaaatacactgcagatgacggtacatccaaaactgcactaaacaaactgggtggcgcagacggcaaaaccgaagttgtttctattggtggtaaaacttacgctgcaagtaaagccgaaggtcacaactttaaagcacagcctgatctggcggaagcggctgctacaaccaccgaaaacccgctgcagaaaattgatgctgctttggcacaggttgacacgttacgttctgacctgggtgcggtacagaaccgtttcaactccgctattaccaacctgggcaacaccgtaaacaacctgacttctgcccgtagccgtatcgaatgctccgactacgcgaccgaagtttccaacatgtctcgcgcgcagattctgcagcaggccggtacctccgttctggcgcaggcgaaccaggttccgcaaaacgtcctctctttactgcgttga ΔFliC (N) -V _L 12.3 ^cys
19 Amino acid sequence MGSQPVLTQSPSVSAAPRQRVTISVSGSNSNIGSNTVNWIQQLPGRAPELLMCDDDLLAPGVSDRFSGSRSGTSASLTISGLQSEDEADYYAATWDDSLNGWVFGGGTKVTVLSAGGSAMGFTANIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAEITQRLNEIDRVSGQTQFNGVKVLAQDNTLTIQVGANDGETIDIDLKQINSQTLGLDTLNVQQKYKVSDTAATVTGYADTTIALDNSTFKASATGLGGTDQKIDGDLKFDDTTGKYYAKVTVTGGTGKDGYYEVSVDKTNGEVTLAGGATSPLTGGLPATATEDVKNVQVANADLTEAKAALTAAGVTGTASVVKMSYTDNNGKTIDGGLAVKVGDDYYSATQNKDGSISINTTKYTADDGTSKTALNKLGGADGKTEVVSIGGKTYAASKAEGHNFKAQPDLAEAAATTTENPLQKIDAALAQVDTLRSDLGAVQNRFNSAITNLGNTVNNLTSARLEHHHHHH 20 Sequence atgggtagtcaaccagtacttacacaaagcccgtctgtgtctgccgctccacgtcagagagtcaccatctcagttagtggttctaattcaaatataggatcgaacacagtgaactggattcagcagttgcctggccgtgccccggagttattaatgtgtgatgatgatttattagcacccggagtatcagatcgcttttcaggaagccgtagtggcactagtgcgtccttaaccattagcgggttacagtctgaagacgaagcggactattacgcagctacgtgggatgatagtttaaatggctgggtttttggtggtggtactaaagttacggtgctgtccgcaggtggtagcgccatgggctttaccgcgaacatcaaaggtctgactcaggcttcccgtaacgctaacgacggtatctccattgcgcagaccactgaaggcgcgctgaacgaaatcaacaacaacctgcagcgtgtgcgtgaactggcggttcagtctgctaacagcaccaactcccagtctgacctcgactccatccaggctgaaatcacccagcgcctgaacgaaatcgaccgtgtatccggccagactcagttcaacggcgtgaaagtcctggcgcaggacaacaccctgaccatccaggttggtgccaacgacggtgaaactatcgatatcgatctgaagcagatcaactctcagaccctgggtctggatacgctgaatgtgcaacaaaaatataaggtcagcgatacggctgcaactgttacaggatatgccgatactacgattgctttagacaatagtacttttaaagcctcggctactggtcttggtggtactgaccagaaaattgatggcgatttaaaatttgatgatacgactggaaaatattacgccaaagttaccgttacggggggaactggtaaagatggctattatgaagtttccgttgataagacgaacggtgaggtgactcttgctggcggtgcgacttccc cgcttacaggtggactacctgcgacagcaactgaggatgtgaaaaatgtacaagttgcaaatgctgatttgacagaggctaaagccgcattgacagcagcaggtgttaccggcacagcatctgttgttaagatgtcttatactgataataacggtaaaactattgatggtggtttagcagttaaggtaggcgatgattactattctgcaactcaaaataaagatggttccataagtattaatactacgaaatacactgcagatgacggtacatccaaaactgcactaaacaaactgggtggcgcagacggcaaaaccgaagttgtttctattggtggtaaaacttacgctgcaagtaaagccgaaggtcacaactttaaagcacagcctgatctggcggaagcggctgctacaaccaccgaaaacccgctgcagaaaattgatgctgctttggcacaggttgacacgttacgttctgacctgggtgcggtacagaaccgtttcaactccgctattaccaacctgggcaacaccgtaaacaacctgacttctgcccgtctcgagcaccaccaccaccaccactga ΔFliC (C) -V _L 12.3 ^cys 21 Amino acid sequence MGFTANIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAEITQRLNEIDRVSGQTQFNGVKVLAQDNTLTIQVGANDGETIDIDLKQINSQTLGLDTLNVQQKYKVSDTAATVTGYADTTIALDNSTFKASATGLGGTDQKIDGDLKFDDTTGKYYAKVTVTGGTGKDGYYEVSVDKTNGEVTLAGGATSPLTGGLPATATEDVKNVQVANADLTEAKAALTAAGVTGTASVVKMSYTDNNGKTIDGGLAVKVGDDYYSATQNKDGSISINTTKYTADDGTSKTALNKLGGADGKTEVVSIGGKTYAASKAEGHNFKAQPDLAEAAATTTENPLQKIDAALAQVDTLRSDLGAVQNRFNSAITNLGNTVNNLTSARLEGGSMGSQPVLTQSPSVSAAPRQRVTISVSGSNSNIGSNTVNWIQQLPGRAPELLMCDDDLLAPGVSDRFSGSRSGTSASLTISGLQSEDEADYYAATWDDSLNGWVFGGGTKVTVLSALEHHHHHH 22 Sequence atgggctttaccgcgaacatcaaaggtctgactcaggcttcccgtaacgctaacgacggtatctccattgcgcagaccactgaaggcgcgctgaacgaaatcaacaacaacctgcagcgtgtgcgtgaactggcggttcagtctgctaacagcaccaactcccagtctgacctcgactccatccaggctgaaatcacccagcgcctgaacgaaatcgaccgtgtatccggccagactcagttcaacggcgtgaaagtcctggcgcaggacaacaccctgaccatccaggttggtgccaacgacggtgaaactatcgatatcgatctgaagcagatcaactctcagaccctgggtctggatacgctgaatgtgcaacaaaaatataaggtcagcgatacggctgcaactgttacaggatatgccgatactacgattgctttagacaatagtacttttaaagcctcggctactggtcttggtggtactgaccagaaaattgatggcgatttaaaatttgatgatacgactggaaaatattacgccaaagttaccgttacggggggaactggtaaagatggctattatgaagtttccgttgataagacgaacggtgaggtgactcttgctggcggtgcgacttccccgcttacaggtggactacctgcgacagcaactgaggatgtgaaaaatgtacaagttgcaaatgctgatttgacagaggctaaagccgcattgacagcagcaggtgttaccggcacagcatctgttgttaagatgtcttatactgataataacggtaaaactattgatggtggtttagcagttaaggtaggcgatgattactattctgcaactcaaaataaagatggttccataagtattaatactacgaaatacactgcagatgacggtacatccaaaactgcactaaacaaactgggtggcgcagacggcaaaaccgaagttgtttctattggtggtaaaacttacgctgcaagta aagccgaaggtcacaactttaaagcacagcctgatctggcggaagcggctgctacaaccaccgaaaacccgctgcagaaaattgatgctgctttggcacaggttgacacgttacgttctgacctgggtgcggtacagaaccgtttcaactccgctattaccaacctgggcaacaccgtaaacaacctgacttctgcccgtctcgagggtggtagcatgggtagtcaaccagtacttacacaaagcccgtctgtgtctgccgctccacgtcagagagtcaccatctcagttagtggttctaattcaaatataggatcgaacacagtgaactggattcagcagttgcctggccgtgccccggagttattaatgtgtgatgatgatttattagcacccggagtatcagatcgcttttcaggaagccgtagtggcactagtgcgtccttaaccattagcgggttacagtctgaagacgaagcggactattacgcagctacgtgggatgatagtttaaatggctgggtttttggtggtggtactaaagttacggtgctgtccgcactcgagcaccaccaccaccaccactga ΔFliC (I) -V _L 12.3 ^cys 23 Amino acid sequence MGFTANIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAEITQRLNEIDRVSGQTQFNGVKVLAQDNTLTIQVGANDGETIDIDLKQINSQTLGLDTLNVQQKYKVSDTAATVTGYADTTIALDNSTFKASATGLGGTDQKIDGDLKFDDTTGKYYAKVTVTGGSGGSMGSQPVLTQSPSVSAAPRQRVTISVSGSNSNIGSNTVNWIQQLPGRAPELLMCDDDLLAPGVSDRFSGSRSGTSASLTISGLQSEDEADYYAATWDDSLNGWVFGGGTKVTVLSAGHMLVPRGSGGSVDGTGKDGYYEVSVDKTNGEVTLAGGATSPLTGGLPATATEDVKNVQVANADLTEAKAALTAAGVTGTASVVKMSYTDNNGKTIDGGLAVKVGDDYYSATQNKDGSISINTTKYTADDGTSKTALNKLGGADGKTEVVSIGGKTYAASKAEGHNFKAQPDLAEAAATTTENPLQKIDAALAQVDTLRSDLGAVQNRFNSAITNLGNTVNNLTSARLEHHHHHH 24 Sequence atgggctttaccgcgaacatcaaaggtctgactcaggcttcccgtaacgctaacgacggtatctccattgcgcagaccactgaaggcgcgctgaacgaaatcaacaacaacctgcagcgtgtgcgtgaactggcggttcagtctgctaacagcaccaactcccagtctgacctcgactccatccaggctgaaatcacccagcgcctgaacgaaatcgaccgtgtatccggccagactcagttcaacggcgtgaaagtcctggcgcaggacaacaccctgaccatccaggttggtgccaacgacggtgaaactatcgatatcgatctgaagcagatcaactctcagaccctgggtctggatacgctgaatgtgcaacaaaaatataaggtcagcgatacggctgcaactgttacaggatatgccgatactacgattgctttagacaatagtacttttaaagcctcggctactggtcttggtggtactgaccagaaaattgatggcgatttaaaatttgatgatacgactggaaaatattacgccaaagttaccgttacggggggatccggtggtagcatgggtagtcaaccagtacttacacaaagcccgtctgtgtctgccgctccacgtcagagagtcaccatctcagttagtggttctaattcaaatataggatcgaacacagtgaactggattcagcagttgcctggccgtgccccggagttattaatgtgtgatgatgatttattagcacccggagtatcagatcgcttttcaggaagccgtagtggcactagtgcgtccttaaccattagcgggttacagtctgaagacgaagcggactattacgcagctacgtgggatgatagtttaaatggctgggtttttggtggtggtactaaagttacggtgctgtccgcaggtcatatgctggtgccgcgcggcagcggtggtagcgtcgacggaactggtaaagatggctattatgaagtttccgttg ataagacgaacggtgaggtgactcttgctggcggtgcgacttccccgcttacaggtggactacctgcgacagcaactgaggatgtgaaaaatgtacaagttgcaaatgctgatttgacagaggctaaagccgcattgacagcagcaggtgttaccggcacagcatctgttgttaagatgtcttatactgataataacggtaaaactattgatggtggtttagcagttaaggtaggcgatgattactattctgcaactcaaaataaagatggttccataagtattaatactacgaaatacactgcagatgacggtacatccaaaactgcactaaacaaactgggtggcgcagacggcaaaaccgaagttgtttctattggtggtaaaacttacgctgcaagtaaagccgaaggtcacaactttaaagcacagcctgatctggcggaagcggctgctacaaccaccgaaaacccgctgcagaaaattgatgctgctttggcacaggttgacacgttacgttctgacctgggtgcggtacagaaccgtttcaactccgctattaccaacctgggcaacaccgtaaacaacctgacttctgcccgtctcgagcaccaccaccaccaccactga

The cloned vector was transformed into E. coli BL21 (DE3) strain and when the OD ₆₀₀ value of the bacteria reached 0.7, 1 mM IPTG was added to induce fusion protein production. After 3 hours of incubation at 37 ° C, E. coli (DE3) was harvested by centrifugation at 4000 rpm for 30 minutes at 4 ° C.

Harvested cell pellets are resuspended in lysis buffer containing 20 mM Tris (pH 8.0), 200 mM NaCl, 10 mM β-mercaptoethanol and 0.1 mM PMSF, followed by a microfluidizer. (microfludizer, Microfluidics, Westwood, Mass.) was used to homogenize. After centrifugation at 4 ° C. at 15,000 rpm for 40 minutes, the proteins were purified by Ni-NTA chelating agarose CL-6B (Incospharm, Daejeon, Korea) and Q-Sepharose (GE Healthcare, Pittsburgh, PA) anion exchange columns. It was. Fractions containing flagellin-V _L 12.3 ^cys fusion protein were obtained and concentrated.

< Experimental Example  1> Preparation of Nanoparticles

The exact concentration of each protein prepared in Examples 2 to 4 was calculated by SDS-PAGE and densitometry. First, the monomer fusion protein prepared in Example 2 was converted into cage-type ferritin self-assembly and I3-01 self-assembled. Was prepared. Nanoparticles were prepared by incorporating the antigen or flagellin prepared in Examples 3 and 4 into the self-assembly by combining the prepared self-assembly with the combinations shown in Table 9 below.

Mixture Type product HTT ^cys -Ftn self-assembly,
ΔFliC (I) -V _L 12.3 ^cys , HA-V _L 12.3 ^cys Ftn-HA-ΔFliC (I) HTT ^cys -Ftn self-assembly,
ΔFliC (I) -V _L 12.3 ^cys , PA-V _L 12.3 ^cys Ftn-PA-ΔFliC (I) HTT ^cys -I3-01 self-assembly,
ΔFliC (I) -V _L 12.3 ^cys , HA-V _L 12.3 ^cys I3-01-HA-ΔFliC (I)

The prepared nanoparticles were further purified by Superdex 200 (GE Healthcare) size exclusion chromatography using DPBS (Dulbecco's phosphate-buffered saline, Welgene) as a buffer. Fractions containing the desired nanoparticles were collected and concentrated, endotoxin was removed using High-Capacity Endotoxin Removal Resin (Pierce, Thermo Scientific, San Jose, Calif.), And the concentrated protein solution was filtered sterilized. Self-assembled nanoparticles were identified through size exclusion chromatography.

As can be seen in the size exclusion chromatography elution profile, PA, HA and flagellin stably bound to ferritin autoassembly, and HA and flagellin also stably bound to I3-01 self-assembly (FIG. 3).

< Experimental Example  2> As an adjuvant Flagellin  Select

< Experimental Example  2-1> With FliC On ΔFliC  According TLR5  Activation measurement

To investigate the effect of the D0 domain in flagellin as an adjuvant, full-length flagellin (FliC) containing all of D0 to D3 and flagellin cleaved from the D0 domain including D1 to D3 TLR5 activation ability of (ΔFliC) was measured. To this end, in the ferritin self-assembly prepared from the monomer of the HTT ^cys- Ftn fusion protein prepared in Example 2-1, V _L 12.3 ^cys -FliC (N) and V _L 12.3 ^cys -ΔFliC (N) prepared in Example 4 ) Were mixed to prepare Ftn-FliC (N) and Ftn-ΔFliC (N).

The procedure for measuring TLR5 activation is as follows. 2 × 10 ⁵ HEK293 cells in 24-well plates were prepared using X-tremeGENE HP DNA transfection reagent (Roche, Basel, Switzerland) at 500 ng of hTLR5 expression vector (pcDNA3.1 / hTLR5), 200 ng pBII firefly NF κB reporter vector and 10 ng pRL-TK Renilla internal control vector were transformed. After 24 hours, Ftn-ΔFliC (N) and Ftn-FliC (N) were treated and further incubated for 18 hours. Relative luciferase activity (RLU, firefly / renilla) of each cell was analyzed according to manufacturer's protocol with dual luciferase reporter assay (Promega, Madison, WI) kit and VICTOR 3 multilabel plate reader (Wallac 1420, Perkin Elmer) Results were normalized to RLU of samples treated with DPBS buffer.

As a result, full-length flagellin (FliC) showed only 1.4-2.3 times more activity than truncated flagellin (ΔFliC) (FIG. 4).

< Experimental Example  2-2> With FliC To ΔFliC  According IgG  Numerical measurement

To compare the immune response according to the in vitro (in vitro) full-length (full-length) Plastic jelrin (FliC) and cutting the Plastic jelrin (ΔFliC). To this end, ferritin self-assembly composed of HTT ^cys- Ftn fusion protein similarly as described in <Experimental Example 2-1>, as described in Table 10, HA-V _L 12.3 ^cys and ΔFliC (N) -V _L 12.3 ^cys or HA-V _L 12.3 ^cys and FliC (N) -V _L 12.3 ^cys were mixed to prepare Ftn-HA-ΔFliC (N) and Ftn-HA-FliC (N).

Mixture Type product HTT ^cys -Ftn self-assembly,
ΔFliC (N) -V _L 12.3 ^cys , HA-V _L 12.3 ^cys Ftn-HA-ΔFliC (N)
(Ftn: HA: ΔFliC = 24: 10: 2.5) HTT ^cys -Ftn self-assembly,
FliC (N) -V _L 12.3 ^cys , HA-V _L 12.3 ^cys Ftn-HA-FliC (N)
(Ftn: HA: FliC = 24: 10: 2.5)

The molar ratio of HA antigen or flagellin bound to ferritin (Table 10) prior to injection into the mice was confirmed using SDS-PAGE and densitometry. The molar ratio of ferritin and HA in the assembled nanoparticles was 24:10.

After obtaining approval from the Chungnam National University Experimental Animal Management Committee, purchased from Daehan Biolink (Eumseong, Korea), 6-week-old female BALB / c mice were divided into two groups of 5 dogs each and then Ftn-HA- Two subcutaneous injections were made on the right side with PBS solution (concentration of HA, 5.2 μg / 100 μl PBS) containing ΔFliC (N) and Ftn-HA-FliC (N).

Serum IgG (Total IgG, IgG ₁ and IgG _2a ) levels after blood sampling from the eye one week before the first immunization and two weeks after the first immunization and priming and boosting as described below. Measured by ELISA. Wells of a 96 well plate (microtiter plate; Komabiotech., Seoul, Korea) were coated overnight at 4 ° C. with 100 μl of 10 μg / ml HA coating buffer (pH 9.6, Komabiotech), followed by wash buffer (0.05% Tween). 1 × PBS) and blocked with PBS containing 1% BSA for 2 hours at room temperature. The collected serum was diluted 1: 100 in PBS containing 0.1% BSA and an additional 10-fold serial diluted sample was added to the BSA-blocked wells. After reacting for 2 hours at room temperature, the plates were washed three times with wash buffer. Conjugated with goat anti-mouse IgG, IgG ₁ , IgG _2a or IgG _2b bound to Horseradish peroxidase (Abcam, Cambridge, UK) was 1: 10,000 in PBS containing 0.1% BSA. Diluted with and added. After reacting for 2 hours at room temperature, the plates were washed again three times with wash buffer and 100 μl TMB developer (Komabiotech) was added. After 10 minutes the reaction was stopped with 100 μl of 0.5 M sulfuric acid and the color was measured at 450 nm with a micro plate reader (Tecan GENios Pro, Mannedorf, Switzerland). The most diluted sample showing OD ₄₅₀ twice as high as pre-immunized mouse serum diluted 1: 100 was defined as the final antibody titer of the antibody.

As a result, serum IgG ₁ levels were similar for both Ftn-HA-ΔFliC (N) and Ftn-HA-FliC (N) (FIG. 5). Upon Ftn-HA-ΔFliC (N) treatment, the amounts of total IgG and IgG _2a in serum showed 2.1 and 2.7 times higher levels, respectively. It was concluded that the D0 domain of flagellin had little effect on the IgG response in the flagellin-ferritin system of the present invention, and in subsequent experiments flagellin without D0 domain was used to make nanoparticles.

< Experimental Example  3> Flagellin  Fusion site ( ΔFliC (C), ΔFliC (I) and ΔFliC Measurement of TLR5 activation according to (N))

In order to identify the optimal fusion site to flagellin (ΔFliC), cage-type ferritin self-assembly was prepared from the monomer of the HTT ^cys- Ftn fusion protein prepared in Example 2-1. In the prepared self-assembly, V _L 12.3 ^cys -ΔFliC (N), V _L 12.3 ^cys -ΔFliC (C) and V _L 12.3 ^cys -ΔFliC (I) prepared in Example 4, respectively, were mixed and Ftn: FliC (ΔFliC ) Nanoparticles of Ftn-ΔFliC (N), Ftn-ΔFliC (C) and Ftn-ΔFliC (I) having a molar ratio of 24:10 were prepared and their TLR5 activation capacity was measured.

TLR5 activation was measured by Ftn-ΔFliC (N), Ftn-ΔFliC (C), Ftn-ΔFliC (I) and unbound ferritin nanoparticles (Ftn) and flagellin (ΔFliC) instead of the nanoparticles of Experimental Example 2-1. Except for using the mixture (Ftn + ΔFliC), it was carried out in the same manner as described in Experimental Example 2-1.

As a result, the unbound mixture of ferritin and flagellin (ΔFliC) showed negligible activity, and all of the flagellin-bound ferritin nanoparticles activated TLR5 in a concentration dependent manner (FIG. 6). Particularly in the case of Ftn-ΔFliC (N) or Ftn-ΔFliC (I), TLR5 was activated 1.3 to 1.9 times higher than Ftn-ΔFliC (C), indicating that the binding of N-terminal or D3 domains of ΔFliC with ferritin It is very effective in stimulating TLR5 signaling pathway. Since the D3 domain is not involved in the interaction with TLR5, in subsequent experiments, ferritin nanoparticles fused with _VL 12.3 ^cys domain between 238G and 238G of ΔFliC were selected.

< Experimental Example  4> Flagellin Combined  Ferritin Nanoparticles  Immune response

Since the flagellin-ferritin nanoparticles in Experimental Example 3 activated TLR5 more effectively than the flagellin-unbound nanoparticles, it was investigated whether the flagellin-ferritin nanoparticles can show a stronger adjuvant response.

To this end, ferritin self-assembly was prepared from the HTT ^cys- Ftn fusion protein prepared in Example 2-1. Ftn-ΔFliC (I), Ftn-HA and Ftn-HA-ΔFliC (I) were prepared by mixing the antigen or flagellin prepared in Examples 3 and 4 with the prepared self-assembly. .

Mixture Type product HTT ^cys -Ftn self-assembly, ΔFliC (I) -V _L 12.3 ^cys Ftn-ΔFliC (I)
(Ftn: ΔFliC = 24:10) HTT ^cys -Ftn self-assembly, HA-V _L 12.3 ^cys Ftn-ha
(Ftn: HA = 24:10) HTT ^cys -Ftn autoassembly, ΔFliC (I) -V _L 12.3 ^cys , HA-V _L 12.3 ^cys Ftn-HA-ΔFliC (I)
(Ftn: HA: ΔFliC =
24: 10: 3.3)

IgG levels in mouse serum were measured by HA, Ftn + ΔFliC (I) + HA, Ftn-ΔFliC (I) + HA, Ftn-HA + ΔFliC (I), Ftn-HA-ΔFliC instead of the nanoparticles of Experimental Example 2-2. Except for using (I), it was carried out in the same manner as described in Experimental Example 2-2. The total concentration of conjugated and non-conjugated HA was 5.2 μg per 100 μl PBS, and the molar ratio of ferritin to HA in the final assembled nanoparticles was 24:10.

Unlike observed in in vitro TLR5 activation assays, Ftn-ΔFliC (I) + HA (HA treated in unbound form) was Ftn + ΔFliC + HA (ΔFliC and HA unbounded form) Were induced a weak serum IgG response in mice to levels similar to the treatment (Fig. 7 (A)). However, Ftn-HA-ΔFliC (I) induced an 16.7 fold higher IgG response than Ftn-HA + ΔFliC (ΔFliC treated in unbound form) (FIG. 7B). These results show that fusion of antigen and flagellin elicits a stronger immune response than simple mixing of antigen and flagellin. In addition, even if HA and flagellin are not directly bound to each other, the simultaneous binding of HA and flagellin to ferritin self-assembly can induce a strong serum antibody response, and the flagellin attached to HA-ferritin nanoparticles Suggests a potent adjuvant.

< Experimental Example  5> necessary for cellular immune response Flagellin  Minimum quantity check

< Experimental Example  5-1> Of HA: ΔFliC  Molar ratio IgG  Numerical measurement

To determine the minimum amount of flagellin in HA-ferritin nanoparticles required for adjuvant activity, the immune response of mice according to the molar ratio of HA to flagellin was examined.

To this end, cage-type ferritin self-assembly was prepared from the HTT ^cys- Ftn fusion protein prepared in Example 2-1. In the prepared self-assembly, V _L 12.3 ^{cys --} ΔFliC (I) and HA-V _L 12.3 ^cys fusion proteins were mixed in the combinations shown in Table 12, and the molar ratio of HA to ΔFliC (I) was 1: 1 to 4: Ftn-HA-ΔFliC (I), which is 1, was prepared.

Mixture Type product HTT ^cys -Ftn self-assembly, HA-V _L 12.3 ^cys Ftn-ha
(Ftn: HA = 24:10) HTT ^cys -Ftn autoassembly, ΔFliC (I) -V _L 12.3 ^cys , HA-V _L 12.3 ^cys
(Mixed HA-V _L 12.3 ^cys ΔFliC (I) -V _L 12.3 ^cys = 3: 1) Ftn-HA-ΔFliC (I)
(Ftn: HA: ΔFliC = 24:10:10) HTT ^cys -Ftn autoassembly, ΔFliC (I) -V _L 12.3 ^cys , HA-V _L 12.3 ^cys
(Mixed HA-V _L 12.3 ^cys ΔFliC (I) -V _L 12.3 ^cys = 6: 1) Ftn-HA-ΔFliC (I)
(Ftn: HA: ΔFliC = 24: 10: 5) HTT ^cys -Ftn autoassembly, ΔFliC (I) -V _L 12.3 ^cys , HA-V _L 12.3 ^cys
(Mixed HA-V _L 12.3 ^cys ΔFliC (I) -V _L 12.3 ^cys = 9: 1) Ftn-HA-ΔFliC (I)
(Ftn: HA: ΔFliC = 24: 10: 3.3) HTT ^cys -Ftn autoassembly, ΔFliC (I) -V _L 12.3 ^cys , HA-V _L 12.3 ^cys
(Mixed HA-V _L 12.3 ^cys ΔFliC (I) -V _L 12.3 ^cys = 12: 1) Ftn-HA-ΔFliC (I)
(Ftn: HA: ΔFliC = 24: 10: 2.5)

IgG levels in mouse serum were measured using Ftn-HA-ΔFliC (I) having a molar ratio of Ftn-HA, HA: ΔFliC (I) of 1: 1 to 4: 1 instead of the nanoparticles of Experimental Example 2-2. , The same method as described in Experimental Example 2-2 was carried out. The total concentration of HA was 5.2 μg per 100 μl PBS and the molar ratio of ferritin to HA in the final assembled nanoparticles was 24:10.

As a result, as shown in Figure 8, the total IgG and IgG ₁ production was not significantly affected even if the molar ratio of HA: ΔFliC (I) 4: 1. However, when the molar ratio of HA to ΔFliC is 4: 1, the level of IgG _2a was 4.8 times lower than that of 1: 1. Therefore, when the molar ratio of HA: ΔFliC was 4: 1, the ratio of IgG ₁ to IgG _2a in serum increased by 4 times. Serum IgG2b levels also decreased by 35.1%.

Since class conversion to immunoglobulin IgG1 requires interleukin-4 (IL-4), and class conversion to IgG2a requires cytokines such as interferon-gamma (IFN-γ), an increase in IgG1 is an increase in Th2 response and IgG2a Is an increase in the Th1 response. The molar ratio of hemagglutinin to flagellin bound to the ferritin self-assembly was found to reduce the amount of flagellin up to 3: 1 without affecting IgG2a class conversion and Th1 response.

< Experimental Example  5-2> Of HA: ΔFliC  Measurement of cytokine secretion according to molar ratio

Class switching of immunoglobulins is primarily due to interactions with B lymphocytes and cytokines such as interferon-gamma (IFN-γ), which is required to produce specific cytokines, specifically interleukin-4 (IL-4) and IgG2a, which are required to make IgG1. Affected by action Therefore, cytokines were measured in spleen cells of mice injected with Ftn-HA or Ftn-HA-ΔFliC nanoparticles prepared in Experimental Example 5-1.

Specifically, mice were repeatedly inoculated with Ftn-HA or Ftn-HA-ΔFliC nanoparticles and splenocytes were separated after 3 weeks. Spleen cells (1 × 10 ⁶ cells) lysed with erythrocytes in RBC lysis buffer (Sigma) were dispensed into 96-well plates and then 139 15-mer peptides at 100 μg / ml concentration in HA CA (H1N1). The mixture was stimulated with 10 μg / ml (PM-INFA-HACal, JPT innovative peptide solutions, Berlin, Germany) and incubated at 37 ° C., 5% CO ₂ for 3 days. Supernatants were obtained and the amounts of INF-γ and IL-4 were measured using the ELISA kit (Komabiotech) according to the manufacturer's protocol.

As shown in FIG. 9, when splenocytes of mice injected with Ftn-HA-ΔFliC nanoparticles were stimulated with HA peptides, IFN-γ secretion was higher in the molar ratio of HA: ΔFliC than spleen cells of Ftn-HA-treated mice. Was 2.8 or 5.4 times higher at 1: 1 or 2: 1, respectively. However, the secretion of IFN-γ was significantly decreased when the molar ratio of HA: ΔFliC was 4: 1. The secretion of IL-4 also increased when ΔFliC was bound to the ferritin self-assembly, but the secretion was not sensitive to the HA: ΔFliC ratio up to 4: 1.

Overall, these results indicate that ferritin nanoparticles bound to HA and flagellin simultaneously stimulate humoral immune and antigen-specific T-cell responses, and that HA versus flagellin is not affected without affecting IgG2a class conversion and Th1 response. It is suggested that the molar ratio can be reduced to 3: 1.

< Experimental Example  5-3> of HA On the number of molecules  According IgG  Numerical check

The effect of serum IgG levels on the number of HA molecules bound to the ferritin self-assembly was investigated. To this end, a cage-type ferritin self-assembly was prepared from the HTT ^cys- Ftn protein prepared in Example 2-1, and the HA was prepared by mixing the V _L 12.3 ^cys fusion protein prepared in Example 3-1 with the prepared self-assembly. Ftn-HA (low) nanoparticles having only 4 molecules and Ftn-HA (high) nanoparticles having 24 molecules of HA were produced and their immunostimulatory activities were compared.

IgG levels in mouse serum were measured by Ftn-HA-ΔFliC (I) with a molar ratio of 3: 1 of Ftn-HA (low), Ftn-HA (high) and HA: ΔFliC (I) instead of the nanoparticles of Experiment 2-2. ) Was carried out in the same manner as described in Experimental Example 2-2.

As a result, as shown in FIG. 10, the Ftn-HA (high) nanoparticles induced stronger total IgG, IgG ₁ , IgG _2a and IgG _2b responses than the Ftn-HA (low) nanoparticles. However, IgG response other than IgG _{1 was significantly lower} than the IgG response of 3: 1 ratio of Ftn-HA-FliC (molar ratio of HA: flagellin in Ftn-HA-FliC = 3: 1). In particular, IgG _2a and IgG _2b levels induced by Ftn-HA-ΔFliC 3: 1 nanoparticles were 1.8 and 7 times higher than those induced by Ftn-HA (high) nanoparticles, respectively. These results suggest that while maximally conjugated HA is useful for inducing a humoral immune response, flagellin binds to ferritin autoassembly in order to produce highly effective nanoparticles with self-adjuvant activity. Indicates that it should be.

<110> THE INDUSTRY & ACADEMIC COOPERATION IN CHUNGNAM NATIONAL UNIVERSITY <120> Nano particle comprising self-assembled ferritin structure          conjugated with antigen peptide and adjuvant and use <130> 2018P-01-002 <160> 42 <170> KoPatentIn 3.0 <210> 1 <211> 165 <212> PRT <213> Escherichia coli <400> 1 Met Leu Lys Pro Glu Met Ile Glu Lys Leu Asn Glu Gln Met Asn Leu   1 5 10 15 Glu Leu Tyr Ser Ser Leu Leu Tyr Gln Gln Met Ser Ala Trp Cys Ser              20 25 30 Tyr His Thr Phe Glu Gly Ala Ala Ala Phe Leu Arg Arg His Ala Gln          35 40 45 Glu Glu Met Thr His Met Gln Arg Leu Phe Asp Tyr Leu Thr Asp Thr      50 55 60 Gly Asn Leu Pro Arg Ile Asn Thr Val Glu Ser Pro Phe Ala Glu Tyr  65 70 75 80 Ser Ser Leu Asp Glu Leu Phe Gln Glu Thr Tyr Lys His Glu Gln Leu                  85 90 95 Ile Thr Gln Lys Ile Asn Glu Leu Ala His Ala Ala Met Thr Asn Gln             100 105 110 Asp Tyr Pro Thr Phe Asn Phe Leu Gln Trp Tyr Val Ser Glu Gln His         115 120 125 Glu Glu Glu Lys Leu Phe Lys Ser Ile Ile Asp Lys Leu Ser Leu Ala     130 135 140 Gly Lys Ser Gly Glu Gly Leu Tyr Phe Ile Asp Lys Glu Leu Ser Thr 145 150 155 160 Leu Asp Thr Gln Asn                 165 <210> 2 <211> 14 <212> PRT <213> Homo sapiens <400> 2 Glu Lys Leu Met Lys Ala Phe Glu Ser Leu Lys Ser Phe Gln   1 5 10 <210> 3 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> HTTcys <400> 3 Glu Lys Leu Met Lys Cys Phe Glu Ser Leu Lys Ser Phe Gln   1 5 10 <210> 4 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> VL12.3 <400> 4 Met Gly Ser Gln Pro Val Leu Thr Gln Ser Pro Ser Val Ser Ala Ala   1 5 10 15 Pro Arg Gln Arg Val Thr Ile Ser Val Ser Gly Ser Asn Ser Asn Ile              20 25 30 Gly Ser Asn Thr Val Asn Trp Ile Gln Gln Leu Pro Gly Arg Ala Pro          35 40 45 Glu Leu Leu Met Tyr Asp Asp Asp Leu Leu Ala Pro Gly Val Ser Asp      50 55 60 Arg Phe Ser Gly Ser Arg Ser Gly Thr Ser Ala Ser Leu Thr Ile Ser  65 70 75 80 Gly Leu Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Ala Ala Thr Trp Asp                  85 90 95 Asp Ser Leu Asn Gly Trp Val Phe Gly Gly Gly Thr Lys Val Thr Val             100 105 110 Leu ser ala         115 <210> 5 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> VL12.3cys <400> 5 Met Gly Ser Gln Pro Val Leu Thr Gln Ser Pro Ser Val Ser Ala Ala   1 5 10 15 Pro Arg Gln Arg Val Thr Ile Ser Val Ser Gly Ser Asn Ser Asn Ile              20 25 30 Gly Ser Asn Thr Val Asn Trp Ile Gln Gln Leu Pro Gly Arg Ala Pro          35 40 45 Glu Leu Leu Met Cys Asp Asp Asp Leu Leu Ala Pro Gly Val Ser Asp      50 55 60 Arg Phe Ser Gly Ser Arg Ser Gly Thr Ser Ala Ser Leu Thr Ile Ser  65 70 75 80 Gly Leu Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Ala Ala Thr Trp Asp                  85 90 95 Asp Ser Leu Asn Gly Trp Val Phe Gly Gly Gly Thr Lys Val Thr Val             100 105 110 Leu ser ala         115 <210> 6 <211> 185 <212> PRT <213> Artificial Sequence <220> <223> HTTcys-Ftn <400> 6 Met Gly Glu Lys Leu Met Lys Cys Phe Glu Ser Leu Lys Ser Phe Gln   1 5 10 15 Gly Gly Ser Arg Met Leu Lys Pro Glu Met Ile Glu Lys Leu Asn Glu              20 25 30 Gln Met Asn Leu Glu Leu Tyr Ser Ser Leu Leu Tyr Gln Gln Met Ser          35 40 45 Ala Trp Cys Ser Tyr His Thr Phe Glu Gly Ala Ala Ala Phe Leu Arg      50 55 60 Arg His Ala Gln Glu Glu Met Thr His Met Gln Arg Leu Phe Asp Tyr  65 70 75 80 Leu Thr Asp Thr Gly Asn Leu Pro Arg Ile Asn Thr Val Glu Ser Pro                  85 90 95 Phe Ala Glu Tyr Ser Ser Leu Asp Glu Leu Phe Gln Glu Thr Tyr Lys             100 105 110 His Glu Gln Leu Ile Thr Gln Lys Ile Asn Glu Leu Ala His Ala Ala         115 120 125 Met Thr Asn Gln Asp Tyr Pro Thr Phe Asn Phe Leu Gln Trp Tyr Val     130 135 140 Ser Glu Gln His Glu Glu Glu Lys Leu Phe Lys Ser Ile Ile Asp Lys 145 150 155 160 Leu Ser Leu Ala Gly Lys Ser Gly Glu Gly Leu Tyr Phe Ile Asp Lys                 165 170 175 Glu Leu Ser Thr Leu Asp Thr Gln Asn             180 185 <210> 7 <211> 558 <212> DNA <213> Artificial Sequence <220> <223> HTTcys-Ftn <400> 7 atgggcgaaa agctgatgaa gtgtttcgag tccctcaagt ccttccaggg tggttctaga 60 atgctgaaac cagaaatgat tgaaaaactt aatgagcaga tgaacctgga actgtactct 120 tcactgcttt atcagcaaat gagcgcctgg tgcagctatc ataccttcga aggtgctgcc 180 gcgttcctgc gccgtcacgc ccaggaagag atgacgcata tgcagcgtct gtttgattac 240 ctgactgata ccggcaattt accgcgtatt aataccgttg aatctccgtt tgctgaatat 300 tcctcacttg atgaattatt ccaggaaacc tataaacacg aacaattaat cacccagaaa 360 attaacgaac tggctcatgc tgcaatgacc aatcaggact acccaacatt taatttcctg 420 cagtggtatg tttctgagca gcatgaagaa gagaaactgt tcaaatcgat tattgataaa 480 ttaagcctgg caggcaaaag cggcgaaggt ctgtatttta tcgacaaaga actctctacc 540 ctcgacacac aaaactaa 558 <210> 8 <211> 513 <212> PRT <213> Influenza A virus <400> 8 Asp Thr Leu Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Asp Thr Val   1 5 10 15 Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr His Ser Val Asn Leu              20 25 30 Leu Glu Asp Lys His Asn Gly Lys Leu Cys Lys Leu Arg Gly Val Ala          35 40 45 Pro Leu His Leu Gly Lys Cys Asn Ile Ala Gly Trp Ile Leu Gly Asn      50 55 60 Pro Glu Cys Glu Ser Leu Ser Thr Ala Ser Ser Trp Ser Tyr Ile Val  65 70 75 80 Glu Thr Pro Ser Ser Asp Asn Gly Thr Cys Tyr Pro Gly Asp Phe Ile                  85 90 95 Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser Val Ser Ser Phe Glu             100 105 110 Arg Phe Glu Ile Phe Pro Lys Thr Ser Ser Trp Pro Asn His Asp Ser         115 120 125 Asn Lys Gly Val Thr Ala Ala Cys Pro His Ala Gly Ala Lys Ser Phe     130 135 140 Tyr Lys Asn Leu Ile Trp Leu Val Lys Lys Gly Asn Ser Tyr Pro Lys 145 150 155 160 Leu Ser Lys Ser Tyr Ile Asn Asp Lys Gly Lys Glu Val Leu Val Leu                 165 170 175 Trp Gly Ile His His Pro Ser Thr Ser Ala Asp Gln Gln Ser Leu Tyr             180 185 190 Gln Asn Ala Asp Thr Tyr Val Phe Val Gly Ser Ser Arg Tyr Ser Lys         195 200 205 Lys Phe Lys Pro Glu Ile Ala Ile Arg Pro Lys Val Arg Asp Gln Glu     210 215 220 Gly Arg Met Asn Tyr Tyr Trp Thr Leu Val Glu Pro Gly Asp Lys Ile 225 230 235 240 Thr Phe Glu Ala Thr Gly Asn Leu Val Val Pro Arg Tyr Ala Phe Ala                 245 250 255 Met Glu Arg Asn Ala Gly Ser Gly Ile Ile Ile Ser Asp Thr Pro Val             260 265 270 His Asp Cys Asn Thr Thr Cys Gln Thr Pro Lys Gly Ala Ile Asn Thr         275 280 285 Ser Leu Pro Phe Gln Asn Ile His Pro Ile Thr Ile Gly Lys Cys Pro     290 295 300 Lys Tyr Val Lys Ser Thr Lys Leu Arg Leu Ala Thr Gly Leu Arg Asn 305 310 315 320 Ile Pro Ser Ile Gln Ser Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe                 325 330 335 Ile Glu Gly Gly Trp Thr Gly Met Val Asp Gly Trp Tyr Gly Tyr His             340 345 350 His Gln Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Leu Lys Ser Thr         355 360 365 Gln Asn Ala Ile Asp Glu Ile Thr Asn Lys Val Asn Ser Val Ile Glu     370 375 380 Lys Met Asn Thr Gln Phe Thr Ala Val Gly Lys Glu Phe Asn His Leu 385 390 395 400 Glu Lys Arg Ile Glu Asn Leu Asn Lys Lys Val Asp Asp Gly Phe Leu                 405 410 415 Asp Ile Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn Glu             420 425 430 Arg Thr Leu Asp Tyr His Asp Ser Asn Val Lys Asn Leu Tyr Glu Lys         435 440 445 Val Arg Ser Gln Leu Lys Asn Asn Ala Lys Glu Ile Gly Asn Gly Cys     450 455 460 Phe Glu Phe Tyr His Lys Cys Asp Asn Thr Cys Met Glu Ser Val Lys 465 470 475 480 Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu Ala Lys Leu Asn                 485 490 495 Arg Glu Glu Ile Asp Gly Val Lys Leu Glu Ser Thr Arg Ile Tyr Gln             500 505 510 Ile     <210> 9 <211> 495 <212> PRT <213> Salmonella typhimurium <400> 9 Met Ala Gln Val Ile Asn Thr Asn Ser Leu Ser Leu Leu Thr Gln Asn   1 5 10 15 Asn Leu Asn Lys Ser Gln Ser Ala Leu Gly Thr Ala Ile Glu Arg Leu              20 25 30 Ser Ser Gly Cys Arg Ile Asn Ser Ala Lys Asp Asp Ala Ala Gly Gln          35 40 45 Ala Ile Ala Asn Arg Phe Thr Ala Asn Ile Lys Gly Leu Thr Gln Ala      50 55 60 Ser Arg Asn Ala Asn Asp Gly Ile Ser Ile Ala Gln Thr Thr Glu Gly  65 70 75 80 Ala Leu Asn Glu Ile Asn Asn Asn Leu Gln Arg Val Arg Glu Leu Ala                  85 90 95 Val Gln Ser Ala Asn Ser Thr Asn Ser Gln Ser Asp Leu Asp Ser Ile             100 105 110 Gln Ala Glu Ile Thr Gln Arg Leu Asn Glu Ile Asp Arg Val Ser Gly         115 120 125 Gln Thr Gln Phe Asn Gly Val Lys Val Leu Ala Gln Asp Asn Thr Leu     130 135 140 Thr Ile Gln Val Gly Ala Asn Asp Gly Glu Thr Ile Asp Ile Asp Leu 145 150 155 160 Lys Gln Ile Asn Ser Gln Thr Leu Gly Leu Asp Thr Leu Asn Val Gln                 165 170 175 Gln Lys Tyr Lys Val Ser Asp Thr Ala Ala Thr Val Thr Gly Tyr Ala             180 185 190 Asp Thr Thr Ile Ala Leu Asp Asn Ser Thr Phe Lys Ala Ser Ala Thr         195 200 205 Gly Leu Gly Gly Thr Asp Gln Lys Ile Asp Gly Asp Leu Lys Phe Asp     210 215 220 Asp Thr Thr Gly Lys Tyr Tyr Ala Lys Val Thr Val Thr Gly Gly Thr 225 230 235 240 Gly Lys Asp Gly Tyr Tyr Glu Val Ser Val Asp Lys Thr Asn Gly Glu                 245 250 255 Val Thr Leu Ala Gly Gly Ala Thr Ser Pro Leu Thr Gly Gly Leu Pro             260 265 270 Ala Thr Ala Thr Glu Asp Val Lys Asn Val Gln Val Ala Asn Ala Asp         275 280 285 Leu Thr Glu Ala Lys Ala Ala Leu Thr Ala Ala Gly Val Thr Gly Thr     290 295 300 Ala Ser Val Val Lys Met Ser Tyr Thr Asp Asn Asn Gly Lys Thr Ile 305 310 315 320 Asp Gly Gly Leu Ala Val Lys Val Gly Asp Asp Tyr Tyr Ser Ala Thr                 325 330 335 Gln Asn Lys Asp Gly Ser Ile Ser Ile Asn Thr Thr Lys Tyr Thr Ala             340 345 350 Asp Asp Gly Thr Ser Lys Thr Ala Leu Asn Lys Leu Gly Gly Ala Asp         355 360 365 Gly Lys Thr Glu Val Val Ser Ile Gly Gly Lys Thr Tyr Ala Ala Ser     370 375 380 Lys Ala Glu Gly His Asn Phe Lys Ala Gln Pro Asp Leu Ala Glu Ala 385 390 395 400 Ala Ala Thr Thr Thr Glu Asn Pro Leu Gln Lys Ile Asp Ala Ala Leu                 405 410 415 Ala Gln Val Asp Thr Leu Arg Ser Asp Leu Gly Ala Val Gln Asn Arg             420 425 430 Phe Asn Ser Ala Ile Thr Asn Leu Gly Asn Thr Val Asn Asn Leu Thr         435 440 445 Ser Ala Arg Ser Arg Ile Glu Cys Ser Asp Tyr Ala Thr Glu Val Ser     450 455 460 Asn Met Ser Arg Ala Gln Ile Leu Gln Gln Ala Gly Thr Ser Val Leu 465 470 475 480 Ala Gln Ala Asn Gln Val Pro Gln Asn Val Leu Ser Leu Leu Arg                 485 490 495 <210> 10 <211> 442 <212> PRT <213> Artificial Sequence <220> <223> truncated flagellin <400> 10 Phe Thr Ala Asn Ile Lys Gly Leu Thr Gln Ala Ser Arg Asn Ala Asn   1 5 10 15 Asp Gly Ile Ser Ile Ala Gln Thr Thr Glu Gly Ala Leu Asn Glu Ile              20 25 30 Asn Asn Asn Leu Gln Arg Val Arg Glu Leu Ala Val Gln Ser Ala Asn          35 40 45 Ser Thr Asn Ser Gln Ser Asp Leu Asp Ser Ile Gln Ala Glu Ile Thr      50 55 60 Gln Arg Leu Asn Glu Ile Asp Arg Val Ser Gly Gln Thr Gln Phe Asn  65 70 75 80 Gly Val Lys Val Leu Ala Gln Asp Asn Thr Leu Thr Ile Gln Val Gly                  85 90 95 Ala Asn Asp Gly Glu Thr Ile Asp Ile Asp Leu Lys Gln Ile Asn Ser             100 105 110 Gln Thr Leu Gly Leu Asp Thr Leu Asn Val Gln Gln Lys Tyr Lys Val         115 120 125 Ser Asp Thr Ala Ala Thr Val Thr Gly Tyr Ala Asp Thr Thr Ile Ala     130 135 140 Leu Asp Asn Ser Thr Phe Lys Ala Ser Ala Thr Gly Leu Gly Gly Thr 145 150 155 160 Asp Gln Lys Ile Asp Gly Asp Leu Lys Phe Asp Asp Thr Thr Gly Lys                 165 170 175 Tyr Tyr Ala Lys Val Thr Val Thr Gly Gly Thr Gly Lys Asp Gly Tyr             180 185 190 Tyr Glu Val Ser Val Asp Lys Thr Asn Gly Glu Val Thr Leu Ala Gly         195 200 205 Gly Ala Thr Ser Pro Leu Thr Gly Gly Leu Pro Ala Thr Ala Thr Glu     210 215 220 Asp Val Lys Asn Val Gln Val Ala Asn Ala Asp Leu Thr Glu Ala Lys 225 230 235 240 Ala Ala Leu Thr Ala Ala Gly Val Thr Gly Thr Ala Ser Val Val Lys                 245 250 255 Met Ser Tyr Thr Asp Asn Asn Gly Lys Thr Ile Asp Gly Gly Leu Ala             260 265 270 Val Lys Val Gly Asp Asp Tyr Tyr Ser Ala Thr Gln Asn Lys Asp Gly         275 280 285 Ser Ile Ser Ile Asn Thr Thr Lys Tyr Thr Ala Asp Asp Gly Thr Ser     290 295 300 Lys Thr Ala Leu Asn Lys Leu Gly Gly Ala Asp Gly Lys Thr Glu Val 305 310 315 320 Val Ser Ile Gly Gly Lys Thr Tyr Ala Ala Ser Lys Ala Glu Gly His                 325 330 335 Asn Phe Lys Ala Gln Pro Asp Leu Ala Glu Ala Ala Ala Thr Thr Thr             340 345 350 Glu Asn Pro Leu Gln Lys Ile Asp Ala Ala Leu Ala Gln Val Asp Thr         355 360 365 Leu Arg Ser Asp Leu Gly Ala Val Gln Asn Arg Phe Asn Ser Ala Ile     370 375 380 Thr Asn Leu Gly Asn Thr Val Asn Asn Leu Thr Ser Ala Arg Ser Arg 385 390 395 400 Ile Glu Cys Ser Asp Tyr Ala Thr Glu Val Ser Asn Met Ser Arg Ala                 405 410 415 Gln Ile Leu Gln Gln Ala Gly Thr Ser Val Leu Ala Gln Ala Asn Gln             420 425 430 Val Pro Gln Asn Val Leu Ser Leu Leu Arg         435 440 <210> 11 <211> 238 <212> PRT <213> Artificial Sequence <220> <223> HTTcys-I3-01 <400> 11 Met His His His His His Gly Gly Ser Gly Gly Ser Glu Lys Leu   1 5 10 15 Met Lys Cys Phe Glu Ser Leu Lys Ser Phe Gln Gly Gly Ser Gly Gly              20 25 30 Ser Met Lys Met Glu Glu Leu Phe Lys Lys His Lys Ile Val Ala Val          35 40 45 Leu Arg Ala Asn Ser Val Glu Glu Ala Lys Lys Lys Ala Leu Ala Val      50 55 60 Phe Leu Gly Gly Val His Leu Ile Glu Ile Thr Phe Thr Val Pro Asp  65 70 75 80 Ala Asp Thr Val Ile Lys Glu Leu Ser Phe Leu Lys Glu Met Gly Ala                  85 90 95 Ile Ile Gly Ala Gly Thr Val Thr Ser Val Glu Gln Cys Arg Lys Ala             100 105 110 Val Glu Ser Gly Ala Glu Phe Ile Val Ser Pro His Leu Asp Glu Glu         115 120 125 Ile Ser Gln Phe Cys Lys Glu Lys Gly Val Phe Tyr Met Pro Gly Val     130 135 140 Met Thr Pro Thr Glu Leu Val Lys Ala Met Lys Leu Gly His Thr Ile 145 150 155 160 Leu Lys Leu Phe Pro Gly Glu Val Val Gly Pro Gln Phe Val Lys Ala                 165 170 175 Met Lys Gly Pro Phe Pro Asn Val Lys Phe Val Pro Thr Gly Gly Val             180 185 190 Asn Leu Asp Asn Val Cys Glu Trp Phe Lys Ala Gly Val Leu Ala Val         195 200 205 Gly Val Gly Ser Ala Leu Val Lys Gly Thr Pro Val Glu Val Ala Glu     210 215 220 Lys Ala Lys Ala Phe Val Glu Lys Ile Arg Gly Cys Thr Glu 225 230 235 <210> 12 <211> 717 <212> DNA <213> Artificial Sequence <220> <223> HTTcys-I3-01 <400> 12 atgcatcatc atcatcatca cggtggaagc ggtggaagcg aaaagctgat gaagtgtttc 60 gagtccctca agtccttcca gggtggtagc ggcggttcta tgaagatgga agagctgttc 120 aagaaacaca agatcgttgc cgtgctgcgt gccaatagtg tggaagaagc gaaaaagaaa 180 gcgctggcgg ttttcctggg cggcgttcat ctgattgaaa ttacctttac cgtgccggat 240 gcggataccg tgattaagga actgagcttt ctgaaggaaa tgggcgcgat tattggtgcg 300 ggcaccgtga ccagcgtgga gcagtgccgt aaagcggtgg aaagtggcgc cgaattcatt 360 gtgagtccgc acctggacga ggaaattagc caattttgca aggagaaggg tgtgttctat 420 atgccaggcg ttatgacccc gaccgaactg gtgaaagcca tgaaactggg ccataccatc 480 ttaaaactgt ttccgggtga ggtggtgggt ccgcagtttg ttaaagcgat gaaaggtccg 540 tttccgaatg tgaaatttgt gccaaccggc ggtgttaatc tggacaatgt gtgcgaatgg 600 ttcaaagcgg gcgtgctggc cgtgggcgtg ggcagcgcgt tagtgaaagg caccccggtg 660 gaagtggcgg aaaaggccaa ggcgttcgtt gagaagattc gtggctgcac cgaataa 717 <210> 13 <211> 644 <212> PRT <213> Artificial Sequence <220> <223> HA-VL12.3cys <400> 13 Ala Asp Pro Asp Thr Leu Cys Ile Gly Tyr His Ala Asn Asn Ser Thr   1 5 10 15 Asp Thr Val Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr His Ser              20 25 30 Val Asn Leu Leu Glu Asp Lys His Asn Gly Lys Leu Cys Lys Leu Arg          35 40 45 Gly Val Ala Pro Leu His Leu Gly Lys Cys Asn Ile Ala Gly Trp Ile      50 55 60 Leu Gly Asn Pro Glu Cys Glu Ser Leu Ser Thr Ala Ser Ser Trp Ser  65 70 75 80 Tyr Ile Val Glu Thr Pro Ser Ser Asp Asn Gly Thr Cys Tyr Pro Gly                  85 90 95 Asp Phe Ile Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser Val Ser             100 105 110 Ser Phe Glu Arg Phe Glu Ile Phe Pro Lys Thr Ser Ser Trp Pro Asn         115 120 125 His Asp Ser Asn Lys Gly Val Thr Ala Ala Cys Pro His Ala Gly Ala     130 135 140 Lys Ser Phe Tyr Lys Asn Leu Ile Trp Leu Val Lys Lys Gly Asn Ser 145 150 155 160 Tyr Pro Lys Leu Ser Lys Ser Tyr Ile Asn Asp Lys Gly Lys Glu Val                 165 170 175 Leu Val Leu Trp Gly Ile His His Pro Ser Thr Ser Ala Asp Gln Gln             180 185 190 Ser Leu Tyr Gln Asn Ala Asp Thr Tyr Val Phe Val Gly Ser Ser Arg         195 200 205 Tyr Ser Lys Lys Phe Lys Pro Glu Ile Ala Ile Arg Pro Lys Val Arg     210 215 220 Asp Gln Glu Gly Arg Met Asn Tyr Tyr Trp Thr Leu Val Glu Pro Gly 225 230 235 240 Asp Lys Ile Thr Phe Glu Ala Thr Gly Asn Leu Val Val Pro Arg Tyr                 245 250 255 Ala Phe Ala Met Glu Arg Asn Ala Gly Ser Gly Ile Ile Ile Ser Asp             260 265 270 Thr Pro Val His Asp Cys Asn Thr Thr Cys Gln Thr Pro Lys Gly Ala         275 280 285 Ile Asn Thr Ser Leu Pro Phe Gln Asn Ile His Pro Ile Thr Ile Gly     290 295 300 Lys Cys Pro Lys Tyr Val Lys Ser Thr Lys Leu Arg Leu Ala Thr Gly 305 310 315 320 Leu Arg Asn Ile Pro Ser Ile Gln Ser Arg Gly Leu Phe Gly Ala Ile                 325 330 335 Ala Gly Phe Ile Glu Gly Gly Trp Thr Gly Met Val Asp Gly Trp Tyr             340 345 350 Gly Tyr His His Gln Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Leu         355 360 365 Lys Ser Thr Gln Asn Ala Ile Asp Glu Ile Thr Asn Lys Val Asn Ser     370 375 380 Val Ile Glu Lys Met Asn Thr Gln Phe Thr Ala Val Gly Lys Glu Phe 385 390 395 400 Asn His Leu Glu Lys Arg Ile Glu Asn Leu Asn Lys Lys Val Asp Asp                 405 410 415 Gly Phe Leu Asp Ile Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu             420 425 430 Glu Asn Glu Arg Thr Leu Asp Tyr His Asp Ser Asn Val Lys Asn Leu         435 440 445 Tyr Glu Lys Val Arg Ser Gln Leu Lys Asn Asn Ala Lys Glu Ile Gly     450 455 460 Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asp Asn Thr Cys Met Glu 465 470 475 480 Ser Val Lys Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu Ala                 485 490 495 Lys Leu Asn Arg Glu Glu Ile Asp Gly Val Lys Leu Glu Ser Thr Arg             500 505 510 Ile Tyr Gln Ile Gly Ser Arg Ser Met Gly Ser Gln Pro Val Leu Thr         515 520 525 Gln Ser Pro Ser Val Ser Ala Ala Pro Arg Gln Arg Val Thr Ile Ser     530 535 540 Val Ser Gly Ser Asn Ser Asn Ile Gly Ser Asn Thr Val Asn Trp Ile 545 550 555 560 Gln Gln Leu Pro Gly Arg Ala Pro Glu Leu Leu Met Cys Asp Asp Asp                 565 570 575 Leu Leu Ala Pro Gly Val Ser Asp Arg Phe Ser Gly Ser Arg Ser Gly             580 585 590 Thr Ser Ala Ser Leu Thr Ile Ser Gly Leu Gln Ser Glu Asp Glu Ala         595 600 605 Asp Tyr Tyr Ala Ala Thr Trp Asp Asp Ser Leu Asn Gly Trp Val Phe     610 615 620 Gly Gly Gly Thr Lys Val Thr Val Leu Ser Ala Gly Ser Ser His His 625 630 635 640 His His His His                 <210> 14 <211> 1934 <212> DNA <213> Artificial Sequence <220> <223> HA-VL12.3cys <400> 14 gcggatcccg acacattgtg cattgggtac catgctaata actcaacaga tacagtagat 60 actgtgttgg aaaaaaacgt aacggtaaca cactcagtga acttactaga agacaaacat 120 aatggaaagt tatgtaaact aagaggggtt gccccattac atctgggtaa atgcaatatt 180 gcagggtgga ttctaggaaa cccagagtgt gagtcactat ccactgcatc aagttggtca 240 tacatcgtgg aaactccaag ctcagataat ggcacatgtt atccgggaga ctttatcgat 300 tatgaggaat taagggaaca gttgagcagt gttagctcgt tcgaaagatt cgagatattc 360 cccaagacat caagttggcc aaatcatgac tccaacaaag gagtaacagc ggcatgccct 420 cacgccggag ctaaatcatt ctacaagaac ctgatttggc tagttaagaa aggcaattct 480 tacccaaaac tgtcaaagtc ttatattaat gacaaaggta aagaagtatt ggttctatgg 540 gggatacatc accccagcac aagcgctgac caacagtcac tataccagaa tgccgatact 600 tacgtatttg tggggagtag cagatattct aaaaagttca agccggaaat tgcaattagg 660 ccgaaggtga gagaccaaga aggtagaatg aattactatt ggacattagt agagccgggt 720 gataaaataa catttgaagc aactggaaat ttggtggttc caagatacgc attcgctatg 780 gagagaaacg ccggtagcgg aatcattata agtgatacac cagtccacga ctgtaacaca 840 acctgccaaa ctcccaaggg agcaataaac acgagcctac cgtttcaaaa tattcaccca 900 attacaatag ggaaatgtcc aaaatatgta aaatcaacaa agttgagact ggcaacaggg 960 ctcagaaaca taccctctat tcagtccaga ggtctatttg gggcaattgc cggattcatc 1020 gaggggggat ggacaggtat ggtcgatggc tggtatgggt accaccatca gaacgagcaa 1080 ggatctggtt acgccgcgat ctaaagtcaa cacaaaatgc aattgatgaa atcacaaata 1140 aggtaaattc agtaatagag aagatgaata cacagttcac agcagtaggc aaagaattta 1200 atcacctgga gaaaagaata gaaaacctca ataaaaaggt agacgatggt ttcttagaca 1260 tttggacata caatgcagaa ctcctagtac tactcgaaaa tgaaaggacg ctggactacc 1320 atgactcaaa cgttaaaaat ctatatgaaa aagtaagatc acagttgaaa aataatgcca 1380 aagagatagg aaatggatgc tttgaatttt accataaatg tgacaataca tgtatggaat 1440 cagttaaaaa tgggacttac gattatccaa agtatagcga agaagctaag ctgaatagag 1500 aagaaataga tggggtaaaa ctagaatcaa ctaggattta tcagattggc tctagaagca 1560 tgggtagtca accagtactt acacaaagcc cgtctgtgtc tgccgctcca cgtcagagag 1620 tcaccatctc agttagtggt tctaattcaa atataggatc gaacacagtg aactggattc 1680 agcagttgcc tggccgtgcc ccggagttat taatgtgtga tgatgattta ttagcacccg 1740 gagtatcaga tcgcttttca ggaagccgta gtggcactag tgcgtcctta accattagcg 1800 ggttacagtc tgaagacgaa gcggactatt acgcagctac gtgggatgat agtttaaatg 1860 gctgggtttt tggtggtggt actaaagtta cggtgctgtc cgcaggcagt agccatcatc 1920 atcatcatca ctaa 1934 <210> 15 <211> 269 <212> PRT <213> Artificial Sequence <220> <223> PA-VL12.3cys <400> 15 Met Gly Phe His Tyr Asp Arg Asn Asn Ile Ala Val Gly Ala Asp Glu   1 5 10 15 Ser Val Val Lys Glu Ala His Arg Glu Val Ile Asn Ser Ser Thr Glu              20 25 30 Gly Leu Leu Leu Asn Ile Asp Lys Asp Ile Arg Lys Ile Leu Ser Gly          35 40 45 Tyr Ile Val Glu Ile Glu Asp Thr Glu Gly Leu Lys Glu Val Ile Asn      50 55 60 Asp Arg Tyr Asp Met Leu Asn Ile Ser Ser Leu Arg Gln Asp Gly Lys  65 70 75 80 Thr Phe Ile Asp Phe Lys Lys Tyr Asn Asp Lys Leu Pro Leu Tyr Ile                  85 90 95 Ser Asn Pro Asn Tyr Lys Val Asn Val Tyr Ala Val Thr Lys Glu Asn             100 105 110 Thr Ile Ile Asn Pro Ser Glu Asn Gly Asp Thr Ser Thr Asn Gly Ile         115 120 125 Lys Lys Ile Leu Ile Phe Ser Lys Lys Gly Tyr Glu Ile Gly Ser Gly     130 135 140 Gly Ser Met Gly Ser Gln Pro Val Leu Thr Gln Ser Pro Ser Val Ser 145 150 155 160 Ala Ala Pro Arg Gln Arg Val Thr Ile Ser Val Ser Gly Ser Asn Ser                 165 170 175 Asn Ile Gly Ser Asn Thr Val Asn Trp Ile Gln Gln Leu Pro Gly Arg             180 185 190 Ala Pro Glu Leu Leu Met Cys Asp Asp Asp Leu Leu Ala Pro Gly Val         195 200 205 Ser Asp Arg Phe Ser Gly Ser Arg Ser Gly Thr Ser Ala Ser Leu Thr     210 215 220 Ile Ser Gly Leu Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Ala Ala Thr 225 230 235 240 Trp Asp Asp Ser Leu Asn Gly Trp Val Phe Gly Gly Gly Thr Lys Val                 245 250 255 Thr Val Leu Ser Ala Leu Glu His His His His His His His             260 265 <210> 16 <211> 810 <212> DNA <213> Artificial Sequence <220> <223> PA-VL12.3cys <400> 16 atgggctttc attatgatag aaataacata gcagttgggg cggatgagtc agtagttaag 60 gaggctcata gagaagtaat taattcgtca acagagggat tattgttaaa tattgataag 120 gatataagaa aaatattatc aggttatatt gtagaaattg aagatactga agggcttaaa 180 gaagttataa atgacagata tgatatgttg aatatttcta gtttacggca agatggaaaa 240 acatttatag attttaaaaa atataatgat aaattaccgt tatatataag taatcccaat 300 tataaggtaa atgtatatgc tgttactaaa gaaaacacta ttattaatcc tagtgagaat 360 ggggatacta gtaccaacgg gatcaagaaa attttaatct tttctaaaaa aggctatgag 420 ataggatccg gtggtagcat gggtagtcaa ccagtactta cacaaagccc gtctgtgtct 480 gccgctccac gtcagagagt caccatctca gttagtggtt ctaattcaaa tataggatcg 540 aacacagtga actggattca gcagttgcct ggccgtgccc cggagttatt aatgtgtgat 600 gatgatttat tagcacccgg agtatcagat cgcttttcag gaagccgtag tggcactagt 660 gcgtccttaa ccattagcgg gttacagtct gaagacgaag cggactatta cgcagctacg 720 tgggatgata gtttaaatgg ctgggttttt ggtggtggta ctaaagttac ggtgctgtcc 780 gcactcgagc accaccacca ccaccactga 810 <210> 17 <211> 634 <212> PRT <213> Artificial Sequence <220> <223> full-length FliC (N) -VL12.3cys <400> 17 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro   1 5 10 15 Arg Gly Ser His Met Gly Ser Gln Pro Val Leu Thr Gln Ser Pro Ser              20 25 30 Val Ser Ala Ala Pro Arg Gln Arg Val Thr Ile Ser Val Ser Gly Ser          35 40 45 Asn Ser Asn Ile Gly Ser Asn Thr Val Asn Trp Ile Gln Gln Leu Pro      50 55 60 Gly Arg Ala Pro Glu Leu Leu Met Cys Asp Asp Asp Leu Leu Ala Pro  65 70 75 80 Gly Val Ser Asp Arg Phe Ser Gly Ser Arg Ser Gly Thr Ser Ala Ser                  85 90 95 Leu Thr Ile Ser Gly Leu Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Ala             100 105 110 Ala Thr Trp Asp Asp Ser Leu Asn Gly Trp Val Phe Gly Gly Gly Thr         115 120 125 Lys Val Thr Val Leu Ser Ala Gly Gly Ser Ala Met Ala Gln Val Ile     130 135 140 Asn Thr Asn Ser Leu Ser Leu Leu Thr Gln Asn Asn Leu Asn Lys Ser 145 150 155 160 Gln Ser Ala Leu Gly Thr Ala Ile Glu Arg Leu Ser Ser Gly Cys Arg                 165 170 175 Ile Asn Ser Ala Lys Asp Asp Ala Ala Gly Gln Ala Ile Ala Asn Arg             180 185 190 Phe Thr Ala Asn Ile Lys Gly Leu Thr Gln Ala Ser Arg Asn Ala Asn         195 200 205 Asp Gly Ile Ser Ile Ala Gln Thr Thr Glu Gly Ala Leu Asn Glu Ile     210 215 220 Asn Asn Asn Leu Gln Arg Val Arg Glu Leu Ala Val Gln Ser Ala Asn 225 230 235 240 Ser Thr Asn Ser Gln Ser Asp Leu Asp Ser Ile Gln Ala Glu Ile Thr                 245 250 255 Gln Arg Leu Asn Glu Ile Asp Arg Val Ser Gly Gln Thr Gln Phe Asn             260 265 270 Gly Val Lys Val Leu Ala Gln Asp Asn Thr Leu Thr Ile Gln Val Gly         275 280 285 Ala Asn Asp Gly Glu Thr Ile Asp Ile Asp Leu Lys Gln Ile Asn Ser     290 295 300 Gln Thr Leu Gly Leu Asp Thr Leu Asn Val Gln Gln Lys Tyr Lys Val 305 310 315 320 Ser Asp Thr Ala Ala Thr Val Thr Gly Tyr Ala Asp Thr Thr Ile Ala                 325 330 335 Leu Asp Asn Ser Thr Phe Lys Ala Ser Ala Thr Gly Leu Gly Gly Thr             340 345 350 Asp Gln Lys Ile Asp Gly Asp Leu Lys Phe Asp Asp Thr Thr Gly Lys         355 360 365 Tyr Tyr Ala Lys Val Thr Val Thr Gly Gly Thr Gly Lys Asp Gly Tyr     370 375 380 Tyr Glu Val Ser Val Asp Lys Thr Asn Gly Glu Val Thr Leu Ala Gly 385 390 395 400 Gly Ala Thr Ser Pro Leu Thr Gly Gly Leu Pro Ala Thr Ala Thr Glu                 405 410 415 Asp Val Lys Asn Val Gln Val Ala Asn Ala Asp Leu Thr Glu Ala Lys             420 425 430 Ala Ala Leu Thr Ala Ala Gly Val Thr Gly Thr Ala Ser Val Val Lys         435 440 445 Met Ser Tyr Thr Asp Asn Asn Gly Lys Thr Ile Asp Gly Gly Leu Ala     450 455 460 Val Lys Val Gly Asp Asp Tyr Tyr Ser Ala Thr Gln Asn Lys Asp Gly 465 470 475 480 Ser Ile Ser Ile Asn Thr Thr Lys Tyr Thr Ala Asp Asp Gly Thr Ser                 485 490 495 Lys Thr Ala Leu Asn Lys Leu Gly Gly Ala Asp Gly Lys Thr Glu Val             500 505 510 Val Ser Ile Gly Gly Lys Thr Tyr Ala Ala Ser Lys Ala Glu Gly His         515 520 525 Asn Phe Lys Ala Gln Pro Asp Leu Ala Glu Ala Ala Ala Thr Thr Thr     530 535 540 Glu Asn Pro Leu Gln Lys Ile Asp Ala Ala Leu Ala Gln Val Asp Thr 545 550 555 560 Leu Arg Ser Asp Leu Gly Ala Val Gln Asn Arg Phe Asn Ser Ala Ile                 565 570 575 Thr Asn Leu Gly Asn Thr Val Asn Asn Leu Thr Ser Ala Arg Ser Arg             580 585 590 Ile Glu Cys Ser Asp Tyr Ala Thr Glu Val Ser Asn Met Ser Arg Ala         595 600 605 Gln Ile Leu Gln Gln Ala Gly Thr Ser Val Leu Ala Gln Ala Asn Gln     610 615 620 Val Pro Gln Asn Val Leu Ser Leu Leu Arg 625 630 <210> 18 <211> 1845 <212> DNA <213> Artificial Sequence <220> <223> full-length FliC (N) -VL12.3cys <400> 18 atgggtagtc aaccagtact tacacaaagc ccgtctgtgt ctgccgctcc acgtcagaga 60 gtcaccatct cagttagtgg ttctaattca aatataggat cgaacacagt gaactggatt 120 cagcagttgc ctggccgtgc cccggagtta ttaatgtgtg atgatgattt attagcaccc 180 ggagtatcag atcgcttttc aggaagccgt agtggcacta gtgcgtcctt aaccattagc 240 gggttacagt ctgaagacga agcggactat tacgcagcta cgtgggatga tagtttaaat 300 ggctgggttt ttggtggtgg tactaaagtt acggtgctgt ccgcaggtgg tagcgccatg 360 gcacaagtca ttaataccaa cagcctgtcg ctgttgaccc agaataacct gaacaaatcc 420 cagtccgctc tgggcaccgc tatcgagcgt ctgtcttcct gcctgcgtat caacagcgcg 480 aaagacgatg cggcaggtca ggcgattgct aaccgtttta ccgcgaacat caaaggtctg 540 actcaggctt cccgtaacgc taacgacggt atctccattg cgcagaccac tgaaggcgcg 600 ctgaacgaaa tcaacaacaa cctgcagcgt gtgcgtgaac tggcggttca gtctgctaac 660 agcaccaact cccagtctga cctcgactcc atccaggctg aaatcaccca gcgcctgaac 720 gaaatcgacc gtgtatccgg ccagactcag ttcaacggcg tgaaagtcct ggcgcaggac 780 aacaccctga ccatccaggt tggtgccaac gacggtgaaa ctatcgatat cgatctgaag 840 cagatcaact ctcagaccct gggtctggat acgctgaatg tgcaacaaaa atataaggtc 900 agcgatacgg ctgcaactgt tacaggatat gccgatacta cgattgcttt agacaatagt 960 acttttaaag cctcggctac tggtcttggt ggtactgacc agaaaattga tggcgattta 1020 aaatttgatg atacgactgg aaaatattac gccaaagtta ccgttacggg gggaactggt 1080 aaagatggct attatgaagt ttccgttgat aagacgaacg gtgaggtgac tcttgctggc 1140 ggtgcgactt ccccgcttac aggtggacta cctgcgacag caactgagga tgtgaaaaat 1200 gtacaagttg caaatgctga tttgacagag gctaaagccg cattgacagc agcaggtgtt 1260 accggcacag catctgttgt taagatgtct tatactgata ataacggtaa aactattgat 1320 ggtggtttag cagttaaggt aggcgatgat tactattctg caactcaaaa taaagatggt 1380 tccataagta ttaatactac gaaatacact gcagatgacg gtacatccaa aactgcacta 1440 aacaaactgg gtggcgcaga cggcaaaacc gaagttgttt ctattggtgg taaaacttac 1500 gctgcaagta aagccgaagg tcacaacttt aaagcacagc ctgatctggc ggaagcggct 1560 gctacaacca ccgaaaaccc gctgcagaaa attgatgctg ctttggcaca ggttgacacg 1620 ttacgttctg acctgggtgc ggtacagaac cgtttcaact ccgctattac caacctgggc 1680 aacaccgtaa acaacctgac ttctgcccgt agccgtatcg aatgctccga ctacgcgacc 1740 gaagtttcca acatgtctcg cgcgcagatt ctgcagcagg ccggtacctc cgttctggcg 1800 caggcgaacc aggttccgca aaacgtcctc tctttactgc gttga 1845 <210> 19 <211> 527 <212> PRT <213> Artificial Sequence <220> <223> truncated FliC (N) -VL12.3cys <400> 19 Met Gly Ser Gln Pro Val Leu Thr Gln Ser Pro Ser Val Ser Ala Ala   1 5 10 15 Pro Arg Gln Arg Val Thr Ile Ser Val Ser Gly Ser Asn Ser Asn Ile              20 25 30 Gly Ser Asn Thr Val Asn Trp Ile Gln Gln Leu Pro Gly Arg Ala Pro          35 40 45 Glu Leu Leu Met Cys Asp Asp Asp Leu Leu Ala Pro Gly Val Ser Asp      50 55 60 Arg Phe Ser Gly Ser Arg Ser Gly Thr Ser Ala Ser Leu Thr Ile Ser  65 70 75 80 Gly Leu Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Ala Ala Thr Trp Asp                  85 90 95 Asp Ser Leu Asn Gly Trp Val Phe Gly Gly Gly Thr Lys Val Thr Val             100 105 110 Leu Ser Ala Gly Gly Ser Ala Met Gly Phe Thr Ala Asn Ile Lys Gly         115 120 125 Leu Thr Gln Ala Ser Arg Asn Ala Asn Asp Gly Ile Ser Ile Ala Gln     130 135 140 Thr Thr Glu Gly Ala Leu Asn Glu Ile Asn Asn Asn Leu Gln Arg Val 145 150 155 160 Arg Glu Leu Ala Val Gln Ser Ala Asn Ser Thr Asn Ser Gln Ser Asp                 165 170 175 Leu Asp Ser Ile Gln Ala Glu Ile Thr Gln Arg Leu Asn Glu Ile Asp             180 185 190 Arg Val Ser Gly Gln Thr Gln Phe Asn Gly Val Lys Val Leu Ala Gln         195 200 205 Asp Asn Thr Leu Thr Ile Gln Val Gly Ala Asn Asp Gly Glu Thr Ile     210 215 220 Asp Ile Asp Leu Lys Gln Ile Asn Ser Gln Thr Leu Gly Leu Asp Thr 225 230 235 240 Leu Asn Val Gln Gln Lys Tyr Lys Val Ser Asp Thr Ala Ala Thr Val                 245 250 255 Thr Gly Tyr Ala Asp Thr Thr Ile Ala Leu Asp Asn Ser Thr Phe Lys             260 265 270 Ala Ser Ala Thr Gly Leu Gly Gly Thr Asp Gln Lys Ile Asp Gly Asp         275 280 285 Leu Lys Phe Asp Asp Thr Thr Gly Lys Tyr Tyr Ala Lys Val Thr Val     290 295 300 Thr Gly Gly Thr Gly Lys Asp Gly Tyr Tyr Glu Val Ser Val Asp Lys 305 310 315 320 Thr Asn Gly Glu Val Thr Leu Ala Gly Gly Ala Thr Ser Pro Leu Thr                 325 330 335 Gly Gly Leu Pro Ala Thr Ala Thr Glu Asp Val Lys Asn Val Gln Val             340 345 350 Ala Asn Ala Asp Leu Thr Glu Ala Lys Ala Ala Leu Thr Ala Ala Gly         355 360 365 Val Thr Gly Thr Ala Ser Val Val Lys Met Ser Tyr Thr Asp Asn Asn     370 375 380 Gly Lys Thr Ile Asp Gly Gly Leu Ala Val Lys Val Gly Asp Asp Tyr 385 390 395 400 Tyr Ser Ala Thr Gln Asn Lys Asp Gly Ser Ile Ser Ile Asn Thr Thr                 405 410 415 Lys Tyr Thr Ala Asp Asp Gly Thr Ser Lys Thr Ala Leu Asn Lys Leu             420 425 430 Gly Gly Ala Asp Gly Lys Thr Glu Val Val Ser Ile Gly Gly Lys Thr         435 440 445 Tyr Ala Ala Ser Lys Ala Glu Gly His Asn Phe Lys Ala Gln Pro Asp     450 455 460 Leu Ala Glu Ala Ala Ala Thr Thr Thr Thr Glu Asn Pro Leu Gln Lys Ile 465 470 475 480 Asp Ala Ala Leu Ala Gln Val Asp Thr Leu Arg Ser Asp Leu Gly Ala                 485 490 495 Val Gln Asn Arg Phe Asn Ser Ala Ile Thr Asn Leu Gly Asn Thr Val             500 505 510 Asn Asn Leu Thr Ser Ala Arg Leu Glu His His His His His His His         515 520 525 <210> 20 <211> 1584 <212> DNA <213> Artificial Sequence <220> <223> truncated FliC (N) -VL12.3cys <400> 20 atgggtagtc aaccagtact tacacaaagc ccgtctgtgt ctgccgctcc acgtcagaga 60 gtcaccatct cagttagtgg ttctaattca aatataggat cgaacacagt gaactggatt 120 cagcagttgc ctggccgtgc cccggagtta ttaatgtgtg atgatgattt attagcaccc 180 ggagtatcag atcgcttttc aggaagccgt agtggcacta gtgcgtcctt aaccattagc 240 gggttacagt ctgaagacga agcggactat tacgcagcta cgtgggatga tagtttaaat 300 ggctgggttt ttggtggtgg tactaaagtt acggtgctgt ccgcaggtgg tagcgccatg 360 ggctttaccg cgaacatcaa aggtctgact caggcttccc gtaacgctaa cgacggtatc 420 tccattgcgc agaccactga aggcgcgctg aacgaaatca acaacaacct gcagcgtgtg 480 cgtgaactgg cggttcagtc tgctaacagc accaactccc agtctgacct cgactccatc 540 caggctgaaa tcacccagcg cctgaacgaa atcgaccgtg tatccggcca gactcagttc 600 aacggcgtga aagtcctggc gcaggacaac accctgacca tccaggttgg tgccaacgac 660 ggtgaaacta tcgatatcga tctgaagcag atcaactctc agaccctggg tctggatacg 720 ctgaatgtgc aacaaaaata taaggtcagc gatacggctg caactgttac aggatatgcc 780 gatactacga ttgctttaga caatagtact tttaaagcct cggctactgg tcttggtggt 840 actgaccaga aaattgatgg cgatttaaaa tttgatgata cgactggaaa atattacgcc 900 aaagttaccg ttacgggggg aactggtaaa gatggctatt atgaagtttc cgttgataag 960 acgaacggtg aggtgactct tgctggcggt gcgacttccc cgcttacagg tggactacct 1020 gcgacagcaa ctgaggatgt gaaaaatgta caagttgcaa atgctgattt gacagaggct 1080 aaagccgcat tgacagcagc aggtgttacc ggcacagcat ctgttgttaa gatgtcttat 1140 actgataata acggtaaaac tattgatggt ggtttagcag ttaaggtagg cgatgattac 1200 tattctgcaa ctcaaaataa agatggttcc ataagtatta atactacgaa atacactgca 1260 gatgacggta catccaaaac tgcactaaac aaactgggtg gcgcagacgg caaaaccgaa 1320 gttgtttcta ttggtggtaa aacttacgct gcaagtaaag ccgaaggtca caactttaaa 1380 gcacagcctg atctggcgga agcggctgct acaaccaccg aaaacccgct gcagaaaatt 1440 gatgctgctt tggcacaggt tgacacgtta cgttctgacc tgggtgcggt acagaaccgt 1500 ttcaactccg ctattaccaa cctgggcaac accgtaaaca acctgacttc tgcccgtctc 1560 gagcaccacc accaccacca ctga 1584 <210> 21 <211> 528 <212> PRT <213> Artificial Sequence <220> <223> truncated FliC (C) -VL12.3cys <400> 21 Met Gly Phe Thr Ala Asn Ile Lys Gly Leu Thr Gln Ala Ser Arg Asn   1 5 10 15 Ala Asn Asp Gly Ile Ser Ile Ala Gln Thr Thr Glu Gly Ala Leu Asn              20 25 30 Glu Ile Asn Asn Asn Leu Gln Arg Val Arg Glu Leu Ala Val Gln Ser          35 40 45 Ala Asn Ser Thr Asn Ser Gln Ser Asp Leu Asp Ser Ile Gln Ala Glu      50 55 60 Ile Thr Gln Arg Leu Asn Glu Ile Asp Arg Val Ser Gly Gln Thr Gln  65 70 75 80 Phe Asn Gly Val Lys Val Leu Ala Gln Asp Asn Thr Leu Thr Ile Gln                  85 90 95 Val Gly Ala Asn Asp Gly Glu Thr Ile Asp Ile Asp Leu Lys Gln Ile             100 105 110 Asn Ser Gln Thr Leu Gly Leu Asp Thr Leu Asn Val Gln Gln Lys Tyr         115 120 125 Lys Val Ser Asp Thr Ala Ala Thr Val Thr Gly Tyr Ala Asp Thr Thr     130 135 140 Ile Ala Leu Asp Asn Ser Thr Phe Lys Ala Ser Ala Thr Gly Leu Gly 145 150 155 160 Gly Thr Asp Gln Lys Ile Asp Gly Asp Leu Lys Phe Asp Asp Thr Thr                 165 170 175 Gly Lys Tyr Tyr Ala Lys Val Thr Val Thr Gly Gly Thr Gly Lys Asp             180 185 190 Gly Tyr Tyr Glu Val Ser Val Asp Lys Thr Asn Gly Glu Val Thr Leu         195 200 205 Ala Gly Gly Ala Thr Ser Pro Leu Thr Gly Gly Leu Pro Ala Thr Ala     210 215 220 Thr Glu Asp Val Lys Asn Val Gln Val Ala Asn Ala Asp Leu Thr Glu 225 230 235 240 Ala Lys Ala Ala Leu Thr Ala Ala Gly Val Thr Gly Thr Ala Ser Val                 245 250 255 Val Lys Met Ser Tyr Thr Asp Asn Asn Gly Lys Thr Ile Asp Gly Gly             260 265 270 Leu Ala Val Lys Val Gly Asp Asp Tyr Tyr Ser Ala Thr Gln Asn Lys         275 280 285 Asp Gly Ser Ile Ser Ile Asn Thr Thr Lys Tyr Thr Ala Asp Asp Gly     290 295 300 Thr Ser Lys Thr Ala Leu Asn Lys Leu Gly Gly Ala Asp Gly Lys Thr 305 310 315 320 Glu Val Val Ser Ile Gly Gly Lys Thr Tyr Ala Ala Ser Lys Ala Glu                 325 330 335 Gly His Asn Phe Lys Ala Gln Pro Asp Leu Ala Glu Ala Ala Ala Thr             340 345 350 Thr Thr Glu Asn Pro Leu Gln Lys Ile Asp Ala Ala Leu Ala Gln Val         355 360 365 Asp Thr Leu Arg Ser Asp Leu Gly Ala Val Gln Asn Arg Phe Asn Ser     370 375 380 Ala Ile Thr Asn Leu Gly Asn Thr Val Asn Asn Leu Thr Ser Ala Arg 385 390 395 400 Leu Glu Gly Gly Ser Met Gly Ser Gln Pro Val Leu Thr Gln Ser Pro                 405 410 415 Ser Val Ser Ala Ala Pro Arg Gln Arg Val Thr Ile Ser Val Ser Gly             420 425 430 Ser Asn Ser Asn Ile Gly Ser Asn Thr Val Asn Trp Ile Gln Gln Leu         435 440 445 Pro Gly Arg Ala Pro Glu Leu Leu Met Cys Asp Asp Asp Leu Leu Ala     450 455 460 Pro Gly Val Ser Asp Arg Phe Ser Gly Ser Arg Ser Gly Thr Ser Ala 465 470 475 480 Ser Leu Thr Ile Ser Gly Leu Gln Ser Glu Asp Glu Ala Asp Tyr Tyr                 485 490 495 Ala Ala Thr Trp Asp Asp Ser Leu Asn Gly Trp Val Phe Gly Gly Gly             500 505 510 Thr Lys Val Thr Val Leu Ser Ala Leu Glu His His His His His His         515 520 525 <210> 22 <211> 1587 <212> DNA <213> Artificial Sequence <220> <223> truncated FliC (C) -VL12.3cys <400> 22 atgggcttta ccgcgaacat caaaggtctg actcaggctt cccgtaacgc taacgacggt 60 atctccattg cgcagaccac tgaaggcgcg ctgaacgaaa tcaacaacaa cctgcagcgt 120 gtgcgtgaac tggcggttca gtctgctaac agcaccaact cccagtctga cctcgactcc 180 atccaggctg aaatcaccca gcgcctgaac gaaatcgacc gtgtatccgg ccagactcag 240 ttcaacggcg tgaaagtcct ggcgcaggac aacaccctga ccatccaggt tggtgccaac 300 gacggtgaaa ctatcgatat cgatctgaag cagatcaact ctcagaccct gggtctggat 360 acgctgaatg tgcaacaaaa atataaggtc agcgatacgg ctgcaactgt tacaggatat 420 gccgatacta cgattgcttt agacaatagt acttttaaag cctcggctac tggtcttggt 480 ggtactgacc agaaaattga tggcgattta aaatttgatg atacgactgg aaaatattac 540 gccaaagtta ccgttacggg gggaactggt aaagatggct attatgaagt ttccgttgat 600 aagacgaacg gtgaggtgac tcttgctggc ggtgcgactt ccccgcttac aggtggacta 660 cctgcgacag caactgagga tgtgaaaaat gtacaagttg caaatgctga tttgacagag 720 gctaaagccg cattgacagc agcaggtgtt accggcacag catctgttgt taagatgtct 780 tatactgata ataacggtaa aactattgat ggtggtttag cagttaaggt aggcgatgat 840 tactattctg caactcaaaa taaagatggt tccataagta ttaatactac gaaatacact 900 gcagatgacg gtacatccaa aactgcacta aacaaactgg gtggcgcaga cggcaaaacc 960 gaagttgttt ctattggtgg taaaacttac gctgcaagta aagccgaagg tcacaacttt 1020 aaagcacagc ctgatctggc ggaagcggct gctacaacca ccgaaaaccc gctgcagaaa 1080 attgatgctg ctttggcaca ggttgacacg ttacgttctg acctgggtgc ggtacagaac 1140 cgtttcaact ccgctattac caacctgggc aacaccgtaa acaacctgac ttctgcccgt 1200 ctcgagggtg gtagcatggg tagtcaacca gtacttacac aaagcccgtc tgtgtctgcc 1260 gctccacgtc agagagtcac catctcagtt agtggttcta attcaaatat aggatcgaac 1320 acagtgaact ggattcagca gttgcctggc cgtgccccgg agttattaat gtgtgatgat 1380 gatttattag cacccggagt atcagatcgc ttttcaggaa gccgtagtgg cactagtgcg 1440 tccttaacca ttagcgggtt acagtctgaa gacgaagcgg actattacgc agctacgtgg 1500 gatgatagtt taaatggctg ggtttttggt ggtggtacta aagttacggt gctgtccgca 1560 ctcgagcacc accaccacca ccactga 1587 <210> 23 <211> 542 <212> PRT <213> Artificial Sequence <220> <223> truncated FliC (I) -VL12.3cys <400> 23 Met Gly Phe Thr Ala Asn Ile Lys Gly Leu Thr Gln Ala Ser Arg Asn   1 5 10 15 Ala Asn Asp Gly Ile Ser Ile Ala Gln Thr Thr Glu Gly Ala Leu Asn              20 25 30 Glu Ile Asn Asn Asn Leu Gln Arg Val Arg Glu Leu Ala Val Gln Ser          35 40 45 Ala Asn Ser Thr Asn Ser Gln Ser Asp Leu Asp Ser Ile Gln Ala Glu      50 55 60 Ile Thr Gln Arg Leu Asn Glu Ile Asp Arg Val Ser Gly Gln Thr Gln  65 70 75 80 Phe Asn Gly Val Lys Val Leu Ala Gln Asp Asn Thr Leu Thr Ile Gln                  85 90 95 Val Gly Ala Asn Asp Gly Glu Thr Ile Asp Ile Asp Leu Lys Gln Ile             100 105 110 Asn Ser Gln Thr Leu Gly Leu Asp Thr Leu Asn Val Gln Gln Lys Tyr         115 120 125 Lys Val Ser Asp Thr Ala Ala Thr Val Thr Gly Tyr Ala Asp Thr Thr     130 135 140 Ile Ala Leu Asp Asn Ser Thr Phe Lys Ala Ser Ala Thr Gly Leu Gly 145 150 155 160 Gly Thr Asp Gln Lys Ile Asp Gly Asp Leu Lys Phe Asp Asp Thr Thr                 165 170 175 Gly Lys Tyr Tyr Ala Lys Val Thr Val Thr Gly Gly Ser Gly Gly Ser             180 185 190 Met Gly Ser Gln Pro Val Leu Thr Gln Ser Pro Ser Val Ser Ala Ala         195 200 205 Pro Arg Gln Arg Val Thr Ile Ser Val Ser Gly Ser Asn Ser Asn Ile     210 215 220 Gly Ser Asn Thr Val Asn Trp Ile Gln Gln Leu Pro Gly Arg Ala Pro 225 230 235 240 Glu Leu Leu Met Cys Asp Asp Asp Leu Leu Ala Pro Gly Val Ser Asp                 245 250 255 Arg Phe Ser Gly Ser Arg Ser Gly Thr Ser Ala Ser Leu Thr Ile Ser             260 265 270 Gly Leu Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Ala Ala Thr Trp Asp         275 280 285 Asp Ser Leu Asn Gly Trp Val Phe Gly Gly Gly Thr Lys Val Thr Val     290 295 300 Leu Ser Ala Gly His Met Leu Val Pro Arg Gly Ser Gly Gly Ser Val 305 310 315 320 Asp Gly Thr Gly Lys Asp Gly Tyr Tyr Glu Val Ser Val Asp Lys Thr                 325 330 335 Asn Gly Glu Val Thr Leu Ala Gly Gly Ala Thr Ser Pro Leu Thr Gly             340 345 350 Gly Leu Pro Ala Thr Ala Thr Glu Asp Val Lys Asn Val Gln Val Ala         355 360 365 Asn Ala Asp Leu Thr Glu Ala Lys Ala Ala Leu Thr Ala Ala Gly Val     370 375 380 Thr Gly Thr Ala Ser Val Val Lys Met Ser Tyr Thr Asp Asn Asn Gly 385 390 395 400 Lys Thr Ile Asp Gly Gly Leu Ala Val Lys Val Gly Asp Asp Tyr Tyr                 405 410 415 Ser Ala Thr Gln Asn Lys Asp Gly Ser Ile Ser Ile Asn Thr Thr Lys             420 425 430 Tyr Thr Ala Asp Asp Gly Thr Ser Lys Thr Ala Leu Asn Lys Leu Gly         435 440 445 Gly Ala Asp Gly Lys Thr Glu Val Val Ser Ile Gly Gly Lys Thr Tyr     450 455 460 Ala Ala Ser Lys Ala Glu Gly His Asn Phe Lys Ala Gln Pro Asp Leu 465 470 475 480 Ala Glu Ala Ala Ala Thr Thr Thr Glu Asn Pro Leu Gln Lys Ile Asp                 485 490 495 Ala Ala Leu Ala Gln Val Asp Thr Leu Arg Ser Asp Leu Gly Ala Val             500 505 510 Gln Asn Arg Phe Asn Ser Ala Ile Thr Asn Leu Gly Asn Thr Val Asn         515 520 525 Asn Leu Thr Ser Ala Arg Leu Glu His His His His His His     530 535 540 <210> 24 <211> 1629 <212> DNA <213> Artificial Sequence <220> <223> truncated FliC (I) -VL12.3cys <400> 24 atgggcttta ccgcgaacat caaaggtctg actcaggctt cccgtaacgc taacgacggt 60 atctccattg cgcagaccac tgaaggcgcg ctgaacgaaa tcaacaacaa cctgcagcgt 120 gtgcgtgaac tggcggttca gtctgctaac agcaccaact cccagtctga cctcgactcc 180 atccaggctg aaatcaccca gcgcctgaac gaaatcgacc gtgtatccgg ccagactcag 240 ttcaacggcg tgaaagtcct ggcgcaggac aacaccctga ccatccaggt tggtgccaac 300 gacggtgaaa ctatcgatat cgatctgaag cagatcaact ctcagaccct gggtctggat 360 acgctgaatg tgcaacaaaa atataaggtc agcgatacgg ctgcaactgt tacaggatat 420 gccgatacta cgattgcttt agacaatagt acttttaaag cctcggctac tggtcttggt 480 ggtactgacc agaaaattga tggcgattta aaatttgatg atacgactgg aaaatattac 540 gccaaagtta ccgttacggg gggatccggt ggtagcatgg gtagtcaacc agtacttaca 600 caaagcccgt ctgtgtctgc cgctccacgt cagagagtca ccatctcagt tagtggttct 660 aattcaaata taggatcgaa cacagtgaac tggattcagc agttgcctgg ccgtgccccg 720 gagttattaa tgtgtgatga tgatttatta gcacccggag tatcagatcg cttttcagga 780 agccgtagtg gcactagtgc gtccttaacc attagcgggt tacagtctga agacgaagcg 840 gactattacg cagctacgtg ggatgatagt ttaaatggct gggtttttgg tggtggtact 900 aaagttacgg tgctgtccgc aggtcatatg ctggtgccgc gcggcagcgg tggtagcgtc 960 gacggaactg gtaaagatgg ctattatgaa gtttccgttg ataagacgaa cggtgaggtg 1020 actcttgctg gcggtgcgac ttccccgctt acaggtggac tacctgcgac agcaactgag 1080 gatgtgaaaa atgtacaagt tgcaaatgct gatttgacag aggctaaagc cgcattgaca 1140 gcagcaggtg ttaccggcac agcatctgtt gttaagatgt cttatactga taataacggt 1200 aaaactattg atggtggttt agcagttaag gtaggcgatg attactattc tgcaactcaa 1260 aataaagatg gttccataag tattaatact acgaaataca ctgcagatga cggtacatcc 1320 aaaactgcac taaacaaact gggtggcgca gacggcaaaa ccgaagttgt ttctattggt 1380 ggtaaaactt acgctgcaag taaagccgaa ggtcacaact ttaaagcaca gcctgatctg 1440 gcggaagcgg ctgctacaac caccgaaaac ccgctgcaga aaattgatgc tgctttggca 1500 caggttgaca cgttacgttc tgacctgggt gcggtacaga accgtttcaa ctccgctatt 1560 accaacctgg gcaacaccgt aaacaacctg acttctgccc gtctcgagca ccaccaccac 1620 caccactga 1629 <210> 25 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> HTTcys Forward primer <400> 25 atcatccatg ggcgaaaagc tgatgaagtg tttc 34 <210> 26 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> HTTcys Reverse primer <400> 26 atcactctag aaccaccctg gaaggacttg ag 32 <210> 27 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Ftn Forward primer <400> 27 tgcggtctag aatgctgaaa ccagaaatga ttgaa 35 <210> 28 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Ftn Reverse primer <400> 28 aggccagcca ctcgagacta gttcattagt tttgtgtgtc 40 <210> 29 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> HTTcys-I3-01 Forward primer <400> 29 attatcatat gcatcatcat catcatcacg gtggaagcgg tggaagc 47 <210> 30 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> HTTcys-I3-01 Reverse primer <400> 30 tgcgcctcga gctattattc ggtgcagcca cgaatc 36 <210> 31 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> HA-VL12.3cys Forward primer <400> 31 aggcctctag aagcatgggt agtcaaccag tactt 35 <210> 32 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> HA-VL12.3cys Reverse primer <400> 32 gtgatgatga tgatgatggc tactgcctgc ggacagcacc gt 42 <210> 33 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> PA-VL12.3cys Forward primer <400> 33 aagagccatg ggctttcatt atgatagaaa taac 34 <210> 34 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> PA-VL12.3cys Reverse primer <400> 34 agcatctcga gtgcggacag caccgtaact tta 33 <210> 35 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> FliC (N) -VL12.3cys Forward primer <400> 35 agcggcatat gggtagtcaa ccagtactta cacaaagccc g 41 <210> 36 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> FliC (N) -VL12.3cys Reverse primer <400> 36 attatgcggc cgctcaacgc agtaaagaga ggacg 35 <210> 37 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> truncated FliC (N) -VL12.3cys Forward primer <400> 37 actggtggta ccatgggtag tcaaccagta ctt 33 <210> 38 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> truncated FliC (N) -VL12.3cys Reverse primer <400> 38 agttactcga gacgggcaga agtcaggttg tt 32 <210> 39 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> truncated FliC (C) -VL12.3cys Forward primer <400> 39 ataatccatg ggctttaccg cgaacatcaa aggt 34 <210> 40 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> truncated FliC (C) -VL12.3cys Reverse primer <400> 40 agcatctcga gtgcggacag caccgtaac 29 <210> 41 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> truncated FliC (I) -VL12.3cys Forward primer <400> 41 ataatccatg ggctttaccg cgaacatcaa aggt 34 <210> 42 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> truncated FliC (I) -VL12.3cys Reverse primer <400> 42 agttactcga gacgggcaga agtcaggttg tt 32

Claims (11)

HTT ^cys Autoassembles of 24 fusion proteins of peptide and ferritin monomers;
Fusion proteins fused to a _VL 12.3 ^cys peptide and an antigen peptide; And
A nanoparticle comprising a fusion molecule of a V _L 12.3 ^cys peptide and an immunoadjuvant,
The antigenic peptide and immune enhancer are the HTT ^cys Peptides with V _L 12.3 ^cys Nanoparticles that are linked to the ferritin self-assembly by binding between peptides.
The method of claim 1,
The HTT ^cys Peptide is a nanoparticle consisting of the amino acid sequence of SEQ ID NO: 3.
The method of claim 1,
V _L 12.3 ^cys Peptide is a nanoparticle consisting of the amino acid sequence of SEQ ID NO: 5.
The method of claim 1,
The HTT ^cys Peptides with V _L 12.3 ^cys The binding between the peptides comprises antigen-antibody binding and disulfide binding.
The method of claim 1,
The immune enhancer is an immune enhancing peptide, nanoparticles.
The method of claim 1,
The HTT ^cys Peptides are fused to the N-terminus of the ferritin monomer, nanoparticles.
The method of claim 5,
V _L 12.3 ^cys The peptide is a nanoparticle, which is fused to or inserted into the N-terminus or C-terminus of an antigenic peptide or an immune enhancing peptide.
The method of claim 5,
Mole ratio of the antigen peptide and the immune enhancing peptide in the nanoparticles is 0.1 ~ 10: 1, nanoparticles.
A vaccine composition comprising the nanoparticles of any one of claims 1 to 8.
The method of claim 9,
Wherein the vaccine increases total IgG levels.
The method of claim 9,
Wherein said vaccine promotes cellular and humoral immunity.

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