KR101130884B1 - Recombinant fusion protein produced by fusing Vibrio vulnificus flagellin and pathogenic antigens and the mucosal vaccine containing the same as an active ingredient - Google Patents

Abstract

The present invention relates to a recombinant fusion protein prepared by fusing a vaccine adjuvant and an antigen of a pathogen and a vaccine for mucosal administration comprising the same, and more particularly, sepsis that is a vaccine adjuvant and a toll-like receptor-5 (TLR-5) agonist. The present invention relates to a recombinant fusion protein prepared by fusing the flagellin of Vibrio vulnificus and an antigenic protein of a pathogen, and a vaccine for mucosal administration comprising the same.

Description

Recombinant fusion protein produced by fusing Vibrio vulnificus flagellin and pathogenic antigens and the mucosal vaccine containing the same as an active ingredient}

The present invention relates to a recombinant fusion protein prepared by fusing a vaccine adjuvant and an antigen of a pathogen and a vaccine for mucosal administration comprising the same, and more particularly, sepsis that is a vaccine adjuvant and a toll-like receptor-5 (TLR-5) agonist. The present invention relates to a recombinant fusion protein prepared by fusing the flagellin of Vibrio vulnificus and an antigenic protein of a pathogen, and a vaccine for mucosal administration comprising the same.

Streptococcus pneumoniae is a Gram-positive diplococci, the most important causative agent of mucosal infections such as pneumonia and otitis media and invasive infections such as meningitis and bacteremia in children and adults. In addition, the recent increase in the frequency of antibiotic resistant pneumococci has increased the need for effective vaccine development. Pneumococci are classified into about 90 serotypes according to the serological properties of the capsular polysaccharides. Among them, about 10 serotypes cause most infectious diseases, and the serotypes vary according to each region, age, and age. have. As a vaccine to prevent pneumococcal disease, a polysaccharide vaccine prepared by mixing polysaccharides extracted from each pneumococcal of each serotype was used, but immunogenicity was observed in children under 2 years of age with the highest risk of invasive pneumococcal infection. And low preventive effects. To compensate for this, a pneumococcal polysaccharide protein-binding vaccine has been developed that combines the polysaccharide and carrier protein of pneumococcal pneumoniae, but this also has a limited disadvantage to cover each serotype.

To overcome these shortcomings of polysaccharide vaccines and polysaccharide protein-binding vaccines, pneumolysin, pneumococcal surface protein A (PsaA) and pneumococcal surface protein A (PspA), which are expressed in the majority of pneumococci Protein antigens such as pneumococcal surface protein A) can be used. Pneumococcal protein vaccines can now be used in the production of vaccines with broad protective immunity against all clinically important pneumococci, and are much easier to manufacture and produce than polysaccharide protein-binding vaccines.

PspA of pneumococci is a well-known virulence factor found on the surface of pneumococci. In animal experiments, PspA is known to show protective immunity against sepsis as well as pneumococcal carriers and respiratory infections. It is becoming.

Flagella is an important component in determining the motility of bacteria and is composed of hook, basal body and filament. Flagella is known to have a function of determining the swimming or swarming motility of bacteria, the taxis of bacteria, and forming a biofilm to determine the adhesion of pathogenic microorganisms. The structural unit protein constituting the flagella filaments is called flagellin, and flagellin is regularly assembled to form filaments. Hayashi et al. Reported that mammalian expression of TLR-5 recognizes Gram-negative and Gram-positive bacteria flagellin and activates NF-κB (Hayashi F, Smith KD, Ozinsky A, Hawn TR, Yi EC, Goodlett DR, Eng JK, Akira S, Underhill DM, Aderem A: Nature 410: 1099-1103, 2001).

Toll-like receptors (TLR) are typical "pattern recognition receptor (PRR: P attern R ecognition R eceptor) ': for as it' (P athogen A ssociated M olecular P attern PAMP) molecules associated with pathogens structure, present in the pathogen It is a recognized receptor and is present on the surface of insects and plant cells as well as mammals. Thirteen types of TLRs have been identified so far, and studies on agonists of each TLR have been actively conducted (Akira S, Uematsu S, Takeuchi O: Cell 124 (4): 783-801, 2006).

PRRs, such as TLRs, are distributed on the cell surface or cytoplasm of host cells, and induce 'innate immune response' by stimulating various PAMPs and further regulate 'adaptive immune response'. Thus, TLR agonists can be suitable targets for the development of various 'immunomodulators', in particular 'vaccine adjuvants'.

Currently used or contemplated for use as a vaccine adjuvant include: 1) mineral salts such as aluminum hydroxide gels, 2) surfactants, 3) bacteria-derived materials, 4) cytokines or hormones, 6) polyanions (polyanion), 7) polyacryl, 8) carriers, 9) living vectors using viruses, and 10) carriers such as mineral oils or liposomes. vehicle). Among these, active research is currently underway, and protein-derived vaccine supplements are attracting much attention, such as cholera toxin (CT) derived from Vibrio cholerae and heat-labile toxin (LT) derived from E. coli. These vaccine adjuvants induce the production of antigen-specific antibodies in mucosal areas and serum and induce B7-2 expression on the surface of antigen presenting cells (APCs). It has been reported that it is a mechanism that promotes co-stimulatory signaling of CD4 + cells. However, since they are exotoxins with high enterotoxicity, studies are being conducted to reduce toxicity and increase adjuvaticity, and in recent clinical trials, facial nerve palsy (when administered with influenza antigens in the nasal cavity) Bells's palsy) increased incidence (Musch M, Zhou W, Rhodes P, Bopp M, Chen RT, LInder T, Spyr C, Steffen R: N. Engl. J. Med. 350: 896-903, 2004) .

Therefore, the present inventors prepared and isolated vaccine adjuvant-antigen fusion recombinant protein (PspA-FlaB or FlaB-PspA) consisting of sepsis Vibrio flagellin FlaB and surface protein PspA of pneumococcal and immunized into the nasal cavity of mouse, As a result of screening the defense against infection, the production of PspA-specific antibodies (IgG and IgA) in serum and mucosal secretions was significantly increased in the group immunized with PspA-FlaB or FlaB-PspA compared to the group immunized with PspA alone. When injected with a lethal dose of pneumococcal, it was confirmed that it shows superior defense immunity and completed the present invention.

Accordingly, it is an object of the present invention to provide a recombinant fusion protein having improved protective immunity against a pathogen and a vaccine for mucosal administration comprising the same.

In order to achieve the above object, the present invention provides a recombinant fusion protein prepared by fusing the flagellin protein of sepsis Vibrio and the protein antigen of the pathogen.

In another aspect, the present invention provides a vaccine for mucosal administration comprising a recombinant fusion protein as an active ingredient.

In the present invention, the recombinant fusion protein prepared by fusing the vaccine adjuvant and the antigen significantly increased the production of PspA-specific antibodies (IgG and IgA) in serum and mucosal secretions, and showed superior protective immunity against lethal pneumococci. Seemed.

Hereinafter, the present invention will be described in more detail.

The inventors of the present inventors have prepared a 'transposon mutant library' in order to screen the host adhesion and invasion factor of sepsis Vibrio in recent PCT WO 2005/070455. Identifying two flagella related operons consisting of 56 genes during the analysis of transposon insertion flanking regions of three clones that have lost adhesion and motility to cells It was. In this process, sepsis vibrioviruses have a total of six flagellin genes ( flaA , flaB, flaF, flaC, flaD, flaE ), and flaB gene is the major constituent of flagellin. The inventors of the present invention are pursuing the possibility of applying the flagellin recombinant protein (FlaB), a component of the sepsis Vibrio polar flagellin, as a 'component vaccine' against sepsis Vibrio, We found that there was an adjuvant effect and we studied to find out. As a result, when the vaccine antigen tetanus toxin is administered to the nasal cavity of experimental animals together with flagellin, flagellin signals the host's TLR5 to activate the immune system, which amplifies the efficacy of the vaccine. Administration of flagellin with immunized mice injected with a lethal dose of tetanus toxin induces 100% protective immunity, demonstrating an excellent mucosal vaccine adjuvant effect (Lee SE, Kim SY, Jeong BC, Kim) YR, Bae SJ, Ahn OS, Lee JJ, Song HC, Kim JM, Choy HE, Chung SS, Kweon MN, Rhee JH .: Infect. Immun. 74: 694-702, 2006).

Among the various TLR agonists, flagellin that stimulates TLR5 is a protein component unlike other TLR agonists (CpG-DNA, mycoplasmal lipopeptide (MALP), etc.), so it is not only possible to synthesize recombinant proteins for continuous quality control. Various recombinant fusion proteins with enhanced TLR5 stimulatory activity can be constructed.

Thus, the production of recombinant fusion proteins consisting of vaccine adjuvant-antigens fused with flagellin and immune epitopes of various diseases (such as infections, cancers or allergies) enables the production of efficient protein vaccines with improved immunity and advantages as protein vaccines. do.

The present invention relates to a recombinant fusion protein prepared by fusing a flagellin protein of sepsis vibrio and a protein antigen of a pathogen.

The flagellin protein used in the present invention is a TLR-5 agonist and is preferably flagellin FlaB of sepsis vibrio having the amino acid sequence of SEQ ID NO: 1, but is not limited thereto.

Protein antigens of the pathogens used in the present invention include alpha-helix domains of surface protein A (PspA) and pneumococcal surface protein A (PsaA) of surface protein A (PspA) of pneumococcal ( Streptococcus pneumoniae ). ; Subunits of influenza virus HA (hemagglutinin) and NA (neuraminidase); And Spike (S) protein of Severe Respiratory Syndrome Virus (SARS virus), but are not limited thereto.

Recombinant protein fusion of sepsis Vibrio flagellin and pneumococcal antigen PspA used in the present invention can be used as a vaccine formulation by itself, and a novel polysaccharide-protein binding vaccine of pneumococcus, which is widely used now, can enhance the efficacy. It can also be used as a conjugation partner.

Recombinant fusion proteins according to the present invention can be prepared by fusing, expressing and separating and purifying flagellin and pathogen antigen proteins of sepsis Vibrio.

In an embodiment of the present invention, the recombinant fusion protein may be FlaB-PspA having an amino acid sequence of SEQ ID NO: 4 or PspA-FlaB having an amino acid sequence of SEQ ID NO: 6.

Flagellin referred to in the present specification is a unit molecule constituting flagella, a cilia that determine the motility of bacteria, and PspA is a protein A present on the surface of pneumococcal and has high antigenicity.

The FlaB-PspA or PspA-FlaB recombinant fusion protein provided by the present invention exhibits superior antibody production and protective immunity compared to the group immunized with PspA alone.

Thus, the FlaB-PspA or PspA-FlaB recombinant fusion protein prepared by the present invention can be used to provide a pneumococcal vaccine with significantly improved immunity than when using only PspA antigen alone.

The present invention also relates to a vaccine for mucosal administration comprising the recombinant fusion protein as an active ingredient. In particular, the present invention may provide a vaccine for nasal mucosa administration, and may be formulated as a vaccine against pneumococcal, influenza virus or acute respiratory syndrome virus (SARS virus).

The vaccine for mucosal administration provided by the present invention may be prepared by conventional methods well known in the art, and may further optionally include various additives that may be used in the manufacture of vaccine for mucosal administration.

In addition, based on the technical configuration of the present invention will be able to provide a vaccine comprising a variety of recombinant fusion protein by fusing the vaccine adjuvant flagellin and antigens for infectious diseases, anti-cancer and various diseases and as an active ingredient .

In one embodiment of the present invention, the process of demonstrating the enhanced immunity of the FlaB-PspA or PspA-FlaB recombinant fusion protein comprises the following steps:

1) preparing and isolating FlaB-PspA of SEQ ID NO: 4 or PspA-FlaB recombinant fusion protein of SEQ ID NO: 6;

2) confirming TLR-5 activation of flagellin in said recombinant fusion protein;

3) identifying an antigen specific immune response after intranasal immunization of the recombinant fusion protein; And

4) measuring host defense ability against wild-type pneumococcal in mice following intranasal immunization of the recombinant fusion protein.

Hereinafter, the content of the present invention will be described in more detail through examples and test examples. These examples are provided only for understanding the contents of the present invention, and the scope of the present invention is not limited to these examples, and modifications, substitutions, and insertions commonly known in the art may be performed. This is also included in the scope of the present invention.

The characteristics of the strains and plasmids used in the present invention are summarized in Table 1. Each manufacturing method and detailed characteristics are indicated in the corresponding examples and test examples.

Strain or
Plasmid Characteristic source Escherichia coli DH5α F- recA 1; restriction negative Existing lab ER2566 F- λ- fhuA2 [lon] ompT lacZ :: T7 gene1 gal sulA1 1 ( mcrC -mrr) 114 :: IS10
R (mcr-73 :: miniTn10-TetS) 2 R (zgb-210 :: Tn10) (TetS) endA1 [dcm] New england
Biolab, Inc. Plasmid pCR2.1 TOPO PCR TA Cloning Vector, Amp ^R , Km ^R Invitrogen pTYB12 Intein Mediated Purification with an Affinity Chitin-binding Tag (IMPACT) expression vector, Amp ^R New england
Biolab, Inc.

<Cultivation and storage of each strain>

E. coli strains used in the following examples and test examples were cultured in LB (Luria Bertani) medium (Difco Co.). Strains used were added to 30% glycerol after incubation and then stored in -80 ℃ cryogenic freezer.

Example 1 Preparation and Isolation of Recombinant Flagellin, PspA and Fusion Proteins

In order to obtain a DNA fragment for N-terminal or C-terminal fusion of the alpha-helix domain portion of the PspA gene of S. pneumoniae as an antigen, PspA-N of SEQ ID NOs: 7 and 8 shown in Table 2 below The PspA - C PCR primer pairs of SEQ ID NOs: 9 and 10 were used to amplify a 0.7 kbp DNA fragment containing the PspA gene for N-terminal fusion or C-terminal fusion. In other words, the PCR reaction using each primer pair was initially denatured at 95 ° C. for 5 minutes, followed by 30 cycles of denaturation at 95 ° C. for 30 seconds, annealing at 65 ° C. for 30 seconds and extension at 72 ° C. for 30 minutes. It carried out on the conditions made to react at 72 degreeC for 10 minutes. The amplified PspA DNA fragment was cloned into PCR 2.1-TOPO cloning vector (Invitrogen Co.) and named pCMM8201 or pCMM8202.

As a vaccine adjuvant, FlaB - N of SEQ ID NOs: 11 and 12 and FlaB - C PCR primer pairs of SEQ ID NOs: 13 and 14 were used to obtain DNA fragments for N-terminal or C-terminal fusion of sepsis vibrio flagellin gene flaB . A 1.1 kbp DNA fragment containing the flaB gene for N-terminal fusion or C-terminal fusion was amplified. In other words, the PCR reaction using each primer pair was initially denatured at 95 ° C. for 5 minutes, followed by 30 cycles of denaturation at 95 ° C. for 30 seconds, annealing at 65 ° C. for 30 seconds and extension at 72 ° C. for 30 minutes. It carried out on the conditions made to react at 72 degreeC for 10 minutes. The flaB DNA fragment thus amplified was cloned into PCR 2.1-TOPO cloning vector (Invitrogen Co.) and named pCMM8203 or pCMM8204.

designation order
number order Remarks PspA - N Forward 7 5'- CAT ATG TCT CCC GTA GCC AGT CAG TCT-3 ' Restriction enzyme Nde I recognition site inserted for cloning is underlined Reverse 8 5'- GTC GAC CTC ATC AAT CTT ATC ACT TAA CTC TTC AAG-3 ' The restriction enzyme Sal I recognition site inserted for cloning is underlined. PspA - C Forward 9 5'- GTC GAC TCT CCC GTA GCC AGT CAG T-3 ' The restriction enzyme Sal I recognition site inserted for cloning is underlined. Reverse 10 5'- CCC GGG TTA CTC ATC AAT CTT ATC ACT TAA CTC TTC-3 ' Restriction enzyme Sma I recognition site inserted for cloning is underlined FlaB - N
Forward 11 5'- CAT ATG ATG GCA GTG AAT GTA AAT ACA AAC GTA GCA-3 ' Restriction enzyme Nde I recognition site inserted for cloning is underlined Reverse 12 5'- GTC GAC GCC TAG TAG ACT TAG CGC T-3 ' The restriction enzyme Sal I recognition site inserted for cloning is underlined. FlaB - C Forward 13 5'- GTC GAC ATG GCA GTG AAT GTA AAT ACA AAC GTA G-3 ' The restriction enzyme Sal I recognition site inserted for cloning is underlined. Reverse 14 5'- CCC GGG TTA GCC TAG TAG ACT TAG CG-3 ' Restriction enzyme Sma I recognition site inserted for cloning is underlined

To prepare the FlaB-PspA fusion protein, the flaB DNA fragment of pCMM8203 was cut with restriction enzymes Nde I and Sal I and cloned into pTYB12 plasmid (New England Biolabs) cut with the same restriction enzyme and named pCMM8205. The PspA DNA fragment of pCMM8202 cut with restriction enzymes Sal I and Sma I was cloned into this plasmid and named pCMM8208.

To prepare the PspA-FlaB fusion protein, the PspA DNA fragment of pCMM8201 was cut with restriction enzymes Nde I and Sal I, cloned into pTYB12 plasmid (New England Biolabs) cut with the same restriction enzyme, and named pCMM8206. The flaB DNA fragment of pCMM8204 cut with restriction enzymes Sal I and Sma I was cloned into this plasmid and named pCMM8207.

These plasmids were placed in ER2566 Escherichia coli by electroporation and 0.5 mM of 5-bromo-indole-3-chloro-isopropyl-β-D-galactopyranoside (IPTG) was added to induce expression. Flasp of SEQ ID NO: 1, PspA of SEQ ID NO: 2 from an intein fusion protein using a chitin bead column and 1,4-dithiothreitol (1,4-DTT) according to the manufacturer's (New England Biolabs Inc.) instructions , FlaB-PspA of SEQ ID NO: 4, and PspA-FlaB proteins of SEQ ID NO: 6 were obtained, and the procedure is shown in detail in FIG. Endotoxins contained in the isolated protein were removed using AffinityPak ^™ Detoxi Gel ^™ Endotoxin Removing gel (Pirece).

Test Example 1 TLR-5 Stimulating Activity of Flagellin-PspA Recombinant Fusion Protein

293-T cells were dispensed in 1 × 10 ⁵ cells in a 24-well plate incubator and cultured overnight, followed by NF-κB-Luc plasmid using Effectene (QIAGEN) (acquired from Professor Kim Jung-mok of the Department of Microbiology, Hanyang University College of Medicine) P3xFlag-hTLR-5 plasmid cloned from TLR-5 gene (obtained from Steven B. Mizel of Wake Forest University School of Medicine, Department of Microbiology and Immunology, USA) and β-galactosidase expression control Plasmids (Clontech) were simultaneously introduced into the cells. After 24 hours of additional culture, the medium was replaced with fresh medium, and the luciferase activity was determined by luminometer (Luminometer) after treatment with FlaB, PspA-FlaB and FlaB-PspA proteins prepared in Example 1 and separated by IMPACT system for a certain time. , Berthold) to determine the degree of NF-κB transcription, the results are shown in FIG.

In the results of FIG. 2, both PspA-FlaB and FlaB-PspA recombinant fusion proteins activate NF-κB transcription, 1.3-fold and 3-fold higher than when treated with FlaB alone, respectively. FlaB in the TLR-5-mediated NF-κB transcriptional activity, as well as the ability was much improved.

[Test Example 2] Protective immunity test of flagellin-PspA recombinant fusion protein

Specific pathogen free (SPF) phosphate buffered saline (PBS) intranasally in 5 week old Balb / c female mice, and 2.5 μg PspA prepared in Example 1, a mixture of 2.5 μg PspA and 4 μg FlaB, 26.5 μg PspA-FlaB and Immunized FlaB-PspA three times at two-week intervals, and 14 days after the last immunization, serum, saliva and vaginal washes of mice were collected and then PspA-specific systemic immune responses. (systemic immune response) and mucosal immune response (mucosal immune response) were measured by the Enzyme linked immunosorbent assay (ELISA) method, and 200 times of lethal dose (LD ₅₀ ) in mice vaccinated three times. The intranasal administration of wild-type Streptococcus pneumoniae D39, which corresponds to the embryo, observed the survival of the mice for 14 days, and the results are shown in FIGS. 3A, 3B and 4.

As can be seen from the results of Figures 3a and 3b, PspA-FlaB (PF) and FlaB than in the control group (PBS) administered only PBS or PspA alone (P) or mice administered PspA and FlaB mixture (P + F) Mice that received the -PspA (FP) recombinant fusion protein showed high PspA antigen-specific systemic and mucosal immune responses.

As can be seen from the results of FIG. 4, 100% of control mice (PBS) immunized with PBS alone died within 3 days, and only 20% of mice survived in PspA-only mice (PspA) and a mixture of PspA and FlaB. When administered (P + F), the survival rate was about 20%, but when the PspA-FlaB (PF) and FlaB-PspA (FP) recombinant fusion proteins were administered nasal, they survived 40% and 60%.

In the present invention, a vaccine adjuvant-antigen fusion recombinant protein vaccine against pneumococcal, which is a vaccine adjuvant using flagellin, which has been shown to have a potent mucosal vaccine adjuvant effect by stimulating TLR-5, as an antigen of surface protein A (PspA) of pneumococcus Vaccine adjuvant-antigen fusion recombinant protein and improved vaccines have been developed that provide much improved immunity than PspA alone. This will suggest a new approach in the development of new paradigms such as 'infectious diseases', 'autoimmune diseases' and 'allergic diseases', as well as 'anti-cancer immunity drugs'. Will provide an important link. Therefore, if the results of the present invention are applied to various vaccine formulations, a great added value can be created.

1 is a schematic diagram showing a process for preparing and isolating PspA, FlaB, PspA-FlaB and FlaB-PspA recombinant proteins.

Figure 2 is a graph showing the results of measuring the TLR-5 stimulating activity of the PspA-FlaB and FlaB-PspA recombinant fusion protein according to the present invention.

Figure 3a and 3b shows the antigen-specific immune response by taking the blood and various mucosal samples of the mouse after immunizing the nasal cavity of the mouse with the PspA, PspA and FlaB mixture, PspA-FlaB and FlaB-PspA recombinant protein according to the present invention It is the result measured by ELISA method.

Figure 4 shows the protection of the host against the lethal dose of S. pneumoniae after administration of Streptococcus pneumoniae D39 after immunizing the nasal cavity of mice with PspA, PspA and FlaB mixture, PspA-FlaB and FlaB-PspA recombinant proteins according to the present invention. The result is.

<110> INDUSTRY FOUNDATION OF CHONNAM NATIONAL UNIVERSITY <120> Recombinant fusion protein produced by fusing Vibrio vulnificus          flagellin and pathogenic antigens and the mucosal vaccine          containing the same as an active ingredient <160> 14 <170> KopatentIn 1.71 <210> 1 <211> 377 <212> PRT <213> Vibrio vulnificus flagellin FlaB <400> 1 Met Ala Val Asn Val Asn Thr Asn Val Ala Ala Met Thr Ala Gln Arg   1 5 10 15 Tyr Leu Asn Asn Ala Asn Ser Ala Gln Gln Thr Ser Met Glu Arg Leu              20 25 30 Ser Ser Gly Phe Lys Ile Asn Ser Ala Lys Asp Asp Ala Ala Gly Leu          35 40 45 Gln Ile Ser Asn Arg Leu Asn Val Gln Ser Arg Gly Leu Asp Val Ala      50 55 60 Val Arg Asn Ala Asn Asp Gly Ile Ser Ile Ala Gln Thr Ala Glu Gly  65 70 75 80 Ala Met Asn Glu Thr Thr Asn Ile Leu Gln Arg Met Arg Asp Leu Ser                  85 90 95 Leu Gln Ser Ala Asn Gly Ser Asn Ser Lys Ser Glu Arg Val Ala Ile             100 105 110 Gln Glu Glu Val Thr Ala Leu Asn Asp Glu Leu Asn Arg Ile Ala Glu         115 120 125 Thr Thr Ser Phe Gly Gly Asn Lys Leu Leu Asn Gly Thr Tyr Gly Thr     130 135 140 Lys Ala Met Gln Ile Gly Ala Asp Asn Gly Glu Ala Val Met Leu Ser 145 150 155 160 Leu Lys Asp Met Arg Ser Asp Asn Val Met Met Gly Gly Val Ser Tyr                 165 170 175 Gln Ala Glu Glu Gly Lys Asp Lys Asn Trp Asn Val Ala Ala Gly Asp             180 185 190 Asn Asp Leu Thr Ile Ala Leu Thr Asp Ser Phe Gly Asn Glu Gln Glu         195 200 205 Ile Glu Ile Asn Ala Lys Ala Gly Asp Asp Ile Glu Glu Leu Ala Thr     210 215 220 Tyr Ile Asn Gly Gln Thr Asp Leu Val Lys Ala Ser Val Gly Glu Gly 225 230 235 240 Gly Lys Leu Gln Ile Phe Ala Gly Asn Asn Lys Val Gln Gly Glu Ile                 245 250 255 Ala Phe Ser Gly Ser Leu Ala Gly Glu Leu Gly Leu Gly Glu Gly Lys             260 265 270 Asn Val Thr Val Asp Thr Ile Asp Val Thr Thr Val Gln Gly Ala Gln         275 280 285 Glu Ser Val Ala Ile Val Asp Ala Ala Leu Lys Tyr Val Asp Ser His     290 295 300 Arg Ala Glu Leu Gly Ala Phe Gln Asn Arg Phe Asn His Ala Ile Ser 305 310 315 320 Asn Leu Asp Asn Ile Asn Glu Asn Val Asn Ala Ser Lys Ser Arg Ile                 325 330 335 Lys Asp Thr Asp Phe Ala Lys Glu Thr Thr Gln Leu Thr Lys Thr Gln             340 345 350 Ile Leu Ser Gln Ala Ser Ser Ser Ile Leu Ala Gln Ala Lys Gln Ala         355 360 365 Pro Asn Ser Ala Leu Ser Leu Leu Gly     370 375 <210> 2 <211> 233 <212> PRT <213> Streptococcus pneumoniae PspA <400> 2 Ser Pro Val Ala Ser Gln Ser Lys Ala Glu Lys Asp Tyr Asp Ala Ala   1 5 10 15 Lys Lys Asp Ala Lys Asn Ala Lys Lys Ala Val Glu Asp Ala Gln Lys              20 25 30 Ala Leu Asp Asp Ala Lys Ala Ala Gln Lys Lys Tyr Asp Glu Asp Gln          35 40 45 Lys Lys Thr Glu Glu Lys Ala Ala Leu Glu Lys Ala Ala Ser Glu Glu      50 55 60 Met Asp Lys Ala Val Ala Ala Val Gln Gln Ala Tyr Leu Ala Tyr Gln  65 70 75 80 Gln Ala Thr Asp Lys Ala Ala Lys Asp Ala Ala Asp Lys Met Ile Asp                  85 90 95 Glu Ala Lys Lys Arg Glu Glu Glu Ala Lys Thr Lys Phe Asn Thr Val             100 105 110 Arg Ala Met Val Val Pro Glu Pro Glu Gln Leu Ala Glu Thr Lys Lys         115 120 125 Lys Ser Glu Glu Ala Lys Gln Lys Ala Pro Glu Leu Thr Lys Lys Leu     130 135 140 Glu Glu Ala Lys Ala Lys Leu Glu Glu Ala Glu Lys Lys Ala Thr Glu 145 150 155 160 Ala Lys Gln Lys Val Asp Ala Glu Glu Val Ala Pro Gln Ala Lys Ile                 165 170 175 Ala Glu Leu Glu Asn Gln Val His Arg Leu Glu Gln Glu Leu Lys Glu             180 185 190 Ile Asp Glu Ser Glu Ser Glu Asp Tyr Ala Lys Glu Gly Phe Arg Ala         195 200 205 Pro Leu Gln Ser Lys Leu Asp Ala Lys Lys Ala Lys Leu Ser Lys Leu     210 215 220 Glu Glu Leu Ser Asp Lys Ile Asp Glu 225 230 <210> 3 <211> 1836 <212> DNA <213> Artificial Sequence <220> <223> Fusion protein FlaB-PspA <400> 3 atggcagtga atgtaaatac aaacgtagca gcaatgacag cacagcgtta cctgaataac 60 gcaaacagcg cacaacaaac ttcgatggag cgtctgtctt caggtttcaa aatcaacagt 120 gcaaaagatg acgcagccgg tctgcaaatc tctaaccgct tgaacgtaca aagtcgcggt 180 ctagacgttg cggtacgtaa cgccaacgac ggtatctcaa tcgcacaaa cgcagaaggt 240 gcgatgaacg agaccaccaa catcctacaa cgtatgcgtg acctatctct acaatccgcg 300 aacggctcaa actcaaaatc agagcgcgtg gcgattcaag aagaagtgac agcattgaat 360 gacgagctaa accgtattgc agaaaccacg tcttttggtg gtaacaagct gctaaacggt 420 acttacggca cgaaagcaat gcaaattggt gcggataacg gtgaagcggt catgctttca 480 ctgaaagaca tgcgctctga caacgtgatg atgggcggcg tgagctacca agctgaagaa 540 ggcaaagaca agaactggaa tgtggccgca ggcgacaacg acttgacgat tgcactgaca 600 gacagctttg gtaacgagca agagatcgaa atcaacgcga aagcgggtga tgacatcgaa 660 gagctagcga cgtacatcaa cggtcaaact gaccttgtaa aagcgtcagt gggtgaaggc 720 ggcaagctac agatctttgc tggtaacaac aaagttcaag gtgaaattgc tttctcaggt 780 agcctagctg gtgaacttgg cctaggcgaa ggcaaaaacg tcacggtaga cacgattgac 840 gtgacaaccg tacaaggtgc gcaagagtcg gtagcgattg tggatgcggc actgaaatac 900 gtagacagcc accgtgcaga gctgggtgca ttccagaacc gtttcaacca tgcaatcagc 960 aacttggaca acatcaacga aaacgtgaac gcgtcgaaga gccgaatcaa agataccgac 1020 ttcgcgaaag aaacgactca gttgaccaag acacaaattc tatcgcaagc atcaagttcc 1080 attcttgcgc aagcgaaaca agcgccaaac tcagcgctaa gtctactagg cgtcgactct 1140 cccgtagcca gtcagtctaa agctgagaaa gactatgatg cagcgaagaa agatgctaag 1200 aatgcgaaaa aagcagtaga agatgctcaa aaggctttag atgatgcaaa agctgctcag 1260 aaaaaatatg acgaggatca gaagaaaact gaggagaaag ccgcgctaga aaaagcagcg 1320 tctgaagaga tggataaggc agtggcagca gttcaacaag cgtatctagc ctatcaacaa 1380 gctacagaca aagccgcaaa agacgcagca gataagatga tagatgaagc taagaaacgc 1440 gaagaagagg caaaaactaa atttaatact gttcgagcaa tggtagttcc tgagccagag 1500 cagttggctg agactaagaa aaaatcagaa gaagctaaac aaaaagcacc agaacttact 1560 aaaaaactag aagaagctaa agcaaaatta gaagaggctg agaaaaaagc tactgaagcc 1620 aaacaaaaag tggatgctga agaagtcgct cctcaagcta aaatcgctga attggaaaat 1680 caagttcata gactagaaca agagctcaaa gagattgatg agtctgaatc agaagattat 1740 gctaaagaag gtttccgtgc tcctcttcaa tctaaattgg atgccaaaaa agctaaacta 1800 tcaaaacttg aagagttaag tgataagatt gatgag 1836 <210> 4 <211> 612 <212> PRT <213> Artificial Sequence <220> <223> Fusion protein FlaB-PspA <400> 4 Met Ala Val Asn Val Asn Thr Asn Val Ala Ala Met Thr Ala Gln Arg   1 5 10 15 Tyr Leu Asn Asn Ala Asn Ser Ala Gln Gln Thr Ser Met Glu Arg Leu              20 25 30 Ser Ser Gly Phe Lys Ile Asn Ser Ala Lys Asp Asp Ala Ala Gly Leu          35 40 45 Gln Ile Ser Asn Arg Leu Asn Val Gln Ser Arg Gly Leu Asp Val Ala      50 55 60 Val Arg Asn Ala Asn Asp Gly Ile Ser Ile Ala Gln Thr Ala Glu Gly  65 70 75 80 Ala Met Asn Glu Thr Thr Asn Ile Leu Gln Arg Met Arg Asp Leu Ser                  85 90 95 Leu Gln Ser Ala Asn Gly Ser Asn Ser Lys Ser Glu Arg Val Ala Ile             100 105 110 Gln Glu Glu Val Thr Ala Leu Asn Asp Glu Leu Asn Arg Ile Ala Glu         115 120 125 Thr Thr Ser Phe Gly Gly Asn Lys Leu Leu Asn Gly Thr Tyr Gly Thr     130 135 140 Lys Ala Met Gln Ile Gly Ala Asp Asn Gly Glu Ala Val Met Leu Ser 145 150 155 160 Leu Lys Asp Met Arg Ser Asp Asn Val Met Met Gly Gly Val Ser Tyr                 165 170 175 Gln Ala Glu Glu Gly Lys Asp Lys Asn Trp Asn Val Ala Ala Gly Asp             180 185 190 Asn Asp Leu Thr Ile Ala Leu Thr Asp Ser Phe Gly Asn Glu Gln Glu         195 200 205 Ile Glu Ile Asn Ala Lys Ala Gly Asp Asp Ile Glu Glu Leu Ala Thr     210 215 220 Tyr Ile Asn Gly Gln Thr Asp Leu Val Lys Ala Ser Val Gly Glu Gly 225 230 235 240 Gly Lys Leu Gln Ile Phe Ala Gly Asn Asn Lys Val Gln Gly Glu Ile                 245 250 255 Ala Phe Ser Gly Ser Leu Ala Gly Glu Leu Gly Leu Gly Glu Gly Lys             260 265 270 Asn Val Thr Val Asp Thr Ile Asp Val Thr Thr Val Gln Gly Ala Gln         275 280 285 Glu Ser Val Ala Ile Val Asp Ala Ala Leu Lys Tyr Val Asp Ser His     290 295 300 Arg Ala Glu Leu Gly Ala Phe Gln Asn Arg Phe Asn His Ala Ile Ser 305 310 315 320 Asn Leu Asp Asn Ile Asn Glu Asn Val Asn Ala Ser Lys Ser Arg Ile                 325 330 335 Lys Asp Thr Asp Phe Ala Lys Glu Thr Thr Gln Leu Thr Lys Thr Gln             340 345 350 Ile Leu Ser Gln Ala Ser Ser Ser Ile Leu Ala Gln Ala Lys Gln Ala         355 360 365 Pro Asn Ser Ala Leu Ser Leu Leu Gly Val Asp Ser Pro Val Ala Ser     370 375 380 Gln Ser Lys Ala Glu Lys Asp Tyr Asp Ala Ala Lys Lys Asp Ala Lys 385 390 395 400 Asn Ala Lys Lys Ala Val Glu Asp Ala Gln Lys Ala Leu Asp Asp Ala                 405 410 415 Lys Ala Ala Gln Lys Lys Tyr Asp Glu Asp Gln Lys Lys Thr Glu Glu             420 425 430 Lys Ala Ala Leu Glu Lys Ala Ala Ser Glu Glu Met Asp Lys Ala Val         435 440 445 Ala Ala Val Gln Gln Ala Tyr Leu Ala Tyr Gln Gln Ala Thr Asp Lys     450 455 460 Ala Ala Lys Asp Ala Ala Asp Lys Met Ile Asp Glu Ala Lys Lys Arg 465 470 475 480 Glu Glu Glu Ala Lys Thr Lys Phe Asn Thr Val Arg Ala Met Val Val                 485 490 495 Pro Glu Pro Glu Gln Leu Ala Glu Thr Lys Lys Lys Ser Glu Glu Ala             500 505 510 Lys Gln Lys Ala Pro Glu Leu Thr Lys Lys Leu Glu Glu Ala Lys Ala         515 520 525 Lys Leu Glu Glu Ala Glu Lys Lys Ala Thr Glu Ala Lys Gln Lys Val     530 535 540 Asp Ala Glu Glu Val Ala Pro Gln Ala Lys Ile Ala Glu Leu Glu Asn 545 550 555 560 Gln Val His Arg Leu Glu Gln Glu Leu Lys Glu Ile Asp Glu Ser Glu                 565 570 575 Ser Glu Asp Tyr Ala Lys Glu Gly Phe Arg Ala Pro Leu Gln Ser Lys             580 585 590 Leu Asp Ala Lys Lys Ala Lys Leu Ser Lys Leu Glu Glu Leu Ser Asp         595 600 605 Lys Ile Asp Glu     610 <210> 5 <211> 1836 <212> DNA <213> Artificial Sequence <220> <223> Fusion protein PspA-FlaB <400> 5 tctcccgtag ccagtcagtc taaagctgag aaagactatg atgcagcgaa gaaagatgct 60 aagaatgcga aaaaagcagt agaagatgct caaaaggctt tagatgatgc aaaagctgct 120 cagaaaaaat atgacgagga tcagaagaaa actgaggaga aagccgcgct agaaaaagca 180 gcgtctgaag agatggataa ggcagtggca gcagttcaac aagcgtatct agcctatcaa 240 caagctacag acaaagccgc aaaagacgca gcagataaga tgatagatga agctaagaaa 300 cgcgaagaag aggcaaaaac taaatttaat actgttcgag caatggtagt tcctgagcca 360 gagcagttgg ctgagactaa gaaaaaatca gaagaagcta aacaaaaagc accagaactt 420 actaaaaaac tagaagaagc taaagcaaaa ttagaagagg ctgagaaaaa agctactgaa 480 gccaaacaaa aagtggatgc tgaagaagtc gctcctcaag ctaaaatcgc tgaattggaa 540 aatcaagttc atagactaga acaagagctc aaagagattg atgagtctga atcagaagat 600 tatgctaaag aaggtttccg tgctcctctt caatctaaat tggatgccaa aaaagctaaa 660 ctatcaaaac ttgaagagtt aagtgataag attgatgagg tcgacatggc agtgaatgta 720 aatacaaacg tagcagcaat gacagcacag cgttacctga ataacgcaaa cagcgcacaa 780 caaacttcga tggagcgtct gtcttcaggt ttcaaaatca acagtgcaaa agatgacgca 840 gccggtctgc aaatctctaa ccgcttgaac gtacaaagtc gcggtctaga cgttgcggta 900 cgtaacgcca acgacggtat ctcaatcgca caaaccgcag aaggtgcgat gaacgagacc 960 accaacatcc tacaacgtat gcgtgaccta tctctacaat ccgcgaacgg ctcaaactca 1020 aaatcagagc gcgtggcgat tcaagaagaa gtgacagcat tgaatgacga gctaaaccgt 1080 attgcagaaa ccacgtcttt tggtggtaac aagctgctaa acggtactta cggcacgaaa 1140 gcaatgcaaa ttggtgcgga taacggtgaa gcggtcatgc tttcactgaa agacatgcgc 1200 tctgacaacg tgatgatggg cggcgtgagc taccaagctg aagaaggcaa agacaagaac 1260 tggaatgtgg ccgcaggcga caacgacttg acgattgcac tgacagacag ctttggtaac 1320 gagcaagaga tcgaaatcaa cgcgaaagcg ggtgatgaca tcgaagagct agcgacgtac 1380 atcaacggtc aaactgacct tgtaaaagcg tcagtgggtg aaggcggcaa gctacagatc 1440 tttgctggta acaacaaagt tcaaggtgaa attgctttct caggtagcct agctggtgaa 1500 cttggcctag gcgaaggcaa aaacgtcacg gtagacacga ttgacgtgac aaccgtacaa 1560 ggtgcgcaag agtcggtagc gattgtggat gcggcactga aatacgtaga cagccaccgt 1620 gcagagctgg gtgcattcca gaaccgtttc aaccatgcaa tcagcaactt ggacaacatc 1680 aacgaaaacg tgaacgcgtc gaagagccga atcaaagata ccgacttcgc gaaagaaacg 1740 actcagttga ccaagacaca aattctatcg caagcatcaa gttccattct tgcgcaagcg 1800 aaacaagcgc caaactcagc gctaagtcta ctaggc 1836 <210> 6 <211> 612 <212> PRT <213> Artificial Sequence <220> <223> Fusion protein PspA-FlaB <400> 6 Ser Pro Val Ala Ser Gln Ser Lys Ala Glu Lys Asp Tyr Asp Ala Ala   1 5 10 15 Lys Lys Asp Ala Lys Asn Ala Lys Lys Ala Val Glu Asp Ala Gln Lys              20 25 30 Ala Leu Asp Asp Ala Lys Ala Ala Gln Lys Lys Tyr Asp Glu Asp Gln          35 40 45 Lys Lys Thr Glu Glu Lys Ala Ala Leu Glu Lys Ala Ala Ser Glu Glu      50 55 60 Met Asp Lys Ala Val Ala Ala Val Gln Gln Ala Tyr Leu Ala Tyr Gln  65 70 75 80 Gln Ala Thr Asp Lys Ala Ala Lys Asp Ala Ala Asp Lys Met Ile Asp                  85 90 95 Glu Ala Lys Lys Arg Glu Glu Glu Ala Lys Thr Lys Phe Asn Thr Val             100 105 110 Arg Ala Met Val Val Pro Glu Pro Glu Gln Leu Ala Glu Thr Lys Lys         115 120 125 Lys Ser Glu Glu Ala Lys Gln Lys Ala Pro Glu Leu Thr Lys Lys Leu     130 135 140 Glu Glu Ala Lys Ala Lys Leu Glu Glu Ala Glu Lys Lys Ala Thr Glu 145 150 155 160 Ala Lys Gln Lys Val Asp Ala Glu Glu Val Ala Pro Gln Ala Lys Ile                 165 170 175 Ala Glu Leu Glu Asn Gln Val His Arg Leu Glu Gln Glu Leu Lys Glu             180 185 190 Ile Asp Glu Ser Glu Ser Glu Asp Tyr Ala Lys Glu Gly Phe Arg Ala         195 200 205 Pro Leu Gln Ser Lys Leu Asp Ala Lys Lys Ala Lys Leu Ser Lys Leu     210 215 220 Glu Glu Leu Ser Asp Lys Ile Asp Glu Val Asp Met Ala Val Asn Val 225 230 235 240 Asn Thr Asn Val Ala Ala Met Thr Ala Gln Arg Tyr Leu Asn Asn Ala                 245 250 255 Asn Ser Ala Gln Gln Thr Ser Met Glu Arg Leu Ser Ser Gly Phe Lys             260 265 270 Ile Asn Ser Ala Lys Asp Asp Ala Ala Gly Leu Gln Ile Ser Asn Arg         275 280 285 Leu Asn Val Gln Ser Arg Gly Leu Asp Val Ala Val Arg Asn Ala Asn     290 295 300 Asp Gly Ile Ser Ile Ala Gln Thr Ala Glu Gly Ala Met Asn Glu Thr 305 310 315 320 Thr Asn Ile Leu Gln Arg Met Arg Asp Leu Ser Leu Gln Ser Ala Asn                 325 330 335 Gly Ser Asn Ser Lys Ser Glu Arg Val Ala Ile Gln Glu Glu Val Thr             340 345 350 Ala Leu Asn Asp Glu Leu Asn Arg Ile Ala Glu Thr Thr Ser Phe Gly         355 360 365 Gly Asn Lys Leu Leu Asn Gly Thr Tyr Gly Thr Lys Ala Met Gln Ile     370 375 380 Gly Ala Asp Asn Gly Glu Ala Val Met Leu Ser Leu Lys Asp Met Arg 385 390 395 400 Ser Asp Asn Val Met Met Gly Gly Val Ser Tyr Gln Ala Glu Glu Gly                 405 410 415 Lys Asp Lys Asn Trp Asn Val Ala Ala Gly Asp Asn Asp Leu Thr Ile             420 425 430 Ala Leu Thr Asp Ser Phe Gly Asn Glu Gln Glu Ile Glu Ile Asn Ala         435 440 445 Lys Ala Gly Asp Asp Ile Glu Glu Leu Ala Thr Tyr Ile Asn Gly Gln     450 455 460 Thr Asp Leu Val Lys Ala Ser Val Gly Glu Gly Gly Lys Leu Gln Ile 465 470 475 480 Phe Ala Gly Asn Asn Lys Val Gln Gly Glu Ile Ala Phe Ser Gly Ser                 485 490 495 Leu Ala Gly Glu Leu Gly Leu Gly Glu Gly Lys Asn Val Thr Val Asp             500 505 510 Thr Ile Asp Val Thr Thr Val Gln Gly Ala Gln Glu Ser Val Ala Ile         515 520 525 Val Asp Ala Ala Leu Lys Tyr Val Asp Ser His Arg Ala Glu Leu Gly     530 535 540 Ala Phe Gln Asn Arg Phe Asn His Ala Ile Ser Asn Leu Asp Asn Ile 545 550 555 560 Asn Glu Asn Val Asn Ala Ser Lys Ser Arg Ile Lys Asp Thr Asp Phe                 565 570 575 Ala Lys Glu Thr Thr Gln Leu Thr Lys Thr Gln Ile Leu Ser Gln Ala             580 585 590 Ser Ser Ser Ile Leu Ala Gln Ala Lys Gln Ala Pro Asn Ser Ala Leu         595 600 605 Ser Leu Leu Gly     610 <210> 7 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for PspA N-terminal <400> 7 catatgtctc ccgtagccag tcagtct 27 <210> 8 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for PspA N-terminal <400> 8 gtcgacctca tcaatcttat cacttaactc ttcaag 36 <210> 9 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for PspA C-terminal <400> 9 gtcgactctc ccgtagccag tcagt 25 <210> 10 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for PspA C-terminal <400> 10 cccgggttac tcatcaatct tatcacttaa ctcttc 36 <210> 11 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for FlaB N-terminal <400> 11 catatgatgg cagtgaatgt aaatacaaac gtagca 36 <210> 12 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for FlaB N-terminal <400> 12 gtcgacgcct agtagactta gcgct 25 <210> 13 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for FlaB C-terminal <400> 13 gtcgacatgg cagtgaatgt aaatacaaac gtag 34 <210> 14 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for FlaB C-terminal <400> 14 cccgggttag cctagtagac ttagcg 26  

Claims (7)

A recombinant fusion protein prepared by fusing the flagellin protein of sepsis Vibrio vulnificus and the protein antigen of a pathogen, wherein the flagellin protein is FlaB, and the protein antigen of the pathogen is surface protein A of Streptococcus pneumoniae. PspA), and the recombinant fusion protein is a FlaB-PspA protein having the amino acid sequence of SEQ ID NO: 4. delete delete delete delete A vaccine for mucosal administration comprising the recombinant fusion protein according to claim 1 as an active ingredient. The vaccine for mucosal administration according to claim 6, wherein the mucosa is nasal mucosa.

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