KR20180043752A - Fusion protein and use thereof - Google Patents

Abstract

The present invention relates to flagellin fusion protein, including chikungunya virus antigen protein and toll-like receptor 5 stimulation protein, and a use thereof. According to the present invention, if the flagellin fusion protein, including chikungunya virus antigen protein and toll-like receptor 5 stimulation protein, is used to diagnose viruses, an antibody in blood is effectively detected to increase the sensitivity and accuracy of the virus diagnosis. Moreover, the fusion protein is able to make a considerable synergy for inducing immunity through a vaccine, as compared to when only the chikungunya virus antigen protein is applied.

Description

[0001] FUSION PROTEIN AND USE THEREOF [0002]

The present invention relates to fusion proteins and uses thereof.

The chikungunya virus is a tropical forest mosquito (Aedes aegypti) or a white mosquito mosquito infected with a 60-70 nm (+) ssRNA virus belonging to the family Togaviridae, genus Alphavirus Aedes albopictus, Tiger mosquito) is known as a virus that causes Chikunguniya fever, an acute febrile disease. 10 to 15% of the infected chickens are asymptomatic when they are infected with the virus, while the rest of infected individuals have a latent period of 1 to 12 days and are exposed to sudden high fever, intermittent chills, headache, nausea, vomiting, severe arthralgia, Rash, etc., and high fever of 39 ~ 40 ℃ is reported to last for several days. In particular, in the case of arthralgia, 30-40% of the infected persons are not only chronic diseases that last for many years, but they are also known as diseases that can lead to meningitis in neonates.

Although the Chikunguniya virus has a very low probability of reinfection when it has immunity after infection, there is no need for a vaccine or diagnostic method for Chikunguniya virus.

Flagella, on the other hand, is an important component that determines the motility of bacteria and consists largely of hooks, basal bodies and filaments. It is known that flagella have a function of determining swelling motility of bacteria, taxis of bacteria, forming biofilm, and determining adherence ability of pathogenic microorganisms. The constituent unit proteins constituting the filament of the flagella are called flagellin, and the plasins are assembled to form filaments. Gram-negative and gram-positive bacterial plasmids with flagella have been reported to activate NF-κB by stimulating TLR5 (toll like receptor 5) (Hayashi F, Smith KD, Ozinsky A, Hawn TR, Yi EC, Goodlett DR , Eng JK, Akira S, Underhill DM, Aderam: Nature 410: 1099-1103, 2001). The TLR5 is known as a pattern recognition receptor (PRP), which recognizes a molecule structure associated with a pathogen, and is known to induce congenital immune responses and further regulate acquired immune responses.

Currently, vaccine adjuvants are being used or are being considered for use: 1) mineral salts such as aluminum hydroxide gel, 2) surfactants, 3) bacterial-derived substances, 4) cytokines or hormones, 6) polyanion, 7) polyacryl, 8) carrier, 9) a living vector using virus, and 10) a vehicle such as mineral oil or liposome. ). However, since the vaccine adjuvant is an exogenous toxin, there is a great risk of side effects, and thus studies for reducing toxicity are being actively carried out. Accordingly, the above-mentioned plasgel can be a suitable target for the development of a vaccine adjuvant, It is in progress.

It is an object of the present invention to provide a fusion protein in which an antigen protein of chikungunya virus and a toll-like receptor 5 stimulating protein are fused and a vector expressing the fusion protein.

It is another object of the present invention to provide information for diagnosing chikungunya virus infection using the fusion protein according to the present invention.

It is another object of the present invention to provide a vaccine for chikungunya virus comprising the fusion protein according to the present invention as an active ingredient.

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

As a result of the study, the present inventors have found that when an antigen protein of chikungunya virus and flagellin, which is a stimulus protein of toll-like receptor 5, is produced as a fusion protein, , And when the plasgelin acts as a vaccine adjuvant to stimulate the toll-like receptor 5 to induce immunological activity, as compared with the case where the chitin shell protein is singly administered, the antibody against the chitin shell coat protein is administered in the same amount of the vaccine (Alum), which is a vaccine adjuvant, is added to the envelope protein to form an antibody amplified from the fusion protein, and a smaller amount of the protein antigen is used as a vaccine As well as the use of IgG and I < RTI ID = 0.0 > gM detection, the present invention has been completed.

In one embodiment of the present invention, a fusion protein comprising a toll-like receptor 5 stimulating protein is provided on one side of a chikungunya virus antigen protein.

In the present invention, the " Toll like receptor 5 "is a typical pattern recognition receptor, which is one of receptors recognizing a molecular structure related to a pathogen present in a pathogen, Like receptor 5 stimulating protein can be used as a vaccine adjuvant by inducing congenital immune response as well as inducing acquired immune response by various stimuli such as flagellin ), But may be included as long as it binds to a pattern recognition receptor and is recognized as an external antigen to induce an immune response.

In the present invention, the plasmin can induce an immune response in an infected host when a homologous bacterium is infected. More specifically, toll-like receptor 5 (TLR5) present on the surface of the cell membrane of the human body induces intracellular signal transduction through interaction with the above-mentioned plasmin, and through this, transcription factor NF-kB To induce innate immune signal activation, as well as to regulate the acquired immune response. Therefore, the fusion protein in which flagellin is bound to one side of the chikungunya virus antigen protein according to the present invention is more effective than the chikungunya virus antigen protein in the case of immunization induction through vaccine And a synergistic effect in the sensitivity and sensitivity of diagnosis of Chikungunya virus using the fusion protein according to the present invention.

In the present invention, the term "fusion protein" means a protein produced by fusion of two or more proteins, and can be produced by a recombinant method. The expression of the fusion protein can be induced by transforming the recombinant gene produced by the above-mentioned recombinant DNA technology into a host cell, but the present invention is not limited thereto. For the purpose of the present invention, the fusion protein may be a fusion protein obtained by expressing a chitinoguania virus antigen protein and a toll-like receptor 5 stimulating protein, in particular, plasgelin, a recombinant gene produced by a recombinant method, in a host cell.

In the present invention, the chiquiniynavirus antigen protein may be a chikunguniya virus envelope antigen protein. The chickungenya virus envelope antigen protein is a protein constituting virus particles together with a nucleic acid, and generally shows specificity as a virus antigen.

In the present invention, the chickungenya virus has E1, E2, E3, and 6K envelope antigen proteins. For the purpose of the present invention, the chickungen virus envelope antigen protein may be the E2 envelope antigen protein represented by SEQ ID NO: 1 , But is not limited thereto. Specifically, the E2 envelope antigen protein represented by SEQ ID NO: 1 may be an antigenic protein consisting of 343 amino acid sequences corresponding to a part of the chicungunya envelope antigen protein represented by SEQ ID NO: 2. The chickungenya virus E2 envelope antigen protein of SEQ ID NO: 2 plays a pivotal role in the formation of the envelope on the viral envelope protrusion but the yield of recombinant protein expression using Escherichia coli strains has been extremely low in the past. However, in the case of expressing the fusion protein using E. coli after binding the plasmin to the E2 envelope antigen protein of SEQ ID NO: 1 according to the present invention, not only the expression efficiency is very high, but also the advantage Lt; / RTI >

In the present invention, the flagellin may be flagellin of the genus Salmonella or the genus Bacillus. Preferably, the plasgelin may be a plasgel derived from Bacillus sp. Bacteria, but is not limited thereto. More preferably, the bacterium is not may be a Salmonella Dublin (salmonella dublin) or Sirius Bacillus (Bacillus cereus), limited.

In the present invention, the term " flagellin "refers to a major protein constituting flagella that provides bacterial motility, and corresponds to the protein most abundantly present in unconjugated bacteria. The plasgelin may generally be composed of 2 to 4 domains, and the domains may be composed of two conserved domains D0 and D1, and diversified domains D2 and D3 having a length and presence or absence . For the purpose of the present invention, the plasmalin of the present invention may include one or more of D0 and D1 domains, and preferably the plasmin protein may be represented by SEQ ID NO: 3, but is not limited thereto. Particularly, since the Bacillus-derived plasgeline is composed of only the D0 and D1 domains, it is possible to selectively activate the innate immune response by the plasmin and minimize the undesired toxicity when the desired individual is inoculated after producing the fusion protein There are advantages.

In one embodiment of the present invention, a histidine-tag may be bound to one side of the fusion protein according to the present invention. When the histidine-tag is bound, there is an advantage that the purified protein can be rapidly purified and the purity of the purified protein can be increased in the host cell after the expression is induced in the host cell by transformation.

In another embodiment of the invention there is provided a polynucleotide encoding said fusion protein according to the invention.

In the present invention, the term "polynucleotide" means a polymer of a nucleotide in which a nucleotide monomer is linked in a long chain by a covalent bond and is a DNA or RNA strand longer than a certain length. Polynucleotide "

The polynucleotide encoding the fusion protein according to the present invention can be obtained by altering the amino acid sequence of the fusion protein expressed from the coding region in consideration of codon degeneracy or a codon preferred in the organism to which the protein is to be expressed Various modifications can be made in the coding region within a range not affecting the expression of the gene in the region other than the coding region and various modifications or modifications can be made within a range that does not affect the expression of the gene in the portion excluding the coding region, .

In another embodiment of the present invention, there is provided an expression vector comprising the polynucleotide according to the present invention. Preferably, the expression vector comprises a gene fragment represented by the nucleotide sequence of SEQ ID NO: 4, but is not limited thereto.

In the present invention, the expression vector means a means for expressing the fusion protein of the present invention in a microorganism by introducing DNA into the host cell. In the production of the expression vector, the type of the host cell to produce the fusion protein An expression control sequence such as a promoter, a terminator, an inhancer and the like and a sequence for membrane targeting or secretion may be appropriately selected depending on the purpose and may be variously combined.

The expression vector of the present invention may include, but is not limited to, a plasmid vector, a cosmid vector, a bacteriophage vector, and a viral vector. Suitable expression vectors include signal sequences or leader sequences for membrane targeting or secretion in addition to expression control elements such as promoter, operator, initiation codon, termination codon, polyadenylation signal and enhancer, and can be prepared variously according to the purpose. The promoter of the expression vector may be constitutive or inducible. An MFN signal sequence and a SUC2 signal sequence in the case where the host is yeast in the signal sequence and an insulin signal sequence, an a-interferon signal sequence and an antibody molecule signal sequence in the case where the host is an animal cell, But is not limited thereto. In addition, the expression vector may include a selection marker for selecting a host cell containing the vector, and may include a replication origin for a replicable expression vector, more preferably a pET49b vector. no.

In another embodiment of the present invention, there is provided a vaccine for Chichungnany virus comprising the fusion protein according to the present invention as an active ingredient.

In the present invention, the vaccine may be prepared by a conventional method well known in the art, and may further include various additives that can be used in the art in the manufacture of a vaccine.

The vaccine according to the present invention may additionally comprise other immunosuppressants, for example consisting of a second group of elements selected from the group consisting of Mg, Ca, Sr, Ba and Ra, Ti, Zr, Hf and Rf A salt of a Group 4 element selected from the group consisting of aluminum, or a hydrate thereof. The salt may preferably be formed with oxides, peroxides, hydroxides, carbonates, phosphates, pyrophosphates, hydrogen phosphates, dihydrogenphosphates, sulphates or silicates. For example, the adjuvants that may be further utilized in the vaccine composition of the present invention include, but are not limited to, magnesium hydroxide, magnesium carbonate hydoxideside pentahydrate, titanium diadoxides, calcium carbonate, barium oxide, barium hydexide, But are not limited to, barium peroxide, barium sulfate, calcium sulfate, calcium pyrophosphate, magnesium carbonate, magnesium oxide, aluminum hydroxide, aluminum phosphate and hydrated aluminum potassium sulfate (Alum).

In addition, the vaccine according to the present invention may further comprise a pharmaceutically acceptable carrier. But are not limited to, for example, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose But are not limited to, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components.

In the present invention, the vaccine may be formulated into a unit dose form by using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by those having ordinary skill in the art to which the present invention belongs. Or by intrusion into a multi-dose container. The formulations may be in the form of solutions, suspensions or emulsions in oils or aqueous media, or in the form of excipients, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.

In the present invention, a suitable dose of the vaccine may be variously prescribed depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate and responsiveness of the patient . On the other hand, the dose of the vaccine according to the present invention may preferably be 0.0001-1000 mg / kg (body weight) per day.

In one embodiment of the present invention, the vaccine comprising the fusion protein as an active ingredient may be administered into the body by intravenous injection, intraarterial injection, intramuscular injection, subcutaneous injection, intraconjugate injection, transdermal delivery or airway inhalation , But is not limited thereto.

In another embodiment of the present invention, there is provided a kit for the diagnosis of chickhunnya virus comprising the fusion protein according to the present invention as an active ingredient. The kit provided by the present invention can measure the level of antigen-antibody complex formation of the fusion protein according to the present invention with an antibody present in a sample of a desired individual, preferably human IgG and / or IgM, But is not limited to. In the case of the diagnosis using the fusion protein of the present invention, there is an advantage that the sensitivity of IgM at the early stage of infection is very high, so that it can be diagnosed very effectively even in the early asymptomatic period immediately after the infection.

However, in the present invention, the term "kit " means a set in which a composition and accessories necessary for a specific purpose are collected. Specifically, the kit may include not only the diagnostic composition according to the present invention, but also tools and reagents commonly used in the field of the present invention, which are used for assaying the level of the antigen-antibody complex formation. Examples of such tools or reagents include, but are not limited to, suitable carriers, markers capable of generating detectable signals, chromophores, solubilizers, buffers, stabilizers, and the like. When the labeling substance is an enzyme, it may include a substrate capable of measuring enzyme activity and a reaction terminator. Examples of the insoluble carrier include polystyrene, polyethylene, polypropylene, polyester, polyvinylpyrrolidone, polyvinylpyrrolidone, polyvinylpyrrolidone, polyvinylpyrrolidone, polyvinylpyrrolidone, polyvinylpyrrolidone, Acrylonitrile, fluorine resin, crosslinked dextran, polysaccharide, polymer such as magnetic fine particles plated with metal on latex, other paper, glass, metal, agarose and combinations thereof.

In the case of using the kit provided by the present invention, the level of antigen-antibody complex formation of the fusion protein is compared from a sample obtained from a desired individual to determine whether or not the desired individual is infected with Chikunguniya virus, Early diagnosis can be used to determine whether the individual patient is actively treated and whether a detailed observation is needed. In addition, the kit of the present invention includes a kit (hereinafter referred to as a 'protein chip kit') containing essential elements necessary for performing protein chip analysis; Or a kit (hereinafter referred to as an " ELISA kit ") containing essential elements necessary for performing an ELISA.

In the present invention, the term "protein chip kit " means a kit in which an antibody capable of reacting with a specific protein is immobilized on a single chip at high density. For the purpose of the present invention, Protein.

The kit for measuring the antigen-antibody complex formation level according to the present invention may include a substrate, a buffer solution, a secondary antibody labeled with a chromogenic enzyme or a fluorescent substance, a chromogenic substrate and the like for immunological detection of the antibody. As the substrate, a nitrocellulose membrane, a 96-well plate synthesized with a polyvinyl resin, a 96-well plate synthesized with a polystyrene resin, a slide glass made of glass, or the like can be used as the substrate, and a peroxidase ), Alkaline phosphatase, and the like can be used. As the fluorescent material, FITC, RITC and the like can be used. As the chromogenic substrate, ABTS (2,2'-azino-bis- (3-ethylbenzothiazoline-6-sulfonic acid)), OPD (Tetramethylbenzidine), and the like can be used. In order to measure the level of antigen-antibody complex formation of the protein, a method of counting the binding of the antibody by nCounter or the like may be used.

In the present invention, the "ELISA kit" refers generally to an enzyme immunoassay method for measuring the amount of an antigen or an antibody using an enzyme-labeled antibody as an indicator. For the purpose of the present invention, ≪ / RTI > The ELISA kit may further comprise a reagent capable of detecting the antibody in the desired individual bound to the fusion protein, for example, a labeled secondary antibody, chromophores, an enzyme conjugated with the antibody and its substrate, Other substances capable of binding to the antibody, and the like.

In another embodiment of the present invention, there is provided a method for providing information for diagnosing Chikungunya virus infection by measuring the level of complex formation between the fusion protein according to the present invention and an antibody present in a sample of a desired individual . Specifically, (a) measuring the level of antigen-antibody complex formation with the fusion protein according to the present invention in a sample separated from the desired individual; And (b) comparing the level of antigen-antibody complex formation measured in step (a) with the level of antigen-antibody complex formation with the fusion protein in a normal control sample. . ≪ / RTI >

Further, in the present invention, the above-mentioned "target object" means an individual who is not sure whether or not Chikunguniya virus infection is present, and which is highly susceptible to infection. The sample isolated from the subject may include, but is not limited to, a tissue of a highly infectious subject, a cell, blood, serum, plasma, saliva, or sputum. But is not limited to, any one or more of IgG and IgM antibodies preferably contained in human serum.

In the present invention, the measurement of the antigen-antibody complex formation level is carried out by measuring the level of antigen-antibody complex formation using the fusion gene according to the present invention as an antigen in a normal control, that is, in an individual not infected with chickhuning virus, Antibody-antibody complex formation levels of the subject suspected of being infected with the virus, that is, the desired individual, can be compared, and the difference in the level of complex formation can be determined to predict whether Chikunguniya virus is infected. Preferably, if the level of antigen-antibody complex formation is higher than that of a normal control sample that is not infected with chichengnyi virus, the subject may be diagnosed as having Chikungunya virus infection, but is not limited thereto .

The term "antigen-antibody complex" in the present invention refers to a combination of the fusion protein according to the present invention and an antibody specific to an antibody present in the desired individual, and the formation level of the antigen- the detection label can be quantitatively measured through the intensity of the signal.

In the present invention, the above-mentioned "antigen-antibody complex formation level measurement" refers to the determination of the presence of an antibody present in a target sample bound to the fusion gene according to the present invention, , The amount of the antibody in the target sample is confirmed using the fusion gene according to the present invention. Analysis methods include Western blot, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis , Immunohistochemical staining, immunoprecipitation assay, complement fixation assay, FACS, protein chip, and the like, but are not limited thereto.

In addition, the above-mentioned "detection label enzyme of the present invention" can be selected from the group consisting of, for example, a complex, ligand, luminescent material, microparticle, redox molecule and radioisotope, no. More specifically, enzymes that can be used as detection labels include, but are not limited to, ß-glucuronidase, ß-D-glucosidase, ß-D-galactosidase, urease, peroxidase or alkaline phosphatase, A glucosoxidase, hexokinase and GDPase, RNase, glucose oxidase and luciferase, phosphofructokinase, phosphoenolpyruvate carboxylase, aspartate aminotransferase, phosphoenolpyruvate decarboxylase Raje, and ß-la Tamaje. But is not limited thereto. Examples of the above-mentioned minerals that can be used include, but are not limited to, fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, alopicocaine, o-phthaldehyde, Do not. The ligand may be a biotin derivative or the like, but is not limited thereto. Examples of the luminescent material include, but are not limited to, acridinium ester, luciferin, luciferase, and the like. The fine particles include, but are not limited to, colloidal gold and colored latex. In addition, the redox molecules include ferrocene, ruthenium complex compounds, Biology hydrogen, quinone, Ti ions, Cs ions, diimide, 1,4-benzoquinone, _{hydroquinone, K 4 W (CN) 8} , [Os (bpy) 3] ²⁺ , [RU (bpy) ₃ ] ²⁺ , [MO (CN) ₈ ] ^{4 -} , and the like. In addition, the radioisotope includes a ^{3 H, 14 C, 32 P} , 35 S, 36 Cl, 51 Cr, 57 Co, 58 Co, 59 Fe, 90 Y, 125 I, 131 I, 186 Re, In It is not limited.

In the case of using the fusion protein in which chitosan-like receptor 5 stimulating protein, which is a toll-like receptor 5 protein, bound to chikungunya virus coat protein according to the present invention is used to detect the virus infection, the antibody in blood is effectively detected , The sensitivity and accuracy of the virus diagnosis can be increased.

In addition, the fusion protein can exert a remarkable synergistic effect when the immunogen induction through the vaccine is performed, as compared with the case where the fusion protein is inoculated with the chickungenya virus antigen protein alone.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic diagram of a plasmid vector for the expression of a Chikungunya-Plazelin fusion protein according to an embodiment of the present invention.
FIG. 2 shows electrophoretic analysis results of chitin-flavin fusion proteins according to an embodiment of the present invention.
FIG. 3 is a graph showing the results of the presence of a chitin-flavin fusion protein as a monomer in a host state according to an embodiment of the present invention.
FIG. 4 shows the results of signal transduction activity through stimulation of Toll like receptor 5 (TLR5) according to an embodiment of the present invention.
FIG. 5 is a schematic diagram of a vaccination induction process according to an embodiment of the present invention.
FIG. 6 (a) is a graph showing the results of immunization of the chickunguniya-plasgelin fusion protein according to one embodiment of the present invention with respect to the number of contact times with respect to the chitin- Lt; / RTI >
7 shows ELISPOT results according to an embodiment of the present invention.
FIG. 8 shows the results of confirming the IgM and IgM responses in patients infected with Chikunkeniya virus and a normal control group according to an embodiment of the present invention.

Hereinafter, the present invention will be described in more detail with reference to Examples. It will be apparent to those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

[ Example  One] Chikunguniya - Plasticine  Production of fusion protein expression plasmid

To prepare a vaccine suitable for Chikungunya virus, a protein fused with an amino acid corresponding to a part of the envelope protein antigen E2 of Chikungunya virus and a plasmin protein was prepared.

In order to prepare the chitinin-plasmin fusion protein, two steps of PCR reaction were carried out. The first amplification reaction was carried out using primers of genomic DNA as templates for 1 hour and 2 primers for denaturation at 95 ° C for 30 seconds, annealing at 60 ° C for 30 seconds, and synthesis conditions at 72 ° C for 60 seconds The denaturation-annealing-synthesis cycle was repeated 25 times to obtain a first reactant. The second amplification reaction was carried out using genomic DNA of Bacillus sirius as a template, denaturation at 95 DEG C for 30 seconds, denaturation at 95 DEG C for 30 seconds using primer No. 3 in Table 1 and primary reaction product as a product of the first reaction Annealing and a cycle of replication reaction at 72 캜 for 1 minute was repeated 25 times to finally obtain a fusion gene of Chikungunya-plasin protein. The fusion gene of Chikungunya-plaslanin protein was inserted into pET49b (Addgene) vector to prepare a protein expression plasmid. The protein expression plasmid was verified through DNA sequencing (macrogen), and as a result, it was confirmed that it was the same as SEQ ID NO: 4. A schematic diagram of the prepared plasmid is shown in Fig.

number Primer sequence One 5'-CAA ATG GTT TCT AAA TTA TTA CAA GCG GCC GC AAG CAC CAA GGA CAA CTT CAA TGT C -3 ' 2 5'-CCT CGA GTT ACC CGG GCC CTT CAG TCG GCA CGG TTAA-3 3 5'-AG GAT CCG ATG AGA ATT AAT ACA AAC ATT AAC AGC ATG CG-3 '

[ Example  2] Chikunguniya - Plasticine  Fusion protein production

The fusion protein was produced through the Chikungunya-Plazelin fusion gene expression plasmid prepared in Example 1 above.

The fusion gene expression plasmid was injected into a chemically treated BL21 (DE3) Escherichia coli BL21 (DE3) by a transformation method and an LB agar (Becton) containing kanamycin antibiotic , Dickinson and Company) plate. A single E. coli colony containing the plasmid vector resistant to antibiotics selected through the above-mentioned culturing process was placed in a liquid medium (LPS) containing 1 mM kanamycin and cultured in a constant temperature shaking incubator at 37 ° C. At this OD ₆₀₀ value of the culture solution to 0.7, 5-Bromo-chloro-indole-3-isopropyl-wool -ß-D- galacto-pyrano seed (IPTG) 3 hours at 37 ℃ After the addition of 1 mM Lt; / RTI > to induce expression of the fusion protein. The E. coli in which the expression of the fusion protein was induced was centrifuged at 4,000 rpm for 20 minutes to obtain cells. A cell lysis of the obtained E. coli cells was performed using a sonicator. The cell lysate was centrifuged to remove other cellular debris and then allowed to bind with Ni-NTA agar beads (Qiagen) at 4 ° C for 2 hours. Then, other proteins that were non-specific binding were first removed using a 10 mM imidazole / 1X phosphate buffer solution (PBS), and finally, 50 mM imidazole / 1X phosphate buffer solution was used to finally remove Nonspecific binding proteins were removed. The chitinin-plasgelin fusion protein bound to the beads was obtained by elution in Ni-NTA agar beads with competitive inhibition using 250 mM imidazole / 1X phosphate buffer. The obtained fusion protein (extraction, elution) was confirmed by SDS-PAGE for size and purity, and the result is shown in FIG.

As shown in FIG. 2, the chitin-flavin fusion protein was observed at about 73 kDa, confirming that the normal fusion protein was produced and expressed properly.

[ Example  3] Chikunguniya - Plasticine  Identification of fusion protein properties

In order to confirm whether the fusion protein produced in Example 2 exists as a monomer, gel filtration chromatography was performed by a conventional method, and the result is shown in FIG.

As shown in FIG. 3, the fusion protein produced in Example 2 was not randomly polymerized in an aqueous solution but formed monomers.

From the above results, it was found that the chitinoguania-flavin fusion protein according to the present invention exists as a monomer and can stimulate TLR5, which is a congenital immunoreceptor (Smith KD, Andersen-Nissen E, Hayashi F, Strobe K, Bergman MA, Barrett SL, Cookson BT, and Aderem A.Toll-like receptor 5 recognizes a conserved site on flagellin required for protofilament formation and bacterial motility Nat Immunol 2003 Dec; 4 (12): 1247-5. .

[ Example  4] Chikunguniya - Plasticine  Of the fusion protein TLR5  Identify stimulating activity

In order to confirm TLR5 stimulation activity of the fusion protein produced in Example 2, the amount of NF-κB activity induced by TLR5 was measured.

HEK293TLR5 cells were seeded in 96-well plates and cultured in DMEM (High glucose with 4500 mg / L D-glucose L-glutamine WELGENE LM 001-07) medium for 12 hours in a constant temperature incubator at 37 ° C and 5% . Plastic handle jelrin protein of Salmonella Dublin (salmonella Dublin) in the said cell culture as a positive control over the stabilization (1), and also processes the fusion protein (2) produced in Example 2 at different concentrations. Alkaline phosphatase substrate yellow (Sigma-Aldrich P7998) was diluted 1/5 into each of the cells treated with the fusion protein and incubated for 30 minutes in the incubator for 30 minutes. The value was measured at the wavelength, and the value is shown in Fig.

As shown in FIG. 4, it was confirmed that the activity of the cell signaling system via TLR5 stimulation can be induced by the fusion protein (2) produced in Example 2 like the control (1).

From the above results, it can be seen that the domain corresponding to a part of the plasmalin according to the present invention can also effectively induce the stimulating activity of TLR5.

[ Example  5] Chikunguniya - Plasticine  Verification of vaccine effect using fusion protein

In order to confirm the vaccine effect of the Chikunguniya-plasgelin fusion protein prepared in Example 2, immunization was carried out according to the method shown in Fig. 5, and then it was verified by ELISA method.

Chikunguniya coat protein (ChiKE2 ⁴²⁶ ) and aluminum potassium sulfate (Alum) purchased from a company as a control in 5-6 week old female BALB / C rats were divided into experimental groups Lt; RTI ID = 0.0 > 10 < / RTI > After the first to third immunizations were induced by the inoculation, serum was obtained from the blood of the individual two weeks later by an ordinary method, and ELISA was performed to confirm the degree of production of IgG.

The ELISA was prepared by adding the chitin coat protein (ChiKE2 ⁴²⁶ ), Alum and the fusion protein antigen of Example 2 at a concentration of 3.0 ug / ml to the coating solution (0.159 g Na2CO3, 0.293 g NaHCO3, 100 ml, pH9.6) After dilution, 100 μl of the solution was added to each well of a 96-well plate and adsorbed at 4 ° C. for one day. After the adsorption of the antigen was completed, the plate was washed four times with PBS. To exclude non-specific binding, PBS containing 5% of normal goat serum was added to each plate and reacted at 37 ° C for 2 hours . Rat sera obtained from the control, Alum and the fusion protein in Example 2 were reacted at room temperature for 1 hour, washed 4 times with PBS, and then incubated with anti-mouse IgG1 After incubation at room temperature for 1 hour, color development was stopped by adding a substrate buffer solution (3,3 ', 5,5'-Tetramethylbenzidine (TMB) and hydrogen peroxide water) in the dark room, adding 2N sulfuric acid, And the results are shown in Figs. 6 and 7.

As shown in FIG. 6 (a), in the case of Chichungonia cotyledon protein (ChiKE2 ⁴²⁶ ) corresponding to the control group, it was confirmed that antibody reaction was increased after immunization three times in the absence of Alum, The antibody response was increased after the immunization. In addition, the amount of the fusion protein of Example 2 was increased according to the number of times of inoculation, and in addition, the group containing the control chitinoguania envelope protein and Alum together (ChiKE2 ⁴²⁶ + Alum), the amount of antibody significantly increased after 4th week.

As shown in FIG. 6 (b), when the antibody response to each antigen was measured, the mice immunized with ChiKE2 protein were found to have SDFlic + E2B, BCFlic + E2B, and BCFlic + E2 proteins And the recombinant E2 protein as well as E2B, a plasgeline fusion protein. Antibody response to the plasgelin antigen was observed in the group immunized with SDFlic + E2B or BCFlic + E2B, a chickungunya-plasgelin fusion protein, and in the group in which only plasgelin was inoculated, whereas Chikunguniya - It was confirmed that the plasgelin fusion protein BCFlic + E2 group does not show antibody reaction to plasgelin. In the group immunized with plasgelin, the recombinant E2 protein, ChiKE2 ⁴²⁶ , did not react with the antibody, while the chitinoguania-plasgelin fusion proteins SDFlic + E2B, BCFlic + E2B, and BCFlic + , Indicating that the chitin-flavin fusion proteins specifically expressed the antibody response. In addition, ChiKE2 ⁴²⁶ , a recombinant chitinase E2 protein, showed no antibody reaction when administered alone, but was mixed with Alum, an immunosuppressant, to induce antibody reaction only when inoculated. On the other hand, Chikunguniya-plasgelin fusion proteins were found to induce the antibody reaction effectively even when administered alone without Alum.

In addition, ELISPOT was performed to confirm the number of immune cells secreting IgG.

The ELISPOT induced 3 rd immunity via inoculation as described above, and the rat spleen was isolated 4 weeks later. The isolated spleen was pulverized into a sterilized slide, and red blood cells were removed to obtain spleen cells. The anti-mouse IgG antibody was diluted to a concentration of 2.0 ug / ml in a coating solution (0.159 g Na ₂ CO _3, 0.293 g NaHCO ₃ , 100 ml, pH 9.6), and then the wells were plated in an ELISPOT 96-well plate And then adsorbed at 4 ° C. for one day. After the antigen-adsorbed plate was washed four times with sterilized PBS, RPMI 1640 culture medium containing 10% fetal bovine serum was added to each well to remove non-specific binding, and the plate was incubated at 37 ° C for 2 hours Lt; / RTI > Then, 5 × 10 ⁶ cells / ml of spleen cells obtained from each mouse spleen was diluted stepwise by 2 folds, added to each well, reacted at 37 ° C. for 4 hours, and washed with PBS. Then streptavidin anti mouse IgG was added and reacted overnight. After washing with PBS, color was developed using AEB reagent, washed thoroughly with tap water, dried, and then the number of spots was measured. The results are shown in Fig.

As shown in FIG. 7, significant increase in antibody-secreting immune cells was observed in the SDFlic + E2B and BCFlic + E2 experimental groups, which are fusion proteins of Chikungunya-plastelin. However, in the case of BCFlic + E2B, the number of immune cells secreting antibody was slightly increased but no significant increase was observed. In addition, it was confirmed that no increase of antibody-secreting immune cells was observed for the recombinant chikungunya protein, ChiKE2 ⁴²⁶ protein and plaslanin protein. From the above results, it was confirmed that the chitinin-flavin fusion protein according to the present invention induces immune cells that effectively produce antibodies.

From the above results, it can be seen that when the chicunguniya-plasgelin fusion protein according to the present invention is used as a vaccine, it can be used as a chikungunya coat protein alone or in a case where an immunological reaction The inventors of the present invention confirmed that the chitinin-flazelin fusion protein according to the present invention can be used as a vaccine having a remarkably increased effect as compared with conventional vaccines.

[ Example  6] Chikunguniya - Plasticine  Verification of diagnostic effect using fusion protein

The diagnostic effect of Chikungunya-Plazelin fusion protein prepared in Example 2 as an antigen for the detection of Chikungunya infection was verified by ELISA method.

The ELISA was performed in the same manner as in Example 5 above. However, the fusion protein prepared in Example 2 was diluted to a concentration of 3.0 μg / ml in a 96-well plate and coated with a final volume of 100 μl. Serum of Chikungunya infection patients and normal The control serum was diluted with buffer to a volume ratio of 1: 300 and reacted with horse radish peroxidase-conjugated Goat anti-Human IgG and IgM antibody for detection. The absorbance was measured at 450 nm as in Example 5 Respectively. The results are shown in Fig.

As shown in FIG. 8, the reaction of the fusion protein (BCFlic-ChiK E2 ³⁴³ ) prepared in Example 2 according to the present invention with the IgG and IgM antibodies of patients infected with Chikungunya was more sensitive than the reaction to chiKE2 ⁴²⁶ Specificity was remarkably high, and it was confirmed that IgM antibody detected at the early stage of infection can be detected very sensitively.

In the above results, when the chitinin-flavin fusion protein according to the present invention is used as an antigen for the diagnosis of Chikunguniya virus infection, a remarkable increase in sensitivity and specificity compared with the use of Eicin alone as a chikungunya antigen protein And it can be confirmed that it can be effectively used for diagnosis of early infection of Chikunkuniya virus.

Although the present invention has been described in detail with reference to the above results, it is to be understood that the scope of the present invention is not limited to these embodiments and that various modifications and changes may be made without departing from the scope of the present invention. It will be obvious to those of ordinary skill in the art.

<110> Industry-Academic Cooperation Foundation, Yonsei University <120> FUSION PROTEIN AND USE THEREOF <130> DPB163726k01 <150> KR 1020160136649 <151> 2016-10-20 <160> 4 <170> KoPatentin 3.0 <210> 1 <211> 343 <212> PRT <213> Chikungunya virus <400> 1 Gly Ser Ser Thr Lys Asp Asn Phe Asn Val Tyr Lys Ala Thr Arg Pro   1 5 10 15 Tyr Leu Ala His Cys Pro Asp Cys Gly Glu Gly His Ser Cys His Ser              20 25 30 Pro Val Ala Leu Glu Arg Ile Arg Asn Glu Ala Thr Asp Gly Thr Leu          35 40 45 Lys Ile Gln Val Ser Leu Gln Ile Gly Ile Lys Thr Asp Asp Ser His      50 55 60 Asp Trp Thr Lys Leu Arg Tyr Met Asp Asn His Met Pro Ala Asp Ala  65 70 75 80 Glu Arg Ala Gly Leu Phe Val Arg Thr Ser Ala Pro Cys Thr Ile Thr                  85 90 95 Gly Thr Met Gly His Phe Ile Leu Ala Arg Cys Pro Lys Gly Glu Thr             100 105 110 Leu Thr Val Gly Phe Thr Asp Gly Arg Lys Ile Ser His Ser Cys Thr         115 120 125 His Pro Phe His His Pro Pro Val Ile Gly Arg Glu Lys Phe His     130 135 140 Ser Arg Pro Gln His Gly Arg Glu Leu Pro Cys Ser Thr Tyr Ala Gln 145 150 155 160 Ser Thr Ala Ala Thr Ala Glu Glu Ile Glu Val His Met Pro Pro Asp                 165 170 175 Thr Pro Asp Arg Thr Leu Met Ser Gln Gln Ser Gly Asn Val Lys Ile             180 185 190 Thr Val Asn Ser Gln Thr Val Arg Tyr Lys Cys Asn Cys Gly Asp Ser         195 200 205 Ser Glu Gly Leu Thr Thr Thr Asp Lys Val Ile Asn Asn Cys Lys Val     210 215 220 Asp Gln Cys His Ala Ala Val Thr Asn His Lys Lys Trp Gln Tyr Asn 225 230 235 240 Ser Pro Leu Val Pro Arg Asn Ala Glu Ser Gly Asp Arg Lys Gly Lys                 245 250 255 Val His Ile Pro Phe Pro Leu Ala Asn Val Thr Cys Arg Val Val Lys             260 265 270 Ala Arg Asn Pro Thr Val Thr Tyr Gly Lys Asn Gln Val Ile Met Leu         275 280 285 Leu Tyr Pro Asp His Pro Thr Leu Leu Ser Tyr Arg Asn Met Gly Glu     290 295 300 Glu Pro Asn Tyr Gln Glu Glu Trp Val Thr His Lys Lys Glu Ile Arg 305 310 315 320 Leu Thr Val Pro Thr Glu Gly Leu Glu Val Thr Trp Gly Asn Asn Glu                 325 330 335 Pro Tyr Lys Tyr Trp Pro Gln             340 <210> 2 <211> 427 <212> PRT <213> Chikungunya virus <400> 2 Gly Ser Ser Thr Lys Asp Asn Phe Asn Val Tyr Lys Ala Thr Arg Pro   1 5 10 15 Tyr Leu Ala His Cys Pro Asp Cys Gly Glu Gly His Ser Cys His Ser              20 25 30 Pro Val Ala Leu Glu Arg Ile Arg Asn Glu Ala Thr Asp Gly Thr Leu          35 40 45 Lys Ile Gln Val Ser Leu Gln Ile Gly Ile Lys Thr Asp Asp Ser His      50 55 60 Asp Trp Thr Lys Leu Arg Tyr Met Asp Asn His Met Pro Ala Asp Ala  65 70 75 80 Glu Arg Ala Gly Leu Phe Val Arg Thr Ser Ala Pro Cys Thr Ile Thr                  85 90 95 Gly Thr Met Gly His Phe Ile Leu Ala Arg Cys Pro Lys Gly Glu Thr             100 105 110 Leu Thr Val Gly Phe Thr Asp Gly Arg Lys Ile Ser His Ser Cys Thr         115 120 125 His Pro Phe His His Pro Pro Val Ile Gly Arg Glu Lys Phe His     130 135 140 Ser Arg Pro Gln His Gly Arg Glu Leu Pro Cys Ser Thr Tyr Ala Gln 145 150 155 160 Ser Thr Ala Ala Thr Ala Glu Glu Ile Glu Val His Met Pro Pro Asp                 165 170 175 Thr Pro Asp Arg Thr Leu Met Ser Gln Gln Ser Gly Asn Val Lys Ile             180 185 190 Thr Val Asn Ser Gln Thr Val Arg Tyr Lys Cys Asn Cys Gly Asp Ser         195 200 205 Ser Glu Gly Leu Thr Thr Thr Asp Lys Val Ile Asn Asn Cys Lys Val     210 215 220 Asp Gln Cys His Ala Ala Val Thr Asn His Lys Lys Trp Gln Tyr Asn 225 230 235 240 Ser Pro Leu Val Pro Arg Asn Ala Glu Ser Gly Asp Arg Lys Gly Lys                 245 250 255 Val His Ile Pro Phe Pro Leu Ala Asn Val Thr Cys Arg Val Val Lys             260 265 270 Ala Arg Asn Pro Thr Val Thr Tyr Gly Lys Asn Gln Val Ile Met Leu         275 280 285 Leu Tyr Pro Asp His Pro Thr Leu Leu Ser Tyr Arg Asn Met Gly Glu     290 295 300 Glu Pro Asn Tyr Gln Glu Glu Trp Val Thr His Lys Lys Glu Ile Arg 305 310 315 320 Leu Thr Val Pro Thr Glu Gly Leu Glu Val Thr Trp Gly Asn Asn Glu                 325 330 335 Pro Tyr Lys Tyr Trp Pro Gln Leu Ser Thr Asn Gly Thr Ala His Gly             340 345 350 His Pro His Glu Ile Ile Leu Tyr Tyr Tyr Glu Leu Tyr Pro Thr Met         355 360 365 Thr Val Val Val Ser Ser Val Ala Ser Phe Ile Leu Ser Ser Val Met     370 375 380 Gly Val Ala Val Gly Met Cys Met Cys Ala Arg Arg Arg Cys Ile Thr 385 390 395 400 Pro Tyr Glu Leu Thr Pro Gly Ala Thr Val Pro Phe Leu Leu Ser Leu                 405 410 415 Ile Cys Cys Ile Arg Thr Ala Lys Ala Val Asp             420 425 <210> 3 <211> 273 <212> PRT <213> Bacillus cereus <400> 3 Met Arg Ile Asn Thr Asn Ile Asn Ser Met Arg Thr Gln Glu Tyr Met   1 5 10 15 Arg Gln Asn Gln Asp Lys Met Asn Thr Ala Met Asn Arg Leu Ser Ser              20 25 30 Gly Lys Ser Ile Asn Ser Ala Ala Asp Asp Ala Ala Gly Leu Ala Ile          35 40 45 Ala Thr Arg Met Arg Ala Lys Glu Gly Gly Leu Asn Val Gly Ala Arg      50 55 60 Asn Thr Gln Asp Ala Met Ser Ala Leu Arg Thr Gly Asp Ala Ala Leu  65 70 75 80 Gly Ser Ile Ser Asn Ile Leu Leu Arg Met Met Asp Leu Ala Thr Gln                  85 90 95 Ala Ala Asn Gly Thr Asn Asn Ala Glu Asp Thr Ala Ser Leu Asp Lys             100 105 110 Glu Tyr Val Ala Leu Lys Asp Glu Ile Asp His Ile Ala Gly Lys Thr         115 120 125 Asn Phe Asn Gly Asn Ser Phe Leu Asp Thr Thr Ala Thr Pro Pro Gly     130 135 140 Lys Asp Ile Glu Ile Gln Leu Ser Asp Ala Ser Gly Asp Thr Met Thr 145 150 155 160 Leu Lys Ala Ile Asp Thr Lys Ser Leu Thr Thr Gly Thr Leu Thr Asn                 165 170 175 Leu Lys Asp Arg Ala Thr Ala Glu Thr Glu Ile Thr Lys Leu Asp Thr             180 185 190 Ala Ile Gln Lys Ile Ala Asp Glu Arg Ala Thr Phe Gly Ser Gln Leu         195 200 205 Asn Arg Leu Asp His Asn Leu Asn Asn Val Thr Ser Gln Ala Thr Asn     210 215 220 Met Ala Ala Ala Ala Ser Gln Ile Glu Asp Ala Asp Met Ala Lys Glu 225 230 235 240 Met Ser Glu Met Thr Lys Phe Lys Ile Leu Asn Glu Ala Gly Ile Ser                 245 250 255 Met Leu Ser Gln Ala Asn Gln Thr Pro Gln Met Val Ser Lys Leu Leu             260 265 270 Gln     <210> 4 <211> 657 <212> PRT <213> Artificial Sequence <220> <223> fusion protein <400> 4 Met Arg Gly Ser His His His His His Gly Met Ala Ser Met Thr   1 5 10 15 Gly Gly Gln Gln Met Gly Arg Asp Leu Tyr Asp Leu Val Pro Arg Gly              20 25 30 Ser Ala Lys Asp Pro Met Arg Ile Asn Thr Asn Ile Asn Ser Met Arg          35 40 45 Thr Gln Glu Tyr Met Arg Gln Asn Gln Asp Lys Met Asn Thr Ala Met      50 55 60 Asn Arg Leu Ser Ser Gly Lys Ser Ile Asn Ser Ala Ala Asp Asp Ala  65 70 75 80 Ala Gly Leu Ala Ile Ala Thr Arg Met Arg Ala Lys Glu Gly Gly Leu                  85 90 95 Asn Val Gly Ala Arg Asn Thr Gln Asp Ala Met Ser Ala Leu Arg Thr             100 105 110 Gly Asp Ala Ala Leu Gly Ser Ile Ser Asn Ile Leu Leu Arg Met Arg         115 120 125 Asp Leu Ala Thr Gln Ala Ala Asn Gly Thr Asn Asn Ala Glu Asp Thr     130 135 140 Ala Ser Leu Asp Lys Glu Tyr Val Ala Leu Lys Asp Glu Ile Asp His 145 150 155 160 Ile Ala Gly Lys Thr Asn Phe Asn Gly Asn Ser Phe Leu Asp Thr Thr                 165 170 175 Ala Thr Pro Pro Gly Lys Asp Ile Glu Ile Gln Leu Ser Asp Ala Ser             180 185 190 Gly Asp Thr Met Thr Leu Lys Ala Ile Asp Thr Lys Ser Leu Thr Thr         195 200 205 Gly Thr Leu Thr Asn Leu Lys Asp Arg Ala Thr Ala Glu Thr Glu Ile     210 215 220 Thr Lys Leu Asp Thr Ala Ile Gln Lys Ile Ala Asp Glu Arg Ala Thr 225 230 235 240 Phe Gly Ser Gln Leu Asn Arg Leu Asp His Asn Leu Asn Asn Val Thr                 245 250 255 Ser Gln Ala Thr Asn Ala Ala Ala Ala Ser Gln Ile Glu Asp Ala             260 265 270 Asp Met Ala Lys Glu Met Ser Glu Met Thr Lys Phe Lys Ile Leu Asn         275 280 285 Glu Ala Gly Ile Ser Met Leu Ser Gln Ala Asn Gln Thr Pro Gln Met     290 295 300 Val Ser Lys Leu Leu Gln Ala Ala Ala Pro Gly Ser Ser Thr Lys Asp 305 310 315 320 Asn Phe Asn Val Tyr Lys Ala Thr Arg Pro Tyr Leu Ala His Cys Pro                 325 330 335 Asp Cys Gly Glu Gly His Ser Cys His Ser Pro Val Ala Leu Glu Arg             340 345 350 Ile Arg Asn Glu Ala Thr Asp Gly Thr Leu Lys Ile Gln Val Ser Leu         355 360 365 Gln Ile Gly Ile Lys Thr Asp Asp Ser His Asp Trp Thr Lys Leu Arg     370 375 380 Tyr Met Asp Asn His Met Pro Ala Asp Ala Glu Arg Ala Gly Leu Phe 385 390 395 400 Val Arg Thr Ser Ala Pro Cys Thr Ile Thr Gly Thr Met Gly His Phe                 405 410 415 Ile Leu Ala Arg Cys Pro Lys Gly Glu Thr Leu Thr Val Gly Phe Thr             420 425 430 Asp Gly Arg Lys Ile Ser His Ser Cys Thr His Pro Phe His His Asp         435 440 445 Pro Pro Val Ile Gly Arg Glu Lys Phe His Ser Arg Pro Gln His Gly     450 455 460 Arg Glu Leu Pro Cys Ser Thr Tyr Ala Gln Ser Thr Ala Ala Thr Ala 465 470 475 480 Glu Glu Ile Glu Val His Met Pro Pro Asp Thr Pro Asp Arg Thr Leu                 485 490 495 Met Ser Gln Gln Ser Gly Asn Val Lys Ile Thr Val Asn Ser Gln Thr             500 505 510 Val Arg Tyr Lys Cys Asn Cys Gly Asp Ser Ser Glu Gly Leu Thr Thr         515 520 525 Thr Asp Lys Val Ile Asn Asn Cys Lys Val Asp Gln Cys His Ala Ala     530 535 540 Val Thr Asn His Lys Lys Trp Gln Tyr Asn Ser Pro Leu Val Pro Arg 545 550 555 560 Asn Ala Glu Ser Gly Asp Arg Lys Gly Lys Val His Ile Pro Phe Pro                 565 570 575 Leu Ala Asn Val Thr Cys Arg Val Val Lys Ala Arg Asn Pro Thr Val             580 585 590 Thr Tyr Gly Lys Asn Gln Val Ile Met Leu Leu Tyr Pro Asp His Pro         595 600 605 Thr Leu Leu Ser Tyr Arg Asn Met Gly Glu Glu Pro Asn Tyr Gln Glu     610 615 620 Glu Trp Val Thr His Lys Lys Glu Ile Arg Leu Thr Val Pro Thr Glu 625 630 635 640 Gly Leu Glu Val Thr Trp Gly Asn Gly Pro Tyr Lys Tyr Trp Pro                 645 650 655 Gln    

Claims (17)

A fusion protein comprising the chikungunya virus coat protein and the Toll-like receptor 5 stimulation protein of SEQ ID NO: 1. The method according to claim 1,
Wherein said toll-like receptor 5 stimulating protein is flagellin. 3. The method of claim 2,
Wherein the plasgelin is a plasmin of Salmonella genus or Bacillus bacteria. The method of claim 3,
Wherein the plasgelin is salmonella dublin or a plasgelin of Bacillus cereus . 3. The method of claim 2,
Wherein the plasgelin comprises at least one domain selected from the group consisting of a plasmid D0 domain and a D1 domain. 3. The method of claim 2,
Wherein the plasgelin is represented by SEQ ID NO: 3. The method according to claim 1,
And a histidine-tag is bound to one side of the fusion protein. 8. A polynucleotide encoding the fusion protein of any one of claims 1 to 7. 9. A recombinant expression vector comprising the polynucleotide of claim 8. 10. The method of claim 9,
Wherein the recombinant expression vector is pET49b. 10. The method of claim 9,
Wherein the recombinant expression vector comprises a gene fragment represented by the nucleotide sequence of SEQ ID NO: 4. A vaccine for Chichungniyya virus comprising the fusion protein of any one of claims 1 to 7 as an active ingredient. 13. The method of claim 12,
Wherein the vaccine is administered intravenously by intravenous injection, intramuscular injection, intramuscular injection, subcutaneous injection, intraconjunctival injection, transdermal delivery or airway inhalation. 8. A kit for the diagnosis of chichengyiya virus comprising the fusion protein of any one of claims 1 to 7 as an active ingredient. (a) measuring the level of antigen-antibody complex formation with the fusion protein of any one of claims 1 to 7 in a sample separated from the desired individual; And
(b) comparing the level of antigen-antibody complex formation measured in step (a) with the level of antigen-antibody complex formation with the fusion protein of any one of claims 1 to 7 in a normal control sample , A method for providing information for diagnosis of Chikunguniya virus infection. 16. The method of claim 15,
Wherein said sample comprises at least one of IgG and IgM in serum. 16. The method of claim 15,
Wherein the level of antigen-antibody complex formation measured in step (a) is higher than the level of antigen-antibody complex formation of the normal control sample measured in step (b), a chikungunya virus infection A method of providing information for diagnosis.

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