KR102006869B1 - N-terminus domain fragments, c-terminus domain fragments and nc fusion protein of ebola virus nucleoprotein, kit for diagnosing ebola virus infection using thereof - Google Patents

Abstract

The present invention relates to N-terminal domain fragments, C-terminal domain fragments and NC fusion proteins of the nucleoprotein of Ebola virus, and a kit for diagnosing Ebola virus infection using the same. The N-terminal domain fragment of the Ebola virus nucleoprotein, the C-terminal domain fragment of the Ebola virus nucleoprotein and the NC fusion protein in which the N-terminal domain fragment and the C-terminal domain fragment of the Ebola virus nucleoprotein are combined, according to the present invention, can be mass-produced in Escherichia coli and effectively produce antibodies from various experimental animals. Accordingly, the N-terminal domain fragment of the Ebola virus nucleoprotein, the C-terminal domain fragment of the Ebola virus nucleoprotein, and the NC fusion protein in which the N-terminal domain fragment and the C-terminal domain fragment of the Ebola virus nucleoprotein are combined can be used as an antigen for diagnosing Ebola virus.

Description

N-terminal domain fragments, C-terminal domain fragments, and NC fusion proteins of the nuclear protein of Ebola virus, and kits for diagnosing Ebola virus infection using the same, C-TERMINUS DOMAIN FRAGMENTS AND NC FUSION PROTEIN OF EBOLA VIRUS NUCLEOPROTEIN , KIT FOR DIAGNOSING EBOLA VIRUS INFECTION USING THEREOF}

The present invention relates to N-terminal domain fragment, C-terminal domain fragment and NC fusion protein of the nuclear protein of Ebola virus, and kit for diagnosing Ebola virus infection using the same.

Ebola is a highly fatal viral hemorrhagic fever disease caused by the infection of Ebola virus. Ebola virus is a member of the Philovirus family that causes fatal hemorrhagic fever, and is a virus having various tubular shapes of about 80 nm in diameter and 700-1,400 nm in length.

There are five subtypes of Ebola virus and there are Zaire, Sudan, Reston, Ivory Coast and Bundibu, depending on the name of the discovery site. Of these, the Zyr is the first subtype and the most frequent subtype. Ebola virus generally refers to the Zyr. In particular, it was confirmed that the West African epidemic in 2014 was caused by a variant of the Zaire type. The Zaire type has the highest mortality rate of 83% among all subtypes since Ebola's first occurrence in 1976.

To date, there are no treatments available to treat the Ebola virus, so only conservative treatments for shock, blood loss and bleeding have been made. For the 2014 West African epidemic Ebola virus infection disease, humanized monoclonal antibodies such as ZMapp were used experimentally. However, the effectiveness and safety of humanized monoclonal antibodies have not been fully demonstrated. At present, Zimab, a representative therapeutic agent known to be effective against Ebola virus, is a combination of three monoclonal antibodies, which is a kind of serum cocktail, which neutralizes the virus by administering antibodies to Ebola virus to the body on a similar principle to serum. (Richard T, et al., 2016). In monkey experiments, Zimab was administered up to 5 days of infection, and primary and booster doses were reported to produce lasting defenses. However, due to the inherent characteristics of Ebola virus, including immune response suppression function and high gene mutation rate, it can be different from monkey experiments using the virus produced by cell culture. Confirmation of effectiveness and stability is necessary.

In addition, TKM-ebola (TKM-ebola) is a therapeutic drug using siRNA that causes RNA interference, and is currently in clinical trials, and has the advantage of being able to mass produce only RNA combinations. However, the long-term effects of nucleic acid preparations have not been confirmed until now because the effects are unclear and genetic material is administered. In addition, various therapeutic agents in the experimental stage have problems, and there is no therapeutic agent for Ebola virus, which has been proven to be effective and stable.

Meanwhile, in the early days of the discovery of the Ebola virus, the development of the Ebola virus vaccine was not carried out because it occurred mainly in Africa, where the frequency of hemorrhagic fever caused by the Ebola virus was not frequent and the industrial investment for the vaccine development was low. Recently, however, it has been raised that hemorrhagic infectious diseases can be used as a means of biological terror, and there is a growing concern that the development of transportation means that the outbreak can occur worldwide. As a result, many countries around the world are supporting the development of faster and more accurate diagnostic methods, antivirals for treatment, and vaccines for prevention of Ebola virus.

 Richard T, et al., A Randomized, Controlled Trial of ZMapp for Ebola Virus Infection. N Engl J Med 2016; 375: 1448-56. DOI: 10.1056 / NEJMoa1604330

It is an object of the present invention to provide a vaccine composition for the prevention or treatment of Ebola virus infection.

In order to achieve the above object, the present invention provides an N-terminal domain fragment of the nuclear protein of Ebola virus.

The present invention also provides a C-terminal domain fragment of the nucleoprotein of Ebola virus.

The present invention also provides an NC fusion protein in which an N-terminal domain fragment and a C-terminal domain fragment of an Ebola virus nucleoprotein are bound.

The present invention also provides a vaccine composition for preventing Ebola virus infection and a kit for diagnosing Ebola virus infection, including an N-terminal domain fragment, C-terminal domain fragment, or NC fusion protein of Ebola virus nucleoprotein.

The present invention also provides a monoclonal antibody that specifically binds to any one selected from the group consisting of N-terminal domain fragments, C-terminal domain fragments and NC fusion proteins of the nuclear protein of Ebola virus.

In addition, the present invention (a) the N-terminal domain fragment of the Ebola virus nucleoprotein, the C-terminal domain fragment of the Ebola virus nucleoprotein and the N-terminal domain fragment and the C-terminal domain fragment of the Ebola virus nucleoprotein are combined Injecting the animal with any one selected from the group consisting of fusion proteins; And (b) provides a method for producing an Ebola virus antibody comprising recovering the antibody from the animal.

The present invention also provides a pharmaceutical composition for the prevention or treatment of diseases caused by Ebola virus infection comprising the antibody as an active ingredient.

The N-terminal domain fragment of the Ebola virus nucleoprotein, the C-terminal domain fragment of the Ebola virus nucleoprotein, and the NC fusion protein in which the N-terminal domain fragment and the C-terminal domain fragment of the Ebola virus nucleoprotein are combined are E. coli. It is possible to mass-produce and to effectively produce antibodies from various experimental animals. Therefore, the N-terminal domain fragment of the Ebola virus nucleoprotein, the C-terminal domain fragment of the Ebola virus nucleoprotein, and the NC fusion protein to which the N-terminal domain fragment and the C-terminal domain fragment of the Ebola virus nucleoprotein are bound It can be used as an antigen for diagnosing Ebola virus.

1 is a result of analyzing the amino acid sequence of Ebola virus nucleoprotein using ESPript 3.0.
Figure 2 shows E. coli expression vector pET28a-Eb_NP_NC which subcloned the nucleotides encoding the N-terminal and C-terminal domain of the Ebola virus nucleoprotein into the pET28a vector.
3a and 3b confirm the obtained N-terminal domain using SDS-PAGE.
4a and 4b confirm the obtained C-terminal domain using SDS-PAGE.
5a and 5b confirm the obtained NC fusion protein using SDS-PAGE.
6 shows the obtained N-terminal domain, C-terminal domain and NC fusion protein using SDS-PAGE.
Figure 7 confirms through ELISA TEST that mice can effectively produce antibodies of N-terminal domain (N), C-terminal domain (C) and NC fusion protein (NC).

In one aspect, the present invention provides an N-terminal domain fragment of the nuclear protein of Ebola virus.

As used herein, the term "ebola virus" refers to a virus that causes acute febrile infection. Fever, muscle pain, nausea, and mouth pain, which are the initial symptoms of Ebola virus infection, are nonspecific symptoms that are difficult to distinguish from other infectious diseases such as typhoid fever, malaria, and Lhasa fever. Clinically evident bleeding symptoms are known to occur in only about one third of all patients. Diagnosis of the Ebola virus can be divided into clinical symptom and laboratory diagnosis. Diagnosis through clinical symptoms cannot be regarded as a specific symptom of Ebola virus infection because eye symptoms such as ocular hyperemia, pain, and skin rashes should be relied on by laboratory diagnostic methods for patients suspected of being infected.

As a laboratory diagnostic method, an antigen detection ELISA, IgG ELISA, nucleic acid polymerase reaction, and virus culture are performed directly and detected in the early stage of infection. In the case of recovery patients, ELISA, immunohistochemistry and PCR for Ebola virus in patient serum have been used. Ebola virus is classified as a biosafety level 4 pathogen because it is highly pathogenic to humans. Therefore, sampling and inspection procedures must be performed in accordance with safety rules established by the BL-4 laboratory.

As used herein, the term “nucleoprotein” is a structural protein that surrounds a viral gene. This is one of the major determinants of species specificity.

The Ebola virus nucleoprotein consists of 739 amino acids represented by SEQ ID NO: 1. In this case, the nuclear protein may be encoded by the nucleotide sequence of SEQ ID NO: 2.

Specifically, the N-terminal domain of the Ebola virus nucleoprotein may be a polypeptide having an amino acid sequence of SEQ ID NO: 3. In this case, the protein may be encoded by the nucleotide sequence of SEQ ID NO: 4. In addition, the N-terminal domain is a polypeptide identical to SEQ ID NO: 3, as well as a polypeptide whose amino acid is substituted by conservative substitution, 80-99%, 85-99%, preferably 90-99% sequence thereof It may include all polypeptides having homology.

In addition, in the present invention, the fragment of the N-terminal domain is 1 to 321, 10 to 270, 20 to 220, 30 to 1 of the amino acid sequence of the amino acid at position 24 to position 344 of SEQ ID NO: 3 It may be 150, 40 to 100 or 50 to 80 contiguous amino acid sequences, preferably 50 to 321 contiguous amino acid sequences.

In addition, the N-terminal domain may be composed of an amino acid residue represented by the sequence of positions 31 to 351 of SEQ ID NO: 1 which is the amino acid sequence of the Ebola virus nucleoprotein.

As used herein, the term "conservative substitution" means that one class of amino acids is replaced with another amino acid of the same class. Conservative substitutions do not alter the structure, function or both of the polypeptides. Classes of amino acids for the purpose of conservative substitutions are hydrophobic (eg Met, Ala, Val, Leu), neutral hydrophilic (eg Cys, Ser, Thr), acidic (eg Asp, Glu) , Basic (eg Asn, Gln, His, Lys, Arg), conformational disruptors (eg Gly, Pro) and aromatics (eg Trp, Tyr, Phe) .

The present invention also provides a C-terminal domain fragment of the nucleoprotein of Ebola virus.

The C-terminal domain of the Ebola virus nucleoprotein may be a polypeptide having the amino acid sequence of SEQ ID NO: 5. In this case, the protein may be encoded by the nucleotide sequence of SEQ ID NO: 6. In addition, the C-terminal domain is a polypeptide identical to SEQ ID NO: 5 as well as a polypeptide whose amino acid is substituted by conservative substitution, 80-99%, 85-99%, preferably 90-99% sequence thereof It may include all polypeptides having homology.

In addition, the fragment of the C-terminal domain in the present invention is 1 to 109, 10 to 90, 20 to 70 or 30 of the amino acid sequence of position 27 to position 135 of SEQ ID NO: 5 And from 50 to 109 contiguous amino acid sequences, preferably from 50 to 109 contiguous amino acid sequences.

In addition, the C-terminal domain may be composed of amino acid residues represented by the sequence of positions 641 to 739 of SEQ ID NO: 1 which is the amino acid sequence of the Ebola virus nucleoprotein.

The present invention also provides an NC fusion protein in which an N-terminal domain fragment and a C-terminal domain fragment of an Ebola virus nucleoprotein are bound.

The N-terminal domain fragment and the C-terminal domain fragment are as described above. In addition, the N-terminal domain fragment and the C-terminal domain fragment may be in various combinations.

In one embodiment, the NC fusion protein in the present invention corresponds to the N-terminal domain and to segment 1 represented by the sequence of amino acid residues 31 to 351 of SEQ ID NO: 1, and to the C-terminal domain and amino acid residue 641 It may be composed of segment 2 represented by the sequence from position to 739 position. Segments 1 and 2 of such an NC fusion protein are those polypeptides having the same or conservative substitutions as previously described or a sequence homology thereof with 80-99%, 85-99%, preferably 90-99%. It can contain everything.

In one embodiment, the NC fusion protein of the Ebola virus nucleoprotein may be a polypeptide having the amino acid sequence of SEQ ID NO: 7. In this case, the NC fusion protein may be encoded by the nucleotide sequence of SEQ ID NO: 8. In addition, the NC fusion protein is a polypeptide identical to SEQ ID NO: 7 as well as a polypeptide whose amino acid is substituted by conservative substitution, 80 to 99%, 85 to 99%, preferably 90 to 99% of the sequence It may include all polypeptides having homology.

In another aspect, the present invention, the N-terminal domain fragment of the Ebola virus nucleoprotein, the C-terminal domain fragment of the Ebola virus nucleoprotein and the N-terminal domain fragment and the C-terminal domain fragment of the Ebola virus nucleoprotein are combined Provided is a vaccine composition for preventing Ebola virus infection comprising any one selected from the group consisting of NC fusion proteins.

The vaccine composition may be in any form known in the art, such as, but not limited to, solid forms suitable for liquid or injectable forms or suspensions. Such formulations may also be emulsified or encapsulated in liposomes or soluble glass, or prepared in the form of aerosols or sprays. They can also be included in transdermal patches. For liquids or injections, it may contain, if necessary, propylene glycol and sodium chloride in an amount sufficient to prevent hemolysis (e.g., about 1%).

The vaccine composition of the present invention may comprise a pharmaceutically acceptable carrier or diluent. Suitable carriers for the vaccine are known to those skilled in the art and may include, but are not limited to, proteins, sugars, and the like. Such carriers can be aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous carriers may be propylene glycol, polyethylene glycol, edible oils such as olive oil, and injectable organic esters such as ethyl oleate.

Aqueous carriers may comprise water, alcohol / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral carriers may include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactically treated Ringer's or fixed oils. Intravenous carriers may include electrolyte supplements such as those based on Ringer's dextrose, liquids and nutritional supplements, and the like. Preservatives and other additives such as antimicrobial agents, antioxidants, chelating agents, inert gases and the like may further be present. Preferred preservatives may include formalin, thimerosal, neomycin, polymyxin B and amphotericin B.

In addition, the vaccine composition may further comprise an adjuvant (adjuvant, immunoadjuvant). The adjuvant may refer to a compound or mixture that enhances the immune response and / or promotes the rate of absorption after inoculation and may include any absorption-promoting agent. Acceptable adjuvants include Freund's complete adjuvant, Freund's incomplete adjuvant, saponins, mineral gels such as aluminum hydroxide, surfactants such as lysocithin, fluoron polyols, polyanions, peptides, oils or hydrocarbon emulsions, keyhole rimpets It may include, but is not limited to, mocyanine, dinitrophenol, and the like.

The vaccine composition of the present invention can be administered via any one of the routes of administration selected from the group consisting of oral, transdermal, intramuscular, intraperitoneal, intravenous, subcutaneous or nasal, preferably administered by injection. Do.

The vaccine composition of the present invention is administered in a pharmaceutically effective amount. As used herein, the term “pharmaceutically effective amount” means an amount sufficient to exhibit a vaccine effect, and an amount that does not cause side effects or serious or excessive immune responses, and the level of an effective dose is a disorder to be treated. The severity of the disorder, the activity of the specific compound, the route of administration, the rate of removal of the protein, the duration of treatment, the drug used in combination with or concurrently with the protein, the age, weight, sex, diet, general health status and medical conditions of the individual It can depend on various factors, including factors.

The present invention relates to a group consisting of an N-terminal domain fragment of Ebola virus nucleoprotein, a C-terminal domain fragment of Ebola virus nucleoprotein, and an NC fusion protein in which an N-terminal domain fragment and C-terminal domain fragment of Ebola virus nucleoprotein are combined. It provides a kit for diagnosing Ebola virus infection comprising any one selected from.

The N-terminal domain fragment, C-terminal domain fragment and NC fusion protein are as described above.

In one embodiment, the N-terminal domain of the Ebola virus nucleoprotein having the amino acid sequence of SEQ ID NO: 3, the C-terminal domain of the Ebola virus nucleoprotein having the amino acid sequence of SEQ ID NO: 5 and the Ebola having the amino acid sequence of SEQ ID NO: 7 The NC fusion protein in which the N-terminal domain fragment and the C-terminal domain fragment of the nuclear protein of the virus are bound can specifically bind to an antibody against Ebola virus, and thus can be applied to an Ebola virus infection diagnosis kit.

The present invention also provides a method for providing Ebola virus infection information comprising adding a sample isolated from a diagnosis target to the Ebola virus infection diagnosis kit.

The term "Ebola virus infection diagnosis kit" is a kit that can quickly diagnose Ebola virus infection and the resulting disease. In one embodiment, the colorant reaction through the antigen-antibody reaction can be confirmed by immersing the strip coated with the antigen in the sample extracted from the patient.

As used herein, the term "disease associated with Ebola virus infection" results from the presence of an Ebola virus, eg, Ebola virus Zaire, Sudan, Cote d'Ivoire or Bundibu-type, in a cell or subject, or Refers to a condition resulting from invasion of cells or subjects by the Ebola virus. Specifically, the term refers to hemorrhagic fever caused by Ebola virus.

In another aspect, the present invention provides a monoclonal antibody that specifically binds to the N-terminal domain fragment of the Ebola virus nucleoprotein.

The term "antibody" is a protein produced by stimulation of an antigen in the immune system, which is a protein that specifically binds to a specific antigen and floats lymph and blood to generate an antigen-antibody reaction. Antigen-antibody responses have high specificity for each antigen. This is because when antibodies are made in B cells of lymphocytes, antibodies produced by specific antigens do not, in principle, react with other antigens. Such high specificity is used for the examination of immunity, allergy, various diseases and types and types of infections.

As used herein, the term "antigen" is a component of the virus that can cause immune function. At this time, the antigen is an N-terminal domain fragment of the Ebola virus nucleoprotein.

In one embodiment of the present invention, when the N-terminal domain of the Ebola virus nucleoprotein of SEQ ID NO: 3 is administered to a subject, it functions as an antigen that induces the formation of immune antibodies. The antibody specifically binds to an N-terminal domain fragment.

The present invention also provides monoclonal antibodies that specifically bind to the C-terminal domain fragment of the Ebola virus nucleoprotein.

In one embodiment of the invention, when the C-terminal domain of the Ebola virus nucleoprotein of SEQ ID NO: 5 is administered to a subject, it acted as an antigen that induces the formation of immune antibodies. At this time, the antigen is a C-terminal domain fragment of the Ebola virus nucleoprotein. In addition, the antibody specifically binds to a C-terminal domain fragment.

The present invention also provides a monoclonal antibody that specifically binds to an NC fusion protein to which an N-terminal domain fragment and a C-terminal domain fragment of an Ebola virus nucleoprotein are bound.

In one embodiment of the present invention, when the NC fusion protein of the Ebola virus nucleoprotein of SEQ ID NO: 7 is administered to an individual, it functions as an antigen that induces the formation of immune antibodies. In this case, the antigen is an NC fusion protein in which an N-terminal domain fragment and a C-terminal domain fragment of an Ebola virus nucleoprotein are bound. In addition, the antibody specifically binds to an NC fusion protein.

According to another aspect of the present invention, (a) the N-terminal domain fragment of Ebola virus nucleoprotein, the C-terminal domain fragment of Ebola virus nucleoprotein and the N-terminal domain fragment and C-terminal domain fragment of Ebola virus nucleoprotein are Injecting the animal with any one selected from the group consisting of bound NC fusion proteins; And (b) recovering the antibody from the animal, the N-terminal domain fragment of Ebola virus nucleoprotein, the C-terminal domain fragment of Ebola virus nucleoprotein, and the N-terminal domain fragment of Ebola virus nucleoprotein and C Provided is a method for producing a monoclonal antibody that specifically binds to any one selected from the group consisting of NC fusion proteins to which terminal domain fragments are bound.

Monoclonal antibodies that specifically bind to N-terminal domain fragments, C-terminal domain fragments or NC fusion proteins of the Ebola virus nucleoprotein of the present invention can be obtained by the above-described antibody production method.

Injection of the N-terminal domain fragment of the Ebola virus nucleoprotein, the C-terminal domain fragment of the Ebola virus nucleoprotein or the N-terminal domain fragment and the C-terminal domain fragment of the Ebola viral nucleoprotein is injected into the skin, It can be done by injection subcutaneously or into muscle tissue.

In one embodiment of the present invention, the N-terminal domain fragment of the Ebola virus nucleoprotein, the C-terminal domain fragment of the Ebola virus nucleoprotein or the N-terminal domain fragment and the C-terminal domain fragment of the Ebola virus nucleoprotein are combined Fusion proteins acted as antigens that effectively induce immune antibody formation when administered to a subject. In this way, the N-terminal domain fragment of the Ebola virus nucleoprotein, the C-terminal domain fragment of the Ebola virus nucleoprotein, or the N-terminal domain fragment and the C-terminal domain fragment of the Ebola virus nucleoprotein are combined with the Ebola virus. It was confirmed that it can be used as an antigen for diagnosing.

The present invention also relates to an N-terminal domain fragment of the Ebola virus nucleoprotein, a C-terminal domain fragment of the Ebola virus nucleoprotein and an NC fusion protein in which the N-terminal domain fragment and the C-terminal domain fragment of the Ebola virus nucleoprotein are combined. It provides a pharmaceutical composition for the prevention or treatment of diseases caused by Ebola virus infection comprising as an active ingredient a monoclonal antibody that specifically binds to any one selected from the group consisting of.

The N-terminal domain fragment of the Ebola virus nucleoprotein, the C-terminal domain fragment of the Ebola virus nucleoprotein, and the N-terminal domain fragment and the C-terminal domain fragment of the Ebola virus nucleoprotein are specifically bound to the NC fusion protein. The preparation of a pharmaceutical composition comprising the antibody may further comprise a suitable carrier, excipient and diluent commonly used.

The pharmaceutical composition may be used in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral formulations, suppositories, and sterile injectable solutions according to conventional methods. The carriers, excipients and diluents that may be contained in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate , Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations may contain at least one excipient such as starch, calcium carbonate, sucrose, or the like. ) Or lactose, gelatin and the like are mixed.

In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. As a suppository base, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerol gelatin and the like can be used.

The preferred dosage of the pharmaceutical composition of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the type of drug, the route of administration and the period of time, but can be appropriately selected by those skilled in the art. However, for the desired effect, the pharmaceutical composition of the present invention is preferably administered at 0.0001 mg / kg to 100 mg / kg, preferably at 0.001 mg / kg to 10 mg / kg. The administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way.

The pharmaceutical composition of the present invention can be administered to mammals such as rats, mice, livestock, humans, and the like in various routes. All modes of administration can be expected, for example by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.

The present invention also provides an expression vector comprising a nucleotide encoding the N-terminal domain of the Ebola virus nucleoprotein, and provides a host cell transfected with the expression vector. The present invention also provides a method for producing an N-terminal domain of Ebola virus nucleoprotein, comprising obtaining an N-terminal domain of Ebola virus nucleoprotein from the transfected host cell.

The present invention also provides an expression vector comprising a nucleotide encoding the C-terminal domain of the Ebola virus nucleoprotein, and provides a host cell transfected with the expression vector. The present invention also provides a method for preparing the C-terminal domain of the Ebola virus nucleoprotein, comprising obtaining the C-terminal domain of the Ebola virus nucleoprotein from the transfected host cell.

The present invention also provides an expression vector comprising an nucleotide encoding an NC fusion protein in which an N-terminal domain fragment and a C-terminal domain fragment of an Ebola virus nucleoprotein are bound, and provides a host cell transfected with the expression vector. do. The present invention also provides an N-terminal domain fragment of the Ebola virus nucleoprotein comprising the step of obtaining an NC fusion protein in which the N-terminal domain fragment and the C-terminal domain fragment of the Ebola virus nucleoprotein are bound from the transfected host cell. Provided is a method for producing an NC fusion protein in which a C-terminal domain fragment is bound.

In one embodiment of the present invention, the nucleotides encoding the N-terminal domain and the C-terminal domain of the Ebola virus nucleoprotein are subcloned into the pET28a vector, inoculated into E. coli, and cultured to the N-terminal domain of the Ebola virus nucleoprotein. An NC fusion protein obtained by binding to the C-terminal domain was obtained.

Hereinafter, the present invention will be described in detail with reference to Examples and Experimental Examples. However, the following examples and experimental examples are illustrative of the present invention, and the content of the present invention is not limited by the following examples and experimental examples.

Example  1. Preparation of N-terminal domain, C-terminal domain and NC fusion protein of Ebolavirus nucleoprotein

To identify sequences of Ebola virus nucleoproteins (NPs) that can be used as diagnostic antigens, amino acids of NPs of various Ebola virus subtypes using structure-based protein sequencing comparison programs (Clustal W & ESPript 3.0) Five of the sequences were compared and analyzed. In addition, the N-terminal domain of NP with conserved sequences between subtypes and the C-terminal domain of NP, the major antigenic determinant, were determined. The sequence analysis results of the N-terminal domain and C-terminal domain of the structure-based Ebola virus NP are shown in FIG. 1.

As shown in FIG. 1, the sequence analysis confirmed the N-terminal domain and the C-terminal domain common to the NPs of various Ebola virus subtypes, and recombinant proteins combining the respective domains were prepared with the following antigens. .

NP of the Ebola virus (Ebola virus / H.sapiens-tc / COD / 1976 / Yambuku-Mayinga, NCBI: J04337.1) represented by SEQ ID NO: 1 used in the following experiment was synthesized in Bioneer Co., Ltd. Same as SEQ ID NO: 2. N-terminal domains (residues 31-351) with amino acid sequences commonly conserved in Ebolavirus NP were subcloneed to the NheI and BamHI sites of the pET28a vector, and Escherichia coli ) overexpressed with BL21 (DE3) pLysS Rosetta strain. In addition, the C-terminal domain (residues 641-739) having the amino acid sequence of the major epitope was subcloned into the EcoRI and XhoI sites of the pET28a vector and overexpressed using the E. coli BL21 (DE3) pLysS Rosetta strain.

To prepare an NC fusion protein (hereinafter referred to as NC) combining the N-terminal and C-terminal domains, fragment 1 is amino acid residues 31-351 corresponding to the N-terminal domain, and fragment 2 is amino acid corresponding to the C-terminal domain. Residues 641-739 were subcloned to attach six histidines to the N-terminus of the pET28a vector protein using NheI and BamHI and EcoRI and XhoI, respectively, and overexpressed using E. coli BL21 (DE3) pLysS Rosetta strain. Each protein was inoculated with Escherichia coli, inoculated with 0.2 mM IPTG between 0.6 and 0.8 at OD 600 nm and incubated at 291K for 16 hours. The E. coli expression vector pET28a-Eb_NP_NC used is shown in FIG. 2.

His-tagged N-terminal domain, C-terminal domain and NC fusion protein were first purified by nickel-affinity chromatography and by HiLoad 16/600 Superdex 200 prep grade gel-filtration chromatography using 2X PBS buffer. Final purification gave purified N-terminal domain, C-terminal domain and NC fusion protein. The results are shown in Figures 3a to 5b.

As shown in Figure 3a to 5b, N-terminal domain, C-terminal domain and NC fusion protein was confirmed on the SDS-PAGE, it was confirmed that purified by chromatographic purity of 95% or more. The base sequence of the NC fusion protein is shown in SEQ ID NO: 8, and the 447 amino acid sequence prepared thereby is shown in SEQ ID NO: 7. Hereinafter, using the prepared N-terminal domain, C-terminal domain, and NC fusion protein as antigens, it was confirmed whether they were useful as an Ebola virus vaccine.

Experimental Example  1. Confirmation of antibody production of N-terminal domain, C-terminal domain and NC fusion protein of Ebola virus nucleoprotein

Example 1 In order to confirm whether the prepared N-terminal domain, C-terminal domain and NC fusion protein antigen effectively induce antibody production, the effect of antibody production was confirmed in mice.

First, pre-immune serum was collected and used as a negative control before injection of the antigens of each of the purified N-terminal, C-terminal and NC fusion proteins into mice. For primary immunization, 200 ug of each purified antigen was mixed with Imject Alum adjuvant (Thermo) and injected into muscle (IM), and after 2 weeks, boosted by mixing with Imject Alum adjuvant (Thermo). .

After 2 weeks, primary blood was collected and the ELISA test was performed. After 2 weeks of boosting, the mice were collected from blood and the final serum was separated to perform the final ELISA test. In the ELISA test, antigen was used 100 ng per well, immune serum was diluted 1: 100 to 1: 1,000,000 ratio with 1XPBS, and anti-mouse IgG-HRP (Sigma) 1: for secondary confirmation. Diluted to 5,000 ratio and used. Color development was detected at an optical density of 450 nm to finally determine whether immune antibodies were generated, which is shown in FIG. 7.

As shown in FIG. 7, the N-terminal, C-terminal, and NC fusion proteins exhibited immune antibody formation effects in all mice used in the experiment, and the NC-fusion protein combining the N-terminus and C-terminus was a single N-. It showed much higher immunity than the terminal and C-terminal domains and had the effect of producing better antibodies. Through this, it was confirmed that the N-terminal domain, C-terminal domain and NC fusion protein can all effectively induce antibodies in the individual.

Therefore, since the N-terminal domain, C-terminal domain and NC fusion protein of the present invention can effectively induce antibodies in an individual by acting as an antigen, it can be expected to be used as a diagnostic antigen of Ebola virus.

<110> Korea Research Institute of Bioscience and Biotechnology <120> N-TERMINUS DOMAIN FRAGMENTS, C-TERMINUS DOMAIN FRAGMENTS AND NC          FUSION PROTEIN OF EBOLA VIRUS NUCLEOPROTEIN, KIT FOR DIAGNOSING          EBOLA VIRUS INFECTION USING THEREOF <130> FPD / 201711-0085 <160> 8 <170> KoPatentIn 3.0 <210> 1 <211> 739 <212> PRT <213> Artificial Sequence <220> <223> Ebola virus NP <400> 1 Met Asp Ser Arg Pro Gln Lys Ile Trp Met Ala Pro Ser Leu Thr Glu   1 5 10 15 Ser Asp Met Asp Tyr His Lys Ile Leu Thr Ala Gly Leu Ser Val Gln              20 25 30 Gln Gly Ile Val Arg Gln Arg Val Ile Pro Val Tyr Gln Val Asn Asn          35 40 45 Leu Glu Glu Ile Cys Gln Leu Ile Ile Gln Ala Phe Glu Ala Gly Val      50 55 60 Asp Phe Gln Glu Ser Ala Asp Ser Phe Leu Leu Met Leu Cys Leu His  65 70 75 80 His Ala Tyr Gln Gly Asp Tyr Lys Leu Phe Leu Glu Ser Gly Ala Val                  85 90 95 Lys Tyr Leu Glu Gly His Gly Phe Arg Phe Glu Val Lys Lys Arg Asp             100 105 110 Gly Val Lys Arg Leu Glu Glu Leu Leu Pro Ala Val Ser Ser Gly Lys         115 120 125 Asn Ile Lys Arg Thr Leu Ala Ala Met Pro Glu Glu Glu Thr Thr Glu     130 135 140 Ala Asn Ala Gly Gln Phe Leu Ser Phe Ala Ser Leu Phe Leu Pro Lys 145 150 155 160 Leu Val Val Gly Glu Lys Ala Cys Leu Glu Lys Val Gln Arg Gln Ile                 165 170 175 Gln Val His Ala Glu Gln Gly Leu Ile Gln Tyr Pro Thr Ala Trp Gln             180 185 190 Ser Val Gly His Met Met Val Ile Phe Arg Leu Met Arg Thr Asn Phe         195 200 205 Leu Ile Lys Phe Leu Leu Ile His Gln Gly Met His Met Val Ala Gly     210 215 220 His Asp Ala Asn Asp Ala Val Ile Ser Asn Ser Val Ala Gln Ala Arg 225 230 235 240 Phe Ser Gly Leu Leu Ile Val Lys Thr Val Leu Asp His Ile Leu Gln                 245 250 255 Lys Thr Glu Arg Gly Val Arg Leu His Pro Leu Ala Arg Thr Ala Lys             260 265 270 Val Lys Asn Glu Val Asn Ser Phe Lys Ala Ala Leu Ser Ser Leu Ala         275 280 285 Lys His Gly Glu Tyr Ala Pro Phe Ala Arg Leu Leu Asn Leu Ser Gly     290 295 300 Val Asn Asn Leu Glu His Gly Leu Phe Pro Gln Leu Ser Ala Ile Ala 305 310 315 320 Leu Gly Val Ala Thr Ala His Gly Ser Thr Leu Ala Gly Val Asn Val                 325 330 335 Gly Glu Gln Tyr Gln Gln Leu Arg Glu Ala Ala Thr Glu Ala Glu Lys             340 345 350 Gln Leu Gln Gln Tyr Ala Glu Ser Arg Glu Leu Asp His Leu Gly Leu         355 360 365 Asp Asp Gln Glu Lys Lys Ile Leu Met Asn Phe His Gln Lys Lys Asn     370 375 380 Glu Ile Ser Phe Gln Gln Thr Asn Ala Met Val Thr Leu Arg Lys Glu 385 390 395 400 Arg Leu Ala Lys Leu Thr Glu Ala Ile Thr Ala Ala Ser Leu Pro Lys                 405 410 415 Thr Ser Gly His Tyr Asp Asp Asp Asp Asp Ile Pro Phe Pro Gly Pro             420 425 430 Ile Asn Asp Asp Asp Asn Pro Gly His Gln Asp Asp Asp Pro Thr Asp         435 440 445 Ser Gln Asp Thr Thr Ile Pro Asp Val Val Val Asp Pro Asp Asp Gly     450 455 460 Ser Tyr Gly Glu Tyr Gln Ser Tyr Ser Glu Asn Gly Met Asn Ala Pro 465 470 475 480 Asp Asp Leu Val Leu Phe Asp Leu Asp Glu Asp Asp Glu Asp Thr Lys                 485 490 495 Pro Val Pro Asn Arg Ser Thr Lys Gly Gly Gln Gln Lys Asn Ser Gln             500 505 510 Lys Gly Gln His Ile Glu Gly Arg Gln Thr Gln Ser Arg Pro Ile Gln         515 520 525 Asn Val Pro Gly Pro His Arg Thr Ile His His Ala Ser Ala Pro Leu     530 535 540 Thr Asp Asn Asp Arg Arg Asn Glu Pro Ser Gly Ser Thr Ser Pro Arg 545 550 555 560 Met Leu Thr Pro Ile Asn Glu Glu Ala Asp Pro Leu Asp Asp Ala Asp                 565 570 575 Asp Glu Thr Ser Ser Le Le Pro Pro Leu Glu Ser Asp Asp Glu Glu Gln             580 585 590 Asp Arg Asp Gly Thr Ser Asn Arg Thr Pro Thr Val Ala Pro Pro Ala         595 600 605 Pro Val Tyr Arg Asp His Ser Glu Lys Lys Glu Leu Pro Gln Asp Glu     610 615 620 Gln Gln Asp Gln Asp His Thr Gln Glu Ala Arg Asn Gln Asp Ser Asp 625 630 635 640 Asn Thr Gln Ser Glu His Ser Phe Glu Glu Met Tyr Arg His Ile Leu                 645 650 655 Arg Ser Gln Gly Pro Phe Asp Ala Val Leu Tyr Tyr His Met Met Lys             660 665 670 Asp Glu Pro Val Val Phe Ser Thr Ser Asp Gly Lys Glu Tyr Thr Tyr         675 680 685 Pro Asp Ser Leu Glu Glu Glu Tyr Pro Pro Trp Leu Thr Glu Lys Glu     690 695 700 Ala Met Asn Glu Glu Asn Arg Phe Val Thr Leu Asp Gly Gln Gln Phe 705 710 715 720 Tyr Trp Pro Val Met Asn His Lys Asn Lys Phe Met Ala Ile Leu Gln                 725 730 735 His His Gln             <210> 2 <211> 2220 <212> DNA <213> Artificial Sequence <220> <223> Ebola virus NP <400> 2 atggattctc gtcctcagaa aatctggatg gcgccgagtc tcactgaatc tgacatggat 60 taccacaaga tcttgacagc aggtctgtcc gttcaacagg ggattgttcg gcaaagagtc 120 atcccagtgt atcaagtaaa caatcttgaa gaaatttgcc aacttatcat acaggccttt 180 gaagcaggtg ttgattttca agagagtgcg gacagtttcc ttctcatgct ttgtcttcat 240 catgcgtacc agggagatta caaacttttc ttggaaagtg gcgcagtcaa gtatttggaa 300 gggcacgggt tccgttttga agtcaagaag cgtgatggag tgaagcgcct tgaggaattg 360 ctgccagcag tatctagtgg aaaaaacatt aagagaacac ttgctgccat gccggaagag 420 gagacaactg aagctaatgc cggtcagttt ctctcctttg caagtctatt ccttccgaaa 480 ttggtagtag gagaaaaggc ttgccttgag aaggttcaaa ggcaaattca agtacatgca 540 gagcaaggac tgatacaata tccaacagct tggcaatcag taggacacat gatggtgatt 600 ttccgtttga tgcgaacaaa ttttctgatc aaatttctcc taatacacca agggatgcac 660 atggttgccg ggcatgatgc caacgatgct gtgatttcaa attcagtggc tcaagctcgt 720 ttttcaggct tattgattgt caaaacagta cttgatcata tcctacaaaa gacagaacga 780 ggagttcgtc tccatcctct tgcaaggacc gccaaggtaa aaaatgaggt gaactccttt 840 aaggctgcac tcagctccct ggccaagcat ggagagtatg ctcctttcgc ccgacttttg 900 aacctttctg gagtaaataa tcttgagcat ggtcttttcc ctcaactatc ggcaattgca 960 ctcggagtcg ccacagcaca cgggagtacc ctcgcaggag taaatgttgg agaacagtat 1020 caacaactca gagaggctgc cactgaggct gagaagcaac tccaacaata tgcagagtct 1080 cgcgaacttg accatcttgg acttgatgat caggaaaaga aaattcttat gaacttccat 1140 cagaaaaaga acgaaatcag cttccagcaa acaaacgcta tggtaactct aagaaaagag 1200 cgcctggcca agctgacaga agctatcact gctgcgtcac tgcccaaaac aagtggacat 1260 tacgatgatg atgacgacat tccctttcca ggacccatca atgatgacga caatcctggc 1320 catcaagatg atgatccgac tgactcacag gatacgacca ttcccgatgt ggtggttgat 1380 cccgatgatg gaagctacgg cgaataccag agttactcgg aaaacggcat gaatgcacca 1440 gatgacttgg tcctattcga tctagacgag gacgacgagg acactaagcc agtgcctaat 1500 agatcgacca agggtggaca acagaagaac agtcaaaagg gccagcatat agagggcaga 1560 cagacacaat ccaggccaat tcaaaatgtc ccaggccctc acagaacaat ccaccacgcc 1620 agtgcgccac tcacggacaa tgacagaaga aatgaaccct ccggctcaac cagccctcgc 1680 atgctgacac caattaacga agaggcagac ccactggacg atgccgacga cgagacgtct 1740 agccttccgc ccttggagtc agatgatgaa gagcaggaca gggacggaac ttccaaccgc 1800 acacccactg tcgccccacc ggctcccgta tacagagatc actctgaaaa gaaagaactc 1860 ccgcaagacg agcaacaaga tcaggaccac actcaagagg ccaggaacca ggacagtgac 1920 aacacccagt cagaacactc ttttgaggag atgtatcgcc acattctaag atcacagggg 1980 ccatttgatg ctgttttgta ttatcatatg atgaaggatg agcctgtagt tttcagtacc 2040 agtgatggca aagagtacac gtatccagac tcccttgaag aggaatatcc accatggctc 2100 actgaaaaag aggctatgaa tgaagagaat agatttgtta cattggatgg tcaacaattt 2160 tattggccgg tgatgaatca caagaataaa ttcatggcaa tcctgcaaca tcatcagtga 2220                                                                         2220 <210> 3 <211> 370 <212> PRT <213> Artificial Sequence <220> <223> N-terminus domain of Ebola virus NP <400> 3 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro   1 5 10 15 Arg Gly Ser His Met Ala Ser Val Gln Gln Gly Ile Val Arg Gln Arg              20 25 30 Val Ile Pro Val Tyr Gln Val Asn Asn Leu Glu Glu Ile Cys Gln Leu          35 40 45 Ile Ile Gln Ala Phe Glu Ala Gly Val Asp Phe Gln Glu Ser Ala Asp      50 55 60 Ser Phe Leu Leu Met Leu Cys Leu His His Ala Tyr Gln Gly Asp Tyr  65 70 75 80 Lys Leu Phe Leu Glu Ser Gly Ala Val Lys Tyr Leu Glu Gly His Gly                  85 90 95 Phe Arg Phe Glu Val Lys Lys Arg Asp Gly Val Lys Arg Leu Glu Glu             100 105 110 Leu Leu Pro Ala Val Ser Ser Gly Lys Asn Ile Lys Arg Thr Leu Ala         115 120 125 Ala Met Pro Glu Glu Glu Thr Thr Glu Ala Asn Ala Gly Gln Phe Leu     130 135 140 Ser Phe Ala Ser Leu Phe Leu Pro Lys Leu Val Val Gly Glu Lys Ala 145 150 155 160 Cys Leu Glu Lys Val Gln Arg Gln Ile Gln Val His Ala Glu Gln Gly                 165 170 175 Leu Ile Gln Tyr Pro Thr Ala Trp Gln Ser Val Gly His Met Met Val             180 185 190 Ile Phe Arg Leu Met Arg Thr Asn Phe Leu Ile Lys Phe Leu Leu Ile         195 200 205 His Gln Gly Met His Met Val Ala Gly His Asp Ala Asn Asp Ala Val     210 215 220 Ile Ser Asn Ser Val Ala Gln Ala Arg Phe Ser Gly Leu Leu Ile Val 225 230 235 240 Lys Thr Val Leu Asp His Ile Leu Gln Lys Thr Glu Arg Gly Val Arg                 245 250 255 Leu His Pro Leu Ala Arg Thr Ala Lys Val Lys Asn Glu Val Asn Ser             260 265 270 Phe Lys Ala Ala Leu Ser Ser Leu Ala Lys His Gly Glu Tyr Ala Pro         275 280 285 Phe Ala Arg Leu Leu Asn Leu Ser Gly Val Asn Asn Leu Glu His Gly     290 295 300 Leu Phe Pro Gln Leu Ser Ala Ile Ala Leu Gly Val Ala Thr Ala His 305 310 315 320 Gly Ser Thr Leu Ala Gly Val Asn Val Gly Glu Gln Tyr Gln Gln Leu                 325 330 335 Arg Glu Ala Ala Thr Glu Ala Glu Gly Ser Glu Phe Glu Leu Arg Arg             340 345 350 Gln Ala Cys Gly Arg Thr Arg Ala Pro Pro Pro Pro Leu Arg Ser         355 360 365 Gly Cys     370 <210> 4 <211> 1113 <212> DNA <213> Artificial Sequence <220> <223> N-terminus domain of Ebola virus NP <400> 4 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60 atggctagcg ttcaacaggg gattgttcgg caaagagtca tcccagtgta tcaagtaaac 120 aatcttgaag aaatttgcca acttatcata caggcctttg aagcaggtgt tgattttcaa 180 gagagtgcgg acagtttcct tctcatgctt tgtcttcatc atgcgtacca gggagattac 240 aaacttttct tggaaagtgg cgcagtcaag tatttggaag ggcacgggtt ccgttttgaa 300 gtcaagaagc gtgatggagt gaagcgcctt gaggaattgc tgccagcagt atctagtgga 360 aaaaacatta agagaacact tgctgccatg ccggaagagg agacaactga agctaatgcc 420 ggtcagtttc tctcctttgc aagtctattc cttccgaaat tggtagtagg agaaaaggct 480 tgccttgaga aggttcaaag gcaaattcaa gtacatgcag agcaaggact gatacaatat 540 ccaacagctt ggcaatcagt aggacacatg atggtgattt tccgtttgat gcgaacaaat 600 tttctgatca aatttctcct aatacaccaa gggatgcaca tggttgccgg gcatgatgcc 660 aacgatgctg tgatttcaaa ttcagtggct caagctcgtt tttcaggctt attgattgtc 720 aaaacagtac ttgatcatat cctacaaaag acagaacgag gagttcgtct ccatcctctt 780 gcaaggaccg ccaaggtaaa aaatgaggtg aactccttta aggctgcact cagctccctg 840 gccaagcatg gagagtatgc tcctttcgcc cgacttttga acctttctgg agtaaataat 900 cttgagcatg gtcttttccc tcaactatcg gcaattgcac tcggagtcgc cacagcacac 960 gggagtaccc tcgcaggagt aaatgttgga gaacagtatc aacaactcag agaggctgcc 1020 actgaggctg agggatccga attcgagctc cgtcgacaag cttgcggccg cactcgagca 1080 ccaccaccac caccactgag atccggctgc taa 1113 <210> 5 <211> 135 <212> PRT <213> Artificial Sequence <220> <223> C-terminus domain of Ebola virus NP <400> 5 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro   1 5 10 15 Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg              20 25 30 Gly Ser Glu Phe Asn Thr Gln Ser Glu His Ser Phe Glu Glu Met Tyr          35 40 45 Arg His Ile Leu Arg Ser Gln Gly Pro Phe Asp Ala Val Leu Tyr Tyr      50 55 60 His Met Met Lys Asp Glu Pro Val Val Phe Ser Thr Ser Asp Gly Lys  65 70 75 80 Glu Tyr Thr Tyr Pro Asp Ser Leu Glu Glu Glu Tyr Pro Pro Trp Leu                  85 90 95 Thr Glu Lys Glu Ala Met Asn Glu Glu Asn Arg Phe Val Thr Leu Asp             100 105 110 Gly Gln Gln Phe Tyr Trp Pro Val Met Asn His Lys Asn Lys Phe Met         115 120 125 Ala Ile Leu Gln His His Gln     130 135 <210> 6 <211> 408 <212> DNA <213> Artificial Sequence <220> <223> C-terminus domain of Ebola virus NP <400> 6 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60 atggctagca tgactggtgg acagcaaatg ggtcgcggat ccgaattcaa cacccagtca 120 gaacactctt ttgaggagat gtatcgccac attctaagat cacaggggcc atttgatgct 180 gttttgtatt atcatatgat gaaggatgag cctgtagttt tcagtaccag tgatggcaaa 240 gagtacacgt atccagactc ccttgaagag gaatatccac catggctcac tgaaaaagag 300 gctatgaatg aagagaatag atttgttaca ttggatggtc aacaatttta ttggccggta 360 atgaatcaca agaataaatt catggcaatc ctgcaacatc atcagtga 408 <210> 7 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> NC fusion protein of Ebola virus NP <400> 7 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro   1 5 10 15 Arg Gly Ser His Met Ala Ser Val Gln Gln Gly Ile Val Arg Gln Arg              20 25 30 Val Ile Pro Val Tyr Gln Val Asn Asn Leu Glu Glu Ile Cys Gln Leu          35 40 45 Ile Ile Gln Ala Phe Glu Ala Gly Val Asp Phe Gln Glu Ser Ala Asp      50 55 60 Ser Phe Leu Leu Met Leu Cys Leu His His Ala Tyr Gln Gly Asp Tyr  65 70 75 80 Lys Leu Phe Leu Glu Ser Gly Ala Val Lys Tyr Leu Glu Gly His Gly                  85 90 95 Phe Arg Phe Glu Val Lys Lys Arg Asp Gly Val Lys Arg Leu Glu Glu             100 105 110 Leu Leu Pro Ala Val Ser Ser Gly Lys Asn Ile Lys Arg Thr Leu Ala         115 120 125 Ala Met Pro Glu Glu Glu Thr Thr Glu Ala Asn Ala Gly Gln Phe Leu     130 135 140 Ser Phe Ala Ser Leu Phe Leu Pro Lys Leu Val Val Gly Glu Lys Ala 145 150 155 160 Cys Leu Glu Lys Val Gln Arg Gln Ile Gln Val His Ala Glu Gln Gly                 165 170 175 Leu Ile Gln Tyr Pro Thr Ala Trp Gln Ser Val Gly His Met Met Val             180 185 190 Ile Phe Arg Leu Met Arg Thr Asn Phe Leu Ile Lys Phe Leu Leu Ile         195 200 205 His Gln Gly Met His Met Val Ala Gly His Asp Ala Asn Asp Ala Val     210 215 220 Ile Ser Asn Ser Val Ala Gln Ala Arg Phe Ser Gly Leu Leu Ile Val 225 230 235 240 Lys Thr Val Leu Asp His Ile Leu Gln Lys Thr Glu Arg Gly Val Arg                 245 250 255 Leu His Pro Leu Ala Arg Thr Ala Lys Val Lys Asn Glu Val Asn Ser             260 265 270 Phe Lys Ala Ala Leu Ser Ser Leu Ala Lys His Gly Glu Tyr Ala Pro         275 280 285 Phe Ala Arg Leu Leu Asn Leu Ser Gly Val Asn Asn Leu Glu His Gly     290 295 300 Leu Phe Pro Gln Leu Ser Ala Ile Ala Leu Gly Val Ala Thr Ala His 305 310 315 320 Gly Ser Thr Leu Ala Gly Val Asn Val Gly Glu Gln Tyr Gln Gln Leu                 325 330 335 Arg Glu Ala Ala Thr Glu Ala Glu Gly Ser Glu Phe Asn Thr Gln Ser             340 345 350 Glu His Ser Phe Glu Glu Met Tyr Arg His Ile Leu Arg Ser Gln Gly         355 360 365 Pro Phe Asp Ala Val Leu Tyr Tyr His Met Met Lys Asp Glu Pro Val     370 375 380 Val Phe Ser Thr Ser Asp Gly Lys Glu Tyr Thr Tyr Pro Asp Ser Leu 385 390 395 400 Glu Glu Glu Tyr Pro Pro Trp Leu Thr Glu Lys Glu Ala Met Asn Glu                 405 410 415 Glu Asn Arg Phe Val Thr Leu Asp Gly Gln Gln Phe Tyr Trp Pro Val             420 425 430 Met Asn His Lys Asn Lys Phe Met Ala Ile Leu Gln His His Gln         435 440 445 <210> 8 <211> 1344 <212> DNA <213> Artificial Sequence <220> <223> NC fusion protein of Ebola virus NP <400> 8 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60 atggctagcg ttcaacaggg gattgttcgg caaagagtca tcccagtgta tcaagtaaac 120 aatcttgaag aaatttgcca acttatcata caggcctttg aagcaggtgt tgattttcaa 180 gagagtgcgg acagtttcct tctcatgctt tgtcttcatc atgcgtacca gggagattac 240 aaacttttct tggaaagtgg cgcagtcaag tatttggaag ggcacgggtt ccgttttgaa 300 gtcaagaagc gtgatggagt gaagcgcctt gaggaattgc tgccagcagt atctagtgga 360 aaaaacatta agagaacact tgctgccatg ccggaagagg agacaactga agctaatgcc 420 ggtcagtttc tctcctttgc aagtctattc cttccgaaat tggtagtagg agaaaaggct 480 tgccttgaga aggttcaaag gcaaattcaa gtacatgcag agcaaggact gatacaatat 540 ccaacagctt ggcaatcagt aggacacatg atggtgattt tccgtttgat gcgaacaaat 600 tttctgatca aatttctcct aatacaccaa gggatgcaca tggttgccgg gcatgatgcc 660 aacgatgctg tgatttcaaa ttcagtggct caagctcgtt tttcaggctt attgattgtc 720 aaaacagtac ttgatcatat cctacaaaag acagaacgag gagttcgtct ccatcctctt 780 gcaaggaccg ccaaggtaaa aaatgaggtg aactccttta aggctgcact cagctccctg 840 gccaagcatg gagagtatgc tcctttcgcc cgacttttga acctttctgg agtaaataat 900 cttgagcatg gtcttttccc tcaactatcg gcaattgcac tcggagtcgc cacagcacac 960 gggagtaccc tcgcaggagt aaatgttgga gaacagtatc aacaactcag agaggctgcc 1020 actgaggctg agggatccga attcaacacc cagtcagaac actcttttga ggagatgtat 1080 cgccacattc taagatcaca ggggccattt gatgctgttt tgtattatca tatgatgaag 1140 gatgagcctg tagttttcag taccagtgat ggcaaagagt acacgtatcc agactccctt 1200 gaagaggaat atccaccatg gctcactgaa aaagaggcta tgaatgaaga gaatagattt 1260 gttacattgg atggtcaaca attttattgg ccggtgatga atcacaagaa taaattcatg 1320 gcaatcctgc aacatcatca gtga 1344

Claims (15)

delete delete delete delete delete delete An NC fusion protein comprising an N-terminal domain fragment and an C-terminal domain fragment of an Ebola virus nucleoprotein, wherein the NC fusion protein comprises the amino acid sequence of SEQ ID NO. delete The method of claim 7, wherein
NC fusion protein consisting of the amino acid sequence of SEQ ID NO: 7, characterized in that encoded by the nucleotide sequence of SEQ ID NO: 8, NC fusion protein. Ebola virus infection prevention vaccine composition comprising the NC fusion protein according to claim 7. Ebola virus infection diagnostic kit comprising the NC fusion protein according to claim 7. delete (a) injecting the NC fusion protein according to claim 7 to an animal other than a human; And
(b) A method for producing a monoclonal antibody that specifically binds to the NC fusion protein according to claim 7, comprising recovering the antibody from the animal. delete delete

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