KR102783435B1 - p30 protein fragment derived from African swine fever virus as recombinant antigen, and uses thereof - Google Patents

Abstract

The present invention provides an isolated polypeptide consisting of an amino acid sequence represented by SEQ ID NO: 1, a vaccine composition against ASFV (African swine fever virus) comprising the polypeptide as an active ingredient, a method for immunizing animals using the polypeptide, and a method, diagnostic reagent and kit for detecting/diagnosing ASFV infection using the polypeptide. The polypeptide consisting of a unique sequence provided by the present invention has significantly superior expression and purification yields compared to natural proteins in viruses, is short in length, and has significantly superior ASFV infection serum detection/diagnosis ability compared to natural proteins in ASFV, and has the potential to be used as a vaccine and can be usefully utilized due to high productivity at an industrial level.

Description

{p30 protein fragment derived from African swine fever virus as recombinant antigen, and uses thereof}

The present invention relates to a p30 protein fragment derived from African swine fever virus (ASFV) and its use, and more specifically, to an isolated polypeptide consisting of an amino acid sequence represented by sequence number 1, a vaccine composition against ASFV comprising the polypeptide as an active ingredient, a method for immunizing animals using the polypeptide, and a method for detecting/diagnosing ASFV infection using the polypeptide, a diagnostic reagent, and a kit.

African swine fever (ASF) is a contagious disease of pigs caused by the African swine fever virus (ASFV) of the Asfarviridae family. It was first reported in Kenya in 1921, and since then, outbreaks have been reported mainly in sub-Saharan Africa. Since 2007, the area of occurrence has begun to expand outside of Africa, including Georgia, Armenia, and Azerbaijan along the Black Sea. In particular, the outbreak has recently spread east to west from Russia, and the number of cases in China and Southeast Asia, which have much exchange with Korea, is rapidly increasing.

African swine fever virus is highly resistant to the general environment, remaining infectious for more than 18 months at room temperature and for more than 6 months in meat products such as ham. It is also highly resistant to heat, losing its infectiousness only when exposed to 56°C for at least 1 hour (Non-patent Document 1).

African swine fever is a serious disease with a mortality rate of up to 100% in pigs, but there is no commercial vaccine for this disease. As African swine fever spreads again in Europe and spreads to the Asian continent, attention is focused on vaccine development research that was not properly conducted in the past. However, due to safety issues, the development of attenuated live vaccines is limited, and previous experimental studies have not shown great effects. There are also many hurdles to overcome in developing an effective product for defense. For example, ASFV is larger and has a complex structure than other viruses, so the effective part for defense is currently being searched for. The gene size of the African swine fever virus is 10 times that of general RNA viruses, and it has many genes. Since the gene is large, there are many types of proteins that can be made from the gene, and the ASFV genome is known to encode at least 151 ORFs. The African swine fever virus has a large number of structural proteins and is much larger than other viruses, being more than 10 times the size of a circovirus and 4-5 times the size of a PRRS virus. Because the African swine fever virus is so large and complex, it is difficult to induce immunity, and an effective vaccine has not yet been developed.

Also, since it is a high-risk infectious disease that requires immediate disposal of pigs, diagnosis is also very important. In order to confirm African swine fever, a diagnostic test from an accredited laboratory is essential. Laboratory diagnostic tests include detecting ASFV in blood and internal organs or detecting antibodies in the serum of infected pigs. Since a vaccine has not yet been developed, a positive result in an antibody test indicates an outdoor infection. If the virus needs to be isolated from blood, a blood sample collected at the time when initial fever symptoms were confirmed is appropriate, and organs such as lymph nodes or spleen should be sent in a refrigerated state. The ideal sample for antibody testing is serum from an infected pig in the recovery period between 8 and 21 days after infection. The method of detecting antibodies using serum from infected pigs can be conducted as a primary screening test using OIE-ELISA (indirect method) and commercialized antibody detection ELISA, and the final evaluation can be conducted through the immunoblotting method (IB), immunoperoxidase test (IPT), and indirect fluorescent antibody method (IFA) (see Non-patent Document 2). Because ASF has a rapid and diverse disease progression and can present asymptomatic infection in pigs, laboratories must perform both antigen and antibody tests to accurately determine infection status.

Therefore, there is an urgent need to develop a commercially available and effective ASFV infection prevention vaccine and ASFV infection diagnosis technology.

Lee, Ji-Yeon, Diagnosis and prevention of African swine fever, Journal of Korean Veterinary Medical Association, Vol. 50_No.11 Nov 2014, 671-673. Daniel Beltran-Alcrudo et al., African swine fever: detection and diagnosis - A manual for veterinarians. FAO Animal Production and Health Manual 2017. No. 19.Rome. Food and Agriculture Organization of the United Nations (FAO). 88 pages.

Accordingly, the inventors of the present invention have made great efforts to provide a commercially available ASFV infection prevention vaccine and ASFV infection diagnosis method, and as a result, have confirmed that a polypeptide composed of a unique sequence provided by the present invention is not only significantly superior in the ASFV infection serum detection/diagnosis ability compared to the natural protein in ASFV, but also has the potential to be used as a vaccine and is also highly productive industrially, thereby completing the present invention.

Accordingly, the purpose of the present invention is to provide an isolated polypeptide comprising an amino acid sequence represented by SEQ ID NO: 1.

Another object of the present invention is to provide a polynucleotide encoding the polypeptide, an expression vector comprising the polynucleotide, and a host cell comprising the expression vector.

Another object of the present invention is to provide a composition for a vaccine against African swine fever virus comprising the above-mentioned polypeptide as an active ingredient.

Another object of the present invention is to provide a method for inducing a protective immune response against African swine fever in an animal, comprising the step of administering to the animal an immunologically effective amount of the polypeptide.

Another object of the present invention is to provide a composition for diagnosing African swine fever virus infection, comprising the polypeptide as an active ingredient.

Another object of the present invention is to provide a diagnostic reagent for determining the presence or absence of antibodies to African swine fever virus, which comprises the above-mentioned polypeptide as an active ingredient, and a diagnostic kit comprising the above-mentioned diagnostic reagent.

Another object of the present invention is to provide a method for detecting African swine fever virus infection serum, comprising the steps of: (a) contacting a sample of an animal with the polypeptide of claim 1; and (b) detecting the presence of antibodies bound to the polypeptide of claim 1 in the sample.

To achieve the above purpose, the present invention provides an isolated polypeptide comprising an amino acid sequence represented by SEQ ID NO: 1.

In order to achieve another object of the present invention, the present invention provides a polynucleotide encoding the polypeptide, an expression vector comprising the polynucleotide, and a host cell comprising the expression vector.

In order to achieve another object of the present invention, the present invention provides a composition for a vaccine against African swine fever virus comprising the above-mentioned polypeptide as an active ingredient.

In order to achieve another object of the present invention, the present invention provides a method for inducing a defensive immune response against African swine fever in an animal, comprising the step of administering to the animal an immunologically effective amount of the polypeptide.

In order to achieve another object of the present invention, the present invention provides a composition for diagnosing African swine fever virus infection comprising the polypeptide as an active ingredient.

In order to achieve another object of the present invention, the present invention provides a diagnostic reagent for determining the presence or absence of an antibody against African swine fever virus, which comprises the polypeptide as an active ingredient, and a diagnostic kit comprising the diagnostic reagent.

In order to achieve another object of the present invention, the present invention provides a method for detecting African swine fever virus infection serum, comprising the steps of: (a) contacting a sample of an animal with the polypeptide of claim 1; and (b) detecting the presence of an antibody bound to the polypeptide of claim 1 in the sample.

The present invention is described in detail below.

As used herein, the term 'antigen' refers to any substance which, upon contact with and introduction into suitable cells, induces a state of sensitivity and/or immune reactivity, and which reacts demonstrably with immune cells and/or antibodies of the subject so sensitized in vivo or in vitro. The term 'antigen' as used herein may be used collectively with the same meaning as the term 'immunogen', and preferably means a molecule comprising one or more epitopes capable of stimulating the host immune system to elicit a secretory, humoral and/or cellular immune response specific to the antigen. In addition, the term 'antigenicity' or 'immunogenicity' as used herein means a property of the antigen or immunogen, and means a property of eliciting a secretory, humoral and/or cellular immune response.

The above term 'immune response' is a self-defense system existing in the animal body, a biological phenomenon that distinguishes various substances or living organisms that invade from the outside from oneself and eliminates these invaders. This surveillance system for self-defense is largely carried out by two mechanisms: humoral immunity and cellular immunity. Humoral immunity is carried out by antibodies existing in the serum, and antibodies play an important role in binding to invading external antigens and eliminating them. On the other hand, cellular immunity is carried out by several types of cells belonging to the lymphatic system, and these cells are responsible for directly destroying invading cells or tissues. Therefore, humoral immunity is mainly effective against external substances such as bacteria, viruses, proteins, and complex carbohydrates existing outside the cell, while cellular immunity exerts its function against various parasites, tissues, intracellular infections, cancer cells, etc. This dual defense system is mainly carried out by two types of lymphocytes, such as B cells and T cells, with B cells producing antibodies and T cells participating in cellular immunity. These immune responses by B cells or T cells are an immune system that reacts to antigens that have once invaded the body, but only when the same type of antigen continues to exist or has invaded repeatedly. Therefore, these immune responses are specific responses to specific antigens. In addition to these antigen-specific immune responses, there is also a type of natural immune response that directly reacts and destroys attacking cells even when the body has not been exposed to any antigen. These immune responses involve neutrophils, macrophages, and NK (natural killer) cells, and are characterized by exhibiting various functions regardless of the type of target cell.

Preferably, the immune response targeted in the present invention includes, but is not limited to, one or more of the effects of producing or activating antibodies, B cells, helper T cells, suppressor T cells, cytotoxic T cells and gamma-delta T cells specifically directed against the antigen or antigens included in the vaccine, inducing a therapeutic or protective immunological response in the host, thereby enhancing resistance to a new infection or reducing the clinical severity of a disease. Preferably, it may be a protective immune response.

The protection is evidenced by a reduction or absence of clinical signs normally exhibited by the infected host, a more rapid recovery time or a lower duration of illness, or a lower virus titer in the tissues or body fluids or excretions of the infected host.

The terms 'polypeptide' and 'protein' as used herein are used in their usual sense, i.e., to mean a polymer of amino acid residues. A polypeptide is not limited to a specific length, but in the context of the present invention may generally refer to a fragment of a full-length protein. The polypeptide or protein may include post-translational modifications, such as glycosylation, acetylation, phosphorylation, etc., and other modifications known in the art (both naturally occurring and non-naturally occurring modifications). The polypeptides and proteins of the present invention may be produced using any of a variety of known recombinant and/or synthetic techniques.

The first (third) letters of amino acids used herein refer to the following amino acids according to the standard abbreviation conventions in the biochemical field: A (Ala): alanine; C (Cys): cysteine; D (Asp): aspartic acid; E (Glu): glutamic acid; F (Phe): phenylalanine; G (Gly): glycine; H (His): histidine; I (IIe): isoleucine; K (Lys): lysine; L (Leu): leucine; M (Met): methionine; N (Asn): asparagine; O (Ply) pyrrolysine; P (Pro): proline; Q (Gln): glutamine; R (Arg): arginine; S (Ser): serine; T (Thr): threonine; U (Sec): selenocysteine, V (Val): valine; W (Trp): tryptophan; Y (Tyr): tyrosine.

The terms 'polynucleotide' and 'nucleic acid' as used herein refer to deoxyribonucleotides (DNA) or ribonucleotides (RNA) in single-stranded or double-stranded form. Unless otherwise limited, known analogs of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally occurring nucleotides are also included.

The term 'expression' in this specification means the production of a protein or nucleic acid in a cell.

The present inventors have newly identified a polypeptide consisting of an amino acid sequence of SEQ ID NO: 1 as a fragment having excellent advantages in terms of detection/diagnosis of ASFV-infected serum (i.e., antibodies to ASFV in serum) from an ASFV p30 protein of Kenya origin, usability as a vaccine, and possibility of mass production for commercial distribution due to excellent expression amount, purification efficiency, and stability. The polypeptide is characterized by having remarkably superior effects in the aforementioned aspects compared to the native protein from which it is derived and polypeptides (fragments) of different lengths or sequence compositions derived from the protein.

Accordingly, the present invention provides an isolated polypeptide comprising an amino acid sequence represented by sequence number 1.

The polypeptide of the present invention is characterized by immunogenicity against African swine fever virus (ASFV), and preferably has immunogenicity specific for ASFV originating in Kenya. Meanwhile, the polypeptide of the present invention is characterized by broad universal immunogenicity that reacts with specimens of ASFV caused by ASFV type 2, which has occurred in Russia, Georgia, etc., as well as ASFV type 6 occurring in Africa, and recently occurring in Central Europe and Asia.

The polypeptides described herein can be produced (produced) by any suitable procedure known to those skilled in the art, i.e., genetic engineering methods, such as recombinant techniques. For example, a nucleic acid encoding the polypeptide or a functional equivalent thereof is produced according to a conventional method. The nucleic acid can be produced by PCR amplification using appropriate primers. Alternatively, the DNA sequence can be synthesized using a standard method known in the art, such as an automatic DNA synthesizer (sold by Biosearch or Applied Biosystems). The produced nucleic acid is inserted into a vector containing one or more expression control sequences (e.g., promoter, enhancer, etc.) operatively linked thereto to control expression of the nucleic acid, and a host cell is transformed with the recombinant expression vector formed thereby. The resulting transformant is cultured in a medium and under conditions suitable for expression of the nucleic acid, and a substantially pure polypeptide expressed by the nucleic acid is recovered from the culture. The above recovery can be performed using a method known in the art (e.g., chromatography). The term "substantially pure polypeptide" as used herein means that the polypeptide according to the present invention does not substantially contain any other protein derived from a host cell.

Genetic engineering methods for synthesizing the polypeptide of the present invention can be found in the following references: Maniatis et al., Molecular Cloning; A laboratory Manual, Cold Spring Harbor laboratory, 1982; Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, N.Y., Second(1998) and Third(2000) Editions; Gene Expression Technology, Method in Enzymology, Genetics and Molecular Biology, Method in Enzymology, Guthrie ＆ Fink(eds.), Academic Press, San Diego, Calif, 1991; and Hitzeman et al., J. Biol. Chem., 255:12073-12080, 1990.

In addition, the polypeptide of the present invention can be produced by chemical synthesis methods known in the art as well as recombinant production methods. Representative methods include, but are not limited to, liquid or solid phase synthesis, fragment condensation, F-MOC or T-BOC chemistry.

In one embodiment, for example, the polypeptides of the present invention can be prepared by direct peptide synthesis using solid-phase techniques (Merrifield, J. Am. Chem. Soc. 85:2149-2154 (1963)). Solid-phase peptide synthesis (SPPS) methods can initiate synthesis by attaching functional units, called linkers, to small porous beads to initiate the peptide chain. Unlike liquid-phase methods, the peptides are covalently bonded to the beads, preventing them from being removed by filtration until they are cleaved by a specific reagent, such as trifluoroacetic acid (TFA). Synthesis is achieved by repeating the cycle of protection, deprotection, and coupling, in which the N-terminal amine of the peptide attached to the solid phase and the N-protected amino acid unit bind, and the newly revealed amine group binds to a new amino acid (cycle, deprotection-wash-coupling-wash). The SPPS method can be performed using microwave technology together, and microwave technology can shorten the time required for coupling and deprotection of each cycle by applying heat during the peptide synthesis process. The heat energy can prevent the expanding peptide chain from folding or forming aggregates and promote chemical bonding.

In addition, the peptide of the present invention can be produced by a liquid phase peptide synthesis method, and the specific method thereof is described in the following references: US Patent No. 5,516,891. In addition, the peptide of the present invention can be synthesized by various methods, such as a method of mixing the solid phase synthesis method and the liquid phase synthesis method, and the production method is not limited to the means described in this specification.

Protein synthesis can be performed using manual techniques or by automation. Automated synthesis can be accomplished, for example, using an Applied Biosystems 431A peptide synthesizer (Perkin Elmer). Alternatively, various fragments can be chemically synthesized separately and combined using chemical methods to produce the desired molecule.

Meanwhile, the scope of the polypeptide of the present invention includes functional equivalents of the polypeptide of the present invention and salts thereof as described above. For example, the 'functional equivalent' refers to a peptide having at least 80%, preferably 90%, and more preferably 95% or more amino acid sequence homology (i.e., identity) with the polypeptide of the present invention as described above, for example, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% sequence homology, and exhibiting substantially the same physiological activity as the polypeptide of the present invention. The term “substantially homogeneous physiological activity” as used above may, but is not limited to, for example, inducing a defensive immune response against ASFV in an animal’s body, or for another example, the ability to detect/diagnose serum from an ASFV-infected animal.

In one embodiment, the functional equivalent of the present invention may be one in which a portion of the amino acid sequence of the above-mentioned polypeptide of the present invention is generated by addition, insertion, substitution (non-conservative or conservative substitution), deletion or a combination thereof. The substitution of the amino acid in the above may preferably be a conservative substitution. Examples of conservative substitutions of naturally occurring amino acids are as follows: aliphatic amino acids (Gly, Ala, Pro), hydrophobic amino acids (Ile, Leu, Val), aromatic amino acids (Phe, Tyr, Trp), acidic amino acids (Asp, Glu), basic amino acids (His, Lys, Arg, Gln, Asn) and sulfur-containing amino acids (Cys, Met). Amino acid exchanges that do not change the overall activity of the molecule are known in the art (H.Neurath, R.L.Hill, The Proteins, Academic Press, New York, 1979). The functional equivalent also includes a variant in which a portion of the amino acid in the amino acid sequence of the polypeptide of the present invention is deleted. The deletion or substitution of the above amino acid is preferably located in a region not directly related to the physiological activity (chemokine activity) of the polypeptide provided by the present invention. In addition, a variant in which several amino acids are added to both ends or within the amino acid sequence of the above polypeptide of the present invention is also included. The added amino acid as mentioned above may be, for example, a histidine tag for protein separation/purification, which may be a '-GSHHHHHH' sequence, and a fragment including the same may preferably be a polypeptide having an amino acid sequence of SEQ ID NO: 3.

In addition, the scope of the above functional equivalents also includes polypeptide derivatives in which some chemical structures of the polypeptide are modified while maintaining the basic backbone and physiological activity of the polypeptide. For example, these include structural modifications to change the stability, storability, volatility, or solubility of the polypeptide of the present invention, and fusion proteins made by fusion with other proteins while maintaining physiological activity.

In one embodiment, the polypeptide of the present invention may be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, etc., as the case may be.

The present invention also provides a polynucleotide encoding the polypeptide of the present invention.

The polynucleotide described above is not particularly limited in its base combination as long as it can encode the polypeptide of the present invention. The polynucleotide may be provided as a nucleic acid molecule in the form of a single-strand or double-strand, including all of DNA, cDNA and RNA sequences.

In one embodiment, for example, the polynucleotide may comprise a base sequence represented by SEQ ID NO: 2.

In another embodiment, the polynucleotide may be comprised of a base sequence represented by SEQ ID NO: 2.

The polypeptide of the present invention can be provided by operably linking a nucleic acid sequence encoding the polypeptide of the present invention to a vector capable of expressing the same. Accordingly, the present invention provides an expression vector or recombinant vector comprising the polynucleotide.

The term 'expression vector' or 'recombinant vector' in the present invention refers to a genetic construct that is capable of expressing a target protein or target RNA in a suitable host cell and includes essential regulatory elements operably linked to enable expression of a gene insert.

The above term 'operably linked' refers to a functional linkage between a nucleic acid expression control sequence and a nucleic acid sequence encoding a target protein or RNA so as to perform a general function. For example, a promoter and a nucleic acid sequence encoding a protein or RNA are operably linked to affect the expression of the encoding nucleic acid sequence. The operably linked sequence with a recombinant vector can be produced using a genetic recombination technique well known in the art, and the site-specific DNA cleavage and linkage uses an enzyme or the like generally known in the art.

In one embodiment, the vector includes, but is not limited to, a plasmid vector, a cosmid vector, a bacteriophage vector, and a viral vector. A suitable expression vector may include, in addition to expression control elements such as a promoter, an operator, an initiation codon, a termination codon, a polyadenylation signal, and an enhancer, a signal sequence or a leader sequence for membrane targeting or secretion, and may be produced in various ways depending on the purpose. The promoter of the vector may be constitutive or inducible. In addition, the expression vector may include a selection marker for selecting a host cell containing the vector, and may include an origin of replication if it is a replicable expression vector.

If the host is a fungus of the genus Escherichia , the signal sequence may include, but is not limited to, a PhoA signal sequence, an OmpA signal sequence, etc. If the host is a fungus of the genus Bacillus, the signal sequence may include an α-amylase signal sequence, a subtilisin signal sequence, etc. If the host is a yeast, the signal sequence may include an MFα signal sequence, a SUC2 signal sequence, etc. If the host is an animal cell, the signal sequence may include an insulin signal sequence, an α-interferon signal sequence, an antibody molecule signal sequence, etc.

The present invention also provides a host cell comprising the expression vector. That is, the present invention provides a transformant (host cell) transformed with the expression vector (recombinant vector).

The above transformation may be performed by any method known to be capable of introducing a nucleic acid into an organism, cell, tissue or organ, and may be performed by selecting a standard technique suitable for the host cell as known in the art. Such methods include, but are not limited to, microprojectile bombardment, electroporation, protoplast fusion, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, stirring using silicon carbide fibers, Agrobacterium-mediated transformation, PEG-mediated fusion, microinjection, liposome-mediated method, dextran sulfate, lipofectamine, heat shock method, and the like.

The above term 'transformant' may be used interchangeably with 'host cell' and means a prokaryotic or eukaryotic cell containing heterologous DNA introduced into the cell by any means (e.g., electroporation, calcium phosphatase precipitation, microinjection, transformation, viral infection, etc.).

In the present invention, the transformant may be any kind of unicellular organism commonly used in the cloning field, such as prokaryotic microorganisms such as various bacteria (e.g., Clostridia genus, Escherichia coli, etc.), lower eukaryotic microorganisms such as yeast, and cells derived from higher eukaryotes including insect cells, plant cells, mammals, etc., but is not limited thereto. Since the amount of protein expression and modification, etc. appear differently depending on the host cell, a person skilled in the art can select and use the host cell most suitable for the purpose. The transformant of the present invention may preferably mean a transformed microorganism. Specifically, but not limited to, the host cell may be a prokaryotic host cell such as Escherichia coli , Bacillus subtilis , Streptomyces , Pseudomonas , Proteus mirabilis or Staphylococcus . Additionally, cells derived from higher eukaryotes, including but not limited to lower eukaryotic cells, insect cells, plant cells, mammals, etc., such as fungi (e.g., Aspergillus ), yeasts (e.g., Pichia pastoris , Saccharomyces cerevisiae , Schizosaccharomyces , Neurospora crassa ), etc., can be used as host cells.

The transformant (or transformed microorganism, host cell) of the present invention may preferably be Escherichia coli ( E. coli ). The Escherichia coli strain of the present invention is not limited thereto, and examples thereof such as Rosetta2 (DE3), C41 (DE3), and SoluBL21 may be used.

In addition, the present invention provides a vaccine composition against African swine fever virus comprising the above-described polypeptide as an active ingredient.

In the present invention, the term 'vaccine' or 'vaccine composition' refers to a composition that stimulates an immune response, and is used interchangeably herein with the term 'immunogenic composition' as having the same meaning. The vaccine includes both a prophylactic vaccine and a therapeutic vaccine. A prophylactic vaccine means one that induces an immune response before exposure to a substance containing an antigen, so as to cause a subject to have a greater immune response when exposed to the antigen, thereby increasing the ability to resist a substance or cell carrying the antigen. A therapeutic vaccine is used in a manner that it is administered to a subject that already has a disease related to the antigen of the vaccine, and the therapeutic vaccine can increase the subject's immune response to the antigen by providing an increased ability to fight the disease or cell carrying the antigen.

In one embodiment, the present invention may provide a vaccine composition for preventing African swine fever virus infection comprising the polypeptide as an active ingredient.

The vaccine composition of the present invention can be administered to mammals, including humans, by any method to induce an immune response. For example, it can be administered orally or parenterally. The parenteral administration method is not limited thereto, but may be administered by percutaneous, intramuscular, intraperitoneal, intravenous, or subcutaneous routes. Preferably, the vaccine may be administered intramuscularly during the first and second vaccinations.

The above vaccines may be in any form known in the art, for example, but not limited to, in the form of liquids and injections, or in solid forms suitable for suspension. These preparations may also be emulsified or encapsulated in liposomes or soluble glasses, or prepared in the form of aerosols or sprays. They may also be incorporated into transdermal patches. In the case of liquids or injections, they may contain, if necessary, propylene glycol and a sufficient amount of sodium chloride to prevent hemolysis (e.g., about 1%).

The vaccine composition of the present invention is characterized by comprising the polypeptide of the present invention as described above, and may additionally comprise one or more selected from the group consisting of pharmaceutically acceptable carriers, diluents, and adjuvants. As used herein, "pharmaceutically acceptable" refers to a non-toxic composition that is physiologically acceptable and does not inhibit the action of an active ingredient when administered to a human, and typically does not cause allergic reactions such as gastrointestinal disorders, dizziness, or similar reactions.

The carrier refers to a material that facilitates delivery of a target substance into a cell or tissue. Pharmaceutically acceptable carriers may further include, for example, carriers for oral administration or carriers for parenteral administration. For example, carriers for parenteral administration may include water, suitable oils, saline, aqueous glucose and glycols, etc. In addition, the carrier may include a dry formulation, such as a coated patch made of titanium or a polymer. Suitable carriers for vaccines are known to those skilled in the art, and include, but are not limited to, proteins, sugars, etc. The carrier may be an aqueous solution, a non-aqueous solution, a suspension, or an emulsion.

In addition, the composition of the present invention may use a structured or amorphous organic or inorganic polymer as an immunoadjuvant to increase immunogenicity. It is generally known that an immunoadjuvant plays a role in promoting an immune response through chemical and physical binding to an antigen. The immunoadjuvants used in this study include a structured aluminum gel, oil emulsion, or double oil emulsion, and immunosol. In addition, various plant-derived saponins, levamisole, CpG dinucleotide, RNA, DNA, LPS, and various types of cytokines were used to promote an immune response. The above-mentioned immune composition can be used as a composition for inducing an optimal immune response by combining various adjuvants and immune response-promoting additives.

These may include, but are not limited to, mineral salt adjuvants (e.g., alum-, calcium-, iron-, zirconium-based salt adjuvants), tensioactive adjuvants (e.g., Quil A, QS-21, other saponins), bacterial-derived adjuvants (e.g., N-acetyl muramyl-L-alanyl-D-isoglutamine (MDP), lipopolysaccharides (LPS), monophosphoryl lipid A, trehalose dimycolate (TDM), DNA, CpGs, bacterial toxins), adjuvant emulsions (e.g., FIA, Montanide, Adjuvant 65, Lipovant), liposomal adjuvants, polymeric adjuvants and carriers, cytokines (e.g., granulocyte-macrophage colony stimulating factor), carbohydrate adjuvants, live antigen delivery systems (e.g., bacteria, viruses), etc.

In addition, stabilizers, inactivators, antibiotics, preservatives, etc. may be used as compositions that can be added to the vaccine. Depending on the route of administration of the vaccine, the vaccine antigen may also be mixed with distilled water, buffer solutions, etc.

Other pharmaceutically acceptable carriers and preparations may be found in reference to the following literature (Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995). Formulations and administration techniques for the vaccines described herein may be provided by reference to the literature (Remington, The Science and Practice of Pharmacy, 22nd ed.).

In addition, a vaccine composition comprising the immunogenic complex protein of the present invention can be used for immunization by administering an effective amount to an individual in need thereof. That is, the present invention provides a method for inducing a protective immune response against African swine fever in an animal, comprising the step of administering an immunogenic effective amount of the polypeptide of the present invention to a pig.

The above 'subject' may mean an animal, preferably a mammal other than a human. In one embodiment, the subject may preferably be a pig.

Therefore, in one embodiment, the present invention provides a method for enhancing immunity of an animal (particularly a pig) against ASFV (i.e., a method for immunizing an animal against ASFV), characterized by administering to the animal (particularly a pig) a polypeptide of claim 1.

The term 'immunization' used herein means that when the immunogenic complex protein according to the present invention is administered to a subject, a secretory, humoral and/or cellular immune response to the immunogenic complex protein is induced in the subject, and through such immunization, a preventive or therapeutic effect against the target disease (particularly ASFV infection in the present invention) is exhibited.

The above 'effective amount' refers to an amount that exhibits a preventive or therapeutic effect of the vaccine composition of the present invention against the target disease (particularly, ASFV infection), and means an amount sufficient for the polypeptide of the present invention to induce secretory, humoral and/or cellular immune responses in the administered subject.

The total effective amount of the polypeptide of the present invention can be administered to a subject as a single dose, or can be administered by a fractionated treatment protocol in which multiple doses are administered over a long period of time. In addition, the content of the effective ingredient can be varied depending on the purpose of administration. The effective dose is determined for each subject by considering various factors such as the type and severity of the target disease, the route of administration, and the number of administrations, as well as the age, weight, health status, sex, disease severity, diet, and excretion rate of the subject requiring administration. Therefore, a person having ordinary knowledge in the relevant field will be able to determine an appropriate effective dose depending on the purpose of administration. In addition, the efficacy of the therapy can be determined by monitoring the efficacy using an assay method that determines the activity of immune cells after administering the protein according to the present invention or a widely known in vivo assay. The pharmaceutical composition of the present invention is not particularly limited in its formulation, route of administration, and method of administration as long as it exhibits the effect of the present invention.

The polypeptide of the present invention described above has a remarkable effect in specifically detecting (diagnosing) ASFV-infected serum, compared to natural proteins and polypeptides of different lengths/sequence configurations derived from the protein. In the present invention, detecting (diagnosing) ASFV-infected serum means confirming the presence or absence and/or amount of anti-ASFV antibodies (antibodies to African swine fever virus) present in the blood, plasma or serum of an individual by binding to the polypeptide of the present invention (forming an antigen-antibody complex). When the presence of anti-ASFV antibodies in the blood, plasma or serum of an individual is detected or/and confirmed, the individual can be determined to be infected with ASFV.

Therefore, the present invention provides a composition for diagnosing African swine fever virus infection comprising the above-described polypeptide as an active ingredient.

In addition, the present invention provides a diagnostic reagent composition for determining the presence or absence of an antibody against African swine fever virus, which comprises the above-described polypeptide as an active ingredient, and a diagnostic kit comprising the diagnostic reagent composition.

In addition, the present invention

(a) a step of contacting a sample of an animal with the polypeptide of the present invention as described above; and

(b) Provided is a method for detecting (diagnosing) African swine fever virus infection serum, comprising a step of detecting the presence of an antibody bound to the polypeptide of the present invention in the sample.

In particular, the polypeptides of the present invention have remarkable specificity for various types of ASFV, and can specifically detect not only ASFV originating in Kenya but also ASFV occurring recently. Therefore, in one embodiment, the present invention may preferably provide a diagnostic reagent composition for determining the presence or absence of an antibody against African swine fever virus, which comprises the polypeptide of the present invention as an active ingredient, and a diagnostic kit comprising the same, and may also provide a method for detecting (diagnosing) ASFV infected serum comprising steps (a) and (b) above.

The term 'sample' in this specification may be a solid sample or a fluid sample, and preferably may be serum, plasma, muscle tissue, whole blood, lymph, spleen or a homogenate thereof.

In the detection or diagnosis according to the present invention, if it is known in the art as a means for detecting an antigen-antibody complex, the method and apparatus can be applied without particular limitation. For example, but not limited thereto, the antigen-antibody complex can be detected by a method such as radial immunodiffusion, immunoprecipitation analysis including immunoelectrophoresis or reverse current electrophoresis, RIA (Radioimmunoassay), competition indirect immunofluorescent assay, ELISA (Enzyme Linked Immunosorbent Assay), or immunochromatic assay. This type of detection is particularly advantageous when the antigen is provided as a soluble protein, and the polypeptide of the present invention satisfies this characteristic. In one embodiment according to the present invention, an ELISA method, particularly a sandwich-type ELISA, is used, and in this case, the detection antibody described below is also used together. It can be understood that the present invention provides a diagnostic kit using the above method, and kit components according to each method are well known in the art.

In one embodiment according to the present invention, for detection of an antigen (polypeptide of the present invention)-antibody complex, the antigen (polypeptide of the present invention) may be labeled with a label. That is, the polypeptide of the present invention may be provided linked (e.g., covalently bound or cross-linked) to a detectable label. The detectable label may be a chromogenic enzyme (e.g., peroxidase, alkaline phosphatase), a radioisotope (e.g., 124I, 125I, 111In, 99mTc, 32P, 35S), a chromophore, biotin, a luminescent or fluorescent substance (e.g., FITC, RITC, rhodamine, Texas Red, fluorescein, phycoerythrin, quantum dots), a magnetic resonance imaging contrast agent (e.g., superparamagnetic iron oxides (SPIO), ultrasuperparamagnetic iron oxides (USPIO)), a gold particle, etc. Similarly, the detectable label may be another antibody epitope, substrate, cofactor, inhibitor or affinity ligand unrelated to ASFV. Such labeling may be performed during the process of synthesizing the polypeptide of the present invention or may be performed additionally to a polypeptide already synthesized.

In one embodiment according to the present invention, a detection antibody (secondary antibody) that detects an antibody (primary antibody, for example, an antibody produced in animal serum) bound to the antigen (polypeptide of the present invention) may be labeled. The detection antibody (secondary antibody) that can be used in the method according to the present invention specifically binds to an immunoglobulin (for example, IgM, IgG) produced in the animal to be diagnosed, and the detection antibody can be labeled with a substance that can be detected visually or using various image detection equipment. This can be understood with reference to the above-described polypeptide labeling substance of the present invention.

In one specific embodiment, the antigen (polypeptide of the present invention) or the detection antibody (secondary antibody) according to the present invention comprises a peroxidase such as horseradish peroxidase, alkaline phosphatase, glucose oxidase, beta-galactosidase, urease, catalase, asparginase, ribonuclease, malate dehydrogenase, staphylococcal nuclease, triose phospate isomerase, glucose-6-phosphate dehydrogenase, glucoamylase, and It may be labeled with an enzyme capable of catalyzing a chemical reaction in the presence of a specific substrate, such as, but not limited to, acetylcholine esterase, which produces a detectable colorimetric reaction or emits light.

In another specific embodiment, the antigen (polypeptide of the present invention) or the detection antibody according to the present invention comprises a chromophore used in virominescence, chemiluminescence, electroluminescence, electrochemiluminescence and photoluminescence that emits light of a different wavelength from the light irradiated upon exposure to light, for example, green fluorescent protein as a protein; and fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, and fluorecamine as an organic compound, but is not limited thereto.

In another specific embodiment, the antigen (the present invention polypeptide) or the detection antibody according to the present invention can be labeled with various radioisotope substances. The detection of the label herein can be performed, for example, by a scintillation counter in the case of a radioisotope, and can be performed, for example, by a method such as spectroscopy, a phosphor imaging device, or a fluorometer in the case of a fluorescent substance. In the case of enzyme labeling, it can be performed by measuring the color product produced by the conversion of the chromogenic substrate by the enzyme in the presence of an appropriate substrate. In addition, detection can be performed by comparing the color of the color product produced by the enzymatic reaction with a suitable standard or control.

In specific embodiments, the material labeling the antigen (polypeptide of the present invention) or the detection antibody according to the present invention includes, but is not limited to, a material including, for example, a chromophore; an enzyme including alkaline phosphatase, biotin, beta-galactosidase or peroxidase; a radioactive substance; or a nanoparticle such as colloidal gold particles or colored latex particles.

The kit of the present invention may additionally include, in addition to the polypeptide of the present invention, a suitable buffer solution or medium for the binding reaction of the polypeptide and the anti-ASFV antibody. In addition, when the polypeptide of the present invention is provided without being directly labeled, another detectable labeling means for labeling the polypeptide may be additionally included in the kit. For example, a secondary antibody labeled with a fluorescent substance of the present invention, a chromogenic substrate, etc. may be additionally included in the kit.

In addition, the polypeptide of the present invention may be provided in a form coated on the surface of a plate. In this case, after treating a sample on the plate and reacting under appropriate conditions, the binding of the polypeptide (antigen) of the present invention to an antibody in the sample on the surface of the plate can be observed to diagnose ASFV infection. The polypeptide of the present invention may be provided attached to a microwell plate such as a 96-well microwell plate, beads or particles including colloidal gold particles or colored latex particles, or a membrane such as cellulose, nitrocellulose, polyethersulfone, polyvinylidene, fluoride, nylon, charged nylon, and polytetrafluoroethylene. A known method can be used for attaching or coating the antigen (polypeptide of the present invention), and for example, reference can be made to those described in the Examples herein.

In a specific embodiment, the diagnostic reagent of the present invention, and a kit including the same, can be used in a sandwich-type immunoassay method, such as ELISA (Enzyme Linked Immuno Sorbent Assay), RIA (Radio Immuno Assay), etc. This method involves adding a specimen to an antigen bound to a solid substrate, such as a bead, membrane, slide, or microwell plate made of glass, plastic (e.g., polystyrene), polysaccharide, nylon, or nitrocellulose, and then qualitatively or quantitatively detecting the antigen by binding to an antibody that is labeled with a labeling substance capable of direct or indirect detection, such as a radioactive substance such as 3H or 125I as described above, a fluorescent substance, a chemiluminescent substance, a hapten, biotin, or digoxigenin, or an enzyme capable of color development or luminescence through reaction with the substrate, such as horseradish peroxidase, alkaline phosphatase, or malate dehydrogenase. Also, immunoassay methods are described in Enzyme Immunoassay, E. T. Maggio, ed., CRC Press, Boca Raton, Florida, 1980; Gaastra, W., Enzyme-linked immunosorbent assay(ELISA), in Methods in Molecular Biology, Vol. 1, Walker, J.M. ed., Humana Press, NJ, 1984, etc. ELISA kits may additionally include reagents capable of detecting bound antibodies, for example, secondary detection antibodies labeled with the above-described substances such as chromophores, enzymes (e.g., conjugated to the antibody), and substrates used for detection.

The polypeptide composed of a unique sequence provided in the present invention has significantly superior expression and purification yields compared to the native protein in the virus, is short in length, and has significantly superior ASFV infection serum detection/diagnosis ability compared to the native protein in ASFV, and has the potential to be used as a vaccine and also has high productivity at the industrial level.

Figure 1 shows the results of comparing the expression level of the Asfv-p30 protein, a polypeptide of the present invention, in a recombinant E. coli cell line with that of the natural protein (Kenya Asfv-p30).
Figure 2 shows the results of purification of Asfv-p30 protein.
Figure 3 shows the results of evaluating the diagnostic utility of the Asfv-p30 protein using the ELISA technique.

The present invention is described in detail below.

However, the following examples are only intended to illustrate the present invention, and the content of the present invention is not limited to the following examples.

Example 1: Discovery of highly immunogenic fragment polypeptides derived from p30 protein of African swine fever virus (ASFV) from Kenya

1-1. Production of Kenya-ASFV-p30 protein-derived fragment polypeptide

Based on the sequence of p30 protein (also referred to as ASFV-p30 in this specification) consisting of an amino acid sequence represented by SEQ ID NO: 5 obtained from Pig/Kenya/KEN-50/1950 ASFV strain, various polypeptide fragments were produced and shown in [Table 1]. This was done in an attempt to overcome the difficulty in producing recombinant proteins at a level for commercial use.

pep. fragment name Sequence information Sequence number Kenya Asfv-p30 W/T STKKKPTITKQELYSLVAADTQLNKALIERIFTSQQKIIQNALKHNQEVIIPPGIKFTVVTVKAKPARQGHNPATGEPIQIKAKPEHKAVKIRALKPVHDMLN Sequence number 5 asfv-p30 TKQELYSLVAADTQLNKALIERIFTSQQKIIQNALKHNQEVIIPPGIKFTVVTVKAKPARQGHNPATGEPIQIKAKPEHKA Sequence number 1

The above proteins and polypeptides were briefly produced by the following methods. DNA encoding Kenya Asfv-p30 W/T polypeptide (SEQ ID NO: 6) was synthesized by request from Macrogen. DNA encoding Asfv-p30 polypeptide (SEQ ID NO: 4) was synthesized by request from Macrogen. Polynucleotides encoding each fragment polypeptide were cloned between the Nde1 and BamH1 restriction sites of pET49b vector (Novagen). Each polypeptide was overexpressed in Escherichia coli strain BL21. E. coli cells transformed with the vector were grown in Luria-Bertani (LB) medium containing 100 ug/ml kanamycin at 37°C until OD600 reached 0.7, and protein expression was induced by 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG). After adding IPTG, the cells were cultured for an additional 3 hours and then centrifuged at 5,000 rpm for 20 minutes to harvest the cells. The harvested cells were resuspended in 50 ml of IB buffer (pH 8.0 Tris 0.1 M, pH 8.0 Ethylenediaminetetraacetic acid 5 mM, phenylmethylsulfonyl fluoride 0.1 mM), sonicated to break the cells, and finally resuspended in Denaturation buffer (Denaturation buffer containing 6 M Guanidine Hydrochloric acid, pH 8.0 Tris 0.1 M, and pH 8.0 Ethylenediaminetetraacetic acid 2.5 mM) and sonicated to break the cells. In the case of Kenya Asfv-p30 W/T, protein expression could not be confirmed visually. For Asfv-p30, centrifuge at 15,000 rpm and measure the concentration at A280. Then, transfer the supernatant to a snake skin tube and dilute to 1 mg/ml in refolding buffer (50 mM NaCl 100 mM Tris pH 8.0 2.5 M EDTA pH 8.0, 0.6 M L-Arginine, 0.2 mM oxidized glutathione, 2 mM reduced glutathione) (10℃, 36 hrs). After refolding, transfer to a snake skin tube and add Urea buffer (20 mM Tris pH 8.0, 100 mM Urea) twice for 12 hours at 4℃, and add 1 X Phosphate-buffered saline (PBS) buffer twice for 12 hours at 4℃. This was centrifuged again at 5,000 rpm for 20 minutes, and the supernatant was treated with Ni-NTA and bound for 1 hour and 30 minutes (4°C). After washing with 1X PBS buffer containing 50 mM Imidazole, it was eluted with 1X PBS buffer containing 250 mM Imidazole and 500 mM Imidazole. The polypeptides were identified using SDS-polyacrylamide gel electrophoresis, and the polypeptides were dissolved in a buffer containing 20 mM HEPES and 150 mM sodium chloride at pH 7.4 (4°C, overnight).

1-2. Productivity evaluation of fragment polypeptides compared to native p30 protein

The productivity of the above various fragment polypeptides was quantitatively compared and evaluated. In the case of other fragment polypeptides (Kenya Asfv-p30 W/T), they were not expressed in E. coli cell lines. However, Asfv-p30 polypeptide was successfully expressed in E. coli and could be purified with high purity. The final yield of expression and purification of the fragment polypeptide of Asfv-p30 (SEQ ID NO: 1) of the present invention was 3 mg protein/1L (LB culture), and the time required for purification (until protein was obtained) was 2.5 to 3 days. Asfv-p30 was successfully expressed in E. coli cell lines, which have the advantage of being able to be produced efficiently in a short period of time, and not only did it show a high yield that could be commercially utilized in the purification process, but the purification period was less than 3 days. The native Kenya Asfv-p30 W/T protein was not expressed in E. coli. In this way, the Asfv-p30 (SEQ ID NO: 1) polypeptide fragment of the present invention can be purified in high yield from an E. coli cell line and exhibits the advantage of a short purification process (see FIGS. 1 and 2).

1-3. Comparative evaluation of the diagnostic ability of fragment polypeptides for ASFV infection in blood compared to native p30 protein

Using the ID Screen® African Swine Fever Indirect Screening test kit from ID.vet, the binding ability of various fragment polypeptides produced in Example 1-1 to antibodies in ASFV-infected serum was comparatively evaluated by the ID (indirect)-ELISA method. Briefly, the method was performed as follows. First, Coating buffer (0.015 M sodium carbonate, 0.035 M sodium bicarbonate, Final pH 9.6) and each antigen (each of various fragment polypeptides produced in Example 1-1, added at a concentration of 2 ug/ml or 4 ug/ml) were added to a 96-well EIA/RIA plate, and the wells were coated with each antigen by incubating at 4°C overnight (16 h). Each well was washed 4 times with 200 ul of PBST buffer (1XPBS + Tween 20 0.05%). 100 ul of primary antibody was added to each well, and incubated at room temperature (22℃) for 1 hour. The primary antibody was treated in the form of ASFV-infected pig serum provided as a positive control in the African Swine Fever Indirect Screening test kit of ID.vet. After washing each well with 200 ul of PBST buffer, 100 ul of secondary antibody was added to each well, and incubated at room temperature for 1 hour. 100 ul of substrate solution was added to each well, light was blocked, and incubated at room temperature for 15 minutes. 100 ul of stop solution was added to each well, and the absorbance (optical density) value was measured at 450 nm.

As a result of the experiment, as shown in Fig. 3, the fragment polypeptide of Asfv-p30 (SEQ ID NO: 1) produced and purified from E. coli showed a high reactivity to ASFV infected serum at a level much higher (~4 times or more) than that of other African swine fever antigen proteins (Asfv-p72). In addition, it showed a reactivity more than 3 times higher than that of the commercial diagnostic reagent provided by ID.vet.

As described above, the present invention relates to the development of a recombinant antigen of a p30 protein fragment derived from African swine fever virus (ASFV) and its use, and more specifically, to an isolated polypeptide consisting of an amino acid sequence represented by SEQ ID NO: 1, a vaccine composition against ASFV comprising the polypeptide as an active ingredient, a method for immunizing animals using the polypeptide, and a method, diagnostic reagent and kit for detecting/diagnosing ASFV infection using the polypeptide. The polypeptide consisting of a unique sequence provided by the present invention is shorter than the native protein in the virus, and has a significantly superior ability to detect/diagnose ASFV infection serum compared to the native protein in ASFV, and has the potential to be used as a vaccine, and also has high productivity at an industrial level, and thus has great potential for industrial applicability.

<110> University industry foundation, Yonsei university wonju campus <120> p30 protein fragment derived from African swine fever virus as recombinant antigen, and uses it <130> NP22-0005 <160> 6 <170> KoPatentIn 3.0 <210> 1 <211> 183 <212> PRT <213> Artificial Sequence <220> <223> Asfv-p30 amino acid sequence <400> 1 Met Pro Glu Val Ile Phe Lys Thr Asp Leu Arg Ala Ser Ser Gln Val 1 5 10 15 Val Phe His Ala Gly Ser Leu Tyr Thr Trp Phe Ser Val Glu Ile Ile 20 25 30 Asn Ser Gly Arg Ile Val Thr Thr Ala Ile Lys Thr Leu Leu Ser Thr 35 40 45 Val Lys Tyr Asp Ile Val Arg Asn Ala Arg Ile Tyr Ala Gly Gln Gly 50 55 60 Tyr Thr Glu Gln Gln Ala Gln Glu Glu Trp Asn Met Ile Leu His Val 65 70 75 80 Leu Phe Glu Glu Glu Thr Val Ser Ser Ala Ser Ser Glu Ser Gly His 85 90 95 Lys Ala Asp Gly His Glu Ile Asn Glu Ser Thr Ser Ser Phe Glu Thr 100 105 110 Leu Phe Glu Gln Glu Pro Ser Ser Glu Thr Leu Lys Asp Thr Lys Leu 115 120 125 Tyr Ala Leu Ala Gln Lys Ala Val Gln His Ile Glu Gln Tyr Gly Lys 130 135 140 Ala Pro Asp Phe Asn Lys Val Ile Arg Ala His Asn Tyr Ile Gln Thr 145 150 155 160 Ile Tyr Gly Ser Pro Leu Lys Glu Glu Glu Lys Glu Glu Val Arg Leu 165 170 175 Met Val Ile Lys Leu Leu Ser 180 <210> 2 <211> 552 <212> DNA <213> Artificial Sequence <220> <223> Asfv-p30 DNA sequence <400> 2 atgccggaag ttatcttcaa aaccgacctg cgtgcttctt ctcaggttgt tttccacgct 60 ggttctctgt acacctggtt ctctgttgaa atcatcaact ctggtcgtat cgttaccacc 120 gctatcaaaa ccctgctgtc taccgttaaa tacgacatcg ttcgtaacgc tcgtatctac 180 gctggtcagg gttacaccga acagcaggct caggaagaat ggaacatgat cctgcacgtt 240 ctgttcgaag aagaaaccgt ttcttctgct tcttctgaat ctggtcacaa agctgacggt 300 cacgaaatca acgaatctac ctcttctttc gaaaccctgt tcgaacagga accgtcttct 360 gaaaccctga aagacaccaa actgtacgct ctggctcaga aagctgttca gcacatcgaa 420 cagtacggta aagctccgga cttcaacaaa gttatccgtg ctcacaacta catccagacc 480 atctacggtt ctccgctgaa agaagaagaa aaagaagaag ttcgtctgat ggttatcaaa 540 ctgctgtctt aa 552 <210> 3 <211> 191 <212> PRT <213> Artificial Sequence <220> <223> Asfv-p30 amino acid sequence with histidine tag <400> 3 Met Pro Glu Val Ile Phe Lys Thr Asp Leu Arg Ala Ser Ser Gln Val 1 5 10 15 Val Phe His Ala Gly Ser Leu Tyr Thr Trp Phe Ser Val Glu Ile Ile 20 25 30 Asn Ser Gly Arg Ile Val Thr Thr Ala Ile Lys Thr Leu Leu Ser Thr 35 40 45 Val Lys Tyr Asp Ile Val Arg Asn Ala Arg Ile Tyr Ala Gly Gln Gly 50 55 60 Tyr Thr Glu Gln Gln Ala Gln Glu Glu Trp Asn Met Ile Leu His Val 65 70 75 80 Leu Phe Glu Glu Glu Thr Val Ser Ser Ala Ser Ser Glu Ser Gly His 85 90 95 Lys Ala Asp Gly His Glu Ile Asn Glu Ser Thr Ser Ser Phe Glu Thr 100 105 110 Leu Phe Glu Gln Glu Pro Ser Ser Glu Thr Leu Lys Asp Thr Lys Leu 115 120 125 Tyr Ala Leu Ala Gln Lys Ala Val Gln His Ile Glu Gln Tyr Gly Lys 130 135 140 Ala Pro Asp Phe Asn Lys Val Ile Arg Ala His Asn Tyr Ile Gln Thr 145 150 155 160 Ile Tyr Gly Ser Pro Leu Lys Glu Glu Glu Lys Glu Glu Val Arg Leu 165 170 175 Met Val Ile Lys Leu Leu Ser Gly Ser His His His His His His 180 185 190 <210> 4 <211> 576 <212> DNA <213> Artificial Sequence <220> <223> Asfv-p30 DNA sequence with histidine tag <400> 4 atgccggaag ttatcttcaa aaccgacctg cgtgcttctt ctcaggttgt tttccacgct 60 ggttctctgt acacctggtt ctctgttgaa atcatcaact ctggtcgtat cgttaccacc 120 gctatcaaaa ccctgctgtc taccgttaaa tacgacatcg ttcgtaacgc tcgtatctac 180 gctggtcagg gttacaccga acagcaggct caggaagaat ggaacatgat cctgcacgtt 240 ctgttcgaag aagaaaccgt ttcttctgct tcttctgaat ctggtcacaa agctgacggt 300 cacgaaatca acgaatctac ctcttctttc gaaaccctgt tcgaacagga accgtcttct 360 gaaaccctga aagacaccaa actgtacgct ctggctcaga aagctgttca gcacatcgaa 420 cagtacggta aagctccgga cttcaacaaa gttatccgtg ctcacaacta catccagacc 480 atctacggtt ctccgctgaa agaagaagaa aaagaagaag ttcgtctgat ggttatcaaa 540 ctgctgtctg gatcccatca tcatcatcat cattaa 576 <210> 5 <211> 193 <212> PRT <213> African swine fever virus <400> 5 Met Lys Met Glu Val Ile Phe Lys Thr Asp Leu Arg Ala Ser Ser Gln 1 5 10 15 Val Val Phe His Ala Gly Ser Leu Tyr Thr Trp Phe Ser Val Glu Ile 20 25 30 Ile Asn Ser Gly Arg Ile Val Thr Thr Ala Ile Lys Thr Leu Leu Ser 35 40 45 Thr Val Lys Tyr Asp Ile Val Arg Asn Ala Arg Ile Tyr Ala Gly Gln 50 55 60 Gly Tyr Thr Glu Gln Gln Ala Gln Glu Glu Trp Asn Met Ile Leu His 65 70 75 80 Val Leu Phe Glu Glu Glu Thr Val Ser Thr Ser Ser Thr Ser Leu Glu 85 90 95 Ser Asn His Glu Thr Asn Gly His Lys Ala Asp Gly His Glu Ile Asn 100 105 110 Glu Cys Thr Ser Ser Phe Glu Thr Leu Phe Glu Gln Glu Pro Ser Ser 115 120 125 Glu Thr Leu Lys Asp Thr Lys Leu Tyr Ala Leu Ala Gln Lys Ala Val 130 135 140 Gln His Ile Glu Gln Tyr Gly Lys Ala Pro Asp Phe Asn Lys Val Ile 145 150 155 160 Arg Ala His Asn Tyr Ile Gln Thr Ile Tyr Gly Ser Pro Leu Lys Glu 165 170 175 Glu Glu Lys Glu Glu Val Arg Leu Met Val Ile Lys Leu Leu Lys Lys 180 185 190 Lys <210> 6 <211> 582 <212> DNA <213> African swine fever virus <400> 6 atgaaaatgg aggtaatctt caaaacggat ttaagagcat cctcacaagt cgtgtttcat 60 gccggtagtc tgtatacctg gttttctgtt gagattatca atagcggtag aatcgttaca 120 accgctataa aaacactgct cagtactgtt aagtatgata ttgtgagaaa tgctcgcatc 180 tatgcaggac agggatatac tgaacagcag gctcaagaag aatggaatat gatcctgcat 240 gtgctgtttg aagaggagac ggtatcaaca tcttcaacat ccttagagag taatcatgaa 300 actaatggtc ataaagcgga tggccatgaa attaatgaat gtacatcatc ctttgaaacg 360 ctgtttgagc aagagccctc atcagagaca ctcaaggaca ccaagctgta tgcgcttgca 420 caaaaggctg tgcaacacat tgaacagtat ggaaaggcac ctgattttaa caaggtcatt 480 agagcacata actatattca aacaatttat ggaagtcctc taaaagagga agaaaaagag 540 gaggtaagac tcatggtcat taaactttta aaaaaaaaaat ag 582

Claims (13)

An isolated polypeptide comprising an amino acid sequence represented by sequence number 1.
An isolated polypeptide according to claim 1, characterized in that the polypeptide is immunogenic against African swine fever virus (ASFV).
An isolated polypeptide, characterized in that in the second paragraph, the ASFV is of Kenya origin.
A polynucleotide encoding the polypeptide of claim 1.
An expression vector comprising the polynucleotide of claim 4.
A cell existing in a human body containing an expression vector of Article 5, and a host cell excluding a human embryo.
A composition for a vaccine against African swine fever virus comprising the polypeptide of claim 1 as an active ingredient.
A vaccine composition in claim 7, wherein the composition further comprises at least one selected from the group consisting of pharmaceutically acceptable carriers, diluents and adjuvants.
A method for inducing a protective immune response against African swine fever in an animal, comprising the step of administering an immunologically effective amount of the polypeptide of claim 1 to an animal other than a human.
A composition for diagnosing African swine fever virus infection, comprising the polypeptide of claim 1 as an active ingredient.
A diagnostic reagent for determining the presence or absence of antibodies to African swine fever virus, comprising the polypeptide of claim 1 as an active ingredient.
A diagnostic kit comprising the diagnostic reagent of Article 11.
(a) a step of contacting a sample of an animal with the polypeptide of claim 1; and
(b) A method for detecting African swine fever virus infection serum, comprising a step of detecting the presence of an antibody bound to the polypeptide of claim 1 among the above samples.