KR20230137070A - 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 African swine fever virus (ASFV) comprising the polypeptide as an active ingredient, a method for immunizing an animal 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 the virus, is short in length, and has significantly superior ASFV infection serum detection/diagnosis ability compared to natural proteins in ASFV. The polypeptide also 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 the amino acid sequence shown in SEQ ID NO: 1, and to an effective method of the polypeptide. It relates to a vaccine composition against ASFV containing it as an ingredient, a method for animal skinning using the polypeptide, and a method for detecting/diagnosing ASFV infection using the polypeptide, diagnostic reagents, and kits.

African swine fever (ASF) is an infectious disease in pigs caused by infection with the African swine fever virus (ASFV), a member of the Asfarviridae family. After its first report in Kenya in 1921, it mainly spread to swine. There have been reports of outbreaks in the sub-Saharan region, and since 2007, the scope of occurrence has begun to increase in regions other than Africa, such as the Black Sea coast of Georgia, Armenia, and Azerbaijan. In particular, the outbreak has recently spread from Russia to the east and west, and the number of cases in China and Southeast Asia, which have a lot of interaction with Korea, is rapidly increasing.

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

Although African swine fever is a serious disease with a mortality rate of up to 100% when infected with pigs, there is no commercialized vaccine for this disease. As African swine fever is spreading again in Europe and has spread to the Asian continent, attention is being focused on vaccine development research, which had not been carried out properly in the past. However, due to safety concerns, the development of live attenuated vaccines is limited, and previous experimental studies have not shown significant effectiveness. There are also many hurdles to overcome in developing a product that is effective in protecting against the Sadok vaccine. For example, ASFV is larger than other viruses and has a more complex structure, so we are looking for effective parts for defense. The gene size of the African swine fever virus is 10 times that of common RNA viruses, and it has many genes. Because 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 the size of the virus itself is much larger than other viruses, being more than 10 times the size of the circovirus and 4 to 5 times the size of the PRRS virus. Because the African swine fever virus is large and complex, it is difficult to immunize against it, and an effective vaccine has not yet been developed.

Diagnosis is also very important because it is a high-risk infectious disease that requires pigs to be disposed of as soon as they occur. To confirm the diagnosis of African swine fever, diagnostic testing by an accredited laboratory is essential. Laboratory diagnostic tests detect ASFV in the blood and internal organs or detect antibodies in the serum of infected pigs. Since a vaccine has not yet been developed, a positive antibody test indicates field infection. If the virus needs to be isolated from blood, a blood sample collected at the time the initial fever symptoms are confirmed is suitable, and organs such as lymph nodes and spleen should be refrigerated. The ideal sample for testing antibodies is serum from an infected pig in the recovery period between 8 and 21 days after infection. The first screening test for antibodies using serum from infected pigs is OIE-ELISA (indirect method) and a commercially available ELISA for antibody detection, followed by immunorubbing (IB), immunoperoxidase test (IPT), and indirect fluorescence. Final evaluation can be performed through the antibody method (IFA) (see Non-Patent Document 2). ASF disease progresses rapidly and in various stages, and asymptotically infected pigs may also exist, so the laboratory must perform both antigen and antibody tests to accurately determine infection.

Accordingly, there is an urgent need to develop commercially available and effective ASFV infection prevention vaccines and ASFV infection diagnosis-related technologies.

Jiyeon Lee, 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 present inventors have made diligent efforts to provide a commercially available ASFV infection prevention vaccine and an ASFV infection diagnosis method. As a result, the polypeptide consisting of the unique sequence provided by the present invention is superior to the natural protein in ASFV. The present invention was completed by confirming that the ASFV infection serum had a significantly superior detection/diagnosis ability, had the potential to be used as a vaccine, and had high industrial productivity.

Therefore, the object of the present invention is to provide an isolated polypeptide consisting of the 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 containing the polynucleotide, and a host cell containing the expression vector.

Another object of the present invention is to provide a composition for a vaccine against African swine fever virus containing the 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 animals, comprising administering an immunologically effective amount of the polypeptide to the animal. .

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 of antibodies against the African swine fever virus containing the polypeptide as an active ingredient and a diagnostic kit containing the diagnostic reagent.

Another object of the present invention is the step of (a) contacting an animal sample with the polypeptide of claim 1; and (b) detecting the presence of an antibody bound to the polypeptide of claim 1 in the sample.

In order to achieve the above object, the present invention provides an isolated polypeptide consisting of the 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 containing the polynucleotide, and a host cell containing 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 containing the above polypeptide as an active ingredient.

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

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 of antibodies to the African swine fever virus containing the polypeptide as an active ingredient and a diagnostic kit containing the diagnostic reagent. .

In order to achieve another object of the present invention, the present invention includes the steps of (a) contacting an animal sample with the polypeptide of claim 1; and (b) detecting the presence of an antibody bound to the polypeptide of claim 1 in the sample.

Hereinafter, the present invention will be described in detail.

In the present invention, the term 'antigen' refers to an antigen that, upon introduction into contact with an appropriate cell, induces a state of sensitivity and/or immunoreactivity and interacts in a demonstrable manner with immune cells and/or antibodies of the subject so sensitized in vivo or in vitro. Refers to all substances that react. In the present invention, the term 'antigen' may be used collectively with the same meaning as the term 'immunogen', and preferably promotes the host immune system to produce a secretory, humoral and/or cellular immune response specific to the antigen. refers to a molecule containing one or more epitopes capable of Additionally, in the present invention, the term 'antigenicity' or 'immunogenicity' refers to the property of the antigen or immunogen, and refers to the property of causing a secretory, humoral and/or cellular immune response.

The term 'immune response' is a self-defense system that exists in the animal's body, and is a biological phenomenon that distinguishes various substances or living things invading from the outside from itself and eliminates these invaders. This surveillance system for self-defense is largely accomplished by two mechanisms: one is humoral immunity, and the other is cellular immunity. Humoral immunity is achieved by antibodies present in serum, and antibodies have an important function of binding to and eliminating invading foreign antigenic substances. Meanwhile, cellular immunity is achieved by several types of cells belonging to the lymphatic system, and these cells are responsible for directly destroying invading cells or tissues. Thus, humoral immunity is mainly effective against external substances such as bacteria, viruses, proteins, and complex carbohydrates that exist 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. B cells produce antibodies, and T cells participate in cellular immunity. The immune response by B cells or T cells is an immune system that responds to antigens that have once invaded the body, but only acts when the same type of antigen continues to exist or has invaded repeatedly. Therefore, this immune response is a specific response to a specific antigen. In addition to these antigen-specific immune responses, there is also a type of natural immune response in the body that directly reacts and destroys attacking cells even when the body has not been exposed to an antigen. This immune response includes neutrophil, macrophage, NK (natural killer) cells, etc. Its characteristic feature is that it exerts a variety of functions regardless of the type of cell being attacked.

Preferably, the target immune response in the present invention is antibodies, B cells, helper T cells, suppressor T cells, cytotoxic T cells, and gamma-delta T cells specifically directed against the antigen or antigens contained in the vaccine. Production or activation of , producing a therapeutic or protective immunological response in the host, including, but not limited to, one or more of the effects of enhancing resistance to new infections or reducing the clinical severity of the 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 or lower duration of recovery, or lower viral titers in the tissues or body fluids or excretions of the infected host.

As used herein, the terms 'polypeptide' and 'protein' are used according to their usual meaning, that is, they mean a polymer of amino acid residues. A polypeptide is not limited to a particular 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 modifications and non-naturally occurring modifications). Polypeptides and proteins of the present invention can be produced using any of a variety of known recombinant and/or synthetic techniques.

As used herein, the single letter (triple letter) of amino acids refers to the following amino acids according to standard abbreviation conventions in the field of biochemistry: 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.

As used herein, the terms 'polynucleotide' and 'nucleic acid' 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.

As used herein, the term 'expression' refers to the production of a protein or nucleic acid in a cell.

The present inventors have developed the Kenya-derived ASFV p30 protein in terms of detection/diagnosis of ASFV infection (i.e., antibodies against ASFV in serum), its usability as a vaccine, and the possibility of mass production for commercial distribution due to its excellent expression level, purification efficiency, and stability. A polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 was newly identified as a fragment with excellent advantages in such areas. The polypeptide is characterized by having a significantly superior effect in the above-mentioned aspects even compared to the natural 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 consisting of the amino acid sequence represented by SEQ ID NO: 1.

The polypeptide of the present invention is characterized by immunogenicity against African swine fever virus (ASFV), and is preferably characterized by specific immunogenicity against ASFV originating from Kenya. On the other hand, the polypeptide of the present invention has a wide range of universal immunogenicity, reacting with ASFV specimens not only from ASFV type 6, which occurs in Africa, but also ASFV type 2, which occurs in Russia, Georgia, etc., and recently, which occurs in Jung-gu and Asia. It is characterized by having

The polypeptides described herein may be prepared (manufactured) 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 its functional equivalent is prepared according to a conventional method. The nucleic acid can be produced by PCR amplification using appropriate primers. Alternatively, DNA sequences can be synthesized using standard methods known in the art, such as automated DNA synthesizers (available from Biosearch or Applied Biosystems). The prepared nucleic acid is inserted into a vector containing one or more expression control sequences (e.g., promoter, enhancer, etc.) that are operatively linked to control the expression of the nucleic acid, and the recombinant formed therefrom is inserted. Transform host cells with expression vectors. The resulting transformant is cultured under media and conditions appropriate for expression of the nucleic acid, and a substantially pure polypeptide expressed by the nucleic acid is recovered from the culture. The recovery can be performed using methods known in the art (eg, chromatography). In the above, 'substantially pure polypeptide' means that the polypeptide according to the present invention substantially does not contain any other proteins derived from host cells.

For genetic engineering methods for polypeptide synthesis of the present invention, refer to the following literature: 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.

Additionally, 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, polypeptides of the invention may be prepared by direct peptide synthesis using solid phase techniques (Merrifield, J. Am. Chem. Soc. 85:2149-2154 (1963)). The solid-phase peptide synthesis (SPPS) method can initiate synthesis by attaching functional units called linkers to small porous beads to connect the peptide chain. Unlike the liquid phase method, the peptide is covalently bonded to the bead and is prevented from falling off during the filtration process until it is cleaved by a specific reactant such as TFA (trifluoroacetic acid). The protection process where the N-terminal amine of the peptide attached to the solid phase binds to the N-protected amino acid unit, the deprotection process, the re-revealed amine group and the new Synthesis occurs by repeating the cycle of coupling process (deprotection-wash-coupling-wash) in which amino acids combine. The SPPS method can be performed using microwave technology, which 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 folding or aggregation of the extended peptide chain and promote chemical bonding.

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

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

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

In one embodiment, the functional equivalent in the present invention may be one produced by addition, insertion, substitution (non-conservative or conservative substitution), deletion, or a combination thereof of some of the amino acid sequences of the polypeptide of the present invention described above. there is. In the above, amino acid substitution 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 overall alter the activity of the molecule are known in the art (H.Neurath, R.L.Hill, The Proteins, Academic Press, New York, 1979). The functional equivalents also include variants in which some amino acids are deleted in the amino acid sequence of the polypeptide of the present invention. The deletion or substitution of the amino acid is preferably located in a region that is not directly related to the physiological activity (chemokine activity) of the polypeptide provided by the present invention. In addition, variants in which several amino acids are added to both ends of the amino acid sequence of the polypeptide of the present invention or within the sequence are also included. The amino acid added above may be, for example, the '-GSHHHHHH' sequence as a histidine tag for protein separation/purification, and the fragment containing this is preferably a polypeptide having the amino acid sequence of SEQ ID NO: 3. It can be.

The scope of the functional equivalents also includes polypeptide derivatives in which part of the chemical structure of the polypeptide is modified while maintaining the basic skeleton of the polypeptide and its physiological activity. For example, this includes structural changes to change the stability, storage, volatility, or solubility of the polypeptide of the present invention, and fusion proteins made by fusing with other proteins while maintaining physiological activity.

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

Additionally, the present invention provides a polynucleotide encoding the polypeptide of the present invention.

The base combination of the polynucleotide is not particularly limited 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 a double strand, including all DNA, cDNA, and RNA sequences.

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

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

The polypeptide of the present invention can be provided by operably linking the 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 containing the above polynucleotide.

In the present invention, the term 'expression vector' or 'recombinant vector' refers to a vector capable of expressing a target protein or target RNA in a suitable host cell, and refers to a gene construct containing essential regulatory elements operably linked to express the gene insert. .

The term 'operably linked' refers to a functional linkage between a nucleic acid expression control sequence and a nucleic acid sequence encoding a desired protein or RNA to perform a general function. For example, a promoter and a nucleic acid sequence encoding a protein or RNA can be operably linked to affect expression of the encoding nucleic acid sequence. Operational linkage with a recombinant vector can be made using genetic recombination techniques well known in the art, and site-specific DNA cutting and ligation can be done using enzymes generally known in the art.

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

The signal sequence includes the PhoA signal sequence and OmpA signal sequence when the host is a genus Escherichia ( Escherichia sp.), and the α-amylase signal sequence and subtilisin signal sequence when the host is a bacterium of the genus Bacillus. In the case of yeast, the MFα signal sequence, SUC2 signal sequence, etc. can be used, and if the host is an animal cell, the insulin signal sequence, α-interferon signal sequence, antibody molecule signal sequence, etc. can be used, but are not limited thereto.

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

The transformation can be performed by any method known to be capable of introducing nucleic acids into an organism, cell, tissue or organ, and can be performed by selecting an appropriate standard technique depending on the host cell, as known in the art. . These methods include microprojectile bombardment, electroporation, protoplast fusion, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, agitation using silicon carbide fibers, agrobacteria-mediated transformation, Includes, but is not limited to, PEG-mediated fusion, microinjection, liposome-mediated method, dextran sulfate, lipofectamine, heat shock method, etc.

The term 'transformant' can be used interchangeably with 'host cell', etc., and can be introduced into a cell by any means (e.g. electric shock method, calcium phosphatase precipitation method, microinjection method, transformation method, virus infection, etc.). refers to a prokaryotic or eukaryotic cell containing heterologous DNA.

In the present invention, the transformant includes all types of single-celled organisms 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 insect cells. , cells derived from higher eukaryotes, including plant cells, mammals, etc., can be used as host cells, but are not limited thereto. Since the expression level and modification of proteins varies 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 refer to a transformed microorganism. Specifically, but not limited thereto, host cells include, for example, Escherichia coli, Bacillus subtilis , Streptomyces , Pseudomonas , and Proteus mirabili. It may be a prokaryotic host cell such as Proteus mirabilis or Staphylococcus . Additionally, fungi (e.g., Aspergillus ), yeast (e.g., Pichia pastoris , Saccharomyces cerevisiae ), Schizosaccharomyces ), Neurospora crassa ( Neurospora crassa ), etc. Cells derived from higher eukaryotes including lower eukaryotes, insect cells, plant cells, mammals, etc. can be used as host cells, but are not limited thereto.

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, but for example, Rosetta2 (DE3), C41 (DE3), SoluBL21, etc. may be used.

Additionally, 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 same meaning as an immunogenic composition. The vaccines include both preventive and therapeutic vaccines. Prophylactic vaccines induce an immune response before exposure to a substance containing the antigen, in order to mount a greater immune response when the subject is exposed to the antigen, thus increasing the ability to resist substances or cells carrying the antigen. means that Therapeutic vaccines are used by administering to an individual who already has a disease related to the antigen of the vaccine. The therapeutic vaccine stimulates the individual's immune response to the antigen by providing an increased ability to fight the disease or cells carrying the antigen. can be increased.

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 to this, but may include administering the vaccine via transdermal, intramuscular, intraperitoneal, intravenous, or subcutaneous routes. Preferably, the vaccine may be administered intramuscularly during the first and second vaccinations.

The vaccine may be in any form known in the art, for example, a solid form suitable for liquid and injection form or suspension, but is not limited thereto. These agents can also be emulsified or encapsulated in liposomes or fusible glasses or prepared in aerosol or spray form. They can also be incorporated into transdermal patches. In the case of liquid or injectable formulations, if necessary, they may contain propylene glycol and a sufficient amount (e.g., about 1%) of sodium chloride to prevent hemolysis.

The vaccine composition of the present invention is characterized by comprising the polypeptide of the present invention described above, and may further include one or more agents selected from the group consisting of pharmaceutically acceptable carriers, diluents, and adjuvants. As used herein, “pharmaceutically acceptable” means a non-toxic composition that is physiologically acceptable and does not inhibit the action of the active ingredient and does not usually cause gastrointestinal upset, allergic reactions such as dizziness, or similar reactions when administered to humans. says

The carrier refers to a substance that facilitates delivery of the target into cells or tissues. 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 solutions, aqueous glucose, glycols, etc. Additionally, the carrier may include dry formulations such as coated patches made of titanium or polymers. 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, or a non-aqueous solution, suspension or emulsion.

Additionally, structured or amorphous organic or inorganic polymers may be used as adjuvants to increase immunogenicity in the composition of the present invention. Immune adjuvants are generally known to play a role in promoting immune responses through chemical and physical binding to antigens. The adjuvants used in this study included amorphous aluminum gel, oil emulsion, double oil emulsion, and immunosol. Additionally, various plant-derived saponins, levamisole, CpG dinucleotide, RNA, DNA, LPS, and various types of cytokines were used to promote immune responses. The above immune composition can be used as a composition for inducing an optimal immune response by combining various adjuvants and additives that promote immune response.

These 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-acetylmuramyl-L-alanyl-D-isoglutamine (MDP), lipopolysaccharide (LPS), monophosphoryl lipid A, trehalose dimycholate (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. may be included.

Additionally, compositions that can be added to the vaccine include stabilizers, inactivators, antibiotics, preservatives, etc. Depending on the route of administration of the vaccine, vaccine antigens may be mixed with distilled water, buffer solutions, etc.

Other pharmaceutically acceptable carriers and agents may be referred to as described in the following literature (Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995). The formulation and administration techniques of the vaccine described herein can be provided by reference to the literature (Remington, The Science and Practice of Pharmacy, 22nd edition), etc.

Additionally, the vaccine composition containing 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 of inducing a protective immune response against African swine fever in animals, comprising administering to a pig an immunologically effective amount of the polypeptide of the present invention described above. .

The ‘subject’ may refer to an animal, preferably a mammal other than a human. In one embodiment, the subject may preferably be a pig.

Accordingly, in one embodiment, the present invention provides a method for enhancing immunity to ASFV in animals (particularly pigs), comprising administering the polypeptide of claim 1 to the animal (particularly, pigs) (i.e., to ASFV Provides a method for immunizing animals against

As used herein, the term 'immunization' refers to the induction of a secretory, humoral and/or cellular immune response to the immunogenic complex protein in the subject when the immunogenic complex protein according to the present invention is administered to the subject. In other words, such immunization produces a preventive or therapeutic effect on the target disease (in particular, ASFV infection in the present invention).

The 'effective amount' is an amount that shows the preventive or therapeutic effect of the vaccine composition of the present invention on the target disease (particularly, ASFV infection), and the polypeptide of the present invention is secreted, humoral and/or cellular in the administered subject. This refers to an amount sufficient to induce a sexual immune response.

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

The above-described polypeptide of the present invention has a remarkable effect in specifically detecting (diagnosing) ASFV infection serum, even when compared to the native protein and polypeptides of different length/sequence composition derived from the protein. In the present invention, detecting (diagnosing) ASFV infection serum means that the polypeptide of the present invention binds to the anti-ASFV antibody (antibody against African swine fever virus) present in the blood, plasma, or serum of an individual (antigen- Antibody complex formation) means confirming the presence or/and amount of the antibody. If the presence of anti-ASFV antibodies is detected or/and confirmed in the blood, plasma, or serum of an individual, the individual may 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 antibodies against African swine fever virus containing the above-described polypeptide as an active ingredient, and a diagnostic kit containing the diagnostic reagent composition.

Additionally, the present invention

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

(b) It provides a method for detecting (diagnosing) serum of African swine fever virus infection, including the 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 from Kenya but also recently emerging ASFV. Therefore, in one embodiment, the present invention preferably provides a diagnostic reagent composition for determining the presence or absence of antibodies against African swine fever virus containing the above-described polypeptide of the present invention as an active ingredient, and a diagnostic kit containing the same. It may also be to provide a method for detecting (diagnosing) ASFV infected serum including the steps (a) and (b).

As used herein, the term 'sample' may be a solid sample or a humoral 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 application, the method and device may be applied without particular limitation as long as it is known in the art as a means for detecting an antigen-antibody complex. For example, but not limited to, antigen-antibody complexes can be tested using radial immunodiffusion, immunoprecipitation assays including immunoelectrophoresis or reverse current electrophoresis, radioimmunoassay (RIA), and competitive indirect immunofluorescent assay. ), ELISA (Enzyme Linked Immunosorbent Assay), or immunochromatic assay. Detection in this manner 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 application, an ELISA method, particularly a sandwich-type ELISA, is used, and in this case, a detection antibody described later is also used. The present invention can be understood as providing 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 application, for detection of an antigen (polypeptide of the present invention)-antibody complex, the antigen (polypeptide of the present invention) may be labeled with a labeling substance. That is, the polypeptide of the present invention may be provided linked (e.g., covalently linked or cross-linked) to a detectable label. The detectable labels include chromogenic enzymes (e.g. peroxidase, alkaline phosphatase), radioisotopes (e.g. 124I, 125I, 111In, 99mTc, 32P, 35S), and chromophores. , biotin, luminescent or fluorescent substances (e.g. FITC, RITC, rhodamine, Texas Red, fluorescein, phycoerythrin, quantum dots) ), magnetic resonance imaging contrast agents (e.g., superparamagnetic iron oxides (SPIO), ultrasuperparamagnetic iron oxides (USPIO)), gold particles, 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 additionally performed on an already synthesized polypeptide.

In one embodiment according to the present application, 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 application specifically binds to immunoglobulin (e.g., IgM, IgG) produced in the animal to be diagnosed, and the detection antibody detects visually or various images. It can be labeled as a substance that can be detected using equipment. This can be understood with reference to the above description of the polypeptide labeling material of the present invention.

In one specific embodiment, the antigen (polypeptide of the present invention) or detection antibody (secondary antibody) according to the present application may be a peroxidase such as horseradish peroxidase or alkaline phosphatase as a labeling substance. phosphatase, glucose oxidase, beta-galactosidase, urease, catalase, asparginase, ribonuclease, horse malate dehydrogenase, staphylococcal nuclease, triose phospate isomerase, glucose-6-phosphate dehydrogenase , glucoamylase, and acetylcholine esterase, which can catalyze a chemical reaction in the presence of a specific substrate and emit a detectable color reaction or light. It is not limited.

In another specific embodiment, the antigen (polypeptide of the present invention) or detection antibody according to the present application is viroluminescence, chemiluminescence, or electroluminescence that emits light of a different wavelength from the light irradiated by light irradiation. Examples of chromophores used in photoluminescence, electrochemiluminescence, and photoluminescence include green fluorescent protein; Organic compounds include fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, and fluorecamine. Including but not limited to this.

In another specific embodiment, the antigen (polypeptide of the invention) or detection antibody according to the present disclosure may be labeled with various radioisotope substances. Detection of the labeling material herein can be performed, for example, by a scintillation counter if the labeling material is a radioisotope, and for example, if the labeling material is a fluorescent material, it can be performed by spectroscopy, a phosphoimaging device, or a fluorescence meter. It can be done by the same method. In the case of labeling with an enzyme, this can be performed by measuring the chromogenic product resulting from conversion of the chromogenic substrate by the enzyme in the presence of an appropriate substrate. In addition, it can be detected by comparing the color of the colored product produced by the enzyme reaction through comparison with an appropriate standard or control group.

In a specific embodiment, the substance that labels the antigen (polypeptide of the present invention) or detection antibody according to the present application may include, for example, a chromophore; Enzymes including alkaline phosphatase, biotin, beta-galactosidase or peroxidase; radioactive substances; or materials containing nanoparticles such as colloidal gold particles or colored latex particles, but are not limited thereto.

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

Additionally, the polypeptide of the present invention may be provided in a form coated on the surface of a plate. In this case, after treating the sample on the plate and reacting under appropriate conditions, ASFV infection can be diagnosed by observing the binding of the polypeptide (antigen) of the present invention on the surface of the plate and the antibody in the sample. The polypeptide of the present invention can be used in a microwell plate such as a 96-well microwell plate, beads or particles containing colloidal gold particles or colored latex particles, or cellulose, nitrocellulose, polyethersulfone, polyvinylidine, fluoride, It may be provided attached to a membrane such as nylon, charged nylon, and polytetrafluoroethylene. The method for attaching or coating the antigen (polypeptide of the present invention) can be a known method, for example, refer to the method described in the Examples herein.

In a specific embodiment, the diagnostic reagent of the present invention, and a kit containing 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 sample to an antigen bound to a solid substrate, such as beads, membranes, slides, or microwell plates made of glass, plastic (e.g., polystyrene), polysaccharide, nylon, or nitrocellulose. , labels capable of direct or indirect detection, such as radioactive substances such as 3H or 125I as described above, fluorescent substances, chemiluminescent substances, haptens, biotin, digoxigenin, etc., or are colored through interaction with a substrate. Alternatively, it can be detected qualitatively or quantitatively through binding to an antibody conjugated to an enzyme such as horseradish peroxidase, alkaline phosphatase, and malate dehydrogenase that can emit light. Additionally, 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 are reagents capable of detecting bound antibodies, e.g. secondary detection antibodies labeled with the above-mentioned substances such as chromophores, enzymes (e.g. conjugated with antibodies), and the substrate used for detection. etc. may be additionally included.

The polypeptide composed of the unique sequence provided by the present invention has significantly superior expression and purification yield compared to the native protein in the virus, is short in length, and is useful for detecting/diagnosing ASFV infection serum even compared to the native protein in ASFV. Not only is its ability significantly superior, but it also has the potential to be used as a vaccine and is highly productive at the industrial level.

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

Hereinafter, the present invention will be described in detail.

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

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

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

Various polypeptide fragments were prepared based on the p30 protein (also referred to as ASFV-p30 herein) sequence consisting of the amino acid sequence shown in SEQ ID NO: 5 obtained from the Pig/Kenya/KEN-50/1950 ASFV strain, It is shown in [Table 1]. We sought to overcome the difficulties in producing recombinant proteins at a level for commercial use.

pep. fragment name Sequence information sequence number Kenya Asfv-p30 W/T STKKKPTITKQELYSLVAADTQLNKALIERIFTSQQKIIQNALKHNQEVIIPPGIKFTVVTVKAKPARQGHNPATGEPIQIKAKPEHKAVKIRALKPVHDMLN SEQ ID NO: 5 Asfv-p30 TKQELYSLVAADTQLNKALIERIFTSQQKIIQNALKHNQEVIIPPGIKFTVVTVKAKPARQGHNPATGEPIQIKAKPEHKA SEQ ID NO: 1

The proteins and polypeptides were produced briefly as follows. DNA encoding Kenya Asfv-p30 W/T polypeptide (SEQ ID NO: 6) was synthesized by Macrogen. DNA encoding Asfv-p30 polypeptide (SEQ ID NO: 4) was synthesized by Macrogen. The polynucleotide encoding each fragment polypeptide was cloned between the Nde1 and BamH1 restriction sites of the pET49b vector (Novagen). Each polypeptide was overexpressed in BL21 strain, an Escherichia coli strain. E. coli cells transformed with the above vector were grown at 37°C using Luria-Bertani (LB) medium containing 100 ug/ml kanamycin until OD600 was 0.7, and 1 mM isopropyl β-D-1. Protein expression was induced by -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 recover the cells. The recovered 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), the cells were broken by sonication, and finally the cells were dissolved in denaturation buffer (6 M Guanidine Hydrochloric Acid). After resuspending in a denaturation buffer containing 0.1 M acid, pH 8.0 Tris, and 2.5 mM Ethylenediaminetetraacetic acid, pH 8.0, the cells were disrupted by sonication. In the case of Kenya Asfv-p30 W/T, protein expression could not be confirmed with the naked eye. In the case of Asfv-p30, centrifuge at 15,000 rpm and measure the concentration at A280, then add the supernatant to a snake skin tube and add 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) and diluted to 1 mg/ml (10℃, 36 hrs). After refolding, transfer to a snake skin tube, add Urea buffer (20mM Tris pH 8.0, 100mM Urea) twice for 12 hours at 4℃, and add 1 Put it in. This was centrifuged again at 5,000 rpm for 20 minutes, and then the supernatant was treated with Ni-NTA and bound for 1 hour and 30 minutes (4°C). After washing using 1X PBS buffer containing 50mM Imidazole, eluted using 1XPBS buffer containing 250mM Imidazole and 500mM Imidazoe. Polypeptides were confirmed using SDS-polyacrylamide gel electrophoresis, and the polypeptides were stored in a buffer containing 20mM HEPES and 150mM sodium chloride at pH 7.4 (4°C, overnight).

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

The productivity of the various fragment polypeptides was quantitatively compared and evaluated. Another fragment polypeptide (Kenya Asfv-p30 W/T) was not expressed in the E. coli cell line. However, Asfv-p30 polypeptide was successfully expressed in E. coli and could be purified to a high degree of 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 (to obtain the protein) was 2.5. It was one to three days. Asfv-p30 was successfully expressed in an E. coli cell line that has the advantage of being efficiently produced in a short period of time, and not only did it show a high yield that could be used commercially during the purification process, but the purification period took less than 3 days. The native Kenya Asfv-p30 W/T protein was not expressed in E. coli. As such, the Asfv-p30 (SEQ ID NO: 1) polypeptide fragment of the present invention can be purified in high yield from E. coli cell lines and shows the advantage of a short purification process (see Figures 1 and 2).

1-3. Comparative evaluation of ASFV infection blood test diagnostic ability of fragment polypeptide compared to native p30 protein

Using ID.vet's ID Screen® African Swine Fever Indirect Screening test kit, the binding ability of various fragment polypeptides prepared in Example 1-1 to antibodies in ASFV infection serum was tested using ID (indirect)-ELISA. Methods were compared and evaluated. Briefly, it was carried out in the following manner. First, Coating buffer (0.015 M Sodium carbonate, 0.035 M Sodium bicarbonate, Final pH 9.6) and each antigen (various fragment polypeptides prepared in Example 1-1, 2 ug/ml or (added at a concentration of 4 ug/ml) was added and incubated overnight (16h) at 4°C to coat each well with each antigen. Each well was washed 4 times using 200 ul of PBST buffer (1XPBS + Tween20 0.05%). 100 ul of primary antibody was added to each well and incubated for 1 hour at room temperature (22°C). The primary antibody was treated with ASFV-infected pig serum provided as a positive control in ID.vet's African Swine Fever Indirect Screening test kit. 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. After adding 100 ul of Substrate solution 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 optical density was measured at 450 nm.

As a result of the experiment, as shown in Figure 3, the fragment polypeptide of Asfv-p30 (SEQ ID NO: 1) produced and purified from E. coli has a very superior level (~4 It showed high reactivity to ASFV-infected serum (more than twice as much). In addition, it showed more than three times higher reactivity compared to the commercial diagnostic reagent provided by ID.vet.

As discussed above, the present invention relates to the development and use of a p30 protein fragment derived from African swine fever virus (ASFV) as a recombinant antigen, and more specifically, to the use of an amino acid sequence represented by SEQ ID NO: 1. It relates to an isolated polypeptide, a vaccine composition against ASFV containing the polypeptide as an active ingredient, an animal immunization method using the polypeptide, and a method for detecting/diagnosing ASFV infection using the polypeptide, diagnostic reagents, and kits. The polypeptide consisting of the unique sequence provided by the present invention is shorter in length compared to the native protein in the virus, and has a significantly better ability to detect/diagnose ASFV infection serum compared to the native protein in ASFV, as well as a vaccine. It has the potential to be used as a product, and its productivity is high at the industrial level, so it has great potential for industrial use.

<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 consisting of the amino acid sequence represented by SEQ ID NO: 1.
The isolated polypeptide according to claim 1, wherein the polypeptide is immunogenic against African swine fever virus (ASFV).
The isolated polypeptide according to claim 2, wherein the ASFV is from Kenya.
A polynucleotide encoding the polypeptide of claim 1.
An expression vector containing the polynucleotide of claim 4.
A host cell containing the expression vector of claim 5.
A composition for a vaccine against African swine fever virus comprising the polypeptide of claim 1 as an active ingredient.
The vaccine composition according to claim 7, wherein the composition further comprises one or more substances selected from the group consisting of pharmaceutically acceptable carriers, diluents and adjuvants.
A method of inducing a protective immune response against African swine fever in an animal, comprising administering an immunologically effective amount of the polypeptide of claim 1 to the animal.
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 of antibodies against African swine fever virus, comprising the polypeptide of claim 1 as an active ingredient.
A diagnostic kit containing the diagnostic reagent of claim 11.
(a) contacting an animal sample with the polypeptide of claim 1; and
(b) A method for detecting African swine fever virus infection serum comprising the step of detecting the presence of an antibody bound to the polypeptide of claim 1 in the sample.