KR102433180B1 - p205 protein fragment derived from African swine fever virus as recombinant antigen, and uses thereof - Google Patents

Abstract

The present invention is an isolated polypeptide comprising an amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 as an isolated polypeptide comprising the first to 120th amino acid sequence from the protein represented by SEQ ID NO: 5, the polypeptide is effective Provided are a vaccine composition for African swine fever virus (ASFV) comprising as a component, an animal immunization method using the polypeptide, a method for detecting/diagnosing ASFV infection using the polypeptide, a diagnostic reagent, and a kit.

Description

African swine fever virus-derived p205 protein fragment and uses thereof {p205 protein fragment derived from African swine fever virus as recombinant antigen, and uses thereof}

The present invention relates to a p205 protein fragment derived from African swine fever virus (ASFV) and uses thereof, and more particularly, an isolated isolated comprising the amino acid sequence of 1 to 120 in the protein represented by SEQ ID NO: 5 An isolated polypeptide consisting of an amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 as a polypeptide, a vaccine composition against ASFV comprising the polypeptide as an active ingredient, a method for animal immunization using the polypeptide, and the polypeptide It relates to a method, diagnostic reagent and kit for detecting/diagnosing ASFV infection using

African swine fever (ASF) is a swine epidemic caused by infection with African swine fever virus (ASFV) belonging to the Asfarviridae family. The occurrence has been reported in sub-Saharan Africa, and since 2007, the range of occurrence has started to increase in areas other than Africa such as Georgia, Armenia, and Azerbaijan along the Black Sea coast. In particular, recently, the outbreak is spreading from east to west in Russia, and the number of cases in China and Southeast Asia, where there are many exchanges with Korea, is increasing rapidly.

African swine fever virus is very resistant to the general environment and maintains its infectivity at room temperature for more than 18 months and meat products such as ham for more than 6 months. It has (non-patent document 1).

Although African swine fever is a serious disease with a mortality rate of 100% when infected with pigs, there is no commercially available vaccine for this disease. As African swine fever spreads again in Europe and spreads to the Asian continent, attention is focused on research on vaccine development, which has not been well progressed in the past. However, due to safety concerns, the development of live attenuated vaccines is limited, and previous experimental studies did not achieve great results. There are many obstacles to overcome in developing a product that is effective for defense against the dead poison vaccine. For example, ASFV has a larger and more complex structure than other viruses, so it is difficult to find an effective site for defense. The gene size of 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 it is known that the ASFV genome normally encodes at least 151 ORFs. African swine fever virus has a large number of structural proteins and the virus itself is much larger than other viruses, making it 10 times larger than circovirus and 4 to 5 times larger than PRRS virus. Because the African swine fever virus is large and complex, immunity is difficult, so an effective vaccine has not yet been developed.

Diagnosis is also very important because it is a high-risk infectious disease that requires the disposal of pigs as soon as they occur. A diagnostic test by an accredited laboratory is essential to confirm the diagnosis of African swine fever. Laboratory diagnostic tests include methods of 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 antibody test indicates outdoor infection. If it is necessary to isolate the virus from the blood, specimens collected at the time fever symptoms are confirmed are suitable. Ideally, the specimen for antibody testing is serum from infected pigs in the recovery phase between 8 and 21 days after infection. The antibody detection method using the serum of infected pigs is the OIE-ELISA (indirect method) and the commercialized ELISA for antibody detection. It can be finally evaluated through the antibody method (IFA) (refer nonpatent literature 2). In SF, the disease progresses rapidly and varies, and there may be inactively infected pigs. Therefore, the laboratory must perform antigen and antibody tests together to accurately determine whether or not the infection is present.

Accordingly, there is an urgent need to develop a commercially available and effective vaccine for preventing ASFV infection and technology related to diagnosis of ASFV infection.

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 intensive efforts to provide a commercially available vaccine for preventing ASFV infection and a method for diagnosing ASFV infection. The present invention was completed by confirming that the ASFV-infected serum detection/diagnostic ability is remarkably excellent, has potential for use as a vaccine, and has high productivity at an industrial level.

Therefore, an object of the present invention, as an isolated polypeptide comprising the first to 120th amino acid sequence in the protein represented by SEQ ID NO: 5, the isolated polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 will provide

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 vaccine composition against African swine fever virus comprising the polypeptide as an active ingredient.

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

Another object of the present invention is to provide a diagnostic reagent for determining the presence of an antibody to African swine fever virus containing the polypeptide as an active ingredient, and a diagnostic kit including the diagnostic reagent.

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

In order to achieve the above object, the present invention is an isolated polypeptide comprising the first to 120th amino acid sequence in the protein represented by SEQ ID NO: 5, consisting of the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 Isolated polypeptides are provided.

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 vaccine composition against African swine fever virus comprising the 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 an animal, comprising administering an immune effective amount of the polypeptide to the animal. A method for inducing an immune response is provided.

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

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

Hereinafter, the present invention will be described in detail.

In the present invention, the term 'antigen' induces a sensitive and/or immune-reactive state as it comes into contact with appropriate cells, and in a way that can be demonstrated with the immune cells and/or antibodies of the sensitized subject in vivo or in vitro. It refers to any substance that reacts. In the present invention, the term 'antigen' may be collectively used with the same meaning as the term 'immunogen', and preferably promotes the host immune system to generate a secretory, humoral and/or cellular immune response specific to the antigen. It refers to a molecule containing one or more epitopes capable of Also, 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 reaction' refers to a self-defense system that exists in an animal's body, and is a biological phenomenon in which various substances or living organisms that invade from the outside are distinguished from themselves and the invaders are removed. The surveillance system for this self-defense is mainly composed of two mechanisms, one is humoral immunity and the other is cellular immunity. Humoral immunity is made by antibodies present in serum, and the antibodies play an important role in binding to and removing invading foreign antigenic substances. On the other hand, cellular immunity is made 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 existing outside cells, and cellular immunity exerts its functions on various parasites, tissues, intracellular infections, and cancer cells. This double defense system is mainly performed 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 these B cells or T cells is an immune system that responds to an antigen that once invaded the body, but acts when the same type of antigen continues to exist or invades repeatedly. Thus, this immune response is a specific response to a specific antigen. In addition to these antigen-specific immune responses, there is also a kind of natural immune response that directly reacts and destroys the attacking cells even when the body has never been exposed to any antigen. It is characterized in that it exerts various functions regardless of the type of attack target cell by this involvement.

Preferably, the immune response desired in the present invention is an antigen or antibodies specifically directed against antigens included in the vaccine, B cells, helper T cells, suppressor T cells, cytotoxic T cells and gamma-delta T cells. production or activation of, and exhibiting a therapeutic or protective immunological response in the host, thereby enhancing resistance to new infections or reducing the clinical severity of the disease. Preferably it may be a protective immune response.

Such protection is evidenced by a reduction or absence of clinical signs normally exhibited by an infected host, a faster recovery time or lower duration, or a lower viral titer in the tissues or body fluids or feces of the infected host.

As used herein, the terms 'polypeptide' and 'protein' are used according to their conventional (conventional) meaning, that is, they refer to 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 contain post-translational modifications, such as glycosylation, acetylation, phosphorylation, etc., and other modifications known in the art (naturally occurring and non-naturally occurring modifications). The polypeptides and proteins of the present invention can be prepared using any of a variety of known recombinant and/or synthetic techniques.

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 those of naturally occurring nucleotides are also included.

One letter (three letters) of amino acids as used herein refers to the following amino acids according to standard abbreviation rules 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 term 'expression' refers to the production of a protein or nucleic acid in a cell.

The present inventors have found that from the pK205R protein of ASFV derived from Italy, ASFV-infected serum (ie, antibody to ASFV in serum) detection/diagnostic aspect, availability as a vaccine, and mass production possibility for commercial dissemination, etc. Fragments having excellent advantages As a new polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, respectively. The polypeptides are characterized in that they have significantly superior effects in the above-described aspects even compared to the native proteins from which they are derived and polypeptides (fragments) of different lengths and sequences derived from the protein.

Accordingly, the present invention provides an isolated polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2. The polypeptide of the present invention is characterized by immunogenicity against African swine fever virus (ASFV), and more preferably, it is characterized in that it has specific immunogenicity from Italy. On the other hand, the polypeptide of the present invention is characterized in that it also has broad immunogenicity that reacts with African swine fever specimens caused by ASFV type 2 infection that has recently occurred in Russia, China, etc.

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

The genetic engineering method for synthesizing the polypeptide of the present invention may be referred to the following documents: 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 to recombinant production methods, the polypeptides of the present invention can also be prepared by chemical synthesis methods known in the art. 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, a polypeptide 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 and inducing them to continue peptide chains. Unlike the liquid phase method, the peptide is covalently bound to the beads and is prevented from falling off by the filtration process until it is cleaved by a specific reactant such as trifluoroacetic acid (TFA). A protection process in which the N-terminal amine of the peptide attached to the solid phase and an N-protected amino acid unit are combined, a deprotection process, a re-discovered amine group and a new As the cycle (cycle, deprotection-wash-coupling-wash) of the coupling process in which amino acids are combined is repeated, synthesis is made. The SPPS method can be performed by using microwave technology together, and the microwave technology can shorten the time required for coupling and deprotection of each cycle by applying heat during the peptide synthesis process. The thermal energy may prevent folding or aggregation of the expanded peptide chain and promote chemical bonding.

In addition, the peptide of the present invention can be prepared by the liquid phase peptide synthesis method, and the specific method thereof is referred 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 a method of mixing the solid phase synthesis method and the liquid phase synthesis method, and the preparation 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, an Applied Biosystems 431A peptide synthesizer (Perkin Elmer). Alternatively, the various fragments can be chemically synthesized separately and combined using chemical methods to produce the molecule of interest.

Meanwhile, the scope of the polypeptide of the present invention includes functional equivalents of the above-described polypeptides of the present invention and salts thereof. For example, the "functional equivalent" refers to having at least 80% or more, preferably 90%, more preferably 95% or more sequence homology (i.e., identity) to 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% , including those having sequence homology of 97%, 98%, 99%, or 100%, and refers to a peptide that exhibits substantially the same physiological activity as the polypeptide of the present invention. In the above, 'substantially homogenous physiological activity' is not limited thereto, but may be, for example, inducing a protective immune response against ASFV in the body of an animal, and as another example, detecting/diagnosing the serum of an ASFV-infected animal It may mean the ability to do something.

In one embodiment, a functional equivalent in the present invention may be one generated by addition, insertion, substitution (non-conservative or conservative substitution), deletion, or a combination thereof in part of the amino acid sequence of the polypeptide of the present invention described above. have. The substitution of amino acids in the above may preferably be a conservative substitution. Examples of conservative substitutions for naturally occurring amino acids are; 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 entirely alter the activity of the molecule are known in the art (H.Neurath, R.L.Hill, The Proteins, Academic Press, New York, 1979). In addition, the functional equivalent includes a variant in which a part of the amino acid is deleted from 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 not directly related to the physiological activity (chemokine activity) of the polypeptide provided in the present invention. Also included are variants in which some amino acids are added at both ends of the amino acid sequence of the polypeptide of the present invention or in the sequence.

In addition, the scope of the functional equivalent includes a polypeptide derivative in which some chemical structure of the polypeptide is modified while maintaining the basic backbone of the polypeptide and its physiological activity. For example, fusion proteins made by fusion with other proteins while maintaining structural changes and physiological activity to change stability, storage, volatility or solubility of the polypeptide of the present invention are included therein.

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

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

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

In one embodiment, for example, the polynucleotide encoding the polypeptide represented by SEQ ID NO: 1 may include the nucleotide sequence represented by SEQ ID NO: 3. In another embodiment, the polynucleotide encoding the polypeptide represented by SEQ ID NO: 1 may consist of the nucleotide sequence represented by SEQ ID NO: 3.

In one embodiment, for example, the polynucleotide encoding the polypeptide represented by SEQ ID NO: 2 may include the nucleotide sequence represented by SEQ ID NO: 4. In another embodiment, the polynucleotide encoding the polypeptide represented by SEQ ID NO: 2 may consist of the nucleotide sequence represented by SEQ ID NO: 4.

A nucleic acid sequence encoding a polypeptide of the present invention may be operatively linked to a vector capable of expressing it to provide the polypeptide of the present invention. The present invention provides an expression vector or a recombinant vector comprising the polynucleotide.

As used herein, 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 comprising essential regulatory elements operably linked to express a 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 may be operably linked to affect expression of the encoding nucleic acid sequence. The operative linkage with the recombinant vector can be prepared using genetic recombination techniques well known in the art, and site-specific DNA cleavage and ligation are performed using enzymes 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. Suitable expression vectors include a signal sequence or leader sequence for membrane targeting or secretion in addition to expression control elements such as promoter, operator, initiation codon, stop codon, polyadenylation signal and enhancer, and can be prepared 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 in the case of an expression vector capable of replication, an origin of replication.

The signal sequence includes a PhoA signal sequence, an OmpA signal sequence, etc. when the host is a bacterium of the genus Escherichia , and an α-amylase signal sequence, a subtilisin signal sequence, etc., when the host is a bacterium of the genus Escherichia. In the case of yeast, the MFα signal sequence, the SUC2 signal sequence, etc. may be used, and in the case of an animal cell, the insulin signal sequence, the α-interferon signal sequence, the antibody molecule signal sequence, etc. may be used, but the present invention is not limited thereto.

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 transformation can be performed by any method known to be capable of introducing a nucleic acid into an organism, cell, tissue or organ, and as is known in the art, it can be performed by selecting an appropriate standard technique according to the host cell. . These methods include microprojectile bombardment, electroporation, protoplast fusion, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, agitation with silicon carbide fibers, agrobacterium-mediated transformation, PEG-mediated fusion, microinjection, liposome-mediated method, dextran sulfate, lipofectamine, thermal shock, and the like.

The term 'transformant' can be used interchangeably with 'host cell' and the like, and is introduced into cells by any means (eg, electroshock method, calcium phosphatase precipitation method, microinjection method, transformation method, virus infection, etc.) It refers to a prokaryotic or eukaryotic cell containing heterologous DNA.

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

The transformant (or transformed microorganism, host cell) of the present invention may preferably be Escherichia coli (E. coli, Escherichia 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.

The present invention also 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, has the same meaning as an immunogenic composition, and is used interchangeably herein. The vaccine includes both prophylactic and therapeutic vaccines. Prophylactic vaccines induce an immune response prior to exposure to a substance containing the antigen, and thus increase the ability of the individual to resist the antigen-carrying substance or cell, in order to induce a greater immune response when the individual is exposed to the antigen. means that Therapeutic vaccine is used in such a way that the vaccine is administered to an individual who already has a disease related to the antigen. The therapeutic vaccine provides an increased ability to fight the disease or cell carrying the antigen, thereby enhancing the immune response of the individual to the antigen. can increase

In one embodiment, the present invention may be to 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 may be administered orally or parenterally. The parenteral administration method is not limited thereto, but may be inoculated by a transdermal, intramuscular, intraperitoneal, intravenous, or subcutaneous route. Preferably, the vaccine may be intramuscularly inoculated during the first and second inoculations.

The vaccine may be in any form known in the art, for example, in the form of solutions and injections, or in solid form suitable for suspension, but is not limited thereto. Such formulations may also be emulsified or encapsulated in liposomes or soluble glass, or may be prepared in the form of an aerosol or spray. They may also be incorporated into transdermal patches. In the case of a solution or injection, it may contain propylene glycol and sodium chloride in an amount sufficient to prevent hemolysis (eg, about 1%) if necessary.

The vaccine composition of the present invention is characterized in that it comprises the above-described polypeptide of the present invention , and may further comprise one or more 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 when administered to humans and does not normally cause allergic reactions such as gastrointestinal disorders, dizziness, or similar reactions. say

The carrier (carrier) refers to a material that facilitates delivery of a target into a cell or tissue. The pharmaceutically acceptable carrier may further include, for example, a carrier for oral administration or a carrier for parenteral administration. In one example, the carrier for parenteral administration may include water, a suitable oil, saline, aqueous glucose and glycol, and the like. The carrier may also include a dry formulation such as a coated patch made of titanium or polymer. Suitable carriers for vaccines are known to those skilled in the art and include, but are not limited to, proteins, sugars, and the like. Such carriers may be aqueous or non-aqueous solutions, suspensions or emulsions.

In addition, the composition of the present invention may be used as an adjuvant for increasing immunogenicity, such as atypical or atypical organic or inorganic polymers. Adjuvants are generally known to promote immune responses through chemical and physical binding to antigens. As the adjuvant used in this study, atypical aluminum gel, oil emulsion, or double oil emulsion and immunosol were used. In addition, various plant-derived saponins, levamisole, CpG dinucleotides, RNA, DNA, LPS, and various types of cytokines were used to promote the immune response. The above immune composition can be used as a composition for inducing an optimal immune response by a combination of various adjuvants and immune response promoting additives.

Without limitation, mineral salt adjuvants (eg, alum-, calcium-, iron-, zirconium-based salt adjuvants), tensioactive adjuvants (eg, Quil A, QS-21, other saponins) , bacterial-derived adjuvants (eg, N-acetyl muramyl-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), liposome adjuvants, polymer adjuvants and carriers, cytokines (e.g. granulocyte-macrophage colony stimulating factor), carbohydrate adjuvants, live antigen delivery systems (eg, bacteria, viruses), and the like.

In addition, as the composition to be added to the vaccine, a stabilizer, an inactivating agent, an antibiotic, a preservative, and the like may be used. Depending on the route of administration of the vaccine, the vaccine antigen may be mixed with distilled water or a buffer solution.

For other pharmaceutically acceptable carriers and agents, reference may be made to those described in Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995). Techniques for formulation and administration of vaccines described herein can be provided by reference to the literature (Remington, The Science and Practice of Pharmacy, 22nd ed.) and the like.

In addition, the 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 administering to the animal an immune effective amount of the polypeptide of the present invention as described above. to provide.

The 'subject' may mean 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 of enhancing immunity to ASFV in an animal (especially a pig) comprising administering to the animal (especially a pig) the polypeptide of claim 1 (i.e. to ASFV a method of immunizing an animal against

As used herein, the term 'immunization' 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 in the subject is induced, through such immunization, a preventive or therapeutic effect on a target disease (in particular, ASFV infection in the present invention) appears.

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

The total effective amount of the polypeptide of the present invention may be administered to a subject in a single dose, or may be administered by a fractionated treatment protocol in which multiple doses are administered for a long period of time. In addition, the content of the active ingredient may vary according to the purpose of administration. The effective dose is determined by considering various factors such as the type and severity of the target disease, the route of administration and the frequency of administration, as well as the age, weight, health status, sex, severity of the disease, diet and excretion rate of the individual requiring administration. Since the effective dosage is determined for the patient, those of ordinary skill in the art will be able to determine an appropriate effective dosage according to the purpose of administration. It can also be determined by monitoring the efficacy of a therapy using an assay that determines the activity of immune cells after administration of 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 the effect of the present invention is exhibited.

The above-described polypeptide of the present invention has a remarkable effect of specifically detecting (diagnosing) ASFV-infected sera, even compared to the native protein and polypeptides of other lengths/sequences derived from the protein. The detection (diagnosis) of ASFV-infected serum in the present invention means that the anti-ASFV antibody (antibody against African swine fever virus) present in the blood, plasma or serum of an individual binds to the polypeptide of the present invention (antigen- Formation of antibody complex) means to check the presence or absence and/or the amount of the antibody. When the presence of an anti-ASFV antibody in the blood, plasma or serum of an individual is detected and/or confirmed, the individual can be determined to be infected with ASFV.

Accordingly, 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 comprising the above-described polypeptide as an active ingredient, and a diagnostic kit comprising the diagnostic reagent composition.

Also, the present invention

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

(b) provides a method for detecting (diagnosing) serum infected with African swine fever virus comprising the step of detecting the presence of the 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 Italian-derived ASFV but also recently occurring ASFV. Therefore, in one embodiment, preferably, the present invention provides a diagnostic reagent composition for determining the presence of an antibody to African swine fever virus comprising the above-described 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 the steps (a) and (b).

As used herein, the term 'sample' may be a solid sample or a bodily fluid sample, preferably serum, plasma, muscle tissue, whole blood, lymph fluid, spleen, or a homogenate thereof.

In the detection or diagnosis according to the present application, the method and apparatus are not particularly limited and can be applied as long as it is known in the art as a means for detecting an antigen-antibody complex. For example, but not limited thereto, the antigen-antibody complex may be subjected to an immunoprecipitation assay including, but not limited to, Radial Immunodiffusion, immunoelectrophoresis or reverse current electrophoresis, RIA (Radioimmunoassay), competition indirect immunofluorescent assay ), ELISA (Enzyme Linked Immunosorbent Assay), or immunochromatic assay. Detection in this way is particularly advantageous when the antigen is provided as a soluble protein, and the polypeptide of the present invention satisfies this property. 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 together. It can be understood that the present invention provides a diagnostic kit using the 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 the antigen (polypeptide of the present invention)-antibody complex, the antigen (polypeptide of the present invention) may be labeled with a labeling material. That is, the polypeptide of the present invention may be provided by being linked (eg, covalently bonded or cross-linked) to a detectable label. The detectable label is a chromogenic enzyme (eg, peroxidase, alkaline phosphatase), a radioactive isotope (eg, 124I, 125I, 111In, 99mTc, 32P, 35S), a chromophore , biotin, luminescent material or fluorescent material (e.g. FITC, RITC, rhodamine, Texas Red, fluorescein, phycoerythrin, quantum dots) ), magnetic resonance imaging contrast agents (eg, superparamagnetic iron oxides (SPIO), ultrasuperparamagnetic iron oxides (USPIO)), gold particles, and the like. 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 the already synthesized polypeptide.

In one embodiment according to the present application, a detection antibody (secondary antibody) that detects an antibody (a primary antibody, for example, an antibody produced in animal serum) bound to the antigen (polypeptide according to the present invention) may be labeled have. The detection antibody (secondary antibody) that can be used in the method according to the present disclosure specifically binds to immunoglobulin (eg, IgM, IgG) produced in an animal to be diagnosed, and the detection antibody detects visual or various images. It can be labeled with a substance that can be detected using the instrument. This can be understood by referring to the above description for the polypeptide labeling material of the present invention.

In one specific embodiment, the antigen (polypeptide of the present invention) or detection antibody (second antibody) according to the present disclosure is a peroxidase such as horseradish peroxidase, alkaline phosphatase as a labeling material. ), glucose oxidase, beta-galactosidase, urease, catalase, asparginase, ribonuclease, malate Dehydrogenase (malate dehydrogenase), staphylococcal nuclease (staphylococcal nuclease), triose phosphate isomerase (triose phosphate isomerase), glucose-6-phosphate dehydrogenase (glucose-6-phosphate dehydrogenase), It may be labeled with an enzyme capable of catalyzing a chemical reaction in the presence of a specific substrate such as glucoamylase and acetylcholine esterase to emit a detectable color reaction or light, but is limited thereto. it's not going to be

In another specific embodiment, the antigen (polypeptide of the present invention) or the detection antibody according to the present application emits light of a wavelength different from the light irradiated by light irradiation, viroluminescence, chemiluminescence, electroluminescence , as a chromophore used in electrochemiluminescence and photoluminescence, for example, green fluorescent protein as a protein; As organic compounds, fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, and fluorecamine including, but not limited to.

In another specific embodiment, the antigen (polypeptide of the present invention) or detection antibody according to the present disclosure may be labeled with various radioisotopes. The detection of the label herein may be performed by, for example, a scintillation counter in the case of a radioisotope, for example, if the label is a fluorescent material, spectroscopy, a phosphoimaging device or a fluorescence meter, etc. This can be done in the same way. In the case of labeling with an enzyme, it can be carried out by measuring a chromogenic product resulting from conversion of a chromogenic substrate by an enzyme in the presence of an appropriate substrate. In addition, it can be detected as a color comparison of the colored product exhibited by the enzymatic reaction through comparison with an appropriate standard or control.

In a specific embodiment, the substance for labeling the antigen (polypeptide of the present invention) or detection antibody according to the present disclosure is, for example, a chromophore; enzymes including alkaline phosphatase, biotin, beta-galactosidase or peroxidase; radioactive material; or materials containing nanoparticles such as colloidal gold particles or colored latex particles, but is not limited thereto.

In addition to the polypeptide of the present invention, the kit of the present invention may further include an appropriate buffer or medium for the binding reaction between the polypeptide and the anti-ASFV antibody. In addition, when the polypeptide of the present invention is directly provided unlabeled, other 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 the form of coating on the surface of the plate. In this case, ASFV infection can be diagnosed by treating the sample on the plate and reacting it under suitable conditions, and then observing the binding of the polypeptide (antigen) of the present invention to the antibody in the sample on the surface of the plate. Such polypeptides of the present invention may be prepared in a microwell plate such as a 96-well microwell plate, beads or particles comprising colloidal gold particles or colored latex particles, or cellulose, nitrocellulose, polyethersulfone, polyvinylidene, fluoride, It can be provided attached to a membrane such as nylon, charged nylon and polytetrafluoroethylene. A method for attaching or coating the antigen (polypeptide of the present invention) may use a known method, for example, reference may be made to the one described in the Examples herein.

In a specific embodiment, the diagnostic reagent of the present invention and a kit including the same may be used in a sandwich immunoassay method such as ELISA (Enzyme Linked Immuno Sorbent Assay), RIA (Radio Immuno Assay), and the like. In this method, a sample is added to an antigen bound to a solid substrate, for example, a bead, a membrane, a slide or a microwell plate made of a solid substrate, such as glass, plastic (eg, polystyrene), polysaccharide, nylon, or nitrocellulose. , a label capable of direct or indirect detection, for example, a radioactive material such as 3H or 125I as described above, a fluorescent material, a chemiluminescent material, hapten, biotin, digoxigenin, etc. Alternatively, it can be detected qualitatively or quantitatively through binding of an antibody conjugated with an enzyme such as horseradish peroxidase, alkaline phosphatase, or malate dehydrogenase capable of luminescence. Immunoassay methods are also 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, et al. The ELISA kit includes a reagent capable of detecting the bound antibody, for example, a secondary detection antibody labeled with the above-mentioned substances such as chromophores, an enzyme (eg, conjugated with an antibody), and a substrate used for detection. and the like may be further included.

The polypeptide composed of the unique sequence provided in the present invention has significantly superior expression and purification yield compared to the native protein in the virus, the length is short, and the detection/diagnosis of ASFV-infected serum even compared to the native protein in ASFV. Not only is the ability remarkably excellent, but it has the potential to be used as a vaccine and also has high productivity at the industrial level.

1 shows the results of confirming the polypeptide by Commassie staining after expression of pK205R protein (also denoted as Italia-asfv-p205) in E. coli, purification, SDS-PAGE electrophoresis, and Commassie staining. The leftmost elution indicates the degree of protein detection immediately after the first purification, and the pK205R protein is degraded during the purification process, and the protein degraded below the pK205R protein can be identified.
Figure 2 shows the Italia-asfv-pep205-1 fragment polypeptide (SEQ ID NO: 1) and Italia-asfv-pep205-2 fragment polypeptide (SEQ ID NO: 2) of the present invention were expressed in E. coli and purified, followed by SDS-PAGE electrolysis Shows the result of confirming the polypeptide by Commassie staining after the electrophoresis. The Italia-asfv-pep205-1 fragment polypeptide (SEQ ID NO: 1) and Italia-asfv-pep205-2 fragment polypeptide (SEQ ID NO: 2) of the present invention were not degraded after purification and remained stable even during storage.
3 shows the effects of Italia-asfv-pep205-1 fragment polypeptide (SEQ ID NO: 1) and Italia-asfv-pep205-2 fragment polypeptide (SEQ ID NO: 2) of the present invention in the detection/diagnosis of ASFV-infected sera. The results of ID-ELISA compared with the native full-length protein (Italia-asfv-p205 native, ie, pK205R protein) are shown.
Figure 4 shows a schematic diagram of the vaccination induction process according to an embodiment of the present invention.
5 shows the results of IgG production by inoculation with African swine fever recombinant proteins (Italia-asfv-pep205-1, Italia-asfv-pep205-2) according to an embodiment of the present invention.

Hereinafter, the present invention will be described in detail.

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

Example 1: Excavation of high-efficiency immunogenic fragment polypeptide derived from Italian-derived African swine fever virus (ASFV) pK205R protein

1-1. Construction of ASFV pK205R protein-derived fragment polypeptide

Based on the sequence of the pK205R protein (NCBI GenBank: AAA65278.1/ Also referred to herein as Italia-asfv-p205) consisting of the amino acid sequence shown in SEQ ID NO: 5 obtained from AFV Malawi/Lil 20-1/1983 strain Thus, various polypeptide fragments were prepared, and some representative examples are shown in Table 1. The pK205R protein has a problem in that the protein is randomly cut (degraded) by itself after expression and purification of the protein due to protein instability. / There are difficulties in application. Accordingly, expression/purification was attempted from various angles in the present invention.

pep. fragment name sequence information SEQ ID NO: Italia-asfv-p205 VEPREQFFQDLLSAVDQQMDTVKNDIKDIMKEKTSFMVSFENFIERYDTMEKNIQDLQNKYEEMAANLMTVMTDTKIQLGAIIAQLEILMINGTPLPAKKTTIKEAMPLPSSNTNNDQTSPPASGKTSETPKKNPTNAMFFTRSEWASSKTFREKEKFLKTGIEQAILMVQMMFFTRSEWASSKTFREKEKFLKFAKDEKAILMV SEQ ID NO:5 Italia-asfv-pep205-1 VEPREQFFQDLLSAVDQQMDTVKNDIKDIMKEKTSFMVSFENFIERYDTMEKNIQDLQNKYEEMAANLMTVMTDTKIQLGAIIAQLEILMINGTPLPAKKTTIKEAMPLPSSNTNNDQTS SEQ ID NO: 1 Italia-asfv-pep205-2 VEPREQFFQDLLSAVDQQMDTVKNDIKDIMKEKTSFMVSFENFIERYDTMEKNIQDLQNKYEEMAANLMTVMTDTKIQLGAIIAQLEILMINGTPLPAKKTTIKEAMPLPSSNTNNDQTSPPASGKTSETPKKNPTNAMFFTRSEWASSKTFREKFLTPEIQAILQAILDEKFLTPEIQAIL SEQ ID NO:2

The proteins and polypeptides were briefly produced in the following way. First, DNA of Italia-asfv-p205 (SEQ ID NO: 6) was synthesized from macrogen. DNAs encoding individual fragment polypeptides, including Italia-asfv-pep205-1 and Italia-asfv-pep205-2, are amplified by PCR (gene amplification technology) from the Italia-asfv-p205 DNA. was obtained in this way. The polynucleotide encoding each fragment polypeptide was cloned between the BamH1 and Sal1 restriction sites of the pET49b vector (Novagen). Each of the polypeptides was overexpressed in the BL21 strain, Escherichia coli strain. E. coli cells transformed with the vector were grown at 37° C. using Luria-Bertani (LB) medium containing 100 ug/ml kanamycin until OD ₆₀₀ became 0.7, and 1 mM isopropyl β-D- Protein expression was induced by 1-thiogalactopyranoside (IPTG). After adding IPTG, incubated for an additional 4 hours, and then centrifuged at 5000 rpm for 20 minutes to recover cells. The recovered cells were resuspended in 50ml of IB buffer (pH8.0 Tris 0.1M, pH8.0 Ethylenediaminetetraacetic acid 5mM, phenylmethylsulfonyl fluoride 0.1mM), and the cells were disrupted by sonication and finally Denaturation buffer (6M Guanidine Hydrochloric acid). , pH8.0 Tris 0.1M and pH8.0 Ethylenediaminetetraacetic acid 2.5mM Denaturation buffer was used), and the cells were disrupted by sonication. Centrifuge at 5,000 rpm, put the supernatant in a Snake skin tube, and put it in 20 mM HEPES pH 7.4 150 mM NaCl buffer at 4 °C for 14 hours. After centrifugation again at 5,000 rpm for 20 minutes, the supernatant was treated with Ni-NTA and combined 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 Imidazoe. Polypeptides were identified using SDS-polyacrylamide gel electrophoresis, and the polypeptides were accommodated in a buffer containing pH7.4 HEPES 20mM and sodium chloride 150mM (4°C, overnight).

1-2. PK205R full-sequence protein versus fragment polypeptide productivity evaluation

The productivity of various fragment polypeptides was quantitatively compared and evaluated. The total amount immediately after culturing a live protein-producing cell line and protein purification is similar to that of other test groups, but Italia-asfv-p205 (pK205R) is very unstable during purification and storage, and random protein degradation occurs, resulting in purification of the total amount. It was impossible to maintain the intact protein state (see FIG. 1).

In order to improve the stability of the recombinant protein, fragments (fragments) of Italia-asfv-p205 that can be obtained and stored in an intact form were screened. As a result, among several fragments, the fragment polypeptides of Italia-asfv-pep205-1 (SEQ ID NO: 1) and Italia-asfv-pep205-2 (SEQ ID NO: 2) of the present invention were produced in a yield similar to that of Italia-asfv-p205, However, it was obtained in a stable form without degradation upon purification and storage (see FIG. 2 ). As such, the fragment polypeptides of the present invention have the characteristic of having significantly increased stability (ie, stability maintained in an intact form during purification process and storage (storage)), unlike the native protein, which shows the advantage of high productivity .

1-3. Comparative evaluation of ASFV infection serodiagnostic ability of fragment polypeptides compared to pK205R full-length protein

Using ID Screen® African Swine Fever Indirect Screening test kit from ID.vet, various fragment polypeptides prepared in Example 1-1 were compared with antibodies in ASFV-infected serum The binding ability was compared and evaluated by the ID (indirect)-ELISA method. Briefly, it was carried out in the following way. First, in a 96 Well EIA/RIA plate, coating buffer (0.015M sodium carbonate, 0.035M sodium bicarbonate, Final pH 9.6) and each antigen (each of the various fragment polypeptides prepared in Example 1-1, 2 ug/ml or 4 ug/ml) was added and incubated overnight (16h) at 4°C to coat the wells with each antigen. Each well was washed 4 times using 200ul of PBST buffer (1XPBS + Tween20 0.05%). 100ul of the primary antibody was added to each well, and then 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 200ul of PBST buffer, 100ul of secondary antibody was added to each well and incubated for 1 hour at room temperature. After adding 100ul of the substrate solution to each well, blocking the light, and incubating at room temperature for 15 minutes. 100ul of Stop solution was added to each well, and the absorbance (Optical Density) value was measured at 450nm.

As a result of the experiment, as shown in FIG. 3 , among several fragments, the fragment polypeptides of Italia-asfv-pep205-1 (SEQ ID NO: 1) and Italia-asfv-pep205-2 (SEQ ID NO: 2) of the present invention were particularly susceptible to ASFV infection. It showed high reactivity to serum.

Specifically, the Italia-asfv-pep205-1 (SEQ ID NO: 1) fragment polypeptide showed reactivity (detection ability) to ASFV-infected sera at an equivalent level compared to the pK205R full-length protein (represented as 'Italia-asfv-p205'). In the case of a fragment polypeptide of Italia-asfv-pep205-2 (SEQ ID NO: 2), it exhibited superior detection ability than pK205R full-length protein against ASFV-infected sera.

Example 2: Verification of vaccine effect using African swine fever protein

In order to confirm the vaccine effect of the African swine fever recombinant protein prepared in Example 1, immunization was performed according to the method shown in FIG. 4 and then verified by the ELISA method.

A mixture of Italia-asfv-pep205-1 and Italia-asfv-pep205-2, recombinant proteins of African swine fever, with Freund's adjuvant was administered to 5-6 week-old female C56BL/6 mice at intervals of 2 weeks. 200 μg per individual was inoculated by subcutaneous immunization. As a control group, PBS and Freund's adjuvant were inoculated in the same manner.

After induced primary to tertiary immunity by the inoculation, and two weeks later, serum was obtained from the subject's blood by a conventional method, and ELISA was performed to confirm the level of IgG production.

The ELISA was performed by adding ASF recombinant proteins (Italia-asfv-pep205-1, Italia-asfv-pep205-2) to the coating solution (Na2CO3 0.159g, NaHCO3 0.293g, per 100ml, pH9.6) at 3.0ug/ml, respectively. After dilution to a concentration of , 100 μl was put into each well of a 96-well plate, and then subjected to an adsorption process at 4° C. for one day. After the antigen adsorption was completed, the plate was washed 4 times using PBS, and then PBS containing 5% of normal goat serum was added to each plate to exclude non-specific binding, and the plate was reacted at 37° C. for 2 hours. . The Freund's adjuvant and the rat serum obtained through the recombinant protein inoculation of Example 1 were diluted 100-fold in PBS and then reacted for 1 hour at room temperature, washed 4 times with PBS, and then washed with PBS for color development. After reacting with enzyme-conjugated anti-mouse IgG1 at room temperature for 1 hour, a substrate buffer (3,3', 5,5'-Tetramethylbenzidine (TMB) and hydrogen peroxide) was added to develop color in the dark, followed by 2N sulfuric acid. was added to stop the color reaction and absorbance was measured at 450 nm, and the results are shown in FIG. 5 .

As shown in FIG. 5 , when inoculated with PBS or adjuvant corresponding to the negative control group, almost no antibody reaction was observed, and the African swine fever recombinant protein (Italia-asfv-p205-2) had an antibody reaction after primary immunization. appeared, and it was confirmed that the antibody response was strongly increased after the second immunization.

Through the above results, it can be seen that when the recombinant protein of African swine fever according to the present invention is used as a vaccine, an immune response can be highly induced.

Although the above results have been described in detail with respect to the present invention above, the scope of the present invention is not limited thereto, and various modifications and variations are possible within the scope without departing from the technical spirit of the present invention described in the claims. It will be apparent to those of ordinary skill in the art.

As described above, the present invention relates to the development and use of a p205 protein fragment derived from African swine fever virus (ASFV) as a recombinant antigen, and more specifically, the first in the protein represented by SEQ ID NO: 5 An isolated polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 as an isolated polypeptide comprising the 120th amino acid sequence, a vaccine composition for ASFV comprising the polypeptide as an active ingredient, and the polypeptide It relates to a method for immunizing an animal using , and a method for detecting/diagnosing ASFV infection using the polypeptide, a diagnostic reagent, and a kit. The polypeptide composed of the unique sequence provided in the present invention is shorter in length compared to the native protein in the virus, and has significantly superior ASFV infection serum detection/diagnostic ability compared to the native protein in ASFV, as well as the vaccine It has the potential to be used as a product, and also has high industrial applicability because of its high productivity at the industrial level.

<110> University industry foundation, Yonsei university wonju campus <120> p205 protein fragment derived from African swine fever virus as recombinant antigen, and uses thereof <130> NP19-0004P <150> KR 10-2019-0069635 <151> 2019-06-12 <160> 6 <170> KoPatentIn 3.0 <210> 1 <211> 120 <212> PRT <213> African swine fever virus <400> 1 Val Glu Pro Arg Glu Gln Phe Phe Gln Asp Leu Leu Ser Ala Val Asp 1 5 10 15 Gln Gln Met Asp Thr Val Lys Asn Asp Ile Lys Asp Ile Met Lys Glu 20 25 30 Lys Thr Ser Phe Met Val Ser Phe Glu Asn Phe Ile Glu Arg Tyr Asp 35 40 45 Thr Met Glu Lys Asn Ile Gln Asp Leu Gln Asn Lys Tyr Glu Glu Met 50 55 60 Ala Ala Asn Leu Met Thr Val Met Thr Asp Thr Lys Ile Gln Leu Gly 65 70 75 80 Ala Ile Ile Ala Gln Leu Glu Ile Leu Met Ile Asn Gly Thr Pro Leu 85 90 95 Pro Ala Lys Lys Thr Thr Ile Lys Glu Ala Met Pro Leu Pro Ser Ser 100 105 110 Asn Thr Asn Asn Asp Gln Thr Ser 115 120 <210> 2 <211> 182 <212> PRT <213> African swine fever virus <400> 2 Val Glu Pro Arg Glu Gln Phe Phe Gln Asp Leu Leu Ser Ala Val Asp 1 5 10 15 Gln Gln Met Asp Thr Val Lys Asn Asp Ile Lys Asp Ile Met Lys Glu 20 25 30 Lys Thr Ser Phe Met Val Ser Phe Glu Asn Phe Ile Glu Arg Tyr Asp 35 40 45 Thr Met Glu Lys Asn Ile Gln Asp Leu Gln Asn Lys Tyr Glu Glu Met 50 55 60 Ala Ala Asn Leu Met Thr Val Met Thr Asp Thr Lys Ile Gln Leu Gly 65 70 75 80 Ala Ile Ile Ala Gln Leu Glu Ile Leu Met Ile Asn Gly Thr Pro Leu 85 90 95 Pro Ala Lys Lys Thr Thr Ile Lys Glu Ala Met Pro Leu Pro Ser Ser 100 105 110 Asn Thr Asn Asn Asp Gln Thr Ser Pro Ala Ser Gly Lys Thr Ser 115 120 125 Glu Thr Pro Lys Lys Asn Pro Thr Asn Ala Met Phe Phe Thr Arg Ser 130 135 140 Glu Trp Ala Ser Ser Lys Thr Phe Arg Glu Lys Phe Leu Thr Pro Glu 145 150 155 160 Ile Gln Ala Ile Leu Asp Glu Gln Phe Ala Asn Lys Thr Gly Ile Glu 165 170 175 Arg Leu His Ala Glu Gly 180 <210> 3 <211> 360 <212> DNA <213> African swine fever virus <400> 3 gttgagccgc gtgagcagtt ctttcaggac ctgttgtccg cagtggatca acagatggac 60 accgtaaaaa atgatattaa ggacattatg aaagagaaaa cctcgtttat ggtgtccttt 120 gaaaacttca ttgagcgtta tgatactatg gagaagaata tccaggactt acagaataaa 180 tacgaagaga tggcagcaaa tttgatgaca gttatgaccg acactaaaat tcagctgggt 240 gcgatcattg ctcaactgga gattcttatg atcaatggta caccacttcc agctaagaaa 300 accactatca aggaagcgat gccgttaccg tcgtcaaata ccaataatga ccaaacctca 360 360 <210> 4 <211> 546 <212> DNA <213> African swine fever virus <400> 4 gttgagccgc gtgagcagtt ctttcaggac ctgttgtccg cagtggatca acagatggac 60 accgtaaaaa atgatattaa ggacattatg aaagagaaaa cctcgtttat ggtgtccttt 120 gaaaacttca ttgagcgtta tgatactatg gagaagaata tccaggactt acagaataaa 180 tacgaagaga tggcagcaaa tttgatgaca gttatgaccg acactaaaat tcagctgggt 240 gcgatcattg ctcaactgga gattcttatg atcaatggta caccacttcc agctaagaaa 300 accactatca aggaagcgat gccgttaccg tcgtcaaata ccaataatga ccaaacctca 360 ccaccagctt ctggcaagac cagtgaaaca cctaagaaaa accctactaa cgcaatgttt 420 ttcacccgtt ctgaatgggc aagctccaag accttccgtg aaaaatttct tacaccagag 480 atccaagcca ttcttgacga acaattcgcc aataaaacag gcatcgagcg tcttcatgcc 540 gagggc 546 <210> 5 <211> 204 <212> PRT <213> African swine fever virus <400> 5 Val Glu Pro Arg Glu Gln Phe Phe Gln Asp Leu Leu Ser Ala Val Asp 1 5 10 15 Gln Gln Met Asp Thr Val Lys Asn Asp Ile Lys Asp Ile Met Lys Glu 20 25 30 Lys Thr Ser Phe Met Val Ser Phe Glu Asn Phe Ile Glu Arg Tyr Asp 35 40 45 Thr Met Glu Lys Asn Ile Gln Asp Leu Gln Asn Lys Tyr Glu Glu Met 50 55 60 Ala Ala Asn Leu Met Thr Val Met Thr Asp Thr Lys Ile Gln Leu Gly 65 70 75 80 Ala Ile Ile Ala Gln Leu Glu Ile Leu Met Ile Asn Gly Thr Pro Leu 85 90 95 Pro Ala Lys Lys Thr Thr Ile Lys Glu Ala Met Pro Leu Pro Ser Ser 100 105 110 Asn Thr Asn Asn Asp Gln Thr Ser Pro Ala Ser Gly Lys Thr Ser 115 120 125 Glu Thr Pro Lys Lys Asn Pro Thr Asn Ala Met Phe Phe Thr Arg Ser 130 135 140 Glu Trp Ala Ser Ser Lys Thr Phe Arg Glu Lys Phe Leu Thr Pro Glu 145 150 155 160 Ile Gln Ala Ile Leu Asp Glu Gln Phe Ala Asn Lys Thr Gly Ile Glu 165 170 175 Arg Leu His Ala Glu Gly Leu Tyr Met Trp Arg Thr Gln Phe Ser Asp 180 185 190 Glu Gln Lys Lys Met Val Lys Glu Met Met Lys Lys 195 200 <210> 6 <211> 612 <212> DNA <213> African swine fever virus <400> 6 gttgagccgc gtgagcagtt ctttcaggac ctgttgtccg cagtggatca acagatggac 60 accgtaaaaa atgatattaa ggacattatg aaagagaaaa cctcgtttat ggtgtccttt 120 gaaaacttca ttgagcgtta tgatactatg gagaagaata tccaggactt acagaataaa 180 tacgaagaga tggcagcaaa tttgatgaca gttatgaccg acactaaaat tcagctgggt 240 gcgatcattg ctcaactgga gattcttatg atcaatggta caccacttcc agctaagaaa 300 accactatca aggaagcgat gccgttaccg tcgtcaaata ccaataatga ccaaacctca 360 ccaccagctt ctggcaagac cagtgaaaca cctaagaaaa accctactaa cgcaatgttt 420 ttcacccgtt ctgaatgggc aagctccaag accttccgtg aaaaatttct tacaccagag 480 atccaagcca ttcttgacga acaattcgcc aataaaacag gcatcgagcg tcttcatgcc 540 gagggccttt acatgtggcg tactcaattc tctgatgagc agaagaagat ggttaaggag 600 atgatgaaaa aa 612

Claims (13)

An isolated polypeptide comprising the first to 120th amino acid sequence in the protein represented by SEQ ID NO: 5, wherein the isolated polypeptide consists of the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2.
The isolated polypeptide of claim 1 , wherein the polypeptide is immunogenic against African swine fever virus (ASFV).
3. The isolated polypeptide of claim 2, wherein the ASFV is of Italian origin.
A polynucleotide encoding the polypeptide of claim 1 .
An expression vector comprising the polynucleotide of claim 4.
A host cell comprising the expression vector of claim 5 .
A vaccine composition against African swine fever virus comprising the polypeptide of claim 1 as an active ingredient.
The vaccine composition of claim 7, wherein the composition further comprises one or more 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 animals other than humans, comprising administering an immune effective amount of the polypeptide of claim 1 to animals other than humans.
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 to African swine fever virus, comprising the polypeptide of claim 1 as an active ingredient.
A diagnostic kit comprising the diagnostic reagent of claim 11 .
(a) contacting the animal sample with the polypeptide of claim 1; and
(b) A method of detecting serum infected with African swine fever virus comprising the step of detecting the presence of an antibody bound to the polypeptide of claim 1 in the sample.

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