WO2020251284A1 - Fragments protéiques p72, p104, p205 dérivés du virus de la peste porcine africaine et leur utilisation - Google Patents

Fragments protéiques p72, p104, p205 dérivés du virus de la peste porcine africaine et leur utilisation Download PDF

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WO2020251284A1
WO2020251284A1 PCT/KR2020/007609 KR2020007609W WO2020251284A1 WO 2020251284 A1 WO2020251284 A1 WO 2020251284A1 KR 2020007609 W KR2020007609 W KR 2020007609W WO 2020251284 A1 WO2020251284 A1 WO 2020251284A1
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polypeptide
asfv
present
swine fever
african swine
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PCT/KR2020/007609
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English (en)
Korean (ko)
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홍민선
전보영
김명일
박재완
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연세대학교 원주산학협력단
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • the present invention relates to a protein fragment of p72, p104, p205 derived from African swine fever virus (ASFV) and a use thereof, and more specifically, to SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 11, and SEQ ID NO: 12.
  • An isolated polypeptide selected from the group consisting of the displayed amino acid sequence, a vaccine composition against ASFV comprising the polypeptide as an active ingredient, an animal immunization method using the polypeptide, and ASFV infection detection/diagnosis using the polypeptide It relates to methods, diagnostic reagents and kits.
  • African swine fever is a swine infectious disease caused by infection with the African swine fever virus (ASFV) belonging to Asfarviridae.
  • ASFV African swine fever virus
  • the African swine fever virus is very resistant to the general environment and maintains its infectivity 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 and loses its infectivity only when exposed at 56°C for at least 1 hour. Have (Non-Patent Document 1).
  • African swine fever is a serious disease with a mortality rate of 100% when infected with pigs, but there is no commercially available vaccine for this disease.
  • African swine fever spreads again in Europe and spread to the Asian continent, attention is being focused on research on the development of vaccines that were not properly progressed in the past.
  • Zadok vaccine also has a lot of acid to overcome in developing effective products for defense.
  • ASFV has a larger and more complex structure than other viruses, so it is looking for 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.
  • African swine fever virus Since the gene is large, there are many types of proteins that can be made from the gene, and the ASFV genome is known to encode at least 151 ORFs. African swine fever virus has a large number of structural proteins and the size of the virus itself is much larger than that of other viruses, which is more than 10 times the size of circovirus and 4 to 5 times the size of PRRS virus. Since the African swine fever virus is large and complex, it is difficult to immunize it, so an effective vaccine has not yet been developed.
  • diagnosis is also very important because it is an infectious disease with a high risk of having to dispose of pigs immediately.
  • a diagnostic test by an accredited laboratory is essential. Laboratory diagnostic tests include detecting ASFV in the blood and internal organs, or detecting antibodies in the serum of infected pigs. Since the vaccine has not yet been developed, a positive test for antibody indicates field infection. If the virus needs to be isolated from the blood, a temporary sample collected at the time when the initial fever symptoms are confirmed is suitable, and organs such as lymph nodes and spleens are requested to be refrigerated. The ideal sample for testing antibodies is serum from infected pigs in the recovery phase between 8 and 21 days after infection.
  • the first screening test is performed using OIE-ELISA (indirect method) and commercially available antibody detection ELISA, and the immunotaxonomy (IB), immunoperoxidase test (IPT), indirect fluorescence.
  • Final evaluation can be performed through the antibody method (IFA) (see Non-Patent Document 2).
  • IFA antibody method
  • Non-Patent Document 1 Ji-yeon Lee, African swine fever diagnosis and prevention, Journal of Korean Veterinary Medical Association, Vol. 50_No. 11 Nov 2014, 671-673.
  • Non-Patent Document 2 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.
  • the polypeptide consisting of a unique sequence provided by the present invention is compared with the native protein in ASFV.
  • the present invention was completed by confirming that the ASFV-infected serum detection/diagnosis ability was remarkably excellent, as well as possible use as a vaccine, and high productivity at an industrial level.
  • an object of the present invention is to provide an isolated polypeptide selected from the group consisting of the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 11, and SEQ ID NO: 12.
  • 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 comprising the polypeptide as an active ingredient.
  • composition for a vaccine against African swine fever virus comprising the polypeptide as an active ingredient.
  • composition for a vaccine against the African swine fever virus consisting essentially of 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 to the animal an immune effective amount of the polypeptide. .
  • Another object of the present invention is to provide a composition for diagnosing African swine fever virus infection comprising the polypeptide as an active ingredient.
  • composition for diagnosing African swine fever virus infection comprising the polypeptide as an active ingredient.
  • composition for diagnosing African swine fever virus infection consisting essentially of the polypeptide as an active ingredient.
  • Another object of the present invention is to provide a diagnostic reagent for determining the presence or absence of an antibody against the African swine fever virus comprising the polypeptide as an active ingredient, and a diagnostic kit comprising the diagnostic reagent.
  • Another object of the present invention is, (a) contacting an animal sample with the polypeptide of the present invention; And (b) detecting the presence of the antibody conjugated to the polypeptide of the present invention in the sample.
  • Another object of the present invention is to provide the use of the above polypeptide for preparing a vaccine formulation against African swine fever virus.
  • Another object of the present invention is to provide a method for treating African swine fever virus comprising administering an effective amount of a composition comprising the polypeptide as an active ingredient to an individual in need thereof.
  • Another object of the present invention is to provide the use of the above polypeptide for preparing a preparation for diagnosing African swine fever virus infection.
  • Another object of the present invention is to provide a method for diagnosing infection of African swine fever virus, comprising administering an effective amount of a composition comprising the above polypeptide as an active ingredient to an individual in need thereof.
  • the present invention provides an isolated polypeptide selected from the group consisting of the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 11, and SEQ ID NO: 12.
  • the present invention provides a polynucleotide encoding the polypeptide, an expression vector containing the polynucleotide, and a host cell containing the expression vector.
  • the present invention provides a composition for a vaccine against African swine fever virus comprising the polypeptide as an active ingredient.
  • composition for a vaccine against the African swine fever virus comprising the polypeptide as an active ingredient.
  • composition for a vaccine against the African swine fever virus consisting essentially of the polypeptide as an active ingredient.
  • the present invention is a method for inducing a protective immune response against African swine fever in an animal, comprising administering an immune effective amount of the polypeptide to the animal. Provides a method of induction.
  • the present invention provides a composition for diagnosing African swine fever virus infection comprising the polypeptide as an active ingredient.
  • compositions for diagnosing African swine fever virus infection comprising the polypeptide as an active ingredient.
  • composition for diagnosing African swine fever virus infection consisting essentially of the polypeptide as an active ingredient.
  • the present invention provides a diagnostic reagent for determining the presence or absence of an antibody against the African swine fever virus containing the polypeptide as an active ingredient, and a diagnostic kit comprising the diagnostic reagent. do.
  • 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 the antibody bound to the polypeptide of the present invention in the sample.
  • a method of treating African swine fever virus comprising administering an effective amount of a composition comprising the polypeptide as an active ingredient to an individual in need thereof.
  • a method for diagnosing infection of African swine fever virus comprising administering an effective amount of a composition comprising the polypeptide as an active ingredient to an individual in need thereof.
  • the term'comprising' is used in the same meaning as'including' or'characterized by', and in the composition or method according to the present invention, specifically Additional components or method steps, etc. not mentioned are not excluded.
  • the term'consisting of' is used in the same way as'consisting of', and means excluding additional elements, steps, or ingredients that are not separately described.
  • the term'essentially consisting of' means that, in the scope of a composition or method, a substance or step that does not substantially affect its basic properties in addition to the described substance or step may be included.
  • treatment refers to a clinical procedure to change the natural process of an individual or cell to be treated, and may be performed to prevent clinical pathology. Desirable effects of treatment include suppression of the occurrence or recurrence of the disease, alleviation of symptoms, reduction of any direct or indirect pathological consequences of the disease, reduction of the rate of disease progression, improvement of the disease state, improvement, alleviation, or improved prognosis Include. Also, the term'prevention' refers to any action that suppresses the onset or delays the progression of a disease.
  • the term'antigen' induces a state of sensitization and/or immunoreactivity upon introduction by contact with appropriate cells, and in a verifiable manner with the immune cells and/or antibodies of the subject thus sensitized in vivo or in vitro.
  • the term'antigen' may be used collectively 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.
  • the term'antigenicity' or'immunogenicity' refers to the property of the antigen or immunogen and means a property that causes a secretory, humoral and/or cellular immune response.
  • the term'immune reaction' is a self-defense system existing in an animal's body, and is a biological phenomenon in which various substances or living organisms invading from the outside are distinguished from oneself and the invader is removed.
  • the surveillance system for self-defense is largely made by two mechanisms, one is humoral immunity and the other is cellular immunity.
  • Humoral immunity is achieved by antibodies present in the serum, and the antibodies play an important role in removing invading foreign antigens by binding them.
  • cellular immunity is made by several types of cells belonging to the lymphatic system, which are responsible for the direct destruction of invading cells or tissues.
  • B cells produce antibodies
  • T cells participate in cellular immunity.
  • This immune response by B cells or T cells is an immune system that responds to antigens once invading the body, but works when the same type of antigen continues to exist or repeatedly invades. Thus, this immune response is a specific response to a specific antigen.
  • immune responses include neutrophil, macrophage, and natural killer (NK) cells. It is characterized by being involved in this and exerting various functions without being limited by the type of target cell.
  • the immune response of the present invention is an antigen contained in the vaccine or an antibody specifically directed against antigens, B cells, helper T cells, suppressor T cells, cytotoxic T cells and gamma-delta T cells.
  • it may be a protective immune response.
  • Such protection is evidenced by a reduction or absence of clinical signs typically exhibited by an infected host, a faster recovery time or a lower duration or a lower viral titer in the tissue or body fluids or excreta of the infected host.
  • the term'protein' is used interchangeably with'polypeptide' or'peptide' and, for example, refers to a polymer of amino acid residues as commonly found in proteins in a natural state.
  • the polypeptide is not limited to a specific length, but may generally refer to a fragment of a full length protein in the context of the present invention.
  • the polypeptide or protein may include post-translational modifications, such as glycosylation, acetylation, phosphorylation, etc., and other modifications known in the art (naturally occurring and non-naturally occurring modifications).
  • Polypeptides and proteins of the present invention can be prepared using any of a variety of known recombinant and/or synthetic techniques.
  • polynucleotide and “nucleic acid” as used herein refer to deoxyribonucleotides (DNA) or ribonucleotides (RNA) in the form of single-stranded or double-stranded. Unless otherwise limited, also includes known analogues of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally occurring nucleotides.
  • One letter (three letters) of amino acids used herein means the following amino acids according to the standard abbreviation regulations 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(Gin): glutamine; R(Arg): arginine; S(Ser): serine; T(Thr): threonine; U(Sec): selenocysteine, V(Val): valine; W(Trp
  • expression means that a protein or nucleic acid is produced in a cell.
  • the present inventors are in terms of detection/diagnosis of ASFV-infected serum (i.e., antibody against ASFV in serum), availability as a vaccine, and mass production for commercial dissemination, As a fragment having an excellent advantage, a polypeptide composed of the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 11, and SEQ ID NO: 12 was newly identified.
  • the polypeptides are characterized by having a remarkably excellent effect in the above-described aspects even when compared to the natural protein from which they are derived and the polypeptides (fragments) of different lengths and sequence configurations derived from the protein.
  • the present invention provides an isolated polypeptide selected from the group consisting of the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 11, and SEQ ID NO: 12.
  • the polypeptide of the present invention is characterized by immunogenicity against African swine fever virus (ASFV), and preferably has an immunogenicity specific to ASFV derived from Kenya or Italy.
  • ASFV African swine fever virus
  • the polypeptide of the present invention has a wide range of immunogenicity that reacts with ASFV type 6 ASFV type 6 issued in Africa, as well as Russia, Georgia, etc., and reacts with ASFV type 2 samples recently occurring in Jung-gu and Asia. It is a feature.
  • 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.
  • 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 an appropriate primer.
  • the DNA sequence may be synthesized by standard methods known in the art, for example, using an automatic DNA synthesizer (available from Biosearch or Applied Biosystems).
  • the produced nucleic acid is operatively linked thereto and inserted into a vector containing one or more expression control sequences (eg, promoters, enhancers, etc.) that control the expression of the nucleic acid, and recombination formed therefrom
  • the host cell is transformed with the expression vector.
  • the resulting transformant is cultured under a medium and conditions suitable for expressing the nucleic acid to recover a substantially pure polypeptide expressed by the nucleic acid from the culture.
  • the recovery can be performed using a method known in the art (eg, chromatography).
  • substantially pure polypeptide means that the polypeptide according to the present invention does not substantially contain any other proteins derived from host cells.
  • the genetic engineering method for synthesizing the polypeptide of the present invention may be referred 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.
  • polypeptide 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.
  • the polypeptide of the present invention can be prepared by direct peptide synthesis using a solid phase technique (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 induce the peptide chain to continue.
  • the peptide covalently binds to the bead and prevents it from being separated by filtration until it is cleaved by a specific reactant such as trifluoroacetic acid (TFA).
  • TFA trifluoroacetic acid
  • the SPPS method may be performed using a microwave technology together, and the microwave technology may 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.
  • the peptide of the present invention can be prepared by the liquid peptide synthesis method, and the specific method thereof is referred to the following documents: US Patent No. 5,516,891.
  • 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 method described herein.
  • Protein synthesis can be performed using manual techniques or by automation. Automated synthesis can be accomplished using, for example, 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.
  • the scope of the polypeptide of the present invention includes the functional equivalents and salts thereof of the polypeptide of the present invention described above.
  • the "functional equivalent” refers to having at least 80% or more, preferably 90%, more preferably 95% or more amino acid group sequence homology (ie, identity) with the aforementioned polypeptide of the present invention.
  • substantially homogeneous physiological activity is not limited thereto, but as an example, it may be to induce a protective immune response against ASFV in the body of an animal, and as another example, detecting/diagnosing serum of an ASFV-infected animal. It could mean something like ability.
  • the functional equivalent in the present invention may be that a part of the amino acid sequence of the polypeptide of the present invention described above is generated by addition, insertion, substitution (non-conservative or conservative substitution), deletion, or a combination thereof. have.
  • Substitution of amino acids in the above may preferably be a conservative substitution. Examples of conservative substitutions of amino acids present in nature 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).
  • the functional equivalent includes a variant in which a part of an 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 by the present invention.
  • variants in which several amino acids are added in the sequence or at both ends of the amino acid sequence of the polypeptide of the present invention are also included.
  • the scope of the functional equivalent includes a polypeptide derivative in which some chemical structures of the polypeptide have been modified while maintaining the basic backbone of the polypeptide and its physiological activity.
  • fusion proteins made by fusion with other proteins while maintaining structural changes and physiological activity to alter the stability, storage, volatility, or solubility of the polypeptide of the present invention are included.
  • the polypeptide of the present invention is modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, etc., depending on the case. ) May be.
  • the present invention provides a polynucleotide encoding (coding) the polypeptide of the present invention.
  • the base combination of the polynucleotide is not particularly limited as long as the polynucleotide can encode the polypeptide of the present invention.
  • the polynucleotide may be provided as a single-stranded or double-stranded nucleic acid molecule including all DNA, cDNA and RNA sequences.
  • the polynucleotide may include a nucleotide sequence represented by SEQ ID NO: 2.
  • polynucleotide may be composed of a nucleotide 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 it.
  • the present invention provides an expression vector or a recombinant vector comprising the polynucleotide.
  • 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. .
  • operably linked refers to a functional linkage between a nucleic acid expression control sequence and a nucleic acid sequence encoding a protein or RNA of interest to perform a general function.
  • a promoter and a nucleic acid sequence encoding a protein or RNA can be operably linked to affect the 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 linkage use enzymes generally known in the art.
  • the vector includes, but is not limited to, a plasmid vector, a cozmid vector, a bacteriophage vector, and a viral vector.
  • Suitable expression vectors include, in addition to expression control elements such as promoters, operators, start codons, stop codons, polyadenylation signals and enhancers, etc., signal sequences or leader sequences for membrane targeting or secretion, and may be variously prepared according to the purpose. .
  • the promoter of the vector can be constitutive or inducible.
  • the expression vector includes a selection marker for selecting a host cell containing the vector, and in the case of a replicable expression vector, it may include an origin of replication.
  • the signal sequence when the host is Escherichia sp., the PhoA signal sequence, OmpA signal sequence, etc., when the host is Bacillus, the ⁇ -amylase signal sequence, subtilisin signal sequence, etc.
  • the MF ⁇ signal sequence, the SUC2 signal sequence, and the like may be used, and in the case of an animal cell, an insulin signal sequence, an ⁇ -interferon signal sequence, an antibody molecule signal sequence, and the like may be used, but are not limited thereto.
  • the present invention provides a host cell comprising the expression vector.
  • the present invention provides a transformant (host cell) transformed with the expression vector (recombinant vector).
  • the transformation may be performed by any method known to be capable of introducing a nucleic acid into an organism, cell, tissue or organ, and as known in the art, appropriate standard techniques may be selected according to the host cell. . These methods include microprojectile bombardment, electroporation, protoplasm fusion, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, agitation using silicon carbide fibers, agrobacterial mediated transformation, PEG-mediated fusion, microinjection, liposome-mediated method, dextran sulfate, lipofectamine, thermal shock method, etc. are included, but are not limited thereto.
  • transformant' can be used interchangeably with'host cells', etc., and introduced into cells by any means (e.g., electric shock method, calcium phosphatase precipitation method, microinjection method, transformation method, virus infection, etc.) It refers to a prokaryotic or eukaryotic cell containing the heterologous DNA.
  • the transformant is all kinds of single-celled organisms commonly used in the field of cloning, such as prokaryotic microorganisms such as various bacteria (eg, Clostridia genus, E. coli, etc.), lower eukaryotic microorganisms such as yeast, and insect cells.
  • prokaryotic microorganisms such as various bacteria (eg, Clostridia genus, E. coli, etc.)
  • lower eukaryotic microorganisms such as yeast
  • insect cells Cells derived from higher eukaryotes including, plant cells, mammals, and the like may be used as host cells, but are not limited thereto.
  • the transformant of the present invention may preferably mean a transforming microorganism.
  • Escherichia coli Escherichia coli
  • Bacillus subtilis Bacillus subtilis
  • Streptomyces Streptomyces
  • Pseudomonas Proteus Mirabilis
  • host cells It may be a prokaryotic host cell such as Proteus mirabilis or Staphylococcus.
  • fungi for example, Aspergillus
  • yeast for example, Pichia pastoris, Saccharomyces cerevisiae
  • Schizosaccharomyces Schizosaccharomyces
  • Neurospora crassa such as lower eukaryotic cells, insect cells, plant cells, and cells derived from higher eukaryotes, including mammals, etc., may be used as host cells, but is not limited thereto.
  • the transformant (or transforming microorganism, host cell) of the present invention may preferably be Escherichia coli (Escherichia coli).
  • Escherichia coli strain of the present invention is not limited thereto, but, for example, Rosetta2 (DE3), C41 (DE3), SoluBL21, and the like may be used.
  • the present invention provides a vaccine composition against African swine fever virus comprising the above-described polypeptide as an active ingredient.
  • the term'vaccine' or'vaccine composition' refers to a composition that stimulates an immune response, and 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, thus increasing the ability to resist an antigen-carrying substance or cell, in order to induce a greater immune response when an individual is exposed to the antigen.
  • Therapeutic vaccine is used by administration to an individual who already has a disease associated with the antigen of the vaccine.
  • the therapeutic vaccine provides an increased ability to fight antigen-carrying diseases or cells, thereby increasing the individual's immune response to the antigen. Can increase.
  • 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.
  • it can be administered orally or parenterally.
  • the parenteral administration method is not limited thereto, but the vaccine may be inoculated by transdermal, intramuscular, intraperitoneal, intravenous, or subcutaneous routes. Preferably, it may be to inoculate the vaccine intramuscularly during the first and second vaccinations.
  • the vaccine may be in any form known in the art, for example, in the form of a solution or an injection or a solid form suitable for suspension, but is not limited thereto.
  • Such formulations may also be emulsified or encapsulated into liposomes or soluble glass, or prepared in the form of aerosols or sprays. They can also be included in transdermal patches.
  • liquids or injections if necessary, they may contain propylene glycol and sodium chloride in an amount sufficient to prevent hemolysis (eg about 1%).
  • 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 selected from the group consisting of pharmaceutically acceptable carriers, diluents and adjuvants.
  • pharmaceutically acceptable is 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 usually cause allergic reactions such as gastrointestinal disorders, dizziness or similar reactions.
  • carrier refers to a material that facilitates delivery of a target object into cells or tissues.
  • the pharmaceutically acceptable carrier may further include, for example, a carrier for oral administration or a carrier for parenteral administration.
  • the carrier for parenteral administration may include water, suitable oil, saline, aqueous glucose and glycol.
  • the carrier may 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.
  • the carrier may be an aqueous solution, or a non-aqueous solution, a suspension or an emulsion.
  • composition of the present invention may be used as an adjuvant for increasing immunogenicity, such as a fixed or atypical organic or inorganic polymer.
  • 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.
  • various plant-derived saponins, levamisol, 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.
  • mineral salt adjuvants e.g., alum-, calcium-, iron-, zirconium-based salt adjuvants
  • tensioactive adjuvants e.g., Quil A, QS-21, other saponins
  • Bacterial-derived adjuvants e.g., N-acetyl muramyl-L-alanyl-D-isoglutamine (MDP), lipid polysaccharide (LPS), monophosphoryl lipid A, trehalose dimycholate (TDM), DNA , CpGs, bacterial toxins
  • adjuvant emulsions e.g., FIA, Montanide, Adjuvant 65, Lipovant
  • liposome adjuvants e.g., polymer adjuvants and carriers
  • cytokines e.g., granulocyte-macrophage colony stimulating factors
  • carbohydrate adjuvants e.g., Live antigen delivery systems
  • compositions to be added to the vaccine stabilizers, inactivators, antibiotics, preservatives, and the like may be used.
  • the vaccine antigen may be mixed with distilled water or a buffer solution.
  • the vaccine composition comprising the immunogenic complex protein of the present invention can be administered to an individual in need thereof in an effective amount and used for immunization. That is, the present invention provides a method for inducing a protective immune response against African swine fever in an animal, comprising the step of administering to a pig an immune effective amount of the polypeptide of the present invention described above. .
  • The'subject' may mean an animal, preferably a mammal other than a human.
  • the subject may preferably be a pig.
  • the present invention is a method for enhancing immunity to ASFV in animals (especially pigs), characterized in that the polypeptide of claim 1 is administered to an animal (especially pigs) (i.e. A method of immunizing animals against).
  • the term'immunization' means that when the immunogenic complex protein according to the present invention is administered to an individual, the secretory, humoral and/or cellular immune response to the immunogenic complex protein is It is caused, and through such immunization, a prophylactic or therapeutic effect for a target disease (in particular, ASFV infection in the present invention) appears.
  • a target disease in particular, ASFV infection in the present invention
  • The'effective amount' is an amount showing a prophylactic or therapeutic effect against a target disease (particularly, ASFV infection) of the vaccine composition of the present invention, and the polypeptide of the present invention is secreted, humoral and/or cell It means an amount sufficient to induce a sexual immune response.
  • the total effective amount of the polypeptide of the present invention may be administered to an individual as a single dose, and may be administered by a fractionated treatment protocol in which multiple doses are administered for a long period of time.
  • the content of the active ingredient may be varied depending on 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 number of administrations, as well as the age, weight, health condition, sex, severity of the disease, diet and excretion rate of the subject in need of administration. Since the effective dose is determined, those skilled in the art will be able to determine the appropriate effective dose according to the purpose of administration.
  • the pharmaceutical composition of the present invention is not particularly limited in its formulation, route of administration, and method of administration as long as it exhibits the effects of the present invention.
  • the above-described polypeptide of the present invention has a remarkable effect of specifically detecting (diagnosing) ASFV-infected serum, even when compared with a native protein and a polypeptide having a different length/sequence structure derived from the protein.
  • detecting (diagnosing) ASFV-infected serum means that the anti-ASFV antibody (antibody against African swine fever virus) present in the individual's blood, plasma or serum is bound to the polypeptide of the present invention (antigen- Antibody complex formation) It means to confirm the presence or absence or/and the amount of the antibody.
  • the presence of an anti-ASFV antibody in the blood, plasma or serum of an individual is detected or/and confirmed, the individual can be determined to be infected with ASFV.
  • the present invention provides a composition for diagnosing African swine fever virus infection comprising the above-described polypeptide as an active ingredient.
  • the present invention provides a diagnostic reagent composition for determining the presence or absence of an antibody against African swine fever virus comprising the aforementioned polypeptide as an active ingredient, and a diagnostic kit comprising the diagnostic reagent composition.
  • the polypeptides of the present invention have remarkable specificity for various types of ASFV, and thus can be specifically detected not only for ASFV derived from Italy but also for ASFV that has recently occurred. Therefore, in one embodiment, preferably, the present invention provides a diagnostic reagent composition for determining the presence or absence of an antibody against the African swine fever virus comprising the polypeptide of the present invention as an active ingredient, and a diagnostic kit comprising the same. In addition, it may be to provide a method for detecting (diagnosing) ASFV infected serum including steps (a) and (b).
  • sample' may be a solid sample or a body fluid sample, and preferably, serum, plasma, muscle tissue, whole blood, lymph fluid, spleen, or a homogenate thereof.
  • antigen-antibody complexes include Radial Immunodiffusion, immunoprecipitation assays including immunoelectrophoresis or reverse current electrophoresis, Radioimmunoassay (RIA), competition indirect immunofluorescent assay. ), ELISA (Enzyme Linked Immunosorbent Assay), or immunochromatic assay (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.
  • an ELISA method in particular, a sandwich type ELISA is used, and in this case, a detection antibody described below is also used together.
  • the present invention can be understood to provide a diagnostic kit using the above method, and kit components according to each method are well known in the art.
  • 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 linking to a detectable label (eg, covalent bond or crosslinking).
  • the detectable label is a chromogenic enzyme (e.g., peroxidase, alkaline phosphatase), a radioactive isotope (e.g., 124I, 125I, 111In, 99mTc, 32P, 35S), chromophore.
  • the detectable label may be another antibody epitope, substrate, cofactor, inhibitor or affinity ligand that is not related to ASFV. Such labeling may be performed during the process of synthesizing the polypeptide of the present invention, or may be performed in addition to the already synthesized polypeptide.
  • a detection antibody that detects an antibody (primary antibody, for example, an antibody produced in animal serum) bound to the antigen (polypeptide according to 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 immunoglobulins (eg, IgM, IgG) generated in the 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 equipment. This can be understood with reference to the foregoing description of the polypeptide labeling material of the present invention.
  • the antigen (polypeptide of the present invention) or the detection antibody (secondary antibody) according to the present invention is a peroxidase such as horseradish peroxidase or alkaline phosphatase as a labeling material.
  • glucose oxidase beta-galactosidase, urease, catalase, asparginase, ribonuclease, malate Dehydrogenase (malate dehydrogenase), staphylococcal nuclease (staphylococcal nuclease), triose phospate isomerase (triose phospate isomerase), glucose-6-phosphate dehydrogenase (glucose-6-phosphate dehydrogenase), It can be labeled with an enzyme capable of releasing light or a detectable color reaction by catalyzing a chemical reaction in the presence of a specific substrate, such as glucoamylase and acetylcholine esterase, but limited to this. It does not become.
  • the antigen (polypeptide of the present invention) or detection antibody according to the present invention is Viroluminescence, Chemilluminescence, and Electroluminescence that emits light of a wavelength different from the irradiated light by irradiation with light.
  • Chromophores used in electrochemical luminescence and photoluminescence for example, green fluorescent protein as a protein;
  • organic compounds fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, and fluorecamine are used as organic compounds. Including, but not limited to.
  • the antigen (polypeptide of the present invention) or detection antibody according to the present application may be labeled with various radioisotope substances.
  • detection of a labeling substance may be performed by, for example, a scintillation counter in the case of a radioisotope.
  • the labeling substance is a fluorescent substance, a spectroscopy, a phosphorimizing device, or a fluorescence measuring instrument It can be done in the same way.
  • labeled with an enzyme it can be carried out by measuring a color-development product exhibited by conversion of the color-development substrate by the enzyme in the presence of an appropriate substrate.
  • it can be detected as a color comparison of the color development product exhibited by an enzymatic reaction through comparison with an appropriate standard or control.
  • the substance for labeling the antigen (polypeptide of the present invention) or the detection antibody according to the present application is, for example, a chromophore; Enzymes including alkaline phosphatase, biotin, beta-galactosidase or peroxidase; Radioactive material; Or a material including nanoparticles such as colloidal gold particles or colored latex particles, but is not limited thereto.
  • the kit of the present invention may further include an appropriate buffer solution or medium for the binding reaction between the polypeptide and the anti-ASFV antibody.
  • other detectable labeling means for labeling the polypeptide may be additionally included in the kit.
  • a secondary antibody labeled with a fluorescent material of the present invention, a color developing substrate, etc. may be additionally included in the kit.
  • polypeptide of the present invention may be provided in a form coated on the surface of a plate.
  • the plate is treated with a sample and reacted under appropriate conditions, and then the binding of the polypeptide (antigen) of the present invention to the antibody in the sample on the surface of the plate can be observed to diagnose ASFV infection.
  • the polypeptide of the present invention is a microwell plate such as a 96 well microwell plate, beads or particles including colloidal gold particles or colored latex particles, or cellulose, nitrocellulose, polyethersulfone, polyvinylidene, fluoride, It may be provided attached to a membrane such as nylon, charged nylon, and polytetrafluoroethylene.
  • a known method may be used, for example, reference may be made to those described in the Examples herein.
  • the diagnostic reagent of the present invention and a kit including the same may be used as a sandwich type immunoassay method such as ELISA (Enzyme Linked Immuno Sorbent Assay), RIA (Radio Immuno Assay), and the like.
  • sandwich type immunoassay method such as ELISA (Enzyme Linked Immuno Sorbent Assay), RIA (Radio Immuno Assay), and the like.
  • This method involves adding a sample to an antigen bound to a solid substrate such as glass, plastic (e.g., polystyrene), polysaccharide, beads, membrane, slide or microwell plate made of nylon or nitrocellulose.
  • Label material 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, a hapten, biotin, digoxigenin, etc., or color development through the action of a substrate Alternatively, it can be detected qualitatively or quantitatively through binding of an enzyme conjugated with an enzyme such as horseradish peroxidase, alkaline phosphatase, and maleate dehydrogenase capable of emitting light.
  • immunoassay methods include Enzyme Immunoassay, E. T.
  • ELISA kit is a reagent capable of detecting bound antibodies, for example, a secondary detection antibody labeled with the above-described substances such as chromophores, enzymes (eg, conjugated with an antibody), and a substrate used for detection. And the like may additionally be included.
  • the polypeptide composed of a unique sequence provided by the present invention has remarkably excellent expression and purification yield compared to the native protein in the virus, has a short length, and detects/diagnoses ASFV infection serum even when compared to the native protein in ASFV. Not only is it remarkably excellent in ability, it has the potential to be used as a vaccine and is also highly productive at the industrial level.
  • Figure 2 shows a schematic diagram of a vaccination induction process according to an embodiment of the present invention.
  • Figure 3 shows the results of IgG production by inoculation of the recombinant protein (Kenya-asfv-pep72-2) of African swine fever according to an embodiment of the present invention.
  • Figure 4 shows the result of confirming the polypeptide by Commassie staining after SDS-PAGE electrophoresis was performed after expressing the natural EP402R protein (also indicated as Kenya-asfv-p104, SEQ ID NO: 3) in E. coli and then purified.
  • Figure 5 shows the result of confirming the polypeptide by Commassie staining after expressing and purifying the Kenya-asfv-pep104-1 fragment polypeptide (SEQ ID NO: 1) of the present invention in E. coli, subjected to SDS-PAGE electrophoresis, and then stained with Commassie.
  • Figure 6 shows the effect of the Kenya-asfv-pep104-1 fragment polypeptide (SEQ ID NO: 1) of the present invention in the detection/diagnosis of ASFV infected serum, its natural full-length protein (Kenya-asfv-p104 natural type, that is, EP402R protein). ) And compared to ID (indirect)-ELISA results.
  • Figure 7 shows the results of IgG production by inoculation of the African swine fever recombinant protein (kenya-asfv-p104) according to an embodiment of the present invention.
  • Figure 8 shows the results of IgG production by inoculation of the African swine fever recombinant protein (kenya-asfv-pep104-1) according to an embodiment of the present invention.
  • FIG. 9 shows the result of confirming the polypeptide by Commassie staining after expressing and purifying the pK205R protein (also referred to as Italia-asfv-p205) in E. coli, subjected to SDS-PAGE electrophoresis.
  • the leftmost elution indicates the degree of protein detection immediately after the first purification, and during the purification process, pK205R protein is degraded, and the protein degraded under the pK205R protein can be identified.
  • FIG. 10 is an 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 Escherichia coli and then purified, SDS-PAGE electrophoresis The results of confirming the polypeptide by Commassie staining after performing the electrophoresis are shown. 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 when stored.
  • Figure 11 shows the effect of 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 in the detection/diagnosis of ASFV infected serum.
  • SEQ ID NO: 1 Italia-asfv-pep205-1 fragment polypeptide
  • SEQ ID NO: 2 Italia-asfv-pep205-2 fragment polypeptide
  • FIG. 12 shows the results of IgG production by inoculation of the African swine fever recombinant protein (Italia-asfv-pep205-1) according to an embodiment of the present invention.
  • FIG. 13 shows the results of IgG production by inoculation of the African swine fever recombinant protein (Italia-asfv-pep205-2) according to an embodiment of the present invention.
  • Example 1 Discovery of highly efficient immunogenic fragment polypeptide derived from Kenya-derived African swine fever virus (ASFV) p72 protein
  • p72 protein (NCBI GenBank: AY578698.1/ in the present specification, also referred to as Kenya-afv-p72) consisting of the amino acid sequence represented by SEQ ID NO: 5 obtained from the ASFV pig/Kenya/KEN-05/1950 strain
  • SEQ ID NO: 5 amino acid sequence represented by SEQ ID NO: 5 obtained from the ASFV pig/Kenya/KEN-05/1950 strain
  • the proteins and polypeptides were briefly produced in the following manner.
  • DNA encoding the fragment polypeptides of Kenya-asfv-pep72-1 and Kenya-asfv-pep72-2 was synthesized by requesting Macrogen.
  • Polynucleotides encoding each fragment polypeptide were cloned between the BamH1 and Sal1 restriction sites of pET49b vector (Novagen).
  • Each polypeptide was overexpressed in the Escherichia coli strain BL21.
  • E. coli cells transformed with the vector were grown using Luria-Bertani (LB) medium containing 100 ug/ml kanamycin at 37° C.
  • LB Luria-Bertani
  • IPTG -thiogalactopyranoside
  • IB buffer pH8.0 Tris 0.1M, pH8.0 Ethylenediaminetetraacetic acid 5mM, phenylmethylsulfonyl fluoride 0.1mM
  • Denaturation buffer 6M Guanidine Hydrochloric acid
  • Polypeptides were identified using SDS-polyacrylamide gel electrophoresis (6M Guanidine Hydrochloric acid, pH8.0 Tris 0.1M and pH8.0 Ethylenediaminetetraacetic acid 2.5mM denaturation buffer), and the polypeptides were pH7.4 HEPES 20mM and Sodium It was accommodated in a buffer containing 150mM chloride (4 °C, overnight).
  • the productivity of various fragment polypeptides was quantitatively compared and evaluated. As a result, among several fragments, it was confirmed that only the fragment polypeptide of the present invention Kenya-asfv-pep72-2 (SEQ ID NO: 1) can be expressed and purified in E. coli, which shows an advantage in terms of productivity (Fig. 1 Reference). In the case of the p72 protein and other fragment polypeptides, the E. coli cell line was not expressed or the yield was extremely low. As a representative comparative example, the Kenya-asfv-pep72-1 polypeptide is shown in FIG. 1, and Kenya-asfv-pep72-1 was not expressed when an E. coli-expressing cell line was used as shown in FIG. 1.
  • the Kenya-asfv-pep72-2 of the present invention can be expressed and purified in an E. coli cell line (Fig. 1).
  • the Kenya-asfv-pep72-2 fragment polypeptide of the present invention had a total yield of about 4 mg/1L (LB culture) for expression and purification.
  • the binding ability of the fragment polypeptide prepared in Example 2-1 to the antibody in the ASFV-infected serum was determined by the ID (indirect)-ELISA method. Comparative evaluation was made. Briefly, it was carried out in the following way. First, the coating buffer (0.015M Sodium carbonate, 0.035M Sodium bicarbonate, Final pH 9.6) and each antigen (each of the fragment polypeptide prepared in Example 1-1, 2 ug/ml or 4 ug/) were placed on a 96Well EIA/RIA plate. ml concentration) was added and incubated overnight (16h) at 4°C, and wells were coated with each antigen.
  • the coating buffer 0.015M Sodium carbonate, 0.035M Sodium bicarbonate, Final pH 9.6
  • each antigen each of the fragment polypeptide prepared in Example 1-1, 2 ug/ml or 4 ug/
  • PBST buffer (1XPBS + Tween20 0.05%). After 100ul of the primary antibody was added to each well, it was incubated for 1 hour at room temperature (22°C). The primary antibody was treated in the form of ASFV-infected pig serum provided as a positive control in the African Swine Fever Indirect Screening test kit of ID.vet. Then, 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 100ul of the substrate solution was added to each well, light was blocked and incubated 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, and the measured values are shown in Table 2 below.
  • negative control 2ug/ml 4ug/ml Kenya-asfv-pep72-2 0.2525 1.1935 1.375
  • the fragment polypeptide of the present invention Kenya-asfv-pep72-2 (SEQ ID NO: 1) exhibited high reactivity to ASFV-infected serum, and Kenya-asfv-pep72-1 (SEQ ID NO: This was not the case in number 3) (results not shown).
  • Example 2 Verification of vaccine effect using African swine fever protein
  • a mixture of Kenya-asfv-pep72-2, a recombinant protein of African swine fever, and Freund's adjuvant in 5-6 week-old female C57BL/6 mice was injected with a subcutaneous immunization of 200 ⁇ g per individual every two weeks. It was inoculated by the method. As a control group, PBS and Freund's adjuvant were inoculated in the same manner.
  • the ELISA was performed by diluting the African swine fever recombinant protein (Kenya-asfv-pep72-2) in a coating solution (0.159g Na2CO3, 0.293g NaHCO3, per 100ml, pH 9.6) to a concentration of 3.0ug/ml, and then 96 wells After putting 100 ⁇ l into each well on the plate, it was adsorbed at 4°C for one day. The plate on which antigen adsorption was completed was washed 4 times using PBS, and then PBS containing 5% normal chlorine serum was added to each plate and reacted at 37°C for 2 hours to exclude non-specific binding. .
  • the Freund's adjuvant and the serum of the rat obtained through the inoculation of the recombinant protein of Example 1 were diluted 100 times in PBS and then reacted at room temperature for 1 hour, followed by washing 4 times with PBS for color development. After reacting with enzyme-conjugated anti-mouse IgG1 for 1 hour at room temperature, a substrate buffer solution (3,3', 5,5'-Tetramethylbenzidine (TMB) and hydrogen peroxide solution) was added in a dark room to develop color, followed by 2N sulfuric acid. The color development reaction was stopped by adding and absorbance was measured at 450 nm, and the results are shown in FIG. 3.
  • a substrate buffer solution (3,3', 5,5'-Tetramethylbenzidine (TMB) and hydrogen peroxide solution
  • Example 3 Discovery of highly efficient immunogenic fragment polypeptide derived from Kenya-derived African swine fever virus (ASFV) EP402R protein
  • the EP402R protein (NCBI GenBank: AJL34072.1) consisting of the amino acid sequence represented by SEQ ID NO: 9 is found to have the same sequence configuration in the ASFV Ken05/Tk1 strain and the BA71V strain. However, it is difficult to produce a recombinant protein at a level for commercial use.
  • Various polypeptide fragments were prepared based on the EP402R protein (also referred to herein as Kenya-asfv-p104) sequence, and some representative examples are shown in Table 3.
  • DNA encoding the Kenya-asfv-p104 polypeptide was synthesized by requesting Macrogen.
  • DNA encoding the Kenya-asfv-pep104-1 polypeptide was obtained by amplifying some gene fragments through PCR (gene amplification process) from the Kenya-asfv-p104 gene (SEQ ID NO: 10).
  • Polynucleotides encoding each fragment polypeptide were cloned between the BamH1 and Sal1 restriction sites of pET49b vector (Novagen). Each of the polypeptides was overexpressed in the Escherichia coli strain BL21.
  • coli cells transformed with the vector were grown using Luria-Bertani (LB) medium containing 100 ug/ml kanamycin at 37° C. until OD600 became 0.7, and 1 mM isopropyl ⁇ -D-1 Protein expression was induced by -thiogalactopyranoside (IPTG). After IPTG was added, the cells were cultured for an additional 4 hours, and then centrifuged at 5000 rpm for 20 minutes to recover the cells.
  • LB Luria-Bertani
  • IPTG -thiogalactopyranoside
  • IB buffer pH8.0 Tris 0.1M, pH8.0 Ethylenediaminetetraacetic acid 5mM, phenylmethylsulfonyl fluoride 0.1mM
  • 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 broken by ultrasonic treatment.
  • Polypeptides were identified using SDS-polyacrylamide gel electrophoresis (6M Guanidine Hydrochloric acid, pH8.0 Tris 0.1M and pH8.0 Ethylenediaminetetraacetic acid 2.5mM denaturation buffer), and the polypeptides were pH7.4 HEPES 20mM and Sodium It was accommodated in a buffer containing 150mM chloride (4 °C, overnight).
  • the productivity of various fragment polypeptides was quantitatively compared and evaluated.
  • the expression amount and purification yield were 1.4 times shorter than that of the EP402R full-length protein (expressed as Kenya-asfv-p104). It was confirmed that the above increased.
  • expression was not performed or the yield was remarkably low.
  • the final yield of expression and purification of the EP402R full-length protein was 7 mg protein /1L (LB culture), and it took a total of 6 days until the purification process (ie, to obtain the protein).
  • Kenya-asfv-pep104-1 SEQ ID NO: 7
  • the final yield of expression and purification of the fragment polypeptide of Kenya-asfv-pep104-1 (SEQ ID NO: 7) of the present invention was significantly increased to 10 mg protein/1L (LB culture), until purification (until the protein was obtained. ) It took 1.5 ⁇ 2 days.
  • Kenya-asfv-pep104-1) not only showed a high yield compared to the natural EP402R protein purification process, but also had a short purification period of 4 days.
  • the results of expression and purification under different conditions are shown in Figs. 4 and 5, and the EP402R full-length protein has a very low purification amount (Fig. 4), but the Kenya-asfv-pep104-1 (SEQ ID NO: 7) fragment polypeptide is The amount of purification is remarkably high (Fig. 5).
  • the expression and purification of EP402R was carried out as a purification method that takes a short time, the yield was significantly lowered, and thus expression and purification were performed in a method that takes a long time.
  • the Kenya-asfv-pep104-1 (SEQ ID NO: 7) polypeptide fragment of the present invention increases the yield of purification and shows the advantage of shortening the purification process (see FIGS. 4 and 5).
  • ID Screen® African Swine Fever Indirect Screening test kit of ID.vet the binding ability of various fragment polypeptides prepared in Example 3-1 to antibodies in ASFV-infected serum was measured by ID (indirect)-ELISA. It was compared and evaluated by the method. Briefly, it was carried out in the following way. First, a 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 3-1, 2 ug/ml or 4) were placed on a 96Well EIA/RIA plate. ug/ml concentration) was added and incubated overnight (16h) at 4°C, and wells were coated with each antigen.
  • a coating buffer 0.015M Sodium carbonate, 0.035M Sodium bicarbonate, Final pH 9.6
  • each antigen each of the various fragment polypeptides prepared in Example 3-1, 2 ug/ml or 4
  • a coating buffer 0.015
  • Each well was washed 4 times using 200ul of PBST buffer (1XPBS + Tween20 0.05%). After 100ul of the primary antibody was added to each well, it was incubated for 1 hour at room temperature (22°C). The primary antibody was treated in the form of ASFV-infected pig serum provided as a positive control in the African Swine Fever Indirect Screening test kit of ID.vet. Then, 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 100ul of the substrate solution was added to each well, light was blocked and incubated 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.
  • the fragment polypeptide of Kenya-asfv-pep104-1 (SEQ ID NO: 7) was similar or similar compared to the EP402R full-length protein (denoted as'Kenya-asfv-p104'). At the same level, it showed high reactivity to ASFV-infected serum.
  • Example 4 Validation of vaccine effect using African swine fever protein
  • the ELISA is a coating solution (Na2CO3 0.159g, NaHCO3 0.293g, per 100ml, pH 9.6) of African swine fever recombinant protein (kenya-asfv-p104, kenya-asfv-pep104-1) at a concentration of 3.0ug/ml, respectively After diluting with, 100 ⁇ l was added to each well in a 96-well plate, and adsorption was performed at 4°C for one day. The plate on which antigen adsorption was completed was washed 4 times using PBS, and then PBS containing 5% normal chlorine serum was added to each plate and reacted at 37°C for 2 hours to exclude non-specific binding. .
  • Example 5 Discovery of highly efficient immunogenic fragment polypeptide derived from Italy-derived African swine fever virus (ASFV) pK205R protein
  • the pK205R protein consisting of the amino acid sequence represented by SEQ ID NO: 15 obtained from the AFV Malawi/Lil 20-1/1983 strain (NCBI GenBank: AAA65278.1/ in this specification, also referred to as Italia-asfv-p205) based on the sequence
  • SEQ ID NO: 15 obtained from the AFV Malawi/Lil 20-1/1983 strain
  • NCBI GenBank: AAA65278.1/ in this specification also referred to as Italia-asfv-p205
  • Table 4 The pK205R protein has a problem in that the protein is randomly cut (degraded) by itself after expressing and purifying the protein due to protein instability, producing a recombinant protein at a level for commercial use and utilizing the purified protein. /There are difficulties in application. Accordingly, expression/purification was attempted in various angles in the present invention.
  • DNA of Italia-asfv-p205 (SEQ ID NO: 16) was synthesized from macrogen. DNAs encoding respective fragment polypeptides, including Italia-asfv-pep205-1 and Italia-asfv-pep205-2, amplify some gene fragments from the Italia-asfv-p205 DNA through PCR (gene amplification). It was obtained in this way. Polynucleotides encoding each fragment polypeptide were cloned between the BamH1 and Sal1 restriction sites of pET49b vector (Novagen). Each polypeptide was overexpressed in the Escherichia coli strain BL21. E.
  • coli cells transformed with the vector were grown using Luria-Bertani (LB) medium containing 100 ug/ml kanamycin at 37° C. until OD600 became 0.7, and 1 mM isopropyl ⁇ -D-1 Protein expression was induced by -thiogalactopyranoside (IPTG). After IPTG was added, the cells were cultured for an additional 4 hours, and then centrifuged at 5000 rpm for 20 minutes to recover the cells.
  • LB Luria-Bertani
  • IPTG -thiogalactopyranoside
  • the productivity of various fragment polypeptides was quantitatively compared and evaluated.
  • the total amount immediately after cultivation and protein purification of the live protein-producing cell line is similar to that of other test groups, but Italia-asfv-p205 (pK205R) is very unstable during the purification and storage process, and random protein degradation occurs, resulting in the purification of the total amount. It was impossible to maintain the intact state of the protein (see Fig. 9).
  • fragments (fragments) of Italia-asfv-p205 that can be obtained and stored in an intact form were screened to improve the stability of the recombinant protein.
  • the fragment polypeptides of Italia-asfv-pep205-1 (SEQ ID NO: 11) and Italia-asfv-pep205-2 (SEQ ID NO: 12) of the present invention in a yield similar to Italia-asfv-p205, However, it was obtained in a stable form without being decomposed upon purification and storage (see FIG. 10).
  • the fragment polypeptides of the present invention have the property of having significantly increased stability (i.e., stability maintained in an intact form during purification and storage (storage)), unlike natural proteins, which shows the advantage of high productivity. .
  • ID Screen® African Swine Fever Indirect Screening test kit of ID.vet the binding ability of various fragment polypeptides prepared in Example 5-1 to antibodies in ASFV-infected serum was measured by ID (indirect)-ELISA. It was compared and evaluated by the method. Briefly, it was carried out in the following way. First, a 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 5-1, 2 ug/ml or 4) were placed on a 96Well EIA/RIA plate. ug/ml concentration) was added and incubated overnight (16h) at 4°C, and wells were coated with each antigen.
  • a coating buffer 0.015M Sodium carbonate, 0.035M Sodium bicarbonate, Final pH 9.6
  • each antigen each of the various fragment polypeptides prepared in Example 5-1, 2 ug/ml or 4
  • a coating buffer 0.015
  • Each well was washed 4 times using 200ul of PBST buffer (1XPBS + Tween20 0.05%). After 100ul of the primary antibody was added to each well, it was incubated for 1 hour at room temperature (22°C). The primary antibody was treated in the form of ASFV-infected pig serum provided as a positive control in the African Swine Fever Indirect Screening test kit of ID.vet. Then, 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 100ul of the substrate solution was added to each well, light was blocked and incubated 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.
  • the fragment polypeptides of Italia-asfv-pep205-1 (SEQ ID NO: 11) and Italia-asfv-pep205-2 (SEQ ID NO: 12) of the present invention are infected with ASFV. It showed high reactivity to serum.
  • Italia-asfv-pep205-1 fragment polypeptide exhibits reactivity (detection ability) to ASFV-infected serum at an equivalent level compared to pK205R full-length protein (denoted as'Italia-asfv-p205').
  • the fragment polypeptide of Italia-asfv-pep205-2 SEQ ID NO: 12
  • the ASFV-infected serum showed better detection ability than the full-length pK205R protein.
  • Example 6 Verification of vaccine effect using African swine fever protein
  • mice Five to six week old female C57BL/6 mice were mixed with the recombinant proteins of African swine fever Italia-asfv-pep205-1 and Italia-asfv-pep205-2 with Freund's adjuvant every two weeks. 200 ⁇ g per individual was inoculated by subcutaneous immunization. As a control group, PBS and Freund's adjuvant were inoculated in the same manner.
  • the ELISA is a coating solution (0.159 g of Na2CO3, 0.293 g of NaHCO3, per 100 ml, pH 9.6) of African swine fever recombinant proteins (Italia-asfv-pep205-1, Italia-asfv-pep205-2), respectively, 3.0 ug/ml
  • adsorption was performed at 4°C for one day.
  • the plate on which antigen adsorption was completed was washed 4 times using PBS, and then PBS containing 5% normal chlorine serum was added to each plate and reacted at 37°C for 2 hours to exclude non-specific binding. .
  • the present invention relates to the development and use of the p72, p104, and p205 protein fragments derived from African swine fever virus (ASFV) as a recombinant antigen, and more specifically, SEQ ID NO: 1, SEQ ID NO. 7, an isolated polypeptide selected from the group consisting of an amino acid sequence represented by SEQ ID NO: 11 and SEQ ID NO: 12, a vaccine composition against ASFV comprising the polypeptide as an active ingredient, and an animal immunization method using the polypeptide, and It relates to a method for detecting/diagnosing ASFV infection using the polypeptide, a diagnostic reagent and a kit.
  • ASFV African swine fever virus
  • the polypeptide consisting of a unique sequence provided by the present invention has a shorter length compared to the native protein in the virus, and has remarkably excellent ability to detect/diagnose ASFV infection serum even compared to the native protein in ASFV, as well as vaccines It has the possibility of being used as a product and also has high industrial applicability because it has high productivity at the industrial level.

Abstract

La présente invention concerne : un polypeptide isolé choisi dans le groupe constitué par les séquences d'acides aminés représentées par SEQ ID NO : 1, SEQ ID NO : 7, SEQ ID NO : 11 et SEQ ID NO : 12 ; une composition de vaccin contre le virus de la peste porcine africaine (ASFV), la composition de vaccin contenant le polypeptide en tant que principe actif ; un procédé d'immunisation d'animal utilisant le polypeptide ; et un procédé de détection/diagnostic d'infection par ASFV, un réactif de diagnostic et un kit utilisant le polypeptide.
PCT/KR2020/007609 2019-06-12 2020-06-11 Fragments protéiques p72, p104, p205 dérivés du virus de la peste porcine africaine et leur utilisation WO2020251284A1 (fr)

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CN113151187A (zh) * 2021-03-26 2021-07-23 南京农业大学 一株非洲猪瘟病毒单克隆抗体杂交瘤细胞及其应用
WO2022140364A3 (fr) * 2020-12-21 2022-08-18 VST LLC dba Medgene Labs Vaccins contre le virus de la peste porcine africaine (ppa)

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* Cited by examiner, † Cited by third party
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WO2022140364A3 (fr) * 2020-12-21 2022-08-18 VST LLC dba Medgene Labs Vaccins contre le virus de la peste porcine africaine (ppa)
CN113151187A (zh) * 2021-03-26 2021-07-23 南京农业大学 一株非洲猪瘟病毒单克隆抗体杂交瘤细胞及其应用

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