A vaccine for preventing African swine fever containing antigenic proteins derived from African swine fever virus

Abstract

The present invention relates to a recombinant vector containing the base sequence of African swine fever virus antigen proteins Lectin, CD2v, p72, p54, p30, p15, p35, E199L, and / or F317L, a transformant transformed with the recombinant vector, and a vaccine composition for preventing African swine fever containing as an active ingredient the Lectin, CD2v, p72, p54, p30, p15, p35, E199L, and / or F317L antigen proteins of African swine fever virus isolated from the transformant, and the like.

Description

[Technical Field]

[0001] The present invention relates to a vaccine for preventing African swine fever, which contains an antigen protein derived from African swine fever virus as an active ingredient.

[0002] The present invention claims priority based on Korean Patent Application No. 10-2021-0114014 filed on August 27, 2021, and Korean Patent Application No. 10-2022-0097525 filed on August 4, 2022, the entire contents of which are incorporated herein by reference in their entirety in their specifications and drawings. [Background technology]

[0003] African swine fever (ASF) is an infectious disease of pigs caused by the African swine fever virus (ASFV), a member of the Asfarviridae family. First reported in Kenya in 1921, ASF cases have been reported primarily in sub-Saharan Africa. Since 2007, the disease has spread to other parts of Africa, including the Black Sea coastal countries of Georgia, Armenia, and Azerbaijan. China, which accounts for roughly half of the world's pig population, suffered enormous property damage and culled approximately 7 million pigs (an estimated 200 million in actuality) when ASF first broke out in 2018. While the disease has been brought under control since then, new cases of ASF were reported in northern China earlier this year. Vietnam has also seen a resurgence of ASF this year, with new cases continuing to be reported across the country, despite the country slaughtering around 36,000 pigs in 2021 alone up until May this year, and over 6 million pigs since 2019. Malaysia, which had previously had no cases, also detected the ASF virus for the first time at the end of February this year.

[0004] In South Korea, ASF cases were first reported at a pig farm in Paju in 2019, with subsequent cases continuing to be reported throughout Gyeonggi and Gangwon provinces. As of September 2020, African swine fever cases were confirmed in wild boars in nine cities and counties in Gyeonggi province, including Paju, Yeoncheon, and Pocheon, and in Gangwon province, including Cheorwon, Hwacheon, Chuncheon, Yanggu, Inje, and Goseong. Of these, Hwacheon in Gangwon province had the highest number of cases with 285, followed by Yeoncheon in Gyeonggi province with 282 and Paju in Gyeonggi province with 98. As a result, extensive fences were installed to prevent the spread of ASF, and active wild boar capture and quarantine measures were implemented.As a result, no ASFV infections were reported for a while after the infection case at the Hwacheon pig farm in Gangwon Province in October 2020, but recently (August 2021), another case of ASF was reported at a pig farm in Gangwon Province.

[0005] African swine fever is a high-risk infectious disease that has a 100% mortality rate when it infects pigs, and as no vaccine has been developed, pigs must be immediately culled when they occur. Therefore, preventing this disease remains a very important issue.

[0006] Furthermore, the remarkable development of molecular biology and genetic engineering technology has also been applied to the plant field, and efforts to produce useful physiologically active substances from plants are steadily continuing. Producing useful substances from plants can significantly reduce production costs and greatly reduce the various sources of contamination (viruses, cancer genes, enterotoxins, etc.) that can occur with conventional methods (methods that synthesize proteins from animal cells or microorganisms and then separate and purify them). Furthermore, unlike animal cells or microorganisms, even at the commercialization stage, it has the advantage of being possible to manage seed stock as seeds.

[0007] As a result of their intensive efforts to develop an antigen for preventing African swine fever, the inventors developed a system that can express African swine fever virus antigen proteins in plants with high efficiency, and confirmed that a specific combination of the antigen proteins can more effectively prevent African swine fever than other combinations of ASFV antigen proteins, thereby completing the present invention. Summary of the Invention [Problem to be solved by the invention]

[0008] African swine fever virus (ASFV) is a large double-stranded DNA virus that replicates in the cytoplasm of infected cells. It causes a fatal hemorrhagic fever in pigs, but also persistently infects its natural hosts, pigs, warthogs, river pigs, and Ornithodoros ticks, which act as vectors without showing any signs of illness. Because this virus causes a fatal hemorrhagic fever in pigs, it is an infectious disease that requires prevention before infection.

[0009] The present invention has been made to address the need for prevention of African swine fever and to resolve the problems of the prior art as described above, and an object of the present invention is to provide a recombinant African swine fever virus-derived recombinant antigen protein that can be efficiently produced using plants and that exhibits high immunogenicity, as well as a vaccine composition containing the same.

[0010] However, the technical problems that the present invention aims to achieve are not limited to those mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the following description. [Means for solving the problem]

[0011] The present invention provides a vaccine composition for preventing African swine fever, comprising a combination of African swine fever virus (ASFV) antigenic proteins as active ingredients, wherein the combination of ASFV antigenic proteins is a combination of one or more selected from the group consisting of lectin, CD2v, p72, p54, and p30 proteins; However, when the combination of ASFV antigenic proteins includes a lectin protein and a CD2v protein, the combination of ASFV antigenic proteins is characterized in that it consists of three or more antigenic proteins.

[0012] In yet another embodiment of the present invention, the vaccine composition may satisfy one or more characteristics selected from the group consisting of, but not limited to: (a) the Lecin protein comprises the amino acid sequence of SEQ ID NO: 1; (b) the CD2v protein comprises the amino acid sequence of SEQ ID NO: 3; (c) the p72 protein comprises the amino acid sequence of SEQ ID NO: 5; (d) the p54 protein comprises the amino acid sequence of SEQ ID NO: 7; and (e) the p30 protein comprises the amino acid sequence of SEQ ID NO:9.

[0013] In yet another embodiment of the present invention, the ASFV antigen protein is produced using a recombinant vector, and the recombinant vector may contain one or more ASFV protein-encoding polynucleotides selected from the group consisting of, but not limited to: (a) a lectin-encoding polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 2; (b) a CD2v-encoding polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 4; (c) a p72-encoding polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 6; (d) a p54-encoding polynucleotide comprising the base sequence represented by SEQ ID NO: 8; and (e) A p30-encoding polynucleotide comprising the base sequence represented by SEQ ID NO: 10.

[0014] In yet another embodiment of the present invention, the recombinant vector may further comprise one or more selected from the group consisting of, but not limited to: (a) a polynucleotide encoding NB of SEQ ID NO: 19 or BiP of SEQ ID NO: 21; (b) a polynucleotide encoding the pFc2 fragment of SEQ ID NO: 27; (c) a polynucleotide encoding the HDEL peptide of SEQ ID NO: 29; and (d) a polynucleotide encoding the M domain of SEQ ID NO: 23 or the polyhistidine tag of SEQ ID NO: 25.

[0015] In yet another embodiment of the present invention, the ASFV antigen protein may be, but is not limited to, produced in a plant transformed with the recombinant vector.

[0016] In yet another embodiment of the present invention, the vaccine composition may further comprise, but is not limited to, an adjuvant.

[0017] In yet another embodiment of the present invention, the adjuvant may be, but is not limited to, a mineral oil or an emulsigen-based adjuvant.

[0018] The present invention also provides a vaccine kit for preventing African swine fever, which comprises the vaccine composition of the present invention.

[0019] The present invention also provides a method for preventing, ameliorating, and / or treating African swine fever, comprising administering a composition according to the present invention (or a combination of one or more proteins selected from the group consisting of lectin, CD2v, p72, p54, and p30 proteins) to an animal other than a human.

[0020] The present invention also provides use of the composition for the prevention, amelioration, and / or treatment of African swine fever.

[0021] The present invention also provides a use of the composition for producing a vaccine for preventing African swine fever.

[0022] The present invention also provides a feed composition for preventing African swine fever, which comprises a combination of ASFV (African swine fever virus, ASFV) antigen proteins as active ingredients, wherein the combination of ASFV antigen proteins is a combination of one or more proteins selected from the group consisting of lectin, CD2v, p72, p54, and p30 proteins; However, when the combination of ASFV antigenic proteins includes a lectin protein and a CD2v protein, the combination of ASFV antigenic proteins is characterized in consisting of three or more antigenic proteins.

[0023] The present invention also provides a polynucleotide encoding a p72 protein comprising the amino acid sequence of SEQ ID NO:5; a polynucleotide encoding a p54 protein comprising the amino acid sequence of SEQ ID NO:7; a polynucleotide encoding a p15 protein comprising the amino acid sequence of SEQ ID NO:11; a polynucleotide encoding a p35 protein comprising the amino acid sequence of SEQ ID NO:13; a polynucleotide encoding an E199L protein comprising the amino acid sequence of SEQ ID NO:15; or a polynucleotide encoding an F317L protein comprising the amino acid sequence of SEQ ID NO:17, The present invention provides a recombinant vector for expressing an antigen protein of African swine fever virus, which is characterized by being expressed in plants.

[0024] In one embodiment of the present invention, the recombinant vector may satisfy one or more characteristics selected from the group consisting of, but not limited to: (a) the polynucleotide encoding the p72 protein comprises the nucleotide sequence represented by SEQ ID NO: 6; (b) the polynucleotide encoding the p54 protein comprises the nucleotide sequence represented by SEQ ID NO: 8; (c) the polynucleotide encoding the p15 protein comprises the nucleotide sequence represented by SEQ ID NO: 12; (d) the polynucleotide encoding the p35 protein comprises the nucleotide sequence represented by SEQ ID NO: 14; (e) the polynucleotide encoding the E199L protein comprises the nucleotide sequence represented by SEQ ID NO: 16; and (f) The polynucleotide encoding the F317L protein comprises the nucleotide sequence shown in SEQ ID NO:18.

[0025] In another embodiment of the present invention, the polynucleotide may further include, but is not limited to, a polynucleotide encoding NB (new chaperone binding protein) of SEQ ID NO: 19 or a polynucleotide encoding BiP (chaperone binding protein) of SEQ ID NO: 21.

[0026] In yet another embodiment of the present invention, the polynucleotide encoding the NB or BiP may be located toward the 5' end of the polynucleotide encoding the p72 protein, p54 protein, p15 protein, p35 protein, E199L protein, or F317L protein, but is not limited to these.

[0027] In yet another embodiment of the present invention, the recombinant vector may further comprise, but is not limited to, a polynucleotide encoding the pFc2 (porcine Fc) fragment of SEQ ID NO:27.

[0028] In yet another embodiment of the present invention, the polynucleotide encoding the pFc2 fragment may be located toward the 3' end of the polynucleotide encoding the p72 protein, p54 protein, p15 protein, p35 protein, E199L protein, or F317L protein, but is not limited to these.

[0029] In yet another embodiment of the present invention, the recombinant vector may further comprise, but is not limited to, a polynucleotide encoding the HDEL (His-Asp-Glu-Leu) peptide of SEQ ID NO:29.

[0030] In yet another embodiment of the present invention, the polynucleotide encoding the HDEL peptide may be located toward the 3' end of the polynucleotide encoding the p72 protein, p54 protein, p15 protein, p35 protein, E199L protein, or F317L protein, but is not limited to these.

[0031] In yet another embodiment of the present invention, the recombinant vector may further comprise, but is not limited to, a polynucleotide encoding NB of SEQ ID NO: 19 or BiP of SEQ ID NO: 21; a polynucleotide encoding the pFc2 fragment of SEQ ID NO: 27; and a polynucleotide encoding the HDEL peptide of SEQ ID NO: 29.

[0032] In yet another embodiment of the present invention, the recombinant vector may be, but is not limited to, a polynucleotide encoding NB or BiP; a polynucleotide encoding p72 protein, p54 protein, p15 protein, p35 protein, E199L protein, or F317L protein; a polynucleotide encoding a pFc2 fragment; and a polynucleotide encoding an HDEL peptide, linked in sequence.

[0033] The present invention also provides a transformant transformed with the recombinant vector according to the present invention.

[0034] In one embodiment of the present invention, the transformant may be, but is not limited to, a plant.

[0035] The present invention also provides a method for producing a recombinant African swine fever virus antigen protein, which comprises the steps of: (S1) transforming a plant body with the recombinant vector according to the present invention; and (S2) A step of isolating and purifying the recombinant antigen protein from the plant body or culture medium.

[0036] In one embodiment of the present invention, the NB-encoding polynucleotide may include the base sequence represented by SEQ ID NO: 20; the BiP-encoding polynucleotide may include the base sequence represented by SEQ ID NO: 22; the pFc2-encoding polynucleotide may include the base sequence represented by SEQ ID NO: 28; and the HDEL-encoding polynucleotide may include, but is not limited to, the base sequence represented by SEQ ID NO: 30. [Effects of the Invention]

[0037] Proteins, particularly antigens, used to prevent viral diseases, including African swine fever, are primarily produced using animal cells rather than bacteria due to issues such as protein folding and glycosylation. However, vaccine production methods using animal cells often require significant costs for expanding facilities for mass production, making production difficult and resulting in expensive antigens. Furthermore, antigens produced using animal cells have drawbacks, such as being difficult to store and being susceptible to contamination by viruses that can infect animals. However, the present invention overcomes these drawbacks by using plants. Unlike animal cells, plant cells are highly unlikely to be contaminated by viruses that can infect animals. Plant cells can be mass-produced at any time if cultivated land is secured, and they can be stored for long periods of time as plants, allowing for stable and inexpensive antigen production.

[0038] The recombinant African swine fever virus antigen protein of the present invention is not only effectively expressed in plants, but also has high water solubility, making it easy to isolate and purify. It also acts as an antigen in the body and exhibits high immunogenicity, making it suitable for use as a novel African swine fever virus vaccine composition. In particular, the present inventors have confirmed through challenge experiments that a vaccine composition containing the five antigen proteins of the present invention (lectin, CD2v, p54, p72, and p30) has a superior preventive effect against African swine fever compared to vaccine compositions further containing other antigen proteins (e.g., p15, p35, E199L, F317L). Therefore, the recombinant vector and vaccine composition of the present invention are expected to be widely used in the livestock industry and other fields. [Brief explanation of the drawings]

[0039] [Figure 1] 1 is a cleavage map showing the sequences of genes for expression of ASFV Lectin, CD2v, p72, p54, p30, p15, p35, E199L, and F317L antigen proteins in the plant body of the present invention. [Figure 2] FIG. 1 shows the results of separating and purifying the ASFV lecin antigen protein, followed by electrophoresis and confirmation by Coomassie blue staining. [Figure 3] FIG. 1 shows the results of separating and purifying the CD2v antigen protein of ASFV, followed by electrophoresis and confirmation by Coomassie blue staining. [Figure 4a] is a photograph of bands obtained by Western blotting to confirm the expression of ASFV p72 antigen protein (DT, Debris Total (total sample before debris removal); T, Total extract (total sample after debris removal); P, pellet fraction; FT, flow-through fraction, the same below). [Figure 4b]This figure shows the results of electrophoresis of protein solutions before and after concentration, after separating and purifying the ASFV p72 antigen protein, followed by Coomassie blue staining (left: p72 protein detection results before concentration; right: p72 protein detection results after concentration). [Figure 5a] 1 is a photograph of bands obtained by Western blotting to confirm the expression of ASFV p54 antigen protein. [Figure 5b] This figure shows the results of electrophoresis of protein solutions before and after concentration, after separating and purifying the ASFV p54 antigen protein, followed by Coomassie blue staining (left: p54 protein detection results before concentration; right: p54 protein detection results after concentration). [Figure 6] FIG. 1 shows the results of separating and purifying the ASFV p30 antigen protein, and examining it by Coomassie blue staining after electrophoresis. [Figure 7a] 1 is a photograph of bands obtained by Western blotting to confirm the expression of ASFV p15 antigen protein. [Figure 7b] FIG. 1 shows the results of separating and purifying the ASFV p15 antigen protein, and examining it by Coomassie blue staining after electrophoresis. [Figure 8] FIG. 1 shows the results of separating and purifying the ASFV p35 antigen protein, and then electrophoresing it and confirming it with Coomassie blue staining. [Figure 9a] 1 is a photograph of bands obtained by Western blotting to confirm the expression of the ASFV E199L antigen protein. [Figure 9b] This figure shows the results of electrophoresis of protein solutions before and after concentration, after separating and purifying the ASFV E199L antigen protein, followed by Coomassie blue staining (left: detection result of E199L protein before concentration; right: detection result of E199L protein after concentration). [Figure 10a]This is a photograph of the bands obtained by Western blotting to confirm the expression of the ASFV F317L antigen protein extracted from 100 g or 1 kg of Nicotiana benthamiana leaves (top: detection results for F317L protein extracted from 100 g of leaves; bottom: detection results for F317L protein extracted from 1 kg of leaves). [Figure 10b] This figure shows the results of separating and purifying the ASFV F317L protein extracted from 100 g or 1 kg of Nicotiana benthamiana leaves, followed by electrophoresis and confirmation by Coomassie blue staining (left: detection results for F317L protein extracted from 100 g of leaves; right: detection results for F317L protein extracted from 1 kg of leaves). [Figure 11] The graph shows the results of a challenge inoculation experiment in which pigs were treated with the five-antigen vaccine (a combination of lectin, CD2v, p54, p72, and p30) of the present invention, the nine-antigen vaccine (a combination of lectin, CD2v, p54, p72, p30, p15, p35, E199L, and F317L), or PBS, and the body temperature and survival rate of the pigs were monitored over time. [Figure 12] The graph shows the results of measuring the viremia levels in the blood of pigs over time after a challenge inoculation experiment in which pigs were treated with the 5-antigen vaccine, 9-antigen vaccine, or PBS according to the present invention. [Figure 13] 1 shows the gene sequence (top) and amino acid sequence (bottom) for expressing a lectin recombinant protein according to one embodiment of the present invention. [Figure 14] 1 shows the gene sequence (top) and amino acid sequence (bottom) for expression of a CD2v recombinant protein according to one embodiment of the present invention. [Figure 15] 1 shows the gene sequence (top) and amino acid sequence (bottom) for expressing p72 recombinant protein according to one embodiment of the present invention. [Figure 16] 1 shows the gene sequence (top) and amino acid sequence (bottom) for expressing p54 recombinant protein according to one embodiment of the present invention. [Figure 17] 1 shows the gene sequence (top) and amino acid sequence (bottom) for expressing p30 recombinant protein according to one embodiment of the present invention. [Figure 18] 1 shows the gene sequence (top) and amino acid sequence (bottom) for expression of p15 recombinant protein according to one embodiment of the present invention. [Figure 19] 1 shows the gene sequence (top) and amino acid sequence (bottom) for expressing p35 recombinant protein according to one embodiment of the present invention. [Figure 20] 1 shows the gene sequence (top) and amino acid sequence (bottom) for expression of E199L recombinant protein according to one embodiment of the present invention. [Figure 21] 1 shows the gene sequence (top) and amino acid sequence (bottom) for expression of F317L recombinant protein according to one embodiment of the present invention. [Figure 22] 1 shows the results of measuring anti-p30 antibody levels on days 0, 7, 14, 21, 28, and 35 after pigs were administered a five-antigen vaccine according to the present invention (a combination of Lectin, CD2v, p54, p72, and p30; "Vax grp1"), a nine-antigen vaccine (a combination of Lectin, CD2v, p54, p72, p30, p15, p35, E199L, and F317L; "Vax grp2"), or PBS ("PBS"). DETAILED DESCRIPTION OF THE INVENTION

[0040] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which this invention belongs. Generally, the nomenclature used herein is well known and commonly used in the art.

[0041] The present inventors have confirmed that highly immunogenic African swine fever virus (ASFV) antigenic proteins, lectin, CD2v, p72, p54, p30, p15, p35, E199L, and F317L, can be efficiently produced and isolated in plants using the genes for these five antigenic proteins. Therefore, the African swine fever virus antigenic proteins of the present invention can be stably and efficiently mass-produced, thereby providing an inexpensive and stable African swine fever virus vaccine. Furthermore, the present inventors have confirmed that a vaccine composition containing the five antigenic proteins, including lectin, CD2v, p72, p54, and p30, exerts excellent preventive effects against African swine fever when administered to pigs, and that the effect is even more pronounced than vaccine compositions that also contain other ASFV antigenic proteins, such as p15, p35, E199L, and F317L. That is, the present invention is significant in that it has discovered an optimal combination of antigen proteins (lectin, CD2v, p72, p54, and p30) that can achieve a more significant preventive effect against African swine fever compared to the prior art.

[0042] Therefore, the present invention provides a recombinant vector for expressing an African swine fever virus (ASFV) antigen protein, comprising a combination of polynucleotides encoding ASFV proteins, wherein the combination of polynucleotides encoding ASFV proteins is one or more combinations selected from the group consisting of: A polynucleotide encoding a lectin protein comprising the amino acid sequence of SEQ ID NO: 1; a polynucleotide encoding a CD2v protein comprising the amino acid sequence of SEQ ID NO: 3; a polynucleotide encoding a p72 protein comprising the amino acid sequence of SEQ ID NO: 5; a polynucleotide encoding a p54 protein comprising the amino acid sequence of SEQ ID NO: 7; a polynucleotide encoding a p30 protein comprising the amino acid sequence of SEQ ID NO: 9; a polynucleotide encoding a p15 protein comprising the amino acid sequence of SEQ ID NO: 11; a polynucleotide encoding a p35 protein comprising the amino acid sequence of SEQ ID NO: 13; A polynucleotide encoding an E199L protein comprising the amino acid sequence of SEQ ID NO: 15; and A polynucleotide encoding the F317L protein comprising the amino acid sequence of SEQ ID NO:17.

[0043] More preferably, the present invention provides a recombinant vector for expressing an African swine fever virus (ASFV) antigen protein, comprising a combination of polynucleotides encoding ASFV proteins, wherein the combination of polynucleotides encoding ASFV proteins is one or more combinations selected from the group consisting of: A polynucleotide encoding a lectin protein comprising the amino acid sequence of SEQ ID NO: 1; a polynucleotide encoding a CD2v protein comprising the amino acid sequence of SEQ ID NO: 3; a polynucleotide encoding a p72 protein comprising the amino acid sequence of SEQ ID NO: 5; A polynucleotide encoding a p54 protein comprising the amino acid sequence of SEQ ID NO: 7; and A polynucleotide encoding a p30 protein comprising the amino acid sequence of SEQ ID NO:9.

[0044] There is no limitation on the order in which the polynucleotides are arranged in the recombinant vector.

[0045] In the present invention, "African swine fever virus" refers to a DNA virus of approximately 200 nm belonging to the Asfarviridae family and the Asfivirus genus, and is the causative agent of African swine fever (ASF). ASFV persistently infects its natural hosts, pigs, warthogs, river boars, and Ornithodoros genus, which belong to the Ardeidae family, and causes fatal hemorrhagic fever upon infection.

[0046] Preferably, the recombinant vector of the present invention comprises polynucleotides encoding the five ASFV proteins (lectin, CD2v, p72, p54, and p30) and does not contain polynucleotides encoding other ASFV proteins. More preferably, the recombinant vector of the present invention does not contain polynucleotides encoding the ASFV antigenic proteins p15, p35, E199L, or F317L.

[0047] The lectin antigen protein can be encoded by a polynucleotide comprising the amino acid sequence of SEQ ID NO: 1 or the nucleotide sequence of SEQ ID NO: 2, and most preferably, can be encoded by a polynucleotide consisting of the amino acid sequence of SEQ ID NO: 1 or the nucleotide sequence of SEQ ID NO: 2, but is not limited to these. Preferably, the lectin protein according to the present invention may include a transmembrane domain, or may be a lectin protein from which the transmembrane domain has been removed.

[0048] Specifically, the polynucleotide encoding the lectin protein may consist of the nucleotide sequence represented by SEQ ID NO: 2, but is not limited to this, and variants of this nucleotide sequence are also included within the scope of the present invention. The nucleic acid molecule of the nucleotide sequence represented by SEQ ID NO: 2 of the present invention encompasses functional equivalents of the nucleic acid molecule that constitutes it, for example, variants in which a portion of the nucleotide sequence of the nucleic acid molecule has been modified by deletion, substitution, or insertion, but which are capable of performing the same functional function as the nucleic acid molecule. Specifically, the polynucleotide encoding the lectin protein may comprise a nucleotide sequence that shares 70% or more, more preferably 80% or more, more preferably 90% or more, and most preferably 95% or more sequence identity with the nucleotide sequence represented by SEQ ID NO: 2. For example, polynucleotides having 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity are included. "Percent sequence identity" for polynucleotides is determined by comparing two optimally aligned sequences and a comparison region, and portions of the polynucleotide sequence in the comparison region may contain additions or deletions (i.e., gaps) compared to a reference sequence (which does not contain additions or deletions) for the optimal alignment of the two sequences.

[0049] Furthermore, the CD2v antigen protein may be encoded by a polynucleotide comprising the amino acid sequence of SEQ ID NO: 3 or the nucleotide sequence of SEQ ID NO: 4, and most preferably consists of the amino acid sequence of SEQ ID NO: 3 or the nucleotide sequence of SEQ ID NO: 4, but is not limited thereto. That is, the polynucleotide encoding the CD2v protein may comprise a nucleotide sequence that has 70% or more, more preferably 80% or more, more preferably 90% or more, and most preferably 95% or more sequence identity with the nucleotide sequence represented by SEQ ID NO: 4. Preferably, the CD2v according to the present invention may comprise the transmembrane domain (TMD) of the CD2v protein or may be a CD2v protein from which the transmembrane domain has been deleted, and preferably may be the N-terminal portion of the CD2v protein from the transmembrane domain.

[0050] Furthermore, the p72 antigen protein may be encoded by a polynucleotide comprising the amino acid sequence of SEQ ID NO: 5 or the nucleotide sequence of SEQ ID NO: 6, and most preferably, the polynucleotide may consist of the amino acid sequence of SEQ ID NO: 5 or the nucleotide sequence of SEQ ID NO: 6, but is not limited thereto. That is, the polynucleotide encoding the p72 protein may comprise a nucleotide sequence having 70% or more, more preferably 80% or more, more preferably 90% or more, and most preferably 95% or more sequence identity with the nucleotide sequence represented by SEQ ID NO: 6.

[0051] Furthermore, the p54 antigen protein may be encoded by a polynucleotide comprising the amino acid sequence of SEQ ID NO: 7 or the nucleotide sequence of SEQ ID NO: 8, most preferably consisting of the amino acid sequence of SEQ ID NO: 7 or the nucleotide sequence of SEQ ID NO: 8, but is not limited thereto. That is, the polynucleotide encoding the p54 protein may comprise a nucleotide sequence having 70% or more, more preferably 80% or more, more preferably 90% or more, and most preferably 95% or more sequence identity with the nucleotide sequence represented by SEQ ID NO: 8. Preferably, the p54 protein of the present invention may be a p54 protein with its transmembrane domain deleted (p54dTM). More preferably, the p54 protein of the present invention may contain the transmembrane domain. Alternatively, the p54 protein may have the transmembrane domain deleted and a linker sequence inserted in its place. The linker sequence may be Gly-Gly-Gly-Gly-Ser.

[0052] Furthermore, the p30 antigen protein may be encoded by a polynucleotide comprising the amino acid sequence of SEQ ID NO: 9 or the nucleotide sequence of SEQ ID NO: 10, and most preferably, the polynucleotide may consist of the amino acid sequence of SEQ ID NO: 9 or the nucleotide sequence of SEQ ID NO: 10, but is not limited thereto. That is, the polynucleotide encoding the p30 protein may comprise a nucleotide sequence having 70% or more, more preferably 80% or more, more preferably 90% or more, and most preferably 95% or more sequence identity with the nucleotide sequence represented by SEQ ID NO: 10.

[0053] Furthermore, the p15, p35, E199L, and F317L antigen proteins may be encoded by a polynucleotide comprising the amino acid sequence of SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, or SEQ ID NO: 17, respectively, or the nucleotide sequence of SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, or SEQ ID NO: 18, respectively, and most preferably may be encoded by a polynucleotide consisting of the amino acid sequence of SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, or SEQ ID NO: 17, respectively, or the nucleotide sequence of SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, or SEQ ID NO: 18, respectively, but are not limited thereto. That is, the polynucleotide encoding the p15, p35, E199L, or F317L antigen protein may comprise a nucleotide sequence having 70% or more, more preferably 80% or more, more preferably 90% or more, and most preferably 95% or more sequence identity to the nucleotide sequence represented by SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, or SEQ ID NO: 18, respectively, respectively.

[0054] Furthermore, the E199L may include the transmembrane region or may exclude the transmembrane region.

[0055] As used herein, a polypeptide (or peptide) consisting of an amino acid sequence represented by a specific SEQ ID NO: is a concept that includes functional equivalents of the polypeptide molecule that constitutes it, such as variants in which a portion of the amino acid sequence of the polypeptide molecule has been modified by deletion, substitution, or insertion, but which can perform the same functional function as the polypeptide molecule. Specifically, the polypeptide represented by the specific SEQ ID NO: may comprise an amino acid sequence that has 70% or more, more preferably 80% or more, more preferably 90% or more, and most preferably 95% or more sequence identity with the amino acid sequence represented by the SEQ ID NO. For example, amino acid sequences having 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% sequence identity are included. "Percent sequence identity" for amino acid sequences is determined by comparing the comparison regions with two optimally aligned sequences, and portions of the amino acid sequence in the comparison region may contain additions or deletions (i.e., gaps) compared to the reference sequence (which does not contain additions or deletions) for the optimal alignment of the two sequences.

[0056] The Lectin, CD2v, p54, p72, p30, p15, p35, E199L, and F317L antigen proteins are antigen proteins of African swine fever virus and can regulate the host immune response mechanism.

[0057] As used herein, the term "polynucleotide" refers to an oligomer or polymer containing two or more linked nucleotides or nucleotide derivatives, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), typically linked together by phosphodiester bonds. Polynucleotide also includes, for example, nucleotide analogs, or DNA and RNA derivatives containing "backbone" bonds other than phosphodiester bonds, such as phosphotriester, phosphoramidate, phosphorothioate, thioester, or peptide bonds (peptide nucleic acids). Polynucleotide includes single-stranded and / or double-stranded polynucleotides, such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), as well as analogs of either RNA or DNA.

[0058] The term "antigen" used in this specification collectively refers to all substances that cause an immune response in the body, and is preferably a virus, chemical substance, bacterium, pollen, cancer cell, or a peptide or protein portion thereof, but is not limited to these, as long as it is a substance that can cause an immune response in the body.

[0059] In one embodiment of the present invention, the recombinant vector may further comprise a polynucleotide encoding a new chaperone binding protein (NB) or chaperone binding protein (BiP) signal peptide, a polynucleotide encoding a porcine pFc2 fragment (porcine Fc fragment), a polynucleotide encoding a His-Asp-Glu-Leu (HDEL) peptide, a polynucleotide encoding an M domain, and / or a polynucleotide encoding a polyhistidine tag.

[0060] In another embodiment of the present invention, the recombinant vector may satisfy one or more of the following characteristics: (a) the polynucleotide encoding the NB or BiP is located 5' towards the polynucleotide encoding the ASFV antigenic protein; (b) the polynucleotide encoding the pFc2 fragment is located toward the 3' end of the polynucleotide encoding the ASFV antigenic protein; (c) the polynucleotide encoding the HDEL peptide is located 3' towards the polynucleotide encoding the ASFV antigenic protein; or (d) The polynucleotide encoding the M domain or the polyhistidine tag is located toward the 5' or 3' end of the polynucleotide encoding the ASFV antigen protein.

[0061] Preferably, the recombinant vector may contain all of the polynucleotide encoding the NB or BiP; the polynucleotide encoding the pFc2 fragment; and the polynucleotide encoding the HDEL peptide. Here, there is no restriction on the order in which each gene is linked, but preferably, the recombinant vector may sequentially link the polynucleotide encoding the NB or BiP; the polynucleotide encoding the Lectin, CD2v, p72, p54, and / or p30 protein; the polynucleotide encoding the pFc2 fragment; and the polynucleotide encoding the HDEL peptide.

[0062] Alternatively, the recombinant vector may contain all of the polynucleotide encoding the NB or BiP; the polynucleotide encoding the pFc2 fragment; and the polynucleotide encoding the HDEL peptide. In this case, there is no restriction on the order in which each gene is linked, but preferably, the recombinant vector may sequentially link the polynucleotide encoding the NB or BiP; the polynucleotide encoding the Lectin, CD2v, p72, p54, p30, p15, p35, E199L, and / or F317L protein; the polynucleotide encoding the pFc2 fragment; and the polynucleotide encoding the HDEL peptide.

[0063] Most preferably, the recombinant vector may contain all of the polynucleotide encoding the NB or BiP; the polynucleotide encoding the pFc2 fragment; the polynucleotide encoding the HDEL peptide; and the polynucleotide encoding the M domain or polyhistidine tag. Here, there is no restriction on the order in which each gene is ligated. Preferably, the recombinant vector may sequentially ligate the polynucleotide encoding the NB or BiP; the M domain or polyhistidine tag; the polynucleotide encoding lectin, CD2v, p72, p54, and / or p30 protein; the polynucleotide encoding the pFc2 fragment; and the polynucleotide encoding the HDEL peptide, or may sequentially ligate the polynucleotide encoding the NB or BiP; the polynucleotide encoding lectin, CD2v, p72, p54, and / or p30 protein; the M domain or polyhistidine tag; the polynucleotide encoding the pFc2 fragment; and the polynucleotide encoding the HDEL peptide.

[0064] Alternatively, the recombinant vector may contain all of the polynucleotide encoding the NB or BiP; the polynucleotide encoding the pFc2 fragment; the polynucleotide encoding the HDEL peptide; and the polynucleotide encoding the M domain or polyhistidine tag. In this case, there is no limitation on the order in which each gene is ligated. Preferably, the recombinant vector contains all of the polynucleotide encoding the NB or BiP; the M domain or polyhistidine tag; and the lectin, CD2v, p72, p54, p30, p15, p35, E199L, and / or F317L protein. Alternatively, a polynucleotide encoding NB or BiP; a polynucleotide encoding Lectin, CD2v, p72, p54, p30, p15 protein, p35 protein, E199L protein, and / or F317L protein; an M domain or polyhistidine tag; a polynucleotide encoding a pFc2 fragment; and a polynucleotide encoding HDEL peptide may be linked in this order.

[0065] In yet another embodiment of the present invention, the polynucleotide encoding the polyhistidine tag is located only toward the 5' end of the polynucleotide encoding lectin and / or the 3' end of the polynucleotide encoding p30 in the ASFV antigen protein gene, and the M domain is located only toward the 5' end of the polynucleotide encoding CD2v in the ASFV antigen protein gene. In yet another embodiment, the polynucleotide encoding the pFc2 fragment is not located toward the 3' end of the polynucleotide encoding p30.

[0066] When linked in the above order, i.e., when the recombinant vector contains the expression cassette shown in the cleavage map of FIG. 1, the recombinant vector according to the present invention comprises the nucleotide sequence represented by SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, or SEQ ID NO: 50, and most preferably consists of the nucleotide sequence represented by SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, or SEQ ID NO: 50, or may comprise a nucleotide sequence having 80% or more, more preferably 90% or more, and more preferably 95% or more sequence identity to the nucleotide sequence represented by SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, or SEQ ID NO: 50, respectively. The base sequences represented by SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, and SEQ ID NO: 50 may be contained in plasmid vectors independent of each other, and a combination of one or more selected from the group consisting of SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, and SEQ ID NO: 50 may be contained in a single plasmid vector.

[0067] As used herein, the term "recombinant vector" refers to a vector capable of expressing a peptide or protein encoded by a heterologous nucleic acid inserted therein, preferably a vector constructed to express a target antigen (in this invention, the ASFV antigens lectin, CD2v, p54, p72, p30, p15, p35, E199L, and / or F317L). The term "vector" refers to any vehicle for introducing and / or transferring bases into a host cell in vitro, ex vivo, or in vivo. It may also be a replication unit to which other DNA fragments are bound, resulting in replication of the fragments bound thereto. A "replication unit" refers to any genetic unit (e.g., a plasmid, phage, cosmid, chromosome, virus, etc.) that functions as an autonomous unit of DNA replication in vivo, i.e., that can replicate under its own control. Preferably, the recombinant vector of the present invention is characterized by its expression in plants.

[0068] The recombinant vector of the present invention may preferably include a promoter, which is a transcription initiation factor to which RNA polymerase binds, an optional operator sequence for regulating transcription, a sequence encoding an appropriate mRNA ribosome binding site, a sequence for regulating the termination of transcription and translation, a terminator, etc., and more preferably may further include a polyhistidine tag (an amino acid motif consisting of at least five or more histidine residues), an endoplasmic reticulum signal peptide (same meaning as endoplasmic reticulum targeting sequence) gene, an endoplasmic reticulum retention signal peptide, a cloning site, etc., and more preferably may further include, in addition to the Fc fragment as a tag, a gene for an additional tag, a selection marker gene such as an antibiotic resistance gene for selecting transformants, etc.

[0069] Representative examples of the tagging genes include Avi tag, Calmodulin tag, polyglutamate tag, E tag, FLAG tag, HA tag, His tag (polyhistidine tag), Myc tag, S tag, SBP tag, IgG-Fc tag, CTB tag, Softag 1 tag, Softag 3 tag, Strep tag, TC tag, V5 tag, VSV tag, and Xpress tag.

[0070] As used herein, the term "Fc fragment" refers to the portion of immunoglobulin digested with papain in which only the heavy chain (H chain) is linked by S-type disulfide bonds and which does not have an antigen-binding site. The Fc fragment of the present invention is preferably a porcine Fc fragment, more preferably the porcine Fc fragment (pFc2) represented by SEQ ID NO: 28, but is not limited thereto. Furthermore, variants of the nucleotide sequence represented by SEQ ID NO: 28 are included within the scope of the present invention as Fc fragments of the present invention. Specifically, the gene may comprise a nucleotide sequence having 90% or more, more preferably 95% or more, and most preferably 98% or more sequence identity with the nucleotide sequence of SEQ ID NO: 28.

[0071] The "cloning site" is a general term for a site inserted for the purpose of linking / separating each gene within a vector. Preferably, the cloning site may be, but is not limited to, a sequence represented by "tctaga," a sequence represented by "ggatcc," a sequence represented by "cccggg," or a sequence represented by "gagctc" in SEQ ID NOs: 2, 4, 6, 8, and 10.

[0072] The "endoplasmic reticulum signal peptide" is a signal peptide located at the N-terminus of a protein, and serves to guide a newly synthesized protein into the endoplasmic reticulum (ER). The endoplasmic reticulum signal peptide according to the present invention is not limited in type or amino acid sequence as long as it is a plant endoplasmic reticulum signal peptide known to those skilled in the art, and is preferably selected from NB (New chaperone binding protein) and BiP (chaperone binding protein).

[0073] The "NB (New chaperone binding protein) gene" is preferably a gene comprising the nucleotide sequence of SEQ ID NO: 20, and most preferably a gene represented by SEQ ID NO: 20, but may also comprise a nucleotide sequence having 80% or more, more preferably 90% or more, and more preferably 95% or more sequence identity to the nucleotide sequence of SEQ ID NO: 20. The "Bip (chaperone binding protein) gene" is preferably a gene comprising the nucleotide sequence of SEQ ID NO: 22, and most preferably a gene represented by SEQ ID NO: 22, but may also comprise a nucleotide sequence having 80% or more, more preferably 90% or more, and more preferably 95% or more sequence identity to the nucleotide sequence of SEQ ID NO: 22. As described above, the NB or BiP gene is used to transport an expressed recombinant protein to the endoplasmic reticulum. When the recombinant protein is expressed, a portion of the sequence may be truncated, leaving only some amino acids, or the entire sequence may be truncated, eliminating the signal peptide sequence.

[0074] The "ER retention signal peptide" is a signal peptide located at the C-terminus of a protein, and when a protein present in the endoplasmic reticulum escapes to the Golgi apparatus via the secretory pathway, it serves to retain the protein back into the endoplasmic reticulum. The ER retention signal peptide of the present invention is not limited in type or amino acid sequence as long as it is a plant ER retention signal peptide known to those skilled in the art, but is preferably selected from the KDEL (Lys-Asp-Glu-Leu) sequence and the HDEL sequence.

[0075] Most preferably, the amino acid sequence of the ER retention signal peptide may be HDEL (His-Asp-Glu-Leu, amino acids represented by SEQ ID NO: 29), or may be encoded by the nucleotide sequence represented by SEQ ID NO: 30. Furthermore, variants of SEQ ID NO: 30 are included within the scope of the endoplasmic reticulum signal peptide of the present invention. Specifically, the gene may contain a nucleotide sequence that has 90% or more, more preferably 95% or more, and most preferably 98% or more sequence identity with the nucleotide sequence represented by SEQ ID NO: 30. The binding site of the endoplasmic reticulum signal peptide is characterized in that it is added (or linked) to the C-terminus of a protein to be expressed or synthesized in plant cells.

[0076] For information regarding the endoplasmic reticulum signal peptide and endoplasmic reticulum retention signal peptide, references such as US20130295065 and WO2009158716 can be referred to.

[0077] Examples of the selectable marker gene include herbicide resistance genes such as glyphosate or phosphinothricin, antibiotic resistance genes such as kanamycin, G418, bleomycin, hygromycin, and chloramphenicol, and the aadA gene. Examples of the promoter include pEMU promoter, MAS promoter, histone promoter, Clp promoter, cauliflower mosaic virus-derived 35S promoter, cauliflower mosaic virus-derived 19S RNA promoter, plant actin protein promoter, ubiquitin protein promoter, CMV (Cytomegalovirus) promoter, SV40 (Simian virus 40) promoter, RSV (Respiratory syncytial virus) promoter, and EF-1α (Elongation factor-1) promoter. Examples of the terminator include the CaMV alpha promoter, pEMU promoter, MAS promoter, histone promoter, Clp promoter, MacT (CaMV 35S+MAS; the 3'-terminal nucleotide of the Mac promoter is replaced with T) promoter, and examples of the terminator include nopaline synthase (NOS), rice amylase RAmy1 A terminator, patholin terminator, the octopine gene terminator of Agrobacterium tumefaciens, the Escherichia coli rrnB1 / B2 terminator, the Arabidopsis thaliana HSP18.2 terminator, and the Arabidopsis thaliana RD29B terminator, but the above-mentioned lists are merely examples and are not limited to these.

[0078] In another aspect, the present invention provides a transformant transformed with the above-mentioned recombinant vector.

[0079] In one embodiment of the present invention, the transformant may be preferably a microorganism such as Escherichia coli, Bacillus, Salmonella, yeast, etc., an insect cell, an animal cell including a human, an animal cell such as a mouse, a rat, a dog, a monkey, a pig, a horse, a cow, etc., an Agrobacterium tumefaciens, a plant, etc., and more preferably a food crop including rice, wheat, barley, corn, a bean, a potato, azuki bean, an oat, and sorghum; Arabidopsis thaliana, Chinese cabbage, radish, chili pepper, strawberry, tomato, watermelon, cucumber, etc. Vegetable crops including cabbage, Japanese melon, pumpkin, leek, onion, and carrot; specialty crops including ginseng, tobacco, Japanese laurel, sesame, sugarcane, sugar beet, perilla, peanut, and rapeseed; fruit trees including apple trees, pear trees, jujubes, peaches, grapes, mandarin oranges, persimmons, plums, apricots, and bananas; and flowering plants including roses, carnations, chrysanthemums, lilies, and tulips, as long as they can be transformed with the vectors of the present invention, but are not limited to these. Most preferably, the transformant may be a Nicotiana plant.

[0080] As used herein, "transformation" collectively refers to the change in the genetic properties of an organism due to injected DNA, and a "transgenic organism" refers to an organism produced by injecting an external gene using a molecular genetic method, preferably an organism transformed with the recombinant expression vector of the present invention. The organism may be any living organism, including microorganisms, eukaryotic cells, insects, animals, and plants, and may preferably be, but is not limited to, Escherichia coli, Salmonella, Bacillus, yeast, animal cells, mice, rats, dogs, monkeys, pigs, horses, cattle, Acrobacterium tumefaciens, and plants.

[0081] As used herein, the term "plant" refers to any plant capable of mass-producing proteins containing the antigens of the present invention. More specifically, the plant may be selected from the group consisting of tobacco, Arabidopsis, corn, rice, soybean, canola, alfalfa, sunflower, sorghum, wheat, sorghum, peanut, tomato, potato, lettuce, and chili pepper, preferably tobacco. The tobacco used in the present invention is a plant of the Nicotiana genus, and is not particularly limited to any species as long as it is capable of overexpressing proteins. The present invention can be carried out by selecting an appropriate variety suited to the transformation method and the purpose of mass-producing the protein. For example, varieties such as Nicotiana benthamiana L. or Nicotiana tabacum cv. xanthi can be used.

[0082] The transformant can be prepared by methods such as transformation, transfection, Agrobacterium-mediated transformation, particle gun bombardment, sonication, electroporation, and PEG (Polyethylene glycol)-mediated transformation, but there is no limitation as long as the method allows for injection of the vector of the present invention.

[0083] In yet another aspect, the present invention provides recombinant proteins for inducing antibodies against African swine fever virus, which are produced using the recombinant vectors of the present invention. That is, the present invention provides lectin, CD2v, p54, p72, p30, p15, p35, E199L, and F317L recombinant antigen proteins, which are produced using the recombinant vectors of the present invention. The antigen proteins may be produced from separate recombinant vectors (i.e., separate nucleic acid molecules), or may be produced from a recombinant vector containing polynucleotides encoding two or more recombinant proteins (i.e., a single nucleic acid molecule). In particular, the present invention primarily aims to provide lectin, CD2v, p54, p72, and p30 recombinant antigen proteins, which are produced using the recombinant vectors of the present invention.

[0084] In one embodiment of the present invention, the lectin, CD2v, p54, p72, p30, p15, p35, E199L, and F317L recombinant antigen proteins may be water-soluble. More specifically, the lectin, CD2v, p54, p72, p30, p15, p35, E199L, and F317L recombinant antigen proteins expressed in plants may be 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% dissolved in the water-soluble fraction.

[0085] In another embodiment of the present invention, the lectin, CD2v, p54, p72, p30, p15, p35, E199L, and F317L recombinant antigen proteins can be isolated and purified to a purity of 85% or more. More specifically, when the lectin, CD2v, p54, p72, p30, p15, p35, E199L, and F317L recombinant antigen proteins are expressed in plants using the recombinant vectors of the present invention, lectin, CD2v, p54, p72, p30, p15, p35, E199L, and F317L recombinant antigen proteins can be obtained with a purity of 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% by conventional isolation and purification methods.

[0086] In yet another aspect, the present invention provides a vaccine composition for preventing African swine fever, comprising a combination of African swine fever virus antigenic proteins as active ingredients, wherein the combination of ASFV antigenic proteins is a combination of one or more selected from the group consisting of lectin, CD2v, p72, p54, p30, p15, p35, E199L, and / or F317L proteins. Preferably, the combination of ASFV antigenic proteins may be a combination of one or more selected from the group consisting of lectin, CD2v, p72, p54, and p30. Most preferably, the combination of ASFV antigenic proteins consists of lectin, CD2v, p72, p54, and p30. The present invention also provides a pharmaceutical composition for preventing or treating African swine fever, comprising a combination of African swine fever virus antigenic proteins as active ingredients, wherein the combination of ASFV antigenic proteins is a combination of one or more proteins selected from the group consisting of lectin, CD2v, p72, p54, p30, p15, p35, E199L, and / or F317L proteins. Preferably, the pharmaceutical composition is for preventing African swine fever.

[0087] Hereinafter, the explanations regarding the vaccine composition of the present invention are equally applicable to the pharmaceutical composition of the present invention, and conversely, the explanations regarding the pharmaceutical composition of the present invention are equally applicable to the vaccine composition of the present invention.

[0088] Preferably, when the combination of ASFV antigenic proteins comprises a lectin protein and a CD2v protein, the combination of ASFV antigenic proteins may consist of three or more antigenic proteins, i.e., a composition comprising a lectin protein and a CD2v protein may further comprise other ASFV antigenic proteins (e.g., p72, p54, and / or p30) in addition to the lectin protein and the CD2v protein.

[0089] Preferably, the ASFV antigen protein contained in the vaccine composition according to the present invention is produced using the recombinant vector according to the present invention. Preferably, the ASFV antigen protein is produced in a plant body using the recombinant vector according to the present invention.

[0090] Furthermore, the vaccine composition according to the present invention is characterized in that it does not contain any ASFV antigenic proteins other than lectin, CD2v, p72, p54, or p30 recombinant proteins. Preferably, the ASFV antigenic proteins other than lectin, CD2v, p72, p54, or p30 recombinant proteins may be one or more selected from the group consisting of p15, p35, E199L, and F317L. That is, in the most preferred embodiment, the vaccine composition according to the present invention is a vaccine composition containing five types of lectin, CD2v, p54, p72, and p30 recombinant antigenic proteins, and is characterized in that it has higher antibody production induction effects against ASFV antigens, higher ASFV infection inhibition effects, and higher African swine fever prevention effects than vaccines that further contain additional antigens (e.g., p15, p35, E199L, F317L). For example, the vaccine composition can induce antibody production against ASFV antigens more rapidly than vaccines that further contain p15, p35, E199L, F317L, etc.

[0091] The present invention also provides a method for preventing or treating African swine fever, comprising administering a vaccine composition or pharmaceutical composition according to the present invention to an individual in need thereof. Preferably, the method for preventing or treating African swine fever according to the present invention is characterized in that the individual is not administered an ASFV antigen protein other than lectin, CD2v, p72, p54, or p30 recombinant protein (e.g., p15, p35, E199L, and / or F317L).

[0092] In one embodiment of the present invention, the vaccine composition may further comprise an adjuvant, preferably a mineral oil or Emulsigen-based adjuvant, more preferably a Drakeol 5 oil-based adjuvant, and most preferably, SEA1, but is not limited to these.

[0093] As used herein, the term "adjuvant" refers to a substance or composition added to a vaccine or pharmaceutically active ingredient to enhance or influence the immune response. Typically, it refers to an immunogen carrier or auxiliary substance and / or other pharmaceutically active substance or composition. Typically, the term "adjuvant" should be interpreted broadly and refers to a wide range of substances or strategies that can enhance the immunogenicity of an antigen incorporated into or administered with the adjuvant. Adjuvants can also be classified as immune potentiators, antigen delivery systems, or combinations thereof, but are not limited to these. Examples of suitable adjuvants include aluminum hydroxide, Freund's complete or incomplete adjuvant, DEAE-dextran, levamisole, PCG, and poly I:C or poly A:U. In one embodiment of the present invention, Drakeol 5 oil-based SEA1 adjuvant was used.

[0094] As used herein, the term "vaccine" refers to a biological preparation containing an antigen that induces an immune response in the body, and is an immunogen that induces immunity in the body by being injected or orally administered to humans or animals for the prevention of infectious diseases. The animal may be a human or a non-human animal, and the non-human animal may include, but is not limited to, pigs, cows, horses, dogs, goats, sheep, etc.

[0095] As used herein, "solubility" refers to the degree to which a target protein or peptide can be dissolved in a solvent suitable for administration to the human body. Specifically, it refers to the degree to which a solute is saturated in a given solvent at a specific temperature. Solubility can be measured by determining the saturation concentration of the solute. For example, an excess amount of the solute is added to a solvent, stirred, and filtered, and the concentration can be measured using, but is not limited to, a UV spectrometer or HPLC. High solubility is advantageous for the separation and purification of recombinant proteins, as it inhibits aggregation of the recombinant protein and helps maintain the physiological or pharmacological activity of the recombinant protein.

[0096] The content of the antigen protein in the vaccine composition or pharmaceutical composition of the present invention can be appropriately adjusted depending on the symptoms of the disease, the progression of the symptoms, the condition of the patient, etc., and may be, for example, 0.0001 to 99.9 wt % or 0.001 to 50 wt % based on the total weight of the composition, but is not limited thereto. The content ratio is based on the dry weight after removal of the solvent.

[0097] The vaccine composition or pharmaceutical composition according to the present invention may further comprise suitable carriers, excipients, and diluents commonly used in the manufacture of pharmaceutical compositions, such as one or more selected from the group consisting of diluents, binders, disintegrants, lubricants, adsorbents, humectants, film coating materials, and controlled-release additives.

[0098] The vaccine composition or pharmaceutical composition according to the present invention can be formulated by a conventional method into the form of a powder, granules, sustained-release granules, enteric-coated granules, liquid, eye drops, elixir, emulsion, suspension, spirits, troches, perfumes, lemonades, tablets, sustained-release tablets, enteric-coated tablets, sublingual tablets, hard capsules, soft capsules, sustained-release capsules, enteric-coated capsules, pills, tinctures, soft extracts, dry extracts, fluid extracts, injections, capsules, perfusion solutions, plasters, lotions, pastes, sprays, inhalants, patches, sterile injection solutions, or external preparations such as aerosols, and the external preparations may be in the form of a cream, gel, patch, spray, ointment, plaster, lotion, liniment, paste, or cataplasm.

[0099] Carriers, excipients and diluents that may be included in vaccine compositions or pharmaceutical compositions according to the invention include lactose, dextrose, sucrose, oligosaccharides, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

[0100] When the formulation is made, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, surfactants, etc.

[0101] Additives for the tablets, powders, granules, capsules, pills, and lozenges according to the present invention include excipients such as corn starch, potato starch, wheat starch, lactose, sucrose, glucose, fructose, D-mannitol, precipitated calcium carbonate, synthetic aluminum silicate, calcium hydrogen phosphate, calcium sulfate, sodium chloride, sodium bicarbonate, purified lanolin, microcrystalline cellulose, dextrin, sodium alginate, methylcellulose, sodium carboxymethylcellulose, kaolin, iodine, colloidal silica gel, hydroxypropyl starch, hydroxypropylmethylcellulose (HPMC), HPMC1928, HPMC2208, HPMC2906, HPMC2910, propylene glycol, casein, calcium lactate, and Primogel; gelatin, gum arabic, ethanol, agar powder, cellulose acetate phthalate, carboxymethylcellulose, calcium carboxymethylcellulose, glucose, purified water, and casein. Binders that can be used include sodium carbonate, glycerin, stearic acid, sodium carboxymethylcellulose, sodium methylcellulose, methylcellulose, microcrystalline cellulose, dextrin, hydroxycellulose, hydroxypropyl starch, hydroxymethylcellulose, purified shellac, starch paste, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl alcohol, and polyvinylpyrrolidone. Other binders that can be used include hydroxypropyl methylcellulose, corn starch, agar powder, methylcellulose, bentonite, hydroxypropyl starch, sodium carboxymethylcellulose, sodium alginate, calcium carboxymethylcellulose, calcium citrate, sodium lauryl sulfate, anhydrous silicic acid, L-hydroxypropyl cellulose, dextran, ion exchange resins, polyvinyl acetate, formaldehyde-treated casein and gelatin, alginic acid, amylose, and guar gum. gum), baking soda, polyvinylpyrrolidone, calcium phosphate, gelling starch, gum arabic, amylovectin, pectin, sodium polyphosphate, ethyl cellulose, white sugar, magnesium aluminum silicate, D-sorbitol solution, light anhydrous silicic acid, and other disintegrants;Lubricants that can be used include calcium stearate, magnesium stearate, stearic acid, hydrogenated vegetable oil, talc, lycopodium, kaolin, petrolatum, sodium stearate, cocoa butter, sodium salicylate, magnesium salicylate, polyethylene glycol (PEG) 4000, PEG 6000, liquid paraffin, hydrogenated soybean oil (Lubri wax), aluminum stearate, zinc stearate, sodium lauryl sulfate, magnesium oxide, macrogol, synthetic aluminum silicate, silicic anhydride, higher fatty acids, higher alcohols, silicone oil, paraffin oil, polyethylene glycol fatty acid ether, starch, sodium chloride, sodium acetate, sodium oleate, DL-leucine, and light silicic anhydride.

[0102] Examples of additives that can be used in the liquid preparation according to the present invention include water, dilute hydrochloric acid, dilute sulfuric acid, sodium citrate, sucrose monostearates, polyoxyethylene sorbitol fatty acid esters (twin esters), polyoxyethylene monoalkyl ethers, lanolin ethers, lanolin esters, acetic acid, hydrochloric acid, aqueous ammonia, ammonium carbonate, potassium hydroxide, sodium hydroxide, prolamine, polyvinylpyrrolidone, ethyl cellulose, and sodium carboxymethylcellulose.

[0103] The syrup of the present invention may contain a solution of sucrose, other sugars or sweeteners, and may optionally contain flavorings, coloring agents, preservatives, stabilizers, suspending agents, emulsifiers, thickeners, etc.

[0104] The emulsion of the present invention can contain purified water, and may contain emulsifiers, preservatives, stabilizers, fragrances, etc. as needed.

[0105] The suspension according to the present invention may contain suspending agents such as acacia, tragacanth, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, microcrystalline cellulose, sodium alginate, hydroxypropylmethylcellulose (HPMC), HPMC1828, HPMC2906, and HPMC2910, and may contain surfactants, preservatives, stabilizers, coloring agents, and fragrances as needed.

[0106] The injections according to the present invention may contain solvents such as distilled water for injection, 0.9% sodium chloride injection, Ringer's injection, dextrose injection, dextrose + sodium chloride injection, PEG, lactated Ringer's injection, ethanol, propylene glycol, non-volatile oils - sesame oil, cottonseed oil, peanut oil, soybean oil, corn oil, ethyl oleate, isopropyl myristate, benzene benzoate; solubilizers such as sodium benzoate, sodium salicylate, sodium acetate, iodine, urethane, monoethylacetamide, butazolidine, propylene glycol, teufels, nicotinamide, hexamine, dimethylacetamide; weak acids and their salts (acetic acid and sodium acetate), weak bases and their salts (ammonia and ammonium acetate), It may also contain buffers such as organic compounds, proteins, albumin, peptone, and gums; isotonic agents such as sodium chloride; stabilizers such as sodium bisulfite (NaHSO3), carbon dioxide gas, sodium metabisulfite (Na2S2O5), sodium sulfite (Na2SO3), nitrogen gas (N2), and ethylenediaminetetraacetic acid; sulfating agents such as sodium bisulfite 0.1%, sodium formaldehyde sulfoxylate, thiourea, disodium ethylenediaminetetraacetic acid, and acetone sodium bisulfite; soothing agents such as benzyl alcohol, chlorobutanol, procaine hydrochloride, glucose, and calcium gluconate; and suspending agents such as sodium CMC, sodium alginate, Tween 80, and aluminum monostearate.

[0107] Suppositories according to the present invention may contain any of the following ingredients: cocoa butter, lanolin, Witepsol, polyethylene glycol, glycerogelatin, methylcellulose, carboxymethylcellulose, a mixture of stearic and oleic acids, Subanal, cottonseed oil, peanut oil, coconut oil, cocoa butter plus cholesterol, lecithin, lanet wax, glycerol monostearate, Tween or Span, Imhausen, Monolen (propylene glycol monostearate), glycerin, Adeps solidus, Buytyrum Tego-G, Cebes Pharma 16, 16), Hexalide Base 95, Cotomar, Hydrokote SP, S-70-XXA, S-70-XX75 (S-70-XX95), Hydrokote 25, Hydrokote 711, Idropostal, Massa Estralium Bases such as estrarium, A, AS, B, C, D, E, I, T), Massa-MF, Maspol, Maspol-15, Neospostal-N, Paramount-B, Sposhiro (OSI, OSIX, A, B, C, D, H, L), suppository base IV type (AB, B, A, BC, BBG, E, BGF, C, D, 299), Spostal (N, Es), Wecoby (W, R, S, M, Fs), and tegester triglyceride base (TG-95, MA, 57) can be used.

[0108] Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations are prepared by mixing the extract with at least one or more excipients, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used.

[0109] Liquid preparations for oral administration include suspensions, oral solutions, emulsions, syrups, etc., and may contain various excipients such as wetting agents, sweeteners, flavorings, preservatives, etc. in addition to the commonly used simple diluents water and liquid paraffin. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Examples of non-aqueous solvents and suspensions that can be used include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

[0110] The vaccine composition or pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, a "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to any medical treatment, and the effective dose level can be determined based on factors including the type and severity of the patient's disease, drug activity, drug sensitivity, administration time, administration route and excretion rate, treatment duration, concurrently used drugs, and other factors well known in the medical field.

[0111] The vaccine composition or pharmaceutical composition according to the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered singly or in multiple doses. It is important to administer an amount that can achieve maximum effect with the minimum amount without side effects, taking into consideration all of the above factors, and this can be easily determined by a person of ordinary skill in the art to which the present invention pertains.

[0112] The vaccine composition or pharmaceutical composition of the present invention can be administered to an individual by a variety of routes. All modes of administration are contemplated, including oral administration, subcutaneous injection, intraperitoneal administration, intravenous injection, intramuscular injection, paraspinal space (intrathecal) injection, sublingual administration, buccal administration, rectal insertion, vaginal insertion, ocular administration, aural administration, nasal administration, inhalation, spraying through the mouth or nose, dermal administration, transdermal administration, etc. Preferably, the composition of the present invention can be administered intramuscularly.

[0113] The vaccine composition or pharmaceutical composition of the present invention is determined by the type of drug as an active ingredient, as well as various related factors such as the disease to be treated, the administration route, the patient's age, sex, weight, and disease severity. Specifically, the effective amount of the composition of the present invention may vary depending on the patient's age, sex, and weight, and is generally 0.001 to 150 mg per kg of body weight, preferably 0.01 to 100 mg, administered daily or every other day, or in 1 to 3 divided doses per day. However, since the amount may increase or decrease depending on the administration route, disease severity, sex, weight, age, etc., the above dosage does not in any way limit the scope of the present invention.

[0114] In the present invention, the term "individual" refers to a subject in need of disease treatment, and more specifically refers to mammals such as human or non-human primates, mice, rats, dogs, cats, horses, and cows. For example, in the present specification, the term "individual" refers to a subject to which the recombinant African swine fever antigen protein of the present invention can be administered, and there is no limitation on the subject.

[0115] In the present invention, "administration" means providing a given composition of the present invention to an individual by any suitable method.

[0116] In the present invention, "prevention" refers to any action that inhibits or delays the onset of a target disease, "treatment" refers to any action that improves or beneficially alters a target disease and its associated metabolic disorder symptoms by administering a pharmaceutical composition according to the present invention, and "amelioration" refers to any action that reduces parameters related to a target disease, such as the severity of symptoms, by administering a composition according to the present invention. For example, in the present specification, "prevention" refers to any action that inhibits or delays the onset of African swine fever by administering a recombinant African swine fever antigen protein according to the present invention. Also, in the present specification, "treatment" refers to any action that improves or beneficially alters symptoms of African swine fever by administering a recombinant African swine fever antigen protein according to the present invention.

[0117] More specifically, the vaccine composition of the present invention can be formulated into oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, as well as sterile injectable solutions, by conventional methods. Conventional diluents or excipients, such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants, can be used for formulation. Solid dosage forms for oral administration include tablets, pills, powders, granules, and capsules, and can be prepared by mixing the lecithin-like emulsifier with at least one or more excipients, such as starch, calcium carbonate, sucrose or lactose, and gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Liquid preparations for oral administration can include suspensions, oral solutions, emulsions, syrups, etc., which may contain various excipients such as wetting agents, sweeteners, flavoring agents, preservatives, etc. in addition to water and liquid paraffin, which are frequently used simple diluents. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous preparations, suspensions, emulsions, and lyophilized preparations. Examples of non-aqueous preparations and suspensions that can be used include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

[0118] Routes of administration of the vaccine compositions of the present invention include, but are not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual, or rectal. Oral or parenteral administration is preferred. The term "parenteral" as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intradural, intralesional, and intracranial injection or infusion techniques. The vaccine compositions of the present invention can also be administered in the form of suppositories for rectal administration.

[0119] The dosage of the vaccine composition or pharmaceutical composition according to the present invention is selected taking into consideration the age, weight, sex, physical condition, etc. of the individual. The amount required to induce an immune protective response in an individual without particular side effects will vary depending on the recombinant protein used as the immunogen and the optional presence of excipients.

[0120] Preferably, Lectin, CD2v, p54, p72, p15, p35, E199L, and / or F317L are present in an amount of 10 μg to 1 mg, 10 to 900 μg, 10 μg to 800 μg, 10 μg to 700 μg, 10 μg to 600 μg, 10 μg to 500 μg, 10 μg to 400 μg, 10 μg to 300 μg, 10 μg to 200 μg, or 50 μg per 2 ml of the vaccine composition according to the present invention. The amount of p30 may be, but is not limited to, 1 μg to 150 μg, 70 μg to 130 μg, 80 μg to 120 μg, or 90 μg to 110 μg, and p30 may be 1 μg to 100 μg, 1 μg to 90 μg, 1 μg to 80 μg, 1 μg to 70 μg, 1 μg to 60 μg, 1 μg to 50 μg, 10 μg to 50 μg, 15 μg to 45 μg, 20 μg to 40 μg, or 25 μg to 35 μg.

[0121] In yet another aspect of the present invention, the present invention provides a vaccine kit for preventing African swine fever, comprising a vaccine composition according to the present invention. The kit may include, in addition to the vaccine composition of the present invention, tools, reagents, etc. commonly used in the art for vaccine administration, without limitation. The kit may also include instructions describing the characteristics and information of the vaccine composition, recombinant vector, etc. according to the present invention, and may also include instructions describing a method for producing the vaccine composition according to the present invention.

[0122] In yet another aspect, the present invention provides a method for producing a recombinant ASFV antigen protein, comprising the steps of: (S1) transforming a plant body with the recombinant vector according to the present invention; and (S2) A step of isolating and purifying the recombinant antigen protein from the plant body or culture medium.

[0123] In yet another aspect, the present invention provides a method for producing a vaccine composition or vaccine kit for preventing African swine fever, the method comprising the steps of: (S1) transforming a plant body with the recombinant vector according to the present invention; (S2) isolating and purifying the recombinant antigen protein from the plant body or culture medium; and (S3) A step of producing a vaccine composition or a vaccine kit using the isolated and purified recombinant antigen protein.

[0124] The "transformation" in step (S1) has been described in detail above. The transformation method in step (S1) is not limited to a specific type, but may preferably be performed using an Agrobacterium-mediated transformation method. Specifically, step (S1) may include the steps of transforming an Agrobacterium strain with a recombinant vector according to the present invention; culturing the transformed Agrobacterium strain; and administering (injecting or inoculating) the cultured strain to a plant. Preferably, the plant may be tobacco.

[0125] The step (S2) of isolating and purifying the recombinant antigen protein can be carried out by any protein isolation and purification method commonly known in the art. Preferably, the protein can be isolated by adding a protein extract solution to the transformed plant. Alternatively, the protein can be purified by chromatography. Preferably, the chromatography can be affinity chromatography, and the ligand used in this step can be, but is not limited to, protein A, Ni-IDA, etc.

[0126] In yet another aspect, the present invention provides a feed composition for preventing African swine fever, comprising a combination of African swine fever virus antigenic proteins as active ingredients, wherein the combination of ASFV antigenic proteins comprises one or more selected from the group consisting of lectin, CD2v, p72, p54, and p30 proteins. Preferably, when the combination of ASFV antigenic proteins comprises lectin protein and CD2v protein, the combination of ASFV antigenic proteins consists of three or more antigenic proteins.

[0127] Preferably, the ASFV antigen protein contained in the feed composition according to the present invention is produced using the recombinant vector according to the present invention. Preferably, the ASFV antigen protein is produced in a plant body using the recombinant vector according to the present invention.

[0128] Preferably, the feed composition according to the present invention is characterized by containing all of the lectin, CD2v, p54, p72, and p30 recombinant antigen proteins as active ingredients, and most preferably, the feed composition is characterized by not further containing ASFV antigen proteins (p15, p35, E199L, and F317L) other than the lectin, CD2v, p72, p54, or p30 recombinant protein.

[0129] Specific examples of the "feed" in the feed composition include, but are not limited to, by-products of pork, beef, chicken, etc., as well as corn, rice, common straw, wild grass, pasture, silage, hay, wild grass, etc., as long as it is feed used for raising livestock. Methods for adding and blending the lectin, CD2v, p54, p72, and p30 recombinant antigen proteins of the present invention to such feed include, but are not limited to, mechanical mixing, adsorption, and occlusion.

[0130] Preferred examples are presented below to aid in understanding the present invention. However, the following examples are provided merely to facilitate understanding of the present invention, and the contents of the present invention are not limited to the following examples.

[0131] [Example] Example 1. Construction of a recombinant vector expressing an antigen of African swine fever virus As shown in the cleavage map in Figure 1, recombinant plant expression vectors were constructed to express the African swine fever virus antigen proteins Lectin, CD2v, p72, p54, p30, p15, p35, E199L, or F317L in plants. More specifically, genetic information for the African swine fever virus Lectin, CD2v, p72, p54, p30, p15, p35, E199L, and F317L proteins was obtained, and the following proteins were synthesized with sequences optimized for expression in plants: Lectin protein-encoding gene (SEQ ID NO: 2), CD2v protein-encoding gene (SEQ ID NO: 4), p72 protein-encoding gene (SEQ ID NO: 6), p54 protein-encoding gene (SEQ ID NO: 8), p30 protein-encoding gene (SEQ ID NO: 10), p15 protein-encoding gene (SEQ ID NO: 12), p35 protein-encoding gene (SEQ ID NO: 14), E199L protein-encoding gene (SEQ ID NO: 16), and F317L protein-encoding gene (SEQ ID NO: 18).

[0132] The specific construction of each recombinant vector is as follows: Recombinant vector for expressing lectin antigen protein A CaMV 35S promoter gene and HSP terminator were inserted into the pCAMBIA1300 vector, and a polynucleotide encoding the NB (new chaperone binding protein) signal peptide (SEQ ID NO: 20), a polynucleotide encoding a polyhistidine tag (SEQ ID NO: 26), a polynucleotide encoding the African swine fever virus lectin antigen recombinant protein (SEQ ID NO: 2), a polynucleotide encoding the pFc2 (porcine Fc) fragment (SEQ ID NO: 28), and a polynucleotide encoding the HDEL (His-Asp-Glu-Leu) peptide (SEQ ID NO: 30) were ligated between them in that order to construct a plant expression vector for the African swine fever virus lectin antigen protein.

[0133] Recombinant vector for expression of CD2v antigen protein The CaMV 35S promoter gene and HSP terminator were inserted into the pCAMBIA1300 vector, and a polynucleotide encoding the NB signal peptide (SEQ ID NO: 20), a polynucleotide encoding the M domain (SEQ ID NO: 24), a polynucleotide encoding the African swine fever virus CD2v antigen recombinant protein (SEQ ID NO: 4), a polynucleotide encoding the pFc2 fragment (SEQ ID NO: 28), and a polynucleotide encoding the HDEL peptide (SEQ ID NO: 30) were ligated between them in that order to construct a plant expression vector for the African swine fever virus CD2v antigen protein.

[0134] Recombinant vector for expressing p72 antigen protein The hygromycin resistance gene, a selection marker of the pCAMBIA1300 vector, was replaced with the human calreticulin 1 gene, and the MacT promoter gene, in which the 3'-terminal nucleotide of the Mac promoter was replaced with T, and the Arabidopsis RD29B terminator were inserted. Between these, a polynucleotide (SEQ ID NO: 22) encoding the BiP (chaperone binding protein) signal peptide, a polynucleotide (SEQ ID NO: 6) encoding the African swine fever virus p72 antigen recombinant protein, a polynucleotide (SEQ ID NO: 28) encoding the pFc2 fragment, and a polynucleotide (SEQ ID NO: 30) encoding the HDEL peptide were ligated in that order to construct a plant expression vector for the African swine fever virus p72 antigen protein.

[0135] Recombinant vector for expressing p54 antigen protein The CaMV 35S promoter gene and HSP terminator were inserted into the pCAMBIA1300 vector, and a polynucleotide encoding the NB signal peptide (SEQ ID NO: 20), a polynucleotide encoding the African swine fever virus p54 antigen recombinant protein (SEQ ID NO: 8), a polynucleotide encoding the pFc2 fragment (SEQ ID NO: 28), and a polynucleotide encoding the HDEL peptide (SEQ ID NO: 30) were ligated between them in that order to construct a plant expression vector for the African swine fever virus p54 antigen protein.

[0136] Recombinant vector for expressing p30 antigen protein A CaMV 35S promoter gene and HSP terminator were inserted into the pCAMBIA1300 vector, and a polynucleotide encoding the NB signal peptide (SEQ ID NO: 20), a polynucleotide encoding the African swine fever virus p30 antigen recombinant protein (SEQ ID NO: 10), a polynucleotide encoding a polyhistidine tag (SEQ ID NO: 26), and a polynucleotide encoding the HDEL peptide (SEQ ID NO: 30) were ligated between them in that order to construct a plant expression vector for the African swine fever virus p30 antigen protein.

[0137] Recombinant vector for expressing p15 antigen protein The CaMV 35S promoter gene and HSP terminator were inserted into the pCAMBIA1300 vector, and a polynucleotide encoding the NB signal peptide (SEQ ID NO: 20), a polynucleotide encoding the African swine fever virus p15 antigen recombinant protein (SEQ ID NO: 12), a polynucleotide encoding the pFc2 fragment (SEQ ID NO: 28), and a polynucleotide encoding the HDEL peptide (SEQ ID NO: 30) were ligated between them in that order to construct a plant expression vector for the African swine fever virus p15 antigen protein.

[0138] Recombinant vector for expressing P35 antigen protein The CaMV 35S promoter gene and HSP terminator were inserted into the pCAMBIA1300 vector, and a polynucleotide encoding the NB signal peptide (SEQ ID NO: 20), a polynucleotide encoding the African swine fever virus p35 antigen recombinant protein (SEQ ID NO: 14), a polynucleotide encoding the pFc2 fragment (SEQ ID NO: 28), and a polynucleotide encoding the HDEL peptide (SEQ ID NO: 30) were ligated between them in that order to construct a plant expression vector for the African swine fever virus p35 antigen protein.

[0139] Recombinant vector for expression of E199L antigen protein The hygromycin resistance gene, which serves as a selection marker in the pCAMBIA1300 vector, was replaced with the turnip crinkle virus coat protein (TCV-CP) gene, and the MacT promoter gene, in which the 3'-terminal nucleotide of the Mac promoter was replaced with T, and the Arabidopsis RD29B terminator were inserted. A polynucleotide encoding the NB signal peptide (SEQ ID NO: 20), a polynucleotide encoding the African swine fever virus E199L antigen recombinant protein (SEQ ID NO: 16), a polynucleotide encoding the pFc2 fragment (SEQ ID NO: 28), and a polynucleotide encoding the HDEL peptide (SEQ ID NO: 30) were ligated between them, in that order, to construct a plant expression vector for the African swine fever virus E199L antigen protein.

[0140] Recombinant vector for expression of F317L antigen protein The hygromycin resistance gene, which serves as a selection marker in the pCAMBIA1300 vector, was replaced with the turnip crinkle virus coat protein (TCV-CP) gene, and the MacT promoter gene, in which the 3'-terminal nucleotide of the Mac promoter was replaced with T, and the Arabidopsis RD29B terminator were inserted. A polynucleotide encoding the NB signal peptide (SEQ ID NO: 20), a polynucleotide encoding the African swine fever virus F317L antigen recombinant protein (SEQ ID NO: 18), a polynucleotide encoding the pFc2 fragment (SEQ ID NO: 28), and a polynucleotide encoding the HDEL peptide (SEQ ID NO: 30) were ligated between them, in that order, to construct a plant expression vector for the African swine fever virus F317L antigen protein.

[0141] Example 2. Confirmation of expression of recombinant ASF virus antigen protein 2-1. Transient expression of plant expression vectors The Agrobacterium strain LBA4404 was transformed using electrophoresis with each of the plant expression recombinant vectors encoding African swine fever antigen proteins (lectin, CD2v, p72, p54, p30, p15, p35, E199L, and F317L) prepared in Example 1. The transformed Agrobacterium was cultured in 5 mL of YEP liquid medium (10 g yeast extract, 10 g peptone, 5 g NaCl, 50 mg / L kanamycin, and 25 mg / L rifampicin) at 28°C for 16 hours with shaking, and 1 mL of the primary culture was inoculated into 50 mL of fresh YEP medium and cultured at 28°C for 6 hours with shaking. The cultured Agrobacteria were harvested by centrifugation (7,000 rpm, 4°C, 5 min) and suspended in infiltration buffer (10 mM MES (pH 5.7), 10 mM MgCl2, 200 μM acetosyringone) to an optical density (OD) of 1.0 at 600 nm. The Agrobacterium suspension was then injected into the abaxial surface of Nicotiana benthamiana leaves using a syringe with the needle removed.

[0142] 2-2. Isolation and purification of ASF virus antigenic proteins To isolate and purify recombinant proteins from the Nicotiana benthamiana leaves prepared in Example 2-1, a protein extract solution was added to Nicotiana benthamiana leaves expressing each recombinant protein. The tissue was disrupted in a blender and then centrifuged at 13,000 rpm at 4°C for 30 minutes to recover the protein extract. To isolate and purify each ASF virus antigen protein from the extract, affinity chromatography was performed using a protein A-Sepharose resin column. The column was loaded with protein A resin and equilibrated with a wash buffer. The recovered protein extract was applied to the column, equilibrated, and then the resin was washed with a wash buffer. Each recombinant protein was eluted with an elution solution. The elution solution containing the recombinant antigen protein was neutralized to the appropriate pH with a neutralizing solution, followed by buffer exchange and concentration using a 30 kDa cutoff filter. The concentration of the isolated and purified recombinant antigen protein was confirmed by electrophoresis (SDS-PAGE) and Coomassie staining.

[0143] The p30 protein was isolated and purified as follows: the protein extract solution was added to p30-expressing Nicotiana benthamiana leaves, the tissue was disrupted in a blender, and the tissue was centrifuged at 13,000 rpm at 4°C for 30 minutes to collect the protein extract. To isolate and purify p30 from the extract, affinity chromatography was performed using a column packed with Ni-IDA resin. First, the column was filled with Ni-IDA resin and equilibrated with a cleaning solution. The collected protein extract was applied to the column, equilibrated, and then the resin was washed with a cleaning solution. The p30 protein was eluted with the elution solution. The elution solution containing the p30 protein was subjected to buffer exchange and concentration using a 10 kDa cutoff filter. The concentration of the isolated and purified p30 protein was confirmed by electrophoresis (SDS-PAGE) followed by Coomassie staining.

[0144] As a result, as shown in Figures 2 to 10b, it was confirmed that all of the African swine fever virus antigen proteins of the present invention were well purified without any significant mutations or modifications compared to the native protein. These results verify that there is no problem of reduced production efficiency due to mutations in sugar structures when proteins are expressed in plants, and confirm that the African swine fever virus recombinant antigen proteins of the present invention can be well produced in plants.

[0145] The conditions and results for the isolation and purification of each recombinant antigen protein are as follows:

[0146] Lectin antigen protein (molecular weight: 40651.82; Figure 2) (1) Protein extraction conditions: 1 kg of Nicotiana benthamiana leaves, 2 L of protein extract (2) Protein separation and purification: Binding for 30 minutes in a column packed with approximately 80 mL of protein A resin, followed by resin down for 20 minutes. Extraction buffer / washing solution: 50 mM Tris-Cl (pH 7.2), 100 mM NaCl, 100 mM sodium sulfite (-W), 0.5% Triton X-100 (-W2), 1.5% PVPP (-W) Elution buffer: 50 mM Sodium citrate (pH 3.0), 100 mM NaCl Neutralization buffer: 1M Tris (neutralized at pH 7.5) -Final buffer: 50mM Sodium citrate, approximately 150mM Tris-Cl, 100mM NaCl, pH 7.5 (3) Final protein concentration (based on Nanodrop): 1.02 μg / μL (4) Final yield (based on Nanodrop): 54 mg / kg

[0147] CD2v antigen protein (molecular weight: 55214.70; Figure 3) (1) Protein extraction conditions: 1 kg of Nicotiana benthamiana leaves, 2 L of protein extract (2) Protein isolation and purification: Binding for 60 minutes in a column packed with approximately 100 mL of protein A resin, followed by resin down for 20 minutes. Extraction / washing solution: 100 mM Tris-Cl (pH 7.5), 154 mM NaCl, 0.5% Triton X-100 (-W2), 100 mM sodium sulfite (-W), 1.5% PVPP (-W) -Eluent: 50mM Sodium citrate (pH3.0), 154mM NaCl Neutralization buffer: 1M Tris (neutralized at pH 7.5) - Final buffer: 50 mM Sodium citrate, 154 mM NaCl, add 1 M Tris-HCl to pH 7.5. (3) Final protein concentration (Nanodrop standard): 1.2μg / μL (4) Final yield (based on Nanodrop): 68.67 mg / kg

[0148] p72 antigen protein (molecular weight: 100405.77 (with an additional 14 kDa due to seven glycosylation sites; Figures 4a and 4b) (1) Protein extraction conditions: 1 kg of Nicotiana benthamiana leaves, 2 L of protein extract (2) Protein isolation and purification: Binding for 60 minutes in a column packed with approximately 100 mL of protein A resin, followed by resin down for 20 minutes. Extraction / washing solution: 100 mM Tris-Cl (pH 7.5), 154 mM NaCl, 0.5% Triton X-100 (-W2), 100 mM sodium sulfite (-W), 1.5% PVPP (-W) -Eluent: 50mM Sodium citrate (pH3.0), 154mM NaCl Neutralization buffer: 1.5 M Tris (neutralized at pH 7.5) - Final buffer: 50 mM Sodium citrate, 154 mM NaCl, add 1 M Tris-HCl to pH 7.5. (3) Final protein concentration (based on Nanodrop): 0.57 μg / μL (4) Final yield (based on Nanodrop): 7.06 mg / kg

[0149] p54 antigen protein (molecular weight: 43956.09 (with an additional 2 kDa due to one glycosylation site); Figures 5a and 5b) (1) Protein extraction conditions: 0.1 kg of Nicotiana benthamiana leaves, 0.2 L of protein extract (2) Protein isolation and purification: Binding for 70 minutes in a column packed with approximately 10 mL of protein A resin, followed by 20 minutes of resin down. -Extract solution: 100mM Tris-Cl (pH7.4), 154mM NaCl, 0.5%Triton X-100(-W2), 100mM Sodium sulfite, 1.5%PVPP, 0.5x PI Washing solution: 100 mM Tris-Cl (pH 7.4), 154 mM NaCl, 0.5% Triton X-100 (-W2) -Eluent: 50mM Sodium citrate (pH3.0), 154mM NaCl Final buffer: 1N NaOH (add 1M Tris-HCl to pH 7.4) (3) Final protein concentration (based on Nanodrop): 1 mg / ml (4) Final yield (based on Nanodrop): 71 mg / kg

[0150] p30 antigen protein (molecular weight: 22993.95; Figure 6) (1) Protein extraction conditions: 1 kg of Nicotiana benthamiana leaves, 2 L of protein extract (2) Protein isolation and purification: Binding for 60 minutes in a column packed with approximately 100 mL of protein A resin, followed by resin down for 20 minutes. -Extract solution: 50mM Tris-Cl (pH8.0), 300mM NaCl, 10mM Imidazole, 0.5%Triton X-100, 50mM Glycine, 100mM Na2SO3, 10mM Ascorbic Acid, 1.5%PVPP -Washing solution: 50mM Tris-Cl (pH7.4), 300mM NaCl, 10mM Imidazole (W1, 2), 100mM Imidazole (W3), 0.5% Triton X-100 (W1) -Eluent: 50mM Tris-Cl (pH7.4), 300mM NaCl, 250mM Imidazole Final buffer: 50mM Tris-Cl pH 7.4, 300mM NaCl, 50mM KCl (3) Final protein concentration (Nanodrop standard): 0.3 μg / μL (volume: 93 ml) (4) Final yield (based on Nanodrop): 27.9 mg / kg

[0151] p15 antigen protein (molecular weight: 44114.34; Figures 7a and 7b) (1) Protein extraction conditions: 1.5 kg of Nicotiana benthamiana leaves, 1 L of protein extract (2) Protein isolation and purification: Binding for 60 minutes in a column packed with approximately 100 mL of protein A resin, followed by resin down for 20 minutes. Extraction / washing solution: 50 mM Tris-Cl (pH 7.2), 100 mM NaCl, 0.5% Triton X-100 (-W2), 100 mM sodium sulfite (-W), 1.5% PVPP (-W), 1 mM PMSF -Eluent: 50mM Sodium citrate (pH3.0), 100mM NaCl Neutralization buffer: 0.5M NaOH (neutralized at pH 7.5) - Final buffer: 50 mM Sodium citrate, 100 mM NaCl, add 1 M Tris-HCl to pH 7.5. (3) Final protein concentration (based on Nanodrop): 0.5 mg / ml (4) Final yield (based on Nanodrop): 98.6 mg / kg

[0152] p35 antigen protein (molecular weight: 61323.42; Figure 8) (1) Protein extraction conditions: 0.5 kg of Nicotiana benthamiana leaves, 1 L of protein extract (2) Protein isolation and purification: Binding for 60 minutes in a column packed with approximately 100 mL of protein A resin, followed by resin down for 20 minutes. Extraction / washing solution: 50 mM Tris-Cl (pH 7.2), 100 mM NaCl, 0.5% Triton X-100 (-W2), 100 mM Sodium sulfite (-W), 15 g PVPP (-W) -Eluent: 50mM Sodium citrate (pH3.0), 100mM NaCl Neutralization buffer: 1 M Tris (neutralized with pH 7.5) - Final buffer: 50 mM Sodium citrate, 100 mM NaCl, add 1 M Tris-HCl to pH 7.5. (3) Final protein concentration (Nanodrop standard): 0.53μg / μL (volume: approx. 110mL) (4) Final yield (based on Nanodrop): 116.6 mg / kg

[0153] E199L antigen protein (molecular weight: 46485.20 (with an additional 4 kDa due to two glycosylation sites; Figures 9a and 9b) (1) Protein extraction conditions: 1 kg of Nicotiana benthamiana leaves, 2 L of protein extract (2) Protein isolation and purification: Binding for 60 minutes in a column packed with approximately 100 mL of protein A resin, followed by resin down for 20 minutes. -Extract solution: 100mM Tris-Cl(pH7.2), 154mM NaCl, 0.5%Triton X-100, 100mM Sodium sulfite, 1.5%PVPP, 1mM PMSF(DMSO) Washing solution: 100 mM Tris-Cl (pH 7.2), 154 mM NaCl, 0.5% Triton X-100 (-W2) -Eluent: 50mM Sodium citrate (pH3.0), 154mM NaCl Neutralization buffer: 0.2N NaOH (neutralized at pH 7.5) (3) Final protein concentration (based on Nanodrop): 1 mg / ml (4) Final yield (based on Nanodrop): 70.3 mg / kg

[0154] F317L antigen protein (molecular weight: 62777.73 (with an additional 6 kDa due to three glycosylation sites); Figures 10a and 10b) (1) Protein extraction conditions: 100 g of Nicotiana benthamiana leaves and 200 mL of protein extract; or 1 kg of Nicotiana benthamiana leaves and 2 L of protein extract. (2) Protein isolation and purification: Binding for 60 minutes in a column packed with approximately 10 mL of protein A resin, followed by 20 minutes of resin down. -Extract solution: 100mM Tris-Cl(pH7.5), 154mM NaCl, 0.5%Triton X-100, 100mM Sodium sulfite, 1.5%PVPP, 1mM PMSF(DMSO) Washing solution: 100 mM Tris-Cl (pH 7.5), 154 mM NaCl, 0.5% Triton X-100 (-W2) -Eluent: 50mM Sodium citrate (pH3.0), 154mM NaCl Neutralization buffer: 0.2N NaOH (neutralized at pH 7.5) (3) Final protein concentration (based on Nanodrop): 1 mg / ml (4) Final yield (based on Nanodrop): 14 mg / kg

[0155] Example 3. Confirmation of the protective effect of a five-antigen or nine-antigen vaccine against ASF virus To confirm the efficacy of administering the ASF virus antigen proteins isolated and purified in Example 2 in preventing ASF virus infection and the stability of the vaccine, a total of 18 pigs were divided into three groups of six each, as shown in Table 1 below, and a protective efficacy test was conducted. Specifically, the animal models were divided into a five-antigen vaccine (combination of lectin, CD2v, p54, p72, and p30) treatment group (G1), a nine-antigen vaccine (combination of lectin, CD2v, p54, p72, p30, p15, p35, E199L, and F317L) treatment group (G2), and a PBS-treated control group (G3). Each ASFV antigen protein, excluding p30, was contained in the vaccine at 100 μg, and p30 was contained at 30 μg. Each pig was administered 2 mL of vaccine per vaccination.

[0156] [Table 1]

[0157] The protective efficacy test schedule is shown in Table 2. The vaccine was prepared by mixing the recombinant protein antigen cocktail with the SEA1 adjuvant and administered intramuscularly twice, three weeks apart. Two weeks after the second injection, two pigs from each group were challenged intramuscularly with African swine fever virus (wild-type ASF virus). The remaining four pigs from each group were kept in the same animal enclosure as the pigs that had been intramuscularly inoculated with African swine fever virus, and were exposed to virus shed by the challenged pigs, inducing horizontal infection (Sentinel). After the challenge, the pigs were observed daily for symptoms, and pigs that showed symptoms of infection (high temperature of 41.1°C or higher) were euthanized early before the end of the test, and serum was collected. Specifically, 3 to 5 mL of serum was collected from the jugular vein or anterior vena cava (using an 18 to 20 G, 1.5 mm needle), and infected tissues such as lung, liver, spleen, kidney, lymph nodes, and pancreas were isolated.

[0158] [Table 2]

[0159] Figure 11 shows the changes in body temperature and survival rates of pigs following challenge inoculation. Two pigs from each group directly challenged with African swine fever virus died around one week after challenge. However, all unchallenged pigs in the group administered the five-antigen vaccine (G1) had body temperatures below 40.5°C and survived for two weeks. In contrast, three unchallenged pigs in the group administered the nine-antigen vaccine (G2) all died after 11 days due to high fever. Of the three unchallenged pigs in the control group (G3), one survived without symptoms, but one died on day 13 due to high fever. The remaining pig developed a high fever from day 11 but survived for the two-week experimental period.

[0160] These results indicate that the five-antigen vaccine according to the present invention (a combination of lectin, CD2v, p54, p72, and p30) has excellent efficacy in preventing classical swine fever virus, and suggest that further combining the p15, p35, E199L, and F317L antigen proteins, which have traditionally been used as ASF virus vaccine antigens (i.e., a nine-antigen vaccine), may actually inhibit the function of the vaccine and reduce its effectiveness in preventing African swine fever.

[0161] Example 4. Confirmation of blood virus levels by challenge vaccination after administration of a five- or nine-antigen vaccine Subsequently, the virus (viremia) levels in the blood of pigs that underwent the challenge inoculation test of Example 3 were measured, and the preventive effect of the vaccine of the present invention against African swine fever virus was confirmed.

[0162] Specifically, to measure the virus levels in the blood, serum samples were extracted from each pig on days 0, 3, 7, 10, and 14 after challenge. Real-time polymerase chain reaction (RT-PCR) was performed using ASF virus-specific primers and probes, and the Ct values ​​for each sample were compared. The ASF virus-specific primer sequences used in the experiment are as follows:

[0163] [Table 3]

[0164] As shown in Figure 12, among pigs administered with the five-antigen vaccine (G1), those contact-challenged with ASF virus had very low levels of ASF virus in their blood until 7 days after challenge (UD). At 10 days after challenge, the virus was detected in some pigs, but at very low levels and was undetectable by 14 days. In contrast, among pigs administered with the nine-antigen vaccine (G2), those contact-challenged with ASF virus had detectable ASF virus from 7 days after challenge, and by 10 days, their blood virus levels had increased, reaching even higher levels than those in the control group at the same time point. This confirmed a lower viral preventative effect compared to those not administered any vaccine. Furthermore, as mentioned above, pigs administered with the nine-antigen vaccine developed a worsening fever 11 days after challenge and were euthanized early.

[0165] The above results demonstrate that the five-antigen vaccine according to the present invention (a combination of lectin, CD2v, p54, p72, and p30) can suppress ASF virus infection and proliferation and achieve excellent efficacy in preventing classical swine fever. In particular, further combining the p15, p35, E199L, and F317L antigen proteins that have traditionally been used in ASF virus vaccine antigens (i.e., a nine-antigen vaccine) actually inhibits the vaccine's effectiveness and fails to prevent viral infection and proliferation in the body at all.

[0166] Example 5. Determination of anti-ASFV antibody levels after administration of a five-antigen or nine-antigen vaccine Next, to confirm the level of serum anti-ASFV antibodies, the level of anti-p30 antibody production over time after vaccination was monitored in all individuals using plates coated with vaccine antigens (on days 0, 7, 14, 21, 28, and 35 after vaccination). In the group administered the five-antigen vaccine, anti-p30 antibody levels were detected from day 21 after vaccination, and high levels of anti-p30 antibody were detected in all vaccinated individuals on days 28 and 35 after vaccination, confirming an average higher antibody response than the group administered the nine-antigen vaccine (Figure 22).

[0167] The above results indicate that both the five- and nine-antigen vaccines of the present invention can induce antibody production against classical swine fever virus antigens, but that the five-antigen vaccine in particular can induce higher levels of antibody production more rapidly than the nine-antigen vaccine.

[0168] As described above, the inventors constructed a vector expressing African swine fever virus-specific proteins for expression in plants, and confirmed that when an ASF virus vaccine incorporating the recombinant protein was administered to pigs, it stably induced the production of antibodies against the ASF virus without any side effects. In particular, pigs administered a five-antigen vaccine combining five antigens (lectin, CD2v, p54, p72, and p30) steadily gained weight after exposure to the virus, did not develop ASFV viremia or fever, and had a 100% survival rate, confirming that the five-antigen vaccine effectively suppresses ASF virus infection and proliferation in the body, thereby suppressing African swine fever. In particular, the antibody production induction effect of the five-antigen vaccine was confirmed to be superior to that of a nine-antigen vaccine (lectin, CD2v, p54, p72, p30, p15, p35, E199L, and F317L), indicating that the combination of the antigen proteins lectin, CD2v, p54, p72, and p30 can achieve superior preventive effects against African swine fever compared to conventional vaccines. Therefore, it is expected that the five-antigen vaccine of the present invention can be used in various ways in novel African swine fever virus vaccine compositions.

[0169] The above description of the present invention is for illustrative purposes only, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical concept or essential features of the present invention. Therefore, the above-described embodiments should be understood to be illustrative in all respects and not limiting. [Industrial Applicability]

[0170] The recombinant African swine fever virus antigen protein of the present invention is not only effectively expressed in plants, but also has high water solubility, making it easy to isolate and purify. It also acts as an antigen in the body and exhibits high immunogenicity, making it suitable for use as a novel African swine fever virus vaccine composition. In particular, the present inventors have confirmed through challenge experiments that a vaccine composition containing five antigen proteins of the present invention (lectin, CD2v, p54, p72, and p30) has a superior preventive effect against African swine fever compared to vaccine compositions further containing other antigen proteins (e.g., p15, p35, E199L, F317L). Therefore, the recombinant vector and vaccine composition of the present invention are expected to be widely used in the livestock industry and other fields.

Claims

1. A vaccine composition for preventing African swine fever, comprising a combination of African swine fever virus (ASFV) antigen proteins as active ingredients, wherein the combination of ASFV antigen proteins consists of Lectin, CD2v, p72, p54, and p30 proteins.

2. The vaccine composition of claim 1, wherein the vaccine composition satisfies one or more characteristics selected from the group consisting of: (a) the Lectin protein comprises the amino acid sequence of SEQ ID NO: 1; (b) the CD2v protein comprises the amino acid sequence of SEQ ID NO:3; (c) the p72 protein comprises the amino acid sequence of SEQ ID NO:5; (d) the p54 protein comprises the amino acid sequence of SEQ ID NO: 7; and (e) the p30 protein comprises the amino acid sequence of SEQ ID NO:

9.

3. The vaccine composition of claim 1, wherein the vaccine composition satisfies one or more characteristics selected from the group consisting of: (a) the lectin protein encoded by a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 2; (b) the CD2v protein encoded by a polynucleotide comprising the base sequence represented by SEQ ID NO: 4; (c) the p72 protein encoded by a polynucleotide comprising the base sequence represented by SEQ ID NO: 6; (d) the p54 protein encoded by a polynucleotide comprising the base sequence represented by SEQ ID NO: 8; and (e) The p30 protein encoded by a polynucleotide comprising the base sequence represented by SEQ ID NO:

10.

4. The vaccine composition of claim 1, further comprising an adjuvant.

5. 5. The vaccine composition according to claim 4, wherein the adjuvant is a mineral oil or emulsigen-based adjuvant.

6. A vaccine kit for preventing African swine fever, comprising the vaccine composition according to any one of claims 1 to 5.

7. A method for preventing African swine fever, comprising administering the vaccine composition of claim 1 to an animal other than a human.

8. 10. Use of the vaccine composition of claim 1 for the prevention of African swine fever.

9. 10. Use of the vaccine composition according to claim 1 for the manufacture of a vaccine for the prevention of African swine fever.

Images