KR20220133632A - Recombinant protein comprising spike protein S1 of PEDV-derived protein and ferritin-derived protein and use thereof - Google Patents

Abstract

The present invention relates to a recombinant protein comprising a protein derived from porcine epidemic diarrhea virus (PEDV) spike protein S1 and a protein derived from ferritin, and a vaccine composition containing the recombinant protein. The recombinant protein of the present invention can form a self-assembling ferritin structure having the protein derived from PEDV spike protein S1, which is an antigenic protein, located on the surface, and thus, specific antibodies against PEDV can be effectively produced by using the vaccine composition comprising the same.

Description

Recombinant protein comprising spike protein S1 of PEDV-derived protein and ferritin-derived protein and use thereof

The present invention relates to a recombinant protein comprising a PEDV (Porcine epidemic diarrhea virus) spike protein S1-derived protein and a ferritin-derived protein, and a vaccine composition comprising the recombinant protein.

Porcine epidemic diarrhea (PED) is an infectious disease in pigs regardless of age caused by infection with the causative agent, porcine epidemic diarrhea virus (PEDV), characterized by vomiting and watery diarrhea. This disease was first reported in Korea in 1992 and then spread nationwide, causing the most damage among the diarrheal diseases of piglets, and is a major disease that causes a lot of economic damage to pig farms.　PEDV, which causes epidemic diarrhea in pigs, mainly proliferates in small intestinal villous cells, denatures or necrosises villous epithelial cells, which accompanies atrophy and exfoliation of the villi, causes malabsorption, and causes persistent watery diarrhea. Regardless of the age of pigs, it causes enteritis and is accompanied by severe diarrhea and dehydration, and even in piglets, the mortality rate reaches 80-90%.

On the other hand, a vaccine is an antigen used to actively immunize animals for the prevention of infection, or a biological product containing the antigen as an active ingredient, which was proposed by French microbiologist L. Pasteur. In particular, a vaccine as a biological agent refers to an immunogen that is administered to an animal for the purpose of preventing an infectious disease to generate immunity in the living body. Normally, when a vaccine is administered, the antibody is made in the living body and immunity is acquired, and once the generated antibody remains in the living body for a relatively long time, even if an infection by the causative organism of the disease occurs, it is possible to defend against it, resulting in disease prevention. it can be

In general, for vaccine preparations, a killed vaccine, an inactivated vaccine, a live vaccine using live bacteria, and an attenuated strain with weakened live bacteria are used for vaccine preparation. There are various types such as attenuated vaccines, bacterial toxoids, or derivatives thereof. Since it must be induced, it is desirable to develop it in a form similar to possible disease agents. For this reason, a live vaccine that uses live cells as it is without any treatment can show the best effect as a vaccine.

However, in the case of a live vaccine, since the disease-causing agent is used while it is alive, it is converted into a toxic strain in the future and has a risk of inducing disease, so it is used only in very few infectious diseases. As an alternative to overcome this disadvantage, an attenuated vaccine with attenuated live cells has been developed and used. has a Although the dead cell vaccine developed for safety in use can solve the above problems in terms of safety, it is common that antigenicity is significantly lower than that of live vaccines or attenuated vaccines. Accordingly, there is a demand for the development of an ideal new vaccine formulation that is safe for use like a dead cell vaccine and has excellent antigenicity like a live vaccine.

Under this background, the present inventors developed a recombinant protein including a PEDV spike protein S1-derived protein and a ferritin-derived protein and a vaccine comprising the same, and completed the present invention by verifying the excellent antigenicity of the vaccine.

One aspect provides a recombinant protein comprising a protein derived from Porcine epidemic diarrhea virus (PEDV) spike protein S1 and a protein derived from ferritin.

Another aspect provides a polynucleotide encoding the recombinant protein.

Another aspect provides a vaccine composition for preventing or treating PEDV infection disease, comprising the recombinant protein as an active ingredient.

Another aspect provides a method for preventing or treating a PEDV-infected disease, comprising administering the vaccine composition to a subject other than a human.

One aspect is to provide a recombinant protein, including a protein derived from Porcine epidemic diarrhea virus (PEDV) spike protein S1 and a protein derived from ferritin.

As used herein, the term "PEDV (Porcine epidemic diarrhea virus)" belongs to the genus Alphacoronavirus, and as a virus having a single-stranded positive-sense RNA as a genome, swine epidemic diarrhea It is known as the causative virus. The genome of PEDV consists of a total of seven open reading frames (ORFs) encoding four structural proteins. The structural protein includes a spike protein (S protein), a membrane protein, an envelope protein, and a nucleocapsid protein.

As used herein, the term "spike protein" is a protrusion-like structure arranged on the surface of a virus, with a length of 10 to 15 nm, which binds to a protein receptor in the cell membrane and penetrates into our body. is in charge of Therefore, it is the spike protein that plays an important role when the virus enters human cells. In particular, the host to which the virus is infected depends on the binding ability of the spike protein. Therefore, the spike protein can be used to develop vaccines, antibodies, and diagnostics for virus treatment.

The spike protein of the PEDV is divided into a spike 1 (spike 1, S1) domain and a spike 2 (spike 2, S2) domain, of which S1 is a site involved in cell adsorption and includes a receptor binding site, and various neutralization Antibody-producing epitopes exist, and in the present invention, they are used as antigenic peptides.

As used herein, the term "ferritin" is a protein that stores iron and is widely present in prokaryotes and eukaryotes. The molecular weight of ferritin is about 500,000 Da, and it is composed of a heavy chain and a light chain. Ferritin is a protein in which 24 monomers (a single monomer or heterogeneous monomer composed of either a heavy chain or a light chain) are gathered to form a huge spherical tertiary structure. In the case of human ferritin, the outer diameter is about 12 nm and the inner diameter is about 8 nm. Ferritin is dispersed as monomers depending on the pH conditions and sometimes forms nanoparticles in which 24 monomers are bound. By using these properties, various substances can be captured in ferritin.

The ferritin-derived protein may be derived from porcine ferritin protein, and specifically, may be derived from a heavy chain of ferritin protein.

The PEDV spike protein S1-derived protein and the ferritin-derived protein may be directly or indirectly linked, and specifically, the PEDV spike protein S1-derived protein and the ferritin-derived protein may be linked through a linker.

The recombinant protein may further include a linker, and specifically, the linker may be positioned between the PEDV spike protein S1-derived protein and the ferritin-derived protein. The linker may be a peptide inserted between a protein and a protein to increase the structural flexibility of these proteins or to enhance the activity of each protein when a recombinant protein is made by linking the PEDV spike protein S1-derived protein and the ferritin-derived protein. have. The linker is not limited as long as it does not inhibit the activity of each protein to be fused and does not cause an unnecessary immune response, but is preferably a peptide linker composed of 1 to 20 amino acids, more preferably 1 to 5 amino acids. , more specifically an SSG peptide linker.

The PEDV-derived protein may be linked to the C-terminus or the N-terminus of the ferritin-derived protein, and specifically, may be linked to the C-terminus of the ferritin-derived protein.

The PEDV spike protein S1-derived protein may include the amino acid sequence of SEQ ID NO: 1, and specifically, may include the amino acid sequence of SEQ ID NO: 1. In addition, not only the amino acid sequence consisting of SEQ ID NO: 1, but also 80% or more, specifically 90% or more, more specifically 95% or more, more specifically 98% or more, most specifically 99% or more of the sequence. As an amino acid sequence showing homology, any amino acid sequence that exhibits substantially the same or corresponding efficacy as the protein is included without limitation. In addition, if it is an amino acid sequence having such homology, it is apparent that amino acid sequences in which some sequences are deleted, modified, substituted or added are also included within the scope of the present invention.

The ferritin-derived protein may include the amino acid sequence of SEQ ID NO: 2, and specifically, may include the amino acid sequence of SEQ ID NO: 2. In addition, not only the amino acid sequence consisting of SEQ ID NO: 2, but also 80% or more, specifically 90% or more, more specifically 95% or more, more specifically 98% or more, most specifically 99% or more of the sequence As an amino acid sequence showing homology, any amino acid sequence that exhibits substantially the same or corresponding efficacy as the protein is included without limitation. In addition, if it is an amino acid sequence having such homology, it is apparent that amino acid sequences in which some sequences are deleted, modified, substituted or added are also included within the scope of the present invention.

The recombinant protein is a protein including the ferritin-conjugated PEDV spike protein S1 domain, and may include the amino acid sequence of SEQ ID NO: 3, and specifically, may include the amino acid sequence of SEQ ID NO: 3. In addition, not only the amino acid sequence consisting of SEQ ID NO: 3, but also 80% or more, specifically 90% or more, more specifically 95% or more, more specifically 98% or more, most specifically 99% or more of the sequence. As an amino acid sequence showing homology, any amino acid sequence that exhibits substantially the same or corresponding efficacy as the protein is included without limitation. In addition, if it is an amino acid sequence having such homology, it is apparent that amino acid sequences in which some sequences are deleted, modified, substituted or added are also included within the scope of the present invention.

The recombinant protein can form a structure capable of presenting the antigenic peptide on the surface of the spherical nanoparticles by conjugating the PEDV spike protein S1 domain, which is an antigenic peptide, to a ferritin-derived protein capable of forming spherical nanoparticles, so that antigenicity and antibody Since it has excellent formation ability, the recombinant protein can be used for preventing infection of PEDV or for use in compositions and methods for preventing or treating PEDV-infected diseases.

As used herein, the term "homology" refers to the degree of similarity between the nucleotide sequence encoding a protein or the amino acid sequence constituting the protein. can have In addition, the homology can be expressed as a percentage according to the degree of correspondence with a given amino acid sequence or base sequence. In the present specification, a homologous sequence having the same or similar activity to a given amino acid sequence or nucleotide sequence is expressed as "% homology". For example, standard software that calculates parameters such as score, identity, and similarity, specifically BLAST 2.0, or hybridization written under defined stringent conditions Appropriate hybridization conditions that can be confirmed by comparing the sequences by experimentation are within the technical scope and are well known to those skilled in the art (eg, J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring). Harbor Laboratory press, Cold Spring Harbor, New York, 1989; F.M. Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York).

Another aspect is to provide a polynucleotide encoding a recombinant protein of the present invention. The same parts as described above also apply to the polynucleotide.

As used herein, the term "polynucleotide" refers to a polymer material to which nucleotides are bound, and refers to DNA encoding genetic information.

In the present invention, the nucleotide sequence constituting the polynucleotide encoding the proteins is not only the nucleotide sequence encoding the amino acid described in each SEQ ID NO: 80% or more, specifically 90% or more, more specifically 95% of the sequence % or more, more specifically 98% or more, and most specifically, if the nucleotide sequence is a nucleotide sequence that shows homology of 99% or more, it is a nucleotide sequence constituting a polynucleotide encoding a protein that exhibits substantially the same or corresponding efficacy as each protein. including without limitation.

In addition, the polynucleotide encoding the proteins is within a range that does not change the amino acid sequence of the protein expressed from the coding region in consideration of codons preferred in the organism in which the protein is to be expressed due to codon degeneracy. Various modifications can be made to the coding region in . Accordingly, the polynucleotide may be included without limitation as long as it is a nucleotide sequence encoding each protein. In addition, if a probe that can be prepared from a known sequence, for example, a sequence encoding a protein having the same activity as the protein by hybridization under stringent conditions with a sequence complementary to all or part of the polynucleotide sequence, is limited may be included without

The "stringent conditions" means conditions that allow specific hybridization between polynucleotides. These conditions are specifically described in the literature (eg, J. Sambrook et al., supra). For example, genes having high homology between genes having homology of 40% or more, specifically 90% or more, more specifically 95% or more, still more specifically 97% or more, and particularly specifically 99% or more homology. Conditions that hybridize with each other and do not hybridize with genes with lower homology, or wash conditions of normal Southern hybridization at 60° C. 1XSSC, 0.1% SDS, specifically 60° C. 0.1XSSC, 0.1% SDS, more specifically As examples, the conditions of washing once, specifically 2 to 3 times, at a salt concentration and temperature equivalent to 68° C. 0.1XSSC, 0.1% SDS can be exemplified.

Hybridization requires that two polynucleotides have complementary sequences, although mismatch between bases is possible depending on the stringency of hybridization. The term "complementary" is used to describe the relationship between nucleotide bases capable of hybridizing to each other. For example, with respect to DNA, adenosine is complementary to thymine and cytosine is complementary to guanine. Accordingly, the present application may also include substantially similar polynucleotide sequences as well as isolated polynucleotide fragments complementary to the overall sequence.

Specifically, polynucleotides having homology can be detected using hybridization conditions including a hybridization step at a Tm value of 55° C. and using the conditions described above. In addition, the Tm value may be 60 °C, 63 °C, or 65 °C, but is not limited thereto and may be appropriately adjusted by those skilled in the art according to the purpose. The appropriate stringency for hybridizing polynucleotides depends on the length of the polynucleotides and the degree of complementarity, and the parameters are well known in the art (see Sambrook et al., supra, 9.50-9.51, 11.7-11.8).

The polynucleotide may be provided in the form of an expression vector.

As used herein, the term "expression vector" is a recombinant vector that can be introduced into a suitable host cell to express a target protein, and refers to a genetic construct comprising essential regulatory elements operably linked to express a gene insert. The term “operably linked” means that a nucleic acid expression control sequence and a nucleic acid sequence encoding a protein of interest are functionally linked to perform a general function. The operative linkage with the recombinant vector can be prepared using genetic recombination techniques well known in the art, and site-specific DNA cleavage and ligation can be facilitated using enzymes generally known in the art. have.

A suitable expression vector of the present invention may contain a signal sequence for membrane targeting or secretion in addition to expression control elements such as promoter, initiation codon, stop codon, polyadenylation signal and enhancer. The start codon and stop codon are generally considered part of the nucleotide sequence encoding the immunogenic target protein, and must be functional in the subject when the genetic construct is administered and must be in frame with the coding sequence. Common promoters can be constitutive or inducible, lac, tac, T3 and T7 promoters for prokaryotic cells, simian virus 40 (SV40) for eukaryotic cells, mouse mammary tumor virus (MMTV) promoter, human immunodeficiency virus (HIV), for example the long terminal repeat (LTR) promoter of HIV, Moloney virus, cytomegalovirus (CMV), Epstein Barr virus (EBV), Loose sarcoma virus (RSV) promoter, as well as β- Promoters derived from actin promoter, human hemoglobin, human muscle creatine, and human metallothionein, but are not limited thereto.

In addition, the expression vector may include a selectable marker for selecting a host cell containing the vector. The selectable marker is for selecting cells transformed with the vector, and markers that confer a selectable phenotype such as drug resistance, auxotrophicity, resistance to cytotoxic agents or expression of surface proteins may be used. In an environment treated with a selective agent, only the cells expressing the selection marker survive, so that the transformed cells can be selected. In addition, when the vector is a replicable expression vector, it may include a replication origin, which is a specific nucleic acid sequence from which replication is initiated.

As a recombinant expression vector for inserting a foreign gene, various types of vectors such as plasmids, viruses, and cosmids may be used. The type of recombinant vector is not particularly limited as long as it functions to express a desired gene and produce a desired protein in various host cells of prokaryotic and eukaryotic cells. A vector capable of producing a large amount of a foreign protein in a form similar to that of a can be used.

To express the recombinant protein of the present invention, various combinations of hosts and vectors can be used. Expression vectors suitable for eukaryotic hosts may include, but are not limited to, expression control sequences derived from SV40, bovine papillomavirus, adenovirus, adenoassociated virus, cytomegalovirus and retrovirus, and the like. Expression vectors that can be used in bacterial hosts are not limited thereto, but Escherichia coli including pcDNA3.1, pET, pRSET, pBluescript, pGEX2T, pUC vector, col E1, pCR1, pBR322, pMB9 or derivatives thereof (Escherichia coli) ) obtained from bacterial plasmids, plasmids having a wider host range such as RP4, phage DNA exemplified by phage lambda derivatives such as λgt10, λgt11 or NM989, and M13 and filamentous single-stranded DNA phage and Other DNA phages, such as, and the like may be included. A 2°C plasmid or a derivative thereof may be used for yeast cells, and pVL941 or the like may be used for insect cells.

Another aspect is to provide a vaccine composition for preventing or treating PEDV infection disease, comprising the recombinant protein of the present invention as an active ingredient. The same parts as those described above also apply to the composition.

As used herein, the term “vaccine” refers to a pharmaceutical composition containing at least one immunologically active ingredient that induces an immunological response in an animal. The immunologically active component of the vaccine may contain appropriate components of live or dead virus (subunit vaccines), whereby these components destroy the whole virus or its growing culture and then the desired construct(s) by synthetic procedures followed by isolation and purification induced by purification steps to obtain It is prepared by induction of the above synthetic process in animals in need of the vaccine by direct incorporation of genetic material using (polynucleotide vaccination). A vaccine may comprise one or more of the elements described above at the same time.

As used herein, the term “prevention” refers to any action that inhibits or delays infection of PEDV and the onset of disease due to the administration of the PEDV vaccine composition.

As used herein, the term “treatment” refers to any action in which the symptoms of a disease already caused by infection of PEDV are improved or beneficial due to administration of the PEDV vaccine composition.

Two or more of the recombinant proteins may be self-assembled to form a structure, and specifically, 24 recombinant proteins may be self-assembled to form a structure in which each of the recombinant proteins is a monomer.

The construct may be a spherical particle, and the antigen peptide, PEDV Spike 1 derived protein, may be located on the outer surface of the construct.

The vaccine composition may further include a pharmaceutically acceptable excipient, diluent or carrier. The "pharmaceutically acceptable excipient, diluent or carrier" may mean an excipient, diluent or carrier that does not inhibit the biological activity and properties of the injected compound without irritating the organism. Here, "pharmaceutically acceptable" means that it does not inhibit the activity of the active ingredient and does not have toxicity beyond what the application (prescription) target can adapt.

Suitable carriers for vaccines are known to those skilled in the art and include, but are not limited to, proteins, sugars, and the like. Such carriers may be aqueous or non-aqueous solutions, suspensions or emulsions. As an adjuvant for increasing immunogenicity, a regular or atypical organic or inorganic polymer may be used. Adjuvants are generally known to promote immune responses through chemical and physical binding to antigens. As an adjuvant, an atypical aluminum gel, an oil emulsion, or a double oil emulsion and an immunosol may be used. In addition, various plant-derived saponins, levamisole, CpG dinucleotides, RNA, DNA, LPS, various types of cytokines, etc. may be used to promote an immune response. The immune composition as described above may be used as a composition for inducing an optimal immune response by a combination of various adjuvants and immune response promoting additives. In addition, as the composition to be added to the vaccine, a stabilizer, an inactivating agent, an antibiotic, a preservative, and the like may be used. Depending on the route of administration of the vaccine, the vaccine antigen may be mixed with distilled water or a buffer solution.

The vaccine composition is formulated in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories or unit dosage ampoules, or injections in multiple dosage forms, respectively, according to a conventional method. can be used for When formulating the vaccine composition, it may be prepared by adding a diluent or excipient such as a generally used filler, extender, binder, wetting agent, disintegrant, or surfactant.

When the vaccine composition is prepared for parenteral use, it may be formulated in the form of injections, transdermal administrations, nasal inhalants and suppositories together with suitable carriers according to methods known in the art. In the case of formulation for injection, suitable carriers include sterile water, ethanol, polyols such as glycerol or propylene glycol, or mixtures thereof, preferably Ringer's solution, PBS (phosphate buffered saline) containing triethanolamine, or sterilization for injection. Water, an isotonic solution such as 5% dextrose, etc. can be used. When formulated for transdermal administration, it may be formulated in the form of an ointment, a cream, a lotion, a gel, an external solution, a pasta agent, a liniment agent, an air roll, and the like. In the case of nasal inhalants, it can be formulated in the form of an aerosol spray using a suitable propellant such as dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, and the like. witepsol), tween 61, polyethylene glycols, cacao fat, laurin fat, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearate, sorbitan fatty acid esters, etc. may be used.

Another aspect provides a method for preventing or treating a PEDV-infected disease comprising administering the vaccine composition to a subject other than a human. The same parts as those described above are equally applied to the above method.

As used herein, the term "individual" refers to a living organism that can be infected with PEDV and can develop a disease due to infected PEDV, preferably a mammal, but is not particularly limited thereto. The mammal may include cattle, horses, sheep, pigs, goats, camels, antelopes, dogs, cats, mice, livestock, and the like, and specifically may be pigs.

As used herein, the term "administration" means introducing a predetermined substance to an individual by an appropriate method, and the administration route of the vaccine composition of the present invention can be administered through any general route as long as it can reach the target tissue. have. Intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, may be administered intrarectally, but is not limited thereto. However, when administered orally, the protein is digestible, so it is preferred that the oral composition be formulated to coat the active agent or to protect it from degradation in the stomach. In addition, the pharmaceutical composition may be administered by any device capable of transporting the active agent to a target cell.

The route of administration of the vaccine composition may be administered through any general route as long as it can reach the target tissue, and specifically, the vaccine composition is for intramuscular administration, subcutaneous administration, intraperitoneal administration, intravenous administration, oral administration , may be selected from the group consisting of compositions for dermal administration, ocular administration, and intracerebral administration.

The vaccine composition may be administered in a pharmaceutically effective amount, and the term "pharmaceutically effective amount" means an amount sufficient to treat or prevent a disease at a reasonable benefit/risk ratio applicable to medical treatment or prevention, , the effective dose level depends on the severity of the disease, the activity of the drug, the patient's age, weight, health, sex, the patient's sensitivity to the drug, the time of administration of the composition of the present invention used, the route of administration and the rate of excretion, the duration of treatment, and the present It can be determined according to factors including drugs used in combination with or concomitantly with the composition of the invention and other factors well known in the medical field. The vaccine composition may be administered alone or in combination with a component known to exhibit a prophylactic or therapeutic effect on known PEDV-infected diseases. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects.

The dosage of the vaccine composition can be determined by those skilled in the art in consideration of the purpose of use, the degree of addiction of the disease, the patient's age, weight, sex, history, or the type of substance used as an active ingredient. For example, the vaccine composition of the present invention may be administered in an amount of about 0.1 ng to about 1,000 mg/kg, preferably, 1 ng to about 100 mg/kg per adult, and the administration frequency of the composition of the present invention is particularly limited thereto. Although not limited, it may be administered once a day or administered several times in divided doses. The dosage or frequency of administration is not intended to limit the scope of the present invention in any way.

The vaccine composition may be administered as an individual therapeutic agent or may be administered in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. and may be administered single or multiple. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, and can be easily determined by those skilled in the art.

The recombinant protein of the present invention can form a ferritin self-assembled structure locating a protein derived from the spike protein S1 of PEDV, an antigen protein, on the surface. have.

1 is a view schematically showing the construction of a recombinant protein of the present invention and the process of forming a nanoparticle structure thereof.
Figure 2 is a view confirming the antibody-forming effect of the vaccine composition comprising the recombinant protein of the present invention.
Figure 3 is a diagram showing the antibody neutralizing ability assay process.
Figure 4 is a view confirming the antibody neutralizing ability of the vaccine composition comprising the recombinant protein of the present invention.

Hereinafter, it will be described in more detail through examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example 1: Recombinant Protein Construction

In order to prepare the recombinant protein of the present invention, the following experiments were performed.

Specifically, as an antigenic peptide capable of inducing a vaccine response to PEDV, a protein derived from the spike protein S1 of PEDV was used, and the amino acid sequence of the protein is described as SEQ ID NO: 1. Next, in order to effectively induce the antibody formation of the antigen peptide, a ferritin-derived protein was used, and the amino acid sequence of the protein was described as SEQ ID NO:2. The PEDV spike protein S1-derived protein was linked to the C-terminus of the ferritin-derived protein using a linker (SSG peptide) to construct a recombinant protein. The amino acid sequence of the recombinant protein is described as SEQ ID NO: 3.

protein amino acid sequence order
number PEDV Spike Protein S1 Derived Protein MRSLIYFWLLLPVLPTLSLPQDVTRCQSTTNFRRFFSKFNVQAPAVVVLGGYLPSMNSSSWYCGTGIETASGVHGIFLSYIDSSQGFEIGISQEPFDPSGYQLYLHKATNGNTNAIARLRISQFPDNKTLGPTVNDVTTGRNCLFNKAIPAYMRDGKDIVVGITWDNDRVTVFADKIYHFYLKNDWSRVATRCYNRRSCAMQYVYTPTYYMLNVTSAGEDGIYYEPCTANCTGYAANVFATDSNGHIPESFSFNNWFLLSNDSTLLHGKVVSNQPLLVNCLLAIPKIYGLGQFFSFNHTMDGVCNGAALDRAPEALRFNINDTSVILAEGSIVLHTALGTNLSFVCSNSSDPHLATFAIPLGATEVPYYCFLIVDTYNSTVYKFLAVLPPTVREIVITKYGDVYVNGFGYLHLGLLDAVTINFTGHGTDDDVSGFWTIDSTNFVDALIEVQGTSIQRILYCDDPVSQLKCSQVAFDLDDGFYPISSRNLLSHEQPISFVTLPSFNDHSFVNITVSASFGDHSGANLVASDTTINGFSSFCVDTRQFTIRLFYNVTSSYGYVSKSQYSNCPFTLQSVNDYLSFSKFCVSTSLLASACTIDLFGYPHFGSGVKFTSLYFQFTEGELITGTPKPLEGVTDVSFMTLDVCTKYTIYGFKGEGIITLTNSSFLAGVYYTSDSGQLLAFKNVTSGAVYSVTPCSFSEQAAYVDDDIVGVISSLSNST One Ferritin-derived protein MTTSCSSQVRQNYHQDSEAAINRQINLELYASYVYLSMSYYFDRDDVALKNFAKYFLHQSHGGRGHAEKLMKLQTQRGARIFLQDIMKPERDDWENGLTAMEFALHVVKNVYQSLLELHKLATDKNDPHLCDFIETHYLHEQVKAIKELGDHITNLHRMSSSS 2 recombinant protein RDDWENGLTAMEFALHVVKNVYQSLLELHKLATDKNDPHLCDFIETHYLHEQVKAIKELGDHITNLHRMGAPEYGMAEYLFDKHTLGSSESSSGMRSLIYFWLLLPVLPTLSLPQDVTRCQSTTNFRRFFSKFNVQAPAVVVLGGYLPSMNSSSWYCGTGIETASGVHGIFLSYIDSSQGFEIGISQEPFDPSGYQLYLHKATNGNTNAIARLRISQFPDNKTLGPTVNDVTTGRNCLFNKAIPAYMRDGKDIVVGITWDNDRVTVFADKIYHFYLKNDWSRVATRCYNRRSCAMQYVYTPTYYMLNVTSAGEDGIYYEPCTANCTGYAANVFATDSNGHIPESFSFNNWFLLSNDSTLLHGKVVSNQPLLVNCLLAIPKIYGLGQFFSFNHTMDGVCNGAALDRAPEALRFNINDTSVILAEGSIVLHTALGTNLSFVCSNSSDPHLATFAIPLGATEVPYYCFLIVDTYNSTVYKFLAVLPPTVREIVITKYGDVYVNGFGYLHLGLLDAVTINFTGHGTDDDVSGFWTIDSTNFVDALIEVQGTSIQRILYCDDPVSQLKCSQVAFDLDDGFYPISSRNLLSHEQPISFVTLPSFNDHSFVNITVSASFGDHSGANLVASDTTINGFSSFCVDTRQFTIRLFYNVTSSYGYVSKSQYSNCPFTLQSVNDYLSFSKFCVSTSLLASACTIDLFGYPHFGSGVKFTSLYFQFTEGELITGTPKPLEGVTDVSFMTLDVCTKYTIYGFKGEGIITLTNSSFLAGVYYTSDSGQLLAFKNVTSGAVYSVTPCSFSEQAAYVDDDIVGVISSLSNST 3

In order to produce the recombinant protein, the polynucleotide encoding it was cloned and introduced into the pCDNA 3.1 myc his vector to prepare a recombinant vector, which was transformed into 293T cells, and then the cells were cultured and purified to obtain a recombinant protein. (FIG. 1).

In the recombinant protein, 24 subunits are gathered by a ferritin-derived protein to form a single spherical structure, and a PEDV spike protein S1-derived protein is located on the surface of the structure.

Example 2: Confirmation of the ability of recombinant protein to produce specific antibody against PEDV spike protein

In order to confirm the ability of the recombinant protein prepared in Example 1 to produce a specific antibody against the PEDV spike protein, the following experiment was performed.

Specifically, Balb/c mice were purchased and divided into the following groups. Each group used 5 mice. The first group was inoculated with PBS alone as a negative control group (N.C.), the second group was inoculated with PEDV virus as a positive control group (P.C.), the third group was inoculated with ferritin protein (Ferritin), and the fourth group was PEDV spike protein. The group inoculated with the S1 protein (PEDV S1) and the fifth group were grouped into the group inoculated with the recombinant protein of the present invention containing the ferritin-conjugated PEDV spike protein S1 domain (Ferritin-PEDV S1).

Inoculation was carried out according to each experimental group and control group, and 2 weeks after the first inoculation, additional inoculation (boosting) was performed. Two weeks after the second inoculation, serum was collected from all mice, and the level of specific antibody production was measured using ELISA (Enzyme-Linked Immunosorbent Assay), and the relative levels were compared based on the level of PEDV S1 production.

As a result of the above experiment, the Ferritin-PEDV S1 group had the best specific antibody production, and it was confirmed that it was about 2.9 times higher than that of the PEDV S1 group. It can be seen that exhibits excellent immunogenicity.

Example 3: Confirmation of Neutralization Ability of Antibodies Constructed Using Recombinant Proteins

In order to confirm the antibody neutralizing ability of the specific antibody against the FEDV spike protein prepared using the recombinant protein of Example 1, the following experiment was performed.

Specifically, serum from mice immunized with porcine ferritin-FEDV S1 protein (Ferritin PEDV S1), the recombinant protein prepared in Example 1, was obtained, incubated with PEDV, and then treated with Vero cells. PEDV can induce apoptosis by infecting the Vero cells, and it was determined whether or not neutralizing antibodies were generated by measuring the degree to which apoptosis did not occur because the cells could not be infected by the neutralizing antibody. In the case of the above experiment, if the neutralizing ability of the antibody in the immunized mouse serum is excellent, PEDV may not infect the cells and the cell viability may be improved. Therefore, the better the antibody neutralizing ability, the higher the cell viability (Fig. 3).

In addition, in the case of PBS, PEDV S1 protein and porcine ferritin-FEDV S2 protein (Ferritin PEDV S2), immunization was performed as described above to obtain serum, and then antibody neutralizing ability was confirmed.

As a result of the above experiment, it was confirmed that the serum of mice immunized with Ferritin PEDV S1 had better cell viability than the serum immunized with S1 protein. can In addition, as it was confirmed that the antibody neutralizing ability was superior to that of Ferritin PEDV S2 including ferritin and S2 portion, when the S1 portion of the PEDV spike protein was bound to ferritin, it was found that the antibody neutralizing ability and vaccine effect were significantly superior (Fig. 4). ).

The foregoing description of the present invention is for illustration, and those of ordinary skill in the art to which the present invention pertains can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.

<110> The Industry & Academic Cooperation in Chungnam National University (IAC) <120> Recombinant protein comprising spike protein S1 of PEDV-derived protein and ferritin-derived protein and use thereof <130> PN136680KR <160> 3 <170> KoPatentIn 3.0 <210> 1 <211> 721 <212> PRT <213> Artificial Sequence <220> <223> PEDV spike S1 <400> 1 Met Arg Ser Leu Ile Tyr Phe Trp Leu Leu Leu Pro Val Leu Pro Thr 1 5 10 15 Leu Ser Leu Pro Gln Asp Val Thr Arg Cys Gln Ser Thr Thr Asn Phe 20 25 30 Arg Arg Phe Phe Ser Lys Phe Asn Val Gln Ala Pro Ala Val Val Val 35 40 45 Leu Gly Gly Tyr Leu Pro Ser Met Asn Ser Ser Ser Trp Tyr Cys Gly 50 55 60 Thr Gly Ile Glu Thr Ala Ser Gly Val His Gly Ile Phe Leu Ser Tyr 65 70 75 80 Ile Asp Ser Ser Gln Gly Phe Glu Ile Gly Ile Ser Gln Glu Pro Phe 85 90 95 Asp Pro Ser Gly Tyr Gln Leu Tyr Leu His Lys Ala Thr Asn Gly Asn 100 105 110 Thr Asn Ala Ile Ala Arg Leu Arg Ile Ser Gln Phe Pro Asp Asn Lys 115 120 125 Thr Leu Gly Pro Thr Val Asn Asp Val Thr Thr Gly Arg Asn Cys Leu 130 135 140 Phe Asn Lys Ala Ile Pro Ala Tyr Met Arg Asp Gly Lys Asp Ile Val 145 150 155 160 Val Gly Ile Thr Trp Asp Asn Asp Arg Val Thr Val Phe Ala Asp Lys 165 170 175 Ile Tyr His Phe Tyr Leu Lys Asn Asp Trp Ser Arg Val Ala Thr Arg 180 185 190 Cys Tyr Asn Arg Arg Ser Cys Ala Met Gln Tyr Val Tyr Thr Pro Thr 195 200 205 Tyr Tyr Met Leu Asn Val Thr Ser Ala Gly Glu Asp Gly Ile Tyr Tyr 210 215 220 Glu Pro Cys Thr Ala Asn Cys Thr Gly Tyr Ala Ala Asn Val Phe Ala 225 230 235 240 Thr Asp Ser Asn Gly His Ile Pro Glu Ser Phe Ser Phe Asn Asn Trp 245 250 255 Phe Leu Leu Ser Asn Asp Ser Thr Leu Leu His Gly Lys Val Val Ser 260 265 270 Asn Gln Pro Leu Leu Val Asn Cys Leu Leu Ala Ile Pro Lys Ile Tyr 275 280 285 Gly Leu Gly Gln Phe Phe Ser Phe Asn His Thr Met Asp Gly Val Cys 290 295 300 Asn Gly Ala Ala Leu Asp Arg Ala Pro Glu Ala Leu Arg Phe Asn Ile 305 310 315 320 Asn Asp Thr Ser Val Ile Leu Ala Glu Gly Ser Ile Val Leu His Thr 325 330 335 Ala Leu Gly Thr Asn Leu Ser Phe Val Cys Ser Asn Ser Ser Asp Pro 340 345 350 His Leu Ala Thr Phe Ala Ile Pro Leu Gly Ala Thr Glu Val Pro Tyr 355 360 365 Tyr Cys Phe Leu Ile Val Asp Thr Tyr Asn Ser Thr Val Tyr Lys Phe 370 375 380 Leu Ala Val Leu Pro Pro Thr Val Arg Glu Ile Val Ile Thr Lys Tyr 385 390 395 400 Gly Asp Val Tyr Val Asn Gly Phe Gly Tyr Leu His Leu Gly Leu Leu 405 410 415 Asp Ala Val Thr Ile Asn Phe Thr Gly His Gly Thr Asp Asp Asp Val 420 425 430 Ser Gly Phe Trp Thr Ile Asp Ser Thr Asn Phe Val Asp Ala Leu Ile 435 440 445 Glu Val Gln Gly Thr Ser Ile Gln Arg Ile Leu Tyr Cys Asp Asp Pro 450 455 460 Val Ser Gln Leu Lys Cys Ser Gln Val Ala Phe Asp Leu Asp Asp Gly 465 470 475 480 Phe Tyr Pro Ile Ser Ser Arg Asn Leu Leu Ser His Glu Gln Pro Ile 485 490 495 Ser Phe Val Thr Leu Pro Ser Phe Asn Asp His Ser Phe Val Asn Ile 500 505 510 Thr Val Ser Ala Ser Phe Gly Asp His Ser Gly Ala Asn Leu Val Ala 515 520 525 Ser Asp Thr Thr Ile Asn Gly Phe Ser Ser Phe Cys Val Asp Thr Arg 530 535 540 Gln Phe Thr Ile Arg Leu Phe Tyr Asn Val Thr Ser Ser Tyr Gly Tyr 545 550 555 560 Val Ser Lys Ser Gln Tyr Ser Asn Cys Pro Phe Thr Leu Gln Ser Val 565 570 575 Asn Asp Tyr Leu Ser Phe Ser Lys Phe Cys Val Ser Thr Ser Leu Leu 580 585 590 Ala Ser Ala Cys Thr Ile Asp Leu Phe Gly Tyr Pro His Phe Gly Ser 595 600 605 Gly Val Lys Phe Thr Ser Leu Tyr Phe Gln Phe Thr Glu Gly Glu Leu 610 615 620 Ile Thr Gly Thr Pro Lys Pro Leu Glu Gly Val Thr Asp Val Ser Phe 625 630 635 640 Met Thr Leu Asp Val Cys Thr Lys Tyr Thr Ile Tyr Gly Phe Lys Gly 645 650 655 Glu Gly Ile Ile Thr Leu Thr Asn Ser Phe Leu Ala Gly Val Tyr 660 665 670 Tyr Thr Ser Asp Ser Gly Gln Leu Leu Ala Phe Lys Asn Val Thr Ser 675 680 685 Gly Ala Val Tyr Ser Val Thr Pro Cys Ser Phe Ser Glu Gln Ala Ala 690 695 700 Tyr Val Asp Asp Asp Ile Val Gly Val Ile Ser Ser Leu Ser Asn Ser 705 710 715 720 Thr <210> 2 <211> 181 <212> PRT <213> Artificial Sequence <220> <223> ferritin <400> 2 Met Thr Thr Ser Cys Ser Ser Gln Val Arg Gln Asn Tyr His Gln Asp 1 5 10 15 Ser Glu Ala Ala Ile Asn Arg Gln Ile Asn Leu Glu Leu Tyr Ala Ser 20 25 30 Tyr Val Tyr Leu Ser Met Ser Tyr Tyr Phe Asp Arg Asp Asp Val Ala 35 40 45 Leu Lys Asn Phe Ala Lys Tyr Phe Leu His Gln Ser His Gly Gly Arg 50 55 60 Gly His Ala Glu Lys Leu Met Lys Leu Gln Thr Gln Arg Gly Ala Arg 65 70 75 80 Ile Phe Leu Gln Asp Ile Met Lys Pro Glu Arg Asp Asp Trp Glu Asn 85 90 95 Gly Leu Thr Ala Met Glu Phe Ala Leu His Val Val Lys Asn Val Tyr 100 105 110 Gln Ser Leu Leu Glu Leu His Lys Leu Ala Thr Asp Lys Asn Asp Pro 115 120 125 His Leu Cys Asp Phe Ile Glu Thr His Tyr Leu His Glu Gln Val Lys 130 135 140 Ala Ile Lys Glu Leu Gly Asp His Ile Thr Asn Leu His Arg Met Gly 145 150 155 160 Ala Pro Glu Tyr Gly Met Ala Glu Tyr Leu Phe Asp Lys His Thr Leu 165 170 175 Gly Ser Ser Glu Ser 180 <210> 3 <211> 815 <212> PRT <213> Artificial Sequence <220> <223> recombinant protein <400> 3 Arg Asp Asp Trp Glu Asn Gly Leu Thr Ala Met Glu Phe Ala Leu His 1 5 10 15 Val Val Lys Asn Val Tyr Gln Ser Leu Leu Glu Leu His Lys Leu Ala 20 25 30 Thr Asp Lys Asn Asp Pro His Leu Cys Asp Phe Ile Glu Thr His Tyr 35 40 45 Leu His Glu Gln Val Lys Ala Ile Lys Glu Leu Gly Asp His Ile Thr 50 55 60 Asn Leu His Arg Met Gly Ala Pro Glu Tyr Gly Met Ala Glu Tyr Leu 65 70 75 80 Phe Asp Lys His Thr Leu Gly Ser Ser Glu Ser Ser Ser Gly Met Arg 85 90 95 Ser Leu Ile Tyr Phe Trp Leu Leu Leu Pro Val Leu Pro Thr Leu Ser 100 105 110 Leu Pro Gln Asp Val Thr Arg Cys Gln Ser Thr Thr Asn Phe Arg Arg 115 120 125 Phe Phe Ser Lys Phe Asn Val Gln Ala Pro Ala Val Val Val Leu Gly 130 135 140 Gly Tyr Leu Pro Ser Met Asn Ser Ser Ser Trp Tyr Cys Gly Thr Gly 145 150 155 160 Ile Glu Thr Ala Ser Gly Val His Gly Ile Phe Leu Ser Tyr Ile Asp 165 170 175 Ser Ser Gln Gly Phe Glu Ile Gly Ile Ser Gln Glu Pro Phe Asp Pro 180 185 190 Ser Gly Tyr Gln Leu Tyr Leu His Lys Ala Thr Asn Gly Asn Thr Asn 195 200 205 Ala Ile Ala Arg Leu Arg Ile Ser Gln Phe Pro Asp Asn Lys Thr Leu 210 215 220 Gly Pro Thr Val Asn Asp Val Thr Thr Gly Arg Asn Cys Leu Phe Asn 225 230 235 240 Lys Ala Ile Pro Ala Tyr Met Arg Asp Gly Lys Asp Ile Val Val Gly 245 250 255 Ile Thr Trp Asp Asn Asp Arg Val Thr Val Phe Ala Asp Lys Ile Tyr 260 265 270 His Phe Tyr Leu Lys Asn Asp Trp Ser Arg Val Ala Thr Arg Cys Tyr 275 280 285 Asn Arg Arg Ser Cys Ala Met Gln Tyr Val Tyr Thr Pro Thr Tyr Tyr 290 295 300 Met Leu Asn Val Thr Ser Ala Gly Glu Asp Gly Ile Tyr Tyr Glu Pro 305 310 315 320 Cys Thr Ala Asn Cys Thr Gly Tyr Ala Ala Asn Val Phe Ala Thr Asp 325 330 335 Ser Asn Gly His Ile Pro Glu Ser Phe Ser Phe Asn Asn Trp Phe Leu 340 345 350 Leu Ser Asn Asp Ser Thr Leu Leu His Gly Lys Val Val Ser Asn Gln 355 360 365 Pro Leu Leu Val Asn Cys Leu Leu Ala Ile Pro Lys Ile Tyr Gly Leu 370 375 380 Gly Gln Phe Phe Ser Phe Asn His Thr Met Asp Gly Val Cys Asn Gly 385 390 395 400 Ala Ala Leu Asp Arg Ala Pro Glu Ala Leu Arg Phe Asn Ile Asn Asp 405 410 415 Thr Ser Val Ile Leu Ala Glu Gly Ser Ile Val Leu His Thr Ala Leu 420 425 430 Gly Thr Asn Leu Ser Phe Val Cys Ser Asn Ser Ser Asp Pro His Leu 435 440 445 Ala Thr Phe Ala Ile Pro Leu Gly Ala Thr Glu Val Pro Tyr Tyr Cys 450 455 460 Phe Leu Ile Val Asp Thr Tyr Asn Ser Thr Val Tyr Lys Phe Leu Ala 465 470 475 480 Val Leu Pro Pro Thr Val Arg Glu Ile Val Ile Thr Lys Tyr Gly Asp 485 490 495 Val Tyr Val Asn Gly Phe Gly Tyr Leu His Leu Gly Leu Leu Leu Asp Ala 500 505 510 Val Thr Ile Asn Phe Thr Gly His Gly Thr Asp Asp Asp Val Ser Gly 515 520 525 Phe Trp Thr Ile Asp Ser Thr Asn Phe Val Asp Ala Leu Ile Glu Val 530 535 540 Gln Gly Thr Ser Ile Gln Arg Ile Leu Tyr Cys Asp Asp Pro Val Ser 545 550 555 560 Gln Leu Lys Cys Ser Gln Val Ala Phe Asp Leu Asp Asp Gly Phe Tyr 565 570 575 Pro Ile Ser Ser Arg Asn Leu Leu Ser His Glu Gln Pro Ile Ser Phe 580 585 590 Val Thr Leu Pro Ser Phe Asn Asp His Ser Phe Val Asn Ile Thr Val 595 600 605 Ser Ala Ser Phe Gly Asp His Ser Gly Ala Asn Leu Val Ala Ser Asp 610 615 620 Thr Thr Ile Asn Gly Phe Ser Ser Phe Cys Val Asp Thr Arg Gln Phe 625 630 635 640 Thr Ile Arg Leu Phe Tyr Asn Val Thr Ser Ser Tyr Gly Tyr Val Ser 645 650 655 Lys Ser Gln Tyr Ser Asn Cys Pro Phe Thr Leu Gln Ser Val Asn Asp 660 665 670 Tyr Leu Ser Phe Ser Lys Phe Cys Val Ser Thr Ser Leu Leu Ala Ser 675 680 685 Ala Cys Thr Ile Asp Leu Phe Gly Tyr Pro His Phe Gly Ser Gly Val 690 695 700 Lys Phe Thr Ser Leu Tyr Phe Gln Phe Thr Glu Gly Glu Leu Ile Thr 705 710 715 720 Gly Thr Pro Lys Pro Leu Glu Gly Val Thr Asp Val Ser Phe Met Thr 725 730 735 Leu Asp Val Cys Thr Lys Tyr Thr Ile Tyr Gly Phe Lys Gly Glu Gly 740 745 750 Ile Ile Thr Leu Thr Asn Ser Ser Phe Leu Ala Gly Val Tyr Tyr Thr 755 760 765 Ser Asp Ser Gly Gln Leu Leu Ala Phe Lys Asn Val Thr Ser Gly Ala 770 775 780 Val Tyr Ser Val Thr Pro Cys Ser Phe Ser Glu Gln Ala Ala Tyr Val 785 790 795 800 Asp Asp Asp Ile Val Gly Val Ile Ser Ser Leu Ser Asn Ser Thr 805 810 815

Claims (12)

A recombinant protein comprising a protein derived from Porcine epidemic diarrhea virus (PEDV) spike protein S1 and a protein derived from ferritin. The recombinant protein of claim 1, wherein the PEDV spike protein S1 derived protein comprises the amino acid sequence of SEQ ID NO: 1. The recombinant protein of claim 1, wherein the ferritin-derived protein comprises the amino acid sequence of SEQ ID NO: 2. The method according to claim 1, The recombinant protein comprising the amino acid sequence of SEQ ID NO: 3, the recombinant protein. A polynucleotide encoding the recombinant protein of any one of claims 1 to 4. A vaccine composition for preventing or treating PEDV infection disease, comprising the recombinant protein of any one of claims 1 to 4 as an active ingredient. The vaccine composition of claim 6 , wherein two or more of the recombinant proteins are self-assembled to form a construct. The vaccine composition of claim 7, wherein the PEDV spike protein S1 derived protein is located on the outer surface of the construct. The method according to claim 6, wherein the vaccine composition is selected from the group consisting of compositions for intramuscular administration, subcutaneous administration, intraperitoneal administration, intravenous administration, intravenous administration, dermal administration, ocular administration, and brain administration, vaccine composition. The vaccine composition of claim 6, wherein the vaccine composition further comprises a pharmaceutically acceptable excipient, diluent or carrier. A method for preventing or treating a PEDV-infected disease comprising administering the composition of claim 6 to a subject other than a human. The method of claim 11 , wherein the subject is a mammal selected from the group consisting of cattle, horses, sheep, pigs, goats, camels, antelopes, dogs, cats, mice and pigs.

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