KR20220133631A - Recombinant protein comprising spike protein of SARS-CoV-2-derived protein and Fc of immunoglobulin-derived protein and use thereof - Google Patents

Abstract

The present invention relates to a recombinant protein including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein S1-derived protein, immunoglobulin fragment crystallizable region (Fc)-derived protein, and SARS-CoV-2 spike protein S2-derived protein, and to a vaccine composition including the recombinant protein. According to the present invention, the recombinant protein can form a tetravalent bispecific antigen protein in which the spike protein S1 and S2 domains of SARS-CoV-2, which are antigen proteins, are presented on both sides of an Fc so that specific antibodies against SARS-CoV-2 can be effectively generated when a vaccine composition including the recombinant protein is used.

Description

Recombinant protein comprising spike protein of SARS-CoV-2-derived protein and Fc of immunoglobulin-derived protein and use thereof

The present invention relates to a recombinant protein comprising a protein derived from Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein S1, a protein derived from fragment crystallizable region (Fc) of immunoglobulin, and a protein derived from SARS-CoV-2 spike protein S2. And it relates to a vaccine composition comprising the recombinant protein.

The 2019 novel Coronavirus (2019-nCoV) is the causative virus of the novel coronavirus infection (Wuhan pneumonia) that occurred in December 2019, and is one of seven coronaviruses that infect humans. It has been reported that the novel coronavirus is transmitted when droplets (saliva) of an infected person penetrate the respiratory tract or the mucous membranes of the eyes, nose, and mouth. Respiratory symptoms such as cough or shortness of breath and pneumonia are the main symptoms, but asymptomatic infections are rare. The World Health Organization (WHO) announced on January 24, 2020 that the transmission power of the novel coronavirus is lower than SARS (severe acute respiratory syndrome), but higher than MERS (Middle East Respiratory Syndrome).

According to the report of the Korea Centers for Disease Control and Prevention, the gene sequence of the novel coronavirus showed the highest homology (89.1%) to the bat-derived coronavirus, 50% to MERS and 77.5% to SARS. Coronaviruses are classified into four genera, including Alpha, Beta, Gamma, and Delta, and the new coronavirus was classified as belonging to the Beta group. There are a total of seven types of human-communicable coronaviruses identified so far, including HCoV 229E, HCoV NL63, HCoV OC43, HCoV HKU1, SARS-CoV, MERS-CoV, and 2019-nCoV.

Coronavirus is one of the viruses that cause the seasonal flu. But it should never be taken lightly. The cause of SARS (severe acute respiratory syndrome) in 2002 and MERS (Middle East Respiratory Syndrome) in 2015 was also caused by coronavirus. The 2019-nCoV discovered this time is also causing great social problems worldwide as the number of patients with severe respiratory syndrome symptoms increases with rapid spread.

When corona virus is observed with a transmission electron microscope (TEM), a structure can be seen in which the protein (spike protein) in the form of projections is tightly attached to the outer surface of the virus membrane. Its shape resembles the sun's corona, hence the name coronavirus. Just as the spikes on the bottom of a soccer shoe prevent slipping, the spike protein binds strongly to the host cell and supports the rapid penetration of the virus into the host cell. Therefore, it can be seen that the spike protein plays an important role in the early infection of the virus into the human body.

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 the 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 have developed a recombinant protein comprising a SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2) spike protein and an Fc-derived protein of immunoglobulin and a vaccine comprising the same, and have excellent antigenicity of the vaccine. The present invention was completed by verification.

One aspect is a recombinant protein comprising a protein derived from Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein S1, a protein derived from fragment crystallizable region (Fc) of immunoglobulin, and a protein derived from SARS-CoV-2 spike protein S2 provides

Another aspect provides a polynucleotide encoding the recombinant protein.

Another aspect provides a vaccine composition for preventing or treating SARS-CoV-2 infection disease, comprising the recombinant protein as an active ingredient.

One aspect is a recombinant protein comprising a protein derived from Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein S1, a protein derived from fragment crystallizable region (Fc) of immunoglobulin, and a protein derived from SARS-CoV-2 spike protein S2 is to provide

As used herein, the term "SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2)" refers to SARS coronavirus-2 or the provisional name 2019 novel coronavirus (2019-nCoV) is a genetic sequence (DNA It is a positive sense single-stranded RNA coronavirus that is contagious to humans and is known to be the cause of coronavirus infection-19. SARS-CoV-2 is the cause of the Coronavirus Infectious Disease-19 epidemic that originated in Wuhan City, China, and has developed into an international public health emergency.

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 SARS-CoV-2 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 S2 is known to play an important role in fusion.

As used herein, the term "immunoglobulin (Ig)" refers to a substance that specifically binds to an antigen and causes an antigen-antibody reaction, and is used in the same sense as 'antibody'. The immunoglobulin may be selected from the group consisting of IgA, IgE, IgM, IgD, IgY, IgNAR, heavy chain antibody, and IgG. The antibody consists of two each of a light chain and a heavy chain, and each chain consists of a variable domain having a variable amino acid sequence and a constant domain having a constant sequence. . In the antibody, an antigen-binding site is located at the end of the three-dimensional structure of the variable region, and this region is formed by a collection of three complementarity determining regions present in each of the light and heavy chains. The complementarity determining region is a region with particularly high amino acid sequence variability among the variable regions, and specific antibodies to various antigens can be found by such high variability. The scope of the present application includes not only complete antibody forms, but also antigen-binding fragments of such antibody molecules.

As used herein, the term "Fc (fragment crystallizable) region" refers to the heavy and light chain variable regions of immunoglobulin, heavy chain constant region 1 (CH1) and light chain constant region (CL1), except for heavy chain constant region 2 (CH2) and heavy chain It refers to the constant region 3 (CH3) portion, and also includes a hinge portion in the heavy chain constant region. Since the antibody Fc region is a biodegradable polypeptide that is metabolized in vivo, it is safe to use as a drug carrier. In addition, since the immunoglobulin Fc region has a relatively low molecular weight compared to the entire immunoglobulin molecule, it is advantageous in terms of preparation, purification, and yield of the conjugate, and the amino acid sequence is different for each antibody. It can also be expected to significantly increase the blood antigen and reduce the possibility of inducing antigenicity in the blood.

The immunoglobulin may be human immunoglobulin, and the Fc region of the immunoglobulin may be an Fc region derived from IgG1, IgG2, IgA, IgD, IgE, IgM, a combination thereof, or a hybrid thereof. , specifically the Fc region of human IgG1.

The SARS-CoV-2 spike protein S1-derived protein, the immunoglobulin Fc-derived protein, and the SARS-CoV-2 spike protein S2-derived protein may be directly or indirectly linked, and specifically, the SARS-CoV-2 spike protein S1 The protein derived from Fc, immunoglobulin Fc-derived protein may be linked through a hinge region, and immunoglobulin Fc-derived protein and SARS-CoV-2 spike protein S2-derived protein may be linked through a linker.

The recombinant protein may further include a hinge region and/or a linker, and specifically, the hinge region may be located between the SARS-CoV-2 spike protein S1-derived protein and the immunoglobulin Fc-derived protein, The linker may be located between the Fc-derived protein of immunoglobulin and the SARS-CoV-2 spike protein S2-derived protein. The hinge region and the linker connect the protein derived from the SARS-CoV-2 spike protein and the protein derived from Fc to increase the structural flexibility of these proteins or enhance the activity of each protein when a recombinant protein is made by connecting the protein and the protein. It may be a peptide that is inserted into The hinge region and the linker are not limited as long as they do not inhibit the activity of each protein to be fused and do not cause an unnecessary immune response, but are preferably 1 to 20 amino acids, more preferably 1 to 5 amino acids. can be

The SARS-CoV-2 spike protein S1 or S2 derived protein may be linked to the C-terminus or N-terminus of the Fc-derived protein of immunoglobulin, and specifically, the SARS-CoV-2 spike protein S1 is derived from the Fc-derived immunoglobulin. At the N-terminus of the protein, SARS-CoV-2 spike protein S2 may be linked to the C-terminus of an Fc-derived protein of immunoglobulin.

The SARS-CoV-2 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 Fc-derived protein of the immunoglobulin 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 SARS-CoV-2 spike protein S2 derived protein may include the amino acid sequence of SEQ ID NO. 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 is a protein comprising the immunoglobulin Fc region-conjugated SARS-CoV-2 spike protein S1 and S2 domains, and may include the amino acid sequence of SEQ ID NO: 4, specifically composed of the amino acid sequence of SEQ ID NO: 4 it may be In addition, not only the amino acid sequence consisting of SEQ ID NO: 4, 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 tetravalent antibody-like structure by inserting a human IgG1 Fc region between the antigenic peptide SARS-CoV-2 spike protein S1 and S2 domains, and thus has excellent antigenicity and antibody-forming ability, the recombinant protein can be used for preventing SARS-CoV-2 infection, or for preventing or treating a SARS-CoV-2 infectious disease, compositions and methods, and the like.

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 above-described conditions. 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 SARS-CoV-2 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 SARS-CoV-2 infection and disease onset due to the administration of the SARS-CoV-2 vaccine composition.

As used herein, the term “treatment” refers to any action in which the symptoms of a disease already caused by SARS-CoV-2 infection are improved or beneficial due to the administration of the SARS-CoV-2 vaccine composition.

Two or more recombinant proteins may be assembled to form a construct. The construct may be a construct similar to that of an immunoglobulin, and specifically may be a tetravalent bispecific construct.

The construct can improve antigenicity by inserting the Fc region of IgG1 between the S1 domain and the S2 domain of the SARS-CoV-2 spike protein, which is an antigenic peptide, to form a tetravalent antibody structure.

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.

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 SARS-CoV-2 infection or infectious disease. 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.

Another aspect provides a method for preventing or treating SARS-CoV-2 infection, or preventing or treating a SARS-CoV-2 infection comprising administering the vaccine composition to a subject. The same parts as those described above are equally applied to the above method.

The subject includes cattle, horses, sheep, pigs, goats, camels, antelopes, dogs, cats, rats, livestock, humans, etc., which are infected with SARS-CoV-2 and are at risk of developing or developing a disease caused by the infection. It may include, without limitation, mammals, farmed fish, and the like.

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 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 is capable of forming a tetravalent bispecific antigen protein in which the spike protein S1 and S2 domains of the antigen protein SARS-CoV-2 are presented on both sides of the Fc, a vaccine composition comprising the same When used, specific antibodies to SARS-CoV-2 can be effectively generated.

1 is a diagram schematically showing the construction of a recombinant protein of the present invention.
Figure 2 is a view confirming the antibody-forming effect 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 SARS-CoV-2, a protein derived from the spike protein S1 and a protein derived from S2 of SARS-CoV-2 were used, and the amino acid sequence of the protein is SEQ ID NO: 1 and 3 was described. Next, in order to effectively induce the antibody formation of the antigen peptide, an Fc-derived protein of IgG was used, and the amino acid sequence of the protein was described as SEQ ID NO:3. The SARS-CoV-2 spike protein S1-derived protein is linked to the N-terminus of the Fc-derived protein using a hinge region, and the SARS-CoV-2 spike protein S2-derived protein is linked to the Fc-derived protein using a linker. It was linked to the C-terminus to construct a recombinant protein. The amino acid sequence of the recombinant protein is described as SEQ ID NO: 4.

protein amino acid sequence order
number Protein derived from spike protein S1 of SARS-CoV-2 MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYE One Fc-derived protein of IgG DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKKNQVSLTCLDSDNGQPREPQVYTLPPSRDELTKKNQVSLTCLDWSDNGQFQSDIKFSDKVSHEDGSGFFYPHDKFSVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTSKFS 2 Protein derived from spike protein S2 of SARS-CoV-2 CDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT 3 recombinant protein DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLDWSDNGQPGHSDIKTSKSPVWSDGSPGQSDIKTSKSPVWSDGSGFFNWYTSKSPVWSDGSGFFNWYVDGVEVHNAKTKPREEQYNSty 4

The recombinant protein can form a structure in the form of a tetravalent bispecific antibody that presents the S1-derived protein and the S2-derived protein of SARS-CoV-2 at both ends (FIG. 1).

Example 2: Confirmation of the ability of recombinant protein to produce specific antibody against SARS-CoV-2 spike protein

In order to confirm the ability of the recombinant protein prepared in Example 1 to produce a specific antibody against the SARS-CoV-2 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 is a negative control group inoculated with PBS only (N.C.), the second group is a group inoculated with SARS-CoV-2 spike protein (SARS-CoV-2 spike), and the third group is an IgG Fc-conjugated SARS-CoV-2 spike. It was classified into a group (S1FcS2) inoculated with the recombinant protein of the present invention containing the protein.

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 specific antibody production level was measured using ELISA (Enzyme-Linked Immunosorbent Assay), and the relative level based on the generation level of the SARS-CoV-2 spike group. were compared.

As a result of the above experiment, it was confirmed that the specific antibody production level was the best in the S1FcS2 group, and about 3.6 times higher than that of the SARS-CoV-2 spike group, the recombinant protein of the present invention than that inoculated with only the SARS-CoV-2 spike protein. It can be seen that when inoculated with a more excellent immunogenicity.

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 of SARS-CoV-2-derived protein and Fc of immunoglobulin-derived protein and use thereof <130> PN136679KR <160> 4 <170> KoPatentIn 3.0 <210> 1 <211> 661 <212> PRT <213> Artificial Sequence <220> <223> SARS-CoV-2 spike s1 <400> 1 Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Val 1 5 10 15 Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser Phe 20 25 30 Thr Arg Gly Val Tyr Pro Asp Lys Val Phe Arg Ser Ser Val Leu 35 40 45 His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp 50 55 60 Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp 65 70 75 80 Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr Glu 85 90 95 Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser 100 105 110 Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile 115 120 125 Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val Tyr 130 135 140 Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val Tyr 145 150 155 160 Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu 165 170 175 Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu Phe 180 185 190 Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr 195 200 205 Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser Ala Leu Glu 210 215 220 Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr 225 230 235 240 Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser 245 250 255 Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro 26 0 265 270 Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala 275 280 285 Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys 290 295 300 Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val 305 310 315 320 Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys 325 330 335 Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala 340 345 350 Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu 355 360 365 Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro 370 375 380 Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe 385 390 395 400 Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gl n Thr Gly 405 410 415 Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys 420 425 430 Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn 435 440 445 Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe 450 455 460 Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys 465 470 475 480 Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly 485 490 495 Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val 500 505 510 Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys 515 520 525 Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn 530 535 540 Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Le u 545 550 555 560 Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala Val 565 570 575 Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe 580 585 590 Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln Val 595 600 605 Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val Pro Val Ala Ile 610 615 620 His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly Ser 625 630 635 640 Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu His Val 645 650 655 Asn Asn Ser Tyr Glu 660 <210> 2 <211> 227 <212> PRT <213> Artificial Sequence <220> <223> IgG Fc <400> 2 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn V al Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 3 <211> 612 <212> PRT <213> Artificial Sequence <220> <223> SARS-CoV-2 spike s2 <400> 3 Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala Ser Tyr Gln Thr Gln 1 5 10 15 Thr Asn Ser Pro Arg Arg Ala Arg Ser Val Ala Ser Gln Ser Ile Ile 20 25 30 Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn Ser Val Ala Tyr Ser Asn 35 40 45 Asn Ser Ile Ala Ile Pro Thr Asn Phe Thr Ile Ser Val Thr Thr Glu 50 55 60 Ile Leu Pro Val Ser Met Thr Lys Thr Ser Val Asp Cys Thr Met Tyr 65 70 75 80 Ile Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu Leu Leu Gln Tyr Gly 85 90 95 Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Thr Gly Ile Ala Val Glu 100 105 110 Gln Asp Lys Asn Thr Gln Glu Val Phe Ala Gln Val Lys Gln Ile Tyr 115 120 125 Lys Thr Pro Pro Ile Lys Asp Phe Gly Gly Phe Asn Phe Ser Gln Ile 130 135 140 Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser Phe Ile Glu Asp Leu 145 150 155 160 Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly Phe Ile Lys Gln Tyr 165 170 175 Gly Asp Cys Leu Gly Asp Ile Ala Ala Arg Asp Leu Ile Cys Ala Gln 180 185 190 Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu Leu Thr Asp Glu Met 195 200 205 Ile Ala Gln Tyr Thr Ser Ala Leu Leu Ala Gly Thr Ile Thr Ser Gly 210 215 220 Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile Pro Phe Ala Met Gln 225 230 235 240 Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr Gln Asn Val Leu Tyr 245 250 255 Glu Asn Gln Lys Leu Ile Ala Asn Gln Phe Asn Ser Ala Ile Gly Lys 260 265 270 Ile Gln Asp Ser Leu Ser Ser Thr Ala Ser Ala Leu Gly Lys Leu Gln 275 280 285 Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn Thr Leu Val Lys Gln 290 295 300 Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val Leu Asn Asp Ile Leu 305 310 315 320 Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gln Ile Asp Arg Leu Ile 325 330 335 Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val Thr Gln Gln Leu Ile 340 345 350 Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn Leu Ala Ala Thr Lys Met 355 360 365 Ser Glu Cys Val Leu Gly Gln Ser Lys Arg Val Asp Phe Cys Gly Lys 370 375 380 Gly Tyr His Leu Met Ser Phe Pro Gln Ser Ala Pro His Gly Val Val 385 390 395 400 Phe Leu His Val Thr Tyr Val Pro Ala Gln Glu Lys Asn Phe Thr Thr 405 410 415 Ala Pro Ala Ile Cys His Asp Gly Lys Ala His Phe Pro Arg Glu Gly 420 425 430 Val Phe Val Ser Asn Gly Thr His Trp Phe Val Thr Gln Arg Asn Phe 435 440 445 Tyr Glu Pro Gln Ile Ile Thr Thr Asp Asn Thr Phe Val Ser Gly Asn 450 455 460 Cys Asp Val Val Ile Gly Ile Val Asn Asn Thr Val Tyr Asp Pro Leu 465 470 475 480 Gln Pro Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys 485 490 495 Asn His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser Gly Ile Asn 500 505 510 Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu Val 515 520 525 Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu Gly Lys 530 535 540 Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Ile Trp Leu Gly Phe Ile 545 550 555 560 Ala Gly Leu Ile Ala Ile Val Met Val Thr Ile Met Leu Cys Cys Met 565 570 575 Thr Ser Cys Cys Ser Cys Leu Lys Gly Cys Cys Ser Cys Gly Ser Cys 580 585 590 Cys Lys Phe Asp Glu Asp Ser Glu Pro Val Leu Lys Gly Val Lys 595 600 605 Leu His Tyr Thr 610 <210> 4 <211> 1503 <212> PRT <213> Artificial Sequence <220> <223> recombinant protein <400> 4 Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Val 1 5 10 15 Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser Phe 20 25 30 Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val Leu 35 40 45 His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp 50 55 60 Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp 65 70 75 80 Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr Glu 85 90 95 Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser 100 105 110 Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile 115 120 125 Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val Tyr 130 135 140 Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val Tyr 145 150 155 160 Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu 165 170 175 Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu Phe 180 185 190 Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr 195 200 205 Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser Ala Leu Glu 210 215 220 Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr 225 230 235 240 Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser 245 250 255 Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro 260 265 270 Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala 275 280 285 Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys 290 295 300 Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val 305 310 315 320 Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys 325 330 335 Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala 340 345 350 Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu 355 360 365 Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro 370 375 380 Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe 385 390 395 400 Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly 405 410 415 Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys 420 425 430 Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn 435 440 445 Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe 450 455 460 Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys 465 470 475 480 Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly 485 490 495 Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val 500 505 510 Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys 515 520 525 Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn 530 535 540 Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu 545 550 555 560 Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala Val 565 570 575 Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe 580 585 590 Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln Val 595 600 605 Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val Pro Val Ala Ile 610 615 620 His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly Ser 625 630 635 640 Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu His Val 645 650 655 Asn Asn Ser Tyr Glu Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 660 665 670 Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 675 680 685 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 690 695 700 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 705 710 715 720 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 725 730 735 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 740 745 750 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 755 760 765 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 770 775 780 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr 785 790 795 800 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 805 810 815 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 820 825 830 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 835 840 845 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 850 855 860 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 865 870 875 880 Ser Leu Ser Leu Ser Pro Gly Lys Ser Ser Gly Cys Asp Ile Pro Ile 885 890 895 Gly Ala Gly Ile Cys Ala Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg 900 905 910 Arg Ala Arg Ser Val Ala Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser 915 920 925 Leu Gly Ala Glu Asn Ser Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile 930 935 940 Pro Thr Asn Phe Thr Ile Ser Val Thr Thr Glu Ile Leu Pro Val Ser 945 950 955 960 Met Thr Lys Thr Ser Val Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser 965 970 975 Thr Glu Cys Ser Asn Leu Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln 980 985 990 Leu Asn Arg Ala Leu Thr Gly Ile Ala Val Glu Gln Asp Lys Asn Thr 995 1000 1005 Gln Glu Val Phe Al a Gln Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile 1010 1015 1020 Lys Asp Phe Gly Gly Phe Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser 1025 1030 1035 1040 Lys Pro Ser Lys Arg Ser Phe Ile Glu Asp Leu Leu Phe Asn Lys Val 1045 1050 1055 Thr Leu Ala Asp Ala Gly Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly 1060 1065 1070 Asp Ile Ala Ala Arg Asp Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu 1075 1080 1085 Thr Val Leu Pro Pro Leu Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr 1090 1095 1100 Ser Ala Leu Leu Ala Gly Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala 1105 1110 1115 1120 Gly Ala Ala Leu Gln Ile Pro Phe Ala Met Gln Met Ala Tyr Arg Phe 1125 1130 1135 Asn Gly Ile Gly Val Thr Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu 1140 1145 1150 Ile Ala Asn Gln Phe Asn Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu 1155 1160 1165 Ser Ser Thr Ala Ser Ala Leu Gly Lys Leu Gln Asp Val Val Asn Gln 1170 1175 1180 Asn Ala Gln Ala Leu Asn Thr Leu Val Lys Gln Leu Ser Ser Asn Phe 1185 1190 1195 1200 Gly Ala Ile Ser Ser Val Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys 1205 1210 1215 Val Glu Ala Glu Val Gln Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln 1220 1225 1230 Ser Leu Gln Thr Tyr Val Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile 1235 1240 1245 Arg Ala Ser Ala Asn Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu 1250 1255 1260 Gly Gln Ser Lys Arg Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met 1265 1270 1275 1280 Ser Phe Pro Gln Ser Ala Pro His Gly Val Val Phe Leu His Val Thr 1285 1290 1295 Tyr Val Pro Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys 1300 1305 1310 His Asp Gly Lys Ala His Phe Pro Arg Glu Gly Val Phe Val Ser Asn 1315 1320 1325 Gly Thr His Trp Phe Val Thr Gln Arg Asn Phe Tyr Glu Pro Gln Ile 1330 1335 1340 Ile Thr Thr Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val Val Ile 1345 1350 1355 1360 Gly Ile Val Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro Glu Leu Asp 1365 1370 1375 Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn His Thr Ser Pro 1380 1385 1390 Asp Val Asp Leu Gly Asp Ile Ser Gly Ile Asn Ala Ser Val Val Asn 1395 1400 1405 Ile Gln Lys Glu Il e Asp Arg Leu Asn Glu Val Ala Lys Asn Leu Asn 1410 1415 1420 Glu Ser Leu Ile Asp Leu Gln Glu Leu Gly Lys Tyr Glu Gln Tyr Ile 1425 1430 1435 1440 Lys Trp Pro Trp Tyr Ile Trp Leu Gly Phe Ile Ala Gly Leu Ile Ala 1445 1450 1455 Ile Val Met Val Thr Ile Met Leu Cys Cys Met Thr Ser Cys Cys Ser 1460 1465 1470 Cys Leu Lys Gly Cys Cys Ser Cys Gly Ser Cys Cys Lys Phe Asp Glu 1475 1480 1485Asp Asp Ser Glu Pro Val Leu Lys Gly Val Lys Leu His Tyr Thr 1490 1495 1500

Claims (11)

A recombinant protein comprising a protein derived from Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein S1, a protein derived from fragment crystallizable region (Fc) of immunoglobulin, and a protein derived from SARS-CoV-2 spike protein S2. The recombinant protein of claim 1, wherein the SARS-CoV-2 spike protein S1 derived protein comprises the amino acid sequence of SEQ ID NO: 1. The recombinant protein of claim 1, wherein the immunoglobulin Fc-derived protein comprises the amino acid sequence of SEQ ID NO: 2. The recombinant protein of claim 1, wherein the SARS-CoV-2 spike protein S2 derived protein comprises the amino acid sequence of SEQ ID NO: 3. The method according to claim 1, The recombinant protein comprising the amino acid sequence of SEQ ID NO: 4, the recombinant protein. A polynucleotide encoding the recombinant protein of any one of claims 1 to 5. A vaccine composition for preventing or treating SARS-CoV-2 infection disease, comprising the recombinant protein of any one of claims 1 to 5 as an active ingredient. The vaccine composition of claim 7 , wherein two or more of the recombinant proteins are assembled to form a construct. The vaccine composition of claim 8, wherein the construct is in the form of a tetravalent bispecific. The method according to claim 7, 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 7, wherein the vaccine composition further comprises a pharmaceutically acceptable excipient, diluent or carrier.

Images