Recombinant spike protein of the novel coronavirus (COVID-19, coronavirus disease 2019) that forms a trimer and a method for mass production of said recombinant spike protein in plants, and a method for producing a vaccine composition based on said recombinant spike protein (Method for producing the trimeric spike protein of the novel coronavirus in plants and its use for vaccination)

Abstract

The present invention relates to a recombinant spike protein of a novel coronavirus that forms a trimer and a method for mass-producing the recombinant spike protein in plants. More specifically, the present invention relates to a method for designing a recombinant gene that expresses a recombinant spike protein of a novel coronavirus that forms a trimer for the purpose of enhancing immunogenicity and effective antigen delivery, and a method for mass-producing the recombinant spike protein in plants.

Description

[Technical field]

[0001] The present invention relates to a recombinant spike protein of a novel coronavirus that forms a trimer and a method for mass-producing the recombinant spike protein in plants, more specifically, to a method for designing a recombinant gene expressing the recombinant spike protein of a novel coronavirus that forms a trimer for the purpose of enhancing immunogenicity and effective antigen delivery, and a method for mass-producing the recombinant spike protein in plants. The present invention also relates to a method for providing an effective vaccine substance against the novel coronavirus using the trimer spike protein produced in plants. [Background technology]

[0002] Recently, the possibility of producing recombinant proteins from plants at low cost has been proposed, and various attempts are being made (Schillberg et al., 2003; Holtz et al., 2015; Marusic et al., 2016). In particular, research is being conducted to confirm the possibility of producing various medical proteins. There are various advantages to producing recombinant proteins from plants, one of which is that there are almost no toxins such as endotoxins present in microorganisms such as E. coli, and there are no pathogens that can infect the human body. It is also known that there are no harmful proteins such as prions, so it is possible to produce recombinant proteins that are safer than animal cells or microorganisms. In addition, the production cost is much cheaper than animal cells, and depending on the cultivation method of plants, it is more economical than microorganisms such as E. coli for large-scale production. In order to realize this possibility, several essential technologies need to be developed. The most important technology is the development of expression vectors to induce high gene expression in plants (Staub et al., 2000; Regnard et al., 2010). Gene expression can be induced in plants through various methods. There are various methods available, including introducing recombinant genes into the genome of the plant body, into the chloroplast genome, and using Agrobacterium to express a gene transiently (Arzola et al., 2011; Werner et al., 2011). The method of introducing recombinant genes into the nuclear genome or chloroplast genome basically involves producing proteins from plants through the process of obtaining transformants. On the other hand, when Agrobacterium is infiltrated into plant tissues and the transient expression of genes is induced to produce proteins, the protein production period is short because the process of producing transformants is not involved, and the protein production level is generally significantly higher than that of protein production by transformants (Arzola et al., 2011). In addition, the expression suppression mechanisms of different genes in plants can be suppressed by co-infiltration with gene silencing suppressors, which can induce a higher protein expression level (Garabagi et al., 2011).However, there is a drawback in that each time transient expression is attempted, Agrobacterium containing a binary vector containing the target gene and Agrobacterium containing a binary vector expressing the p38 gene silencer must be cultured separately, and then they must be mixed in an appropriate ratio and co-cultured. In particular, culturing two types of Agrobacterium is limited in terms of time and cost.

[0003] Coronaviruses are one of the three major viruses that cause colds in humans, along with adenoviruses and rhinoviruses. They are RNA viruses with a gene size of 27-32kb that can infect humans in various ways. When viewed under an electron microscope, the surface of the virus particles protrudes like a crown, and the name was derived from the Latin word "corona," which means crown. Coronaviruses account for 10-30% of colds in adults that occur mainly in the cold winter, and their main symptoms are a cold accompanied by headache, sore throat, and cough. Since first being discovered in chickens in the 1930s, coronaviruses have been found in animals such as dogs, pigs, and birds, and in the 1960s in humans. Coronaviruses have been found in both animals and humans, and as the area of ​​human activity becomes more widespread, viruses that were only prevalent among animals can undergo genetic mutations for survival and be transmitted to humans. Examples of this include SARS (bats and civets), MERS (bats and camels), and the new coronavirus (presumed to be caused by bats). Coronaviruses discovered so far are classified into four genera: alpha, beta, gamma, and delta. Here, alpha is again divided into types 1a and 1b, and beta is divided into types 2a, 2b, 2c, and 2d. Of these, alpha and beta infect humans and animals, while gamma and delta infect animals. There are seven types of human-transmitting coronaviruses identified to date, including HCoV 229E, HCoV NL63, HCoV OC43, HCoV HKU1, SARS-CoV, MERS-CoV, and SARS-CoV-2. Of these, four (229E, OC43, NL63, and HKU1) only cause mild cold-like symptoms. However, SARS-CoV (Severe Acute Respiratory Syndrome), MERS-CoV (Middle East Respiratory Syndrome), and SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) can cause severe respiratory diseases such as severe pneumonia, resulting in many deaths.

[0004] The novel coronavirus (COVID-19) is a new type of coronavirus (SARS-CoV-2) that first emerged in Wuhan, China in December 2019 and has since spread throughout China and the world. The novel coronavirus has a very high transmission rate and is particularly contagious. After an incubation period of approximately 2 to 14 days (estimated), infection with the novel coronavirus leads to respiratory symptoms such as fever (37.5°C), cough, and dyspnea, as well as pneumonia, as the main symptoms, although asymptomatic cases of infection are not uncommon. The spike (S) protein of the novel coronavirus (COVID-19) is a type 1 membrane glycoprotein that contains a very large ectodomain region, a single transmembrane domain (TMD), and a short cytoplasmic tail. The above-mentioned spike protein, like the spike (S) proteins of other coronaviruses, exists as a trimer on the surface of the virus, and has a receptor binding region necessary for the virus to enter the host cell and a fusion peptide that induces fusion between the viral membrane and the organelle membrane upon entry into the cell, and is believed to play a role in inducing neutralizing antibodies against the above-mentioned spike protein in the natural host. The above-mentioned corona spike protein exists as a trimer on the surface of the viral membrane. Therefore, it is thought that the trimeric spike protein acts as an antigen, and the trimer is also important for inducing neutralizing antibodies. Similarly, in the case of influenza viruses, the HA protein exists as a trimer on the surface of the virus, and the formation of this trimer is said to be highly antigenic.

[0005] Although recombinant proteins are safe, they have the disadvantages of low immunogenicity and high production costs compared to live viruses. Therefore, in order to effectively prevent disease using recombinant proteins that are safe, it is essential to produce highly immunogenic recombinant protein vaccines that can induce various immune responses and high immune responses, and recombinant proteins that can be produced to be effectively delivered are also required.

[0006] They tried to develop a vaccine using the recombinant spike protein of the new coronavirus. During infection with the new coronavirus, the spike protein undergoes a process involving two subunits, S1 and S2, with the region that binds to the receptor required for infection being present in S1, and S2 having a fusion peptide. Therefore, it is not easy to predict what effect it will have on protective immunity when S1, S2, or the full length is used as an antigen. Since S1 has an RBD, it can induce antibodies that bind to the RBD, and through the binding of such antibodies, it will be possible to prevent viral infection by interfering with receptor binding. On the other hand, since S1 contains the part where the most mutations are introduced, it may not be possible to cover a wide variety of variants. In addition, in the case of S2, it is predicted that the induced antibodies will not be able to prevent the virus from binding to the receptor, but they will be able to generate antibodies that interfere with the fusion step. In the case of the full length, since it contains both parts, it will be possible to induce antibodies that interfere with the binding of the virus to the receptor and antibodies that interfere with the fusion process. However, it may be more difficult to produce proteins to be used as antigens compared to S1 and S2.

[0007] Based on these various considerations, we attempted to construct a recombinant gene to produce the spike protein as an antigen from plants. In order to mass-produce the recombinant protein, we attempted to create a recombinant protein using the full-length ectodomain excluding the TMD and cytoplasmic domain. In particular, we attempted to create a trimer using only the ectodomain, which is the form in which the spike protein exists on the surface of the virus, to enhance antigenicity. For this purpose, we used a fragment (named SfΔ(TMD-CT)) containing a total of 1198 residues from the 16th amino acid to the 1213th amino acid of the spike (spike; (s)) of the new coronavirus. In addition, we attempted to construct an expression system from plants using the coronavirus spike protein, including the S1 subunit (named S1s) containing the N-terminus (16th amino acid) without a leader sequence to subdomain 2 (SD2) (681st amino acid); and a segment without the TMD and cytoplasmic tail region present in the C-terminal region of the S2 subunit, which is considered to be less prone to mutation among spike proteins (a total of 532 residues, including the portion from 682nd amino acid to 1213th amino acid, named [S2sΔ(TMD-CT)]). We attempted to create these two types of recombinant proteins produced from plants and develop them as vaccines by using them as antigenic substances. The full-length spike protein forms a trimer when present on the surface of the virus, but we presumed that if recombinant proteins were made using the ectodomain without the TMD and cytoplasmic region of the gene encoding the spike protein, or the portion encoding S2 without the TMD and cytoplasmic domain, the above recombinant proteins would not easily form trimers. We attempted to develop a technology to produce these two types of recombinant proteins in the form of trimers from plants for use as vaccines. A binary vector capable of expressing the recombinant spike protein thus produced was constructed to highly express the vector in plants.

[0008] Another object of the present invention is to provide a vaccine composition by isolating and purifying these proteins produced in plant cells. In order to increase the yield of proteins produced in plant cells, an appropriate buffer composition is important. Since these proteins are large and highly glycosylated, various buffer compositions must be compared and analyzed to ensure the optimal buffer composition for solubilization. The proteins were then isolated and purified by Ni2+-NTA affinity column chromatography and size-exclusion gel filtration column chromatography using the His tag present at the C-terminus.

[0009] The various spike protein trimers produced in this way can be used to immunize mice and hamsters to confirm the degree of antibody induction, and the level of protective ability (PRNT50) of the induced antibodies can be confirmed. In addition, since hamsters have a certain degree of susceptibility to the new coronavirus, it is possible to confirm the degree of inhibition of viral proliferation by actually challenging them with the new coronavirus. Through this process, it is possible to select the optimal vaccine candidate substance. Summary of the Invention [Problem to be solved by the invention]

[0010] The present invention has been made to solve the above problems, and an object of the present invention is to provide (i) a gene encoding a protein lacking the transmembrane domain to the C-terminus in the full-length coronavirus spike protein (SfΔ(TMD-CT); or an S1 subunit protein (S1s) including the N-terminus excluding the leader sequence to subdomain 2 (SD2) in the coronavirus spike protein; or a protein lacking the transmembrane domain to the C-terminus in the S2 subunit of the coronavirus spike protein, S2sΔ(TMD-CT); and (ii) a gene encoding a protein in the trimerization motif region of mouse coronin 1 (mCor1); a gene encoding five histidine residues for separation and purification; and a gene capable of being accumulated in the endoplasmic reticulum of plants. The present invention provides a recombinant vector for producing recombinant coronavirus spike proteins SfΔ(TMD-CT):mCor1:Hisx5:HDEL (designated Sfull-t), S1:mCor1:Hisx5:HDEL (designated S1s-t), or S2Δ(TMD-CT):mCor1:Hisx5:HDEL (designated S2s-t) that form trimers, which contain a gene encoding an ER retention motif, HDEL; and a recombinant vector for producing SfΔ(TMD-CT):Hisx5:HDEL (designated Sfull-m), S1:Hisx5:HDEL (designated S1s-m), or S2Δ(TMD-CT):Hisx5:HDEL (designated S2s-m) proteins that do not have mCor as controls.

[0011] Another object of the present invention is to provide a method for producing a recombinant spike protein of a coronavirus that forms a trimer from a plant, comprising the steps of: (a) producing the recombinant vector; (b) introducing the recombinant vector into an organism to produce a transformed organism; (c) culturing the transformed organism; (d) infiltrating the culture into a plant; and

[0012] (e) disrupting the plant to obtain the recombinant coronavirus spike protein that forms a trimer.

[0013] Another object of the present invention is to provide conditions for expressing recombinant proteins derived from the spike protein, SfΔ(TMD-CT):mCor1:Hisx5:HDEL (designated Sfull-t), S1s:mCor1:Hisx5:HDEL (designated S1s-t) or S2sΔ(TMD-CT):mCor1:Hisx5:HDEL (designated S2s-t), in plant cells and then isolating and purifying them highly efficiently from plant extracts.

[0014] Another object of the present invention is to provide a vaccine composition that effectively protects against the new coronavirus using two of these multiple spike-derived recombinant proteins, SfΔ(TMD-CT):mCor1:Hisx5:HDEL and S2sΔ(TMD-CT):mCor1:Hisx5:HDEL. [Means for solving the problem]

[0015] The present invention provides a recombinant vector for producing recombinant spike protein Sfull-t or S2s-t of a novel coronavirus that forms a trimer, comprising: (i) a gene encoding a protein SfΔ(TMD-CT) that lacks the transmembrane domain to the C-terminus in the coronavirus spike protein; or a protein (S1s) that contains the S1 subunit of the coronavirus spike protein; or a protein S2sΔ(TMD-CT) that lacks the TMD to the C-terminus in the S2 subunit of the coronavirus spike protein; and (ii) a gene encoding a protein at the trimerization motif site of coronin 1 (mCor1); a gene encoding five histidine residues for separation and purification; and a gene encoding HDEL, an ER retention motif that allows the protein to be accumulated in the endoplasmic reticulum of plants.

[0016] The coronavirus is any one selected from the group consisting of SARS (SARS-coronavirus), MERS (MERS-coronavirus) and novel coronavirus (SARS-coronavirus-2).

[0017] The protein lacking the transmembrane domain to the C-terminus in the spike protein of the above-mentioned novel coronavirus can include the amino acid sequence of SEQ ID NO:2.

[0018] A gene encoding a protein that lacks the transmembrane domain through to the C-terminus of the spike protein of the above-mentioned novel coronavirus can include the base sequence of SEQ ID NO:1.

[0019] In the spike protein of the above-mentioned novel coronavirus, the S1 subunit, "the protein comprising amino acids 16 to 681" (S1s), can include the amino acid sequence of SEQ ID NO:4.

[0020] The gene encoding the S1 subunit of the spike protein of the above-mentioned new coronavirus, "the protein comprising the 16th amino acid to the 681st amino acid," can include the base sequence of SEQ ID NO:3.

[0021] In the spike protein of the above-mentioned novel coronavirus, the protein comprising amino acids 682 to 1213 of the S2 subunit may include the amino acid sequence of SEQ ID NO:6.

[0022] The gene encoding a protein comprising amino acids 682 to 1213 of the S2 subunit of the spike protein of the above-mentioned novel coronavirus can include the base sequence of SEQ ID NO:5.

[0023] The trimerization motif site protein of coronin 1 (mCor1) may include the amino acid sequence of SEQ ID NO:8.

[0024] The gene encoding the protein of the trimerization motif site of coronin 1 (mCor1) can include the base sequence of SEQ ID NO:7.

[0025] The gene encoding the 5'-UTR can include the base sequence of SEQ ID NO:14.

[0026] The present invention also provides a recombinant vector for producing a recombinant spike protein of a coronavirus that forms a trimer, which further comprises a gene encoding a Hisx5 tagged protein in the above recombinant vector.

[0027] The present invention also provides a recombinant vector for producing a recombinant spike protein of a coronavirus that forms a trimer, which further comprises a gene encoding a protein with an HDEL motif in the above recombinant vector.

[0028] The HDEL motif protein can include the amino acid sequence of SEQ ID NO:13.

[0029] The gene encoding the protein of the HDEL motif domain can include the base sequence of SEQ ID NO:12.

[0030] The above recombinant vector may additionally include any promoter selected from the group consisting of a 35S promoter derived from cauliflower mosaic virus, a 19S RNA promoter derived from cauliflower mosaic virus, a Mac promoter, a plant actin protein promoter, and a ubiquitin protein promoter.

[0031] The present invention also provides a transformed organism transformed with the above recombinant vector.

[0032] The transformed organism may be a prokaryote or a eukaryote.

[0033] The present invention may provide a method for producing a recombinant spike protein of a coronavirus that forms a trimer from a plant, the method comprising the steps of: (a) producing the recombinant vector; (b) introducing the recombinant vector into an organism to produce a transformed organism; (c) culturing the transformed organism; (d) infiltrating the culture into a plant; and (e) disrupting the plant to obtain the recombinant coronavirus spike protein that forms a trimer.

[0034] The present invention can provide a method for producing a vaccine candidate substance through a process of separating and purifying Sfull-t and S2s-t proteins from a plant extract using Ni2+-NTA affinity column chromatography and size exclusion column chromatography.

[0035] Another object of the present invention is to provide a vaccine composition that effectively protects against the novel coronavirus using these two spike proteins, Sfull-t and S2s-t, which can be provided in a vaccine composition with or without an adjuvant, with the amount of protein ranging from 1 microgram to 30 micrograms. Effect of the Invention

[0036] The recombinant spike protein of the novel coronavirus that forms a trimer of the present invention is a recombinant protein that forms a trimer with a trimer motif of mouse coronin 1 to increase immunity, includes HDEL at the C-terminus so that the protein accumulates in the endoplasmic reticulum of the plant and can be produced in large quantities from the plant, and includes a gene encoding an Se protein composed of the ectodomain of the spike protein of the novel coronavirus lacking the transmembrane domain to the C-terminus (ectodomain; 1198 residues in total from the 16th amino acid to the 1213th amino acid) or a protein comprising the 16th amino acid to the 681st amino acid of the S1 subunit of the coronavirus spike protein (666 residues in total) or a protein comprising the 682nd amino acid to the 1213th amino acid of the S2 subunit of the coronavirus spike protein lacking the transmembrane domain to the C-terminus (532 residues in total).

[0037] The vaccine composition includes a vaccine composition composed of Sfull-t or S2s-t recombinant protein of the S protein of the novel coronavirus produced from plants. The vaccine composition may be composed of only plant-produced proteins, or may contain an adjuvant such as alum. [Brief description of the drawings]

[0038] [Figure 1] 1 is a diagram showing the construction of multiple recombinant vectors used in the present invention. [Diagram 2] The results of Western blot analysis with anti-His antibody and Coomassie brilliant blue staining of total extracts of Nicotiana benthamiana expressing His-tagged, HDEL-bearing SfΔ(TMD-CT), S1s, and S2sΔ(TMD-CT) proteins and the three proteins additionally containing mCor1 are shown. [Diagram 3]Sfull-t and S2s-t were separated and purified from Nicotiana benthamiana extract using Ni2+-NTA affinity column chromatography, developed by SDS / PAGE, and analyzed by Western blotting using anti-His antibody. The membranes were then stained again with Coomassie brilliant blue. [Figure 4] Sfull-t separated from Nicotiana benthamiana extract using Ni2+-NTA affinity column chromatography was sliced ​​using size-exclusion column chromatography, and the fractions were run on SDS / PAGE and stained with Coomassie brilliant blue. [Diagram 5] Sfull-t and Sfull-m were isolated from Nicotiana benthamiana extract using Ni2+-NTA affinity column chromatography, purified with pure water, and sliced ​​using size-exclusion column chromatography. The slices were then subjected to Western blot analysis using an anti-His antibody. The isolated and purified Sfull-t was negatively stained, and the protein morphology was confirmed using an electron microscope, confirming that it forms a trimer. [Figure 6] S2s-t separated from Nicotiana benthamiana extract using Ni2+-NTA affinity column chromatography was sliced ​​using size-exclusion column chromatography, and the fractions were run on SDS / PAGE and stained with Coomassie brilliant blue. [Figure 7] The results of immunogenicity evaluation of the novel coronavirus plant vaccine candidate substance in experimental animals (Balb / c mice) are shown below. For this purpose, humoral immune response analysis (Fig. 7B), neutralizing antibody induction analysis (Fig. 7C, D), and cell-mediated immune response analysis (Fig. 7E) were performed, and the results are shown. [Figure 8]In an experiment to evaluate the protective efficacy of a candidate novel coronavirus plant vaccine substance in experimental animals (TG mice), each immunization antigen was administered intramuscularly twice, two weeks apart. To confirm the production of antibodies against the immunization antigen, blood was collected before immunization, and two weeks after the primary immunization and secondary immunization. The results of immunogenicity evaluation, such as the production of antibodies (IgGs) in the serum and the production of neutralizing antibodies, are shown (Figure 8A), as well as the results of an analysis of antibody induction by plant vaccine immunization (Figure 8B), and an analysis of the survival rate and tissue titers after challenge of hamsters with plant vaccine immunization (Figures 8C, D, E). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0039] The present invention will be described in detail below.

[0040] As a membrane protein of the novel coronavirus, the spike protein, which is considered to be important for cell penetration, is considered to be a candidate group of vaccines that can prevent infection with the novel coronavirus. In the present invention, we have attempted to create various recombinant proteins based on this spike protein and develop them into vaccines using them. The full length of the spike protein forms a trimer when present on the surface of the virus, but we presumed that if a recombinant protein is created using the entire spike gene or a part that encodes only the ectodomain, the recombinant protein will not form a trimer. Therefore, in order to use these recombinant proteins as vaccines, we believe that it is important for the protein to form a trimer as when it is present on the surface of the virus, and we have attempted to develop a technology that maintains the trimer even when they are present in a soluble form. In the present invention, we have attempted to develop a technology that induces the formation of a trimer when recombinant proteins of the entire ectodomain of the spike protein [SfΔ(TMD-CT)], S1s, or the S2 region without the TMD and C-terminus [S2sΔ(TMD-CT)] are expressed and produced from plants. For this purpose, we constructed a construct in which the ectodomain (Se; 1198 residues from the 16th to 1213th amino acids), which lacks the spike transmembrane domain to the C-terminus of the mass-produced novel coronavirus spike protein, the portion including the N-terminus to the SD2 domain (S1s; 666 residues from the 16th to 681st amino acids), or the portion of the S2 subunit lacking the TMD and C-terminus (S2sΔ(TMD-CT); 532 residues from the 682nd to 1213th amino acids) was fused to the trimer motif of mouse coronin 1, which forms a trimer structure, and additionally linked to the portion encoding Hisx5 and HDEL. As a control for these recombinant proteins, a construct without mCor1 was constructed. We constructed a binary vector that can express recombinant genes containing the above-mentioned various spike proteins in plants.

[0041] The present invention can provide a recombinant vector for producing a recombinant coronavirus spike protein that forms a trimer, comprising: (i) a gene encoding a protein lacking the amino acid sequence from the transmembrane domain to the C-terminus in the coronavirus spike protein (SfΔ(TMD-CT)); or an S1 subunit protein (S1s) comprising the N-terminus to subdomain 2 (SD2) in the coronavirus spike protein; or a protein lacking the amino acid sequence from the transmembrane domain to the C-terminus in the S2 subunit of the coronavirus spike protein (S2sΔ(TMD-CT)); and (ii) a gene encoding a protein in the trimerization motif region of coronin 1 (mCor1).

[0042] In order to develop a vaccine using recombinant spike protein of the new coronavirus, we tried to create a recombinant protein using the ectodomain of the spike protein without the transmembrane domain to the C-terminus (full-length ectodomain of S; from the 16th amino acid to the 1213th amino acid, a total of 1198 residues), or the portion including the N-terminus to subdomain 2 (SD2) (from the 16th amino acid to the 681st amino acid, a total of 666 residues), or the ectodomain of S2 (from the 682nd amino acid to the 1213th amino acid, a total of 532 residues), and use this to develop a vaccine. The full-length spike protein forms a trimer when present on the virus surface, but if the whole or part of the ectodomain portion of the gene encoding the spike protein is used to express the recombinant protein, it is predicted that trimers will not form easily, and that the immunogenicity will be low due to the lack of formation of the trimer. Therefore, in the present invention, we attempted to create a recombinant protein of the entire spike protein in the same trimer form as the full-length spike protein that is originally present on the surface of the virus, or the S1c ectodomain from the N-terminus to the SD2 domain, or the S2 ectodomain.To this end, we selected the novel coronavirus (COVID-19) and constructed a construct by fusing 32 residues from the 122nd amino acid to the 153rd amino acid of mouse coronin 1 (mCor1) using a linker with 12 amino acid residues.

[0043] Next, a His tag with five His residues was fused to the recombinant proteins for separation and purification, and finally, an HDEL motif was fused to the recombinant proteins for accumulation in the ER to complete the constructs (SfΔ(TMD-CT):mCor1:Hisx5:HDEL, S1s:mCor1:Hisx5:HDEL, S2sΔ(TMD-CT):mCor1:Hisx5:HDEL, Figure 1). We also constructed constructs lacking mCor1 in the above three types of spike recombinant proteins to be used as controls.

[0044] The coronavirus is any one selected from the group consisting of SARS (SARS-coronavirus), MERS (MERS-coronavirus) and novel coronavirus (SARS-coronavirus-2).

[0045] The protein lacking the transmembrane domain to the C-terminus in the spike protein of the above-mentioned novel coronavirus can include the amino acid sequence of SEQ ID NO:2.

[0046] The gene encoding the protein lacking the transmembrane domain to the C-terminus in the spike protein of the novel coronavirus may include the nucleotide sequence of SEQ ID NO: 1, and specifically, the gene may include a nucleotide sequence having a sequence homology of 70% or more, more preferably 80% or more, even more preferably 90% or more, and most preferably 95% or more with the nucleotide sequence of SEQ ID NO: 1. The "% sequence homology" for a polynucleotide is determined by comparing the comparison region with two optimally aligned sequences, and a portion of the polynucleotide sequence in the comparison region may include additions or deletions (i.e., gaps) compared to the reference sequence (which does not include additions or deletions) for the optimal alignment of the two sequences.

[0047] In the spike protein of the above-mentioned new coronavirus, the S1s protein including the N-terminus to subdomain 2 (SD2) can include the amino acid sequence of SEQ ID NO:4.

[0048] The gene encoding the S1s protein including the N-terminus to subdomain 2 (SD2) in the spike protein of the novel coronavirus may include the nucleotide sequence of SEQ ID NO: 3, and specifically, the gene may include a nucleotide sequence having sequence homology of 70% or more, more preferably 80% or more, even more preferably 90% or more, and most preferably 95% or more with the nucleotide sequence of SEQ ID NO: 3. The "% sequence homology" for a polynucleotide is determined by comparing the comparison region with two optimally aligned sequences, and a portion of the polynucleotide sequence in the comparison region may include additions or deletions (i.e., gaps) compared to the reference sequence (which does not include additions or deletions) for the optimal alignment of the two sequences.

[0049] In the spike protein of the novel coronavirus, the S2 protein is represented by the sequence 6 The amino acid sequence may include:

[0050] The trimerization motif region of the coronin 1 (mCor1) protein is represented by the following sequence: 8 The amino acid sequence may include:

[0051] The gene encoding the trimer motif site protein of coronin 1 (mCor1) is represented by SEQ ID NO: 7 Specifically, the gene can include the base sequence of SEQ ID NO: 7 The "percent sequence identity" for a polynucleotide may include a base sequence having at least 70% sequence identity with the base sequence of each of the two sequences, more preferably at least 80%, even more preferably at least 90%, and most preferably at least 95%. The "percent sequence identity" for a polynucleotide is determined by comparing a comparison region with two optimally aligned sequences, and the portion of the polynucleotide sequence in the comparison region may contain additions or deletions (i.e., gaps) compared to the reference sequence (which does not contain additions or deletions) for the optimal alignment of the two sequences.

[0052] The above recombinant genes were introduced into pTEX1, a plant expression vector, to create a plant expression vector. Six types of ectodomain recombinant protein genes were constructed from the spike gene of the new coronavirus (Figure 1), and these were introduced into a plant (Nicotiana benthamiana) to induce recombinant protein expression. To confirm the expression of proteins in leaf extracts of Nicotiana benthamiana into which these genes were introduced, Western blot analysis was performed using an anti-His antibody. As shown in Figure 2, Sfull was confirmed at a position of approximately 180 kD. It was determined that this was larger than the calculated protein position in order to form the N-glycosylated spike protein. S1 and S2 originated from positions 100 kD and 75 kD, respectively, and since these two proteins were also larger than the calculated size, it was determined that they would also be N-glycosylated (Figure 2).

[0053] To separate these two proteins, we prepared a total extract from Nicotiana benthamiana and purified it using Ni2+-NTA affinity column chromatography. We then purified it by SDS / PAGE and stained it with Coomassie Brilliant Blue. We confirmed that the recombinant proteins of the two spike proteins could be separated and purified (Figure 3).

[0054] The above recombinant vector may additionally include any promoter selected from the group consisting of the 35S promoter derived from cauliflower mosaic virus, the 19S RNA promoter derived from cauliflower mosaic virus, the Mac promoter, a plant actin protein promoter and a ubiquitin protein promoter, preferably the Mac promoter, and more preferably the MacT promoter.

[0055] The MacT promoter may be a promoter in which the 3'-terminal base A of the Mac promoter base sequence is replaced with T, and the MacT promoter is represented by SEQ ID NO: 15 Specifically, the gene can include the base sequence of SEQ ID NO: 15The "percent sequence identity" for a polynucleotide may include a base sequence having at least 70%, more preferably at least 80%, even more preferably at least 90%, and most preferably at least 95% sequence identity with the base sequence of each of the two best aligned sequences. The "percent sequence identity" for a polynucleotide is determined by comparing a comparison region with two best aligned sequences, and the portion of the polynucleotide sequence in the comparison region may contain additions or deletions (i.e., gaps) compared to the reference sequence (which does not contain additions or deletions) for the best aligned sequence of the two sequences.

[0056] The recombinant vector may further comprise a termination site of RD29B-t, the termination site of RD29B-t being represented by SEQ ID NO: 16 Specifically, the gene can include the base sequence of SEQ ID NO: 16 The "percent sequence identity" for a polynucleotide may include a base sequence having at least 70% sequence identity with the base sequence of each of the two sequences, more preferably at least 80%, even more preferably at least 90%, and most preferably at least 95%. The "percent sequence identity" for a polynucleotide is determined by comparing a comparison region with two optimally aligned sequences, and the portion of the polynucleotide sequence in the comparison region may contain additions or deletions (i.e., gaps) compared to the reference sequence (which does not contain additions or deletions) for the optimal alignment of the two sequences.

[0057] By including the BiP signal sequence and HDEL, an ER retention signal, at the N-terminus and C-terminus of the gene for the recombinant protein, respectively, it is possible to obtain the effect of inducing high concentration accumulation in the ER (endoplasmic reticulum). The recombinant vector can additionally include any one selected from the group consisting of BiP (chaperone binding protein) and HDEL (His-Asp-Glu-Leu) peptide, and the BiP (chaperone binding protein) is represented by SEQ ID NO: 11 and HDEL (His-Asp-Glu-Leu) is represented by the sequence 12 It can include the base sequence:

[0058] Recombinant refers to a cell that replicates a heterologous nucleic acid, expresses the nucleic acid, or expresses a peptide, heterologous peptide, or protein encoded by a heterologous nucleic acid. Recombinant cells can express genes or segments of genes in sense or antisense form that are not found in the native form of the cell. Recombinant cells can also express genes that are found in the native state of the cell, but that have been altered and reintroduced into the cell by artificial means.

[0059] The term "recombinant expression vector" refers to bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses or different vectors. Generally, any plasmids and vectors can be used as long as they are replicated and stabilized in the host. The important characteristics of the expression vectors are that they have an origin of replication, a promoter, a marker gene and a translation control element. The recombinant expression vectors and expression vectors containing appropriate transcription / translation control signals can be constructed by methods well known to those skilled in the art. The methods include in vitro recombinant DNA techniques, DNA synthesis techniques and in vivo recombination techniques.

[0060] A preferred example of a recombinant vector of the present invention is a Ti plasmid vector, which, when present in a suitable host, is capable of transferring a part of itself, the so-called T-region, to a plant cell. Different types of Ti plasmid vectors are currently utilized to transfer hybrid DNA sequences in plant cells, or in protoplasts from which new plants are produced, allowing the hybrid DNA to be properly inserted into the plant genome. Particularly preferred forms of Ti plasmid vectors are the so-called binary vectors, as claimed in EP0120516B1 and U.S. Pat. No. 4,940,838. Other suitable vectors that can be utilized to introduce the DNA according to the present invention into a plant host can be selected from viral vectors, such as defective plant viral vectors, which can be derived from double-stranded plant viruses (e.g., CaMV) and single-stranded viruses, geminiviruses, etc. The use of such vectors is advantageous, especially when it is difficult to properly transform the plant host.

[0061] The present invention also provides a transformed organism transformed with the above recombinant vector.

[0062] The transformed organisms may be prokaryotes or eukaryotes, examples of which include yeast (Saccharomyces cerevisiae), fungi such as E. coli, insect cells, human cells (e.g., CHO cell lines (Chinese Hamster Ovary), W138, BHK, COS-7, 293, HepG2, 3T3, RIN and MDCK cell lines) and plant cells, preferably Agrobacterium. In the case of insect cells, human cells, etc., the gene encoding the trimer-forming recombinant spike protein can be expressed using an expression vector required for expression in these various animal cells.

[0063] The vector of the present invention can be delivered into a host cell by the CaCl2 method, the Hanahan method (Hanahan, D., J. Mol. Biol., 166:557-580 (1983)), electroporation, etc., when the host cell is a prokaryotic cell. In addition, when the host cell is a eukaryotic cell, the vector can be injected into the host cell by microinjection, calcium phosphate coprecipitation, electroporation, liposome-mediated transfection, DEAE-dextran, gene barmbardment, etc.

[0064] The present invention may provide a method for producing a recombinant spike protein of a coronavirus that forms a trimer from a plant, the method comprising the steps of: (a) producing the recombinant vector; (b) introducing the recombinant vector into an organism to produce a transformed organism; (c) culturing the transformed organism; (d) infiltrating the culture into a plant; and (e) disrupting the plant to obtain the recombinant coronavirus spike protein that forms a trimer.

[0065] The method of infiltrating the plant includes, but is not limited to, chemical cell method, vacuum or syringe infiltration, most preferably syringe infiltration.

[0066] The plant may be selected from food crops including rice, wheat, barley, corn, beans, potatoes, wheat, red beans, oats, and sorghum; vegetable crops including Arabidopsis, Chinese cabbage, radish, chili pepper, strawberry, tomato, watermelon, cucumber, cabbage, Japanese melon, pumpkin, leek, onion, and carrot; specialty crops including ginseng, tobacco, cotton, sesame, sugarcane, sugar beet, perilla, peanut, and oilseed rape; fruit trees including apples, pears, jujubes, peaches, grapes, mandarin oranges, persimmons, plums, apricots, and bananas; and ornamental plants including roses, carnations, chrysanthemums, lilies, and tulips.

[0067] The spike protein of the novel coronavirus produced from plants can be separated and purified by Ni2+-NTA affinity column chromatography using the C-terminal His tag, and the isolated protein can be further separated again by size exclusion gel chromatography to increase the purity.

[0068] Another object of the present invention is to provide a vaccine composition using the spike proteins of these plant-produced Corona-19. The spike protein trimers Sfull-t and S2s-t can be used with or without alum to form a vaccine composition.

[0069] In the following, preferred examples are shown to aid in understanding the present invention. However, the following examples are provided to facilitate understanding of the present invention, and the contents of the present invention are not limited to the following examples. Example 1 Design of a gene encoding a trimer-forming recombinant spike protein of the novel coronavirus To induce the expression of the novel coronavirus surface protein spike (S) in a soluble form in the ER lumen, the ectodomain of the spike protein lacking the transmembrane domain (TMD) to the C-terminus (ectodomain; 1198 residues in total from the 16th amino acid to the 1213th amino acid, SfΔ(TMD-CT)), the S1 subunit region including the N-terminus to the SD2 domain (666 residues in total from the 16th amino acid to the 681st amino acid), or the S2 ectodomain region (562 residues in total from the 682nd amino acid to the 1213th amino acid) were obtained. The 5' end of the novel coronavirus spike gene was fused with an ER targeting signal obtained from Arabidopsis thaliana protein BiP to enable ER targeting. To induce trimer formation of the recombinant spike protein thus created, a motif that induces the formation of homogenous trimers from a protein called mouse m-coronin 1 was fused to the C-terminus of these spike proteins by a linker, and a Hisx5 tag and an ER retention motif, HDEL, were sequentially fused to the C-terminus of mCor1 to purify the spike protein and accumulate it highly in the ER. macT was used for expression, and the end of Rd29b was used as a transcription terminator. The above transcription terminator was confirmed to show high transcription efficiency as a result of previous research. The base sequences used in the experiment are shown in Table 1 below (Figure 1).

[0070] These constructs were then introduced into pTEX to finally construct nine plant expression vectors. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] Example 2 Expression and characterization of the gene encoding the trimer-forming recombinant spike protein of the novel coronavirus

[0071] Six constructs displayed in Figure 1 [SfΔ(TMD-CT):mCor1:Hisx5:HDEL(Sfull-t), SfΔ(TMD-CT):Hisx5:HDEL(Sfull-m), S1s:mCor1:Hisx5:HDEL(S1s-t), S1s:Hisx5:HDEL(S1s-m), S2sΔ(TMD-CT):mCor1:Hisx5:HDEL(S2s-t), S2sΔ(TMD-CT):Hisx5:HEDL(S2s-m)] were induced to express through transient expression in leaves of 4-5 week-old Nicotiana benthamiana plants using the vacuum infiltration method. Infiltrated leaves were harvested 5 days after infiltration (dpi), completely ground in liquid nitrogen, and dissolved in 3 volumes of buffer. Total soluble proteins from the infiltrated leaf extracts were subjected to SDS-PAGE and then Western blot analysis. The spike (S) recombinant proteins were identified by anti-His antibody at approximately 180 kD for Sfull-t and Sfull-m, and at 100 kD for S1s-t and S1s-m. S2s-t and S2s-m were identified at 75 kD. Comparing the positions of the construct without mCor and the protein with mCor1, the protein was slightly smaller than that without mCor1, and these differences are considered to be due to mCor1. In addition, all six recombinant proteins were larger than the calculated size, which was determined to be due to N-glycosylation (Figure 2). The same membrane was stained with Coomassie Brilliant Blue to confirm the bands, and the expression levels of these recombinant proteins were estimated to be approximately 20-50 μg / g fresh weight in the infiltrated leaves based on the band intensity. Example 3 Isolation and purification of the trimer-forming recombinant spike protein of the new coronavirus

[0072] The recombinant proteins of two types of novel coronavirus S proteins, Sfull-t and S2s-t, were expressed in Nicotiana benthamiana, and separated and purified using a Ni2+-NTA affinity column. After developing them by SDS / PAGE, Western blotting was performed using an anti-His antibody to confirm the separation and purification. The membrane was then stained with Coomassie Brilliant Blue. Through this, it was confirmed that the recombinant proteins derived from the two types of spike proteins could be separated and purified in a pure form (Figure 3). Example 4 Separation and purification of trimer-forming recombinant Sfull-t protein by size-exclusion column chromatography

[0073] The recombinant proteins of Sfull-t separated and purified by Ni2+-NTA affinity column were concentrated and then separated and purified again by size exclusion column chromatography. The sections from 7 to 20 were developed by SDS / PAGE and Western blotted with anti-His antibody to confirm the position of the recombinant proteins of Se and the amount of eluted proteins. The membrane was then stained with Coomassie Brilliant Blue to confirm contamination of other protein bands (Figure 4). Example 5 Confirmation of the difference in size-exclusion column cleavage of recombinant proteins of the novel coronavirus SfΔ(TMD-CT) with and without mCor1, and formation of trimers of Sfull-t recombinant protein by mCor1

[0074] After isolating and purifying proteins from Nicotiana benthamiana leaf extracts expressing Sfull-t and Sfull-m using a Ni2+-NTA affinity column (Figure 5A, B), the proteins were sectioned using size-exclusion column chromatography (Figure 6C), and the sections were purified using SDS / PAGE, and then analyzed by Western blotting using these anti-His antibodies (Figure 5D). After negative staining of the separated Sfull-t protein, the protein morphology was observed using an electron microscope to confirm the formation of trimers (Figure 5E). Example 6 Separation and purification of the trimer-forming recombinant S2e protein by size-exclusion column chromatography

[0075] The recombinant S2s-t proteins separated and purified using a Ni2+-NTA affinity column were concentrated and then separated and purified again using size-exclusion column chromatography. The sections from number 7 to number 20 obtained from the column were developed using SDS / PAGE, and Western blotting was performed using an anti-His antibody to confirm the position and amount of the recombinant S2s-t proteins eluted (Figure 6). Example 7 Immunogenicity evaluation of novel coronavirus plant vaccine candidate substances in experimental animals

[0076] (1) Immunogenicity evaluation of novel coronavirus plant vaccine candidate substances in experimental animals (mice, Balb / c) Animal experiments to evaluate the immunogenicity of the manufactured plant vaccine were carried out using 6-week-old female mice (Balb / c), five mice per experimental group, and immunization was carried out at four different doses (1, 5, 15, 30μg). Each immunization antigen (Sfull-t) was administered intramuscularly three times at two-week intervals, and blood samples were taken before immunization and two weeks after the first, second, and third immunizations to confirm the production of antibodies against the immunization antigen, and immunogenicity evaluations were carried out, including the production of antibodies (IgGs) in the serum and the production of neutralizing antibodies. At this time, the negative control group was immunized with PBS.

[0077] 1) Humoral immune response analysis The immunogenicity of the plant vaccine was evaluated by ELISA to see if antigen-specific antibodies were produced in the serum. After two or three mouse immunizations, sufficient IgG titers were observed, and it was confirmed that the vaccine produced specific antibodies for both S1 and S2, which are subdomains of the S antigen, with a particularly high titer for S2. It was also confirmed that the group immunized with an immune adjuvant (aluminum hydroxide) had slightly higher IgG and neutralizing antibody titers than the group that was not administered the adjuvant.

[0078] 2) Neutralizing antibody induction analysis After isolating serum from the blood of mice immunized with the manufactured plant vaccine, the neutralizing ability of antibodies against SARS-CoV-2 was confirmed using the plaque reduction neutralizing antibody test (PRNT), which is known as the "gold standard" among the methods commonly used to measure virus neutralizing antibodies. To analyze the titer of neutralizing antibodies, the SARS-CoV-2 virus (BetaCoV / Korea / KCDC03 / 2020) provided by the National Pathogen Resource Bank was used to treat mouse serum (antibodies) with the virus and confirm the change in infection rate. In addition, a cross-reaction analysis against the recently reported British variant (B.1.1.7) and South African variant (B.1.351) was also performed.

[0079] As with the IgG production results, the induction of neutralizing antibodies showed higher titers of neutralizing antibodies in the group immunized with the adjuvant than in the group injected with the immunizing antigen alone, and it was confirmed that the serum collected after immunization with a lower concentration showed a higher titer of Chinese antibodies. It was also confirmed that no neutralizing antibodies were induced in the PBS group, which was the negative control group (Figure 7C). It was also confirmed that cross-reactions were observed against the British and South African variants, which have recently become a problem (Figure 7D).

[0080] 3) Cell-mediated immune response analysis The cell-mediated immune response induced by plant vaccine immunization was analyzed by ELISPOT assay. A commercial IFN-γ ELISPOT kit was used for the ELISPOST analysis. It was confirmed that the number of spleen cells secreting IFN-γ increased in the plant vaccine immunized group compared to the control group (PBS group), and that this was not significantly proportional to the amount of antigen. Furthermore, no increase was observed due to the immune adjuvant. In conclusion, it was confirmed that plant vaccine immunization induces both humoral and cellular immune responses (Figure 7E). Example 8 Evaluation of protective efficacy of novel coronavirus plant vaccine candidate substances in experimental animals (TG mice)

[0081] Animal experiments to evaluate the protective efficacy of the plant vaccine produced according to the present invention used 6-week-old transgenic mice (B6.Cg-Tg(K18-ACE2)2Primn / J) expressing the SARS-CoV-2 human receptor ACE2, with 14 mice per experimental group (12 mice in the control group), and immunization was performed at two different doses (15 and 30 μg). Each immunization antigen was administered intramuscularly twice at 2-week intervals, and blood samples were taken before immunization, 2 weeks after the first immunization, and 2 weeks after the second immunization to confirm the production of antibodies against the immunization antigen, and immunogenicity evaluations such as the production of antibodies (Igs) and neutralizing antibodies in the serum were performed. At this time, the negative control group was immunized with PBS. Virus challenge inoculation was performed 28 days after the first immunization, with nasal infection at a concentration of 2x104 pfu / mouse, and the survival rate and tissue titers were measured (Figure 8A).

[0082] 1) Analysis of antibody induction by plant vaccine immunization After isolating serum from the blood of mice immunized with the plant vaccine, ELISA was used to confirm whether antigen-specific antibodies were produced in the serum. It was confirmed that sufficient Ig titers were produced after two immunizations, and that the vaccine produced specific antibodies for both S1 and S2 subdomains of the S antigen. It was also confirmed that the group immunized simultaneously with an immune adjuvant (aluminum hydroxide) had higher Ig titers than the group that was not administered the adjuvant (Figure 8B).

[0083] 2) Survival rate and tissue titer analysis following challenge of hamsters with plant vaccines 28 days after the first immunization, the virus was intranasally infected to confirm the protective ability against SARS-CoV-2 infection in the immunogenic transgenic mice, and body weight, temperature, and clinical symptoms were observed for 14 days after infection (Figure 8C). After 2 weeks of infection, body weight changes were measured, and the hamsters immunized with the plant vaccine showed less weight loss than the negative control PBS group. Analysis of the survival rate of the plant vaccine immunized group showed that 100% of the 15 and 30 μg immunized groups survived, and the 15 and 30 μg and immune adjuvant co-administration groups showed a survival rate of 80%. The survival rate of the control (PBS) immunized group was confirmed to be one out of three (33%) (Figure 8D). To confirm the viral growth pattern and histopathological findings in the lung tissue following the virus challenge, autopsies were performed 3, 5, and 7 days after infection, and the viral titers in the dissected lung tissue were measured. In the plant vaccine immunized group, suppression of viral growth was confirmed in proportion to the administered dose compared to the control group, and no virus was detected in the group co-administered with 30 μg and immune adjuvant on day 7 (Figure 8E).

Claims

1. (i) a protein lacking the amino acid sequence from the transmembrane domain to the C-terminus in the coronavirus spike protein; A protein comprising the amino acid sequence from the N-terminus to subdomain 2 (SD2) of the coronavirus spike protein; or A gene encoding a protein lacking the amino acid sequence from the transmembrane domain to the C-terminus in subunit 2 of the coronavirus spike protein; and (ii) a gene encoding a protein at the trimerization motif site of coronin 1 (mCor1); The trimerization motif region protein of coronin 1 (mCor1) comprises the amino acid sequence of SEQ ID NO:8; A recombinant vector for producing a water-soluble recombinant spike protein of a coronavirus that forms a trimer.

2. The recombinant vector for producing a water-soluble recombinant spike protein of the trimer-forming coronavirus of claim 1, wherein the coronavirus is any one selected from the group consisting of SARS (SARS-coronavirus), MERS (MERS-coronavirus) and novel coronavirus (SARS-coronavirus-2).

3. A recombinant vector for producing a water-soluble recombinant spike protein of a coronavirus that forms a trimer as described in claim 2, wherein the protein lacking the amino acid sequence from the transmembrane domain to the C-terminus in the spike protein of the new coronavirus has the amino acid sequence of SEQ ID NO:

2.

4. A recombinant vector for producing a water-soluble recombinant spike protein of a coronavirus that forms a trimer as described in claim 2, wherein the gene encoding a protein that lacks the amino acid sequence from the transmembrane domain to the C-terminus in the spike protein of the new coronavirus includes the base sequence of SEQ ID NO:

1.

5. A recombinant vector for producing a water-soluble recombinant spike protein of a coronavirus that forms a trimer as described in claim 2, wherein the protein comprising the N-terminus to subdomain 2 (SD2) of the spike protein of the new coronavirus comprises the amino acid sequence of SEQ ID NO:

4.

6. A recombinant vector for producing a water-soluble recombinant spike protein of the coronavirus that forms a trimer as described in claim 2, wherein the gene encoding a protein including the N-terminus to subdomain 2 (SD2) of the spike protein of the new coronavirus includes the base sequence of SEQ ID NO:

3.

7. A recombinant vector for producing a water-soluble recombinant spike protein of a coronavirus that forms a trimer as described in claim 1, wherein the gene encoding the protein of the trimer motif region of coronin 1 (mCor1) comprises the base sequence of SEQ ID NO:

7.

8. The recombinant vector for producing a water-soluble recombinant spike protein of a trimer-forming coronavirus according to claim 1, wherein the vector is a binary vector.

9. 2. The recombinant vector for producing the water-soluble recombinant spike protein of a trimer-forming coronavirus according to claim 1, wherein the recombinant vector further comprises any one of promoters selected from the group consisting of a 35S promoter derived from cauliflower mosaic virus, a 19S RNA promoter derived from cauliflower mosaic virus, a Mac promoter, a plant actin protein promoter, and a ubiquitin protein promoter.

10. A transformed organism transformed with the recombinant vector according to claim 1.

11. The transformant according to claim 10, wherein the transformed organism is a prokaryote or a eukaryote.

12. (a) producing the recombinant vector of claim 1; (b) introducing the recombinant vector into an organism to produce a transformed organism; (c) culturing the transformed organism; (d) infiltrating a plant with a culture of said culture; and (e) crushing the plant to obtain a recombinant spike protein of coronavirus that forms a trimer; A method for producing a water-soluble recombinant spike protein of a trimer-forming coronavirus from plants.

13. A recombinant protein produced by the method for producing a water-soluble recombinant spike protein of a trimer-forming coronavirus from a plant according to claim 12.

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