RU2850189C2 - Recombinant hybrid protein derived from the hr region of the sars-cov-2 s2 protein, and use of the recombinant hybrid protein - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: the invention relates to the field of biotechnology, namely to a recombinant hybrid protein from the HR region of the SARS-CoV-2 S2 protein, which consists of a truncated HR1 protein obtained from the HR1 region of the SARS-CoV-2 S2 protein, a truncated HR2 protein obtained from the 1H2 region, and a binding peptide conjugating them, as well as to a vector expressing the recombinant hybrid protein from the HR region of the SARS-CoV-2 S2 protein.

EFFECT: the invention is effective in the manufacture of a medicinal product for the prevention or treatment of SARS-CoV-2, in the manufacture of a reagent for the detection of the SARS-CoV-2 antigen, as an immunogen in the manufacture of a vaccine against SARS-CoV-2, and as an immunogen for the production of antibodies to SARS-CoV-2.

10 cl, 14 dwg, 1 tbl, 13 ex

Description

Field of technology

The present invention relates to a recombinant fusion protein of a novel coronavirus, in particular to a recombinant fusion protein obtained from the heptad repeat (HR) region of the S2 protein of SARS-COV-2, and its use.

State of the art

SARS-CoV-2 is an enveloped, single-stranded, positive-strand RNA virus with a linear genome. It belongs to the subfamily Orthocoronavirinae of the family Coronaviridae of the order Nidovirales and is a new member of the genus β-coronavirus. The novel coronavirus was discovered in 2019 and subsequently caused a worldwide pandemic. It can be transmitted from person to person during the incubation period, spreads rapidly, and can cause a variety of mild, moderate, and severe symptoms, affecting the respiratory tract and other organs of infected people. The virus is highly pathogenic. On February 12, 2020, the World Health Organization officially designated the disease caused by SARS-CoV-2 COVID-19.

The novel coronavirus genome is 26-30 kbp in size and contains several open reading frames that can encode four major structural proteins and 16 nonstructural proteins. Its major structural proteins are the spike protein (S), membrane protein (M), envelope protein (E), and nucleocapsid protein (N). The spike protein (S) is a trimer and plays a key role in viral entry into host cells. The spike protein monomer consists of the S1 and S2 proteins. When the virus approaches a host cell, the RBD domain of the S1 protein binds to the host cell's ACE2 receptor, inducing viral adsorption and cell entry. At the same time, the S1 and S2 proteins separate. The S2 protein contains two important functional domains: HR1 and HR2. These two structural domains interact with each other to form six supercoiled HR structures from the HR1-HR2 trimer, further reducing the distance between the viral membrane and the cellular membrane, which promotes the fusion of the viral membrane and the cellular membrane and the entry of the virus into the cell.

Currently, research and development of new protein vaccines and antibodies against the novel coronavirus are primarily focused on the RBD domain of the S1 protein of the novel coronavirus. However, only a small number of studies have been conducted on the HR1 and HR2 domains of the S2 protein. The HR1 and HR2 domains can mediate the fusion of the virus and the host membrane and are among the targets for the development of drugs and vaccines against the novel coronavirus. The complexity of developing drugs and vaccines against the novel coronavirus infection stems from the need to determine how to simulate the natural conformation of the HR1 and HR2 domains before their fusion, how to inhibit the binding of HR1 and HR2, and how to block the fusion of the novel coronavirus and the cell membrane. There are no relevant patents or literature on this topic. The fusion protein described in Application No. CN202011346373.6, "Fusion Protein for Transmembrane Expression of Novel Coronavirus S2 Antigen, Recombinant Vector, Recombinant Dendritic Cell, and Uses Therefor," comprises a CD4 signal peptide, a novel coronavirus S2 protein antigen, a FLAG tag (peptide sequence), and a CD4 transmembrane domain linked in sequence. The present invention utilizes a completely different strategy.

Brief summary of the invention

The aim of the present invention is to obtain a stable recombinant fusion protein derived from the HR region of the S2 protein of a novel coronavirus and its use.

The present invention uses the following technical solution:

A recombinant fusion protein HR121 or HR212 from the HR region of the S2 protein of a novel coronavirus, which consists of a truncated HR1 protein derived from the HR1 region of the S2 protein of a novel coronavirus, a truncated HR2 protein derived from the HR2 region, and a linking peptide conjugating the two above-mentioned proteins. The structure of the HR121 protein is HR1-linker1-HR2-linker2-HR1; the structure of the HR212 protein is HR2-linker2-HR1-linker1-HR2, wherein the amino acid sequence of HR1 is presented in SEQ ID NO: 1 or is a sequence in which one or more amino acids are replaced, deleted or inserted into the amino acid sequence presented in SEQ ID NO: 1. The amino acid sequence of HR2 is presented in SEQ ID NO: 2 or is a sequence in which one or more amino acids are replaced, deleted or inserted into the amino acid sequence presented in SEQ ID NO: 2, and the amino acid sequence of linker 1 and linker 2 are presented in SEQ ID NO: 3-4, respectively.

In addition, the amino acid sequence of the recombinant fusion protein HR121 of the novel coronavirus is presented in SEQ ID No. 5.

In addition, the amino acid sequence of the recombinant fusion protein HR212 of the novel coronavirus is presented in SEQ ID No. 6.

In addition, the nucleotide sequence encoding HR121 is presented in SEQ ID NO: 7.

In addition, the mRNA sequence encoding HR121 is shown in SEQ ID NO: 23.

In addition, the nucleotide sequence encoding HR212 is presented in SEQ ID NO: 8.

In addition, the mRNA sequence encoding HR212 is shown in SEQ ID NO: 27.

The present invention also includes a vector for expressing a recombinant fusion protein consisting of the HR region of the S2 protein of the novel coronavirus, which is characterized in that the eHR121 framework vector, the HR212 framework vector, and the HR121 framework vector and the HR212 framework vector contain the coding DNA of SEQ ID NO. 7 and 8, respectively, and the coding mRNA of SEQ ID NO. 23 and 27, respectively. In another preferred embodiment of the present invention, said vector is selected from an mRNA vaccine vector or an adenovirus vector.

The present invention also relates to the use of a recombinant fusion protein obtained from the HR region of the S2 protein of a novel coronavirus in the manufacture of an antigen detection reagent and a medicinal product for the prevention or treatment of a novel coronavirus infection.

In addition, the present invention also relates to the use of a recombinant fusion protein obtained from the HR region of the S2 protein of the novel coronavirus as an immunogen in the manufacture of a vaccine and antibodies to the novel coronavirus.

Due to the source of the HR region of the S2 protein of the novel coronavirus, the recombinant HR121 and HR212 proteins of the novel coronavirus proposed in the present invention can mimic the intermediate conformation of the fusion of the novel coronavirus with the membrane and act in the fusion step of the novel coronavirus with the cell membrane. As a new target for inhibiting the replication of the novel coronavirus, it is significantly different from the recombinant RBD protein (acting in the adsorption step of the novel coronavirus) derived from the S1 protein of the novel coronavirus, which is currently used. In addition, these two proteins can induce the formation of high-affinity neutralizing antibodies against the novel coronavirus in animals, and their antibody activity and animal protective effect are equivalent to those of the antibodies induced by the RBD protein, which is currently used (Nature. 2020 Oct; 586 (7830): 572-577 doi: 10.1038/s41586-020-2599-8).

In another preferred embodiment, the fusion protein of the present invention can be used directly or indirectly.

In another preferred embodiment, direct use includes administration in the form of recombinant protein vaccines, and indirect use includes administration in the form of nucleic acid-based vaccines (DNA or RNA vaccines), recombinant viral vector vaccines (such as adenovirus vectors, poxvirus vectors, adeno-associated viral vectors, herpes simplex virus-based vectors, cytomegalovirus vectors), etc.

Compared with the prior art, the advantageous effects of the present invention are:

1. The recombinant fusion protein of the novel coronavirus proposed in the present invention can form and maintain a stable dimeric structure, imitate the conformation of the intermediate state of fusion of the novel coronavirus with the membrane, and can be used as a starting material for detecting the fusion process of the novel coronavirus with the membrane.

2. The recombinant protein of the new coronavirus proposed in the present invention has significant activity against the new coronavirus and has broad prospects for application in the field of developing protein drugs for the prevention or treatment of the new coronavirus infection.

3. The recombinant protein of the novel coronavirus proposed in this invention exhibits good immunogenicity, can induce high-affinity neutralizing antibodies to the novel coronavirus infection in animals, and protect animals susceptible to the novel coronavirus infection, such as golden hamsters, from viral infection. It has broad potential for use in the development of a vaccine against the novel coronavirus infection and antibodies to the novel coronavirus.

4. The recombinant protein of the new coronavirus and the induced neutralizing antibody to the new coronavirus proposed in the present invention are effective against the new coronavirus and have a broad spectrum of action.

Description of drawings

Figure 1 is a structural diagram of the recombinant fusion proteins HR121 and HR212 of the novel coronavirus; among them, the fusion protein HR121 formed by connecting HR1-linker1-HR2-linker2-HR1; HR212 formed by connecting HR2-linker2-HR1-linker1-HR2.

Figure 2 is a diagram of the SDS-PAGE results of the recombinant fusion proteins HR121 and HR212 of the novel coronavirus, in which Figure 2A is a diagram of the electrophoresis results of HR121. M (marker): 170 kDa, 130 kDa, 100 kDa, 70 kDa, 55 kDa, 40 kDa, 35 kDa, 25 kDa, 15 kDa, 10 kDa (from top to bottom); 1: HR121 (20 μg); 2: HR121 (50 μg); 3: HR121 (50 μg); Figure 2B is a diagram of the electrophoresis results of HR212. M (marker): 170 kDa, 130 kDa, 100 kDa, 70 kDa, 55 kDa, 40 kDa, 35 kDa, 25 kDa, 15 kDa, 10 kDa (from top to bottom); 1: HR212 (10 μg); 2: HR212 (20 μg); 3: HR212 (50 μg).

Figure 3 shows the secondary structure of the recombinant fusion proteins HR121 and HR212 of the novel coronavirus, determined by circular dichroism analysis. Among them, Fig. 3A shows the structure of the HR121 protein forming an α-helix; Fig. 3B shows the structure of the HR212 protein forming an α-helix.

Figure 4 is a diagram of the activity of the recombinant fusion proteins of the novel coronavirus HR121 and HR212 against the novel coronavirus.

Figure 5 is a graph of the antibody titer of rabbits immunized with recombinant fusion proteins HR121 and HR212 of the novel coronavirus.

Figure 6 shows that both rabbit antisera against the recombinant novel coronavirus fusion proteins HR121 and HR212 can effectively inhibit the entry of the novel coronavirus into 293T-ACE2 cells.

Figure 7 is a diagram of the activity of rabbit serum against the novel coronavirus after immunization of rabbits with recombinant proteins of the novel coronavirus HR121 and HR212.

Figure 8 is a diagram of the activity of rabbit antibodies against the novel coronavirus after immunization of rabbits with recombinant proteins of the novel coronavirus HR121 and HR212.

Figure 9 shows that after immunization of hamsters with the recombinant protein of the novel coronavirus HR121, effective inhibition of hamster lung tissue infection caused by the novel coronavirus can be achieved.

Figure 10 is a schematic comparison of the sequences of the other three HR121 variants and three HR212 variants of the novel coronavirus with different amino acids resulting from deletions in the HR1 and HR2 regions.

Figure 11 shows the SDS-PAGE results of other three recombinant HR121 proteins and three recombinant HR212 proteins with different amino acid sequences resulting from deletions in the HR1 and HR2 regions of the novel coronavirus; M: marker: 100 kDa, 70 kDa, 40 kDa, 25 kDa, 20 kDa, 15 kDa, 10 kDa (from top to bottom); 1: HR121-1 (20 μg); 2: HR121-2 (20 μg); 3: HR121-3 (20 μg); 4: HR212-1 (20 μg); 5: HR212-2 (20 μg); 6: HR212-3 (20 μg).

Figure 12 shows that the other three recombinant HR121 proteins and three recombinant HR212 proteins with different amino acid sequences resulting from deletions in the HR1 and HR2 regions of the novel coronavirus also have inhibitory activity against the novel coronavirus.

Figure 13 shows that the rabbit antiserum against the recombinant protein of the novel coronavirus HR121 effectively inhibited the entry of 23 mutant pseudovirus strains of the novel coronavirus into 293T-ACE2 cells.

Figure 14 shows that after immunization of hamsters with the recombinant protein of the novel coronavirus HR121, it is possible to effectively inhibit the infection of hamster lung tissue caused by the mutant strain of the novel coronavirus Omicron BA.2.

Detailed description

As a result of extensive and intensive research, the inventor developed a class of recombinant proteins based on the HR region of the S2 protein of the novel coronavirus (such as recombinant proteins HR121 and HR212 of the novel coronavirus) that can mimic the intermediate conformation of membrane fusion of the novel coronavirus and block the process of virus-cell membrane fusion. The fusion protein of the present invention exhibits good immunogenicity, can induce high-affinity neutralizing antibodies in animals, and exhibits excellent activity against the novel coronavirus. Thus, it can be used to develop drugs for the treatment of infections caused by the novel coronavirus. This is the basis for the present invention.

In a preferred embodiment, neutralizing antibodies induced by the fusion protein of the present invention are effective against the novel coronavirus and exhibit a broad spectrum of activity. At the cellular level, neutralizing antibodies induced by the fusion protein are active against many mutant strains of the SARS-CoV-2 virus that infiltrate cells and can protect animals susceptible to the novel coronavirus from infection with mutant strains of the virus (such as Omicron BA.2). In a preferred embodiment, the mutant SARS-CoV-2 virus strain is selected from the group consisting of: S477N, E484K, A222V, N439K, K417N, D614G, D839Y, B.1.617, B.1.617.1, B.1.617.2.V2, B.1.429, B.1.525, B.1.526, B.1.1.7, B.1.351, B.1.1.28, B.1.617.2, C.37, B.1.621, B.1.1.529 (Omicron BA.1), Omicron BA.2, Omicron BA.3 and Omicron BA.4/5 from pCDNA3.1-SARS-CoV-2 V-2-Spike-Δ 18.

Terms

To better understand the contents of this application, definitions of the terms used are provided. When used in this application, each of the following terms has the meaning set forth below unless otherwise expressly defined herein. Other definitions are described in detail throughout the application.

The term "about" may refer to a value or composition within the tolerance of a particular value or composition, as determined by one skilled in the art, which will depend in part on how the value or composition is measured or determined. For example, in the context of this document, the expression "about 100" includes all values from 99 to 101 (e.g., 99.1; 99.2; 99.3; 99.4, etc.).

In this context, the terms "comprising" or "including" may be open, semi-closed, or closed. In other words, such terms also include "consisting essentially of" or "consisting of."

In this context, the term "effective dose" generally refers to an amount of an immunogen that can induce a protective immune response sufficient to induce immunity to prevent and/or alleviate the course of an infection or disease and/or reduce the severity of at least one symptom of an infection or disease.

In this context, the term "protective immune response" or "protective response" refers to an immunogen-mediated immune response against an infectious agent or disease that has been demonstrated in vertebrate animals (such as humans) and that prevents or alleviates the course of the infection or reduces the severity of at least one symptom of the disease.

The recombinant fusion protein of the novel coronavirus according to the present invention

In the present invention, the "fusion protein of the present invention", "the antigen protein of the present invention", "the immunogenic peptide of the present invention", "the recombinant fusion protein of the present invention", "the recombinant fusion protein of the novel coronavirus of the present invention" and "the recombinant protein of the novel coronavirus of the present invention" can be used interchangeably, all of them refer to a recombinant fusion protein obtained from the HR region of the S2 protein of the novel coronavirus. The fusion protein of the present invention comprises: the amino acid sequences of HR1 or HR2 or sequences with a substitution (e.g., a conservative amino acid substitution), deletion or insertion of one or more amino acids. It should be understood that this term includes not only one type of fusion protein of the present invention, but also a set of proteins (or a set of polypeptides) formed by several fusion proteins of the present invention.

In a preferred embodiment, the hybrid protein of the present invention comprises a structure shown in the formulas below:

HR1-linker1-HR2-linker2-HR1 (formula I),

HR2-linker2-HR1-linker1-HR2 (formula II);

wherein HR1 is a truncated HR1 region protein or a sequence thereof in which one or more amino acids are replaced, deleted or inserted;

HR2 is a truncated HR2 region protein or its sequence in which one or more amino acids are substituted, deleted, or inserted;

the amino acid sequences of linker 1 and linker 2 are shown in SEQ ID No. 3 or 4.

In another preferred embodiment, the truncated protein from the HR1 region or its sequence in which one or more amino acids are replaced, deleted or inserted comprises:

(1) The amino acid sequence shown in SEQ ID No. 1;

(2) Compared to the amino acid sequence of HR1 (SEQ ID NO: 1), an amino acid sequence in which one or more amino acids (e.g., 5-10 amino acids, 7-12 amino acids, 9-14 amino acids, 11-16 amino acids, or 13-18 amino acids) are deleted from the N-terminus; and/or one or more amino acids (e.g., 10-15 amino acids, 14-19 amino acids, 18-23 amino acids, 20-25 amino acids, 22-27 amino acids, 24-29 amino acids, 28-31 amino acids, or 30-35 amino acids) are deleted from the C-terminus.

In a preferred embodiment, the amino acid sequence of HR1 is selected from the group consisting of:

(1) SEQ ID No. 1 (HR1);

(2) SEQ ID No. 11 (HR1- ;

(3) SEQ ID NO. 12 (HR1- ; or

(4) SEQ ID NO. 13 (HR1- .

In another preferred embodiment, the truncated protein from the HR2 region or its sequence in which one or more amino acids are replaced, deleted or inserted comprises:

(1) The amino acid sequence shown in SEQ ID No. 2;

(2) Compared to the amino acid sequence of HR2 (SEQ ID NO: 2), an amino acid sequence in which one or more amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 12 amino acids) are deleted from the N-terminus; and/or one or more amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8 amino acids) are deleted from the C-terminus.

In a preferred embodiment, the amino acid sequence of HR2 is selected from the group consisting of:

(1) SEQ ID No. 2 (HR2);

(2) SEQ ID NO. 14 (HR2- ;

(3) SEQ ID NO. 15 (HR2- ; or

(4) SEQ ID NO. 16 (HR2- .

In a preferred embodiment, the amino acid sequence of the recombinant fusion protein of the new coronavirus (HR121) is presented in SEQ ID No. 5; the amino acid sequence of the recombinant fusion protein of the new coronavirus (HR212) is presented in SEQ ID No. 6.

In a preferred embodiment, the recombinant fusion protein of the novel coronavirus further comprises an amino acid sequence selected from the group consisting of:

(1) HR121-1: Connection HR1- Linker1- HR2- Linker2- HR1- ;

(2) HR121-2: Connection HR1- Linker1- HR2- Linker2- HR1- ;

(3) HR121-3: HR1- connection Linker1- HR2- Linker2- HR1- ;

(4) HR212-1: HR2- connection Linker2- HR1- Linker1- HR2- ;

(5) HR212-2: HR2- connection Linker1- HR1- Linker2- HR2- ;

(6) HR212-3: HR2- connection Linker1- HR1- Linker2- HR2- .

In a preferred embodiment, conservative amino acid substitution is carried out in accordance with Table A.

Table A

The original amino acid residue Representative substitutions Preferred replacement Ala (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp (D) Glu Glu Cys (C) Ser Ser Gln (Q) Asn Asn Glu (E) Asp Asp Gly (G) Pro; Ala Ala His (H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val; Met; Ala; Phe Leu Leu (L) Ile; Val; Met; Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe; Ile Leu Phe (F) Leu; Val; Ile; Ala; Tyr Leu Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr; Ser Phe Val (V) Ile; Leu; Met; Phe; Ala Leu

In the present invention, the fusion protein also includes other forms such as pharmaceutically acceptable salts or conjugates.

In this context, a set of proteins described by the term "set of proteins" consists of at least two hybrid proteins of the present invention or their derivative sequences (formed as a result of substitution, deletion or insertion).

In addition, the protein set may further include other antigenic peptides or proteins of the SARS-CoV-2 coronavirus, in addition to SEQ ID Nos: 5 and 6.

In this context, the term "isolated" refers to the separation of a substance from its original environment (if it is a natural substance, then the original environment is the natural environment). For example, a protein from living cells in its natural state is not isolated and purified, but the same protein is isolated and purified if it is separated from other substances that exist in their natural state.

The hybrid protein of the present invention may be a recombinant protein or a synthetic protein, preferably a synthetic protein.

In the present invention, if the sequence of the fusion protein is short (for example, ≤70 amino acids, more preferably ≤60 amino acids), the corresponding peptide sequence can be directly synthesized using chemical methods.

If the sequence of the fusion protein of the present invention is long or the fusion protein of the present invention is produced by protein fusion, the corresponding peptide sequences can also be produced in large quantities using a recombinant DNA-based method. This typically involves cloning the sequence encoding the antigenic polypeptide or its fusion protein into a vector, followed by transfer into cells, and then isolating the corresponding antigenic polypeptide or fusion protein from dividing host cells using conventional methods.

Based on the sequence information provided, skilled artisans can use available cloning technology to generate nucleic acid sequences or vectors suitable for transduction into cells.

The vector may be a plasmid or a virus. A viral vector is capable of entering cells. However, non-viral vectors, such as plasmids, may be combined with reagents to facilitate uptake of the viral vectors by target cells. Such reagents include polycationic substances. Optionally, a delivery system, such as a liposome-based delivery system, may be used. The vector used in the present invention is, in a preferred embodiment, suitable for in vivo or in vitro use.

In a preferred embodiment, the vector will contain one or more regulatory sequences for directing the expression of nucleic acid sequences in a target cell. A regulatory sequence may include a promoter, enhancer, transcription termination signal, polyadenylation sequence, origin of replication, nucleic acid restriction site, and homologous recombination site, which can be operably linked to the nucleic acid sequence. The vector may also include selectable tags, for example, to direct vector expression in a growth system (such as a bacterial cell) or in a target cell.

Pharmaceutical composition

The present invention also provides a pharmaceutical composition. The pharmaceutical composition of the present invention can be therapeutic or prophylactic (e.g., vaccines, vaccine compositions, or immunogenic compositions). The pharmaceutical composition of the present invention comprises an effective amount of fusion proteins or sets of proteins, nucleotide molecules, vectors, and/or vaccines for host cells of the present invention or immune cells activated by the fusion protein of the present invention (such as dendritic cells sensitized by the fusion protein of the present invention or T cells induced by dendritic cells), as well as at least one pharmaceutically acceptable carrier, diluent, or excipient.

In the present invention, these medicinal compositions contain immune antigens (including a fusion protein, a set of peptides, or derivatives thereof according to the present invention) and are typically combined with "pharmaceutically acceptable carriers," including any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition. Examples of suitable carriers include, but are not limited to, proteins, lipid aggregates (such as oil droplets or liposomes), and the like. These carriers are well known to those skilled in the art. Furthermore, these carriers can function as immunostimulants ("adjuvants").

In a preferred embodiment, the carrier is a pharmaceutically acceptable carrier. These carriers include any carrier that does not induce the production of harmful antibodies in individuals receiving the composition. In a preferred embodiment, the pharmaceutically acceptable carrier comprises a liquid, preferably water, a saline solution, or a buffer. The carrier may further comprise an excipient, preferably a filler, lubricant, glidant, wetting agent, or emulsifier, a buffer for pH adjustment, etc.

Furthermore, the pharmaceutical composition of the present invention may further comprise additional adjuvants, such as vaccine adjuvants. Typical vaccine adjuvants include, but are not limited to, the following types: inorganic adjuvants such as aluminum hydroxide, alum, etc.; synthetic adjuvants such as synthetic double-stranded polynucleotides (double-stranded polyadenosinic acid, polyuridic acid), levamisole, isoprinosine, etc.; oil emulsions such as Freund's adjuvant, peanut oil emulsion adjuvant, mineral oil, vegetable oil, etc. Examples of known adjuvants include, but are not limited to: complete Freund's adjuvant, incomplete Freund's adjuvant, aluminum hydroxide adjuvant, lipopolysaccharide (LPS), RIBI adjuvant, MF-59, SAS adjuvant, MF59 adjuvant, QS-21 adjuvant, poly I:C and other TLR ligands, GM-CSF, IL-2, IL-3, IL-7, IL-11, IL-12, IL-18, IL-21, etc.

Typically, a pharmaceutical composition (vaccine composition or immunogenic composition) can be formulated into an injectable preparation, such as a solution or suspension. It can also be formulated into a solid form suitable for reconstitution into a solution or suspension, or as a liquid excipient prior to injection. To enhance the adjuvant effect, the preparation can also be emulsified or encapsulated in liposomes.

In another preferred embodiment of the present invention, said pharmaceutical composition may further comprise a vaccine component derived from one or more pathogens selected from the group consisting of other viruses and combinations thereof. The vaccine component comprises an inactivated strain, an attenuated strain, or a protein, nucleic acid, etc.

The resulting pharmaceutical combination may be administered by conventional routes, including (but not limited to) intramuscular administration, intradermal administration, subcutaneous administration, nasal instillation, nebulization, administration into the reproductive tract, rectum, oral administration, or any combination thereof.

When using the pharmaceutical composition, a safe and effective amount of the fusion protein or peptide set of the present invention is administered to a human subject. The safe and effective amount is typically at least about 1 μg peptide/kg body weight and, in most cases, no more than about 8 mg peptide/kg body weight. The preferred dose is about 1 μg to 1 mg peptide/kg body weight. Of course, the specific dose will also depend on other factors, such as the route of administration and the patient's health status, which are within the discretion of a qualified physician.

Bid

It should be understood that the fusion protein of the present invention can be used directly in the form of a protein (such as a recombinant protein-based vaccine) or indirectly. For example, said indirect use includes common methods in this field. For example, it is administered to a subject in need thereof in the form of a nucleic acid-based vaccine (DNA or RNA vaccine), a recombinant viral vector vaccine (such as an adenovirus vector, a poxvirus vector, an adeno-associated viral vector, a herpes simplex virus vector, a cytomegalovirus vector), etc. A preferred vector vaccine is an adeno-associated viral vector vaccine.

In some embodiments of the invention, the vaccine is a nucleic acid-based vaccine (DNA or RNA vaccine), a recombinant protein subunit vaccine, a recombinant viral vector vaccine, a recombinant vaccine with vector expression in bacterial cells, a vaccine based on virus-like particles, a vaccine based on nanoparticles, and a vaccine with viral vector expression in cells.

Preferably, the vaccine of the present invention may be a recombinant protein vaccine, a recombinant RNA vaccine, a recombinant viral vector vaccine (such as an adenovirus vector, a poxvirus vector, an adeno-associated viral vector, a herpes simplex virus vector, a cytomegalovirus vector), a recombinant bacterial cell-expressing vector vaccine, a recombinant yeast cell-expressing vector vaccine, or a recombinant virus-like particle-based vaccine.

In some embodiments of the invention, the vaccine of the present invention is selected from a recombinant protein vaccine, a recombinant RNA vaccine, a recombinant adenovirus vector, a recombinant poxvirus vector, or a combination of one or two or more of the above.

In some embodiments of the invention, one or more vaccines are used for vaccination, such as co-vaccination or sequential vaccination.

In some embodiments of the invention, one or more vaccines and other vaccines against a new coronavirus are used for vaccination, for example, other vaccines include vaccines against coronavirus infections based on the S or S1 protein, including the following viruses, but not limited to, SARS CoV-2, SARS-CoV, MERS-CoV, HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, Bat-CoV, etc.

In some embodiments, the vaccine is a recombinant protein vaccine. In some embodiments, the vaccine comprises a combination of a nucleic acid-based vaccine (DNA or RNA vaccine) and a recombinant human cell-based vector vaccine.

In one aspect of the present invention, there is provided a method for producing a vaccine against the novel coronavirus SARS-CoV-2, which comprises the following steps:

(a) Obtaining a hybrid protein, nucleotide molecule, vector and/or host cell according to the present invention;

(b) Combining the active substance provided in (a) with an immunologically or pharmaceutically acceptable carrier.

The vaccine of the present invention can be used to induce an immunostimulatory protective immune response to prevent and/or mitigate the consequences of associated diseases caused by the novel coronavirus SARS-CoV-2.

In another preferred embodiment, the associated disease caused by the novel coronavirus SARS-CoV-2 is selected from the group consisting of: respiratory tract infection, pneumonia and its complications, and a combination thereof.

In a preferred embodiment, the vaccine of the present invention can prevent, eliminate or reduce the severity of infection with a new coronavirus or alleviate at least one of its symptoms, such as respiratory symptoms (such as nasal congestion, sore throat, hoarseness), headache, cough, sputum, fever, wheezing, shortness of breath, pneumonia caused by infection, severe acute respiratory syndrome, renal failure, etc.

In one aspect, the present invention also provides a method for preventing or treating infection caused by the novel coronavirus SARS CoV-2, which comprises the step of administering a fusion protein, nucleotide molecule, vector, host cell and/or vaccine of the present invention to a subject in need thereof.

The technical solution of the present invention is further illustrated below by means of experiments.

I. Materials and reagents:

1. Basic materials

Virus: a new isolated clinical strain of coronavirus (SARS-CoV-2) (National Microbiological Data Center, serial number: NMDCN0000HUI); a new isolated clinical strain of coronavirus Omicron BA.2 (National Microbiological Data Center, serial number: UB1663744906574);

Novel coronavirus pseudovirus (pseudovirus was produced by transfection of 293FT cells using VSVΔG- *G and pcDNA3.1-SARS-CoV-2-envΔ18);

Plasmid amplification strain: E. coli DH5α competent cells; protein expression strain: E. coli BL21 (DE3) competent cells (Beijing Novozan).

2. Basic reagents

LB medium, 2XYT medium (Sigma);

DMEM medium, FCS (Gibco);

JetPRIME transfection reagent (Polyplus Transfer, Illkirch, France)

Kanamycin (Sigma Aldrich, St. Louis, USA)

Endotoxin-free small plasmid extraction kit, large plasmid extraction kit (Tiangen, Beijing)

TaKaRa BCA Protein Assay Kit (TaKaRa Bio, Beijing)

CCK8 reagent kit (Beyotime, Shanghai)

Roche Viral RNA Extraction Kit (Roche Diagnostics, Mannheim, Germany)

Renilla Luciferase Assay Kit (Promega, Madison, USA)

Trizol reagent (Invitrogen, California, USA)

Protein A Antibody Purification Kit (BBI Life Science, Shanghai)

Complete and incomplete Freund's adjuvant (Sigma Aldrich, USA)

Aluminum hydroxide-based adjuvant (Alhydrogel® 2% adjuvant, Invivogen, USA)

2. Examples

Example 1: Construction of recombinant protein expression vectors of the novel coronavirus SARS-CoV-2 pET-30a-SARS-CoV-2-HR121 and pET-30a-SARS-CoV-2-HR212

1.1 Construction of pET-30a-SARS-CoV-2-HR121 vector:

The nucleotide sequence (SEQ ID NO: 7) of the HR121 protein of the novel coronavirus was genetically synthesized (Shanghai Jierui Biological Engineering Co., Ltd.) and inserted between the EcoRI (GAATTC) and XhoI (CTCGAG) cleavage sites of the pET-30a vector.

The synthesized pET-30a-SARS-CoV-2-HR121 vector was transformed into competent E. coli DH5α cells. After culturing at 37°C for 12 hours in liquid LB medium containing 50 μg/ml kanamycin, a small amount of plasmid was extracted for subsequent use.

1.2 Construction of pET-30a-SARS-CoV-2-HR212 vector:

The nucleotide sequence (SEQ ID NO: 8) of the HR212 protein of the novel coronavirus was genetically synthesized (Shanghai Jierui Biological Engineering Co., Ltd.) and inserted between the EcoRI (GAATTC) and XhoI (CTCGAG) cleavage sites of the pET-30a vector.

The synthesized pET-30a-SARS-CoV-2-HR212 vector was transformed into competent E. coli DH5α cells and cultured in liquid LB medium containing 50 μg/ml kanamycin at 37°C for 12 hours, then a small amount of plasmids was extracted for subsequent use.

The structures of HR121 and HR212 are shown in Figure 1.

Example 2: Induced expression and purification of recombinant proteins HR121 and HR212 of the novel coronavirus SARS-CoV-2

2.1. Competent BL21(DE3) cells were transformed with plasmid pET-30a-SARS-CoV-2-HR121 or pET-30a-SARS-CoV-2-HR212 according to the conventional transformation method;

2.2. A single colony was selected and cultured in a shaker in 5 ml of 2×YT medium containing kanamycin at 37°C until turbidity was achieved. 100 µl of the bacterial solution were inoculated into 100 ml of 2×YT medium at 180 rpm, 37°C, for a total of 500 ml, and cultured in a shaker overnight (12 h).

2.3 A 0.238 g sample of isopropyl-β-D-1-thiogalactopyranoside (IPTG) was dissolved in 1 ml of water, 100 μl of IPTG solution was added to each 100 ml of water, placed on a shaker overnight at 140 rpm at 20°C (12 hours);

2.4 The bacterial solution was collected and resuspended in 5 ml of 20 mmol/l imidazole solution for every 100 ml of bacterial solution;

2.5 Ultrasonic fragmentation was performed with 1-second pulses and 1-second pauses at 216,000 joules. During ultrasonication, samples should be placed in an ice bath to prevent protein damage at high temperatures. After ultrasonication (approximately 10-20 minutes), the turbid bacterial fluid clears;

2.6 10% Triton X 100 was added to the sonicated bacterial solution to achieve a final concentration of 1% and placed on ice for 30 min;

2.7 The solution was centrifuged at 12,000 rpm for 30 minutes, the supernatant was transferred to a new centrifuge tube, the process was repeated, and then the supernatant was transferred to a new centrifuge tube;

2.8 The sample was loaded onto a column with nickel resin (GE Ni Sepharose TM6 Fast Flow; art. no.: 17-5318-06), before loading the column, the column was washed with a 10-fold volume of 20 mmol/L imidazole solution;

2.9 After loading the sample, the column was washed with 20 times the column volume (40 ml) of 20 mmol/l imidazole solution to remove impurities;

2.10 The column volume (10 ml) was washed 5 times with 100 mmol/l imidazole solution to remove impurities;

2.11 After washing, the target protein was eluted with 25-fold column volume (50 ml) of 200 mmol/l imidazole solution;

2.12 The target protein was concentrated in batches using a 15 mL tube to concentrate molecules with an MW greater than 10 KD. After sample loading, the 200 mmol/L imidazole solution was replaced with PBS. That is, after sample concentration, the PBS solution was added to the concentration tube and the tube was centrifuged to replace the 200 mmol/L imidazole solution. This process was repeated three times to ensure that the protein solubilization liquid was PBS;

2.13 Approximately 1.5 ml of the concentrated protein solution was placed in a 1.5 ml EP tube, and the protein concentration was measured using a BCA assay kit. The solution was divided into smaller tubes to avoid repeated freeze-thaw cycles.

Example 3: Analysis of recombinant proteins HR121 and HR212 of the novel coronavirus SARS-CoV-2 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)

3.1 The amount of purified protein was determined using the BCA assay kit.

3.2 Fifty micrograms of HR121 and HR212 proteins were collected and separated by electrophoresis on a 10% SDS-PAGE gel. After electrophoresis, Coomassie brilliant blue was used for staining and destaining. The results of analysis and detection are shown in Fig. 2. The size of the HR121 protein is approximately 28 kDa, and the size of the HR212 protein is approximately 24 kDa.

Example 4: Secondary structure analysis of recombinant proteins HR121 and HR212 of the novel coronavirus by circular dichroism spectroscopy

4.1. Construction of the expression vector and purification of recombinant HR1 and HR2 proteins of the novel coronavirus: The nucleotide coding sequence of HR1 of the novel coronavirus (SEQ ID NO: 9) and the nucleotide coding sequence of HR2 of the novel coronavirus (SEQ ID NO: 10) were inserted between the EcoRI (GAATTC) and XhoI (CTCGAG) restriction sites of the pET-30a vector, respectively. Recombinant HR1 and HR2 proteins of the novel coronavirus were obtained by protein purification according to the method of Example 2.

4.2 Jasco spectrophotometer (model J-815) was used to detect the absorbance of 1 μmol/L HR121, 1 1 μmol/L HR121 +1 1 μmol/L MHR2, 1 1 μmol/L HR212, 1 1 μmol/L HR212 +1 1 μmol/L HR2. The binding ability of HR121 protein and HR2 protein was detected; the binding ability of HR212 protein and HR1 protein was detected.

The results of the analysis and detection are shown in Fig. 3. The HR121 protein can bind to the HR2 protein, forming a stable α-helical structure. The HR212 protein can bind to the HR1 protein, forming a stable α-helical structure. Therefore, both of these proteins can be used to develop new reagents for coronavirus antigen detection.

Example 5: Detection of the activity of recombinant proteins HR121 and HR212 of the novel coronavirus against the novel coronavirus

The antiviral effect on cells was determined using the CCK8 method.

5.1 Vero E6 cells were inoculated with 100 µl of 5× ¹⁰⁵ /ml Vero E6 cells per well in a 96-well cell culture plate and cultured overnight at 37°C in a CO2 incubator.

5.2. Then, 50 µl of SARS-CoV-2 virus at an MOI of 0.1 was added to each well to infect the cells, and 50 µl of recombinant HR121 and HR212 proteins of the novel coronavirus were added to each well. After incubation for 2 hours, the medium was replaced with 200 µl of fresh medium containing serially diluted recombinant HR121 and HR212 fusion proteins of the novel coronavirus.

5.3 After 72 hours of culture at 37°C in a _CO2 incubator, CCK8 reagent was added to the culture supernatant and the OD450 value was read on an enzyme-linked immunosorbent assay (ELISA) reader to quantify the cytopathic effects induced by SARS-CoV-2 and the protective effects of HR121 and HR212 on cell injury.

The results of the analysis and detection are shown in Fig. 4. As shown in Fig. 4 (A), HR121 could inhibit the cytopathic effect against Vero-E6 induced by the novel coronavirus, and the _EC50 of HR121 protein for protection against the cytopathic effect caused by SARS-CoV-2 was 0.891 μmol/L; as shown in Fig. 4 (B), HR212 could inhibit the cytopathic effect against Vero-E6 induced by the novel coronavirus, and the _EC50 of HR212 protein for protection against the cytopathic effect of SARS CoV-2 was 0.583 μmol/L. This indicates that both fusion proteins have good activity against SARS-CoV-2.

Example 6 Immunization of adult New Zealand White rabbits with recombinant proteins HR121 and HR212 of the novel coronavirus

6.1. Primary immunization: The recombinant proteins of the novel coronavirus HR121 or HR212 obtained in Example 3 were diluted in PBS solution to a protein concentration of 200 μg/ml. An equal volume of Freund's complete adjuvant was added, mixed with the protein solution to form a white latex, and each rabbit was immunized using a volume of 1 ml by subcutaneous administration at several points (the protein content was 100 μg/rabbit).

6.2. Booster immunization: Two booster immunizations were administered on days 21 and 42. The recombinant proteins of the novel coronavirus HR121 or HR212 obtained in Example 3 were diluted with PBS to a protein concentration of 300 μg/ml, an equal volume of incomplete Freund's adjuvant was added, the mixture was mixed with the protein solution to form a white latex, and each rabbit was immunized using a volume of 1 ml by subcutaneous administration at several sites (the protein content was 150 μg/rabbit). A negative control group of immunizations was also created, which did not receive the protein.

6.3 On day 56, blood was collected from the hearts of immunized rabbits, serum was separated, packaged and stored at -80°C.

Example 7: Determination of serum antibody titers to recombinant proteins HR121 and HR212 of the new coronavirus in rabbits

7.1 1 μg/ml HR121 or HR212 protein was dissolved in ELISA adsorption buffer, applied at 100 μl/well and incubated at 4°C overnight or at 37°C for 2 hours;

7.2 The plate was washed with ELISA wash buffer 3 times for 3 minutes, 100 µl of PBS-5% dry skim milk was added to each well and the plate was sealed at 37°C for 2 hours;

7.3 The plate was washed as above, 100 µl of diluted serum was added to each well, 37°C/2 hours;

7.4 The plate was washed as above and 100% diluted 1:5000 sheep anti-rabbit IgG HRP-labeled antibodies (or anti-mouse IgG HRP-labeled antibodies) were added at 37°C for 1 hour; the plate was washed as above and the OPD substrate reaction solution was added. After 10 minutes, the reaction was quenched with 2 M sulfuric acid;

7.5 Using an ELISA reader, the optical density was measured at 490/630 nm.

7.6 According to the ELISA results, the OD value of the sample >0.12 + the OD value of the negative control was considered positive.

The results of the analysis and detection are shown in Figure 5, and the antibody titers to the HR121 and HR212 proteins in rabbit serum were in both cases between 10 ⁶ and 10 ⁷ . This indicates that both proteins can induce high antibody titers in the animal body.

Example 8: Determination of the titer of antibodies to SARS-CoV-2 pseudovirus neutralized by rabbit antiserum to recombinant proteins HR121 and HR212 of the new coronavirus.

(1) Determination of antibody titers to the pseudovirus of the original SARS CoV-2 strain neutralized by rabbit antiserum to the recombinant proteins HR121 and HR212 of the new coronavirus

8.1 Production and titration of the original novel coronavirus pseudovirus: According to the JetPRIME transfection reagent instructions, 10 μg of pCDNA3.1-SARS-CoV-2-Spike-Δ 18 plasmids and 500 μl of JetPRIMEBuffer were mixed; 20 μl of JetPRIMEBuffer were added; the mixture was mixed and incubated at room temperature for 10 min. The mixture was added dropwise to 239T cells in a T75 flask, and the culture medium was discarded after 12 hours of cultivation at 37°C and 5% _CO2 . The cells were infected with VSVΔG-VSVG pseudovirus at an MOI of 1, washed three times with PBS 6 hours after infection, replaced with fresh DMEM medium, and continued culturing. The supernatant containing SARS-CoV-2 pseudovirus was collected after 24 and 48 hours. The cells were centrifuged at 1000 rpm for 10 minutes, and the supernatant was collected, packaged, and stored at -80°C. Viral titer was measured using 239T-ACE2 cells.

8.2 Assay of HR121- and HR212-immunized rabbit sera for SARS-CoV-2 pseudovirus neutralization: 100 µl of 239T-ACE2 (2 × 10 ⁵ cells/mL) per well were incubated for 12 h in a 96-well cell culture plate, 50 µl of serial dilutions of anti-HR121 or anti-HR212 serum solution were incubated in another 96-well cell culture plate (three wells per dilution), and 50 µl of SARS-CoV-2 pseudovirus at an MOI of 0.1 per well were added. Positive control wells without protein and negative control wells without virus were used simultaneously. After incubation at 37°C for 1 h, the virus and protein mixture was added to the cells. After 48 hours of culture at 37°C in a _CO2 incubator, the fluorescence intensity of SARS-CoV-2 pseudovirus was determined using a Renilla luciferase activity assay kit, and the _EC50 value of rabbit antiserum after immunization with HR121 or HR212 was calculated.

The analysis and detection results are shown in Fig. 6. The _EC50 value of the rabbit antiserum after immunization with HR121, which neutralizes the SARS-CoV-2 pseudovirus, corresponds to a serum dilution of 11,536 times, and the _EC50 value of the rabbit antiserum after immunization with HR212, which neutralizes the SARS-CoV-2 pseudovirus, corresponds to a serum dilution of 636 times. This indicates that antibodies to these two proteins may have good activity against the entry of the SARS-CoV-2 virus into cells.

(2) Determination of antibody titers to the pseudovirus of various SARS CoV-2 mutants neutralized by rabbit antiserum to the recombinant HR121 protein of the new coronavirus

8.3 Production and titration of pseudoviruses of various mutants of the novel coronavirus: according to the operating instructions of the JetPRIME transfection reagent, 10 μg of pCDNA3.1-SARS-CoV-2-Spike-Δ18 plasmids containing 23 new coronavirus mutants: S477N, E484K, A222V, N439K, K417N, D614G, D839Y, B.1.617, B.1.617.1, B.1.617.2.V2, B.1.429, B.1.525, B.1.526, B.1.1.7, B.1.351, B.1.1.28, B.1.617.2, C.37, B.1.621, B.1.1.529 (Omicron BA. 1), Omicron BA.2, Omicron BA.3, and Omicron BA.4/5 were mixed well with 500 μl of jetPRIME buffer, and 20 μl of jetPRIME reagent were added, mixed, and incubated at room temperature for 10 minutes. The mixture was added dropwise to 239T cells in a T75 culture flask. The cells were incubated at 37°C and 5% _CO2 for 12 hours. The culture fluid was discarded, and pseudovirus with the Delta-G-VSV protein was added at an MOI of 1 for infection. The cells were washed 3 times with PBS 6 hours after infection, then the medium was replaced with fresh DMEM for further culturing, and the supernatants containing different SARS-CoV-2 pseudoviruses were collected after 24 and 48 hours. The cells were centrifuged at 1000 rpm for 10 minutes, and the supernatant was collected, packaged, and stored at -80°C. Viral titer was measured using 239T-ACE2 cells.

8.4 Assay of HR121-immunized rabbit sera to assess SARS-CoV-2 pseudovirus neutralization capacity: 100 µl of 239T-ACE2 (2 × 10 ⁵ cells/mL) per well were incubated for 12 h in a 96-well cell culture plate, 50 µl of serial dilutions of the anti-HR121 serum solution were incubated in another 96-well cell culture plate (three wells per dilution), and 50 µl of SARS-CoV-2 pseudovirus at an MOI of 0.1 per well were added. Positive control wells without protein and negative control wells without virus were used simultaneously. After incubation at 37°C for 1 h, the virus and protein mixture was added to the cells. After 48 hours of culture at 37°C in a _CO2 incubator, the fluorescence intensity of SARS-CoV-2 pseudovirus was determined using a Renilla luciferase activity assay kit, and the _EC50 value of the rabbit antiserum after immunization with HR121 was calculated.

The results of the analysis and detection are shown in Fig. 13. The EC values₅₀rabbit antiserum to HR121 for neutralization of various SARS-CoV-2 pseudoviruses are equal to: 14,663-fold dilution of serum for S477N, 8,824-fold dilution of serum for E484K, 36,164-fold dilution of serum for A222V, 20,174-fold dilution of serum for N439K, 9,025-fold dilution of serum for K417N, 5,986-fold dilution of serum for D614G, 12,087-fold dilution of serum for D839Y, 15,199-fold dilution of serum for B.1.617.1, 7,132-fold dilution of serum for B.1.617.2, 9,023-fold dilution of serum for B.1.617.2 V2, 433,390-fold serum dilution for B.1.429, 2,498-fold serum dilution for B.1.525, 1,801-fold serum dilution for B.1.526, 28,969-fold serum dilution for B.1.1.7 (alpha), 3,462-fold serum dilution for B.1.351 (beta), 1,899-fold serum dilution for B.1.1.28 (gamma), 15,013-fold serum dilution for B1.617.2 (delta), 6,276-fold serum dilution for C.37 (lambda), 4,250-fold serum dilution for B.1.621 (mu).

In particular, activity against the mutant Omicron strain was high, with serum dilutions of 6616 times for B.1.1.529 (Omicron BA.1), 2621 times for Omicron BA.2), 1983 times for Omicron BA.3, and 4984 times for Omicron BA.4/5. This indicates that the antibody to this protein has good activity against various mutant strains of SARS-CoV-2 viruses that penetrate cells.

Example 9: Titers of rabbit antiserum against recombinant proteins HR121 and HR212 of the novel coronavirus to neutralize the SARS-CoV-2 virus

In a 48-well cell culture plate, 200 μl of HPAEpiC (5 × 10 ⁵ particles/ml) were added per well for 12 h, the supernatant was discarded, 100 μl of DMEM medium with serial dilutions of rabbit antiserum against HR121 or HR212 were added per well (three wells per dilution), and then 100 μl of SARS-CoV-2 virus was added at an MOI of 1. Positive control wells without protein and negative control wells without virus were used simultaneously. After incubation at 37°C for 2 h, the supernatant was discarded and the cells were washed with PBS three times, then 200 μl of DMEM medium with serial dilutions of rabbit antiserum against HR121 or HR212 were added. After incubation at 37°C in a _CO2 incubator for 48 hours, the supernatant was collected, and RNA was extracted from the culture supernatant using a Roche viral RNA extraction kit. Quantitative PCR was used to detect viral RNA in the supernatant. The following primers were used:

SARS-CoV-2-NF: 5'-GGGAACTTTCCTGCTAGAAT-3' (SEQ ID No. 17);

SARS-CoV-2-NR: 5'-CAGACATTTGCTCAAGCTG-3' (SEQ ID No. 18);

Probe for SARS-CoV-2 N gene: FAM-TTGCTGCTGCTTGACAGATT-TAMRA-3' (SEQ ID no. 19).

EC ₅₀ values for anti-rabbit serum to HR121 or HR212 were calculated based on the viral RNA content.

The analysis and detection results are shown in Fig. 7. The _EC50 value of the rabbit antiserum against the HR121 protein for neutralizing the SARS-CoV-2 virus corresponded to a serum dilution of 8515 times. The _EC50 value of the rabbit antiserum against the HR212 protein for neutralizing the SARS-CoV-2 virus corresponded to a serum dilution of 1308 times. This indicates that the rabbit antiserum against these two proteins has good activity against SARS-CoV-2 virus replication.

Example 10: Purification of rabbit antibodies against recombinant fusion proteins HR121 and HR212 of the novel coronavirus and determination of their activity against SARS CoV-2 virus replication.

10.1 Purification of rabbit antibodies against recombinant HR121 and HR212 proteins of novel coronavirus: Rabbit antiserum against HR121 and HR212 proteins was purified using Protein A antibody purification kit to obtain polyclonal rabbit IgG antibodies against HR121 and HR212.

10.2 Determination of inhibitory activity of rabbit anti-HR121 and anti-HR212 antibodies against SARS-CoV-2 virus replication: The detection method is the same as that in Example 9, except that the test sample “serially diluted rabbit antiserum against HR121 or HR212” was replaced by “serially diluted rabbit anti-HR121 or anti-HR212 antibodies”.

The results of the analysis are shown in Figure 8, and the _EC50 value of rabbit antibodies against the HR121 protein for neutralizing the SARS-CoV-2 virus is 1.52 μg/mL. The _EC50 value of rabbit antibodies against the HR212 protein for neutralizing the SARS-CoV-2 virus is 39.22 μg/mL. This indicates that antibodies to these two proteins exhibit good activity against SARS-CoV-2 virus replication.

Example 11: Protective effect of recombinant HR121 protein of novel coronavirus as a vaccine in golden hamsters infected with SARS CoV-2 virus

(1) Protective effect of recombinant HR121 protein of the novel coronavirus as a vaccine in golden hamsters infected with the original SARS-CoV-2 virus strain

11.1. Eight-week-old golden hamsters were immunized with the recombinant HR121 protein of the novel coronavirus. Primary immunization: the recombinant HR121 protein obtained in Example 3 was diluted in PBS to achieve a protein concentration of 100 μg/ml. The same volume of Freund's complete adjuvant was added and mixed with the protein solution to form a white latex. Each hamster was immunized with 0.3 ml by subcutaneous administration at several points (the protein content was 15 μg/animal). Booster immunization: two booster immunizations were performed on days 14 and 28. The recombinant HR121 protein obtained in Example 3 was diluted in PBS to achieve a protein concentration of 100 μg/ml. The same volume of incomplete Freund's adjuvant was added and mixed with the protein solution to form a white latex. Each rabbit was immunized with 0.3 ml by subcutaneous injection at several points (protein content was 15 μg/animal). A negative control group without protein and a negative control group without adjuvant were also created.

11.2. Protective effect on hamsters: Hamsters were infected with SARS-CoV-2 virus at a dose of TCID50 = 10 ⁴ on day 42 and euthanized 3 days after infection. Lung tissue was extracted with Trizol reagent. The content of viral genomic RNA (gRNA) in the supernatant was determined by quantitative PCR. Primers for qPCR were:

SARS-CoV-2-NF: 5'-GGGAACTTTCCTGCTAGAAT-3' (SEQ ID No. 17);

SARS-CoV-2-NR: 5'-CAGACATTTGCTCAAGCTG-3' (SEQ ID No. 18);

Probe for SARS-CoV-2 N gene: FAM-TTGCTGCTGCTTGAGATT-TAMRA-3' (SEQ ID No. 19).

The protective effect of HR121 against SARS-CoV-2-infected hamsters was calculated based on the viral RNA content.

As shown in Figure 9, immunization with HR121 protein can protect golden hamsters from SARS-CoV-2 infection, thus it is a potential protein vaccine to prevent SARS-CoV-2 novel coronavirus infection.

(2) Protective effect of the recombinant HR121 protein of the novel coronavirus as a vaccine in golden hamsters infected with the mutant Omicron BA.2 strain of the SARS-CoV-2 virus

11.3. Eight-week-old golden hamsters were immunized with the recombinant HR121 protein of the novel coronavirus. Primary immunization: the recombinant HR121 protein obtained in Example 3 was diluted in PBS solution to achieve a protein concentration of 100 μg/mL. The same volume of Freund's complete adjuvant or aluminum hydroxide adjuvant was added and mixed with the protein solution to form a white latex, and each hamster was immunized with 0.3 ml (protein content 15 μg/animal) by subcutaneous administration at several sites (Freund's adjuvant group) or by intramuscular injection (aluminum hydroxide adjuvant group). Booster immunization: A double booster immunization was performed on days 14 and 28. The recombinant HR121 protein obtained in Example 3 was diluted with PBS solution to achieve a protein concentration of 100 μg/mL. The same volume of incomplete Freund's adjuvant or aluminum hydroxide adjuvant was added and mixed with the protein solution to form a white latex, and each rabbit was immunized with 0.3 ml (protein content 15 μg/animal) by subcutaneous administration at several sites (Freund's adjuvant group) or by intramuscular injection (aluminum hydroxide adjuvant group). A negative control group that did not receive protein was also created.

11.4 Protective effect on hamsters: Hamsters were infected with SARS-CoV-2 virus at a dose of TCID50 = 10 ⁴ on day 42 and euthanized 3 days after infection. Lung tissue was extracted with Trizol reagent. The viral RNA content in the supernatant was determined by quantitative PCR. Primers for quantitative PCR of viral genomic RNA (gRNA) were:

SARS-CoV-2-NF: 5'-GGGAACTTTCCTGCTAGAAT-3' (SEQ ID No. 17);

SARS-CoV-2-NR: 5'-CAGACATTTGCTCAAGCTG-3' (SEQ ID No. 18);

Probe for SARS-CoV-2 N gene: 5'-FAM-TTGCTGCTTGACAGATT-TAMRA-3' (SEQ ID no. 19).

Viral subgenomic RNA (representing a replicating virus, sgRNA). Primers for quantitative PCR are:

SARS-CoV-2-EF: 5'-CGATCTTTGTAGATTGTTCTC-3' (SEQ ID no. 20);

SARS-CoV-2-ER: 5'-ATATTGCAGCAGTACGCACACA-3' (SEQ ID No. 21);

SARS-CoV-2-E probe: 5'-FAM-CGAAGCGCAGTAAGGATGGCTAGTGT-TAMRA-3' (SEQ ID no. 22).

The protective effect of HR121 against SARS-CoV-2-infected hamsters was calculated based on the content of gRNA and sgRNA.

The results of the analysis and detection are shown in Fig. 14. After immunization of golden hamsters with HR121 protein, it can effectively protect golden hamsters from infection with the mutant strain of SARS CoV-2 Omicron BA.2, thus it is a potential protein vaccine to prevent infection with the mutant strain of the novel coronavirus SARS CoV-2.

Example 12: Construction and purification of expression vectors for HR121 and HR212 protein variants with partial deletion of amino acid sequences in the HR1 and HR2 regions

Modification of the amino acid sequence of HR1 or HR2: 3 other HR121 proteins with missing amino acids: HR121-1, HR121-2, HR121-3 and 3 other HR212 proteins: HR212-1, HR212-2, HR212-3. Expression and purification method were the same as HR121 and HR212 in Example 2.

Based on the amino acid sequence of HR1 (SEQ ID NO: 1) and the amino acid sequence of HR2 (SEQ ID NO: 2), partial deletions of the amino acid sequences of the HR1 and HR2 regions were performed, resulting in HR1- , HR1- and HR1- , respectively. Amino acid sequences of HR1- , HR1- and HR1- are presented in SEQ ID No. 11-13, and the amino acid sequences of HR2- , HR2- and HR2- are presented in SEQ ID Nos. 14-16. A comparison of the amino acid sequences with deletions is shown in Figure 10.

Based on these HR1 and HR2 sequences, three other HR121 proteins and three HR212 proteins were constructed. They were named HR121-1, HR121-2, HR121-3 and HR212-1, HR212-2, HR212-3, respectively. Among them, the amino acid sequence of HR121-1 is formed as follows: HR1- Linker1-HR2- Linker2-HR1- ; the amino acid sequence of HR121-2 is formed as follows: HR1- Linker1-HR2- Linker2-HR1- ; the amino acid sequence of HR121-3 is formed as follows: HR1- Linker1-HR2- Linker2-HR1- The amino acid sequence of HR212-1 is formed as follows: HR2 - Linker2-HR1 - Linker1-HR2 ; the amino acid sequence of HR212-2 is formed as follows: HR2- - Linker1-HR1- - Linker2-HR2- ; the amino acid sequence of HR212-3 is formed as follows: HR2- - Linker1-HR1- - Linker2-HR2- .

After the nucleotide sequences encoding them were linked to the pET-30a vector, the proteins were purified according to the method described in Examples 1-3, and the results of SDS-PAGE of the purified proteins are shown in Fig. 11. Among them, the size of the HR121-1 protein is about 18 kDa, the size of the HR121-2 protein is about 19 kDa, and the size of the HR121-3 protein is about 19 kDa. The size of the HR212-1 protein is about 18 kDa; the size of the HR212-2 protein is about 19 kDa; and the size of the HR212-3 protein is about 19 kDa.

Example 13: Variants of the HR121 and HR212 proteins with partial deletion of amino acid sequences in the HR1 and HR2 regions also have antiviral activity.

According to the method described in Example 5, the activity of three HR121 proteins and three HR212 proteins against the new coronavirus was studied, and it was found that other HR121 and HR212 proteins with a partial deletion of the amino acid sequence in the HR1 and HR2 regions also have antiviral activity. The results are shown in Fig. 12, where the EC ₅₀ value of HR121-1 activity against the new coronavirus is 0.672 μmol/L, the EC50 value of HR121-2 is 0.614 μmol/L, and the EC50 value of HR121-3 is <1 μmol/L. The EC ₅₀ value of HR212-1 activity against the new coronavirus is 0.157 μmol/L. The EC ₅₀ value of HR212-2 is 0.128 μmol/L. The EC ₅₀ value of HR212-3 is 0.736 μmol/L. The other three recombinant HR121 proteins and three recombinant HR212 proteins with deletions of different amino acid sequences in the HR1 and HR2 regions of the novel coronavirus also have inhibitory activity against the novel coronavirus.

The above results confirm that three other recombinant HR121 proteins and three recombinant HR212 proteins, with different amino acid sequences resulting from deletions in the HR1 and HR2 regions of the novel coronavirus, can also mimic the intermediate state of membrane fusion of the novel coronavirus and inhibit the replication of the novel coronavirus. Furthermore, they can act as immunogens, inducing the production of strong neutralizing antibodies and exhibiting good immunogenicity.

The recombinant HR121 or HR212 protein of the novel coronavirus obtained in the present invention is derived from the HR region of the S2 protein of the novel coronavirus and can mimic the intermediate conformation of membrane fusion of the novel coronavirus. These two proteins act during the membrane fusion step of the novel coronavirus and exhibit specific activity against the novel coronavirus, which is expected to be used to develop new broad-spectrum reagents for detecting the novel coronavirus and prophylactic or therapeutic agents.

The recombinant HR121 or HR212 protein of the novel coronavirus as an immunogen can induce the formation of high-affinity neutralizing antibodies against the novel coronavirus in rabbits. After immunization of golden hamsters with the recombinant HR121 protein of the novel coronavirus, the golden hamsters can be significantly protected from the novel coronavirus infection. The antibody activity and the protective effect of animals are comparable to those of antibodies induced by a vaccine based on the RBD of the S1 protein of the novel coronavirus (Nature. 2020 Oct; 586(7830):572-577. doi: 10.1038/s41586-020-2599-8). Thus, the recombinant HR121 or HR212 protein of the novel coronavirus SARS-CoV-2 obtained in the present invention can potentially be used to develop a new broad-spectrum vaccine based on the proteins of the novel coronavirus.

In conclusion, it is expected that the recombinant protein HR121 or HR212 of the novel coronavirus in some examples of the invention will be used as a raw material for the production of new reagents for detecting coronavirus, drugs, vaccines and antibodies.

The sequences according to the present invention were as follows:

SEQ ID No. 1 Amino acid sequence of HR1

TQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVE

SEQ ID No. 2 Amino acid sequence of HR2

DVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKY

SEQ ID No. 3 linker 1 GGSGG

SEQ ID No. 4 linker 2 SGGRGG

SEQ ID No. 5 Amino acid sequence of HR121

TQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEGGSGGDVDLGDISGINASVVNIQKEID RLNEVAKNLNESLIDLQELGKYSGGRGGTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVE

SEQ ID No. 6 Amino acid sequence of HR212

DVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYSGGRGGTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEGGSGGDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKY

SEQ ID No. 7 Nucleotide sequence encoding HR121

Acacagaatgttctctatgagaaccaaaaattgattgccaaccaatttaatagtgctattggcaaaattcaagactcactttcttccacagcaagtgcacttggaaaacttcaagatgtggtcaaccaaaatgcacaagctttaaacacgcttgtta aacaacttagctccaattttggtgcaatttcaagtgttttaaatgatatcctttcacgtcttgacaaagttgagggaggaagcggaggagatgttgatttaggtgacatctctggcattaatgcttcagttgtaaacattcaaaaagaaattgaccgc ctcaatgaggttgccaagaatttaaatgaatctctcatcgatctccaagaacttggaaagtatagcggaggaagaggaggaacacagaatgttctctatgagaaccaaaaattgattgccaaccaatttaatagtgctattggcaaaattcaagact cactttcttccacagcaagtgcacttggaaaacttcaagatgtggtcaaccaaaatgcacaagctttaaacacgcttgttaaacaacttagctccaattttggtgcaatttcaagtgttttaaatgatatcctttcacgtcttgacaaagttgagtaa

SEQ ID No. 8 Nucleotide sequence encoding HR212

Gatgttgatttaggtgacatctctggcattaatgcttcagttgtaaacattcaaaaagaaattgaccgcctcaatgaggttgccaagaatttaaatgaatctctcatcgatctccaagaacttggaaagtat agcggaggaagaggaggaacacagaatgttctctatgagaaccaaaaattgattgccaaccaatttaatagtgctattggcaaaattcaagactcactttcttccacagcaagtgcacttggaaaacttcaag atgtggtcaaccaaaatgcacaagctttaaacacgcttgttaaacaacttagctccaattttggtgcaatttcaagtgttttaaatgatatcctttcacgtcttgacaaagttgagggaggaagcggaggaga tgttgatttaggtgacatctctggcattaatgcttcagttgtaaacattcaaaaagaaattgaccgcctcaatgaggttgccaagaatttaaatgaatctctcatcgatctccaagaacttggaaagtattaa

SEQ ID No. 9 Nucleotide sequence encoding HR1

Acacagaatgttctctatgagaaccaaaaattgattgccaaccaatttaatagtgctattggcaaaattcaagactcactttcttccacagcaagtgcacttggaaaacttcaag atgtggtcaaccaaaatgcacaagctttaaacacgcttgttaaacaacttagctccaattttggtgcaatttcaagtgttttaaatgatatcctttcacgtcttgacaaagttgag

SEQ ID No. 10 Nucleotide sequence encoding HR2

Gatgttgatttaggtgacatctggcattaatgcttcagttgtaaacattcaaaaagaaattgaccgcctcaatgaggttgccaagaatttaaatgaatctcatcgatctccaagaacttggaaagtat

SEQ ID No. 11 Amino acid sequence of HR1-

QKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQN

SEQ ID No. 12 Amino acid sequence of HR1-

KLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

SEQ ID No. 13 Amino acid sequence of HR1-

ANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLV

SEQ ID No. 14 Amino acid sequence of HR2-

GINASVVNIQKEIDRLNEVAKNLNESLIDLQELG

SEQ ID No. 15 Amino acid sequence of HR2-

VDLGDISGINASVVNIQKEIDRLNEVAKNLNESLID

SEQ ID No. 16 Amino acid sequence of HR2-

DISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL

SEQ ID No. 17 GGGGAACTTCTCCTGCTAGAAT SARS-CoV-2-NF

SEQ ID No. 18 SARS-CoV-2-NR CAGACATTTTGCTCTCAAGCTG

SEQ ID No. 19 Probe for the N-gene of SARS-CoV-2 TTGCTGCTGCTTGACAGATT

SEQ ID No. 20 SARS-CoV-2-EF CGATCTCTTGTAGATCTGTTCTC

SEQ ID No. 21 SARS-CoV-2-ER ATATTGCAGCAGTACGCACACA

SEQ ID No. 22 Probe for the E-gene of SARS-CoV-2

CGAAGCGCAGTAAGGATGGCTAGTGT

SEQ ID No. 23: mRNA sequence encoding HR121

AUGACACAGAAUGUUCUCUAUGAGAACCAAAAAUUGAUUGCCAACCAAUUUAAUAGUGCUAUUGGCAAAAUUCAAGACUCACUUUCUUCCACAGCAAGUGCACUUGGAAAACUUCAAGAUGUGGUCAACCAAAAUGCACAAGCUUUUAAACACGCUUGU UAAACAACUUAGCUCCAAUUUUGGUGCAAUUUCAAGUGUUUUAAAUGAUAUCCUUUCACGUCUUGACAAAGUUGAGGGAGGAAGCGGAGGAGAUGUUGAUUUAGGUGACAUCUCUGGCAUUAAUGCUUCAGUUGUAAACAUUCAAAAAAGAAAUUGACC GCCUCAAUGAGGUUGCCAAGAAUUUAAAUGAAUCUCUCAUCGAUCUCCAAGAACUUGGAAAGUAUAGCGGAGGAAGAGGAGGAACACAGAAUGUUCUCUAUGAGAACCAAAAAUUGAUUGCCAACCAAUUUAAUAGUGCUAUUGGCAAAAUUCAAGAC UCACUUUCUUCCACAGCAAGUGCACUUGGAAAACUUCAAGAUGUGGUCAACCAAAAUGCACAAGCUUUAAACACGCUUGUUAAACAACUUAGCUCCAAUUUUGGUGCAAUUUCAAGUGUUUUAAAUGAUAUCCUUUCACGUCUUGACAAAGUUGAGUAA

SEQ ID No. 24: mRNA sequence encoding HR121-1

AUGCAAAAAUUGAUUGCCAACCAAUUUAAUAGUGCUAUUGGCAAAAUUCAAGACUCACUUUCUUCCACAGCAAGUGCACUUGGAAAACUUCAAGAUGUGGUCAACCAAAAUGGAGGAAGCGGAGGAGGCAUUAAUGCUUCAGUUGUAAACAUUCAAAAAGAAAUUGACCGCCUCAAUG AGGUUGCCAAGAAUUUAAAUGAAUCUCUCAUCGAUCUCCAAGAACUUGGAAGCGGAGGAAGAGGAGGACAAAAAUUGAUUGCCAACCAAUUUAAUAGUGCUAUUGGCAAAAUUCAAGACUCACUUUCUUCCACAGCAAGUGCACUUGGAAAACUUCAAGAUGUGGUCAACCAAAAUUAA

SEQ ID No. 25: mRNA sequence encoding HR121-2

AUGAAAUUGAUUGCCAACCAAUUUAAUAGUGCUAUUGGCAAAAUUCAAGACUCACUUUCUUCCACAGCAAGUGCACUUGGAAAACUUCAAGAUGUGGUCAACCAAAAUGCACAAGCUUUAAACACGCUUGGAGGAAGCGGAGGAGUUGAUUUAGGUGACAUCUCUGGCAUUAAUGCUUCAGUUGUAAACAUUCAAAAAAAG AAAUUGACCGCCUCAAUGAGGUUGCCAAGAAUUUAAAUGAAUCUCUCAUCGAUAGCGGAGGAAGAGGAGGAAAAUUGAUUGCCAACCAAUUUAAUAGUGCUAUUGGCAAAAUUCAAGACUCACUUUCUUCCACAGCAAGUGCACUUGGAAAACUUCAAGAUGUGGUCAACCAAAAUGCACAAGCUUUAAACACGCUUUAA

SEQ ID No. 26: mRNA sequence encoding HR121-3

AUGGCCAACCAAUUUAAUAGUGCUAUUGGCAAAAUUCAAGACUCACUUUCUUCCACAGCAAGUGCACUUGGAAAACUUCAAGAUGUGGUCAACCAAAAUGCACAAGCUUUUAAACACGCUUGUUAAACAAGGAGGAAGCGGAGGAGACAUCUCUGGCAUUAAUGCUUCAGUUGUAAACAUUCAAAAAAGAAAUUGACCGCC UCAAUGAGGUUGCCAAGAAUUUAAAUGAAUCUCUCAUCGAUCUCCAAGAACUUAGCGGAGGAAGAGGAGGAGCCAACCAAUUUAAUAGUGCUAUUGGCAAAAUUCAAGACUCACUUUCUUCCACAGCAAGUGCACUUGGAAAACUUCAAGAUGUGGUCAACCAAAAUGCACAAGCUUUUAAACACGCUUGUUAAACAAUAA

SEQ ID No. 27: mRNA sequence encoding HR212

AUGGAUGUUGAUUUAGGUGACAUCUCUGGCAUUAAUGCUUCAGUUGUAAACAUUCAAAAAAGAAAUUGACCGCCUCAAUGAGGUUGCCAAGAUUUAAAUGAAUCUCUCAUCGAUCUCCAAGAACUUGGAAAGU AUAGCGGAGGAAGAGGAGGAACACAGAAUGUUCUCUAUGAGAACCAAAAAUUGAUUGCCAACCAAUUUAAUAGUGCUAUUGGCAAAAUUCAAGACUCACUUUCUUCCACAGCAAGUGCACUUGGAAAACUUCAA GAUGUGGUCAACCAAAAUGCACAAGCUUUAAACACGCUUGUUAAACAACUUAGCUCCAAUUUUGGUGCAAUUUCAAGUGUUUUAAAUGAUAUCCUUUCACGUCUUGACAAAGUUGAGGGAGGAAGCGGAGGAG AUGUUGAUUUAGGUGACAUCUCUGGCAUUAAUGCUUCAGUUGUAAACAUUCAAAAAGAAAUUGACCGCCUCAAUGAGGUUGCCAAGAUUUAAAUGAAUCUCUCAUCGAUCUCCAAGAACUUGGAAAGUAUUAA

SEQ ID No. 28: mRNA sequence encoding HR212-1

AUGGGCAUUAAUGCUUCAGUUGUAAACAUUCAAAAAAGAAAUUGACCGCCUCAAUGAGGUUGCCAAGAAUUUAAAUGAAUCUCUCAUCGAUCUCCAAGAACUUGGAAGCGGAGGAAGAGGAGGACAAAAAUUGAUUGCCAACCAAUUUAAUAGUGCUAUUGGCAAAAUUCAAGACU CACUUUCUUCCACAGCAAGUGCACUUGGAAAACUUCAAGAUGUGGUCAACCAAAAUGGAGGAAGCGGAGGAGGCAUUAAUGCUUCAGUUGUAAACAUUCAAAAAAGAAAUUGACCGCCUCAAUGAGGUUGCCAAGAAUUUAAAUGAAUCUCUCAUCGAUCUCCAAGAACUUGGAUAA

SEQ ID No. 29: mRNA sequence encoding HR212-2

AUGGUUGAUUUAGGUGACAUCUCUGGCAUUAAUGCUUCAGUUGUAAACAUUCAAAAAAGAAAUUGACCGCCUCAAUGAGGUUGCCAAGAAUUUAAAUGAAUCUCUCAUCGAUAGCGGAGGAAGAGGAGGAAAAUUGAUUGCCAACCAAUUUAAUAGUGCUAUUGGCAAAAUUCAAGACUCACUUUCUUCCA CAGCAAGUGCACUUGGAAAACUUCAAGAUGUGGUCAACCAAAAUGCACAAGCUUUAAACACGCUUGGAGGAAGCGGAGGAGUUGAUUUAGGUGACAUCUCUGGCAUUAAUGCUUCAGUUGUAAACAUUCAAAAAAGAAAUUGACCGCCUCAAUGAGGUUGCCAAGAAUUUAAAUGAAUCUCUCAUCGAUUAA

SEQ ID No. 30: mRNA sequence encoding HR212-3

AUGGACAUCUCUGGCAUUAAUGCUUCAGUUGUAAACAUUCAAAAAAGAAAUUGACCGCCUCAAUGAGGUUGCCAAGAAUUUAAAUGAAUCUCUCAUCGAUCUCCAAGAACUUAGCGGAGGAAGAGGAGGAGCCAACCAAUUUAAUAGUGCUAUUGGCAAAAUUCAAGACUCACUUUCUUCCACAGCAAGUG CACUUGGAAAACUUCAAGAUGUGGUCAACCAAAAUGCACAAGCUUUAAACACGCUUGUUAAACAAGGAGGAAGCGGAGGAGACAUCUCUGGCAUUAAUGCUUCAGUUGUAAACAUUCAAAAAAGAAAUUGACCGCCUCAAUGAGGUUGCCAAGAAUUUAAAUGAAUCUCUCAUCGAUCUCCAAGAACUUUAA

Those skilled in the art will readily understand that the foregoing is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modification, equivalent substitution, and improvement made in accordance with the spirit and principle of the present invention are within the scope of protection of the present invention. For example, the deletion or modification of the amino acid of the HR1 or HR2 peptide segment in the above embodiment, the replacement of the host cell with a yeast cell, an Escherichia coli cell, or a mammalian cell, etc., so that the appropriate carrier backbone is selected in accordance with the host cell, the expression of the recombinant protein of the new coronavirus HR121 or HR212 similar to the above experimental examples, and the use of the recombinant protein of the new coronavirus HR121 or HR212 as a vaccine or the production of antibodies thereto are within the scope of protection of the invention.

--->

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<ApplicantNameLatin>ETERNIVAX BIOMEDICAL, INC</ApplicantNameLatin>

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</InventionTitle>

<InventionTitle languageCode="en">Recombinant fusion protein derived

from HR region of S2 protein of SARS-COV-2 and application of

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<INSDSeq_sequence>SGGRGG</INSDSeq_sequence>

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<INSDSeq_sequence>TQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALN

TLVKQLSSNFGAISSVLNDILSRLDKVEGGSGGDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLID

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atagtgctattggcaaaattcaagactcactttcttccacagcaagtgcacttggaaaacttcaagatgt

ggtcaaccaaaatgcacaagctttaaacacgcttgttaaacaacttagctccaattttggtgcaatttca

agtgttttaaatgatatcctttcacgtcttgacaaagttgagggaggaagcggaggagatgttgatttag

gtgacatctctggcattaatgcttcagttgtaaacattcaaaaagaaattgaccgcctcaatgaggttgc

caagaatttaaatgaatctctcatcgatctccaagaacttggaaagtatagcggaggaagaggaggaaca

cagaatgttctctatgagaaccaaaaattgattgccaaccaatttaatagtgctattggcaaaattcaag

actcactttcttccacagcaagtgcacttggaaaacttcaagatgtggtcaaccaaaatgcacaagcttt

aaacacgcttgttaaacaacttagctccaattttggtgcaatttcaagtgttttaaatgatatcctttca

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ttcaaaaagaaattgaccgcctcaatgaggttgccaagaatttaaatgaatctctcatcgatctccaaga

acttggaaagtatagcggaggaagaggaggaacacagaatgttctctatgagaaccaaaaattgattgcc

aaccaatttaatagtgctattggcaaaattcaagactcactttcttccacagcaagtgcacttggaaaac

ttcaagatgtggtcaaccaaaatgcacaagctttaaacacgcttgttaaacaacttagctccaattttgg

tgcaatttcaagtgttttaaatgatatcctttcacgtcttgacaaagttgagggaggaagcggagaggagat

gttgatttaggtgacatctctggcattaatgcttcagttgtaaacattcaaaaagaaattgaccgcctca

atgaggttgccaagaatttaaatgaatctctcatcgatctccaagaacttggaaagtattaa</INSDSe

q_sequence>

</INSDSeq>

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<SequenceData sequenceIDNumber="9">

<INSDSeq>

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<INSDQualifier id="q102">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>The nucleotide sequence encoding

HR1</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q103">

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</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>acacagaatgttctctatgagaaccaaaaattgattgccaaccaattta

atagtgctattggcaaaattcaagactcactttcttccacagcaagtgcacttggaaaacttcaagatgt

ggtcaaccaaaatgcacaagctttaaacacgcttgttaaacaacttagctccaattttggtgcaatttca

agtgttttaaatgatatcctttcacgtcttgacaaagttgag</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="10">

<INSDSeq>

<INSDSeq_length>132</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..132</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q104">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>The nucleotide sequence encoding

HR2</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q105">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>gatgttgatttaggtgacatctctggcattaatgcttcagttgtaaaca

ttcaaaaagaaattgaccgcctcaatgaggttgccaagaatttaaatgaatctctcatcgatctccaaga

acttggaaagtat</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="11">

<INSDSeq>

<INSDSeq_length>36</INSDSeq_length>

<INSDSeq_moltype>AA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..36</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>protein</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q106">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>Amino acid sequence of

HR1-1</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q107">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>QKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQN</INSDSeq_seq

uence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="12">

<INSDSeq>

<INSDSeq_length>42</INSDSeq_length>

<INSDSeq_moltype>AA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..42</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>protein</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q108">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>Amino acid sequence of

HR1-2</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q109">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>KLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL</INSDS

eq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="13">

<INSDSeq>

<INSDSeq_length>40</INSDSeq_length>

<INSDSeq_moltype>AA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..40</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>protein</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q110">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>Amino acid sequence of

HR1-3</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q111">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>ANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLV</INSDSeq

_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="14">

<INSDSeq>

<INSDSeq_length>34</INSDSeq_length>

<INSDSeq_moltype>AA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..34</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>protein</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q112">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>Amino acid sequence of

HR2-1</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q113">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>GINASVVNIQKEIDRLNEVAKNLNESLIDLQELG</INSDSeq_seque

nce>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="15">

<INSDSeq>

<INSDSeq_length>36</INSDSeq_length>

<INSDSeq_moltype>AA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..36</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>protein</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q114">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>Amino acid sequence of

HR2-2</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q115">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>VDLGDISGINASVVNIQKEIDRLNEVAKNLNESLID</INSDSeq_seq

uence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="16">

<INSDSeq>

<INSDSeq_length>36</INSDSeq_length>

<INSDSeq_moltype>AA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..36</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>protein</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q116">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>Amino acid sequence of

HR2-3</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q117">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>DISGINASVNIQKEIDRLNEVAKNLNESLIDLQEL</INSDSeq_seq

uence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="17">

<INSDSeq>

<INSDSeq_length>22</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..22</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q118">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>SARS-CoV-2-NF</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q119">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>ggggaacttctcctgctagaat</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="18">

<INSDSeq>

<INSDSeq_length>22</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..22</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q120">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>SARS-CoV-2-NR</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q121">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>cagacattttgctctcaagctg</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="19">

<INSDSeq>

<INSDSeq_length>20</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..20</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q122">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>SARS-CoV-2-N-probe</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q123">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>ttgctgctgcttgacagatt</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="20">

<INSDSeq>

<INSDSeq_length>23</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..23</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q124">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>SARS-CoV-2-EF</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q125">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>cgatctcttgtagatctgttctc</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="21">

<INSDSeq>

<INSDSeq_length>22</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..22</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q126">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>SARS-CoV-2-ER</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q127">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>atattgcagcagtacgcacaca</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="22">

<INSDSeq>

<INSDSeq_length>26</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..26</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q128">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>SARS-CoV-2-E-probe</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q129">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>cgaagcgcagtaaggatggctagtgt</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="23">

<INSDSeq>

<INSDSeq_length>633</INSDSeq_length>

<INSDSeq_moltype>RNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..633</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other RNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q130">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>The mRNA sequence encoding

HR121</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q131">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>atgacacagaatgttctctatgagaaccaaaaattgattgccaaccaat

ttaatagtgctattggcaaaattcaagactcactttcttccacagcaagtgcacttggaaaacttcaaga

tgtggtcaaccaaaatgcacaagctttaaacacgcttgttaaacaacttagctccaattttggtgcaatt

tcaagtgttttaaatgatatcctttcacgtcttgacaaagttgagggaggaagcggaggagatgttgatt

taggtgacatctctggcattaatgcttcagttgtaaacattcaaaaagaaattgaccgcctcaatgaggt

tgccaagaatttaaatgaatctctcatcgatctccaagaacttggaaagtatagcggaggaagaggagga

acacagaatgttctctatgagaaccaaaaattgattgccaaccaatttaatagtgctattggcaaaattc

aagactcactttcttccacagcaagtgcacttggaaaacttcaagatgtggtcaaccaaaatgcacaagc

tttaaacacgcttgttaaacaacttagctccaattttggtgcaatttcaagtgttttaaatgatatcctt

tcacgtcttgacaaagttgagtaa</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="24">

<INSDSeq>

<INSDSeq_length>357</INSDSeq_length>

<INSDSeq_moltype>RNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..357</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other RNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q132">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>The mRNA sequence encoding

HR121-1</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q133">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>atgcaaaaattgattgccaaccaatttaatagtgctattggcaaaattc

aagactcactttcttccacagcaagtgcacttggaaaacttcaagatgtggtcaaccaaaatggaggaag

cggaggaggcattaatgcttcagttgtaaacattcaaaaagaaattgaccgcctcaatgaggttgccaag

aatttaaatgaatctctcatcgatctccaagaacttggaagcggaggaagaggaggacaaaaattgattg

ccaaccaatttaatagtgctattggcaaaattcaagactcactttcttccacagcaagtgcacttggaaa

acttcaagatgtggtcaaccaaaattaa</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="25">

<INSDSeq>

<INSDSeq_length>399</INSDSeq_length>

<INSDSeq_moltype>RNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..399</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other RNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q134">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>The mRNA sequence encoding

HR121-2</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q135">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>atgaaattgattgccaaccaatttaatagtgctattggcaaaattcaag

actcactttcttccacagcaagtgcacttggaaaacttcaagatgtggtcaaccaaaatgcacaagcttt

aaacacgcttggaggaagcggaggagttgatttaggtgacatctctggcattaatgcttcagttgtaaac

attcaaaaagaaattgaccgcctcaatgaggttgccaagaatttaaatgaatctctcatcgatagcggag

gaagaggaggaaaattgattgccaaccaatttaatagtgctattggcaaaattcaagactcactttcttc

cacagcaagtgcacttggaaaacttcaagatgtggtcaaccaaaatgcacaagctttaaacacgctttaa

</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="26">

<INSDSeq>

<INSDSeq_length>399</INSDSeq_length>

<INSDSeq_moltype>RNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..399</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other RNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q136">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>The mRNA sequence encoding

HR123-1</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q137">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>atggccaaccaatttaatagtgctattggcaaaattcaagactcacttt

cttccacagcaagtgcacttggaaaacttcaagatgtggtcaaccaaaatgcacaagctttaaacacgct

tgttaaacaaggaggaagcggaggagacatctctggcattaatgcttcagttgtaaacattcaaaaagaa

attgaccgcctcaatgaggttgccaagaatttaaatgaatctctcatcgatctccaagaacttagcggag

gaagaggagggagccaaccaatttaatagtgctattggcaaaattcaagactcactttcttccacagcaag

tgcacttggaaaacttcaagatgtggtcaaccaaaatgcacaagcttttaaacacgcttgttaaacaataa

</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="27">

<INSDSeq>

<INSDSeq_length>534</INSDSeq_length>

<INSDSeq_moltype>RNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..534</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other RNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q138">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>The mRNA sequence encoding

HR212</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q139">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>atggatgttgatttaggtgacatctctggcattaatgcttcagttgtaa

acattcaaaaagaaattgaccgcctcaatgaggttgccaagaatttaaatgaatctctcatcgatctcca

agaacttggaaagtatagcggaggaagaggaggaacacagaatgttctctatgagaaccaaaaattgatt

gccaaccaatttaatagtgctattggcaaaattcaagactcactttcttccacagcaagtgcacttggaa

aacttcaagatgtggtcaaccaaaatgcacaagctttaaacacgcttgttaaacaacttagctccaattt

tggtgcaatttcaagtgttttaaatgatatcctttcacgtcttgacaaagttgagggaggaagcggagga

gatgttgatttaggtgacatctctggcattaatgcttcagttgtaaacattcaaaaagaaattgaccgcc

tcaatgaggttgccaagaatttaaatgaatctctcatcgatctccaagaacttggaaagtattaa</INS

DSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="28">

<INSDSeq>

<INSDSeq_length>351</INSDSeq_length>

<INSDSeq_moltype>RNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..351</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other RNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q140">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>The mRNA sequence encoding

HR212-1</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q141">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>atgggcattaatgcttcagttgtaaacattcaaaaagaaattgaccgcc

tcaatgaggttgccaagaatttaaatgaatctctcatcgatctccaagaacttggaagcggaggaagagg

aggacaaaaattgattgccaaccaatttaatagtgctattggcaaaattcaagactcactttcttccaca

gcaagtgcacttggaaaacttcaagatgtggtcaaccaaaatggaggaagcggaggaggcattaatgctt

cagttgtaaacattcaaaaagaaattgaccgcctcaatgaggttgccaagaatttaaatgaatctctcat

cgatctccaagaacttggataa</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="29">

<INSDSeq>

<INSDSeq_length>381</INSDSeq_length>

<INSDSeq_moltype>RNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..381</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other RNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q142">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>The mRNA sequence encoding

HR212-2</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q143">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>atggttgatttaggtgacatctctggcattaatgcttcagttgtaaaca

ttcaaaaagaaattgaccgcctcaatgaggttgccaagaatttaaatgaatctctcatcgatagcggagg

aagaggaggaaaattgattgccaaccaatttaatagtgctattggcaaaattcaagactcactttcttcc

acagcaagtgcacttggaaaacttcaagatgtggtcaaccaaaatgcacaagctttaaacacgcttggag

gaagcggagggagttgatttaggtgacatctctggcattaatgcttcagttgtaaacattcaaaaagaaat

tgaccgcctcaatgaggttgccaagaatttaaatgaatctctcatcgattaa</INSDSeq_sequence

>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="30">

<INSDSeq>

<INSDSeq_length>381</INSDSeq_length>

<INSDSeq_moltype>RNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..381</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other RNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q144">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>The mRNA sequence encoding

HR212-3</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q145">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>atggacatctctggcattaatgcttcagttgtaaacattcaaaaagaaa

ttgaccgcctcaatgaggttgccaagaatttaaatgaatctctcatcgatctccaagaacttagcggagg

aagaggagggaccaccaatttaatagtgctattggcaaaattcaagactcactttcttccacagcaagt

gcacttggaaaacttcaagatgtggtcaaccaaaatgcacaagctttaaacacgcttgttaaacaaggag

gaagcggaggagacatctctggcattaatgcttcagttgtaaacattcaaaaagaaattgaccgcctcaa

tgaggttgccaagaatttaaatgaatctctcatcgatctccaagaactttaa</INSDSeq_sequence

>

</INSDSeq>

</SequenceData>

</ST26SequenceListing>

<---

Claims (15)

1. A recombinant fusion protein from the HR region of the S2 protein of SARS-CoV-2, characterized in that the recombinant fusion protein of SARS-CoV-2 consists of a truncated HR1 protein obtained from the HR1 region of the S2 protein of SARS-CoV-2, a truncated HR2 protein obtained from the 1H2 region, and a binding peptide conjugating them; the protein structure of the recombinant SARS-CoV-2 fusion protein is HR1-linker1-HR2-linker2-HR1 or HR2-linker2-HR1-linker1-HR2; wherein the amino acid sequence of HR1 is as shown in SEQ ID No. 1, 11, 12 or 13; the amino acid sequence of HR2 is as shown in SEQ ID NO: 2, 14, 15 or 16; the amino acid sequences of linker 1 and linker 2 are shown in SEQ ID NOs 3-4, respectively, and The recombinant protein has anti-SARS-CoV-2 activity and immunogenicity to induce an immune response against SARS-CoV-2. 2. A recombinant hybrid protein from the HR region of the S2 protein of SARS-CoV-2 according to claim 1, wherein the amino acid sequence of the recombinant hybrid protein of SARS-CoV-2 is presented in SEQ ID No. 5. 3. A recombinant hybrid protein from the HR region of the S2 protein of SARS-CoV-2 according to claim 1, wherein the amino acid sequence of the recombinant hybrid protein of SARS-CoV-2 is presented in SEQ ID No. 6. 4. A recombinant hybrid protein from the HR region of the S2 protein of SARS-CoV-2 according to claim 2, wherein the nucleotide sequence encoding the recombinant hybrid protein is presented in SEQ ID No. 7. 5. A recombinant hybrid protein from the HR region of the S2 protein of SARS-CoV-2 according to claim 3, wherein the nucleotide sequence encoding the recombinant hybrid protein is presented in SEQ ID No. 8. 6. A vector expressing a recombinant fusion protein from the HR region of the S2 protein of SARS-CoV-2, wherein the vector comprises the nucleotide sequences of SEQ ID No. 7 or SEQ ID No. 8. 7. Use of a recombinant hybrid protein from the HR region of the S2 protein of SARS-CoV-2 according to any of claims 1-5 in the manufacture of a medicinal product for the prevention or treatment of SARS-CoV-2. 8. Use of a recombinant fusion protein from the HR region of the S2 protein of SARS-CoV-2 according to any one of claims 1-5 in the manufacture of a reagent for detecting a SARS-CoV-2 antigen. 9. Use of a recombinant hybrid protein from the HR region of the S2 protein of SARS-CoV-2 according to any one of claims 1-5 as an immunogen in the manufacture of a vaccine against SARS-CoV-2. 10. Use of a recombinant fusion protein from the HR region of the S2 protein of SARS-CoV-2 according to any one of claims 1-5 as an immunogen for producing antibodies to SARS-CoV-2.