Recombinant fusion proteins derived from the HR region of the novel coronavirus S2 protein and uses thereof

Abstract

The present invention discloses a recombinant fusion protein derived from the HR region of the novel coronavirus S2 protein, and its use. This type of novel coronavirus recombinant fusion protein is a recombinant fusion protein obtained by linking two conserved amino acid sequences HR1 and HR2 related to membrane fusion of the novel coronavirus membrane protein S2 protein via a linking peptide. The recombinant fusion protein can be induced and expressed in E. coli, has high expression levels, and is easy to purify at the same time. The novel coronavirus recombinant fusion protein provided by the present invention can form and maintain a stable dimer structure, simulate the three-dimensional structure of the novel coronavirus membrane fusion intermediate state, and can be used as a detection material for detecting the novel coronavirus membrane fusion process. It has excellent anti-new coronavirus activity and excellent immunogenicity, and is expected to be widely used in the development of drugs for preventing or treating novel coronavirus proteins, as well as the development of novel coronavirus vaccines and anti-new coronavirus antibodies.

Description

[Technical field]

[0001] The present invention relates to a novel coronavirus recombinant fusion protein, in particular to a recombinant fusion protein derived from the HR region of the novel coronavirus S2 protein and uses thereof. [Background technology]

[0002] The novel coronavirus (SARS-CoV-2) is an enveloped, single-stranded, positive-sense RNA virus with a linear genome, belonging to the β-genus novel coronaviruses in the family Coronaviridae, family Coronaviridae, order Nidovirales. The novel coronavirus was discovered in 2019 and has since caused a pandemic around the world. It is a highly pathogenic pathogen that has the characteristics of human-to-human transmission during the incubation period, spreads rapidly, and can cause various symptoms in the lungs and airways of infected individuals. On February 12, 2020, the World Health Organization officially named the disease caused by SARS-CoV-2 as COVID-19.

[0003] The size of the novel coronavirus genome is 26 kb to 30 kb and contains multiple nascent reading frames that can code for four major structural proteins and 16 nonstructural proteins. Its major structural proteins are the coat spike protein (S), transmembrane protein (M) and small membrane protein (E) and nucleocapsid protein (N). Here, the coat spike protein (S) is a trimer and plays an important role in the virus invading the host cell. The coat spike protein monomer is composed of S1 protein and S2 protein. When the virus approaches the host cell, the RBD domain of the S1 protein binds to the ACE2 receptor of the host cell together, inducing the virus adsorption and entry into the cell, and at the same time, the S1 protein and the S2 protein are cleaved. The S2 protein has two important functional domains, HR1 and HR2. These two domains then bind to each other to form a six-HR helix structure of the HR1-HR2 trimer, further shortening the distance between the viral membrane and the cell membrane, promoting the mutual fusion of the viral membrane and the cell membrane, and inducing the virus to enter the cell.

[0004] At present, the development of vaccines and antibodies against novel coronavirus proteins mainly targets the RBD domain of the novel coronavirus S1 protein, and there are few studies on the HR1 and HR2 domains of the S2 protein. The HR1 and HR2 domains can mediate the fusion of the virus with the host membrane, and are one of the design targets of novel coronavirus drugs and vaccines. However, how to simulate the natural pre-fusion conformation of the HR1 and HR2 domains, inhibit the binding of HR1 and HR2, and block the fusion process of novel coronavirus with the cell membrane is currently a challenge in the design of anti-new coronavirus drugs and novel coronavirus vaccines. At present, no relevant patents or literature have been searched. The fusion protein disclosed in Patent No. CN202011346373.6 "Fusion protein, recombinant vector, recombinant dendritic cell and use thereof for transmembrane expression of novel coronavirus antigen S2" includes a CD4 signal peptide, a novel coronavirus antigen S2 protein, a Flag tag sequence and a CD4 transmembrane domain linked in sequence. Compared with the present invention, a completely different strategy is adopted. Summary of the Invention [Problem to be solved by the invention]

[0005] The object of the present invention is to provide a stable recombinant fusion protein of the HR region of novel coronavirus S2 protein and its use. [Means for solving the problem]

[0006] The technical solutions adopted in the present invention are as follows: The recombinant fusion protein HR121 or HR212 derived from the HR region of the novel coronavirus S2 protein is composed of a truncated protein HR1 derived from the HR1 region of the novel coronavirus S2 protein, a truncated protein HR2 derived from the HR2 region, and a connecting peptide that couples the two, and the protein structure of the HR121 is HR1-Linker1-HR2-linker2-HR1, and the protein structure of the HR212 is HR2-Linker2-HR1-linker1-HR2, wherein the HR1 amino acid sequence is as shown in SEQ ID NO.1 or contains a sequence in which one or more amino acids have been substituted, deleted or inserted in the amino acid sequence shown in SEQ ID NO.1, the HR2 amino acid sequence is as shown in SEQ ID NO.2 or contains a sequence in which one or more amino acids have been substituted, deleted or inserted in the amino acid sequence shown in SEQ ID NO.2, and the amino acid sequences of Linker1 and Linker2 are shown in SEQ ID NOs.3 to 4, respectively.

[0007] Furthermore, the amino acid sequence of the novel coronavirus recombinant fusion protein HR121 is as shown in SEQ ID NO.5. Furthermore, the amino acid sequence of the novel coronavirus recombinant fusion protein HR212 is as shown in SEQ ID NO.6. Furthermore, the nucleotide sequence encoding said HR121 is as set forth in SEQ ID NO.7.

[0008] Furthermore, the mRNA sequence encoding said HR121 is as shown in SEQ ID NO.23. Furthermore, the nucleotide sequence encoding said HR212 is as set forth in SEQ ID NO.8. Furthermore, the mRNA sequence encoding said HR212 is as shown in SEQ ID NO.27.

[0009] The present invention further provides vectors expressing recombinant fusion proteins derived from the HR region of novel coronavirus S2 protein, including HR121 backbone vector and HR212 backbone vector, which respectively comprise the DNA coding sequence set forth in SEQ ID NO.7, 8 and the mRNA coding sequence set forth in SEQ ID NO.23, 27. In another preferred embodiment, the vector is selected from an mRNA vaccine vector or an adenovirus vector.

[0010] The present invention further provides the use of a recombinant fusion protein derived from the HR region of the novel coronavirus S2 protein in the preparation of an antigen detection reagent and a drug for preventing or treating novel coronavirus. The present invention further provides the use of a recombinant fusion protein derived from the HR region of the novel coronavirus S2 protein as an immunogen in the preparation of novel coronavirus vaccines and antibodies.

[0011] Because they are derived from the HR region of the novel coronavirus S2 protein, the novel coronavirus recombinant proteins HR121 and HR212 provided by the present invention can simulate the three-dimensional structure of the novel coronavirus membrane fusion intermediate state and can be acted upon at the novel coronavirus membrane fusion stage. As a new target for inhibiting novel coronavirus replication, they are clearly distinguished from the currently reported recombinant RBD protein derived from the novel coronavirus S1 protein (acted upon at the novel coronavirus adsorption stage). And these two proteins can induce high-affinity neutralizing novel coronavirus antibodies in animals, and their antibody activity and animal protection effect are comparable to the antibody activity induced by the currently reported RBD protein (Nature.2020 Oct;586(7830):572-577.doi:10.1038 / s41586-020-2599-8.).

[0012] In another preferred embodiment, the fusion protein of the present invention can be administered directly or indirectly. In another preferred embodiment, the direct administration includes administration in the form of a recombinant protein vaccine, or the indirect administration includes administration in the form of a nucleic acid vaccine (DNA vaccine or RNA vaccine), a recombinant viral vector vaccine (e.g., an adenovirus vector, a poxvirus vector, an adeno-associated virus vector, a herpes simplex virus vector, a cytomegalovirus vector), or the like. Effect of the Invention

[0013] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. The novel coronavirus recombinant fusion protein provided by the present invention can form and maintain a stable dimer structure, simulate the three-dimensional structure of the novel coronavirus membrane fusion intermediate state, and can be used as a detection material for detecting the novel coronavirus membrane fusion process. 2. The novel coronavirus recombinant protein provided by the present invention has excellent anti-new coronavirus activity and is expected to be widely used in the development of protein drugs for preventing or treating the novel coronavirus. 3. The novel coronavirus recombinant protein provided by the present invention has excellent immunogenicity, induces high-affinity novel coronavirus neutralizing antibodies in animals, and protects animals susceptible to novel coronavirus infection, such as golden hamsters, from viral infection. It is expected to be widely used in the development of novel coronavirus vaccines and anti-new coronavirus antibodies. 4. The novel coronavirus neutralizing antibodies induced by the novel coronavirus recombinant proteins provided by the present invention have broad-spectrum anti-new coronavirus activity. [Brief description of the drawings]

[0014] [Figure 1]Schematic structure of the novel coronavirus recombinant fusion proteins HR121 and HR212, in which the HR121 fusion protein is linked by HR1-linker1-HR2-linker2-HR1, and the HR212 fusion protein is linked by HR2-linker2-HR1-linker1-HR2. [Diagram 2] SDS-PAGE analysis of novel coronavirus recombinant fusion proteins HR121 and HR212, where Part A of Figure 2 is the HR121 electrophoretogram. M (marker): 170kDa, 130kDa, 100kDa, 70kDa, 55kDa, 40kDa, 35kDa, 25kDa, 15kDa, 10kDa (from top to bottom), 1: HR121 (20μg), 2: HR121 (50μg), 3: HR121 (50μg), Part B of Figure 2 is the HR212 electrophoretogram. M (marker): 170kDa, 130kDa, 100kDa, 70kDa, 55kDa, 40kDa, 35kDa, 25kDa, 15kDa, 10kDa (from top to bottom), 1: HR212 (10μg), 2: HR212 (20μg), 3: HR212 (50μg). [Diagram 3] Circular dichroism spectroscopy secondary structure diagram of novel coronavirus recombinant fusion proteins HR121 and HR212, where part A of Fig. 3 is a diagram showing that HR121 can form an α-helical structure, and part B of Fig. 3 is a diagram showing that HR212 can form an α-helical structure. [Figure 4] FIG. 1 shows the anti-coronavirus activity of novel coronavirus recombinant fusion proteins HR121 and HR212. [Diagram 5] Figure 1 shows antibody titers after immunization of rabbits with novel coronavirus recombinant fusion proteins HR121 and HR212. [Figure 6] We show that rabbit antisera to the novel coronavirus recombinant proteins HR121 and HR212 can all effectively inhibit the entry of novel coronavirus pseudovirus into 293T-ACE2 cells. [Figure 7]This figure shows the anti-coronavirus activity of rabbit serum after immunization of rabbits with the novel coronavirus recombinant proteins HR121 and HR212. [Figure 8] This figure shows the anti-new coronavirus activity of rabbit antibodies after immunization of rabbits with new coronavirus recombinant proteins HR121 and HR212. [Figure 9] After immunizing hamsters with the novel coronavirus recombinant protein HR121, we show that it can effectively inhibit the lung tissue of hamsters infected with the novel coronavirus. [Figure 10] This is a sequence comparison diagram of three other HR121s and three HR212s of novel coronaviruses with different amino acid deletions in the HR1 and HR2 regions. [Figure 11] SDS-PAGE electrophoresis of three other HR121 and three HR212 recombinant proteins with different amino acid sequence deletions in the novel coronavirus HR1 and HR2 regions; 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] Three other HR121 and three HR212 recombinant proteins with different amino acid sequence deletions in the novel coronavirus HR1 and HR2 regions are also shown to have activity inhibiting the novel coronavirus. [Figure 13] This shows that rabbit antiserum to the novel coronavirus recombinant protein HR121 effectively inhibits the entry of 23 types of novel coronavirus mutant pseudoviruses into 293T-ACE2 cells. [Figure 14] We show that after immunizing hamsters with the novel coronavirus recombinant protein HR121, infection of hamster lung tissue by the novel coronavirus Omicron mutant strain BA.2 can be effectively inhibited. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] Through extensive and thorough research, the present inventors have designed recombinant proteins (such as novel coronavirus recombinant proteins HR121 and HR212) based on the HR region of the novel coronavirus S2 protein, which can simulate the three-dimensional structure of the novel coronavirus membrane fusion intermediate state and block the fusion process between the virus and the cell membrane. The fusion protein of the present invention has excellent immunogenicity, can induce high-affinity neutralizing antibodies in animals, has excellent anti-new coronavirus activity, and can be used in the development of drugs related to the novel coronavirus. Based on this, the present invention has been completed.

[0016] Preferably, the neutralizing antibodies induced by the fusion proteins of the present invention have a broad spectrum of anti-coronavirus capabilities, and at the cellular level, the induced neutralizing antibodies have a range of activities against anti-SARS-CoV-2 mutant viruses that invade cells, and protect animals susceptible to the novel coronavirus from infection with mutant viruses (e.g., Omicron BA.2). Preferably, the anti-SARS-CoV-2 mutant virus is selected from the group of pCDNA3.1-SARS-CoV-2-Spike-Δ18: 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.

[0017] term To facilitate understanding of this disclosure, certain terms are first defined. As used herein, unless otherwise specified herein, each of the following terms shall have the meaning indicated below. Other definitions are set forth throughout the invention.

[0018] The term "about" can refer to a particular value or configuration within an acceptable error range as determined by one of ordinary skill in the art, which depends in part on how the value or configuration is measured or measured. For example, as used herein, the phrase "about 100" includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0019] As used herein, the terms "containing" or "comprising" can be open, semi-closed and closed. In other words, the terms also include "consisting essentially of" or "consisting of."

[0020] As used herein, the term "effective amount" generally refers to an amount of an immunogen capable of inducing a protective immune response sufficient to induce immunity to prevent and / or ameliorate an infection or disease and / or reduce at least one symptom of an infection or disease.

[0021] As used herein, the term "protective immune response" or "protective response" refers to an immunogen-mediated immune response exhibited by a vertebrate (e.g., a human) against an infectious agent or disease that prevents or ameliorates infection or reduces at least one of the disease symptoms.

[0022] Novel coronavirus recombinant fusion protein of the present invention In the present invention, the terms "fusion protein of the present invention", "antigenic protein of the present invention", "immunogenic peptide of the present invention", "recombinant fusion protein ... and "recombinant protein of the present invention" are used interchangeably and refer to a recombinant fusion protein derived from the HR region of the novel coronavirus S2 protein in the present invention. The fusion protein of the present invention comprises an HR1 or HR2 amino acid sequence or a substitution thereof (e.g., conservative amino acid substitution), deletion or insertion of one or more amino acid sequences. It should be understood that the terms include not only one fusion protein of the present invention, but also a protein collection or a peptide collection (or a peptide combination) formed by multiple fusion proteins of the present invention.

[0023] Preferably, the fusion protein of the present invention comprises the structure shown in the following formula: (Formula I):HR1-Linker1-HR2-linker2-HR1 (Formula II):HR2-Linker2-HR1-linker1-HR2 wherein HR1 is a truncated protein of the HR1 region or a sequence in which one or more amino acids have been substituted, deleted or inserted, HR2 is a truncated protein of the HR2 region or a sequence in which one or more amino acids have been substituted, deleted or inserted, The amino acid sequences of Linker1 and Linker2 are as shown in SEQ ID NO.3 or 4.

[0024] In another preferred embodiment, the truncated protein of the HR1 region or the sequence in which one or more amino acids have been substituted, deleted or inserted is (1) an amino acid sequence as set forth in SEQ ID NO.1; (2) An amino acid sequence in which, compared to the amino acid sequence of HR1 (SEQ ID NO. 1), one or more amino acids are deleted from the N-terminus (e.g., 5 to 10 amino acids, 7 to 12 amino acids, 9 to 14 amino acids, 11 to 16 amino acids, or 13 to 18 amino acids are deleted) and / or one or more amino acids are deleted from the C segment (e.g., 10 to 15 amino acids, 14 to 19 amino acids, 18 to 23 amino acids, 20 to 25 amino acids, 22 to 27 amino acids, 24 to 29 amino acids, 28 to 31 amino acids, or 30 to 35 amino acids are deleted).

[0025] Preferably, the amino acid sequence of HR1 is (1) SEQ ID NO. 1 (HR1), (2) SEQ ID NO. 11(HR1- <1> ), (3) SEQ ID NO. 12(HR1- <2> ),or (4) SEQ ID NO. 13(HR1- <3> ).

[0026] In another preferred embodiment, the truncated protein of the HR2 region or the sequence in which one or more amino acids have been substituted, deleted or inserted is (1) an amino acid sequence as set forth in SEQ ID NO.2; (2) An amino acid sequence that, compared to the amino acid sequence of HR2 (SEQ ID NO.2), has one or more amino acids deleted from the N-terminus (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, or 12 amino acids deleted) and / or one or more amino acids deleted from the C segment (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 amino acids deleted).

[0027] Preferably, the amino acid sequence of HR2 is (1) SEQ ID NO. 2 (HR2), (2) SEQ ID NO.14(HR2- <1> ), (3) SEQ ID NO. 15(HR2- <2> ),or (4) SEQ ID NO.16(HR2- <3> ).

[0028] Preferably, the amino acid sequence of the novel coronavirus recombinant fusion protein (HR121) is as shown in SEQ ID NO.5, or the amino acid sequence of the novel coronavirus recombinant fusion protein (HR212) is as shown in SEQ ID NO.6.

[0029] Preferably, the novel coronavirus recombinant fusion protein comprises: (1)HR1- <1> -Linker1-HR2- <1> -linker2-HR1- <1> HR121-1, which was linked with (2)HR1- <2> -Linker1-HR2- <2> -linker2-HR1- <2> HR121-2, which was connected by (3)HR1- <3> -Linker1-HR2- <3> -linker2-HR1- <3> HR121-3, which was linked with (4)HR2 <1> -Linker2-HR1 <1> -linker1-HR2- <1> HR212-1, which was linked to (5)HR2- <2> -Linker1-HR1- <2> -linker2-HR2- <2> HR212-2, which was linked with (6)HR2- <3> -Linker1-HR1- <3> -linker2-HR2- <3> HR212-3 linked by .

[0030] Preferably, the conservative amino acid substitutions are made according to Table A. [Table 1]

[0031] In the present invention, the fusion protein of the present invention also includes other forms such as pharma- ceutically acceptable salts or complexes. As used herein, the term "protein assembly" refers to a protein assembly consisting of at least two of the fusion proteins of the invention or their derivative sequences (substitutions, deletions or insertions). Furthermore, the protein collection also includes antigenic peptides or proteins of other coronaviruses SARS-CoV-2 other than SEQ ID NO.:5, 6.

[0032] As used herein, the term "isolated" refers to a substance being separated from its original environment (for a naturally occurring substance, the original environment is the natural environment). For example, a protein in its natural state within a living cell is not isolated and purified, but the same protein is isolated and purified if it is separated from other substances present in the natural state.

[0033] The fusion protein of the present invention can be a recombinant or synthetic protein, preferably a synthetic protein. In the present invention, when the sequence of the fusion protein is short (eg, ≦70 aa, more preferably ≦60 aa), the relevant peptide sequence can be directly synthesized using chemical methods.

[0034] If the sequence of the fusion protein of the invention is long or if the fusion protein of the invention is provided in the form of a fusion protein, recombinant methods can also be used to obtain large amounts of the relevant peptide sequence, typically by cloning the coding sequence encoding said antigenic polypeptide or its fusion protein into a vector, transfecting cells, and then isolating the relevant antigenic polypeptide or fusion protein from the grown host cells by conventional methods.

[0035] With the sequence information provided, one skilled in the art can use available cloning techniques to generate suitable nucleic acid sequences or vectors for transduction into cells.

[0036] The vector may be a plasmid or a virus. Viral vectors have the ability to enter cells. However, non-viral vectors, such as plasmids, can be complexed with agents to facilitate uptake of the viral vector by target cells. Such agents include polycationic agents. Alternatively, delivery systems such as liposome-based delivery systems can be used. The vectors for use in the present invention are preferably suitable for in vivo or in vitro use.

[0037] The vector preferably comprises one or more regulatory sequences for directing the expression of the nucleic acid sequence in the target cell. The regulatory sequence may comprise a promoter, an enhancer, a transcription termination signal, a polyadenylation sequence, a replication origin, a nucleic acid restriction site, and a homologous recombination site operably linked to the nucleic acid sequence. The vector may further comprise a selectable marker to determine the expression of the vector in a propagation system (e.g., bacterial cells) or in a target cell.

[0038] Pharmaceutical Compositions The present invention further provides a pharmaceutical composition. The pharmaceutical composition of the present invention can be for therapeutic or prophylactic use (e.g., a vaccine, a vaccine composition or an immunogenic composition). The pharmaceutical composition of the present invention comprises an effective amount of a fusion protein or protein assembly, a nucleotide molecule, a vector and / or a host cell vaccine of the present invention, or an immune cell activated with the fusion protein of the present invention (e.g., a dendritic cell sensitized with the fusion protein of the present invention or a T cell induced with the dendritic cell), and at least one pharma- ceutically acceptable carrier, diluent or excipient.

[0039] In the present invention, these pharmaceutical compositions comprise an immunogenic antigen (including the fusion protein, peptide aggregate or derivative thereof of the present invention), usually in combination with a "pharmaceutical acceptable carrier", which includes any carrier that does not itself induce the production of antibodies harmful to the individual to whom the composition is administered. Examples of suitable carriers include, but are not limited to, proteins, lipid aggregates (e.g., oil droplets or liposomes), and the like. These carriers are well known to those skilled in the art. These carriers can also act as immune stimulants ("adjuvants").

[0040] Preferably, the carrier is a pharma- ceutically acceptable carrier. These carriers include any carrier that does not itself induce the production of antibodies harmful to the individual to whom the composition is administered. In a preferred embodiment, the pharma- ceutically acceptable carrier comprises a liquid, preferably water, saline or buffer. The carrier may further comprise auxiliary substances, preferably fillers, lubricants, glidants, wetting or emulsifying agents, pH buffering substances, and the like.

[0041] Furthermore, the pharmaceutical composition of the present invention may further contain an additional adjuvant such as a vaccine adjuvant. Representative vaccine adjuvants include (but are not limited to) inorganic adjuvants such as aluminum hydroxide and alum, synthetic double-stranded polynucleotides (double-stranded polyadenylic acid, uridylic acid), levamisole, isoprinosine, and other synthetic adjuvants, Freund's adjuvant, peanut oil emulsion adjuvant, mineral oil, vegetable oil, and other oils. 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 complexes, GM-CSF, IL-2, IL-3, IL-7, IL-11, IL-12, IL-18, IL-21, and the like.

[0042] Typically, the pharmaceutical composition (vaccine composition or immunogenic composition) can be prepared as an injectable, such as a liquid solution or suspension, or in a solid form suitable for solution or suspension in liquid vehicles prior to injection. To enhance adjuvant effect, the formulation can also be emulsified or encapsulated in liposomes.

[0043] In another preferred embodiment, the pharmaceutical composition may also include vaccine components derived from one or more pathogens selected from the group consisting of other viruses, or combinations thereof, including inactivated strains, attenuated strains, or proteins, nucleic acids, etc.

[0044] The prepared pharmaceutical compositions can be administered by conventional routes, including, but not limited to, intramuscular inoculation, intradermal inoculation, subcutaneous inoculation, intranasal instillation, aerosol inhalation, genital tract, rectal, oral administration or any combination thereof.

[0045] When using a pharmaceutical composition, a safe and effective amount of the fusion protein or peptide assembly of the present invention is administered to a human, where the safe and effective amount is usually at least about 1 μg peptide / kg body weight, and in most cases does not exceed about 8 mg peptide / kg body weight, and preferably the dose is about 1 μg to 1 mg peptide / kg body weight. Of course, the specific dose should also take into account factors such as the route of administration and the health status of the patient, all of which are within the skill of a skilled physician.

[0046] use It should be understood that the fusion protein of the present invention can be directly administered in the form of protein (e.g., recombinant protein vaccine) or indirectly administered. For example, the indirect administration includes methods conventionally used in the art. For example, it is administered to a subject in need in the form of a nucleic acid vaccine (DNA vaccine or RNA vaccine), a recombinant virus vector vaccine (e.g., adenovirus vector, poxvirus vector, adeno-associated virus vector, herpes simplex virus vector, cytomegalovirus vector), etc. Preferably, it is an adeno-associated virus vector vaccine.

[0047] In some embodiments, the vaccine is a nucleic acid vaccine (DNA or RNA vaccine), a recombinant protein subunit vaccine, a recombinant viral vector vaccine, a recombinant bacterial vector vaccine, a virus-like particle vaccine, a nanoparticle vaccine, or a cell-vectored vaccine.

[0048] Preferably, the vaccine of the present invention may be a recombinant protein vaccine, a recombinant RNA vaccine, a recombinant viral vector vaccine (e.g., an adenovirus vector, a poxvirus vector, an adeno-associated virus vector, a herpes simplex virus vector, a cytomegalovirus vector), a recombinant bacterial vector vaccine, a recombinant yeast vector vaccine, or a recombinant virus-like particle vaccine.

[0049] In some embodiments, the vaccine of the present invention is selected from a recombinant protein vaccine, a recombinant RNA vaccine, a recombinant adenoviral vector, a recombinant poxvirus vector, or a combination of one or more thereof. In some embodiments, one or more of the vaccines are used in a vaccination, such as a combination vaccination or a sequential vaccination.

[0050] In some embodiments, one or more of the vaccines are used to vaccinate in conjunction with other vaccines against a novel coronavirus, e.g., the other vaccines include a vaccine against coronavirus S or S1, e.g., the S or S1 is derived from a source including, but not limited to, SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, bat-CoV, etc.

[0051] In some embodiments, the vaccine is a recombinant protein vaccine. In some embodiments, the vaccine comprises a combination of a nucleic acid vaccine (DNA or RNA vaccine) and a recombinant human cell vector vaccine.

[0052] One aspect of the present invention provides a method for preparing a vaccine against the novel coronavirus SARS-CoV-2, the method comprising: (a) providing a fusion protein, a nucleotide molecule, a vector and / or a host cell of the invention; (b) combining the active agent provided in (a) with an immunologically or pharma- ceutically acceptable carrier.

[0053] The vaccine of the present invention is used to induce a protective immune response that enhances immunity to prevent and / or mitigate 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 of respiratory tract infections, pneumonia and its complications, or a combination thereof.

[0054] Preferably, the vaccines herein are capable of preventing, eliminating or alleviating at least one of the following symptoms in a subject: COVID-19 infection or respiratory symptoms (e.g., stuffy nose, sore throat, hoarseness), headache, cough, phlegm, fever, rales, asthma, difficulty breathing, pneumonia due to infection, severe acute respiratory syndrome, renal failure, etc.

[0055] One aspect of the present invention further provides a method for preventing or treating the novel coronavirus SARS-CoV-2, said method comprising administering to a subject in need thereof a fusion protein, a nucleotide molecule, a vector, a host cell and / or a vaccine of the present invention.

[0056] The technical solution of the present invention will be further illustrated below in conjunction with the experiments. 1. Materials and Reagents: 1. Main Ingredients Viruses: SARS-CoV-2 clinical isolates (National Microbiology Data Center of China, sequence number: NMDCN0000HUI), SARS-CoV-2 Omicron BA.2 clinical isolates (National Microbiology Data Center of China, sequence number: UB1663744906574), Novel coronavirus pseudovirus (pseudovirus obtained by co-transfecting and packaging VSVΔG-*G and pcDNA3.1-SARS-CoV-2-envΔ18 into 293FT cells); Plasmid amplification strain: E. coli DH5α competent strain, protein expression strain: E. coli BL21(DE3) competent strain (Beijing Vazyme).

[0057] 2. Main Reagents LB medium, 2xYT medium (Sigma), DMEM medium, FBS (Gibco), jetPRIME transfection reagent (polyplus-transfection, Illkirch, France) Kanamycin (Sigma-Aldrich, St Louis, USA) Endotoxin-free plasmid miniprep, Plasmid MaxPrep Kit (Tiangen, Beijing) TaKaRa BCA Protein Assay Kit (TaKaRa Bio, Beijing) CCK8 Kit (Beyotime, Shanghai) Roche viral RNA extraction kit (Roche Diagnostics, Mannheim, Germany) Renilla Luciferase kit (Promega, Madison, USA) Trizol reagent (Invitrogen, CA, USA) Protein A Packing Purification Kit (BBI life science, Shanghai) Complete and incomplete Freund's adjuvant (Sigma-Aldrich, USA) Aluminum adjuvant (AlhydrogelR adjuvant 2%, Invivogen, USA)

[0058] II. Example Example 1: Construction of novel coronavirus SARS-CoV-2 recombinant protein expression vectors 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 novel coronavirus HR121 was gene synthesized and introduced between the EcoRI (GAATTC) and XhoI (CTCGAG) restriction sites of the pET-30a vector (Shanghai Generay Biotechnology Co., Ltd.). Transform the synthesized pET-30a-SARS-CoV-2-HR121 vector into E. coli DH5α competent. After culturing in liquid LB medium containing 50 μg / mL kanamycin at 37 °C for 12 hours, extract and stockpile a small amount of plasmid.

[0059] 1.2. Construction of pET-30a-SARS-CoV-2-HR212 vector: The nucleotide sequence (SEQ ID NO.8) of the novel coronavirus HR212 was gene synthesized and introduced between the EcoRI (GAATTC) and XhoI (CTCGAG) restriction sites of the pET-30a vector (Shanghai Generay Biotechnology Co., Ltd.). The synthesized pET-30a-SARS-CoV-2-HR212 vector is transformed into E. coli DH5α competent and cultured in liquid LB medium containing 50 μg / mL kanamycin at 37 °C for 12 hours, after which a small amount of plasmid is extracted and stored. The structures of HR121 and HR212 are as shown in FIG.

[0060] Example 2. Inducible expression and purification of novel coronavirus SARS-CoV-2 recombinant proteins HR121 and HR212 2.1. Transform the plasmid pET-30a-SARS-CoV-2-HR121 or pET-30a-SARS-CoV-2-HR212 into BL21(DE3) competent cells according to conventional transformation methods; 2.2. Select the monocloning, use 2xYT medium containing 5mL of kan, place the medium at 37°C and shake the bacteria until it becomes cloudy, inoculate 100μL of the bacterial solution into 100mL of 2xYT medium, inoculate a total of 500mL, shake the bacteria at 180rpm at 37°C overnight (12 hours), 2.3. Weigh out 0.238 g of IPTG and dissolve in 1 mL of water. Add 100 µL of IPTG solution for every 100 mL. Shake at 140 rpm at 20 °C overnight (12 hours). 2.4. Collect the bacterial solution and resuspend it in 20 mM imidazole solution, and resuspend it in 5 mL of 20 mM imidazole solution for every 100 mL of bacterial solution. 2.5. Destroy with ultrasound, ultrasonic 1 second and stop 1 second, 216000 Joules, pay attention to the ice bath during ultrasonic treatment to prevent the temperature from becoming too high and destroying the protein, and ultrasonic treatment will make the cloudy bacterial liquid slightly transparent (about 10 to 20 minutes of ultrasonic treatment), 2.6. Add 10% Tritonx100 to the sonicated bacterial solution until the final concentration is 1%, and place on ice for 30 minutes. 2.7.1 Centrifuge at 2000 rpm for 30 minutes, pour the supernatant into a new centrifuge tube, repeat once, pour the supernatant into a new centrifuge tube, 2.8. Load the sample onto a nickel column (GE Ni SepharoseTM6 Fast Flow, Catalog No.: 17-5318-06), wash the column with 10 times the volume of 20 mM imidazole solution before loading onto the column, and then load onto the column after washing; 2.9. After loading the sample, wash with 20 column volumes (40 mL) of 20 mM imidazole solution to remove impurity proteins. 2. Wash with 10.5 column volumes (10 mL) of 100 mM imidazole solution to remove impurity proteins. 2.11. After washing, elute the target protein using 25 column volumes (50 mL) of 200 mM imidazole solution. 2.12. Use 15mL 10KD concentrating tubes to concentrate the target protein in batches. After loading the samples, replace the 200mM imidazole solution with PBS solution. That is, after concentrating the samples, add PBS solution to the concentrating tube, centrifuge, and replace with 200mM imidazole solution, and repeat three times to ensure that the protein dissolution solution is PBS. 2.13. Aspirate approximately 1.5 mL of concentrated protein solution into a 1.5 mL EP tube and quantify the protein concentration using a BCA kit. Divide into smaller tubes to avoid repeated freezing and thawing.

[0061] Example 3. SDS-PAGE protein electrophoresis analysis of novel coronavirus SARS-CoV-2 recombinant proteins HR121 and HR212 3.1. Quantify the purified protein with a BCA kit. 3.2. Take 50μg of HR121 and HR212 protein respectively and separate them by electrophoresis using 10% SDS-PAGE gel. After electrophoresis, stain and destain using Coomassie Brilliant Blue method. The analysis and detection results are as shown in Figure 2, the size of HR121 protein is about 28kDa, and the size of HR212 protein is about 24kDa.

[0062] Example 4. Secondary structure analysis of circular dichroism spectroscopy of novel coronavirus recombinant proteins HR121 and HR212 4.1. Construction of expression vector for novel coronavirus recombinant proteins HR1 and HR2 and purification of the proteins: The novel coronavirus HR1 nucleotide coding sequence (SEQ ID NO.9) and the novel coronavirus HR2 nucleotide coding sequence (SEQ ID NO.10) are ligated between the EcoRI (GAATTC) and XhoI (CTCGAG) restriction sites of the pET-30a vector, respectively, and the proteins are purified according to the method of Example 2 to obtain novel coronavirus recombinant proteins HR1 and HR2.

[0063] 4.2. Use a Jasco spectropolarimeter (model J-815) to detect the light absorption of 1 μM HR121, 1 μM HR121 + 1 μM HR2, 1 μM HR212, and 1 μM HR212 + 1 μM HR2, respectively, to detect the binding ability of HR121 protein to HR2 protein, and the binding ability of HR212 protein to HR1 protein.

[0064] The analysis and detection results are shown in Figure 3. HR121 protein can bind with HR2 protein to form a stable α-helical structure. HR212 protein can bind with HR1 protein to form a stable α-helical structure, so these two proteins can be used to prepare a novel coronavirus antigen detection reagent.

[0065] Example 5. Detection of the anti-coronavirus activity of novel coronavirus recombinant proteins HR121 and HR212 The CCK8 method is used to detect the antiviral activity of cells. 5.1. Seed 100 μl of 5×105 cells / mL Vero E6 cells into each well of a 96-well cell culture plate and culture overnight in a CO2 incubator at 37° C.

[0066] 5.2. Then, add 50μl of SARS-CoV-2 virus with MOI of 0.1 to each well to infect the cells, and at the same time, add 50μl of serially diluted novel coronavirus recombinant proteins HR121 and HR212 to each well. After incubating for 2 hours, replace the medium with fresh medium containing 200μl of serially diluted novel coronavirus recombinant fusion proteins HR121 and HR212.

[0067] 5.3.After culturing in a CO2 incubator at 37℃ for 72 hours, add CCK8 reagent to the culture supernatant and read the OD450 value with a microplate reader to quantitatively analyze the cytopathic effect caused by SARS-CoV-2 and the protective effect of HR121 and HR212 against cytopathic effect.

[0068] The analysis and detection results are shown in Figure 4. Figure 4(A) shows that HR121 can inhibit the Vero-E6 cell degeneration induced by the new coronavirus, and the HR121 protein has an EC 50 It was found that HR212 protects against the cytopathic effect caused by SARS-CoV-2. Figure 4(B) shows that HR212 inhibits the Vero-E6 cell degeneration induced by the novel coronavirus, and the HR212 protein has an EC 50 It can be seen that the fusion proteins protect against the cytopathic effect caused by SARS-CoV-2, indicating that these two fusion proteins have excellent anti-SARS-CoV-2 activity.

[0069] Example 6. Procedure for immunizing adult New Zealand white rabbits with novel coronavirus recombinant proteins HR121 and HR212 6.1. First immunization: Dilute the novel coronavirus recombinant protein HR121 or HR212 prepared in Example 3 with PBS solution so that the protein concentration is 200 μg / mL. Add an equal amount of complete Freund's adjuvant and mix evenly with the protein solution to form a white emulsion, and administer 1 ml to each rabbit for subcutaneous multi-point immunization (protein content is 100 μg / rabbit).

[0070] 6.2. Booster immunization: Two booster immunizations are performed on the 21st and 42nd days. The novel coronavirus recombinant protein HR121 or HR212 prepared in Example 3 is diluted with PBS solution to a protein concentration of 300 μg / mL. An equal amount of Freund's incomplete adjuvant is added and mixed evenly with the protein solution to form a white emulsion, and 1 ml is administered subcutaneously to each rabbit (protein content is 150 μg / rabbit). At the same time, a negative control immunization group without protein is set up. 6.3. On day 56, collect blood from the heart of the immunized rabbits and collect serum, which is stored in aliquots at -80°C.

[0071] Example 7. Measurement of rabbit serum antibody titers against novel coronavirus recombinant proteins HR121 and HR212 7.1.1 μg / ml HR121 or HR212 protein was dissolved in 100 μl / well of ELISA coating buffer and coated overnight at 4° C. or coated for 2 hours at 37° C. 7.2. Wash 3 times with ELISA wash buffer for 3 min each time, add 100 μl of PBS-5% nonfat dry milk to each well, seal the plate at 37° C. for 2 hours, 7.3. Wash the plate as above and add 100 μl of diluted serum to each well at 37°C / 2 hours. 7.4. Wash the plate as above, add 100 μl of 1:5000 diluted goat anti-rabbit IgG-HRP (or anti-mouse IgG-HRP) for 1 hour at 37°C, wash the plate as above, add OPD substrate reaction solution, and after 10 minutes, stop the reaction with 2M sulfuric acid. 7.5.Measure the OD490 / 630nm value using an ELISA device. 7.6. According to the ELISA reading, sample OD value > 0.12 + negative control OD value is positive.

[0072] The analysis and detection results are shown in Figure 5, and the anti-rabbit serum titers of HR121 and HR212 proteins are all between 106 and 107. These two proteins are all capable of inducing higher titers of antibodies in animals.

[0073] Example 8. Measurement of neutralizing SARS-CoV-2 pseudovirus antibody titers in rabbit antisera against novel coronavirus recombinant proteins HR121 and HR212 (1) Measurement of neutralizing SARS-CoV-2 original strain pseudovirus antibody titers in rabbit antisera against the novel coronavirus recombinant proteins HR121 and HR212 8.1. Preparation and virus titer of novel coronavirus original strain pseudovirus: According to the instruction manual of the transfection reagent jetPRIME, homogenously mix 10μg of plasmid containing pCDNA3.1-SARS-CoV-2-Spike-Δ18 with 500μL of jetPRIMEBuffer, add 20μL of jetPRIMERegent and mix, mix homogenously, leave at room temperature for 10 minutes, drop the mixture into 239T cells in a T75 culture bottle, culture at 37℃ and 5% CO2 for 12 hours, discard the culture medium, add VSVΔG-VSVG pseudovirus with MOI=1 to infect, wash with PBS three times after 6 hours of infection, replace with fresh DMEM medium and continue to culture, and obtain the supernatant containing SARS-CoV-2 pseudovirus after 24 hours and 48 hours. After centrifugation at 1000rpm / min for 10 minutes, collect the supernatant and store it in portions at -80℃. Virus titers are measured using 239T-ACE2 cells.

[0074] 8.2.Measurement of the ability of HR121 and HR212 rabbit sera to neutralize SARS-CoV-2 pseudovirus: Add 100 μl of 239T-ACE2 (2 × 105 cells / ml) to each well of a 96-well cell culture plate and culture for 12 hours, then add 50 μl of serially diluted HR121 or HR212 anti-rabbit serum solution to another 96-well cell culture plate, each dilution has three replicate wells, and add 50 μl of SARS-CoV-2 pseudovirus with MOI of 0.1 to each well. At the same time, set up a positive control well without protein and a negative control well without virus. Add the virus and protein mixture to the cells after incubation at 37 °C for 1 hour. After culturing in a CO2 incubator at 37 °C for 48 hours, use Renilla Luciferase kit to detect the fluorescence expression intensity of SARS-CoV-2 pseudovirus and the EC of HR121 or HR212 anti-rabbit serum. 50 Calculate the value.

[0075] The analysis and detection results are shown in Figure 6, which shows the EC of HR121 protein rabbit antiserum to neutralize SARS-CoV-2 pseudovirus.50 Values ​​are 11536-fold serum dilution. EC of HR212 protein rabbit antiserum for neutralizing SARS-CoV-2 pseudovirus 50 The values ​​are at 636-fold serum dilution, indicating that antibodies to these two proteins may have superior activity against the SARS-CoV-2 virus invading cells.

[0076] (2) Measurement of neutralizing pseudovirus antibody titers against various mutant strains of SARS-CoV-2 against the novel coronavirus recombinant protein HR121 in rabbit antisera 8.3. Preparation and viral titer of various mutant pseudoviruses of the novel coronavirus: Following the instruction manual of the transfection reagent jetPRIME, 23 mutant pseudoviruses of the novel coronavirus: 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, B1.526, B1.17, B1.351, B1.1.28, B1.617.2, C.37, B1.621, B1.1.529 (Omicron BA.1), Omicron BA.2, Omicron BA.3 and Omicron BA.4 were prepared using the following kit: Mix pCDNA3.1-SARS-CoV-2-Spike-Δ18 plasmid containing BA.4 / 5 with 10 μg and 500 μL of jetPRIMEBuffer, add 20 μL of jetPRIMERegent and mix. After mixing, let stand at room temperature for 10 minutes. Drop the mixture into 239T cells in a T75 culture bottle. After culturing for 12 hours at 37 ° C and 5% CO2, discard the culture medium, add VSVΔG-VSVG pseudovirus with MOI = 1 to infect, wash with PBS three times after 6 hours of infection, replace with fresh DMEM medium and continue culturing, and obtain the supernatant containing each SARS-CoV-2 pseudovirus after 24 hours and 48 hours. Centrifuge at 1000 rpm / min for 10 minutes, collect the supernatant after centrifugation, and store it in portions at -80 ° C. Measure the virus titer in 239T-ACE2 cells.

[0077] 8.4.Measurement of the ability of HR121 rabbit serum to neutralize SARS-CoV-2 pseudovirus: Add 100μl of 239T-ACE2 (2×105 cells / ml) to each well of a 96-well cell culture plate and culture for 12 hours, then add 50μl of serially diluted HR121 anti-rabbit serum solution to another 96-well cell culture plate, each dilution has three replicate wells, and add 50μl of SARS-CoV-2 pseudovirus with MOI of 0.1 to each well. At the same time, set up a positive control well without protein, and a negative control well without virus. Add the virus and protein mixture to the cells after incubation at 37℃ for 1 hour. After culturing in a CO2 incubator at 37℃ for 48 hours, detect the fluorescence expression intensity of SARS-CoV-2 pseudovirus with Renilla Luciferase kit and determine the EC of HR121 anti-rabbit serum. 50 Calculate the value.

[0078] The analysis and detection results are shown in Figure 13, which shows the EC of HR121 protein rabbit antiserum to neutralize various pseudoviruses of SARS-CoV-2. 50 The values ​​are as follows: S477N, 14663-fold serum dilution, E484K, 8824-fold serum dilution, A222V, 36164-fold serum dilution, N439K, 20174-fold serum dilution, K417N, 9025-fold serum dilution, D614G, 5986-fold serum dilution, D839Y, 12087-fold serum dilution, B.1.617, 15199-fold serum dilution, B.1.617.1, 7132-fold serum dilution, B.1.617.2.V2, 9023-fold serum dilution, B.1.429, 43339-fold serum dilution. 0x serum dilution, B.1.525, 2498x serum dilution, B.1.526, 1801x serum dilution, B.1.1.7 (Alpha), 28969x serum dilution, B.1.351 (Beta), 3462x serum dilution, B.1.1.28 (Gamma), 1899x serum dilution, B1.617.2 (Delta), 15013x serum dilution, C.37 (Lambda), 6276x serum dilution, B.1.621 (Mu), 4250x serum dilution.

[0079] In particular, activity against Omicron mutant strains is also very high: B.1.1.529 (Omicron BA.1) is 6616 times serum dilution, Omicron BA.2 is 2621 times serum dilution, Omicron BA.3 is 1983 times serum dilution, and Omicron BA.4 / 5 is 4984 times serum dilution. Antibodies to this protein show excellent activity against various mutant strains of SARS-CoV-2 that invade cells.

[0080] Example 9. Measurement of antiserum titers of rabbit serum against novel coronavirus recombinant proteins HR121 and HR212 that neutralize SARS-CoV-2 virus Add 200 μl of HPAEpiC (5 × 105 cells / ml) to each well of a 48-well cell culture plate and culture for 12 hours, discard the supernatant, add 100 μl of serially diluted HR121 or HR212 anti-rabbit serum in DMEM medium to each well, each dilution has three replicate wells, and then add 100 μl of SARS-CoV-2 virus with an MOI of 1. At the same time, set up a positive control well without protein, and a negative control well without virus. Discard the supernatant after incubating at 37 ° C for 2 hours, wash the cells three times with PBS, and add 200 μl of serially diluted HR121 or HR212 anti-rabbit serum in DMEM medium. After culturing in a CO2 incubator at 37 ° C for 48 hours, take the supernatant and extract the RNA in the culture supernatant with a Roche viral RNA extraction kit. Quantitative PCR method is used to detect the viral RNA content in the supernatant, and the primers for quantitative PCR are: SARS-CoV-2-NF:5'-GGGGAACTTCTCCTGCTAGAAT-3' (SEQ ID NO.17), SARS-CoV-2-NR:5'-CAGACATTTTGCTCTCAAGCTG-3' (SEQ ID NO.18), SARS-CoV-2-N-probe:FAM-TTGCTGCTGCTTGACAGATT-TAMRA-3' (SEQ ID NO.19).

[0081] EC of HR121 or HR212 anti-rabbit serum based on viral RNA content 50 Calculate the value. The analysis and detection results are shown in Figure 7. The EC of HR121 protein anti-rabbit serum to neutralize SARS-CoV-2 virus. 50 The EC value for neutralizing the SARS-CoV-2 virus by anti-rabbit serum against the HR212 protein is 8515 times the serum dilution value. 50 The values ​​are at serum dilution times 1308. The rabbit antisera to these two proteins show excellent anti-SARS-CoV-2 viral replication activity.

[0082] Example 10. Purification of novel coronavirus recombinant fusion protein HR121 and HR212 rabbit antibodies and determination of their inhibitory activity against SARS-CoV-2 virus replication 10.1. Purification of novel coronavirus recombinant protein HR121 and HR212 rabbit antibodies: Using a Protein A packing purification kit, the anti-rabbit serum of HR121 and HR212 proteins is passed through a column and purified to obtain HR121 and HR212 anti-rabbit IgG polyclonal antibodies.

[0083] 10.2. Measurement of the inhibitory activity of HR121 and HR212 rabbit antibodies against SARS-CoV-2 virus replication: The detection method is the same as in Example 9, except that the detection sample “serial diluted HR121 or HR212 anti-rabbit serum” is changed to “serial diluted HR121 or HR212 rabbit antibody.”

[0084] The analysis and detection results are shown in Figure 8, which shows that HR121 protein rabbit antibody neutralizes SARS-CoV-2 virus. 50 The EC value for rabbit antibody to HR212 protein neutralizes the SARS-CoV-2 virus is 1.52 μg / mL. 50 The value was 39.22 μg / mL, indicating that the antibodies against these two proteins have excellent anti-SARS-CoV-2 viral replication activity.

[0085] Example 11: Protective effect of novel coronavirus recombinant protein HR121 as a vaccine against golden hamsters infected with SARS-CoV-2 virus (1) Protective effect of the novel coronavirus recombinant protein HR121 as a vaccine against golden hamsters infected with the original strain of the SARS-CoV-2 virus 11.1. 8-week-old golden hamsters are immunized with the novel coronavirus recombinant protein HR121. First immunization: The novel coronavirus recombinant protein HR121 prepared in Example 3 is diluted with PBS solution to a protein concentration of 100 μg / mL. An equal amount of complete Freund's adjuvant is added and mixed evenly with the protein solution to form a white emulsion, and each hamster is subcutaneously immunized with 0.3 ml of the solution (protein content is 15 μg / wing). Booster immunization: Two booster immunizations are performed on the 14th and 28th days. The novel coronavirus recombinant protein HR121 prepared in Example 3 is diluted with PBS solution to a protein concentration of 100 μg / mL. An equal amount of incomplete Freund's adjuvant is added and mixed evenly with the protein solution to form a white emulsion, and each hamster is subcutaneously immunized with 0.3 ml of the solution (protein content is 15 μg / wing). At the same time, a negative control immunization group without protein and a negative control group without adjuvant are set up.

[0086] 11.2. Protective effect on hamsters: Hamsters were challenged with SARS-CoV-2 virus at a viral dose of TCID50=104 on day 42, and sacrificed 3 days after the challenge. Lung tissue was taken and lung tissue RNA was extracted with Trizol reagent. Quantitative PCR was used to detect viral genomic RNA (gRNA) content in the supernatant, and the primers for quantitative PCR were: SARS-CoV-2-NF:5'-GGGGAACTTCTCCTGCTAGAAT-3' (SEQ ID NO.17), SARS-CoV-2-NR:5'-CAGACATTTTGCTCTCAAGCTG-3' (SEQ ID NO.18), SARS-CoV-2-N-probe:FAM-TTGCTGCTGCTTGACAGATT-TAMRA-3' (SEQ ID NO.19).

[0087] Calculate the protective effect of HR121 on hamsters infected with SARS-CoV-2 virus based on viral RNA content. The analysis and detection results are shown in Figure 9. After immunizing golden hamsters, the HR121 protein can protect them from SARS-CoV-2 infection, making it an excellent candidate protein vaccine for preventing infection with the new coronavirus SARS-CoV-2.

[0088] (2) Protective effect of the novel coronavirus recombinant protein HR121 as a vaccine against golden hamsters infected with the SARS-CoV-2 virus mutant Omicron BA.2 11.3. 8-week-old golden hamsters are immunized with the novel coronavirus recombinant protein HR121. First immunization: The novel coronavirus recombinant protein HR121 prepared in Example 3 is diluted with PBS solution to a protein concentration of 100 μg / mL. An equal amount of complete Freund's adjuvant or aluminum adjuvant is added and mixed evenly with the given protein solution to form a white emulsion, and each hamster is immunized with 0.3 ml by subcutaneous injection (Freund's adjuvant group) or intramuscular injection (aluminum adjuvant group) (protein content is 15 μg / bird). Booster immunization: Two booster immunizations are performed on the 14th and 28th days. The novel coronavirus recombinant protein HR121 prepared in Example 3 is diluted with PBS solution to a protein concentration of 100 μg / mL. Add an equal amount of Freund's incomplete adjuvant or aluminum adjuvant and mix evenly with the protein solution to form a white emulsion, and immunize each hamster with 0.3 ml of the emulsion by subcutaneous injection (Freund's adjuvant group) or intramuscular injection (aluminum adjuvant group) (protein content is 15 μg / hamster). At the same time, set up a negative control immunization group that does not contain protein.

[0089] 11.4. Protective effect on hamsters: Hamsters were challenged with SARS-CoV-2 virus at a viral dose of TCID50=104 on day 42, and sacrificed 3 days after the challenge. Lung tissue was taken and lung tissue RNA was extracted with Trizol reagent. Quantitative PCR was used to detect viral RNA content in the supernatant, and the primers for quantitative PCR were: SARS-CoV-2-NF:5'-GGGGAACTTCTCCTGCTAGAAT-3' (SEQ ID NO.17), SARS-CoV-2-NR:5'-CAGACATTTTGCTCTCAAGCTG-3' (SEQ ID NO.18), SARS-CoV-2-N-probe:5'-FAM-TTGCTGCTGCTTGACAGATT-TAMRA-3' (SEQ ID NO.19).

[0090] Viral subgenomic RNA (representing the replicating virus, sgRNA). Primers for quantitative PCR were: SARS-CoV-2-EF:5'-CGATCTCTTGTAGATCTGTTCTC-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).

[0091] Calculate the protective effect of HR121 on hamsters infected with SARS-CoV-2 virus based on the viral gRNA and sgRNA content. The analysis and detection results are shown in Figure 14. After immunizing golden hamsters, the HR121 protein can effectively protect golden hamsters from infection with the SARS-CoV-2 virus mutant Omicron BA.2, making it an excellent candidate protein vaccine for preventing infection with the new coronavirus SARS-CoV-2 mutant strain.

[0092] Example 12. Construction and purification of expression vectors of other HR121 and HR212 in which the amino acid sequences of the HR1 and HR2 regions were partially deleted Modifications to HR1 or HR2 amino acid sequence: Three other HR121 proteins with partial amino acid deletion: HR121-1, HR121-2, HR121-3 and three other HR212 proteins: HR212-1, HR212-2, HR212-3. The expression and purification methods are the same as those of HR121 and HR212 in Example 2.

[0093] Based on the HR1 amino acid sequence (SEQ ID NO.1) and the HR2 amino acid sequence (SEQ ID NO.2), partial deletions were made to the amino acid sequences of the HR1 and HR2 regions, and the deleted sequences were designated HR1- <1> , HR1- <2> , HR1- <3> and the HR1- <1> , HR1- <2> , HR1- <3> The amino acid sequences of are as shown in SEQ ID NOs. 11 to 13, and HR2- <1> , HR2- <2> , HR2- <3> The amino acid sequences of these are shown in SEQ ID NOs. 14 to 16. A comparison of the deleted amino acid sequences is shown in FIG.

[0094] Based on these HR1 and HR2 sequences, three other HR121 proteins and three HR212 proteins are constructed, which are designated HR121-1, HR121-2, HR121-3, and HR212-1, HR212-2, HR212-3, respectively. Here, the amino acid sequence of HR121-1 is: <1> -Linker1-HR2- <1> -linker2-HR1- <1> The amino acid sequence of HR121-2 is <2> -Linker1-HR2- <2> -linker2-HR1- <2> The amino acid sequence of HR121-3 is <3> -Linker1-HR2- <3> -linker2-HR1- <3> The amino acid sequence of HR212-1 is <1> -Linker2-HR1 <1> -linker1-HR2 <1> The amino acid sequence of HR212-2 is <2> -Linker1-HR1- <2> -linker2-HR2- <2> The amino acid sequence of HR212-3 is <3> -Linker1-HR1- <3> -linker2-HR2- <3> are connected with

[0095] After ligating their nucleotide coding sequences into pET-30a vector, the proteins were purified according to the methods of Examples 1 to 3, and the SDS-PAGE electrophoretic patterns of the purified proteins are shown in Figure 11. Here, the size of HR121-1 protein is about 18 kDa, the size of HR121-2 protein is about 19 kDa, and the size of HR121-3 protein is about 19 kDa. The size of HR212-1 protein is about 18 kDa, the size of HR212-2 protein is about 19 kDa, and the size of HR212-3 protein is about 19 kDa.

[0096] Example 13: HR121 and HR212, which are deletions of parts of the amino acid sequences of the HR1 and HR2 regions, also have antiviral activity According to the method of Example 5, the anti-new coronavirus activity of the three HR121 and three HR212 was detected, and it was found that the other HR121 and HR212, which had a partial deletion of the amino acid sequence of the HR1 and HR2 regions, still had antiviral activity. The results are shown in Figure 12, where the EC 50 The EC value for HR121-2 was 0.672 μM. 50 The EC value for HR121-3 was 0.614 μM. 50 The EC value is <1 μM. 50 The EC value for HR212-2 was 0.157 μM. 50 The EC value for HR212-3 was 0.128 μM. 50 The value is 0.736 μM. Three other HR121 and three HR212 recombinant proteins, which have various amino acid sequences deleted from the HR1 and HR2 regions of the novel coronavirus, also have the activity of inhibiting the novel coronavirus.

[0097] The above results indicate that the other three HR121 and three HR212 recombinant proteins, which have various amino acid sequences deleted in the HR1 and HR2 regions of the new coronavirus, can also simulate the three-dimensional structure of the membrane fusion intermediate state of the new coronavirus, have the activity of inhibiting the replication of the new coronavirus, and can induce the production of strong neutralizing antibodies as immunogens, showing excellent immunogenicity.

[0098] The novel coronavirus recombinant protein HR121 or HR212 prepared by the present invention is derived from the HR region of the novel coronavirus S2 protein and can simulate the three-dimensional structure of the intermediate state of novel coronavirus membrane fusion. These two proteins are adopted in the membrane fusion stage of the novel coronavirus and have certain anti-new coronavirus activity themselves, and are expected to be used as novel broad-spectrum anti-new coronavirus detection reagent raw materials and preventive or therapeutic drugs.

[0099] The use of the novel coronavirus recombinant protein HR121 or HR212 as an immunogen can induce high-affinity neutralizing novel coronavirus antibodies in rabbits. After immunizing golden hamsters with the novel coronavirus recombinant protein HR121, the golden hamsters can be essentially protected from novel coronavirus infection. The antibody activity and animal protection effect are equivalent to the antibody activity induced by the currently reported RBD vaccine derived from the novel coronavirus S1 protein (Nature.2020 Oct;586(7830):572-577.doi:10.1038 / s41586-020-2599-8.). Therefore, the novel coronavirus SARS-CoV-2 recombinant protein HR121 or HR212 prepared by the present invention has the potential to be developed as a novel broad-spectrum novel coronavirus protein vaccine.

[0100] Regardless, in some embodiments of the present invention, the novel coronavirus recombinant protein HR121 or HR212 is expected to be used for preparing novel novel coronavirus detection reagent materials, drugs, vaccines and antibodies.

[0101] The sequence of the present invention is as follows: SEQ ID NO.1: HR1 amino acid sequence TQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVE SEQ ID NO.2: HR2 amino acid sequence DVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKY SEQ ID NO.3:Linker1 GGSGG SEQ ID NO.4:Linker2 SGGRGG

[0102] SEQ ID NO.5: Amino acid sequence of HR121 TQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEGGSGGDVDLGDISGINASVVNIQKEID RLNEVAKNLNESLIDLQELGKYSGGRGGTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVE

[0103] SEQ ID NO.6: Amino acid sequence of HR212 DVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYSGGRGGTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEGGSGGDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKY

[0104] SEQ ID NO.7: Nucleotide sequence encoding HR121 Acacagaatgttctctatgagaaccaaaaattgattgccaaccaatttaatagtgctattggcaaaattcaagactcactttcttccacagcaagtgcacttggaaaacttcaagatgtggtcaaccaaaatgcacaagctttaaacacgcttgtta aacaacttagctccaattttggtgcaatttcaagtgttttaaatgatatcctttcacgtcttgacaaagttgagggaggaagcggaggatgttgatttaggtgacatctctggcattaatgcttcagttgtaaacattcaaaaagaaattgaccgc ctcaatgaggttgccaagaatttaaatgaatctctcatcgatctccaagaacttggaaagtatagcggaggaagaggaggaacacagaatgttctctatgagaaccaaaaattgattgccaaccaatttaatagtgctattggcaaaattcaagact cactttcttccacagcaagtgcacttggaaaacttcaagatgtggtcaaccaaaatgcacaagctttaaacacgcttgttaaacaacttagctccaattttggtgcaatttcaagtgttttaaatgatatcctttcacgtcttgacaaagttgagtaa

[0105] SEQ ID NO.8: Nucleotide sequence encoding HR212 gatgttgatttaggtgacatctctggcattaatgcttcagttgtaaacattcaaaaagaaattgaccgcctcaatgaggttgccaagaatttaaatgaatctctcatcgatctccaagaacttggaaagtat agcggaggaagaggaggaacacagaatgttctctatgagaaccaaaaattgattgccaaccaatttaatagtgctattggcaaaattcaagactcactttcttccacagcaagtgcacttggaaaacttcaag atgtggtcaaccaaaatgcacaagctttaaacacgcttgttaaacaacttagctccaattttggtgcaatttcaagtgttttaaatgatatcctttcacgtcttgacaaagttgagggaggaagcggaggaga tgttgatttaggtgacatctctggcattaatgcttcagttgtaaacattcaaaaagaaattgaccgcctcaatgaggttgccaagaatttaaatgaatctctcatcgatctccaagaacttggaaagtattaa

[0106] SEQ ID NO.9: Nucleotide sequence encoding HR1 Acacagaatgttctctatgagaaccaaaaattgattgccaaccaatttaatagtgctattggcaaaattcaagactcactttcttccacagcaagtgcacttggaaaacttcaag atgtggtcaaccaaaatgcacaagctttaaacacgcttgttaaacaacttagctccaattttggtgcaatttcaagtgttttaaatgatatcctttcacgtcttgacaaagttgag

[0107] SEQ ID NO.10: Nucleotide sequence encoding HR2 Gatgttgatttaggtgacatctctggcattaatgcttcagttgtaaacattcaaaaagaaattgaccgcctcaatgaggttgccaagaatttaaatgaatctctcatcgatctccaagaacttggaaagtat

[0108] SEQ ID NO.11:HR1- <1> Amino acid sequence QKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQN SEQ ID NO.12:HR1- <2> Amino acid sequence KLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL SEQ ID NO.13:HR1- <3> Amino acid sequence ANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLV

[0109] SEQ ID NO.14:HR2- <1> Amino acid sequence GINASVVNIQKEIDRLNEVAKNLNESLIDLQELG SEQ ID NO.15:HR2- <2> Amino acid sequence VDLGDISGINASVVNIQKEIDRLNEVAKNLNESLID SEQ ID NO.16:HR2- <3> Amino acid sequence DISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL

[0110] SEQ ID NO.17:SARS-CoV-2-NF GGGGAACTTCTCCTGCTAGAAT SEQ ID NO.18:SARS-CoV-2-NR CAGACATTTTGCTCTCAAGCTG SEQ ID NO.19:SARS-CoV-2-N-probe TTGCTGCTGCTTGACAGATT

[0111] SEQ ID NO.20:SARS-CoV-2-EF CGATCTCTTGTAGATCTGTTCTC SEQ ID NO.21:SARS-CoV-2-ER ATATTGCAGCAGTACGCCACACA SEQ ID NO.22:SARS-CoV-2-E-probe CGAAGCGCAGTAAGGATGGCTAGTGT

[0112] SEQ ID NO.23:HR121 AUGACACAGAUGUUCUCUAUGAACCAAAAAUUGUCCAACCAAUUUAAUAGUGCUAUUGGCAAAAUUCAAGACUCACUUUCUUCCAGCAAGUGCACUUGGAAAACUUCAAGAUGUGGGUCAAACCAAAUAGCACAAGCUUUAAACACGCUUGUUAAACAACUUAGCUCCAAUUUUGGUGGUGCAAUUUUUAAUGAUAUUCCUUCACGUCUUGACAAAGUUGAGGAAGCGGAGGAGGAUGUGUGUGAUUUAUGGUGACAUCUCUGGCAUUAAUAUAGCUUCUGGCAUUAAUUGCUUCAGGUUGUAACAUAUAGAUUGACAUUGAC GCCUCAAUGAGGUUGCCAAGAAUUUAAUUGAAUCUCUCAUCGAUCUCCAAGAACUUGGAAAGUAUAGCGGAGGAAGAGGAGGAAACACAGAAUGUUCUCUAUUGGAGAACCAAAAAUUGAUUGCCAACCAAUUUAAUAGUGCUAUUGGCAAAAUUCAAGACUCACUUUCUUCCAGCAAGUGCACUUGGAAACUUCAAGAUGUGGCUACCAAAAUGCACAAAGCUUUAAACACGCUUGUUAAACAACUUAGCUCCAAUUUUGGUGCAAUUUUUAAUGAUAUAUUGGUGCUAUUCACGUCUUGACAAAGUUGAGUA

[0113] SEQ ID NO.24: mRNA sequence encoding HR121-1 AUGCAAAAAUUGAUUGCCAACCAAUUUAAUAGUGCUAUUGGCAAAAUUCAAGACUCACUUUCUUCCACAGCAAGUGCACUUGGAAAACUUCAAGAUGUGGUCAACCAAAAUGGAGGAAGCGGAGGAGGCAUUAAUGCUUCAGUUGUAAACAUUCAAAAAGAAAUUGACCGCCUCAAUG AGGUUGCCAAGAAUUUAAAUGAAUCUCUCAUCGAUCCAAGAACUUGGAAGCGGAGGAAGAGGAGGACAAAAAUUGAUUGCCAACCAAUUUAAUAGUGCUAUUGGCAAAAUUCAAGACUCACUUUCUUCCACAGCAAGUGCACUUGGAAAACUUCAAGAUGUGGUCAACCAAAAUUAA

[0114] SEQ ID NO.25: mRNA sequence encoding HR121-2 AUGAAAUUGAUUGCCAACCAAUUUAAUAGUGCUAUUGGCAAAAUUCAAGACUCACUUUUCCACAGCAAGUGCACUUGGAAAACUUCAAGAUGUGGUCAACCAAAAUGCACAAGCUUUAAACACGCUUGGAGGAAGCGGAGGAGUUGAUUUAGGUGACAUCUCUGGCAUUAUGCUUCAGUUGUAAACAUUCAAAAAG AAAUUGACCGCCUCAAUGAGGUUGCCAAGAAUUUAAAUGAAUCUCUCAUCGAUAGCGGAGGAAGAGGAGGAAAAUUGAUUGCCAACCAAUUUAAUAGUGCUAUUGGCAAAAUUCAAGACUCACUUUCUUCCACAGCAAGUGCACUUGGAAAACUUCAAGAUGUGGUCAACCAAAAUGCACAAGCUUUAAACACGCUUUAA

[0115] SEQ ID NO.26: mRNA sequence encoding HR121-3 AUGGCCAACCAAUUUAAUAGUGCUAUUGGCAAAAUUCAAGACUCACUUUCUUCCAGCAAGUGCACUUGGAAAACUUCAAGAUGUGUGGUCAACCAAAAUGCACAAGCUUUAAACACGCUUGGUUAAACAAGGAGGAAGCGGAGAGACACUCUCUGGCAUUAAUAUGCUUCAGGUUGUAACAUCAAAAGAAAUUGACCGCUCUCUUGGCAUUAAUAGUGGUGCCAAGAAUUUAAUAUAUUGAUAUAUAUAUUGAUCAAGACUCUCUCAUCGAUCUACGCAAAGACUUAGCGGAGGAGGAGGCCAACCAAUUUAAUAUAGUGCUAUUGGCAAAAUUCAAGACUCUCUUCCACAGCAAGUGCACUUGGAAAACUUUUCCACAGUGCACUUGGAAAACUUCAAAUGCACAAGCUUUAAACACGCUUGUAUAAACAAUA

[0116] SEQ ID NO.27:HR212ををををかすする mRNA sequence AUGGAUGUUGAUUAGGUGACAUCUCUGGCAUUAAUUGCUUCAGUUGAAACAUUCAAAAAGAAAUUGACCGCCUCAAUGAGGUUGCCAAGAAUUUAAUUGAUAUAUUGAUAUUGAUAUUGCCAACGAUCUUCCAAGAACUUGGAAAGUAUAUUGCCAACCAAGAUGUUCUCUAUAUUGGAACCAAAAUAUUGUCCAACCAAUUUAAUAGUGCUAUUGGCAAAAUAUAUGACUCUAUUGGCAAAAUUGCUAUUGGCAAAAUUGCUAUUGGCAAAAUUGCACAAAGCUUAAACACGCUUGGUUAAAAACAACUUAGCCUAUUUGGUGGUCAAUUUUAAUUGAUUCCUUCACGUCUUGUAAAUUGAUAUUGAUUCCUUUCACGUUGACAAAGUUGAGGAAGCGGAGGAGAUGUUGAUUGAUUAAUUGUGCAAUUAAUUGAAUUGACCGCAUGAGGUUGCAAUUAAUUGAAUUGACCGCAUGAGGUUGCAAUUAAUUGAAUUGACCGCAUGAGGUUGCAAUUAAUUGAAUUGACCGCAUGAGGUUGCAAUUAAUUGAAUUGACCGCAUGAGGUUGCAAUUAAUUGAAUUGACCGCAUGAGGUUGCAAUUUAAUUGACCAAAAUUUAAUUGAAUUGAAUUGACCGCAUGAGGUUGCCAAAAUUUAAUUGAAUUGAAUUGACCGCAUGAGGUUGCCAAAAUUUA

[0117] SEQ ID NO.28:HR212-1ををフェするする mRNA sequence AUGGGCAUUAAUGCUUCAGUUGUAACAUUCAAAAAGAAAUUGACCGCCUCAAUGAGGUUGCCAAGAAUUUAAUUGAUAUAUUGAUAUUGCAUCUGAUCUCCACCAAGAACUUGGAAGCGGAAGGAGGAGGACAAAAAUUGAUUGCCAACCAAUUUAAUAGUGCUAUUGGCAAAAUUCAAGACUCACUUUUCCAGCAAAGUGCACUUGGAAAACUUCAAGAUGUGUGGUCAACCAAAAAUUGAGGAAGCGGAGGGAGCAUUAAUAUUGCUUCAGUAUACAUUCAAAAAGAAAUUGACCGCCUCAAAUAGAGGUUGUCCAAAAUUUAAUAUAUUGAAUUGAAUAUUGAAUUGAAUUGAAUUGACCGCCUCAAAUAGAGGUUGCCAAAUUUAAUAUUGA

[0118] SEQ ID NO.29:HR212-2をををフェするする mRNA sequence AUGGUUGAUUAGGUGACAUCUCUGGCAUUAAUUGCUUCAGUUGUAACAUUCAAAAAGAAAUUGACCGCCUCAAUGAGGUUGCCAAGAAUUUAAUAUUGAUAUUGCCAAAGAUUUAUGAUAUAUUGCCAACGAUAUAUUGAUUGCCAACCAAUUUAAUAGUGCUAUUGGCAAAAUUCAAGACUCACUUUCUUCCAGCAAGUGCACUUGGAAACUUUCAAAUGCACAAGCUUUAAACACGCUUGGAGGAGAGCGGAGGAGGUUGAUUGAUUAUGGUGACAUCUCUGGCAUUAAUAUGCUUCAGGUAUAUACAUUGCUUCAGCUAUCAAAUGCUUCAGCUUCAAAAAGAAAUUGACCGCUUGCAUCUGGCAUUAAUAGCUUCAGCUUCGCAAUAUUUAAUGAUGAAUUGACCGCUUGCAAUAUAGAGAGGUUGUCCAAGAAUUUAAUUGA

[0119] SEQ ID NO.30:HR212-3ををフェするする mRNA sequence AUGGACAUCUCUGGCAUUAAUGCUUCAGUUGAAACAUUCAAAAAGAAAUUGACCGCCUCAAAUGAGUUGCCAAGAAUUUAAUUGAUAUAUUGAUAUAUUGAUAUAUAUAUUGCUAAUCGCAACAGACCUUAUAUGCGGAAGGAGGAGGCCAACCAAUUUAAUAGUGCUAUUGGCAAAAUUCAAGACUCACUUUCUUCCAGCAAGUGCACUUGGAAACUUCAAAUGCACAAGCUUUAAACACGCUUGUAAAAACAAGGAGGAAGCGGAGACAUCUCUGGCAUUAAUAUUGCUUCAGUAACAUAUCAAAAAGAAAUUGACCGCCUCAAAUAGAGGAGUUGUCCAAAAUUUAAUAUAUAGAUUGACCGCCUCAAAUAGAGAGGUUGUCCAAAAUUUAAUAUAUUGAAUUGAAUUGACCGCCUCAAAUAGAGAGGUUGUCCAAAAUUUAAUAUUGAAUUGAAUUGACCGCCUCAAAUAGAGAGGUUGUCCAAAAUUUAAUUGAAUUGAAUUGACCGCCUCAAAUAGAGAGGUUGUCCAAAAUUUAAUUGAAUUGAAUUGACCGCCUCAAAUAGAGGUUGUCCAAAAUUUAAUUGAAUUGAAUUGACCGCCUCAAAUAGAGGUUGUCCAAAAUUUAAUUGAAUUGAAUUGACCGCCUCAAAUGAAUUGACCGUCCAAGAGAUUCUCCAAGAGAUUUAAA

[0120] Those skilled in the art can easily understand that the above description is only a preferred embodiment of the present invention, and does not limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principle of the present invention are all within the scope of protection of the present invention, for example, when the amino acids of the HR1 or HR2 peptide segment in the above examples are deleted or modified, the host cell is changed to yeast cells, E. coli, mammalian cells, etc., and the corresponding backbone vector is selected according to the host cell, and the novel coronavirus recombinant protein HR121 or HR212 is expressed as in the above experimental examples, and the novel coronavirus recombinant protein HR121 or HR212 is used as a vaccine or an antibody is prepared, etc., are all within the scope of the claims of the present invention.

Claims

1. A recombinant fusion protein derived from the HR region of the novel coronavirus S2 protein, comprising: The novel coronavirus recombinant fusion protein HR121 or HR212 is composed of a truncated protein HR1 derived from the HR1 region of the novel coronavirus S2 protein, a truncated protein HR2 derived from the HR2 region, and a linking peptide coupling the two, the protein structure of the HR121 being HR1-Linker1-HR2-Linker2-HR1, and the protein structure of the HR212 being HR2-Linker2-HR1-Linker1-HR2, wherein the HR1 amino acid sequence is as shown in SEQ ID NO. 1 or includes a sequence in which one or more amino acids are substituted, deleted or inserted in the amino acid sequence of SEQ ID NO. 1, and the HR2 amino acid sequence is as shown in SEQ ID NO. 2 or includes a sequence in which one or more amino acids are substituted, deleted or inserted in the amino acid sequence of SEQ ID NO. The recombinant fusion protein is derived from the HR region of the novel coronavirus S2 protein, and is characterized in that the amino acid sequence of Linker1 and Linker2 is represented by SEQ ID NO. 3-4, respectively, and includes a sequence in which one or more amino acids are substituted, deleted or inserted in the amino acid sequence represented by SEQ ID NO.

2.

2. The amino acid sequence of the novel coronavirus recombinant fusion protein HR121 is as shown in SEQ ID NO.

5. A recombinant fusion protein derived from the HR region of the novel coronavirus S2 protein described in claim 1.

3. The amino acid sequence of the novel coronavirus recombinant fusion protein HR212 is as shown in SEQ ID NO.

6. A recombinant fusion protein derived from the HR region of the novel coronavirus S2 protein described in claim 1.

4. The nucleotide sequence encoding said HR121 is as shown in SEQ ID NO.

7. A recombinant fusion protein derived from the HR region of the novel coronavirus S2 protein described in claim 2.

5. The nucleotide sequence encoding said HR212 is as shown in SEQ ID NO.

8. A recombinant fusion protein derived from the HR region of the novel coronavirus S2 protein described in claim 3.

6. A vector expressing a recombinant fusion protein derived from the HR region of a novel coronavirus S2 protein, comprising: The vectors are HR121 backbone vectors and HR212 backbone vectors, each of which contains a nucleotide sequence as described in claims 4 and 5. Vectors expressing recombinant fusion proteins derived from the HR region of the novel coronavirus S2 protein.

7. Use of a recombinant fusion protein derived from the HR region of the novel coronavirus S2 protein described in any one of claims 1 to 5 in the preparation of an antigen detection reagent and a drug for the prevention or treatment of novel coronavirus.

8. Use of a recombinant fusion protein derived from the HR region of the novel coronavirus S2 protein described in any one of claims 1 to 5 as an immunogen in the preparation of a novel coronavirus vaccine and antibody.

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