CN114702556A - Coronavirus RBD variant and application thereof - Google Patents

Abstract

The present invention relates to a protein comprising a coronavirus spike glycoprotein RBD variant, which has mutations at amino acid positions K417, L452, T478, E484, and N501, as compared to a coronavirus spike glycoprotein RBD wild-type protein. The coronavirus spike glycoprotein RBD variant has the potential of preventing infection of wild strains and variant strains of coronavirus, the vaccine prepared by using the RBD variant protein can widely prevent diseases caused by novel coronavirus, the preparation method is simple, the large-scale production of the novel coronavirus vaccine is facilitated, and the application prospect is good.

Description

Coronavirus RBD variant and application thereof

Technical Field

The invention relates to the field of biological medicine, in particular to a coronavirus RBD variant and application thereof.

Background

SARS-CoV-2 virus can replicate rapidly after invading human cells. However, because it is a single-stranded RNA virus, the error rate is high during replication, and the SARS-CoV-2 is very easy to generate gene mutation and gene recombination due to the wide range of coronavirus hosts and the genome structure of the coronavirus.

In late 12 months of 2020, a new SARS-CoV-2 variant, Alpha (lineage B.1.1.7), was reported in the UK based on whole genome sequencing of samples. Alpha variants include 17 mutations in the viral genome, of which there are 8 mutations in the spike protein (S protein) (Δ 69-70 deletion, Δ 144 deletion, N501Y, a570D, P681H, T716I, S982A, D1118H).

A new variant Beta of the SARS-CoV-2 lineage, (i.e., B.1.351 lineage or 501Y.V2) with multiple spike protein mutations was found in south Africa 10.2020, resulting in a second wave COVID-19 infection. The Beta variant contained 9 mutations in the spike protein (L18F, D80A, D215G, R246I, K417N, E484K, N501Y, D614G and a701V), of which 3 mutations (K417N, E484K and N501Y) are located in the RBD and increase the binding affinity to the ACE2 receptor.

The b.1.1.28 variant, also known as the 501y.v3 or Gamma lineage variant, was discovered in brazil in 12 months 2020 and first in the us in 1 month 2021. The Gamma variant contained 10 mutations in the spike protein (L18F, T20N, P26S, D138Y, R190S, H655Y, T1027IV1176, K417T, E484K and N501Y), of which three mutations (L18F, K417N, E484K) were located in the RBD, similar to the Beta (b.1.351) variant.

The first discovery in india in 10 months 2020, a variant B.1.617, in which infection has been detected in several countries at present, has further evolved three different mutations: b.1.617.1, B.1.617.2(Delta), and B.1.617.3.

The variation strain of Omicron (B.1.1.529) contains 32 mutation sites on spike protein, wherein 15 mutations in RBD region are G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493K, G496S, Q498R, N501Y and Y505H.

Mutation and base deletion of the spike protein of coronavirus make SARS-CoV-2 show abundant genetic diversity. It has been demonstrated that mutation of SARS-CoV-2 can lead to increased infectivity, increased toxicity or altered clinical manifestations, and even that some variants can escape immunologically, resulting in a vaccine or specific antibody with reduced protection against the body.

There is a need for a new candidate vaccine against constantly mutated SARS-CoV-2 with a broad spectrum of coronaviruses.

Disclosure of Invention

In view of the technical problems in the prior art, the present invention provides a protein comprising a coronavirus spike glycoprotein RBD variant, which has a mutation at one or more amino acid positions of K417, L452, T478, E484, and N501, as compared with a wild-type coronavirus spike glycoprotein RBD protein.

A protein comprising an RBD variant of the coronavirus spike glycoprotein as described above, wherein the RBD variant protein comprises one or more of the following amino acid mutations: K417N, L452R, T478K, E484Q/K and N501Y.

A protein comprising a coronavirus spike glycoprotein RBD variant as described above, wherein: (1) the amino acid sequence of the RBD variant protein is selected from the group consisting of: (a1) SEQ ID NO: 1; (a2) the amino acid sequence of SEQ ID NO:1 by substituting, deleting and/or adding one or more amino acids; and (a3) the truncation of (a1) or (a 2); or (2) the amino acid sequence of the RBD variant protein is selected from the group consisting of: (b1) SEQ ID NO: 3; (b2) consisting of SEQ ID NO:3 by substituting, deleting and/or adding one or more amino acids; and (b3) the truncation of (b1) or (b 2); or (3) the amino acid sequence of the RBD variant protein is selected from the group consisting of: (c1) SEQ ID NO: 5; (c2) consisting of SEQ ID NO:5 by substituting, deleting and/or adding one or more amino acids; and a truncation of (c3) (c1) or (c 2).

A protein comprising a variant of coronavirus spike glycoprotein RBD as described above, the amino acid sequence of which is encoded by a coding gene, wherein: (1) the coding gene is selected from: (d1) SEQ ID NO: 2; (d2) and SEQ ID NO:2, and the encoded protein is more than 80% homologous with the coronavirus spike glycoprotein RBD variant protein; and a truncation of (d3) (d1) or (d 2); or (2) the encoding gene is selected from: (e1) SEQ ID NO: 4; (e2) and SEQ ID NO:4, and the encoded protein is more than 80% homologous with the coronavirus spike glycoprotein RBD variant protein; and (e3) the truncation of (e1) or (e 2); or (3) the encoding gene is selected from: (f1) SEQ ID NO: 6; (f2) and SEQ ID NO:6, and the coded protein is more than 80 percent homologous with the coronavirus spike glycoprotein RBD variant protein; and a truncated body of (f3) (f1) or (f 2).

An isolated nucleic acid molecule encoding a protein comprising a coronavirus spike glycoprotein RBD variant as set forth in any preceding claim.

A recombinant vector comprising: a protein comprising a coronavirus spike glycoprotein RBD variant as defined in any of the above, or a nucleic acid molecule as defined above; and an expression vector.

The recombinant vector as described above, wherein the expression vector is selected from one or more of the following vectors: pPICZ alpha A vector, pPIC9 vector, pPIC9K vector, pPICZ alpha B vector, pET series vector, pGEX series vector, pMAL series vector, pQE series vector, pBADmycHis series vector, pTrcHis series vector, pTXB series, T series vector and the modified vector of the above vectors.

A fused cell comprising: a recombinant vector as described above; and an expression cell.

The above-mentioned fusion cell, the host cell including but not limited to mammalian cells, Escherichia coli, yeast cells.

The yeast cell is Pichia pastoris which is genetically modified through a glycosylation modification way, is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, and has the preservation number of CGMCC No. 19488.

A method of producing a protein comprising a variant of coronavirus spike glycoprotein RBD as defined above or a protein expressed by a nucleic acid molecule as defined above, comprising: obtaining a coding gene; transforming the coding gene into an expression cell; expressing the RBD variant protein in an expressing cell; and purifying the RBD variant protein.

The method as described above, further comprising: constructing a recombinant vector containing the coding gene; and transforming the recombinant vector into an expression cell.

A vaccine composition comprising: at least one immunogenic fragment or an immunogenic composition comprising said immunogenic fragment; wherein the immunogenic fragment is a protein comprising a coronavirus spike glycoprotein RBD variant as defined in any one of the above, or an RBD variant protein encoded by a nucleic acid molecule as defined above; and an adjuvant.

The vaccine composition as described above, wherein the adjuvant is selected from one or more of the following components: aluminium salts, such as aluminium hydroxide or aluminium phosphate, calcium salts, iron salts, zinc salts, acylated tyrosines, acylated sugars, cationically or anionically derivatised polysaccharides, polyphosphazenes, preferential inducers of Th1 type responses, monophosphoryl lipid A or derivatives thereof, such as 3-de-O-acylated monophosphoryl lipid A (3D-MPL), combinations of saponin derivatives, QS21, 3D-MPL, tocopherol, Tween 80, sorbitol trioleate, squalene and CpG.

The vaccine composition as described above, wherein the adjuvant comprises aluminum hydroxide and CpG; the mixing mass ratio of the immunogenic fragment to the CpG and the aluminum hydroxide is 1:10: 20.

A method of making a vaccine comprising: (1) providing at least one immunogenic fragment based on the coronavirus spike glycoprotein or an immunogenic composition comprising said immunogenic fragment; wherein the immunogenic fragment is a protein comprising a coronavirus spike glycoprotein RBD variant as defined in any one of the above, or an RBD variant protein encoded by a nucleic acid molecule as defined above; and (2) mixing the immunogenic fragment or the immunogenic composition of (1) with a pharmaceutically acceptable adjuvant.

A method of treating and/or preventing a new coronavirus infection comprising administering to a subject a vaccine composition as described above or a vaccine prepared as described above.

Use of a protein as defined above comprising a coronavirus spike glycoprotein RBD variant or a nucleic acid molecule as defined above for the preparation of a medicament or vaccine for the prevention of a disease caused by a coronavirus.

A protein comprising a variant of the coronavirus spike glycoprotein RBD as described above, said coronavirus being a β coronavirus or a variant thereof.

The protein comprising the coronavirus spike glycoprotein RBD variant as described above, wherein the beta coronavirus is a 2019 novel coronavirus (SARS-CoV-2).

As mentioned above containing coronavirus spike glycoprotein RBD variant protein, the Beta coronavirus variant is SARS-CoV-2 virus variant, which includes but not limited to Alpha variant, Beta variant, Gamma variant, Delta variant, Kappa variant, Lambda variant, Delta variant, Omicron variant.

The coronavirus spike glycoprotein RBD variant has the potential of preventing infection of wild strains and variant strains of coronavirus, the vaccine prepared by using the RBD variant protein can prevent diseases caused by novel coronavirus in a broad spectrum, the preparation method is simple, the large-scale production of the novel coronavirus vaccine is facilitated, and the application prospect is good.

Drawings

Preferred embodiments of the present invention will be described in further detail below with reference to the accompanying drawings, in which:

FIG. 1 is a schematic structural diagram of the spike glycoprotein (S protein) of a novel coronavirus according to one embodiment of the present invention;

FIG. 2 is a diagram of a screening gel electrophoresis analysis of pPICZ α -M5 expression vector according to one embodiment of the present invention;

FIG. 3 is a drawing of a linearized electrophoresis analysis of pPICZ α -M5 expression vector BglII according to one embodiment of the present invention;

FIG. 4 is a CGMCC19488/pPICZ α -M5 clone screening map according to one embodiment of the invention;

FIG. 5 is a graph of different induction time electrophoretic detection of CGMCC19488/pPICZ α -M5 according to one embodiment of the invention;

FIG. 6 is a SDS-PAGE pattern of a purified sample of M5 according to one embodiment of the invention;

FIG. 7A is a mouse serum anti-RBDwt antibody titer 14 days after hyperimmunization according to one embodiment of the invention;

figure 7B is a mouse serum anti-M5 antibody titer after 14 days of hyperimmunization according to one embodiment of the invention;

FIG. 8A shows the results of a neutralization assay for wild-strain pseudoviruses of coronaviruses according to one embodiment of the invention;

FIG. 8B shows the results of a neutralization assay for wild-strain pseudoviruses of coronaviruses according to one embodiment of the present invention;

FIG. 8C shows the results of a coronavirus Beta variant pseudovirus neutralization assay according to one embodiment of the present invention;

FIG. 8D shows the results of a pseudovirus neutralization assay according to one embodiment of the present invention;

FIG. 8E is the results of a coronavirus Delta variant pseudovirus neutralization assay according to one embodiment of the present invention;

FIG. 8F is the results of a coronavirus Delta variant pseudovirus neutralization assay according to one embodiment of the present invention;

FIG. 8G shows the results of a neutralization test of a coronavirus Omicron variant pseudovirus according to an embodiment of the present invention;

FIG. 8H shows the results of a neutralization test of the coronavirus Omicron variant pseudovirus according to an embodiment of the present invention;

FIG. 9A is the results of an in vitro IFN- γ cytokine assay according to one embodiment of the invention;

FIG. 9B is an in vitro IL-2 cytokine assay according to one embodiment of the present invention;

FIG. 9C is the results of an in vitro IL-4 cytokine assay according to one embodiment of the present invention; and

FIG. 9D is the results of an in vitro IL-5 cytokine assay according to one embodiment of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

In the following detailed description, reference is made to the accompanying drawings that form a part hereof and in which is shown by way of illustration specific embodiments of the application. In the drawings, like numerals describe substantially similar components throughout the different views. Various specific embodiments of the present application are described in sufficient detail below to enable those skilled in the art to practice the teachings of the present application. It is to be understood that other embodiments may be utilized and structural and logical changes may be made to the embodiments of the present application.

All expression systems of the invention are available in the open literature. It will be understood by those skilled in the art that even if the same bacteria or species are different in their origin, growth, etc., and the like, but their functions are substantially the same, the bacteria or cells mentioned in the present invention may include modified forms of these bacteria or cells.

The terms used in this application have the following meanings:

in this application, the term "comprising" is used in a generic sense to mean including, summarizing, containing or encompassing. In some cases, the meaning of "being", "consisting of … …" is also indicated.

The term "about" generally refers to a variation in the range of 0.5% -10% above or below the stated value, such as a variation in the range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below the stated value.

The term "Coronavirus" refers to a virus belonging to the phylogenetic group of the order of the nested viruses (Nidovirales) Coronaviridae (Coronaviridae) genus of Coronaviridae (Coronaviridae). There are 4 species of coronaviruses, namely, alpha coronavirus, beta coronavirus, gamma coronavirus, and delta coronavirus. Wherein the alpha and beta coronaviruses are mainly derived from human and other animals, especially mammals, such as bat, pig, cat, dog, rat, cow, horse; the delta and gamma coronaviruses are mainly from birds, such as chicken, duck, goose, sparrow, and pigeon.

Wherein, the human coronavirus (HCoV) of the beta genus comprises HCoV-OC43, HCoV-HKU1, severe acute respiratory syndrome coronavirus (SARS-CoV) Middle East respiratory syndrome coronavirus (Middle East respiratory syndrome coronavirus, MERS-CoV) and 2019 novel coronavirus (2019-nCoV or SARS-CoV-2).

2019 novel coronaviruses (2019-nCoV or SARS-CoV-2, causing novel coronavirus pneumonia COVID-19) are the 7 th coronavirus which is known to infect humans at present, and the remaining 6 are HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV (causing severe acute respiratory syndrome) and MERS-CoV (causing middle east respiratory syndrome), respectively.

The terms "new coronavirus", "SARS-CoV-2", "2019-nCoV" all refer to a coronavirus designated SARS-CoV-2 by the International Committee for viral Classification on month 11 of 2020.

Variants of the novel coronavirus SARS-CoV-2 known to date include, but are not limited to, Alpha, Beta, Gamma, Delta, Epsilon, Zeta, Eta, Theta, Iota, Iota, Kappa, Lambda, Delta, Omicron. The glycoprotein vaccines of the present application relate to SARS-CoV-2 and variants thereof, including but not limited to the variants described above.

As used herein, a "variant" is a protein or protein having an altered configuration as compared to the wild-type protein, or an amino acid or nucleotide sequence obtained by substitution, addition or deletion of one or more sites of the sequence, or an amino acid or nucleotide sequence obtained by truncation of the sequence.

GISAID uses the whole genome sequence of strain hCoV-1 (EPI _ ISL _402124) as a formal reference sequence. The WIV04 strain is generally defined as the wild type strain or the original strain. In this application, the term "SARS-CoV-2 wild-type strain" generally refers to the WIV04 strain.

The term "vaccine" or "vaccine composition" refers to a vaccine that is directed against the pathogenic agent of a disease or its associated proteins (polypeptides, peptides), polysaccharides or nucleic acids, in one or more components, directly or via a carrier, and after immunization into the body, induces specific humoral and/or cellular immunity, thereby conferring immunity against the disease.

The genetic engineering vaccine is a vaccine prepared by cloning and expressing protective immunogenic fragment genes by using a DNA recombination technology and using an expressed immunogenic fragment product or a recombinant per se. When preparing the genetic engineering vaccine, the DNA recombination biotechnology is used to directionally insert natural or artificially synthesized genetic materials into bacteria, saccharomycetes or mammalian cells so as to fully express the genetic materials, and the vaccine is prepared after purification. By applying gene engineering technology, subunit vaccine, stable attenuated vaccine and multivalent vaccine capable of preventing several diseases can be prepared.

The glycoprotein vaccine is one kind of genetic engineering vaccine. In some embodiments, the glycoprotein vaccines of the present application include an immunogenic fragment and an adjuvant. In some embodiments, the glycoprotein vaccines of the present application include an immunogenic composition and an adjuvant.

The term "immunogenic composition" generally refers to a subunit composition. In the present application, a subunit composition is a composition in which the components have been isolated and purified to at least 50%, at least 60%, 70%, 80%, 90% purity, prior to mixing the components to form an antigenic composition. For example, the subunit composition may be an aqueous solution of a water-soluble protein. For example, the subunit composition may comprise a detergent. For example, the subunit composition may comprise a non-ionic, zwitterionic or ionic detergent. For example, the subunit composition may comprise lipids. In some cases, immunogenic fragments are included in the immunogenic composition. For example, in some cases, the immunogenic fragment can be an RBD or a functionally active fragment thereof. In some cases, the immunogenic fragment can be an RBD variant or a functionally active fragment thereof.

Further, the term "immunogenic fragment" refers to a substance that stimulates the body to produce a (specific) immune response and that binds to the immune response product antibodies and sensitized lymphocytes in vitro and in vivo to produce an immune effect (specific reaction). According to one embodiment of the application, the immunogenic fragment is a coronavirus S protein receptor binding domain variant RBD. Further, in some embodiments, the immunogenic fragment is the sequence of SEQ ID NO:1 and/or SEQ ID NO:3 and/or SEQ ID NO:5 or a protein thereof.

In the present application, the immunogenic composition may further comprise an adjuvant. The term "adjuvant" refers to a non-specific immunopotentiator that, when injected or pre-injected into a body with an immunogenic fragment, enhances the body's immune response to the immunogenic fragment or alters the type of immune response. The present application is not limited to adjuvants. For example, the adjuvant may comprise an aluminium salt (such as aluminium hydroxide gel (alum) or aluminium phosphate), but may also be a salt of calcium, iron or zinc, or may be an insoluble suspension of acylated tyrosine or acylated sugars, cationically or anionically derivatised polysaccharides or polyphosphazenes. For example, the immunogenic composition may also be selected to be a preferential inducer of a Th1 type response. For example, a preferential inducer of a Th1 type response may comprise monophosphoryl lipid A or a derivative thereof. For example, the adjuvant may be a combination of monophosphoryl lipid A (e.g., 3-de-O-acylated monophosphoryl lipid A (3D-MPL)) and an aluminum salt. An adjuvant enhancement system may comprise a combination of monophosphoryl lipid a and a saponin derivative, in particular the combination of QS21 and 3D-MPL as disclosed in WO94/00153, or a composition in which QS21 is quenched with cholesterol to make it less reactive as disclosed in WO 96/33739. For example, the adjuvant may also be an adjuvant as described in WO95/17210, which contains QS21, 3D-MPL and tocopherol in an oil-in-water emulsion. For example, the adjuvant may be tween 80, sorbitol trioleate and squalene mixed and then microfluidized under high pressure to form a uniform emulsion of small drops. For example, the adjuvant may comprise unmethylated CpG of the oligonucleotide (WO 96/02555).

According to one embodiment of the present application, the glycoprotein vaccine has a mixture of coronavirus S protein receptor binding region, CpG and aluminum hydroxide at a mass ratio of 1:10: 20.

The term "subject" refers to a human or selected animal, and may even be a cell of a human or selected animal, vaccinated with a vaccine or vaccine composition, or otherwise administered with a medicament to prevent or treat a coronavirus infection.

The term "neutralizing antibody" refers to an antibody secreted by B lymphocytes, which is a soluble protein. When pathogenic microorganisms (such as viruses) invade the body, the body produces corresponding antibodies. Invasion of target cells by pathogenic microorganisms requires binding of specific molecules (e.g., S protein) expressed by the pathogen itself to receptors on the target cells in order to infect and further amplify the cells. The neutralizing antibody is capable of binding to an immunogenic fragment on the surface of a pathogenic microorganism, thereby preventing the pathogenic microorganism from adhering to a target cell receptor and preventing its invasion into the cell.

The term "immune escape" refers to the ability of an immunosuppressive pathogen to antagonize, block, and suppress the body's immune response through its structural and non-structural products. The method comprises the following steps: 1. antigenic variation neutralizing immunogenic fragments of pathogens, which can frequently undergo sustained mutations, escape the neutralizing and blocking effects of established anti-infective immune antibodies, leading to the presence of infection. Such as immune escape due to persistent variation of new coronaviruses. 2. Persistent infection intracellular pathogens can be hidden in cells to be in a dormant state, escape from attack of cellular immunity and humoral immunity, survive for a long time and form persistent infection. For example, the human body is infected with hepatitis B virus in the infant period without establishing human immunity, so that the immunity is escaped.

The terms "Spike protein", "S protein", "Spike glycoprotein" refer to a coronavirus capsid surface glycoprotein. Belongs to type I membrane protein, is modified by N-glycosylation, and is composed of 1300 amino acids in monomer, which is polymerized to form homotrimer after being folded. SARS-COV-2 binds to the ACE2 receptor via the S protein and invades the cell. The S protein consists of 1213 amino acids and contains a transmembrane region, including a fragment of the capsid surface glycoprotein from coronavirus from the N-terminus or from amino acid position 14 up to at least 1213 amino acids, or the corresponding region from other SARS viruses. The S1 protein is subunit 1 of S protein, and mainly refers to the fragment from the N-terminal or 14 th amino acid to 685 th amino acid.

FIG. 1 is a schematic representation of the structure of the spike glycoprotein (S protein) of the novel coronavirus according to one embodiment of the present application. As shown in fig. 1, the S protein monomer is composed of an N-terminal S1 subunit and a C-terminal S2 subunit, mediating fusion between the receptor of the host cell and the membrane of the novel coronavirus protein. Wherein the S1 subunit is responsible for binding to a host cell receptor and both the N-terminus and the C-terminus of the S1 domain are capable of binding to a host receptor; the S2 subunit is responsible for fusion with the host cell membrane. After uptake of the new coronavirus by the host cell, the S2 subunit is cleaved close to the S2 site of the fusion peptide by the host protease, triggering a conformational change in the S protein, and thus S2 subunit mediated membrane fusion (ref: lexandra C wells, M Alejandra Totortici, Brandon Frenz, Joost Snijder, Wentao Li, F elix A Rey, Frank DiMaio, Berend-Jan Bosch & David Veeseler. Glycan shield and epitope mask of a coronavir protein obtained by dependent b-electron microscopic serology. Nature Structural & Molecular biology. volume 23, pages 899-905 (2016)).

The term "fusion peptide" refers to a polypeptide located on the S2 subunit of the S protein described above. As shown in fig. 1, the S2 subunit of S virus contains a number of key molecules, including multiple fusion peptides and two conserved peptide repeats (HRs), to drive fusion between the virus and the host cell. HRs can trimerize into coiled structures, bringing the viral envelope and host cell bilayers into close proximity. The fusion peptide further mediates membrane fusion.

The terms "membrane protein", "transmembrane protein" refer to an integral membrane protein, or an integral transmembrane protein. According to the Singer classification, integral membrane proteins are of the following 6 types: type I membrane proteins have a single transmembrane spanning, with the N-terminus of the polypeptide chain being outside the cell membrane and the C-terminus being inside the cell, such as immunoglobulin superfamily (IgSF) members. The novel coronavirus S protein related to the invention also belongs to type I membrane protein. Type II membrane proteins have a single transmembrane spanning, with the C-terminus of the polypeptide chain outside the cell membrane and the N-terminus inside the cell, such as molecules that are members of the tumor necrosis factor superfamily. Type III membrane proteins are polypeptide chains that have multiple transmembrane spanning, varying from 2 to 7, for example, the tetraspanin (TM4-SF) and the seven transmembrane receptor superfamily (STR-SF). Chemokine receptors are STM-SF, also known as G Protein-Coupled receptors (GPCRs). Type IV membrane proteins are membrane proteins composed of multiple transmembrane subunits. The polypeptide chain of type V membrane proteins is linked to the lipid bilayer of the cell membrane with Glycosylphosphatidylinositol (GPI). Such as GPI-linked CD16, CD55, CD58, and the like. Type VI membrane proteins are polypeptide chains that have one end attached to the cytoplasmic membrane in the GPI form and one or more transmembrane events at the other end, such as membrane bridge proteins.

The term "glycosylation modification" refers to the process by which glycosylation of a protein is one of the most common post-translational modifications of a protein, and is the process by which sugars are transferred to proteins and to specific amino acid residues on proteins by glycosyltransferases to form glycosidic bonds. The glycosylation pattern of proteins can be largely divided into two categories: n-glycosylation and O-glycosylation. Here, the "O-glycosylation modification" means that an O-sugar chain is covalently linked to the free OH group of serine or threonine of a protein. The O-glycosylation site has no conserved sequence, and the sugar chain has no fixed core structure, and the composition can be a monosaccharide or huge sulfonated polysaccharide. The term "N-glycosylation modification" as used herein means that an N-sugar chain is modified by the free-NH reaction with aspartic acid in a protein₂The groups are covalently linked.

The term "ACE 2" refers to angiotensin converting enzyme 2 receptor, which is a host cell surface binding body to the spike glycoprotein of the novel coronavirus, and is a key binding site of the novel coronavirus (SARS-CoV-2). ACE2 is a type I transmembrane glycoprotein comprising an N-terminal ectodomain composed of two alpha helices. The novel coronavirus receptor binding domain RBD binds to the N-terminus of the host cell receptor using its external domain.

The term "RBD" refers to the Receptor Binding Domain of the novel coronavirus (Receptor Binding Domain) which is located in the S1 subunit of the S protein as shown in FIG. 1. Further, in the present application, the RBD may be a peptide fragment between SARS-CoV-2 spike protein (S protein) 310 and 560 amino acids or a variant thereof, and may also be truncated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 20, 25 or 30 amino acids from the N-terminal or C-terminal, or extended by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 20, 25 or 30 amino acids from the N-terminal or C-terminal. In the present application, the RBD may be a peptide fragment between SARS-CoV-2 spike protein (S protein) 319-541 amino acids or a variant thereof, and may also be suitably truncated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 20, 25 or 30 amino acids from the N-or C-terminus, or extended by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 20, 25 or 30 amino acids from the N-or C-terminus. In the present application, the RBD may be a peptide fragment between SARS-CoV-2 spike protein (S protein) 319-534 amino acids or a variant thereof, and may also be suitably truncated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 20, 25 or 30 amino acids from the N-or C-terminus, or extended by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 20, 25 or 30 amino acids from the N-or C-terminus. In the present application, the extended amino acid may be the amino acid of the RBD itself, or may be an amino acid tag added to obtain a specific function, such as adding an amino acid sequence at the C-terminus for proper protein deposition. In the present application, "variant" generally refers to a sequence that differs from a reference sequence by the inclusion of one or more differences (mutations). The difference may be a substitution, deletion or insertion of one or more amino acids.

The terms "RBD variant", "term M5", and term "M5K" refer to a novel coronavirus receptor binding domain variant, as distinguished from the wild type (RBDwt, RBD wild type) of RBD. In some embodiments, the new variant coronavirus differs from the wild-type strain by 1 or more amino acids. In some embodiments, the RBD variant has a mutation in the amino acid sequence at any one or more of K417, L452, T478, E484, and N501, as compared to RBDwt. Further, in some embodiments, the RBD variant has an amino acid sequence with any one or more of mutations at K417N, L452R, T478K, E484Q/K, and N501Y, as compared to RBDwt. According to one embodiment of the present application, M5 is a coronavirus spike protein RBD variant having mutation sites of K417N, L452R, T478K, E484Q, and N501Y. According to one embodiment of the present application, M5K is a coronavirus spike protein RBD variant having mutation sites of K417N, L452R, T478K, E484K, and N501Y.

The terms "RBD variant glycoprotein", and "RBD variant glycoprotein" refer to a glycoprotein produced by expression of an RBD variant in a vector. In some embodiments, it can be prepared by recombinant expression of e.coli, mammalian cells, yeast cells. In some embodiments, the RBD variant glycoprotein is prepared using glycoengineered yeast; preferably, the RBD variant glycoprotein is prepared by using yeast CGMCC No.19488 (i.e., Pichia pastoris genetically modified through glycosylation modification pathway, which is a strain with the preservation number of CGMCC No.19488 and is preserved in the China general microbiological culture Collection center). The present application does not limit the manner of obtaining the RBD variant glycoprotein, and RBD variant glycoproteins obtained in any other manner consistent with the structure, function, etc. of the present invention are also within the scope of the present invention.

According to one embodiment of the invention, the amino acid sequence of the M5 variant of the RBD of SARS-CoV-2 is: (a1) as shown in SEQ ID NO:1, or a fragment thereof. In some embodiments, the amino acid sequence of the M5 variant of the RBD of SARS-CoV-2 is: (a2) SEQ ID NO:1 by substitution, deletion and/or addition of one or more amino acid residues. In some embodiments, the amino acid sequence of the M5 variant of the RBD of SARS-CoV-2 is: (a3) (a1) and a truncation of the sequence shown in (a 2). In some embodiments, the amino acid sequence set forth in (a2) (a3) is identical to SEQ ID NO:1 is a protein with the same conformation or the same function. In other embodiments, the M5 variant of SARS-CoV-2 RBD is a variant of SEQ ID NO:1 has a homology of 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more, and has the same conformation or the same function.

According to one embodiment of the invention, the amino acid sequence of the M5K variant of the RBD of SARS-CoV-2 is: (b1) as shown in SEQ ID NO:3, respectively. In some embodiments, the amino acid sequence of the M5K variant of the RBD of SARS-CoV-2 is: (b2) SEQ ID NO:3 by substitution, deletion and/or addition of one or more amino acid residues. In some embodiments, the amino acid sequence of the M5K variant of the RBD of SARS-CoV-2 is: (b3) (b1) and a truncation of the sequence shown in (b 2). In some embodiments, the amino acid sequence set forth in (b2) (b3) is identical to SEQ ID NO:3 is a protein with the same conformation or the same function. In other embodiments, M5 variant K of the RBD of SARS-CoV-2 is a variant of the amino acid sequence of SEQ ID NO:3 has a homology of 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more, and has the same conformation or the same function.

According to one embodiment of the invention, the amino acid sequence of M5 variant of RBD of SARS-CoV-2 includes histag, specifically: (c1) as shown in SEQ ID NO:5, respectively. In some embodiments, the amino acid sequence of the M5 variant of the RBD of SARS-CoV-2 is followed by histag, specifically: (c2) SEQ ID NO:5 by substitution, deletion and/or addition of one or more amino acid residues. In some embodiments, the amino acid sequence of the M5 variant of the RBD of SARS-CoV-2 is followed by histag, specifically: (c3) (c1) and a truncation of the sequence shown in (c 2). In some embodiments, the amino acid sequence set forth in (c2) (c3) is identical to SEQ ID NO:5 is a protein with the same conformation or the same function. In other embodiments, the M5 variant of SARS-CoV-2 RBD is a variant of SEQ ID NO:5 has a homology of 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more, and has the same conformation or the same function.

According to one embodiment of the present invention, the 3 amino acid sequences shown in SEQ ID NO 1, SEQ ID NO 3 and SEQ ID NO 5 are designed and mutated based on a portion of the S protein of SARS-CoV-2"Wuhan-Hu-1" isolate with GenBank number MN 908947.3.

In some embodiments, the term "homology" in the above proteins refers to the identity of amino acid sequences. In some embodiments, the identity of the amino acid sequences can be determined using homology search sites on the internet, such as the BLAST web page of the NCBI home website. For example, in the advanced BLAST2.1, by using blastp as a program, setting the value of Expect to 10, setting all filters to OFF, using BLOSUM62 as a Matrix, setting Gap existence cost, Per residual Gap cost, and Lambda ratio to 11, 1, and 0.85 (default values), respectively, and performing a calculation by searching for the identity of a pair of amino acid sequences, a value (%) of identity can be obtained.

The term "vector" generally refers to a nucleic acid molecule capable of self-replication in a suitable host, which transfers the inserted nucleic acid molecule into and/or between host cells. The vector may include a vector mainly for inserting a DNA or RNA into a cell, a vector mainly for replicating a DNA or RNA, and a vector mainly for expression of transcription and/or translation of a DNA or RNA. The vector also includes vectors having a plurality of the above-described functions. The vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, the vector will produce the desired expression product by culturing a suitable host cell containing the vector.

In some embodiments of the present invention, the term "recombinant vector" is specifically a recombinant vector obtained by cloning the gene encoding the coronavirus S protein receptor binding domain variant (M5 or M5K) into an expression vector, such as pPICZ α A vector (which contains a cleavage site, e.g., the cleavage sites are XhoI and NotI). In other embodiments, the expression vector is selected from one or more of the following vectors: pPIC9, pPIC9K, pPICZ alpha B, pPICZ alpha B vector, pET series vector, pGEX series vector, pMAL series vector, pQE series vector, pBADmycHis series vector, pTrcHis series vector, pTXB series, T series vector and other vectors and the modified vector of the above vectors. The present application does not limit the manner of obtaining the recombinant vector, and the recombinant vector obtained by other manners is also within the scope of the present application.

The terms "coding gene", "nucleic acid molecule" refer to a Ribonucleotide (RNA) or Deoxyribonucleotide (DNA) sequence that can encode a peptide chain of a particular amino acid. In some embodiments, the encoding gene or nucleic acid molecule can be obtained by whole gene synthesis, PCR amplification, chemical synthesis, and the like. The present application is not limited to the manner in which the encoding gene and nucleic acid molecule are obtained.

In some embodiments, the gene or nucleic acid molecule encoding a coronavirus S protein receptor binding domain variant (M5 or M5K) may be a nucleic acid molecule encoding a polypeptide of SEQ ID NO: 1. SEQ ID NO: 3. SEQ ID NO: 5. SEQ ID NO:1 or SEQ ID NO:3 or SEQ ID NO:5 by substitution, deletion and/or addition of one or more amino acid residues, and the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:3 or SEQ ID NO:5 and a truncation of the sequence shown in SEQ ID NO:1 or SEQ ID NO:3 or SEQ ID NO:5 has at least 99%, at least 95%, at least 90%, at least 85% or at least 80% homology with the protein represented by 5.

According to one embodiment of the invention, the gene sequence (DNA sequence) encoding the RBD M5 variant protein of SAR-CoV-2 is: (d1) the amino acid sequence of SEQ ID NO: 2. In some embodiments, the gene sequence encoding the RBD M5 variant protein of SARS-CoV-2 is: (d2) SEQ ID NO:2 by replacing one or more bases, deleting and/or adding 3 times of bases. In some embodiments, the gene sequence of the RBD M5 variant protein of SARS-CoV-2 is: (d3) (d1) and a truncation of the sequence shown in (d 2). In some embodiments, the nucleic acid sequence set forth in (d2) (d3) is identical to SEQ ID NO:2 and encoding a coronavirus S protein receptor binding domain variant (M5), or a DNA molecule which hybridizes under stringent conditions with the DNA molecule of SEQ ID NO. 2 and encodes a coronavirus S protein Receptor Binding Domain (RBD).

According to one embodiment of the invention, the gene sequence (DNA sequence) encoding the RBD M5K variant protein of SAR-CoV-2 is: (e1) SEQ ID NO:4, respectively. In some embodiments, the gene sequence encoding the RBD M5K variant protein of SARS-CoV-2 is: (e2) the amino acid sequence of SEQ ID NO:4 by replacing one or more bases, deleting and/or adding 3 times of bases. In some embodiments, the RBD M5K variant protein of SARS-CoV-2 has the gene sequence: (e3) (e1) and a truncation of the sequence shown in (e 2). In some embodiments, the nucleic acid sequence set forth in (e2) (e3) is identical to SEQ ID NO:4 and encoding coronavirus S protein receptor binding domain variant (M5K), a nucleic acid sequence encoding a protein that is more than 80% homologous to the coronavirus spike glycoprotein RBD variant protein, or a DNA molecule that hybridizes under stringent conditions with the DNA molecule of SEQ ID NO:4 and encodes coronavirus S protein Receptor Binding Domain (RBD).

According to one embodiment of the invention, the RBD M5 variant protein of SAR-CoV-2 is followed by histag. According to one embodiment of the invention, the gene sequence (DNA sequence) encoding the RBD M5 variant protein of SAR-CoV-2 is: (f1) SEQ ID NO: 6. In some embodiments, the gene sequence encoding the RBD M5 variant protein of SARS-CoV-2 is: (f2) SEQ ID NO:6 by replacing one or more bases, deleting and/or adding 3 times of bases. In some embodiments, the gene sequence of the RBD M5 variant protein of SARS-CoV-2 is: (f3) (f1) and (f 2). In some embodiments, the nucleic acid sequence set forth in (f2) (f3) is identical to SEQ ID NO:6, or a DNA molecule which has a homology of 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more and encodes a coronavirus S protein receptor binding domain variant (M5), or a DNA molecule which hybridizes under stringent conditions with the DNA molecule of SEQ ID NO. 6 and encodes a coronavirus S protein Receptor Binding Domain (RBD).

According to one embodiment of the invention, the nucleotide sequences shown in SEQ ID NO 2, SEQ ID NO 4 and SEQ ID NO 6 are according to SEQ ID NO: 1. the amino acid sequence of SEQ ID NO:3 and SEQ ID NO 5 are obtained by codon optimization. Further, in one embodiment of the present application, the nucleotide sequences shown in SEQ ID NO 2, SEQ ID NO 4 and SEQ ID NO 6 are DNA fragments obtained by whole gene synthesis. In the present application, the manner of obtaining the nucleotide sequence is not limited, and the nucleotide sequence obtained in any other manner is also within the scope of the present application.

In some embodiments, in the description of genes, the term "homology" refers to the identity of nucleotide sequences. In some embodiments, the identity of the nucleotide sequences may be determined using homology search sites on the internet, such as the BLAST web page of the NCBI home website. For example, in the advanced BLAST2.1, by using blastp as a program, setting the value of Expect to 10, setting all filters to OFF, using BLOSUM62 as a Matrix, setting Gap existence cost, Per residual Gap cost, and Lambda ratio to 11, 1, and 0.85 (default values), respectively, and performing a calculation by searching for the identity of a pair of nucleotide sequences, a value (%) of identity can be obtained.

In some embodiments, the term "homology of 95% or more" in the above proteins and genes may be at least 96%, 97%, 98% identity. The term "90% or more identity" may be at least 91%, 92%, 93%, 94% identity. The term "homology of 85% or more" may be at least 86%, 87%, 88%, 89% identity. The term "homology of 80% or more" may be at least 81%, 82%, 83%, 84% identity.

The term "DNA molecule hybridization" refers to a DNA molecule having complementary base sequences, forming a stable double-stranded region by forming hydrogen bonds or the like between base pairs. Before the hybridization of DNA molecules, the DNA molecules of two organisms are extracted from the cells, and the double-stranded DNA molecules are separated into single strands by heating or increasing the pH, a process known as denaturation. Then, the DNA single strands of the two organisms are put together and hybridized, wherein the DNA single strand of one organism is labeled with an isotope in advance. Double-stranded regions can be formed if complementary portions are present between two biological DNA molecules. Since the isotope is detected with high sensitivity, even a one-part-per-million double-stranded region is formed between two kinds of biological DNA molecules, it can be detected.

In some embodiments of the present application, the DNA molecule hybridization is performed under "stringent conditions". In some embodiments, stringent conditions may be: DNA molecules (e.g., as shown in SEQ ID NO:2 or SEQ ID NO:4 or SEQ ID NO: 6) were incubated at 50 ℃ in 7% Sodium Dodecyl Sulfate (SDS), 0.5M NaPO₄Hybridizing with a mixed solution of 1mM EDTA; rinse at 50 ℃ in 2 XSSC, 0.1% SDS. In other embodiments, stringent conditions may also be: DNA molecules (e.g., as shown in SEQ ID NO:2 or SEQ ID NO:4 or SEQ ID NO: 6) were subjected to conditions of 50 ℃ in 7% SDS, 0.5M NaPO₄And 1mM EDTA, and rinsed at 50 ℃ in 1 XSSC, 0.1% SDS. In other embodiments, stringent conditions may also be: DNA molecules (e.g., as shown in SEQ ID NO:2 or SEQ ID NO:4 or SEQ ID NO: 6) were subjected to conditions of 50 ℃ in 7% SDS, 0.5M NaPO₄And 1mM EDTA, and rinsed at 50 deg.C in 0.5 XSSC, 0.1% SDS. In other embodiments, stringent conditions may also be: DNA molecules (e.g., as shown in SEQ ID NO:2 or SEQ ID NO:4 or SEQ ID NO: 6) were subjected to conditions of 50 ℃ in 7% SDS, 0.5M NaPO₄And 1mM EDTA, and rinsed at 50 deg.C in 0.1 XSSC, 0.1% SDS. In other embodiments, stringent conditions may also be: DNA molecules (e.g., as shown in SEQ ID NO:2 or SEQ ID NO:4 or SEQ ID NO: 6) were subjected to conditions of 50 ℃ in 7% SDS, 0.5M NaPO₄And 1mM EDTAHybridization in solution, rinsing in 0.1 XSSC, 0.1% SDS at 65 ℃. In other embodiments, stringent conditions may also be: DNA molecules (e.g., as shown in SEQ ID NO:2 or SEQ ID NO:4 or SEQ ID NO: 6) were hybridized at 65 ℃ in a solution of 6 XSSC, 0.5% SDS, and then washed once each with 2 XSSC, 0.1% SDS and 1 XSSC, 0.1% SDS. The reaction conditions for hybridization of DNA molecules are not limited in the present application.

The term "host cell" generally refers to an individual cell, cell line or cell culture that may or may already contain a plasmid or vector comprising a nucleic acid molecule described herein, or that is capable of expressing an antibody or antigen-binding fragment thereof described herein. The host cell may comprise progeny of a single host cell. Due to natural, accidental, or deliberate mutation, the progeny cell may not necessarily be identical in morphology or in genome to the original parent cell, but may be capable of expressing the fusion protein described herein. Host cells can be obtained by transfecting cells in vitro with the vectors described herein. The host cell may be a prokaryotic cell (e.g., E.coli) or a eukaryotic cell (e.g., a yeast cell, such as a COS cell, a Chinese Hamster Ovary (CHO) cell, a HeLa cell, a HEK293 cell, a COS-1 cell, an NS0 cell, or a myeloma cell). In some embodiments, the host cell is a mammalian cell. For example, the mammalian cell may be a CHO cell.

The terms "recombinant expression cell", "recombinant cell" refer to a cell having a foreign gene integrated into its genome, or a host cell containing an expression vector in vivo. In the present invention, the recombinant expression cells may be cells in which a gene encoding a novel coronavirus M5 variant or M5K variant is integrated into the genome, and further glycoprotein of M5 variant or M5K variant is synthesized and prepared. In some embodiments, the gene encoding the coronavirus S protein receptor binding domain variant (M5 or M5K) can be obtained by introducing the gene into Escherichia coli, a mammalian cell, a yeast cell, or Pichia pastoris genetically modified by a glycosylation modification pathway.

In some embodiments of the present application, the recombinant expression cells are prepared by introducing the genes encoding the coronavirus S protein receptor binding domain variant (M5 or M5K) into host cells such as Escherichia coli, mammalian cells, yeast cells, or Pichia pastoris genetically modified through glycosylation modification pathways, in the form of a recombinant vector. The present application does not limit the manner of obtaining the recombinant expression cells, and the recombinant expression cells or recombinant cells of the present invention obtained in other manners are also within the scope of the present invention.

In some embodiments, the method of purifying to obtain the novel coronavirus S protein receptor binding domain variant M5 or M5K comprises: and (3) sequentially carrying out cation exchange chromatography, hydrophobic chromatography, G25 desalting and anion exchange chromatography on the supernatant obtained by culturing the recombinant expression cells. Further, in other examples, M5-histag or M5K-histag (sequence obtained after addition of a flexible linker and 6 histidine tags after RBD) can also be obtained by nickel affinity chromatography purification.

Further, in some embodiments, the method of purifying a coronavirus S protein receptor binding domain variant comprises: capturing target protein of the supernatant of the culture group expression cells through a CaptoMMC chromatographic column, and then eluting through a buffer solution containing 1M NaCl to obtain a crude sample containing the target protein; then, the crude sample is purified by a hydrophobic chromatography column Phenyl HP, the elution peak sample containing the target protein is desalted by a G25 chromatography column, then the hybrid protein is adsorbed by an anion exchange chromatography column Source30Q, and the flow-through solution is the target protein. In some embodiments, the protein of interest is a coronavirus S protein receptor binding domain with a mammalian glycoform structure N-sugar chain modification.

According to research reports, a plurality of mutations are found in a new crown variant strain, and each mutation has different understandings, for example, a K417N mutation site is found in a Beta variant strain, and the mutation at the site is found to possibly lead to the enhancement of the binding capacity of the virus to an ACE2 receptor; the N501Y mutation site appears in Alpha, Beta and Gamma variant strains; the Omicron variant contains an E484A mutation site, and the E484K mutation site which is found in the Beta variant before is considered as an important mutation causing immune escape, but the Omicron contains 32 mutation sites in the S protein, wherein 15 mutations in the RBD region exist, so that the information is richer.

Current studies have shown that SARS-CoV-2 invades human cells via the receptor ACE2 (references: Danel Wrapp, Nianshoung Wang, Kizmekia S.Corbett.A.Goldgith, Ching-Lin Hsieh, Olubukola Abiona, Barney S.Graham, Jason S.Mclellan.Cryo-EM structure of the 2019-nCoV spike in the prediction compatibility. SCIENCE.19 Feb 2020.Vol 367, Issue 6483. pp.1260-1263.). Based on the vaccine research of the full-length SARS-CoV-2S protein, the S protein can induce the organism to generate a neutralizing antibody aiming at SARS-CoV-2, and the RBD region of the SARS-CoV-2S protein is used as an independent structural domain, can form correct conformation, contains a plurality of spatial structure dependent antigen epitopes, and is one of the main studied antigens of subunit vaccines. Namely, the RBD is one of the tested antigens except for the S1 region, the S2 region, the full-length S region and the nucleoprotein. However, the SARS-CoV-2S protein RBD has two potential N-glycosylation sites, and the correct glycoform structure plays an important role in maintaining the natural conformation and immunogenicity of the RBD.

The invention provides several new designed RBD variant glycoproteins, wherein the M5 variant contains 5 amino acid mutations, namely K417N, L452R, T478K, E484Q and N501Y; the M5K variant contains 5 amino acid mutations, K417N, L452R, T478K, E484K, N501Y, respectively. These mutations occur in thousands of mutation sites that have been found by sequencing, but not from a particular variant.

The present invention will now be illustrated by way of examples. The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice of the present invention, as will be apparent to those skilled in the art. All publications and other references mentioned herein are incorporated by reference in their entirety. In case of inconsistencies, the present specification, including definitions, will prevail. The materials, methods, and examples are illustrative only and not intended to be limiting.

pPICZ α A, GS115 Pichia pastoris is a product of Invitrogen corporation. The Pichia pastoris 19488 strain is preserved in China general microbiological culture Collection center with the preservation number of CGMCC 19488.

The Q5 enzyme, Taq enzyme, dNTPs, restriction enzyme, T4 ligase and the like used in the experiment are purchased from NEB company, and pfu enzyme, kit and DH5 alpha competent cells are products of Beijing Quanjin Limited company. Total gene synthesis, nucleotide synthesis, primer synthesis, sequencing and the like are provided by Shanghai Biotechnology service, Inc.

SARS-CoV-2(2019-nCoV) Spike RBD-Fc Recombinant Protein (40592-V02H) is a product of Beijing Yi Qiao Shenzhou Biotechnology Co., Ltd; the goat anti-rabbit IgG secondary antibody (SAB3700885) is a product of Sigma company; the goat anti-mouse IgG secondary antibody (ab205719) is a product of abcam company; BglII restriction enzyme is a product of NEB corporation. Pseudoviruses were purchased from Nanjing Novozam.

The Capto MMC chromatography media used in the experiments, Phenyl HP, G25, Source30Q, were all available from GE Healthcare.

The RBD variant of the present application is illustrated by taking the M5 variant as an example.

Example 1M 5 Gene acquisition and construction of recombinant Yeast strains

Firstly, obtaining M5 gene and constructing yeast expression vector

According to the 319 th to 534 th amino acids (R319-V534) of the S protein of SARS-CoV-2 'Wuhan-Hu-1' isolate K417N, L452R, T478K, E484Q and N501Y loci, a sequence SEQ ID NO 1 is obtained, then codon optimization is carried out, and a coding gene sequence SEQ ID N O2 which is assigned as SARS-CoV-2M5 and delegated to the whole gene synthesis of prokaryotae organism limited is inserted between XhoI and NotI enzyme cutting sites of pPICZ alpha A vector, so as to obtain a recombinant expression vector pPICZ alpha-M5, namely an M5 protein expression vector. The constructed vector was subjected to PCR using 5 'AOX and 3' AOX as primers, followed by analysis by 1% agarose gel electrophoresis. FIG. 2 is a diagram of a screening gel electrophoresis analysis of pPICZ α -M5 expression vector according to one embodiment of the present invention. The analysis result is shown in FIG. 2, and the size of the positive control band is consistent with that of the expression vector pPICZ alpha-M5 successfully constructed. And (4) sequencing the clones which are analyzed to be positive by electrophoresis, wherein the sequencing results are consistent with theories.

Secondly, the recombinant expression vector pPICZ alpha-M5 is transformed into yeast CGMCC No.19488

Yeast were streaked on YPD plates for recovery and isolation of single clones. Selecting recovered monoclonal antibody, inoculating into YPD liquid culture medium, culturing in test tube to logarithmic phase, taking 1ml, transferring into 100ml YPD shake flask, shake-culturing at 25 deg.C and 200rpm to OD₆₀₀Centrifuging at 1500g and 4 deg.C for 5min to 1.3-1.5, discarding supernatant, resuspending with equal volume of precooled distilled water, centrifuging at 1500g and 4 deg.C for 5min, discarding supernatant, and repeating the step for 3 times; resuspend it with equal volume of precooled 1M sorbitol, centrifuge at 1500g 4 ℃ for 5min, discard the supernatant and repeat this step 3 times. The above cell pellet washed with 3 times of distilled water and 3 times of sorbitol was suspended by adding 1ml of 1M sorbitol, and 100. mu.l of each pellet was dispensed into a sterile centrifuge tube and stored at-80 ℃.

About 10 mu g of the constructed expression plasmid pPICZ alpha-M5 is subjected to single-point linearization by using a restriction enzyme BglII, and the restriction enzyme digestion system (50 mu L) is as follows: the expression plasmid pPICZ alpha-M543 mu L, BglII 2 mu L, 10 XNEB 3.1 buffer 5 mu L, after cutting enzyme at 37 ℃ for 1h, sampling, separating by 1% agarose gel electrophoresis, analyzing whether the plasmid is completely linearized. FIG. 3 is a drawing of a linearized electrophoresis analysis of pPICZ α -M5 expression vector BglII according to one embodiment of the present invention. The separation results are shown in FIG. 3, and the completely linearized digested product was subjected to fragment recovery using a DNA fragment recovery kit of the centrifugal column type, and finally the linearized plasmid was eluted with 15. mu.L of pure water.

Taking the linearized expression plasmid pPICZ alpha-M515 mu L, adding 100 mu L of pichia pastoris (preservation number is CGMCC No.19488) to the electric shock transformation competent cells, mixing the mixture evenly, transferring the mixture into a precooled 0.2cm electric transfer cup, and placing the cup on ice for 5 min. According to the requirements of yeast electrotransfer manual, 900 microliter of precooled 1M sorbitol is quickly added after 2kV voltage electric shock, and is transferred into a clean test tube and is placed in an incubator at 25 ℃ for standing for 2 hours. Then, 1ml of YPD liquid medium without antibiotic addition was added thereto, and shake-cultured at 25 ℃ and 200rpm for 3 to 4 hours. The bacterial liquid obtained by shaking culture is taken out, 300 mu L of the bacterial liquid is coated on a YPD plate with screening resistance of Zeocin, and the bacterial liquid is inversely cultured for 60 to 72 hours at the temperature of 25 ℃.

Screening of recombinant expression strains

After the coated plate had grown single clones, 6 single clones were randomly picked and inoculated onto new YPD/Zeocin plates, and incubated in an inverted incubator at 25 ℃. After bacterial colony grows out, inoculating the bacterial colony into 3ml YPD/Zeocin liquid culture medium, carrying out shake culture at 25 ℃ and 200rpm, after bacterial liquid grows to be thick, transferring the bacterial colony into 3ml BMGY culture medium according to the inoculation amount of 5% (volume percentage content), carrying out shake culture at 25 ℃ and 200rpm in the culture medium, and supplementing 0.5% (V/V) methanol for induction every 12 hours after 48 hours. After 48h of induction, the culture supernatants were collected at 12000rpm for 3min for SDS-PAGE detection.

FIG. 4 is a clone screen of CGMCC19488/pPICZ α -M5 according to one embodiment of the invention. The results are shown in FIG. 4. As can be seen, SDS-PAGE analysis shows that 1#, 2#, 4#, 10#, 11#, and 12# clones have different levels of protein expression, wherein 10# has higher expression level and fewer background bands, and is selected as the next experimental clone strain and named as CGMCC19488/pPICZ alpha-M5.

Example 2 expression and purification of recombinant M5 glycoprotein

Firstly, culturing a recombinant strain CGMCC19488/pPICZ alpha-M5

The positive clones identified in example 1 (i.e., recombinant strain CGMCC 19488/pPICZ. alpha. -M5) were picked and inoculated into YPD/Zeocin liquid medium, cultured at 25 ℃ and 200rpm to OD₆₀₀15-20 percent of the total amount of the culture medium is inoculated to BMGY culture medium in an inoculation amount of 5% (V/V), the BMGY culture medium is cultured at 25 ℃ and 200rpm for 24 hours, then methanol with the volume percentage of 0.5 percent is added to induce the expression of M5, the expression is induced once every 12 hours, samples are taken to detect the expression condition, and culture supernatant is collected by centrifugation after 36 hours of induction.

FIG. 5 is a graph of IDECGMCC19488/pPICZ α -M5 electrophoresis at different induction times according to an embodiment of the invention. SDS-PAGE detection of different induction times is shown in FIG. 5. As can be seen, the expression level of the target protein is increased along with the increase of the induction time.

Purification of di, M5 protein

1. Cation exchange chromatography

Adjusting the pH of the culture supernatant which is induced to express for 36 hours in the first step to 5.5, and purifying the culture supernatant by using a Capto MMC chromatography medium, wherein the mobile phase comprises the following components:

a: 20mM pH5.5 PB (phosphate buffer);

B：100mM pH8.5 Tris-HCl+1M NaCl。

after the end of the loading, the column was equilibrated with A and then eluted with B.

2. Hydrophobic chromatography

Purifying a sample purified by Capto MMC by using Phenyl HP, eluting hybrid protein by using 40% (volume percentage content) B, eluting target protein by using 20% (volume percentage content) B, wherein the mobile phase comprises the following components:

a: 20mM pH7.5Tris-HCl +1M AS (ammonium sulfate);

B：20mM pH7.5 Tris-HCl

3. desalination by G25

Desalting Phenyl HP purified sample with G25 chromatography medium, collecting protein sample, and mobile phase composition: 20mM Tris-HCl pH 8.5.

4. Anion exchange chromatography

The desalted sample was purified using Source30Q chromatography media with mobile phase composition:

A：20mM pH8.5 Tris-HCl；

B：20mM pH8.5 Tris-HCl+1M NaCl。

after the end of the loading, the column was equilibrated with A and then eluted with B.

5. Cation exchange chromatography

After Source30Q the sample was adjusted to pH 6.5 with HCl and subsequently purified with a Source 30S column with mobile phase composition:

A：20mM pH6.5 PB

B：20mM pH7.0 PB+1M NaCl

the M5 protein can be captured by Capto MMC through SDS-PAGE electrophoresis; after the sample is purified by the Source30Q, the target protein flows through, and almost all the hetero-protein is adsorbed on the Source30Q chromatographic medium. FIG. 6 is an SDS-PAGE pattern of a purified sample of M5 according to one embodiment of the invention. The results of SDS-PAGE are shown in FIG. 6.

Example 3 purification of mammalian cells expressing recombinant M5-histag protein and M5-histag protein

Adding a flexible joint and 6 histidine tags at the C-terminal end of SEQ ID NO.1 to obtain a sequence SEQ ID NO. 5 and a corresponding coding gene sequence SEQ ID NO. 6, carrying out Nhel/Notl double enzyme digestion on the complete target gene shown in SEQ ID NO. 6, and then connecting the complete target gene to a eukaryotic expression vector subjected to the same enzyme digestion to obtain a recombinant vector pcDNA3.1/M5; the recombinant vector is transformed into escherichia coli, plasmid amplification is carried out according to a conventional method, and then plasmids are extracted by using a reagent kit of Tiangen biology, Inc.

Preparing a DNA-liposome mixture according to a Lipofectin kit manual, transfecting Chinese hamster CHO cells, and incubating for 2h at 37 ℃; the culture medium was changed to DMEM medium containing 10% BSF, and the culture was continued for 48 hours. Cloning and screening with Neomycin resistance, separating transfected cells from culture flask at 1 × 10⁵The cells/well are added into a 96-well plate, the transfected cells are continuously cultured in a DMEM medium (with 10% BSF) containing 500 mu g/mL Neomycin, and after 7 days, the cells forming clones are selected and are amplified and cultured into a 6-well plate.

The NEO resistant clones were cultured at 1.5X 10⁵Cell density in/mL in a flask containing 5% CO₂Culturing at 37 deg.C for 72h in incubator, and collecting supernatant to obtain M5 protein. The obtained supernatant was identified and analyzed for M5-histag protein content.

Through identification, both SEQ ID NO. 5 and SEQ ID NO. 6 can express the novel coronavirus M5-histag protein, the protein expressed by SEQ ID NO. 5 is concentrated through a 10kDa membrane package, meanwhile, a low-salt buffer solution is used for replacing a culture medium in the protein, and then a 10kDa ultrafiltration tube is used for further concentration. After dilution, the concentrate can be purified by nickel affinity chromatography for further use.

Example 4, M5 mouse immunization experiment

Immunization methods have been disclosed in various documents, such as replication of animal models of human diseases, compiled by the Lei-talent, published by the national institutes of health. In particular asThe following: 75 female Balb/c mice aged 6-8 weeks old are taken and randomly divided into the following 6 groups: 10 μ g RBDwt immunization group (n ═ 10), 2.5 μ g RBDwt immunization group (n ═ 10), 10 μ g M5 immunization group (n ═ 10), 2.5 μ g M5 immunization group (n ═ 15), adjuvant group (CpG + al (oh)₃) (n-15) and saline group (NS) (n-15). Wherein group 1-2 are prepared RBDwt glycoprotein expressed by CGMCC19488, and respectively contain 10 μ g RBDwt or 2.5 μ g RBDwt and 100 μ g Al (OH) in volume of 100 μ l ₃50 ug of CpG was mixed with normal saline to prepare vaccine. Group 3-4M 5 is the M5 glycoprotein expressed in CGMCC19488 prepared above, and contains 10 μ g M5 or 2.5 μ g M5 and 100 μ g Al (OH) in volume of 100 μ l ₃50 ug of CpG was mixed with normal saline to prepare vaccine. Group 5 adjuvant groups 100. mu.g Al (OH)₃50 ug of CpG was adjuvanted with physiological saline. Group 6 is normal saline, i.e. normal saline group. Each group was immunized with 100. mu.l of muscle on days 0 and 14, and blood was collected on day 28, and 5 cytokines were measured 10 days after the second immunization in each of 4-6 groups.

The antibody titer against RBD in the sera of the groups of mice was determined by indirect ELISA. The RBDwt and M5 expressed by CGMCC19488 prepared in the previous stage are used for plate wrapping, and other operation steps are shown in the finely compiled molecular biology experimental manual [ M ] scientific Press, 2008.

Fig. 7A-7B are serum anti-RBDwt, M5 antibody titers in mice after 14 days of hyperimmunization according to one embodiment of the invention. The results are shown in FIGS. 7A-7B. As can be seen from the figure: 1-4 RBDwt and M5 immune groups generate specific antibodies for resisting various variant RBD, and the antibody titer can reach 1: 1000000, while the control group had only 1: 100.

example 5 virus neutralization assay

In example 3, 1-5 groups of mice were serum-collected 14 days after the second immunization, incubated at 56 ℃ for 30min, and diluted with physiological saline at a given dilution. Virus neutralization assays were performed according to conventional methods (ref: Nie J, Li Q, Wu J, et al.Quantionantification of SARS-CoV-2neutral antibody by a particulate virus-based assay Nat Protoc. 2020; 15(11):3699-3715. doi:10.1038/s 41596-020-0394-5). The method comprises the following steps:

1. preparing cells: HEK293-ACE2 cells (Vazyme, nanjing, cat # DD1401) were digested.

2. Serum dilution: the dilution was carried out by diluting with DMEM medium (Gibco, cat # C11995500BT) containing 10% FBS (Excell, cat # FND100) and 1% diabody (Vitrent, cat # 450-.

3. Filtering the serum: the mixture was filtered through a 0.22 μm filter.

4. Gradient dilution: the first well was added with 150. mu.L of diluted filtered serum, 50. mu.L were transferred to 100. mu.L of medium (3-fold dilution) in the next dilution well, and a total of 6 dilutions (containing the first well) were set. Virus control wells were filled with 100 μ L of medium and cell control wells with 150 μ L of medium.

5. And (3) diluting the virus: according to the TCID of the virus (SARS-CoV-2-Fluc WT: Vazyme, cat # DD 1702; SARS-CoV-2-Fluc B.1.1.529: Vazyme, cat # DD 1768; SARS-CoV-2-Fluc B.1.617.2: Vazyme, cat # DD 1754; SARS-CoV-2-Fluc 501 Y.V2-1: Vazyme, cat # DD1741)₅₀Value, diluted to 2X 10⁴TCID50/mL。

6. And (3) neutralization reaction: mu.L of virus dilutions (50-fold final dilution of the first well in groups 1-4: WT, 501Y.V2-1, B.1.617.2, 30-fold final dilution of the first well in group B.1.1.529; 30-fold final dilution of the first well in group 5: wild strain WT, Beta (501Y.V2-1), Delta (B.1.617.2), and Omicron (B.1.1.529)) were added to the sample wells and virus control wells, respectively, mixed by shaking, and neutralized at 37 ℃ for 1 hour.

7. Adding cells: dilute cells to 2 × 10⁴cells/50. mu.L, adding 50. mu.L of cell diluent into each well, shaking and mixing, culturing in a CO2 incubator at 37 ℃ for 48 hours, taking out the cell plate, balancing to room temperature, adding a reporter gene reagent for detection (Vazyme, cat # DD1201), and recording data.

FIGS. 8A-8H show the results of a pseudovirus neutralization assay according to one embodiment of the present invention. Wherein, the pseudovirus of FIG. 8A-FIG. 8B is coronavirus wild strain; FIGS. 8C-8D show pseudoviruses of the variant strain of coronavirus Beta; FIGS. 8E-8F are pseudoviruses of coronavirus Delta variants; FIGS. 8G to 8H show pseudoviruses of the Omicron variant coronavirus. As shown in FIGS. 8A-8H, for the neutralizing activity of SARS-CoV-2-Fluc WT pseudovirus, the titers of the high-dose and low-dose groups of the serum of the RBDwt immune group are higher than those of the M5 immune group, and the difference between the high-dose groups is more significant;

the neutralizing activity to SARS-CoV-2-Fluc Beta (501Y.V2-1) pseudovirus, the serum of the RBDwt immune group has no significant difference with the serum of the M5 immune group;

the neutralizing activity to SARS-CoV-2-Fluc Delta (B.1.617.2) pseudovirus, the serum of the RBDwt immune group has no significant difference with the serum of the M5 immune group;

the neutralizing activity to SARS-CoV-2-Fluc Omicron (B.1.1.529) pseudovirus is obviously lower in the serum of the RBDwt immune group than in the serum of the M5 immune group.

From the above studies, it was found that not only can the M5 variant vaccine effectively neutralize the SARS-CoV2 wild strain pseudovirus, but we also surprisingly found that the variant vaccine can also neutralize novel coronavirus Beta, Delta variants, and newly emerged Omicron variants, i.e., M5 can neutralize a variety of "cut-off variants" (VOCs) defined by WHO, and that the neutralizing activity of M5 is 10 regardless of the pseudovirus^2.2-10^2.7This makes M5 vaccine a potential broad spectrum candidate for a new coronavirus.

Example 6 cytokine detection

Spleen cells were taken 10 days after the second immunization from 5 mice in 4-6 groups in example 3, and a negative control group (3 duplicate wells), an experimental group (3 duplicate wells), and a positive control group (2 duplicate wells) were set for the experiment. The ELISpot assay was performed according to the conventional method (ref: X Liu et al. identification of T Cell Epitopes in the Spike Glycoprotein of spring attack reactivity. J. Immunol.2021 Jun 1; 206(11):2527-2535. doi: 10.4049/jimunol.2000922). The method comprises the following steps:

1. ELISpot plates (Mabtech) pre-coated with antibodies (IFN-. gamma., IL-2, IL-4 and IL-5) were washed 4 times with sterile PBS at 200. mu.L/well;

2. adding Blocking buffer (1640 culture solution + 10% FBS + 1% P/S)200 μ L/well, sealing and balancing at room temperature for more than 30 min;

3. discard the plate liquid, add 100 μ L of non-irritant (DMSO at the same volume with peptide pool) per well of negative control; adding 100 μ L SARS-CoV-2S protein full-length peptide library (2 μ g/mL) into each well of experimental group, adding 100 μ L Canavalia bean protein A (ConA) (8 μ g/mL) into each well of IFN-gamma, IL-2, IL-4 positive control group; IL-5 positive control group 100. mu.L PMA (100ng/mL) + Ionomycin (1. mu.g/mL) per well;

4. mouse spleen cells were expressed at 2X 10⁵Each 100 μ L of the total extract is mixed with the extract (IL-5 is 5 × 10)⁵One/100. mu.L), the plate was placed at 37 ℃ in 5% CO₂Culturing for 36h in an incubator;

5. discarding the mixed solution of the in-plate stimulus and cells, washing the plate 5 times with PBS, 200 μ L/well;

6. diluting the enzyme-labeled detection antibody to 1 mu g/mL and 100 mu L/well with PBS (PBS-0.5% FBS) containing 0.5% FBS, and incubating for 2h at room temperature in a dark place;

7. wash the plate 5 times with PBS, 200. mu.L/well; Streptavidin-ALP conjugate (Streptavidin-ALP) (1:1000) was diluted with PBS-0.5% FBS and incubated at room temperature for 1 h;

washing the plate 5 times with PBS, 200 μ L/well; after drying, adding 100 μ L substrate color development solution (0.45 μm filtration) into each well, developing for 2-15min, and stopping with deionized water when spots appear clearly; and (5) airing the bottom of the plate, and carrying out full-automatic spot image acquisition and counting on an ELISPOT plate reading instrument.

FIGS. 9A-9D show in vitro cytokine assay results, according to one embodiment of the present invention. As shown in FIGS. 9A to 9D, we induced spleen cells of mice with the full-length peptide library of SARS-CoV-2S protein as a stimulator, and observed that IFN-. gamma.IL-2, IL-4 and IL-5 were significantly higher in the 2.5. mu. g M5 immune group than in the adjuvant and NS groups. Of the 4 cytokines tested, the M5 immunization group induced the highest IL-2 values, the induced IL-4 levels were lower relative to the adjuvant and NS groups, and the induced IL-5 values were the lowest, suggesting that the M5 induced cytokine levels may be more inclined to the Th1 type. In general, the M5 vaccine induces high-level Th1 cellular immune response, and can accelerate the elimination of intracellular microorganisms such as bacterial viruses.

The above embodiments are provided only for illustrating the present invention and not for limiting the present invention, and those skilled in the art can make various changes and modifications without departing from the scope of the present invention, and therefore, all equivalent technical solutions should fall within the scope of the present invention.

SEQUENCE LISTING

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Claims (16)

1. A protein comprising a variant of coronavirus spike glycoprotein RBD, which protein has mutations at amino acid positions K417, L452, T478, E484, and N501 as compared to a wild-type protein of coronavirus spike glycoprotein RBD.

2. A protein comprising a coronavirus spike glycoprotein RBD variant according to claim 1, wherein the RBD variant protein comprises one or more of the following amino acid mutations: K417N, L452R, T478K, E484Q/K and N501Y.

3. A protein comprising a coronavirus spike glycoprotein RBD variant according to claim 1 or 2, wherein:

(1) the amino acid sequence of the RBD variant protein is selected from the group consisting of:

(a1)SEQ ID NO：1；

(a2) SEQ ID NO:1 by substituting, deleting and/or adding one or more amino acids; and

(a3) a truncation of (a1) or (a 2); or alternatively

(2) The amino acid sequence of the RBD variant protein is selected from the group consisting of:

(b1)SEQ ID NO：3；

(b2) consisting of SEQ ID NO:3 by substituting, deleting and/or adding one or more amino acids; and

(b3) a truncation of (b1) or (b 2); or

(3) The amino acid sequence of the RBD variant protein is selected from the group consisting of:

(c1)SEQ ID NO：5；

(c2) consisting of SEQ ID NO:5 by substituting, deleting and/or adding one or more amino acids; and

(c3) truncated form of (c1) or (c 2).

4. A protein comprising a coronavirus spike glycoprotein RBD variant according to claim 1 or 2, the amino acid sequence of which is encoded by a coding gene, wherein:

(1) the coding gene is selected from:

(d1)SEQ ID NO：2；

(d2) and SEQ ID NO:2, and the encoded protein is more than 80% homologous with the coronavirus spike glycoprotein RBD variant protein; and

(d3) a truncation of (d1) or (d 2); or alternatively

(2) The coding gene is selected from:

(e1)SEQ ID NO：4；

(e2) and SEQ ID NO:4, and the encoded protein is more than 80% homologous with the coronavirus spike glycoprotein RBD variant protein; and

(e3) a truncation of (e1) or (e 2); or

(3) The coding gene is selected from:

(f1)SEQ ID NO：6；

(f2) and SEQ ID NO:6, and the coded protein is more than 80 percent homologous with the coronavirus spike glycoprotein RBD variant protein; and

(f3) a truncated body of (f1) or (f 2).

5. An isolated nucleic acid molecule encoding a protein comprising a coronavirus spike glycoprotein RBD variant according to any one of claims 1-3.

6. A recombinant vector comprising: a protein comprising a coronavirus spike glycoprotein RBD variant according to any one of claims 1-4 or a nucleic acid molecule according to claim 5; and

an expression vector.

7. A fused cell comprising:

the recombinant vector of claim 6; and

an expression cell.

8. A method for producing a protein comprising a coronavirus spike glycoprotein RBD variant according to any one of claims 1 to 4 or a protein expressed by a nucleic acid molecule according to claim 5, comprising:

obtaining a coding gene;

transforming the coding gene into an expression cell;

expressing the RBD variant protein in an expressing cell; and

purifying the RBD variant protein.

9. The method of manufacturing according to claim 8, further comprising:

constructing a recombinant vector containing the coding gene; and

transforming the recombinant vector into an expression cell.

10. A vaccine composition comprising:

at least one immunogenic fragment or an immunogenic composition comprising said immunogenic fragment; wherein the immunogenic fragment is a protein comprising a coronavirus spike glycoprotein RBD variant according to any one of claims 1-4 or an RBD variant protein encoded by a nucleic acid molecule according to claim 5; and

an adjuvant.

11. The vaccine composition of claim 10, wherein the adjuvant comprises aluminum hydroxide and CpG; the mixing mass ratio of the immunogenic fragment to the CpG and the aluminum hydroxide is 1:10: 20.

12. A method of making a vaccine comprising:

(1) providing at least one immunogenic fragment based on the coronavirus spike glycoprotein or an immunogenic composition comprising said immunogenic fragment; wherein the immunogenic fragment is the RBD variant protein of any one of claims 1-4 or the RBD variant protein encoded by the nucleic acid molecule of claim 5; and

(2) mixing the immunogenic fragment or the immunogenic composition of (1) with a pharmaceutically acceptable adjuvant.

13. Use of a protein comprising a coronavirus spike glycoprotein RBD variant according to any one of claims 1 to 4 or a nucleic acid molecule according to claim 5 for the preparation of a medicament or vaccine for the prevention of a disease caused by a coronavirus.

14. A protein according to any one of claims 1 to 4 comprising a variant of the spike glycoprotein RBD of a coronavirus which is a β coronavirus or a variant thereof.

15. The protein comprising a coronavirus spike glycoprotein RBD variant according to claim 14, wherein the beta coronavirus or variant thereof is 2019 novel coronavirus (SARS-CoV-2) or a variant thereof.

16. A protein comprising a coronavirus spike glycoprotein RBD variant according to claim 15, the SARS-CoV-2 virus variants including but not limited to Alpha variant, Beta variant, Gamma variant, Delta variant, Kappa variant, Lambda variant, Delta variant, Omicron variant.

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