CN113461787A

CN113461787A - Recombinant novel coronavirus S-RBD trimer protein, and preparation method and application thereof

Info

Publication number: CN113461787A
Application number: CN202110464788.1A
Authority: CN
Inventors: 李启明; 梁宇; 苏计国; 杜丽芳; 侯俊伟; 张学峰; 张靖; 靳玉琴; 唐芳; 刘兆明; 韩子泊; 雷泽华; 张�浩; 邵帅; 侯亚楠; 马智静; 陈实; 郑凡; 刘宁
Original assignee: China National Biotec Research Institute Co ltd
Current assignee: China National Biotec Research Institute Co ltd
Priority date: 2021-04-28
Filing date: 2021-04-28
Publication date: 2021-10-01
Also published as: CN115246874A

Abstract

The invention discloses a recombinant novel coronavirus S-RBD trimer protein, which is composed of 308-603 amino acid segments of a trimer-shaped novel coronavirus S protein RBD region. The vaccine prepared by the invention takes S-RBD tripolymer protein as antigen, is supplemented with adjuvant, immunizes organism, can generate high-titer protective neutralizing antibody aiming at novel coronavirus, and can be used for treating and/or preventing novel coronavirus (SARS-CoV-2) infection and/or novel coronavirus diseases.

Description

Recombinant novel coronavirus S-RBD trimer protein, and preparation method and application thereof

Technical Field

The invention relates to the field of biomedicine, in particular to a novel recombinant coronavirus S-RBD trimer protein, a preparation method and application thereof.

Background

SARS-CoV-2, belonging to the order Nedovirales (Nidovirales), Coronaviridae (Coronaviridae), the subfamily orthocoronaviridae, the genus Betaconoviridus, the subfamily Sarbecovirus, the SARS-like virus species, single-stranded positive-strand RNA viruses, has an envelope with a genome of about 29.9kb in overall length, encodes mostly non-structural proteins, is involved in functions such as viral replication and translation, and encodes structural proteins for a small part of the sequence, such as: spike proteins (S), M proteins (membrane proteins), E proteins (envelope proteins) and N proteins (nucleo proteins), in addition to several accessory proteins: 3a,3b, p6,7a,7b,8b,9b and orf14, all of which are involved in viral assembly. S, M and the E protein constitute the viral envelope, the major surface antigen for the virus to elicit an immune response. Wherein the S protein is a transmembrane glycoprotein with a molecular weight of about 150kDa, and forms an outstanding homotrimer on the surface of the virus. S consists of two functional subunits, cleaved at the boundary between the S1 and S2 subunits (S1/S2 cleavage site), which remain non-covalently associated in the pre-fusion conformation. The S2 subunit is also composed of multiple domains, whose function is primarily to mediate fusion of the virus with the host cell. The distal S1 subunit is structurally divided into four distinct domains: the vaccine comprises NTD, RBD, CTD1 and CTD2, wherein RBD is a receptor binding domain and is mainly responsible for binding with angiotensin converting enzyme 2 (ACE 2) on the surface of a host cell, so that the virus is mediated to infect the host cell, and therefore S protein and RBD are main targets for development of current genetic engineering vaccines.

Up to now, there are 7 vaccines on the market globally approved, BNT162b2 and mRNA-1273 for the american authorization for Emergency Use (EUA), AZD1222 for the uk authorization for Emergency Use (EUA), 3 new crown inactivated vaccines, conyoto adenovirus vector vaccines and smart fly recombinant protein vaccines, respectively, for chinese midlings (beijing and wuhan) and beijing kexing, respectively, for indian authorization for Emergency Use (EUA), and for russia, "satellite V", in addition to dozens of vaccines in different stages of clinical studies. The high safety and effectiveness of the recombinant vaccine developed by using the genetic engineering technology are widely proved, and in addition, the existing new coronavirus variant strains continuously appear and the proportion continuously rises, so that the protective effect of the existing vaccine and neutralizing antibody on a specific mutant strain is greatly reduced, and the worry of each community on the trend of new coronary epidemics and the effectiveness of the vaccine and medicaments is caused.

Therefore, it has become an urgent task to develop a recombinant vaccine that can produce protective neutralizing antibodies against novel coronaviruses at high titers.

Disclosure of Invention

In one aspect, the present invention provides a recombinant novel coronavirus S-RBD trimer protein, which overcomes the defect of the prior art that a recombinant vaccine capable of generating protective neutralizing antibodies against the novel coronavirus with high titer is lacked.

The technical scheme provided by the invention is as follows:

a recombinant novel coronavirus S-RBD trimeric protein, wherein the trimeric protein consists of 308-603 amino acid segments of an RBD region of a trimeric novel coronavirus S protein.

Based on the structural characteristics of the S-RBD region of the novel coronavirus, the invention designs a brand-new fusion protein by a computational biology method, the protein comprises three RBD structural domains, a trimer form with stable antigen conformation is formed under the condition that no exogenous connecting arm or other irrelevant components are introduced, the trimerization of the S-RBD protein is realized, the S-RBD trimer protein is recombined, expressed and purified by using a genetic engineering technology and then mixed with an adjuvant to prepare a vaccine, and an immune animal can generate a protective neutralizing antibody aiming at the high titer of the novel coronavirus, so that the novel coronavirus (SARS-CoV-2) infection and/or the novel coronavirus disease (COVID-19) can be treated and/or prevented. In addition, the RBD region has clear function and clear structure, is responsible for recognizing the ACE region of a receptor cell, and simultaneously has clear function aiming at the Antibody generated by the RBD, thereby avoiding the Enhancement effect of inducing the organism to generate Antibody Dependence (ADE) to the maximum extent.

The trimeric proteins described in the present invention can be formed by self-assembly of three polypeptide subunits of identical sequence. In an embodiment of the present invention, a suitable Linker (Linker) or spacer, which may be an oligopeptide or a polypeptide, may be included between the three identical sequences, and the function of the Linker may be to increase flexibility.

Preferably, however, in one embodiment of the invention, the primary structure of the trimeric protein is such that three of the amino acid fragments are linked in N-terminal to C-terminal order.

More preferably, in one embodiment of the present invention, the amino acid sequence of the fusion protein is as shown in SEQ ID No.1 or a sequence having more than 95% homology thereto.

In the above embodiments, the trimeric proteins of the invention form a trimeric form with a stable antigen conformation, possibly without the introduction of any foreign linker arms or other extraneous components.

The above-mentioned sequence having 95% or more homology thereto refers to an amino acid sequence having 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of the fusion protein. The amino acid sequence of the fusion protein described in the present specification can be randomly or engineered by a person skilled in the art in a suitable manner to obtain, for example, better affinity and/or dissociation properties, and such mutated amino acid sequences are included in the scope of the present invention.

In another aspect of the present invention, there is provided a fusion protein comprising the recombinant novel coronavirus S-RBD trimer protein as described above.

Preferably, in an embodiment of the present invention, the fusion protein further comprises one or more selected from a signal peptide, a tag, and an immune enhancing peptide. The effect of the signal peptide may be to facilitate expression of the protein; the tag may be, for example, a Flag tag, enhanced green fluorescent protein (eGFP), glutathione mercaptotransferase (GST), etc., which may be useful for detection, purification, isolation, etc. The above functional sequences may be used in any combination.

In another aspect of the present invention, there is provided a nucleic acid molecule comprising a nucleotide sequence encoding the above recombinant novel coronavirus S-RBD trimer protein, or encoding the above fusion protein.

Preferably, in one embodiment of the present invention, the codon of the trimeric protein is optimized by the inventors, and the obtained nucleotide sequence is shown as SEQ ID No.2 or a sequence having more than 95% homology thereto.

The above-mentioned sequence having 95% or more homology thereto refers to a nucleotide sequence having 95%, 96%, 97%, 98% or 99% identity to the nucleotide sequence.

The nucleic acid molecule can be prepared by a known technique such as chemical synthesis or PCR amplification based on the nucleotide sequence. In general, the codons encoding the amino acids of the domains described above can be optimized to optimize their expression in a host cell. The information on the above-mentioned nucleotide sequence can be obtained by searching a database such as a known literature or NCBI (https:// www.ncbi.nlm.nih.gov /).

In another aspect of the invention, there is provided a vector comprising the nucleic acid molecule described above.

In the present invention, the vector may be a linear vector or a cyclic vector. The vector may be a non-viral vector such as a plasmid, a viral vector, or a vector using a transposon. The vector can contain regulatory sequences such as a promoter, a terminator and the like, and marker sequences such as a drug resistance gene, a reporter gene and the like.

Preferably, in one embodiment of the present invention, the vector is an expression vector for the nucleic acid molecule of the present invention.

In another aspect of the present invention, there is provided a host cell comprising the nucleic acid molecule or the vector as described above.

Preferably, in an embodiment of the present invention, the host cell is escherichia coli, a yeast cell, an insect cell, or a mammalian cell;

more preferably, in one embodiment of the invention, the host cell is a CHO cell.

In another aspect of the present invention, there is provided a method for preparing the recombinant novel coronavirus S-RBD trimer protein or the fusion protein, comprising the steps of:

step A) preparing the nucleic acid molecule, constructing the expression vector, and transforming or transfecting the expression vector into the host cell;

step B) protein expression using the product of step A);

and C) purifying the expression product obtained in the step B) to obtain the recombinant novel coronavirus S-RBD tripolymer protein or fusion protein.

Wherein, the nucleic acid molecule in the step A) comprises a nucleotide sequence for coding the recombinant novel coronavirus S-RBD trimeric protein or coding the fusion protein.

Preferably, in one embodiment of the present invention, the nucleotide sequence is as shown in SEQ ID No.2 or a sequence having 95% or more homology thereto.

The nucleic acid molecules may be prepared according to the nucleotide sequences described in the present specification using any suitable molecular biological method.

Wherein, the nucleotide sequence can be constructed in the corresponding expression vector of the host cell by any suitable method for constructing the expression vector in step A).

The expression vector is then transformed or transfected into the host cell. Preferably, in one embodiment of the present invention, the inventors transfect the CHO cell expression vector into 293FT cells or CHO cells after constructing it to construct a recombinant cell line.

Wherein, the protein expression in the step B) can express the recombinant protein according to different expression systems. Further, in one embodiment of the present invention, the inventors screened a cell line capable of stably secreting and expressing the S-RBD trimer protein or fusion protein by limiting dilution.

Wherein the purification in step C) may be by any suitable method. For example, salting out, precipitation, dialysis or ultrafiltration, molecular sieve chromatography, ion exchange chromatography, hydrophobic chromatography, affinity chromatography, and the like. Preferably, in one embodiment of the present invention, the S-RBD trimer protein or the fusion protein is purified by ion exchange and hydrophobic chromatography.

Of course, according to the prior art, a process of collecting the target protein, for example, collecting the supernatant of a cell culture solution rich in the target protein, is also included before the purification step; the host cells after the target protein has been expressed are subjected to a post-disruption collection process, and the disruption may be performed by any suitable disruption method, such as ultrasonication, repeated freeze-thaw disruption, chemical treatment, and the like. The above collection of host cells is also understood to be within the scope of such purification.

In another aspect of the present invention, there is provided a use of said recombinant novel coronavirus S-RBD trimer protein, said fusion protein, said nucleic acid molecule, said vector or said host cell for the preparation of a medicament for the treatment and/or prevention of a novel coronavirus infection and/or a disease caused by a novel coronavirus.

The disease caused by the novel coronavirus is preferably novel coronavirus pneumonia (COVID-19).

In another aspect of the invention, a vaccine is provided, and the vaccine comprises the recombinant novel coronavirus S-RBD trimer protein or the fusion protein and an adjuvant.

In an embodiment of the present invention, the vaccine is a recombinant protein vaccine (or a genetically engineered subunit vaccine). Further, in other embodiments of the present invention, the vaccine may be a genetic engineering vector vaccine, or may be a nucleic acid vaccine comprising the nucleotide sequence described in the present specification.

Any suitable adjuvant may be included in the vaccines of the present invention. Preferably, however, in an embodiment of the invention, the adjuvant is aluminium hydroxide, aluminium phosphate, MF59 or CpG. More preferably, the adjuvant is aluminum hydroxide.

In another aspect of the present invention, there is provided a method for preparing the vaccine, wherein the purified recombinant novel coronavirus S-RBD trimer protein or the fusion protein is mixed with the adjuvant.

In another aspect of the invention, a genetically engineered vector vaccine is provided, which comprises the above-mentioned nucleic acid molecule.

In the embodiment of the present invention, the vector in the genetically engineered vector vaccine may be a replicative or non-replicative vector, such as adenovirus, vaccinia virus, vibrio cholerae, salmonella, bacillus calmette-guerin, etc.

In another aspect of the present invention, there is provided a nucleic acid vaccine comprising a nucleotide sequence encoding the recombinant novel coronavirus S-RBD trimer protein described above, or encoding the fusion protein described above.

The nucleic acid vaccine may be a DNA vaccine or an RNA vaccine, in particular an mRNA vaccine.

In another aspect of the invention there is provided the use of a vaccine as described above in the treatment and/or prevention of a novel coronavirus infection and/or a disease caused by a novel coronavirus.

In another aspect of the invention, a pharmaceutical composition is provided, which comprises the vaccine, and a pharmaceutically acceptable carrier.

The pharmaceutically acceptable carrier may be any pharmaceutically acceptable additive, for example, physiological saline, cell culture medium, glucose, water for injection, glycerol, amino acids and their compositions, stabilizers, surfactants, preservatives, isotonic agents, and the like.

The pharmaceutical composition of the present invention can also be used in combination with other drugs for treating and/or preventing a novel coronavirus infection and/or a disease caused by a novel coronavirus at an effective and safe dose.

In another aspect of the invention, there is provided a method of eliciting an immune response against a novel coronavirus or treating a novel coronavirus infection in a subject by administering an effective dose of said vaccine or said pharmaceutical composition to said subject.

The subject may be a human or other animal.

The administration may be intramuscular, intraperitoneal or subcutaneous.

The invention has the beneficial effects that:

the vaccine prepared by the invention takes S-RBD tripolymer protein as antigen, is supplemented with adjuvant, immunizes organism, can generate high-titer protective neutralizing antibody aiming at novel coronavirus, and can be used for treating and/or preventing novel coronavirus (SARS-CoV-2) infection and/or novel coronavirus diseases.

Drawings

FIG. 1 is a structural analysis diagram of S protein of the novel coronavirus in example 1 of the present invention, wherein A is the monomeric structure of S protein (based on the coordinate file of PDB code 6zgg, and plotted by UCSF Chimera software), and the structural unit S1 comprises NTD, RBD, SD1 and SD2 domains; b is the RBD and ACE2 receptor complex structure (based on the PDB code as the coordinate file of 6m0j, drawn by UCSF Chimera software);

FIG. 2 is a drawing showing the design of S-RBD trimer protein in example 1 of the present invention, wherein A is the S-RBD monomer protein structure; b is S-RBD tripolymer protein structure;

FIG. 3 is an SDS-PAGE of the S-RBD trimer protein purified in example 2 of the present invention, wherein lane 1 shows the S-RBD trimer protein purified; lanes 2-7 are S-RBD trimer protein during purification; lane M is protein marker (molecular weight standards: kDa: 250, 150, 100, 70, 50, 40, 30, 20, 15, 10, 5);

FIG. 4 is a Western-blot identification chart of the S-RBD trimer protein purified in example 2 of the present invention, in which lane 1 is a negative control; lane 2 is the purified S-RBD trimer protein; lane M is protein marker (molecular weight standards: kDa: 250, 130, 100, 70, 55, 35, 25, 15, 105);

FIG. 5 is a graph showing the binding of the purified S-RBD trimer protein to MM43 and MM57 neutralizing monoclonal antibodies in example 3 of the present invention;

FIG. 6 is a graph showing the results of serum-specific IgG titer detection in example 5 of the present invention;

FIG. 7 is a graph showing the results of measurement of the serum-neutralizing antibody titer against wild viruses in example 5 of the present invention;

FIG. 8 is a graph showing the result of measurement of serum-specific IgG titer in comparative example 1 of the present invention;

FIG. 9 is a graph showing the results of detection of the serum neutralizing antibody titer against the wild virus in comparative example 1 of the present invention.

DESCRIPTION OF THE SEQUENCES

SEQ ID No.1 is an amino acid sequence of the recombinant novel coronavirus S-RBD trimer protein in the embodiment of the invention;

SEQ ID No.2 is a nucleotide sequence of the recombinant novel coronavirus S-RBD trimer protein in the embodiment of the invention;

SEQ ID No.3 is the amino acid sequence of the novel coronavirus S-RBD dimer protein in the comparative example of the present invention;

SEQ ID No.4 shows the nucleotide sequence of the novel coronavirus S-RBD dimer protein in the comparative example of the present invention.

Detailed Description

The invention discloses a recombinant novel coronavirus S-RBD trimer protein, a preparation method and application thereof, and can be realized by appropriately improving process parameters by taking the contents as reference by a person skilled in the art. It is specifically intended that all such alterations and modifications which are obvious to those skilled in the art are deemed to be incorporated herein by reference, and that the techniques of the invention may be practiced and applied by those skilled in the art without departing from the spirit and scope of the invention.

In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art. Some terms appearing in the present invention are explained below.

The term "novel coronavirus", SARS-CoV-2, belonging to the order Nedovirales (Nidovirales), Coronaviridae (Coronaviridae), the subfamily orthocoronaviruses, the genus Betaconoviridae, the subgenus Sarbecovirus, the SARS-like virus species, a single-stranded positive-strand RNA virus, having an envelope with a genome of about 29.9kb in length, encoding for the most part non-structural proteins, involved in the functions of viral replication and translation, and a small part of the sequence encoding structural proteins, such as: spike proteins (S), M proteins (membrane proteins), E proteins (envelope proteins) and N proteins (nucleo proteins), in addition to several accessory proteins: 3a,3b, p6,7a,7b,8b,9b and orf14, all of which are involved in viral assembly. S, M and the E protein constitute the viral envelope and are the major surface antigens of the virus to elicit an immune response. Wherein the S protein is a transmembrane glycoprotein with a molecular weight of about 150kDa, and forms prominent homotrimers on the surface of the virus. S consists of two functional subunits, cleaved at the boundary between the S1 and S2 subunits (S1/S2 cleavage site), which remain non-covalently associated in the pre-fusion conformation. The S2 subunit is also composed of multiple domains, whose function is primarily to mediate fusion of the virus with the host cell. The distal S1 subunit is structurally divided into four distinct domains: the antigen-binding protein S and the antigen-binding protein RBD are main targets for current genetic engineering vaccine development, and are NTD, RBD, CTD1 and CTD2, wherein RBD is a receptor binding domain and is mainly responsible for binding with receptor angiotensin converting enzyme 2 (ACE 2) on the surface of a host cell so as to mediate virus infection on the host cell.

The term "trimeric form" is a type of quaternary structure of proteins. The quaternary Structure of a protein refers to the number and arrangement of protein subunits relative to each other (Chou, Kuo-Chen; Cai, Yu-Dong. Predicting protein quaternary Structure by pseudo amino acid composition. proteins: Structure, Function, and bioinformatics.1November 2003,53(2): 282-289.). Three protein subunits are contained in the protein, namely the trimer form.

The term "primary structure" is a linear sequence of amino acids in a peptide or protein. Conventionally, the primary structure of a protein is reported from the amino-terminus (N) to the carboxy-terminus (C).

The term "fusion protein" refers to an expression product of one, two or more genes obtained by recombinant DNA techniques. The fusion protein technology is a purposeful gene fusion and protein expression method for obtaining a large number of standard fusion proteins, and a novel target protein with multiple functions can be constructed and expressed by utilizing the fusion protein technology.

The term "vector" is a nucleic acid delivery vehicle into which a polynucleotide may be inserted. When a vector is capable of providing for expression of a protein encoded by an inserted polynucleotide, the vector is referred to as an expression vector. The vector may be introduced into a host cell by transformation, transduction, or transfection, and the genetic material elements carried thereby are expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to: a plasmid; phagemid; a cosmid; artificial chromosomes such as Yeast Artificial Chromosomes (YACs), Bacterial Artificial Chromosomes (BACs), or artificial chromosomes (PACs) derived from P1; bacteriophage such as lambda phage or M13 phage, animal virus, etc. Animal viruses that may be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papilloma viruses, papilloma polyoma vacuolatum viruses (e.g., SV 40). A vector may contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may contain a replication initiation site.

The term "host cell" is a cell into which a nucleic acid molecule has been introduced by molecular biological techniques. These techniques include transfection of viral vectors, transformation with plasmid vectors, and introduction of naked DNA by electroporation, lipofection, and particle gun acceleration.

The term "treating" refers to reducing the likelihood of disease pathology, reducing the occurrence of disease symptoms, e.g., to the extent that a subject has a longer survival period or reduced discomfort. Treatment can refer to the ability of a therapy to reduce the symptoms, signs, or causes of a disease when administered to a subject. Treatment also refers to alleviation or reduction of at least one clinical symptom and/or inhibition or delay of progression of a disorder and/or prevention or delay of the onset of a disease or disorder.

The term "subject" refers to any human or other animal, particularly other mammals, that is being prevented, treated, diagnosed. Other mammals may include, for example, dogs, cats, cows, horses, sheep, pigs, goats, rabbits, rats, guinea pigs, mice, and the like.

In order to make those skilled in the art better understand the technical solution of the present invention, the following detailed description of the present invention is provided with reference to specific embodiments.

Example 1: structural biology-based design and recombination of novel coronavirus S-RBD trimer protein

Through analyzing the spatial structure of the natural S protein trimer (as shown in figure 1) and calculating the spatial distance between the N end and the C end of the RBD structure domain, the result shows that under the condition of not introducing an exogenous vector or a sequence, the structural characteristics of the RBD can be utilized to realize the trimerization, meanwhile, a larger spatial barrier cannot exist, and a receptor binding site and a main neutralizing antibody epitope cannot be shielded. Based on the sequence, a sequence fragment (308-603 amino acids) of the RBD region of the S protein of the novel coronavirus (Genebank number: MN908947.3) is intercepted, and the distance between the N end and the C end is

Three same RBD regions are connected in series end to form a new fusion protein, the amino acid sequence is shown as SEQ ID NO.1, and polymer structure prediction software (AIDA) and molecular dynamics simulation (GROMACS) are utilized to show that the fusion protein contains three independent RBD structural domains to form a trimer form with stable antigen conformation (shown as figure 2), namely the recombinant novel coronavirus S-RBD trimer protein.

Example 2: expression, purification and identification of recombinant novel coronavirus S-RBD trimer protein

According to the codon preference of a CHO cell expression system, carrying out codon optimization on the amino acid sequence of SEQ ID NO.1, constructing a CHO cell expression vector, transfecting the CHO cell expression vector to 293FT cells or CHO cells to construct a recombinant cell strain, and screening by a limiting dilution method to obtain the cell strain capable of stably secreting and expressing S-RBD trimer protein.

After series of chromatographic purifications, the S-RBD tripolymer protein with the purity of more than or equal to 90 percent is obtained. The SDS-PAGE detection result is shown in figure 3, the molecular weight of the protein is between 100 kD and 150kD, and part of product related substances such as dimeric protein and monomeric protein can be seen. The purified protein is electrophoresed by SDS-PAGE and then electrically transferred to a PVDF membrane, Western-blot identification is carried out by using an RBD specific antibody (manufacturer: Beijing Yi Qiao Shen science and technology Co., Ltd.; product number: 40591-T62; dilution: 2000 times) (the result is shown in figure 4), and the purified protein can be combined with the RBD specific antibody and has good immunogenicity.

Example 3: biological analysis of combination of recombinant novel coronavirus S-RBD trimer protein and neutralizing monoclonal antibody

Diluting the purified S-RBD tripolymer protein to 1 mu g/ml, 0.5 mu g/ml, 0.25 mu g/ml, 0.125 mu g/ml, 0.0625 mu g/ml, 0.03125 mu g/ml, 0.015625 mu g/ml, 0.007813 mu g/ml and 100 mu l/well, coating the protein on a 96-well enzyme label plate, and taking blank wells as negative controls at 4 ℃ for 8-12 h; after washing the plate with the PBST solution, adding a sealing solution, and sealing for 3h at 37 ℃; after the PBST solution is washed, a diluted MM43 monoclonal antibody (the manufacturer: Beijing Yi Qiao Shen science and technology Limited; the product number: 40591-MM43, the dilution degree: 2000 times) or an MM57 monoclonal antibody (the manufacturer: Beijing Yi Qiao Shen science and technology Limited; the product number: 40592-MM57, the dilution degree: 2000 times) is respectively added into the PBST solution, 100 mul/hole is incubated for 1h at 37 ℃; after the plate is washed by the PBST solution, diluted HEP-labeled goat anti-mouse IgG (manufacturer: Beijing Zhonghua Jinqiao Biotech Co., Ltd.; product number: ZB-2305; dilution: 10000 times) is added, 100. mu.l/well is incubated at 37 ℃ for 1 h; after the PBST solution is washed, adding a developing solution (manufacturer: Wantai biology) A and B in sequence, developing for 5-10 min at room temperature, and adding a stopping solution C; the reading values of double wavelengths (OD450nm and 630nm) are carried out on a microplate reader, the cutoff value is determined, a protein concentration-absorbance value curve is drawn, and the result is shown in figure 5, and the S-RBD tripolymer protein and the MM43 and MM57 neutralizing monoclonal antibodies have good binding activity.

Example 4: preparation of recombinant coronavirus vaccine

Diluting the purified S-RBD tripolymer protein to 2 times of target antigen concentration, mixing and adsorbing the purified S-RBD tripolymer protein with 1.2mg/ml aluminum hydroxide adjuvant according to a ratio of 1:1 (w/w), stirring the mixture on a magnetic stirrer for 40-120 min at a rotating speed of 200-300 rpm to obtain a semi-finished vaccine product, wherein the content of residual protein in supernatant is less than 10% of the content of total protein, and each bottle of the semi-finished vaccine product is aseptically subpackaged according to the loading of 0.5ml to obtain the finished vaccine product.

Example 5: immunological effect evaluation of recombinant novel coronavirus vaccine

The recombinant novel coronavirus vaccine prepared in example 4 was intraperitoneally injected into BALB mice, 0.5 ml/mouse, according to the animal test protocol shown in Table 1. The ELISA method is used for detecting the titer of the specific IgG antibody aiming at the S-RBD protein in the immune serum, the titer of the neutralizing antibody is detected by using a wild virus micro-neutralization test, the mouse can be stimulated to generate a high titer antibody level after 3-needle immunization, the serum specific IgG result is shown in figure 6, the GMT value of the IgG antibody aiming at the RBD protein reaches 120 ten thousand, the wild virus micro-neutralization is shown in figure 7, and the GMT value of the neutralizing antibody aiming at the wild virus is 707.

Serum specific IgG antibody detection: detecting the level of a specific IgG antibody aiming at the S-RBD protein in the immune serum by adopting an ELISA method, diluting the RBD protein (purchased from Beijing Yiqiao Shenzhou science and technology Co., Ltd., with the product number of 40592-V08B) to 1 mu g/ml, coating the RBD protein in an enzyme label plate by 100 mu l/hole, adding the serum after being sealed, and determining the cut-off value by utilizing an enzyme-labeled antibody (mouse source) to detect a signal, wherein the highest dilution multiple of the positive serum is the specific IgG antibody titer of the serum sample.

Wild virus micro neutralization assay: the test was carried out in a BSL 3-grade laboratory, the strain was 2020XN4276, serially diluted sera were incubated with equal volume of virus (100TCID50), neutralized in an incubator at 37 ℃ for 2 hours, and added to a sample containing Vero-E6 cells (2X 10)⁵/ml) cell culture plate, simultaneously setting cell control and virus control, culturing in 37 deg.C incubator for 3-5 days, observing cytopathic condition, calculating serum dilution factor at 50% infection inhibition rate by Reed-Muench method, that is neutralizing antibody titer of the serum sample。

TABLE 1 animal test protocol for immunogenicity evaluation of S-RBD trimer protein

Comparative example 1: comparison of S-RBD trimer protein and dimer protein

Intercepting new type coronavirus (Genebank number: MN908947.3) S protein RBD region sequence segments (319-537 amino acids), connecting two same RBD region segments in series end to form a dimer fusion protein, wherein the amino acid sequence is shown as SEQ ID NO.3, performing codon optimization on the amino acid sequence of SEQ ID NO.3 according to the codon preference of a CHO cell expression system, constructing a CHO cell expression vector, transfecting the CHO cell to 293FT cells to obtain a cell strain capable of expressing dimer protein, performing series chromatography purification to obtain S-RBD dimer protein with the purity of more than or equal to 95%, mixing and adsorbing the S-RBD dimer protein with 1.2mg/ml aluminum hydroxide adjuvant according to a ratio of 1:1 (w/w) to prepare a dimer protein immune substance, preparing the trimer protein immune substance by the same process, injecting immune BALB/c mice by abdominal cavity, the antigen content is 6.0 mu g/dose, the aluminum adjuvant content is 0.30 mg/dose, 3 immunization is carried out according to 0w, 1w and 2w, serum is collected and separated 2w after the whole immunization, the titer of specific IgG antibody aiming at S-RBD protein in the serum after the immunization is detected by an ELISA method, the detection result is shown in figure 8, the titer of neutralizing antibody is detected by a wild virus micro-neutralization test, the detection result is shown in figure 9, the antibody level generated by the tripolymer protein is equivalent to the protein level of the dipolymer, and the difference of the specific IgG antibody and the titer of the neutralizing antibody has no statistical difference.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

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Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn

165 170 175

Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe

180 185 190

Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu

195 200 205

Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys

210 215 220

Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn Gly

225 230 235 240

Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu Pro

245 250 255

Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala Val Arg

260 265 270

Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe Gly

275 280 285

Gly Val Ser Val Ile Thr Pro Gly Thr Val Glu Lys Gly Ile Tyr Gln

290 295 300

Thr Ser Asn Phe Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro

305 310 315 320

Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg

325 330 335

Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val

340 345 350

Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys

355 360 365

Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn

370 375 380

Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile

385 390 395 400

Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro

405 410 415

Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp

420 425 430

Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys

435 440 445

Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln

450 455 460

Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe

465 470 475 480

Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln

485 490 495

Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala

500 505 510

Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys

515 520 525

Val Asn Phe Asn Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu

530 535 540

Ser Asn Lys Lys Phe Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala

545 550 555 560

Asp Thr Thr Asp Ala Val Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp

565 570 575

Ile Thr Pro Cys Ser Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr

580 585 590

Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val Gln Pro Thr

595 600 605

Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly

610 615 620

Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg

625 630 635 640

Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser

645 650 655

Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu

660 665 670

Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg

675 680 685

Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys Ile Ala

690 695 700

Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala

705 710 715 720

Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr

725 730 735

Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp

740 745 750

Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val

755 760 765

Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro

770 775 780

Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe

785 790 795 800

Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr

805 810 815

Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn Gly Leu Thr

820 825 830

Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu Pro Phe Gln

835 840 845

Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala Val Arg Asp Pro

850 855 860

Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe Gly Gly Val

865 870 875 880

Ser Val Ile Thr Pro Gly Thr

885

<210> 2

<211> 2664

<212> DNA

<213> Artificial

<400> 2

atgacagtgg agaagggcat ctatcagaca agcaatttta gggtacaacc tacagagtct 60

attgtgagat tcccaaatat caccaacctt tgtccttttg gagaggtctt taatgccaca 120

agatttgcct ctgtgtatgc ctggaacaga aagagaattt caaattgtgt tgctgactat 180

agtgtgctct ataactctgc tagctttagc accttcaagt gctatggagt gagcccaacc 240

aagctgaatg acctgtgttt taccaatgtg tatgctgatt cctttgtgat cagaggagat 300

gaggttagac agattgcccc tggccaaact ggtaaaattg ctgactacaa ctacaagctg 360

cctgatgact tcactggctg tgtgattgcc tggaacagca ataatctgga ctctaaggta 420

gggggtaact ataattacct gtacagattg ttcagaaaaa gcaaccttaa gccttttgag 480

agagacattt ccacagagat atatcaagct ggctctaccc cttgtaatgg agtggaaggt 540

ttcaactgct attttcctct gcagagctat ggctttcagc ccaccaatgg agtgggctat 600

cagccttaca gagtggtggt tctgtctttt gagctcctgc atgcccctgc cacagtgtgt 660

ggccctaaaa agagcaccaa cctggtaaag aacaaatgtg tcaattttaa tttcaatggc 720

cttactggca ctggagtgct aacagagtcc aataagaagt tcctaccttt tcaacagttt 780

ggcagagata ttgctgacac cactgatgct gtgagagatc ctcaaaccct ggagatctta 840

gacatcaccc cctgcagctt tggaggagtg tctgtgataa ctcctggcac tgtggaaaag 900

ggcatttacc aaacctccaa cttcagagtg caacccacag agagcattgt gagatttccc 960

aatattacca acctttgccc ttttggtgaa gtgtttaatg ccacaagatt tgcctctgtg 1020

tatgcctgga atagaaagag aatcagcaac tgtgtagctg actactctgt tttgtataat 1080

tcagcttcat tctcaacctt caagtgttat ggagtgtccc ctaccaagct aaatgattta 1140

tgctttacca atgtctatgc tgatagcttt gtgatcagag gtgatgaggt tagacaaatt 1200

gcccctggac agactggcaa gattgctgac tacaattata aactgcctga tgacttcact 1260

ggctgtgtta tagcctggaa cagcaacaac ctggacagta aggttggagg caactacaac 1320

tatctgtata ggctgtttag aaagagcaat ctaaagccat ttgagagaga catatcaaca 1380

gagatctatc aagctggcag caccccttgc aatggagtgg agggatttaa ctgctatttc 1440

ccacttcaga gttatggctt tcagcctaca aatggagtgg gatatcagcc ttatagagtg 1500

gttgtattat catttgagct actgcatgcc cctgcaacag tttgtgggcc taaaaaaagc 1560

actaatctgg tgaaaaacaa gtgtgtgaat ttcaacttta atggcctgac tggcactgga 1620

gtcctgacag agagcaataa gaagtttcta ccttttcagc aatttggcag agatattgct 1680

gacaccactg atgctgtgag agaccctcag acactggaaa tcctggacat aaccccttgt 1740

tcttttggag gagtgtctgt gatcactcct ggcacagtgg agaagggcat atatcagaca 1800

agcaacttca gagtgcagcc tacagagagc attgtgagat tccctaacat caccaacctg 1860

tgcccttttg gagaggtgtt caatgccaca agatttgcct ctgtgtatgc ctggaataga 1920

aagagaatca gcaactgtgt ggctgactac tctgtgctgt acaactctgc tagcttcagc 1980

accttcaagt gctatggagt gagccctacc aagctgaatg acctgtgctt caccaatgtg 2040

tatgctgaca gctttgtgat cagaggagat gaggtgagac agattgcccc tggacagact 2100

ggcaagattg ctgactacaa ctacaagctg cctgatgact tcactggctg tgtgattgcc 2160

tggaacagca acaacctgga cagcaaggtg ggaggcaact acaactacct gtacagactg 2220

ttcagaaaga gcaacctgaa gccttttgag agagacatca gcacagagat ctaccaagct 2280

ggcagcaccc cttgcaatgg agtggagggc ttcaactgct acttccctct gcagagctat 2340

ggctttcagc ctaccaatgg agtgggctat cagccttaca gagtggtggt gctgagcttt 2400

gagctgctgc atgcccctgc cacagtgtgt ggccctaaaa agagcaccaa cctggtgaag 2460

aacaagtgtg tgaacttcaa cttcaatggc ctgactggca ctggagtgct gacagagagc 2520

aacaagaagt tcctgccttt tcagcagttt ggcagagaca ttgctgacac cacagatgct 2580

gtgagagacc ctcagaccct ggagatcctg gacatcaccc cttgcagctt tggaggagtg 2640

tctgtgatca cccctggcac ctga 2664

<210> 3

<211> 439

<212> PRT

<213> Artificial

<400> 3

Met Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr

1 5 10 15

Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser

20 25 30

Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr

35 40 45

Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly

50 55 60

Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala

65 70 75 80

Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly

85 90 95

Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe

100 105 110

Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val

115 120 125

Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu

130 135 140

Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser

145 150 155 160

Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln

165 170 175

Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg

180 185 190

Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys

195 200 205

Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Arg Val Gln Pro

210 215 220

Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe

225 230 235 240

Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn

245 250 255

Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn

260 265 270

Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys

275 280 285

Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile

290 295 300

Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys Ile

305 310 315 320

Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile

325 330 335

Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn

340 345 350

Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg

355 360 365

Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly

370 375 380

Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln

385 390 395 400

Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser

405 410 415

Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser

420 425 430

Thr Asn Leu Val Lys Asn Lys

435

<210> 4

<211> 1320

<212> DNA

<213> Artificial

<400> 4

atgagagtgc agcccacaga gtcaattgtg agattcccca atatcactaa cctgtgcccc 60

tttggggagg tgttcaatgc cacaagattt gcttctgtgt atgcctggaa tagaaagaga 120

atcagtaact gtgttgctga ctactctgtg ctgtacaatt ctgcctcttt cagcacattc 180

aaatgttatg gggttagccc caccaaacta aatgacctgt gcttcaccaa tgtgtatgct 240

gacagctttg tgatcagagg tgatgaagtg agacaaattg cccctgggca gactggcaag 300

atagctgact acaattacaa gctgcctgat gactttactg gctgtgtgat tgcctggaat 360

agcaataacc tggacagcaa ggtggggggc aactataact acctatacag attgtttaga 420

aagtccaatt tgaaaccctt tgagagagat atcagcactg agatctatca agctggcagc 480

accccctgta atggggtgga ggggttcaac tgctatttcc ccttacagag ctatggcttt 540

cagcccacca atggggtggg ctatcagccc tacagagtgg tggtgctgtc ttttgaacta 600

cttcatgccc ctgccacagt gtgtggcccc aaaaaaagca caaacctggt gaaaaacaag 660

agagtgcagc caacagagag cattgtgaga ttccctaaca taaccaatct gtgtcccttt 720

ggtgaggttt ttaatgctac tagatttgcc tctgtgtatg cctggaacag aaaaagaatc 780

tcaaactgtg tggctgacta ttcagtcctc tacaactcag ctagctttag caccttcaaa 840

tgctatggtg tcagtcccac caaactgaat gacctttgtt tcaccaatgt ctatgctgac 900

agctttgtga tcagagggga tgaagtgaga caaatagccc ctgggcagac tggcaagata 960

gctgattaca actataaact gcctgatgat ttcactggct gtgtcattgc ctggaactca 1020

aataatctag acagcaaggt aggtggcaat tataactacc tctataggct gtttaggaaa 1080

agtaacctga aaccctttga gagagacatt agcacagaga tttatcaagc tggctcaaca 1140

ccctgtaatg gtgtggaggg ctttaactgc tacttccccc tgcagagcta tggctttcag 1200

ccaaccaatg gagtgggcta tcagccctat agagtagttg tgctgagctt tgagctgctg 1260

catgcccctg ccacagtgtg tggccctaag aaaagcacca acttggtcaa aaataagtga 1320

Claims

1. A recombinant novel coronavirus S-RBD trimer protein, wherein the trimer protein is composed of amino acid segments 308-603 of an RBD region of the novel coronavirus S protein in a trimer form.

2. The recombinant novel coronavirus S-RBD trimer protein as claimed in claim 1, wherein the trimer protein has a primary structure in which three of said amino acid segments are linked in an N-terminal to C-terminal order;

preferably, the amino acid sequence of the recombinant novel coronavirus S-RBD trimer protein is shown as SEQ ID No.1 or a sequence with more than 95% homology with the amino acid sequence.

3. A fusion protein comprising the recombinant novel coronavirus S-RBD trimer protein of claim 1 or 2.

4. The fusion protein of claim 3, further comprising one or more selected from a signal peptide, a tag, and an immune enhancing peptide.

5. A nucleic acid molecule comprising a nucleotide sequence encoding the recombinant novel coronavirus S-RBD trimer protein of claim 1 or 2, or encoding the fusion protein of claim 3 or 4;

preferably, the nucleotide sequence is shown as SEQ ID No.2 or a sequence with more than 95% homology with the nucleotide sequence.

6. A vector comprising the nucleic acid molecule of claim 5.

7. A host cell comprising the nucleic acid molecule of claim 5 or the vector of claim 6;

preferably, the host cell is escherichia coli, a yeast cell, an insect cell, or a mammalian cell;

more preferably, the host cell is a CHO cell.

8. The method for producing a recombinant novel coronavirus S-RBD trimer protein according to claim 1 or 2 or a fusion protein according to claim 3 or 4, which comprises the steps of:

step A) preparing the nucleic acid molecule of claim 5, constructing the expression vector of claim 6, transforming or transfecting the expression vector into the host cell of claim 7;

step B) protein expression using the product of step A);

9. Use of the recombinant novel coronavirus S-RBD trimer protein according to claim 1 or 2, the fusion protein according to claim 3 or 4, the nucleic acid molecule according to claim 5, the vector according to claim 6 or the host cell according to claim 7 for the preparation of a medicament for the treatment and/or prevention of a novel coronavirus infection and/or a novel coronavirus-caused disease.

10. A recombinant protein vaccine comprising the recombinant novel coronavirus S-RBD trimer protein of claim 1 or 2 or the fusion protein of claim 3 or 4, and an adjuvant.

11. The recombinant protein vaccine of claim 10, wherein the adjuvant is aluminum hydroxide, aluminum phosphate, MF59, or CpG;

preferably, the adjuvant is aluminum hydroxide.

12. The method for preparing a recombinant protein vaccine according to claim 10 or 11, wherein the purified recombinant novel coronavirus S-RBD trimer protein or the fusion protein is mixed with the adjuvant.

13. A genetically engineered vector vaccine comprising the nucleic acid molecule of claim 5.

14. A nucleic acid vaccine comprising a nucleotide sequence encoding the recombinant novel coronavirus S-RBD trimer protein of claim 1 or 2 or encoding the fusion protein of claim 3 or 4.

15. Use of a vaccine according to claim 10, 11, 13 or 14 for the treatment and/or prevention of a novel coronavirus infection and/or a disease caused by a novel coronavirus.

16. A pharmaceutical composition comprising the vaccine of claim 10, 13 or 14, and a pharmaceutically acceptable carrier.

17. A method of eliciting an immune response against a novel coronavirus or treating a novel coronavirus infection in a subject, wherein an effective amount of the vaccine of claim 10, 13 or 14 or the pharmaceutical composition of claim 16 is administered to the subject.