CN111939250B

CN111939250B - Vaccine for preventing COVID-19 and preparation method thereof

Info

Publication number: CN111939250B
Application number: CN202010827992.0A
Authority: CN
Inventors: 程振国; 王尧河
Original assignee: Zhengzhou University
Current assignee: Zhengzhou University
Priority date: 2020-08-17
Filing date: 2020-08-17
Publication date: 2022-07-29
Anticipated expiration: 2040-08-17
Also published as: CN111939250A

Abstract

The vaccine for preventing COVID-19, the nucleotide sequence of the antigen of the novel vaccine is SEQ NO. 1, the amino acid sequence is SEQ NO. 2, and the antigen of the vaccine comprises two functional parts: an S protein receptor binding domain capable of inducing a specific neutralizing antibody and a T cell-associated N protein truncated peptide segment capable of inducing activation of effector T cells; the vaccine of the invention has the following characteristics: the T cell related N protein truncated peptide fragment has weak generation capacity of inducing an N protein antibody, a vaccine vaccinator and a new coronary infected patient can be identified by using the N antibody, and the vaccine antigen does not induce the generation of the N protein antibody, so that lung injury can be reduced, and the method is safer; the cell vaccine of the present invention has low cost and can induce the generation of virus characteristic neutralizing antibody and T cell immune response.

Description

Vaccine for preventing COVID-19 and preparation method thereof

Technical Field

The invention belongs to the technical field of genetic engineering and molecular immunology, and particularly relates to a vaccine for preventing COVID-19 and a preparation method thereof.

Background

SARS-CoV2, also known as 2019-nCoV, is a novel RNA virus of the sub-genus Sarbecovirus of the family Coronaviridae, resulting in a global prevalence of 2019 novel coronavirus pneumonia (COVID-19). By 8 months and 10 days, the number of virus infections is more than 1986 thousands and the number of deaths is more than 73 thousands. At present, 139 candidate vaccines are evaluated in preclinical, and 25 candidate vaccines are in clinical evaluation or clinical trials, wherein partial vaccines such as recombinant adenovirus vector vaccines and inactivated vaccines enter phase II clinical trials and show encouraging results, however, the antigen design of the current various vaccines still has some defects and needs to be further optimized. For example, the existence of high neutralizing antibody of adenovirus type 5 (Ad 5) in part of human bodies inhibits the curative effect of the Ad 5-based new crown vaccine; the fire-fighting vaccine can induce an organism to generate a large amount of non-neutralizing antibodies, potential safety risks exist, and meanwhile, N protein antibodies generated by the vaccine stimulated organism interfere with serological diagnosis of infected patients.

Spike protein (S protein) is a key protein on the surface of SARS-CoV2, and is mainly composed of two parts, S1 and S2. Wherein the Receptor Binding Domain (RBD) in S1 can be combined with host cell surface receptor angiotensin converting enzyme 2 (ACE 2) to trigger the conformation change of S protein homotrimer and the fusion between virus and host cell membrane, so that the virus enters the host cell and replicates and proliferates, therefore, the S protein is the main target of current vaccine design and antiviral therapy. However, recent serological results showed that the serum of patients with COVID-19 contained high titers of S1-specific antibodies, whereas antibodies directed against the S protein RBD were low. Meanwhile, partial SARS-CoV-2 infected persons cannot generate long-lasting antibody of S protein, which suggests that the vaccine aiming at the S protein alone may have the risk of losing effect, and the vaccine design still needs to be improved. Antibody-dependent enhanced infection (ADE) refers to a phenomenon in which some virus-specific antibodies (usually non-neutralizing antibodies) bind to viruses and then bind to specific cells expressing FcR (Fc receptor) through the Fc fragment of the antibody, thereby enhancing viral infection, and is closely related to the safety of antiviral vaccines. Studies have shown that the Receptor Binding Domain (RBD) of SARS-CoV2 elicits a potent neutralization response without significant ADE phenomenon, suggesting that the receptor binding domain is an ideal candidate region for the development of S protein-based vaccines.

Humoral immunity (particularly the production of neutralizing antibodies) and T cell immunity are the main defense responses of the body to control and eliminate infectious pathogenic microorganisms. COVID-19 clinical data indicate that there are a large number of specific T cells against the SARS-CoV2 antigen in PBMCs of convalescent patients, indicating that enhancing the specific antiviral T cell response will help prevent SARS-CoV2 infection. Nucleocapsid protein (N) is one of the most abundant proteins expressed after SARS-CoV2 infection, and is highly homologous with SARS-CoV N protein. It was found that SARS-CoV infected patients have a large number of memory T cells against the virus N protein, which can exist in vivo for a long period of time and can cross-react with the N protein of SARS-CoV 2. The SARS related research also shows that the inoculation of N protein vaccine can induce body to produce specific antiviral antibody and T cell immune reaction, inhibit virus infection, and suggest that N protein can induce body to produce strong immune reaction and may be used as potential vaccine target. However, animal experiments show that when SARS-CoV full-length N protein is used as a vaccine, the body can be induced to generate a large amount of N protein antibodies, which causes the aggravation of mouse lung injury, and suggest that N protein-based vaccines need to overcome the problem of N antibody generation.

Disclosure of Invention

In order to solve the problems, the invention provides a vaccine for preventing COVID-19 and a preparation method thereof.

The invention mainly provides a chimeric vaccine against COVID-19 and a preparation method thereof, and provides a new effective selection for preventing novel coronavirus or highly homologous variant.

The vaccine for preventing COVID-19 has an antigen with a nucleotide sequence of SEQ NO. 1 and an amino acid sequence of SEQ NO. 2.

The vaccine antigen mainly comprises three parts, namely a nucleotide sequence SEQ NO 3 for coding a receptor binding structural domain of SARS-CoV2, a nucleotide sequence SEQ NO 4 for coding self-cutting peptide T2A, and a nucleotide sequence SEQ NO 5 for coding an N protein T cell reaction peptide segment.

The polynucleotide for coding the receptor binding structural domain of SARS-CoV2 is optimized by codon, and the amino acid sequence is SEQ NO. 6; the amino acid sequence of the self-cutting peptide T2A is SEQ NO. 7; the amino acid sequence of the N protein T cell reaction peptide segment is SEQ NO. 8.

The receptor binding structural domain of the code SARS-CoV2 is RBD, the reaction peptide segment of the code N protein T cell is Ntap.

A method for preparing a vaccine for preventing COVID-19 as described above, comprising the steps of:

(1) Firstly, constructing a lentiviral vector capable of secreting and expressing GP96-hFc, infecting and screening a cell line of HEK293T capable of stably secreting and expressing GP96-hFc, wherein the nucleotide sequence is shown as SEQ NO. 9, and the protein sequence is shown as SEQ NO. 10;

(2) constructing a lentivirus vector which can be used for carrying out chimeric expression on an S protein receptor binding structural domain of SARS-CoV2 and a truncated N protein T cell related peptide fragment, and screening out a cell line of HEK293T which can secrete and express GP96-hFc and simultaneously carry out chimeric expression on the binding structural domain of SARS-CoV2 and the truncated N protein T cell related peptide fragment on the basis of the step 1);

(3) collecting the cells obtained in step 2) under aseptic conditions, washing with sterile physiological saline or PBS, and adjusting the cell concentration to 1X 10 ⁶ ~1×10 ⁷ The concentration is 100 mu L, and the vaccine for preventing COVID-19 is obtained.

The vaccine for preventing COVID-19 in the step (3) is live cells, mitomycin B or radioactive ray treated cells and cell lysate.

The cell line of HEK293T can be replaced by homologous or heterologous cells that satisfy antigen expression.

A method for immunizing a vaccine for preventing COVID-19 as described above, comprising administering to an individual a cell vaccine containing a vaccine antigen for preventing COVID-19 subcutaneously or intramuscularly, one or more times, said vaccine for preventing COVID-19 being preserved at-80 ℃ or in liquid nitrogen for use before injection.

The vaccine is used for preparing a medicament for preventing SARS-CoV2 virus.

The invention relates to an immunization method against SARS-CoV2, which comprises subcutaneous (or intramuscular) injection of cell vaccine containing antigen of the invention to individual, furthermore, the administration can be once or many times, the specific implementation situation can change or adjust the immunization times or time points according to the specific actual situation.

The fusion antigen constructed by the invention can be further processed into DNA vaccine, RNA vaccine, protein vaccine or recombinant virus vaccine.

The nucleocapsid protein T cell reaction peptide segment contained in the invention has high conservative property and high homology with SARS-CoV virus, and simultaneously the peptide segment can induce an organism to generate T cell immunoreaction, and the antibody induction capability is weak.

The nucleocapsid protein T cell reaction peptide fragment antibody contained in the invention has weak induction capability and can be used for differential diagnosis of vaccinees and SARS-CoV2 infection.

The invention also provides the application of the effective novel cell vaccine in preventing novel coronavirus SARS-CoV2 virus. The cell expresses fused vaccine antigen, and the secreted GP96-hFc (fusion protein of GP96 and human IgG1 Fc fragment) is used as immunity increasing agent.

The invention has the beneficial effects that: the antigen of the vaccine of the invention comprises two functional parts: an S protein receptor binding domain capable of inducing a specific neutralizing antibody and a T cell-associated N protein truncated peptide segment capable of inducing activation of effector T cells; the vaccine of the invention has the following characteristics: the generation capacity of the T cell related N protein truncated peptide fragment for inducing the N protein antibody is weak, a vaccine vaccinator and a new corona infected patient can be identified by using the N antibody, the vaccine antigen does not induce the generation of the N protein antibody, the lung injury can be reduced, and the safety is higher; the cell vaccine of the invention has low cost and can induce the generation of virus specific neutralizing antibody and T cell immune response.

Drawings

FIG. 1 is a bioinformatics analysis of S protein of SARS-CoV2, wherein (A) shows the major functional domains of the S protein; (B) potential B cell epitope distribution predicted based on SARS-CoV2 RBD 3D structure; (C) potential linear B cell epitope distribution of SARS-CoV 2S full-length protein; (D) the left panel shows the position of the potential B-cell antigens of SARS-CoV (blue) and SARS-CoV2 (green) in the 3D structure (SARS-CoV antigen labeled purple, SARS-CoV2 antigen labeled red), the right panel is a model of the interaction between the binding domain of the SARS-CoV2 receptor and the ACE2 receptor, and the yellow portion is the key site for binding of the RBD region to the receptor ACE 2.

FIG. 2 is a comparison of SARS-CoV2 and SARS-CoV, (A) homology analysis of the S1 and S2 domains of SARS-CoV2 and SARS-nCoV spike protein; (B) the distribution of the SARS-CoV2 and SARS-nCoV spike protein at different sites. (C) Potential B cell epitope distribution predicted based on SARS-CoV RBD 3D structure; (D) the SARS-CoV S full-length protein has potential linear B cell epitope distribution.

FIG. 3 (A) expression of plasmids with the spike protein and nucleocapsid protein of the SARS-CoV2 wild sequence in human HEK-293T; (B) SARS-CoV2 spike protein wild type sequence and codon optimized (opt) sequence expression.

FIG. 4 homology analysis of SARS-CoV2 and SARS-nCoV nucleocapsid protein.

FIG. 5 SARS-CoV2 nucleocapsid protein (N) epitope analysis, wherein (A) N protein is a potential B cell epitope; (B) potential MHCI binding peptide distribution of N protein; (C) functional domain of SARS-CoV N protein and its antibody surface bitmap.

FIG. 6 expression of chimeric vaccine antigen and antibody recognition features, wherein (A) is a skeletal schematic of a chimeric vaccine of SARS-CoV 2; (B) the recognition characteristics of different antibodies against SARS-CoV2 derived proteins and chimeric vaccine antigens were analyzed using SARS-CoV2 recovered patient antiserum, commercial polyclonal antibodies against SARS-CoV2 RBD, and commercial antibodies against SARS-CoV2 nucleocapsid.

FIG. 7 shows the recognition characteristics of different COVID-19 patient convalescent sera (5 cases) against the SARS-COV2 derived protein and the chimeric vaccine antigen.

Fig. 8 demonstrates binding of 3 polypeptides of the Ntap region to human HLA-a 0201 molecules using T2 cells.

FIG. 9 validation of antigen expression of a novel cell-based fusion vaccine, wherein (A) expression of gp96-Fc protein in a chimeric cell vaccine (C-Vac) was detected using immunocytochemistry experiments; (B) Western-Blot was used to examine the recognition characteristics of different antibodies against the chimeric cell vaccine (C-Vac) antigen.

FIG. 10 evaluation of the effectiveness and safety of the chimeric vaccine (C-Vac) using the golden hamster. Immunization of syrian hamsters with different treated HEK293T cell vaccines (untreated live cells, mitomycin C treated cells and freeze-thaw cell lysates), evaluation of neutralizing antibody expression by a pseudovirus infection assay at day 7 (a) and day 21 (day 14 for second immunization) after immunization (B); (C) hematoxylin-eosin (HE) to detect hamster lung changes at day 7 of primary immunization and (D) to assess virus-specific T cell responses using the size of the tumor of hamster-derived kidney cells BHK21 expressing the fusion antigen.

Detailed Description

The vaccine for preventing COVID-19 has the antigen with the nucleotide sequence of SEQ NO. 1 and the amino acid sequence of SEQ NO. 2.

The polynucleotide for coding the receptor binding structural domain of SARS-CoV2 is optimized by codon, and the amino acid sequence is SEQ NO. 6; the self-cutting peptide T2A has an amino acid sequence of SEQ NO. 7; the amino acid sequence of the N protein T cell reaction peptide segment is SEQ NO. 8.

(1) firstly, constructing a lentiviral vector capable of secreting and expressing GP96-hFc, infecting and screening a cell line of HEK293T capable of stably secreting and expressing GP96-hFc, wherein the nucleotide sequence of GP96-hFc is shown as SEQ NO. 9, and the protein sequence is shown as SEQ NO. 10;

(2) constructing a lentivirus vector which can be used for chimeric expression of a receptor binding structural domain of SARS-CoV2 and a truncated N protein T cell related peptide fragment, and screening out a cell line of HEK293T which can secrete and express GP96-hFc and simultaneously chimeric expression of the receptor binding structural domain of SARS-CoV2 and the truncated N protein T cell related peptide fragment on the basis of the step 1);

(3) Collecting the cells obtained in step 2) under aseptic conditions, washing with sterile physiological saline or PBS, and adjusting the cell concentration to 1X 10 ⁶ ~1×10 ⁷ And 100 mu L of the vaccine is used for obtaining the vaccine for preventing the COVID-19.

Example 1: bioinformatic analysis of the S protein of SARS-CoV2

The S protein of SARS-CoV2 can be divided into a plurality of functional structural domains by referring to SARS-CoV related research and UniProt public data; it can be seen that the S protein is mainly composed of S1 and S2 subunits (fig. 1A), in which the Receptor Binding Domain (RBD) in S1 can be bound to angiotensin converting enzyme 2 (ACE 2) to trigger the conformational change of homotrimer, while the S2 subunit can further form six-helix bundle to promote the fusion of virus and host cell membrane. The potential 3D structure-based B cell antigen of the SARS-CoV 2S protein receptor binding domain was analyzed using the Discospe software (FIG. 1B), and the potential linear B cell epitope of the SARS-CoV2 whole S protein was analyzed using the IEDB database (FIG. 1C), which showed less structure-based B antigen and more abundant linear B cell antigen. The structure of the RBD domain from SARS-CoV2 (PDB: 6 LZG) and SARS-CoV (PDB: 2 GHW) was compared using 3D-Match (http: www.softberry.commberry.phtml) and was shown using Discovery Studio to find that the potential 3D B cell antigen (SARSCoV 2: Red, SARS-CoV: purple) localized in the receptor binding domain was predominantly localized in the region where the receptor binding domain interacted with the ACE2 receptor (FIG. 1D).

By using MegAlign software analysis to show that the S1 subunit homology between SARS-CoV and SARS-CoV-2 is lower than the S2 subunit (67.1% vs 90%) (FIG. 2A), further labeling for differential amino acids (red) (FIG. 2B) reveals that the residues of SARS-CoV and SARS-CoV-2 are very different, suggesting that S protein-based vaccines alone may have reduced vaccine protection due to mutations at the RBD locus point. FIG. 2C is a Discospe software analysis of potential 3D structure-based B cell antigens of the SARS-CoV S protein receptor binding domain, and FIG. 2D is an analysis of potential linear B cell epitopes of SARS-CoV whole S protein using the IEDB database, and it was found that the RBD of SARS-CoV2 is less immunogenic than the RBD of SARS-CoV, which may be the reason for such widespread of SARS-CoV 2.

Example 2: verification of SARS-CoV2 spike protein and nucleocapsid protein expression

The S protein gene of SARS-COV (SEQ NO: 11) and the nucleocapsid protein of SARS-COV2 (SEQ NO: 12) were synthesized by Sangon Biotech according to the NCBI database published sequences ((MN 908947.3) and constructed into pcDNA3.1-his tag (hygro) vector the codon-optimized SARS-CoV2 plasmid was purchased from Sino biological and the expression of PCDNA3.1-his-opt S, PCDNA3.1-his-opt S1, PCDNA3.1-his-opt NRBD (from N-terminus to RBD domain) was constructed using the following primers, respectively.

Spike-optF (codon optimized): ACTTAATTAAGCCACCATGTTTGTGTTCCTGGTGCTGCT the flow of the air in the air conditioner,

Spike-opt-R：TAACCGGTGGTGTAGTGCAGTTTCACTCCTTTCA；

Spike-optS1-R：TAACCGGTGCTGTTGGTCTGGGTCTGGTAGG；

Spike-optNRBD-R：TAACCGGTTCCATTGAAGTTGAAGTTCACACACTT；

the corresponding expression plasmid is transfected into HEK-293T cells by using PEI, and the expression of the corresponding gene is detected by using a Western Blot method. Western Blot procedure as follows:

cells were washed twice on ice with phosphate buffered saline, and cells were lysed on ice with RIPA lysis buffer containing 1% protease inhibitor (50 mM tris.hcl, pH 7.4, 0.1% SDS, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA) for 10 minutes, centrifuged at 13000 rpm for 30 minutes at 4 ℃ to clarify the cell lysate, then 25 ug total protein was separated by 10% SDS-PAGE and transferred to PVDF membrane (Millipore). Membranes were blocked with 5% skim milk and incubated with primary antibody, TBST washed 3 times, following the antibody manufacturer's instructions, then incubated with horseradish peroxidase-conjugated secondary antibody (1: 5000, ZSBIO) for 1 hour at room temperature, then membranes were washed 3 times with TBST and detected by Enhanced Chemiluminescence (ECL) system (Thermo pierce, usa). The antibodies used included mouse anti-histidine tag mAb (Abmart, M20001S), HRP goat anti-mouse IgG (ZSBIO, ZB-5305).

FIG. 3A shows the expression of SARS-CoV2 wild sequence plasmid (Vector: pcDNA3.1-his Vector, Spike: S protein, N: N protein), and FIG. 3B shows the expression of different Spike and its derivative sequence plasmids (Spike-WT: wild type S protein, Spike-OPT: S expression plasmid after codon optimization, S1-OPT: S1 expression plasmid after codon optimization, NRBD-OPT: expression plasmid after codon optimization from the initial N-terminal of Spike to RBD region). The results show that the expression of the S protein can hardly be detected in human 293T cells by using the original sequence of the S gene, and the expression of the S protein is obviously increased after the codon is optimized, which indicates that the codon-optimized S gene is very important for designing SARS-COV-2 vaccine.

Example 3: bioinformatic analysis of the N protein of SARS-CoV2

Homology analysis was performed on the nucleocapsid protein (N protein) between SARS-CoV2 and SARS-nCoV using MegAlign. The N protein was shown to have 91.2% homology between SARS-CoV-2 and SARS-CoV amino acids (FIG. 4), which supports the results that the convalescent serum of SARS-CoV has high cross-reactivity with the serum of a patient newly infected with SARS-CoV-2, suggesting that N protein-based vaccines may have cross-protective effects.

The potential B cell epitopes of N protein (fig. 5A) and potential MHC I binding peptides of N protein (fig. 5B) were predicted from the IEDB database and the results showed that there were two regions of N protein with fewer B cell epitopes but abundant histocompatibility complex 1 (MHC I) binding T cell epitopes. FIG. 5C is a functional domain of SARS-CoV N protein and its antibody surface map, which shows that the antibody against amino acid 341 (second region) 212-protein polypeptide in SARS patients is reduced, and this result is consistent with the analysis result in FIG. 4.

Example 4: construction of chimeric vaccine antigen and antigen expression verification

The framework of the chimeric vaccine for SARS-CoV2 was designed based on the above analytical data (FIG. 6A), RBD: s protein receptor binding domain (316-541 aa), Ntap: t cell associated peptide of protein N (211-339 aa). The vaccine consists of a receptor binding domain of an S protein and an N protein T cell response peptide fragment (Ntap) which induce a specific neutralizing antibody.

A T2A cleavage peptide and a truncated N protein T cell-associated peptide fragment (synthesized by Sangon Biotech) with a codon optimized RBD domain (including a signal peptide) were synthesized and inserted into a modified pLenti6-puromycin vector to construct a recombinant lentiviral vector. The antibody recognition characterization of SARS-CoV 2-derived proteins and C-Vac antigen was verified using the SARS-CoV2 recovered patient antisera and commercial antibodies against SARS-CoV2 RBD or nucleocapsid (FIG. 6B), the method is referenced in example 3. The result shows that the chimeric vaccine antigen can be well expressed, the SARS-CoV2 RBD protein and the full-length N protein can induce the organism to generate the antibody, and the commercialized polyclonal antibody can not well identify the Ntap, which proves that the induction capability of the Ntap antibody is weak.

In addition, Western Blot assays using convalescent serum patients of five additional patients with COVID-19 were found to have high reactivity antibodies against the intact N protein in the convalescent serum of COVID-19 patients, without significant recognition of the S protein and T cell-associated Ntap (fig. 7), all of which were provided by the first subsidiary hospital of zheng state university and approved by the ethical committee of zheng state university.

The antibody used is detailed below: rabbit anti-RBD PAb (polyclonal antibody) (Sino Biological, # 40592-T62), rabbit anti-nucleoapsid PAb (Sino Biological, # 40588-T62), HRP goat anti-rabbit IgG (ZSBIO, ZB-5301), HRP goat anti-human IgG (ZSBIO, ZB-2304).

Example 5: verification of binding ability of Ntap-derived polypeptide and human MHC I by flow cytometry

The affinity between HLA-a 0201 and the peptide was assessed using T2 cells that do not express HLA DR and are negative for class II Major Histocompatibility (MHC) molecules as tool cells. Three potential binding polypeptides HLA-a × 0201 from Ntap (LALLLLDRLNQL, RLNQLESKM, GMSRIGMEV), a positive binding peptide from HER2 (KIFGSLAFL) and a negative binding peptide from MUC1 (SAPDTRPAP) were synthesized separately (all peptides were synthesized by gill chemical limited) and verified as described below.

The method comprises the following steps: mixing 100 μ L of 1 × 10 ⁵ T2 cells (FBS-free IMEM medium resuspended) were platedPlanting the polypeptides in a 96-well round bottom plate, and then adding the polypeptides diluted by a multiple ratio with the maximum concentration of 100 mu M and incubating for 4 hours; the cells were then washed twice with pre-chilled PBS and stained with mouse anti-human HLA-A2-FITC antibody (Abcam) for 30 min on ice; after two washes with pre-chilled PBS, the fluorescence intensity of the cells was analyzed using BD FACSAria (BD Biosciences Immunocytometry Systems).

The results show that both epitope peptides derived from Ntap (LLLDRLNQL and GMSRIGMEV) and a positive peptide derived from HER2 (KIFGSLAFL) are able to bind to T2 cells, whereas RLNQESKM peptide and a negative peptide derived from MUC1 (SAPDTRPAP) do not bind to T2 cells (fig. 8). Sequence alignment also showed that, compared to SARS-CoV (RLNQESKV) (which has been shown to bind to HLA-A0201), the peptide RLNQESKM derived from SARS-CoV2 has a mutation at a site that may affect the binding of the peptide to HLA-A0201 molecules.

Example 6: construction of COVID-19 novel cell vaccine

(1) Construction of a plasmid 3.1-GP96-hFc (SEQ NO: 9) capable of secreting and expressing GP96-hFc was performed synthetically, a plasmid was transfected with Higene (apple, China), and selected with 200. mu.g/ml hygromycin B (Invitrogen), a 293T-GP96-hFc overexpression cell line was constructed, followed by detection of the expression of the fusion protein using HRP goat anti-human IgG (ZSBIO, ZB-2304) and DAB color kit (Mixin, Fuzhou) and immunocytochemistry (FIG. 9A).

(2) Synthesis of a lentivirus plasmid expressing the vaccine antigen (SEQ NO: 1) and coating of the lentivirus expressing the vaccine antigen according to the following steps: 2 x 10 to ⁶ HEK293T cells were seeded into 10 cm cell culture dishes and the plasmid mixture (including 25. mu.g of the plasmid of interest, 8. mu.g of psPAX2, 4. mu.g of pMD2.G and 40. mu.L of 2mg/ml PEI) was co-transfected; fresh medium was replaced after 6 hours, the lentiviral-containing supernatant was collected after 48 hours, filtered through a 0.22 μm filter (Millipore) and stored in aliquots at-80 ℃. The 293T-GP96-hFc cells constructed in the step 1) were inoculated into a 24-well plate, then 1 ml of lentivirus supernatant containing vaccine antigens and 5. mu.g/ml polybrene (Sigma) were added, the fresh medium was replaced after 12 hours, and 5. mu.g/ml purine was added 72 hours after infection For selection of stable cell lines, an ansamycin (selelck) was used. The expression of the vaccine antigen is detected by using a Western Blot method (FIG. 9B), and the chimeric HEK293T cell vaccine (C-Vac) can be seen to stably express the vaccine fusion antigen.

Example 7: evaluation of therapeutic Effect of COVID-19 novel cell vaccine

A number of articles have reported that Syrian hamsters (also known as golden hamsters) support infection and replication of SARS-CoV2 and are ideal animal models. 36 female syrian hamsters of 12 weeks of age were purchased from experimental animal technology ltd, viton, beijing and randomized into 4 groups (n = 9). 1 st to 4 th groups were inoculated with 1X 10 seed respectively ⁷ 293T-GP96-hFc control cells, live 293T-C-Vac cells, 293T-C-Vac cells treated with 5. mu.g/ml Mitomycin (MCE) for 4h, and 293T-C-Vac vaccine cells freeze-thaw lysate. On day 7 post-immunization, three hamsters per group were sacrificed and animal serum samples were collected and stored at-80 ℃ while lung tissue was fixed in 10% neutral buffered formalin and made paraffin specimens. The remaining animals were boosted on day 14 using the same protocol as the prime and serum was harvested on day 21 (day 7 after boost). The remaining hamsters were used 5X 10 on day 45 ⁶ Hamster BHK21 cells expressing RBD-Ntap (C-Vac antigen) (constructed as in example 5) were subjected to tumor-bearing experiments and the allograft volume was measured to assess vaccine-specific cytotoxic T cell effects. All animals used in this study were housed under SPF-grade conditions at the experimental animal center of zhengzhou university and were treated according to the ethical provisions of the animal experiments.

(1) Encapsulation of SARS-CoV2 pseudovirus and hamster serum neutralizing antibody assay

2 x 10 to ⁶ HEK293T cells were seeded into 10 cm cell culture dishes and co-transfected with 20. mu.g of lenti-Luc (a luciferase-expressing lentiviral plasmid), 10. mu.g of psPAX and 10. mu.g of 3.1-opt S-d18 (expressing the terminal 18 amino acid residues of the deleted S protein), and SARS-CoV2 pseudovirus was collected as described for the lentivirus method. Hamster sera from vaccine immunizations and control groups were heat inactivated at 56 ℃ for 30 minutes and were separated from 1: 25 start the dilution by multiple. Then will be fixedQuantitative pseudovirus (50. mu.L) was incubated with 50. mu.L of diluted serum for 1 hour at 37 ℃ in a cell incubator and infected with 1X 10 ⁴ Hamster BHK21 expressing human hACE 2. At 72 hours post-infection, cells were measured for relative luciferase activity using the luciferase assay system (Promega) and the GloMax Discover detector (Promega). Serum neutralization titers were calculated using 50% RLU signal compared to control cells (ID 50). As shown in fig. 10, all vaccinated groups expressing the fusion antigen were effective in inducing specific neutralizing antibodies on day 7 of a single immunization (fig. 10A) and on day 7 after the second booster immunization (day 21 after the first immunization) (fig. 10B).

(2) Evaluation of the Effect of vaccines on the Lung of golden hamster by hematoxylin and eosin staining

Cutting a lung tissue paraffin specimen of a hamster into 6 mu m sections, operating according to a standard hematoxylin-eosin staining procedure, and photographing after a neutral resin is sealed to analyze lung tissue change. The results showed that no significant lung changes were observed in all animals, suggesting higher safety of the novel covi-19 cell vaccine (fig. 10C).

(3) Homogeneous tumor-bearing experiment analysis of virus specific T cell response after vaccination

Subcutaneous injection of 5X 10 into the remaining hamsters on day 45 after primary immunization ⁶ Hamster BHK-21 expressing the fusion vaccine antigen (constructed as in example 6) was assayed for tumor volume at day 70. The syrian hamster significantly reduced the allogeneic cell tumorigenic volume following immunization with the live cell vaccine and the split cell vaccine as described in fig. 10D, indicating that the chimeric vaccine of the invention can stimulate the body to produce a viral antigen-specific T cell immune response.

Many vaccines against SARS-CoV2 are currently being developed in preclinical studies and clinical trials, but most vaccines are directed against spike protein only. In the present invention, we have developed a novel chimeric vaccine (C-Vac) targeting the Receptor Binding Domain (RBD) of the S protein and the truncated peptide of the nucleocapsid protein (N) that induces T cell activation. The vaccine can effectively induce specific neutralizing antibody and specific T cell effect aiming at virus protein, has lower risk of inducing antibody-dependent infection enhancement and N protein antibody-mediated immunotoxicity, and has higher safety. The C-Vac of the invention contains high conservative Ntap, is expected to provide long-term immune protection for a novel coronavirus and provides certain protection for SARS-CoV2 and mutants thereof. Meanwhile, the Ntap peptide segment contained in the vaccine disclosed by the invention has weak generation capacity of inducing an N protein antibody, and a vaccine vaccinee and a new coronary infected patient can be identified by using the N protein antibody. The cell vaccine of the invention has low cost and can effectively induce and generate virus characteristic neutralizing antibody and T cell immune response.

SEQUENCE LISTING

<110> Zhengzhou university

<120> a novel vaccine for preventing COVID-19 and a preparation method thereof

<130> 2020

<160> 10

<170> PatentIn version 3.3

<210> 1

<211> 1206

<212> DNA

<213> nucleotide sequence SEQ NO:1

<400> 1

atggacatga gggtgcccgc ccagctgctg ggcctgctgc tgctgtggct gaggggcgcc 60

aggtgcgtcc aaccaacaga gagcattgtg aggtttccaa acatcaccaa cctgtgtcca 120

tttggagagg tgttcaatgc caccaggttt gcctctgtct atgcctggaa caggaagagg 180

attagcaact gtgtggctga ctactctgtg ctctacaact ctgcctcctt cagcaccttc 240

aagtgttatg gagtgagccc aaccaaactg aatgacctgt gtttcaccaa tgtctatgct 300

gactcctttg tgattagggg agatgaggtg agacagattg cccctggaca aacaggcaag 360

attgctgact acaactacaa actgcctgat gacttcacag gctgtgtgat tgcctggaac 420

agcaacaacc tggacagcaa ggtgggaggc aactacaact acctctacag actgttcagg 480

aagagcaacc tgaaaccatt tgagagggac atcagcacag agatttacca ggctggcagc 540

acaccatgta atggagtgga gggcttcaac tgttactttc cactccaatc ctatggcttc 600

caatcaacca atggagtggg ctaccaacca tacagggtgg tggtgctgtc ctttgaactg 660

ctccatgccc ctgccacagt gtgtggacca aagaagagca ccaacctggt gaagaacaag 720

tgtgtgaact tcaacttcaa tggacgtagg aagcgaggat caggcgaggg cagaggaagt 780

cttctaacat gcggtgacgt gcaggagaat cccggccctg gcaatggcgg tgatgctgct 840

cttgctttgc tgctgcttga cagattgaac cagcttgaga gcaaaatgtc tggtaaaggc 900

caacaacaac aaggccaaac tgtcactaag aaatctgctg ctgaggcttc taagaagcct 960

cggcaaaaac gtactgccac taaagcatac aatgtaacac aagctttcgg cagacgtggt 1020

ccagaacaaa cccaaggaaa ttttggggac caggaactaa tcagacaagg aactgattac 1080

aaacattggc cgcaaattgc acaatttgcc cccagcgctt cagcgttctt cggaatgtcg 1140

cgcattggca tggaagtcac accttcggga acgtggttga cctacacagg tgccatcaaa 1200

ttgtaa 1206

<210> 2

<211> 401

<212> PRT

<213> amino acid sequence SEQ NO 2

<400> 2

Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp

1 5 10 15

Leu Arg Gly Ala Arg Cys Val Gln Pro Thr Glu Ser Ile Val Arg Phe

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

Cys Val Asn Phe Asn Phe Asn Gly Arg Arg Lys Arg Gly Ser Gly Glu

245 250 255

Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Gln Glu Asn Pro Gly

260 265 270

Pro Gly Asn Gly Gly Asp Ala Ala Leu Ala Leu Leu Leu Leu Asp Arg

275 280 285

Leu Asn Gln Leu Glu Ser Lys Met Ser Gly Lys Gly Gln Gln Gln Gln

290 295 300

Gly Gln Thr Val Thr Lys Lys Ser Ala Ala Glu Ala Ser Lys Lys Pro

305 310 315 320

Arg Gln Lys Arg Thr Ala Thr Lys Ala Tyr Asn Val Thr Gln Ala Phe

325 330 335

Gly Arg Arg Gly Pro Glu Gln Thr Gln Gly Asn Phe Gly Asp Gln Glu

340 345 350

Leu Ile Arg Gln Gly Thr Asp Tyr Lys His Trp Pro Gln Ile Ala Gln

355 360 365

Phe Ala Pro Ser Ala Ser Ala Phe Phe Gly Met Ser Arg Ile Gly Met

370 375 380

Glu Val Thr Pro Ser Gly Thr Trp Leu Thr Tyr Thr Gly Ala Ile Lys

385 390 395 400

Leu

<210> 3

<211> 744

<212> DNA

<213> codon optimized RBD synthetic sequence SEQ NO:3

<400> 3

atggacatga gggtgcccgc ccagctgctg ggcctgctgc tgctgtggct gaggggcgcc 60

aggtgcgtcc aaccaacaga gagcattgtg aggtttccaa acatcaccaa cctgtgtcca 120

tttggagagg tgttcaatgc caccaggttt gcctctgtct atgcctggaa caggaagagg 180

attagcaact gtgtggctga ctactctgtg ctctacaact ctgcctcctt cagcaccttc 240

aagtgttatg gagtgagccc aaccaaactg aatgacctgt gtttcaccaa tgtctatgct 300

gactcctttg tgattagggg agatgaggtg agacagattg cccctggaca aacaggcaag 360

attgctgact acaactacaa actgcctgat gacttcacag gctgtgtgat tgcctggaac 420

agcaacaacc tggacagcaa ggtgggaggc aactacaact acctctacag actgttcagg 480

aagagcaacc tgaaaccatt tgagagggac atcagcacag agatttacca ggctggcagc 540

acaccatgta atggagtgga gggcttcaac tgttactttc cactccaatc ctatggcttc 600

caatcaacca atggagtggg ctaccaacca tacagggtgg tggtgctgtc ctttgaactg 660

ctccatgccc ctgccacagt gtgtggacca aagaagagca ccaacctggt gaagaacaag 720

tgtgtgaact tcaacttcaa tgga 744

<210> 4

<211> 75

<212> DNA

<213> Synthesis of T2A sequence SEQ NO 4

<400> 4

cgtaggaagc gaggatcagg cgagggcaga ggaagtcttc taacatgcgg tgacgtgcag 60

gagaatcccg gccct 75

<210> 5

<211> 387

<212> DNA

<213> N protein T reaction peptide fragment synthetic sequence SEQ NO 5

<400> 5

ggcaatggcg gtgatgctgc tcttgctttg ctgctgcttg acagattgaa ccagcttgag 60

agcaaaatgt ctggtaaagg ccaacaacaa caaggccaaa ctgtcactaa gaaatctgct 120

gctgaggctt ctaagaagcc tcggcaaaaa cgtactgcca ctaaagcata caatgtaaca 180

caagctttcg gcagacgtgg tccagaacaa acccaaggaa attttgggga ccaggaacta 240

atcagacaag gaactgatta caaacattgg ccgcaaattg cacaatttgc ccccagcgct 300

tcagcgttct tcggaatgtc gcgcattggc atggaagtca caccttcggg aacgtggttg 360

acctacacag gtgccatcaa attgtaa 387

<210> 6

<211> 250

<212> PRT

<213> RBD amino acid sequence SEQ NO 6

<400> 6

Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp

1 5 10 15

Leu Arg Gly Ala Arg Cys Leu Glu Val Gln Pro Thr Glu Ser Ile Val

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His

210 215 220

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

225 230 235 240

Asn Lys Cys Val Asn Phe Asn Phe Asn Gly

245 250

<210> 7

<211> 25

<212> PRT

<213> T2A amino acid sequence SEQ NO 7

<400> 7

Arg Arg Lys Arg Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys

1 5 10 15

Gly Asp Val Gln Glu Asn Pro Gly Pro

20 25

<210> 8

<211> 128

<212> PRT

<213> N protein T reaction peptide amino acid sequence SEQ NO 8

<400> 8

Gly Asn Gly Gly Asp Ala Ala Leu Ala Leu Leu Leu Leu Asp Arg Leu

1 5 10 15

Asn Gln Leu Glu Ser Lys Met Ser Gly Lys Gly Gln Gln Gln Gln Gly

20 25 30

Gln Thr Val Thr Lys Lys Ser Ala Ala Glu Ala Ser Lys Lys Pro Arg

35 40 45

Gln Lys Arg Thr Ala Thr Lys Ala Tyr Asn Val Thr Gln Ala Phe Gly

50 55 60

Arg Arg Gly Pro Glu Gln Thr Gln Gly Asn Phe Gly Asp Gln Glu Leu

65 70 75 80

Ile Arg Gln Gly Thr Asp Tyr Lys His Trp Pro Gln Ile Ala Gln Phe

85 90 95

Ala Pro Ser Ala Ser Ala Phe Phe Gly Met Ser Arg Ile Gly Met Glu

100 105 110

Val Thr Pro Ser Gly Thr Trp Leu Thr Tyr Thr Gly Ala Ile Lys Leu

115 120 125

<210> 9

<211> 3102

<212> DNA

<213> GP96-hFc nucleotide sequence SEQ NO 9

<400> 9

atgagggccc tgtgggtgct gggcctctgc tgcgtcctgc tgaccttcgg gtcggtcaga 60

gctgacgatg aagttgatgt ggatggtaca gtagaagagg atctgggtaa aagtagagaa 120

ggatcaagga cggatgatga agtagtacag agagaggaag aagctattca gttggatgga 180

ttaaatgcat cacaaataag agaacttaga gagaagtcgg aaaagtttgc cttccaagcc 240

gaagttaaca gaatgatgaa acttatcatc aattcattgt ataaaaataa agagattttc 300

ctgagagaac tgatttcaaa tgcttctgat gctttagata agataaggct aatatcactg 360

actgatgaaa atgctctttc tggaaatgag gaactaacag tcaaaattaa gtgtgataag 420

gagaagaacc tgctgcatgt cacagacacc ggtgtaggaa tgaccagaga agagttggtt 480

aaaaaccttg gtaccatagc caaatctggg acaagcgagt ttttaaacaa aatgactgaa 540

gcacaggaag atggccagtc aacttctgaa ttgattggcc agtttggtgt cggtttctat 600

tccgccttcc ttgtagcaga taaggttatt gtcacttcaa aacacaacaa cgatacccag 660

cacatctggg agtctgactc caatgaattt tctgtaattg ctgacccaag aggaaacact 720

ctaggacggg gaacgacaat tacccttgtc ttaaaagaag aagcatctga ttaccttgaa 780

ttggatacaa ttaaaaatct cgtcaaaaaa tattcacagt tcataaactt tcctatttat 840

gtatggagca gcaagactga aactgttgag gagcccatgg aggaagaaga agcagccaaa 900

gaagagaaag aagaatctga tgatgaagct gcagtagagg aagaagaaga agaaaagaaa 960

ccaaagacta aaaaagttga aaaaactgtc tgggactggg aacttatgaa tgatatcaaa 1020

ccaatatggc agagaccatc aaaagaagta gaagaagatg aatacaaagc tttctacaaa 1080

tcattttcaa aggaaagtga tgaccccatg gcttatattc actttactgc tgaaggggaa 1140

gttaccttca aatcaatttt atttgtaccc acatctgctc cacgtggtct gtttgacgaa 1200

tatggatcta aaaagagcga ttacattaag ctctatgtgc gccgtgtatt catcacagac 1260

gacttccatg atatgatgcc taaatacctc aattttgtca agggtgtggt ggactcagat 1320

gatctcccct tgaatgtttc ccgcgagact cttcagcaac ataaactgct taaggtgatt 1380

aggaagaagc ttgttcgtaa aacgctggac atgatcaaga agattgctga tgataaatac 1440

aatgatactt tttggaaaga atttggtacc aacatcaagc ttggtgtgat tgaagaccac 1500

tcgaatcgaa cacgtcttgc taaacttctt aggttccagt cttctcatca tccaactgac 1560

attactagcc tagaccagta tgtggaaaga atgaaggaaa aacaagacaa aatctacttc 1620

atggctgggt ccagcagaaa agaggctgaa tcttctccat ttgttgagcg acttctgaaa 1680

aagggctatg aagttattta cctcacagaa cctgtggatg aatactgtat tcaggccctt 1740

cccgaatttg atgggaagag gttccagaat gttgccaagg aaggagtgaa gttcgatgaa 1800

agtgagaaaa ctaaggagag tcgtgaagca gttgagaaag aatttgagcc tctgctgaat 1860

tggatgaaag ataaagccct taaggacaag attgaaaagg ctgtggtgtc tcagcgcctg 1920

acagaatctc cgtgtgcttt ggtggccagc cagtacggat ggtctggcaa catggagaga 1980

atcatgaaag cacaagcgta ccaaacgggc aaggacatct ctacaaatta ctatgcgagt 2040

cagaagaaaa catttgaaat taatcccaga cacccgctga tcagagacat gcttcgacga 2100

attaaggaag atgaagatga taaaacagtt ttggatcttg ctgtggtttt gtttgaaaca 2160

gcaacgcttc ggtcagggta tcttttacca gacactaaag catatggaga tagaatagaa 2220

agaatgcttc gcctcagttt gaacattgac cctgatgcaa aggtggaaga agagcccgaa 2280

gaagaacctg aagagacagc agaagacaca acagaagaca cagagcaaga cgaagatgaa 2340

gaaatggatg tgggaacaga tgaagaagaa gaaacagcaa aggaatctac agctgaagct 2400

agcgagccca aatcttgtga caaaactcac acatgcccac cgtgcccagc acctgaactc 2460

ctggggggac cgtcagtctt cctcttcccc ccaaaaccca aggacaccct catgatctcc 2520

cggacccctg aggtcacatg cgtggtggtg gacgtgagcc acgaagaccc tgaggtcaag 2580

ttcaactggt acgtggacgg cgtggaggtg cataatgcca agacaaagcc gcgggaggag 2640

cagtacaaca gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca ggactggctg 2700

aatggcaagg agtacaagtg caaggtctcc aacaaagccc tcccagcccc catcgagaaa 2760

accatctcca aagccaaagg gcagccccga gaaccacagg tgtacaccct gcccccatcc 2820

cgggatgagc tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctatccc 2880

agcgacatcg ccgtggagtg ggagagcaat gggcagccgg agaacaacta caagaccacg 2940

cctcccgtgc tggactccga cggctccttc ttcctctaca gcaagctcac cgtggacaag 3000

agcaggtggc agcaggggaa cgtcttctca tgctccgtga tgcatgaggg tctgcacaac 3060

cactacacgc agaagagcct ctccctgtct ccgggtaaat aa 3102

<210> 10

<211> 1033

<212> PRT

<213> GP96-hFc amino acid sequence SEQ NO 10

<400> 10

Met Arg Ala Leu Trp Val Leu Gly Leu Cys Cys Val Leu Leu Thr Phe

1 5 10 15

Gly Ser Val Arg Ala Asp Asp Glu Val Asp Val Asp Gly Thr Val Glu

20 25 30

Glu Asp Leu Gly Lys Ser Arg Glu Gly Ser Arg Thr Asp Asp Glu Val

35 40 45

Val Gln Arg Glu Glu Glu Ala Ile Gln Leu Asp Gly Leu Asn Ala Ser

50 55 60

Gln Ile Arg Glu Leu Arg Glu Lys Ser Glu Lys Phe Ala Phe Gln Ala

65 70 75 80

Glu Val Asn Arg Met Met Lys Leu Ile Ile Asn Ser Leu Tyr Lys Asn

85 90 95

Lys Glu Ile Phe Leu Arg Glu Leu Ile Ser Asn Ala Ser Asp Ala Leu

100 105 110

Asp Lys Ile Arg Leu Ile Ser Leu Thr Asp Glu Asn Ala Leu Ser Gly

115 120 125

Asn Glu Glu Leu Thr Val Lys Ile Lys Cys Asp Lys Glu Lys Asn Leu

130 135 140

Leu His Val Thr Asp Thr Gly Val Gly Met Thr Arg Glu Glu Leu Val

145 150 155 160

Lys Asn Leu Gly Thr Ile Ala Lys Ser Gly Thr Ser Glu Phe Leu Asn

165 170 175

Lys Met Thr Glu Ala Gln Glu Asp Gly Gln Ser Thr Ser Glu Leu Ile

180 185 190

Gly Gln Phe Gly Val Gly Phe Tyr Ser Ala Phe Leu Val Ala Asp Lys

195 200 205

Val Ile Val Thr Ser Lys His Asn Asn Asp Thr Gln His Ile Trp Glu

210 215 220

Ser Asp Ser Asn Glu Phe Ser Val Ile Ala Asp Pro Arg Gly Asn Thr

225 230 235 240

Leu Gly Arg Gly Thr Thr Ile Thr Leu Val Leu Lys Glu Glu Ala Ser

245 250 255

Asp Tyr Leu Glu Leu Asp Thr Ile Lys Asn Leu Val Lys Lys Tyr Ser

260 265 270

Gln Phe Ile Asn Phe Pro Ile Tyr Val Trp Ser Ser Lys Thr Glu Thr

275 280 285

Val Glu Glu Pro Met Glu Glu Glu Glu Ala Ala Lys Glu Glu Lys Glu

290 295 300

Glu Ser Asp Asp Glu Ala Ala Val Glu Glu Glu Glu Glu Glu Lys Lys

305 310 315 320

Pro Lys Thr Lys Lys Val Glu Lys Thr Val Trp Asp Trp Glu Leu Met

325 330 335

Asn Asp Ile Lys Pro Ile Trp Gln Arg Pro Ser Lys Glu Val Glu Glu

340 345 350

Asp Glu Tyr Lys Ala Phe Tyr Lys Ser Phe Ser Lys Glu Ser Asp Asp

355 360 365

Pro Met Ala Tyr Ile His Phe Thr Ala Glu Gly Glu Val Thr Phe Lys

370 375 380

Ser Ile Leu Phe Val Pro Thr Ser Ala Pro Arg Gly Leu Phe Asp Glu

385 390 395 400

Tyr Gly Ser Lys Lys Ser Asp Tyr Ile Lys Leu Tyr Val Arg Arg Val

405 410 415

Phe Ile Thr Asp Asp Phe His Asp Met Met Pro Lys Tyr Leu Asn Phe

420 425 430

Val Lys Gly Val Val Asp Ser Asp Asp Leu Pro Leu Asn Val Ser Arg

435 440 445

Glu Thr Leu Gln Gln His Lys Leu Leu Lys Val Ile Arg Lys Lys Leu

450 455 460

Val Arg Lys Thr Leu Asp Met Ile Lys Lys Ile Ala Asp Asp Lys Tyr

465 470 475 480

Asn Asp Thr Phe Trp Lys Glu Phe Gly Thr Asn Ile Lys Leu Gly Val

485 490 495

Ile Glu Asp His Ser Asn Arg Thr Arg Leu Ala Lys Leu Leu Arg Phe

500 505 510

Gln Ser Ser His His Pro Thr Asp Ile Thr Ser Leu Asp Gln Tyr Val

515 520 525

Glu Arg Met Lys Glu Lys Gln Asp Lys Ile Tyr Phe Met Ala Gly Ser

530 535 540

Ser Arg Lys Glu Ala Glu Ser Ser Pro Phe Val Glu Arg Leu Leu Lys

545 550 555 560

Lys Gly Tyr Glu Val Ile Tyr Leu Thr Glu Pro Val Asp Glu Tyr Cys

565 570 575

Ile Gln Ala Leu Pro Glu Phe Asp Gly Lys Arg Phe Gln Asn Val Ala

580 585 590

Lys Glu Gly Val Lys Phe Asp Glu Ser Glu Lys Thr Lys Glu Ser Arg

595 600 605

Glu Ala Val Glu Lys Glu Phe Glu Pro Leu Leu Asn Trp Met Lys Asp

610 615 620

Lys Ala Leu Lys Asp Lys Ile Glu Lys Ala Val Val Ser Gln Arg Leu

625 630 635 640

Thr Glu Ser Pro Cys Ala Leu Val Ala Ser Gln Tyr Gly Trp Ser Gly

645 650 655

Asn Met Glu Arg Ile Met Lys Ala Gln Ala Tyr Gln Thr Gly Lys Asp

660 665 670

Ile Ser Thr Asn Tyr Tyr Ala Ser Gln Lys Lys Thr Phe Glu Ile Asn

675 680 685

Pro Arg His Pro Leu Ile Arg Asp Met Leu Arg Arg Ile Lys Glu Asp

690 695 700

Glu Asp Asp Lys Thr Val Leu Asp Leu Ala Val Val Leu Phe Glu Thr

705 710 715 720

Ala Thr Leu Arg Ser Gly Tyr Leu Leu Pro Asp Thr Lys Ala Tyr Gly

725 730 735

Asp Arg Ile Glu Arg Met Leu Arg Leu Ser Leu Asn Ile Asp Pro Asp

740 745 750

Ala Lys Val Glu Glu Glu Pro Glu Glu Glu Pro Glu Glu Thr Ala Glu

755 760 765

Asp Thr Thr Glu Asp Thr Glu Gln Asp Glu Asp Glu Glu Met Asp Val

770 775 780

Gly Thr Asp Glu Glu Glu Glu Thr Ala Lys Glu Ser Thr Ala Glu Ala

785 790 795 800

Ser Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro

805 810 815

Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

820 825 830

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

835 840 845

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

850 855 860

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

865 870 875 880

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

885 890 895

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

900 905 910

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

915 920 925

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu

930 935 940

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

945 950 955 960

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

965 970 975

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

980 985 990

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

995 1000 1005

Phe Ser Cys Ser Val Met His Glu Gly Leu His Asn His Tyr Thr

1010 1015 1020

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

1025 1030

Claims

1. A vaccine for preventing COVID-19, comprising: the nucleotide sequence of the antigen of the vaccine is SEQ ID NO. 1, and the amino acid sequence is SEQ ID NO. 2.

2. The vaccine of claim 1, wherein the vaccine is for preventing COVID-19, and comprises: the vaccine antigen consists of three parts, namely a nucleotide sequence SEQ ID NO. 3 for coding a receptor binding structural domain of SARS-CoV2, a nucleotide sequence SEQ ID NO. 4 for coding self-cutting peptide T2A, and a nucleotide sequence SEQ ID NO. 5 for coding an N protein T cell reaction peptide segment.

3. The vaccine of claim 2, wherein the vaccine is for preventing COVID-19, and wherein: the polynucleotide for coding the receptor binding structural domain of SARS-CoV2 is optimized by codon, and the amino acid sequence is SEQ ID NO. 6; the amino acid sequence of the self-cutting peptide T2A is SEQ ID NO. 7; the amino acid sequence of the N protein T cell reaction peptide segment is SEQ ID NO. 8.

4. A vaccine for preventing COVID-19 according to claim 2 or 3, wherein: the receptor binding structural domain of the code SARS-CoV2 is RBD, and the reaction peptide segment of the code N protein T cell is Ntap.

5. A method of preparing a vaccine according to claim 1 or 2 for the prevention of covi-19, wherein: the method comprises the following steps:

(1) firstly, constructing a lentiviral vector capable of secreting and expressing GP96-hFc, infecting and screening a cell line of HEK293T capable of stably secreting and expressing GP96-hFc, wherein the nucleotide sequence of GP96-hFc is shown as SEQ ID NO. 9, and the protein sequence is shown as SEQ ID NO. 10;

(2) constructing a lentivirus vector which can be used for chimeric expression of a receptor binding structural domain of SARS-CoV2 and a T cell response peptide fragment of an N protein, and screening out a cell line of HEK293T which can secrete and express GP96-hFc and simultaneously chimeric expression of the receptor binding structural domain of SARS-CoV2 and the T cell response peptide fragment of the N protein on the basis of the step 1);

6. The method of claim 5, wherein the vaccine is administered in the form of a vaccine that prevents COVID-19, wherein the vaccine comprises: the vaccine for preventing COVID-19 in the step (3) is live cells, mitomycin B or radioactive ray treated cells and cell lysate.

7. The method of claim 5, wherein the vaccine is administered in the form of a vaccine that prevents COVID-19, wherein the vaccine comprises: the cell line of HEK293T is homologous or heterologous cell satisfying antigen expression.

8. Use of the vaccine of claim 1 in the manufacture of a medicament for the prevention of SARS-CoV2 virus.

9. Use of the vaccine according to claim 8 for the preparation of a medicament for the prevention of SARS-CoV2 virus, characterized in that: the vaccine is further processed into a DNA vaccine, an RNA vaccine, a protein vaccine or a recombinant virus vaccine.