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

Abstract

A 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 of the present invention comprises two functional parts: an inducible specific The S protein receptor binding domain of the neutralizing antibody and the T cell-related N protein truncated peptide that can induce the activation of effector T cells; the vaccine of the present invention has the following characteristics: the T cell-related N protein truncated peptide induces the N protein The ability to generate antibodies is weak, and N antibodies can be used to identify vaccine recipients and patients with new crown infection. The vaccine antigen does not induce the production of N protein antibodies, which can reduce lung damage and is safer; the cellular vaccine of the present invention is low in cost and can induce the production of viruses. Characterized neutralizing antibodies and T cell immune responses.

Description

A 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 in particular relates to a vaccine for preventing COVID-19 and a preparation method thereof.

Background technique

SARS-CoV2, also known as 2019-nCoV, is a novel RNA virus belonging to the Sarbecovirus subgenus of the family Coronaviridae, which has caused the global epidemic of the 2019 novel coronavirus pneumonia (COVID-19). As of August 10, the number of global virus infections has exceeded 19.86 million and the death toll has exceeded 730,000. Currently, 139 candidate vaccines are under preclinical evaluation, and 25 vaccine candidates are in clinical evaluation or clinical trials. Some of them, such as recombinant adenovirus vector vaccines and inactivated vaccines, have entered Phase II clinical trials and have shown encouraging results. However, there are still some deficiencies in the antigen design of various vaccines at present, which need to be further optimized. For example, some people have high neutralizing antibodies against adenovirus type 5 (Ad5), which inhibits the efficacy of Ad5-based new crown vaccines; fire-fighting vaccines can induce the body to produce a large number of non-neutralizing antibodies, which has potential safety risks, and the vaccine stimulates the body to produce Antibodies to the N protein can interfere with the serological diagnosis of infected patients.

Spike protein (S protein) is a key protein on the surface of SARS-CoV2, mainly composed of S1 and S2. Among them, the receptor binding domain (RBD) in S1 can bind to the host cell surface receptor-angiotensin-converting enzyme 2 (ACE2), triggering the conformational change of the S protein homotrimer and the fusion between the virus and the host cell membrane , allowing the virus to enter the host cell and replicate and multiply, so the S protein is the main target of current vaccine design and antiviral therapy. However, recent serological results have shown that sera from COVID-19 patients contain high titers of S1-specific antibodies, while antibodies against the S protein RBD are low. At the same time, some SARS-CoV-2 infected patients cannot produce long-term antibodies to the S protein, suggesting that vaccines targeting the S protein alone may have a risk of deactivation, and the vaccine design still needs to be improved. Antibody-dependent infection enhancement (ADE) means that after some virus-specific antibodies (usually non-neutralizing antibodies) bind to the virus, the antibody Fc fragment binds to specific cells expressing FcR (Fc receptor), thereby enhancing the ability of virus infection. phenomenon, which is closely related to the safety of antiviral vaccines. Studies have shown that the receptor binding domain (RBD) of SARS-CoV2 can elicit an efficient neutralization response without obvious ADE phenomenon, suggesting that the receptor binding domain is an ideal candidate region for the development of S protein-based vaccines.

Humoral immunity (especially 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 showed that a large number of specific T cells targeting SARS-CoV2 antigens were present in PBMCs of convalescent patients, suggesting that enhancing specific antiviral T cell responses would help prevent SARS-CoV2 infection. Nucleocapsid protein (N) is one of the most abundantly expressed proteins after SARS-CoV2 infection and is highly homologous to the SARS-CoV N protein. The study found that there are a large number of memory T cells targeting the virus N protein in SARS-CoV-infected patients. These T cells can exist in the body for a long time and can cross-react with the N protein of SARS-CoV2. SARS-related studies have also shown that inoculation of N protein vaccine can induce the body to produce specific antiviral antibodies and T cell immune responses, and inhibit viral infection, suggesting that N protein can induce a strong immune response in the body and can be used as a potential vaccine target. However, animal experiments have found that the use of the full-length N protein of SARS-CoV as a vaccine can induce the body to produce a large number of N protein antibodies, leading to aggravated lung damage in mice, suggesting that N protein-based vaccines need to overcome the problem of N antibody production.

SUMMARY OF THE INVENTION

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

The present invention mainly provides a chimeric vaccine against COVID-19 and a preparation method thereof, and provides a new and effective choice for the prevention of novel coronaviruses or highly homologous variants.

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

The vaccine antigen is mainly composed of three parts, the nucleotide sequence SEQ NO:3 encoding the receptor binding domain of SARS-CoV2, the nucleotide sequence SEQ NO:4 encoding the self-cleaving peptide T2A, and the N protein encoding The nucleotide sequence of the T cell response peptide is SEQ NO:5.

The polynucleotide encoding the receptor binding domain of SARS-CoV2 is codon-optimized, and the amino acid sequence is SEQ NO: 6; the amino acid sequence of the self-cleaving peptide T2A is SEQ NO: 7; the amino acid of the N protein T cell response peptide segment The sequence is SEQ NO:8.

The receptor binding domain encoding SARS-CoV2 is RBD, and the T cell response peptide encoding N protein is Ntap.

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

(1) First, construct a lentiviral vector that can secrete and express GP96-hFc, infect and screen out a cell line that can stably secrete and express HEK293T expressing GP96-hFc. The nucleotide sequence is shown in SEQ NO: 9, and the protein sequence is shown in SEQ NO: 10;

(2) Construct a lentiviral vector that can chimerically express the S protein receptor binding domain of SARS-CoV2 and the truncated N protein T cell-related peptide segment, and screen out on the basis of step 1) that can secrete and express GP96- hFc chimeric HEK293T cell line expressing both the binding domain of SARS-CoV2 and a truncated N protein T cell-associated peptide;

(3) Collect the cells obtained in step 2) under sterile conditions, wash with sterile saline or PBS, and adjust the cell concentration to 1×10 ⁶ ~1×10 ⁷ /100 μL to obtain a vaccine for preventing COVID-19 .

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

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

An immunization method for a vaccine for preventing COVID-19 as described above, subcutaneously or intramuscularly injecting a cell vaccine containing a vaccine antigen for preventing COVID-19, and administering one or more times, said vaccine for preventing COVID-19 Vaccines should be stored at -80°C or in liquid nitrogen for later use.

A vaccine as described above is preparing a medicine for preventing SARS-CoV2 virus.

An immunization method for SARS-CoV2 of the present invention comprises subcutaneously (or intramuscularly) injecting a cell vaccine containing the antigen of the present invention into an individual. Further, the administration can be administered once or multiple times. The specific implementation can be based on The specific actual situation changes or adjusts the number of immunizations or the immunization time point.

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

The nucleocapsid protein T cell response peptide contained in the present invention is highly conservative and highly homologous to the SARS-CoV virus, and at the same time, the peptide can induce the body to produce T cell immune response, and the antibody inducing ability is weak.

The nucleocapsid protein T cell response peptide segment antibody contained in the present invention has weak inducing ability, and can be used for differential diagnosis between vaccine recipients and SARS-CoV2 infection.

The invention also provides the use of an effective novel cell vaccine in preventing the novel coronavirus SARS-CoV2 virus. Fusion vaccine antigens are expressed in the cells, and secreted GP96-hFc (a fusion protein of GP96 and human IgG1 Fc fragment) is used as an immune enhancer.

The beneficial effects of the present invention are: the antigen of the vaccine of the present invention comprises two functional parts: the S protein receptor binding domain that can induce specific neutralizing antibodies and the T cell-related N protein truncated peptide that can induce the activation of effector T cells The vaccine of the present invention has the following characteristics: the T cell-related N protein truncated peptide segment has a weak ability to induce the production of N protein antibodies, the N antibody can be used to identify vaccine recipients and patients with new crown infection, and the vaccine antigen does not induce the production of N protein antibodies , can reduce lung damage and is safer; the cell vaccine of the invention is low in cost, and can induce virus-specific neutralizing antibodies and T cell immune responses.

Description of drawings

Figure 1 shows the bioinformatics analysis of the S protein of SARS-CoV2, in which (A) shows the main functional domains of the S protein; (B) the distribution of potential B cell epitopes predicted based on the 3D structure of the SARS-CoV2 RBD; ( C) Distribution of potential linear B-cell epitopes of SARS-CoV2 S full-length protein; (D) Left panel shows the location of potential B-cell antigens of SARS-CoV (blue) and SARS-CoV2 (green) in 3D structure (SARS-CoV-2) The CoV antigen is marked in purple, and the SARS-CoV2 antigen is marked in red), the right picture is the interaction model between the SARS-CoV2 receptor binding domain and the ACE2 receptor, and the yellow part is the key site where the RBD region binds to the receptor ACE2 .

Figure 2 shows the comparison between SARS-CoV2 and SARS-CoV, (A) homology analysis of S1 and S2 functional domains of SARS-CoV2 and SARS-nCoV spike proteins; (B) SARS-CoV2 and SARS-nCoV spike Distribution of different sites in the protein. (C) Distribution of potential B-cell epitopes predicted based on the 3D structure of SARS-CoV RBD; (D) distribution of potential linear B-cell epitopes of SARS-CoV S full-length protein.

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

Figure 4 Homology analysis of SARS-CoV2 and SARS-nCoV nucleocapsid proteins.

Figure 5 Epitope analysis of SARS-CoV2 nucleocapsid protein (N), in which (A) potential B cell epitopes of N protein; (B) distribution of potential MHCI-binding peptides of N protein; (C) function of SARS-CoV N protein Domain and its antibody epitope map.

Figure 6 Antigen expression and antibody recognition characteristics of chimeric vaccines, in which (A) is the backbone pattern of chimeric vaccines against SARS-CoV2; (B) uses SARS-CoV2 to restore patient antiserum and anti-SARS-CoV2 RBDs are commercially available Clonal antibodies and commercial antibodies against the SARS-CoV2 nucleocapsid were analyzed for the recognition characteristics of different antibodies to SARS-CoV2-derived proteins and chimeric vaccine antigens.

Figure 7 shows the identification characteristics of SARS-COV2-derived proteins and chimeric vaccine antigens in convalescent sera from different COVID-19 patients (5 cases).

Figure 8 Validation of the binding of three peptides in the Ntap region to human HLA-A*0201 molecules using T2 cells.

Figure 9 Validation of cell-based novel fusion vaccine antigen expression, in which (A) the expression of gp96-Fc protein in chimeric cell vaccine (C-Vac) was detected by immunocytochemistry; (B) different antibodies were detected by Western-Blot Identification of chimeric cellular vaccine (C-Vac) antigens.

Figure 10 Evaluation of chimeric vaccine (C-Vac) efficacy and safety using golden hamsters. Syrian hamsters were immunized with different treatments of HEK293T cell vaccine (untreated live cells, mitomycin C-treated cells, and frozen-thawed cell lysate), assessed by pseudovirus infection test on day 7 post-immunization (A) and day 21 (second immunization was performed on day 14) (B) neutralizing antibody expression; (C) hematoxylin-eosin (HE) detection of hamster lung changes on day 7 of primary immunization; (D) use of fusion antigen expressing Virus-specific T cell responses were assessed by tumor size in hamster-derived kidney cells BHK21.

Detailed ways

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

The vaccine antigen is mainly composed of three parts, the nucleotide sequence SEQ NO:3 encoding the receptor binding domain of SARS-CoV2, the nucleotide sequence SEQ NO:4 encoding the self-cleaving peptide T2A, and the N protein encoding The nucleotide sequence of the T cell response peptide is SEQ NO:5.

The polynucleotide encoding the receptor binding domain of SARS-CoV2 is codon-optimized, and the amino acid sequence is SEQ NO: 6; the amino acid sequence of the self-cleaving peptide T2A is SEQ NO: 7; the amino acid of the N protein T cell response peptide segment The sequence is SEQ NO:8.

The receptor binding domain encoding SARS-CoV2 is RBD, and the T cell response peptide encoding N protein is Ntap.

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

(1) First, construct a lentiviral vector that can secrete and express GP96-hFc, infect and screen a cell line that can stably secrete and express HEK293T expressing GP96-hFc. The nucleotide sequence of GP96-hFc is shown in SEQ NO: 9 , the protein sequence is shown in SEQ NO: 10;

(2) Construct a lentiviral vector capable of chimerically expressing the receptor binding domain of SARS-CoV2 and the truncated N protein T cell-related peptide, and screened on the basis of step 1) that can secrete and express GP96-hFc at the same time. A cell line HEK293T chimerically expressing the receptor binding domain of SARS-CoV2 and a truncated N protein T cell-associated peptide;

(3) Collect the cells obtained in step 2) under sterile conditions, wash with sterile saline or PBS, and adjust the cell concentration to 1×10 ⁶ ~1×10 ⁷ /100 μL to obtain a vaccine for preventing COVID-19 .

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

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

An immunization method for a vaccine for preventing COVID-19 as described above, subcutaneously or intramuscularly injecting a cell vaccine containing a vaccine antigen for preventing COVID-19, and administering one or more times, said vaccine for preventing COVID-19 Vaccines should be stored at -80°C or in liquid nitrogen for later use.

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

By referring to SARS-CoV-related research and UniProt public data, the S protein of SARS-CoV2 can be divided into multiple functional domains; it can be seen that the S protein is mainly composed of S1 and S2 subunits (Figure 1A), in which the receptor in S1 binds The binding of the RBD domain (RBD) to angiotensin-converting enzyme 2 (ACE2) triggers a conformational change in the homotrimer, while the S2 subunit can further form a six-helix bundle to facilitate the fusion of the virus with the host cell membrane. Potential 3D structure-based B-cell antigens of the SARS-CoV2 S protein receptor-binding domain were analyzed using Discospe software (Fig. 1B), and the IEDB database was used to analyze potential linear B-cell epitopes of the SARS-CoV2 whole S protein (Fig. 1C), It can be seen that structure-based B antigens are less, while linear B cell antigens are more abundant. The structures of the RBD domains from SARS-CoV2 (PDB: 6LZG) and SARS-CoV (PDB: 2GHW) were compared using 3D-Match (http:www.softberry.commberry.phtml) and revealed using Discovery Studio Potential 3D B-cell antigens located in the receptor-binding domain (SARSCoV2: red, SARS-CoV: purple) were mainly located in the region where the receptor-binding domain interacts with the ACE2 receptor (Fig. 1D).

Differential amino acids were further labeled (red) by analysis using MegAlign software showing that the S1 subunit is less homologous than the S2 subunit (67.1% vs. 90%) between SARS-CoV and SARS-CoV-2 (Fig. 2A) (Fig. 2B) It can be seen that the residues of SARS-CoV and SARS-CoV-2 are very different, suggesting that the vaccine based solely on the S protein will weaken the protective effect of the vaccine due to the mutation of the RBD region. Figure 2C shows the potential 3D structure-based B cell antigens of the receptor binding domain of the SARS-CoV S protein analyzed by Discospe software, and Figure 2D shows the potential linear B cell epitopes of the SARS-CoV whole S protein analyzed using the IEDB database. SARS can be found The RBD of -CoV2 is less immunogenic than that of SARS-CoV, which may be the reason why SARS-CoV2 is so widespread.

Example 2: Validation 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 sequence published in the NCBI database ((MN908947.3), and constructed to pcDNA3.1 -his tag (hygro) vector. The codon-optimized SARS-CoV2 plasmid was purchased from Sino Biological. The following primers were used to construct PCDNA3.1-his-optS, PCDNA3.1-his-optS1, PCDNA3.1-his- optNRBD (from N terminus to RBD domain) expression.

Spike-optF (codon optimized): ACTTAATTAAGCCACCATGTTTGTGTTCCTGGTGCTGCT,

Spike-opt-R:TAACCGGTGGTGTAGTGCAGTTTCACTCCTTTTCA;

Spike-optS1-R: TAACCGGTGCTGTTGGTCTGGGTCTGGTAGG;

Spike-optNRBD-R:TAACCGGTTCCATTGAAGTTGAAGTTCACACACTT;

The corresponding expression plasmids were transfected into HEK-293T cells using PEI, and the expression of corresponding genes was detected by Western Blot method. Western Blot steps are as follows:

Cells were washed twice with phosphate-buffered saline on ice and lysed with RIPA lysis buffer (50 mM Tris HCl, pH 7.4, 0.1% SDS, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA) containing 1% protease inhibitors. ) Cells were lysed for 10 min on ice, cell lysates were clarified by centrifugation at 13000 rpm for 30 min at 4°C, and 25 ug total protein was separated by 10% SDS-PAGE and transferred to PVDF membranes (Millipore). Membranes were blocked with 5% nonfat milk and incubated with primary antibody according to the antibody manufacturer's instructions, washed 3 times with TBST, and then incubated with horseradish peroxidase-conjugated secondary antibody (1:5000, ZSBIO) at room temperature for 1 After 1 h, the membranes were washed 3 times with TBST and detected by an enhanced chemiluminescence (ECL) system (Thermopierce, USA). Antibodies used included mouse anti-histidine-tagged mAb (Abmart, M20001S), HRP goat anti-mouse IgG (ZSBIO, ZB-5305).

Figure 3A shows the expression of SARS-CoV2 wild sequence plasmid (Vector: pcDNA3.1-his vector, Spike: S protein, N: N protein), and Figure 3B shows the expression of different Spike and its derived sequence plasmids (Spike-WT: wild Type S protein, Spike-OPT: codon-optimized S expression plasmid, S1-OPT: codon-optimized S1 expression plasmid, NRBD-OPT: codon-optimized expression plasmid from spike start N-terminal to RBD region) . The results showed that the expression of the S protein could hardly be detected in human 293T cells using the original sequence of the S gene, while the expression of the S protein was significantly increased after codon optimization, indicating that the codon-optimized S gene is useful for the design of SARS-COV-2. Vaccines are very important.

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

Homology analysis of the nucleocapsid protein (N protein) between SARS-CoV2 and SARS-nCoV using MegAlign. It was shown that the amino acid homology of N protein between SARS-CoV-2 and SARS-CoV was 91.2% (Fig. 4), which supports that the convalescent sera of SARS-CoV and the sera of patients newly infected with SARS-CoV-2 have the same The results of high cross-reactivity suggest that N protein-based vaccines may have cross-protective effects.

The potential B cell epitopes of the N protein (Fig. 5A) and the potential MHCI-binding peptides of the N protein (Fig. 5B) were predicted by the IEDB database, and the results showed that there were two regions in the N protein with fewer B cell epitopes, but with Abundant histocompatibility complex 1 (MHC I) binds T cell epitopes. Figure 5C is a map of the functional domain of SARS-CoV N protein and its antibody epitope. It can be seen that the antibodies against amino acids 212-341 of the N protein polypeptide (the second region) decreased in SARS patients. This result is consistent with the analysis results in Figure 4. Consistent.

Example 4: Construction of Chimeric Vaccine Antigen and Validation of Antigen Expression

Based on the above analysis data, the backbone of SARS-CoV2 chimeric vaccine was designed (Fig. 6A), RBD: receptor binding domain of S protein (316-541aa), Ntap: T cell-associated peptide of N protein (211-339aa). The vaccine consists of the receptor-binding domain of the S protein that induces specific neutralizing antibodies and the N protein T-cell response peptide (Ntap).

A codon-optimized RBD domain (containing a signal peptide), a T2A cleavage peptide, and a truncated N protein T cell-associated peptide (synthesized by Sangon Biotech) were synthesized and inserted into the modified pLenti6-puromycin vector to construct recombinant Lentiviral vector. Antibody recognition characterization of SARS-CoV2-derived proteins and C-Vac antigens was validated using SARS-CoV2 recovered patient antisera and commercial antibodies against SARS-CoV2 RBD or nucleocapsid (Figure 6B), as described in Example 3. The results showed that the chimeric vaccine antigen could be expressed well, and the SARS-CoV2 RBD protein and the full-length N protein could induce the body to produce antibodies, while the commercial polyclonal antibodies did not recognize Ntap well, which confirmed that the Ntap antibody induction ability was weak.

In addition, Western Blot testing using convalescent sera from five other COVID-19 patients found that convalescent sera from COVID-19 patients had highly reactive antibodies against the intact N protein, but no antibodies against S protein and T cells were found. Related Ntap was significantly identified (Figure 7), and all patient sera were provided by the First Affiliated Hospital of Zhengzhou University and approved by the Ethics Committee of Zhengzhou University.

The antibodies used are listed below: Rabbit anti-RBD PAb (polyclonal antibody) (Sino Biological, #40592-T62), Rabbit anti-Nucleocapsid 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 the binding ability of Ntap-derived polypeptides to 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 major histocompatibility (MHC) class II molecules as tool cells. Three potential binding peptides (LALLLLDRLNQL, RLNQLESKM, GMSRIGMEV) of HLA-A*0201 derived from Ntap, one positive binding peptide from HER2 (KIFGSLAFL) and one negative binding peptide from MUC1 (SAPDTRPAP) were synthesized respectively (all peptides) All synthesized by Gill Biochemical Co., Ltd.), and verified by the following method.

The method was as follows: 100 μL of 1×10 ⁵ T2 cells (resuspended in IMEM medium without FBS) were seeded into a round-bottom 96-well plate, followed by the addition of a doubling-diluted peptide with a maximum concentration of 100 μM and co-incubated for 4 hours; Cells were washed twice with pre-cooled PBS and stained with mouse anti-human HLA-A2-FITC antibody (Abcam) for 30 minutes on ice; after two washes with pre-cooled PBS, cells were analyzed for fluorescence using BD FACSAria (BD Biosciences Immunocytometry Systems). strength.

The results showed that the two epitope peptides derived from Ntap (LLLDRLNQL and GMSRIGMEV) and the positive peptide derived from HER2 (KIFGSLAFL) were both able to bind to T2 cells, while the RLNQESKM peptide and the negative peptide derived from MUC1 (SAPDTRPAP) did not bind to T2 cells. combined (Figure 8). The sequence alignment results also showed that, compared with SARS-CoV (RLNQESKV) (which has been confirmed to bind to HLA-A*0201), the peptide RLNQESKM derived from SARS-CoV2 has a site mutation, and the The variation may affect the binding of the peptide to the HLA-A*0201 molecule.

Example 6: Construction of a novel cellular vaccine for COVID-19

(1) Synthetically constructed the expression plasmid 3.1-GP96-hFc (SEQ NO: 9) capable of secreting and expressing GP96-hFc, transfected the plasmid with Higene (Applygen, China), and selected with 200 μg/ml hygromycin B (Invitrogen), The 293T-GP96-hFc overexpression cell line was constructed, and then the expression of the fusion protein was detected by HRP goat anti-human IgG (ZSBIO, ZB-2304) and DAB chromogenic kit (Fuzhou Maixin) and immunocytochemical methods (Fig. 9A). .

(2) Synthesize and construct a lentiviral plasmid expressing the vaccine antigen (SEQ NO: 1), and coat the lentivirus expressing the vaccine antigen according to the following steps: inoculate 2 × 10 ⁶ HEK293T cells into a 10 cm cell culture dish, and Co-transfect the plasmid mixture (including 25 μg target plasmid, 8 μg psPAX2, 4 μg pMD2.G, and 40 μL 2 mg/ml PEI); replace the medium with fresh medium after 6 hours, collect the lentivirus-containing supernatant after 48 hours, 0.22 μm filter (Millipore) aliquots were stored at -80°C after filtration. The 293T-GP96-hFc cells constructed in step 1) were inoculated into 24-well plates, and then 1 ml of lentiviral supernatant containing vaccine antigens and 5 μg/ml polybrene (Sigma) were added. 5 μg/ml puromycin (Selleck) was added 72 h after infection to screen for stable cell lines. The expression of vaccine antigens was detected by Western Blot method (Fig. 9B), which showed that the chimeric HEK293T cell vaccine (C-Vac) could stably express vaccine fusion antigens.

Example 7: Evaluation of the efficacy of a novel cellular vaccine for COVID-19

Several articles have reported that the Syrian hamster (also known as the golden hamster) can support the infection and replication of SARS-CoV2 and is an ideal animal model. Thirty-six 12-week-old female Syrian hamsters were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd. and randomly divided into 4 groups (n = 9). Groups 1-4 were inoculated with 1×10 ⁷ 293T-GP96-hFc control cells, live 293T-C-Vac cells, 293T-C-Vac cells treated with 5 μg/ml mitomycin (MCE) for 4 h, and 293T-C-Vac vaccine cell freeze-thaw lysate. On the 7th day after immunization, three hamsters in each group were sacrificed, and animal serum samples were collected and stored at -80°C, while lung tissues were fixed in 10% neutral buffered formalin and made into paraffin specimens. The remaining animals were boosted on day 14 with the same protocol as the prime, and serum was harvested on day 21 (day 7 post-boost). On the 45th day, the remaining hamsters were subjected to a tumor-bearing experiment with 5×10 ⁶ hamster cells BHK21 cells expressing RBD-Ntap (C-Vac antigen) (the construction method was the same as that in Example 5), and the volume of the allograft was measured and evaluated. Vaccine-specific cytotoxic T cell effects. All animals used in this study were housed under SPF-grade conditions in the Experimental Animal Center of Zhengzhou University and handled in accordance with the regulations of animal experimentation ethics.

(1) Coating of SARS-CoV2 pseudovirus and determination of neutralizing antibody in hamster serum

2 x 10 ⁶ HEK293T cells were seeded into 10 cm cell culture dishes and treated with 20 μg lenti-Luc (lentiviral plasmid expressing luciferase), 10 μg psPAX and 10 μg 3.1-optS-d18 (expressing deletion of S protein end 18 amino acid residues) were co-transfected, and the SARS-CoV2 pseudovirus was collected with reference to the lentiviral method. The hamster sera of the vaccine-immunized group and the control group were heat-inactivated at 56°C for 30 minutes, and were doubling dilution starting from 1:25. A fixed amount of pseudovirus (50 μL) was then incubated with 50 μL of diluted serum for 1 h in a 37°C cell incubator and infected 1×10 ⁴ hamster BHK21 expressing human hACE2. The relative luciferase activity of cells was measured 72 hours after infection using the Luciferase Assay System (Promega) and the GloMax Discover Assay (Promega). Serum neutralization titers (ID50) were calculated using the 50% RLU signal compared to control cells. As shown in Figure 10, all vaccinated groups expressing fusion antigens were effective on day 7 of a single immunization (Figure 10A) and on day 7 after a second booster immunization (day 21 after the first immunization) (Figure 10B) Induce specific neutralizing antibodies.

(2) Hematoxylin and eosin staining to evaluate the effect of vaccine on the lungs of golden hamsters

The paraffin specimens of hamster lung tissue were cut into 6 µm sections, followed by standard hematoxylin-eosin staining procedures, and photographs were taken to analyze the changes of lung tissue after sealing with neutral resin. The results showed that no significant lung changes were observed in all animals, suggesting that the novel COVID-19 cellular vaccine has high safety (Fig. 10C).

(3) Analysis of virus-specific T cell responses after vaccination by homologous tumor-bearing experiments

On the 45th day after the primary immunization, the remaining hamsters were subcutaneously injected with 5×10 ⁶ hamster BHK-21 expressing the fusion vaccine antigen (the construction method was the same as that of Example 6), and the tumor volume was measured on the 70th day. As shown in Fig. 10D, after immunization with live cell vaccine and split cell vaccine, Syrian hamsters significantly reduced the tumor volume of allogeneic cells, indicating that the chimeric vaccine of the present invention can stimulate the body to produce virus antigen-specific T cell immune responses.

Many vaccines against SARS-CoV2 are currently being developed in preclinical research and clinical trials, but most target only the spike protein. In the present invention, we developed a novel chimeric vaccine (C-Vac) with the receptor binding domain (RBD) of the S protein and a truncated peptide of the nucleocapsid protein (N) that can induce T cell activation segment is the target. The vaccine can effectively induce specific neutralizing antibodies against viral proteins and specific T cell effects, and has a lower risk of induced antibody-dependent infection enhancement and N protein antibody-mediated immunotoxicity, and has high safety. The C-Vac of the present invention contains highly conserved Ntap, and is expected to provide the body with long-term immune protection against novel coronaviruses, and will provide certain protection for SARS-CoV2 and its mutants. At the same time, the Ntap peptide segment contained in the vaccine of the present invention has a weak ability to induce the production of N protein antibodies, and the N protein antibodies can be used to identify vaccine recipients and patients with new crown infection. The cell vaccine of the invention has low cost and can effectively induce virus-specific neutralizing antibodies and T cell immune responses.

SEQUENCE LISTING

<110> Zhengzhou University

<120> A novel vaccine for preventing COVID-19 and its preparation method

<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> T2A synthetic 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-responsive peptide synthesis 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-reactive 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

atgaggggccc 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 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 (9)

1. A vaccine for preventing COVID-19, characterized in that: 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. A vaccine for preventing COVID-19 according to claim 1, characterized in that: the vaccine antigen consists of three parts, the nucleotide sequence SEQ ID NO of the receptor binding domain encoding SARS-CoV2 : 3, the nucleotide sequence SEQ ID NO: 4 encoding the self-cleaving peptide T2A, and the nucleotide sequence SEQ ID NO: 5 encoding the N protein T cell response peptide segment. 3. The vaccine for preventing COVID-19 according to claim 2, wherein the polynucleotide encoding the receptor binding domain of SARS-CoV2 is codon-optimized, and the amino acid sequence is SEQ ID NO. : 6; the amino acid sequence of the self-cleaving peptide T2A is SEQ ID NO: 7; the amino acid sequence of the N protein T cell response peptide segment is SEQ ID NO: 8. 4. A vaccine for preventing COVID-19 according to claim 2 or 3, characterized in that: the described encoding SARS-CoV2 receptor binding domain is RBD, and the described encoding N protein T cell response peptide The segment is Ntap. 5. a preparation method of the vaccine of preventing COVID-19 as claimed in claim 1 or 2, is characterized in that: comprise the following steps: (1) First, construct a lentiviral vector that can secrete and express GP96-hFc, infect and screen a cell line that can stably secrete and express HEK293T expressing GP96-hFc. The nucleotide sequence of said GP96-hFc is shown in SEQ ID NO: 9. Shown, the protein sequence is shown in SEQID NO:10; (2) Construct a lentiviral vector capable of chimerically expressing the receptor binding domain of SARS-CoV2 and the N protein T cell response peptide, and screened on the basis of step 1) to secretely express GP96-hFc and simultaneously express chimerically HEK293T cell line with receptor binding domain of SARS-CoV2 and N protein T cell response peptide; (3) Collect the cells obtained in step 2) under sterile conditions, wash with sterile saline or PBS, and adjust the cell concentration to 1×10 ⁶ ~1×10 ⁷ /100 μL to obtain a vaccine for preventing COVID-19 . 6. The preparation method of a vaccine for preventing COVID-19 according to claim 5, wherein the vaccine for preventing COVID-19 in the step (3) is live cells, mitomycin B or Radiation treatment of cells and cell lysates. 7 . The method for preparing a vaccine for preventing COVID-19 according to claim 5 , wherein the cell line of HEK293T is homologous or heterologous cells that satisfy antigen expression. 8 . 8. A use of the vaccine as claimed in claim 1 in the preparation of a medicine for preventing SARS-CoV2 virus. 9. The use of the vaccine according to claim 8 in the preparation of a medicine for preventing SARS-CoV2 virus, wherein the vaccine is further processed into a DNA vaccine, an RNA vaccine, a protein vaccine or a recombinant virus vaccine.

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