A novel coronavirus recombinant subunit antigen and its preparation method and application

Abstract

The present invention relates to the field of biopharmaceutical technology, and in particular to a novel coronavirus recombinant subunit antigen and a preparation method and application thereof. The antigen is a novel coronavirus recombinant protein, named HR1 910-1011-L6-HR2P. The novel coronavirus recombinant protein provided by the present invention is highly conservative for coronaviruses and novel coronavirus mutants, and contains abundant effective T cell epitopes, which can stimulate the body to produce antigen-specific antibodies and cellular immune responses. The recombinant protein of the present invention can use a prokaryotic expression system as an expression vector, has the advantages of high yield, low production cost, and a state similar to a physiological state, and is suitable for large-scale preparation of novel coronavirus vaccines.

Description

Technical Field

[0001] The present invention relates to the field of biopharmaceutical technology, and in particular to a novel coronavirus recombinant subunit antigen and a preparation method and application thereof. Technical Background

[0002] The novel coronavirus (SARS-CoV-2, hereinafter referred to as SARS2) recognizes epithelial cells expressing ACE2 receptors and enters host cells in the following process: the polymotif on the S1-S2 boundary is cleaved when the virus matures in the infected cell, but the S2′ site is cleaved after angiotensin-converting enzyme 2 (ACE2) binds to the target cell. Virus binding to ACE2 causes conformational changes in the S1 subunit and exposes the S2′ cleavage site in the S2 subunit. Cleavage of the S2′ site exposes the fusion peptide (FP), and the dissociation of S1 and S2 causes a dramatic conformational change in the S2 subunit, especially HR1, which pushes the fusion peptide forward into the target membrane and initiates membrane fusion. The fusion between the viral membrane and the cell membrane forms a fusion pore, through which the viral RNA is released into the cytoplasm of the host cell for uncoating and replication.

[0003] SARS-CoV-2 can induce humoral and cellular immune responses in the host after infecting human respiratory epithelial cells. Infected epithelial cells degrade viral particles and present them to cytotoxic CD8 + T cells. CD8 + T cells recognize viral proteins through TCR-MHC I interactions and release cytotoxic granules such as granzyme B and perforin to act on infected cells. In addition, antigen-presenting cells present viral peptides to CD4 through TCR-MHC II interactions. + T cells (Th0). Once the antigen peptide is recognized, Th0 cells mainly polarize toward Th1, release IFN-γ to eliminate the virus, and release Th2 to trigger humoral-mediated immune response and antibody secretion against SARS-CoV-2 virus.

[0004] At present, vaccination is the most effective public intervention measure to prevent novel coronavirus infection. At present, the global vaccine research and development routes mainly include the following: virus vaccines represented by inactivated vaccines and live attenuated vaccines; recombinant subunit vaccines with S protein, RBD, virus-like particles or peptides as antigens; DNA or RNA nucleic acid vaccines and vector vaccines including non-replicating and replicating types. However, with the spread of the new coronavirus, more variants have been discovered. The new coronavirus variants that are currently of concern include the B.1.1.7 (Alpha) variant first discovered in the UK, the B.1.351 (Beta) variant discovered in South Africa, the P.1 (Gamma) variant discovered in Brazil, the B.1.617.2 (Delta) variant discovered in India, and the B.1.1.529 (Omicron) mutant discovered in many countries (https: / / www.who.int / en / activities / tracking-SARS-CoV-2-variants / ). Early vaccine development mainly targeted the spike region of SARS-CoV-2, because the receptor binding domain in this region binds to the ACE2 receptor on the surface of the host cell and mediates the virus's invasion of the host. However, as mutants mutate at key sites of the S protein, immune escape and other phenomena occur, and some mutants have stronger transmission capabilities and even cause breakthrough infections in people who have been vaccinated with the new coronavirus vaccine, which brings new challenges to the development of existing vaccines and the prevention and treatment of the new coronavirus.

[0005] In order to cope with the continuous mutation of the SARS-CoV-2 virus, the development of a safer and more effective SARS-CoV-2 vaccine is currently a more urgent issue. Summary of the invention

[0006] In order to solve the problems in the prior art, one of the objects of the present invention is to provide a recombinant subunit antigen of a new coronavirus.

[0007] The present invention adopts the following technical solutions:

[0008] A recombinant subunit antigen of a novel coronavirus, wherein the antigen is a recombinant protein of a novel coronavirus, named HR1 910-1011-L6-HR2P, whose amino acid sequence is shown in SEQ ID NO:1, and whose nucleotide sequence is shown in SEQ ID NO:2.

[0009] A second object of the present invention is to provide an expression vector comprising the nucleotide sequence described above.

[0010] A third object of the present invention is to provide a host cell comprising the expression vector as described above.

[0011] The fourth object of the present invention is to provide the use of the above-mentioned new coronavirus recombinant subunit antigen in the preparation of a vaccine for preventing the original strain and / or variant strain of the new coronavirus.

[0012] The fifth object of the present invention is to provide the use of the above-mentioned new coronavirus recombinant subunit antigen in the preparation of a kit for diagnosing new coronavirus infection.

[0013] The sixth object of the present invention is to provide a vaccine comprising a recombinant subunit antigen of the new coronavirus as described above.

[0014] The seventh object of the present invention is to provide a kit for diagnosing novel coronavirus infection, which comprises a novel coronavirus recombinant subunit antigen as described above.

[0015] The present invention finally provides a method for preparing a recombinant subunit antigen of a novel coronavirus as described above, comprising the following steps:

[0016] Step 1. The nucleotide sequence of HR1 910-1011-L6-HR2P is used as the target gene and assembled with the pET30a plasmid, and then transferred into Rosetta competent cells to obtain expression bacteria containing the target gene; the nucleotide sequence of HR1 910-1011-L6-HR2P is shown in SEQ ID NO: 2;

[0017] Step 2. The expression bacteria containing the target gene are broken after IPTG induction, and the desired new coronavirus recombinant protein is obtained by chromatography purification.

[0018] Preferably, in step 1, the target gene is inserted between the restriction sites Sal I and Not I of the pET30a plasmid by mixing the target gene PCR recovery product and the pET30a plasmid PCR recovery product at a molar ratio of 4:1, adding Gibson assembly enzyme, and incubating in a 50°C water bath for 15 minutes.

[0019] Preferably, in step 2, the IPTG induction process is as follows: after the expression bacteria containing the target gene are cultured in LB containing Kana for 4-5 hours, IPTG is added in an amount of 500 μL bacterial solution: 1 mM IPTG, and the culture is continued for 3-4 hours, and then the bacterial solution is aspirated and centrifuged to obtain the induced bacteria.

[0020] The beneficial effects of the present invention are:

[0021] The new coronavirus recombinant protein provided by the present invention is highly conservative against coronaviruses and new coronavirus mutant strains, contains rich effective T cell epitopes, has good immunogenicity, and can stimulate the body to produce antigen-specific antibodies and cellular immune responses.

[0022] The recombinant protein of the present invention can utilize a prokaryotic expression system as an expression vector, and has the advantages of high yield, low production cost, high protein purity, and a state similar to the physiological state, and is suitable for large-scale preparation of new crown vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The antigen epitope map provided by the present invention, in which A is a schematic diagram of antigen protein expression; B is the T cell epitope map of SARS-CoV-2 Spike.

[0024] Figure 2 Figure 1 is the purification diagram and SDS-PAGE results of the prepared recombinant protein, wherein Figure A is the Ni affinity column chromatography purification diagram of the supernatant after ultrasonic disruption of the bacteria; Figure B is the hydrophobic interaction chromatography (HIC column) purification diagram of the target protein collected after TEV enzyme cleavage; Figure C is the ion exchange chromatography (Q column) purification diagram after hydrophobic column purification; Figure D is the gel filtration chromatography (24mL molecular sieve) purification diagram of the target protein; and Figure E is the SDS-PAGE identification diagram of the final target protein.

[0025] Figure 3 is the level of antigen-specific antibodies in the serum of immunized mice. Figure A is the ELISA curve of the immunized target protein antigen and the serum after PBS. Each point is the OD at different dilution gradients. 450 nm A is the reading at the immunization target protein antigen and the titer bar graph of the serum calculated after PBS.

[0026] Figure 4 CD4 + and CD8 + T cell activation level, Figure A is the CD4 + CD69 + The percentage of T cells; B is the percentage of CD4 + CD69 + The average fluorescence intensity of T cells; C is the CD8 + CD69 + The percentage of T cells in spleen tissue is D: CD8 + CD69 + The average fluorescence intensity of T cells; E is the CD4 + PD-1 + The percentage of T cells; F is the percentage of CD4+ PD-1 + The average fluorescence intensity of T cells; G is the CD8 + PD-1 + The percentage of T cells; H is the percentage of CD8 + PD-1 + Average fluorescence intensity of T cells. HR2P marked in the figure is the abbreviation of HR1910-1011-L6-HR2P.

[0027] Figure 5 CD4 + and CD8 + T cell activation level, Figure A is CD4 + CD69 + The percentage of T cells; B is the percentage of CD4 + CD69 + The average fluorescence intensity of T cells; C is the average fluorescence intensity of CD8 + CD69 + The percentage of T cells; D is the percentage of CD8 + CD69 + The average fluorescence intensity of T cells; E is the CD4 + PD-1 + The percentage of T cells; F is the percentage of CD4 + PD-1 + The average fluorescence intensity of T cells; G is the average fluorescence intensity of CD8 + PD-1 + The percentage of T cells; H is the percentage of CD8 + PD-1 + Mean fluorescence intensity of T cells. HR2P marked in the figure is the abbreviation of HR1 910-1011-L6-HR2P.

[0028] Figure 6 Figure 2 shows the levels of B cell maturation-related cells in the lymph nodes of immunized mice. Figure A shows the percentage of follicular helper T cells (Tfh) in lymph node tissue; Figure B shows the percentage of germinal center B cells (GC B) in lymph node tissue. The HR2P marked in the figures are the abbreviations of HR1 910-1011-L6-HR2P.

[0029] Figure 7 Figure 2 is the level of B cell maturation-related cells in the spleen of immunized mice, where Figure A is the percentage of follicular helper T cells (Tfh) in spleen tissue; Figure B is the percentage of germinal center B cells (GC B) in spleen tissue. The HR2P marked in the figure is the abbreviation of HR1 910-1011-L6-HR2P.

[0030] Figure 8 CD8 + The level of intracellular IFN-γ production in T cells after 24 hours, where A is CD4 + IFN-γ + The percentage of T cells; Figure B shows CD4 + IFN-γ + Mean fluorescence intensity (MFI) of T cells; Figure C shows CD8 + IFN-γ + The percentage of T cells; D shows CD8 + IFN-γ + Mean fluorescence intensity (MFI) of T cells. DETAILED DESCRIPTION

[0031] For ease of understanding, the technical solution of the present invention is described in more detail below in conjunction with embodiments.

[0032] Example 1

[0033] The novel coronavirus recombinant protein provided by the present invention is selected from the reported SARS-CoV-2Spik genome sequence (Genbank: MT019533.1) HR1 and its derivative domain (910-1011aa) and HR2 domain (1167-1203aa), and the two parts are connected using a 6-amino acid linker (SGGRGG). After that, we codon-optimized the spliced ​​gene and sent it to General Bio (Anhui) Co., Ltd. for synthesis, labeled as HR1 910-1011-L6-HR2P, Figure 1 A in the figure is a schematic diagram of antigen protein expression.

[0034] The gene sequence of HR1 910-1011-L6-HR2P protein is shown in SEQ ID NO.1, and the amino acid sequence of the protein is shown in SEQ ID NO.2.

[0035] The T cell epitopes of SARS-CoV-2 Spike detected by T cell experiments that have been reported were downloaded from the IEDB database (https: / / www.iedb.org / ), and these T cell epitopes were analyzed and summarized using ImmunomeBrowser (http: / / tools.iedb.org / immunomebrowser / ), and the following were obtained: Figure 1 B shows the Spike T cell epitope map. It can be seen from the figure that the amino acid position of the target protein has a relatively rich T cell epitope.

[0036] By comparing the S protein of the original SARS-CoV-2 strain with the variants prevalent at various stages and six other coronavirus strains that infect humans, it can be seen that the selected S910-1011 and S1167-1203 amino acid regions are highly conserved.

[0037] Example 2

[0038] Preparation method of novel coronavirus recombinant protein

[0039] Step 1. Insert the target gene of the DNA sequence of HR1 910-1011-L6-HR2P protein between Sal I and Not I in the ET30a plasmid. The specific steps are: add 0.1pmoL of the target gene PCR recovery product and 0.025pmoL of the vector PCR recovery product, add 1μL of 5× Gibson assembly enzyme, and then add pure water to 5μL. After mixing, incubate in a 50℃ water bath for 15min and then add it to DH5α competent cells. The transformation method is as follows: slowly thaw 50μL of competent bacterial solution on ice, add the above Gibson assembly product, flick the tube wall, and then stand on ice for 15min, then heat shock in a 42℃ water bath for 90s, and then place on ice for 3min. Add 500 μL of non-resistant LB medium to the clean bench, shake the bacteria in a shaker at 37°C and 220 rpm / min for 45 minutes, then take 100 μL in the clean bench and evenly spread it on the Kana-containing resistant plate with coating beads, and culture it upside down in a 37°C incubator overnight.

[0040] Step 2. Pick a single clone into LB containing Kana, shake the cells at 37℃220rpm / min for 4-5h, then take 500μL of the cell solution into a 1.5mL EP tube as a non-induced control, add 1mM IPTG to the test tube and continue shaking for 3-4h. Then take 200μL of the cell solution and centrifuge it, remove the culture medium, add 50μL of pure water and an appropriate amount of SDS-loading buffer, incubate at 100℃ for 10min, centrifuge at 12000rpm for 5min, take 5μL of the sample and run SDS-PAGE.

[0041] Step 3. Purification of the recombinant protein of the new coronavirus The specific process is as follows: add an appropriate amount of Nibinding buffer (20mM Tris 8.5, 250mM NaCl, 14mM imidazole) to the collected bacteria and then perform ultrasonic disruption, and then centrifuge at high speed to obtain the supernatant for nickel column affinity chromatography purification, and use Ni elution buffer (20mM Tris 8.5, 250mM NaCl, 400mM imidazole) for gradient elution. Take the protein solution at the peak position, add 5mM DTT, 1mM EDTA, and 1 / 10 molar ratio of TEV protease, mix and let it stand at 4 degrees for 4h. Dialyze overnight into 5L dialysate (20mM Tris 8.5, 250mM NaCl) without imidazole. On the second day, use the same Ni binding buffer and Ni elution buffer again for nickel column affinity chromatography. Collect the protein in the flow-through and add 1M (NH 4 ) 2 SO 4 Then, the mixture was purified by hydrophobic column and buffer A1 (20 mM Tris8.5, 1 M (NH 4 ) 2 SO 4 ) Equilibrate the hydrophobic column and load the protein sample, then elute with buffer B1 (20mM Tris 8.5). Collect the protein eluted from 100% B and continue to purify using anion exchange column. Equilibrate the anion exchange column with buffer A2 (20mM Tris 8.5) and load the protein sample, then gradient elute with buffer B2 (20mM Tris 8.5, 1M NaCl). After collecting the protein at the peak position, use 1×PBS as the buffer to identify the state of the purified protein by 24mL molecular sieve. Collect the protein at the trimer position, i.e., the new coronavirus recombinant protein HR1 910-1011-L6-HR2P, concentrate it in an ultrafiltration centrifuge tube, and then aliquot and freeze it at -80°C.

[0042] Verification of the prepared novel coronavirus recombinant protein HR1 910-1011-L6-HR2P:

[0043] The pET30a plasmid was provided by Professor Jin Tengchuan's research group at the School of Life Sciences and Medicine, University of Science and Technology of China; Kana antibiotics, LB medium, etc. were purchased from Yuze Biotechnology; gene synthesis, primer synthesis, cloning and sequencing, etc. were provided by General Biotechnology (Anhui) Co., Ltd.; the enzymes used in PCR were purchased from Takara; Gibson assembly enzyme was purchased from Yisheng Biotechnology Co., Ltd.;

[0044] The process is as follows:

[0045] 1) Using the gene fragment of the recombinant protein HR1 910-1011-L6-HR2P obtained by gene synthesis, the target gene was cloned into the pET30a plasmid using Gibson assembly, transformed into E. coli DH5α competent cells, plated and cultured overnight, positive bacteria were screened, sequenced, PCR and enzyme digestion were used for identification, and the correct pET30a-target protein plasmid was obtained. The upstream and downstream PCR primers for identifying the target gene fragment are:

[0046] Upstream primer sequence:

[0047] 5'-ttatttccaaggttctgtcgacGGTGTTACCCAGAATGTGCTGTATGAAAATCAG-3' (SEQ IDNO.3);

[0048] Downstream primer sequence:

[0049] 5'-ctTGCGGCCgcCAGTTCCTGCAGATCAATCAGGC-3' (SEQ ID NO. 4).

[0050] 2) Transform the plasmid into the competent cell of Rosetta expression bacteria, pick out the single clones from the grown ones and put them into LB containing Kana, shake the cells at 37℃220rpm / min for 4-5h, then take 500μL of the cell solution into a 1.5mL EP tube as the uninduced control, add 1mM IPTG into the test tube and continue to shake the cells for 3-4h. Then take 200μL of the cell solution and centrifuge it, remove the culture medium, add 50μL of pure water and appropriate amount of SDS-loading buffer, incubate at 100℃ for 10min, centrifuge at 12000rpm for 5min, take 5μL of the sample and run SDS-PAGE.

[0051] 3) After the clones with good expression in small scale were expanded, they were transferred to large volume culture medium for culture. After OD600 reached 1.2, the temperature was lowered to 16℃ and 0.1mM IPTG was added to induce expression overnight. On the second day, the cells were collected after centrifugation at 5000rpm / min for 10min.

[0052] 4) Purify the target protein. Figure 2 The collected cells were subjected to nickel column affinity chromatography ( Figure 2 A) purification, followed by TEV protease cleavage and nickel column affinity chromatography. The flow-through protein was collected and added with 1M (NH 4 ) 2 SO 4 Then use hydrophobic column ( Figure 2 B) was used for purification, and the protein eluted with 20 mM Tris 8.5 was then purified using an ion exchange column ( Figure 2 C) purification, and the state of the purified protein was subjected to 24 mL molecular sieve ( Figure 2 Each purified protein was identified by SDS-PAGE ( Figure 2 Middle E), the results show that a trimeric protein with higher purity was obtained.

[0053] Example 3

[0054] The following materials were selected to study the immune properties of the novel coronavirus recombinant protein: flow cytometry antibodies were purchased from biolegend, HPR-labeled goat anti-mouse IgG was purchased from Sangon, and 6-8 week-old female C57BL / 6 mice were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.

[0055] 1. The specific process of the experiment is as follows:

[0056] S1. Immunity

[0057] At 0, 2, and 4 weeks, 6-8 week old mice were intraperitoneally injected with 0.5 mL of solution containing 20 μg of recombinant protein, 20 μg of CpG, and 100 μg of Al(OH) 3 For vaccines with adjuvants, a PBS control group was also set up.

[0058] S2. Specific IgG antibody detection

[0059] 14 days after the last immunization, blood was collected from the eyeballs of mice. After standing at room temperature for 2 hours, the upper serum was centrifuged at 5000rpm for 5 minutes and stored at -20℃. Antigen was coated on the ELISA plate at room temperature for 2 hours at 0.2μg / well. Then, the protein that was not bound to the plate was washed three times with 1×PBS, and 5% skim milk in 1×PBS was added to block at room temperature for 2 hours. Serum dilution solution with a 50-fold starting gradient was added and continuously diluted in 11 gradients in three times. After incubation at room temperature at 110rpm / min for 1 hour, it was washed three times with 1×PBST (containing 0.1% Tween-20). HRP-goat anti-mouse IgG diluted at 1:10000 was added and incubated at room temperature at 110rpm / min for 1 hour. After washing three times with 1×PBST (containing 0.1% Tween-20), 100μL / well TMB colorimetric solution was added and reacted in the dark for 7 minutes. Then 50μL / well 1M H 2 SO 4 Stop the reaction and select OD450 nm on the microplate reader to detect the results.

[0060] S3. Spleen and Lymph Node Management

[0061] 14 days after the last immunization, mice were killed by neck dislocation. After the surface of mice was disinfected with alcohol, they were placed in a biosafety cabinet for mouse dissection. The spleen was removed and temporarily soaked in 1×PBS. First, the spleen was cut into several small pieces with scissors and placed on a moistened 70μm steel yarn filter. The spleen was gently ground vertically up and down with the hard head of the syringe core. During the grinding process, the 70μm steel yarn filter was continuously rinsed with pre-cooled 1×PBS, and the ground spleen single cells were promptly rinsed into the collection container to reduce the mechanical damage of spleen cells. When only white mucosal tissue remained in the spleen tissue, the grinding could be stopped. The spleen single cell suspension was centrifuged at 800g and 4℃ for 5 minutes. Then the supernatant was discarded, and the cells were lysed with red blood cell lysis buffer (Biyuntian, C3702) for 5-10 minutes. The supernatant was removed by centrifugation, and the cells were washed once with 1×PBS. Finally, the spleen cells were resuspended with an appropriate amount of 1×PBS.

[0062] Isolate the bilateral inguinal lymph nodes of mice and temporarily place them in pre-cooled 1× PBS. Place the lymph nodes on a moistened 70μm steel yarn filter and gently grind the lymph nodes vertically up and down with the hard tip of the syringe core. During the grinding process, rinse the ground lymph nodes with pre-cooled 1× PBS continuously and collect the lymph node single cells into the collection container. When only white mucosal tissue remains in the lymph nodes, the grinding can be stopped. Centrifuge the lymph node single cell suspension at 800g and 4℃ for 5 minutes. Then discard the supernatant and resuspend the lymph node single cells with an appropriate amount of 1× PBS.

[0063] S4. Detection of immune cells in spleen and lymph nodes

[0064] First, 1-2 × 10 6Single cells were prepared from spleen and lymph nodes and added to U-bottom 96-well plates. The cells were then washed once with 1×PBS, the supernatant was discarded, and the Fc receptors were blocked with anti-mouse CD16 / CD32 monoclonal antibodies for 20 minutes on ice. Then all blocked cells were added with external standard antibodies and labeled on ice for 1 hour in the dark. Anti-mouse antibodies include: Percp / cy5.5-anti-mouse CD3, Pacific blue-anti-mouse CD4, BV510-anti-mouse CD8, FITC-anti-mouse CD44, APC / Cy7-anti-mouse CD62L, APC-anti-mouseCD69, PE / Cy7-anti-mouse PD-1, PE-anti-mouse CXCR5, PE-anti-mouse B220, APC / Cy7-anti-mouse CD19, BV421-anti-mouse CD38, FITC-anti-mouse GL7, Percp / cy5.5-anti-mouse Fas. Finally, the mouse cells were washed once with 1×PBS, then resuspended with an appropriate volume of 1×PBS, and each cell was detected with a three-laser Cytoflex flow cytometer, and the flow data were analyzed with FlowJo v10 data analysis software.

[0065] S5. Peptide stimulation experiment of spleen T cells

[0066] First, 1-2 × 10 6 Single cells from the spleen were taken out and added to U-bottom 96-well plates. 5 μg / mL protein and 1 μg / mL anti-mouse CD28 antibody of different groups were added to each well. Incubate at 37°C with 5% CO 2Culture in the incubator for 24 hours. At the 18th hour, add the protein transport inhibitor monensin to inhibit the secretion of cytokines to the extracellular space. Centrifuge the 96-well plate after 24 hours, then wash the cells once with 1×PBS, discard the supernatant, block the Fc receptor with anti-mouse CD16 / CD32 monoclonal antibody, and block on ice for 20 minutes. Then add external standard antibodies to all blocked cells and label them on ice in the dark for 1 hour. Anti-mouse antibodies include: APC / Cy7-anti-mouse CD45, FITC-anti-mouse CD4, BV510-anti-mouseCD8. Add 100μL PBS and centrifuge at 500g for 5min. After removing the supernatant, add 100μL / well of fixative and fix on ice for 30min. After centrifugation at 500g for 5min, remove the supernatant and add 100μL / well of membrane permeabilization solution and place on ice for 15min. Then centrifuge and add the internal standard antibody BV421-anti-mouse-IFN-γ prepared in the membrane permeabilization solution and place on ice for 1 hour. Supplement 100 μL PBS and centrifuge at 500g for 5 minutes, then add 100 μL PBS and resuspend, and then detect each cell with a three-laser Cytoflex flow cytometer, and analyze the flow data with FlowJo v10 data analysis software.

[0067] 2. Results Analysis

[0068] 1) Analysis of T cell epitope abundance and sequence conservation of S910-1011-L6-HR2P protein

[0069] After mapping the T cell epitopes of the currently reported SARS-CoV-2 Spike using ImmunomeBrowser, it was found that the abundance of T cell epitopes was relatively high in the HR1 and its derivative domains (910-1011aa) and the HR2 domain (1167-1203aa), indicating that there are relatively abundant T cell epitopes in this segment. Then, the S protein of the original SARS-CoV-2 strain was compared with the variants prevalent at various stages, as well as the other six coronavirus strains that infect humans, and it was found that the selected S910-1011 and S1167-1203 amino acid regions were highly conserved.

[0070] 2) Detection results of specific IgG antibodies stimulated by the novel coronavirus recombinant protein in mice

[0071] The results of the detection of antigen HR1 910-1011-L6-HR2P in the serum of mice immunized three times are as follows Figure 3 As shown, it can be seen that the immunized group is significantly higher than the control group, indicating that the recombinant protein provided by the present invention can induce strong humoral immunity and produce higher antibody levels after immunizing mice with the recombinant protein antigen, suggesting that the prepared vaccine can produce strong specific antibody response and has good application prospects.

[0072] 3) Test results of cellular immune response stimulated by the novel coronavirus recombinant protein in mice

[0073] The differences in cell subsets in the spleen and lymph nodes of mice after three immunizations were detected. The results are as follows Figure 4-8 As shown, the spleen of mice in the immunized antigen group ( Figure 4 ) and lymph nodes ( Figure 5 ) + and CD8 + There was a significant increase in T cell activation. In addition, in the lymph nodes ( Figure 6 ) and spleen ( Figure 7 ) and other secondary immune organs, compared with the control group, GC B cells, Tfh cells and other cell subsets that are crucial for B cell affinity maturation were significantly increased in the immune antigen group. Figure 8 , antigen-specific stimulation of mouse spleen lymphocytes showed that the antigen stimulation significantly increased CD8 + The production of IFN-γ in T cells effectively activates antigen-specific T cell immune responses.

[0074] In summary, the HR1 910-1011-L6-HR2P provided by the present invention is used as the antigen of the recombinant subunit vaccine. This region is highly conserved in both coronavirus and novel coronavirus mutant strains, and is rich in multiple T cell epitopes, which can fully activate the body's humoral immunity and T cell immunity.

[0075] The above implementation modes are only used to illustrate the technical solutions of the present invention, but not to limit the present invention. Although the present invention has been described in detail with reference to the above implementation modes, those skilled in the art should understand that any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A novel coronavirus recombinant subunit antigen, characterized in that: The antigen is a recombinant protein of the new coronavirus, named HR1 910-1011-L6-HR2P, whose amino acid sequence is shown in SEQ ID NO:1, and whose nucleotide sequence is shown in SEQ ID NO:

2.

2. An expression vector comprising the nucleotide sequence according to claim 1. A host cell comprising the expression vector according to claim 2.

4. Use of a recombinant subunit antigen of a novel coronavirus as described in claim 1 in the preparation of a vaccine for preventing the original strain and / or variant strain of the novel coronavirus.

5. Use of a recombinant subunit antigen of a novel coronavirus as described in claim 1 in the preparation of a kit for diagnosing novel coronavirus infection.

6. A vaccine comprising a recombinant subunit antigen of a new coronavirus as described in claim 1.

7. A kit for diagnosing novel coronavirus infection, comprising a novel coronavirus recombinant subunit antigen as described in claim 1.

8. A method for preparing a recombinant subunit antigen of a novel coronavirus as claimed in claim 1, characterized in that: The following steps are involved: Step 1. The nucleotide sequence of HR1 910-1011-L6-HR2P is used as the target gene and assembled with the pET30a plasmid, and then transferred into Rosetta competent cells to obtain expression bacteria containing the target gene; the nucleotide sequence of HR1 910-1011-L6-HR2P is shown in SEQ ID NO: 2; Step 2. The expression bacteria containing the target gene are broken after IPTG induction, and the desired new coronavirus recombinant protein is obtained by chromatography purification.

9. The method for preparing a recombinant subunit antigen of a novel coronavirus according to claim 8, characterized in that: In step 2, the target gene is inserted between the restriction sites Sal I and NotI of the pET30a plasmid by mixing the target gene PCR recovery product and the pET30a plasmid PCR recovery product at a molar ratio of 4:1, adding Gibson assembly enzyme, and incubating at 50° C. in a water bath for 15 min.

10. The method for preparing a recombinant subunit antigen of a novel coronavirus according to claim 8, characterized in that: In step 2, the IPTG induction process is as follows: after the expression bacteria containing the target gene are cultured in LB containing Kana for 4-5 hours, IPTG is added in an amount of 500 μL bacterial solution: 1 mM IPTG, and the culture is continued for 3-4 hours, and then the bacterial solution is aspirated and centrifuged to obtain the induced bacteria.

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