CN117924437A - Novel coronavirus recombinant subunit antigen and preparation method and application thereof - Google Patents

Abstract

The invention relates to the technical field of biological pharmacy, in particular to a novel coronavirus recombinant subunit antigen, a preparation method and application thereof. The antigen is a novel coronavirus recombinant protein, named HR 1-910-1011-L6-HR 2P. The novel coronavirus recombinant protein provided by the invention has high conservation to coronaviruses and novel coronavirus mutant strains, contains abundant effective T cell epitopes, and can stimulate organisms to generate antigen-specific antibodies and cellular immune responses. The recombinant protein can use a prokaryotic expression system as an expression vector, has the advantages of high yield, low production cost, similar state with physiological state and the like, and is suitable for preparing new crown vaccines on a large scale.

Description

Novel coronavirus recombinant subunit antigen and preparation method and application thereof

Technical Field

The invention relates to the technical field of biological pharmacy, in particular to a novel coronavirus recombinant subunit antigen, a preparation method and application thereof.

Technical Field

Novel coronaviruses (SARS-CoV-2, hereinafter referred to as SARS 2) recognize epithelial cells expressing the ACE2 receptor and enter host cells as follows: the multiple motifs on the S1-S2 border are cleaved when the virus matures in the infected cell, but the S2' site is cleaved after binding of angiotensin converting enzyme 2 (ACE 2) to the target cell. Binding of the virus 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 dissociation of S1 and S2 results in a dramatic change in the conformation of the S2 subunit, especially HR1, thereby pushing the fusion peptide forward into the target membrane and initiating membrane fusion. Fusion between the viral membrane and the cell membrane forms a fusion pore through which viral RNA is released into the cytoplasm of the host cell for uncoating and replication.

SARS-CoV-2 can induce host to generate humoral and cellular immune response after infecting human respiratory epithelial cells. The infected epithelial cells degrade the viral particles and present them to cytotoxic CD8 ⁺ T cells. CD8 ⁺ T recognizes viral proteins via TCR-MHC I interactions, releasing cytotoxic particles such as granzyme B and perforin, and acts on infected cells. In addition, antigen presenting cells present viral polypeptides to CD4 ⁺ T cells (Th 0) via TCR-MHC II interactions. Once the antigen polypeptide is recognized, th0 cells are polarized primarily toward Th1, releasing IFN- γ to eliminate the virus, and releasing Th2 triggers humoral-mediated immune responses and antibody secretion against the SARS-CoV-2 virus.

Currently, vaccination is the most effective common intervention to prevent new coronavirus infections. The current global route for vaccine development mainly includes the following: viral vaccines represented by inactivated vaccines and attenuated live vaccines; recombinant subunit vaccines with S protein, RBD, virus-like particle or polypeptide as antigen; nucleic acid vaccines of the DNA or RNA class, including non-replicating and replicating vector vaccines, and the like. However, with the prevalence of novel coronaviruses, variants have been found more frequently. The new crown variants of major interest at present include the first B.1.1.7 (Alpha) variants found in the United kingdom, the B.1.351 (Beta) variants found in south Africa, the P.1 (Gamma) variants found in Brazil, the B.1.617.2 (Delta) variants found in India and the B.1.1.529 (Omicron) variants (https:// www.who.int/en/activators/tracking-SARS-CoV-2-variants /). Prophase vaccines were developed that primarily target the spike protein (spike) region of SARS-CoV-2, as the receptor binding domain (receptor binding domain) of this region binds to host cell surface ACE2 receptors and thus mediates viral invasion into the host. However, along with mutation of the variant at the key site of the S protein, immune escape and other phenomena occur, and some variant has stronger transmission capability, even breakthrough infection occurs to people vaccinated with the novel coronavirus, which brings new challenges to development of the existing vaccine and prevention and treatment of the novel coronavirus.

In order to cope with the continuous variation of SARS-CoV-2 virus, the development of a safer and more effective SARS-CoV-2 vaccine is a more urgent problem at present.

Disclosure of Invention

In order to solve the problems in the prior art, an object of the present invention is to provide a novel coronavirus recombinant subunit antigen.

The invention adopts the following technical scheme:

A novel coronavirus recombinant subunit antigen is a novel coronavirus recombinant protein, named HR 1-1011-L6-HR 2P, and has an amino acid sequence shown as SEQ ID NO.1 and a nucleotide sequence shown as SEQ ID NO. 2.

It is a second object of the present invention to provide an expression vector comprising the nucleotide sequence as described above.

It is a further object of the present invention to provide a host cell comprising an expression vector as described above.

The fourth object of the present invention is to provide the application of the novel coronavirus recombinant subunit antigen in preparing a vaccine for preventing novel coronavirus original strain and/or variant strain.

The fifth object of the present invention is to provide the use of the novel coronavirus recombinant subunit antigen in the preparation of a kit for diagnosing novel coronavirus infection.

It is a sixth object of the present invention to provide a vaccine comprising a novel coronavirus recombinant subunit antigen as described above.

The seventh object of the present invention is to provide a kit for diagnosing a novel coronavirus infection comprising a novel coronavirus recombinant subunit antigen as described above.

The invention finally provides a preparation method of the novel coronavirus recombinant subunit antigen, which comprises the following steps:

Step 1, assembling a target gene of a nucleotide sequence of HR1910-1011-L6-HR2P with a pET30a plasmid, and transferring into Rosetta competent cells to obtain an expression bacterium containing the target gene; the nucleotide sequence of HR1910-1011-L6-HR2P is shown as SEQ ID NO. 2;

And 2, crushing the target gene-containing expression bacteria after IPTG induction, and purifying by chromatography to obtain the novel required coronavirus recombinant protein.

Preferably, in the step 1, the target gene is inserted between the cleavage 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 according to the mass ratio of 4:1, adding Gibson enzyme, and incubating for 15min under the water bath condition of 50 ℃.

Preferably, in the step 2, the IPTG induction process is as follows: culturing the target gene-containing expression bacteria in Kana-containing LB for 4-5h, adding IPTG with the amount of 1mM IPTG in 500 mu L of bacterial liquid, culturing for 3-4h, sucking bacterial liquid, and centrifuging to obtain induced bacterial body.

The invention has the beneficial effects that:

The novel coronavirus recombinant protein provided by the invention has high conservation to coronaviruses and novel coronavirus mutant strains, contains abundant effective T cell epitopes, has good immunogenicity, and can stimulate organisms to generate antigen-specific antibodies and cellular immune responses.

The recombinant protein can be used as an expression vector by using a prokaryotic expression system, has the advantages of high yield, low production cost, high protein purity, similar state with physiological state and the like, and is suitable for preparing new crown vaccines on a large scale.

Drawings

FIG. 1 is an epitope map provided by the invention, wherein A is a schematic diagram of antigen protein expression; b is the T cell epitope map of SARS-CoV-2 Spike.

FIG. 2 is a purification chart and SDS-PAGE result of the prepared recombinant protein, wherein the A chart is a Ni affinity column chromatography purification chart of the supernatant after ultrasonic disruption of the thallus; panel B shows a purification pattern of hydrophobic interaction chromatography (HIC column) of the target protein collected after TEV cleavage; panel C is an ion exchange chromatography (Q column) purification profile performed after hydrophobic column purification; panel D shows a gel filtration chromatography (24 mL molecular sieve) purification map of the target protein; e is an SDS-PAGE identification of the final target protein.

FIG. 3 shows the levels of antigen-specific antibodies in serum of immunized mice, wherein A is an ELISA graph of the immunized target protein antigen and the serum after PBS, and each point is the reading at OD _{450 nm} under different dilution gradients; b is a histogram of titers calculated for the immunized protein antigen of interest and serum after PBS.

FIG. 4 is a graph of CD4 ⁺ and CD8 ⁺ T cell activation levels in spleen of immunized mice, where A is the percentage of CD4 ⁺CD69⁺ T cells in spleen tissue; b is the average fluorescence intensity of CD4 ⁺CD69⁺ T cells in spleen tissue; c is the percentage of CD8 ⁺CD69⁺ T cells in spleen tissue; d is the average fluorescence intensity of CD8 ⁺CD69⁺ T cells in spleen tissue; e is the percentage of CD4 ⁺PD-1⁺ T cells in spleen tissue; f is the average fluorescence intensity of CD4 ⁺PD-1⁺ T cells in spleen tissue; g is the percentage of CD8 ⁺PD-1⁺ T cells in spleen tissue; h is the average fluorescence intensity of CD8 ⁺PD-1⁺ T cells in spleen tissues, and the HR2P marked in the figure is the abbreviation of HR 1-1011-L6-HR 2P.

FIG. 5 shows the activation levels of CD4 ⁺ and CD8 ⁺ T cells in lymph nodes of immunized mice, wherein A is the percentage of CD4 ⁺CD69⁺ T cells in lymph node tissue; b is the average fluorescence intensity of CD4 ⁺CD69⁺ T cells in lymph node tissue; c is the percentage of CD8 ⁺CD69⁺ T cells in lymph node tissue; d is the average fluorescence intensity of CD8 ⁺CD69⁺ T cells in lymph node tissue; e is the percentage of CD4 ⁺PD-1⁺ T cells in lymph node tissue; f is the average fluorescence intensity of CD4 ⁺PD-1⁺ T cells in lymph node tissue; g is the percentage of CD8 ⁺PD-1⁺ T cells in lymph node tissue; h is the average fluorescence intensity of CD8 ⁺PD-1⁺ T cells in lymph node tissue, and HR2P is abbreviated as HR 1-910-1011-L6-HR 2P in the figure.

FIG. 6 is a graph of B cell maturation associated cell levels in lymph nodes of immunized mice, wherein graph A is the percentage of follicular helper T cells (Tfh) in lymph node tissue; panel B shows the percentage of germinal center B cells (GC B) in lymph node tissue, and the HR2P is abbreviated as HR 1-910-1011-L6-HR 2P.

FIG. 7 is B cell maturation associated cell levels in the spleen of immunized mice, wherein Panel A is the percentage of follicular helper T cells (Tfh) in spleen tissue; b is the percentage of germinal center B cells (GC B) in spleen tissue, and the HR2P is abbreviated as HR1 910-1011-L6-HR2P in the figures.

FIG. 8 is the level of intracellular IFN-gamma production 24h after antigen stimulation of CD8 ⁺ T cells in the spleen of immunized mice, wherein Panel A is the percentage of CD4 ⁺IFN-γ⁺ T cells; panel B shows the Mean Fluorescence Intensity (MFI) of CD4 ⁺IFN-γ⁺ T cells; panel C is the percentage of CD8 ⁺IFN-γ⁺ T cells; panel D shows the Mean Fluorescence Intensity (MFI) of CD8 ⁺IFN-γ⁺ T cells.

Detailed Description

In order to facilitate understanding, the technical scheme of the present invention will be described in more detail with reference to examples.

Example 1

The novel coronavirus recombinant protein provided by the invention is characterized in that HR1 and a derivative domain (910-1011 aa) and HR2 domain (1167-1203 aa) thereof are selected from reported SARS-CoV-2Spik genome sequence (Genbank: MT 019533.1), and the two parts are connected by utilizing linker (SGGRGG) of 6 amino acids. Then we performed codon optimization on the spliced genes and sent to general biology (Anhui) Inc. for synthesis, labeled HR 1-910-1011-L6-HR 2P, and FIG. 1A is a schematic representation of antigen protein expression.

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

The currently reported T cell epitopes of SARS-CoV-2Spike detected by using T cell experiment are downloaded in IEDB database (https:// www.iedb.org /), and then ImmunomeBrowser (http:// tools. IEDB. Org/immunomebrowser /) is used for analyzing and summarizing the T cell epitopes, so that a Spike T cell epitope map shown in figure 1B is obtained. The figure shows that the amino acid position of the target protein has rich T cell epitopes.

Comparing the S protein of SARS-CoV-2 original strain with the variants popular in each stage and the coronavirus strains of six other infected people, it can be seen that the selected S910-1011 and S1167-1203 amino acid regions have higher conservation.

Example 2

Preparation method of novel coronavirus recombinant protein

Step 1. DNA sequence of HR1 910-1011-L6-HR2P protein the gene of interest was inserted between Sal I and Not I in the ET30a plasmid. The method comprises the following specific steps: the target gene PCR recovered product of 0.1pmoL and the vector PCR recovered product of 0.025pmoL were added, 1. Mu.L of 5 XGibson enzyme was added, and then purified water was added to make up to 5. Mu.L. After mixing, the mixture was incubated in a water bath at 50℃for 15min and then added to DH 5. Alpha. Competent cells. The transformation method is as follows: slowly thawing 50 mu L of competent bacterial liquid on ice, adding the Gibson assembly product, flicking the tube wall, standing on ice for 15min, heat-shocking in a water area pot at 42 ℃ for 90s, and standing on ice for 3min. 500. Mu.L of the non-resistant LB medium was added to the super-clean bench, and the mixture was shaken in a shaker at 37℃at 220rpm/min for 45min, then 100. Mu.L of the mixture was uniformly coated with coating beads on a Kana-containing resistant plate in the super-clean bench, and the mixture was cultured in an incubator at 37℃overnight with inversion.

Step 2, selecting a monoclonal antibody, shaking the monoclonal antibody in a shaking table containing Kana at a temperature of 37 ℃ and at a speed of 220rpm/min for 4-5 hours, taking 500 mu L of bacterial liquid into a 1.5mL EP tube as an uninduced control, adding 1mM IPTG into the tube, and continuing shaking the bacterial antibody for 3-4 hours. Then, after centrifugation of 200. Mu.L of each of the bacterial solutions was performed, the medium was removed, 50. Mu.L of pure water and an appropriate amount of SDS-loading buffer were added, and after centrifugation at 12000rpm for 5min at 100℃in a metal bath, 5. Mu.L of the sample was subjected to SDS-PAGE.

Step 3. The purification of the novel coronavirus recombinant protein is specifically as follows: adding a proper amount of Ni binding buffer (20mM Tris 8.5, 250mM NaCl,14mM imidazole) into the collected thalli, performing ultrasonic crushing treatment, centrifuging at a high speed, taking the supernatant, performing nickel column affinity chromatography purification, and performing gradient elution by using Ni elution buffer (20mM Tris 8.5, 250mM NaCl,400mM imidazole). Adding 5mM DTT,1mM EDTA,1/10 molar ratio TEV protease into the protein solution at the peak position, mixing, standing for enzyme digestion for 4 hours at 4 degrees. Dialyzed overnight into 5L of dialysate without imidazole (20 mM Tris 8.5, 250mM NaCl). The next day was again subjected to nickel column affinity chromatography using the same Ni binding buffer and Ni elution buffer. Collecting protein in the flow-through, adding 1M (NH ₄)₂SO₄, then purifying by using a hydrophobic column, balancing a hydrophobic column by using buffer A1 (20 mM Tris 8.5,1M (NH ₄)₂SO₄), then eluting by using buffer B1 (20 mM Tris 8.5), collecting protein eluted by 100% B, continuing purifying by using an anion exchange column, balancing an anion exchange column by using buffer A2 (20 mM Tris 8.5), loading protein, then gradient eluting by using buffer B2 (20mM Tris 8.5,1M NaCl), collecting protein at peak position, then carrying out 24mL molecular sieve identification by using 1 XPBS as buffer, collecting protein at trimer position, namely novel coronavirus recombinant protein HR1 910-1011-L6-HR2P, concentrating by ultrafiltration, and then split charging in a freeze-80 ℃.

The verification of the prepared novel coronavirus recombinant protein HR1 910-1011-L6-HR 2P:

The pET30a plasmid is provided by the professor task group of the university of Chinese science and technology student science and medical department Jin Tengchuan; kana antibiotics, LB medium, etc. to the rainy organism; gene synthesis, primer synthesis, clone sequencing, etc. are provided by general biology (Anhui) Inc.; enzymes used for PCR were purchased to Takara; gibson enzyme was purchased from Holly Biolabs;

The process is as follows:

1) The gene fragment of recombinant protein HR1 910-1011-L6-HR2P obtained by gene synthesis is used, the obtained target gene is cloned into pET30a plasmid by Gibson assembly, and is transformed into E.coli DH5 alpha competent cells, and the target gene is plated and cultured overnight, positive bacteria are screened, and the correct pET30 a-target protein plasmid is obtained through sequencing, PCR and enzyme digestion identification. The PCR upstream and downstream primers for identifying the target gene fragment are as follows:

Upstream primer sequence:

5’-ttatttccaaggttctgtcgacGGTGTTACCCAGAATGTGCTGTATGAAAATCAG-3’(SEQ ID NO.3)；

downstream primer sequence:

5’-ctTGCGGCCgcCAGTTCCTGCAGATCAATCAGGC-3’(SEQ ID NO.4)。

2) The plasmid is transformed into Rosetta expression bacteria competence, the monoclonal is selected from the grown monoclonal and put into LB containing Kana, and the bacteria are shaken for 4-5 hours in a shaking table at 37 ℃ and 220rpm/min, then 500 mu L of bacterial liquid is taken into a 1.5mL EP tube as non-induced control, and after 1mM IPTG is added into the test tube, the bacteria are continuously shaken for 3-4 hours. Then, after centrifugation of 200. Mu.L of each of the bacterial solutions was performed, the medium was removed, 50. Mu.L of pure water and an appropriate amount of SDS-loading buffer were added, and after centrifugation at 12000rpm for 5min at 100℃in a metal bath, 5. Mu.L of the sample was subjected to SDS-PAGE.

3) The clone with better small-scale expression is transferred into a large-volume culture medium for culture after the OD600 is as long as 1.2, the temperature is reduced to 16 ℃, and 0.1mM IPTG is added for overnight induction of expression. The cells were collected after centrifugation at 5000rpm/min for 10min the next day.

4) Purifying the target protein. As shown in FIG. 2, the collected cells were purified by nickel column affinity chromatography (FIG. 2A), and then subjected to nickel column affinity chromatography again after being digested with TEV protease. Collecting the protein, adding 1M (NH ₄)₂SO₄, purifying with hydrophobic column (B in FIG. 2), eluting with 20mM Tris 8.5, purifying with ion exchange column (C in FIG. 2), collecting main peak position, and identifying the purified protein by 24mL molecular sieve (D in FIG. 2), and performing SDS-PAGE identification (E in FIG. 2) of each purified protein, wherein the result shows that the obtained trimeric protein has higher purity.

Example 3

The immunity of the novel coronavirus recombinant protein is researched by selecting the following materials, wherein the flow antibody is purchased from biolegend company, the HPR marked goat anti-mouse IgG is purchased from the biological company, and the 6-8 week old female C57BL/6 mouse is purchased from Jiangsu Ji Yikang biological technology company.

1. The specific procedure of the experiment is as follows:

S1, immunization

The young rats of 6-8 weeks old were intraperitoneally injected with 0.5mL of a vaccine containing 20. Mu.g recombinant protein, 20. Mu.g CpG and 100. Mu.g Al (OH) ₃ adjuvant at weeks 0, 2 and 4, and PBS control groups were additionally provided.

S2, specific IgG antibody detection

The mice were collected with eyeball blood after 14 days of the last immunization, and after standing at room temperature for 2 hours, centrifuged at 5000rpm for 5min, the upper serum was taken out and stored at-20 ℃. The room temperature coated antigen 0.2. Mu.g/well in ELISA plate for 2h. Proteins not bound to the plates were then washed three times with 1 XPBS and blocked for 2h at room temperature with 5% skimmed milk added to 1 XPBS. Serum dilutions were added which serially diluted 11 gradients at a triple gradient with a 50-fold initial gradient, incubated at 110rpm/min for 1h at room temperature and washed 3 times with 1 XPBST (containing 0.1% Tween-20). HRP-goat anti-mouse IgG diluted 1:10000 was added and incubated at 110rpm/min for 1h at room temperature. After washing 3 times with 1 XPBST (containing 0.1% Tween-20), 100. Mu.L/well TMB color development solution was added and the reaction was protected from light for 7min. The reaction was then stopped by adding 50. Mu.L/well 1M H ₂SO₄. The detection result of the wavelength at OD450 nm is selected on an enzyme label instrument.

S3 spleen and lymph node treatment

After 14 days of the last immunization, the mice are killed by cervical scission, the surfaces of the mice are sterilized by alcohol, and then the mice are placed in a biosafety cabinet for dissection. Spleens were removed and briefly immersed in 1×pbs. Firstly, cutting the spleen into a plurality of small sections by scissors, placing the small sections on a wet 70 mu m steel yarn filter screen, lightly grinding the spleen vertically upwards and downwards by using a hard head part of a syringe core, flushing the 70 mu m steel yarn filter screen by precooling 1X PBS continuously in the grinding process, flushing the grinded spleen single cells into a collecting container in time, and reducing the mechanical damage of the spleen cells. Grinding can be suspended when spleen tissue only remains white mucosal tissue. Spleen single cell suspension 800g was centrifuged at 4℃for 5 minutes. The supernatant was then discarded, red blood cell lysate (Biyun Tian, C3702) was centrifuged to remove the supernatant for 5-10 min, the cells were washed once with 1 XPBS, and finally spleen cells were resuspended with an appropriate amount of 1 XPBS.

The mice were isolated from bilateral inguinal lymph nodes and placed temporarily in pre-chilled 1 XPBS. Lymph nodes were placed on a wet 70 μm steel gauze screen, the lymph nodes were gently ground vertically up and down with the hard head portion of the syringe core, and during the grinding, the ground lymph nodes were washed uninterruptedly with pre-chilled 1×pbs, and lymph node single cells were collected in a collection container. Grinding can be suspended when the lymph nodes only leave white mucosal tissue. The lymphonodic single cell suspension 800g,4 ℃ centrifugal 5 minutes. The supernatant was then discarded and the lymphoid cells were resuspended in an appropriate amount of 1 XPBS.

S4, spleen and lymph node immunocyte detection

First, single cells prepared from spleen and lymph node were removed and added to U-bottom 96-well plates, respectively, according to the standard of 1-2X 10 ⁶ cells per well. Cells were then washed 1 time with 1 XPBS, the supernatant discarded, the Fc receptor blocked with anti-murine CD16/CD32 monoclonal antibody and blocked on ice for 20 minutes. All blocked cells were then added to the external standard antibody and marked on ice protected from light for 1 hour. Anti-mouse antibody included ：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-mouse CD69,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, each cell of the mice was washed once with 1 XPBS, then resuspended with an appropriate volume of 1 XPBS, each cell detected with a three laser Cytoflex flow cytometer, and the flow data analyzed with FlowJo v10 data analysis software.

S5 polypeptide stimulation experiment of spleen T cells

First, spleen single cells were removed and added to U-bottom 96-well plates, respectively, according to the standard of 1-2X 10 ⁶ cells per well. Different groups of 5. Mu.g/mL protein and 1. Mu.g/mL anti-mouse CD28 antibody were added to each well. Culturing in a 5% CO ₂ incubator at 37℃for 24h. The addition of the protein transport inhibitor monensin at 18h inhibited cytokine secretion outside the cell. After 24h, the 96-well plates were centrifuged, and the cells were washed 1 XPBS, the supernatant was discarded, and the Fc receptor was blocked with anti-mouse CD16/CD32 monoclonal antibody, and blocked on ice for 20 minutes. All blocked cells were then added to the external standard antibody and marked on ice protected from light for 1 hour. The anti-mouse antibodies include: APC/Cy7-anti-mouse CD45, FITC-anti-mouse CD4, BV510-anti-mouse CD8. Centrifuge 500g for 5min with 100. Mu.L PBS, remove supernatant and add 100. Mu.L/well of fixative onto ice for 30min. After centrifugation for 500g of 5min, the supernatant was removed and placed on 100. Mu.L/Kong Pomo liquid ice for 15min. And then centrifugally adding an internal standard antibody BV 421-anti-mouse-IFN-gamma prepared by membrane rupture liquid, and standing for 1h on ice. Centrifuge 500g for 5min with 100. Mu.L PBS, then re-suspend with 100. Mu.L PBS, and check each cell with a three laser Cytoflex flow cytometer and analyze the flow data with FlowJo v10 data analysis software.

2. Analysis of results

1) T cell epitope abundance and sequence conservation analysis of S910-1011-L6-HR2P protein

The use of ImmunomeBrowser to map the currently reported T cell epitope of SARS-CoV-2Spike reveals a relatively high abundance of T cell epitopes in HR1 and its derivative domains (910-1011 aa) and HR2 domains (1167-1203 aa), indicating a relatively rich T cell epitope in this segment. Then comparing the S protein of SARS-CoV-2 original strain with the variants popular in each stage and other six coronavirus strains of infected person, the conservation of the selected S910-1011 and S1167-1203 amino acid regions is higher.

2) Specific IgG antibody detection result of novel coronavirus recombinant protein excited in mouse

The detection result of the antigen HR 1-1011-L6-HR 2P in the serum of the three immunized mice is shown in figure 3, and the immune group is obviously higher than the control group, which shows that the recombinant protein antigen provided by the invention can cause strong humoral immunity after immunizing the mice, generate higher antibody level, and indicate that the prepared vaccine can generate strong specific antibody reaction, thereby having better application prospect.

3) Cell immune response detection result of novel coronavirus recombinant protein excited in mouse

Cell subpopulations of spleen and lymph nodes were examined three times after immunization, and as shown in fig. 4-8, CD4 ⁺ and CD8 ⁺ T cell activation was significantly increased in spleen (fig. 4) and lymph nodes (fig. 5) of mice immunized with the antigen group. In addition, in secondary immune organs such as lymph nodes (fig. 6) and spleen (fig. 7), cell subsets critical for B cell affinity maturation, such as GC B cells, tfh cells, were significantly increased in the immune antigen group compared to the control group. Referring to fig. 8, antigen-specific stimulation of mouse spleen lymphocytes can be seen to significantly increase IFN- γ production in CD8 ⁺ T cells, effectively activating antigen-specific T cell immune responses.

In conclusion, the HR1 910-1011-L6-HR2P provided by the invention is used as an antigen of a recombinant subunit vaccine, has higher conservation in coronavirus and novel coronavirus mutant strains, is rich in various T cell epitopes, and can fully activate humoral immunity and T cell immunity of organisms.

The above embodiments are only for illustrating the technical scheme of the present invention, and are not limiting to the present invention; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (10)

1. A novel coronavirus recombinant subunit antigen is characterized in that the antigen is a novel coronavirus recombinant protein, named HR 1-910-1011-L6-HR 2P, the amino acid sequence of which is shown as SEQ ID NO.1, and the nucleotide sequence of which is shown as SEQ ID NO. 2.

2. An expression vector comprising the nucleotide sequence of claim 1.

3. A host cell comprising the expression vector of claim 2.

4. Use of a novel coronavirus recombinant subunit antigen according to claim 1 for the preparation of a vaccine for the prevention of novel coronavirus original and/or variant strains.

5. Use of a novel coronavirus recombinant subunit antigen according to claim 1 for the preparation of a kit for diagnosing a novel coronavirus infection.

6. A vaccine comprising a novel coronavirus recombinant subunit antigen of claim 1.

7. A kit for diagnosing a novel coronavirus infection comprising a novel coronavirus recombinant subunit antigen of claim 1.

8. A method of preparing a novel coronavirus recombinant subunit antigen of claim 1, comprising the steps of:

Step 1, assembling a target gene of a nucleotide sequence of HR1910-1011-L6-HR2P with a pET30a plasmid, and transferring into Rosetta competent cells to obtain an expression bacterium containing the target gene; the nucleotide sequence of HR1910-1011-L6-HR2P is shown as SEQ ID NO. 2;

And 2, crushing the target gene-containing expression bacteria after IPTG induction, and purifying by chromatography to obtain the novel required coronavirus recombinant protein.

9. The method for preparing a novel coronavirus recombinant subunit antigen according to claim 8, wherein in step 2, the target gene is inserted between the cleavage sites Sal I and NotI of the pET30a plasmid, the method comprises: mixing the target gene PCR recovery product and the pET30a plasmid PCR recovery product according to the mass ratio of 4:1, adding Gibson enzyme, and incubating for 15min under the water bath condition of 50 ℃.

10. The method of claim 8, wherein in step 3, IPTG induction is performed by: culturing the target gene-containing expression bacteria in Kana-containing LB for 4-5h, adding IPTG with the amount of 1mM IPTG in 500 mu L of bacterial liquid, culturing for 3-4h, sucking bacterial liquid, and centrifuging to obtain induced bacterial body.