Sars-related coronavirus universal antigen and preparation method and application thereof

Abstract

The application provides a SARS-related coronavirus universal antigen and a preparation method and application thereof, and belongs to the technical field of immunity. The candidate antigen prepared by the application has higher S309 antibody and detection serum binding activity, the titer of serum obtained by immunizing mice with the candidate antigen has higher cross-binding activity to VOCs than that of template antigen immunized serum; meanwhile, the candidate antigen immunized serum has the ability to block the combination of VOCs and ACE2. Therefore, the candidate antigen prepared by the application can become a potential universal antigen or combination for inducing cross-neutralization activity for preventing SARS-related coronavirus infection. The strategy of preparing the candidate antigen with cross-neutralization immunogenicity based on DNA Shuffling and membrane mutation antigen screening technology is expected to be applied to universal vaccine research and development. The candidate universal antigen prepared by the application has the potential to be developed into a SARS-related coronavirus universal vaccine.

Description

Technical Field

[0001] The present invention relates to the field of immunotechnology, and in particular to a universal antigen of SARS-related coronaviruses and a preparation method and application thereof. Background Art

[0002] The SARS virus (SARS-CoV) and the novel coronavirus (SARS-CoV-2) belong to the same family, Coronaviridae, and are classified as two subtypes within the genus Betacoronavirus, group B. Both viruses are collectively referred to as SARS-related coronaviruses. Infection with both viruses can cause respiratory illness, with primary symptoms including fever, fatigue, lung infection, and difficulty breathing. Severe cases can be fatal. SARS has a higher mortality rate, reaching 30%-40%.

[0003] The World Health Organization (WHO) has designated some highly infective, immune-evading variants of the novel coronavirus as "Variants of Concern (VOCs)," including Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), and Omicron (B.1.1.529). Omicron is further divided into five sublineages: BA.1, BA.2, BA.3, BA.4, and BA.5. BA.5 has formed multiple evolutionary branches, including BF.7, while BA.2 has further formed multiple evolutionary branches, including BA.2.75, BA.2.86, and XBB. With the continuous emergence of new variants of the novel coronavirus and the increasing immune-evasion phenomenon, the continued effectiveness of existing coronavirus vaccines and antibody drugs faces severe challenges. Therefore, it is crucial to develop highly effective, broad-spectrum coronavirus vaccines to protect against the current epidemic and other coronaviruses that may emerge in the future.

[0004] The receptor binding domain (RBD) on the S protein of both SARS-CoV and SARS-CoV-2 is the part that directly binds to the host cell receptor, and the sequence homology of the two is 73%, which plays an important role in the process of virus adsorption and entry into host cells. The RBD region has abundant antigen epitopes, and more than 90% of neutralizing antibodies are targeted at the RBD region. The neutralizing antibodies induced can block the binding of the virus to angiotensin-converting enzyme 2 (ACE2) receptors, cause effective humoral and cellular immune responses, and produce immune protection reactions. At the same time, compared with the full-length S protein, RBD can avoid the potential risk of antibody-dependent enhancement of infection caused by non-neutralizing antibodies, which makes RBD a key target for vaccine design. However, RBD has certain limitations in immune response. Some studies have shown that the RBD monomer has weak ability to stimulate the body's immune response, and it is difficult to produce enough protective antibodies using RBD monomer as an antigen, but the immune effect of RBD can be enhanced to some extent through some strategies, such as increasing the size of the antigen or making RBD multimerization.

[0005] In the prior art, vaccines are difficult to produce effective broad-spectrum cross-recognition activity against constantly mutating coronaviruses, which is still a problem to be solved. SUMMARY

[0006] The purpose of the present application is to provide a SARS-related coronavirus universal antigen and a preparation method and application thereof, which can provide a potential candidate universal antigen for the prevention of SARS-related coronavirus.

[0007] In order to achieve the above-mentioned purpose of the application, the present application provides the following technical solutions:

[0008] The present application provides a SARS-related coronavirus antigen C-11, and the amino acid sequence of the antigen C-11 is shown as SEQ ID NO. 1.

[0009] The present application also provides a coding gene of the above-mentioned SARS-related coronavirus antigen C-11, and the nucleotide sequence of the coding gene is shown as SEQ ID NO. 3.

[0010] The present application also provides a SARS-related coronavirus candidate antigen 8-C, and the amino acid sequence of the antigen 8-C is shown as SEQ ID NO. 2.

[0011] The present application also provides a coding gene of the above-mentioned SARS-related coronavirus antigen C-8, and the nucleotide sequence of the coding gene is shown as SEQ ID NO. 4.

[0012] The application also provides application of the SARS coronavirus antigen C-11, the SARS coronavirus candidate antigen 8-C or the coding gene in preparation of a vaccine for SARS coronavirus.

[0013] The application also provides application of the SARS coronavirus antigen C-11, the SARS coronavirus candidate antigen 8-C or the coding gene in preparation of a vaccine for SARS coronavirus.

[0014] The application also provides application of the SARS coronavirus antigen C-11, the SARS coronavirus candidate antigen 8-C or the coding gene in development of a diagnostic kit or a diagnostic reagent for SARS coronavirus.

[0015] The application also provides a biological material related to the SARS coronavirus antigen C-11 or the SARS coronavirus candidate antigen 8-C, which is an expression cassette, a recombinant vector or a recombinant bacterium containing the coding gene.

[0016] The application also provides a preparation method of the SARS coronavirus antigen C-11 or the SARS coronavirus candidate antigen 8-C, which comprises the following steps:

[0017] The template antigen gene fragment is obtained by PCR amplification, and is cut into small fragments of about 50-100 bp by DNase I, and then the small fragments are amplified by primer-free PCR with the homologous regions as primers, and finally the rearranged antigen is obtained by primer PCR with the homologous fragments of 100 bp at both ends of all template antigen sequences as primers.

[0018] The antigen gene fragment is constructed into a membrane expression vector by a homologous recombination method to form a recombinant membrane mutant antigen plasmid, the recombinant membrane mutant antigen plasmid is transfected into 293T cells, the antibody or detection serum with broad-spectrum cross-binding activity is selected, and the antigen is screened by comprehensively comparing three groups of data of binding percentage, fluorescence intensity average and median.

[0019] The antigen gene fragment is constructed into a secretory vector by a homologous recombination method to obtain a recombinant secretory antigen plasmid, and the recombinant secretory antigen plasmid is transfected into target cells, and then the antigen is purified.

[0020] Preferably, the secretory vector takes pcDNA3.1 as a starting vector, takes the first 15 amino acids of the S protein of SARS-CoV-2 Omicron wild type as a signal peptide, contains 62 transmembrane region amino acids at the C terminal and a 6×His label.

[0021] The application has the following beneficial effects:

[0022] The present invention utilizes DNA Shuffling technology to effectively simulate viral evolution and perform gene rearrangement on the RBD and CTD1 regions of SARS-related coronaviruses. During the rearrangement process, the key mutation sites of different strains can be optimized and combined, overcoming the shortcomings of existing technologies that can only design vaccines based on existing viral gene sequences, and preparing antigens that can cross-recognize multiple strains.

[0023] The present invention selects the RBD region as the sequence for gene rearrangement and appropriately extends the RBD sequence to the CTD1 region (319-591aa), which can enhance the ability of the RBD monomer to stimulate the body to produce an immune response to a certain extent, thereby producing sufficient protective antibodies.

[0024] The membrane mutant antigen screening system of the present invention can express rearranged antigens on the cell membrane using membrane expression vectors, and perform high-throughput and rapid screening of antigens by flow cytometry, overcoming the problem of the existing technology that requires large-scale expression and purification of antigens before screening. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Schematic diagram of the nucleotides E08, C-11, 11-D, and 8-C obtained for DNA shuffling;

[0026] Figure 2 The amino acid sequence alignment results of candidate antigens 8-C and C-11 with the template antigen;

[0027] Figure 3 This is the map of the mutant membrane antigen expression plasmid;

[0028] Figure 4 The cross-binding activity results of S309 antibody and test serum with different VOCs RBD, where A: cross-binding of S309; ​​B: cross-binding of test serum;

[0029] Figure 5 Schematic diagram of flow cytometry analysis of membrane-displayed mutant antigens;

[0030] Figure 6 Statistical results of the binding data of rearranged antigen and S309 detected by flow cytometry, where A: Percentage of binding of rearranged antigen to S309; ​​B: Mean fluorescence intensity of binding of rearranged antigen to S309; ​​C: Median fluorescence intensity of binding of rearranged antigen to S309;

[0031] Figure 7 The data are statistical results of the binding of rearranged antigens and test serum by flow cytometry, where A: the percentage of binding of rearranged antigens to test serum; B: the mean fluorescence intensity of binding of rearranged antigens to test serum; C: the median fluorescence intensity of binding of rearranged antigens to test serum;

[0032] Figure 8 This is a schematic diagram of the electrophoresis results of flow cytometry screening of antigen proteins;

[0033] Figure 9 Statistical graphs of the binding activity between antigen and S309 antibody and test serum after flow cytometry screening, where A: Flow cytometry screening of the binding activity between antigen and S309 antibody; B: Flow cytometry screening of the binding activity between antigen and test serum;

[0034] Figure 10 Flow cytometry screening of the binding activity between antigen and ACE2 protein;

[0035] Figure 11 To screen the binding titer of serum from Bal b / c mice immunized with antigen by flow cytometry;

[0036] Figure 12 Flow cytometry screening of cross-binding activity between antigen immune serum and VOCs, including: A: comparison of cross-binding activity between 8-C and WT immune serum; B: comparison of cross-binding activity between C-11 and WT immune serum; C: comparison of cross-binding activity between 11-D and WT immune serum; D: comparison of cross-binding activity between E08 and WT immune serum;

[0037] Figure 13 The specific sera binding titers of Bal b / c mice immunized with aluminum adjuvant and CPG-1018 adjuvant for candidate antigens;

[0038] Figure 14 Comparison of the cross-binding activity of candidate antigens and WT antigen using aluminum adjuvant and CPG-1018 adjuvant, among which A: 8-C cross-binding activity with WT antigen immune serum; B: C-11 cross-binding activity with WT antigen immune serum;

[0039] Figure 15 It is the blocking effect of candidate antigen immune serum on the binding of VOCs to ACE2, among which A: the blocking effect of 8-C antigen immune serum on the binding of VOCs to ACE2; B: the blocking effect of C-11 antigen immune serum on the binding of VOCs to ACE2. DETAILED DESCRIPTION

[0040] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0041] The sequence information involved in the embodiments of the present invention is as follows:

[0042] SEQ ID NO.1

[0043] C-11:

[0044] RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWERKRISNCVADYSVLY

[0045] NSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIAD

[0046] YNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDIST

[0047] EIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHA

[0048] PATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIA

[0049] DTTDAVRDPQTLEILDITPCS

[0050] SEQ ID NO.2

[0051] 8-C:

[0052] ITNLCPFGEVFNATKFPSVYAWERKRISNCVADYSVLYNSTFFSTFKCYGV

[0053] SATKLNDLCFSNVYADSFVIRGDEVRQIAPGQTGVIADYNYKLPDDFTGC

[0054] VIAWNSNKLDSKPSGNYNYLYRLFRKSKLKPFERDISTEIYQAGNMPCNG

[0055] VAGPNCYSPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTN

[0056] LVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTL

[0057] EILDITPCS

[0058] SEQ ID NO.3

[0059] C-11:

[0060] AGAGTGCAGCCCACCGAGAGCATTGTGAGGTTTCCCAATATCACCAACCTGTGCCCCTTCGGCGAAGTGTTCAACGCCACCAGGTTTGCATCTGTCTATGCCTGGGAGAGGAAAAGGATCAGTAATTGCGTGGCAGACTACAGCGTGCTGTACAACTCCGCATCCTTCAGCACCTTTAAGTGCTATGGCGTGTCCCCCACCAAGCTGAACGACCTGTGTTTTACCAACGTGTACGCCGACTCCTTCGTGATTAGAGGCGACGAGGTGCGGCAGATCGCCCCCGGACAGACAGGCAACATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGCGTGATTGCCTGGAACTCCAACAATCTGGACTCTAAGGTGGGGGGGAACTACAACTACCTCTACAGACTGTTCAGGAAGTCCAACCTGAAGCCATTCGAACGGGACATCAGCACAGAGATTTACCAGGCTGGCAGCACACCATGTAATGGAGTGGAGGGATTTAACTGCTACTTCCCACTGCAGTCCTACGGCTTCCAGCCCACCAACGGCGTGGGATACCAGCCCTACCGGGT GGTGGTGCTGTCCTTCGAGCTGCTGCACGCACCCGCCACCGTGTGCGGACCTAAGAAGTCCACCAACCTGGTGAAAAACAAATGCGTGAACTTCAATTTTAACGGCCTGACCGGCACCGGCGTGCTGACCGAATCCAACAAAAAGTTCCTGCCCTTTCAGCAGTTCGGCAGAGACATCGCCGACACCACCGACGCCGTGAGGGACCCTCAGACCCTGGAGATCCTGGACATTACCCCCTGCTCC

[0061] SEQ ID NO.4

[0062] 8-C:

[0063] ATCACCAACCTGTGTCCATTTGGAGAGGTGTTCAATGCCACCAAGTTTCCATCTGTCTATGCCTGGGAGAGGAAGAGGATTAGCAACTGTGTGGCTGACTACTCTGTGCTCTACAACAGCACCTTCTTCAGCACCTTCAAGTGTTATGGAGTGTCTGCCACCAAACTGAATGACCTGTGTTTCAGCAATGTCTATGCTGACTCCTTTGTGATTAGGGGAGATGAGGTGAGACAGATTGCCCCTGGACAAACAGGAGTGATTGCTGACTACAACTACAAACTGCCTGATGACTTCACAGGCTGTGTGATTGCCTGGAACAGCAATAAACTGGATTCCAAACCTTCTGGTAACTACAACTACTTGTACCGGTTGTTCCGGAAGAGCAAACTGAAACCATTCGAGCGAGACATTTCCACTGAAATCTATCAGGCTGGTAATATGCCTTGCAATGGTGTTGCTGGTCCAAACTGTTACAGTCCACTGCAGTCTTACGGATTTCGGCCTACTTATGGCGTGGGACACCAGCCTTACAGGGTGGTGGTGCTGTCCTTTGAACTGCTCCATGCCCCTGCCACAGTGTGTGGACCAAAGAAGAGCACCAACTTGGTCAAGAACAAGTGTGTGAACTTTAATTTCAACGGACTGACTGGTACCGGCGTGTTGACCGAAAGCAACAAGAAGTTTCTGCCATTCCAGCAGTTCGGCAGAGACATCGCCGACACCACCGACGCCGTGCGGGACCCTCAGACCCTGGAGATTCTGGACATTACACCTTGCTCC

[0064] 实施例1利用DNA Shuffling技术制备SARS相关冠状病毒重排抗原库

[0065] Obtaining template antigen gene fragments: A total of 7 template antigens were selected for testing in this Example 1, namely the RBD and CTD1 sequences of SARS, SARS-CoV-2WT, Beta, Delta, Omicron BA.1, BF.7, and XBB.1.5. All template fragments were synthesized separately onto the pcDNA3.1(+) plasmid. The amplification primers for the template antigen gene fragments were homologous sequences outside the 150bp of the RBD and CTD1 sequences. The primer sequences are shown in Table 1. Vazyme 2×Phanta Flash Master Mix was used to amplify the RBD and CTD1 fragments. The fragment amplification system and reaction conditions are shown in Table 2. The template antigen gene fragments were purified and recovered by agarose gel electrophoresis to completely remove residual plasmid templates, primers, and non-specific amplification products. The concentration of the recovered product was determined as the template fragment used in the next step.

[0066] Table 1 PCR primers for DNA Shuffling template fragment amplification

[0067]

[0068] Table 2 DNA Shuffling fragment amplification conditions

[0069]

[0070] DNase I digestion of template fragments: The total amount of starting template fragments for the DNA shuffling experiment in Example 1 was 2-5 μg, with a 1:1 ratio between the seven homologous gene fragments. Using the Promega Corporation RQ1 RNase-Free DNase Kit (Cat. #M6101), DNase I was diluted to 0.1 U / μL. Reaction components were added in the order shown in Table 3:

[0071] Table 3 Template fragment enzyme digestion system

[0072]

[0073]

[0074] Mix thoroughly and gently by pipetting, then incubate at 37°C. After 2 and 4 minutes, sample 50 μL of the sample into a new PCR tube. Add 5 μL of Stop Solution and immediately incubate at 65°C for 10 minutes to inactivate DNase I. Purify the digested product by electrophoresis on a 2% agarose gel to recover a small fragment of approximately 50-100 bp. Measure the concentration of the recovered product and use it as the recovered fragment in the next step.

[0075] Primerless PCR: Add 0.5 μg of recovered fragment, 50 μL of Vasyme 2× Phanta Flash Master Mix, and 2 μL of DMSO to a PCR tube and make up to 100 μL with ddH2O. Perform primerless PCR according to the following PCR parameters: Step 1: 98°C for 30 s; Step 2: 98°C for 10 s, 60°C for 5 s, 57°C for 5 s, 54°C for 5 s, 51°C for 5 s, 48°C for 5 s, 45°C for 5 s, 42°C for 5 s, and 72°C for 5 s, for a total of 40 cycles; Step 3: 72°C for 1 min.

[0076] Primed PCR: The primerless PCR amplification product was diluted 100-fold and 200-fold, and used as the PCR template in this step, along with the original primerless PCR amplification product. The PCR amplification primers were homologous to sequences within 100 bp of the RBD and CTD1 ends of the pcDNA3.1(+) plasmid. The primer sequences are shown in Table 4. The fragment amplification system and reaction conditions are shown in Table 5.

[0077] Table 4 DNA Shuffling Primerless PCR Primers

[0078]

[0079] Table 5 DNA Shuffling Primer PCR Reaction System

[0080]

[0081]

[0082] The PCR product was subjected to agarose gel electrophoresis to recover a fragment of 819 bp in size. A (adenine) base was added to the 3' end of the recovered DNA fragment. The A addition reaction system was shown in Table 6 and the reaction was carried out at 72°C for 20 min. The A addition product was recovered from the gel and ligated to the pMD TM The reaction system for the 19-T vector was as shown in Table 7, and the reaction was carried out at 16°C for 30 minutes. The ligation product was transformed into competent E. coli cells. 20 μL of X-Gal and 10 μL of IPTG were applied to an ampicillin-resistant culture dish and incubated at room temperature for 30 minutes. The transformed bacterial solution was evenly spread on the dish and incubated at 37°C for 12-16 hours. White colonies were selected for sequencing.

[0083] Table 6 Reaction system for adding A to the end of recovered fragments

[0084]

[0085] Table 7 Blue-white spot screening system

[0086]

[0087]

[0088] Results: The sequencing results were compared with the template sequences using BioEdit software, and the fragments with complete and sufficient rearrangement were selected. A total of 42 rearranged antigens were obtained, named as: E-3, G12, E08, H02, G06, F-1, F01, E10, C06, C-11, F10, A01, E04, F02, C03, C-2, B10, D12, G-5, D10, 2-4, 2-5, 6-1, 8-8, 9-3, 9-4, 11-5, 10-H, 3-E, 5-B, 8-D, 1-C, 1-F, 12-C, 7-E, 4-H, 8-F, 4-G, 4-F, 2-B, 6-C, 11-D. First, the nucleotide sequence characteristics of the rearranged antigens were analyzed, for example, as shown in Figure 1 E08, C-11, 11-D and 8-C, it can be seen that the rearranged antigens are composed of nucleotide fragments of different sizes from different template antigens. Then, the amino acid composition of the rearranged antigens was analyzed, for example, as shown in 8-C and C-11, it can be seen that 8-C contains fragments of amino acid sites K15, P17, E23, T41, F42, A53, S62 and V86 from SARS, fragments of amino acid sites K109, P114, S115, K129, N146, A153, P155, S159 from XBB.1.5, and fragments of amino acid sites R167, Y170, H174 from B.1.1.529, BF.7 or XBB.1.5. In addition, the amino acid site M147 of the candidate universal antigen 8-C is a newly added mutation site compared with the 7 template antigens. And the C-11 antigen contains fragments of amino acid site E36 from SARS and fragments of amino acid site N99 from Beta, B.1.1.529, BF.7 or XBB.1.5 by rearrangement.

[0089] Example 2 Flow cytometry screening of rearranged antigens using mutant membrane antigen expression plasmid

[0090] The rearranged antigen sequences obtained by DNA Shuffling technology in Example 1 were constructed into the membrane mutant expression vector designed in the application, and the vector plasmid map is shown in Figure 3 The plasmid containing the rearranged antigen after construction was transfected into 293T cells, and the correct expression of the antigen was confirmed by Western Blot experiment using Anti-6xHis tag(HRP) antibody.

[0091] The S309 antibody used in the flow cytometry screening of rearranged antigens in this Example 2 is derived from the literature: [Pinto, D., Park, YJ., Beltramello, M. et al. Cross-neutralization of SARS-CoV-2 by a human monoclonal SARS-CoV antibody. Nature 583, 290-295 (2020).], which is derived from the convalescent serum of SARS-infected patients and has been shown to have neutralizing activity against SARS-CoV-2 and various mutant strains; this Example also uses a test serum to screen rearranged antigens, which is a serum that was used in the early laboratory to sequentially immunize Bal b / c mice with two injections of SARS-CoV-2 B.1.351 (S1-2P) and B.1.618 (S2-2P) proteins and one injection of SARS protein, and then immunized with one injection of Omicron BA.1 trimer antigen. The cross-binding activity of the S309 antibody and the test serum to VOCs was verified, and the results are as follows: Figure 4 As shown. The S309 antibody has high binding activity against SARS and SARS-CoV-2 WT, and also has some binding activity against other VOCs. However, it has no binding activity against the BA.1 antigen and has low binding activity against the BF.7 antigen. The test serum exhibits some binding to antigens from various mutant strains. The use of the S309 antibody and the test serum ensures effective cross-binding activity screening for rearranged antigens and VOCs.

[0092] In Example 2, rearranged antigens were screened by flow cytometry. The specific implementation method is as follows: (1) Cell collection: 293T cells were collected into 1.5 mL EP tubes after 48 hours of transfection, centrifuged at 4800 rpm for 3 minutes, and the supernatant was discarded. Then, 1 mL of PBS was added to the test serum tube and the cells were gently shaken to mix. After centrifugation at 4800 rpm for 3 minutes, the PBS was discarded and the washing was repeated 3 times. (2) Blocking nonspecific sites: 3% BSA was added to the washed cells at 200 μL / tube, gently shaken to mix, and incubated at 4°C for 1 hour. (3) Primary antibody incubation: The blocked cells were washed 3 times, and the antigen to be screened was diluted at a ratio of 1:250. The S309 antibody was diluted at a concentration of 10 μg / ml. After dilution, 100 μL / tube was added to the EP tube, gently shaken to mix, and incubated at 4°C for 1 hour. (4) Secondary antibody incubation: Wash the cells three times, add the fluorescently labeled APC-antiMouse IgG antibody corresponding to the test serum and the PE-antiHuman IgG antibody corresponding to the S309 antibody at a dilution ratio of 1:10, 50 μL / tube, gently shake and mix, and incubate at 4°C for 30 minutes. (5) Cell resuspension: Wash the cells three times, add 500 μL of PBS and resuspend. Filter the cells with a 200-mesh sieve to remove cell clumps. (6) Flow cytometer detection: Calibrate the optical and liquid paths of the flow cytometer to ensure that its CV value is less than 2%. Use the same type control tube to set the fluorescent staining positive and negative dividing line, adjust the fluorescence compensation, and then detect the unknown sample. During the detection process, adjust the voltage and fluorescence compensation according to the instrument analysis software, and read the number of particles in the cell population of 10,000 in each tube. (7) Data analysis: The flow cytometry gating strategy first removes cell debris and dead cells based on the size of forward scattered light (FSC) and side scattered light (SSC), and then gates based on FSC-A and FSC-H to remove adhesion bodies. In the experimental design, the pcDNA3.1 empty vector plasmid was transfected as a negative control. This vector should not bind to fluorescent IgG antibodies. On this basis, cells that bind to fluorescently labeled IgG antibodies were selected from the cells that removed adhesion bodies, and their percentages were divided. Take the flow cytometry detection graph of 8-C binding to S309 antibody as an example, as shown in Figure 2. Figure 5 The final test results are the percentage of cells within the cell population with a fluorescent signal, as well as the mean and median of the cell fluorescence intensity. Using the WT antigen test data as a benchmark, the data for other antigens were divided by the WT data to obtain the fold distribution data. A vertical distribution histogram was then created. The data was analyzed using Graphpad Prism 9.5 software to select rearranged antigens with strong binding ability.

[0093] Results: Four candidate antigens, E08, 11-D, C-11, and 8-C, were screened. These four antigens had strong binding to S309 antibody and test serum and were selected as candidate antigens. Specific statistical data are shown in the figure. Figure 6 and Figure 7 .

[0094] Example 3 Expression, purification and identification of flow cytometry screening antigens

[0095] After screening the high binding antigens by flow cytometry at the gene level, it is necessary to obtain the antigen protein for further verification. In this Example 3, the four antigen sequences screened in Example 2 were recombined into an expression vector with a secretory peptide, and after transfection into 293F, Ni was used to 2+ The antigens were purified by affinity chromatography, and the purity of the four antigen proteins was identified by SDS-PAGE and Coomassie brilliant blue staining.

[0096] Results: The protein molecular size of the candidate universal antigens is between 35-40kDa, close to the estimated molecular weight, and the purity of the purified protein is >95%, which can be used for subsequent screening and animal immunization. The purity of the candidate antigen protein is verified as follows Figure 8 shown.

[0097] Example 4 Detection of Binding Activity between Candidate Antigens and S309 Antibodies or Test Serum

[0098] The ability of the purified four antigens to bind to the antibodies used for screening antigens is a further verification of antigenicity. Therefore, in this Example 4, indirect ELISA was used to simultaneously test the binding activity of the candidate antigens and the seven template antigens with the S309 antibody and the test serum to compare whether the rearranged antigens can enhance the binding activity. The specific implementation method is as follows: (1) Antigen coating: The candidate antigen was diluted with PBS to a concentration of 2 μg / mL, and 100 μL per well was coated at 4°C overnight. (2) Blocking: The plate was washed 3 times with PBST (PBS containing 0.05% Tween 20), 100 μL of 3% BSA diluted with PBST was added to each well, and the plate was blocked at 37°C for 2 hours. (3) Sample addition: The plate was washed 3 times, and the test serum was diluted 3-fold with PBS starting from 1:200, and the S309 antibody was diluted 3-fold with 10 μg / mL. 100 μL / well was added to the ELISA plate and incubated at 37°C for 1 hour. (4) Detection antibody incubation: Wash the plate 5 times, dilute the enzyme-labeled secondary antibody with 1% BSA at a ratio of 1:10,000, add 100 μL / well to the plate, and incubate at 37°C for 1 hour. (5) Color development: Wash the plate 5 times, mix TMB color development solution A and solution B at a ratio of 1:1, add 100 μL to each well, and develop in the dark for 15 minutes. (6) Stop and read: Add 50 μL of stop solution to each well, read at 450 nm and 630 nm using a microplate reader, and analyze the data using Graphpad Prism 9.5 software.

[0099] Results: All four candidate antigen proteins had high binding activity with the screening S309 antibody and the test serum. The statistical results are shown in Figure 9 .

[0100] Example 5 Flow cytometry screening of antigen binding ability to ACE2

[0101] Screening of antigens can effectively bind to ACE2 receptor is another direction of antigenicity verification, this embodiment 5 uses flow cytometry to verify the binding ability of candidate antigens to ACE2 protein. The specific implementation is as follows, (1) cell preparation: 293T-ACE2 stable strain cell; (2) cell collection: collect the cells after 48h of subculture into 1.5ml EP tube, centrifuge at 4800rpm for 3min, then discard the supernatant, then add 1ml PBS to the tube and mix gently, centrifuge at 4800rpm for 3min, then discard the PBS, repeat the washing for 3 times; (3) primary antibody incubation: dilute the candidate antigen at a starting concentration of 5μg / ml with 3 times gradient, then add it into the EP tube, mix with equal volume of cells, then mix gently, incubate at 4℃ for 1h; (4) secondary antibody incubation: wash the cells after primary antibody incubation for 3 times, then add the APC-anti His antibody with fluorescence label at a dilution ratio of 1:50, 50μl / tube, mix gently, then incubate at 4℃ for 10min; (5) cell resuspension: add 500μl PBS to resuspend the incubated cells. Filter the cells with 200 mesh screen to remove cell clumps; (6) flow cytometry detection: calibrate the light path and liquid path of the flow cytometer, and determine that the CV value is less than 2%. Use the same type of control tube to set the fluorescence staining negative and positive boundary line, adjust the fluorescence compensation, then detect the unknown sample. During the detection process, adjust the voltage and fluorescence compensation according to the instrument analysis software, and read 10000 particles in each cell group; (7) data analysis: the final detection result of this instrument is the mean fluorescence intensity of the cells with fluorescence signal in the cell group.

[0102] Results: Four kinds of candidate antigen proteins can bind to ACE2 protein, and the binding ability is higher than that of WT antigen protein, the data statistical results are shown in Figure 10 , select E08, 11-D, C-11, 8-C candidate antigens for subsequent animal immunization.

[0103] Example 6 Screening of antigen immune serum titer detection

[0104] Immunogenicity screening of the four antigens screened is an important means to evaluate the effectiveness of candidate antigens. The detection of serum titer after immunization is a preliminary screening method of immunogenicity. In this embodiment 6, four candidate antigens and WT antigen were immunized in Bal b / c mice, and the specific scheme is as follows: using aluminum hydroxide adjuvant, the immunization method of hind leg muscle injection was used, the protein immunization dose of each mouse was 10 μg, and the physiological saline was used to make up to 25 μL, then mixed with the adjuvant 1:1 uniformly, and finally the injection dose of each mouse was 50 μL. The second immunization was carried out at an interval of 14 days, and the blood sampling for detecting the immune serum titer was carried out at 14 days after the second immunization. At the same time, 7 kinds of template antigen proteins of SARS, SARS-CoV-2 WT, Beta, Delta, BA.1, BF.7 and XBB.1.5 were used for cross-binding activity detection with immune serum. The specific implementation scheme is shown in the indirect ELISA detection method in Example 4.

[0105] Results: The average EC50 of C-11 and 8-C candidate antigen immunization groups were 4591 and 4312 respectively, which were 4.56 times and 4.27 times of the control group WT, respectively, which caused a strong immune response, and the data analysis and statistical results are shown in Figure 11 . At the same time, C-11 and 8-C candidate antigens also produced cross-binding activity, and the data analysis and statistical results are shown in Figure 12 .

[0106] Example 7 Serum titer detection after immunization of candidate antigens with aluminum adjuvant and CPG-1018 adjuvant

[0107] After comparison in Example 5, the serum titer and cross activity of 8-C and C-11 antigens compared with WT antigen were improved to a certain extent. In this embodiment, 8-C and C-11 antigens and WT antigen were immunized in Bal b / c mice using aluminum adjuvant and CPG adjuvant, and the specific scheme is as follows: using aluminum hydroxide adjuvant 25 μL, CPG-1018 adjuvant 10 ul, the antigen immunization dose of each mouse was 10 μg, and the physiological saline was used to make up to 15 μL, then mixed with the two kinds of adjuvants uniformly, and the immunization method of hind leg muscle injection was used, the injection dose of each mouse was 50 μL. The second immunization was carried out at an interval of 14 days, and the blood sampling for detecting the immune serum titer was carried out at 14 days after the second immunization. At the same time, 7 kinds of template antigen proteins of SARS, SARS-CoV-2 WT, Beta, Delta, BA.1, BF.7 and XBB.1.5 were used for cross-binding activity detection with immune serum. The specific implementation scheme is shown in the indirect ELISA detection method in Example 4.

[0108] Results: The serum titer after antigen immunization was significantly improved compared with the serum titer of aluminum adjuvant alone in Example Five, and the improvement of 8-C and C-11 antigens was more obvious, and the data analysis and statistical results are shown in Figure 13 The cross-binding activity of 8-C and C-11 antigens to VOCs after immunization was also analyzed, and the statistical results of the cross-binding activity improvement data are also available. Figure 14 .

[0109] Example 8 Verification of the Blocking Activity of Candidate Antigen Immune Serum on VOCs Binding to ACE2

[0110] Whether the serum antibodies produced after candidate antigen immunization can effectively block the binding of the virus to ACE2 can evaluate the effectiveness of the antigen. Therefore, this example uses the 8-C and C-11 antigen immune serum in Example 7 to detect the VOCs protein binding to ACE2 blocking activity. The specific implementation plan is as follows: (1) Cell preparation: 293T-ACE2 cells after 48 hours of passage were cultured at 1×10 6 The cells were divided into 1.5mL EP tubes and washed three times. (2) Incubation of VOCs protein with immune serum: RBD proteins of SARS and 6 SARS-CoV-2 VOC strains were pre-incubated with serum. In this example, the immune serum was diluted at 1:20, and the VOCs protein was diluted 2-fold starting from 4μg / mL, and 60μL of serum and protein were mixed. At the same time, unimmunized mouse serum was mixed with 60μL of each of the 7 VOCs proteins as a negative control and incubated at 4℃ for 40min. (3) Incubation of protein serum mixture with ACE2 cells: 100μL / tube of protein serum mixture was added to 293T-ACE2 cells, gently shaken to mix, and incubated at 4℃ for 1h. (4) Incubation with detection antibody: After incubation, the cells were washed three times, and the fluorescently labeled APC-anti His antibody was added at a dilution ratio of 1:20, 50μL / tube, gently shaken to mix, and incubated at 4℃ for 15min. (5) Cell resuspension and flow cytometry: After washing the cells three times, resuspend them in 500 μL of PBS and filter them with a 200-mesh sieve before flow cytometry. The mean fluorescence intensity was statistically analyzed to calculate the blocking rate of immune serum on the binding of various VOCs proteins to ACE2: blocking rate = (mean fluorescence intensity of negative control serum - mean fluorescence intensity of candidate antigen immune serum) / mean fluorescence intensity of negative control serum × 100%. The blocking rates of alloantigen immune serum were averaged for comparison, and the data were analyzed using Graphpad Prism 9.5 software.

[0111] Results: Both groups of immune sera showed that they could effectively block the binding of different variant proteins to ACE2-expressing cells. Among them, the 8-C immune serum not only showed high blocking activity against WT and Delta variants with a blocking rate of more than 50%, but also had certain blocking activity against the newer novel coronavirus strains Omicron series BF.7 and XBB.1.5 variants. The C-11 immune serum showed high blocking activity against SARS, WT, and Delta variants, and was also able to effectively block BF.7 and XBB.1.5 variants. The statistical results of the data analysis are shown in Figure 15 .

[0112] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A SARS-related coronavirus antigen C-11, characterized in that The amino acid sequence of the antigen C-11 is shown in SEQ ID NO.

1.

2. The gene encoding the SARS-related coronavirus antigen C-11 according to claim 1, characterized in that The nucleotide sequence of the coding gene is shown in SEQ ID NO.

3.

3. A SARS-related coronavirus candidate antigen 8-C, characterized in that The amino acid sequence of antigen 8-C is shown in SEQ ID NO.

2.

4. The gene encoding the SARS-related coronavirus antigen C-8 according to claim 3, characterized in that The nucleotide sequence of the coding gene is shown in SEQ ID NO.

4.

5. Use of the SARS-related coronavirus antigen C-11 according to claim 1, the SARS-related coronavirus candidate antigen 8-C according to claim 3, or the encoding gene according to claim 2 or 4 in the preparation of a SARS-related coronavirus vaccine.

6. Use of the SARS-associated coronavirus antigen C-11 according to claim 1, the SARS-associated coronavirus candidate antigen 8-C according to claim 3, or the encoding gene according to claim 2 or 4 in the preparation of SARS-associated coronavirus detection antibodies or therapeutic antibodies.

7. Use of the SARS-related coronavirus antigen C-11 according to claim 1, the SARS-related coronavirus candidate antigen 8-C according to claim 3, or the encoding gene according to claim 2 or 4 in the preparation of a SARS-related coronavirus diagnostic kit or diagnostic reagent.

8. A biological material related to the SARS-associated coronavirus antigen C-11 according to claim 1 or the SARS-associated coronavirus candidate antigen 8-C according to claim 3, characterized in that: The biological material is an expression cassette, a recombinant vector or a recombinant bacterium containing the coding gene according to claim 2 or 4.

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