General antigen for SARS (severe acute respiratory syndrome) related coronavirus as well as preparation method and application of general antigen

Abstract

The invention provides an SARS (severe acute respiratory syndrome) related coronavirus universal antigen as well as a preparation method and application thereof, and belongs to the technical field of immunity. The prepared candidate antigen has high binding activity to an S309 antibody and detected serum, and the cross binding activity of the titer of serum obtained by immunizing a mouse with the candidate antigen to VOCs is higher than that of template antigen immunized serum; meanwhile, the candidate antigen immune serum has the capability of blocking the combination of VOCs and ACE2. Therefore, the candidate antigen prepared by the invention can become a potential universal antigen or combination for preventing SARS-related coronavirus infection and inducing cross neutralization activity. The strategy of preparing candidate antigens with cross-neutralization immunogenicity based on DNA Shuffling and a membrane mutation antigen screening technology is expected to be applied to research and development of universal vaccines. The candidate universal antigen prepared by the invention has the potential of being developed into an 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 a SARS-related coronavirus and a preparation method and application thereof. Background Art

[0002] SARS-CoV and SARS-CoV-2 are both subtypes of the Coronavirus family and the genus Betacoronavirus, group B, and are collectively referred to as SARS-related coronaviruses. Both viruses can cause respiratory diseases, with the main symptoms being fever, fatigue, lung infection, and breathing difficulties. Severe cases can lead to death. SARS has a higher mortality rate, reaching 30%-40%.

[0003] The World Health Organization (WHO) has named some highly infective and 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 such as BF.7, and BA.2 has formed multiple evolutionary branches such as BA.2.75, BA.2.86, and XBB. With the continuous emergence of new variants of the novel coronavirus and the increasing immune escape phenomenon, the continued effectiveness of existing novel coronavirus vaccines and antibody drugs is facing severe challenges. Therefore, it is of great significance to develop an efficient and broad-spectrum coronavirus vaccine to resist the current epidemic and other coronaviruses that may appear 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. The sequence homology between the two is 73%, and it plays an important role in the process of virus adsorption and entry into host cells. The RBD region has rich antigenic epitopes. More than 90% of neutralizing antibodies target the RBD region. The induced neutralizing antibodies can block the binding of the virus to the angiotensin-converting enzyme 2 (ACE2) receptor, induce effective humoral and cellular immune responses, and produce immune protection responses. At the same time, compared with the full-length S protein, RBD can avoid the potential risk of antibody-dependent infection enhancement 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 ability of RBD monomers to stimulate the body to produce immune responses is weak, and it is difficult to produce sufficient protective antibodies using RBD monomers as antigens. However, the immune effect of RBD can be enhanced to a certain extent through some strategies, such as increasing the size of the antigen or polymerizing RBD.

[0005] With existing technologies, it is difficult for vaccines to produce effective broad-spectrum cross-recognition activity against constantly mutating coronaviruses, and this remains a problem to be solved. Summary of the invention

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

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

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

[0009] The present invention also provides a gene encoding the SARS-related coronavirus antigen C-11, the nucleotide sequence of which is shown in SEQ ID NO.3.

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

[0011] The present invention also provides a gene encoding the SARS-related coronavirus antigen C-8, characterized in that the nucleotide sequence of the encoding gene is shown in SEQ ID NO.4.

[0012] The present invention also provides the use of the above-mentioned SARS-related coronavirus antigen C-11, SARS-related coronavirus candidate antigen 8-C or encoding gene in the preparation of SARS coronavirus-related virus vaccines.

[0013] The present invention also provides the use of the above-mentioned SARS-related coronavirus antigen C-11, SARS-related coronavirus candidate antigen 8-C or encoding gene in the preparation of SARS-related coronavirus detection antibodies or therapeutic antibodies.

[0014] The present invention also provides the use of the above-mentioned SARS-related coronavirus antigen C-11, SARS-related coronavirus candidate antigen 8-C or encoding gene in developing a SARS-related coronavirus diagnostic kit or diagnostic reagent.

[0015] The present invention also provides biological materials related to the above-mentioned SARS-related coronavirus antigen C-11 or SARS-related coronavirus candidate antigen 8-C, wherein the biological materials are expression cassettes, recombinant vectors or recombinant bacteria containing the above-mentioned encoding genes.

[0016] The present invention also provides a method for preparing the above-mentioned SARS-related coronavirus antigen C-11 or the above-mentioned SARS-related coronavirus candidate antigen 8-C, comprising the following steps:

[0017] The template antigen gene fragments were obtained by PCR amplification, and were cut into small fragments of about 50-100 bp using DNase I. The small fragments were amplified by primer-free PCR using homologous regions as primers. Finally, the homologous fragments outside 100 bp at both ends of all template antigen sequences were used as primers for primer-loaded PCR to obtain rearranged antigens.

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

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

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

[0021] Beneficial effects of the present invention:

[0022] The present invention utilizes DNA Shuffling technology to effectively simulate virus 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 shortcoming of the prior art that vaccines can only be designed for 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, so as to produce sufficient protective antibodies.

[0024] The membrane mutant antigen screening system of the present invention can express rearranged antigens on the cell membrane using a membrane expression vector, and perform high-throughput and rapid screening of antigens by flow cytometry, thus overcoming the problem of the prior art that antigens need to be expressed and purified in large quantities before screening. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Schematic representation of the nucleotides of 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 mutant membrane antigen expression plasmid map;

[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 The data statistics of the binding of rearranged antigens to S309 detected by flow cytometry, where A: the binding percentage of rearranged antigens to S309; ​​B: the mean fluorescence intensity of the binding of rearranged antigens to S309; ​​C: the median fluorescence intensity of the binding of rearranged antigens to S309;

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

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

[0033] Fig. 9 It is a statistical diagram of the binding activity of flow cytometry screening antigens and S309 antibodies and test serum, where A: flow cytometry screening antigen and S309 antibody binding activity; B: flow cytometry screening antigen and test serum binding activity;

[0034] Fig.10 The results of flow cytometry screening of antigen-ACE2 protein binding activity;

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

[0036] Fig.12 The cross-binding activity between antigen immune serum and VOCs was screened by flow cytometry, 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] Fig.13 The specific sera binding titers of Bal b / c mice immunized with aluminum adjuvant and CPG-1018 adjuvant for candidate antigens;

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

[0039] Fig.15 It is the blocking effect of candidate antigen immune serum on the binding of VOCs to ACE2, where 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 in conjunction with the embodiments, but they should not be construed as limiting the protection scope 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] Example 1 Preparation of a SARS-related coronavirus rearranged antigen library using DNA Shuffling technology

[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 are homologous sequences outside 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 a template fragment for 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 template fragment: The total amount of the starting template fragment of the DNA Shuffling experiment in Example 1 is 2-5 μg, and the ratio between the 7 homologous gene fragments is 1:1. Use the Promega Corporation RQ1 RNase-FreeDNase (Cat.#M6101) kit to dilute DNase I to 0.1U / μL. Add the reaction components in the order of Table 3:

[0071] Table 3 Template fragment digestion system

[0072]

[0073]

[0074] After mixing thoroughly and gently, incubate at 37°C. Take 50 μL of the sample at 2 and 4 minutes into a new PCR tube, add 5 μL of Stop Solution and immediately incubate at 65°C for 10 minutes to inactivate DNase I. Perform agarose gel electrophoresis on the digested product in 2% agarose gel to purify and recover small fragments of about 50-100 bp. Determine the concentration of the recovered product to use as the recovered fragment in the next step.

[0075] Primer-free PCR: Add 0.5 μg of recovered fragment, 50 μL Vasyme 2× Phanta Flash Master Mix, 2 μL DMSO, and ddHO into a PCR tube. 2 The mixture was filled to 100 μL with 0.05% HO. Primer-free PCR was performed according to the following PCR reaction parameters: Program segment 1: 98°C for 30 s; Program segment 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, 72°C for 5 s, for a total of 40 cycles; Program segment 3: 72°C for 1 min.

[0076] Primed PCR: The primer-free PCR amplification product was diluted 100-fold and 200-fold, and the primer-free PCR amplification product stock solution was used as the PCR template in this step. The PCR amplification primers were homologous sequences 100 bp outside the ends of RBD and CTD1 on the pcDNA3.1(+) plasmid. The primer sequences are shown in Table 4, and 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, and an 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 by gel electrophoresis and ligated to pMD TM 19-T vector, the reaction system is shown in Table 7, and the reaction was carried out at 16°C for 30 min. The ligation product was transformed into E. coli competent cells, 20 μL X-Gal and 10 μL IPTG were applied to an ampicillin-resistant culture dish, and the culture was placed at room temperature for 30 min. The transformed bacterial solution was evenly applied to the culture dish, and cultured at 37°C for 12-16 h, and white colonies were selected for sequencing.

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

[0084]

[0085] Table 7 Blue-white spot screening system

[0086]

[0087]

[0088] Results: The sequencing results were compared with the template sequence using BioEdit software, and complete and fully rearranged fragments were selected. A total of 42 rearranged antigens were obtained, named as follows: 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, taking E08, C-11, 11-D and 8-C as examples. Figure 1 As shown, it can be clearly seen that the rearranged antigen is composed of nucleotide fragments of different sizes from different template antigens. Then the amino acid composition of the rearranged antigen was analyzed. Taking 8-C and C-11 as examples, it can be seen that the fragments of 8-C containing amino acid sites K15, P17, E23, T41, F42, A53, S62 and V86 are from SARS, the fragments containing amino acid sites K109, P114, S115, K129, N146, A153, P155, S159 are from XBB.1.5, and the fragments containing amino acid sites R167, Y170, H174 are 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 to the 7 template antigens. The C-11 antibody is rearranged so that the antigen contains a fragment of the amino acid site E36 from SARS, and a fragment of the amino acid site N99 from Beta, B.1.1.529, BF.7 or XBB.1.5.

[0089] Example 2 Screening of rearranged antigens by flow cytometry using mutant membrane antigen expression plasmids

[0090] The rearranged antigen sequence obtained by DNA Shuffling technology in Example 1 was constructed into the membrane mutation expression vector designed by the present invention. The vector plasmid map is shown in FIG. Figure 3 As shown, the constructed plasmid containing the rearranged antigen 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 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 proven to have neutralizing activity against SARS-CoV-2 and multiple mutant strains; this example also uses a test serum to screen rearranged antigens, which is a serum that was used 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 in the early laboratory, 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 to SARS and SARS-CoV-2WT, and also has some binding activity to other VOCs, but has no binding activity to BA.1 antigen, and has low binding activity to BF.7 antigen. The test serum has some binding to various mutant antigens. The use of S309 antibody and test serum can ensure effective cross-binding activity screening of rearranged antigens and VOCs.

[0092] In this Example 2, rearranged antigens were screened by flow cytometry. The specific implementation method is as follows: (1) Cell collection: 293T cells were collected into a 1.5 mL EP tube after transfection for 48 hours, centrifuged at 4800 rpm for 3 minutes, and the supernatant was discarded. Then 1 mL PBS was added to the test serum tube and the cells were gently shaken and mixed. After centrifugation at 4800 rpm for 3 minutes, the PBS was discarded and the washing was repeated 3 times. (2) Blocking non-specific sites: 3% BSA was added to the washed cells, 200 μL / tube, gently shaken and mixed, and incubated at 4°C for 1 hour. (3) Primary antibody incubation: The blocked cells were washed 3 times, and the antigen used for screening 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 and mixed, and incubated at 4°C for 1 hour. (4) Secondary antibody incubation: Wash the cells three times, add the APC-antiMouse IgG antibody corresponding to the fluorescently labeled detection serum and the PE-anti Human IgG antibody corresponding to the S309 antibody at a dilution ratio of 1:10, 50μL / tube, gently shake and mix, and incubate at 4℃ for 30min. (5) Cell resuspension: Wash the cells three times, add 500μL PBS and resuspend. Filter the cells with a 200-mesh screen to remove cell clumps. (6) Flow cytometer detection: Calibrate the optical path and liquid path of the flow cytometer to determine 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 10,000 particles in the cell population 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 are selected from the cells that have removed adhesion bodies, and their percentages are divided. Take the flow cytometry detection diagram of 8-C binding to S309 antibody as an example, as shown in Figure 1. Figure 5 As shown. The final test results are the percentage of cells with fluorescent signals in the cell population, the mean and median of the cell fluorescence intensity. Taking the WT antigen test result data as the benchmark 1, the data of other antigens are divided by the WT data to obtain the multiple distribution data and then make a vertical distribution bar graph. Graphpad Prism 9.5 software is used to analyze the data and 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. The specific data statistics are shown in Figure 6 and Figure 7 .

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

[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 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 35-40 kDa, 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 of Candidate Antigens with S309 Antibodies or Test Serum

[0098] The fact that the purified four antigens can bind to the antibodies used to screen the antigens is a further verification of the antigenicity. Therefore, in this Example 4, indirect ELISA is used to detect the binding activity of the candidate antigens and the seven template antigens with the S309 antibody and the test serum at the same time, so as to compare whether the rearranged antigens can enhance the binding activity. The specific implementation method is as follows: (1) Antigen coating: dilute the candidate antigen with PBS to a concentration of 2 μg / mL, and coat the plate with 100 μL per well at 4°C overnight. (2) Blocking: wash the plate 3 times with PBST (PBS containing 0.05% Tween 20), add 100 μL of 3% BSA diluted with PBST to each well, and block at 37°C for 2h. (3) Sample addition: wash the plate 3 times, dilute the test serum with PBS at a 1:200 dilution and 3-fold gradient dilution, and dilute the S309 antibody at a concentration of 10 μg / mL and 3-fold gradient dilution, 100 μL / well is added to the ELISA plate, and incubate at 37°C for 1h. (4) Detection antibody incubation: Wash the plate 5 times, dilute the enzyme-labeled secondary antibody with 1% BSA at a ratio of 1:10000, 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 color for 15 minutes in the dark. (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 have high binding activity with the screening S309 antibody and the test serum. The statistical results are shown in Fig. 9 .

[0100] Example 5 Flow cytometry screening of antigens and ACE2 binding ability detection

[0101] Screening antigens that can effectively bind to the ACE2 receptor is another direction of antigenicity verification. In this Example 5, flow cytometry was used to verify the binding ability of candidate antigens to the ACE2 protein. The specific implementation method is as follows: (1) Cell preparation: 293T-ACE2 stable transfected cells; (2) Cell collection: Collect the cells after 48 hours of passage into a 1.5 ml EP tube, centrifuge at 4800 rpm for 3 minutes, discard the supernatant, then add 1 ml PBS to the tube and gently shake the cells to mix, centrifuge at 4800 rpm for 3 minutes, discard the PBS, and repeat washing 3 times; (3) Primary antibody incubation: The candidate antigen is diluted in a gradient ratio of 3 times the starting concentration of 5 μg / ml, and after dilution, it is added to the EP tube, 100 μl / tube, mixed with an equal volume of cells, and gently shaken to mix, and incubated at 4°C for 1 hour; (4) Secondary antibody incubation: After the cells are washed three times after primary antibody incubation, the fluorescently labeled APC-anti His antibody is added at a dilution ratio of 1:50, 50 μl / tube, and gently shaken to mix, and incubated at 4°C for 10 minutes; (5) Cell resuspension: After the incubated cells are washed, 500 μl of PBS is added and resuspended. Filter the cells with a 200-mesh screen to remove cell clumps; (6) Flow cytometer detection: Calibrate the optical and liquid paths of the flow cytometer to determine that its CV value is less than 2%. Use the same type control tube to set the fluorescent staining positive and negative boundary, adjust the fluorescence compensation, and then detect unknown samples. During the detection process, adjust the voltage and fluorescence compensation according to the instrument analysis software, and read 10,000 particles in each cell group; (7) Data analysis: The final detection result obtained by this instrument is the average fluorescence intensity of cells with fluorescent signals in the cell group.

[0102] Results: All four candidate antigen proteins can bind to ACE2 protein, and their binding ability is higher than that of WT antigen protein. The statistical results are shown in Fig.10 , E08, 11-D, C-11, and 8-C candidate antigens were selected for subsequent animal immunization.

[0103] Example 6 Screening Antigen Immune Serum Titer Detection

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

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

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

[0107] After comparison in Example 5, the serum titer and cross-activity of 8-C and C-11 antigens have been improved to a certain extent compared with those of WT antigens. In this embodiment, aluminum adjuvants and CPG adjuvants were used to immunize Bal b / c mice with 8-C and C-11 antigens and WT antigens. The specific scheme is as follows: 25 μL of aluminum hydroxide adjuvant and 10 ul of CPG-1018 adjuvant were used, and the antigen immunization dose for each mouse was 10 μg. The antigen was fixed to 15 μL with normal saline and then fully mixed with the two adjuvants. The immunization method was hind leg muscle injection, and the injection dose for each mouse was 50 μL. The second immunization was performed at an interval of 14 days, and blood was collected 14 days after the second immunization to detect the titer of the immune serum. At the same time, a total of 7 template antigen proteins, including SARS, SARS-CoV-2WT, Beta, Delta, BA.1, BF.7 and XBB.1.5, were used to detect cross-binding activity 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 higher than that of the serum titer using aluminum adjuvant alone in Example 5, and the increase was more obvious for 8-C and C-11 antigens. The specific statistical results of data analysis are shown in Fig.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. Fig.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 blocking activity of VOCs protein binding to ACE2. 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 from 4μg / mL, and 60μL of serum and protein were mixed. At the same time, non-immune mouse serum was mixed with 60μL of each of the 7 VOCs proteins as a negative control and incubated at 4℃ for 40min. (3) Co-incubation of protein serum mixture with ACE2 cells: 100μL / tube of protein serum mixture was added to 293T-ACE2 cells, gently shaken and mixed, and incubated at 4℃ for 1h. (4) Incubation with detection antibody: After the cells were washed three times after incubation, the fluorescently labeled APC-anti His antibody was added at a dilution ratio of 1:20, 50μL / tube, gently shaken and mixed, and incubated at 4℃ for 15min. (5) Cell resuspension and flow cytometry: After washing the cells three times, add 500 μL of PBS to resuspend and filter with a 200-mesh screen for flow cytometry detection. The mean fluorescence intensity was statistically analyzed to calculate the blocking rate of immune serum on the binding of various VOCs proteins to ACE 2. 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 epidemic strains, the BF.7 and XBB.1.5 variants of the Omicron series. The C-11 immune serum showed high blocking activity against SARS, WT, and Delta variants, and was also able to effectively block the BF.7 and XBB.1.5 variants. The statistical results of the data analysis are shown in Fig.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 principle 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 coding gene of 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-associated 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 described in claim 1, the SARS-related coronavirus candidate antigen 8-C described in claim 3, or the encoding gene described in claim 2 or 4 in the preparation of a SARS coronavirus-related virus vaccine.

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

7. Use of the SARS-related coronavirus antigen C-11 described in claim 1, the SARS-related coronavirus candidate antigen 8-C described in claim 3, or the encoding gene described in claim 2 or 4 in developing 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.

9. A method for preparing 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 following steps are involved: The template antigen gene fragments were obtained by PCR amplification, and were cut into small fragments of about 50-100 bp using DNase I. The small fragments were amplified by primer-free PCR using homologous regions as primers. Finally, the homologous fragments outside 100 bp at both ends of all template antigen sequences were used as primers for primer-loaded PCR to obtain rearranged antigens. The gene fragment of the rearranged antigen is constructed into a membrane expression vector by homologous recombination method to form a recombinant membrane mutant antigen plasmid, the recombinant membrane mutant antigen plasmid is transfected into 293T cells, antibodies or test serum with broad-spectrum cross-binding activity are selected to screen the rearranged antigen, and the antigen is screened by comprehensively comparing the three groups of data of binding percentage, mean and median of fluorescence intensity; The antigen gene fragment is constructed onto a secretory vector by homologous recombination to obtain a recombinant secretory antigen plasmid. After the recombinant secretory antigen plasmid is transfected into the target cells, the antigen is purified.

10. The preparation method according to claim 9, characterized in that: The secretory vector uses pcDNA3.1 as the starting vector, the first 15 amino acids of the wild-type S protein of SARS-CoV-2Omicron as the signal peptide, and contains 62 amino acids in the C-terminal transmembrane region and a 6×His tag.

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