A novel coronavirus s protein specific b cell antigen epitope polypeptide and application thereof
By synthesizing and modifying a novel coronavirus S protein-specific B cell antigen epitope peptide, the difficulties in detection and prevention caused by immune escape mutations in novel coronavirus variants have been overcome, achieving highly efficient detection and immune protection.
Patent Information
- Application Number
- CN202310202291.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-03-06
- Publication Date
- 2026-03-17
- Estimated Expiration
- 2043-03-06
AI Technical Summary
Existing vaccines and therapeutic antibodies have reduced immune efficacy against variants of the novel coronavirus due to immune escape mutations, making it difficult to effectively detect and prevent novel coronavirus infection.
We designed and synthesized a novel coronavirus S protein-specific B cell antigen epitope peptide, formed a fusion sequence through chemical modification and coupling with a carrier protein, and used recombinant vectors and recombinant microbial cells for expression to prepare diagnostic kits and vaccines, thereby enhancing detection and immunization effects.
It improves the specificity and accuracy of the test, reduces the false positive rate, enhances the immune induction ability of the vaccine, and can effectively detect and prevent novel coronavirus infection.
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Figure CN116410272B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of molecular immunology technology, specifically relating to a novel coronavirus S protein-specific B cell antigenic epitope polypeptide and its application. Background Technology
[0002] The novel coronavirus (SARS-CoV-2) is an RNA virus with a high mutation rate. From the initial outbreak strain to the present, hundreds of variant strains have emerged, including dozens identified by the World Health Organization as VOCs (variants of concern). These mutations alter the transmission and infection characteristics of the virus, particularly the mutation of certain important antigenic epitopes, affecting the efficacy of existing vaccines and, in particular, the therapeutic effects of certain epitope-specific therapeutic antibodies. This immune escape mutation of the virus poses a significant challenge to vaccine and therapeutic antibody research. The preparation of monoclonal antibodies with broad-spectrum neutralizing activity and the identification of highly conserved neutralizing antigenic epitopes provide an important theoretical basis for the development of broad-spectrum protective vaccines and therapeutic antibodies. This invention focuses on the antigenic epitope research of a monoclonal antibody with broad-spectrum neutralizing activity targeting the S protein (RBD domain), obtaining a highly conserved antigenic epitope. Furthermore, this invention illustrates the application of conserved neutralizing antigenic epitopes in diagnostic and vaccine research. Summary of the Invention
[0003] The purpose of this invention is to provide a novel coronavirus S protein polypeptide antigen that has immune protection function or serological diagnostic value.
[0004] This invention provides a novel coronavirus S protein-specific B cell antigenic epitope polypeptide, the amino acid sequence of which is shown in SEQ ID NO:1 or SEQ ID NO:2.
[0005] Further specified, chemical modifications are made to the C-terminus and / or N-terminus of the amino acid sequence shown in SEQ ID NO.1 or SEQ ID NO.2.
[0006] Further specifying, the C-terminus is chemically modified by carboxylation or amidation; the N-terminus is chemically modified by aminoation or acetylation.
[0007] Further specifying, the amino acid sequence shown in SEQ ID NO.1 or SEQ ID NO.2 is linked with the coupling protein KLH, BSA or OVA to form a fusion sequence.
[0008] Further specifying, the amino acid sequence shown in SEQ ID NO.1 or SEQ ID NO.2 utilizes cysteine to form a cyclic peptide.
[0009] This invention provides a gene encoding the above-mentioned novel coronavirus S protein-specific B cell antigenic epitope polypeptide.
[0010] The present invention provides a recombinant vector carrying the above-mentioned coding gene.
[0011] The present invention provides a recombinant microbial cell carrying the above-mentioned gene or the above-mentioned antigenic epitope polypeptide.
[0012] This invention provides a novel coronavirus vaccine, the protein sequence of which is shown in SEQ ID NO.4.
[0013] The present invention provides the use of the above-mentioned antigenic epitope polypeptide, the above-mentioned encoding gene, the above-mentioned recombinant vector or the above-mentioned recombinant microbial cell in the preparation of a kit for diagnosing or detecting the novel coronavirus or an antibody against the novel coronavirus S protein, or in the preparation of a vaccine for the prevention or treatment of novel coronavirus infection.
[0014] Beneficial effects: 1. Using the novel coronavirus S protein-specific B cell antigen epitope polypeptide or polypeptide-carrier protein conjugate of the present invention as an antigen can detect novel coronavirus antibodies or anti-novel coronavirus polypeptide antibodies.
[0015] 2. The novel coronavirus S protein-specific B cell antigen epitope polypeptide of the present invention has stronger specificity and can reduce the false positive rate of detection results.
[0016] 3. The novel coronavirus S protein-specific B-cell antigenic epitope or its conjugate of the present invention, as an antigen, has higher antigen purity and can overcome the non-specific reactions caused by residual bacterial proteins in the purification of genetically engineered antigens.
[0017] 4. The novel coronavirus S protein-specific B-cell antigen epitope or its conjugate of the present invention can be used as an antigen to detect SARS-CoV-2 antibodies or anti-novel coronavirus polypeptide antibodies. The produced antigen can be automated, ensuring consistent quality and more stable detection results.
[0018] 5. The novel coronavirus S protein antigenic epitope polypeptide of the present invention, when coupled with a carrier protein or fused with other antigen proteins for expression, can be used as a vaccine antigen to induce the production of novel coronavirus polypeptide antibodies in immunized pigs. Attached Figure Description
[0019] Figure 1 Electrophoretic analysis for the expression and purification of the novel coronavirus (SARS-CoV-2) RBD protein.
[0020] Figure 2Electrophoretic analysis results of RBD short peptide fusion expressed proteins. M is the protein molecular weight standard; 1-14 are short peptide fusion proteins of MBP-P1 to MBP-P14; 15 is MBP protein expressed by empty vector.
[0021] Figure 3 The image shows the results of the Western blot reaction between the RBD peptide fusion protein and the monoclonal antibody. M is the protein molecular weight standard; 1-14 are short peptide fusion proteins from MBP-P1 to MBP-P14; 15 is the empty vector expression of MBP protein.
[0022] Figure 4 Electrophoretic analysis results of expression and carrier protein expression and purification for peptide vaccine preparation. 1 is the protein molecular weight standard; 2 is the purified epitope protein EP-SpyTag; 3 and 4 are the carrier protein expression samples before and after purification, respectively. Detailed Implementation
[0023] Example 1. Eukaryotic expression and purification of the novel coronavirus (SARS-CoV-2) RBD protein
[0024] Referring to the S gene sequence of the SARS-CoV-2 Omicron mutant strain (GISAID database gene sequence number: EPI_ISL_7160037), the gene sequence encoding the RBD region was codon-optimized. A signal peptide gene sequence (atgaagttctcctgggtgatgttcttcctgatggccgtggtgaccggcgtgaactcc) was added to the 5' end, and two 6×His tag coding sequences were added to the 3' end to form an expression cassette. The optimized expression cassette gene sequence is shown in SEQ ID NO.3, and the codon-optimized gene sequence is named opti-O-RBD. The opti-O-RBD gene was synthesized by a biotechnology company and cloned into the eukaryotic expression vector pCAGneo (Hua et al., 2014. Generation and characterization of a new mammalian cell line continuously expressing virus-like particles of Japanese encephalitis virus for a subunit vaccine candidate. DOI:10.1186 / 1472-6750-14-62) between the SacI and BglII restriction sites to construct the recombinant expression plasmid pCAG-opti-O-RBD. The recombinant plasmid pCAG-opti-O-RBD was transfected into BHK-21 cells. After selection culture in G418 medium, a stable expression cell line B-ORBD was obtained. After expansion culture of the stable expression cell line, the cell culture supernatant was harvested. The cell expression supernatant was purified by affinity chromatography using Ni-NTA resin. SDS-PAGE electrophoresis analysis of the purified RBD protein showed that the purified S protein (…) was obtained. Figure 1 ).
[0025] Example 2. Replication and preparation of S protein monoclonal antibody
[0026] Using the recombinant protein RBD obtained in Example 1 as an immunogen, 6-week-old BALB / c mice were immunized. Hybridoma fusion of splenic lymphocytes was performed, and after limiting dilution cloning, a hybridoma cell line secreting specifically targeting the SARS-CoV-2 RBD protein was obtained, named 22.9-1. This monoclonal antibody was identified as IgG1 / κ. Western blot analysis showed that all monoclonal antibodies specifically reacted with the SARS-CoV-2 RBD protein, and indirect immunofluorescence assays also showed that all monoclonal antibodies could recognize the SARS-CoV-2 RBD protein expressed by the cells. This also indicates that the antigenic epitope recognized by this monoclonal antibody is a linear antigenic epitope located on the surface of the S protein, and its antigenic epitope sequence can be further identified.
[0027] Example 3. Screening and Identification of Antigenic Epitopes
[0028] A series of SARS-CoV-2 RBD protein antigenic peptide fragments were expressed using partially overlapping peptide fusion, and indirect ELISA was used to screen monoclonal antibodies for antigenic epitopes. Based on the SARS-CoV-2 RBD protein amino acid sequence, a series of peptides were designed that overlapped with each other and covered the full length of the SARS-CoV-2 RBD protein. Based on each peptide amino acid sequence (overlapping peptide sequence), a pair of DNA strands were designed and synthesized. These DNA strands were inserted into the expression vector pMAL-c5X and fused with MBP protein for expression. SDS-PAGE electrophoresis analysis of the fused short peptides showed that all peptides were expressed. Figure 2 The reactivity of the fusion-expressed recombinant proteins with SARS-CoV-2 RBD-specific monoclonal antibodies was analyzed to identify their linear antigenic epitopes. The specific procedure is as follows:
[0029] Design of peptide sequences → Synthesis of DNA strands encoding the peptide sequences → Construction of peptide fusion expression vectors → Induction of expression → Coating of fusion protein onto ELISA plates → Immunoreactivity assay with monoclonal antibodies → Analysis of SARS-CoV-2S protein-specific antigenic epitopes. Overlapping peptide sequences and ELISA results are shown in Table 1. Further Western blot analysis of the fusion expression peptide with monoclonal antibodies was performed for validation, and the results are as follows: Figure 3 As shown, the P6 peptide fusion protein can be specifically recognized by monoclonal antibodies.
[0030] Table 1. ELISA results of the overlapping polypeptide fusion protein reacting with SARS-CoV-2RBD-specific monoclonal antibody.
[0031] polypeptide number Peptide position (AA) polypeptide sequence OD450 P1 1-30 VQPTESIVRFPNITNLCPFDEVFNATRFAS 0.081 P2 16-45 LCPFDEVFNATRFASVYAWNRKRISNCVAD 0.077 P3 31-60 VYAWNRKRISNCVADYSVLYNLAPFFTFKC 0.071 P4 46-75 YSVLYNLAPFFTFKCYGVSPTKLNDLCFTN 0.117 P5 61-90 YGVSPTKLNDLCFTNVYADSFVIRGDEVRQ 0.095 P6 76-105 VYADSFVIRGDEVRQIAPGQTGNIADYNYK 3.137 P7 91-120 IAPGQTGNIADYNYKLPDDFTGCVIAWNSN 0.145 P8 106-135 LPDDFTGCVIAWNSNKLDSKVSGNYNYLYR 0.143 P9 121-150 KLDSKVSGNYNYLYRLFRKSNLKPFERDIS 0.081 P10 136-165 LFRKSNLKPFERDISTEIYQAGNKPCNGVA 0.082 P11 151-180 TEIYQAGNKPCNGVAGFNCYFPLRSYSFRP 0.143 P12 166-195 GFNCYFPLRSYSFRPTYGVGHQPYRVVVLS 0.151 P13 181-210 TYGVGHQPYRVVVLSFELLHAPATVCGPKK 0.096 P14 196-221 FELLHAPATVCGPKKSTNLVKNKCVN 0.075
[0032] Note: The coating antigen is a fusion expression protein of a polypeptide and MBP, and the polypeptide sequences are shown in the table. The OD value is the average OD value of the three replicate wells.
[0033] The above experiments show that the linear antigenic epitopes recognized by this monoclonal antibody are mainly concentrated between amino acid residues 76-105. Further N-terminal and C-terminal deletion mutations were performed on the region recognized by the monoclonal antibody, followed by fusion expression with MBP. The expressed fusion proteins were then subjected to ELISA to accurately locate the antigenic epitopes. The deleted mutant peptide sequence was fused with MBP protein for expression, and then subjected to ELISA with the monoclonal antibody. The results of the reaction between monoclonal antibody 22.9-1 and the mutant peptide fusion protein are shown in Table 2. The results indicate that the core sequence of the antigenic epitope recognized by the monoclonal antibody is... D EV R QIAPGQ. This dominant epitope region has the potential to be used as a diagnostic antigen and to induce the body to produce immune antibodies.
[0034] Table 2. Results of ELISA reaction between truncated epitope peptide fusion protein and SARS-CoV-2 RBD-specific monoclonal antibody 22.9-1
[0035]
[0036]
[0037] Note: The coating antigen is a fusion expression protein of a polypeptide and MBP, and the polypeptide sequences are shown in the table. The OD value is the average OD value of the three replicate wells.
[0038] Mutation analysis of antigenic epitopes
[0039] Because there are different mutant strains of the novel coronavirus, in order to analyze whether the antigenic epitope identified in this invention is conserved, the S protein sequences of all VOC variants published by the WHO and those recently circulating in my country and around the world were compared. It was found that the antigenic epitope identified in this invention has only 4 mutations in all strains, corresponding to mutations at positions 405 and 408 in the original strain S protein sequence. Moreover, the sequences of all current VOC strains are highly consistent. This invention further fused the mutant epitope peptide for expression. The expressed mutant epitope peptide was analyzed by ELISA with the neutralizing monoclonal antibody 22.9-1. The results are shown in Table 3, indicating that neutralizing the two mutations in the epitope does not affect the reactivity of the neutralizing monoclonal antibody. Therefore, the neutralizing epitope targeted by this monoclonal antibody is highly conserved. This further indicates that the neutralizing antigenic epitope sequence is D(N)EVR(S)QIAPGQ.
[0040] Table 3. Results of ELISA reaction between truncated epitope peptide fusion protein and SARS-CoV-2K205R-specific monoclonal antibody pK205R-10
[0041]
[0042] Note: The coating antigen is a fusion expression protein of a polypeptide and MBP, and the polypeptide sequences are shown in the table. The OD value is the average OD value of the three replicate wells.
[0043] Example 4. Synthesis of B-cell antigenic epitope polypeptide of SARS-CoV-2S protein.
[0044] 1. Based on the results of antigen epitope screening and identification in Example 3, the amino acid sequence of the K205R-specific B-cell antigen epitope was synthesized using solid-phase peptide synthesis technology. To ensure sufficient exposure of the antigen epitope antibody binding site, the core epitope sequence was extended by 1 to 2 amino acid residues towards the N-terminus and C-terminus, respectively. The epitope sequence GDEVRQIAPGQTG was synthesized using an automated peptide synthesizer or artificial peptide synthesis method. The solid-phase resin used was Fmoc-protected amino acid Wang resin or other resins. After the resin was swollen with DMF and protected with pipeidine gel Fmoc, Fmoc-protected amino acids were added according to the amino acid sequence, and acylation was performed in the presence of HBTU. After acylation, the mixture was washed, and then the second Fmoc-amino acid was added for acylation, followed by washing. This process was repeated, starting from the C-terminus of the peptide sequence and proceeding sequentially to the N-terminus to synthesize a complete peptide chain. After synthesis, appropriate reagents were selected based on the different amino acids in the peptide chain, and the peptide chain was cleaved using the TFA method. The TFA peptide was precipitated with cold diethyl ether, purified by desalting, and identified by LC-MS and HPLC analysis before use. Throughout the synthesis process, the completion of each amino acid acylation reaction can be tested using the Kaiser method or TMBS method. To facilitate the linkage between the epitope peptide and the carrier protein, a cysteine residue can be added to the N-terminus of the peptide sequence during peptide synthesis. Based on the solid-phase peptide synthesis technique described above, the antigenic epitope peptide shown in SEQ NO.1 was synthesized.
[0045] 2. The gene sequence encoding the S protein antigenic epitope polypeptide is shown in SEQ ID NO.4. The sequence shown in SEQ ID NO.4 is inserted into any one of the pET series, Duet series, pGEX series, pHY300, pHY300PLK, pPIC3K or pPIC9K series to obtain a recombinant vector. The antigenic epitope polypeptide shown in SEQ NO.1 is obtained by expressing the recombinant vector.
[0046] Example 5. Chemical modification product of the B-cell antigenic epitope polypeptide of the novel coronavirus SARS-CoV-2S protein.
[0047] The polypeptide fragment (SEQ NO.1) synthesized according to the method of Example 4 was modified with a natural amino group at the N-terminus and a natural carboxyl group at the C-terminus.
[0048] N-terminal acetylation modification method: Following the peptide synthesis in Example 4 until the coupling of the last Fmoc protecting amino acid, the N-terminal Fmoc protecting group was removed, followed by the following steps: 150 μl of acetic anhydride and 20 μl of LEIPEA were dissolved in 4.8 ml of DMF, thoroughly mixed, and cooled in an ice bath. The mixture was then added to the peptide synthesis reaction tube and reacted for 5 min. The mixture was then washed twice, successively with 5 ml of DMF and dichloromethane (DCM), 5 min each time. After drying under nitrogen, the side chain protecting groups were removed, cleaved, purified, and identified using conventional methods.
[0049] C-terminal amidation modification method: Rink resin should be used for the synthesis of C-terminal amidated peptides. When executing the synthesis program, the resin should be swollen with DMF for 20 min, and then the synthesis program should be executed starting from the first amino acid at the C-terminus, similar to the previous method using Fmoc-wang resin.
[0050] The product obtained by modifying the C-terminus of the amino acid sequence shown in SEQ ID NO.1 to carboxylation or amidation was obtained using the above method;
[0051] The N-terminus of the amino acid sequence shown in SEQ ID NO.1 is chemically modified to produce an amino or acetylated product.
[0052] The product is obtained by chemically modifying the N-terminus of the amino acid sequence shown in SEQ ID NO.1 to amino or acetylate, and by chemically modifying the C-terminus to carboxylate or amidate.
[0053] Obtaining the modified product increases the stability of the antigen, and in applications detecting antibodies against SARS-CoV-2S protein, the results are more stable than those using only the unmodified peptide with the amino acid sequence shown in SEQ ID NO. 1. The modified product, when used to make a vaccine, has comparable therapeutic and preventative efficacy against novel coronavirus infection to the vaccine made with the amino acid sequence shown in SEQ ID NO. 1.
[0054] Example 6. Conjugation products of the B-cell antigenic epitope polypeptide of the novel coronavirus SARS-CoV-2S protein with carrier proteins KLH and BSA
[0055] 4 mg of carrier protein KLH or BSA was dissolved in 0.5 ml of PBS (pH 7.2) buffer containing 5 mM EDTA, and 1.0 mg of Sulfo-SMCC linker was added. After thorough dissolution, the solution was incubated at room temperature for 60 min or at 37°C for 30 min. The Sulfo-SMCC-treated carrier protein was desalted and purified using a Sephadex G-25 column or dialysis, and the protein content was determined by the BCA method before use.
[0056] Weigh 4 mg of the epitope polypeptide synthesized in Example 4, add 0.5 ml of double-distilled water to dissolve the polypeptide, then mix the dissolved polypeptide with the carrier protein treated with Sulfo-SMCC, incubate at 4°C for 2 hours or react overnight, dispense for use, and obtain the fusion protein of the antigen epitope polypeptide coupled with the carrier protein KLH shown in SEQ NO.1 and the fusion protein of the antigen epitope polypeptide coupled with the carrier protein BSA shown in SEQ NO.1.
[0057] The antigenic epitope polypeptide shown in SEQ NO.1 was linked to the carrier protein OVA to obtain a fusion protein.
[0058] The three fusion proteins described above, when used as antigens in vaccines, exhibit a stronger antibody-inducing effect than the antigenic epitope peptide shown in SEQ NO. 1. In applications for detecting antibodies against SARS-CoV-2S protein, the detection results are comparable in stability and accuracy to those using only the amino acid sequence shown in SEQ ID NO. 1.
[0059] Example 7. Products obtained by self-ligation of the B-cell antigenic epitope of the novel coronavirus SARS-CoV-2S protein through oxidation.
[0060] 1. Cysteine-containing peptides can form self-links through the oxidation of thiol groups. A cysteine residue is added to the N-terminus of the amino acid sequence shown in SEQ NO.1. Generally, a DMSO-mediated oxidation reaction is used to achieve this linking. Depending on the acidity or basicity of the peptide, the oxidation reaction can be carried out under slightly acidic or slightly alkaline conditions, yielding cyclic peptides.
[0061] Oxidation reaction under slightly acidic conditions: The peptide was dissolved in acetic acid of appropriate concentration, with the peptide concentration ranging from 0.5 to 1.5 mM and the final concentration of acetic acid not exceeding 5%. The pH of the peptide solution was adjusted to approximately 6.0 using (NH4)2CO3. 10-20% DMSO was added, and the reaction was carried out at 25°C for 5-25 h. The progress of the oxidation reaction could be monitored by HPLC. Then, the oxidized peptide was purified by preparative reversed-phase HPLC using 1% TFA water and acetonitrile as the mobile phase.
[0062] Oxidation reaction under slightly alkaline conditions: The peptide was dissolved in 0.01M phosphate buffer (pH 7.5) to a final concentration of 1.0 mM, and then DMSO was added to a final concentration of 1%. The reaction was carried out overnight at 25°C. The extent of the oxidation reaction could be monitored by HPLC. Then, the oxidized peptide was purified by preparative reversed-phase HPLC using 1% TFA water and acetonitrile as the mobile phase to obtain the cyclic peptide formed by the antigenic epitope peptide shown in SEQ NO.1.
[0063] 2. Self-link the N-terminus and C-terminus of the amino acid sequence shown in SEQ ID NO.1 to form peptide bonds and obtain a cyclic peptide segment.
[0064] The cyclic peptides obtained in both steps 1 and 2 can increase the stability of the antigen. In applications detecting anti-SARS-CoV-2S protein antibodies, the detection results are more stable than those using only the linear peptide with the amino acid sequence shown in SEQ ID NO. 1. In applications detecting anti-SARS-CoV-2S protein antibodies, the detection results are comparable in stability and accuracy to those using only the amino acid sequence shown in SEQ ID NO. 1.
[0065] Example 8. Indirect ELISA detection of anti-SARS-CoV-2 antibodies using B-cell antigenic epitope peptides and peptide-coupled protein antigens of the novel coronavirus SARS-CoV-2 S protein.
[0066] The SARS-CoV-2S protein B-cell antigen epitope peptide (SEQ NO.1) and the conjugate of the epitope peptide (SEQ NO.1) obtained in Example 6 and the carrier protein (KLH) were used as coating antigens to coat 96-well polystyrene ELISA plates. The antigen was diluted to a final concentration of 4 μg / ml with pH 9.6, 0.1M carbonate buffer, and 100 μl / well was added to each well. The plates were incubated overnight at 4°C. The plates were then washed three times with PBST (300 μl / well) containing 0.05% Tween 20. The plates were then blocked with PBST containing 1% BSA (200 μl / well) at 37°C for 2 hours. After blocking, the plates were washed three times with PBST. The plates were then used immediately for detection or stored at -20°C for later use.
[0067] Antibody detection procedure: Add 100 μl / well of the serum to be tested (qualitative detection serum diluted 100-fold, antibody titer detection serum serially diluted, and positive serum control, negative serum control, and blank control without serum added), incubate at 37℃ for 1 h, wash 3 times with PBST buffer for 3 minutes each time; add 100 μl / well of horseradish peroxidase-labeled goat anti-pig IgG, incubate at 37℃ for 1 h, wash 4 times with PBST buffer for 3 minutes each time; add 100 μl / well of TMB chromogenic solution, incubate at room temperature for 10 minutes and observe the colorimetric reaction; after sufficient color development, add 100 μl / well of 2M sulfuric acid to terminate the colorimetric reaction; measure the absorbance (OD450) at 450 nm using an ELISA reader; determine the results.
[0068] When determining the results: the absorbance of the blank control and negative serum wells is less than or equal to 0.25, and the absorbance of the positive serum control wells is greater than 0.4. The P / N value is calculated as (OD value of the detection well - OD value of the blank control well) / (OD value of the negative serum - OD value of the blank control well). A P / N value equal to or greater than 2 is considered positive. The antibody titer of the sample serum is the maximum dilution factor of the positive serum.
[0069] Ten SARS-CoV-2 antibody-positive serum samples and ten SARS-CoV-2 antibody-negative serum samples were selected and indirect ELISA was performed using epitope synthetic peptide and synthetic peptide-coupled protein KLH as coating antigens to detect the antigen detection effect.
[0070] The experimental results are shown in Tables 4, 5 and 6.
[0071] Table 4. Serum detection results of the synthesized epitope peptide SEQ ID NO.1
[0072]
[0073]
[0074] Note: The coating antigen is the synthetic peptide of antigenic epitope SEQ ID NO.1, 4 μg / ml. The positive serum titer is 800.
[0075] Table 5. Serum Results of Epitope Peptide SEQ ID NO.1 Conjugated with KLH Antigen
[0076]
[0077] Note: The coating antigen is the synthetic peptide of antigenic epitope SEQ ID NO.1, 5 μg / ml. The positive serum titer is 800.
[0078] Table 6. Results of Detection of Porcine Serum Using Peptides and Peptide Conjugates as Antigens
[0079]
[0080] Note: The polypeptide antigen is SEQ ID NO.1, and the polypeptide conjugate antigen is the conjugate of SEQ NO.1 and the carrier protein (KLH).
[0081] Example 9. Preparation of a vaccine based on the B-cell antigen epitope peptide of the novel coronavirus SARS-CoV-2S protein.
[0082] References for SpyCatcher003-mi3 and SpyTag self-assembled nanoparticle systems (Keeble AH, Turkki P, Stokes S, Khairil Anuar INA, Rahikainen R, VP, et al. Approaching infinite affinity through engineering of peptide-protein interaction. ProcNatl Acad Sci USA 2019:201909653.). The peptide sequence encoding the antigenic epitope shown in SEQ ID NO.1 was repeated and linked with GGSGG or GPG, and a SpyTag sequence and a 6×His tag were added to the C-terminus. The combined expressed protein sequence is shown in SEQ ID NO.4. The gene encoding SEQ ID NO.4 was optimized for prokaryotic expression codons, and the optimized gene sequence is SEQ ID NO.5. The SEQ ID NO.5 gene was artificially synthesized and cloned into the NcoI and XhoI restriction sites of the expression plasmid pET28a to construct the expression plasmid pET28-EP-SpyTag. After the expression plasmid was verified by sequence determination, it was transformed into competent cells of expression bacterium BL21(DE3). Single colonies were picked and cultured for expansion, and expression was induced with 0.3 mmol / L IPTG at 30℃ for 4 h. The induced bacterial cells were centrifuged, washed, and then lysed by sonication. After centrifugation at 12000 rpm for 10 min, the supernatant was collected for expression purification. The fusion protein was purified by affinity chromatography using a nickel column. The carrier protein SpyCatcher003-mi3 was expressed and purified according to the reference (Rahikainen R, Rijal P, Tan TK, Wu H, Andersson AC, Barrett JR, et al. Overcoming Symmetry Mismatch in Vaccine Nanoassembly through Spontaneous Amidation. Angew Chem Int Ed 2021; 60:321–30.). Figure 3 and Figure 4 As shown, both the epitope fusion protein and the carrier protein were expressed in soluble protein form. After purification, the two proteins were mixed at a molar ratio of 1:1 and naturally coupled at room temperature for 12 hours. After dialysis purification, the antigen solution was prepared with PBS to a concentration of 1 mg / ml. 20% (v / v) of aluminum hydroxide adjuvant was added to the antigen solution, and the mixture was refrigerated for later use.
[0083] Example 10. Immunization Trial of SARS-CoV-2 S Protein B Cell Antigen Epitope Nanoparticle Vaccine
[0084] The antigenic epitope nanoparticle vaccine prepared in Example 9 was used for animal immunization experiments. The specific steps were as follows: Six-week-old BALb / c mice were randomly divided into two groups of five mice each. The first group was the immunization group, with each mouse receiving an intramuscular injection of 0.1 ml of vaccine in its leg. A booster immunization was administered 28 days after the initial immunization, using the same dose and method. The second group was the control group, receiving a saline injection at the same time as the first group. Blood samples were collected from the mice before immunization, 28 days after immunization, and 42 days after immunization to separate serum. Indirect ELISA was used to detect S protein antigenic epitope-specific antibodies. The antigenic epitope peptide synthesized in Example 4 was used as the coating antigen, and indirect ELISA was used to detect serum antibodies in the experimental animals. The method for detecting antibodies in the experimental animals using the S protein antigenic epitope peptide was as described in Example 8. The results are shown in Table 7. Mice in the vaccine-immunized group produced antigenic epitope peptide-specific antibodies after immunization, and the antibody titer increased significantly after the booster immunization, while no specific antibodies were produced in the unimmunized control group.
[0085] Table 7. Results of Antibody Detection in Pigs Using the Antigen Epitope Peptide Fusion Protein Immunoassay
[0086]
[0087] SEQ ID NO.1 (Amino acid sequence of novel coronavirus S protein antigenic epitope polypeptide)
[0088] DEVRQIAPGQ;
[0089] SEQ ID NO.2 (Amino acid sequence of novel coronavirus S protein antigenic epitope polypeptide)
[0090] NEVSQIAPGQ;
[0091] SEQ ID NO.3 (Codon-optimized gene sequence encoding SARS-CoV-2S protein)
[0092] atgaagttctcctgggtgatgttcttcctgatggccgtggtgaccggcgtgaactccgtgcagcccaccgagtccatcgtgcgcttccccaacatcaccaacctgtgc
[0093] cccttcgacgaggtgttcaacgccacccgcttcgcctccgtgtacgcctggaaccgcaagcgcatctccaactgcgtggccgactactccgtgctgtacaacctggc
[0094] ccccttcttcaccttcaagtgctacggcgtgtcccccaccaagctgaacgacctgtgcttcaccaacgtgtacgccgactccttcgtgatccgcggcgacgaggtgcgccagatcgcccccggccagaccggcaacatcgccgactacaactacaagctgcccgacgacttcaccggctgcgtgatcgcctggaactccaacaagctggactccaaggtgtccggcaactacaactacctgtaccgcctgttccgcaagtccaacctgaagcccttcgagcgcgacatctccaccgagatctaccaggccggcaacaagccctgcaacggcgtggccggcttcaactgctacttccccctgcgctcctactccttccgccccacctacggcgtgggccaccagccctaccgcgtggtggtgctgtccttcgagctgctgcacgcccccgccaccgtgtgcggccccaagaagtccaccaacctggtgaagaacaagtgcgtgaacCTCGAGggcggcggcagcggcggcggcagccaccaccaccaccaccacggcggcagcggcggccaccaccaccaccaccactaa;
[0095] SEQ ID NO.4 (Epitope Peptide Fusion Protein EP-SpyTag Sequence)
[0096] DEVRQIAPGQGGSGGDEVRQIAPGQGGSGGDEVRQIAPGQGGSGGDEVRQIAPGQGGSGGDEVRQIAPGQGGSGGDEVRQIAPGQGGSGGDEVRQIAPGQGGSGGRGVPHIVMVDAYKRYKGGSGGHHHHHH;
[0097] SEQ ID NO.5 (Gene sequence of antigenic epitope fusion protein)
[0098] gatgaagtgcgccagattgccccgggccagggcggctccggcggcgatgaagtgcgccagattgccccgggccagggcggctccggcggcgatgaagtgcgccagattgccccgggccagggcggctccggcggcgatgaagtgcgccagattgccccgggccagggcggctccggcggcgatgaagtgcgccagattgccccgggccagggcggctccggcggcgatgaagtgcgccagattgccccgggccagggcggctccggcggcgatgaagtgcgccagattgccccgggccagggcggctccggcggccgcggcgtgccgcatattgtgatggtggatgcctataaacgctataaaggcggctccggcggccatcatcatcatcatcattaa。
Claims
1. An antigenic epitope polypeptide of SARS-CoV-2 S protein specific B cell, characterized in that, The D at position 1 is replaced by N, and the R at position 4 is replaced by S in the amino acid sequence shown in SEQ ID NO. 1, as shown in SEQ ID NO.
2.
2. A novel coronavirus S protein specific B cell antigen epitope polypeptide, the C-terminus of the amino acid shown in SEQ ID NO. 2 is chemically modified to carboxylation or amidation; the N-terminus is chemically modified to amination or acetylation.
3. A novel coronavirus S protein specific B cell antigen epitope polypeptide, the amino acid sequence shown in SEQ ID NO. 2 is linked to a coupling protein KLH, BSA or OVA to form a fusion sequence.
4. A gene encoding the novel coronavirus S protein specific B cell antigen epitope polypeptide of claim 1.
5. A recombinant vector, characterized in that, The recombinant vector carries the encoding gene of claim 4.
6. A recombinant microbial cell, characterized in that, Carrying the gene of claim 4 or expressing the antigen epitope polypeptide of claim 1.
7. Use of the antigen epitope polypeptide shown in SEQ ID NO. 1, the antigen epitope polypeptide of any one of claims 1-3, the encoding gene of claim 4, the recombinant vector of claim 5 or the recombinant microbial cell of claim 6 in the preparation of a kit for diagnosing or detecting a novel coronavirus or anti-novel coronavirus S protein serum.