A broad-spectrum reactive single-chain antibody of human beta coronavirus nucleocapsid protein and application thereof

Abstract

The application discloses a kind of broad-spectrum reactivity single-chain antibody of human beta coronavirus nucleocapsid N protein and application thereof, it is related to biological medicine manufacturing, gene engineering medicine and vaccine manufacturing technical field, antigen epitope is located in the peptide segment of sequence SEQ ID No.15;The broad-spectrum reactivity single-chain antibody includes heavy chain variable region and light chain variable region;The antibody of the application is the antibody for beta coronavirus homologous sequence, is used as the capture antibody of virus antigen detection, that is, it can be used for the recognition capture of all viruses of the same genus, and then combined with another high specificity antibody to form sandwich mode, realize the specific detection of certain subtype target virus antigen;The scFv antibody of the application has strong reaction with five virus N antigens, which shows that the scFv antibody can capture five beta coronavirus N proteins, and can be used for the capture of currently discovered human beta coronavirus N protein, and may recognize other human beta coronavirus N proteins discovered in the future in the future.

Description

Technical Field

[0001] This invention relates to the fields of biopharmaceutical manufacturing, genetically engineered drugs and vaccine manufacturing technology, specifically a broad-spectrum reactive single-chain antibody against human β-coronavirus nucleocapsid N protein and its applications. Background Technology

[0002] Beta coronaviruses are a significant group of viruses that seriously threaten human public health. Currently known subtypes that can infect humans include HCoV-OC43, HCoV-HKU1, SARS-CoV-1, MERS-CoV, and SARS-CoV-2. Among these, SARS-CoV-1, MERS-CoV, and SARS-CoV-2 have caused several major global epidemics, resulting in severe public health crises; the other two subtypes mostly cause mild upper respiratory tract infections and are common respiratory viruses in clinical practice. Furthermore, a large number of unknown beta coronaviruses lurk in natural hosts such as bats, and the risk of cross-species transmission remains constant. Therefore, developing rapid and broad-spectrum detection technologies for beta coronaviruses is of great significance for routine clinical testing and early prevention and control of emerging epidemics.

[0003] Current virus diagnostic technologies mainly include nucleic acid testing, antibody testing, and antigen testing. Nucleic acid testing offers high accuracy but relies on specialized laboratories and sophisticated equipment, is cumbersome to operate, and has a long testing cycle, making it difficult to meet the needs of rapid on-site screening. Antibody testing only reflects the body's past infection status and cannot determine real-time active infection, thus it is not suitable for early screening. In contrast, antigen testing is simple to operate, fast to operate, and less dependent on equipment, enabling immediate on-site testing, making it the preferred technology for grassroots screening and emergency testing. Among coronavirus detection targets, the N protein is highly expressed in infected cells and its sequence is relatively conserved in β-coronaviruses, making it a core preferred target for antigen detection.

[0004] Current coronavirus antigen detection systems generally employ a double-antibody sandwich immunoassay. The core idea is to utilize two specific antibodies targeting different epitopes to achieve accurate detection of a single viral subtype, with high subtype specificity as the core research and development goal, strictly avoiding cross-reactivity between subtypes. Currently, detection technology based on N protein-specific antibodies is relatively mature, and several detection reagents have been commercialized, with detection sensitivity exceeding 90% and specificity exceeding 95%. Existing research has screened multiple SARS-CoV-2 specific monoclonal antibodies and fragment antibodies, which can accurately identify a single strain, do not cross-react with other respiratory viruses, and can meet the needs for accurate detection of a single viral subtype.

[0005] However, existing technologies have significant shortcomings: current testing reagents are all single-subtype specific detectors, lacking a universal detection method that covers the entire β-coronavirus genus, and thus cannot meet the screening and early warning needs of unknown emerging strains. Existing research has confirmed that broad-spectrum cross-reactive antibodies have the ability to recognize multiple subtype viruses, which has been validated in the field of immunotherapy. Although a few studies in the field of antigen detection have obtained partially cross-reactive N protein antibodies that can recognize a few β-coronavirus subtypes, the coverage of these antibodies is limited, failing to achieve broad-spectrum detection of the entire β-coronavirus genus, and they have not yet been applied to the construction of a universal detection system.

[0006] Therefore, there is an urgent need for a broad-spectrum and universal β-coronavirus detection technology that can not only achieve accurate detection of a single known subtype, but also serve as a universal capture antibody for broad-spectrum screening, providing new and adaptable technical support for clinical detection of β-coronavirus, monitoring of zoonotic viruses, and early prevention and control of emerging epidemics. Summary of the Invention

[0007] To address the aforementioned problems, the present invention aims to provide a broad-spectrum reactive single-chain antibody targeting the nucleocapsid N protein of human β-coronavirus and its applications. This invention develops a broad-spectrum reactive antibody targeting a conserved homologous sequence of the nucleocapsid N protein of human β-coronaviruses, serving as a universal capture antibody for viral antigen detection. This antibody can capture viral antigens of any subtype within the human β-coronavirus genus. When combined with another subtype-specific detection antibody, forming a sandwich pattern, specific detection of a specific β-coronavirus subtype can be achieved.

[0008] To achieve the above objectives, the present invention employs the following technical solution: A broad-spectrum reactive single-chain antibody (scFv antibody) against the human β-coronavirus nucleocapsid N protein targets the antigenic epitope located in the peptide of sequence SEQ ID No. 15. The broad-spectrum reactive single-chain antibody includes a heavy chain variable region and a light chain variable region. The heavy chain variable region includes HCDR1, HCDR2, and HCDR3, and the light chain variable region includes LCDR1, LCDR2, and LCDR3. The amino acid sequence of LCDR1 is SEQ ID No. 3, the amino acid sequence of LCDR2 is EVS, and the amino acid sequence of LCDR3 is SEQ ID No. 4. The amino acid sequence of HCDR1 is SEQ ID No. 5, the amino acid sequence of HCDR2 is SEQ ID No. 6, and the amino acid sequence of HCDR3 is SEQ ID No. 7.

[0009] Preferably, the amino acid sequence of the light chain variable region is SEQ ID No. 1, and the amino acid sequence of the heavy chain variable region is SEQ ID No. 2.

[0010] Preferably, the broad-spectrum reactive single-chain antibody is a monoclonal antibody.

[0011] The present invention also includes a method for preparing a broad-spectrum reactive single-chain antibody against the human β-coronavirus nucleocapsid N protein, comprising the following steps: ① The amino acid sequences of five β-coronavirus N proteins were compared and analyzed using analytical tools. Homologous peptides were selected for peptide synthesis to obtain synthetic peptides. The five beta coronaviruses mentioned are SARS-CoV-2, SARS-CoV-1, MERS-CoV, HCoV-HKU1, and HCoV-OC43. The amino acid sequence of the homologous peptide is SEQ ID No. 15; ② The synthesized peptide was coupled with the carrier protein KLH and used as an immunogen to immunize mice. After three booster immunizations, antiserum with good titer was obtained. The immunized mice were sorted for B cells, cloned, and expression plasmids were constructed. HEK293 cells were transfected for cell culture. Cell clones with high antibody titers were selected for sequencing to obtain the base sequences of the light chain variable region and the heavy chain variable region. ③ The scFv-pcDNA3.4 recombinant vector was constructed and expressed in CHO cells. The supernatant was purified by affinity column to obtain scFv antibody with a purity greater than 95%.

[0012] The present invention also includes the nucleic acid encoded by the broad-spectrum reactive single-chain antibody against the above-mentioned human β-coronavirus nucleocapsid N protein.

[0013] The present invention also includes the application of a broad-spectrum reactive single-chain antibody against human β-coronavirus nucleocapsid N protein in the preparation of β-coronavirus antigen detection products.

[0014] The present invention has the following advantages over the prior art: This invention features a novel design concept. Typically, coronavirus antigen detection reagents employ a double-antibody sandwich antigen method, requiring highly specific antibodies that do not cross-react with antigens of other subtypes of the same species. However, this invention takes the opposite approach, developing antibodies targeting homologous sequences of viruses of the same genus (β-coronaviruses) as capture antibodies for viral antigen detection. This allows for the recognition and capture of all viruses of the same genus. Furthermore, by combining these antibodies with another highly specific antibody to form a sandwich pattern, specific detection of a target viral antigen of a specific subtype can be achieved.

[0015] The single-chain antibody (scFv antibody) against the human β-coronavirus nucleocapsid N protein of this invention is a broad-spectrum reactive antibody that can be used as a capture antibody for β-coronavirus antigen detection. By comparing the amino acid sequences of the nucleocapsid N protein of five currently discovered human β-coronaviruses, this invention found that the homologous sequence NP peptide is highly conserved, indicating that the NP peptide is a proprietary peptide sequence of the human β-coronavirus N protein. Therefore, the scFv antibody developed in this invention can not only be used for capturing currently discovered human β-coronavirus proteins, but also has the high potential to recognize other newly discovered β-coronavirus N proteins in the future.

[0016] This invention uses a sandwich ELISA method to verify the reactivity of scFv antibody with the N antigens of five β-coronaviruses, including the nucleocapsid N proteins of SARS-CoV-2, SARS-CoV-1, MERS-CoV, HCoV-HKU1, and HCoV-OC43. The sandwich ELISA results show that scFv antibody has a strong reaction with the N antigens of the above five viruses, indicating that scFv antibody can capture the N proteins of all five β-coronaviruses. Attached Figure Description

[0017] Figure 1 The results of partial amino acid sequence alignment of the nucleocapsid N protein of five human β-coronaviruses; Figure 2 Three-dimensional structure diagram of the -NH2 terminus region of the N protein in the nucleocapsid of β-coronavirus; Figure 3 MS molecular weight determination results of the synthesized peptides; Figure 4 The results are the HPLC purity identification results of the synthesized peptides; Figure 5 The results of SDS-PAGE identification of the synthesized peptide before and after coupling with BSA; Figure 6 The image shows the results of ELISA detection of titers in immunized mice. Figure 7 A flow cytometry scatter plot of single B cells sorted; Figure 8 Figure showing the verification results of PCR amplification of light chain genes in a 96-well plate; Figure 9 Figure showing the verification results of PCR amplification of heavy chain genes in a 96-well plate; Figure 10 The image shows the enzyme digestion identification results of the scFv expression vector. Figure 11 The image shows the SDS-PAGE analysis results of the purified scFv antibody. Figure 12 The graph shows the capture performance of scFv antibodies against five β-coronavirus N proteins as verified by sandwich ELISA. Detailed Implementation

[0018] The purpose of this invention is to provide a broad-spectrum reactive single-chain antibody against the nucleocapsid N protein of human β-coronavirus and its applications. The invention will be further described below with reference to specific embodiments. Example 1

[0019] I. Peptide design and synthesis; First, the amino acid sequences of the nucleocapsid N protein of five β-coronaviruses—SARS-CoV-2, SARS-CoV-1, MERS-CoV, HCoV-HKU1, and HCoV-OC43—were obtained from the NCBI database. Clustal Omega was used to perform BLAST alignment of the amino acid sequences of these five β-coronavirus nucleocapsid N proteins. The amino acid sequences of SARS-CoV-2 showed 90% and 48.5% homology with SARS-CoV-1 and MERS-CoV, respectively, but only 38.9% and 36.8% homology with HCoV-HKU1 and HCoV-OC43, respectively. However, a peptide (NP) located approximately 75–110 AA from the -NH2 terminus of all five N proteins was found to have high sequence homology. This homologous peptide sequence was selected as a candidate hapten. Figure 1 As shown, the shaded area indicates the selected homologous peptide sequence; the amino acid sequence of the homologous peptide is SEQ ID No. 15; In addition, the three-dimensional spatial structure of the N protein was analyzed using PyMOL software. Figure 2 As can be seen, the NP peptide sequences are all located on the surface of the viral N protein structure; therefore, the NP sequence was selected as the target peptide. After modifying the -COOH terminus of the NP peptide with -Cys, peptide synthesis was performed, where the Cys cysteine ​​residue provides free -SH for coupling with the carrier protein. Figure 3 and Figure 4 As can be seen, the molecular weight and purity of the synthesized peptides were good, as identified by MS and HPLC. The synthesized peptide haptens were conjugated to KLH and BSA carrier proteins using the SMCC method, with NP-KLH serving as the immunogen for immunizing mice, and NP-BSA serving as the detection antigen for subsequent antibody screening. Figure 5 As shown, M is the protein marker, Lane 1 is the peptide-BSA conjugate band, and Lane 2 is the naked BSA band. It can be seen that the molecular weight of the conjugated NP-BSA is significantly larger than that of the naked BSA, indicating that the peptide and carrier protein are well conjugated and can be used as an immunogenic antigen or detection antigen.

[0020] II. Immunization of BALB / c mice; A synthesized peptide conjugated with KLH carrier protein was used as an immunogen, emulsified with Freund's (incomplete) adjuvant at a 1:1 volume ratio, and Balb / c mice were immunized with an appropriate immunization dose. The specific immunization procedure is shown in Table 1. One week after the third booster immunization, mouse antiserum was collected via tail vein blood collection, and the antiserum titer was detected using indirect enzyme-linked immunosorbent assay (ELISA) to assess whether the immunized mice could be sacrificed for spleen harvesting after three booster immunizations.

[0021] Table 1. Immunization schedule for Balb / c mice

[0022] III. Determination of antiserum titer by indirect enzyme-linked immunosorbent assay (ELISA); The titer of antiserum from NP-KLH-immunized mice was determined using an ELISA method. NP-BSA (2.0 μg / ml) was coated in 96-well plates and incubated overnight at 4°C. After washing with PBST, 5% skim milk powder was added, and the plates were blocked at 37°C for 2 hours. After washing with PBST, mouse antiserum was added, with the first well diluted 2000-fold, followed by a series of serially diluted concentrations. The plates were incubated at 37°C for 1 hour. After washing with PBST, HRP-labeled goat anti-mouse secondary antibody (1:10000 dilution) was added, and the reaction was incubated at 37°C for 1 hour. The reaction was terminated by adding 2M H2SO4 after TMB color development. The absorbance at 450 nm was measured using a microplate reader; an OD value greater than 2.1 times that of the negative control was considered positive.

[0023] Between each step above, wash the microplate with washing buffer (20mM PBS, pH 7.4, 0.05% Tween-20), pat dry the washing buffer, and then add the sample solution for the next step.

[0024] Two mice were immunized in this experiment. The negative control was the result of the antiserum from the mice before immunization. Figure 6 As shown, the titer of the mouse antiserum after immunization was significantly higher than that before immunization, and the titer reached 1:256000, indicating a good immunization effect. This suggests that the NP peptide design is reasonable, has good immunogenicity, and that immunized mice can produce a sufficient number of antigen-specific B cells for subsequent antibody cloning screening.

[0025] IV. B cell sorting; Immunized mice were euthanized by cervical dislocation and placed on a sterile operating table. The mice were dissected, their abdominal walls opened, and the spleen was removed. The spleen was rinsed in a culture dish containing 5 mL of 0.01 M PBS (pH=7.4) buffer, and the surrounding connective tissue was removed. A 40 µm cell filter was placed on a 50 mL centrifuge tube containing 0.01 M PBS (pH=7.4), and the spleen was transferred onto the filter. The spleen was gently crushed to obtain a cell suspension. The suspension was centrifuged at 1500 rpm / min for 10 minutes, the supernatant was discarded, 3 mL of erythrocyte lysis buffer was added, and the cells were incubated on ice for 12 minutes. Then, 0.01 M PBS (pH=7.4) was added, and the suspension was centrifuged at 1500 rpm / min for 10 minutes. The supernatant was discarded, and the cells were resuspended in 1 mL of sorting buffer (1×PBS + 2% FBS + 2 mM EDTA). The resuspended cell solution was loaded onto a flow cytometer, and the antigen-affinity memory B cells were sorted into 96-well plates by detecting the fluorescence signals of the antigen protein and the fluorescence signals after B cell staining.

[0026] The prepared B cell suspension was analyzed using a flow cytometer by detecting the fluorescence signals of the antigen proteins and the fluorescence signals after B cell staining. Figure 7 As can be seen, the X-axis Comp-PE-A is the PE fluorescence channel, representing B cell surface antigen markers (such as antigen PE fluorescence markers). The higher the value, the stronger the cell binding to the target antigen. The Y-axis Comp-APC-A is the APC fluorescence channel, representing B cell-specific markers (such as CD19 / CD27 APC fluorescence). The higher the value, the higher the expression of B cell characteristic molecules. The Q2 quadrant among the four quadrants is positive for both APC and PE fluorescence. It is the antigen-specific positive B cell population for target sorting, accounting for 4.09% of the total cells. The positive enrichment ratio is moderate, and the antigen-affinity single B cells are sorted into 96-well plates.

[0027] V. Cloning of B-cell genes and construction of expression plasmids; B cells sorted into 96-well plates were lysed using a dedicated lysis buffer to release total RNA, which was then reverse transcribed to obtain cDNA. Using the cDNA as a template, PCR amplification was performed in two separate 96-well plates using specific primers for the antibody light chain (VL) and heavy chain (VH) to obtain the variable region sequences of the antibody light and heavy chains. The amplification products from each well of the 96-well plate were then subjected to agarose gel electrophoresis. Figure 8 and Figure 9As shown, each lane in the light and heavy chain gel image corresponds to each well of a 96-well plate. Lane M is the DNA molecular weight standard, and Lanes 1-96 correspond to the amplification products in the 96 wells of the plate, respectively. ★ indicates amplification wells where both light and heavy chain genes are present. For a candidate positive clone, both VH and VL bands from the same well must be present simultaneously. Positive wells with both VH and VL bands are selected, and the cloned genes are ligated into the pcDNA3.4 plasmid, transformed into Top10 cloning bacteria, and monoclonal bacteria are obtained. Colony PCR is used to screen for candidate antibody clones with paired light and heavy chains. Candidate antibody clones are then cultured in a shake culture, and finally, endotoxin-free plasmid extraction is performed.

[0028] VI. High-throughput antibody expression and positive clone screening sequencing; Pairs of light and heavy chain pcDNA3.4 plasmids were simultaneously transfected into single wells of HEK293 cells in a 96-well plate. Cell culture was performed, and cell supernatant was collected after 5 days of growth. The antibody titer in the cell expression supernatant was detected by indirect ELISA, using the same method as mouse antiserum ELISA. Cell clones with high antibody titers were selected and sent to a third party for first-generation sequencing to obtain the base sequences of the variable regions of the light and heavy chains.

[0029] Indirect ELISA was used to detect antibody expression in cell supernatants. As shown in Tables 2 and 3, using BSA-peptide as the coating antigen, 10 cell clones with high antibody titers were screened using indirect ELISA. The four cell clones with the highest antibody titers were then selected, and their reactivity with the full-length N protein of β-coronavirus was further verified using indirect ELISA. As shown in Tables 4 and 5, all four selected cell clones reacted with the N protein of five β-coronaviruses. The antibodies in the supernatants of cell lines 1M1E4 and 2M1C9 showed stronger reactivity with the N protein; therefore, these two cell lines will be selected for sequencing of the antibody light and heavy chain variable regions.

[0030] Table 2. Arrangement of cell lines for peptide screening by indirect ELISA on the microplate.

[0031] Table 3. Antibody expression in candidate cell line supernatants detected by indirect ELISA peptide assay

[0032] Note: The ELISA coating antigen was peptide-BSA 2 μg / ml; the positive control was 1:1000 diluted immunized mouse antiserum; the negative control was untransfected blank HEK293 cell culture supernatant; the blank control was PBS buffer.

[0033] Table 4. Arrangement of full-length N protein screening cell lines in ELISA plates

[0034] Table 5. Antibody expression in the supernatant of candidate clonal cell lines as detected by indirect ELISA.

[0035] Note: The ELISA coating antigen was 2 μg / ml of full-length β-coronavirus N protein; the blank control was 2% BSA.

[0036] Antibody variable region sequencing results: Based on the ELISA screening results, two positive clones, 2M1C9 and 1M1E4, were selected and sent to a third-party sequencing company for light and heavy chain variable region sequencing. The amino acid sequences of the light and heavy chain variable regions of the two antibodies are as follows: The amino acid sequence of the variable region of the light chain of the 2M1C9 antibody is SEQ ID No. 1; The amino acid sequence of the variable region of the heavy chain of the 2M1C9 antibody is SEQ ID No. 2; The amino acid sequence of the 2M1C9 antibody LCDR1 is SEQ ID No. 3; The amino acid sequence of the 2M1C9 antibody LCDR2 is EVS; The amino acid sequence of the 2M1C9 antibody LCDR3 is SEQ ID No. 4; The amino acid sequence of the 2M1C9 antibody HCDR1 is SEQ ID No. 5; The amino acid sequence of the 2M1C9 antibody HCDR2 is SEQ ID No. 6; The amino acid sequence of the 2M1C9 antibody HCDR3 is SEQ ID No. 7.

[0037] The amino acid sequence of the variable region of the light chain of the 1M1E4 antibody is SEQ ID No. 8; The amino acid sequence of the 1M1E4 antibody LCDR1 is SEQ ID No. 9; The amino acid sequence of the 1M1E4 antibody LCDR2 is KVS; The amino acid sequence of the 1M1E4 antibody LCDR3 is SEQ ID No. 10; The amino acid sequence of the variable region of the heavy chain of the 1M1E4 antibody is SEQ ID No. 11; The amino acid sequence of the 1M1E4 antibody HCDR1 is SEQ ID No. 12; The amino acid sequence of the 1M1E4 antibody HCDR2 is SEQ ID No. 13; The amino acid sequence of the 1M1E4 antibody HCDR3 is SEQ ID No. 14.

[0038] VII. Synthesis, expression, and purification of the scFv gene; The VH and VL genes obtained from sequencing were ligated using a (GGGGS)3 short peptide. The 5' end was modified with an expression signal peptide and an EcoRI restriction site, and the 3' end was modified with a -His purification tag and a BamHI restriction site. Codon optimization was performed, and the gene was synthesized and ligated with the pcDNA3.4 plasmid to obtain the scFv-pcDNA3.4 recombinant plasmid. This plasmid was transformed into Top10 bacteria for expression plasmid amplification culture, and positive clones were identified by restriction enzyme digestion. Figure 10 As shown, Lanes 1 and 3 are the scFv-pcDNA3.4 recombinant plasmids of 1M1E4 and 2M1C9 clones, respectively; Lanes 2 and 4 are the scFv-pcDNA3.4 recombinant plasmids of 1M1E4 and 2M1C9 clones digested with EcoRI and BamHi restriction endonucleases, respectively; Lane M is a DNA molecular weight standard, with molecular sizes from top to bottom as follows: 10000, 6000, 4000, 2000, 1500, 1000, 750, 500, 250, and 100 bp. It can be seen that the 2M1C9 and 1M1E4 recombinant expression vectors were successfully constructed.

[0039] Positive clone plasmids were extracted and transfected into CHO cells for transient expression. After 48 hours of culture, the expression supernatant was collected, centrifuged at 12,000 rpm for 10 minutes, cell debris was removed, and SDS-PAGE analysis was performed to analyze the expression of scFv antibody in the supernatant. Figure 11 As shown, no scFv expression band was observed in the supernatant of the 1M1E4 clone (Lane 1), while a clear scFv expression band was observed in the supernatant of the 2M1C9 clone (Lane 2). This indicates that the 1M1E4 clone failed to express scFv, while the 2M1C9 clone successfully expressed scFv.

[0040] Ni was sampled from the supernatant of the 2M1C9 clone expression fluid. 2+ scFv antibody purification was performed using metal chelate affinity chromatography, followed by elution with 250 nM imidazole solution. The eluted sample was then replaced with PBS 7.4 buffer (containing 5% sucrose) for preservation. Antibody purity was analyzed using SDS-PAGE. Figure 11As shown, M represents the protein molecular weight standards, with molecular weights from top to bottom being 180, 130, 95, 72, 55, 43, 33, 25, and 17 kDa. Lane 3 is the BSA control, Lane 4 is purified scFv (reduced denaturation treatment), and Lane 5 is purified scFv (non-reduced denaturation treatment). It can be seen that the purity of the scFv antibody can reach over 95%. The subsequent step will be to use a sandwich ELISA method to identify the antigenic reactivity of the scFv antibody with the β-coronavirus N protein. The full genome sequence of the scFv of the 2M1C9 antibody is shown in SEQ ID No. 16; The full genome sequence of the scFv-pcDNA3.4 recombinant plasmid of the 2M1C9 antibody is shown in SEQ ID No. 17.

[0041] 8. Performance verification of purified scFv antibody; (1) Sandwich ELISA was used to verify the capture performance of scFv antibody against five β-coronavirus N proteins; The capture performance of scFv antibody against N protein was verified using a sandwich ELISA method. The scFv antibody, as the capture antibody, was first coated onto the ELISA plate. Commercially available rabbit polyclonal antibodies against MERS-CoV, HCoV-OC43, and HCoV-HKU1 N proteins, and HRP-labeled antibodies against SARS-CoV-1 and SARS-CoV-2 N proteins were used as detection antibodies. Different concentration gradients of MERS-CoV, HCoV-OC43, HCoV-HKU1, SARS-CoV-1, and SARS-CoV-2 N proteins were sandwiched between the plates, forming a "sandwich" reaction pattern. The capture ability of the scFv antibody against different concentrations of N protein was then demonstrated by the HRP-catalyzed TMB color reaction. Specifically: 10.0 μg / ml scFv antibody was coated into 96-well plates and incubated overnight at 4°C. After washing with PBST, 5% skim milk powder was added, and the plates were blocked at 37°C for 2 h. After washing with PBST, MERS-CoV, HCoV-OC43, HCoV-HKU1, SARS-CoV-1, and SARS-CoV-2 N proteins were added to columns 1-5, with the first well containing 5 μg / ml. A series of concentration gradients were then applied column-wise, and the plates were incubated at 37°C for 1 h. After washing with PBST, rabbit polyclonal antibodies against MERS-CoV, HCoV-OC43, and HCoV-HKU1 N proteins were added to the first three columns, and the plates were incubated at 37°C for 1 h. Then, HRP-labeled anti-rabbit secondary antibody was added. HRP-labeled SARS-CoV-1 and SARS-CoV-2 N protein antibodies were added to columns 4-5, and the plates were reacted at 37°C for 1 h. After TMB color development, add 2M H2SO4 to terminate the reaction. Measure the absorbance at 450nm using an ELISA reader. A positive result is considered to have an OD value greater than 2.1 times that of the negative control.

[0042] Depend on Figure 12 As can be seen, the reaction trend of scFv antibody with different concentrations of N protein shows a linear gradient change, and the detection limit can reach 0.156 μg / ml, indicating that scFv antibody has good capture ability for the N protein of all five β coronaviruses.

[0043] (2) Detection reactivity of scFv antibody to real clinical samples Throat swabs from SARS-CoV-2 infected individuals were selected as real clinical specimens for testing. Following the methods described above, the sandwich ELISA method was used to verify the capture performance of the prepared scFv antibody against the nucleocapsid N protein in real clinical specimens. As shown in Table 6, the antibody exhibited a certain reactivity towards specimens positive for novel coronavirus nucleic acid and could distinguish between positive and negative specimens. The nucleic acid detection results of the clinical specimens were determined using a fluorescent RT-PCR kit from ShuoShi Biotechnology.

[0044] Table 6. Sandwich ELISA method to verify the reactivity of scFv antibodies to clinical specimens.

[0045] This invention compares the amino acid sequences of five β-coronavirus N proteins and selects homologous peptide sequences as haptens for developing capture antibodies. Clustal Omega sequence alignment revealed that the amino acid sequence near the -NH2 terminus (approximately 75-110 AA) of the five N proteins corresponds to the peptide (NP) in SEQ ID No. 15, exhibiting high sequence homology. This 12-amino acid peptide possesses both strong immunogenicity and ensures the uniqueness of the antigenic epitope. Furthermore, PyMOL analysis of the NP's spatial distribution within the protein structure shows that the selected peptide sequence is close to the -NH2 terminus, located on the conformational surface of the N protein, avoiding steric hindrance in subsequent antigen-antibody reactions. Therefore, the NP sequence is an ideal targeting peptide. Cysteine ​​residues are modified at the -COOH terminus of the NP peptide to provide -SH for subsequent coupling reactions. The synthesized peptide is coupled to the carrier protein KLH, and mice are immunized with this as an immunogen. After three booster immunizations, antiserum with good titers is obtained. Using single-cell flow cytometry, memory B cells were screened, amplified, and recombinantly expressed. Ten positive cell lines reacting with the BSA peptide were identified. Two cell clones showing strong reactivity with the N proteins of five β-coronaviruses were then selected, and their VH and VL variable region sequences were obtained through sequencing. The scFv-pcDNA3.4 recombinant expression vector was successfully constructed. The supernatant from CHO cell expression was purified using an affinity column to obtain scFv antibodies with a purity greater than 95%.

[0046] This invention employs a sandwich ELISA method to verify the reactivity of scFv antibodies with the N antigens of five beta coronaviruses, including the nucleocapsid N proteins of SARS-CoV-2, SARS-CoV-1, MERS-CoV, HCoV-HKU1, and HCoV-OC43. The sandwich ELISA results show that the scFv antibodies exhibit strong reactivity with the N antigens of these five viruses, indicating that the scFv antibodies can capture the N proteins of all five beta coronaviruses. Furthermore, comparison of the amino acid sequences of the nucleocapsid N proteins of the five currently discovered human beta coronaviruses reveals a high degree of conservation of their homologous NP peptide sequences, indicating that the NP peptide sequence is a proprietary peptide sequence of the human beta coronavirus N protein. Therefore, the scFv antibody developed in this invention can not only be used to capture the currently discovered human beta coronavirus N proteins but also has the potential to identify other newly discovered beta coronavirus N proteins in the future, possessing significant technological reserve value.

Claims

1. A broad-spectrum reactive single-chain antibody against the nucleocapsid N protein of human β-coronavirus, characterized in that: The targeted antigenic epitope is located in the peptide segment of sequence SEQ ID No. 15; the broad-spectrum reactive single-chain antibody includes a heavy chain variable region and a light chain variable region; the heavy chain variable region includes HCDR1, HCDR2, and HCDR3, and the light chain variable region includes LCDR1, LCDR2, and LCDR3; the amino acid sequence of LCDR1 is SEQ ID No. 3, the amino acid sequence of LCDR2 is EVS, and the amino acid sequence of LCDR3 is SEQ ID No. 4; the amino acid sequence of HCDR1 is SEQ ID No. 5, the amino acid sequence of HCDR2 is SEQ ID No. 6, and the amino acid sequence of HCDR3 is SEQ ID No.

7.

2. The broad-spectrum reactive single-chain antibody against the human β-coronavirus nucleocapsid N protein according to claim 1, characterized in that: The amino acid sequence of the light chain variable region is SEQ ID No. 1, and the amino acid sequence of the heavy chain variable region is SEQ ID No.

2.

3. The broad-spectrum reactive single-chain antibody against the human β-coronavirus nucleocapsid N protein according to claim 1, characterized in that: The broad-spectrum reactive single-chain antibody is a monoclonal antibody.

4. The nucleic acid encoded by the broad-spectrum reactive single-chain antibody against the human β-coronavirus nucleocapsid N protein as described in any one of claims 1 to 2.

5. The use of the broad-spectrum reactive single-chain antibody against the human β-coronavirus nucleocapsid N protein according to any one of claims 1 to 2, characterized in that: Application in the preparation of β-coronavirus antigen detection products.

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