Peptide specifically binding to coronavirus nucleoprotein

Abstract

Disclosed are a peptide, an antibody or an antigen-binding fragment thereof, a nucleic acid, a recombinant expression vector, a cell, a substance for detecting porcine epidemic diarrhea virus, a one-step diagnostic kit for porcine epidemic diarrhea, and a one-step diagnostic method for porcine epidemic diarrhea. Disclosed are a peptide, an antibody or an antigen-binding fragment thereof, a nucleic acid, a recombinant expression vector, a cell, a substance for detecting SARS-CoV-2 nucleoprotein, a one-step SARS-CoV-2 diagnostic kit, and a one-step SARS-CoV-2 diagnostic method.

Description

A peptide that specifically binds to the coronavirus nucleoprotein

[0001] The present invention relates to a peptide that specifically binds to a coronavirus nucleoprotein, an antibody or antigen-binding fragment thereof comprising the same, a nucleic acid, a recombinant expression vector, a cell, a substance for detecting a coronavirus, a diagnostic kit, a diagnostic method, and the like. The coronavirus may include porcine infectious diarrhea virus (PEDV) and / or SARS-CoV-2.

[0002] Porcine infectious diarrhea virus (PEDV) is a virus belonging to the genus Alphacoronavirus in the coronavirus family, which can cause enteric diseases such as vomiting, anorexia, diarrhea, and dehydration in pigs. Like other coronaviruses, this virus has a large, positive-sense RNA genome (excluding polyA). The open reading frames (ORFs) contain four structural proteins: a glycosylated spike protein (S), a glycosylated membrane protein (M), an envelope protein (E), and an RNA-binding nucleocapsid protein (N). The S protein can be composed of an N-terminal S1 region for virus-receptor binding and a C-terminal S2 region for membrane fusion with the host cell. The entire S protein is known to promote the induction of neutralizing antibodies in the host and has been used as a target for PEDV diagnosis. Traditional methods for PEDV diagnosis primarily involved the RT-PCR method using various primers. The cutoff value for PEDV diagnosis has been reported to be Ct=24.2-37.6 in 5-year-old pigs, corresponding to 560-0.0056 TCID50 / ml. Other analytical methods for PEDV detection have also been reported, including enzyme-linked immunosorbent assay (ELISA), electrochemical devices such as potentiometry, amperometric, and impedometric transducers, and lateral flow immunoassays. In the present invention, a one-step immunoassay for PEDV based on Fv-antibody and switching peptide is proposed, which provides PEDV analysis results simply by mixing the sample without any additional reaction steps.

[0003] * PCR: Polymerase chain reaction, ELISA: Enzyme-linked immunosorbent assay. Fv-antibodies represent the binding pocket of immunoglobulin G (IgG), which is composed of three complementarity determining regions (CDRs) and four framework regions (FRs) in the heavy chain (VH) of IgG. Fv-antibodies with specific binding affinities were selected from an Fv-antibody library expressed on the outer membrane of Escherichia coli. The Fv-antibody library was prepared by randomizing the CDR3 containing 11 amino acid residues through site-directed mutagenesis. The prepared Fv-antibody library was expressed on the outer membrane of E. coli using autodisplay technology. The autodisplayed Fv-antibodies were reported to have more than 105 Fv-antibodies / E. coli, and the transformation rate was measured to be more than 90% among the total E. coli population. The diversity of the autodisplayed Fv-antibody library was measured to be more than 106 clones / library. To date, Fv antibodies against the spike proteins of SARS-CoV-1 and SARS-CoV-2 have been screened from Fv antibody libraries. In the present invention, Fv antibodies against the S protein of PEDV were screened from an Fv antibody library using the receptor binding domain (RBD) of the S protein as a screening probe.

[0004] Fv antibodies were used in a one-step immunoassay based on switching peptides for the detection of PEDV without a washing step. In this assay, analytical results are obtained by mixing samples with Fv antibodies conjugated with switching peptides. The switching peptides are synthesized with amino acid sequences that are part of the IgG light chain and can specifically bind to the framework region (FR) of Fv antibodies (VH). When the S protein of PEDV binds to the Fv antibodies, the switching peptides are quantitatively released from the Fv antibodies. This is because the binding affinity of the PEDV S protein is stronger than that of the switching peptides to Fv antibodies. Since the switching peptides are labeled with a fluorescent dye, a fluorescent signal can be detected depending on the concentration of PEDV. This one-step immunoassay has been used to detect hepatitis B, foodborne pathogens, ochratoxin A, influenza virus, and SARS-CoV-2.

[0005] Meanwhile, SARS-CoV-2 is classified as a beta-coronavirus with a large RNA genome. The virus expresses four structural proteins: a glycosylated spike protein (S), a glycosylated membrane protein (M), an inclusion protein (E), and an RNA-binding nucleocapsid protein (NP). The S protein is expressed on the viral surface and mediates SARS-CoV-2 transmission by interacting with the ACE2 receptor on host cells. Specifically, this interaction occurs through the receptor binding domain (RBD) of the S protein, where mutations are known to generate variants. For this reason, the S protein has limited application in antigen-based diagnostics of COVID-19. NP binds to genomic RNA to form a complex and is involved in the viral assembly process. NP is the most abundant structural protein, with over 90% amino acid sequence similarity, and is known to be highly conserved and have a low mutation rate compared to the S protein. Glycosylated NPs are known to be useful in vaccine development, as they can induce the activation of cytotoxic CD8+ T cells. For this reason, glycosylated NPs have become a target for antigen-based diagnosis and vaccine development for SARS-CoV-2. In the present invention, a one-step immunoassay based on Fv antibodies and switching peptides for SARS-CoV-2 NPs has been developed.

[0006] Fv antibodies represent the antigen-binding domain of immunoglobulin G (IgG) and consist of a complementarity-determining region (CDR) and a framework region (FR3). Fv antibodies against SARS-CoV-2 NP were screened from a Fv antibody library with random amino acid sequences of 11 amino acid residues in CDR3 through site-specific mutagenesis. The Fv antibody library was automatically displayed on the outer membrane of Escherichia coli with a diversity of more than 10^6 clones per library. E. coli clones harboring Fv antibodies against NP were screened using probes targeting the N-terminal and C-terminal regions of SARS-CoV-2 NP. The screened Fv antibodies were expressed as soluble proteins, and these prepared Fv antibodies were used to develop a one-step immunoassay for SARS-CoV-2. Recently, a one-step immunoassay based on switching peptides was reported to analyze target analytes without washing or reaction steps. The switching peptide has an amino acid sequence corresponding to the light chain framework region of IgG and binds to the framework region (FR) of the Fv antibody. In the one-step immunoassay, the switching peptide was previously conjugated to the Fv antibody. Based on the difference in binding affinity (KD), the switching peptide (fluorescent label) was quantitatively separated from the Fv antibody depending on the concentration of the target analyte (SARS-CoV-2). Therefore, the fluorescent signal was generated depending on the concentration of the target analyte (SARS-CoV-2).

[0007] The present invention aims to provide an effective method and tool for the rapid and accurate detection of porcine epidemic diarrhea virus (PEDV) infection. In particular, the invention provides a method for more efficiently diagnosing and managing PEDV infection by developing novel peptides and antibodies or antigen-binding fragments thereof that specifically bind to the spike protein of PEDV.

[0008] In addition, one object of the present invention is to provide a novel peptide, antibody or antigen-binding fragment thereof, nucleic acid, recombinant expression vector, and cell that specifically bind to the SARS-CoV-2 nucleoprotein (NP). Through this, a one-step SARS-CoV-2 diagnostic kit and diagnostic method capable of effectively detecting SARS-CoV-2 NP can be provided. Specifically, in the present invention, Fv antibodies against the NP of SARS-CoV-2 were screened from an Fv antibody library. For the screening of the Fv antibody library, the N-terminal domain (NTD) and C-terminal domain (CTD) of the NP were used as probes. The Fv antibodies screened against the NTD and CTD were used in a one-step immunoassay using a switching peptide. Finally, a one-step immunoassay for the detection of SARS-CoV-2 using an actual virus sample was demonstrated.

[0009] In one aspect, the present invention provides a peptide that specifically binds to porcine epidemic diarrhea virus (PEDV), the peptide comprising at least one peptide sequence selected from the group consisting of the peptide sequence of SEQ ID NO: 3, the peptide sequence of SEQ ID NO: 4, the peptide sequence of SEQ ID NO: 5, and the peptide sequence of SEQ ID NO: 6.

[0010] In another aspect, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to porcine epidemic diarrhea virus (PEDV), comprising an antibody or antigen-binding fragment thereof selected from the group consisting of a variable region comprising CDR1 of SEQ ID NO: 1, CDR2 of SEQ ID NO: 2, and CDR3 of SEQ ID NO: 3; a variable region comprising CDR1 of SEQ ID NO: 1, CDR2 of SEQ ID NO: 2, and CDR3 of SEQ ID NO: 4; a variable region comprising CDR1 of SEQ ID NO: 1, CDR2 of SEQ ID NO: 2, and CDR3 of SEQ ID NO: 5; and a variable region comprising CDR1 of SEQ ID NO: 1, CDR2 of SEQ ID NO: 2, and CDR3 of SEQ ID NO: 6.

[0011] In one embodiment, the antibody or antigen-binding fragment thereof may comprise a heavy chain variable region comprising HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, and HCDR3 of SEQ ID NO: 3; a heavy chain variable region comprising HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, and HCDR3 of SEQ ID NO: 4; a heavy chain variable region comprising HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, and HCDR3 of SEQ ID NO: 5; and a heavy chain variable region comprising HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, and HCDR3 of SEQ ID NO: 6.

[0012] In one embodiment, the antibody or antigen-binding fragment thereof may be in the form of a single domain antibody.

[0013] In another aspect, the present invention provides a nucleic acid encoding the peptide or the antibody or an antigen-binding fragment thereof.

[0014] In another aspect, the present invention provides a recombinant expression vector comprising the nucleic acid.

[0015] In another aspect, the present invention provides a cell transformed with the recombinant expression vector.

[0016] In one embodiment, the cell may comprise one or more cells selected from the group consisting of animal cells, plant cells, yeast, Escherichia coli, and insect cells.

[0017] In one embodiment, monkey kidney cells 7 (COS7) cells, NSO cells, SP2 / 0 cells, Chinese hamster ovary (CHO) cells, W138, baby hamster kidney (BHK) cells, MDCK, myeloma cell lines, HuT 78 cells and HEK293 cells, Escherichia coli, Bacillus subtilis, Streptomyces sp., Pseudomonas sp., Proteus mirabilis or Staphylococcus sp., Aspergillus sp., Pichiapastoris, Saccharomyces cerevisiae, Schizosaccharomyces sp.) and Neurospora crassa.

[0018] In another aspect, the present invention provides a substance for detecting porcine epidemic diarrhea virus, comprising: the antibody or an antigen-binding fragment thereof; a switching peptide capable of specifically and reversibly binding to at least one of the first to fourth framework regions (FR1, FR2, FR3 and FR4) of a heavy chain or a light chain of the antibody or an antigen-binding fragment thereof; and a marker bound to the switching peptide.

[0019] In one embodiment, the antibody or antigen-binding fragment thereof may be single-stranded.

[0020] In one embodiment, the marker may be a fluorescent marker.

[0021] In another aspect, the present invention provides a one-step swine epidemic diarrhea diagnostic kit comprising a substance for detecting the porcine epidemic diarrhea virus.

[0022] In another aspect, the present invention provides a one-step swine epidemic diarrhea diagnosis method, comprising the step of mixing a substance for detecting swine epidemic diarrhea virus in the one-step swine epidemic diarrhea diagnosis kit with a sample to be detected and then identifying the marker.

[0023] In one embodiment, the marker may be prepared as a fluorescent marker.

[0024] In the present invention, Fv antibodies against the spike protein of porcine infectious diarrhea virus (PEDV) were screened from an Fv antibody library. The expressed receptor binding domain (RBD) of the PEDV S protein was used for screening the Fv antibody library. Target clones were screened from the Fv antibody library, and the selected Fv antibodies were expressed as soluble proteins. The expressed Fv antibodies were used in a one-step immunoassay based on a switching peptide. Finally, a one-step immunoassay for the detection of not only the RBD but also real PEDV samples was demonstrated.

[0025] In another aspect, the present invention provides a peptide that specifically binds to the SARS-CoV-2 nucleoprotein, the peptide comprising at least one peptide sequence selected from the group comprising the peptide sequence of SEQ ID NO: 7 and the peptide sequence of SEQ ID NO: 8.

[0026] In another aspect, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to the SARS-CoV-2 nucleoprotein, comprising an antibody or antigen-binding fragment thereof selected from the group consisting of a variable region comprising CDR1 of SEQ ID NO: 1, CDR2 of SEQ ID NO: 2, and CDR3 of SEQ ID NO: 7; and a variable region comprising CDR1 of SEQ ID NO: 1, CDR2 of SEQ ID NO: 2, and CDR3 of SEQ ID NO: 8.

[0027] In one embodiment, the antibody or antigen-binding fragment thereof may comprise a heavy chain variable region comprising HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, and HCDR3 of SEQ ID NO: 7; a heavy chain variable region comprising HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, and HCDR3 of SEQ ID NO: 8;

[0028] In one embodiment, the antibody or antigen-binding fragment thereof may be in the form of a single domain antibody.

[0029] In another aspect, the present invention provides a nucleic acid encoding the peptide or the antibody or an antigen-binding fragment thereof.

[0030] In another aspect, the present invention provides a recombinant expression vector comprising the nucleic acid.

[0031] In another aspect, the present invention provides a cell transformed with the recombinant expression vector.

[0032] In one embodiment, the cell may comprise one or more cells selected from the group consisting of animal cells, plant cells, yeast, Escherichia coli, and insect cells.

[0033] In one embodiment, the cell is selected from the group consisting of monkey kidney cells 7 (COS7), NSO cells, SP2 / 0 cells, Chinese hamster ovary (CHO) cells, W138, baby hamster kidney (BHK) cells, MDCK, myeloma cell lines, HuT 78 cells, and HEK293 cells, Escherichia coli, Bacillus subtilis, Streptomyces sp., Pseudomonas sp., Proteus mirabilis or Staphylococcus sp., Aspergillus sp., Pichiapastoris, Saccharomyces cerevisiae, and Schizosaccharomaces. It may comprise one or more cells selected from the group comprising Schizosaccharomyces sp. and Neurospora crassa.

[0034] In another aspect, the present invention provides a substance for detecting SARS-CoV-2 nucleoprotein, comprising: the antibody or an antigen-binding fragment thereof; a switching peptide capable of specifically and reversibly binding to one or more of the first to fourth framework regions (FR1, FR2, FR3, and FR4) of the heavy chain or light chain of the antibody or an antigen-binding fragment thereof; and a marker bound to the switching peptide.

[0035] In one embodiment, the antibody or antigen-binding fragment thereof may be single-stranded.

[0036] In one embodiment, the marker may be a fluorescent marker.

[0037] In another aspect, the present invention provides a one-step SARS-CoV-2 diagnostic kit comprising a substance for detecting the SARS-CoV-2 nucleoprotein.

[0038] In another aspect, the present invention provides a one-step SARS-CoV-2 diagnostic method, comprising the step of mixing a substance for detecting SARS-CoV-2 nucleoprotein in the one-step SARS-CoV-2 diagnostic kit with a sample to be detected and then identifying the marker.

[0039] In one embodiment, the marker may be prepared as a fluorescent marker.

[0040] The effect of the present invention is that it can effectively improve the management and control of porcine epidemic diarrhea virus by providing an innovative diagnostic method and kit that can rapidly and accurately detect PEDV infection. In particular, the present invention provides a one-step diagnostic method that can be used without a specialized laboratory or complex equipment, thereby enabling rapid diagnosis and timely response in the field. Furthermore, the present invention can accurately detect the SARS-CoV-2 nucleoprotein using peptides, antibodies, antigen-binding fragments, and diagnostic methods with improved sensitivity and specificity, thereby enabling rapid diagnosis even in the early stages of SARS-CoV-2 infection. Furthermore, the simple one-step diagnostic method provides rapid results without complex experimental procedures and can be effectively utilized for large-scale population screening and surveillance in various settings such as hospitals, laboratories, and field diagnostic centers.

[0041] Figure 1 is an autodisplayed Fv library with randomized CDR3 regions used for Fv antibody screening against the spike protein of porcine epidemic diarrhea virus (PEDV), preparation of an Fv library with randomized CDR3 regions in VH using site-directed mutagenesis.

[0042] Figure 2 shows an autodisplayed Fv-library with randomized CDR3 regions used for Fv-antibody screening against the spike protein of porcine epidemic diarrhea virus (PEDV), and expression of the Fv-library on the outer membrane of E. coli using an autodisplay vector (SDS-PAGE of autodisplayed Fv-variants, control strain with CDR1 and CDR2, and intact E. coli).

[0043] Figure 3 shows the expression of selected Fv-antibodies as soluble proteins and the measurement of binding constants (Kd).

[0044] Figure 4 shows the binding constant (Kd) measurements of Fv antibodies (Anti-RBD-1, Anti-RBD-2, Anti-RBD-3, Anti-RBD-4) from four selected clones to the RBD antigen using an SPR biosensor, expressing the selected Fv antibodies as soluble proteins and measuring the binding constant (Kd).

[0045] Figure 5 shows the RBD antigen sequence of PEDV.

[0046] Figure 6 shows the fluorescence signal compared to the cutoff line between the library E. coli and the control strain by selection of Fv variants (clones) with affinity for the porcine epidemic diarrhea virus (PEDV) spike protein.

[0047] Figure 7 shows selection of Fv variants (clones) with affinity for the spike protein of porcine epidemic diarrhea virus (PEDV), with target clone selection through genetic sequence analysis of the CDR3 region in flow cytometry.

[0048] Figure 8. Measurement of binding constant (Kd) from quantitative binding analysis using a flow cytometer in flow cytometry screening for Fv variants (clones) with affinity for the spike protein of porcine epidemic diarrhea virus (PEDV).

[0049] Figure 9 shows a target clone selection procedure using magnetic beads with immobilized RBD antigen for Fv-variant (clone) selection with affinity for the spike protein of porcine epidemic diarrhea virus (PEDV).

[0050] Figure 10 shows selection of Fv variants (clones) with affinity for the spike protein of porcine epidemic diarrhea virus (PEDV), with target clone selection through genetic sequence analysis of the CDR3 region in magnetic selection.

[0051] Figure 11. Measurement of binding constant (Kd) from quantitative binding analysis using flow cytometry in magnetic sorting for selection of Fv variants (clones) with affinity for the spike protein of porcine epidemic diarrhea virus (PEDV).

[0052] Figure 12 illustrates the principle of a uniform one-step immunoassay, a switching peptide that specifically binds to the antigen-binding pocket of IgG. The binding pocket of IgG is composed of a heavy chain including complementarity-determining regions (CDRs) and framework regions (FRs).

[0053] Figure 13 shows the principle of a uniform one-step immunoassay, measuring the binding constant (Kd) of Fv antibodies (Anti-RBD-1, Anti-RBD-2, Anti-RBD-3, Anti-RBD-4) from four selected clones against a switching peptide using an SPR biosensor.

[0054] Figure 14 is a one-step immunoassay based on a switching peptide, and the configuration of a one-step immunoassay based on a switching peptide.

[0055] Figure 15 is a one-step immunoassay based on a switching peptide, and detection of RBD antigen using a one-step immunoassay based on a switching peptide.

[0056] Figure 16 RT-PCR analysis of a real PEDV sample.

[0057] Figure 17 is a lateral flow immunoassay of PEDV samples using a one-step immunoassay based on a switching peptide for PEDV detection.

[0058] Figure 18 is a PEDV detection using a one-step immunoassay based on a switching peptide. PEDV detection using a one-step immunoassay based on a switching peptide.

[0059] Figure 19 Molecular docking analysis of the interaction between the RBD region of the PEDV spike protein (UniProt ID: Q91AV1) and Fv antibodies (Anti-RBD-1, Anti-RBD-2, Anti-RBD-3, Anti-RBD-4).

[0060] Figure 20 shows the protein structure of APN (or ACE2) as a result of analysis of amino acid similarity between Fv antibodies (Anti-RBD-1, Anti-RBD-2, Anti-RBD-3, Anti-RBD-4) and APN (or ACE2).

[0061] Figure 21 is an analysis of amino acid similarity between Fv antibodies (Anti-RBD-1, Anti-RBD-2, Anti-RBD-3, Anti-RBD-4) and APN (or ACE2), showing similar amino acid sequence regions of APN (or ACE2) and Fv antibodies.

[0062] Figure 22 shows the mutual binding site between Fv-antibody and RBD antigen.

[0063] Figure 23a is a diagram of the outer membrane expression of the Fv-antibody library in E. coli.

[0064] Figure 23b is a diagram showing the expression of NTD and CTD.

[0065] Figure 24a is a diagram illustrating the interaction between the Fv-antibody library and the NTD-GFP probe using a flow cytometer.

[0066] Figure 24b is a diagram comparing the fluorescence signals resulting from the interaction of the Fv-antibody library, the NTD-GFP probe, and E. coli.

[0067] Figure 24c is a diagram showing three clones that were finally selected from among the Fv-antibody clones with high binding affinity for the NTD-GFP probe.

[0068] Figure 24d is a diagram showing the binding affinity of the selected clones (clone number 14 for NTD and clone number 1 for CTD).

[0069] Figure 25a is a diagram showing the expression and structure of selected Fv antibodies.

[0070] Figure 25b is a diagram showing the binding and dissociation profiles of Fv-antibodies immobilized on the gold surface of an SPR biochip.

[0071] Figure 25c is a diagram showing the results of measuring the binding affinity of an Fv antibody using an SPR biosensor.

[0072] Figure 25d is a diagram showing the affinity between Fv-antibody and NTD, CTD, and NP.

[0073] Figure 26a is a diagram showing a one-step immunoassay based on expressed Fv-antibodies and switching peptides.

[0074] Figure 26b is a diagram showing the results of a one-step immunoassay based on Fv-antibody (anti-NTD).

[0075] Figure 26c is a diagram showing the results of a one-step immunoassay based on Fv-antibody (anti-CTD).

[0076] Figure 26d is a diagram comparing the amino acid sequence similarity between the NTD and CTD of SARS-CoV-2 and those of other coronaviruses.

[0077] Figure 27a is a diagram examining the interaction between NP of SARS-CoV-2 and anti-NTD Fv antibody.

[0078] Figure 27b is a diagram examining the interaction between NP of SARS-CoV-2 and anti-CTD Fv antibody.

[0079] Hereinafter, embodiments of the present invention will be described in detail with reference to the attached drawings. The present invention may be modified in various ways and may take various forms. Therefore, specific embodiments are illustrated in the drawings and described in detail in the text. However, this is not intended to limit the present invention to a specific disclosed form, but should be understood to include all modifications, equivalents, and substitutes included in the spirit and technical scope of the present invention. In describing each drawing, similar reference numerals are used to indicate similar components. In the attached drawings, the dimensions of structures are shown larger than actual size to ensure clarity of the present invention.

[0080] The terminology used in this application is only used to describe specific embodiments and is not intended to limit the present invention. The singular expression includes plural expression unless the context clearly indicates otherwise. In this application, it should be understood that the terms "comprise" or "have" are intended to specify the presence of a feature, number, step, operation, component, or combination thereof described in the specification, but do not exclude in advance the possibility of the presence or addition of one or more other features, numbers, steps, operations, components, or combinations thereof. In the context of this specification, the term "about" or the like can mean about ± 1%, about ± 2%, about ± 3%, about ± 4%, about ± 5%, about ± 6%, about ± 7%, about ± 8%, about ± 9%, or about ± 10% of a numerical value described in the specification.

[0081] Additionally, the description of one aspect of the present invention may be applied identically or similarly to the same or similar configurations or terms in the description of other aspects.

[0082] Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and will not be interpreted in an idealized or overly formal sense unless explicitly defined herein.

[0083] A peptide according to an embodiment of the present invention is a peptide that specifically binds to porcine epidemic diarrhea virus (PEDV), and may include one or more peptide sequences selected from the group consisting of a peptide sequence of SEQ ID NO: 3, a peptide sequence of SEQ ID NO: 4, a peptide sequence of SEQ ID NO: 5, and a peptide sequence of SEQ ID NO: 6.

[0084] In the context of this specification, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 may refer to:

[0085] Sequence number 3: YGPSIVEQTDV

[0086] Sequence number 4: YVGLGPGTVDF

[0087] Sequence number 5: YETVRGNGTDV

[0088] Sequence number 6: YGVLRRGCLDF

[0089] The peptide according to an embodiment of the present invention has a function of specifically binding to porcine epidemic diarrhea virus (PEDV), and may optionally include one or more of the peptide sequences of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6. These sequences are defined as YGPSIVEQTDV, YVGLGPGTVDF, YETVRGNGTDV, and YGVLRRGCLDF, respectively, and they have a structure capable of specifically binding to a specific protein structure or surface antigen of PEDV. The selection of such peptide sequences provides a wide range of detection possibilities for various mutations of PEDV, thereby enabling more accurate and reliable PEDV diagnosis.

[0090] In the context of this specification, porcine epidemic diarrhea virus (PEDV) is a virus belonging to the coronavirus family and the genus Alphacoronavirus. This virus can infect pigs and cause an enteric disease with symptoms such as vomiting, anorexia, diarrhea, and dehydration. Diagnosis and management of PEDV are crucial for animal disease management and prevention, and the development of effective diagnostic tools is essential for this purpose. The present invention provides a one-step diagnostic method based on a peptide that specifically interacts with the spike protein of PEDV, enabling rapid and accurate diagnosis of PEDV infection. This peptide has affinity for a specific region of the spike protein of PEDV, enabling sensitive and specific detection of the presence of PEDV. This can be a valuable tool for pig farmers and animal health professionals, enabling the early diagnosis and appropriate response to PEDV infection.

[0091] Meanwhile, the antibody or antigen-binding fragment thereof according to an embodiment of the present invention may include an antibody or antigen-binding fragment thereof that specifically binds to porcine epidemic diarrhea virus (PEDV), and may include a variable region comprising CDR1 of SEQ ID NO: 1, CDR2 of SEQ ID NO: 2, and CDR3 of SEQ ID NO: 3; a variable region comprising CDR1 of SEQ ID NO: 1, CDR2 of SEQ ID NO: 2, and CDR3 of SEQ ID NO: 4; a variable region comprising CDR1 of SEQ ID NO: 1, CDR2 of SEQ ID NO: 2, and CDR3 of SEQ ID NO: 5; and a variable region comprising CDR1 of SEQ ID NO: 1, CDR2 of SEQ ID NO: 2, and CDR3 of SEQ ID NO: 6.

[0092] In the context of this specification, sequence numbers 1 and 2 may mean:

[0093] Sequence number 1: TYGIQ

[0094] Sequence number 2: WIHAGTGGTKYSRKFQG

[0095] In the context of the present specification, the antibody or antigen-binding fragment may comprise sequences of CDR1, CDR2, and CDR3 that provide specific binding to the spike protein of porcine epidemic diarrhea virus (PEDV). SEQ ID NO: 1 (TYGIQ) and SEQ ID NO: 2 (WIHAGTGGTKYSRKFQG) each represent specific CDR regions of the antibody, and combinations of these can generate various antibodies or antigen-binding fragments that can interact with various variants of PEDV. Such antibodies or antigen-binding fragments enable rapid and effective detection of PEDV and can be used as components of diagnostic kits.

[0096] In one embodiment, the antibody or antigen-binding fragment thereof may comprise a heavy chain variable region comprising HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, and HCDR3 of SEQ ID NO: 3; a heavy chain variable region comprising HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, and HCDR3 of SEQ ID NO: 4; a heavy chain variable region comprising HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, and HCDR3 of SEQ ID NO: 5; and a heavy chain variable region comprising HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, and HCDR3 of SEQ ID NO: 6.

[0097] In the context of this specification, the meaning of HCDR can refer to a highly variable portion of the complementarity determining region (CDR) located in the heavy chain of an antibody. The specificity and binding ability of an antibody can be largely determined by the amino acid sequence in this HCDR region. Generally, antibodies have two types of polypeptide chains, namely, a light chain and a heavy chain. Each chain contains three CDR regions, of which HCDR can be a CDR located in the heavy chain. HCDR1, HCDR2, HCDR3, etc. each refer to a specific portion within the CDR of the heavy chain, and these can play an important role in binding an antibody to a specific antigen. These HCDR regions are key elements that increase the diversity and specificity of antibodies, allowing the antibody to bind to an antigen with high specificity.

[0098] In one embodiment, the antibody or antigen-binding fragment thereof may be in the form of a single-domain antibody. In the context of this specification, a single-domain antibody refers to an antibody composed of a single polypeptide chain, rather than the complex structure of a traditional antibody. Such single-domain antibodies are small in size and structurally simple, have high target accessibility, and can be easily produced. These antibodies can provide high specificity and binding capacity for specific antigens, including clustered region (CDR) regions. These small and flexible antibodies have the ability to bind to antigens in small spaces that are difficult for traditional antibodies to access, and may have various potential applications in biomedical and diagnostic fields. In particular, they can be used as effective tools for the specific detection of viral constituent proteins, such as porcine epidemic diarrhea virus (PEDV), and can also be utilized as a core component of diagnostic kits or biosensor systems.

[0099] Meanwhile, the nucleic acid according to an embodiment of the present invention may encode the peptide or the antibody or an antigen-binding fragment thereof. In the context of this specification, the meaning of coding refers to the process by which a specific DNA or RNA sequence determines the amino acid sequence of a specific protein or peptide. This refers to the process by which genetic information is translated into a protein, and through this process, the genetic sequence can determine the structure and function of a specific protein. According to an embodiment of the present invention, the specific nucleic acid may encode a peptide or antibody or an antigen-binding fragment thereof that specifically binds to porcine epidemic diarrhea virus (PEDV), thereby providing the information necessary for producing a specific protein or peptide. This coding process can play an important role in various applications, such as the development of diagnostic kits, biosensors, therapeutics, or vaccines, through recombinant DNA technology. In other words, the nucleic acid can serve as a blueprint for the production of a specific protein or peptide used to detect or defend against viral infections.

[0100] Meanwhile, a recombinant expression vector according to an embodiment of the present invention may include the nucleic acid. In the context of this specification, a recombinant expression vector refers to a DNA molecule engineered for gene expression. It can serve to transfer a gene into a new host cell and cause the gene to be translated into a protein. A recombinant expression vector contains a specific gene or DNA sequence, thereby inducing the production of a specific protein in the host cell. Such a vector typically includes a promoter, an origin for gene replication, a selection marker, and other regulatory sequences for the expression of the inserted gene. According to an embodiment of the present invention, a recombinant expression vector may include a nucleic acid encoding a peptide or antibody or an antigen-binding fragment thereof that specifically binds to porcine epidemic diarrhea virus (PEDV), thereby facilitating the mass production of a specific protein or peptide. This has important applications in the biomedical and diagnostic fields, and may enable the efficient production of proteins or peptides that can be used to detect or defend against viral infections.

[0101] Meanwhile, cells according to embodiments of the present invention can be transformed with the recombinant expression vector. In the context of this specification, "transformation" refers to the process of introducing exogenous genetic material into a cell. Through this process, the cell acquires new genetic characteristics, which can lead to changes in the physiological, morphological, or genetic characteristics of the cell. Transformation can be widely used in gene expression, protein production, and biotechnology research and applications. Cells according to embodiments of the present invention can be transformed with a recombinant expression vector. Such vectors contain a gene encoding the amino acid sequence of a specific protein or peptide, and can induce expression of the protein or peptide by delivering it into the cell. This process can play a key role in various fields, including genetic manipulation, biomedical research, protein engineering, and the development of therapeutics and vaccines. For example, cells transformed with a recombinant expression vector containing a gene encoding a peptide or antibody or antigen-binding fragment thereof that specifically binds to porcine epidemic diarrhea virus (PEDV) could play an important role in detecting or defending against viral infections, enabling mass production of the protein or peptide.

[0102] In one embodiment, the cell may comprise one or more cells selected from the group consisting of animal cells, plant cells, yeast, Escherichia coli, and insect cells. In one embodiment, the cell is selected from the group consisting of monkey kidney cells 7 (COS7), NSO cells, SP2 / 0 cells, Chinese hamster ovary (CHO) cells, W138, baby hamster kidney (BHK) cells, MDCK, myeloma cell lines, HuT 78 cells, and HEK293 cells, Escherichia coli, Bacillus subtilis, Streptomyces sp., Pseudomonas sp., Proteus mirabilis or Staphylococcus sp., Aspergillus sp., Pichiapastoris, Saccharomyces cerevisiae, and Schizosaccharomaces. It may comprise one or more cells selected from the group comprising Schizosaccharomyces sp. and Neurospora crassa.

[0103] The selection of the above cells may be based on a cell type suitable for the expression and production of a specific protein or peptide. Among various cell types, monkey kidney cell 7 (COS7), NSO cells, SP2 / 0 cells, Chinese hamster ovary (CHO) cells, W138 cells, baby hamster kidney (BHK) cells, MDCK cells, myeloma cell lines, HuT 78 cells, and HEK293 cells are all of human or animal origin, and they can be widely used for the expression and production of specific proteins. In addition, bacterial types such as Escherichia coli, Bacillus subtilis, Streptomyces, Pseudomonas, Proteus mirabilis, or Staphylococcus can also be used for protein production. In addition, fungal or yeast cells such as Aspergillus, Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomaces, and Neurospora crassa may also be suitable for protein production. These cells can be used to purify, modify, or optimize the functional activity of proteins based on their individual characteristics. Each cell type is selected based on its production and functional properties for specific proteins or peptides, which can be crucial for efficient protein production in biomedical and diagnostic applications.

[0104] Meanwhile, a substance for detecting porcine epidemic diarrhea virus (PEDV) according to an embodiment of the present invention may include: the antibody or an antigen-binding fragment thereof; a switching peptide capable of specifically and reversibly binding to one or more of the first to fourth framework regions (FR1, FR2, FR3 and FR4) of the heavy chain or light chain of the antibody or an antigen-binding fragment thereof; and a marker bound to the switching peptide.

[0105] In the context of this specification, a "switching peptide" refers to a short amino acid sequence that can specifically and reversibly bind to one or more of the first through fourth framework regions (FR1, FR2, FR3, and FR4) of the heavy or light chain of a specific antibody or antigen-binding fragment thereof. Such switching peptides can be used to alter the structural and functional properties of an antibody, thereby modulating its binding ability or specificity. Switching peptides can act as an "on and off" switch to activate the antibody upon binding to it, which can contribute to enhancing the efficiency and accuracy of antibody-based therapeutics or diagnostic tools. The switching peptides can be conjugated with a label, which can facilitate the detection or tracking of the antibody-peptide complex. The label can take various forms, such as a fluorescent substance, a radioisotope, an enzyme, or another biochemical substance, and can thereby detect the presence or activity of the antibody. For example, using a marker conjugated to a switching peptide, the location or amount of antibodies that specifically bind to porcine epidemic diarrhea virus (PEDV) can be accurately measured, which may play an important role in the diagnosis and monitoring of viral infections.

[0106] In one embodiment, the antibody or antigen-binding fragment thereof may be single-stranded. As described above, the single-stranded antibody or antigen-binding fragment can provide high binding specificity and sensitivity to a specific antigen, and its small size and simple structure can facilitate its manufacture and use. A single-stranded antibody or antigen-binding fragment may utilize only one of the variable regions of a traditional double-stranded antibody, or may be composed of a specifically designed single polypeptide chain. This single-stranded structure reduces the size and complexity of the antibody, allowing it to more easily access antigen-binding sites that are otherwise inaccessible. This may be particularly advantageous for binding to specific viral proteins or small molecules. The use of a single-stranded antibody or antigen-binding fragment according to an embodiment of the present invention may play a significant role in, for example, a material for detecting porcine epidemic diarrhea virus (PEDV). This single-stranded structure can specifically bind to specific viral proteins, providing more accurate and sensitive diagnostic results. This contributes to the early detection and monitoring of viral infections, which can provide crucial information for public health management and infectious disease response strategies.

[0107] In one embodiment, the marker may be a fluorescent marker. In the context of this specification, a "fluorescent marker" refers to a fluorescent substance that binds to a specific antibody or antigen-binding fragment thereof, thereby enabling the visual detection of the presence or activity of the antibody. Such fluorescent markers can enable the precise measurement of the location or amount of the antibody through a fluorescent signal generated when the antibody binds to a specific antigen. The use of fluorescent markers can be particularly useful in diagnostic applications. For example, by conjugating a fluorescent marker to an antibody that specifically binds to porcine epidemic diarrhea virus (PEDV), the location where the antibody interacts with the virus can be precisely identified and visualized. This provides accurate information for the diagnosis and monitoring of viral infections, which can aid in the early detection of infections and the establishment of appropriate therapeutic measures.

[0108] Fluorescent markers according to embodiments of the present invention can be selected from a variety of fluorescent materials, which emit light in response to light of a specific wavelength. These fluorescent signals can be observed and analyzed using specialized fluorescence microscopes or other fluorescence detection devices, making them valuable tools for laboratory diagnostics, field testing, and clinical research.

[0109] Meanwhile, a one-step swine epidemic diarrhea diagnostic kit according to an embodiment of the present invention may include a substance for detecting porcine epidemic diarrhea virus (PEDV). In the context of this specification, a "one-step swine epidemic diarrhea diagnostic kit" refers to a diagnostic tool designed to rapidly and accurately detect porcine epidemic diarrhea virus (PEDV). This diagnostic kit may include the above-mentioned substance for detecting porcine epidemic diarrhea virus (PEDV), thereby enabling simple and rapid diagnosis of virus infection. The core component of the one-step swine epidemic diarrhea diagnostic kit may be a complex comprising a specific antibody or antigen-binding fragment thereof, a switching peptide, and a fluorescent marker. This complex can specifically react with porcine epidemic diarrhea virus (PEDV), allowing rapid detection of infection. This diagnostic kit is particularly designed for ease of use in the field, and can be used without complex laboratory equipment or specialized skills. The user can collect a sample, apply it to the diagnostic kit, and obtain results, and determine infection based on the presence or absence of a fluorescent signal. The advantages of these one-step diagnostic kits lie in their rapid results and ease of use. This can play a crucial role in public health management and infectious disease response strategies, especially during pandemics. These kits are essential for early detection and appropriate treatment, and can help prevent the spread of the virus.

[0110] The above kit does not exclude the addition of other components. Examples of other components that the kit may additionally include to realize diagnostic utility include a sample preparation kit, a nucleic acid extraction kit, an amplification kit, and various detection and analysis tools. These additional components may be necessary to optimize each step of the diagnostic process and obtain faster and more accurate results. For example, a sample preparation kit may provide tools that allow the user to easily prepare and process various types of samples, such as blood, saliva, and nasal swabs. This can play a crucial role in improving diagnostic accuracy by ensuring proper sample collection and storage. A nucleic acid extraction kit may be used to extract viral RNA or DNA from a sample. This process is the first step in detecting the viral genetic material and may be essential for an accurate diagnosis. An amplification kit is used to amplify the extracted nucleic acid, which allows for a more definitive confirmation of the presence of the virus. In particular, when using PCR (polymerase chain reaction) technology, even very small amounts of viral genetic material can be detected. Additionally, various detection and analysis tools can be used to read and interpret the signals from antibodies conjugated to fluorescent markers. These tools can be provided as both laboratory-based and portable equipment, enabling rapid on-site diagnosis. Thus, a one-step swine epidemic diarrhea diagnostic kit can provide an efficient method for rapid and accurate detection of viral infections by combining various components and tools.

[0111] Meanwhile, a one-step swine epidemic diarrhea diagnosis method according to an embodiment of the present invention may include a step of mixing a substance for detecting porcine epidemic diarrhea virus (PEDV) in the one-step swine epidemic diarrhea diagnosis kit with a sample to be detected, and then identifying the marker. In one embodiment, the marker may be prepared as a fluorescent marker.

[0112] A peptide according to an embodiment of the present invention is a peptide that specifically binds to the SARS-CoV-2 nucleoprotein, and may include one or more peptide sequences selected from the group comprising the peptide sequence of SEQ ID NO: 7 and the peptide sequence of SEQ ID NO: 8.

[0113] In the context of this specification, SEQ ID NO: 7 and SEQ ID NO: 8 may mean:

[0114] Sequence number 7: CGPHLVVVDDF

[0115] Sequence number 8: DPEGGQHRLDF

[0116] In the context of this specification, a peptide may refer to a short amino acid sequence that specifically binds to the SARS-CoV-2 nucleoprotein (NP). These peptides have specific sequences, such as SEQ ID NO: 7 (CGPHLVVVDDF) and SEQ ID NO: 8 (DPEGGQHRLDF). These peptide sequences are optimized for binding to SARS-CoV-2 NP, allowing for the specific detection of the virus. These peptides can be utilized in diagnostic kits or other biosensor systems to rapidly and accurately diagnose SARS-CoV-2 infection. These peptides can be utilized to generate or alter a signal upon binding to the virus, enabling the detection and quantification of the virus.

[0117] In the context of this specification, nucleoprotein may refer to a core structural protein of a virus that coats and protects the RNA genome of the virus. In particular, the nucleoprotein (Nucleocapsid protein, NP) of SARS-CoV-2 may play a crucial role in the stability and replication of the virus particle. This protein binds to viral RNA to form the nucleocapsid, which may be essential for the survival and replication of the virus. Nucleoprotein is also an important target for diagnosis and research because it has a relatively low mutation rate compared to other viral structural proteins, allowing it to be used as a diagnostic marker. Peptides or antibodies that can specifically detect and bind to nucleoprotein could be very useful tools for diagnosing SARS-CoV-2 infection.

[0118] Meanwhile, the antibody or antigen-binding fragment thereof according to an embodiment of the present invention may include an antibody or antigen-binding fragment thereof that specifically binds to the SARS-CoV-2 nucleoprotein, and may include a variable region comprising CDR1 of SEQ ID NO: 1, CDR2 of SEQ ID NO: 2, and CDR3 of SEQ ID NO: 7; and a variable region comprising CDR1 of SEQ ID NO: 1, CDR2 of SEQ ID NO: 2, and CDR3 of SEQ ID NO: 8.

[0119] In the context of this specification, sequence numbers 1 and 2 may mean:

[0120] Sequence number 1: TYGIQ

[0121] Sequence number 2: WIHAGTGGTKYSRKFQG

[0122] In the context of this specification, an antibody or antigen-binding fragment may refer to a portion of an immunoglobulin that can specifically bind to the SARS-CoV-2 nucleoprotein (NP). They may have a variable region consisting of sequences including SEQ ID NO: 1 (TYGIQ) and SEQ ID NO: 2 (WIHAGTGGTKYSRKFQG). The variable region includes complementarity determining regions (CDRs) that determine the binding specificity of the antibody, and in this case, CDR1, CDR2, and CDR3 of SEQ ID NO: 7 (CDR3: CGPHLVVVDDF) or SEQ ID NO: 8 (CDR3: DPEGGQHRLDF) may be applicable. Such antibodies or antigen-binding fragments can be used to bind to SARS-CoV-2 NP to identify or quantify the presence of the virus.

[0123] In the context of this specification, SARS-CoV-2 is used as an abbreviation for Severe Acute Respiratory Syndrome Coronavirus 2. This virus was first discovered in late 2019 and caused the global COVID-19 pandemic. SARS-CoV-2 is an RNA virus belonging to the genus Beta-coronavirus that can infect multiple species, including humans, primarily causing respiratory symptoms. The virus is known to cause infection by binding to the ACE2 receptor on host cells, particularly through the spike protein located on its surface. Diagnosis, treatment, and prevention of SARS-CoV-2 are major global public health concerns, and the present invention can make a significant contribution to the effective detection and diagnosis of this virus.

[0124] In one embodiment, the antibody or antigen-binding fragment thereof may comprise a heavy chain variable region comprising HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, and HCDR3 of SEQ ID NO: 7; a heavy chain variable region comprising HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, and HCDR3 of SEQ ID NO: 8;

[0125] In the context of this specification, the meaning of HCDR can refer to a highly variable portion of the complementarity determining region (CDR) located in the heavy chain of an antibody. The specificity and binding ability of an antibody can be largely determined by the amino acid sequence in this HCDR region. Generally, antibodies have two types of polypeptide chains, namely, a light chain and a heavy chain. Each chain contains three CDR regions, of which HCDR can be a CDR located in the heavy chain. HCDR1, HCDR2, HCDR3, etc. each refer to a specific portion within the CDR of the heavy chain, and these can play an important role in binding an antibody to a specific antigen. These HCDR regions are key elements that increase the diversity and specificity of antibodies, allowing the antibody to bind to an antigen with high specificity.

[0126] In one embodiment, the antibody or antigen-binding fragment thereof may be in the form of a single-domain antibody. In the context of this specification, a single-domain antibody refers to an antibody composed of a single polypeptide chain, rather than the complex structure of a traditional antibody. Such single-domain antibodies are small in size and structurally simple, have high target accessibility, and can be easily produced. These antibodies can provide high specificity and binding capacity for specific antigens, including clustered region (CDR) regions. These small and flexible antibodies have the ability to bind to antigens in small spaces that are difficult for traditional antibodies to access, and may have various potential applications in biomedical and diagnostic fields. In particular, they can be used as effective tools for specifically detecting viral components such as the SARS-CoV-2 nucleoprotein. They can also be utilized as core components of diagnostic kits or biosensor systems.

[0127] Meanwhile, the nucleic acid according to an embodiment of the present invention may encode the peptide or the antibody or an antigen-binding fragment thereof. In the context of this specification, coding refers to the process by which a specific DNA or RNA sequence determines the amino acid sequence of a specific protein or peptide. This refers to the process by which genetic information is translated into a protein, and through this process, the genetic sequence can determine the structure and function of a specific protein. According to an embodiment of the present invention, a specific nucleic acid may encode a peptide or antibody or an antigen-binding fragment thereof that specifically binds to the SARS-CoV-2 nucleoprotein (NP), thereby providing the information necessary to produce a specific protein or peptide. This coding process can play an important role in various applications, such as the development of diagnostic kits, biosensors, therapeutics, or vaccines, through recombinant DNA technology. In other words, the nucleic acid can serve as a blueprint for the production of a specific protein or peptide used to detect or defend against viral infection.

[0128] Meanwhile, a recombinant expression vector according to an embodiment of the present invention may include the nucleic acid. In the context of this specification, a recombinant expression vector refers to a DNA molecule engineered for gene expression. It can serve to transfer a gene into a new host cell and cause the gene to be translated into a protein. A recombinant expression vector contains a specific gene or DNA sequence, thereby inducing the production of a specific protein in the host cell. Such a vector may typically include a promoter, an origin for gene replication, a selection marker, and other regulatory sequences for the expression of the inserted gene. According to an embodiment of the present invention, a recombinant expression vector may include a nucleic acid encoding a peptide or antibody or antigen-binding fragment thereof that specifically binds to the SARS-CoV-2 nucleoprotein (NP), thereby facilitating the mass production of a specific protein or peptide. This has important applications in the biomedical and diagnostic fields, and may enable the efficient production of proteins or peptides that can be used to detect or defend against viral infections.

[0129] Meanwhile, cells according to embodiments of the present invention can be transformed with the recombinant expression vector. In the context of this specification, "transformation" refers to the process of introducing exogenous genetic material into a cell. Through this process, the cell acquires new genetic characteristics, which can lead to changes in the physiological, morphological, or genetic characteristics of the cell. Transformation can be widely used in gene expression, protein production, and biotechnology research and applications. Cells according to embodiments of the present invention can be transformed with a recombinant expression vector. Such vectors contain a gene encoding the amino acid sequence of a specific protein or peptide, and can induce expression of the protein or peptide by delivering it into the cell. This process can play a key role in various fields, including genetic manipulation, biomedical research, protein engineering, and the development of therapeutics and vaccines. For example, cells transformed with a recombinant expression vector containing a gene encoding a peptide, antibody, or antigen-binding fragment thereof that specifically binds to the SARS-CoV-2 nucleoprotein (NP) could play an important role in detecting or defending against viral infection by enabling mass production of the protein or peptide.

[0130] In one embodiment, the cell may comprise one or more cells selected from the group consisting of animal cells, plant cells, yeast, Escherichia coli, and insect cells. In one embodiment, the cell is selected from the group consisting of monkey kidney cells 7 (COS7), NSO cells, SP2 / 0 cells, Chinese hamster ovary (CHO) cells, W138, baby hamster kidney (BHK) cells, MDCK, myeloma cell lines, HuT 78 cells, and HEK293 cells, Escherichia coli, Bacillus subtilis, Streptomyces sp., Pseudomonas sp., Proteus mirabilis or Staphylococcus sp., Aspergillus sp., Pichiapastoris, Saccharomyces cerevisiae, and Schizosaccharomaces. It may comprise one or more cells selected from the group comprising Schizosaccharomyces sp. and Neurospora crassa.

[0131] The selection of the above cells may be based on a cell type suitable for the expression and production of a specific protein or peptide. Among various cell types, monkey kidney cell 7 (COS7), NSO cells, SP2 / 0 cells, Chinese hamster ovary (CHO) cells, W138 cells, baby hamster kidney (BHK) cells, MDCK cells, myeloma cell lines, HuT 78 cells, and HEK293 cells are all of human or animal origin, and they can be widely used for the expression and production of specific proteins. In addition, bacterial types such as Escherichia coli, Bacillus subtilis, Streptomyces, Pseudomonas, Proteus mirabilis, or Staphylococcus can also be used for protein production. In addition, fungal or yeast cells such as Aspergillus, Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomaces, and Neurospora crassa may also be suitable for protein production. These cells can be used to purify, modify, or optimize the functional activity of proteins based on their individual characteristics. Each cell type is selected based on its production and functional properties for specific proteins or peptides, which can be crucial for efficient protein production in biomedical and diagnostic applications.

[0132] Meanwhile, a substance for detecting SARS-CoV-2 nucleoprotein according to an embodiment of the present invention may include: the antibody or an antigen-binding fragment thereof; a switching peptide capable of specifically and reversibly binding to one or more of the first to fourth framework regions (FR1, FR2, FR3, and FR4) of the heavy chain or light chain of the antibody or an antigen-binding fragment thereof; and a marker bound to the switching peptide.

[0133] In the context of this specification, a "switching peptide" refers to a short amino acid sequence that can specifically and reversibly bind to one or more of the first through fourth framework regions (FR1, FR2, FR3, and FR4) of the heavy or light chain of a specific antibody or antigen-binding fragment thereof. Such switching peptides can be used to alter the structural and functional properties of an antibody, thereby modulating its binding ability or specificity. Switching peptides can act as an "on and off" switch to activate the antibody upon binding to it, which can contribute to enhancing the efficiency and accuracy of antibody-based therapeutics or diagnostic tools. The switching peptides can be conjugated with a label, which can facilitate the detection or tracking of the antibody-peptide complex. The label can take various forms, such as a fluorescent substance, a radioisotope, an enzyme, or another biochemical substance, and can thereby detect the presence or activity of the antibody. For example, using a marker conjugated to a switching peptide, the location or amount of antibodies that specifically bind to the SARS-CoV-2 nucleoprotein can be accurately measured, which could play an important role in the diagnosis and monitoring of viral infections.

[0134] In one embodiment, the antibody or antigen-binding fragment thereof may be single-stranded. As described above, the single-stranded antibody or antigen-binding fragment can provide high binding specificity and sensitivity to a specific antigen, and its small size and simple structure can facilitate its manufacture and use. A single-stranded antibody or antigen-binding fragment may utilize only one of the variable regions of a traditional double-stranded antibody, or may be composed of a specifically designed single polypeptide chain. This single-stranded structure reduces the size and complexity of the antibody, allowing it to more easily access antigen-binding sites that are otherwise inaccessible. This may be particularly advantageous for binding to specific viral proteins or small molecules. The use of a single-stranded antibody or antigen-binding fragment according to an embodiment of the present invention may play a significant role, for example, in a material for detecting the SARS-CoV-2 nucleoprotein. This single-stranded structure can specifically bind to specific viral proteins, providing more accurate and sensitive diagnostic results. This contributes to the early detection and monitoring of viral infections, which can provide crucial information for public health management and infectious disease response strategies.

[0135] In one embodiment, the marker may be a fluorescent marker. In the context of this specification, "fluorescent marker" refers to a fluorescent substance that binds to a specific antibody or antigen-binding fragment thereof, enabling the visual detection of the presence or activity of the antibody. Such fluorescent markers can enable the precise measurement of the location or amount of the antibody through a fluorescent signal generated when the antibody binds to a specific antigen. The use of fluorescent markers can be particularly useful in diagnostic applications. For example, by conjugating a fluorescent marker to an antibody that specifically binds to the SARS-CoV-2 nucleoprotein, the location where the antibody interacts with the virus can be precisely identified and visualized. This provides accurate information for the diagnosis and monitoring of viral infections, which can aid in the early detection of infections and the establishment of appropriate therapeutic measures.

[0136] Fluorescent markers according to embodiments of the present invention can be selected from a variety of fluorescent materials, which emit light in response to light of a specific wavelength. These fluorescent signals can be observed and analyzed using specialized fluorescence microscopes or other fluorescence detection devices, making them valuable tools for laboratory diagnostics, field testing, and clinical research.

[0137] Meanwhile, a one-step SARS-CoV-2 diagnostic kit according to an embodiment of the present invention may include a substance for detecting the SARS-CoV-2 nucleoprotein. In the context of the present specification, a "one-step SARS-CoV-2 diagnostic kit" refers to a diagnostic tool designed to rapidly and accurately detect the SARS-CoV-2 nucleoprotein. This diagnostic kit may include the above-mentioned substance for detecting the SARS-CoV-2 nucleoprotein, thereby enabling simple and rapid diagnosis of viral infection. A key component of the one-step SARS-CoV-2 diagnostic kit may be a complex comprising a specific antibody or an antigen-binding fragment thereof, a switching peptide, and a fluorescent marker. This complex can specifically react with the SARS-CoV-2 nucleoprotein, thereby enabling rapid detection of infection. This diagnostic kit is particularly designed to be easy to use in the field and can be used without requiring complex laboratory equipment or specialized skills. Users can collect a sample, apply it to a diagnostic kit, and obtain results. The presence or absence of a fluorescent signal determines whether they are infected. The advantages of these one-step diagnostic kits lie in their rapid results and ease of use. This can play a crucial role in public health management and infectious disease response strategies, especially during pandemics. These diagnostic kits are essential for early detection and appropriate treatment, and can help prevent the spread of the virus.

[0138] The above kit does not exclude the addition of other components. Examples of other components that the kit may additionally include to realize diagnostic utility include a sample preparation kit, a nucleic acid extraction kit, an amplification kit, and various detection and analysis tools. These additional components may be necessary to optimize each step of the diagnostic process and obtain faster and more accurate results. For example, a sample preparation kit may provide tools that allow the user to easily prepare and process various types of samples, such as blood, saliva, and nasal swabs. This can play a crucial role in improving diagnostic accuracy by ensuring proper sample collection and storage. A nucleic acid extraction kit may be used to extract viral RNA or DNA from a sample. This process is the first step in detecting the viral genetic material and may be essential for an accurate diagnosis. An amplification kit is used to amplify the extracted nucleic acid, which allows for a more definitive confirmation of the presence of the virus. In particular, when using PCR (polymerase chain reaction) technology, even very small amounts of viral genetic material can be detected. Additionally, various detection and analysis tools can be used to read and interpret the signals from antibodies conjugated to fluorescent markers. These tools can be provided as both laboratory-based and portable devices, enabling rapid on-site diagnosis. Thus, a one-step SARS-CoV-2 diagnostic kit can provide an efficient method for rapid and accurate detection of viral infections by combining various components and tools.

[0139] Meanwhile, a one-step SARS-CoV-2 diagnostic method according to an embodiment of the present invention may include a step of mixing a substance for detecting SARS-CoV-2 nucleoprotein in the one-step SARS-CoV-2 diagnostic kit with a sample to be detected, and then identifying the marker. In one embodiment, the marker may be prepared as a fluorescent marker.

[0140] Hereinafter, embodiments of the present invention will be described. However, the embodiments described below are merely some embodiments of the present invention, and the scope of the present invention is not limited to the embodiments described below.

[0141] Summary of the Example

[0142] In the present invention, we propose a one-step immunoassay based on Fv antibodies and switching peptides for porcine infectious diarrhea virus (PEDV). This method requires only sample mixing and does not require additional reaction or washing steps. Fv antibodies with binding affinity for the PEDV spike protein were selected from an Fv antibody library using the receptor binding domain (RBD) of the spike protein as a screening probe. Four types of Fv antibodies with binding affinity for the RBD antigen were expressed as soluble proteins, and the affinity constants (Kd) were calculated using an isothermal model to be 142 nM (Anti-RBD-1), 149 nM (Anti-RBD-2), 101 nM (Anti-RBD-3), and 83 nM (Anti-RBD-4). The one-step immunoassay for PEDV detection consisted of a displacement immunoassay using a fluorescently labeled switching peptide. A one-step immunoassay based on switching peptide was performed using PEDV, and the limits of detection (LOD) values ​​for PEDV detection were measured as Ct = 41.5 (~8.6 copies / μL) (Anti-RBD-1), Ct = 40.7 (~14.5 copies / μL) (Anti-RBD-2), Ct = 38.1 (~94.3 copies / μL) (Anti-RBD-3), and Ct = 39.3 (~38.7 copies / μL) (Anti-RBD-4). Compared with the conventional lateral flow immunoassay (Ct = 33.0), the one-step immunoassay was judged to have a significantly improved LOD for PEDV detection. Finally, the interactions between the selected Fv-antibodies and the PEDV RBD were investigated using docking simulations and compared with the amino acid sequences of the host cell receptors aminopeptidase N (APN) and angiotensin-converting enzyme-2 (ACE-2).

[0143] Materials and Methods

[0144] ingredient

[0145] High-salt Luria-Bertani (LB) medium and LB agar were purchased from Duchefa Co., Haarlem, the Netherlands. Bovine serum albumin (BSA) and Tween® 20 were purchased from Sigma-Aldrich, Seoul, Korea. Ni-NTA magnetic beads, high-fidelity Phusion polymerase, and PCR reagents were purchased from Thermo Fisher Scientific Inc., Waltham, MA, USA. PCR cleanup and NucleoSpin gel kits were purchased from Macherey-Nagel Co., Düren, Germany. Primers were synthesized by BIONICS CO., Seoul, Korea. Klenow (exo-) polymerase was purchased from New England Biolabs, Ipswich, MA, USA. The plasmid for the RBD of porcine infectious diarrhea virus (PEDV) tagged with green fluorescent protein (Superfolder GFP; sGFP) was custom synthesized by Cosmogenetech, Seoul, Korea. PEDV was provided by Chungnam National University, Daejeon, Korea. Fv-antibody peptides were synthesized at 90% purity by Peptron Co., Daejeon, Korea. Amicon® Ultra 0.5 mL filters with a cutoff of 10 kDa were purchased from Millipore Co., Darmstadt, Germany. Phosphate-buffered saline (PBS) (20X) was purchased from CUREBIO Co., Seoul, Korea. A commercial lateral flow immunoassay kit for PEDV detection was purchased from MEDIAN diagnostics, Gangwon-do, Korea.

[0146] Preparation and screening of Fv-antibody libraries

[0147] As previously reported, Fv-antibody libraries were synthesized and autodisplayed (Jung et al., 2023; Lee et al., 2021; Sung et al., 2022a; Sung et al., 2022b). To synthesize the Fv-antibody library in the CDR3 region via site-directed mutagenesis, a single-stranded forward primer containing a randomized CDR3 sequence (75 bp) was mixed with the corresponding reverse primer (22 bp), as shown in Figure 1 (Dao-Pin et al., 1987; Fortier et al., 2005; Jung et al., 2021a; Jung et al., 2021b; Jung et al., 2021c; Jung et al., 2023; Lee et al., 2021; Sung et al., 2022a; Sung et al., 2022b). The specific primer sequences used for the Fv-antibody library are summarized in Table 1.

[0148] Primer typeOligonucleotide sequenceRandomized forward primer (75 bases)5'-GTCTATTATTGCGCTCGT 1 KRYVNN 7 VNNVNN 13 VNNVNN 19 VNNVNN 25 VNNGAT 31 KWYTGGGGTCAAGGTACTACGGTTACG-3'Corresponding reverse primer (22 bases)3'-CCCAGTTCCATGATGCCAATGC-5'Composition of nucleotides at each positionN = A, C, G, T / R = A, G / K = G, T / Y = C, T / W = A, T / V = A, C, G

[0149] The Fv-antibody library plasmid was constructed using a PCR machine as described in the subsequent steps. Subsequently, the forward primer (2 μL) containing the randomized CDR3 sequence and the reverse primer (2 μL) were combined using NEB buffer (4 μL) and deionized water (32 μL) (heated at 95°C for 5 min, then cooled to 36°C at a cooling rate of -0.3°C / s). For the extension step, Klenow (exo-) polymerase (3 μL), NEB buffer (16 μL), 10 mM dNTPs (8 μL), and deionized water (133 μL) were added to the product of the above step (40 μL), and the reaction was performed at 37°C for 15 min. After inactivating the polymerase reaction at 75°C for 20 min, the double-stranded primers of the Fv-antibody library containing the randomized CDR3 region obtained in this step were purified using a PCR cleanup kit and NucleoSpin® gel. The Fv-antibody library plasmid was prepared in a total volume of 50 μL by mixing the pST009 template plasmid (150 ng), the double-stranded primer of the purified Fv-antibody library (140 ng), 10 mM dNTPs (1 μL), HF buffer (10 μL), Phusion high-fidelity polymerase (0.5 μL), and DW. The PCR reaction was then performed using the following steps: (1) initial denaturation at 98°C for 1 min, (2) denaturation at 98°C for 30 s, (3) binding at 68°C for 1 min, (4) extension at 72°C for 5 min, (5) repeating steps (2)-(4) for 30 cycles, and (6) termination at 72°C for 10 min. After the PCR reaction step, the template plasmid (pST009) was digested with DpnI restriction enzyme at 37°C for 16 h. Finally, the Fv-antibody library plasmid was filtered using an Amicon® Ultra filter with a cutoff of 100 kDa. E. of Fv-antibodies with different sequences in the CDR3 region.Autodisplay in the outer membrane of E. coli BL21(DE3) was performed by electroporation of electrocompetent cells with the prepared Fv-antibody library plasmid, as shown in Fig. 2 (Bong et al. 2018; Dower et al. 1988; Jose and Meyer 2007; Jung et al. 2021b; Jung et al. 2023; Lee et al. 2021; Lopez-Rojas et al. 2023; Sung et al. 2022a; Sung et al. 2022b).

[0150] Autodisplay of Fv antibody libraries on the outer membrane of E. coli

[0151] Autodisplay of Fv antibodies on the outer membrane of Escherichia coli was performed as previously reported (Park et al., 2011; Yoo et al., 2011). The transformed E. coli cells were cultured in high-salt Luria-Bertani (LB) medium containing 50 μg / mL kanamycin antibiotic at 37°C with shaking at 200 rpm for 16 h. Then, 50 μL of the cultured E. coli cells were inoculated into 5 mL of LB medium containing 4 μL of 99% β-mercaptoethanol, kanamycin (50 μg / mL), and ethylenediaminetetraacetic acid (5 μM), and the mixture was shaken at 150 rpm until the optical density at 600 nm reached 0.5. To induce Fv-antibody expression, 1 mM isopropylthio-β-galactoside (IPTG) was added to the E. coli culture and incubated at 30°C and 150 rpm for 3 h.

[0152] Screening of target Fv-antibody variants (clones)

[0153] Selection of target E. coli harboring autodisplayed Fv-antibodies with specific binding activity to the RBD antigen was performed in the following steps: (1) Ni-NTA magnetic beads (10 μL) and RBD antigen were mixed with 0.1% PBST (0.1% Tween 20) for 1 h. (2) The magnetic beads bound to monoclonal antibodies were sorted using a magnet, and the unbound RBD antigen was washed five times with 0.1% PBST and PBS. (3) The RBD-bound magnetic beads were mixed with E. coli harboring the autodisplayed Fv-antibody library (100 μL, OD600 = 1.0) and rotated at 20 rpm at 23°C for 1 h. (4) The E. coli-bound magnetic beads were separated using a magnet and washed ten times with 0.1% PBST. (5) The magnetic beads bound to the isolated E. coli were resuspended in LB medium (50 μL), and then spread on an agar plate containing 50 μg / mL of kanamycin antibiotic to obtain E. coli clones (Jung et al. 2021b; Kim et al. 2023b; Mair et al. 2019). The binding properties of E. coli with autodisplayed Fv antibodies to the RBD antigen were evaluated using a FACSCalibur flow cytometer (Becton-Dickinson, Franklin Lakes, NJ, USA). The E. coli with the selected autodisplayed Fv antibodies were mixed with the RBD antigen for 1 h while gently rotating at 20 rpm. After centrifugation (3000 × g, 3 min), the E. coli were washed twice with PBS, and the quantitative binding of E. coli with autodisplayed Fv antibodies to the RBD antigen was performed using a flow cytometer.

[0154] Expression of Fv antibodies

[0155] The plasmid encoding the open reading frame of the Fv antibody having binding activity to the RBD antigen was synthesized by Peptron Co., Daejeon, Korea, and is shown in Figure 3. The amino acid sequence of the Fv antibody consists of CDRs and FRs, which are summarized in Table 2.

[0156] RegionAmino acid (N → C-term)Oligonucleotide sequence (5' → 3')Frame-1EVQLVESAAEVRRPGASVKITCKASGYSFSGAA GTG CAG CTC GTG GAA AGC GCT GCC GAA GTT CGG CGT CCT GGG GCT AGC GTG AAG ATC ACC TGC AAA GCG TCC GGC TAT TCA TTC AGCCDR1TYGIQACC TAT GGG ATT CAGFrame-2WMRQAPGQRPEWLGTGG ATG CGC CAA GCG CCA GGC CAG CGT CCG GAA TGG CTT GGGCDR2WIHAGTGGTKYSRKFQGTGG ATA CAT GCA GGC ACA GGT GGG ACT AAG TAC TCG CGC AAA TTT CAG GGTFrame-3RITITRDTSANTVYLDLNSLTSEDTAVYYCARCGC ATT ACT ATC ACC CGT GAT ACC AGC GCG AAT ACC GTC TAT CTG GAT CTG AAC TCT CTG ACA TCG GAG GAT ACG GCC GTC TAT TAT TGC GCT CGTCDR3(Template)DKVTVWACQDNGAC AAA GTT ACA GTC TGG GCT TGT CAG GAT AATFrame-4WGQGTTVTVSSTGG GGT CAA GGT ACT ACG GTT ACG GTC AGC AGT

[0157] Fv antibodies were expressed in Escherichia coli by transforming a custom plasmid into competent cells. The transformed E. coli were cultured in 100 mL of high-salt LB medium containing 1 mM IPTG and 30 μg / mL carbenicillin at 30°C for 16 h. The E. coli pellet was collected by centrifugation (3,000 × g for 3 min) and then resuspended in 20 mL of binding buffer (5 mM Tris-HCl, 0.5 mM EDTA, 1 M NaCl) containing 6 M urea. The resuspended E. coli was then sonicated using an ultrasonic reactor (Vibracell VCX-130, Sonics, USA). The cell lysate was centrifuged at 25,000 × g for 10 min. The Fv antibody in the supernatant was purified using a His-tag purification column from Roche (Bassel, Switzerland) with elution buffer (binding buffer containing 6 M urea and 500 mM imidazole). The purified Fv antibody was dialyzed at 50 rpm at 4°C for 16 h to remove urea and imidazole (Jung et al., 2021b; Jung et al., 2021c; Jung et al., 2023; Lee et al., 2021; Spriestersbach et al., 2015; Sung et al., 2022a).

[0158] Measurement of binding affinity of Fv antibodies

[0159] Surface plasmon resonance (SPR) measurements of autodisplayed Fv antibodies obtained from RBD antigens were measured using an SPR biosensor from i-Cluebio, Seongnam, Korea. SPR assays were prepared using purified Fv antibodies on gold-coated BK-7 glass. Gold-coated BK-7 glass was prepared using the following steps: BK-7 glass (1 × 1 cm2) was washed with ethanol, isopropanol, and deionized water. Then, a 2 nm titanium adhesion layer was coated, and gold was sputtered using a 48 nm K575X at Quorum Emitech, Kent, UK (Bong et al., 2018; Jung et al., 2023; Kim et al., 2022c). Figure 4 shows that the SPR chip was incubated with purified Fv antibodies (10 μg / mL) at 23°C for 1 h. After washing with PBS, blocking with BSA (1 mg / mL) was performed for 30 minutes. After washing again with PBS, RBD antigen (1–500 nM) was flowed for 15 minutes (10 μL / min) as a binding step. Finally, PBS was flowed for 15 minutes (10 μL / min) as a separation step. SPR measurements were performed by processing the sample under flow conditions and recording the SPR signal according to the amount of bound analyte after the separation step. The SPR signal was calculated from the dose-response curve of the sample with the solution protein, which was performed using the isothermal model:

[0160] R = (Rmax[Ag]) / (Kd+[Ag])

[0161] Here, R represents the SPR signal (Rmax = maximum SPR signal), [Ag] represents the solution protein concentration, and Kd represents the affinity constant. The SPR signal results were fitted using Hill's equation (Bong et al. 2020; Kim et al. 2022c).

[0162] One-step immunoassay for PEDV (immunoassay)

[0163] A one-step immunoassay for quantitative analysis of PEDV was performed using Fv antibodies. For the one-step immunoassay, Fv antibodies (10 μg / mL, 100 μL) were immobilized on microplates. After washing twice with PBS, blocking with BSA (1 mg / mL) was performed for 30 minutes. After washing twice more with PBS, fluorescently labeled switching peptide L1 (1 μM, 100 μL) was conjugated to the immobilized Fv antibodies for 1 hour. In response to positive samples containing the corresponding antigen, the pre-bound switching peptide (containing a fluorescent label) was released depending on the concentration of the antigen in the sample. In negative samples without the corresponding antigen, there was no change in fluorescence. Fluorescence signals were measured using a Victor5 Fluorometer (PerkinElmer Co., Massachusetts, USA) at an excitation wavelength of 488 nm and an emission wavelength of 512 nm. The limit of detection (LOD) was determined as the mean intensity plus three standard deviations (3σ) from the blank sample (PBS), and the dynamic range was calculated using a linear correlation with the log concentration of PEDV. Additionally, the assay results were compared with a commercial lateral flow immunoassay. The commercial lateral flow immunoassay kit for PEDV detection was purchased from MEDIAN Diagnostics, Gangwon-do, South Korea (Kim et al., 2023a; Kim et al., 2022a; Kim et al., 2022b; Kim et al., 2022c; Lee et al., 2022; Sung et al., 2022a).

[0164] Results and Discussion

[0165] Fv antibody screening against PEDV RBD

[0166] Porcine infectious diarrhea virus (PEDV) is classified as an alphacoronavirus. It is known that this virus infects host cells through the interaction between the spike protein (SP) on the viral surface and the aminopeptidase N (APN) receptor on the outer membrane of the host cell (Lee et al., 2011; Li et al., 2007; Lin et al., 2022; Liu et al., 2015). Fv antibodies with binding affinity to the spike protein of PEDV have been screened from Fv antibody libraries using the receptor-binding domain (RBD) of the spike protein as a screening probe (Dao-Pin et al., 1987; Fortier et al., 2005; Jung et al., 2021c; Lee et al., 2022; Lee et al., 2021; Sung et al., 2022a; Sung et al., 2022b). As shown in Figure 5, the RBD is composed of 129 amino acid residues (21.2 kDa), including a portion of the spike protein (amino acids 582 to 781) (Deng et al., 2016; Lee et al., 2011). Additionally, superfolder green fluorescent protein (sGFP, 26.9 kDa) and a His-tag are incorporated into the structure of the RBD antigen (49 kDa). Fv antibodies against the RBD antigen were screened from an autodisplayed Fv antibody library using a flow cytometer. When the RBD antigen reacted with the Fv antibody library, E. coli cells with higher fluorescence signals than the baseline were observed, as shown in Figure 6. In the control strain, the number of E. coli cells in the high fluorescence region (i.e., screening gate) was significantly lower than that of the baseline. These results indicate that the RBD antigen bound to E. coli cells through interaction with the CDR3 region of the Fv antibody. E. coli cells isolated from the screening gate were cultured on agar plates. As shown in Figure 7, seven colonies (clones) were randomly selected, and the selected Fv-antibody library clones were expressed on the outer membrane of E. coli using autodisplay technology (Park et al. 2015; Yoo et al. 2015).These clones were then tested against the RBD antigen. Two of these clones exhibited significantly higher binding affinity than the other clones and the control strain (containing only CDR1 and CDR2); their nucleic acid sequences are listed in Table 3.

[0167] Screened cloneNucleotideCDR3 sequenceKd value (FACS)Anti-RBD-13-TACGGACCAAGTATTGTAGAACAAACAGATGTT-5 1 YGPSI 6 VEQTD 11 V22 nMAnti-RBD-23-TACGTGGGTCTTGGGCCGGGGACCGTTGATTTC-5 1 YVGLG 6 PGTVD 11 F20 nMAnti-RBD-33-TACGAGACCGTACGCGGCAACGGCACGGATGTC-5 1 YETVR 6 GNGTD 11 V16 nMAnti-RBD-43-TACGGCGTCCTTCGGCGGGGCTGCCTTGATTTC-5 1 YGVLR 6 RGCLD 11 F35 nM

[0168] These strong fluorescent signals indicate that the RBD antigen specifically bound to the autodisplayed Fv antibody of the selected clone. Furthermore, the specific binding of the RBD antigen was achieved through the CDR3 of the selected clone. The binding affinity of the RBD antigen was measured using a flow cytometer after treating E. coli cells of the screened clones with the RBD antigen. As shown in Figure 8, the fluorescent signal increased quantitatively in a concentration-dependent manner when reacted with the two selected clones at RBD antigen concentrations ranging from 1.2 to 300 nM. In contrast, the fluorescent signal remained at the baseline in a concentration-dependent manner for the two selected clones containing only GFP. These results indicate that the RBD antigen (but not GFP) specifically bound to the autodisplayed Fv antibody of the selected clone. From the dose-response curves of these two clones, the affinity constants (Kd) were estimated to be 22 nM (clone anti-RBD-1) and 20 nM (clone anti-RBD-2) using an isothermal model. Fv antibodies against the RBD antigen were selected from the autodisplayed Fv antibody library using magnetic bead screening. For the screening of Fv antibodies, the RBD antigen was immobilized on magnetic beads through the interaction of the His-tag of the magnetic beads with Ni-NTA. After the Fv antibody library was treated with the magnetic beads, the E. coli cells bound to the magnetic beads were cultured on agar plates, as shown in Figure 9. Seven colonies (clones) were randomly selected, and the selected Fv antibody library clones were expressed on the outer membrane of E. coli using the autodisplay technique (Park et al., 2015; Yoo et al., 2015). These clones were then reacted with the RBD antigen, as shown in Figure 10. Two of these clones exhibited significantly higher binding affinity compared to the other clones and to a control strain containing only CDR1 and CDR2. Finally, both clones were determined to possess the target sequence after nucleic acid sequencing, and the nucleic acid sequences are listed in Table 2.These strong fluorescent signals indicate that the RBD antigen specifically bound to the autodisplayed Fv antibody of the selected clone. Furthermore, the specific binding of the RBD antigen was achieved through the CDR3 of the selected clone. The binding affinity of the RBD antigen was measured using a flow cytometer after treating E. coli cells of the screened clones with the RBD antigen or only GFP. As shown in Figure 11, the fluorescent signal increased quantitatively in a concentration-dependent manner when reacted with the two selected clones at RBD antigen concentrations ranging from 1.2 to 300 nM. In contrast, the fluorescent signal remained at the baseline in a concentration-dependent manner for the two selected clones containing only GFP. These results indicate that the RBD antigen (but not GFP) specifically bound to the autodisplayed Fv antibody of the selected clone. From the dose-response curves of these two clones, the affinity constants (Kd) were measured to be 16 nM (clone anti-RBD-3) and 35 nM (clone anti-RBD-4) using an isothermal model.

[0169] One-step immunoassay based on switching peptides

[0170] Four types of selected Fv antibodies with binding affinity to RBD antigen were expressed as soluble proteins as shown in Figure 3. The Fv antibodies consisted of three CDRs and four FRs (Marillet et al. 2017), including a selected CDR3 sequence (13 kDa) for binding to RBD antigen. These antibodies were expressed as fusion proteins with GFP (27 kDa) at the N-terminus and a His-tag at the C-terminus. The fusion proteins containing GFP had the following advantages for the expression of Fv antibodies: (1) Fv antibodies had limited solubility, but the fusion proteins containing GFP had much improved solubility (Jung et al. 2023; Liu et al. 2019; Pedelacq et al. 2006), and (2) GFP could be effectively used to immobilize on the metal surface of biosensors (Jung et al. 2023). The immobilization efficiency of GFP was measured similarly to that of BSA, a blocking protein frequently used in immunoassays. The expression of four types of Fv-antibodies as fusion proteins with a molecular weight of approximately 40 kDa was confirmed by SDS-PAGE. After immobilizing the Fv-antibodies on the gold surface of the SPR biosensor, the affinity constants (Kd) of the four expressed Fv-antibodies (Anti-RBD-1, Anti-RBD-2, Anti-RBD-3, and Anti-RBD-4) toward the RBD antigen were measured. After treating with various concentrations of RBD antigen, the association and dissociation profiles were measured as shown in Figure 4. The affinity constants (Kd) were measured separately for the four types of Fv-antibodies by treating with RBD antigen in the concentration range of 1 to 500 nM. The affinity constants (Kd) were calculated using the isothermal model to be 142 nM (Anti-RBD-1), 149 nM (Anti-RBD-2), 101 nM (Anti-RBD-3), and 83 nM (Anti-RBD-4).

[0171] For the one-step immunoassay, a switching peptide was synthesized with an amino acid sequence of a portion of the light chain of IgG, which could specifically bind to the frame region (FR) of the Fv antibody (VH), as shown in Figure 12 (Bong et al., 2021; Kim et al., 2022a; Kim et al., 2022b; Kim et al., 2022c; Lee et al., 2022). When the S protein of PEDV bound to the Fv antibody, the switching peptide could be quantitatively released from the Fv antibody because the S protein of PEDV bound to the Fv antibody more strongly than the switching peptide in a pre-bound state. Since the switching peptide was labeled with a fluorescent dye, a fluorescent signal could be detected depending on the concentration of PEDV. The affinity constants (Kd) for the switching peptides were measured separately by treating four types of Fv-antibodies in the concentration range of 1 - 20 μM, and as shown in Fig. 13, the affinity constants (Kd) were calculated to be 1.19 μM (Anti-RBD-1), 1.24 μM (Anti-RBD-2), 1.23 μM (Anti-RBD-3), and 1.74 μM (Anti-RBD-4) using the isothermal model.

[0172] To realize a one-step immunoassay based on switching peptide, several prerequisites were necessary: ​​(1) the Fv antibody must remain bound to the switching peptide until antigen processing, and (2) the switching peptide must be quantitatively released from the Fv antibody immediately after antigen processing. The difference in the binding constant (Kd) between the switching peptide (Kd = 1.19 - 1.74 μM) and the target antigen (RBD antigen, Kd = 83 - 149 nM) indicated that the switching peptide could be quantitatively released from the immobilized Fv antibody when the target antigen binds to the immobilized Fv antibody.

[0173] The one-step immunoassay for PEDV detection was constructed using a displacement immunoassay. In this immunoassay, the switching peptide dissociates from the Fv antibody and binds to it as soon as the immobilized Fv antibody binds to the target antigen. The switching peptide bound to the immobilized Fv antibody, and as shown in Figure 14, upon reaction with the RBD antigen (or PEDV), the switching peptide dissociated from the immobilized Fv antibody, and the fluorescence signal of the released switching peptide was detected in the reaction solution. The first step in constructing the switching peptide-based one-step immunoassay was to determine the optimal switching peptide concentration. This result indicated that the optimal switching peptide concentration for the one-step immunoassay was 2 μM. The LOD of the one-step immunoassay was determined by determining the switching peptide concentration based on the optimal switching peptide concentration. As shown in Fig. 15, the fluorescence signal quantitatively increased when the target antigen (RBD antigen) was treated with the Fv antibody conjugated to the switching peptide at concentrations ranging from 1 pg / mL to 100 ng / mL. In addition, the LOD values ​​for the detection of the target antigen (RBD antigen) were measured as 7.1 pg / mL (Anti-RBD-1), 155.7 pg / mL (Anti-RBD-2), 1.3 ng / mL (Anti-RBD-3), and 522.1 ng / mL (Anti-RBD-4), which were three times the standard deviation of the fluorescence signal of the blank sample (n = 3). These results demonstrate that RBD antigen (or PEDV) can be detected using the switching peptide-based one-step immunoassay.

[0174] A one-step immunoassay based on the switching peptide was performed using PEDV. The Ct values ​​of the PEDV samples were calculated as described in Figure 16. The assay results were compared with a conventional lateral flow immunoassay for PEDV. Figure 17 shows that the conventional lateral flow assay for PEDV was performed over a 4.9-fold dilution range (Ct value = 30.3) to a 630-fold dilution range (Ct value = 37.3), and a separate signal band for PEDV was observed on the test strip depending on the antigen concentration. The same antigen samples were used in a one-step immunoassay based on the switching peptide. As shown in Figure 18, the one-step immunoassay provided quantitative results based on PEDV over a 100-fold dilution range (Ct value = 34.6) to a 22220-fold dilution range (Ct value = 45.8). The LOD values ​​for PEDV detection were measured as Ct = 41.5 (approximately 8.6 copies / μL) (Anti-RBD-1), Ct = 40.7 (approximately 14.5 copies / μL) (Anti-RBD-2), Ct = 38.1 (approximately 94.3 copies / μL) (Anti-RBD-3), and Ct = 39.3 (approximately 38.7 copies / μL) (Anti-RBD-4), which were three times the standard deviation of the fluorescence signal by the blank sample (n = 3). Compared with the conventional lateral flow immunoassay (Ct = 33.0), the one-step immunoassay was found to have significantly improved LODs for PEDV detection: 362-fold improved LOD (Anti-RBD-1), 208-fold improved LOD (Anti-RBD-2), 34-fold improved LOD (Anti-RBD-3), and 78-fold improved LOD (Anti-RBD-4) compared to the lateral flow immunoassay.

[0175] Docking analysis of Fv antibodies and receptors

[0176] The one-step immunoassay results showed that the four Fv antibodies sensitively detected PEDV RBD. To investigate the interactions between the selected Fv antibodies and PEDV RBD, docking simulations were performed using AutoDock Vina software developed by Scripps Research (La Jolla, CA, USA) (Eberhardt et al. 2021; Goodsell et al. 1996; Sung et al. 2022b; Trott and Olson 2010). These interactions were visualized and analyzed using PyMOL software version 2.5 (DeLano Scientific LLC, South San Francisco, CA, USA) and Discovery Studio Visualizer version 2020 (Dassault Systemes BIOVIA, San Diego, CA, USA). The RBD structure of PEDV (UniProt ID: Q91AV1) was obtained from UniProt (www.uniprot.org) and interacted with the CDR3 regions of four selected Fv-antibodies.

[0177] As shown in Figure 19, the interaction between RBD and anti-RBD-1 was analyzed with a negative Gibbs free energy of -9.6 Kcal / mol. Among the amino acids of RBD, F2, F39, and G73 were designated as those contributing to hydrophobic interactions, and C16, T40, K41, F74, and Y92 were designated as those contributing to hydrogen bonds. The interaction between RBD and anti-RBD-2 occurred in a different RBD region from that of anti-RBD-1, and was analyzed with a negative Gibbs free energy of -10.2 Kcal / mol. The amino acids of RBD participating in hydrophobic interactions were designated as K41, I87, A89, V91, Y93, L101, and A102, and G90 and Y92 were designated as those participating in hydrogen bonds. The interaction between RBD and anti-RBD-3 occurred in a different RBD region from that of the previous Fv-antibody, and was analyzed with a negative Gibbs free energy of -9.2 Kcal / mol. The amino acids of RBD involved in hydrophobic interactions were I87 and P116, and G90, T94, S95, S113, and T115 were assigned to participate in hydrogen bonds. The interaction between RBD and anti-RBD-4 also occurred in a different RBD region from that of the previous Fv-antibody, and was analyzed with a negative Gibbs free energy of -9.2 Kcal / mol. The amino acids of RBD involved in hydrophobic interactions were I79 and L137, and N139, N144, and T145 were assigned to participate in hydrogen bonds. The amino acids interacting with RBD are summarized in Table 4.

[0178] Fv-antibodyInteraction amino acidat PEDV RBDDocking energy (kcal / mol)Anti-RBD-1F2, C 16 , F 39 , T 40 , K 41 , G 73 , F 74 , Y 92-9.6Anti-RBD-2K 41 , I 87 , A 81 , G 90 , V 91 , Y 92 , Y 93 , L 101 , A 102 -10.2Anti-RBD-3I 87 , G 90 , T 94 , S 95 , S 113 , T 115 , P 116 -9.2Anti-RBD-4D 64 , I 97 , L 137 , N 139 , N 144 , T 145 ,-9.2

[0179] These results demonstrated that the four Fv antibodies specifically interacted with different regions of the RBD. Considering the LOD of conventional RT-PCR, a one-step immunoassay based on these four Fv antibodies and switching peptides was considered applicable to the medical diagnosis of PEDV. The binding affinities measured in docking simulations were similar to the measured binding affinities of the four Fv antibodies in Gibbs free energy and were considered comparable to the Kd measured using an SPR biosensor (Fig. 4). Receptors for most human coronaviruses have been identified. Although extensive research on coronaviruses is underway, the identification of the receptor that triggers infection by PEDV, classified as an alphacoronavirus, remains an unknown area requiring continued research (Lin et al. 2022). In recent studies, aminopeptidase N (APN), commonly known as the tentorium antigen CD13 in the field of immunology, has been highlighted as a potential PEDV receptor (Barnieh et al. 2021; Delmas et al. 1992; Tresnan et al. 1996; Yeager et al. 1992). Furthermore, angiotensin-converting enzyme-2 (ACE-2) has also been highlighted as a PEDV receptor, as heparin sulfate induces a conformational change in the S protein RBD, facilitating viral binding to ACE-2 (Clausen et al. 2020; Milewska et al. 2014; Wang et al. 2023). The binding affinity between the PEDV spike protein and the APN receptor on host cells was reported to be 216–6,518 nM, and the binding affinity between the SARS-CoV-2 spike protein and the ACE2 receptor on host cells was reported to be 4.7–10.0 nM (Lan et al. 2020; Walls et al. 2020; Zahradnik et al. 2021).However, whether pAPN and ACE2 regulate the entry of PEDV remains a matter of debate (Ji et al. 2018). To elucidate the infection pathway of PEDV in porcine cells, amino acid sequence similarity analysis between selected Fv antibodies and aminopeptidase N (APN) was performed using BLAST (https: / blast.ncbi.nlm.nih.gov / Blast.cgi), and the results are shown in Figure 20. In the amino acid sequence analysis, the identity and similarity between Fv antibodies and APN (or ACE2) in terms of the percentage of identical amino acid residues (red) and the percentage of amino acid residues with similar chemical properties (blue) were analyzed for four types of Fv antibodies (Ji et al. 2018). Amino acid sequence similarity analysis between the selected Fv antibodies and angiotensin-converting enzyme-2 (ACE2) was also performed using BLAST (https: / blast.ncbi.nlm.nih.gov / Blast.cgi), and the results are shown in Figure 21. This amino acid sequence analysis also analyzed the identity (red) and similarity (blue) of the four Fv antibodies. These results showed that the binding affinities between the four Fv antibodies and the RBD antigen were comparable to the binding affinities between the coronavirus and its receptor on host cells. Therefore, the expressed Fv antibodies are considered applicable for the detection of PEDV. Figure 22 depicts the interaction site between the Fv antibody and the RBD antigen.

[0180] Table 5 shows the amino acid sequence of the switching peptide (L1-peptide).

[0181] Switching peptideAmino acid sequenceL1-peptideTYLEW 5 YPQKP 10 GQSPK 15 LLIYK 20

[0182] conclusion

[0183] Fv antibodies with binding affinity to the PEDV spike protein were screened from a Fv antibody library using the receptor binding domain (RBD) of the spike protein as a screening probe. Flow cytometry analysis revealed that two clones exhibited significantly higher binding affinities than the other clones, with affinity constants (Kd) of 22 nM (clone anti-RBD-1) and 20 nM (clone anti-RBD-2), respectively, using the isoterm model. Two other clones also exhibited significantly higher binding affinities than the other clones using RBD-immobilized magnetic beads, with affinity constants (Kd) of 16 nM (clone anti-RBD-3) and 35 nM (clone anti-RBD-4), respectively. Four types of Fv antibodies with binding affinity to the RBD antigen were expressed as soluble proteins, and the affinity constants (Kd) were calculated to be 142 nM (anti-RBD-1), 149 nM (anti-RBD-2), 101 nM (anti-RBD-3), and 83 nM (anti-RBD-4) using the isoterm model. The one-step immunoassay for PEDV detection consisted of a displacement immunoassay using a fluorescently labeled switching peptide. A one-step immunoassay based on switching peptide was performed using PEDV, and the limits of detection (LOD) values ​​for PEDV detection were measured as Ct = 41.5 (approximately 8.6 copies / μL) (anti-RBD-1), Ct = 40.7 (approximately 14.5 copies / μL) (anti-RBD-2), Ct = 38.1 (approximately 94.3 copies / μL) (anti-RBD-3), and Ct = 39.3 (approximately 38.7 copies / μL) (anti-RBD-4). Compared with the conventional lateral flow immunoassay (Ct = 33.0), the one-step immunoassay had significantly improved LODs for PEDV detection: 362-fold (anti-RBD-1), 208-fold (anti-RBD-2), 34-fold (anti-RBD-3), and 78-fold (anti-RBD-4).Finally, the interactions between the selected Fv-antibodies and the PEDV RBD were investigated using docking simulations, and the amino acid sequences of the Fv-antibodies were compared with receptors on host cells, such as aminopeptidase N (APN) and angiotensin-converting enzyme-2 (ACE-2).

[0184] Summary of the Example

[0185] Fv antibodies against the nucleocapsid protein (NP) of SARS-CoV-2 were screened from an Fv antibody library, and a one-step immunoassay for the detection of SARS-CoV-2 using real virus samples was demonstrated. The Fv antibody library was prepared through site-specific mutagenesis of the CDR3 region, which consists of 11 amino acids, and had a diversity of 3 × 10^5 clones per library. To screen target E. coli from the Fv antibody library, the expressed probes (NTD-GFP and CTD-GFP) were reacted with the Fv antibody library, respectively. Two clones for each probe were selected as final clones through oligonucleotide sequencing. The screened Fv antibodies with binding affinities to the NTD and CTD were expressed as soluble proteins, and the affinity constants (KD) were calculated to be 25.4 nM for the NTD and 26.9 nM for the CTD. The expressed Fv-antibodies were used in a one-step immunoassay based on a switching peptide conjugated to a fluorescent dye (FAM). The one-step immunoassay based on Fv-antibodies can be used for the linear detection of SARS-CoV2 NP, with limits of detection measured as 9.6 nM (438 ng / mL) for anti-NTD and 14.1 nM (639 ng / mL) for anti-CTD. For the demonstration of the one-step immunoassay on real samples, NATtrol™ SARS-CoV-2 samples from Zeptometrix, Buffalo, NY, USA were used, and the limits of detection were measured as 29.7 copies / mL (Ct = 39.5, n = 3) using anti-NTD Fv-antibody and 117.8 copies / mL (Ct = 38.0, n = 3) using anti-CTD Fv-antibody. The measured LOD for SARS-CoV-2 detection using a one-step immunoassay based on switching peptides was considered suitable for medical diagnosis of COVID-19.Finally, the interactions between the screened Fv-antibodies and SARS-CoV-2 NPs were investigated using docking simulations.

[0186] Materials and Methods

[0187] ingredient

[0188] SARS-CoV-2 NTD, CTD, and Fv-antibodies (anti-NTD and anti-CTD) fused to superfolder green fluorescent protein (sfGFP) (Liu et al., 2019; Jung et al., 2023) were custom synthesized by Cosmogenetech (Seoul, Korea). SARS-CoV-2 NPs were provided by Optolane (Seongnam, Korea). NATtrol™ SARS-CoV-2, SARS-CoV-1, MERS-CoV, CoV-229E strain reagents (106 copies / mL, Ct=25.0) and NATtrol™ negative control reagent were purchased from Zeptometrics (Buffalo, NY, USA) (Bong et al., 2020, 2021b; Jung et al., 2021a; Jung et al., 2021b; Wei et al., 2021; Park et al., 2022a; Jung et al., 2023). FAM-labeled L2-switching peptide (1VYYCF 6QGSHV 11PFTK) was synthesized by Peptron (Daejeon, Korea). 96-well microplates (Maxisorp) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Bovine serum albumin (BSA) was purchased from Sigma-Aldrich Korea (Seoul, Korea).

[0189] Screening of Fv-antibody libraries

[0190] Preparation of Fv-antibody library plasmids and autodisplay technology on the outer membrane of E. coli have been described in previous reports (Jung et al., 2021c; Jung et al., 2021d; Lee et al., 2021; Sung et al., 2022a; Sung et al., 2022b; Jung et al., 2023). As shown in Fig. 24a, target E. coli cells with binding activity to SARS-CoV-2 NTD (or CTD) were selected from the autodisplayed Fv-antibody library through the following steps: (1) GFP-labeled SARS-CoV-2 NTD (or CTD) (2 μM) was incubated with the autodisplayed Fv-antibody library (100 μL, OD600nm=0.5) at 37 °C for 1 h. (2) After washing with 0.01% PBST and PBS, the target E. coli (n=500) were sorted using a FACSCalibur™ flow cytometer (Becton-Dickinson, Franklin Lakes, NJ, USA) as shown in Fig. 24b. (3) The sorted E. coli clones for SARS-CoV-2 NTD (or CTD) were spread on agar plates. (4) To confirm the binding activity, the sorted E. coli clones (n=20) were mixed with SARS-CoV-2 NTD (or CTD) (2 μM). (5) The selected E. coli clones with binding activity to SARS-CoV-2 NTD (or CTD) were selected using a flow cytometer as shown in Fig. 24c. (6) The binding constant (KD) of the selected E. coli clones was measured using a flow cytometer. Selected E. coli clones were mixed with SARS-CoV-2 NTD (or CTD) at a concentration range of 31-2,000 nM for 1 h at 37 °C, as shown in Fig. 24d.

[0191] Expression and purification of Fv antibodies

[0192] Plasmids (pJY009 and pJY010) were custom synthesized by Cosmogenetech (Seoul, Korea) to contain the open reading frame (ORF) of an Fv antibody with binding activity to the SARS-CoV-2 NTD (or CTD), as shown in Figure 25a. The oligonucleotide sequences and amino acid sequences of the CDR3 region are summarized in Table 6.

[0193] ScreenedcloneOligonucleotide sequence(33 bp)CDR3 sequence(11 mer)NTD3- TGC GGC CCG CAT CTG GTT GTG GTT GAT GAT TTC-5 1 CGPHL 6 VVVDD 11 FCTD3- GAC CCA GAA GGT GGT CAA CAC CGG CTG GAT TTT-5 1 DPEGG 6 QHRLD 11 F

[0194] Fv antibodies with binding activity to SARS-CoV-2 NTD (or CTD) were purified through the following steps: (1) To express Fv antibodies using Escherichia coli, BL21(DE3) competitor cells were transformed with custom plasmids (pJY009 and pJY010). (2) The transformed E. coli were cultured in 10 mL Luria-Bertani (Duchefa, Haarlem, Netherlands) medium supplemented with 1 mM isopropyl β-D-1 thiogalactopyranoside (IPTG) at 30°C for 16 h. (3) The E. coli were collected by centrifugation (2,000 g for 2 min) and resuspended in 20 mL of binding buffer (3 M urea, 5 mM Tris-HCl, and 0.5 M NaCl). (4) E. coli was sonicated using an ultrasonic reactor (Vibracell VCX-130, Sonics, USA), and the E. coli lysate was centrifuged at 22,500 g for 5 min. (5) Fv-antibody (supernatant) was purified using a His-tag column (Roche, Basel, Switzerland). (6) The purified Fv-antibody was dialyzed at 4 °C for 16 h to remove urea and imidazole. Measurement of Fv-antibody binding using surface plasmon resonance

[0195] The binding constants (KD) of Fv antibodies to SARS-CoV-2 NTD, CTD, and NP were measured using a SPR biosensor from i-Cluebio (Seongnam, Korea). The SPR chip (BK-7 glass) was sputter-coated with a 2 nm titanium layer and a 48 nm gold layer as an adhesive layer.

[0196] As shown in Figure 25b, SPR measurements were performed using Fv antibodies immobilized on a gold surface. The SPR chip was incubated with Fv antibodies (20 μg / mL) at 37°C for 1 h. After washing with PBS, the SPR chip was blocked with BSA (1 mg / mL) for 1 h. After washing with PBS, samples of SARS-CoV-2 NTD, CTD, and NP (6–100 nM) were injected into the flow cell for the binding step (flow rate of 25 μL / min for 10 min). Subsequently, PBS was injected into the flow cell for the dissociation step (flow rate of 25 μL / min for 10 min), as shown in Figure 25c.

[0197] The resonance units (RU) of the SPR signal were calculated by fitting the dose-response curves of samples containing SARS-CoV-2 NTD, CTD, and NP to an isothermal model:

[0198] R = (Rmax [Ag]) / (Kd+[Ag])

[0199] Here, R is RU, Rmax is the maximum RU, [Ag] is the concentration of the sample, and Kd is the binding constant (Bong et al., 2020; Bong et al., 2021a).

[0200] One-step immunoassay (immunoassay)

[0201] The one-step immunoassay for SARS-CoV-2 NP and NATtrol™ SARS-CoV-2, SARS-CoV-1, MERS-CoV, and CoV-229E strain reagents was performed as follows: (1) Fv antibodies (20 μg / mL, 100 μL / well) with binding activity to SARS-CoV-2 NTD and CTD were incubated in 96-well microplates (Maxisorp) at 4°C for 16 h (see Figure 26a). (2) After washing with PBS, the microplates were blocked with BSA (1 mg / mL, 150 μL) for 1 h. (3) After washing with PBS, FAM-labeled L2 switching peptide (200 nM, 100 μL / well) was added to each well and incubated at 37°C for 1 h. (4) After washing with PBS, diluted SARS-CoV-2 NP (7.8-500.0 nM) and NATtrol™ reagent (100 μL / well) were incubated for 1 h at a range of dilutions from 100-fold (Ct=31.6) to 24,300-fold (Ct=39.6). (5) The supernatant was transferred to another microplate. (6) As shown in Figures 26b and 26c, the fluorescence intensity of the L2 switching peptide (supernatant) was measured using Victor X5 from PerkinElmer Inc. (Waltham, MA, USA).

[0202] Results and Discussion

[0203] Preparation and screening of Fv-antibody libraries

[0204] Using the amino acid sequence of the VH region of an anti-peroxidase antibody as a template, a Fv-antibody library was prepared through site-directed mutagenesis of the 11-amino acid CDR3 region. The Fv-antibody library prepared in this way was inserted into an autoexpression vector, and the Fv-antibody library was expressed in the outer membrane of Escherichia coli (see Figure 23a). The diversity of the Fv-antibody library was measured through random selection of the population, and the diversity of approximately 3 x 10^5 clones per library was estimated through oligonucleotide sequencing of the CDR3 region.

[0205] To screen for Fv antibodies, the N-terminal domain (NTD) and C-terminal domain (CTD) were prepared as probes. The NP of SARS-CoV-2 consists of 419 amino acids, and the NTD and CTD are known to function as RNA-binding domains for RNA genome packaging. As shown in Figure 23b, the NTD was expressed as a fusion protein (41.8 kDa) with the NP sequence of amino acids 48-174 (127 amino acids, 13.9 kDa) and green fluorescent protein (GFP). The CTD was also expressed as a fusion protein (40.1 kDa) with the NP sequence of amino acids 247-364 (118 amino acids, 13.2 kDa) and green fluorescent protein (GFP).

[0206] To select target E. coli from the Fv-antibody library, the expressed probes (NTD-GFP and CTD-GFP) were each reacted with the Fv-antibody library. Target E. coli with binding affinity to the probes were then identified using flow cytometry, as shown in Figure 24a. When the Fv-antibody library reacted with both probes, a strong fluorescent signal was observed for E. coli bound to the probes (see Figure 24b). For the control strain, in which only CDR1 and CDR2 were automatically labeled, no high fluorescent signal was observed compared to the results from the Fv-antibody library (flow cytometry dot plot). These results indicate that (1) target clones with binding affinity to the NTD (or CTD) exist in the Fv-antibody library, and (2) the binding affinity for the target clones originates from the CDR3 of the target clones.

[0207] Two signal regions with different fluorescence signals were selected as gate regions for flow cytometry, and target clones were isolated and cultured on agar plates. Among the NTD-binding clones selected on the agar plates, 20 clones were analyzed using NTD-GFP as a probe, and three clones (numbered 2, 10, and 14) were selected as having high binding affinity to the NTD, as shown in Figure 24c. Through oligonucleotide sequence analysis, clone 10 was confirmed to have the same sequence as the template (initial CDR3 sequence) before site-directed mutagenesis, and clone 2 was selected as the final clone with binding affinity to the NTD. Among the CTD-binding clones selected on the agar plates, 20 clones were analyzed using CTD-GFP as a probe, and three clones (numbered 1, 13, and 19) were selected as having high binding affinity to the CTD, as shown in Figure 24c. Oligonucleotide sequence analysis confirmed that clone 13 had a sequence identical to the template (initial CDR3 sequence) prior to site-directed mutagenesis, and clone 1 was selected as the final clone with binding affinity for the CTD. The oligonucleotide sequences and corresponding amino acid sequences of CDR3 are summarized in Table 6.

[0208] The binding affinities of the selected clones (clone no. 14 for NTD and clone no. 1 for CTD) were measured by treating NTD-GFP and CTD-GFP at concentrations ranging from 31.3 to 2,000.0 nM, as shown in Fig. 24d. Using flow cytometry, the affinity constants (KD) for the NTD clone were calculated to be 0.77 μM and 0.91 μM for the CTD clone using an isothermal model. In the case of the control strain in which only CDR1 and CDR2 were autolabeled, the fluorescence signals due to the binding of the two probes were observed to be insignificant compared to those of the selected clones. These results demonstrate that (1) the clones selected from the Fv-antibody library have positive binding affinity for NTD (and CTD), and (2) the binding affinity for the target clone is derived from CDR3 of the target clone.

[0209] One-step immunoassay based on switching peptides

[0210] The selected Fv antibodies with binding affinity for the NTD and CTD were expressed as soluble proteins according to the oligonucleotide sequences of CDR3 and their corresponding amino acid sequences, which are summarized in Table 6. As shown in Figure 25a, the Fv antibody (13.8 kDa) was co-expressed with GFP in fusioNP (40.8 kDa). As previously reported, the Fv protein was co-expressed with GFP for the following reasons: (1) co-expression of GFP can significantly enhance the solubility of the Fv antibody (Pedelacq et al., 2006; Liu et al., 2019) and (2) GFP can be effectively immobilized on the metal surface of a biosensor (Jung et al., 2023). The binding affinity of the expressed Fv antibody was measured using an SPR biosensor. As shown in Figure 25b, the Fv antibody was immobilized on the gold surface of the SPR biochip, and the binding of the Fv antibody was measured through the binding and dissociation profiles of the two probes (NTD and CTD). As shown in Figure 25c, the affinity constants (KD) of 25.4 nM for the NTD and 26.9 nM for the CTD were calculated by treating the two probes in the concentration range of 6–100 nM using an isothermal model. When co-expressed GFP was treated on the same SPR biosensor immobilized with the Fv antibody, the signal was observed at the baseline level. The remarkably high affinity for both probes and the NP indicates that the Fv antibody can be effectively used for the detection of SARS-CoV-2.

[0211] The expressed Fv antibody was used in a one-step immunoassay based on a switching peptide. The switching peptide was synthesized through self-assembly using the amino acid sequence of the FR that constitutes the binding pocket of IgG. As mentioned above, the expressed Fv antibody corresponds to the VH of IgG, and the FR in the expressed Fv antibody (VH) has affinity for the FR in the IgG light chain (VL). This VL FR amino acid sequence was synthesized into a peptide, which is called a switching peptide because it quantitatively dissociates upon antigen (target analyte) binding to the CDR. As shown in Figure 26a, the switching peptide, labeled with a fluorescent dye (FAM), binds to the expressed Fv antibody and dissociates upon analyte binding.

[0212] When using expressed Fv antibodies with affinity for the NTD and CTD of NP, fluorescence signals were quantitatively observed. As shown in Figure 26b, the Fv antibody (anti-NTD)-based one-step immunoassay was able to linearly detect SARS-CoV2 NP over a concentration range of 7.9–500.0 nM, with a detection limit of 9.6 nM (438 ng / mL). For Fv antibody (anti-CTD), linear detection of SARS-CoV2 NP was possible over the same concentration range of 7.9–500.0 nM, with a detection limit of 14.1 nM (639 ng / mL).

[0213] To demonstrate the one-step immunoassay for real samples, NATtrol™ SARS-CoV-2 samples provided by Zeptometrix (Buffalo, NY, USA) were used after being diluted to a concentration range of 4.1 × 10^1 - 1.0 × 10^4 copies / mL. As shown in Fig. 26c, the Fv-antibody (anti-NTD)-based one-step immunoassay showed linear detection of SARS-CoV2 NP, with a detection limit of 29.7 copies / mL (Ct = 39.5, n = 3). In the case of Fv-antibody (anti-CTD), linear detection of SARS-CoV2 NP was possible at the same concentration, with a detection limit of 117.8 copies / mL (Ct = 38.0, n = 3). Considering that the cutoff value of conventional RT-PCR for positive COVID-19 test was reported to be Ct=34-35, the measured detection limit for SARS-CoV-2 detection using a switching peptide-based one-step immunoassay is expected to be suitable for medical diagnosis of COVID-19.

[0214] To compare the sensitivity to different coronaviruses, SARS-CoV-1, MERS-CoV, and CoV-229E, these were used in a one-step immunoassay. As shown in Figure 26c, in the one-step immunoassay based on Fv antibodies (anti-NTD and anti-CTD), a lower sensitivity was observed than that for SARS-CoV-2, and this sensitivity decreased in the order of SARS-CoV-1, MERS-CoV, and CoV-229E for both Fv antibodies. To explain this sensitivity trend, the NP amino acid sequences of these coronaviruses were analyzed using BLAST (https: / blast.ncbi.nlm.nih.gov / Blast.cgi). In amino acid sequence analysis, the identity of two proteins refers to the proportion of identical amino acid residues, and the similarity of two proteins refers to the proportion of amino acid residues with similar chemical properties (polar, nonpolar, acidic, basic, etc.). As shown in Figure 26d, the NTD of SARS-CoV-2 was measured to have the highest identity (92%) and similarity (96%) with SARS-CoV-1. These two parameters were analyzed to decrease in the same trend as the sensitivity in Figure 26c. The CTD of SARS-CoV-2 also showed the same trend in identity and similarity with other coronaviruses as described for the NTD. These results demonstrate that the similarity in amino acid sequences between coronavirus NPs determines the binding affinity of SARS-CoV-2 NPs to Fv antibodies.

[0215] The one-step immunoassay results demonstrated that two types of Fv antibodies could sensitively detect NP. To investigate the interactions between the selected Fv antibodies and SARS-CoV-2 NP, docking simulations were performed using AutoDock Vina software provided by The Scripps Research (La Jolla, CA, USA) (Goodsell et al., 1996; Trott and Olson, 2010; Eberhardt et al., 2021). The interactions were visualized and analyzed using PyMOL software version 2.5 from DeLano Scientific LLC (South San Francisco, CA, USA) and Discovery Studio Visualizer version 2020 from Dassault Systemes BIOVIA (San Diego, CA, USA). The NP structure of SARS-CoV-2 (PDB ID: 8FD5) was obtained from the PDB (https: / www.rcsb.org / ), and its interactions with two types of Fv antibodies against the NTD and CTD were analyzed. The interaction between the NP and the anti-NTD was analyzed with a negative Gibbs free energy of -6.4 Kcal / mol, as shown in Figure 27a. The amino acids in the RBD for hydrophobic interactions were assigned to ALA50, TRP52, PHE53, TYR111, and PRO117, and those for hydrogen bonding were assigned to PHE53 and GLY116. The interaction between the NP and the anti-CTD was analyzed with a negative Gibbs free energy of -6.0 Kcal / mol, as shown in Figure 27b. The amino acids in RBD for hydrophobic interactions were assigned to TRP330, and those for hydrogen bonding were assigned to THR247, SER250, SER255, ARG259, ARG262, THR271, SER327, THR329, and TRP330.These results show that (1) the two types of Fv-antibodies had specific interactions with different regions of the NP representing different epitopes, and (2) these Fv-antibodies formed stable bonds with the NP due to negative Gibbs free energy.

[0216] conclusion

[0217] The Fv-antibody library was prepared through site-directed mutagenesis of the CDR3 region consisting of 11 amino acids, and the Fv-antibody library was expressed on the outer membrane of Escherichia coli (E. coli) with a diversity of 3 x 105 / library. To select target E. coli from the Fv-antibody library, the expressed probes (NTD-GFP and CTD-GFP) were reacted separately with the Fv-antibody library, respectively. Two clones for each probe were selected as final clones through oligonucleotide sequencing. The binding constant (KD) of the NTD clone was calculated to be 0.77 μM using the isothermal model, and that of the CTD clone was calculated to be 0.91 μM through flow cytometry analysis. The selected Fv-antibodies that bind to the NTD and CTD were expressed as soluble proteins according to the oligonucleotide sequences of the CDR3 of each clone and the corresponding amino acid sequences. The binding constants (KD) were calculated to be 25.4 nM for NTD and 26.9 nM for CTD using an isothermal model. The expressed Fv-antibodies were used in a one-step immunoassay based on a switching peptide conjugated to a fluorescent dye (FAM), which dissociated upon binding of the analyte. The one-step immunoassay based on Fv-antibodies could be used for the linear detection of SARS-CoV2 NP, with detection limits of 9.6 nM (438 ng / mL) using anti-NTD Fv-antibodies and 14.1 nM (639 ng / mL) using anti-CTD Fv-antibodies. To demonstrate the one-step immunoassay for real samples, SARS-CoV-2 samples were used in a medical matrix called NATtrol™ SARS-CoV-2 from Zeptometrix (Buffalo, NY, USA), with a concentration range of 4.1 x 10 1 - 1.0 x 10 4copies / mL. The one-step immunoassay based on Fv antibody (anti-NTD) showed linear detection of SARS-CoV2 NP, with a detection limit of 29.7 copies / mL (Ct = 39.5, n = 3). For anti-CTD Fv antibody, linear detection of SARS-CoV2 NP at the same concentration was possible, with a detection limit of 117.8 copies / mL (Ct = 38.0, n = 3). Due to the measured detection limit for SARS-CoV-2 detection using the one-step immunoassay based on switching peptide, it is considered suitable for medical diagnosis of COVID-19. Docking simulations were used to investigate the interactions between the screened Fv-antibodies and SARS-CoV-2 NP, revealing that (1) the two types of Fv-antibodies had specific interactions with the NP at different regions representing different epitopes, and (2) these Fv-antibodies formed stable binding from negative Gibbs free energy.

[0218] At least a portion of the sequence list used in this specification is as shown in Table 7 below.

[0219] SequenceIDNumberSequenceNameMoleculeTypeOrganismSequence1CDR1AAsynthetic constructTYGIQ2CDR2AAsynthetic constructWIHAGTGGTKYSRKFQG3CDR3_PEDV1AAsynthetic constructYGPSIVEQTDV4CDR3_PEDV2AAsynthetic constructYVGLGPGTVDF5CDR3_PEDV3AAsynthetic constructYETVRGNGTDV6CDR3_PEDV4AAsynthetic constructYGVLRRGCLDF7CDR3_NTDAAsynthetic constructCGPHLVVVDDF8CDR3_CTDAAsynthetic constructDPEGGQHRLDF

[0220] Although the present invention has been described above with reference to preferred embodiments thereof, it will be understood by those skilled in the art that various modifications and changes may be made to the present invention without departing from the spirit and scope of the present invention as set forth in the claims below.

[0221] National Research and Development Project that supported this invention

[0222] - Assignment ID (10 digits): 1465038753

[0223] - Assignment number: RS-2022-KH128357

[0224] - Ministry name: Ministry of Health and Welfare

[0225] - Project Management (Professional) Organization Name: Korea Health Industry Development Institute

[0226] - Research Project Name: Rapid Universal Vaccine Technology Development

[0227] Research Project Name: Research on a Vaccine Screening Platform Capable of Rapidly Responding to Mutant Viruses

[0228] - Contribution rate: 1 / 1

[0229] - Project implementation organization name (host organization): Yonsei University Industry-Academic Cooperation Foundation

[0230] Research period: January 1, 2023 - December 31, 2023

Claims

1. A peptide that specifically binds to the nucleoprotein of porcine epidemic diarrhea virus (PEDV) or SARS-CoV-2, Comprising at least one peptide sequence selected from the group comprising the peptide sequence of SEQ ID NO: 3, the peptide sequence of SEQ ID NO: 4, the peptide sequence of SEQ ID NO: 5, the peptide sequence of SEQ ID NO: 6, the peptide sequence of SEQ ID NO: 7 and the peptide sequence of SEQ ID NO:

8. Peptide.

2. An antibody or an antigen-binding fragment thereof that specifically binds to the nucleoprotein of porcine epidemic diarrhea virus (PEDV) or SARS-CoV-2, A variable region comprising CDR1 of SEQ ID NO: 1, CDR2 of SEQ ID NO: 2, and CDR3 of SEQ ID NO: 3; A variable region comprising CDR1 of SEQ ID NO: 1, CDR2 of SEQ ID NO: 2, and CDR3 of SEQ ID NO: 4; A variable region comprising CDR1 of sequence number 1, CDR2 of sequence number 2, and CDR3 of sequence number 5; and A variable region comprising CDR1 of SEQ ID NO: 1, CDR2 of SEQ ID NO: 2, and CDR3 of SEQ ID NO: 6; A variable region comprising CDR1 of sequence number 1, CDR2 of sequence number 2, and CDR3 of sequence number 7; and A variable region comprising CDR1 of SEQ ID NO: 1, CDR2 of SEQ ID NO: 2, and CDR3 of SEQ ID NO: 8; Including a group selected from the group consisting of: An antibody or an antigen-binding fragment thereof.

3. In paragraph 2, The above antibody or antigen-binding fragment thereof, A heavy chain variable region comprising HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, and HCDR3 of SEQ ID NO: 3; A heavy chain variable region comprising HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, and HCDR3 of SEQ ID NO: 4; A heavy chain variable region comprising HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, and HCDR3 of SEQ ID NO: 5; A heavy chain variable region comprising HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, and HCDR3 of SEQ ID NO: 6; A heavy chain variable region comprising HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, and HCDR3 of SEQ ID NO: 7; and A heavy chain variable region comprising HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, and HCDR3 of SEQ ID NO: 8; Including a group selected from the group consisting of: An antibody or an antigen-binding fragment thereof.

4. In paragraph 2, The above antibody or antigen-binding fragment thereof is in the form of a single domain antibody, An antibody or an antigen-binding fragment thereof.

5. Coding a peptide according to paragraph 1 or an antibody or antigen-binding fragment thereof according to paragraph 2; Nucleic acid.

6. Containing nucleic acid according to Article 5, Recombinant expression vector.

7. Transformed with a recombinant expression vector according to Article 6, cell.

8. In paragraph 7, The above cell comprises one or more cells selected from the group including animal cells, plant cells, yeast, E. coli and insect cells. cell.

9. In paragraph 8, The above cells are monkey kidney cells 7 (COS7), NSO cells, SP2 / 0 cells, Chinese hamster ovary (CHO) cells, W138, baby hamster kidney (BHK) cells, MDCK, myeloma cell lines, HuT 78 cells, and HEK293 cells, Escherichia coli, Bacillus subtilis, Streptomyces sp., Pseudomonas sp., Proteus mirabilis or Staphylococcus sp., Aspergillus sp., Pichiapastoris, Saccharomyces cerevisiae, Schizosaccharomyces sp.) and Neurospora crassa, comprising one or more cells selected from the group consisting of cell.

10. An antibody or antigen-binding fragment thereof according to paragraph 2; A switching peptide capable of specifically and reversibly binding to one or more of the first to fourth framework regions (FR1, FR2, FR3 and FR4) of the heavy or light chain of the antibody or antigen-binding fragment thereof; and A marker bound to the above switching peptide; comprising; A substance for detection of porcine epidemic diarrhea virus or SARS-CoV-2 nucleoprotein.

11. In paragraph 10, The above antibody or antigen-binding fragment thereof is single-stranded, A substance for detection of porcine epidemic diarrhea virus or SARS-CoV-2 nucleoprotein.

12. In paragraph 10, The above marker is a fluorescent marker, A substance for detection of porcine epidemic diarrhea virus or SARS-CoV-2 nucleoprotein.

13. A substance containing a substance for detecting porcine epidemic diarrhea virus or SARS-CoV-2 nucleoprotein according to Article 10. One-step swine epidemic diarrhea or SARS-CoV-2 diagnostic kit.

14. A step of mixing a substance for detecting porcine epidemic diarrhea virus or SARS-CoV-2 nucleoprotein in a one-step porcine epidemic diarrhea or SARS-CoV-2 diagnostic kit according to Article 13 with a sample to be detected, and then confirming the marker; One-step swine epidemic diarrhea or SARS-CoV-2 diagnostic method.

15. In paragraph 14, The above marker is prepared as a fluorescent marker. One-step swine epidemic diarrhea or SARS-CoV-2 diagnostic method.

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