A kit and method for detecting SARS-CoV-2 neutralizing antibodies

Abstract

The present application relates to a kit and method for detecting SARS-CoV-2 neutralizing antibodies. The present application relates to the field of biomedicine, and provides a detection kit and method for detecting SARS-CoV-2 neutralizing antibodies, which uses protein tags to modify RBD and ACE2 to make them have better stability and reactivity, thereby improving the detection sensitivity.

Description

[0001] This application is a divisional application of invention patent application No. 2020112552126, filed on November 11, 2020, and entitled “A kit and method for detecting SARS-CoV-2 neutralizing antibodies”. Technical Field

[0002] The present application relates to the field of biomedicine, and specifically to a kit and method for detecting SARS-CoV-2 neutralizing antibodies. Background Art

[0003] The SARS-CoV-2 surface spike protein is an important receptor binding site on the surface of the coronavirus. It can bind to the virus-specific receptors on the cell surface, mediating the fusion of the viral outer membrane and the cell, viral adsorption and membrane penetration. The coronavirus S protein receptor binding domain (RBD) can bind to angiotensin-converting enzyme 2 (ACE2) on the surface of the host cell and enable the virus to enter the host cell.

[0004] When SARS-CoV-2 invades the body, it stimulates the body to produce neutralizing antibodies with protective effects. Neutralizing antibodies are a soluble protein secreted by adaptive immune response cells that have the function of recognizing viral surface proteins and blocking their binding to specific receptors on the surface of human cells, thereby exerting an antiviral effect. The amount of neutralizing antibodies is an important indicator of the immune protection effect of vaccines and an important basis for vaccine evaluation and quality control.

[0005] The existing detection methods for SARS-CoV-2 mainly include nucleic acid detection and antibody detection. The main methods include fluorescent PCR, enzyme-linked immunosorbent assay, colloidal gold chromatography, and chemiluminescence. Among them, nucleic acid detection has high sensitivity, strong specificity, and high accuracy, but the operation requirements are relatively high and the detection time is long. Antibody detection has high sensitivity and strong specificity, but IgM / IgG antibodies will not appear until 7-14 days after infection. Moreover, these methods can only detect whether the patient is in the infection period, and cannot confirm whether the patient is at risk of reinfection after recovery, or whether the general public is at risk of exposure and infection. Therefore, new methods for detecting SARS-CoV-2 are urgently needed. Summary of the invention

[0006] The present application provides a kit and method for detecting SARS-CoV-2 neutralizing antibodies, which have one or more of the following properties: after RBD, ACE2 receptor protein or its functionally active fragment is fused with Fc, compared with the protein of natural structure, 1) it has better stability and reactivity; 2) the detection sensitivity is improved; 3) the clinical efficacy of COVID-19 vaccine can be judged and the individual autoimmune effect can be evaluated; 4) whether there is a risk of SARS-CoV-2 reinfection can be judged; and 5) the reference antibody has a good neutralizing effect and can well simulate the natural form of neutralizing antibodies in the human body.

[0007] On the one hand, the present application provides a detection kit for SARS-CoV-2 neutralizing antibodies, comprising:

[0008] 1) SARS-CoV-2 spike protein or a functionally active fragment thereof; and

[0009] 2) A competitive binder, which comprises a substance that can compete with the SARS-CoV-2 neutralizing antibody for binding to the SARS-CoV-2 spike protein or a functionally active fragment thereof.

[0010] In certain embodiments, the SARS-CoV-2 spike protein or a functionally active fragment thereof comprises a receptor binding domain (RBD).

[0011] In certain embodiments, the SARS-CoV-2 spike protein or a functionally active fragment thereof comprises a full-length SARS-CoV-2 spike protein.

[0012] In certain embodiments, the SARS-CoV-2 spike protein or a functionally active fragment thereof comprises a trimeric full-length SARS-COV-2 spike protein.

[0013] In certain embodiments, the SARS-CoV-2 spike protein or a functionally active fragment thereof comprises a receptor binding region RBD, and the RBD is directly or indirectly connected to the Fc region.

[0014] In certain embodiments, the RBD is fused in frame to the Fc.

[0015] In certain embodiments, the Fc region is selected from the group consisting of rFc, mFc, and hFc.

[0016] In certain embodiments, the SARS-CoV-2 spike protein or a functionally active fragment thereof comprises the amino acid sequence shown in any one of SEQ ID NOs: 6-11.

[0017] In certain embodiments, the concentration of the SARS-CoV-2 spike protein or a functionally active fragment thereof is 50 ng / ml-4000 ng / ml.

[0018] In certain embodiments, the SARS-CoV-2 spike protein or a functionally active fragment thereof is immobilized on a solid support.

[0019] In certain embodiments, the competitive binder comprises an ACE2 receptor protein or a functionally active fragment thereof.

[0020] In certain embodiments, the ACE2 receptor protein or a functionally active fragment thereof comprises a human ACE2 receptor protein or a functionally active fragment thereof.

[0021] In certain embodiments, the ACE2 receptor protein or a functionally active fragment thereof comprises an extracellular domain ECD.

[0022] In certain embodiments, the ACE2 receptor protein or a functionally active fragment thereof is directly or indirectly linked to the Fc region.

[0023] In certain embodiments, the ACE2 receptor protein or a functionally active fragment thereof is fused in frame to the Fc region.

[0024] In certain embodiments, the Fc region is selected from the group consisting of rFc, mFc, and hFc.

[0025] In certain embodiments, the competitive binder comprises the amino acid sequence shown in any one of SEQ ID NOs: 1-5.

[0026] In certain embodiments, the competitive binding agent comprises one or more labels that are capable of directly or indirectly generating a signal indicating the presence and / or amount of the competitive binding agent.

[0027] In certain embodiments, the label comprises horseradish peroxidase (HRP) or an HRP-like enzyme.

[0028] In certain embodiments, the concentration of the competitive binder is 100 ng / ml-2000 ng / ml.

[0029] In certain embodiments, the kit further comprises a calibrator comprising one or more reference antibody molecules or antigen-binding fragments thereof that specifically recognize the SARS-CoV-2 spike protein or a functionally active fragment thereof.

[0030] In certain embodiments, the one or more reference antibody molecules or antigen-binding fragments thereof specifically recognize at least two different epitopes on the SARS-CoV-2 spike protein or a functionally active fragment thereof.

[0031] In certain embodiments, the one or more reference antibody molecules or antigen-binding fragments thereof specifically recognize at least three different epitopes on the SARS-CoV-2 spike protein or a functionally active fragment thereof.

[0032] In certain embodiments, the reference antibody comprises a heavy chain variable region VH and a light chain variable region VL; wherein: the VH comprises HCDR1, HCDR2 and HCDR3 of the amino acid sequences shown in SEQ ID NOs:30-32, and the VL comprises LCDR1, LCDR2 and LCDR3 of the amino acid sequences shown in SEQ ID NOs:33-35; the VH comprises HCDR1, HCDR2 and HCDR3 of the amino acid sequences shown in SEQ ID NOs:36-38, and the VL comprises LCDR1, LCDR2 and LCDR3 of the amino acid sequences shown in SEQ ID NOs:39-41; or the VH comprises HCDR1, HCDR2 and HCDR3 of the amino acid sequences shown in SEQ ID NOs:42-44, and the VL comprises LCDR1, LCDR2 and LCDR3 of the amino acid sequences shown in SEQ ID NOs:45-47.

[0033] In certain embodiments, the reference antibody comprises a heavy chain variable region VH and a light chain variable region VL; wherein: the VH comprises the amino acid sequence shown in SEQ ID NO:21, and the VL comprises the amino acid sequence shown in SEQ ID NO:22; the VH comprises the amino acid sequence shown in SEQ ID NO:24, and the VL comprises the amino acid sequence shown in SEQ ID NO:25; or the VH comprises the amino acid sequence shown in SEQ ID NO:27, and the VL comprises the amino acid sequence shown in SEQ ID NO:28.

[0034] In certain embodiments, the reference antibodies include antibodies 32-1, 12-5 and 12-8.

[0035] In certain embodiments, the concentration of the reference antibody is 50 ng / ml-1000 ng / ml.

[0036] In certain embodiments, the SARS-CoV-2 spike protein or a functionally active fragment thereof comprises one or more markers that are capable of directly or indirectly generating a signal indicating the presence and / or amount of the SARS-CoV-2 spike protein or a functionally active fragment thereof.

[0037] In certain embodiments, the label comprises horseradish peroxidase (HRP) or an HRP-like enzyme.

[0038] On the other hand, the present application provides a method for detecting SARS-CoV-2 neutralizing antibodies in a sample, which comprises: using the kit.

[0039] On the other hand, the present application provides a method for detecting SARS-CoV-2 neutralizing antibodies in a sample, comprising:

[0040] 1) Encapsulating the SARS-CoV-2 spike protein or its functionally active fragment on a solid support;

[0041] 2) adding the sample to be tested in step 1) to react with the SARS-CoV-2 spike protein or its functionally active fragment;

[0042] 3) adding the competitive binding agent to step 2);

[0043] 4) detecting SARS-CoV-2 neutralizing antibodies in the sample to be tested;

[0044] Wherein, the competitive binder comprises a substance that can compete with the SARS-CoV-2 neutralizing antibody for binding to the SARS-CoV-2 spike protein or its functionally active fragment; the SARS-CoV-2 spike protein or its functionally active fragment comprises a receptor binding domain RBD; the competitive binder comprises an ACE2 receptor protein or its functionally active fragment.

[0045] In certain embodiments, the SARS-CoV-2 spike protein or a functionally active fragment thereof comprises a full-length SARS-CoV-2 spike protein.

[0046] In certain embodiments, the SARS-CoV-2 spike protein or a functionally active fragment thereof comprises a trimeric full-length SARS-COV-2 spike protein.

[0047] In certain embodiments, the SARS-CoV-2 spike protein or a functionally active fragment thereof comprises a receptor binding region RBD, and the RBD is directly or indirectly connected to the Fc region.

[0048] In certain embodiments, the RBD is fused in frame to the Fc.

[0049] In certain embodiments, the Fc region is selected from the group consisting of rFc, mFc, and hFc.

[0050] In certain embodiments, the SARS-CoV-2 spike protein or a functionally active fragment thereof comprises the amino acid sequence shown in any one of SEQ ID NOs: 6-11.

[0051] In certain embodiments, the concentration of the SARS-CoV-2 spike protein or a functionally active fragment thereof is 50 ng / ml-4000 ng / ml.

[0052] In certain embodiments, the SARS-CoV-2 spike protein or a functionally active fragment thereof is immobilized on a solid support.

[0053] In certain embodiments, the competitive binder comprises an ACE2 receptor protein or a functionally active fragment thereof.

[0054] In certain embodiments, the ACE2 receptor protein or a functionally active fragment thereof comprises a human ACE2 receptor protein or a functionally active fragment thereof.

[0055] In certain embodiments, the ACE2 receptor protein or a functionally active fragment thereof comprises an extracellular domain ECD.

[0056] In certain embodiments, the ACE2 receptor protein or a functionally active fragment thereof is directly or indirectly linked to the Fc region.

[0057] In certain embodiments, the ACE2 receptor protein or a functionally active fragment thereof is fused in frame to the Fc region.

[0058] In certain embodiments, the Fc region is selected from the group consisting of rFc, mFc, and hFc.

[0059] In certain embodiments, the competitive binder comprises the amino acid sequence shown in any one of SEQ ID NOs: 1-5.

[0060] In certain embodiments, the competitive binding agent comprises one or more labels that are capable of directly or indirectly generating a signal indicating the presence and / or amount of the competitive binding agent.

[0061] In certain embodiments, the label comprises horseradish peroxidase (HRP) or an HRP-like enzyme.

[0062] In certain embodiments, the concentration of the competitive binder is 100 ng / ml-2000 ng / ml.

[0063] In certain embodiments, the kit further comprises a calibrator comprising one or more reference antibody molecules or antigen-binding fragments thereof that specifically recognize the SARS-CoV-2 spike protein or a functionally active fragment thereof.

[0064] In certain embodiments, the one or more reference antibody molecules or antigen-binding fragments thereof specifically recognize at least two different epitopes on the SARS-CoV-2 spike protein or a functionally active fragment thereof.

[0065] In certain embodiments, the one or more reference antibody molecules or antigen-binding fragments thereof specifically recognize at least three different epitopes on the SARS-CoV-2 spike protein or a functionally active fragment thereof.

[0066] In certain embodiments, the reference antibody comprises a heavy chain variable region VH and a light chain variable region VL; wherein: the VH comprises HCDR1, HCDR2 and HCDR3 of the amino acid sequences shown in SEQ ID NOs:30-32, and the VL comprises LCDR1, LCDR2 and LCDR3 of the amino acid sequences shown in SEQ ID NOs:33-35; the VH comprises HCDR1, HCDR2 and HCDR3 of the amino acid sequences shown in SEQ ID NOs:36-38, and the VL comprises LCDR1, LCDR2 and LCDR3 of the amino acid sequences shown in SEQ ID NOs:39-41; or the VH comprises HCDR1, HCDR2 and HCDR3 of the amino acid sequences shown in SEQ ID NOs:42-44, and the VL comprises LCDR1, LCDR2 and LCDR3 of the amino acid sequences shown in SEQ ID NOs:45-47.

[0067] In certain embodiments, the reference antibody comprises a heavy chain variable region VH and a light chain variable region VL; wherein: the VH comprises the amino acid sequence shown in SEQ ID NO:21, and the VL comprises the amino acid sequence shown in SEQ ID NO:22; the VH comprises the amino acid sequence shown in SEQ ID NO:24, and the VL comprises the amino acid sequence shown in SEQ ID NO:25; or the VH comprises the amino acid sequence shown in SEQ ID NO:27, and the VL comprises the amino acid sequence shown in SEQ ID NO:28.

[0068] In certain embodiments, the reference antibodies include antibodies 32-1, 12-5 and 12-8.

[0069] In certain embodiments, the concentration of the reference antibody is 50 ng / ml-1000 ng / ml.

[0070] In certain embodiments, the sample is selected from the group consisting of urine, saliva, serum, plasma, nasopharyngeal aspirate, and bronchial lavage fluid.

[0071] In certain embodiments, the sample is derived from a subject.

[0072] In certain embodiments, the subject is a recovered COVID-19 patient, a healthy subject, and / or a subject who has received COVID-19 vaccine treatment.

[0073] On the other hand, the present application provides a method for detecting SARS-CoV-2 neutralizing antibodies in a sample, comprising:

[0074] 1) Encapsulating ACE2 receptor protein or its functionally active fragment on a solid support;

[0075] 2) mixing and incubating the sample to be tested with the SARS-CoV-2 spike protein or its functionally active fragment to obtain an incubation product;

[0076] 3) adding the incubated product of step 2) to step 1);

[0077] 4) detecting SARS-CoV-2 neutralizing antibodies in the sample to be tested;

[0078] The SARS-CoV-2 spike protein or a functionally active fragment thereof comprises a receptor binding domain RBD, and the RBD comprises one or more markers, which can directly or indirectly generate a signal showing the presence and / or content of the RBD.

[0079] In certain embodiments, the label comprises horseradish peroxidase (HRP) or an HRP-like enzyme.

[0080] In certain embodiments, the RBD is directly or indirectly linked to the Fc region.

[0081] In certain embodiments, the RBD is fused in frame to the Fc.

[0082] In certain embodiments, the Fc region is selected from the group consisting of rFc, mFc, and hFc.

[0083] In certain embodiments, the SARS-CoV-2 spike protein or a functionally active fragment thereof comprises the amino acid sequence shown in SEQ ID NO:7.

[0084] In certain embodiments, the SARS-CoV-2 spike protein or a functionally active fragment thereof comprises the amino acid sequence shown in SEQ ID NO:8.

[0085] In certain embodiments, the SARS-CoV-2 spike protein or a functionally active fragment thereof comprises the amino acid sequence shown in SEQ ID NO:9.

[0086] In certain embodiments, the SARS-CoV-2 spike protein or a functionally active fragment thereof comprises the amino acid sequence shown in SEQ ID NO:10.

[0087] In certain embodiments, the SARS-CoV-2 spike protein or a functionally active fragment thereof comprises the amino acid sequence shown in SEQ ID NO:11.

[0088] In certain embodiments, the concentration of the SARS-CoV-2 spike protein or a functionally active fragment thereof is 50 ng / ml-4000 ng / ml.

[0089] In certain embodiments, the ACE2 receptor protein or a functionally active fragment thereof is directly or indirectly linked to the Fc region.

[0090] In certain embodiments, the ACE2 receptor protein or a functionally active fragment thereof is fused in frame to the Fc region.

[0091] In certain embodiments, the Fc region is selected from the group consisting of rFc, mFc, and hFc.

[0092] In certain embodiments, the ACE2 receptor protein or a functionally active fragment thereof comprises the amino acid sequence shown in SEQ ID NO:2.

[0093] In certain embodiments, the ACE2 receptor protein or a functionally active fragment thereof comprises the amino acid sequence shown in SEQ ID NO:3.

[0094] In certain embodiments, the ACE2 receptor protein or a functionally active fragment thereof comprises the amino acid sequence shown in SEQ ID NO:4.

[0095] In certain embodiments, the concentration of the ACE2 receptor protein or a functionally active fragment thereof is 100 ng / ml-2000 ng / ml.

[0096] In certain embodiments, it comprises a sample diluent having a pH of 6.0-9.6.

[0097] In certain embodiments, the detection is obtained by comparison with a calibrator, wherein the calibrator comprises one or more reference antibody molecules or antigen-binding fragments thereof that specifically recognize the SARS-CoV-2 spike protein or a functionally active fragment thereof; the reference antibody comprises a heavy chain variable region VH and a light chain variable region VL, wherein: the VH comprises HCDR1, HCDR2 and HCDR3 of the amino acid sequence shown in SEQ ID NOs:30-32, and the VL comprises LCDR1, LCDR2 and LCDR3 of the amino acid sequence shown in SEQ ID NOs:33-35; the VH comprises HCDR1, HCDR2 and HCDR3 of the amino acid sequence shown in SEQ ID NOs:36-38, and the VL comprises LCDR1, LCDR2 and LCDR3 of the amino acid sequence shown in SEQ ID NOs:39-41; or the VH comprises HCDR1, HCDR2 and HCDR3 of the amino acid sequence shown in SEQ ID NOs:42-44, and the VL comprises LCDR1, LCDR2 and LCDR3 of the amino acid sequence shown in SEQ ID NOs:45-47.

[0098] In certain embodiments, the detection is obtained by comparison with a calibrator, wherein the calibrator comprises one or more reference antibody molecules or antigen-binding fragments thereof that specifically recognize the SARS-CoV-2 spike protein or a functionally active fragment thereof; the reference antibody comprises a heavy chain variable region VH and a light chain variable region VL; wherein: the VH comprises the amino acid sequence shown in SEQ ID NO:21, and the VL comprises the amino acid sequence shown in SEQ ID NO:22; the VH comprises the amino acid sequence shown in SEQ ID NO:24, and the VL comprises the amino acid sequence shown in SEQ ID NO:25; or the VH comprises the amino acid sequence shown in SEQ ID NO:27, and the VL comprises the amino acid sequence shown in SEQ ID NO:28.

[0099] In certain embodiments, the reference antibodies include antibodies 32-1, 12-5 and 12-8.

[0100] In certain embodiments, the concentration of the reference antibody is 50 ng / ml-1000 ng / ml.

[0101] In certain embodiments, the sample to be tested is selected from the group consisting of urine, saliva, serum, plasma, whole blood, nasopharyngeal aspirate, and bronchial lavage fluid.

[0102] In certain embodiments, the sample to be tested is derived from a subject.

[0103] In certain embodiments, the subject is a recovered COVID-19 patient, a healthy subject, and / or a subject who has received COVID-19 vaccine treatment.

[0104] Those skilled in the art can easily perceive other aspects and advantages of the present application from the detailed description below. In the detailed description below, only exemplary embodiments of the present application are shown and described. As will be appreciated by those skilled in the art, the content of the present application enables those skilled in the art to modify the disclosed specific embodiments without departing from the spirit and scope of the invention to which the present application relates. Accordingly, the description in the drawings and specification of the present application is merely exemplary and not restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0105] The features and advantages of the inventions of the present application can be better understood by referring to the exemplary embodiments and drawings described in detail below. A brief description of the drawings is as follows:

[0106] Figure 1 What is shown is the trend of the change in the content of the neutralizing antibodies simulated by the calibrator described in this application in the human body;

[0107] Figure 2 Shown is the ROC curve for the Cutoff value described in this application. DETAILED DESCRIPTION

[0108] The following is an explanation of the implementation of the present invention by means of specific embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0109] Definition of terms

[0110] In this application, the term "sample" generally refers to any substance containing or assumed to contain antibodies, especially SARS-CoV-2 neutralizing antibodies. "Sample" can be of natural or synthetic origin and can be obtained by any means known to those skilled in the art. "Sample" can be a tissue or liquid sample separated from a subject, including but not limited to urine, saliva, serum, plasma, whole blood, nasopharyngeal aspirate, and bronchial lavage fluid. The sample can be a research sample and a clinical sample. The sample can also be a blood sample for transfusion or treatment. The sample can also be synthetic and include but not limited to in vitro cell culture components, including but not limited to conditioned medium, recombinant cells, and cell components. In this application, the terms "sample" and "specimen" can be used interchangeably.

[0111] In this application, the term "detect" generally means to find the presence or occurrence of something.

[0112] In this application, the term "antigen" generally refers to a substance that prompts the production of antibodies and can cause an immune response. They can also be used for diagnostic or patient selection or characterization purposes.

[0113] In the present application, the term "antibody" generally refers to a polypeptide molecule that can specifically recognize and / or neutralize a specific antigen. For example, an antibody may comprise an immunoglobulin consisting of at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, and includes any molecule comprising an antigen-binding portion thereof. The term "antibody" includes monoclonal antibodies, antibody fragments or antibody derivatives, including but not limited to human antibodies (fully human antibodies), humanized antibodies, chimeric antibodies, single-chain antibodies (e.g., scFv), and antibody fragments that bind to antigens (e.g., Fab, Fab' and (Fab)2 fragments). The term "antibody" also includes all recombinant forms of antibodies, such as antibodies expressed in prokaryotic cells, unglycosylated antibodies, and any antibody fragments and derivatives thereof that bind to antigens described herein. Each heavy chain may be composed of a heavy chain variable region (VH) and a heavy chain constant region. Each light chain may be composed of a light chain variable region (VL) and a light chain constant region. The VH and VL regions can be further divided into hypervariable regions called complementarity determining regions (CDRs), which are interspersed in more conserved regions called framework regions (FRs). Each VH and VL can be composed of three CDRs and four FR regions, which can be arranged in the following order from amino terminus to carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens.

[0114] In the present application, the term "antigen-binding fragment" generally refers to one or more fragments of an antibody that specifically binds to an antigen. The antigen-binding function of an antibody can be achieved by a full-length fragment of the antibody. The antigen-binding function of an antibody can also be achieved by a heavy chain fragment comprising a fragment of Fv, ScFv, dsFv, Fab, Fab' or F(ab')2, or a light chain fragment comprising a fragment of Fv, ScFv, dsFv, Fab, Fab' or F(ab')2. (1) Fab fragment, i.e., a monovalent fragment composed of VL, VH, CL and CH domains; (2) F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bond at the hinge region; (3) Fd fragment composed of VH and CH domains; (4) Fv fragment composed of the VL and VH domains of a single antibody arm; (5) dAb fragment composed of the VH domain (Ward et al., (1989) Nature 341: 544-546); (6) isolated complementarity determining regions (CDRs) and (7) a combination of two or more isolated CDRs optionally linked by a linker. In addition, it can also include a monovalent single-chain molecule Fv (scFv) formed by pairing VL and VH (see Bird et al. (1988) Science 242: 423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. 85: 5879-5883). The "antigen binding portion" may also include an immunoglobulin fusion protein, which comprises a binding domain selected from the following: (1) a binding domain polypeptide fused to an immunoglobulin hinge region polypeptide; (2) an immunoglobulin heavy chain CH2 constant region fused to the hinge region; and (3) an immunoglobulin heavy chain CH3 constant region fused to the CH2 constant region.

[0115] In this application, the term "subject" generally refers to any organism, including but not limited to mammals such as mice, rats, dogs, guinea pigs, ferrets, rabbits, and primates. In this application, the subject can be a human, a pet, or a livestock animal. In this application, the subject can be a recovered COVID-19 patient, a healthy subject, and / or a subject who has been treated with a COVID-19 vaccine.

[0116] In this application, the term "RBD" refers to the receptor binding domain of the SARS-CoV-2 spike protein (S protein). In this application, the RBD may be a mutant or truncate of the SARS-CoV-2 spike protein (S protein). In this application, the truncate generally refers to anything less than the whole. For example, the truncate is less than at least 60%, 65%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the amino acid sequence of the reference sequence. In this application, "mutant" generally refers to a sequence that is different from the reference sequence because it contains one or more differences (mutations). The difference may be a substitution, deletion or insertion of one or more amino acids. In this application, a truncate or mutant may be naturally occurring or non-naturally occurring. Non-naturally occurring truncations or mutants may be generated using techniques known in the art.

[0117] In the present application, the term "SARS-CoV-2 spike protein or a functionally active fragment thereof" generally refers to the capsid surface glycoprotein of the coronavirus. SARS-COV-2 binds to the ACE2 receptor through the SARS-CoV-2 spike protein and invades cells. The SARS-CoV-2 spike protein contains a transmembrane region comprising at least a 1213 amino acid fragment from the capsid surface glycoprotein of the coronavirus from the N-terminus or from the 14th amino acid, or from the corresponding region of other SAS viruses. In the present application, the SARS-CoV-2 spike protein can be a trimeric full-length SARS-CoV-2 spike protein. In the present application, the SARS-CoV-2 spike protein can be a full-length SARS-CoV-2 spike protein.

[0118] In this application, the term "ACE2 receptor protein or a functionally active fragment thereof" generally refers to a functional receptor of a coronavirus spike protein. In some embodiments, the ACE2 receptor protein may be a human ACE2 receptor protein. For example, the ACE2 receptor protein sequence may be found in Uniprot accession number Q9BYF1.

[0119] In this application, the term "functionally active fragment" generally refers to a fragment of a larger polypeptide or polynucleotide that retains the same activity or ability as its larger counterpart. The activity level of the functionally active fragment can be the same as the activity of the larger counterpart, or less or greater than it. For example, a functionally active fragment of the SARS-CoV-2 spike protein can be a polypeptide that consists of fewer amino acids than the full-length SARS-CoV-2 spike protein, but can still retain the activity of the full-length SARS-CoV-2 spike protein. For another example, a functionally active fragment of the human ACE2 receptor protein can be a polypeptide that consists of fewer amino acids than the full-length human ACE2 receptor protein, but can still retain the activity of the full-length human ACE2 receptor protein.

[0120] In this application, the term "Fc region" or "Fc" generally refers to the C-terminal region of an immunoglobulin heavy chain, which contains at least a portion of a constant region. The Fc region includes a native sequence Fc region and a variant Fc region. In some embodiments, the Fc region may be directly or indirectly connected to the SARS-CoV-2 spike protein. In some embodiments, the Fc region may be fused in frame with the SARS-CoV-2 spike protein. In some embodiments, the Fc region may be directly or indirectly connected to the ACE2 receptor protein. In some embodiments, the Fc region may be fused in frame with the ACE2 receptor protein. In this application, the Fc region includes a human or non-human (e.g., rat, mouse, rabbit) Fc region. For example, the Fc region may include a rabbit Fc region (rFc). For example, the Fc region may include a mouse Fc region (mFc). For example, the Fc region may include a human Fc region (hFc).

[0121] In the present application, "in-frame fusion" generally refers to connecting two or more open reading frames (ORFs) to form a continuous longer ORF in a manner that maintains the correct reading frame of the original ORF. Therefore, the resulting "fusion polypeptide" is a single protein comprising two or more fragments, and the two or more fragments correspond to the polypeptide encoded by the original ORF (the fragment is usually not connected in this way in nature). "Fusion site" refers to the sequence where two or more fragments are connected together. In some cases, the fusion site can be a sequence identical to a sequence in two or more fragments to be connected. In some cases, the fusion site may further include a vacant fragment that is different from any of the sequences of the two or more fragments to be connected. DETAILED DESCRIPTION OF THE INVENTION

[0123] Detection Kits

[0124] On the one hand, the present application provides a detection kit for SARS-CoV-2 neutralizing antibodies, which may include:

[0125] 1) SARS-CoV-2 spike protein or a functionally active fragment thereof; and

[0126] 2) A competitive binder, which comprises a substance that can compete with the SARS-CoV-2 neutralizing antibody for binding to the SARS-CoV-2 spike protein or a functionally active fragment thereof.

[0127] In the present application, the SARS-CoV-2 spike protein or its functionally active fragment may contain a receptor binding region RBD.

[0128] In the present application, the SARS-CoV-2 spike protein or its functionally active fragment may comprise the full-length SARS-CoV-2 spike protein.

[0129] In the present application, the SARS-CoV-2 spike protein or its functionally active fragment may comprise a trimeric full-length SARS-CoV-2 spike protein.

[0130] For example, the SARS-CoV-2 spike protein or its functionally active fragment may comprise the amino acid sequence shown in SEQ ID NO: 10. For example, the SARS-CoV-2 spike protein or its functionally active fragment may comprise the amino acid sequence shown in SEQ ID NO: 11.

[0131] In the present application, the SARS-CoV-2 spike protein or a functionally active fragment thereof may comprise a receptor binding region RBD, and the RBD may be directly or indirectly connected to the Fc region.

[0132] In the present application, the RBD may be fused in frame with the Fc. In the present application, the Fc may be selected from the group consisting of rFc, mFc and hFc.

[0133] In the present application, the RBD may be directly or indirectly linked to the Fc region. In the present application, the Fc may be rFc.

[0134] In the present application, the RBD may be directly or indirectly connected to the Fc region. In the present application, the Fc may be mFc.

[0135] In the present application, the RBD may be directly or indirectly connected to the Fc region. In the present application, the Fc may be hFc.

[0136] For example, the SARS-CoV-2 spike protein or its functionally active fragment may comprise the amino acid sequence shown in SEQ ID NO: 7. For example, the SARS-CoV-2 spike protein or its functionally active fragment may comprise the amino acid sequence shown in SEQ ID NO: 8. For example, the SARS-CoV-2 spike protein or its functionally active fragment may comprise the amino acid sequence shown in SEQ ID NO: 9.

[0137] In the present application, the concentration of the SARS-CoV-2 spike protein or its functionally active fragment may be 50ng / ml-4000ng / ml. For example, the concentration of the SARS-CoV-2 spike protein or its functionally active fragment may be 50ng / ml, 100ng / ml, 200ng / ml, 300ng / ml, 400ng / ml, 800ng / ml, 900ng / ml, 1000ng / ml, 1500ng / ml, 2000ng / ml, 2500ng / ml, 3000ng / ml, 3500ng / ml or 4000ng / ml.

[0138] In the present application, the SARS-CoV-2 spike protein or a functionally active fragment thereof can be immobilized on a solid support.

[0139] In the present application, the solid support may be a reaction plate or an ELISA plate (a reaction plate or an ELISA plate suitable for ELISA assay).

[0140] In the present application, the competitive binder may comprise an ACE2 receptor protein or a functionally active fragment thereof.

[0141] In the present application, the competitive binder may comprise a known RBD neutralizing antibody.

[0142] In the present application, the ACE2 receptor protein may include human ACE2 receptor protein or a functionally active fragment thereof.

[0143] In the present application, the ACE2 receptor protein or a functionally active fragment thereof may comprise an extracellular domain ECD.

[0144] In the present application, the ACE2 receptor protein or a functionally active fragment thereof may be directly or indirectly connected to the Fc region.

[0145] In the present application, the ACE2 receptor protein or a functionally active fragment thereof may be fused in-frame with the Fc region.

[0146] In the present application, the Fc region may be selected from the group consisting of rFc, mFc and hFc.

[0147] In the present application, the Fc region may be rFc. In the present application, the Fc region may be mFc. In the present application, the Fc region may be hFc.

[0148] In the present application, the competitive binder may comprise an amino acid sequence shown in any one of SEQ ID NOs: 1-5.

[0149] For example, the competitive binding agent may include the amino acid sequence shown in SEQ ID NO: 2. For example, the competitive binding agent may include the amino acid sequence shown in SEQ ID NO: 3. For example, the competitive binding agent may include the amino acid sequence shown in SEQ ID NO: 4.

[0150] In the present application, the competitive binding agent may include one or more labels that can directly or indirectly generate a signal showing the presence and / or content of the competitive binding agent. For example, the label may include horseradish peroxidase (HRP) or an enzyme similar to HRP. For example, the label may be horseradish peroxidase (HRP), alkaline phosphatase (AP), glucose oxidase or β-galactosidase β-Galactosidase. In some embodiments, the label may be horseradish peroxidase (HRP).

[0151] In the present application, the competitive binding agent may include horseradish peroxidase (HRP) or an HRP-like enzyme and one or more labels selected from the group consisting of: AVI tag, biotin, fluorescein and streptavidin.

[0152] For example, the competitive binding agent may comprise HRP, AVI tag, streptavidin.

[0153] In the present application, the concentration of the competitive binding agent may be 100ng / ml-2000ng / ml. For example, the concentration of the competitive binding agent may be 100ng / ml, 200ng / ml, 300ng / ml, 400ng / ml, 500ng / ml, 600ng / ml, 700ng / ml, 800ng / ml, 1000ng / ml, 1500ng / ml, 1800ng / ml, 1900ng / ml or 2000ng / ml.

[0154] In the present application, the kit may include a sample diluent, and the pH of the sample diluent may be 6.0-9.6. For example, the pH of the sample diluent may be 6.0, 6.1, 6.2, 6.3, 6.5, 7.0, 7.5, 7.8, 8.0, 8.5, 9.0, 9.5 or 9.6.

[0155] For example, the pH of the sample diluent may be 6.0.

[0156] In the present application, the kit also includes a calibrator, which may contain one or more reference antibody molecules or antigen-binding fragments thereof that specifically recognize the SARS-CoV-2 spike protein or its functionally active fragment.

[0157] In the present application, the one or more reference antibody molecules or antigen-binding fragments thereof can specifically recognize at least two different epitopes on the SARS-CoV-2 spike protein or a functionally active fragment thereof.

[0158] In the present application, the one or more reference antibody molecules or antigen-binding fragments thereof can specifically recognize at least 3 different epitopes on the SARS-CoV-2 spike protein or its functionally active fragment.

[0159] On the other hand, the present application provides a SARS-CoV-2 neutralizing antibody detection kit, which is different from the aforementioned kit in that the SARS-CoV-2 spike protein or its functionally active fragment may contain one or more markers, and the markers may directly or indirectly generate a signal showing the presence and / or content of the competitive binding agent. For example, the competitive binding agent may not contain a marker.

[0160] Reference Antibody

[0161] In the present application, the reference antibodies may include antibodies 32-1, 12-5 and 12-8.

[0162] For example, the reference antibody or antigen-binding fragment thereof described in the present application may comprise a heavy chain variable region VH and a light chain variable region VL, wherein the VH comprises HCDR1, HCDR2 and HCDR3, and the VL comprises LCDR1, LCDR2 and LCDR3. Wherein, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:30 or a variant thereof; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:31 or a variant thereof; the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:32 or a variant thereof; the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO:33 or a variant thereof; the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:34 or a variant thereof; the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:35 or a variant thereof. For example, the reference antibody or antigen-binding fragment thereof may comprise antibody 32-1 or an antibody having the same HCDR1-3 and LCDR1-3 therewith. In the present application, the VH may comprise the amino acid sequence shown in SEQ ID NO:21 or a variant thereof; and the VL may comprise the amino acid sequence shown in SEQ ID NO:22 or a variant thereof. In the present application, the nucleic acid sequence encoding VH may include the sequence shown in SEQ ID NO: 12; and the nucleic acid sequence encoding VL may include the sequence shown in SEQ ID NO: 13. For example, the reference antibody or its antigen-binding fragment may include antibody 32-1 or an antibody having the same light chain variable region and heavy chain variable region. For example, the reference antibody or its antigen-binding fragment described in the present application may include a heavy chain constant region CH and a light chain constant region CL. In the present application, the CH may include the amino acid sequence shown in SEQ ID NO: 29 or a variant thereof; and the CL may include the amino acid sequence shown in SEQ ID NO: 23 or a variant thereof. In the present application, the nucleic acid sequence encoding CH may include the sequence shown in SEQ ID NO: 20; and the nucleic acid sequence encoding CL may include the sequence shown in SEQ ID NO: 14. For example, the reference antibody or its antigen-binding fragment may include antibody 32-1 or an antibody having the same heavy chain constant region and light chain constant region.

[0163] In some embodiments, the antibody described in the present application may be 32-1. The HCDR1, HCDR2 and HCDR3 of antibody 32-1 may respectively comprise the amino acid sequences shown in SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32; VH may comprise the amino acid sequence shown in SEQ ID NO: 21; LCDR1, LCDR2 and LCDR3 may respectively comprise the amino acid sequences shown in SEQ ID NO: 33, SEQ ID NO: 34 and SEQ ID NO: 35; VL may comprise the amino acid sequence shown in SEQ ID NO: 22.

[0164] For example, the reference antibody or antigen-binding fragment thereof described in the present application may comprise a heavy chain variable region VH and a light chain variable region VL, wherein the VH may comprise HCDR1, HCDR2 and HCDR3, and the VL may comprise LCDR1, LCDR2 and LCDR3. Wherein, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:36 or a variant thereof; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:37 or a variant thereof; the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:38 or a variant thereof; the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO:39 or a variant thereof; the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:40 or a variant thereof; the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:41 or a variant thereof. For example, the reference antibody or antigen-binding fragment thereof may comprise antibody 12-5 or an antibody having the same HCDR1-3 and LCDR1-3 as antibody 12-5. In the present application, the VH may comprise the amino acid sequence shown in SEQ ID NO:24 or a variant thereof; and the VL may comprise the amino acid sequence shown in SEQ ID NO:25 or a variant thereof. In the present application, the nucleic acid sequence encoding VH may comprise the sequence shown in SEQ ID NO:15; and the nucleic acid sequence encoding VL may comprise the sequence shown in SEQ ID NO:16. For example, the reference antibody or its antigen-binding fragment may include antibody 12-5 or an antibody having the same light chain variable region and heavy chain variable region. For example, the reference antibody or its antigen-binding fragment described in the present application may comprise a heavy chain constant region CH and a light chain constant region CL. In the present application, the CH may comprise the amino acid sequence shown in SEQ ID NO:29 or a variant thereof; and the CL may comprise the amino acid sequence shown in SEQ ID NO:26 or a variant thereof. In the present application, the nucleic acid sequence encoding CH may comprise the sequence shown in SEQ ID NO:20; and the nucleic acid sequence encoding CL may comprise the sequence shown in SEQ ID NO:17. For example, the reference antibody or antigen-binding fragment thereof may include antibody 12-5 or an antibody having the same heavy chain constant region and light chain constant region therewith.

[0165] In some embodiments, the antibody described in the present application may be 12-5. The HCDR1, HCDR2 and HCDR3 of antibody 12-5 may respectively comprise the amino acid sequences shown in SEQ ID NO:36, SEQ ID NO:37 and SEQ ID NO:38; VH may comprise the amino acid sequence shown in SEQ ID NO:24; LCDR1, LCDR2 and LCDR3 may respectively comprise the amino acid sequences shown in SEQ ID NO:39, SEQ ID NO:40 and SEQ ID NO:41; VL may comprise the amino acid sequence shown in SEQ ID NO:25.

[0166] For example, the reference antibody or antigen-binding fragment thereof described in the present application may comprise a heavy chain variable region VH and a light chain variable region VL, wherein the VH comprises HCDR1, HCDR2 and HCDR3, and the VL comprises LCDR1, LCDR2 and LCDR3. Wherein, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:42 or a variant thereof; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:43 or a variant thereof; the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:44 or a variant thereof; the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO:45 or a variant thereof; the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:46 or a variant thereof; the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:47 or a variant thereof. For example, the reference antibody or antigen-binding fragment thereof may comprise antibody 12-8 or an antibody having the same HCDR1-3 and LCDR1-3 therewith. In the present application, the VH may comprise the amino acid sequence shown in SEQ ID NO:27 or a variant thereof; and the VL may comprise the amino acid sequence shown in SEQ ID NO:28 or a variant thereof. In the present application, the nucleic acid sequence encoding VH may include the sequence shown in SEQ ID NO: 18; and the nucleic acid sequence encoding VL may include the sequence shown in SEQ ID NO: 19. For example, the reference antibody or its antigen-binding fragment may include antibody 12-8 or an antibody having the same light chain variable region and heavy chain variable region. For example, the reference antibody or its antigen-binding fragment described in the present application may include a heavy chain constant region CH and a light chain constant region CL. In the present application, the CH may include the amino acid sequence shown in SEQ ID NO: 29 or a variant thereof; and the CL may include the amino acid sequence shown in SEQ ID NO: 23 or a variant thereof. In the present application, the nucleic acid sequence encoding CH may include the sequence shown in SEQ ID NO: 20; and the nucleic acid sequence encoding CL may include the sequence shown in SEQ ID NO: 14. For example, the reference antibody or its antigen-binding fragment may include antibody 12-8 or an antibody having the same heavy chain constant region and light chain constant region.

[0167] In some embodiments, the antibody described in the present application may be 12-8. The HCDR1, HCDR2 and HCDR3 of antibody 12-8 may comprise the amino acid sequences shown in SEQ ID NO:42, SEQ ID NO:43 and SEQ ID NO:44, respectively; VH may comprise the amino acid sequence shown in SEQ ID NO:27; LCDR1, LCDR2 and LCDR3 may comprise the amino acid sequences shown in SEQ ID NO:45, SEQ ID NO:46 and SEQ ID NO:47, respectively; and VL may comprise the amino acid sequence shown in SEQ ID NO:28.

[0168] In the present application, the concentration of the reference antibody can be 50ng / ml-1000ng / ml. For example, the concentration of the reference antibody can be 50ng / ml, 60ng / ml, 70ng / ml, 80ng / ml, 90ng / ml, 100ng / ml, 200ng / ml, 300ng / ml, 400ng / ml, 500ng / ml, 600ng / ml, 700ng / ml, 800ng / ml, 900ng / ml and 1000ng / ml.

[0169] Detection Methods

[0170] On the other hand, the present application provides a method for detecting SARS-CoV-2 neutralizing antibodies in a sample, which may include:

[0171] The described kit was used.

[0172] On the other hand, the present application provides a method for detecting SARS-CoV-2 neutralizing antibodies in a sample, which may include:

[0173] 1) Encapsulating SARS-CoV-2 spike protein or its functionally active fragment on a solid support;

[0174] 2) adding the sample to be tested in step 1) to react with the SARS-CoV-2 spike protein or its functionally active fragment;

[0175] 3) adding the competitive binding agent to step 2);

[0176] 4) detecting SARS-CoV-2 neutralizing antibodies in the sample to be tested;

[0177] Among them, the competitive binder may include a substance that can compete with the SARS-CoV-2 neutralizing antibody for binding to the SARS-CoV-2 spike protein or its functionally active fragment; the SARS-CoV-2 spike protein or its functionally active fragment may include a receptor binding region RBD; the competitive binder may include ACE2 receptor protein or its functionally active fragment.

[0178] In some embodiments, the SARS-CoV-2 spike protein or its functionally active fragment may comprise a full-length SARS-CoV-2 spike protein. In some embodiments, the SARS-CoV-2 spike protein or its functionally active fragment may comprise a trimeric full-length SARS-COV-2 spike protein.

[0179] In some embodiments, the RBD may be directly or indirectly linked to the Fc region. In some embodiments, the RBD may be fused in frame to the Fc.

[0180] In some embodiments, the Fc region can be selected from the group consisting of rFc, mFc, and hFc. For example, the Fc region can be rFc. For example, the Fc region can be mFc. For example, the Fc region can be hFc.

[0181] In some embodiments, the SARS-CoV-2 spike protein or a functionally active fragment thereof may comprise the amino acid sequence shown in SEQ ID NO:7.

[0182] In some embodiments, the SARS-CoV-2 spike protein or a functionally active fragment thereof may comprise the amino acid sequence shown in SEQ ID NO:8.

[0183] In some embodiments, the SARS-CoV-2 spike protein or a functionally active fragment thereof may comprise the amino acid sequence shown in SEQ ID NO:9.

[0184] In some embodiments, the SARS-CoV-2 spike protein or a functionally active fragment thereof may comprise the amino acid sequence shown in SEQ ID NO:10.

[0185] In some embodiments, the SARS-CoV-2 spike protein or a functionally active fragment thereof may comprise the amino acid sequence shown in SEQ ID NO:11.

[0186] In some embodiments, the concentration of the SARS-CoV-2 spike protein or its functionally active fragment may be 50ng / ml-4000ng / ml. For example, it may be 50ng / ml-3500ng / ml, 50ng / ml-3000ng / ml, 50ng / ml-2000ng / ml, 50ng / ml-1500ng / ml, 50ng / ml-1000ng / ml, 50ng / ml-900ng / ml or 50ng / ml-800ng / ml. For example, the concentration of the SARS-CoV-2 spike protein or its functionally active fragment can be 50ng / ml, 100ng / ml, 200ng / ml, 300ng / ml, 400ng / ml, 800ng / ml, 900ng / ml, 1000ng / ml, 1500ng / ml, 2000ng / ml, 2500ng / ml, 3000ng / ml, 3500ng / ml or 4000ng / ml.

[0187] In some embodiments, the ACE2 receptor protein or a functionally active fragment thereof may comprise a human ACE2 receptor protein or a functionally active fragment thereof.

[0188] In some embodiments, the ACE2 receptor protein or a functionally active fragment thereof may comprise an extracellular domain ECD.

[0189] In some embodiments, the ACE2 receptor protein or a functionally active fragment thereof may be directly or indirectly linked to the Fc region.

[0190] In some embodiments, the ACE2 receptor protein or a functionally active fragment thereof may be fused in frame with the Fc region.

[0191] In some embodiments, the Fc region is selected from the group consisting of rFc, mFc, and hFc. For example, it is rFc. For example, it is mFc. For example, it is hFc.

[0192] In some embodiments, the ACE2 receptor protein or a functionally active fragment thereof may comprise the amino acid sequence shown in SEQ ID NO:2.

[0193] In some embodiments, the ACE2 receptor protein or a functionally active fragment thereof may comprise the amino acid sequence shown in SEQ ID NO:3.

[0194] In some embodiments, the ACE2 receptor protein or a functionally active fragment thereof may comprise the amino acid sequence shown in SEQ ID NO:4.

[0195] In some embodiments, the ACE2 receptor protein or a functionally active fragment thereof may contain one or more markers, which can directly or indirectly generate a signal indicating the presence and / or content of the ACE2 receptor protein or a functionally active fragment thereof.

[0196] In some embodiments, the label may include horseradish peroxidase (HRP) or an HRP-like enzyme.

[0197] In some embodiments, the reaction concentration of the ACE2 receptor protein or its functionally active fragment may be 100ng / ml-2000ng / ml. For example, it may be 100ng / ml-1500ng / ml, 100ng / ml-1000ng / ml, 100ng / ml-800ng / ml, 500ng / ml-2000ng / ml, 500ng / ml-1500ng / ml, 500ng / ml-1000ng / ml or 500ng / ml-800ng / ml. For example, it may be 200ng / ml, 300ng / ml, 400ng / ml, 500ng / ml, 600ng / ml, 700ng / ml, 800ng / ml, 900ng / ml, 1000ng / ml, 1500ng / ml or 2000ng / ml.

[0198] In some embodiments, the coating concentration of the RBD can be 50ng / ml-4000ng / ml. For example, it can be 50ng / ml-3500ng / ml, 50ng / ml-3000ng / ml, 50ng / ml-2000ng / ml, 50ng / ml-1500ng / ml, 50ng / ml-1000ng / ml, 50ng / ml-900ng / ml or 50ng / ml-800ng / ml. For example, it can be 50ng / ml, 60ng / ml, 70ng / ml, 80ng / ml, 90ng / ml, 100ng / ml, 200ng / ml, 300ng / ml, 400ng / ml, 500ng / ml, 600ng / ml, 700ng / ml, 800ng / ml, 900ng / ml or 1000ng / ml.

[0199] In some embodiments, when the coating concentration of the RBD may be 50 ng / ml-4000 ng / ml, the reaction concentration of the ACE2 receptor protein or its functionally active fragment may be 500 ng / ml-1500 ng / ml.

[0200] In some embodiments, it may include a sample diluent, and the pH of the sample diluent may be 6.0-9.6.

[0201] In some embodiments, it may also include a calibrator, which may contain one or more reference antibody molecules or antigen-binding fragments thereof that specifically recognize the SARS-CoV-2 spike protein or a functionally active fragment thereof.

[0202] In some embodiments, the one or more reference antibody molecules or antigen-binding fragments thereof can specifically recognize at least two different epitopes on the SARS-CoV-2 spike protein or a functionally active fragment thereof.

[0203] In some embodiments, the one or more reference antibody molecules or antigen-binding fragments thereof can specifically recognize at least 3 different epitopes on the SARS-CoV-2 spike protein or a functionally active fragment thereof.

[0204] In some embodiments, the reference antibodies may include antibodies 32-1, 12-5, and 12-8.

[0205] In some embodiments, the concentration of the reference antibody may be 50 ng / ml-1000 ng / ml.

[0206] In another aspect, the present application provides a method for detecting SARS-CoV-2 neutralizing antibodies in a sample, which may include:

[0207] 1) Encapsulating ACE2 receptor protein or its functionally active fragment on a solid support;

[0208] 2) mixing and incubating the sample to be tested with the SARS-CoV-2 spike protein or its functionally active fragment to obtain an incubation product;

[0209] 3) adding the incubated product of step 2) to step 1);

[0210] 4) detecting SARS-CoV-2 neutralizing antibodies in the sample to be tested;

[0211] The SARS-CoV-2 spike protein or a functionally active fragment thereof may comprise a receptor binding domain (RBD), and the RBD may comprise one or more markers capable of directly or indirectly generating a signal showing the presence and / or content of the RBD.

[0212] In some embodiments, the label may include horseradish peroxidase (HRP) or an HRP-like enzyme.

[0213] In some embodiments, the RBD may be linked directly or indirectly to the Fc region.

[0214] In some embodiments, the RBD may be fused in frame to the Fc.

[0215] In some embodiments, the Fc region can be selected from the group consisting of rFc, mFc and hFc.

[0216] In some embodiments, the SARS-CoV-2 spike protein or a functionally active fragment thereof may comprise the amino acid sequence shown in SEQ ID NO:7.

[0217] In some embodiments, the SARS-CoV-2 spike protein or a functionally active fragment thereof may comprise the amino acid sequence shown in SEQ ID NO:8.

[0218] In some embodiments, the SARS-CoV-2 spike protein or a functionally active fragment thereof may comprise the amino acid sequence shown in SEQ ID NO:9.

[0219] In some embodiments, the SARS-CoV-2 spike protein or a functionally active fragment thereof may comprise the amino acid sequence shown in SEQ ID NO:10.

[0220] In some embodiments, the SARS-CoV-2 spike protein or a functionally active fragment thereof may comprise the amino acid sequence shown in SEQ ID NO:11.

[0221] In some embodiments, the concentration of the SARS-CoV-2 spike protein or a functionally active fragment thereof can be 50 ng / ml-4000 ng / ml.

[0222] In some embodiments, the ACE2 receptor protein or a functionally active fragment thereof may be directly or indirectly linked to the Fc region.

[0223] In some embodiments, the ACE2 receptor protein or a functionally active fragment thereof may be fused in frame with the Fc region.

[0224] In some embodiments, the Fc region can be selected from the group consisting of rFc, mFc and hFc.

[0225] In some embodiments, the ACE2 receptor protein or a functionally active fragment thereof may comprise the amino acid sequence shown in SEQ ID NO:2.

[0226] In some embodiments, the ACE2 receptor protein or a functionally active fragment thereof may comprise the amino acid sequence shown in SEQ ID NO:3.

[0227] In some embodiments, the ACE2 receptor protein or a functionally active fragment thereof may comprise the amino acid sequence shown in SEQ ID NO:4.

[0228] In some embodiments, the coating concentration of the ACE2 receptor protein or its functionally active fragment may be 1 μg / ml-20 μg / ml. For example, it may be 1 μg / ml-15 μg / ml, 1 μg / ml-10 μg / ml, 1 μg / ml-8 μg / ml, 1 μg / ml-7 μg / ml, 1 μg / ml-6 μg / ml or 1 μg / ml-5 μg / ml. For example, it may be 2 μg / ml-10 μg / ml, 2 μg / ml-8 μg / ml, 2 μg / ml-6 μg / ml, 3 μg / ml-10 μg / ml, 3 μg / ml-8 μg / ml or 3 μg / ml-6 μg / ml. For example, it may be 1 μg / ml, 2 μg / ml, 3 μg / ml, 4 μg / ml, 5 μg / ml, 6 μg / ml, 7 μg / ml, 8 μg / ml, 9 μg / ml, 10 μg / ml, 15 μg / ml or 20 μg / ml.

[0229] In some embodiments, the reaction concentration of the RBD may be 10 ng / ml-200 ng / ml. For example, it may be 10 ng / ml-150 ng / ml, 10 ng / ml-100 ng / ml, 10 ng / ml-80 ng / ml, 10 ng / ml-50 ng / ml, 10 ng / ml-40 ng / ml, 10 ng / ml-30 ng / ml, or 10 ng / ml-20 ng / ml. For example, it may be 20 ng / ml-150 ng / ml, 20 ng / ml-100 ng / ml, 20 ng / ml-80 ng / ml, 20 ng / ml-50 ng / ml, 20 ng / ml-40 ng / ml, or 20 ng / ml-30 ng / ml. For example, it can be 10 ng / ml, 20 ng / ml, 30 ng / ml, 40 ng / ml, 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 400 ng / ml, 90 ng / ml or 100 ng / ml.

[0230] In some embodiments, when the coating concentration of the ACE2 receptor protein or its functionally active fragment may be 1 μg / ml-10 μg / ml, the reaction concentration of the RBD may be 20 ng / ml-150 ng / ml.

[0231] In some embodiments, when the coating concentration of the ACE2 receptor protein or its functionally active fragment can be 3 μg / ml-6 μg / ml, the reaction concentration of the RBD can be 20 ng / ml-40 ng / ml.

[0232] In some embodiments, it may include a sample diluent, and the pH of the sample diluent may be 6.0-9.6.

[0233] In some embodiments, it may also include a calibrator, which may contain one or more reference antibody molecules or antigen-binding fragments thereof that specifically recognize the SARS-CoV-2 spike protein or a functionally active fragment thereof.

[0234] In some embodiments, the one or more reference antibody molecules or antigen-binding fragments thereof can specifically recognize at least two different epitopes on the SARS-CoV-2 spike protein or a functionally active fragment thereof.

[0235] In some embodiments, the one or more reference antibody molecules or antigen-binding fragments thereof can specifically recognize at least 3 different epitopes on the SARS-CoV-2 spike protein or a functionally active fragment thereof.

[0236] In some embodiments, the reference antibodies may include antibodies 32-1, 12-5, and 12-8.

[0237] In some embodiments, the concentration of the reference antibody can be 50 ng / ml-1000 ng / ml.

[0238] In the present application, the sample can be natural or synthetic in origin, and can be obtained by any means known to those skilled in the art. For example, the sample can be a tissue or liquid sample separated from a subject, including but not limited to urine, saliva, serum, plasma, whole blood, nasopharyngeal aspirate and bronchial lavage fluid. For example, the sample can be a research sample and a clinical sample. For example, the sample can also be a blood sample for transfusion or treatment. For example, the sample can also be synthetic and include but not limited to in vitro cell culture components, including but not limited to conditioned medium, recombinant cells and cell components. In the present application, the terms "sample" and "specimen" can be used interchangeably.

[0239] In this application, the sample is derived from a subject. In this application, the subject generally refers to any organism, including but not limited to mammals, such as mice, rats, dogs, guinea pigs, ferrets, rabbits and primates. In this application, the subject can be a human, a pet or a livestock. In this application, the subject can be a recovered COVID-19 patient, a healthy subject and / or a subject who has received a COVID-19 vaccine.

[0240] In some embodiments, the method may include the following steps:

[0241] Step 1, coating antigen: dilute the SARS-CoV-2 spike protein or its functionally active fragment with coating solution, and add the diluted SARS-CoV-2 spike protein or its functionally active fragment into the enzyme-labeled wells of the reaction plate.

[0242] Step 2, add the sample to be tested and incubate for 30-60 minutes (for example, incubate for 30 minutes, incubate for 35 minutes, incubate for 40 minutes, incubate for 45 minutes, incubate for 50 minutes, incubate for 55 minutes or incubate for 60 minutes).

[0243] Step three, add competitive binding agent, and incubate for 30-60 minutes (e.g., incubate for 30 minutes, incubate for 35 minutes, incubate for 40 minutes, incubate for 45 minutes, incubate for 50 minutes, incubate for 55 minutes, or incubate for 60 minutes).

[0244] Step 4: color development and end reading.

[0245] In some embodiments, the sample to be tested can be incubated in an environment of 35°C-40°C (for example, the incubation temperature can be 35°C, 35.5°C, 36°C, 36.5°C, 36.6°C, 36.7°C, 36.8°C, 36.9°C, 37°C, 37.1°C, 37.2°C, 37.3°C, 37.4°C, 37.5°C, 37.6°C, 37.7°C, 37.8°C, 37.9°C, 38°C, 38.5°C, 39°C, 39.5°C or 40°C).

[0246] In some embodiments, the competitive binders may be incubated in a 35°C-40°C environment (e.g., the incubation temperature may be 35°C, 35.5°C, 36°C, 36.5°C, 36.6°C, 36.7°C, 36.8°C, 36.9°C, 37°C, 37.1°C, 37.2°C, 37.3°C, 37.4°C, 37.5°C, 37.6°C, 37.7°C, 37.8°C, 37.9°C, 38°C, 38.5°C, 39°C, 39.5°C, or 40°C).

[0247] In some embodiments, the color development process can be carried out in an environment of 35°C-40°C (for example, 35°C, 35.5°C, 36°C, 36.5°C, 36.6°C, 36.7°C, 36.8°C, 36.9°C, 37°C, 37.1°C, 37.2°C, 37.3°C, 37.4°C, 37.5°C, 37.6°C, 37.7°C, 37.8°C, 37.9°C, 38°C, 38.5°C, 39°C, 39.5°C or 40°C).

[0248] Some embodiments of the present invention:

[0249] 1. A detection kit for SARS-CoV-2 neutralizing antibodies, comprising:

[0250] 1) SARS-CoV-2 spike protein or a functionally active fragment thereof; and

[0251] 2) A competitive binder, which comprises a substance that can compete with the SARS-CoV-2 neutralizing antibody for binding to the SARS-CoV-2 spike protein or a functionally active fragment thereof.

[0252] 2. A kit according to embodiment 1, wherein the SARS-CoV-2 spike protein or a functionally active fragment thereof comprises a receptor binding domain (RBD).

[0253] 3. A kit according to any one of embodiments 1-2, wherein the SARS-CoV-2 spike protein or a functionally active fragment thereof comprises a full-length SARS-CoV-2 spike protein.

[0254] 4. A kit according to any one of embodiments 1-3, wherein the SARS-CoV-2 spike protein or a functionally active fragment thereof comprises a trimeric full-length SARS-COV-2 spike protein.

[0255] 5. A kit according to any one of embodiments 2-4, wherein the RBD is directly or indirectly connected to Fc.

[0256] 6. A kit according to embodiment 5, wherein the RBD is fused in frame with the Fc.

[0257] 7. The kit of any one of embodiments 5-6, wherein the Fc region is selected from the group consisting of rFc, mFc and hFc.

[0258] 8. A kit according to embodiment 7, wherein the SARS-CoV-2 spike protein or a functionally active fragment thereof comprises the amino acid sequence shown in SEQ ID NO:7.

[0259] 9. The kit according to embodiment 7, wherein the SARS-CoV-2 spike protein or a functionally active fragment thereof comprises the amino acid sequence shown in SEQ ID NO:8.

[0260] 10. The kit according to embodiment 7, wherein the SARS-CoV-2 spike protein or a functionally active fragment thereof comprises the amino acid sequence shown in SEQ ID NO:9.

[0261] 11. A kit according to embodiment 3, wherein the SARS-CoV-2 spike protein or a functionally active fragment thereof comprises the amino acid sequence shown in SEQ ID NO:10.

[0262] 12. The kit according to embodiment 4, wherein the SARS-CoV-2 spike protein or a functionally active fragment thereof comprises the amino acid sequence shown in SEQ ID NO:11.

[0263] 13. A kit according to any one of embodiments 1-12, wherein the competitive binder comprises an ACE2 receptor protein or a functionally active fragment thereof.

[0264] 14. A kit according to embodiment 13, wherein the ACE2 receptor protein or a functionally active fragment thereof comprises a human ACE2 receptor protein or a functionally active fragment thereof.

[0265] 15. A kit according to any one of embodiments 13-14, wherein the ACE2 receptor protein or a functionally active fragment thereof comprises an extracellular domain ECD.

[0266] 16. A kit according to any one of embodiments 13-15, wherein the ACE2 receptor protein or a functionally active fragment thereof is directly or indirectly connected to the Fc region.

[0267] 17. A kit according to embodiment 16, wherein the ACE2 receptor protein or a functionally active fragment thereof is fused in frame with the Fc region.

[0268] 18. The kit of any one of embodiments 16-17, wherein the Fc region is selected from the group consisting of rFc, mFc and hFc.

[0269] 19. A kit according to embodiment 18, wherein the competitive binder comprises the amino acid sequence shown in SEQ ID NO:2.

[0270] 20. A kit according to embodiment 18, wherein the competitive binder comprises the amino acid sequence shown in SEQ ID NO:3.

[0271] 21. A kit according to embodiment 18, wherein the competitive binder comprises the amino acid sequence shown in SEQ ID NO:4.

[0272] 22. A kit according to embodiment 13, wherein the competitive binder comprises one or more labels that are capable of directly or indirectly generating a signal indicating the presence and / or amount of the competitive binder.

[0273] 23. The kit of embodiment 22, wherein the label comprises horseradish peroxidase (HRP) or an HRP-like enzyme.

[0274] 24. The kit according to any one of embodiments 1-23, comprising a sample diluent, wherein the pH of the sample diluent is 6.0-9.6.

[0275] 25. A kit according to any one of embodiments 1-24, further comprising a calibrator, wherein the calibrator comprises one or more reference antibody molecules or antigen-binding fragments thereof that specifically recognize the SARS-CoV-2 spike protein or a functionally active fragment thereof.

[0276] 26. A kit according to embodiment 25, wherein the one or more reference antibody molecules or antigen-binding fragments thereof specifically recognize at least 2 different epitopes on the SARS-CoV-2 spike protein or a functionally active fragment thereof.

[0277] 27. A kit according to embodiment 25, wherein the one or more reference antibody molecules or antigen-binding fragments thereof specifically recognize at least 3 different epitopes on the SARS-CoV-2 spike protein or a functionally active fragment thereof.

[0278] 28. The kit according to any one of embodiments 25-27, wherein the reference antibody comprises a heavy chain variable region VH and a light chain variable region VL; wherein:

[0279] The VH comprises HCDR1, HCDR2 and HCDR3 of the amino acid sequences shown in SEQ ID NOs: 30-32, and the VL comprises LCDR1, LCDR2 and LCDR3 of the amino acid sequences shown in SEQ ID NOs: 33-35;

[0280] The VH comprises HCDR1, HCDR2 and HCDR3 of the amino acid sequences shown in SEQ ID NOs: 36-38, and the VL comprises LCDR1, LCDR2 and LCDR3 of the amino acid sequences shown in SEQ ID NOs: 39-41; or

[0281] The VH comprises HCDR1, HCDR2 and HCDR3 of the amino acid sequences shown in SEQ ID NOs: 42-44, and the VL comprises LCDR1, LCDR2 and LCDR3 of the amino acid sequences shown in SEQ ID NOs: 45-47.

[0282] 29. The kit according to any one of embodiments 25-28, wherein the reference antibody comprises a heavy chain variable region VH and a light chain variable region VL; wherein:

[0283] The VH comprises the amino acid sequence shown in SEQ ID NO: 21, and the VL comprises the amino acid sequence shown in SEQ ID NO: 22;

[0284] The VH comprises the amino acid sequence shown in SEQ ID NO: 24, and the VL comprises the amino acid sequence shown in SEQ ID NO: 25; or

[0285] The VH comprises the amino acid sequence shown in SEQ ID NO:27, and the VL comprises the amino acid sequence shown in SEQ ID NO:28.

[0286] 30. The kit of any one of embodiments 25-29, wherein the reference antibodies include antibodies 32-1, 12-5, and 12-8.

[0287] 31. The kit of any one of embodiments 25-30, wherein the concentration of the reference antibody is 50 ng / ml-1000 ng / ml.

[0288] 32. A kit according to any one of embodiments 1-31, wherein the SARS-CoV-2 spike protein or a functionally active fragment thereof comprises one or more markers that are capable of directly or indirectly generating a signal indicating the presence and / or amount of the SARS-CoV-2 spike protein or a functionally active fragment thereof.

[0289] 33. A kit according to embodiment 32, wherein the label comprises horseradish peroxidase (HRP) or an HRP-like enzyme.

[0290] 34. A method for detecting SARS-CoV-2 neutralizing antibodies in a sample, comprising:

[0291] Use the kit described in embodiment 1 or 13.

[0292] 35. A method for detecting SARS-CoV-2 neutralizing antibodies in a sample, comprising:

[0293] 1) Encapsulating SARS-CoV-2 spike protein or its functionally active fragment on a solid support;

[0294] 2) adding the sample to be tested in step 1) to react with the SARS-CoV-2 spike protein or its functionally active fragment;

[0295] 3) adding the competitive binding agent to step 2);

[0296] 4) detecting SARS-CoV-2 neutralizing antibodies in the sample to be tested;

[0297] Wherein, the competitive binding agent comprises a substance that can compete with the SARS-CoV-2 neutralizing antibody for binding to the SARS-CoV-2 spike protein or its functionally active fragment;

[0298] The SARS-CoV-2 spike protein or a functionally active fragment thereof comprises a receptor binding domain RBD; the competitive binder comprises an ACE2 receptor protein or a functionally active fragment thereof.

[0299] 36. The method of embodiment 35, wherein the SARS-CoV-2 spike protein or a functionally active fragment thereof comprises a full-length SARS-CoV-2 spike protein.

[0300] 37. A method according to any one of embodiments 35-36, wherein the SARS-CoV-2 spike protein or a functionally active fragment thereof comprises a trimeric full-length SARS-COV-2 spike protein.

[0301] 38. A method according to any one of embodiments 35-37, wherein the RBD is directly or indirectly connected to the Fc region.

[0302] 39. A method according to embodiment 38, wherein the RBD is fused in frame with the Fc.

[0303] 40. The method of any one of embodiments 38-39, wherein the Fc region is selected from the group consisting of rFc, mFc, and hFc.

[0304] 41. The method of embodiment 40, wherein the SARS-CoV-2 spike protein or a functionally active fragment thereof comprises the amino acid sequence shown in SEQ ID NO:7.

[0305] 42. The method of embodiment 40, wherein the SARS-CoV-2 spike protein or a functionally active fragment thereof comprises the amino acid sequence shown in SEQ ID NO:8.

[0306] 43. The method of embodiment 40, wherein the SARS-CoV-2 spike protein or a functionally active fragment thereof comprises the amino acid sequence shown in SEQ ID NO:9.

[0307] 44. The method of embodiment 36, wherein the SARS-CoV-2 spike protein or a functionally active fragment thereof comprises the amino acid sequence shown in SEQ ID NO:10.

[0308] 45. The method of embodiment 37, wherein the SARS-CoV-2 spike protein or a functionally active fragment thereof comprises the amino acid sequence shown in SEQ ID NO:11.

[0309] 46. ​​The method of any one of embodiments 35-45, wherein the concentration of the SARS-CoV-2 spike protein or a functionally active fragment thereof is 50 ng / ml-4000 ng / ml.

[0310] 47. A method according to any one of embodiments 35-46, wherein the ACE2 receptor protein or a functionally active fragment thereof comprises a human ACE2 receptor protein or a functionally active fragment thereof.

[0311] 48. A method according to embodiment 47, wherein the ACE2 receptor protein or a functionally active fragment thereof comprises an extracellular domain ECD.

[0312] 49. A method according to any one of embodiments 47-48, wherein the ACE2 receptor protein or a functionally active fragment thereof is directly or indirectly linked to the Fc region.

[0313] 50. A method according to embodiment 49, wherein the ACE2 receptor protein or a functionally active fragment thereof is fused in-frame with the Fc region.

[0314] 51. The method of any one of embodiments 49-50, wherein the Fc region is selected from the group consisting of rFc, mFc, and hFc.

[0315] 52. A method according to embodiment 51, wherein the ACE2 receptor protein or a functionally active fragment thereof comprises the amino acid sequence shown in SEQ ID NO:2.

[0316] 53. A method according to embodiment 51, wherein the ACE2 receptor protein or a functionally active fragment thereof comprises the amino acid sequence shown in SEQ ID NO:3.

[0317] 54. A method according to embodiment 51, wherein the ACE2 receptor protein or a functionally active fragment thereof comprises the amino acid sequence shown in SEQ ID NO:4.

[0318] 55. A method according to any one of embodiments 35-54, wherein the ACE2 receptor protein or a functionally active fragment thereof comprises one or more markers, which are capable of directly or indirectly generating a signal showing the presence and / or content of the ACE2 receptor protein or a functionally active fragment thereof.

[0319] 56. A method according to embodiment 55, wherein the label comprises horseradish peroxidase (HRP) or an HRP-like enzyme.

[0320] 57. A method according to any one of embodiments 35-56, wherein the concentration of the ACE2 receptor protein or its functionally active fragment is 100ng / ml-2000ng / ml.

[0321] 58. The method of any one of embodiments 35-57, comprising a sample diluent having a pH of 6.0-9.6.

[0322] 59. The method of any one of embodiments 35-58, further comprising a calibrator comprising one or more reference antibody molecules or antigen-binding fragments thereof that specifically recognize the SARS-CoV-2 spike protein or a functionally active fragment thereof.

[0323] 60. A method according to embodiment 59, wherein the one or more reference antibody molecules or antigen-binding fragments thereof specifically recognize at least 2 different epitopes on the SARS-CoV-2 spike protein or a functionally active fragment thereof.

[0324] 61. A method according to embodiment 59, wherein the one or more reference antibody molecules or antigen-binding fragments thereof specifically recognize at least 3 different epitopes on the SARS-CoV-2 spike protein or a functionally active fragment thereof.

[0325] 62. The method according to any one of embodiments 59-61, wherein the reference antibody comprises a heavy chain variable region VH and a light chain variable region VL; wherein:

[0326] The VH comprises HCDR1, HCDR2 and HCDR3 of the amino acid sequences shown in SEQ ID NOs: 30-32, and the VL comprises LCDR1, LCDR2 and LCDR3 of the amino acid sequences shown in SEQ ID NOs: 33-35;

[0327] The VH comprises HCDR1, HCDR2 and HCDR3 of the amino acid sequences shown in SEQ ID NOs: 36-38, and the VL comprises LCDR1, LCDR2 and LCDR3 of the amino acid sequences shown in SEQ ID NOs: 39-41; or

[0328] The VH comprises HCDR1, HCDR2 and HCDR3 of the amino acid sequences shown in SEQ ID NOs: 42-44, and the VL comprises LCDR1, LCDR2 and LCDR3 of the amino acid sequences shown in SEQ ID NOs: 45-47.

[0329] 63. The method according to any one of embodiments 59-62, wherein the reference antibody comprises a heavy chain variable region VH and a light chain variable region VL; wherein:

[0330] The VH comprises the amino acid sequence shown in SEQ ID NO: 21, and the VL comprises the amino acid sequence shown in SEQ ID NO: 22;

[0331] The VH comprises the amino acid sequence shown in SEQ ID NO: 24, and the VL comprises the amino acid sequence shown in SEQ ID NO: 25; or

[0332] The VH comprises the amino acid sequence shown in SEQ ID NO:27, and the VL comprises the amino acid sequence shown in SEQ ID NO:28.

[0333] 64. The method of any one of embodiments 59-63, wherein the reference antibodies include antibodies 32-1, 12-5, and 12-8.

[0334] 65. The method of any one of embodiments 59-64, wherein the concentration of the reference antibody is 50 ng / ml-1000 ng / ml.

[0335] 66. The method of embodiment 35, wherein the sample is selected from the group consisting of urine, saliva, serum, plasma, whole blood, nasopharyngeal aspirate, and bronchial lavage fluid.

[0336] 67. A method according to embodiment 66, wherein the sample originates from a subject.

[0337] 68. A method according to embodiment 67, wherein the subject is a recovered COVID-19 patient, a healthy subject and / or a subject who has received COVID-19 vaccine treatment.

[0338] 69. A method for detecting SARS-CoV-2 neutralizing antibodies in a sample, comprising:

[0339] 1) Encapsulating ACE2 receptor protein or its functionally active fragment on a solid support;

[0340] 2) mixing and incubating the sample to be tested with the SARS-CoV-2 spike protein or its functionally active fragment to obtain an incubation product;

[0341] 3) adding the incubated product of step 2) to step 1);

[0342] 4) detecting SARS-CoV-2 neutralizing antibodies in the sample to be tested;

[0343] The SARS-CoV-2 spike protein or a functionally active fragment thereof comprises a receptor binding domain RBD, and the RBD comprises one or more markers, which can directly or indirectly generate a signal showing the presence and / or content of the RBD.

[0344] 70. The method of embodiment 69, wherein the label comprises horseradish peroxidase (HRP) or an HRP-like enzyme.

[0345] 71. A method according to any one of embodiments 69-70, wherein the RBD is directly or indirectly connected to the Fc region.

[0346] 72. A method according to embodiment 71, wherein the RBD is fused in frame with the Fc.

[0347] 73. The method of any one of embodiments 71-72, wherein the Fc region is selected from the group consisting of rFc, mFc, and hFc.

[0348] 74. The method of embodiment 73, wherein the SARS-CoV-2 spike protein or a functionally active fragment thereof comprises the amino acid sequence shown in SEQ ID NO:7.

[0349] 75. The method of embodiment 73, wherein the SARS-CoV-2 spike protein or a functionally active fragment thereof comprises the amino acid sequence shown in SEQ ID NO:8.

[0350] 76. A method according to embodiment 73, wherein the SARS-CoV-2 spike protein or a functionally active fragment thereof comprises the amino acid sequence shown in SEQ ID NO:9.

[0351] 77. The method of embodiment 69, wherein the SARS-CoV-2 spike protein or a functionally active fragment thereof comprises the amino acid sequence shown in SEQ ID NO:10.

[0352] 78. A method according to embodiment 69, wherein the SARS-CoV-2 spike protein or a functionally active fragment thereof comprises the amino acid sequence shown in SEQ ID NO:11.

[0353] 79. The method of any one of embodiments 69-78, wherein the concentration of the SARS-CoV-2 spike protein or a functionally active fragment thereof is 50 ng / ml-4000 ng / ml.

[0354] 80. A method according to embodiments 69-79, wherein the ACE2 receptor protein or a functionally active fragment thereof is directly or indirectly connected to the Fc region.

[0355] 81. A method according to embodiment 80, wherein the ACE2 receptor protein or a functionally active fragment thereof is fused in-frame with the Fc region.

[0356] 82. The method of any one of embodiments 80-81, wherein the Fc region is selected from the group consisting of rFc, mFc, and hFc.

[0357] 83. A method according to embodiment 82, wherein the ACE2 receptor protein or a functionally active fragment thereof comprises the amino acid sequence shown in SEQ ID NO:2.

[0358] 84. A method according to embodiment 82, wherein the ACE2 receptor protein or a functionally active fragment thereof comprises the amino acid sequence shown in SEQ ID NO:3.

[0359] 85. A method according to embodiment 82, wherein the ACE2 receptor protein or a functionally active fragment thereof comprises the amino acid sequence shown in SEQ ID NO:4.

[0360] 86. A method according to any one of embodiments 69-85, wherein the concentration of the ACE2 receptor protein or its functionally active fragment is 100ng / ml-2000ng / ml.

[0361] 87. The method of any one of embodiments 69-86, comprising a sample diluent having a pH of 6.0-9.6.

[0362] 88. The method according to any one of embodiments 69-87, wherein the detection is obtained by comparison with a calibrator, wherein the calibrator comprises one or more reference antibody molecules or antigen-binding fragments thereof that specifically recognize the SARS-CoV-2 spike protein or a functionally active fragment thereof; the reference antibody comprises a heavy chain variable region VH and a light chain variable region VL, wherein:

[0363] The VH comprises HCDR1, HCDR2 and HCDR3 of the amino acid sequences shown in SEQ ID NOs: 30-32, and the VL comprises LCDR1, LCDR2 and LCDR3 of the amino acid sequences shown in SEQ ID NOs: 33-35;

[0364] The VH comprises HCDR1, HCDR2 and HCDR3 of the amino acid sequences shown in SEQ ID NOs: 36-38, and the VL comprises LCDR1, LCDR2 and LCDR3 of the amino acid sequences shown in SEQ ID NOs: 39-41; or

[0365] The VH comprises HCDR1, HCDR2 and HCDR3 of the amino acid sequences shown in SEQ ID NOs: 42-44, and the VL comprises LCDR1, LCDR2 and LCDR3 of the amino acid sequences shown in SEQ ID NOs: 45-47.

[0366] 89. The method according to embodiment 88, wherein the detection is obtained by comparison with a calibrator, wherein the calibrator comprises one or more reference antibody molecules or antigen-binding fragments thereof that specifically recognize the SARS-CoV-2 spike protein or a functionally active fragment thereof; the reference antibody comprises a heavy chain variable region VH and a light chain variable region VL; wherein:

[0367] The VH comprises the amino acid sequence shown in SEQ ID NO: 21, and the VL comprises the amino acid sequence shown in SEQ ID NO: 22;

[0368] The VH comprises the amino acid sequence shown in SEQ ID NO: 24, and the VL comprises the amino acid sequence shown in SEQ ID NO: 25; or

[0369] The VH comprises the amino acid sequence shown in SEQ ID NO:27, and the VL comprises the amino acid sequence shown in SEQ ID NO:28.

[0370] 90. The method of any one of embodiments 88-89, wherein the reference antibodies include antibodies 32-1, 12-5, and 12-8.

[0371] 91. The method of any one of embodiments 88-90, wherein the concentration of the reference antibody is 50 ng / ml-1000 ng / ml.

[0372] 92. The method according to embodiment 69, wherein the sample to be tested is selected from the group consisting of urine, saliva, serum, plasma, whole blood, nasopharyngeal aspirate and bronchial lavage fluid.

[0373] 93. A method according to embodiment 92, wherein the sample to be tested originates from a subject.

[0374] 94. A method according to embodiment 93, wherein the subject is a recovered COVID-19 patient, a healthy subject and / or a subject who has received COVID-19 vaccine treatment.

[0375] Without intending to be bound by any theory, the following examples are merely intended to illustrate the fusion protein, preparation method and use of the present application, and are not intended to limit the scope of the present invention.

[0376] Example

[0377] Example 1 ELISA test method for SARS-CoV-2 neutralizing antibodies

[0378] 1.1 Activation of horseradish peroxidase (HRP): Dissolve horseradish peroxidase (HRP) powder in ddH2O to prepare 10 mg / ml horseradish peroxidase (HRP) solution, dissolve sodium periodate powder in ddH2O to prepare 0.1 M sodium periodate solution, mix horseradish peroxidase (HRP) solution and sodium periodate solution at a volume ratio of 1:0.51, incubate at 2-8°C in dark for 1 hour; add 0.26 times the volume of horseradish peroxidase (HRP) solution of ethylene glycol, incubate at 2-8°C in dark for 20 minutes; centrifuge in 50 mL ultrafiltration tube at 6000-7500 rpm for 10-20 minutes; centrifuge 3-5 times with 1 mM acetate buffer; store at -80°C.

[0379] 1.2 Horseradish peroxidase (HRP) labeled human ACE2 receptor protein: add human ACE2 receptor protein and activated horseradish peroxidase (HRP) at a mass ratio of 1:4, add sodium carbonate buffer (pH 9.6) with a volume of 1 / 5 of the total volume of human ACE2 receptor protein and horseradish peroxidase (HRP), and place it at room temperature away from light for 1 hour; add 1 / 10 volume of horseradish peroxidase (HRP) reducing agent, and place it at room temperature away from light for 1 hour; add 1 / 10 volume of horseradish peroxidase (HRP) terminator, and place it at room temperature away from light for 30 minutes, then it can be used or stored at 4°C.

[0380] 1.3 Coating antigen: Use 1x coating solution (10x coating solution is 15.9g / LNa2CO3+29.3g / LNaHCO3, diluted to 1x with ddH2O for use) to dilute the RBD antigen to 0.3μg / mL and mix thoroughly. Add the RBD antigen dilution solution to the enzyme-labeled wells of the reaction plate, 100μL per well. Cover the reaction plate and place it at 4℃ for overnight coating. The upper layer is liquid, and the coated RBD antigen is adsorbed on the reaction plate.

[0381] 1.4 Blocking: Use 1x washing solution (20x washing solution is 2.4g / L KH2PO4+29g / L NaHPO4·12H2O+160g / LNaCl+4g / L KCl+10mL / L Tween-20+2.24g / LP300, diluted to 1x with ddH2O) to wash the reaction plate once with 350μL per well to wash off the coating solution, pat the washing solution in the reaction plate dry on absorbent paper, and quickly add 100-200μL blocking solution (104.5g / L 10x PBS+10g / L Casein+50g / L sucrose+2.24g / L P300), place in a 37℃ incubator, and incubate for 2h; pour off the blocking solution, spin dry with a plate shaker or pat dry on absorbent paper, and then you can use the reaction plate. If not used immediately, the reaction plate should be placed in a sealed bag and stored at 2-8℃ with a desiccant pack. The desiccant pack should be located at the bottom of the reaction plate and should not touch the wells of the reaction plate.

[0382] 1.5 Add the sample to be tested: take out all components including the serum to be tested in advance and equilibrate to room temperature. After the reaction plate is equilibrated to room temperature, take it out of the sealed bag, add 80 μL of sample diluent (0.02M PBS+0.9% NaCl+0.5% BSA+0.1% Tween-20+0.2% P300, pH7.4) to each well, then add 20 μL of calibrator (calibrator is an antibody with different concentrations, the concentrations are 1000 ng / mL, 800 ng / mL, 400 ng / mL, 200 ng / mL, 100 ng / mL and 50 ng / mL, see Example 6 for details), 20 μL of serum to be tested to the corresponding wells to which the sample diluent has been added, attach the plate sticker, oscillate on a micro-oscillator for 60 seconds to mix the liquid in the wells evenly, place in a 37°C incubator, and incubate for 0.5 hours.

[0383] 1.6 Add enzyme-labeled conjugate: Use sample diluent to dilute ACE2-HRP at 1:3000 (initial concentration is 5 mg / mL), carefully peel off the plate sticker, add 100 μL of diluted ACE2-HRP to each well, replace the plate sticker and stick it on, oscillate on a micro-oscillator for 60 seconds to mix the liquid in the well evenly, place in a 37°C incubator, and incubate for 0.5 hours.

[0384] 1.7 Wash the plate: Carefully peel off the plate sticker and rinse the reaction plate six times with 1x washing solution (it is recommended to use a plate washer, with 350μL of solution added to each well). Finally, pat dry on absorbent paper or spin dry with a plate spinner.

[0385] 1.8 Color development: Mix color development solution A (2.1g / L citric acid monohydrate + 8.448g / L sodium acetate + 0.5g / L urea peroxide + 0.08g / L EDTA + 2mL / L P300) and solution B (10mL / L 100x basic buffer (105.6g / L citric acid monohydrate + 10.3g / L sodium acetate + 2mL / L P300) + 100mL / L glycerol + 0.5g / L TMB hydrochloride + 1.67mL / L Na2S2O3 (20mg / mL) + 2mL / L P300) in a volume ratio of 1:1 to form a color development working solution. Add 100μL of the color development working solution to each well, replace the plate sticker and stick it well, and place it at 37℃ in the dark for 10 minutes.

[0386] 1.9 Reading:

[0387] Carefully peel off the plate sticker, add 50 μL of stop solution (252.5 g / L citric acid monohydrate + 26.6 mL / L concentrated sulfuric acid) to each well to stop the reaction, and use an ELISA reader at 450 nm single wavelength to measure the absorbance of each well.

[0388] 1.10 Determination of critical value: The test serum was detected by the above method. When the sample inhibition rate was >15%, the sample was determined to be positive. When the sample inhibition rate was <15%, the sample was determined to be negative. Inhibition rate = (1-absorbance value of the test sample / absorbance value of the negative control) x 100%. The inhibition rate reflects the neutralization efficiency of the antibodies in the test serum.

[0389] Example 2 ELISA test method for SARS-CoV-2 neutralizing antibodies

[0390] The steps and corresponding reagents not particularly described in this example are the same as those in Example 1.

[0391] 1.1 Activation of horseradish peroxidase (HRP): Dissolve horseradish peroxidase (HRP) powder in ddH2O to prepare 10mg / ml horseradish peroxidase (HRP) solution, dissolve sodium periodate powder in ddH2O to prepare 0.1M sodium periodate solution, mix horseradish peroxidase (HRP) solution and sodium periodate solution at a volume ratio of 1:0.51, and store in dark at 2-8 degrees for 1 hour; add 0.26 times the volume of horseradish peroxidase (HRP) solution of ethylene glycol, and store in dark at 2-8 degrees for 20 minutes; centrifuge in a 50ml ultrafiltration tube at 6000-7500rpm for 10-20 minutes; centrifuge 3-5 times with 1mM acetate buffer; and store at -80 degrees.

[0392] 1.2 Horseradish peroxidase (HRP) labeling of RBD protein: add RBD protein and activated horseradish peroxidase (HRP) at a mass ratio of 1:4, add sodium carbonate buffer (pH 9.6) with a volume of 1 / 5 of the total volume of RBD protein and horseradish peroxidase (HRP), and place at room temperature away from light for 1 hour; add reducing agent with a volume of 1 / 10 horseradish peroxidase (HRP), and place at room temperature away from light for 1 hour; add terminator with a volume of 1 / 10 horseradish peroxidase (HRP), and place at room temperature away from light for 30 minutes. It can be used or stored at 4°C.

[0393] 1.3 Coating protein: Use 1x coating solution to dilute ACE2 protein to 0.3 μg / mL and mix thoroughly. Add ACE2 protein dilution solution to the enzyme-labeled wells of the reaction plate, 100 μL per well. Cover the reaction plate and place it at 4°C for overnight coating. The upper layer is liquid. The coated ACE2 protein is adsorbed on the reaction plate.

[0394] 1.4 Blocking: Rinse the reaction plate once with 350 μL of 1x washing solution per well to wash away the coating solution, pat the washing solution in the reaction plate dry on absorbent paper, quickly add 100-200 μL of blocking solution to each well, place in a 37°C incubator, and incubate for 2 hours; pour out the blocking solution, spin dry with a plate spinner or pat dry on absorbent paper, and then you can use the reaction plate. If not used immediately, the reaction plate should be placed in a sealed bag and stored at 2-8°C with a drying bag. The drying bag should be located at the bottom of the reaction plate and cannot touch the wells of the reaction plate.

[0395] 1.5 Add the samples to be tested: Take out all components including samples in advance and equilibrate to room temperature. After the reaction plate is equilibrated to room temperature, take it out of the sealed bag. After the samples are diluted twice with sample diluent, add 10 μL of positive control, negative control and a series of samples with different dilution multiples to each well, and add 90 μL of sample diluent.

[0396] 1.6 Add enzyme-labeled conjugate: Use sample diluent to dilute RBD-HRP to the required concentration (initial concentration is 5 mg / mL), add 100 μL of diluted RBD-HRP to each well, attach the plate sticker, oscillate on a micro-oscillator for 60 seconds to mix the liquid in the well evenly, place in a 37°C incubator, and incubate for 0.5 h.

[0397] 1.7 Transfer: Carefully peel off the plate sticker and transfer 100 μL of sample / control reaction solution from the 96-well PCR plate to the corresponding wells of the reaction plate. Stick the plate sticker and place in a 37°C incubator for 0.5 h.

[0398] 1.8 Wash the plate: Carefully peel off the plate sticker and rinse the reaction plate six times with 1x washing solution (it is recommended to use a plate washer, with 350μL of solution added to each well). Finally, pat dry on absorbent paper or spin dry with a plate spinner.

[0399] 1.9 Color development: Mix color development solution A and solution B in a volume ratio of 1:1 to form a color development working solution, add 100 μL of color development working solution to each well, replace the plate sticker and stick it well, and place it at 37°C in the dark for 10 minutes.

[0400] 1.10 Reading: Carefully peel off the plate sticker, add 50 μL of stop solution to each well to stop the reaction, and use an ELISA reader at 450 nm single wavelength to measure the absorbance of each well.

[0401] 1.11 Determination of titer value: When the sample is tested using the above method, if the inhibition rate of the sample at a certain dilution factor is greater than 15%, and the inhibition rate of the sample at the next dilution gradient of the dilution factor is less than 15%, then the dilution factor is the titer of the sample. The titer is the maximum dilution that can still produce a positive result, reflecting the potency of the antibody.

[0402] Example 3 Effect of different pH sample dilutions on test results

[0403] The non-specific binding of serum samples was reduced by changing the pH of the sample diluent. As shown in Table 1, pH 6.0, pH 7.8, and pH 9.6 sample diluents were used respectively, and according to the detection steps of Example 1, RBD-mFc coated plates and horseradish peroxidase (HRP) were used to label ACE2-mFc, and non-SARS-CoV-2 infected human serum samples, i.e., negative serum, were detected, and the inhibition rates of positive serum diluted with sample diluents of different pH and the inhibition rates of negative serum were compared. The results show that the universal sample diluent of pH 6.0 can reduce the effect of non-specific binding in negative serum on the reaction without affecting the detection of positive samples.

[0404] Table 1 Effects of different pH sample dilutions on test results

[0405]

[0406] Example 4 Effects of different molecular forms of RBD and ACE2 on detection results

[0407] According to the established ELISA detection method (the operating steps are the same as in Example 1 or Example 2), horseradish peroxidase (HRP)-labeled RBD-His and RBD-mFc were added to the ACE2-mFc plate, and horseradish peroxidase (HRP)-labeled ACE2-His, ACE2-hFc, ACE2-mFc and ACE2-rFc were added to the RBD-His plate. Two replicates were performed for each sample, and 0 μg / mL RBD neutralizing antibody and 1 μg / mL RBD neutralizing antibody were detected respectively. The results are shown in Table 2. The signal-to-noise ratio and stability of RBD and ACE2 with Fc tags were significantly improved compared to His-tagged RBD and ACE2.

[0408] Table 2 Effects of RBD and ACE2 with different labels on detection results

[0409]

[0410] Example 5 Effect of RBD and ACE2 dosage on test results

[0411] 5.1 Using five reaction plates with different RBD-mFc concentrations, ACE2-mFc-HRP was diluted at two dilution ratios of 1:2000 and 1:4000. According to the operating procedures of Example 1, 25 COVID-19 vaccine sera, i.e., positive sera and 25 negative sera were detected to compare the sensitivity, specificity and linear range under different RBD-mFc coating concentrations and ACE2-mFc-HRP dilution ratios.

[0412] 5.2 Use reaction plates with five different full-length S protein concentrations and follow the same operating steps as 5.1 to compare the sensitivity, specificity, and linear range at different full-length S protein coating concentrations and ACE2-mFc-HRP dilution ratios.

[0413] 5.3 Use reaction plates with five different trimeric S protein concentrations and the same operating steps as 5.1 to compare the sensitivity, specificity and linear range under different trimeric S protein coating concentrations and ACE2-mFc-HRP dilution ratios.

[0414] The results showed that the detection sensitivity could be significantly improved by optimizing the coating concentration of RBD-mFc or full-length S protein or trimeric S protein, as shown in Tables 3-5.

[0415] Table 3 Results of RBD-mFc and ACE2-mFc-HRP dosage optimization

[0416]

[0417] Table 4 Optimization results of full-length S protein and ACE2-mFc-HRP dosage

[0418]

[0419] Table 5 Optimization results of the dosage of trimer S protein and ACE2-mFc-HRP

[0420]

[0421] Optimization of ACE2-mFc coating amount and RBD-mFc-HRP dosage

[0422] Using three reaction plates with different ACE2-mFc concentrations, RBD-mFc-HRP was diluted at five dilution ratios of 500 ng / ml, 250 ng / ml, 125 ng / ml, 62.5 ng / ml and 30 ng / ml. According to the operating steps of Example 2, the gradient dilution concentration of the antibody was detected, and the differences in the detection lines under different ACE2-mFc coating concentrations and RBD-mFc-HRP dilution ratios were compared.

[0423] The results showed that the detection sensitivity could be significantly improved by optimizing the coating concentration of ACE2-mFc and the concentration of RBD-mFc-HRP. The results are shown in Table 6.

[0424] Table 6 Optimization of ACE2-mFc coating amount and RBD-HRP dosage

[0425]

[0426] Example 6 Selection of Calibrators

[0427] Multiple strains of natural human neutralizing antibodies were screened out through single B cells, and they were verified to have good neutralizing effects through real virus verification, which can well simulate the natural form of neutralizing antibodies in the human body. Three antibodies with different epitopes were screened out and mixed to prepare standard products, and six antibody concentrations of calibrators were prepared. The antibody concentrations of C.1 to C.6 were 1000ng / mL, 800ng / mL, 400ng / mL, 200ng / mL, 100ng / mL and 50ng / mL, respectively. According to the operating steps of Example 1, the absorbance values ​​of the six antibody concentrations of the standard products were detected at 450nm. Using Graphpad 6.02 version software, a graph was drawn based on the antibody concentration and absorbance value of the calibrator. According to the results, it is inferred that its reactivity (reaction slope) is closer to the real sample, and it can more realistically simulate the trend of changes in the content of neutralizing antibodies in the human body, such as Figure 1 shown.

[0428] Example 7 Determination of Cutoff Value

[0429] The cut-off value is the value of the sample being tested, which is used to determine whether the result is above or below the clinical or analytical decision point. The setting of the cut-off value gives a brief combination of sensitivity and specificity. The cut-off value was determined based on the evaluation results of 27 recovered patient sera, 1452 non-COVID-infected human serum samples, and 72 vaccine sera (one-third placebo), and analyzed using SPSS software. Based on this cut-off value, according to the operating steps of Example 1, the sensitivity of the test kit of the present application is >90% and the specificity is >99%. The consistency of the vaccine effect evaluation with the real virus experiment results is 95%. Sensitivity: that is, sensitivity, which indicates the detection rate of positive samples. For example, 0.977 means that 99.7% of positive samples can be correctly judged as positive results. Specificity: indicates the detection rate of negative samples. For example, 0.991 means that 99.1% of negative samples can be correctly judged as negative results. The test variable results are shown in Table 7, and the receiver operating characteristic curve (receiver operating characteristic curve, or ROC curve) is shown in Table 7. Figure 2 The curve coordinates and detection results are shown in Table 8.

[0430] Table 7 Detection variable results

[0431]

[0432] Table 8 Curve coordinates and test variable results

[0433] Sequence Listing <110> Nanjing Novozymes Biotech Co., Ltd. <120> A kit and method for detecting SARS-CoV-2 neutralizing antibodies <160> 47 <170> SIPOSequenceListing 1.0 <210> 1 <211> 723 <212> PRT <213> Artificial Sequence <400> 1 Gln Ser Thr Ile Glu Glu Gln Ala Lys Thr Phe Leu Asp Lys Phe Asn 1 5 10 15 His Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp Asn 20 25 30 Tyr Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn Ala 35 40 45 Gly Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Leu Ala Gln 50 55 60 Met Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu Gln Leu 65 70 75 80 Gln Ala Leu Gln Gln Asn Gly Ser Ser Val Leu Ser Glu Asp Lys Ser 85 90 95 Lys Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser Thr 100 105 110 Gly Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu Glu 115 120 125 Pro Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu Arg 130 135 140 Leu Trp Ala Trp Glu Ser Trp Arg Ser Glu Val Gly Lys Gln Leu Arg 145 150 155 160 Pro Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg Ala 165 170 175 Asn His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu Val 180 185 190 Asn Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln Leu Ile Glu Asp 195 200 205 Val Glu His Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu His 210 215 220 Ala Tyr Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile Ser 225 230 235 240 Pro Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly Arg 245 250 255 Phe Trp Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys Pro 260 265 270 Asn Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala Gln 275 280 285 Arg Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu Pro 290 295 300 Asn Met Thr Gln Gly Phe Trp Glu Asn Ser Met Leu Thr Asp Pro Gly 305 310 315 320 Asn Val Gln Lys Ala Val Cys His Pro Thr Ala Trp Asp Leu Gly Lys 325 330 335 Gly Asp Phe Arg Ile Leu Met Cys Thr Lys Val Thr Met Asp Asp Phe 340 345 350 Leu Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala Tyr 355 360 365 Ala Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe His 370 375 380 Glu Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys His 385 390 395 400 Leu Lys Ser Ile Gly Leu Leu Ser Pro Asp Phe Gln Glu Asp Asn Glu 405 410 415 Thr Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly Thr 420 425 430 Leu Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe Lys 435 440 445 Gly Glu Ile Pro Lys Asp Gln Trp Met Lys Lys Trp Trp Glu Met Lys 450 455 460 Arg Glu Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr Tyr 465 470 475 480 Cys Asp Pro Ala Ser Leu Phe His Val Ser Asn Asp Tyr Ser Phe Ile 485 490 495 Arg Tyr Tyr Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu Ala Leu 500 505 510 Cys Gln Ala Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile Ser 515 520 525 Asn Ser Thr Glu Ala Gly Gln Lys Leu Phe Asn Met Leu Arg Leu Gly 530 535 540 Lys Ser Glu Pro Trp Thr Leu Ala Leu Glu Asn Val Val Gly Ala Lys 545 550 555 560 Asn Met Asn Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe Thr 565 570 575 Trp Leu Lys Asp Gln Asn Lys Asn Ser Phe Val Gly Trp Ser Thr Asp 580 585 590 Trp Ser Pro Tyr Ala Asp Gln Ser Ile Lys Val Arg Ile Ser Leu Lys 595 600 605 Ser Ala Leu Gly Asp Lys Ala Tyr Glu Trp Asn Asp Asn Glu Met Tyr 610 615 620 Leu Phe Arg Ser Ser Val Ala Tyr Ala Met Arg Gln Tyr Phe Leu Lys 625 630 635 640 Val Lys Asn Gln Met Ile Leu Phe Gly Glu Glu Asp Val Arg Val Ala 645 650 655 Asn Leu Lys Pro Arg Ile Ser Phe Asn Phe Phe Val Thr Ala Pro Lys 660 665 670 Asn Val Ser Asp Ile Ile Pro Arg Thr Glu Val Glu Lys Ala Ile Arg 675 680 685 Met Ser Arg Ser Arg Ile Asn Asp Ala Phe Arg Leu Asn Asp Asn Ser 690 695 700 Leu Glu Phe Leu Gly Ile Gln Pro Thr Leu Gly Pro Pro Asn Gln Pro 705 710 715 720 Pro Val Ser <210> 2 <211> 954 <212> PRT <213> Artificial Sequence <400> 2 Gln Ser Thr Ile Glu Glu Gln Ala Lys Thr Phe Leu Asp Lys Phe Asn 1 5 10 15 His Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp Asn 20 25 30 Tyr Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn Ala 35 40 45 Gly Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Leu Ala Gln 50 55 60 Met Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu Gln Leu 65 70 75 80 Gln Ala Leu Gln Gln Asn Gly Ser Ser Val Leu Ser Glu Asp Lys Ser 85 90 95 Lys Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser Thr 100 105 110 Gly Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu Glu 115 120 125 Pro Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu Arg 130 135 140 Leu Trp Ala Trp Glu Ser Trp Arg Ser Glu Val Gly Lys Gln Leu Arg 145 150 155 160 Pro Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg Ala 165 170 175 Asn His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu Val 180 185 190 Asn Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln Leu Ile Glu Asp 195 200 205 Val Glu His Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu His 210 215 220 Ala Tyr Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile Ser 225 230 235 240 Pro Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly Arg 245 250 255 Phe Trp Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys Pro 260 265 270 Asn Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala Gln 275 280 285 Arg Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu Pro 290 295 300 Asn Met Thr Gln Gly Phe Trp Glu Asn Ser Met Leu Thr Asp Pro Gly 305 310 315 320 Asn Val Gln Lys Ala Val Cys His Pro Thr Ala Trp Asp Leu Gly Lys 325 330 335 Gly Asp Phe Arg Ile Leu Met Cys Thr Lys Val Thr Met Asp Asp Phe 340 345 350 Leu Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala Tyr 355 360 365 Ala Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe His 370 375 380 Glu Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys His 385 390 395 400 Leu Lys Ser Ile Gly Leu Leu Ser Pro Asp Phe Gln Glu Asp Asn Glu 405 410 415 Thr Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly Thr 420 425 430 Leu Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe Lys 435 440 445 Gly Glu Ile Pro Lys Asp Gln Trp Met Lys Lys Trp Trp Glu Met Lys 450 455 460 Arg Glu Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr Tyr 465 470 475 480 Cys Asp Pro Ala Ser Leu Phe His Val Ser Asn Asp Tyr Ser Phe Ile 485 490 495 Arg Tyr Tyr Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu Ala Leu 500 505 510 Cys Gln Ala Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile Ser 515 520 525 Asn Ser Thr Glu Ala Gly Gln Lys Leu Phe Asn Met Leu Arg Leu Gly 530 535 540 Lys Ser Glu Pro Trp Thr Leu Ala Leu Glu Asn Val Val Gly Ala Lys 545 550 555 560 Asn Met Asn Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe Thr 565 570 575 Trp Leu Lys Asp Gln Asn Lys Asn Ser Phe Val Gly Trp Ser Thr Asp 580 585 590 Trp Ser Pro Tyr Ala Asp Gln Ser Ile Lys Val Arg Ile Ser Leu Lys 595 600 605 Ser Ala Leu Gly Asp Lys Ala Tyr Glu Trp Asn Asp Asn Glu Met Tyr 610 615 620 Leu Phe Arg Ser Ser Val Ala Tyr Ala Met Arg Gln Tyr Phe Leu Lys 625 630 635 640 Val Lys Asn Gln Met Ile Leu Phe Gly Glu Glu Asp Val Arg Val Ala 645 650 655 Asn Leu Lys Pro Arg Ile Ser Phe Asn Phe Phe Val Thr Ala Pro Lys 660 665 670 Asn Val Ser Asp Ile Ile Pro Arg Thr Glu Val Glu Lys Ala Ile Arg 675 680 685 Met Ser Arg Ser Arg Ile Asn Asp Ala Phe Arg Leu Asn Asp Asn Ser 690 695 700 Leu Glu Phe Leu Gly Ile Gln Pro Thr Leu Gly Pro Pro Asn Gln Pro 705 710 715 720 Pro Val Ser Gly Gly Gly Ser Val Pro Arg Asp Cys Gly Cys Lys Pro 725 730 735 Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro Pro 740 745 750 Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr Cys 755 760 765 Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser Trp 770 775 780 Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg Glu 785 790 795 800 Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile Met 805 810 815 His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn Ser 820 825 830 Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly 835 840 845 Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln 850 855 860 Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe Phe 865 870 875 880 Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu 885 890 895 Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe 900 905 910 Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly Asn 915 920 925 Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His Thr 930 935 940 Glu Lys Ser Leu Ser His Ser Pro Gly Lys 945 950 <210> 3 <211> 955 <212> PRT <213> Artificial Sequence <400> 3 Gln Ser Thr Ile Glu Glu Gln Ala Lys Thr Phe Leu Asp Lys Phe Asn 1 5 10 15 His Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp Asn 20 25 30 Tyr Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn Ala 35 40 45 Gly Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Leu Ala Gln 50 55 60 Met Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu Gln Leu 65 70 75 80 Gln Ala Leu Gln Gln Asn Gly Ser Ser Val Leu Ser Glu Asp Lys Ser 85 90 95 Lys Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser Thr 100 105 110 Gly Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu Glu 115 120 125 Pro Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu Arg 130 135 140 Leu Trp Ala Trp Glu Ser Trp Arg Ser Glu Val Gly Lys Gln Leu Arg 145 150 155 160 Pro Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg Ala 165 170 175 Asn His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu Val 180 185 190 Asn Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln Leu Ile Glu Asp 195 200 205 Val Glu His Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu His 210 215 220 Ala Tyr Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile Ser 225 230 235 240 Pro Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly Arg 245 250 255 Phe Trp Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys Pro 260 265 270 Asn Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala Gln 275 280 285 Arg Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu Pro 290 295 300 Asn Met Thr Gln Gly Phe Trp Glu Asn Ser Met Leu Thr Asp Pro Gly 305 310 315 320 Asn Val Gln Lys Ala Val Cys His Pro Thr Ala Trp Asp Leu Gly Lys 325 330 335 Gly Asp Phe Arg Ile Leu Met Cys Thr Lys Val Thr Met Asp Asp Phe 340 345 350 Leu Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala Tyr 355 360 365 Ala Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe His 370 375 380 Glu Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys His 385 390 395 400 Leu Lys Ser Ile Gly Leu Leu Ser Pro Asp Phe Gln Glu Asp Asn Glu 405 410 415 Thr Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly Thr 420 425 430 Leu Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe Lys 435 440 445 Gly Glu Ile Pro Lys Asp Gln Trp Met Lys Lys Trp Trp Glu Met Lys 450 455 460 Arg Glu Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr Tyr 465 470 475 480 Cys Asp Pro Ala Ser Leu Phe His Val Ser Asn Asp Tyr Ser Phe Ile 485 490 495 Arg Tyr Tyr Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu Ala Leu 500 505 510 Cys Gln Ala Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile Ser 515 520 525 Asn Ser Thr Glu Ala Gly Gln Lys Leu Phe Asn Met Leu Arg Leu Gly 530 535 540 Lys Ser Glu Pro Trp Thr Leu Ala Leu Glu Asn Val Val Gly Ala Lys 545 550 555 560 Asn Met Asn Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe Thr 565 570 575 Trp Leu Lys Asp Gln Asn Lys Asn Ser Phe Val Gly Trp Ser Thr Asp 580 585 590 Trp Ser Pro Tyr Ala Asp Gln Ser Ile Lys Val Arg Ile Ser Leu Lys 595 600 605 Ser Ala Leu Gly Asp Lys Ala Tyr Glu Trp Asn Asp Asn Glu Met Tyr 610 615 620 Leu Phe Arg Ser Ser Val Ala Tyr Ala Met Arg Gln Tyr Phe Leu Lys 625 630 635 640 Val Lys Asn Gln Met Ile Leu Phe Gly Glu Glu Asp Val Arg Val Ala 645 650 655 Asn Leu Lys Pro Arg Ile Ser Phe Asn Phe Phe Val Thr Ala Pro Lys 660 665 670 Asn Val Ser Asp Ile Ile Pro Arg Thr Glu Val Glu Lys Ala Ile Arg 675 680 685 Met Ser Arg Ser Arg Ile Asn Asp Ala Phe Arg Leu Asn Asp Asn Ser 690 695 700 Leu Glu Phe Leu Gly Ile Gln Pro Thr Leu Gly Pro Pro Asn Gln Pro 705 710 715 720 Pro Val Ser Gly Gly Gly Ser Ala Pro Ser Thr Cys Ser Lys Pro Thr 725 730 735 Cys Pro Pro Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro 740 745 750 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 755 760 765 Cys Val Val Val Asp Val Ser Gln Asp Asp Pro Glu Val Gln Phe Thr 770 775 780 Trp Tyr Ile Asn Asn Glu Gln Val Arg Thr Ala Arg Pro Pro Leu Arg 785 790 795 800 Glu Gln Gln Phe Asn Ser Thr Ile Arg Val Val Ser Thr Leu Pro Ile 805 810 815 Ala His Gln Asp Trp Leu Arg Gly Lys Glu Phe Lys Cys Lys Val His 820 825 830 Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Arg 835 840 845 Gly Gln Pro Leu Glu Pro Lys Val Tyr Thr Met Gly Pro Pro Arg Glu 850 855 860 Glu Leu Ser Ser Arg Ser Val Ser Leu Thr Cys Met Ile Asn Gly Phe 865 870 875 880 Tyr Pro Ser Asp Ile Ser Val Glu Trp Glu Lys Asn Gly Lys Ala Glu 885 890 895 Asp Asn Tyr Lys Thr Thr Pro Ala Val Leu Asp Ser Asp Gly Ser Tyr 900 905 910 Phe Leu Tyr Ser Lys Leu Ser Val Pro Thr Ser Glu Trp Gln Arg Gly 915 920 925 Asp Val Phe Thr Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 930 935 940 Thr Gln Lys Ser Ile Ser Arg Ser Pro Gly Lys 945 950 955 <210> 4 <211> 959 <212> PRT <213> Artificial Sequence <400> 4 Gln Ser Thr Ile Glu Glu Gln Ala Lys Thr Phe Leu Asp Lys Phe Asn 1 5 10 15 His Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp Asn 20 25 30 Tyr Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn Ala 35 40 45 Gly Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Leu Ala Gln 50 55 60 Met Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu Gln Leu 65 70 75 80 Gln Ala Leu Gln Gln Asn Gly Ser Ser Val Leu Ser Glu Asp Lys Ser 85 90 95 Lys Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser Thr 100 105 110 Gly Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu Glu 115 120 125 Pro Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu Arg 130 135 140 Leu Trp Ala Trp Glu Ser Trp Arg Ser Glu Val Gly Lys Gln Leu Arg 145 150 155 160 Pro Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg Ala 165 170 175 Asn His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu Val 180 185 190 Asn Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln Leu Ile Glu Asp 195 200 205 Val Glu His Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu His 210 215 220 Ala Tyr Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile Ser 225 230 235 240 Pro Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly Arg 245 250 255 Phe Trp Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys Pro 260 265 270 Asn Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala Gln 275 280 285 Arg Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu Pro 290 295 300 Asn Met Thr Gln Gly Phe Trp Glu Asn Ser Met Leu Thr Asp Pro Gly 305 310 315 320 Asn Val Gln Lys Ala Val Cys His Pro Thr Ala Trp Asp Leu Gly Lys 325 330 335 Gly Asp Phe Arg Ile Leu Met Cys Thr Lys Val Thr Met Asp Asp Phe 340 345 350 Leu Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala Tyr 355 360 365 Ala Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe His 370 375 380 Glu Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys His 385 390 395 400 Leu Lys Ser Ile Gly Leu Leu Ser Pro Asp Phe Gln Glu Asp Asn Glu 405 410 415 Thr Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly Thr 420 425 430 Leu Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe Lys 435 440 445 Gly Glu Ile Pro Lys Asp Gln Trp Met Lys Lys Trp Trp Glu Met Lys 450 455 460 Arg Glu Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr Tyr 465 470 475 480 Cys Asp Pro Ala Ser Leu Phe His Val Ser Asn Asp Tyr Ser Phe Ile 485 490 495 Arg Tyr Tyr Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu Ala Leu 500 505 510 Cys Gln Ala Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile Ser 515 520 525 Asn Ser Thr Glu Ala Gly Gln Lys Leu Phe Asn Met Leu Arg Leu Gly 530 535 540 Lys Ser Glu Pro Trp Thr Leu Ala Leu Glu Asn Val Val Gly Ala Lys 545 550 555 560 Asn Met Asn Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe Thr 565 570 575 Trp Leu Lys Asp Gln Asn Lys Asn Ser Phe Val Gly Trp Ser Thr Asp 580 585 590 Trp Ser Pro Tyr Ala Asp Gln Ser Ile Lys Val Arg Ile Ser Leu Lys 595 600 605 Ser Ala Leu Gly Asp Lys Ala Tyr Glu Trp Asn Asp Asn Glu Met Tyr 610 615 620 Leu Phe Arg Ser Ser Val Ala Tyr Ala Met Arg Gln Tyr Phe Leu Lys 625 630 635 640 Val Lys Asn Gln Met Ile Leu Phe Gly Glu Glu Asp Val Arg Val Ala 645 650 655 Asn Leu Lys Pro Arg Ile Ser Phe Asn Phe Phe Val Thr Ala Pro Lys 660 665 670 Asn Val Ser Asp Ile Ile Pro Arg Thr Glu Val Glu Lys Ala Ile Arg 675 680 685 Met Ser Arg Ser Arg Ile Asn Asp Ala Phe Arg Leu Asn Asp Asn Ser 690 695 700 Leu Glu Phe Leu Gly Ile Gln Pro Thr Leu Gly Pro Pro Asn Gln Pro 705 710 715 720 Pro Val Ser Gly Gly Gly Ser Glu Pro Lys Ser Cys Asp Lys Thr His 725 730 735 Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 740 745 750 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 755 760 765 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 770 775 780 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 785 790 795 800 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 805 810 815 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 820 825 830 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 835 840 845 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 850 855 860 Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 865 870 875 880 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 885 890 895 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 900 905 910 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 915 920 925 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 930 935 940 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 945 950 955 <210> 5 <211> 740 <212> PRT <213> Artificial Sequence <400> 5 Gln Ser Thr Ile Glu Glu Gln Ala Lys Thr Phe Leu Asp Lys Phe Asn 1 5 10 15 His Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp Asn 20 25 30 Tyr Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn Ala 35 40 45 Gly Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Leu Ala Gln 50 55 60 Met Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu Gln Leu 65 70 75 80 Gln Ala Leu Gln Gln Asn Gly Ser Ser Val Leu Ser Glu Asp Lys Ser 85 90 95 Lys Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser Thr 100 105 110 Gly Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu Glu 115 120 125 Pro Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu Arg 130 135 140 Leu Trp Ala Trp Glu Ser Trp Arg Ser Glu Val Gly Lys Gln Leu Arg 145 150 155 160 Pro Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg Ala 165 170 175 Asn His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu Val 180 185 190 Asn Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln Leu Ile Glu Asp 195 200 205 Val Glu His Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu His 210 215 220 Ala Tyr Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile Ser 225 230 235 240 Pro Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly Arg 245 250 255 Phe Trp Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys Pro 260 265 270 Asn Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala Gln 275 280 285 Arg Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu Pro 290 295 300 Asn Met Thr Gln Gly Phe Trp Glu Asn Ser Met Leu Thr Asp Pro Gly 305 310 315 320 Asn Val Gln Lys Ala Val Cys His Pro Thr Ala Trp Asp Leu Gly Lys 325 330 335 Gly Asp Phe Arg Ile Leu Met Cys Thr Lys Val Thr Met Asp Asp Phe 340 345 350 Leu Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala Tyr 355 360 365 Ala Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe His 370 375 380 Glu Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys His 385 390 395 400 Leu Lys Ser Ile Gly Leu Leu Ser Pro Asp Phe Gln Glu Asp Asn Glu 405 410 415 Thr Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly Thr 420 425 430 Leu Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe Lys 435 440 445 Gly Glu Ile Pro Lys Asp Gln Trp Met Lys Lys Trp Trp Glu Met Lys 450 455 460 Arg Glu Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr Tyr 465 470 475 480 Cys Asp Pro Ala Ser Leu Phe His Val Ser Asn Asp Tyr Ser Phe Ile 485 490 495 Arg Tyr Tyr Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu Ala Leu 500 505 510 Cys Gln Ala Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile Ser 515 520 525 Asn Ser Thr Glu Ala Gly Gln Lys Leu Phe Asn Met Leu Arg Leu Gly 530 535 540 Lys Ser Glu Pro Trp Thr Leu Ala Leu Glu Asn Val Val Gly Ala Lys 545 550 555 560 Asn Met Asn Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe Thr 565 570 575 Trp Leu Lys Asp Gln Asn Lys Asn Ser Phe Val Gly Trp Ser Thr Asp 580 585 590 Trp Ser Pro Tyr Ala Asp Gln Ser Ile Lys Val Arg Ile Ser Leu Lys 595 600 605 Ser Ala Leu Gly Asp Lys Ala Tyr Glu Trp Asn Asp Asn Glu Met Tyr 610 615 620 Leu Phe Arg Ser Ser Val Ala Tyr Ala Met Arg Gln Tyr Phe Leu Lys 625 630 635 640 Val Lys Asn Gln Met Ile Leu Phe Gly Glu Glu Asp Val Arg Val Ala 645 650 655 Asn Leu Lys Pro Arg Ile Ser Phe Asn Phe Phe Val Thr Ala Pro Lys 660 665 670 Asn Val Ser Asp Ile Ile Pro Arg Thr Glu Val Glu Lys Ala Ile Arg 675 680 685 Met Ser Arg Ser Arg Ile Asn Asp Ala Phe Arg Leu Asn Asp Asn Ser 690 695 700 Leu Glu Phe Leu Gly Ile Gln Pro Thr Leu Gly Pro Pro Asn Gln Pro 705 710 715 720 Pro Val Ser Gly Ser Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys Ile 725 730 735 Glu Trp His Glu 740 <210> 6 <211> 223 <212> PRT <213> Artificial Sequence <400> 6 Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn 1 5 10 15 Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val 20 25 30 Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser 35 40 45 Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val 50 55 60 Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp 65 70 75 80 Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln 85 90 95 Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr 100 105 110 Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly 115 120 125 Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys 130 135 140 Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr 145 150 155 160 Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser 165 170 175 Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val 180 185 190 Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly 195 200 205 Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe 210 215 220 <210> 7 <211> 455 <212> PRT <213> Artificial Sequence <400> 7 Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn 1 5 10 15 Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val 20 25 30 Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser 35 40 45 Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val 50 55 60 Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp 65 70 75 80 Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln 85 90 95 Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr 100 105 110 Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly 115 120 125 Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys 130 135 140 Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr 145 150 155 160 Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser 165 170 175 Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val 180 185 190 Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly 195 200 205 Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Gly 210 215 220 Gly Gly Gly Ser Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys 225 230 235 240 Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys 245 250 255 Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val 260 265 270 Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp 275 280 285 Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe 290 295 300 Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp 305 310 315 320 Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe 325 330 335 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys 340 345 350 Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys 355 360 365 Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp 370 375 380 Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys 385 390 395 400 Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser 405 410 415 Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr 420 425 430 Cys Ser Val Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser 435 440 445 Leu Ser His Ser Pro Gly Lys 450 455 <210> 8 <211> 456 <212> PRT <213> Artificial Sequence <400> 8 Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn 1 5 10 15 Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val 20 25 30 Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser 35 40 45 Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val 50 55 60 Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp 65 70 75 80 Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln 85 90 95 Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr 100 105 110 Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly 115 120 125 Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys 130 135 140 Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr 145 150 155 160 Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser 165 170 175 Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val 180 185 190 Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly 195 200 205 Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Gly 210 215 220 Gly Gly Gly Ser Ala Pro Ser Thr Cys Ser Lys Pro Thr Cys Pro Pro 225 230 235 240 Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Pro 245 250 255 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 260 265 270 Val Asp Val Ser Gln Asp Asp Pro Glu Val Gln Phe Thr Trp Tyr Ile 275 280 285 Asn Asn Glu Gln Val Arg Thr Ala Arg Pro Pro Leu Arg Glu Gln Gln 290 295 300 Phe Asn Ser Thr Ile Arg Val Val Ser Thr Leu Pro Ile Ala His Gln 305 310 315 320 Asp Trp Leu Arg Gly Lys Glu Phe Lys Cys Lys Val His Asn Lys Ala 325 330 335 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Arg Gly Gln Pro 340 345 350 Leu Glu Pro Lys Val Tyr Thr Met Gly Pro Pro Arg Glu Glu Leu Ser 355 360 365 Ser Arg Ser Val Ser Leu Thr Cys Met Ile Asn Gly Phe Tyr Pro Ser 370 375 380 Asp Ile Ser Val Glu Trp Glu Lys Asn Gly Lys Ala Glu Asp Asn Tyr 385 390 395 400 Lys Thr Thr Pro Ala Val Leu Asp Ser Asp Gly Ser Tyr Phe Leu Tyr 405 410 415 Ser Lys Leu Ser Val Pro Thr Ser Glu Trp Gln Arg Gly Asp Val Phe 420 425 430 Thr Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 435 440 445 Ser Ile Ser Arg Ser Pro Gly Lys 450 455 <210> 9 <211> 460 <212> PRT <213> Artificial Sequence <400> 9 Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn 1 5 10 15 Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val 20 25 30 Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser 35 40 45 Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val 50 55 60 Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp 65 70 75 80 Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln 85 90 95 Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr 100 105 110 Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly 115 120 125 Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys 130 135 140 Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr 145 150 155 160 Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser 165 170 175 Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val 180 185 190 Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly 195 200 205 Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Gly 210 215 220 Gly Gly Gly Ser Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro 225 230 235 240 Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 245 250 255 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 260 265 270 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 275 280 285 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 290 295 300 Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 305 310 315 320 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 325 330 335 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 340 345 350 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 355 360 365 Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 370 375 380 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 385 390 395 400 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 405 410 415 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 420 425 430 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 435 440 445 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 450 455 460 <210> 10 <211> 1195 <212> PRT <213> Artificial Sequence <400> 10 Gln Cys Val Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr 1 5 10 15 Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser 20 25 30 Ser Val Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn 35 40 45 Val Thr Trp Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys 50 55 60 Arg Phe Asp Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala 65 70 75 80 Ser Thr Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr 85 90 95 Leu Asp Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn 100 105 110 Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu 115 120 125 Gly Val Tyr Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe 130 135 140 Arg Val Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln 145 150 155 160 Pro Phe Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu 165 170 175 Arg Glu Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser 180 185 190 Lys His Thr Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser 195 200 205 Ala Leu Glu Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg 210 215 220 Phe Gln Thr Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp 225 230 235 240 Ser Ser Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr 245 250 255 Leu Gln Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile 260 265 270 Thr Asp Ala Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys 275 280 285 Thr Leu Lys Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn 290 295 300 Phe Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr 305 310 315 320 Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser 325 330 335 Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr 340 345 350 Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly 355 360 365 Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala 370 375 380 Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly 385 390 395 400 Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe 405 410 415 Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val 420 425 430 Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu 435 440 445 Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser 450 455 460 Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln 465 470 475 480 Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg 485 490 495 Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys 500 505 510 Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe 515 520 525 Asn Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys 530 535 540 Lys Phe Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr 545 550 555 560 Asp Ala Val Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro 565 570 575 Cys Ser Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser 580 585 590 Asn Gln Val Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val Pro 595 600 605 Val Ala Ile His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser 610 615 620 Thr Gly Ser Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala 625 630 635 640 Glu His Val Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly 645 650 655 Ile Cys Ala Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala Arg 660 665 670 Ser Val Ala Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly Ala 675 680 685 Glu Asn Ser Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn 690 695 700 Phe Thr Ile Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr Lys 705 710 715 720 Thr Ser Val Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys 725 730 735 Ser Asn Leu Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg 740 745 750 Ala Leu Thr Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu Val 755 760 765 Phe Ala Gln Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe 770 775 780 Gly Gly Phe Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser 785 790 795 800 Lys Arg Ser Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala 805 810 815 Asp Ala Gly Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala 820 825 830 Ala Arg Asp Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu 835 840 845 Pro Pro Leu Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu 850 855 860 Leu Ala Gly Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala 865 870 875 880 Leu Gln Ile Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile 885 890 895 Gly Val Thr Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala Asn 900 905 910 Gln Phe Asn Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser Thr 915 920 925 Ala Ser Ala Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln 930 935 940 Ala Leu Asn Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile 945 950 955 960 Ser Ser Val Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala 965 970 975 Glu Val Gln Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln 980 985 990 Thr Tyr Val Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser 995 1000 1005 Ala Asn Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser 1010 1015 1020 Lys Arg Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro 1025 1030 1035 1040 Gln Ser Ala Pro His Gly Val Val Phe Leu His Val Thr Tyr Val Pro 1045 1050 1055 Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His Asp Gly 1060 1065 1070 Lys Ala His Phe Pro Arg Glu Gly Val Phe Val Ser Asn Gly Thr His 1075 1080 1085 Trp Phe Val Thr Gln Arg Asn Phe Tyr Glu Pro Gln Ile Ile Thr Thr 1090 1095 1100 Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val Val Ile Gly Ile Val 1105 1110 1115 1120 Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro Glu Leu Asp Ser Phe Lys 1125 1130 1135 Glu Glu Leu Asp Lys Tyr Phe Lys Asn His Thr Ser Pro Asp Val Asp 1140 1145 1150 Leu Gly Asp Ile Ser Gly Ile Asn Ala Ser Val Val Asn Ile Gln Lys 1155 1160 1165 Glu Ile Asp Arg Leu Asn Glu Val Ala Lys Asn Leu Asn Glu Ser Leu 1170 1175 1180 Ile Asp Leu Gln Glu Leu Gly Lys Tyr Glu Gln 1185 1190 1195 <210> 11 <211> 1224 <212> PRT <213> Artificial Sequence <400> 11 Gln Cys Val Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr 1 5 10 15 Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser 20 25 30 Ser Val Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn 35 40 45 Val Thr Trp Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys 50 55 60 Arg Phe Asp Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala 65 70 75 80 Ser Thr Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr 85 90 95 Leu Asp Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn 100 105 110 Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu 115 120 125 Gly Val Tyr Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe 130 135 140 Arg Val Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln 145 150 155 160 Pro Phe Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu 165 170 175 Arg Glu Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser 180 185 190 Lys His Thr Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser 195 200 205 Ala Leu Glu Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg 210 215 220 Phe Gln Thr Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp 225 230 235 240 Ser Ser Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr 245 250 255 Leu Gln Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile 260 265 270 Thr Asp Ala Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys 275 280 285 Thr Leu Lys Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn 290 295 300 Phe Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr 305 310 315 320 Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser 325 330 335 Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr 340 345 350 Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly 355 360 365 Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala 370 375 380 Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly 385 390 395 400 Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe 405 410 415 Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val 420 425 430 Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu 435 440 445 Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser 450 455 460 Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln 465 470 475 480 Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg 485 490 495 Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys 500 505 510 Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe 515 520 525 Asn Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys 530 535 540 Lys Phe Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr 545 550 555 560 Asp Ala Val Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro 565 570 575 Cys Ser Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser 580 585 590 Asn Gln Val Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val Pro 595 600 605 Val Ala Ile His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser 610 615 620 Thr Gly Ser Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala 625 630 635 640 Glu His Val Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly 645 650 655 Ile Cys Ala Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala Arg 660 665 670 Ser Val Ala Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly Ala 675 680 685 Glu Asn Ser Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn 690 695 700 Phe Thr Ile Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr Lys 705 710 715 720 Thr Ser Val Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys 725 730 735 Ser Asn Leu Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg 740 745 750 Ala Leu Thr Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu Val 755 760 765 Phe Ala Gln Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe 770 775 780 Gly Gly Phe Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser 785 790 795 800 Lys Arg Ser Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala 805 810 815 Asp Ala Gly Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala 820 825 830 Ala Arg Asp Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu 835 840 845 Pro Pro Leu Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu 850 855 860 Leu Ala Gly Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala 865 870 875 880 Leu Gln Ile Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile 885 890 895 Gly Val Thr Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala Asn 900 905 910 Gln Phe Asn Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser Thr 915 920 925 Ala Ser Ala Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln 930 935 940 Ala Leu Asn Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile 945 950 955 960 Ser Ser Val Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala 965 970 975 Glu Val Gln Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln 980 985 990 Thr Tyr Val Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser 995 1000 1005 Ala Asn Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser 1010 1015 1020 Lys Arg Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro 1025 1030 1035 1040 Gln Ser Ala Pro His Gly Val Val Phe Leu His Val Thr Tyr Val Pro 1045 1050 1055 Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His Asp Gly 1060 1065 1070 Lys Ala His Phe Pro Arg Glu Gly Val Phe Val Ser Asn Gly Thr His 1075 1080 1085 Trp Phe Val Thr Gln Arg Asn Phe Tyr Glu Pro Gln Ile Ile Thr Thr 1090 1095 1100 Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val Val Ile Gly Ile Val 1105 1110 1115 1120 Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro Glu Leu Asp Ser Phe Lys 1125 1130 1135 Glu Glu Leu Asp Lys Tyr Phe Lys Asn His Thr Ser Pro Asp Val Asp 1140 1145 1150 Leu Gly Asp Ile Ser Gly Ile Asn Ala Ser Val Val Asn Ile Gln Lys 1155 1160 1165 Glu Ile Asp Arg Leu Asn Glu Val Ala Lys Asn Leu Asn Glu Ser Leu 1170 1175 1180 Ile Asp Leu Gln Glu Leu Gly Lys Tyr Glu Gln Gly Ser Gly Tyr Ile 1185 1190 1195 1200 Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr Val Arg Lys Asp Gly Glu 1205 1210 1215 Trp Val Leu Leu Ser Thr Phe Leu 1220 <210> 12 <211> 345 <212> DNA <213> Artificial Sequence <400> 12 gaggtgcagc tggtggagtc tgggggaggc ttggtccagc ctggggggtc cctgagactc 60 tcctgtacag cctctgaaat caacgtcagt agcaactaca tgaactgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcagtt ctttatgccg gtggtaccac atactacgca 180 gactccgtga aggacaggtt caccatctcc agagacaatt ccaagaacac gctgtatctt 240 caaatgaaca acctgagagc cgaggacacg gctttctatt actgtgcgag agaggcctat 300 gcttttgata tctggggcca agggacaatg gtcaccgtct cttca 345 <210> 13 <211> 324 <212> DNA <213> Artificial Sequence <400> 13 gccatccagt tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60 atcacttgcc gggccagtca gggcattagc agttatttag cctggtatca gcaaaaacca 120 gggaaagccc ctaagctcct gatctatgct gcatccactt tgcaaagtgg ggtcccatca 180 aggttcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct 240 gaagattttg caacttatta ctgtcaactg cttaatagtt accctccgag caccttcggc 300 caagggacac gactggagat taaa 324 <210> 14 <211> 321 <212> DNA <213> Artificial Sequence <400> 14 cgaactgtgg ctgcaccatc tgtcttcatc ttcccgccat ctgatgagca gttgaaatct 60 ggaactgcct ctgttgtgtg cctgctgaat aacttctatc ccagagaggc caaagtacag 120 tggaaggtgg ataacgccct ccaatcgggt aactcccagg agagtgtcac agagcaggac 180 agcaaggaca gcacctacag cctcagcagc accctgacgc tgagcaaagc agactacgag 240 aaacacaaag tctacgcctg cgaagtcacc catcagggcc tgagctcgcc cgtcacaaag 300 agcttcaaca ggggagagtg t 321 <210> 15 <211> 381 <212> DNA <213> Artificial Sequence <400> 15 gaggtgcaac tggtggagtc tgggggaggt gtggtacggc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt cacctttggt gattatgcca tgagctgggt ccgccaagct 120 ccagagaagg ggctggagtg ggtctctggt attaattgga atggtggtag gacaggttat 180 gcggactctg tgaagggccg attcaccatc tccagagaca acgccaagaa ctccctgtat 240 ctgcaaatga acagtctgag agccgaggac acggccttgt attactgtgc gagatatctt 300 cagcactcat attttgactt gttattacct aatgatgctt ttgatatctg gggccaaggg 360 acaatggtca ccgtctcttc a 381 <210> 16 <211> 324 <212> DNA <213> Artificial Sequence <400> 16 gaaattgtgg tgacgcagtc tccaggcacc ctgtctttgt ctccggggga aagagccacc 60 gaaattgtgg tgacgcagtc tccaggcacc ctgtctttgt ctccggggga aagagccacc 60 ctctcctgca gggccagtca gagtgttagc agcaactact tagcctggta ccagcagaaa 120 ctctcctgca gggccagtca gagtgttagc agcaactact tagcctggta ccagcagaaa 120 cctggccagg ctcccaggct cctcatctat ggtgcatcca gcagggccac tggcatccca 180 cctggccagg ctcccaggct cctcatctat ggtgcatcca gcagggccac tggcatccca 180 gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag aagactggag 240 gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag aagactggag 240 gccgaagatt ttgcagtgta ttactgtcaa cagtatggga gctcaccgct cactttcggc 300 gccgaagatt ttgcagtgta ttactgtcaa cagtatggga gctcaccgct cactttcggc 300 ggagggacca aggtggagat caaa 324 ggagggacca aggtggagat caaa 324 <210> 17<210> 17 <211> 321<211> 321 <212> DNA<212> DNA <213> 人工序列(Artificial Sequence)<213> Artificial Sequence <400> 17 <400> 17 cgaactgtgg ctgcaccatc tgtcttcatc ttcccgccat ctgatgagca gttgaaatct 60 cgaactgtgg ctgcaccatc tgtcttcatc ttcccgccat ctgatgagca gttgaaatct 60 ggaactgcct ctgttgtgtg cctgctgaat aacttctatc ccagagaggc caaagtacag 120 ggaactgcct ctgttgtgtg cctgctgaat aacttctatc ccagagaggc caaagtacag 120 tggaaggtgg ataacgccct ccaatcgggt aactcccagg agagtgtcac agagcaggac 180 tggaaggtgg ataacgccct ccaatcgggt aactcccagg agagtgtcac agagcaggac 180 agcaaggaca gcacctacag cctcagcagc accctgacgc tgagcaaagc agactacgag 240 agcaaggaca gcacctacag cctcagcagc accctgacgc tgagcaaagc agactacgag 240 aaacacaaac tctacgcctg cgaagtcacc catcagggcc tgagctcgcc cgtcacaaag 300 aaacacaaac tctacgcctg cgaagtcacc catcagggcc tgagctcgcc cgtcacaaag 300 agcttcaaca ggggagagtg t 321 <210> 18 <211> 366 <212> DNA <213> Artificial Sequence <400> 18 caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcggagac cctgtccctc 60 acctgcactg tctctggtgg ctccatcagt agttactact ggagctggat ccggcagccc 120 ccggggaggg gactggagtg gattgggtat atccattaca gtgggagcac caactacaac 180 ccctccctca ggagtcgact caccatatca gtagacacgt ccaagaacca gttctccctg 240 aagctgagct ccgtgaccgc tgcggacacg gccgtgtatt attgtgcgag agatcagggg 300 tatagcagtg gctggcctga tgcttttgat atctggggcc aagggacaat ggtcaccatc 360 tcttca 366 <210> 19 <211> 321 <212> DNA <213> Artificial Sequence <400> 19 gaaattgtgt tgacacagtc tccagccacc ctgtctttgt ctccagggga aagagccacc 60 ctctcctgca gggccagtca gagtgttagc agctacttag cctggtacca acagaaacct 120 ggccaggctc ccaggctcct catctatgat gcatccaaca gggccactga catcccagcc 180 aggttcagtg acagtgggtc tgggacagac ttcactctca ccatcaccag cctagagcct 240 gaagattttg cagtttatta ctgtcagcag cgtagcaact ggcctgggac gttcggccaa 300 gggaccaagg tggaaatcaa a 321 <210> 20 <211> 990 <212> DNA <213> Artificial Sequence <400> 20 gcctccacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 60 ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120 tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180 ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 240 tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 300 aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 360 ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 420 gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 480 tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 540 agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 600 gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 660 aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggatgag 720 ctgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 780 gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 840 ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 900 cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 960 cagaagagcc tctccctgtc tccgggtaaa 990 <210> 21 <211> 115 <212> PRT <213> Artificial Sequence <400> 21 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Ala Ser Glu Ile Asn Val Ser Ser Asn 20 25 30 Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Val Leu Tyr Ala Gly Gly Thr Thr Tyr Tyr Ala Asp Ser Val Lys 50 55 60 Asp Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Asn Leu Arg Ala Glu Asp Thr Ala Phe Tyr Tyr Cys Ala 85 90 95 Arg Glu Ala Tyr Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr 100 105 110 Val Ser Ser 115 <210> 22 <211> 108 <212> PRT <213> Artificial Sequence <400> 22 Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Leu Leu Asn Ser Tyr Pro Pro 85 90 95 Ser Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100 105 <210> 23 <211> 107 <212> PRT <213> Artificial Sequence <400> 23 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 24 <211> 127 <212> PRT <213> Artificial Sequence <400> 24 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Arg Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Gly Asp Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Asn Trp Asn Gly Gly Arg Thr Gly Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Arg Tyr Leu Gln His Ser Tyr Phe Asp Leu Leu Leu Pro Asn Asp 100 105 110 Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 115 120 125 <210> 25 <211> 108 <212> PRT <213> Artificial Sequence <400> 25 Glu Ile Val Val Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Asn 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Arg Arg Leu Glu 65 70 75 80 Ala Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro 85 90 95 Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 26 <211> 107 <212> PRT <213> Artificial Sequence <400> 26 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Leu Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 27 <211> 122 <212> PRT <213> Artificial Sequence <400> 27 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 苏氨酸 亮氨酸 丝氨酸 亮氨酸 苏氨酸 半胱氨酸 苏氨酸 缬氨酸 丝氨酸 甘氨酸 甘氨酸 丝氨酸 异亮氨酸 丝氨酸 丝氨酸 酪氨酸 20 25 30 酪氨酸 色氨酸 丝氨酸 色氨酸 异亮氨酸 精氨酸 谷氨酰胺 脯氨酸 脯氨酸 甘氨酸 精氨酸 甘氨酸 亮氨酸 谷氨酸 色氨酸 异亮氨酸 35 40 45 甘氨酸 酪氨酸 异亮氨酸 组氨酸 酪氨酸 丝氨酸 甘氨酸 丝氨酸 苏氨酸 天冬酰胺 酪氨酸 天冬酰胺 脯氨酸 丝氨酸 亮氨酸 精氨酸 50 55 60 丝氨酸 精氨酸 亮氨酸 苏氨酸 异亮氨酸 丝氨酸 缬氨酸 天冬氨酸 苏氨酸 丝氨酸 赖氨酸 天冬酰胺 谷氨酰胺 苯丙氨酸 丝氨酸 亮氨酸 65 70 75 80 赖氨酸 亮氨酸 丝氨酸 丝氨酸 缬氨酸 苏氨酸 丙氨酸 丙氨酸 天冬氨酸 苏氨酸 丙氨酸 缬氨酸 酪氨酸 酪氨酸 半胱氨酸 丙氨酸 85 90 95 精氨酸 天冬氨酸 谷氨酰胺 甘氨酸 酪氨酸 丝氨酸 丝氨酸 甘氨酸 色氨酸 脯氨酸 天冬氨酸 丙氨酸 苯丙氨酸 天冬氨酸 异亮氨酸 色氨酸 100 105 110 甘氨酸 谷氨酰胺 甘氨酸 苏氨酸 甲硫氨酸 缬氨酸 苏氨酸 异亮氨酸 丝氨酸 丝氨酸 115 120 <210> 28 <211> 107 <212> PRT <213> Artificial Sequence <400> 28 谷氨酸 异亮氨酸 缬氨酸 亮氨酸 苏氨酸 谷氨酰胺 丝氨酸 脯氨酸 丙氨酸 苏氨酸 亮氨酸 丝氨酸 亮氨酸 丝氨酸 脯氨酸 甘氨酸 1 5 10 15 谷氨酸 精氨酸 丙氨酸 苏氨酸 亮氨酸 丝氨酸 半胱氨酸 精氨酸 丙氨酸 丝氨酸 谷氨酰胺 丝氨酸 缬氨酸 丝氨酸 丝氨酸 酪氨酸 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Asp Ile Pro Ala Arg Phe Ser Asp 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Thr Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Gly 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 29 <211> 330 <212> PRT <213> Artificial Sequence <400> 29 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 30 <211> 8 <212> PRT <213> Artificial Sequence <400> 30 Glu Ile Asn Val Ser Ser Asn Tyr 1 5 <210> 31 <211> 7 <212> PRT <213> Artificial Sequence <400> 31 Leu Tyr Ala Gly Gly Thr Thr 1 5 <210> 32 <211> 9 <212> PRT <213> Artificial Sequence <400> 32 Ala Arg Glu Ala Tyr Ala Phe Asp Ile 1 5 <210> 33 <211> 6 <212> PRT <213> Artificial Sequence <400> 33 Gln Gly Ile Ser Ser Tyr 1 5 <210> 34 <211> 3 <212> PRT <213> Artificial Sequence <400> 34 Ala Ala Ser 1 <210> 35 <211> 10 <212> PRT <213> Artificial Sequence <400> 35 Gln Leu Leu Asn Ser Tyr Pro Pro Ser Thr 1 5 10 <210> 36 <211> 8 <212> PRT <213> Artificial Sequence <400> 36 Gly Phe Thr Phe Gly Asp Tyr Ala 1 5 <210> 37 <211> 8 <212> PRT <213> Artificial Sequence <400> 37 Ile Asn Trp Asn Gly Gly Arg Thr 1 5 <210> 38 <211> 20 <212> PRT <213> Artificial Sequence <400> 38 Ala Arg Tyr Leu Gln His Ser Tyr Phe Asp Leu Leu Leu Pro Asn Asp 1 5 10 15 Ala Phe Asp Ile 20 <210> 39 <211> 7 <212> PRT <213> Artificial Sequence <400> 39 Gln Ser Val Ser Ser Asn Tyr 1 5 <210> 40 <211> 3 <212> PRT <213> Artificial Sequence <400> 40 Gly Ala Ser 1 <210> 41 <211> 9 <212> PRT <213> Artificial Sequence <400> 41 Gln Gln Tyr Gly Ser Ser Pro Leu Thr 1 5 <210> 42 <211> 8 <212> PRT <213> Artificial Sequence <400> 42 Gly Gly Ser Ile Ser Ser Tyr Tyr 1 5 <210> 43 <211> 7 <212> PRT <213> Artificial Sequence <400> 43 Ile His Tyr Ser Gly Ser Thr 1 5 <210> 44 <211> 16 <212> PRT <213> Artificial Sequence <400> 44 Ala Ala Ala Gly Gly Thr Ser Ser Gly Thr Pro Ala Ala Pro Ala Ile 1 5 10 15 <210> 45 <211> 6 <212> PRT <213> Artificial Sequence <400> 45 Gln Ser Val Ser Ser Tyr 1 5 <210> 46 <211> 3 <212> PRT <213> Artificial Sequence <400> 46 Asp Ala Ser 1 <210> 47 <211> 9 <212> PRT <213> Artificial Sequence <400> 47 Gln Gln Arg Ser Asn Trp Pro Gly Thr 1 5

Claims

1. A method for detecting SARS-CoV-2 neutralizing antibodies in a sample, comprising: 1) Encapsulating ACE2 receptor protein or its functionally active fragment on a solid support; 2) mixing and incubating the sample to be tested with the SARS-CoV-2 spike protein or its functionally active fragment to obtain an incubation product; 3) adding the incubated product of step 2) to step 1); 4) detecting SARS-CoV-2 neutralizing antibodies in the sample to be tested; Wherein the SARS-CoV-2 spike protein or a functionally active fragment thereof comprises a receptor binding region RBD, the RBD is directly or indirectly connected to the Fc region and fused in frame with the Fc, the Fc region is selected from rFc, mFc and hFc, and the RBD comprises one or more markers, which are capable of directly or indirectly generating a signal showing the presence and / or content of the RBD; The amino acid sequence of the SARS-CoV-2 spike protein or its functionally active fragment is shown in any one of SEQ ID NOs: 7-11; The amino acid sequence of the ACE2 receptor protein or its functionally active fragment is as described in any one of SEQ ID NOs: 2-4; The method is used for non-disease diagnosis or treatment purposes.

2. The method according to claim 1, wherein the label comprises HRP or an HRP-like enzyme.

3. The method according to claim 1, wherein the concentration of the SARS-CoV-2 spike protein or its functionally active fragment is 50 ng / ml-4000 ng / ml.

4. The method according to claim 1, wherein the concentration of the ACE2 receptor protein or its functionally active fragment is 100 ng / ml-2000 ng / ml.

5. The method according to claim 1, comprising a sample diluent, wherein the pH of the sample diluent is 6.0-9.

6.

6. The method of claim 1, wherein the detection is obtained by comparison with a calibrator, wherein the calibrator comprises one or more reference antibody molecules or antigen-binding fragments thereof that specifically recognize the SARS-CoV-2 spike protein or a functionally active fragment thereof; the reference antibody comprises a heavy chain variable region VH and a light chain variable region VL, wherein: The amino acid sequences of HCDR1, HCDR2 and HCDR3 of the VH are shown in SEQ ID NOs: 30-32, respectively; the amino acid sequences of LCDR1, LCDR2 and LCDR3 of the VL are shown in SEQ ID NOs: 33-35, respectively; The amino acid sequences of HCDR1, HCDR2 and HCDR3 of the VH are shown in SEQ ID NOs: 36-38, respectively, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 of the VL are shown in SEQ ID NOs: 39-41, respectively; or The amino acid sequences of HCDR1, HCDR2 and HCDR3 of the VH are shown in SEQ ID NOs: 42-44, respectively, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 of the VL are shown in SEQ ID NOs: 45-47, respectively.

7. The method according to claim 1, wherein the detection is obtained by comparison with a calibrator, wherein the calibrator comprises one or more reference antibody molecules or antigen-binding fragments thereof that specifically recognize the SARS-CoV-2 spike protein or a functionally active fragment thereof; the reference antibody comprises a heavy chain variable region VH and a light chain variable region VL; wherein: The amino acid sequence of the VH is shown in SEQ ID NO: 21, and the amino acid sequence of the VL is shown in SEQ ID NO: 22; The amino acid sequence of the VH is shown in SEQ ID NO: 24, and the amino acid sequence of the VL is shown in SEQ ID NO: 25; or The amino acid sequence of the VH is shown in SEQ ID NO:27, and the amino acid sequence of the VL is shown in SEQ ID NO:

28.

8. The method according to claim 6 or 7, wherein the concentration of the reference antibody is 50 ng / ml-1000 ng / ml.

9. The method according to claim 1, wherein the sample to be tested is selected from the group consisting of urine, saliva, serum, plasma, whole blood, nasopharyngeal aspirate and bronchial lavage fluid.

10. The method according to claim 9, wherein the sample to be tested is derived from a subject.

11. The method according to claim 10, wherein the subject is a recovered COVID-19 patient, a healthy subject and / or a subject who has received COVID-19 vaccine treatment.

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