JP2017145246A - Antibody against mers coronavirus, method for detecting mers coronavirus using the antibody, and kit containing the antibody - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an antibody that enables comprehensive and specific detection of MERS coronavirus but does not detect its closely related coronavirus including SARS, and to provide a method for simply detecting MERS coronavirus, accurately and simply.SOLUTION: According to the present invention, there is provided a monoclonal antibody specifically recognizing the nucleocapsid protein of MERS coronavirus, a method for detecting MERS coronavirus using the antibody, and a kit containing the antibody.SELECTED DRAWING: None

Description

  The present invention relates to a monoclonal antibody against the nucleocapsid protein of MERS coronavirus, a method for detecting MERS coronavirus using the antibody, and a kit containing the antibody.

  Coronavirus is an enveloped single-stranded (+) RNA virus having a diameter of 60 to 220 nm, and is known to cause respiratory symptoms when infecting humans. MERS (Middle East Respiratory Syndrome) coronavirus and SARS (Severe Acute Respiratory Syndrome) coronaviruses that are highly pathogenic as coronaviruses, human coronavirus NL63, human coronavirus 229E, human coronavirus OC43 and humans that are less pathogenic Coronavirus HKU1 and the like are known, and coronavirus is also known to be a causative virus for colds.

  MERS coronavirus is a new type of coronavirus whose existence was confirmed in 2012. The WHO (World Health Organization) has announced that it is prevalent in more than 20 countries, mainly in the Middle East and South Korea, and that it will cause an extremely high fatality rate of over 30%. Although the main source of infection to humans is thought to be camels, the exact route of infection has not yet been clarified. Even today, the MERS epidemic continues and there is no antiviral drug against MERS coronavirus. Therefore, in order to prevent the spread of infection, it is extremely important to detect MERS patients at an early stage by a rapid test method.

As a method for detecting MERS coronavirus, genetic testing such as real-time reverse transcription PCR is mainly used. This makes it possible to accurately determine the presence or absence of MERS coronavirus, but genetic testing requires a special and expensive device that detects fluorescence in real time during PCR, and to obtain results, several Since time is required, there is a problem in that it lacks practicality in early detection in places where facilities such as infection sites and prevention sites are not sufficient.
Therefore, in recent years, development of antibodies against MERS coronavirus has been promoted for the purpose of developing a test method having accuracy, rapidity, and simplicity.

  For example, Non-Patent Document 1 discloses a hydrophilic region of a protein constituting a nucleocapsid of MERS coronavirus isolated from dromedary (hereinafter, also referred to as nucleocapsid protein, simply NP, and particularly MERS-derived protein is also referred to as MERS-NP). By synthesizing 5 oligopeptides P1 to P5 consisting of about 20 to 40 amino acids in sequence and immunizing mice with each oligopeptide conjugated to bovine serum albumin (BSA) as an antigen, MERS-NP It has been reported that monoclonal antibodies have been obtained. And the antibody obtained by using as an antigen 2 types of those 5 types, ie, the 22-40th or 164-202nd oligopeptide (P1 or P3) in the amino acid sequence of MERS-NP, in the immunochromatography method, It has been reported that MERS-NP derived from the dromedary camel was detected without detecting NP derived from bovine coronavirus (BCV), canine coronavirus (CCV) and feline coronavirus (FCV).

  Non-Patent Document 2 discloses that against MERS-NP by immunizing a mouse with an antigen expressed and purified from full-length MERS-NP derived from a specific strain of clinically isolated MERS coronavirus. It has been reported that 24 monoclonal antibodies have been obtained. It was reported that 12 of the antibodies reacted with the specific MERS-NP in the enzyme-linked immunosorbent assay (ELISA method) but did not react with human coronavirus 229E and OC43-derived NP. ing.

Song D., Ha G., Eltahir Y., et al., Development and validation of a rapid immunochromatographic assay for detection of Middle East respiratory syndrome coronavirus antigen in dromedary camels, J Clin Microb (2015) 53: 178-182. Chen Y., Chan K.-H., Kang Y., et al., A sensitive and specific antigen detection assay for Middle East respiratory syndrome coronavirus, Emerg Microbes Infect (2015) 4: e26.

Whether the antibody capable of detecting a specific MERS-NP described in Non-Patent Documents 1 and 2 can detect any MERS-NP against other various MERS coronavirus strains. The epitope of the antibody has not been clarified specifically. Furthermore, in the non-patent literature, the specificity of the antibody against a related coronavirus that may infect humans is not fully examined. In other words, all of the antibodies described in the non-patent literature can detect various MERS coronavirus strains comprehensively, not related coronaviruses, and specifically detect only MERS coronaviruses. It was not made clear whether or not.
Accordingly, an object of the present invention is to provide an antibody capable of comprehensively and specifically detecting MERS coronavirus, and thereby to provide a method for accurately, quickly and conveniently detecting MERS coronavirus. And

  The inventors of the present invention made use of MERS-NP amino acid sequences derived from various strains of MERS coronavirus, and these amino acid sequences and NP amino acid sequences derived from various strains of SARS and other coronaviruses. As a result of the original analysis, the present inventors have found for the first time a region that is highly conserved among the amino acid sequences of MERS-NP and low in identity with the amino acid sequences of NPs of closely related coronaviruses including SARS. And in view of the said subject, it came to complete this invention by producing the antibody which recognizes the area | region of this amino acid sequence as an epitope.

That is, the present invention relates to the following.
[1] An antibody or a fragment thereof that specifically binds to a protein constituting a nucleocapsid of MERS (Middle East Respiratory Syndrome) coronavirus, and the epitope recognized by the antibody and the fragment thereof is represented by SEQ ID NO: 1. The antibody or fragment thereof, which is present at positions 208 to 413 in the amino acid sequence.
[2] The antibody or fragment thereof according to [1], wherein the epitope is at positions 208 to 212 or 364 to 413 of the amino acid sequence represented by SEQ ID NO: 1.
[3] The antibody or fragment thereof according to [1] or [2], wherein the antibody is a monoclonal antibody.
[4] An antibody or fragment thereof produced by a hybridoma having a receipt number of NITE AP-02156 or NITE AP-02157.
[5] The antibody or fragment thereof according to any one of [1] to [4], wherein the fragment is a Fab fragment, Fab / c fragment, Fv fragment, Fab ′ fragment or F (ab ′) ₂ fragment.

[6] A method for detecting MERS coronavirus from a sample, comprising the step of contacting the sample with the antibody or fragment thereof according to any one of [1] to [5].
[7] The method according to [6], wherein the sample is a body fluid containing cells and / or secretions derived from a living body.
[8] The method according to [6] or [7], further comprising the step of detecting MERS coronavirus by at least one immunoassay selected from the group consisting of ELISA, immunochromatography and filter antigen assay. Method.
[9] A kit for detecting MERS coronavirus, comprising the antibody or fragment thereof according to any one of [1] to [5].
[10] The kit according to [9], which is in the form of an immunochromato strip.

Since the antibody of the present invention or a fragment thereof is highly conserved in the amino acid sequence of MERS-NP and recognizes a region having low homology with the amino acid sequence of NP of a closely related coronavirus as an epitope, various MERS coronaviruses The strain is comprehensively detected, and the related coronavirus containing SARS is not detected, and only the MERS coronavirus can be specifically detected.
Currently, there are only genetic tests such as real-time reverse transcription PCR method for detecting MERS coronavirus. However, according to the detection method according to the present invention, immunological measurement methods such as ELISA method, immunochromatography method and filter antigen assay method are available. Thus, the MERS coronavirus can be detected accurately, quickly and simply, and thus can be used for a test for preventing the spread of MERS coronavirus infection.

FIG. 1 shows an outline of a method for producing a monoclonal antibody against MERS-NP. FIG. 2 shows the results of MERS-NP secondary structure prediction, functional domain analysis and mutation analysis. FIG. 3 shows the results of homology analysis of N protein between MERS and other human coronaviruses. FIG. 4 shows the structures of mutants prepared for the epitope analysis of an antibody, in which a specific sequence is deleted in the amino acid sequence of MERS-NP. FIG. 5 shows the results of Western blotting in which epitopes recognized by each antibody were analyzed using each deletion mutant of MERS-NP. FIG. 6 shows the epitope region of an antibody in the three-dimensional structure of MERS-NP.

FIG. 7 shows the results of Western blotting in which the specificity of each antibody was analyzed using recombinant N proteins of MERS and other human coronaviruses. FIG. 8 shows the detection results of antigen protein by antigen supplement ELISA. FIG. 9 shows a schematic diagram of an immunochromatographic strip for MERS-NP detection. FIG. 10 shows the detection result of the antigen protein using the immunochromato strip for MERS-NP detection.

FIG. 11 shows an image of the instrument used in the filter antigen assay for MERS-NP detection. FIG. 12 shows the detection result by fluorescence of the antigen protein using the filter antigen assay for MERS-NP detection. FIG. 13 shows the detection results of antigen proteins contained in virus-like particles of MERS coronavirus by antigen supplement ELISA. FIG. 14 shows the detection results of antigen proteins contained in the MERS coronavirus virus-like particles using an immunochromato strip for MERS-NP detection.

Hereinafter, the present invention will be described in detail based on preferred embodiments of the present invention.
The “nucleocapsid protein” in the present invention is also referred to as “N protein”. In particular, the N protein of MERS coronavirus is, for example, the amino acid sequence represented by SEQ ID NO: 1 or the amino acid sequence disclosed in GenBank Accession Number (NC — 019843), etc. And is also referred to herein as “MERS-NP”. The amino acid sequence of MERS-NP is the amino acid sequence of SEQ ID NO: 1, preferably 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, Has sequence identity of 98% or more or 99% or more.

  The present invention provides an antibody or a fragment thereof that specifically binds to a protein that constitutes the nucleocapsid of MERS coronavirus, and an antibody that recognizes an epitope at positions 208 to 413 of the amino acid sequence represented by SEQ ID NO: 1. Or a fragment thereof. The reason why the present inventors focused on the 208th to the 413th is that, as a result of mutation analysis of MERS-NP derived from MERS coronavirus 113 strain (see FIG. 2), the ratio of mutant strains is 5% or less. An antibody is specific and universal for MERS against a region excluding the serine-arginine rich (SR rich) motif conserved in SARS and the like, that is, the 208-413th region of the amino acid sequence It is because it discovered that it couple | bonds. Furthermore, as a result of analyzing the homology between MERS-NP and nucleocapsid protein of other related coronaviruses including SARS (see FIG. 3), the present inventors have found that the amino acid sequence of MERS-NP (SEQ ID NO: 1) Such homology was found to be very low or 0% in the 208-212th region and the 364-413th region. Therefore, the present invention preferably provides an antibody or fragment thereof that recognizes an epitope in the region of 208-212 or 364-413 of the amino acid sequence represented by SEQ ID NO: 1.

<Antibody>
The antibody of the present invention is preferably a monoclonal antibody. When there are a plurality of monoclonal antibodies that recognize epitopes at the 208-413th, preferably 208-212th or 364-413th positions of the amino acid sequence represented by SEQ ID NO: 1, a composition comprising a mixture of each monoclonal antibody ( It may be a so-called polyclonal antibody).

  The hybridoma producing the antibody was received on November 6, 2015 at the National Institute of Technology and Evaluation, Patent Microorganisms Deposit Center (Postal Code 292-0818, 2-5-8 Kazusa Kamashi, Kisarazu City, Chiba Prefecture, Japan). It has been deposited under the Budapest Treaty as NITE AP-02156 and NITE AP-02157.

  As described below, the hybridoma producing the monoclonal antibody according to the present invention can be obtained by the method of Keller and Milstein (Koehler, G. and Milstein, C. (1975) Nature 256, 495-497) or a modified method thereof. . That is, after immunizing animals such as mice, rats, rabbits and chickens using the protein prepared by a suitable method, the B cells obtained from the animals and myeloma cells are fused, and the hybridoma obtained is used for the purpose. The hybridoma that produces and secretes the antibody can be selected and isolated. Hereinafter, in one aspect, the above-described hybridoma according to the present invention, a method for preparing a monoclonal antibody, and characteristics of the monoclonal antibody will be described in detail.

(1) Preparation of antigen protein Recombinant protein synthesized by a genetic engineering technique can be used as an antigen, but it is desired to have a high purity and a shape that retains the same three-dimensional structure as in vivo. It is. Protein synthesis by genetic engineering techniques is mainly divided into two types: methods using living cells such as bacteria such as E. coli and yeast, insect cells and animal cells, and methods using these cell extracts and the like. Cell lines exist. In general, a virus-derived protein may be toxic to cells, and expression using a living cell is often difficult. Therefore, a cell-free protein synthesis method is desirable. In particular, a protein synthesis system using a wheat germ extract is an expression system derived from a eukaryotic cell. In addition to being able to synthesize a protein that has been correctly folded, the translation activity is extremely high. It is known that it can be recovered with high purity. Therefore, it is preferable to use a cell-free protein synthesis system utilizing a wheat germ extract for the preparation of the antigenic protein used in the present invention.

(2) Immunization of animals and preparation of hybridoma cells As immunized animals, 6-week-old female BALB / c mice can be preferably used, but mice of other strains can also be used. The immunization schedule and antigen concentration are chosen so that a sufficient amount of antigen-stimulated lymphocytes are formed. For example, it is represented by SEQ ID NO: 1 synthesized by a cell-free protein synthesis system containing MERS-NP, that is, a wheat germ extract described in detail below, together with an adjuvant suitable for the abdominal cavity, footpad, and ridge of a mouse. 10 to 300 μg / mouse of protein consisting of positions 122 to 413 of the amino acid sequence is administered. Thereafter, every few days to several weeks, the same antigen used for the first immunization is administered several times. After the second immunization, blood is collected from a vein and the antibody titer in the blood is examined. The antibody titer can be suitably measured by the ELISA method.

  The spleen and lymph nodes are removed from the immunized mouse, and a B cell suspension is prepared therefrom. This is fused with an appropriate mouse myeloma cell using an appropriate fusion promoter. Myeloma cells are preferably those of the same species as the animal from which B cells were obtained, and those that do not produce antibodies are preferred.

  In a separate container, the mixture of unfused B cells, unfused myeloma cells and fused hybridoma cells is cultured for a time sufficient to kill the unfused cells in a selective medium that does not support the unfused myeloma cells. . The medium is drug resistant (eg 8-azaguanine resistant) and does not support unfused myeloma cells, eg HAT medium. Since unfused B cells are non-neoplastic cells, unfused B cells and unfused myeloma cells die after a certain time in this selective medium. Since the fused cells have both the tumor characteristics of myeloma cells and the properties of B cells, they survive in a selective medium.

(3) Screening and cloning of hybridoma cells After the hybridoma is detected by the above operation, the culture supernatant is collected and screened for antibodies against the immunized MERS-NP. This screening can be suitably performed by, for example, ELISA method, Western blot method, immunostaining method and the like. In this screening, it is important to screen for a clone having high reactivity and specificity with the antigen. The hybridoma producing the target antibody is cloned by an appropriate method such as limiting dilution.
(4) Preparation of monoclonal antibody Two methods can be used to obtain a desired antibody from cloned cells. One is a method of culturing a hybridoma in an appropriate medium for a certain period of time, and a monoclonal antibody produced by the hybridoma can be obtained from the culture supernatant. The second method is to inoculate the hybridomas into the peritoneal cavity of mice with isogenic or semi-isogenic genes. After a certain time, the desired monoclonal antibody produced by the hybridoma can be obtained from the blood and ascites of the inoculated mouse.

  The monoclonal antibody thus obtained can be purified by methods such as salting out using ammonium sulfate, sodium sulfate, etc., ion exchange chromatography, gel filtration, affinity chromatography, or a combination of these methods. it can.

The antibody of the invention is a complete immunoglobulin molecule, preferably IgM, IgD, IgE, IgA or IgG, more preferably IgG1, IgG2a, IgG2b, IgG3 or IgG4, while fragments of the antibody of the invention , Including Fab fragments or portions of such immunoglobulin molecules, such as V-, VH- or CDR-regions, but fragments of the antibodies of the invention also specifically recognize MERS-NP to the same extent as the antibodies of the invention. Can be combined. In addition, antibodies include modified and / or altered antibodies, such as chimeric and humanized antibodies. The antibodies of the present invention also include modified or altered monoclonal or polyclonal antibodies, as well as recombinantly or synthetically produced or synthesized antibodies. Fragments of antibodies of the present invention also include antibody fragments and portions thereof, such as separated light and heavy chains, Fab, Fab / c, Fv, Fab ′, F (ab ′) ₂ . The antibodies of the present invention also include antibody derivatives such as bifunctional antibodies and antibody constructs such as single chain Fvs (scFv), bispecific scFvs or antibody fusion proteins.

<Inspection method>
The present invention also provides a method of detecting MERS coronavirus from a sample, comprising contacting the sample with an antibody against MERS-NP or a fragment thereof.

  Examples of biological samples in the present invention include human or animal blood, serum, plasma, urine, semen, spinal fluid, saliva, sweat, tears, ascites or amniotic fluid; mucus; feces; organs such as blood vessels or liver; A biological sample that may contain MERS-NP, such as cells or extracts thereof, and is preferably a subject, preferably the oral cavity, tonsils, nasal cavity, pharynx, larynx, trachea, Examples include cells and secretions from bronchi and lungs, nasal swabs, throat swabs, gargles, sputum, tracheal aspirates, bronchoalveolar lavage fluids, and the like. A method for collecting these specimens is not particularly limited, and a known method can be adopted. Specifically, a method using a cotton swab is frequently used.

  Any measurement method of MERS-NP in the test method of the present invention can be employed as long as it is a normal immunological measurement method. Here, as immunological measurement methods, enzyme immunoassay (ELISA, EIA), fluorescence immunoassay (FIA), radioimmunoassay (RIA), luminescence immunoassay (LIA), enzyme antibody method, fluorescent antibody Method, immunochromatography method (immunochromatography method), filter antigen assay method, immunoturbidimetric method, latex turbidimetric method, latex agglutination reaction measurement method, hemagglutination reaction method, particle agglutination reaction method and the like. In particular, the immunological assay method for MERS-NP of the present invention is preferably an ELISA method, an immunochromatography method and a filter antigen assay method capable of measuring MERS-NP accurately, rapidly and simply.

  When measuring by immunochromatography, for example, as shown in FIGS. 9, 10 and 14, the antibody against MERS-NP of the present invention and the anti-mouse immune immunoglobulin antibody are immobilized at different sites, that is, a test line and a control line, respectively. An immunochromatographic strip is used. The sample pad of the immunochromatographic strip is impregnated in advance with an antibody against MERS-NP labeled with colloidal gold particles, latex particles, fluorescent particles, magnetic particles or the like. The sample dropped on the sample pad is developed by capillary action, and when it reaches the test line, only MERS-NP in the sample reacts, and when it reaches the control line, only the antibody against MERS-NP labeled with gold colloid or the like Reacts. The other components in the sample move to the water absorption pad without reacting. MERS-NP in the sample can be quantified by measuring the color density, reflection intensity, absorbance, fluorescence intensity, magnetic intensity, etc. of the test line and control line with an immunochromatographic reader.

  The present invention also provides a diagnostic agent comprising an antibody against MERS-NP or a fragment thereof. The present invention further provides a diagnostic method comprising the step of contacting such an antibody or fragment thereof with a sample.

<Inspection kit>
The present invention provides a kit for detecting MERS coronavirus comprising an antibody against MERS-NP or a fragment thereof.

  Any measuring method of MERS-NP in the test kit of the present invention can be adopted as long as it is a normal immunological measuring method. Here, as immunological measurement methods, enzyme immunoassay (ELISA, EIA), fluorescence immunoassay (FIA), radioimmunoassay (RIA), luminescence immunoassay (LIA), enzyme antibody method, fluorescent antibody Method, immunochromatography method (immunochromatography method), filter antigen assay method, immunoturbidimetric method, latex turbidimetric method, latex agglutination reaction measurement method, hemagglutination reaction method, particle agglutination reaction method and the like. Particularly, the MERS-NP immunoassay method of the present invention is preferably an ELISA method, an immunochromatography method and a filter antigen assay method having different characteristics. The ELISA method is excellent in sensitivity and quantification, and can process multiple samples at the same time. However, it takes time until the determination. Although the immunochromatography method is generally inferior to the ELISA method in sensitivity and quantitativeness, it can be quickly and easily inspected. Although the filter antigen assay method is comparable in sensitivity and quantification to the ELISA method and can be rapidly examined, the operation is complicated compared with the immunochromatography method.

  The test kit of the present invention can adopt any form used in the immunological measurement method described above, but preferably an immunochromatographic strip (for example, capable of measuring MERS-NP accurately, quickly and simply) FIG. 9).

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated further in detail, this invention is not limited to these Examples.

[Example 1] Production of monoclonal antibody against MERS-NP A monoclonal antibody against MERS-NP was produced by the following steps. Specifically, a MERS-NP partial protein was prepared by a wheat cell-free protein synthesis system, and this was used as an antigen to immunize mice. B cells were extracted from the immunized mice and fused with myeloma cells. From the obtained hybridoma cells, cells producing an antibody that reacts with MERS-NP were selected, and a monoclonal antibody was purified from the culture supernatant of the hybridoma cells. Details are shown below.

(1) Preparation of antigen protein In order to prepare the antigen protein, a cDNA base sequence encoding the nucleocapsid protein of the prototype strain of MERS shown in SEQ ID NO: 2 was synthesized (consigned to Genewiz). The gene sequence is optimized based on the amino acid sequence for codon synthesis for human protein synthesis. Using this as a template, the 364th to 1242nd DNA fragments of the cDNA sequence encoding the 122th to 413th region in the MERS-NP amino acid sequence were amplified by PCR. First, 5 μL of 1 μM forward primer (SEQ ID NO: 3), 5 μL of 1 μM reverse primer (SEQ ID NO: 4), 1 μL of 10 ng / μL DNA vector inserted with a synthetic gene (pUC57 (Gene With)), PrimeSTAR (registered trademark) ) Max DNA Polymerase (Takara Bio Inc.) 25 μL and ultrapure water 14 μL were mixed to prepare a PCR reaction mixture of 50 μL in total. Next, PCR reaction was performed with the settings shown in Table 1.

The obtained DNA fragment and the pEU-E01-His-TEV-MCS-N1 vector (Cell Free Science) were cleaved with the restriction enzyme EcoRV and purified by agarose gel electrophoresis. Inserted. The inserted DNA vector was transformed into Escherichia coli JM109 strain and inoculated into ampicillin-containing LB medium. After growing E. coli, a DNA vector was prepared from the cells using a plasmid purification kit (Promega or Roche).
MRNA is transcribed from the prepared DNA vector using SP6 RNA polymerase, MERS-NP protein is prepared according to the instruction manual of Cell Free Science WEPRO7240 kit, and His-tag is added from the translation reaction solution using Ni column. The recombinant MERS-NP (122-413) protein was purified. The series of processes of transcription, synthesis, and purification was performed using an automatic synthesizer (Cell Free Science).

(2) Immunization of mice and preparation of hybridoma cells 300 μg of purified antigen protein was mixed with Freund's adjuvant and emulsified, and BALB / c mice were immunized by multiple subcutaneous injections. One month after immunized mice, B cells were collected and fused with SP2 / 0 myeloma cells using a polyethylene glycol solution. The fused hybridoma cells were seeded in a 96-well microplate with a selection medium.

(3) Screening and cloning of hybridoma cells Next, the reactivity of monoclonal antibodies produced by each hybridoma to MERS-NP was examined by ELISA and Western blotting. In the ELISA method, first, recombinant MERS-NP (122-413) protein to which His-tag was added was immobilized on an ELISA plate. After blocking the plate, the culture supernatant of hybridoma was added to the plate for reaction, and then a peroxidase-labeled goat anti-mouse immunoimmunoglobulin antibody was added for reaction. Thereafter, a peroxidase substrate solution was added to cause color development, and the absorbance was measured. In Western blotting, recombinant MERS-NP (122-413) protein to which His-tag was added was first added to 2 × SDS sample buffer (125 mM Tris-HCl, 4% SDS, 20% glycerol, 0.01% bromo). Phenol blue, 10% 2-mercaptoethanol) was mixed and heat-treated, and subjected to electrophoresis on a polyacrylamide gel. The gel after electrophoresis was transferred to a PVDF membrane. After blocking the membrane with a 2% skim milk solution, the culture supernatant of the hybridoma was added and reacted, and reacted with a peroxidase-labeled goat anti-mouse immunoimmunoglobulin antibody. Thereafter, a peroxidase substrate solution was added to emit light and detected. By cloning the hybridomas identified in these screens, 7 types of hybridoma cells (# 5, # 13, # 20, # 25, # 29, # 45 and # 45 and # 45) that produce monoclonal antibodies highly reactive to the antigen protein are used. 46) was obtained.

(4) Preparation of monoclonal antibody Each hybridoma was cultured in a serum-free / protein-free culture solution, and the culture supernatant was collected. From the collected culture supernatant, a monoclonal antibody produced by each hybridoma cell was purified using a protein A column (AcroSep Hyper DF, Nippon Pole). First, 120 mL of the culture supernatant was diluted 2-fold with an equilibration / washing buffer (1.5 M Glycine, 3M NaCl, pH 8.9), and equilibrated with the equilibration / washing buffer (volume 1). 0.04 mL) to capture the antibody on the protein A carrier, and the component non-specifically adsorbed on the carrier was washed away with 10 mL of the equilibration / washing buffer. Next, the antibody adsorbed on the column was eluted with 10 mL of elution buffer (100 mM sodium citrate, pH 3.0). The recovered eluent was concentrated using Amicon Ultra (Merck Millipore). The concentration of the obtained purified antibody was quantified by the UV absorption method at 280 nm, and the purity as a protein was analyzed using gel filtration HPLC (TSKGEL-G3000SWXL, Tosoh Corporation). The results are summarized in Table 2.

[Example 2] Analysis of epitopes recognized by each monoclonal antibody As shown in Fig. 4, six recombinant proteins (deficient mutants) each lacking each region of the amino acid sequence of MERS-NP were prepared. The epitope recognized by each monoclonal antibody was examined by comparing the reactivity with.
In order to prepare each recombinant protein, a wheat cell-free expression vector pEU-E01-His-TEV-MERS-NP (122) encoding the amino acid sequence at positions 122 to 413 of MERS-NP shown in SEQ ID NO: 1. -413) (SEQ ID NO: 22) was prepared by the method described in Example 1 (1), and using this as a template, 122-168th, 165-212th, 208-253rd of the MERS-NP amino acid sequence , 250-312th, 299-363rd, and 355-413th amino acid sequence regions were deleted from the cDNA fragments encoding the respective expression vectors. The production method of each DNA vector is shown in detail below.

PCR was performed using a wheat cell-free expression vector pEU-E01-His-TEV-MERS-NP (122-413) and the primer combinations shown in Table 3. First, 1 μL of 1 μM forward primer, 1 μL of 1 μM reverse primer, 1 μL of 25 pg / μL template DNA vector, 5 μL of PrimeSTAR (registered trademark) Max DNA Polymerase (Takara Bio Inc.), and 2 μL of ultrapure water are mixed in a total volume of 10 μL. A reaction mixture was prepared. Next, PCR reaction was performed with the settings shown in Table 1.

After completion of PCR, 2 μL of the reaction solution was transformed into E. coli JM109 strain and inoculated into ampicillin-containing LB medium. After growing E. coli, a DNA vector was prepared from the cells using a plasmid purification kit (Promega or Roche).
MRNA was synthesized from the prepared DNA vector using SP6 RNA polymerase, and each recombinant protein was synthesized by a wheat embryo cell-free protein synthesis system according to the method of using Cell Free Science's WEPRO7240 kit.

The epitope of each monoclonal antibody was analyzed by Western blotting using each obtained protein. The results are shown in FIG. The 5 clones # 5, # 20, # 25, # 29 and # 45 did not react only with the mutants in which the 355th to 413th defects were deleted among the 6 types of MERS-NP deletion mutants produced. From this, it was revealed that the 364th to 413rd regions were recognized. On the other hand, the clones # 13 and # 46 do not react with the two mutants lacking 165th to 212th and 208th to 253rd of MERS-NP. Was shown to recognize.
Next, it was investigated which site in the three-dimensional structure of MERS-NP the epitope of these antibodies. First, the three-dimensional structure of MERS-NP was constructed as a dimer by the homology modeling method using Modeler with the three-dimensional structure (1SSK, 2CJR) of SARS-NP registered in PDBj as a template. As the amino acid region where the template structure was not registered, the amino acid region whose structure was predicted by I-TASSER and QUARK was used. Then, the epitope of each antibody on the molecular surface of the constructed three-dimensional structure was analyzed by UCSF Chimera. The results are shown in FIG. 6 (in the figure, the 208th to 212th areas are indicated as “208-212” and the 364th to 413th areas are indicated as “364-413”). As a result, it was speculated that the antibody epitope was a surface portion in MERS-NP.

[Example 3] Confirmation of specificity of each monoclonal antibody Does each monoclonal antibody specifically recognize MERS-NP without reacting with NP of other human coronaviruses, SARS, HKU1, OC43, 229E and NL63? The recombinant protein was synthesized and examined.
In order to prepare each recombinant protein, first, cDNA base sequences encoding nucleocapsid proteins of MERS, SARS, HKU1, OC43, 229E and NL63 were synthesized (consigned to Genewiz). Each sequence is shown in SEQ ID NO: 2, 17, 18, 19, 20 and 21, respectively. The gene sequence is optimized based on the amino acid sequence for codon synthesis for human protein synthesis.

A vector in which each synthesized artificial gene is inserted (pUC57 (Genewith)) cleaved with restriction enzymes KpnI and XhoI (NEB) is purified by agarose gel electrophoresis, and then DNA-ligated enzyme (Takara Bio). ) To insert each DNA fragment into the pEU-E01-His-TEV-MCS-N2 vector. The inserted DNA vector was transformed into Escherichia coli JM109 strain and inoculated into ampicillin-containing LB medium. After growing E. coli, a DNA vector was prepared from the cells using a plasmid purification kit (Promega or Roche).
Each recombinant protein was synthesized by the same procedure as that described in Example 2.
Using each of the obtained proteins, the specificity of each monoclonal antibody was analyzed in the same procedure as the Western blot method described in Example 2. The results are shown in FIG. All of # 5, # 13, # 20, # 25, # 29, # 45 and # 46 did not react with human coronavirus NPs other than MERS, and specifically recognized only MERS-NP.

[Example 4] Detection of MERS-NP by antigen capture ELISA Antigen capture ELISA was carried out using # 20 and # 46 having different antigen recognition sites among the seven types of monoclonal antibodies obtained in the present invention.
Anti-MERS-NP antibody # 46 was diluted to 2.5 μg / mL with carbonate buffer (0.05 M sodium carbonate, pH 9.6), immobilized on an ELISA plate, and blocked with 2% skim milk solution. After washing the plate with PBS-T, the recombinant MERS-NP (122-413) protein serially diluted 2-fold with PBS-T from 10 ng / mL was dispensed into the wells for reaction. The plate was washed with PBS-T and then reacted with peroxidase-labeled anti-MERS-NP antibody # 20. After the plate was washed with PBS-T, a color was developed by adding a peroxidase substrate solution (KPL), and the absorbance at wavelengths of 450 nm / 630 nm was measured. The results are shown in FIG. Recombinant MERS-NP (122-413) protein could be quantitatively detected up to 0.625 ng / mL.

[Example 5] Detection of MERS-NP by immunochromatostrip Among the seven monoclonal antibodies obtained in Example 1, immunochromatostrips were prepared using # 20 and # 46 having different antigen recognition sites.
First, with 50 mM phosphate buffer (pH 8.0), the final concentration of anti-MERS-NP antibody # 46 is 1.0 mg / mL and the final concentration of anti-mouse immune immunoglobulin antibody is 0.125 mg / mL. Prepared. Next, as shown in FIG. 9, anti-MERS-NP antibody # 46 was applied to the test line of the nitrocellulose membrane, and anti-mouse immunoimmunoglobulin antibody was applied to the control line on the line using an antibody coating apparatus (Musashi Engineering Co., Ltd.). Later it was dried. After blocking the dried membrane, it was washed with pure water and dried to obtain an immunochromatography membrane.

Next, the final concentration of anti-MERS-NP antibody # 20 was adjusted to 0.2 mg / mL with 50 mM phosphate buffer (pH 8.0). After sensitizing the antibody solution to gold colloidal particles, 0.5% sodium caseinate solution was added for blocking. Thereafter, the colloidal gold solution was washed and then applied to a glass fiber pad and dried under reduced pressure to obtain a conjugate pad.
The above-mentioned immunochromatography membrane and conjugate pad were bonded and cut as shown in FIG. 9 to obtain an immunochromatographic strip.
Recombinant MERS-NP (122-413) serially diluted with 20 ng / mL of the developing solution (20 mM sodium phosphate, 150 mM NaCl, 1% nonidet (registered trademark) P-40, pH 7.4) on an immunochromato strip. ) After providing the protein solution, the coloration on the test line was visually observed after about 15 minutes. The results are shown in FIG. Recombinant MERS-NP (122-413) protein could be detected rapidly up to 5 ng / mL with a simple operation.

[Example 6] Color development detection of MERS-NP using filter antigen assay A filter antigen assay method was performed using # 29 and # 13 having different antigen recognition sites among the seven types of monoclonal antibodies obtained in the present invention. did.
After anti-MERS-NP antibody (# 29) was spotted on a membrane filter (cellulose mixed ester, outer shape 13 mm, pore size 0.45 μm, Merck Millipore) equivalent to 0.5 μg, the membrane was naturally dried. The membrane on which the antibody was immobilized was immersed in Block Ace solution (DS Pharma Biomedical), and then excess blocking solution was washed away with PBS-T. Then, as shown in FIG. 11, it was mounted on a commercially available filter holder (Merck Millipore).

  Next, the 1, 10, 100, 1000 ng / mL recombinant MERS-NP (122-413) protein solution of the sample was passed through the filter holder from the syringe side to capture the antibody on the filter, and then PBS-T The solution was passed through and washed. Furthermore, after passing anti-MERS-NP antibody # 13 fluorescently labeled (IRDye 700DX, LI-COR), PBS-T was continuously passed. Finally, the filter membrane was taken out from the filter holder, and the amount of the fluorescence labeled antibody captured on the membrane after the sandwich system was established was measured with a fluorescence scanner (Odyssey CLx, LI-COR). As a result, as shown in FIG. 12, 1 ng / mL recombinant MERS-NP (122-413) protein could be quantitatively detected by this method.

[Example 7] Detection of MERS coronavirus virus-like particles by antigen capture ELISA By the antigen capture ELISA described in Example 4 developed in the present invention, MERS-NP in virus particles as well as recombinant proteins can be detected. In order to confirm this, virus-like particles (VLP) were prepared and examined. VLP has nucleocapsid protein (NP), membrane (M) and envelope (E), but does not have spike (S) or single-stranded (+) RNA, so it does not infect or proliferate. A particle having a structure similar to the external structure. The antibody exhibits an antigen-antibody reaction against VLPs as well as an antigen-antibody reaction against naturally occurring viruses.
In order to prepare VLP, first, cDNA base sequences encoding NP, M and E of the prototype strain of MERS were synthesized (Gene With). Each sequence is shown in SEQ ID NO: 2, 23 and 24, respectively. The gene sequence is optimized based on the amino acid sequence for codon synthesis for human protein synthesis.
A vector in which each synthesized artificial gene was inserted (pUC57 (Genewith)) cut with restriction enzymes KpnI and XhoI (NEB) was purified by agarose gel electrophoresis. Thereafter, the DNA fragment encoding NP by DNA ligation enzyme (Takara Bio Inc.) is converted to pCAG-Neo (Wako Pure Chemical Industries, Ltd.), and the DNA fragments encoding M and E are pcDNA3.1 (Thermo Fisher Scientific) Inserted into. The inserted DNA vector was transformed into Escherichia coli JM109 strain and inoculated into ampicillin-containing LB medium. After growing E. coli, a DNA vector was prepared from the cells using a plasmid purification kit (Promega or Roche).

The prepared DNA vector was introduced into HEK293T cells using polyethyleneimine (Polyscience). The transfected cells were cultured for 40 hours to release VLP into the culture solution, and the culture supernatant was collected. The culture supernatant was centrifuged at 1000 × g for 10 minutes, and then the supernatant was filtered through a polyvinylidene fluoride filter (Millipore) having a pore size of 0.45 μm. The filtrate was overlaid with 20% (w / v) sucrose in a centrifuge tube and ultracentrifuged at 141,000 × g for 3 hours. VLPs were collected by suspending the resulting precipitate in PBS. The prepared VLP was confirmed by transmission electron microscopic analysis to form particles having a size of about 80 to 120 nm, approximately the same size as the virus particles.
Detection was attempted by the antigen capture ELISA described in Example 4 using this VLP. The VLP was prepared so that the concentration of MERS-NP contained in the VLP was equivalent to about 11 ng / mL, and it was serially diluted with PBS-T two times at a time, and then the nonidet so that the final concentration was 1%. What added (trademark) P-40 was used. The results are shown in FIG. MRS-NP in VLP could be quantitatively detected to about 0.7 ng / mL by antigen capture ELISA using anti-MERS-NP antibodies # 20 and # 46.

[Example 8] Detection of virus-like particles of MERS coronavirus by immunochromatostrip It was examined whether not only recombinant protein but also VLP of MERS coronavirus could be detected by immunochromatostrip described in Example 5 developed in the present invention. VLP was prepared in the same manner as in Example 7, and the concentration of MERS-NP contained in VLP with a developing solution (20 mM sodium phosphate, 150 mM NaCl, 1% nonidet (registered trademark) P-40, pH 7.4). Was prepared so as to be equivalent to about 40 ng / mL, and it was serially diluted two-fold with a developing solution. The immunochromatographic strip was prepared by the method described in Example 5 and then used in a housing case. The results are shown in FIG. MERS-NP in VLP could be quantitatively detected to about 5 ng / mL by immunochromatographic strip using anti-MERS-NP antibodies # 20 and # 46.

  The present invention comprehensively detects various MERS coronavirus strains, does not detect related coronaviruses, and can specifically detect only MERS coronaviruses. Further, according to the present invention, the MERS coronavirus can be detected accurately, quickly and simply, which can be used for a test for preventing the spread of MERS coronavirus infection.

Claims (10)

  An antibody or a fragment thereof that specifically binds to a protein constituting a nucleocapsid of MERS (Middle Eastern Respiratory Syndrome) coronavirus, wherein the epitope recognized by the antibody and the fragment is an amino acid sequence represented by SEQ ID NO: 1 The said antibody or its fragment which exists in 208-413th.   The antibody or fragment thereof according to claim 1, wherein the epitope is located at positions 208 to 212 or 364 to 413 in the amino acid sequence represented by SEQ ID NO: 1.   The antibody or fragment thereof according to claim 1 or 2, wherein the antibody is a monoclonal antibody.   An antibody or fragment thereof produced by a hybridoma having a receipt number of NITE AP-02156 or NITE AP-02157. The antibody or fragment thereof according to any one of claims 1 to 4, wherein the fragment is a Fab fragment, Fab / c fragment, Fv fragment, Fab 'fragment or F (ab') ₂ fragment.   A method for detecting MERS coronavirus from a sample, comprising the step of contacting the sample with the antibody or fragment thereof according to any one of claims 1 to 5.   The method according to claim 6, wherein the sample is a body fluid containing cells and / or secretions derived from a living body.   The method according to claim 6 or 7, further comprising the step of detecting MERS coronavirus by at least one immunoassay selected from the group consisting of an ELISA method, an immunochromatography method and a filter antigen assay method.   A kit for detecting MERS coronavirus, comprising the antibody or fragment thereof according to any one of claims 1 to 5.   The kit according to claim 9, which is in the form of an immunochromatographic strip.