KR102019008B1 - A method for detecting mers coronavirus using mers coronavirus nucleocapsid fusion protein - Google Patents

Abstract

The present invention relates to a nucleocapsid fusion protein comprising N-terminal and C-terminal domain fragments of the MERS coronavirus nucleocapsid protein, a method for detecting MERS coronavirus using a monoclonal antibody derived from the fusion protein and a detection kit to be used therefor. According to the present invention, the presence of MERS coronavirus-related antibody in the target sample can be effectively confirmed, and the MERS coronavirus infection can be quickly diagnosed.

Description

A METHOD FOR DETECTING MERS CORONAVIRUS USING MERS CORONAVIRUS NUCLEOCAPSID FUSION PROTEIN}

The present invention provides a method for detecting MERS coronavirus specific antibody using NC fusion protein comprising N-terminal and C-terminal domain fragments of MERS coronavirus nucleocapsid (NC) protein and monoclonal antibodies derived from the fusion protein thereof. A detection kit for use therewith. According to the present invention, the presence of MERS coronavirus-related antibody in the target sample can be effectively confirmed, and the MERS coronavirus infection can be quickly diagnosed.

The Middle East Respiratory Syndrome Coronavirus ("MERS-CoV", Middle East Respiratory Syndrome Coronavirus), was first discovered in Saudi Arabia in 2012 and has been intensive in the Middle East. It is known to be similar to SARS coronavirus (SARS-CoV, Severe Respiratory Syndrome Coronavirus).

MERS (MERS) is a respiratory infection caused by MERS-CoV, with 1,980 deaths and 699 deaths in 13 countries from September 2012 to May 2017, particularly in the Arabian Peninsula in the Middle East. In South Korea, a massive outbreak occurred in 2015, resulting in 186 confirmed cases, of which 38 were reported to have died.

MERS develops at various ages, from infants to the elderly. The incubation period is at least 2 days to 14 days, and mainly includes respiratory symptoms such as cough, shortness of breath, shortness of breath, and sputum with fever. Digestive symptoms may also be present. Unlike SARS, however, it is accompanied by acute renal failure. The death toll is six times higher than SARS.

The mortality rate is 20-46%, but it can be over 50% depending on age. In particular, it is known that infection rate is high and poor prognosis in people with underlying diseases (diabetes, kidney failure, chronic lung disease, immunodeficiency diseases, etc.). To date, antiviral agents for MERS have not been developed, and the treatment is mainly focused on symptoms, and in severe cases, a ventilator or hemodialysis may be required.

Although no clear source of infection and route of infection has been identified, it is reported that infection is more likely through contact with camels in the Middle East and that it can be spread by close contact between people. Reported.

MERS-CoV is a beta coronavirus among the coronaviruses. The genes vary in length but correspond to about 30 kb and have 11 open reading frames (ORFs). Structural proteins of MERS-CoV include S (spike), E (envelope), M (matrix) and NC (nucleocapsid) proteins.

Diagnosis of MERS-CoV is classified into molecular test and serology test. The most commonly used diagnostics are molecular diagnostics, by PCR or gene sequencing using viral gene specific primers from a nasal swap sample. The serological diagnosis of MERS-CoV uses the principle of the antigen-antibody response, and includes antibody diagnostic ELISA (enzyme-linked immunosorbent assay) or microneutralization assay for S antigen or NC antigen. The antibody diagnostic kit developed by Euroimmune (Germany) is used for diagnosing MERS-CoV using an Indirect ELISA method based on purified S1 antigen. However, indirect ELISA has a disadvantage in that the secondary antibody must be applied separately according to the target animal.

Meanwhile, as some previous studies related to MERS-CoV, Korean Patent No. 1593641 discloses an antibody that recognizes Mers Coronavirus Nucleocapsid (MERS-CoV ΔNC) and a diagnostic kit using the same. Patent No.1782862 discloses a monoclonal antibody against MERS-CoV ΔNC and a hybridoma cell line producing the same, and discloses a sandwich ELISA kit using them. However, to date, there has been no disclosure of Blocking ELISA kits based on MERS-CoV ΔNC based fusion proteins.

Given the continuing threat to human health and high mortality rates, continuous serological testing of risk groups is necessary to continuously monitor the risk of recurrence of MERS, and ELISA techniques for detecting MERS-CoV-specific antibodies. Improvement is required.

The present invention aims to provide a foundation that can respond quickly to human infections that may occur in the future through the production of antigens and monoclonal antibodies based on a relatively high conservative and enriched nucleocapsid fusion protein in MERS coronavirus.

In order to quickly determine whether there is MERS-CoV infection, an ELISA technique capable of detecting MERS-CoV-specific antibodies may be considered. However, in the case of Indirect ELISA, which is commercially available, there are limitations in application such as requiring a separate secondary antibody depending on the individual. Therefore, a method that can be applied to various individuals through an antibody detection method through competition with one type of detection antibody is proposed. I needed to develop.

Accordingly, the present inventors have developed a blocking ELISA-based MERS-CoV antibody detection technique to develop a technique that can be used in both human and human animal serum. Specifically, in view of the stability of standard positive / negative control serum for serum diagnostic application of ELISA technique, we developed a blocking ELISA technique that does not require the use of human-derived serum as a standard positive / negative control, and in fact, MERS patients. We attempted to evaluate the effectiveness of this technique based on serum and control serum.

Accordingly, in the present invention, an effective detection method for identifying the presence of MERS-CoV antibody in the target serum, in particular Blocking, using a mers coronavirus (MERS-CoV) nucleocapsid (NC) -based fusion protein and a monoclonal antibody derived therefrom It is aimed to provide an ELISA.

The present invention

(a) the detection of the nucleocapsid fusion protein comprising the N-terminal domain fragment and the C-terminal domain fragment of the nucleocapsid protein of MERS coronavirus as the antigen for detection, and the sample to be fixed to the solid phase support Reacting with a dragon antigen;

(b) reacting the monoclonal antibody specifically binding to the nucleocapsid fusion protein and conjugated with the detection label with the detection antigen immobilized on a solid phase support; And

(c) measuring the level of the detection label of the monoclonal antibody that antigen-antibodies to the detection antigen

It characterized in that it comprises a, mers coronavirus detection method.

In one embodiment, the N-terminal domain of the nucleocapsid fusion protein may be a polypeptide having an amino acid sequence of SEQ ID NO: 4. At this time, the N-terminal domain fragment of the nucleocapsid fusion protein may be one having a sequence of 50 to 134 consecutive amino acids of the amino acid sequence of position 36 to position 169 of SEQ ID NO.

In one embodiment, the C-terminal domain of the nucleocapsid fusion protein may be a polypeptide having an amino acid sequence of SEQ ID NO: 6. At this time, the C-terminal domain fragment of the nucleocapsid fusion protein may be one having a sequence of 50 to 117 consecutive amino acid sequence of the amino acid position of position 246 to position 362 of SEQ ID NO: 6.

In one embodiment, the nucleocapsid fusion protein may be one having an amino acid sequence of SEQ ID NO: 8. In another embodiment, the nucleocapsid protein may be encoded by the nucleotide sequence of SEQ ID NO: 7.

In one embodiment, the monoclonal antibody may be conjugated with Horseradish peroxidase (“HRP”) as a detection label.

In one embodiment, the detection method may use a blocking ELISA method (FIG. 1).

The present invention also provides

Nucleocapsid fusion proteins including N-terminal domain fragments and C-terminal domain fragments of the nucleocapsid protein of MERS coronavirus; And

It provides a kit for mers coronavirus detection for use in the blocking ELISA method, characterized in that it comprises a monoclonal antibody specifically bound to the nucleocapsid fusion protein and conjugated with a detection label. do. In one embodiment, the monoclonal antibody may be conjugated with horseradish peroxidase as a detection label.

When using the MERS-CoV ΔNC-based fusion protein of the present invention and a monoclonal antibody derived from the antigen, the presence or absence of the MERS-CoV antibody in the target serum can be effectively confirmed.

In particular, when the nucleocapsid fusion protein according to the present invention is used as a coating antigen, and a blocking ELISA is performed using a monoclonal antibody specific for the antigen conjugated to HRP as a detection marker, as a detection factor, MERS- in the target sample is used. Comparable presence of CoV specific antibodies can be confirmed. In addition, the method according to the present invention has a higher sensitivity and specificity than the ELISA-based antibody diagnostic technique, which is commercially available at a considerably high price, and thus shows a superior diagnostic effect.

Furthermore, since the Blocking ELISA of the present invention does not use a host specific secondary antibody, it can be utilized in a wider range of human, as well as intermediate host or mediated animal serum, compared to the existing Indirect ELISA, MERS coronavirus You can quickly check for infection.

Accordingly, the kit according to the present invention is expected to be able to secure market competitiveness as a leader by distributing to domestic as well as overseas MERS generated or likely.

Figure 1 shows a step-by-step schematic diagram of Blocking ELISA for the detection of MERS-CoV-specific antibodies in the target serum using MERS-CoV ΔNC-based fusion protein and monoclonal antibody (conjugated with HRP as detection marker) .
FIG. 2 shows a diagram of a plasmid construct for the preparation of a MERS-CoV ΔNC based fusion protein used in Example 1. FIG. The MERS-CoV-ΔNC gene was introduced into the pET28a (+) vector.
Figure 3 shows the results of purification after expressing the MERS-CoV ΔNC-based fusion protein in E. coli Rosetta (DE3) pLysS.
Figure 4 shows the results confirming the monoclonal antibody prepared from MERS-CoV ΔNC-based fusion protein.
5 is a schematic diagram showing a process of conjugating HRP as a detection label to monoclonal antibody IgG.
Figure 6 is a graph showing the results confirmed the specific binding of MERS-CoV ΔNC-based fusion protein antigen and monoclonal antibody.
FIG. 7 is a graph showing that MERS-CoV ΔNC based fusion protein antigens do not react nonspecifically with human coronavirus antibodies other than MERS-CoV antibodies.
8 is a graph showing the results of confirming the optimization conditions of the Blocking ELISA of the present invention for human serum. A represents experimental data according to the change of MCoV-ΔNC antigen coating concentration, B represents experimental data according to the change of human serum dilution concentration, and C represents experimental data according to the change of HRP-conjugated anti-ΔNC IgG dilution concentration.
Figure 9 is a graph showing the results according to the change in HRP-conjugated anti-ΔNC IgG dilution concentration in the optimization conditions of the present invention Blocking ELISA for mouse serum.
10 is a graph showing the detection results of Blocking ELISA of the present invention for 50 human serum (1-50), positive antiserum (ΔNC), negative negative serum (mouse negative serum).
11 is a graph showing the detection results of Euroimmune's commercialization kit for 50 human negative serum (1-50), positive antiserum (ΔNC), negative negative serum (mouse negative serum).
12 is a graph showing the detection result of Alpha diagnostic's commercialization kit for 50 human negative serum (1-50), positive antiserum against ΔNC, and negative negative serum.
FIG. 13 shows bones of mouse antiserum against ΔNC, mouse negative serum, 50 human negative serum (1-50), and 7 human positive serum (4503-4509) of MERS complete It is a graph which shows the detection result of the Blocking ELISA of this invention.
14 is a graph showing the detection results of the commercialization kit of Euroimmune for human positive serum (P05 and P09) and negative control (sigma).
15 is a graph showing the detection results of Blocking ELISA according to the present invention for human positive serum (P05 and P09) and negative control (sigma).

1. MERS-CoV ΔNC based fusion protein and polynucleotide encoding the same

In the present invention, a fusion protein (hereinafter, NC fusion protein) of an N-terminal domain fragment and a C-terminal domain fragment of the mers coronavirus nucleocapsid (MERS-CoV ΔNC) protein can be used.

MERS-CoV is a positive-sense, single-stranded RNA virus of the genus Betacoronavirus and is known to be a very fatal cause of severe acute respiratory infections. The viral genome is capped, polyadenylated, and stacked with nucleocapsid proteins. MERS-CoV virions include viral epidermis with large spike glycoproteins.

The MERS-CoV genome, like most coronaviruses, contains a replica gene in two-thirds (2/3) of the genome, and structural genes in one-third of the genome. It has a common dielectric structure. The MERS-CoV genome encodes a set of classical structural protein genes of 5'-spike (S) -epidermal (E) -membrane (M) and nucleoside (NC) -3 '.

The NC protein of the MERS-CoV structural protein is a 413 amino acid long protein that shares high homology with the N protein of other coronaviruses. The NC protein is relatively highly conserved, and because it is most produced among viral structural proteins in infected cells, it can induce strong humoral and cellular immune responses. For example, the nucleocapsid protein (413 amino acids) of the MERS-CoV (Human betacoronavirus 2cEMC / 2012) may have a nucleotide sequence of SEQ ID NO: 1.

In the present invention, the NC protein of the MERS-CoV structural protein was selected, thereby producing a MERS-CoV Δ NC based fusion protein.

Specifically, the N-terminal domain of the nucleocapsid protein of MERS-CoV may be a polypeptide having an amino acid sequence of SEQ ID NO: 4. In this case, the protein may be encoded by the nucleotide sequence of SEQ ID NO: 3. In addition, in the present invention, the fragment of the N-terminal domain is 1 to 134, 10 to 120, 30 to 100, or 50 of the amino acid sequence of amino acid position 36 to position 169 of SEQ ID NO: 4 May be from 80 to 80 contiguous amino acid sequences.

The C-terminal domain of the nucleocapsid protein of MERS-CoV may be a polypeptide having an amino acid sequence of SEQ ID NO: 6. In this case, the protein may be encoded by the nucleotide sequence of SEQ ID NO: 5. In addition, in the present invention, the fragment of the C-terminal domain is 1 to 117, 10 to 100, 20 to 80, or 30 of the amino acid sequence of position 3246 to position 362 of SEQ ID NO: 6 May be from 50 to 50 contiguous amino acid sequences.

The NC fusion protein of the nucleocapsid protein of MERS-CoV has the amino acid sequence of SEQ ID NO: 8 and includes functional equivalents thereof. The term "functional equivalent" means at least 70%, preferably 80%, more preferably 90%, more preferably at least 70% of the amino acid sequence represented by SEQ ID NO: 8 as a result of the addition, substitution, or deletion of the amino acid. Refers to a polypeptide having 95% or more of sequence homology and exhibiting substantially homogeneous physiological activity with the polypeptide represented by SEQ ID NO: 8.

Polypeptides having the amino acid sequence of SEQ ID NO: 8 of the present invention include polypeptides having their native amino acid sequence as well as amino acid sequence variants thereof are also included within the scope of the present invention. A variant of the polypeptide of SEQ ID NO: 8 refers to a polypeptide having a sequence in which the polypeptide natural amino acid sequence of SEQ ID NO: 8 and one or more amino acid residues have a different sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof.

Amino acid exchange in proteins and peptides that do not alter the activity of the molecule as a whole is known in the art (H. Neurath, R. L. Hill, The Proteins, Academic Press, New York, 1979). The most commonly occurring exchanges are amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thy / Phe, Ala / Exchange between Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, Asp / Gly. In some cases, it may be modified by phosphorylation, sulfation, acetylation, glycosylation, methylation, farnesylation, or the like.

In one embodiment, the MERS-CoV ΔNC-based fusion protein of the present invention exhibits a specific antigen-antibody response when there is a MERS-CoV-related antibody in a target sample, thereby detecting MERS-CoV or diagnosing infection thereof. Can be used as

According to one embodiment of the invention, the fusion protein may be encoded by the nucleotide sequence of the polynucleotide of SEQ ID NO.

2. Recombinant Expression Vector

Fusion proteins of the present invention can be produced by recombinant expression vectors comprising the polynucleotides.

As used herein, a "recombinant expression vector" refers to a gene construct capable of expressing a protein of interest in a suitable host cell and comprising a gene construct comprising essential regulatory elements operably linked to express the gene insert.

As used herein, the term “operably linked” refers to a functional link between a nucleic acid expression control sequence and a nucleic acid sequence encoding a protein of interest to perform a general function. For example, a promoter and a nucleic acid sequence encoding a protein can be operably linked to affect the expression of the encoding nucleic acid sequence. Operative linkage with recombinant expression vectors can be prepared using genetic recombination techniques well known in the art, and site-specific DNA cleavage and ligation employs enzymes and the like generally known in the art.

Expression vectors usable in the present invention include, but are not limited to, plasmid vectors, cosmid vectors, bacteriophage vectors, viral vectors, and the like. Suitable expression vectors include membrane targeting or in addition to expression control sequences such as promoters, operators, initiation codons, termination codons, polyadenylation sigsal, enhancers, etc. It may be prepared in various ways according to the purpose, including a signal sequence (leadcr sesuence) or a signal sequence for secretion.

The promoter of an expression vector can be constitutive or inducible. In addition, the expression vector includes a selection marker for selecting a host cell containing the vector, and in the case of an expression vector capable of replication, includes a replication origin.

The regulatory sequence may be a promoter, enhancer, initiation codon, stop codon or polyadenylation signal. Regulatory sequences direct the desired expression of the desired nucleic acid in many host cells at all times, direct the expression of the desired nucleic acid only in specific tissue cells (e.g., tissue specific regulatory sequences), and Directing expression by a particular signal is included (eg, inducible regulatory sequences). The design of the expression vector can vary depending on factors such as the choice of host cell to be transformed and the level of protein expression desired.

According to one embodiment of the present invention, the vector comprises a gene construct in which a polynucleotide encoding the MERS-CoV ΔNC fusion protein is operably linked to a regulatory sequence. It can be an expression vector capable of expression.

According to a preferred embodiment of the present invention, pET28a (+) is used as the expression vector backbone, and the MERS-CoV ΔNC-derived gene is inserted into the vector to express the expression plasmid "pET28a (+)-MCoV-ΔNC. "Was prepared. The plasmid pET28a (+)-MCoV-ΔNC construct is shown in FIG. 2.

In one embodiment, the amino acid residues 36-169 of the N-terminal domain of the MERS-CoV nucleocapsid protein represented by SEQ ID NO: 4 can be subcloned to the NdeI and BamHI sites of the pET28a vector. In addition, amino acid residues 246-362 of the C-terminal domain of the MERS-CoV nucleocapsid protein represented by SEQ ID NO: 6 can be subcloned to the EcoRI and XhoI sites of pET28a.

Vectors of the present invention can be prepared by standard recombinant DNA techniques, which include, for example, blunt and adhesion term ligation, restriction enzyme treatment to provide appropriate ends, to prevent inadequate binding. Phosphate group removal by alkaline post stay treatment and enzymatic linkage by T4 DNA ligase. The vector of the present invention can be prepared by recombining the MERS-CoV ΔNC-derived DNA of the present invention into a vector containing an appropriate regulatory sequence. The vector containing the regulatory sequence can be purchased or produced commercially, in one embodiment of the present invention used pET28a (+).

3. Transformant

A transformant transformed with the recombinant expression vector may be used for expression of the fusion protein of the present invention.

The transformant of the present invention may be preferably E. coli , more preferably E. coli Rosetta (DE3) pLysS. The E. coli can be expressed in large quantities by transforming E. coli with a recombinant expression vector of the present invention, for example, pET28a (+)-MCoV-ΔNC. In one embodiment of the present invention, a transformant transformed with the recombinant expression vector pET28a (+)-MCoV-ΔNC to E. coli rosetta (DE3) pLysS.

For transformation, any method of introducing a nucleic acid into a host cell may be used without limitation, and may be performed by transformation techniques known to those skilled in the art. Preferably, microprojectile bombardment, electroporation, electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, PEG-mediated fusion, microimjection And liposome-mediated methods and the like, but are not limited thereto.

The present invention also provides a method for producing a MERS-CoV ΔNC fusion protein comprising culturing the transformant.

The preparation method is carried out by culturing the transformant under appropriate medium and conditions such that the polynucleotide encoding the MERS-CoV ΔNC fusion protein of the present invention is expressed in the recombinant expression vector introduced into the transformant of the present invention. . Methods for culturing the transformant to express the fusion protein are known in the art, for example inoculating them in a suitable medium in which the transforming bacteria can grow and then incubating them in the medium for incubation and Expression of the protein can be induced by culturing in the presence of conditions such as isopropyl-β-D-thiogalactoside (IPTG), a gene expression inducing agent. Upon completion of the culture, substantially pure fusion proteins can be recovered from the culture. As used herein, the term "substantially pure" means that the sequence of the fusion protein of the invention and the polynucleotide encoding it are substantially free of other proteins derived from host cells.

Recovery of the fusion protein expressed in the transgenic bacteria can be carried out through various separation and purification methods known in the art, and typically centrifuged cell lysate to remove cell debris, culture impurities, etc. After separation, precipitation, for example, salting out (ammonium sulfate precipitation and sodium phosphate precipitation), solvent precipitation (protein fraction precipitation using acetone, ethanol and the like) and the like can be carried out, dialysis, electrophoresis and various column chromatography. And the like. As the chromatography, techniques such as ion exchange chromatography, gel-penetration chromatography, HPLC, reverse phase-HPLC, affinity column chromatography, and ultrafiltration may be used alone or in combination.

4. Monoclonal Antibodies

The monoclonal antibody used in the present invention is characterized in that it specifically binds to the MERS-CoV ΔNC fusion protein.

The term “specifically binding” is commonly known to those skilled in the art, and specifically, antigens and antibodies can specifically interact to form antigen-antibody complexes and also to undergo an immunological response. Can mean.

As used herein, the term “monoclonal antibody” means that the antibody molecule is made to consist of a single molecule. Monoclonal antibodies have specificity that responds only to antigens with the same epitope, and also show affinity only for specific epitopes.

In one embodiment of the invention, the monoclonal antibody may be produced in a hybridoma cell line. The hybridoma cells can be prepared using methods known in the art. For example, the hybridoma cell was prepared by hybridizing an immunogen MERS-CoV ΔNC fusion protein to an animal and fusing B cells, an antibody-producing cell derived from the immunized animal, with myeloma cells. Next, it can be prepared by a method of selecting a hybridoma producing a monoclonal antibody that specifically binds to the MERS-CoV ΔNC fusion protein. The immunized animal can use an animal such as goat, sheep, morphot, rat or rabbit as well as the mouse used in the examples.

As a method for immunizing the immunized animal, a method already known in the art may be used. For example, when immunizing mice, 1 to 100 µg of an immunogen is emulsified once with an adjuvant such as physiological saline and / or Freund's adjuvant to subcutaneously abdomen of the immunized animal. Or 2-6 inoculations every 2-5 weeks intraperitoneally.

After immunizing the immunized animals, spleens or lymph nodes are extracted after 3-5 days of final immunization, and according to cell fusion methods already known in the art, B cells contained in their tissues in the presence of a fusion promoter are myeloma. To fuse with the cell. The fusion promoter may use a material such as polyethylene glycol (PEG), for example. The myeloma cells are described, for example, in P3U1, NS-1, P3x63. Mouse derived cells such as Ag 8.653, Sp2 / 0-Ag14, and rat derived cells such as AG1, AG2 can be used. In addition, the cell fusion method known in the art, for example, B cells and myeloma cells are mixed at a ratio of 1: 1-10: 1, to which a PEG having a molecular weight of 1,000-6,000 is added at a concentration of 10-80%. For example, the method may be performed by incubating at 30-37 ° C. for 1-10 minutes.

The obtained hybridomas are cultured to obtain monoclonal antibodies. There is no restriction | limiting in particular in the culture method, According to the objective, it can select suitably, For example, the said hybridoma cell line is intraperitoneally of the mouse which previously received pristane (2,6,10,14- tetramethylpentadecane). The method of transplanting and culturing is mentioned. For example, the monoclonal antibody can be obtained from the ascites obtained by recovering ascites 10 to 14 days after transplantation into the abdominal cavity of the mouse.

In addition, the hybridomas can be selected, for example, by culturing in a selective medium such as HAT medium, in which only hybridomas are viable, and measuring the antibody activity in the hybridoma culture supernatant using a method such as ELISA. .

The monoclonal antibody produced by the hybridoma may be used without purification, but in order to obtain the best results, it is recommended to use it by purifying with high purity (for example, 95% or more) according to a method well known in the art. desirable. Such purification techniques can be separated from the culture medium or ascites using, for example, purification methods such as gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, affinity chromatography and the like.

On the other hand, in a preferred embodiment, the monoclonal antibody may be an IgG immunoglobulin isotype.

5. MERS-CoV Detection Kit and Detection Method

The present invention provides a MERS-CoV detection kit comprising the MERS-CoV ΔNC fusion protein and the monoclonal antibody.

In the MERS-CoV detection kit of the present invention, the presence of virus from a biological sample using recombinant antigen proteins and antibodies, such as a tool for collecting a sample or applying a detection reagent, a reagent or a device for confirming antigen-antibody binding, and the like. Tools, devices or reagents may be further included that can be included in conventional detection or diagnostic kits to confirm.

The kit of the present invention can be used to diagnose MERS-CoV infection by detecting MERS-CoV according to an immunoassay method. Such immunoassays can be performed according to various immunoassays or immunostaining protocols developed in the prior art. The immunoassay or immunostaining format may be, for example, radioimmunoassay, radioimmunoprecipitation, immunoprecipitation, enzyme-linked immunoassay (ELISA), flow cytometry, immunofluorescence immunoaffinity purification, immunochromatography (immunochromatography) and the like. Preferably, the kit of the present invention may be for blocking ELISA.

In addition, the present invention provides a MERS-CoV detection method comprising the step of contacting the fusion protein with the monoclonal antibody and the sample to be tested using the MERS-CoV detection kit, to detect the antigen-antibody complex formed therefrom to provide. In one embodiment, the detection method of the present invention comprises the steps of: (a) reacting the test antigen with the detection antigen immobilized on a solid phase support wherein the fusion protein is a detection antigen; (b) reacting the monoclonal antibody with the detection antigen immobilized on a solid phase support; And (c) measuring the level of the detection label of the monoclonal antibody that antigen-antibodies to the detection antigen.

Preferably, the detection method of the present invention may be a blocking ELISA method. In a specific embodiment, each well of a plate coated with the fusion protein of the present invention is reacted with a sample collected from a test subject, eg, a suspected MERS patient, and added with HRP-conjugated monoclonal antibody, followed by TMB. (3,3 ', 5,5'-tetramethylbenzidine) A substrate solution may be treated in each well to measure the color reaction by absorbance.

More specifically, as shown in Figure 1, if there is a MERS-CoV-related antibody in the sample to be tested, first react the sample on a plate coated with the MERS-CoV ΔNC antigen and then remove the non-binding elements. HRP-conjugated monoclonal antibody was reacted, and then the MERS-CoV-associated antibody and the MERS-CoV ΔNC antigen were bound to the sample to be tested, thereby reducing the extent of binding of the HRP-conjugated monoclonal antibody to the antigen. do. In this case, compared with the negative control group (that is, when there is no MERS-CoV in the sample to be tested), the color reaction by the HRP enzyme is reduced, and it is possible to confirm whether MERS-CoV in the sample to be tested therefrom.

The sample to be tested may be a biological material derived from an individual. The subject may be a vertebrate. The vertebrate may be a mammal. The mammal may be a primate, including a human and a non-human primate, a camel, or a rodent, including a mouse and a rat. The sample may be taken or isolated from an individual, such as nasal swabs, nasal aspirates, nasopharyngeal swabs, nasopharyngeal aspirates, blood or Blood constituent, bodily fluid, saliva, sputum, or a combination thereof.

As used herein, the term “antigen-antibody complex” refers to a combination of a MERS-CoV ΔNC antigen in a sample and a monoclonal antibody according to the present invention that recognizes the antigen, and the antigen-antibody complex may be obtained by colormetric method, electrical By any method selected from the group consisting of electrochemical method, fluorimetric method, luminometry, particle counting method, visual assessment and scintillation counting method Can be detected. By this detection, MERS-CoV can be qualitatively or quantitatively analyzed.

Monoclonal antibodies can be conjugated with various detection labels to detect antigen-antibody complexes of the invention. Specific examples of detection labels include chemicals (eg biotin), enzymes (alkaline phosphatase, β-galactosidase, horseradish peroxidase and cytochrome P450), radioactive substances (eg C14). , I125, P32, and S35), fluorescent materials (eg, fluorescein), luminescent materials, chemiluminescent, and fluorescence resonance energy transfer (FRET). Preferably, the detection label is Horseradish peroxidase (HRP).

Methods of conjugating detection labels to such monoclonal antibodies are known in the art. For example, when HRP is used as a detection label, HRP-conjugated monoclonal antibodies can be prepared by conjugating HRP to a monoclonal antibody of the present invention by a known Nakane method.

Hereinafter, the present invention will be described in more detail with reference to the following examples, but the following examples are for illustrative purposes only and are not intended to limit the scope of the present application.

Example 1

MERS-CoV ΔNC-based Fusion Protein Gene Cloning

Polynucleotides derived from the MERS-CoV ΔNC fusion protein of the present invention were amplified and inserted into the pET28a (+) plasmid vector and cloned (referred to as pET28a (+)-MCoV-ΔNC). The structure of the produced vector is shown in FIG.

Example 2

Expression and Purification of MERS-CoV ΔNC Fusion Protein Using E. Coli Expression System

PET28a (+)-MCoV-ΔNC gene prepared in Example 1 was transformed into E. coli Rosetta (DE3) pLysS, and the protein was expressed using 0.2 mN IPTG and cultured in large quantities. After filtering off the supernatant of the product, it was purified using an equilibrium Ni-NTA column. The results are shown in FIG.

Example 3

Preparation of HRP-conjugated monoclonal antibody IgG from MERS-CoV ΔNC fusion protein

In order to produce the monoclonal antibody, the MERS-CoV ΔNC fusion protein produced in Example 2 was inoculated into mice to establish a monoclonal antibody-producing cell line.

After culturing the cell line inoculated into the mouse abdominal cavity to obtain ascites containing a large amount of antibody, monoclonal antibodies were purified therefrom. Monoclonal antibody IgG was purified from mouse ascites using protein A resin, and 300 ul of mouse ascites contained 0.5 mg / ml of monoclonal antibody IgG. After purification, 100 ug / 200 ul of monoclonal antibody IgG was obtained. 4 shows the results of confirming purified monoclonal antibody IgG by comparing proteins after purification of mouse ascites protein before purification.

Purified monoclonal antibody IgG was conjugated with HRP to obtain HRP-conjugated monoclonal antibody IgG. A schematic diagram of the specific bonding process is shown in FIG. 5. 200 ug of HRP was mixed with 50 ug of purified monoclonal antibody IgG and reacted for 3 hours at room temperature where light was blocked. 50 ug / 100 ul of HRP-conjugated IgG was obtained.

Example 4

Confirmation of specific binding of MERS-CoV ΔNC fusion protein to monoclonal antibody

In this example, ELISA was performed to confirm whether the HRP-conjugated IgG obtained in Example 3 specifically binds to the actual MERS-CoV ΔNC fusion protein antigen. MERS-CoV ΔNC fusion protein purified antigen was coated on an ELISA plate, the HRP-conjugated monoclonal antibody was reacted, washed and reacted with a substrate to measure absorbance at 450 nm. As a result, it was confirmed that HRP-conjugated IgG specifically reacted with the substrate until diluted to a concentration of 1: 10000. Specific data is shown in FIG. 6.

Example 5

Nonspecific Response Experiments Using Mouse Serum Immunized with Human Coronavirus

In this example, it was confirmed that the MERS-CoV ΔNC fusion protein antigen obtained in Example 2 did not exhibit a nonspecific response to other antibodies, for example, human coronavirus antibodies, which were not MERS-CoV antibodies.

Human coronavirus 229E, OC43 strain was immunized to mice to obtain mouse serum from it. ΔNC antigen-based Indirect ELISA was performed using the obtained mouse serum and HRP-conjugated IgG obtained in Example 3, and the results are shown in FIG. 7. Mouse sera obtained from the human coronavirus 229E, OC43 strain hardly reacted with the MERS-CoV ΔNC fusion protein antigen, while reacting at very high levels with the HRP-conjugated IgG of Example 3.

From this, the MERS-CoV ΔNC fusion protein antigen of the present invention was confirmed to have a low nonspecific response to other human coronavirus antibodies.

Example 6 Blocking ELISA Optimization Conditions Using Human Serum and Mouse Serum

Optimization conditions for performing Blocking ELISA using human MERS-positive serum and mouse serum from MERS patients, ie antigen coating concentration, serum dilution concentration, and dilution of HRP-conjugated anti-MERS-CoV ΔNC mouse IgG The concentration was determined.

Blocking ELISA analysis of human serum by varying antigen coating concentration, serum dilution concentration, and dilution concentration of HRP-conjugated anti-MERS-CoV ΔNC mouse IgG, respectively, is shown in FIG. 8. Optimization conditions are as follows.

● MCoV-ΔNC antigen coating concentration: 200 ng

● human serum dilution concentration: 1/10

HRP-conjugated anti-ΔNC IgG dilution concentration: 1/20000 (25ng / ml)

Optimization conditions for mouse serum are as follows, the results of the HRP-conjugated anti-ΔNC IgG dilution concentration is shown in FIG.

● MCoV-ΔNC antigen coating concentration: 200 ng

Mouse serum dilution concentration: 1/5

HRP-conjugated anti-ΔNC IgG dilution concentration: 1/10000 (50ng / ml)

In order to select a suitable solution as the blocking solution, non-specific responses were evaluated when using Skim milk, BSA, and Horse serum. As a result, horse serum was found to minimize the non-specific response in MERS-negative human serum, which was used as an optimized blocking solution.

The optimized protocol of Blocking ELISA for MERS-CoV ΔNC based on the above results is shown in Table 1. In addition, the schematic diagram for Blocking ELISA is as shown in FIG.

NC blocking ELISA protocol optimization Mouse Human △ NC antigen coating concentration 200ng (overnight) Blocking with 10% horse serum (1hr, RT) Washing with PBS-T (0.05% Tween 20 in 1X PBS) (X2) 5-fold dilution of mouse serum (1hr, RT) Treatment with 10-fold dilution of human serum (1hr, RT) Washing with PBS-T (X3) HRP-conjugated anti-ΔNC mouse IgG diluted 1: 10,000 (1hr, RT) HRP-conjugated anti-ΔNC mouse IgG 1: 20,000 diluted for treatment (1hr, RT) Washing with PBS-T (X4) TMB substrate solution treatment (15min, RT) TMB stop solution treatment Absorbance measurement

Example 7

Detection Sensitivity and Specificity Evaluation of Blocking ELISA of the Present Invention Using Human Negative Serum

Detection sensitivity and specificity for human negative sera (i.e., sera from humans not infected with MERS) when using the Blocking ELISA of the present invention as compared to the previously commercialized MERS ELISA kit were evaluated.

In 2002, 13 years before the outbreak of MERS in Korea in 2015, 50 human negative sera were distributed from the pathogenic virus bank. This human sample was obtained on the basis of the clinical trial exemption certificate issued after the deliberation by the Ministry of Health and Welfare.

Based on the Blocking ELISA optimization protocol established in Example 6, the response to the mouse antiserum against ΔNC, mouse negative serum and 50 human negative serum was evaluated. The results are shown in FIG.

The 50 human negative serum O.D values were 1.43-1.60, which was higher than the mouse negative serum O.D value of 1.31. On the other hand, the mouse positive serum had an O.D value of 0.26, which is much lower than the human negative serum or mouse negative serum. From the blocking ELISA results of the present invention, it was confirmed that all of the tested human negative serum MERS-CoV antigen is negative.

As a comparative experiment, 50 human negative serum, positive control and negative control groups were evaluated in the same manner as above using the existing commercialization kit.

Data obtained using Euroimmune's Indirect ELISA commercialization kit based on MERS CoV-S1 is shown in FIG. 11. The positive control / negative control-based ratio value was negative when 0 <ratio <0.7 and positive when 1.5 <ratio <3.9. Of the 50 human negative sera, 49 were confirmed as negative, and sample 22 had a false positive value of 0.728, which was not included in the positive criterion (1.5 <ratio <3.9) but was higher than the negative criterion (0 <ratio <0.7). It was judged to belong to the range.

Data obtained using a commercialization kit from Alpha diagnostic is shown in FIG. 12. While the positive control group and the negative control group were obtained as expected, the human control samples showed O.D values of 1.0 or more, indicating higher values than the positive control group, indicating that the non-specific response was very severe. Accordingly, this commercialization kit would be difficult to use as a control group in the future.

The data of the kit of the present invention, Euroimmune, Alpha Diagnostics, commercialization kit for 50 human negative sera is summarized and shown in Table 2 below.

Alpha Diagnostics' commercialization kit was confirmed that the non-specific reaction occurs severely, when using the Blocking ELISA of the present invention, the competitive reaction did not occur in all 50 human negative serum, whereas one of the commercialization kit of Euroimmune The false positive result was confirmed in the negative sample. From this, it can be seen that the Blocking ELISA kit of the present invention has superior detection sensitivity and specificity than the conventional commercialization kit.

Example 8

Detection Sensitivity Evaluation of Blocking ELISA of the Present Invention Using Human Positive Serum of MERS Complete

In this Example, the sensitivity of detection for MERS completeness serum (human positive serum) when the Blocking ELISA of the present invention was used was compared with the conventionally commercialized MERS ELISA kit.

As in Example 7, 50 human negative serum and 7 MERS complete sera were obtained based on the clinical trial review exemption certificate. Based on the Blocking ELISA optimization protocol established in Example 6, positive antiserum against (ΔNC), negative negative serum (mouse negative serum), 50 human negative serum (1-50), and MERS cured serum 7 Responses to dogs (4503-4509) were evaluated. Using the measured absorbance value OD, percent inhibition (PI) was calculated as follows. The results are shown in FIG. Positive PI2 values above 50 were determined as positive.

(1) Percent Inhibition (PI1) = {1- (OD test sample / OD negative control)} * 100

(2) Percent Inhibition (PI2) = [{(OD negative control-OD test sample) / OD positive control} / OD negative control] * 100

As a result of analysis based on 7 MERS completely cured samples and 50 human negative serum samples, the Blocking ELISA of the present invention was determined to be negative because the PI2 value of all 50 samples of human serum was measured to be less than 50. The remaining samples, except for 4503 and 4509 serum samples, showed positive PI2 values of 50 or more.

As a comparative experiment, it was evaluated in the same manner as above, using a commercialization kit of Euroimmune based on MERS CoV-S1. For Euroimmune, the positive control / negative control-based ratio values included 4503 and 4509 samples in the positive standard (1.1 <ratio) out of 7 MERS patient sera, and negative values for 4505 and 4506 samples. A higher value than (ratio <0.8) was considered to be within the false positive range.

The data from the Blocking ELISA kit of the present invention and the commercialization kit from Euroimmune is summarized in Table 3 below.

 As shown in Table 3, in the case of using the kit of the present invention, five samples out of seven samples of MERS patients serum were determined to be positive, and only two samples were positive compared to the conventional commercialization kit evaluated as positive. It can be seen that the detection rate is improved to show a better diagnostic effect.

Example 9

Sensitivity Evaluation of Human Positive Serum Using Euroimmune's Commercialization Kit and Kit of the Present Invention

Using Euroimmune's commercialization kit based on MERS CoV-S1 and the Blocking ELISA kit of the present invention, human positive serum (P05 and P09) was diluted and its sensitivity compared to the OD value for the negative control (sigma). Evaluated through. Results according to the commercialization kit of Euroimmune are shown in FIG. 14. In addition, the results using the Blocking ELISA kit of the present invention is shown in Figure 15.

From the above experimental results, P05 serum was confirmed to be positive among two human positive serum, and P09 serum was confirmed to be negative. Also, in sensitivity, the Euroimmune commercialization kit showed positive results for human-positive serum diluted 16-fold, whereas the kit of the present invention confirmed positive results for human-positive serum diluted 128-fold. Accordingly, it was confirmed that the sensitivity of the kit of the present invention is better than the conventional commercialization kit.

<110> Korea Centers for Disease Control and Prevention <120> A METHOD FOR DETECTING MERS CORONAVIRUS USING MERS CORONAVIRUS          NUCLEOCAPSID FUSION PROTEIN <130> P180053 <160> 8 <170> KoPatentIn 3.0 <210> 1 <211> 1242 <212> DNA <213> Artificial Sequence <220> <223> MERS-CoV nucleocapsid protein <400> 1 atggcatccc ctgctgcacc tcgtgctgtt tcctttgccg ataacaatga tataacaaat 60 acaaacctat ctcgaggtag aggacgtaat ccaaaaccac gagctgcacc aaataacact 120 gtctcttggt acactgggct tacccaacac gggaaagtcc ctcttacctt tccacctggg 180 cagggtgtac ctcttaatgc caattctacc cctgcgcaaa atgctgggta ttggcggaga 240 caggacagaa aaattaatac cgggaatgga attaagcaac tggctcccag gtggtacttc 300 tactacactg gaactggacc cgaagcagca ctcccattcc gggctgttaa ggatggcatc 360 gtttgggtcc atgaagatgg cgccactgat gctccttcaa cttttgggac gcggaaccct 420 aacaatgatt cagctattgt tacacaattc gcgcccggta ctaagcttcc taaaaacttc 480 cacattgagg ggactggagg caatagtcaa tcatcttcaa gagcctctag cttaagcaga 540 aactcttcca gatctagttc acaaggttca agatcaggaa actctacccg cggcacttct 600 ccaggtccat ctggaatcgg agcagtagga ggtgatctac tttaccttga tcttctgaac 660 agactacaag cccttgagtc tggcaaagta aagcaatcgc agccaaaagt aatcactaag 720 aaagatgctg ctgctgctaa aaataagatg cgccacaagc gcacttccac caaaagtttc 780 aacatggtgc aagcttttgg tcttcgcgga ccaggagacc tccagggaaa ctttggtgat 840 cttcaattga ataaactcgg cactgaggac ccacgttggc cccaaattgc tgagcttgct 900 cctacagcca gtgcttttat gggtatgtcg caatttaaac ttacccatca gaacaatgat 960 gatcatggca accctgtgta cttccttcgg tacagtggag ccattaaact tgacccaaag 1020 aatcccaact acaataagtg gttggagctt cttgagcaaa atattgatgc ctacaaaacc 1080 ttccctaaga aggaaaagaa acaaaaggca ccaaaagaag aatcaacaga ccaaatgtct 1140 gaacctccaa aggagcagcg tgtgcaaggt agcatcactc agcgcactcg cacccgtcca 1200 agtgttcagc ctggtccaat gattgatgtt aacactgatt ag 1242 <210> 2 <211> 413 <212> PRT <213> Artificial Sequence <220> <223> MERS-CoV nucleocapsid protein <400> 2 Met Ala Ser Pro Ala Ala Pro Arg Ala Val Ser Phe Ala Asp Asn Asn   1 5 10 15 Asp Ile Thr Asn Thr Asn Leu Ser Arg Gly Arg Gly Arg Asn Pro Lys              20 25 30 Pro Arg Ala Ala Pro Asn Asn Thr Val Ser Trp Tyr Thr Gly Leu Thr          35 40 45 Gln His Gly Lys Val Pro Leu Thr Phe Pro Pro Gly Gln Gly Val Pro      50 55 60 Leu Asn Ala Asn Ser Thr Pro Ala Gln Asn Ala Gly Tyr Trp Arg Arg  65 70 75 80 Gln Asp Arg Lys Ile Asn Thr Gly Asn Gly Ile Lys Gln Leu Ala Pro                  85 90 95 Arg Trp Tyr Phe Tyr Tyr Thr Gly Thr Gly Pro Glu Ala Ala Leu Pro             100 105 110 Phe Arg Ala Val Lys Asp Gly Ile Val Trp Val His Glu Asp Gly Ala         115 120 125 Thr Asp Ala Pro Ser Thr Phe Gly Thr Arg Asn Pro Asn Asn Asp Ser     130 135 140 Ala Ile Val Thr Gln Phe Ala Pro Gly Thr Lys Leu Pro Lys Asn Phe 145 150 155 160 His Ile Glu Gly Thr Gly Gly Asn Ser Gln Ser Ser Ser Arg Ala Ser                 165 170 175 Ser Leu Ser Arg Asn Ser Ser Arg Ser Ser Ser Gln Gly Ser Arg Ser             180 185 190 Gly Asn Ser Thr Arg Gly Thr Ser Pro Gly Pro Ser Gly Ile Gly Ala         195 200 205 Val Gly Gly Asp Leu Leu Tyr Leu Asp Leu Leu Asn Arg Leu Gln Ala     210 215 220 Leu Glu Ser Gly Lys Val Lys Gln Ser Gln Pro Lys Val Ile Thr Lys 225 230 235 240 Lys Asp Ala Ala Ala Ala Lys Asn Lys Met Arg His Lys Arg Thr Ser                 245 250 255 Thr Lys Ser Phe Asn Met Val Gln Ala Phe Gly Leu Arg Gly Pro Gly             260 265 270 Asp Leu Gln Gly Asn Phe Gly Asp Leu Gln Leu Asn Lys Leu Gly Thr         275 280 285 Glu Asp Pro Arg Trp Pro Gln Ile Ala Glu Leu Ala Pro Thr Ala Ser     290 295 300 Ala Phe Met Gly Met Ser Gln Phe Lys Leu Thr His Gln Asn Asn Asp 305 310 315 320 Asp His Gly Asn Pro Val Tyr Phe Leu Arg Tyr Ser Gly Ala Ile Lys                 325 330 335 Leu Asp Pro Lys Asn Pro Asn Tyr Asn Lys Trp Leu Glu Leu Leu Glu             340 345 350 Gln Asn Ile Asp Ala Tyr Lys Thr Phe Pro Lys Lys Glu Lys Lys Gln         355 360 365 Lys Ala Pro Lys Glu Glu Ser Thr Asp Gln Met Ser Glu Pro Pro Lys     370 375 380 Glu Gln Arg Val Gln Gly Ser Ile Thr Gln Arg Thr Arg Thr Arg Pro 385 390 395 400 Ser Val Gln Pro Gly Pro Met Ile Asp Val Asn Thr Asp                 405 410 <210> 3 <211> 546 <212> DNA <213> Artificial Sequence <220> <223> N-terminus of MERS-CoV nucleocapsid protein <400> 3 atgggcagca gccatcatca ttatcatcac agcagcggcc tggtgccgcg cggcagccat 60 atggcaccaa ataacactgt ctcttggtac actgggctta cccaacacgg gaaagtccct 120 cttacctttc cacctgggca gggtgtacct cttaatgcca attccacccc tgcgcaaaat 180 gctgggtatt ggcggagaca ggacagaaaa attaataccg ggaatggaat taagcaactg 240 gctcccaggt ggtacttcta ctacactgga actggacccg aagcagcact cccattccgg 300 gctgttaagg atggcatcgt ttgggtccat gaagatggcg ccactgatgc tccttcaact 360 tttgggacgc ggaaccctaa caatgattca gctattgtta cacaattcgc tcccggtact 420 aagcttccta aaaacttcca cattgagggg actggaggca atagtggatc cgaattcgag 480 ctccgtcgac aagcttgcgg ccgcactcga gcaccaccac caccaccact gagatccggc 540 tgctaa 546 <210> 4 <211> 181 <212> PRT <213> Artificial Sequence <220> <223> N-terminus of MERS-CoV nucleocapsid protein <400> 4 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro   1 5 10 15 Arg Gly Ser His Met Ala Pro Asn Asn Thr Val Ser Trp Tyr Thr Gly              20 25 30 Leu Thr Gln His Gly Lys Val Pro Leu Thr Phe Pro Pro Gly Gln Gly          35 40 45 Val Pro Leu Asn Ala Asn Ser Thr Pro Ala Gln Asn Ala Gly Tyr Trp      50 55 60 Arg Arg Gln Asp Arg Lys Ile Asn Thr Gly Asn Gly Ile Lys Gln Leu  65 70 75 80 Ala Pro Arg Trp Tyr Phe Tyr Tyr Thr Gly Thr Gly Pro Glu Ala Ala                  85 90 95 Leu Pro Phe Arg Ala Val Lys Asp Gly Ile Val Trp Val His Glu Asp             100 105 110 Gly Ala Thr Asp Ala Pro Ser Thr Phe Gly Thr Arg Asn Pro Asn Asn         115 120 125 Asp Ser Ala Ile Val Thr Gln Phe Ala Pro Gly Thr Lys Leu Pro Lys     130 135 140 Asn Phe His Ile Glu Gly Thr Gly Gly Asn Ser Gly Ser Glu Phe Glu 145 150 155 160 Leu Arg Arg Gln Ala Cys Gly Arg Thr Arg Ala Pro Pro Pro Pro                 165 170 175 Leu Arg Ser Gly Cys             180 <210> 5 <211> 462 <212> DNA <213> Artificial Sequence <220> <223> C-terminus of MERS-CoV nucleocapsid protein <400> 5 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccag 60 atggctagca tgactggtgg acagcaaatg ggtcgcggat ccgaattcgc taaaaataag 120 atgcgccaca agcgcacttc caccaaaagt ttcaacatgg tgcaagcttt tggtcttcgc 180 ggaccaggag acctccaggg aaactttggt gatcttcaat tgaataaact cggcactgag 240 gacccacgtt ggccccaaat tgctgagctt gctcctacag ccagtgcttt tatgggtatg 300 tcgcaattta aacttaccca tcagaacaat gatgatcatg gcaaccctgt gtacttcctt 360 cggtacagtg gagccattaa acttgaccca aagaatccca actacaataa gtggttggag 420 cttcttgagc aaaatattga tgcctacaaa accttccctt ga 462 <210> 6 <211> 153 <212> PRT <213> Artificial Sequence <220> <223> C-terminus of MERS-CoV nucleocapsid protein <400> 6 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro   1 5 10 15 Arg Gly Ser Gln Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg              20 25 30 Gly Ser Glu Phe Ala Lys Asn Lys Met Arg His Lys Arg Thr Ser Thr          35 40 45 Lys Ser Phe Asn Met Val Gln Ala Phe Gly Leu Arg Gly Pro Gly Asp      50 55 60 Leu Gln Gly Asn Phe Gly Asp Leu Gln Leu Asn Lys Leu Gly Thr Glu  65 70 75 80 Asp Pro Arg Trp Pro Gln Ile Ala Glu Leu Ala Pro Thr Ala Ser Ala                  85 90 95 Phe Met Gly Met Ser Gln Phe Lys Leu Thr His Gln Asn Asn Asp Asp             100 105 110 His Gly Asn Pro Val Tyr Phe Leu Arg Tyr Ser Gly Ala Ile Lys Leu         115 120 125 Asp Pro Lys Asn Pro Asn Tyr Asn Lys Trp Leu Glu Leu Leu Glu Gln     130 135 140 Asn Ile Asp Ala Tyr Lys Thr Phe Pro 145 150 <210> 7 <211> 780 <212> DNA <213> Artificial Sequence <220> <223> NC fusion protein of recombination MERS-CoV nucleocapsid protein <400> 7 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60 atggcaccaa ataacactgt ctcttggtac actgggctta cccaacacgg gaaagtccct 120 cttacctttc cacctgggca gggtgtacct cttaatgcca attccacccc tgcgcaaaat 180 gctgggtatt ggcggagaca ggacagaaaa attaataccg ggaatggaat taagcaactg 240 gctcccaggt ggtacttcta ctacactgga actggacccg aagcagcact cccattccgg 300 gctgttaagg atggcatcgt ttgggtccat gaagatggcg ccactgatgc tccttcaact 360 tttgggacgc ggaaccctaa caatgattca gctattgtta cacaattcgc tcccggtact 420 aagcttccta aaaacttcca cattgagggg actggaggca atagtggatc cgaattcgct 480 aaaaataaga tgcgccacaa gcgcacttcc accaaaagtt tcaacatggt gcaagctttt 540 ggtcttcgcg gaccaggaga cctccaggga aactttggtg atcttcaatt gaataaactc 600 ggcactgagg acccacgttg gccccaaatt gctgagcttg ctcctacagc cagtgctttt 660 atgggtatgt cgcaatttaa acttacccat cagaacaatg atgatcatgg caaccctgtg 720 tacttccttc ggtacagtgg agccattaaa cttgacccaa agaatcccaa ctacaataag 780                                                                          780 <210> 8 <211> 276 <212> PRT <213> Artificial Sequence <220> <223> NC fusion protein of recombination MERS-CoV nucleocapsid protein <400> 8 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro   1 5 10 15 Arg Gly Ser His Met Ala Pro Asn Asn Thr Val Ser Trp Tyr Thr Gly              20 25 30 Leu Thr Gln His Gly Lys Val Pro Leu Thr Phe Pro Pro Gly Gln Gly          35 40 45 Val Pro Leu Asn Ala Asn Ser Thr Pro Ala Gln Asn Ala Gly Tyr Trp      50 55 60 Arg Arg Gln Asp Arg Lys Ile Asn Thr Gly Asn Gly Ile Lys Gln Leu  65 70 75 80 Ala Pro Arg Trp Tyr Phe Tyr Tyr Thr Gly Thr Gly Pro Glu Ala Ala                  85 90 95 Leu Pro Phe Arg Ala Val Lys Asp Gly Ile Val Trp Val His Glu Asp             100 105 110 Gly Ala Thr Asp Ala Pro Ser Thr Phe Gly Thr Arg Asn Pro Asn Asn         115 120 125 Asp Ser Ala Ile Val Thr Gln Phe Ala Pro Gly Thr Lys Leu Pro Lys     130 135 140 Asn Phe His Ile Glu Gly Thr Gly Gly Asn Ser Gly Ser Glu Phe Ala 145 150 155 160 Lys Asn Lys Met Arg His Lys Arg Thr Ser Thr Lys Ser Phe Asn Met                 165 170 175 Val Gln Ala Phe Gly Leu Arg Gly Pro Gly Asp Leu Gln Gly Asn Phe             180 185 190 Gly Asp Leu Gln Leu Asn Lys Leu Gly Thr Glu Asp Pro Arg Trp Pro         195 200 205 Gln Ile Ala Glu Leu Ala Pro Thr Ala Ser Ala Phe Met Gly Met Ser     210 215 220 Gln Phe Lys Leu Thr His Gln Asn Asn Asp Asp His Gly Asn Pro Val 225 230 235 240 Tyr Phe Leu Arg Tyr Ser Gly Ala Ile Lys Leu Asp Pro Lys Asn Pro                 245 250 255 Asn Tyr Asn Lys Trp Leu Glu Leu Leu Glu Gln Asn Ile Asp Ala Tyr             260 265 270 Lys Thr Phe Pro         275

Claims (15)

(a) the detection of the nucleocapsid fusion protein comprising the N-terminal domain fragment and the C-terminal domain fragment of the nucleocapsid protein of MERS coronavirus as the antigen for detection, and the sample to be fixed to the solid phase support Reacting with a dragon antigen;
(b) reacting the monoclonal antibody specifically binding to the nucleocapsid fusion protein and conjugated with the detection label with the detection antigen immobilized on a solid phase support; And
(c) measuring the level of the detection label of the monoclonal antibody that antigen-antibodies to the detection antigen
To include, wherein the nucleocapsid fusion protein, characterized in that having the amino acid sequence of SEQ ID NO: 8, mers coronavirus detection method.
delete delete delete delete delete According to claim 1, wherein the nucleocapsid fusion protein is characterized in that the encoded by the nucleotide sequence of SEQ ID NO: 7, Mers coronavirus detection method.
The method according to claim 1, wherein the detection label is Horseradish peroxidase, Mers coronavirus detection method.
The method according to claim 1, wherein the detection method is characterized by using a blocking enzyme immunoassay (Blocking ELISA) method, mers coronavirus detection method.
Nucleocapsid fusion proteins including N-terminal domain fragments and C-terminal domain fragments of the nucleocapsid protein of MERS coronavirus; And
Monoclonal antibody that specifically binds to the nucleocapsid fusion protein and is conjugated with a detection label
Includes, wherein the nucleocapsid fusion protein is characterized in that having an amino acid sequence of SEQ ID NO: 8, mers coronavirus detection kit for use in blocking enzyme-linked immunoassay method.
delete delete delete delete The kit for mers coronavirus detection according to claim 10, wherein the monoclonal antibody is conjugated with horseradish peroxidase as a detection label.

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