KR20190044006A - An antibody against MERS-CoV and method of determining titration for antibody to MERS-CoV using the same - Google Patents

Abstract

The present invention relates to a recombinant spike protein antigen of a Middle East respiratory syndrome coronavirus (MERS-CoV). More specifically, the present invention relates to a recombinant expression vector comprising a receptor binding domain (RBD) which is a part of a spike (S) protein of the MERS-CoV, a recombinant baculovirus produced by co-transfecting the recombinant expression vector with a baculovirus DNA, a recombinant spike protein fragment (RBD) and a monoclonal antibody which are obtained by transfecting a host cell with the recombinant baculovirus, and a method for producing the same. In addition, the present invention relates to an immunologic technique-based technology for measuring an antibody titer of a virus by using a recombinant MERS-CoV S-RBD protein.

Description

The present invention relates to an antibody against coronavirus of the Middle East Respiratory Syndrome and to a method for measuring antibody titer using the same, and more particularly, to an antibody against MERS-CoV using MERS-

The present invention relates to an antibody against the coronavirus of the respiratory syndrome of the Middle East, and a method for measuring the antibody titer using the antibody.

The Middle East Respiratory Syndrome Coronavirus (MERS-CoV) is the first virus found in the Middle East region of Saudi Arabia in 2012. It is a member of the Coroviridae family and is a member of the Coronavirus (SARS-CoV) Respiratory syndrome coronavirus).

As with SARS-CoV, MERS-CoV has a one-week latency period, and it has respiratory symptoms such as coughing, dyspnea, shortness of breath and sputum with high fever as well as headache, chills, runny nose and muscle aches, Symptoms of digestive problems such as vomiting, abdominal pain and diarrhea can also occur. However, unlike SARS-CoV, MERS-CoV is accompanied by acute renal failure, and the mortality rate is more than six times higher than that of SARS. To date, antiviral agents for the treatment of MERS-CoV have not been developed yet, and the treatment of the symptoms is predominant. In severe cases, respiratory or artificial hemodialysis may be required.

Although there is no clear infectious source and infection route of MERS-CoV, it has been reported that it is highly likely to be infected through contact with camel in the Middle East, and it is possible to spread by close contact between humans. In some cases, the virus detected in the infected person in the Middle East and the virus detected in the camel in which the infected person was raised may coincide. However, prevention is not easy because of the custom of eating camel meat or drinking milk in the Middle East.

Although the development of vaccines against MERS-CoV has been promoted so far, quantitative antibody titers after vaccination have not been established. Therefore, there is a need for a sophisticated, standardized, and efficient evaluation system that can confirm the antibody production of vaccine candidates against MERS-CoV.

In order to solve the above problems, it is an object of the present invention to provide a binding molecule that is specific for the Middle East Respiratory Syndrome Coronavirus (MERS-CoV), and the binding molecule is an antibody or a binding fragment thereof.

It is an object of the present invention to provide a method for determining the antigen of MERS-CoV (Middle Respiratory Syncytial Virus) (MERS-CoV) for determining antibody titer on a sample as a binding molecule and a plate to which the method is applied.

Another object of the present invention is to provide a diagnostic kit for determining the antigen of the Middle Respiratory Syncytial Coronavirus (MERS-CoV) comprising the binding molecule.

It is yet another object of the present invention to provide a method for determining the antigen of the Middle Respiratory Syncytial Coronavirus (MERS-CoV) on a sample of interest as the binding molecule.

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

Hereinafter, various embodiments described herein will be described with reference to the drawings. In the following description, for purposes of complete understanding of the present invention, various specific details are set forth, such as specific forms, compositions and processes, and the like. However, certain embodiments may be practiced without one or more of these specific details, or with other known methods and forms. In other instances, well-known processes and techniques of manufacture are not described in any detail, in order not to unnecessarily obscure the present invention. Reference throughout this specification to " one embodiment " or " embodiment " means that a particular feature, form, composition, or characteristic described in connection with the embodiment is included in at least one embodiment of the invention. Accordingly, the appearances of the phrase " in one embodiment " or " an embodiment " in various places throughout this specification are not necessarily indicative of the same embodiment of the present invention. In addition, a particular feature, form, composition, or characteristic may be combined in any suitable manner in one or more embodiments.

Unless defined otherwise within the present invention, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

In one embodiment of the present invention, the binding molecule is a binding molecule specific for the middle respiratory syndrome coronavirus (MERS-CoV) comprising the amino acid sequence of SEQ ID NO: 1, wherein the binding molecule is an antibody or a binding fragment thereof, The binding molecule has the CDR1 of the heavy chain of the variable region represented by SEQ ID NO: 2, the CDR2 of the heavy chain of the variable region is represented by SEQ ID NO: 3, the CDR3 of the heavy chain of the variable region is the binding molecule of SEQ ID NO: 4, The molecule is represented by SEQ ID NO: 5 in the light chain of the variable region, the CDR2 of the light chain of the variable region is represented by SEQ ID NO: 6, and the CDR3 of the variable region light chain is represented by SEQ ID NO:

In one embodiment of the invention, there is provided a method comprising determining an antigen of a Middle Respiratory Syncytial Coronavirus (MERS-CoV) to determine antibody titer on a desired sample with the binding molecule, wherein the determination of the method 1 is a method for determining the amount of antigen to be applied, and the antigen of the above-mentioned Middle East Respiratory Syndrome Coronavirus (MERS-CoV) provides a method represented by SEQ ID NO: 1.

In one embodiment of the present invention, there is provided a plate for determining the antigen of the Middle Respiratory Syncytial Coronavirus (MERS-CoV) for determining antibody titer on a sample of interest with the binding molecule, wherein the plate comprises one or more wells (MERS-CoV) is a plate coated with an antigen of Middle East Respiratory Syndrome coronavirus at 25 to 500 ng / well per well, and the antigen of the above-mentioned Middle Respiratory Syndrome Coronavirus (MERS-CoV) to provide.

In one embodiment of the invention, there is provided a diagnostic kit for determining the antigen of a Middle Respiratory Syncytial Coronavirus (MERS-CoV) comprising said binding molecule. According to another embodiment of the present invention, the diagnostic kit can be used to quantify the antibody titer against MERS-CoV using a sample as the reference material after contacting the protein with the antibody or chimeric antibody.

In one embodiment of the invention, there is provided a method for determining the antigen of the Middle Respiratory Syncytial Coronavirus (MERS-CoV) on a sample of interest with the binding molecule.

In one embodiment of the present invention, a diagnostic kit for determining an antibody comprising the antigen of the Middle Respiratory Syncytial Coronavirus (MERS-CoV) as shown in SEQ ID NO: 1 or the Middle Respiratory Syndrome coronavirus (MERS Lt; RTI ID = 0.0 > (CoV). ≪ / RTI >

In the present specification, the term "antibody titer" refers to a value obtained by measuring the amount of an antibody contained in a serum. For example, the antibody titer is low before being exposed to a germ having a specific antigen. However, After antibody production, antibody titer is measured high.

As used herein, " variable region CDR " is determined in a conventional manner according to a system designed by Kabat et al. (Kabat et al., Sequences of Proteins of Immunological Interest (5th), National Institutes of Health, Bethesda, MD (1991)). Although the CDR crystal used in the present invention uses the Kabat method, a binding molecule comprising a CDR determined according to other methods such as the IMGT method, Chothia method, AbM method and the like is also included in the present invention.

As used herein, " binding molecule " may be an antibody or fragment thereof. In addition, the binding molecule may be, but is not limited to, a Fab fragment, an Fv fragment, a diabody, a chimeric antibody, a humanized antibody, or a human antibody. In one embodiment of the invention, there is provided a fully human antibody that binds to an antigen. As used herein, antibodies are used in their broadest sense and specifically include multispecific antibodies (e. G., Bispecific antibodies) formed from intact monoclonal antibodies, polyclonal antibodies, two or more intact antibodies, Lt; RTI ID = 0.0 > biologically < / RTI > Antibodies are proteins produced by the immune system that can recognize and bind specific antigens. In its structural aspect, the antibody typically has a Y-shaped protein consisting of four amino acid chains (heavy chain of two variable regions and light chain of two variable regions). Each antibody has two regions, mainly a variable region and a constant region. The variable region located at the distal end of the arm of Y binds and interacts with the target antigen. The variable region comprises a complementarity determining region (CDR) that recognizes and binds to a specific binding site on a specific antigen. The constant region in the tail of Y is recognized and interacted by the immune system. Target antigens typically have multiple binding sites called epitopes, which are recognized by CDRs on multiple antibodies. Each antibody that specifically binds to a different epitope has a different structure. Thus, an antigen can have one or more corresponding antibodies. The invention also encompasses functional variants of the antibody. Antibodies are considered functional variants of the antibodies of the invention if the variants can compete with the antibodies of the invention for specific binding to the antigen or its G protein. Functional variants include, but are not limited to, derivatives that are substantially similar in primary structural sequence, such as in-vitro or in-vivo modifications, chemical and / or biochemical agents , Which are not found in the parental monoclonal antibodies of the present invention. Such modifications include, for example, acetylation, acylation, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, crosslinking, disulfide bond formation, glycosylation, hydroxylation, methylation, oxidation, And phosphorylation. A functional variant may optionally be an antibody comprising an amino acid sequence that contains substitutions, insertions, deletions, or combinations of one or more amino acids compared to the amino acid sequence of the parent antibody. Moreover, functional variants may comprise truncated forms of the amino acid sequence at one or both of the amino terminus or the carboxy terminus. The functional variants of the present invention may have the same, different, higher or lower binding affinity as the parent antibody of the invention, but still bind to the antigen or its G protein. In one example, the amino acid sequence of the variable region, including but not limited to the framework structure, the hypervariable region, particularly the CDR3 region, can be modified. Generally, the heavy chain region of the light chain or variable region of the variable region comprises three hypervariable regions and more conserved regions, i.e., the framework region (FR), which comprises three CDR regions. The hypervariable region comprises an amino acid residue from the CDR and an amino acid residue from the hypervariable loop. Functional variants falling within the scope of the present invention are those that comprise about 50% to about 99%, about 60% to about 99%, about 80% to about 99%, about 90% to about 99% Or about 97% to 99% amino acid sequence homology. Gap or Bestfit known to those skilled in the art of computer algorithms may be used to optimally align the amino acid sequences to be compared and to define similar or identical amino acid residues. Functional variants can be obtained by, but not limited to, altering the parent antibody or a portion thereof by known general molecular biology methods, including PCR methods, mutagenesis using oligonucleotides and partial mutagenesis, or by organic synthesis.

In the present specification, " MERS-CoV " is a kind of beta-coronavirus among coronaviruses, and has a gene length of about 30 kb and 11 open reading frames (ORFs). The structural proteins of the Middle East Respiratory Syndrome coronavirus (MERS-CoV) are S (Spike), E (Envelope), M (Matrix) and NP (Nucleocapsid) proteins. The S (Spike) protein of MERS-CoV is a class I membrane fusion protein corresponding to the major E (envelope) protein on the CoV surface. The S protein consists of an N-terminal S1 domain containing the receptor binding domain (RBD) and an S2 domain responsible for virus-cell fusion. The receptors for MERS-CoV were identified as dipeptidyl peptidase 3 (DPP4, CD26) and the MERS-CoV RBD consists of the core domain co-crystallized with human DPP4 protein and interacts with blades 4 and 5 of DPP4. That is, the MERS-RBD sequence of SEQ ID NO: 1 contains RBD and contains the 358th-606th sequence of the MERS-CoV S protein.

One embodiment of the invention comprises a receptor binding domain (RBD) region of the spike (S) protein of MERS-CoV, wherein the N-terminus of the receptor binding domain region comprises a signal peptide sequence for insect cell gp67 and a C- Lt; RTI ID = 0.0 > MERS-CoV < / RTI > S1-RBD with amino acids containing several histidines. The recombinant MERS-CoV S1-RBD contains amino acid residues 358 to 606 of the entire spike (S) protein sequence, and the N- The signal peptide sequence for insect cell gp67 is connected to the end, and several histidine sequences are contained at the C-terminus.

In one embodiment of the present invention, the polymorphic promoter of baculovirus is one of the powerful promoters that operate in insect cells, regulating the expression of a polymorphic gene, one of the genes expressing baculovirus. The signal peptide located downstream of the promoter allows the protein encoded by the gene of the foreign protein additionally inserted into the vector to be expressed to a large extent outside the host cell. When a large amount of the protein is expressed outside the cell, the separation and purification of the protein can be carried out using a culture solution of the host cell at a high yield and as a fixing agent. The sequence encoding the signal peptide is a gp67 signal peptide sequence, and the signal peptide is cleaved during the process of expressing a large amount of the protein outside the cell, and several amino acids may be included in the process. This is an additional sequence commonly found in recombinant proteins made using expression vectors containing signal peptides, including three N-terminal amino acids of SEQ ID NO: 1 of the present invention.

One embodiment of the present invention binds several histines to the C-terminus of MERS-CoV S1-RBD for easy separation and purification of recombinant MERS-CoV S1-RBD. The reason for binding histidine is not only that the binding process through gene cloning is simple, but also because the size of histidine is small, it does not affect the reaction of MERS-CoV S1-RBD with antibodies in serum and also causes interaction with other proteins This is because the possibility is low. In one embodiment of the present invention, a gene is extended using 6 histidine primers to prepare a recombinant MERS-CoV S1-RBD having an amino acid sequence comprising 6 histines at the C-terminal. The recombinant MERS-CoV S1-RBD may consist of the amino acid sequence shown in SEQ ID NO: 1, but is not limited thereto.

One embodiment of the present invention relates to a method for measuring the titer of a virus antibody in serum bound to the MERS-CoV S1-RBD, comprising the step of coating a microplate with a MERS-CoV S1-RBD recombinant protein. Antibody titers can be quantified using a chimeric antibody made by combining a variable region of a monoclonal antibody produced using a recombinant MERS-CoV S1-RBD antigen protein with a human immunoglobulin constant region as a reference material. In the present invention, the amino acid sequences represented by SEQ ID NOS: 2, 3 and 4 are heavy chain variable regions CDR1, CDR2 and CDR3 of the variable region of the monoclonal antibody, and the amino acid sequences represented by SEQ ID NOS: 5, 6 and 7 are monoclonal antibodies The light chain variable regions CDR1, CDR2, and CDR3 of the variable region. The amino acid sequence of SEQ ID NO: 8 is the entire heavy chain variable region of the variable region of the monoclonal antibody and the amino acid sequence of SEQ ID NO: 9 is the entire light chain variable region of the variable region of the monoclonal antibody.

According to the present invention, the recombinant MERS-CoV S1-RBD protein is overexpressed as a water-soluble protein in recombinant baculovirus, making it possible to easily purify it with high purity. The recombinant MERS-CoV S1-RBD protein antigen thus obtained can be used to construct indirect enzyme immunoassay which can measure viral antibody titer in serum in a simple and rapid manner.

According to the present invention, an indirect enzyme immunoassay method capable of quantitatively measuring the titer of a virus antibody in serum can be constructed using a chimeric antibody that binds to the MERS-CoV S1-RBD protein.

In addition, since the antibody titer against MERS virus in the serum can be quantitatively measured, it provides a quantitative test method for verifying the efficacy of the MERS-CoV vaccine candidate substance, and can also rapidly measure the antibody titer of the vaccine at the time of vaccination Can be used as a diagnostic kit.

Figure 1 relates to the pFastBac-MERS S1 RBD vector for the production of the recombinant baculovirus of the present invention.
Figure 2 (a) shows the results of 10% SDS-PAGE analysis of the expression of the MERS recombinant S1-RBD protein of the present invention.
FIG. 2 (b) shows the results of 10% SDS-PAGE after purification of the MERS recombinant S1-RBD protein of the present invention.
Figure 3 (a) shows the sensitivity test results of monoclonal antibodies produced using the MERS recombinant S1-RBD antigen protein of the present invention.
FIG. 3 (b) shows cross-sectional results of monoclonal antibodies produced using the MERS recombinant S1-RBD antigen protein of the present invention.
FIG. 3 (c) shows the isotype test results of the monoclonal antibody produced using the MERS recombinant S1-RBD antigen protein of the present invention.
4 (a) shows the binding difference between the MERS recombinant S1-RBD antigen protein of the present invention and the MERS recombinant S1 (1 to 751) protein of the full-length sequence.
FIG. 4 (b) shows the result of setting the antigen application amount of the MERS recombinant S1-RBD protein of the present invention.
FIG. 5 shows MERS antibody titer measurements of sera from mice immunized with MERS S1 antigen and negative control / positive control of indirect enzyme immunoassay using the MERS recombinant S1-RBD protein of the present invention.
FIG. 6 shows the results of confirming the linearity of the monoclonal antibody of FIG. 3 in the indirect enzyme immunoassay using the MERS recombinant S1-RBD protein of the present invention.

Hereinafter, the present invention will be described in more detail with reference to Examples. It will be apparent to those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

[Example 1] Preparation of recombinant MERS-CoV S1-RBD protein expression vector

Genes at positions corresponding to the nucleotide sequences 1072 to 1818 containing the receptor binding domain (RBD) of the MERS-CoV EMC / 2012 (Gene Bank No. JX869059) gene sequence were obtained from Macrogen (Seoul, Korea). The gene was amplified by applying pfu DNA polymerase as a template. The forward primer of the S1-RBD gene is 5 'CAG GAT CCC TCT GGA GTC TAC TCT GTG TCC TCC T' 3 and the reverse primer is 5 'GGT GAT GGT GAT GAT GGT ATT CCA CAC AGT TGC C' 3. The gene for the above primers was amplified using a gene amplifier and the conditions were as follows. That is, denaturation was carried out at 94 캜 for 5 minutes, and then repeated 25 times with the basic cycles of 94 캜 for 30 seconds, 60 캜 for 30 seconds, and 72 캜 for 50 seconds. And finally extended at 72 ° C for 10 minutes. The forward primer of the gene is 5'CAG GAT CCC TCT GGA GTC TAC TCT GTG TCC TCC T'3 and the reverse primer is 5'GAC TCG AGG GCG GCC GCT The gene was amplified using CAG TGG TGA TG'3. The amplified gene was cloned into a pFastBac vector (Invitrogen), a baculovirus expression vector to which a cysteine peptide of gp67 was bound, to prepare a recombinant baculovirus vector. The schematic diagram of the recombinant baculovirus expression vector is shown in FIG. 1 and named pFastBac / MERS-S1-RBD. The thus constructed expression vector was transformed into E. coli DH10Bac cells containing baculovirus gene bacmid DNA. The recombinant MERS-CoV S1-RBD protein expressed by the recombinant expression vector pFastBac / MERS-S1-RBD is represented by the amino acid sequence of SEQ ID NO: 1.

[Example 2] Preparation and expression of recombinant baculovirus

E. coli DH10Bac (Invitrogen, USA) containing DNA (BacON, bMON14272) of AcNPV, a kind of baculovirus, was used for this example. The bacmid has the lacZ gene and a transposition site in the gene. On the other hand, in the movement vector pFastBac used in Example 1, there is a transposon, and the MERS-CoV S1-RBD gene to be expressed therebetween is inserted. The pBastBac / MERS-S1-RBD prepared in Example 1 was transformed into E. coli DH10Bac to induce the translocation of the backmade and pFastBac / MERS-S1-RBD in E. coli, and then the antibiotic of gentamycin, tetracycline and kanamycin And LB plates containing X-gal and IPTG, and only colorless colonies were selected. Then, the colorless colonies were cultured in a test tube containing LB medium for 24 hours, and then recombinant Bacmid was isolated using plasmid extraction method. The presence or absence of the MERS-CoV S1-RBD gene in the isolated recombinant backmids was confirmed by PCR.

On the other hand, 1 × 10 ⁶ insect cells Sf9 (Invitrogen, USA) were fixedly cultured in a 60 mm culture dish for 1 hour under the condition of 27 ° C. culture. Then, the medium was removed and the recombinant backmade and cell pectin (Invitrogen, USA) was placed in the culture dish with Sf-900 II SFM (GIBCO, USA) medium. After 5 hours, the medium in the culture dish was removed and fresh 2 ml of Sf-900 II SFM medium was added and cultured for 72 hours. Next, 2 ml of the medium in which the insect cells were grown was re-infected with recombinant baculovirus in growing Sf9 cells in a flask containing 20 ml of Sf-900 II SFM medium to increase the virus concentration. After culturing for 72 hours, 20 ml of the amplified recombinant baculovirus was recovered in a flask, centrifuged (1000 rpm, 5 minutes), and the supernatant was recovered again in a new 50 ml tube and refrigerated.

Since the protein secreted by the cell is known to be more expressed in High Five cells than in Sf9 cells (Biotech. Bioengin., 63: 255-262 (1999)), 100 ml of Sf-900 II SFM medium The cultured Hi-Five cells (Invitrogen, USA) were transfected with the recombinant baculovirus obtained in Example 2 and cultured for 72 hours to induce protein expression. 200 ml of the Hi-Five cell culture, which had been cultured for 3 days through the above-described virus infection step, was centrifuged at 4 ° C (4000 rpm, 7 minutes).

[Example 3] Purification of recombinant MERS-CoV S1-RBD protein

The Hi-Five cells of Example 2 were cultured for 72 hours, and then 200 ml of the cell culture medium was harvested. Then, recombinant MERS-CoV S1-RBD secreted extracellularly was purified as follows.

To concentrate the cell culture, 150 g of a magnetic rod and ammonium sulfate were added to a beaker containing the supernatant, and the mixture was stirred for 30 minutes using a s magnetic stirrer. When the ammonium sulfate reached saturation, which was no longer soluble, the supernatant was centrifuged at 4 ° C (4000 rpm, 7 minutes). The centrifuged supernatant was collected in a 500 ml beaker, and the protein precipitated on the centrifuge bottle wall was dissolved in 30 ml buffer solution and placed in a 50 ml tube. The supernatant collected by centrifugation was centrifuged (4000 rpm, 7 min) at 4 ° C for one more time, and the supernatant was discarded. The protein precipitated in the centrifuged bottle was re-dissolved in the previously dissolved buffer solution, tube.

Then, 2 ml of the nickel resin was flowed through 10 ml of the buffer solution to activate the resin. The enriched cell culture fluid in the activated resin was poured over the resin wall. When the concentrated cell culture solution spread evenly throughout the resin, the non-specific protein was removed by washing twice with 10 ml of buffer solution. To separate the recombinant MERS-CoV S1-RBD protein bound to the nickel resin, the eluate was sequentially harvested in 1 ml portions while inserting the buffer solution into the column. In order to confirm the color of the purified product, Bradford protein assay (Bio-Rad, USA) was used to confirm the color. The blue-colored tablets were analyzed by SDS-PAGE for protein presence. FIG. 2 (a) shows the result of sequential SDS-PAGE of the tablets harvested in the above order.

Only the purified product exhibiting recombinant MERS-CoV S1-RBD protein on SDS-PAGE was dialyzed overnight at 4 < 0 > C. The dialyzed recombinant MERS-CoV S1-RBD protein was concentrated using Centricon (Amicon, USA) and the protein concentration was quantitated using EPOCH (BioTek, USA) instrument.

FIG. 2 (b) shows the SDS-PAGE results of loading the purified recombinant MERS-CoV S1-RBD protein in the final column. Thus, it can be confirmed that the recombinant MERS-CoV S1-RBD protein of the present invention can be purified through a simple purification process.

[Example 4] Production of recombinant MERS-CoV S1-RBD monoclonal antibody

The recombinant MERS-CoV S1-RBD antigen protein purified in Example 3 was immunized with BALB / c mouse through the sole or abdomen injection, and blood was taken from the mouse eyes to select an immunized mouse, .

Antibody production was carried out using the conventional method of Kohler and Milstein, and cell fusion was carried out. Specific methods are as follows:

B lymphocytes were isolated from well-immunized BALB / c mice by harvesting spleen or lymph node, and myeloma cells and myeloma cells, which were cancer cells deficient in hypoxanthine guanine phosphoribosyl transferase (HGPRT) B cells were fused with polyethylene glycol (PEG), and the cells participating in the fusion were selected using a culture medium supplemented with hypoxanthine aminopterin thymidine (HAT). Converted MERS-CoV S1-RBD antigen-specific fused cells were selected by enzyme-linked immunosorbent assay (ELISA) using the culture medium of the surviving wells in HAT-supplemented culture. After the selected clones are mass-cultured and hybrid cells treated with DMEM medium not supplemented with PBS or Bovine Serum (FBS) are injected into the abdominal cavity of the mice, if ascites is formed in the mouse abdominal cavity, The purified antibody was purified using Protein G or A beads. The sensitivity, cross reactivity and isotyping of the final screened monoclonal antibody AT2F7 were analyzed (see FIG. 3). Further, the antibody is defined by the following SEQ ID NOS: 1 to 9.

SEQ ID NO: 1 (Antigen) ADPSGVYSVS SFEAKPSGSV VEQAEGVECD FSPLLSGTPP QVYNFKRLVF TNCNYNLTKL

LSLFSVNDFT CSQISPAAIA SNCYSSLILD YFSYPLSMKS DLSVSSAGPI SQFNYKQSFS

NPTCLILATV PHNLTTITKP LKYSYINKCS RLLSDDRTEV PQLVNANQYS PCVSIVPSTV

WEDGDYYRKQ LSPLEGGGWL VASGSTVAMT EQLQMGFGIT VQYGTDTNSV CPKLEFANDT

KIASQLGNCV EYHHHHHH

SEQ ID NO: 2: (VH CDR1): GFTFSSYT

SEQ ID NO: 3: (VH CDR2): ISSGGSYT

SEQ ID NO: 4: (VH CDR3): TRDLYDGYFYYAMDY

SEQ ID NO: 5: (VL CDR1): QSLLNSSNQKNY

SEQ ID NO: 6: (VL CDR2): FAS

SEQ ID NO: 7 (VL CDR3): QQHYSTPWT

SEQ ID NO: 8 (VH full):

EVKLVESGGGLVKPGGSLKLSCAASGFTFSSYTMSWVRQTPEKRLEWVATISSGGSYTYYPDSVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCTRDLYDGYFYYAMDYWGQGTSVTVSS

SEQ ID NO: 9 (VL full):

DIVMTQSPSSLAMSVGQKVTMSCKSSQSLLNSSNQKNYLAWYQQKPGQSPKLLVYFASTKYSGVPDRFIGSGSGTDFTLTISSVLAEDLADYFCQQHYSTPWTFGGGTKLEIK

[Example 5] Setting of antigen application amount of recombinant MERS-CoV S1-RBD protein

The conditions for the antigen application amount were established using the recombinant MERS-CoV S1-RBD protein antigen purified in Example 3 and the monoclonal antibody AT2F7 prepared in Example 4. That is, as compared with the MERS (No. 1 to No. 751) of the full-length sequence, it was possible to confirm a more appropriate binding in the MERS-RBD sequence of SEQ ID NO: 1 (positions 358 to 606) The binding of the antibody and the antigen was confirmed at 25-500 ng / well of the S1-RBD protein antigen (see FIG. 4B). The amount of recombinant MERS-CoV S1-RBD protein antigen in indirect enzyme immunoassay was determined as 100 ng / well.

[Example 6] Measurement of antibody titer of indirect enzyme immunoassay using recombinant MERS S1-RBD protein

Mice immunized and boosted with a vaccine candidate substance (plasmid DNA) expressing the protein of MERS virus were obtained and the serum of the mouse was obtained and the production of the anti-MERS antibody in the serum was measured by the indirect enzyme immunoassay of the present invention Respectively. (See FIG. 5). In addition, as the monoclonal antibody AT2F7 showed linearity according to the concentration, the standard in the indirect enzyme immunoassay system established by the present invention (See FIG. 6). This confirms that quantitative analysis of MERS antibody titers is possible through the production of chimeric antibodies of monoclonal antibody AT2F7.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

<110> Atgen          Industry-Academic Cooperation Foundation, Yonsei University <120> An antibody against MERS-CoV and method of determining titration          for antibody to MERS-CoV using the same <130> DPB173846k01 <150> KR 10-2017-0135754 <151> 2017-10-19 <160> 9 <170> KoPatentin 3.0 <210> 1 <211> 258 <212> PRT <213> Artificial Sequence <220> <223> antigen <400> 1 Ala Asp Pro Ser Gly Val Ser Ser Val Ser Ser Phe Glu Ala Lys Pro   1 5 10 15 Ser Gly Ser Val Val Glu Gln Ala Glu Gly Val Glu Cys Asp Phe Ser              20 25 30 Pro Leu Leu Ser Gly Thr Pro Pro Gln Val Tyr Asn Phe Lys Arg Leu          35 40 45 Val Phe Thr Asn Cys Asn Tyr Asn Leu Thr Lys Leu Leu Ser Leu Phe      50 55 60 Ser Val Asn Asp Phe Thr Cys Ser Gln Ile Ser Pro Ala Ala Ile Ala  65 70 75 80 Ser Asn Cys Tyr Ser Ser Leu Ile Leu Asp Tyr Phe Ser Tyr Pro Leu                  85 90 95 Ser Met Lys Ser Asp Leu Ser Val Ser Ser Ala Gly Pro Ile Ser Gln             100 105 110 Phe Asn Tyr Lys Gln Ser Phe Ser Asn Pro Thr Cys Leu Ile Leu Ala         115 120 125 Thr Val Pro His Asn Leu Thr Thr Ile Thr Lys Pro Leu Lys Tyr Ser     130 135 140 Tyr Ile Asn Lys Cys Ser Arg Leu Leu Ser Asp Asp Arg Thr Glu Val 145 150 155 160 Pro Gln Leu Val Asn Ala Asn Gln Tyr Ser Pro Cys Val Ser Ile Val                 165 170 175 Pro Ser Thr Val Trp Glu Asp Gly Asp Tyr Tyr Arg Lys Gln Leu Ser             180 185 190 Pro Leu Glu Gly Gly Gly Trp Leu Val Ala Ser Gly Ser Thr Val Ala         195 200 205 Met Thr Glu Gln Leu Gln Met Gly Phe Gly Ile Thr Val Gln Tyr Gly     210 215 220 Thr Asp Thr Asn Ser Val Cys Pro Lys Leu Glu Phe Ala Asn Asp Thr 225 230 235 240 Lys Ile Ala Ser Gln Leu Gly Asn Cys Val Glu Tyr His His His                 245 250 255 His His         <210> 2 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> VH CDR1 <400> 2 Gly Phe Thr Phe Ser Ser Tyr Thr   1 5 <210> 3 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> VH CDR2 <400> 3 Ile Ser Ser Gly Ser Ser Tyr Thr   1 5 <210> 4 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> VH CDR3 <400> 4 Thr Arg Asp Leu Tyr Asp Gly Tyr Phe Tyr Tyr Ala Met Asp Tyr   1 5 10 15 <210> 5 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> VL CDR1 <400> 5 Gln Ser Leu Leu Asn Ser Ser Asn Gln Lys Asn Tyr   1 5 10 <210> 6 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> VL CDR2 <400> 6 Phe Ala Ser   One <210> 7 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> VL CDR3 <400> 7 Gln Gln His Tyr Ser Thr Pro Trp Thr   1 5 <210> 8 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> VH full <400> 8 Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly   1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr              20 25 30 Thr Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val          35 40 45 Ala Thr Ile Ser Ser Gly Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Soy      50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr  65 70 75 80 Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys                  85 90 95 Thr Arg Asp Leu Tyr Asp Gly Tyr Phe Tyr Tyr Ala Met Asp Tyr Trp             100 105 110 Gly Gln Gly Thr Ser Val Thr Val Ser Ser         115 120 <210> 9 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> VL full <400> 9 Asp Ile Val Met Thr Gln Ser Ser Ser Leu Ala Met Ser Val Gly   1 5 10 15 Gln Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser              20 25 30 Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln          35 40 45 Ser Pro Lys Leu Leu Val Tyr Phe Ala Ser Thr Lys Tyr Ser Gly Val      50 55 60 Pro Asp Arg Phe Ile Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr  65 70 75 80 Ile Ser Ser Val Leu Ala Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln                  85 90 95 His Tyr Ser Thr Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile             100 105 110 Lys    

Claims (14)

A binding molecule specific for the Middle A respiratory syndrome coronavirus (MERS-CoV) comprising the amino acid sequence of SEQ ID NO: 1, wherein the binding molecule is an antibody or a binding fragment thereof. The method according to claim 1,
CDR1 of the heavy chain of the variable region is represented by SEQ ID NO: 2,
CDR2 of the heavy chain of the variable region is represented by SEQ ID NO: 3,
CDR2 of the heavy chain of the variable region is represented by SEQ ID NO: 4
Binding molecule. 3. The method of claim 2,
CDR1 of the light chain of the variable region is represented by SEQ ID NO: 5,
CDR2 of the light chain of the variable region is represented by SEQ ID NO: 6,
CDR2 of the light chain of the variable region is represented by SEQ ID NO: 7,
Binding molecule. 4. A method comprising determining an antigen of a Middle Respiratory Syncytial Coronavirus (MERS-CoV) to determine antibody titer on a sample of interest as a binding molecule of any one of claims 1 to 3. 5. The method of claim 4,
Wherein the determining step determines the application amount of the antigen. 5. The method of claim 4,
The antigen of the Middle East Respiratory Syndrome coronavirus (MERS-CoV) is represented by SEQ ID NO: 1. A plate for determining the antigen of a Middle Respiratory Syndrome Coronavirus (MERS-CoV) for determining antibody titer on a sample of interest as a binding molecule of any one of claims 1 to 3. 8. The method of claim 7,
Wherein the plate comprises one or more wells. 9. The method of claim 8,
Plates coated with 25-500 ng / well of Middle A respiratory syndrome coronavirus antigen per well. 10. The method of claim 9,
The antigen of the Middle East Respiratory Syndrome coronavirus (MERS-CoV) is the plate shown in SEQ ID NO: 1. A diagnostic kit for determining the antigen of a Middle Respiratory Syncytial Coronavirus (MERS-CoV) comprising a binding molecule according to any one of claims 1 to 3. A method for determining the antigen of a Middle Respiratory Syndrome Coronavirus (MERS-CoV) on a sample of interest as a binding molecule of any one of claims 1 to 3. A diagnostic kit for determining an antibody comprising an antigen of the Middle Respiratory Syncytial Coronavirus (MERS-CoV) as shown in SEQ ID NO: 1. A method for determining an antibody comprising the antigen of the Middle Respiratory Syndrome Coronavirus (MERS-CoV) as shown in SEQ ID NO: 1.

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