KR20190000009A - A method for producing MERS antigen using Drosophila melanogastor S2 - Google Patents

Abstract

The present invention relates to a method for producing a receptor binding domain of MERS virus spike protein (MERS-RBD) as a MERS antigen protein and a method for producing an antibody for the antigen. The method for producing the MERS-RBD antigen comprises the following steps: preparing a transformed cell by introducing an expression vector comprising nucleic acid encoding the MERS-RBD of MERS virus spike protein into a Drosophila melanogaster S2 cell; and culturing the transformed cell in a culture medium.

Description

A method for producing Mers antigen protein using Drosophila melanogaster S2 (Drosophila melanogastor S2)

The present invention relates to a method for producing a receptor binding domain (MERS-RBD) antigen of a mervivirus spike protein as a mers antigen protein, and a method for producing an antibody against said antigen.

Middle East Respiratory Syndrome (MERS) is a disease caused by the infection of MERS-CoV virus (MERS-CoV) with an average latency of about 5 days and is infected by close contact. The main symptoms are similar to those of the cold, with high fever, coughing, shortness of breath, and chronic diseases or immunosuppression, resulting in multiple organ failure. MERS is mainly in Middle East countries such as Saudi Arabia, Lebanon and Jordan, and has been spread to USA and Europe by those who have returned from these countries. In Korea, the first patients were reported in May 2015, and 186 patients died and 38 deaths occurred by January 2016, resulting in a death rate of 20.4%. Therefore, there is a need to develop a technique for diagnosing and treating the infection of Mers-Coronavirus.

The method of producing foreign protein using Drosophila melanogaster S2 cells has a high protein expression level, and the expressed protein is expressed in post-translational modification (glycosylation, phosphorylation, addition of fatty acid) similar to that expressed in animal cells, Proteins can be simultaneously expressed or discharged outside the cells. Thus, it is easy to produce viral envelope proteins and virus-like particles of a complex component, and it is possible to separate and purify and mass-produce them, thus being easily applicable to commercialization. In the past, studies on a method for producing a muss antigen protein using an animal cell expression system or a recombinant baculovirus have been conducted (Vaccine 2014, 32: 5975-5982; Cell Res. 2013, 23: 986-993) Indrosophila melanogaster S2 ( Drosophila melanogastor S2) cells have not been reported to date.

Under these circumstances, the present inventors have made intensive efforts to develop a method capable of producing an antigen of MERS-CoV. As a result, they have found that the receptor binding domain (MERS-RBD) of the MERS- The present inventors completed the present invention by producing a monoclonal antibody that specifically binds to secreted MERS-RBD.

One object of the present invention is to provide an expression vector comprising a nucleic acid encoding the receptor binding domain (MERS-RBD) of the Mers virus spike protein, with a Drosophila melanogaster ) S2 cells to produce transformed cells; And culturing the transfected cells in a culture medium. The present invention also provides a method for producing MERS-RBD antigen.

Another object of the present invention is to provide a method for producing MERS-RBD antibody comprising the step of producing a monoclonal antibody against the prepared MERS-RBD antigen.

Another object of the present invention is to provide a MERS-RBD antigen, a nucleic acid molecule encoding the MERS-RBD antigen, a vector comprising the MERS-RBD antigen, and a transformed cell comprising the vector.

This will be described in detail as follows. On the other hand, each description and embodiment disclosed in the present invention can be applied to each other description and embodiment. That is, all combinations of various elements disclosed in the present invention fall within the scope of the present invention. Further, the scope of the present invention is not limited by the detailed description described below.

One aspect of the present invention for achieving the above object is a method for expressing an expression vector comprising a nucleic acid encoding a receptor binding domain (MERS-RBD) of a Mers virus spike protein with a Drosophila melanogaster ) S2 cells to produce transformed cells; And culturing the transformed cells in a culture medium.

In the present invention, mers virus refers to Middle respiratory syndrome-coronavirus (MERS-CoV). The clinical characteristics of a mers virus infection in humans vary from asymptomatic infection to very severe pneumonia and can lead to death from acute respiratory distress syndrome, septic shock, and multi-organ dysfunction. Therefore, it is necessary to use the vaccine for the respiratory syndrome of the Middle East, or the antigen production technique for the mers virus to produce the antibody.

Mers virus shares similarity with bat corona viruses HKU4 and HKU5. The virus uses spike proteins to interact with cell receptors for entry into target cells. In particular, Mers virus can bind dipeptidyl peptidase 4 (DPP4) in human epithelium and endothelial cells via the receptor binding domain of the spike protein, and the receptor binding domain is a receptor binding subdomain that interacts with DPP4 And a core. Accordingly, one embodiment of the present invention is a method for producing the receptor binding domain (MERS-RBD) of the Mers virus spike protein as an antigen.

In the present invention, the spike protein of the Mervis virus may be, for example, but is not limited to, NCBI accession number AKL59401. The MERS-RBD may be composed of the 377-588th amino acid of the Mers virus spike protein, and the nucleic acid encoding the MERS-RBD may be the nucleotide sequence of SEQ ID NO: 2, but is not limited thereto. In a specific embodiment of the present invention, Drosophila < RTI ID = 0.0 > MBS-RBD wild-type sequence (SEQ ID NO: 1) and 25.9% sequence identity (SEQ ID NO: 2) were obtained in order to express MERS-RBD antigen in melanogaster S2 cells.

The method for producing MERS-RBD antigen of the present invention comprises the step of producing transformed cells expressing MERS-RBD antigen and the step of producing MERS-RBD antigen from the transformed cells. Hereinafter, each step will be described in detail.

First, the step of preparing a transformant cell expressing the MERS-RBD antigen comprises the step of introducing an expression vector containing a nucleic acid encoding MERS-RBD into Drosophila melanogaster S2 cells. Drosophila melanogaster S2 cells have a high expression level of exogenous proteins, and proteins expressed in these cells undergo a post-translational modification (glycosylation, phosphorylation, addition of fatty acid, etc.) similar to the original protein. In addition, since the expressed protein can be easily released from the cell, it is very easy to mass-produce and separate and purify an exogenous protein, which is advantageous for mass production of useful foreign protein. However, there has been no report on the possibility of expressing MERS-RBD in Drosophila melanogaster S2 cells.

As used herein, the term " vector " refers to an artificial DNA molecule having a genetic material that is capable of expressing a gene of interest in a suitable host, and specifically includes a DNA construct containing a nucleotide sequence operably linked to a suitable regulatory sequence it means. The regulatory sequence may include any operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence regulating the termination of transcription and translation, in addition to the promoter of the invention capable of initiating transcription But is not limited thereto.

The vector used in the present invention is not particularly limited as long as it is replicable in the host cell, and any vector known in the art can be used. Examples of commonly used vectors include plasmids, cosmids, viruses and bacteriophages in their natural or recombinant state. For example, pWE15, M13, λBL3, λBL4, λIXII, λASHII, λAPII, λt10, λt11, Charon4A, and Charon21A can be used as a phage vector or cosmid vector, and pMT, pBR, , pBluescriptII system, pGEM system, pTZ system, pCL system, pET system and the like can be used. However, the vectors that can be used in the present invention are not limited thereto, and any vectors available for expression in Drosophila melanogaster S2 cells are included in the scope of the present invention.

In the present invention, the expression vector may further include a secretion signal sequence. The protein secretion signal induces secretion of the expressed protein from the vector into the cell, for example, but not exclusively, a BiP secretion signal sequence.

In the present invention, the term " transformation " means introducing an expression vector containing a nucleic acid encoding MERS-RBD according to the present invention into a host cell. In addition, the transfected target gene may be inserted into the chromosome of the host cell or located outside the chromosome as long as it can be expressed in the host cell.

The method of transforming the vector of the present invention includes any method of introducing a nucleic acid into a cell and can be carried out by selecting a suitable standard technique as known in the art. For example, electroporation, calcium phosphate (CaPO ₄ ) precipitation, calcium chloride (CaCl ₂ ) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, A lithium acetate-DMSO method, and the like, but are not limited thereto.

Next, the step of producing MERS-RBD antigen comprises culturing the transfected cells in a culture medium.

The term " culturing " in the present invention means culturing the transfected cells in an appropriately controlled environment. In the present invention, the culturing may be performed according to the appropriate culture medium and culture conditions of the Drosophila melanogaster S2 cells known in the art. The culture medium and other culture conditions used in the culture of the present invention are not particularly limited, but specifically, they may be cultured under aerobic conditions in an ordinary medium containing a suitable carbon source, nitrogen source, phosphorus, inorganic compound, amino acid and / pH and the like.

In the present invention, the carbon sources include carbohydrates such as glucose, fructose, sucrose, maltose, mannitol, sorbitol and the like; Alcohols such as sugar alcohol, glycerol, pyruvic acid, lactic acid, citric acid and the like; But are not limited to, amino acids such as organic acids, glutamic acid, methionine, lysine, and the like. Natural organic nutrients such as starch hydrolyzate, molasses, black strap molasses, rice winter, casserole, sugar cane residue and corn steep liquor can be used, and specifically include glucose and sterilized pretreated molasses Molasses) can be used, and other suitable amounts of carbon sources can be used without limitation. These carbon sources may be used alone or in combination of two or more.

Examples of the nitrogen source include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate; An organic nitrogen source such as amino acids such as glutamic acid, methionine, glutamine and the like, peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolyzate, fish or its degradation products, defatted soybean cake, Can be used. These nitrogen sources may be used alone or in combination of two or more, but the present invention is not limited thereto.

Such personnel may include potassium phosphate, potassium phosphate, or the corresponding sodium-containing salts, and the like. Examples of the inorganic compound include sodium chloride, calcium chloride, magnesium chloride, iron sulfate, manganese sulfate, calcium carbonate, and the like, but may also include amino acids, vitamins and / or suitable precursors. These media or precursors may be added to the culture in a batch or continuous manner, but are not limited thereto.

In the present invention, a compound such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid and the like may be added to the culture in a suitable manner during the culture of the transformed cells to adjust the pH of the culture. In addition, foaming can be suppressed by using a defoaming agent such as fatty acid polyglycol ester during the culture. Also, in order to maintain the aerobic state of the culture, oxygen or oxygen-containing gas may be injected into the culture, or nitrogen, hydrogen or carbon dioxide gas may be injected without injecting gas to maintain anaerobic and microorganism conditions.

The temperature of the culture may be from 15 캜 to 35 캜, more specifically from 25 캜 to 30 캜, but is not limited thereto. The incubation period can be continued until the desired amount of useful material is obtained.

Since the transfected cells express the MERS-RBD antigen, the MERS-RBD antigen can be contained in the transformed cells or in the culture medium by being cultured in the culture medium. In a specific example of the present invention, cells transformed to express the MERS-RBD antigen were cultured to confirm that MERS-RBD protein of about 30 kDA size was present in the cell culture medium and cell culture medium. Especially, in the BiP signal sequence And the MERS-RBD protein was secreted out of the cell to confirm that most of the MERS-RBD protein was present in the cell culture medium (FIG. 3). Thus, the MERS-RBD protein is produced, accumulated in the cell or secreted, and is present in the cell culture medium.

For example, the method for producing MERS-RBD antigen of the present invention may further comprise the step of recovering the MERS-RBD antigen from the culture medium in which the transfected cells have been cultured. When the antigen is obtained from the culture medium, since the cell is not dissolved and the protein is isolated, there is an advantage that the manufacturing process can be simplified and the manufacturing cost can be lowered in the production of MERS-RBD antigen.

The step of recovering the MERS-RBD antigen can be carried out by culturing the desired MERS-RBD antigen from the culture medium using a suitable method known in the art according to the cell culture method of the present invention, for example, batch, continuous, or fed- Can be recovered. For example, centrifugation, filtration, affinity chromatography and the like can be used, and suitable methods known in the art can be used in combination.

The recovering step may include a separation step and / or a purification step.

Another aspect of the present invention relates to a method of screening for an expression vector comprising a nucleic acid encoding a receptor binding domain (MERS-RBD) of a Mers virus spike protein, using a Drosophila melanogaster ) S2 cells to produce transformed cells; Culturing the transformed cells in a culture medium; Recovering the MERS-RBD antigen from the culture medium in which the transformed cells have been cultured; And producing a monoclonal antibody against the MERS-RBD antigen.

The process of expressing MERS-RBD in Drosophila melanogaster S2 cells and recovering MERS-RBD antigen is as described above. The present invention provides a method for preparing an antibody against MERS-RBD using the MERS-RBD antigen prepared by the above method.

As used herein, the term " monoclonal antibody " refers to a single molecule composition of antibody molecules obtained in a substantially identical population of antibodies, which monoclonal antibodies exhibit a single binding specificity and affinity for a particular epitope. The monoclonal antibody of the present invention is an antibody that uses the MERS-RBD prepared in the present invention as an antigen, and has the property of binding to an epitope contained in the MERS-RBD. The method for producing the monoclonal antibody in the present invention is not particularly limited, and a person skilled in the art can make use of the MERS-RBD antigen prepared by the method of the present invention, Antibodies can be prepared.

In a specific embodiment of the present invention, lymph node cells are obtained from a mouse injected with the recombinant MERS-RBD protein thus prepared and fused with the myeloma cells to produce five MERS-RBD protein-specific antibodies. Cell lines (C1F2, 2F9, D2G2, D4E3, 1E9) were obtained. Each monoclonal antibody isolated and purified from the cell lines has a high binding capacity for the recombinant MERS-RBD protein (FIG. 5).

Another embodiment of the present invention is an antigenic protein consisting of a receptor binding domain (MERS-RBD) fragment of a mers virus spike protein consisting of the 377-588 amino acid of a mers virus spike protein, A nucleic acid molecule comprising the nucleotide sequence, a vector comprising the nucleic acid molecule, and a transformed cell comprising the vector.

The nucleic acid molecules composed of the nucleotide sequences of MERS-RBD and SEQ ID NO: 2 are as described above.

A typical artisan can be included in an appropriate vector for the purpose of expressing, carrying, amplifying, or transforming a nucleic acid molecule composed of the nucleotide sequence of SEQ ID NO: 2 of the present invention. Can be manufactured. Therefore, the vector may vary depending on its purpose, and a person skilled in the art can appropriately select a known vector according to the purpose of using it. The transformed cell may be a microorganism, an insect cell, or a mammalian cell. Like the vector, the transformed cell may be appropriately selected depending on the purpose to be used by a person skilled in the art, and the kind thereof is not particularly limited. The cells may be isolated cells.

The recombinant mers virus antigen protein MERS-RBD and the mers specific monoclonal antibody produced in the transformed insect cell Drosophila melanogaster S2 cells obtained in the present invention can be utilized in the development of a diagnostic kit for the diagnosis of mers virus . In addition, the recombinant mers virus antigen protein MERS-RBD and the mers specific monoclonal antibody can be used as a vaccine material and an antibody for treatment that can prevent the infection of mers viruses. It is expected to be used as a method to cope with the middle respiratory syndrome caused by mers virus infection by diagnosing infection of mers virus, vaccine material for prevention of mers virus infection, and developing antibody for treatment.

Figure 1 compares the nucleotide sequences of the Mers viral spike protein receptor binding domain (MERS-RBD) and the mers virus spike protein receptor binding domain (S2-MERS-RBD) optimized to the insect cell codon usage frequency to be.
Fig. 2 is a schematic diagram of a recombinant pMT / BiP-MERS-RBD-V5-His plasmid.
FIG. 3 is a graph showing the activity of recombinant MERS-RBD protein in intracellular fraction and medium fraction of Drosophila melanogaster S2 cells transformed with the recombinant pMT / BiP-MERS-RBD-V5-His plasmid The presence of anti-V5 antibody results in western blot analysis.
FIG. 4 shows the results of the affinity chromatography using a Ni-NTA resin in a culture medium of Drosophila melanogaster S2 cells transformed with the recombinant pMT / BiP-MERS-RBD-V5-His plasmid. After separation and purification, SDS-PAGE was followed by silver staining.
FIG. 5 shows the binding ability of Mers virus monoclonal antibody produced in a hybridoma cell line to the recombinant mers virus antigen protein MERS-RBD by ELISA.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are intended to illustrate the present invention, and the scope of the present invention is not limited to these examples.

Example  One: Mers  The receptor binding domain of the virus spike protein ( MERS -RBD) gene

The present inventors used the receptor binding domain (RBD) (amino acid residues 377 to 588) of MERS-Cov / KOR / KNIH / 002_05_2015 spike protein (NCBI accession number, AKL59401) as a Drosophila melanogaster (S2-MERS-RBD; SEQ ID NO: 2) for the receptor binding domain (MERS-RBD) of a known mers virus spike protein in order to express it in S2 ( Drosophila melanogastor S2) Respectively.

In the S2-MERS-RBD sequence (SEQ ID NO: 2), 471 nucleotide sequences of the 636 nucleotide sequences coding for MERS-RBD were identical to wild type (SEQ ID NO: 1) (74.1% sequence similarity) (SEQ ID NO: 25.9%) (Fig. 1).

Example  2: Drosophila Melano Gastor  S2 cell culture

Drosophila melanogaster S2 cells are one of the cell lines belonging to the genus Drosophila. The expression level of foreign proteins is high, and the proteins expressed in the cells express post-translational modifications (glycosylation, phosphorylation, fatty acid addition, etc.) It goes through. In addition, since the expressed protein can be easily released from the cell, it is very easy to mass-produce and separate and purify an exogenous protein, which is advantageous for mass production of useful foreign protein. However, no method has been reported so far for producing recombinant MERS-RBD protein using Drosophila melanogaster S2 cells.

In the present invention, Drosophila melanogaster S2 cells were cultured at 27 ° C using protein-free culture medium for production of recombinant protein and Hyclone ^™ SFX insect cell culture medium (Invitregen).

Example  3: Construction of recombinant plasmid

The pMT / BiP-V5-His plasmid vector (36 kb; Invitrogen) was used for the production of recombinant plasmids for the expression of recombinant MERS-RBD protein. The plasmid vector has a methallothionein promoter, a cell secretory signal BiP sequence of a recombinant protein, a V5 epitope tag, and a polyhistidine site.

The S2-MERS-RBD DNA fragment (S2-MERS-RBD) of SEQ ID NO: 2 obtained in Example 1 was cloned into the Bgl II and Apa I restriction sites of the pMT / BiP-V5- And the recombinant plasmid vector for expression of Drosophila melanogaster S2 cell expression was obtained and designated pMT / BiP-MERS-RBD-V5-His (FIG. 2).

Example  4: Transformation Drosophila Melano Gastor  S2 cell establishment

The recombinant pMT / BiP-MERS-RBD-V5-His plasmid was used to establish the Drosophila melanogaster S2 cells transformed with the recombinant pMT / BiP-MERS-RBD-V5-His plasmid vector prepared in Example 3 PCoHygro plasmid vector (0.3 μg) containing the vector (2.7 μg) and the hygromycin resistance gene was mixed with 100 μl of Grace serum-free medium (Invitrogen), and 100 μl of Grace (Invitrogen) mixed with 10 μl of Cellfectin II reagent Mixed with serum-free medium and allowed to react at room temperature for 30 minutes. The mixture of pMT / BiP-MERS-RBD-V5-His and pCoHygro plasmid vector / Cellfectin II reagent was added to the wells of 6-well plates in which 3 x 10 ⁶ Drosophila melanogaster S2 cells were dispensed. After culturing, the cells were exchanged with HyClone ^™ SFX insect cell culture medium and cultured for 48 hours. Then, the cell culture medium was replaced with HyClone ^™ SFX insect cell culture medium containing 300 μg / ml hygromycin at intervals of 3 to 4 days, cultured for about 45 days, and resistant to hygromycin, and pMT / BiP-MERS-RBD-V5-His plasmid vector was stably transformed.

Example  5: Stably transfected Drosophila Melano Gastor  Recombination in S2 cells MERS - RBD  Identification of Protein Expression

To confirm expression of the recombinant MERS-RBD protein in the Drosophila insect cells in which the recombinant pMT / BiP-MERS-RBD-V5-His plasmid was stably transfected, HyClone ^™ SFX insect cell culture medium containing 500 μM CuSO ₄ The cell extract and culture medium were collected from the cell culture for 4 days, and the presence of the recombinant MERS-RBD protein was confirmed by Western blot analysis using anti-V5 antibody.

As a result, the recombinant MERS-RBD protein was expressed at a size of about 30 kDa and was found to exist in the cell and the cell culture medium (FIG. 3). This result implies that the recombinant MERS-RBD protein is stably expressed in transformed Drosophila melanogaster S2 cells. In addition, the recombinant MERS-RBD protein of the present invention may be secreted out of the cell by the BiP signal sequence, and most of the recombinant MERS-RBD protein expressed in the transformed Drosophila melanogaster S2 cells is present in the cell culture medium (Fig. 3, comparison of lane 3 and 4). This means that the recombinant MERS-RBD protein is efficiently secreted out of the cell by the BiP signal sequence.

Example  6: Recombination MERS - RBD  Separation of protein purification

To isolate and purify the recombinant MERS-RBD protein, the culture medium was recovered from the cell culture medium for 4 days in HyClone ^™ SFX insect cell culture medium containing 500 μM CuSO ₄ , and the affinity chromatography method using Ni-NTA resin Recombinant MERS-RBD protein was isolated and purified from the culture medium. The purity of the isolated and purified recombinant MERS-RBD protein was confirmed by silver staining after SDS-PAGE (FIG. 4).

Example  7: Recombination MERS - RBD  Protein animal immunity and lymph node cell securing

The recombinant MERS-RBD protein was injected into the food pad of BALB / c mice to induce an immune response. In order to promote the immune response by the recombinant MERS-RBD protein, a complete Freund's adjuvant (CFA) was mixed with the recombinant MERS-RBD protein at a ratio of 1: 1 (v / v) For immunization, IFA (Incomplete Freund's adjuvant) was mixed with recombinant MERS-RBD protein at a ratio of 1: 1 (v / v). Immunization was carried out three times at intervals of one week and 200 μg of recombinant MERS-RBD protein was injected per mouse in one immunization.

Three days after the final immunization, mice were sacrificed using CO ₂ gas and lymph nodes were removed from the popliteal. The extracted lymph nodes were washed with RPMI1640 medium, unilamellated with glue, and washed three times with RPMI1640 medium to obtain hybridoma cells producing recombinant MERS-RBD protein-specific monoclonal antibody And to the cell fusion experiments with SP2 / 0 myeloma cells.

Example  8: SP2 / 0 myeloma cell culture

SP2 / 0 myeloma cells lacking the HGPRT (hypoxanthine-guanine phosphoribosyltransferase) gene were cultured at 37 ° C in RPMI 1640 medium containing 10% Fetal Bovine Serum (FBS).

Example  9: Cell fusion and recombination MERS - RBD  Production of protein-specific antibodies Single clone Hybridoma  Cell securing

SP2 / 0 myeloma cells were washed with RPMI1640 medium and mixed with lymph node cells recovered from immunized mice with recombinant MERS-RBD protein at 1: 5 and centrifuged to precipitate. After removing the supernatant, the precipitated cells were lightly kneaded and mixed well, then 1 ml of 50% PEG1500 solution was slowly added over 1 minute. The RPMI medium was sequentially instilled with 1 ml per 30 seconds, 3 ml per 30 seconds, and 17 ml per minute and reacted for 5 minutes to induce fusion of cells. After completion of the reaction, the reaction mixture was centrifuged at 1,200 rpm for 5 minutes, suspended in RPMI1640 medium containing HAT (hypoxanthine-aminopterin-thymidine), and the resultant was added to a 96-well plate and cultured.

When the cells were grown at the bottom of the wells by 60-70% after cell fusion, the cell culture medium was recovered and the activity of the antibody against the recombinant MERS-RBD protein was measured by indirect ELISA. Wells with antibody titer greater than 1 for the recombinant MERS-RBD protein were selected and used for secondary screening. The antibody titer of the recombinant MERS-RBD protein was measured by indirect ELISA method in the cell culture obtained from the secondary screening, and well cells with high potency and good cell status were selected and used for culturing for cell line secreting. The cells of the wells selected through the secondary screening were diluted to a concentration of 1 cell / well on a 96-well plate, and the cells were divided and cultured to produce five hybridoma cell lines (C1F2 , 2F9, D2G2, D4E3, and 1E9).

Example  10: Monoclonal antibody  Production and refining

Hybridoma cell lines (5 x 10 ⁶ cells / ml) were injected into the peritoneal cavity of BALB / c mice in 1 ml increments. Ten days later, the ascites was collected. Centrifuged at 4,000 rpm for 10 minutes, and the supernatant was recovered to separate and purify the antibody from the ascites. The ascites was dialyzed against PBS (phosphate buffered saline, pH 7.4), the buffer was exchanged, and adsorbed onto the column bound to Protein A / G. The column was washed with PBS to remove non-adsorbed material, and the antibody bound to Protein A / G was eluted with 100 mM glycerin solution (pH 2.8). The eluted antibody was neutralized by adding 1 M Tris solution (pH 9.0) to 1/10 volume of the eluate. The purified antibody was dialyzed against PBS buffer solution, concentrated using a centrifuge tube, and quantified using a UV spectrometer.

Example  11: Recombination MERS - RBD  Measurement of binding activity of monoclonal antibodies against proteins

The binding ability of the purified monoclonal antibody to the recombinant MERS-RBD protein was confirmed by an indirect ELISA method. The recombinant MERS-RBD protein was diluted to a concentration of 1 μg / ml in a 0.05 M carbonate-bicarbonate buffer solution (pH 9.7) and dispensed into a 96-well plate at a rate of 100 μl per well, followed by overnight reaction at 4 ° C Respectively. The cells were washed three times with PBST (PBS containing 0.05% Tween-20), and 100 μl of the monoclonal antibody diluted 1/10 successively from 10 μg / ml to 1 ng / ml was dispensed per well. Lt; / RTI > After washing three times with PBST solution, anti-mouse IgG antibody conjugated with horse radish peroxidase (HRP) was diluted 1 / 7,000 times and 100 ㎕ per well was dispensed and reacted at 37 캜 for 1 hour. After washing three times with PBST solution, 100 μl of substrate (TMB) solution was added per well to induce the enzyme reaction. After the reaction was maintained at room temperature for 5 minutes, 50 μl of 1 NH ₂ SO ₄ solution was added to stop the reaction, and the absorbance at 450 nm was measured using a microplate reader. As a result, it was confirmed that the monoclonal antibodies isolated and purified from the five hybridoma cell lines (C1F2, 2F9, D2G2, D4E3, and 1E9) all had high binding ability to the recombinant MERS-RBD protein (FIG.

From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all aspects and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention without departing from the scope of the present invention as defined by the appended claims.

<110> UNIVERSITY-INDUSTRY COOPERATION GROUP OF KYUNG HEE UNIVERSITY          Boreda biotech <120> A method for producing MERS antigen using Drosophila melanogaster          S2 <130> P17-041-KHU <160> 2 <170> KoPatentin 3.0 <210> 1 <211> 636 <212> DNA <213> Artificial Sequence <220> <223> MERS-RBD wild-type sequence <400> 1 caggctgaag gtgttgaatg tgatttttca cctcttctgt ctggcacacc tcctcaggtt 60 tataatttca agcgtttggt ttttaccaat tgcaattata atcttaccaa attgctttca 120 cttttttctg tgaatgattt tacttgtagt caaatatctc cagcagcaat tgctagcaac 180 tgttattctt cactgatttt ggattatttt tcatacccac ttagtatgaa atccgatctc 240 agtgttagtt ctgctggtcc aatatcccag tttaattata aacagtcctt ttctaatccc 300 acatgtttga ttttagcgac tgttcctcat aaccttacta ctattactaa gcctcttaag 360 tacagctata ttaacaagtg ctctcgtctt ctttctgatg atcgtactga agtacctcag 420 ttagtgaacg ctaatcaata ctcaccctgt gtatccattc tcccatccac tgtgtgggaa 480 gacggtgatt attataggaa acaactatct ccacttgaag gtggtggctg gcttgttgct 540 agtggctcaa ctgttgccat gactgagcaa ttacagatgg gctttggtat tacagttcaa 600 tatggtacag acaccaatag tgtttgcccc aagctt 636 <210> 2 <211> 636 <212> DNA <213> Artificial Sequence <220> <223> MERS-RBD modified sequence <400> 2 caggctgaag gtgttgagtg cgatttcagt cctctcctta gcggtacgcc tccacaagtt 60 tacaacttca agcgtctcgt gttcaccaac tgcaactaca acctgaccaa gctcctctcg 120 ctcttcagcg tgaacgactt cacgtgctcg caaatcagcc cagcagctat cgccagtaac 180 tgctacagtt cgcttatcct ggactacttc tcgtacccgc tgagtatgaa gtccgacctg 240 agtgtgagca gcgccggtcc aatatcccag tttaactaca agcagtcctt tagcaatccc 300 acgtgcctga tcctggcgac ggtaccgcac aatttgacca ccataaccaa accgctgaag 360 tacagctaca ttaataagtg ctcccgcctg ctgtccgacg atcgaaccga ggtgccgcaa 420 cttgtaaatg ccaatcagta ttcgccctgt gtgtccattc tgccctccac tgtgtgggag 480 gatggagatt attatcggaa acagctgtcc cccttggagg gaggaggctg gttggtggcg 540 agcggctcga cagtcgccat gactgaacag ttgcagatgg gctttggcat tacagtccag 600 tatggcactg atacaaattc ggtctgtccc aaattg 636

Claims (13)

Preparing a transformed cell by introducing an expression vector containing a nucleic acid encoding a receptor binding domain (MERS-RBD) of a Mers virus spike protein into Drosophila melanogaster S2 cells; And
And culturing the transformed cells in a culture medium. 2. The method of claim 1, wherein the MERS-RBD is comprised of the 377-588 amino acid of a Mervis virus spike protein. 2. The method of claim 1, wherein the nucleic acid encoding MERS-RBD comprises the nucleotide sequence of SEQ ID NO: 2. 2. The method of claim 1, wherein the expression vector further comprises a secretion signal sequence. The method of claim 1, further comprising recovering the MERS-RBD antigen from a culture medium in which the transformed cells have been cultured. Preparing a transformed cell by introducing an expression vector containing a nucleic acid encoding a receptor binding domain (MERS-RBD) of a Mers virus spike protein into Drosophila melanogaster S2 cells;
Culturing the transformed cells in a culture medium;
Recovering the MERS-RBD antigen from the culture medium in which the transformed cells have been cultured; And
And producing a monoclonal antibody against said MERS-RBD antigen. 7. The method of claim 6, wherein the MERS-RBD is comprised of the 377-588th amino acid of a mers virus spike protein. 7. The method of claim 6, wherein the nucleic acid encoding MERS-RBD comprises the nucleotide sequence of SEQ ID NO: 2. 7. The method of claim 6, wherein the expression vector further comprises a secretion signal sequence. (MERS-RBD) fragment of a mers virus spike protein consisting of the 377-588 amino acid of the Mers virus spike protein. A nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 2 encoding the antigen protein of claim 10. 12. A vector comprising the nucleic acid molecule of claim 11. A transformed cell comprising the vector of claim 12.

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