KR100506485B1 - Fusion cells producing monoclonal antibodies against T7 bacteriophage GENE9 protein and monoclonal antibodies produced thereby - Google Patents

Abstract

The present invention provides a fusion cell for producing a monoclonal antibody against T7 bacteriophage Gene9 protein, produced by cell fusion of splenocytes obtained from mice immunized with T7 bacteriophage Gene9 protein with myeloma cells, and monoclonal produced therefrom. Relates to raw antibodies.

Description

Fusion Cells Producing Monoclonal Antibodies to T7 Bacteriophage Genie9 Protein and Monoclonal Antibodies Produced thereby

The present invention is directed to novel fusion cells (monbridoma) producing monoclonal antibodies against T7 bacteriophage Gene9 protein (hereinafter referred to as "G9 protein") and monoclonal antibodies produced thereby. More specifically, the fusion cells producing monoclonal antibodies against the G9 protein and their fusion cells, which are produced by cell fusion of spleen cells of mice immunized with G9 protein with myeloma cells. It relates to a monoclonal antibody produced by.

T7 bacteriophages are medium sized E. coli phages that consist of a head containing a 26 × 10 ⁶ molecular weight DNA and a very short tail attached to the head. T7 DNA has been identified all 39,936bp, characterized by clustering (cluster) according to the function and time of expression.

The G9 protein is a coat assembly protein of T7 bacteriophage, and has a molecular weight of about 34,000 consisting of 306 amino acids. The G9 protein is an acidic protein having an isoelectric point of about 4.2 and has a very high solubility, so that even when expressed at high concentration in E. coli , it is known that it does not form an inclusion body due to its high solubility (Howell et al. , Journal of Biological Chemistry 264, p15268, 1989]. The formation of inclusion bodies is a factor that makes it difficult to express high concentrations of proteins, and this property can be used as a fusion partner of G9 proteins to express high concentrations of some proteins that are difficult to express. To be. That is, by combining the N-terminus of the protein of interest with the C-terminus of the G9 protein and expressing it in E. coli by a genetic recombination method, a fusion protein of the two proteins can be obtained, and then the fusion protein. It can be obtained in high concentration by adding a protease to isolate the desired protein. In the above process, when a monoclonal antibody to the G9 protein is present, the desired protein can be purified very easily. That is, the monoclonal antibody against the G9 protein is immobilized on a support such as Sephadex or Sepharose, and then passed through the E. coli extract to attach only the fusion protein and remove the remaining proteins. Proteolytic enzymes were added to the fusion protein thus obtained to cleave the G9 protein and the target protein, and then re-passed through the support to which the monoclonal antibody was fixed. Only the G9 protein was attached and the target protein was easily passed through to obtain a pure state. You can do it.

Therefore, the use of a monoclonal antibody that specifically binds to the G9 protein facilitates the purification of the fusion protein with the G9 protein and the desired protein from the fusion protein. Fusion cells producing ronal antibodies and monoclonal antibodies produced thereby will be of great utility.

On the other hand, antibodies produced in vivo by antigen stimulation are usually mixtures of antibodies (polyclonal antibodies) having various binding properties to antigens, but some antibody-producing cells produce only one type of antibody. Bar monoclonal antibody means a homogeneous antibody having the same structure formed by cells (single clones) that have grown from a single antibody producing cell. A method for preparing cells that produce monoclonal antibodies was developed in 1975 by Kohler and Milstein. This method involves fusion of B-lymphocytes obtained from the spleen or lymph nodes of an animal immunized with a particular antigen with myeloma cells, which are normal cells with the ability to produce monoclonal antibodies and myeloma cells become cancerous. As cells obtained from B-lymphocytes, which do not have the ability to produce immunoglobulins but have abnormal proliferative capacity to continue growing, consequently, the fusion cells of these cells can produce monoclonal antibodies of B-lymphocytes. The ability to grow and to continue to grow without killing myeloma cells.

When cell fusion is completed, the fusion cells, non-fusion splenocytes and myeloma cells are mixed in the medium in which the fusion is performed. Therefore, only fusion cells need to be selected. Cells are grown in HAT medium for this purpose. Purine (adenine, guanine) is biosynthesized through two pathways, the endogenous pathway and the salvage pathway, so wild pathogens are not lethal if they lose either pathway. HAT medium contains hypoxantine, aminopterin and thymidine, which inhibit the endogenes pathway's action by blocking the endogenes pathway's action of dihydrofolate reductase (DHFR). Therefore, non-fused myeloma cells that lack the salvage pathway's hypoxanthine phosphoribosyl transferase (HGPRT) (and thus are resistant to toxic base analogs (thioguanine or azaguuanine)) are also known as wild strains. Unlike, HAT medium does not grow, but dies, non-fused splenocytes die naturally after 1 to 2 weeks after separation, so that only the fusion cells grow in the medium, allowing selection of the fusion cells.

Monoclonal antibodies have several advantages over polyclonal antibodies, such as the need for multiple administrations of the antigen in the antibody production phase and the need for high purity of the antigen to be administered. Thus, monoclonal antibodies are currently used in a variety of immunological studies, particularly in the analysis, identification and purification of proteins such as ELISA (Enzyme Linked Immunosorbent Assay) or Western blots.

In conclusion, monoclonal antibodies that specifically bind to G9 proteins as described above are useful for the analysis, identification, purification of proteins, and especially for purification of proteins containing G9 proteins by chromatography by immobilization on a column. To date, no fusion cells producing monoclonal antibodies against the G9 protein and monoclonal antibodies produced thereby have not been known at all.

Accordingly, the present inventors have conducted continuous research to obtain monoclonal antibodies that specifically bind to G9 protein. As a result, the present inventors have produced fusion cells by cell fusion of splenocytes and myeloma cells of mice immunized with G9 protein. The present invention was completed by successfully culturing the monoclonal antibody against the G9 protein.

It is therefore an object of the present invention to provide novel fusion cells producing monoclonal antibodies against G9 protein, which are prepared by cell fusion of splenocytes obtained from mice immunized with G9 protein with myeloma cells.

Another object of the present invention is to provide a monoclonal antibody against the G9 protein produced by the fusion cells.

It is another object of the present invention to provide a method for purifying a protein comprising a G9 protein by specifically binding the monoclonal antibody with a G9 protein site.

The present invention provides fusion cells producing monoclonal antibodies against G9 protein, which are prepared by cell fusion of splenocytes obtained from mice immunized with G9 protein with myeloma cells. In the present invention, the fusion cell is preferably CJ-M146 of Accession No. KCLRF-BP-00019.

The present invention also provides a monoclonal antibody against the G9 protein produced by the fusion cells. The monoclonal antibody may be one that selectively recognizes the N-terminal portion of the G9 protein.

The present invention also provides a method for purifying a protein comprising a G9 protein, wherein the monoclonal antibody specifically binds to a G9 protein site.

Hereinafter, the present invention will be described in more detail.

In the present invention, mice are immunized twice with G9 protein over a period of time in order to produce fusion cells producing monoclonal antibodies that specifically bind to G9 protein. The G9 protein may be one produced by genetic recombination methods.

Splenocytes were taken from immunized mice and mixed with 8-azaguanine resistant plasma cell NS-O (Galfre, G and Milstein, C. Methods Enzymol. 73, p3, 1981) and 50% PEG (polyethylene glycol) Cell fusion reactions were performed by treatment with a solution and a 50% PEG and 20% DMSO (dimethyl sulfoxide) mixed solution. PEG acts to enhance intercellular fusion.

After completion of the cell fusion reaction, cells were cultured in MEM-added DMEM medium to select only the fusion cells, and the monoclonal antibody was subjected to high range by investigating whether an antibody to G9 protein was present in each culture by using an ELISA method. Isolate the cells producing horizontally However, since the primary clones thus obtained contain not only antibody-producing cells but also other cells, only antibody-producing cells are isolated by subcloning method using soft agarose. Isolate fusion cells whose antibody production stability is confirmed.

The present inventors named the isolated fused cells as CJ-M146, which was established in August 4, 1998 by the Korean Cell Line Research Foundation, affiliated with the Seoul National University College of Medicine, an international depository institution under the Treaty of Budapest. It was deposited and was given accession number KCLRF-BP-00019.

Monoclonal antibodies against the G9 protein are obtained by culturing the fusion cell CJ-M146 obtained above in a suitable medium and separating it from the medium using ammonium sulfate precipitation and column chromatography. The monoclonal antibody obtained according to the present invention can be usefully used for the analysis, identification and purification of proteins, such as attached to a column and used for the purification of a protein including G9 protein by chromatography, or used in Western blot. have.

EXAMPLE

Hereinafter, the configuration and operation of the present invention will be described in more detail with reference to Examples, but this is not intended to limit the scope of the present invention in any way.

Example 1 Preparation of Fusion Cell CJ-M146

In order to obtain purified G9 protein, the expression vector pSR9 cloned with the G9 gene was overexpressed by transformation into E. coli BL21 (Howell et al., Journal of Biological Chemistry 264, p15268, 1989). Subsequently, ammonium sulfate precipitation (25-55%) was carried out, followed by dialysis under conditions of 10 mM Tris buffer and pH 7.2, and NaCl concentration gradient of 0.14 to 0.5 under conditions of 10 mM Tris and pH 7.2. Q Sepharose column chromatography eluting with M yielded a peak of 0.3 M, followed by dialysis under conditions of 10 mM Na-citrate buffer, pH 5.2, 10 mM Na-citrate buffer, pH 5.2, NaCl 0- SP column chromatography eluting with a concentration gradient of 0.2 M gave a peak of 0.1 M portion to obtain purified G9 protein of 95% purity or more.

Mice were first immunized with the purified G9 protein and a second immunization thereafter one month thereafter, and after 3 days thereafter, about 10 ³ splenocytes were taken from the mice and washed with DMEM medium. In addition, about 3.8 × 10 ⁷ plasma cells NS-O showing 8-azaguanine resistance were taken and washed with the same medium. After these two cells were mixed together, the mixture was treated with 0.5 ml of 50% PEG solution (pH 9.0) for 30 seconds, and then treated with 50% PEG and 20% DMSO solution for 30 seconds to perform cell fusion reaction.

Cells were then washed three times with DMEM medium and added to 96-well plates to 2.5 × 10 ⁴ cells per well. At this time, DMEM containing a certain amount of HAT (hypoxanthine 10 ^-4 M, aminopterin 6 x 10 ^-7 M, thymidine 1.6 x 10 ^-6 M) and 5% calf serum was used as a culture medium. Two weeks after the cell fusion reaction, the presence of a monoclonal antibody against G9 protein was examined in the culture medium in each well. An ELISA method was used to detect very small amounts of antibodies. About 1,000 fusion cells were obtained through the above method, from which 30 fusion cells producing monoclonal antibodies to G9 protein at high titers were isolated.

However, since the primary clone contains not only antibody-producing cells but also other cells, the antibody-producing cells may be diluted due to overgrowth of other cells. Therefore, since the antibody-producing cells should be separated from the well in which the clones grow as quickly as possible, the soft agarose method was used as the subcloning method. That is, 0.8% agarose was added to the culture solution containing HAT, and primary clones were added thereto and cultured for 2 weeks. Two weeks later, 20 cells were randomly selected to confirm the production of monoclonal antibodies against the G9 protein, and fusion cells of which antibody production stability was confirmed for more than 6 months were isolated from the fusion cells.

As mentioned above, the present inventors named the isolated fusion cells as CJ-M146 and, on August 4, 1998, deposited internationally with the Korea Cell Line Research Foundation, affiliated with the Seoul National University College of Medicine, Accession No. KCLRF-BP- 00019 was granted.

Example 2 Preparation of Monoclonal Antibody Specifically Binding to G9 Protein

Fusion cell CJ-M146 prepared according to the method of Example 1 was cultured in RPMI1640 medium containing 5% calf serum. The culture solution was collected by 1 L and centrifuged at 10,000 rpm (Sorval) for 10 minutes at 4 ° C to remove cells. The supernatant was adjusted to pH 8 with 2M Tris buffer and ammonium sulfate was added to 50% to precipitate the antibody in solution. The precipitate was collected by centrifugation and dissolved in 50 ml buffer (50 mM Tris-HCl + 100 mM NaCl, pH 7.2) and diafiltration with the same buffer. The dialysis filtrate was passed after equilibrating the DEAE Affi-Gel Blue (Bio Rad) column with 100 ml of the same buffer. The column was also washed with 100 ml of the same buffer and eluted with 70 ml of eluent (50 mM Tris-HCl + 500 mM NaCl, pH 7.2) to obtain the purified antibody protein. The monoclonal antibody obtained here was used for the purification of the following G9 proteins.

Example 3 Preparation of Sepharose 4B Column Attached with Monoclonal Antibody

6 liters of 1 mM hydrochloric acid was poured into Sepharose 4B (Pharmacia), which was activated with 30 g of dried CNBr, and swelled. The gel was washed three times with buffer (0.1 M NaHCO ₃ + 0.5 M NaCl, pH 8.3) and then placed in 150 ml of the same buffer. The monoclonal antibody against the G9 protein obtained from the fusion cell CJ-M146 was purified and added to 450 mg, the total volume was adjusted to 200 ml and shaken at 4 ° C. overnight. The supernatant was decanted and added to hinder buffer (1M ethanolamine, pH 8.0) and left at 4 ° C. for 16 hours.

Example 4 Purification of G9 Protein by Sepharose 4B Column Attached with Monoclonal Antibody

As a starting material, a G9 protein prepared by a gene recombination method was used. That is, after culturing E. coli transformed with the G9 gene, cells were obtained by centrifugation and the supernatants obtained by crushing cells were used as protein solutions (Example 1).

After filling 50 ml of Sepharose 4B with monoclonal antibody to the G9 protein obtained from the fusion cell CJ-M146 in a column according to the method of Example 3, buffer solution (50 mM sodium phosphate + 0.15 M NaCl, pH) was added. 7.0) Equilibrate with 0.3 L. 1 L of the protein solution obtained above was passed through a column, washed with 300 ml of a buffer solution (50 mM sodium phosphate + 0.5 M NaCl, pH 7.0), and the protein was eluted with 32 ml of eluent (0.1 M citric acid). The pH was then adjusted to 8.0.

Protein content in the solution was measured according to the Bradford method (Bradford, MM, Anal. Biochem. 72, pp248-254, 1976) using serum proteins as a reference material, and the degree of purification was measured by Laemli method (document [ Laemmli, UK, Nature 227, pp680-683, 1970]) was confirmed by Coomassie brilliant blue (CBB) staining by electrophoresis on SDS polyacrylamide gel. The results are shown in FIG. In FIG. 1, lane 1 shows the results of electrophoresis of the expressed G9 protein, and lane 2 shows the results of electrophoresis of the purified G9 protein with a monoclonal antibody against the G9 protein produced by the fusion cell CJ-M146. .

Example 5 Western Blot Experiments Using Monoclonal Antibodies

Western blot experiments were performed by methods such as Tobin et al. (Towbin et al., PNAS 76, p4350, 1979). In other words, the purified solution containing the G9 protein was electrophoresed on 8-16% gradient SDS-polyacrylamide gel (Novex) and then electrophoresed on nitrocellulose paper (25mM Tris + 192mM glycine + 20% methanol, pH 8.3). After nitrocellulose paper and gel were assembled, the mixture was added to a blot chamber, filled with the same buffer solution, and a current of 200 mA was passed for 6 hours. After removing the nitrocellulose paper, it was added to the buffer solution (0.1mM sodium phosphate + 0.15M NaCl + 3% BSA, pH 7.2) and allowed to stand at room temperature for 1 hour, and then again to the G9 protein produced by the fusion cell CJ-M146. It was added to the same buffer containing the monoclonal antibody and left for 2 hours at room temperature.

After washing several times with the same buffer solution for 30 minutes, anti-mouse serum to which horse radish peroxidase was attached was added and left at room temperature for 30 minutes for color development. The result is shown in FIG. 2 (lane 1).

Example 6 Western Blot Experiment on N-Terminal Site of G9 Protein

The protease Lys-C was used to digest the G9 protein, leaving only 1 to 164 protein fragments of the G9 protein. This site was western blotted according to the method of Example 5. The result is shown in FIG. 2 (lane 2). As shown in FIG. 2, since the fusion cell CJ-M146 exhibited a positive response, it was confirmed that the monoclonal antibody produced by the fusion cell CJ-M146 recognized the N-terminal region of the G9 protein.

Example 7 Isotype Determination of Monoclonal Antibodies

In order to identify the antibody isotype of CJ-M146, an experiment was performed using Immunopure Monoclonal Antibody Isotyping Kit I of Pierce (USA). Experimental method is as follows.

50 μl of the coating antibody in the kit was added to each well and left at 4 ° C. overnight. The supernatant was removed and 125 μl of blocking solution (0.5% BSA / PBS) was added, left at 37 ° C. for 1 hour, and washed. 50 μl of cell culture was then added to each well and left for 1 hour at 37 ° C. before washing. Then 50 μl of subclass-specific anti-mouse immunoglobulin was added to each well, left at 37 ° C. for 1 hour, and washed four times. Thereafter, goat anti-rabbit IgG conjugated with horse radish peroxidase was added to each well and left at 37 ° C for 1 hour. After washing, 100 μl of ABTS substrate solution was added to each well and allowed to stand at room temperature for about 30 minutes to measure absorbance at 405 nm. As a result, it was confirmed that the monoclonal antibody produced by the fusion cell CJ-M146 is of IgG ₃ type.

The fusion cells and monoclonal antibodies produced thereby according to the present invention can be usefully used for the isolation, identification and purification of proteins, particularly for purification of proteins comprising G9 proteins by chromatography by attachment to columns.

1 shows SDS-polyacrylamide gel electrophoresis results of Gene9 protein purified with expressed Gene9 protein and monoclonal antibody produced by fusion cell CJ-M146, respectively; And

Fig. 2 shows the results of Western blot using monoclonal antibodies produced by the fusion cell CJ-M146 of the expressed Gene9 protein and the N-terminal sites (amino acids 1-164) of the Gene9 protein, respectively.

Claims (1)

Splenocytes obtained from mice immunized with T7 bacteriophage Gene9 protein are produced from fusion cell CJ-M146 (Accession No. KCLRF-BP-00019) prepared by cell fusion with myeloma cells and 1 of the T7 bacteriophage Gene9 protein. A monoclonal antibody against the T7 bacteriophage Gene9 protein, which selectively recognizes amino acids from No. 164.