KR100233962B1 - Monoclonal antibody recognizing phospholipase c-beta4 and hybridoma cell line secreting sameh - Google Patents

Abstract

The present invention provides a monoclonal antibody protein capable of specifically recognizing and binding to a phospholipase C-beta (β) 4 isoenzyme, a fusion cell line secreting the antibody, and the monoclonal antibody protein using the fusion cell line. The present invention relates to a method of preparation, wherein the monoclonal antibodies of the present invention have specific reactivity to phospholipase C-beta4 isoenzyme of not only bovine as well as all mammals, but also to regulate physiological activity, differentiation and growth regulation of mammalian cells. Can be used as a good means of basic biology research.

Description

Monoclonal antibodies recognizing phospholipase C-beta4 and fusion cell lines secreting them

The present invention relates to a monoclonal antibody capable of specifically recognizing phospholipase C-beta4 and a fusion cell line secreting the same. More specifically, phospholipase C-beta 4, one of the isoenzymes of phospholipase C, an enzyme that plays a pivotal role in intracellular signal transduction, can be easily and sensitively detected and quantified from biological or pathological samples. It relates to a monoclonal antibody as a means, a fusion cell line capable of mass production in a homogeneous manner, and a method for producing the monoclonal antibody using these fusion cell lines.

The monoclonal antibodies of the present invention (hereinafter referred to as 'antibodies of the present invention') have the property of specifically reacting only with phospholipase C-beta4 enzymes in mammals, and the physiological activity, differentiation and It can be used as a good means of basic biology research to study growth regulation.

Many physiological phenomena occurring in cells are affected by various signals transmitted from the cell's external environment: neurotransmitters, hormones and growth factors. These signals bind to specific receptors present in the cell membrane, which are transferred into the cell to create a second messenger. Phospholipase C, which is specific for phosphoinositide, decomposes phosphatidylinositol 4,5-bisphosphate by receiving an external signal, thereby diacyl as a secondary transporter. Produces glycerol and inositol 1,4,5-trisphosphate, which are activated intracellularly by activating protein kinase C and releasing calcium ions into the cytoplasm It is a central enzyme in the signal transduction system that regulates phenomena, particularly cell secretion, muscle contraction, sensory mechanisms, and cell growth and differentiation (Nishizuka, Y., Science, 233, 305 (1986)).

It has been found that phospholipase C enzymes have 10 types of isoenzymes (beta1, beta2, beta3, beta4, gamma1, gamma2, delta1, delta2, delta3 and delta4) to date. The nucleotide sequence and amino acid sequence of cDNA to this have been found.

The unique structural properties of phospholipase C isoenzymes are associated with specific mechanisms of enzymatic activity via receptors. Β-type isozymes, which are known to be present in many brains, are among the Gαq groups (Taylor, S, J., Chae, HZ, Rhee, SG, and Exton, JH, Nature, 350) among GTP-binding proteins (G proteins). , 516 (1991)) or βγ subunits (Katz, A., Wu, D., and Simon, MI, Nature, 360, 686 (1992)). It is present at the C-terminal site and βγ subunits are known to interact with and activate the N-terminal site of phospholipase C-β, and these G proteins are associated with receptors having seven transmembrane domains. It is proposed to convey a signal. As such, phospholipase C enzyme is widely known as an important enzyme involved in various physiological phenomena of cells, such as growth, differentiation, metabolism, excitement, secretion, and contraction. These results are specific to each phospholipase C isoenzyme. It is speculated that different cells have developed different mechanisms for precisely transmitting and regulating signals in connection with receptors and mediators. These studies also suggest the possibility of different types of phospholipase C isozymes varying between tissues and cells.

The cerebellum is the main site where the signals of the brain and the nerves of motor organs are connected, transmitted and regulated, making it a good model for studying neurotransmission and hormone action. Despite many attempts to elucidate neurotransmission and hormone action through studies based on the cerebellum model, specific mechanisms at the cellular or molecular level have not been established to date. Recently, the researchers found that there is a new isoenzyme of phospholipase C, a key enzyme for signal transduction, and isolated and purified it (Min, DS, Kim, DM, Seo, JK, Suh, PG). , and Ryu, SH, J. Biol. Chem. 268, 12207 (1993)), and their complementary nucleic acids were cloned and named phospholipase C-beta4 (Kim, MJ, Bahk, YI, Min, DS, Lee, SJ, Ryu, SH, and Suh, PG, Biochem. Biophys.Res.Commun. (1993)).

Phospholipase C-beta4 also has a high degree of primary structural similarity to norp A phospholipase C, which is known to be essential for light signal transduction in Drosophila, and is known to be present in bovine retinas (Ferreira, PA, Shortridge, RD, Pak, WL). Proc. Natl. Acad. Sci., USA., 90, 6042 (1993). Therefore, phospholipase C-beta4 is thought to have both a physiological function for light signaling in the retina and a function for neurotransmission in the cerebellum, and thus the interest in its role is increasing. Moreover, the present inventors have reported that phospholipase C-beta4 is activated in connection with G-proteins present in the cell membrane fraction of C6Bu-1, glioma cells (Min, DS, Kim, Y., Lee). , YH, Suh, PG, and Ryu, SH, FEBS letter (1993)), phospholipase C-beta4 is further reported to be inactivated by G-proteins known to date. The physiological function of beta4 and the regulatory mechanisms of signal transduction are thought to be very complex.

As the results of these studies were reported, the importance of molecular studies on the signaling and regulatory mechanisms through phospholipase C-beta4 isozymes began to be recognized. In order to conduct studies on phospholipase C-beta4 isozymes present in trace amounts in cells, immunoblots using monoclonal antibodies that specifically recognize phospholipase C-beta4 isozymes should be used.

In order to develop a monoclonal antibody capable of specifically recognizing and binding only a phospholipase C-beta4 isozyme, the present inventors purely isolated and purified phospholipase C-beta4 isozyme, After immunizing Balb / c), spleens are isolated from these cells and fused with V653 cells, which are myeloma cells of mice, thereby releasing the monoclonal antibody while allowing for continuous cell culture. The present invention was completed by developing a method for producing a large amount of homogeneous antibody from the culture medium by culturing the cell line.

An object of the present invention is to provide a monoclonal antibody that specifically recognizes phospholipase C-beta4, a fusion cell line capable of producing the same, and a method for mass production of the monoclonal antibody by culturing the fusion cell line.

Another object of the present invention is to provide a method for detecting phospholipase C-beta4 using the monoclonal antibody.

Figure 1 shows the results of the immune blot for phospholipase C-beta4 of the monoclonal antibody of the present invention.

Figure 2 shows the results confirmed by immunoblot the specificity for phospholipase C-beta4 of the monoclonal antibody of the present invention.

Figure 3 shows the results of confirming the tissue-specific distribution of the mouse of the phospholipase C-beta4 by immunoblot using the monoclonal antibody of the present invention.

In order to achieve the above object, the present invention provides a monoclonal antibody protein capable of specifically recognizing and binding to a phospholipase C-beta4 isozyme, a fusion cell line which can be continuously cultured while secreting the monoclonal antibody protein, and Monoclonal antibody capable of specifically recognizing and binding to the phospholipase C-beta4 isoenzyme, which comprises the steps of culturing the fusion cell line or peritoneal culture, and then chromatography the supernatant or the peritoneal fluid of the cell culture. It provides a method of manufacturing.

In another aspect, the present invention provides a method for detecting and measuring phospholipase C-beta4 isozyme, characterized by using the monoclonal antibody protein.

The antibody of the present invention can be prepared as follows. In other words, a part of the complementary nucleic acid to phospholipase C-beta4 was made into a glutathione transferase fusion protein, overexpressed using an E. coli overexpression system, and then purified and purified from the extract of E. coli. Glutathione transferase phospholipase C-beta4 fusion protein was immunized to mice, and splenocytes and myeloma cells of the immunized mice were fused to obtain a hybrid cell line, and then the cells were cloned to clone phospholipase C. Monoclonal antibodies can be selected for production of antibodies that can only recognize beta4 isoenzymes and then cultured to produce large amounts of monoclonal antibodies.

Immunization of mice is performed according to conventional methods. That is, the phospholipase C-beta4 isozyme as an antigen is administered intraperitoneally, intravenously, subcutaneously, several times every 14 to 21 days, allowed to rest for one month, and then the antigen is administered again to give the total dose to mice. 80 to 200 µg per sugar. As a conventional adjuvant, Freund's complete adjuvant and incomplete adjuvant can be used as needed. Splenocytes are obtained 3 days after the final administration of the immune antigen and used as immune cells. Mouse myeloma cells used for cell fusion are those that have a survival rate of 95% or more and are grown in the mid-log phase of logarithmic growth.

Cell fusion between immunized spleen cells and myeloma cells is known or modified by, for example, Kohler's and Milstein's methods (Kohler, G. and Milstein, Nature, 256, 495 (1975)). Is performed by them. In order to mix the immunized cells and myeloma cells in a ratio of 1: 10 to 1: 5, and to promote fusion, polyethylene glycol having a molecular weight of about 1500 can be added (Boehringer Mannheim Cat. No. 783641). have. Such medium is generally used with fetal calf serum. The fused cells can be isolated by culturing in a conventional selection medium such as HAT medium (containing hypoxanthine, aminopterin, thymidine) to destroy unfused cells and selectively growing the fused cells.

The fusion cells thus obtained can be treated with known restriction dilution methods (Current Protocols in Immunology, 1991, Wiley Interscience) to obtain individual clones that produce the desired antibodies, resulting in the production of monoclonal antibodies. That is, first, the fusion cell population containing the fusion cell clones that produce the desired antibody is propagated to the middle of the logarithmic growth stage in HT medium of 24 well-plate, and then the dilution and culturing are continued step by step to produce monoclonal antibody. Clonal fusion cells are cloned.

Clones of fusion cells producing the desired antibody can be searched by enzyme immunoassay, Antibodies: A Laboratory Manual, Cold Spring Harbor, NY (1988), and in addition to conventional methods, namely plaque methods Methods such as the spot method, aggregation method, Ouchterlony method and radiolabeled immunoassay can be used to search for clones producing the desired antibody (Hybridoma Methods & Monoclonal Antibodies, Presearch). and Development, pp 30-53 (1982).

Mass production of the monoclonal antibody of the present invention can be carried out as follows. First, the fusion cell line producing the monoclonal antibody is cultured in DMEM medium containing 20% fetal bovine serum, and then the culture supernatant is collected by centrifugation. Alternatively, about 10 days after injecting the fusion cell line cultured as above into 1 x 107 cells / cell in the abdominal cavity of a mouse injected with 1 ml of pristane (2,6,10,14-tetramethyl pentadecane) in advance. The ascites is collected and then centrifuged to obtain a plurality of supernatants, which can be easily purified using conventional methods such as affinity chromatography.

The monoclonal antibodies of the present invention thus obtained have specific reactivity to phospholipase C-beta4 isoenzyme in all mammals as well as cattle, and signaling and regulatory mechanisms of phospholipase C-beta4 in cells It can be used as a good means of studying.

Hereinafter, the present invention will be described in more detail based on the following examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited thereto.

Example 1 Preparation of Fusion Cell Lines Secreting Monoclonal Antibodies to Phospholipase C-beta4 Isozyme

(Step 1) Immunity

For immunization, mice of Balb / c seeds 6 to 8 weeks of age (obtained from Korea Research Institute of Chemical Technology) were used. After overexpression using E. coli overexpression system, the same amount of complete Freund's adjuvant was added to 20 ㎍ of purified glutathione transferase phospholipase C-beta4 fusion protein to make a suspension, and 200 ㎕ per mouse. Thickly injected intraperitoneally. In the same manner, two injections at two-week intervals were allowed to rest for one month, followed by 500 μl of saline containing 25 μg of glutathione transferase phospholipase C-beta4 fusion protein (antigen) three days before cell fusion. Intraperitoneal injection.

(Step 2) Cell Fusion and Culture

Three days after the last immunization with the antigen, the spleens of the immunized mice were removed and washed several times with 10 ml of DMEM medium (Gibco BRL, Cat. No. 430-2800 EC) containing washing solution (10 mM Hepes, pH 7.2). Washed. Plasma cells were extracted from the spleen by dropping 10 ml of the washing medium drop by pressing on a 200 mesh (mesh) wire mesh. As a cell fusion countermeasure, V653 cells, which are myeloma cells of Valv mice, maintained by passage culture were used. The cells were cultured in 8-azaguanine medium (DMEM medium containing 20 μg / ml of 8-azaguanine, 20% fetal bovine serum and 50 μg / ml gentamycin) in logarithmic growth phase. Harvest by centrifugation when dividing into mid-log phase (1-4 x 105 cells / ml), suspended in 10 ml of washing medium, diluted 50-fold, and then hemocytometer (hemocytometer) The number was measured. Cell fusion was performed by a slightly modified method of Kohler and Milstein (Kohler, G. and Milstein, Nature, 256, 495 (1975)). Transfer 1 x 108 splenocytes and 1-2 x 107 V653 myeloma cells to a 50 ml centrifuge tube and wash them with the wash solution (DMEM medium containing 10 mM Hepes), which was then warmed to 37 ° C. beforehand. One 50% polyethylene glycol 1500 (PEG 1500, Boehriger Mannheim, Cat. No. 783641) was added dropwise to fuse. Cells precipitated by centrifugation were cultured in HAT medium (containing hypoxanthine, aminopterin, thymidine) to select transformed cells by fusion. Antibody screening was performed about 2 weeks after cell fusion.

(Step 3) Antibody Search

Search for fusion cells was performed using a secondary antibody. 50 µl of pure purified glutathione transferase (GST) protein and glutathione transferase-phospholipase C-beta4 fusion protein are respectively 5 µg / ml on different 96-well polystyrene plates. And adsorbed to the bottom of the plate. Take 50 μl of medium from the cell culture plate in which the fused cells are growing and place them in each well of the plate where glutathione transferlace (GST) protein and glutathione transferlace-phospholipase C-beta4 fusion protein are adsorbed, respectively. After inducing antibody binding, 50 μl of a solution of 1 μg / ml peroxidase-bound goat anti-mouse immunoglobulin G (peroxidase conjugated goat anti-mouse IgG) was added back to each well for binding. 50 μl peroxidase substrate solution (10 mg / ml acid and 0.02% hydrogen peroxide dissolved in 100 mM phosphate-citric acid buffer (pH 4.4)) was added to the wells, and the glutathione translace protein was adsorbed. A fusion cell that did not occur in the plate and showed a positive result only on the plate to which glutathione trasperase-phospholipase C-beta4 fusion protein was adsorbed was used as a fusion cell that produced an antibody against phospholipase C-beta4. Decided.

(Step 4) Cloning of Fusion Cells

For fusion cell cloning, first, after fusion of cells, the positive fusion cell group that searches for the fusion cells is transferred to a 24-well plate, and the cells are grown in HT medium so that the growth of the cells reaches the middle of the logarithmic growth phase. Incubation was continued cloning. As a result of the positive reaction, the clone specifically recognized only phospholipase C-beta4 as shown in Table 1 below, and named one of these as a fusion cell line β4-22-9. It was deposited on January 16, 1997 as Accession No. KCTC 0294BP to Gene Bank of Korea Institute of Biotechnology.

Example 2: Production of Monoclonal Antibodies Specific for Phospholipase C-beta4 Isozymes

(Step 1) Production of monoclonal antibodies through in vitro mass culture of fusion cell lines

The fusion cell line secreting monoclonal antibodies was adjusted to carbon dioxide at 5% in DMEM medium containing 20% fetal bovine serum and incubated at 37 ° C. When the cells were sufficiently grown and the color of the culture medium was yellow, the culture was continued by adding 10 times the fresh medium. By continuously increasing the cell number in this manner, the cells were allowed to grow sufficiently, centrifuged at 1000 x g for 10 minutes to remove the cells, and monoclonal antibodies in the supernatant were used.

(Step 2) Production of monoclonal antibody from ascites of mice by injecting fusion cell lines into mouse abdominal cavity

Fusion cells secreting monoclonal antibodies against phospholipase C-beta4 isoenzyme were cultured in the same manner as in (step 1) above, and then harvested and harvested 1 ml of pristane (2,6) 1 week before. 1 x 107 cells per horse were injected into the abdominal cavity of Valv mice with 10,14-tetramethyl pentadecane. After about 10 days from this, a plurality of 6-8 ml per horse were collected and centrifuged at 5000 x g for 10 minutes to obtain the supernatant.

(Step 3) Purification of the produced monoclonal antibody

The culture supernatant obtained in the above (Step 2) can be used for purification as it is. The plural supernatants obtained in (Step 2) were added with the same amount of binding buffer (ImmunoPure Binding buffer from Pierce), centrifuged at 1000 X g for 10 minutes, the supernatant was taken, and the protein A-Sepharose was previously equilibrated with the binding buffer. 4B column (protein A-sepharose 4B column, Pierce, Cat. No 20334) was loaded. After loading it was washed with binding buffer approximately 5 times the gel volume. The antibody bound to the column was eluted with an elution buffer (Pierce's ImmunoPure elution buffer), which is twice the volume of the gel, dialyzed in physiological saline, and then aliquoted in an appropriate amount and stored at -20 ° C.

Example 3: Specificity Assay for Phospholipase C-beta4 Isozyme of Monoclonal Antibodies

In order to assay the specificity of the monoclonal antibodies of the present invention, immunoblot and enzyme immunoassay were used. Each purified sample was electrophoresed on an 8% SDS-polyacrylamide gel to immobilize the isolated protein by electrophoresis on Coomassie blue staining (FIG. 1A) and nitrocellulose membrane. Blots (FIGS. 1B and C) were performed. To stain proteins with Coomassie Blue, electrophoresis-separated gels were stained with Coomassie Brilliant Blue per ml of solution (ethanol, acetic acid, distilled water 5: 1: 4). R-250) was dissolved in 0.1%) for 30 minutes and then decolorized with a destaining solution (ethanol, glacial acetic acid, distilled water mixed at a ratio of 5: 2: 15).

To perform the immunoblot, the margins of the nitrocellulose membrane were combined with a 5% skim milk solution, followed by 5% of the primary antibody solution (culture containing monoclonal antibody to the phospholipase C-beta4 isozyme of the present invention). Diluted 1/5 with physiological saline containing skim milk powder and reacted at room temperature for 4 hours. At the end of the reaction, the primary antibody solution was removed, washed three times for 10 minutes with a wash solution (0.05% Tween 20 dissolved in physiological saline), and 0.5 mg / ml goat anti-mouse immunoglobulin with peroxidase. (KPL, Co. 141807) was reacted for 2 hours with a nitrocellulose membrane in a solution diluted 5000-fold with physiological saline containing 5% skim milk powder, and then washed in the same manner as the primary antibody. Color development was performed by adding a blot film to ECL substrate solution (Amersham).

1 is a result of an immunoblot against phospholipase C-beta4 of the monoclonal antibody of the present invention, wherein (A) is a pure purified glutathione transferase (GST) protein and glutathione transferase-phospholipase C-beta Coomassie blue staining of the fusion protein, (B) is the result of the immunoblot using an antibody against glutathione transferase protein, (C) is a result of the immunoblot using the antibody of the present invention. In addition, Sample No. 1 is a purified glutathione transferase protein, and Sample No. 2 represents glutathione transferase-phospholipase C-beta4 fusion protein, respectively.

Figure 2 shows the specificity for the phospholipase C-beta4 of the monoclonal antibody of the present invention by immunoblot, the antibody of the present invention does not recognize other phospholipase C enzymes and only purified phosphate in the cerebellum It can be seen that only the lipase C-beta4 protein is specifically recognized. Sample numbers 1 to 7 represent isozymes of phospholipase C-beta1, beta2, beta4, gamma1, gamma2, delta1 and delta2, respectively. Using the enzyme immunoassay, the antibody was a homogeneous protein of immunoglobulin G1 produced in mice against phospholipase C-beta4 enzymes as shown in the results in Table 1 and other phospholipase C enzymes were recognized. It can be seen that only the phospholipase C-beta4 protein purified in the cerebellum is specifically recognized.

Example 4: Measurement of phosphopyrease C-beta4 isozyme distribution by tissues of mice using monoclonal antibodies

After killing the rats, the tissues were immediately removed and the grinding buffer (20 mM Tris hydrochloride, pH 7.6, 1 mM EDTA, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 0.1 mM dithiothreitol, 10 μg / ml leupetin, 10占 // ml aprotinin) was added and ground, and the supernatant obtained by centrifugation at 500 xg for 5 minutes was centrifuged at 10,000 xg for 1 hour. The obtained precipitate was extracted with 3M salt, dialyzed and used for measurement. Samples of dialysis mouse tissues were centrifuged to obtain supernatant, electrophoresis by loading a certain amount of total protein into the gel, and then the proteins separated from the gel was transferred to the nitrocellulose membrane. Immunoblotting was performed using the antibody of the present invention in the same manner as in Example 3.

Figure 3 is a result of confirming the tissue-specific distribution of the mouse of the phospholipase C-beta4 by immunoblot using the monoclonal antibody of the present invention, wherein the sample number 1 is a phospholipase C- purified from the cerebellum of bovine Beta4 isoenzyme, 2 eyes, 3 thymus, 4 thyroid, 5 cerebellum, 6 cerebral, 7 heart, 8 lung, 9 liver, 10 spleen, 11 kidney, 12 pancreas 13 represents the uterus and 14 represents the testicle. Using this antibody, phospholipase C-beta4 isoenzyme is mainly present in the brain. Especially, the protein having a molecular weight of 13,500, the same size as the phospholipase C-beta4 isoenzyme purified from cerebellar tissue, is the most abundant. It confirmed that there was. These results can be seen to confirm the specificity of the antibody.

As described above, the monoclonal antibody of the present invention has specific reactivity to not only bovine but also phospholipase C-beta4 isozymes of other mammals, and is a basic biology for studying the physiological activity, differentiation and growth regulation of mammalian cells. It can be used as a good means of research.

Claims (5)

A monoclonal antibody protein capable of specifically recognizing and binding to phospholipase C-beta4 isoenzyme. A fusion cell line that can be continuously cultured while secreting the antibody of claim 1. The fusion cell line β4-22-9 (KCTC 0294BP) according to claim 2. Cell culture or intraperitoneal culture of the fusion cell line of claim 2 or 3, followed by chromatography of the supernatant or peritoneal fluid of the cell culture, specifically recognizing and binding to the phospholipase C-beta4 isoenzyme Method for producing a monoclonal antibody that can be. A method for detecting and measuring phospholipase C-beta4 isozyme, using the monoclonal antibody protein of claim 1.

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