KR101142418B1 - Antibody against romo1, hybridoma producing the same and uses thereof - Google Patents

Abstract

The present invention relates to an antibody against Romo1 protein that specifically binds to Romo1 protein by recognizing one or more of epitopes between 8 and 20 and epitopes between 44 and 56 of SEQ ID NO: 1. It relates to a hybridoma producing the same and its use.

Description

Antibodies against the RMO protein, hybridomas producing the same, and uses thereof {ANTIBODY AGAINST ROMO1, HYBRIDOMA PRODUCING THE SAME AND USES THEREOF}

The present invention relates to an antibody against Romo1 (Reactive Oxygen Species Modulator 1) protein having the amino acid sequence set forth in SEQ ID NO: 1 and its use. Specifically, the present invention recognizes one or more of epitopes existing between 8 and 20 and 44 through 56 of SEQ ID NO: 1 to Romo1 protein that specifically binds to Romo1 protein. An antibody, a hybridoma producing the same, and a use thereof.

Excessive increase in free radicals in cells causes various diseases. In particular, non-ideally increased free radicals cause chronic inflammation, which can cause cancer, aging, arthritis, sclerosis, Alzheimer's disease and diabetes. It acts as a major cause of diabetes, fibrosis, and inflammatory bowel diseases.

The production of free radicals is mainly due to ROS leakage in mitochondria, which induce various diseases of the human body. Although mitochondria-derived free radicals are closely related to disease, the mechanism of their production is not well understood. In this regard, Romo1, a protein located in mitochondria, has recently been discovered, and it has been reported to play an important role in generating free radicals in mitochondria. (Chung Y.M. et al., Biochem Biophys Res Commun. 347: 649-655, 2006)

A representative example of the abnormal increase in free radicals involved in the disease is TNF-alpha induced disease. TNF-alpha is an important factor that causes inflammation. If the amount is uncontrolled and continuously increases or the signaling pathway is excessively activated, it causes chronic inflammation, and this chronic inflammation causes TNF-alpha induced disease. At this time, examples of diseases induced by TNF-alpha include sepsis, diabetes, osteoporosis, agraft rejection, multiple sclerosis, and rheumatoid arthritis. ), Inflammatory bowel diseases, ischemia / reperfusion injury, pulmonary fibrosis, neuronal degeneration (ischemic stroke, Alzheimer's disease, Parkinson's disease), etc. (Balkwill F. et al) , Nature 431: 405-406, 2004; Chen G and David VG Science 296: 1634, 2002).

Recently we found that TNF-alpha produces free radicals through Romo1 and induces cell death and filed a patent in July 2009 (Korean Patent Application No. 10-2009-0062255). That is, when Romo1 is increased quantitatively by external stress, excessive free radical production and induction of apoptosis by TNF-alpha are promoted, resulting in chronic inflammation and TNF-alpha induced disease. Therefore, when an antibody against Romo1 is developed, a disease induced by TNF-alpha can be diagnosed in advance by detecting the Romo1 protein using the antibody.

Conventionally known antibodies against Romo1 include polyclonal antibodies produced and sold by the SIGMA-ALDRICH company in the United States. The antibody was produced by injecting rabbits with 31 amino acid sequences of the amino acid sequences of Romo1 protein described in SEQ ID NO: 1. However, the antibody can be used only for immunohistochemistry, but cannot be used for Western blotting, etc., and thus the use of the antibody is limited. In addition, even when analyzed by immunohistochemistry, it is shown that the Romo1 protein is located in the nucleus when the antibody is used. The Romo1 protein is reported to be located in the mitochondria.

Therefore, there is an urgent need in the art for the development of new antibodies that can be used in a variety of analytical methods or detection methods, including Western blotting, while having high specificity for Romo1.

The present invention is to solve the problems of the prior art as described above, the object of the present invention has a high specificity for Romo1 protein, Western blotting, immunofluorescence, immunohistochemistry (staining) method, enzyme immunoassay ( The present invention provides a new Romo1 antibody which can be used in various analytical methods or detection methods such as ELISA) to obtain accurate experimental results.

Another object of the present invention is to provide a hybridoma that produces the antibody against Romo1.

Still another object of the present invention is to provide a composition and diagnostic kit for diagnosing the use of the antibody against Romo1, in particular TNF-alpha induced disease comprising the antibody.

In order to achieve the above object, the present invention provides an antibody against the Romo1 protein having the amino acid sequence set forth in SEQ ID NO: 1, produced by a hybridoma with accession number KCLRF-BP-00226.

The present invention also provides a hybridoma of Accession No. KCLRF-BP-00226 for producing the above antibody.

In another aspect, the present invention provides a composition and diagnostic kit for diagnosing TNF-alpha induced disease, comprising the above antibody.

The present invention also provides a method for detecting Romo1 protein, which is a marker of TNF-alpha induced disease, using the above antibody.

The antibody of the present invention can specifically recognize the Romo1 protein, and can be used without limitation in various kinds of assays or detection methods including Western blotting, immunofluorescence, immunohistochemistry, enzyme immunoassay (ELISA), and the like. Therefore, it is useful for accurately preventing and diagnosing diseases caused by abnormal expression of Romo1, such as diseases induced by Romo1 and TNF-alpha. In addition, the antibodies of the present invention can be used to specifically recognize, detect, and isolate Romo1 protein in a sample.

Figure 1 shows the results of the antigenic analysis of the amino acid sequence (SEQ ID NO: 1) of the Romo1 protein.
Figure 2 is a Western blotting result of measuring the Romo1 protein using the Romo1 antibody of the present invention obtained in Example 2 after transfection of a HeLa cell line with a Romo1 siRNA to reduce the amount of Romo1 protein.
Figure 3 is a Western blotting result of measuring the Romo1 protein using a polyclonal antibody against Romo1 purchased from Sigma-Aldrich after reducing the amount of Romo1 protein by injecting Romo1 siRNA into the HeLa cell line.
Figure 4 shows the results of immunofluorescence experiments staining the expression of Romo1 protein using an antibody against the Romo1 protein.
FIG. 5 shows that Romo1 binds to RIP, TRADD, TRAF2, and FADD proteins, which are complex II constituent proteins that transmit apoptosis signals in the TNF-alpha signaling pathway when TNF-alpha is treated by treating TNF-alpha in HEK 293 cells. Western blotting results show that.

Hereinafter, the configuration and operation of the present invention will be described in detail.

The present invention provides an antibody against the Romo1 protein, specifically an antibody against the Romo1 protein having the amino acid sequence set forth in SEQ ID NO: 1, produced by a hybridoma with accession number KCLRF-BP-00226.

* SEQ ID NO 1:

MPVAVGPYGQSQPSCFDRVKMGFVMGCAVGMAAGALFGTFSCLRIGMRGRELMGGIGKTMMQSGGTFGTFMAIGMGIRC

As used herein, the term “Romo1 protein” includes the Romo1 protein itself, its recombinant protein and its artificial or mutant variants, as well as the natural forms of Romo1 protein and functional equivalents thereof.

Romo1 protein is the first inventors of the world to identify its function. Initially, the protein was found to be involved in anticancer drug resistance and was named Chemp-1 (Chemoresistant Protein-1), but subsequent studies found that the protein also produced free radicals. (Chung YM et al., Biochem Biophys Res Commun. 347: 649-655, 2006).

Following the above studies, we have recently discovered that Romo1 plays an important role in its intermediate signaling pathway when TNF-alpha induces inflammatory diseases (Korean Patent Application No. 10-2009-0062255). Thus, the inventors have realized that Romo1 is available as a new target in the diagnosis of diseases induced by TNF-alpha, and on the basis of such findings produced antibodies to the Romo1 protein.

The antibody against the Romo1 protein according to the present invention recognizes one or more of epitopes existing between 8 and 20 and epitopes existing between 44 and 56 in the sequence of SEQ ID NO: 1 (amino acid sequence of the Romo1 protein). Specifically bind to Romo1 protein.

The amino acid sequence (YGQSQPSCFDRVK; peptide 1) and the amino acid sequence 44 to 56 (RIGMRGRELMGGI; peptide 2) of SEQ ID NO: 1, the present inventors analyzed the antigenicity (antigenicity) against the amino acid sequence of Romo1 protein As a result, in the parts with the highest antigenicity, the present inventors prepared the antibodies according to the present invention using these peptides. Therefore, the antibody according to the present invention has high binding specificity for the peptide 1, the peptide 2 or the fragment comprising the same, and the Romo1 protein.

Meanwhile, the antibody according to the present invention is an antibody against one antigen, that is, the Romo1 protein, and includes not only the whole antibody form but also a functional fragment of the antibody molecule.

The total antibody is a structure having two full length light chains and two full length heavy chains, each of which is linked by heavy and disulfide bonds. The heavy chain constant region has gamma (γ), mu (μ), alpha (α), delta (δ) and epsilon (ε) types, and subclasses gamma 1 (γ1), gamma 2 (γ2), and gamma 3 ( γ3), gamma 4 (γ4), alpha 1 (α1) and alpha 2 (α2). The constant regions of the light chains have kappa (κ) and lambda (λ) types.

The functional fragment of an antibody molecule means the fragment which has antigen binding function, and includes Fab, F (ab '), F (ab') 2, Fv, etc. Fab in the antibody fragment has a variable region of the light and heavy chains, a constant region of the light chain and the first constant region (CH1) of the heavy chain, and has one antigen binding site. Fab 'differs from Fab in that it has a hinge region comprising at least one cysteine residue at the C terminus of the heavy chain CH1 domain. In the F (ab ') 2 antibody, a cysteine residue in the hinge region of Fab' forms a disulfide bond. Fv is a minimal antibody fragment having only a heavy chain variable region and a light chain variable region. Recombinant techniques for generating Fv fragments are disclosed in WO 88/10649, WO 88/106630, WO 88/07085 and WO 88/09344. It is. dsFv (double-chain Fv) is non-covalently linked to the heavy chain variable region and the light chain variable region, scFv (short chain Fv) is generally covalently linked to the variable region of the heavy chain and short-chain variable region through a peptide linker or C Directly connected at the end can form a dimer-like structure, such as dsFv.

Such antibody fragments can be obtained using proteolytic enzymes (e.g., the whole antibody can be restrictionly cut with papain to obtain a Fab, and cleavage with pepsin can yield an F (ab ') 2 fragment). Preferably it can be produced through genetic recombination techniques.

Such antibodies to the Romo1 protein of the present invention can be made by a general fusion method (fusion method). To make hybridomas that secrete antibodies of the invention, one of the two cell populations to be fused uses cells from an immunologically suitable host animal, such as a mouse injected with a Romo1 protein or peptide fragment thereof, One population uses cancer or myeloma cell lines. These two populations of cells are fused by methods known in the art, such as polyethylene glycol, and then antibody-producing cells are propagated by standard tissue culture methods. Hybridomas capable of producing homogeneous populations by cloning by the limited dilution technique and then producing antibodies specific for Romo1 protein or peptide fragments thereof in vitro or in vivo according to standard techniques Incubate in large quantities.

The antibodies produced by the hybridomas may be used in the state of cell culture containing the same without purification, or may be purified and used according to methods well known in the art. Purification of the antibody can be carried out from the culture medium or ascites using purification methods such as gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, affinity chromatography, and the like.

The hybridoma producing the antibody according to the present invention was named Romo1-AS-1 and was deposited on January 5, 2010 to which accession number KCLRF-BP-00226 was assigned.

Meanwhile, the present inventors reduced the amount of Romo1 protein by transfection with a Hemo cell line, and then analyzed by Western blotting using the antibody of the present invention, unlike Sigma-Aldrich's existing antibody, It was confirmed that the antibody of the invention can analyze the Romo1 protein well even by Western blotting. In addition, as a result of examining the intracellular expression of Romo1 protein by immunofluorescence using the antibody of the present invention, it was confirmed that the Romo1 protein is located in the mitochondria as known in the art.

Therefore, the antibody against Romo1 of the present invention can be freely used for various analytical methods and detection methods such as Western blotting and immunofluorescence as well as immunohistochemistry and enzyme immunoassay, to effectively detect or isolate Romo1 protein.

Furthermore, the present inventors confirmed that when Romo1 siRNA was used to reduce Romo1 expression, an increase in free radicals produced by TNF-alpha was blocked and cell death by TNF-alpha was reduced. These experimental results show that Romo1 is an important mediator of reactive oxygen production by TNF-alpha and apoptosis by TNF-alpha. Apoptosis induced by TNF-alpha is directly linked to inflammatory diseases induced by TNF-alpha (Leist M and Jaattela M. Nat. Rev. Mol. Cell Biol. 2: 589598, 2001). According to the results, Romo1 can be a diagnostic marker for diseases induced by TNF-alpha.

Accordingly, the present invention provides a composition and diagnostic kit for diagnosing a variety of diseases, preferably TNF-alpha-induced diseases, caused by aberrant expression of Romo1 or resulting in aberrant expression of Romo1, including the antibody against the Romo1 protein. to provide.

Specifically, examples of the TNF-alpha induced disease include sepsis, diabetes, osteoporosis, transplant rejection, multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease, ischemic disease, pulmonary fibrosis, neurodegenerative disease, and the like. It is not limited.

The diagnostic compositions and diagnostic kits of the invention include other antibodies, such as polyclonal antibodies, chimeric-antibodies, humanized-antibodies, monoclones, as long as the antibodies of the invention, as well as the Romo1 protein can be selectively recognized. Not only raw antibodies, but also fragments of monoclonal antibodies can be used. Such antibody fragments include F (ab ') 2, Fab, Fab', Fv fragments and the like.

In addition, the diagnostic composition and diagnostic kit of the present invention may include a tool / reagent used for immunological analysis. Such tools / reagents include suitable carriers, labeling substances capable of producing detectable signals, solubilizers, detergents and the like. In addition, when the labeling substance is an enzyme, it may include a substrate capable of measuring enzyme activity and a reaction terminator.

Suitable carriers include, but are not limited to, soluble carriers such as physiologically acceptable buffers known in the art, such as PBS; Insoluble carriers such as polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, fluorine resins, crosslinked dextran, polysaccharides, polymers such as magnetic fine particles plated on latex, other paper, glass, metal , Agarose and combinations thereof.

Assay systems for use in the diagnostic compositions and diagnostic kits of the invention include, but are not limited to, ELISA plates, dip-stick devices, immunochromatographic test strips, radiosegmented immunoassay devices, flow-through ) Devices and the like.

The present invention also provides a method for detecting a marker of TNF-alpha induced disease by contacting the antibody with a biological sample to detect antigen-antibody complex formation.

In the present invention, the term "marker of TNF-alpha-induced disease" refers to a substance capable of detecting the inflammatory disease induced by TNF-alpha through detection and comparison in tissues. For the purposes of the present invention, “markers of TNF-alpha induced disease” in compositions, diagnostic kits and methods comprising or using the antibodies of the invention refer to the Romo1 protein.

As used herein, the term “biological sample” includes but is not limited to tissues, cells, whole blood, serum, plasma, cerebrospinal fluid, tissue autopsy samples (brain, skin, lymph nodes, spinal cord, etc.), cell culture supernatants, or supernatants. It doesn't work. The biological sample may be collected from various animals such as humans, rabbits, pigs, mice, and the like, but is not limited thereto. These biological samples can be used to diagnose TNF-alpha induced diseases by reacting with or without the engineered antibodies of the present invention.

As used herein, the term “antigen-antibody complex” refers to a combination of the protein and an antibody that recognizes it to identify the presence or absence of a Romo1 protein in a biological sample. Preferably the antigen-antibody complex is a Romo1 protein-complex of the antibody according to the invention.

Formation and detection of such antigen-antibody complexes include enzyme immunoassay (ELISA), western blotting, immunofluorescence, immunohistochemistry staining, flow cytometry, immunocytochemistry Method, radioimmunoassay (RIA), immunoprecipitation assay, immunodiffusion assay, complement fixation assay, protein chip or kit, and the like, but are not limited thereto. Does not. Preferably, western blotting, enzyme immunosorbent method, protein chip or kit is used.

Labels that enable qualitative or quantitative measurement of antigen-antibody complex formation include, but are not limited to, enzymes, fluorescent materials, ligands, luminescent materials, microparticles, redox molecules, and radioisotopes. .

Enzymes available as detection labels include β-glucuronidase, β-D-glucosidase, β-D-galactosidase, urease, peroxidase, alkaline phosphatase, acetylcholinesterase, glucose oxy Multidase, Hexokinase and GDPase, RNase, Glucose Oxidase and Luciferase, Phosphofructokinase, Phosphoenolpyruvate Carboxylase, Aspartate Aminotransferase, Phosphopyruvate Decarboxylase, β- Latamases and the like, but are not limited thereto. Fluorescent materials include, but are not limited to, fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde, fluorescamine, and the like. Ligands include, but are not limited to, biotin derivatives. Luminescent materials include, but are not limited to, acridinium ester, luciferin, luciferase, and the like. Microparticles include, but are not limited to, colloidal gold, colored latex, and the like. Redox molecules include ferrocene, ruthenium complex, biologen, quinone, Ti ion, Cs ion, diimide, 1,4-benzoquinone, hydroquinone, K ₄ W (CN) ₈ , [Os (bpy) ₃ ] ²⁺ , [RU (bpy) ₃ ] ²⁺ , [MO (CN) ₈ ] ^4-, and the like. Radioisotopes include, but are not limited to, ³ H, ¹⁴ C, ³² P, ³⁵ S, ³⁶ Cl, ⁵¹ Cr, ⁵⁷ Co, ⁵⁸ Co, ⁵⁹ Fe, ⁹⁰ Y, ¹²⁵ I, ¹³¹ I, ¹⁸⁶ Re, and the like.

Hereinafter, the configuration of the present invention will be described with reference to Examples, but the scope of the present invention is not necessarily limited thereto.

Example 1 Antigenicity and Epitope Determination of Romo1 Amino Acid Sequences

For the amino acid sequence of the Romo1 protein set forth in SEQ ID NO: 1, the Jameson-Wolf antigenicity index values were obtained using dnastar software (DNASTAR) to analyze the parts of Romo1 that are expected to be antigenic (Jameson and Wolf). , 1988 BA Jameson and H. Wolf, The antigenic index: a novel algorithm for predicting antigenic determinants, CABIOS 4 (1988), pp. 181186).

As a result, as shown in Figure 1, the amino acid sequence (YGQSQPSCFDRVK; peptide 1) and the amino acid sequence of the 44 to 56 amino acid sequence (RIGMRGRELMGGI; peptide 2) of SEQ ID NO: 1 is the highest appear. Therefore, these portions were selected as epitopes, and peptides were synthesized, respectively.

Example 2: Preparation of Hybridoma Cell Line Producing Antibodies to Romo1 Protein and Purification of the Antibodies

<2-1> Preparation of hybridoma cell line and confirmation of antibody generation (antigen-antibody reactivity)

Two peptides (peptide 1 and peptide 2) selected as epitopes in Example 1 were injected subcutaneously into BALB / c mice. In the first injection, two peptides (10 μg each) were injected with Complete Freund's Adjuvant (Sigma-Aldrich). IFA (Incomplete Freund's adjuvant; Sigma-Aldrich) was then further injected three times at two week intervals with the peptide. After 14 days, only peptides were further injected without adjuvant, and again 3 days later, splenocytes were fused with mouse myeloma cells (ATCC CRL-1645). After fused cells were cultured using HAT medium (Sigma-Aldrich) in a 96-well plate, hybridoma cell lines producing antibodies against Romo1 protein were selected using the ELISA method as follows.

First, the peptide 1 and peptide 2 were separately coated in wells of a 96-well plate at 3 ug / ml, respectively, and then 100 μl of the hybridoma cell culture obtained above was continuously diluted 1/10 for each well to each well. Added. After three washes, 50 μl of HRP-conjugated goat anti-mouse immunoglobulin (IgG) (Fc-specific) antibody with secondary antibody; Pierce] was then incubated at 37 ℃ for 1 hour. After washing three more times, 0.2 M citrate-PO ₄ buffer (pH 5.0) containing 0.04% ortho-phenylenediamine dihydrochloride and 0.012% H ₂ O ₂ was added. Then, after stopping the reaction with 2.5 MH ₂ SO ₄ solution, the absorbance at a wavelength of 490 nm was measured using an ELISA reader (SOFTmaxPRO; Molecular Devices, United States).

The results are shown in Tables 1 and 2 below. Table 1 shows the result of measuring the absorbance at 490 nm wavelength by coating peptide 1 (amino acid sequence No. 8-20) on a 96-well plate and adding a hybridoma cell culture solution obtained from mice. From the results of Table 1, the experimental cultures showed higher absorbance than the comparative hybridoma cultures obtained from mice not injected with the Romo1 peptide, indicating that the antibody to peptide1, ie, the Romo1 protein, containing the same, was properly produced. .

Comparative hybridomas Romo1 Antibody Production Hybridomas 10 ^ 4 * 0.144 2.601

(*: Indicates that the culture was diluted 10,000-fold)

Table 2 shows the result of measuring the absorbance at 490 nm wavelength by coating peptide 2 (amino acid sequence No. 44-56) on a 96-well plate and adding a hybridoma cell culture solution obtained from the mouse. As shown in the results of Table 2, similar to the experimental results for the peptide 1, the culture medium, the absorbance was higher than the comparative hybridoma culture solution obtained in the mice not injected with the Romo1 peptide, the peptide 2, that is, the Romo1 protein containing the same It can be seen that the antibody against.

Comparative hybridomas Romo1 Antibody Production Hybridomas 10 ^ 4 0.144 1.701

The hybridomas tested in the above were selected as the Romo1 antibody-producing hybridomas of the present invention, and were assigned the accession number KCLRF-BP-00226.

<2-2> Purification of Antibody Against Romo1 Protein

The cells selected with the hybridoma of the present invention were cultured for 5 days [basic medium: DMEM (Gibco 11995-065), complete medium: DMEM with 10% (v / v) FBS], and then the cell culture medium was 2,500 rpm. The supernatant was collected by centrifugation at 4 ° C. for 5 minutes and used for antibody purification. 60 μl of Protein G-agarose (Sigma-Aldrich) was then washed twice with NETN buffer (20 mM Tris buffer (pH 8.0), 100 mM NaCl, 1 mM EDTA, 0.5% NP-40). It was. The washing was centrifuged (5,000 rpm, 1 minute, 4 ° C.) to precipitate protein G-agarose, and the supernatant was discarded. This method was repeated once more. Then, protein G-agarose was washed twice again using 400 μl 20 mM sodium phosphate buffer. 400 μl of the cell culture solution was added to the precipitate containing the protein G-agarose and the antibody was adsorbed at 4 ° C. for 2 hours. Antibody-adsorbed protein G-agarose was washed twice using PBS buffer and centrifugation. After washing, 30 μl of glycine buffer (pH 3.0) was added to protein G-agarose to which the antibody was adsorbed to separate the antibody from protein G-agarose. After centrifugation, the supernatant was taken and used as an antibody against Romo1.

The following experiment was performed using the antibody against the Romo1 protein obtained above.

Experimental Example 1: Western blotting using an antibody against Romo1 protein

<1-1> Cell Culture

Cervical cancer cells (HeLa) and human embryonic kidney cells (HEK 293) were purchased from the American Cell Line Bank (ATCC). These cells were cultured in DMEM / F12 culture medium (GIBCO / BRL, Grand Island, NY) containing 10% fetal calf serum, penicillin 100 units / ml, and 100 μg / ml streptomycin.

<1-2> Intracellular Injection of Romo1 siRNA-Reduced Expression of Romo1 Protein

HeLa cells were aliquoted into 6 well plates at 1 × 10 ⁵ , and cultured. After 24 hours, HeLa cells were washed once with serum-free medium containing no antibiotics. Then, control siRNA, Romo1 siRNA (100 nM) were added to the cells in 5 μl of lipofectamine. After 6 hours, the cells were washed with serum-free medium twice and then serum medium was added.

Romo1 siRNA base sequences are as follows: 5'-CUA CGG ACA GUC CCA GCC A (dTdT) -3 '(sense) and 5'-UGG CUG GGA CUG UCC GUA G (dTdT) -3' (antisense).

<1-3> western blotting

Cancer cells were harvested and RIPA buffer (NaCl 150 mM, 1% Nonidet P-40, 0.5%) containing a phosphate inhibitor (1 mM sodium ortho vanadate, NaF 30 mM, phenyl methylsulfonyl fluoride 1 mM, NaPPi 30 mM) Deoxycholic acid, 0.1% SDS, Tris 50 mM, pH 8.0). Protein concentration was determined by the Virorad Protein Assay Kit (BioRad, Hercules, CA). 50 μg of each of the 10% sodium dodecyl sulfate-polyacrylamide gel was loaded and electrophoresed, and then transferred to the nitrocellulose membrane. The membrane was blocked at room temperature with PBS-T buffer (50 mM Tris, pH8.0, 150 mM NaCl, 0.1% Tween-20) containing 5% skim milk, and Romo1 to see protein expression. The antibody was reacted for 1 hour and then washed three times with PBS-T buffer. Subsequently, a secondary antibody (Sigma-Aldrich), which is bound to horse radish peroxidase (HRP) and binds to an antibody made in rats, was reacted and detected by an ECL detection kit (Amersham, USA).

<1-4> Comparative Experiment

Meanwhile, the same experiment was performed using the polyclonal antibody against Romo1 purchased from Sigma-Aldrich for comparison.

The experimental results are shown in FIGS. 2 and 3.

2 is a result of Western blotting of Romo1 protein measured using a Romo1 antibody of the present invention obtained in Example 2 after transfection of a HeLa cell line with a Romo1 siRNA to reduce the amount of Romo1 protein. Romo1 protein was not reduced in expression by injecting control siRNA into the comparative group using the Romo1 antibody of the present invention. 2, it can be seen that the antibody of the present invention can detect or analyze Romo1 protein well by Western blotting method.

Figure 3 is a Western blotting result of measuring the Romo1 protein using a polyclonal antibody against Romo1 purchased from Sigma-Aldrich after reducing the amount of Romo1 protein by injecting Romo1 siRNA into the HeLa cell line. In this case as well, the control siRNA was injected into the comparative group, and the expression of Romo1 protein, which was not reduced, was measured using the polyclonal antibody. From the results of FIG. 3, it can be seen that the conventional polyclonal antibody against Romo1 does not detect Romo1 protein by Western blotting. At this time, the band shown at 11 kDa is a nonspecific band.

Experimental Example 2: Immunofluorescence Using Antibody Against Romo1 Protein

Cover glass coated with 0.1% gelatin was prepared and placed in a 6 well plate. HeLa cell lines were dispensed at 2 × 10 ⁴ cells / well and incubated for one day. The cultured cells were washed with PBS and then fixed with 4% formaldehyde PBS for 10 minutes at room temperature and washed with PBS. After incubation on ice for 10 minutes with 1 ml of a permeabilisation solution (0.1% Triton X-100 in 0.1% sodium citrate), it was washed twice with PBS again. It was then blocked with 0.1% BSA / 0.1% Tx-100 / PBS solution for 30 minutes and washed twice with PBS. Antibody treatment was performed by diluting the Romo1 antibody obtained in Example 2 to 1: 200 with 1% BSA / PBS-Tx (0.1%) for 60 minutes in a 37 ° C. incubator. Then washed twice with PBS. Subsequently, the anti-mouse IgG was diluted 1: 250 with 1% BSA / PBS-Tx (0.1%), incubated in a 37 ° C. incubator for 60 minutes, and washed twice with PBS. To stain the nucleus, DAPI (biotium, CA, # 40011) was incubated for 15 minutes at room temperature in which light was blocked at a concentration of 2 μg / ml, and the cells were washed with PBS. 20 μl of mounting medium (H-1000, Vector shield, Vector Laboratories, Calif.) Was placed on the slide glass and covered with the cover glass where the cells were fixed. The cover glass was mounted on the slide glass with a transparent nail polish, dried and observed with an Olympus LX71 microscope.

The results are shown in FIG. 4 is a result of immunofluorescence experiment staining the expression of Romo1 protein using a Romo1 antibody. In Figure 4 A is a result showing the location of the mitochondria by staining the mitochondria using Mito-G. B is a result showing the Romo1 protein staining using an antibody to the Romo1 protein. C is the result of staining the nucleus with DAPI and overlapping the A and B pictures. As can be seen from the results of FIG. 4, the antibody of the present invention detects Romo1 protein well by immunofluorescence assay.

As a result, the antibody of the present invention has a high binding specificity for Romo1 protein, and can be used without limitation for various analytical methods such as Western blotting, immunofluorescence, ELISA, and the like, thereby analyzing, detecting, or isolating the expression of Romo1 protein. It can be seen that it can be effectively used in the required technical field.

Experimental Example 3: Identification of the role of Romo1 as an intermediate mediator in TNF-alpha induced free radical increase and cell death

<3-1> Cell Culture and Intracellular Injection of Romo1 siRNA

It carried out similarly to <1-1> and <1-2> of Experimental Example 1.

<3-2> Free radical measurement

Cover glass coated with 0.1% gelatin was prepared and placed in a 6 well plate. Cells were dispensed 2 x 10 ⁴ per well. After incubation for 24 hours, CHX and TNF-alpha were treated. In order to quantify the active oxygen, the active oxygen probe (probe) DCF-DA (2,7-dichlorofluorescein diacetate) was blocked for 30 minutes in foil at a concentration of 10 μM, treated with cells in a 37 ℃ thermostat, and then Cells were washed twice with PBS. 20 μl of mounting medium (H-1000, Vector shield, Vector Laboratories, Calif.) Was placed on the slide glass and the cover glass on which the cells were fixed was covered. The cover glass was mounted on a slide glass with a transparent nail polish, dried and observed with an Olympus LX71 microscope. Free radicals were measured using Metamorph software (Universal Imaging, Westchester, PA).

<3-3> Cell death rate measurement after TNF-alpha treatment

Dispense the cells in 6 well plates by 1 x 10 ⁵ and incubate. After 24 hours, wash the cells with serum-free medium containing no antibiotics, and then control siRNA, Romo1 siRNA (100 nM), and 5 μl of lipofectamine. Was added to the cells. After 6 hours, the cells were washed twice with serum-free medium and then cultured with serum medium. After 24 hours in 6 well plate After removing the cells by treating the trypsin-EDTA, it was dispensed to ensure that the 2 ml culture medium can be 2 x 10 ⁴ cells. After treatment with TNF-alpha and CHX, the number of living cells was measured by mixing with trypan blue (0.4%).

<3-4> Immunoprecipitation Assay and Western Blotting

Romo1-Flag hybrid genes linked to Flag DNA to Romo1 were injected into cells and harvested to contain phosphate inhibitors (1 mM sodium ortho vanadate, NaF 30 mM, phenyl methylsulfonyl fluoride 1 mM, NaPPi 30 mM). Solubilized in RIPA buffer (NaCl 150 mM, 1% Nonidet P-40, 0.5% deoxycholic acid, 0.1% SDS, Tris 50 mM, pH 8.0). Protein concentration was determined by the Virorad Protein Essay Kit (BioRad, Hercules, CA). Flag antibody was added to bind to Romo1-Flag protein. Protein A agarose was added to the flag antibody bound to Romo1-Flag to form a complex of Romo1-Flag protein, Romo1-Flag antibody and protein A agarose. The complex was precipitated by centrifugation (4 ° C. 1 min, 5,000 rpm). After washing three times with PBS (phosphate-buffered saline) buffer solution, separated on SDS-polyacrylamide gel, the proteins were transferred to nitrocellulose membrane and RIP, TRADD, TRAF2, FADD, Bcl-xL, and Flag proteins, respectively. Western blotting was performed using the antibody.

The experimental results are as follows.

Table 3 shows the result of blocking the increase of free radicals by TNF-alpha (20 ng / ml) when Romo1 expression is reduced using Romo1 siRNA in cervical cancer cells (HeLa). This is a result of measuring the amount of active oxygen after observing using a fluorescence microscope after treatment with TNF-alpha. In Table 3, the amount of active oxygen not treated with TNF-alpha was set to 1, and the amount of active oxygen generation with time after treatment with TNF-alpha was relatively shown. Butylated hydroxyanisole (BHA) was used as a positive control as an antioxidant.

Control siRNA: Experimental results in which a control siRNA 100 nM was injected into cells.

Romo1 siRNA: Experimental results injecting Romo1 siRNA (100 nM) into cells.

BHA: Experimental results of treatment of cells 30 minutes prior to treatment with TNF-alpha at a concentration of 100 uM BHA.

Cycloheximide (CHX) was treated with TNF-alpha cells at a concentration of 10 μg / ml 30 minutes before treatment.

time Control siRNA Romo1 siRNA BHA 0 1.0 1.0 1.0 15 minutes 1.0 1.1 1.1 30 minutes 1.2 1.3 1.1 1 hours 1.4 1.3 1.1 2 hours 1.5 1.1 1.2 4 hours 2.1 1.2 1.0 6 hours 2.5 1.3 1.0

Table 4 shows the result that when the expression of Romo1 was reduced by Romo1 siRNA in human embryonic kidney cells (HEK 293), increased free oxygen was blocked by TNF-alpha (20 ng / ml). After treatment with TNF-alpha, the amount of active oxygen was measured after observing using a fluorescence microscope. In Table 4, the amount of active oxygen not treated with TNF-alpha was set to 1, and the relative change in the amount of active oxygen generation with time after treatment with TNF-alpha was shown. BHA was used as a positive control as an antioxidant.

Control siRNA: Experimental results in which a control siRNA 100 nM was injected into cells.

Romo1 siRNA: Experimental results injecting Romo1 siRNA (100 nM) into cells.

BHA: Experimental results of treatment of cells 30 minutes prior to treatment with TNF-alpha at a concentration of 100 μM BHA.

Cycloheximide (CHX) was treated with TNF-alpha cells at a concentration of 10 μg / ml 30 minutes before treatment.

time Control siRNA Romo1 siRNA BHA 0 1.0 1.0 1.0 15 minutes 1.2 1.2 1.3 30 minutes 1.3 1.3 1.4 1 hours 1.5 1.1 1.1 2 hours 2.1 1.2 1.2 4 hours 2.4 1.2 1.1 6 hours 2.8 1.2 0.8

Table 5 shows the result of using cell counting experiments that cell death induced by TNF-alpha is blocked when Romo1 expression is reduced using Romo1 siRNA in HeLa cells. In Table 5, the number of cells not treated with TNF-alpha was set to 1, and the relative change in cell number with time after treatment with TNF-alpha was shown. BHA was used as a positive control as an antioxidant.

Control siRNA: Experimental results in which a control siRNA 100 nM was injected into cells.

Romo1 siRNA: Experimental results injecting Romo1 siRNA (100 nM) into cells.

BHA: Experimental results of treatment of cells 30 minutes prior to treatment with TNF-alpha at a concentration of 100 μM BHA.

Cycloheximide (CHX) was treated with TNF-alpha cells at a concentration of 10 μg / ml 30 minutes before treatment.

time Control siRNA Romo1 siRNA BHA 0 1.00 1.00 1.00 3 hours 0.65 0.92 0.90 6 hours 0.29 0.63 0.68 9 hours 0.11 0.50 0.52

FIG. 5 shows that Romo1 binds to RIP, TRADD, TRAF2, and FADD proteins, which are complex II constituent proteins that transmit apoptosis signals in the TNF-alpha signaling pathway when TNF-alpha is treated by treating TNF-alpha in HEK 293 cells. The results show that using the Western blotting experiment technique. Romo1-Flag hybrid genes incorporating Flag DNA to Romo1 were injected into cells, and then Romo1-Flag protein was precipitated using Flag antibodies. Western blotting was performed over time after TNF-alpha treatment using RIP, TRADD, TRAF2, and FADD antibodies to identify proteins that bind Romo1 in response to TNF-alpha signals. Lysate is a result of Western blotting of cell extract without precipitation. β-actin shows that the same amount of protein was used in western blotting.

The experimental results show that Romo1 is an important mediator of free radical production by TNF-alpha and apoptosis by TNF-alpha. In addition, when treated with TNF-alpha, complex II (combination of RIP, TRADD, TRAF2, and FADD proteins) is formed in the cell death pathway, and the complex II induces cell death. Experimental results show that complex II binds to Romo1 located in the mitochondria and transmits TNF-alpha signals. Therefore, it can be seen from these results that Romo1 can be a diagnostic marker for diseases induced by TNF-alpha, and the antibody can be diagnosed and prevented in advance by using an antibody against Romo1 according to the present invention.

Korea Cell Line Research Foundation KCLRFBP00226 20100105

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Claims (10)

An antibody against Romo1 (Reactive Oxygen Species Modulator 1) protein having the amino acid sequence set forth in SEQ ID NO: 1 produced by hybridoma with accession number KCLRF-BP-00226.
delete Hybridoma of Accession No. KCLRF-BP-00226 for producing the antibody of claim 1.
delete delete delete delete A method of detecting Romo1 protein, which is a marker of TNF-alpha induced disease by contacting the antibody of claim 1 with a biological sample to detect antigen-antibody complex formation.
The method of claim 8,
Said TNF-alpha induced disease is selected from the group consisting of sepsis, diabetes, osteoporosis, transplant rejection, multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease, ischemic disease, pulmonary fibrosis and neuronal degenerative disease.
The method of claim 8,
The antigen-antibody complex is a complex of Romo1 protein-antibody of claim 1,
Detection of the complex formation may include enzyme immunoassay (ELISA), western blotting, immunofluorescence, immunohistochemistry staining, flow cytometry, immunocytochemistry, radioimmunoassay. A method characterized in that it is carried out by RIA, immunoprecipitation assay, immunodiffusion assay, complement fixation assay or protein chip.

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