JP4834826B2 - Monoclonal antibody recognizing histone H3 and H4 linker regions and hybridoma producing the same - Google Patents

Description

  The present invention relates to an anti-histone monoclonal antibody that recognizes the internal structure L2 linker region of histones H3 and H4, and a hybridoma producing the same.

  Histones have high homology across species and are not easily recognized as foreign substances in vivo, and thus have low antigenicity. Conventionally, it has been difficult to produce antibodies against histones by the usual method of immunizing animals. However, it is known that many anti-histone antibodies exist in the serum of patients with systemic lupus Erythematosus (SLE), an autoimmune disease. In addition to histones in many sera of autoimmune diseases, antibodies against HMG protein, ribonucleoprotein, RNA polymerase, tRNA synthetase, topoisomerase, etc., single-stranded DNA, double-stranded DNA, Z- It has already been reported that antibodies that recognize DNA, double-stranded RNA, rRNA, poly ADP ribose and the like are included.

  It has not yet been elucidated how autoantibodies found in patients with these autoimmune diseases are produced, but it is expected that nuclear degradation products of dead cells serve as antigen stimuli. Nucleosomes, one of the nuclear degradation products, have also been reported to stimulate T cells that help create these antibodies.

  Thus, in many cases, anti-histone antibodies have often been obtained by purification using serum donated from SLE patients. In this case, antibody features such as antibody titer, antigen characteristics, affinity for antigen, antigen-antibody reaction rate, etc. are markedly different in patient serum lots, making it difficult to stably supply antibodies of the same standard. Met. In Japan, it is difficult to obtain serum from patients with SLE, and reliance on imports for all of these biological samples has been problematic in terms of ethics.

  In recent years, spleen cells have been prepared from autoimmune disease model mice (MRL / lpr mice and NZB / NZW mice), and anti-histone monoclonal antibodies have also been prepared using them. However, the number of clones of anti-histone monoclonal antibodies obtained by this method is limited, and the types of monoclonal antibodies obtained are also limited. In most cases, antibodies against linker histones such as H1 and antibodies recognizing the amino terminal region not covered by DNA in the nucleosome structure are almost all. Furthermore, since conventional anti-histone monoclonal antibody technology has been mainly directed to the development of antibodies that recognize differences in amino acid modification of the amino terminal region of histones, the antibodies cannot examine the internal structure of nucleosomes. .

  On the other hand, antibodies that recognize the histone internal region, especially those that recognize the histone region where DNA and core histones bind to each other in the nucleosome structure, do not recognize changes in the amino terminus of the histone. It is a useful means for detection, and can be used for various applications described later. Accordingly, an object of the present invention is to provide an antibody that recognizes a histone region in which a DNA and a core histone bind to each other in a nucleosome structure, as a monoclonal antibody, regardless of the means for separating from serum or the like in the above-described conventional technology. For this reason, an object of the present invention is to provide a hybridoma capable of producing the antibody easily and in large quantities.

As a result of intensive studies, the present inventors have obtained a fusion of an antibody-producing cell obtained by immunization with a fragment of nuclear formalin immobilized from an animal cell as an antigen and a myeloma cell. By screening hybridomas, the present inventors have completed the present invention by producing a high-rate antibody that recognizes a histone region where DNA and core histone bind to each other in a nucleosome structure, and having found a clone that is highly proliferative. Is.
That is, the present invention relates to the following (1) and (2).

(1) An anti-histone monoclonal antibody produced by hybridoma, FERMP-19369 strain, which is obtained by hybridoma method, using a fragment of nuclear formalin immobilized isolated from animal cells as an antigen, and contains histones H3 and H4. Anti-histone monoclonal antibody that specifically recognizes the structural L2 linker region.

(2) The anti-histone monoclonal antibody according to (1) above, wherein the nuclear formalin-immobilized product isolated from animal cells is a nuclear formalin-immobilized product separated from HeLa cells.

(3) It is obtained by fusing antibody-producing cells obtained by immunization with myeloma cells using a fragment of nuclear formalin immobilized isolated from animal cells as an antigen, and claim 1 A hybridoma producing the anti-histone monoclonal antibody described in 1., FERMP-19369 strain.

(4) The hybridoma according to (3) above, wherein the nuclear formalin immobilized isolated from animal cells is a nuclear formalin immobilized isolated from HeLa cells.

(5) The hybridoma according to (3) above, wherein the antibody-producing cells are spleen cells.

(6) The hybridoma according to (5) above, wherein the spleen cells and myeloma cells are mouse-derived cells.

  The anti-histone monoclonal antibody of the present invention recognizes the L2 linker region of various histones H3 and H4, including humans, and can be applied to a wide range of uses such as separation, purification and quantification of various histones and nucleosomes. It is a thing. Further, the hybridoma of the present invention can efficiently produce the above monoclonal antibody, and the present invention is extremely useful in cells, gene manipulation techniques and the like.

Hereinafter, the present invention will be described in more detail.
The anti-histone monoclonal antibody of the present invention can be obtained by a hybridoma method using a fragment of nuclear formalin immobilized from animal cells as an antigen.
The present invention will be described in more detail. The present invention will be described in more detail with reference to the production method based on a hybridoma method using a fragment of HeLa cell nuclear formalin immobilized product as an antigen.
The antigen to be used is one obtained by separating and purifying nuclei from HeLa cells, fixing the intranuclear structure with formaldehyde, and then crushing and fragmenting with ultrasonic waves.
Immunization with the antigen was performed in vitro using splenocytes extracted from mice according to the method of Boss (Boss, BD (1984) Brain Res., 291, 193-196).

  The Boss method is an in vitro immunization method for producing antibodies, instead of immunizing antigens to normal mice and rabbits. The advantages of this method are (1) immunization with a small amount of antigen, (2) short immunization period, and (3) immune tolerance and immunosuppression observed when sensitized to normal animals. It can be avoided and can be produced without depending on antigenicity. (4) It is possible to immunize even highly toxic antigens. (5) An antibody that is harmful to immunized animals is also created. It is possible.

  In order to induce antibody production of B cells, antigen presentation and signal crosstalk by macrophages and T cells are required, but splenocytes contain these cells including B cells. Therefore, spleen cells are prepared from mice, antigen-stimulated with N-acetyl-muramyl-L-alanyl-D-isoglutamine as an adjuvant in a serum-free medium, and immunized in the medium.

  Subsequently, the immunized spleen cells and myeloma cells are fused with polyethylene glycol, and the hybridomas are selected with a HAT medium by a conventional method. Further, the culture supernatant of each clone of the hybridomas obtained by the dilution method is added to the nucleus of the antigen. A positive hybridoma clone is obtained by measurement by enzyme immunoassay (ELISA) using a formalin-immobilized product. Furthermore, for the obtained clones, the recognition antigen was determined by ELISA using the DNA fraction, nucleosome fraction, histone fraction, and nuclear matrix fraction obtained by subdividing the HeLa cell nucleus, and positive specifically for the histone fraction. A hybridoma clone showing a reaction is obtained. Thereafter, clones having good antibody production ability and proliferation are further screened to obtain the anti-histone monoclonal antibody-producing hybridoma of the present invention.

   The anti-histone monoclonal antibody-producing hybridoma of the present invention is deposited under the accession number FERM P-19369 at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology.

  The anti-histone monoclonal antibody of the present invention obtained from the above hybridoma specifically reacts with the histone H3 and the linker region L2 of H4, and does not react with the amino terminus of these histones, the L1 or a helix region. It does not react with other histones H1, H2A and H2B.

Since the anti-histone monoclonal antibody of the present invention specifically recognizes not only human but also chicken and many internal structures L2 linker regions of histones H3 and H4, for example, affinity using the anti-histone monoclonal antibody of the present invention By performing chromatography, the core histones and histone H3 / H4 tetramers, as well as histones H3 and H4, can be easily separated and purified from various cell nuclear disruptions regardless of the amino acid modification in the amino terminal region. Extremely versatile. In eukaryotic cells, it is known that the chromatin structure changes in regions where gene transcription, replication, repair, etc. are performed, and staining the cell nucleus using the anti-histone antibody Thus, the region where the chromatin structure is changed and the nucleosome structure is being collapsed can be directly observed and identified. Furthermore, after the chromatin structure is immobilized with formalin, affinity chromatography is performed using the anti-histone antibody, whereby the region in which the chromatin structure is changed and the nucleosome structure is decaying can be specifically recovered.
Although the above has mainly described the case of using a hybridoma of a mouse spleen cell and a mouse myeloma cell, the present invention is not limited to this, and uses a mouse-derived antibody-producing cell and a human-derived myeloma cell. An anti-histone monoclonal antibody can be obtained by applying a conventionally known hybridoma method such as a hybridoma method or a hybridoma method using mouse-derived antibody-producing cells and rat-derived myeloma cells.

Further, the anti-histone monoclonal antibody of the present invention can be used for various purposes in addition to the above-mentioned uses. These are exemplified below.
(A) Preparation of model animal for research of autoimmune disease It is known that anti-histone antibodies exist in human autoimmune disease, particularly in serum of SLE patients. Similarly, anti-histone antibodies are present in serum in model mice (NZB x NZW) F1, MRL, BXSB and the like of these diseases. There is a correlation between the amount of anti-histone antibody in the serum and clinical symptoms, and it is considered that anti-histone antibody is responsible for some kind of autoimmune disease (Suwa, A. and Mimori, T., Nippon Rinsho (1999), 57, 403-405). It has also been suggested that anti-histone antibodies found in SLE act on signal transduction systems by reacting with cell surface proteins, particularly cell surface proteins of B lymphocyte progenitor cells (Raz, E. et al., Eur J. Immunol. (1993), 23, 383-390, Mecheri, S. et al., Mol. Immunol. (1993), 30, 549-557). Therefore, by inoculating, for example, a mouse with the anti-histone monoclonal antibody established this time, a model mouse for studying autoimmune disease can be prepared.

(B) Evaluation of cytotoxic substances When cultured cells are treated with various cytotoxic substances, the cells die. At this time, by quantifying the intranuclear degradation products containing histones released from the dead cell nuclei into the culture medium by using the anti-histone monoclonal antibody, the viable cell rate (death rate) can be measured. In this way, cytotoxic substances can be evaluated.

(C) Regulation of transcription reaction in cells In the nucleus, gene DNA takes a chromatin structure to suppress transcription. By expressing the anti-histone antibody in the cell, histone H3 and H4 can bind to the antibody and suppress formation of a nucleosome structure. Thus, it is considered that gene DNA can always activate transcription because it can maintain a transcribable state.

(D) Inhibition of estrogen cell proliferation The effect of estrogen on cells includes a pathway through high-binding estrogen receptors (Type I) and a pathway through binding to other proteins with low binding (Type II). In particular, it has been reported that the type II pathway is very important for cell proliferation (Markaverich, BM, et al., Endocrinol. (1981), 109, 62-69). Furthermore, it was reported that Type II is a pathway through the binding of histone H4 (Shoulars, K. et al., Biochem. Biophys. Res. Commun. (2002), 296, 1083-1090). Therefore, by expressing the anti-histone antibody in the cell, it becomes possible to inhibit the activation of estrogen via the Type II pathway and suppress the cell proliferation.

(E) Production of various histone antibodies by modifying the anti-histone monoclonal antibody of the present invention Currently, means for producing antibodies with different specificities by genetic modification of antibodies are being studied, including, for example, antibody-producing cells In some cases, the antigen recognizability is regulated by extracting the monoclonal antibody mRNA from the antibody and artificially introducing a mutation into the antigen recognition site (Variable region). By extracting anti-histone monoclonal antibody mRNA from the hybridoma of the present invention and adding mutations, it may be possible to create antibodies that specifically recognize other histone-like proteins such as H2A, H2B, and H1. It is possible to provide a test sample that is extremely useful in the study of antibody modification techniques.

(1) Preparation of antigen
Suspend 1 x 108 HeLa cells in 10 mM Tris-HCl (pH 7.5), 10 mM NaCl, 3 mM MgCl solution, homogenize in the presence of NP-40, and then precipitate the nucleus by centrifugation at 1000 x g for 5 minutes Separate and purify. Suspended purified nuclei in 5 ml of 1% formaldehyde solution and reacted at room temperature for 20 minutes to immobilize the intranuclear structure including DNA. This immobilized nuclear fraction was fragmented by sonication for 2 minutes until the average DNA was 3 kbp, and this was used as an antigen.

(2) Cell fusion and cloning 5 x 108 spleen cells were prepared from 8-week-old female mice, and N-acetyl-muramyl- was prepared according to the Boss method (Boss, BD (1984) Brain Res., 291, 193-196). Immunization was performed in vitro using the previously prepared antigen in the presence of L-alanyl-D-isoglutamine. After 3 days of sensitization culture, 6 x 107 spleen cells and 6 x 106 8-azaguanine-resistant mouse myeloma cells, P3U1 (P3-X63-Ag8-U1), were mixed and subjected to sedimentation after centrifugation. Cell fusion was carried out by adding 50% polyethylene glycol (average molecular weight 4000) and allowing it to react gently for 2 minutes. The obtained hybridoma-containing solution was implanted in a 96-well microculture plate and cultured for 2 weeks using a HAT medium. The culture supernatant of wells in which growth has been observed is collected, screened by enzyme-linked immunosorbent assay (ELISA), and the cells in the wells that are positive for the whole fraction of HeLa cell-immobilized nuclei used as antigens are cloned. It was. Cells in ELISA-positive wells were seeded on a 96-well microculture plate at an average of 0.1-1 per well. Colony formation was observed 2 weeks after the start of culture, and the thus obtained colonies were screened and cloned in the same manner as described above to obtain hybridomas (clones) producing antibodies exhibiting the desired reactivity.

(3) Screening Screening for obtaining the desired clones that produce anti-histone antibodies among the hybridomas obtained in (2) above was performed using the ELISA method. That is, the nuclear fraction used for the antigen is further fractionated into a DNA fraction, a nucleosome fraction, a nuclear matrix fraction, and a sulfuric acid extraction fraction containing mainly histones (100 ml / well at a concentration of 10 mg / ml). Was immobilized on a 96-well microplate (4 ° C, overnight), then the culture supernatant was reacted in wells blocked with BSA (room temperature, 90 minutes). After washing, peroxidase-labeled anti-mouse IgG + IgM antibody Was added, allowed to react at room temperature for 60 minutes, and then detected by a color development system using o-phenylenediamine as a substrate.

(4) Preparation of anti-histone antibody-producing hybridoma and acquisition of the anti-DNA antibody According to the above (2), 20 hybridomas capable of producing an immobilized nuclear antigen recognizing antibody could be established. By conducting the screening of 3), 8 anti-histone antibody-producing hybridomas positive for the above histone fraction were established, and among them, the hybridoma is considered to be the most excellent in terms of balance in hybridoma growth and antibody production ability. The resulting hybridoma (FERM P-19369) was grown in a serum-free medium or in the abdominal cavity of pristane-treated mice, and the culture supernatant or ascites was collected. These were precipitated with ammonium sulfate and then purified and recovered by HPLC hydroxyapatite column chromatography to obtain the anti-histone monoclonal antibody of the present invention.

(5) Characteristics of anti-histone monoclonal antibody (a) Substrate specificity of anti-histone monoclonal antibody Various concentrations of HeLa cell gene DNA, histone fraction, matrix fraction, and nucleosome were immobilized on a 96-well microplate (4. C, all day and night), then blocked with BSA, and reacted with 100 ml of anti-histone monoclonal antibody (1 mg / ml) added to each well (room temperature, 90 minutes). After washing, a peroxidase-labeled anti-mouse IgG + IgM antibody was further added and reacted at room temperature for 60 minutes, and then the absorbance at 492 nm was measured using a color development system using o-phenylenediamine as a substrate. The results are shown in FIG. As shown, this anti-histone monoclonal antibody was found to recognize only the histone fraction.

(B) Reactive histone fraction proteins for each histone were separated by SDS-polyacrylamide gel electrophoresis and Western blotted onto a nitrocellulose membrane. After blocking with 2% BSA, add 10 ml of this anti-histone monoclonal antibody (1 mg / ml), react at room temperature for 1 hour, and after washing, add peroxidase-labeled anti-mouse IgG + IgM antibody for 60 minutes at room temperature. Next, the histone protein which reacts was detected using the luminescent system which used ECL as the substrate. The results are shown in FIG.
As shown in the figure, this anti-histone monoclonal antibody recognizes both human and chicken histones H3 and H4 as well.

(C) Recognition region in histone protein Based on the amino acid sequence of histone protein, a region having high homology to histones H3 and H4 and low homology to H2A and H2B was searched using a computer. A helix (Ile-Arg-Arg-Leu-Ala-Arg-Arg-Gly) and an L2 linker (Thr-Glu-His-Ala-Lys-Arg-Lys-Thr-Val-Thr) were found. These peptides were expressed in E. coli as fusion proteins with dehydrofolate reductase, purified, separated by SDS-polyacrylamide gel electrophoresis, and Western blotted onto a nitrocellulose membrane. After blocking with 2% BSA, add 10 ml of this anti-histone monoclonal antibody (1 mg / ml), react at room temperature for 1 hour, and after washing, add peroxidase-labeled anti-mouse IgG + IgM antibody for 60 minutes at room temperature. Next, the reaction protein was detected using a luminescence system using ECL as a substrate. The results are shown in FIG. From these results, it was found that the antibody was specifically bound to the L2 linker. From the above results, it was found that this anti-histone monoclonal antibody recognizes the L2 linker region of histones H3 and H4.

It is a graph which shows the result of having tested the histone recognition property of the anti-histone monoclonal antibody of this invention about each nuclear component, gene DNA, a histone fraction, a matrix fraction, and a nucleosome. It is a figure which shows the result of having tested the binding property of the anti-histone monoclonal antibody of this invention with respect to each histone protein. It is a figure which shows the result of having tested the reactivity of the anti-histone monoclonal antibody of this invention with respect to the combination protein containing the peptide fragment by which the coupling | bonding was estimated by Western blot analysis.

Claims (6)

An anti-histone monoclonal antibody produced by hybridoma, FERMP-19369 strain, obtained by hybridoma method using a fragment of nuclear formalin immobilized isolated from animal cells as an antigen, and an internal structure L2 linker of histones H3 and H4 Anti-histone monoclonal antibody that specifically recognizes the region. 2. The anti-histone monoclonal antibody according to claim 1, wherein the nuclear formalin immobilized isolated from animal cells is a nuclear formalin immobilized isolated from HeLa cells. The fusion product of antibody-producing cells obtained by immunization and myeloma cells using, as an antigen, a fragment of nuclear formalin-immobilized material separated from animal cells, A hybridoma producing an anti-histone monoclonal antibody, FERMP-19369 strain. The hybridoma according to claim 3, wherein the nuclear formalin immobilized isolated from animal cells is a nuclear formalin immobilized isolated from HeLa cells.   The hybridoma according to claim 3, wherein the antibody-producing cells are spleen cells.   The hybridoma according to claim 5, wherein the spleen cells and myeloma cells are cells derived from a mouse.

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