KR20110019495A - Antibody specific for bubr1 - Google Patents

Abstract

The present invention relates to an antibody against BubR1, and more particularly to an antibody capable of specifically recognizing only acetylated BubR1, and a kit and method for detecting acetylation of BubR1 protein using the same. The BubR1 protein is acetylated at the 250th lysine in the whole mitotic cycle of the cell division cycle, and the acetylated BubR1 protein acts as a molecular switch that allows the cells to be divided to have a complete genome. The antibody of the present invention can be usefully used for measuring cell division checkpoint activity as a measure of the degree of acetylation of BubR1, detecting, diagnosing, anticancer drug screening methods or cell division cycle control studies based on cell division abnormalities.

BubR1, acetylation, cell division checkpoint, antibody

Description

Antibody specific for BubR1}

The present invention relates to the field of antibodies, specifically to measure the degree of acetylation of BubR1, one of the proteins that acts at checkpoints in the cell division cycle, more particularly antibodies that specifically bind to acetylated BubR1, including BubR1. The present invention relates to a cell division checkpoint activity detection kit and a detection method.

The most important thing in cell division is to accurately transmit the same genetic information to each daughter cell. In particular, the chromosome separation time, which is transferred to each daughter cell after genetic information is replicated, that is, the transition period from the middle to the late stage of cell division is the peak of cell division, and the spindle assembly checkpoint ( Spindle Assembly Checkpoint (SAC) (Taylor SS, Scott MI, Holland AJ (2004) The spindle checkpoint: a quality control mechanism which ensures accurate chromosome segregation. Chromosome Res 12: 599-616). Normally, cell division usually ends within 30 minutes, but if one chromosome is not properly attached to spindles, cell division is delayed for up to 16 hours. SAC activated by SAC inhibits Anaphase Promoting Complex (APC / C), which is necessary for the progression of late cell division (Yu H (2002) Regulation of APC-Cdc20 by the spindle checkpoint.Cur Opin Cell Biol 14: 706-714). APC / C is a multiplex E3 ligase that destroys securin and cyclin B (Yu, ibid ). If SAC does not work properly, cells die or produce diploids, which are known to be important features of cancer cells (Kops GJ, Weaver BA, Cleveland DW (2005) On the road to cancer: aneuploidy and the mitotic checkpoint Nat Rev Cancer 5: 773-785; Pihan GA et al (1999) Cancer Biology 9: 289-302). In addition, incorrect SAC can lead to rapid and difficult to treat Chromosomal Instability Cancer (CIN) (Hannahan D et). al , Cell 100: 57-70 (2000); Li R et al. PNAS 97: 3236-3241 (2000). Because cancer cells divide indefinitely, controlling cell division is a shortcut to cancer treatment. To this end, the development of a substance that can check the interaction of various factors at the cell division checkpoint, the regulatory mechanism between them and the activity of these checkpoints. need. BubR1 is a factor known to be important in inhibiting APC / C by directly binding to APC / C among factors constituting SAC, a cell division checkpoint (Yu, ibid ). However, the exact APC / C regulatory mechanism of this BubR1 protein and antibodies that specifically recognize it is not known.

Herein, BubR1 protein, which can inhibit the production of diploid by timely controlling the activity of Anaphase Promoting Complex (APC / C), functions as a molecular switch in SAC through acetylation / deacetylation. To find and specifically detect them.

The present invention provides antibodies that specifically recognize acetylated BubR1 protein.

The invention also provides an antibody that specifically recognizes a BubR1 protein derived from human ( Homo sapiens ), Mus musculus , Gallus gallus or Xenopus laevis . .

The present invention also discloses that the 250th lysine residue for human origin, the 243th lysine residue for Musmusculus, the 240th lysine residue for Gallus Gallus, and the 237th lysine residue for Genoese Laebis are acetylated, Or an antibody that specifically recognizes BubR1 in which each of the lysine residues is substituted with glutamine.

The present invention also provides antibodies which do not exhibit cross-reactivity with BubR1 protein that is not acetylated and with BubR1 protein in which the lysine residues are substituted with arginine.

The invention also relates to a polypeptide having the amino acid sequence of SEQ ID NO: 1 in Table 1 wherein the lysine residues are acetylated; A polypeptide having the amino acid sequence of SEQ ID NO: 2 of Table 1 wherein the lysine residues are acetylated; A polypeptide having the amino acid sequence of SEQ ID NO: 3 of Table 1 wherein the lysine residues are acetylated; A polypeptide having the amino acid sequence of SEQ ID NO: 4 of Table 1 wherein the lysine residues are acetylated; A polypeptide having the amino acid sequence of SEQ ID NO: 5 of Table 1 wherein the lysine residues are acetylated; A polypeptide having the amino acid sequence of SEQ ID NO: 6 of Table 1 wherein the lysine residues are acetylated; A polypeptide having the amino acid sequence of SEQ ID NO: 7 of Table 1 wherein the lysine residues are acetylated; A polypeptide having the amino acid sequence of SEQ ID NO: 8 of Table 1 wherein the lysine residues are acetylated; And a polypeptide having an amino acid sequence in which each lysine residue of each sequence of SEQ ID NOS: 1-8 is substituted with glutamine.

The invention also provides antibodies that do not exhibit cross-reactivity to polypeptides comprising each sequence of non-acetylated SEQ ID NOs: 1-8 or a sequence in which each lysine residue of each sequence is substituted with arginine.

The invention also provides an antibody wherein said antibody is polyclonal or monoclonal.

The invention also provides an antibody wherein the antibody comprises a fragment of an antibody selected from the group consisting of Fv, ScFv, Fab, Fab 'and F (ab') 2.

The present invention also provides an antibody wherein the antibody further comprises a marker.

The invention also provides antibodies wherein the antibodies can be obtained by animal serum, cell culture or recombinant DNA techniques.

The present invention also provides a kit for detecting the degree of acetylation of BubR1 protein in a sample, comprising the antibody of the present application.

The invention also provides a kit wherein said kit further comprises a second antibody labeled with a marker.

The present invention also provides a kit in which the sample contained in the kit is derived from cancer tissue.

The invention also comprises the steps of separating the sample from the subject; Contacting said sample with an antibody according to the invention; And it provides a method for detecting the degree of acetylation of BubR1 protein, comprising determining the degree of acetylation of BubR1 protein.

The invention will now be described more clearly in the following detailed description, claims and drawings.

In one aspect the invention relates to antibodies that specifically recognize / bind acetylated BubR1 protein. In one embodiment of the present invention, the BubR1 protein is not limited thereto, but is derived from human ( Homo sapiens ), Mus musculus , Gallus gallus , or Xenopus laevis ( Xenopus laevis ). to be. Each protein sequence is known as NP_001202 in humans, NP_033903 in M. musculus , NP_989734 in G. gallus , NP_001079357 in X. laevis , and conservative mutations in the sequence. Sequences comprising sequence variants that produce functionally equivalent results are also within the scope of the present invention.

BubR1 (budding uninhibited by benzimidazoles 1 beta) is a protein that plays an important role in SAC and cell division checkpoint signaling, and is known to play an important role in binding and regulation of the APC / C-Cdc20 complex, but its specific mechanism is unknown. (Yu, ibid ). Herein it is identified that BubR1 protein is acetylated at the premature stage of the cell division cycle by PCAF [known as P300 / CBP-associated factor (PCAF), or lysine acetyltransferase 2B (KAT2B)] at certain residues to function at cell division checkpoints. Choi et al. EMBO Journal 28: 2077-2089 (2009), which is incorporated herein in its entirety by reference]. That is, acetylation of BubR1 protein is required for the activity of SAC, and the acetylated BubR1 is not degraded by the APC / C-Cdc20 complex that degrades the protein through ubiquitination, thereby controlling the entry point of cell division. (Choi et al, ibid ). In the present invention, acetylation sites important for the regulation of SAC in BubR1 have been identified through BubR1 sequence comparison and a series of deletion experiments on BubR1 protein (see Examples and Choi et al. ). BubR1 is acetylated at the 250th lysine residue (based on the human sequence) in the whole mitotic cycle of the cell division cycle, and acetylated BubR1 inhibits degradation by APC / C-Cdc20 to regulate the progression to the end of cell division. BubR1 with residues substituted with glutamine (K250Q BubR1) has the same effect as acetylated and is not acetylated when substituted with arginine (Choi et. al , ibid ).

In other words, BubR1 acts as a molecular switch that regulates cell division in SAC through acetyl / non-acetylation, and acetylated BubR1 inhibits cell division by inhibiting degradation by APC / C. Unconjugated BubR1 is degraded by APC / C to induce progression to the end of cell division. Thus, in one embodiment the antibody of the invention is a 250th lysine residue for human origin, or a corresponding residue in another species, for example 243 lysine residue for M. musculus , 240th lysine for G. gallus The residue, X. laevis , specifically recognizes a BubR1 protein in which the 237th lysine residue is acetylated or each lysine residue is substituted with glutamine (see Examples 4-6). Such an antibody of the present invention is a very specific antibody which does not exhibit cross-reactivity with respect to the non-acetylated BubR1 protein and the BubR1 protein which is substituted with arginine and not acetylated.

Antibodies of the invention that specifically recognize the above-mentioned acetylated BubR1 or K250Q BubR1 exhibiting equivalent effects can be used to measure the degree of acetylation of BubR1 in cells and tissues, which is associated with cancer due to non-ideal cell division. It can be used as an indicator to measure the occurrence of the same disease, the progress of cancer and the prognosis of cancer, and can be widely used in fields such as cell division cycle research and cancer drug development. .

Antibodies that can specifically recognize the acetylated BubR1 protein of the invention can be prepared using the following polypeptides as antigens. Human ( Homo sapiens ), the 250 th position based on the BubR1 protein sequence NP_001202, and in other species the position of the corresponding lysine, for example, but not limited thereto, for M. musculus the 243 th residue based on NP_033903, G. gallus In the case of X, laevis has 240 residues based on NP_989734, 237th lysine residues based on NP_001079357 in X. laevis has 5 residues each, in particular each of the four residue sequences, the lysine residues are acetylated The polypeptide may be prepared as an antigen.

In one embodiment, the antibody of the present invention may be produced by using a polypeptide having an amino acid sequence of human-derived GGAL (K-Ac) APSQ (SEQ ID NO: 1) in which lysine residues are acetylated. In another embodiment, an antibody of the present invention can be produced by using a polypeptide having an amino acid sequence of human-derived VGGAL (K-Ac) APSQN (SEQ ID NO: 2) in which lysine residues are acetylated. In another embodiment, an antibody of the present invention may be prepared by using each polypeptide having an amino acid sequence of SEQ ID NOs 3 to 8 described in Table 1 below, wherein the lysine residue is acetylated as an antigen.

Amino acid sequence origin SEQ ID NO: 1 GGAL K APSQ H. sapiens SEQ ID NO: 2 VGGAL K APSQN H. sapiens SEQ ID NO: 3 GGAL K APGQ M. musculus SEQ ID NO: 4 VGGAL K APGQS M. musculus SEQ ID NO: 5 GDAL K ATNQ G. gallus SEQ ID NO: 6 VGDAL K ATNQN G. gallus SEQ ID NO: 7 GDSI K SRPQ X. laevis SEQ ID NO: 8 VGDSI K SRPQG X. laevis

G: Glycine; A: Alanine; L: Leucine; K: Lysine; P: Proline; S: Serine; Q: Glutamine; V: Valine; N: Asparagine; D: Aspartic Acid; I: Isoleucine; R: Arginine; T: Threonine; Bold K: acetylation site.

In general, the use of full-length protein has the advantage of inducing an effective antibody response when producing antibodies, but antibodies to epitopes of various sites can be generated to specifically recognize a site where a specific residue is modified as in the case of the present invention. The probability of obtaining an antibody can be very low. On the other hand, when a short peptide fragment is used, effective antibody production is very difficult when preparing the antibody. However, the present invention reproducibly obtains an antibody that specifically recognizes acetylated BubR1 using the peptide of the short fragment as shown in the above table as an antigen. In the case of using a total of 13 amino acid peptides having 6 residues on the left and right as lysine as antigens, cross-reactivity was also observed for unacetylated BubR1, indicating that the specificity for acetylated BubR1 was reduced (see Example). .

In one embodiment of the present invention, an antibody that specifically recognizes acetylated BubR1 was obtained using a peptide having a sequence derived from humans as an antigen (see Example 3). Thus, a person skilled in the art will be able to obtain an antibody that specifically recognizes acetylated BubR1 using peptides having different sequences and sequences from different animals described in the table above as reference with reference to the methods described herein.

Thus, in another aspect the invention provides a polypeptide having the amino acid sequence of SEQ ID NO: 1 of Table 1 wherein the lysine residues are acetylated; A polypeptide having the amino acid sequence of SEQ ID NO: 2 of Table 1 wherein the lysine residues are acetylated; A polypeptide having the amino acid sequence of SEQ ID NO: 3 of Table 1 wherein the lysine residues are acetylated; A polypeptide having the amino acid sequence of SEQ ID NO: 4 of Table 1 wherein the lysine residues are acetylated; A polypeptide having the amino acid sequence of SEQ ID NO: 5 of Table 1 wherein the lysine residues are acetylated; A polypeptide having the amino acid sequence of SEQ ID NO: 6 of Table 1 wherein the lysine residues are acetylated; A polypeptide having the amino acid sequence of SEQ ID NO: 7 of Table 1 wherein the lysine residues are acetylated; A polypeptide having the amino acid sequence of SEQ ID NO: 8 of Table 1 wherein the lysine residues are acetylated; And a polypeptide having an amino acid sequence in which each lysine residue of each sequence of SEQ ID NOS: 1-8 is substituted with glutamine.

In the present invention, proteins, polypeptides, and peptides are used interchangeably as molecules in which amino acid residues are linked by peptide bonds.

The antibody of the present invention has specific binding ability only to the acetylated BubR1 protein and other amino acids in which the acetylation site has an effect equivalent to acetylation, for example, the BubR1 protein in which the acetylation site is substituted with glutamine, It is an antibody that specifically recognizes only acetylated BubR1 that does not exhibit cross-reactivity to the unized BubR1 protein. Specifically, BubR1 in which the lysine residue is substituted with a BubR1 protein that is not acetylated or a 250-lysine lysine residue (or a corresponding position in another species) of the human sequence, which is important for the role of BubR1 as a molecular switch. There is no cross-reactivity for proteins.

Antibodies of the invention include complete antibodies, antigen-binding fragments of antibody molecules, and antibodies or fragments functionally equivalent to these. In addition, the antibody of the present invention may be of the IgG, IgM, IgD, IgE, IgA or IgY type, and may be of the IgGl, IgG2, IgG3, IgG4, IgA1 or IgA2 class or subclass thereof. Antibodies of the invention include monoclonal, polyclonal, chimeric, single chain, bispecific, amatogenic and humanized antibodies and active fragments thereof.

A complete antibody is a structure having two light chains and two 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 (Cellular and Molecular Immunology, Wonsiewicz, MJ, Ed., Chapter 45, pp. 41-50, WB Saunders Co. Philadelphia, PA (1991); Nisonoff, A., Introduction to Molecular Immunology, 2nd Ed., Chapter 4, pp. 45-65, Sinauer Associates, Inc., Sunderland, MA (1984)).

Examples of active fragments of the antibody that bind to the antigen include Fv, ScFv, Fab, Fab 'and F (ab') 2. Fab in the antibody fragment has a structure having a variable region of the light and heavy chains, a constant region of the light chain and the first constant region of the heavy chain (CH1) has one antigen binding site. Fab 'differs from Fab in that it has a hinge region comprising one or more cysteine residues at the C-terminus of the heavy chain CH1 domain. F (ab ') 2 antibodies are produced when the cysteine residues of the hinge region of Fab' form disulfide bonds. Recombinant techniques for generating Fv fragments with minimal antibody fragments in which sFv has only a heavy chain variable region and a light chain variable region are disclosed in PCT International Publication Nos. WO88 / 10649, WO 88/106630 and the like. Double-chain Fv is a non-covalent bond in which the heavy chain variable region and the light chain variable region are linked, and sFv (single-chain Fv) is generally covalently linked to the variable region of the heavy chain and the short chain variable region through a peptide linker. Or a C-terminus directly connected to form a dimer structure such as a double-chain Fv. Such antibody fragments can be obtained using proteolytic enzymes (e.g., restriction digestion of the entire antibody with papain yields Fab and cleavage with pepsin yields F (ab ') 2 fragments). It can be produced through recombinant technology. Details on this method can be found in, for example, Khaw, BA et al. J. Nucl. Med. 23: 1011-1019 (1982); Rousseaux et al. See Methods Enzymology, 121: 663-69, Academic Press (1986).

The term “functionally equivalent antibody” is any of the types, classes mentioned above as antibodies that can specifically recognize at least one major functional property, namely acetylated BubR1 or BubR1, including mutations exhibiting an acetylated effect. Or fragments, including antibodies produced by a variety of methods such as phage display, any individual and cells such as bacteria, insect cells, mammalian cells, etc., which can produce the desired antibody.

Antibodies of the invention can be obtained by animal serum, cell culture or recombinant DNA techniques.

In one embodiment of the invention the antibody of the invention is a monoclonal antibody. Methods of making monoclonal antibodies are known in the art and can be prepared through traditional cloning and cell fusion methods. Antigens are typically administered (eg subcutaneously or intraperitoneally) to antigens engineered to produce wild type or inbred mice (BALB / c or C57BL / 6), rats, rabbits or other mammalian or wild type or human antibodies. Administration). The antigen may be administered alone or in combination with an adjuvant, and the antigen may be introduced in the form of a fusion protein capable of being expressed in a vector or inducing a DNA or immune response. Fusion proteins include peptides capable of inducing an immune response and include, for example, carrier proteins such as beta galactosides, glutathione, S-transferases, kiholimpethemocyanin (KLH) and fetal bovine serum. . After boosting the animals 2-3 times with these antibodies, the spleens are evicted and fused in the presence of a myeloma cell line (eg SP2 / O from ATCC, USA) and a fusion promoter such as polyethylene glycol to generate a hybridoma cell line. For more information see Kohler and Milstein. Nature 256: 495-497 (1975) and Harlow and Lane. Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1988).

In one embodiment of the invention the above-described antigenic peptides of the invention are in the form of an antigen composition comprising an adjuvant, for example 1 to 15 times, in particular 2 to 10 times, by subcutaneous or intraperitoneal injection to an animal, for example Balb / c mouse. , In particular 1 to 10 weeks, in particular 1 to 6 weeks, more particularly 1 to 4 weeks, more particularly 2 to 3 weeks at a frequency of 3 to 7 times. Serum is collected after an appropriate time, for example 3 to 10 days, especially 4 to 8 days, more particularly 5 to 6 days after boosting, followed by purification methods such as known methods such as affinity chromatography and dialysis. The immunogenicity of the antigen is determined using methods such as Western blot or assays such as Enzyme-Linked Immunosorbent Assay (ELISA).

An antigen according to the present invention, ie, an antigen having a sequence of any one of SEQ ID NOs: 1 to 8 where the lysine residues are acetylated, exhibits a high immune response in the animal. ELISA assays can be used to produce hybridomas by selecting individuals that test positive in dilutions of 1: 4000 to 1: 6000, especially 1: 4500 to 1: 5500. Hybridoma cells are cultured in a medium comprising hypoxanthine, aminopterin and thymidine (HAT) using the conventional methods described in the documents cited above for the selection of hybridomas producing the desired antibody. Selected clones were then specifically reacted with wild-type acetylated / non-acetylated BubR1 protein and BubR1 protein where the acetylation site was substituted with glutamine to specifically bind lysine residues to acetylated BubR1 protein. The monoclonal antibody is selected. Hybridomas producing the antibody can be passaged or cryopreserved for later use. Preferred monoclonal antibodies in one embodiment of the invention are of IgG type.

In another embodiment of the invention, the antibody of the invention is polyclonal. The antigenic composition of the present invention may be used in suitable animals such as, for example, mice, rats, rabbits, goats, sheep or cattle several times, for example 1 to 10 times, especially 2 to 7 times, especially 2 to 4 times, 1 to 5 times. After administration at weekly intervals, especially at intervals of 2 to 4 weeks, serum was collected from the animals to determine whether the acetylated / non-acetylated BubR1 protein and the acetylated site were specifically reacted with glutamine-substituted BubR1 protein. Monoclonal antibodies are selected in which the lysine residues specifically bind to the acetylated BubR1 protein.

The antigen of the present invention may comprise a carrier. The carrier serves to expose / present the antigen to the immune system of the animal in which the antigen is produced by incorporation / combination with the antigenic peptide of the present invention. Such carriers include, but are not limited to, maltose binding protein (MBP), keyholepethemocyanin (KLH), fetal bovine serum (BSA), ovalbumin (OVA) and other adjuvants. In one embodiment of the invention the antigen of the invention comprises KLH. In another embodiment the KLH is conjugated to the N-terminus of the antigen. Various adjuvants known in the art can be used to produce the antibodies of the invention. Examples include Freund's incomplete adjuvant, Freund's complete adjuvant, TITERMAX (polyoxyethylene-polyoxypropylene copolymer adjuvants, CytRx Corporation), modified lipid adjuvant (Chiron Corporation), saponin derivative adjuvant (Cambridge Biotech), dextran sulfate, Alum, aluminum hydroxide, and aluminum phosphate include, but are not limited to. In one embodiment of the invention a Freund's complete adjuvant is used. In another embodiment of the present invention a Freund's incomplete adjuvant is used. In other embodiments of the invention, Freund's complete and incomplete adjuvants are used.

The antibodies of the present invention can be applied to a variety of fields based on the detection of acetylated BubR1 protein as described above. Such antigen-antibody binding can be detected using a label bound to the antibody or antibody fragment, for which the antibody of the invention is directly labeled with various markers that emit a detectable signal, or It may be indirectly labeled using a recognizing substance. In the former case, various compounds known in the art may be used as markers, and these markers include enzymes, radioisotopes, fluorescent materials, luminescent and colloidal gold particles, latex beads, and the like. Include, but are not limited to, chromogens. Kits for labeling antibodies with such markers are commercially available (for example Zenon labeling kit from Invitrogen). The latter case can be carried out using a labeled substance having affinity for the antibody such as labeled Protein A or G or a second antibody which recognizes the antibody of the invention. For example, the second antibody is conjugated with biotin and such antibody-biotin conjugate is detected with labeled avidin or streptavidin. Likewise, the second antibody can be conjugated to the hapten and the antibody-hapten conjugate can be detected using a labeled anti-hapten antibody.

Those skilled in the art will be able to select appropriate methods for the detection of antibody-antigen binding according to the invention. Methods of attaching such markers to antibodies or antibody fragments are known in the art. See, eg, Kennedy, J. H., et al. Clin. Chim. Acta 70: 1-31 (1976), and Schurs, A. H. W. M., et al. Clin. See Chim Acta 81: 1-40 (1977). Examples of the hapten coupling method include a glutaaldehyde method, a periodate method, a dimaleimide method, and the like.

In another aspect, the present invention provides a kit or method for measuring the degree of acetylation of BubR1 protein in a sample (sample, biological sample), or a kit or method for measuring the degree of cell division checkpoint activation. The kit of the present invention includes a container containing one or more antibodies according to the present invention, and a method for using the antibody, which method may be used to determine the presence and extent of acetylated BubR1 by performing an antibody-antigen reaction in a biological sample. Explain. The method of the present invention comprises the steps of: separating the sample from the subject; Contacting said sample with an antibody according to the invention; And determining the degree of acetylation of the BubR1 protein.

Biological samples of the invention include, but are not limited to, cells, tissues or body fluids derived from various proliferative diseases, including, for example, cancers, including solid and hematological cancers. In one embodiment of the invention the sample used in the kits and methods is from cancer. Antigen-antibody using the antibody according to the present invention to determine the degree of acetylation of BubR1 protein in a sample derived from such a disease (or a series of specimens according to variables such as time course / treatment drug / dose of the disease under treatment). Measure through reaction and compare with each other. Reduced degrees of acetylation compared to normal tissues may be found in tissues such as cancer and indicate abnormal cell division, and normal levels of acetylation will indicate normal cell division. Those skilled in the art will be able to determine the degree of acetylation of BubR1 compared to normal samples, and these results can be usefully applied in the development of cancer, the progression of cancer, or the prognosis after cancer treatment. The antibodies used in the kits or methods of the invention can be labeled directly or indirectly with appropriate markers such as the enzymes or radioactive materials described above.

Hereinafter, the present invention will be described in detail by the following examples. However, the following examples are illustrative of the present invention, and the contents of the present invention are not limited by the following examples.

The present invention may be practiced using conventional techniques within the skill of those skilled in the art of cell biology, cell culture, molecular biology, gene transformation techniques, microbiology, DNA recombination techniques, immunology, unless otherwise noted. See also the following books and literature for a more detailed description of general techniques. For general methods of molecular biology and biochemistry, see Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., Harbor Laboratory Press 2001); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. Eds., John Wiley & Sons 1999); DNA Cloning, Volumes I and II (Glover ed., 1985); Oligonucleotide Synthesis (Gait ed., 1984); Nucleic Acid Hybridization (Hames and Higgins eds. 1984); Transcription And Translation (Hames and Higgins eds. 1984); Culture Of Animal Cells (Freshney and Alan, Liss, Inc., 1987); Gene Transfer Vectors for Mammalian Cells (Miller and Calos, eds.); Current Protocols in Molecular Biology and Short Protocols in Molecular Biology, 3rd Edition (Ausubel et al., Eds.); And Recombinant DNA Methodology (Wu, ed., Academic Press). See also Gene Transfer Vectors For Mammalian Cells (Miller and Calos, eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al., Eds.); Immobilized Cells And Enzymes (IRL Press, 1986); Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (Weir and Blackwell, eds., 1986); Protein Methods (Bollag et al., John Wiley & Sons 1996); Non-viral Vectors for Gene Therapy (Wagner et al. Eds., Academic Press 1999); Viral Vectors (Kaplitt & Loewy eds., Academic Press 1995); Immunology Methods Manual (Lefkovits ed., Academic Press 1997); And Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons 1998). For general techniques regarding cell culture and medium, see Large Scale Mammalian Cell Culture (Hu et al., Curr. Opin. Biotechnol. 8 (1997), 148); Serum-free Media (Kitano, Biotechnology 17 (1991), 73); Large Scale Mammalian Cell Culture (Curr. Opin. Biotechnol. 2 (1991), 375); And Suspension Culture of Mammalian Cells (Birch et al., Bioprocess Technol. 19 (1990), 251).

The experiment described in the following example is described by Choi et al. As described in the description of the drawings of EMBO Journal 28: 2077-2089 (2009) and in the Experimental Methods and Materials and Supplemental Information sections, various reagents, cloning vectors and kits used in the experiments are generally BioRad. , Stratagene, Invitrogen, Sigma-Aldrich, and ClonTech et al. And are also described in the literature.

Example 1 Determination of Acetylation Sites of BubR1 Protein

In order to determine the acetylation site of BubR1, the following experiment was conducted.et al.It was performed as described in EMBO Journal 28: 2077-2089 (2009). In summary, a series of plasmids expressing BubR1 and full-length BubR1 lacking a specific region of the BubR1 protein (see FIG. 1A) was selected.et alIt was produced according to the method described in (Supplemental information, Material and Method). 10 μg of each plasmid containing each type of wild-type full-length BubR1 (BubR1 FL) or each mutation missing a specific moiety was transfected into 10 μg of PCAF expressing plasmid and 293T cells. After performing immunoprecipitation (IP) using an anti-Ac-K (αAcK) antibody that specifically recognizes acetylated lysine on the extract of the cells, experiments were performed with western blot (WB) with 9E10 antibody or vice versa. Was performed. The results are described in FIG. 1 B. Mutants ΔBR1, ΔBR2, ΔBR3 and full-length BubR1 were acetylated (marked with *), but the remaining mutants were not acetylated, indicating that the acetylation site was between residues 1-322. "Input" in Figure 1 indicates that each mutant was used in the same amount.

Subsequently, the ΔBR2 (1-514) region was further divided into five regions (see FIG. 2A) to prepare a series of plasmids expressed by the GST protein fusion protein. After in vitro acetylation in the presence or absence, Western blot was performed using anti-Ac-K and anti-GST antibodies. As shown in FIG. 2B, acetylation was detected only at the ΔBR2-3 (221-299) site.

Each of the five lysine residues (see FIG. 2C) contained in the ΔBR2-3 (221-299) site (see FIG. 3A) or two combinations thereof (see FIG. 3B) expresses a mutant substituted with arginine. Each plasmid was constructed, expressed in E. coli, purified, and subjected to in vitro acetylation in the presence or absence of PCAF, followed by Western blotting using anti-Ac-K and anti-GST antibodies. In vitro acetylation was performed using 0.05 μCi [1- ¹⁴ C] Acetyl-CoA, followed by SDS-PAGE gel electrophoresis, followed by exposure to an image plate of BAS 2500 phospher imager (Fujifilm). It confirmed again. Only when the 250 th lysine was substituted with arginine as shown in Figure 3, no acetylation was observed and in all other cases acetylation was observed. From this, the 250th lysine residue was determined as an acetylation site, and the full-length BubR1 isolated from insect cells was analyzed by mass spectrometry before and after acetylation using PCAF (+42 Da). Results indicate acetylation at lysine 250 (results not shown, see Choi et al ). The experiment was performed as described in Choi et al . BubR1 is acetylated at full weight as described in Example 3 below (see FIGS. 4B and C).

Example 2: Preparation of Antibodies

A. Construction of Antigen Peptides

The acetylation site determined in Example 1 was used as the antigen. The left and right sequences were selected with lysine residues in between for efficient antibody production. Peptides having GGAL (K-Ac) APSQ (SEQ ID NO: 1, Ac: acetylation) as antigens conjugated with KLH at the N and C termini were prepared (Peptron Corporation Korea). A peptide having a KLH conjugated to the N terminus was prepared having the RVGGAL (K-Ac) APSQNR sequence as an antigen. The purity of the used peptides analyzed by mass spectrometry was greater than 90%.

B. Preparation of Antibodies-Rabbit

Each of the three peptides was subcutaneously administered in 1 mg doses in three rabbits (purchased from Orient Bio, Yac: NZW (KBL), 2 months old) raised in SPF (Specific Pathogen Free) conditions (Seoul National University Animal Breeding Room). Injection. After that, three boosting sessions were conducted every three weeks. In the initial immunization, a total of 1 ml of antigen was injected by mixing the Freund's complete adjuvant 1: 1 with the antigen in a volume ratio, and in the case of boosting, the Freund's incomplete adjuvant was mixed with the antigen in a 1: 1 volume ratio. Injection with 1 ml of antigen. One week after last boost 50 ml of whole blood was collected from each rabbit and purified by the following method.

The collected whole blood was centrifuged at 4 ° C. and 3000 rpm for 1 hour to separate serum, and purified using a 0.45 μm filter. Serum was stored frozen at -80 ° C until use. Subsequently, 7 ml of binding buffer (0.5 M NaCl, 0.2 M Tris.HCl (pH 7.8), 0.1% Tween) was added to 1 ml of the serum, followed by recombinant protein comprising 1 to 322 residues of GST fused BubR1 (GST-BubR1). 1-322, Example 1, see FIG. 1, Choi et al .) 5 mg was added and reacted at 4 ° C. for 13 hours to remove the antibody recognizing the unacetylated BubR1 protein. The antibody was then isolated from the serum by affinity chromatography. For chromatography, first GGAL (K-Ac) APSQ was coupled to CNBr activated beads (Sigma Aldrich, C9210) according to the manufacturer's recommendations, and 1.5 ml of the primary purified serum was adjusted to adjust pH and salt concentration. 4M NaCl, 0.24 ml of 1 M Tris HCl pH7.8, 0.12 ml of 10% Tween 20 and 0.14 ml of distilled water were added. Then, 1 ml of the beads were washed once with a binding buffer (0.5 M NaCl, 0.2 M Tris.HCl (pH 7.8), 0.1% Tween), mixed with 8 ml of the first purified serum, and returned at 4 ° C. 13 The reaction was carried out for a time. Pass the bead-serum suspension through a polyprep chromatography column (0.5 ml beads / column, BioRad) (flow through collected separately and stored separately at 4 ° C), wash the column twice with 5 ml binding buffer and bind antibody Was collected into a 1.5 ml tube containing 20 μl of 1 M sodium phosphate (pH 8.0) using 0.2 ml of 100 mM Glycine (pH2.8) as the elution buffer, and this was repeated 10 times for the same column. The collected fractions were combined, and some of them were taken and subjected to protein quantitation using the Bradford method (Zor, T. and Selinger, Z, Anal. Biochem. 236: 302-8 (1996), purchased from BioRad). Afterwards, the concentration of the collected fractions was adjusted to 1 mg / ml (addition of binding buffer) and dialyzed using 3 ml of 10,000 MWCO slide-a-lyzer (Perbio) as recommended by the manufacturer, followed by binding of 500 ml and 1 L at 4 ° C., respectively. Dialysis was performed twice in a buffer for 30 minutes. The concentration of the antibody thus purified was 1 mg / ml.

C. Preparation of Antibodies-Mice

100 ㎍ of each of the three peptides were intraperitoneally injected into mice bred under SPF conditions (BALB / c, 2 months old) in the animal breeding room of Seoul National University.

After that, three boosting sessions were conducted every three weeks. During initial immunization, 100 μl of antigen was injected by mixing Freund's complete adjuvant 1: 1 with the antigen in the volume ratio, and in the case of boosting, Freund's incomplete adjuvant was mixed with the antigen in a 1: 1 volume ratio. A total of 100 μl of antigen was injected. One week after last boost 2 ml of whole blood was collected from each mouse and purified in the same manner as above. The concentration of purified antibody was 1 mg / ml.

Example  3: Acetylated BubR1  Confirm specificity of antibody

In all the experiments below, an antibody prepared using GGAL (K-Ac) APSQ (KLH conjugated at the N-terminus) (SEQ ID NO: 1) was used, and an antibody using RVGGAL (K-Ac) APSQNR as an antigen and Antibodies against GGAL (K-Ac) APSQ conjugated with KLH at the C-terminus were not used because they lacked the specificity of antibodies recognizing acetylated BubR1 or did not produce antibodies effectively (results not shown).

A. In the rabbit  Specificity of Prepared Antibodies

In order to confirm the specificity of the antibody prepared in Example 2B for acetylated BubR1, the following experiment was conducted by Choi et al. It was performed as described in EMBO Journal 28: 2077-2089 (2009).

In summary, the recombinant proteins ΔBR2-3, ΔBR2-4 and K250R (250th lysine substituted with arginine) produced as in Example 1 were prepared in vitro as in Example 1 with or without PCAF. Using the anti-Ac-K250 antibody prepared in Example 2B after acetylation using GACAL (K-Ac) APSQ (including KLH at the N-terminus) as an anti-Ac-K antibody, anti-GST antibody and antigen Western blot was performed. The results are described in FIG. 4A. As shown in FIG. 4A, the antibody (anti-Ac-K250) of the present invention specifically recognized only BubR1 in which the 250th residue included an acetylated lysine residue, and ΔBR2-4 and K250RΔBR2- that were not acetylated even with the same BubR1. 3 protein was not recognized at all and showed no cross-reactivity. This demonstrates the superior specificity of the antibodies of the invention. Anti-GST shows that all proteins were used in the same amount.

In addition, immunoprecipitation (IP) was performed with anti-BubR1 antibody, anti-AcK antibody, and anti-Ac-K250 antibody to the extracts of interstitial and intermediate HeLa cells, followed by Western blot with anti-BubR1 antibody. The results are described in FIG. 4B. As shown in FIG. 4B, acetylation at K250 occurs specifically at the mid-dividing stage and the antibody of the present invention correctly recognizes only BubR1 and does not exhibit cross-reactivity with BubR1 that is not acetylated.

Furthermore, in order to find out exactly when BubR1 is acetylated, heLa cells were treated with nocodazole (nocodazole, 200ng / ml) to stop cell division in the whole uterus (time point 0). The cells were then washed and sampled at the indicated time points while allowing the cell cycle to proceed in the presence of CHX (Cycloheximide, 100µg / ml), followed by Western anti-Actin, anti-Ac-K250 and anti-BubR1 antibodies. Blots were performed. The results are shown in Figure 4C, it can be seen that the acetylation of BubR1 is concentrated in the cell division prematuration, after which the acetylated BubR1 was not detected and the amount of unacetylated BubR1 gradually decreased. The anti-Ac-K250 antibody of the invention specifically detected only acetylated BubR1. Anti-Actin results show that the same amount of sample was used. Further, the same experiment was performed in the presence and absence of trichostatin A (TSA), a pan-HDAC (histon deacetylase) inhibitor, after cell counting. The results are described in FIG. 4D. The left panel shows that BubR1 is acetylated at full weight, and the right panel shows that this acetylation phenomenon continues after premiddle when treated with TSA. This suggests that the target of TSA is acetylated BubR1.

 The above experiment demonstrates that BubR1 is acetylated at full weight using an acetylated anti-Ac-K250 antibody while simultaneously showing the specificity of the acetylated BubR1 of the anti-Ac-K250 antibody.

B. Specificity of Antibodies Prepared in Mice

In order to confirm the specificity of the antibody prepared in Example 2C for acetylated BubR1, the following experiment was performed in the same manner as described in Example 3A (experiment using in vitro acetylated nocodazole), except that the antibody Western blot was performed using the one shown in FIG. 5. The results are described in FIG. 5 and obtained in accordance with FIG. 4. As shown in FIG. 5A, the anti-Ac-K250 BubR1 antibody prepared in the mouse specifically recognized only the acetylated BubR1 protein and did not respond to the non-acetylated BubR1 like the antibody prepared in the rabbit. As shown in the figure, only acetylated BubR1 of the full weight group was specifically recognized.

C. Specificity of Antibodies to Acetylated BubR1 Protein and K250Q BubR1 Protein Intracellularly

Intracellular of the antibody prepared in Example 2B (in vivoIn order to confirm the specificity for acetylated BubR1 inet al.It was performed as described in EMBO Journal 28: 2077-2089 (2009).

In summary, 293T cells were transfected with the plasmids described in FIG. 6A (see Example 1), and then treated with MG132 and nocodazole to inhibit degradation of BubR1 protein in the mid-term and immunoprecipitation using 9E10 antibody. IP b) followed by Western blot with the indicated antibodies. The results are shown in FIG. 6A, wherein the anti-Ac-K250 antibody of the present invention specifically recognizes BubR1 protein that is acetylated in vitro as well as BubR1 protein that is topically expressed and acetylated in cells (FIG. 5). K250Q BubR1 protein with 250th lysine (K) residue substituted with glutamine (Q) was recognized, but not K250R mutant that could not be acetylated. This also proves that the K250Q mutant has the same effect as acetylated. pcDNA3myc was used as a negative control as a parent vector containing no BubR1 DNA.

Next, HeLa cells were transfected with plasmid containing Myc-tagged BubR1 or K250R. Subsequently, the cells were synchronized with nocodazole and the medium cells were removed by gently tapping the cell culture vessel (mitotic shake off). The cells were then collected and Western blots were performed using each antibody described in FIG. 6B using the cell extracts obtained therefrom. The results are described in FIG. 6B. As shown in FIG. 6B, the concentration of wild type (WT) BubR1 decreases in mitosis (compare + CHX, 0, 2 and 5h) but increases with TSA treatment (+ TSA and TSA + CHX). In contrast, for K250R BubR1 there is no change in amount after 2 h, indicating that the K250R protein is not stabilized by TSA, suggesting the non-acetylation of K250R.

K250Q (or corresponding equivalent position in different species), with 250th lysine (or corresponding equivalent position in different species), acetylated BubR1 or equivalent effect as described in the detailed description herein, including Examples Antibodies of the present invention that specifically recognize BubR1 can be used to measure the degree of acetylation of BubR1 in cells and tissues to determine the occurrence of diseases such as cancer due to non-ideal cell division, the progression of cancer and the prognosis of cancer treatment. It can be used as an indicator that can be widely used in fields such as cell division cycle research and cancer-related disease drug development.

1 is a schematic diagram (A) of a deletion mutant for determining the acetylation site of BubR1 protein, the result of immunoprecipitation and Western blot using acetylated mutant protein (B).

FIG. 2 shows additional mutant schematics (A) and Western blots using acetylated / nonacetylated mutant proteins to more accurately determine the acetylation site (ΔBR2) determined in FIG. 1 (B). Here is shown the sequence comparison (C) between the lysine residues contained in ΔBR2-3, which are shown to contain acetylated sites and their different species.

FIG. 3 is a residue level determination of the acetylation site of ΔBR2-3 determined in FIG. 2, where each lysine residue of ΔBR2-3 is substituted with arginine (A) or a lysine at a position different from the 250th lysine residue. The results of western blot using double mutant (B) with residues substituted with arginine are shown.

4 is a western blot showing that the anti-Ac-K250 antibody according to one embodiment of the present invention specifically recognizes only acetylated BubR1.

5 is a Western blot showing that the anti-Ac-K250 antibody according to another embodiment of the present invention specifically recognizes only acetylated BubR1.

6 shows the results of Western blot showing that anti-Ac-K250 antibody specifically recognizes K250Q BubR1, a mutant that mimics intracellular acetylated BubR1 and acetylated BubR1 (A) And Western blot results (B) showing that K250R, an acetylatable mutant, is not stabilized by TSA.

Claims (16)

An antibody that specifically recognizes acetylated BubR1 protein. The antibody of claim 1, wherein the BubR1 protein is derived from human ( Homo sapiens ), Mus musculus , Gallus gallus or Xenopus laevis . The method of claim 2, wherein the BubR1 protein is the 250th lysine residue for human origin, the 243th lysine residue for Musmusculus, the 240th lysine residue for Gallus Gallus, and the 237th lysine for Genoa Laebis The residue is acetylated or each lysine residue is substituted with glutamine. The antibody of claim 3, wherein the antibody does not exhibit cross-reactivity with BubR1 protein that is not acetylated and with BubR1 protein in which the lysine residue is substituted with arginine. The antibody of claim 1, wherein the antibody is a polyclonal or monoclonal antibody. The antibody of claim 5, wherein the antibody comprises a fragment of an antibody selected from the group consisting of Fv, ScFv, Fab, Fab 'and F (ab') 2. The antibody of claim 1, wherein the antibody further comprises a marker. 8. The antibody of claim 7, wherein the marker is an enzyme, radioisotope, fluorescent material, luminescent material or chromophoric material. The antibody of claim 1, wherein the antibody is obtained by animal serum, cell culture or recombinant DNA technology. A polypeptide having the amino acid sequence of SEQ ID NO: 1 of Table 1 wherein the lysine residue is acetylated; A polypeptide having the amino acid sequence of SEQ ID NO: 2 of Table 1 wherein the lysine residues are acetylated; A polypeptide having the amino acid sequence of SEQ ID NO: 3 of Table 1 wherein the lysine residues are acetylated; A polypeptide having the amino acid sequence of SEQ ID NO: 4 of Table 1 wherein the lysine residues are acetylated; A polypeptide having the amino acid sequence of SEQ ID NO: 5 of Table 1 wherein the lysine residues are acetylated; A polypeptide having the amino acid sequence of SEQ ID NO: 6 of Table 1 wherein the lysine residues are acetylated; A polypeptide having the amino acid sequence of SEQ ID NO: 7 of Table 1 wherein the lysine residues are acetylated; A polypeptide having the amino acid sequence of SEQ ID NO: 8 of Table 1 wherein the lysine residues are acetylated; And An antibody against an antigen selected from the group consisting of a polypeptide having an amino acid sequence in which each lysine residue of each sequence of SEQ ID NOs: 1 to 8 is substituted with glutamine. The antibody of claim 10, wherein the antibody does not exhibit cross-reactivity to a polypeptide comprising each sequence of SEQ ID NOs: 1 to 8 that is not acetylated or a sequence in which each lysine residue of each sequence is substituted with arginine. . The antibody of claim 10, wherein the antibody is a polyclonal or monoclonal antibody. Kit for detecting the degree of acetylation of BubR1 protein in a sample comprising the antibody according to any one of claims 1 to 12. The kit of claim 13, wherein the kit further comprises a second antibody labeled with a marker. The kit of claim 14, wherein the sample is derived from cancer tissue. Separating the sample from the subject; Contacting said sample with an antibody according to any one of claims 1 to 12; And  A method for detecting acetylation of BubR1 protein, comprising determining the degree of acetylation of BubR1 protein.

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