WO2005073373A1 - Method of collecting damaged dna fragment - Google Patents

Method of collecting damaged dna fragment Download PDF

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Publication number
WO2005073373A1
WO2005073373A1 PCT/JP2005/001085 JP2005001085W WO2005073373A1 WO 2005073373 A1 WO2005073373 A1 WO 2005073373A1 JP 2005001085 W JP2005001085 W JP 2005001085W WO 2005073373 A1 WO2005073373 A1 WO 2005073373A1
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Prior art keywords
dna
dna fragment
antibody
oxidative stress
damaged
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PCT/JP2005/001085
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French (fr)
Japanese (ja)
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Shinya Toyokuni
Shinya Akatsuka
Osamu Nikaido
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Shirankai Kyoto University Faculty Of Medicine Alumni Association Inc.
Nikken Seil Corporation
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Priority to JP2005517480A priority Critical patent/JPWO2005073373A1/en
Publication of WO2005073373A1 publication Critical patent/WO2005073373A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6804—Nucleic acid analysis using immunogens
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism

Definitions

  • the present invention relates to a method for selective collection of damaged DNA, and more particularly, to a method for selectively selecting a DNA fragment corresponding to a DNA region damaged by the stress in a genome exposed to oxidative stress. And the use of DNA fragments collected in this way to pray for genomic damage.
  • Oxidative stress is a concept that means the amount of free radicals / active oxygen generated minus the erasing ability-defense ability * repair ability of injury, etc., for humans. It has been clarified to be related to lifestyle-related diseases such as arteriosclerosis and cancer.
  • Nucleic acids such as DNA are also target molecules for free radicals and active oxygen, but guanine, one of the purine bases among nucleic acid bases, is known to be easily damaged. Hydroxyradiation, singlet oxygen, or the modified base formed by photodynamic reaction include the hydroxylated 8-position of 2, -deoxyguanosine and 8-hydroxy-2'-deoxyguanosine ( Non-patent document 1). When 8-hydroxy-2'-deoxyguanosine is present in DNA, it can cause a transversion mutation (gene mutation) from GC (guanine-cytosine) to TA (thymine-adenine) during its replication. (Non-patent document 2) [Shibutani et al., Nature 349: 431, 1991].
  • 8-hydroxy-2'-deoxyguanosine has been used as a marker for DNA damage, which is a factor that causes senescence, cell mutation and canceration.
  • 8-hydroxydeoxyguanosine excreted in urine has been used as a marker that reflects the onset and disease state, such as the occurrence of cancer, management of diabetic patients, and monitoring after radiation exposure. The measurements were performed using thin-layer chromatography, high-performance liquid chromatography, electrochemical detection (HPLC-ECD), gas chromatography mass spectrometry, etc.
  • Kits for immunoassay using monoclonal antibodies against xyguanosine (8_ ⁇ HdG) Patent Document 1, Non-patent Document 3 have also been used. Antibodies to 8_ ⁇ HdG are frequently used to identify cells with a high content of 8-hydroxyguanine in the nucleus and play a crucial role in a wide range of fields such as medicine, pharmacy, and agriculture .
  • Patent Document 1 Patent No. 3091974
  • Non-Patent Document 1 Kasai, Mutat. Res. 387: 147, 1997
  • Non-patent document 2 Takatani et al., Nature 349: 431, 1991
  • Non-Patent Document 3 Toyokuni et al., Lab. Invest. 76: 365, 1997
  • the present invention specifies a region / site on a gene (DNA) susceptible to damage by stress. More specifically, to selectively collect DNA fragments containing one or more damaged nucleosides from a population of DNA fragments obtained from a sample. It is intended to provide a way to:
  • the present inventors succeeded in selectively collecting damaged DNA fragments by immunoprecipitation for the first time, and completed the present invention. That is, the present invention is as follows.
  • a method for collecting DNA fragments in a region containing DNA which has been damaged by oxidative stress comprising extracting DNA from a sample, fragmenting the DNA, and modifying the modified nucleoside or the modified nucleoside.
  • a method comprising incubating with a primary antibody specific for a polynucleotide containing a nucleoside, recovering a precipitated complex, and recovering a DNA fragment damaged by the complex.
  • modified nucleoside is selected from 8-hydroxy-2′-deoxyguanosine, cyclobutane-type pyrimidinin dimer, and acrolein-added 2′-deoxynucleoside.
  • the present invention it is possible for the first time to collect a DNA fragment containing a damaged nucleoside. Noh.
  • a means for identifying and analyzing a damaged or easily damaged region on the gene can be obtained.
  • protective means against genetic injury and by identifying sites (regions) that are susceptible to injury, if the site is a region specific to the type of cancer or organ, diagnosis of those sites can be made. It can contribute to future preventive treatment by elucidating the mechanism of onset of cancer and the like due to DNA damage, and understanding the genetic characteristics of each individual.
  • FIG. 1 shows the amount of DNA fragments collected from a methylene blue-treated genomic DNA sample using an antibody against 8-OHdG.
  • FIG. 3 shows the amount of DNA fragments collected from a DNA sample obtained from rat plasma using an antibody against 8-OHdG.
  • FIG. 4 shows the amount of DNA fragments collected from a UV-C-treated genomic DNA sample using an antibody against CPD.
  • FIG. 5 shows the amount of DNA fragments collected from DNA extracted from mouse kidney under oxidative stress load (6 hours after administration of Fe_NTA) using an antibody against acrolein-added 1′-deoxyadenosine.
  • modified nucleoside refers to a nucleoside damaged by oxidation stress caused by ultraviolet light, radiation, a chemical substance, or the like.
  • the 8'-position of 2'-deoxyguanosine is hydroxyl 8-hydroxy-2'-deoxyguanosine (hereinafter also referred to as “8_OHdG”), 8-nitroguanosine with 8-position guanosine nitrated, and cyclobutane-type pyrimidine dimer (hereinafter “CPD”). ), Nucleosides modified by reaction with acrolein, and the like.
  • Cyclobutane pyrimidine dimer is most frequently produced by ultraviolet light. This is one of the DNA damages that are caused.
  • acrolein is one of the unsaturated aldehydes formed by peroxidation of membrane lipids, and is widely detected in the environment and also found in cells under oxidative stress [Uchida et al. (Uchida et al.) , J. Biol. Chem. 273: 16058, 1998]. Since acrolein reacts with highly reactive nucleic acids to form adducts, it is known that nucleosides react with acrolein to form adducts under oxidative stress.
  • the present invention is directed to any nucleoside that is modified by reacting with acrolein under oxidative stress.
  • acrolein-added 2'-dexoxynucleoside is preferred for acrolein-added 2'-dexoxy.
  • Adenosine is particularly preferred.
  • an antibody specific to a modified nucleoside or a polynucleotide containing the modified nucleoside include polyclonal antibodies and monoclonal antibodies.
  • Antibodies to the modified deoxynucleoside can be prepared by a conventional method using a complex of a suitable carrier (for example, limpet hemocyanin (KLH)) and a synthetic modified deoxynucleoside as a hapten antigen as immunogen DNA. Can be manufactured. A polynucleotide (DNA fragment) containing the modified nucleoside is recognized by an antibody against the modified deoxynucleoside.
  • KLH limpet hemocyanin
  • an animal is immunized with the immunogen DNA prepared as described above.
  • the administration is performed by intravenously, subcutaneously or intraperitoneally administering an appropriate amount to a mammal (eg, rat, mouse, egret, human, etc.).
  • the interval between immunizations is not particularly limited, and is 1 to 10 times, preferably 4 to 5 times at intervals of several days to several weeks, preferably at intervals of 23 weeks.
  • the antibody titer is also measured 7 to 10 days after the final immunization, and blood is collected on the day showing the highest antibody titer to obtain antiserum.
  • the antibody titer can be measured by enzyme immunoassay (ELISA), radioimmunoassay (RIA), immunohistochemical staining, and the like.
  • ammonium sulfate precipitation ion exchange chromatography Purification can be carried out by appropriately selecting a known method such as luffy, gel filtration, affinity chromatography and the like, or by combining them.
  • an animal is immunized with immunogenic DNA. Immunization is performed by administering a suitable amount to mammals (for example, rats, mice, etc.) intravenously, subcutaneously or intraperitoneally.
  • mammals for example, rats, mice, etc.
  • the interval between immunizations is not particularly limited, and the immunization is performed at least for four to five times at intervals of several days to several weeks, preferably at intervals of two to three weeks.
  • antibody-producing cells are collected. Spleen cells are preferred as antibody-producing cells.
  • Myeloma cells used for cell fusion between antibody-producing cells such as spleen cells and myeloma cells may be cells derived from animals such as mice and commonly available cell lines. . As a cell line to be used, it has drug selectivity, cannot survive in a HAT selection medium (including hypoxanthine, aminopterin and thymidine) in an unfused state, and can survive only in a state fused to antibody-producing cells. Are preferred.
  • specific examples of myeloma cells include mouse myeloma cell lines such as PSUI, P3X63-Ag, and X63Ag8.653.
  • Antibody-producing cells and myeloma cells are mixed at a predetermined ratio (e.g., 3: 1) in animal cell culture medium such as serum-free DMEM or RPMI-1640 medium, and then mixed in the presence of a cell fusion promoter. Alternatively, it is performed by an electric noise treatment (for example, electoral port correction).
  • the cells are cultured using, for example, a HAT medium containing hypoxanthine (100 / im), aminopterin (0.4 ⁇ ) and thymidine (16 ⁇ ), and the cells that grow can be obtained as hybridomas.
  • Screening for the presence of the target antibody in the culture supernatant of the proliferated hybridomas is carried out by enzyme-linked immunosorbent assay (EIA) or the like. Cloning of the fused cells that have finally confirmed antibody production is performed by limiting dilution, etc., and finally, hybridomas, which are monoclonal antibody-producing cells, are established.
  • a usual cell culture method or the like can be employed.
  • a mouse is immunized using a complex of synthetic 8_OHdG and KLH as an immunogen, a hybridoma is prepared, and the obtained hybridoma is used.
  • a mouse monoclonal antibody against 8-hydroxy-2, -deoxyguanosine (clone N45.1 (Niken Zile), hereinafter referred to as “ ⁇ 45.1”) was prepared by preparing ascites fluid and fractionating and purifying it by ammonium sulfate salting out. (Name).
  • TD II-2 [Mori et al., Photochem Photobiol 54: 225, 1991] is known as a monoclonal antibody against clobtan-type pyrimidine dimer (CPD).
  • CPD clobtan-type pyrimidine dimer
  • 8_nitroguanosine have been provided (Clone # N02G52, Dojin).
  • monoclonal antibodies against the reaction product of acrolein with 2'-deoxyadenosine can be prepared by methods known in the literature [Kawai et al. (Kawai et al.), J. Biol. Chem. 278:
  • the DNA fragment having oxidative stress injury is specifically recognized by the mouse monoclonal antibody N45.1 against 8_OHdG. Therefore, they are precipitated as antigen-antibody complexes.
  • the immunoprecipitation method is well known in the art as an experimental technique for separating and purifying an antigen protein corresponding to a specific antibody.
  • a DNA fragment containing 8-hydroxydeoxyguanosine can be separated from a miscellaneous population of DNA fragments by immunoprecipitation using N45.1 under the conditions described below.
  • the amount of the injured DNA fragment containing the modified deoxynucleoside collected in this manner was determined by the sample used for preparing the original DNA fragment population as the starting material. The degree of oxidative stress is reflected, and the oxidative stress level of a living body can be appropriately evaluated by the method of the present invention.
  • each of the DNA fragments collected by the method of the present invention can be cloned using an appropriate vector-host system.
  • Such vectors are capable of replicating in a host. Is not particularly limited so long as it is, for example, plasmid DNA, phage DNA, etc.
  • plasmid DNA examples include a plasmid derived from Escherichia coli (eg, pBR322), a plasmid derived from Bacillus subtilis (eg, pUB110), a plasmid derived from yeast (eg, YEp13), and the like.
  • I phage eg, gtlO
  • animal viruses such as retrovirus, adenovirus or vaccinia virus, and insect virus vectors such as baculovirus can be used.
  • the purified DNA fragment is cleaved with an appropriate restriction enzyme, inserted into a restriction enzyme site or a multiple cloning site of the vector DNA, and ligated to the vector. Is adopted.
  • a transformant can be obtained by introducing the above-described recombinant vector into a host suitable for expressing a DNA fragment.
  • the host is not particularly limited.
  • bacteria of the genus Escherichia such as Escherichia coli
  • bacteria of the genus Bacillus such as Bacillus subtilis
  • yeasts such as Saccharomyces cerevisiae
  • COS cells And animal cells such as CHO cells, and insect cells such as Sf9 and Sf21.
  • a method for introducing a recombinant vector into a host is also known in the art, and examples thereof include a method using calcium ions, an electoporation method, a calcium phosphate method, and a lipofection method.
  • Culture of the transformant is performed according to a usual method used for culturing a host, and a medium and culture conditions suitable for each host are also known.
  • a microbial host is suitable for the present invention.
  • the medium contains a carbon source, a nitrogen source, inorganic salts, and the like that can be assimilated by the microorganism to efficiently culture the transformant.
  • Either a natural medium or a synthetic medium may be used as long as the medium can be used.
  • Cultivation is usually carried out at 37 ° C for 812 hours under aerobic conditions such as shaking culture or aeration and stirring culture.
  • the cloning in the present invention can also be carried out using a commercially available cloning kit.
  • a commercial kit for blunt-end PCR product cloning (Invitrogen; Zero Blunt TOPO PCR Cloning Kit for Sequencing) is available. is there.
  • the nucleotide sequence of each cloned collected DNA fragment is decoded to obtain sequence information, and the genome database is queried to determine the position of the chromosome corresponding to the damaged DNA fragment in the whole genome sequence. be able to.
  • the injured DNA fragment is mapped onto the chromosome as a single point.
  • mapping for example, there is a method of creating a distribution map based on the positional coordinate information on the chromosome obtained from the genome database of Celera.
  • the following steps employ a commonly used method.
  • a very small amount of the target substance is contained in the DNA (generally 100% of the amount of the sample DNA as a starting material) (About 1 / 1000th of the damaged DNA), so the special treatment described below was adopted and the first successful collection of the damaged DNA was achieved.
  • any sample containing DNA such as cultured cells, tissue cells of experimental animals, and blood collected from the human body
  • Preparation of DNA from specimens can be carried out in the usual way. It is necessary to select conditions under which DNA (deoxynucleoside) is not artificially modified in the process. For example, in order to suppress the production of 8-hydroxydeoxyguanosine to a very small amount, a deproteinization treatment using an organic solvent such as phenol cannot be applied as a DNA purification operation. Therefore, it is effective to add a chaotropic agent (such as sodium iodide) and suppress the co-precipitation phenomenon of DNA and protein, thereby omitting the phenol treatment.
  • a chaotropic agent such as sodium iodide
  • Specimen strength Fragment the obtained DNA by restriction enzyme treatment.
  • Select the type of restriction enzyme to be used so that the average length of the DNA fragment after enzyme digestion is about 1,000 bases.
  • Such restriction enzymes vary depending on the target (animal, plant, tissue, etc.).
  • Haelll recognition base sequence: GG ICC
  • the time of the restriction enzyme reaction is kept to a necessary minimum (1 hour) in order to reduce the generation of modified DNA (eg, 8-OHdG) as an artifact.
  • the solvent, temperature, etc. for the restriction enzyme reaction differ depending on the restriction enzyme used. To completely digest the sample DNA in one hour, it is desirable to add as many restriction enzymes as possible to the reaction system.
  • the immunoprecipitation reaction can be performed by the following method.
  • conditions were selected under which the antibody stably forms a complex with the antigen and precipitates.
  • a fragment containing 8_OHdG is separated from a heterogeneous population of DNA fragments.
  • PBS phosphate buffered saline, pH 7.4
  • the amount of antibody to be used depends on the amount of DNA and the amount of 8-OH dG contained therein (expected value), but it is added so that the antibody molecule is in excess with respect to the DNA molecule.
  • the number of antibody molecules is adjusted to be about 50 times or more, preferably 300 times or more, and more preferably 625 times or more with respect to one DNA fragment (one molecule).
  • the upper limit of the antibody molecule for the DNA fragment can be appropriately determined by those skilled in the art according to the purpose and the reaction system to be used. Generally, when a sample obtained by fragmenting genomic DNA extracted from mammalian tissue cells under physiological conditions into an average lkb length is used as a sample, the weight ratio of antibody to fragmented DNA is 0.08: 1, preferably 0.48: 1, most preferably about 1: 1.
  • DNA and antibody are combined in a test tube in a buffer, for example PBS, at a concentration of 0.01-0.05 / ⁇ ⁇ / ⁇ 1, preferably 0.01-0.02 zig / ⁇ , and this is rotated on a vertical surface. And mix each component. This mixing operation is performed at a low temperature (4 ° C) and continues for 1 to 3 hours.
  • bead particles having a molecule having an affinity for mouse immunoglobulin G bound to the surface thereof are added to the mixture obtained in (1).
  • carrier particles include Sepharose and agarose beads bound to Protein A or Protein G, and antibodies derived from other animals against mouse immunoglobulin G.
  • Combined magnetic beads eg, Dynabeads
  • proteins such as antibodies and restriction enzymes remain together with DNA.
  • the protease Proteinase K
  • the protease is allowed to act at 37 ° C for 1 hour in a TE buffer.
  • phenol-chloroform extraction and ethanol precipitation are performed, and the damaged DNA is purified and concentrated.
  • the following method is used to determine the amount of the damaged DNA fragment containing the modified deoxynucleoside (eg, 8_OHdG) collected by the method of the present invention.
  • the amount of DNA fragments damaged by oxidative stress is small, and the amount of DNA fragments collected by the method of the present invention is extremely small (about 100 minutes of the amount of sample DNA as a starting material). About 1 / 1000th of 1).
  • the residual level of 8-hydroxyhydroxyguanosine in the genomic DNA of mammalian cells is on the order of one per million guanine. Therefore, the spectrophotometer measurement method usually used for DNA quantification cannot be applied because the concentration is below the detection limit concentration.
  • the present inventors have succeeded in detecting and quantifying a trace amount of DNA by using a bromide reagent.
  • the quantification procedure is as follows.
  • the standard DNA solution of known concentration and the collected damaged DNA fragment solution are mixed with appropriate amounts of bromide solution.
  • the mixed solution of DNA-brominated medium is placed as droplets on an ultraviolet irradiation device, and the intensity of the fluorescence by ultraviolet irradiation is compared between the standard solution and the sample solution to determine the target damaged DNA content.
  • the damaged DNA collected in an appropriate vector-host system is cloned, for example, by the method described above, and the fragment population is separated as separate clones.
  • the base sequence (part of) is determined for each clone. Based on this sequence, a publicly available genome database can be searched to obtain detailed genomic information on the chromosome, a site having a sequence that matches the DNA fragment, and its surroundings. According to this analysis method, it is possible to comprehensively identify the remaining site of DNA damage due to oxidative stress over the entire genome.
  • the present invention is based on the combination of immunoprecipitation and a monoclonal antibody against a modified deoxynucleoside (e.g., 8-OHdG). And a method for selectively isolating and identifying the same.
  • the “optimal condition” can be determined based on the following points.
  • the amount of the antibody is adjusted as described above so as to efficiently recognize the damaged DNA fragment contained in the fragmented DNA population and form a complex.
  • Thorough washing was performed to completely remove non-target fragments non-specifically attached to or in proximity to the beads without antigen-antibody reaction. A total of eight washes were performed using four types of buffers with different surfactant compositions.
  • DNA is quantified using a spectrophotometer, utilizing the absorption characteristics of nucleic acids.However, since the amount of damaged DNA is lower than the detection sensitivity of a normal spectrophotometer, it is mixed with a bromide reagent. It is quantified by the fluorescence signal intensity of the mixed droplet.
  • Genomic DNA was prepared from kidney tissue of mice (16 weeks old, male C57BLZ6). DNA extraction from tissue cells was performed using the sodium iodide method (Wang et al., Nucleic Acids Res. 22: 1774, 1994), a commercial reagent kit (Wako Pure Chemical Industries; DNA Extractor WB Kit). It was implemented according to the attached instructions. During the extraction process, keep the sample at a low temperature as much as possible to suppress the new generation of 8-hydroxyguanine (ie, 8-hydroxy-2'-deoxyguanosine as a modified deoxynucleoside) during the extraction process. (On ice) and light-shielded. The buffers were saturated with argon gas, and 0.1M ImM desferrioxamine was added to inactivate the catalytic iron.
  • the mouse genomic DNA obtained in the above (1) was digested with the restriction enzyme Haelll at 37 ° C in 10 mM Tris-HCl (H7.5), 10 mM MgC12, 1 mM dithiothreitol and 50 mM NaCl, and the fragment was digested. It has become.
  • the reaction system was adjusted so that the enzyme could be completely cleaved in one hour.
  • the genomic DNA extracted was introduced artificially into the sample, and samples having different contents of 8-hydroxydeoxyguanosine were prepared.
  • guanine in the DNA is reduced to 8-hydroxydeoxy. It can be artificially changed to ciguanosine. Residual levels of 8-hydroxydeoxyguanosine in the genomic DNA of mammalian cells are usually on the order of one per million guanine.
  • Table 1 8_OHdG content of genomic DNA treated with methylene blue and light
  • the amount of PBS to be initially added was adjusted so that the total volume of the mixed system was 900 ⁇ l. This tube was stirred for 3 hours using a rotary shaker in a cold room (4 ° C).
  • Magnetic beads (Dynabeads M-280) conjugated with a secondary antibody (She-marked anti-Mouse IgG) manufactured by Dynal were used as a carrier for sedimentation.
  • a magnetic bead suspension (PBS containing 0.1% of plasma albumin) was used.
  • (Medium) (100 / il) was added to the mixed system, and the stirring operation was continued for another 3 hours by using a rotary shaker at a low temperature.After completion of the binding reaction, the beads were washed sequentially with four kinds of buffers.
  • composition of each buffer is as follows:
  • Solubilization buffer 140 mM NaCl
  • Solubilization buffer 500mM NaCl
  • 0.1% sodium deoxycholate 0.1% sodium deoxycholate, ImM EDTA, 50mM Hepes-KOH (pH7.5), 500mM NaCl, 1% TritonX-100
  • washing was carried out twice in each buffer in the order of 1 to 4 described above. After washing, the beads were suspended in 80 ⁇ l of an elution buffer (10 mM EDTA, 1% SDS, 50 mM Tris-HCl (pH 8.0)). The collected DNA fragments were eluted by heat treatment at 65 ° C for 15 minutes. The beads were then separated from the solution components using a magnet. The operation of adding the elution buffer ⁇ heating ⁇ separation was repeated once.
  • an elution buffer (10 mM EDTA, 1% SDS, 50 mM Tris-HCl (pH 8.0)
  • Fig. 1 shows the results when the initial DNA fragments to be subjected to immunoprecipitation were treated with 0, 5, 10, 50 ⁇ M methylene blue to cause artificial damage.
  • the vertical axis of the figure represents the amount of damaged DNA fragments collected by the method of the present invention from DNA samples in which the content of 8-—dG was increased stepwise. The results indicate that the amount of damaged DNA fragments collected depends on the amount of 8-hydroxydeoxyguanosine in the starting DNA sample.
  • Fe-NTA Iron nitrite triacetic acid
  • oxidative stress agent Iron nitrite triacetic acid
  • DNA extraction from kidney tissue and its fragmentation, and subsequent collection and purification of 8-hydroxyguanine-containing fragments were performed as described in Example 1.
  • the DNA cloning kit used in the following step (3) before purifying the collected DNA fragment, remove the 5'-terminal phosphate group from the DNA fragment Added a fostase treatment. That is, the collected DNA fragments were treated with a small intestine alkaline phosphatase (Takara Bio) at 50 ° C. for 30 minutes.
  • a commercial kit for blunt-end PCR product cloning (Invitrogen; Zero Blunt TOPO PCR Cloning Kit for Sequencing) was used.
  • the collected DNA fragment was inserted into a plasmid vector (pCR4Blunt-TOPO) according to the instructions attached to the kit, E. coli was transformed, and the resulting transformant was cultured on an agar plate. This was used as the library of the collected fragment clone.
  • Escherichia coli colonies were sequentially picked up from the agar plate of the above (3), and the nucleotide sequence of the DNA fragment clone was comprehensively determined using an autosequencer (ABI PRISM377).
  • FIG. 2 shows the mapping result of the remaining 8-hydroxyguanine under the oxidative stress load state.
  • the points indicated by black squares in the figure indicate the origin of each collected fragment on the chromosome. ing.
  • the DNA content in plasma is extremely low as compared with the kidney tissue used in Examples 1 and 2.
  • the amount of DNA obtained from blood (5-7 ml) collected from a single rat is shown in Table 2 below.
  • the plasma DNA obtained in the above (1) was fragmented in the same manner as described in Example 1, and the DNA fragment containing 8-hydroxydeoxyguanosine was collected and purified by immunoprecipitation.
  • the initial amount of DNA to be subjected to immunoprecipitation was 1.5 / ig.
  • 1.5 ⁇ of DNA was used from the combined extracted DNA of three animals. This initial amount of DNA is about one-third of the amount in Example 1.
  • FIG. 3 shows the results of collecting fragments containing 8-hydroxydeoxyguanosine from DNA in rat plasma by the method of the present invention.
  • Ultraviolet irradiation is also considered to be one of the oxidative stresses, and cyclobutane-type pyrimidine dimer (CPD) is one of the most frequently generated DNA damage by ultraviolet light
  • TDM-2 [Mori et al, Photochem Photobiol 54: 225, 1991] is a monoclonal antibody against CPD.
  • the DNA extracted from the tissue was irradiated with UVC (254 nm) to prepare a sample in which CPD was artificially introduced into the DNA fragment.
  • UVC 254 nm
  • SPECTROLINKER XL-100 UV crosslinker
  • Example 2 In immunoprecipitation with Example 1 the procedure, it was used TDM- 2, 20 ⁇ ⁇ as the primary antibody.
  • FIG. 4 shows the results of collecting CPD-containing DNA fragments by the method of the present invention. These results indicate that the amount of collected DNA fragments depends on the amount of UV irradiation.
  • Oxidative damage also includes peroxidation of lipids.
  • Acrolein is one of the unsaturated aldehydes formed by the peroxidation of membrane lipids, and is also found in cells under oxidative stress. (Uchida et al., Supra). Acrolein reacts with nucleic acids to form adducts.
  • mAb21 is a monoclonal antibody to the reaction of acrolein with 2'-deoxyadenosine
  • Each of 12-week-old and 17-week-old male C57BLZ6 mice was administered Fe-NTA in an amount equivalent to 3 mg of iron per kg of mouse body weight. Mice were sacrificed and extracted 6 hours after administration for S storage sample. Samples were also taken from untreated mice of the same age for controls. As in Example 1, genomic DNA was extracted from kidney tissue using a sodium iodide kit.
  • Kidney genomic DNA was fragmented in the same manner as described in Example 1. Further, according to the procedure of Example 1, the acrolein-added fragment was collected and purified by immunoprecipitation. However, as a primary antibody, 2 ⁇ g of a monoclonal antibody (mAb21) against a reaction product of acrolein and 2′-deoxyadenosine was used.
  • mAb21 monoclonal antibody against a reaction product of acrolein and 2′-deoxyadenosine
  • a genomic location (or gene) that is susceptible to damage is searched using the sequence information of the damaged DNA. It becomes possible.
  • a gene that is susceptible to injury is a candidate for a gene that is susceptible to mutation, and such information provides, for example, insight into the earliest stages of carcinogenesis. Therefore, in the end, information that is essential for establishing cancer prevention measures It can be used as basic information for deriving.

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Abstract

It is intended to provide a method of selectively collecting a DNA fragment in a region damaged by oxidative stress from a DNA-containing sample characterized by comprising extracting DNA from the sample, fragmenting the DNA, incubating with a primary antibody specific to a modified nucleoside having been modified by the damage or a polynucleotide containing the modified nucleoside as described above, collecting a complex thus precipitated, and then collecting a damaged DNA fragment from the complex.

Description

明 細 書  Specification
傷害を受けた DNA断片の収集法  Collection of damaged DNA fragments
技術分野  Technical field
[0001] 本発明は、傷害を受けた DNAの選択的な収集法に関し、さらに詳しくは酸化ストレ スに曝されたゲノム中、該ストレスで傷害を受けた DNA領域に相当する DNA断片を 選択的に収集する方法、そのようにして収集した DNA断片を用レ、るゲノム傷害の解 祈に関する。  The present invention relates to a method for selective collection of damaged DNA, and more particularly, to a method for selectively selecting a DNA fragment corresponding to a DNA region damaged by the stress in a genome exposed to oxidative stress. And the use of DNA fragments collected in this way to pray for genomic damage.
背景技術  Background art
[0002] 放射線、紫外線、大気汚染あるいは種々の化学物質などの環境ストレスによる生体 傷害の初期過程、遺伝子の発現調節や生体情報伝達、種々の疾患の発生やそれら の制御、等における活性酸素'フリーラジカルの影響が世界的に注目されている。特 に、老化や発がんの要因の一つとして活性酸素'フリーラジカルが関与する酸化スト レスによる遺伝子の損傷が様々な弊害をもたらすことから、その防御機構の研究、さ らには防御方法の開発研究も行われている。  [0002] Active oxygen-free in the initial process of biological injury due to environmental stresses such as radiation, ultraviolet rays, air pollution and various chemical substances, regulation of gene expression and biological information transmission, generation of various diseases and their control, etc. The effects of radicals are receiving worldwide attention. In particular, since gene damage caused by oxidative stress involving active oxygen 'free radicals as one of the factors of aging and carcinogenesis causes various adverse effects, research on its defense mechanism and development of defense methods Research is also being done.
[0003] 酸化ストレスとは、フリーラジカル ·活性酸素の発生量から、それらに対する消去能 力-防御能力 *傷害の修復能力などを差し引いたものを意味する概念であり、ヒトにお レ、ては動脈硬化症、がんなどの生活習慣病等に関係することが明らかとなっている。  [0003] Oxidative stress is a concept that means the amount of free radicals / active oxygen generated minus the erasing ability-defense ability * repair ability of injury, etc., for humans. It has been clarified to be related to lifestyle-related diseases such as arteriosclerosis and cancer.
DNAに代表される核酸もフリーラジカル '活性酸素の標的分子であるが、核酸を構 成する塩基のうち、プリン塩基の一つ、グァニンは傷害されやすいことが知られている 。ヒドロキシラジ力ノレ、一重項酸素あるいは光力学反応によって生成する修飾塩基と して、 2，ーデォキシグアノシンの 8位ヒドロキシル化体、 8—ヒドロキシ— 2'—デォキシグ ァノシンが記載されている（非特許文献 1)。この、 8—ヒドロキシー 2 '—デォキシグアノ シンが DNA中に存在すると、その複製過程で GC (グァニンーシトシン)から TA (チミ ン—アデニン)のトランスバージョン変異 (遺伝子変異)を引き起こす可能性があること が記載されている（非特許文献 2) [潞谷ら（Shibutani et al.) , Nature 349: 431, 1991] 。従って、 8—ヒドロキシー 2 '—デォキシグアノシンは老化、細胞の突然変異及び癌化 などを引き起こす要因である DNA損傷のマーカーとして用いられてレ、る。 [0004] 従来から、尿中に排泄される 8—ヒドロキシデォキシグアノシンは、癌の発生、糖尿病 患者の管理、放射線被曝後の監視など、発症や病勢を反映するマーカーとして使用 されており、その測定は、薄層クロマトグラフィー、高速液体クロマトグラフィーおよび 電気化学検出器 (HPLC— ECD)、ガスクロマトグラフィー質量分析計などを使用して 行われていたが、合成 8—ヒドロキシ _2 '—デォキシグアノシン（8_〇HdG)に対するモ ノクローナル抗体 (特許文献 1 ,非特許文献 3)を用レ、るィムノアッセィ用のキットも利 用されるようになっている。 8_〇HdGに対する抗体は、核内で 8—ヒドロキシグァニン 含有量の高い細胞を同定するために高頻度に使用され、医学、薬学、農学などの広 範な分野で極めて大きな役割を果たしている。 Nucleic acids such as DNA are also target molecules for free radicals and active oxygen, but guanine, one of the purine bases among nucleic acid bases, is known to be easily damaged. Hydroxyradiation, singlet oxygen, or the modified base formed by photodynamic reaction include the hydroxylated 8-position of 2, -deoxyguanosine and 8-hydroxy-2'-deoxyguanosine ( Non-patent document 1). When 8-hydroxy-2'-deoxyguanosine is present in DNA, it can cause a transversion mutation (gene mutation) from GC (guanine-cytosine) to TA (thymine-adenine) during its replication. (Non-patent document 2) [Shibutani et al., Nature 349: 431, 1991]. Therefore, 8-hydroxy-2'-deoxyguanosine has been used as a marker for DNA damage, which is a factor that causes senescence, cell mutation and canceration. [0004] Conventionally, 8-hydroxydeoxyguanosine excreted in urine has been used as a marker that reflects the onset and disease state, such as the occurrence of cancer, management of diabetic patients, and monitoring after radiation exposure. The measurements were performed using thin-layer chromatography, high-performance liquid chromatography, electrochemical detection (HPLC-ECD), gas chromatography mass spectrometry, etc. Kits for immunoassay using monoclonal antibodies against xyguanosine (8_ΔHdG) (Patent Document 1, Non-patent Document 3) have also been used. Antibodies to 8_〇HdG are frequently used to identify cells with a high content of 8-hydroxyguanine in the nucleus and play a crucial role in a wide range of fields such as medicine, pharmacy, and agriculture .
[0005] しかし、従来法では動植物から得た生体サンプルや病理標本、あるいはゲノム DN Aなど多様な混合物における 8—ヒドロキシー 2 '—デォキシグアノシン（8—ヒドロキシデ ォキシグアノシン）の検出および量の多寡の評価は可能である力 ゲノム情報と結び つけて、例えば遺伝子上の傷害に対する感受性の高い部位を特定することはできな 力つた。そのような感受性の高い部位を特定し、その構造 (配列等）を解析することに よって、例えば、遺伝子変異を回避し細胞の癌化を予防することが可能と考えられる 。また、そのような効果を有する化合物の研究開発にも寄与しうる。し力しながら、 DN Aは蛋白とは性質が全く異なるために、たとえ 8— OHdGに対するモノクローナル抗体 を用いても、ァフィ二ティークロマトグラフィーによって、特異的に 8— OHdG含む長鎖 の DNAを収集することは困難であった。ポストゲノム時代の現在、損傷を受けた DN A、即ち 8— OHdGを含むゲノム DNA断片を特異的に収集する簡便で経済的な方 法が強く求められている。  [0005] However, according to the conventional method, the detection and amount of 8-hydroxy-2'-deoxyguanosine (8-hydroxydeoxyguanosine) in biological samples and pathological specimens obtained from animals and plants, or various mixtures such as genomic DNAs. The power of assessing the potential of harmfulness In the context of genomic information, for example, it was not possible to identify sites that are highly susceptible to genetic damage. By identifying such a highly sensitive site and analyzing its structure (sequence and the like), for example, it is considered possible to avoid gene mutation and prevent canceration of cells. It can also contribute to the research and development of compounds having such effects. However, since DNA is completely different from protein in nature, even if a monoclonal antibody against 8-OHdG is used, long-chain DNA containing 8-OHdG is specifically collected by affinity chromatography. It was difficult to do. In the post-genome era, there is a strong need for a simple and economical method for specifically collecting genomic DNA fragments containing damaged DNA, ie, 8-OHdG.
[0006] 特許文献 1 :特許第 3091974号  Patent Document 1: Patent No. 3091974
非特許文献 1 :葛西、 Mutat. Res. 387: 147, 1997  Non-Patent Document 1: Kasai, Mutat. Res. 387: 147, 1997
非特許文献 2 :潞谷ら、 Nature 349: 431, 1991  Non-patent document 2: Takatani et al., Nature 349: 431, 1991
非特許文献 3 :豊國ら、 Lab. Invest. 76: 365, 1997  Non-Patent Document 3: Toyokuni et al., Lab. Invest. 76: 365, 1997
発明の開示  Disclosure of the invention
発明が解決しょうとする課題  Problems to be solved by the invention
[0007] 本発明はストレスによる損傷を受けやすい遺伝子（DNA)上の領域/部位を特定し 、解析する手段を提供することを目的としており、より直接的には、検体より得られた DNA断片の集団から、損傷を受けた 1またはそれ以上のヌクレオシドを含む DNA断 片を選択的に収集する方法を提供することを目的としている。 [0007] The present invention specifies a region / site on a gene (DNA) susceptible to damage by stress. More specifically, to selectively collect DNA fragments containing one or more damaged nucleosides from a population of DNA fragments obtained from a sample. It is intended to provide a way to:
課題を解決するための手段  Means for solving the problem
[0008] 本発明者らは上記課題を解決するために鋭意研究した結果、初めて、免疫沈降法 により傷害された DNA断片を選択的に収集することに成功し、本発明を完成した。 即ち、本発明は以下の通りである。 [0008] As a result of intensive studies to solve the above problems, the present inventors succeeded in selectively collecting damaged DNA fragments by immunoprecipitation for the first time, and completed the present invention. That is, the present invention is as follows.
(1) DNAを含む試料中の酸化ストレスにより傷害された領域の DNA断片を収集す る方法であって、試料力 DNAを抽出し、断片化した後、傷害により修飾された修飾 ヌクレオシドまたは該修飾ヌクレオシドを含むポリヌクレオチドに特異的な一次抗体と インキュベートし、沈降した複合体を回収し、該複合体力 傷害された DNA断片を 回収することを特徴とする方法。  (1) A method for collecting DNA fragments in a region containing DNA which has been damaged by oxidative stress, comprising extracting DNA from a sample, fragmenting the DNA, and modifying the modified nucleoside or the modified nucleoside. A method comprising incubating with a primary antibody specific for a polynucleotide containing a nucleoside, recovering a precipitated complex, and recovering a DNA fragment damaged by the complex.
(2)断片化により得られた修飾ヌクレオシドを含む DNA断片 1個に対して抗体分子 の数が 50倍以上である、 （1)記載の方法。  (2) The method according to (1), wherein the number of antibody molecules is 50 times or more per DNA fragment containing the modified nucleoside obtained by fragmentation.
(3)修飾ヌクレオシドが 8—ヒドロキシー 2'—デォキシグアノシン、シクロブタン型ピリミジ ンニ量体およびァクロレイン付加 2'—デォキシヌクレオシドから選択される、（1)又は (2)記載の方法。  (3) The method according to (1) or (2), wherein the modified nucleoside is selected from 8-hydroxy-2′-deoxyguanosine, cyclobutane-type pyrimidinin dimer, and acrolein-added 2′-deoxynucleoside.
(4)ァクロレイン付加 2，-デォキシヌクレオシドがァクロレイン付加 2，-デォキシアデノ シンである、（1)一（3)のレ、ずれかに記載の方法。  (4) The method according to (1) or (3), wherein the acrolein-added 2, -deoxynucleoside is acrolein-added 2, -deoxyadenosine.
(5)抗体がモノクローナル抗体である（1)一（4)のいずれかに記載の方法。  (5) The method according to any one of (1) to (4), wherein the antibody is a monoclonal antibody.
(6)上記（1)一 (5)のレ、ずれかに記載の方法によって回収した DNA断片を臭化工 チジゥム法によって定量し、その多寡により当該 DNA試料の酸化ストレスによる傷害 の程度を評価する方法。  (6) The DNA fragments recovered by the method described in (1) or (5) above are quantified by the bromination method and the degree of damage of the DNA sample due to oxidative stress is evaluated based on the quantity. Method.
(7)上記（1)一 (5)のレ、ずれかに記載の方法によって回収した DNA断片をクロー二 ングし、塩基配列を決定し、その配列情報と既知のゲノムの配列情報とを比較するこ とにより、酸化ストレスに対して感受性の遺伝子領域を同定する方法。  (7) Cloning the DNA fragment recovered by the method described in (1) or (5) above, determining the base sequence, and comparing the sequence information with the sequence information of a known genome. A method for identifying a gene region sensitive to oxidative stress.
発明の効果  The invention's effect
[0009] 本発明により、傷害されたヌクレオシドを含む DNA断片を収集することが初めて可 能となった。こうして得られた DNA断片をクローニングすることにより、遺伝子上で傷 害された、または傷害され易い領域を同定し、解析する手段を得ることができる。その 結果、遺伝子傷害に対する防御手段の開発が可能となり、また傷害を受けやすい部 位 (領域)を同定することにより、該部位が癌の種類や臓器に特異的な領域であれば 、それらの診断に用いることが可能となり、 DNA傷害による癌等の発症メカニズムの 解明、さらには各個人の遺伝子面での特徴を把握することで将来の予防治療にも貢 献しうる。 According to the present invention, it is possible for the first time to collect a DNA fragment containing a damaged nucleoside. Noh. By cloning the DNA fragment thus obtained, a means for identifying and analyzing a damaged or easily damaged region on the gene can be obtained. As a result, it becomes possible to develop protective means against genetic injury, and by identifying sites (regions) that are susceptible to injury, if the site is a region specific to the type of cancer or organ, diagnosis of those sites can be made. It can contribute to future preventive treatment by elucidating the mechanism of onset of cancer and the like due to DNA damage, and understanding the genetic characteristics of each individual.
図面の簡単な説明  Brief Description of Drawings
[0010] [図 1]メチレンブルー処理したゲノム DNA試料より、 8— OHdGに対する抗体を使って 収集した DNA断片量を示す。  [0010] FIG. 1 shows the amount of DNA fragments collected from a methylene blue-treated genomic DNA sample using an antibody against 8-OHdG.
[図 2]酸化ストレス負荷状態（Fe-NTA投与後 6時間）のマウス腎より抽出した DNAより 収集した 8-ヒドロキシグァニン含有断片について、個々の断片のゲノム内での位置を 決定した結果を示す。  [Figure 2] The results of determining the position of each fragment in the genome of 8-hydroxyguanine-containing fragments collected from DNA extracted from mouse kidney under oxidative stress load (6 hours after administration of Fe-NTA) Show.
[図 3]ラット血漿から得た DNA試料より、 8— OHdGに対する抗体を使って収集した D NA断片量を示す。  FIG. 3 shows the amount of DNA fragments collected from a DNA sample obtained from rat plasma using an antibody against 8-OHdG.
[図 4]UV— C処理したゲノム DNA試料より、 CPDに対する抗体を使って収集した DN A断片量を示す。  FIG. 4 shows the amount of DNA fragments collected from a UV-C-treated genomic DNA sample using an antibody against CPD.
[図 5]酸化ストレス負荷状態（Fe_NTA投与後 6時間）のマウス腎より抽出した DNAより 、ァクロレイン付加一 2 '—デォキシアデノシンに対する抗体を使って収集した DNA断 片量を示す。  FIG. 5 shows the amount of DNA fragments collected from DNA extracted from mouse kidney under oxidative stress load (6 hours after administration of Fe_NTA) using an antibody against acrolein-added 1′-deoxyadenosine.
発明を実施するための最良の形態  BEST MODE FOR CARRYING OUT THE INVENTION
[0011] 本明細書中、「修飾ヌクレオシド」とは、紫外線、放射線、化学物質等による酸化スト レスによって傷害を受けたヌクレオシドを意味し、例えば、 2 ' -デォキシグアノシンの 8 位がヒドロキシル化された 8-ヒドロキシー 2 '—デォキシグアノシン、以下、（「8_OHdG 」とも呼称）、グアノシンの 8位がニトロ化された 8-ニトログアノシン、シクロブタン型ピリミ ジン二量体（以下、「CPD」と呼称）、ァクロレインとの反応により修飾されたヌクレオシ ド等を挙げること力 Sできる。 [0011] As used herein, the term "modified nucleoside" refers to a nucleoside damaged by oxidation stress caused by ultraviolet light, radiation, a chemical substance, or the like. For example, the 8'-position of 2'-deoxyguanosine is hydroxyl 8-hydroxy-2'-deoxyguanosine (hereinafter also referred to as “8_OHdG”), 8-nitroguanosine with 8-position guanosine nitrated, and cyclobutane-type pyrimidine dimer (hereinafter “CPD”). ), Nucleosides modified by reaction with acrolein, and the like.
なお、シクロブタン型ピリミジン二量体 (CPD)は、紫外線によって最も高頻度に生 成される DNA傷害の一つである。また、ァクロレインは膜脂質の過酸化によって生成 する不飽和アルデヒドの一つであり、広く環境中に検出されると同時に、酸化ストレス 下の細胞内にも認められる [内田ら（Uchida et al.) , J. Biol. Chem.273: 16058, 1998] 。ァクロレインは非常に反応性が高ぐ核酸と反応して付加物を形成するので、酸化 ストレス下でヌクレオシドがァクロレインと反応して付加物を形成することが知られてい る。本発明は、酸化ストレス下でァクロレインと反応して修飾される任意のヌクレオシド を対象としている力 本発明方法にとって、ァクロレイン付加 2 ' -デォキシヌクレオシド が好ましぐァクロレイン付加 2 ' -デォキシアデノシンが特に好ましい。 Cyclobutane pyrimidine dimer (CPD) is most frequently produced by ultraviolet light. This is one of the DNA damages that are caused. In addition, acrolein is one of the unsaturated aldehydes formed by peroxidation of membrane lipids, and is widely detected in the environment and also found in cells under oxidative stress [Uchida et al. (Uchida et al.) , J. Biol. Chem. 273: 16058, 1998]. Since acrolein reacts with highly reactive nucleic acids to form adducts, it is known that nucleosides react with acrolein to form adducts under oxidative stress. The present invention is directed to any nucleoside that is modified by reacting with acrolein under oxidative stress.For the method of the present invention, acrolein-added 2'-dexoxynucleoside is preferred for acrolein-added 2'-dexoxy. Adenosine is particularly preferred.
[0012] 「修飾ヌクレオシドまたは該修飾ヌクレオシドを含むポリヌクレオチドに特異的な抗体 」としては、ポリクローナル抗体、モノクローナル抗体が挙げられる。 [0012] Examples of "an antibody specific to a modified nucleoside or a polynucleotide containing the modified nucleoside" include polyclonal antibodies and monoclonal antibodies.
ポリクローナル及びモノクローナル抗体の製造法は、既知である。例えば、 Methods for producing polyclonal and monoclonal antibodies are known. For example,
Antibodies; A Laboratory Manual, Lane, H, D.ら編，し old Spring Harbor Laboratory Press出版 New York 1989年、 Kohlerら, Nature, 256:495- 497 (1975)及び Eur. J. Immunol. 6:511-519 (1976)； Milsteinら， Nature 266: 550-552 (1977)； Koprowskiら、 米国特許第 4, 172, 124号)等を参照。修飾デォキシヌクレオシドに対する抗体は、適 当な担体 (例えば、カサガイへモシァニン (KLH) )等）とハプテン抗原として合成修 飾デォキシヌクレオシドとの複合体を免疫原 DNAとし、通常の方法で製造することが できる。修飾ヌクレオシドを含むポリヌクレオチド（DNA断片）は、上記修飾デォキシ ヌクレオシドに対する抗体により認識される。 Antibodies; A Laboratory Manual, Lane, H, D. et al., Edited by old Spring Harbor Laboratory Press New York 1989, Kohler et al., Nature, 256: 495-497 (1975) and Eur. J. Immunol. 6: 511. -519 (1976); Milstein et al., Nature 266: 550-552 (1977); Koprowski et al., US Patent No. 4,172,124) and the like. Antibodies to the modified deoxynucleoside can be prepared by a conventional method using a complex of a suitable carrier (for example, limpet hemocyanin (KLH)) and a synthetic modified deoxynucleoside as a hapten antigen as immunogen DNA. Can be manufactured. A polynucleotide (DNA fragment) containing the modified nucleoside is recognized by an antibody against the modified deoxynucleoside.
[0013] ポリクローナル抗体 [0013] polyclonal antibody
ポリクローナル抗体を得るには、前記のようにして調製した免疫原 DNAを用いて動 物を免疫する。哺乳動物（例えばラット、マウス、ゥサギ、ヒトなど）に静脈内、皮下又 は腹腔内に適当量を投与することにより行う。また、免疫の間隔は特に限定されず、 数日から数週間間隔、好ましくは 2 3週間間隔で、 1一 10回、好ましくは 4一 5回で ある。最終の免疫日力も 7— 10日後に抗体価を測定し、最大の抗体価を示した日に 採血し、抗血清を得る。抗体価の測定は、酵素免疫測定法 (ELISA)、放射性免疫測 定法 (RIA)、免疫組織染色法等により行うことができる。  To obtain a polyclonal antibody, an animal is immunized with the immunogen DNA prepared as described above. The administration is performed by intravenously, subcutaneously or intraperitoneally administering an appropriate amount to a mammal (eg, rat, mouse, egret, human, etc.). The interval between immunizations is not particularly limited, and is 1 to 10 times, preferably 4 to 5 times at intervals of several days to several weeks, preferably at intervals of 23 weeks. The antibody titer is also measured 7 to 10 days after the final immunization, and blood is collected on the day showing the highest antibody titer to obtain antiserum. The antibody titer can be measured by enzyme immunoassay (ELISA), radioimmunoassay (RIA), immunohistochemical staining, and the like.
抗血清から抗体の精製が必要とされる場合は、硫安塩析法、イオン交換クロマトグ ラフィー、ゲル濾過、ァフィ二ティークロマトグラフィーなどの公知の方法を適宜選択し て、又はこれらを組み合わせることにより精製することができる。 If purification of the antibody from antiserum is required, ammonium sulfate precipitation, ion exchange chromatography Purification can be carried out by appropriately selecting a known method such as luffy, gel filtration, affinity chromatography and the like, or by combining them.
[0014] モノクローナル抗体  [0014] Monoclonal antibody
モノクローナル抗体を得るには、免疫原 DNAを用いて動物を免疫する。免疫は、 哺乳動物（例えばラット、マウスなど）に静脈内、皮下又は腹腔内に適当量を投与す ることにより行う。免疫の間隔は特に限定されず、数日から数週間間隔、好ましくは 2 一 3週間間隔で、最低 4一 5回行う。そして、最終免疫後、抗体産生細胞を採集する 。抗体産生細胞としては、脾臓細胞が好ましい。  To obtain a monoclonal antibody, an animal is immunized with immunogenic DNA. Immunization is performed by administering a suitable amount to mammals (for example, rats, mice, etc.) intravenously, subcutaneously or intraperitoneally. The interval between immunizations is not particularly limited, and the immunization is performed at least for four to five times at intervals of several days to several weeks, preferably at intervals of two to three weeks. After the final immunization, antibody-producing cells are collected. Spleen cells are preferred as antibody-producing cells.
[0015] 脾臓細胞等の抗体産生細胞とミエローマ細胞との細胞融合に用いるミエローマ細 胞としては、マウスなどの動物由来の細胞であって一般に入手可能な株化細胞を使 用すること力 Sできる。使用する細胞株として、薬剤選択性を有し、未融合の状態では HAT選択培地（ヒポキサンチン、アミノプテリン及びチミジンを含む）で生存できず、 抗体産生細胞と融合した状態でのみ生存できる性質を有するものが好ましい。例え ば、ミエローマ細胞の具体例としては PSUI、 P3X63-Ag、 X63Ag8.653などのマウスミ エローマ細胞株が挙げられる。血清を含まない DMEM、 RPMI-1640培地などの動物 細胞培養用培地中に、抗体産生細胞とミエローマ細胞とを所定の割合 (例えば 3 : 1) で混合し、細胞融合促進剤存在の下で、あるいは電気ノ^レス処理 (例えばエレクト口 ポレーシヨン）により行う。  [0015] Myeloma cells used for cell fusion between antibody-producing cells such as spleen cells and myeloma cells may be cells derived from animals such as mice and commonly available cell lines. . As a cell line to be used, it has drug selectivity, cannot survive in a HAT selection medium (including hypoxanthine, aminopterin and thymidine) in an unfused state, and can survive only in a state fused to antibody-producing cells. Are preferred. For example, specific examples of myeloma cells include mouse myeloma cell lines such as PSUI, P3X63-Ag, and X63Ag8.653. Antibody-producing cells and myeloma cells are mixed at a predetermined ratio (e.g., 3: 1) in animal cell culture medium such as serum-free DMEM or RPMI-1640 medium, and then mixed in the presence of a cell fusion promoter. Alternatively, it is performed by an electric noise treatment (for example, electoral port correction).
[0016] 次いで、例えばヒポキサンチン（100 /i m)、アミノプテリン（0.4 μ πι)及びチミジン（16 β Μ)を含む HAT培地を用いて培養し、生育する細胞をハイプリドーマとして得ること ができる。増殖したハイプリドーマの培養上清中に、 目的とする抗体が存在するか否 かを酵素抗体法 (EIA)等でスクリーニングする。最終的に抗体産生を確認した融合 細胞のクローニングは、限界希釈法等により行い、最終的に単クローン抗体産生細 胞であるハイプリドーマを樹立する。樹立したハイプリドーマから単クローン抗体を採 取するには、通常の細胞培養法等を採用することができる。抗体の精製が必要とされ る場合は、硫安分画法、イオン交換クロマトグラフィー、ァフィ二ティーク口マトグラフィ 一、ゲルクロマトグラフィーなどの公知の方法を適宜に選択して、又はこれらの方法を 組み合わせることにより精製することができる。 [0017] 具体的には、特許文献 1に記載されているように、合成 8_OHdGと KLHとの複合 体を免疫原としてマウスを免役し、ハイプリドーマを作製し、得られたハイプリドーマを 用いて腹水を調製し、硫酸アンモニゥム塩析法により分画精製する方法によって 8— ヒドロキシー 2，—デォキシグアノシンに対するマウスモノクローナル抗体（クローン N45 .1 (日研ザイル）、以下、「Ν45· 1」と呼称）が得られる。 [0016] Next, the cells are cultured using, for example, a HAT medium containing hypoxanthine (100 / im), aminopterin (0.4 µπι) and thymidine (16βΜ), and the cells that grow can be obtained as hybridomas. Screening for the presence of the target antibody in the culture supernatant of the proliferated hybridomas is carried out by enzyme-linked immunosorbent assay (EIA) or the like. Cloning of the fused cells that have finally confirmed antibody production is performed by limiting dilution, etc., and finally, hybridomas, which are monoclonal antibody-producing cells, are established. To collect a monoclonal antibody from the established hybridoma, a usual cell culture method or the like can be employed. If antibody purification is required, use known methods such as ammonium sulfate fractionation, ion exchange chromatography, affinity chromatography, gel chromatography, etc., or combine these methods. Can be purified. [0017] Specifically, as described in Patent Document 1, a mouse is immunized using a complex of synthetic 8_OHdG and KLH as an immunogen, a hybridoma is prepared, and the obtained hybridoma is used. A mouse monoclonal antibody against 8-hydroxy-2, -deoxyguanosine (clone N45.1 (Niken Zile), hereinafter referred to as “Ν45.1”) was prepared by preparing ascites fluid and fractionating and purifying it by ammonium sulfate salting out. (Name).
また、クロブタン型ピリミジン二量体（CPD)に対するモノクローナル抗体として TD Μ—2 [森ら（Mori et al.)， Photochem Photobiol 54: 225, 1991]が知られている。さら に、 8_ニトログアノシンに対する抗体も提供されている（Clone#N02G52、同仁)。 さらに、ァクロレインと 2 '—デォキシアデノシンとの反応物に対するモノクローナル抗 体も文献既知の方法で作製できる [河井ら（Kawai et al.)， J. Biol. Chem. 278:  Also, TD II-2 [Mori et al., Photochem Photobiol 54: 225, 1991] is known as a monoclonal antibody against clobtan-type pyrimidine dimer (CPD). In addition, antibodies to 8_nitroguanosine have been provided (Clone # N02G52, Dojin). In addition, monoclonal antibodies against the reaction product of acrolein with 2'-deoxyadenosine can be prepared by methods known in the literature [Kawai et al. (Kawai et al.), J. Biol. Chem. 278:
50346, 2003]  50346, 2003]
[0018] <方法 >  [0018] <Method>
本発明方法の概略を、主として 8— OHdGとそのモノクローナル抗体 N45. 1を用い て説明するが、当業者ならば、本発明は任意の修飾デォキシヌクレオシドまたは修飾 デォキシヌクレオシドを含む DNAに対する抗体を用いて実施可能であることを理解 するであろう。  Although the outline of the method of the present invention will be mainly described using 8-OHdG and its monoclonal antibody N45.1, those skilled in the art will understand that the present invention relates to any modified deoxynucleoside or DNA containing a modified deoxynucleoside. It will be appreciated that this can be done using antibodies.
[0019] 酸化ストレス傷害を有する DNA断片は、 8_OHdGに対するマウスモノクローナル 抗体 N45. 1により特異的に認識される。従って、抗原抗体複合物としてこれらを沈 降させる。免疫沈降法は、特定の抗体に対応する抗原蛋白質を分離、精製するため の実験技術として当該技術分野で周知である。 8-ヒドロキシデォキシグアノシンを含 む DNA断片は、 N45. 1を用いて、後述する条件下で免疫沈降することにより、雑多 な DNA断片の集団から分離することができる。  [0019] The DNA fragment having oxidative stress injury is specifically recognized by the mouse monoclonal antibody N45.1 against 8_OHdG. Therefore, they are precipitated as antigen-antibody complexes. The immunoprecipitation method is well known in the art as an experimental technique for separating and purifying an antigen protein corresponding to a specific antibody. A DNA fragment containing 8-hydroxydeoxyguanosine can be separated from a miscellaneous population of DNA fragments by immunoprecipitation using N45.1 under the conditions described below.
[0020] このようにして収集された、修飾デォキシヌクレオシドを含む傷害を受けた DNA断 片の量の多寡は、出発物質である元の DNA断片集団の調製に供された検体が受け た酸化ストレスの程度を反映しており、本発明方法によって生体の酸化ストレスレべ ルを適切に評価することができる。  [0020] The amount of the injured DNA fragment containing the modified deoxynucleoside collected in this manner was determined by the sample used for preparing the original DNA fragment population as the starting material. The degree of oxidative stress is reflected, and the oxidative stress level of a living body can be appropriately evaluated by the method of the present invention.
[0021] さらに、本発明方法によって収集された DNA断片の各々を、適当なベクター-宿主 系によりクローニングすることができる。そのようなベクターは、宿主中で複製可能なも のであれば特に限定されず、例えばプラスミド DNA、ファージ DNA等が挙げられる [0021] Further, each of the DNA fragments collected by the method of the present invention can be cloned using an appropriate vector-host system. Such vectors are capable of replicating in a host. Is not particularly limited so long as it is, for example, plasmid DNA, phage DNA, etc.
[0022] プラスミド DNAとしては、大腸菌由来のプラスミド（例えば pBR322等）、枯草菌由来 のプラスミド (例えば pUB 110等）、酵母由来のプラスミド (例えば YEp 13等）などが挙げ られ、ファージ DNAとしては; Iファージ（例えば gtlO等）が挙げられる。また、レトロ ウィルス、アデノウイルス又はワクシニアウィルスなどの動物ウィルスやバキュ口ウィル スなどの昆虫ウィルスベクターを用いることもできる。 Examples of the plasmid DNA include a plasmid derived from Escherichia coli (eg, pBR322), a plasmid derived from Bacillus subtilis (eg, pUB110), a plasmid derived from yeast (eg, YEp13), and the like. I phage (eg, gtlO). Also, animal viruses such as retrovirus, adenovirus or vaccinia virus, and insect virus vectors such as baculovirus can be used.
ベクターに DNA断片を揷入するには、まず、精製した DNA断片を適当な制限酵 素酵素で切断して、ベクター DNAの制限酵素部位又はマルチクローニングサイトに 揷入してベクターに連結する方法などが採用される。  In order to insert a DNA fragment into a vector, first, the purified DNA fragment is cleaved with an appropriate restriction enzyme, inserted into a restriction enzyme site or a multiple cloning site of the vector DNA, and ligated to the vector. Is adopted.
[0023] 上記の組換えベクターを、 DNA断片の発現に適した宿主中に導入することにより 形質転換体を得ることができる。宿主は特に限定されるものではなぐ例えばェシエリ ヒア ·コリ（Escherichia coli)等の Escherichia属、バチルス ·ズブチリス（Bacillus subtilis )等のバチルス属の細菌、サッカロミセス'セレピシェ（Saccharomyces cerevisiae)等 の酵母、 COS細胞、 CHO細胞等の動物細胞、及び Sf9、 Sf21等の昆虫細胞が挙げら れる。  A transformant can be obtained by introducing the above-described recombinant vector into a host suitable for expressing a DNA fragment. The host is not particularly limited. For example, bacteria of the genus Escherichia such as Escherichia coli, bacteria of the genus Bacillus such as Bacillus subtilis, yeasts such as Saccharomyces cerevisiae, and COS cells And animal cells such as CHO cells, and insect cells such as Sf9 and Sf21.
また、組換えベクターの宿主への導入方法も当該技術分野で既知であり、カルシゥ ムイオンを用いる方法、エレクト口ポレーシヨン法、リン酸カルシウム法、リポフエクショ ン法等が挙げられる。  In addition, a method for introducing a recombinant vector into a host is also known in the art, and examples thereof include a method using calcium ions, an electoporation method, a calcium phosphate method, and a lipofection method.
[0024] 形質転換体の培養は、宿主の培養に用いられる通常の方法に従って行われ、それ ぞれの宿主に適した培地、培養条件も既知である。なお、本発明にとっては微生物 宿主が適しており、そのような場合の培地としては、微生物が資化し得る炭素源、窒 素源、無機塩類等を含有し、形質転換体の培養を効率的に行うことができる培地で あれば、天然培地、合成培地のいずれを用いてもよい。培養は、通常、振盪培養又 は通気攪拌培養などの好気的条件下、 37°Cで 8 12時間行う。  [0024] Culture of the transformant is performed according to a usual method used for culturing a host, and a medium and culture conditions suitable for each host are also known. A microbial host is suitable for the present invention.In such a case, the medium contains a carbon source, a nitrogen source, inorganic salts, and the like that can be assimilated by the microorganism to efficiently culture the transformant. Either a natural medium or a synthetic medium may be used as long as the medium can be used. Cultivation is usually carried out at 37 ° C for 812 hours under aerobic conditions such as shaking culture or aeration and stirring culture.
[0025] 本発明におけるクローニングは市販のクローニングキットを用いても実施でき、その ようなキットとして平滑末端 PCR産物クローユングのための巿販キット（インビトロジェ ン； Zero Blunt TOPO PCR Cloning Kit for Sequencing)がある。 [0026] クローニングされた個々の収集 DNA断片について塩基配列を解読して配列情報 を得、ゲノムデータベースを照会することにより、全ゲノム配列中の当該傷害 DNA断 片に一致する染色体の位置を決定することができる。実際には、傷害 DNA断片をマ 一力一として染色体上へマッピングする。染色体上に傷害 DNAの場所を位置付ける (マッピング）方法として、例えば、 Celera社のゲノムデータベースより入手できる染色 体上での位置座標情報をもとに分布地図を作成する方法がある。 [0025] The cloning in the present invention can also be carried out using a commercially available cloning kit. As such a kit, a commercial kit for blunt-end PCR product cloning (Invitrogen; Zero Blunt TOPO PCR Cloning Kit for Sequencing) is available. is there. [0026] The nucleotide sequence of each cloned collected DNA fragment is decoded to obtain sequence information, and the genome database is queried to determine the position of the chromosome corresponding to the damaged DNA fragment in the whole genome sequence. be able to. In practice, the injured DNA fragment is mapped onto the chromosome as a single point. As a method of mapping the location of the damaged DNA on the chromosome (mapping), for example, there is a method of creating a distribution map based on the positional coordinate information on the chromosome obtained from the genome database of Celera.
[0027] 酸化ストレスによる病態の研究では、フリーラジカル '活性酸素の標的となって傷害 を受けやすい遺伝子又は遺伝子の領域を同定することが必須である。本発明の方法 によって収集された DNA断片は、そのような目的にとって極めて有用な情報を提供 する。  [0027] In the study of pathological conditions due to oxidative stress, it is essential to identify a gene or a region of a gene that is a target of free radicals and active oxygen and is susceptible to injury. DNA fragments collected by the method of the invention provide extremely useful information for such purposes.
[0028] 傷害 DNAの収集法  [0028] Method for collecting damaged DNA
本発明の方法による DNA断片の収集は、一般に、以下の工程は通常用いられる 方法を採用している力 目的物が DNA中に極微量 (一般に出発物質としての試料 D NAの量の 100分の 1一 1,000分の 1程度）しか存在しない傷害 DNAであることから 、後述の特別な処置を採用し、初めて傷害 DNAの収集に成功した。  In the collection of DNA fragments according to the method of the present invention, generally, the following steps employ a commonly used method.A very small amount of the target substance is contained in the DNA (generally 100% of the amount of the sample DNA as a starting material) (About 1 / 1000th of the damaged DNA), so the special treatment described below was adopted and the first successful collection of the damaged DNA was achieved.
[0029] A)試料 DNAの調製  A) Preparation of sample DNA
検体としては、培養細胞、実験動物の組織細胞、人体から採取した血液など DNA を含有するあらゆる試料を使うことが可能である。検体からの DNAの調製は、通常の 方法で行うことができる力 その過程で人為的に DNA (デォキシヌクレオシド）が修飾 されない条件を選択する必要がある。例えば、 8—ヒドロキシデォキシグアノシンの生 成を極少量に抑制するためには、 DNAの精製操作として、フエノール等の有機溶媒 を使用した除蛋白処理を適用することができない。従って、カオトロピック剤（ヨウ化ナ トリウムなど）を添加し、 DNAと蛋白質の共沈現象を抑制することにより、フヱノール 処理を省略する方法が有効である。  As the specimen, any sample containing DNA, such as cultured cells, tissue cells of experimental animals, and blood collected from the human body, can be used. Preparation of DNA from specimens can be carried out in the usual way. It is necessary to select conditions under which DNA (deoxynucleoside) is not artificially modified in the process. For example, in order to suppress the production of 8-hydroxydeoxyguanosine to a very small amount, a deproteinization treatment using an organic solvent such as phenol cannot be applied as a DNA purification operation. Therefore, it is effective to add a chaotropic agent (such as sodium iodide) and suppress the co-precipitation phenomenon of DNA and protein, thereby omitting the phenol treatment.
[0030] B) DNAの断片化  [0030] B) DNA fragmentation
検体力 得た DNAを制限酵素処理により断片化する。酵素消化後の DNA断片の 平均長が、 1， 000塩基程度となるように、使用する制限酵素の種類を選択する。そ のような制限酵素は対象 (動物、植物、組織など）により異なるが、例えば、制限酵素 Haelll (認識塩基配列： GG I CC)は上記要件を満たしている。この制限酵素処理の 場合、人為産物としての修飾 DNA (例、 8— OHdG)の生成をより少なくするため、制 限酵素反応の時間は必要最小限（1時間）に抑える。制限酵素反応の溶媒、温度等 は使用する制限酵素により異なる。 1時間で完全に試料 DNAを消化するために、可 能な限り多くの制限酵素を反応系に添加することが望ましい。 Specimen strength Fragment the obtained DNA by restriction enzyme treatment. Select the type of restriction enzyme to be used so that the average length of the DNA fragment after enzyme digestion is about 1,000 bases. Such restriction enzymes vary depending on the target (animal, plant, tissue, etc.). Haelll (recognition base sequence: GG ICC) satisfies the above requirements. In the case of this restriction enzyme treatment, the time of the restriction enzyme reaction is kept to a necessary minimum (1 hour) in order to reduce the generation of modified DNA (eg, 8-OHdG) as an artifact. The solvent, temperature, etc. for the restriction enzyme reaction differ depending on the restriction enzyme used. To completely digest the sample DNA in one hour, it is desirable to add as many restriction enzymes as possible to the reaction system.
[0031] C)免疫沈降 [0031] C) Immunoprecipitation
免疫沈降反応は以下の方法で行うことができる。本発明の対象である修飾デォキ シヌクレオシドを含む傷害 DNA断片を効率よく沈降させるために、抗体が抗原と安 定的に複合物を形成し、沈降する条件を選択した。  The immunoprecipitation reaction can be performed by the following method. In order to efficiently precipitate the damaged DNA fragment containing the modified deoxynucleoside, which is the object of the present invention, conditions were selected under which the antibody stably forms a complex with the antigen and precipitates.
(1)抗原-抗体反応  (1) Antigen-antibody reaction
DNA断片の雑多な集団から例えば 8_OHdGを含む断片だけを分離する。適当な 容量の PBS (リン酸緩衝生理的食塩水、 pH7.4)に、断片化した DNAとモノクローナ ル抗体 (Ν45· 1)を添加する。使用する抗体量は、 DNA量とそれに含まれる 8— OH dG量 (予想値）に依存するが、抗体分子が DNA分子に対して過剰になるように加え る。 DNA断片 1個（1分子）に対して抗体分子が約 50倍以上、好ましくは 300倍以上 、より好ましくは 625倍以上になるように調整する。 DNA断片に対する抗体分子の上 限は当業者が目的や使用する反応系に応じて適宜決定することができる。一般に、 生理的条件下にある哺乳動物の組織細胞より抽出したゲノム DNAを平均 lkb長に 断片化したものを試料とする場合、抗体と断片化 DNAの重量比は 0.08 : 1、好ましく は 0.48 : 1、最も好ましくは約 1： 1である。 DNAと抗体を試験管内で緩衝液、例えば PBS中、濃度 0.01— 0·05 /ι §/ μ 1、好ましくは 0.01— 0.02 zi g/ μ ΐで昆合し、これ を鉛直面にて回転し、各成分を混和させる。この混和操作は低温 (4°C)で行レ、、 1一 3時間続ける。 For example, only a fragment containing 8_OHdG is separated from a heterogeneous population of DNA fragments. Add the fragmented DNA and monoclonal antibody (Ν45.1) to an appropriate volume of PBS (phosphate buffered saline, pH 7.4). The amount of antibody to be used depends on the amount of DNA and the amount of 8-OH dG contained therein (expected value), but it is added so that the antibody molecule is in excess with respect to the DNA molecule. The number of antibody molecules is adjusted to be about 50 times or more, preferably 300 times or more, and more preferably 625 times or more with respect to one DNA fragment (one molecule). The upper limit of the antibody molecule for the DNA fragment can be appropriately determined by those skilled in the art according to the purpose and the reaction system to be used. Generally, when a sample obtained by fragmenting genomic DNA extracted from mammalian tissue cells under physiological conditions into an average lkb length is used as a sample, the weight ratio of antibody to fragmented DNA is 0.08: 1, preferably 0.48: 1, most preferably about 1: 1. DNA and antibody are combined in a test tube in a buffer, for example PBS, at a concentration of 0.01-0.05 / ι § / μ 1, preferably 0.01-0.02 zig / μΐ, and this is rotated on a vertical surface. And mix each component. This mixing operation is performed at a low temperature (4 ° C) and continues for 1 to 3 hours.
[0032] (2)ビーズへの結合反応 (2) Binding reaction to beads
抗原-抗体複合体を沈降させる担体として、例えば、マウス免疫グロブリン Gに親和 性をもつ分子をその表面に結合したビーズ粒子を、 （1)で得られた混和液に添加す る。このような担体粒子として、 Protein Aまたは Protein Gを結合させたセファロースお よびァガロースのビーズ、マウス免疫グロブリン Gに対する他の動物由来の抗体を結 合させた磁気ビーズ (例、 Dynabeads)などが利用可能である。ビーズの添加後、さら に 1一 3時間、 4°Cで混和操作を続ける。 As a carrier for precipitating the antigen-antibody complex, for example, bead particles having a molecule having an affinity for mouse immunoglobulin G bound to the surface thereof are added to the mixture obtained in (1). Such carrier particles include Sepharose and agarose beads bound to Protein A or Protein G, and antibodies derived from other animals against mouse immunoglobulin G. Combined magnetic beads (eg, Dynabeads) are available. After adding the beads, continue mixing at 4 ° C for another 13 hours.
[0033] (3)収集した傷害 DNAの分離  (3) Separation of collected damaged DNA
PBSを界面活性剤を含む洗浄バッファーと交換し、新しレ、バッファーと混和させるこ とによりビーズを洗浄する。この操作を 5回以上繰り返す。洗い終わったビーズを溶出 バッファ一中、 65°Cで 10 30分加熱する。ビーズ粒子力、ら収集 DNAが遊離するの で、溶液成分だけを遠心分離やマグネットを使う方法等によって回収する。  Replace the PBS with a washing buffer containing a detergent, and wash the beads by mixing with a fresh buffer. Repeat this operation 5 times or more. Heat the washed beads in an elution buffer at 65 ° C for 10 30 minutes. Since the collected DNA is released from the bead particle force, only the solution components are collected by centrifugation or a method using a magnet.
[0034] D)収集した傷害 DNAの精製  D) Purification of collected damaged DNA
上記 B)で得た回収溶液には、 DNAとともに抗体や制限酵素などの蛋白質が残存 してレ、る。蛋白成分を除去するために、 TEバッファ一中で、蛋白分解酵素 Proteinase Kを 37°C、 1時間作用させる。さらに、常法に従い、フヱノール-クロロフオルム抽出、 エタノール沈殿を行い、傷害 DNAを精製'濃縮する。  In the recovered solution obtained in B), proteins such as antibodies and restriction enzymes remain together with DNA. In order to remove protein components, the protease, Proteinase K, is allowed to act at 37 ° C for 1 hour in a TE buffer. Further, according to a conventional method, phenol-chloroform extraction and ethanol precipitation are performed, and the damaged DNA is purified and concentrated.
[0035] E)収集した傷害 DNAの定量  E) Quantification of collected damaged DNA
本発明の方法によって収集した修飾デォキシヌクレオシド (例、 8_OHdG)を含む 傷害 DNA断片量の多寡を調べるには、下記の方法を用いる。  The following method is used to determine the amount of the damaged DNA fragment containing the modified deoxynucleoside (eg, 8_OHdG) collected by the method of the present invention.
一般に、酸化ストレスで傷害される DNA断片量は少量であり、本発明の方法によつ ても収集される DNA断片の量は極めて微量である（出発物質としての試料 DNAの 量の約 100分の 1一 1，000分の 1程度）。哺乳動物細胞のゲノム DNAにおける 8—ヒ ドロキシデォキシグアノシンの残存レベルは、およそ 100万グァニンにつき 1個のォ ーダ一である。そのため、通常 DNAの定量に使われる分光光度計による測定法は、 検出限界濃度以下となるため適用することができない。本発明者らは、臭化工チジゥ ムを使って、微量の DNAを検出 ·定量することに成功した。定量の手順は以下の通り である。濃度既知の標準 DNA溶液と収集した傷害 DNA断片溶液をそれぞれ適量 の臭化工チジゥムと混合する。 DNA-臭化工チジゥムの混合液を液滴として紫外線 照射装置の上に並べ、紫外線照射による蛍光の強度を標準液と試料液で比較して、 目的の傷害 DNA含有量を定める。  Generally, the amount of DNA fragments damaged by oxidative stress is small, and the amount of DNA fragments collected by the method of the present invention is extremely small (about 100 minutes of the amount of sample DNA as a starting material). About 1 / 1000th of 1). The residual level of 8-hydroxyhydroxyguanosine in the genomic DNA of mammalian cells is on the order of one per million guanine. Therefore, the spectrophotometer measurement method usually used for DNA quantification cannot be applied because the concentration is below the detection limit concentration. The present inventors have succeeded in detecting and quantifying a trace amount of DNA by using a bromide reagent. The quantification procedure is as follows. The standard DNA solution of known concentration and the collected damaged DNA fragment solution are mixed with appropriate amounts of bromide solution. The mixed solution of DNA-brominated medium is placed as droplets on an ultraviolet irradiation device, and the intensity of the fluorescence by ultraviolet irradiation is compared between the standard solution and the sample solution to determine the target damaged DNA content.
[0036] F)収集した傷害 DNAのマッピング  F) Mapping of collected damaged DNA
本発明の方法によって得られる傷害を受けた DNA断片の収集液中に含まれてい る個々の DNA断片について、全ゲノム配列における当該断片の由来場所を同定す るには、例えば以下の方法で行う。 Contained in the collection solution of damaged DNA fragments obtained by the method of the present invention. In order to identify the origin of the fragment in the whole genome sequence of each individual DNA fragment, for example, the following method is used.
まず、適当なベクター-宿主系で収集した傷害 DNAを、例えば上記の方法でクロ 一ユングし、断片集団を別々のクローンとして分離する。次に、各クローンについて、 その塩基配列（の一部）を決定する。この配列をもとに、公開されているゲノムデータ ベースを検索し、染色体上で、当該 DNA断片と一致する配列を有する部位、その周 辺の詳細なゲノム情報を得ることができる。本解析法によれば、酸化ストレスによる D NA傷害の残存部位を全ゲノムにわたって網羅的に同定することが可能となる。 本発明は、免疫沈降法と修飾デォキシヌクレオシド (例、 8-OHdG)に対するモノク ローナル抗体とを組合わせ、最適な条件を設定することにより、初めてゲノム DNAに 存在する極微量の傷害 DNA断片を選択的に単離し、同定する方法を提供するもの である。  First, the damaged DNA collected in an appropriate vector-host system is cloned, for example, by the method described above, and the fragment population is separated as separate clones. Next, the base sequence (part of) is determined for each clone. Based on this sequence, a publicly available genome database can be searched to obtain detailed genomic information on the chromosome, a site having a sequence that matches the DNA fragment, and its surroundings. According to this analysis method, it is possible to comprehensively identify the remaining site of DNA damage due to oxidative stress over the entire genome. The present invention is based on the combination of immunoprecipitation and a monoclonal antibody against a modified deoxynucleoside (e.g., 8-OHdG). And a method for selectively isolating and identifying the same.
ここで、「最適な条件」は以下の点に基づいて決定することができる。  Here, the “optimal condition” can be determined based on the following points.
(a)免疫沈降に際して、断片化 DNA集団中に含まれる傷害された DNA断片を効率 よく認識し複合体を形成するよう、抗体の量を上記のごとく調整する。  (a) At the time of immunoprecipitation, the amount of the antibody is adjusted as described above so as to efficiently recognize the damaged DNA fragment contained in the fragmented DNA population and form a complex.
(b)また、抗体と担体ビーズの量が一致するよう、それぞれの添加量を調整する。 DN A断片も担体ビーズに非特異的に吸着するが、 目的とする傷害 DNA断片の含有割 合が極めて低いため、ビーズ 1個に対して抗体 (分子）の数が少ないと、非特異的吸 着の影響が無視できない。逆に抗体が多すぎると、 目的断片と抗体の複合体のうち ビーズと結合できないものがあらわれ、それらは洗い流されてしまうことになるので、 回収量がさらに少なくなる。  (b) Further, the addition amounts of each are adjusted so that the amounts of the antibody and the carrier beads match. The DNA fragment also adsorbs nonspecifically to the carrier beads, but the content of the target damaged DNA fragment is extremely low. Therefore, if the number of antibodies (molecules) per bead is small, the nonspecific adsorption will occur. The effects of wearing cannot be ignored. Conversely, if there are too many antibodies, some of the target fragment-antibody complexes will be unable to bind to the beads and will be washed away, further reducing the amount of recovery.
(c)抗原 -抗体複合体の沈殿後、徹底的に洗浄する。  (c) Thoroughly wash after precipitation of the antigen-antibody complex.
抗原一抗体反応せずに非特異的にビーズに付着または近接している非目的断片を 完全に除去するために洗浄操作を徹底した。界面活性剤の組成を変えた 4種類のバ ッファーを使レ、、計 8回の洗浄を行った。  Thorough washing was performed to completely remove non-target fragments non-specifically attached to or in proximity to the beads without antigen-antibody reaction. A total of eight washes were performed using four types of buffers with different surfactant compositions.
(d)微少な回収 DNAを定量するために特別な検出法を使用する。  (d) Use a special detection method to quantify minute recovered DNA.
通常、 DNAの定量は、核酸の吸光特性を利用して、分光光度計により行うが、傷 害 DNAの量は通常の分光光度計の検出感度以下であるため、臭化工チジゥムと混 ぜた液滴の蛍光シグナル強度により定量する。 Normally, DNA is quantified using a spectrophotometer, utilizing the absorption characteristics of nucleic acids.However, since the amount of damaged DNA is lower than the detection sensitivity of a normal spectrophotometer, it is mixed with a bromide reagent. It is quantified by the fluorescence signal intensity of the mixed droplet.
(e)操作中にグァニンが修飾を受けて 8-ヒドロキシグァニンが生成しないよう、断片を 分離するまでは、遮光、遮酸素および低温 (氷上）の状態に維持する。また、フリーラ ジカル反応の起点となる鉄イオンをキレート剤デスフェリォキサミンで除去する。  (e) Keep the cells protected from light, oxygen and cold (on ice) until the fragments are separated, so that guanine is not modified during the operation to generate 8-hydroxyguanine. In addition, iron ions that are the starting point of the free radical reaction are removed with the chelating agent desferioxamine.
[0038] 以下、実施例により本発明をさらに具体的に説明する。但し、本発明はこれらの実 施例にその技術的範囲が限定されるものではない。 Hereinafter, the present invention will be described more specifically with reference to examples. However, the technical scope of the present invention is not limited to these embodiments.
実施例 1 マウスゲノム DNA断片集団からの 8—ヒドロキシデォキシグアノシン含有断 片の収集  Example 1 Collection of 8-hydroxydeoxyguanosine-containing fragments from a mouse genomic DNA fragment population
(1)試料 DNAの調製  (1) Preparation of sample DNA
ゲノム DNAをマウス（16週齢、雄性 C57BLZ6)の腎組織より調製した。組織細胞 からの DNA抽出は、よう化ナトリウム法 [ワンら（Wang et al.)， Nucleic Acids Res. 22: 1774， 1994]のための巿販試薬キット（和光純薬工業； DNA Extractor WB Kit)を用 レ、、添付の指示書に従い、実施した。なお、抽出過程における 8—ヒドロキシグァニン (即ち、修飾デォキシヌクレオシドとしては 8—ヒドロキシー 2 '—デォキシグアノシン）の 新たな生成を抑えるため、抽出操作中は、できるかぎり、試料を低温 (氷上)および遮 光の状態に保った。また、緩衝液類はアルゴンガスで飽和し、触媒性鉄を不活性化 するために 0. ImMデスフェリォキサミン (desferrioxamine)を添加した。  Genomic DNA was prepared from kidney tissue of mice (16 weeks old, male C57BLZ6). DNA extraction from tissue cells was performed using the sodium iodide method (Wang et al., Nucleic Acids Res. 22: 1774, 1994), a commercial reagent kit (Wako Pure Chemical Industries; DNA Extractor WB Kit). It was implemented according to the attached instructions. During the extraction process, keep the sample at a low temperature as much as possible to suppress the new generation of 8-hydroxyguanine (ie, 8-hydroxy-2'-deoxyguanosine as a modified deoxynucleoside) during the extraction process. (On ice) and light-shielded. The buffers were saturated with argon gas, and 0.1M ImM desferrioxamine was added to inactivate the catalytic iron.
[0039] (2) DNAの断片化 (2) DNA fragmentation
上記（1)で得られたマウスゲノム DNAを、 10mM Tris-HCl ( H 7.5)、 lOmM MgC12 、 1 mMジチオスレィトール及び 50mM NaCl中、 37°Cで制限酵素 Haelllにより切断処 理し、断片化した。酵素反応時間 1時間で完全に切断できるように、反応系を調節し た。  The mouse genomic DNA obtained in the above (1) was digested with the restriction enzyme Haelll at 37 ° C in 10 mM Tris-HCl (H7.5), 10 mM MgC12, 1 mM dithiothreitol and 50 mM NaCl, and the fragment was digested. It has become. The reaction system was adjusted so that the enzyme could be completely cleaved in one hour.
[0040] (3) 8—ヒドロキシデォキシグアノシンの導入  [0040] (3) Introduction of 8-hydroxyhydroxyguanosine
本発明の方法によって収集された DNA断片の量の多寡力 S、元の DNA断片集団 における 8—ヒドロキシデォキシグアノシンの含有量を反映することを確認するため、 組織力、ら抽出したゲノム DNAに人為的に 8—ヒドロキシデォキシグアノシンを導入し、 8—ヒドロキシデォキシグアノシンの含有量がそれぞれ異なる試料を準備した。 DNA をメチレンブルーと光で処理することにより、 DNA中のグァニンを 8—ヒドロキシデォキ シグアノシンに人工的に変化させることができる。哺乳動物細胞のゲノム DNAにおけ る 8—ヒドロキシデォキシグアノシンの残存レベルは、通常、およそ 100万グァニンに つき 1個のオーダーである。同ゲノム DNAをメチレンブルーで処理すれば、最高で 対グァニン比 2%のレベルまで 8—ヒドロキシデォキシグアノシンを増加させることがで きる [シュナイダーら（Schneider et al), Nucleic Acids Res. 18: 631-635， 1990]。 96 穴マイクロタイターのゥエルに 100 xg/ml DNA、 5 50μΜ メチレンブルー、 0.1 mMデスフェリォキサミン、 10mM Tris (pH 8.0)力 なる反応系を設定した。マ イクロタイタ一.プレートから 12cm上に 60W電球を設置し、 30分間の光照射により反 応を促した。本処理後のゲノム DNAの 8—ヒドロキシデォキシグアノシン含有量を HPLC- ECD法により実測した。結果を表 1に示す。 In order to confirm that the amount of DNA fragments collected by the method of the present invention reflects the content of 8-hydroxyhydroxyguanosine in the original DNA fragment population, the genomic DNA extracted Was introduced artificially into the sample, and samples having different contents of 8-hydroxydeoxyguanosine were prepared. By treating the DNA with methylene blue and light, guanine in the DNA is reduced to 8-hydroxydeoxy. It can be artificially changed to ciguanosine. Residual levels of 8-hydroxydeoxyguanosine in the genomic DNA of mammalian cells are usually on the order of one per million guanine. Treatment of the same genomic DNA with methylene blue can increase 8-hydroxydoxyguanosine to levels up to 2% guanine [Schneider et al., Nucleic Acids Res. 18: 631 -635, 1990]. A reaction system containing 100 xg / ml DNA, 550 µm methylene blue, 0.1 mM desferrioxamine, and 10 mM Tris (pH 8.0) was set in a 96-well microtiter well. Microtiter 1. A 60 W light bulb was placed 12 cm above the plate, and the reaction was stimulated by irradiating with light for 30 minutes. The 8-hydroxydeoxyguanosine content of the genomic DNA after this treatment was actually measured by the HPLC-ECD method. Table 1 shows the results.
表 1:メチレンブルーと光により処理したゲノム DNAの 8_OHdG含有量  Table 1: 8_OHdG content of genomic DNA treated with methylene blue and light
[表 1]  [table 1]
メチレンブル一灌 i グァェン 105個当たりの Methylene bull irrigation i guan 10 per 5
'ス 8— OHdGの数  'S 8—number of OHdG
無 処 理 6  No processing 6
5 μΜ 1 62  5 μΜ 1 62
1 0 μΜ 1 82  1 0 μΜ 1 82
50 μΜ 333  50 μΜ 333
[0042] (4)免疫沈降 (4) Immunoprecipitation
1. 5 mlチューブに 0.1%のゥシ血漿アルブミンを含む PBSを分注し、（3)で調製 した断片化ゲノム DNA (20 xg)と一次抗体（モノクローナル抗体 N45. 1、 日研ザ ィル）（20 zg)をカロえた。最初に入れておく PBSの量は、混合系全体の容量が 900 μ 1となるように調整した。このチューブを低温室（4°C)においてロータリーシェーカー を用いて 3時間撹拌した。沈降用の担体として、 Dynal社製の二次抗体（She印 anti-Mouse IgG結合済み磁気ビーズ（Dynabeads M-280)を使用した。磁気ビーズ懸 濁液（0.1%のゥシ血漿アルブミンを含む PBS中）（100 /il)を混合系に追加した後、 さらに 3時間、低温でのロータリーシェーカーによる撹拌操作を継続した。結合反応 の終了後、ビーズを 4種類のバッファーを用いて順次洗浄した。  1. Dispense PBS containing 0.1% of plasma albumin into a 5 ml tube, and use the fragmented genomic DNA (20 xg) prepared in (3) and the primary antibody (monoclonal antibody N45.1, NIKEN ZYL) ) (20 zg) calories. The amount of PBS to be initially added was adjusted so that the total volume of the mixed system was 900 μl. This tube was stirred for 3 hours using a rotary shaker in a cold room (4 ° C). Magnetic beads (Dynabeads M-280) conjugated with a secondary antibody (She-marked anti-Mouse IgG) manufactured by Dynal were used as a carrier for sedimentation.A magnetic bead suspension (PBS containing 0.1% of plasma albumin) was used. (Medium) (100 / il) was added to the mixed system, and the stirring operation was continued for another 3 hours by using a rotary shaker at a low temperature.After completion of the binding reaction, the beads were washed sequentially with four kinds of buffers.
[0043] 各バッファーの組成は次の通りである：  [0043] The composition of each buffer is as follows:
1.可溶化バッファー（140mM NaCl) ;0.1%デォキシコール酸ナトリウム， ImM EDTA , 50mM Hepes-KOH (pH7.5) , 140mM NaCl, 1% TritonX-100 1. Solubilization buffer (140 mM NaCl); 0.1% sodium deoxycholate, ImM EDTA , 50mM Hepes-KOH (pH7.5), 140mM NaCl, 1% TritonX-100
2.可溶化バッファー（500mM NaCl) ; 0.1%デォキシコール酸ナトリウム， ImM EDTA , 50mM Hepes-KOH (pH7.5) , 500mM NaCl, 1% TritonX—100  2. Solubilization buffer (500mM NaCl); 0.1% sodium deoxycholate, ImM EDTA, 50mM Hepes-KOH (pH7.5), 500mM NaCl, 1% TritonX-100
3.洗浄バッファー； 0.1%デォキシコール酸ナトリウム， ImM EDTA, 250mM LiCl, 0.5% Nonidet P-40, lOmM Tris-HCl (pH8.0)  3.Washing buffer; 0.1% sodium deoxycholate, ImM EDTA, 250mM LiCl, 0.5% Nonidet P-40, lOmM Tris-HCl (pH8.0)
[0044] 4. TEバッファー  [0044] 4. TE buffer
上記 1一 4の順に、各バッファーにっき 2回ずつ洗浄を実施した。洗浄後、 80 μ 1の溶 出バッファー（lOmM EDTA, 1% SDS， 50mM Tris- HC1 (pH8.0) )にビーズを懸濁した 。 65°C、 15分間の加熱処理により収集した DNA断片を溶離させた。次いで、マグネ ットを使って溶液成分からビーズを分離した。溶出バッファーの追加→加熱→分離の 操作をもう一度繰り返した。  Washing was carried out twice in each buffer in the order of 1 to 4 described above. After washing, the beads were suspended in 80 μl of an elution buffer (10 mM EDTA, 1% SDS, 50 mM Tris-HCl (pH 8.0)). The collected DNA fragments were eluted by heat treatment at 65 ° C for 15 minutes. The beads were then separated from the solution components using a magnet. The operation of adding the elution buffer → heating → separation was repeated once.
[0045] (5)収集 DNAの精製 ( 5 ) Purification of collected DNA
上記（4)の最終回収物を Proteinase K (Invitrogen)で 37°C、 1時間処理した後、フ ェノール-クロロフオルム抽出およびエタノール沈殿による DNAの精製'濃縮操作を 定法に従って実施した。  After treating the final recovered product of the above (4) with Proteinase K (Invitrogen) at 37 ° C. for 1 hour, phenol-chloroform extraction and purification of DNA by ethanol precipitation′concentration operation were performed according to a standard method.
[0046] (6)結果  [0046] (6) Results
免疫沈降に供する初期 DNA断片を 0, 5, 10, 50 μ Mのメチレンブルーで処理し て人為的に傷害を起こした場合の結果を図 1に示す。図の縦軸は、段階的に 8— ΟΗ dGの含有量を増加させた DNA試料から、本発明の方法によって収集した傷害 DN A断片の量を表している。この結果は、収集した傷害を受けた DNA断片の量は、出 発物質である DNA試料の 8-ヒドロキシデォキシグアノシン量に依存することを示して いる。  Fig. 1 shows the results when the initial DNA fragments to be subjected to immunoprecipitation were treated with 0, 5, 10, 50 µM methylene blue to cause artificial damage. The vertical axis of the figure represents the amount of damaged DNA fragments collected by the method of the present invention from DNA samples in which the content of 8-—dG was increased stepwise. The results indicate that the amount of damaged DNA fragments collected depends on the amount of 8-hydroxydeoxyguanosine in the starting DNA sample.
[0047] 実施例 2 鉄二トリ口三酢酸 (Fe— NTA)による酸化ストレス負荷状態でのゲノム内 8_ ヒドロキシグァニン分布の解析  Example 2 Analysis of 8_-Hydroxyguanine Distribution in Genome under Oxidative Stress Loading by Iron Tri-Triacetate (Fe-NTA)
(1)実験動物に対する酸化ストレスの人為的負荷  (1) Artificial load of oxidative stress on experimental animals
鉄二トリ口三酢酸（以下、 Fe— NTAと記す）を酸化ストレス負荷剤として使用した。こ の生体傷害物質を齧歯類の腹腔内に投与すると、腎臓特異的に酸化ストレスが発生 する [豊國ら（Toyokuni et al.) , Proc. Natl. Acad. Sci. USA 91: 2616, 1994] 0 12—1 3週齢の雄 C57BL/6マウスに、マウス kg体重あたり鉄 3mg相当量の Fe_NTAを 投与した。臓器試料は、投与 6時間後にマウスを屠殺し、摘出した。 Iron nitrite triacetic acid (hereinafter referred to as Fe-NTA) was used as an oxidative stress agent. When this biotoxic substance is intraperitoneally administered to rodents, oxidative stress is generated specifically in the kidney [Toyokuni et al., Proc. Natl. Acad. Sci. USA 91: 2616, 1994] 0 12—1 Three-week-old male C57BL / 6 mice were administered Fe_NTA in an amount equivalent to 3 mg of iron per kg of mouse body weight. Mice were sacrificed and removed 6 hours after administration.
[0048] (2)酸化ストレス傷害 DNA断片の収集  [0048] (2) Collection of oxidative stress injury DNA fragments
腎組織からの DNA抽出とその断片化、およびそれに続く 8—ヒドロキシグァニン含 有断片の収集'精製は、実施例 1に記載した通りに実施した。但し、次の（3)の手順 で使用する DNAクローニングキットへの適用要件に適合するために、収集した DNA 断片の精製操作の前に、当該 DNA断片の 5 '末端リン酸基を除去するアルカリフォ スターゼ処理を追加した。即ち、収集した DNA断片を、ゥシ小腸アルカリフォスファタ ーゼ（タカラバイオ）で 50°C、 30分間処理した。  DNA extraction from kidney tissue and its fragmentation, and subsequent collection and purification of 8-hydroxyguanine-containing fragments were performed as described in Example 1. However, in order to meet the requirements for application to the DNA cloning kit used in the following step (3), before purifying the collected DNA fragment, remove the 5'-terminal phosphate group from the DNA fragment Added a fostase treatment. That is, the collected DNA fragments were treated with a small intestine alkaline phosphatase (Takara Bio) at 50 ° C. for 30 minutes.
[0049] (3) DNA断片のクローニング  [0049] (3) Cloning of DNA fragment
収集した DNA断片のクローニングには、平滑末端 PCR産物クローユングのための 巿販キット（インビトロジェン； Zero Blunt TOPO PCR Cloning Kit for Sequencing)を 用いた。キットに添付の指示書に従って収集 DNA断片をプラスミドベクター（ pCR4Blunt-TOPO)に組み込み、大腸菌を形質転換し、得られた形質転換体を寒天 プレート上で培養した。これを収集断片クローンのライブラリ一とした。  For the cloning of the collected DNA fragments, a commercial kit for blunt-end PCR product cloning (Invitrogen; Zero Blunt TOPO PCR Cloning Kit for Sequencing) was used. The collected DNA fragment was inserted into a plasmid vector (pCR4Blunt-TOPO) according to the instructions attached to the kit, E. coli was transformed, and the resulting transformant was cultured on an agar plate. This was used as the library of the collected fragment clone.
[0050] (4) DNA断片の塩基配列決定  (4) Determination of base sequence of DNA fragment
上記（3)の寒天プレートより大腸菌コロニーを順次拾い上げ、オートシークェンサ一 (ABI PRISM377)を用いて DNA断片クローンの塩基配列決定を網羅的に実施した。  Escherichia coli colonies were sequentially picked up from the agar plate of the above (3), and the nucleotide sequence of the DNA fragment clone was comprehensively determined using an autosequencer (ABI PRISM377).
[0051] (5)マウスゲノム'データベースの検索  [0051] (5) Searching the mouse genome 'database
上記（3)で得られた塩基配列を検索の条件として、 Celera社データベース（Celera Discovery System)の BLAST検索を実施した。検索結果から、該 DNA断片の一致す るゲノム部位について染色体上での位置情報および付近 0.5Mb範囲の塩基配列デ ータと遺伝子リストを取得した。これらの個々の解析情報を、酸化ストレス負荷状態（F e— NTA投与後 6時間）および無処置状態の検体より収集した DNA断片について集 積し、それぞれの状態における 8—ヒドロキシグァニン残存点の染色体へのマッピング ならびに 8—ヒドロキシグァニン集中部位周辺の特性分析を実施した。解析結果として 、酸化ストレス負荷状態における 8-ヒドロキシグァニン残存点のマッピング結果を図 2 に示す。図中の黒い四角で示した点は染色体上での各収集断片の由来位置を表し ている。 Using the base sequence obtained in (3) above as a search condition, a BLAST search of Celera's database (Celera Discovery System) was performed. From the search results, positional information on the chromosome of the genomic site corresponding to the DNA fragment, and nucleotide sequence data and a gene list in the vicinity of 0.5 Mb were obtained. These individual analysis information were collected for DNA fragments collected from samples in the oxidative stress-loaded state (6 hours after administration of Fe-NTA) and untreated, and the remaining points of 8-hydroxyguanine in each state were collected. Chromosome mapping and characterization around 8-hydroxyguanine concentration sites were performed. As an analysis result, FIG. 2 shows the mapping result of the remaining 8-hydroxyguanine under the oxidative stress load state. The points indicated by black squares in the figure indicate the origin of each collected fragment on the chromosome. ing.
[0052] 実施例 3 ラット血漿中の DNAからの酸化ストレスによる傷害 DNA断片の収集  Example 3 Collection of Damaged DNA Fragment due to Oxidative Stress from DNA in Rat Plasma
(1)血漿からの DNA抽出  (1) DNA extraction from plasma
11週令の Wistarラットに、ラット kg体重あたり鉄 10mg相当量の Fe—ΝΤΑを腹腔 内投与し、腎臓における酸化ストレス傷害を惹起した。 24時間経過後、眼窩静脈叢 採血法により、 5— 7mlのラット血液を採取した。遠心分離後、血漿成分を試料として 回収した。血漿からの DNA抽出は、実施例 1と同様、よう化ナトリウム法による巿販キ ットを用いて実施した。  11-week-old Wistar rats were intraperitoneally administered Fe-II in an amount equivalent to 10 mg of iron per kg of rat body weight to induce oxidative stress injury in the kidney. After 24 hours, 5 to 7 ml of rat blood was collected by the orbital venous plexus blood sampling method. After centrifugation, plasma components were collected as a sample. DNA extraction from plasma was performed using a sales kit according to the sodium iodide method, as in Example 1.
[0053] 血漿における DNA含有量は、実施例の 1および 2で使用した腎組織と比べて極め て少ない。ラット 1匹力も採った血液（5— 7ml)より得られた DNAの量は以下の表 2 に記載の通りである。  [0053] The DNA content in plasma is extremely low as compared with the kidney tissue used in Examples 1 and 2. The amount of DNA obtained from blood (5-7 ml) collected from a single rat is shown in Table 2 below.
[0054] [表 2] 処置条件  [Table 2] Treatment conditions
抽出 D NA量 （μ g )  Extracted DNA amount (μg)
無処置 （1 ) 0 . 2 5  No treatment (1) 0.25
無処置 （2 ) 0 . 4 3  No treatment (2) 0.43
無処置 （3 ) 1 . 0 3  No treatment (3) 1.0 3
F e—N T A投与 1 . 5 6  F e—N T A administration 1.5 6
※Fe— NTA投与の条件下では、傷害組織において多数の細胞が壊死あるいはァ ポトーシスに陥るため、血漿中の DNAレベルが上昇する。 * Under the condition of Fe-NTA administration, plasma DNA levels increase due to necrosis or apoptosis of many cells in the injured tissue.
[0055] (2)酸化ストレス傷害断片の収集 (2) Collection of Oxidative Stress Injury Fragments
上記（1)で得た血漿 DNAを、実施例 1に記載の方法と同様に断片化し、免疫沈降 により 8—ヒドロキシデォキシグアノシン含有 DNA断片を収集 '精製した。但し、免疫 沈降に供する初期 DNA量は 1.5 /i gとした。無処置条件の試料については 3匹分の 抽出 DNAを合わせたものから、 1.5 μ §の DNAを用いた。この初期 DNA量は、実施 例 1のときの量の約 13分の 1である。  The plasma DNA obtained in the above (1) was fragmented in the same manner as described in Example 1, and the DNA fragment containing 8-hydroxydeoxyguanosine was collected and purified by immunoprecipitation. However, the initial amount of DNA to be subjected to immunoprecipitation was 1.5 / ig. For samples under untreated conditions, 1.5 μ of DNA was used from the combined extracted DNA of three animals. This initial amount of DNA is about one-third of the amount in Example 1.
[0056] (3)結果 [0056] (3) Result
ラット血漿中の DNAから、本発明の方法によって 8—ヒドロキシデォキシグアノシン 含有断片を収集した結果を図 3に示す。この結果から、 Fe— NTA投与により酸化スト レスを負荷したラットのほうが無処置のラットよりも、その血漿中の DNAにおいて 8—ヒ ドロキシデォキシグアノシンの含有量が高くなつていることがうかがえる。すなわち、本 発明方法によれば、生体全体での酸化ストレスの程度は、直接傷害を受けた組織か ら抽出した DNAを用いなくても、血漿から得られる DNAを用いて評価することができ ることが明らかになった。 FIG. 3 shows the results of collecting fragments containing 8-hydroxydeoxyguanosine from DNA in rat plasma by the method of the present invention. These results indicate that rats loaded with oxidative stress by administration of Fe-NTA had a higher DNA concentration in their plasma than did untreated rats. It can be seen that the content of droxidideoxyguanosine is increasing. That is, according to the method of the present invention, the degree of oxidative stress in the whole organism can be evaluated using DNA obtained from plasma without using DNA directly extracted from injured tissue. It became clear.
[0057] 実施例 4 別種の酸化的 DNA傷害を含有する断片の収集（1) Example 4 Collection of Fragments Containing Different Kinds of Oxidative DNA Damage (1)
(1)シクロブタン型ピリミジン二量体  (1) cyclobutane-type pyrimidine dimer
一般に紫外線照射も酸化ストレスの一つと考えられており、シクロブタン型ピリミジン 二量体 (CPD)は、紫外線によって最も高頻度に生成される DNA傷害の一つである Ultraviolet irradiation is also considered to be one of the oxidative stresses, and cyclobutane-type pyrimidine dimer (CPD) is one of the most frequently generated DNA damage by ultraviolet light
。 TDM—2 [森ら（Mori et al) , Photochem Photobiol 54: 225, 1991]は、 CPDに対す るモノクローナル抗体である。 . TDM-2 [Mori et al, Photochem Photobiol 54: 225, 1991] is a monoclonal antibody against CPD.
[0058] (2)ゲノム DNA断片集団の調製 (2) Preparation of Genomic DNA Fragment Population
実施例 1と同様にして制限酵素 Haelllによって切断された、マウスゲノム DNAを調 製した。  Mouse genomic DNA cut with the restriction enzyme Haelll was prepared in the same manner as in Example 1.
[0059] (3)紫外線処理 (3) UV treatment
組織から抽出した DNAに UVC (254nm)を照射し、人為的に CPDを DNA断片 中に導入した試料を作成した。 96穴マイクロタイターのゥエルに分注した 100 /i g/ mlの DNA溶液に対し、 UVクロスリンカ一（SPECTROLINKER XL-100)を用いて 25 The DNA extracted from the tissue was irradiated with UVC (254 nm) to prepare a sample in which CPD was artificially introduced into the DNA fragment. Using a UV crosslinker (SPECTROLINKER XL-100) for the DNA solution of 100 / ig / ml dispensed into a 96-well microtiter well
4nmUVCを照射した。 Irradiated with 4 nm UVC.
[0060] (4)免疫沈降 (4) Immunoprecipitation
実施例 1と同手順の免疫沈降において、一次抗体として TDM— 2、 20 μ §を使用し た。 In immunoprecipitation with Example 1 the procedure, it was used TDM- 2, 20 μ § as the primary antibody.
[0061] (5)結果  [0061] (5) Results
本発明の方法によって、 CPDを含有する DNA断片を収集した結果を図 4に示す。 この結果から、収集 DNA断片の量は紫外線照射量に依存してレ、ることが分力、る。  FIG. 4 shows the results of collecting CPD-containing DNA fragments by the method of the present invention. These results indicate that the amount of collected DNA fragments depends on the amount of UV irradiation.
[0062] 実施例 5 別種の酸化的 DNA傷害を含有する断片の収集（2) Example 5 Collection of Fragments Containing Another Kind of Oxidative DNA Damage (2)
(1)ァクロレイン（Acrolein)  (1) Acrolein
酸化的な傷害は、脂質の過酸化も含まれる。ァクロレインは、膜脂質の過酸化によ つて生成する不飽和アルデヒドのひとつであり、酸化ストレス下の細胞内にも認めら れる（内田ら、前掲)。ァクロレインは、核酸と反応して付加物を形成する。 mAb21は、 ァクロレインと 2 '—デォキシアデノシンとの反応物に対するモノクローナル抗体であるOxidative damage also includes peroxidation of lipids. Acrolein is one of the unsaturated aldehydes formed by the peroxidation of membrane lipids, and is also found in cells under oxidative stress. (Uchida et al., Supra). Acrolein reacts with nucleic acids to form adducts. mAb21 is a monoclonal antibody to the reaction of acrolein with 2'-deoxyadenosine
(河井ら、前掲)。 (Kawai et al., Supra).
[0063] (2)腎組織からの DNA抽出 (2) DNA extraction from kidney tissue
12週齢および 17週齢の雄 C57BLZ6マウス 1匹ずつに、マウス kg体重あたり鉄 3 mg相当量の Fe— NTAを投与した。 S蔵器試料は、投与 6時間後にマウスを屠殺し、摘 出した。対照のために、同じ週齢の無処置のマウスからも試料を採取した。実施例 1 と同様に、よう化ナトリウム法によるキットを用いて、腎組織からゲノム DNAを抽出した  Each of 12-week-old and 17-week-old male C57BLZ6 mice was administered Fe-NTA in an amount equivalent to 3 mg of iron per kg of mouse body weight. Mice were sacrificed and extracted 6 hours after administration for S storage sample. Samples were also taken from untreated mice of the same age for controls. As in Example 1, genomic DNA was extracted from kidney tissue using a sodium iodide kit.
[0064] (3)酸化ストレス傷害断片の収集 (3) Collection of Oxidative Stress Injury Fragments
腎ゲノム DNAを、実施例 1に記載の方法と同様に断片化した。さらに、実施例 1の 手順に準じて、免疫沈降によるァクロレイン付加断片の収集及び精製を実施した。但 し、一次抗体としてァクロレインと 2 '—デォキシアデノシンとの反応物に対するモノクロ ーナル抗体 (mAb21) 2 μ gを使用した。  Kidney genomic DNA was fragmented in the same manner as described in Example 1. Further, according to the procedure of Example 1, the acrolein-added fragment was collected and purified by immunoprecipitation. However, as a primary antibody, 2 μg of a monoclonal antibody (mAb21) against a reaction product of acrolein and 2′-deoxyadenosine was used.
[0065] (4)結果  [0065] (4) Results
本発明の方法によって、ァクロレインの付カ卩した DNA断片を収集した結果を、図 5 に示す。 12週齢のマウスのゲノム DNAからは免疫沈降を 2度行レ、、 17週齢のマウス 力 得たデータも含めた、これら 3点を 1群として、収集 DNA量の統計的な有意差を 検討した（ρ = 0· 0132)。この結果から、 Fe— NTA投与により酸化ストレスを負荷し たマウスの方が、無処置のマウスよりも、腎ゲノム DNAにおけるァクロレインの付加量 が高くなつていると考えられる。実施例 4、 5により、酸化的 DNA傷害に特異的に結 合する抗体一般に対して、本発明の方法が適用可能であることが示された。  FIG. 5 shows the results of collecting DNA fragments to which acrolein was added by the method of the present invention. Immunoprecipitation was performed twice from genomic DNA of 12-week-old mice, and these three points, including data obtained from 17-week-old mice, were treated as a group to determine the statistically significant Were studied (ρ = 0.0132). From these results, it is considered that the mice loaded with oxidative stress by the administration of Fe-NTA have a higher amount of acrolein added to the kidney genomic DNA than the untreated mice. Examples 4 and 5 show that the method of the present invention is applicable to general antibodies that specifically bind to oxidative DNA damage.
産業上の利用可能性  Industrial applicability
[0066] 酸化ストレスに曝されて傷害された DNA断片を特異的に収集することにより、その 傷害された DNAの配列情報を用いて、傷害を受けやすいゲノム上の場所 (あるいは 遺伝子）を検索することが可能となる。傷害を受けやすい遺伝子とは、変異しやすい 遺伝子の候補であり、そのような情報は、例えば、発がんの最も初期の段階に対する 知見を与える。よって、最終的には、がん予防手段の確立において必須となる情報を 導きだすための基礎情報として利用し得る。 [0066] By specifically collecting DNA fragments that have been damaged by exposure to oxidative stress, a genomic location (or gene) that is susceptible to damage is searched using the sequence information of the damaged DNA. It becomes possible. A gene that is susceptible to injury is a candidate for a gene that is susceptible to mutation, and such information provides, for example, insight into the earliest stages of carcinogenesis. Therefore, in the end, information that is essential for establishing cancer prevention measures It can be used as basic information for deriving.
また、血清あるいは血漿内には、破壊された細胞の DNAが微量ながら存在するこ とがわかっており、臓器によって、傷害を受けやすいゲノム部位が異なれば、少量の 血液から、体内のどの臓器において、活性酸素が発生し酸化ストレスに曝されている かを簡単に判定することが可能となる（例えば実施例 3参照）。  It is also known that a small amount of the DNA of destroyed cells is present in serum or plasma. In addition, it is possible to easily determine whether active oxygen is generated and exposed to oxidative stress (for example, see Example 3).

Claims

請求の範囲 The scope of the claims
[1] DNAを含む試料中の酸化ストレスにより傷害された領域の DNA断片を収集する 方法であって、試料から DNAを抽出し、断片化した後、傷害により修飾された修飾ヌ クレオシドまたは該修飾ヌクレオシドを含むポリヌクレオチドに特異的な一次抗体とィ ンキュペートし、沈降した複合体を回収し、該複合体から傷害された DNA断片を回 収することを特徴とする方法。  [1] A method for collecting a DNA fragment in a region containing a DNA, which is damaged by oxidative stress, comprising extracting DNA from the sample, fragmenting the sample, and then modifying the modified nucleoside or the modified nucleoside by the damage A method comprising incubating with a primary antibody specific for a polynucleotide containing a nucleoside, collecting a precipitated complex, and recovering a damaged DNA fragment from the complex.
[2] 断片化により得られた修飾ヌクレオシドを含む DNA断片 1個に対して抗体分子の 数が 50倍以上である、請求項 1記載の方法。  [2] The method according to claim 1, wherein the number of antibody molecules is 50 times or more per DNA fragment containing the modified nucleoside obtained by fragmentation.
[3] 修飾ヌクレオシドが 8—ヒドロキシ— 2'—デォキシグアノシン、シクロブタン型ピリミジン 二量体およびァクロレイン付加 2' -デォキシヌクレオシドから選択される、請求項 1又 は 2記載の方法。  [3] The method according to claim 1 or 2, wherein the modified nucleoside is selected from 8-hydroxy-2'-deoxyguanosine, cyclobutane-type pyrimidine dimer, and acrolein-added 2'-deoxynucleoside.
[4] ァクロレイン付加 2，-デォキシヌクレオシドがァクロレイン付加 2，-デォキシアデノシ ンである、請求項 3記載の方法。  [4] The method according to claim 3, wherein the acrolein-added 2, -deoxynucleoside is acrolein-added 2, -deoxyadenosine.
[5] 抗体がモノクローナル抗体である請求項 1一 4のいずれかに記載の方法。 [5] The method according to any one of claims 14 to 14, wherein the antibody is a monoclonal antibody.
[6] 請求項 1一 5のいずれかに記載の方法によって回収した DNA断片を臭化工チジゥ ム法によって定量し、その多寡により当該 DNA試料の酸化ストレスによる傷害の程 度を評価する方法。 [6] A method of quantifying a DNA fragment recovered by the method according to any one of claims 11 to 5 by a bromide chemical method, and evaluating the degree of damage of the DNA sample due to oxidative stress based on the amount thereof.
[7] 請求項 1一 5のいずれかに記載の方法によって回収した DNA断片をクローニング し、塩基配列を決定し、その配列情報と既知のゲノムの配列情報とを比較することに より、酸化ストレスに対して感受性の遺伝子領域を同定する方法。  [7] Oxidative stress by cloning the DNA fragment recovered by the method according to any one of claims 11 to 5, determining the nucleotide sequence, and comparing the sequence information with the sequence information of a known genome. A method for identifying a gene region sensitive to
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WO2010084998A1 (en) * 2009-01-26 2010-07-29 Kyushu University, National University Corporation A method of predicting the efficacy of a drug
CN103212066A (en) * 2013-03-14 2013-07-24 曹毅 Preparation method of cyclobutane pyrimidine dimmer photolyase liposome
CN108530539A (en) * 2018-03-20 2018-09-14 北京博雅捷康生物科技有限公司 The antibody of pyrimidine dimer caused by one group of identification DNA is irradiated by ultraviolet light
CN108530539B (en) * 2018-03-20 2021-08-13 北京博雅捷康生物科技有限公司 Group of antibodies for recognizing pyrimidine dimers of DNA caused by ultraviolet irradiation

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