KR100917695B1 - Polynucleotides Sequence Associated with Apoptosis Induced from Cleavage of Caspase-7, Inhibitor or Inducer of the Apoptosis, and The Screening Method thereof - Google Patents

Abstract

The present invention relates to a polynucleotide sequence related to apoptosis derived from caspase-7 cleavage, an inhibitor of apoptosis, an inducing factor, and a method for screening thereof, wherein the polynucleotide sequence represented by SEQ ID NO: 1, (b) ( a polynucleotide sequence having at least 70% homology over the entire length of the DNA of a), or (c) a hybridized polynucleotide sequence under conditions stringent on the polynucleotide sequence of (a) or (b), And (d) a sequence or RNA sequence complementary to the sequences (a), (b) or (c), and inhibition, induction factors, and screening methods thereof using the same.

Apoptosis, Caspase-7

Description

Polynucleotides Sequence Associated with Apoptosis Induced from Cleavage of Caspase-7, Inhibitor or Inducer of the Apoptosis, and The Screening Method

The present invention relates to a novel nucleotide sequence involved in apoptosis and related apoptosis suppression or inducer and screening method thereof, and more particularly, a polynucleotide involved in apoptosis caused by cleavage of caspase-7. The present invention relates to a method for screening a sequence, an apoptosis inhibitor or an inducer, an inhibitor or apoptosis factor.

Analysis of the human genome has revealed many genes whose function is unknown. Large-scale expression systems and RNA interference (RNAi) libraries have been developed to elucidate the role of these genes. For example, large gene overexpression screens show that novel apoptosis inducers (Albayrak and Grimm, 2003; Koenig-Hoffman et al., 2005; Mannherz et al., 2006), and knockdown of gene expression using RNAi libraries ), New tumor suppressors, cell cycle regulators (Kittler et al., 2004) and apoptosis inducers (MacKeigan et al., 2005) were identified (Kolfschoten et al., 2005; Westbrook et al., 2005).

However, large scale gene expression studies require screening of cell phenotypes performed in a large scale manner. In order to speed up the analysis of phenotypes, subcellular image-based screening technology using an automated fluorescence microscope has been developed. They determine the intensity and location of the fluorescence signal, which makes it possible to measure the effect on cells in large numbers of samples (Gasparri et al., 2004; Lovborg et al., 2004).

In this regard, the present inventors have temporarily overexpressed 938 hypothetical cDNAs to identify modulators of novel apoptosis, and a novel polynucleus involved in apoptosis by measuring apoptosis using an automated fluorescence microscope. Cleotidi was selected.

Accordingly, an object of the present invention is to provide a novel polynucleotide sequence involved in apoptosis, a method for screening apoptosis inhibitors or inducers, apoptosis inhibitors or inducers, and the like.

In order to achieve the above object, the present invention provides a purified or isolated polynucleotide sequence which is involved in apoptosis and comprises a polynucleotide sequence selected by the following sequence groups.

(a) a polynucleotide sequence represented by SEQ ID NO: 1,

(b) a polynucleotide sequence having at least 70% homology over the full length of the DNA of (a), or

(c) a polynucleotide sequence hybridized under conditions stringent to the polynucleotide sequence of (a) or (b), and

(d) a sequence or RNA sequence complementary to the sequence (a), (b) or (c).

The present invention provides an apoptosis inducing agent comprising the polynucleotide sequence, and / or a polypeptide expressed from the polynucleotide.

The apoptosis inducer according to the present invention cancer, autoimmune diseases, viral infections, endocrine diseases, organ hyperplasia, restenosis after angioplasty surgery, recurrence after cancer resection surgery, transplant organ rejection, immune failure, neurodegenerative disease, ischemic heart disease, For the treatment of diseases selected from the group consisting of radiation disorders, ultraviolet disorders, addictive disorders, nutritional disorders, inflammatory diseases, ischemic neuropathy, diabetic neuropathy, vascular disease, respiratory disease and cartilage disease.

The present invention is a candidate that inhibits or promotes cell death as compared to the case where the cells expressing any one of the genes of the present invention are cultured in the presence of a candidate drug and, if necessary, are cultured in the absence of the candidate drug. It provides a method for screening an apoptosis inhibitor or inducer comprising the step of finding an agent.

The present invention provides apoptosis suppression or inducer found by the screening method.

The present invention provides a recombinant vector comprising the polynucleotide sequence of the present invention.

The present invention includes a host cell transformed with the vector of the present invention.

The present invention provides animals other than humans, including the transformed cells according to the present invention.

The present invention includes an apoptosis inhibitor comprising an antibody or antisense polynucleotide that specifically binds to the polynucleotide described above.

The present invention provides a polynucleotide sequence involved in apoptosis caused by cleavage of caspase-7, a method for screening apoptosis or inducer, an apoptosis inhibitor or inducer, and is effective in a disease caused by the apoptosis. Treatment can be provided.

Hereinafter, the content of the present invention will be described in detail.

The polynucleotide used for the present invention includes a nucleotide sequence identical or substantially identical to the nucleotide sequence shown in SEQ ID NO: 1 (named C10orf61 ). As the "substantially identical nucleotide sequence" shown in SEQ ID NO: 1, about 70% or more, preferably about 80% or more, even more preferably about 90% or more, and particularly preferably, the nucleotide sequence shown in SEQ ID NO: 1 Preferably, a nucleotide sequence having a homology of about 95% or more, and most preferably about 98% or more.

A base sequence substantially identical to the base sequence shown in SEQ ID NO: 1 has the ability to express apoptosis factors that induce cell death by causing cleavage of caspase-7. Such ability is shown to be homogeneous in nature (eg, physiologically or pharmacologically), but there are some differences in quantitative factors such as the degree of apoptosis activity obtained by expression or the molecular weight of the protein, for example, With respect to the activity, an error within the range of about 0.01 to 100 times, preferably about 0.1 to 10 times, more preferably 0.5 to 2 times can be tolerated.

In addition, the polynucleotide according to the present invention includes, for example, a DNA containing the nucleotide sequence shown in SEQ ID NO: 1, or a base sequence hybridized under DNA stringing conditions with a DNA having substantially the same nucleotide sequence. do. As the DNA capable of hybridizing under the conditions stringent with the nucleotide sequence shown in SEQ ID NO: 1, for example, at least about 50%, preferably about 60% of the complementary sequence of the nucleotide sequence shown in SEQ ID NO: 1 As mentioned above, more preferably at least about 70%, particularly preferably at least about 80% and most preferably at least about 90% DNA containing a base sequence having homology is used. Hybridization is performed according to a method known per se or a method equivalent thereto, for example, the method described in Molecular Cloning 2nd Edition (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). I can do it. In addition, when using a commercially available library, hybridization can be performed in accordance with the method as described in the attached instruction manual. Hybridization can be performed preferably in accordance with stringent conditions. Stringent conditions refer to conditions of, for example, sodium concentration of about 19 to 40 mM, preferably about 19 to 20 mM, and temperature of about 50 to 70 ° C, preferably about 60 to 65 ° C. . In particular, a sodium concentration of about 19 mM and a temperature of about 65 ° C are preferred.

DNA expression vectors containing any of the polynucleotides according to the invention can be prepared, for example, by obtaining the desired DNA fragment and linking the DNA fragment downstream of the promoter in a suitable expression vector. As an expression vector, E. coli-derived extranuclear genes (eg pBR322, pBR325, pUC12, pUC13); Bacillus subtilis genes (eg pUB110, pTP5, pC194); Yeast-derived extranuclear genes (eg pSH19, pSH15); λ sterilization; Bacteriophages such as viruses; animal viruses such as RNA tumor viruses and baculoviruses; pA1-11, pXT1, pRc / CMV, pRc / RSV, pcDNAI / Neo and the like can be used.

As the promoter, any promoter may be used as long as it is a suitable promoter known to be usable for host cells used for gene expression. For example, when the host cell is an animal cell, an SRα promoter, an SV40 promoter, an LTR promoter, a CMV (site megalovirus) promoter, an HSV-TK promoter, or the like can be used. Especially, a CMV promoter, SR alpha promoter, etc. are preferable. When the host cell is Escherichia spp, trp promoter, lac promoter, recA promoter, λPL promoter, lpp promoter, T7 promoter and the like can be used. When the host cell is Bacillus sp., SPO1 promoter, SPO2 promoter, penP promoter and the like can be used. When the host cell is a yeast, a PHO5 promoter, a PGK promoter, a GAP promoter, an ADH promoter, or the like can be used. If the host cell is an insect cell, the P10 promoter can be used.

In addition to the above, an expression vector may contain an enhancer, a splicing signal, a poly A-addition signal, a selection marker, an SV40 replication origin (SV40ori), or the like, as necessary. As a selection marker, a dihydrofolate reductase (dhfr) gene, an ampicillin resistance gene, a neomycin resistance gene, etc. are mentioned, for example.

A transformant containing any one of the polynucleotides according to the present invention can be produced by transforming a host cell with an expression vector containing the polynucleotide according to a known method. As the host cell, for example, Escherichia spp., Bacillus spp., Yeast, insect cells, insects, animal cells and the like can be used.

Transformation can be performed according to a well-known method according to the kind of host cell. Escherichia spp. Include, for example, Proceedings of the National Academy of Sciences (Proc. Natl. Acad. Sci. USA), 69, 2110 (1972) or Gene, 17, 107 (1982), and the like. Transformation can be performed according to the methods described. Bacillus sp. Can be transformed according to the method described in Molecular & General Genetics, Vol. 168, 111 (1979) and the like, for example. Yeasts are described, for example, in Methods in Enzymology, Vol. 194, 182-187 (1991), Proc. Natl. Acad. Sci. Transformation can be carried out according to the methods described in USA, 75, 1929 (1978) and the like. Insect cells and insects can be transformed according to the methods described, for example, in Bio / Technology, 6, 47-55 (1988) and the like. Animal cells can be transformed according to the methods described, for example, in Virology, Vol. 52, 456 (1973).

Cultivation of the transformant can be carried out according to a known method depending on the type of host cell. For example, when the host cell cultures a transformant which is Escherichia spp. Or Bacillus spp., A liquid culture medium is preferable as the culture medium used for the culture. In addition, the culture medium preferably contains a carbon source, a nitrogen source, an inorganic material, and the like necessary for the growth of the transformant. Here, as the carbon source, for example, glucose, dextrin, soluble starch, sucrose and the like; As the nitrogen source, for example, inorganic or organic substances such as ammonium salts, nitrates, peptone, soybean paste; For example, calcium chloride and magnesium chloride. In addition, yeast extract, vitamins, growth promoting factors and the like may be added to the culture medium. The pH of the culture medium is preferably about 5-8.

As a culture medium when the host cell cultures transformants of Escherichia spp., For example, M9 culture medium containing glucose and kazamino acid (Miller, Journal of Experiments in Molecular Genetics, 431-343) , Cold Spring Harbor Laboratory, New York 1972. Cultivation of transformants in which the host cell is Escherichia spp. Is usually carried out at about 15 to 43 ° C. for about 3 to 24 hours. At this time, ventilation or stirring may be performed. Cultivation of the transformant whose host cell is Bacillus sp. Is usually performed at about 30 to 40 ° C. for about 6 to 24 hours. At this time, ventilation or stirring may be performed. As a culture medium in the case of culturing a transformant in which the host cell is a yeast, for example, a Burkholder minimum culture [Bostian, K. L. et al., Proc. Natl. Acad. Sci. USA, Vol. 77, 4505 (1980)]. The pH of the culture medium is preferably about 5-8. The culture is usually performed at about 20 ° C to 35 ° C, and performed for about 24 to 72 hours. As needed, you may perform ventilation and stirring. As a culture medium when the host cell cultures an insect cell or a transformant that is an insect, for example, 10% immobilized to Grace's Insect Medium (Grace, TCC, Nature, 195, 788 (1962)). What suitably added additives, such as a bovine serum, can be used. The pH of the culture medium is preferably about 6.2 to 6.4. The culture is usually performed at about 27 ° C. for about 3-5 days. If necessary, ventilation or agitation may be performed. As a culture medium in the case of culturing a transformant whose host cell is an animal cell, for example, MEM culture medium containing about 5-20% fetal bovine serum [Science, 122, 501 (1952)], DMEM Culture Medium [Virology, Volume 8, 396 (1959)], RPMI 1640 Culture Medium [The Journal of the American Medical Association, Volume 199, 519 (1967)], 199 Culture Medium [Proceeding of the Society for the Biological Medicine, 73] Vol. 1 (1950)] may be used. The pH of the culture medium is preferably about 6-8. The culture is usually carried out at about 30 ° C to 40 ° C, for about 15 to 60 hours. If necessary, ventilation or agitation may be performed.

The apoptosis inducer of the present invention can be separated and purified from the culture obtained by culturing the transformant according to a method known per se. For example, when the inducer of the present invention is extracted from culture cells or cells, the cells or cells collected from the culture by a known method are suspended in an appropriate buffer solution, and the cells or cells are sonicated by ultrasound, lysozyme and / or freeze-thawing. After destroying, the method of obtaining the crystal extract of soluble protein by centrifugation or filtration, etc. can be used suitably. At this time, the buffer solution may contain a protein denaturant such as urea or guanidine hydrochloride, or a surfactant such as Triton X-100.

The isolation and purification of the apoptosis inducer according to the present invention thus obtained can be carried out according to a method known per se. As such a method, a method using solubility such as salting out or a solvent precipitation method; a method using mainly molecular weight differences such as dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis; charge such as ion exchange chromatography A method using specific affinity such as affinity chromatography; a method using hydrophobicity such as reverse phase high performance liquid chromatography; a method using difference of isoelectric point such as isoelectric electrophoresis. These methods may be combined as appropriate.

When the apoptosis inducer obtained according to the present invention is a vitreous body, the vitreous can be converted into a salt according to a method known per se or a method similar thereto, and when a form of a salt can be obtained, a self-known body is known. The salt can be converted into a vitreous or other salt by the method described below or by a method similar thereto.

The presence of apoptosis inducer obtained according to the present invention can be confirmed by enzyme immunoassay or Western blotting using a specific antibody.

The antibody against the apoptosis inducer or salt thereof obtained according to the present invention may be either a polyclonal antibody or a monoclonal antibody. These antibodies can be prepared according to a known method of producing antibodies or antiserum using an apoptosis factor obtained by the present invention as an antigen. As the apoptosis inducer of the present invention to be used as an antigen, any of the apoptosis inducing factor of the present invention, a partial peptide thereof or a salt thereof may be used.

As a polynucleotide (hereinafter referred to as an antisense polynucleotide) containing a base sequence complementary to the base sequence encoding the apoptosis inducer of the present invention or a part thereof, a base sequence completely complementary to the base sequence encoding the apoptosis inducer Or a base having a substantially complementary nucleotide sequence or part thereof and having a function of inhibiting translation of the protein from RNA encoding apoptosis inducing factor. Substantially complementary nucleotide sequences include base sequences encoding apoptosis inducers or partial peptides thereof, and base sequences capable of hybridizing under physiological conditions of cells expressing the protein, more specifically, these inducers Or at least about 70%, preferably at least about 80%, more preferably at least about 90%, most preferably about 95 with respect to the overlapping portion between the complementary chains of the base sequences encoding the partial peptides. The base sequence etc. which have homology of% or more are mentioned.

Antisense polynucleotides of the present invention can be designed and synthesized based on cloned or determined base sequence information of the polynucleotide of the present invention. Such polynucleotides can inhibit the replication or expression of genes encoding apoptosis inducers. That is, the antisense polynucleotide of the present invention can hybridize with RNA transcribed from a gene encoding an apoptosis inducer, thereby inhibiting the synthesis (processing) or the function (translation into protein) of mRNA.

The target region of the antisense polynucleotide of the present invention is hybridized by the antisense polynucleotide, and as a result, if the translation of apoptosis inducer is inhibited, the length thereof is not particularly limited. The arrangement may be partial or partial, and the short may be about 15 bp and the long may be an entire arrangement of mRNA or initial transcription product. In view of ease of synthesis and antigenicity, oligonucleotides composed of about 15 to about 30 bp are preferred, but not limited thereto. Specifically, for example, a 5 ′ terminal hairpin loop, a 5 ′ terminal 6bp repeat, a 5 ′ terminal untranslated region, a polypeptide translation start codon, a protein code region, an ORF translation start codon, Any region within the gene can be selected as a target, such as the 3 ′ terminal untranslated region, 3 ′ terminal Palindrom region, and 3 ′ terminal hairpin loop. For example, it is also preferable to make the intron part of the said gene into a target region.

Furthermore, the antisense polynucleotides of the present invention not only inhibit the translation into proteins by hybridizing with mRNAs or early transcription products encoding apoptosis inducers, but also bind triplex by combining with genes encoding apoptosis inducers. It may form and inhibit the transcription of RNA.

The antisense polynucleotide may be RNA, DNA, or modified nucleic acid (RNA, DNA). Specific examples of the modified nucleic acid include, but are not limited to, sulfur derivatives and thiophosphate derivatives of nucleic acids, and those having resistance to degradation of polynucleotide amides and oligonucleotide amides. For example, a ribozyme that can specifically cleave an mRNA or gene initial transcription product encoding an apoptosis inducer in the interior of the code region (in the case of the initial transcription product includes an intron portion). Antisense polynucleotides. In addition, siRNAs complementary to the mRNA encoding the apoptosis inducer or a partial sequence in the code region of the gene initial transcription product (in the case of the initial transcription product include an intron portion) may also be included in the antisense polynucleotide of the present invention. have. The phenomenon called so-called RNA interference (RNAi), which degrades mRNA complementary to the RNA when SiRNA is introduced into cells, has been known as nematodes, insects, plants, etc., but recently, it has been confirmed that this phenomenon also occurs in mammalian cells. [Nature, 411 (6836): 494-498 (2001)] has attracted attention as an alternative technique of ribozyme. Antisense oligonucleotides and ribozymes of the present invention determine target regions of mRNA or initial transcription products based on cDNA sequence or genomic DNA sequence information encoding the proteins of the present invention, and use commercially available DNA / RNA autosynthesizers, It can manufacture by synthesizing the sequence complementary to this. siRNA synthesizes the sense strand and the antisense strand with a DNA / RNA automatic synthesizer, respectively, and denatures in a suitable annealing buffer, for example, at about 90 to about 95 ° C. for about 1 minute, and then at about 30 to about 70 ° C. It can manufacture by annealing for 1 to about 8 hours. Further, longer two-stranded polynucleotides can be produced by synthesizing complementary oligonucleotide strands alternately and annealed and then ligated with ligase.

Apoptosis inducers or activating peptides according to the invention cause the induction of apoptosis by cleaving caspase-7. Thus, 1) apoptosis inducer or activating peptide according to the present invention, 2) apoptosis inducer or polynucleotide encoding the activating peptide of the present invention, 3) a base sequence encoding apoptosis inducer or part thereof Polynucleotide, 4) antibody of the present invention, 5) antisense polynucleotide of the present invention, 6) inhibitory peptide of the present invention can be used as a prophylactic / treatment agent for a disease associated with inhibition of apoptosis.

As described above, the apoptosis inducer according to the present invention has a function of inducing apoptosis by cleaving caspase-7, and thus apoptosis inducer or nucleic acid (eg, gene, mRNA, etc.) encoding the same in vivo. In case of abnormality or deficiency or abnormally decreased expression level, or induction of apoptosis by any other factor excessively, for example, cancer, autoimmune disease, viral infection, It develops various diseases such as endocrine diseases, blood diseases and organ hyperplasia. Therefore, if there is a patient who cannot expect to eliminate unnecessary cells or abnormal cells due to the reduction or other factors of apoptosis inducing factor, a) apoptosis inducing factor or activating peptide or salt thereof is administered to the patient. By supplementing the amount or by b) (i) administering DNA encoding an apoptosis inducer or activating peptide to the patient and expressing it in the target cell, or (ii) inducing apoptosis in a single isolated target cell. After introducing and expressing a DNA encoding a factor or an activating peptide, the cell is transplanted into the patient, thereby increasing the amount of apoptosis inducing factor in the patient's body, thereby causing abnormal cells or unnecessary cells. Apoptosis resulting from cleavage of caspase-7 can be induced.

In the case of using the apoptosis inducing factor or the activating peptide or the salt thereof according to the present invention as the prophylactic and therapeutic agent, it can be formulated according to known means. On the other hand, when the polynucleotide encoding the apoptosis inducing factor or the activating peptide of the present invention is used as the prophylactic and therapeutic agent, the polynucleotide alone or suitable for RNA tumor virus vectors, adenovirus vectors, adenovirus-associated virus vectors, etc. After insertion into the vector, it can be sanctioned according to known means. For example, a) apoptosis inducer or the activating peptide or salt thereof of the present invention, or b) apoptosis inducing factor or the polynucleotide encoding the activating peptide of the present invention may be purified, capsule, microcapsules or the like as necessary. It can be used orally or parenterally in the form of an injection.

As an additive that can be mixed with tablets, capsules and the like, for example, binders such as gelatin, corn starch, tragant, gum arabic, excipients such as crystalline cellulose, swelling agents such as corn starch, gelatin, alginic acid, Lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, flavoring agents such as peppermint, akamono oil or cherry, and the like can be used. When the dosage form is a capsule, the material of the above type may further contain a liquid carrier such as oil or fat. As the aqueous solution for injection, for example, isotonic solutions (e.g., D-sorbitol, D-mannitol, sodium chloride, etc.) containing physiological saline, glucose, and other auxiliary agents are used, and suitable dissolution aids, for example, For example, you may use together with alcohol (for example, ethanol), poly alcohol (for example, propylene glycol, polyethylene glycol), nonionic surfactant (for example, polysorbate 80 TM, HCO-50). As an oily liquid, sesame oil, soybean oil, etc. may be used, for example, and it may be used together with benzyl benzoate, benzyl alcohol, etc. which are dissolution aids.

The prophylactic and therapeutic agents according to the present invention include, for example, buffers (e.g., phosphate buffers, sodium acetate buffers), analgesic agents (e.g. benzalconium chloride, procaine hydrochloride, etc.), stabilizers (e.g., For example, human serum albumin, polyethylene glycol, etc.), a preservative (for example, benzyl alcohol, phenol, etc.), antioxidant, etc. may be combined. The prepared injection solution is usually filled in a suitable ampoule.

The sanctions obtained in this way are safe and low-toxic, so for example humans or other warm-blooded animals (e.g., rats, mice, hamsters, rabbits, sheep, goats, pigs, cattle, horses, cats, dogs, monkeys). , Chimpanzees, birds, etc.). The dose of the apoptosis inducer or the activating peptide of the present invention may vary depending on the administration target, the target organ, the symptoms, the administration method, and the like. About 0.1 mg to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg. In the case of parenteral administration, the single dose varies depending on the administration target, target organ, symptom, administration method, and the like, but for example, in the form of an injection, for example, about 60 kg in adults, about one day It is convenient to administer about 0.01-30 mg, preferably about 0.1-20 mg, and more preferably about 0.1-10 mg. In the case of other animals, the amount converted in terms of 60 kg can be administered.

The dosage of the apoptosis inducer or polynucleotide encoding the activating peptide of the present invention may vary depending on the subject, organ, symptom, method of administration, etc., but in the case of oral administration, for example, in a 60 kg adult In the case of about 0.1 mg to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg per day. In the case of parenteral administration, the single dose varies depending on the administration target, the target organ, the symptoms, the administration method, and the like, but, for example, in the form of an injection, for example, about 0.01 mg / day for a 60 kg adult It is convenient to administer about 30 mg, preferably about 0.1-20 mg, and more preferably about 0.1-10 mg. In the case of other animals, the amount converted in terms of 60 kg can be administered.

The antibody according to the present invention may specifically inhibit the apoptosis by specifically binding to apoptosis inducer to prevent cleavage of caspase-7. On the other hand, the antisense polynucleotide of the present invention inhibits the expression of apoptosis inducing factor, and can similarly reduce the apoptosis induction promoting action.

When the antibody of the present invention is used as the apoptosis inhibitor or the like, it can be prepared by dissolving it in an appropriate concentration in water or an appropriate buffer (eg, phosphate buffer, PBS, tris hydrochloric acid buffer, etc.). Moreover, you may mix | blend a preservative, a stabilizer, a reducing agent, a tonicity agent etc. which are normally used as needed. On the other hand, when the antisense polynucleotide of the present invention is used as the apoptosis inhibitor or inhibitor, the polynucleotide is inserted alone or in a suitable vector such as an RNA tumor virus vector or adenovirus vector, and then the above transformation method ( For example, a liposome method, an electroporation method, etc. can be introduce | transduced into a cell.

The dosage of the antibody of the present invention varies depending on the administration target, target organ, symptom, administration method, etc., but in the case of oral administration, for example, about 0.1 mg to 100 mg per day in a 60 kg adult, Preferably it is about 1.0-50 mg, More preferably, it is about 1.0-20 mg. In the case of parenteral administration, the single dose varies depending on the administration target, the target organ, the symptoms, the administration method, and the like, but for example, in the form of an injection, for example, in a 60 kg adult, about one day It is convenient to administer about 0.01-30 mg, preferably about 0.1-20 mg, and more preferably about 0.1-10 mg. In the case of other animals, the amount converted in terms of 60 kg can be administered.

The dosage of the antisense polynucleotide of the present invention varies depending on the administration target, target organ, symptom, and administration method, but in the case of oral administration, for example, about 0.1 mg to 100 per day in a 60 kg adult, for example. mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg. In the case of parenteral administration, the single dose varies depending on the administration target, the target organ, the symptoms, the administration method, and the like, but, for example, in the form of an injection, for example, about 0.01 mg / day for a 60 kg adult It is convenient to administer about 30 mg, preferably about 0.1-20 mg, and more preferably about 0.1-10 mg. In the case of other animals, the amount converted in terms of 60 kg can be administered.

The present invention also provides a method for screening apoptosis inducer material using an apoptosis inducer or an activating peptide or salt thereof. As an example of this screening method, the substance which changes the expression level of an apoptosis inducer can be screened by using the sense and antisense polynucleotide of this invention as a probe, or using the antibody of this invention. That is, the present invention, for example, (i) apoptosis in non-human mammals, b) certain organs, c) tissue or cells isolated from organs, or (ii) apoptosis included in transformants or the like. A method for screening a substance for changing the expression level of apoptosis inducer by measuring the mRNA amount or protein amount of a factor is provided.

For example, measurement of mRNA amount or protein amount of apoptosis inducing factor can be specifically carried out as follows.

For normal or disease model non-human mammals (eg, mice, rats, rabbits, sheep, pigs, cattle, cats, dogs, monkeys, etc.), drugs (eg, TNF-α, IL-1, Fas) Blood, or certain organs (e.g., brain, liver, kidneys, etc.) after a certain period of time after giving a physicochemical stress (e.g. UV, free radicals, ischemia, etc.) Obtain tissue or cells isolated from organs.

The mRNA contained in the obtained cell can be quantified by extracting mRNA from a cell or the like by a common method, for example, by using a technique such as RT-PCR, or a known Northern blot. It can also be quantified by interpretation. On the other hand, the content of apoptosis inducer can be quantified by extracting proteins from cells and the like by conventional methods, for example, by Western blot analysis.

By the above screening method, a substance which increases the expression level of apoptosis inducing factor can be selected as a substance for inducing apoptosis induction and a substance which decreases the expression level of apoptosis inducing factor as an activation inhibitory substance of apoptosis induction factor.

Although an Example is given to the following and this invention is demonstrated to it further more concretely, the scope of the present invention is not limited by them.

Example 1 Expression and Function Confirmation of Apoptosis Inducing Factor I

cDNA  Expression plasmid

Of the 7,385 full-length genes in the Korean UniGene Information database (KUGI; http://kugi.kribb.re.kr ), 4,782 are present in mammalian expression vectors (Kim et al., 2004; Oh et al., 2004). . We used these hypothetical cDNA clones on the screen. Their coding sites were PCR amplified to generate C10orf61 (SEQ ID NO: 1), MGC26717 (SEQ ID NO: 2), and FLJI3855 (SEQ ID NO: 3) in C-terminal GFP or myc tagged form. For C10orf61, 5'-GCGAATTCATGGATTCTAATGGA-3 '(SEQ ID NO: 4) was used as the forward primer, and 5'-GCGGTACCCATAGTCTCTAGGTT-3' (SEQ ID NO: 5) was used as the reverse primer. For MGC26717, 5'-GCCTCGAGATGTCGGGACTCAGC-3 '(SEQ ID NO: 6) was used as the forward primer and 5'-GCGGATCCAACTTGGTCCCCAGT-3' (SEQ ID NO: 7) as the reverse primer. For FLJ13855, 5'-CGCTCGAGATGTCCATTTATAAG-3 '(SEQ ID NO: 8) was used as the forward primer and 5'-CGGGATCCAACCCTCAGGCTCCC-3' (SEQ ID NO: 9) was used as the reverse primer. PCR-amplified C10orf61 was subcloned into the EcoRI and KpnI sites of pEGFP / N3 (Invitrogen, USA) and pcDNA3.1 / myc-His (-) (Invitrogen, USA). PCR products of MGC26717 and FLJ13855 were subcloned into the XhoI and BamHI sites of pEGFP / N3 and pcDNA3.1 / myc-His (-). DNA sequences of all the plasmids made after subcloning were confirmed. Expression of C10orf61-myc was not detected by western blot with antimyc antibody. However, C10orf61-GFP was easily detected using anti-GFP antibody.

Cell culture and Transfection

HeLa cells were maintained at 37 ° C. in a humidified cell incubator containing 5% CO ₂ and incubated in DMEM supplemented with 10% fetal bovine serum (FBS) and antibodies (JBI, Korea). For transfection, 7.5 × 10 ³ HeLa cells per well were seeded in 96 well plates, and 1.4 × 10 ⁵ HeLa cells per well were seeded in 6 well plates. After 16-18 hours (the cells reached about 70% confluence) they were transfected with Lipofectamine Plus ^™ (Invitrogen). Transfection was performed with 100 ng cDNA per well for 96 well plates and 1 μg cDNA per well for 6 well plates.

Lentivirus  Vector building

Kim Yeon-su (Inje University, Korea) was constructed in the recombinant lentiviral vector. Myc-tagged C10orf61, MGC26717 and FLJ13855 were inserted separately by the IRES system into the polycloning site of the lentiviral vector (LentiH1.2) containing the hCMV promoter and EGFP-zeosin fusion protein, and the recombinant vector was incorporated by VectorCore A. Produced by (Korea). Three plasmids, a delivery vector expressing the VSV-G vector, and a gag-pol expression vector were coatfected into 293T cells in a 1: 1: 1 molar ratio using Lipofectamine PlusTM. Culture supernatants containing viral vector particles were harvested 48 hours after transfection, clarified with 0.45 μm membrane filter (Nalge Nunc International, USA) and immediately stored at −70 ° C.

Their titers were approximately 3-5 × 10 ⁷ transduction units without further enrichment. Approximately 1 × 10 ⁵ HeLa cells were seeded per well of 12 well plates for transduction. After 16-18 hours, transduction was performed with 300 μl of lentiviral (1˜1.7 × 10 ⁷ transduction units) using polybrain (Invitrogen).

Nuclear  Blast image

Cells were stained with 5 μM Hoechst 33342 (Molecular Probes, USA) 28-30 hours after transfection and nuclear morphology was analyzed using an automated In Cell Analyzer 1000 fluorescence microscope (GE, Cardiff, UK). Three fields of fluorescence images per well were obtained and the total number of nuclei (approximately 500 cells per transfection) was automatically counted approximately using the object intensity module (GE), and the number of dead nuclei was manually counted.

DNA  Fraction ELISA

HeLa cells cultured in 6-well plates were transfected with cDNA as mentioned above and obtained and measured for DNA fraction using the Cell Death Detection ELISA system (Roche, USA). Cytoplasmic fractions (20,000 × g) obtained from approximately 1 × 10 ⁴ cells were incubated in microtiter plates coated with antihistone antibodies followed by a second anti-DNA antibody bound to peroxidase. Percent DNA fractions were compared to control cells into which the vector was introduced (Oh et al., 2004).

Weston Blotting

Cells were lysed in 1% SDS, 100 mM Tris / pH8.0 and immediately heated to 100 ° C. Protein was dissolved in either 9% or 12% Tris / Tricine gel, transferred to nitrocellulose membrane (Bio-Rad, USA) and reacted with primary antibody. Anti-caspase 7 was purchased from BD (USA) and anti-RARP from Cell Signaling (USA). Anti-GFP was purchased from Zymed (USA) and anti-actin was purchased from Sigma (USA). Antimyc and secondary antibodies, antirabbit IgG-HRP and antimouse IgG-HRP were purchased from Santa Cruz Biotechnology (USA). Antigens were visualized using ECL solution (Pierce, USA).

Cell viability measurement

Approximately 1 × 10 ⁵ HeLa cells seeded in 12 well plates were transduced with the lentiviruses encoding C10orf61, MGC26717 or FLJ13855 as mentioned above. 48 hours after transduction, cells were harvested, resuspended in fresh medium and counted for viable cell count in hematopoiesis using trypanblue staining. Subsequently 6 × 10 ⁴ cells were plated in 60 mm dishes and incubated for 4 days. Cells were fixed with 10% formaldehyde and stained with 1% crystal violet. The dish was dried and the image was captured with a flatbed scanner.

Protein domains Homomorphic  Confirm

Protein domains and homology were identified using the SMART tool in the EMBL database (http://smart.embl-heidelberg.de).

Experiment result

To identify the genes of novel apoptosis inducers, 938 hypothetical cDNA clones were transiently expressed in HeLa cells. The latter cells were easily cultured and used for transfection with high efficiency. Moreover, apoptosis can be detected with reliability and selectivity. Apoptosis was measured by capturing and analyzing images of nuclei stained with Hoechst 33342 using an automated fluorescence microscope 28-30 hours after transfection.

All 27 genes resulted in stimulation with a greater than three times the number of dead nuclei (FIG. 1A). According to DNA sequencing, the seven genes were full-length, and these sequences were completely identical to the annotated sequences, while the remaining 20 genes were usually partial clones with the C terminus deleted. We selected three sequences C10orf61 (SEQ ID NO: 1), MGC26717, and FLJ13855 identified as full-length genes with the highest rate of apoptosis (Table 1). Images of representative Hoechst stained nuclei of HeLa cells transfected with these three genes are shown in FIG. 1B.

Table 1 Genes with Assumed Proapoptotic Activity

Approval number sign Gene name Magnification in Apoptosis Nucleus NM_015631.2 C10orf61 Chromosome 10 Open reading frame 61 4.7 NM_173824.2 MGC26717 Assumption Protein MGC26717 3.9 NM_023079.2 FLJ13855 Assumption Protein FLJ13855 3.7

To confirm the results obtained from this screen, we performed viability measurements. As can be seen in Figure 2A, cell survival was significantly reduced by transfection by these trigenes. Using DNA fractional ELISAs, we found that a decrease in cell viability was derived from apoptosis (FIG. 2B). DNA fractions increased almost 2-2.5 fold in cells transfected with C10orf61, MGC26717, and FLJ13855, comparable to a 3-fold increase in apoptosis caused by BAX plasmid as a positive control.

To substantiate this finding, we introduced Western blot to determine the level of caspase-7, a running caspase (FIG. 2C). Transfection by apoptosis gene candidate induced caspase-7 cleavage and Western blot with antibodies against poly- (ADP-ribose) -polymerase (PARP), a downstream target of execution caspase Cleavage of PARP was also caused (FIG. 2D).

To confirm whether the introduced gene was really expressed, we generated a GFP tagged form of FLJ13855 and stained the nucleus transfected with this construct with Hoechst (FIG. 3A). Apoptotic nuclei were counted only in GFP expressing cells, and these results indicate that FLJ13855 expressing HeLa cells had an approximately 5-fold increase in apoptosis. Positive control Bax-GFP induced 89.3% apoptosis, whereas Sec61β, a protein unrelated to apoptosis, produced similar apoptosis as that of the vector control. These results show that apoptosis caused by FLJ13855 is specific for that gene.

For both C10orf61 and FLJ13855, we performed western blot with anti-caspase-7 to detect apoptosis (FIG. 3C) and blotted with anti-GFP to confirm these expressions. Slight cleavage of procaspase-7 was noted in the transfected GFP vector control, consistent with low apoptosis levels in these cells. The level of cleaved caspase-7 was increased in cells transfected with C10orf61-GFP and FLJ13855-GFP, which was confirmed to cause cell death due to overexpression of C10orf61 and FLJ13855.

To rule out the possibility that this apoptosis has resulted from overexpression of large amounts of genes by transfection reagents used, we have made C10orf61, MGC26717, and FLJ13855 in the C-terminus myc-tagged forms, which are referred to as lentiviruses. Subcloned into the vector. GFP expression levels in the transduced cells showed that these viruses efficiently infected HeLa cells. We did not test whether the introduced cells caused apoptosis, but we found that a large number of living cells were significantly reduced (FIG. 3D).

To determine the function of C10orf61, MGC26717, and FLJ13855, we searched for homolog-like protein domains. The amino acid sequence of MGC26717 had no apparent similarity with known proteins or domains. C10orf61 corresponds to the recently named Tectonic3 isoform b, a splicing variant of Tectonic3. Mammalian Tectonics had two homologs, Tect2 and Tect3, and their homology to Tectonic was 49% and 58%, respectively (Reiter et al., 2006). Tectonic is essential for both activation and inhibition of hedgehog signaling (Reiter et al., 2006), but the mechanisms involved should be determined. Hedgehog signals act as positive and negative regulators of the cell cycle (Hutchin et al., 2005; Shkumatava and Neumann, 2005). Whether apoptosis induced by C10orf61 is associated with cell cycle regulation mediated by hedgehog signaling should be further studied.

Example 2 Expression and Function Confirmation of Apoptosis Inducing Factor Ⅱ

Polypeptides functionally equivalent to the polypeptides (C10orf61, MGC26717, and FLJ13855) according to Example 1 of the present invention can be prepared by those skilled in the art, for example, by introducing a mutation into an amino acid sequence in the polypeptide. As a method of introducing a mutation, for example, site-specific mutation induction method (Current Protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. Jhon Wily & Sons Section 8.1-8.5) and the like. In addition, such a polypeptide may be caused by amino acid variation in nature. In the present invention, one or several amino acids are substituted, deleted, inserted, and / or added to the final amino acid sequence obtained in Example 1, as long as they have the same function as the polypeptides obtained in Example 1. Other polypeptides that can be obtained are also included. The number of mutations and the mutation sites of amino acids in the polypeptide are not particularly limited as long as their function is maintained. The number of variations is typically within 10% of the total amino acid, preferably within 5% of the total amino acid, and even more preferably within 1% of the total amino acid. The amino acid to be substituted is preferably an amino acid having properties similar to those of the amino acid before substitution from the viewpoint of the function, storage and maintenance of the polypeptide. For example, since Ala, Val, Leu, Ile, Pro, Met, Phe, and Trp are all classified as nonpolar amino acids, they are considered to have similar properties. Moreover, Gly, Ser, Thr, Cys, Tyr, Asn, Gln is mentioned as non-chargeability. Moreover, Asp and Glu are mentioned as acidic amino acid. Moreover, Lys, Arg, His is mentioned as alkaline amino acid.

Moreover, the polypeptide functionally equivalent to the polypeptide of this invention can also be isolated using the hybridization technique or gene amplification technique well-known to those skilled in the art. That is, those skilled in the art, using the hybridization technology (Current Protocols in Molecular Biology edit.Ausubel et al. (1987) Publish. Jhon Wily & Sons Section 6.3-6.4), the base sequence encoding the polypeptide obtained in Example 1 Or isolating a polynucleotide having a high homology with this based on a part thereof, and obtaining a functionally equivalent polypeptide from the polynucleotide corresponds to conventional techniques. In the present invention, a polynucleotide encoding these proteins and a polypeptide encoded by a polynucleotide hybridizing as long as they have a function equivalent to that of the polypeptide of the present invention are also included.

The stringent conditions of hybridization for isolating polynucleotides encoding functionally equivalent polypeptides have already been mentioned above. In general, as hybridization conditions become more difficult, isolation of DNA having high homology with probe arrays can be expected. Polypeptides isolated using this hybridization technique generally have high homology to the amino acid sequence or the nucleotide sequence encoding the polypeptide as compared to the polypeptide of the present invention obtained in Example 1. High homology refers to at least 70%, preferably at least 80%, more preferably at least 90%, more preferably at least 95%, particularly preferably at least 98%. The homology can be determined using the BLAST2 search algorithm (Altschul, S.F. et al, 1997 Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25: 3389-3402.).

According to the conditions described above, the inventors have 70%, 80%, 90%, 95%, and 98% homology, respectively. A portion of the codon was modified to convert to to Met, and a portion of Phe was modified to a portion of the codon to transform to Trp. The resulting gene was expressed as a polypeptide by the same method as in Example 1, and each of the obtained polypeptides was named C10orf61-1 to 5, respectively.

Using these C10orf61-1-5, as a result of confirming the function of the caspase-7 cleavage and apoptosis in the same manner as in Example 1, it was confirmed that the C10orf61 had the same apoptosis activity.

1 shows screen results for a novel gene with a proapoptotic phenotype according to the present invention.

A. HeLa cells seeded in 96 well plates were transfected with 938 hypothetical genes. After incubation for 28-30 hours, cells were stained with Hoechst 33342 and apoptosis was measured using an In Cell Analyzer 1000. The rate of apoptosis in cells transfected with the vector was determined at 1.

Nuclear images of HeLa cells stained with Hoechst transfected with B. pCNS vector (control), C10orf61, MGC26717, and FLJ13855 were captured using In Cell Analyzer 1000. These representative images from three fields per well are shown.

2 shows apoptosis activity test results of candidate genes selected according to the present invention.

A. Cell viability was measured 36 hours after transfection of HeLa cells with pCNS vector (control), C10orf61, MGC26717, and FLJ13855. NT refers to HeLa cells that are not transfected. Cells were fixed and visualized with crystal violet to visualize live cells. Asterisks indicate wells that have not been seeded into cells.

B. HeLa cells were transfected with Bax plasmid with pCNS vectors (control), C10orf61, MGC26717, and FLJ13855 plasmid, or positive control, and cell death was measured using DNA fractionation ELISA. The results show the mean and standard deviation of three independent experiments.

Lysates from HeLa cells transfected with the plasmids described in C. B were isolated with 12% Tris / Tricine polyacrylamide gels, and the western blots were used for caspase with activated fast mobility. Seventh-7 was detected. Non-specific cross reaction bands (NS) were used as load controls. The results are at least three independent representative experiments.

Lysates from HeLa cells transfected with the plasmids described in D. B were separated with 9% Tris / Tricine polyacrylamide gels, and Western blot was used to detect PARP and cleaved PARP. Actin was used as the load control. The results are at least three independent representative experiments.

3 is a result of the expression test of candidate genes selected according to the present invention.

A. HeLa cells are transiently transfected with a GFP vector (GFP), FLJ13855 (FLJ-GFP) tagged with GFP, Sec61β (Sec61-GFP) tagged with GFP, or Bax (Bax-GFP) tagged with GFP It was. Nuclei were stained with Hoechst 33342 and GFP and Hoechst images were captured using an automated fluorescence microscope. Representative images from three wells are shown.

B. The percentage of apoptosis nuclei was calculated from the number of GFP expressing cells with apoptosis nuclei in 3 wells per construct.

C. HeLa cells were not transfected or transfected with GFP, C10orf61-GFP, or FLJ13855-GFP. * Indicates expression of C10orf61-GFP and ** indicates expression of FLJ13855-GFP. Western blot was used to detect procaspase-7 and activated fast moving caspase-7. Actin was used as the load control.

D. Left panel: HeLa cells transduced with control lentiviral (control), C10orf61, MGC26717, or FLJ13855 were counted and the same number of cells plated in 60 mm dishes. After 4 days, viable cells were detected using crystal violet.

Right panel: Cell lysates obtained 2 days after transduction were separated on 9% polyacrylamide Tris / Tricine gel and blotted with antimyc. Short and long exposure of the blot. * Indicates expression of MGC26717-myc and ** indicates expression of FLJ13855-myc.

<110> Korea Research Institute of Bioscience and Biotechnology <120> Polynucleotides Sequence Associated with Apoptosis Induced from          Cleavage of Caspase-7, Inhibitor or Inducer of the Apoptosis, and          The Screening Method <160> 9 <170> KopatentIn 1.71 <210> 1 <211> 2825 <212> DNA <213> Homo sapiens <400> 1 ggttgccagg caacggaggc acggcccggc ccgcgttaag gaggagggcg cagaccgaag 60 gacactgaaa gagctgtaac aaccccactt tcgattggtt gaagagctct cagccttctc 120 atgagccaat gagaagaggc acgcggatgg cgtcagacgc tatgcgactc ctcccaccca 180 cgctctggca atgcgattgg agaccgcgga ggcctacgtc ggacccggag gccctgaatg 240 ccccatgcgc accccacagc tcgcgctcct gcaagtgttc tttctggtgt tccccgatgg 300 cgtccggcct cagccctctt cctccccatc aggggcagtg cccacgtctt tggagctgca 360 gcgagggacg gatggcggaa ccctccagtc cccttcagag gcgactgcaa ctcgcccggc 420 cgtgcctgga ctccctacag tggtccctac tctcgtgact ccctcggccc ctgggaatag 480 gactgtggac ctcttcccag gtgagggaaa acgttgtggg ggtggaaact tacttctgag 540 atctaaaatt ctgtgagcaa agttgagctg ccacaaagca ggagtggggc tgtcggaaga 600 aggagggtga gcacctggga gagtttgggg atcttcatcc cactcactct gtgtcgtact 660 ttcatagtct taccgatctg tgtctgtgac ttgactcctg gagcctgcga tataaattgc 720 tgctgcgaca gggactgcta tcttctccat ccgaggacag ttttctcctt ctgccttcca 780 ggcagcgtaa ggtcttcaag ctgggtttgt gtagacaact ctgttatctt caggagtaat 840 tccccgtttc cttcaagagt tttcatggat tctaatggaa tcaggcagtt ttgtgtccat 900 gtgaacaact caaacttaaa ctatttccag aagcttcaaa aggtcaatgc aaccaacttc 960 caggccctgg ctgcagagtt tggaggcgaa tcattcactt caacattcca aactcaatca 1020 ccaccatctt tttacagggc tggggacccc attcttactt acttccccaa gtggtctgta 1080 ataagcttgc tgagacaacc tgcaggagtt ggagctgggg gactctgtgc tgaaagcaat 1140 cctgcaggtt tcctagagag taaaagtaca acttgcactc gttttttcaa gaacctggct 1200 agtagctgta ccttggattc agccctcaat gctgcctctt actataactt cacagtctta 1260 aaggttccaa gaagcatgac tgatccacag aatatggagg tcacctatga gatagagacc 1320 aatgggactt ttggaatcca gaaagtttct gtcagtttgg gacaaaccaa cctgactgtt 1380 gagccaggcg cttccttaca gcaacacttc atccttcgct tcagggcttt tcaacagagc 1440 acagctgctt ctctcaccag tcctagaagt gggaatcctg gctatatagt tgggaagcca 1500 ctcttggctc tgactgatga tataagttac tcaatgaccc tcttacagag ccagggtaat 1560 ggaagttgct ctgttaaaag acatgaagtg cagtttggag tgaatgcaat atctggatgc 1620 aagctcaggt tgaagaaggc agactgcagc cacttgcagc aggagattta tcagactctt 1680 catggaaggc ccagaccaga gtatgttgcc atctttggta atgctgaccc agcccagaaa 1740 ggagggtgga ccaggatcct caacaggcac tgcagcattt cagctataaa ctgtacttcc 1800 tgctgtctca taccagtttc cctggagatc caggtattgt gggcatatgt aggtctcctg 1860 tccaacccgc aagctcatgt atcaggagtt cgattcctat accagtgcca gtctatacag 1920 gattctcagc aagttacaga agtatctttg acaactcttg tgaactttgt ggacattacc 1980 cagaagccac agcctccaag gggccaaccc aaaatggact ggaaatggcc attcgacttc 2040 tttcccttca aagtggcatt cagcagagga gtattctctc aaaaatgctc agtctctccc 2100 atccttatcc tgtgcctctt actacttgga gttctcaacc tagagactat gtgaagaaaa 2160 gaaaataatc agatttcagt tttccctatg agaaactctg aggcagccac ttatcttggc 2220 taaatagaac ctcacctgct catgaccaga gagcatttag gataatagag gacctaactg 2280 aaggaatcct tgtatatgaa aggagttatt ttagaaaagc aataaaaata ttttattcat 2340 catagctctc tgctttgggc tctgcaggcc accagataca catgaggccc ctacttctca 2400 agctgggaag gccaagagcc ttccttcagc ctttctggtt atgttacacc tagctgaatg 2460 tttacaaggt ctggatccat cagccctcag gcacagttgg gccaagcaga aagagagaaa 2520 cacttctgct gtcaccttga atgaactcag gaatagcttc cctctggact gtagaggagc 2580 taactgtttg gaacagaaaa ctgctggctg ttgattttgt ctggttcctt tgccaacatc 2640 tgggcacacc ctttgcccag acacgagtgg ggaaagcagt tctttctcct cagtttccaa 2700 agtaaatggg gaatcccagc tttcttttct actagcaaat gaccctacca tttatttctg 2760 cctttttctt ccgttcattg tgaggaaaaa taaaactggt tgagaaaaaa aaaaaaaaaa 2820 aaaaa 2825 <210> 2 <211> 1382 <212> DNA <213> Homo sapiens <400> 2 aagaaaggca cttccggtgt ctgttgccag gcgcgggccc agtgggccgt aggggcgaca 60 ttgttgccgt cgtctttccc cccccagtcc cggggatgga gatgtcggga ctcagctttt 120 cagagatgga gggctgccgt aacctacttg gcctactgga caacgacgag atcatggccc 180 tatgcgacac cgtcaccaac cgcctggtgc agcctcagga ccgccaagat gctgttcatg 240 caatattagc atacagtcaa agtgcagaag aacttctgag gcgtagaaaa gtccaccgag 300 aagttatatt taagtacttg gcaacacagg ggattgttat acctccagct actgaaaaac 360 acaatcttat tcagcatgca aaagattact ggcaaaagca accacaactg aaattgaagg 420 aaacgccaga gccagttaca aagacagagg acatccacct atttcaacag caggtgaaag 480 aagataaaaa agctgaaaaa gttgattttc gtcgcctagg agaagaattc tgtcattggt 540 tctttggact tcttaattct cagaatcctt ttctaggacc acctcaagat gaatggggac 600 cacagcactt ctggcatgat gtgaagctta ggttttatta caacacatca gaacaaaatg 660 ttatggacta ccatggagca gaaatcgtga gccttcgttt gctgtcacta gtaaaagaag 720 aatttctttt tctcagcccc aacctagatt cacatggact gaaatgtgca tcttctcctc 780 atgggctggt tatggttgga gttgctggga ctgtccatcg aggaaacact tgtttgggca 840 tttttgaaca aatttttgga ctcatccgct gcccttttgt ggagaatact tggaaaatca 900 aatttatcaa cctgaaaatt atgggagaga gttcccttgc tcctggaaca ttaccgaaac 960 catctgttaa atttgaacaa agtgatctag aggcctttta taatgtaatc actgtatgtg 1020 gtaccaatga agtacgacat aatgtaaagc aggcttcgga tagtggaact ggggaccaag 1080 tttgaggtag tggaaatgag acattgctga acaaaagaga actgggttta cctgaccctc 1140 taaagcgcta agtactgtca gcctgaaaaa aatcttctat acagaaactc ttccaaatac 1200 tatatcagta atgtctgaat gatttcagat gtgaaaattg acatatttta gttgaaatac 1260 ctttctggac tacagactta catatcatgt gaatacttac ctatttctac ccgagttgca 1320 gcaagtattc tgaaagctta atgcaaataa atcccacttt agatcctaaa aaaaaaaaaa 1380 aa 1382 <210> 3 <211> 3101 <212> DNA <213> Homo sapiens <400> 3 acactctggc ccggttctcg gtggtgcggg agcgggcggg agcagcggcc gctctggtcg 60 gcggacgtgc tgccgagtag tcccggaagc gaagcagcga tggcggagag tccgactgag 120 gaggcggcaa cggcgggcgc cggggcggcg ggccccgggg cgagcagcgt tgctggtgtt 180 gttggcgtta gcggcagcgg cggcgggttc gggccgcctt tcctgccgga tgtgtgggcg 240 gcggcggcgg cagcgggcgg ggccgggggc ccggggagcg gcctggctcc gctgcccggg 300 ctcccgccct cagccgctgc ccacggggcc gcgctgctta gccactggga ccccacgctc 360 agctccgact gggacggcga gcgcaccgcg ccgcagtgtc tactccggat caagcgggat 420 atcatgtcca tttataagga gcctcctcca ggaatgttcg ttgtacctga tactgttgac 480 atgactaaga ttcatgcatt gatcacaggc ccatttgaca ctccttatga agggggtttc 540 ttcctgttcg tgtttcggtg tccgcccgac tatcccatcc acccacctcg ggtcaaactg 600 atgacaacgg gcaataacac agtgaggttt aaccccaact tctaccgcaa tgggaaagtc 660 tgcttgagta ttctaggtac atggactgga cctgcctgga gcccagccca gagcatctcc 720 tcagtgctca tctctatcca gtccctgatg actgagaacc cctatcacaa tgagcccggc 780 tttgaacagg agagacatcc aggagacagc aaaaactata atgaatgtat ccggcacgag 840 accatcagag ttgcagtctg tgacatgatg gaaggaaagt gtccctgtcc tgaaccccta 900 cgaggggtga tggagaagtc ctttctggag tattacgact tctacgaggt ggcctgcaaa 960 gatcgcctgc accttcaagg ccaaactatg caggaccctt ttggagagaa gcggggccac 1020 tttgactacc agtccctctt gatgcgcctg ggactgatac gtcagaaagt gctggagagg 1080 ctccataatg agaatgcaga aatggactct gatagcagtt catctgggac agagacagac 1140 cttcatggga gcctgagggt ttagaccctg ctcccatctc cccttccccc actcaagagt 1200 cccagcagaa tcccttcccc ccaccccagg gatggagagg cactgtgtat ctccctccag 1260 actcgaagtc atcctgcaag atggcaagaa ccaagcaagc tccgatccca gggtgtggga 1320 gtgggggcct gttcccggtc tgacctcctt ggcactggag catctggggc ttcgttcatc 1380 cattcatccc gtatcagggg ccaaggtacc tttacaggag cacctagagc gagggccttt 1440 ggcaaaaaca aaacaaccaa cacacctctc cacagggcca gctccttagg gataagtgga 1500 agatggaaat tgcaattcca agagggagtg tgcccaaatg atttatgggg atacctggaa 1560 gggagcttgg ggtgggggct gtctgtgaca cttaagcagt ctgggtggtt gtctatttgt 1620 ctgtcttcag tcttgaagca gggcttccca atgccctttt cctccctgcc ttccttcccc 1680 cattatttcc cacaggccag cataattttg tttttcctaa tttatagtca ctgttctaga 1740 cagaccaaag agaaggaaca gtggtggagt ctaggctgct gatcagtaag ctttacctag 1800 cacctgagca cctttctccc ctcccctctt tcctcaccct tttctagatg taagacagaa 1860 agtaaatgtg actgggactt aaccaaggtc ttggtaaagc ctgcatggca ccgtaagaag 1920 ctgaaaatac tgtttgttcc cgcaatcact gatttgaaaa gttcccaaca caggcagctg 1980 ctgtgtatat gggattagag ccactacata gaatagtctc ttacagattt tcataaatac 2040 tagtcacaat aagggtattt ttcttggggg tggagtaagg gggagactga tgctagtcct 2100 tgttgtattt tgttgggctg tccttgtgta ttttcacccc agcctgtagt cctcctcact 2160 tcaaccccag ggatttttgg ggagcaaggg tagccaatgg cagagggggt tggggctggg 2220 actctggagg ctcctcccct tctttctctt ccttccgcct cccccgtgcc cccagctgct 2280 cttgtcactg tctctgatgg gtatttgcct ggctttgttg cttctctatc tgtatttagc 2340 tgcagtgatc ctttagctgg ttggctcaga aaaaaaaaaa tgtgctttag gtgccctgta 2400 atcctgggca tcaagggaat ccatccttcc cctttttgat atgttctccc cgtacttcca 2460 gatttattgt tatggctccc agtgggtatt ggcgattctt gtgatgcagg gcctcagtca 2520 gtgtccagcc atgcataagg gagaggatag tgtgtacctg ccctgccctc tgctatgaag 2580 gtctctgcct tgtggatcat gggactcccc ttggaggatc tgtgcaaagg ggggctgggc 2640 acaaaggaga atgtcctatt tgggagggca ggaagcaaag gaactggaca gggattggtg 2700 ggcttgggga acggaagttt atcttggata cccttgaaga ggctgggtct cttcacatga 2760 agatcgaaaa gggaccctgc ttccaatttc cctcttccat tcctcgagct actccagggc 2820 ttagaagaat gctcttggtc tgtgggtcca gtgttgtctg tcatccattt aagtgttccc 2880 actttcaagt gacaatcctc tccttggccc tgccataggg cagagcatgt ctggcatagc 2940 agcctgactt ttatgcccta atcttgagtt gaggaaatat atgcacagga gtcaaagaga 3000 tgtctttata tctgactgta tataaatgaa gtttttttgt tttttttgtt ttcctttttg 3060 gtgcaataaa gtttgttttg gcagaaggag aaaaaaaaaa a 3101 <210> 4 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for C10orf61 <400> 4 gcgaattcat ggattctaat gga 23 <210> 5 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for C10orf61 <400> 5 gcggtaccca tagtctctag gtt 23 <210> 6 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for MGC26717 <400> 6 gcctcgagat gtcgggactc agc 23 <210> 7 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for MGC26717 <400> 7 gcggatccaa cttggtcccc agt 23 <210> 8 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for FLJ13855 <400> 8 cgctcgagat gtccatttat aag 23 <210> 9 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for FLJ13855 <400> 9 cgggatccaa ccctcaggct ccc 23  

Claims (9)

delete An apoptosis inducing agent comprising a polypeptide expressed from the polynucleotide represented by SEQ ID NO: 1. The method of claim 2, wherein the apoptosis inducing agent is cancer, autoimmune disease, viral infection, endocrine disease, organ hyperplasia, restenosis after angioplasty, recurrence after cancer resection surgery, organ transplant rejection, immune failure, neurodegenerative disease, ischemic heart disease Cell death for the treatment of diseases selected from the group consisting of radiation disorders, ultraviolet radiation disorders, addictive disorders, nutritional disorders, inflammatory diseases, ischemic neuropathy, diabetic neuropathy, vascular disease, respiratory disease and cartilage disease Inducers. delete delete delete delete delete delete

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