CN101273132A

CN101273132A - Compositions and methods for treating breast cancer

Info

Publication number: CN101273132A
Application number: CNA2006800353952A
Authority: CN
Inventors: 中村佑辅; 片桐丰雅; 中鹤修一
Original assignee: Oncotherapy Science Inc
Current assignee: Oncotherapy Science Inc
Priority date: 2005-07-25
Filing date: 2006-07-21
Publication date: 2008-09-24
Also published as: EP1920056A1; WO2007013574A1; JP2009502113A

Abstract

The invention features a method for inhibiting growth of a cancer cell by contacting the cell with a composition of a siRNA that inhibits expression of A2254 or A5623. Methods of treating cancer are also within the invention. The invention also features products, including nucleic acid sequences and vectors as well as to compositions comprising them, useful in the provided methods. The invention also provides a method for inhibiting growth of a tumor cell, for example a breast cancer cell, as well as a method for identifying compounds that reduce or prevent the binding between A5623 and A2254. Moreover, the present invention relates to methods of treatment or prevention of cancer, particularly breast cancer, comprising administering a compound that inhibits binding between A2254 and A5623 as well as to compositions comprising such binding inhibitors.

Description

The composition and the method that are used for the treatment of mammary cancer

Present patent application requires the U.S. Provisional Patent Application serial number No.60/702 of submission on July 25th, 2005,446 rights and interests, and this paper quotes its full content as a reference.

Technical field

The present invention relates to bio-science field, more clearly, relate to the cancer research field.Especially, the present invention relates to comprise the composition of the nucleic acid that can suppress A2254 or the expression of A5623 encoding gene.In some embodiments, this compound is corresponding to the siRNA (siRNA) from the subsequence of these genes.The invention still further relates to and be used to identify method and the test kit that can be used for treating with the compound of preventing cancer.And, the invention still further relates to the particularly method of mammary cancer of treatment or preventing cancer, comprise using and suppress bonded compound between A2254 and the A5623, the invention still further relates to the composition that comprises these binding inhibitors.

Background technology

Mammary cancer (BRC) is a kind of disease of complexity, it is characterized in that in a large amount of genes the accumulation (Nathanson KL., et al., Nat Med, 7 (5): 552-6,2001) of heredity and epigenetic change.The multistep model that mammary cancer takes place, be that normal cell is via atypia ductal hyperplasia, ductal carcinoma in situ (DCIS) and the conversion in wetting property (invasive) duct carcinoma stages such as (IDC), similar to the oncogenesis stage in its hetero-organization, cutting handset system is still unknown really but advance mammary cancer.Obviously, cause the branching factor that former hair-cream gland cancer takes place, develops and shifts to become the development goal that prevents and treat the more good tool of this class disease probably.

The gene expression pattern that obtains by the cDNA microarray analysis can provide quite a large amount of information for the essence that characterizes every kind of cancer; The application prospect of these information is that it passes through potentiality (Petricoin, E.F., 3rd, et al., Nat Genet, 32 Suppl:474-479,2002 that the development of new medicine improves neoplastic disease clinical treatment strategy; Bange J., et al., Nat Med, 7 (5): 548-52,2001).Be with this purpose, we have analyzed the expression pattern of 77 routine breast tumor, comprise by Laser Microbeam micro-dissection (LMM) and represent 27,12 DCIS and 69 IDC (Nishidate T of the combined method purifying of the cDNA microarray of 648 genes, et al., Int J Oncol.2004 Oct; 25 (4) 797-819).The important information that the data of these experiments not only can provide relevant breast tumor to take place is for identifying that its product can be used as diagnostic markers and/or also has very much value as the candidate gene of the molecular target of treatment mammary cancer.

Disclosure of the Invention

The present invention is based on following discovery, i.e. A2254 and the A5623 remarkable high expression level of crossing in breast cancer cell, and the expression of inhibition A2254 or A5623 can effectively suppress multiple cancer cells, comprises the cell growth of the cancer cells that BRC relates to.The invention that the application describes promptly is to be based in part on this discovery.

In order to separate the recruit's target that is used for the treatment of mammary cancer, the inventor is used in combination the full genomic expression pattern that mammary cancer case before 77 climacteric has been studied in cDNA array and Laser Microbeam micro-dissection.In the gene that raises, the inventor has identified to be censured to the A2254 of kinesin family member 2C (KIF2C) and censured and has been the A5623 of division of cytoplasm albumen regulatory factor (PRC1) 1, being expressed in the mammary cancer case that the inventor can obtain expression data of they has rise in 44 examples in 60 examples and 37 examples in 58 examples respectively.Sxemiquantitative RT-PCR subsequently and Northern engram analysis confirm, comprises that with normal human subject tissue the breast duct cell compares with normal breast, and A2254 and A5623 be remarkable mistake high expression level in clinical mammary cancer sample and breast cancer cell line.Immunocytochemical stain shows that external source A2254 and A5623 are positioned in the tenuigenin and/or tenuigenin device (cytoplasmic apparatus) of COS7 cell.Especially, external source A5623 is found in the median fiber network of COS7 cell.

Handle breast cancer cell with siRNA (siRNAs) and effectively suppressed the expression of A2254 and A5623, and suppress cell/tumor growth of breast cancer cell, T47D and HBC5 respectively.In addition, the inventor finds, A2254[and A5623] the instantaneous high expression level of crossing can promote cut to measure the migration of cell in (scratch assay) in the HT1080 cell, shows that these genes all bring into play keying action (data not shown) in cell migration.These discoveries show, breast tumor take place or shift in may relate to A2254 and A5623 crosses high expression level, and may be specific treatment patient with breast cancer's strategy likely.

And the inventor proves that A5623 can combine with A2254 by the distolateral part of its N, and during anaphase of cell division and division of cytoplasm, A5623 and A2254 are positioned intermediate altogether.Immunocytochemical stain shows that in breast cancer cell, external source A2254 is positioned the mitotic spindle utmost point in early stage, and the commitment in mid-term and later stage is positioned whole mitotic spindle then.These discoveries show that the mixture of A2254 and A5623 may participate in the breast tumor generation, and may be specific therapy patient with breast cancer's strategies likely.

A2254 is censured the 2C for the kinesin family member, it has two kinds of different variants of transcribing, constitute by 21 and 20 exons respectively, corresponding to A2254V1 (GenBank accession number NO:AB264115, SEQ ID NO:35,36) and A2254V2 (GenBank accession number NO:AY026505, SEQ ID NO:37,38) (Fig. 3 A, last drawing).Therefore, in the present invention, A2254 comprises A2254V1 and A2254V2.The exons 1 of V1 variant and 2 is respectively 185bp and 94bp, and the V2 variant does not have the

exons

1 and 2 of V1, and has a new exon that is made of 346bp as exons 1.Last exon (extron 20) of V2 variant is than the short 537bp of 3 ' end of last exon (exon 2 1) of V1 variant.The full length cDNA sequence of A2254V1 and A2254V2 variant contains 2886 and 2401 Nucleotide respectively.The ORF of these variants starts from each exons 1.Finally, V1 and V2 transcript 725 and 671 amino acid of encoding respectively.

A5623 is censured is division of cytoplasm albumen regulatory factor 1, and it also has three kinds of different variants of transcribing, and is made of 15,14 and 14 exons respectively, corresponding to A5623V1 (Genbank accession number NO:NM_003981; SEQ ID NO:39,40), A5623V2 (Genbank accession number NO:NM_199413; SEQ ID NO:41,42) and A5623V3 (Genbank accession number NO:NM_199414; SEQ ID NO:43,44) (Fig. 3 B, last drawing).Therefore, A5623 comprises A5623V1, A5623V2 and A5623V3 among the present invention.The exons 13 and 14 of V1 has selectable variation, and all the other exons are all identical in all variants.The V2 variant does not have the exons 14 of V1, in the end produces new early stopping codon in an exon.The exons 14 of V3 is deleted fully, and the exons 13 of V3 also in the end produces a new early stopping codon in an exon at the exons 13 short 77bp of 3 ' end than V1.The full length cDNA sequence of A5623V1, A5623V2 and A5623V3 variant is made of 3128,3091 and 3011 Nucleotide respectively.The ORF of these variants starts from each exons 1.Finally, V1, V2 and V3 transcript 620,606 and 566 amino acid of encoding respectively.

The invention provides cytostatic method.Such method is arranged in method provided herein, and they comprise makes cell contact with the composition that comprises the siRNA (siRNA) that suppresses A2254 or A5623 expression.The present invention also provides the method that is used for suppressing experimenter's growth of tumour cell.These methods comprise to the experimenter uses the composition that contains with the siRNA (siRNA) of hybridizing from the sequence specific of A2254 or A5623.Another aspect of the present invention provides the method that is used for suppressing biological sample cell A2254 or A5623 genetic expression.Described expression of gene can be suppressed by double-stranded ribonucleotide (RNA) molecule of introducing the amount be enough to suppress A2254 or A5623 genetic expression in cell.Another aspect of the present invention comprises the product of nucleotide sequence and carrier, and the composition that comprises them, and they are useful on, for example, and method provided herein.Such siRNA molecule is arranged in product provided herein, and they suppress the character of these genetic expressions when having in being introduced in the cell of expressing A2254 or A5623 gene.Such molecule is arranged in this quasi-molecule, and they comprise sense strand and antisense strand, and wherein sense strand comprises the ribonucleoside acid sequence corresponding with A2254 or A5623 target sequence, and wherein this antisense strand comprises and described sense strand complementary ribonucleoside acid sequence.The have justice and the antisense strand of molecule are hybridized the formation duplex molecule each other.

The further aspect of the present invention is the method that screening can be used for the compound of treatment or preventing cancer, particularly mammary cancer, and described method comprises the steps:

(a) in the presence of test compounds, the A2254-that comprises the A5623 polypeptide is contacted in conjunction with the polypeptide in territory with the A5623-that comprises the A2254 polypeptide in conjunction with the polypeptide in territory;

(b) combination between the detection polypeptide; With

(c) select to suppress bonded test compounds between polypeptide.

The A2254-that the technician can determine the A5623 polypeptide by the method that is used for determining the protein-interacting territory known in the art in conjunction with the A5623-of territory or A2254 polypeptide in conjunction with the territory.For example, the interaction territory can be determined by interaction territory mapping (interaction domain mapping).For example, express different piece and the application examples such as the yeast two-hybrid system screening interaction of A2254 or A5623 polypeptide respectively.By the interaction that yeast two-hybrid system is identified, can as interacting on immunoprecipitation and/or the post, be proved conclusively by application examples such as biochemical measurement.Meaning without limits that identify in the above-mentioned territory that is used to interact and/or the method for mapping is determined known in the artly is used to interact that identify in the territory and the method for mapping because the technician can easily use other.

Preferably, that uses in the screening method of bonded inhibitor between A5623 polypeptide and A2254 polypeptide contains the polypeptide that A2254-combines the territory, comprise the A5623 polypeptide, for example A5623V1 (SEQ IDNO:40), A5623V2 (SEQ ID NO:42) or A5623V3 (SEQ ID NO:44).

In aspect another is preferred, that uses in the screening method of binding inhibitors between A5623 polypeptide and A2254 polypeptide contains the polypeptide that A5623-combines the territory, comprise the A2254 polypeptide, for example A2254V1 (SEQ ID NO:36) or A2254V1 (SEQ ID NO:38).

Another aspect of the present invention is the test kit that is used to screen the compound of treatment or Breast Cancer Prevention, and wherein this test kit comprises:

(a) comprise the polypeptide of the A2254-of A5623 polypeptide in conjunction with the territory;

(b) comprise the polypeptide of the A5623-of A2254 polypeptide in conjunction with the territory; With

(c) be used to detect the reagent of polypeptide interphase interaction.

Preferably, the described A2254-of containing comprises the A5623 polypeptide in conjunction with the polypeptide in territory, and the perhaps described A5623-of containing comprises the A2254 polypeptide in conjunction with the polypeptide in territory.

In one aspect of the method, the present invention relates to be used for the treatment of or prevent the method for mammary cancer among the experimenter.Wherein this method comprises the step of bonded compound between the suppressed A5623 polypeptide of drug administration significant quantity and the A2254 polypeptide.

And, the invention still further relates to and be used for the treatment of or the composition of Breast Cancer Prevention, wherein said composition comprises bonded compound and medicine acceptable carrier between the inhibition A5623 polypeptide of medicine effective quantity and the A2254 polypeptide.

As used herein, term " organism " is meant any live body by at least one cellularity.The organism that lives can simply arrive for example single eukaryotic cells, and is perhaps complicated to Mammals, comprises the mankind.

As used herein, term " biological sample " is meant a part (body fluid for example of whole organism or its tissue, cell or integral part, include but are not limited to blood, mucus, lymph liquid, synovia, cerebrospinal fluid, saliva, amniotic fluid, amnion bleeding of the umbilicus, urine, vaginal secretion and seminal fluid)." biological sample " further refers to from homogenate, lysate, extract, cell culture or the tissue culture of the part preparation of whole organism or its cell, tissue or integral part, perhaps its fraction or part.At last, " biological sample " refers to contain cellular component, the medium of protein or nucleic acid molecule for example, nutrient broth that the biological example body is bred therein or gel.

The present invention is a feature with cytostatic method.Make the composition contact inhibition cell growth of cell and A2254 or A5623 siRNA (siRNA).Cell further contacts with the transfection toughener.Cell is in external, body or exsomatize and provide.The experimenter is a Mammals, for example people, inhuman primate, mouse, rat, dog, cat, horse or ox.Cell is the breast duct cell.Perhaps cell is tumour cell (cancer cells just), for example cell carcinoma (carcinoma) cell or adenocarcinoma cell.For example, cell is a breast cancer cell.The meaning of " cell growth inhibiting " is processed cell and the untreated cell lower or viability decline of specific growth rate mutually.The cell growth is measured by proliferation assay known in the art.

The meaning of term " siRNA " is the double stranded rna molecule that stops the said target mrna translation.Use comprises that with the standard technique that siRNA is incorporated into cell wherein DNA is the technology of the template of transcribe rna.SiRNA includes adopted A2254 or A5623 nucleotide sequence, antisense A2254 or A5623 nucleotide sequence, perhaps both.SiRNA can comprise two complementary molecules, perhaps can be fabricated make single transcript have simultaneously from target gene justice and complementary antisense sequences arranged, hair clip for example, hair clip causes the generation of microRNA (miRNA) in some embodiments.

SiRNA produces minimizing with A2254 or the A5623 that combining of A2254 or A5623 transcript causes cell in the target cell.The length of oligonucleotide is at least about 10 Nucleotide, and can be the same long with naturally occurring A2254 or A5623 transcript.Preferably, the length of oligonucleotide is about 25 Nucleotide of about 19-.Most preferably, the length of oligonucleotide is less than about 75, about 50 or about 25 Nucleotide.Suppress the interior A2254 of mammalian cell or the A2254 of A5623 expression or the example of A5623 siRNA oligonucleotide and comprise the oligonucleotide that contains target sequence respectively, wherein said target sequence for example is respectively the Nucleotide of SEQ ID NO:21 or 25, or 29 or 33 Nucleotide.

It is known (for example see, U.S. Patent No. 6,506,559, this paper quotes its full content as a reference) that design has the method that suppresses the double-stranded RNA of genetic expression ability in the target cell.For example, can be from the Ambion site (http://www.ambion.co.jp/techlib/tb/tb_506.html) obtain to be used to design the computer program of siRNA.This computer program can obtain from Ambion company, is used for siRNA synthetic nucleotide sequence according to following Scheme Choice.

Select the siRNA target spot

1. the AUG initiator codon from transcript begins scan A A dinucleotide sequence downstream.Write down 19 adjacent Nucleotide of the appearance of each AA and 3 ' side thereof as potential siRNA target spot.Tuschl etc. do not recommend zone (within 75 bases) the design siRNA at 5 ' and 3 ' non-translational region (UTR) and contiguous initiator codon in Targeted mRNA degradation by double-stranded RNA in vitro.Genes Dev13 (24): 3191-7 (1999), regulate combination of proteins site because may more be rich in here.Conjugated protein and/or the translation initiation complex of UTR may disturb the combination of siRNA endonuclease enzyme complex.

2. potential target spot and suitable genome database (people, mouse, rat etc.) are compared, get rid of outside considering with the remarkable homologous target sequence of other encoding sequences any.BLAST (Altschul SF, et al., Nucleic Acids Res.1997 are used in suggestion; 25:3389-402; J Mol Biol.1990; 215:403-10.), it is found in the NCBI server: Www.ncbi.nlm.nih.gov/BLAST/

3. select qualified target sequence to be used to synthesize.Usually on the length of gene to be assessed, select several target sequences.

The present invention also comprises the isolated nucleic acid molecule of the nucleotide sequence that contains target sequence, described target sequence is SEQ ID NO:21,25,29 and 33 Nucleotide for example, perhaps with the nucleic acid molecule of the nucleic acid array complementation of SEQ ID NO:21,25,29 and 33 Nucleotide.As used herein, " isolating nucleic acid " is the nucleic acid that shifts out from its former environment (for example, if exist naturally, i.e. physical environment), thereby artificially (synthetically) changed its state of nature.In the present invention, isolating nucleic acid comprises DNA, RNA and derivative thereof.When isolating nucleic acid is the RNA or derivatives thereof, should in nucleotide sequence, base " t " be replaced with " u ".As used herein, term " complementation " is meant Watson-Crick or the Hoogsteen base pairing between the nucleic acid molecule nucleotide units, and term " combination " is meant physics or chemical interaction between two nucleic acid or compound or associated nucleic acid or compound or their combination.The complementary nucleic acid sequence is hybridized under appropriate condition, forms the stable duplex that contains seldom or do not have mispairing.For the purposes of the present invention, two sequences with 5 or still less mispairing are considered to complementary.And the sense strand of separating nucleotide of the present invention and antisense strand can form double chain nucleotide or hairpin ring structure by hybridization.In preferred embodiments, the mispairing number of this duplex in per 10 couplings is no more than 1.In the complete complementary particularly preferred embodiment of duplex chain, this duplex does not have mispairing.For A2254 or A5623, the length of nucleic acid molecule is respectively less than 2886 or 3128 Nucleotide.For example, the length of nucleic acid molecule is less than about 500, about 200 or about 75 Nucleotide.The present invention also comprises the carrier that contains one or more nucleic acid described herein, and the cell that contains this carrier.Isolating nucleic acid of the present invention can be used for the siRNA of anti-A2254 or A5623, the DNA of this siRNA that perhaps encodes.When nucleic acid was used for siRNA or its coding DNA, sense strand preferably was longer than about 19 Nucleotide, more preferably is longer than 21 Nucleotide.

The present invention is based in part on following discovery, the gene of promptly encode A2254 or A5623 in mammary cancer (BRC) than in non-carcinous mammary tissue, crossing high expression level.The cDNA length of A2254V1 and A2254V2 is respectively 2886 and 2401 Nucleotide.The cDNA length of A5623V1, A5623V2 and A5623V3 is respectively 3128,3091 and 3011 Nucleotide.The nucleic acid of A2254V1 and A2254V2 and peptide sequence respectively as SEQ ID NO:35 and 36, and SEQ ID NO:37 and 38 shown in.The nucleic acid of A5623V1, A5623V2 and A5623V3 and peptide sequence respectively as SEQ ID NO:39 and 40, SEQ ID NO:41 and 42 and SEQ ID NO:43 and 44 shown in.Sequence data can also obtain from following accession number.

A2254：AY026505

A5623：NM_003981、NM_199413、NM_199414

Cytostatic method

The present invention relates to cell growth inhibiting, i.e. growth of cancer cells by the expression that suppresses A2254 or A5623.Perhaps, the present invention relates to by suppressing combining and cell growth inhibiting of A5623 polypeptide and A2254 polypeptide, it is growth of cancer cells, the particularly growth of mammary cancer, because finding two peptide species can interact, and think their interaction in oncogenesis, particularly mammary cancer is brought into play crucial effects in taking place.Therefore, the present invention relates to treat or prevent the means and the method for mammary cancer among the experimenter with combining of A2254 polypeptide by suppressing the A5623 polypeptide.And the present invention has imagined the binding inhibitors of a kind of A5623 polypeptide and A2254 polypeptide, is used as treatment or preventing cancer, particularly the medicine of treatment or Breast Cancer Prevention.

The expression of A2254 or A5623 is suppressed by the siRNA (siRNA) of for example selectively targeted A2254 or A5623 gene.A2254 or A5623 target comprise SEQ ID NO:21,25 for example, 29 and 33 Nucleotide.

In the nonmammalian cell, double-stranded RNA (dsRNA) shows that genetic expression is had strong and specific reticent effect, and this is called RNA and disturbs (RNAi) (Sharp PA.Genes Dev.1999; 13 (2): 139-41.).DsRNA is processed into the dsRNA of 20-23 Nucleotide by a kind of enzyme of the RNA of containing enzyme III motif, is called siRNA (siRNA).SiRNA is with the selectively targeted complementary mRNA of multicomponent nucleic acid enzymes mixture (Hammond SM et al., Nature.2000; 404 (6775): 293-6; Hannon GJ.Nature.2002; 418 (6894): 244-51.).In mammalian cell, constitute, have 19 complementary nucleotides and 3 ' end incomplementarity thymidine or the dimeric siRNA of uridine by 20 or 21 aggressiveness dsRNA and show that having gene specific strikes low (knockdown) effect, and the overall situation that can not cause genetic expression changes (Elbashir SM et al., Nature.2001; 411 (6836): 494-8.).In addition, the plasmid that contains small nuclear rna (snRNA) U6 or polymerase III H1-RNA promotor can effectively produce this short rna and raise type III family rna plymerase iii, thereby can suppress its said target mrna (Miyagishi M.et al., Nat Biotechnol.2002 by composing type; 20 (5): 497-500.; Brummelkamp TR, et al., Science.296 (5567): 550-3,2002.).

By making the growth of composition contact inhibition cell of cell and the siRNA that contains A2254 or A5623.Further cell is contacted with transfection reagent.Suitable transfection reagent is known in this area.The meaning of " cell growth inhibiting " is, compares with the cell that is not exposed to composition, and the rate of propagation of cell is lower, and viability descends.The cell growth is measured with methods known in the art, for example the MTT cell proliferating determining.

The siRNA of A2254 or A5623 points to the single target of A2254 or A5623 gene order.Perhaps siRNA points to a plurality of targets of A2254 or A5623 gene order.For example, composition contains 2,3,4 or 5 or the A2254 of more a plurality of target sequences or the siRNA of A5623 that points to A2254 or A5623." A2254 or A5623 target sequence " is meant the identical nucleotide sequence of a part with A2254 or A5623 gene.Target sequence can comprise 5 ' untranslated (UT) district, open reading-frame (ORF) (ORF) or the 3 ' non-translational region of human A2254 or A5623 gene.Perhaps siRNA is upstream or the downstream regulon complementary nucleotide sequence with A2254 or A5623 genetic expression.The example of upstream and downstream regulon comprise in conjunction with the transcription factor of A2254 or A5623 gene promoter, with A2254 or interactional kinases of A5623 polypeptide or Phosphoric acid esterase, A2254 or A5623 promotor or enhanser.With the A2254 of said target mrna hybridization or the siRNA of A5623, by combining with the mRNA transcript that is generally strand, thereby disturb translation, and then the expression of interferencing protein, reduction or inhibition are by the generation of the A2254 or the A5623 polypeptide product of A2254 or A5623 genes encoding by this.Therefore, siRNA molecule of the present invention can be identified under stringent condition Yu from the mRNA of A2254 or A5623 gene or the ability of cDNA specific hybrid by it.For the purposes of the present invention, term " hybridization " or " specific hybrid " are used to represent the ability of two nucleic acid molecule in " under the tight hybridization conditions " hybridization.Phrase " tight hybridization conditions " is meant that under this condition typically in the complex mixture of nucleic acid, nucleic acid molecule and its target sequence are hybridized and with other sequences detectable hybridization do not taken place.Stringent condition is a sequence dependent, can be different under different environment.Long more sequence is at high more temperature generation specific hybrid.In following document, can obtain the detailed guidance of nucleic acid hybridization: Tijssen, Techniques in Biochemistry andMolecular Biology--Hybridization with Nucleic Probes, " Overview of principlesof hybridization and the strategy of nucleic acid assays " (1993).Usually, for specific sequence, under the ionic strength pH that limits, stringent condition is selected specific heat melting temperature(Tm) (T _m) low about 5-10 ℃.T _mBe under equilibrium state, with the temperature (at the ionic strength that limits, pH and nuclear concentration) of 50% in the probe of target complement sequence and target sequence hybridization (because the excessive existence of target sequence, therefore at T _mThe time, 50% probe is occupied).Stringent condition can also be realized by adding destabilizing agent such as methane amide.For selectivity or specific hybrid, positive signal is 2 times of background at least, preferably 10 of background hybridization times.The example of tight hybridization conditions can be as follows: 50% methane amide, 5x SSC and 1%SDS, and 42 ℃ of incubations, perhaps 5x SSC, 1%SDS, 65 ℃ of incubations clean in 50 ℃ of 0.2x SSC and 0.1%SDS.

The length of siRNA of the present invention is less than about 500, about 200, about 100, about 50 or about 25 Nucleotide.Preferably, the length of siRNA is about 25 Nucleotide of about 19-.The exemplary nucleotide sequence that is used to produce A2254 or A5623siRNA comprises that respectively the sequence of Nucleotide of SEQ ID NO:21 or 25 or 29 or 33 is as target sequence.And, in order to improve the inhibition activity of siRNA, can add Nucleotide " u " at 3 ' end of target sequence antisense strand.The number of " u " that is added is about at least 2, about 10 of general about 2-, about 5 of preferably about 2-." u " that adds forms strand at 3 ' end of siRNA antisense strand.

Cell is any expression or the cell of crossing high expression level A2254 or A5623.Cell is an epithelial cell, for example the breast duct cell.Perhaps, cell is a tumour cell, for example cancer, gland cancer, blastoma, leukemia, myelomatosis or sarcoma.Cell is a mammary cancer.

The siRNA of A2254 or A5623 is being introduced directly in the cell in conjunction with the form of mRNA transcript.Perhaps, the DNA of the siRNA of coding A2254 or A5623 is in the carrier.

Carrier be by, for example, to allow two chains all to express the mode of (transcribing), A2254 or A5623 target sequence are cloned into the expression vector with adjusting sequence flank, that can be operatively connected that is arranged in A2254 or A5623 sequence and (Lee, N.S., et al., (2002) the Nature Biotechnology 20:500-5) that produces by dna molecular.Transcribe by first promotor (for example being positioned at the promoter sequence of cloned DNA 3 ' side) with the RNA molecule of A2254 or A5623mRNA antisense, A2254 or A5623mRNA are transcribed by second promotor (for example being positioned at 5 ' side promoter sequence of cloned DNA) for sense strand RNA molecule.There are justice and antisense strand to hybridize in vivo, produce the siRNA construct that is used for reticent A2254 or A5623 gene.That utilizes perhaps that two constructs produce the siRNA constructs has justice and an antisense strand.Clone's A2254 or A5623 codified have for example construct of hair clip of secondary structure, and wherein single transcript has adopted and the complementary antisense sequences of having from target gene simultaneously.

Can there be the ring sequence that constitutes by any nucleotide sequence between justice and the antisense sequences having, to form hairpin ring structure.Therefore, the present invention also provides has general formula 5 '-siRNA of [A]-[B]-[A ']-3 ', and wherein [A] is and corresponding ribonucleoside acid sequence from the sequence of the mRNA of A2254 or A5623 or cDNA specific hybrid.In preferred embodiments, [A] is such ribonucleoside acid sequence, and it is corresponding to the sequence that is selected from SEQ ID NO:21,25,29 and 33 Nucleotide.

[B] be the ribonucleoside acid sequence that constitutes by 3-23 Nucleotide and

[A '] be ribonucleoside acid sequence by the complementary sequence formation of [A].

Zone [A] and [A '] hybridization form the ring that is made of zone [B] then.The length of ring sequence is preferably about 23 Nucleotide of about 3-.The ring sequence can be selected (http://www.ambion.com/techlib/tb/tb_506.html) from the group that for example is made of following sequence.And the ring sequence that is made of 23 Nucleotide also provides active siRNA (Jacque, J.M.et al., (2002) Nature 418:435-8.).

CCC, CCACC or CCACACC:Jacque, J.M.et al., (2002) Nature, 418:435-8.

UUCG：Lee、N.S.et?al.，(2002)Nature?Biotechnology?20：500-5；Fruscoloni，P.et?al.，(2003)Proc.Natl.Acad.Sci.USA?100(4)：1639-44.

UUCAAGAGA：Dykxhoorn、D.M.et?al.，(2003)Nature?ReviewsMolecular?Cell?Biology?4：457-67.

For example, it is as follows to have a preferred siRNA of hairpin ring structure of the present invention.In following array structure, the ring sequence can be selected from CCC, UUCG, CCACC, CCACACC and UUCAAGAGA.Preferred ring sequence is UUCAAGAGA (being " ttcaagaga " in DNA).

GAGAAGAAGGCCCAGAACU-[B]-AGUUCUGGGCCUUCUUCUC (to target sequence SEQ ID NO:21)

CUCUAGGACUUGCAUGAUU-[B]-AAUCAUGCAAGUCCUAGAG (to target sequence SEQ ID NO:25)

GGAAAGACUCAUCAAAAGC-[B]-GCUUUUGAUGAGUCUUUCC (to target sequence SEQ ID NO:29)

GCAUAUCCGUCUGUCAGAA-[B]-UUCUGACAGACGGAUAUGC (to target sequence SEQ ID NO:33).

The adjusting sequence that is positioned at A2254 or A5623 sequence flank is identical or different, thereby their expression can be conditioned independently or be conditioned with time or space mode.For example contain in the carrier from small nuclear rna (snRNA) U6 promotor or people H1 RNA promoter for RNA polymerase III transcriptional units by A2254 or A5623 gene template are cloned into, thereby in cell, transcribe siRNA.For carrier is incorporated in the cell, can use the transfection toughener.FuGENE (RocheDiagnostices), Lipofectamine 2000 (Invitrogen), Oligofectamine (Invitrogen) and Nucleofector (Wako pure Chemical) can be used as the transfection toughener.

According to standard method, reduce the ability (for example, using cell line of mammary gland) that A2254 in the tumour cell or A5623 produce as mammary cancer (BRC) clone at the vitro test oligonucleotide with many part complementary of the mRNA oligonucleotide of A2254 or A5623.Use the specific antibody of A2254 or A5623 or other to detect strategy, detect the reduction of comparing with A2254 or A5623 gene product cultured cells when not having the candidate set compound in the cell that candidate siRNA composition contacts.For external based on cell mensuration or cell-less measurement in reduce the sequence that A2254 or A5623 produce, test the retarding effect of its cell growth subsequently.For cytostatic sequence in external mensuration, its generation reduction and growth of tumour cell of testing in rat or mouse body with A2254 or A5623 in the animal body of confirming to suffer from malignant tumour is reduced based on cell.

The method of treatment malignant tumour

Suffer from A2254 or the too high patient who is expressed as the tumour of feature of A5623 by the siRNA treatment of using A2254 or A5623.SiRNA treatment is used to suppress to suffer from or for example expression of A2254 or A5623 in patient's body of mammary cancer of dangerous generation.These patients are identified by the standard method of concrete tumor type.The diagnosis of mammary cancer (BRC) is by for example CT, MRI, ERCP, MRCP, computer tomography or ultrasonic wave.If treatment causes clinical benefit, for example A2254 or A5623 express reduction among the experimenter, and perhaps the size of tumour, morbidity (prevalence) or metastatic potential reduce, and then treatment is effective.Prophylactically applying when treatment, " effectively " is meant that treatment can postpone or stop tumour formation, or the symptom of the clinical symptom of prevention or ameliorate tumor.Validity is determined in conjunction with the known method that is used to diagnose or treats the specific tumors type.

The execution of siRNA treatment is by using siRNA to the experimenter, and it is used is standard vector and/or the gene delivery system that utilizes code book invention siRNA, for example by carrying synthetic siRNA molecule.Typically, will synthesize siRNA molecular chemistry stabilization to prevent the nucleic acid in vivo enzyme liberating.The method of the RNA molecule of preparation chemical stabilization is being known in the art.Typically, this quasi-molecule contains through the skeleton modified and Nucleotide to prevent the rnase effect.Other modifications also are possible, and for example cholesterol link coupled siRNA shows the pharmacological property (Song et al.Nature Med.9:347-51 (2003)) that improves.Suitable gene delivery system can comprise liposome, receptor-mediated delivery system or virus vector, for example simplexvirus, retrovirus, adenovirus and adeno-associated virus and other.The therapeutic nucleic acids composition is formulated in the medicine acceptable carrier.Therapeutic composition can also comprise aforesaid gene delivery system.The medicine acceptable carrier is the biocompatibility solvent that is fit to be applied to animal, for example physiological saline.The amount that the treatment significant quantity of compound is to produce medically ideal results---for example being treated A2254 or A5623 gene product generation reduction, cell growth as propagation reduction in the animal, perhaps tumor growth in vivo reduction---.

Parenteral admin for example intravenously, subcutaneous, intramuscular and intraperitoneal transport way can be used for carrying the siRNA composition of A2254 or A5623.In order to treat breast tumor, be useful to the direct infusion of abdomen artery, splenic artery or arteria hepatica communis.

Any one patient's dosage depends on multiple factor, comprises patient's stature, body surface area, age, specific nucleic acid, sex, time of administration and approach, general health situation to be administered and the other drug of using simultaneously.The intravenous administration dosage of nucleic acid is about 10 of nucleic acid molecule ⁶-10 ²²Individual copy.

Polynucleotide are by the standard method administration, for example by be expelled to tissue such as muscle or skin between the matter space, be incorporated into the recycle system or body cavity, perhaps by sucking or being blown into.Polynucleotide with the acceptable liquid vehicle of medicine for example the liquid vehicle of water-based or part water-based inject or flow to animal by other modes.Polynucleotide combine with liposome (for example positively charged ion or anionic liposome).Polynucleotide comprise by target cell expresses required genetic information, for example promotor.

The patient who perhaps suffers from tumour, particularly breast tumor can suppress between A5623 polypeptide and the A2254 polypeptide bonded compound and treated by using.Be not subject to theory, we believe that the A5623 polypeptide and the interactional inhibitor of A2254 polypeptide can change, influence each other, regulate, disturb, destroy combining of combining of A5623 polypeptide and A2254 polypeptide or A2254 polypeptide and A5623 polypeptide.Thereby, believe that two kinds of protein all no longer can resemble them and interact in cancer cells.Therefore, further believe, two kinds of protein are in oncogenesis---during particularly mammary cancer takes place, as suggested in the inventor's the observations---and the keying action of being brought into play can be blocked, thereby can realize the cancer particularly prevention or the treatment of mammary cancer.

Unless otherwise defined, the same meaning of the common understanding of those of ordinary skill institute in the field under the meaning of whole technology used herein and scientific terminology and the present invention.Although can use method and the material similar or of equal value with material in practice or test when of the present invention, hereinafter still describe suitable method and material to method disclosed herein.The full content that this paper quotes whole publications, patent application, patent and other reference mentioned herein as a reference.As contradictory, then to comprise that this specification sheets in being defined in is as the criterion.In addition, material, method and embodiment just illustrate, and without any limited significance.

Reduce or suppress the generation and the evaluation of bonded compound between A5623 and the A2254

The evidence that provides in the consideration present embodiment, one aspect of the present invention relates to the evaluation that reduces or prevent bonded test compounds between A5623 and the A2254.

Be used for determining that A5623/A2254 bonded method comprises any method that is used for determining two protein-protein interactions.Other method has hereinafter been described.These mensuration include, but are not limited to traditional method, for example crosslinked, co-immunoprecipitation and by gradient or chromatographic column copurification.In addition, can use based on the zymic genetic system and monitor protein-protein interaction, this system such as Fields and its co-worker describe (Fields and Song, Nature 340:245-6 (1989); Chien et al., Proc.Natl.Acad.Sci.USA 88,9578-82 (1991)), and as Chevray and Nathans disclosed (Proc.Natl.Acad.Sci.USA 89:5789-93 (1992)).Many transcriptional activators, for example yeast GALA is made of two physically separated adjusting territories, and one is served as DNA in conjunction with the territory, the effect of another performance transcription activating domain.Formerly the yeast expression system of describing in the publication (generally being called " two-hybrid system ") is exactly to utilize this character, and adopt two hybrid proteins, a middle target protein combines the territory with the DNA of GAL4 and merges therein, and in another, candidate's activator and activation domain merge.The reconstruction of GAL4 activity by protein-protein interaction depended in the expression of GAL1-lacZ reporter gene under the control of GALA activated promotor.The bacterium colony that contains the interaction polypeptide can be detected with the chromogenic substrate of beta-galactosidase enzymes.Complete test kit (MATCHMAKER with protein-protein interaction between two specified proteins of double cross technical evaluation ^TM) can buy from Clontech.This system can also be extended for the territory mapping of the interactional albumen of participation specific protein and locate for these vital amino-acid residues that interacts.

Although the application should be mentioned that " A5623 " or " A2254 ", should be appreciated that when the two interaction is analyzed or operated, can replace these proteinic total length copies with the bound fraction of these protein one or both of.By with the standard deletion analysis of A5623 and/or mutagenesis to identify fragment in conjunction with A2254, can easily identify A5623 fragment in conjunction with A2254.Can use similar analysis to identify the A5623 associativity fragment of A2254.

As disclosed herein, any test compounds comprises for example protein (comprising antibody), mutain (mutein), polynucleotide, nucleic acid, aptamer and polypeptide and the little organic molecule of non-peptide, all can be used as test compounds of the present invention.Test compounds can be separated from natural origin, synthetic or reorganization preparation, perhaps their arbitrary combination.

For example, peptide can save with " Solid Phase Peptide Synthesis (solid-phase peptide is synthetic) " of being write by G.Barany and R.B.Merrifield in " Peptides " second edition of E.Gross and J.Meienhoffer chief editor, Academic Press, New York, N.Y., the synthetic preparation of the solid phase technique of describing among the pp.100-118 (1980).Similarly, nucleic acid also can be used Beaucage, S.L. ， ﹠amp; Iyer, R.P. (1992) Tetrahedron, 48,2223-311; With Matthes et al., EMBO J., the solid phase technique of describing among the 3:801-5 (1984) is synthesized.

When identifying inhibiting peptide, modify peptide of the present invention with various amino acid analog things or alpha-non-natural amino acid, in vivo stable particularly useful of raising peptide.Stability can be measured with several different methods.For example, peptase and various Biomedia as human plasma and serum, have been used to stable testing.See for example Verhoef et al., Eur.J.Drug Metab Pharmacokinet.11:291-302 (1986).Other useful peptides known in the art are modified and are comprised glycosylation and acetylize.

Reorganization and chemical synthesising technology all can be used for producing test compounds of the present invention.For example, can be by being inserted into the nucleic acid that produces test compounds in the suitable carriers, it can afterwards be expressed in competent cell in transfection.Perhaps, can utilize round pcr or in suitable host, express amplification of nucleic acid (to see for example Sambrook et al., Molecular Cloning:A Laboratory Manual, 1989, Cold Spring Harbor Laboratory, New York, USA).

Peptide and protein can also be expressed with recombinant technology well known in the art, for example by transforming proper host cell with the recombinant DNA construction body, and as Morrison, J.Bact., 132:349-51 (1977); With Clark-Curtiss ﹠amp; Curtiss, Methods in Enzymology, 101:347-362 (Wu et al., eds, 1983) is described.

Anti-A5623 and anti-A2254 antibody

In aspect more of the present invention, test compounds is anti-A5623 or anti-A2254 antibody.In some embodiments, antibody is chimeric antibody, includes but are not limited to humanized antibody.In some cases, antibody embodiment of the present invention combines A5623 or A2254 on the interface of these protein and other protein bound.In some embodiments, under physiological condition, the K of these antibodies A5623 or A2254 _aBe at least about 10 ⁵Mol ^-1, 10 ⁶Mol ^-1Perhaps bigger, 10 ⁷Mol ^-1Perhaps bigger, 10 ⁸Mol ^-1Perhaps bigger or 10 ⁹Mol ^-1Perhaps bigger.These antibody can be buied from commercial source, and for example Chemicon company (Temecula Calif.) perhaps can for example use the A5623 of purifying or A2254 albumen basically, and for example people's albumen or its fragment excited generation originally as immunity.Method by given immunogen preparing mono-clonal and polyclonal antibody is being known in the art.See for example PCT/US02/07144 (WO/03/077838) about specific immunogen purifying antibody technology and authentication method, this paper quotes its content as a reference.Form affinity column with for example antibody affinity matrix and come the method for antibody purification being known in the art, and can commercially obtain (AntibodyShop, Copenhagen, Denmark).Can destroy the evaluation of A5623/A2254 bonded antibody, use the testing method of the general test compound that hereinafter describes in detail to carry out.

Conversion enzyme (converting enzymes)

The conversion enzyme can be used as test compounds of the present invention.In the context of the present invention, the conversion enzyme is that both carry out the molecular catalyst of covalency posttranslational modification to one of A5623 or A2254 or they.Conversion enzyme of the present invention will be with one or more amino-acid residues of mode covalent modification A5623 described below and/or A2254: it causes by the structure of modified protein other structure being taken place and changes, thereby perhaps changes by combining between the A5623/A2254 binding site molecule point chemistry of modified protein or structure interference A5623 and the A2254.Disturb the combination between two molecules, be meant with respect at 30 ℃, ionic strength 0.1 with there is not K between the albumen that records under the condition of washing agent _a, make in conjunction with K _aReduce at least 25%, 30%, 40%, 50%, 60%, 70% or more.Conversion enzyme example of the present invention comprises kinases, Phosphoric acid esterase, Ntn hydrolase, acetylase, Glycosylase etc.

Make up the test compounds library

Although being structured in of test compounds library is known in the art, this part still provides at the characterization test compound and has made up the library of these compounds, be used to screen the interactional effective inhibitor of A5623/A2254 extra guidance.Further guidance about library of compounds hereinafter is provided.

The molecule modeling

To the molecular structure of compound and/or treat that to suppress target molecule be the knowledge of the molecular structure of A5623 and A2254, made things convenient for the structure in test compounds library with destination properties.Prescreen is suitable for one of method of the further test compounds of assessing, is that microcomputer modelling is carried out in the interaction of test compounds and its target.In the present invention, the interactional modeling of A5623 and A2254 provides the understanding to the own details that interacts, and has pointed out and disturbed this interactional possibility strategy, comprises interactional potential molecule inhibitor.

The microcomputer modelling technology is for the visual of the three-dimensional atomic structure of selected molecule and provide with the appropriate design of the new compound of this interaction of molecules may.The three-dimensional data that typically depend on to come that make up from the x-ray crystal analysis of selected molecule or NMR imaging.Molecular dynamics needs field of force data.How computer graphics system connects target molecule for the prediction new compound, and the structure of experimental implementation compound and target molecule provides possibility with the specificity of optimizing integration.In order to predict producing tiny change in molecule one or both of is which type of to molecule-compound interaction, need molecular mechanics software and computation-intensive computer, the interface of user-friendly, the menu-drive between their usually related molecular designing programs and the user.

Above an example of the general molecule modeling of describing is to be made of Polygen Corporation, Waltham, Mass CHARMm and QUANTA program.CHARMm carries out energy minimization and molecular dynamics function.QUANTA carries out structure, graphical modeling and analysis of the molecular structure.QUANTA for the analysis of molecule interbehavior, visual, modification and interaction structure provide may.

Lot of documents has been summarized the microcomputer modelling with the interactional medicine of specified protein, Rotivinen for example, et al.Acta Pharmaceutica Fennica 97,159-166 (1988); Ripka, NewScientist 54-57 (Jun.16,1988); McKinlay and Rossmann, Annu.Rev.Pharmacol.Toxiciol.29,111-122 (1989); Perry and Davies, Prog Clin BiolRes.291:189-93 (1989); Lewis and Dean, Proc.R.Soc.Lond B Biol Sci.236,125-40 and 141-62 (1989); And, Askew, et al. are arranged, J.Am.Chem.Soc.111,1082-90 (1989) about the model acceptor of nucleic acid component.

The computer program of other screenings and pattern description chemical substance is BioDesign Inc. for example, Pasadena, Calif., Allelix Inc., Mississauga, Ontario, Canada and Hypercube Inc., Cambridge, companies such as Ontario obtain.See for example DesJarlais et al. (1988) J.Med.Chem.31:722-9; Meng et al. (1992) J.Computer Chem.13:505-24; Meng et al. (1993) Proteins 17:266-78; Shoichet et al. (1993) Science 259:1445-50.

In case identified the interactional supposition inhibitor of A5623/A2254, just can make up the variant of any number according to the chemical structure use combinatorial chemistry technique of the supposition inhibitor of having identified, as mentioned below.Can use method of the present invention to screen for final supposition inhibitor or " test compounds " library, disturb A5623/A2254 bonded test compounds in this library to identify.

Combinatorial chemistry is synthetic

The combinatorial library of test compounds can be used as the part that the rational drug of the knowledge that relates to the core texture that exists in known A5623/A2254 interaction inhibitor designs program and produces.This method makes the library keep reasonably size, is convenient to high flux screening.Perhaps can pass through the simple synthetic whole arrangements that constitute the molecule family in this storehouse and make up simply, particularly Duan polymer molecule library.The example of a kind of method in back is to be 6 libraries that amino acid whose whole peptides constitute by length.This peptide library can comprise each six amino acid series arrangement.Such library is called linear combination chemistry library.

The preparation in combinatorial chemistry library is known in those skilled in the art, and can produce by chemistry or biosynthesizing.Combinatorial chemical library includes, but are not limited to peptide library (seeing for example United States Patent (USP) 5,010,175, Furka, Int.J.Pept.Prot.Res.37:487-93 (1991) and Houghten et al., Nature354:84-6 (1991)).Also can adopt other to be used to produce the chemistry in Chemical Diversity storehouse.This class chemistry comprises, but be not limited only to: peptide (for example PCT discloses WO No. 91/19735), the peptide that is encoded (for example PCR discloses WO No. 93/20242), biological at random oligomer (for example PCT discloses WO92/00091 number), (for example United States Patent (USP) the 5th for Benzodiazepine (benzodiazepine), 288, No. 514), diversomers such as hydantoins (hydantoins), Benzodiazepines (benzodiazepines) and dipeptides (dipeptides) (DeWitt et al., Proc.Natl.Acad.Sci.USA 90:6909-13 (1993)), vinylogy (vinylogous) polypeptide (Hagihara et al., J.Amer.Chem.Soc.114:6568-70 (1992)), non-peptide peptide mimics (Hirschmann et al. with glucose skeleton, J.Amer.Chem.Soc.114:9217-8 (1992)), the similar organic synthesis of little library of compounds (Chen et al., J.Amer.Chem.Soc.116:2661 (1994)), oligomerization carbamate (oligocarbamates) (Cho et al., Science 261:1303 (1993)), and/or peptidyl phosphonate or ester (peptidylphosphonate) (Campbell et al., J.Org.Chem.59:658 (1994)), nucleic acid library (is seen Ausubel, andSambrook, all see above), for example United States Patent (USP) 5 (is seen in the peptide nucleic acid(PNA) library, 539,083), antibody library (is seen for example Vaughan et al., Nature Biotechnology, 14 (3): 309-14 (1996) and PCT/US96/10287), for example Liang et al. (is seen in the sugar library, Science, 274:1520-2 (1996) and United States Patent (USP) 5,593,853), little organic molecule library (is for example seen, Benzodiazepine, Gordon EM.CurrOpin Biotechnol.1995 Dec 1; 6 (6): 624-31.; Isoprenoid, United States Patent (USP) 5,569,588; Thiazoline ketone (thiazanones) and metathiazole quinoline ketone (metathiazanones, United States Patent (USP) 5,549,974; Pyrrolidines, United States Patent (USP) 5,525,735 and 5,519,134; Morpholino compounds, United States Patent (USP) 5,506,337; Benzodiazepine, 5,288,514 etc.).Hereinafter provide about combinatorial chemistry is synthetic and further instructed.

Phage display

Another kind method uses recombinant phage to produce the library.Use " phage method " (Scott andSmith, Science 249:386-90,1990; Cwirla, et al, Proc.Natl.Acad.Sci., 87:6378-82,1990; Devlin et al., Science, 249:404-6,1990) can make up very large library (for example 10 ⁶-10 ⁸Individual chemical entities).Second method is mainly used chemical process, and the example is Geysen method (Geysen et al., Molecular Immunology 23:709-15,1986; Geysen etal.J.Immunologic Method 102:259-74,1987) and people's such as Fodor method (Science251:767-73,1991).(the 14th international biological chemistry meeting, the 5th volume, summary FR:013,1988 such as Furka; Furka, Int.J.Peptide Protein Res.37:487-493,1991), Houghten (U.S. Patent No. 4,631, in December, 211,1986 is open) and (U.S. Patent No. 5,010 such as Rutter, on April 23rd, 175,1991 is open) method that produces the peptide mixt that can be tested as agonist or antagonist described.

The equipment of preparation combinatorial library can commercially obtain (to see for example 357MPS, 390MPS, Advanced Chem Tech, Louisville KY, Symphony, Rainin, Woburn, MA, 433A Applied Biosystems, Foster City, CA, 9050 Plus, Millipore, Bedford, MA).In addition, a large amount of combinatorial library itself also can commercially obtain (see for example ComGenex, Princeton, N.J., Tripos, Inc., St.Louis, MO, 3D Pharmaceuticals, Exton, PA, MartekBiosciences, Columbia, MD, etc.).

The test compounds library screening

Screening method of the present invention can provide efficiently and fast evaluation at having the test compounds of disturbing A5623/A2254 bonded high likelihood.Usually, any definite test compounds disturbs the method for A5623/A2254 binding ability all to be applicable to the present invention.For example, can adopt the competitiveness of ELISA pattern and noncompetitive to suppress to measure.Should carry out control experiment with the maximum binding capacity of determining system (for example in the example hereinafter, bonded A5623 is contacted with A2254, and the amount of definite and A5623 bonded A2254).The further guidance of filler test library of compounds is provided below.

The competitive assay pattern

Competitive assay can be used for screening test compounds of the present invention.As an example, the competitive ELISA pattern can comprise and solid support bonded A5623 (or A2254).Bonded A5623 (or A2254) is total to incubation with A2254 (or A5623) and test compounds.Make test compounds and/or A2254 (or A5623) in conjunction with A5623 (or A2254) through time enough, clean substrate to remove not bond material.Determine amount then with A5623 bonded A2254.This can for example use to have A2254 (or A5623) kind of detectable label as label with any realization the in the several different methods known in the art, and the substrate that has cleaned is contacted with anti-A2254 (or A5623) antibody of mark.To disturb A2254/A5623 bonded ability to be inversely proportional to test compounds with the amount of A5623 (or A2254) bonded A2254 (or A5623).The mark that---includes but are not limited to antibody---about protein is put down in writing as following document: Harlow; Lane, Antibodies, A LaboratoryManual (1988).

In a kind of version, with affinity tag mark A5623 (or A2254).Then, the A5623 (or A2254) of mark with test compounds and A2254 (or A5623) incubation, is carried out immunoprecipitation then.Use anti-A2254 (or A5623) antibody that immunoprecipitate is carried out the Western trace then.The same with the competitive assay pattern of front, be found with amount and the test compounds of A5623 (or A2254) bonded A2254 (or A5623) and disturb A2254/A5623 bonded ability to be inversely proportional to.

Non-competing mensuration form

Be unsuitable for the test compounds library that utilizes competitive assay to be screened immediately for making up pattern, those libraries as described herein, noncompetitive may also be useful in conjunction with measuring as initial screening.The example in above-mentioned library be the phage display storehouse (see for example Barret, et al. (1992) Anal.Biochem 204,357-364).

The useful part of phage library is to produce fast a variety of different recombinant peptides of workload.Phage library is unsuitable for competitive assay of the present invention, but can screen efficiently with non-competing pattern, determines which kind of recombinant peptide test compounds is in conjunction with A5623 or A2254.Can prepare the test compounds that has been accredited as associativity then, and be screened with the competitive assay pattern.The generation of phage and cell display libraries and screening are being known in the art, and discussion is arranged in following document for example: Ladner et al., and WO 88/06630; Fuchs et al. (1991) Biotechnology 9:1369-72; Goward et al. (1993) TIBS 18:136-40; Charbit et al. (1986) EMBO J 5,3029-37; Cull et al. (1992) PNAS USA 89:1865-9; Cwirla, et al. (1990) Proc.Natl.Acad.Sci.U.S.A.87,6378-82.

Exemplary noncompetitive is measured and is followed the program similar to above-mentioned competitive assay, does not just add wherein a kind of component (A5623 or A2254).Yet, be the combination of test compounds because non-competing pattern determines to A5623 or A2254, need determine that test compounds is in conjunction with the two ability of A5623 and A2254 for each material standed for.Therefore, for example, can determine test compounds and fixing combining of A5623 through the following steps: wash unconjugated test compounds; From the test compounds of upholder elution of bound, subsequently by mass spectrum for example, protein determination (Bradford or Lowry measure, or the deciding of 280nm absorbancy) is analyzed eluate.Perhaps can omit elution step, determine the combination of test compounds by the variation of organic layer spectroscopic properties on the monitoring support surface.The method of monitoring surperficial spectroscopic properties comprises, but be not limited to absorbancy, reflectivity, transparence, double refraction, refractive index, diffraction, surface plasma body resonant vibration, ellipsometric measurement, resonant mirror (resonant mirror) technology, coupled wave of grating waveguide technology and multipole resonance spectrum, all these are known to those skilled in the art.But the test compounds of applying marking also in mensuration, thereby save elution step.In this case, flush away not behind the bond material with the direct ratio that is combined into of the amount of upholder bonded mark and test compounds.

Develop a large amount of known robot systems, be used for the solution phase chemistry.These systems comprise automatic workstation, as the Takeda Chemical Industries (Osaka of company, Japan) Kai Fa automatic synthesizer, and many robot system (Zymate II, Zymark company, Hopkinton, Mass. that utilizes robot arm; Orca, Hewlett Packard, Palo Alto, Calif.), their simulated personnel's that learn a skill manual synthetic operation.Top any equipment all is suitable for utilizing the present invention.For the character and the enforcement of the modification (if any) that it can be worked as described herein these equipment is done, be conspicuous for those skilled in the relevant art.In addition, can commercially obtain a large amount of combinatorial library and (see for example ComGenex, Princeton, N.J., Asinex, Moscow, Ru, Tripos, Inc., St.Louis, MO, ChemStar, Ltd, Moscow, RU, 3DPharmaceuticals, Exton, PA, Martek Biosciences, Columbia, MD, etc.).

The screening of conversion enzyme

So, can under non-competing pattern, be measured with the cofactor and the cosubstrate of conversion enzyme spcificity to be determined to the test compounds of conversion enzyme.Behind the given conversion enzyme type to be studied, these cofactors and cosubstrate it is known to those skilled in the art that.

An exemplary screening procedure that is used to change enzyme comprises at first in the presence of cofactor and cosubstrate, preferably under physiological condition, A5623 and/or A2254 are contacted with the conversion enzyme, and wherein said cofactor and cosubstrate are essential for the proteic covalent modification of characteristic of conversion enzyme.Test the ability (being the combination of A5623) of adorned its binding partners of protein binding then to A2254.More adorned then albumen combines and right the combining of unmodified contrast with its binding partners, determines aforesaid necessary K _aChange and whether be achieved.

In order when carry out measuring, to be convenient to proteinic detection, can utilize the technology of well known to a person skilled in the art with one or more albumen with above-mentioned detectable label mark in addition.

Screening method

Above-mentioned screening embodiment is suitable for high-throughput and determines to be suitable for the further test compounds of research.Especially, screening of the present invention preferably includes following detection step:

(b) combination between the detection polypeptide; With

(c) select to suppress bonded test compounds between polypeptide.

Perhaps to just adding the test compounds studied, and monitor the propagation of this processing cell with respect to the propagation of the control group that test compounds is not provided in proliferating cells.According to the instruction that this paper provided, the clone that is suitable for the filler test compound is conspicuous to those skilled in the art.

For the body build-in test, test compounds can be applied to acceptable animal model.Gene is imported in the zooblast to express alien gene, can be according to any method, electroporation (Chu for example, et al., Nucleic Acids Res 15:1311-26 (1987)), calcium phosphate method (Chen and Okayama, Mol Cell Biol 7:2745-52 (1987)), deae dextran method (Lopata et al., Nucleic AcidsRes 12:5707-17 (1984), lipofectamine (lipofectin) method (Derijard B, et al.Cell 7:1025-37 (1994); Lamb et al., Nature Genetics 5:22-30 (1993): Rabindran et al., Science 259:230-4 (1993)) wait and implement.Gene can be expressed the protein that merges with label (for example HA or Myc).

Investigate the Subcellular Localization of A5623 and A2254 with immunohistochemical staining.With label-A5623, label-A2254 or its combination transfectional cell.Can utilize for example fluorescent microscope acquisition positioning image of microscope.

In preferred embodiments, can be by A2254 and A5623 expression carrier be transfected into the cell that suitable clone obtains to express simultaneously A2254 and A5623.For example, can use HEK293, SW480 or COS7 cell as clone.And detection reagent is preferably discerned the antibody of A2254.Perhaps, when A2254 or A5623 are expressed as fusion rotein with label, but the antibody that can use the label that any identification of protein merges is as detection reagent.In test kit of the present invention, antibody can be used fluorescent agent (for example FITC, TAMRA or GFP) mark.

Especially, as described herein, the inventor observes the A5623 polypeptide and the A2254 polypeptide interacts.Therefore, believe that the interaction of two peptide species takes place in cancer, particularly mammary cancer is brought into play keying action in taking place.Therefore, we are intended to screening and can be used for the treatment or the compound of preventing cancer, particularly mammary cancer, and it suppresses interaction or its opposite interaction of A5623 polypeptide and A2254 polypeptide.That is, in the presence of test compounds, the A2254-that comprises the A5623 polypeptide is contacted in conjunction with the polypeptide in territory with the A5623-that comprises the A2254 polypeptide in conjunction with the polypeptide in territory, detect the combination between two polypeptide, and select to suppress bonded test compounds between two polypeptide.Certainly, as selection, can also be in the presence of test compounds, make comprise A2254 polypeptide A 5623-in conjunction with the polypeptide in territory with comprise the polypeptide of A5623 polypeptide A 2254-and contact in conjunction with the territory.

Preferably, contain A2254 and comprise the A5623 polypeptide, contain A5623 and comprise the A2254 polypeptide in conjunction with the polypeptide in territory in conjunction with the polypeptide in territory.

Term " makes ... contact " and is included under the existence of test compounds, by known technology in the field of use test compound and method, the A2254-that comprises the A5623 polypeptide is contacted in conjunction with the polypeptide in territory with the A5623-that comprises the A2254 polypeptide in conjunction with the polypeptide in territory, the A5623-that comprises the A2254 polypeptide is contacted in conjunction with the polypeptide in territory with the polypeptide A 2254-that comprises A5623 in conjunction with the polypeptide in territory.

For example, can use the binding inhibitors that mensuration or cell-less measurement based on cell screen A5623 polypeptide and A2254 polypeptide.In a example, can use to express to contain A5623 in conjunction with the polypeptide in territory with contain A2254 and screen in conjunction with the cell of the polypeptide in territory based on raji cell assay Raji.In an example of cell-less measurement, can use purifying partially or completely contain A5623 in conjunction with the polypeptide in territory and partially or completely purifying contain the polypeptide of A2254 in conjunction with the territory.Certainly, also can use to express and contain A5623 in conjunction with the polypeptide in territory with contain the extract of A2254 in conjunction with the cell of the polypeptide in territory.

The further example of test determination and method has above been described.

Term " test compounds " or " compound to be tested " are meant as A5623 and the interactional supposition inhibitor of A2254, molecule or material or compound or composition or the medicament that will be tested by one or more screening methods of the present invention, or its arbitrary combination.Test compounds can be any chemical substance, for example inorganic chemistry material, organic chemicals, protein, peptide, sugar, lipid or its arbitrary combination, compound perhaps described herein, composition or medicament any.Should be appreciated that term " test compounds " when being used for the context of the invention, can and term " test molecule ", " test substances ", " potential material standed for ", " material standed for " or term mentioned above exchange and use.

Therefore, we have imagined little peptide or class peptide molecule be used for the interacting screening method of inhibitor.This little peptide or class peptide molecule in conjunction with and occupy A5623 in conjunction with territory or A2254 in conjunction with the two interaction territory of the interaction territory in one of territory or they, make A5623 or A2254 can not be touched by A2254 polypeptide or A5623 polypeptide by this respectively in conjunction with the territory.And, imagine any biological or chemical composition or material and all can be used as the interaction inhibitor.The inhibit feature of inhibitor can be measured with methods known in the art.These methods comprise interacting to be measured, as immune precipitation determination, ELISA, RIA.Equally, the potential candidate molecules or the candidate molecules mixture that when screening A5623, preferably will use with A2254 combination or opposite bonded inhibitor, can be, typically, the material of chemistry or biogenetic derivation, compound or composition, it is that nature exists and/or synthesizes, recombinates and/or the chemistry generation.Therefore, candidate molecules can be with A5623 and/or the A2254 polypeptide combines and/or interacting proteins, protein fragments, peptide, amino acid and/or its derivative, perhaps other compounds, for example ion.The synthetic compound library can be from Maybridge Chemical Co. (Trevillet, Cornwall, UK), Comgenex (Princeton, N.J.), Brandon Associates (Merrimack, N.H.) and Microsource (New Milford.Conn.) be purchased.Rare chemical library can (Milwaukee Wis.) obtains from Aldrich.Perhaps, the natural compounds library of bacterium, fungi, plant and animal form of extract can obtain from for example Pan Laboratories (Bothell.Wash.) or MycoSearch (N.C.), perhaps can easily produce.In addition, the library of natural and synthetic generation and compound can easily be modified by conventional chemical, physics or biochemical method.

In addition, being created in of chemical library is known in the art.For example, use combinatorial chemistry to produce the library of compounds that will in mensuration described herein, be screened.The combinatorial chemistry library is by making up the set of a large amount of chemistry " component unit " (building block) reagent by the various chemical compounds of chemosynthesis or biosynthesizing generation.For example, the formation of linear combination chemistry library such as polypeptide libraries is to make up the peptide that produces given length by making up every kind of possible amino acid.By the combined hybrid of this chemical component unit, can synthesize millions of kinds of compounds in theory.For example, a reviewer thinks, system, mix 100 interchangeable chemical component units in combination, can synthesize 100,000,000 kinds of tetramerization compounds or 10,000,000,000 kinds of pentamer compounds (Gallop A, et al.Journal ofMedicinal Chemistry, 37 (9) 1233-51 (1994)) in theory.Also can use other chemical libraries known in the art, comprise natural product libraries.In case combinatorial library produces, just it is screened, seek compound with expectation biological property.

In the context of the present invention, the SCREENED COMPOUND library is to identify the compound of performance A5623 and the effect of A2254 polypeptide binding inhibitors.For example, at first, produce the small molecules library with combinatorial library formation method well known in the art.United States Patent (USP) NOs.5,463,564 and 5,574,656 is exactly two such instructions.Compound to the library screens the compound that has desired structure and functional property with evaluation then.U.S. Patent No. 5,684,711 have discussed the method that is used to screen the library.Illustrate screening process: will contain A2254 in conjunction with the polypeptide in territory with contain A5623 and mix mutually in conjunction with the polypeptide in territory and the compound in the library, and make it interact with each other.Further observe the combination whether described compound suppresses this two peptide species then.As an example, as long as two peptide species interact and can produce fluorescent signal, and this signal test compounds suppress between polypeptide in conjunction with the time can reduce and/or disappear, then can use the FRET technology.Correspondingly, two peptide species all can comprise the label that is suitable for FRET.This class label is well known in the art.

The feature of each library compound is encoded, make that the compound of show activity can be analyzed in the polypeptide bonded suppresses, and the common trait of all cpds of having identified can be separated and its be combined in the further iteration (interation) in library.In case screened a library of compounds, promptly be used in and show in the first round screening that having the anti-active chemical component unit that interacts generates follow-up library.Make in this way, candidate compound iteration subsequently will have the essential 26S Proteasome Structure and Function feature of increasing inhibitory phase mutual effect, up to finding one group to suppressing between two peptide species in conjunction with the inhibitor with high degree of specificity.Can further test these compounds then as animal security and the effectiveness the during medicine in the Mammals for example.Be easy to recognize that this specific screening method is learned just for example.Other method is known in those skilled in the art.

For example, candidate's medicament comprises the chemical substance of numerous species, but typically they are organic molecules, is preferably molecular weight about 50 and less than about 2500 dalton, preferably less than about 750 dalton, more preferably less than about 350 daltonian organic molecules.

Candidate's medicament also may comprise and the interaction of the protein structure necessary functional group of hydrogen bond particularly, typically comprises at least one amido, carbonyl, hydroxyl or carboxyl, preferably at least two functionalized chemical groups.Fragrance or poly aromatic structure that candidate agent often contains carbocyclic ring or heterocycle structure and/or replaced by one or more above-mentioned functions groups.

The Exemplary types of candidate's medicament can comprise heterocycle, peptide, carbohydrate, steroid etc.These compounds can be accepted to modify to improve effectiveness, stability, drug compatibility etc.Can utilize the structure of medicament to identify, produce or screen other medicament.For example; when identifying peptide reagent; can in all sorts of ways they are modified in order to improve its stability; for example use particularly D-L-Ala of alpha-non-natural amino acid such as D-amino acid; make amino or C-terminal functionalization; for example carry out acidylate or alkylation, carry out esterification or amidation etc. for carboxyl for amino.Other stabilization methods can comprise to be sealed, and it is medium for example to be encapsulated in liposome.

As mentioned above, candidate's medicament also in biomolecules, comprises in peptide, amino acid, carbohydrate, lipid acid, steroid, purine, pyrimidine, nucleic acid and derivative, analog or its combination being found.Candidate's medicament comprises the library of synthetic or natural compounds from source acquisition widely.For example, a lot of methods are arranged, comprise the expression of randomized oligonucleotide and oligopeptides, can be used for synthesizing diversified organic compound and biomolecules with orientation at random.Perhaps, the natural compounds library of bacterium, fungi, plant and animal form of extract is existing available or can easily produces.In addition, library and compound natural or synthetic generation can easily be modified by conventional chemical, physics and biochemical method, and can be used for producing combinatorial library.Can carry out orientation or chemically modified at random to known pharmacological agent, for example acidylate, alkanisation, esterification, amidation etc. are to produce analog.Other test compounds can be fit, antibody, affybody, trinectin or anticalin, or similar compound.Certainly, to specific fit, the antibody of A5623 or A2254, affybody, trinectin or anticalin self may be bonded inhibitor between A5623 and the A2254, therefore can easily be used for the treatment of or preventing cancer, particularly the method for mammary cancer.

Can be used for prevention or treatment cancer, particularly mammary cancer by the method institute compounds identified that is used to screen A5623 polypeptide and A2254 polypeptide binding inhibitors as herein described.Therefore, inhibitor has such character: directly or indirectly combine the territory with A2254 and/or A5623 interacts in conjunction with the territory, by this, as described herein, influence the interaction in these territories, they no longer can particularly be interacted in the breast cancer cell like that as at cancer cells.

For example, inhibitor can be anti-A2254 in conjunction with territory or the A5623 antibody in conjunction with the territory.The another kind of example of this inhibitor is affybody, fit, anticalin or trinectin.

Screening and treatment test kit

In one embodiment, the invention provides to be used to screen and can be used for treating or the goods or the test kit of Breast Cancer Prevention, wherein this test kit comprises: (a) A2254-of A5623 polypeptide is in conjunction with the territory; (b) A5623-of A2254 polypeptide is in conjunction with the territory; (c) detect the reagent of this two peptide species interphase interaction.As discussed above, contain A2254 can comprise total length in conjunction with the polypeptide in territory A5623 polypeptide or its A2254 bound fraction.Similarly, contain A5623 can comprise total length in conjunction with the polypeptide in territory A2254 polypeptide or its A5623 bound fraction.

In the further embodiment of the present invention, goods and test kit are provided, it contains the material that can be used for treating pathological condition described herein.These goods can comprise the container and the label of medicament described herein.Suitable containers comprises for example bottle, bottle and test tube.Container can form with various materials, for example glass or plastics.In the context of the present invention, container holds the composition with promoting agent, and this promoting agent can effectively be treated for example mammary cancer of cell proliferation disorders.In one embodiment, the promoting agent in the composition is to be accredited as to destroy A5623/A2254 bonded test compounds (for example antibody, small molecules etc.) in vivo.Label on the container should indicate said composition, and to be used for the treatment of one or more be the situation of feature with the abnormal cell proliferation.Label can also provide to be used and Monitoring techniques, for example guidance of the techniques described herein.

Except said vesse, test kit of the present invention can also randomly comprise and holds second container that medicine can be accepted thinner.It may further include other commercial and user perspectives and sees desirable material, comprises other buffers, thinner, Filters, syringe needle, syringe and has the specification sheets of instruction.

If desired, composition may reside in packing or the distribution device, and packing or distribution device can comprise one or more unit dosage that contain activeconstituents.Packing can for example comprise metal or plastic foil, for example blister pack.Packing or partitioning device can be put together with using to instruct.Can also prepare the composition of in the consistency pharmaceutical carrier, preparing that comprises The compounds of this invention, be placed in the suitable containers, and mark is used for the treatment of the label of specifying illness.

The accompanying drawing summary

Fig. 1 has shown in the tumour cell (3T, 31T, 149T, 175T, 431T, 453T, 491T, 554T, 571T, 709T, 772T and 781T), breast cancer cell line (HBC4, HBC5, HBL100, HCC1937, MCF7, MDA-MB-231, SKBR3, T47D, YMB1) (following drawing) and the health adult tissue that derive from patient with breast cancer's (go up drawing) (A) A2254 and (B) the sxemiquantitative RT-PCR result of A5623 expression.

Fig. 2 has shown in various people tissue (going up drawing) and breast cancer cell line and normal people vital organ (following drawing) (A) A2254 and (B) the Northern engram analysis photo of A5623 transcript.

Fig. 3 has shown (A) A2254 and (B) genome structure of A5623.A2254 has 2 different variants, censures to be that V1 and V2, A5623 also have 3 different variants (V1, V2 and V3).Last figure; Genome structure, figure below; To every kind of specific sxemiquantitative RT-PCR result of variant.

Fig. 4 A is the proteic heterogenous expression of A2254 that shows by the Western engram analysis.Fig. 4 B has shown the proteic Subcellular Localization of A2254.Fig. 4 C has shown A5623V1, A5623V2 and the proteic heterogenous expression of A5623V3 by the Western engram analysis.Fig. 4 D-F has shown (D) A5623V1, (E) A5623V2 and (F) the proteic Subcellular Localization of A5623V3.

Fig. 5 has shown the growth inhibitory effect that is designed for the siRNA (siRNAs) that A2254 expresses in the reduction breast cancer cell.Fig. 5 A has shown the sxemiquantitative RT-PCR of the endogenous expression by inhibitation system of A2254 in breast cancer cell line T47D (left drawing) and HBC5 (right drawing).GAPDH is as internal contrast.What Fig. 5 B showed is that MTT measures, and it shows by striking the number that the A2254 that hangs down in T47D (left drawing) and HBC5 (right drawing) cell has reduced colony.What Fig. 5 C showed is colony forming assay, and it shows by striking the number that the A2254 that hangs down in T47D (left drawing) and HBC5 (right drawing) cell has reduced colony.

Fig. 6 has shown the growth inhibitory effect that is designed for the siRNA (siRNAs) that A5623 expresses in the reduction breast cancer cell.Fig. 6 A has shown the sxemiquantitative RT-PCR of the endogenous expression by inhibitation system of A5623 in breast cancer cell line T47D (left drawing) and HBC5 (right drawing).GAPDH is as internal contrast.What Fig. 6 B showed is that MTT measures, and it shows by striking the number that the A5623 that hangs down in T47D (left drawing) and HBC5 (right drawing) cell has reduced colony.What Fig. 6 C showed is colony forming assay, and it shows by striking the number that the A5623 that hangs down in T47D (left drawing) and HBC5 (right drawing) cell has reduced colony.

Fig. 7.A5623 and the A2254 expression in breast cancer cell line and tissue slice.Fig. 7 A, the comparison in the endogenous A5623 in the breast cancer cell line and A2254 expression and the HMEC clone, it carries out the Western engram analysis with anti-A5623 antibody or anti-A2254 antibody and investigates.Fig. 7 B, in breast cancer cell line, endogenous A5623 albumen or the Subcellular Localization of A2254 albumen in the cell cycle.Use the anti--A5623 polyclonal antibody or the anti--A2254 polyclonal antibody (green) of affinity purification, HBC4, HBC5 and MCF-7 cell are carried out the immunohistochemical staining of A5623, the HBC5 cell is carried out the immunohistochemical staining of A2254, and with DAPI (blueness) dyeing to distinguish nucleus (seeing material and method).The location of A5623 in the white arrow indication cell intermediate in latter stage.Fig. 7 C, the immunohistochemical staining result of mammary cancer and healthy tissues section (normal galactophore tissue, lung, heart, liver, kidney and testis).Endogenous A5623 albumen is with resisting-the A5623 antibody staining.Can detect expression hardly in the normal galactophore tissue (sample No.10441), but cancer cells is all dyeed strongly in the cancerous tissue of all researchs, comprises reality tubular carcinoma (sold-tubular carcinoma) (sample No.234), nipple tubular carcinoma (papillotubular carcinoma) (sample No.240) and inocarcinoma (sample No.179).Representative diagram comes from the microscopic examination of original magnification x 200.

Interaction between Fig. 8 A5623 and A2254.Fig. 8 A, A5623 that detects by sxemiquantitative RT-PCR and A2254 are at breast cancer cell line (HBC4, HBC5, HBL100, HCC1937, MCF7, MDA-MB-231, SKBR3, T47D, YMB1) and health adult tissue (N, normal breast vessel cell; MG, mammary gland; LUN, lung; LIV, liver; HEA, heart; KID, kidney; And BM, marrow) in expression.Fig. 8 B, C, the co-immunoprecipitation of A5623 and A2254.To from transfection the cell lysate of HA label A 2254 and Flag label A 5623 proteic COS7 cells with anti-HA or anti-Flag immunoprecipitation.Immunoprecipitate carries out immunoblotting with monoclonal anti HA or anti-Flag antibody.Fig. 8 D, endogenous A5623 or the A2254 Subcellular Localization in the stably express cell.Left side drawing is presented in the cell of stably express A2254, and endogenous A5623 albumen (redness) and external source A2254 albumen (green) is the location altogether, and right drawing is presented at and stablize in the A5623 cell, and endogenous A2254 (redness) and external source A5623 (green) locate altogether.External source A2254 albumen (redness) is located (right figure) altogether with external source A5623 albumen.

Promotes growth or the immersional wetting of Fig. 9 external source A2254 in the NIH3T3 cell.Fig. 9 A expresses high level or the cell of medium level external source A2254 or the Western engram analysis of analog carrier cells transfected.Confirmed the external source introducing that A2254 expresses with anti-HA tag monoclonal antibody.Contrast as last sample with beta-actin.Fig. 9 B, the growth of NIH3T3-A2254 cells in vitro.With A2254 (NIH3T3-A2254-#1 ,-#2 ,-#3 and-#4) and stand-in (NIH3T3-simulation-#1 ,-#2 ,-#3) rotaring copolymering NIH 3 T 3 cell, measured by MTT.Fig. 9 C, Matrigel soak into to measure confirm NIH3T3-A2254--#3 and-#4 and NIH3T3-simulation-#1 can strengthen infiltration.Computation migration is wrapped by the cell number of filter membrane by Matrigel.The each same form three holes are carried out 3 times and are measured.

Implement optimal mode of the present invention

To further describe the present invention in the following embodiments, they do not limit the scope by claims of the present invention.

[general method]

Clone and clinical material

MCF-7 HBC4, HBC5, MDA-MB-231 are so kind as to give by doctor Yamori (Japanese Foundation For Cancer Research, Tokyo), and BT-549, MCF-7, T47D, SKBR3, HCC1937, MDA-MB-435S, YMB1, HBL100 and COS7 obtain from ATCC.All cell cultures are in suitable medium, and promptly BT-549, HBC4, HBC5, SKBR3, T47D, YMB1 and HCC1937 are with RPMI-1640 (Sigma, St.Louis, MO) (containing the 2mM L-glutaminate); The Eagle substratum (Invitrogen, Carlsbad, CA) that HBL100, COS7 modify with Dulbecco; MCF-7 is with containing 0.1mM indispensable amino acid (Roche), the EMEM (Sigma) of 1mM Sodium.alpha.-ketopropionate (Roche), 0.01mg/ml Regular Insulin (Sigma), MDA-MB-231 and MDA-MB-435S L-15 (Roche).Every kind of substratum all adds 10% foetal calf serum (Cansera) and 1% microbiotic/anti-mycotic agent solution (Sigma).MDA-MB-231 and MDA-MB-435S cell maintain 37 ℃ of no CO ₂Moistening atmosphere in.Other clone maintains 37 ℃ and contains 5%CO ₂Moistening atmosphere in.Clinical sample (mammary cancer and normal breast conduit) obtains from the surgery sample, and has obtained all patients' relevant informed consent.

The new mankind gene of the spot representative of A2254 and A5623 separates on our the cDNA microarray

In order to detect the gene that in mammary cancer, is raised usually, to on the microarray 27, the graphic screening of whole expression of 648 genes, to select respectively i＞50%) mammary cancer case before whole 77 menopause, ii) 69 infitrating ductal carcinoma, the genes of expression ratio＞3.0 in the case of iii) 31 highly low differentiation infringements, iv) 14 medium low differentiation infringements or v) 24 low differentiation infringements.In whole 493 genes that in tumour cell, show as rise, we are conceived to the internal indicator number gene for A2254 and A5623, because its expression ratio in surpassing 50% the mammary cancer case that information is provided is greater than 3.0, and it crosses low the expression in the healthy tissues that comprises heart, lung, liver and marrow by health adult tissue's expression pattern discovery.

Sxemiquantitative RT-PCR analyzes

We extract total RNA from each colony of laser capture cell, carry out amplification and reverse transcription based on T7 then, as mentioned before (Ono K, et al., Cancer Res., 60,5007-11,2000).We as quantitative internal contrast, have prepared the suitable dilution of every kind of strand cDNA by monitoring glyceraldehyde-3-phosphate dehydrogenase (GAPDH), are used for pcr amplification subsequently.The PCR primer sequence is as follows:

5 '-CGACCACTTTGTCAAGCTCA-3 ' (SEQ ID NO; 1) and

5 '-GGTTGAGCACAGGGTACTTTATT-3 ' (SEQ ID NO; 2) be used for GAPDH;

5 '-AACTTAGAGGTGGGAGCAG-3 ' (SEQ ID NO:45) and

5 '-CACAACCATGCCTTACTTTATC-3 ' (SEQ ID NO:46) is used for β 2MG;

5 '-ACTCTAGGACTTGCATGATTGCC-3 ' (SEQ ID NO; 3) and

5 '-TGGGTGTCAAACCAAACAGA-3 ' (SEQ ID NO; 4) be used for A2254V1,

5 '-GTTAGAACTTGTTTCCTCCTCCG-3 ' (SEQ ID NO; 5) and

5 '-ATCCTCAATGGTATTTCAGC-3 ' (SEQ ID NO; 6) be used for A2254V2,

5 '-GTGGTCCTAGGAGACTTGGTTTT-3 ' (SEQ ID NO; 7) and

5 '-TACATGCATACCCCCAACAA-3 ' (SEQ ID NO; 8) be used for the total zone of A5623,

5 '-GCTTCAGCGAGAACTTTC-3 ' (SEQ ID NO; 9) and

5 '-CAACTGTAACACTCATTCACATC-3 ' (SEQ ID NO; 10) be used for A5623V1,

5 '-CTATTCTGAGTTTGCGCGAGAAC-3 ' (SEQ ID NO; 11) and

5 '-CAACTGTAACACTCATTCACATC-3 ' (SEQ ID NO; 10) be used for A5623V2,

5 '-CATCCTGAGTGCGAGAACTTTC-3 ' (SEQ ID NO; 12) and

5 '-CAACTGTAACACTCATTCACATC-3 ' (SEQ ID NO; 10) be used for A5623V3.

Northern-blot analyzes

According to the specification sheets of manufacturers, from all breast cancer cell lines, extracted total RNA with Rneasy test kit (QIAGEN).After DNase I (Nippon Gene, Osaka, Japan) processing, with the specification sheets separating mRNA of mRNA purification kit (Amersham Biosciences) according to manufacturers.With every kind of mRNA of 1-μ g equal portions with from normal adult mammary gland (Biochain), lung, heart, liver, kidney, marrow (BD, Clontech, Palo Alto, CA) isolating poly-A (+) RNA separates on 1% sex change sepharose together, and transfers to (mammary cancer-Northern trace) on the nylon membrane.With mammary cancer-organize the Northern trace with the people (Clontech, Palo Alto is CA) with [α more ³²P]-A2254 of dCTP-mark and the hybridization of A5623PCR product, this PCR product is by RT-PCR preparation (seeing below).Prehybridization, hybridization and cleaning are carried out in recommendation according to supplier.Trace uses intensifying screen-80 ℃ of radioautograph 14 days.Specific probe at A2254 (548bp) and A5623 (454bp) uses following primer sets to prepare by PCR:

5 '-ACTCTAGGACTTGCATGATTGCC-3 ' (SEQ ID NO; 3) and

5 '-TGGGTGTCAAACCAAACAGA-3 ' (SEQ ID NO; 4) be used for the total zone of A2254,

5 '-GTGGTCCTAGGAGACTTGGTTTT-3 ' (SEQ ID NO; 7) and

5 '-TACATGCATACCCCCAACAA-3 ' (SEQ ID NO; 8) be used for the total zone of A5623.

In addition, the specific probe of A2254V1 (350bp) prepares by PCR with following Auele Specific Primer group: 5 '-CTGGAAGAGCAGGCTAGCAG-3 ' (SEQ ID NO; 13) and

5′-GCTGCGGAGAAAGCTCTATG-3′(SEQ?ID?NO；14)。

The structure of expression vector

In order to make up A5623 and A2254 expression vector, with KOD-Plus archaeal dna polymerase (Toyobo, Osaka, Japan) entire coded sequence by pcr amplification PRC1 cDNA.Primer sets is

The A5623-forward, 5 '-CCG GAATTCTCCGCCATGAGGAGAAGTGA-3 ' (SEQID NO:47) (underscore is represented the EcoRI site) and

A5623-is reverse, 5 '-TTGCCG CTCGAGGGACTGGATGTTGGTTGAA-3 ' (SEQ ID NO:48) (underscore is represented the XhoI site),

The A2254-forward, 5 '-CCG GAATTCATGGCCATGGACTCGTCG-3 ' (SEQ IDNO:49) and

A2254-is reverse, 5 '-GCTCCG CTCGAGCTGGGGCCGTTTCTT-3 ' (SEQ IDNO:50).The PCR product is inserted among the EocRI and XhoI site of pCAGGS-nHA or pCAGGS-Flag expression vector.

Anti--A5623-specific polyclonal antibody and anti--A2254-specific polyclonal antibody

Use pET21 carrier (Novagen, Madison, WI) preparation plasmid, these plasmids are designed to express respectively two fragments (86-239 and 124-239 amino acids) of two fragments of A5623 (179-360 and 234-360 amino acids) or A2254, and wherein said segmental C end has the epi-position of His label.Express recombinant peptide in intestinal bacteria (Escherichia coli) BL21codon-plus strain (Stratagene, La Jolla, CA) respectively, and carry out purifying according to supplier's experimental program with Ni-NTA resin agarose (Qiagen).The recombinant protein of purifying is mixed, and immunization is in the rabbit body then.With immune serum on affinity column according to the standard method purifying.Anti--A5623 the antibody or the anti-A2254 antibody of affinity purification are used for Western trace, immunoprecipitation and immunocyte dyeing, as mentioned below.We confirm that by the Western engram analysis these antibody can be distinguished endogenous A5623 albumen or the A2254 albumen in the specific recognition MCR7 breast cancer cell.

Immunocytochemical stain

In order to check endogenous A5623 albumen or the Subcellular Localization of A2254 albumen in breast cancer cell line MCF7, HBC4 and HBC5, we are with every hole 1 * 10 ⁵Individual cell inoculation cell (Lab-Tek II chamber slide, Nalgen Nunc International, Naperville, IL).Behind the incubation 24 hours, cell is fixed 15 minutes with the PBS (-) that contains 4% Paraformaldehyde 96, and handles 2.5 minutes so that it is permeable with the PBS (-) that contains 0.1%Triton X-100 in 4 ℃.Subsequently, covered cell 12 hours with the PBS (-) that contains 3%BSA,, use the anti-A5623 polyclonal antibody of rabbit of dilution in 1: 1000 or the anti-A2254 polyclonal antibody incubation of dilution in 1: 1000 subsequently with the sealing non-specific hybridization at 4 ℃.After PBS (-) cleaning, with Alexa488-link coupled anti-rabbit second antibody (MolecularProbe, Eugene, OR) dyeing of cell with dilution in 1: 1000.With 4 ', 6 '-diamidino-2-phenylindone two hydrochloric acid (4 ', 6 '-(DAPI) pair cell nuclear counterstaining of diamidine-2 '-phenylindolendihydrochrolide).(Leica, Tokyo Japan) obtain fluoroscopic image down at TCS SP2AOBS.

The Western engram analysis

We with FuGENE 6 transfection reagents (Roche) according to manufacturer specification respectively with 1 μ gpCAGGS-A2254-HA, pCAGGS-A5623V1-HA, pCAGGS-A5623V2-HA or pCAGGS-A5623V3-HA transient transfection in the COS7 cell.Cell lysate is separated on the 10%SDS polyacrylamide gel, and transfers on the nitrocellulose filter, then with 1: 1000 the dilution mouse anti HA antibody (Roche) incubation as first antibody.Behind sheep anti mouse IgG-HRP (Amersham Biosciences) incubation as second antibody, (AmershamBiosciences) makes signal visual with the ECL test kit.

And, in order to detect endogenous A5623 albumen or the A2254 albumen among breast cancer cell line (BT-474, BT-549, HBC4, HBC5, HBL-100, MCF-7, MDA-MB-231, SKBR3 and T47D) and the HMEC (people's mammary epithelial cell), cell is being contained 0.1% proteinase inhibitor mixture III (Calbiochem, San Diego, CA) cracking in the lysis buffer (50mM Tris-HCl, pH 8.0/150mMNaCl/0.5%NP-40).Estimate total protein concentration with protein determination kit (Bio-Rad, Hercules, CA), then albumen is mixed with the SDS-sample-loading buffer, boil, be loaded into the 10%SDS-PAGE gel then.Behind the electrophoresis, with the albumen point sample to nitrocellulose filter (GE Healthcare).To contain proteic film and seal, with anti-A5623 polyclonal antibody or anti-A2254 polyclonal antibody incubation, be used to detect endogenous A5623 albumen or A2254 albumen then with confining liquid.Make protein band visual at last with film and HRP link coupled second antibody incubation, and by ECL detection reagent (GE Healthcare).Beta-actin is experimentized as last sample contrast.

Immunocytochemical stain

In order to check the Subcellular Localization of A2254 or A5623V1, A5623V2 and A5623V3, we for all 4 constructs with every hole 1 * 10 ⁵Individual cell inoculation COS7 cell.After 24 hours, use FuGENE 6 transfection reagents (Roche) according to manufacturer specification respectively with 1 μ gpCAGGS-A2254-HA, pCAGGS-A5623V1-HA, pCAGGS-A5623V2-HA or pCAGGS-A5623V3-HA transient transfection COS7 cell.Then, with the PBS fixed cell 15 minutes that contains 4% Paraformaldehyde 96, and with 4 ℃ of processing of the PBS that contains 0.1%Triton X-100 2.5 minutes so that it is permeable.Subsequently, in 4 ℃ cell is covered 12 hours with the sealing non-specific hybridization with the PBS that contains 3%BSA.Then, with every kind of rat anti HA antibody (Roche) incubation that is fabricated the COS7 cell of body transfection with dilution in 1: 1000.After cleaning with PBS, all transfectional cells are dyeed with Alexa594-link coupled Chinese People's Anti-Japanese Military and Political College mouse second antibody (Molecular Probe) of diluting at 1: 1000.With 4 ', 6-diamidino-2-phenylindone two hydrochloric acid (4 ', 6 '-(DAPI) pair cell nuclear counterstaining of diamidine-2 '-phenylindolendihydrochrolide).(Japan) microscopically obtains fluoroscopic image for Leica, Tokyo at TCS SP2AOBS.

Make up A2254 and A5623 specificity-siRNA expression vector with psiU6X3.0

We have set up RNAi system based on carrier according to previous report (WO2004/076623) with psiU6BX siRNA expression vector.By preparing siRNA expression vector in the BbsI site of the double chain oligonucleotide in the table 1 being cloned into the psiU6BX3.0 carrier at A2254 (psiU6BX-A2254) and A5623 (psiU6BX-A5623).Control plasmid---the psiU6BX-simulation is by preparing in the BbsI site of following double chain oligonucleotide being cloned into the psiU6BX3.0 carrier:

5 '-CACCGAAGCAGCACGACTTCTTCTTCAAGAGAGAAGAAGTCGTGCTGCTTC-3 ' (SEQ ID NO; 15) and

5’-AAAAGAAGCAGCACGACTTCTTCTCTCTTGAAGAAGAAGTCGTGCTGCTTC-3’(SEQ?ID?NO；16)。

The oligonucleotide sequence of siRNA

Oligonucleotide sequence at A2254 and A5623 siRNA is as follows.

Table 1. is at the oligonucleotide sequence of A2254 and A5623 siRNA

Underscore indication specific siRNA sequence.

The gene silencing effect of A2254 and A5623

MCF-7 T47D and HBC5 are coated on the 10-cm culture dish (1 * 10 ⁶Individual cell/ware), and with FuGENE6 reagent carry out transfection with psiU6BX-simulation (as negative control), psiU6BX-A2254 or psiU6BX-A5623 according to supplier's recommendation (Roche).After every kind of construct transfection 7 days, from the total RNA of cell extraction, then with the above-mentioned low effect of striking of confirming siRNA at the Auele Specific Primer in A2254 and the total zone of A5623 by sxemiquantitative RT-PCR.Primer as the GAPDH of internal contrast is as follows:

5 '-CGACCACTTTGTCAAGCTCA-3 ' (SEQ ID NO; 1) and

5′-GGTTGAGCACAGGGTACTTTATT-3′(SEQ?ID?NO；2)。In addition, the transfectant that uses T47D and HBC5 expression of cell lines siRNA was cultivated 28 days containing on the selection substratum of 0.7mg/ml Xin Meisu.With 4% Paraformaldehyde 96 fixing after, with transfectional cell Giemsa solution-dyed, with the formation of assessment colony.Carrying out MTT measures with quantitative cell viability.Cultivate in containing the substratum of Xin Meisu after 12 days, (Sigma), concentration is 0.5mg/ml to add MTT solution (3-(4,5-dimethylthiazole-2-yl)-2,5-phenylbenzene tetrazolium bromide).37 ℃ of incubations added acid-SDS (0.01N HCl/10%SDS) after 2.5 hours; Violent mixing suspension, 37 ℃ are incubated overnight with dissolving mazarine crystal then.Measure the 570nm absorbancy with Microplate Reader 550 (BioRad).

Immunoprecipitation and Western trace

With cell cracking in lysis buffer (50mM Tris-HCL (pH 8.0), 150mM NaCL, 0.5%NP-40 and proteinase inhibitor mixture group III (Calbiochem, San Diego, CA)).With the total protein of equivalent and 1mg rat anti HA antibody (Roche) or mouse anti Flag antibody (Santa Cruz) at 4 ℃ of incubations 1 hour altogether.(CA) incubation 1 hour altogether cleans with lysis buffer then for Zymed Laboratories, South San Francisco with immunocomplex and Protein G-Sepharose.Co-precipitation albumen separates by SDS-PAGE.

The isolating protein transduction of SDS-PAGE is moved on on the nitrocellulose filter, be total to incubation with rat anti HA antibody or mouse anti Flag antibody (Santa Cruz) then, after the second antibody with coupling HRP was total to incubation, (Amersham Biosciences) made signal visual with the ECL test kit.

Set up the NIH3T3 cell of stably express A5623 or A2254

As mentioned above, with FUGENE6 A5623 or A2254 expression vector or analog carrier transfection are entered the NIH3T3 cell.With cells transfected incubation in containing 0.9mg/ml Geneticin (G418) substratum (Invitrogen).To clone the NIH3T3 cell and carry out subclone by limiting dilution.The A5623 of band HA label or the expression of A2254 are assessed by the Western engram analysis with anti-HA monoclonal antibody.At last, set up several clones, and called after A5623-NIH3T3 or A2254-NIH3T3.

In order to study the growth promoting function of A2254, we with 4 kinds independently A2254-NIH3T3 cell and 3 kinds independently the MOCK-NIH3T3 cell inoculated 5000 cells for every kind, and measure by MTT and to write down cell number every day, through 6 days.These experiments are by carrying out in triplicate.

And we have studied the ability of A2254-NIH3T3 cell (A2254-NIH3T3-3 and-4) infiltration by matrigel.In brief, we with 2 kinds independently A2254-NIH3T3 cell (A2254-NIH3T3-3 and-4) and a kind of independently simulation-NIH3T3 cell inoculated 10000 cells respectively, and cell cultivated in containing the DMEM of 10%FCS converges.Cell is collected by trypsin treatment, in the DMEM of not increase serum or proteinase inhibitor, cleaned, and with 1 * 10 ⁵The concentration of individual cell/mL is suspended among the DMEM.Before the preparation cell suspension, at room temperature with DMEM to Matrigel matrix (Bection Dickinson Labware, Bedford, MA) drying layer rehydration 2 hours.Each the bottom cell that soaks into cell to 24 hole Matrigel adds the DMEM that contains 10%FBS, and adds 0.5mL (5 * 10 to the insertion sheet (insert) of each top cell ⁴Individual cell) cell suspension.With plate 37 ℃ of incubations 22 hours.Behind the incubation, cell is handled, the cell that soaks into the insertion sheet by matrigel bag quilt fix, and is dyeed with Giemsa, above step is all according to the explanation of supplier (Bection Dickinson Labware).

Immunohistochemical staining

Graphic with the expression of anti-A5623 rabbit polyclonal antibody research A5623 albumen in mammary cancer and healthy tissues.In brief, with paraffin-embedded sample with dimethylbenzene and Ethanol Treatment, and with protein blocking reagent (Dako Cytomation, Carpinteria CA) seal.Add the polyclonal antibody in the antibody dilution solution (1: 100), use substrate-chromogen (DAKO liquid DAB chromogen, DakoCytomation) dyeing then.At last, tissue sample is used brazilwood extract dyeing to distinguish nucleus and tenuigenin.

[result]

Identify that A2254 and A5623 are the up-regulated gene in the breast cancer cell

When we with the cDNA microarray analysis of represent 27,648 Human genomes from 77 menopause before during patient with breast cancer's the gene expression pattern of cancer cells, we have identified 493 general genes of rise in breast cancer cell.In them, we have paid close attention to following two kinds of genes: a kind of internal number is A2254, is censured (the SEQ ID NO into kinesin family member 2C (KIF2C); 35,36), another kind of internal number is A5623, is censured to be division of cytoplasm albumen regulon 1 (Genebank accession number No.NM_003981; SEQ ID NO; 39,40).On microarray, compare with the normal breast vessel cell, A2254 and A5623 expression of gene respectively in 60 routine mammary cancer 44 the example and 58 routine mammary cancer in 37 examples in to some extent the rising.In order to confirm the expression of these up-regulated genes, we have carried out sxemiquantitative RT-PCR and have analyzed comparison mammary cancer sample and contain expression level between the health adult tissue of normal breast vessel cell.At first, we find, with normal breast vessel cell and health adult tissue, comprise that mammary gland, lung, heart, liver, kidney and marrow compares, being expressed in of A2254 raise to some extent in 7 examples in the 12 routine clinical mammary cancer samples (low differentiated) (Figure 1A, last drawing).In addition, this gene is all crossed high expression level (Figure 1A, following drawing) in whole 9 kinds of breast cancer cell lines.Then, we also find, with health adult tissue, normal breast vessel cell (Figure 1B particularly, last drawing) compares, being expressed in 7 examples in the 12 routine mammary cancer samples (low differentiated) of A5623 raises to some extent, and all crosses high expression level (Figure 1B, following drawing) in whole 9 kinds of breast cancer cell lines that we check.

In order to check that further these expression of gene are graphic, we have carried out Northern engram analysis (see material and method) as probe to various human tissue and breast cancer cell line with the cDNA fragment of A2254 and A5623.A2254 does not have in the health adult tissue except that testis and thymus gland and expresses or express and can't detect (Fig. 2 A, last drawing), and compares with other healthy tissuess except that marrow, its mistake high expression level (Fig. 2 A, following drawing) surprisingly in breast cancer cell line.A5623 also only expresses (Fig. 2 B, last drawing) in testis, and compares with other healthy tissuess except that marrow, particularly compares the remarkable high expression level (Fig. 2 B, following drawing) of crossing in all breast cancer cell lines with normal human mammary.Therefore, we pay close attention to the specific expressed transcript of these mammary cancer.

The genome structure of A2254 and A5623

In order to obtain the global cDNA sequence of A2254 and A5623, we carry out RT-PCR with breast cancer cell line T47D as template.A2254 is made of 21 exons, censures to kinesin family member 2C (KIF2C), is positioned on the karyomit(e) 1p34.1.The full length mRNA sequence of A2254 comprises 2886 Nucleotide, 725 amino acid of encoding.

A2254 has two kinds of different variants of transcribing, they are made of 21 and 20 exons respectively, correspond respectively to A2254V1 (GenBank accession number NO:AB264115, SEQ ID NO:35,36) and A2254V2 (GenBank accession number NO:AY026505, SEQ ID NO:37,38) (Fig. 3 A, last drawing).The exons 1 of V1 variant and 2 is respectively 185bp and 94bp, and the V2 variant does not have the exons 1 and 2 of V1, and has a new exon that is made of 346bp as exons 1.Last exon (extron 20) of V2 variant is than the short 537bp of 3 ' end of last exon (exon 2 1) of V1 variant.The full length cDNA sequence of A2254V1 and A2254V2 variant contains 2886 and 2401 Nucleotide respectively.The ORF of these variants starts from each exons 1.At last, V1 and V2 transcript 725 and 671 amino acid of encoding respectively.In order to confirm that further every kind of variant expression in mammary cancer sample and health adult tissue is graphic, we carry out sxemiquantitative RT-PCR with the primer sets of every kind of variant of identification.As a result, we find that compare with the expression of V2 variant, A2254 V1 variant is mainly expressed in breast cancer cell, and the V2 variant is only expressed (Fig. 3 A in testis; Following drawing).Therefore, we pay close attention to A2254 V1 variant.

A5623 also has three kinds of different variants of transcribing, and they are made of 15,14 and 14 exons respectively, correspond respectively to A5623V1 (Genbank accession number NO:NM_003981; SEQ ID NO:39,40), A5623V2 (Genbank accession number NO:NM_199413; SEQ ID NO:41,42) and A5623V3 (Genbank accession number NO:NM_199414; SEQ ID NO:43,44) (Fig. 3 B, last drawing).The exons 13 and 14 of V1 has selectable version, and it is identical that all the other exons keep in all variants.The V2 variant does not have the exons 14 of V1, in the end produces new premature termination codon in an exon.The exons 14 of V3 is lacked fully, and the exons 13 of V3 than the exons 13 of V1 at the short 77bp of 3 ' end, in the end also produce a new premature termination codon in an exon.The full length cDNA sequence of A5623V1, A5623V2 and A5623V3 variant is made of 3128,3091 and 3011 Nucleotide respectively.The ORF of these variants starts from each exons 1.At last, V1, V2 and V3 transcript 620,606 and 566 amino acid of encoding respectively.In order further to confirm the expression pattern of every kind of variant in mammary cancer sample and health adult tissue, we have carried out sxemiquantitative RT-PCR with the primer sets of every kind of variant of identification.As a result, we find that compare with health adult tissue, all variants are crossed high expression level (Fig. 3 B at breast cancer cell cell camber; Following drawing).Therefore, we have further carried out functional analysis to whole variants of A5623.

The Subcellular Localization of A2254 and A5623

In order further to investigate the feature of A2254 and A5623, we have checked the Subcellular Localization of these gene products in the COS7 cell.At first, the plasmid transient transfection that will express A2254 albumen (pCAGGS-A2254-HA) when us is in the COS7 cell time, and we have observed the 81KD-A2254 albumen (Fig. 4 A) with expection size by the Western engram analysis.In addition, immunocytochemical stain shows that external source A2254 is positioned at (Fig. 4 B) under the plasma membrane in transfectional cell.Surprisingly, the positive signal in the immunocytochemical stain disappears in the film of cell-cell attachment, points out this gene to bring into play keying action in cell-cell interaction or cell polarity.

Then, the plasmid transient transfection that to express A5623 V1, V2 and V3 albumen (pCAGGS-A5623-HA) again when us has been observed external source A5623 V1, V2 and the V3 albumen (Fig. 4 C) with expection size separately in COS7 and T47D cell the time by the Western engram analysis.And immunocytochemical stain shows that whole misfolded proteins of A5623 all are positioned tenuigenin device (Fig. 4 D, E, F) as median fiber in transfectional cell, and prompting A5623 may also bring into play keying action in cell-cell interaction.

We have developed the polyclonal antibody of anti-A5623 or A2254, then by the Western engram analysis, as experiment contrast, A5623 albumen or A2254 albumen have been studied from the endogenous expression in the cell lysate of breast cancer cell line BT-474, BT-549, HBC4, HBC5, HBL-100, MCF-7, MDA-MB-231, SKBR3 and T47D with HMEC (people's mammary epithelial cell).All breast cancer cell line has shown that all high-caliber A5623 or A2254 express, and the HMEC cell shows very faint expression.With anti-A5623 polyclonal antibody breast cancer cell line HBC4, HBC5 and MCF7 are carried out the immunocytochemical assay demonstration subsequently, endogenous A5623 mainly is positioned in the tenuigenin and/or nucleus device of medium cell.Especially, observe the median fiber network that endogenous A5623 is positioned at all breast cancer cell lines.When the mitotic division of cell experience, A5623 takes place to distribute significantly again.In early stage, it and mitotic spindle be the location extremely altogether, and commitment from the mid-term to the later stage and whole mitotic spindle are located altogether then.Till the middle and later periods, A5623 concentrates in the later stage of cell spindle body intermediate zone becomes a series of arrowbands (Fig. 7 B).With anti-A2254 polyclonal antibody breast cancer cell line, HBC5 are carried out immunocytochemical stain subsequently and show, endogenous A2254 is observed in the tenuigenin device that mainly is positioned medium cell, although external source A2254 is positioned under the plasma membrane of transfectional cell.When the mitotic division of cell experience, the A2254 experience distributes significantly again.Although A2254 still is positioned in medium cell in the tenuigenin, A2254 concentrates in the later stage of cell spindle body intermediate zone becomes a series of arrowbands (Fig. 7 B).At last, this protein accumulation is at the intermediate of cell in latter stage.These find prompting, (Mollinari C, et al.JCell Biol, 2002 in breast cancer cell and previously described HeLa cell; 157; 1175-86.; Mollinari C, et al.Mol Biol Cell.2005; 16; 1043-55.), A5623 and A2254 play a significant role in division of cytoplasm.

For the further expression of research A5623 in mammary cancer and healthy tissues section, we have carried out immunohistochemical staining with anti-A5623 antibody.We confirm that the mammary cancer three kinds of different tissues hypotypes is in the tenuigenin of comedocarcinoma, ductus papillaris cancer and inocarcinoma and the nucleus strong dyeing to be arranged, but it is expressed in and almost detects in the normal galactophore tissue less than (Fig. 7 C, last drawing).And the result is consistent with the Northen engram analysis, detects its expression in testis, and there is no expression (Fig. 7 C, following drawing) in heart, lung, liver and kidney.

Be designed to reduce the growth inhibitory effect of the siRNA (siRNA) that A2254 and A5623 express

In order to assess the growth promoting function of A2254 and A5623, we are means to disturb (RNAi) technology (seeing above) based on the RNA of Mammals carrier, strike to show the expression that A2254 and A5623 cross endogenous A2254 and A5623 among the breast cancer cell line T47D of high expression level and the HBC5 low.By sxemiquantitative RT-PCR experimental check the expression level of A2254 and A5623.As illustrated in Figures 5 and 6, compare with contrast siRNA construct (psiU6BX-simulation), these two genes siRNA construct separately---A2254 (si2 and si5) and A5623 (si1 and si2) specific siRNA, significantly suppressed separately expression of gene (Fig. 5 A, 6A).In order to confirm the cell growth-inhibiting of A2254 and A5623 specific siRNA s, we have implemented respectively, and colony forms and MTT measures.A2254 (si2 and si5) (Fig. 5 B, C) and A5623 (si1 and si2) (Fig. 6 B, the C) introducing of construct have suppressed the growth of T47D and HBC5 cell, and be consistent with the result who above reduces expression.Each result has obtained the checking of 3 independent experiments.These find prompting, and A2254 and A5623 have important function in the breast cancer cell growth.

The carcinogenic activity of A2254

In order further to confirm the growth promoting function of A2254, we have set up the NIH3T3-derived cell (NIH3T3-A2254-1 ,-2 ,-3 and-4 cells) of stable expression of exogenous A2254.The Western engram analysis shows in three clones that derive (NIH3T3-A2254-1 ,-2 and-3) high-caliber external source A2254 albumen, and low-level A2254 (Fig. 9 A) is arranged in a clone (A2254-4).Subsequently MTT measures and shows, the high expression level cell growth of crossing of external source A2254 does not have and significantly improves (Fig. 9 B).These find prompting, although singly be that the A2254 gene is crossed high expression level and do not had growth-promoting activity, the survival that this gene product lacks for breast cancer cell has material impact.

And, because our first observed is positioned under the plasma membrane of transfectional cell to external source A2254,, we soak into to measure with definite A2254 in cell mobility, whether to bring into play certain effect so carrying out matrigel.The NIH3T3-derived cell of stable expression of exogenous A2254 (NIH3T3-A2254-3 and-4 cells) is by the infiltration of matrigel, significantly improve than simulation stable transfected cells (NIH3T3-simulation), and show that according to Western trace result this raising depends on A2254 protein expression (Fig. 9 C), this shows that the A2254 gene may also have keying action in tumor cell invasion.

The interaction of A5623 and A2254

In order further to study the physiological function of A5623 in breast cancer cell, we have attempted identifying its interaction protein.We find that kinesin family member 2C/ mitotic division-kinetochore-associated drives albumen (KIF2c/MCAK) albumen (A2254) is possible candidate A5623-interaction protein, because this albumen is known at late-anaphase or be positioned in the intermediate in the cell latter stage or near the shrunk ring, and the intermediate zone form and division of cytoplasm in play a role.In addition, report A5623 and several kinesin family proteins interact (Kurasawa Y, et al.EMBO J 2004; 23; 3237-48.; ZhuC, et al.Proc Natl Acad Sci USA 2005; 102; 343-8.; Gruneberg U, et al.2006; 172; 363-72.).

We are at first graphic by the expression of sxemiquantitative RT-PCR analysis and research A2254 in the mammary cancer case, find that A2254 and A5623 raise (Fig. 8 A) altogether in the mammary cancer case.Subsequently, we carry out the co-immunoprecipitation experiment with cotransfection to Flag-label A 5623 and HA-label A 2254 in the COS7 cell.Utilize anti-Flag and anti-HA antibody, we confirm Flag-label A 5623 and HA-label A 2254 co-precipitation (Fig. 8 B), and HA-label A 2254 conversely with Flag-label A 5623 coprecipitated label (Fig. 8 C), show this two kinds of protein-interactings.

Subsequently, we have set up the NIH3T3-derived cell (NIH3T3-A5623 and NIH3T3-A2254 cell) of stable expression of exogenous A5623 and A2254.Immunocytochemical assay shows, the external source A2254 of endogenous mouse A5623 and stably express is positioned the intermediate zone altogether at late-anaphase and forms thing (Fig. 8 D in the NIH3T3 cell, left side drawing), the external source A5623 of endogenous A2254 and stably express is positioned the intermediate zone altogether at late-anaphase and forms thing (Fig. 8 D) in the NIH3T3 cell.Although also need further to study endogenous A5623 and the common location of endogenous A2254 in breast cancer cell, these results point out the structure picture (conformation) of A5623 and A2254 to bring into play keying action in the division of cytoplasm of cancer cells strongly.

[discussion]

The accurate expression pattern of the mammary cancer by utilizing full genome cDNA microarray technology, we have separated new gene A 2254 and A5623, compare with health adult tissue, and they are the remarkable high expression level of crossing in breast cancer cell.

And the Northern engram analysis shows, being expressed in any health adult tissue that is checked except that testis and marrow of A5623 and A2254 almost can not detect.And the immunohistochemical staining experiment that utilizes anti-A5623 polyclonal antibody or anti-A2254 polyclonal antibody to carry out shows that clearly being expressed in breast cancer tissue's section of A5623 or A2254 raised.These results show that this gene may become the valuable target of exploitation mammary cancer carcinostatic agent.

Our immunocytochemical stain experiment shows that A5623 is positioned tenuigenin and/or nucleus in intermitotic breast cancer cell, be positioned the intermediate zone at late-anaphase, is positioned shrunk ring in latter stage.

Further, we confirm, handle breast cancer cell with siRNA, have effectively suppressed the expression of all three kinds of target gene A2254 and A5623, and have significantly suppressed breast cancer cell/growth of tumor.These find prompting, and A2254 and 5623 may bring into play keying action in growth of tumour cell propagation, and can be used as the target that is used to develop cancer therapy drug with prospect.

We confirm, strike low endogenous A5623 by siRNA and lure division of cytoplasm failure in the breast cancer cell into, cause coenocytic accumulation and necrocytosis subsequently.These find prompting, and A5623 also plays a role in the division of cytoplasm of breast cancer cell.A5623 it is reported also and several kinesin family proteins interact (Kurasawa Y, et al.EMBO J 2004; 23; 3237-48.; Zhu C, et al.ProcNatl Acad Sci USA 2005; 102; 343-8.; Gruneberg U, et al.2006; 172; 363-72.).According to report, A5623 can with several molecules, for example relevant KIF4 or KIF14 interact (Kurasawa Y, et al.EMBO J 2004 with mitotic division incident, particularly division of cytoplasm; 23; 3237-48.; Zhu C, et al.Proc Natl Acad Sci USA 2005; 102; 343-8.).Yet in our whole mammary cancer expression pattern, KIF4 and KIF14 all do not express (data not shown) in mammary cancer.Therefore, we further are tested and appraised the interaction protein of A5623 and have investigated its biological action in breast cancer cell, and identify that A2254 (kinesin family member 2C/ mitotic division-kinetochore associated drives albumen) albumen is as candidate's interaction protein, because this albumen is known at late-anaphase or be positioned in the intermediate in the cell latter stage or near the shrunk ring, and the intermediate zone form and division of cytoplasm in play a role.As shown in Figure 8, we confirmed in the cell cycle, particularly interacted and were total in the body of A2254 and A5623 in the intermediate when latter stage, cell cytoplasm divided and locate.Their evidence promptings in the mammary cancer case, their interaction is brought into play keying action in mammary cancer takes place.And we have determined the land (51-70 and 200-490 amino acid) (data not shown) of A5623 and A2254.Although also need further to analyze the function of A5623, the data that provided should help more in depth to understand the generation of mammary cancer and the novel method of treatment of exploitation mammary cancer.

Industrial usability

The inventor has proved siRNA (siRNA) cell growth inhibiting of selectively targeted A2254 or A5623 gene.Therefore, this novel siRNA is the useful target that is used to develop cancer therapy drug.For example, blocking-up A2254 or A5623 express or stop its active medicament useful asticancer agents, especially for the carcinostatic agent of treatment mammary cancer (BRC).

Although the present invention is described in detail with reference to its specific embodiment, those skilled in the art obviously knows under the prerequisite that does not deviate from spirit and scope of the invention can make variations and modifications.

Sequence table

＜110〉Oncotherapy Science Inc (ONCOTHERAPY SCIENCE, INC.)

＜120〉be used for the treatment of the composition and the method for mammary cancer

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<400>28

aaaaggaaag?actcatcaaa?agctctcttg?aagcttttga?tgagtctttc?c 51

<210>29

<211>19

<212>DNA

＜213〉artificial

<220>

＜223〉siRNA target sequence

<400>29

ggaaagactc?atcaaaagc 19

<210>30

<211>47

<212>DNA

＜213〉artificial

<220>

＜223〉siRNA hairpin structure

<400>30

ggaaagactc?atcaaaagct?tcaagagagc?ttttgatgag?tctttcc 47

<210>31

<211>51

<212>DNA

＜213〉artificial

<220>

＜223〉be used for the artificial synthetic oligonucleotide of siRNA

<400>31

caccgcatat?ccgtctgtca?gaattcaaga?gattctgaca?gacggatatg?c 51

<210>32

<211>51

<212>DNA

＜213〉artificial

<220>

＜223〉be used for the artificial synthetic oligonucleotide of siRNA

<400>32

aaaagcatat?ccgtctgtca?gaatctcttg?aattctgaca?gacggatatg?c 51

<210>33

<211>20

<212>DNA

＜213〉artificial

<220>

＜223〉siRNA target sequence

<400>33

gcatatccgt?ctgtcagaat 20

<210>34

<211>47

<212>DNA

＜213〉artificial

<220>

＜223〉siRNA hairpin structure

<400>34

gcatatccgt?ctgtcagaat?tcaagagatt?ctgacagacg?gatatgc 47

<210>35

<211>2886

<212>DNA

＜213〉people (Homo sapiens)

<220>

<221>CDS

<222>(116)..(2290)

<400>35

acgcttgcgc?gcgggattta?aactgcggcg?gtttacgcgg?cgttaagact?tcgtagggtt 60

agcgaaattg?aggtttcttg?gtattgcgcg?tttctcttcc?ttgctgactc?tccga?atg 118

Met

1

gcc?atg?gac?tcg?tcg?ctt?cag?gcc?cgc?ctg?ttt?ccc?ggt?ctc?gct?atc 166

Ala?Met?Asp?Ser?Ser?Leu?Gln?Ala?Arg?Leu?Phe?Pro?Gly?Leu?Ala?Ile

5 10 15

aag?atc?caa?cgc?agt?aat?ggt?tta?att?cac?agt?gcc?aat?gta?agg?act 214

Lys?Ile?Gln?Arg?Ser?Asn?Gly?Leu?Ile?His?Ser?Ala?Asn?Val?Arg?Thr

20 25 30

gtg?aac?ttg?gag?aaa?tcc?tgt?gtt?tca?gtg?gaa?tgg?gca?gaa?gga?ggt 262

Val?Asn?Leu?Glu?Lys?Ser?Cys?Val?Ser?Val?Glu?Trp?Ala?Glu?Gly?Gly

35 40 45

gcc?aca?aag?ggc?aaa?gag?att?gat?ttt?gat?gat?gtg?gct?gca?ata?aac 310

Ala?Thr?Lys?Gly?Lys?Glu?Ile?Asp?Phe?Asp?Asp?Val?Ala?Ala?Ile?Asn

50 55 60 65

cca?gaa?ctc?tta?cag?ctt?ctt?ccc?tta?cat?ccg?aag?gac?aat?ctg?ccc 358

Pro?Glu?Leu?Leu?Gln?Leu?Leu?Pro?Leu?His?Pro?Lys?Asp?Asn?Leu?Pro

70 75 80

ttg?cag?gaa?aat?gta?aca?atc?cag?aaa?caa?aaa?cgg?aga?tcc?gtc?aac 406

Leu?Gln?Glu?Asn?Val?Thr?Ile?Gln?Lys?Gln?Lys?Arg?Arg?Ser?Val?Asn

85 90 95

tcc?aaa?att?cct?gct?cca?aaa?gaa?agt?ctt?cga?agc?cgc?tcc?act?cgc 454

Ser?Lys?Ile?Pro?Ala?Pro?Lys?Glu?Ser?Leu?Arg?Ser?Arg?Ser?Thr?Arg

100 105 110

atg?tcc?act?gtc?tca?gag?ctt?cgc?atc?acg?gct?cag?gag?aat?gac?atg 502

Met?Ser?Thr?Val?Ser?Glu?Leu?Arg?Ile?Thr?Ala?Gln?Glu?Asn?Asp?Met

115 120 125

gag?gtg?gag?ctg?cct?gca?gct?gca?aac?tcc?cgc?aag?cag?ttt?tca?gtt 550

Glu?Val?Glu?Leu?Pro?Ala?Ala?Ala?Asn?Ser?Arg?Lys?Gln?Phe?Ser?Val

130 135 140 145

cct?cct?gcc?ccc?act?agg?cct?tcc?tgc?cct?gca?gtg?gct?gaa?ata?cca 598

Pro?Pro?Ala?Pro?Thr?Arg?Pro?Ser?Cys?Pro?Ala?Val?Ala?Glu?Ile?Pro

150 155 160

ttg?agg?atg?gtc?agc?gag?gag?atg?gaa?gag?caa?gtc?cat?tcc?atc?cgt 646

Leu?Arg?Met?Val?Ser?Glu?Glu?Met?Glu?Glu?Gln?Val?His?Ser?Ile?Arg

165 170 175

ggc?agc?tct?tct?gca?aac?cct?gtg?aac?tca?gtt?cgg?agg?aaa?tca?tgt 694

Gly?Ser?Ser?Ser?Ala?Asn?Pro?Val?Asn?Ser?Val?Arg?Arg?Lys?Ser?Cys

180 185 190

ctt?gtg?aag?gaa?gtg?gaa?aaa?atg?aag?aac?aag?cga?gaa?gag?aag?aag 742

Leu?Val?Lys?Glu?Val?Glu?Lys?Met?Lys?Asn?Lys?Arg?Glu?Glu?Lys?Lys

195 200 205

gcc?cag?aac?tct?gaa?atg?aga?atg?aag?aga?gct?cag?gag?tat?gac?agt 790

Ala?Gln?Asn?Ser?Glu?Met?Arg?Met?Lys?Arg?Ala?Gln?Glu?Tyr?Asp?Ser

210 215 220 225

agt?ttt?cca?aac?tgg?gaa?ttt?gcc?cga?atg?att?aaa?gaa?ttt?cgg?gct 838

Ser?Phe?Pro?Asn?Trp?Glu?Phe?Ala?Arg?Met?Ile?Lys?Glu?Phe?Arg?Ala

230 235 240

act?ttg?gaa?tgt?cat?cca?ctt?act?atg?act?gat?cct?atc?gaa?gag?cac 886

Thr?Leu?Glu?Cys?His?Pro?Leu?Thr?Met?Thr?Asp?Pro?Ile?Glu?Glu?His

245 250 255

aga?ata?tgt?gtc?tgt?gtt?agg?aaa?cgc?cca?ctg?aat?aag?caa?gaa?ttg 934

Arg?Ile?Cys?Val?Cys?Val?Arg?Lys?Arg?Pro?Leu?Asn?Lys?Gln?Glu?Leu

260 265 270

gcc?aag?aaa?gaa?att?gat?gtg?att?tcc?att?cct?agc?aag?tgt?ctc?ctc 982

Ala?Lys?Lys?Glu?Ile?Asp?Val?Ile?Ser?Ile?Pro?Ser?Lys?Cys?Leu?Leu

275 280 285

ttg?gta?cat?gaa?ccc?aag?ttg?aaa?gtg?gac?tta?aca?aag?tat?ctg?gag 1030

Leu?Val?His?Glu?Pro?Lys?Leu?Lys?Val?Asp?Leu?Thr?Lys?Tyr?Leu?Glu

290 295 300 305

aac?caa?gca?ttc?tgc?ttt?gac?ttt?gca?ttt?gat?gaa?aca?gct?tcg?aat 1078

Asn?Gln?Ala?Phe?Cys?Phe?Asp?Phe?Ala?Phe?Asp?Glu?Thr?Ala?Ser?Asn

310 315 320

gaa?gtt?gtc?tac?agg?ttc?aca?gca?agg?cca?ctg?gta?cag?aca?atc?ttt 1126

Glu?Val?Val?Tyr?Arg?Phe?Thr?Ala?Arg?Pro?Leu?Val?Gln?Thr?Ile?Phe

325 330 335

gaa?ggt?gga?aaa?gca?act?tgt?ttt?gca?tat?ggc?cag?aca?gga?agt?ggc 1174

Glu?Gly?Gly?Lys?Ala?Thr?Cys?Phe?Ala?Tyr?Gly?Gln?Thr?Gly?Ser?Gly

340 345 350

aag?aca?cat?act?atg?ggc?gga?gac?ctc?tct?ggg?aaa?gcc?cag?aat?gca 1222

Lys?Thr?His?Thr?Met?Gly?Gly?Asp?Leu?Ser?Gly?Lys?Ala?Gln?Asn?Ala

355 360 365

tcc?aaa?ggg?atc?tat?gcc?atg?gcc?tcc?cgg?gac?gtc?ttc?ctc?ctg?aag 1270

Ser?Lys?Gly?Ile?Tyr?Ala?Met?Ala?Ser?Arg?Asp?Val?Phe?Leu?Leu?Lys

370 375 380 385

aat?caa?ccc?tgc?tac?cgg?aag?ttg?ggc?ctg?gaa?gtc?tat?gtg?aca?ttc 1318

Asn?Gln?Pro?Cys?Tyr?Arg?Lys?Leu?Gly?Leu?Glu?Val?Tyr?Val?Thr?Phe

390 395 400

ttc?gag?atc?tac?aat?ggg?aag?ctg?ttt?gac?ctg?ctc?aac?aag?aag?gcc 1366

Phe?Glu?Ile?Tyr?Asn?Gly?Lys?Leu?Phe?Asp?Leu?Leu?Asn?Lys?Lys?Ala

405 410 415

aag?ctg?cgc?gtg?ctg?gag?gac?ggc?aag?caa?cag?gtg?caa?gtg?gtg?ggg 1414

Lys?Leu?Arg?Val?Leu?Glu?Asp?Gly?Lys?Gln?Gln?Val?Gln?Val?Val?Gly

420 425 430

ctg?cag?gag?cat?ctg?gtt?aac?tct?gct?gat?gat?gtc?atc?aag?atg?ctc 1462

Leu?Gln?Glu?His?Leu?Val?Asn?Ser?Ala?Asp?Asp?Val?Ile?Lys?Met?Leu

435 440 445

gac?atg?ggc?agc?gcc?tgc?aga?acc?tct?ggg?cag?aca?ttt?gcc?aac?tcc 1510

Asp?Met?Gly?Ser?Ala?Cys?Arg?Thr?Ser?Gly?Gln?Thr?Phe?Ala?Asn?Ser

450 455 460 465

aat?tcc?tcc?cgc?tcc?cac?gcg?tgc?ttc?caa?att?att?ctt?cga?gct?aaa 1558

Asn?Ser?Ser?Arg?Ser?His?Ala?Cys?Phe?Gln?Ile?Ile?Leu?Arg?Ala?Lys

470 475 480

ggg?aga?atg?cat?ggc?aag?ttc?tct?ttg?gta?gat?ctg?gca?ggg?aat?gag 1606

Gly?Arg?Met?His?Gly?Lys?Phe?Ser?Leu?Val?Asp?Leu?Ala?Gly?Asn?Glu

485 490 495

cga?ggc?gca?gac?act?tcc?agt?gct?gac?cgg?cag?acc?cgc?atg?gag?ggc 1654

Arg?Gly?Ala?Asp?Thr?Ser?Ser?Ala?Asp?Arg?Gln?Thr?Arg?Met?Glu?Gly

500 505 510

gca?gaa?atc?aac?aag?agt?ctc?tta?gcc?ctg?aag?gag?tgc?atc?agg?gcc 1702

Ala?Glu?Ile?Asn?Lys?Ser?Leu?Leu?Ala?Leu?Lys?Glu?Cys?Ile?Arg?Ala

515 520 525

ctg?gga?cag?aac?aag?gct?cac?acc?ccg?ttc?cgt?gag?agc?aag?ctg?aca 1750

Leu?Gly?Gln?Asn?Lys?Ala?His?Thr?Pro?Phe?Arg?Glu?Ser?Lys?Leu?Thr

530 535 540 545

cag?gtg?ctg?agg?gac?tcc?ttc?att?ggg?gag?aac?tct?agg?act?tgc?atg 1798

Gln?Val?Leu?Arg?Asp?Ser?Phe?Ile?Gly?Glu?Asn?Ser?Arg?Thr?Cys?Met

550 555 560

att?gcc?acg?atc?tca?cca?ggc?ata?agc?tcc?tgt?gaa?tat?act?tta?aac 1846

Ile?Ala?Thr?Ile?Ser?Pro?Gly?Ile?Ser?Ser?Cys?Glu?Tyr?Thr?Leu?Asn

565 570 575

acc?ctg?aga?tat?gca?gac?agg?gtc?aag?gag?ctg?agc?ccc?cac?agt?ggg 1894

Thr?Leu?Arg?Tyr?Ala?Asp?Arg?Val?Lys?Glu?Leu?Ser?Pro?His?Ser?Gly

580 585 590

ccc?agt?gga?gag?cag?ttg?att?caa?atg?gaa?aca?gaa?gag?atg?gaa?gcc 1942

Pro?Ser?Gly?Glu?Gln?Leu?Ile?Gln?Met?Glu?Thr?Glu?Glu?Met?Glu?Ala

595 600 605

tgc?tct?aac?ggg?gcg?ctg?att?cca?ggc?aat?tta?tcc?aag?gaa?gag?gag 1990

Cys?Ser?Asn?Gly?Ala?Leu?Ile?Pro?Gly?Asn?Leu?Ser?Lys?Glu?Glu?Glu

610 615 620 625

gaa?ctg?tct?tcc?cag?atg?tcc?agc?ttt?aac?gaa?gcc?atg?act?cag?atc 2038

Glu?Leu?Ser?Ser?Gln?Met?Ser?Ser?Phe?Asn?Glu?Ala?Met?Thr?Gln?Ile

630 635 640

agg?gag?ctg?gag?gag?aag?gct?atg?gaa?gag?ctc?aag?gag?atc?ata?cag 2086

Arg?Glu?Leu?Glu?Glu?Lys?Ala?Met?Glu?Glu?Leu?Lys?Glu?Ile?Ile?Gln

645 650 655

caa?gga?cca?gac?tgg?ctt?gag?ctc?tct?gag?atg?acc?gag?cag?cca?gac 2134

Gln?Gly?Pro?Asp?Trp?Leu?Glu?Leu?Ser?Glu?Met?Thr?Glu?Gln?Pro?Asp

660 665 670

tat?gac?ctg?gag?acc?ttt?gtg?aac?aaa?gcg?gaa?tct?gct?ctg?gcc?cag 2182

Tyr?Asp?Leu?Glu?Thr?Phe?Val?Asn?Lys?Ala?Glu?Ser?Ala?Leu?Ala?Gln

675 680 685

caa?gcc?aag?cat?ttc?tca?gcc?ctg?cga?gat?gtc?atc?aag?gcc?tta?cgc 2230

Gln?Ala?Lys?His?Phe?Ser?Ala?Leu?Arg?Asp?Val?Ile?Lys?Ala?Leu?Arg

690 695 700 705

ctg?gcc?atg?cag?ctg?gaa?gag?cag?gct?agc?aga?caa?ata?agc?agc?aag 2278

Leu?Ala?Met?Gln?Leu?Glu?Glu?Gln?Ala?Ser?Arg?Gln?Ile?Ser?Ser?Lys

710 715 720

aaa?cgg?ccc?cag?tgacgactgc?aaataaaaat?ctgtttggtt?tgacacccag 2330

Lys?Arg?Pro?Gln

725

cctcttccct?ggccctcccc?agagaacttt?gggtacctgg?tgggtctagg?cagggtctga 2390

gctgggacag?gttctggtaa?atgccaagta?tgggggcatc?tgggcccagg?gcagctgggg 2450

agggggtcag?agtgacatgg?gacactcctt?ttctgttcct?cagttgtcgc?cctcacgaga 2510

ggaaggagct?cttagttacc?cttttgtgtt?gcccttcttt?ccatcaaggg?gaatgttctc 2570

agcatagagc?tttctccgca?gcatcctgcc?tgcgtggact?ggctgctaat?ggagagctcc 2630

ctggggttgt?cctggctctg?gggagagaga?cggagccttt?agtacagcta?tctgctggct 2690

ctaaaccttc?tacgcctttg?ggccgagcac?tgaatgtctt?gtactttaaa?aaaatgtttc 2750

tgagacctct?ttctacttta?ctgtctccct?agagtcctag?aggatcccta?ctgttttctg 2810

ttttatgtgt?ttatacattg?tatgtaacaa?taaagagaaa?aaataaaaaa?aaaaaaaaaa 2870

aaaaaaaaaa?aaaaaa 2886

<210>36

<211>725

<212>PRT

＜213〉people (Homo sapiens)

<400>36

Met?Ala?Met?Asp?Ser?Ser?Leu?Gln?Ala?Arg?Leu?Phe?Pro?Gly?Leu?Ala

1 5 10 15

Ile?Lys?Ile?Gln?Arg?Ser?Asn?Gly?Leu?Ile?His?Ser?Ala?Asn?Val?Arg

20 25 30

Thr?Val?Asn?Leu?Glu?Lys?Ser?Cys?Val?Ser?Val?Glu?Trp?Ala?Glu?Gly

35 40 45

Gly?Ala?Thr?Lys?Gly?Lys?Glu?Ile?Asp?Phe?Asp?Asp?Val?Ala?Ala?Ile

50 55 60

Asn?Pro?Glu?Leu?Leu?Gln?Leu?Leu?Pro?Leu?His?Pro?Lys?Asp?Asn?Leu

65 70 75 80

Pro?Leu?Gln?Glu?Asn?Val?Thr?Ile?Gln?Lys?Gln?Lys?Arg?Arg?Ser?Val

85 90 95

Asn?Ser?Lys?Ile?Pro?Ala?Pro?Lys?Glu?Ser?Leu?Arg?Ser?Arg?Ser?Thr

100 105 110

Arg?Met?Ser?Thr?Val?Ser?Glu?Leu?Arg?Ile?Thr?Ala?Gln?Glu?Asn?Asp

115 120 125

Met?Glu?Val?Glu?Leu?Pro?Ala?Ala?Ala?Asn?Ser?Arg?Lys?Gln?Phe?Ser

130 135 140

Val?Pro?Pro?Ala?Pro?Thr?Arg?Pro?Ser?Cys?Pro?Ala?Val?Ala?Glu?Ile

145 150 155 160

Pro?Leu?Arg?Met?Val?Ser?Glu?Glu?Met?Glu?Glu?Gln?Val?His?Ser?Ile

165 170 175

Arg?Gly?Ser?Ser?Ser?Ala?Asn?Pro?Val?Asn?Ser?Val?Arg?Arg?Lys?Ser

180 185 190

Cys?Leu?Val?Lys?Glu?Val?Glu?Lys?Met?Lys?Asn?Lys?Arg?Glu?Glu?Lys

195 200 205

Lys?Ala?Gln?Asn?Ser?Glu?Met?Arg?Met?Lys?Arg?Ala?Gln?Glu?Tyr?Asp

210 215 220

Ser?Ser?Phe?Pro?Asn?Trp?Glu?Phe?Ala?Arg?Met?Ile?Lys?Glu?Phe?Arg

225 230 235 240

Ala?Thr?Leu?Glu?Cys?His?Pro?Leu?Thr?Met?Thr?Asp?Pro?Ile?Glu?Glu

245 250 255

His?Arg?Ile?Cys?Val?Cys?Val?Arg?Lys?Arg?Pro?Leu?Asn?Lys?Gln?Glu

260 265 270

Leu?Ala?Lys?Lys?Glu?Ile?Asp?Val?Ile?Ser?Ile?Pro?Ser?Lys?Cys?Leu

275 280 285

Leu?Leu?Val?His?Glu?Pro?Lys?Leu?Lys?Val?Asp?Leu?Thr?Lys?Tyr?Leu

290 295 300

Glu?Asn?Gln?Ala?Phe?Cys?Phe?Asp?Phe?Ala?Phe?Asp?Glu?Thr?Ala?Ser

305 310 315 320

Asn?Glu?Val?Val?Tyr?Arg?Phe?Thr?Ala?Arg?Pro?Leu?Val?Gln?Thr?Ile

325 330 335

Phe?Glu?Gly?Gly?Lys?Ala?Thr?Cys?Phe?Ala?Tyr?Gly?Gln?Thr?Gly?Ser

340 345 350

Gly?Lys?Thr?His?Thr?Met?Gly?Gly?Asp?Leu?Ser?Gly?Lys?Ala?Gln?Asn

355 360 365

Ala?Ser?Lys?Gly?Ile?Tyr?Ala?Met?Ala?Ser?Arg?Asp?Val?Phe?Leu?Leu

370 375 380

Lys?Asn?Gln?Pro?Cys?Tyr?Arg?Lys?Leu?Gly?Leu?Glu?Val?Tyr?Val?Thr

385 390 395 400

Phe?Phe?Glu?Ile?Tyr?Asn?Gly?Lys?Leu?Phe?Asp?Leu?Leu?Asn?Lys?Lys

405 410 415

Ala?Lys?Leu?Arg?Val?Leu?Glu?Asp?Gly?Lys?Gln?Gln?Val?Gln?Val?Val

420 425 430

Gly?Leu?Gln?Glu?His?Leu?Val?Asn?Ser?Ala?Asp?Asp?Val?Ile?Lys?Met

435 440 445

Leu?Asp?Met?Gly?Ser?Ala?Cys?Arg?Thr?Ser?Gly?Gln?Thr?Phe?Ala?Asn

450 455 460

Ser?Asn?Ser?Ser?Arg?Ser?His?Ala?Cys?Phe?Gln?Ile?Ile?Leu?Arg?Ala

465 470 475 480

Lys?Gly?Arg?Met?His?Gly?Lys?Phe?Ser?Leu?Val?Asp?Leu?Ala?Gly?Asn

485 490 495

Glu?Arg?Gly?Ala?Asp?Thr?Ser?Ser?Ala?Asp?Arg?Gln?Thr?Arg?Met?Glu

500 505 510

Gly?Ala?Glu?Ile?Asn?Lys?Ser?Leu?Leu?Ala?Leu?Lys?Glu?Cys?Ile?Arg

515 520 525

Ala?Leu?Gly?Gln?Asn?Lys?Ala?His?Thr?Pro?Phe?Arg?Glu?Ser?Lys?Leu

530 535 540

Thr?Gln?Val?Leu?Arg?Asp?Ser?Phe?Ile?Gly?Glu?Asn?Ser?Arg?Thr?Cys

545 550 555 560

Met?Ile?Ala?Thr?Ile?Ser?Pro?Gly?Ile?Ser?Ser?Cys?Glu?Tyr?Thr?Leu

565 570 575

Asn?Thr?Leu?Arg?Tyr?Ala?Asp?Arg?Val?Lys?Glu?Leu?Ser?Pro?His?Ser

580 585 590

Gly?Pro?Ser?Gly?Glu?Gln?Leu?Ile?Gln?Met?Glu?Thr?Glu?Glu?Met?Glu

595 600 605

Ala?Cys?Ser?Asn?Gly?Ala?Leu?Ile?Pro?Gly?Asn?Leu?Ser?Lys?Glu?Glu

610 615 620

Glu?Glu?Leu?Ser?Ser?Gln?Met?Ser?Ser?Phe?Asn?Glu?Ala?Met?Thr?Gln

625 630 635 640

Ile?Arg?Glu?Leu?Glu?Glu?Lys?Ala?Met?Glu?Glu?Leu?Lys?Glu?Ile?Ile

645 650 655

Gln?Gln?Gly?Pro?Asp?Trp?Leu?Glu?Leu?Ser?Glu?Met?Thr?Glu?Gln?Pro

660 665 670

Asp?Tyr?Asp?Leu?Glu?Thr?Phe?Val?Asn?Lys?Ala?Glu?Ser?Ala?Leu?Ala

675 680 685

Gln?Gln?Ala?Lys?His?Phe?Ser?Ala?Leu?Arg?Asp?Val?Ile?Lys?Ala?Leu

690 695 700

Arg?Leu?Ala?Met?Gln?Leu?Glu?Glu?Gln?Ala?Ser?Arg?Gln?Ile?Ser?Ser

705 710 715 720

Lys?Lys?Arg?Pro?Gln

725

<210>37

<211>2401

<212>DNA

＜213〉people (Homo sapiens)

<220>

<221>CDS

<222>(349)..(2364)

<400>37

gggggtaact?ggggaagttt?ctgggcccac?aagaaaatgt?aggtagtctt?agggttaggt 60

ctcagctgtc?aggaacttgc?ccctgcccat?aagatcctaa?agggccccca?tttgactctc 120

accagacagt?tagaacttgt?ttcctcctcc?gtgtcagcca?tcaagaggtg?cttggggggc 180

tgtgcccagc?aggacctcac?tgcccagcag?atcagcaggg?gagccaagtg?gcctagatct 240

gctgtggagt?acccgactgt?ttgcctgcct?gtctgccctc?ctcttcacct?cattctcatc 300

actgacgtct?accattggct?tggaaatcag?aaacccatat?ccatctag?atg?att?gat 357

Met?Ile?Asp

1

ttt?gat?gat?gtg?gct?gca?ata?aac?cca?gaa?ctc?tta?cag?ctt?ctt?ccc 405

Phe?Asp?Asp?Val?Ala?Ala?Ile?Asn?Pro?Glu?Leu?Leu?Gln?Leu?Leu?Pro

5 10 15

tta?cat?ccg?aag?gac?aat?ctg?ccc?ttg?cag?gaa?aat?gta?aca?atc?cag 453

Leu?His?Pro?Lys?Asp?Asn?Leu?Pro?Leu?Gln?Glu?Asn?Val?Thr?Ile?Gln

20 25 30 35

aaa?caa?aaa?cgg?aga?tcc?gtc?aac?tcc?aaa?att?cct?gct?cca?aaa?gaa 501

Lys?Gln?Lys?Arg?Arg?Ser?Val?Asn?Ser?Lys?Ile?Pro?Ala?Pro?Lys?Glu

40 45 50

agt?ctt?cga?agc?cgc?tcc?act?cgc?atg?tcc?act?gtc?tca?gag?ctt?cgc 549

Ser?Leu?Arg?Ser?Arg?Ser?Thr?Arg?Met?Ser?Thr?Val?Ser?Glu?Leu?Arg

55 60 65

atc?acg?gct?cag?gag?aat?gac?atg?gag?gtg?gag?ctg?cct?gca?gct?gca 597

Ile?Thr?Ala?Gln?Glu?Asn?Asp?Met?Glu?Val?Glu?Leu?Pro?Ala?Ala?Ala

70 75 80

aac?tcc?cgc?aag?cag?ttt?tca?gtt?cct?cct?gcc?ccc?act?agg?cct?tcc 645

Asn?Ser?Arg?Lys?Gln?Phe?Ser?Val?Pro?Pro?Ala?Pro?Thr?Arg?Pro?Ser

85 90 95

tgc?cct?gca?gtg?gct?gaa?ata?cca?ttg?agg?atg?gtc?agc?gag?gag?atg 693

Cys?Pro?Ala?Val?Ala?Glu?Ile?Pro?Leu?Arg?Met?Val?Ser?Glu?Glu?Met

100 105 110 115

gaa?gag?caa?gtc?cat?tcc?atc?cgt?ggc?agc?tct?tct?gca?aac?cct?gtg 741

Glu?Glu?Gln?Val?His?Ser?Ile?Arg?Gly?Ser?Ser?Ser?Ala?Asn?Pro?Val

120 125 130

aac?tca?gtt?cgg?agg?aaa?tca?tgt?ctt?gtg?aag?gaa?gtg?gaa?aaa?atg 789

Asn?Ser?Val?Arg?Arg?Lys?Ser?Cys?Leu?Val?Lys?Glu?Val?Glu?Lys?Met

135 140 145

aag?aac?aag?cga?gaa?gag?aag?aag?gcc?cag?aac?tct?gaa?atg?aga?atg 837

Lys?Asn?Lys?Arg?Glu?Glu?Lys?Lys?Ala?Gln?Asn?Ser?Glu?Met?Arg?Met

150 155 160

aag?aga?gct?cag?gag?tat?gac?agt?agt?ttt?cca?aac?tgg?gaa?ttt?gcc 885

Lys?Arg?Ala?Gln?Glu?Tyr?Asp?Ser?Ser?Phe?Pro?Asn?Trp?Glu?Phe?Ala

165 170 175

cga?atg?att?aaa?gaa?ttt?cgg?gct?act?ttg?gaa?tgt?cat?cca?ctt?act 933

Arg?Met?Ile?Lys?Glu?Phe?Arg?Ala?Thr?Leu?Glu?Cys?His?Pro?Leu?Thr

180 185 190 195

atg?act?gat?cct?atc?gaa?gag?cac?aga?ata?tgt?gtc?tgt?gtt?agg?aaa 981

Met?Thr?Asp?Pro?Ile?Glu?Glu?His?Arg?Ile?Cys?Val?Cys?Val?Arg?Lys

200 205 210

cgc?cca?ctg?aat?aag?caa?gaa?ttg?gcc?aag?aaa?gaa?att?gat?gtg?att 1029

Arg?Pro?Leu?Asn?Lys?Gln?Glu?Leu?Ala?Lys?Lys?Glu?Ile?Asp?Val?Ile

215 220 225

tcc?att?cct?agc?aag?tgt?ctc?ctc?ttg?gta?cat?gaa?ccc?aag?ttg?aaa 1077

Ser?Ile?Pro?Ser?Lys?Cys?Leu?Leu?Leu?Val?His?Glu?Pro?Lys?Leu?Lys

230 235 240

gtg?gac?tta?aca?aag?tat?ctg?gag?aac?caa?gca?ttc?tgc?ttt?gac?ttt 1125

Val?Asp?Leu?Thr?Lys?Tyr?Leu?Glu?Asn?Gln?Ala?Phe?Cys?Phe?Asp?Phe

245 250 255

gca?ttt?gat?gaa?aca?gct?tcg?aat?gaa?gtt?gtc?tac?agg?ttc?aca?gca 1173

Ala?Phe?Asp?Glu?Thr?Ala?Ser?Asn?Glu?Val?Val?Tyr?Arg?Phe?Thr?Ala

260 265 270 275

agg?cca?ctg?gta?cag?aca?atc?ttt?gaa?ggt?gga?aaa?gca?act?tgt?ttt 1221

Arg?Pro?Leu?Val?Gln?Thr?Ile?Phe?Glu?Gly?Gly?Lys?Ala?Thr?Cys?Phe

280 285 290

gca?tat?ggc?cag?aca?gga?agt?ggc?aag?aca?cat?act?atg?ggc?gga?gac 1269

Ala?Tyr?Gly?Gln?Thr?Gly?Ser?Gly?Lys?Thr?His?Thr?Met?Gly?Gly?Asp

295 300 305

ctc?tct?ggg?aaa?gcc?cag?aat?gca?tcc?aaa?ggg?atc?tat?gcc?atg?gcc 1317

Leu?Ser?Gly?Lys?Ala?Gln?Asn?Ala?Ser?Lys?Gly?Ile?Tyr?Ala?Met?Ala

310 315 320

tcc?cgg?gac?gtc?ttc?ctc?ctg?aag?aat?caa?ccc?tgc?tac?cgg?aag?ttg 1365

Ser?Arg?Asp?Val?Phe?Leu?Leu?Lys?Asn?Gln?Pro?Cys?Tyr?Arg?Lys?Leu

325 330 335

ggc?ctg?gaa?gtc?tat?gtg?aca?ttc?ttc?gag?atc?tac?aat?ggg?aag?ctg 1413

Gly?Leu?Glu?Val?Tyr?Val?Thr?Phe?Phe?Glu?Ile?Tyr?Asn?Gly?Lys?Leu

340 345 350 355

ttt?gac?ctg?ctc?aac?aag?aag?gcc?aag?ctg?cgc?gtg?ctg?gag?gac?ggc 1461

Phe?Asp?Leu?Leu?Asn?Lys?Lys?Ala?Lys?Leu?Arg?Val?Leu?Glu?Asp?Gly

360 365 370

aag?caa?cag?gtg?caa?gtg?gtg?ggg?ctg?cag?gag?cat?ctg?gtt?aac?tct 1509

Lys?Gln?Gln?Val?Gln?Val?Val?Gly?Leu?Gln?Glu?His?Leu?Val?Asn?Ser

375 380 385

gct?gat?gat?gtc?atc?aag?atg?ctc?gac?atg?ggc?agc?gcc?tgc?aga?acc 1557

Ala?Asp?Asp?Val?Ile?Lys?Met?Leu?Asp?Met?Gly?Ser?Ala?Cys?Arg?Thr

390 395 400

tct?ggg?cag?aca?ttt?gcc?aac?tcc?aat?tcc?tcc?cgc?tcc?cac?gcg?tgc 1605

Ser?Gly?Gln?Thr?Phe?Ala?Asn?Ser?Asn?Ser?Ser?Arg?Ser?His?Ala?Cys

405 410 415

ttc?caa?att?att?ctt?cga?gct?aaa?ggg?aga?atg?cat?ggc?aag?ttc?tct 1653

Phe?Gln?Ile?Ile?Leu?Arg?Ala?Lys?Gly?Arg?Met?His?Gly?Lys?Phe?Ser

420 425 430 435

ttg?gta?gat?ctg?gca?ggg?aat?gag?cga?ggc?gca?gac?act?tcc?agt?gct 1701

Leu?Val?Asp?Leu?Ala?Gly?Asn?Glu?Arg?Gly?Ala?Asp?Thr?Ser?Ser?Ala

440 445 450

gac?cgg?cag?acc?cgc?atg?gag?ggc?gca?gaa?atc?aac?aag?agt?ctc?tta 1749

Asp?Arg?Gln?Thr?Arg?Met?Glu?Gly?Ala?Glu?Ile?Asn?Lys?Ser?Leu?Leu

455 460 465

gcc?ctg?aag?gag?tgc?atc?agg?gcc?ctg?gga?cag?aac?aag?gct?cac?acc 1797

Ala?Leu?Lys?Glu?Cys?Ile?Arg?Ala?Leu?Gly?Gln?Asn?Lys?Ala?His?Thr

470 475 480

ccg?ttc?cgt?gag?agc?aag?ctg?aca?cag?gtg?ctg?agg?gac?tcc?ttc?att 1845

Pro?Phe?Arg?Glu?Ser?Lys?Leu?Thr?Gln?Val?Leu?Arg?Asp?Ser?Phe?Ile

485 490 495

ggg?gag?aac?tct?agg?act?tgc?atg?att?gcc?acg?atc?tca?cca?ggc?ata 1893

Gly?Glu?Asn?Ser?Arg?Thr?Cys?Met?Ile?Ala?Thr?Ile?Ser?Pro?Gly?Ile

500 505 510 515

agc?tcc?tgt?gaa?tat?act?tta?aac?acc?ctg?aga?tat?gca?gac?agg?gtc 1941

Ser?Ser?Cys?Glu?Tyr?Thr?Leu?Asn?Thr?Leu?Arg?Tyr?Ala?Asp?Arg?Val

520 525 530

aag?gag?ctg?agc?ccc?cac?agt?ggg?ccc?agt?gga?gag?cag?ttg?att?caa 1989

Lys?Glu?Leu?Ser?Pro?His?Ser?Gly?Pro?Ser?Gly?Glu?Gln?Leu?Ile?Gln

535 540 545

atg?gaa?aca?gaa?gag?atg?gaa?gcc?tgc?tct?aac?ggg?gcg?ctg?att?cca 2037

Met?Glu?Thr?Glu?Glu?Met?Glu?Ala?Cys?Ser?Asn?Gly?Ala?Leu?Ile?Pro

550 555 560

ggc?aat?tta?tcc?aag?gaa?gag?gag?gaa?ctg?tct?tcc?cag?atg?tcc?agc 2085

Gly?Asn?Leu?Ser?Lys?Glu?Glu?Glu?Glu?Leu?Ser?Ser?Gln?Met?Ser?Ser

565 570 575

ttt?aac?gaa?gcc?atg?act?cag?atc?agg?gag?ctg?gag?gag?aag?gct?atg 2133

Phe?Asn?Glu?Ala?Met?Thr?Gln?Ile?Arg?Glu?Leu?Glu?Glu?Lys?Ala?Met

580 585 590 595

gaa?gag?ctc?aag?gag?atc?ata?cag?caa?gga?cca?gac?tgg?ctt?gag?ctc 2181

Glu?Glu?Leu?Lys?Glu?Ile?Ile?Gln?Gln?Gly?Pro?Asp?Trp?Leu?Glu?Leu

600 605 610

tct?gag?atg?acc?gag?cag?cca?gac?tat?gac?ctg?gag?acc?ttt?gtg?aac 2229

Ser?Glu?Met?Thr?Glu?Gln?Pro?Asp?Tyr?Asp?Leu?Glu?Thr?Phe?Val?Asn

615 620 625

aaa?gcg?gaa?tct?gct?ctg?gcc?cag?caa?gcc?aag?cat?ttc?tca?gcc?ctg 2277

Lys?Ala?Glu?Ser?Ala?Leu?Ala?Gln?Gln?Ala?Lys?His?Phe?Ser?Ala?Leu

630 635 640

cga?gat?gtc?atc?aag?gcc?tta?cgc?ctg?gcc?atg?cag?ctg?gaa?gag?cag 2325

Arg?Asp?Val?Ile?Lys?Ala?Leu?Arg?Leu?Ala?Met?Gln?Leu?Glu?Glu?Gln

645 650 655

gct?agc?aga?caa?ata?agc?agc?aag?aaa?cgg?ccc?cag?tga?cgactgcaaa 2374

Ala?Ser?Arg?Gln?Ile?Ser?Ser?Lys?Lys?Arg?Pro?Gln

660 665 670

taaaaatctg?tttggttcca?aaaaaaa 2401

<210>38

<211>671

<212>PRT

＜213〉people (Homo sapiens)

<400>38

Met?Ile?Asp?Phe?Asp?Asp?Val?Ala?Ala?Ile?Asn?Pro?Glu?Leu?Leu?Gln

1 5 10 15

Leu?Leu?Pro?Leu?His?Pro?Lys?Asp?Asn?Leu?Pro?Leu?Gln?Glu?Asn?Val

20 25 30

Thr?Ile?Gln?Lys?Gln?Lys?Arg?Arg?Ser?Val?Asn?Ser?Lys?Ile?Pro?Ala

35 40 45

Pro?Lys?Glu?Ser?Leu?Arg?Ser?Arg?Ser?Thr?Arg?Met?Ser?Thr?Val?Ser

50 55 60

Glu?Leu?Arg?Ile?Thr?Ala?Gln?Glu?Asn?Asp?Met?Glu?Val?Glu?Leu?Pro

65 70 75 80

Ala?Ala?Ala?Asn?Ser?Arg?Lys?Gln?Phe?Ser?Val?Pro?Pro?Ala?Pro?Thr

85 90 95

Arg?Pro?Ser?Cys?Pro?Ala?Val?Ala?Glu?Ile?Pro?Leu?Arg?Met?Val?Ser

100 105 110

Glu?Glu?Met?Glu?Glu?Gln?Val?His?Ser?Ile?Arg?Gly?Ser?Ser?Ser?Ala

115 120 125

Asn?Pro?Val?Asn?Ser?Val?Arg?Arg?Lys?Ser?Cys?Leu?Val?Lys?Glu?Val

130 135 140

Glu?Lys?Met?Lys?Asn?Lys?Arg?Glu?Glu?Lys?Lys?Ala?Gln?Asn?Ser?Glu

145 150 155 160

Met?Arg?Met?Lys?Arg?Ala?Gln?Glu?Tyr?Asp?Ser?Ser?Phe?Pro?Asn?Trp

165 170 175

Glu?Phe?Ala?Arg?Met?Ile?Lys?Glu?Phe?Arg?Ala?Thr?Leu?Glu?Cys?His

180 185 190

Pro?Leu?Thr?Met?Thr?Asp?Pro?Ile?Glu?Glu?His?Arg?Ile?Cys?Val?Cys

195 200 205

Val?Arg?Lys?Arg?Pro?Leu?Asn?Lys?Gln?Glu?Leu?Ala?Lys?Lys?Glu?Ile

210 215 220

Asp?Val?Ile?Ser?Ile?Pro?Ser?Lys?Cys?Leu?Leu?Leu?Val?His?Glu?Pro

225 230 235 240

Lys?Leu?Lys?Val?Asp?Leu?Thr?Lys?Tyr?Leu?Glu?Asn?Gln?Ala?Phe?Cys

245 250 255

Phe?Asp?Phe?Ala?Phe?Asp?Glu?Thr?Ala?Ser?Asn?Glu?Val?Val?Tyr?Arg

260 265 270

Phe?Thr?Ala?Arg?Pro?Leu?Val?Gln?Thr?Ile?Phe?Glu?Gly?Gly?Lys?Ala

275 280 285

Thr?Cys?Phe?Ala?Tyr?Gly?Gln?Thr?Gly?Ser?Gly?Lys?Thr?His?Thr?Met

290 295 300

Gly?Gly?Asp?Leu?Ser?Gly?Lys?Ala?Gln?Asn?Ala?Ser?Lys?Gly?Ile?Tyr

305 310 315 320

Ala?Met?Ala?Ser?Arg?Asp?Val?Phe?Leu?Leu?Lys?Asn?Gln?Pro?Cys?Tyr

325 330 335

Arg?Lys?Leu?Gly?Leu?Glu?Val?Tyr?Val?Thr?Phe?Phe?Glu?Ile?Tyr?Asn

340 345 350

Gly?Lys?Leu?Phe?Asp?Leu?Leu?Asn?Lys?Lys?Ala?Lys?Leu?Arg?Val?Leu

355 360 365

Glu?Asp?Gly?Lys?Gln?Gln?Val?Gln?Val?Val?Gly?Leu?Gln?Glu?His?Leu

370 375 380

Val?Asn?Ser?Ala?Asp?Asp?Val?Ile?Lys?Met?Leu?Asp?Met?Gly?Ser?Ala

385 390 395 400

Cys?Arg?Thr?Ser?Gly?Gln?Thr?Phe?Ala?Asn?Ser?Asn?Ser?Ser?Arg?Ser

405 410 415

His?Ala?Cys?Phe?Gln?Ile?Ile?Leu?Arg?Ala?Lys?Gly?Arg?Met?His?Gly

420 425 430

Lys?Phe?Ser?Leu?Val?Asp?Leu?Ala?Gly?Asn?Glu?Arg?Gly?Ala?Asp?Thr

435 440 445

Ser?Ser?Ala?Asp?Arg?Gln?Thr?Arg?Met?Glu?Gly?Ala?Glu?Ile?Asn?Lys

450 455 460

Ser?Leu?Leu?Ala?Leu?Lys?Glu?Cys?Ile?Arg?Ala?Leu?Gly?Gln?Asn?Lys

465 470 475 480

Ala?His?Thr?Pro?Phe?Arg?Glu?Ser?Lys?Leu?Thr?Gln?Val?Leu?Arg?Asp

485 490 495

Ser?Phe?Ile?Gly?Glu?Asn?Ser?Arg?Thr?Cys?Met?Ile?Ala?Thr?Ile?Ser

500 505 510

Pro?Gly?Ile?Ser?Ser?Cys?Glu?Tyr?Thr?Leu?Asn?Thr?Leu?Arg?Tyr?Ala

515 520 525

Asp?Arg?Val?Lys?Glu?Leu?Ser?Pro?His?Ser?Gly?Pro?Ser?Gly?Glu?Gln

530 535 540

Leu?Ile?Gln?Met?Glu?Thr?Glu?Glu?Met?Glu?Ala?Cys?Ser?Asn?Gly?Ala

545 550 555 560

Leu?Ile?Pro?Gly?Asn?Leu?Ser?Lys?Glu?Glu?Glu?Glu?Leu?Ser?Ser?Gln

565 570 575

Met?Ser?Ser?Phe?Asn?Glu?Ala?Met?Thr?Gln?Ile?Arg?Glu?Leu?Glu?Glu

580 585 590

Lys?Ala?Met?Glu?Glu?Leu?Lys?Glu?Ile?Ile?Gln?Gln?Gly?Pro?Asp?Trp

595 600 605

Leu?Glu?Leu?Ser?Glu?Met?Thr?Glu?Gln?Pro?Asp?Tyr?Asp?Leu?Glu?Thr

610 615 620

Phe?Val?Asn?Lys?Ala?Glu?Ser?Ala?Leu?Ala?Gln?Gln?Ala?Lys?His?Phe

625 630 635 640

Ser?Ala?Leu?Arg?Asp?Val?Ile?Lys?Ala?Leu?Arg?Leu?Ala?Met?Gln?Leu

645 650 655

Glu?Glu?Gln?Ala?Ser?Arg?Gln?Ile?Ser?Ser?Lys?Lys?Arg?Pro?Gln

660 665 670

<210>39

<211>3128

<212>DNA

＜213〉people (Homo sapiens)

<220>

<221>CDS

<222>(158)..(2020)

<400>39

gcttcgcccc?gtggcgcggt?ttgaaatttt?gcggggctca?acggctcgcg?gagcggctac 60

gcggagtgac?atcgccggtg?tttgcgggtg?gttgttgctc?tcggggccgt?gtggagtagg 120

tctggacctg?gactcacggc?tgcttggagc?gtccgcc?atg?agg?aga?agt?gag?gtg 175

Met?Arg?Arg?Ser?Glu?Val

1 5

ctg?gcg?gag?gag?tcc?ata?gta?tgt?ctg?cag?aaa?gcc?cta?aat?cac?ctt 223

Leu?Ala?Glu?Glu?Ser?Ile?Val?Cys?Leu?Gln?Lys?Ala?Leu?Asn?His?Leu

10 15 20

cgg?gaa?ata?tgg?gag?cta?att?ggg?att?cca?gag?gac?cag?cgg?tta?caa 271

Arg?Glu?Ile?Trp?Glu?Leu?Ile?Gly?Ile?Pro?Glu?Asp?Gln?Arg?Leu?Gln

25 30 35

aga?act?gag?gtg?gta?aag?aag?cat?atc?aag?gaa?ctc?ctg?gat?atg?atg 319

Arg?Thr?Glu?Val?Val?Lys?Lys?His?Ile?Lys?Glu?Leu?Leu?Asp?Met?Met

40 45 50

att?gct?gaa?gag?gaa?agc?ctg?aag?gaa?aga?ctc?atc?aaa?agc?ata?tcc 367

Ile?Ala?Glu?Glu?Glu?Ser?Leu?Lys?Glu?Arg?Leu?Ile?Lys?Ser?Ile?Ser

55 60 65 70

gtc?tgt?cag?aaa?gag?ctg?aac?act?ctg?tgc?agc?gag?tta?cat?gtt?gag 415

Val?Cys?Gln?Lys?Glu?Leu?Asn?Thr?Leu?Cys?Ser?Glu?Leu?His?Val?Glu

75 80 85

cca?ttt?cag?gaa?gaa?gga?gag?acg?acc?atc?ttg?caa?cta?gaa?aaa?gat 463

Pro?Phe?Gln?Glu?Glu?Gly?Glu?Thr?Thr?Ile?Leu?Gln?Leu?Glu?Lys?Asp

90 95 100

ttg?cgc?acc?caa?gtg?gaa?ttg?atg?cga?aaa?cag?aaa?aag?gag?aga?aaa 511

Leu?Arg?Thr?Gln?Val?Glu?Leu?Met?Arg?Lys?Gln?Lys?Lys?Glu?Arg?Lys

105 110 115

cag?gaa?ctg?aag?cta?ctt?caa?gag?caa?gat?caa?gaa?ctg?tgc?gaa?att 559

Gln?Glu?Leu?Lys?Leu?Leu?Gln?Glu?Gln?Asp?Gln?Glu?Leu?Cys?Glu?Ile

120 125 130

ctt?tgt?atg?ccc?cac?tat?gat?att?gac?agt?gcc?tca?gtg?ccc?agc?tta 607

Leu?Cys?Met?Pro?His?Tyr?Asp?Ile?Asp?Ser?Ala?Ser?Val?Pro?Ser?Leu

135 140 145 150

gaa?gag?ctg?aac?cag?ttc?agg?caa?cat?gtg?aca?act?ttg?agg?gaa?aca 655

Glu?Glu?Leu?Asn?Gln?Phe?Arg?Gln?His?Val?Thr?Thr?Leu?Arg?Glu?Thr

155 160 165

aag?gct?tct?agg?cgt?gag?gag?ttt?gtc?agt?ata?aag?aga?cag?atc?ata 703

Lys?Ala?Ser?Arg?Arg?Glu?Glu?Phe?Val?Ser?Ile?Lys?Arg?Gln?Ile?Ile

170 175 180

ctg?tgt?atg?gaa?gaa?tta?gac?cac?acc?cca?gac?aca?agc?ttt?gaa?aga 751

Leu?Cys?Met?Glu?Glu?Leu?Asp?His?Thr?Pro?Asp?Thr?Ser?Phe?Glu?Arg

185 190 195

gat?gtg?gtg?tgt?gaa?gac?gaa?gat?gcc?ttt?tgt?ttg?tct?ttg?gag?aat 799

Asp?Val?Val?Cys?Glu?Asp?Glu?Asp?Ala?Phe?Cys?Leu?Ser?Leu?Glu?Asn

200 205 210

att?gca?aca?cta?caa?aag?ttg?cta?cgg?cag?ctg?gaa?atg?cag?aaa?tca 847

Ile?Ala?Thr?Leu?Gln?Lys?Leu?Leu?Arg?Gln?Leu?Glu?Met?Gln?Lys?Ser

215 220 225 230

caa?aat?gaa?gca?gtg?tgt?gag?ggg?ctg?cgt?act?caa?atc?cga?gag?ctc 895

Gln?Asn?Glu?Ala?Val?Cys?Glu?Gly?Leu?Arg?Thr?Gln?Ile?Arg?Glu?Leu

235 240 245

tgg?gac?agg?ttg?caa?ata?cct?gaa?gaa?gaa?aga?gaa?gct?gtg?gcc?acc 943

Trp?Asp?Arg?Leu?Gln?Ile?Pro?Glu?Glu?Glu?Arg?Glu?Ala?Val?Ala?Thr

250 255 260

att?atg?tct?ggg?tca?aag?gcc?aag?gtc?cgg?aaa?gcg?ctg?caa?tta?gaa 991

Ile?Met?Ser?Gly?Ser?Lys?Ala?Lys?Val?Arg?Lys?Ala?Leu?Gln?Leu?Glu

265 270 275

gtg?gat?cgg?ttg?gaa?gaa?ctg?aaa?atg?caa?aac?atg?aag?aaa?gtg?att 1039

Val?Asp?Arg?Leu?Glu?Glu?Leu?Lys?Met?Gln?Asn?Met?Lys?Lys?Val?Ile

280 285 290

gag?gca?att?cga?gtg?gag?ctg?gtt?cag?tac?tgg?gac?cag?tgc?ttt?tat 1087

Glu?Ala?Ile?Arg?Val?Glu?Leu?Val?Gln?Tyr?Trp?Asp?Gln?Cys?Phe?Tyr

295 300 305 310

agc?cag?gag?cag?aga?caa?gct?ttt?gcc?cct?ttc?tgt?gct?gag?gac?tac 1135

Ser?Gln?Glu?Gln?Arg?Gln?Ala?Phe?Ala?Pro?Phe?Cys?Ala?Glu?Asp?Tyr

315 320 325

aca?gaa?agt?ctg?ctc?cag?ctc?cac?gat?gct?gag?att?gtg?cgg?tta?aaa 1183

Thr?Glu?Ser?Leu?Leu?Gln?Leu?His?Asp?Ala?Glu?Ile?Val?Arg?Leu?Lys

330 335 340

aac?tac?tat?gaa?gtt?cac?aag?gaa?ctc?ttt?gaa?ggt?gtc?cag?aag?tgg 1231

Asn?Tyr?Tyr?Glu?Val?His?Lys?Glu?Leu?Phe?Glu?Gly?Val?Gln?Lys?Trp

345 350 355

gaa?gaa?acc?tgg?agg?ctt?ttc?tta?gag?ttt?gag?aga?aaa?gct?tca?gat 1279

Glu?Glu?Thr?Trp?Arg?Leu?Phe?Leu?Glu?Phe?Glu?Arg?Lys?Ala?Ser?Asp

360 365 370

cca?aat?cga?ttt?aca?aac?cga?gga?gga?aat?ctt?cta?aaa?gaa?gaa?aaa 1327

Pro?Asn?Arg?Phe?Thr?Asn?Arg?Gly?Gly?Asn?Leu?Leu?Lys?Glu?Glu?Lys

375 380 385 390

caa?cga?gcc?aag?ctc?cag?aaa?atg?ctg?ccc?aag?ctg?gaa?gaa?gag?ttg 1375

Gln?Arg?Ala?Lys?Leu?Gln?Lys?Met?Leu?Pro?Lys?Leu?Glu?Glu?Glu?Leu

395 400 405

aag?gca?cga?att?gaa?ttg?tgg?gaa?cag?gaa?cat?tca?aag?gca?ttt?atg 1423

Lys?Ala?Arg?Ile?Glu?Leu?Trp?Glu?Gln?Glu?His?Ser?Lys?Ala?Phe?Met

410 415 420

gtg?aat?ggg?cag?aaa?ttc?atg?gag?tat?gtg?gca?gaa?caa?tgg?gag?atg 1471

Val?Asn?Gly?Gln?Lys?Phe?Met?Glu?Tyr?Val?Ala?Glu?Gln?Trp?Glu?Met

425 430 435

cat?cga?ttg?gag?aaa?gag?aga?gcc?aag?cag?gaa?aga?caa?ctg?aag?aac 1519

His?Arg?Leu?Glu?Lys?Glu?Arg?Ala?Lys?Gln?Glu?Arg?Gln?Leu?Lys?Asn

440 445 450

aaa?aaa?cag?aca?gag?aca?gag?atg?ctg?tat?ggc?agc?gct?cct?cga?aca 1567

Lys?Lys?Gln?Thr?Glu?Thr?Glu?Met?Leu?Tyr?Gly?Ser?Ala?Pro?Arg?Thr

455 460 465 470

cct?agc?aag?cgg?cga?gga?ctg?gct?ccc?aat?aca?ccg?ggc?aaa?gca?cgt 1615

Pro?Ser?Lys?Arg?Arg?Gly?Leu?Ala?Pro?Asn?Thr?Pro?Gly?Lys?Ala?Arg

475 480 485

aag?ctg?aac?act?acc?acc?atg?tcc?aat?gct?acg?gcc?aat?agt?agc?att 1663

Lys?Leu?Asn?Thr?Thr?Thr?Met?Ser?Asn?Ala?Thr?Ala?Asn?Ser?Ser?Ile

490 495 500

cgg?cct?atc?ttt?gga?ggg?aca?gtc?tac?cac?tcc?ccc?gtg?tct?cga?ctt 1711

Arg?Pro?Ile?Phe?Gly?Gly?Thr?Val?Tyr?His?Ser?Pro?Val?Ser?Arg?Leu

505 510 515

cct?cct?tct?ggc?agc?aag?cca?gtc?gct?gct?tcc?acc?tgt?tca?ggg?aag 1759

Pro?Pro?Ser?Gly?Ser?Lys?Pro?Val?Ala?Ala?Ser?Thr?Cys?Ser?Gly?Lys

520 525 530

aaa?aca?ccc?cgt?act?ggc?agg?cat?gga?gcc?aac?aag?gag?aac?ctg?gag 1807

Lys?Thr?Pro?Arg?Thr?Gly?Arg?His?Gly?Ala?Asn?Lys?Glu?Asn?Leu?Glu

535 540 545 550

ctc?aac?ggc?agc?atc?ctg?agt?ggt?ggg?tac?cct?ggc?tcg?gcc?ccc?ctc 1855

Leu?Asn?Gly?Ser?Ile?Leu?Ser?Gly?Gly?Tyr?Pro?Gly?Ser?Ala?Pro?Leu

555 560 565

cag?cgc?aac?ttc?agc?att?aat?tct?gtt?gcc?agc?acc?tat?tct?gag?ttt 1903

Gln?Arg?Asn?Phe?Ser?Ile?Asn?Ser?Val?Ala?Ser?Thr?Tyr?Ser?Glu?Phe

570 575 580

gcg?aag?gat?ccg?tcc?ctc?tct?gac?agt?tcc?act?gtt?ggg?ctt?cag?cga 1951

Ala?Lys?Asp?Pro?Ser?Leu?Ser?Asp?Ser?Ser?Thr?Val?Gly?Leu?Gln?Arg

585 590 595

gaa?ctt?tca?aag?gct?tcc?aaa?tct?gat?gct?act?tct?gga?atc?ctc?aat 1999

Glu?Leu?Ser?Lys?Ala?Ser?Lys?Ser?Asp?Ala?Thr?Ser?Gly?Ile?Leu?Asn

600 605 610

tca?acc?aac?atc?cag?tcc?tga?gaagccctga?tcagtcaacc?agctgtggct 2050

Ser?Thr?Asn?Ile?Gln?Ser

615 620

tcctgtgcct?agactggacc?taattatatg?ggggtgactt?tagtttttct?tcagcttagg 2110

cgtgcttgaa?accttggcca?ggttccatga?ccatgggcct?aacttaaaga?tgtgaatgag 2170

tgttacagtt?gaaagcccat?cataggttta?gtggtcctag?gagacttggt?tttgacttat 2230

atacatgaaa?agtttatggc?aagaagtgca?aattttagca?tatggggcct?gacttctcta 2290

ccacataatt?ctacttgctg?aagcatgatc?aaagcttgtt?ttatttcacc?actgtaggaa 2350

aatgattgac?tatgcccatc?cctgggggta?attttggcat?gtatacctgt?aactagtaat 2410

taacatcttt?tttgtttagg?catgttcaat?taatgctgta?gctatcatag?ctttgctctt 2470

acctgaagcc?ttgtccccac?cacacaggac?agccttcctc?ctgaagagaa?tgtctttgtg 2530

tgtccgaagt?tgagatggcc?tgccctactg?ccaaagaggt?gacaggaagg?ctgggagcag 2590

ctttgttaaa?ttgtgttcag?ttctgttaca?cagtgcattg?ccctttgttg?ggggtatgca 2650

tgtatgaaca?cacatgcttg?tcggaacgct?ttctcggcgt?ttgtcccttg?gctctcatct 2710

cccccattcc?tgtgcctact?ttgcctgagt?tcttctaccc?ccgcagttgc?cagccacatt 2770

gggagtctgt?ttgttccaat?gggttgagct?gtctttgtcg?tggagatctg?gaactttgca 2830

catgtcacta?ctggggaggt?gttcctgctc?tagcttccac?gatgaggcgc?cctctttacc 2890

tatcctctca?atcactactc?ttcttgaagc?actattattt?attcttccgc?tgtctgcctg 2950

cagcagtact?actgtcaaca?tagtgtaaat?ggttctcaaa?agcttaccag?tgtggacttg 3010

gtgttagcca?cgctgtttac?tcatacagta?cgtgtcctgt?ttttaaaata?tacaattatt 3070

cttaaaaata?aattaaaatc?tgtatactta?catttcaaaa?agaaaaaaaa?aaaaaaaa 3128

<210>40

<211>620

<212>PRT

＜213〉people (Homo sapiens)

<400>40

Met?Arg?Arg?Ser?Glu?Val?Leu?Ala?Glu?Glu?Ser?Ile?Val?Cys?Leu?Gln

1 5 10 15

Lys?Ala?Leu?Asn?His?Leu?Arg?Glu?Ile?Trp?Glu?Leu?Ile?Gly?Ile?Pro

20 25 30

Glu?Asp?Gln?Arg?Leu?Gln?Arg?Thr?Glu?Val?Val?Lys?Lys?His?Ile?Lys

35 40 45

Glu?Leu?Leu?Asp?Met?Met?Ile?Ala?Glu?Glu?Glu?Ser?Leu?Lys?Glu?Arg

50 55 60

Leu?Ile?Lys?Ser?Ile?Ser?Val?Cys?Gln?Lys?Glu?Leu?Asn?Thr?Leu?Cys

65 70 75 80

Ser?Glu?Leu?His?Val?Glu?Pro?Phe?Gln?Glu?Glu?Gly?Glu?Thr?Thr?Ile

85 90 95

Leu?Gln?Leu?Glu?Lys?Asp?Leu?Arg?Thr?Gln?Val?Glu?Leu?Met?Arg?Lys

100 105 110

Gln?Lys?Lys?Glu?Arg?Lys?Gln?Glu?Leu?Lys?Leu?Leu?Gln?Glu?Gln?Asp

115 120 125

Gln?Glu?Leu?Cys?Glu?Ile?Leu?Cys?Met?Pro?His?Tyr?Asp?Ile?Asp?Ser

130 135 140

Ala?Ser?Val?Pro?Ser?Leu?Glu?Glu?Leu?Asn?Gln?Phe?Arg?Gln?His?Val

145 150 155 160

Thr?Thr?Leu?Arg?Glu?Thr?Lys?Ala?Ser?Arg?Arg?Glu?Glu?Phe?Val?Ser

165 170 175

Ile?Lys?Arg?Gln?Ile?Ile?Leu?Cys?Met?Glu?Glu?Leu?Asp?His?Thr?Pro

180 185 190

Asp?Thr?Ser?Phe?Glu?Arg?Asp?Val?Val?Cys?Glu?Asp?Glu?Asp?Ala?Phe

195 200 205

Cys?Leu?Ser?Leu?Glu?Asn?Ile?Ala?Thr?Leu?Gln?Lys?Leu?Leu?Arg?Gln

210 215 220

Leu?Glu?Met?Gln?Lys?Ser?Gln?Asn?Glu?Ala?Val?Cys?Glu?Gly?Leu?Arg

225 230 235 240

Thr?Gln?Ile?Arg?Glu?Leu?Trp?Asp?Arg?Leu?Gln?Ile?Pro?Glu?Glu?Glu

245 250 255

Arg?Glu?Ala?Val?Ala?Thr?Ile?Met?Ser?Gly?Ser?Lys?Ala?Lys?Val?Arg

260 265 270

Lys?Ala?Leu?Gln?Leu?Glu?Val?Asp?Arg?Leu?Glu?Glu?Leu?Lys?Met?Gln

275 280 285

Asn?Met?Lys?Lys?Val?Ile?Glu?Ala?Ile?Arg?Val?Glu?Leu?Val?Gln?Tyr

290 295 300

Trp?Asp?Gln?Cys?Phe?Tyr?Ser?Gln?Glu?Gln?Arg?Gln?Ala?Phe?Ala?Pro

305 310 315 320

Phe?Cys?Ala?Glu?Asp?Tyr?Thr?Glu?Ser?Leu?Leu?Gln?Leu?His?Asp?Ala

325 330 335

Glu?Ile?Val?Arg?Leu?Lys?Asn?Tyr?Tyr?Glu?Val?His?Lys?Glu?Leu?Phe

340 345 350

Glu?Gly?Val?Gln?Lys?Trp?Glu?Glu?Thr?Trp?Arg?Leu?Phe?Leu?Glu?Phe

355 360 365

Glu?Arg?Lys?Ala?Ser?Asp?Pro?Asn?Arg?Phe?Thr?Asn?Arg?Gly?Gly?Asn

370 375 380

Leu?Leu?Lys?Glu?Glu?Lys?Gln?Arg?Ala?Lys?Leu?Gln?Lys?Met?Leu?Pro

385 390 395 400

Lys?Leu?Glu?Glu?Glu?Leu?Lys?Ala?Arg?Ile?Glu?Leu?Trp?Glu?Gln?Glu

405 410 415

His?Ser?Lys?Ala?Phe?Met?Val?Asn?Gly?Gln?Lys?Phe?Met?Glu?Tyr?Val

420 425 430

Ala?Glu?Gln?Trp?Glu?Met?His?Arg?Leu?Glu?Lys?Glu?Arg?Ala?Lys?Gln

435 440 445

Glu?Arg?Gln?Leu?Lys?Asn?Lys?Lys?Gln?Thr?Glu?Thr?Glu?Met?Leu?Tyr

450 455 460

Gly?Ser?Ala?Pro?Arg?Thr?Pro?Ser?Lys?Arg?Arg?Gly?Leu?Ala?Pro?Asn

465 470 475 480

Thr?Pro?Gly?Lys?Ala?Arg?Lys?Leu?Asn?Thr?Thr?Thr?Met?Ser?Asn?Ala

485 490 495

Thr?Ala?Asn?Ser?Ser?Ile?Arg?Pro?Ile?Phe?Gly?Gly?Thr?Val?Tyr?His

500 505 510

Ser?Pro?Val?Ser?Arg?Leu?Pro?Pro?Ser?Gly?Ser?Lys?Pro?Val?Ala?Ala

515 520 525

Ser?Thr?Cys?Ser?Gly?Lys?Lys?Thr?Pro?Arg?Thr?Gly?Arg?His?Gly?Ala

530 535 540

Asn?Lys?Glu?Asn?Leu?Glu?Leu?Asn?Gly?Ser?Ile?Leu?Ser?Gly?Gly?Tyr

545 550 555 560

Pro?Gly?Ser?Ala?Pro?Leu?Gln?Arg?Asn?Phe?Ser?Ile?Asn?Ser?Val?Ala

565 570 575

Ser?Thr?Tyr?Ser?Glu?Phe?Ala?Lys?Asp?Pro?Ser?Leu?Ser?Asp?Ser?Ser

580 585 590

Thr?Val?Gly?Leu?Gln?Arg?Glu?Leu?Ser?Lys?Ala?Ser?Lys?Ser?Asp?Ala

595 600 605

Thr?Ser?Gly?Ile?Leu?Asn?Ser?Thr?Asn?Ile?Gln?Ser

610 615 620

<210>41

<211>3091

<212>DNA

＜213〉people (Homo sapiens)

<220>

<221>CDS

<222>(158)..(1978)

<400>41

gcttcgcccc?gtggcgcggt?ttgaaatttt?gcggggctca?acggctcgcg?gagcggctac 60

gcggagtgac?atcgccggtg?tttgcgggtg?gttgttgctc?tcggggccgt?gtggagtagg 120

tctggacctg?gactcacggc?tgcttggagc?gtccgcc?atg?agg?aga?agt?gag?gtg 175

Met?Arg?Arg?Ser?Glu?Val

1 5

ctg?gcg?gag?gag?tcc?ata?gta?tgt?ctg?cag?aaa?gcc?cta?aat?cac?ctt 223

Leu?Ala?Glu?Glu?Ser?Ile?Val?Cys?Leu?Gln?Lys?Ala?Leu?Asn?His?Leu

10 15 20

cgg?gaa?ata?tgg?gag?cta?att?ggg?att?cca?gag?gac?cag?cgg?tta?caa 271

Arg?Glu?Ile?Trp?Glu?Leu?Ile?Gly?Ile?Pro?Glu?Asp?Gln?Arg?Leu?Gln

25 30 35

aga?act?gag?gtg?gta?aag?aag?cat?atc?aag?gaa?ctc?ctg?gat?atg?atg 319

Arg?Thr?Glu?Val?Val?Lys?Lys?His?Ile?Lys?Glu?Leu?Leu?Asp?Met?Met

40 45 50

att?gct?gaa?gag?gaa?agc?ctg?aag?gaa?aga?ctc?atc?aaa?agc?ata?tcc 367

Ile?Ala?Glu?Glu?Glu?Ser?Leu?Lys?Glu?Arg?Leu?Ile?Lys?Ser?Ile?Ser

55 60 65 70

gtc?tgt?cag?aaa?gag?ctg?aac?act?ctg?tgc?agc?gag?tta?cat?gtt?gag 415

Val?Cys?Gln?Lys?Glu?Leu?Asn?Thr?Leu?Cys?Ser?Glu?Leu?His?Val?Glu

75 80 85

cca?ttt?cag?gaa?gaa?gga?gag?acg?acc?atc?ttg?caa?cta?gaa?aaa?gat 463

Pro?Phe?Gln?Glu?Glu?Gly?Glu?Thr?Thr?Ile?Leu?Gln?Leu?Glu?Lys?Asp

90 95 100

ttg?cgc?acc?caa?gtg?gaa?ttg?atg?cga?aaa?cag?aaa?aag?gag?aga?aaa 511

Leu?Arg?Thr?Gln?Val?Glu?Leu?Met?Arg?Lys?Gln?Lys?Lys?Glu?Arg?Lys

105 110 115

cag?gaa?ctg?aag?cta?ctt?caa?gag?caa?gat?caa?gaa?ctg?tgc?gaa?att 559

Gln?Glu?Leu?Lys?Leu?Leu?Gln?Glu?Gln?Asp?Gln?Glu?Leu?Cys?Glu?Ile

120 125 130

ctt?tgt?atg?ccc?cac?tat?gat?att?gac?agt?gcc?tca?gtg?ccc?agc?tta 607

Leu?Cys?Met?Pro?His?Tyr?Asp?Ile?Asp?Ser?Ala?Ser?Val?Pro?Ser?Leu

135 140 145 150

gaa?gag?ctg?aac?cag?ttc?agg?caa?cat?gtg?aca?act?ttg?agg?gaa?aca 655

Glu?Glu?Leu?Asn?Gln?Phe?Arg?Gln?His?Val?Thr?Thr?Leu?Arg?Glu?Thr

155 160 165

aag?gct?tct?agg?cgt?gag?gag?ttt?gtc?agt?ata?aag?aga?cag?atc?ata 703

Lys?Ala?Ser?Arg?Arg?Glu?Glu?Phe?Val?Ser?Ile?Lys?Arg?Gln?Ile?Ile

170 175 180

ctg?tgt?atg?gaa?gaa?tta?gac?cac?acc?cca?gac?aca?agc?ttt?gaa?aga 751

Leu?Cys?Met?Glu?Glu?Leu?Asp?His?Thr?Pro?Asp?Thr?Ser?Phe?Glu?Arg

185 190 195

gat?gtg?gtg?tgt?gaa?gac?gaa?gat?gcc?ttt?tgt?ttg?tct?ttg?gag?aat 799

Asp?Val?Val?Cys?Glu?Asp?Glu?Asp?Ala?Phe?Cys?Leu?Ser?Leu?Glu?Asn

200 205 210

att?gca?aca?cta?caa?aag?ttg?cta?cgg?cag?ctg?gaa?atg?cag?aaa?tca 847

Ile?Ala?Thr?Leu?Gln?Lys?Leu?Leu?Arg?Gln?Leu?Glu?Met?Gln?Lys?Ser

215 220 225 230

caa?aat?gaa?gca?gtg?tgt?gag?ggg?ctg?cgt?act?caa?atc?cga?gag?ctc 895

Gln?Asn?Glu?Ala?Val?Cys?Glu?Gly?Leu?Arg?Thr?Gln?Ile?Arg?Glu?Leu

235 240 245

tgg?gac?agg?ttg?caa?ata?cct?gaa?gaa?gaa?aga?gaa?gct?gtg?gcc?acc 943

Trp?Asp?Arg?Leu?Gln?Ile?Pro?Glu?Glu?Glu?Arg?Glu?Ala?Val?Ala?Thr

250 255 260

att?atg?tct?ggg?tca?aag?gcc?aag?gtc?cgg?aaa?gcg?ctg?caa?tta?gaa 991

Ile?Met?Ser?Gly?Ser?Lys?Ala?Lys?Val?Arg?Lys?Ala?Leu?Gln?Leu?Glu

265 270 275

gtg?gat?cgg?ttg?gaa?gaa?ctg?aaa?atg?caa?aac?atg?aag?aaa?gtg?att 1039

Val?Asp?Arg?Leu?Glu?Glu?Leu?Lys?Met?Gln?Asn?Met?Lys?Lys?Val?Ile

280 285 290

gag?gca?att?cga?gtg?gag?ctg?gtt?cag?tac?tgg?gac?cag?tgc?ttt?tat 1087

Glu?Ala?Ile?Arg?Val?Glu?Leu?Val?Gln?Tyr?Trp?Asp?Gln?Cys?Phe?Tyr

295 300 305 310

agc?cag?gag?cag?aga?caa?gct?ttt?gcc?cct?ttc?tgt?gct?gag?gac?tac 1135

Ser?Gln?Glu?Gln?Arg?Gln?Ala?Phe?Ala?Pro?Phe?Cys?Ala?Glu?Asp?Tyr

315 320 325

aca?gaa?agt?ctg?ctc?cag?ctc?cac?gat?gct?gag?att?gtg?cgg?tta?aaa 1183

Thr?Glu?Ser?Leu?Leu?Gln?Leu?His?Asp?Ala?Glu?Ile?Val?Arg?Leu?Lys

330 335 340

aac?tac?tat?gaa?gtt?cac?aag?gaa?ctc?ttt?gaa?ggt?gtc?cag?aag?tgg 1231

Asn?Tyr?Tyr?Glu?Val?His?Lys?Glu?Leu?Phe?Glu?Gly?Val?Gln?Lys?Trp

345 350 355

gaa?gaa?acc?tgg?agg?ctt?ttc?tta?gag?ttt?gag?aga?aaa?gct?tca?gat 1279

Glu?Glu?Thr?Trp?Arg?Leu?Phe?Leu?Glu?Phe?Glu?Arg?Lys?Ala?Ser?Asp

360 365 370

cca?aat?cga?ttt?aca?aac?cga?gga?gga?aat?ctt?cta?aaa?gaa?gaa?aaa 1327

Pro?Asn?Arg?Phe?Thr?Asn?Arg?Gly?Gly?Asn?Leu?Leu?Lys?Glu?Glu?Lys

375 380 385 390

caa?cga?gcc?aag?ctc?cag?aaa?atg?ctg?ccc?aag?ctg?gaa?gaa?gag?ttg 1375

Gln?Arg?Ala?Lys?Leu?Gln?Lys?Met?Leu?Pro?Lys?Leu?Glu?Glu?Glu?Leu

395 400 405

aag?gca?cga?att?gaa?ttg?tgg?gaa?cag?gaa?cat?tca?aag?gca?ttt?atg 1423

Lys?Ala?Arg?Ile?Glu?Leu?Trp?Glu?Gln?Glu?His?Ser?Lys?Ala?Phe?Met

410 415 420

gtg?aat?ggg?cag?aaa?ttc?atg?gag?tat?gtg?gca?gaa?caa?tgg?gag?atg 1471

Val?Asn?Gly?Gln?Lys?Phe?Met?Glu?Tyr?Val?Ala?Glu?Gln?Trp?Glu?Met

425 430 435

cat?cga?ttg?gag?aaa?gag?aga?gcc?aag?cag?gaa?aga?caa?ctg?aag?aac 1519

His?Arg?Leu?Glu?Lys?Glu?Arg?Ala?Lys?Gln?Glu?Arg?Gln?Leu?Lys?Asn

440 445 450

aaa?aaa?cag?aca?gag?aca?gag?atg?ctg?tat?ggc?agc?gct?cct?cga?aca 1567

Lys?Lys?Gln?Thr?Glu?Thr?Glu?Met?Leu?Tyr?Gly?Ser?Ala?Pro?Arg?Thr

455 460 465 470

cct?agc?aag?cgg?cga?gga?ctg?gct?ccc?aat?aca?ccg?ggc?aaa?gca?cgt 1615

Pro?Ser?Lys?Arg?Arg?Gly?Leu?Ala?Pro?Asn?Thr?Pro?Gly?Lys?Ala?Arg

475 480 485

aag?ctg?aac?act?acc?acc?atg?tcc?aat?gct?acg?gcc?aat?agt?agc?att 1663

Lys?Leu?Asn?Thr?Thr?Thr?Met?Ser?Asn?Ala?Thr?Ala?Asn?Ser?Ser?Ile

490 495 500

cgg?cct?atc?ttt?gga?ggg?aca?gtc?tac?cac?tcc?ccc?gtg?tct?cga?ctt 1711

Arg?Pro?Ile?Phe?Gly?Gly?Thr?Val?Tyr?His?Ser?Pro?Val?Ser?Arg?Leu

505 510 515

cct?cct?tct?ggc?agc?aag?cca?gtc?gct?gct?tcc?acc?tgt?tca?ggg?aag 1759

Pro?Pro?Ser?Gly?Ser?Lys?Pro?Val?Ala?Ala?Ser?Thr?Cys?Ser?Gly?Lys

520 525 530

aaa?aca?ccc?cgt?act?ggc?agg?cat?gga?gcc?aac?aag?gag?aac?ctg?gag 1807

Lys?Thr?Pro?Arg?Thr?Gly?Arg?His?Gly?Ala?Asn?Lys?Glu?Asn?Leu?Glu

535 540 545 550

ctc?aac?ggc?agc?atc?ctg?agt?ggt?ggg?tac?cct?ggc?tcg?gcc?ccc?ctc 1855

Leu?Asn?Gly?Ser?Ile?Leu?Ser?Gly?Gly?Tyr?Pro?Gly?Ser?Ala?Pro?Leu

555 560 565

cag?cgc?aac?ttc?agc?att?aat?tct?gtt?gcc?agc?acc?tat?tct?gag?ttt 1903

Gln?Arg?Asn?Phe?Ser?Ile?Asn?Ser?Val?Ala?Ser?Thr?Tyr?Ser?Glu?Phe

570 575 580

gcg?cga?gaa?ctt?tca?aag?gct?tcc?aaa?tct?gat?gct?act?tct?gga?atc 1951

Ala?Arg?Glu?Leu?Ser?Lys?Ala?Ser?Lys?Ser?Asp?Ala?Thr?Ser?Gly?Ile

585 590 595

ctc?aat?tca?acc?aac?atc?cag?tcc?tga?gaagccctga?tcagtcaacc 1998

Leu?Asn?Ser?Thr?Asn?Ile?Gln?Ser

600 605

agctgtggct?tcctgtgcct?agactggacc?taattatatg?ggggtgactt?tagtttttct 2058

tcagcttagg?cgtgcttgaa?accttggcca?ggttccatga?ccatgggcct?aacttaaaga 2118

tgtgaatgag?tgttacagtt?gaaagcccat?cataggttta?gtggtcctag?gagacttggt 2178

tttgacttat?atacatgaaa?agtttatggc?aagaagtgca?aattttagca?tatggggcct 2238

gacttctcta?ccacataatt?ctacttgctg?aagcatgatc?aaagcttgtt?ttatttcacc 2298

actgtaggaa?aatgattgac?tatgcccatc?cctgggggta?attttggcat?gtatacctgt 2358

aactagtaat?taacatcttt?tttgtttagg?catgttcaat?taatgctgta?gctatcatag 2418

ctttgctctt?acctgaagcc?ttgtccccac?cacacaggac?agccttcctc?ctgaagagaa 2478

tgtctttgtg?tgtccgaagt?tgagatggcc?tgccctactg?ccaaagaggt?gacaggaagg 2538

ctgggagcag?ctttgttaaa?ttgtgttcag?ttctgttaca?cagtgcattg?ccctttgttg 2598

ggggtatgca?tgtatgaaca?cacatgcttg?tcggaacgct?ttctcggcgt?ttgtcccttg 2658

gctctcatct?cccccattcc?tgtgcctact?ttgcctgagt?tcttctaccc?ccgcagttgc 2718

cagccacatt?gggagtctgt?ttgttccaat?gggttgagct?gtctttgtcg?tggagatctg 2778

gaactttgca?catgtcacta?ctggggaggt?gttcctgctc?tagcttccac?gatgaggcgc 2838

cctctttacc?tatcctctca?atcactactc?ttcttgaagc?actattattt?attcttccgc 2898

tgtctgcctg?cagcagtact?actgtcaaca?tagtgtaaat?ggttctcaaa?agcttaccag 2958

tgtggacttg?gtgttagcca?cgctgtttac?tcatacagta?cgtgtcctgt?ttttaaaata 3018

tacaattatt?cttaaaaata?aattaaaatc?tgtatactta?catttcaaaa?agaaaaaaaa 3078

aaaaaaaaaa?aaa 3091

<210>42

<211>606

<212>PRT

＜213〉people (Homo sapiens)

<400>42

Met?Arg?Arg?Ser?Glu?Val?Leu?Ala?Glu?Glu?Ser?Ile?Val?Cys?Leu?Gln

1 5 10 15

Lys?Ala?Leu?Asn?His?Leu?Arg?Glu?Ile?Trp?Glu?Leu?Ile?Gly?Ile?Pro

20 25 30

Glu?Asp?Gln?Arg?Leu?Gln?Arg?Thr?Glu?Val?Val?Lys?Lys?His?Ile?Lys

35 40 45

Glu?Leu?Leu?Asp?Met?Met?Ile?Ala?Glu?Glu?Glu?Ser?Leu?Lys?Glu?Arg

50 55 60

Leu?Ile?Lys?Ser?Ile?Ser?Val?Cys?Gln?Lys?Glu?Leu?Asn?Thr?Leu?Cys

65 70 75 80

Ser?Glu?Leu?His?Val?Glu?Pro?Phe?Gln?Glu?Glu?Gly?Glu?Thr?Thr?Ile

85 90 95

Leu?Gln?Leu?Glu?Lys?Asp?Leu?Arg?Thr?Gln?Val?Glu?Leu?Met?Arg?Lys

100 105 110

Gln?Lys?Lys?Glu?Arg?Lys?Gln?Glu?Leu?Lys?Leu?Leu?Gln?Glu?Gln?Asp

115 120 125

Gln?Glu?Leu?Cys?Glu?Ile?Leu?Cys?Met?Pro?His?Tyr?Asp?Ile?Asp?Ser

130 135 140

Ala?Ser?Val?Pro?Ser?Leu?Glu?Glu?Leu?Asn?Gln?Phe?Arg?Gln?His?Val

145 150 155 160

Thr?Thr?Leu?Arg?Glu?Thr?Lys?Ala?Ser?Arg?Arg?Glu?Glu?Phe?Val?Ser

165 170 175

Ile?Lys?Arg?Gln?Ile?Ile?Leu?Cys?Met?Glu?Glu?Leu?Asp?His?Thr?Pro

180 185 190

Asp?Thr?Ser?Phe?Glu?Arg?Asp?Val?Val?Cys?Glu?Asp?Glu?Asp?Ala?Phe

195 200 205

Cys?Leu?Ser?Leu?Glu?Asn?Ile?Ala?Thr?Leu?Gln?Lys?Leu?Leu?Arg?Gln

210 215 220

Leu?Glu?Met?Gln?Lys?Ser?Gln?Asn?Glu?Ala?Val?Cys?Glu?Gly?Leu?Arg

225 230 235 240

Thr?Gln?Ile?Arg?Glu?Leu?Trp?Asp?Arg?Leu?Gln?Ile?Pro?Glu?Glu?Glu

245 250 255

Arg?Glu?Ala?Val?Ala?Thr?Ile?Met?Ser?Gly?Ser?Lys?Ala?Lys?Val?Arg

260 265 270

Lys?Ala?Leu?Gln?Leu?Glu?Val?Asp?Arg?Leu?Glu?Glu?Leu?Lys?Met?Gln

275 280 285

Asn?Met?Lys?Lys?Val?Ile?Glu?Ala?Ile?Arg?Val?Glu?Leu?Val?Gln?Tyr

290 295 300

Trp?Asp?Gln?Cys?Phe?Tyr?Ser?Gln?Glu?Gln?Arg?Gln?Ala?Phe?Ala?Pro

305 310 315 320

Phe?Cys?Ala?Glu?Asp?Tyr?Thr?Glu?Ser?Leu?Leu?Gln?Leu?His?Asp?Ala

325 330 335

Glu?Ile?Val?Arg?Leu?Lys?Asn?Tyr?Tyr?Glu?Val?His?Lys?Glu?Leu?Phe

340 345 350

Glu?Gly?Val?Gln?Lys?Trp?Glu?Glu?Thr?Trp?Arg?Leu?Phe?Leu?Glu?Phe

355 360 365

Glu?Arg?Lys?Ala?Ser?Asp?Pro?Asn?Arg?Phe?Thr?Asn?Arg?Gly?Gly?Asn

370 375 380

Leu?Leu?Lys?Glu?Glu?Lys?Gln?Arg?Ala?Lys?Leu?Gln?Lys?Met?Leu?Pro

385 390 395 400

Lys?Leu?Glu?Glu?Glu?Leu?Lys?Ala?Arg?Ile?Glu?Leu?Trp?Glu?Gln?Glu

405 410 415

His?Ser?Lys?Ala?Phe?Met?Val?Asn?Gly?Gln?Lys?Phe?Met?Glu?Tyr?Val

420 425 430

Ala?Glu?Gln?Trp?Glu?Met?His?Arg?Leu?Glu?Lys?Glu?Arg?Ala?Lys?Gln

435 440 445

Glu?Arg?Gln?Leu?Lys?Asn?Lys?Lys?Gln?Thr?Glu?Thr?Glu?Met?Leu?Tyr

450 455 460

Gly?Ser?Ala?Pro?Arg?Thr?Pro?Ser?Lys?Arg?Arg?Gly?Leu?Ala?Pro?Asn

465 470 475 480

Thr?Pro?Gly?Lys?Ala?Arg?Lys?Leu?Asn?Thr?Thr?Thr?Met?Ser?Asn?Ala

485 490 495

Thr?Ala?Asn?Ser?Ser?Ile?Arg?Pro?Ile?Phe?Gly?Gly?Thr?Val?Tyr?His

500 505 510

Ser?Pro?Val?Ser?Arg?Leu?Pro?Pro?Ser?Gly?Ser?Lys?Pro?Val?Ala?Ala

515 520 525

Ser?Thr?Cys?Ser?Gly?Lys?Lys?Thr?Pro?Arg?Thr?Gly?Arg?His?Gly?Ala

530 535 540

Asn?Lys?Glu?Asn?Leu?Glu?Leu?Asn?Gly?Ser?Ile?Leu?Ser?Gly?Gly?Tyr

545 550 555 560

Pro?Gly?Ser?Ala?Pro?Leu?Gln?Arg?Asn?Phe?Ser?Ile?Asn?Ser?Val?Ala

565 570 575

Ser?Thr?Tyr?Ser?Glu?Phe?Ala?Arg?Glu?Leu?Ser?Lys?Ala?Ser?Lys?Ser

580 585 590

Asp?Ala?Thr?Ser?Gly?Ile?Leu?Asn?Ser?Thr?Asn?Ile?Gln?Ser

595 600 605

<210>43

<211>3011

<212>DNA

＜213〉people (Homo sapiens)

<220>

<221>CDS

<222>(158)..(1858)

<400>43

gcttcgcccc?gtggcgcggt?ttgaaatttt?gcggggctca?acggctcgcg?gagcggctac 60

gcggagtgac?atcgccggtg?tttgcgggtg?gttgttgctc?tcggggccgt?gtggagtagg 120

tctggacctg?gactcacggc?tgcttggagc?gtccgcc?atg?agg?aga?agt?gag?gtg 175

Met?Arg?Arg?Ser?Glu?Val

1 5

ctg?gcg?gag?gag?tcc?ata?gta?tgt?ctg?cag?aaa?gcc?cta?aat?cac?ctt 223

Leu?Ala?Glu?Glu?Ser?Ile?Val?Cys?Leu?Gln?Lys?Ala?Leu?Asn?His?Leu

10 15 20

cgg?gaa?ata?tgg?gag?cta?att?ggg?att?cca?gag?gac?cag?cgg?tta?caa 271

Arg?Glu?Ile?Trp?Glu?Leu?Ile?Gly?Ile?Pro?Glu?Asp?Gln?Arg?Leu?Gln

25 30 35

aga?act?gag?gtg?gta?aag?aag?cat?atc?aag?gaa?ctc?ctg?gat?atg?atg 319

Arg?Thr?Glu?Val?Val?Lys?Lys?His?Ile?Lys?Glu?Leu?Leu?Asp?Met?Met

40 45 50

att?gct?gaa?gag?gaa?agc?ctg?aag?gaa?aga?ctc?atc?aaa?agc?ata?tcc 367

Ile?Ala?Glu?Glu?Glu?Ser?Leu?Lys?Glu?Arg?Leu?Ile?Lys?Ser?Ile?Ser

55 60 65 70

gtc?tgt?cag?aaa?gag?ctg?aac?act?ctg?tgc?agc?gag?tta?cat?gtt?gag 415

Val?Cys?Gln?Lys?Glu?Leu?Asn?Thr?Leu?Cys?Ser?Glu?Leu?His?Val?Glu

75 80 85

cca?ttt?cag?gaa?gaa?gga?gag?acg?acc?atc?ttg?caa?cta?gaa?aaa?gat 463

Pro?Phe?Gln?Glu?Glu?Gly?Glu?Thr?Thr?Ile?Leu?Gln?Leu?Glu?Lys?Asp

90 95 100

ttg?cgc?acc?caa?gtg?gaa?ttg?atg?cga?aaa?cag?aaa?aag?gag?aga?aaa 511

Leu?Arg?Thr?Gln?Val?Glu?Leu?Met?Arg?Lys?Gln?Lys?Lys?Glu?Arg?Lys

105 110 115

cag?gaa?ctg?aag?cta?ctt?caa?gag?caa?gat?caa?gaa?ctg?tgc?gaa?att 559

Gln?Glu?Leu?Lys?Leu?Leu?Gln?Glu?Gln?Asp?Gln?Glu?Leu?Cys?Glu?Ile

120 125 130

ctt?tgt?atg?ccc?cac?tat?gat?att?gac?agt?gcc?tca?gtg?ccc?agc?tta 607

Leu?Cys?Met?Pro?His?Tyr?Asp?Ile?Asp?Ser?Ala?Ser?Val?Pro?Ser?Leu

135 140 145 150

gaa?gag?ctg?aac?cag?ttc?agg?caa?cat?gtg?aca?act?ttg?agg?gaa?aca 655

Glu?Glu?Leu?Asn?Gln?Phe?Arg?Gln?His?Val?Thr?Thr?Leu?Arg?Glu?Thr

155 160 165

aag?gct?tct?agg?cgt?gag?gag?ttt?gtc?agt?ata?aag?aga?cag?atc?ata 703

Lys?Ala?Ser?Arg?Arg?Glu?Glu?Phe?Val?Ser?Ile?Lys?Arg?Gln?Ile?Ile

170 175 180

ctg?tgt?atg?gaa?gaa?tta?gac?cac?acc?cca?gac?aca?agc?ttt?gaa?aga 751

Leu?Cys?Met?Glu?Glu?Leu?Asp?His?Thr?Pro?Asp?Thr?Ser?Phe?Glu?Arg

185 190 195

gat?gtg?gtg?tgt?gaa?gac?gaa?gat?gcc?ttt?tgt?ttg?tct?ttg?gag?aat 799

Asp?Val?Val?Cys?Glu?Asp?Glu?Asp?Ala?Phe?Cys?Leu?Ser?Leu?Glu?Asn

200 205 210

att?gca?aca?cta?caa?aag?ttg?cta?cgg?cag?ctg?gaa?atg?cag?aaa?tca 847

Ile?Ala?Thr?Leu?Gln?Lys?Leu?Leu?Arg?Gln?Leu?Glu?Met?Gln?Lys?Ser

215 220 225 230

caa?aat?gaa?gca?gtg?tgt?gag?ggg?ctg?cgt?act?caa?atc?cga?gag?ctc 895

Gln?Asn?Glu?Ala?Val?Cys?Glu?Gly?Leu?Arg?Thr?Gln?Ile?Arg?Glu?Leu

235 240 245

tgg?gac?agg?ttg?caa?ata?cct?gaa?gaa?gaa?aga?gaa?gct?gtg?gcc?acc 943

Trp?Asp?Arg?Leu?Gln?Ile?Pro?Glu?Glu?Glu?Arg?Glu?Ala?Val?Ala?Thr

250 255 260

att?atg?tct?ggg?tca?aag?gcc?aag?gtc?cgg?aaa?gcg?ctg?caa?tta?gaa 991

Ile?Met?Ser?Gly?Ser?Lys?Ala?Lys?Val?Arg?Lys?Ala?Leu?Gln?Leu?Glu

265 270 275

gtg?gat?cgg?ttg?gaa?gaa?ctg?aaa?atg?caa?aac?atg?aag?aaa?gtg?att 1039

Val?Asp?Arg?Leu?Glu?Glu?Leu?Lys?Met?Gln?Asn?Met?Lys?Lys?Val?Ile

280 285 290

gag?gca?att?cga?gtg?gag?ctg?gtt?cag?tac?tgg?gac?cag?tgc?ttt?tat 1087

Glu?Ala?Ile?Arg?Val?Glu?Leu?Val?Gln?Tyr?Trp?Asp?Gln?Cys?Phe?Tyr

295 300 305 310

agc?cag?gag?cag?aga?caa?gct?ttt?gcc?cct?ttc?tgt?gct?gag?gac?tac 1135

Ser?Gln?Glu?Gln?Arg?Gln?Ala?Phe?Ala?Pro?Phe?Cys?Ala?Glu?Asp?Tyr

315 320 325

aca?gaa?agt?ctg?ctc?cag?ctc?cac?gat?gct?gag?att?gtg?cgg?tta?aaa 1183

Thr?Glu?Ser?Leu?Leu?Gln?Leu?His?Asp?Ala?Glu?Ile?Val?Arg?Leu?Lys

330 335 340

aac?tac?tat?gaa?gtt?cac?aag?gaa?ctc?ttt?gaa?ggt?gtc?cag?aag?tgg 1231

Asn?Tyr?Tyr?Glu?Val?His?Lys?Glu?Leu?Phe?Glu?Gly?Val?Gln?Lys?Trp

345 350 355

gaa?gaa?acc?tgg?agg?ctt?ttc?tta?gag?ttt?gag?aga?aaa?gct?tca?gat 1279

Glu?Glu?Thr?Trp?Arg?Leu?Phe?Leu?Glu?Phe?Glu?Arg?Lys?Ala?Ser?Asp

360 365 370

cca?aat?cga?ttt?aca?aac?cga?gga?gga?aat?ctt?cta?aaa?gaa?gaa?aaa 1327

Pro?Asn?Arg?Phe?Thr?Asn?Arg?Gly?Gly?Asn?Leu?Leu?Lys?Glu?Glu?Lys

375 380 385 390

caa?cga?gcc?aag?ctc?cag?aaa?atg?ctg?ccc?aag?ctg?gaa?gaa?gag?ttg 1375

Gln?Arg?Ala?Lys?Leu?Gln?Lys?Met?Leu?Pro?Lys?Leu?Glu?Glu?Glu?Leu

395 400 405

aag?gca?cga?att?gaa?ttg?tgg?gaa?cag?gaa?cat?tca?aag?gca?ttt?atg 1423

Lys?Ala?Arg?Ile?Glu?Leu?Trp?Glu?Gln?Glu?His?Ser?Lys?Ala?Phe?Met

410 415 420

gtg?aat?ggg?cag?aaa?ttc?atg?gag?tat?gtg?gca?gaa?caa?tgg?gag?atg 1471

Val?Asn?Gly?Gln?Lys?Phe?Met?Glu?Tyr?Val?Ala?Glu?Gln?Trp?Glu?Met

425 430 435

cat?cga?ttg?gag?aaa?gag?aga?gcc?aag?cag?gaa?aga?caa?ctg?aag?aac 1519

His?Arg?Leu?Glu?Lys?Glu?Arg?Ala?Lys?Gln?Glu?Arg?Gln?Leu?Lys?Asn

440 445 450

aaa?aaa?cag?aca?gag?aca?gag?atg?ctg?tat?ggc?agc?gct?cct?cga?aca 1567

Lys?Lys?Gln?Thr?Glu?Thr?Glu?Met?Leu?Tyr?Gly?Ser?Ala?Pro?Arg?Thr

455 460 465 470

cct?agc?aag?cgg?cga?gga?ctg?gct?ccc?aat?aca?ccg?ggc?aaa?gca?cgt 1615

Pro?Ser?Lys?Arg?Arg?Gly?Leu?Ala?Pro?Asn?Thr?Pro?Gly?Lys?Ala?Arg

475 480 485

aag?ctg?aac?act?acc?acc?atg?tcc?aat?gct?acg?gcc?aat?agt?agc?att 1663

Lys?Leu?Asn?Thr?Thr?Thr?Met?Ser?Asn?Ala?Thr?Ala?Asn?Ser?Ser?Ile

490 495 500

cgg?cct?atc?ttt?gga?ggg?aca?gtc?tac?cac?tcc?ccc?gtg?tct?cga?ctt 1711

Arg?Pro?Ile?Phe?Gly?Gly?Thr?Val?Tyr?His?Ser?Pro?Val?Ser?Arg?Leu

505 510 515

cct?cct?tct?ggc?agc?aag?cca?gtc?gct?gct?tcc?acc?tgt?tca?ggg?aag 1759

Pro?Pro?Ser?Gly?Ser?Lys?Pro?Val?Ala?Ala?Ser?Thr?Cys?Ser?Gly?Lys

520 525 530

aaa?aca?ccc?cgt?act?ggc?agg?cat?gga?gcc?aac?aag?gag?aac?ctg?gag 1807

Lys?Thr?Pro?Arg?Thr?Gly?Arg?His?Gly?Ala?Asn?Lys?Glu?Asn?Leu?Glu

535 540 545 550

ctc?aac?ggc?agc?atc?ctg?agt?gcg?aga?act?ttc?aaa?ggc?ttc?caa?atc 1855

Leu?Asn?Gly?Ser?Ile?Leu?Ser?Ala?Arg?Thr?Phe?Lys?Gly?Phe?Gln?Ile

555 560 565

tga?tgctacttct?ggaatcctca?attcaaccaa?catccagtcc?tgagaagccc 1908

tgatcagtca?accagctgtg?gcttcctgtg?cctagactgg?acctaattat?atgggggtga 1968

ctttagtttt?tcttcagctt?aggcgtgctt?gaaaccttgg?ccaggttcca?tgaccatggg 2028

cctaacttaa?agatgtgaat?gagtgttaca?gttgaaagcc?catcataggt?ttagtggtcc 2088

taggagactt?ggttttgact?tatatacatg?aaaagtttat?ggcaagaagt?gcaaatttta 2148

gcatatgggg?cctgacttct?ctaccacata?attctacttg?ctgaagcatg?atcaaagctt 2208

gttttatttc?accactgtag?gaaaatgatt?gactatgccc?atccctgggg?gtaattttgg 2268

catgtatacc?tgtaactagt?aattaacatc?ttttttgttt?aggcatgttc?aattaatgct 2328

gtagctatca?tagctttgct?cttacctgaa?gccttgtccc?caccacacag?gacagccttc 2388

ctcctgaaga?gaatgtcttt?gtgtgtccga?agttgagatg?gcctgcccta?ctgccaaaga 2448

ggtgacagga?aggctgggag?cagctttgtt?aaattgtgtt?cagttctgtt?acacagtgca 2508

ttgccctttg?ttgggggtat?gcatgtatga?acacacatgc?ttgtcggaac?gctttctcgg 2568

cgtttgtccc?ttggctctca?tctcccccat?tcctgtgcct?actttgcctg?agttcttcta 2628

cccccgcagt?tgccagccac?attgggagtc?tgtttgttcc?aatgggttga?gctgtctttg 2688

tcgtggagat?ctggaacttt?gcacatgtca?ctactgggga?ggtgttcctg?ctctagcttc 2748

cacgatgagg?cgccctcttt?acctatcctc?tcaatcacta?ctcttcttga?agcactatta 2808

tttattcttc?cgctgtctgc?ctgcagcagt?actactgtca?acatagtgta?aatggttctc 2868

aaaagcttac?cagtgtggac?ttggtgttag?ccacgctgtt?tactcataca?gtacgtgtcc 2928

tgtttttaaa?atatacaatt?attcttaaaa?ataaattaaa?atctgtatac?ttacatttca 2988

aaaagaaaaa?aaaaaaaaaa?aaa 3011

<210>44

<211>566

<212>PRT

＜213〉people (Homo sapiens)

<400>44

Met?Arg?Arg?Ser?Glu?Val?Leu?Ala?Glu?Glu?Ser?Ile?Val?Cys?Leu?Gln

1 5 10 15

Lys?Ala?Leu?Asn?His?Leu?Arg?Glu?Ile?Trp?Glu?Leu?Ile?Gly?Ile?Pro

20 25 30

Glu?Asp?Gln?Arg?Leu?Gln?Arg?Thr?Glu?Val?Val?Lys?Lys?His?Ile?Lys

35 40 45

Glu?Leu?Leu?Asp?Met?Met?Ile?Ala?Glu?Glu?Glu?Ser?Leu?Lys?Glu?Arg

50 55 60

Leu?Ile?Lys?Ser?Ile?Ser?Val?Cys?Gln?Lys?Glu?Leu?Asn?Thr?Leu?Cys

65 70 75 80

Ser?Glu?Leu?His?Val?Glu?Pro?Phe?Gln?Glu?Glu?Gly?Glu?Thr?Thr?Ile

85 90 95

Leu?Gln?Leu?Glu?Lys?Asp?Leu?Arg?Thr?Gln?Val?Glu?Leu?Met?Arg?Lys

100 105 110

Gln?Lys?Lys?Glu?Arg?Lys?Gln?Glu?Leu?Lys?Leu?Leu?Gln?Glu?Gln?Asp

115 120 125

Gln?Glu?Leu?Cys?Glu?Ile?Leu?Cys?Met?Pro?His?Tyr?Asp?Ile?Asp?Ser

130 135 140

Ala?Ser?Val?Pro?Ser?Leu?Glu?Glu?Leu?Asn?Gln?Phe?Arg?Gln?His?Val

145 150 155 160

Thr?Thr?Leu?Arg?Glu?Thr?Lys?Ala?Ser?Arg?Arg?Glu?Glu?Phe?Val?Ser

165 170 175

Ile?Lys?Arg?Gln?Ile?Ile?Leu?Cys?Met?Glu?Glu?Leu?Asp?His?Thr?Pro

180 185 190

Asp?Thr?Ser?Phe?Glu?Arg?Asp?Val?Val?Cys?Glu?Asp?Glu?Asp?Ala?Phe

195 200 205

Cys?Leu?Ser?Leu?Glu?Asn?Ile?Ala?Thr?Leu?Gln?Lys?Leu?Leu?Arg?Gln

210 215 220

Leu?Glu?Met?Gln?Lys?Ser?Gln?Asn?Glu?Ala?Val?Cys?Glu?Gly?Leu?Arg

225 230 235 240

Thr?Gln?Ile?Arg?Glu?Leu?Trp?Asp?Arg?Leu?Gln?Ile?Pro?Glu?Glu?Glu

245 250 255

Arg?Glu?Ala?Val?Ala?Thr?Ile?Met?Ser?Gly?Ser?Lys?Ala?Lys?Val?Arg

260 265 270

Lys?Ala?Leu?Gln?Leu?Glu?Val?Asp?Arg?Leu?Glu?Glu?Leu?Lys?Met?Gln

275 280 285

Asn?Met?Lys?Lys?Val?Ile?Glu?Ala?Ile?Arg?Val?Glu?Leu?Val?Gln?Tyr

290 295 300

Trp?Asp?Gln?Cys?Phe?Tyr?Ser?Gln?Glu?Gln?Arg?Gln?Ala?Phe?Ala?Pro

305 310 315 320

Phe?Cys?Ala?Glu?Asp?Tyr?Thr?Glu?Ser?Leu?Leu?Gln?Leu?His?Asp?Ala

325 330 335

Glu?Ile?Val?Arg?Leu?Lys?Asn?Tyr?Tyr?Glu?Val?His?Lys?Glu?Leu?Phe

340 345 350

Glu?Gly?Val?Gln?Lys?Trp?Glu?Glu?Thr?Trp?Arg?Leu?Phe?Leu?Glu?Phe

355 360 365

Glu?Arg?Lys?Ala?Ser?Asp?Pro?Asn?Arg?Phe?Thr?Asn?Arg?Gly?Gly?Asn

370 375 380

Leu?Leu?Lys?Glu?Glu?Lys?Gln?Arg?Ala?Lys?Leu?Gln?Lys?Met?Leu?Pro

385 390 395 400

Lys?Leu?Glu?Glu?Glu?Leu?Lys?Ala?Arg?Ile?Glu?Leu?Trp?Glu?Gln?Glu

405 410 415

His?Ser?Lys?Ala?Phe?Met?Val?Asn?Gly?Gln?Lys?Phe?Met?Glu?Tyr?Val

420 425 430

Ala?Glu?Gln?Trp?Glu?Met?His?Arg?Leu?Glu?Lys?Glu?Arg?Ala?Lys?Gln

435 440 445

Glu?Arg?Gln?Leu?Lys?Asn?Lys?Lys?Gln?Thr?Glu?Thr?Glu?Met?Leu?Tyr

450 455 460

Gly?Ser?Ala?Pro?Arg?Thr?Pro?Ser?Lys?Arg?Arg?Gly?Leu?Ala?Pro?Asn

465 470 475 480

Thr?Pro?Gly?Lys?Ala?Arg?Lys?Leu?Asn?Thr?Thr?Thr?Met?Ser?Asn?Ala

485 490 495

Thr?Ala?Asn?Ser?Ser?Ile?Arg?Pro?Ile?Phe?Gly?Gly?Thr?Val?Tyr?His

500 505 510

Ser?Pro?Val?Ser?Arg?Leu?Pro?Pro?Ser?Gly?Ser?Lys?Pro?Val?Ala?Ala

515 520 525

Ser?Thr?Cys?Ser?Gly?Lys?Lys?Thr?Pro?Arg?Thr?Gly?Arg?His?Gly?Ala

530 535 540

Asn?Lys?Glu?Asn?Leu?Glu?Leu?Asn?Gly?Ser?Ile?Leu?Ser?Ala?Arg?Thr

545 550 555 560

Phe?Lys?Gly?Phe?Gln?Ile

565

<210>45

<211>19

<212>DNA

＜213〉artificial

<220>

＜223〉be used for the artificial sequence synthesized primer of RT-PCR

<400>45

aacttagagg?tgggagcag 19

<210>46

<211>22

<212>DNA

＜213〉artificial

<220>

＜223〉be used for the artificial sequence synthesized primer of RT-PCR

<400>46

cacaaccatg?ccttacttta?tc 22

<210>47

<211>29

<212>DNA

＜213〉artificial

<220>

＜223〉be used for the artificial sequence synthesized primer of PCR

<400>47

ccggaattct?ccgccatgag?gagaagtga 29

<210>48

<211>31

<212>DNA

＜213〉artificial

<220>

＜223〉be used for the artificial sequence synthesized primer of PCR

<400>48

ttgccgctcg?agggactgga?tgttggttga?a 31

<210>49

<211>27

<212>DNA

＜213〉artificial

<220>

＜223〉be used for the artificial sequence synthesized primer of PCR

<400>49

ccggaattca?tggccatgga?ctcgtcg 27

<210>50

<211>27

<212>DNA

＜213〉artificial

<220>

＜223〉be used for the artificial sequence synthesized primer of PCR

<400>50

gctccgctcg?agctggggcc?gtttctt 27

Claims

1. the method for mammary cancer among treatment or the prevention experimenter, comprise to described experimenter and use composition, described composition contains the siRNA (siRNA) that suppresses A2254 (SEQ ID NO:35 or 37) or A5623 (SEQ IDNO:39,41 or 43) expression.

2. the process of claim 1 wherein that described siRNA includes phosphorothioate odn sequence and anti sense nucleotide sequence, described anti sense nucleotide sequence is hybridized with the sequence specific from A2254 or A5623.

3. the method for claim 2, wherein said siRNA comprises ribonucleoside acid sequence as described below as target sequence: this ribonucleoside acid sequence is corresponding to being selected from SEQ ID NO:21,25,29 and 33 sequence.

4. the method for claim 3, wherein said siRNA has general formula 5 '-[A]-[B]-[A ']-3 ', and wherein [A] is and the corresponding ribonucleoside acid sequence of selecting from SEQ ID NO:21,25,29 and 33 Nucleotide of sequence;

[B] is the ribonucleotide ring sequence that is made of 3-23 Nucleotide,

5. the process of claim 1 wherein that described composition comprises the transfection toughener.

6. duplex molecule, comprise sense strand and antisense strand, wherein sense strand comprises and the corresponding ribonucleoside acid sequence of selecting from SEQ IDNO:21,25,29 and 33 of target sequence, wherein antisense strand comprises and described sense strand complementary ribonucleoside acid sequence, wherein said sense strand and described antisense strand are hybridized each other and are formed described duplex molecule, and wherein said duplex molecule suppresses described expression of gene in being introduced in the cell of expressing A2254 or A5623 gene the time.

7. the duplex molecule of claim 6, wherein said target sequence comprise coming about at least 10 continuous nucleotides of the nucleotide sequence of selecting in SEQ ID NO:35,37,39,41 and 43.

8. the duplex molecule of claim 7, wherein said target sequence comprise coming the nucleotide sequence in SEQ ID NO:35,37,39,41 and 43, selected about 19 to about 25 continuous nucleotides.

9. the duplex molecule of claim 8, wherein said duplex molecule is single ribonucleotide transcript, comprises the sense strand and the antisense strand that connect by strand ribonucleoside acid sequence.

10. the duplex molecule of claim 7, wherein this duplex molecule is the oligonucleotide of length less than about 100 Nucleotide.

11. the duplex molecule of claim 10, wherein this duplex molecule is the oligonucleotide of length less than about 75 Nucleotide.

12. the duplex molecule of claim 11, wherein this duplex molecule is the oligonucleotide of length less than about 50 Nucleotide.

13. the duplex molecule of claim 12, wherein this duplex molecule is the oligonucleotide of length less than about 25 Nucleotide.

14. the duplex molecule of claim 13, wherein this duplex molecule is that length is the oligonucleotide of about 25 Nucleotide of about 19-.

15. the carrier of the duplex molecule of the claim 7 of encoding.

16. the carrier of claim 15, this vector encoded transcript wherein, described transcript has secondary structure and comprises sense strand and antisense strand.

17. the carrier of claim 16, wherein this transcript further comprises the strand ribonucleoside acid sequence that connects described sense strand and described antisense strand.

18. carrier that comprises polynucleotide, these polynucleotide comprise the combination of sense strand nucleic acid and antisense strand nucleic acid, wherein said sense strand nucleic acid comprises SEQ ID NO:21,25,29 and 33 nucleotide sequence, and described antisense strand nucleic acid is by constituting with sense strand complementary sequence.

19. the carrier of claim 18, wherein said polynucleotide have general formula 5 '-[A]-[B]-[A ']-3 '

Wherein [A] is SEQ ID NO:21,25,29 and 33 nucleotide sequence; [B] is the nucleotide sequence that is made of 3-23 Nucleotide; [A '] be and [A] complementary nucleotide sequence.

20. one kind is used for the treatment of or the pharmaceutical composition of Breast Cancer Prevention, comprises siRNA (siRNA) and medicine acceptable carrier as the medicine effective quantity of activeconstituents, wherein this siRNA can suppress the expression of A2254 or A5623.

21. the pharmaceutical composition of claim 20, wherein this siRNA comprises from SEQ ID NO:21,25,29 and 33 nucleotide sequences of selecting as target sequence.

22. the composition of claim 21, wherein this siRNA has general formula 5 '-[A]-[B]-[A ']-3 '

Wherein [A] is the ribonucleoside acid sequence corresponding to SEQ ID NO:21,25,29 and 33 nucleotide sequence; [B] is the ribonucleoside acid sequence that is made of 3-23 Nucleotide; [A '] be and [A] complementary ribonucleoside acid sequence.

23. a screening is used for the treatment of or the method for the compound of Breast Cancer Prevention, described method comprises the steps:

(b) combination between the detection polypeptide; With

(c) select to suppress bonded test compounds between polypeptide.

24. the method for claim 23, wherein this contains A2254-and comprises the A5623 polypeptide in conjunction with the polypeptide in territory.

25. according to the method for claim 23, wherein this contains A5623-and comprises the A2254 polypeptide in conjunction with the polypeptide in territory.

26. screening can be used for treating or the test kit of the compound of Breast Cancer Prevention, wherein this test kit comprises:

(a) comprise the polypeptide of A5623 polypeptide A 2254-in conjunction with the territory;

(b) comprise the polypeptide of A2254 polypeptide A 5623-in conjunction with the territory; With

(c) be used to detect the reagent of polypeptide interphase interaction.

27. according to the test kit of claim 26, wherein this contains A2254-and comprises the A5623 polypeptide in conjunction with the polypeptide in territory.

28. according to the test kit of claim 26, wherein this contains A5623-and comprises the A2254 polypeptide in conjunction with the polypeptide in territory.

29. be used for the treatment of or prevent the method for mammary cancer among the experimenter, wherein this method comprises the step of the inhibition A5623 polypeptide and the A2254 polypeptide bonded compound of drug administration significant quantity.

30. be used for the treatment of or the composition of Breast Cancer Prevention, wherein said composition comprises inhibition A5623 polypeptide and the A2254 polypeptide bonded compound and the medicine acceptable carrier of medicine effective quantity.