CN101586155A - Method for diagnosing non-small cell lung cancers - Google Patents

Method for diagnosing non-small cell lung cancers Download PDF

Info

Publication number
CN101586155A
CN101586155A CNA2008101867066A CN200810186706A CN101586155A CN 101586155 A CN101586155 A CN 101586155A CN A2008101867066 A CNA2008101867066 A CN A2008101867066A CN 200810186706 A CN200810186706 A CN 200810186706A CN 101586155 A CN101586155 A CN 101586155A
Authority
CN
China
Prior art keywords
nsc
gene
cell
lung cancer
expression
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CNA2008101867066A
Other languages
Chinese (zh)
Inventor
中村佑辅
醍醐弥太郎
中鹤修一
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Oncotherapy Science Inc
Original Assignee
Oncotherapy Science Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Oncotherapy Science Inc filed Critical Oncotherapy Science Inc
Publication of CN101586155A publication Critical patent/CN101586155A/en
Pending legal-status Critical Current

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1135—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against oncogenes or tumor suppressor genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57423—Specifically defined cancers of lung
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/50—Determining the risk of developing a disease

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • Biomedical Technology (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Hematology (AREA)
  • Microbiology (AREA)
  • Urology & Nephrology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Pathology (AREA)
  • Medicinal Chemistry (AREA)
  • Oncology (AREA)
  • Biophysics (AREA)
  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Hospice & Palliative Care (AREA)
  • Food Science & Technology (AREA)
  • Toxicology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Epidemiology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Plant Pathology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

Disclosed are methods for detecting non-small cell lung cancer using differentially expressed genes. Furthermore, novel human genes whose expression is elevated in non-small cell lung cancer compared to no-cancerous tissues are provided. Also disclosed are methods of identifying compounds for treating and preventing non-small cell lung cancer.

Description

The diagnostic method of nonsmall-cell lung cancer
The application be that September 22, application number in 2003 are 03825506.5 the applying date, exercise question divides an application for the application for a patent for invention of " diagnostic method of nonsmall-cell lung cancer ".
The application relates to the USSN60/414 of submission on September 30th, 2002, the USSN60/451 that on February 28th, 673,2003 submitted to, and the USSN 60/466 that on April 28th, 374 and 2003 submitted to is hereby incorporated by.
Invention field
The present invention relates to field of biology, relate more specifically to the cancer research field.Particularly, the present invention relates to the diagnostic method of nonsmall-cell lung cancer, and the gene that raises or reduced of the expression in this class cancerous cells.
Background technology
Lung cancer is one of human modal mortality tumour.Reported that now many and lung cancer take place and develop relevant heredity to change.Although hereditary variation can help risk that prognosis and prediction shift or to the replying of some treatments, can not provide gratifying result (Clin Cancer Res 6:4055-63 (2000) for the clinical diagnosis of nonsmall-cell lung cancer (NSCLC) usually about the information of the molecule marker of single or limited quantity; Niklinski et al., Lung Cancer.34 Suppl 2:S53-8 (2001); Watine, Bmj 320:379-80 (2000)).Nonsmall-cell lung cancer (NSCLC) is present modal type, accounts for nearly 80% (Society, A.C.Cancer Facts and Figures 2001,2001.) of whole lung tumors.The progress of coming in multiple-pattern although (multi-modality) treatment, but total 10-annual survival rate still remain on 10% low-level, this mainly is (Fry because most of NSCLC just can be diagnosed up to late period, W.A., Phillips, J.L., and Menck, H.R.Ten-year survey oflung cancer treatment and survival in ho spitals in the United States:a nationalcancer data base report, Cancer.86:1867-76., 1999.).Although be considered to the reference standard of NSCLC treatment based on the chemotherapy regimen of platinum, but those medicines only can be with (Chemotherapy in non-small cell lung cancer:ameta-analysis using updated data on individual patients from 52 randomisedclinical trials.Non-small Cell Lung Cancer Collaborative Group of about 6 weeks of advanced NSCLC patient's prolonged survival period, Bmj.311:899-909., 1995.).Now just studying the multiple targeted therapy method of this disease, comprising tyrosine kinase inhibitor, but only in the patient of limited quantity, obtained satisfied effect up to now, and some recipients even serious adverse effects (Kris M occurred, N.R., Herbst RS A phaseII trial of ZD1839 (' Iressa ') in advanced non-small cell lung cancer (NSCLC) patients who had failed platinum-and docetaxel-based regimens (IDEAL 2)., Proc Am Soc Clin Oncol.21:292a (A1166), 2002).
The cDNA microarray technology can obtain comprehensive gene expression atlas from normal and malignant cell, and genetic expression in the malignant cell and genetic expression in the corresponding normal cell compared (Okabe et al., Cancer Res 61:2129-37 (2001); Kitahara et al., Cancer Res 61:3544-9 (2001); Lin et al., Oncogene 21:4120-8 (2002); Hasegawa et al., CancerRes 62:7012-7 (2002)).This method can disclose the complex characteristics of cancer cells, and helps to understand the mechanism of carcinogenesis.Identify in the tumour those and go genes of regulating can be more accurately and the individual cancer of diagnosis exactly, thus the treatment target (Bienz and Clevers, Cell103:311-20 (2000)) that exploitation makes new advances.In order to disclose the genesis mechanism of tumour from the genome angle, and find the target molecules of diagnosis usefulness and the medicine that exploitation makes new advances, the inventor has carried out analyzing (Okabe et al., CancerRes 61:2129-37 (2001) with the cDNA microarray of 23040 genomic constitutions to the expression map of tumour cell; Kitahara et al., Cancer Res 61:3544-9 (2001); Lin et al., Oncogene 21:4120-8 (2002); Hasegawa et al., Cancer Res 62:7012-7 (2002)).
Promoted the evaluation of the molecular target of Anti-tumor agent for disclosing experiment that mechanism of carcinogenesis designs.For example, be initially the farnesyl tranfering enzyme (farnexyltransferase) that suppresses the growth-signal pathway relevant and design with Ras (FTI) inhibitor can effectively treat Ras-dependent tumors in the animal model, wherein the activation of Ras depends on translation back farnesylation (He et al., Cell 99:335-45 (1999)).Used Anti-tumor medicine and the combination of anti--HER2 monoclonal antibody trastuzumab that the people is carried out clinical experiment, with antagonism antagonism proto-oncogene acceptor HER2/neu; And patient with breast cancer's clinical response and total survival rate (Lin et al., Cancer Res 61:6345-9 (2001)) have been improved.A kind of tyrosine kinase inhibitor STI-571 that can selectivity suppresses the bcr-abl fusion rotein has been developed and has been used for the treatment of chronic lymphocytic leukemia (chronic myelogenous leukemia), and wherein the activation of the composition of bcr-abl Tyrosylprotein kinase plays an important role in white corpuscle transforms.The medicament of these kinds is designed to suppress the carcinogenic activity (Fujita et al., Cancer Res61:7722-6 (2001)) of concrete gene product.Therefore, the gene product that raises usually in the cancerous cells can be as the potential target of development of new cancer therapy drug.
Proved CD8 +Cytotoxic T cell (CTL) is discerned the epitope peptide of the tumor associated antigen of presenting from MHC I quasi-molecule (TAA), and the dissolving tumour cell.Since finding MAGE family first example, many other TAA (Boon, Int JCancer 54:177-80 (1993) have been found with immunological method as TAA; Boon and van der Bruggen, J Exp Med 183:725-9 (1996); Van der Bruggen etc., Science 254:1643-7 (1991); Brichard etc., J ExpMed 178:489-95 (1993); Kawakami etc., J Exp Med 180:347-52 (1994)).Some TAA that have been found that are in the clinical development stage as the immunotherapy target spot at present.The TAA that has been found that up to now comprises MAGE (van der Bruggen etc., Science 254:1643-7 (1991)), gp100 (Kawakami etc., J Exp Med 180:347-52 (1994)), SART (Shichijo etc., J Exp Med 187:277-88 (1998)) and NY-ESO-1 (Chen etc., Proc Natl Acad SciUSA 94:1914-8 (1997)).On the other hand, the certified gene product of especially crossing expression in tumour cell can be used as the immunoreactive target spot of inducing cell and is identified.This gene product comprises p53 (Umano etc., Brit J Cancer 84:1052-7 (2001)), HER2/neu (Tanaka etc., Brit JCancer 84:94-9 (2001)), CEA (Nukaya etc., Int J Cancer 80:92-7 (1999)) or the like.
Although in about the basis of TAA and clinical study, obtained important progress (Rosenbeg etc., Nature Med 4:321-7 (1998); Mukherji etc., Proc Natl Acad Sci USA 92:8078-82 (1995); Hu etc., Cancer Res 56:2479-83 (1996)), have only the candidate TAA of limited quantity can treat gland cancer, comprise colorectal carcinoma.A large amount of expression in cancer cells, the TAA that its expression simultaneously is limited in the cancer cells is the material standed for (candidate) of immunotherapy target spot likely.In addition, can promote clinical use (Boon and can der Bruggen, the J Exp Med 183:725-9 (1996) of peptide vaccination strategy in the broad variety cancer to inducing evaluation effective and the novel TAA that specificity antineoplastic immunity reacts to be expected; Van der Bruggen etc., Science 254:1643-7 (1991); Brichard etc., JExp Med 178:489-95 (1993); Kawakami etc., J Exp Med 180:347-52 (1994); Shichijo etc., J Exp Med 187:277-88 (1998); Chen etc., Proc Natl Acad Sci USA94:1914-8 (1997); Harris, J Natl Cancer Inst 88:1442-5 (1996); Butterfield etc., Cancer Res 59:3134-42 (1999); Vissers etc., Cancer Res 59:5554-9 (1999); Vander Burg etc., J Immunol 156:3308-14 (1996); Tanaka etc., Cancer Res 57:4465-8 (1997); Fujie etc., Int J Cancer 80:169-72 (1999); Kikuchi etc., Int J Cancer 81:459-66 (1999); Oiso etc., Int J Cancer 81:387-94 (1999)).
Existing report repeats to point out, peptide stimulated peripheral mononuclear cells (PBMC) from concrete healthy donors produces response and the IFN-γ of generation conspicuous level to this peptide, but in chromium-51 release experiment hardly with HLA-A24 or-A0201 restriction map performance is at cytotoxicity (Kawano etc., the Cance Res 60:3550-8 (2000) of tumour cell; Nishizaka etc., Cancer Res 60:4830-7 (2000); Tamura etc., Jpn J Cancer Res 92:762-7 (2001)).But HLA-A24 and HLA-A0201 are a kind of HLA allelotrope common in Japanese and Caucasian (Date etc., Tissue Antigens 47:93-101 (1996); Kondo etc., J Immunol 155:4307-12 (1995); Kubo etc., J Immunol 152:3913-24 (1994); Imanishi etc., Proceeding of the eleventh International Hictocompatibility Workshop andConference Oxford University Press, Oxford, 1065 (1992); Williams etc., TissueAntigen 49:129 (1997)).Therefore, be specially adapted to treat cancer among Japanese and the Caucasian by the cancer antigen peptide that these HLA presented.In addition, the known result who typically uses the high density peptide at external evoked low-affinity CTL, the use of described high density peptide is gone up at antigen presenting cell (APC) and is generated high-level specific peptide/MHC mixture, described mixture will effectively activate these CTL (Alexander-Miller etc., Proc Natl Acad Sci USA 93:4102-7 (1996)).
Summary of the invention
The present invention is based on discovery with nonsmall-cell lung cancer gene expression related pattern, for example squamous cell carcinoma, gland cancer is (promptly for described lung cancer, acinus (acinar), corpora mammillaria (papillary) and bronchovesicular (bronchoalveolar)), large cell carcinoma (that is, giant cells and hyaline cell), adenosquamous carcinoma and undifferentiated carcinoma.
Those genes of differential expression in nonsmall-cell lung cancer are referred to as " nonsmall-cell lung cancer-genes involved ", " NSC nucleic acid " or " NSC polynucleotide " in this application, and the polypeptide of these genes encodings is called " NSC polypeptide " or " NSC albumen ".Among the application, the differential expression in nonsmall-cell lung cancer shows that the expression level of gene in non-small cell lung cancer cell is different from the expression level in normal cell.Normal cell is available from lung tissue.
The invention provides the tendentious method of suffering from nonsmall-cell lung cancer among a kind of diagnosis or the definite experimenter, this method is undertaken by nonsmall-cell lung cancer-Expression of Related Genes level in the biological sample of measuring the patient source.Nonsmall-cell lung cancer-genes involved comprises that for example nonsmall-cell lung cancer-genes involved comprises, for example, and NSC 1-1448 (referring to table 1-3).Compare with the normal control level of this gene, the variation of this gene expression dose for example raises or reduces, and shows that this experimenter suffers from or easily suffers from nonsmall-cell lung cancer.
" normal control level " is meant detected gene expression dose the colony of or the known individuality of not suffering from nonsmall-cell lung cancer individual from normal health.Control level is single expression pattern or the multiple expression pattern that is derived from single reference group.Different with " normal control level ", " control level " is detected gene expression dose from disease state background (that is cancer or non-cancer) known individuality or population of individuals.Therefore, control level can be measured based on expression of gene level in normal health individuality, known colony of not suffering from the individuality of nonsmall-cell lung cancer, nonsmall-cell lung cancer patient or the described patient colony.Be called " non--the small cell lung cancer control level " with the corresponding control level of gene expression dose among nonsmall-cell lung cancer patient or the patient group.In addition, described control level can be the database of expression pattern of the cell of this Pretesting.
In the biological subject sample among the detected NSC807-1448 any one or one group of (panel) expression of gene level raise compared with the normal control level and show: this experimenter (having obtained described sample from described experimenter) suffers from or easily suffers from nonsmall-cell lung cancer.On the contrary, detecting any one or one group of expression of gene level among the NSC 1-806 in the biological subject sample reduces then compared with the normal control level and show: this experimenter suffers from or easily suffers from nonsmall-cell lung cancer.Optional, the nonsmall-cell lung cancer control level of any one or one group of nonsmall-cell lung cancer-Expression of Related Genes level and same or same group gene in the biological sample can be compared.
Compare the genetic expression rising with control level or reduced by 10%, 25%, 50% or more.Perhaps, genetic expression is compared with control level increases or has reduced 1,2,5 or more times.Can measure described expression by hybridization (for example on chip or the array) situation that detects nonsmall-cell lung cancer-genes involved probe and be derived between patient's the genetic transcription thing of biological sample.The described patient's of being derived from biological sample can be derived from the experimenter, for example known or doubtful any sample of suffering from the patient of nonsmall-cell lung cancer.For example, described biological sample can be the tissue that contains phlegm, blood, serum, blood plasma or pneumonocyte.
The present invention also provide a kind of nonsmall-cell lung cancer with reference to expression map, wherein contain gene expression dose mode charts two or more among the NSC1-1448.
The present invention further also provides and has been used for identifying and can (for example suppresses or improve the non-small cell lung cancer associated gene, NSC 1-1448) the expression or the method for active compound, this method contacts with test-compound by making the subject cell of expressing the non-small cell lung cancer associated gene, and measures this nonsmall-cell lung cancer Expression of Related Genes level or activity is carried out.Described subject cell can be a for example pulmonary epithelial cells of pneumonocyte.Expression level is compared reduction and is shown that then this test-compound is the inhibitor of described nonsmall-cell lung cancer Expression of Related Genes or function with the normal control level of this gene.Therefore, if a kind of compound is suppressed the expression level of nonsmall-cell lung cancer-genes involved NSC 807-1448 compared with the normal control level, this compound just is expected to alleviate the symptom of nonsmall-cell lung cancer so.Otherwise compare expression level with the normal control level of this gene or active the rising then shows: described test-compound is the reinforce of described nonsmall-cell lung cancer-Expression of Related Genes or function.The compound that expection can improve nonsmall-cell lung cancer-genes involved NSC 1-806 expression level can alleviate the symptom of nonsmall-cell lung cancer.
In addition, the present invention also provides a kind of screening to be used for the treatment of or to prevent the method for nonsmall-cell lung cancer.This method comprises: the NSC polypeptide is contacted with test-compound, filter out then to combine with described NSC polypeptide and maybe can change the bioactive test-compound of described NSC polypeptide.In addition, the present invention also provides a kind of screening to be used for the treatment of or to prevent the method for the compound of nonsmall-cell lung cancer, the cells contacting that this method comprises the steps: to make test-compound and a kind of NSC of expression albumen or imported a kind of carrier, this carrier contains the NSC gene transcription control region that is positioned at the reporter gene upstream, filters out the test-compound that can change NSC albumen or the coded protein expression level of described reporter gene then.According to these methods, when using NSC 807-1448 encoded polypeptides or express cell by NSC 807-1448 or NSC 807-1448 transcription regulatory region encoded protein, expection makes the downtrod compared with the normal control level test-compound of described biological activity or expression level can alleviate the symptom of nonsmall-cell lung cancer.Optional, when using NSC 1-806 encoded polypeptides or express the coded proteic cell of the transcription regulatory region of NSC 1-806 or NSC1-806, the raise test-compound of described expression level of expection can alleviate the symptom of nonsmall-cell lung cancer.
The present invention also provides a kind of test kit, wherein contains two or more detection reagent, this reagent can combine with one or more NSC nucleic acid or can with gene product (for example, mRNA and the polypeptide) combination of NSC nucleic acid.The present invention also provide can with the array of one or more NSC nucleic acid bonded polynucleotide.
The present invention also provides treatment or prevention nonsmall-cell lung cancer method and this method compositions for use in addition.Methods of treatment comprises the method for nonsmall-cell lung cancer among treatment or the prevention experimenter, this method perhaps contains antibody or its segmental composition that can combine and suppress described polypeptide function with this coded by said gene polypeptide and carries out by use the composition that contains antisense, short interfering rna (siRNA) or the ribozyme that can reduce gene NSC 807-1448 expression to this experimenter.
The present invention also comprises vaccine and inoculation method.For example, thereby implement the method for treatment or prevention nonsmall-cell lung cancer, contain polynucleotide NSC807-1448 encoded polypeptides or its immunocompetence fragment in the described vaccine by use vaccine to this experimenter.The immunocompetence fragment is a kind of length and polypeptide that when importing body can induce immune response shorter than total length native protein.For example, the immunocompetence fragment comprises the polypeptide of at least 8 residues of length, and described polypeptide is immune stimulatory cell such as T cell or B cell in vivo.The mensuration that immunocyte stimulates can (for example, IL-2) processing (elaboration) or antibody generate and carry out by detecting cell proliferation, cytokine.
Other methods of treatment comprises: use a kind of active compound that can improve gene NSC 1-806 expression level or the coded polypeptide of gene NSC 1-806 to the experimenter.Optional, can treat or prevent nonsmall-cell lung cancer by using the polynucleotide among the NSC 1-806 (for example, be contained in the carrier polynucleotide) or the polypeptide of this polynucleotide encoding.In addition, the invention provides the method that is used for the treatment of or prevents nonsmall-cell lung cancer, wherein use the compound of selecting by screening method of the present invention.
On the other hand, the invention provides a kind of pure basically polypeptide, it comprises aminoacid sequence shown in the SEQ ID NO:2.Can described aminoacid sequence be undergone mutation by substituting, lack, insert and/or adding at least 1,2,3,5,10,25,50,100 or 200 amino acid, still keep proteic one or more biologic activity of forming by aminoacid sequence shown in the SEQ ID NO:2 as long as have the polypeptide of described aminoacid sequence.Described mutant polypeptide and comprise between the polypeptide of aminoacid sequence shown in the SEQ ID NO:2 and have at least 85%, 90%, 95% or 99% identity.In addition the present invention also comprise a kind of by can with the polypeptide of the polynucleotide encoding of nucleic acid array hybridizing shown in the SEQ ID NO:1.Described polynucleotide rigorous, moderate is rigorous or low rigorous condition under with nucleotide sequence hybridization shown in the SEQ ID NO:1.
The term that uses among the application " rigorous (hybridization) condition " is meant that probe, primer, oligonucleotide will be hybridized with target sequence under such condition, and not with other any sequence hybridization.Rigorous condition is a sequence-dependent, because of the difference of environment different.The temperature of longer sequence generation specific hybrid is higher than shorter sequence.Usually, the temperature of rigorous condition is set at the bit sequencing and is listed in low about 5 ℃ of the ionic strength of regulation and the pyrolysis chain temperature (Tm) under the pH condition.Tm is meant such temperature, (under specific ion intensity, pH and nucleic acid concentration), when this temperature reaches balance, with the probe of target complement sequence 50% with target sequence hybridization.Because described target sequence is excessive usually, when Tm reached balance, 50% probe was occupied.Usually, rigorous condition is such condition, wherein during pH 7.0-8.3, salt concn is less than about 1.0M sodium ion, be generally about 0.01-1.0M sodium ion (or other salt), and temperature is at least about 30 ℃ for short probe, primer or Nucleotide (for example, 10-50 Nucleotide), is at least about 60 ℃ for long probe, primer or Nucleotide.Rigorous condition can also by add destabilizing agent for example methane amide realize.
The present invention further also provides the isolating polynucleotide of the polypeptide of the present invention of encoding.In this application, isolating polynucleotide are meant such polynucleotide, its structure and any naturally occurring polynucleotide or incomplete same above the natural any fragment of genome polynucleotide that exists of 3 separate gene with span.Therefore, this term comprises, (a) DNA for example, and it has the partial sequence of naturally occurring natural gene group dna molecular in the organism genome, and wherein said DNA is natural to be present in the described organism; (b) insert carrier or insert polynucleotide among protokaryon or the eukaryotic gene group DNA, this inserted mode makes the molecule of acquisition and any naturally occurring carrier or genomic dna inequality; (c) isolating molecule is as the fragment or the restriction fragment of cDNA, genomic fragment, polymerase chain reaction (PCR) generation; (d) recombinant nucleotide sequence, it is the part of heterozygous genes (being the gene of encoding fusion protein).Preferably, the identity of nucleotide sequence is at least 65%, 70% shown in this isolated nucleic acid molecule and the SEQ ID NO:1,75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher.When isolating polynucleotide were longer or more identical than the length of canonical sequence (for example SEQID NO:1), described relatively was to carry out with the total length of canonical sequence.Shorter than canonical sequence (for example SEQ ID NO:1) when isolating polynucleotide, described relatively is (get rid of homology and calculate required any ring (loop)) of carrying out with the fragment of the equal length of canonical sequence.
In addition, the present invention also provides a kind of carrier, wherein contains the described nucleic acid of one or more the application, and a kind of cell that contains carrier of the present invention or nucleic acid.The invention still further relates to the host cell that has transformed with the carrier that contains one of above-mentioned polynucleotide.
The present invention also provides by cultivation and has comprised the method that the cell that contains shown in the SEQ ID NO:1 carrier that separates polynucleotide prepares the described polypeptide of the application.
On the other hand, the invention provides a kind of can with polypeptide shown in the SEQ ID NO:2 or its fragments specific bonded antibody.Described antibody can be mono-clonal or polyclonal antibody.Partly, the present invention also provides polynucleotide (for example antisense DNA), ribozyme and the siRNA (siRNA) with polynucleotide complementation of the present invention or antisense.Described polynucleotide constructs can be used for detecting polynucleotide of the present invention, i.e. diagnosing non-small cell lung cancer perhaps is used for the treatment of or prevents this disease.
The present invention further provides antisense polynucleotides, it has SEQ ID NO:423,425,427,429,431,433,435,437,439,441,443,445,447,449,451,453,455,457,459,461,463,465,467,469,471,473,475,477,479,481,483,485,487,489,491,493,495,497,499,501,503,505,507,509,511,513,515,517,519,521,523,525,527,529, or the nucleotide sequence shown in 531.Proved that the polynucleotide with one of these nucleotide sequences can both effectively suppress the focus formation (focus formation) of NSCLC clone.
In addition, the present invention also provides has SEQ ID NO:533, and 534,535,536,537,538,539,540,541,542,543,544,545,546,547,548,549, the siRNA of nucleotide sequence shown in 550,551 or 552.Proved that the siRNA with arbitrary above-mentioned these nucleotide sequences can both suppress the cell viability of NSCLC clone effectively.
The application should provide a kind of pharmaceutical composition that uses arbitrary antisense polynucleotides or siRNA treatment nonsmall-cell lung cancer, and the method for using said composition treatment or prevention nonsmall-cell lung cancer.
The present invention also provides the pharmaceutical composition that is used for the treatment of nonsmall-cell lung cancer, and it contains the polynucleotide of polypeptide, its function equivalent or coding said polypeptide or its function equivalent of aminoacid sequence shown in a kind of SEQ of having ID NO:2.The polynucleotide that comprise in the described composition can be introduced carrier and expression in vivo.
The expectation pharmaceutical composition of the present invention mechanism (course) but the growth of anticancer.Described pharmaceutical composition can be used to comprise the Mammals of people and domestic Mammals (domesticated mammal).
In addition, the invention provides the method that NSC polypeptide (for example, NSC 807-1448) by using rise or its immune fragment or polynucleotides encoding them are come inducing antitumor immunity.
Unless otherwise indicated, all scientific and technical terminologies among the application all has and the identical implication of one skilled in the art's common sense of the present invention.Although with the method for describing among the application and materials similar or the method that is equal to and material all can be used for implementing or check the present invention, hereinafter still still be that suitable method and material are described.Whole publications of quoting among the application, patent application, patent and its full content of other reference are hereby incorporated by.If any conflict, comprise that with this specification sheets definition is as the criterion.In addition, described material, method and embodiment are for explanation better, and are not intended to extremely limit.
It should be understood that above-mentioned summary of the invention and following detailed description part all are embodiment preferred, and never constitute restriction the present invention or alternate embodiment of the present invention.
The accompanying drawing summary
Fig. 1 is illustrated to be the photo of trace, has shown with 200 genes crossing in lung carcinoma cell of sxemiquantitative RT-PCR confirmation to express.Lung carcinoma cell passes through the LCM method available from patients with lung cancer.
Fig. 2 illustrates the growth-inhibiting effect of antisense S-oligonucleotide in lung cancer cell line, and described antisense S-oligonucleotide is used to suppress NSC 810, and NSC 811, NSC 812, and NSC 825, and NSC 841, NSC 857, and NSC 859, and NSC 893, NSC 905, and NSC 947, and NSC 956, NSC 994, NSC 1075, and NSC 1107, NSC 1191 and NSC 1389.Fig. 2 illustrates the MTT result of experiment, has shown NSC 810-AS, NSC 811-AS1, NSC 811-AS2, NSC 811-AS4, NSC812-AS1, NSC 812-AS2, NSC 825-AS1, NSC 825-AS3, NSC 825-AS5, NSC841-AS4, NSC 841-AS5, NSC 857-AS3, NSC 857-AS4, NSC 859-AS2, NSC859-AS3, NSC 859-AS5, NSC 893-AS1, NSC 893-AS2, NSC 905-AS2, NSC905-AS3, NSC 905-AS5, NSC 947-AS1, NSC 947-AS2, NSC 947-AS3, NSC947-AS4, NSC 956-AS1, NSC 956-AS2, NSC 994-AS1, NSC 994-AS3, NSC994-AS4, NSC 994-AS5, NSC 1075-AS5, NSC 1107-AS1, NSC 1107-AS4, NSC 1191-AS2, NSC 1191-AS4, the restraining effect of NSC 1191-AS5 and NSC 1389-AS cell growth.
Fig. 3 illustrates siRNA (NSC 807-si1, NSC 810-si1, NSC 825-si1, NSC825-si2, NSC 841-si1, NSC 841-si2, NSC 903-si1, NSC 903-si2, NSC 956-si1, NSC 956-si2, NSC 994-si1, NSC 1107-si1, NSC 1107-si2, NSC 1107-si3, NSC 1107-si4, NSC 1107-si5, NSC 1191-si2, NSC 1246-si2 and NSC 1389-si2) to the growth-inhibiting effect of lung cancer cell line.Fig. 3 A illustrates the A549 cell with the carrier transfection of expressing contrast siRNA or target-siRNA is carried out the MTT result of experiment.Fig. 3 B is illustrated to be to carrying out the MTT result of experiment with the LC319 cell of the carrier transfection of expressing contrast-siRNA or target-siRNA.What Fig. 3 C showed is the Photomicrograph of using time lapse imaging (timelapse imaging) analysis of siRNA transfection-LC319 cell.Fig. 3 D is illustrated to be the result of flow cytometry, has shown the cell cycle collection of illustrative plates of siRNA cells transfected.Fig. 3 E is a photo, and the result that it shows the Western trace that detects with two kinds of different monoclonal antibodies shows the expression of native protein in the LC319 cell and situation about being suppressed by siRNA.Fig. 3 F, G and H show cytochrome c oxidase (CCO) activity in the A549 cell and situation about being suppressed by COX17 RNAi thereof.Fig. 3 F is the synoptic diagram of CCO determination of activity.That Fig. 3 G shows is the result of Western trace, and it utilizes mouse-anti human mitochondrion monoclonal antibody (MAB1273; CHEMICON, Temecula CA) has confirmed classification by the A549 cell of COX17 RNAi transfection, the tenuigenin fraction of this cell and plastosome fraction.Fig. 3 H is presented at after the transfection 2 or 5 days, suppresses endogenous COX17 gene and causes that CCO is active to be reduced.
Fig. 4 is a photo, by many-organize the northern engram analysis to NSC 807, NSC 810, NSC811, NSC 822, and NSC 825, and NSC 841, and NSC 849, NSC 855, and NSC 859, and NSC 885, and NSC 895, NSC 903, and NSC 904, and NSC 905, and NSC 915, NSC 948, and NSC 956, and NSC 994, and NSC 1000, NSC 1066, and NSC 1075, and NSC 1107, and NSC 1113, NSC 1131, and NSC 1141, and NSC 1164, and NSC 1183, NSC 1201, and NSC 1240, and NSC 1246, NSC1254, NSC 1265, and NSC 1277, NSC 1295, and NSC 1306, and NSC 1343, NSC 1362, and NSC 1389, and NSC 1399, NSC 1406, the analysis that NSC 1413 and the expression of NSC 1420 in the different people tissue are carried out.
Fig. 5 A is a photo, demonstration is observed by immunocytochemical assay, NSC 849, NSC 855, NSC 895, and NSC 915, and NSC 948, NSC 1000, NSC 1103, and NSC 1164, and NSC 1201, NSC 1288, NSC 1295, and NSC 1389, and NSC 1420 and NSC 1441 are in the Subcellular Localization of COS-7 cell, wherein said COS-7 cell transfecting has the NSC-expression vector of c-myc-His mark, and this experiment uses anti--His monoclonal antibody and rhodamine link coupled second anti--mouse IgG antibody to be used for colour developing.Nucleus is redyed with DAPI.Fig. 5 B shows photo, and it shows the NSC 895 to excretory c-myc mark in the substratum, and NSC 1164 and NSC 1295 carry out the result of Western engram analysis.
What Fig. 6 showed is the influence of NSC-gene pairs with the COS-7 cell growth of the expression vector stable transfection that adds the c-myc-His mark.Fig. 6 a shows by the detected NSC 810 of Western trace, NSC 841 and NSC 1389 expression in stable transfection COS-7 cell.What Fig. 6 b showed is the influence of NSC 810, NSC 841 and 1389 pairs of COS-7 cell growths of NSC.To express 2 or the 3 kind of independent transformant of high-level NSC 810 (COS7-TTK-1 and 2), NSC 841 (NIH3T3-URLC2-3 and 5) and NSC1389 (COS-7-NMU-2,3 and 5) and contrast (simulation) by cultivating in triplicate.By MTT measuring cell viability.
Fig. 7 has shown the influence by the NMU cell growth of autocrine (autocrine) systems measurement.What Fig. 7 A showed is the autocrine experimental result of NMU.Per 48 hours, add the activity form (NMU-25) and BSA (contrast) albumen of 25 amino acid polypeptides of NMU to independent COS-7 cell.Added back 7 days, with MTT experiment counting cells number.Fig. 7 B shows is anti--NMU antibody to the growth-inhibiting effect of the COS-7 cell handled through NMU-25.What Fig. 7 C showed is the growth-inhibiting effect of anti--NMU antibody to the COS-7 cell of endogenous expression NMU.
Fig. 8 shows the result of Western engram analysis, and it has confirmed TTK albumen expressing excessively in NSCLC clone A549, LC319 and NCI-H522.
Fig. 9 shows with anti--NSC 947 antibody, resists-NSC 1164 antibody, resists-NSC 1295 antibody and anti--NSC 1389 antibody, the NSC in the clinical sample that comprises gland cancer, squamous cell carcinoma and normal lung 947, NSC 1164, NSC 1295 and NSC 1389 is carried out the result of immunohistochemical staining.
Detailed Description Of The Invention
" one " who uses among the application, " a kind of " reach " described " and refer to " extremely except other has special explanation Few one (kind) ".
The present invention is based in part on the following discovery: at former cancerous lung tissue taking from patients with lung cancer In the pneumonocyte, the expression pattern of several genes changes. By comprehensive cDNA microarray system Identified the difference of gene expression dose.
23040 genes have been carried out the cDNA microarray analysis, suffered to select at non-small cell lung cancer The common mistake expressed or repressed gene among the person. It is of the present invention poor to find that 1448 genes present The opposite sex is expressed. Wherein, 642 genes are raised, and 806 genes are reduced.
The base that above-mentioned microarray analysis is identified by antisense S-oligonucleotides and/or siRNA technology Because further screening, to identify the marquis of the target that can be used as exploitation medicine or immunotherapy Select gene. Antisense S-oligonucleotides and siRNA are short synthetic DNA/RNA sequences, can with target The hybridization of specific mrna chain (Jansen and Zangemeister-Wittke, Lancet that gene pairs is answered Oncol 3:672-83 (2002); Brummenlkamp et al., Science 296:550-3 (2002)). By being combined with mRNA, ASON stops the target gene translation to become albumen, thus blocking-up institute State gene work (Jansen and Zangemeister-Wittke, Lancet Oncol 3:672-83 (2002)). On the contrary, siRNA is a kind of sequence-specific double-stranded RNA, can with its transfered cell The gene function that causes giving birth to (epigenetic) after the noninheritedness knocks out, thereby makes in the described target gene Null mutation carries out phenocopy (phenocopy) (Brummenlkamp et al., Science 296:550-3 (2002)). The expression database of this integrated gene that utilizes non-small cell lung cancer and up-regulated gene Rear life knocks out the combined method of (epigenetic knock-down), for Rapid identification and the assessment target molecule with Carry out personalized treatment a kind of strong means are provided. Now identified and to have regulated NSCLC The gene of Growth of Cells, propagation and/or survival. These gene codes are in autocrine, cell cycle/growth With the albumen that plays a role in the signal transduction, the perhaps product of Unknown Function.
The gene of the differential expression of identifying among the application can be used for diagnosis, and exploitation is used for suppressing non-The gene target type therapy of ED-SCLC.
It is general that expression in Patients with Non-small-cell Lung is subjected to regulating the gene of (that is, raise or reduce) Draw together in table 1-3, be referred to as " non--ED-SCLC-related gene (non-small cell lung among the application Cancer-associated genes) ", " the NSC gene ", " NSC nucleic acid " or " NSC polynucleotides ", The polypeptide of these gene codes is called " NSC polypeptide " or " NSC albumen ". Unless otherwise indicated, " NSC " refers to the disclosed arbitrary sequence of the application (for example, NSC 1-1448). These genes before this Through describing, provide in the lump its database login number.
Figure A20081018670600151
Figure A20081018670600171
Figure A20081018670600191
Figure A20081018670600201
Figure A20081018670600211
Figure A20081018670600221
Figure A20081018670600231
Figure A20081018670600241
Figure A20081018670600251
Figure A20081018670600261
Figure A20081018670600271
Figure A20081018670600281
Figure A20081018670600291
Figure A20081018670600301
Figure A20081018670600311
Figure A20081018670600321
Figure A20081018670600331
Figure A20081018670600341
Figure A20081018670600351
Figure A20081018670600361
Figure A20081018670600391
Figure A20081018670600411
Figure A20081018670600421
Figure A20081018670600431
Figure A20081018670600441
Figure A20081018670600461
Figure A20081018670600471
Figure A20081018670600491
Figure A20081018670600501
Figure A20081018670600511
Figure A20081018670600521
Figure A20081018670600531
Figure A20081018670600561
Figure A20081018670600571
Figure A20081018670600581
Figure A20081018670600591
Figure A20081018670600601
Figure A20081018670600621
The gene (〉=* 5, 〉=50% case) that table 2 raises
Figure A20081018670600622
Figure A20081018670600631
Figure A20081018670600641
Figure A20081018670600651
Figure A20081018670600661
Figure A20081018670600671
Figure A20081018670600691
Figure A20081018670600701
Figure A20081018670600711
Figure A20081018670600721
Figure A20081018670600731
Figure A20081018670600741
Figure A20081018670600761
Figure A20081018670600771
Figure A20081018670600781
Figure A20081018670600791
Figure A20081018670600801
Figure A20081018670600811
Figure A20081018670600831
Figure A20081018670600841
Figure A20081018670600851
Figure A20081018670600861
Figure A20081018670600881
Figure A20081018670600891
Figure A20081018670600901
Figure A20081018670600911
Figure A20081018670600931
Figure A20081018670600941
Figure A20081018670600961
Figure A20081018670600971
Figure A20081018670600981
Figure A20081018670600991
Figure A20081018670600992
Figure A20081018670601001
Figure A20081018670601011
Figure A20081018670601021
The diagnosis of nonsmall-cell lung cancer
Can use the biological sample of taking from the experimenter,, determine that this experimenter suffers from nonsmall-cell lung cancer or has the proneness of suffering from nonsmall-cell lung cancer by measuring various NSC expression of gene levels.
At least one that the present invention includes in the listed NSC sequence of his-and-hers watches 1-3 measured (for example, detecting) until the expression level of all sequences.
According to the present invention the genetic transcription thing of nonsmall-cell lung cancer-genes involved is detected, to measure described expression of gene level.Can be on the sequence information basis that known array GenBank database login (entry) provides, the technology of using those of ordinary skills to know detects and measures described non-small cell lung cancer associated gene.The genetic transcription thing that described method detects comprises transcribes and translation product, for example mRNA and albumen.For example: can use with the login of the corresponding sequence library of NSC polynucleotide in the sequence construct probe be used for the probe in detecting NSC mRNA that analyzes by for example Northern blot hybridization.Hybridization in probe and the experimenter's biological sample between the genetic transcription thing can also be carried out on the DNA array.The preferred a large amount of NSC expression of gene of the array detection level of using.As another example, in detection method such as reverse transcriptional PCR (RT-PCR) for example, can be used for the primer that specific amplification NSC polynucleotide use with described sequence construct based on amplification.In addition, can also analyze the expression level of NSC according to the biological activity or the amount of described gene coded protein.The method that can be used for measuring protein content comprises method of immunity.
Any biomaterial can be as the biological sample that is used to measure expression level, as long as can detect the NSC gene in this sample, comprising population of test (that is, taking from experimenter's tissue sample).Preferably, contain pneumonocyte (cell that obtains from lung) in the described biological sample.Genetic expression can also for example be measured in the sputum at blood, other body fluid of serum.In addition, described given the test agent can also be the cell of purifying self-organization.
Be used for according to the method for the invention diagnosing non-small cell lung cancer at the experimenter be preferably Mammals, comprise people, non-human primate, mouse, rat, dog, cat, horse and cow.
With one or more NSC expression of gene levels in the biological sample with compare with reference to the level of expression of gene described in the sample.Describedly comprise the known cell of one or more parameters with reference to sample, that is, and carcinous or non-cancerous cells.Describedly should take from and similarly organized by given the test agent source tissue that product come with reference to sample.Optional, can also be according to measuring the contrast expression level from the molecular information database of cell, wherein location parameter or condition are known.
Whether the pattern of gene expression dose shows and exists NSCLC to depend on composition with reference to cell mass in the biological sample.For example, when the reference cell colony was made up of non-cancerous cells, the described level of gene expression dose and reference is approximate in the biological subject sample showed that described biological subject sample is non-cancer.Otherwise when the control cells group was made up of cancerous cells, the described level of gene expression dose and reference is approximate in the biological subject sample showed that described biological subject sample comprises cancerous cells.
Can and a plurality ofly compare the biological subject sample with reference to sample.The known parameters of described multiple each with reference in the sample can be inequality.Therefore, can and knownly contain for example comparing of non-small cell lung cancer cell with given the test agent, and compare with reference to sample with known second of for example non--nonsmall-cell lung cancer (the non-non-small cell lung cancer) cell (normal cell) that contains simultaneously with reference to sample.
According to the present invention, to one or more the nonsmall-cell lung cancer-genes involveds in the biological sample for example the expression of NSC 1-1448 measure, and compare with the normal control level of homologous genes." the normal control level " is meant the nonsmall-cell lung cancer-Expression of Related Genes collection of illustrative plates that sees usually in the biological sample of not suffering from nonsmall-cell lung cancer colony to term.The NSC gene expression dose can be measured simultaneously in contrast and experimenter's the biological sample, and perhaps the normal control level can be measured by statistical method based on the gene expression dose from the sample of control group collection is before this analyzed the result that obtains.Be derived from the biological sample of patient tissue samples nonsmall-cell lung cancer-Expression of Related Genes level and raise compared with the normal control level or reduce, show that this experimenter suffers from or easily suffers from nonsmall-cell lung cancer.For example, compared with the normal control level, the expression level of NSC 807-1448 raises in the biological subject sample, shows that this this experimenter suffers from or easily suffers from nonsmall-cell lung cancer.Otherwise the expression level of NSC 1-806 reduces compared with the normal control level in the biological subject sample, shows that this experimenter suffers from or easily suffers from nonsmall-cell lung cancer.
When in the biological subject sample between NSC expression of gene level and the control level difference surpass 1.0,1.5,2.0,5.0,10.0 or more times the time, can think that variation has taken place NSC expression of gene level in this biological subject sample.Optional, compare with control level, NSC expression of gene level raises or reduces at least 50%, 60%, 80%, 90% or more for a long time in the biological subject sample, just can think that variation has taken place NSC expression of gene level in this biological subject sample.
Can make gene expression difference stdn between given the test agent and the standard model, described reference examples such as housekeeping gene (housekeeping gene) according to contrast.For example, the contrast polynucleotide can comprise that those known its expression levels do not have the polynucleotide of difference between carcinous and non-cancerous cells.The contrast polynucleotide expression level that is tried to examine in the reference sample can be used for the stdn of detected NSC gene expression dose.The crt gene that uses among the present invention comprises beta-actin, glyceraldehyde 3 phosphate desaturase and ribosomal protein P1.
The NSC gene of the differential expression of identifying among the application also can be used for monitoring the therapeutic process of nonsmall-cell lung cancer.In these methods, obtain the biological subject sample from the experimenter who accepts the nonsmall-cell lung cancer treatment.If desired, can before the described treatment, during or afterwards different time points obtain a plurality of biological subject samples from the experimenter.Then, one or more NSC expression of gene in the sample are measured, and be in comparing of the nonsmall-cell lung cancer known state that also is not exposed to described treatment with reference to sample.
If describedly do not comprise non-small cell lung cancer cell with reference to sample, biological subject sample and show then that with reference to NSC expression of gene level in the sample is approximate described treatment is effective.But NSC expression of gene level shows then that at given the test agent with reference to the difference in the sample clinical effectiveness or prognosis are very poor.
Term " effectively " is meant that described treatment causes: the expression decreased of the gene (NSC 807-1448) that pathologic raises, the expression of the gene (NSC 1-806) of pathologic downward modulation raises, and perhaps the size of nonsmall-cell lung cancer, sickness rate or metastatic potential reduce among the experimenter.When a kind of treatment is a pre-property when giving, " effectively " means that described treatment postpones or stoped the generation of nonsmall-cell lung cancer or alleviated the clinical symptom of nonsmall-cell lung cancer.The evaluation of described nonsmall-cell lung cancer can use the standard clinical method to finish.In addition, can combine with any currently known methods that is used to diagnose or treat nonsmall-cell lung cancer the validity of treatment is measured.For example, come diagnosing non-small cell lung cancer from histopathology or by the discriminating symptom, described symptom is chronic cough (chronic cough) for example, hoarse (hoarseness), hemoptysis (coughing up blood) is lost weight, poor appetite, breathe hard (shortness of breath) asthma (wheezing), Fa Zuo bronchitis or pneumonia and pectoralgia repeatedly.
In addition, the present invention's method of being used for diagnosing non-small cell lung cancer also can be used for coming from patient's biological sample NSC expression of gene level by reference source assessment of cancer patient's prognosis.Optional, thus can in each phase process of disease, measure the prognosis situation of evaluating the patient to the level of expression of gene described in the biological sample.
Compared with the normal control level, the expression level reduction of rising of the expression level of NSC 807-1448 or NSC 1-806 shows prognosis mala.Compared with the normal control level, the expression level of NSC 807-1448 or NSC 1-806 is similar to and then shows this patient prognosis bona.Preferably, can evaluate experimenter's prognosis situation by the expression map that compares NSC 807-1448 or NSC 1-806.
Expression map
The present invention also provides a kind of nonsmall-cell lung cancer with reference to expression map, wherein the pattern of two or more gene expression doses among the NSC 1-1448.Described expression map can be used as the contrast of following purposes: diagnosing non-small cell lung cancer or suffer from described disease proneness, and the monitor therapy process, and this disease experimenter's prognosis is suffered from evaluation.
Evaluation can suppress or strengthen the compound of nonsmall-cell lung cancer-related gene expression
Can suppress nonsmall-cell lung cancer-related gene expression or active compound by following method evaluation: the subject cell of expression nonsmall-cell lung cancer-genes involved is contacted with test-compound, measure this nonsmall-cell lung cancer-Expression of Related Genes level or activity.It is expressed to reduce compared with the normal control level and shows that this compound is the supressor of this nonsmall-cell lung cancer-genes involved.When the nonsmall-cell lung cancer-genes involved of expressing in the described subject cell is the gene that raises, can be used for suppressing nonsmall-cell lung cancer according to this method compounds identified.
Optional, can nonsmall-cell lung cancer-related gene expression or the active compound identification enhanser for this gene will be improved by following operation: the population of test of expression nonsmall-cell lung cancer-genes involved is contacted with test-compound, measure this nonsmall-cell lung cancer-Expression of Related Genes level or activity.When the nonsmall-cell lung cancer-genes involved of expressing in the described subject cell is the gene of downward modulation, can be used for suppressing nonsmall-cell lung cancer according to this method compounds identified.
Described subject cell can be a cell mass, comprises any cell, as long as this cell expressing target nonsmall-cell lung cancer-genes involved.For example, described subject cell contains epithelial cell, and described cell can be derived from lung tissue, blood, serum or phlegm.Described subject cell can be a kind of immortal cell line that is derived from non-small cell lung cancer cell.Optional, described subject cell can be with the cell of NSC gene transfection or use regulating and controlling sequence (for example promotor) cells transfected of NSC gene that described regulating and controlling sequence is operably connected to reporter gene.
SCREENED COMPOUND
Can use albumen or this gene transcription regulation and control zone of NSC gene, this genes encoding, screening can change the bioactive compound of described genetic expression or described genes encoding polypeptide.This compound can be used as the medicine that can be used in treatment or prevention nonsmall-cell lung cancer.
Therefore, the present invention also provides a kind of method of using polypeptide screening of the present invention to be used for the treatment of or to prevent the compound of nonsmall-cell lung cancer.Contain the following step in the embodiment of this screening method: test-compound is contacted with polypeptide of the present invention; (b) detect the activity that combines between polypeptide of the present invention and the described test-compound; And (c) select can with polypeptide bonded compound of the present invention.
The polypeptide that screening is used can be the albumen of a kind of recombinant polypeptide or a kind of natural origin or the peptide of its part.The polypeptide that is used for contacting with test-compound can be, for example, the polypeptide of purifying, soluble albumen are with carrier-bound form or the fusion rotein that merges with other polypeptide.
Can be as screening in conjunction with the proteic method of NSC polypeptide, many methods well known to those skilled in the art all can be used.Described screening can be implemented by for example immuno-precipitation method (immunoprecipitation), particularly is to use following manner.Can be by the following gene of expressing the arbitrary described NSC polypeptide of coding in the zooblast etc. that operates in: described gene is inserted the expression vector of alien gene, such as pSV2neo, pcDNA I, pcDNA3.1, pCAGGS and pCD8.The promotor of described expression usefulness can be normally used arbitrary promotor, comprise, for example, SV40 early promoter (Rigby inWilliamson (ed.), Genetic Engineering, vol.3.Academic Press, London, 83-141 (1982)), EF-α promotor (Kim et al., Gene 91:217-23 (1990)), CAG promotor (Niwa et al., Gene 108:193-200 (1991)), RSV LTR promotor (Cullen, Methodsin Enzymology 152:684-704 (1987)), SR α promotor (Takebe et al., Mol Cell Biol8:466 (1988)), instant early stage (immediate early) promotor (the Seed and Aruffo of CMV, Proc Natl Acad Sci USA 84:3365-9 (1987)), SV40 late promoter (Gheysen andFiers, J Mol Appl Genet 1:385-94 (1982)), adenovirus promoter (Kaufman et al., Mol Cell Biol 9:946 (1989)), HSV TK promotor or the like.The zooblast that described gene is imported expression alien gene can be finished with arbitrary method, for example, electroporation method (Chu 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., NucleicAcids Res 12:5707-17 (1984); Sussman and Milman, Mol Cell Biol 4:1642-3 (1985)), lipofection (Lipofectin) method (Derijard, B Cell 7:1025-37 (1994); Lambet al., Nature Genetics 5:22-30 (1993): Rabindran et al., Science 259:230-4 (1993)) etc.Can also be the fusion rotein that contains the monoclonal antibody recognition site with described NSC expression of polypeptides, can be undertaken by N or the C-end of the epi-position of known this monoclonal antibody of specificity being introduced described polypeptide.Epitope-antibody system (experimental medicine 13:85-90 (1995)) that can commodity in useization.Can utilize its multiple clone site and express to have for example carrier of the fusion rotein of beta-galactosidase enzymes, maltose binding protein, glutathione S-transferase, green fluorescent protein (GFP) etc., can obtain from the commercial channel.
In addition, also reported the fusion rotein that is only prepared by several little epi-positions of forming to ten several (dozen) amino acid by introducing, wherein the characteristic of NSC polypeptide is unlikely changes because of fusion.Epi-position is polyhistidyl (His-mark) for example, influenza virus hemagglutinin (influenza aggregate) HA, people C-myc mark, vesicular stomatitis virus (Vesicular stomatitis virus) glycoprotein (VSV-GP), T7 gene 10 albumen (T7-mark), human herpes simplex vicus (human simple herpes virus) glycoprotein (HSV-mark), E-mark (going the epi-position on the mono-clonal phage) etc., and the monoclonal antibody of discerning them can be used as all that the epitope-antibody system screens can be in conjunction with the albumen (experimental medicine 13:85-90 (1995)) of described NSC polypeptide.
In immuno-precipitation, thereby form immunocomplex by in cell lysate, adding these antibody with suitable stain remover preparation.Described immunocomplex by described NSC polypeptide, have and can form with the polypeptide and the antibody of this polypeptide binding ability.Except that the antibody that uses anti-above-mentioned epi-position, immunoprecipitation can also use the antibody of anti-NSC polypeptide to implement, and described anti-NSC polypeptide antibody can be used method for preparing.
When described antibody is a kind of mouse IgG antibody, can precipitate immunocomplex with for example albumin A agarose or Protein G agarose.If described NSC polypeptide is prepared to having epi-position for example during the fusion rotein of GST, can use the mode identical to form immunocomplex with using anti-NSC polypeptide antibody, at this moment to use can with these epitope specificity bonded material, for example gsh-sepharose 4B.
Immunoprecipitation can by according to or carry out according to the method for record in the document (Harlow and Lane, Antibodies, 511-52, Cold Spring Harbor Laboratory publications, New York (1988)).
Usually use SDS-PAGE that the albumen of immunoprecipitation is analyzed, the conjugated protein gel of suitable concn that can use is analyzed by this proteic molecular weight.Owing to be difficult to detection with the normal dyeing rule as examining Macchiavello's staining or silver dyeing with NSC polypeptide bonded albumen, thereby can improve described proteic detection sensitivity: cell cultures is being contained radio isotope by following method 35The S-methionine(Met) or 35In the substratum of S-halfcystine, the albumen in the described cell of mark detects this albumen then.Albumen can directly be used SDS-polyacrylamide gel purifying, can measure its sequence then when this molecular weight of albumen is known.
Use any NSC polypeptide screening can use Western engram analysis (Skolnik et al, Cell 65:83-90 (1991)) with the proteic method of this polypeptide bonded.Particularly, can obtain by following method can be in conjunction with the albumen of NSC polypeptide: use phage vector (for example ZAP), express and to prepare the cDNA library with the proteic cell of NSC polypeptide bonded, tissue, organ (for example, tissue is as testis and prostate gland) or cultured cells (for example LNCaP, PC3, DU145) from expectation; This protein expression on the LB-agarose, on filter membrane, is made NSC polypeptide and the reaction of above-mentioned filter membrane through purifying and mark with this proteopexy, detect according to described mark then and express and the proteic plaque of NSC polypeptide bonded.Can carry out mark to the NSC polypeptide with following method: utilize combining between vitamin H and the avidin, perhaps use can specificity in conjunction with the antibody of this NSC polypeptide, perhaps peptide that merges with the NSC polypeptide or polypeptide are (for example, GST).Also can use the method for marks such as adopting radio isotope or fluorescence.
Optional, in another embodiment of screening method of the present invention, can use two-hybrid system (" MATCHMAKER two-hybrid system ", " Mammalian MATCHMAKER double cross measure test kit ", " MATCHMAKER single crosses system " (Clontech); " HybriZAP double cross carrier system " (Stratagene); With reference to " Dalton and Treisman, Cell 68:597-612 (1992) ", " Fields and Sternglanz, Trends Genet 10:286-92 (1994) ").
In two-hybrid system,, and be expressed in the yeast cell NSC polypeptide and SRF-land or the fusion of GAL4-land.Express and the proteic cell preparation cDNA of NSC polypeptide bonded library from expection, thus make this library when expressing with VP16 or the fusion of GAL4 transcription activating zone.Then the cDNA library is imported above-mentioned yeast cell, go out to be derived from the cDNA (when can be with polypeptide bonded protein expression of the present invention during in yeast cell, the two combination can activate reporter gene makes positive colony to detect) in described library from the clone and separate of test positive.Can prepare described cDNA encoded protein by above-mentioned isolating cDNA is imported intestinal bacteria and expresses described albumen.
With regard to reporter gene, except that the HIS3 gene, also can use for example Ade2 gene, lacZ gene, CAT gene, luciferase gene etc.
Can also use the affinity chromatography screening with NSC polypeptide bonded compound.For example, the NSC polypeptide can be fixed in the carrier of affinity column, will comprise then and can be added on this chromatography column with the proteic test-compound of NSC polypeptide bonded.Test-compound among the application can be, for example cell extract, cell lysate etc.Behind the sample, cylinder is washed on described test-compound, so just can prepare the compound that combines with the NSC polypeptide.
When test-compound is a kind of albumen, the proteic aminoacid sequence that obtains is analyzed, then based on the synthetic oligo DNA of this sequence, make the DNA that probe screening cDNA library obtains encoding said proteins with this oligo DNA.
Can will utilize the biosensor (biosensor) of surface plasmon resonance phenomenon (surface plasmon resonancephenomenon) bonded compound among the present invention to be detected or quantitative means with work.When using this class biosensor, the interaction between NSC polypeptide and the test-compound can be used as a kind of surface plasmon resonance phenomenon and carries out Real Time Observation, only use the polypeptide of minute quantity and do not need mark (for example, BIAcore, Pharmacia).Therefore, can use biosensor such as BIAcore to testing and assessing between NSC polypeptide and the test-compound in conjunction with situation.
The method of bonded molecule takes place in screening when immobilized NSC polypeptide is exposed to synthetic compound or crude substance storehouse or random phage peptide display libraries, and according to combinatorial chemistry technique (Wrighton etc., Science 273:458-64 (1996); Verdine, Nature 384:11-13 (1996); Hogan, Nature 384:17-9 (1996)) screening method that utilizes high-throughput to separate with NSC albumen (comprising agonist and antagonist) bonded albumen and compound is well known to those skilled in the art.
In addition, the invention provides the method that a kind of NSC of utilization polypeptide screening is used for the treatment of or prevents the compound of nonsmall-cell lung cancer, may further comprise the steps:
(a) test-compound is contacted with the NSC polypeptide;
(b) biological activity of NSC polypeptide in the detection step (a); With
(c) selecting to make when not having test-compound detected polypeptide biological activity to compare NSC polypeptide biological activity is suppressed or the enhanced compound.
Because having, one of NSC1-1448 encoded protein promotes the outgrowth activity of non-small cell lung cancer cell, thus can SCREENED COMPOUND with this activity as index, described compound promoted or suppress this activity of one of these albumen.
Available any polypeptide screens as long as they have the proteic biological activity of NSC.This biological activity comprises the proteic cell proliferative activity of people of one of NSC 807-1448 coding.For example, also can use the people's albumen and the function equivalence polypeptide thereof of one of NSC 807-1448 coding.This peptide species can be by cell endogenous or exogenous expression.
Compound by this screening and separating is the agonist or the antagonist of NSC polypeptide." agonist " refers to the molecule by the function that combines activation NSC polypeptide with it to term.Term " antagonist " refers to by combine the molecule of inhibition NSC polypeptide function with it.And the compound by this screening and separating is to suppress NSC polypeptide and molecule (comprising DNA and albumen) interactional candidate compound in vivo.
When the biological activity that is detected in the inventive method is hyperplasia, can for example followingly detect: the cell of NSC polypeptide (for example NSC 807-1448) is expressed in preparation, there is the described cell of cultivation under the situation of test-compound, measure hyperplasia speed, measure the cell cycle etc., and the mensuration colony forms activity.
Go through as preamble, by control NSC expression of gene level, we can control the morbidity and the process of nonsmall-cell lung cancer.Therefore, can identify the compound that is used for the treatment of or prevents nonsmall-cell lung cancer by as a token of screening with the NSC gene expression dose.In the context of the present invention, this screening comprises, for example following step:
A) with test-compound and the cells contacting of expressing one or more NSC gene; With
B) screening make NSC 807-1448 gene one or more expression level detect described level during with this test-compound not and compare the compound that reduces or raise.
The cell of expressing at least a NSC gene comprises, for example the clone of setting up from nonsmall-cell lung cancer; This cell can be used for the above-mentioned screening method of the present invention (A549 for example, NCI-H226, NCI-H522, LC319).Estimate described expression level according to method well known to those skilled in the art.In the screening method, select to reduce the candidate agent of the compound of at least a NSC gene expression dose as treatment or prevention nonsmall-cell lung cancer.
Perhaps, screening method of the present invention may further comprise the steps:
A) make test-compound and the cells contacting that has imported carrier, this carrier contains the transcriptional regulatory district of one or more marker gene and the reporter gene of expressing under this transcriptional regulatory district control, and wherein one or more marker gene is NSC 1-1448,
B) activity of the described reporter gene of mensuration; With
C) when described reporter gene is the gene (for example NSC 807-1448) that raises, the compound of selecting feasible described compared with the control reporter gene expression level to reduce, or when described reporter gene is the gene (for example NSC 1-806) of downward modulation, select the compound that makes that described compared with the control reporter gene expression level raises.
The reporter gene and the host cell that are fit to are known in the art.Screening required report construct can utilize the transcriptional regulatory district of marker gene to prepare.If the transcriptional regulatory district of marker gene is well known by persons skilled in the art, can utilize the sequence information preparation report construct of front.When the transcriptional regulatory district of marker gene is undetermined, can separate the nucleotide fragments that contains the transcriptional regulatory district from genomic library according to the nucleotide sequence information of this marker gene.
Any test-compound, for example, cell extract, cell culture supernatant, microbial fermentation product, marine organism extract, plant milk extract, purifying or rough albumen, peptide, non-peptide compound, synthetic micromolecular compound and natural compounds all can be used in the screening method of the present invention.Test-compound of the present invention can use one of several different methods in the combinatorial library known in the art to obtain, described library comprises (1) biology library, (2) parallel solid phase of spatial positioning or liquid phase library (spatiallyaddressable parallel solid phase or solution phase libraries) (3) need the synthetic library method of deconvolution (deconvolution), and the synthetic library method of affinity chromatography screening is used in (4) " pearl one compound (one bead one compound) " library method and (5).Use the biology library method of affinity chromatography screening to only limit to peptide library, and other four kinds of methods are applicable to peptide, non-peptide oligomer or compound small molecules library (Lam (1997) Anticancer Drug Des.12:145).The method in synthetic molecules library see following document (DeWitt etc. (1993) Proc.Natl.Acad.Sci.USA 90:6909 for example; Erb etc. (1994) Proc.Natl.Acad.Sci.USA 91:11422; Zuckermann etc. (1994) J.Med.Chem.37:2678; Cho etc. (1993) Science 261:1303; Carell etc. (1994) Angew.Chem.Int.Ed.Engl.33:2059; Carell etc. (1994) Angew.Chem.Int.Ed.Engl.33:2061; Gallop etc. (1994) J.Med.Chem.37:1233).Library of compounds can be present in solution (referring to Houghten (1992) Bio/Techniques 13:412) or pearl (Lam (1991) Nature 354:82), chip (Fodor (1993) Nature 364:555), bacterium (United States Patent (USP) 5,223,409), spore (United States Patent (USP) 5,571,698; 5,403,484, and 5,223, and 409), plasmid (Cull etc. (1992) Proc.Natl.Acad.Sci.USA 89:1865) or phage (Scott and Smith (1990) Science 249:386; Delvin (1990) Science 249:404; Cwirla etc. (1990) Proc.Natl.Acad.Sci.USA 87:6378; Felici (1991) J.Mol.Biol.222:301; U.S. Patent application 2002103360).According to screening method of the present invention, being exposed to cell or proteic test-compound can be individualized compound or combination of compounds.When use in the screening method of the present invention be combination of compounds the time, described compound can be successively or contact simultaneously.
By screening method isolated compound of the present invention is the material standed for that promotes or suppress the medicine of NSC polypeptide active, can be used for the disease for the treatment of or preventing to cause because of cell hyperplastic disease, such as nonsmall-cell lung cancer.The part-structure of the compound that obtains with screening method of the present invention because add, disappearance and/or replace variation has taken place, such compound is also included within the compound that obtains with screening method of the present invention.Can effective stimulus (for example hang down expression (under-expressed) gene, NSC 1-806) or (for example can effectively suppress the gene of expressing, NSC 807-1448) compound of Biao Daing is considered to have clinical effectiveness, and can further measure the growth that can it stop cancer cells in animal model or the study subject.
Selection is suitable for concrete individual nonsmall-cell lung cancer medicine
The difference that genes of individuals is formed can cause the difference of the relative capacity of the various medicines of its metabolism.A kind of can be as anti-non-small cell lung cancer drug and by metabolic compound in the experimenter, can change by inducible gene expression pattern in this experimenter's cell and show, described to change into by the gene expression pattern with carcinous status flag be the gene expression pattern with non-carcinous status flag.Therefore, the NSC gene of disclosed differential expression makes it possible to select the treatment of inferring or the preventative inhibition of the nonsmall-cell lung cancer that is fit to concrete experimenter by detect candidate compound in being derived from selected experimenter's subject cell (or population of test) among the application.
In order to identify the anti-non-small cell lung cancer drug that is suitable for concrete experimenter, subject cell or the population of test that is derived from this experimenter is exposed to this therapeutical agent, measure one or more NSC1-1448 expression of gene then.
Described subject cell is the non-small cell lung cancer cell of expressing the non-small cell lung cancer associated gene, contains the non-small cell lung cancer cell of expressing the non-small cell lung cancer associated gene in the perhaps described population of test.Preferably, described subject cell is that a kind of epithelial cell or described population of test contain epithelial cell.For example, described subject cell or population of test are incubated under the condition that has drug candidate to exist, measure the gene expression pattern of described subject cell or cell mass then, and with one or more the contrast collection of illustrative plates compare, for example, nonsmall-cell lung cancer with reference to expression map or non--nonsmall-cell lung cancer lung cancer with reference to expression map.
With the control cells faciation ratio that contains nonsmall-cell lung cancer, among the NSC 807-1448 one or more expression reduce or NSC1-806 in one or more expression raise and show that described medicine is medicable.
Described testing drug can be arbitrary compound or composition.For example:, described testing drug is a kind of immunomodulator.
Test kit
The present invention also provides a kind of test kit, wherein contains the NSC-detection reagent, for example, a kind of can specificity in conjunction with or identify the nucleic acid of one or more NSC polynucleotide.Described can specificity in conjunction with or the nucleic acid of identifying one or more NSC polynucleotide for example, with part NSC polynucleotide complementary oligonucleotide sequence, or can with the coded polypeptide bonded of NSC polynucleotide antibody.Described reagent can be packaged together in the test kit.Described reagent, such as nucleic acid or antibody (be combined on the solid substrate or with being used for that it is bonded to reagent packing separately on the described matrix), contrast agents (positive and/or negative) and/or detect described nucleic acid or means (means) that antibody is used, be preferably packaged in the container separately.The specification sheets (for example, literal, tape, VCR, CD-ROM etc.) of implementing described mensuration usefulness can be included in the described test kit.The mensuration mode of described test kit can be Northern hybridization known in the art or sandwich ELISA.
For example, the NSC detection reagent is fixed in solid substrate and for example forms at least one NSC detection site on the porous band.The mensuration of described porous band or surveyed area can comprise a large amount of detection site, and each detection site contains a kind of NSC detection reagent.Test strip can also comprise feminine gender and/or positive control site.Optional, control site can be positioned at the band that separates with described test strip.Randomly, described different detection site can comprise the immobilized reagent of different amounts, and promptly the content in first detection site is higher, and the amount in the site subsequently is lower.When adding the biological subject sample, the number of sites of demonstration detectable signal provides the quantitative target of the NSC content that is present in the sample.Can described detection site be set to arbitrary suitable detected shape, be generally bar shaped or across the spot of test strip width.
Optional, described test kit comprises a kind of nucleic acid primer array, wherein contains one or more NSC polynucleotide sequences.Nucleic acid on the described array can specificity be identified one or more polynucleotide sequences that NSC 1-1448 represents.2,3,4,5,6,7,8,9,10,15,20,25,40 or 50 or more kinds of expression of gene of the gene of NSC 1-1448 representative can be by identifying with the level that combines of array test band or chip.Described substrate array can be positioned on " chip " that solid substrate for example for example describes in the U.S. Pat 5,744,305.
Array and plural form (Pluralities)
The present invention also comprises a kind of nucleic acid primer array, wherein contains one or more NSC polynucleotide sequences.One or more polynucleotide sequences that nucleic acid specificity ground on the described array is represented corresponding to NSC 1-1448.NSC 1-1448 represents 2,3 in the gene, 4,5,6,7,8,9,10,15,20,25,40 50 or more kinds of expression of gene level can identify with combining of described array by detecting nucleic acid.
The present invention also comprises a kind of isolating nucleic acid plural form (being the mixture of two or more nucleic acid).Described nucleic acid is in liquid phase or solid phase, for example, is fixed in for example nitrocellulose membrane of solid support.Described plural form comprises one or more polynucleotide of NSC 1-1448 representative.According to another embodiment of the present invention, described plural form comprises 2,3,4,5,6,7,8,9,10,15,20,25,40 in the polynucleotide of NSC 1-1448 representative or 50 or more kinds of polynucleotide.
Chip
The DNA chip is a kind of device of simultaneously many expression of gene levels being compared be convenient to.For example can utilize among " Microarray BiochipTechnology " (Mark Schena, Eaton publishes, 2000) disclosed method etc. based on the structure of the expression map of DNA chip.
Contain in the DNA chip and detect process immobilization height-density probe that a large amount of genes are used.Therefore, just can determine many expression of gene levels simultaneously by the single-wheel analysis.That is, the expression map of sample can be measured with the DNA chip.Method based on the DNA chip of the present invention comprises the following steps:
(1) synthetic and corresponding aRNA of marker gene or cDNA;
(2) with the probe hybridization of above-mentioned aRNA or cDNA and marker gene; And
(3) aRNA or the cDNA of detection and described probe hybridization carry out quantitatively its mRNA then.
ARNA is meant with RNA polymerase and transcribes the RNA that obtains from template cDNA.Be used for to buy based on the aRNA transcript reagent box of the expression map of DNA chip.Use described test kit, can use the T7 RNA polymerase to be the synthetic aRNA of template with the cDNA that is connected with the T7 promotor.On the other hand, can be by using random primer, using from mRNA synthetic cDNA is that template is passed through pcr amplification cDNA.
Optional, contain the probe of putting on described chip in the described DNA chip, be used to detect the probe of marker gene of the present invention.The number of the marker gene of point on described DNA chip without limits.For example, can from marker gene of the present invention, select 5% or more, preferred 20% or more, how preferred 50% or more, even more preferably 70% or more.Can be on described DNA chip with any other gene and described marker gene one starting point.For example, the probe points of gene that can expression level is almost constant is on described DNA chip.When the measurement result that is intended between more a plurality of chips or different the mensuration, can be with such gene with the measurement result stdn.
Be selected each marker gene designing probe, then with described probe points on the DNA chip.Described probe can be for example to contain the oligonucleotide of 5-50 nucleotide residue.The method that is used for synthetic described oligonucleotide on the DNA chip is known to those skilled in the art.Long DNA can be synthetic by PCR or chemical process.Be used for the method on slide glass also to be that those skilled in the art are known with PCR or other method synthetic length dna point.The DNA chip that obtains by aforesaid method can be used for diagnosing nonsmall-cell lung cancer of the present invention.
The DNA chip for preparing is contacted with aRNA, then the hybridisation events between above-mentioned probe and the aRNA is detected.Can use fluorochrome label aRNA in advance.Can use fluorescence dye such as Cy3 (redness) and Cy5 mark aRNA such as (greens).To use different fluorochrome labels respectively from the aRNA of experimenter and contrast.Can calculate the difference of expression level between the said two devices according to the difference of above-mentioned strength of signal.Fluorescence dye signal on the DNA chip can detect with scanning device, analyzes with special program then.For example, the Suite from Affymetrix is the software package that a kind of DNA chip analysis is used.
Be used for the treatment of or prevent the method for nonsmall-cell lung cancer
The invention provides a kind of method that is used for the treatment of, alleviates or prevent experimenter's nonsmall-cell lung cancer.The experimenter that the treatment compound can prevent or therapeutic suffers from or easily suffer from nonsmall-cell lung cancer (or to nonsmall-cell lung cancer sensitivity).NSC 1-1448 expresses or active horizontal abnormality is identified described experimenter with the routine clinical method or by detecting.Before manifesting tangible disease clinical symptom, prevent administration, thereby stop or postpone the process of disease or illness.
Described methods of treatment comprises the expression of one or more gene products that improve gene or/and function, the expression of described gene in non-small cell lung cancer cell with compare reduction (" low-express gene ") in the expression of normal cell (from the tissue identical) with types of organization that non-small cell lung cancer cell is originated.In these methods, with the described experimenter of compounds for treating of significant quantity, this compound can make the expression amount of one or more low genes (NSC 1-806) of expressing among the described experimenter raise.The administration of this compound can be general or locality.Described treatment compound comprises: the polypeptide product of low gene of expressing, or its bioactive fragment; Be positioned at the nucleic acid of the low expressing gene of coding in expression regulation element downstream, this controlling element makes described gene to express in non-small cell lung cancer cell; And can make endogenous be present in the compound (that is turning down the compound of the expression of gene of expression) that the described expression of gene level in the non-small cell lung cancer cell improves.Use the effect that described treatment compound can resist the gene of unusual low expression in experimenter's pneumonocyte, improve this experimenter's clinical condition.Described compound can obtain by the above-mentioned screening method of the present invention.
Described method also comprises the expression of one or more gene products of gene or/and function, the expression of described gene in non-small cell lung cancer cell with compare rising (" gene of mistake-expression ") in the expression of normal cell (from the tissue identical) with types of organization that non-small cell lung cancer cell is originated.Described expression can suppress with arbitrary method known in the art.For example, with the described experimenter of compounds for treating of significant quantity, this compound can make that one or more expression amounts of crossing expressing gene (NSC 807-1448) reduce among the described experimenter.The administration of this compound can be general or locality.Described treatment compound comprises the compound (promptly reducing the compound of the expression of gene of expressing) that can make endogenous be present in the described expression of gene level reduction in the non-small cell lung cancer cell.Use described treatment compound and can resist unusual effect of crossing expressing gene in experimenter's pneumonocyte, improve this experimenter's clinical condition.Described compound can obtain by the above-mentioned screening method of the present invention.
The active compound that can regulate the albumen (NSC1-1448) that is used for the treatment of or prevents nonsmall-cell lung cancer of the present invention comprises albumen, these proteic natural cognate ligands, peptide, peptide mimics and other small molecules.
Optional, can suppress described expression of crossing the gene (NSC 807-1448) of expressing with following method: use a kind of can the inhibition or the described nucleic acid of crossing the expression of gene of expressing of antagonism to the experimenter.Can disturb antisense oligonucleotide, siRNA or the ribozyme of the expression of gene of expressing to can be used for suppressing the described expression of gene of expressing of crossing.
As mentioned above, the antisense-oligonucleotide corresponding with arbitrary nucleotide sequence among the NSC 807-1448 can be used for reducing the expression level of described NSC 807-1448.Antisense-the oligonucleotide corresponding with the NSC 807-1448 of up-regulated in the nonsmall-cell lung cancer can be used for treatment or prevention nonsmall-cell lung cancer.Particularly the corresponding mRNA combination plays a role, thereby suppresses described gene transcription or translation, promotes the degraded of mRNA, and/or suppresses the proteic expression of NSC nucleotide coding, and finally suppresses described proteic function.Term in this application " antisense-oligonucleotide " comprises and the complete complementary Nucleotide of target sequence and the Nucleotide of a place or many places mispairing is arranged, if described antisense-oligonucleotide can with described target sequence specific hybrid.For example, antisense-oligonucleotide of the present invention comprises having SEQ IDNO:423,425,427,429,431,433,435,437,439,441,443,445,447,449,451,453,455,457,459,461,463,465,467,469,471,473,475,477,479,481,483,485,487,489,491,493,495,497,499,501,503,505,507,509,511,513,515,517,519,521, nucleotide sequence shown in 523,525,527,529 and 531 has proved that now these nucleotide sequences can both effectively suppress the transforming focus formation of NSCLC clone.In addition, antisense-oligonucleotide of the present invention comprises the polynucleotide with following characteristics: have at least 70% or higher with nucleotide sequence shown in arbitrary NSC807-1448 at least 15 continuous nucleotide scopes, be preferably 80% or higher, more preferably 90% or higher, even more preferably 95% or higher homology.Described homology can use algorithm known in the art to measure.In addition, the derivative of described antisense oligonucleotide or modified outcome also can be used as antisense oligonucleotide of the present invention.Described modified outcome comprises that for example low alkyl group phosphonic acid ester (low alkyl phosphnote modification) is modified, for example methylphosphonate-type (methyl-phosphonate type) or ethyl phosphine acid esters-type (ethyl-phosphonate-type), thiophosphoric acid modification (phosphorothioate) and phosphamide (phosphoroamidate) modification.
Described antisense-oligonucleotide and derivative thereof play a role to the cell that generates NSC 807-1448 proteins encoded by following process: combine with the DNA or the mRNA of encoding said proteins, suppress transcribing or translating of they, promote the degraded of described mRNA and suppress described proteic expression, thereby described proteic function is suppressed.
Can antisense-oligonucleotide and derivative thereof be prepared into external preparation, for example liniment (liniment) or plaster (poultice) by mixing with the suitable base materials of relative described derivative non-activity.
Antisense-oligonucleotide of the present invention can suppress at least a NSC albumen of arbitrary coding among the NSC 807-1448, thereby can be used for suppressing described proteic biological activity.
The polynucleotide that can suppress one or more gene products of expressing gene also comprise siRNA (siRNA), and the proteic nucleotide sequence of NSC that containing among this RNA encoded expresses is the sense strand nucleic acid of NSC 807-1448 and combining of antisense strand nucleic acid for example.Term " siRNA " is meant a kind of double-stranded RNA that can stop the said target mrna translation.The routine techniques of siRNA transfered cell be can be used for treatment of the present invention or prevention, comprise those wherein DNA be the technology of rna transcription template.Make single transcript have simultaneously from adopted sequence of having of target gene and complementary antisense sequences thereby make up siRNA, described target gene is hairpin structure for example.
Described method can be used for suppressing the wherein gene expression of cells of NSC expression of gene rise.The NSC protein yield that causes this cell that combines of NSC genetic transcription thing reduces in described siRNA and the target cell.The length of described oligonucleotide is at least 10 Nucleotide, also can be isometric with natural generation transcript.Preferably, the length of described oligonucleotide is 19-25 Nucleotide.Most preferably, described oligonucleotide length is less than 75,50 or 25 Nucleotide.The preferred siRNA of the present invention comprises having SEQ ID NO:533,534,535,536,537,538,539,540,541,542,543,544,545,546,547,548,549, and the polynucleotide as target sequence of nucleotide sequence shown in 550,551 or 552.
In addition, described siRNA nucleotide sequence can design computer programming with siRNA, and this program can obtain from Ambion website (http://www.ambion.com/techlib/misc/sirna_finder.html).Based on following scheme, select the nucleotide sequence of described siRNA by computer program:
The selection of siRNA target site:
1. from the AUG initiation codon of target transcript, AA dinucleotide sequence is sought in scanning downstream.Write down the appearance of each AA, close on 3 ' 19 Nucleotide as possible siRNA target site.Suggestions such as Tuschl do not relate at 5 ' and 3 ' non-translational region (UTR) and be close to the siRNA in the zone (75 base within) of initiator codon, because aforementioned region may comparatively be rich in the modulability protein binding site, be designed to thus can disturb combining of the conjugated protein and/or translation initiation complex of UTR at these regional endonucleases and the mixture of siRNA.
2. described potential target position and human genome database are compared, do not consider and any target sequence of the remarkable homologous of other encoding sequence.Utilize BLAST to carry out the homology search, BLAST can find at the NCBI server, and network address is Www.ncbi.nlm.nih.gov/BLAST/
3. select qualified target sequence to be used to synthesize.In the Ambion website, assess along several preferred target sequences of this gene Selection.
Described siRNA can suppress the proteic expression of NSC of expression, suppressed described proteic biological activity thereby can be used in.Therefore, the composition that contains described siRNA can be used for treatment or prevention nonsmall-cell lung cancer.
The nucleic acid that can suppress one or more gene products of the gene of expressing also comprises the ribozyme of crossing the gene (NSC 807-1448) of expressing at described.
Described ribozyme can suppress the proteic expression of expression NSC, suppressed described proteic biological activity thereby can be used in.Therefore, the composition that contains described ribozyme can be used for treatment or prevention nonsmall-cell lung cancer.
Usually, ribozyme is divided into big ribozyme and little ribozyme.Big ribozyme is the enzyme of cracking phosphatase nucleic acid ester bond.After the macronucleus enzyme reaction, reaction site is made up of 5 '-phosphate group and 3 '-oh group.Big ribozyme is further divided into (1) catalysis guanosine at the group I of the transesterification of 5 '-splice site introne RNA; (2) catalysis is via the group II introne RNA of lasso trick sample (lariat-like) structure through the two-step reaction self-splicing; (3) by the RNA assembly of hydrolysis at the ribonuclease P of 5 ' site cutting tRNA precursor.On the other hand, little ribozyme is compared less (about 40bp) with big ribozyme, and small nut enzyme cutting RNA produces 5 '-hydroxyl and 2 '-3 ' annular phosphate.Hammerhead ribozyme (Koizumi etc., (1988) FEBS LErr 228:225) and hair clip type ribozyme (Buzayan, (1986) Nature 323:349; Kikuchi and Sasaki, (1992), Nucleic Acids Res 19:6751) all be included in the little ribozyme.Design and the method that makes up ribozyme be as known in the art (referring to Koizumi etc., FEBS Lett 228:225 (1988); Koizumi etc., Nucleic Acids Res 17:7059 (1989); Kikuchi and Sasaki, (1992), and suppressed the proteic ribozyme of NSC of expressing and also can make up Nucleic AcidsRes 19:6751), according to the sequence information (SEQ ID NO:1,3 or 5) of the proteic nucleotide sequence of the described NSC of coding and the ordinary method for preparing ribozyme.
Described ribozyme can suppress the proteic expression of expression NSC, thereby can be used in the described proteic biological activity of inhibition.Therefore, the composition that contains described ribozyme can be used for treatment or prevention nonsmall-cell lung cancer.
Optional, by use can in conjunction with or can suppress the compound of described gene product function, suppress the described function of crossing one or more gene products of the gene of expressing.For example, described compound is a kind of product bonded antibody that can cross the gene of expression with one or more.
The present invention relates to antibody specific and be segmental purposes at a kind of antibody or this antibody of up-regulated gene proteins encoded.In this application, term " antibody " is meant a kind of immunoglobulin molecules with specificity structure, this structure can with the molecule that wherein contains the antigen (that is the product of the gene of rise) that is useful on synthetic this antibody or with interact with its closely-related antigen-specific (combination).In conjunction with the antibody of crossing the NSC Nucleotide of expressing can be arbitrary form, for example mono-clonal or polyclonal antibody, comprise by with the described polypeptide immune animal antiserum(antisera) that obtains of rabbit for example the polyclone of all kinds and monoclonal antibody, people's antibody and the humanized antibody by the gene recombination preparation.
In addition, antibody of the present invention can be the antibody of antibody fragment or modification, as long as it is in conjunction with one or more NSC albumen of the present invention.For example, described antibody fragment can be Fab, F (ab ') 2, Fv or strand Fv (scFv), wherein the Fv fragment of H chain and L chain is connected (Huston etc., ProcNatl Acad Sci USA 85:5879-83 (1988)) by suitable joint.More specifically, antibody fragment available enzyme such as papoid or pepsin antibody and generate.Perhaps can make up the gene of this antibody fragment of coding, be inserted into suitable expression vector, and in the host cell that is fit to, express (referring to, as Co etc., JImmunol 152:2968-76 (1994); Better and Horwitz, Methods Enzymol 178:476-96 (1989); Pluckthun and Skerra, Methods Enzymol 178:497-515 (1989); Lamoyi, Methods Enzymol 121:652-63 (1986); Rousseaux etc., MethodsEnzymol 121:663-9 (1986); Bird and Walker, Trends Biotechnol 9:132-7 (1991)).
Antibody can by with various molecules, connect as polyoxyethylene glycol (PEG) and to modify.The invention provides the antibody of this modification.The also antibody that can obtain to modify by chemically modified antibody.These modifying method are this area routines.
Perhaps, antibody of the present invention can be used as chimeric antibody and obtains, described chimeric antibody variable region from the non-human antibody and constant region from people's antibody; Perhaps obtain as humanized antibody, described humanized antibody comprises the complementary determining region (CDR) from the non-human antibody, from the framework region (FR) and the constant region of people's antibody.Utilize known technology to prepare this antibody.
The invention provides a kind of method that is used for the treatment of or prevents nonsmall-cell lung cancer, this method is used the antibody that resisted the NSC polypeptide of expressing.According to described method, use the antibody of the anti-described NSC polypeptide of pharmacy effective dose.The antibody that resisted the NSC polypeptide of expressing is used with the dosage that is enough to weaken described NSC protein-active.Optional, can be used for medicine with tumor cell specific cell surface marker bonded antibody and send.Therefore, for example, use be enough to destroy tumour cell dosage, the cytotoxic factor coupling type resisted the antibody of the NSC polypeptide of expressing.
The invention still further relates to a kind of method of in the experimenter, treating or preventing nonsmall-cell lung cancer, this method comprises: described experimenter is used a kind of vaccine, the immunocompetence fragment that contains the polypeptide or the described polypeptide of the nucleic acid encoding that is selected from NSC 807-1448 in this vaccine, perhaps coding said polypeptide or its segmental polynucleotide.The administration of described polypeptide inducing antitumor immunity in subject.Therefore, the present invention further also provides a kind of method that is used for inducing antitumor immunity.Described polypeptide or its immunocompetence fragment can be used as the vaccine at nonsmall-cell lung cancer.Under the certain situation, described albumen or its fragment can be used with TXi Baoshouti (TCR) bonded form or by the form that antigen presenting cell (APC) is presented.Because DC has the strong ability of offering, therefore in APC, preferably use DC.
Among the present invention, " at the vaccine of nonsmall-cell lung cancer " is meant the material that can induce the Anti-tumor immunity when those are in animal body is gone in inoculation or suppress the nonsmall-cell lung cancer immunity.Usually, antineoplastic immune comprises immunne responses such as following:
-induce cellulotoxic lymphocyte at tumour,
-induce the antibody that can discern tumour and
The generation of-inducing antitumor cytokine.
Therefore, if concrete albumen can be induced in these immunne responses any one when inoculating in the animal body, so just can think that this albumen has antineoplastic immune and induces effectiveness.A kind of albumen to antineoplastic immune induce can by in the body or the observation in vitro host immune system detect at this proteic replying.
For example, the method for detection inducing cytotoxic T lymphocyte is known.The effect that enters the foreign matter process antigen presenting cell (APC) of live body is presented to T cell and B cell.The T cell that the antigen that APC is presented in the antigen-specific mode responds is owing to be subjected to this antigenic stimulation, and (or cytotoxic T cell CTL), is bred (being called t cell activation herein) subsequently to be divided into cytotoxic T cell.Therefore, concrete peptide can be assessed by by APC peptide being presented to the T cell inducing of CTL, and detects inducing CTL.In addition, APC can activate the CD4+T cell, CD8+T cell, scavenger cell, eosinophilic granulocyte and NK cell.Because the CD4+T cell is also very important to antineoplastic immune, so the antineoplastic immune inducing action of peptide can utilize the activating effect of these cells to estimate as indicator.
Is known in the art with dendritic cell (DC) as the method that APC estimates the inducing action of CTL.DC is a kind of representative APC, and its CTL induced activity is the strongest in APC.In the method, tried polypeptide and at first contacted, then this DC and T cells contacting with DC.Contact the back if detecting the T cell has cellulotoxic effect to interested cell with DC, illustrate that then being tried polypeptide has the inducing cytotoxic T cell activity.The anti-tumor activity of CTL for example can utilize, 51The dissolving of the tumour cell of Cr-mark detects as indication.Perhaps, utilize 3H-thymidine assimilating activity or LDH (lactose desaturase)-discharge as indicator, the method for estimating the tumour cell degree of injury also is known in the art.
Except DC, peripheral blood lymphocytes (PBMC) also can be used as APC.There is report when having GM-CSF and IL-4, to cultivate PBMC and can promotes inducing of CTL.Similarly, when having keyhole limpet hemocyanin (keyhole limpet hemocyanin), cultivate PBMC and also can induce CTL (KLH) with IL-7.
The polypeptide that tried that has the CTL induced activity through these methods confirmations is the polypeptide that possesses DC activating effect and CTL induced activity subsequently.Therefore, the polypeptide of inducing antitumor cell CTL can be used as the vaccine of anti-nonsmall-cell lung cancer.In addition, the vaccine that also can be used as anti-nonsmall-cell lung cancer by the APC that contacts the CTL ability that has obtained to induce anti-nonsmall-cell lung cancer with this polypeptide.In addition, owing to APC has obtained the vaccine that Cytotoxic CTL also can be used as anti-nonsmall-cell lung cancer to presenting of polypeptide antigen.This utilization is called the cellular immunization therapy by the method for the anti-tumor immunotherapy nonsmall-cell lung cancer that APC and CTL cause.
Usually, when using polypeptide to carry out the cellular immunization therapy, known unite to use to have the multiple polypeptides of different structure and they are contacted with DC can increase CTL inductive efficient.Therefore, when stimulating DC with protein fragments, it is favourable using the segmental mixture of broad variety.
Perhaps, the antineoplastic immune inducing action of polypeptide can confirm by observing the inducing antitumor production of antibodies.For example, when in laboratory animal, having induced the antibody of anti-polypeptide with polypeptide immune, and the growth of tumour cell, propagation or shift when being suppressed by these antibody, determine that then this polypeptide has the ability of inducing antitumor immunity.
By giving vaccine-induced antineoplastic immune of the present invention, and inducing of antineoplastic immune makes and can treat and prevent nonsmall-cell lung cancer.Treatment cancer or the morbidity of prevention nonsmall-cell lung cancer comprise following arbitrary steps, such as the growth that suppresses the NSCLC cell, and the degeneration of NSCLC cell and the appearance that suppresses the NSCLC cell.Reduce nonsmall-cell lung cancer patient experimenter's mortality ratio, reduce the NSC mark in the blood, alleviate the detectable symptom of following nonsmall-cell lung cancer etc., all be included in the effect of treatment or prevention nonsmall-cell lung cancer.This treatment or preventive effect are preferably significant on the statistics.For example, under observation, significance level is 5% or when lower, and the treatment or the preventive effect of the vaccine of nonsmall-cell lung cancer and the control group that do not give vaccine are compared.As, Student ' s t-check, graceful-Whitney U-check or ANOVA can be used to carry out statistical study.
Above-mentioned have immunocompetent albumen or coding these proteic polynucleotide or carrier can be united with adjuvant.Adjuvant refer to when with have immunocompetent albumen when giving (or giving in succession) simultaneously, can promote at this proteic immunoreactive compound.Adjuvant comprises Toxins,exo-, cholera, the Salmonellas toxin, and aluminium etc., but be not limited to these.In addition, vaccine of the present invention can be united with suitable pharmaceutically acceptable carrier.This carrier is sterilized water for example, physiological saline, phosphate buffered saline buffer, nutrient solution etc.And if necessary, vaccine can comprise stablizer, suspensoid, sanitas, tensio-active agent etc.Vaccine general or topical.Give vaccine and can be single-dose or strengthen by multiple dosing.
When using APC or CTL as vaccine of the present invention, can be for example by in vitro method treatment or prevention nonsmall-cell lung cancer.More specifically, collect and just to receive treatment or the experimenter's of preventive therapy PBMC, under isolated condition, this cell is contacted with described polypeptide, induce after APC or the CTL, give the experimenter with this cell again.Thereby also can induce APC by under isolated condition, the carrier of coding said polypeptide being imported PBMC.Can before administration, be cloned in external evoked APC or CTL.By the clone target cell is had the cell of high damagine activity and makes its growth, the cellular immunization therapy can be carried out more effectively.In addition, isolating by this way APC and CTL can be used for the cellular immunization therapy, and described therapy is not only at the experimenter in described cell source, also at the disease of other experimenter's similar type.
Be used for the treatment of or prevent the pharmaceutical composition of nonsmall-cell lung cancer
The invention provides the composition that is used for the treatment of or prevents nonsmall-cell lung cancer, wherein contain the compound that useful the method for the invention is selected, this method is used for screening can change nonsmall-cell lung cancer-Expression of Related Genes or active compound.
When being administered to the people and other Mammals (for example is used for the treatment of cell hyperplastic disease by screening method isolated compound of the present invention, nonsmall-cell lung cancer) time, can be mixed with formulation with the direct administration of described separating compound or with the conventional medicine formulation method, wherein said Mammals such as mouse, rat, cavy, rabbit, cat, dog, sheep, pig, ox, monkey, baboon or chimpanzee etc.The formulation of the present composition comprises the formulation that is suitable for oral, rectum, nasal cavity, part (comprising cheek and hypogloeeis), vagina or parenteral (comprising through intramuscular, subcutaneous and intravenously) administration, perhaps is suitable for by sucking or be blown into the formulation of administration.Can be randomly with preparation packing in isolating dose unit.
Be suitable for oral formulation and comprise capsule, cachet (cachet) or tablet, each all contains the activeconstituents of predetermined amount.Formulation also comprises powder, particle, solution, suspension or emulsion.Can select form administration with medicine sugar-pill (bolus electuary) or ointment.Tablet for oral use and capsule can comprise habitual vehicle for example tackiness agent, filler, lubricant, disintegrating agent or wetting agent.Can choose wantonly with one or more preparation compositions, by compacting or the molded tablet of making.Compressed tablet (compressedtablet) can be made by for example powder of free-flowing form or particulate activeconstituents are pressed in the suitable machine, and described activeconstituents also can be chosen wantonly and a kind of wedding agent, lubricant, inert diluent, lubricant, surfactivity or dispersant.Molded tablet (molded tablet) can be carried out molded forming with the powder compounds after wetting by the inertia liquid diluent in suitable machine.Described tablet can wrap quilt with method well known in the art.The form of oral fluid preparation can be that for example, moisture or butyraceous suspension, solution, emulsion, syrup or elixir perhaps can be anhydrous products, before use water or other suitable carrier reprovision.Described liquid preparation can comprise habitual additive for example suspension agent, emulsifying agent, anhydrous carrier (comprising edible oil) or sanitas.Can choose the described tablet of preparation wantonly makes activeconstituents wherein can slowly or controlledly discharge in vivo.The packing of tablet can comprise mensal single tablet.The formulation of these preparation of traditional Chinese medicine or dosage make that these prepared products are the described suitable dose that gets in the scope.
But the formulation of administered parenterally comprises: moisture and anhydrous aseptic parenteral solution, wherein contain antioxidant, damping fluid, fungistat and make formulation and solute that purpose recipient's blood etc. is opened; And moisture and no aqueous suspensions, wherein can comprise suspension agent and viscosifying agent.Described formulation may reside in unitary dose or the multi-dose container, for example Mi Feng ampoule and bottle, and can be kept under lyophilize (freeze-drying) condition, only need before use to add aseptic liquid carrier for example salt solution, water for injection, promptly join promptly and use.Optional, described formulation can be the form of continuous infusion.Instant injection liquid of joining and suspension can form with above-mentioned sterilized powder, particle and tablet preparation.
The formulation of rectal administration agent comprises the suppository that has standard vector, for example theobroma oil (cocoa butter) or polyoxyethylene glycol.The formulation that oral cavity local medication for example uses through cheek or sublingual administration comprises lozenge (lozenges), and the activeconstituents that wherein contains is present in perfuming base (flavored base) for example in sucrose and gum arabic (acacia) or the tragacanth (tragacath); And pastille (pastille), the activeconstituents that wherein contains is present in base for example gelatinum, glycerine, in sucrose or the gum arabic.For the intranasal administration of activeconstituents, can use liquid spray or dispersible powder or drops.Drops can be formulated with moisture or non-aqueous (non-aqueous) base, wherein also contains one or more dispersion agents, solubilizing agent or suspension agent.
For by inhalation, described composition want can be expediently from insufflator, spraying gun (nebulizer), pressurized package (pressured package) or send other convenient utensil of aerosol spray and discharge.Pressurized package can comprise a kind of suitable propelling agent for example Refrigerant 12 (dichlorodifluoromethane), trichlorofluoromethane (trichlorofluromethane), dichloro tetrafluoro ethane (dichiorotetrafluoroethane), carbonic acid gas or other suitable gas.With regard to pressurized aerosol, can determine dose unit by a kind of valve that can quantitatively send is provided.
Optional, with regard to sucking or be blown into administration, described composition can adopt the form of dry powder composite, and for example, activeconstituents and suitable powder base be the powdered mixture of lactose or starch for example.Described powder composition for example can unit dosage exists in capsule, cartridge case (cartridge), gelatinum or the foaming cartridge bag (blister pack), and described powder can pass through sucker or therefrom administration of insufflator.
Other formulation comprises implantable apparatus (implantable device) and adhesive patch (adhesivepatch); It discharges healing potion.
When needs, can adopt the above-mentioned formulation that can continue release of active ingredients through improving.Described pharmaceutical composition also can contain other activeconstituents, for example antimicrobial medicament (antimicrobial agent), immunosuppressor or sanitas.
Should be understood that the composition of mentioning except that last mask body that preparation of the present invention also can comprise other the habitual medicament of this area according to the purpose preparation type, for example, is suitable for oral preparation and can comprises sweetener.
Preferred unit dose formulations is the activeconstituents of effective dose or those preparations of its suitable part of containing as described below.
For above-mentioned every kind of disease, described composition for example polypeptide and organic compound can be by the oral or injection consumption administration with the about 250mg/kg/ of about 0.1-days.Adult's dosage range is generally the about 17.5g/ of about 5mg-days, is preferably the about 10g/ of about 5mg-days, most preferably is the about 3g/ of about 100mg-days.The present invention can comprise such amount easily with tablet or other unit dosage that discrete unit provides, make that described composition is effective when the multiple of described dosage or described dosage, for example each unit contains and has an appointment 5 milligrams-Yue 500 milligrams, is generally about 100 milligrams-Yue 500 milligrams.
The dosage that adopts depends on many factors, comprises experimenter's age and sex, concrete illness and the severity thereof that treat.In addition, route of administration can also change with illness and severity thereof.
The present invention further also provides a kind of composition that is used for the treatment of or prevents nonsmall-cell lung cancer, wherein contains the activeconstituents that can suppress to be selected from arbitrary genetic expression among the NSC 807-1448.Described activeconstituents can be a kind of antisense-oligonucleotide, at the siRNA or the ribozyme of described gene, the derivative of perhaps described antisense-oligonucleotide, siRNA or ribozyme is such as expression vector etc.Described activeconstituents can be made external preparation by mixing with a kind of suitable base materials at described derivative non-activity, such as liniment or plaster.
In addition, if desired, can described activeconstituents be formulated as tablet, powder, particle, capsule, liposome methods, injection liquid, solution, nasal drop and lyophilized medication by adding vehicle, isotonic agent, solubilizing agent, sanitas, analgesic agent etc.These can utilize preparation to contain the common method preparation of nucleic acid drug.
Preferably, described antisense-oligonucleotide derivative, siRNA derivative or ribozyme derivative can pass through at the affected part direct drug injection, thereby perhaps give the patient by being injected into blood vessel arrival affected part.In addition, can also add mounting medium to described composition and be used to increase persistence and film-permeability.The embodiment of mounting medium comprises liposome, poly-L-Methionin, lipid, cholesterol, fat transfection and derivative thereof.
Described composition dosage can suitably be adjusted according to the patient body situation, uses with required amount.For example, can use 0.1-100mg/kg, the dosage of preferred 0.1-50mg/kg.
Another embodiment of the present invention is a kind of composition that is used for the treatment of or prevents nonsmall-cell lung cancer, wherein contains the anti-antibody that is selected from the arbitrary coded by said gene polypeptide of NSC 807-1448, or can with the fragment of described polypeptide bonded antibody.
Although can be because of the different differences to some extent of symptom, but when normal adult (60 kilograms of weight) oral medication, being used for the treatment of or preventing the antibody of nonsmall-cell lung cancer or its segmental dosage is about 0.1 milligram-Yue 100 mg/day, be preferably about 1.0 milligrams-Yue 50 mg/day, more preferably about 1.0 milligrams-Yue 20 mg/day.
When by injection to normal adult (60 kilograms of weight) with gi tract outside during the mode administration, although can be because of the different differences to some extent of patient disease, disease symptoms and medication, generally speaking, the intravenous injection amount is about 0.01 milligram-Yue 30 mg/day, be preferably about 20 mg/day of about 0.1-, about 10 mg/day of more preferably about 0.1-.In addition, with regard to other animal, can use amount by the conversion of 60kg body weight.
Polypeptide
According to the present invention, new people's gene URLC1 (NSC 905) is provided, the expression of this gene in nonsmall-cell lung cancer with in corresponding non-cancer tissue, comparing remarkable rising.
URLC 1 (NSC 905) a kind of TUDOR structural domain of encoding.The TUDOR structural domain has RNA combination and nucleic acid combined function.The nucleotide sequence of this gene is shown in SEQ ID NO:1, and the aminoacid sequence of this genes encoding is shown in SEQ ID NO:2.
T these genes just makes cancer cells have carcinogenic activity, so the effective means that to suppress these proteic activity can be a kind of treatment cancer, particularly nonsmall-cell lung cancer.
The invention provides the Novel Human gene, comprise the nucleotide sequence and degeneracy body and the mutant that are selected from SEQ ID NO:1, these genes encodings NSC albumen comprises the aminoacid sequence shown in SEQ ID NO:2, or its functional equivalent.Hereinafter, the polypeptide of these genes encodings is referred to as NSC albumen.The example of the proteic polypeptide function equivalent of NSC comprises, for example, and with the homologous protein of corresponding other organism of described people NSC albumen, and the proteic mutant of people NSC.
Among the present invention, term " function equivalent " is meant such desired polypeptides, and they have the activity that can promote cell proliferation the same with any NSC albumen, and can give the cancer cells carcinogenic activity.Can judge whether desired polypeptides has cell-proliferation activity by following method: the DNA of the desired polypeptides of will encoding imports the cell of the corresponding polypeptide of encoding, and the propagation or the colony that detect described cell then form active rising.
Prepare that to specify the method for proteic function equivalence polypeptide be well known by persons skilled in the art, be included in the currently known methods that imports sudden change in the albumen.For example, those skilled in the art can import suitable sudden change by site-directed mutagenesis in the aminoacid sequence of one of these albumen, prepare the proteic function equivalence polypeptide of human NSC (Hashimoto-Gotoh etc., Gene 152:271-5 (1995); Zoller and Smith, Methods Enzymol 100:468-500 (1983); Kramer etc., Nucleic Acids Res.12:9441-9456 (1984); Kramer and Fritz, Methods Enzymol 154:350-67 (1987); Kunkel, Proc Natl Acad Sci USA 82:488-92 (1985); Kunkel, Methods Enzymol85:2763-6 (1988)).Amino acid mutation also can spontaneous generation.Polypeptide of the present invention comprises the albumen with human NSC Argine Monohydrochloride sequence, and wherein one or more amino acid is undergone mutation, the mutant polypeptide of generation and human NSC protein function equivalence.The quantity of mutating acid is normally 10 or still less in this mutant, and preferred 6 or still less, most preferably 3 or still less.
The albumen of known mutations or modification keeps original biological activity, described albumen is to have by one or more amino-acid residue to concrete aminoacid sequence to replace, disappearance, albumen (Mark etc., the Proc Natl Acad Sci USA 81:5662-6 (1984) of the aminoacid sequence that forms modified in insertion and/or interpolation; Zoller and Smith, Nucleic Acids Res 10:6487-500 (1982); Dalbadie-McFarland etc., Proc Natl Acad Sci USA 79:6409-13 (1982)).
The amino-acid residue that is suddenlyd change preferably sports different amino acid, and the character of its amino acid side chain is (being that conserved amino acid replaces process) of guarding.The character of amino acid side chain for example hydrophobic amino acid (A, I, L, M, F, P, W, Y, V), hydrophilic amino acid (R, D, N, C, E, Q, G, H, K, S, T), and side chain has the following function group or has total character: aliphatic lateral chain (G, A, V, L, I, P); Contain hydroxyl side chain (S, T, Y); Contain sulphur atom side chain (C, M); Contain carboxylic acid and acid amides side chain (D, N, E, Q); Contain base side chain (R, K, H); Contain aromatic base side chain (H, F, Y, W).Notice that the letter in the bracket is meant amino acid whose one-letter code.
The example that has added the polypeptide of one or more amino-acid residue in the proteic aminoacid sequence of people NSC is to comprise the proteic fusion rotein of people NSC.Fusion rotein is people NSC albumen and other peptide or proteic syzygy, and is included in scope of the present invention.Available technology well known to those skilled in the art prepares fusion rotein, as the proteic DNA of code book contriver NSC is connected with coding other peptide or proteic DNA, makes the encoder block coupling, with this fusion dna insertion expression vector and express in host cell.For the peptide that merges with albumen of the present invention or albumen without limits.
Can be used as the known peptide that merges with NSC albumen of the present invention and comprise, for example FLAG (Hopp etc., Biotechnology 6:1204-10 (1988)), 6 * the His that contains 6 His (Histidine) residue, 10 * His, influenza hemagglutinin (Influenza agglutinin) is (HA), people c-myc fragment, VSP-GP fragment, p18HIV fragment, the T7-mark, HSV-mark, E-mark, the SV40 antigen fragment, lck mark, alpha-tubulin fragment, the B-mark, PROTEIN C fragment and analogue.Can comprise for example GST (glutathione-S-transferase), influenza hemagglutinin (HA), constant region for immunoglobulin, beta-galactosidase enzymes, MBP (maltose binding protein) etc. with the albumen that albumen of the present invention merges.
Fusion rotein can be prepared as follows: will encode above-mentioned fusogenic peptide or the proteic DNA of buying, and with the DNA fusion of code book invention NSC polypeptide, and the fusion dna of expressing preparation.
The method of known another separation function polypeptide of equal value for example is in this area, uses the method (Sambrook etc., Molecular Cloning 2nd ed.9.47-9.58, Cold Spring HarborLab.Press (1989)) of hybridization technique.Those skilled in the art can separate and NSC albumen (being SEQID NO:1) height homologous DNA at an easy rate, and from separated DNA the proteic function equivalence polypeptide of separation of human NSC.NSC albumen of the present invention comprises by such DNA encoded polypeptide, all or part of hybridization of described NDA and the proteic dna sequence dna of coding people NSC, and polypeptide of the present invention and the proteic function equivalence of people NSC.These polypeptide comprise the Mammals homologue corresponding with the albumen of derived from human (for example, monkey, mouse, rabbit and cow genome encoded polypeptides).When separating from animal with coding people NSC protein D NA height homologous cDNA, the concrete preferred tissue that uses from lung cancer.
Those skilled in the art can conventional select to be used for to separate the hybridization conditions of the DNA of coding people NSC protein function polypeptide of equal value.For example, hybridize following carrying out: at 68 ℃ of prehybridization 30min or longer, add label probe with " Rapid-hyb damping fluid ", 68 ℃ of insulation 1h or longer.Washing step subsequently can carry out in for example low rigorous condition.Hanging down rigorous condition is, for example 42 ℃, and 2 * SSC, 0.1%SDS, or preferred 50 ℃, 2 * SSC, 0.1%SDS.More preferably, adopt high rigorous condition.High rigorous condition is, for example room temperature, in 2 * SSC, 3 each 20min of washing among the 0.01%SDS, 37 ℃ subsequently, in 1 * SSC, 3 each 20min of washing among the 0.1%SDS, 50 ℃ then, in 1 * SSC, 0.1%SDS washs 2 each 20min.Though some factors such as temperature and salt concn can influence the preciseness of hybridization, those skilled in the art can these factors of appropriate selection to obtain necessary rigorous degree.
Can utilize gene amplification method such as polymerase chain reaction (PCR) to replace hybridization, separate the DNA of coding NSC protein function polypeptide of equal value, use sequence information (SEQ ID NO:1) synthetic primer according to this proteic DNA of coding.
Common and the proteic aminoacid sequence height of the people NSC homology by the proteic function equivalence polypeptide of people NSC of above-mentioned hybridization technique or the isolating dna encoding of gene amplification technology." height homology " is often referred to 40% homology or higher, preferred 60% or higher, more preferably 80% or higher, even preferred 95% or higher.The homology of polypeptide can be determined with the algorithm of " Wilbur and Lipman, Proc Natl Acad Sci USA80:726-30 (1983) ".
Amino acid sequence of polypeptide of the present invention, molecular weight, iso-electric point, the existence of sugar chain or disappearance, or form may morph, and this depends on the purification process of the preparation employed cell of this polypeptide or host or use.But, the functional equivalent that needs only it is in people NSC albumen of the present invention, and it just belongs to scope of the present invention.
Available method well known to those skilled in the art prepares polypeptide of the present invention with the form of recombinant protein or native protein.Recombinant protein is prepared as follows: with the DNA of code book invention polypeptide (for example, the DNA that contains the nucleotide sequence of SEQID NO:1) inserts suitable expression vector, this carrier is imported proper host cell, obtain extract, use the post that is fixed with anti-protein antibodies of the present invention on it or unite a plurality of above-mentioned posts of use, thereby this extract is carried out the described polypeptide of chromatography purifying, described chromatography such as ion exchange chromatography, reversed phase chromatography, gel-filtration or affinity chromatography.
When polypeptide of the present invention was expressed as the fusion rotein that merges with glutathione-S-transferase albumen or has added the recombinant protein of a plurality of Histidines in host cell (for example zooblast and intestinal bacteria), the recombinant protein of this expression can be used gsh post or ni-sepharose purification.Perhaps, when polypeptide of the present invention was expressed as the albumen of c-myc, polyhistidine or FLAG mark, it can use anti-c-myc, His or FLAG antibody test and purifying respectively.
Behind this fusion rotein of purifying, also may remove the zone different by excising zymoplasm or factor Xa as required with being tried polypeptide.
Separating natural albumen can adopt method known to those skilled in the art, for example contacts with affinity column, wherein combines with the tissue of expressing polypeptide of the present invention or the extract of cell with the protein bound antibody of following NSC.Described antibody can be polyclonal antibody or monoclonal antibody.
The present invention also comprises the partial peptide of polypeptide of the present invention.This partial peptide contains the proteic specific amino acid of NSC of the present invention, and by at least 7 amino acid, preferred 8 or amino acids more, more preferably 9 or more amino acids form.Described partial peptide can be used for, and for example prepares the proteic antibody of anti-NSC of the present invention, and proteic promotor of NSC of the present invention (accelerator) or inhibitor are screened in screening and the protein bound compound of NSC of the present invention.
Can pass through gene engineering method, known peptide synthetic method, or prepare partial peptide of the present invention with suitable peptide enzymic digestion polypeptide of the present invention.Synthetic for peptide, can use that for example solid-phase peptide is synthetic or the liquid phase peptide is synthetic.
In addition, the invention provides the proteic polynucleotide of code book invention NSC.NSC albumen of the present invention can be used in vivo or the NSC albumen of external preparation the invention described above, perhaps is applied to the gene therapy of disease, and described disease is owing to the genetic abnormality of the gene of code book invention polypeptide.Can utilize any one form of polynucleotide of the present invention, NSC albumen of the present invention or its Equivalent as long as it is encoded, described form comprises mRNA, RNA, cDNA, genomic dna, the polynucleotide of chemosynthesis.Polynucleotide of the present invention comprise DNA and the degenerate sequence thereof that contains given nucleotide sequence, as long as the dna encoding NSC albumen of the present invention or its Equivalent that form.
Available method known to those skilled in the art prepares polynucleotide of the present invention.For example, polynucleotide of the present invention can be prepared as follows: preparation cDNA library from express the proteic cell of NSC of the present invention, hybridize as probe with the partial sequence (as SEQ ID NO:1) of DNA of the present invention.The cDNA library can be according to Sambrook etc., Molecular Cloning, the method preparation that Cold Spring Harbor LaboratoryPress (1989) describes; Perhaps also can use the cDNA library that to buy.The cDNA library also can be prepared as follows: extract RNA from express the proteic cell of NSC of the present invention, the dna sequence dna according to the present invention (as SEQ ID NO:1) synthesizes few DNA, is that primer carries out PCR with this widow DNA, the proteic cDNA of amplification coding NSC of the present invention.
In addition, the nucleotide sequence of the cDNA that obtains by checking order, the translation district of definite cDNA coding that can be conventional also just can easily obtain the proteic aminoacid sequence of NSC of the present invention.And the cDNA that usefulness obtains or its part can isolation of genomic DNA as probe screening-gene group DNA library.
More specifically, can at first be tried the proteic cell of NSC, be prepared mRNA in tissue or the organ from expressing the present invention.With known method separating mRNA; For example use guanidine ultracentrifugation (guanidineultracentrifugation) (Chirgwin etc., Biochemistry 18:5294-9 (1979)) or AGPC method (Chomczynski and Sacchi, Anal Biochem 162:156-9 (1987)) prepare total RNA.In addition, with mRNA purification kit (Pharmacia) purified mRNA from total RNA.Perhaps, with QuickPrep mRNA purification kit (Pharmacia) direct purification mRNA.
Utilize reversed transcriptive enzyme from the synthetic cDNA of the mRNA that obtains.The synthetic of cDNA can be used the test kit that can buy, as the AMV reversed transcriptive enzyme first chain cDNA synthetic agent box (Seikagaku Kogyo).Perhaps, synthetic and amplification cDNA (Frohman etc., Proc Natl Acad SciUSA 85:8998-9002 (1988) with 5 '-RACE method; Belyavsky etc., Nucleic Acids Res 17:2919-32 (1989)), wherein use primer as herein described, 5 '-Ampli FINDER RACE test kit (Clontech) and polymerase chain reaction (PCR).
Prepare required dna fragmentation and be connected from the PCR product with carrier DNA.Recombinant vectors is used for transformed into escherichia coli etc., and prepares required recombinant vectors from the colony of selecting.By the nucleotide sequence of the required DNA of ordinary method checking, for example dideoxy nucleotide chain cessation method (dideoxynucleotide chain termination).
The codon of considering the host cell that is used for expressing utilizes frequency, and the nucleotide sequence of design polynucleotide of the present invention makes it more effectively express (Grantham etc., Nucleic Acids Res 9:43-74 (1981)).Useful commercial test kit or ordinary method change the sequence of polynucleotide of the present invention.For example, synthetic oligonucleotide or suitable polynucleotide passage are inserted in the available constraints enzymic digestion, increase joint, or insert initiator codon (ATG) and/or terminator codon (TAA, TGA or TAG) changes described sequence.
Particularly, polynucleotide of the present invention comprise the DNA of the nucleotide sequence that contains SEQ ID NO:1.
In addition, the invention provides polynucleotide, it is under rigorous condition and have the multi-nucleotide hybrid of nucleotide sequence shown in the SEQ ID NO:1, and encodes above-mentioned and polypeptide NSC protein function equivalence of the present invention.Those skilled in the art can suitably select rigorous condition.For example, can adopt low rigorous condition.More preferably, can adopt high rigorous condition.These conditions are identical with the described condition of preamble.Preferred cDNA of the above-mentioned DNA that is used to hybridize or chromosomal DNA.
Carrier and host cell
The present invention also provides the carrier that has wherein inserted the above-mentioned polynucleotide of the present invention.Carrier of the present invention is used for keeping polynucleotide of the present invention at host cell and specifically is DNA, is used to express NSC albumen of the present invention, or is used to give polynucleotide of the present invention and carries out gene therapy.
When with intestinal bacteria as host cell and in intestinal bacteria (JM109 for example, DH5 α, HB101 or XL1Blue) a large amount of amplification and when producing carrier, this carrier should have " the replication origin ori " that increases in intestinal bacteria, and the colibacillary marker gene that is used to screen conversion is (for example, by for example ampicillin, tsiklomitsin, kantlex, the drug resistant gene that medicines such as paraxin screen).For example, can use the M13 serial carrier, pUC serial carrier, pBR322, pBluescript, pCR-Script or the like.In addition, also can use pGEM-T, pDIRECT and pT7 carry out subclone and extract cDNA and above-mentioned carrier.When utilizing preparing carriers NSC albumen of the present invention, expression vector is particularly useful.For example, the expression vector at expression in escherichia coli should possess above-mentioned character so that increase in intestinal bacteria.When with intestinal bacteria such as JM109, DH5 α, HB101 or XL1Blue are during as host cell, and carrier should have promotor, as lacZ promotor (Ward etc., Nature 341:544-6 (1989); FASEB J 6:2422-7 (1992)), araB promotor (Better etc., Science 240:1041-3 (1988)), or T7 promotor etc., they can be in intestinal bacteria the required gene of effectively expressing.In this respect, can use for example pGEX-5X-1 (Pharmacia), " QIAexpress system " (Qiagen), pEGFP and pET (BL21 of host's preferred expression T7 RNA polymerase in this case) replace above-mentioned carrier.In addition, this carrier also can comprise the signal peptide sequence of protein excretion.Instructing the NSC protein excretion is pelB signal sequence (Lei etc., J Bacteriol 169:4379 (1987)) to the example of the signal peptide sequence of colibacillus periplasm.The method of described carrier importing target host cell is comprised, for example Calcium Chloride Method and electroporation.
Except intestinal bacteria, for example also can utilize mammalian expression vector (as pcDNA3 (Invitrogen) and pEGF-BOS (Nucleic Acids Res 18 (17): 5322 (1990)), pEF, pCDM8), the insect cell expression carrier is (as " Bac-to-BAC baculovirus expression system " (GIBCO BRL), pBacPAK8), plant expression vector is (as pMH1, pMH2), the animal virus expression vector (as pHSV, pMV, pAdexLcw), retrovirus expression vector (as pZIpneo), Yeast expression carrier (pichia spp (as " Pichia) the expression test kit " (Invitrogen), pNV11, SP-Q01), Bacillus subtilus (Bacillus subtilis) expression vector (as pPL608, pKTH50) prepares polypeptide of the present invention.
For at zooblast such as CHO, express described carrier in COS or the NIH3T3 cell, this carrier should have expresses necessary promotor in these cells, SV40 promotor (Mulligan etc. for example, Nature 277:108 (1979)), the MMLV-LTR promotor, EF1 α promotor (Mizushima etc., Nucleic Acids Res 18:5322 (1990)), CMV promotor etc., and preferably have the marker gene that is used to screen transformant (as, through medicine (for example Xin Meisu, the drug resistant gene of G418) selecting).The known carrier that possesses these character comprises, for example pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV and pOP13.
Preparation NSC albumen
The present invention also provides preparation NSC of the present invention proteic method.Can prepare described NSC albumen by cultivating host cell, described host cell contains and comprises the proteic expression carrier of the described NSC of coding.Employing method is as required expressed described stable gene, increases the copy number of this gene in the cell simultaneously.For example, the carrier (as pCHO I) that will contain complementary DHFR gene imports the Chinese hamster ovary celI that the nucleic acid route of synthesis is lacked, and is increased by methotrexate (MTX) then.In addition, when the transient expression gene, can adopt following method: the carrier (pcD etc.) that will contain the SV40 replication origin is transformed into the COS cell, and this cell chromosome comprises SV40T antigen presentation gene.
As mentioned above the NSC albumen of the present invention of Huo Deing can be in host cell or outside (as substratum) separate, and purifying is the polypeptide of pure substantially homogeneous.The used term " pure substantially " of the given polypeptide of this paper is meant that this polypeptide separates with other biomacromolecule basically.Substantially pure polypeptide refers to that dry weight at least 75% (as at least 80,85,95, or 99%) is pure.Measure purity with suitable standard method, described method is column chromatography for example, and polyacrylamide gel electrophoresis or HPLC analyze.The method of polypeptide separation and purification is not limited to any concrete grammar; In fact, can adopt any standard method.
For example, can suitably select and unite the use column chromatography, filter, ultrafiltration, the salt precipitation, solvent deposition, solvent extraction, distillation, immunoprecipitation, the SDS-polyacrylamide gel electrophoresis, the iso-electric point electrophoresis, dialysis and recrystallization are with the described NSC albumen of separation and purification.
The example of chromatography comprises, affinity chromatography for example, ion exchange chromatography, hydrophobic chromatography, gel-filtration, reversed phase chromatography, adsorption chromatography etc. (Strategies for Protein Purification andCharacterization:A Laboratory Course Manual.Ed.Daniel R.Marshak etc., ColdSpring Harbor Laboratory Press (1996)).These chromatography can liquid chromatography (LC) such as HPLC and FPLC carry out.Therefore, the invention provides the highly purified polypeptide of method for preparing.
Before or after purifying, handle NSC albumen of the present invention, can choose wantonly and modify or the described polypeptide of excalation with suitable protein modified enzyme.Useful protein modified enzyme is including, but not limited to trypsinase, Chymotrypsin, lysyl endopeptidase, protein kinase, Glycosylase or the like.
Antibody
The invention provides protein bound antibody with NSC of the present invention.Antibody of the present invention can arbitrary form such as monoclonal antibody or polyclonal antibody use, and comprise the antiserum(antisera) that obtains with NSC protein immune animal of the present invention such as rabbit, all types of polyclones and monoclonal antibody, people's antibody and the humanized antibody for preparing by gene recombination.
Can be to obtain antibody with NSC albumen of the present invention from any animal kind as antigen, but preferably from Mammals such as people, mouse or rat, more preferably obtains antibody from the people.NSC albumen from the people can obtain from Nucleotide as herein described or aminoacid sequence.As described herein, the polypeptide as immunizing antigen can be complete NSC albumen or the proteic partial peptide of NSC.Partial peptide comprises, proteic aminoterminal of NSC for example of the present invention (N) or carboxyl terminal (C) fragment.
Herein, antibody is defined as the albumen that reacts with the proteic total length of NSC of the present invention or its fragment.
Code book is invented NSC albumen or its segmental gene is inserted into known expression vector, transform host cell as herein described with this carrier then.With the arbitrary standards method in host cell or outside reclaim required NSC albumen or its fragment, and subsequently with described NSC albumen or its fragment as antigen.Perhaps, to express the proteic whole cell of this NSC or its lysate or chemically synthesized polypeptide as antigen.
With any Mammals of described antigen immune, but the preferred consistency of considering and being used for the parental cell of cytogamy.Usually use Rodentia (Rodentia), the animal of Lagomorpha (Lagomorpha) or primates (Primates).Rodent comprises, as mouse, and rat and hamster.Lagomorph comprises rabbit.Primate comprises, for example catarrhine (Catarrhini) (the Eastern Hemisphere monkey (old world monkey)) is as Macaca fascicularis, rhesus monkey (rhesus monkey), baboon (sacred baboon) and chimpanzee (chimpanzee).
Method with antigen-immunized animal is as known in the art.Abdominal injection or subcutaneous injection antigen are the standard methods of immune animal.More specifically, can be with an amount of phosphate buffered saline buffer (PBS), dilution and suspension antigens such as physiological saline.If desired, antigen suspension and an amount of standard adjuvant such as Fu Shi (Freund ' s) Freund's complete adjuvant is mixed, form emulsion, give Mammals then.Preferably, will with the administration in per 4 to 21 days of an amount of Fu Shi Freund's complete adjuvant antigens mixed for several times.Also can use suitable carriers to carry out immunity.After the above immunity, with the increase of antibody quantity required in the standard method check serum.
The proteic polyclonal antibody of NSC of the present invention can be prepared as follows: collect blood from the animal (this animal via detects antibody required its serum to be increased) through immunity, with method separation of serum from described blood of any conventional.Polyclonal antibody comprises the serum that contains polyclonal antibody and can separate the fraction that contains this polyclonal antibody from described serum.For example utilize the affinity column with NSC albumen coupling of the present invention, prepare immunoglobulin G or M from only discerning the proteic fraction of NSC of the present invention, subsequently further with albumin A or this fraction of Protein G column purification.
Be the preparation monoclonal antibody, from the animal that increases with described antigen immune and as required antibody horizontal the above-mentioned serum after testing, collect immunocyte, and carry out cytogamy.The immunocyte that is used for cytogamy is preferably available from spleen.Other treats that the preferred parental cell that merges with above-mentioned immunocyte comprises, Mammals myelomatosis cell for example, and the characteristic that more preferably possesses acquisition is so that with the myeloma cell of drug screening fused cell.
According to known method.Method as (Galfre and Milstein, Methods Enzymol 73:3-46 (1981)) such as Milstein merges above-mentioned immunocyte and myeloma cell.
By in Standard Selection substratum such as HAT substratum (containing xanthoglobulin, the substratum of aminopterin and thymidine) etc., cultivating the hybridoma of selecting the cytogamy gained.Usually, this time will be enough to make all other cells (nonfused cell) death except required hybridoma to cell culture to several weeks cultured continuously a couple of days in the HAT substratum.Then, produce the hybridoma of required antibody with screening of standard limiting dilution assay and clone.
Except the above-mentioned method for preparing hybridoma with the antigen immune non-human animal, the lymphocyte of human lymphocyte such as ebv infection also can be expressed the proteic cell of NSC or its lysate and carry out immunity at external use NSC albumen.Then, merged such as U266 with the myeloma cell that can blur splitted (indefinitedividing), people source by the lymphocyte of immunity, with the acquisition generation can with the hybridoma (unexamined Japanese patent application (UnexaminedPublished Japanese Patent Application No.) is Sho 63-17688 (JP-A)) of the protein bound required people's antibody of described NSC.
The hybridoma that obtains is transplanted to the abdominal cavity of mouse subsequently, and extracting ascites.The monoclonal antibody that obtains can be passed through, for example ammonium sulfate precipitation, albumin A or Protein G post, DEAE ion exchange chromatography or coupling the proteic affinity column of NSC of the present invention carry out purifying.Antibody of the present invention not only can be used for purifying and detect NSC albumen of the present invention, can also be as proteic candidate's agonist of NSC of the present invention and antagonist.In addition, this antibody can be applicable to the Antybody therapy of NSC protein related diseases of the present invention, and described disease comprises nonsmall-cell lung cancer.When the antibody that will obtain gave human body (Antybody therapy), preferred people's antibody or humanized antibody were to reduce immunogenicity.
For example, such as NSC albumen, the immunity that comes of expressing the proteic cell of NSC or its lysate has the transgenic animal in human immunoglobulin gene storehouse with antigen.Collect the cell that produces antibody from this animal then, itself and myeloma cell are merged the acquisition hybridoma, resist people's antibody of described polypeptide (referring to WO92-03918 from this hybridoma preparation, WO93-2227, WO94-02602, WO94-25585, WO96-33735 and WO96-34096).
Perhaps, make the immunocyte that produces antibody as lymphocyte immortalization, be used to prepare monoclonal antibody through immunity with oncogene.
So the monoclonal antibody that obtains also can utilize genetic engineering technique pass through recombinant methods (referring to, as Borrebaeck and Larrick, Therapeutic Monoclonal Antibodies, published in the United Kingdom by MacMillan Publishers LTD (1990)).For example, can be from immunocyte, come the DNA of this antibody of clones coding as the hybridoma that produces antibody or through the lymphocyte of immunity, this DNA is inserted suitable carriers, and change host cell over to and prepare recombinant antibodies.The present invention also provides the recombinant antibodies of preparation as mentioned above.
In addition, antibody of the present invention can be the antibody of antibody fragment or modification, as long as it is in conjunction with one or more NSC albumen of the present invention.For example, described antibody fragment can be Fab, F (ab ') 2, Fv or strand Fv (scFv), wherein the Fv fragment of H chain and L chain is connected (Huston etc., ProcNatl Acad Sci USA 85:5879-83 (1988)) by suitable joint.More specifically, antibody fragment available enzyme such as papoid or pepsin antibody and generate.Perhaps can make up the gene of this antibody fragment of coding, be inserted into suitable expression vector, and in the host cell that is fit to, express (referring to, as Co etc., JImmunol 152:2968-76 (1994); Better and Horwitz, Methods Enzymol 178:476-96 (1989); Pluckthun and Skerra, Methods Enzymol 178:497-515 (1989); Lamoyi, Methods Enzymol 121:652-63 (1986); Rousseaux etc., MethodsEnzymol 121:663-9 (1986); Bird and Walker, Trends Biotechnol 9:132-7 (1991)).
Antibody can by with various molecules, connect as polyoxyethylene glycol (PEG) and to modify.The invention provides the antibody of this modification.The also antibody that can obtain to modify by chemically modified antibody.These modifying method are this area routines.
Perhaps, antibody of the present invention can be used as chimeric antibody and obtains, described chimeric antibody variable region from the non-human antibody and constant region from people's antibody; Perhaps obtain as humanized antibody, described humanized antibody comprises the complementary determining region (CDR) from the non-human antibody, from the framework region (FR) and the constant region of people's antibody.By utilizing known technology to prepare this antibody.
The antibody of Huo Deing can purifying becomes homogeneous as mentioned above.For example, carry out the separation and purification of antibody to be used for common proteic separation purification method.For example, antibody can be by suitably selecting and uniting and use column chromatography to separate and purifying, described chromatography is such as affinity chromatography, filter, ultrafiltration is saltoutd, dialysis, SDS-polyacrylamide gel electrophoresis and isoelectrofocusing (Antibodies:A Laboratory Manual.Ed Harlow and David Lane, Cold Spring Harbor Laboratory (1988)) etc., but be not limited thereto.Albumin A post and Protein G post can be used as affinity column.Available exemplary albumin A post comprises, as Hyper D, and POROS and Sepharose F.F. (Pharmacia).
Except affinity chromatography, the example of chromatography also comprises, as ion exchange chromatography, hydrophobic chromatography, gel-filtration, reversed phase chromatography, adsorption chromatography etc. (Strategies for Protein Purification andCharacterization:A Laboratory Course Manual.Ed.Daniel R.Marshak etc., ColdSpring Harbor Laboratory Press (1996)).The chromatography process can be by liquid chromatography (LC) such as enforcements such as HPLC and FPLC.
For example, by the mensuration absorbancy, Enzyme Linked Immunoadsorbent Assay (ELISA), enzyme immunoassay (EIA), radioimmunoassay (RIA) and/or immunofluorescence are measured the antigen-binding activity of antibody of the present invention.Among the ELISA, antibody of the present invention is fixed on the flat board, and NSC albumen of the present invention is added on the flat board, applies the sample (such as the culture supernatant or the antibody purified of the cell that produces antibody) that contains required antibody then.Then apply the identification first antibody and also use the second antibody of enzyme such as alkali phosphatase enzyme mark, the described flat board of incubation.After the washing, add enzyme substrates such as p-nitrophenyl phosphate, measure the antigen-binding activity of absorbancy with assess sample to flat board.The proteic fragment of NSC also can be used as the combination activity that antigen is estimated antibody as C-terminal or N-terminal fragment.According to the present invention, available BIAcore (Pharmacia) estimates the activity of antibody.
Aforesaid method can detect or measure NSC albumen of the present invention, and this is to contain the proteic sample of NSC of the present invention by antibody of the present invention being exposed to infer, and detects or measure the immunocomplex that antibody and albumen form.
Because detection of the present invention or measure the proteic method of NSC can specific detection or measure described albumen, so this method can be used for the various proteic experiments of having used.
Providing of following example is in order to set forth the present invention, to help those of ordinary skills to implement and utilize the present invention.Described embodiment in no case should be construed as limiting scope of the present invention.
Unless otherwise indicated, all scientific and technical terminologies among the application all has and the identical implication of one skilled in the art's common sense of the present invention.Although with describe similar among the application or the method that is equal to and material all can be used for implementing or check the present invention, hereinafter still still be that suitable method and material are described.Arbitrary patent, patent application and the publication quoted among the application are hereby incorporated by.
Implement best mode of the present invention
The present invention elaborates by the following example, but the present invention is in no way limited to these embodiment.
To available from illing tissue's epithelial cell of nonsmall-cell lung cancer (for example, from) thereby and the tissue of healthy tissues assess and identify at the morbid state gene of differential expression in the nonsmall-cell lung cancer for example.Described experimental implementation is as follows.
[embodiment 1] universal method
(1) patient and tissue sample
Abide by the informed consent principle, from 37 patients having accepted pulmonay lobectomy (lobectomy) (15 women and 22 male sex, 46-79 year; Mean age is 66.0) obtain (primary) cancerous lung tissue of former generation.From case taking, obtain clinical information, each tumour is diagnosed according to histopathology hypotype and pathologist's classification; 22 in 37 tumours classify as gland cancer, and 14 is SCC, and remaining 1 is adenosquamous carcinoma.Judge the clinical stage of each tumour according to UICC TNM grade scale.All samples is freezing immediately and be embedded into TissueTek OCT medium (medium) (Sakura, Tokyo Japan), are stored in-80 ℃ then.
(2) laser-capture micro-dissections (laser-capture microdissection), RNA extracts and base In the RNA of T7 amplification
Use laser-the capture sample selective collection cancer cells of micro-dissections (Kitahara et al., Cancer Res 61:3544-9 (2001)) from being preserved.Finish the extraction of total RNA and based on the amplification of T7 according to previously described method (Okabe et al., Cancer Res 61:2129-37 (2001)).Probe in contrast, the normal people's lung that in kind increases gathers (A) RNA (CLONTECH).From every part of aliquots containig that is 2.5-μ g of the RNA (aRNA) of the amplification of every kind of cancerous tissue and contrast, under the condition that Cy5-dCTP and Cy3-dCTP exist, carry out reverse transcription respectively.
(3) preparation of cDNA microarray
In order to obtain to be used for the cDNA of point sample on slide glass, each gene is carried out RT-PCR amplification (Kitahara et al., Cancer Res 61:3544-9 (2001)) with previously described method.With Microarray Spotter Generation III (Amersham Biosciences) with PCR product point sample on slide.4,608 genes are put on single slide glass in bipartite mode.The different slide glass (23,040 genes altogether) of preparation 5 covers, every suit is all put identical 52 housekeeping genes and 2 kinds of negative control genes.
(4) acquisition of hybridization and data
Hybridize, wash and detect (Yanagawa et al., Neoplasia 3:395-401 (2001)) according to description before this.Adjust the fluorescence intensity of the Cy5 (tumour) and the Cy3 (contrast) of each target spot point (target spot), make the average Cy3/Cy5 ratio of described 52 housekeeping genes equal 1.The data that are derived from low signal intensity are unreliable.Therefore, measure the cutoff value (cut-off value) of strength of signal on each slide glass.When the strength of signal of sending when Cy3 and Cy5 dyestuff all is lower than this cutoff value, such gene is got rid of outside further analyzing.
(5) according to gene-expression map 37 NSCLC are carried out cluster analysis (cluster analysis)
All use hierarchical clustering (hierachical clustering) method to analyze to gene and tumour.In order to obtain the reproducible cluster of described 37 samples, select 899 genes, these genes have obtained trust data in 95% experiment, and its standard deviation of expressing the ratio variation is greater than 1.0.Use the software (" Cluster " and " Tree View ") of M.Eisen establishment to analyze, this software can obtain (http://genome-www5.stanford.edu/Micro Array/SMD/restech.html) from the website.At application group set algorithm (clustering aligorithm) before, the fluorescence ratio of each spot is converted into logarithmic value, concentrates (median-centere) to get rid of deviation of experiment to the data of each sample to median then.
[embodiment 2] utilize clinical correlated expression pattern identified gene in the non-small cell lung cancer cell
Use is used two-dimentional hierarchical clustering Algorithm Analysis sample and intergenic similarity from the data of the expression map acquisition of all 37 NSCLC samples.When Cy3-or Cy5-fluorescence intensity are lower than described cutoff value, such gene is got rid of outside further analyzing, (yanagawa etal., Neoplasia 3:395-401 (2001)) selects at the gene that is tried to have obtained in the case confidence values more than 95% as previously mentioned.The gene that to observe (observed) standard deviation＜1.0 also forecloses.To doing further to analyze by this 899 genes holding back filter paper.
On sample axle (transverse axis), will be divided into 2 main groups (major group) according to their expression map from 39 duplicate samples (2 cases are made the reproducibility and the reliability of duplicate replicate(determination) with the confirmatory experiment method) of 37 cases.Dendrogram has shown the similarity between the individual patient chart expression patterns.Branch is short more, and similarity is just high more.2 the repetition cases (No. 6 and No. 12) that are labeled in independent experiment and hybridize are the most approaching in same group.According to described analysis, be that described gene hives off and is that adjacent several rows of gene, wherein every row's gene are that identical and described gene is put on the different position of slide glass (data not shown).In 37 cases, 22 gland cancer case branches have been gone into a main group, and 14 SCC case branches have been gone into another main group.Unique glandular scale cell carcinoma (No. 25) has divided to go into SCC group.Obviously, gland cancer it seems to have specific and different gene expression atlas with SCC, and this may disclose the molecular characterization of nosetiology difference.
In order to search for the gene of down-regulated expression among the NSCLC, to the expression in the NSCLC more than 70% reduced＜0.2 times or lower gene screen.Identified the gene of 806 down-regulated expressions in NSCLC, these genes may have the tumor suppression function thereby may can be used in the future gene therapy (referring to, table 1).Identified the gene of 582 rises altogether, its Cy5/Cy3 ratio in the NSCLC more than 50% greater than 5.0 (tables 2).In this table, expressing high 5 times gene in the NSCLC more than 70% is the potential diagnostic flag, and the gene of crossing expression that has 5 times in the case more than 50% is the potential target of medicine.As the target of medicine, the gene that data are present in the case of 33%-50% also is selected, and measured the high 5 times expression of in the NSCLC more than 90% demonstration 60 in gene (table 3).Further the standard of selecting is as follows: detectable tumor marker in (1) serum: only show the gene of expressing in people's testis, ovary and 4 parts of fetal tissues; (2) detectable tumor marker in the phlegm: in airway tissue (that is, lung, tracheae and sialisterium), do not have the gene of expressing; And the target of (3) treatment: in the most important internal organs of people such as liver and kidney, do not have the gene of expressing.Utilization contains the cDNA microarray of 23,040 people's genes, has obtained the healthy tissues distributed data of these genes from the expression map of 25 parts of adults and 4 parts of fetus people tissues.
[embodiment 3] are used to suppress the evaluation of molecular target of non-small cell lung cancer cell growth and qualitative
For the recruit's target that can regulate growth of cancer cells, propagation and survival to those is identified and qualitative, following application antisense S-oligonucleotide choice of technology target gene.
(1) evaluation of full length sequence
Use Marathon cDNA amplification kit (BD Biosciences Clontech, Palo Alto, CA, USA), according to the method that the manufacturer recommends,, the full length sequence of the higher gene of Cy5/Cy3 signal intensity rate is measured by 5 ' rapid amplifying of database screening and cDNA.With gene-specific reverse primers in the described test kit and AP1 primer, from the synthetic cDNA template of people's testis mRNA (BDBiosciences Clontech).(CA USA), illustrates the mensuration nucleotide sequence according to the manufacturer for Applied Biosystems, Foster City with ABI PRISM 3700 dna sequencing instrument.
(2) Northern engram analysis
The corresponding of every kind of gene experimentally studying at microarray will be selected to 32The PCR product of P mark, with the people many-organize trace (BD Biosciences Clontech) to hybridize.Carry out prehybridization, hybridization and washing according to the method that the manufacturer recommends.Under-80 ℃ with described trace on intensifying screen radioautograph 24-168 hour.
For tissue distribution and the size of measuring every kind of gene, end user cDNA carries out the people as probe and organizes northern engram analysis (Fig. 4) more, and the result who obtains from every kind of gene is as follows:
NSC 807: single (single) 4.4kb mRNA is found in placenta and testis;
NSC 810: single 3.1kb mRNA is found in testis;
NSC 811:2.4 and 2.7kb mRNA are found in placenta and tongue, and have detected weak expression in kidney, liver, suprarenal gland, bladder, brain (entirely), lymphoglandula, prostate gland, stomach, Tiroidina and tracheae;
NSC 822: single 1.3kb mRNA is found in the heart, liver and testis;
NSC 825: single 4.3kb mRNA is found in testis and spinal cord;
The weak expression of NSC 841:2.8kb transcript is found in the heart, suprarenal gland, brain (complete (whole)), lymphoglandula, spinal cord, stomach, Tiroidina, tongue and tracheae;
NSC 849: single 1.4kb mRNA is found in placenta, prostate gland and tracheae;
NSC 855:3.6kb mRNA is found in placenta, prostate gland and tracheae;
The weak expression of NSC 859:2.1kb transcript is found in skeletal muscle and lymphoglandula;
NSC 885: single 5.0kb mRNA is found in testis;
NSC 895: single 1.5kb mrna is found in placenta, stomach and tracheae;
NSC 903: single 2.7kb mRNA is found in testis, and has detected weak expression in thymus gland, small intestine, colon and marrow;
NSC 904: single 4.4kb mRNA is found in testis and skeletal muscle;
NSC 905: single 2.5kb mRNA is found in the heart, skeletal muscle, liver, stomach and tongue, and has detected weak expression in placenta and Tiroidina;
NSC 915: single 1.5kb mRNA is found in testis;
NSC 948: single 3.8kb mRNA is found in kidney, liver, placenta, stomach, Tiroidina, tongue and tracheae;
NSC 956: single 2.1kb mRNA is found in the heart, skeletal muscle, testis, stomach, Tiroidina and suprarenal gland, and has detected weak expression in liver, pancreas, thymus gland, prostate gland and spinal cord;
NSC 994: single 3.3kb mRNA is found in skeletal muscle and testis, and has detected weak expression in the heart, liver and pancreas;
NSC 1000: single 3.5kb mRNA is found in brain, pancreas, prostate gland and testis, and has detected weak expression in stomach, spinal cord and suprarenal gland;
NSC 1066: single 3.6kb mRNA is found in skeletal muscle and testis;
NSC 1075: single 1.9kb mRNA is found in testis;
NSC 1107: single 2.2kb mRNA is found in testis;
The transcript of NSC 1131:1.6kb and 1.4kb is found in testis;
NSC 1141: single 2.9kb mRNA is found in placenta, and has detected weak expression in skeletal muscle and testis;
NSC1164: single 5.2kb mRNA is found in brain and suprarenal gland;
NSC 1183: single 2.0kb mRNA is found in the skeletal muscle and the heart;
NSC 1201: the 7.8kb transcript of the weak expression of finding in the heart, skeletal muscle, spinal cord, prostate gland, testis, Tiroidina, spleen, lymphoglandula, tracheae and the suprarenal gland;
NSC 1240: the 5.7kb transcript of the weak expression of finding in stomach, spinal cord and the lymphoglandula;
NSC 1246: single 1.4kb mRNA is found in testis;
NSC 1254: single 3.0kb mRNA is found in testis;
The weak expression of NSC 1265:3.0kb transcript is found in the stomach;
NSC 1277: single 1.8kb mRNA is found in testis;
NSC 1295: single 3.5kb mRNA is found in white corpuscle, lymphoglandula and marrow;
NSC 1306: single 7.4kb mRNA is found in the heart and skeletal muscle;
NSC 1343: single 4.7kb mRNA is found in placenta and skeletal muscle;
NSC 1362: single 3.6kb mRNA is found in brain and full brain;
NSC 1389: single 0.9kb mRNA is found in tongue;
NSC 1399: single 0.9kb mRNA is found in placenta;
NSC 1406: single 2.4kb mRNA is found in the heart, skeletal muscle and prostate gland;
NSC 1413: single 4.0kb mRNA is found in liver and prostate gland;
NSC 1420: single 2.8kb mRNA is found in testis.
(2) semiquantitative RT-PCR analyzes
The raise situation of 5 times or more times of mRNA expression level among the NSCLC more than 50% is verified (Akashi et al.Int J Cancer 88:873-80 (2000)) as mentioned above with semiquantitative RT-PCR.(MD USA), according to the method that the manufacturer recommends, extracts total RNA from cultured cells and clinical tissue for Life Technologies, Inc.Gaithersburg to use Trizol reagent.(Roche Diagnostics, Basel Switzerland) handle the DNase I that uses of the RNA that extracts, and (Life Technologies, Inc.) reverse transcription is the cDNA of strand with Superscript II ThermoScript II to use oligomerization (dT) 12-18 primer then., each strand cDNA suitably diluted be used for subsequent P CR amplification as quantitatively contrast by monitoring beta-actin (ACTB) or beta-2-microglobulin (microglobulin) gene (B2M).Institute responds and all carries out on GeneAmp PCR system 9700 (Applied Biosystems), uses following scheme: 94 ℃ of initial sex change 2min; Then, 58-62 ℃, 30 seconds and 72 ℃, 45 seconds, carry out 18 (for ACTB or B2M) or 25-30 circulation (for every kind of gene of the present invention) with 94 ℃, 30 seconds.Described primer sequence sees Table 4.
Table 4: the primer sequence that is used for sxemiquantitative RT-PCR experiment
Figure A20081018670601421
Figure A20081018670601431
Figure A20081018670601441
Figure A20081018670601451
Figure A20081018670601471
Figure A20081018670601481
Figure A20081018670601491
Figure A20081018670601501
Figure A20081018670601511
Figure A20081018670601521
Figure A20081018670601531
Figure A20081018670601541
Figure A20081018670601551
Figure A20081018670601561
Each expression of gene (Fig. 2) is verified with semiquantitative RT-PCR from the cancerous tissue that patients with lung cancer obtains.The result who obtains is as follows:
6 in NSC 807:9 NSCLC case the NSC807 up-regulated occurred;
6 in NSC 810:10 NSCLC case the NSC810 up-regulated occurred;
The NSC811 up-regulated has all appearred in NSC 811:9 NSCLC case;
The NSC812 up-regulated has all appearred in NSC 812:15 NSCLC case;
The NSC816 up-regulated has all appearred in NSC 816:8 NSCLC case;
8 in NSC 820:9 NSCLC case NSC 820 up-regulateds occurred;
3 in NSC 822:10 NSCLC case the NSC822 up-regulated occurred;
The NSC824 up-regulated has all appearred in NSC 824:9 NSCLC case;
The NSC825 up-regulated has all appearred in NSC 825:12 NSCLC case;
7 in NSC 830:10 NSCLC case NSC 830 up-regulateds occurred;
7 in NSC 837:9 NSCLC case NSC 837 up-regulateds occurred;
9 in NSC 840:10 NSCLC case NSC 840 up-regulateds occurred;
9 in NSC 841:11 NSCLC case NSC 841 up-regulateds occurred;
7 in NSC 842:8 NSCLC case NSC 842 up-regulateds occurred;
9 in NSC 846:10 NSCLC case NSC 846 up-regulateds occurred;
7 in NSC 849:10 NSCLC case NSC 849 up-regulateds occurred;
The NSC850 up-regulated has all appearred in NSC 850:7 NSCLC case;
8 in NSC 853:10 NSCLC case NSC 853 up-regulateds occurred;
The NSC854 up-regulated has all appearred in NSC 854:7 NSCLC case;
10 in NSC 855:11 NSCLC case NSC 855 up-regulateds occurred;
The NSC857 up-regulated has all appearred in NSC 857:8 NSCLC case;
The NSC859 up-regulated has all appearred in NSC 859:8 NSCLC case;
5 in NSC 861:7 NSCLC case NSC 861 up-regulateds occurred;
NSC 864: sxemiquantitative RT-PCR has confirmed that NSC 864 up-regulateds have all appearred in 10 NSCLC cases;
10 in NSC 870:11 NSCLC case NSC 870 up-regulateds occurred;
12 in NSC 871:13 NSCLC case NSC 871 up-regulateds occurred;
9 in NSC 872:12 NSCLC case NSC 872 up-regulateds occurred;
NSC 881 up-regulateds have all appearred in NSC 881:10 NSCLC case;
7 in NSC 882:10 NSCLC case NSC 882 up-regulateds occurred;
The NSC884 up-regulated has all appearred in NSC 884:9 NSCLC case;
The NSC885 up-regulated has all appearred in NSC 885:8 NSCLC case;
7 in NSC 889:8 NSCLC case NSC 889 up-regulateds occurred;
7 in NSC 893:9 NSCLC case NSC 893 up-regulateds occurred;
5 in NSC 895:6 NSCLC case the NSC895 up-regulated occurred;
5 in NSC 898:6 NSCLC case the NSC898 up-regulated occurred;
The NSC901 up-regulated has all appearred in NSC 901:14 NSCLC case;
7 in NSC 902:8 NSCLC case NSC 902 up-regulateds occurred;
9 in NSC 903:10 NSCLC case NSC 903 up-regulateds occurred;
7 in NSC 904:10 NSCLC case NSC 904 up-regulateds occurred;
The NSC905 up-regulated has all appearred in NSC 905:13 NSCLC case;
9 in NSC 909:13 NSCLC case NSC 909 up-regulateds occurred;
The NSC912 up-regulated has all appearred in NSC 912:7 NSCLC case;
The NSC915 up-regulated has all appearred in NSC 915:9 NSCLC case;
The NSC917 up-regulated has all appearred in NSC 917:9 NSCLC case;
8 in NSC 920:10 NSCLC case NSC 920 up-regulateds occurred;
The NSC921 up-regulated has all appearred in NSC 921:8 NSCLC case;
The NSC924 up-regulated has all appearred in NSC 924:8 NSCLC case;
10 in NSC 929:12 NSCLC case NSC 929 up-regulateds occurred;
9 in NSC 930:10 NSCLC case NSC 930 up-regulateds occurred;
9 in NSC 933:10 NSCLC case NSC 933 up-regulateds occurred;
7 in NSC 934:8 NSCLC case NSC 934 up-regulateds occurred;
The NSC936 up-regulated has all appearred in NSC 936:8 NSCLC case;
9 in NSC 938:10 NSCLC case NSC 938 up-regulateds occurred;
2 in NSC 940:10 NSCLC case NSC 940 up-regulateds occurred;
NSC 944 up-regulateds have all appearred in NSC 944:10 NSCLC case;
9 in NSC 947:10 NSCLC case NSC 947 up-regulateds occurred;
8 in NSC 948:10 NSCLC case NSC 948 up-regulateds occurred;
The NSC956 up-regulated has all appearred in NSC 956:8 NSCLC case;
7 in NSC 957:8 NSCLC case NSC 957 up-regulateds occurred;
NSC 958 up-regulateds have all appearred in NSC 958:10 NSCLC case;
NSC 963 up-regulateds have all appearred in NSC 963:10 NSCLC case;
The NSC964 up-regulated has all appearred in NSC 964:8 NSCLC case;
10 in NSC 965:11 NSCLC case NSC 965 up-regulateds occurred;
3 in NSC 966:8 NSCLC case NSC 966 up-regulateds occurred;
7 in NSC 970:12 NSCLC case NSC 970 up-regulateds occurred;
9 in NSC 972:10 NSCLC case NSC 972 up-regulateds occurred;
3 in NSC 973:9 NSCLC case NSC 973 up-regulateds occurred;
9 in NSC 974:10 NSCLC case NSC 974 up-regulateds occurred;
NSC 975 up-regulateds have appearred in NSC 975:12 NSCL case;
7 in NSC 980:8 NSCLC case NSC 980 up-regulateds occurred;
8 in NSC 984:9 NSCLC case NSC 984 up-regulateds occurred;
The NSC989 up-regulated has all appearred in NSC 989:9 NSCLC case;
4 in NSC 990:8 NSCLC case NSC 990 up-regulateds occurred;
3 in NSC 991:10 NSCLC case NSC 991 up-regulateds occurred;
The NSC994 up-regulated has all appearred in NSC 994:8 NSCLC case;
12 in NSC 1000:13 NSCLC case the NSC1000 up-regulated occurred
The NSC1002 up-regulated has all appearred in NSC 1002:8 NSCLC case;
NSC 1003 up-regulateds have all appearred in NSC 1003:10 NSCLC case;
NSC 1012 up-regulateds have appearred in NSC 1012:8 NSCLC case;
NSC 1015 up-regulateds have appearred in NSC 1015:10 NSCLC case;
8 in NSC 1016:9 NSCLC case NSC 1016 up-regulateds occurred;
3 in NSC 1018:6 NSCLC case NSC 1018 up-regulateds occurred;
7 in NSC 1023:12 NSCLC case NSC 1023 up-regulateds occurred;
7 in NSC 1026:9 NSCLC case NSC 1026 up-regulateds occurred;
5 in NSC 1027:8 NSCLC case NSC 1027 up-regulateds occurred;
5 in NSC 1030:6 NSCLC case NSC 1030 up-regulateds occurred;
5 in NSC 1034:8 NSCLC case NSC 1034 up-regulateds occurred;
The NSC1037 up-regulated has all appearred in NSC 1037:9 NSCLC case;
6 in NSC 1038:7 NSCLC case NSC 1038 up-regulateds occurred;
4 in NSC 1047:6 NSCLC case NSC 1047 up-regulateds occurred;
NSC 1049:6 NSCLC case all detected NSC 1049 up-regulateds;
The NSC1052 up-regulated has all appearred in NSC 1052:8 NSCLC case;
The NSC1057 up-regulated has all appearred in NSC 1057:8 NSCLC case;
8 in NSC 1058:10 NSCLC case NSC 1058 up-regulateds occurred;
8 in NSC 1059:9 NSCLC case NSC 1059 up-regulateds occurred;
The NSC1064 up-regulated has all appearred in NSC 1064:13 NSCLC case;
8 in NSC 1066:10 NSCLC case NSC 1066 up-regulateds occurred;
NSC 1067 up-regulateds have all appearred in NSC 1067:10 NSCLC case;
NSC 1071 up-regulateds have all appearred in NSC 1071:10 NSCLC case;
7 in NSC 1072:10 NSCLC case NSC 1072 up-regulateds occurred;
The NSC1075 up-regulated has all appearred in NSC 1075:9 NSCLC case;
8 in NSC 1077:11 NSCLC case NSC 1077 up-regulateds occurred;
8 in NSC 1078:9 NSCLC case NSC 1078 up-regulateds occurred;
10 in NSC 1086:11 NSCLC case NSC 1086 up-regulateds occurred;
6 in NSC 1089:9 NSCLC case NSC 1089 up-regulateds occurred;
3 in NSC 1090:7 NSCLC case NSC 1090 up-regulateds occurred;
7 in NSC 1103:8 NSCLC case NSC 1103 up-regulateds occurred;
8 in NSC 1107:9 NSCLC case NSC 1107 up-regulateds occurred;
8 in NSC 1109:9 NSCLC case NSC 1109 up-regulateds occurred;
10 in NSC 1113:11 NSCLC case NSC 1113 up-regulateds occurred;
8 in NSC 1116:9 NSCLC case NSC 1116 up-regulateds occurred;
NSC 1125 up-regulateds have appearred in NSC 1125:10 NSCLC case;
2 in NSC 1131:6 NSCLC case have detected NSC 1131 up-regulateds;
NSC 1133 up-regulateds have all appearred in NSC 1133:10 NSCLC case;
8 in NSC 1136:9 NSCLC case NSC 1136 up-regulateds occurred
6 in NSC 1141:10 NSCLC case the NSC1141 up-regulated occurred;
9 in NSC 1142:11 NSCLC case have detected NSC 1142 up-regulateds;
1 in NSC 1157:11 NSCLC case NSC 1157 up-regulateds occurred;
9 in NSC1162:10 NSCLC case the NSC1162 up-regulated occurred;
The NSC1164 up-regulated has all appearred in NSC 1164:7 NSCLC case;
8 in NSC 1167:9 NSCLC case NSC 1167 up-regulateds occurred
3 in NSC 1169:7 NSCLC case NSC 1169 up-regulateds occurred;
5 in NSC 1173:7 NSCLC case NSC 1173 up-regulateds occurred;
8 in NSC 1176:9 NSCLC case NSC 1176 up-regulateds occurred;
NSC 1183:10 NSCLC case all detected NSC 1183 up-regulateds;
8 in NSC 1184:9 NSCLC case NSC 1184 up-regulateds occurred;
5 in NSC 1185:6 NSCLC case have detected NSC 1185 up-regulateds;
7 in NSC 1191:8 NSCLC case NSC 1191 up-regulateds occurred;
5 in NSC 1195:9 NSCLC case NSC 1195 up-regulateds occurred;
The NSC1196 up-regulated has all appearred in NSC 1196:6 NSCLC case;
The NSC1201 up-regulated has all appearred in NSC 1201:9 NSCLC case;
7 in NSC 1205:9 NSCLC case NSC 1205 up-regulateds occurred;
8 in NSC 1207:10 NSCLC case NSC 1207 up-regulateds occurred;
9 in NSC 1210:10 NSCLC case NSC 1210 up-regulateds occurred;
7 in NSC 1214:9 NSCLC case NSC 1214 up-regulateds occurred;
9 in NSC 1234:10 NSCLC case NSC 1234 up-regulateds occurred;
6 in NSC 1236:8 NSCLC case NSC 1236 up-regulateds occurred;
5 in NSC 1237:6 NSCLC case the NSC1237 up-regulated occurred;
6 in NSC 1238:7 NSCLC case the NSC1238 up-regulated occurred;
The NSC1240 up-regulated has all appearred in NSC 1240:7 NSCLC case;
4 in NSC 1242:7 NSCLC case the NSC1242 up-regulated occurred;
6 in NSC 1246:10 NSCLC case the NSC1246 up-regulated occurred;
5 in NSC 1247:8 NSCLC case NSC 1247 up-regulateds occurred;
NSC 1250 up-regulateds have appearred in NSC 1250:8 NSCLC case;
NSC 1254 up-regulateds have appearred in NSC 1254:10 NSCLC case;
4 in NSC 1265:5 NSCLC case have detected NSC 1265 up-regulateds;
5 in NSC 1273:6 NSCLC case have detected NSC 1273 up-regulateds;
NSC 1277 up-regulateds have all appearred in NSC 1277:10 NSCLC case;
The NSC1279 up-regulated has all appearred in NSC 1279:7 NSCLC case;
6 in NSC 1288:9 NSCLC case NSC 1288 up-regulateds occurred;
6 in NSC 1289:9 NSCLC case NSC 1289 up-regulateds occurred;
NSC 1290 up-regulateds have all appearred in NSC 1290:10 NSCLC case;
The NSC1292 up-regulated has all appearred in NSC 1292:8 NSCLC case;
4 in NSC 1293:6 NSCLC case have detected NSC 1293 up-regulateds;
7 in NSC 1294:8 NSCLC case NSC 1294 up-regulateds occurred;
5 in NSC 1295:7 NSCLC case NSC 1295 up-regulateds occurred;
5 in NSC 1299:6 NSCLC case have detected NSC 1299 up-regulateds;
The NSC1302 up-regulated has all appearred in NSC 1302:7 NSCLC case;
5 in NSC 1306:6 NSCLC case have detected NSC 1306 up-regulateds;
7 in NSC 1309:8 NSCLC case NSC 1309 up-regulateds occurred;
9 in NSC 1310:10 NSCLC case NSC 1310 up-regulateds occurred;
6 in NSC 1315:9 NSCLC case NSC 1315 up-regulateds occurred;
5 in NSC 1320:9 NSCLC case NSC 1320 up-regulateds occurred;
NSC 1323 up-regulateds have all appearred in NSC 1323:10 NSCLC case;
2 in NSC 1325:9 NSCLC case NSC 1325 up-regulateds occurred;
The NSC1328 up-regulated has all appearred in NSC 1328:9 NSCLC case;
The NSC1337 up-regulated has all appearred in NSC 1337:9 NSCLC case;
3 in NSC 1345:8 NSCLC case NSC 1345 up-regulateds occurred;
6 in NSC 1350:8 NSCLC case NSC 1350 up-regulateds occurred;
3 in NSC 1353:10 NSCLC case the NSC1353 up-regulated occurred;
6 in NSC 1362:7 NSCLC case the NSC1362 up-regulated occurred;
NSC 1371 up-regulateds have all appearred in NSC 1371:10 NSCLC case;
The NSC1375 up-regulated has all appearred in NSC 1375:8 NSCLC case;
5 in NSC 1377:8 NSCLC case NSC 1377 up-regulateds occurred;
8 in NSC 1378:9 NSCLC case NSC 1378 up-regulateds occurred;
8 in NSC 1384:11 NSCLC case NSC 1384 up-regulateds occurred;
8 in NSC 1389:9 NSCLC case NSC 1389 up-regulateds occurred;
8 in NSC 1390:10 NSCLC case NSC 1390 up-regulateds occurred;
The NSC1391 up-regulated has all appearred in NSC 1391:8 NSCLC case;
6 in NSC 1394:10 NSCLC case the NSC1394 up-regulated occurred;
4 in NSC 1395:7 NSCLC case the NSC1395 up-regulated occurred;
8 in NSC 1398:9 NSCLC case the NSC1398 up-regulated occurred;
4 in NSC 1399:10 NSCLC case the NSC1399 up-regulated occurred;
6 in NSC 1403:8 NSCLC case NSC 1403 up-regulateds occurred;
NSC 1406 up-regulateds have all appearred in NSC 1406:10 NSCLC case;
NSC 1407 up-regulateds have all appearred in NSC 1407:10 NSCLC case;
5 in NSC 1410:10 NSCLC case the NSC1410 up-regulated occurred;
6 in NSC 1412:9 NSCLC case the NSC1412 up-regulated occurred;
3 in NSC 1417:7 NSCLC case the NSC1417 up-regulated occurred;
NSC 1420:7 NSCLC case all detected the NSC1420 up-regulated;
4 in NSC 1422:10 NSCLC case the NSC1422 up-regulated occurred;
5 in NSC 1424:6 NSCLC case the NSC1424 up-regulated occurred;
4 in NSC 1435:8 NSCLC case the NSC1435 up-regulated occurred;
The NSC1436 up-regulated has all appearred in NSC 1436:7 NSCLC case;
The NSC1439 up-regulated has all appearred in NSC 1439:8 NSCLC case;
8 in NSC 1440:9 NSCLC case the NSC1440 up-regulated occurred;
9 in NSC 1441:11 NSCLC case the NSC1441 up-regulated occurred;
4 in NSC 1444:6 NSCLC case the NSC1444 up-regulated occurred;
6 in NSC 1445:7 NSCLC case the NSC1445 up-regulated occurred;
The NSC1447 up-regulated has all appearred in NSC 1447:7 NSCLC case.
(3) antisense S-oligonucleotide is measured
The corresponding 3-5 for preparing every kind of gene is to oppositely (contrast) and antisense S-oligonucleotide are right.Four kinds of NSCLC clone A549, NCI-H226, NCI-H522 and/or LC319 are plated on 6-hole flat board or 10cm culture dish, use lipofectin reagent (Life Technologies, Inc, Inc.), use and the synthetic accordingly above-mentioned clone of S-oligonucleotide transfection of every kind of gene, the clone after the transfection was kept in the substratum that contains 10% foetal calf serum 2 days.Then, with the fixing above-mentioned cell of 100% methyl alcohol, and use the Giemsa solution-dyed.Antisense S-oligonucleotide at 26 genes suppresses focus formation compared with the control.Therefore, cell was grown, is bred and/or the survival reduction after the inhibition of these genes showed as transfection.Effectively the sequence of (effective) antisense S-oligonucleotide and contrast reverse oligonucleotide is shown in table 5.Carry out in triplicate MTT experiment, adopted methods known in the art (Akashi et al. (2000) Int.J.Cancer 88:873-80).Method and result that each gene pairs is answered are as follows:
NSC 810:TTKEffective antisense S-oligonucleotide (SEQ IDNO:423) and reverse S-oligonucleotide (contrast) (SEQ ID NO:424) have been synthesized corresponding to TTK.Described S-oligonucleotide all is transfected into NSCLC clone A549 and the LC319 that expresses TTK with highest level.After the transfection two days, by the MTT experiment as seen, described antisense-S oligonucleotide obviously suppressed cell proliferation (Fig. 2) compared with the control.This shows, caused cell growth, propagation and/or survival to reduce the inhibition of TTK.This result has also obtained focus and has formed the confirmation (data do not provide) of testing (focus formation) (dyeing with Giemsa).
NSC 811:SDC1Synthesized corresponding to the effective antisense S-of 3 couple of SDC1 oligonucleotide (AS1 (SEQ ID NO:425), AS2 (SEQ ID NO:427) and AS4 (SEQ ID NO:429)) and reverse S-oligonucleotide (contrast) (R1 (SEQ ID NO:426), R2 (SEQ ID NO:428) and R4 (SEQNO:430)), they have been each separately transfected into the NSCLC clone A549 that expresses SDC1 with highest level.After the transfection two days, see obviously that by the MTT experiment these antisenses-S oligonucleotide has obviously suppressed cell proliferation (Fig. 2) compared with the control.Therefore as seen, the inhibition of SDC1 has caused weakening of cell growth, propagation and/or survival.This result has also obtained focus and has formed the confirmation (data do not provide) of testing (dyeing with Giemsa).
NSC 812:NMBSynthesized corresponding to the effective antisense S-of 2 couple of NMB oligonucleotide (AS1 (SEQ ID NO:431) and AS2 (SEQ ID NO:433)) and reverse S-oligonucleotide (contrast) (R1 (SEQ ID NO:432) and R2 (SEQ ID NO:434)).Every kind of S-oligonucleotide all is transfected into NSCLC clone A549, NCI-H226 and the NCI-H522 that expresses NMB with highest level.After the transfection two days, by the MTT experiment as seen, these antisenses-S oligonucleotide obviously suppressed cell proliferation (Fig. 2) compared with the control.Therefore as seen, the inhibition of NMB has caused the reduction of cell growth, propagation and/or survival.These results have also obtained focus and have formed the confirmation (data do not provide) of testing (dyeing with Giemsa).
NSC 816:PIR51Effective antisense S-oligonucleotide AS1 (SEQ ID NO:435) and reverse S-oligonucleotide (contrast) R1 (SEQ ID NO:436) have been synthesized corresponding to RIR51.Described S-oligonucleotide all is transfected into NSCLC clone A549, NCI-H226 and the NCI-H522 that expresses NMB with highest level.After the transfection two days, form experiment (dyeing) as seen by focus with Giemsa, these antisenses-S oligonucleotide has obviously suppressed cell proliferation (data do not provide) compared with the control.This shows, the inhibition of PIR51 has been caused the reduction of cell growth, propagation and/or survival.
NSC 825:ANLN: synthesized corresponding to the effective antisense S-of 3 couple of ANLN oligonucleotide (AS1 (SEQ ID NO:437), AS3 (SEQ ID NO:439) and AS5 (SEQ ID NO:441)) and reverse S-oligonucleotide (contrast) (R1 (SEQ ID NO:438), R3 (SEQ ID NO:440) and R5 (SEQID NO:442)).Every kind of S-oligonucleotide all is transfected into NSCLC clone A549, NCI-H226 and the NCI-H522 that expresses ANLN with highest level.After the transfection two days, by the MTT experiment as seen, these antisenses-S oligonucleotide obviously suppressed cell proliferation (Fig. 2) compared with the control.This shows, the inhibition of ANLN has been caused the reduction of cell growth, propagation and/or survival.
NSC 841:URLC2Synthesized corresponding to the effective antisense S-of 2 couple of URLC2 oligonucleotide (AS4 (SEQ ID NO:443) and AS5 (SEQ ID NO:445)) and reverse S-oligonucleotide (contrast) (R4 (SEQ ID NO:444) and R5 (SEQ ID NO:446)).Every kind of S-oligonucleotide all is transfected into NSCLC clone A549, NCI-H226 and the NCI-H522 that expresses URLC2 with highest level.After the transfection two days, by the MTT experiment as seen, these antisenses-S oligonucleotide obviously suppressed cell proliferation (Fig. 2) compared with the control.This shows, the inhibition of URLC2 has been caused the reduction of cell growth, propagation and/or survival.This result has also obtained focus and has formed the confirmation (data do not provide) of testing (dyeing with Giemsa).
NSC 857:TIGD5Synthesized corresponding to the effective antisense S-of 2 couple of TIGD5 oligonucleotide (AS3 (SEQ ID NO:447) and AS4 (SEQ ID NO:449)) and reverse S-oligonucleotide (contrast) (R3 (SEQ ID NO:448) and R4 (SEQ ID NO:450)).Every kind of S-oligonucleotide all is transfected into NSCLC clone A549, NCI-H226 and the NCI-H522 that expresses TIGD5 with highest level.After the transfection two days, by the MTT experiment as seen, these antisenses-S oligonucleotide obviously suppressed cell proliferation (Fig. 2) compared with the control.This shows, the inhibition of TIGD5 has been caused the reduction of cell growth, propagation and/or survival.This result has also obtained focus and has formed the confirmation (data do not provide) of testing (dyeing with Giemsa).
NSC 859:URLC3Synthesized corresponding to the effective antisense S-of 3 couple of URLC3 oligonucleotide (AS2 (SEQ ID NO:451), AS3 (SEQ ID NO:453) and AS5 (SEQ ID NO:455)) and reverse S-oligonucleotide (contrast) (R2 (SEQ ID NO:452) and R5 (SEQ ID NO:456)).Every kind of S-oligonucleotide all is transfected into NSCLC clone A549, NCI-H226 and the NCI-H522 that expresses URLC3 with highest level.After the transfection two days, by the MTT experiment as seen, these antisenses-S oligonucleotide obviously suppressed cell proliferation (Fig. 2) compared with the control.This shows, the inhibition of URLC3 has been caused the reduction of cell growth, propagation and/or survival.This result has also obtained focus and has formed the confirmation (data do not provide) of testing (dyeing with Giemsa).
NSC 885:BAG5Synthesized corresponding to the effective antisense S-of 2 couple of BAG5 oligonucleotide (AS1 (SEQ ID NO:457) and AS2 (SEQ ID NO:459)) and reverse S-oligonucleotide (R1 (SEQID 458), R3 (SEQ ID NO:448) and R2 (SEQ ID 460)).Every kind of S-oligonucleotide all is transfected into NSCLC clone A549, NCI-H226 and the NCI-H522 that expresses BAG5 with highest level.After the transfection two days, form experiment (dyeing) as seen by focus with Giemsa, these antisenses-S oligonucleotide has obviously suppressed cell proliferation (data do not provide) compared with the control.This shows, the inhibition of BAG5 has been caused the reduction of cell growth, propagation and/or survival.
NSC 893:MPHOSPH1Synthesized corresponding to the effective antisense S-of 2 couple of MPHOSPH1 oligonucleotide (AS1 (SEQ ID NO:461) and AS2 (SEQ ID NO:463)) and reverse S-oligonucleotide (R1 (SEQ ID NO:462) and R2 (SEQ ID NO:464)).Every kind of S-oligonucleotide all is transfected into NSCLC clone A549, NCI-H226 and the NCI-H522 that expresses MPHOSPH1 with highest level.After the transfection two days, by the MTT experiment as seen, these antisenses-S oligonucleotide obviously suppressed cell proliferation (Fig. 2) compared with the control.This shows, the inhibition of MPHOSPH1 has been caused the reduction of cell growth, propagation and/or survival.This result has also obtained focus and has formed the confirmation (data do not provide) of testing (dyeing with Giemsa).
NSC 905:URLC1Synthesized corresponding to the effective antisense S-of 3 couple of URLC1 oligonucleotide (AS2 (SEQ ID NO:465), AS3 (SEQ ID NO:467) and AS5 (SEQ ID NO:469)) and reverse S-oligonucleotide (contrast) (R2 (SEQ ID NO:466), R3 (SEQ ID NO:468) and R5 (SEQ ID NO:470)).Every kind of S-oligonucleotide all is transfected into NSCLC clone A549, NCI-H226 and the NCI-H522 that expresses URLC1 with highest level.After the transfection two days, by the MTT experiment as seen, these antisenses-S oligonucleotide obviously suppressed cell proliferation (Fig. 2) compared with the control.This shows, the inhibition of URLC1 has been caused the reduction of cell growth, propagation and/or survival.This result has also obtained focus and has formed the confirmation (data do not provide) of testing (dyeing with Giemsa).
NSC 909:FLJ10468A pair of effective antisense S-oligonucleotide AS1 (SEQ ID NO:471) and reverse S-oligonucleotide (contrast) R1 (SEQ ID NO:472) have been synthesized corresponding to FLJ10468.Every kind of S-oligonucleotide all is transfected into NSCLC clone A549, NCI-H226 and the NCI-H522 that expresses FLJ10468 with highest level.After the transfection two days, form experiment (dyeing) as seen by focus with Giemsa, these antisenses-S oligonucleotide has obviously suppressed cell proliferation (data do not provide) compared with the control.This shows, the inhibition of FLJ10468 has been caused the reduction of cell growth, propagation and/or survival.
NSC 920:CHAF1ASynthesized corresponding to the effective antisense S-of 2 couple of CHAF1A oligonucleotide (AS1 (SEQ ID NO:473) and AS4 (SEQ ID NO:459)) and reverse S-oligonucleotide (R1 (SEQ ID NO:474) and R4 (SEQ ID NO:476)).Every kind of S-oligonucleotide all is transfected into NSCLC clone A549, NCI-H226 and the NCI-H522 that expresses CHAF1A with highest level.After the transfection two days, form experiment (dyeing) as seen by focus with Giemsa, these antisenses-S oligonucleotide has obviously suppressed cell proliferation (data do not provide) compared with the control.This shows, the inhibition of CHAF1A has been caused the reduction of cell growth, propagation and/or survival.
NSC 947:PKP3Synthesized corresponding to the effective antisense S-of 4 couple of PKP3 oligonucleotide (AS1 (SEQ ID NO:477), AS2 (SEQ ID NO:479), AS3 (SEQ ID NO:481) and AS4 (SEQ ID NO:483)) and reverse S-oligonucleotide (contrast) ((R1 (SEQ ID NO:478), R2 (SEQ ID NO:480), R3 (SEQ ID 482) and R4 (SEQ ID NO:484)).Every kind of S-oligonucleotide all is transfected into NSCLC clone A549 and the LC319 that expresses PKP3 with highest level.After the transfection two days, by the MTT experiment as seen, these antisenses-S oligonucleotide obviously suppressed cell proliferation (Fig. 2) compared with the control.This shows, the inhibition of PKP3 has been caused the reduction of cell growth, propagation and/or survival.This result has also obtained focus and has formed the confirmation (data do not provide) of testing (dyeing with Giemsa).
NSC 956:SIAHBP1Synthesized corresponding to the effective antisense S-of 2 couple of SIAHBP1 oligonucleotide (AS1 (SEQ ID NO:485) and AS2 (SEQ ID NO:487)) and reverse S-oligonucleotide (contrast) (R1 (SEQ ID NO:486) and R2 (SEQ ID NO:488)).Every kind of S-oligonucleotide all is transfected into NSCLC clone A549, NCI-H226 and the NCI-H522 that expresses SIAHBP1 with highest level.After the transfection two days, by the MTT experiment as seen, these antisenses-S oligonucleotide obviously suppressed cell proliferation (Fig. 2) compared with the control, and this shows the growth that the inhibition of SIAHBP1 has been reduced cell, propagation and/or survive.This result has also obtained focus and has formed the confirmation (data do not provide) of testing (dyeing with Giemsa).
NSC 994:DKFZP434E2318Synthesized corresponding to the effective antisense S-of 4 couple of DKFZP434E2318 oligonucleotide ((AS1 (SEQ ID NO:489), AS3 (SEQ ID NO:491), AS4 (SEQ ID NO:493) and AS5 (SEQ ID NO:495)) and reverse S-oligonucleotide (contrast) ((R1 (SEQ ID NO:490), R3 (SEQ ID NO:492), R4 (SEQ ID 494) and R5 (SEQID NO:496)).Every kind of S-oligonucleotide all is transfected into NSCLC clone A549, NCI-H226 and the NCI-H522 that expresses DKFZP434E2318 with highest level.After the transfection two days, by the MTT experiment as seen, these antisenses-S oligonucleotide obviously suppressed cell proliferation (Fig. 2) compared with the control.This shows, the inhibition of DKFZP434E2318 has been reduced cell growth, propagation and/or survival.This result has also obtained focus and has formed the confirmation (data do not provide) of testing (dyeing with Giemsa).
NSC 1075:URLC4A pair of effective antisense S-oligonucleotide AS5 (SEQ ID NO:497) and reverse S-oligonucleotide (contrast) R1 (SEQ ID NO:498) have been synthesized corresponding to URLC4.Every kind of S-oligonucleotide all is transfected into NSCLC clone A549, NCI-H226 and the NCI-H522 that expresses URLC4 with highest level.After the transfection two days, see obviously that by the MTT experiment described antisense-S oligonucleotide has obviously suppressed cell proliferation (Fig. 2) compared with the control.This shows, the inhibition of URLC4 has been caused the reduction of cell growth, propagation and/or survival.Described result has also obtained focus and has formed the confirmation (data do not provide) of testing (dyeing with Giemsa).
NSC 1107:URLC8Synthesized corresponding to the effective antisense S-of 2 couple of URLC8 oligonucleotide (AS1 (SEQ ID NO:499) and AS4 (SEQ ID NO:501)) and reverse S-oligonucleotide (contrast) (R1 (SEQ ID NO:500) and R4 (SEQ ID NO:502)), every pair of S-oligonucleotide has been each separately transfected into the NSCLC clone A549 that expresses URLC8 with highest level.After the transfection two days, by the MTT experiment as seen, these antisenses-S oligonucleotide obviously suppressed cell proliferation (Fig. 2) compared with the control.This shows, the inhibition of URLC8 has been caused the reduction of cell growth, propagation and/or survival.This result has also obtained focus and has formed the confirmation (data do not provide) of testing (dyeing with Giemsa).
NSC 1113:URLC5Synthesized corresponding to the effective antisense S-of 2 couple of URLC5 oligonucleotide (AS1 (SEQ ID NO:503) and AS2 (SEQ ID NO:505)) and reverse S-oligonucleotide (R1 (SEQ ID NO:504) and R2 (SEQ ID NO:506)).Every kind of S-oligonucleotide all is transfected into NSCLC clone A549, NCI-H226 and the NCI-H522 that expresses URLC5 with highest level.After the transfection two days, form experiment (dyeing) as seen by focus with Giemsa, described antisense-S oligonucleotide obviously suppresses cell proliferation (data do not provide) compared with the control.This shows the growth that the inhibition of URLC5 has been reduced cell, propagation and/or survival.
NSC 1131:SYNJ2BPA pair of effective antisense S-oligonucleotide (AS1 (SEQ ID NO:507)) and reverse S-oligonucleotide (contrast) (R1 (SEQ IDNO:508)) have been synthesized corresponding to SYNJ2BP.Every kind of S-oligonucleotide all is transfected into clone A549 and the NCI-H226 that expresses SYNJ2BP with highest level.After the transfection two days, form experiment (dyeing) as seen by focus with Giemsa, described antisense-S oligonucleotide obviously suppresses cell proliferation (data do not provide) compared with the control.This shows the growth that the inhibition of SYNJ2BP has been reduced cell, propagation and/or survival.
NSC 1142:NAPGA pair of effective antisense S-oligonucleotide (AS1 (SEQ ID NO:509)) and reverse S-oligonucleotide (contrast) (R1 (SEQ ID NO:510)) have been synthesized corresponding to NAPG.Every kind of S-oligonucleotide all is transfected into NSCLC clone A549, NCI-H226 and the NCI-H522 that expresses NAPG with highest level.After the transfection two days, form experiment (dyeing) as seen by focus with Giemsa, described antisense-S oligonucleotide obviously suppresses cell proliferation (data do not provide) compared with the control.This shows the growth that the inhibition of NAPG has been reduced cell, propagation and/or survival.
NSC 1183:BYSLSynthesized corresponding to the effective antisense S-of 3 couple of BYSL oligonucleotide (AS1 (SEQ ID NO:511), AS2 (SEQ ID NO:513) and AS3 (SEQ ID NO:515)) and reverse S-oligonucleotide (contrast) (R1 (SEQ ID NO:512), R2 (SEQ ID NO:514) and R3 (SEQ ID NO:516)).Every kind of S-oligonucleotide all is transfected into NSCLC clone A549, NCI-H226 and the NCI-H522 that expresses BYSL with highest level.After the transfection two days, form experiment (dyeing) as seen by focus with Giemsa, these antisenses S-oligonucleotide has obviously suppressed cell proliferation (data do not provide) compared with the control, and this shows the growth that the inhibition of BYSL has been reduced cell, propagation and/or survive.
NSC 1185:URLC6Synthesized corresponding to the effective antisense S-of 2 couple of URLC6 oligonucleotide (AS4 (SEQ ID NO:517) and AS6 (SEQ ID NO:519)) and reverse S-oligonucleotide (contrast) (R4 (SEQ ID NO:518) and R6 (SEQ ID NO:520)).Every kind of S-oligonucleotide all is transfected into clone A549 and the NCI-H226 that expresses URLC6 with highest level.After the transfection two days, form experiment (dyeing) as seen by focus with Giemsa, these antisenses S-oligonucleotide has obviously suppressed cell proliferation (data do not provide) compared with the control, and this shows the growth that the inhibition of URLC6 has been reduced cell, propagation and/or survive.
NSC 1191:COX17Synthesized corresponding to the effective antisense S-of 3 couple of COX17 oligonucleotide (AS2 (SEQ ID NO:521), AS4 (SEQ ID NO:523) and AS5 (SEQ ID NO:525)) and reverse S-oligonucleotide (contrast) (R2 (SEQ ID NO:522), R4 (SEQ ID NO:524) and R5 (SEQ ID NO:526)).Every kind of S-oligonucleotide all is transfected into NSCLC clone A549, NCI-H226 and the NCI-H522 that expresses COX17 with highest level.After the transfection two days, by the MTT experiment as seen, these antisenses-S oligonucleotide obviously suppressed cell proliferation (Fig. 2) compared with the control, and this shows the growth that the inhibition of COX17 has been reduced cell, propagation and/or survive.This result has also obtained focus and has formed the confirmation (data do not provide) of testing (dyeing with Giemsa).
NSC 1273:FLJ32549Synthesized corresponding to the effective antisense S-of 2 couple of FLJ32549 oligonucleotide (AS1 (SEQ ID NO:527) and AS2 (SEQ ID NO:529)) and reverse S-oligonucleotide (R1 (SEQ ID NO:528) and R2 (SEQ ID NO:530)).Every kind of S-oligonucleotide all is transfected into NSCLC clone A549, NCI-H226 and the NCI-H522 that expresses FLJ32549 with highest level.After the transfection two days, form experiment (dyeing) as seen by focus with Giemsa, described antisense-S oligonucleotide obviously suppresses cell proliferation (data do not provide) compared with the control.This shows the growth that the inhibition of FLJ32549 has been reduced cell, propagation and/or survival.
NSC 1389:NMUA pair of effective antisense S-oligonucleotide AS (SEQ ID NO:531) and reverse S-oligonucleotide (contrast) R (SEQ ID NO:532) have been synthesized corresponding to NMU.Every kind of S-oligonucleotide all is transfected into NSCLC clone A549 and the LC319 that expresses NMU with highest level.After the transfection two days, by the MTT experiment as seen, described antisense-S oligonucleotide obviously suppressed cell proliferation (Fig. 2) compared with the control, and this shows the growth that the inhibition of NMU has been reduced cell, propagation and/or survive.This result has also obtained focus and has formed the confirmation (data do not provide) of testing (dyeing with Giemsa).
Table 5 is the right sequence of S-oligonucleotide effectively
Figure A20081018670601701
Figure A20081018670601711
Figure A20081018670601721
Figure A20081018670601731
(4) RNA interference experiment
PsiH1BX3.0 is a kind of RNAi system based on carrier, and it can instruct the synthetic of siRNA in the mammalian cell (siRNA), uses this system to suppress the expression of every kind of native gene in the NSCLC cell.Designed 5 kinds of carriers, it is used to instruct synthetic at 5 of every kind of gene target sequence different 19-base pair double chain nucleotides.(CA USA) is transfected into described carrier four kinds of NSCLC clones for Invitrogene, Carlsbad, and the efficient of described transfection has reached more than the 60-90% to use Lipofectamin 2000 reagent.Under the condition that has proper concn Geneticin (geneticin) to exist (G418) cultivated 5-9 days with cell.Measure cell number or cell viability with Giemsa dyeing and/or MTT experiment in triplicate mode.
(5) flow cytometry
Cell is carried out bed board with the concentration of 5 * 105 cells/100-millimeter ware, use the above-mentioned described cell of siRNA-expression vector transfection then.24-48 hour, trypsin digestion and cell was collected in the PBS kind, and fixed 30 minutes with 70% cold ethanol.(MO) after the processing, 50 μ g/ml propidium diiodides (propidium iodide) among the usefulness PBS are (Sigma-Aldrich) to cell dyeing for Sigma ChemicalCo.-Aldrich, St.Louis with 100 μ g/ml RNAe.Carry out flow cytometry on Becton Dickinson FACScan, (Topsham ME) analyzes for Verity Software House, Inc. to use ModFit software then.Measure and be in cell cycle G0/G1, S and the nuclear in G2/M stage and the shared per-cent of Asia-G1 group at least in 20,000 static (ungated) cell.Also utilize bonded Flow cytometry apoptosis based on annexin V.
(6)RNAi
For the new molecular target that can regulate growth of cancer cells, propagation and survival to those is identified and qualitative, use the psiH1BX3.0 carrier to implement the RNA perturbation technique, suppress the endogenous expression of the corresponding candidate gene selected by aforesaid method.The concrete grammar and the result of every kind of candidate gene are as follows:
NSC 807:KOC1: designed 3 kinds of effective carriers, described carrier can instruct the synthetic of the double-stranded sequence of the 19-base pair of gene shown in the target, and these carriers are transfected into NSCLC clone A549 and LC319.After the transfection 5 days, compared with the control, with RNAi carrier colony (colony) number (Fig. 3 A and 3B) that imported this gene inhibition, this showed the growth that the inhibition of KOC1 has been reduced cell, propagation and/or survival.This result has also obtained focus and has formed the confirmation (data do not provide) of testing (Giemsa dyeing).
NSC 810:TTK: designed 3 kinds of effective carriers, described carrier can instruct the synthetic of the double-stranded sequence of the 19-base pair of gene shown in the target, and these carriers are transfected into NSCLC clone A549 and LC319.After the transfection 5 days, find by the MTT experiment: compared with the control, the RNAi carrier is imported this gene obviously suppressed cell proliferation (Fig. 3 A and 3B), this showed the growth that the inhibition of TTK has been reduced cell, propagation and/or survival.This result has also obtained focus and has formed the confirmation (data do not provide) of testing (Giemsa dyeing).To make further check, per 24 or 48 days document images with microscopy (microscopy) with the LC319 cell of effective TTK RNAi transfection.The LC319 cell of described TTK RNAi transfection shows multinucleation (multi-nucleated) cell phenotype, complete necrocytosis (complete cell death) occurs, and show monokaryonization (mono-nucleated) cell phenotype (Fig. 3 C) with above-mentioned EGFP RNAi cells transfected.The Western engram analysis of-TTK monoclonal antibody anti-by using, detect natural TTK albumen in the LC319 cell expression and TTK RNAi to the inhibition (Fig. 3 E) of its expression.
NSC 825:ANLN: designed 2 kinds of effective carriers, described carrier can instruct the synthetic of the double-stranded sequence of the 19-base pair of gene shown in the target, and these carriers are transfected into NSCLC clone A549 and LC319.After the transfection 5 days, find by the MTT experiment: compared with the control, the RNAi carrier is imported this gene obviously suppressed cell proliferation (Fig. 3 A and 3B), this showed the growth that the inhibition of ANLN has been reduced cell, propagation and/or survival.This result has also obtained focus and has formed the confirmation (data do not provide) of testing (Giemsa dyeing).LC319 cell with any transfection among above-mentioned 2 kinds of effective ANLN RNAi has the multinucleation cell phenotype, and complete necrocytosis occurs, and shows monokaryon cell phenotype (Fig. 3 C) with the cell of EGFP RNAi transfection.By the cell cycle collection of illustrative plates of flow cytometry mensuration ANLN RNAi cells transfected, the cell cycle of display abnormality and polyploidy (＞4N dna content) (Fig. 3 D).
NSC 841:URLC2: having designed 2 kinds, to instruct with this gene be the double-stranded sequence synthetic of the 19-base pair effective carrier of target, and these carriers are transfected into NSCLC clone A549 and LC319.After the transfection 5 days, find by the MTT experiment: compared with the control, the importing of these RNAi carriers of this gene obviously suppressed cell proliferation (Fig. 3 A and 3B), and this shows the growth that the inhibition of URLC2 has been reduced cell, propagation and/or survival.This result has also obtained focus and has formed the confirmation (data do not provide) of testing (Giemsa dyeing).After described siRNA transfection, carried out flow cytometry in 24 hours.As a result, detect the Asia-G1 group who finds the LC319 cell and increased by 28%.Above-mentioned URLC2-siRNA has reduced apoptosis significantly, and the flow cytometry experiment that apoptosis also is combined into the basis in order to annexin V has been done and evaluated (Fig. 3 D).
NSC 903:URLC9: designed 2 kinds of effective carriers, described carrier can instruct the synthetic of the double-stranded sequence of the 19-base pair of gene shown in the target, and these carriers are transfected into NSCLC clone A549 and LC319.After the transfection 5 days, compared with the control, the importing of these RNAi carriers of this gene suppressed colony number (Fig. 3 A and 3B) significantly, and this shows the growth that the inhibition of URLC9 has been reduced cell, propagation and/or survival.This result has also obtained focus and has formed the confirmation (data do not provide) of testing (Giemsa dyeing).
NSC 956:SIAHBP1: designed 2 kinds of effective carriers, described carrier can instruct the synthetic of the double-stranded sequence of the 19-base pair of gene shown in the target, and these carriers are transfected into NSCLC clone A549 and LC319.After the transfection 5 days, find by the MTT experiment: compared with the control, the RNAi carrier is imported this gene obviously suppressed cell proliferation (Fig. 3 A and 3B), this showed the growth that the inhibition of SIAHBP1 has been reduced cell, propagation and/or survival.This result has also obtained focus and has formed the confirmation (data do not provide) of testing (Giemsa dyeing).
NSC 994:DKFZP434E2318: designed 2 kinds of effective carriers, described carrier can instruct the synthetic of the double-stranded sequence of the 19-base pair of gene shown in the target, and these carriers are transfected into NSCLC clone A549 and LC319.After the transfection 5 days, find by the MTT experiment: compared with the control, the importing of the RNAi carrier (No.1) of this gene obviously suppressed cell proliferation (Fig. 3 A and 3B), and this shows the growth that the inhibition of DKFZP434E2318 has been reduced cell, propagation and/or survival.This result has also obtained focus and has formed the confirmation (data do not provide) of testing (Giemsa dyeing).
NSC 1107:URLC8: designed 5 kinds of effective carriers, described carrier can instruct the synthetic of the double-stranded sequence of the 19-base pair of gene shown in the target, and these carriers are transfected into NSCLC clone A549 and LC319.After the transfection 5 days, compared with the control, the importing of these RNAi carriers of this gene suppressed colony number (Fig. 3 A and 3B) significantly, and this shows the growth that the inhibition of URLC8 has been reduced cell, propagation and/or survival.This result has also obtained focus and has formed the confirmation (data do not provide) of testing (Giemsa dyeing).
NSC 1191:COX17: designed 2 kinds of effective carriers, described carrier can instruct the synthetic of the double-stranded sequence of the 19-base pair of gene shown in the target, and these carriers are transfected into NSCLC clone A549 and LC319.After the transfection 5 days, find by the MTT experiment: compared with the control, the importing of the RNAi carrier (No.2) of this gene obviously suppressed cell proliferation (Fig. 3 A and 3B), and this shows the growth that the inhibition of COX17 has been reduced cell, propagation and/or survival.This result has also obtained focus and has formed the confirmation (data do not provide) of testing (Giemsa dyeing).
NSC 1246:SUPT3H: designed 3 kinds of effective carriers, described carrier can instruct the synthetic of the double-stranded sequence of the 19-base pair of gene shown in the target, and these carriers are transfected into NSCLC clone A549.After the transfection 5 days, find by the MTT experiment: compared with the control, the importing of the RNAi carrier (No.2) of this gene obviously suppressed cell proliferation (Fig. 3 A and 3B), and this shows the growth that the inhibition of SUPT3H has been reduced cell, propagation and/or survival.This result has also obtained focus and has formed the confirmation (data do not provide) of testing (Giemsa dyeing).
NSC 1389:NMU: designed 2 kinds of effective carriers, described carrier can instruct the synthetic of the double-stranded sequence of the 19-base pair of gene shown in the target, and these carriers are transfected into NSCLC clone A549 and LC319.After the transfection 5 days, find by the MTT experiment: compared with the control, the importing of the RNAi carrier (No.2) of this gene obviously suppressed cell proliferation (Fig. 3 A and 3B), and this shows the growth that the inhibition of NMU has been reduced cell, propagation and/or survival.This result has also obtained focus and has formed the confirmation (data do not provide) of testing (Giemsa dyeing).Then, after the siRNA transfection 24 hours, carried out flow cytometry.As a result, detect the Asia-G1 group who finds the LC319 cell and increased by 34.5% (Fig. 3 D).
NSC 1395:FBN2: designed a kind of effective carrier, described carrier can instruct the synthetic of the double-stranded sequence of the 19-base pair of gene shown in the target, and this carrier is transfected into NSCLC clone A549 and LC319.After the transfection 5 days, form experiment (Giemsa dyeing) discovery by focus: compared with the control, the importing of the RNAi carrier (No.2) of this gene obviously suppressed cell proliferation (data do not provide).
(7) cytochrome c oxidase activity
Cytochrome c oxidase in the A549 cell (CCO) activity and described activity are measured by the situation that COX17 suppresses.Fig. 3 F illustrates the active method of CCO of measuring.Particularly, with digitonin (digotonin) with cell be divided into plastosome and other fraction (Wako, Osaka, Japan).With cytochrome c (63mM) at damping fluid (10mM Tris, 0.2mM EDTA, 0.05%n-dodecyl-b-D-maltoside, pH7.6) in 12.5mM L (+)-xitix room temperature (18 ℃) insulation 30 minutes, ferric cytochrome c is converted into ferrous cytochrome c.Then, 37 ℃ add the 2ml said mixture with 20 microlitre 1mg/ml mitochondrial protein solution.Measure the CCO activity of above-mentioned reaction at the 550nm place.
In order to illustrate whether above-mentioned natural COX17 albumen has cytochrome c oxidase (CCO) activity in the people NSCLC cell, effective COX17 RNAi carrier is transfected into the A549 clone that detects the active usefulness of CCO.After the transfection 2-5 days, COX is active to be weakened owing to endogenous COX17 gene is suppressed.This result has confirmed the importance (Fig. 3 F, G and H) of CCO activity in people NSCLC of COX17 performance.
The sequence of synthetic oligonucleotide that plays the RNAi effect is as shown in table 6.Identified 30 genes, it is suppressed owing to being suppressed growth, propagation and the survival that will cause cell by antisense S-oligonucleotide and/or RNAi transfection.
Table 6: the sequence that plays the synthetic oligonucleotide of RNAi effect
Figure A20081018670601781
(8) immunocytochemical assay
In order to prepare the albumen of c-myc-His mark, synthesized carrier, described carrier contains the gene that coding is positioned at the c-myc-His epitope sequences (LDEESILKQE-HHHHHH) of every kind of PROTEIN C end, and the synthetic carrier is transfected into the COS-7 cell.With the PBS that contains 4% Paraformaldehyde 96 fixing heavily be laid on trough of belt slide glass (chamber slides) through the COS-7 of transient transfection cell, handle at 4 ℃ with the PBS that contains 0.1%Triton X-100 then and made that these cells can penetratingization in 3 minutes.Above-mentioned cell at room temperature uses lock solution (2% bovine serum albumin PBS solution) to cover 30 minutes, seals nonspecific antibody-binding site.Then, be incubated above-mentioned cell with mouse anti-c-myc antibody (with 1: 800 times of dilution of confining liquid).With coupling have the goat of FITC anti--the mouse second antibody dyes to above-mentioned antibody, places observation ECLIPSE E800 microscope (Nikon) under.For the expression of albumen in transfected cell of verifying the c-myc-mark, carry out Western-trace (Shiratsuchi etal., Biochem Biophys Res Commun 247:597-604 (1998)) as described before.
(9) location of the product of potential target gene in mammalian cell
In order to study these cellular localization of candidate gene encoded protein in mammalian cell, with pcDNA3.1 (+)/c-myc-His transfection COS-7 cell, pcDNA3.1 (+)/c-myc-His is a plasmid, and it contains c-myc-His-epitope sequences (LDEESILKQE-HHHHHH) gene that coding is positioned at every kind of PROTEIN C end.With anti--c-myc antibody, detect 24 kinds of albumen at different subcellular locations.These albumen some of them expression in transfected cell have obtained the checking (Fig. 5 A) of immunoblot experiment.
(10) select to stride the target of film/secreted protein as anti--cancer treatment and diagnosis
Select 14 kinds of energy to cross the film/secreted protein of striding of expression in tumor cell surface.Expect that these albumen are acceptor-targets/based on the cancer therapy of antibody and the good targets of diagnostic method.The expression and the cellular localization of albumen some of them described in the transfected COS-7 cell have been verified with immunocytochemical assay.
In order to measure every kind of proteic Subcellular Localization of described coded by said gene, with the proteic plasmid transfection COS-7 cell of expressing c-myc-His or Flag mark.The result who every kind of gene is carried out immunocytochemical assay is as follows:
NSC 807:KOC1: KOC1/c-myc-His albumen is mainly seen in (detected in) tenuigenin (cytoplasm) (data not shown).
NSC 810:TTK: TTK/c-myc-His albumen is mainly seen in nucleus (nucleus) (data not shown).
NSC 825:ANLN: ANLN-myc-His albumen is mainly seen in nucleus and tenuigenin (data not shown).
NSC 841:URLC2: URLC2/c-myc-His albumen is mainly seen in nucleus and tenuigenin (data not shown).
NSC 849:GJB5: GJB5/c-myc-His albumen is mainly seen in cytoplasmic membrane (cytoplasmicmembrane), and (Fig. 5 is a).
NSC 855:LNIR: LNIR/c-myc-His albumen is mainly seen in cytoplasmic membrane, and (Fig. 5 is a).
NSC 895:FAM3D: FAM3D/c-myc-His albumen is mainly seen in kytoplasm particle (cytoplasmic granule), golgi body (golgi) and cytoplasmic membrane, and (Fig. 5 is a).The Western trace has detected the secretion (Fig. 5 B) of FAM3D in substratum.Therefore, can think that FAM3D is a kind of secreted protein.
NSC 903:URLC9: URLC9/c-myc-His albumen is mainly seen in nucleus (data not shown).
NSC 915:URLC10: URLC10/c-myc-His albumen is mainly seen in kytoplasm particle and golgi body, and appears at cytoplasmic membrane surface (Fig. 5 A) with the spot form.
NSC 948:TASK-2: TASK-2/c-myc-His albumen is mainly seen in cytoplasmic membrane (Fig. 5 A).
NSC 956:SIAHBP1: SIAHBP1/c-myc-His albumen is mainly seen in tenuigenin (data not shown).
NSC 994:DKFZp434E2318: DKFZp434E2318/c-myc-His albumen is mainly seen in tenuigenin (data not shown).
NSC 1000:PSK-1: PSK-1/c-myc-His albumen is mainly seen in cytoplasmic membrane (Fig. 5 A).
NSC 1103:KCNK1: KCNK1/c-myc-His albumen is mainly seen in cytoplasmic membrane (Fig. 5 A).
NSC 1107:URLC8: URLC8/c-myc-His albumen is mainly seen in nucleus (data not shown).
NSC 1164:NPTX1: NPTX1/c-myc-His albumen is mainly seen in kytoplasm particle (Fig. 5 A).The Western trace has detected the secretion (Fig. 5 B) of NPTX1 in substratum.Therefore, can think that NPTX1 is a kind of secreted protein.
NSC 1191:COX17: COX17/c-myc-His albumen is mainly seen in plastosome (data not shown).
NSC 1201:SLC7A1: SLC7A1/c-myc-His albumen is mainly seen in cytoplasmic membrane and golgi body (Fig. 5 A).
NSC 1246:SUPT3H: SUPT3H/c-myc-His albumen is mainly seen in nucleus and tenuigenin (data not shown).
NSC 1288:PTGFRN: PTGFRN/c-myc-His albumen is mainly seen in cytoplasmic membrane and golgi body (Fig. 5 A).
NSC 1295:ADAM8: ADAM8/c-myc-His albumen is mainly seen in cytoplasmic membrane (Fig. 5 A).The Western trace has detected the secretion (Fig. 5 B) of form (cleavaged form) in substratum of three kinds of cuttings of ADAM8.Therefore, can think that ADAM8 is a kind of secreted protein.
NSC 1389:NMU: NMU/c-myc-His albumen is mainly seen in golgi body, and is the form (Fig. 5 A) of secretory protein.
NSC 1420:CHDOL: CHDOL/c-myc-His albumen is mainly seen in cytoplasmic membrane and golgi body (Fig. 5 A).
NSC 1441:HSCOV: HSNOV/c-myc-His albumen is mainly seen in cytoplasmic membrane and golgi body (Fig. 5 A).
(12) cell growth measurement and bacterium colony form and measure
Set up stable transfectant according to ordinary method.Particularly;, this cell was cultivated 14 days with Geneticin (G418) after (pcDNA3.1) be transfected into the COS-7 cell at the plasmid that will express described target gene (pcDNA3.1/myc-His) or this gene complementation chain (pcDNA3.1-antisense) or simulation plasmid.Then, select colony, detect described expression of gene by the Western trace.Verified that by immunostaining stable transfection of being set up is monoclonal (data not shown) with anti-c-myc antibody.Stable transfection of COS-7 cell is inoculated in 6-hole microtiter plate (5 * 10 4Individual cells/well), in adding the substratum of the antibiotic 10%FBS of containing, keep 24,48,72,96,120 and 144 hours then.At each time point, with cell counting test kit (WAKO) or by MTT experimental evaluation cell-proliferation activity.
(13) the cell growth measurement of stable transformant and autocrine are measured
NSC 810:TTKIn order to measure the influence of TTK, set up the COS-7 cell (COS-7-TTK1 and 2) of expressing external source TTK, and its growing state and control cells with the analog carrier transfection have been compared mammalian cell growth.As shown in Figure 6, according to the proteic expression level of pcDNA3.1-TTK-c-myc-His, the growth of COS-7-TTK cell is accelerated than remarkable with the growth phase of control cells.This experimental result has obtained the checking of 3 parts of independent experiments.Described COS-7-TTK cell also shows the obvious tendency (data not shown) of formation greater than the cell colony of control cells colony.
NSC 841:URLC2In order to measure the influence of URLC2, set up the NIH3T3 cell (NIH3T3-URLC2,3 and 5) of expressing external source URLC2, and its growing state and control cells with analog carrier (NIH3T3-simulation) transfection have been compared mammalian cell growth.As shown in Figure 6, according to the proteic expression level of pcDNA3.1-URLC2-myc-His, the growth of NIH3T3-URLC2 cell significantly improves with comparing of control cells.This experimental result has obtained the checking of 3 parts of independent experiments.Described NIH3T3-URLC2 cell also shows the obvious tendency (data not shown) of formation greater than the cell colony of control cells colony.
NSC 1389:NMUIn order to measure the influence of NMU to mammalian cell growth, set up the COS-7 cell (COS-7-NMU-2,3 and 5) of expressing external source NMU, and with their growing state with antisense strand or analog carrier (COS-7-AS-1 and 2; The COS-7-simulation) growth of the control cells of transfection compares.As shown in Figure 6, according to the proteic expression level of pcDNA3.1-NMU-c-myc/His, the growth of COS-7-NMU cell significantly improves with the growth phase of control cells ratio.This experimental result has obtained the checking of four parts of independent experiments.Described COS-7-NMU cell also shows the obvious tendency (data not shown) of formation greater than the cell colony of control cells colony.This result showed that the NMU that expresses had conversion effectiveness (transforming effect) for mammalian cell.
(14) autocrine is measured
In order to confirm the autocrine function of NMU in the cell growth, in the substratum of the activity form (NMU-25) of 25 amino acid polypeptides that contain NMU, the final concentration of NMU-25 in substratum is 1 μ g～50 μ g (3 μ M～15 μ M/ml) with the COS-7 cell cultures.With the substratum of the bovine serum albumin that contains same concentrations (BSA) in contrast.In 7 days by a definite date time, added described polypeptide or BSA, and continued 7 days in per 48 hours.Time point at the 24th, 48,72,96,120 and 144 hour detects cell viability by the MTT experiment.In order to verify the growth-promoting effect of NMU albumen to the COS-7 cell, adding final concentration in the substratum that contains 3 μ M/ml NMU-25 is anti--NMU antibody of 0.5 μ M～7.5 μ M/ml.
Found that the COS-7 cell that together is incubated with NMU-25 is grown greatlyyer than the COS-7 cell that together is incubated with BSA and be faster, and shows as dose-dependently mode (Fig. 7 A).
Then, in the COS-7 cell culture that contains 3 μ M/mlNMU-25, add anti--NMU antibody that final concentration is 0.5 μ M～7.5 μ M/ml.The MTT experimental result finds, grows more slowly with the COS-7 cell that NMU-25 and anti--NMU antibody is incubated altogether than control cells, and shows as dose-dependently mode (Fig. 7 B).
In addition, express the anti--NMU antibody that adds same concentrations in the LC319 cell culture of endogenous NMU to mistake.The MTT experimental result finds, grows more slowly with anti--LC319 cell that NMU antibody together is incubated than control cells, and shows as dose-dependently mode (Fig. 7 C).
(15) immunohistochemical analysis
In order to measure the expression of described albumen in comprising the clinical tissue sample of normal lung and NSCLC, use ENVISION+Kit/HRP (DAKO) to section statining.Particularly, after endogenous peroxidase and protein blocking reaction (protein blocking reaction), add Anti-Human's antibody, handle described tissue sample with the anti--rabbit igg of HRP mark as second antibody then as first antibody.Then, add chromogen as the substrate of tissue sample being redyed with phenodin.
In order to verify TTK albumen expressing excessively in NSCLC, at first, by the described albumen (Fig. 8) among Western engram analysis evaluation NSCLC clone A549, LC319 and the NCI.Then, every kind of gene is carried out immunohistochemical staining, as described below:
NSC 947:PKP3With anti--PKP3 antibody NSCLC (squamous cell carcinoma) sample that obtains with surgical operation is carried out immunohistochemical staining, this tissue sample has been frozen and has been embedded in the OCT substratum.The tenuigenin major part of all neoplasmic tissue sample is all by anti--PKP3 antibody staining, and normal lung tissue is not colored (Fig. 9).
NSC 1164:NPTX1With anti--NPTX1 antibody the NSCLC sample that obtains with surgical operation is carried out immunohistochemical staining, this tissue sample has been frozen and has been embedded in the OCT substratum.The tenuigenin major part of all neoplasmic tissue sample is all by anti--NPTX1 antibody staining, and normal lung tissue is not colored (Fig. 9).
NSC 1295:ADAM8With anti--ADAM8 antibody the NSCLC sample that obtains with surgical operation is carried out immunohistochemical staining, this tissue sample has been frozen and has been embedded in the OCT substratum.All neoplasmic tissue sample are dyeed by force by anti--ADAM8 antibody, and normal lung tissue is only by dyeing (Fig. 9) slightly.
NSC 1389:NMUWith anti--NMU antibody the NSCLC sample that obtains with surgical operation is carried out immunohistochemical staining, this tissue sample has been frozen and has been embedded in the OCT substratum.The tenuigenin major part of all neoplasmic tissue sample is all by anti--NMU antibody staining, and normal lung tissue is not colored.In the gland cancer sample, NMU is mainly seen in vessel cell and the perinuclear squamous cell carcinoma, specifically is (Fig. 9) in the kytoplasm particle.
(16) total length of target gene order-checking, Northern trace and sxemiquantitative RT-PCR analyze
Make up by the tabulation of crossing the gene of expressing of the expression in 50% above NSCLC being compared 5 times of risings with the expression in 34 parts of healthy tissuess, select the target of 642 candidate genes as tumor marker or treatment, selected these specific gene expressions in NSCLC, and except be not most important for survival or can substituted germinal tissue or the fetus organ healthy tissues in do not express.By the full length sequence of the described target gene of EST screening assay, and with their gene expression patterns in tumour and healthy tissues of sxemiquantitative RT-PCR checking.
Found a kind of new gene URLC1.Its nucleotide sequence and amino acid sequence coded thereof have following SEQ ID NO in sequence table:
The nucleotide sequence aminoacid sequence
URLC1?SEQ?ID?NO：1 SEQ?ID?NO：2
Aforesaid method is summarized as follows the result that each gene of the present invention obtains:
NSC 807:KOC1A kind of hnRNA K-of this genes encoding homology (KH) structural domain and a kind of RNA identification motif (RRM) structural domain.The KH structural domain has the IGF2 with IGF-II) 5 ' the UTR bonded function of leading 3 ' mRNA, therefore can suppress the translation of IGF-II between the growth period in late period.This gene is lower at normal tissue expression, but high expression level in NSCLC, and suppress feasible growth, propagation and/or the survival through cells transfected of this gene and weaken, these show that all this gene can be as the target of new diagnostic flag and new drug and immunotherapy.
NSC 810:TTKA kind of S TKc of this genes encoding structural domain.The albumen of this coded by said gene is phosphorylation on Serine, Threonine and tyrosine, and this phosphorylation may be relevant with cell proliferation.This gene is lower at normal tissue expression, but high expression level in NSCLC, and suppress feasible growth, propagation and/or the survival through cells transfected of this gene and weaken, these show that all this gene can be as the target of new diagnostic flag and new drug and immunotherapy.According to the present invention, be expressed in TTK albumen in the stable transfectant has promoted the COS-7 cell in the dose-dependently mode growth.This result shows that the expression of crossing of TTK has short (transforming) effect that transforms for mammalian cell.These data show: TTK is a kind of new oncogene of NSCLC, can set up the treatment means likely of treatment lung cancer by target TTK.
NSC 811:SDC1A kind of band of inferring 4.1 homologue binding motif (4.1m) structural domains of this genes encoding.The albumen of this genes encoding is a kind of cell surface protein glycan, and syndecan is a kind of integrated membranin that serves as extracellular matrix receptor.It belongs to strides film heparan sulfate proteoglycan (heparan sulfate proteoglycan) colony.This gene is lower at normal tissue expression, but high expression level in NSCLC, and suppress feasible growth, propagation and/or the survival through cells transfected of this gene and weaken, these show that all this gene can be as the target of new diagnostic flag and new drug and immunotherapy.
NSC 812:NMBA kind of signal peptide of this genes encoding and a kind of membrane spaning domain.The albumen of this genes encoding can be brought into play the function of neuromedin (neuromedin) B, and it is a member of bombesin (bombesin) family, is a kind of autocrine growth factor of lung cancer.This gene is lower at normal tissue expression, but high expression level in NSCLC, and suppress feasible growth, propagation and/or the survival through cells transfected of this gene and weaken, these show that all this gene can be as the target of new diagnostic flag and new drug and immunotherapy.
NSC 816:PIR51The albumen of this genes encoding is positioned nucleus, and does not find any structure territory in this albumen.This albumen can serve as DNA-and RNA-is conjugated protein; And interact with the RAD51 recombinase protein that participates in the DNA reorganization and repair.This gene is lower at normal tissue expression, but high expression level in NSCLC, and suppress feasible growth, propagation and/or the survival through cells transfected of this gene and weaken, these show that all this gene can be as the target of new diagnostic flag and new drug and immunotherapy.
NSC 825:ANLNA kind of PH structural domain of these genes encodings, have now found that its several function of inferring: (1) combines with the β/γ subunit of heterotrimeric G protein; (2) with lipid for example, phosphatidylinositols-4, the combination of 5-bisphosphate; (3) combine with the Ser/Thr residue of phosphorylation; And (4) mechanism by a kind of the unknown is attached on the film.A kind of actin binding protein of this genes encoding, this albumen and cleavage furrow (cleavage furrow) albumen for example septins interact, and can play a role in division of cytoplasm (cytokenesis).This gene is lower at normal tissue expression, but high expression level in NSCLC, and suppress feasible growth, propagation and/or the survival through cells transfected of this gene and weaken, these show that all this gene can be as the target of new diagnostic flag and new drug and immunotherapy.
NSC 841:URLC2A kind of Jmjc structural domain of this genes encoding (the structural domain family of part cupin metalloenzyme (metalloenzyme)).The albumen of this genes encoding may be a kind of enzyme of Unknown Function, and this kind of enzyme can be regulated chromatin reconstruct (reorganization) process.This gene is lower at normal tissue expression, but high expression level in NSCLC, and suppress feasible growth, propagation and/or the survival through cells transfected of this gene and weaken, these show that all this gene can be as the target of new diagnostic flag and new drug and immunotherapy.According to this experiment, the apoptosis of LC319 cell has been induced in the inhibition of URLC2.And, be expressed in URLC2 albumen in the stable transfectant has promoted the NIH3T3 cell in the dose-dependently mode growth.This result showed that the URLC2 that expresses had transformation for mammalian cell.These data show that URLC2 is a kind of new oncogene of NSCLC, and prompting can be by being that focus is set up a kind of treatment means likely for the treatment of lung cancer with URLC2.
NSC 849:GJB5A kind of gap junction protein of this genes encoding, β 5 (connecting albumen (connexin) 31.1).GJB5 connects a member of protein family (β-type (i group) subfamily).Gap connects and is made up of the closelypacked paired transmembrane channel connexon of cluster (connexon) according to reports, low molecular weight substance by it from a cellular invasion extremely in abutting connection with cell.Connexon is formed by connecting the albumen sexamer.Detect by immunocytochemical assay, find that the albumen of this genes encoding mainly is present in cytoplasmic membrane.In NSCLC, this shows that this gene can be used as the treatment target of diagnostic flag (that is, in the diagnosis of using serum or phlegm) and NSCLC to this gene at the lower but high expression level of normal tissue expression.
NSC 855:LNIRThis genes encoding signal peptide, immunoglobulin (Ig), immunoglobulin (Ig) C2 structural domain and a kind of membrane spaning domain.The transmembrane protein of this genes encoding is crossed and is expressed on the tumor cell surface, but is not expressed in normal cell.Therefore, this gene is the good target of the treatment or the diagnosis of receptor targeted.
NSC 857:TIGD5A kind of kinetochore of this genes encoding albumen (CentromereProtein) B (CENP-B).CENP-B is that a kind of DNA-that is positioned to the kinetochore is conjugated protein.In 125 residues of this albumen N-end, exist the DNA-binding domains, this structural domain can be in conjunction with corresponding 17bp CENP-B frame sequence.In terminal 59 residues of C-, CENP-B has a dimerization structural domain.The CENP-B dimer both can be in conjunction with two isolated DNA molecule, also can be in conjunction with two CENP-B frames that are positioned on the dna molecular, and wherein the insertion fragment of DNA (intervening stretch) forms ring structure.This gene belongs to the tigger subfamily of the transposon pogo superfamily of people DNA-mediation.It is relevant with DNA transposon in seeing fungi and nematode (nematode) to belong to the albumen of this subfamily, and the relation between Tc1 and the sailer transposase enzyme (mariner transposase) is far away.The albumen of this genes encoding also is very similar to mainly (major) Mammals kinetochore protein B.The definite function of this gene or the unknown.This gene is lower at normal tissue expression, but high expression level in NSCLC, and suppress feasible growth, propagation and/or the survival through cells transfected of this gene and weaken, these show that all this gene can be as the target of new diagnostic flag and new drug and immunotherapy.
NSC 859:URLC3This gene arbitrary known structural domain of not encoding, the albumen of this genes encoding has 70% similarity on 56 amino acid scopes and between eukaryotic translation initiation factor 3 subunits (people).This subunit is in conjunction with the 40s rrna, can promote combine (the passing through similarity) of methionyl-tRNAi and mRNA.This gene is lower at normal tissue expression, but high expression level in NSCLC, and suppress feasible growth, propagation and/or the survival through cells transfected of this gene and weaken, these show that all this gene can be as the target of new diagnostic flag and new drug and immunotherapy.
NSC 885:BAG5A kind of BAG structural domain of this genes encoding.Therefore, the albumen of this genes encoding is a member of BAG1-associated protein family.This gene is lower at normal tissue expression, but high expression level in NSCLC, and suppress feasible growth, propagation and/or the survival through cells transfected of this gene and weaken, these show that all this gene can be as the target of new diagnostic flag and new drug and immunotherapy.
NSC 893:MPHOSP1A kind of KISc structural domain of this genes encoding and microtubule-dependent molecular motor (motor), its in the born of the same parents of organoid, transport and cell fission in play a significant role.The albumen of this genes encoding belongs to kinesin (kenesin)-sample protein family, with the guanosine triphosphate gtp of rab6a and rab6b)-the combining form interaction.This albumen can serve as Golgi membrane and relevant vesicle is transported required motor (motor) along the retreating property (retrograde) that rab6 regulates.Microtubule anode-the guiding that has this albumen moves (microtubule plus end-directed motility), M stage of cell cycle by phosphorylation.This gene is lower at normal tissue expression, but high expression level in NSCLC, and suppress feasible growth, propagation and/or the survival through cells transfected of this gene and weaken, these show that all this gene can be as the target of new diagnostic flag and new drug and immunotherapy.
NSC 895:FAM3DA kind of N-end of this genes encoding has the albumen of signal peptide structural domain, and this albumen is considered to a kind of secreted protein, and let it be to the greatest extent, and function is still waiting to illustrate.In immunocytochemical assay, this albumen is mainly seen in kytoplasm particle and golgi body, and this shows that this albumen is secretor type.In NSCLC, this shows that this gene can be as the diagnostic flag (that is, in the diagnosis of using serum or phlegm) of NSCLC to this gene at the lower but high expression level of normal tissue expression.
NSC 898:URLC7The albumen of this genes encoding is positioned nucleus, and does not have any known structural domain in this albumen.This gene is lower at normal tissue expression, but high expression level in NSCLC, and suppress feasible growth, propagation and/or the survival through cells transfected of this gene and weaken, these show that all this gene can be as the target of new diagnostic flag and new drug and immunotherapy.
NSC 903:URLC9The albumen of this genes encoding is positioned nucleus, and does not have any known structural domain in this albumen.This gene is lower at normal tissue expression, but high expression level in NSCLC, and suppress feasible growth, propagation and/or the survival through cells transfected of this gene and weaken, these show that all this gene can be as the target of new diagnostic flag and new drug and immunotherapy.
NSC 905:URLC1A kind of TUDOR structural domain of these genes encodings has now found that its several function of inferring: (1) RNA-combination; And (2) nucleic acid combination.This gene is lower at normal tissue expression, but high expression level in NSCLC, and suppress feasible growth, propagation and/or the survival through cells transfected of this gene and weaken, these show that all this gene can be as the target of new diagnostic flag and new drug and immunotherapy.
NSC 909:FLJ10468The albumen of this genes encoding is positioned nucleus, and does not have any known structural domain in this albumen.This gene is lower at normal tissue expression, but high expression level in NSCLC, and suppress feasible growth, propagation and/or the survival through cells transfected of this gene and weaken, these show that all this gene can be as the target of new diagnostic flag and new drug and immunotherapy.
NSC 915:URLC102 membrane spaning domains of this genes encoding.The albumen of this genes encoding has a zone that has low similarity with GML.Find that by immunocytochemical assay this albumen mainly is present in kytoplasm particle and golgi body, and appears on the cytoplasmic membrane with the spot form.The transmembrane protein of inferring this genes encoding is crossed and is expressed on the tumor cell surface, but is not expressed in normal cell.Therefore, this albumen can be as the treatment of receptor targeted or the good target of diagnosis.
NSC 920:CHAF1ADo not detect the arbitrary known structural domain of this genes encoding.The albumen of this genes encoding has 150kDa subunit chromatin assembly factor 1, and it will help histone H 3 acetylize H4 to deposit on the DNA that is duplicating.This gene is lower at normal tissue expression, but high expression level in NSCLC, and suppress feasible growth, propagation and/or the survival through cells transfected of this gene and weaken, these show that all this gene can be as the target of new diagnostic flag and new drug and immunotherapy.
NSC 947:PKP3White (the catenin)-sample repeat unit structure territory (ARM) of a kind of tatou of this genes encoding (armadillo)/beta-catenin.The tatou repeating unit is a kind of about 40 amino acid long tandem repetitive sequence motifs, and it at first sees in the fruit bat sections polarity gene tatou (armadillo).Be to have found similar repetition in polyposis colibacillus (APC) the tumor suppressor gene albumen of connecting-type plaque albuminous plasue globin (junctional plaqueprotein plakoglobin), adenoma and a lot of other albumen in vain at Mammals tatou homologue beta-catenin afterwards.The albumen of this genes encoding can be brought into play the effect of plakophillin 3, and the mediation protein-protein interaction is a member of tatou protein family.This gene is lower at normal tissue expression, but high expression level in NSCLC, and suppress feasible growth, propagation and/or the survival through cells transfected of this gene and weaken, these show that all this gene can be as the target of new diagnostic flag and new drug and immunotherapy.Immunohistochemical staining shows: PKP3 is dyeed by force in the tenuigenin of squamous cell cancer cells.These data show that PKP3 can be used as a kind of effective treatment and the diagnosis target of treatment lung cancer.
NSC 948:TASK-2A kind of ion transport thing of this genes encoding structural domain, signal peptide (SOSUI).A kind of albumen of this genes encoding, this albumen belong to the potassium channel protein superfamily that contains two pore-forming P-structure territories.The mRNA of this gene mainly is expressed in cortex section far away (distal) tubule and the collecting tubule of kidney.The albumen of this genes encoding pH to external world is extremely sensitive, and this point shows in conjunction with its expression pattern: this gene plays a significant role in the potassium transhipment of kidney.The transmembrane protein of this genes encoding is crossed and is expressed on the tumor cell surface, but is not expressed in normal cell.Therefore, this albumen can be as the treatment of receptor targeted or the good target of diagnosis.
NSC 956:SIAHBP1A kind of RNA identification of this genes encoding motif (RRM) structural domain, known this structural domain is a kind of nucleic acid binding domains.The albumen of this genes encoding is that a kind of Ro RNS-is conjugated protein.The function of activation Ro RNPs thereby it and Ro RNPs interact.This albumen also with upstream element far away (FUSE) and a kind of ternary complex of the conjugated protein formation of FUSE-.It can be by suppressing the c-myc reporter in conjunction with FUSE.Transcription factor IIH also is this proteic target, thereby this albumen can suppress activated transcribing.This gene is also relevant with xeroderma pitmentosum (xerodermapigmentosum disorder).There are two kinds of optional splicing transcript variants in this gene, the different isotype of described variant coding.There is a plurality of adenosine acidifyings site on this gene.This gene is lower at normal tissue expression, but high expression level in NSCLC, and suppress feasible growth, propagation and/or the survival through cells transfected of this gene and weaken, these show that all this gene can be as the target of new diagnostic flag and new drug and immunotherapy.
NSC 991:DOLPP1A kind of signal peptide of this genes encoding and a kind of acid phosphatase homologue structural domain.The transmembrane protein of this genes encoding is crossed and is expressed on the tumor cell surface, but is not expressed in normal cell.Therefore, this albumen can be as the treatment of receptor targeted or the good target of diagnosis.
NSC 994:DKFZP434E2318This genes encoding BTB/POZ structural domain and Kelch structural domain.The BTB/POZ structural domain is the protein-protein interaction motif.The BTB/POZ structural domain can mediate with poly-dimerization (homomeric dimerization), and can also mediate assorted poly-dimerization in some cases.Inhibition is transcribed in the mediation of the POZ structural domain of several zinc finger proteins, and with the histon deacetylase (HDAC) that comprises N-CoR and SMART altogether-component interaction of repressor complex body.The Kelch structural domain is a kind of β propeller (propeller) structural domain that participates in protein-protein interaction, has some and is similar to the oxidasic enzymatic activity of gycolate.This gene is lower at normal tissue expression, but high expression level in NSCLC, and suppress feasible growth, propagation and/or the survival through cells transfected of this gene and weaken, these show that all this gene can be as the target of new diagnostic flag and new drug and immunotherapy.
NSC 1000:PSK-1This genes encoding signal peptide, CUB structural domain, Sushi structural domain (SCR repeating unit) and a kind of membrane spaning domain.The albumen height of this genes encoding is similar to mouse Sez6, and mouse 7 Sez6 are a kind of attachment proteins albumen, and it contains 5 sushi (SCR) structural domains and an extracellular CUB structural domain.The transmembrane protein of inferring this genes encoding is crossed and is expressed on the tumor cell surface, but is not expressed in normal cell.Therefore, this albumen can be as the treatment of receptor targeted or the good target of diagnosis.
NSC 1066:MCM8This genes encoding keeps the relevant ATPase (AAA) of (MCM) structural domain with various kinds of cell active structure domain and minichromosome.In healthy tissues, these show that all this gene can be as the target of new diagnostic flag and new drug and immunotherapy to this gene at the lower but high expression level of normal tissue expression.
NSC 1075:URLC4Do not detect arbitrary known structure territory of this genes encoding.This gene is lower at normal tissue expression, but high expression level in NSCLC, and suppress feasible growth, propagation and/or the survival through cells transfected of this gene and weaken, these show that all this gene can be as the target of new diagnostic flag and new drug and immunotherapy.
NSC 1103:KCNK1A kind of albumen of this genes encoding, this albumen belong to the potassium channel protein superfamily that contains two pore-forming P-structure territories.The product that does not show this gene is a kind of function passage.Other non-PFP for performance function channel activity may be for necessity.Infer this transmembrane protein and cross and be expressed on the tumor cell surface, but be not expressed in normal cell.Therefore, this albumen can be as the treatment of receptor targeted or the good target of diagnosis.
NSC 1107:URLC8A kind of double-stranded RNA binding motif of this genes encoding (DSRM) structural domain.This gene is lower at normal tissue expression, but high expression level in NSCLC, and suppress feasible growth, propagation and/or the survival through cells transfected of this gene and weaken, these show that all this gene can be as the target of new diagnostic flag and new drug and immunotherapy.
NSC 1113:URLC5Do not detect arbitrary known structure territory of this genes encoding.This gene is lower at normal tissue expression, but high expression level in NSCLC, and suppress feasible growth, propagation and/or the survival through cells transfected of this gene and weaken, these show that all this gene can be as the target of new diagnostic flag and new drug and immunotherapy.
NSC 1131:SYNJ2BPA kind of PDZ membrane spaning domain of this genes encoding.The albumen of this genes encoding may be a kind of signal protein of target film, and it contains the PDZ structural domain.This gene is lower at normal tissue expression, but high expression level in NSCLC, and suppress feasible growth, propagation and/or the survival through cells transfected of this gene and weaken, these show that all this gene can be as the target of new diagnostic flag and new drug and immunotherapy.
NSC 1141:URLC119 membrane spaning domains of this genes encoding.The transmembrane protein of this genes encoding is crossed and is expressed on the tumor cell surface, but is not expressed in normal cell.Therefore, this albumen can be as the treatment of receptor targeted or the good target of diagnosis.
NSC 1142:NAPGDo not detect the known structure territory of this genes encoding.Inferring between proteic sequence of 312-amino acid people and the ox γ SNAP of NAPG coding has 95% identity.The protein mediated thrombocyte exocytosis of NAPG and control the film fusion event of this process.This gene is lower at normal tissue expression, but high expression level in NSCLC, and suppress feasible growth, propagation and/or the survival through cells transfected of this gene and weaken, these show that all this gene can be as the target of new diagnostic flag and new drug and immunotherapy.
NSC 1164:NPTX1A kind of Pentaxin/C-reactive protein of this genes encoding.NPTX1 is the member of neurone (neuronal) pentraxin gene family.Neurone pentraxin 1 is similar to rat NP1 gene, and latter's venom toxin of encoding is conjugated protein.Immunocytochemical assay mainly detects the albumen of this genes encoding in the kytoplasm particle.In healthy tissues, this shows that this gene can be used as the treatment target of diagnostic flag (that is, in the diagnosis of using serum or phlegm) and NSCLC to this gene at the lower but high expression level of normal tissue expression.Immunohistochemical staining shows: NPTX1 is dyeed by force in the tenuigenin of adenocarcinoma cell.These data show that NPTX1 can be used as a kind of effective treatment and the diagnosis target of treatment lung cancer.
NSC 1183:BYSLDo not detect the known structure territory of this genes encoding.The albumen of this genes encoding has the function of bystin, and itself and trophinin (TRO) and TASTIN form a kind of cell adhesion molecule complex body, and this complex body is implanted significant for the embryo.This gene is lower at normal tissue expression, but high expression level in NSCLC, and suppress feasible growth, propagation and/or the survival through cells transfected of this gene and weaken, these show that all this gene can be as the target of new diagnostic flag and new drug and immunotherapy.
NSC 1185:URLC6A kind of zinc of this genes encoding refers to RNA identification motif structural domain.This gene is lower at normal tissue expression, but high expression level in NSCLC, and suppress feasible growth, propagation and/or the survival through cells transfected of this gene and weaken, these show that all this gene can be as the target of new diagnostic flag and new drug and immunotherapy.
NSC 1191:COX17The albumen of this genes encoding is positioned plastosome intermembranous space (mitochondrial intermemebrane space) (passing through similarity), and copper can be transported to plastosome.And this albumen may be that Terminal oxidase is expressed required.This gene is lower at normal tissue expression, but high expression level in NSCLC, and suppress feasible growth, propagation and/or the survival through cells transfected of this gene and weaken, these show that all this gene can be as the target of new diagnostic flag and new drug and immunotherapy.
NSC 1201:SLC7A114 membrane spaning domains of this genes encoding.The albumen height of this genes encoding is similar to mouse Rec-1 (Atrc 1), its performance cationic amino acid transporter albumen (cationicamino acid transporter) (environmental (ecotropic) retrovirus acceptor), transhipment arginine, Methionin and ornithine pass through plasma membrane.By immunocytochemical assay, this albumen is mainly seen in cytoplasmic membrane and golgi body.The transmembrane protein of this genes encoding is crossed and is expressed on the tumor cell surface, but is not expressed in normal cell.Therefore, this albumen can be as the treatment of receptor targeted or the good target of diagnosis.
NSC 1240:FLJ001594 membrane spaning domains of this genes encoding.The transmembrane protein of this genes encoding is crossed and is expressed on the tumor cell surface, but is not expressed in normal cell.Therefore, this albumen can be as the treatment of receptor targeted or the good target of diagnosis.
NSC 1246:SUPT3HThis genes encoding transcript starts factor-alpha D, 18kD subunit.Contain this gene coded protein comprises Spt3 yeast transcription factor and people's transcript initiation factors α D in interior family 18kD subunit (TF α D-18).Crystal structure determination shows that it is that a kind of atypical histone is folding.In healthy tissues, these show that all this gene can be as the target of new diagnostic flag and new drug and immunotherapy to this gene at the lower but high expression level of normal tissue expression.
NSC 1254:FLJ10815A kind of film amino acid transporter of striding of this genes encoding.This is striden the film district and all finds in comprising many amino acid transporters of UNC-47 and MTR.UNC-47 a kind of vesica shape (vesicular) aminobutyric acid (GABA) translocator (VGAT) of encoding.It is logical that the proteic function of this genes encoding is similar to amino acid/plant growth hormones slightly
Permease (auxin permease) is the protein called membrane transporters of family (AAAP).The transmembrane protein of this genes encoding is crossed and is expressed on the tumor cell surface, but is not expressed in normal cell.Therefore, this albumen can be as the treatment of receptor targeted or the good target of diagnosis.
NSC 1265:SLC28A2This genes encoding Na+ dependency nucleoside transporter.The albumen of this genes encoding can be brought into play the effect of sodium-link coupled nucleoside transporter 2, its Transshipment Permitted purine nucleoside and uridine.The transmembrane protein of this genes encoding is crossed and is expressed on the tumor cell surface, but is not expressed in normal cell.Therefore, this albumen can be as the treatment of receptor targeted or the good target of diagnosis.
NSC 1273:FLJ32549Do not detect arbitrary known structure territory of this genes encoding.This gene is lower at normal tissue expression, but high expression level in NSCLC, and suppress feasible growth, propagation and/or the survival through cells transfected of this gene and weaken, these show that all this gene can be as the target of new diagnostic flag and new drug and immunotherapy.
NSC 1288:PTGFRNThis genes encoding signal peptide, 6 immunoglobulin domains and a membrane spaning domain.The albumen of this genes encoding is by reducing the acceptor number but not suppresses combining of prostaglandin(PG) f2-α (pgf2-α) and its specificity fp acceptor by reducing affinity constant.This albumen is taken a fancy to and being deenergized and the coupling of prostaglandin(PG) f2-α function of receptors.Detect by immunocytochemical assay, this albumen is mainly seen in cytoplasmic membrane and golgi body.The transmembrane protein of this genes encoding is crossed and is expressed on the tumor cell surface, but is not expressed in normal cell.Therefore, this albumen can be as the treatment of receptor targeted or the good target of diagnosis.
NSC 1292:C17orf263 membrane spaning domains of this genes encoding, zinc translocator structural domain and signal peptide (SOSUI).The transmembrane protein of this genes encoding is crossed and is expressed on the tumor cell surface, but is not expressed in normal cell.Therefore, this albumen can be as the treatment of receptor targeted or the good target of diagnosis.
NSC 1295:ADAM8This genes encoding snake removes integrin homologous protein, the former peptide of Reprolysin family and Reprolysin (M12B) family zinc metalloprotein enzyme.The member of ADAM family is the cell surface protein with unique texture, and this structure contains potential and adheres to and the proteolytic enzyme structural domain.The extracellular region of ADAM8 shows the remarkable amino-acid sequence homology with echidnotoxin, wherein contains metalloprotease and removes the integrin structural domain.This gene normal tissue expression lower but in NSCLC high expression level, this shows that this gene can be used as the treatment target of diagnostic flag (that is, in the diagnosis of using serum or phlegm) and NSCLC.Immunohistochemical staining shows: ADAM8 is dyeed by force in the adenocarcinoma cell.These data show that ADAM8 can be used as a kind of effective treatment and the diagnosis target of treatment lung cancer.
NSC 1306:ABCA4This genes encoding signal peptide and AAA structural domain.The film of this genes encoding-conjugated protein is the member of ATP-linking frame (ABC) translocator superfamily.The various molecules of described ABC protein transport are by epicyte and intracellular membrane.The ABC gene is divided into 7 kinds of different subfamilies (ABC1, MDR/TAP, MRP, ALD, OABP, GCN20 and White).The albumen of this genes encoding is the member of ABC1 subfamily.The member of ABC1 subfamily comprises the unique main ABC subfamily that only comes across in the many cells eukaryote.This albumen is a kind of retina-specificity abc transport albumen, and it uses N-retinylidene-PE as substrate.This albumen only is expressed in the retina photosensory cell, shows that the essential molecule of this gene product mediation is by the photosensory cell film.The sudden change of this gene sees diagnosis and suffers from the patient of Stargardt disease, with retinitis pigmentosa (retinis pigmentosa)-19 and macular degeneration the age-relevant 2 (macular degeneration age-related 2) are relevant.The transmembrane protein of this genes encoding is crossed and is expressed on the tumor cell surface, but is not expressed in normal cell.Therefore, this albumen can be as the treatment of receptor targeted or the good target of diagnosis.
NSC 1343:GPR49This genes encoding signal peptide is rich in leucic reparation N-end structure territory and 7 transmembrane receptors (Visual purple family).The albumen of this genes encoding belongs to G albumen-link coupled receptor family, and this family member has the extracellular region territory that the leucine acceptor is rich in big comprising.The transmembrane protein of this genes encoding is crossed and is expressed on the tumor cell surface, but is not expressed in normal cell.Therefore, this albumen can be as the treatment of receptor targeted or the good target of diagnosis.
NSC 1362:SCAMP54 membrane spaning domains of this genes encoding.The transmembrane protein of inferring this genes encoding is crossed and is expressed on the tumor cell surface, but is not expressed in normal cell.Therefore, this albumen can be as the treatment of receptor targeted or the good target of diagnosis.
NSC 1389:NMUThis genes encoding NMU structural domain.The same with most of bioactive peptides, the albumen of this genes encoding is by forming by proteolytic treatment than larger precursor albumen.This proteic mature peptide length is 8-25 residue, and its C-end is by amidation.This albumen stimulated muscle is shunk, particularly GI muscle, and suppress to ingest.This gene is lower at normal tissue expression, but high expression level in NSCLC, and suppress feasible growth, propagation and/or the survival through cells transfected of this gene and weaken, these show that all this gene can be as the target of new diagnostic flag and new drug and immunotherapy.According to this experiment, the stable conversion body is secreted into the NMU albumen of substratum, or is added into the NMU peptide activity form in the substratum, promotes the growth of COS-7 cell in the dose-dependently mode.According to immunohistochemical staining, NMU albumen is all dyeed by force in the tenuigenin of adenocarcinoma cell and squamous cancer cell.These data show that NMU may be a kind of important autocrine growth factor of NSCLC, and prompting can develop effective treatment of a kind of treatment lung cancer and diagnosis policy with NMU ligand-receptor systems center.In addition, the LC319 apoptosis is induced in the inhibition of NMU.And, to compare with contrast LC319 cell, anti--NMU antibody suppresses the proteic inhibition induced growth of NMU.These results show that lung cancer can use the siRNA of antibody or target NMU to treat.
NSC 1395:FBN2This genes encoding calcium-in conjunction with EGF-sample (EGF CA) structural domain and an EGF sample (non-classified subfamily) structural domain.The effect of the albumen performance fibrillin 2 of this genes encoding, fibrillin 2 is a kind of extracellular matrix proteins, can regulate the formation and the maintenance of the outer micro-fibril of born of the same parents.The sudden change of FBN2 can cause congenital congenital contracture dolichostenomelia (congeintal contractualarachnodactyly).This gene is lower at normal tissue expression, but high expression level in NSCLC, and suppress feasible growth, propagation and/or the survival through cells transfected of this gene and weaken, these show that all this gene can be as the target of new diagnostic flag and new drug and immunotherapy.
NSC 1420:CHDOLA kind of I type membranin that has carbohydrate recognition structure territory of this genes encoding, this structural domain is the feature of C-type phytohemagglutinin in its born of the same parents' outside part.In other albumen, this structural domain involved in sugar albumen and external source contain the cell endocytic of sugared cause of disease.This machine mainly is positioned at nuclear week district because of encoded protein.By immunocytochemical assay, this albumen is mainly seen in cytoplasmic membrane and golgi body.The transmembrane protein of inferring this genes encoding is crossed and is expressed on the tumor cell surface, but is not expressed in normal cell.Therefore, this albumen can be as the treatment of receptor targeted or the good target of diagnosis.
NSC 1441:HSNOV1This genes encoding integrated (integral) membranin DFU6 structural domain.Detect by immunocytochemical assay, find that the albumen of this genes encoding mainly is present in cytoplasmic membrane and golgi body.The transmembrane protein of this genes encoding is crossed and is expressed on the tumor cell surface, but is not expressed in normal cell.Therefore, this albumen can be as the treatment of receptor targeted or the good target of diagnosis.
Industrial applicibility
Dissect and the combination of genome range cDNA array by laser-capture, the gene expression analysis that the described nonsmall-cell lung cancer of the application is carried out has identified the concrete gene that can be used as prevention and treat the nonsmall-cell lung cancer target.Expression according to the subgroup of the gene of these differential expressions the invention provides the molecular diagnosis mark that is used to identify or detect nonsmall-cell lung cancer.
The described method of the application also can be used for identifying other molecular target that can be used for preventing, diagnosing and treat nonsmall-cell lung cancer.The data of the application report make to the understanding of nonsmall-cell lung cancer more comprehensively, help to develop new diagnosis policy, and provide clue for the molecular target of identifying medicine and prevention medicament.These information make has had more deep understanding to oncogenesis, and provides enlightenment for exploitation diagnosis, treatment reach final prevention nonsmall-cell lung cancer New Policy.
Although in conjunction with specific embodiments of the present invention the present invention is described in detail, those skilled in the art obviously understand under the prerequisite that does not break away from essence of the present invention and scope can carry out various changes and modification to the present invention.
Sequence table
＜110〉Oncotherapy Science Inc (ONCOTHERAPY SCIENCE, INC.)
JAPAN?AS?REPRESENTED?BY?THE?PRESIDENT?OF?THE?UNIVERSITY?OF?TOKYO
＜120〉diagnostic method of nonsmall-cell lung cancer
<130>ONC-A0212Y2P
<150>US?60/414,673
<151>2002-09-30
<150>US?60/451,374
<151>2003-02-28
<150>US?60/466,100
<151>2003-04-28
<160>552
<170>PatentIn?version?3.1
<210>1
<211>2235
<212>DNA
＜213〉people (Homo sapiens)
<400>1
ggcacgaggc?ggcggaggcg?gcggcggcgg?cggcgatggc?agcggaccct?gagcgagctt 60
gagggctcgg?acccagctcc?ctcccgcgaa?accttgggcg?gatccggcgc?tgcggcccca 120
gctcgctccg?ctcctgctcc?ctccccggcc?gctgcctggg?cggaggcaga?ggcagaggcc 180
cgggctggcc?gccctgctcg?tgccccagct?cggccccgga?cggcccggct?gctgtgcaga 240
gaggaggccg?agtcggtagt?gaaaagagaa?tactgaagaa?taggatctca?agatgagtaa 300
aaagccccca?aatcgccctg?gaatcacttt?tgagattggt?gctcgtttgg?aggcactgga 360
ctacttacaa?aaatggtatc?catcacgaat?tgaaaaaatt?gactatgagg?agggcaagat 420
gttggtccat?tttgagcgct?ggagtcatcg?ttatgatgag?tggatttact?gggatagcaa 480
tagattgcga?ccccttgaga?gaccagcact?aagaaaagaa?gggctaaaag?atgaggaaga 540
tttctttgat?tttaaagctg?gagaagaagt?tctggctcgt?tggacagact?gtcgctatta 600
ccctgccaag?attgaagcaa?ttaacaaaga?aggaacattt?acagttcagt?tttatgatgg 660
agtaattcgt?tgtttaaaaa?gaatgcacat?taaagccatg?cccgaggatg?ctaaggggca 720
ggtgaaatcc?cagcatccac?taagctggtg?ttgtcctatc?gacccagctg?gatcgtgtaa 780
ccagtctatg?ggaagtgagg?attggatagc?tttagtcaaa?gcagctgctg?cagctgcagc 840
caagaacaaa?acagggagta?aacctcgaac?cagcgctaac?agcaataaag?ataaggataa 900
agatgagaga?aagtggttta?aagtaccttc?aaagaaggag?gaaacttcaa?cttgtatagc 960
cacaccagac?gtagagaaga?aggaagatct?gcctacatct?agtgaaacat?ttggacttca 1020
tgtagagaac?gttccaaaga?tggtctttcc?acagccagag?agcacattat?caaacaagag 1080
gaaaaataat?caaggcaact?cgtttcaggc?aaagagagct?cgacttaaca?agattactgg 1140
tttgttggca?tccaaagctg?ttggggttga?tggtgctgaa?aaaaaggaag?actacaatga 1200
aacagctcca?atgctggagc?aggcgatttc?acctaaacct?caaagtcaga?aaaaaaatga 1260
agctgacatt?agcagttctg?ccaacactca?gaaacctgca?ctgttatcct?caactttgtc 1320
ttcagggaag?gctcgcagca?agaaatgcaa?acatgaatct?ggagattctt?ctgggtgtat 1380
aaaaccccct?aaatcaccac?tttccccaga?attaatacaa?gtcgaggatt?tgacgcttgt 1440
atctcagctt?tcttcttcag?tgataaataa?aactagtcct?ccacagcctg?tgaatccccc 1500
tagacctttc?aagcatagtg?agcggagaag?aagatctcag?cgtttagcca?ccttacccat 1560
gcctgatgat?tctgtagaaa?aggtttcttc?tccctctcca?gccactgatg?ggaaagtatt 1620
ctccatcagt?tctcaaaatc?agcaagaatc?ttcagtacca?gaggtgcctg?atgttgcaca 1680
tttgccactt?gagaagctgg?gaccctgtct?ccctcttgac?ttaagtcgtg?gttcagaagt 1740
tacagcaccg?gtagcctcag?attcctctta?ccgtaatgaa?tgtcccaggg?cagaaaaaga 1800
ggatacacag?atgcttccaa?atccttcttc?caaagcaata?gctgatggaa?gaggagctcc 1860
agcagcagca?ggaatatcga?aaacagaaaa?aaaagtgaaa?ttggaagaca?aaagctcaac 1920
agcatttggt?atcaggagtt?gggatttctc?agcactgcta?atgaagatcc?cctcttatag 1980
tccaataagc?ttatcaggac?ttccagagtc?atgacatgaa?cagtttaatt?gaacccatcc 2040
actctgggca?ggtgactgga?atagctgatt?aaaacataaa?tgctgctttt?aggttaacca 2100
caaaggaaca?actcaggatc?agtcgtgatt?gctaaagtat?tcacttttat?cttttaggta 2160
agagaaaaga?aaaagataag?gaaagaagag?agaagagaga?caaagatcac?tacagaccaa 2220
aacagaagaa?gaaga 2235
<210>2
<211>573
<212>PRT
＜213〉people (Homo sapiens)
<400>2
Met?Ser?Lys?Lys?Pro?Pro?Asn?Arg?Pro?Gly?Ile?Thr?Phe?Glu?Ile?Gly
1 5 10 15
Ala?Arg?Leu?Glu?Ala?Leu?Asp?Tyr?Leu?Gln?Lys?Trp?Tyr?Pro?Ser?Arg
20 25 30
Ile?Glu?Lys?Ile?Asp?Tyr?Glu?Glu?Gly?Lys?Met?Leu?Val?His?Phe?Glu
35 40 45
Arg?Trp?Ser?His?Arg?Tyr?Asp?Glu?Trp?Ile?Tyr?Trp?Asp?Ser?Asn?Arg
50 55 60
Leu?Arg?Pro?Leu?Glu?Arg?Pro?Ala?Leu?Arg?Lys?Glu?Gly?Leu?Lys?Asp
65 70 75 80
Glu?Glu?Asp?Phe?Phe?Asp?Phe?Lys?Ala?Gly?Glu?Glu?Val?Leu?Ala?Arg
85 90 95
Trp?Thr?Asp?Cys?Arg?Tyr?Tyr?Pro?Ala?Lys?Ile?Glu?Ala?Ile?Asn?Lys
100 105 110
Glu?Gly?Thr?Phe?Thr?Val?Gln?Phe?Tyr?Asp?Gly?Val?Ile?Arg?Cys?Leu
115 120 125
Lys?Arg?Met?His?Ile?Lys?Ala?Met?Pro?Glu?Asp?Ala?Lys?Gly?Gln?Val
130 135 140
Lys?Ser?Gln?His?Pro?Leu?Ser?Trp?Cys?Cys?Pro?Ile?Asp?Pro?Ala?Gly
145 150 155 160
Ser?Cys?Asn?Gln?Ser?Met?Gly?Ser?Glu?Asp?Trp?Ile?Ala?Leu?Val?Lys
165 170 175
Ala?Ala?Ala?Ala?Ala?Ala?Ala?Lys?Asn?Lys?Thr?Gly?Ser?Lys?Pro?Arg
180 185 190
Thr?Ser?Ala?Asn?Ser?Asn?Lys?Asp?Lys?Asp?Lys?Asp?Glu?Arg?Lys?Trp
195 200 205
Phe?Lys?Val?Pro?Ser?Lys?Lys?Glu?Glu?Thr?Ser?Thr?Cys?Ile?Ala?Thr
210 215 220
Pro?Asp?Val?Glu?Lys?Lys?Glu?Asp?Leu?Pro?Thr?Ser?Ser?Glu?Thr?Phe
225 230 235 240
Gly?Leu?His?Val?Glu?Asn?Val?Pro?Lys?Met?Val?Phe?Pro?Gln?Pro?Glu
245 250 255
Ser?Thr?Leu?Ser?Asn?Lys?Arg?Lys?Asn?Asn?Gln?Gly?Asn?Ser?Phe?Gln
260 265 270
Ala?Lys?Arg?Ala?Arg?Leu?Asn?Lys?Ile?Thr?Gly?Leu?Leu?Ala?Ser?Lys
275 280 285
Ala?Val?Gly?Val?Asp?Gly?Ala?Glu?Lys?Lys?Glu?Asp?Tyr?Asn?Glu?Thr
290 295 300
Ala?Pro?Met?Leu?Glu?Gln?Ala?Ile?Ser?Pro?Lys?Pro?Gln?Ser?Gln?Lys
305 310 315 320
Lys?Asn?Glu?Ala?Asp?Ile?Ser?Ser?Ser?Ala?Asn?Thr?Gln?Lys?Pro?Ala
325 330 335
Leu?Leu?Ser?Ser?Thr?Leu?Ser?Ser?Gly?Lys?Ala?Arg?Ser?Lys?Lys?Cys
340 345 350
Lys?His?Glu?Ser?Gly?Asp?Ser?Ser?Gly?Cys?Ile?Lys?Pro?Pro?Lys?Ser
355 360 365
Pro?Leu?Ser?Pro?Glu?Leu?Ile?Gln?Val?Glu?Asp?Leu?Thr?Leu?Val?Ser
370 375 380
Gln?Leu?Ser?Ser?Ser?Val?Ile?Asn?Lys?Thr?Ser?Pro?Pro?Gln?Pro?Val
385 390 395 400
Asn?Pro?Pro?Arg?Pro?Phe?Lys?His?Ser?Glu?Arg?Arg?Arg?Arg?Ser?Gln
405 410 415
Arg?Leu?Ala?Thr?Leu?Pro?Met?Pro?Asp?Asp?Ser?Val?Glu?Lys?Val?Ser
420 425 430
Ser?Pro?Ser?Pro?Ala?Thr?Asp?Gly?Lys?Val?Phe?Ser?Ile?Ser?Ser?Gln
435 440 445
Asn?Gln?Gln?Glu?Ser?Ser?Val?Pro?Glu?Val?Pro?Asp?Val?Ala?His?Leu
450 455 460
Pro?Leu?Glu?Lys?Leu?Gly?Pro?Cys?Leu?Pro?Leu?Asp?Leu?Ser?Arg?Gly
465 470 475 480
Ser?Glu?Val?Thr?Ala?Pro?Val?Ala?Ser?Asp?Ser?Ser?Tyr?Arg?Asn?Glu
485 490 495
Cys?Pro?Arg?Ala?Glu?Lys?Glu?Asp?Thr?Gln?Met?Leu?Pro?Asn?Pro?Ser
500 505 510
Ser?Lys?Ala?Ile?Ala?Asp?Gly?Arg?Gly?Ala?Pro?Ala?Ala?Ala?Gly?Ile
515 520 525
Ser?Lys?Thr?Glu?Lys?Lys?Val?Lys?Leu?Glu?Asp?Lys?Ser?Ser?Thr?Ala
530 535 540
Phe?Gly?Ile?Arg?Ser?Trp?Asp?Phe?Ser?Ala?Leu?Leu?Met?Lys?Ile?Pro
545 550 555 560
Ser?Tyr?Ser?Pro?Ile?Ser?Leu?Ser?Gly?Leu?Pro?Glu?Ser
565 570
<210>3
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>3
taaatggctt?caggagactt?cag 23
<210>4
<211>24
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>4
ggttttaaat?gcagctccta?tgtg 24
<210>5
<211>34
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>5
atggaatccg?aggatttaag?tggcagagaa?ttga 34
<210>6
<211>37
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>6
tttttttccc?cttttttttt?caaaagtctt?ggaggat 37
<210>7
<211>20
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>7
gcttcctcct?ggaaattgac 20
<210>8
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>8
tctactgtac?agggaaaaac?cca 23
<210>9
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>9
agtcgtggtt?cagaagttac?agc 23
<210>10
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>10
tctcttacca?aatgctgttg?agc 23
<210>11
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>11
catctggcat?ttctgctctc?tat 23
<210>12
<211>25
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>12
ctcagggaaa?ggagaataaa?agaac 25
<210>13
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>13
gaagtatcaa?aactccgctg?tca 23
<210>14
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>14
atgctgagta?gacatgcaga?tga 23
<210>15
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>15
cggtatgcta?atgaagatgg?aga 23
<210>16
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>16
cacagggtat?cagcaactgt?gta 23
<210>17
<211>22
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>17
agaagtatct?gagcccctga?tg 22
<210>18
<211>21
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>18
gtctaacctc?ccagctgttc?c 21
<210>19
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>19
gctgcgtagc?ttacagactt?agc 23
<210>20
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>20
aaggcgttta?aaggtgatag?gtg 23
<210>21
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>21
gtttgcaacc?aggagataca?aag 23
<210>22
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>22
gctgtgaggt?acaacaaatc?aca 23
<210>23
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>23
cctcctttcc?ctagagactc?aat 23
<210>24
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>24
agaagcaaca?gcaagaccac?tac 23
<210>25
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>25
ttgcctatga?aagataggtc?ctg 23
<210>26
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>26
gttttaatgc?ccagatagca?cag 23
<210>27
<211>21
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>27
aggagaagtt?ggaggtggaa?a 21
<210>28
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>28
cagatgaaag?atccaaattc?caa 23
<210>29
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>29
tccacgactt?cttattctcc?ttg 23
<210>30
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>30
catttctttt?agggactggg?gta 23
<210>31
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>31
gagaaactga?agtcccagga?aat 23
<210>32
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>32
ctgatacttc?cattcgcttc?aac 23
<210>33
<211>20
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>33
agctaagcca?tgaggtaggg 20
<210>34
<211>21
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>34
cgcatgtgtg?ttcttctatg?a 21
<210>35
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>35
ccaagacagg?cagagtaggt?aaa 23
<210>36
<211>22
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>36
cattttcatt?gtgatcagcc?ag 22
<210>37
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>37
tgtatggggg?attacctaca?cac 23
<210>38
<211>21
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>38
aaaggagcac?aacaaacatg?c 21
<210>39
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>39
tgtccaagga?gtctgaagtt?ctc 23
<210>40
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>40
cttgccacca?tacctttatt?ctg 23
<210>41
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>41
cgagagagta?ggagttgagg?tga 23
<210>42
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>42
cagaaatcca?gcagatttca?gac 23
<210>43
<211>20
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>43
gaacaggtgg?ctgtgttcct 20
<210>44
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>44
atagaatcaa?gtggtgtgct?tcg 23
<210>45
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>45
ctgagacttt?gagtccttgg?gag 23
<210>46
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>46
ttcctcattt?ctctcagtaa?ccg 23
<210>47
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>47
aacaatgcaa?agtagtgctc?ctc 23
<210>48
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>48
gctgaacttc?tttatgctct?tcg 23
<210>49
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>49
acctttgatt?ttagactgag?ggc 23
<210>50
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>50
acactgggtt?gtgtgttatt?tcc 23
<210>51
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>51
atgagcctct?catccatgtc?ttt 23
<210>52
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>52
agtaagagtc?tgcctgagac?acg 23
<210>53
<211>21
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>53
agaaaatggg?ggtgcaagta?g 21
<210>54
<211>22
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>54
taaccaaatt?aacacgtgct?gg 22
<210>55
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>55
agaaaagttg?gagaagatga?ggg 23
<210>56
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>56
gccacctctg?tgagagagtc?taa 23
<210>57
<211>21
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>57
agaactagtg?tgaccccacc?c 21
<210>58
<211>22
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>58
gcttgccttt?tcccttagta?gg 22
<210>59
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>59
agggaaatga?agacaggaga?act 23
<210>60
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>60
gagacacggc?ttaagaagtt?ttg 23
<210>61
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>61
gcttgtaaag?tcctcggaaa?gtt 23
<210>62
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>62
atctcaactc?tgcatcatct?ggt 23
<210>63
<211>22
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>63
ataagagaaa?tattggccat?cg 22
<210>64
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>64
gcaagcgtaa?gagactggtt?tta 23
<210>65
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>65
caaatattag?gtggagccaa?cac 23
<210>66
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>66
tagatcacct?tggcaaagaa?cac 23
<210>67
<211>22
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>67
acacacagag?aggaggaagt?ct 22
<210>68
<211>22
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>68
gagtctttat?ggagctgtgt?ca 22
<210>69
<211>21
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>69
caggccaagt?gattttaatg?g 21
<210>70
<211>22
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>70
caatacagga?tgcaagttcc?aa 22
<210>71
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>71
acagcccaga?cacaaacaaa?tac 23
<210>72
<211>20
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>72
accccattct?ctccacagac 20
<210>73
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>73
tacaggccag?gatagaaaca?ctc 23
<210>74
<211>22
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>74
gttcaaatat?tgaaagggcc?ac 22
<210>75
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>75
agttatgggt?tcctgtgtgc?tta 23
<210>76
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>76
aaaggcctgt?tcacaagcta?agt 23
<210>77
<211>21
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>77
ctcgtgaagc?ctcagatgtc?c 21
<210>78
<211>21
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>78
ctccaccgaa?aagacccatt?c 21
<210>79
<211>21
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>79
agcgtacacc?ctctgcactt?g 21
<210>80
<211>25
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>80
tttgctgtat?ggtatgtact?caagg 25
<210>81
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>81
cagaagagag?aggagagaac?acg 23
<210>82
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>82
gaggttatct?ctgatggaac?caa 23
<210>83
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>83
cttgaagaag?aacttccaga?cga 23
<210>84
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>84
aatgttctaa?agatgagagg?ggg 23
<210>85
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>85
aggaggctgc?tggtacaaat?act 23
<210>86
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>86
gcaggaaata?cagcaggaac?ata 23
<210>87
<211>22
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>87
attcattctg?gaccaaagat?cc 22
<210>88
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>88
tctactgtgg?acaagaagcc?tgt 23
<210>89
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>89
gtgatctctt?caaggtcaac?tgc 23
<210>90
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>90
ccagatgaga?tgataaggca?aag 23
<210>91
<211>22
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>91
attcgctact?gcaatttaga?gg 22
<210>92
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>92
gtttaatgca?acaggtgaca?acg 23
<210>93
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>93
cacttggatt?ccttgcttgt?tac 23
<210>94
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>94
gggaaaaagt?atgcaacact?cag 23
<210>95
<211>22
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>95
aggcgatgac?ctgaaggtac?tg 22
<210>96
<211>22
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>96
caataggcca?gcaatctcaa?ta 22
<210>97
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>97
aggttctgat?ccgtttccat?atc 23
<210>98
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>98
atctttacat?cctcagtgtt?ggc 23
<210>99
<211>22
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>99
gaagacaaat?ggtgtccaca?aa 22
<210>100
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>100
ccactggaag?ttttcttcgt?aca 23
<210>101
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>101
ttcgttctct?cctctcctct?ctt 23
<210>102
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>102
ggcagcagta?caacaatcta?agc 23
<210>103
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>103
cagcacagag?taggtgaaca?cag 23
<210>104
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>104
cctcagtaca?ttttcaaccc?atc 23
<210>105
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>105
aggatgatga?ggatgactga?aga 23
<210>106
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>106
gaatgggcct?ctatctggta?tct 23
<210>107
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>107
tgtgtctcat?ctgtgaactg?ctt 23
<210>108
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>108
ttcgtgttac?ggtatatcct?gct 23
<210>109
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>109
cttctgttcc?gtaaactcct?tga 23
<210>110
<211>22
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>110
caattgtgta?ctccaaaccc?aa 22
<210>111
<211>22
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>111
gcccttccaa?cttgtcctta?ac 22
<210>112
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>112
gcctctttat?tcccatctcc?tta 23
<210>113
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>113
gaacagatca?ctggtttacc?tcg 23
<210>114
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>114
atctttcagt?aacagacctc?ccc 23
<210>115
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>115
acaagatggc?tagctcaaaa?gtg 23
<210>116
<211>22
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>116
caacacgtgg?tggttctaat?tt 22
<210>117
<211>22
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>117
atgcaggacg?gtaacttcct?gc 22
<210>118
<211>21
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>118
tgggcccagg?aagtcctcct?t 21
<210>119
<211>22
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>119
cccaacatgt?gaagacagtg?at 22
<210>120
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>120
cctgtccacc?tcatgtttta?ttg 23
<210>121
<211>21
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>121
gctgaagtgt?acgaccagga?g 21
<210>122
<211>21
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>122
cacctttatc?cgcactgtag?g 21
<210>123
<211>22
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>123
aaagctgatg?aggacagacc?ag 22
<210>124
<211>22
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>124
ggcagaggca?caatcatttt?ag 22
<210>125
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>125
gaagagaatg?caggtgttga?gtt 23
<210>126
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>126
gtccacagca?ttcataaaac?agg 23
<210>127
<211>22
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>127
ctcctcagtg?tccacacttc?aa 22
<210>128
<211>21
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>128
gttacttgca?gccaaaagca?g 21
<210>129
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>129
agtctctccttt?tcagacat?ccc 23
<210>130
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>130
tccataaagt?cagaccagca?gtt 23
<210>131
<211>21
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>131
ccttctggga?ggacagactt?t 21
<210>132
<211>25
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>132
tttctcttca?ttagacttgg?cctct 25
<210>133
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>133
aacctagcct?cccttcaaac?tta 23
<210>134
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>134
gagacaggat?ggaaaaatct?gtg 23
<210>135
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>135
cctttcctga?cccttttagt?ctt 23
<210>136
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>136
caaatcctgt?atttctcaca?ggc 23
<210>137
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>137
gaaaaaggag?agcatcttgg?act 23
<210>138
<211>21
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>138
aaaggaaaat?gcttccgttc?c 21
<210>139
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>139
taatgtagga?tgacaggctc?tcc 23
<210>140
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>140
ccaattgtat?aaaggctctt?ccc 23
<210>141
<211>21
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>141
aggacaacgt?cagctctcct?g 21
<210>142
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>142
tccactattc?cacccacagt?aac 23
<210>143
<211>21
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>143
gaccgagagt?ccagcatttt?t 21
<210>144
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>144
actgaacaga?gcagacagaa?acc 23
<210>145
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>145
ctgctgttat?taccccattc?aag 23
<210>146
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>146
gtgagtgaca?gatggcaatt?aca 23
<210>147
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>147
ctcgggtaga?atttgatgac?aac 23
<210>148
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>148
gctggtaaag?caggtgtaaa?aga 23
<210>149
<211>21
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>149
ccctgacaac?atcaactggt?c 21
<210>150
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>150
gtccaccttc?gcttttattg?agt 23
<210>151
<211>22
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>151
ctctctgccc?aatgataagg?ag 22
<210>152
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>152
gaaactttct?ctcctcactg?ctc 23
<210>153
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>153
cagaagtttt?gaggactgaa?ctg 23
<210>154
<211>22
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>154
ccgacctacc?ttccctagaa?at 22
<210>155
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>155
ggggttttga?aggatgtgta?ctt 23
<210>156
<211>22
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>156
tatgaggcca?ttctgcacat?ta 22
<210>157
<211>22
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>157
gggagtatga?agtttccatc?tg 22
<210>158
<211>24
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>158
ggatgctggt?ttatttactg?tagg 24
<210>159
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>159
aatatggaat?ccctacccac?agt 23
<210>160
<211>22
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>160
tttgacttca?caacttcatg?gg 22
<210>161
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>161
gaggccattt?tagttctgag?gtt 23
<210>162
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>162
ctttactgca?tatggattct?ggg 23
<210>163
<211>22
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>163
tcaacctcaa?gt?taaaggaa?cg 22
<210>164
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>164
agggaaaagt?agagacaaat?ggg 23
<210>165
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>165
tctaggcaaa?gtggaagtca?aag 23
<210>166
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>166
ctcctagaga?aatgggttgg?att 23
<210>167
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>167
atacactgaa?tgtggaagaa?ccg 23
<210>168
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>168
gggcacacaa?tttcatgtag?tct 23
<210>169
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>169
agacattgca?taccagctct?cat 23
<210>170
<211>24
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>170
cctttacttc?cttcacttta?agcc 24
<210>171
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>171
gtaacaaacg?ccaccttaca?ctc 23
<210>172
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>172
ttctgttctt?gcaactgagt?cct 23
<210>173
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>173
acctccagta?aaagtttctt?ccg 23
<210>174
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>174
gtaaattcag?cttcaaaccc?tgg 23
<210>175
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>175
cattgagcct?tctctgatca?ctc 23
<210>176
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>176
gcactgttac?agatagtctg?ggg 23
<210>177
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>177
tatcagtaac?tgctccgtgt?tca 23
<210>178
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>178
ggtctgtcat?tgaccaaaac?atc 23
<210>179
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>179
tcctgaataa?aggcctagta?ccc 23
<210>180
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>180
aaaccagaat?ccaacactac?cct 23
<210>181
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>181
gagccctctc?cacatttcta?ttt 23
<210>182
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>182
acactgaaac?gtgatgggta?act 23
<210>183
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>183
gacatgagtc?cgaaacaact?acc 23
<210>184
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>184
atgagactgt?accaaatgat?ggc 23
<210>185
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>185
tgatacctgg?aggcttatct?gag 23
<210>186
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>186
gactcagtag?ccagttgaag?gaa 23
<210>187
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>187
tcttgcaggt?ctggctattt?tag 23
<210>188
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>188
tatatttaag?gagcacccct?tcc 23
<210>189
<211>22
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>189
acaagcaagt?gcattttcag?tc 22
<210>190
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>190
gaacagggta?gccattaaca?caa 23
<210>191
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>191
tctatcatcg?acgtctacca?caa 23
<210>192
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>192
gctactcttt?gtggctttct?tca 23
<210>193
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>193
gcatgatcat?agacgtcttt?tcc 23
<210>194
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>194
gatgaactca?ctgaagtcca?cct 23
<210>195
<211>21
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>195
tctccaggac?aagatcaagg?a 21
<210>196
<211>22
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>196
gttttatttc?cagcatgttc?cc 22
<210>197
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>197
agagctgatc?aagttcatgt?gtg 23
<210>198
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>198
acatagcaag?ttcgagtttc?tgc 23
<210>199
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>199
gagctggagg?taggaataca?ggt 23
<210>200
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>200
caatagtttg?gcttggtgta?agg 23
<210>201
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>201
ctcctctgca?tgcacttaac?ttt 23
<210>202
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>202
gagagtttaa?tgttgtggga?agg 23
<210>203
<211>22
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>203
ccgggcaata?aagtaactct?tg 22
<210>204
<211>25
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>204
gtatttgtct?gtatgcctac?atctg 25
<210>205
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>205
tctgcgtatc?ttgagtgctt?aca 23
<210>206
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>206
acagagatgt?ggtggtgcta?gtt 23
<210>207
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>207
agcagaggat?cagagctttc?ttt 23
<210>208
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>208
agaaaaggtg?tgaacagagt?tgc 23
<210>209
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>209
agagccatag?aaactgctcc?tct 23
<210>210
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>210
cataactgca?tagacagcac?gtc 23
<210>211
<211>21
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>211
tacctgctct?atgtgggtgc?t 21
<210>212
<211>25
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>212
cctcagaact?ctcagtttat?tcctg 25
<210>213
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>213
ataagccaca?gagacaaacc?aga 23
<210>214
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>214
gggaggttat?tttcacagaa?cac 23
<210>215
<211>22
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>215
gagttcctgt?ctctctgcca?ac 22
<210>216
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>216
taatatacaa?gggctcaacc?gag 23
<210>217
<211>21
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>217
gtcatagctg?tgtcctgggt?c 21
<210>218
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>218
ctattttatc?cccatggcag?agt 23
<210>219
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>219
cagatattct?gtatgctgga?ggg 23
<210>220
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>220
ccatctcaga?agggctttat?ttc 23
<210>221
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>221
gatttccata?cttcgggaga?aac 23
<210>222
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>222
tatcagatgc?cacacatacg?aga 23
<210>223
<211>22
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>223
atggaacaaa?gaagctgtga?cc 22
<210>224
<211>22
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>224
gggtacatgc?aaaccagtac?ac 22
<210>225
<211>20
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>225
tgaacagttt?gctggtcttg 20
<210>226
<211>20
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>226
aatgtcaggt?tggggagtta 20
<210>227
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>227
ttctggacag?acggagagac?tac 23
<210>228
<211>22
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>228
agtgatgaca?tacccctggt?tc 22
<210>229
<211>22
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>229
caagacttct?cagatccttg?gg 22
<210>230
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>230
actcacatgt?ggaagtgttc?ctt 23
<210>231
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>231
tcaagcaata?tgaagtaggg?ctc 23
<210>232
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>232
aacacaaatg?tcccgtgtaa?gtc 23
<210>233
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>233
ctgcctctta?ctcgtcacag?ttt 23
<210>234
<211>22
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>234
tgacttcttt?gaagtgaagg?ct 22
<210>235
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>235
ccctagtttt?tgtagctgtc?gaa 23
<210>236
<211>22
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>236
gatcacatgc?caagaacaca?at 22
<210>237
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>237
ctacgtacct?gggtgcctat?atc 23
<210>238
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>238
gtcctcttat?aaggctcact?ccc 23
<210>239
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>239
gatgttagag?actccttcac?cca 23
<210>240
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>240
cggtattctt?aacacatctt?gcc 23
<210>241
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>241
gtgtctgcgt?atctacctga?acg 23
<210>242
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>242
ataactctgt?cttcgtgagc?tgg 23
<210>243
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>243
gtatttggct?tactgtccca?aac 23
<210>244
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>244
ctaggaagaa?atcatgctgg?gtt 23
<210>245
<211>22
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>245
cagtttgagc?aagcaaaaga?tg 22
<210>246
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>246
cggatatccc?taatctattc?cca 23
<210>247
<211>22
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>247
gacagtatag?ctgcccttgc?tc 22
<210>248
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>248
aagcagtggg?gtagagtcag?aac 23
<210>249
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>249
acagaagaag?ctacctcagg?tgt 23
<210>250
<211>22
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>250
ctagcggaag?acaattcaga?ac 22
<210>251
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>251
taaccttgat?agaagaacct?tgg 23
<210>252
<211>22
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>252
gcaaatgaga?caaaattggg?ac 22
<210>253
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>253
atctccactc?tacggccttt?tac 23
<210>254
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>254
taatgactta?aacaccagca?cgg 23
<210>255
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>255
gtgttctcct?aatcccagaa?cct 23
<210>256
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>256
aagagttgtg?gcctattacc?tcc 23
<210>257
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>257
tggtcctact?aagagaatgc?agc 23
<210>258
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>258
agccattagg?aaaaagagca?gag 23
<210>259
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>259
gactgctata?ctccaactct?ggg 23
<210>260
<211>24
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>260
gccaaagaca?tggtttagtc?atac 24
<210>261
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>261
acactgagct?ttaatggctg?aag 23
<210>262
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>262
tccacagtga?cctgacacaa?tag 23
<210>263
<211>22
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>263
gtcctcattc?cctttctgtt?cc 22
<210>264
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>264
ctgttttctt?tcaacctgca?ctc 23
<210>265
<211>21
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>265
aagagaggcc?agaaactgag?c 21
<210>266
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>266
aactagcagc?tttattgccc?ttc 23
<210>267
<211>20
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>267
gtggacatct?aattgaggcc 20
<210>268
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>268
gaagatcttc?cactagtaat?att 23
<210>269
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>269
cagaggactc?tgatgaagaa?agc 23
<210>270
<211>22
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>270
tttccacaaa?cgctaagaga?ac 22
<210>271
<211>21
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>271
atgtctgctc?cgtgagtgtc?t 21
<210>272
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>272
gcaaatccta?ctttcaactg?cac 23
<210>273
<211>22
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>273
gccttaaagc?tggacacaga?ag 22
<210>274
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>274
ctccagacac?cattgcttaa?atc 23
<210>275
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>275
agactttaaa?atcccacctg?gac 23
<210>276
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>276
cacccagcct?tctctttatt?ttc 23
<210>277
<211>21
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>277
aggggattct?ggaactgaat?g 21
<210>278
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>278
ttataccgag?gagatgggaa?agt 23
<210>279
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>279
gttgcagtac?caatcctttc?ttg 23
<210>280
<211>24
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>280
gtcctatgtt?aatttccacc?aagc 24
<210>281
<211>21
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>281
tatccagagg?gtgtccctga?c 21
<210>282
<211>24
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>282
gttctttaat?gacagttcaa?gggg 24
<210>283
<211>22
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>283
atcggatcga?tattacacag?ca 22
<210>284
<211>22
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>284
cccatcaggg?aaacaaagat?ta 22
<210>285
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>285
tgcatctgta?acttcaggag?gat 23
<210>286
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>286
tccatcaact?tacctatcga?tgc 23
<210>287
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>287
aaacctacga?acgccttttc?tac 23
<210>288
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>288
ggtatcacag?gagcaccaat?aaa 23
<210>289
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>289
ctttctgttg?ctttcccagt?aga 23
<210>290
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>290
ttgatacatt?acactggtgg?cag 23
<210>291
<211>21
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>291
acccacagaa?ctgggagtga?g 21
<210>292
<211>22
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>292
attttactgc?agaaacgggt?tg 22
<210>293
<211>24
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>293
gatggggaaa?ctatgactaa?tgac 24
<210>294
<211>25
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>294
ggtatcaata?aagcccagat?attcc 25
<210>295
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>295
ttagtggatc?tggctcttct?tgt 23
<210>296
<211>21
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>296
caggcacatc?acagttgtca?c 21
<210>297
<211>22
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>297
gatttggaac?ttggaaggag?tg 22
<210>298
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>298
acttcagtca?cccaaaacaa?cag 23
<210>299
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>299
cgggaggatt?gtaagatact?gtg 23
<210>300
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>300
acttctcatg?agttcagcct?cag 23
<210>301
<211>22
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>301
gtgagtattc?ctccgttagc?tt 22
<210>302
<211>22
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>302
cagggagaag?agaaaacatc?ac 22
<210>303
<211>20
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>303
agctgaagct?gactgtgtct 20
<210>304
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>304
aggcacagac?ggtattgttg?tag 23
<210>305
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>305
gactttcaaa?caacccagtg?tct 23
<210>306
<211>22
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>306
ctctagccag?cttcttcctc?ac 22
<210>307
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>307
ggtcttcata?cgctgtactt?gct 23
<210>308
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>308
tatgccttca?ctgatccacc?tac 23
<210>309
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>309
tcctgtggaa?atagaactgt?cgt 23
<210>310
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>310
cacaaagttc?aaggaagcag?tct 23
<210>311
<211>22
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>311
aaggttctct?accgcctcaa?gt 22
<210>312
<211>21
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>312
ctgaacacac?cgtggcttta?t 21
<210>313
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>313
aagaagccac?cactattcct?ctc 23
<210>314
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>314
cctgaaggac?tgaaaaggtc?ata 23
<210>315
<211>22
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>315
cctgtctcca?aaggaaaaac?aa 22
<210>316
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>316
ctcagtttca?tcaagtcctt?tgc 23
<210>317
<211>22
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>317
agcgaggaga?actcttgaaa?tc 22
<210>318
<211>22
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>318
gtgtcccacc?atagaaaact?tc 22
<210>319
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>319
gaagccagcc?tactccttct?tac 23
<210>320
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>320
tagcattcac?agagcaggag?att 23
<210>321
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>321
catatgtgga?ggtgctgtgt?aaa 23
<210>322
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>322
gtctacagtt?agacagggaa?gcc 23
<210>323
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>323
gacagctctt?ggatccctat?ttt 23
<210>324
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>324
agagtgaact?tgcatctgtt?cct 23
<210>325
<211>22
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>325
gtgtgtgtac?gtgtctccag?gt 22
<210>326
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>326
cagacaagat?agctgactct?ccc 23
<210>327
<211>21
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>327
gaagtctggg?ggtgttggtc?t 21
<210>328
<211>27
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>328
ataaagactt?gtctagactc?cactggg 27
<210>329
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>329
gaacagtgtt?tggtctggaa?tgt 23
<210>330
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>330
ggatatgaga?aaggaaggca?agt 23
<210>331
<211>22
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>331
atcgtgagca?tcatcagaga?ag 22
<210>332
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>332
agacacacag?acaaacatgc?aga 23
<210>333
<211>22
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>333
gcactaccca?gacatcttcg?ag 22
<210>334
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>334
tgggtggcaa?gtctaatcta?ttc 23
<210>335
<211>22
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>335
gatccgaaga?aactggctac?tg 22
<210>336
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>336
aggtcctgct?ctctttgtcc?tat 23
<210>337
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>337
gagtcttccc?cattttcagt?cat 23
<210>338
<211>22
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>338
ctacatttat?gtggcacgaa?gg 22
<210>339
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>339
ctttggctta?tttacagagc?tgg 23
<210>340
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>340
aggaggctaa?aggcaatgaa?tag 23
<210>341
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>341
gagtcttgaa?gctctgtttg?gtg 23
<210>342
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>342
aacatcctga?cagtcatcca?cat 23
<210>343
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>343
agtgtctgca?accttgcttt?aac 23
<210>344
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>344
agtccagggc?ataaaaccta?aac 23
<210>345
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>345
atgtgtgtgt?gttcatcttc?cag 23
<210>346
<211>22
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>346
atccattttc?tcacaagcag?tg 22
<210>347
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>347
ggggaatata?tatcctctgt?ggc 23
<210>348
<211>22
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>348
aaaaacaact?gaggtgatgg?gt 22
<210>349
<211>21
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>349
atgaaggaca?gcaaccagtt?c 21
<210>350
<211>21
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>350
caatgctggt?ttattcccca?t 21
<210>351
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>351
gtgaaaaagt?ggaatgcagt?agc 23
<210>352
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>352
ttaggtaaca?gcagggaaag?tca 23
<210>353
<211>21
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>353
cagtcctgtg?actcaactca?a 21
<210>354
<211>22
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>354
cgagtttcac?ctcagctctt?ct 22
<210>355
<211>22
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>355
ggatgtagca?atctccacca?gt 22
<210>356
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>356
gttcaaacac?tcactgaaga?gcc 23
<210>357
<211>21
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>357
ctccactcgc?tcttccaaca?c 21
<210>358
<211>22
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>358
ctttttacct?tcgtgcacct?tt 22
<210>359
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>359
gacagcaaag?tcttgactcc?ttc 23
<210>360
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>360
aaagtggctg?ggagtaaggt?atc 23
<210>361
<211>22
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>361
agggcacaca?ttcatctttg?ta 22
<210>362
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>362
gttaccaaag?acagacacat?tgg 23
<210>363
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>363
ctcagcaaga?gaagaaccgt?tta 23
<210>364
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>364
ccacttagaa?atcgaatacg?tcc 23
<210>365
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>365
tacccaagtc?agaaagactc?tgc 23
<210>366
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>366
ggtggccttc?tctcaaaatt?agt 23
<210>367
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>367
cgctgataat?attcctcgtc?cta 23
<210>368
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>368
agtttttaga?gtttcagggg?gtc 23
<210>369
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>369
ctccctaggg?gtagactctt?ctg 23
<210>370
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>370
gagactaggc?ctcttttctg?gat 23
<210>371
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>371
ttccagctat?tcttcagatg?ctc 23
<210>372
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>372
tatatggcag?gtttgtgtgt?ctg 23
<210>373
<211>20
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>373
atgctgcgaa?cagagagctg 20
<210>374
<211>20
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>374
aatgaaccct?gctgaccttc 20
<210>375
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>375
tgagtctcct?cttggtgatt?ctg 23
<210>376
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>376
ggaagagcaa?agagagcttc?atc 23
<210>377
<211>21
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>377
gctcaagtcc?aaacagcact?c 21
<210>378
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>378
acatacacag?ggacgctgta?aac 23
<210>379
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>379
tcctagggga?ctcttgagct?tag 23
<210>380
<211>22
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>380
ataaataggt?acccgtgagc?cc 22
<210>381
<211>22
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>381
tatgtgctac?ccacaacacc?tc 22
<210>382
<211>22
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>382
gtttgagagg?aacaaccagg?ag 22
<210>383
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>383
agtcttggtt?tacctgtggt?gac 23
<210>384
<211>21
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>384
aaaacaaaac?cccagaaacc?c 21
<210>385
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>385
gggactactg?tgttttgctg?ttc 23
<210>386
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>386
tgaggtcata?gatttcaagg?cac 23
<210>387
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>387
taatagtacc?agccatcgct?cag 23
<210>388
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>388
atcctacggc?tttattgaca?cct 23
<210>389
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>389
cagccagttc?tcagacactt?agg 23
<210>390
<211>21
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>390
gtactcgagc?catctggcct?t 21
<210>391
<211>22
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>391
acttttgtgg?tgtccccaag?ta 22
<210>392
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>392
ctgtgtaccc?tttacccatt?cct 23
<210>393
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>393
actagagaaa?tgaggggcgt?atc 23
<210>394
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>394
atctctaacc?aaacatcgta?gcg 23
<210>395
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>395
ctgaggcagc?tttatttcct?aca 23
<210>396
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>396
actggtgggg?ttacataacc?ttt 23
<210>397
<211>25
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>397
ggtagtgaaa?tatggacaaa?ggaca 25
<210>398
<211>22
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>398
acttctgcca?tgtcgtcttt?tt 22
<210>399
<211>22
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>399
acaaagagga?gaaggctgac?ct 22
<210>400
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>400
ctcctcgctg?ggtagaacta?act 23
<210>401
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>401
ggaaggaaag?gaactacgaa?atc 23
<210>402
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>402
gttaaaagga?gcacagggac?ata 23
<210>403
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>403
ctccttactt?gtgggatcaa?atg 23
<210>404
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>404
atgtgctaga?attacagccc?tga 23
<210>405
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>405
aggagctgag?tgtgttagag?gtg 23
<210>406
<211>22
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>406
ataaacctgg?atgctgacgc?tc 22
<210>407
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>407
agacctaagt?ctggaacaga?gcc 23
<210>408
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>408
ctacagcact?catttggaaa?agg 23
<210>409
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>409
ttggtcctcc?tctgtttcat?aga 23
<210>410
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>410
gcttctcccc?agttacaaga?gac 23
<210>411
<211>21
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>411
gtactgaagg?acctgccaag?g 21
<210>412
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>412
gggaaagcca?gctttattga?gta 23
<210>413
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>413
agttttggat?gactctgctc?aag 23
<210>414
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>414
ggcatttacg?agcattatct?gac 23
<210>415
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>415
cagtttcagt?cccaggtcat?act 23
<210>416
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>416
ggcatactct?ttggtgagaa?atg 23
<210>417
<211>22
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>417
ctaccctgaa?ggggaagaaa?ag 22
<210>418
<211>22
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>418
aacacaccct?acatccaagg?tc 22
<210>419
<211>22
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>419
cttcagagga?aatctcccag?tc 22
<210>420
<211>22
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>420
ggcgttatct?cgttgtactc?gt 22
<210>421
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>421
aaagctgaat?acagaaggca?ctg 23
<210>422
<211>23
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the primer sequence of the synthetic of RT-PCR
<400>422
tttactgaca?ggtggtgaaa?ggt 23
<210>423
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>423
taaatcctcg?gattccat 18
<210>424
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>424
taccttaggc?tcctaaat 18
<210>425
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>425
cgccgcgcgc?ctcatgct 18
<210>426
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>426
tcgtactccg?cgcgccgc 18
<210>427
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>427
cggccgcact?caccggca 18
<210>428
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>428
acggccactc?acgccggc 18
<210>429
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>429
acgactgctt?gaaagagg 18
<210>430
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>430
ggagaaagtt?cgtcagca 18
<210>431
<211>19
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>431
agtgcactcg?gatcttgct 19
<210>432
<211>19
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>432
tcgttctagg?ctcacgtga 19
<210>433
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>433
gcctcctgta?ctggattt 18
<210>434
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>434
tttaggtcat?gtcctccg 18
<210>435
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>435
ttgactggtt?tcttatgt 18
<210>436
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>436
tgtattcttt?ggtcagtt 18
<210>437
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>437
ctccgtaaac?ggatccat 18
<210>438
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>438
tacctaggca?aatgcctc 18
<210>439
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>439
cggatccatc?gccccagg 18
<210>440
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>440
ggaccccgct?acctaggc 18
<210>441
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>441
accaaagacg?catcatca 18
<210>442
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>442
actactacgc?agaaacca 18
<210>443
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>443
ccctcgattc?ctccgagt 18
<210>444
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>444
tgagcctcct?tagctccc 18
<210>445
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>445
aactgccaca?cagtagta 18
<210>446
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>446
atgatgacac?accgtcaa 18
<210>447
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>447
atcctcgctg?tccagggc 18
<210>448
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>448
cgggacctgt?cgctccta 18
<210>449
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>449
cgtccaggtg?cagccact 18
<210>450
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>450
tcaccgacgt?ggacctgc 18
<210>451
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>451
gttcccattc?aagaacat 18
<210>452
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>452
tacaagaact?tacccttg 18
<210>453
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>453
catgagtgat?ggtggctc 18
<210>454
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>454
ctcggtggta?gtgagtac 18
<210>455
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>455
cctctcccat?ggcttcaa 18
<210>456
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>456
aacttcggta?ccctctcc 18
<210>457
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>457
ggacaggaac?caatgtac 18
<210>458
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>458
catgtaacca?aggacagg 18
<210>459
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>459
acaatacaat?gtgacaag 18
<210>460
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>460
gaacagtgta?acataaca 18
<210>461
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>461
agattccatt?ctgcaaac 18
<210>462
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>462
caaacgtctt?accttaga 18
<210>463
<211>19
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>463
gattaaaatt?agattccat 19
<210>464
<211>19
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>464
taccttagat?taaaattag 19
<210>465
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>465
catcttgaga?tcctattc 18
<210>466
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>466
cttatcctag?agttctac 18
<210>467
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>467
tgggggcttt?ttactcat 18
<210>468
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>468
tactcatttt?tcgggggt 18
<210>469
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>469
aggtacttta?aaccactt 18
<210>470
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>470
ttcaccaaat?ttcatgga 18
<210>471
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>471
aggagccatg?gcgctcgg 18
<210>472
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>472
ggctcgcggt?accgagga 18
<210>473
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>473
gcaatccatg?gctgtggc 18
<210>474
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>474
cggtgtcggt?acctaacg 18
<210>475
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>475
aataattacc?ttgtatta 18
<210>476
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>476
tacctaacgt?ttctatct 18
<210>477
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>477
gaagttaccg?tcctgcat 18
<210>478
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>478
tacgtcctgc?cattgaag 18
<210>479
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>479
gcaggaagtt?accgtcct 18
<210>480
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>480
tcctgccatt?gaaggacg 18
<210>481
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>481
gttgttgagc?acagctat 18
<210>482
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>482
tatcgacacg?agttgttg 18
<210>483
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>483
gaagtcctcc?ttccgata 18
<210>484
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>484
atagccttcc?tcctgaag 18
<210>485
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>485
ccgtcgccat?cttgcgtc 18
<210>486
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>486
ctgcgttcta?ccgctgcc 18
<210>487
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>487
tatggtcgcc?gtcgccat 18
<210>488
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>488
taccgctgcc?gctggtat 18
<210>489
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>489
ggactgcatg?gtggagat 18
<210>490
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>490
tagaggtggt?acgtcagg 18
<210>491
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>491
catggtggag?atggcgac 18
<210>492
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>492
cagcggtaga?ggtggtac 18
<210>493
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>493
agcagggctg?cagaatgg 18
<210>494
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>494
ggtaagacgt?cgggacga 18
<210>495
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>495
tgctcttgaa?gtcgggac 18
<210>496
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>496
cagggatgaa?gttctcgt 18
<210>497
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>497
gcagttgaga?tgattatt 18
<210>498
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>498
ttattagtag?agttgacg 18
<210>499
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>499
caaaatcatt?tcctcctc 18
<210>500
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>500
ctcctccttt?actaaaac 18
<210>501
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>501
cgggccacca?tcacggaa 18
<210>502
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>502
aaggcactac?caccgggc 18
<210>503
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>503
acgattcatt?gctgcctt 18
<210>504
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>504
ttccgtcgtt?acttagca 18
<210>505
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>505
acacaagaca?cgattcat 18
<210>506
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>506
tacttagcac?agaacaca 18
<210>507
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>507
atccactctt?ccgttcat 18
<210>508
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>508
tacttgcctt?ctcaccta 18
<210>509
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>509
agccgccatc?tccacagt 18
<210>510
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>510
tgacacctct?accgccga 18
<210>511
<211>19
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>511
cttgttcatg?aacatctct 19
<210>512
<211>19
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>512
tctctacaag?tacttgttc 19
<210>513
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>513
tggcaggagg?gttcttgt 18
<210>514
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>514
tacttgttct?tgggagga 18
<210>515
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>515
caggcctacc?tggcagga 18
<210>516
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>516
aggacggtcc?atccggac 18
<210>517
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>517
accgcttacg?gttggctg 18
<210>518
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>518
gtcggttggc?attcgcca 18
<210>519
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>519
tctgaagaaa?atagatca 18
<210>520
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>520
actagataaa?agaagtct 18
<210>521
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>521
gtcaaccaga?cccggcat 18
<210>522
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>522
tacggcccag?accaactg 18
<210>523
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>523
tctcctttct?cgatcata 18
<210>524
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>524
atagtagctc?tttcctct 18
<210>525
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>525
attccttgtg?ggcctcaa 18
<210>526
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>526
aactccgggt?gttcctta 18
<210>527
<211>17
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>527
cccatgcgag?ctgcgcc 17
<210>528
<211>17
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>528
ccgcgtcgag?cgtaccc 17
<210>529
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>529
agtgataaac?agaaagcg 18
<210>530
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>530
gcgaaagaca?aatagtga 18
<210>531
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>531
tatcctcgac?tttgactt 18
<210>532
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the S-oligonucleotide sequence of the synthetic of antisense method
<400>532
ttcagtttca?gctcctat 18
<210>533
<211>19
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the oligonucleotide sequence of the synthetic of RNAi
<400>533
ggaccaagct?agacaagca 19
<210>534
<211>19
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the oligonucleotide sequence of the synthetic of RNAi
<400>534
acagtgttcc?gctaagtga 19
<210>535
<211>19
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the oligonucleotide sequence of the synthetic of RNAi
<400>535
ccagttgagt?cgacatctg 19
<210>536
<211>19
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the oligonucleotide sequence of the synthetic of RNAi
<400>536
gcagcagata?ccatcagtg 19
<210>537
<211>20
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the oligonucleotide sequence of the synthetic of RNAi
<400>537
cgcagctgcg?aagtgttgta 20
<210>538
<211>19
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the oligonucleotide sequence of the synthetic of RNAi
<400>538
gatacgaaag?cagctgcga 19
<210>539
<211>19
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the oligonucleotide sequence of the synthetic of RNAi
<400>539
gagcgattca?tcttcatca 19
<210>540
<211>19
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the oligonucleotide sequence of the synthetic of RNAi
<400>540
ctgcaattga?ggctccttc 19
<210>541
<211>19
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the oligonucleotide sequence of the synthetic of RNAi
<400>541
gagtgtgctg?gtgaagcag 19
<210>542
<211>19
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the oligonucleotide sequence of the synthetic of RNAi
<400>542
gatcaagtcc?tgcacactg 19
<210>543
<211>19
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the oligonucleotide sequence of the synthetic of RNAi
<400>543
cgtgctagca?gctgcgtgt 19
<210>544
<211>19
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the oligonucleotide sequence of the synthetic of RNAi
<400>544
tgaggtgctc?agcacagtg 19
<210>545
<211>19
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the oligonucleotide sequence of the synthetic of RNAi
<400>545
cggaggatct?catgaccac 19
<210>546
<211>19
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the oligonucleotide sequence of the synthetic of RNAi
<400>546
gattcgcatc?ctgccatcg 19
<210>547
<211>19
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the oligonucleotide sequence of the synthetic of RNAi
<400>547
cagtattcgg?acatagagg 19
<210>548
<211>19
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the oligonucleotide sequence of the synthetic of RNAi
<400>548
caccaagtac?tgcttgtgc 19
<210>549
<211>19
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the oligonucleotide sequence of the synthetic of RNAi
<400>549
ggagaagaac?actgtggac 19
<210>550
<211>19
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the oligonucleotide sequence of the synthetic of RNAi
<400>550
gacaaattga?gtggcagca 19
<210>551
<211>19
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the oligonucleotide sequence of the synthetic of RNAi
<400>551
gagattcaga?gtggacgaa 19
<210>552
<211>19
<212>DNA
＜213〉artificial
<220>
＜223〉be used for the oligonucleotide sequence of the synthetic of RNAi
<400>552
gagagcaatg?aggatgact 19

Claims (13)

1. diagnose the nonsmall-cell lung cancer among the experimenter or suffer from the tendentious method of nonsmall-cell lung cancer for one kind, this method comprises the nonsmall-cell lung cancer Expression of Related Genes level in the biological sample that is derived from this experimenter of measuring, wherein said non-small cell lung cancer associated gene is NSC 810, and wherein said level is compared with the normal control level of this gene to raise or reduce and shown that this experimenter suffers from or easily suffers from nonsmall-cell lung cancer.
2. the described method of claim 1, wherein said rising is than described normal control level height at least 10%.
3. the described method of claim 1, wherein said level are with the arbitrary method mensuration that is selected from down group:
(1) mRNA of detection non-small cell lung cancer associated gene;
(2) detect the related gene coded albumen of nonsmall-cell lung cancer; And
(3) detect the coded proteic biological activity of non-small cell lung cancer associated gene.
4. the described method of claim 1, wherein said level are to measure by the hybridization between the genetic transcription thing that detects non-small cell lung cancer associated gene probe and described biological sample from the patient.
5. the described method of claim 4, wherein said hybridization step is implemented on the DNA array.
6. the described method of claim 1, wherein said biological sample comprises phlegm or blood.
7. method that screening is used for the treatment of or prevents the compound of nonsmall-cell lung cancer, this method comprises the following steps:
(1) test-compound is contacted with the polynucleotide encoded polypeptide of NSC 810;
(2) measure the activity that combine between described polypeptide and the test-compound; And
(3) select and described polypeptide bonded compound.
8. method that screening is used for the treatment of or prevents the compound of nonsmall-cell lung cancer, this method comprises the following steps:
(a) test-compound is contacted with the polynucleotide encoded polypeptide of NSC 810;
(b) biological activity of the polypeptide of detection step (a); And
(c) select such compound, the biological activity of the coded polypeptide of polynucleotide that described compound makes NSC 810 during with this test-compound not detected biological activity compare and be suppressed.
9. the described method of claim 8, wherein said biological activity is a cell-proliferation activity.
10. method that screening is used for the treatment of or prevents the compound of nonsmall-cell lung cancer, this method comprises the following steps:
(1) makes the cells contacting of test-compound and presentation markup gene NSC 810; And
(2) select the compound of the expression level that can reduce NSC 810 marker gene.
11. the described method of claim 10, wherein said cell are the NSCLC cell.
12. a screening is used for the treatment of or prevents the method for the compound of nonsmall-cell lung cancer, this method comprises the following steps::
(1) make test-compound and the cells contacting that has imported a kind of carrier, this carrier contains the transcription regulatory region of marker gene NSC 810 and the reporter gene of expressing under described transcription regulatory region control;
(2) activity of the described reporter gene of mensuration; And
(3) select the compound that the expression level that makes described reporter gene reduces compared with the control.
13. a test kit, wherein contain can with NSC810 gene or its encoded polypeptides bonded detection reagent.
CNA2008101867066A 2002-09-30 2003-09-22 Method for diagnosing non-small cell lung cancers Pending CN101586155A (en)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US41467302P 2002-09-30 2002-09-30
US60/414,673 2002-09-30
US60/451,374 2003-02-28
US60/466,100 2003-04-28

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
CNA038255065A Division CN1705753A (en) 2002-09-30 2003-09-22 Method for diagnosing non-small cell lung cancers

Publications (1)

Publication Number Publication Date
CN101586155A true CN101586155A (en) 2009-11-25

Family

ID=35578308

Family Applications (3)

Application Number Title Priority Date Filing Date
CN2006100738054A Expired - Fee Related CN1854313B (en) 2002-09-30 2003-09-22 Method for diagnosing non-small cell lung cancers
CNA038255065A Pending CN1705753A (en) 2002-09-30 2003-09-22 Method for diagnosing non-small cell lung cancers
CNA2008101867066A Pending CN101586155A (en) 2002-09-30 2003-09-22 Method for diagnosing non-small cell lung cancers

Family Applications Before (2)

Application Number Title Priority Date Filing Date
CN2006100738054A Expired - Fee Related CN1854313B (en) 2002-09-30 2003-09-22 Method for diagnosing non-small cell lung cancers
CNA038255065A Pending CN1705753A (en) 2002-09-30 2003-09-22 Method for diagnosing non-small cell lung cancers

Country Status (1)

Country Link
CN (3) CN1854313B (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102243240A (en) * 2010-05-11 2011-11-16 中国科学院上海生命科学研究院 Non-small cell lung cancer molecular marker and its application
CN106148525A (en) * 2011-05-23 2016-11-23 细胞信号科技公司 ROS kinases in pulmonary carcinoma

Families Citing this family (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8541183B2 (en) 2007-09-11 2013-09-24 Cancer Prevention And Cure, Ltd. Methods of identification, assessment, prevention and therapy of lung diseases and kits thereof
ES2703714T3 (en) * 2009-03-12 2019-03-12 Cancer Prevention & Cure Ltd Methods of identification, evaluation, prevention and therapy of pulmonary diseases and kits thereof, including the identification, evaluation, prevention and therapy of diseases on the basis of gender
MY148542A (en) * 2009-06-15 2013-04-30 Cancer Res Initiatives Foundation A method for the assessment of cancer in a biological sample obtained from a subject
EP2617730B1 (en) * 2010-09-17 2018-03-07 National Institute of Advanced Industrial Science And Technology Lung cancer differential marker
CA2943636C (en) 2014-03-25 2020-06-09 Quest Diagnostics Investments Incorporated Detection of gene fusions by intragenic differential expression (ide) using average cycle thresholds
EP2933639A1 (en) * 2014-04-16 2015-10-21 Deutsches Krebsforschungszentrum Stiftung des Öffentlichen Rechts S100p and Hyaluronic acid as biomarkers for metastatic breast cancer
CN115097133A (en) * 2015-09-09 2022-09-23 私募蛋白质体操作有限公司 Methods for developing personalized drug treatment plans and targeted drug development based on proteomic profiles
CN107974503A (en) * 2016-10-20 2018-05-01 上海透景诊断科技有限公司 Multiple lung cancer related genes methylate combined detection kit, associated detecting method and application
US20190360051A1 (en) * 2017-02-17 2019-11-28 Stichting Vumc Swarm intelligence-enhanced diagnosis and therapy selection for cancer using tumor- educated platelets
EP3607089A4 (en) 2017-04-04 2020-12-30 Lung Cancer Proteomics, LLC Plasma based protein profiling for early stage lung cancer prognosis
CN107574248A (en) * 2017-09-28 2018-01-12 郑州大学第附属医院 A kind of the non-small cell lung cancer auxiliary diagnosis based on GALNT2 genes, prognostic evaluation kit and its application method
CN109971853A (en) * 2019-03-14 2019-07-05 中国科学院北京基因组研究所 One kind molecular marker relevant to Diagnosis of Non-Small Cell Lung and its application
CN112129938B (en) * 2019-06-25 2023-08-18 中国科学院分子细胞科学卓越创新中心 Use of UDP-Glc in lung cancer metastasis assessment
CN112213492B (en) * 2019-07-09 2024-04-30 复旦大学 Application of CLIC4 in preparation of preparation for treating nasopharyngeal carcinoma by radiation
CN110484628B (en) * 2019-07-31 2022-06-14 扬州大学 Molecular marker related to golden black chicken abdominal fat character and application
CN111983233B (en) * 2020-08-17 2021-05-11 江苏省人民医院（南京医科大学第一附属医院） Antibody composition for identifying cancer stem cell components in gastric poorly differentiated adenocarcinoma and application thereof
CN112379095A (en) * 2020-11-02 2021-02-19 无锡市人民医院 Type 2diabetes patient lung adenocarcinoma diagnosis marker PZP and ELISA kit thereof
CN112433051B (en) * 2020-11-23 2022-07-08 四川大学华西医院 Application of platelet activating factor acetylhydrolase detection reagent in preparation of lung cancer screening kit
CN112535734A (en) * 2021-01-15 2021-03-23 辽宁省肿瘤医院 Application of reagent for inhibiting expression of LncRNA-RP5 in preparation of medicine for treating small cell lung cancer
CN113721020B (en) * 2021-09-14 2022-06-24 大连医科大学附属第二医院 Application of CTSF (cytokine-induced killer) in non-small cell lung cancer diagnosis
CN114085832B (en) * 2021-10-25 2024-05-17 南通大学附属医院 SiRNA molecules for inhibiting PRR14 gene
CN115109799A (en) * 2022-07-26 2022-09-27 华中科技大学同济医学院附属协和医院 Fusion plasmid of small molecular peptide and application of fusion plasmid in antitumor drugs

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU776600B2 (en) * 1999-07-28 2004-09-16 Genentech Inc. Compositions and methods for the treatment of tumors

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11099188B2 (en) 2006-01-20 2021-08-24 Cell Signaling Technology, Inc. ROS kinase in lung cancer
CN102243240A (en) * 2010-05-11 2011-11-16 中国科学院上海生命科学研究院 Non-small cell lung cancer molecular marker and its application
CN102243240B (en) * 2010-05-11 2014-08-27 中国科学院上海生命科学研究院 Non-small cell lung cancer molecular marker and its application
CN106148525A (en) * 2011-05-23 2016-11-23 细胞信号科技公司 ROS kinases in pulmonary carcinoma
CN106148525B (en) * 2011-05-23 2021-01-12 细胞信号科技公司 ROS kinase in lung cancer

Also Published As

Publication number Publication date
CN1854313A (en) 2006-11-01
CN1705753A (en) 2005-12-07
CN1854313B (en) 2010-10-20

Similar Documents

Publication Publication Date Title
KR101075158B1 (en) - method for diagnosing non-small cell lung cancers
CN101586155A (en) Method for diagnosing non-small cell lung cancers
CN1977052B (en) Double-chain molecule and vector related with KIF 11 gene
TW200922626A (en) Cancer-related genes, CDCA5, EPHA7, STK31 and WDHD1
CN101278059A (en) Method for diagnosing and treating renal cell carcinoma
CN101855346A (en) PKIB and NAALADL2 for target genes of prostate cancer therapy and diagnosis
CN101273131A (en) Pancreatic cancer related gene CST6 and GABRP
CN101273271A (en) Genes and polypeptides relating to prostate cancers
CN101528768A (en) Genes and polypeptides relating to breast cancers
CN102597235A (en) Lung and esophageal cancer related gene ADAMTS18
JP2008505601A (en) Genes and polypeptides related to prostate cancer

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
C02 Deemed withdrawal of patent application after publication (patent law 2001)
WD01 Invention patent application deemed withdrawn after publication

Application publication date: 20091125