JP2002536995A - Genes associated with colon disease - Google Patents

Abstract

  (57) [Summary] The present invention provides colorectal cancer genes and polypeptides encoding them. The present invention also provides expression vectors, host cells, and antibodies. Further, the present invention provides a method for diagnosing or treating a colon disease, and a method for preventing the disease.

Description

DETAILED DESCRIPTION OF THE INVENTION

TECHNICAL FIELD The present invention relates to diseases of the colon identified by co-expression with colon cancer genes,
In particular, it relates to seven genes associated with colorectal cancer. The present invention also relates to the diagnosis and prognosis, prevention, treatment and treatment of colon diseases using these biomolecules.

BACKGROUND OF THE INVENTION Colorectal cancer is the third leading cause of cancer death in the United States. There are more than 100,000 new cases each year and 50,000 patients die. The main reason for death is that early diagnosis of the disease is not possible (Wanebo (1993) Colorectal Cancer , M
osby, St Louis MO). Several genes have been found that are involved in or regulate the growth of colon cells, but many other genes have not yet been identified. The ability to identify novel genes that are highly expressed in cells of the diseased colon could provide an opportunity for earlier diagnosis and a target for the development of therapeutics.

[0003] The present invention relates to a novel composition useful for diagnosis and prognosis, treatment, prevention and evaluation of treatment of colon diseases, ie, a colon disease identified by co-expression with a gene expressed in colorectal cancer. Providing seven related genes can meet the needs in the art.

SUMMARY OF THE INVENTION One embodiment of the present invention provides a substantially purified polynucleotide comprising a gene that is co-expressed with one or more known colon cancer genes in a plurality of biological samples. . Known colon cancer genes are preferably carbonic anhydrases I and II, IV (CA I,
II, and IV), carcinoembryonic antigen family protein (cea), colorectal cancer tumor associated antigen (CO-029), down-regulation in adenoma (dra), fatty acid binding protein (fabp), galectin (galectin: galec) Glutathione peroxidase (gp
x2), guanylin (guanyl), cytokeratin 8 and 20 (ker 8 and 20), cadherin (cadher), and intestinal mucin (muc-2). In a preferred embodiment, (a) a polynucleotide sequence selected from SEQ ID NO: 1-7 (SEQ ID NO: 1 through SEQ ID NO: 7); (b) a polypeptide of SEQ ID NO: 8 or 9 (C) a polynucleotide sequence having at least 75% identity to the polynucleotide sequence of (a) or (b), (d) the (a) or (b),
(c) a polynucleotide sequence complementary to the polynucleotide sequence, (e) at least 10, preferably at least 18 contiguous sequences of the polynucleotide sequence of (a) or (b), (c), (d) A polynucleotide sequence comprising the nucleotides, or (f)
Under stringent conditions, polynucleotides that hybridize to the polynucleotides (a) or (b), (c), (d), and (e) above are included. The present invention further provides an expression vector containing any of the above-described polynucleotides and a host cell containing the expression vector. Further, the present invention relates to altered expression of a gene co-expressed with one or more known colorectal cancer genes, comprising administering to a patient an effective amount of the above-described polynucleotide for the treatment or prevention of a disease. The present invention provides a method for treating and preventing a disease or symptom.

[0005] In a second embodiment of the present invention, there is provided a substantially purified polypeptide comprising a gene product of a gene that is co-expressed with one or more known colon cancer genes in a plurality of biological samples. Known colon cancer genes are carbonic anhydrase I and II, IV,
It can be selected from the gene group consisting of the carcinoembryonic antigen family protein, colorectal cancer tumor-associated antigen, down-regulation in adenoma, fatty acid binding protein, galectin, glutathione peroxidase, guanilin, cytokeratins 8 and 20, cadherin, and intestinal mucin it can. In a preferred embodiment, (a) SEQ ID N
O: polypeptide sequences of 8 and 9, (b) a polypeptide sequence having 85% or more identity with the polypeptide sequence of (a), and (c) a polypeptide sequence of (a) or (b) Polypeptide sequences comprising at least 6 consecutive amino acids of
The present invention further provides an antibody that specifically binds to any of the above-described polypeptides. Further, the present invention relates to a method for treating or preventing a disease or condition associated with a change in the expression of a gene co-expressed with one or more known colorectal cancer genes, wherein the amount is an amount effective for treating or preventing the disease. A method for treating or preventing the disease or condition, which comprises administering the antibody to a patient.

In another embodiment of the present invention, there is provided a pharmaceutical composition comprising the polynucleotide of claim 2 or the polypeptide of claim 3 together with a suitable pharmaceutical carrier. In a further embodiment of the present invention, there is provided a method for treating or preventing a disease or condition associated with alteration of the expression of a gene co-expressed with one or more colon cancer genes, the method being effective for treating or preventing the disease. A method for treating or preventing the above-mentioned diseases and conditions, which comprises administering a proper amount of such a composition to a patient.

[0007] In a further embodiment of the present invention, there is provided a method for diagnosing a disease or condition associated with a change in expression of a gene co-expressed with one or more known colorectal cancer genes, wherein each of the known colorectal cancer genes is , Carbonic anhydrase I and II, IV, carcinoembryonic antigen family protein, colorectal cancer tumor associated antigen, down-regulation in adenoma, fatty acid binding protein, galectin, glutathione peroxidase, guanylin, cytokeratins 8 and 20, cadherin, intestine Selected from a group of genes consisting of mucins,
A method for diagnosing the disease or condition is provided. The method comprises the steps of (a) providing a sample containing one or more co-expressed genes, and (b) conditions under which one or more hybridization complexes are effectively formed. Hybridizing the polynucleotide of the co-expressed gene, (c) detecting a hybridization complex, (d) the level of the hybridization complex and the level of the hybridization complex in a non-lesion sample The step of comparing, wherein a change in the level of one or more hybridization complexes in the diseased sample relative to the level of hybridization complexes in the non-lesioned sample is correlated with the presence of a colon disease or condition. The steps are included.

Further, the present invention provides antibodies and antibody fragments having specificity for any of the above-mentioned polypeptides, immunoconjugates, and methods for treating or preventing colon diseases and symptoms thereof.

BRIEF DESCRIPTION OF SEQUENCE LISTING This sequence listing provides specific colorectal cancer gene sequences comprising the polynucleotide sequences of SEQ ID NOs: 1-7 and the polypeptide sequences of SEQ ID NOs: 8 and 9. Show. Each sequence can be identified by the sequence identification number (SEQ ID NO) and the Incyte clone number in which the sequence was first identified.

In the present specification, the singular forms “a” and “the” include plural referents unless the context clearly dictates otherwise. Note that Thus, for example, reference to "a host cell" includes a plurality of such host cells, and reference to "an antibody" includes reference to one or more antibodies and their equivalents well known to those of skill in the art. Also represent.

Definitions “NSEQ” generally refers to a polynucleotide sequence of the invention that includes SEQ ID NOs: 1-7. “PSEQ” generally refers to a polypeptide sequence of the invention that includes SEQ ID NO: 8 and SEQ ID NO: 9.

A “fragment” is a nucleic acid sequence preferably 20 nucleic acids in length, more preferably 40 nucleic acids in length, most preferably 60 nucleic acids in length, for example SEQ ID NO: 1-7 Of nucleic acids 1-50 and 51-400, 401-4,000, 4,001-12,000, etc.

“Gene” refers to a partial or complete coding sequence of a gene and its 5 ′ or 3 ′ untranslated region. The gene can be arranged on a sense strand or an antisense strand (complementary sequence).

“Colon cancer gene” refers to a gene having an expression pattern similar to that of a known colon cancer gene useful for diagnosis, treatment, prognosis, and prevention of colon diseases, particularly colon cancer and other diseases associated with abnormal cell proliferation. Point to. “Known colon cancer gene” refers to an already identified sequence that is useful for diagnosis and treatment, prognosis, and prevention of colon disease. That is, the novel gene is usually expressed at higher levels (ie, transcript-rich) in diseased or cancerous colon tissue than in non-lesioned colon or any other tissue.

“Polynucleotide” refers to a nucleic acid molecule or sequence, an oligonucleotide, a nucleotide, or any fragment thereof. A "polynucleotide" can be DNA or RNA of genomic or synthetic origin, can be double-stranded or single-stranded, has specific activity when bound to a hydrocarbon or lipid, protein, or other substance, or is useful. The composition. "Oligonucleotide" is substantially equivalent to the term amplimer and primers, oligomers, elements, probes.

“Polypeptide” refers to any naturally occurring or synthetic amino acid molecule or sequence, oligopeptides, peptides, proteins, and portions thereof.

A “portion of a peptide sequence” is preferably at least 5 to 15 amino acids in length, preferably at least 10 amino acids in length, for example,
Refers to peptide sequences that retain certain biological or immunological activities of SEQ ID NO: 8 and fragments of SEQ ID NO: 9.

“Sample” is used in its broadest sense. Samples containing nucleic acids include body fluids, extracts from cells and chromosomes, organelles, and membranes isolated from cells, genomic DNA or RNA, cDNA in solution or bound to a substrate, cells, and tissues. , Tissue prints, and the like.

“Substantially purified” is a nucleic acid or amino acid sequence that has been removed from its natural environment and is isolated or separated and contains at least about 60%, preferably at least about 60%, of another naturally occurring composition. Refers to about 75%, most preferably about 90% removed.

“Substrate” refers to any suitable solid or semi-solid support to which a polynucleotide or polypeptide binds, such as a membrane or filter, chip, slide, wafer, fiber, magnetic or non-magnetic bead, gel, Capillaries and other tubes, plates, polymers, microparticles are included, and the surface morphology of those substrates is walls and grooves, pins, channels, pores, and the like.

“Variant” refers to a polynucleotide sequence branched from SEQ ID NO: 1-7, or SE
Refers to polypeptide sequences branched from Q ID NOs: 8 and 9. Branching of a polynucleotide sequence can occur due to mutational changes, such as deletion or addition or substitution of one or more nucleotides, and also by changing the codon reading frame. Each of these mutations can occur alone or in combination, and can occur once or more than once in a sequence. Variants of the polypeptide include SEQ ID NOs: 8 and 9
Sequences having at least one structural or functional characteristic of

(Invention) The present invention provides a method for identifying biomolecules associated with a particular disease or regulatory pathway, intracellular site, type of cell, type of tissue, or type. In particular, diagnosis, prognosis, treatment, prevention and treatment of colon diseases including, but not limited to, colorectal cancer and metastatic colorectal cancer, atrophic gastritis, cholecystitis, Coulomb disease, irritable bowel syndrome, ulcerative colitis, etc. Provided is a method for identifying a gene that is useful for evaluation of a gene.

This method first identifies polynucleotides expressed in a plurality of cDNA libraries. Identified polynucleotides include genes with known or unknown functions expressed in specific disease processes or intracellular sites, types of cells, types of tissues, and types. The expression pattern of a gene having a known function is compared with the expression pattern of a gene having an unknown function to determine whether or not a predetermined simultaneous expression probability threshold is satisfied. This comparison can identify a group of polynucleotides that have high co-expression probabilities for the novel gene. The high co-expression probability is preferably correlated with a predetermined co-expression probability threshold of less than 0.001, more preferably less than 0.00001.

Polynucleotides include cDNA libraries from various species including, but not limited to, eukaryotes such as humans and mice, rats, dogs, monkeys, plants, yeasts, and prokaryotes such as bacteria and viruses. Obtained from. These polynucleotides include, but are not limited to, expressed gene sequence fragments (ESTs) and constructed polynucleotide sequences, full-length gene coding regions, promoters, introns, enhancers, 5 'untranslated regions, 3' untranslated regions. Can be selected from a variety of sequences, including In order to obtain statistically useful analytical results, these polynucleotides should be at least 3
Must be expressed in one cDNA library.

The cDNA library used in the co-expression analysis of the present invention includes an adrenal gland, a bile duct,
Blood cells, blood vessels, bone marrow, brain, bronchi, cartilage, chromaffin system, colon, connective tissue, cultured cells, embryonic stem cells, endocrine glands, epithelium, esophagus, fetus, ganglion, heart, hypothalamus, immune system, intestine, Islets of Langerhans, kidney, larynx, liver, lungs, lymph nodes, muscles, nerves, ovaries, pancreas, penis, peripheral nervous system, phagocytic cells, placenta, pleura, prostate, salivary glands, seminal vesicles, skeleton, spleen, stomach, testis, It can be obtained from the thymus, tongue, ureter, uterus and the like. The number of cDNA libraries selected can range from as few as 3 to more than 10,000. The number of cDNA libraries is preferably 500 or more.

In a preferred embodiment, genes are constructed to reflect related sequences, such as sequence fragments constructed from one transcript. Construction of this polynucleotide sequence can be performed using a variety of sequences including, but not limited to, ESTs or extended sequences, shotgun sequences. In a most preferred embodiment, the polynucleotide sequence has the name “System and Methods for A” filed May 25, 1999.
nalyzing Biomolecular Sequences ", obtained from human sequences constructed using the algorithm disclosed in US patent application Ser. No. 09 / 276,534. It is to be noted that the description of the patent application is incorporated herein by reference.

[0027] The different expression of the polynucleotide may be based on experimentation, but not limited to:
It can be evaluated by methods including differential display by spatial fixation or gel electrophoresis, gene mismatch scanning, difference analysis, and transcription imaging. Furthermore, different expression can be evaluated by microarray technology. These methods can be used alone or in combination.

Known colon cancer genes can be selected based on their use as diagnostic or prognostic markers or therapeutic targets. Each of these known colorectal oncogenes includes carbonic anhydrase I and II, IV, proteins of the carcinoembryonic antigen family, colorectal cancer tumor-associated antigens, down-regulation in adenomas, fatty acid binding proteins, galectins, glutathione peroxidase, guanylin, cytochromes It is preferably selected from the gene group consisting of keratins 8 and 20, cadherin and intestinal mucin.

A method for identifying a novel gene having a statistically useful co-expression pattern with a known colorectal cancer gene is as follows. First, the presence or absence of the gene in the cDNA library is determined. That is, at least one cDNA fragment corresponding to the gene
If detected in a cDNA sample from the NA library, the gene is present in the library; if the corresponding cDNA fragment is not detected in the sample, the gene is not present in the library.

Next, the probability of co-expression by chance is measured using a probability-based method to evaluate the significance of gene co-expression. Probability methods include the Fisher exact test (Fisher e
xact test) or chi-square test or kappa test (kappa test) can be used. These tests and their applications are well known to those skilled in the art and are given in the general statistical literature (Agresti (1990) Categorical Data Analysis , John Wil
ey & Sons, New York NY; Rice (1988) Mathematical Statistics and Data Analysis , Duxbury Press, Pacific Grove CA). Bonferroni correction (Rice, see above
p 384) can be combined with one of the stochastic methods for correcting the statistical results of one gene for many other genes. In a preferred embodiment, the probability of chance is measured by Fisher's exact test, and the threshold of probability of chance is preferably set to less than 0.001, more preferably set to less than 0.00001.

To determine whether the two genes A and B have similar co-expression patterns, a vector of developmental data can be created as shown in Table 1. When a gene occurred at least once in the library, it was expressed as 1;

[0032]

[Table 1] The occurrence data in Table 1 for a set of genes can be represented in a 2x2 contingency table.

[0033]

[Table 2] Table 2 shows co-occurrence data for gene A and gene B in a total of 30 libraries. Both gene A and gene B occur 10 times in the library. Table 2 shows 1) the number of times that both genes A and B are present in the library, 2) the number of times that both genes A and B are not present in the library, 3) the number of times that gene A is present and gene B is not present, 4 ) Represents the number of times gene B is present and gene A is not. The upper left column shows the number of times two genes occurred simultaneously in the library, and the right center column shows the number of times neither gene appeared in the library. The diagonal column is the number of times one gene occurred and the other did not. Eight occurrences of both genes A and B occur, and 18 do not occur both. The number of times the gene A appears and the gene B appears is two times, and the number of times the gene B appears and the gene A does not appear is two times. The probability (p-value) that the above relationship is caused by chance calculated using Fisher's exact test is 0.0003. If the p-value is less than 0.01, the relationship is generally considered significant (Agresti, supra; Rice, supra).

This method of assessing the probability of co-expression of two genes makes several assumptions. This method assumes that each library is independent and sampled identically. However, in practice, the selected cDNA libraries are not completely independent, as more than one library may be obtained from a single patient or a single tissue. Also, since a variable number of cDNAs can be sequenced from each library, they are not sampled exactly the same. The number of cDNAs sequenced is usually between 5,000 and 10,000 per library.
It is in the range of 0. In addition, Fisher's exact co-expression probability requires Bonferroni correction for multiple statistical tests to calculate each gene for the other 41,419 genes constructed.

Using the method of the present invention, several novel genes closely related or co-expressed with novel genes specific for colorectal cancer were identified. These known colorectal oncogenes include carbonic anhydrase I and II, IV, proteins of the carcinoembryonic antigen family, colorectal cancer tumor-associated antigens, down-regulation in adenomas, fatty acid binding proteins, galectins, glutathione peroxidase, guanylin, cytokeratin 8
And 20, cadherin and intestinal mucin. As shown in Table 6, the expression of the seven novel genes is directly or indirectly related to the expression of known colorectal cancer genes. Therefore, these novel genes can be used for diagnosis, treatment, prognosis and prevention of colon diseases, or for evaluation of treatment of colon diseases. In addition, the gene products of these seven novel genes can be potential therapeutic proteins or targets for colon disease.

Thus, in one embodiment, the invention includes a polynucleotide sequence comprising the sequence of SEQ ID NO: 1-7. The methods of the present invention show that these seven polynucleotides have a strong co-expression relationship to known colon cancer genes and to each other. The present invention also includes variants or complements of the polynucleotide sequence, or contiguous 18 nucleotide sequences of the above-described sequences. The nucleotide sequence variant is NSEQ
And at least about 75%, preferably about 85%, and most preferably at least about 95
% Polynucleotide sequence identity.

Using NSEQ or encoded PSEQ, GenBank primates (pri) and rodents (rod)
, Mammals (mam), vertebrates (vrtp), eukaryotes (eukp) database, SwissProt,
You can search against BLOCKS (Bairoch et al. (1997) Nucleic Acids Res 25: 217-221), PFAM, or other databases containing previously identified and annotated motifs and sequences, gene function. Search by primary sequence pattern with secondary structure gap penalty (Smith et al. (1992) Protein Engineering 5: 35-51) and Basic Local Alignment Search Tool (BLAST; Altschul (1993)
J Mol Evol 36: 290-300; Altnehul et al. (1990) J Mol Biol 215: 403-410), BL
OCKS (Henikoff and Henikoff (1991) Nucleic Acids Research 19: 6565-6572)
, Hidden Markov Models (HMM; Eddy (1996) Cur Opin Str Biol 6: 361-365; So
nnhammer et al. (1997) Proteins 28: 405-420)
The nucleotide and amino acid sequences can be manipulated or analyzed. These databases, algorithms, and other methods are well known to those skilled in the art and are described in Ausubel
Et al. (1997; Short Protocols in Molecular Biology , John Wiley & Sons, New Y
ork NY, unit 7.7) and Meyers (1995; Molecular Biology and Biotechnolony ,
Wiley VCH, New York NY, P 856-853).

The present invention also includes polynucleotide sequences that are capable of hybridizing to SEQ ID NOs: 1-7 and fragments thereof under stringent conditions. Stringent conditions are defined by salt concentration and temperature, and other chemicals and conditions well known to those skilled in the art. Suitable conditions can be selected, for example, by changing the concentration of salts in the pre-hybridization and hybridization, washing solutions, or by changing the temperature of hybridization and washing. Several substrates can be added and the temperature can be reduced by adding formamide to the prehybridization and hybridization reactions.

Hybridization was performed in 5 × SSC containing 1% sodium dodecyl sulfate (SDS).
A low stringency condition at 60 ° C. in a buffer such as can result in the formation of a complex of two nucleic acid sequences containing some mismatches. Subsequent washes were performed to leave only hybridization of complexes of perfectly complementary sequences.
It is performed at a high stringency in a buffer such as 0.2xSSC containing 0.1% SDS at 45 ° C (medium stringency) or 68 ° C (high stringency). Background signal is SDS or Sarcosyl, Triton X-l0
It can be reduced using a surfactant such as 0 and / or a blocking agent such as salmon sperm DNA. The hybridization method is Ausubel (supra, units 2.8-2.11,
3.18-3.19 and 4-6-4.9) and Sambrook et al. (1989; Molecular Cloning. A Laboratory Manual , Cold Spring Harbor Press, Plainview NY).

Extension of NSEQ can be performed using partial nucleotide sequences and various PCR-based methods known to those skilled in the art for detecting upstream sequences such as promoters and other regulatory elements ( See, for example, Dieffenbach and Dveksler (1995) PCR Primer, a Laboratory Manual , Cold Spring Harbor Press, Plainview NY).

Further, extending the sequence using an XL-PCR kit (PE Biosystems, Foster City CA) and nested primers, a commercially available cDNA (Life Technologies, Rockville MD) or a genomic library (Clontech, Palo Alto CA) Is also possible. All of
In the PCR method, OLIGO 4.06 primer analysis software (National Bioscie
primers using commercially available software, such as C.nces, Plymouth MN) or other suitable program, to obtain primers that are about 18-30 nucleotides in length, have a GC content of about 50%, and are about 68-72 ° C. Is designed to form a hybridization complex at a temperature of.

In another embodiment of the present invention, NSEQ can be cloned into a recombinant DNA molecule that induces expression of PSEQ or a structural or functional fragment thereof in a suitable host cell. Due to the degeneracy of the genetic code, another DNA sequence encoding a substantially identical or functionally equivalent amino acid sequence can be generated and used to express the polypeptide encoded by NSEQ. . The nucleotide sequences of the present invention may be altered using a variety of methods well known in the art for a variety of purposes, including, but not limited to, controlling cloning and processing, or altering the expression of a gene product. be able to. Nucleotide sequences can be recombined using DNA shuffling with random fragments or PCR reconstruction of gene fragments and synthetic oligonucleotides. For example, oligonucleotide-mediated site-directed mutagenesis can be used to introduce mutations that result in new restriction sites or altered glycosylation patterns, codon preference changes, and splice variants.

To express a biologically active protein, NSEQ or a derivative thereof is converted to a suitable expression vector, ie, a vector containing elements necessary for the regulation of transcription and translation of a coding sequence introduced into a particular host. It is possible to introduce These elements include regulatory sequences such as enhancers and constitutive promoters, inducible promoters, 5 'and 3' untranslated regions. Such expression vectors can be prepared using methods well known to those skilled in the art. These methods include in vitro recombinant DNA
Techniques and synthetic techniques, including in vivo genetic recombination (see, eg, Sambrook, supra, and Ausubel, supra).

[0044] NSEQ can be expressed using a variety of expression vector / host cell systems. These include, but are not limited to, recombinant bacteriophages, or plasmids, microorganisms such as bacteria transformed with cosmid DNA expression vectors,
Includes yeast transformed with yeast expression vectors, insect cell lines infected with baculovirus vectors, plant cell lines transformed with viral or bacterial expression vectors, and animal cell lines. For long-term production of recombinant proteins in mammalian systems, stable expression in cell lines is preferred. For example, NSEQ can be introduced into a cell line using an expression vector. This expression vector contains
The origin of replication of the virus and / or endogenous expression elements, selectable marker genes on the same or different vectors or visible marker genes may be included. The present invention is not limited by the vector or host cell used.

In general, host cells containing an NSEQ that expresses a PSEQ can be identified by various methods well known to those skilled in the art. These methods include, but are not limited to, DNA-DNA or DNA-RNA hybridization, including, but not limited to, membrane or solution for detection and quantification of nucleic acid or protein sequences, or chip-based techniques, PC
R amplification, protein bioassay or immunoassay techniques are included. Immunological methods for detecting and measuring PSEQ expression using either specific polyclonal or monoclonal antibodies are well known in the art. Such technologies include:
Includes enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA), fluorescence activated cell sorter (FACS).

A host cell transformed with NSEQ can be cultured under conditions suitable for protein expression and recovery from cultured cells. This protein produced by the recombinant cell will be secreted or remain intracellular depending on the sequence and / or the vector used. As will be apparent to those skilled in the art, expression vectors containing NSEQ can be designed to include a signal sequence that directs secretion of proteins that penetrate prokaryotic or eukaryotic cell membranes.

Furthermore, the host cell strain can be selected based on its ability to regulate the expression of the inserted sequence or to modify the expressed protein into a desired form. Such peptide modifications include, but are not limited to, acetylation and carboxylation, glycosylation, phosphorylation,
Includes lipidation and acylation. Post-translational processing that cleaves the "prepro" form of the protein can be used to target, fold and / or
Or the activity can be revealed. A variety of host cells (eg, CHO and HeLa, MDCK, HEK293) with specific and characteristic mechanisms for post-translational activity
, W138) are available from the American Type Culture Collection (ATCC, Manasas VA), and those that accurately modify and process the expressed protein are selected.

In another embodiment of the present invention, a natural or modified nucleic acid sequence, a recombinant nucleic acid sequence is linked to a heterologous sequence, and the fusion protein comprising the heterologous protein portion is translated in any of the host systems described above. To be done. Such a heterologous protein moiety facilitates purification of the fusion protein using a commercially available affinity matrix. Such portions include, but are not limited to, glutathione S
Includes transferase and maltose binding proteins, thioredoxin, calmodulin binding peptides, 6-His, FLAG, c-myc, hemagglutinin, monoclonal antibody epitope.

In another embodiment, the entire nucleic acid sequence or a portion thereof is synthesized by chemical or enzymatic methods well known in the art (Caruthers et al. (1980) Nucl Acids Symp Ser (7) 21
5-233; Ausubel, supra). For example, various solid-phase techniques (Roberge et al . (1995) Sc
ience 269: 202-204), and automatic synthesis is possible by using an apparatus such as ABI 431A peptide synthesizer (PE Biosystems). If desired, the amino acid sequence can be altered during synthesis and / or combined with other protein sequences to produce protein variants.

In another embodiment, the invention includes a substantially purified polypeptide comprising the amino acid sequence of SEQ ID NOs: 8 and 9, or a fragment thereof.

(Diagnosis and Treatment) The polynucleotide sequence may be used for, but not limited to, colorectal cancer and metastatic colorectal cancer, atrophic gastritis, cholecystitis, Coulomb disease, irritable bowel syndrome, ulcerative colitis, etc. And diagnosis, prognosis, treatment, prevention and treatment of colon diseases, including:

In a preferred embodiment, the polynucleotide sequence is used for diagnostic purposes to determine the presence or absence and overexpression of the protein. The polynucleotide is at least 18 nucleotides in length and may be composed of complementary RNA and DNA molecules, branched nucleic acids, and / or peptide nucleic acids (PNA). Alternatively, the polynucleotide is used to detect and quantify the expression of a gene in a sample in which NSEQ expression is associated with a disease. In a further alternative, NSEQ can be used to detect polymorphisms in disease-associated genes. These polymorphisms can be detected in the cDNA transcript.

The specificity of the probe will depend on whether it was generated from a unique or regulatory region or from a conserved motif. Due to both the specificity of the probe and the stringency of hybridization and amplification in diagnosis (maximum, high, medium, low), this probe can only identify naturally occurring, completely complementary sequences or allelic variants or related sequences. It is decided whether to identify. Desirably, probes designed to detect related sequences have at least 75% sequence identity with any nucleic acid sequence encoding PSEQ.

A method for preparing a hybridization probe includes cloning a nucleic acid sequence into a vector to prepare an mRNA probe. Such vectors, which are commercially available and well known in the art, can be used to synthesize RNA probes in vitro with the addition of a suitable RNA polymerase and labeled nucleotides. Hybridization probes include, but are not limited to, ³² P or ^{3 5} radionuclides such as S, avidin / enzyme labels such as alkaline phosphatase coupled to the probe by biotin coupling systems, various reporter groups, including fluorescent labels Can be incorporated. Labeled polynucleotide sequences can be used in Southern or Northern analysis, dot plots, other membrane-based techniques, PCR techniques, microarrays to detect changes in PSEQ expression using samples from patients.

NSEQ can be labeled by standard methods and added to a sample from a patient under conditions suitable for the formation and detection of hybridization complexes. After incubation, the sample is washed and the signal associated with the formation of the hybrid complex is quantified and compared to a standard value. Standard values are usually obtained from any control sample that is not infected with the disease. If the amount of signal in the patient sample differs from the standard value, a change in expression value in the sample indicates the presence of the disease. Qualitative and quantitative methods of comparing the hybridization complex formed in a patient sample to a standard value are well known in the art.

[0056] Such assays can also be used to evaluate the efficacy of a particular treatment regimen in animal studies or clinical trials, or to monitor patient treatment. Once the disease is confirmed and treatment has begun, hybridization or amplification assays are performed periodically to determine if the patient's expression level has begun to approach that of a healthy patient. The data obtained by serial assays can confirm the efficacy of treatment over a period ranging from days to years.

The polynucleotide can be used for diagnosis of various diseases related to the colon. These diseases include, but are not limited to, colorectal and metastatic colorectal cancer, atrophic gastritis, cholecystitis, Coulomb disease, irritable bowel syndrome, ulcerative colitis, and the like.

This polynucleotide can be used as a target in a microarray. Microarrays can be used to simultaneously monitor the expression patterns of numerous genes and identify splice variants and mutations, polymorphisms. The information obtained from the analysis of expression patterns can be used to determine gene function, understand the principles of genes in diseases, diagnose diseases, and develop and monitor the activity of therapeutic agents used to treat diseases. . Microarrays can also be used to detect genetic diversity (SNP) at the genomic level, ie, single nucleotide polymorphisms that can characterize a particular population.

In yet another alternative, the polynucleotides can be used to generate hybridization probes useful for mapping naturally occurring genomic sequences. in situ
Fluorescence hybridization (FISH) is performed by Heinz-Ulrich et al. (In: Meyers, et al., Pp.
965-968) and other physical chromosome mapping techniques.

In another embodiment, an antibody or antibody fragment comprising an antibody binding site that specifically binds to PSEQ can be used in the diagnosis of a disease characterized by reduced or over-expressed PSEQ. Various protocols for measuring PSEQ, including ELISA and RIA, FACS, are well known in the art and provide a basis for diagnosing altered or abnormal expression levels. Standard values for the expression of PSEQ were determined by combining a sample obtained from a healthy patient, preferably a human, with the antibody of PSEQ under conditions suitable for complex formation. The yield of complex formed is quantified by various methods, preferably by photometry. The amount of PSEQ expressed in the lesion sample is compared to a standard value. Deviations between standard values and patient values provide indices for diagnosing and monitoring disease. Alternatively, for binding to this protein, a competitive drug screening assay can be used in which a neutralizing antibody capable of specifically binding to the PSEQ competes with the test compound. Antibodies can be used to detect the presence of any peptide that shares one or more antigenic determinants with a PSEQ. In one embodiment, the anti-PSEQ antibody of the present invention can be used to treat, monitor, or evaluate the treatment of a disease of the colon, especially colon cancer.

In another embodiment, NSEQ or its complementary sequence can be used to express mRNA or protein, or conversely, block transcription or translation of mRNA for therapeutic purposes.
The expression vector can be prepared using a retrovirus, an adenovirus, a herpes or vaccinia virus, a bacterial plasmid, or the like. These vectors can be used to deliver nucleotide sequences to specific target organs, tissues and cell populations. Using methods well known to those skilled in the art, a vector expressing the nucleic acid sequence or its complementary sequence can be prepared (e.g., Maulik other (1997) Molec ular Biotechnology, Therapeutic Applications and Strategies, Wiley-Liss,
New York NY.). Alternatively, NSEQ or its complement can be used for somatic or stem cell gene therapy. It is possible to introduce the vector in vivo and in vitro, in ex vivo. For ex vivo treatment, the vector is introduced into stem cells harvested from a patient, and the resulting recombinant cells are clonally expanded and autografted to the same patient. Transfection or liposome injection, transport of NSEQ by a polycation amino polymer can be accomplished by methods well known in the art (eg, Goldman et al. (1997) Nature Biotechnology).
15: 462-466). In addition, the endogenous expression of NSEQ
It can be inactivated using homologous recombination methods that insert into the coding region of Q or another suitable target region (see, eg, Thomas et al. (1987) Cell 51: 503-512).

[0062] Cells or tissues can be transformed with a vector containing NSEQ to express the lost protein or replace it with a nonfunctional protein. Similarly, cells can be transformed with a vector made to express the complement of NSEQ.
Overexpression of PSEQ can also be down-regulated. The complementary or antisense sequence may consist of an oligonucleotide of the transcription start site, preferably about nucleotides -10 to +10 of ATG. Similarly, it can be inhibited using This triplex method is useful because it inhibits the unwinding ability of the double helix sufficient for binding of polymerases, transcription factors and regulatory molecules. Recent advances in therapy using triple helical DNA have been described in the literature (eg, Gee
In: Huber and Carr (1994) Molecular and Immunologic Approaches .Futura
Publishing, Mt. Kisco NY, pp 163-177).

[0063] Ribozymes, which are RNA enzyme molecules, can be used to catalyze the cleavage of mRNA and can reduce the level of certain mRNAs, such as mRNAs containing a polynucleotide sequence of the invention (eg, Rossi (1994) Current Biology 4: 469-471). Ribozymes can cleave mRNA at specific cleavage sites. Alternatively, the ribozyme is targeted
mRNA at a position designated by a flanking region that forms a base pair complementary to mRNA
NA can be cleaved. The production and production of ribozymes is well known in the art and
rs (supra).

[0064] RNA molecules can be modified to increase intracellular stability and increase half-life. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5 'and / or 3' end of the molecule, phosphorothioate or 2'O methyl instead of the phosphodiester bond that backs the molecule. To form the spine. Alternatively, similar modified forms of adenine and cytidine, guanine, thymine, and uridine that are not readily recognized by endogenous endonucleases, as well as acetyl- and methyl-, thio-, as well as inosine, queosine, and wibbosine, Bases that are not of the same nature.

Furthermore, an antagonist or antibody that specifically binds to PSEQ can be administered to a patient for treating or preventing a disease associated with colorectal cancer. Antagonists, antibodies and fragments thereof may be used directly to inhibit protein activity or indirectly to deliver a therapeutic to cells or tissues that express PSEQ. Of antibodies and antagonists
An immunoconjugate comprising a PSEQ binding site and a therapeutic agent can also be administered to a patient for the treatment or prevention of a disease. The therapeutic agents include, but are not limited to, abrin, ricin, doxorubicin, daunorubicin, taxol, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxy anthracin dione (dihydroxy anthracin dione).
), Actinomycin D, diphtheria toxin, pseudomonas exotoxin A and 4
0, it is possible to use a cytotoxic agent selected from the group consisting of radioisotopes and glucocorticoids.

[0066] Antibodies to PSEQ can be produced by methods well known in the art. Such antibodies include, but are not limited to, polyclonal and monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, fragments produced by Fab expression libraries. Neutralizing antibodies that inhibit dimer formation are particularly useful in therapy. Monoclonal antibodies of PSEQ can be produced by any method that produces antibody molecules by continuous culture in a medium. These techniques include, but are not limited to, hybridoma and human B cell hybridoma technology, EBV hybridoma technology. In addition, techniques developed for the production of chimeric antibodies can be used (eg, Pound (1998) Immunochemical Protocols , Met
hods Mol Biol, Vol 80). Alternatively, the disclosed techniques for making single-chain antibodies can be used. Antibody fragments that contain sites that specifically bind to PSEQ can also be generated. Various immunoassays can be used to identify an antibody with the desired specificity. Various protocols for competitive binding assays or immunoradiometric assays using either polyclonal or monoclonal antibodies with established specificity are well known in the art.

Further, an agonist of PSEQ can be administered to a patient for the treatment or prevention of a disease associated with a decrease in PSEQ expression, longevity or activity.

Another embodiment of the present invention involves administering a pharmaceutical or sterile composition with a pharmaceutically acceptable carrier for any of the treatments described above. Such pharmaceutical compositions may consist of PSEQ or antibodies, mimetics or agonists, antagonists, and inhibitors of the polypeptides. The composition can be administered alone or with at least one other agent. The additional agent includes a stabilizer that can be incorporated into any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline and buffered saline, dextrose, water. The composition can be administered to the patient alone or with other drugs or hormones.

The pharmaceutical compositions used in the present invention can be administered using various routes. This route includes oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual, or rectal Is not limited to these.

In addition to the active ingredient, these pharmaceutical compositions also include suitable pharmaceutically acceptable additives, including excipients and adjuvants, which facilitate the conversion of the active compound into pharmaceutically acceptable agents. A possible carrier may be included. Detailed techniques for formulation and administration are described in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing, PA).

The therapeutically effective dosage of any composition can be evaluated initially either in cell culture assays or in animal models such as mice, rats, rabbits, dogs, pigs, etc. . In addition, a suitable concentration range and administration route can be determined using an animal model. Based on such information, an effective dose and administration route for a human can be determined.

A medically effective dose refers to that amount of the active ingredient that ameliorates the symptoms or condition. Medicinal efficacy and toxicity are compared by calculating the ED ₅₀ (50% of the population has a medical effect on taking) and the LD ₅₀ (50% of the population is fatal on taking) statistics For example, it can be determined by standard preparation methods in cell culture or animal experiments. Administering all of the above pharmaceutical compositions to any subject in need of such treatment, including dogs and cats, cattle, horses, rabbits, monkeys, and most preferably humans Can be.

EXAMPLES It is to be understood that this invention is not limited to the particular devices, machines, materials, and methods described. Although particular embodiments have been described, the invention can be embodied using equivalent embodiments. The scope of the present invention is limited only by the appended claims and not by the described embodiments. The examples described below are intended to illustrate but not limit the invention.

1 Preparation of cDNA Library The COLNTUT16 cDNA library, which identified the clone 2790708 of Insight, was
It was made from colon tumor tissue taken during a left hemicolectomy of an aged Caucasian male. Pathologically, a sessile mass located 3 cm away from the distal margin showed invasive grade 2 adenocarcinoma. The tumor had reached the surface of the lamina propria from submucosa. The lesion did not extend to the margin of the resection. One of the nine regional lymph nodes was affected by metastatic adenocarcinoma. The patient had bloody stools and changes in bowel status. History included thrombophlebitis and inflammatory polyarthritis, inflammatory prostate disease, and depression. Also,
He had undergone rectal and vasectomy and spinal canal examinations. His family history included his brother's malignant colon tumor. COLNNOTO8 c where Incyte clone 1843578 was identified
DNA libraries are from the same patient.

The frozen tissue was subjected to a Polytron PT-3000 homogenizer (PT-3000; Brinkmann Instrument).
ments, Westbury NY), and homogenized and dissolved in TRIZOL reagent (1 g of tissue / 10 ml of TRIZOL; Life Technologies) which is a single-phase solution of phenol and guanidine isothiocyanate. After a brief incubation on ice, chloroform was added (1: 5 v / v) and the mixture was centrifuged. Transfer the chloroform phase to a new tube, precipitate the RNA with isopropanol, resuspend in DEPC-treated water,
For 25 minutes with DNAse. RNA is converted to acidic phenol chloroform (pH 4
.7) and precipitated with 0.3 M sodium acetate and 2.5 volumes of ethanol. MRNA was isolated using the OLIGOTEX kit (QIAGEN, Valencia CA), and a cDNA library was prepared using this mRNA.

MRNA was processed according to the recommended protocol of the SUPERSCRIPT plasmid system (Life Technologies). These cDNAs were applied to a SEPHAROSE CL4B column (Amersham Pha
rmacia Biotech, Piscataway NJ)
Was ligated to the pINCY1 plasmid (Incyte Pharmaceuticals, Palo Alto CA).
This plasmid was introduced into DH5α competent cells (Life Technologies).

2 Isolation and Sequencing of cDNA Clones Plasmid DNA was released from cells and purified using REAL Prep 96 plasmid kit (Qiagen). This kit can simultaneously purify 96 samples in a 96-well block using a multi-channel reagent dispenser. The recommended protocol was followed except for the following: 1) Bacteria were cultured in 1 ml of sterile Terrific Broth (Life Technologies) containing 25 mg / L carbenicillin and 0.4% glycerol. 2)
After inoculation, the culture was incubated for 19 hours and at the end of the incubation the cells were lysed in 0.3 ml of lysis buffer. 3) After isopropanol precipitation, the plasmid DNA pellet was resuspended in 0.1 ml of distilled water, and then the sample was transported to a 96-well block and stored at 4 ° C.

The cDNA was prepared using a combination of MICROLAB 2200 (Hamilton, Reno NV) and a DNA ENGINE thermal cycler (PTC200; MJ Research, Watertown MA). cDNA
Can be used with ABI PRISM 377 DNA Sequencing System (PE Biosyssems) or MEGABA
Sequencing was performed using the SE 1000 sequencing system (Molecular Dynamics, Sunnyvale CA) according to the method of Sanger et al. (1975, J. Mol. Biol. 94: 441f).

Most of the sequences described herein use standard ABI protocols and ABI kits (Cat
Nos. 79345. 79339, 79340, 79357, 79355; PE Biosystems). Solution volumes were used at concentrations between 0.25x and 1.0x. Some of the sequences described herein were made using Amersham Pharmacia Biotech solutions and dyes.

3 Selection, Construction, and Characterization of Sequences The sequences used for coexpression analysis were EST sequences, 5 ′ and 3 ′ longread sequences (longread sequences).
sequences) and the full-length coding sequence. The selected construction sequences were expressed in at least three cDNA libraries.

The construction method is described below. The EST sequence chromatogram was processed and verified. P
HRED (Ewing et al . (1998) Genuine Res 8: 175-185; Ewing and Green (1998)
Quality scores were determined using Genome Res 8: 186-194) and the edited sequences were added to a relational management based data system (RDBMS). Using BLAST,
Clustering was performed with a product score of 50. All clusters of two or more sequences form a bin, with each bin containing a resident sequence representing one transcribed gene.

Construction of the component sequences in each bin was performed using a modification of the publicly available public DNA fragment construction program Phrap (Green, University of Washington).
, Seattle WA). Bins with a local base pairing alignment of 82% between any consensus sequences were combined.

The consensus sequence for each bin was screened against public databases such as NCBI GBpri and GenPept to annotate the bins.
Annotation process includes FAST against GenBank's gbpri database
n screens included. Hits with an identity of 75% or more and an alignment length of 100 base pairs or more were recorded as homologous hits. The remaining unannotated sequences were screened against GenPept by FASTx. Those with an E value of 10 ^-8 or less were recorded as homologous hits.

The sequences were then continuously reclustered using Cross-Match and BLASTn, programs for fast comparison and database search (Green, supra) of protein and nucleic acid sequences. BLAS of sequence with consensus sequence with score over 150
All T alignments were realigned using Cross-Match. This sequence was combined with the consensus sequence in a local alignment with greater than 82% identity.
Added to bins with the highest Smith-Waterman scores (Smith et al., Supra). Unmatched sequences were transferred to a new bin and the construction process was performed on the new bin.

4 Co-Expression Analysis of Known Colorectal Cancer Genes To identify new genes closely related to colon diseases, 14 known colorectal cancer genes were selected. These known colorectal oncogenes include carbonic anhydrase I and II, IV, proteins of the carcinoembryonic antigen family, colorectal cancer tumor-associated antigens, down-regulation in adenomas, fatty acid binding proteins, galectins, glutathione peroxidase, guanylin, cytokeratin 8 And 20, cadherin and intestinal mucin. Table 4 shows the colorectal cancer genes examined by this analysis and their functions.

[0086]

[Table 4] From a total of 41,419 constructed gene sequences, seven novel genes closely related to 14 known colorectal cancer genes were identified. First, the degree of relevance was determined by probability value using a cutoff p-value less than 0.00001. The sequences were further examined to ensure that the genes that passed the probability value test were closely related to the novel colorectal cancer genes.
By repeating this process many times, the gene which was originally 41,419 finally became seven colon cancer-related genes. Details of the expression patterns of 14 known colorectal cancer genes and 7 novel colorectal cancer genes are shown in Tables 5 and 6.

[0087]

[Table 5]

[Table 6] Genes co-expressed with 14 known colorectal cancer genes were examined and 7 novel genes that were significantly co-expressed were identified. Each of the seven novel genes co-expressed with at least one of the 14 known genes with a p-value less than 10e-05. Seven new genes co-expressed with 14 known genes are shown in Table 6. The values shown in Table 6 are p-values (-log p) for co-expression of the two genes. The identified novel genes are indicated by Incyte clone numbers, and the known genes are indicated in the table in abbreviated form as shown in Example 5. All genes in Table 5 have been assigned identification numbers 1 to 14.

5. Using a novel gene co-expression analysis method associated with colon disease, has a close relationship with 14 known colon cancer genes,
That is, seven novel genes that were co-expressed were identified.

The nucleic acids comprising the consensus sequence of SEQ ID NOs: 1-7 of the present invention were clones 1580553 and 1843578, 1961467, 2296694, 2516888, 2790708, 3233, respectively, of Incyte clones.
It was initially identified from 5282 and was constructed according to 3 of this example. Analysis for SEQ ID NO: 1-7 according to 7 of this example was performed by BLAST and other motif searches. SEQ ID NOs: 1-7 were translated and sequence identity was determined by comparison to known sequences. SEQ ID NOs: 8 and 9 of the present invention were each encoded by the nucleic acids of SEQ ID NOs: 6-8. SEQ ID NOs: 8 and 9 were analyzed using BLAST and other motif search tools described in Example 6. The analysis results of the novel gene are as follows.

SEQ ID NO: 1 (Incyte clone 1580553) is 219 nucleotides long and has approximately 74% identity to the nucleic acid sequence of mouse mucin and glycoprotein (g2583092). SEQ ID NO: 2 (Incyte clone 2296694) contains nucleotide 252
And is absent in any of the public databases described herein. SEQ ID NO: 3 (Insight Clone 2516888) has nucleotide 28
It is five long and there are no known homologs in any of the public databases described herein. SEQ ID NO: 4 (Incyte clone 2790708) is 1,010 nucleotides in length and is 1 nucleotide from nucleotide 107789 of human chromosome 9
It has about 56% identity to the nucleic acid sequence up to position 08777 (g2564750). SEQ ID
NO: 5 (Incyte Clone 3252882) is 2,616 nucleotides long and is a mouse calcium-sensitive chloride conductance protein (mouse calcium se
a colon-specific gene CS having about 64% identity to the nucleic acid sequence encoding nsitive chloride conductance protein (g3925280) and having a length of 878 nucleotides
It has 70% identity to a portion of the G5 cDNA. SEQ ID NO: 6 (Incyte clone 1843578) is 795 nucleotides in length and has about 64% identity to the nucleic acid sequence encoding the mouse calcium-sensitive chloride conductance protein (g3925280). SEQ ID NO: 7 (Incyte clone 1961467) is 2,225 nucleotides in length and has about 6% identity to the human gene signature HUMG507792. SEQ ID NO: 8, which has 115 amino acids encoded by SEQ ID NO: 6, has no known homologs in any of the public databases described herein. Motif analysis of SEQ ID NO: 8 revealed a potential phosphorylation site at S83 residue. SEQ ID NO: 9, which has 90 amino acids encoded by SEQ ID NO: 7, has no known homologs in any of the public databases described herein. Motif analysis of SEQ ID NO: 9 revealed five potential phosphorylation sites at T10 and T6, T21, S66, S86 residues.

[0091] 6 colon disease gene and homologous resistant retrieval polynucleotide sequences SEQ ID NO for the protein encoded by them: 1-7 and the polypeptide sequences SEQ ID NO: and 8 and 9 to the database obtained from such GenBank and SwissProt Searched against. In these databases containing previously identified and annotated sequences, BLAST (Altschul, supra) was used to search for similar regions. Sequence matches were searched for by BLAST and only those that met a probability threshold of 10 ^-25 or less for nucleotide sequences and 10 ^-8 or less for polypeptide sequences were reported.

[0092] Polypeptide sequences were also analyzed against known motif patterns according to MOTIFS and protocols based on SPSCAN, BLIMPS, HMM. MOTIFS (Geneti
cs Computer Group, Madison WI), Prosite Dictionary of Protein Si
Search polypeptide sequences for patterns that match those defined in tes and Patterns (Bairoch, supra) and display the found sequence pattern and the corresponding bibliography. Search for potential signal peptide sequences by SPSCAN (Genetics Computer Group) using the weighted matrix method (Nielsen et al . (1997) Prot Eng 10: 1-6). Hits with a score of 5 or more were taken into account. BLOCKS (PROSITE) by BLIMPS using a weighted matrix analysis algorithm
Database (compiled from Henikoff. Supra; Bairoch, supra) and composed of short amino acid segments or blocks of 3 to 60 amino acids in length)
And PRINTS (SwissProt and GenBank, PIR, NRL-3
D (Attwood, et al. (1997) J. Chem Inf Comput Sci 37: 417-424) and a sequence between the polypeptide sequence and the polypeptide sequence based on a non-overlapping sequence obtained from the polypeptide sequence. Search for similarities. For the purpose of the present invention, BLIMPS searches report cutoff scores of 1000 or more and cutoff probability values satisfying 1.0 × 10 ⁻³ . Using a probabilistic HMM-based protocol, the gene family of protein sequences (Eddy, supra; Sonnh
ammer, supra). Known protein families with a cut-off score of 10 to 50 bits and more than 500 were selected for use in the present invention.

7 Probe Labeling and Hybridization Analysis Blotting A polynucleotide sequence is isolated from an organism and immobilized on a solid substrate suitable for standard nucleic acid hybridization protocols by one of the methods described below. Mix the target nucleic acid mixture with 1x TAE (40 mM Tris acetate and 2 mM ethylenediamine 4).
Separate by electrophoresis on a 0.7% agarose gel in an acetic acid (EDTA) buffer and transfer to a nylon membrane by capillary transport using 20x sodium citrate (SSC). Alternatively, the target nucleic acids are individually ligated to a vector and introduced into a bacterial host cell to create a library. The target nucleic acids are arranged on the blot by one of the following methods. In the first method, bacterial cells containing individual clones are mechanically selected and arranged on a nylon membrane. The nylon membrane is placed on a bacterial growth medium, LB agar containing carbenicillin, and incubated at 37 ° C. for 16 hours. Bacterial colonies are denatured, neutralized and digested with proteolytic enzyme K. Nylon membrane with STRATALINKER UV-cro
The DNA is cross-linked to the membrane by UV irradiation with an sslinker (Stratagene, La Jolla CA).

In a second method, the PCR is repeated 30 times using primers complementary to the vector sequence adjacent to the insert to amplify the target nucleic acid from the bacterial vector. The amplified target nucleic acid is purified using SEPHACRYL-400 (Amersham Pharmacia Biotech). The purified target nucleic acid is mechanically aligned on a glass microscope slide. The slides were coated with 0.05% aminopropylsilane (Sigma-Aldrich, St Louis MO) and cured at 110 ° C. Arrays of glass slides (microarrays) are UV irradiated with STRATALINKER UV crosslinks (Stratagene).

Preparation of Probe A cDNA probe sequence is prepared from an mRNA template. 5 μg of mRNA is mixed with 1 μg of random primer (Life Technologies), incubated at 70 ° C. for 10 minutes and lyophilized. The lyophilized sample was combined with the dNTP mixture and [a- ³² P] d
Resuspend in 50 μl of 1 × first strand buffer (cDNA Synthesis system; Life Technologies) containing CTP, dithiothreitol, MMLV reverse transcriptase (Stratagene) and incubate at 42 ° C. for 1-2 hours. After incubation, the probe is diluted with 42 μl of distilled water and heated at 95 ° C. for 3 minutes, then cooled on ice. Probe mRNA
Is removed by alkali decomposition. Neutralize this probe and use PROBEQUANT G-50 Mi
The denatured mRNA and unincorporated nucleotides are removed using croColumn (Amersham Pharmacia Biotech). C probe instead of the radionuclide ^[32 P] dCTP
Labeling can be performed with a y3-dCTP or Cy5-dCTP fluorescent marker (Amersham Pham? acia Biotech).

Hybridization Hybridization was performed using 0.5M sodium phosphate (pH 7.2) and 7% SDS, 1 mM
Performed at 65 ° C. in a hybridization buffer containing EDTA. After incubating the blot in hybridization buffer for at least 2 hours at 65 ° C., the buffer is replaced with 10 ml of fresh buffer containing the probe sequence. After incubating at 65 ° C for 18 hours, the hybridization buffer was removed, and the conditions were continuously increased up to 40 mM sodium phosphate and 1% SDS, 1 mM EDTA at 65 ° C while gradually increasing the stringency. Rinse the blot. To detect the signal generated by the radiolabeled probe hybridized to the membrane, the blot is exposed to a PHOSPHORIMAGER cassette (Molecular Dynamics) and the images are analyzed using IMAGEQUANT data analysis software (Molecular Dynamics). The blot is examined by confocal laser microscopy to detect the signal generated by the fluorescent probe hybridized to the microarray, and the images are modified and analyzed using GEMTOOLS gene expression analysis software (Incyse Pharmaceuticals). 8. Production of Specific Antibodies Rabbits were immunized with rabbits using SEQ ID NOs: 8 and 9 or fragments thereof, substantially purified by polyacrylamide gel electrophoresis or other purification techniques, according to the standard protocol described in Pound (supra). To produce an antibody.

[0097] Alternatively, the amino acid sequence is analyzed using LASERGENE software (DNASTAR, Madison WI) to determine regions of high immunogenicity, and the corresponding oligopeptides are synthesized and used by those skilled in the art. To produce antibodies. Methods for selecting appropriate epitopes, such as those near the C-terminus or in adjacent hydrophilic regions, are well known in the art (see, eg, Ausubel, 1995, supra, Chapter 11). Typically, oligopeptides about 15 residues in length were synthesized by Fmoc chemistry using an ABI 431A peptide synthesizer from Applied Biosystems (PE Biosystems) and N-maleimidobenzoyl-N-hydroxysuccinimide ester (Ausubel, supra). To enhance immunogenicity by binding to keyhole limpet hemocyanin (KLH Sigma-Aldrich) by reaction with Rabbits are immunized with the oligopeptide-KLH conjugate in Freund's complete adjuvant. To test the anti-peptide activity of the obtained antiserum, for example, the peptide is bound to plastic, blocked with 1% BSA, reacted with rabbit antiserum, washed, and further labeled with radioactive iodine. React with anti-rabbit IgG.

[Sequence list]

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification code FI Theme coat ゛ (Reference) A61K 48/00 A61P 35/00 4C085 A61P 1/00 C07K 14/47 4C086 35/00 14/78 4H045 C07K 14 / 47 14/82 14/78 16/32 14/82 C12N 1/15 16/32 1/19 C12N 1/15 1/21 1/19 9/08 1/21 9/88 5/10 C12Q 1/68 A 9/08 C12N 15/00 ZNAA 9/88 5/00 A C12Q 1/68 A61K 37/02 (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB , GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG ), AP (GH, GM, KE, LS, M , SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB , BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU , SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Folkmas, Wayne 94025, California, United States Menlo Park # 1 Roble Avenue 783 (72 Inventor Kringler, Todd M. San Carlos Dovercourt, CA 94070, California, United States of America 28 (72) Inventor Lal, Pleity 95054, Santa Clara Las Drive, California 2382 F-term (reference) 4B024 AA01 AA12 BA08 BA36 CA04 CA09 HA14 4B050 CC03 DD11 LL01 LL03 4B063 QA01 QA13 QA18 QQ08 QQ43 QR32. NA10 NA14 ZA66 ZB26 4H045 AA10 AA11 AA30 BA10 CA41 DA75 DA86 EA28 EA51 FA74

Claims (16)

[Claims] 1. A substantially purified polynucleotide comprising a gene that is co-expressed with one or more known colon cancer genes in a plurality of biological samples, wherein each of the known colon cancer genes comprises: Carbonic anhydrase I and II, IV (CA I, II, IV)
, A protein of the carcinoembryonic antigen family (cea), a colorectal cancer tumor-associated antigen (CO-029
), Down-regulation in adenoma (dra), fatty acid binding protein (fabp),
Galectin (galectin), glutathione peroxidase (gpx2), guanylin (guanylin: guan), cytokeratins 8 and 20 (ker 8 and 20), cadherin (cad
her) and a gut mucin (muc-2). 2. A polynucleotide sequence selected from the group consisting of (a) SEQ ID NO: 1 to SEQ ID NO: 7; and (b) a polynucleotide sequence selected from the group consisting of SEQ ID NO: 8 and SEQ ID NO: 9. (C) at least 75% of the polynucleotide sequence of (a) or (b),
(D) a polynucleotide sequence complementary to the polynucleotide sequence of (a) or (b), (c); and (e) a polynucleotide sequence of (a) or (b), (C) a polynucleotide sequence comprising at least 18 consecutive nucleotides of the polynucleotide sequence of (d); and (f) under stringent conditions, said (a) or (b), (c)
(D) a polynucleotide that hybridizes with the polynucleotide of (e);
The polynucleotide of claim 1, comprising a polynucleotide sequence selected from the group consisting of: 3. A substantially purified polypeptide comprising a gene product of a gene that is co-expressed with one or more known colon cancer genes in a plurality of biological samples, wherein each of said known colon cancer genes is Are carbonic anhydrases I and II, IV (CA I, II
, IV), a protein of the carcinoembryonic antigen family (cea), a colorectal cancer tumor-associated antigen (
CO-029), down-regulation in adenomas (dra), fatty acid binding protein (f
abp), galectin (galec), glutathione peroxidase (gpx2), guanylin (
guan), cytokeratin 8 and 20 (ker 8 and 20), cadherin (cadher), intestinal mucin
A polypeptide selected from the group consisting of (muc-2). 4. A polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 8 and SEQ ID NO: 9, and (b) at least 85% of the polypeptide sequence of (a). A polypeptide sequence selected from the group consisting of: a polypeptide having identity; and (c) a polypeptide sequence comprising at least 6 consecutive amino acids of the polypeptide sequence of (a) or (b). 4. The polypeptide of claim 3, wherein: 5. An expression vector comprising the polynucleotide of claim 2. 6. A host cell comprising the expression vector according to claim 5. 7. A pharmaceutical composition comprising the polynucleotide of claim 2 together with a suitable pharmaceutical carrier. 8. A pharmaceutical composition comprising the polypeptide of claim 3 together with a suitable pharmaceutical carrier. 9. An antibody or antibody fragment comprising an antigen-binding site that specifically binds to the polypeptide of claim 4. 10. An immunoconjugate comprising the antigen-binding site of the antibody or antibody fragment of claim 9 conjugated to a therapeutic agent. 11. A method for diagnosing a disease or a symptom thereof associated with a change in expression of a gene co-expressed with one or more known colorectal cancer genes, wherein each of the known colorectal cancer genes comprises carbonic anhydrase I and II, IV (CA I, II, IV), carcinoembryonic antigen family protein (cea), colorectal cancer tumor associated antigen (CO-029), down-regulation in adenoma (dra), fatty acid binding protein (fabp), Galectin (galec),
Selected from the group consisting of glutathione peroxidase (gpx2), guanylin (guan), cytokeratins 8 and 20 (kers 8 and 20), cadherin (cadher), and intestinal mucin (muc-2); (a) a biological sample And (b) under conditions to form one or more hybridization complexes,
3. hybridizing the polynucleotide of claim 2 with the biological sample; (c) detecting the hybridization complex; (d) hybridizing the level of the hybridization complex with a non-lesioned sample. Comparing the level of the complex of the biological sample with the level of the complex of the non-lesioned sample correlates with the presence of the disease or its symptoms. A diagnostic method characterized by including the following steps: 12. A method for treating or preventing a patient in need of treatment or prevention of a disease associated with a change in expression of a gene that is co-expressed with one or more known colorectal cancer genes, the method comprising: Genes are carbonic anhydrase I and II, IV
(CA I, II, IV), carcinoembryonic antigen family protein (cea), colorectal cancer tumor-associated antigen (CO-029), down-regulation in adenoma (dra), fatty acid binding protein (fabp), galectin (galec) ), Glutathione peroxidase (gpx2)
, Guanilin (guan), cytokeratin 8 and 20 (ker 8 and 20), cadherin (cadh
er), selected from a group of genes consisting of intestinal mucin (muc-2), the method comprising administering to the patient an amount of the pharmaceutical composition of claim 7, which is effective for treating or preventing the disease. A characteristic treatment or prevention method. 13. A method for treating or preventing a patient in need of treatment or prevention of a disease associated with a change in expression of a gene co-expressed with one or more known colorectal cancer genes, the method comprising treating each of the known colorectal cancers Genes are carbonic anhydrase I and II, IV
(CA I, II, IV), carcinoembryonic antigen family protein (cea), colorectal cancer tumor-associated antigen (CO-029), down-regulation in adenoma (dra), fatty acid binding protein (fabp), galectin (galec) ), Glutathione peroxidase (gpx2)
, Guanilin (guan), cytokeratin 8 and 20 (ker 8 and 20), cadherin (cadh
er), selected from a group of genes consisting of intestinal mucin (muc-2), the method comprising administering to the patient an effective amount of the pharmaceutical composition of claim 8 for treating or preventing the disease. A characteristic treatment or prevention method. 14. A method for treating or preventing a patient in need of treatment or prevention of a disease associated with a change in the expression of a gene that is co-expressed with one or more known colorectal cancer genes, the method comprising: Genes are carbonic anhydrase I and II, IV
(CA I, II, IV), carcinoembryonic antigen family protein (cea), colorectal cancer tumor-associated antigen (CO-029), down-regulation in adenoma (dra), fatty acid binding protein (fabp), galectin (galec) ), Glutathione peroxidase (gpx2)
, Guanilin (guan), cytokeratin 8 and 20 (ker 8 and 20), cadherin (cadh
er), selected from a group of genes consisting of intestinal mucin (muc-2), comprising administering to the patient an antibody or antibody fragment of claim 9 in an amount effective to treat or prevent the disease. A method of treatment or prevention characterized by the following. 15. A method for treating or preventing a patient in need of treatment or prevention of a disease associated with a change in the expression of a gene that is co-expressed with one or more known colorectal cancer genes, the method comprising: Genes are carbonic anhydrase I and II, IV
(CA I, II, IV), carcinoembryonic antigen family protein (cea), colorectal cancer tumor-associated antigen (CO-029), down-regulation in adenoma (dra), fatty acid binding protein (fabp), galectin (galec) ), Glutathione peroxidase (gpx2)
, Guanilin (guan), cytokeratin 8 and 20 (ker 8 and 20), cadherin (cadh
er), selected from a group of genes consisting of intestinal mucin (muc-2), comprising administering to the patient an effective amount of the immune complex of claim 10 for treating or preventing the disease. A characteristic treatment or prevention method. 16. A method for treating or preventing a patient in need of treatment or prevention of a disease associated with a change in the expression of a gene that is co-expressed with one or more known colorectal cancer genes, the method comprising: Genes are carbonic anhydrase I and II, IV
(CA I, II, IV), carcinoembryonic antigen family protein (cea), colorectal cancer tumor-associated antigen (CO-029), down-regulation in adenoma (dra), fatty acid binding protein (fabp), galectin (galec) ), Glutathione peroxidase (gpx2)
, Guanilin (guan), cytokeratin 8 and 20 (ker 8 and 20), cadherin (cadh
er), selecting from the group of genes consisting of intestinal mucin (muc-2), administering to the patient an effective amount of the polynucleotide sequence of claim 2 for treating or preventing the disease. A characteristic treatment or prevention method.