KR20090052976A - Stomach-specific gene phs11s15230 associated with development of stomach - Google Patents

Abstract

The present invention provides a PHS11S15230 protein specifically expressed in human stomach tissue, a gene encoding the protein, a recombinant vector comprising the gene, a transformant transformed with the recombinant vector, an antibody against the protein, the gene The present invention relates to a marker for diagnosing gastric related diseases comprising a microarray for diagnosing gastric related diseases comprising the gene or the antibody, and a method for diagnosing gastric diseases of a mammal using the gene.

Stomach specific gene, PHS11S15230 protein, marker, microarray

Description

Gastric tissue-specific gene PHS11S15230 associated with development of stomach}

The present invention relates to a gastric tissue-specific gene PHS11S15230 involved in gastric function, more specifically, a PHS11S15230 protein specifically expressed in human gastric tissue, a gene encoding the protein, a recombinant vector comprising the gene, A transformant transformed with the recombinant vector, an antibody to the protein, a marker for diagnosing a stomach related disease comprising the gene, a microarray for diagnosing a stomach related disease comprising the gene or the antibody and the The present invention relates to a method for diagnosing gastric diseases of mammals using genes.

The stomach, one of the body's most important digestive organs, is the beginning of the digestive tract, and cancer develops very well because it is the first, easiest, longest contact with carcinogens in food. The stomach wall is composed of four layers of the mucosal layer, the submucosal layer, the muscle layer and the intestinal layer from the inside. Hydrochloric acid in the gastric juice kills bacteria and prevents food from being damaged. Therefore, since the stomach is easily exposed to disease, much research is being conducted. In addition, the stomach is a very strong acid-secreting place, which is made to eliminate the germs associated with the disease, but sometimes the condition becomes excessive, melting the stomach to become a disease. In order to diagnose such a disease, since the stomach specific gene is diagnosed with a molecular marker, the importance of the stomach specific gene is very important.

Therefore, the inventors of the present invention, while studying the above specific genes, revealed that the PHS11S15230 gene is specifically expressed in the tissues, and completed the present invention.

In order to solve the above problems, the present invention provides a PHS11S15230 protein that is specifically expressed in gastric tissue.

The present invention also provides a gene encoding the protein.

The present invention also provides a recombinant vector comprising the gene.

The present invention also provides a transformant transformed with the vector.

The present invention also provides an antibody against the protein.

The present invention also provides a marker for diagnosing gastric related diseases comprising the gene.

The present invention also provides a microarray for diagnosing gastric related diseases comprising the gene or the antibody.

The present invention also provides a method for diagnosing gastrointestinal related diseases in mammals using the gene.

Gastric tissue specific genes are essential for the production of the stomach, and it is important to reveal information on protein-protein interactions, pathways, or regulatory networks involved in development. But analyzing individual genes to reveal this information is very time consuming.

By providing the fact that the PHS11S15230 gene is specifically expressed in gastric tissue, the present inventors can easily access information such as protein-protein networks or pathways for the production of gastric tissue, and the fact that the PHS11S15230 gene is gastric specific gene This can reduce false positives in diagnostic studies of stomach-related diseases. In addition, the PHS11S15230 gene can be used as a marker in the diagnosis of the development of gastric related diseases, and when the drug is designed as a therapeutic agent for disease symptoms, the protein target for PHS11S15230 may be reduced. This allows for faster treatment at an early stage.

In order to achieve the above object, the present invention provides a PHS11S15230 protein that is specifically expressed in the tissue consisting of the amino acid sequence represented by SEQ ID NO: 2.

Prediction of tissue-specific genes, such as DNA chips, is not possible to distinguish whether the gene is actually involved in tissue formation mechanisms, housekeeping genes, or experimental errors due to numerous errors in experiments. However, if the candidate group is identified as a tissue-specific gene by performing an Audic-Claverie Test based on the EST (Expressed Sequence Tag) or using an experiment such as RT-PCR, the candidate may be judged as a genetic influence factor. Reliability is increased. The inventors have provided higher reliability for candidate groups of genes involved in tissue development by examining candidate groups of genes involved in tissue development and examining whether they contain tissue specific information.

Accordingly, the present invention provides a PHS11S15230 protein that is specifically expressed in gastric tissue. The range of PHS11S15230 protein according to the present invention includes proteins having the amino acid sequence represented by SEQ ID NO: 2 and functional equivalents of the proteins. "Functional equivalent" means at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 70% of the amino acid sequence represented by SEQ ID NO: 2 as a result of the addition, substitution, or deletion of the amino acid Is 95% or more of sequence homology, and refers to a protein that exhibits substantially homogeneous physiological activity with the protein represented by SEQ ID NO: 2.

The PHS11S15230 protein according to the invention is preferably of human origin, but is not limited thereto.

The PHS11S15230 protein according to the present invention can be used as a target for therapeutic drugs for stomach related diseases. Examples of gastric diseases include, but are not limited to, chronic gastritis, acute gastritis, gastric ulcers, gastric cancer, and indigestion. In the case of designing a drug as a diagnostic agent for gastric related disease symptoms, the drug target can be reduced with PHS11S15230 protein, which enables faster diagnosis at the onset of onset.

The present invention also provides a gene encoding the PHS11S15230 protein. Genes of the invention include both genomic DNA and cDNA encoding the PHS11S15230 protein. Preferably, the gene of the present invention may include the nucleotide sequence represented by SEQ ID NO: 1.

In addition, variants of the above nucleotide sequences are included within the scope of the present invention. Specifically, the gene comprises a nucleotide sequence having at least 70%, more preferably at least 80%, even more preferably at least 90%, most preferably at least 95% homology with the nucleotide sequence of SEQ ID NO: 1 can do. The "% sequence homology" for a polynucleotide is identified by comparing two optimally arranged sequences with a comparison region, wherein part of the polynucleotide sequence in the comparison region is the reference sequence (addition or deletion) for the optimal alignment of the two sequences. It may include the addition or deletion (ie, gap) compared to).

The present invention also provides a recombinant vector comprising the gene according to the present invention. The recombinant vector is preferably a recombinant animal expression vector.

The term “recombinant” refers to a cell in which a cell replicates a heterologous nucleic acid, expresses the nucleic acid, or expresses a protein encoded by a peptide, a heterologous peptide, or a heterologous nucleic acid. Recombinant cells can express genes or gene fragments that are not found in their natural form in either the sense or antisense form. Recombinant cells can also express genes found in natural cells, but the genes have been modified and reintroduced into cells by artificial means.

The term “vector” is used to refer to a DNA fragment (s), a nucleic acid molecule, that is delivered into a cell. Vectors can replicate DNA and be reproduced independently in host cells. The term "carrier" is often used interchangeably with "vector". The term “expression vector” refers to a recombinant DNA molecule comprising a coding sequence of interest and a suitable nucleic acid sequence necessary to express a coding sequence operably linked in a particular host organism.

In the expression vector of the present invention, the promoter may include a constitutive promoter and an inducible promoter. Examples of constitutive promoters are apes early virus 40 (SV40) early promoter, juvenile breast as well as human genetic promoters such as acute early cytomegalovirus (CMV) promoter, actin promoter, myosin promoter, hemoglobin promoter, and muscle creatine promoter. Tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat sequence (LTR) promoter, molony virus promoter, avian leukemia virus promoter, Epstein-Barr virus acute early promoter, Raus sarcoma virus promoter Including but not limited to. Examples of inducible promoters include, but are not limited to, metallothionine promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

In the expression vector of the present invention, the transcription terminator may include any transcription terminator known in the art. For example, human growth hormone polyadenylation signals, bovine growth hormone polyadenylation signals and SV40 viral polyadenylation signals may be included.

The present invention also provides a transformant transformed with the recombinant vector according to the present invention. The transformant is preferably a mammal. Although preferably human, pets such as dogs or cats, livestock such as cattle, sheep, pigs, horses, and experimental animals such as rats, white papers, guinea pigs, and the like may also be included. In the present invention, a method of transforming a mammal with a recombinant vector includes a micro-injection method (Capecchi, MR, Cell, 22: 479 (1980)), calcium phosphate precipitation method (Graham, FL et al., Virology, 52: 456 ( 1973)), electroporation (Neumann, E. et al., EMBO J., 1: 841 (1982)), liposome-mediated transformation (Wong, TK et al., Gene, 10:87 (1980) ), DEAE-dextran treatment (Gopal, Mol. Cell Biol., 5: 1188-1190 (1985)), and gene bombardment (Yang et al., Proc. Natl. Acad. Sci., 87: 9568- 9572 (1990)), and the like.

The invention also provides an antibody against the PHS11S15230 protein according to the invention. The antibody of the present invention can be produced by a conventional method from the obtained protein by cloning each gene into an expression vector according to a conventional method to obtain a protein encoded by the PHS11S15230 gene. This includes partial peptides that can be made from the protein, and the partial peptide of the present invention includes at least 7 amino acids, preferably 9 amino acids, more preferably 12 or more amino acids. The form of the antibody of the present invention is not particularly limited and a part thereof is included in the antibody of the present invention and all immunoglobulin antibodies are included as long as they are polyclonal antibody, monoclonal antibody or antigen-binding. Furthermore, the antibody of this invention also contains special antibodies, such as a humanized antibody.

In addition, the present invention is PHS11S15230 according to the present invention Provided are markers for diagnosing stomach related diseases, including genes. PHS11S15230 of the present invention Gene may preferably have a nucleotide sequence represented by SEQ ID NO: 1, but is not limited thereto. Examples of gastric diseases include, but are not limited to, chronic gastritis, acute gastritis, gastric ulcers, gastric cancer, and indigestion. By measuring the severity of the disease associated with the amount of expression of the gene, it is possible to establish a correlation between the expression level of the gene and the severity of the disease. Based on the established correlations, only the expression level of the gene can be measured to predict and diagnose the stomach related diseases of the subject. Since the gene can be used as a marker for identifying or diagnosing a genetic tendency of the above-related diseases, such information can be used to diagnose and perform treatment before the disease occurs.

The present invention may also provide a kit for diagnosing gastric related diseases comprising sense and antisense primers of the gene. By performing PCR amplification using the sense and antisense primers of the gene, the disease can be diagnosed through the generation of a desired product. PCR conditions, sense and antisense primer lengths can be modified based on what is known in the art.

Kits of the invention may also comprise probes complementary to the gene. Hybridization may be performed using a probe complementary to the gene, and the disease may be diagnosed through hybridization. Selection of suitable probes and hybridization conditions can be modified based on what is known in the art.

The present invention also provides a microarray for diagnosing gastric related diseases comprising the PHS11S15230 gene according to the present invention or an antibody against the PHS11S15230 protein of the present invention. PHS11S15230 of the present invention Gene may preferably have a nucleotide sequence represented by SEQ ID NO: 1, but is not limited thereto. Examples of gastric diseases include, but are not limited to, chronic gastritis, acute gastritis, gastric ulcers, gastric cancer, and indigestion. By measuring the severity of the disease associated with the amount of expression of the gene, it is possible to establish a correlation between the expression level of the gene and the severity of the disease. Based on the established correlations, only the expression level of the gene can be measured to predict and diagnose the stomach related diseases of the subject. The microarray of the present invention can be easily produced by a manufacturing method commonly used in the art.

In the microarray of the present invention, "microarray" means that a polynucleotide or an antibody group is immobilized at a high density on a substrate, and the polynucleotide or antibody group means a microarray each immobilized in a predetermined region. Such microarrays are well known in the art. Microarrays are disclosed, for example, in US Pat. Nos. 5,445,934 and 5,744,305, the contents of which are incorporated herein by reference.

The term "substrate" refers to any substrate to which a polynucleotide or antibody probe can be coupled under conditions that retain hybridization properties and keep the background level of hybridization low. Typically, the substrate may be a microtiter plate, membrane (eg, nylon or nitrocellulose) or microspheres (beads) or chips. Prior to application or immobilization on the membrane, nucleic acid probes can be modified to promote immobilization or to improve hybridization efficiency. Such modifications may include homopolymer tailing, coupling with different reactive functional groups such as aliphatic groups, NH ₂ groups, SH groups and carboxyl groups, or coupling with biotin, hapten or protein.

The present invention also provides a method of diagnosing gastrointestinal related diseases in a mammal, comprising measuring the expression of the PHS11S15230 gene according to the present invention. PHS11S15230 of the present invention Gene may preferably have a nucleotide sequence represented by SEQ ID NO: 1, but is not limited thereto. Examples of gastric diseases include, but are not limited to, chronic gastritis, acute gastritis, gastric ulcers, gastric cancer, and indigestion. By measuring the severity of the disease associated with the amount of expression of the gene, it is possible to establish a correlation between the expression level of the gene and the severity of the disease. Based on the established correlations, only the expression level of the gene can be measured to diagnose a stomach-related disease of the subject. The mammal is preferably a human, but may also include non-human animals including pets such as dogs or cats, livestock such as cattle, sheep, pigs, horses, and experimental animals such as rats, white papers, guinea pigs, and the like.

Hereinafter, the present invention will be described in more detail with reference to Examples. Since these examples are only for illustrating the present invention, the scope of the present invention is not to be construed as being limited by these examples.

Example

Example  One: TISA  Establishment of Gastric Specific Gene Candidates Using Database

Noh SJ 2006 [DNA Res 13 (5) 229-43] published a TISA (Tissue-specific Alternative Splicing) database containing information on genes, transcript structure, alternative splicing events and tissue specificity. TISA is a database that identifies tissue-specific genes by clustering EST data including the information of the expressed tissue into a genetic map and verifying that the number of ESTs of the corresponding TISA genes is statistically significant. If there was a gene predicted as a tissue-specific gene in TISA, it was extracted as a candidate group of the tissue-specific gene.

Example  2: of gastric specific gene candidates RT - PCR  Experiment

As total RNA, human total RNA of Biochain and Staratagene was used. There are 15 different types of RNA tissue used, including small intestine, stomach, testis, thymus, thyroid, bone marrow, brain and liver. (liver), colon (colon), kidney (kidney), lung (lung), mammary gland (ovary), ovary (prostate), pancreas (pancreas). Roche Co. CDNA was synthesized according to the Ltd RT-PCR kit manual and PCR was performed. The primers used were as follows, all designed through eprimer3 primer design software and in-house python wrapping script. PCR conditions were finally extended for 72 min 10 min after 35 cycles of pre-denatured 94 ° C. 3 min, followed by 94 ° C. 30 sec, annealing 58-60 ° C. 30 sec, extension 72 ° C. 1 min. 1% agarose gel electrophoresis was performed to confirm the PCR product.

Primer sequence:

Forward primer: 5'-ATGGAGGGCCTGGAAGTATTG-3 '(SEQ ID NO: 3)

Reverse primer: 5'-AAGTGCCAATGCACCACACT-3 '(SEQ ID NO: 4)

Figure 2 shows the RT-PCR results of the PHS11S15230 gene. As can be seen in Figure 2, it was found that the PHS11S15230 gene is rarely expressed in other tissues, but specifically expressed in the stomach tissues.

Example  3: UniProt Reference of Gastric Specific Gene Candidates

UniProt (http://www.ebi.uniprot.org) was developed in 2002 as an EBI / SIB Swiss-Prot, TrEMBL database PIR-Protein Sequence Database (PIR-PSD). It is a way to combine the contents into a UniProt consortium and provide them with mutual cooperation. The consortium provides comprehensive, accurate information about protein sequences, and maintains an accessible interface despite providing many references.

In order to confirm which protein candidates are specifically linked to which proteins and functions in UniProt, the sequence data of these specific gene candidates was applied to BLAST alignment provided by UniProt's website, and the identity for accuracy. Only data with values greater than 95% were adopted.

Example  4: STRING Analysis of Protein Interaction in Gastric Specific Gene Candidates

STRING (Search Tool for the Retrieval of Interacting Proteins; http://string.embl.de) is a database that provides comprehensive information on protein interactions.In 2000, Snel B 2000 Nucleic Acids Res 28 (18) 3442 Version 7.1 was released in 2007 as a continual upgrade since its release by -4.

In order to confirm the protein interaction information of the above specific gene candidate group, the human sequence in STRING is determined by the candidate data and our BLAST alignment (identity> = 84% and coverage> = 60%), Corresponding protein interaction information was extracted.

Figure 3 shows protein interaction information of the PHS11S15230 gene prepared using STRING. In FIG. 3, red dots represent proteins aligned with 100% identity and 100% coverage on PHS11S15230 and BLAST alignment.

This gene has an antigen-binding effect, functions as an immune response, and forms a plasma membrane. On the other hand, since the stomach has a function of protecting and blocking from food and constant stimulation from the outside, it is assumed that the antigen binding and immune response functions are more developed than other tissues.

1 is a schematic of a system for analyzing gastric specific genes.

Figure 2 shows the RT-PCR results of the PHS11S15230 gene.

Figure 3 shows protein interaction information of the PHS11S15230 gene prepared using STRING.

<110> Korea Research Institute of Bioscience and Biotechnology <120> Stomach-specific gene PHS11S15230 associated with development of          stomach <130> PN07093 <160> 4 <170> KopatentIn 1.71 <210> 1 <211> 1180 <212> DNA <213> Homo sapiens <220> <221> gene (222) (1) .. (1180) <223> Human PHS11S15230 gene <400> 1 gtttgctctg agataccagt ctcgctcctg cccagcccgg gcggctgccc ttgggtgctc 60 ccttccctgc ccgacaccca gaccgacctt gaccgcccac ctggcaggag caggacagga 120 cggccggacg cggccatggc cgagctcccg gggccctttc tctgcggggc cctgctaggc 180 ttcctgtgcc tgagtgggct ggccgtggag gtgaaggtac ccacagagcc gctgagcacg 240 cccctgggga agacagccga gctgacctgc acctacagca cgtcggtggg agacagcttc 300 gccctggagt ggagctttgt gcagcctggg aaacccatct ctgagtccca tccaatcctg 360 tacttcacca atggccatct gtatccaact ggttctaagt caaagcgggt cagcctgctt 420 cagaaccccc ccacagtggg ggtggccaca ctgaaactga ctgacgtcca cccctcagat 480 actggaacct acctctgcca agtcaacaac ccaccagatt tctacaccaa tgggttgggg 540 ctaatcaacc ttactgtgct ggttcccccc agtaatccct tatgcagtca gagtggacaa 600 acctctgtgg gaggctctac tgcactgaga tgcagctctt ccgagggggc tcctaagcca 660 gtgtacaact gggtgcgtct tggaactttt cctacacctt ctcctggcag catggttcaa 720 gatgaggtgt ctggccagct cattctcacc aacctctccc tgacctcctc gggcacctac 780 cgctgtgtgg ccaccaacca gatgggcagt gcatcctgtg agctgaccct ctctgtgacc 840 gaaccctccc aaggccgagt ggccggagct ctgattgggg tgctcctggg cgtgctgttg 900 ctgtcagttg ctgcgttctg cctggtcagg ttccagaaag agagggggaa gaagcccaag 960 gagacatatg ggggtagtga ccttcgggag gatgccatcg ctcctgggat ctctgagcac 1020 acttgtatga gggctgattc tagcaagggg ttcctggaaa gaccctcgtc tgccagcacc 1080 gtgacgacca ccaagtccaa gctccctatg gtcgtgtgac ttctcccgat ccctgagggc 1140 ggtgaggggg aatatcaata attaaagtct gtgggtacca 1180 <210> 2 <211> 327 <212> PRT <213> Homo sapiens <220> <221> PEPTIDE (222) (1) .. (327) <223> Human PHS11S15230 protein <400> 2 Met Ala Glu Leu Pro Gly Pro Phe Leu Cys Gly Ala Leu Leu Gly Phe   1 5 10 15 Leu Cys Leu Ser Gly Leu Ala Val Glu Val Lys Val Pro Thr Glu Pro              20 25 30 Leu Ser Thr Pro Leu Gly Lys Thr Ala Glu Leu Thr Cys Thr Tyr Ser          35 40 45 Thr Ser Val Gly Asp Ser Phe Ala Leu Glu Trp Ser Phe Val Gln Pro      50 55 60 Gly Lys Pro Ile Ser Glu Ser His Pro Ile Leu Tyr Phe Thr Asn Gly  65 70 75 80 His Leu Tyr Pro Thr Gly Ser Lys Ser Lys Arg Val Ser Leu Leu Gln                  85 90 95 Asn Pro Pro Thr Val Gly Val Ala Thr Leu Lys Leu Thr Asp Val His             100 105 110 Pro Ser Asp Thr Gly Thr Tyr Leu Cys Gln Val Asn Asn Pro Pro Asp         115 120 125 Phe Tyr Thr Asn Gly Leu Gly Leu Ile Asn Leu Thr Val Leu Val Pro     130 135 140 Pro Ser Asn Pro Leu Cys Ser Gln Ser Gly Gln Thr Ser Val Gly Gly 145 150 155 160 Ser Thr Ala Leu Arg Cys Ser Ser Ser Glu Gly Ala Pro Lys Pro Val                 165 170 175 Tyr Asn Trp Val Arg Leu Gly Thr Phe Pro Thr Pro Ser Pro Gly Ser             180 185 190 Met Val Gln Asp Glu Val Ser Gly Gln Leu Ile Leu Thr Asn Leu Ser         195 200 205 Leu Thr Ser Ser Gly Thr Tyr Arg Cys Val Ala Thr Asn Gln Met Gly     210 215 220 Ser Ala Ser Cys Glu Leu Thr Leu Ser Val Thr Glu Pro Ser Gln Gly 225 230 235 240 Arg Val Ala Gly Ala Leu Ile Gly Val Leu Leu Gly Val Leu Leu Leu                 245 250 255 Ser Val Ala Ala Phe Cys Leu Val Arg Phe Gln Lys Glu Arg Gly Lys             260 265 270 Lys Pro Lys Glu Thr Tyr Gly Gly Ser Asp Leu Arg Glu Asp Ala Ile         275 280 285 Ala Pro Gly Ile Ser Glu His Thr Cys Met Arg Ala Asp Ser Ser Lys     290 295 300 Gly Phe Leu Glu Arg Pro Ser Ser Ala Ser Thr Val Thr Thr Thr Lys 305 310 315 320 Ser Lys Leu Pro Met Val Val                 325 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> PCR primers <400> 3 atggagggcc tggaagtatt g 21 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> PCR primers <400> 4 aagtgccaat gcaccacact 20  

Claims (11)

A PHS11S15230 protein specifically expressed in gastric tissue, consisting of the amino acid sequence represented by SEQ ID NO: 2. The protein of claim 1, which is used as a target of therapeutic drug for stomach related diseases. The gene encoding the PHS11S15230 protein of claim 1. The gene of claim 3, wherein the gene consists of a nucleotide sequence represented by SEQ ID NO: 1. Recombinant vector comprising the gene of claim 3. A transformant transformed with the recombinant vector of claim 5. An antibody against the protein of claim 1. A marker for diagnosing a stomach related disease, comprising the nucleotide sequence represented by SEQ ID NO: 1. A microarray for diagnosing stomach related diseases, comprising the gene of claim 4 or the antibody of claim 7. A method for diagnosing gastrointestinal related diseases in a mammal, comprising measuring the expression of the gene of claim 4 in the mammal. The method of claim 10, wherein the mammal is a non-human animal.

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