KR20120023877A - Liver specific gene ws-h10m06630 - Google Patents

Abstract

PURPOSE: A protein which is specifically expressed in liver tissue, WS-H10M06630, and a gene encoding the same are provided to diagnose diseases and reduce side effect of drug. CONSTITUTION: A protein specifically expressed in liver tissue, WS-H10M06630, contains an amino acid sequence of sequence number 2. A gene encoding the protein has a base of sequence number 1. A recombinant vector contains the gene encoding the protein. A transformant is prepared by transforming with the recombinant vector. A microarray for diagnosing liver-related diseases contains the gene or an antibody to WS-H10M06630 protein.

Description

Liver specific gene WS-H10M066630 {Liver specific gene WS-H10M06630}

The present invention relates to a liver tissue specific gene WS-H10M06630, and more particularly to a WS-H10M06630 protein specifically expressed in liver tissue, a gene encoding the protein, a recombinant vector comprising the gene, and the recombinant vector. A microarray for diagnosing liver related diseases, comprising transformed transformants and antibodies to said genes and said proteins.

The liver plays a central role in metabolism and has numerous functions such as carbohydrate storage, red blood cell breakdown, protein synthesis, and detoxification. To this end, the liver consists of specialized cellular tissues and secretes unique enzymes, signaling agents, and regulators.

Tissue-specific genes can be used as markers for diagnosing diseases by tracking their changes, or they can be themselves targets for the treatment of diseases, or they can act as mediators to help drugs act on specific tissues.

Korean Patent No. 10-0389109 discloses a gene sequence involved in tissue specific expression in the liver or pancreas, and Korean Patent Publication No. 2010-0044623 discloses a liver tissue specific gene MML2, but the present invention Is different from the WS-H10M06630 gene.

Therefore, the inventors of the present invention, while studying a specific gene of the liver, revealed that the WS-H10M06630 gene is specifically expressed in liver tissue, and completed the present invention.

In order to solve the above problems, the present invention provides a WS-H10M06630 protein that is specifically expressed in liver 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 recombinant vector.

The present invention also provides a microarray for diagnosing liver related diseases, including the gene and antibodies to the protein.

To know the safety and effectiveness of the drug metabolism in the liver, it is important to identify information on protein-protein interactions, pathways, or regulatory networks involved in the metabolic activity of drug metabolism. However, analyzing and generating individual genes to reveal such information takes a very long time to identify genes involved in drug metabolic activity.

The present inventors provide the fact that the WS-H10M06630 gene involved in drug metabolism activity is specifically expressed in liver tissue, allowing researchers to easily access information such as protein-protein networks or pathways for drug metabolism activity. The fact that the H10M06630 gene is a liver specific gene can provide valuable information about drug safety and efficacy. In addition, the WS-H10M06630 gene can be used as a marker for the diagnosis of drug metabolism activity, which can be diagnosed more easily, and the side effects of the drug can be reduced with the protein for WS-H10M06630. It is possible.

1 is a schematic of a system for analyzing liver specific expressed genes.
Figure 2 shows the RT-PCR results of the WS-H10M06630 gene (Si: small intestine, St: stomach, Te: testicles, Th: thymus, Tr: thyroid, Bm: bone marrow, Br: brain, Li: liver, Co: Large intestine, Ki: kidney, Lu: lung, Mg: mammary gland, Ov: ovary, Pa: pancreas, Pr: prostate).
Figure 3 shows protein interaction information of the WS-H10M06630 gene prepared using STRING.

In order to achieve the above object, the present invention provides a WS-H10M06630 protein that is specifically expressed in liver tissue, consisting of the amino acid sequence represented by SEQ ID NO: 2. The range of WS-H10M06630 protein according to the present invention includes a protein having the amino acid sequence represented by SEQ ID NO: 2 and a functional equivalent of the protein. "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.

Prediction of tissue-specific genes through methods such as DNA chips does not make it possible to distinguish whether they are actually tissue-specific genes or experimental errors due to the numerous errors present in the experiment.

However, if a candidate group is identified as a tissue-specific gene using an experiment such as RT-PCR based on a DNA chip, the candidate group can be judged as a genetic influence, thereby increasing the reliability of the candidate group. The inventors have provided higher reliability for candidate groups of tissue specific genes by examining candidate groups of genes involved in tissue development and examining whether they contain tissue specific information.

The WS-H10M06630 protein according to the present invention is preferably derived from human, but is not limited thereto.

The WS-H10M06630 protein according to the present invention can be used as a target of therapeutic drugs for liver related diseases. Examples of liver-related diseases include, but are not limited to, hepatitis, liver cancer, fatty liver, cirrhosis, and the like. When designing drugs as diagnostics for liver-related disease symptoms, the drug target can be reduced with WS-H10M06630 protein, allowing for faster diagnosis early onset.

The present invention also provides a gene encoding the WS-H10M06630 protein. Genes of the invention include both genomic DNA and cDNA encoding the WS-H10M06630 protein. Preferably, the gene of the present invention may include the nucleotide sequence shown in SEQ ID NO: 1.

Variants of the above base sequences are also 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, heterologous peptide or heterologous nucleic acid. The recombinant cell can express a gene or a gene fragment that is not found in the natural form of the cell in one of 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), nucleic acid molecule, which is transferred into a cell. The vector replicates the DNA and can be independently regenerated in the host cell. 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. Promoters, enhancers, termination signals and polyadenylation signals available in eukaryotic cells are known.

In the present invention, any promoter known in the art may be used as a promoter of the expression vector. For example, the immediate early promoter of cytomegalovirus, the promoter of SV40 (SV 40 late promoter and SV 40 early promoter), the tk promoter of herpes simplex virus (HSV), and the Adeno 2 major late promoter PAdml ), Adenovirus 2 early promoter (PAdE2), p19 promoter of human parvo virus-associated virus (AAV) and metallothionein promoter of mouse, MT promoter and MMTV LTR promoter, etc. may be used, but are not limited thereto. It is not.

In the present invention, the transcription terminator of the expression vector 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 a microarray for diagnosing the development of liver related diseases, comprising the WS-H10M06630 gene according to the invention. The microarray of the present invention can be easily prepared by a manufacturing method commonly used in the art using the marker of the present invention.

In the microarray of the present invention, "microarray" refers to a microarray in which a group of polynucleotides is immobilized to a high density on a substrate, and each of the polynucleotide groups is 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 an oligonucleotide 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 invention also provides a microarray for diagnosing the development of liver related diseases, comprising an antibody of the WS-H10M06630 protein according to the invention. The microarray of the present invention may refer to the aforementioned microarray except that the antibody of the present invention is immobilized in place of the polynucleotide in the microarray.

The antibody of the present invention can be produced by a conventional method from the protein obtained by cloning each gene into an expression vector according to a conventional method to obtain a protein encoded by the WS-H10M06630 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.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only intended to illustrate the invention, so the scope of the invention is not to be construed as limited by these examples.

Example  One: DNA  Chip experiment

It was observed that the expression gradually increased over time in the liver after drug treatment. DNA chip experiments were performed using Affymetrix: Human Genome U133A 2.0 Array, and Quintet [Choe JK 2005 LNCS 3642 392] and Genowiz (Ocimum Biosolutions) were used to examine the genes involved in the metabolic changes. A list of significant genes of time flow state was extracted.

Table 1 below shows the number of significant genes over time as a result of DNA chip experiments.

time Significant gene (TG / WT) Liver Specific Related Significant Genes 6 hours 1521 (845/676) 65 12 hours 1220 (639/581) 52 24 hours 1533 (720/813) 41

In Table 1, TG is a group treated with drugs, and WT represents a group not treated with drugs. As can be seen in Table 1, the WS-H10M06630 gene was shown to be up-regulated only at 6 hours.

Example  2: TISA  Establishment of Liver Specific Gene Candidates Using Database

Noh SJ (DNA Res (2006) 13 (5); 229-43) published a Tissue-specific Alternative Splicing (TISA) database containing information on genes, transcript structure, alternative splicing events and tissue specificity. In order to check whether there are significant genes extracted from the TISA experiments in the TISA, check whether the Refseq information of the TISA genes is the same as the Refseq corresponding to the probe on the DNA chip, and connect the TISA database with the DNA chip. Among the liver specific genes predicted at, the genes significantly expressed on the DNA chip were extracted as candidate groups of the liver specific genes.

Example  3: of liver specific gene candidates RT - PCR  Experiment

There are 20 different types of tissues: mammary gland, kidney, bone marrow, thyroid, colon, pancreas, kidney, liver, lung, brain, ovary, prostate, thymus, stomach, testes, heart and small intestine. RNA was obtained by purchasing products of Clontech (www.clontech.com) of each organization. About 100ul of cDNA was obtained by RT-PCR using Standard Two-step RT-PCR- & GO ™ (Q-biogene, Cat # RT2ST100) from the obtained RNA 1ug. Primers for these genes to identify liver specific genes (forward primer: 5'-GGCATTACTGACTTCCGTGCTA-3 '(SEQ ID NO: 3) and reverse primers: 5'-TGTGCAGTGACCTGAACAAC-3' (SEQ ID NO: 4)) (PCR product size 719 bp) and PCR was performed using cDNA for the 20 tissues obtained above. PCR conditions were last extended for 30 min of predenatured 95 ° C. 5 min, followed by 30 cycles of 95 ° C. 1 min, annealing 58 ° C. 1 min, extension 72 ° C. 1 min, and 72 ° C. 5 min. 1% agarose gel electrophoresis was performed to confirm the PCR product.

Figure 2 shows the RT-PCR results of the WS-H10M06630 gene. As can be seen in Figure 2, it was found that the WS-H10M06630 gene is rarely expressed in other tissues, but specifically expressed in the liver.

Example  4: UniProt Reference of Liver 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 identify which protein candidates appear to be specific to the liver, and which functions are linked to which proteins on UniProt, the sequence data of the liver specific gene candidates was applied to the BLAST alignment provided by UniProt's website. Only data with values greater than or equal to% are taken.

Example  5: STRING Analysis of Protein Interaction in Liver 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 (Nucleic Acids Res (2000) 28 ( 18); version 8.3 was released in 2010 as a continual upgrade since its release by 3442-4).

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

Figure 3 shows protein interaction information of the WS-H10M06630 gene prepared using STRING. In FIG. 3, red dots represent proteins aligned with 100% identity and 100% coverage on the BLAST alignment with WS-H10M06630.

<110> KOREA FOOD RESEARCH INSTITUTE <120> Liver specific gene WS-H10M06630 <130> PN10171 <160> 4 <170> KopatentIn 1.71 <210> 1 <211> 1924 <212> DNA <213> Homo sapiens <400> 1 tgaaccagca attaataaca ctttttattt agcacattca caaaagaaat ggatctaaat 60 tatcattcat taatcacttt tctgtgtttt acagtgataa catattaaaa agtcagcatt 120 agaaaagtat tagcatatgc agcaattaat acaagtgtta cagagtatga ataattgcag 180 atatatttgt gcagtgacct gaacaactct ccttaatgga gtttggatgc ttatgggata 240 ttgagtgaat gggaagattt gatgagaggt cagagaagac ataatagtgg gaatgtcctt 300 gataaaaaaa gagttgcagg tgatagcaga tcggcagcca gatgggctag cattcttcag 360 acagggatga agcagatctg gtatgagggt ggcagagaaa caatcccttt ggtaactgca 420 gtagtattga ggttctttaa atcatcaaca gatttcaggt taaagttctg taaaattgtg 480 gttagaaata aaaatagctc catgcgggca agtccttctc ctgcacaaat tcgttttcct 540 gctgagaaag gcatgaagta gtcacttttc ttaaagttgc cattcttatc tagaaagtgg 600 ccagggtcaa agatatttgg attaggaaat tctttgtcat catgtagcac ggaagtcagt 660 aatgccatta tggttgtgcc cttggggatg aggtagtttc tgaacttagt atcagtggtc 720 actgcatggg gcacaccggt ggggacaagg tcactgtatc tctggatctc gtgcactaca 780 gcatcagtgt aaggcatgtg gctcctatcc tgcatgcagg ggctcctgtg tctgccaatt 840 acatgatcaa tctcttcctg gactttagct gtgacctctg ggtgcttcag caggagcagg 900 agtccatatc tcagagtggt gcttgttgtc tctgttccag caacaaatag atcagctaca 960 gtgccaacca agttttcaat attgaattct gacttttggt tgtccttttc ctgctccatt 1020 ttgatcagga agcaatcgat aaagtcccga ggattgttaa catccagtga tgcttggtgt 1080 tcttttactt tctccctaat gtaacttcgt gtaagagcaa catttttaag cactttgttg 1140 tgagttcctg ggaaacaatc aatgagtaga gggaaattat tgcagacctg gatccatggg 1200 gagttcagaa tcctgaagtt ttcattgaat cttttcatca gggtgagaaa attctgatct 1260 ttataatcaa atcgtttctg gaaaacaacg gagcagatca cattgcaggg agcacagccc 1320 aggatgaaag tgggatcaca gggtgaagcc ttggtttttc tcaactcctc cacaaggcag 1380 tgagcttcct cttgaacacg gtcctcaatg ctcctcttcc ccatcccaaa attccgcaag 1440 gttgtgaggg agaaacgccg gatctccttc catctctttc cattgctgga aatgattcca 1500 agtcctttag taattctttg agatattggg gaattgcctc ttccagaaaa ctcctctcca 1560 ttatcaatca gggcttcctt cactgcctca tatccatgaa acaccactat gggattcatg 1620 ccaaaataca cggtgaacac aggaccatag acttttgaga aattggtgaa agatttgcag 1680 atgtccttaa catctatctg tagcatattt ccaataatag gaagaggagt ggggccagga 1740 gggagcttcc ttctcctaca gctctgtctc cagagtgaaa agagaagcat aaaagagaga 1800 cacagcacca ggaccacaaa aggttccatt gaagccttct cttcttatta agacagctgt 1860 gagcttgcac tccaaagttt ttataacact ccctgctaat ttagtgtgtg tctctttgac 1920 atgt 1924 <210> 2 <211> 490 <212> PRT <213> Homo sapiens <400> 2 Met Glu Pro Phe Val Val Leu Val Leu Cys Leu Ser Phe Met Leu Leu   1 5 10 15 Phe Ser Leu Trp Arg Gln Ser Cys Arg Arg Arg Lys Leu Pro Pro Gly              20 25 30 Pro Thr Pro Leu Pro Ile Ile Gly Asn Met Leu Gln Ile Asp Val Lys          35 40 45 Asp Ile Cys Lys Ser Phe Thr Asn Phe Ser Lys Val Tyr Gly Pro Val      50 55 60 Phe Thr Val Tyr Phe Gly Met Asn Pro Ile Val Val Phe His Gly Tyr  65 70 75 80 Glu Ala Val Lys Glu Ala Leu Ile Asp Asn Gly Glu Glu Phe Ser Gly                  85 90 95 Arg Gly Asn Ser Pro Ile Ser Gln Arg Ile Thr Lys Gly Leu Gly Ile             100 105 110 Ile Ser Ser Asn Gly Lys Arg Trp Lys Glu Ile Arg Arg Phe Ser Leu         115 120 125 Thr Thr Leu Arg Asn Phe Gly Met Gly Lys Arg Ser Ile Glu Asp Arg     130 135 140 Val Gln Glu Glu Ala His Cys Leu Val Glu Glu Leu Arg Lys Thr Lys 145 150 155 160 Ala Ser Pro Cys Asp Pro Thr Phe Ile Leu Gly Cys Ala Pro Cys Asn                 165 170 175 Val Ile Cys Ser Val Val Phe Gln Lys Arg Phe Asp Tyr Lys Asp Gln             180 185 190 Asn Phe Leu Thr Leu Met Lys Arg Phe Asn Glu Asn Phe Arg Ile Leu         195 200 205 Asn Ser Pro Trp Ile Gln Val Cys Asn Asn Phe Pro Leu Leu Ile Asp     210 215 220 Cys Phe Pro Gly Thr His Asn Lys Val Leu Lys Asn Val Ala Leu Thr 225 230 235 240 Arg Ser Tyr Ile Arg Glu Lys Val Lys Glu His Gln Ala Ser Leu Asp                 245 250 255 Val Asn Asn Pro Arg Asp Phe Ile Asp Cys Phe Leu Ile Lys Met Glu             260 265 270 Gln Glu Lys Asp Asn Gln Lys Ser Glu Phe Asn Ile Glu Asn Leu Val         275 280 285 Gly Thr Val Ala Asp Leu Phe Val Ala Gly Thr Glu Thr Thr Ser Thr     290 295 300 Thr Leu Arg Tyr Gly Leu Leu Leu Leu Leu Lys His Pro Glu Val Thr 305 310 315 320 Ala Lys Val Gln Glu Glu Ile Asp His Val Ile Gly Arg His Arg Ser                 325 330 335 Pro Cys Met Gln Asp Arg Ser His Met Pro Tyr Thr Asp Ala Val Val             340 345 350 His Glu Ile Gln Arg Tyr Ser Asp Leu Val Pro Thr Gly Val Pro His         355 360 365 Ala Val Thr Thr Asp Thr Lys Phe Arg Asn Tyr Leu Ile Pro Lys Gly     370 375 380 Thr Thr Ile Met Ala Leu Leu Thr Ser Val Leu His Asp Asp Lys Glu 385 390 395 400 Phe Pro Asn Pro Asn Ile Phe Asp Pro Gly His Phe Leu Asp Lys Asn                 405 410 415 Gly Asn Phe Lys Lys Ser Asp Tyr Phe Met Pro Phe Ser Ala Gly Lys             420 425 430 Arg Ile Cys Ala Gly Glu Gly Leu Ala Arg Met Glu Leu Phe Leu Phe         435 440 445 Leu Thr Thr Ile Leu Gln Asn Phe Asn Leu Lys Ser Val Asp Asp Leu     450 455 460 Lys Asn Leu Asn Thr Thr Ala Val Thr Lys Gly Ile Val Ser Leu Pro 465 470 475 480 Pro Ser Tyr Gln Ile Cys Phe Ile Pro Val                 485 490 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Forward primer <400> 3 ggcattactg acttccgtgc ta 22 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer <400> 4 tgtgcagtga cctgaacaac 20

Claims (7)

WS-H10M06630 protein specifically expressed in liver tissue, consisting of the amino acid sequence represented by SEQ ID NO: 2. The gene encoding the WS-H10M06630 protein of claim 1. The gene according to claim 2, wherein the gene consists of a nucleotide sequence represented by SEQ ID NO: 1. A recombinant vector comprising the gene of claim 2. A transformant transformed with the recombinant vector of claim 4. Microarray for diagnosing liver related diseases, comprising a gene consisting of the nucleotide sequence of SEQ ID NO: 1. A microarray for diagnosing liver related diseases, comprising an antibody against the protein of claim 1.

Images