KR101032860B1 - Pancreas specific gene MMP1 - Google Patents

Abstract

The present invention provides a microarray for diagnosing pancreatic related diseases including MMP1 (Mus musculus Pancreas 1) protein, a gene encoding the protein, the gene or an antibody to the protein, which are specifically expressed in pancreatic tissue of a mouse, A primer set for diagnosing pancreatic related diseases consisting of primers for the gene, a kit for diagnosing pancreatic related diseases including the primer set, and a method for diagnosing pancreatic related diseases in mammals using the primer set. .

Pancreatic specific gene, MMP1 protein, microarray, primer set, kit

Description

Pancreas specific gene MMP1

The present invention relates to a pancreatic tissue-specific gene MMP1 involved in the function of the pancreas, and more particularly to MMP1 protein, a gene encoding the protein, the gene or an antibody against the protein, specifically expressed in pancreatic tissue of a mouse. A microarray for diagnosing pancreatic related diseases, comprising a primer set for diagnosing pancreatic related diseases consisting of primers for the gene, a kit for diagnosing pancreatic related diseases comprising the primer set, and using the primer set The present invention relates to a method for diagnosing pancreatic related diseases in mammals.

The pancreas secretes digestive enzymes to promote the breakdown of carbohydrates, proteins and fats that pass through the stomach and produces important hormones such as insulin and glucagon. Without sufficient secretion of digestive enzymes from the pancreas, absorption of nutrients is insufficient. Insulin and glucagon secretion regulate blood glucose levels. Research on the mechanisms that regulate the production and secretion of these enzymes and hormones has become an important topic in relation to various diseases such as nutrition, diabetes and obesity. Pancreatic diseases include pancreatic cancer and pancreatitis. Pancreatic 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.

Accordingly, the present inventors have studied the specific genes of the pancreas, revealed that the MMP1 gene is specifically expressed in pancreatic tissue, and completed the present invention.

In order to solve the above problems, the present invention provides MMP1 protein specifically expressed in pancreatic tissue.

The present invention also provides a gene encoding the protein.

The present invention also provides a microarray for diagnosing a pancreatic related disease comprising an antibody to the gene or the protein.

The present invention also provides a primer set for diagnosing pancreatic related diseases consisting of primers for the gene.

The present invention also provides a kit for diagnosing pancreatic related diseases comprising the primer set.

The present invention also provides a method for diagnosing pancreatic related diseases in mammals using the primer set.

The present inventors provide the fact that the MMP1 gene is specifically expressed in pancreatic tissue, allowing researchers to easily access information such as protein-protein networks, pathways or transcriptional regulation of genes, and the fact that the MMP1 gene is a pancreatic specific gene. This can reduce false positives in diagnostic studies of pancreas-related diseases. In addition, the MMP1 gene can be used as a marker for the diagnosis of pancreatic diseases, and when the drug is designed as a therapeutic agent for disease symptoms, the protein target for MMP1 can be reduced. This allows for faster treatment at an early stage.

In order to achieve the above object, the present invention provides an MMP1 protein specifically expressed in pancreatic tissue, consisting of the amino acid sequence 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 the candidate group is identified as a tissue-specific gene by performing an Audic-Claverie Test based on the EST (Expressed Sequence Tag) and using experiments such as RT-PCR, the candidate group can be judged as a genetic influence factor. Reliability is increased. 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.

Thus, the present invention provides MMP1 protein that is specifically expressed in pancreatic tissue. The range of MMP1 proteins 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 MMP1 protein according to the invention is preferably derived from, but not limited to, a mouse.

MMP1 protein according to the present invention can be used as a target of therapeutic drugs for pancreatic related diseases. Examples of pancreatic diseases include, but are not limited to, pancreatic cancer, pancreatitis, diabetes, and the like. When designing drugs as diagnostics for pancreatic-related disease symptoms, drug targets can be reduced with MMP1 protein, enabling faster diagnosis at the onset of the onset.

The present invention also provides a gene encoding the MMP1 protein. Genes of the invention include both genomic DNA and cDNA encoding the MMP1 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 invention also provides an antibody against the MMP1 protein according to the invention. The antibody of the present invention can be produced by a conventional method from a protein obtained by cloning a gene into an expression vector according to a conventional method to obtain a protein encoded by the MMP1 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.

The present invention also provides a marker for diagnosing pancreatic related diseases, comprising the MMP1 gene according to the present invention. MMP1 gene of the present invention preferably may have a nucleotide sequence represented by SEQ ID NO: 1, but is not limited thereto. Examples of pancreatic diseases include, but are not limited to, pancreatic cancer, pancreatitis, diabetes, and the like. By measuring the severity of the disease associated with the pancreas according to the expression amount 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 pancreatic diseases. Since the gene can be used as a marker for identifying or diagnosing a genetic trend of a pancreas-related disease, it is possible to diagnose and perform treatment before the disease occurs by using such information.

The invention also provides a microarray for diagnosing pancreatic related diseases comprising an MMP1 gene according to the invention or an antibody against the MMP1 protein of the invention. MMP1 gene of the present invention preferably may have a nucleotide sequence represented by SEQ ID NO: 1, but is not limited thereto. Examples of pancreatic diseases include, but are not limited to, pancreatic cancer, pancreatitis, diabetes, and the like. By measuring the severity of the disease associated with the pancreas according to the expression amount 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 pancreatic diseases. 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 primer set for diagnosing pancreatic related diseases consisting of sense and antisense primers of the MMP1 protein coding gene of the present invention. The sense and antisense primers may be preferably composed of the nucleotide sequence of SEQ ID NO: 3 and 4, respectively. Examples of pancreatic diseases include, but are not limited to, pancreatic cancer, pancreatitis, diabetes, and the like. After separating the whole RNA from the tissue, and performing the RT-PCR using the primer set to measure the severity of the pancreatic-related disease according to the amount of the RT-PCR product to be amplified by measuring the amount and disease of the RT-PCR product Correlation of severity of Based on the established correlations, only the presence and quantity of RT-PCR products can be measured to predict and diagnose pancreatic related diseases in the subject.

In the present invention, "primer" refers to a single stranded oligonucleotide sequence that is complementary to the nucleic acid strand to be copied and may serve as a starting point for the synthesis of the primer extension product. The length and sequence of the primer should allow to start the synthesis of the extension product. The specific length and sequence of the primers will depend on the primer utilization conditions such as temperature and ionic strength as well as the complexity of the DNA or RNA target required.

As used herein, oligonucleotides used as primers may also include nucleotide analogues such as phosphorothioate, alkylphosphothioate or peptide nucleic acid, or It may comprise an intercalating agent.

The present invention also provides a primer set according to the present invention; And kits for diagnosing pancreatic related diseases, including reagents required for RT-PCR. In the kit of the present invention, the reagents required for the RT-PCR may be, for example, but not limited to, a forward primer and a reverse primer, a reverse transcriptase, a ribonuclease inhibitor, a heat resistant polymerase, a reaction buffer, and the like, and a reaction buffer Silver includes both reverse transcriptase and PCR buffer, and heat resistant polymerase may be EF Taq polymerase and the like, and reverse transcriptase is a reverse transcriptase originating from various sources, for example, avian myeloblastosis virus-derived reverse transcriptase. (avian myeloblastosis virus-derived virus reverse transcriptase (AMV RTase)), mouse leukemia virus-derived virus reverse transcriptase (MuLV RTase) and Rouse-associated virus 2 reverse transcriptase 2 reverse transcriptase (RAV-2 RTase).

The kit may also include instructions. The guide is a printed document that explains how to use the kit, such as how to prepare reverse transcription buffer and PCR buffer, and the reaction conditions presented. The instructions include brochures in the form of pamphlets or leaflets, labels affixed to the kit, and instructions on the surface of the package containing the kit. In addition, the guide includes information that is disclosed or provided through electronic media such as the Internet.

The present invention also provides

Isolating total RNA from the mammalian sample;

Amplifying a target sequence by using the isolated whole RNA as a template and performing RT-PCR using a primer set according to the present invention; And

It provides a method for diagnosing a pancreatic related disease in a mammal, comprising detecting the amplification product.

After separating the whole RNA from the tissue, and performing the RT-PCR using the primer set to measure the severity of the pancreatic-related disease according to the amount of the RT-PCR product to be amplified by measuring the amount and disease of the RT-PCR product Correlation of severity of Based on the established correlations, only the presence and quantity of RT-PCR products can be measured to predict and diagnose pancreatic related diseases in the subject. Examples of pancreatic diseases include, but are not limited to, pancreatic cancer, pancreatitis, diabetes, and the like.

As a method for separating total RNA from a mammalian sample of the present invention, a method known in the art may be used, for example, a phenol extraction method may be used. Using the isolated whole RNA as a template, a target sequence can be amplified by performing RT-PCR using a primer set according to an embodiment of the present invention as a primer. Such PCR methods are well known in the art, and commercially available kits may be used.

In the method of the present invention, the amplified target sequence may be labeled with a detectable labeling substance. In one embodiment, the labeling material may be, but is not limited to, a material that emits fluorescence, phosphorescence, or radioactivity. Preferably, the labeling substance is Cy-5 or Cy-3. When amplifying the target sequence, PCR is performed by labeling Cy-5 or Cy-3 at the 5'-end of the primer to allow the target sequence to be labeled with a detectable fluorescent labeling substance. In addition, the label using the radioactive material may add radioactive isotopes such as ³² P or ³⁵ S to the PCR reaction solution when PCR is performed, and the amplification product may be incorporated into the amplification product while the amplification product is synthesized. The oligonucleotide primer set used to amplify the target sequence is as described above.

The method of the present invention includes detecting the amplification product. Detection of the amplification product may be performed through DNA chip, gel electrophoresis, radioactivity measurement, fluorescence measurement or phosphorescence measurement, but is not limited thereto. As one of the methods for detecting amplification products, gel electrophoresis can be performed. Gel electrophoresis may use agarose gel electrophoresis or acrylamide gel electrophoresis depending on the size of the amplification product. In addition, in the fluorescence measurement method, PCR is performed by labeling Cy-5 or Cy-3 at the 5'-end of a primer, and a target sequence is labeled with a detectable fluorescent labeling material. It can be measured. In addition, radioactivity measuring method is to add a radioactive isotope such as ³² P or ³⁵ S to the PCR reaction solution to label the amplification product when performing PCR, and then radioactive measuring apparatus, for example, Geiger counter or liquid flash The radioactivity can be measured using a liquid scintillation counter.

In the method of the present invention, the mammal is preferably a human, but may also include non-human animals including pets such as dogs and cats, cattle such as cattle, sheep, pigs, horses, and experimental animals such as rats, white papers, guinea pigs, and the like. have.

The present invention also provides a method for diagnosing pancreatic related diseases in a mammal, comprising measuring the expression of the MMP1 gene according to the present invention. MMP1 gene of the present invention preferably may have a nucleotide sequence represented by SEQ ID NO: 1, but is not limited thereto. Examples of pancreatic diseases include, but are not limited to, pancreatic cancer, pancreatitis, diabetes, and the like. By measuring the severity of the disease associated with the pancreas according to the expression level 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 correlation, only the expression level of the gene can be measured to diagnose pancreatic related diseases in 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. 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

Example 1: Establishment of Pancreatic Specific Gene Candidates Using TISA 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 containing information on 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 pancreatic tissue specific gene in TISA, it was extracted as a candidate group of pancreatic tissue specific gene.

Example 2: RT-PCR experiment of pancreatic specific gene candidate group

Total RNA was used as mouse total RNA of Biochain. There are 15 different types of RNA tissue used, including muscle, stomach, testis, thymus, heart, cerebellum, cerebral and liver. ), Colon, kidney, lung, spleen, ovary, pancreas and uterus. 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'-GTCCAATCATGACCCTGAGC-3 '(SEQ ID NO: 3)

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

Figure 2 shows the RT-PCR results of the MMP1 gene. As can be seen in Figure 2, it was found that the MMP1 gene is rarely expressed in other tissues, but specifically expressed in pancreatic tissue.

Example 3: Reference of Pancreatic Specific Gene Candidates with UniProt

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 are specific to the pancreas and which functions are linked to UniProt, the sequence data of the pancreatic specific gene candidates was applied to the BLAST alignment provided by UniProt's website, and the identity for accuracy. Only data with values greater than 95% were adopted.

The results of the BLAST showed a 100% identity with mouse FK506-binding protein 11 (201/201) and 90% with human FK506-binding protein 11. Human FKBP11 has been reported to have relatively high expression levels in the pancreas, secretory organs such as the stomach, pituitary gland, and salivary glands.

1 depicts a system for analyzing pancreatic specific genes.

Figure 2 shows the RT-PCR results of the MMP1 gene.

<110> Korea Research Institute of Bioscience and Biotechnology <120> Pancreas specific gene MMP1 <130> PN08205 <160> 4 <170> KopatentIn 1.71 <210> 1 <211> 785 <212> DNA <213> Mouse <400> 1 aaagaaaggg gtgtgggggg gggcagtacc gagtgaaaaa tacaacgtct tgttaaaaac 60 cactatttat ttcttcttac tcttgtttcg tttctcctct tttagcttct ttttggagac 120 tttgggtctg ctggcctttc tgtacaggtg atacccgatc agccccagga gcgccggcac 180 catggcgatg cctaccagag gcaaaatgct ctttagcagc ttctgccagt agttggctcg 240 aatcaaagcg atcagctcca catcgtactg caccactgca tcggctggga tagatggcgg 300 gtaccctcgc tttccataag ccaggtgaga aggaatgact gctcttcgct tctctcccac 360 acacatgtcc aaaaggctct gctccagacc tggaatcacc tgcttttggc cgagttctat 420 gaccagagga tctctggtca gagaagtgtc aataatgcgt ccatctacca aactgcccgt 480 gtagtgtatg tggagcgtgt ctccaatggc agcagattcc gtgcacgatt cagggggctg 540 caccagggtc tccacttgga gggtccggac aggactttcg gtttctggcc cagcctcagc 600 ccggcacacc gccccactga acagtagcag cagtaacagc tggagtggta ggagcaaagg 660 gctcagggtc atgattggac cgggtcagag caccaggata ccagggcagg ctgttcgggt 720 gcacgcgacc aggacgggtt ccctcactcc aggccagact ggtatgggcg ggttttcaag 780 gcgct 785 <210> 2 <211> 201 <212> PRT <213> Mouse <400> 2 Met Thr Leu Ser Pro Leu Leu Leu Pro Leu Gln Leu Leu Leu Leu Leu   1 5 10 15 Leu Phe Ser Gly Ala Val Cys Arg Ala Glu Ala Gly Pro Glu Thr Glu              20 25 30 Ser Pro Val Arg Thr Leu Gln Val Glu Thr Leu Val Gln Pro Pro Glu          35 40 45 Ser Cys Thr Glu Ser Ala Ala Ile Gly Asp Thr Leu His Ile His Tyr      50 55 60 Thr Gly Ser Leu Val Asp Gly Arg Ile Ile Asp Thr Ser Leu Thr Arg  65 70 75 80 Asp Pro Leu Val Ile Glu Leu Gly Gln Lys Gln Val Ile Pro Gly Leu                  85 90 95 Glu Gln Ser Leu Leu Asp Met Cys Val Gly Glu Lys Arg Arg Ala Val             100 105 110 Ile Pro Ser His Leu Ala Tyr Gly Lys Arg Gly Tyr Pro Pro Ser Ile         115 120 125 Pro Ala Asp Ala Val Val Gln Tyr Asp Val Glu Leu Ile Ala Leu Ile     130 135 140 Arg Ala Asn Tyr Trp Gln Lys Leu Leu Lys Ser Ile Leu Pro Leu Val 145 150 155 160 Gly Ile Ala Met Val Pro Ala Leu Leu Gly Leu Ile Gly Tyr His Leu                 165 170 175 Tyr Arg Lys Ala Ser Arg Pro Lys Val Ser Lys Lys Lys Leu Lys Glu             180 185 190 Glu Lys Arg Asn Lys Ser Lys Lys Lys         195 200 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer <400> 3 gtccaatcat gaccctgagc 20 <210> 4 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer <400> 4 atggcgatgc ctaccagag 19  

Claims (9)

delete delete A microarray for diagnosing pancreatic related diseases comprising a gene encoding an MMP1 protein specifically expressed in pancreatic tissue, consisting of an amino acid sequence represented by SEQ ID NO: 2. The microarray of claim 3, wherein the gene is formed of a nucleotide sequence represented by SEQ ID NO: 1. A microarray for diagnosing pancreatic related diseases comprising an amino acid sequence represented by SEQ ID NO: 2, comprising an antibody against MMP1 protein specifically expressed in pancreatic tissue. A primer set for diagnosing pancreatic related diseases consisting of the nucleotide sequences of SEQ ID NOs: 3 and 4. A primer set according to claim 6; And a reagent necessary for RT-PCR. Isolating total RNA from the mammalian sample; Amplifying a target sequence by using the isolated whole RNA as a template and performing RT-PCR using the primer set according to claim 6; And And detecting the amplification product. The method of claim 8, wherein the mammal is a non-human animal.

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