WO2001009320A1 - Gene codant pour une nouvelle proteine de type facteur de ribosylation adp (arf) - Google Patents

Gene codant pour une nouvelle proteine de type facteur de ribosylation adp (arf) Download PDF

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WO2001009320A1
WO2001009320A1 PCT/JP2000/005067 JP0005067W WO0109320A1 WO 2001009320 A1 WO2001009320 A1 WO 2001009320A1 JP 0005067 W JP0005067 W JP 0005067W WO 0109320 A1 WO0109320 A1 WO 0109320A1
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protein
dna
seq
sequence
present
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PCT/JP2000/005067
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English (en)
Japanese (ja)
Inventor
Toshio Ota
Takao Isogai
Koji Hayashi
Kaoru Saito
Jun-Ichi Yamamoto
Shizuko Ishii
Tomoyasu Sugiyama
Ai Wakamatsu
Keiichi Nagai
Tetsuji Otsuki
Ryo Tokioka
Shigeo Ihara
Hiroki Nakae
Tetsuo Nishikawa
Koichi Kimura
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Helix Research Institute
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to novel MF-like proteins, their genes, and their production and use.
  • ADP-ribosylation factor-like protein is a gene that has been widely identified from various organisms as an ADP-ribosylation factor family member gene (Boman, AL et al. , (1995) Trends in Biological Science 20: 147-150).
  • ARF-like protein is a GTP-binding protein like ARF, but unlike ARF, it has a weak ADP-ribosylation-promoting activity and is known to be localized on the membrane in cells. Its exact physiological function has not yet been determined, but recently it has been shown that ARF-like proteins are deeply involved in protein secretion from cells (Huang CF et al., (1999) J. Biol. Chem.
  • ARF-like proteins are related. Therefore, a novel gene encoding ARF-like protein is considered to be important as a target for drug development for diagnosis, prevention and treatment of these diseases. Disclosure of the invention
  • the present invention has been made in view of such circumstances, and an object of the present invention is to provide a novel ARF-like protein, a gene thereof, and a production method and use thereof. is there.
  • the present inventors isolated a plurality of full-length cDNAs from a human 10-week-old fetal tissue cDNA library by an oligocap method originally developed to isolate full-length cDNAs.
  • the nucleotide sequence of one of the isolated cDNAs was determined and its structural analysis revealed significant homology with the known ARF, indicating that the cDNA encodes a novel ARF-like protein.
  • This clone was named “(: -HEMBA1001174”). Since ARF is involved in biological functions such as intracellular vesicle transport in cells, C-HEMBA1001174 is a disease associated with these functions. It is considered to be an important target for the development of drugs for diagnosing and preventing or treating these diseases.
  • the present invention relates to a novel ARF-like protein C-HEMBA1001174 and a DNA encoding the protein, and their production and use.
  • a polynucleotide comprising at least 15 nucleotides complementary to DNA consisting of the nucleotide sequence of SEQ ID NO: 1 or a complementary strand thereof,
  • step (c) selecting a compound that increases or decreases the DNA expression level according to (1), which is measured in step (b) and compared to the case where the test sample is not contacted, according to (1), (12) The DNA according to (1) or (2), the protein or peptide according to (3), the vector according to (4), or the screening according to (9) or (11).
  • a pharmaceutical composition comprising a compound isolated by
  • the present invention provides a novel protein “C-HEMBA1001174”.
  • the amino acid sequence of human C-HEMBA1001174 protein contained in the protein of the present invention is shown in SEQ ID NO: 2, and the nucleotide sequence of cDNA encoding the protein is shown in SEQ ID NO: 1.
  • the C-HEMBA1001174 gene encodes a 179 amino acid protein with significant homology to the ADP-ribosylation factor (ARF) family.
  • ARF plays an important role in the intracellular vesicle trafficking process and functions as a major regulator of protein transport from ER cells to the Golgi apparatus.
  • the C-HEMBA1001174 protein is considered to have such a function in vivo due to its structural characteristics. Therefore, it is expected to develop a drug targeting the C-HEMBA1001174 gene and its protein to diagnose diseases related to protein secretion and reabsorption functions and to prevent or treat such diseases.
  • the C-HEMBA1001174 protein can be prepared as a recombinant protein or as a natural protein.
  • the recombinant protein can be prepared, for example, by introducing a vector into which an MA encoding the protein of the present invention has been inserted into an appropriate host cell and purifying the protein expressed in the transformant as described below. It is.
  • a natural protein can be prepared, for example, using an affinity column to which an antibody against the protein of the present invention described below is bound (Current Protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons Section 16.1-16.19).
  • the antibody used for affinity purification may be a polyclonal antibody or a monoclonal antibody.
  • the present invention includes a protein functionally equivalent to the human-derived C-HEMBA1001174 protein identified in this example.
  • Such proteins include, for example, mutants, homologs, and variants of the C-HEMBA1001174 protein shown in SEQ ID NO: 2.
  • “functionally equivalent” means that the target protein has the same biological or biochemical function as the C-HEMBA1001174 protein.
  • Such functions include the function of binding to GTP and the function of regulating vesicle transport, which is localized on the membrane in cells.
  • Whether the target protein is functionally equivalent to the protein of the present invention is determined by introducing a DNA encoding the protein of interest into a yeast in which the gene of the present invention is defective or temperature-sensitive. It can be evaluated by confirming whether or not the functions can be complemented.
  • proteins functionally equivalent to the protein identified in this example, for example, by introducing a mutation into an amino acid sequence in the protein (for example, by site-directed mutagenesis (Current Protocols). (1987) Publish. John Wiley & Sons Section 8.1-8.5)) in Molecular Biology edit.
  • Such proteins may also be generated by amino acid mutations in nature.
  • one or more amino acids may be substituted, deleted, inserted and / or added in the amino acid sequence (SEQ ID NO: 2) as long as the protein has a function equivalent to the protein identified in this example. Different proteins are included depending on the factors.
  • the number and location of amino acid mutations in proteins are not limited as long as their functions are maintained.
  • the number of mutations is typically within 30 amino acids, preferably within 10 amino acids, and more preferably within 5 amino acids (eg, within 3 amino acids).
  • the substituted amino acid is preferably an amino acid having properties similar to the amino acid before substitution from the viewpoint of maintaining the function of the protein. For example, Ala, Val, Leu, I Since le, Pro, Met, Phe, and Trp are all classified as non-polar amino acids, they are considered to have similar properties.
  • examples of the non-charger include Gly, Ser, Thr, Cys, Tyr, Asn, and Gin.
  • acidic amino acids include Asp and Glu
  • basic amino acids include Lys, Arg, and His.
  • Proteins functionally equivalent to the proteins identified in this example can be obtained using the current hybridization techniques known to those skilled in the art (Current Protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons Section). 6.3-6.4) or can be isolated using gene amplification technology (PCR) (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons Section 6.1-6.4) It is. That is, those skilled in the art can use a DNA encoding the protein identified in the present example (SEQ ID NO: 1) or a part thereof as a probe, or an oligonucleotide that specifically hybridizes with the DNA as a primer.
  • DNA that hybridizes with DNA can be isolated. Further, based on the isolated DNA, a protein encoded by the DNA can be prepared.
  • the present invention includes proteins encoded by DNAs that hybridize with DNAs encoding these proteins, as long as they have the same function as the proteins identified in this example. Examples of organisms for isolating functionally equivalent proteins include, but are not limited to, vertebrates such as humans, mice, rats, magpies, bushes, and magpies.
  • the stringent conditions of hybridization for isolating DNA that encodes a functionally equivalent protein are usually about lxSSC, 0.1% SDS, 37.C. Is about 0.5xSSC, 0.13 ⁇ 4SDS, 42 ° C, and the more severe condition is about 0.1xSSC, O.SDS, 65 ° C.
  • the more severe the hybridization conditions the more probe arrangement It can be expected that a DNA having high homology with the DNA will be isolated.
  • the combination of the SSC, SDS, and temperature conditions described above is merely an example, and those skilled in the art will appreciate that the determination of the stringency of the hybridization can be performed in the above manner. By appropriately combining other factors (eg, probe concentration, probe length, hybridization reaction time, etc.), it is possible to achieve the same stringency as described above.
  • a protein isolated using such a hybridization technique or a gene amplification technique usually has higher homology in its amino acid sequence than the protein of the present invention described in SEQ ID NO: 2.
  • High homology refers to sequence identity of at least 50% or more, more preferably 70% or more, and even more preferably 90% or more (eg, 95% or more).
  • Amino acid sequence homology can be determined by homology search using BLAST X.
  • the present invention also provides a partial peptide of the protein of the present invention.
  • the partial peptide of the protein of the present invention can be used, for example, for preparing an antibody that binds to the protein of the present invention.
  • the partial peptide of the present invention comprises an amino acid sequence of at least 7 amino acids, preferably 9 amino acids or more, more preferably 12 amino acids or more, more preferably 15 amino acids or more.
  • the partial peptide of the present invention can be produced, for example, by a genetic engineering technique, a known peptide synthesis method, or by cleaving the protein of the present invention with an appropriate peptide.
  • the present invention also provides a DNA encoding the protein of the present invention.
  • the form of the DNA of the present invention is not particularly limited as long as it can encode the protein of the present invention, and includes genomic DNA, chemically synthesized DNA, and the like in addition to cDNA.
  • a DNA having an arbitrary nucleotide sequence based on the degeneracy of the genetic code is included.
  • the DNA encoding the protein of the present invention is prepared by a hybridization method using the DNA sequence of SEQ ID NO: 1 or a part thereof as a probe, or a primer designed based on the information of these DNA sequences. It can be isolated by a conventional method such as the gene amplification method (PCR) used.
  • PCR gene amplification method
  • the present invention also provides a vector into which a DNA encoding the protein of the present invention has been inserted.
  • the vector of the present invention is not particularly limited as long as it can stably maintain the inserted DNA.
  • the cloning vector is preferably a pBluescript vector (Stratagene).
  • an expression vector is particularly useful.
  • an expression vector for example, pBEST vector for in vitro expression
  • pET vector Invitrogen
  • PME18S-FL3 vector GenBank Accession No. AB009864
  • the pME18S vector can be suitably used. Insertion of the DNA encoding the protein of the present invention into a vector is carried out by a conventional method, for example, ligase anti / heart using a restriction enzyme site (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons. Section 11.4-11.11).
  • the present invention also provides a transformant having a DNA encoding the protein of the present invention or a vector into which the DNA has been inserted.
  • the host cell into which the vector of the present invention is introduced is not particularly limited, and various host cells can be used depending on the purpose. Host cells can be used, for example, for the production of the proteins of the invention.
  • Production systems for protein production include in vitro and in vivo production systems. In vitro production systems include production systems that use eukaryotic cells and production systems that use prokaryotic cells. When eukaryotic cells are used, for example, animal cells, plant cells, and fungal cells can be used as hosts.
  • the host cells of the present invention also include cells intended for use in analyzing the function of the C-HEMBA1001174 protein and screening for a function inhibitor or function promoter using the C-HEMBA1001174 protein.
  • the introduction of a vector into a host cell can be performed, for example, by the calcium phosphate precipitation method or the electropulse perforation method (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley
  • the present invention also provides a polynucleotide comprising at least 15 nucleotides complementary to MA consisting of the nucleotide sequence of SEQ ID NO: 1 or a complementary strand thereof.
  • the “complementary strand” refers to one strand of the double-stranded DNA composed of A: T and G: C base pairs with respect to the other strand.
  • the term “complementary” is not limited to a case where the sequence is completely complementary in at least 15 contiguous nucleotide regions, and is at least 70%, preferably at least 80%, more preferably 90%, and still more preferably It suffices to have at least 95% homology on the base sequence.
  • the algorithm for determining homology may use the one described in the present specification.
  • Such a polynucleotide can be used as a probe for detecting and isolating MA encoding the protein of the present invention, and as a primer for amplifying the DNA of the present invention.
  • a primer When used as a primer, it usually has a chain length of 15 to 100 bases, preferably 15 to 35 bases, and more preferably 20 to 30 bases.
  • MA having at least a part or all of the sequence of the DNA of the present invention and having a chain length of at least 15 bases is used.
  • the 3 'region must be complementary, but a restriction enzyme recognition sequence or a tag can be added to the 5' region.
  • the polynucleotide of the present invention includes an antisense for suppressing the expression of the C-HEMBA1001174 protein of the present invention.
  • the antisense has a chain length of at least 15 bp or more, preferably 100 bp, more preferably 500 bp or more, and preferably has a chain length of 2000 bp or less in order to cause an antisense effect.
  • Such an antisense can be obtained, for example, by the phosphorothioate method (Stein, 1988) based on the sequence information of MA described in SEQ ID NO: 1 (Stein, 1988). ) Can be prepared.
  • the DNA of the present invention and its antisense can be applied to, for example, gene therapy.
  • Alheimer for example, is considered as a disease targeted for gene therapy using D of the present invention.
  • the ex vivo method or the in vivo method using viral vectors such as retrovirus vectors, adenovirus vectors, and adeno-associated virus vectors or non-viral vectors such as liposomes is used. It may be administered to a patient by an in vivo method or the like.
  • the present invention also provides an antibody that binds to the protein of the present invention.
  • the form of the antibody of the present invention is not particularly limited, and includes a polyclonal antibody, a monoclonal antibody, and a part thereof having antigen-binding properties. It also includes all classes of antibodies. Furthermore, the antibodies of the present invention also include special antibodies such as humanized antibodies.
  • the antibody of the present invention can be obtained by synthesizing an oligonucleotide corresponding to an amino acid sequence and immunizing a rabbit according to a conventional method (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons. Section 11.12-11.13)
  • mice were immunized using a protein expressed and purified in E. coli according to a conventional method, and spleen cells were isolated. It can be obtained from hybridoma cells obtained by cell fusion of myeloma cells (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons. Section 11.4 to 11.11).
  • Antibodies that bind to the protein of the present invention may be used, for example, for the examination and diagnosis of abnormal expression or structural abnormality of these proteins, in addition to the purification of the protein of the present invention. Specifically, proteins are extracted from, for example, tissues, blood, or cells, and the presence or absence of abnormal expression or structure is examined through detection of the protein of the present invention by Western blotting, immunoprecipitation, or ELISA. Can be diagnosed.
  • Antibodies that bind to the protein of the present invention may be used for the purpose of treating diseases related to the protein of the present invention.
  • a human antibody or a humanized antibody is preferred because of its low immunogenicity.
  • Human antibodies are immune Mice in which the system has been replaced with that of a human (see, for example, “Functional transplant of meg abase human immunoglobul in loci recapitulates human antiboay response in mice, Mendez, MJ et al. (1997) Nat. Genet. 15: 146-156”). ) Can be prepared.
  • a humanized antibody can be prepared by genetic recombination using the hypervariable region of a monoclonal antibody (Methods in Enzymology 203, 99-121 (1991)).
  • the present invention also provides a method for screening for a compound that binds to the protein of the present invention.
  • This screening method comprises: (a) a step of bringing a test sample into contact with the protein of the present invention or a partial peptide thereof; (b) a step of detecting a binding activity between the protein or a partial peptide thereof and the test sample; (C) selecting a compound having an activity of binding to the protein or a partial peptide thereof.
  • Specific methods include, for example, a method of contacting and purifying a test sample with an affinity column of the protein of the present invention, a method using a two-hybrid system, a West Western blotting method, and a combinatorial chemistry technique. Many known methods such as a method using high-throughput screening can be used. Screening can also be performed by using a measuring device such as BIACORE (Pharmacia) to evaluate the binding between the protein of the present invention and a test compound.
  • the test sample used for the screening is not limited to these, but includes, for example, a cell extract, an expression product of a gene library, a synthetic low molecular compound, a synthetic peptide, a natural compound, and the like.
  • the compound isolated by this screening is a candidate for a compound (agonist, angonist) that promotes or inhibits the activity of the protein of the present invention.
  • it is a candidate for a compound that inhibits the interaction between the protein of the present invention and a molecule that interacts with the protein in vivo.
  • the present invention also provides a method for diagnosing a disease caused by abnormal expression of the gene of the present invention. This diagnosis is expected to be effective in the diagnosis of diseases caused by abnormal protein secretion and reabsorption, such as cystic fibrosis and diabetes.
  • the diagnosis of the present invention can be performed by preparing a cell sample from a patient and detecting the expression level or mutation of the gene of the present invention in the cell.
  • the cell sample prepared from the patient may be appropriately selected according to the type of the disease to be diagnosed. For example, cells derived from kidney tissue or muscle tissue can be suitably used.
  • Expression of the gene of the present invention in cells can be detected by a method known to those skilled in the art. Examples of such a method include, but are not limited to, Northern blotting and RT-PCR.
  • Northern blotting method RNA from cells to be tested is purified, electrophoresed in an agarose gel, and then blotted on a membrane. The expression of the gene of the present invention is measured based on the presence or absence and density of a band that is hybridized and appears specifically.
  • RT-PCR method RNA of a cell to be tested is purified and converted into cDNA using reverse transcriptase, and this is designated as ⁇ , and a sequence characteristic of the gene of the present invention is used as a primer to obtain the gene of the present invention.
  • the cDNA derived from the transcript is amplified by PCR. Since the amount of cDNA amplified by this is considered to be proportional to the amount of type II cDNA and, consequently, the amount of the transcript of the gene of the present invention, the amount of DNA fragment amplified by this PCR is determined by electrophoresis. By using this method, the expression level of the gene of the present invention can be measured.
  • the mutation of the gene of the present invention can be detected by a method of directly determining the nucleotide sequence by PCR or the like.
  • the DNA of the cell to be tested is prepared, and the MA of the cell is amplified into a ⁇ type using the sequence characteristic of the gene of the present invention as a primer to amplify the gene of the present invention on the chromosome of the cell.
  • the presence or absence of a mutation can be determined by confirming the nucleotide sequence.
  • the present invention provides a method for preventing or treating a disease caused by the DNA abnormality of the present invention.
  • a method for screening a candidate compound for a drug is suitable for screening candidate compounds for drugs for preventing or treating diseases caused by abnormal protein secretion and reabsorption functions such as cystic fibrosis and diabetes. It is considered to be.
  • a test sample is brought into contact with a cell in which the gene of the present invention is expressed, the expression level of the gene of the present invention in the cell is measured, and compared with the case where the test sample is not contacted.
  • the test sample can be carried out by selecting a compound that increases or decreases the expression level of the gene of the present invention in the cells contacted with the test sample.
  • the test sample is not particularly limited. Chemistry—Applying a group of compounds obtained by technology (Tetrahedron (1995) 51, 8135-8137) or the phage display method (J. Mol. Biol. (1991) 222, 30-310). The created random peptide group can be used.
  • culture supernatants of microorganisms and natural components derived from plants and marine organisms are also targets for screening.
  • Other examples include, but are not limited to, biological tissue extracts, cell extracts, expression products of gene libraries, synthetic low molecular weight compounds, synthetic peptides, and natural compounds.
  • Examples of cells used for screening include, but are not limited to, C0S7 cells.
  • the expression of the gene of the present invention in the cells can be measured by a method known to those skilled in the art, for example, the Northern blotting method, the RT-PCR method, and the like, as in the above-mentioned diagnosis.
  • a compound capable of altering the expression of the gene of the present invention is expected to act, for example, in regulating the expression level of the gene of the present invention in patients with the above-mentioned diseases.
  • the gene of the present invention its protein, a compound that regulates the expression of the gene, or a compound that regulates the activity of the protein is used as a drug
  • the drug itself is used as a drug.
  • it can also be formulated and used by a known pharmaceutical method.
  • it may be used in the form of a formulation by appropriately combining with a pharmacologically acceptable carrier or medium, specifically, sterile water, physiological saline, vegetable oil, emulsifier, suspending agent and the like.
  • Administration to a patient can be performed by a method known to those skilled in the art, such as intraarterial injection, intravenous injection, and subcutaneous injection.
  • the dose varies depending on the weight and age of the patient, the administration method, and the like, but those skilled in the art can appropriately select an appropriate dose.
  • the DNA When DNA is used as a therapeutic agent, the DNA may be incorporated into a gene therapy vector and administered to a patient.
  • the dose and administration method vary depending on the patient's weight, age, symptoms, and the like, but those skilled in the art will be able to select as appropriate.
  • the present invention provides a DNA chip on which the DNA or polynucleotide of the present invention is arranged.
  • the “DNA chip” means a DNA chip substrate such as glass or silicon having a size of 1 to 10 cm 2, in which a large number of DNAs are precisely arranged in a predetermined order. This makes it possible to analyze a large amount of DNA and RNA in a short time.
  • the DNA chip can be manufactured as follows. First, a partial sequence specific to the gene of the present invention is selected as a probe. This is spotted, for example, on a commonly supplied DNA chip substrate of glass or silicon having a size of 1 to 10 cm 2 using a microlayer.
  • FIG. 1 shows the alignment of the amino acid sequences of C-HEMBA1001174 and a known protein.
  • FIG. 2 is a continuation of FIG.
  • FIG. 3 is a continuation of FIG.
  • FIG. 4 is a continuation of FIG. BEST MODE FOR CARRYING OUT THE INVENTION
  • MRNA was extracted from human 10-week-old fetal tissues by the method described in Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989). Furthermore, poly (A) + RNA was purified using an oligo (dT) cellulose column (Collaborative labs) according to the method described in Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989). .
  • a cDNA library was prepared by an oligocap method [M. Maruyama and S. Sugano, Gene, 138: 171-174 (1994)].
  • an oligocaplinker synthetic RNA
  • an oligo (dT) adapter consisting of the sequence represented by SEQ ID NO: 4
  • the literature [Suzuki 'Kano, Protein Nucleic Acid Enzyme, 41: 197-201 (1996), Y.
  • the direction of the cDNA was determined and cloned into the vector PME18SFL3 (GenBank AB009864) cut with Dral II to prepare a cDNA library.
  • the cloned site of pME18SFL3 is an asymmetric Dral II site, and a complementary Sfil site is added to the end of the cDNA fragment. It is inserted unidirectionally downstream of the promoter.
  • Example 2 Analysis of cDNA clone derived from cDNA library prepared from human 10-week-old fetal tissue
  • a part of the cDNA library prepared in Example 1 was introduced into Escherichia coli DH10B by electroporation using Genepulsa-1 (manufactured by Biorad). Transformants were selected by culturing on LB agar medium containing 50 g / ml ampicillin. These transformants were converted to ampicillin. The cells were cultured overnight in an LB medium, and plasmid was extracted using a plasmid automatic extractor PI100 (manufactured by Kurabo Industries).
  • Plasmid DNA of clones obtained from these transformants was subjected to a sequencing reaction using a DNA sequencing reagent (BigDye Terminator Cycle Sequencing FS Ready Reaction Kit, manufactured by PE Biosystems) according to the manual, followed by DNA sequencing.
  • the nucleotide sequence from the 5, 5 or 3 'end of each cDNA clone was analyzed using a sensor (ABI PRISM 377, manufactured by PE Biosystems).
  • ME761FW represented by SEQ ID NO: 7 is used for determination of the base sequence from the 5 'end.
  • ME1250RV represented by SEQ ID NO: 8 is used as a sequencing primer. Using.
  • 5 ′ terminal sequence data of the clone sequence was characterized based on the following method.
  • Human mRNA sequence ⁇ identity to human EST sequence Comparison of 5'-end length Identity of 5'-end and 3'-end sequences of clone sequences to mRNA sequences of human and other organisms Indicates that the length of the comparison sequence with each sequence is 200 bases or more Were considered identical in all cases. The identity to the human EST sequence was considered to be identical if the length of the comparison sequence portion with the 5 'terminal sequence was 200 bases or more and 90 or more matches.
  • the results of ATGpr [A. Salamov, T. Nishikawa, ⁇ . ⁇ . Swindells. Asse ssing protein coding region integrity in cDNA sequencing projects. Bioin formatics 14: 384-390 (1998)] were used to predict the full length. .
  • the ATGprl value is a value that predicts the possibility of the full length from the calculated value. The higher the ATGprl value, the higher the possibility of the full length.
  • the maximum ATGprl value and maximum ATGpr2 value indicate the maximum ATGprl value and ATGpr2 value predicted from all the start codons contained in the 5 'terminal sequence of the clone sequence, and these values are used for characterization. Was.
  • the 3′-end sequence was determined by homology search using GenBank. For the human EST sequence, the sequence was determined to be identical when the length of the comparison sequence portion with the 5 'terminal sequence was 90% or more over 200 bases or more. The number of EST sequences was directly used for characterization and used as an index of novelty. Clones having 5′-terminal sequences and 3′-terminal sequences that are not identical to not only mRNA sequences but also EST sequences are genes encoding novel sequences. Similarly, clones having the same 5 'terminal sequence or 3' terminal sequence with a small number of EST sequences were also determined to be cDNA clones encoding the novel sequence.
  • the class 1 was regarded as a cluster identical to the mRNA sequence.
  • the class 1 is non-full-length with respect to the mRNA sequence or human EST sequence.
  • the maximum value of the number of EST sequences is calculated for each of the 5'-terminal sequences and 3'-terminal sequences included in class I, and the number of identical EST sequences of the 5'-terminal sequence is the same as the EST sequence of the 3'-terminal sequence in class U. Number.
  • At least one clone contains a clone with high novelty and full length.
  • the full-length rate is low, but the full-length class still contains new clones.
  • Those clones that contained only one clone in the same cluster were selected. When a plurality of clones were included in the same cluster and there were a plurality of clones having an ATGprl value of more than 0.3, a clone having a larger ATGprl value was selected. When multiple clones were included in the same class, there were multiple clones with an ATGprl value of 0.3 or less, and if the ATGpr2 value was greater than 0.3, the clone with the larger ATGpr2 value was selected.
  • the clone is Selected. If multiple clones are included in the same cluster and selection by ATGpr value as above is not possible In this case, a longer clone was selected on the 5 'end side by assembling using the 5' end sequence and the human EST sequence. Sequencher (Gene Codes) was used for assembling, and if it could not be determined by assembling a part, all the target clones were judged to be full length.
  • the nucleotide sequence is mainly determined by the primer walking by the dideoxy-minine method using a custom-synthesized DNA primer (using a custom-synthesized DNA primer and following a sequencing reaction using a DNA sequencing reagent manufactured by PE Biosystem according to the manual. (Analysis of the DNA base sequence using the company's sequencer).
  • the full-length nucleotide sequence was finally determined by completely overlapping the partial nucleotide sequence determined by the above method.
  • a deduced amino acid sequence was determined from the determined nucleotide sequence of the full-length cDNA.
  • CDNA clone C-HEMBA1001174 is an example of a cDNA clone derived from a human 10-week-old fetal tissue, which was selected as described in (1) to (10) and judged to be highly likely to be new and full-length. Is shown in SEQ ID NO: 1.
  • the amino acid sequence of the gene product encoded by cDNA clone C-HEMBA1001174 deduced from the full-length nucleotide sequence is shown in SEQ ID NO: 2.
  • C-HEMBA1001174 (SEQ ID NO: 1) was searched using the in-house GenBank database-based BLA ST program. As a result, as shown in Figs. 1 to 4, C-HEMBA1001174 is a rat reported in the literature (Breiner, M. et al., (1988) Biochem. Biophys. Acta. 1308: 1-6). Showed 80% similarity in amino acid sequence of ADP-ribosylation factor-like protein. In addition, the amino acid sequence of human ADP-ribosylation factor-like protein reported in GenBank (Song, H. et al., (1999) Direct submission to GenBank) Columns showed 79% similarity. Thus, it was about 80% identical to rat and human ADP-ribosylating factor-like proteins. Therefore, C-HEMBA1001174 is a new human protein that is different from the conventional ADP ribosylation factor-like protein.
  • the present invention provides a novel ARF-like protein (C-HEMBA1001174), a gene encoding the protein, a vector containing the gene, a transformant containing the vector, and a method for producing the protein. . It is suggested that the cDNA of the present invention is related to a disease in which protein secretion and abnormal reabsorption function occur. For this reason, the gene or protein of the present invention is effective for the development of diagnostic markers and pharmaceuticals for these diseases.

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Abstract

Grâce à un procédé Oligocap mis initialement au point pour isoler des ADNc pleine longueur, il est possible d'isoler une pluralité d'ADNc pleine longueur dans une banque d'ADNc tirés d'un tissus foetal humain de 10 semaines. Parmi ces ADNc, on isole un clone (C-HEMBA1001174) codant pour une protéine de type ARF. On suppose que le clone C-HEMBA1001174 possède des fonctions biologiques, notmment de transport vésiculaire intracellulaire. On s'attend donc à ce que ce clone serve de cible importante pour le diagnostic de maladies en rapport avec une anomalie de ces fonctions et pour la mise au point de médicaments destinés au traitement ou à la prévention de telles maladies.
PCT/JP2000/005067 1999-07-29 2000-07-28 Gene codant pour une nouvelle proteine de type facteur de ribosylation adp (arf) WO2001009320A1 (fr)

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JP11/248036 1999-07-29
JP24803699 1999-07-29
JP30025399 1999-08-27
JP11/300253 1999-08-27
US15959099P 1999-10-18 1999-10-18
US60/159,590 1999-10-18
JP2000/118776 2000-01-11
JP2000118776 2000-01-11
US18332200P 2000-02-17 2000-02-17
US60/183,322 2000-02-17

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004044592A1 (fr) * 2002-11-11 2004-05-27 Evotec Neurosciences Gmbh Utilisation diagnostique et therapeutique de la proteine et du arl7 dans la maladie d'alzheimer
CN100368435C (zh) * 2006-02-21 2008-02-13 中国科学技术大学 一个Arf6激活蛋白及其编码基因与应用
US9920123B2 (en) 2008-12-09 2018-03-20 Genentech, Inc. Anti-PD-L1 antibodies, compositions and articles of manufacture
CN111662912A (zh) * 2020-06-01 2020-09-15 云南省烟草农业科学研究院 一种烟草NtARF6基因突变体及分子鉴定方法和应用

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102009005912B4 (de) * 2009-01-23 2010-11-25 Sponeta Gmbh Anordnung zur Verriegelung einer Tischtennisplatte

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Publication number Priority date Publication date Assignee Title
US5856160A (en) * 1997-03-31 1999-01-05 Incyte Pharmaceuticals, Inc. Mitochondrial adenylate kinase

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
BOBAK D.A. ET AL.: "Molecular cloning, characterization and expression of human ADP-ribosylation factors", PROC. NATL. ACAD. SCI,. USA, vol. 86, 1989, pages 6101 - 6105, XP002933785 *
BREINER M. ET AL.: "Cloning of a novel member (ARL5) of the ARF-family of Ras-related GTPases", BIOCHIM. BIOPHYS. ACTA, vol. 1308, 1996, pages 1 - 6, XP002933784 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004044592A1 (fr) * 2002-11-11 2004-05-27 Evotec Neurosciences Gmbh Utilisation diagnostique et therapeutique de la proteine et du arl7 dans la maladie d'alzheimer
CN100368435C (zh) * 2006-02-21 2008-02-13 中国科学技术大学 一个Arf6激活蛋白及其编码基因与应用
US9920123B2 (en) 2008-12-09 2018-03-20 Genentech, Inc. Anti-PD-L1 antibodies, compositions and articles of manufacture
CN111662912A (zh) * 2020-06-01 2020-09-15 云南省烟草农业科学研究院 一种烟草NtARF6基因突变体及分子鉴定方法和应用

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