WO2002072628A1 - Polypeptide physiologiquement actif et polynucleotide le codant - Google Patents

Polypeptide physiologiquement actif et polynucleotide le codant Download PDF

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Publication number
WO2002072628A1
WO2002072628A1 PCT/JP2002/002184 JP0202184W WO02072628A1 WO 2002072628 A1 WO2002072628 A1 WO 2002072628A1 JP 0202184 W JP0202184 W JP 0202184W WO 02072628 A1 WO02072628 A1 WO 02072628A1
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WIPO (PCT)
Prior art keywords
polypeptide
seq
polynucleotide
rat
amino acid
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PCT/JP2002/002184
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English (en)
Japanese (ja)
Inventor
Naohisa Ishikawa
Hidetsugu Murakami
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Japan Science And Technology Corporation
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Priority to JP2002571541A priority Critical patent/JPWO2002072628A1/ja
Publication of WO2002072628A1 publication Critical patent/WO2002072628A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention of this application relates to a polypeptide having a physiological activity and a polynucleotide encoding the polypeptide. More specifically, the invention of this application relates to a novel bioactive polypeptide having a physiological activity similar to the pathogenic bacterium toxin and useful for elucidation of cytotoxicity and development of therapeutic drugs, and a large amount of this polypeptide. It relates to a polynucleotide useful for production and the like.
  • VT verotoxin
  • the inventors of the present application have developed a method for isolating and purifying the toxin (VT1, VT2) of Escherichia coli 0157 and testing for its infection (j. Mass Spectr. 32: 1140-). 1 142, 1997; Aichi Medical Journal No. 1483: 3-5, 1996; Aichi Prefectural Institute of Public Health annual report dai25: 36, 1997). Using these toxins, we have also succeeded in producing anti-VT1 and anti-VT2 antibodies from rabbits (The Japanese Biochemical Society, Biochemistry, Vol. 72, No. 8, 2000). Infection of pathogenic Escherichia coli can cause dramatic changes in infected individuals even if the amount of venom toxin produced is extremely small.
  • the existence of specific receptors for this substance has been postulated. If the functions of these physiological substances and their receptors are elucidated, the cytotoxicity of verotoxin It is expected that it will be possible to understand the effects and develop a fundamental treatment for the pathogenic Escherichia coli infection. However, at present, there is no known bioactive substance or receptor.
  • the invention of this application has been made in view of the circumstances described above, and comprises a novel in vivo physiologically active substance having a physiological activity similar to that of verotoxin such as pathogenic Escherichia coli 0157, and encoding this substance. It is an object to provide a polynucleotide.
  • a purified rat derived from a rat having the amino acid sequence of SEQ ID NO: 2 and having biological activity is provided.
  • FIG. 1 is a graph showing the amount of mRNA transcribed from the polynucleotide of the present invention for each organ of a rat.
  • FIG. 2 shows the results of in situ hybridization in which the amount of mRNA of the polynucleotide of the present invention in rat liver was measured.
  • FIG. 3 shows the state of a rat to which the synthetic oligopeptide of the present invention was administered intraventricularly.
  • FIG. 4 shows the change in blood pressure of a rat injected intravenously with the synthetic rigopeptide of the present invention.
  • FIG. 5 shows the passage change of cell morphology cultured in the presence of the oligopeptide of the present invention.
  • FIG. 6 shows an electrophoretic image of DNA extracted from the cultured cells shown in Figure 5.
  • the polypeptide of the invention (1) is an isolated and purified rat protein having an amino acid sequence of SEQ ID NO: 2, which is similar to or similar to a toxin produced by pathogenic Escherichia coli. It has the same physiological activity (cytotoxicity).
  • the polypeptide of the invention (1) may be isolated from a rat tissue, prepared by a peptide by chemical synthesis based on the amino acid sequence of SEQ ID NO: 2, or obtained by encoding a polynucleotide encoding the amino acid sequence of SEQ ID NO: 2.
  • RNA is prepared by in vitro transcription from a vector having the polynucleotide (the protein translation region of cDNA: ORF) of the invention (3), and the resulting RNA is subjected to invitro translation by using this as a cyclized form.
  • the protein can be expressed in the mouth.
  • the polynucleotide When the polynucleotide is recombined into an appropriate expression vector by a known method, the polynucleotide encodes in prokaryotic cells such as Escherichia coli and Bacillus subtilis, and eukaryotic cells such as yeast, insect cells, and mammalian cells. Protein can be expressed in large amounts.
  • prokaryotic cells such as Escherichia coli and Bacillus subtilis
  • eukaryotic cells such as yeast, insect cells, and mammalian cells. Protein can be expressed in large amounts.
  • the polypeptide of the invention (1) is produced by expressing DNA by in vitro translation
  • the polynucleotide of the invention (3) is inserted into a vector having an RNA polymerase promoter to prepare a recombinant vector
  • this vector is added to an in vitro translation system such as a heron reticulocyte lysate or a wheat germ extract containing an RNA polymerase corresponding to the promoter, the polypeptide of the invention (1) is produced at the in vitro mouth. be able to.
  • RNA polymerase promoter examples include T7, T3, SP6 and the like.
  • vectors containing these RNA polymerase promoters include pCMV-SPORT, pKA1, pCDM8, pT3 / T718, ⁇ 7 / 319, pBluescript II and the like.
  • An expression vector in which the polynucleotide of the invention (3) is recombined is prepared in a vector, the host cell is transformed with the expression vector, and the resulting transformant is cultured.
  • Polypeptides encoding nucleotides can be mass-produced in microorganisms. At this time, a protein fragment containing an arbitrary region can be obtained by adding a start codon and a stop codon before and after the arbitrary translation region for expression. Alternatively, it can be expressed as a fusion protein with another protein. By cleaving the fusion protein with an appropriate protease, only the protein portion encoded by the polynucleotide can be obtained.
  • Examples of expression vectors for Escherichia coli include a pUC system, a pBluescript I pET expression system, a pGEX expression system, and a pQE expression system.
  • the polypeptide of the invention (1) is produced by expressing DNA in a eukaryotic cell
  • the polynucleotide of the invention (3) has a promoter, a splicing region, a poly (A) addition site, and the like. If the recombinant vector is prepared by inserting it into an eukaryotic cell expression vector and introduced into eukaryotic cells, the polypeptide of the invention (1) can be produced in eukaryotic cells.
  • expression vectors examples include pKA1, pCDM8, pSVK3, pMSG, pSV and pBK-CMV, pBK-RSV, EBV vector, pRS, pYES2, and the like. If plND / V5-His, pFLAG-CMV-2, pEGFP-N1, pEGFP-C1, etc. are used as expression vectors, they can be expressed as fusion proteins with various tags such as His tag, FLAG tag, and GFP. Can also.
  • eukaryotic cells monkey kidney cells COS7, cultured mammalian cells such as Chinese hamster ovary cells CHO, budding yeast, fission yeast, silkworm cells, African toad frog egg cells and the like are generally used, and the polypeptide of the invention (1) is used. Any eukaryotic cell can be used as long as it can express E. coli.
  • known methods such as an electroporation method, a calcium phosphate method, a liposome method, and a DEAE dextran method can be used.
  • the target polypeptide can be isolated and purified from the culture by a combination of known separation procedures. For example, treatment with a denaturant such as urea or a surfactant, ultrasonic treatment, enzyme elimination, salting-out / solvent precipitation, dialysis, centrifugation, ultrafiltration, gel filtration, SDS-PAGE, isoelectric focusing Examples include electrophoresis, ion exchange chromatography, hydrophobic chromatography, affinity chromatography, and reversed-phase chromatography.
  • a denaturant such as urea or a surfactant
  • ultrasonic treatment enzyme elimination
  • salting-out / solvent precipitation dialysis, centrifugation, ultrafiltration, gel filtration, SDS-PAGE
  • isoelectric focusing examples include electrophoresis, ion exchange chromatography, hydrophobic chromatography, affinity chromatography, and reversed-phase chromatography.
  • the polypeptide of the invention (1) includes a peptide fragment (5 or more amino acid residues) containing any partial amino acid sequence of the amino acid sequence represented by SEQ ID NO: 2. These peptide fragments can be used as antigens for producing antibodies.
  • the polypeptide of the invention (1) also includes a fusion protein with any other protein. For example, a protein fusion with Dalphin Chin-S-transferase (GST) and green fluorescent protein (GFP) can be exemplified. Further, the polypeptide of the invention (1) may undergo various modifications in the cell after being translated. Therefore, modified proteins are also included in the scope of the protein of the invention (1).
  • invention (2) is a purified polynucleotide encoding the polypeptide of the invention (1), and includes a rat genomic DNA, its mRNA and cDNA (specifically, a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1), Their complementary strands are included.
  • the polynucleotide of the invention (3) is a polynucleotide (cDNA) containing a base sequence constituting the translation region (ORF) of SEQ ID NO: 1. Since the polypeptide of the invention (.1) is expressed in any cell, a rat cDNA library prepared from rat cells is screened using an oligonucleotide probe synthesized based on the nucleotide sequence of SEQ ID NO: 1. By doing so, the same clone as the polynucleotide of the invention (3) can be easily obtained. Alternatively, the target cDNA can also be synthesized by RT-PCR using these oligonucleotides as primers and using mRNA isolated from rat cells as type III.
  • polypeptides in which one or more amino acids have been added, deleted and / or substituted by other amino acids resulting from these changes also have the same physiological activity as Vero toxin. It is included in the range of the polypeptide of (1).
  • Invention (4) is a polynucleotide in which the polynucleotide consisting of SEQ ID NO: 1 or a partially continuous sequence thereof is hybridized under stringent conditions, and comprises all mammals, including humans, other than rats. Some regions of genomic DNA (genomic DNA fragments), including their mRNA and cDNA.
  • the stringent condition includes the nucleotide sequence of SEQ ID NO: 1 or a partially continuous sequence thereof (for example, 10 bp or more). Conditions that allow for selective and detectable specific binding of the polynucleotide to genomic DNA. Stringent conditions are defined by salt concentration, organic solvent (eg, formamide), temperature, and other known conditions.
  • a stringent salt concentration is usually about 750 mM or less NaCI and about 75 mM or less trisodium citrate, more preferably about 500 mM or less NaCI and about 50 mM or less trisodium citrate, Most preferably, it is about 250 mM or less for NaCI and about 25 mM or less for trisodium citrate.
  • a stringent organic solvent concentration is at least about 35% formamide, most preferably at least about 50%.
  • Stringent temperature conditions are about 30 ° C. or higher, more preferably about 37 ° C. or higher, and most preferably about 42 ° C. or higher.
  • stringent salt conditions for washing are preferably about 30 mM NaCI or less and about 3 mM trisodium citrate, most preferably about 15 mM NaCI or less and about 1.5 mM trisodium citrate. It is.
  • Stringent temperature conditions for washing are about 25 ° C. or higher, more preferably about 42 ° C. or higher, and most preferably about 68 ° C. or higher.
  • the polynucleotide of the invention (4) may be, for example, a genome prepared from genomic DNA other than rat by the above-described stringing method using the above-mentioned polynucleotide as a probe, It can be isolated by screening libraries and cDNA libraries.
  • the polypeptide of the invention (5) is a non-rat animal-derived polypeptide produced from the polynucleotide of the invention (4), and is 50% or more of the rat-derived 'polypeptide of the invention (1), Preferably, the polypeptide has an amino acid homology of 75% or more, most preferably 90% or more, and has the same physiological activity as the polypeptide of the invention (1).
  • the polypeptide of the invention (5) can be obtained by the same known recombinant DNA technique as that of the polypeptide of the invention (1). Can be obtained.
  • the oligopeptide of the invention (6) is a part of the polypeptide of the invention (1) and is a peptide consisting of 8 amino acid residues of SEQ ID NOS: 3, 4 and 5, respectively. These oligonucleotides have the same physiological activity as the polypeptide of the invention (1), as shown in Examples described later, and can be used for elucidation of cytotoxicity and development of drugs and the like. These oligopeptides can be prepared by a known peptide synthesis method.
  • the antibody of the invention (7) can be obtained from serum after immunizing an animal using the polypeptide of the invention (1) as an antigen.
  • an antigen a peptide chemically synthesized based on the amino acid sequence of SEQ ID NO: 2 or a polypeptide protein expressed in eukaryotic cells or prokaryotic cells can be used. Alternatively, it can be prepared by introducing the above expression vector for eukaryotic cells into an animal muscle or skin by injection or gene gun, and then collecting serum (for example, see Japanese Patent Application Laid-Open No. 7-313187). No. invention).
  • a mouse, a rat, a heron, a goat, a chicken, and the like are used.
  • CDNA was prepared by converting mRNA isolated from rat brain into type II, and this was converted to double strand Then, it was integrated into an Agt11 vector to create a rat brain cDNA library. Next, after packaging this cDNA library, the cells are infected into host Escherichia coli and cultured on an agar medium to form plaques, and the proteins contained in the plaques are transferred to a double-cell membrane to create replicas. The replica was reacted with an anti-VT2 rabbit antibody, and the procedure of detecting an anti-VT2 antibody-positive plaque using a peroxidase-labeled anti-rabbit IgG goat antibody was repeated at least three times to purify the cDNA. The purified cDNA was amplified and its nucleotide sequence was determined.
  • the obtained cDNA clone had the nucleotide sequence of SEQ ID NO: 1.
  • sequence of 6 amino acid residues from the N-terminus of the obtained 5 kDa peptide was determined, and the ORF region of the cloned cDNA was determined.
  • the immunization schedule consisted of three inoculations of three antigens every four weeks. After confirming the production of Egret antibody by the ELISA method, finally, three kinds of antigens were inoculated into each Egret, boosted, and antiserum was collected. From this antiserum, the IgG fraction was purified by affinity chromatography using protein G-Sepharose to obtain a rabbit antibody to each of the rival peptides of SEQ ID NOS: 3, 4, and 5.
  • Each of the obtained antibodies recognizes the N-terminal side, the central part, and the C-terminal side of the polypeptide having the amino acid sequence of SEQ ID NO: 2. It is possible to confirm more accurately.
  • Polypeptide expression distribution A PCR primer was synthesized based on the nucleotide sequence (sequence number 1) of the polynucleotide obtained in Example 1, and the mRNA expression level in various rat organs was determined by the well-known RT-PCT method. did.
  • Figure 1 shows the data obtained by quantifying one actin as a control and standardizing the value to 1.0.
  • this polypeptide is expressed systemically in rats, but is highly expressed in the central nervous system (cerebrum, hypothalamus, cerebellum, and medulla oblongata), especially in the spinal cord.
  • the central nervous system Cerebrum, hypothalamus, cerebellum, and medulla oblongata
  • the least expressed site was the skeletal muscle.
  • the expression level increased with aging.
  • Sprague-Dawley rats were bred for a long period of time, and liver specimens of rats 2-4 months, 6 months, 12 months, 18 months, 24 months and 30 months old were prepared.
  • 'In situ hybridization was performed on each specimen.
  • Figure 3 shows the rat after waking from anesthesia.
  • the rat was unable to move around, remained immobile for the next two weeks, and lost weight due to inability to eat and drink.
  • motor function recovered.
  • the invention of this application provides a novel polypeptide having a bioactivity equivalent or similar to that of verotoxin of pathogenic Escherichia coli, and a polynucleotide encoding this polypeptide.
  • This polypeptide is useful for elucidating the cytotoxicity of Vero toxin and developing therapeutics against pathogenic Escherichia coli infection.
  • the physiological activity of this polypeptide is deeply involved in neurodegeneration, as shown by the inhibition of motor function and neuronal apoptosis. For this reason, this polypeptide greatly contributes to elucidation of the pathogenesis of neurodegenerative diseases and development of therapeutic drugs.
  • this polypeptide since this polypeptide is present in vascular endothelial cells and has an effect of increasing blood pressure, it can be used for elucidation of the active mechanism of essential hypertension and for development of new antihypertensive agents.

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
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  • Gastroenterology & Hepatology (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

L'invention concerne un polypeptide de rat de séquence en acides aminés représentée par la SEQ ID NO:2 et possédant une activité physiologique similaire à la vérotoxine de la bactérie entéropathogène Escherichia coli, ainsi qu'un polynucléotide codant pour ce polypeptide et possédant la séquence de base représentée par la SEQ ID NO:1. Ce polypeptide sert à clarifier la cytotoxicité de la vérotoxine et à développer des procédés thérapeutiques contre l'infection par E. coli entéropathogène. En outre, l'activité physiologique de ce polypeptide se rapporte étroitement à la neurodégénérescence observée dans la dépression motrice et dans l'apoptose. Ainsi, ce polypeptide contribue largement à la clarification du mécanisme de démarrage de maladies neurodégénératives et donc au développement de remèdes. De plus, ce polypeptide se trouve dans les cellules endothéliales vasculaires et provoque une élévation de la tension sanguine. Il est ainsi possible de l'utiliser dans la clarification du mécanisme d'activité de l'hypertension intrinsèque et dans le développement de nouveaux hypotenseurs.
PCT/JP2002/002184 2001-03-08 2002-03-08 Polypeptide physiologiquement actif et polynucleotide le codant WO2002072628A1 (fr)

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JP2002571541A JPWO2002072628A1 (ja) 2001-03-08 2002-03-08 生理活性を有するポリペプチドとこのポリペプチドをコードするポリヌクレオチド

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JP2001-65469 2001-03-08
JP2001065469 2001-03-08

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Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
TANAKA T. ET AL.: "Genome-wide expression profiling of mid-gestation placenta and embryo using a 15,000 mouse developmental cDNA microarray", PROC. NATL. ACAD. SCI. USA, vol. 97, no. 16, 2000, pages 9127 - 9132, XP002951807 *
WATANABE H. ET AL.: "cDNA cloning of the immunologically cross-reactive substance to anti-verotoxin antibodies from rat central nerves", THE JAPANESE JOURNAL OF PHARMACOLOGY, vol. 85, 1 March 2001 (2001-03-01), XP002951806 *

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