WO2001047980A1 - Nouveau polypeptide, dihydrodipicolinate synthase 9, et polynucleotide codant pour ce polypeptide - Google Patents

Nouveau polypeptide, dihydrodipicolinate synthase 9, et polynucleotide codant pour ce polypeptide Download PDF

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Publication number
WO2001047980A1
WO2001047980A1 PCT/CN2000/000715 CN0000715W WO0147980A1 WO 2001047980 A1 WO2001047980 A1 WO 2001047980A1 CN 0000715 W CN0000715 W CN 0000715W WO 0147980 A1 WO0147980 A1 WO 0147980A1
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WO
WIPO (PCT)
Prior art keywords
polypeptide
polynucleotide
synthase
dihydrodioxin
sequence
Prior art date
Application number
PCT/CN2000/000715
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English (en)
Chinese (zh)
Inventor
Yumin Mao
Yi Xie
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Shanghai Biowindow Gene Development Inc.
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Publication date
Application filed by Shanghai Biowindow Gene Development Inc. filed Critical Shanghai Biowindow Gene Development Inc.
Priority to AU23419/01A priority Critical patent/AU2341901A/en
Publication of WO2001047980A1 publication Critical patent/WO2001047980A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)
    • 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 also relates to an antibody capable of specifically binding to a polypeptide of the invention.
  • the invention also relates to a method for in vitro detection of a disease or susceptibility to disease associated with abnormal expression of dihydrodiopterin synthase 9 protein, which comprises detecting a mutation in the polypeptide or a sequence encoding a polynucleotide thereof in a biological sample, or Detection of the amount or biological activity of a polypeptide of the invention in a biological sample.
  • “Deletion” refers to the deletion of one or more amino acids or nucleotides in an amino acid sequence or nucleotide sequence.
  • substantially pure means substantially free of other proteins, lipids, carbohydrates or other substances with which it is naturally associated.
  • Those skilled in the art can purify dihydrodioxin synthase 9 using standard protein purification techniques. Basically pure dihydrodioxin synthase 9 produces a single main band on a non-reducing polyacrylamide gel. The purity of the dihydrodioxin synthase 9 polypeptide can be analyzed by amino acid sequence.
  • Antisense refers to a nucleotide sequence that is complementary to a particular D or RNA sequence.
  • the "antisense strand” refers to a nucleic acid strand that is complementary to the “sense strand”.
  • polynucleotide sequence of the gene of the present invention or various DNA fragments and the like obtained as described above can be measured by a conventional method such as dideoxy chain termination method (Sanger et al. PNAS, 1977, 74: 5463-5467). Such polynucleotide sequences can also be determined using commercial sequencing kits and the like. In order to obtain the full-length cDNA sequence, sequencing needs to be repeated. Sometimes it is necessary to determine the cDNA sequence of multiple clones in order to splice into a full-length cDNA sequence.
  • a polynucleotide encoding dihydrodiopterin synthase 9 or a recombinant vector containing the polynucleotide can be transformed or introduced into a host cell to form a genetically engineered host cell containing the polynucleotide or the recombinant vector.
  • the term "host cell” refers to a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples are: E.
  • Transformation of a host cell with a DM sequence according to the present invention or a recombinant vector containing the DNA sequence can be performed by conventional techniques well known to those skilled in the art.
  • the host is a prokaryote such as E. coli
  • competent cells capable of DNA uptake can be in the exponential growth phase were harvested, treated with (1 2 method used in the step are well known in the art. Alternatively, it is a MgCl 2. If If necessary, transformation can also be performed by electroporation.
  • the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, or conventional mechanical methods such as microinjection, electroporation, and liposome packaging Wait.
  • the abnormal expression of the dihydrodiopterin synthase 9 of the present invention will produce various diseases, especially organic acidemia and amino acid metabolism deficiency diseases. These diseases include, but are not limited to:
  • Antibodies against dihydrodioxin synthase 9 can be used in immunohistochemical techniques to detect dihydrodioxin synthase 9 in biopsy specimens.
  • Monoclonal antibodies that bind to dihydrodioxin synthase 9 can also be labeled with radioisotopes and injected into the body to track their location and distribution. This radiolabeled antibody can be used as a non-invasive diagnostic method to locate tumor cells and determine whether there is metastasis. Antibodies can also be used to design immunotoxins that target a particular part of the body. For example, dihydropyridine synthase 9 high-affinity monoclonal antibodies can covalently bind to bacterial or plant toxins (such as diphtheria toxin, ricin, ormosine, etc.).
  • a common method is to attack the amino group of an antibody with a sulfhydryl crosslinker such as SPDP and bind the toxin to the antibody through the exchange of disulfide bonds.
  • This hybrid antibody can be used to kill dihydrodioxin synthase 9 positive Cell.
  • Polynucleotides encoding dihydropyridine synthase 9 are useful in the diagnosis of diseases related to dihydropyridine synthase 9.
  • a polynucleotide encoding dihydrodioxin synthase 9 can be used to detect the expression of dihydrodioxin synthase 9 or the abnormal expression of dihydrodioxin synthase 9 in a disease state.
  • the DNA sequence encoding dihydrodioxin synthase 9 can be used to hybridize biopsy specimens to determine the expression of dihydrodioxin synthase 9.
  • Hybridization techniques include Souter hern blotting, Nor thern blotting, and in situ hybridization.
  • Fluorescent in situ hybridization of cDNA clones with metaphase chromosomes allows precise chromosomal localization in one step.
  • FISH Fluorescent in situ hybridization
  • Dye terminate cycle react ion sequencing kit Perkin-Elmer
  • ABI 377 automatic sequencer Perkin-Elmer
  • the determined cDNA sequence was compared with the existing public D sequence database (Genebank), and it was found that the cDNA sequence of one of the clones 0139g08 was new DNA.
  • the inserted cDNA fragments contained in this clone were determined in both directions by synthesizing a series of primers.
  • Primerl 5-CTCAGTAGTATCAGTGTGGGACTG -3 '(SEQ ID NO: 3)
  • Amplification reaction conditions A reaction volume of 50 ⁇ 1 contains 50 mmol / L KC1, 10 mmol / L Tr is-HC1, pH 8.5, 1.5 mmol / L MgCl 2 , 200 mol / L dNTP, 1 Opmol primer, 1U Taq DNA polymerase (Clontech).
  • the reaction was performed on a PE9600 DNA thermal cycler (Perkin-Elmer) for 25 cycles under the following conditions: 94 ° C 30sec; 55 ° C 30sec; 72 ° C 2min.
  • RT-PCR set ⁇ -act in as a positive control and template blank as a negative control.
  • the amplified product was purified using a QIAGEN kit and ligated to a pCR vector (Invitrogen) using a TA cloning kit. DNA sequence analysis results showed that the DNA sequence of the PCR product was exactly the same as 1-2017bp shown in SEQ ID NO: 1.
  • Example 4 Analysis of the expression of dihydrodiopterin synthase 9 gene by Nor thern blot
  • RNA extraction in one step [Ana l. Biochem 1982, 162, 156-159] 0
  • This method involves acid guanidinium thiocyanate-chloroform extraction. That is, the tissue is homogenized with 4M guanidine isothiocyanate-25mM sodium citrate, 0.2M sodium acetate (pH4.0), and 1 volume of phenol and 1/5 volume of chloroform-isoamyl alcohol (49: 1) Centrifuge after mixing. Aspirate the aqueous layer, add isopropanol (0.8 vol) and centrifuge the mixture to obtain RNA precipitate. The resulting RNA pellet was washed with 70% ethanol, dried and dissolved in water.
  • RNA containing 20raM 3- (N- morpholino) propanesulfonic acid (pH7 0.) - subjected to electrophoresis on a 1.2% agarose gel 5mM sodium acetate -ImM EDTA-2 2M formaldehyde. It was then transferred to a nitrocellulose membrane. A- 32 P dATP was used to prepare 32 P-labeled DNA probes by random primers. The DNA probe used was the PCR amplified dihydrodioxin synthase 9 coding region sequence (285bp to 533bp) shown in FIG. 1.
  • a 32P-labeled probe (about 2 x 10 6 cpm / ml) was hybridized with a nitrocellulose membrane to which RNA was transferred at 42 ° C overnight in a solution containing 50% formamide-25mM KH 2 P0 4 (pH7. 4) -5 x SSC-5 x Denhardt's solution and 20 ( ⁇ g / ml salmon sperm DNA. After hybridization, the filter was washed in 1 x SSC-0.1% SDS at 55 ° C for 30 minutes. Then Phosphor Imager was used for analysis and quantification.
  • Example 5 In vitro expression, isolation and purification of recombinant dihydrodioxin synthase 9
  • a peptide synthesizer (product of PE company) was used to synthesize the following peptides specific to dihydrodioxin synthase 9: NH2-Met-Phe-Ser-Phe-Tyr-Phe-Ser-Phe-Leu-Phe-Phe-Leu -Arg-Trp-Ser- C00H (SEQ ID NO: 7).
  • the polypeptide is coupled to hemocyanin and bovine serum albumin to form a complex, respectively.
  • the preferred range of probe size is 18-50 nucleotides
  • a total of 4,000 polynucleotide sequences of various full-length cDNAs are used as the target DM, including the polynucleotide of the present invention. They were respectively amplified by PCR. After purification, the concentration of the amplified product was adjusted to about 500 ng / ul, and spotted on a glass medium with a Cartesian 7500 spotter (purchased from Cartesian Company, USA). The distance between them is 280 ⁇ . The spotted slides were hydrated and dried, cross-linked in a UV cross-linker, and dried after elution to fix the DNA on the glass slides to prepare chips. The specific method steps have been reported in the literature. The sample post-processing steps in this embodiment are:
  • Total mRM was extracted from normal liver and liver cancer in one step, and mRNA was purified with Oligotex mRNA Midi Kit (purchased from QiaGen).
  • the fluorescent reagent Cy3dUTP (5-Amino- propargyl-2-- deoxyur idine 5'-triphate coupled to Cy3 f luorescent dye (purchased from Amersham Pharaac ia Biotech) was used to label the mRNA of normal liver tissue
  • the fluorescent reagent Cy5dUTP (5-Amino-propargy 1-2 ⁇ -deoxyuri dine 5'-tr iphate Coupled to Cy5 f luorescent dye (purchased from Amersham Phamacia Biotech) was used to label liver cancer tissue mRNA, and the probe was prepared after purification.
  • the probes from the two types of tissues were hybridized with the chip in a UniHyb TM Hybridization Solution (purchased from TeleChem) hybridization solution for 16 hours.
  • the washing solution (lx SSC, 0.2% SDS) After washing, scan with a ScanArray 3000 scanner (purchased from General Scanning, USA).
  • the scanned images are analyzed and processed with Imagene software (Biodi scovery, USA) to calculate the Cy3 of each point. / Cy5 ratio, the ratio of which is less than 0.5 and greater than 2 is considered to be a gene with differential expression.

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  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne un nouveau polypeptide, une dihydrodipicolinate synthase 9, et un polynucléotide codant pour ce polypeptide ainsi qu'un procédé d'obtention de ce polypeptide par des techniques recombinantes d'ADN. L'invention concerne en outre les applications de ce polypeptide dans le traitement de maladies, notamment des tumeurs malignes, de l'hémopathie, de l'infection par VIH, de maladies immunitaires et de diverses inflammations. L'invention concerne aussi l'antagoniste agissant contre le polypeptide et son action thérapeutique ainsi que les applications de ce polynucléotide codant pour la dihydrodipicolinate synthase 9.
PCT/CN2000/000715 1999-12-27 2000-12-25 Nouveau polypeptide, dihydrodipicolinate synthase 9, et polynucleotide codant pour ce polypeptide WO2001047980A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU23419/01A AU2341901A (en) 1999-12-27 2000-12-25 A novel polypeptide-dihydrodipicolinat synthase 9 and the polynucleotide encoding said polypeptide

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN99125792.8 1999-12-27
CN 99125792 CN1301845A (zh) 1999-12-27 1999-12-27 一种新的多肽——二氢二砒啶合成酶9和编码这种多肽的多核苷酸

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CN (1) CN1301845A (fr)
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5773691A (en) * 1992-03-19 1998-06-30 E. I. Du Pont De Nemours And Company Chimeric genes and methods for increasing the lysine and threonine content of the seeds of plants
WO1999054483A1 (fr) * 1998-04-17 1999-10-28 Hokko Chemical Industry Co., Ltd. Gene de la dihydrodipicolinate synthase du riz et adn associe

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5773691A (en) * 1992-03-19 1998-06-30 E. I. Du Pont De Nemours And Company Chimeric genes and methods for increasing the lysine and threonine content of the seeds of plants
WO1999054483A1 (fr) * 1998-04-17 1999-10-28 Hokko Chemical Industry Co., Ltd. Gene de la dihydrodipicolinate synthase du riz et adn associe

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AU2341901A (en) 2001-07-09
CN1301845A (zh) 2001-07-04

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