WO2001088111A1 - Deshydrogenase 50, polypeptide humain, et polynucleotide la codant - Google Patents

Deshydrogenase 50, polypeptide humain, et polynucleotide la codant Download PDF

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WO2001088111A1
WO2001088111A1 PCT/CN2001/000695 CN0100695W WO0188111A1 WO 2001088111 A1 WO2001088111 A1 WO 2001088111A1 CN 0100695 W CN0100695 W CN 0100695W WO 0188111 A1 WO0188111 A1 WO 0188111A1
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polypeptide
polynucleotide
human dehydrogenase
sequence
seq
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Chinese (zh)
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Yumin Mao
Yi Xie
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Shanghai Biowindow Gene Development Inc
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Shanghai Biowindow Gene Development Inc
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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
    • C12N9/0004—Oxidoreductases (1.)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders

Definitions

  • the present invention belongs to the field of biotechnology. Specifically, the present invention describes a new polypeptide ⁇ A dehydrogenase 50, and a polynucleotide sequence encoding the polypeptide. The invention also relates to a preparation method and application of the polynucleotide and the polypeptide.
  • G6PD catalyzes the dehydrogenation of glucose-6-phosphate to 6-phosphate gluconolactone.
  • the reaction uses DP as the hydrogen acceptor to form NADPH.
  • G6PD is a regulatory enzyme that regulates the pathway of pentose phosphate and catalyzes the irreversible reaction. Its product ribose-5-phosphate is an essential raw material for nucleic acid synthesis;
  • GMP reductase relies on NADPH and catalyzes an irreversible reaction to make GMP into hypoxanthine nucleotide (IMP), and IMP and NH 2 can be converted to adenine nucleotide (AMP) to ensure the balance of G and A in the cell;
  • IMP hypoxanthine nucleotide
  • AMP adenine nucleotide
  • IMPDH catalyzes hypoxanthine nucleotides (IMP) to xanthine nucleotides (XMP). This reaction depends on NAD, which is the rate-limiting reaction of GTP de novo synthesis. Inhibition of IMP dehydrogenase activity will lead to the termination of DM synthesis.
  • G6PD exists as a tetramer in various tissues of the human body.
  • the pentose phosphate pathway regulated by G6PD can form NADPH, which provides the reducing power required for various biosynthetic metabolisms. Therefore, most patients can tolerate the treatment of drugs, while innate genetic deficiency6 -Patients with phosphoglucose dehydrogenase develop yellow gall bladder, black urine, and decreased hemoglobin after several days of oxidizing drugs such as sulfa and aspirin. Because there are no mitochondria in the red blood cells and lack of NADPH, the reduced glutathione levels in the cells cannot be maintained, leading to the destruction of the membrane structure and the symptoms of hemolysis and anemia.
  • Type I is ubiquitin and most of them are found in normal cells.
  • Type II is mainly found in malignant tumor cells. IMPDH activity changes in cancer cells, so some inhibitors and metabolic analogs can play an antitumor role.
  • a current effective method for treating leukemia is to inhibit the activity of IMPDH with drugs such as bacteriocin adenine diphosphate, which results in the inhibition of cell growth and induces cell differentiation.
  • drugs such as bacteriocin adenine diphosphate
  • IMPDH is a rate-limiting enzyme for GTP de novo synthesis, it regulates the secretion of extracellular insulin and the synthesis of intracellular DM and other important functions of ⁇ -cells. Changing the expression and activity of MPDH will change ⁇ -cell replication, cell cycle progression, and cell differentiation. This effect can be mediated directly by GTP or indirectly by a small amount of GTPase.
  • UMPDH Hypoxanthine nucleotide dehydrogenase
  • GMP reductase has many homologous sequences with GMP reductase, in which One is the phosphate-binding site- ⁇ / ⁇ barrel structure (beta / a lpha-barre l, phosphate-binding site).
  • This tertiary structure is composed of about 200 amino acids and is found in many proteins, such as cells.
  • pigment C another common sequence of IMPDH and GMP reductase is an active region centered on cysteine [LIVM]-[RK ⁇ -[LIVM]-G- [LIVM]-GXGS- [LIVM] -CX -T, cysteine C is an active residue.
  • E-XMP covalent enzyme-linked intermediate
  • the human dehydrogenase 50 protein plays an important role in regulating important functions of the body such as cell division and embryo development, and it is believed that a large number of proteins are involved in these regulatory processes, so it has been necessary to identify more involved in these processes Human dehydrogenase 50 protein, especially the amino acid sequence of this protein is identified. Isolation of the new human dehydrogenase 50 protein encoding gene also provides a basis for research to determine the role of this protein in health and disease states. This protein may form the basis for the development of diagnostic and / or therapeutic drugs for diseases, so it is important to isolate its coding DNA. Object of the invention
  • Another object of the invention is to provide a polynucleotide encoding the polypeptide.
  • Another object of the present invention is to provide a recombinant vector containing a polynucleotide encoding human dehydrogenase 50.
  • Another object of the present invention is to provide a genetically engineered host cell comprising a polynucleotide encoding a human dehydrogenase 50.
  • the present invention also relates to a method for detecting a disease or susceptibility to disease associated with abnormal expression of the human dehydrogenase 50 protein in vitro, which comprises detecting a mutation in the polypeptide or a sequence encoding a polynucleotide thereof in a biological sample, or detecting a biological sample The amount or biological activity of a polypeptide of the invention.
  • polynucleotide encoding a polypeptide refers to a polynucleotide comprising the polypeptide and a polynucleotide comprising additional coding and / or non-coding sequences.
  • the probe used for hybridization is homologous to any part of the polynucleotide of the present invention, and its length is at least 10 nucleotides, preferably at least 30 nucleotides, more preferably At least 50 nucleotides, preferably at least 100 nucleotides.
  • the length of the probe is usually within 2000 nucleotides, preferably within 1000 nucleotides.
  • the probe used herein is usually a DM sequence chemically synthesized based on the gene sequence information of the present invention.
  • the genes or fragments of the present invention can of course be used as probes.
  • DNA probes can be labeled with radioisotopes, luciferin, or enzymes (such as alkaline phosphatase).
  • a method for amplifying DM / RNA by PCR is preferably used to obtain the gene of the present invention.
  • the RACE method RACE-Rapid Amplification of cDNA Ends
  • the primers used for PCR can be appropriately based on the polynucleotide sequence information of the present invention disclosed herein Select and synthesize using conventional methods.
  • the amplified DM / RNA fragment can be isolated and purified by conventional methods such as by gel electrophoresis.
  • the present invention also relates to a vector comprising the polynucleotide of the present invention, and a host cell genetically engineered using the vector of the present invention or directly using a human dehydrogenase 50 coding sequence, and a method for producing a polypeptide of the present invention by recombinant technology.
  • DM sequence can be operably linked to an appropriate promoter in an expression vector to guide mRNA synthesis.
  • promoters are: the lac or trp promoter of E.
  • the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture.
  • selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture.
  • GFP fluorescent protein
  • tetracycline or ampicillin resistance for E. coli.
  • the polynucleotide sequence of the present invention can be used to express or produce recombinant human dehydrogenase 50 by conventional recombinant DNA technology (Science, 1984; 224: 1431). Generally there are the following steps:
  • the medium used in the culture may be selected from various conventional mediums. Culture is performed under conditions suitable for host cell growth. After the host cells have grown to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.
  • a suitable method such as temperature conversion or chemical induction
  • the recombinant polypeptide may be coated in a cell, expressed on a cell membrane, or secreted outside the cell. If necessary, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. These methods include, but are not limited to: conventional 'renaturation treatment, protein precipitant treatment (salting out method), centrifugation, osmolytic bacteria, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, Ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • conventional 'renaturation treatment protein precipitant treatment (salting out method), centrifugation, osmolytic bacteria, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, Ion exchange chromatography, high performance
  • polypeptides of the present invention as well as antagonists, agonists and inhibitors of the polypeptides, can be directly used in the treatment of diseases, for example, they can treat malignant tumors, adrenal deficiency, skin diseases, various types of inflammation, HIV infection, and immune diseases.
  • G6PD deficiency People with G6PD deficiency are often accompanied by neonatal jaundice, non-erythrocyte hemolytic anemia, and various other types of anemia (Cooper et al, 1972); endemic malaria is often found in areas with a high incidence of G6PD deficiency (Krasnopo l et al, 1980).
  • the type II IMPDH mainly appears in malignant tumor cells, indicating that IMPDH plays an important role in the expression of tumor suppressor genes and proto-oncogenes such as P53, c-myc and Ki-ras.
  • IMPDH inhibitors can induce apoptosis and provide new ideas for the prevention and treatment of tumors (Jayaram et al, 1999).
  • GMP reductase is involved in GTP and its related signaling system.
  • Human dehydrogenase 50 sequences, protein expression products and corresponding antibodies, repressors, Inhibitors and so on have great application value in the diagnosis and treatment of related genes, as well as the auxiliary treatment of drugs and rehabilitation and health care.
  • the invention also provides screening compounds to identify increasing (agonist) or repressing (antagonist) human dehydrogenases
  • Anti-human dehydrogenase 50 antibodies can be used in immunohistochemistry to detect human dehydrogenase 50 in biopsy specimens.
  • the antibodies of the present invention can be used to treat or prevent diseases related to human dehydrogenase 50.
  • Administration of an appropriate dose of antibody can stimulate or block the production or activity of human dehydrogenase 50.
  • the invention also relates to a diagnostic test method for quantitative and localized detection of human dehydrogenase 50 levels.
  • tests are well known in the art and include FISH assays and radioimmunoassays.
  • the level of human dehydrogenase 50 detected in the test can be used to explain the importance of human dehydrogenase 50 in various diseases and to diagnose diseases in which human dehydrogenase 50 plays a role.
  • the recombinant gene therapy vector can be used for treating diseases caused by abnormal expression or activity of human dehydrogenase 50.
  • Virus-derived expression vectors such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus, etc. can be used to transfer a polynucleotide encoding human dehydrogenase 50 into a cell.
  • Methods for constructing a recombinant viral vector carrying a polynucleotide encoding human dehydrogenase 50 can be found in existing literature (Sambrook, et al.).
  • the recombinant polynucleotide encoding human dehydrogenase ⁇ 50 can be packaged into liposomes and transferred into cells.
  • Methods for introducing a polynucleotide into a tissue or cell include: directly injecting the polynucleotide into a tissue in vivo; or introducing the polynucleotide into a cell in vitro through a vector (such as a virus, phage, or plasmid), and then transplanting the cell Into the body and so on.
  • a vector such as a virus, phage, or plasmid
  • Oligonucleotides including antisense RM and DNA
  • ribozymes that inhibit human dehydrogenase 50 mRNA are also within the scope of the present invention.
  • a ribozyme is an enzyme-like RM molecule that can specifically decompose a specific RNA. Its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target RNA for endonucleation.
  • Antisense RM and DM and ribozymes can be obtained using any existing RNA or DNA synthesis techniques, such as solid-phase phosphoramide chemistry The technology of synthesizing oligonucleotides has been widely used.
  • Antisense RNA molecules can be obtained by in vitro or in vivo transcription of a DNA sequence encoding the MA. This DNA sequence has been integrated downstream of the RNA polymerase promoter of the vector. In order to increase the stability of a nucleic acid molecule, it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the ribonucleoside linkages should use phosphate thioester or peptide bonds instead of phosphodiester bonds.
  • a part or all of the polynucleotides of the present invention can be used as probes to be fixed on a microarray or a DNA chip (also called a "gene chip") for analyzing differential expression analysis of genes and genetic diagnosis in tissues.
  • Human dehydrogenase 50-specific primers can also be used to detect human dehydrogenase 50 transcripts by in vitro amplification of A-polymerase chain reaction (RT-PCR).
  • PCR localization of somatic hybrid cells is a quick way to localize DNA to specific chromosomes.
  • oligonucleotide primers of the present invention in a similar manner, a set of fragments from a specific chromosome or a large number of genomic clones can be used to achieve sublocalization.
  • Other similar strategies that can be used for chromosomal localization include in situ hybridization, chromosome pre-screening with labeled flow sorting, and pre-selection of hybridization to construct a chromosome-specific c-threat library.
  • the difference in cDNA or genomic sequence between the affected and unaffected individuals needs to be determined. If a mutation is observed in some or all diseased individuals and the mutation is not observed in any normal individuals, the mutation may be the cause of the disease. Comparing affected and unaffected individuals usually involves first looking for structural changes in chromosomes, such as deletions or translocations that are visible at the chromosomal level or detectable with cDNA sequence-based PCR. According to the resolution capabilities of current physical mapping and gene mapping technology, the cDNA accurately mapped to the chromosomal region associated with the disease can be one of 50 to 500 potentially pathogenic genes (assuming 1 megabase mapping resolution) Capacity and each 20kb corresponds to a gene).
  • the polypeptides, polynucleotides and mimetics, agonists, antagonists and inhibitors of the present invention can be used in combination with a suitable pharmaceutical carrier.
  • suitable pharmaceutical carrier can be water, glucose, ethanol, salts, buffers, glycerol, and combinations thereof.
  • the composition comprises a safe and effective amount of the polypeptide or antagonist, and carriers and excipients which do not affect the effect of the drug. These compositions can be used as drugs for the treatment of diseases.
  • the pharmaceutical composition can be administered in a convenient manner, such as by a topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal route of administration.
  • Human dehydrogenase 50 is administered in an amount effective to treat and / or prevent a particular indication.
  • the amount and dosage range of human dehydrogenase 50 administered to a patient will depend on many factors, such as the mode of administration, the health conditions of the person to be treated, and the judgment of the diagnostician. Examples
  • Total RM of human fetal brain was extracted by one-step method with guanidine isothiocyanate / phenol / chloroform.
  • Poly (A) raRNA was isolated from total RNA using Quik mRNA Isolat ion Kit (product of Qiegene). 2ug poly (A) mRNA is reverse transcribed to form cDNA. Smar t cDM cloning kit (purchased from Citronech) was used. The 0 ⁇ fragment was inserted into the multicloning site of pBSK (+) vector (Clontech), and transformed into DH5a. The bacteria formed a CDM library.
  • Dye terminate cycle reaction ion sequencing kit Perkin-Elmer
  • ABI 377 automatic sequencer Perkin-Elmer
  • the inserted cDNA fragments contained in this clone were determined in both directions by synthesizing a series of primers.
  • the 0271e01 clone contains a full-length CDM of 1611bp (as shown in Seq ID N0: l), and has a 1370bp open reading frame (0RF) from lOlbp to l ⁇ lbp, encoding a new protein (such as Seq ID NO: 2).
  • This clone pBS_0271e01 and the encoded protein was named human dehydrogenase 50.
  • Example 2 Cloning of a gene encoding human dehydrogenase 50 by RT-PCR
  • CDM was synthesized by reverse transcription reaction using total RNA from fetal brain cells as a template and ol igo-dT as a primer. After purification with Qiagene's kit, PCR was performed with the following primers:
  • Primer 1 5,-GGAAACTGTCGGGAGAAGTTCTGC-3 '(SEQ ID NO: 3)
  • Pr iraer2 5'- TTCTTCAAAAATTTATTATTTATT-3 '(SEQ ID NO: 4)
  • Pr imerl is a forward sequence located at the 5th end of SEQ ID NO: 1, starting at lbp;
  • Conditions for the amplification reaction 50 mmol / L KCl, 10 mmol / L Tri s-HCl pH 8.5, 1.5 mmol / L MgCl 2 , 200 mol / L dNTP, 1 Opmol primer, 1U Taq DM polymerase (Clontech).
  • the reaction was performed on a PE OO 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.
  • RNA extraction in one step involves acid guanidinium thiocyanate phenol-chloroform extraction. That is, the tissue was 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.
  • 32P-labeled probes (approximately 2 x 1 Q 6 cpm / ml) were hybridized with RM-transferred nitrocellulose membrane in a solution at 42 ° C overnight, the solution Contains 50% formamide-25mM KH 2 P0 4 (pH7. 4) -5 x SSC-5 x Denhardt's solution and 200 ⁇ ⁇ / ⁇ 1 salmon sperm DM. After hybridization, the filter is placed in 1 x SSC-0. 13 ⁇ 4SDS in Wash at 55 ° C for 30 min. Then, use Phosphor Imager for analysis and quantification.
  • Example 4 In vitro expression, isolation and purification of recombinant human dehydrogenase 50
  • Primer 3 5 '-CATGCTAGCATGGCGGCGGCACTGAAGTGTCTA-3' (Seq ID No:)
  • Primer 4 5 '-CCCGAATTCTCAACTCTCAGAGGGATCCTGAGC-3' (Seq ID No: 6)
  • the 5 'ends of these two primers contain Nhel and EcoRI restriction sites, respectively , followeded by the coding sequences of the 5 ,, and 3 'ends of the gene of interest, respectively.
  • the Nhel and EcoRI digestion sites correspond to the expression vector plasmid pET- 2 8b (+) (Novagen, Cat. No. 69865. 3) Selective endonuclease site.
  • the PCR reaction was performed using the pBS-0271e01 plasmid containing the full-length target gene as a template.
  • the PCR reaction conditions were as follows: 10 pg of pBS-0271e01 plasmid in a total volume of 50 ⁇ 1, Primer-3 and Primer-4 were lpmol, Advantage polymerase Mix (Clontech) 1 ⁇ 1, respectively. Cycle parameters: 94. C 20s, 60. C 30s, 68 ° C 2 min, a total of 25 cycles. Nhel and EcoRI were used to double digest the amplified product and plasmid P ET-28 (+), respectively, and large fragments were recovered and ligated with T4 ligase.
  • the ligation product was transformed into the colibacillus DH5 cx by the calcium chloride method. After being cultured on LB plates containing kanamycin (final concentration 30 ⁇ ⁇ / ⁇ 1) overnight, positive clones were selected by colony PCR method and sequenced. A positive clone (pET-0271e01) with the correct sequence was selected, and the recombinant plasmid was transformed into E. coli BL21 (DE3) plySs (N 0vag en company product) by the calcium chloride method.
  • NH2-Met-Ala-Ala-Ala-Leu-Lys-Cys-Leu-Leu-Thr-Leu-Gly-Arg-Trp-Cys-C00H (SEQ ID NO: 7).
  • the polypeptide is coupled to hemocyanin and bovine serum albumin to form a complex, respectively.
  • hemocyanin and bovine serum albumin For the method, see: Avrameas, et al. I let unochemi s try, 1969; 6: 43. Rabbits were immunized with 4 mg of the hemocyanin polypeptide complex plus complete Freund's adjuvant, and 15 days later, the hemocyanin polypeptide complex plus incomplete Freund's adjuvant was used to boost immunity once.
  • the titer of antibody in rabbit serum was determined by ELISA using a titer plate coated with 15 ⁇ ⁇ / ⁇ 1 bovine serum albumin polypeptide complex.
  • Total IgG was isolated from antibody-positive rabbit serum using protein A-Sepharose.
  • the peptide was bound to a cyanogen bromide-activated Sepharos B column and the anti-peptide antibody was separated from the total IgG by affinity chromatography. Immunoprecipitation demonstrated that the purified antibody could specifically bind to human dehydrogenase 50.
  • Example 6 Application of the polynucleotide fragment of the present invention as a hybridization probe
  • Suitable oligonucleotide fragments selected from the polynucleotides of the present invention are used as hybridization probes in a variety of ways.
  • the probes can be used to hybridize to genomic or cDNA libraries of normal tissue or pathological tissue from different sources to It is determined whether it contains the polynucleotide sequence of the present invention and a homologous polynucleotide sequence is detected.
  • the probe can be used to detect the polynucleotide sequence of the present invention or its homologous polynucleotide sequence in normal tissue or pathology. Whether the expression in tissue cells is abnormal.
  • the purpose of this embodiment is to select a suitable oligonucleotide fragment from the polynucleotide SEQ ID NO: 1 of the present invention as a hybridization probe, and to identify whether some tissues contain the polynucleoside of the present invention by a filter hybridization method.
  • Filter hybridization methods include dot blotting, Southern blotting, Northern blotting, and copying methods. They all use the same steps of hybridization after fixing the polynucleotide sample to be tested on the filter.
  • the sample-immobilized filter is first pre-hybridized with a probe-free hybridization buffer, so that the non-specific binding site of the sample on the filter is saturated with the carrier and the synthetic polymer.
  • the pre-hybridization solution is then replaced with a hybridization buffer containing the labeled probe and incubated to hybridize the probe to the target nucleic acid.
  • the unhybridized probes are removed by a series of membrane washing steps Off.
  • This embodiment utilizes higher-intensity washing conditions (such as lower salt concentration and higher temperature) to reduce the hybridization background and retain only strong specific signals.
  • the probes used in this embodiment include two types: the first type of probes are oligonucleotide fragments that are completely the same as or complementary to the polynucleotide SEQ ID NO: 1 of the present invention; the second type of probes are partially related to the present invention
  • the polynucleotide SEQ ID NO: 1 is the same or complementary oligonucleotide fragment.
  • the dot blot method is used to fix the sample on the filter membrane. Under the high-intensity washing conditions, the first type of probe and the sample have the strongest hybridization specificity and are retained.
  • oligonucleotide fragments from the polynucleotide SEQ ID NO: 1 of the present invention for use as hybridization probes should follow the following principles and several aspects to be considered:
  • the preferred range of probe size is 18-50 nucleotides
  • the GC content is 30% -70%, and the non-specific hybridization increases when it exceeds;
  • Those that meet the above conditions can be used as primary selection probes, and then further computer sequence analysis, including the primary selection probe and its source sequence region (ie, SEQ ID NO: 1) and other known genomic sequences and their complements For homology comparison of the regions, if the homology with the non-target molecular region is greater than 85% or there are more than 15 consecutive bases, the primary probe should not be used generally;
  • Probe 1 which belongs to the first type of probe, is completely homologous or complementary to the gene fragment of SEQ ID NO: 1 (41Nt):
  • Probe 2 which belongs to the second type of probe, is equivalent to the replacement mutant sequence of the gene fragment of SEQ ID NO: 1 or its complementary fragment (41Nt):
  • PBS phosphate buffered saline
  • step 8-13 are only used when contamination must be removed, otherwise step 14 can be performed directly.
  • NC membrane nitrocellulose membrane
  • Gene chip or DNA microarray is a new technology that many national laboratories and large pharmaceutical companies are currently developing and developing. It refers to the orderly and high-density arrangement of a large number of target gene fragments on glass, The data is compared and analyzed on a carrier such as silicon using fluorescence detection and computer software to achieve the purpose of fast, efficient, and high-throughput analysis of biological information.
  • the polynucleotide of the present invention can be used as target DNA for gene chip technology for high-throughput research of new gene functions; search for and screen new tissue-specific genes, especially new genes related to diseases such as tumors; diagnosis of diseases such as hereditary diseases .
  • the specific method steps have been reported in the literature. For example, refer to the literature DeRis i, JL, Lyer, V. & Brown, P. 0. (1997) Science 278, 680-686. And the literature Hel le, RA, Schema , M., Chai, A., Shalom, D., (1997) PNAS 94: 2150
  • a total of 4,000 polynucleotide sequences of various full-length cDNAs are used as target DMs, including the polynucleotides of the present invention. They were respectively amplified by PCR, and the concentration of the amplified product was adjusted to about 500 ng / ul after purification. The spots were spotted on a sloped glass medium using a Cartesian 7500 spotter (purchased from Cartesian Company, USA). The distance between them is 280 ⁇ . The spotted slides were hydrated, dried, and cross-linked in an ultraviolet cross-linker. After elution, the DM was fixed on a glass slide to prepare chips. The specific method steps There are many reports in the literature. The sample post-processing steps in this embodiment are:
  • the probes from the two types of tissues were hybridized with the chip in a UniHyb TM Hybridizat ion Solut ion (purchased from TeleChem) hybridization solution for 16 hours, and washed with a washing solution (lx SSC, 0.2 ° / oSDS) at room temperature. Scanning was then performed with a ScanArray 3000 scanner (purchased from General Scanning, USA). The scanned images were analyzed and processed with Imagene software (Biodiscovery, USA) to calculate the Cy3 / Cy5 ratio of each point.
  • the above specific tissues are thymus, testis, muscle, spleen, lung, skin, thyroid, liver, PMA + Ecv304 cell line, PMA-Ecv304 cell line, non-starved L02 cell line, L02 cell line stimulated by arsenic for 1 hour, L02 cell line stimulated by arsenic for 6 hours prostate, heart, lung cancer, fetal bladder, fetal small intestine, fetal large intestine, fetal thymus, fetal muscle, fetal liver, fetal kidney, fetal spleen, fetal brain, Fetal lung and fetal heart.

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Abstract

L'invention concerne un polypeptide humain, une déshydrogénase 50, le polynucléotide qui le code et un procédé de production de ce polypeptide à l'aide de technologies de recombinaison de l'ADN. Elle concerne aussi des méthodes d'utilisation du polypeptide pour le traitement de troubles variés, notamment de tumeurs malignes, d'hémopathie, d'infection par le VIH, de maladies immunologiques, d'inflammations variées, et d'autres. Elle concerne encore un antagoniste du polypeptide et son action thérapeutique. Elle concerne enfin l'utilisation du polynucléotide codant la déshydrogénase 50.
PCT/CN2001/000695 2000-05-09 2001-05-08 Deshydrogenase 50, polypeptide humain, et polynucleotide la codant Ceased WO2001088111A1 (fr)

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AU70443/01A AU7044301A (en) 2000-05-09 2001-05-08 A new polypeptide- human dehydrogenase 50 and the polynucleotide encoding it

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CN00115630.6 2000-05-09
CN00115630A CN1322826A (zh) 2000-05-09 2000-05-09 一种新的多肽——人脱氢酶50和编码这种多肽的多核苷酸

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998031815A2 (fr) * 1997-01-17 1998-07-23 Incyte Pharmaceuticals, Inc. Sous-unites de nadh deshydrogenase
US5925543A (en) * 1997-09-12 1999-07-20 Incyte Pharmaceuticals, Inc. Isolated polynucleotide sequence encoding NADH dehydrogenase B17 subunit
US5976804A (en) * 1997-12-12 1999-11-02 Incyte Pharmaceuticals, Inc. NADH dehydrogenase PDSW subunit

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998031815A2 (fr) * 1997-01-17 1998-07-23 Incyte Pharmaceuticals, Inc. Sous-unites de nadh deshydrogenase
US5925543A (en) * 1997-09-12 1999-07-20 Incyte Pharmaceuticals, Inc. Isolated polynucleotide sequence encoding NADH dehydrogenase B17 subunit
US5976804A (en) * 1997-12-12 1999-11-02 Incyte Pharmaceuticals, Inc. NADH dehydrogenase PDSW subunit

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
CARNINCI P. AND HAYASHIZAKI Y.: "High-efficiency full-length cDNA cloning", METH. ENZYMOL., vol. 303, 1999, pages 19 - 44 *

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