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WO2001094593A1 - Nouveau polypeptide, glycero-3-phosphate deshydrogenase humaine 11, et polynucleotide codant ce polypeptide - Google Patents

Nouveau polypeptide, glycero-3-phosphate deshydrogenase humaine 11, et polynucleotide codant ce polypeptide Download PDF

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Publication number
WO2001094593A1
WO2001094593A1 PCT/CN2001/000766 CN0100766W WO0194593A1 WO 2001094593 A1 WO2001094593 A1 WO 2001094593A1 CN 0100766 W CN0100766 W CN 0100766W WO 0194593 A1 WO0194593 A1 WO 0194593A1
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Prior art keywords
polypeptide
polynucleotide
phosphate dehydrogenase
sequence
human
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PCT/CN2001/000766
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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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Priority to AU87503/01A priority Critical patent/AU8750301A/en
Publication of WO2001094593A1 publication Critical patent/WO2001094593A1/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
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0008—Oxidoreductases (1.) acting on the aldehyde or oxo group of donors (1.2)

Definitions

  • the present invention belongs to the field of biotechnology. Specifically, the present invention describes a new polypeptide, ⁇ -glycerol 3 phosphate dehydrogenase 11, and a polynucleotide sequence encoding the polypeptide. The invention also relates to a preparation method and application of the polynucleotide and polypeptide.
  • the content of sugar in blood and related tissues in the body is maintained at a normal level, and the abnormal content of the sugar will cause the abnormal function of the relevant tissues in the human body.
  • the stability of blood glucose content is regulated by various hormones. Insulin is the main hormone that promotes anabolic metabolism in the body and regulates blood glucose stability. Insulin secretion and its content in the body are regulated by some related enzymes. Mitochondria glycerol 3-phosphate dehydrogenase in pancreatic ⁇ -cell line regulates glucose-stimulated insulin secretion in the body.
  • the abnormal action of this enzyme will affect the abnormal secretion of insulin in the body, and then affect the instability of blood glucose content in the body, that is, it will trigger a series of diseases related to glucose metabolism disorders, such as type II diabetes [Ferrer J., Aoki M et a l., 1996, Diabetes, 45 (2): 262-266].
  • Glycerol 3 phosphate dehydrogenase has been cloned from many different organisms in many different sources.
  • Gong et al. Cloned a mitochondrial FAD-associated glycerol 3 phosphate dehydrogenase from mice, which is one of two enzymes in the glycerol phosphate shuttle system, and has a higher expression in brown adipose tissue.
  • the protein sequence contains a FAD binding domain.
  • the enzyme consists of about 17 exons, and the first exon has three different transcripts under the transcriptional expression of different promoters.
  • exon 1A is expressed in the brain
  • exon 1B is expressed in various tissues
  • exon 1C is mainly in tissues such as the testis Express.
  • the expression of all these different transcripts in the body are regulated by thyroid hormones in the receptor. Abnormal expression of these transcripts will usually lead to abnormal blood glucose levels in the organism, and then cause various related metabolic disorders [Gong DW, Bi S. et a l., 1998, DNA Cel l Biol, 17 (3): 301-309].
  • mitochondrial glycerol 3-phosphate dehydrogenase plays a very important regulatory role in related metabolic pathways in vivo.
  • This enzyme regulates glucose-stimulated insulin secretion in the body. Insulin is the main hormone that promotes anabolic metabolism and regulates blood glucose stability. Abnormal secretion of the enzyme will lead to abnormal blood glucose levels in the body, and then cause related metabolic disorders. It can be seen that this enzyme is closely related to the occurrence of some sugar metabolism disorders such as type I and diabetes in living organisms and various tumors and cancers related thereto.
  • the protein can also be used for the diagnosis and treatment of various related diseases mentioned above.
  • the human glycerol 3 phosphate dehydrogenase 11 protein plays an important role in regulating important functions of the body such as cell division and embryonic development, and it is believed that a large number of proteins are involved in these regulatory processes, so the identification of more participation has been required in the field. During these processes, the human glycerol 3-phosphate dehydrogenase 11 protein was specifically identified for its amino acid sequence. Isolation of the new human glycerol 3 phosphate dehydrogenase 11 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 is a large number of proteins are involved in these regulatory processes, so the identification of more participation has been required in the field. During these processes, the human glycerol 3-phosphate dehydrogenase 11 protein was specifically identified for its amino acid sequence. Isolation of the new
  • An object of the present invention is to provide an isolated novel polypeptide, human glycerol 3 phosphate dehydrogenase 11 and fragments, analogs and derivatives thereof.
  • 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 glycerol triphosphate dehydrogenase 11.
  • Another object of the present invention is to provide a method for producing human glycerol triphosphate dehydrogenase 11.
  • Another object of the present invention is to provide a polypeptide-human glycerol 3-phosphate dehydrogenase 11 directed to the present invention. Antibodies.
  • Another object of the present invention is to provide mimetic compounds, antagonists, agonists, and inhibitors against the polypeptide of the present invention, human glycerol 3 phosphate dehydrogenase 11.
  • Another object of the present invention is to provide a method for diagnosing and treating diseases associated with abnormalities in human glycerol 3 phosphate dehydrogenase 11. Summary of invention
  • the present invention relates to an isolated polypeptide, which is of human origin and comprises: a polypeptide having the amino acid sequence of SEQ ID No. 2, or a conservative variant, biologically active fragment or derivative thereof.
  • the polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • the invention also relates to an isolated polynucleotide comprising a nucleotide sequence or a variant thereof selected from the group consisting of:
  • sequence of the polynucleotide is one selected from the group consisting of: (a) a sequence having positions 137-445 in SEQ ID NO: 1; and (b) a sequence having 1-1836 in SEQ ID NO: 1 Sequence of bits.
  • the present invention further relates to a vector, particularly an expression vector, containing the polynucleotide of the present invention; a host cell genetically engineered with the vector, including a transformed, transduced or transfected host cell; Host cell and method of preparing the polypeptide of the present invention by recovering the expression product.
  • 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 screening compounds that mimic, activate, antagonize or inhibit the activity of human glycerol 3 phosphate dehydrogenase 11 protein, which comprises utilizing the polypeptide of the invention.
  • the invention also relates to compounds obtained by this method.
  • the present invention also relates to a method for detecting a disease or disease susceptibility related to abnormal expression of human glycerol 3 phosphate dehydrogenase 11 protein in vitro, comprising detecting a mutation in the polypeptide or a polynucleotide sequence encoding the same in a biological sample, or Detection of the amount or biological activity of a polypeptide of the invention in a biological sample.
  • the invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a polypeptide of the invention or a mimetic thereof, an activator, an antagonist or an inhibitor, and a pharmaceutically acceptable carrier.
  • the present invention also relates to the preparation of the polypeptide and / or polynucleotide of the present invention for the treatment of type 2 diabetes, diabetic complications, other types of diabetes or other diseases caused by abnormal expression of human glycerol 3 phosphate dehydrogenase 11 use.
  • Other aspects of the invention will be apparent to those skilled in the art from the disclosure of the techniques herein.
  • FIG. 1 is a comparison diagram of gene chip expression profiles of human glycerol 3 phosphate dehydrogenase 11 and human glycerol 3 phosphate dehydrogenase according to the present invention.
  • the upper graph is a graph of the expression profile of human glycerol 3 phosphate dehydrogenase 11 and the lower graph is the graph of the expression profile of human glycerol 3 phosphate dehydrogenase 11.
  • 1 indicates fetal kidney
  • 2 indicates fetal large intestine
  • 3 indicates fetal small intestine
  • 4 indicates fetal muscle
  • 5 indicates fetal brain
  • 6 indicates fetal bladder
  • 7 indicates unstarved L02
  • 8 indicates L02 +, lhr, As 3+
  • 9 indicates ECV304 PMA-
  • 10 means ECV304 PMA +
  • 11 means fetal liver
  • 12 means normal liver
  • 13 means thyroid
  • 14 means skin
  • 15 means fetal lung
  • 16 means lung
  • 17 means lung cancer
  • 18 means fetal spleen
  • 19 means spleen
  • 20 Indicates prostate
  • 21 indicates fetal heart
  • 22 indicates heart
  • 23 indicates muscle
  • 24 indicates testis
  • 25 indicates fetal thymus
  • 26 indicates thymus.
  • FIG. 2 shows the polyacrylamide gel electrophoresis (SDS-PAGE) of human glycerol 3 phosphate dehydrogenase 11 isolated.
  • lKDa is the molecular weight of the protein.
  • the arrow indicates the isolated protein band.
  • Nucleic acid sequence refers to an oligonucleotide, a nucleotide or a polynucleotide and a fragment or part thereof, and may also refer to a genome or a synthetic DM or RNA, they can be single-stranded or double-stranded, representing the sense or antisense strand.
  • amino acid sequence refers to an oligopeptide, peptide, polypeptide or protein sequence and fragments or portions thereof.
  • amino acid sequence in the present invention relates to the amino acid sequence of a naturally occurring protein molecule, such "polypeptide” or “protein” does not mean to limit the amino acid sequence to a complete natural amino acid related to the protein molecule .
  • a “variant" of a protein or polynucleotide refers to an amino acid sequence having one or more amino acids or nucleotide changes or a polynucleotide sequence encoding it.
  • the changes may include deletions, insertions or substitutions of amino acids or nucleotides in the amino acid sequence or nucleotide sequence.
  • Variants can have "conservative" changes, in which the amino acid substituted has a structural or chemical property similar to the original amino acid, such as replacing isoleucine with leucine.
  • Variants can also have non-conservative changes, such as replacing glycine with tryptophan.
  • “Deletion” refers to the deletion of one or more amino acids or nucleotides in an amino acid sequence or nucleotide sequence.
  • “Insertion” or “addition” refers to an alteration in the amino acid sequence or nucleotide sequence that results in an increase in one or more amino acids or nucleotides compared to a naturally occurring molecule.
  • “Replacement” refers to the replacement of one or more amino acids or nucleotides with different amino acids or nucleotides.
  • Bioactivity refers to a protein that has the structure, regulation, or biochemical function of a natural molecule.
  • immunologically active refers to the ability of natural, recombinant or synthetic proteins and fragments thereof to induce a specific immune response and to bind specific antibodies in a suitable animal or cell.
  • An "agonist” refers to a molecule that, when combined with human glycerol 3 phosphate dehydrogenase 11, causes a change in the protein to regulate the activity of the protein.
  • An agonist may include a protein, a nucleic acid, a carbohydrate, or any other molecule that binds human glycerol 3 phosphate dehydrogenase 11.
  • Antagonist refers to a molecule that can block or regulate the biological or immunological activity of human glycerol 3 phosphate dehydrogenase 11 when combined with human glycerol 3 phosphate dehydrogenase 11.
  • Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates or any other molecule that can bind human glycerol 3-phosphate dehydrogenase 11.
  • Regular refers to a change in the function of human glycerol 3-phosphate dehydrogenase 11, including an increase or decrease in protein activity, a change in binding characteristics, and any other biological properties, functions, or immunity of human glycerol 3-phosphate dehydrogenase 11. Change of nature.
  • Those skilled in the art can purify human glycerol 3 phosphate dehydrogenase 11 using standard protein purification techniques11
  • the substantially pure human glycerol 3 phosphate dehydrogenase 11 can generate a single main band on a non-reducing polyacrylamide gel.
  • the purity of the human glycerol 3 phosphate dehydrogenase 11 polypeptide can be analyzed by amino acid sequence.
  • Complementary refers to the natural binding of polynucleotides by base-pairing under conditions of acceptable salt concentration and temperature.
  • sequence C-T-G-A
  • complementary sequence G-A-C-T.
  • the complementarity between two single-stranded molecules may be partial or complete.
  • the degree of complementarity between nucleic acid strands has a significant effect on the efficiency and strength of hybridization between nucleic acid strands.
  • “Homology” means the degree of complementarity can be partial or complete homology.
  • "Partial homology” refers to a partially complementary sequence that at least partially inhibits hybridization of a fully complementary sequence to a target nucleic acid. This inhibition of hybridization can be detected by performing hybridization (Southern imprinting or Northern blotting, etc.) under conditions of reduced stringency. Substantially homologous sequences or hybridization probes can compete and inhibit the binding of fully homologous sequences to the target sequence under conditions of reduced stringency. This does not mean that the conditions of reduced stringency allow non-specific binding, because the conditions of reduced stringency require that the two sequences bind to each other as a specific or selective interaction.
  • Percent identity means the sequence is the same in two or more amino acid or nucleic acid sequence comparisons Similar percentages.
  • the percent identity can be determined electronically, such as by the MEGALIGN program (Lasergene sof tware package, DNASTAR, Inc., Madi son Wis.).
  • the MEGALIGN program can compare two or more sequences according to different methods, such as the Clus ter method (Higgins, DG and PM Sharp (1988) Gene 73: 237-244). 0
  • the Clus ter method groups each group by checking the distance between all pairs. The sequences are arranged in clusters. The clusters are then assigned in pairs or groups.
  • the percent identity between two amino acid sequences such as sequence A and sequence B is calculated by the following formula:
  • the percent identity between nucleic acid sequences can also be determined by the Clus ter method or by methods known in the art such as Jotun Hein (Hein J., (199.0) Methods in enzymology 183: 625-645).
  • Similarity refers to the degree of identical or conservative substitutions of amino acid residues at corresponding positions in the alignment of amino acid sequences.
  • Amino acids used for conservative substitution for example, negatively charged amino acids may include aspartic acid and glutamic acid; positively charged amino acids may include lysine and arginine; having an uncharged head group is Similar hydrophilic amino acids may include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; serine and threonine; phenylalanine and tyrosine.
  • Antisense refers to a nucleotide sequence that is complementary to a particular DNA or RNA sequence.
  • Antisense strand refers to a nucleic acid strand that is complementary to a “sense strand.”
  • Derivative refers to HFP or a chemical modification of its nucleic acid. This chemical modification may be a substitution of a hydrogen atom with a fluorenyl, acyl or amino group. Nucleic acid derivatives can encode polypeptides that retain the main biological properties of natural molecules. '
  • Antibody refers to a complete antibody molecule and its fragments, such as Fa,? ( ⁇ ') 2 and?, which specifically bind to the epitope of human glycerol 3 phosphate dehydrogenase 11.
  • a “humanized antibody” refers to an antibody in which the amino acid sequence of a non-antigen binding region is replaced to become more similar to a human antibody, but still retains the original binding activity.
  • isolated refers to the removal of a substance from its original environment (for example, its natural environment if it is naturally occurring).
  • a naturally-occurring polynucleotide or polypeptide is not isolated when it is present in a living thing, but the same polynucleotide or polypeptide is separated from some or all of the substances that coexist with it in the natural system.
  • Such a polynucleotide may be part of a certain vector, or such a polynucleotide or polypeptide may be part of a certain composition. Since the carrier or composition is not part of its natural environment, they are still isolated.
  • isolated refers to the separation of a substance from its original environment (if it is a natural substance, the original environment is the natural environment).
  • Polynucleoside in its natural state Acids and polypeptides are not isolated and purified, but the same polynucleotides or polypeptides are isolated and purified if they are separated from other substances in their natural state.
  • isolated human glycerol 3-phosphate dehydrogenase 11 means that human glycerol 3-phosphate dehydrogenase 11 is substantially free of other proteins, lipids, sugars, or other substances with which it is naturally associated.
  • Those skilled in the art can purify human glycerol 3 phosphate dehydrogenase 11 using standard protein purification techniques. Substantially pure peptides produce a single main band on a non-reducing polyacrylamide gel. The purity of the human glycerol 3 phosphate dehydrogenase 11 peptide can be analyzed by amino acid sequence.
  • the present invention provides a new polypeptide, human glycerol 3 phosphate dehydrogenase 11, which is basically composed of the amino acid sequence shown in SEQ ID NO: 2.
  • the polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide, and preferably a recombinant polypeptide.
  • the polypeptides of the invention can be naturally purified products, or chemically synthesized products, or produced using recombinant techniques from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, higher plants, insects, and mammalian cells). Depending on the host used in the recombinant production protocol, the polypeptide of the invention may be glycosylated, or it may be non-glycosylated. Polypeptides of the invention may also include or exclude starting methionine residues.
  • the present invention also includes fragments, derivatives and analogs of human glycerol 3-phosphate dehydrogenase 11.
  • fragment refers to a polypeptide that substantially maintains the same biological function or activity of the human glycerol 3 phosphate dehydrogenase 11 of the present invention.
  • a fragment, derivative or analog of the polypeptide of the present invention may be: (I) one in which one or more amino acid residues are replaced with conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and A substituted amino acid may or may not be encoded by a genetic codon; or ( ⁇ ) a type in which a group on one or more amino acid residues is replaced by another group to include a substituent; or ( ⁇ ⁇ ) A type in which the mature polypeptide is fused with another compound (such as a compound that extends the half-life of the polypeptide, such as polyethylene glycol); or (IV) a type in which the additional amino acid sequence is fused into the mature polypeptide Sequence (such as a leader sequence or a secreted sequence or a sequence used to purify this polypeptide or a protein sequence).
  • conservative amino acid residues preferably conservative amino acid residues
  • a substituted amino acid may or may not be encoded by a genetic codon
  • ⁇ a type in which
  • the present invention provides an isolated nucleic acid (polynucleotide), which basically consists of a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • the polynucleotide sequence of the present invention includes the nucleotide sequence of SEQ ID NO: 1.
  • Polynucleotides of the invention are found from a CDM library of human fetal brain tissue. It contains a full-length polynucleotide sequence of 1836 bases, and its open reading frames 137-445 encode 102 amino acids.
  • the polynucleotide of the present invention may be in the form of DM or RNA.
  • DNA forms include cDNA, genomic DNA, or synthetic DM.
  • DNA can be single-stranded or double-stranded.
  • DM can be coded or non-coded.
  • the coding region sequence encoding a mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO: 1 or a degenerate variant.
  • a "degenerate variant” refers to a nucleic acid sequence encoding a protein or polypeptide having SEQ ID NO: 2 but different from the coding region sequence shown in SEQ ID NO: 1 in the present invention.
  • the polynucleotide encoding the mature polypeptide of SEQ ID NO: 2 includes: only the coding sequence of the mature polypeptide; the coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence of the mature polypeptide (and optional additional coding sequences); Coding sequence.
  • polynucleotide encoding a polypeptide refers to a polynucleotide comprising the polypeptide and a polynucleotide comprising additional coding and / or non-coding sequences.
  • the invention also relates to variants of the polynucleotides described above, which encode polypeptides or fragments, analogs and derivatives of polypeptides having the same amino acid sequence as the invention.
  • Variants of this polynucleotide can be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants.
  • an allelic variant is an alternative form of a polynucleotide that may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially change the function of the polypeptide it encodes .
  • the invention also relates to a polynucleotide that hybridizes to the sequence described above (having at least 50%, preferably 70% identity, between the two sequences).
  • the present invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the present invention under stringent conditions.
  • stringent conditions means: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2xSSC, 0.1% SDS, 60; or (2) during hybridization Add denaturants, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficol l, 42 ° C, etc .; or (3) only the identity between the two sequences Crosses occur at least 95% and more preferably 97%.
  • the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO: 2.
  • nucleic acid fragments that hybridize to the sequences described above.
  • a "nucleic acid fragment” contains at least 10 nucleotides in length, preferably at least 20-30 nucleotides, more preferably at least 50-60 nucleotides, and most preferably at least 100 nuclei. Glycylic acid or more. Nucleic acid fragments can also be used in nucleic acid amplification techniques (such as PCR) to identify and / or isolate polynucleotides encoding human glycerol 3 phosphate dehydrogenase 11.
  • polypeptides and polynucleotides in the present invention are preferably provided in an isolated form and are more preferably purified to homogeneity.
  • the specific polynucleotide sequence encoding human glycerol 3 phosphate dehydrogenase 11 of the present invention can be obtained by various methods.
  • polynucleotides are isolated using hybridization techniques well known in the art. These technologies include but are not Limitations are: 1) hybridization of probes to genomic or cDNA libraries to detect homologous polynucleotide sequences, and 2) antibody screening of expression libraries to detect cloned polynucleotide fragments with common structural characteristics.
  • the DNA fragment sequence of the present invention can also be obtained by the following methods: 1) isolating the double-stranded DM sequence from the DM of the genome; 2) chemically synthesizing the DM sequence to obtain the double-stranded DM of the polypeptide.
  • genomic DM is the least commonly used. Direct chemical synthesis of DNA sequences is often the method of choice. The more commonly used method is the isolation of cDNA sequences.
  • the standard method for isolating the cDNA of interest is to isolate mRM from donor cells that overexpress the gene and perform reverse transcription to form a plasmid or phage cDNA library.
  • Various methods have been used to extract mRNA, and kits are also commercially available (Qiagene).
  • the construction of a CDM library is also a common method (Sambrook, et al., Molecular Cloning, A Laboratory Manua, Cold Spring Harbor Laboratory. New York, 1989).
  • Commercially available cDNA libraries are also available, such as different cDNA libraries from Clontech. When polymerase reaction technology is used in combination, even very small expression products can be cloned.
  • the genes of the present invention can be selected from these cDNA libraries by conventional methods. These methods include (but are not limited to): (l) DNA-DNA or DNA-RNA hybridization; (2) the presence or absence of marker gene functions; (3) determining the level of transcripts of human glycerol 3 phosphate dehydrogenase 11; (4) Detecting the protein product of gene expression by immunological technology or measuring biological activity. The above methods can be used singly or in combination.
  • 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 here is generally a DNA 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).
  • immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA) can be used to detect the protein product expressed by the human glycerol 3-phosphate dehydrogenase 11 gene.
  • ELISA enzyme-linked immunosorbent assay
  • a method (Sa iki, et al. Sc; 1985; 230: 1350-1354) using PCR technology to amplify DNA / RM is preferred for obtaining 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 DNA / RNA fragments can be isolated and purified by conventional methods such as by gel electrophoresis.
  • polynucleotide sequence of the gene of the present invention or various DNA fragments and the like obtained as described above can be determined by a conventional method such as dideoxy chain termination method (Sanger et al. PMS, 1977, 74: 5463-5467). Such polynucleotide sequences can also be determined using commercial sequencing kits and the like. To obtain 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 CDM sequence.
  • the present invention also relates to a vector comprising a polynucleotide of the present invention, and a host cell produced by genetic engineering using the vector of the present invention or directly using a human glycerol 3 phosphate dehydrogenase 11 coding sequence, and the recombinant technology to produce the polypeptide of the present invention Methods.
  • a polynucleotide sequence encoding human glycerol 3 phosphate dehydrogenase 11 can be inserted into a vector to constitute a recombinant vector containing the polynucleotide of the present invention.
  • vector refers to bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses or other vectors well known in the art.
  • Vectors suitable for use in the present invention include, but are not limited to: T7 promoter-based expression vectors (Rosenberg, et al.
  • any plasmid and vector can be used to construct a recombinant expression vector.
  • An important feature of expression vectors is that they usually contain an origin of replication, a promoter, a marker gene, and translational regulatory elements.
  • Methods known to those skilled in the art can be used to construct an expression vector containing a DM sequence encoding human glycerol 3 phosphate dehydrogenase 11 and appropriate transcription / translation regulatory elements. These methods include in vitro recombinant DM technology, DNA synthesis technology, in vivo recombination technology, etc. (Sambroook, et al. Molecular Cloning, a Laboratory Manual, Cold Spin Harbor Laboratory. New York, 1989).
  • the DNA sequence can be operably linked to an appropriate promoter in an expression vector to guide mRNA synthesis. Representative examples of these promoters are: the lac or trp promoter of E.
  • the expression vector also includes a ribosome binding site for translation initiation, a transcription terminator, and the like. Insertion of enhancer sequences into the vector will enhance its transcription in higher eukaryotic cells. Enhancers are cis-acting factors expressed by DM, usually about 10 to 300 base pairs, which act on promoters to enhance gene transcription. Examples include SV40 enhancers of 100 to 270 base pairs on the late side of the origin of replication, polyoma enhancers and adenovirus enhancers on the late side of the origin of replication.
  • 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 Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.
  • 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 Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.
  • a polynucleotide encoding human glycerol 3 phosphate dehydrogenase 11 or a recombinant vector containing the polynucleotide can be transformed or transduced 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.
  • coli Streptomyces
  • bacterial cells such as Salmonella typhimurium
  • fungal cells such as yeast
  • plant cells such as fly S2 or Sf9
  • animal cells such as CH0, COS or Bowes melanoma cells.
  • 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 absorbing DM can be harvested after the exponential growth phase and treated with the CaCl 2 method. The steps used are well known in the art. Alternatively, MgCl 2 is used. If necessary, transformation can also be performed by electroporation.
  • the host is a eukaryote, the following DM transfection methods can be used: calcium phosphate co-precipitation method, or conventional mechanical methods such as microinjection, electroporation, and liposome packaging.
  • polynucleotide sequence of the present invention can be used to express or produce recombinant human glycerol 3 phosphate dehydrogenase 11 (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. 3 ⁇ 4
  • 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, osmotic disruption, 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, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid
  • polypeptide of the present invention and the antagonists, agonists and inhibitors of the polypeptide can be directly used in the treatment of diseases Therapy, for example, can treat malignant tumors, adrenal deficiency, skin diseases, various types of inflammation, HIV infection and immune diseases.
  • the content of sugar in the blood and related tissues of the human body should be maintained at a normal level. Abnormalities in its content will cause abnormal functions of the relevant tissues in the human body.
  • the stability of blood glucose is regulated by various hormones such as insulin and thyroid hormone. Among them, insulin is the main hormone that promotes anabolic metabolism in the body and regulates blood sugar stability. Insulin secretion and its content in the body are regulated by some related enzymes. Mitochondrial glycerol 3-phosphate dehydrogenase in the pancreatic P cell line regulates glucose-stimulated insulin secretion in the body. The abnormal action of this enzyme will affect the abnormal secretion of insulin in the body, and then affect the instability of blood sugar content in the body, that is, trigger a series of diseases related to glucose metabolism disorders, such as type II diabetes.
  • the glycerol 3-phosphate dehydrogenase gene sequence has multiple exons, and its exons have different transcripts under the transcriptional expression of different promoters.
  • the expression of these different transcripts in the body are all regulated by thyroid hormones in the receptor. Abnormal expression of these transcripts will usually lead to abnormal blood glucose levels in the body, and then cause various related metabolic disorders.
  • mitochondrial glycerol 3-phosphate dehydrogenase plays a very important regulatory role in related metabolic pathways in vivo.
  • This enzyme regulates glucose-stimulated insulin secretion in the body, and insulin is the main hormone that promotes anabolic metabolism and regulates blood glucose stability in the body. Abnormal secretion will lead to abnormal blood glucose content in the body, and then cause related metabolic disorders. It can be seen that this enzyme is closely related to the occurrence of some sugar metabolism disorders such as type II diabetes and various tumors and cancers in the organism.
  • the protein can also be used for the diagnosis and treatment of various related diseases mentioned above.
  • the expression profile of the polypeptide of the present invention is consistent with the expression profile of human mitochondrial glycerol 3 phosphate dehydrogenase, and the two have similar biological functions. It regulates the glucose-stimulated insulin secretion process in the body, and then regulates the body's blood glucose stability. Its abnormal expression directly affects the human mitochondrial glycerol 3 phosphate dehydrogenase in vivo, and then affects the related insulin secretion in the body. And normal levels of blood sugar in the body, and cause related diseases.
  • human glycerol 3 phosphate dehydrogenase 11 of the present invention will produce various diseases, especially type II diabetes, diabetic complications, and other types of diabetes, including but not limited to: non-obese non-insulin Dependent diabetes, obese non-insulin dependent diabetes, type I diabetes, impaired glucose tolerance, gestational diabetes, ketoacidosis, diabetic heart disease, sugar Urinary cardiovascular disease, diabetic nephropathy, diabetic eye disease, diabetic neuropathy, diabetic skin, muscle, and joint disease
  • polypeptides of the present invention can be directly used in the treatment of diseases, for example, they can treat various diseases, especially type I and diabetes, complications of diabetes, and other types of diabetes.
  • the invention also provides methods for screening compounds to identify agents that increase (agonist) or suppress (antagonist) human glycerol 3 phosphate dehydrogenase 11.
  • Agonists enhance human glycerol 3 phosphate dehydrogenase 11 to stimulate biological functions such as cell proliferation, while antagonists prevent and treat disorders related to excessive cell proliferation, such as various cancers.
  • mammalian cells or a membrane preparation expressing human glycerol 3 phosphate dehydrogenase 11 can be cultured with labeled human glycerol 3 phosphate dehydrogenase 11 in the presence of a drug. The ability of the drug to increase or block this interaction is then determined.
  • Antagonists of human glycerol 3-phosphate dehydrogenase 11 include antibodies, compounds, receptor deletions, and the like that have been screened. Antagonists of human glycerol 3 phosphate dehydrogenase 11 can bind to human glycerol 3 phosphate dehydrogenase 11 and eliminate its function, or inhibit the production of the polypeptide, or bind to the active site of the polypeptide so that the polypeptide cannot function biological functions.
  • human glycerol 3 phosphate dehydrogenase 11 When a compound is selected as an antagonist, human glycerol 3 phosphate dehydrogenase 11 can be added to the bioanalytical assay, and the effect of the compound on the interaction between human glycerol 3 phosphate dehydrogenase 11 and its receptor can be determined. Whether the compound is an antagonist. Receptor deletions and analogs that act as antagonists can be screened in the same way as for screening compounds described above. Polypeptide molecules capable of binding to human glycerol 3 phosphate dehydrogenase 11 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. During screening, 11 molecules of human glycerol 3 phosphate dehydrogenase should generally be labeled.
  • the present invention provides a method for producing antibodies using polypeptides, and fragments, derivatives, analogs or cells thereof as antigens. These antibodies can be polyclonal or monoclonal antibodies.
  • the invention also provides antibodies directed against the human glycerol 3 phosphate dehydrogenase 11 epitope. These antibodies include (but are not limited to): polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, and fragments generated from Fab expression libraries.
  • Polyclonal antibodies can be produced by injecting human glycerol 3 phosphate dehydrogenase 11 directly into immunized animals (such as rabbits, mice, rats, etc.).
  • immunized animals such as rabbits, mice, rats, etc.
  • adjuvants can be used to enhance the immune response, including but not limited to Freund's Adjuvant, etc.
  • Techniques for preparing monoclonal antibodies to human glycerol 3 phosphate dehydrogenase 11 include, but are not limited to, hybridoma technology (Kohler and Mistein. Nature, 1975, 256: 495-497), triple tumor technology, human beta-cell hybridoma Technology, EBV-hybridoma technology, etc.
  • Chimeric antibodies that bind human constant regions to non-human-derived variable regions can be produced using existing techniques (Morrison et al, PNAS, 1985, 81: 6851).
  • the existing technology for producing single chain antibodies (US Pat No. 4946778) can also be used to produce single chain antibodies against human glycerol 3 phosphate dehydrogenase 11.
  • Antibodies against human glycerol 3 phosphate dehydrogenase 11 can be used in immunohistochemical techniques to detect human glycerol 3 phosphate dehydrogenase 11 in biopsy specimens.
  • Monoclonal antibodies that bind to human glycerol 3 phosphate dehydrogenase 11 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.
  • human glycerol 3-phosphate dehydrogenase 11 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 thiol cross-linking agent such as SPDP and bind the toxin to the antibody through the exchange of disulfide bonds.
  • This hybrid antibody can be used to kill human glycerol 3 phosphate dehydrogenase 11 positive Cell.
  • the antibodies of the present invention can be used to treat or prevent diseases related to human glycerol triphosphate dehydrogenase 11.
  • Administration of an appropriate dose of the antibody can stimulate or block the production or activity of human glycerol triphosphate dehydrogenase 11.
  • the invention also relates to a diagnostic test method for quantitative and localized detection of human glycerol 3 phosphate dehydrogenase 11 levels.
  • tests are well known in the art and include FISH assays and radioimmunoassays.
  • the level of human glycerol 3 phosphate dehydrogenase 11 detected in the test can be used to explain the importance of human glycerol 3 phosphate dehydrogenase 11 in various diseases and to diagnose the role of human glycerol 3 phosphate dehydrogenase 11 disease.
  • polypeptide of the present invention can also be used for peptide mapping analysis.
  • the polypeptide can be specifically cleaved by physical, chemical or enzymatic analysis, and subjected to one-dimensional or two-dimensional or three-dimensional gel electrophoresis analysis, and more preferably mass spectrometry analysis.
  • the polynucleotide encoding human glycerol 3 phosphate dehydrogenase 11 can also be used for a variety of therapeutic purposes.
  • Gene therapy technology can be used to treat abnormal cell proliferation, development, or metabolism caused by the non-expression or abnormal / inactive expression of human glycerol 3-phosphate dehydrogenase 11.
  • Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated human glycerol 3 phosphate dehydrogenase 11 to inhibit endogenous human glycerol 3 phosphate dehydrogenase 11 activity.
  • a mutated human glycerol 3 phosphate dehydrogenase 11 may be a shortened human glycerol 3 phosphate dehydrogenase 11 that lacks a signaling functional domain, and although it can bind to a downstream substrate, it lacks signaling activity. Therefore, the recombinant gene therapy vector can be used for treating diseases caused by abnormal expression or activity of human glycerol 3 phosphate dehydrogenase 11.
  • Virus-derived expression vectors such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus, etc.
  • a polynucleotide encoding human glycerol 3 phosphate dehydrogenase 11 can be used to transfer a polynucleotide encoding human glycerol 3 phosphate dehydrogenase 11 into a cell.
  • a method for constructing a recombinant viral vector carrying a polynucleotide encoding human glycerol 3 phosphate dehydrogenase 11 can be found in the existing literature (Sambrook, et al.).
  • recombination coding The polynucleotide of human glycerol 3-phosphate dehydrogenase 11 can be packaged into liposomes and transferred into cells.
  • Methods for introducing a polynucleotide into a tissue or cell include: injecting the polynucleotide directly 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 glycerol 3 phosphate dehydrogenase 11 mRNA are also within the scope of the present invention.
  • a ribozyme is an enzyme-like RNA molecule that can specifically decompose a specific RM. Its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target RM to perform endonucleation.
  • Antisense RNA, DNA, and ribozymes can be obtained using any existing RNA or DNA synthesis technology, such as solid-phase phosphoramidite chemical synthesis to synthesize oligonucleotides.
  • Antisense RNA molecules can be obtained by in vitro or in vivo transcription of a DM sequence encoding the RM. This DNA sequence is integrated downstream of the RM polymerase promoter of the vector. In order to increase the stability of the nucleic acid molecule, it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the linkage between ribonucleosides using phosphate thioester or peptide bonds instead of phosphodiester bonds.
  • the polynucleotide encoding human glycerol triphosphate dehydrogenase 11 can be used for the diagnosis of diseases related to human glycerol triphosphate dehydrogenase 11.
  • the polynucleotide encoding human glycerol 3 phosphate dehydrogenase 11 can be used to detect the expression of human glycerol 3 phosphate dehydrogenase 11 or abnormal expression of human glycerol 3 phosphate dehydrogenase 11 in a disease state.
  • the DM sequence encoding human glycerol 3 phosphate dehydrogenase 11 can be used to hybridize biopsy specimens to determine the expression of human glycerol 3 phosphate dehydrogenase 11.
  • Hybridization techniques include Southern blotting, Nor thern blotting, and in situ hybridization. These techniques and methods are publicly available and mature, and the relevant kits are commercially available. Some 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 known as a "gene chip") for analyzing differential expression analysis and gene diagnosis of genes in tissues.
  • Human glycerol 3 phosphate dehydrogenase 11 specific primers can be used for RNA-polymerase chain reaction (RT-PCR) in vitro amplification to detect human glycerol 3 phosphate dehydrogenase 11 transcription products.
  • Human glycerol 3-phosphate dehydrogenase 11 gene can also be used to diagnose human glycerol 3-phosphate dehydrogenase 11-related diseases.
  • Human glycerol 3 phosphate dehydrogenase 11 mutations include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to normal wild-type human glycerol 3 phosphate dehydrogenase 11 DNA sequences. Mutations can be detected using existing techniques such as Southern blotting, DNA sequence analysis, PCR and in situ hybridization. In addition, mutations may affect protein expression. Therefore, Northern blotting and Western blotting can be used to indirectly determine whether a gene is mutated.
  • sequences of the invention are also valuable for chromosome identification. This sequence will specifically target a specific position on a human chromosome and can hybridize to it. At present, the specificity of each gene on the chromosome needs to be identified Site. Currently, only a few chromosome markers based on actual sequence data (repeating polymorphisms) are available for labeling chromosome positions. According to the present invention, in order to associate these sequences with disease-related genes, an important first step is to locate these DNA sequences on a chromosome.
  • PCR primers (preferably 15-35bp) are prepared based on cDNA, and the sequences can be located on chromosomes. These primers were then used for PCR screening of somatic hybrid cells containing individual human chromosomes. Only those heterozygous cells containing the human gene corresponding to the primer will produce amplified fragments.
  • 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 chromosome-specific cDNA libraries.
  • Fluorescent in situ hybridization of cDNA clones with metaphase chromosomes allows precise chromosomal localization in one step.
  • FISH Fluorescent in situ hybridization
  • 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 the chromosomes, such as deletions or translocations that are visible at the chromosomal level or detectable with cDM 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 present invention also provides a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the present invention.
  • containers there can be medicines manufactured, used or sold by Instructions given by the government regulatory agency for the product or biological product, which reflects the permission of the government regulatory agency for production, use, or sale to be administered to the human body.
  • the polypeptides of the invention can be used in combination with other therapeutic compounds.
  • 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 glycerol 3 phosphate dehydrogenase 11 is administered in an amount effective to treat and / or prevent a specific indication.
  • the amount and range of human glycerol 3 phosphate dehydrogenase 11 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 human fetal brain RNA was extracted by one-step method with guanidine isothiocyanate / phenol / chloroform.
  • Ion Ki t Qiegene Co.
  • Quik mRM Isolat isolated from total RNA poly (A) mRNA 0 2ug poly (A) mRNA is formed by reverse transcription cDNA.
  • the Smart cDNA cloning kit purchased from Clontech i cDNA fragment was inserted into the multicloning site of pBSK (+) vector (Clontech)) to transform DH5 ⁇ to form a cDNA library.
  • Dye terminate cycle react ion sequencing ki t Perkin-Elmer
  • ABI 377 automatic sequencer Perkin-Elmer
  • the determined cDNA sequence was compared with the existing public DM sequence database '(Genebank) By comparison, it was found that the cDNA sequence of one of the clones 0339 (105 was a new DM.
  • a series of primers were synthesized to determine the inserted cDNA fragment in both directions.
  • CDNA was synthesized using fetal brain cell total RNA as a template and ol igo-dT as a primer for reverse transcription reaction. After purification using Qiagene's kit, the following primers were used for PCR amplification: Pr imer 1: 5,-GCCACCACCACTCTCACTCCACCC —3, (SEQ ID NO: 3)
  • Pr imer2 5'- GCAACAGCATAATACATCTGTTTA -3 '(SEQ ID NO: 4)
  • Pr imerl is a forward sequence located at the 5th end of SEQ ID NO: 1, starting at lbp;
  • Pr imer 2 is the 3, terminal reverse sequence of SEQ ID NO: 1.
  • Amplification conditions 50 mmol / L KCl, 10 mmol / L Tris-HCl pH 8.50, 1.5 mmol / L MgCl 2 , 200 ⁇ ol / L dNTP, l Opmol primers in a reaction volume of 50 ⁇ 1 , 1U of 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.
  • ⁇ -act in was set as a positive control and template blank was set 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.
  • DM sequence analysis results showed that the DNA sequence of the PCR product was exactly the same as l-1836bp shown in SEQ ID NO: 1.
  • Example 3 Northern blot analysis of human glycerol 3 phosphate dehydrogenase 11 gene expression Total RNA was extracted in one step [Anal. Biochera 1987, 162, 156-159]. This method involves acid guanidinium thiocyanate phenol-chloroform extraction.
  • the tissue was homogenized with 4M guanidine isothiocyanate-25mM sodium citrate, 0.2M acetic acid ((pH4.0)), and 1 volume of phenol and 1/5 volume of chloroform-isoamyl alcohol (49: 1), mix and centrifuge. Aspirate the aqueous layer, add isopropanol (0.8 vol) and centrifuge the mixture to obtain RNA precipitate. Wash the obtained RNA precipitate with 70% ethanol, dry and dissolve in water. 20 ⁇ RNA , Perform electrophoresis on a 1.2% agarose gel containing 20 mM 3- (N-morpholino) propanesulfonic acid (pH 7.0)-5 mM sodium acetate-ImM EDTA-2.
  • A- 32 P dATP was used to prepare a 32 P-labeled DNA probe by a random primer method.
  • the DNA probe used was the PCR amplified human glycerol 3 phosphate dehydrogenase 11 coding region shown in Figure 1 Sequence (137bp to 445bp).
  • 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% formazan Amide-25mM KH 2 P0 4 (pH7.
  • Pr imer 3 5'- CATGCTAGCATGAGGAGTTTTTATAAGATTGCG -3, (Seq ID No: 5)
  • Pr imer 4 5'- CATGGATCCCTAGGTCCCCCGCCCCTGGCGCCC -3, (Seq ID No: 6)
  • These two primers contain Nhel and BamHI digestion respectively 5 'end of the target gene And the 3 'end coding sequence, the Nhel and BamHI restriction sites correspond to the selective endonuclease sites on the expression vector plasmid pET-28b (+) (Novagen, Cat. No. 69865. 3).
  • PCR was performed using the pBS-0339d05 plasmid containing the full-length target gene as a template.
  • the PCR reaction conditions were as follows: 10 pg of pBS— 0339d05 plasmid, Primer — 3 and Primer — 4 points in a total volume of 50 ⁇ 1, and lpmol, Advantage polymerase Mix (Clontech) 1 ⁇ 1. Cycle parameters: 94. C 20s, 60 ° C 30s, 68 ° C 2 rain, a total of 25 cycles. Nhel and BamHI were used to double digest the amplified product and plasmid pET-28 (+), respectively, and large fragments were recovered and ligated with T4 ligase. The ligation product was transformed into the coliform bacteria DH5 ⁇ by the calcium chloride method.
  • the following peptides specific for human glycerol 3 phosphate dehydrogenase 11 were synthesized using a peptide synthesizer (product of PE company): NH2-Phe-Trp-Lys-Val-Gly-Leu-I le-Ser-Gly-Thr-Val- Phe-Val-I le-Leu-0H (SEQ ID NO: 7).
  • the polypeptide is coupled with hemocyanin and bovine serum albumin to form a complex, respectively.
  • Selecting suitable oligonucleotide fragments from the polynucleotides of the present invention has various uses as hybridization probes, such as using the probes to hybridize to genomic or cDNA libraries of normal tissues or pathological tissues from different sources.
  • the probe may further be used to detect the polynucleotide sequence of the present invention or a homologous polynucleotide sequence thereof in normal tissue or Whether the expression in pathological 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 using a filter hybridization method.
  • Filter hybridization methods include dot blotting, Southern imprinting, Northern blotting, and copying methods. They all use the same steps to immobilize the polynucleotide sample to be tested on the filter.
  • the sample-immobilized filter is first pre-hybridized with a probe-free hybridization buffer to saturate the non-specific binding site of the sample on the filter with the carrier and the synthesized polymer.
  • the pre-hybridization solution is then replaced with a hybridization buffer containing labeled probes and incubated to hybridize the probes to the target nucleic acid.
  • the unhybridized probes are removed by a series of membrane washing steps.
  • This embodiment uses higher-intensity washing conditions (such as lower salt concentration and higher temperature), so that the hybridization background is reduced and only strong specific signals are retained.
  • the probes used in this example include two types: the first type of probes are oligonucleotide fragments that are completely identical or complementary to the polynucleotide SEQ ID NO: 1 of the present invention; the second type of probes are partially The same or complementary oligonucleotide fragment of the polynucleotide SEQ ID NO: 1 of the present invention.
  • 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
  • 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 The regions are compared for homology. If the homology with the non-target molecular region is greater than 85% or there are more than 15 consecutive bases, then the primary probe should not be used;
  • 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 (medulla) of the gene fragment of SEQ ID NO: 1 or its complementary fragment:
  • PBS phosphate buffered saline
  • step 8-13 are only used when contamination must be removed, otherwise step 14 can be performed directly.
  • NC membranes nitrocellulose membranes
  • Two NC membranes are required for each probe, so that they can be used in the following experimental steps.
  • the film was washed with high-strength conditions and strength conditions, respectively.
  • the sample membrane was placed in a plastic bag, and 3 to 10 mg of prehybridization solution (lOxDenhardt-s; 6xSSC, 0.1 lrag / ml CT DNA (calf thymus DNA)) was added. After sealing the bag, shake at 68 ° C for 2 hours.
  • prehybridization solution lOxDenhardt-s; 6xSSC, 0.1 lrag / ml CT DNA (calf thymus DNA)
  • Gene chip or gene microarray is a new technology currently being developed by many national laboratories and large pharmaceutical companies. 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 rapid, efficient, and high-throughput analysis of biological information.
  • the polynucleotide of the present invention can be used as a target DM for gene chip technology for high-throughput research of new gene functions; finding and screening for tissue specificity New genes, especially those related to diseases such as tumors; Diagnosis of diseases, such as hereditary diseases.
  • a total of 4,000 polynucleotide sequences of various full-length cDMs are used as target DMs, including the polynucleotides of the present invention. They were amplified by PCR respectively. After purification, the concentration of the obtained amplified product was adjusted to about 500 ng / ul, and spotted on a glass medium using 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 DM on the glass slide to prepare chips. The specific method steps have been reported in the literature. The post-sampling processing steps of this embodiment are:
  • Total mRM was extracted from human mixed tissue and specific tissues (or stimulated cell lines) in one step, and mRM was purified with Oligotex raRNA Midi Kit (purchased from QiaGen).
  • the fluorescent test J Cy3dUTP (5-Amino-propargyl-2'-deoxyuridine 5'-triphate coupled, to Cy3 f luorescent dye, purchased from Amersham Phamacia Biotech) was used to label the mRNA of human mixed tissue, and the fluorescent reagent Cy5dUTP ( 5-Amino-propargyl-2'-deoxyuridine 5'-triphate coupled to Cy5 fluorescent dye, purchased from Amershara Phamacia Biotech, was used to label the mRNA of specific tissues (or stimulated cell lines) of the body, and probes were prepared after purification.
  • Cy3dUTP 5-Amino-propargyl-2'-deoxyuridine 5'-triphate coupled, to Cy3 f luorescent dye, purchased from Am
  • 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 nouveau polypeptide, une glycéro-3-phosphate déshydrogénase humaine 11, et un polynucléotide codant 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 du diabète de type II, des complications liées au diabète et d'autres types de diabètes. L'invention concerne aussi l'antagoniste agissant contre le polypeptide et son action thérapeutique ainsi que les applications de ce polynucléotide codant la glycéro-3-phosphate déshydrogénase humaine 11.
PCT/CN2001/000766 2000-05-16 2001-05-14 Nouveau polypeptide, glycero-3-phosphate deshydrogenase humaine 11, et polynucleotide codant ce polypeptide WO2001094593A1 (fr)

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CN 00115698 CN1323891A (zh) 2000-05-16 2000-05-16 一种新的多肽——人甘油3磷酸脱氢酶11和编码这种多肽的多核苷酸
CN00115698.5 2000-05-16

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CN110029092B (zh) * 2019-04-29 2020-09-04 江南大学 一种3-磷酸甘油醛脱氢酶及其应用

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Title
DATABASE GENBANK [online] 23 November 1995 (1995-11-23), ARCARI P. ET AL., retrieved from GI:31645 accession no. NCBI Database accession no. CAA25833.1 *
DATABASE GENBANK [online] 9 April 1998 (1998-04-09), HALL L., retrieved from GI:3046742 accession no. NCBI Database accession no. CAA06501.1 *

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