WO2001048168A1 - Nouveau polypeptide, gamma1-pyroline-5-hydroxy reductase 14, et polynucleotide codant pour ce polypeptide - Google Patents

Nouveau polypeptide, gamma1-pyroline-5-hydroxy reductase 14, et polynucleotide codant pour ce polypeptide Download PDF

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Publication number
WO2001048168A1
WO2001048168A1 PCT/CN2000/000695 CN0000695W WO0148168A1 WO 2001048168 A1 WO2001048168 A1 WO 2001048168A1 CN 0000695 W CN0000695 W CN 0000695W WO 0148168 A1 WO0148168 A1 WO 0148168A1
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polynucleotide
polypeptide
pyrroline
hydroxyreductase
sequence
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PCT/CN2000/000695
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English (en)
French (fr)
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Yumin Mao
Yi Xie
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Shanghai Biowindow Gene Development Inc.
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Priority to AU23415/01A priority Critical patent/AU2341501A/en
Publication of WO2001048168A1 publication Critical patent/WO2001048168A1/zh

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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.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0026—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on CH-NH groups of donors (1.5)
    • C12N9/0028—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on CH-NH groups of donors (1.5) with NAD or NADP as acceptor (1.5.1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention belongs to the field of biotechnology. Specifically, the present invention describes a novel polypeptide, gammal-pyrroline-5-hydroxyreductase 14, and a polynucleotide sequence encoding the polypeptide. The invention also relates to a preparation method and application of the polynucleotide and the polypeptide.
  • Proline plays a very important physiological function in the body.
  • Proline residues are post-translationally modified to produce 4-hydroxy-L-proline, which is an important component of certain proteins, especially collagen. It is well known that collagen is involved in many biological processes such as maintaining the strength of bone tendons, blood vessels, cartilage, and skin in the body, and is one of the important components in the body.
  • ⁇ ⁇ -pyrroline-5-hydroxyreductase catalyzes the last step in the biosynthesis process from glutamic acid to proline in vivo, which is NAD (P) -dependent 1-pyrroline-5-hydroxy oxidation Is proline.
  • NAD NAD
  • P -dependent 1-pyrroline-5-hydroxy oxidation Is proline.
  • the abnormal expression of this enzyme will directly affect the synthesis of proline in the organism, and then affect the synthesis of some relevant important proteins, that is, affect some important physiological processes.
  • ⁇ -Pyrroline-5-hydroxyreductase is widely distributed in the biological world, and it exists from eukaryotes, archaea, and eukaryotes.
  • the enzyme has a high sequence similarity in these organisms, and the C-terminus of ⁇ ⁇ -pyrroline-5-hydroxyreductase contains a conserved consensus sequence fragment as follows: [PALF]-X (2, 3) — [LIV] — X (3)-[LI VM]-[STAC]-[STV] -X- [GAN] -GXTX (2)-[AG]-[LIV] -X (2)-[ LMF]-[DENQK]; ⁇ 1 -pyrroline-5-hydroxyreductase from all different sources contains this conserved sequence fragment, which may be the active center of the enzyme, and mutations at this site will lead to the loss of the enzyme It affects the final synthesis of proline and affects a series of related biological processes. This protein is closely related to the
  • gamma l-pyrroline-5-hydroxyreductase 14 protein plays an important role in important functions of the body as described above, and it is believed that a large number of proteins are involved in these regulatory processes, there has been a need in the art to identify more involved in these processes ga makes al-pyrroline-5 -hydroxyreductase 14 protein, especially identifying this protein Amino acid sequence.
  • the isolation of the novel gammal-pyrroline-5-hydroxyreductase 14 protein encoding gene also provides a basis for the study 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.
  • Another object of the invention is to provide a polynucleotide encoding the polypeptide.
  • Another object of the present invention is to provide mimic compounds, antagonists, agonists, and inhibitors directed to the polypeptide of the present invention, gammal-pyrroline-5-hydroxyreductase 14.
  • Another object of the present invention is to provide a method for diagnosing and treating diseases related to the abnormality of gammal-pyrroline-5-hydroxyreductase 14. 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 835-1218 in SEQ ID NO: 1; and (b) a sequence having 1-1629 in SEQ ID NO: 1 Sequence of bits.
  • the invention further relates to a vector, in particular an expression vector, containing a polynucleotide of the invention.
  • the vector genetically engineered host cell includes a transformed, transduced or transfected host cell; a method for preparing a polypeptide of the present invention comprising culturing the host cell and recovering an expression product.
  • the invention also relates to an antibody capable of specifically binding to a polypeptide of the invention.
  • the present invention also relates to a method for screening compounds that mimic, activate, antagonize or inhibit the activity of gammal-pyrroline-5 hydroxyreductase 14 protein, which comprises utilizing the polypeptide of the present invention.
  • the invention also relates to compounds obtained by this method.
  • the present invention also relates to a method for detecting a disease or susceptibility to disease associated with abnormal expression of gammal-pyrroline-5-hydroxyreductase 14 protein in vitro, which comprises detecting the polypeptide in a biological sample or its coding polynucleotide sequence. Mutates, or detects 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 use of the polypeptide and / or polynucleotide of the present invention in the preparation of a medicament for treating cancer, developmental disease or immune disease or other diseases caused by abnormal expression of gammal-pyrroline-5-hydroxyreductase .
  • Figure 1 is a comparison of the amino acid sequence homology of the characteristic proteins of gammal-pyrroline-5-hydroxyreductase 14 of the present invention between 14-102 and a total of 89 amino acids and domains ga cryptoa l-pyrroline-5-hydroxyreductase .
  • the upper sequence is gamma l-pyrroline-5-hydroxyreductase 14, and the lower sequence is the characteristic protein domain of gammal-pyrroline-5-hydroxyreductase.
  • " And ":” and ".” Indicate that the probability of the same amino acid appearing between the two sequences decreases in sequence.
  • Figure 2 is an isolated gammal- reductase pyrroline-5-hydroxy-polyacrylamide gel electrophoresis (SDS-PAGE) 0 14kDa protein having a molecular weight of 14. 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 protein or polynucleotide “variant” 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 substituted amino acid has a structural or chemical property similar to the original amino acid, such as the replacement of 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 means that a change in the amino acid sequence or nucleotide sequence results in an increase in one or more amino acids or nucleotides compared to a molecule that exists in nature.
  • 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 gammal-pyrroline-5 -hydroxyreductase 14, can cause the protein to change, thereby regulating the activity of the protein.
  • An agonist may include a protein, a nucleic acid, a carbohydrate, or any other molecule that can bind to al-pyrroline-5-hydroxyreductase 14.
  • Antagonist refers to a biological activity that can block or regulate gamma l-pyrroline-5-hydroxyreductase 14 when combined with gamma l-pyrroline-5-hydroxyreductase 14. Or immunologically active molecules. Antagonists and inhibitors can include proteins, nucleic acids, carbohydrates, or any other molecule that can bind gamma l-pyrroline-5-hydroxyreductase 14.
  • Regular refers to a change in the function of ga cryptoa l-pyrroline-5-hydroxyreductase 14, including an increase or decrease in protein activity, a change in binding characteristics, and gamma l-pyrroline-5-hydroxyreductase 14 Of any other biological, functional or immune properties.
  • substantially pure means substantially free of other proteins, lipids, sugars or other substances with which it is naturally associated.
  • Those skilled in the art can purify garama l-pyrroline-5 -hydroxyreductase 14 using standard protein purification techniques.
  • the substantially pure gammal-pyrroline-5-hydroxyreductase 14 produces a single main band on a non-reducing polyacrylamide gel.
  • the purity of garama l-pyrroline-5-hydroxyreductase 14 polypeptide can be analyzed by amino acid sequence.
  • “Complementary” or “complementary” refers to polynucleotides that naturally bind through base-pairing under conditions of acceptable salt concentration and temperature.
  • the sequence "C-T-G-A” can be combined with the complementary sequence "GA-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” refers to the degree of complementarity and can be partially homologous or completely homologous.
  • 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 blotting or Nor thern blotting, etc.) under conditions of reduced stringency.
  • Substantially homologous sequences or hybridization probes can compete and inhibit the binding of completely 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 refers to the percentage of sequences that are identical or similar in the comparison of two or more amino acid or nucleic acid sequences. Electronic methods can be used to determine the percentage of identity, such as through the MEGALIGN program (Lasergene sof tware package, DNASTAR, Inc., Madi son Wis.) 0 MEGALIGN program can compare two or more according to different methods, such as the C lus ter method Sequence (Hi gg ins, DG and PM Sharp (1988) Gene 73: 237-244). The C l uster method arranges each group of sequences into clusters by checking the distance between all pairs. 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 number of matching residues between the sequence and the sequence S
  • the number of residues in the sequence-the number of spacer residues in the sequence-the number of spacer residues in the sequence ⁇ can also be determined by Cluster method or using methods known in the art such as Jotun He in. in J., (1990) Methods in enzymo logy 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 substitutions for example, negatively charged amino acids may include aspartic acid and glutamic acid; positively charged amino acids may include lysine and arginine; 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 DM or RNA sequence.
  • the "antisense strand” refers to a nucleic acid strand that is complementary to the “sense strand”.
  • Derivative refers to HFP or a chemical modification of its nucleic acid. This chemical modification may be the replacement of a hydrogen atom with an alkyl, acyl or amino group. Nucleic acid derivatives can encode to retain the main biological properties of natural molecules Of peptides.
  • Antibody refers to a complete antibody molecule and its fragments, such as Fa,? ( ⁇ ') 2 and?, Which specifically bind to the epitope of gammal-pyrroline-5-hydroxyreductase 14.
  • 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 matter 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 animal, but the same polynucleotide or polypeptide is separated from some or all of the substances that coexist in the natural system.
  • Such a polynucleotide may be part of a vector, or such a polynucleotide or polypeptide may be part of a 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).
  • polynucleotides and polypeptides in a natural state in a living cell are not isolated and purified, but the same polynucleotides or polypeptides are separated and purified if they are separated from other substances existing in the natural state. .
  • isolated gammal-pyrroline-5 hydroxyreductase 14 means that gammal-pyrroline-5 hydroxyreductase 14 is substantially free of other proteins, lipids, carbohydrates or others that are naturally associated with it. substance. Those skilled in the art can purify gamraa l-pyrroline-5-hydroxyreductase 14 using standard protein purification techniques. Substantially pure polypeptides can produce a single main band on a non-reducing polyacrylamide gel. The purity of the gamma l-pyrroline-5-hydroxyreductase I 4 polypeptide can be analyzed by amino acid sequence.
  • the present invention provides a new polypeptide, al-pyrroline-5-hydroxy reductase 14, which basically consists 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 present invention can be naturally purified products or chemically synthesized products, or can be produced from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insects, and mammalian cells) using recombinant techniques. 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 invention also includes fragments, derivatives and analogs of gamraal-pyrroline-5-hydroxyreductase 14.
  • fragment refers to a polypeptide that substantially maintains the same biological function or activity of al-pyrroline-5-hydroxyreductase 14 of the present invention.
  • a fragment, derivative or analog of the polypeptide of the present invention may be: (I) a kind in which one or more amino acid residues are substituted with conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and the substitution Amino acids can Therefore, it may or may not be encoded by the genetic code; or ( ⁇ ) such a type in which a group on one or more amino acid residues is substituted by another group to include a substituent; or (III) such a type Wherein the mature polypeptide is fused with another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol); or (IV) a polypeptide sequence in which an additional amino acid sequence is fused into a mature polypeptide
  • 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 a nucleotide sequence of SEQ ID NO: 1.
  • the polynucleotide of the present invention is found from a cDNA library of human fetal brain tissue. It contains a total nucleotide sequence of 1629 bases, and its open reading frame 835-1218 encodes 127 amino acids.
  • This polypeptide has the characteristic sequence of the characteristic protein of gammal-pyrroline-5-hydroxyreductase. It can be deduced that the characteristic protein of gammal-pyrroline-5-hydroxyreductase 14 has the characteristic Structure and function.
  • the polynucleotide of the present invention may be in the form of DM or RNA.
  • DNA forms include cDNA, genomic DNA or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be coding or non-coding.
  • 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 that includes the polypeptide and a polynucleotide that includes 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.
  • This polynucleotide variant can be a naturally occurring allelic variant or a non-naturally occurring variant.
  • 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 701 ⁇ 2 identity, between the two sequences).
  • the invention particularly relates to polynucleosides according to the invention under stringent conditions Acid hybridizable polynucleotide.
  • “strict conditions” means: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2xSSC, 0.1% SDS, 6 (TC; or (2) added during hybridization) Use a denaturant, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42 ° C, etc .; or (3) the identity between the two sequences is at least 95% Above, it is more preferable that the hybridization occurs at 97% or more.
  • 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 gammal-pyrroline-5-hydroxyreductase 14.
  • 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 the gammal-pyrroline-5-hydroxyreductase 14 of the present invention can be obtained by various methods.
  • polynucleotides are isolated using hybridization techniques well known in the art. These techniques include, but are not limited to: 1) hybridization of probes to genomic or cDNA libraries to detect homologous polynucleotide sequences, and 2) antibody screening of expression libraries to detect cloned polynucleosides with common structural characteristics Acid fragments.
  • the DM fragment sequence of the present invention can also be obtained by the following methods: 1) isolating the double-stranded DNA sequence from the genomic DNA; 2) chemically synthesizing the DNA sequence to obtain the double-stranded DNA of the polypeptide.
  • genomic DNA isolation 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 mRNA from donor cells that overexpress the gene and perform reverse transcription to form a plasmid or phage cDNA library.
  • mRNA extraction There are many mature techniques for mRNA extraction, and kits are also commercially available (Qiagene).
  • the construction of cDNA libraries is also a common method (Sambrook, et al., Molecular Cloning, A Laboratory Manual, 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.
  • 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) determination of the transcript of gammal-pyrroline-5-hydroxyreductase 14 (4) Detecting the protein product of gene expression by immunological techniques or measuring biological activity. The above methods can be used alone 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 usually 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.
  • DM 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 gammal-pyrroline-5-hydroxyreductase 14 gene.
  • ELISA enzyme-linked immunosorbent assay
  • a method for amplifying DNA / RNA using PCR technology is preferably used to obtain the gene of the present invention.
  • the RACE method RACE-rapid cDNA end rapid amplification method
  • 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 measured by a conventional method such as dideoxy chain termination method (Sanger et al. PNAS, 1977, 74: 5463-5467). Such polynucleotide sequences can also be determined using commercial sequencing kits and the like. In order to obtain the full-length cDNA sequence, sequencing needs to be repeated. Sometimes it is necessary to determine the cDNA sequence of multiple clones in order to splice into a full-length cDNA sequence.
  • 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 garamal-pyrroline-5-hydroxyreductase 14 coding sequence, and the recombinant technology to produce the present invention.
  • a method of inventing the polypeptide is not limited 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 garamal-pyrroline-5-hydroxyreductase 14 coding sequence, and the recombinant technology to produce the present invention.
  • a polynucleotide sequence encoding gammal-pyrroline-5-hydroxyreductase 14 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 recombinant expression vectors.
  • An important feature of expression vectors is that they usually contain an origin of replication, a promoter, a marker gene, and translational regulatory elements.
  • the expression vector also includes a ribosome binding site and a transcription terminator for translation initiation. Insertion of enhancer sequences into the vector will enhance its transcription in higher eukaryotic cells. Enhancers are cis-acting factors for DNA expression, usually about 10 to 300 base pairs, which act on promoters to enhance gene transcription. Illustrative examples include SV40 enhancers of 100 to 270 base pairs on the late side of the origin of replication, polyoma enhancers on the late side of the origin of replication, and adenoviral enhancers.
  • 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.
  • a polynucleotide encoding gammal-pyrroline-5-hydroxyreductase 14 or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to constitute a genetic engineering containing the polynucleotide or the recombinant vector.
  • Host cells 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.
  • Escherichia coli, Streptomyces bacterial cells such as Salmonella typhimurium
  • fungal cells such as yeast
  • plant cells insect cells
  • fly S2 or Sf9 animal cells
  • animal cells such as CH0, COS or Bowes melanoma cells.
  • Transformation of a host cell with a DNA sequence described in the present invention or a recombinant vector containing the DNA sequence can be performed using 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 may be in exponential growth phase were harvested after the treatment with (Method 12, using the procedure well known in the art. Alternatively, it is a MgCl 2. If If necessary, transformation can also be performed by electroporation.
  • the host is a eukaryotic organism, the following DM transfection methods can be used: calcium phosphate co-precipitation method, or conventional mechanical methods such as microinjection, electroporation, and liposome packaging Wait.
  • the polynucleotide sequence of the present invention can be used to express or produce recombinant gammal-pyrroline-5-hydroxyreductase 14 (Science, 1984; 224: 1431). Average 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, 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
  • polypeptides of the present invention can be directly used in the treatment of diseases, for example, they can be used to treat malignant tumors, adrenal deficiency, skin diseases, various types of inflammation, HIV infection, and immunological diseases.
  • Proline plays a very important physiological function in the body. Proline residues are modified after translation to produce 4-hydroxy-L-proline, which is an important component of certain proteins, especially collagen. Collagen is involved in many biological processes such as maintaining the strength of tendons, blood vessels, cartilage, and skin, as well as tissue fibrosis and inflammation repair.
  • ⁇ ⁇ -pyrroline-5-hydroxy reductase (P5CR) catalyzes the last step in the biosynthesis process from glutamic acid to proline in the body, which directly affects the synthesis of proline in the body, and then affects some related Synthesis of important proteins.
  • the ⁇ 1-pyrroline-5-hydroxyreductase-specific conserved sequence is required to form its active raot if. It can be seen that the abnormal expression of the specific ⁇ ⁇ -pyrroline-5-hydroxyreductase mot if will cause the function of the polypeptide containing the mot if of the present invention to be abnormal, thereby leading to abnormal glutamate metabolism and proline metabolism. Abnormal, abnormal metabolism of proline makes collagen synthesis abnormal. Because collagen is of great significance in the process of tissue fibrosis and inflammatory repair, the abnormal expression of this polypeptide can cause fibrosis in some tissues, which can cause abnormal inflammatory repair process, which can lead to excessive scarring, narrowing of the cavity, and unhealed wounds. And other phenomena.
  • abnormal expression of gamma l-pyrroline-5-hydroxyreductase I 4 of the present invention will produce Various diseases, especially amino acid metabolism deficiency diseases, organic acidemia, tissue fiber pathology, and diseases related to abnormal inflammation and repair, these diseases include, but are not limited to:
  • Amino acid metabolism deficiency diseases glutamate metabolism deficiency disease, proline and hydroxyproline metabolism deficiency disease, phenylketonuria, tyrosine metabolism deficiency disease, tryptophan metabolism deficiency disease, branch amino acid metabolism deficiency disease, Glycine metabolism deficiency disease, Proline and hydroxyproline metabolism deficiency disease, Metabolism deficiency disease of urea cycle, Histidine metabolism deficiency disease, Lysine metabolism deficiency disease
  • Tissue fibropathology fibroids, nodular fasciitis, proliferative fasciitis, myofibromatosis, fibrosarcoma, fibrous histiocytoma, reticular histiocytoma, fibromatosis such as keloids, diffuse interstitial lung Qualitative disorders, selenium lung, cirrhosis, benign prostatic hyperplasia
  • stenosis of various tissue channels such as pyloric stenosis, tracheal stenosis after injury, mitral valve stenosis, aortic stenosis, pulmonary artery stenosis, constrictive pericarditis, pancreatic cystic fibrosis, pyelonephritis
  • Organic acidemia propionic acidemia, methylmalonic aciduria, isovalerate, combined carboxylase deficiency, glutarate type I
  • Abnormal expression of gamma l-pyrroline-5-hydroxyreductase 14 of the present invention may also cause certain genetic diseases and diseases of the immune system.
  • polypeptide of the present invention and the antagonists, agonists and inhibitors of the polypeptide can be directly used in the treatment of diseases, for example, it can treat various diseases, especially amino acid metabolism deficiency disease, organic acidemia, tissue fiber pathology, and inflammatory repair abnormalities. Related diseases, certain genetic diseases and immune system diseases.
  • the invention also provides methods for screening compounds to identify agents that increase (agonist) or inhibit (antagonist) gamma 1 -pyrroline-5-hydroxyreductase 1 4.
  • Agonists increase gamma l-pyrroline-5-hydroxyreductase 14 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 gamma l-pyrroline-5-hydroxyreductase 14 can be cultured with labeled gamma l-pyrroline-5-hydroxyreductase 14 in the presence of a drug. The ability of the drug to increase or block this interaction is then determined.
  • Antagonists of gamma l-pyrroline-5-hydroxyreductase 14 include antibodies, compounds, receptor deletions, and the like that have been screened. Antagonists of gamma l-pyrroline-5-hydroxyreductase 14 can bind to gamma l-pyrroline-5-hydroxyreductase 14 and eliminate its function, or inhibit the production of the polypeptide, or the activity of the polypeptide Site binding prevents the polypeptide from performing its biological function.
  • gamma l-pyrroline-5-hydroxyreductase 14 can be added to the bioanalytical assay. The effect of this interaction is used to determine whether the compound is an antagonist. Screening compounds using the above In the same way, receptor deletions and analogs that can act as antagonists can be screened.
  • Peptide molecules capable of binding to gammal-pyrroline-5-hydroxyreductase 14 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. During screening, gammal-pyrroline-5-hydroxyreductase 14 molecules 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 against the gamma l-pyrroline-5-hydroxyreductase 14 epitope. These antibodies include (but are not limited to): polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, and fragments produced by Fab expression libraries.
  • Polyclonal antibodies can be produced by injecting gamma l-pyrroline-5-hydroxyreductase 14 directly into immunized animals (such as rabbits, mice, rats, etc.).
  • immunized animals such as rabbits, mice, rats, etc.
  • a variety of adjuvants can be used to enhance the immune response, including It is not limited to Freund's adjuvant and the like.
  • Techniques for preparing monoclonal antibodies to garamal-pyrroline-5-hydroxyreductase 14 include, but are not limited to, hybridoma technology (Kohler and Miste in. Nature, 1975, 256: 495-497), triple tumor technology , Human B-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 U.S. Pat No. 4946778, can also be used to produce single chain antibodies against gammal-pyrroline-5-hydroxyreductase 14.
  • Anti-gamma l-pyrroline-5-hydroxyreductase 14 antibodies can be used in immunohistochemistry to detect garamal-pyrroline-5-hydroxyreductase 14 in biopsy specimens.
  • Monoclonal antibodies that bind to gammal-pyrroline-5-hydroxyreductase I 4 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.
  • gammal-pyrroline-5hydroxyreductase 14 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 crosslinker such as SPDP and bind the toxin to the antibody through the exchange of disulfide bonds.
  • This hybrid antibody can be used to kill the reduction of gammal-pyrroline-5-hydroxy Enzyme 14 positive cells.
  • the antibodies of the present invention can be used to treat or prevent diseases related to garamal-pyrroline-5-hydroxyreductase 14. Administration of an appropriate dose of the antibody can stimulate or block the production or activity of gamma l-pyrroline-5-hydroxyreductase 14.
  • the invention also relates to a diagnostic test method for quantitative and localized detection of gammal-pyrroline-5-hydroxyreductase 14 levels.
  • tests are well known in the art and include FI SH assays and radioimmunoassays.
  • the levels of gamma l-pyrroline-5-hydroxyreductase 14 detected in the test can be used to explain the importance of gatnma l-pyrroline-5-hydroxyreductase 14 in various diseases and to diagnose gamma l- Diseases in which pyrroline-5-hydroxyreductase 14 functions.
  • 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.
  • Polynucleotides encoding gamraa l-pyrroline-5-hydroxyreductase 14 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 absence or abnormal / inactive expression of gamma l-pyrroline-5-hydroxyreductase M.
  • Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated gamma l-pyrroline-5-hydroxyreductase 14 to inhibit endogenous gamma l-pyrroline-5-hydroxyreductase 14 activity.
  • a mutated gamma l-pyrroline-5 hydroxyreductase 14 may be a shortened gamraa l-pyrroline-5 hydroxyreductase 14 that lacks a signaling domain, although it can interact with downstream substrates. Binding, but lacks signaling activity. Therefore, the recombinant gene therapy vector can be used to treat diseases caused by abnormal expression or activity of gamma l-pyrroline-5-hydroxyreductase 14.
  • Virus-derived expression vectors such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus, etc.
  • polynucleotide of enzyme 14 can be used to transfer a polynucleotide encoding gamma l-pyrroline-5-hydroxyreductase 14 into cells .
  • a method for constructing a recombinant viral vector carrying a polynucleotide encoding gamma l-pyrroline-5-hydroxyreductase 14 can be found in the existing literature (Sambrook, etal.). 0 Another recombinant encoding gamma l-pyrroline-5-hydroxy reduction
  • the polynucleotide of enzyme 14 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 RNA and DM
  • ribozymes that inhibit gamma l-pyrroline-5-hydroxyreductase 14 mRNA are also within the scope of the present invention.
  • a ribozyme is an enzyme-like RNA molecule that specifically decomposes specific RNA. Its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target RNA for endonucleation.
  • Antisense RNA, DNA, and ribozymes can be obtained by any existing RNA or DNA synthesis technology, such as the technology for the synthesis of oligonucleotides by solid-phase phosphoramidite chemical synthesis has been widely used.
  • Antisense RNA molecules can be obtained by in vitro or in vivo transcription of a DNA sequence encoding the RNA. This DNA sequence has been integrated downstream of the vector's RNA polymerase promoter. 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 polynucleotide encoding gamma l-pyrroline-5-hydroxyreductase 14 can be used for the diagnosis of diseases related to gammal-pyrroline-5-hydroxyreductase 14.
  • the polynucleotide encoding gammal-pyrroline-5-hydroxyreductase 14 can be used to detect the expression of gamma l-pyrroline-5-hydroxyreductase 14 or the reduction of gamma l-pyrroline-5-hydroxy in disease states Abnormal expression of enzyme 14.
  • the DNA sequence encoding gamma l-pyrroliline-5 hydroxyreductase 14 can be used to hybridize biopsy specimens to determine the expression of gammal-pyrrolinline 5-hydroxyreductase 14.
  • Hybridization techniques include Southern blotting, Nor thern blotting, and in situ hybridization. These techniques and methods are publicly available and mature, and related kits are commercially available.
  • a part or all of the polynucleotides of the present invention can be used as probes to be fixed on a micro array or a DNA chip (also called a "gene chip") for analyzing differential expression analysis of genes and genetic diagnosis in tissues.
  • Gamraal-pyrroline-5-hydroxyreductase 14 specific primers for RNA-polymerase chain reaction (RT-PCR) in vitro amplification can also detect the gammal-pyrroline-5-hydroxyreductase transcription products.
  • Detecting mutations in the gamma l-pyrroline-5-hydroxyreductase 14 gene can also be used to diagnose gammal-pyrroline-5-hydroxyreductase 14-related diseases.
  • the forms of gammal-pyrroline-5-hydroxyreductase 14 mutations include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to the normal wild-type gamma l-pyrroline-5-hydroxyreductase 14 DNA sequence. . Mutations can be detected using existing techniques such as Southern imprinting, DM sequence analysis, PCR and in situ hybridization. In addition, mutations may affect protein expression. Therefore, Nor thern 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. Currently, specific sites for each gene on the chromosome need to be identified. Currently, only a few chromosome markers based on actual sequence data (repeating polymorphisms) are available for marking 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 according to cDM, 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 (FISH) of cDNA clones with metaphase chromosomes can be refined in one step Perform chromosomal mapping accurately.
  • 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 individual, the mutation may be the cause of the disease. Comparing affected and unaffected individuals usually involves first looking for structural changes in the chromosome, 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 that 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.
  • a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the present invention.
  • these containers there may be instructional instructions given by government agencies that manufacture, use, or sell pharmaceuticals or biological products, which reminders permit their administration on the human body by government agencies that produce, use, or sell them.
  • 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.
  • gammal-pyrroline-5-hydroxyreductase 14 is administered in an amount effective to treat and / or prevent a specific indication.
  • the amount and range of gammal-pyrroline-5-hydroxyreductase 14 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
  • 2ug poly (A) mRNA is reverse transcribed to form cDNA.
  • a Smart cDNA cloning kit purchased from Clontech was used to insert the cDNA fragment into the multicloning site of the pBSK (+) vector (Clontech) to transform DH5 ⁇ to form a cDNA library.
  • Dye terminate cycle react ion sequencing kit Perkin-Elmer
  • ABI 377 automatic sequencer Perkin-Elmer
  • the sequence of the gamraal-pyrroline-5-hydroxyreductase 14 of the present invention and its encoded protein sequence ⁇ 1 J were used in a profile scan program (Basic local al ignment search tool) in GCG [Altschul, SF et al. J Mol. Biol. 1990; 215: 403-10], domain analysis was performed in databases such as prosite.
  • This hair The garanal-pyrroline-5-hydroxyreductase 14 shown in Figure 14 has homology with the characteristic protein of the domain gammal-pyrroline-5-hydroxyreductase at 14-102. The results of the homology are shown in Figure 1.
  • the homology rate is 6%.
  • the score is 3.83; the threshold is 3.77.
  • Example 3 Cloning of the gene encoding gammal-pyrroline-5-hydroxyreductase 14 by RT-PCR method.
  • Total RNA from fetal brain cells was used as a template, and oligo-dT was used as a primer for reverse transcription reaction to synthesize cDNA.
  • a Qiagene kit was used. After purification, PCR amplification was performed with the following primers:
  • Primerl 5'- CCTGATATATCCATTTTGCTTCCC —3 '(SEQ ID NO: 3)
  • Primer2 5'- ATTTTCAAACTTTATTTACAACTG -3 '(SEQ ID NO: 4)
  • Primerl is a forward sequence starting at lbp at the 5 ′ end of SEQ ID NO: 1;
  • Primer2 is the 3 'end reverse sequence in SEQ ID NO: 1.
  • Amplification reaction conditions 50 ⁇ l / L KC1, 10 ⁇ l / L Tris-HC1, ⁇ 8.5, 1.5 mmol / L MgCl 2 , 200 ⁇ 1 / ⁇ dNTP, lOpmol primer, 1U in a reaction volume of 50 ⁇ 1 Taq DM polymerase (Clontech).
  • the reaction was performed on a PE9600 DNA thermal cycler (Perkin-Elmer) under the following conditions for 25 cycles: 94 ° C 30sec; 55 ° C 30sec; 72 ° C 2min.
  • RT-PCR set ⁇ -act in as a positive control and template blank as a negative control.
  • the amplified product was purified using a QIAGEN kit, and ligated to a pCR vector (Invitrogen product) using a TA cloning kit.
  • the DNA sequence analysis results showed that the DM sequence of the PCR product was exactly the same as the 1- 1629bp shown in SEQ ID NO: 1.
  • Example 4 Analysis of the expression of gammal-pyrroline-5-hydroxyreductase 14 gene by Northern blotting method Total RNA was extracted in one step [Anal. Biochem 1987, 162, 156-159] 0 This method includes acid guanidinium thiocyanate- Chloroform extraction.
  • the tissue is homogenized with 4M guanidine isothiocyanate-25mM sodium citrate, 0.2M sodium acetate (pH4.0), and 1 time 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. The resulting RNA pellet was washed with 70% ethanol, dried and dissolved in water.
  • Electrophoresis was performed on a 1.2% agarose gel containing 2 g of RNA on 20 mM 3- (N-morpholino) propanesulfonic acid (pH 7.0)-5 mM sodium acetate-1 mM EDTA-2.2M formaldehyde. Then transferred to nitrocellulose.
  • cc- 32 P dATP with 32 P- DNA probes prepared by random primer labeled SYSTEM.
  • the DNA probes used for PCR amplification shown in FIG gammal- pyrroline-5 Hydroxyreductase 14 coding region sequence (835bp to 1218bp).
  • 32P-labeled probe (about 2 x 10 6 cpm / ml) was hybridized with a nitrocellulose membrane to which RNA was transferred in a solution at 42 ° C overnight, This solution contains 50% formamide-25mM KH 2 P0 4 ( ⁇ 7 ⁇ 4)-5 xSSC- 5 x Denhardt, s solution and 200 ⁇ ⁇ / ⁇ 1 salmon sperm DNA. After hybridization, the filter is placed at 1 X SSC- 0.1% SDS Wash at 55 ° C for 30 min. Then, analyze and quantify with Phosphor Imager.
  • Example 5 In vitro expression, isolation and purification of recombinant gammal-pyrroline-5-hydroxyreductase 14 Based on the sequence of the coding region shown in SEQ ID NO: 1 and Figure 1, a pair of specific amplification primers were designed, the sequence is as follows :
  • Primer3 5'- CCCCATATGATGCCGGGGCAGGAGGACGAGGGT -3 '(Seq ID No: 5)
  • Primer4 5'- CATGGATCCTTACAAGCATGCTCCCATCACACA -3' (Seq ID No: 6)
  • These two primers contain Ndel and BamHI restriction sites, respectively.
  • the coding sequences of the 5 'and 3' ends of the gene of interest are followed, respectively.
  • the Ndel and BamHI restriction sites correspond to the selectivity within the expression vector plasmid pET-28b (+) (Novagen, Cat. No. 69865.3). Digestion site.
  • the PCR reaction was performed using the pBS-0217e08 plasmid containing the full-length target gene as a template.
  • the PCR reaction conditions were as follows: a total volume of 50 ⁇ 1 containing 10 pg of pBS — 0217e08 plasmid, 10 ⁇ mol of primer Primer 3 and Primer 4 respectively, and 1 ⁇ 1 of Advantage polymerase Mix (Clontech). Cycle parameters: 94 ° C 20s, 60 ° C 30s, 68 ° C 2 min, a total of 25 cycles. Nde I and BamH I 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 DH5cx by the calcium chloride method. After being cultured overnight on LB plates containing kanamycin (final concentration 3 ( ⁇ g / ml)), positive clones were selected by colony PCR method and sequenced. The correct positive clone (pET-0217e08) was used to transform the recombinant plasmid into E. coli BL21 (DE3) plySs (product of Novagen) by calcium chloride method.
  • the polypeptide is coupled with hemocyanin and bovine serum albumin to form a complex, respectively.
  • hemocyanin and bovine serum albumin For methods, see: Avrameas, et al. Imnumochemistry, 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.
  • 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 imprinting, Nor thern blotting, and copying methods. They all use the same steps to fix the polynucleotide sample to be tested on the filter and then hybridize.
  • 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) 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 for use as hybridization probes from the polynucleotide SEQ ID NO: 1 of the present invention 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 probes, and then further computer sequence analysis, including Including the primary selected probe with its source sequence region (ie, SEQ ID NO: 1) and other known genomic sequences and their complementary regions, if the homology with non-target molecular region is greater than 85% Or if more than 15 consecutive bases are identical, the primary probe should not be used in general;
  • SEQ ID NO: 1 source sequence region
  • other known genomic sequences and their complementary regions if the homology with non-target molecular region is greater than 85% Or if more than 15 consecutive bases are identical, the primary probe should not be used in general;
  • Probe 1 (probel), which belongs to the first type of probe, is completely homologous to or homologous 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 membranes nitrocellulose membranes
  • Two NC membranes are required for each probe for subsequent experiments.
  • the film is washed with high-strength conditions and strength conditions, respectively.
  • the sample membrane was placed in a plastic bag, and 3-10 mg of prehybridization solution (lOxDenhardt's; 6xSSC, 0.1 mg / ml CT DNA (calf thymus DNA)) was added. After closing the bag, 68. C water bath for 2 hours.
  • prehybridization solution lOxDenhardt's; 6xSSC, 0.1 mg / ml CT DNA (calf thymus DNA)
  • 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 large numbers 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 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 methods and steps have been reported in the literature. For example, see DeRisi, J. L., Lyer, V. & Brown, P.0.
  • a total of 4,000 polynucleotide sequences of various full-length cDNAs are used as target DNA, including the polynucleotide of the present invention. They were amplified by PCR respectively. After purification, the concentration of the amplified product was adjusted to about 500 ng / ul, and spotted on a glass medium with a Cartesian 7500 spotting instrument (purchased from Cartesian, USA). The distance is 280 ⁇ . The spotted slides were hydrated and dried, cross-linked in a UV cross-linker, and dried after elution to fix the DNA on the glass slides to prepare chips. The specific method steps have been reported in the literature. The sample post-processing steps in this embodiment are:
  • Total mRNA was extracted from normal liver and liver cancer by one-step method, and mRNA was purified with Oligotex mRNAMidi Kit (purchased from QiaGen).
  • the fluorescent reagent Cy3dUTP (5- Amino- propargy 1-2 '-deoxyur i dine) was separately reverse-transcribed. 5'-tr iphate coupled to Cy3 fluorescent dye, purchased (Amersham Phamacia Biotsch company) labeled mRNA of normal liver tissue, Cy5dUTP (5-Amino-propargyl-2'-deoxyur idine 5'-triphate coupled to Cy5 fluorescent dye, purchased from Amersham Phamacia Biotech) labeled liver cancer mRNA The probe was prepared after purification. For specific steps and methods, see
  • Probes from the two types of tissues and the chip were hybridized in a UniHyb TM Hybridization Solution (purchased from TeleChem) hybridization solution for 16 hours, washed with a washing solution (1 x SSC, 0.2% SDS) at room temperature, and then scanned with ScanArray 3000.
  • Scanner purchased from General Scanning Company, USA
  • the scanned image is processed with Imagene software (Biodiscovery Company, USA) for data analysis, and the Cy3 / Cy5 ratio of each point is calculated.
  • the points whose ratio is less than 0.5 and greater than 2 are considered Genes with differential expression.

Description

一种新的多肽—— gammal- 咯啉 -5 -羟某还原酶 14和编码这种多肽的多核苷酸 抟术领域
本发明属于生物技术领域， 具体地说，本发明描述了一种新的多肽一 gammal - 吡咯啉 -5-羟基还原酶 14 , 以及编码此多肽的多核苷酸序列。 本发明还涉及此多核 苷酸和多肽的制备方法和应用。 技术背景
在生物的氨基酸代谢及合成过程中， 谷氨酸代谢中的碳和氮被用于脯氨酸及 精氨酸的生物合成。 脯氨酸在生物体内起着十分重要的生理学功能， 脯氨酸残基 经翻译后修饰生成 4-羟基- L-脯氨酸， 该氨基酸为某些蛋白尤其是胶原蛋白的重要 组分。 而众所周知， 胶原蛋白在生物体内涉及维持骨腱、 血管、 软骨和皮肤强度 等众多的生物学过程， 是生物体中的重要组成成分之一。
δ ΐ-吡咯啉 -5-羟基还原酶 （P5CR ) 在生物体内催化从谷氨酸到脯氨酸的生物 合成过程的最后一步， 即 NAD ( Ρ ) 依赖的 1-吡咯啉 -5-羟基氧化为脯氨酸。 该酶 的表达异常将直接影响生物体内脯氨酸的合成， 进而影响一些相关的重要蛋白质 的合成， 即影响一些重要的生理过程。
1990 年， De l auney A. J.等人从大肠杆菌中克隆得到了一 δ 1-吡咯啉 -5-羟基 还原酶， 该酶的序列与其它来源的 δ ΐ-吡咯啉 -5-羟基还原酶亦有较高的同源性， 研究发现，该酶在生物体内可能通过渗透调节来调节其表达 [De launey A. J. ， Verma D. P. , Mo l Gen Gene t, 221: 299-305]„
δ ΐ-吡咯啉 -5-羟基还原酶在生物界的分布非常广泛， 从真细菌、 古细菌到真 核生物均存在该酶。 该酶在这些生物体内均有着较高的序列相似性， δ ΐ-吡咯啉- 5-羟基还原酶的 C末端均含有如下所示的保守的一致性序列片段： [PALF] - X ( 2 , 3 ) — [LIV]— X ( 3 ) - [LI VM] - [STAC] - [STV] -X- [GAN] -G-X-T-X ( 2 ) - [AG] - [LIV] -X ( 2 ) - [LMF] - [DENQK]； 所有不同来源的 δ 1 -吡咯啉- 5-羟基还原酶均含有这一保 守的序列片段， 该序列片段可能是酶活性中心， 该位点的突变将导致酶的失活， 从而影响脯氨酸的最终合成及影响一系列相关的生物学过程。 该蛋白在生物体内 与一些脯氨酸代谢紊乱相关的疾病、 胶原蛋白相关疾病的发生密切相关。
由于如上所述 gamma l-吡咯啉 -5-羟基还原酶 14 蛋白在机体重要功能中起重 要作用， 而且相信这些调节过程中涉及大量的蛋白， 因而本领域中一直需要鉴定 更多参与这些过程的 ga讓 al-吡咯啉 -5 -羟基还原酶 14蛋白， 特别是鉴定这种蛋白 的氨基酸序列。 新 gammal-吡咯啉 -5-羟基还原酶 14蛋白编码基因的分离也为研究 确定该蛋白在健康和疾病状态下的作用提供了基础。 这种蛋白可能构成开发疾病 诊断和 /或治疗药的基础， 因此分离其编码 DNA是非常重要的。 发明目的
本发明的一个目的是提供分离的新的多肽—— gammal-吡咯啉 -5-羟基还原酶 14以及其片段、 类似物和衍生物。
本发明的另一个目的是提供编码该多肽的多核苷酸。
本发明的另一个目的是提供含有编码 ga隱 al-吡咯啉 -5-羟基还原酶 14 的多 核苷酸的重组载体。
本发明的另一个目的是提供含有编码 gammal-吡咯啉 -5-羟基还原酶 14 的多 核苷酸的基因工程化宿主细胞。
本发明的另一个目的是提供生产 gammal-吡咯啉 -5-羟基还原酶 14的方法。 本发明的另一个目的是提供针对本发明的多肽—— ga隱 al-吡咯啉 - 5-羟基还原 酶 14的抗体。
本发明的另一个目的是提供了针对本发明多肽—— gammal-吡咯啉 -5-羟基还原 酶 14的模拟化合物、 拮抗剂、 激动剂、 抑制剂。
本发明的另一个目的是提供诊断治疗与 gammal-吡咯啉 -5-羟基还原酶 14 异 常相关的疾病的方法。 发明概要
本发明涉及一种分离的多肽， 该多肽是人源的， 它包含： 具有 SEQ ID No. 2 氨基酸序列的多肽、 或其保守性变体、 生物活性片段或衍生物。 较佳地， 该多肽 是具有 SEQ ID NO: 2氨基酸序列的多肽。
本发明还涉及一种分离的多核苷酸， 它包含选自下组的一种核苷酸序列或其 变体：
(a)编码具有 SEQ ID No. 2氨基酸序列的多肽的多核苷酸；
(b)与多核苷酸（a)互补的多核苷酸；
(c)与（a)或（b)的多核苷酸序列具有至少 70¼相同性的多核苷酸。
更佳地， 该多核苷酸的序列是选自下组的一种： （a)具有 SEQ ID NO: 1 中 835-1218位的序列； 和（b)具有 SEQ ID NO: 1中 1-1629位的序列。
本发明另外涉及一种含有本发明多核苷酸的载体， 特别是表达载体； 一种用 该载体遗传工程化的宿主细胞， 包括转化、 转导或转染的宿主细胞； 一种包括培 养所述宿主细胞和回收表达产物的制备本发明多肽的方法。
本发明还涉及一种能与本发明多肽特异性结合的抗体。
本发明还涉及一种筛选的模拟、 激活、 拮抗或抑制 gammal-吡咯啉 - 5-羟基还 原酶 14蛋白活性的化合物的方法， 其包括利用本发明的多肽。 本发明还涉及用该 方法获得的化合物。
本发明还涉及一种体外检测与 gammal-吡咯啉 -5-羟基还原酶 14蛋白异常表 达相关的疾病或疾病易感性的方法， 包括检测生物样品中所述多肽或其编码多核 苷酸序列中的突变， 或者检测生物样品中本发明多肽的量或生物活性。
本发明也涉及一种药物组合物， 它含有本发明多肽或其模拟物、 激活剂、 拮 抗剂或抑制剂以及药学上可接受的载体。
本发明还涉及本发明的多肽和 /或多核苷酸在制备用于治疗癌症、 发育性疾病 或免疫性疾病或其它由于 gammal-吡咯啉 -5-羟基还原酶 表达异常所引起疾病 的药物的用途。
本发明的其它方面由于本文的技术的公开， 对本领域的技术人员而言是显而 易见的。 附图说明
下列附图用于说明本发明的具体实施方案， 而不用于限定由权利要求书 所界定的本发明范围。
图 1是本发明 gammal-吡咯啉 -5-羟基还原酶 14在 14- 102共 89个氨基酸和结 构域 ga隱 a l-吡咯啉 -5-羟基还原酶特征蛋白的氨基酸序列同源性比较图。 上方 序列是 gamma l-吡咯啉 -5-羟基还原酶 14， 下方序列是 gammal-吡咯啉 -5-羟基还 原酶特征蛋白结构域。 " | " 和 " ： " 及 ". " 表示在两个序列间同一氨基酸 出现的概率依次减小。
图 2 为分离的 gammal-吡咯啉 -5-羟基还原酶 14 的聚丙烯酰胺凝胶电泳图 ( SDS-PAGE )0 14kDa为蛋白质的分子量。 箭头所指为分离出的蛋白条带。 发明内容
本说明书和权利要求书中使用的下列术语除非特别说明具有如下的含义： "核酸序列" 是指寡核苷酸、 核苷酸或多核苷酸及其片段或部分， 也可以指 基因组或合成的 DM或 RNA, 它们可以是单链或双链的， 代表有义链或反义链。 类 似地， 术语 "氨基酸序列" 是指寡肽、 肽、 多肽或蛋白质序列及其片段或部分。 当本发明中的 "氨基酸序列" 涉及一种天然存在的蛋白质分子的氨基酸序列时， 这种 "多肽" 或 "蛋白质" 不意味着将氨基酸序列限制为与所述蛋白质分子相关 的完整的天然氨基酸。
蛋白质或多核苷酸 "变体" 是指一种具有一个或多个氨基酸或核苷酸改变的 氨基酸序列或编码它的多核苷酸序列。 所述改变可包括氨基酸序列或核苷酸序列 中氨基酸或核苷酸的缺失、 插入或替换。 变体可具有 "保守性" 改变， 其中替换 的氨基酸具有与原氨基酸相类似的结构或化学性质， 如用亮氨酸替换异亮氨酸。 变体也可具有非保守性改变， 如用色氨酸替换甘氨酸。
"缺失" 是指在氨基酸序列或核苷酸序列中一个或多个氨基酸或核苷酸的缺 失。
"插入" 或 "添加" 是指在氨基酸序列或核苷酸序列中的改变导致与天然存 在的分子相比， 一个或多个氨基酸或核苷酸的增加。 "替换" 是指由不同的氨基酸 或核苷酸替换一个或多个氨基酸或核苷酸。
"生物活性" 是指具有天然分子的结构、 调控或生物化学功能的蛋白质。 类 似地， 术语 "免疫学活性" 是指天然的、 重组的或合成蛋白质及其片段在合适的 动物或细胞中诱导特定免疫反应以及与特异性抗体结合的能力。
"激动剂" 是指当与 gammal-吡咯啉 -5 -羟基还原酶 14结合时， 一种可引起该 蛋白质改变从而调节该蛋白质活性的分子。 激动剂可以包括蛋白质、 核酸、 碳水 化合物或任何其它可结合 ga画 al-吡咯啉 -5-羟基还原酶 14的分子。
"拮抗剂" 或 "抑制物" 是指当与 gamma l-吡咯啉 -5-羟基还原酶 14结合时， 一种可封闭或调节 gamma l-吡咯啉 -5-羟基还原酶 14的生物学活性或免疫学活性的 分子。 拮抗剂和抑制物可以包括蛋白质、 核酸、 碳水化合物或任何其它可结合 gamma l-吡咯啉 -5-羟基还原酶 14的分子。
"调节" 是指 ga隱 a l-吡咯啉 -5-羟基还原酶 14的功能发生改变， 包括蛋白质 活性的升高或降低、 结合特性的改变及 gamma l-吡咯啉 -5-羟基还原酶 14的任何其 它生物学性质、 功能或免疫性质的改变。
"基本上纯" 是指基本上不含天然与其相关的其它蛋白、 脂类、 糖类或其它 物质。 本领域的技术人员能用标准的蛋白质纯化技术纯化 garama l-吡咯啉 -5 -羟基 还原酶 14。 基本上纯的 gammal-吡咯啉 -5-羟基还原酶 14 在非还原性聚丙烯酰胺 凝胶上能产生单一的主带。 garama l-吡咯啉 -5-羟基还原酶 14 多肽的纯度可用氨基 酸序列分析。 "互补的" 或 "互补" 是指在允许的盐浓度和温度条件下通过碱基配对的多 核苷酸天然结合。 例如， 序列 "C- T- G- A" 可与互补的序列 "G-A- C- T" 结合。 两 个单链分子之间的互补可以是部分的或全部的。 核酸链之间的互补程度对于核酸 链之间杂交的效率及强度有明显影响。
"同源性" 是指互补的程度， 可以是部分同源或完全同源。 "部分同源" 是指 一种部分互补的序列， 其至少可部分抑制完全互补的序列与靶核酸的杂交。 这种 杂交的抑制可通过在严格性程度降低的条件下进行杂交（ Southern印迹或 Nor thern 印迹等） 来检测。 基本上同源的序列或杂交探针可竟争和抑制完全同源的序列与 靶序列在严格性程度降低的条件下的结合。 这并不意味严格性程度降低的条件允 许非特异性结合， 因为严格性程度降低的条件要求两条序列相互的结合为特异性 或选择性相互作用。
"相同性百分率" 是指在两种或多种氨基酸或核酸序列比较中序列相同或相 似的百分率。 可用电子方法测定相同性百分率， 如通过 MEGALIGN程序 （ Lasergene sof tware package, DNASTAR, Inc. , Madi son Wi s. )0 MEGALIGN程序可根据不同的 方法， 如 C l us ter法比较两种或多种序列（Hi gg ins, D. G. 和 P. M. Sharp (1988) Gene 73: 237-244)。 C l us ter法通过检査所有配对之间的距离将各组序列排列成簇。 然后将各簇以成对或成组分配。 两个氨基酸序列如序列 A和序列 B之间的相同性百 分率通过下式计算： 序列 与序列 S之间匹配的残基个数
序列 的残基数 -序歹 中间隔残基数 -序列 ^中间隔残基数 也可以通过 C lus ter法或用本领域周知的方法如 Jotun He in 测定核酸序列之 间的相同性百分率（He in J. , (1990) Methods in enzymo logy 183: 625—645)。
"相似性" 是指氨基酸序列之间排列对比时相应位置氨基酸残基的相同或保 守性取代的程度。 用于保守性取代的氨基酸,例如带负电荷的氨基酸可包括天冬氨 酸和谷氨酸； 带正电荷的氨基酸可包括赖氨酸和精氨酸； 具有不带电荷的头部基 团有相似亲水性的氨基酸可包括亮氨酸、 异亮氨酸和缬氨酸； 甘氨酸和丙氨酸； 天冬酰胺和谷氨酰胺； 丝氨酸和苏氨酸； 苯丙氨酸和酪氨酸。
"反义"是指与特定的 DM或 RNA序列互补的核苷酸序列。 "反义链"是指与 "有 义链" 互补的核酸链。
"衍生物" 是指 HFP或编码其核酸的化学修饰物。 这种化学修饰物可以是用烷 基、 酰基或氨基替换氢原子。 核酸衍生物可编码保留天然分子的主要生物学特性 的多肽。
"抗体" 是指完整的抗体分子及其片段， 如 Fa、 ？(^') 2及? ， 其能特异性 结合 gammal-吡咯啉 -5-羟基还原酶 14的抗原决定簇。
"人源化抗体" 是指非抗原结合区域的氨基酸序列被替换变得与人抗体更为 相似， 但仍保留原始结合活性的抗体。
"分离的" 一词指将物质从它原来的环境 （例如， 若是自然产生的就指其天 然环境） 之中移出。 比如说， 一个自然产生的多核苷酸或多肽存在于活动物中就 是没有被分离出来， 但同样的多核苷酸或多肽同一些或全部在自然系统中与之共 存的物质分开就是分离的。 这样的多核苷酸可能是某一载体的一部分， 也可能这 样的多核苷酸或多肽是某一组合物的一部分。 既然载体或组合物不是它天然环境 的成分， 它们仍然是分离的。
如本发明所用， "分离的" 是指物质从其原始环境中分离出来 （如果是天然的 物质， 原始环境即是天然环境）。 如活体细胞内的天然状态下的多聚核苷酸和多肽 是没有分离纯化的， 但同样的多聚核苷酸或多肽如从天然状态中同存在的其他物 质中分开， 则为分离纯化的。
如本文所用， "分离的 gammal-吡咯啉 - 5-羟基还原酶 14" 是指 gammal-吡 咯啉- 5-羟基还原酶 14 基本上不含天然与其相关的其它蛋白、 脂类、 糖类或其 它物质。 本领域的技术人员能用标准的蛋白质纯化技术纯化 gamraa l-吡咯啉- 5 - 羟基还原酶 14。 基本上纯的多肽在非还原聚丙烯酰胺凝胶上能产生单一的主 带。 gamma l-吡咯啉 -5-羟基还原酶 I 4多肽的纯度能用氨基酸序列分析。
本发明提供了一种新的多肽—— ga瞧 al-吡咯啉 -5-羟基还原酶 14 , 其基本上是 由 SEQ ID NO: 2所示的氨基酸序列组成的。 本发明的多肽可以是重组多肽、 天然多 肽、 合成多肽， 优选重组多肽。 本发明的多肽可以是天然纯化的产物， 或是化学 合成的产物， 或使用重组技术从原核或真核宿主（例如， 细菌、 酵母、 高等植物、 昆虫和哺乳动物细胞）中产生。 根据重组生产方案所用的宿主， 本发明的多肽可以 是糖基化的， 或可以是非糖基化的。 本发明的多肽还可包括或不包括起始的甲硫 氨酸残基。
本发明还包括 gamraal-吡咯啉 -5-羟基还原酶 14 的片段、 衍生物和类似物。 如本发明所用， 术语 "片段"、 "衍生物" 和 "类似物" 是指基本上保持本发明的 ga瞧 al-吡咯啉 -5-羟基还原酶 14相同的生物学功能或活性的多肽。 本发明多肽的 片段、 衍生物或类似物可以是： （ I ) 这样一种， 其中一个或多个氨基酸残基被保 守或非保守氨基酸残基 （优选的是保守氨基酸残基） 取代， 并且取代的氨基酸可 以是也可以不是由遗传密码子编码的； 或者 （ Π ) 这样一种， 其中一个或多个氨 基酸残基上的某个基团被其它基团取代包含取代基； 或者 （I I I ) 这样一种， 其中 成熟多肽与另一种化合物 （比如延长多肽半衰期的化合物， 例如聚乙二醇） 融合； 或者 （ IV ) 这样一种， 其中附加的氨基酸序列融合进成熟多肽而形成的多肽序列
(如前导序列或分泌序列或用来纯化此多肽的序列或蛋白原序列）。 通过本文的阐 述， 这样的片段、 衍生物和类似物被认为在本领域技术人员的知识范围之内。
本发明提供了分离的核酸 （多核苷酸）， 基本由编码具有 SEQ ID NO: 2 氨基 酸序列的多肽的多核苷酸组成。 本发明的多核苷酸序列包括 SEQ ID NO: 1 的核苷 酸序列。 本发明的多核苷酸是从人胎脑组织的 cDNA文库中发现的。 它包含的多核 苷酸序列全长为 1629个碱基， 其开放读框 835-1218编码了 127个氨基酸。 此多 肽具有 gammal-吡咯啉 -5-羟基还原酶特征蛋白的特征序列， 可推断出该 gammal - 吡咯啉 -5-羟基还原酶 14 具有 gammal-吡咯啉 -5-羟基还原酶特征蛋白所代表的结 构和功能。
本发明的多核苷酸可以是 DM形式或是 RNA形式。 DNA形式包括 cDNA、 基因 组 DNA或人工合成的 DNA。 DNA可以是单链的或是双链的。 DNA可以是编码链或非 编码链。 编码成熟多肽的编码区序列可以与 SEQ ID N0: 1 所示的编码区序列相同 或者是简并的变异体。 如本发明所用， "简并的变异体" 在本发明中是指编码具有 SEQ ID NO: 2的蛋白质或多肽， 但与 SEQ ID NO: 1所示的编码区序列有差别的核酸 序列。
编码 SEQ ID NO: 2 的成熟多肽的多核苷酸包括： 只有成熟多肽的编码序列； 成熟多肽的编码序列和各种附加编码序列； 成熟多肽的编码序列 （和任选的附加 编码序列） 以及非编码序列。
术语 "编码多肽的多核苷酸" 是指包括编码此多肽的多核苷酸和包括附加编 码和 /或非编码序列的多核苷酸。
本发明还涉及上述描述多核苷酸的变异体， 其编码与本发明有相同的氨基酸 序列的多肽或多肽的片断、 类似物和衍生物。 此多核苷酸的变异体可以是天然发 生的等位变异体或非天然发生的变异体。 这些核苷酸变异体包括取代变异体、 缺 失变异体和插入变异体。 如本领域所知的， 等位变异体是一个多核苷酸的替换形 式， 它可能是一个或多个核苷酸的取代、 缺失或插入， 但不会从实质上改变其编 码的多肽的功能。
本发明还涉及与以上所描述的序列杂交的多核苷酸 （两个序列之间具有至少 50%, 优选具有 70½的相同性）。 本发明特别涉及在严格条件下与本发明所述多核苷 酸可杂交的多核苷酸。 在本发明中， "严格条件" 是指： （1)在较低离子强度和较 高温度下的杂交和洗脱， 如 0.2xSSC， 0.1%SDS，6(TC;或（2)杂交时加用变性剂， 如 50%(v/v)甲酰胺， 0.1%小牛血清 /0. l%Ficoll, 42°C等； 或（3)仅在两条序列之间 的相同性至少在 95%以上，更好是 97%以上时才发生杂交。 并且， 可杂交的多核苷 酸编码的多肽与 SEQ ID NO: 2所示的成熟多肽有相同的生物学功能和活性。
本发明还涉及与以上所描述的序列杂交的核酸片段。 如本发明所用， "核酸片 段"的长度至少含 10 个核苷酸， 较好是至少 20-30 个核苷酸， 更好是至少 50-60 个核苷酸， 最好是至少 100个核苷酸以上。 核酸片段也可用于核酸的扩增技术（如 PCR)以确定和 /或分离编码 gammal-吡咯啉 -5-羟基还原酶 14的多核苷酸。
本发明中的多肽和多核苷酸优选以分离的形式提供， 更佳地被纯化至均质。 本发明的编码 gammal-吡咯啉 -5-羟基还原酶 14 的特异的多核苷酸序列能 用多种方法获得。 例如， 用本领域熟知的杂交技术分离多核苷酸。 这些技术包 括但不局限于： 1)用探针与基因组或 cDNA 文库杂交以检出同源的多核苷酸序 列， 和 2)表达文库的抗体筛选以检出具有共同结构特征的克隆的多核苷酸片 段。
本发明的 DM片段序列也能用下列方法获得： 1)从基因组 DNA分离双链 DNA 序列； 2)化学合成 DNA序列以获得所述多肽的双链 DNA。
上述提到的方法中， 分离基因组 DNA 最不常用。 DNA 序列的直接化学合成 是经常选用的方法。 更经常选用的方法是 cDNA序列的分离。 分离感兴趣的 cDNA 的标准方法是从高表达该基因的供体细胞分离 mRNA 并进行逆转录， 形成质粒 或噬菌体 cDNA文库。 提取 mRNA 的方法已有多种成熟的技术， 试剂盒也可从商 业途径获得（Qiagene)。 而构建 cDNA 文库也是通常的方法（Sambrook, et al.， Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory. New York, 1989)。还可得到商业供应的 cDNA文库，如 Clontech公司的不同 cDNA 文库。 当结合使用聚合酶反应技术时， 即使极少的表达产物也能克隆。
可用常规方法从这些 cDNA 文库中筛选本发明的基因。 这些方法包括（但不 限于）： （l)DNA-DNA 或 DNA-RNA 杂交； （2)标志基因功能的出现或丧失； （3)测 定 gammal-吡咯啉 -5-羟基还原酶 14的转录本的水平； （4)通过免疫学技术或测 定生物学活性， 来检测基因表达的蛋白产物。 上述方法可单用， 也可多种方法 联合应用。
在第（1)种方法中， 杂交所用的探针是与本发明的多核苷酸的任何一部分 同源， 其长度至少 10 个核苷酸， 较好是至少 30个核苷酸， 更好是至少 50 个 核苷酸， 最好是至少 100 个核苷酸。 此外， 探针的长度通常在 2000 个核苷酸 之内， 较佳的为 1000 个核苷酸之内。 此处所用的探针通常是在本发明的基因 序列信息的基础上化学合成的 DNA 序列。 本发明的基因本身或者片段当然可以 用作探针。 DM探针的标记可用放射性同位素， 荧光素或酶（如碱性磷酸酶）等。
在第（4)种方法中， 检测 gammal-吡咯啉 -5-羟基还原酶 14基因表达的蛋白 产物可用免疫学技术如 Western 印迹法、 放射免疫沉淀法、 酶联免疫吸附法 (ELISA)等。
应 用 PCR 技术 扩增 DNA/RNA 的 方 法 （Saiki, et al. Science 1985;230: 1350-1354)被优选用于获得本发明的基因。 特别是很难从文库中得 到全长的 cDNA时， 可优选使用 RACE法（RACE- cDNA末端快速扩增法），用于 PCR 的引物可根据本文所公开的本发明的多核苷酸序列信息适当地选择， 并可用常 规方法合成。 可用常规方法如通过凝胶电泳分离和纯化扩增的 DNA/RNA片段。
如上所述得到的本发明的基因， 或者各种 DNA 片段等的多核苷酸序列可用 常规方法如双脱氧链终止法（Sanger et al. PNAS, 1977, 74： 5463- 5467)测 定。 这类多核苷酸序列测定也可用商业测序试剂盒等。 为了获得全长的 cDNA 序列， 测序需反复进行。 有时需要测定多个克隆的 cDNA 序列， 才能拼接成全 长的 cDNA序列。
本发明也涉及包含本发明的多核苷酸的载体， 以及用本发明的载体或直接 用 garamal-吡咯啉 -5-羟基还原酶 14 编码序列经基因工程产生的宿主细胞， 以 及经重组技术产生本发明所述多肽的方法。
本发明中， 编码 gammal-吡咯啉 -5-羟基还原酶 14 的多核苷酸序列可插入 到载体中， 以构成含有本发明所述多核苷酸的重组载体。 术语 "载体" 指本领 域熟知的细菌质粒、 噬菌体、 酵母质粒、 植物细胞病毒、 哺乳动物细胞病毒如 腺病毒、 逆转录病毒或其它载体。 在本发明中适用的载体包括但不限于： 在细 菌中表达的基于 T7 启动子的表达载体（Rosenberg, et al. Gene, 1987, 56: 125)； 在哺乳动物细胞中表达的 pMSXND表达载体（Lee and Nathans, J Bio Chem. 263: 3521， 1988)和在昆虫细胞中表达的来源于杆状病毒的载体。 总之， 只要能在宿主体内复制和稳定， 任何质粒和载体都可以用于构建重组表达载 体。 表达载体的一个重要特征是通常含有复制起始点、 启动子、 标记基因和翻 译调控元件。
本领域的技术人员熟知的方法能用于构建含编码 gammal-吡咯啉 -5-羟基还 原酶 14 的 DNA序列和合适的转录 /翻译调控元件的表达载体。 这些方法包括体 外重组 DNA技术、 DNA合成技术、体内重组技术等（Sambroook, et al. Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory. New York, 1989)。 所述的 DNA序列可有效连接到表达载体中的适当启动子上， 以指导 mRNA 合成。 这些启动子的代表性例子有： 大肠杆菌的 lac或 trp启动子； λ噬菌体 的 PL启动子； 真核启动子包括 CMV立即早期启动子、 HSV胸苷激酶启动子、 早 期和晚期 SV40启动子、 反转录病毒的 LTRs 和其它一些已知的可控制基因在原 核细胞或真核细胞或其病毒中表达的启动子。 表达载体还包括翻译起始用的核 糖体结合位点和转录终止子等。 在载体中插入增强子序列将会使其在高等真核 细胞中的转录得到增强。 增强子是 DNA 表达的顺式作用因子， 通常大约有 10 到 300 个碱基对， 作用于启动子以增强基因的转录。 可举的例子包括在复制起 始点晚期一侧的 100 到 270个碱基对的 SV40增强子、 在复制起始点晚期一侧 的多瘤增强子以及腺病毒增强子等。
此外， 表达载体优选地包含一个或多个选择性标记基因， 以提供用于选择 转化的宿主细胞的表型性状， 如真核细胞培养用的二氢叶酸还原酶、 新霉素抗 性以及绿色荧光蛋白（GFP) , 或用于大肠杆菌的四环素或氨苄青霉素抗性等。
本领域一般技术人员都清楚如何选择适当的载体 /转录调控元件 （如启动 子、 增强子等） 和选择性标记基因。
本发明中， 编码 gammal-吡咯啉 - 5-羟基还原酶 14 的多核苷酸或含有该多 核苷酸的重组载体可转化或转导入宿主细胞， 以构成含有该多核苷酸或重组载 体的基因工程化宿主细胞。 术语 "宿主细胞" 指原核细胞， 如细菌细胞； 或是 低等真核细胞， 如酵母细胞； 或是高等真核细胞， 如哺乳动物细胞。 代表性例 子有： 大肠杆菌， 链霉菌属； 细菌细胞如鼠伤寒沙门氏菌； 真菌细胞如酵母； 植物细胞； 昆虫细胞如果蝇 S2或 Sf9; 动物细胞如 CH0、 COS或 Bowes 黑素瘤 细胞等。
用本发明所述的 DNA序列或含有所述 DNA序列的重组载体转化宿主细胞可 用本领域技术人员熟知的常规技术进行。 当宿主为原核生物如大肠杆菌时， 能 吸收 DM 的感受态细胞可在指数生长期后收获， 用 (12法处理， 所用的步骤 在本领域众所周知。 可供选择的是用 MgCl2。 如果需要， 转化也可用电穿孔的 方法进行。 当宿主是真核生物， 可选用如下的 DM转染方法： 磷酸钙共沉淀法， 或者常规机械方法如显微注射、 电穿孔、 脂质体包装等。
通过常规的重组 DNA技术， 利用本发明的多核苷酸序列可用来表达或生产 重组的 gammal-吡咯啉 -5-羟基还原酶 14 (Science, 1984; 224： 1431)。 一般 来说有以下步骤：
(1) 用本发明的编码人 gammal-吡咯啉 -5-羟基还原酶 14 的多核苷酸（或 变异体）， 或用含有该多核苷酸的重组表达载体转化或转导合适的宿主细胞；
(2) 在合适的培养基中培养宿主细胞；
(3) 从培养基或细胞中分离、 纯化蛋白质。
在步骤 （2 ) 中， 根据所用的宿主细胞， 培养中所用的培养基可选自各种 常规培养基。 在适于宿主细胞生长的条件下进行培养。 当宿主细胞生长到适当 的细胞密度后， 用合适的方法（如温度转换或化学诱导）诱导选择的启动子， 将 细胞再培养一段时间。
在步骤 （ 3 ) 中， 重组多肽可包被于细胞内、 或在细胞膜上表达、 或分泌 到细胞外。 如果需要， 可利用其物理的、 化学的和其它特性通过各种分离方法 分离和纯化重组的蛋白。 这些方法是本领域技术人员所熟知的。 这些方法包括 但并不限于： 常规的复性处理、 蛋白沉淀剂处理（盐析方法）、 离心、 渗透破菌、 超声波处理、 超离心、 分子筛层析（凝胶过滤）、 吸附层析、 离子交换层析、 高 效液相层析（HPLC)和其它各种液相层析技术及这些方法的结合。
本发明的多肽以及该多肽的拮抗剂、 激动剂和抑制剂可直接用于疾病治 疗， 例如， 可治疗恶性肿瘤、 肾上腺缺乏症、 皮肤病、 各类炎症、 HIV 感染和免 疫性疾病等。
在氨基酸代谢及合成过程中， 谷氨酸代谢中的碳和氮被用于脯氨酸及精氨酸 的生物合成。 脯氨酸在生物体内起着十分重要的生理学功能， 脯氨酸残基经翻译 后修饰生成 4-羟基- L -脯氨酸， 该氨基酸为某些蛋白尤其是胶原蛋白的重要组分。 而胶原蛋白在生物体内涉及维持骨腱、 血管、 软骨和皮肤强度以及组织纤维化及 炎症修复等众多的生物学过程。 δ ΐ-吡咯啉 -5-羟基还原酶 （P5CR ) 在生物体内催 化从谷氨酸到脯氨酸的生物合成过程的最后一步， 它直接影响生物体内脯氨酸的 合成， 进而影响一些相关的重要蛋白质的合成。
δ 1-吡咯啉 -5-羟基还原酶特异保守序列是形成其活性 raot if 所必需。 由此可 见， 特异的 δ ΐ-吡咯啉 -5-羟基还原酶 mot if 的表达异常， 将致使本发明的含此 mot if 的多肽的功能异常， 从而导致谷氨酸代谢异常、 脯氨酸代谢异常， 脯氨酸代 谢异常使胶原蛋白合成异常。 由于胶原蛋白在组织纤维化及炎症修复过程中具有 重要意义， 故此多肽的表达异常可使某些组织纤维化， 可使炎症修复过程出现异 常， 可出现瘢痕过多、 腔道狭窄、 伤口不愈等现象。
由此可见， 本发明的 gamma l-吡咯啉 -5-羟基还原酶 I 4 的表达异常将产生 各种疾病尤其是氨基酸代谢缺陷病、 有机酸血症、 组织纤维病理病变、 炎症修复 异常相关疾病， 这些疾病包括但不限于：
氨基酸代谢缺陷病： 谷氨酸代谢缺陷病， 脯氨酸和羟脯氨酸代谢缺陷病， 苯 丙酮尿症， 酪氨酸代谢缺陷性症， 色氨酸代谢缺陷病， 分支氨基酸代谢缺陷病， 甘氨酸代谢缺陷病， 脯氨酸和羟脯氨酸代谢缺陷病， 尿素循环的代谢缺陷病， 组 氨酸代谢缺陷病， 赖氨酸代谢缺陷病
组织纤维病理病变： 纤维瘤， 结节性筋膜炎， 增生性筋膜炎， 肌纤维瘤病， 纤维肉瘤， 纤维组织细胞瘤， 网状组织细胞瘤， 纤维瘤病如瘢痕疙瘩， 肺弥漫性 间质性疾患， 硒肺， 肝硬化， 前列腺增生症
炎症修复异常相关疾病： 各种组织腔道的狭窄如幽门狭窄、 损伤后气管狭窄、 二尖瓣狭窄、 主动脉狭窄、 肺动脉狭窄、 缩窄性心包炎、 胰腺囊性纤维化、 肾盂 肾炎
有机酸血症： 丙酸血症， 甲基丙二酸尿症， 异戊酸血症， 联合羧化酶缺陷， 戊二酸血症 I型
本发明的 gamma l-吡咯啉 -5-羟基还原酶 14 的表达异常还将产生某些遗传 性疾病及免疫系统疾病等。
本发明的多肽以及该多肽的拮抗剂、 激动剂和抑制剂可直接用于疾病治 疗， 例如， 可治疗各种疾病尤其是氨基酸代谢缺陷病、 有机酸血症、 组织纤维病 理病变、 炎症修复异常相关疾病， 某些遗传性疾病及免疫系统疾病等。
本发明也提供了筛选化合物以鉴定提高（激动剂）或阻遏（拮抗剂） gamma l - 吡咯啉 -5-羟基还原酶 1 4 的药剂的方法。 激动剂提高 gamma l -吡咯啉 -5-羟基还 原酶 14 刺激细胞增殖等生物功能， 而拮抗剂阻止和治疗与细胞过度增殖有关 的紊乱如各种癌症。 例如， 能在药物的存在下， 将哺乳动物细胞或表达 gamma l - 吡咯啉 -5-羟基还原酶 14 的膜制剂与标记的 gamma l-吡咯啉 -5-羟基还原酶 14 一起培养。 然后测定药物提高或阻遏此相互作用的能力。
gamma l-吡咯啉 -5-羟基还原酶 14 的拮抗剂包括筛选出的抗体、 化合物、 受体缺失物和类似物等。 gamma l-吡咯啉 -5-羟基还原酶 14 的拮抗剂可以与 gamma l -吡咯啉 -5-羟基还原酶 14 结合并消除其功能， 或是抑制该多肽的产生， 或是与该多肽的活性位点结合使该多肽不能发挥生物学功能。
在筛选作为拮抗剂的化合物时， 可以将 gamma l -吡咯啉 -5-羟基还原酶 14 加入生物分析测定中， 通过测定化合物对 gamma l-吡咯啉 -5-羟基还原酶 14 和 其受体之间相互作用的影响来确定化合物是否是拮抗剂。 用上述筛选化合物的 同样方法， 可以筛选出起拮杭剂作用的受体缺失物和类似物。 能与 gammal-吡 咯啉 -5-羟基还原酶 14 结合的多肽分子可通过筛选由各种可能组合的氨基酸结 合于固相物组成的随机多肽库而获得。 筛选时， 一般应对 gammal-吡咯啉 -5-羟 基还原酶 14分子进行标记。
本发明提供了用多肽， 及其片段、 衍生物、 类似物或它们的细胞作为抗原 以生产抗体的方法。 这些抗体可以是多克隆抗体或单克隆抗体。 本发明还提供 了针对 gamma l-吡咯啉 -5-羟基还原酶 14抗原决定簇的抗体。 这些抗体包括（但 不限于）： 多克隆抗体、 单克隆抗体、 嵌合抗体、 单链抗体、 Fab片段和 Fab表 达文库产生的片段。
多克隆抗体的生产可用 gamma l-吡咯啉 -5-羟基还原酶 14 直接注射免疫动 物 （如家兔， 小鼠， 大鼠等） 的方法得到， 多种佐剂可用于增强免疫反应， 包 括但不限于弗氏佐剂等。 制备 garamal-吡咯啉 - 5-羟基还原酶 14 的单克隆抗体 的技术包括但不限于杂交瘤技术 （Koh l er and Mi l s te in. Na ture, 1975, 256: 495-497) , 三瘤技术， 人 B-细胞杂交瘤技术， EBV-杂交瘤技术等。 将人恒 定区和非人源的可变区结合的嵌合抗体可用已有的技术生产（Morr i son et a l , PNAS, 1985, 81: 6851)。而已有的生产单链抗体的技术（U. S. Pa t No. 4946778) 也可用于生产抗 gammal-吡咯啉 -5-羟基还原酶 14的单链抗体。
抗 gamma l-吡咯啉 -5-羟基还原酶 14 的抗体可用于免疫组织化学技术中， 检测活检标本中的 garamal-吡咯啉 -5-羟基还原酶 14。
与 gammal-吡咯啉 -5-羟基还原酶 I 4 结合的单克隆抗体也可用放射性同位 素标记， 注入体内可跟踪其位置和分布。 这种放射性标记的抗体可作为一种非 创伤性诊断方法用于肿瘤细胞的定位和判断是否有转移。
抗体还可用于设计针对体内某一特殊部位的免疫毒素。 如 gammal-吡咯啉- 5-羟基还原酶 14 高亲和性的单克隆抗体可与细菌或植物毒素（如白喉毒素， 蓖 麻蛋白， 红豆碱等）共价结合。 一种通常的方法是用巯基交联剂如 SPDP , 攻击 抗体的氨基， 通过二硫键的交换， 将毒素结合于抗体上， 这种杂交抗体可用于 杀灭 gammal-吡咯啉 -5-羟基还原酶 14阳性的细胞。
本发明中的抗体可用于治疗或预防与 garamal-吡咯啉 -5-羟基还原酶 14 相 关的疾病。 给予适当剂量的抗体可以刺激或阻断 gamma l-吡咯啉 -5-羟基还原酶 14的产生或活性。
本发明还涉及定量和定位检测 gammal-吡咯啉 -5-羟基还原酶 14 水平的诊 断试验方法。 这些试验是本领域所熟知的， 且包括 FI SH 测定和放射免疫测定。 试验中所检测的 gamma l-吡咯啉 -5-羟基还原酶 14水平， 可以用作解释 gatnma l- 吡咯啉 -5-羟基还原酶 14在各种疾病中的重要性和用于诊断 gamma l-吡咯啉- 5 - 羟基还原酶 14起作用的疾病。
本发明的多肽还可用作肽谱分析， 例如， 多肽可用物理的、 化学或酶进行 特异性切割， 并进行一维或二维或三维的凝胶电泳分析，更好的是进行质谱分 析。
编码 gamraa l-吡咯啉 - 5-羟基还原酶 14 的多核苷酸也可用于多种治疗目 的。 基因治疗技术可用于治疗由于 gamma l-吡咯啉 -5-羟基还原酶 M 的无表达 或异常 /无活性表达所致的细胞增殖、发育或代谢异常。 重组的基因治疗载体（如 病毒载体）可设计用于表达变异的 gamma l-吡咯啉 -5-羟基还原酶 14， 以抑制内 源性的 gamma l-吡咯啉 -5-羟基还原酶 14 活性。 例如， 一种变异的 gamma l -吡 咯啉- 5-羟基还原酶 14 可以是缩短的、 缺失了信号传导功能域的 gamraa l-吡咯 啉- 5-羟基还原酶 14， 虽可与下游的底物结合， 但缺乏信号传导活性。 因此重 组的基因治疗载体可用于治疗 gamma l-吡咯啉 -5-羟基还原酶 14 表达或活性异 常所致的疾病。 来源于病毒的表达载体如逆转录病毒、 腺病毒、 腺病毒相关病 毒、 单纯疱疹病毒、 细小病毒等可用于将编码 gamma l-吡咯啉 -5-羟基还原酶 14 的多核苷酸转移至细胞内。 构建携带编码 gamma l-吡咯啉 -5-羟基还原酶 14 的 多核苷酸的重组病毒载体的方法可见于已有文献（Sambrook，e t a l . ) 0 另外重 组编码 gamma l-吡咯啉 -5-羟基还原酶 14 的多核苷酸可包装到脂质体中转移至 细胞内。
多核苷酸导入组织或细胞内的方法包括： 将多核苷酸直接注入到体内组织 中； 或在体外通过载体（如病毒、 噬菌体或质粒等）先将多核苷酸导入细胞中， 再将细胞移植到体内等。
抑制 gamma l-吡咯啉 -5-羟基还原酶 14 mRNA 的寡核苷酸（包括反义 RNA和 DM)以及核酶也在本发明的范围之内。 核酶是一种能特异性分解特定 RNA 的酶 样 RNA分子， 其作用机制是核酶分子与互补的靶 RNA特异性杂交后进行核酸内 切作用。 反义的 RNA和 DNA及核酶可用已有的任何 RNA或 DNA合成技术获得， 如固相磷酸酰胺化学合成法合成寡核苷酸的技术已广泛应用。 反义 RNA 分子可 通过编码该 RNA的 DNA序列在体外或体内转录获得。 这种 DNA序列已整合到载 体的 RNA 聚合酶启动子的下游。 为了增加核酸分子的稳定性， 可用多种方法对 其进行修饰， 如增加两侧的序列长度， 核糖核苷之间的连接应用磷酸硫酯键或 肽键而非磷酸二酯键。 编码 gamma l-吡咯啉 - 5-羟基还原酶 14 的多核苷酸可用于与 gammal-吡咯 啉- 5-羟基还原酶 14的相关疾病的诊断。 编码 gammal-吡咯啉 -5-羟基还原酶 14 的多核苷酸可用于检测 gamma l-吡咯啉 -5-羟基还原酶 14 的表达与否或在疾病 状态下 gamma l-吡咯啉 -5-羟基还原酶 14 的异常表达。 如编码 gamma l-吡咯啉- 5-羟基还原酶 14的 DNA序列可用于对活检标本进行杂交以判断 gammal-吡咯啉 - 5-羟基还原酶 14 的表达状况。 杂交技术包括 Southern 印迹法、 Nor thern 印 迹法、 原位杂交等。 这些技术方法都是公开的成熟技术， 相关的试剂盒都可从 商业途径得到。 本发明的多核苷酸的一部分或全部可作为探针固定在微阵列 (Mi croarray)或 DNA 芯片（又称为 "基因芯片" ）上， 用于分析组织中基因的差 异表达分析和基因诊断。 用 gamraal-吡咯啉 -5-羟基还原酶 14 特异的引物进行 RNA-聚合酶链反应（RT- PCR)体外扩增也可检测 gammal-吡咯啉 -5-羟基还原酶 的转录产物。
检测 gamma l-吡咯啉 -5-羟基还原酶 14 基因的突变也可用于诊断 gammal - 吡咯啉 -5-羟基还原酶 14 相关的疾病。 gammal-吡咯啉 -5-羟基还原酶 14 突变 的形式包括与正常野生型 gamma l-吡咯啉 -5-羟基还原酶 14 DNA 序列相比的点 突变、 易位、 缺失、 重组和其它任何异常等。 可用已有的技术如 Southern 印 迹法、 DM 序列分析、 PCR 和原位杂交检测突变。 另外， 突变有可能影响蛋白 的表达， 因此用 Nor thern印迹法、 Wes tern印迹法可间接判断基因有无突变。
本发明的序列对染色体鉴定也是有价值的。 该序列会特异性地针对某条人 染色体具体位置并且可以与其杂交。 目前， 需要鉴定染色体上的各基因的具体 位点。 现在， 只有很少的基于实际序列数据（重复多态性）的染色体标记物可用 于标记染色体位置。 根据本发明， 为了将这些序列与疾病相关基因相关联， 其 重要的第一步就是将这些 DNA序列定位于染色体上。
简而言之， 根据 cDM制备 PCR引物（优选 15- 35bp) , 可以将序列定位于染色 体上。 然后， 将这些引物用于 PCR筛选含各条人染色体的体细胞杂合细胞。 只 有那些含有相应于引物的人基因的杂合细胞会产生扩增的片段。
体细胞杂合细胞的 PCR定位法， 是将 DNA定位到具体染色体的快捷方法。 使 用本发明的寡核苷酸引物， 通过类似方法， 可利用一组来自特定染色体的片段 或大量基因组克隆而实现亚定位。 可用于染色体定位的其它类似策略包括原位 杂交、 用标记的流式分选的染色体预筛选和杂交预选， 从而构建染色体特异的 cDNA库。
将 cDNA克隆与中期染色体进行荧光原位杂交（FISH) , 可以在一个步骤中精 确地进行染色体定位。此技术的综述参见 Verma等， Human Chromosomes: a Manual of Basic Techniques, Pergamon Press, New York(1988)。
一旦序列被定位到准确的染色体位置， 此序列在染色体上的物理位 置就可以与基因图数据相关联。 这些数据可见于V.Mckusick,Mendelian Inheritance in Man (可 通 过 与 Johns Hopkins University Welch Medical L i br a ry联机获得）。 然后可通过连锁分析， 确定基因与业已 定位到染色体区域上的疾病之间的关系。
接着， 需要测定患病和未患病个体间的 cDNA或基因组序列差异。 如 果在一些或所有的患病个体中观察到某突变， 而该突变在任何正常个 体中未观察到， 则该突变可能是疾病的病因。 比较患病和未患病个体， 通常涉及首先寻找染色体中结构的变化， 如从染色体水平可见的或用 基于 cDNA序列的 PCR可检测的缺失或易位。 根据目前的物理作图和基因 定位技术的分辨能力， 被精确定位至与疾病有关的染色体区域的 cDNA， 可以是 50至 500个潜在致病基因间之一种（假定 1兆碱基作图分辨能力和 每 20kb对应于一个基因）。
可以将本发明的多肽、 多核苷酸及其模拟物、 激动剂、 拮抗剂和抑 制剂与合适的药物载体组合后使用。 这些载体可以是水、 葡萄糖、 乙 醇、 盐类、 缓冲液、 甘油以及它们的组合。 组合物包含安全有效量的 多肽或拮抗剂以及不影响药物效果的载体和赋形剂。 这些组合物可以 作为药物用于疾病治疗。
本发明还提供含有一种或多种容器的药盒或试剂盒， 容器中装有一 种或多种本发明的药用组合物成分。 与这些容器一起， 可以有由制造、 使用或销售药品或生物制品的政府管理机构所给出的指示性提示， 该 提示反映出生产、 使用或销售的政府管理机构许可其在人体上施用。 此外， 本发明的多肽可以与其它的治疗化合物结合使用。
药物组合物可以以方便的方式给药， 如通过局部、 静脉内、 腹膜内、 肌内、 皮下、 鼻内或皮内的给药途径。 gammal-吡咯啉 - 5-羟基还原酶 14 以有效地治疗和 /或预防具体的适应症的量来给药。 施用于患者的 gammal-吡咯啉 -5-羟基还原酶 14 的量和剂量范围将取决于许多因素， 如给药方式、 待治疗者的建康条件和诊断医生的判断。 实施例
下面结合具体实施例， 进一步阐述本发明。 应理解， 这些实施例仅 用于说明本发明而不用于限制本发明的范围。 下列实施例中未注明具 体条件的实验方法， 通常按照常规条件如 Sambrook 等人， 分子克隆： 实验室手册（New York: Cold Spring Harbor Laboratory Press, 1989) 中所述的条件， 或按照制造厂商所建议的条件。
实施例 1 gammal-吡咯啉 - 5-羟基还原酶 14 的克隆
用异硫氰酸胍 /酚 /氯仿一步法提取人胎脑总 RNA。 用 Quik mRNA Isolation Kit ( Qiegene 公司产品 ） 从总 RNA中分离 po 1 y (A) mRNA。
2ug poly (A) mRNA经逆转录形成 cDNA。 用 Smart cDNA克隆试剂盒 （ 购 自 Clontech ) 将 cDNA片段定向插入到 pBSK (+)载体（Clontech公司产品） 的多克隆位点上，转化 DH5 α，细菌形成 cDNA文库。用 Dye terminate cycle react ion sequencing ki t (Perkin - Elmer公司产品） 和 ABI 377自动测 序仪（Perkin- Elmer公司）测定所有克隆的 5 '和 3'末端的序列。 将测定 的 cDNA序列与 已有的公共 DNA序列数据库 （ Genebank ) 进行比较， 结果 发现其中一个克隆 0217e08的 cDNA序列为新的 DNA。 通过合成一系列引 物对该克隆所含的插入 cDNA片段进行双向测定。 结果表明， 0217e08克 隆所含的全长 cDNA为 1629bp (如 Seq ID NO: 1所示） ， 从第 835bp至 1218bp 有一个 384bp的开放阅读框架 （ 0RF) ， 编码一个新的蛋白质 （如 Seq ID N0: 2所示 ） 。 我们将此克隆命名为 pBS- 0217e08, 编码的蛋白质命名为 garamal-吡咯啉 - 5-羟基还原酶 14。 实施例 2 cDNA 克隆的结构域分析
将本发 明 的 gamraal-吡咯啉 -5-羟基还原 酶 14的序 列及其编 码的蛋白序歹1 J ,用 GCG中的 profile scan程序 （Basic local al ignment search tool) [Altschul, SF et al. J. Mol. Biol.1990; 215: 403-10] , 在 p r o s i t e 等 数 据 库 进 行 结 构 域 分 析 。 本 发 明的 garanal-吡咯啉 - 5-羟基还原酶 14在 14- 102与结构域 gammal-吡咯啉 - 5-羟基还 原酶特征蛋白有同源， 同源结果示于图 1， 同源率为 6%， 得分为 3.83; 阈值为 3.77。 实施例 3 用 RT-PCR方法克隆编码 gammal-吡咯啉 -5-羟基还原酶 14的基因 用胎脑细胞总 RNA为模板，以 oligo- dT为引物进行逆转录反应合成 cDNA,用 Qiagene的试剂盒纯化后，用下列引物进行 PCR扩增：
Primerl: 5'- CCTGATATATCCATTTTGCTTCCC —3' (SEQ ID NO: 3)
Primer2: 5'- ATTTTCAAACTTTATTTACAACTG -3' (SEQ ID NO: 4)
Primerl为位于 SEQ ID NO: 1的 5'端的第 lbp开始的正向序列；
Primer2为 SEQ ID NO: 1的中的 3'端反向序列。
扩增反应的条件： 在 50μ1的反应体积中含有 50隱 ol/L KC1， 10隱 ol/L Tris- HC1, ρΗ8· 5, 1.5mmol/L MgCl2， 200μηο1/ί dNTP, lOpmol引物， 1U的 Taq DM聚合酶 (Clontech公司产品）。 在 PE9600型 DNA热循环仪（Perkin- Elmer公司)上按下列条件 反应 25个周期： 94°C 30sec; 55°C 30sec; 72°C 2min。 在 RT- PCR时同时设 β -act in 为阳性对照和模板空白为阴性对照。 扩增产物用 QIAGEN公司的试剂盒纯化， 用 TA 克隆试剂盒连接到 pCR载体上 （ Invitrogen公司产品） 。 DNA序列分析结果表明 PCR 产物的 DM序列与 SEQ ID NO: 1所示的 1- 1629bp完全相同。 实施例 4 Northern 印迹法分析 gammal-吡咯啉 -5-羟基还原酶 14基因的表达 用一步法提取总 RNA[Anal. Biochem 1987, 162, 156-159] 0 该法包括酸性硫 氰酸胍苯酚 -氯仿抽提。 即用 4M异硫氰酸胍 -25mM柠檬酸钠， 0.2M乙酸钠 （ pH4.0 ) 对组织进行匀浆， 加入 1倍体积的苯酚和 1/5体积的氯仿-异戊醇 （49: 1 ) , 混合 后离心。 吸出水相层， 加入异丙醇 （ 0.8体积） 并将混合物离心得到 RNA沉淀。 将 得到的 RNA沉淀用 70%乙醇洗涤， 干燥并溶于水中。 用 2(^g RNA, 在含 20mM 3- ( N- 吗啉代 ) 丙磺酸 (pH7.0) - 5mM乙酸钠 - ImM EDTA-2.2M甲醛的 1.2%琼脂糖凝胶上进 行电泳。 然后转移至硝酸纤维素膜上。 用 cc- 32P dATP通过随机引物法制备 32P-标记 的 DNA探针。所用的 DNA探针为图 1所示的 PCR扩增的 gammal-吡咯啉 -5-羟基还原酶 14 编码区序列（835bp至 1218bp)。 将 32P-标记的探针 （约 2 χ 106cpm/ml ) 与转移了 RNA 的硝酸纤维素膜在一溶液中于 42°C杂交过夜， 该溶液包含 50%甲酰胺- 25mM KH2P04 (ρΗ7·4 ) - 5 xSSC- 5 xDenhardt，s溶液和 200με/ιη1鲑精 DNA。 杂交之后， 将滤膜在 1 X SSC- 0.1%SDS中于 55。C洗 30min。 然后， 用 Phosphor Imager进行分析和定量。 实施例 5 重组 gammal-吡咯啉 -5-羟基还原酶 14的体外表达、 分离和纯化 根据 SEQ ID N0: 1和图 1所示的编码区序列， 设计出一对特异性扩增引物， 序 列如下：
Priraer3: 5'- CCCCATATGATGCCGGGGCAGGAGGACGAGGGT -3' ( Seq ID No: 5 ) Primer4: 5'- CATGGATCCTTACAAGCATGCTCCCATCACACA -3' ( Seq ID No: 6 ) 此两段引物的 5'端分别含有 Ndel和 BamHI酶切位点， 其后分别为目的基因 5'端 和 3'端的编码序列 ， Ndel和 BamHI酶切位点相应于表达载体质粒 pET- 28b(+) (Novagen公司产品， Cat. No.69865.3)上的选择性内切酶位点。 以含有全长 目的基因的 pBS-0217e08质粒为模板， 进行 PCR反应。 PCR反应条件为： 总体积 50μ1 中含 pBS— 0217e08质粒 10pg、 引物 Primer—3和 Pr imer_4分别为 10pmol、 Advantage polymerase Mix (Clontech公司产品） 1μ1。 循环参数： 94°C 20s, 60°C 30s, 68°C 2 mi n，共 25个循环。 用 Nde I和 BamH I分别对扩增产物和质粒 pET- 28 (+)进行双酶切，分 别回收大片段，并用 T4连接酶连接。 连接产物转化用氯化钙法大肠杆细菌 DH5cx,在 含卡那霉素 （终浓度 3(^g/ml ) 的 LB平板培养过夜后， 用菌落 PCR方法筛选阳性克 隆， 并进行测序。 挑选序列正确的阳性克隆 （pET-0217e08)用氯化钙法将重组质 粒转化大肠杆菌 BL21(DE3)plySs(Novagen公司产品）。 在含卡那霉素 （终浓度 30μΒ/ηι1 ) 的 LB液体培养基中， 宿主菌 BL21 ( PET-0217e08 ) 在 37°C培养至对数生 长期， 加入 IPTG至终浓度 lmmol/L, 继续培养 5小时。 离心收集菌体， 经超声波破 菌，离心收集上清液， 用能与 6个组氨酸 （6His- Tag) 结合的亲和层析柱 His. Bind Quick Cartridge ( Novagen公司产品） 进行层析， 得到了纯化的目的蛋白 gammal- 吡咯啉 -5-羟基还原酶 14。 经 SDS-PAGE电泳， 在 14kDa处得到一单一的条带（图 2 ) 。 将该条带转移至 PVDF膜上用 Edams水解法进行 N-端氨基酸序列分析， 结果 N-端 15个 氨基酸与 SEQ ID NO: 2所示的 N-端 15个氨基酸残基完全相同。 实施例 6 抗 gammal-吡咯啉 -5-羟基还原酶 14抗体的产生
用多肽合成仪 （PE公司产品） 合成下述 gammal-吡咯啉 _5 -羟基还原酶 14特异 性的多肽：
NH2-Met-Pr o-G 1 y-G 1 n-G 1 u-A s -G 1 u-G ly-Ala-Leu-Tr -Thr-G 1 n-G ln-Ser- COOH (SEQ ID NO: 7)。 将该多肽分别与血蓝蛋白和牛血清白蛋白耦合形成复合物， 方法参见： Avrameas, et al. Imnumochemistry, 1969; 6: 43。 用 4mg上述血蓝蛋白 多肽复合物加上完全弗氏佐剂免疫家兔， 15天后再用血蓝蛋白多肽复合物加不完 全弗氏佐剂加强免疫一次。 采用经 15 g/ml牛血清白蛋白多肽复合物包被的滴定板 做 ELISA测定兔血清中抗体的滴度。 用蛋白 A-Sepharose从抗体阳性的家兔血清中 分离总 IgG。 将多肽结合于溴化氰活化的 Sepharose4B柱上， 用亲和层析法从总 I gG 中分离抗多肽抗体。 免疫沉淀法证明纯化的抗体可特异性地与 gamma l-吡咯啉- 5 - 羟基还原酶 14结合。 实施例 7 本发明的多核苷酸片段用作杂交探针的应用
从本发明的多核苷酸中挑选出合适的寡核苷酸片段用作杂交探针有多方面的 用途， 如用该探针可与不同来源的正常组织或病理组织的基因组或 cDNA文库杂交 以鉴定其是否含有本发明的多核苷酸序列和检出同源的多核苷酸序列，进一步还可 用该探针检测本发明的多核苷酸序列或其同源的多核苷酸序列在正常组织或病理 组织细胞中的表达是否异常。
本实施例的目的是从本发明的多核苷酸 SEQ ID NO: 1 中挑选出合适的寡核苷 酸片段用作杂交探针， 并用滤膜杂交方法鉴定一些组织中是否含有本发明的多核 苷酸序列或其同源的多核苷酸序列。 滤膜杂交方法包括斑点印迹法、 Southern 印 迹法、 Nor thern 印迹法和复印方法等， 它们都是将待测的多核苷酸样品固定在滤 膜上后使用基本相同的步骤杂交。 这些相同的步骤是： 固定了样品的滤膜首先用 不含探针的杂交缓冲液进行预杂交， 以使滤膜上样品的非特异性的结合部位被载 体和合成的多聚物所饱和。 然后预杂交液被含有标记探针的杂交缓冲液替换， 并 保温使探针与靶核酸杂交。 杂交步骤之后， 未杂交上的探针被一系列洗膜步骤除 掉。 本实施例利用较高强度的洗膜条件 （如较低盐浓度和较高的温度）， 以使杂交 背景降低且只保留特异性强的信号。 本实施例选用的探针包括两类： 第一类探针 是完全与本发明的多核苷酸 SEQ ID NO: 1相同或互补的寡核苷酸片段； 第二类探 针是部分与本发明的多核苷酸 SEQ ID NO: 1相同或互补的寡核苷酸片段。 本实施 例选用斑点印迹法将样品固定在滤膜上， 在较高强度的的洗膜条件下， 第一类探 针与样品的杂交特异性最强而得以保留。
一、 探针的选用
从本发明的多核苷酸 SEQ ID NO: 1 中选择寡核苷酸片段用作杂交探针， 应遵 循以下原则和需要考虑的几个方面：
1 , 探针大小优选范围为 18-50个核苷酸；
2 , GC含量为 30%- 70%, 超过则非特异性杂交增加；
3 , 探针内部应无互补区域；
4 , 符合以上条件的可作为初选探针， 然后进一步作计算机序列分析， 包 括将该初选探针分别与其来源序列区域 （即 SEQ ID NO: 1 ) 和其它已知的基因组 序列及其互补区进行同源性 较， 若与非靶分子区域的同源性大于 85%或者有超过 15个连续碱基完全相同， 则该初选探针一般就不应该使用；
5， 初选探针是否最终选定为有实际应用价值的探针还应进一步由实验确 定。
完成以上各方面的分析后挑选并合成以下二个探针：
探针 1 (probel ), 属于第一类探针， 与 SEQ ID NO: 1 的基因片段完全同源 或互朴 (41Nt )
5'- TGCCGGGGCAGGAGGACGAGGGTGCGCTGTGGACACAGCAG -3' ( SEQ ID NO: 8 ) 探针 2 ( probe2 ), 属于第二类探针， 相当于 SEQ ID NO: 1 的基因片段或其 互补片段的替换突变序列 (41Nt):
5'- TGCCGGGGCAGGAGGACGAGAGTGCGCTGTGGACACAGCAG -3' ( SEQ ID NO: 9) 与以下具体实验步骤有关的其它未列出的常用试剂及其配制方法请参考文 献： DM PROBES G. Η· Keller;M.M.Manak; Stockton Press, 1989 (USA)以及更常 用的分子克隆实验手册书籍如 《分子克隆实验指南》（1998年第二版） [美]萨姆布 鲁克等著， 科学出版社。
样品制备
1， 从新鲜或冰冻组织中提取 DNA
步骤： 1 ) 将新鲜或新鲜解冻的正常肝组织放入浸在冰上并盛有磷酸盐缓冲液 (PBS) 的平皿中。 用剪刀或手术刀将组织切成小块。 操作中应保持组织湿润。 2) 以 lOOOg离心切碎组织 10分钟。 3)用冷匀浆缓冲液 ( 0.25mol/L蔗糖； 25讓 ol/L Tris-HCl, pH7.5; 25mmol/L NaCl; 25mmol/L MgCl2 ) 悬浮沉淀 （大约 lOml/g )。 4) 在 4°C 用电动匀浆器以全速匀浆组织悬液， 直至组织被完全破碎。 5) lOOOg 离心 10分钟。 6)用重悬细胞沉淀（每 0. lg最初组织样品加 l-5tnl ), 再以 1000g离心 10分钟。 7)用裂解缓冲液重悬沉淀 （每 0. lg最初组织样品加 lral ), 然后接以下 的苯酚抽提法。
2， DNA的苯酚抽提法
步骤： 1 )用 1- 10ml冷 PBS 洗细胞， 1000g离心 10分钟。 2 )用冷细胞裂解 液重悬浮沉淀的细胞 U X 108细胞 /ml ) 最少应用 lOOul 裂解缓冲液。 3)加 SDS 至终浓度为 1%, 如果在重悬细胞之前将 SDS 直接加入到细胞沉淀中， 细胞可能会 形成大的团块而难以破碎， 并降低总产率。 这一点在抽提 >107细胞时特别严重。 4) 加蛋白酶 K至终浓度 200ug/ml。 5 ) 50°C保温反应 1小时或在 37°C轻轻振摇过夜。 6 )用等体积苯酚： 氯仿： 异戊醇 （ 25: 24： 1 )抽提， 在小离心机管中离心 10分 钟。 两相应清楚分离， 否则重新进行离心。 7) 将水相转移至新管。 8) 用等体积 氯仿： 异戊醇 （24： 1 )抽提， 离心 10分钟。 9 )将含 DM的水相转移至新管。 然 后进行 DNA的纯化和乙醇沉淀。
3， DNA的纯化和乙醇沉淀
步骤： 1 ) 将 1/10体积 2mol/L醋酸钠和 2倍体积冷 100%乙醇加到 DNA溶液 中， 混匀。 在- 20°C放置 1 小时或过夜。 2) 离心 10分钟。 3)小心吸出或倒出乙 醇。 4)用 70%冷乙醇 500ul洗涤沉淀， 离心 5分钟。 5)小心吸出或倒出乙醇。 用 500ul 冷乙醇洗涤沉淀， 离心 5 分钟。 6) 小心吸出或倒出乙醇， 然后在吸水纸上 倒置使残余乙醇流尽。 空气干燥 10-15 分钟， 以使表面乙醇挥发。 注意不要使沉 淀完全干燥， 否则较难重新溶解。 7) 以小体积 TE或水重悬 DNA 沉淀。 低速涡旋 振荡或用滴管吹吸， 同时逐渐增加 TE, 混合至 DM充分溶解， 每 1-5 χ 106细胞所 提取的大约加 lul。
以下第 8_13步骤仅用于必须除去污染时， 否则可直接进行第 14步骤。
8)将 RNA酶 A加到 DNA溶液中， 终浓度为 100ug/ml , 37°C保温 30分钟。 9) 加入 SDS和蛋白酶 K, 终浓度分别为 0.5%和 100ug/ml。 37°C保温 30分钟。 10) 用等体积的苯酚： 氯仿： 异戊醇 （ 25: 24： 1 ) 抽提反应液， 离心 10 分钟。 11 ) 小心移出水相， 用等体积的氯仿： 异戊醇 （24: 1 ) 重新抽提， 离心 10分钟。 12) 小心移出水相， 加 1 0体积 2mol/L醋酸钠和 2.5体积冷乙醇， 混匀置 - 20°C 1小 时。 13) 用 70%乙醇及 100%乙醇洗涤沉淀， 空气干燥， 重悬核酸， 过程同第 3 - 6 步骤。 14 ) 测定 A26。和 A28。以检测 DNA的纯度及产率。 15 )分装后存放于 -20° (。
样膜的制备
1 )取 4 x 2 张适当大小的硝酸纤维素膜 （NC 膜）， 用铅笔在其上轻轻标出 点样位置及样号， 每一探针需两张 NC膜， 以便在后面的实验步骤中分别用高强度 条件和强度条件洗膜 。
2 ) 吸取及对照各 15微升， 点于样膜上， 在室温中晾干。
3 ) 置于浸润有 0. lmol/L NaOH, 1.5mol/L NaCl的滤纸上 5分钟 (两次 ), 晾干置于浸润有 0.5mol/L Tris-HCl ( pH7.0 ), 3mol/L NaCl 的滤纸上 5分钟 （两 次）， 晾干。
4) 夹于干净滤纸中， 以铝箔包好， 60- 80。C真空干燥 2小时。
探针的标记
1 )3μ1 Probe( 0. IOD/Ιθμΐ )，加入 2 lKinase缓冲液， 8-10 uCi γ-32Ρ- dATP+2U Kinase, 以补加至终体积 20μ1。
2 ) 37 °C 保温 2小时。
3)加 1/5体积的溴酚蓝指示剂 （BPB)。
4 ) 过 Sephadex G-50柱。
5 ) 至有 32P-Probe洗出前开始收集第一峰 （可用 Monitor监测）。
6 ) 5滴 /管， 收集 10- 15管。
7 )用液体闪烁仪监测同位素量。
8 )合并第一峰的收集液后即为所需制备的 "P-Probe (第二峰为游离 γ-32Ρ - dATP )。
预杂交
将样膜置于塑料袋中， 加入 3- 10mg 预杂交液 （ lOxDenhardt's; 6xSSC, 0. lmg/ml CT DNA (小牛胸腺 DNA ) )。 封好袋口后， 68。C水浴摇 2小时。
杂交
将塑料袋剪去一角， 加入制备好的探针， 封好袋口后， 42°C水洛摇过夜。 洗膜：
高强度洗膜：
1 )取出已杂交好的样膜。
2 ) 2xSSC, 0.1%SDS中， 40。C洗 15分钟 （ 2次）。
3 ) 0. lxSSC, 0.1%SDS中， 40°C洗 15分钟 ( 2次）。
4 ) 0. lxSSC, 0.1%SDS中， 55°C洗 30分钟 （ 2次）， 室温晾干。 低强度洗膜-.
1 )取出已杂交好的样膜。
2 ) 2xSSC， 0.1%SDS中， 37°C洗 15分钟 ( 2次）。
3 ) 0. lxSSC, 0.1%SDS中， 37°C洗 15分钟 （ 2次）。
4) 0. lxSSC, 0.1%SDS中， 40°C洗 15分钟 （ 2次）， 室温晾干。
X -光自显影：
-70°C, X-光自显影 （压片时间根据杂交斑放射性强弱而定）。
实验结果：
采用低强度洗膜条件所进行的杂交实验， 以上两个探针杂交斑放射性强弱没 有明显区别； 而采用高强度洗膜条件所进行的杂交实验， 探针 1 的杂交斑放射性 强度明显强于另一个探针杂交斑的放射性强度。 因而可用探针 1 定性和定量地分 析本发明的多核苷酸在不同组织中的存在和差异表达。 实施例 8 DNA Microarray
基因芯片或基因微矩阵 （DNA Microarray)是目前许多国家实验室和大制药 公司都在着手研制和开发的新技术， 它是指将大量的靶基因片段有序地、 高密度 地排列在玻璃、 硅等载体上， 然后用荧光检测和计算机软件进行数据的比较和分 析， 以达到快速、 高效、 高通量地分析生物信息的目的。 本发明的多核苷酸可作 为靶 DNA 用于基因芯片技术用于高通量研究新基因功能； 寻找和筛选组织特异性 新基因特别是肿瘤等疾病相关新基因； 疾病的诊断， 如遗传性疾病。 其具体方法 步骤在文献中已有多种报道， 如可参阅文献 DeRisi, J. L. , Lyer, V. &Brown, P.0.
(1997) Science 278, 680-686.及文献 Helle, R. A.， Schema, M.， Chai, A. , Shalom, D.，
(1997) PNAS 94: 2150-2155.
(一） 点样
各种不同的全长 cDNA共计 4000条多核苷酸序列作为靶 DNA,其中包括本发明 的多核苷酸。 将它们分别通过 PCR 进行扩增， 纯化所得扩增产物后将其浓度调到 500ng/ul左右， 用 Cartesian 7500点样仪（购自美国 Cartesian公司）点于玻璃介 质上， 点与点之间的距离为 280μηι。 将点样后的玻片进行水合、 干燥， 置于紫外 交联仪中交联， 洗脱后干燥使 DNA 固定在玻璃片上制备成芯片。 其具体方法步骤 在文献中已有多种报道。 本实施例的点样后处理步骤是：
1. 潮湿环境中水合 4小时；
2. 0.2%SDS洗涤 1分钟；
3. ddH20洗涤两次， 每次 1分钟；
4. NaBH4封闭 5分钟；
5. 95°C水中 2分钟；
6. 0.2%SDS洗涤 1分钟；
7. ddH20冲洗两次；
8. 凉干， 25°C储存于暗处备用。
(二）探针标记
用一步法分别从正常肝与肝癌中抽提总 mRNA,并用 OligotexmRNAMidi Kit (购 自 QiaGen 公司）纯化 mRNA，通过反转录分别将荧光试剂 Cy3dUTP (5- Amino- propargy 1-2' -deoxyur i dine 5'-tr iphate coupled to Cy3 fluorescent dye, 购 自 Amersham Phamacia Biotsch 公司）标记正常肝组织的 mRNA, 用荧光试剂 Cy5dUTP (5-Amino-propargyl-2'-deoxyur idine 5'-triphate coupled to Cy5 fluorescent dye, 购自 Amersham Phamacia Biotech公司）标记肝癌组织 mRNA, 经纯化后制备出探针。 具体步骤参照及方法见
Schena, M.， Shalon, D. , Heller, R. (1996) Proc. Natl. Acad. Sci. US Vol.93: 10614-10619.
Schena, M. , Shalon, Dari. , Davis, R. W. (1995) Science.270 (20)： 467-480. (三） 杂交
分别将来自以上两种组织的探针与芯片一起在 UniHyb™ Hybridization Solution (购自 TeleChem 公司）杂交液中进行杂交 16 小时， 室温用洗涤液 （1 χ SSC, 0.2%SDS ) 洗涤后用 ScanArray 3000扫描仪 （购自美国 General Scanning公 司）进行扫描， 扫描的图象用 Imagene软件 （美国 Biodiscovery公司） 进行数据 分析处理， 算出每个点的 Cy3/Cy5 比值， 该比值小于 0.5 大于 2 的点被认为是表 达有差异的基因。
实验结果表明， Cy3 signal=9321.82 (取四次实验的平均值） ， Cy5 加税 signal=3768.1 (取四次实验的平均值） ， Cy3/Cy5=2.473878，本发明的多核苷酸在 以上两种组织中的表达有明显差异， 表明本发明的多核苷酸与肝癌相关。

Claims

权利要求
1、 一种分离的多肽 - gammal-吡咯啉 -5-羟基还原酶 14 , 其特征在于它包含 有： SEQ ID NO: 2所示的氨基酸序列的多肽、 或其多肽的活性片段、 类似物或衍生 物。
2、 如权利要求 1所述的多肽， 其特征在于所述多肽、 类似物或衍生物的氨基 酸序列具有与 SEQ ID NO: 2所示的氨基酸序列至少 95%的相同性。
3、 如权利要求 2所述的多肽， 其特征在于它包含具有 SEQ ID NO: 2所示的氨 基酸序列的多肽。
4、 一种分离的多核苷酸， 其特征在于所述多核苷酸包含选自下组中的一种：
(a) 编码具有 SEQ ID NO: 2 所示氨基酸序列的多肽或其片段、 类似物、 衍 生物的多核苷酸；
(b) 与多核苷酸 ) 互补的多核苷酸； 或
(c) 与 （a )或 （b ) 有至少 70%相同性的多核苷酸。
5、 如权利要求 4 所述的多核苷酸， 其特征在于所述多核苷酸包含编码具 有 SEQ ID NO: 2所示氨基酸序列的多核苷酸。
6、如权利要求 4所述的多核苷酸，其特征在于所述多核苷酸的序列包含有 SEQ ID NO: 1 中 835- 1218位的序列或 SEQ ID NO: 1中 1-1629位的序列。
7、 一种含有外源多核苷酸的重组载体， 其特征在于它是由权利要求 4-6 中的 任一权利要求所述多核苷酸与质粒、 病毒或运载体表达载体构建而成的重组载体。
8、 一种含有外源多核苷酸的遗传工程化宿主细胞， 其特征在于它是选自于下 列一种宿主细胞：
(a) 用权利要求 7所述的重组载体转化或转导的宿主细胞； 或
(b) 用权利要求 4- 6中的任一权利要求所述多核苷酸转化或转导的宿主细 胞。
9、 一种具有 gamma l-吡咯啉 -5-羟基还原酶 14活性的多肽的制备方法， 其特 征在于所述方法包括：
(a) 在表达 ga隱 al-吡咯啉 - 5-羟基还原酶 14 条件下， 培养权利要求 8 所述 的工程化宿主细胞；
(b) 从培养物中分离出具有 ga麵 al-吡咯啉 -5-羟基还原酶 14活性的多肽。
10、 一种能与多肽结合的抗体，其特征在于所述抗体是能与 gamma l-吡咯啉- 5- 羟基还原酶 14特异性结合的抗体。
π、 一类模拟或调节多肽活性或表达的化合物， 其特征在于它们是模拟、 促 进、 拮抗或抑制 gammal-吡咯啉 -5-羟基还原酶 14的活性的化合物。
12、 如权利要求 11所述的化合物， 其特征在于它是 SEQ ID N0: 1所示的多核 苷酸序列或其片段的反义序列。
13、 一种权利要求 11 所述化合物的应用， 其特征在于所述化合物用于调节 gammal-吡咯啉 -5-羟基还原酶 14在体内、 体外活性的方法。
14、 一种检测与权利要求 1-3 中的任一权利要求所述多肽相关的疾病或疾病 易感性的方法， 其特征在于其包括检测所述多肽的表达量， 或者检测所述多肽的 活性， 或者检测多核苷酸中引起所述多肽表达量或活性异常的核苷酸变异。
15、 如权利要求 1-3 中的任一权利要求所述多肽的应用， 其特征在于它应用 于筛选 gammal-吡咯啉 -5-羟基还原酶 14的模拟物、 激动剂， 拮抗剂或抑制剂； 或 者用于肽指紋图谱鉴定。
16、 如权利要求 4-6 中的任一权利要求所述的核酸分子的应用， 其特征在于 它作为引物用于核酸扩增反应， 或者作为探针用于杂交反应， 或者用于制造基因 芯片或微阵列。
17、 如权利要求 1-6及 11中的任一权利要求所述的多肽、 多核苷酸或化合物 的应用， 其特征在于用所述多肽、 多核苷酸或其模拟物、 激动剂、 拮抗剂或抑制 剂以安全有效剂量与药学上可接受的载体组成作为诊断或治疗与 gammal-吡咯啉- 5 -羟基还原酶 14异常相关的疾病的药物组合物。
18、 权利要求 1-6及 11 中的任一权利要求所述的多肽、 多核苷酸或化合物的 应用， 其特征在于用所述多肽、 多核苷酸或化合物制备用于治疗如恶性肿瘤， 血 液病， HIV感染和免疫性疾病和各类炎症的药物。
PCT/CN2000/000695 1999-12-27 2000-12-25 Nouveau polypeptide, gamma1-pyroline-5-hydroxy reductase 14, et polynucleotide codant pour ce polypeptide WO2001048168A1 (fr)

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CN 99125800 CN1301851A (zh) 1999-12-27 1999-12-27 一种新的多肽——gammal－吡咯啉－5－羟基还原酶14和编码这种多肽的多核苷酸
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