WO2001085903A2 - A novel polypeptide- human protein 9 which contains a p5cr characteristic sequence fragment and a polynucleotide encoding the same - Google Patents

A novel polypeptide- human protein 9 which contains a p5cr characteristic sequence fragment and a polynucleotide encoding the same Download PDF

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Publication number
WO2001085903A2
WO2001085903A2 PCT/CN2001/000590 CN0100590W WO0185903A2 WO 2001085903 A2 WO2001085903 A2 WO 2001085903A2 CN 0100590 W CN0100590 W CN 0100590W WO 0185903 A2 WO0185903 A2 WO 0185903A2
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Prior art keywords
polypeptide
polynucleotide
p5cr
sequence
characteristic sequence
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PCT/CN2001/000590
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English (en)
French (fr)
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WO2001085903A3 (fr
Inventor
Yumin Mao
Yi Xie
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Biowindow Gene Development Inc. Shanghai
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Priority to AU70433/01A priority Critical patent/AU7043301A/en
Publication of WO2001085903A2 publication Critical patent/WO2001085903A2/zh
Publication of WO2001085903A3 publication Critical patent/WO2001085903A3/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals

Definitions

  • the present invention belongs to the field of biotechnology. Specifically, the present invention describes a new polypeptide, a human protein 9 containing a characteristic sequence fragment of P5CR, and a polynucleotide sequence encoding the polypeptide. The invention also relates to a preparation method and application of the polynucleotide and the polypeptide. Background technique
  • 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-hydroxy reductase is widely distributed in the biological world, and it exists in 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)-[LIVM]-[STAC]-[STV] -X- [GAN] -GXTX (2)-[AG]-[LIV] -X (2)-[LMF ]-[DENQK]; ⁇ 1-pyrroline-5-hydroxy reductase 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 inactivation of the enzyme Thus, it affects the final synthesis of proline and affects a series of related biological processes.
  • This protein is closely related to the
  • the human protein 9 protein containing the characteristic sequence fragment of P5CR plays an important role in important functions in the body, and it is believed that a large number of proteins are involved in these regulatory processes. Therefore, identification in the art has been required. More people involved in these processes have protein 9 protein containing P5CR characteristic sequence fragments, especially the amino acid sequence of this protein is identified. Isolation of the protein 9 protein encoding gene containing the characteristic sequence fragment of P5CR in newcomers also provides a basis for research to determine the role of this protein in health and disease states. This protein may form the basis for the development of diagnostic and / or therapeutic drugs for diseases, so it is important to isolate its coding for DM. Disclosure of invention
  • Another object of the invention is to provide a polynucleotide encoding the polypeptide.
  • Another object of the present invention is to provide a recombinant vector containing a polynucleotide encoding a human protein 9 containing a fragment containing a characteristic sequence of P5CR.
  • Another object of the present invention is to provide mimic compounds, antagonists, agonists, and inhibitors directed to protein 9 of a polypeptide of the present invention, which contains a P5CR characteristic sequence fragment.
  • Another object of the present invention is to provide a method for diagnosing and treating diseases related to abnormalities of human protein 9 containing a characteristic sequence fragment of P5CR.
  • 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 1188 to 1433 in SEQ ID NO: 1; and (b) a sequence having positions 1 to 3878 in SEQ ID NO: 1 Sequence of bits.
  • the present invention further relates to a vector, particularly an expression vector, containing the polynucleotide of the present invention; a host cell genetically engineered with the vector, including a transformed, transduced or transfected host cell; A method of preparing the polypeptide of the present invention by culturing the host cell and recovering the expressed product.
  • the invention also relates to an antibody capable of specifically binding to a polypeptide of the invention.
  • the invention also relates to a method for screening a compound that mimics, activates, antagonizes or inhibits the activity of a protein 9 protein containing a characteristic sequence fragment of P5CR, which comprises utilizing the polypeptide of the invention.
  • the invention also relates to compounds obtained by this method.
  • the invention also relates to a method for detecting a disease or disease susceptibility related to abnormal expression of protein 9 protein containing human P5CR characteristic sequence fragments in vitro, which comprises detecting mutations in the polypeptide or a polynucleotide sequence encoding the same in a biological sample. Or detecting 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 polypeptides and / or polynucleotides of the present invention in the preparation of a medicament for treating cancer, developmental or immune diseases, or other diseases caused by abnormal expression of protein 9 containing human P5CR characteristic sequence fragments. .
  • Nucleic acid sequence refers to an oligonucleotide, a nucleotide or a polynucleotide and a fragment or part thereof, and may also refer to a genomic or synthetic DNA 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” or “addition” refers to an alteration in the amino acid sequence or nucleotide sequence that results in an increase in one or more amino acids or nucleotides compared to a naturally occurring molecule. "Replacement” refers to the replacement of one or more amino acids or nucleotides with different amino acids or nucleotides.
  • Biological activity refers to a protein that has the structure, regulation, or biochemical function of a natural molecule.
  • the term “immunologically active” refers to the ability of natural, recombinant, or synthetic proteins and fragments thereof to induce a specific immune response and to bind specific antibodies in a suitable animal or cell.
  • An "agonist” refers to a molecule that, when combined with human protein 9 containing a characteristic sequence fragment of P5CR, 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 binds to human protein 9 containing a P5CR characteristic sequence fragment.
  • Antagonist refers to a biological or immunological activity that can block or modulate human protein 9 containing P5CR characteristic sequence fragments when combined with human protein 9 containing P5CR characteristic sequence fragments.
  • Molecule Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates, or any other molecule that binds to human protein 9 containing P5CR characteristic sequence fragments.
  • Regular refers to a change in the function of human protein 9 containing a characteristic sequence fragment of P5CR, including an increase or decrease in protein activity, a change in binding characteristics, and any other biological properties of human protein 9 containing a characteristic sequence fragment of P5CR. , 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 use standard protein purification techniques to purify human proteins containing P5CR characteristic sequence fragments.
  • a substantially pure human protein 9 containing P5CR characteristic sequence fragments can generate a single main band on a non-reducing polyacrylamide gel.
  • the purity of human protein 9 containing characteristic sequence fragments of P5CR can be analyzed by amino acid sequence .
  • Complementary refers to the natural binding of a nucleotide by base-pairing under conditions of acceptable salt concentration and temperature.
  • sequence "C-T-G-A” can be combined with the complementary sequence "G-A-C-T”.
  • the complementarity between two single-stranded molecules may be partial or complete.
  • the degree of complementarity between nucleic acid strands has a significant effect on the efficiency and strength of hybridization between nucleic acid strands.
  • “Homology” 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 specifically or selectively.
  • 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. The percent identity can be determined electronically, such as by the MEGALIGN program (La sergene sof tware package, DNASTAR, Inc., Madi son Wis.). The MEGALIGN program can be Methods such as the Clus ter method compare two or more sequences (Higg ins, DG and PM Sharp (1988) Gene 73: 237-244). 0 The Clus ter method arranges groups 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 sequence A and sequence X 100
  • the percent identity between nucleic acid sequences can also be determined by the Clus ter method or by a method known in the art such as Jotun He in. (He in J., (1990) Methods in erazumology 183: 625-645) 0
  • 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; having an uncharged head group is Similar hydrophilic amino acids may include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; serine and threonine; phenylalanine and tyrosine.
  • Antisense refers to a nucleotide sequence that is complementary to a particular DNA or RNA sequence.
  • Antisense strand refers to a nucleic acid strand that is complementary to the “sense strand”.
  • Derivative refers to a chemical modification of HFP or a nucleic acid encoding it. Such a chemical modification may be the replacement of a hydrogen atom with an alkyl group, an acyl group or an amino group. Nucleic acid derivatives can encode polypeptides that retain the main biological characteristics of natural molecules.
  • Antibody refers to a complete antibody molecule and its fragments, such as Fa,? ( ⁇ ') 2 and?, which can specifically bind to human epitopes of protein 9 containing a characteristic sequence fragment of P5CR.
  • a “humanized antibody” refers to an antibody in which the amino acid sequence of a non-antigen binding region is replaced to become more similar to a human antibody, but still retains the original binding activity.
  • isolated refers to the removal of a substance from its original environment (for example, its natural environment if it occurs naturally).
  • 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 with it 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 natural Matter, 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 human P5CR characteristic sequence fragment-containing protein 9 refers to human P5CR characteristic sequence fragment-containing protein 9 that is substantially free of other proteins, lipids, sugars, or other substances with which it is naturally associated.
  • Those skilled in the art can use standard protein purification techniques to purify human proteins containing P5CR characteristic sequence fragments 9.
  • Substantially pure polypeptides can produce a single main band on a non-reducing polyacrylamide gel.
  • the purity of the human protein 9 polypeptide containing the characteristic sequence fragment of P5CR can be analyzed by amino acid sequence.
  • the present invention provides a new polypeptide, a human protein 9 containing a characteristic sequence fragment of P5CR, 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.
  • polypeptide of the invention may be glycosylated, or it may be non-glycosylated.
  • the polypeptides of the invention may also include or exclude the initial methionine residue.
  • the invention also includes fragments, derivatives and analogs of human protein 9 containing fragments characteristic of the P5CR sequence.
  • fragment refers to a polypeptide that substantially maintains the same biological function or activity of the human P5CR characteristic sequence-containing fragment 9 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
  • the amino acid may or may not be encoded by a genetic codon; 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 in which a mature polypeptide is fused to another compound (such as a compound that extends the half-life of a polypeptide, such as polyethylene glycol); or (IV) a type of polypeptide sequence in which an additional amino acid sequence is fused into a mature polypeptide (such as the leader sequence or secreted sequence or the sequence used to purify this polypeptide or protease sequence)
  • an additional amino acid sequence is fused into a mature polypeptide (such as the leader sequence or secreted sequence or the sequence used to purify
  • the present invention provides an isolated nucleic acid (polynucleotide), which basically consists of a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • the polynucleotide sequence of the present invention includes the nucleotide sequence of SEQ ID NO: 1.
  • the polynucleotide of the present invention is found from a cDNA library of human fetal brain tissue. It contains a polynucleotide sequence of 3878 bases in length and its open reading frame (1188-1433) encodes 81 amino acids.
  • This polypeptide has the characteristic sequence of P5CR. It can be deduced that the human protein 9 containing the characteristic sequence fragment of P5CR has the structure and function represented by P5CR.
  • the polynucleotide of the present invention may be in the form of DNA or RM.
  • 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 the 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 70% identity between the two sequences).
  • the present invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the present invention under stringent conditions.
  • "strict conditions” means: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2xSSC, 0.1 ° /.
  • 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, most preferably at least 100 nuclei. Glycylic acid or more. Nucleic acid fragments can also be used in nucleic acid amplification techniques (such as PCR) to identify and / or isolate polynucleotides encoding human protein 9 containing P5CR characteristic sequence fragments.
  • 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 of the present invention encoding human protein 9 containing a characteristic sequence fragment of P5CR 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 genomic or cDNA libraries with probes to detect homologous polynucleotide sequences Columns, and 2) antibody screening of expression libraries to detect cloned polynucleotide fragments having common structural characteristics.
  • the DNA 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 DM 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.
  • 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 human transcripts of protein 9 containing P5CR characteristic sequence fragments Level; (4) detecting protein products of gene expression by immunological techniques or measuring biological activity. The above methods can be used singly or in combination.
  • the probe used for hybridization is homologous to any part of the polynucleotide of the present invention, and its length is at least 10 nucleotides, preferably at least 30 nucleotides, more preferably At least 50 nucleotides, preferably at least 100 nucleotides.
  • the length of the probe is usually within 2000 nucleotides, preferably within 1000 nucleotides.
  • the probe used here is generally a DNA sequence chemically synthesized based on the gene sequence information of the present invention.
  • the genes or fragments of the present invention can of course be used as probes.
  • DNA probes can be labeled with radioisotopes, luciferin, or enzymes (such as alkaline phosphatase).
  • the protein product of human protein 9 gene containing P5CR characteristic sequence fragments can be detected by immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA).
  • immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA).
  • a method using PCR technology to amplify DNA / RNA is preferably used to obtain the gene of the present invention.
  • the RACE method RACE-Rapid Amplification of cDNA Ends
  • the primers used for PCR can be appropriately based on the polynucleotide sequence information of the present invention disclosed herein. Select and synthesize using conventional methods.
  • the amplified 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 DM fragments and the like obtained as described above can be determined 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 must 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 produced by genetic engineering using the vector of the present invention or directly using a human protein 9 coding sequence containing a characteristic sequence fragment of P5CR, and recombinant technology to produce the Said method of polypeptide.
  • a polynucleotide sequence encoding a human protein 9 containing a characteristic sequence fragment of P5CR can be inserted into a vector to constitute a recombinant vector containing the polynucleotide of the present invention.
  • vector refers to bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors well known in the art.
  • Vectors suitable for use in the present invention include, but are not limited to: T7 promoter-based expression vectors (Rosenberg, et al.
  • any plasmid and vector can be used to construct a recombinant expression vector.
  • An important feature of expression vectors is that they usually contain an origin of replication, a promoter, a marker gene, and translational regulatory elements.
  • Methods known to those skilled in the art can be used to construct expression vectors containing a DNA sequence encoding a human protein 9 fragment containing a characteristic sequence of P5CR and suitable transcription / translation regulatory elements. These methods include in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombination technology (Sambroook, et al. Mo l ecu l ar C l on i ng, a Labora tory Manua l, co ld Spr ing Harbor Labora tory. New York, 1989).
  • the DNA sequence can be operably linked to an appropriate promoter in the expression vector to guide mRNA synthesis. Representative examples of these promoters are: the l ac or trp promoter of E.
  • the expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. 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. Examples include 100 to 270 base pairs of SV40 enhancer at a later stage of the replication initiation point, polyoma enhancer and adenovirus enhancer at the late side of the replication initiation point.
  • 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 for eukaryotic cell culture. And green fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.
  • selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance for eukaryotic cell culture.
  • GFP green fluorescent protein
  • tetracycline or ampicillin resistance for E. coli.
  • a polynucleotide encoding human protein 9 containing a characteristic sequence fragment of P5CR or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to form a genetic engineering containing the polynucleotide or the recombinant vector.
  • Host cell refers to a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples are: E.
  • coli Streptomyces
  • bacterial cells such as Salmonella typhimurium
  • fungal cells such as yeast
  • plant cells insect cells
  • fly S 2 or Sf 9 animal cells
  • animal cells such as CH0, COS or Bowes s melanoma cells Wait.
  • 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 DNA can be harvested after the exponential growth phase and treated with the 0.1 (12 ) method. The steps used are well known in the art.
  • MgC l 2 If necessary, transformation can also be performed by electroporation.
  • the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, or conventional mechanical methods such as microinjection, electroporation, and lipids. Body packaging, etc.
  • the polynucleotide sequence of the present invention can be used to express or produce recombinant human P5CR-containing characteristic sequence fragment protein 9 (Scence, 1984; 224: 1431). Generally speaking, 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.
  • recombinant proteins can be isolated and purified by various separation methods using their 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 chromat
  • FIG. 1 is a comparison diagram of the amino acid sequence homology of 78 amino acids from 2-79 in the protein 9 containing the characteristic sequence fragment of the P5CR of the present inventors and the characteristic domain of the P5CR.
  • the upper sequence is human protein 9 containing the characteristic sequence fragment of P5CR, and the lower sequence is the characteristic domain of P5CR.
  • Identical amino acids are represented by single-character amino acids between the two sequences, and similar amino acids are represented by "+”.
  • Figure 2 shows the polyacrylamide gel electrophoresis of human protein 9 containing P5CR characteristic sequence fragments.
  • Total human fetal brain RNA was extracted by one-step method with guanidine isothiocyanate / phenol / chloroform.
  • Poly (A) mRNA was isolated from total RNA using Quik mRNA Isolat ion Kit (product of Qiegene). 2ug poly (A) mRNA is reverse transcribed to form cDNA.
  • the Smar t cDNA cloning kit (purchased from CI on tech) was used to insert the 00 fragment into the multiple cloning site of pBSK (+) vector (Clontech) to transform DH5a. The bacteria formed a cDNA library.
  • Dye terminate cycle reaction ion sequencing kit product of Perkin-Elmer
  • ABI 377 automatic sequencer Perkin-Elmer
  • the determined cDNA sequence was compared with the existing public DNA sequence database (Genebank), and it was found that the cDNA sequence of one of the clones 1007hl 0 was new DNA.
  • a series of primers were synthesized to determine the inserted cDNA fragments of the clone in both directions.
  • the sequence of the human protein 5 containing the characteristic sequence fragment of P5CR of the present invention and the protein sequence encoded by the same were subjected to a profile scan program (Basiclocal Alignment search tool) in GCG [Altschul, SF et al. J. Mol. Biol. 1990 215: 403-10], performing domain analysis in databases such as prosite.
  • the human 9 containing the characteristic sequence fragment of P5CR of the present invention is homologous to the domain P5CR from 2-79.
  • the homology result is shown in Fig. 1.
  • the homology rate is 0.07, the score is 3.69; the threshold value is 3.50.
  • Example 3 Cloning of a gene encoding human P5CR-containing characteristic sequence fragment 9 by RT-PCR
  • CDNA was synthesized using fetal brain total RNA as a template and oligo-dT as a primer for reverse transcription reaction. After purification with Qiagene's kit, the following primers were used for PCR amplification:
  • Primerl 5-GGAGAAGGAGCCAATAAAGAAAAA-3 '(SEQ ID NO: 3)
  • Primer 2 5 '-GCATGGTAAAGGAATTATTGAGGG- 3' (SEQ ID NO: 4)
  • Primerl is a forward sequence located at the 5th end of SEQ ID NO: 1, starting at lbp;
  • Primer2 is the 3 'end reverse sequence in SEQ ID NO: 1.
  • CI (pH8.5), 1.5mmol / L MgCl 2 , 200 ⁇ mol / L dNTP, lOpmol primer, 1U Taq DNA polymerase (C 1 out ech company product).
  • the reaction was performed on a PE9600 DNA thermal cycler (Perkinn Elmer Co., Ltd.) under the following conditions for 25 cycles: 94 ° C 30sec; 55 ° C 30sec; 72 ° C 2min 0 Simultaneously set ⁇ -during RT-PCR Act in is a positive control and template blank is 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.
  • Example 4 Analysis of expression of human protein 9 gene containing P5CR characteristic sequence fragments by Northern blot method: Total RM extraction in one step [Anal. Biochem 1987, 162, 156-159] 0
  • This method includes acid guanidinium thiocyanate -Chloroform extraction. That is, the tissue is homogenized with 4M guanidinium isothiocyanate-25mM sodium citrate, 0.2M sodium acetate (pH4.0), and 1 volume of phenol and 1/5 volume of chloroform-isoamyl alcohol (49: 1 ), Mix and centrifuge.
  • RNA precipitate Aspirate the aqueous layer, add isopropanol (0.8 vol) and centrifuge the mixture to obtain RNA precipitate. The obtained RM precipitate was washed with 70% ethanol, dried and dissolved in water. Using 20 ⁇ ⁇ RNA, electrophoresis was performed on a 1.2% agarose gel containing 20 mM 3- (N-morpholino) propanesulfonic acid (pH 7.0)-5 mM sodium acetate-1 EDTA-2.2M formaldehyde. It was then transferred to a nitrocellulose membrane. Preparation 32 P- DNA probe labeled with a- 32 P dATP by random priming method.
  • the DNA probe used was the sequence of the protein 9 coding region (1188 bp to 1433 bp) of the human P5CR-containing characteristic sequence fragment amplified by PCR shown in FIG. 1.
  • Transfer the 32P-labeled probe (approximately 2 x 10 6 cpm / mi) with RNA-nitrocellulose membrane was hybridized overnight at 42 ° C in a solution containing 50% formamide- 25 mM ⁇ 2 PO 4 ( ⁇ 7 ⁇ 4)-5 x SSC- 5 x Denhardt, s solution and 200 g / ml salmon sperm DNA. After hybridization, the filter was washed in 1 x SSC-0.1% SDS at 55 ° C for 30 min. Then, Phosphor Imager was used for analysis and quantification.
  • Example 5 In vitro expression, isolation and purification of recombinant human protein 9 containing a characteristic sequence fragment of P5CR
  • Priraer3 5-CATGCTAGCATGGTATCTTCAGCTCTGACATCT-3 '(Seq ID No: 5)
  • Primer4 5'-CCCGAATTCCTAACTGTATACAGAACTAAAAGG-3' (Seq ID No: 6)
  • These two primers contain Nhel and EcoRI digestion sites at the 5 'ends, respectively.
  • the coding sequences of the 5 'and 3' ends of the gene of interest are respectively followed by the Nhel and EcoRI restriction sites corresponding to the selective endonuclease sites on the expression vector plasmid pET28b (+) (Novagen, Cat. No. 69865.3). point.
  • the pBS-1007hl0 plasmid containing the full-length target gene was used as a template for the PCR reaction.
  • the PCR reaction conditions are as follows: a total volume of 50 ⁇ l contains 10 pg of pBS-1007hl0 plasmid, primers Primer-3 and Primer- 4 points; j is lpmol, Advantage polymerase Mix (Clontech) 1 ⁇ 1. Cycle parameters: 94 ° C 20s, 60 ° C 30s, 68 ° C 2 min, a total of 25 cycles. Nhel and EcoRI were used to double digest the amplified product and plasmid pET-28 (+), respectively, and large fragments were recovered and ligated with T4 ligase.
  • Ligation products were transformed by the calcium chloride method Escherichia bacteria DH5CC, overnight (final concentration of 30 ⁇ ⁇ / ⁇ ⁇ 1) LB plates, screened by colony PCR positive clones containing kanamycin method, and sequenced. A positive clone (pET-1007hlO) with the correct sequence was selected, and the recombinant plasmid was transformed into E. coli BL21 (DE3) plySs (product of Novagen) using the calcium chloride method.
  • the host bacteria BL21 (pET-1007hl0) was cultured at 37 ° C to the logarithmic growth phase, and IPTG was added to the final concentration lmraol / L, Continue incubation for 5 hours. The bacteria were collected by centrifugation, and the supernatant was collected by centrifugation. The supernatant was collected by centrifugation. The affinity chromatography column His. Bind Quick Cartridge (product of Novagen) was used to obtain 6 histidine (6His-Tag). The purified protein 9 containing P5CR characteristic sequence fragment was purified.
  • a peptide 9-specific peptide containing human P5CR characteristic sequence fragments was synthesized using a peptide synthesizer (product of PE company): NH2- et-Val-Ser-Ser-Ala-Leu-Thr-Ser-Leu-Leu-Asn-Arg-Arg-Pro-Asn- C00H (SEQ ID NO: 7).
  • the polypeptide is coupled to hemocyanin and bovine serum albumin to form a complex, respectively.
  • 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, Northern blotting, and copying methods. They all use the same steps to immobilize the polynucleotide sample to be tested on the filter.
  • the sample-immobilized filter is first pre-hybridized with a probe-free hybridization buffer to saturate the non-specific binding site of the sample on the filter with the carrier and the synthesized polymer.
  • the pre-hybridization solution is then replaced with a hybridization buffer containing labeled probes and incubated to hybridize the probes to the target nucleic acid.
  • the unhybridized probes are removed by a series of membrane washing steps.
  • This embodiment uses higher-intensity washing conditions (such as lower salt concentration and higher temperature), so that the hybridization background is reduced and only strong specific signals are retained.
  • the probes used in this 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 fragment selected from the polynucleotide SEQ ID NO: 1 of the present invention for use as a hybridization probe shall be Following the following principles and several aspects to consider:
  • the preferred range of probe size is 18-50 nucleotides
  • Those that meet the above conditions can be used as primary selection probes, and then further computer sequence analysis, including the primary selection probe and its source sequence region (ie, SEQ ID NO: 1) and other unknown genomic sequences and their complements The regions are compared for homology. If the homology with the non-target molecular region is greater than 85% or there are more than 15 consecutive bases, the primary probe should not be used;
  • Probe 1 which belongs to the first type of probe, is completely homologous or complementary to the gene fragment of SEQ ID NO: 1 (41Nt):
  • Probe 2 which belongs to the second type of probe, is equivalent to the replacement mutation sequence (41Nt) of the gene fragment or its complementary fragment of SEQ ID NO: 1:
  • 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 32 P-Probe (the second peak is free ⁇ - 32P -dATP) is prepared.
  • the sample membrane was placed in a plastic bag, and 3-10 mg of prehybridization solution (10xDenhardt's; 6xSSC, 0. lrag / ml was added)
  • probe 1 can be used to qualitatively and quantitatively analyze the presence and differential expression of the polynucleotide of the present invention in different tissues.
  • polypeptides of the present invention can be directly used in the treatment of diseases, for example, they can treat malignant tumors, adrenal deficiency, skin diseases, various types of inflammation, HIV infection, and immune diseases. '
  • 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 bone tendons, blood vessels, cartilage, and skin in the body, and is one of the important components in the body.
  • ⁇ ⁇ -Pyrroline-5-hydroxyreductase (P5CR) catalyzes the last step in the biosynthesis process from glutamic acid to proline in the organism. 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.
  • the characteristic sequence of the delta-pyrrololine-5-hydroxyreductase protein family is necessary to form its biological activity.
  • the polypeptide of the present invention is a polypeptide containing a characteristic sequence of this protein family. Abnormal expression will lead to abnormal biosynthesis of proline, and then affect the synthesis of certain proteins, especially collagen, and affect bone tendons, blood vessels, cartilage and The function of tissues and organs such as the skin and related diseases.
  • the abnormal expression of the protein 9 containing the characteristic sequence fragment of P5CR of the present invention will produce various diseases, especially glutamate metabolism disease, connective tissue disease, various tumors, developmental disorders, inflammation, and immunity.
  • Diseases including but not limited to:
  • Connective tissue diseases rheumatic fever, systemic lupus erythematosus, dermatomyositis, polymyositis, sclerosis, mixed connective tissue disease, panniculitis, sarcoidosis, vasculitis, purine metabolic disease, gout
  • Tumors of various tissues gastric cancer, liver cancer, breast cancer, leukemia, lymphoma, thyroid tumor, uterine fibroids, neurofibromas, colon cancer, melanoma, bladder cancer, endometrial cancer, colon cancer, laryngeal cancer, trachea Tumor, fibroid, fibrosarcoma
  • developmental disorders congenital abortion, cleft palate, limb loss, limb differentiation disorder, atrial septal defect, neural tube defect, congenital hydrocephalus, congenital glaucoma or cataract, mental retardation, brain development disorder, skin, fat and Muscular dysplasia, bone and joint dysplasia, various metabolic defects, sexual retardation
  • Inflammation chronic active hepatitis, sarcoidosis, polymyositis, chronic rhinitis, chronic gastritis, cerebrospinal multiple sclerosis, glomerulonephritis, myocarditis, cardiomyopathy, atherosclerosis, gastric ulcer, cervicitis, Various infectious inflammations
  • Immune diseases Systemic lupus erythematosus, rheumatoid arthritis, bronchial asthma, urticaria, specific dermatitis, post-infection myocarditis, scleroderma, myasthenia gravis, Guillain-Barre syndrome, common variable immunodeficiency disease , Primary B-lymphocyte immunodeficiency disease, Acquired immunodeficiency syndrome
  • the abnormal expression of the protein 9 containing the characteristic sequence fragment of P5CR of the present invention will also produce certain hereditary, hematological diseases and the like.
  • the 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 glutamate metabolism diseases, connective tissue diseases, various tumors, and development disorders. , Inflammation, immune diseases, certain hereditary, blood diseases, etc.
  • the invention also provides methods for screening compounds to identify agents that enhance (agonist) or suppress (antagonist) human protein 9 containing P5CR characteristic sequence fragments.
  • Agonists increase human proteins containing P5CR characteristic sequence fragments to stimulate biological functions such as cell proliferation, while antagonists prevent and treat disorders related to excessive cell proliferation, such as various cancers.
  • a mammalian cell or a membrane preparation expressing human P5CR characteristic sequence fragment-containing protein 9 can be cultured with the labeled human P5CR characteristic sequence fragment-containing protein 9 in the presence of a drug. The ability of the drug to increase or block this interaction is then determined.
  • Antagonists of human protein 9 containing a characteristic sequence fragment of P5CR include antibodies, compounds, receptor deletions, and the like that have been screened. Antagonists of human protein 9 containing a characteristic sequence fragment of P5CR can bind to human protein 9 containing a characteristic sequence fragment of P5CR and eliminate its function, or inhibit the production of the polypeptide, or bind to the active site of the polypeptide such that The polypeptide cannot perform biological functions.
  • human protein 9 containing a characteristic sequence fragment of P5CR can be added to a bioanalytical assay, and by determining the compound's interaction with human protein 5 containing a characteristic sequence fragment of P5CR and its receptor, Influence to determine if a compound is an antagonist.
  • Receptor deletions and analogs that act as antagonists can be screened in the same manner as described above for screening compounds.
  • Polypeptide molecules capable of binding to human protein 9 containing a characteristic sequence fragment of P5CR can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. During screening, generally, 9 molecules of human proteins containing P5CR characteristic sequence fragments should be labeled.
  • the present invention provides a method for producing an antibody using a polypeptide, a fragment, a derivative, an analog thereof, or a cell thereof as an antigen.
  • These antibodies can be polyclonal or monoclonal antibodies.
  • the present invention also provides an antibody against a human protein 9 epitope containing a P 5 CR characteristic sequence fragment.
  • These antibodies include (but 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 directly injecting humans containing protein P5CR characteristic sequence fragment 9 into immunized animals (such as rabbits, mice, rats, etc.).
  • immunized animals such as rabbits, mice, rats, etc.
  • adjuvants can be used to enhance the immune response, including but not limited to Freund's adjuvant, etc.
  • Techniques for preparing a human monoclonal antibody containing protein 9 fragment characteristic of the P5CR sequence 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 variable regions can be produced using known 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 human protein 9 containing P5CR characteristic sequence fragments.
  • Antibodies against protein 9 containing P5CR characteristic sequence fragments can be used in immunohistochemical techniques to detect human protein 5 containing P5CR characteristic sequence fragments in biopsy specimens.
  • Monoclonal antibodies that bind to human protein 9 containing a characteristic sequence fragment of P5CR 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.
  • humans containing high-affinity monoclonal antibodies to the P5CR characteristic sequence fragments can covalently bind to bacterial or plant toxins (such as diphtheria toxin, ricin, ormosine, etc.).
  • a common method is to attack the amino group of an antibody with a thiol cross-linking agent such as SPDP and bind the toxin to the antibody through the exchange of disulfide bonds.
  • This hybrid antibody can be used to kill human proteins containing P5CR characteristic sequence fragments. 9 positive cells.
  • the antibodies of the present invention can be used to treat or prevent diseases related to human protein 9 containing P5CR characteristic sequence fragments. Administration of an appropriate dose of the antibody can stimulate or block the production or activity of human protein 9 containing a P5CR characteristic sequence fragment.
  • the present invention also relates to a diagnostic test method for quantitatively and locally detecting the level of protein 9 containing a characteristic sequence fragment of P5CR.
  • tests are well known in the art and include FISH assays and radioimmunoassays.
  • the level of protein 9 of human P5CR characteristic sequence fragments detected in the test can be used to explain the importance of human P5CR characteristic sequence fragment-containing protein 9 in various diseases and to diagnose human P5CR characteristic sequence fragments Protein 9 plays a role in the disease.
  • polypeptide of the present invention can also be used for peptide mapping analysis.
  • the polypeptide can be specifically cleaved by physical, chemical or enzymatic analysis, and subjected to one-dimensional or two-dimensional or three-dimensional gel electrophoresis analysis, and more preferably mass spectrometry analysis.
  • Polynucleotides encoding human protein 9 with a characteristic sequence fragment of P5CR can also be used for a variety of therapeutic purposes of.
  • Gene therapy technology can be used to treat abnormal cell proliferation, development, or metabolism caused by the non-expression or abnormal / inactive expression of protein 9 containing human P5CR characteristic sequence fragments.
  • Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated human P5CR characteristic sequence fragment protein 9 to inhibit endogenous human P5CR characteristic sequence fragment protein 9 activity.
  • a mutated human P5CR characteristic sequence-containing protein 9 may be a shortened human P5CR characteristic sequence-containing protein 9 lacking a signaling domain, although it can bind to downstream substrates, but lacks Signaling activity.
  • the recombinant gene therapy vector can be used for treating diseases caused by abnormal expression or activity of protein 9 of the characteristic sequence fragment of P5CR in humans.
  • Virus-derived expression vectors such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus and the like can be used to transfer a polynucleotide encoding human protein 9 containing a characteristic sequence fragment of P5CR into a cell.
  • a method for constructing a recombinant viral vector carrying a polynucleotide encoding a human protein 9 fragment containing a characteristic sequence of P5CR can be found in the existing literature (Sambrook, et al.).
  • a recombinant polynucleotide encoding human protein 9 containing a characteristic sequence fragment of P5CR 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 DNA
  • ribozymes that inhibit human protein 9 mRM containing P5CR characteristic sequence fragments are also within the scope of the present invention.
  • a ribozyme is an enzyme-like RNA molecule that can specifically decompose a specific RM. Its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target 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 phosphate amide chemical synthesis, which is 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 the nucleic acid molecule, it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the phosphorothioate or peptide bond instead of the phosphodiester bond is used for the ribonucleoside linkage.
  • the polynucleotide encoding human protein 9 containing a characteristic sequence fragment of P5CR can be used for the diagnosis of diseases related to human protein 9 containing a characteristic sequence fragment of P5CR.
  • Polynucleotides encoding human P5CR characteristic sequence fragments containing protein 9 can be used to detect the expression of human P5CR characteristic sequence fragments containing protein 9 or human disease containing P5CR characteristic sequence fragments of abnormal expression of human 9 .
  • a DNA sequence encoding human protein 9 containing a characteristic sequence fragment of P5CR can be used to hybridize a biopsy specimen to determine the expression status of human protein 5 containing a characteristic sequence fragment of P5CR.
  • Hybridization techniques include Southern blotting, Nor thern blotting, in situ hybridization, and the like. These technical methods are all mature technologies that are publicly available. Obtained from commercial sources. Part or all of the polynucleotides of the present invention can be immobilized on a microarray as probes
  • RNA-polymerase chain reaction in vitro amplification of human protein 9 specific primers containing P5CR characteristic sequence fragments can also be used to detect human protein 5 containing P5CR characteristic sequence fragments.
  • Detecting mutations in the protein 9 gene containing human P5CR characteristic sequence fragments can also be used to diagnose human protein 9 containing P5CR characteristic sequence fragments related diseases.
  • the form of human protein 9 mutations containing P5CR characteristic sequence fragments includes point mutations, translocations, deletions, recombinations, and any other abnormalities compared to the normal wild-type human P5CR characteristic sequence containing protein 9 DNA sequences. Mutations can be detected using existing techniques such as Southern blotting, DNA sequence analysis, PCR and in situ hybridization. In addition, the mutation may affect the expression of the protein. Therefore, the Nor thern mark method and Western blot method can be used to indirectly determine whether the gene is mutated.
  • the sequences of the invention are also valuable for chromosome identification.
  • the sequence specifically targets a specific position on a human chromosome and can hybridize to it.
  • specific sites for each gene on the chromosome need to be identified.
  • only a few chromosome markers based on actual sequence data are available for marking chromosome positions.
  • an important first step is to locate these DNA sequences on a chromosome.
  • PCR primers (preferably 15-35bp) are prepared based on cDNA, and the sequences can be located on chromosomes. These primers were then used for PCR screening of somatic hybrid cells containing individual human chromosomes. Only those heterozygous cells containing the human gene corresponding to the primer will produce amplified fragments.
  • PCR localization of somatic hybrid cells is a quick way to localize DNA to specific chromosomes.
  • oligonucleotide primers of the present invention in a similar manner, a set of fragments from a specific chromosome or a large number of genomic clones can be used to achieve sublocalization.
  • Other similar strategies that can be used for chromosomal localization include in situ hybridization, chromosome pre-screening with labeled flow sorting, and pre-selection of hybridization to construct chromosome-specific cDNA libraries.
  • Fluorescent in situ hybridization of cDNA clones with metaphase chromosomes allows precise chromosomal localization in one step.
  • FISH Fluorescent in situ hybridization
  • the physical location of the sequence on the chromosome can be correlated with the genetic map data. These data can be found in, for example, V. Mckusick, Mendel i an Inher i tance in Man (available online with Johns Hopkins University Welch Medical Library). Linkage analysis can then be used to determine the relationship between genes and diseases that have been mapped to chromosomal regions. Next, the differences in cDNA or genomic sequences between the affected and unaffected individuals need to be determined. If a mutation is observed in some or all diseased individuals and the mutation is not observed in any normal individuals, the mutation may be the cause of the disease.
  • Comparing affected and unaffected individuals usually involves first looking for structural changes in the chromosome, such as deletions or translocations that are visible at the chromosomal level or detectable using cDNA sequence-based PCR.
  • the cDNA accurately mapped to the chromosomal region associated with the disease can be one of 50 to 500 potentially pathogenic genes (assuming 1 megabase mapping resolution) Capacity and each 20kb corresponds to a gene).
  • the polypeptides, polynucleotides and mimetics, agonists, antagonists and inhibitors of the present invention can be used in combination with a suitable pharmaceutical carrier.
  • suitable pharmaceutical carrier can be water, glucose, ethanol, salts, buffers, glycerol, and combinations thereof.
  • the composition comprises a safe and effective amount of the polypeptide or antagonist, and carriers and excipients which do not affect the effect of the drug. These compositions can be used as drugs for the treatment of diseases.
  • the invention also provides a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the invention.
  • a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the invention.
  • these containers there may be instructional instructions given by government agencies that manufacture, use, or sell pharmaceuticals or biological products, which prompts permission for administration on the human body by government agencies that produce, use, or sell.
  • the polypeptides of the invention can be used in combination with other therapeutic compounds.
  • the pharmaceutical composition can be administered in a convenient manner, such as by a topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal route of administration.
  • Human protein 9 containing P5CR characteristic sequence fragments is administered in an amount effective to treat and / or prevent a specific indication.
  • the amount and range of protein 9 containing P5CR characteristic sequence fragments in a human being 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.

Description

一种新的多肽一一人含 P5CR特征性序列片段的蛋白 9和编码这种多肽的多核苷酸 技术领域
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一一人含 P5CR 特征性序列片段的蛋白 9 , 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。 背景技术
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 launey 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 Genet, 221: 299-305] 0
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 ) - [LIVM] - [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、 运动系统疾病及一些相关的皮肤病的发生密 切相关。
由于如上所述人含 P5CR特征性序列片段的蛋白 9蛋白在机体内重要功能中起重 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人含 P5CR特征性序列片段的蛋白 9蛋白， 特别是鉴定这种蛋 白的氨基酸序列。 新人含 P5CR特征性序列片段的蛋白 9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 DM是非常重要的。 发明的公开
本发明的一个目的是提供分离的新的多肽一一人含 P5CR 特征性序列片段的蛋 白 9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人含 P5CR特征性序列片段的蛋白 9 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人含 P5CR特征性序列片段的蛋白 9 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人含 P5CR特征性序列片段的蛋白 9的方法。 本发明的另一个目的是提供针对本发明的多肽一一人含 P5CR特征性序列片段 的蛋白 9的抗体。 „
本发明的另一个目的是提供了针对本发明多肽一一人含 P5CR特征性序列片段 的蛋白 9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人含 P5CR特征性序列片段的蛋白 9异 常相关的疾病的方法。
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 70%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 1188- 1433位的序列; 和（b)具有 SEQ ID NO: 1 中 1 - 3878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、 拮抗或抑制人含 P5CR特征性序列片段 的蛋白 9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人含 P5CR特征性序列片段的蛋白 9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人含 P5CR 特征性序列片段的蛋白 9 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。
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 DNA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" 是指当与人含 P5CR特征性序列片段的蛋白 9 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人含 P5CR特征性序列片段的蛋白 9的分子。
"拮抗剂" 或 "抑制物" 是指当与人含 P5CR特征性序列片段的蛋白 9结合时， 一种可封闭或调节人含 P5CR特征性序列片段的蛋白 9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人 含 P5CR特征性序列片段的蛋白 9的分子。
"调节" 是指人含 P5CR特征性序列片段的蛋白 9的功能发生改变， 包括蛋白质 活性的升高或降低、 结合特性的改变及人含 P5CR特征性序列片段的蛋白 9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人含 P5CR 特征性序列片段的蛋 白 9。 基本上纯的人含 P5CR特征性序列片段的蛋白 9在非还原性聚丙烯酰胺凝胶 上能产生单一的主带。 人含 P5CR特征性序列片段的蛋白 9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 靶序列在的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 （La sergene sof tware package, DNASTAR, Inc. , Madi son Wi s. )。 MEGALIGN程序可根据不同的 方法如 Clus ter法比较两种或多种序列（Higg ins, D. G. 和 P. M. Sharp (1988) Gene 73: 237-244) 0 Clus ter法通过检查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 A与序列 B之间匹配的残基个数 X 100
(序列 A的残基数一序列 A中间隔残基数一序列 B中间隔残基数） 也可以通过 Clus 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 erazumology 183: 625-645) 0
"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 DNA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或编码其的核酸的化学修饰物。 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 合人含 P5CR特征性序列片段的蛋白 9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， "分离的人含 P5CR特征性序列片段的蛋白 9" 是指人含 P5CR 特征性序列片段的蛋白 9 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人含 P5CR特征性 序列片段的蛋白 9。 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。 人含 P5CR特征性序列片段的蛋白 9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一一人含 P5CR特征性序列片段的蛋白 9 , 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。
本发明还包括人含 P5CR特征性序列片段的蛋白 9 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 人含 P5CR特征性序列片段的蛋白 9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 3878个碱基， 其开放读框（1188-1433 )编码了 81个氨基酸。 此多 肽具有 P5CR的特征序列，可推断出该人含 P5CR特征性序列片段的蛋白 9具有 P5CR 所代表的结构和功能。 本发明的多核苷酸可以是 DNA形式或是 RM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 70%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, 60°C ;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%Ficol l , 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 /或分离编码人含 P5CR特征性序列片段的蛋白 9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。 本发明的编码人含 P5CR特征性序列片段的蛋白 9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 DNA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。 DM 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 定人含 P5CR 特征性序列片段的蛋白 9 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。 DNA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， 检测人含 P5CR特征性序列片段的蛋白 9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 DM 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 用人含 P5CR特征性序列片段的蛋白 9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， 编码人含 P5CR特征性序列片段的蛋白 9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 a l . Gene, 1987， 56: 125)； 在哺乳动物细胞中表达的 pMSXND表达载体（Lee and Na thans , J Bi o Chera. 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人含 P 5 CR特征性序列片段 的蛋白 9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 a l . Mo l ecu l ar C l on i ng, a Labora tory Manua l , co l d Spr ing Harbor Labora tory. 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 l ac或 t rp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 点晚期一恻的 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， 编码人含 P5CR特征性序列片段的蛋白 9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 S 2或 Sf 9 ; 动物细胞如 CH0、 COS或 Bowe s 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 DNA 的感受态细胞可在指数生长期后收获， 用 0 ( 12法处理， 所用的步骤 在本领域众所周知。 可供选择的是用 MgC l 2。 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 DNA 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 重组的人含 P5CR特征性序列片段的蛋白 9 (Sc i ence , 1984 ; 224： 1431)。 一般 来说有以下步骤：
(1) .用本发明的编码人 人含 P 5CR特征性序列片段的蛋白 9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。 附图的简要说明
下列附图用于说明本发明的具体实施方案， 而不用于限定由杈利要求书所 界定的本发明范围。
图 1是本发明人含 P5CR特征性序列片段的蛋白 9在 2-79共 78个氨基酸和 P5CR 特征结构域的氨基酸序列同源性比较图。 上方序列是人含 P5CR特征性序列片段 的蛋白 9， 下方序列是 P 5 CR的特征结构域。 相同氨基酸在两个序列间用单字符 氨基酸表示， 相似氨基酸用 "+" 表示。
图 2为分离的人含 P5CR特征性序列片段的蛋白 9 的聚丙烯酰胺凝胶电泳图
( SDS-PAGE )0 9KDa为蛋白质的分子量。 箭头所指为分离出的蛋白条带。 实现本发明的最佳方式
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 Labora tory 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 : 人含 P5 CR特征性序列片段的蛋白 9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 Isolat ion Ki t ( Qiegene 公司产品） 从总 RNA中分离 poly (A) mRNA。 2ug poly (A) mRNA经逆转录 形成 cDNA。用 Smar t cDNA克隆试剂盒（购自 CI on tech )将00 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 a , 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 1007hl 0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， 1007hl O克隆所含的全长 cDNA为 3878bp (如 Seq ID N0: l 所示） ， 从第 1188bp至 1433bp有一个 246bp的开放阅读框架 （ 0RF ) , 编码一个新 的蛋白质 （如 Seq ID NO: 2所示） 。 我们将此克隆命名为 pBS- 1007hl 0 , 编码的蛋 白质命名为人含 P5CR特征性序列片段的蛋白 9。 实施例 2: cDNA 克隆的结构域分析
将本发明的人含 P5CR特征性序列片段的蛋白 9的序列及其编码的蛋白序列， 用 GCG中的 profile scan程序 (Basiclocal Alignment search tool) [Altschul, SF et al. J. Mol. Biol. 1990; 215: 403-10] , 在 prosite等数据库进行结构域分析。 本发明的人含 P5CR特征性序列片段的蛋白 9在 2-79与结构域 P5CR有同源， 同源结果 示于图 1， 同源率为 0.07, 得分为 3.69; 阈值为 3.50。 实施例 3: 用 RT- PCR方法克隆编码人含 P5CR特征性序列片段的蛋白 9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-GGAGAAGGAGCCAATAAAGAAAAA-3' (SEQ ID NO: 3)
Primer 2: 5 '-GCATGGTAAAGGAATTATTGAGGG- 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 50μ 1的反应体积中含有 50瞧 ol/L KC1,10隱 ol/L Tris-
CI, (pH8.5), 1.5mmol/L MgCl2, 200 μ mol/L dNTP, lOpmol引物， 1U的 Taq DNA聚合 酶（C 1 out ech公司产品）。 在 PE9600型 DNA热循环仪（Per k i n- E lme r公司）上按下列条 件反应 25个周期： 94°C 30sec; 55°C 30sec; 72°C 2min0 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） 。 DM序列分析结果表明 PCR 产物的 DNA序列与 SEQ ID NO: 1所示的 1- 3878bp完全相同。 实施例 4: Northern 印迹法分析人含 P5CR特征性序列片段的蛋白 9基因的表达： 用一步法提取总 RM[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 RM沉淀用 70%乙醇洗涤， 干燥并溶于水中。 用 20μ§ RNA, 在含 20mM 3- ( N- 吗啉代） 丙磺酸 (pH7.0) - 5mM乙酸钠 - 1 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 a-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扩增的人含 P5CR特征性序列片段的蛋 白 9编码区序列（1188bp至 1433bp)。 将 32P -标记的探针 （约 2 x 106cpm/mi ) 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 ΚΗ2Ρ04 (ρΗ7·4) - 5 x SSC- 5 xDenhardt，s溶液和 200 g/ml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: 重组人含 P5CR特征性序列片段的蛋白 9的体外表达、 分离和纯化
根据 SEQ ID NO: 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-CATGCTAGCATGGTATCTTCAGCTCTGACATCT-3' ( Seq ID No: 5 ) Primer4: 5'-CCCGAATTCCTAACTGTATACAGAACTAAAAGG-3' (Seq ID No: 6 ) 此两段引物的 5'端分别含有 Nhel和 EcoRI酶切位点， 其后分别为目的基因 5'端 和 3'端的编码序列， Nhel和 EcoRI酶切位点相应于表达载体质粒 pET28b(+) (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- 1007hl0质粒为模板， 进行 PCR反应。 PCR反应条件为： 总体积 50 μ 1中含 pBS- 1007hl0 质粒 10pg、 引物 Pr imer-3和 Primer- 4分另 !j为 lOpmol、 Advantage polymerase Mix (Clontech公司产品） 1 μ 1。 循环参数： 94°C 20s, 60°C 30s, 68°C 2 min,共 25个 循环。 用 Nhel和 EcoRI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 DH5CC,在含卡那霉素 (终浓度 30μ§/ηι1 ) 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-1007hlO)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 30 μ g/ml ) 的 LB 液体培养基中， 宿主菌 BL21 (pET- 1007hl0)在 37°C培养至对数生长期， 加入 IPTG 至终浓度 lmraol/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， 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人含 P5CR特征性序列片段 的蛋白 9。 经 SDS- PAGE电泳， 在 9KDa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 抗人含 P5CR特征性序列片段的蛋白 9抗体的产生
用多肽合成仪 （PE公司产品）合成下述人含 P5CR特征性序列片段的蛋白 9特异 性的多肽： NH2- et-Val-Ser-Ser-Ala-Leu-Thr-Ser-Leu-Leu-Asn-Arg-Arg-Pro-Asn- C00H (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， 方法参见： Avrameas, et al. Immunochemi s try, 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。 釆用经 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 Sepha rose4B柱上，用亲和层析法从总 IgG 列片段的蛋白 9结合。 、 ^ 一 ' 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、 Northern 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 )和其它巳知的基因组序 列及其互补区进行同源性比较， 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 或互补（41Nt) ：
5'-TGGTATCTTCAGCTCTGACATCTCTCCTAAATCGTAGACCA-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-TGGTATCTTCAGCTCTGACACCTCTCCTAAATCGTAGACCA-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： DNA PROBES G. H. 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； 25mmol/L Tris-HCl, pH7.5; 25mmol/LnaCl; 25隱 ol/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-5ml)， 再以 lOOOg离心 10分钟。 7)用裂解缓冲液重悬沉淀（每 O. lg最初组织样品加 lml), 然后接以下 的苯酚抽提法。
2, DM的苯酚抽提法
步骤： 1 )用 1- 10ml冷 PBS洗细胞， lOOOg离心 10分钟。 2)用冷细胞裂解 液重悬浮沉淀的细胞 （ l x l 08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， 并降低的总产率。 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 DNA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小时或至过夜。 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 DNA充分溶解， 每 1- 5 χ 10δ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 1/10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°C。 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. Imol/LNaOH, 1.5mol/LNaCl的滤纸上 5分钟 （两次）， 晾干置 于浸润有 0.5mol/L Tris-HCl ( pH7.0 )， 3raol/LNaCl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 ) 3 μ lProbe ( 0.10D/10 μ 1 ),加入 2 μ I inase缓冲液， 8-1。 uCi γ-32Ρ- dATP+2U Kinase, 以补加至终体积 20 μ1。
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 32P- 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（10xDenhardt's;6xSSC， 0. lrag/ml
CT DNA (小牛胸腺 DM)。）， 封好袋口后， 68。C水洛摇 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水浴摇过夜。 洗膜：
高强度洗膜：
1 ) 取出巳杂交好的样膜。
2 ) 2xSSC， 0.1%SDS中， 40°C洗 15分钟 （ 2次）。
3 ) 0. IxSSC, 0.1%SDS中 , 40°C洗 15分钟 ( 2次）。
4) 0. IxSSC, 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, 0.1¾SDS中， 37°C洗 15分钟 （ 2次）。
3 ) 0. IxSSC, 0.1%SDS中， 37°C洗 15分钟 （ 2次）。
4) 0. IxSSC, 0.1 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： 釆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； 而釆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。 工业实用性
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。 '
在机体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， 是机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。 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。
δ ΐ -吡咯啉- 5-羟基还原酶蛋白家族的特征性序列是形成其生物活性所必需。 本发明的多肽是含此蛋白家族的特征性序列的多肽， 其表达异常将导致脯氨 酸的生物合成的异常， 进而影响某些蛋白尤其是胶原蛋白的合成， 影响骨腱、 血 管、 软骨和皮肤等组织器官的功能， 并产生相关的疾病。
由此可见， 本发明的人含 P5CR特征性序列片段的蛋白 9 的表达异常将产生 各种疾病尤其是谷氨酸代谢病、 结締组织疾病、 各种肿瘤、 发育紊乱症、 炎症、 免疫性疾病， 这些疾病包括但不限于：
结締组织疾病： 风湿热， 系统性红斑狼疮， 皮肌炎， 多发性肌炎， 硬化症， 混 合性结締组织病， 脂膜炎， 结节病， 血管炎， 嘌呤代谢疾病， 痛风
各种组织的肿瘤： 胃癌， 肝癌， 乳腺癌， 白血病， 淋巴瘤， 甲状腺肿瘤， 子宫 肌瘤， 神经纤维瘤， 结肠癌， 黑色素瘤， 膀胱癌， 子宫内膜癌， 结肠癌， 喉癌， 气管肿瘤， 纤维瘤， 纤维肉瘤
发育紊乱症： 先天性流产， 腭裂， 肢体缺如， 肢体分化障碍， 房间隔缺损， 神 经管缺陷， 先天性脑积水， 先天性青光眼或白内障， 精神发育迟缓， 脑发育障碍， 皮肤、 脂肪和肌肉发育不良性疾病， 骨与关节发育不良性疾病， 各种代谢缺陷病， 性发育迟缓症
炎症： 慢性活动性肝炎， 结节病， 多肌炎， 慢性鼻炎， 慢性胃炎， 脑脊髓多发 性硬化， 肾小球性肾炎， 心肌炎， 心肌病， 动脉粥样硬化， 胃溃疡， 子宫颈炎， 各种感染性炎症
免疫性疾病： 系统性红斑狼疮， 类风湿性关节炎， 支气管哮喘， 荨麻疹， 特异 性皮炎， 感染后心肌炎， 硬皮病， 重症肌无力， 格林-巴利综合症， 普通易变免疫 缺陷病， 原发性 B淋巴细胞免疫缺陷病， 获得性免疫缺陷综合症
本发明的人含 P5CR特征性序列片段的蛋白 9 的表达异常还将产生某些遗传性， 血液性疾病等。
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谷氨酸代谢病、 结締组织疾病、 各种肿瘤、 发育紊 乱症、 炎症、 免疫性疾病， 某些遗传性， 血液性疾病等。
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）人含 P5CR 特征性序列片段的蛋白 9的药剂的方法。 激动剂提高人含 P5CR特征性序列片段 的蛋白 9 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人含 P5CR 特征性序列片段的蛋白 9的膜制剂与标记的人含 P5CR特征性序列片段的蛋白 9 一起培养。 然后测定药物提高或阻遏此相互作用的能力。
人含 P5CR特征性序列片段的蛋白 9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。 人含 P5CR特征性序列片段的蛋白 9的拮抗剂可以与人 含 P5CR特征性序列片段的蛋白 9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， 可以将人含 P5CR特征性序列片段的蛋白 9 加入生物分析测定中， 通过测定化合物对人含 P5CR特征性序列片段的蛋白 9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， 可以筛选出起拮抗剂作用的受体缺失物和类似物。 能与人含 P5CR特 征性序列片段的蛋白 9 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， 一般应对人含 P5CR特征性序列 片段的蛋白 9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 了针对人含 P 5 CR特征性序列片段的蛋白 9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人含 P5CR特征性序列片段的蛋白 9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。 制备人含 P5CR特征性序列片段的蛋白 9的单克隆抗体 的技术包括但不限于杂交瘤技术 （Kohl 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巳有的技术生产（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) 也可用于生产抗人含 P5CR特征性序列片段的蛋白 9的单链抗体。
抗人含 P5CR特征性序列片段的蛋白 9的抗体可用于免疫组织化学技术中， 检测活检标本中的人含 P5CR特征性序列片段的蛋白 9。
与人含 P5CR特征性序列片段的蛋白 9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。 如人含 P5CR特征性 序列片段的蛋白 9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 杀灭人含 P5CR特征性序列片段的蛋白 9阳性的细胞。
本发明中的抗体可用于治疗或预防与人含 P5CR特征性序列片段的蛋白 9相 关的疾病。 给予适当剂量的抗体可以刺激或阻断人含 P5CR特征性序列片段的蛋 白 9的产生或活性。
本发明还涉及定量和定位检测人含 P5CR特征性序列片段的蛋白 9水平的诊 断试验方法。 这些试验是本领域所熟知的， 且包括 FISH测定和放射免疫测定。 试验中所检测的人含 P5CR特征性序列片段的蛋白 9水平，可以用作解释人含 P5CR 特征性序列片段的蛋白 9在各种疾病中的重要性和用于诊断人含 P5CR特征性序 列片段的蛋白 9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。
编码人含 P5CR 特征性序列片段的蛋白 9 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人含 P5CR特征性序列片段的蛋白 9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人含 P5CR 特征性序列片段的蛋白 9 , 以抑制 内源性的人含 P5CR特征性序列片段的蛋白 9活性。 例如， 一种变异的人含 P5CR 特征性序列片段的蛋白 9可以是缩短的、 缺失了信号传导功能域的人含 P5CR特 征性序列片段的蛋白 9 , 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人含 P5CR特征性序列片段的蛋白 9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人含 P5CR特征性序列片段的蛋 白 9的多核苷酸转移至细胞内。 构建携带编码人含 P5CR特征性序列片段的蛋白 9 的多核苷酸的重组病毒载体的方法可见于已有文献（Sambrook, et a l . )。 另外 重组编码人含 P5CR特征性序列片段的蛋白 9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。
抑制人含 P5CR特征性序列片段的蛋白 9 mRM的寡核苷酸（包括反义 RNA和 DNA)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 RM 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巳广泛应用。 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。
编码人含 P5CR特征性序列片段的蛋白 9的多核苷酸可用于与人含 P5CR特 征性序列片段的蛋白 9的相关疾病的诊断。 编码人含 P5CR特征性序列片段的蛋 白 9的多核苷酸可用于检测人含 P5CR特征性序列片段的蛋白 9的表达与否或在 疾病状态下人含 P5CR特征性序列片段的蛋白 9 的异常表达。 如编码人含 P5CR 特征性序列片段的蛋白 9的 DNA序列可用于对活检标本进行杂交以判断人含 P5CR 特征性序列片段的蛋白 9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。 用人含 P5CR特征性序列片段的蛋白 9特异的引物进行 RNA-聚合酶链反应（RT- PCR)体外扩增也可检测人含 P5CR特征性序列片段的蛋白 9的转录产物。
检测人含 P5CR特征性序列片段的蛋白 9基因的突变也可用于诊断人含 P5CR 特征性序列片段的蛋白 9相关的疾病。 人含 P5CR特征性序列片段的蛋白 9突变 的形式包括与正常野生型人含 P5CR特征性序列片段的蛋白 9 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、 DNA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 染色体具体位置且并可以与其杂交。 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 cDNA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 Manua l of Bas ic Techniques, Pergamon Pres s, 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。 这些数据可见于例如， V. Mckus ick, Mendel i an Inher i tance in Man (可通过与 Johns Hopkins Univers i ty Welch Medica l Library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。 人含 P5CR特征性序列片段的蛋白 9以有效地治 疗和 /或预防具体的适应症的量来给药。 施用于患者的人含 P5CR 特征性序列片 段的蛋白 9 的量和剂量范围将取决于许多因素， 如给药方式、 待治疗者的健康 条件和诊断医生的判断。

Claims

权 利 要 求 书
1、 一种分离的多肽-人含 P5CR特征性序列片段的蛋白 9 , 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 （a ) 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 1188-1433位的序列或 SEQ ID NO: 1中 1-3878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 杈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、 一种具有人含 P5CR 特征性序列片段的蛋白 9 活性的多肽的制备方法， 其特征 在于所述方法包括：
(a) 在表达人含 P5CR特征性序列片段的蛋白 9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人含 P5CR特征性序列片段的蛋白 9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人含 P5CR 特征性序列 片段的蛋白 9特异性结合的抗体。
11、 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、 拮抗或抑制人含 P5CR特征性序列片段的蛋白 9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人含 P5CR 特征性序列片段的蛋白 9在体内、 体外活性的方法。
14、 一种检测与杈利要求 1-3 中的任一杈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 选人含 P5CR特征性序列片段的蛋白 9 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
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人含 P5CR 特征性序列 片段的蛋白 9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/CN2001/000590 2000-04-27 2001-04-23 A novel polypeptide- human protein 9 which contains a p5cr characteristic sequence fragment and a polynucleotide encoding the same WO2001085903A2 (en)

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Applications Claiming Priority (2)

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CN 00115482 CN1320635A (zh) 2000-04-27 2000-04-27 一种新的多肽——人含p5cr特征性序列片段的蛋白9和编码这种多肽的多核苷酸
CN00115482.6 2000-04-27

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KR101838308B1 (ko) * 2010-02-22 2018-03-13 큐알엔에이, 인크. 피롤린-5-카르복실레이트 환원효소 1(ｐｙｃｒ1)에 대한 천연 안티센스 전사체의 억제에 의한 ｐｙｃｒ1과 관련된 질환의 치료
CN111529694A (zh) * 2020-05-11 2020-08-14 昆明医科大学第一附属医院 人吡咯林-5-羧酸还原酶作为制备治疗皮肤创伤药物的应用

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GENE vol. 172, no. 1, 1996, pages 149 - 153 *
J. BIOL. CHEM. vol. 271, no. 16, 1996, pages 9795 - 9800 *
MOL. GEN. GENET. vol. 221, no. 3, 1990, pages 299 - 305 *
NUCLEIC ACIDS RES. vol. 10, no. 23, 1982, pages 7701 - 7714 *

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