WO2002000821A2 - Nouveau polypeptide, proteine humaine plp 39, et polynucleotide codant ce polypeptide - Google Patents

Nouveau polypeptide, proteine humaine plp 39, et polynucleotide codant ce polypeptide Download PDF

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Publication number
WO2002000821A2
WO2002000821A2 PCT/CN2001/001019 CN0101019W WO0200821A2 WO 2002000821 A2 WO2002000821 A2 WO 2002000821A2 CN 0101019 W CN0101019 W CN 0101019W WO 0200821 A2 WO0200821 A2 WO 0200821A2
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polypeptide
polynucleotide
protein
plp protein
human
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PCT/CN2001/001019
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Chinese (zh)
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WO2002000821A3 (fr
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Yumin Mao
Yi Xie
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Biowindow Gene Development Inc. Shanghai
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Priority to AU93645/01A priority Critical patent/AU9364501A/en
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Publication of WO2002000821A3 publication Critical patent/WO2002000821A3/fr

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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention belongs to the field of biotechnology. Specifically, the present invention describes a new polypeptide, a human PLP protein 39, and a polynucleotide sequence encoding the polypeptide. The invention also relates to the preparation method and application of the polynucleotide and polypeptide. Background technique
  • the PLP protein family is a class of membrane proteins represented by Myelin proteolipid protein (PLP, lipophilin), which mainly exists in neural tissues and participates in the formation and maintenance of the multilayer structure of myelin sheaths. Its main members are shown in Table 1.
  • the high-level structure of the members of the PLP protein family contains four hydrophobic transmembrane domains (TM) and two conserved sequences (Motif), one of which is located at the beginning of the first TM region at the N-terminus and the second conserved sequence
  • TM transmembrane domains
  • Motif conserved sequences
  • the extracellular loop region between the three and four TM regions contains a Cys that forms a disulfide bond.
  • PLP protein is the main component of the central nervous system myelin protein, accounting for about 50% of its total protein.
  • the gene encoding the human PLP protein is located in the long arm 22 subregion (Xq22) of the X chromosome, which is about 17 Kb in length, and contains 7 exons and 6 introns.
  • the encoded PLP protein has a total of 276 amino acids, and its homology is close to 100% in mammals. It also shows high conservation in other species such as horn sharks and African claw frogs.
  • the precursor mRM is spliced in two different ways to form two mature mRNAs, which encode the PLP and 20 proteins, respectively. Compared with PLP, except for DM20, which deleted 35 amino acids at positions 116-150, the rest of the sequence was completely identical to PLP.
  • PLP protein is one of the most hydrophobic proteins in nature. Its primary structure contains 276 amino acids, of which about 50% are hydrophobic amino acids. The peptide folds to form four hydrophobic transmembrane domains (TM). ) And 5 hydrophilic extra-membrane loops, PLP is chimeric in the cell membrane through TM. Fourteen cysteine, five are free sulfhydryl, 'embedded in hydrophobic TM I and III, four form two disulfide bonds: Cys200-Cys219 and Cysl83-Cys227, and the remaining 6 Cys are on the long chain Fatty acids are covalently bonded to form thiolipid bonds.
  • the structure of DM20 is similar to that of PLP, with 35 amino acids deleted in loop III.
  • PLP / DM20 protein function 1. Maintain and stabilize the multilayer structure of central myelin sheath: central nervous system
  • the myelin structure is a multi-layer structure formed by tightly bonding the cell membranes of the oligodendrocytes.
  • PLP / DM20 is mainly produced by oligodendrocytes in the central nervous system. It is the main component of myelin proteins, together with Myel in basic protein (MBP). Formation and stability play an important role.
  • MBP basic protein
  • PLP may a. Be involved in the composition of ion channels; b. Be associated with signal transduction.
  • PLP familial middle lobe sclerosis
  • PMD familial white matter disease
  • X-chromosome-coupled type 2 spastic paraplegics X-1 inked spas t ic parapleg ia type 2, SPG2.
  • the morphology of PMD is mainly manifested by the absence of myelin sheaths in the central nervous system, a decrease in the number of mature oligodendrocytes, clinical manifestations of nystagmus, mental retardation, tonicity, ataxia, and symptoms starting to appear in the first year after birth.
  • SPG2 symptoms are similar to PMD, but to a lesser extent and occur later.
  • Mutations in PLP genes in other species such as mice and dogs can also cause symptoms similar to PMD.
  • PLP gene mutations and related diseases are shown in Table 2.
  • PLP gene mutations such as point mutations, deletions, frameshifts, nonsense mutations, and gene duplications, can cause PMD or SPG2 lesions.
  • the misfolded polypeptide chain produced by the mutant PLP gene aggregates in the endoplasmic reticulum, and the resulting toxic effect may be the main reason for the destruction of the central nervous system myelin sheath structure. This may also be the reason for the loss of PLP protein caused by clinically nonsense mutations, and the symptoms of PMD manifested as milder than those caused by missense mutations.
  • the gene encoding the polypeptide of the present invention contains the PLP protein family Mot if, based on the conservation of the mot if, combined with the analysis of the amino acid composition and hydrophobicity of the new gene by GCG software (including more hydrophobic amino acids, strong hydrophobicity), it is speculated that The protein encoded by this gene may also be a membrane protein involved in the formation of neural tissue or other specific structures (such as ion channels).
  • polypeptide of the present invention was deduced to be identified as a novel human PLP protein.
  • the human PLP protein 39 protein plays an important role in regulating important functions of the body, such as cell division and embryonic development, and it is believed that a large number of proteins are involved in these regulatory processes, so it has been necessary to identify more people involved in these processes PLP protein 39 protein, especially the amino acid sequence of this protein is identified.
  • the isolation of the new human PLP protein 39 protein encoding gene also provides a basis for the study to determine the role of this protein in health and disease states. This protein may form the basis for the development of diagnostic and / or therapeutic drugs for disease 1 and it is therefore important to isolate its coding DNA. 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 PLP protein 39.
  • Another object of the present invention is to provide a genetically engineered host cell containing a polynucleotide encoding a human PLP protein 39.
  • Another object of the present invention is to provide a method for producing human PLP protein 39.
  • Another object of the present invention is to provide an antibody against the polypeptide-human PLP protein 39 of the present invention.
  • Another object of the present invention is to provide mimic compounds, antagonists, agonists, and inhibitors directed to the polypeptide of the present invention-human PLP protein 39.
  • Another object of the present invention is to provide a method for diagnosing and treating diseases associated with abnormalities in human PLP protein 39.
  • 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:
  • the sequence of the polynucleotide is one selected from the group consisting of: (a) a sequence having positions 381-1439 in SEQ ID NO: 1; and (b) a sequence having 1-3201 in SEQ ID NO: 1 Sequence of bits.
  • the invention further relates to a vector, in particular an expression vector, containing the polynucleotide of the invention; a host cell genetically engineered with the vector, including a transformed, transduced or transfected host cell; and a method comprising culturing said Host cell and method of preparing the polypeptide of the present invention by recovering the expression product.
  • the invention also relates to an antibody capable of specifically binding to a polypeptide of the invention.
  • the invention also relates to a method for screening compounds that mimic, activate, antagonize or inhibit the activity of human PLP protein 39 protein, 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 susceptibility to disease associated with abnormal expression of human PLP protein 39 protein in vitro, which comprises detecting a mutation in the polypeptide or a sequence encoding a polynucleotide thereof in a biological sample, or detecting a mutation in a biological sample The amount or biological activity of a polypeptide of the invention.
  • the invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a polypeptide of the invention or a mimetic thereof, an activator, an antagonist or an inhibitor, and a pharmaceutically acceptable carrier.
  • the present invention also relates to the use of the polypeptide and / or polynucleotide of the present invention in the preparation of a medicament for treating cancer, developmental disease or immune disease or other diseases caused by abnormal expression of human PLP protein 39.
  • 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 “variant" of a protein or polynucleotide refers to an amino acid sequence having one or more amino acids or nucleotide changes or a polynucleotide sequence encoding it.
  • the changes may include deletions, insertions or substitutions of amino acids or nucleotides in the amino acid sequence or nucleotide sequence.
  • Variants can have "conservative" changes, in which the amino acid substituted has a structural or chemical property similar to the original amino acid, such as replacing isoleucine with leucine.
  • Variants can also have non-conservative changes, such as replacing glycine with tryptophan.
  • “Deletion” refers to the deletion of one or more amino acids or nucleotides in an amino acid sequence or nucleotide sequence.
  • “Insertion” or “addition” refers to changes in the amino acid sequence or nucleotide sequence that result in The molecule is increased compared to one or more amino acids or nucleotides. “Replacement” refers to the replacement of one or more amino acids or nucleotides with different amino acids or nucleotides.
  • Bioactivity refers to a protein that has the structure, regulation, or biochemical function of a natural molecule.
  • immunologically active refers to the ability of natural, recombinant or synthetic proteins and fragments thereof to induce a specific immune response in appropriate animals or cells and to bind to specific antibodies.
  • An "agonist” most refers to a molecule that, when combined with human PLP protein 39, causes a change in the protein to regulate the activity of the protein.
  • An agonist may include a protein, a nucleic acid, a carbohydrate, or any other molecule that binds human PLP protein 39.
  • Antagonist refers to a molecule that, when combined with human PLP protein 39, can block or modulate the biological or immunological activity of human PLP protein 39.
  • Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates, or any other molecule that binds human PLP protein 39.
  • Regular refers to a change in the function of human PLP protein 39, including an increase or decrease in protein activity, a change in binding properties, and any other biological, functional, or immune change in human PLP protein 39.
  • substantially pure is meant substantially free of other proteins, lipids, sugars or other substances with which it is naturally associated.
  • Those skilled in the art can purify human PLP protein 39 using standard protein purification techniques.
  • the substantially pure human PLP protein 39 produces a single main band on a non-reducing polyacrylamide gel.
  • the purity of the human PLP protein 39 polypeptide can be analyzed by amino acid sequence.
  • Complementary refers to the natural binding of polynucleotides by base-pairing under conditions of acceptable salt concentration and temperature.
  • sequence C-T-G-A
  • complementary sequence G-A-C-T.
  • the complementarity between two single-stranded molecules may be partial or complete.
  • the degree of complementarity between nucleic acid strands has a significant effect on the efficiency and strength of hybridization between nucleic acid strands.
  • “Homology” 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 imprinting or Nor thern blotting, etc.) under conditions of reduced stringency.
  • Substantially homologous sequences or hybridization probes can compete and inhibit the binding of fully homologous sequences to the target sequence under conditions of reduced stringency. This does not mean that conditions with reduced stringency allow non-specific binding, because conditions with reduced stringency require that the two sequences bind to each other as either specific or selective interactions.
  • 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 (Lasergenes of tware package, DNASTAR, Inc., Mad is on Wi s.). MEGALIGN The program can compare two or more sequences according to different methods such as the Clus ter method (Higgins, DG and PM Sharp (1988) Gene 73: 237-244). 0 The Clus ter method compares groups of sequences by checking the distance between all pairs. Arranged in clusters. The clusters are then assigned in pairs or groups. The percent identity between two amino acid sequences such as sequence A and sequence B is calculated by the following formula:
  • the number of residues matching between sequence A and sequence X 100
  • the number of residues in sequence A-the number of spacer residues in sequence A-the number of spacer residues in sequence B can also be determined by the Clus ter method or by methods known in the art For example, Jotun He in determines the percent identity between nucleic acid sequences (Hein J., (1990) Methods in emzumology 183: 625-645). "Similarity" refers to the amino acid residues at the corresponding positions in the alignment of amino acid sequences. Degree of identical or conservative substitutions.
  • Amino acids used for conservative substitutions may include aspartic acid and glutamic acid; positively charged amino acids may include lysine and arginine; Amino acids with similar hydrophilicity in charged head groups may include leucine, isoleucine, and valine; glycine and alanine; asparagine and glutamine; serine and threonine; benzene Alanine and tyrosine.
  • Antisense refers to a nucleotide sequence that is complementary to a particular DNA or RM sequence.
  • Antisense strand refers to a nucleic acid strand that is complementary to a “sense strand.”
  • Derivative refers to a chemical modification of HFP or a nucleic acid encoding it. This chemical modification may be a substitution of a hydrogen atom with a fluorenyl, acyl or amino group. Nucleic acid derivatives can encode polypeptides that retain the main biological properties of natural molecules.
  • Antibody refers to a complete antibody molecule and its fragments, such as Fa,? ( ⁇ ') 2 and? It can specifically bind to the epitope of human PLP protein 39.
  • a “humanized antibody” refers to an antibody in which the amino acid sequence of a non-antigen binding region is replaced to become more similar to a human antibody, but still retains the original binding activity.
  • isolated refers to the removal of a substance from its original environment (for example, its natural environment if it is naturally occurring).
  • a naturally-occurring polynucleotide or polypeptide is not isolated when it is present in a living thing, but the same polynucleotide or polypeptide is separated from some or all of the substances that coexist with it in the natural system.
  • Such a polynucleotide may be part of a certain vector, or such a polynucleotide or polypeptide may be part of a certain composition. Since the carrier or composition is not part of its natural environment, they are still isolated.
  • isolated refers to the separation of a substance from its original environment (if it is a natural substance, the original environment is the natural environment).
  • polynucleotides and polypeptides in a natural state in a living cell are not isolated and purified, but the same polynucleotides or polypeptides coexist in a natural state Separated in other materials, it is isolated and purified.
  • isolated human PLP protein 39 means that human PLP protein 39 is substantially free of other proteins, lipids, sugars, or other substances with which it is naturally associated. Those skilled in the art can purify human PLP protein 39 using standard protein purification techniques. Substantially pure polypeptides produce a single main band on non-reducing polyacrylamide gels. The purity of the human PLP. Protein 39 polypeptide can be analyzed by amino acid sequence.
  • the present invention provides a new polypeptide, human PLP protein 39, which is basically composed of the amino acid sequence shown in SEQ ID NO: 2.
  • the polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide, and preferably a recombinant polypeptide.
  • the polypeptides of the present invention can be naturally purified products or chemically synthesized products, or can be produced from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insects, and mammalian cells) using recombinant techniques. Depending on the host used in the recombinant production protocol, the polypeptide of the invention may be glycosylated, or it may be non-glycosylated. Polypeptides of the invention may also include or exclude starting methionine residues.
  • the invention also includes fragments, derivatives and analogs of human PLP protein 39.
  • fragment refers to a polypeptide that substantially retains the same biological function or activity of the human PLP protein 39 of the present invention.
  • a fragment, derivative, or analog of the polypeptide of the present invention may be: (I) a type in which one or more amino acid residues are replaced with conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and the substitution is The amino acid may or may not be encoded by the genetic code; or (II) such a type in which a group on one or more amino acid residues is substituted by other groups 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) As explained herein, such fragments, derivatives and analogs are considered to be within the knowledge of those skilled in the art.
  • 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 full-length polynucleotide sequence of 3,201 bases, and its open reading frames 381-1439 encode 352 amino acids. According to the comparison of gene chip expression profiles, it was found that this peptide has a similar expression profile with human PLP protein, and it can be deduced that the human PLP protein 39 has similar functions to human PLP protein.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • DNA forms include cDNA, genomic DNA, or synthetic DM.
  • 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" is in the present invention It 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.
  • the polynucleotide encoding the mature polypeptide of SEQ ID NO: 2 includes: only the coding sequence of the mature polypeptide; the coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence of the mature polypeptide (and optional additional coding sequences); Coding sequence.
  • polynucleotide encoding a polypeptide refers to a polynucleotide comprising the polypeptide and a polynucleotide comprising additional coding and / or non-coding sequences.
  • the invention also relates to variants of the polynucleotides described above, which encode polypeptides or fragments, analogs and derivatives of polypeptides having the same amino acid sequence as the invention.
  • Variants of this polynucleotide can be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants.
  • an allelic variant is an alternative form of a polynucleotide that may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially change the function of the polypeptide it encodes .
  • the present 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% SDS, 60 ° C; or (2) Add denaturants during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Fi co ll, 42 ° C, etc .; or (3) only between two sequences Hybridization occurs only when the identity is at least 95%, more preferably 97%.
  • the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO: 2.
  • nucleic acid fragments that hybridize to the sequences described above.
  • a "nucleic acid fragment” contains at least 10 nucleotides in length, preferably at least 20-30 nucleotides, more preferably at least 50-60 nucleotides, most preferably at least 100 More than nucleotides.
  • Nucleic acid fragments can also be used in nucleic acid amplification techniques, such as PCR, to identify and / or isolate polynucleotides encoding human PLP protein 39.
  • polypeptides and polynucleotides in the present invention are preferably provided in an isolated form and are more preferably purified to homogeneity.
  • the specific polynucleotide sequence encoding the human PLP protein 39 of the present invention can be obtained by various methods.
  • polynucleotides are isolated using hybridization techniques well known in the art. These techniques include, but are not limited to: 1) hybridization of probes to genomic or cDNA libraries to detect homologous polynucleotide sequences, and 2) antibody screening of expression libraries to detect cloned polynucleosides with common structural characteristics Acid fragments.
  • the DNA fragment sequence of the present invention can also be obtained by the following methods: 1) isolating the double-stranded DM sequence from the genomic DNA; 2) chemically synthesizing the DNA sequence to obtain the double-stranded DNA of the polypeptide.
  • genomic DNA isolation is the least commonly used. Direct chemical synthesis of DNA sequences is often the method of choice. The more commonly used method is the isolation of cDNA sequences.
  • the standard method for isolating 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. There are many mature techniques for extracting niRNA, and kits are also commercially available (Qi agene).
  • 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) measuring the level of human PLP protein 39 transcripts; (4) passing Immunological techniques or assays for biological activity to detect gene-expressed protein products. The above methods can be used singly or in combination.
  • the probe used for hybridization is homologous to any part of the polynucleotide of the present invention, and its length is at least 10 nucleotides, preferably at least 30 nucleotides, more preferably At least 50 nucleotides, preferably at least 100 nucleotides.
  • the length of the probe is usually within 2000 nucleotides, preferably within 1000 nucleotides.
  • the probe used here is generally a DNA sequence chemically synthesized based on the gene sequence information of the present invention.
  • the genes or fragments of the present invention can of course be used as probes.
  • DNA probes can be labeled with radioisotopes, luciferin, or enzymes (such as alkaline phosphatase).
  • immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA) can be used to detect the protein product of human PLP protein 39 gene expression.
  • ELISA enzyme-linked immunosorbent assay
  • a method (Sa ik i, et al. Science 1985; 230: 1 350- 1 354) 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 for PCR may be appropriately based on the polynucleotide sequence information of the present invention disclosed herein. Select and synthesize using conventional methods.
  • the amplified DNA / RNA fragments can be isolated and purified by conventional methods such as by gel electrophoresis.
  • polynucleotide sequence of the gene of the present invention or various DNA fragments and the like obtained as described above can be determined by a conventional method such as dideoxy chain termination method (Sanger et al. PNAS, 1977, 74: 546 3- 5467). Such polynucleotide sequences can also be determined using commercial sequencing kits and the like. In order to obtain the full-length cDNA sequence, the sequencing must be repeated. Sometimes it is necessary to determine the cDM sequences of multiple clones in order to splice into full-length cDM sequences.
  • 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 the coding sequence of human PLP protein 39, and the present invention is produced by recombinant technology A method of inventing the polypeptide.
  • a polynucleotide sequence encoding the human PLP protein 39 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 (Ros enberg, etal.
  • 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 human PLP protein 39 and appropriate 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 Cl on ing, 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 lac or trp promoter of E.
  • the expression vector also includes a ribosome binding site and a transcription terminator for translation initiation. Insertion of enhancer sequences into the vector will enhance its transcription in higher eukaryotic cells. Enhancers are cis-acting factors for DNA expression, usually about 10 to 300 base pairs, which act on promoters to enhance gene transcription. Illustrative examples include SV40 enhancers of 100 to 270 base pairs on the late side of the origin of replication, polyoma enhancers on the late side of the origin of replication, and adenoviral enhancers.
  • the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture.
  • selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture.
  • GFP fluorescent protein
  • tetracycline or ampicillin resistance for E. coli.
  • a polynucleotide encoding human PLP protein 39 or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to constitute a genetically engineered host cell containing the polynucleotide or the recombinant vector.
  • the term "host cell” refers to a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast Mother cells; or higher eukaryotic cells, such as mammalian cells. Representative examples are: E.
  • coli Streptomyces
  • bacterial cells such as Salmonella typhimurium
  • fungal cells such as yeast
  • plant cells such as fly S2 or Sf 9
  • animal cells such as CH0, COS, or Bowes melanoma cells.
  • Transformation of a host cell with a DM sequence according to the present invention or a recombinant vector containing the DNA sequence can be performed by conventional techniques well known to those skilled in the art.
  • the host is a prokaryote such as E. coli
  • competent cells capable of absorbing DM may be harvested after exponential growth phase, treated with CaC l 2 method used in steps well known in the art. The alternative is to use MgC l 2 .
  • transformation can also be performed by electroporation.
  • the following DNA transfection methods can be used: calcium phosphate co-precipitation method, or conventional mechanical methods such as microinjection, electroporation, and liposome packaging.
  • the polynucleotide sequence of the present invention can be used to express or produce recombinant human PLP protein 39 (Scence, 1984; 224: 14 31). Generally there are the following steps:
  • the medium used in the culture may be selected from various conventional mediums. Culture is performed under conditions suitable for host cell growth. After the host cells have grown to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.
  • a suitable method such as temperature conversion or chemical induction
  • the recombinant polypeptide may be coated in a cell, expressed on a cell membrane, or secreted outside the cell.
  • recombinant proteins can be separated 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 chromatography
  • FIG. 1 is a comparison diagram of gene chip expression profiles of the human PLP protein 39 and the human PLP protein of the present invention. Pictured above are people
  • PLP protein 39 expression profile chart the following figure is a human PLP protein expression profile chart.
  • Figure 2 shows the polyacrylamide gel electrophoresis (SDS-PAGE) of the isolated human PLP protein 39. 39kDa is the molecular weight of the protein. The arrow indicates the isolated protein band. The best way to implement the invention
  • the determined cDNA sequence was compared with the existing public DNA sequence database (Genebank), and it was found that the cDNA sequence of one clone 0063b02 was new DNA.
  • the inserted cDNA fragments contained in this clone were determined in both directions by synthesizing a series of primers.
  • the results show that the 0063b02 clone contains a full-length cDNA of 3201bp (as shown in Seq ID NO: 1), and has a 1059bp open reading frame (0RF) from 381bp to 1439bp, encoding a new protein (such as Seq ID NO : Shown in 2).
  • This clone pBS-0063b02 and the encoded protein was named human PLP protein 39.
  • CDNA was synthesized using fetal brain cell total RNA as a template and ol igo-dT as a primer for reverse transcription reaction.
  • Pr imerl 5'- GTTGACGTACATATATATTCAGAA -3 '(SEQ ID NO: 3)
  • Pr imer2 5'- GTTGATGTATTATTTATTATGTAA -3 '(SEQ ID NO: 4)
  • Pr imerl is a forward sequence located at the 5th end of bp in SEQ ID NO: 1; Primer2 is the 3, terminal reverse sequence of SEQ ID NO: 1.
  • Amplification conditions 50 mmol / L KC1, 10 mmol / L Tris-Cl, (pH 8.5), 1.5 mmol / L MgCl 2 , 200 ⁇ mol / L dNTP, lOpmol primers in a 50 ⁇ 1 reaction volume, 1U of Taq DNA polymerase (Clontech).
  • the reaction was performed on a PE9600 DNA thermal cycler (Perkin-Elraer) for 25 cycles under the following conditions: 94 ° C 30sec; 55 ° C 30sec; 72. C 2rain.
  • 3-actin was used as a positive control and template blank was used as a negative control.
  • the amplified product was purified using a QIAGEN kit and ligated to a pCR vector using a TA cloning kit (Invitrogen product). DNA sequence The analysis results showed that the DNA sequence of the PCR product was exactly the same as that of 1-3201 bp shown in SEQ ID NO: 1.
  • Example 3 Northern blot analysis of human PLP protein 39 gene expression:
  • RNA extraction in one step [Anal. Biochera 1987, 162, 156-159] 0
  • This method involves acid guanidinium thiocyanate-chloroform extraction. That is, the tissue is homogenized with 4M guanidine isothiocyanate-25mM sodium citrate, 0.2M sodium acetate (pH4.0), and 1 time volume of phenol and 1/5 volume of chloroform-isoamyl alcohol (49: 1 ) And centrifuge after mixing. Aspirate the aqueous layer, add isopropanol (0.8 vol) and centrifuge the mixture to obtain RNA precipitate. The resulting RNA pellet was washed with 70% ethanol, dried and dissolved in water.
  • the 32P- labeled probe (about 2x l0 6 cpm / ml) and RNA was transferred to a nitrocellulose membrane overnight at 42 ° C in a hybridization solution, the solution comprising 50% formamide -25mM KH 2 P0 4 ( P H7.4) -5 x SSC-5 x Denhardt's solution and 200 g / ml salmon sperm DNA.
  • the filters were placed at 1 x SSC-0.1 ° /. Wash in SDS at 55 ° C for 30 min. Then, Phosphor Imager was used for analysis and quantification.
  • Example 4 In vitro expression, isolation and purification of recombinant human PLP protein 39
  • Primer3 5 '-CCCCATATGATGCATACCCAGCCTCTCAAAGAA- 3' (Seq ID No: 5)
  • Priraer4 5 '-CATGGATCCTTACTTAATGGCTTCTCCATTCTT- 3' (Seq ID No: 6)
  • the 5 'ends of these two primers contain Mel and BaraHI restriction sites, respectively. Following are the coding sequences of the 5 ,, and 3 'ends of the gene of interest, respectively.
  • the Ndel and BamHI digestion sites correspond to the selectivity within the expression vector plasmid pET-28b (+) (Novagen, Cat. No. 69865.3). Digestion site.
  • the PCR reaction was performed using the pBS-0063b02 plasmid containing the full-length target gene as a template.
  • the PCR reaction conditions are: a total volume of 50 ⁇ 1 containing 10 pg of pBS- 0063b02 plasmid, primers! ⁇ ! ⁇ ; ⁇ And! ⁇ ! ! ⁇ -Separate! !! For: ⁇ !! ⁇ , Advantage polymerase Mix (Clontech) 1 ⁇ 1. Cycle parameters: 94 ° C 20s, 60 ° C 30s, 68. C 2 min, a total of 25 cycles.
  • the amplified product and plasmid pET-28 (+) were double digested with Mel and BaraHI, respectively, and large fragments were recovered and ligated with T4 ligase.
  • the ligation product was transformed into E. coli DH5a by the calcium chloride method. After being cultured overnight on LB plates containing kanamycin (final concentration 30 g / ml), positive clones were selected by colony PCR method and sequenced. Selected positive clones with the correct sequence (P ET-0063b02) recombinant plasmids by the calcium chloride method to transform E. coli BL 2 l (DE3) plySs ( Novagen Co.).
  • the host strain BL21 (pET-0063b02) was at 37. C. Cultivate to logarithmic growth phase, add IPTG to a final concentration of 1 ol / L, and continue to cultivate for 5 hours. The cells were collected by centrifugation, and the supernatant was collected by centrifugation. The supernatant was collected by centrifugation, and the layer was layered with His s. Bind Quick Cartr idge (product of Novagen) with an affinity chromatography column capable of binding to 6 histidines (6His-Tag). Analysis to obtain purified human protein PLP protein 39.
  • the polypeptide is coupled to hemocyanin and bovine serum albumin to form a complex, respectively.
  • hemocyanin and bovine serum albumin For the method, see: Avraraeas, et al. Immunochemi s try, 1969; 6: 43. Rabbits were immunized with 4 mg of the hemocyanin peptide complex plus complete Freund's adjuvant, and 15 days later, the hemocyanin peptide complex plus incomplete Freund's adjuvant was used to boost immunity once.
  • a titer plate coated with a 15 g / ml bovine serum albumin peptide complex was used as an ELISA to determine antibody titers in rabbit serum.
  • Protein A-Sepharose was used to isolate total IgG from antibody-positive rabbit serum.
  • the peptide was bound to a cyanogen bromide-activated Sepharose4B column, and anti-peptide antibodies were separated from the total IgG by affinity chromatography.
  • the immunoprecipitation method proved that the purified antibody could specifically bind to human PLP protein 39.
  • Example 6 Application of the polynucleotide fragment of the present invention as a hybridization probe
  • Suitable oligonucleotide fragments selected from the polynucleotides of the present invention are used as hybridization probes in a variety of ways.
  • the probes can be used to hybridize to genomic or cDNA libraries of normal tissue or pathological tissue from different sources to It is determined whether it contains the polynucleotide sequence of the present invention and a homologous polynucleotide sequence is detected.
  • the probe can be used to detect the polynucleotide sequence of the present invention or its homologous polynucleotide sequence in normal tissue or pathology. Whether the expression in tissue cells is abnormal.
  • the purpose of this embodiment is to select suitable oligonucleotide fragments from the polynucleotide SEQ ID NO: 1 of the present invention for use as hybridization probes, and to identify whether some tissues contain the multinucleus of the present invention by using a filter hybridization method.
  • Filters 3 ⁇ 4 include dot blots, Southern blots, Northern blots, and photocopying. They are basically the same steps used to fix the polynucleotide sample to be tested on the filter.
  • the sample-immobilized filter is first pre-treated with a probe-free buffer solution, so that the non-specific binding site of the sample on the filter is saturated with the carrier and the synthesized polymer.
  • the pre-hybrid solution is then replaced with a buffer solution containing labeled probes and incubated with the probes and target nucleic acids.
  • the probes on the substrate are removed by a series of membrane washing steps. In this embodiment, higher-intensity washing conditions (such as lower salt concentration and higher temperature) are used to reduce the heterogeneous background and retain only strong specific signals.
  • the probes used in this embodiment include two types: the first type of probes are oligonucleotide fragments that are completely the same as or complementary to the polynucleotide SEQ ID NO: 1 of the present invention; the second type of probes are partially related to the present invention
  • the polynucleotide SEQ ID NO: 1 is the same or complementary oligonucleotide fragment.
  • a dot blot is used to fix the sample on the filter membrane. Under the condition of higher intensity washing of the membrane, the first type of probe and the sample have the strongest specificity and are retained.
  • oligonucleotide fragments from the polynucleotide SEQ ID NO: 1 of the present invention for use as hybrid probes should follow the following principles and considerations:
  • the preferred range of probe size is 18-50 nucleotides
  • GC content is 30% -70%, more than non-specific ⁇ 'li: increase;
  • 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 (probel), which belongs to the first class of probes, is completely homologous or complementary to the gene fragment of SEQ ID NO: 1 (41Nt): '
  • Probe 2 (probe2), which belongs to the second type of probe, is equivalent to the replacement mutant sequence (41Nt) of the gene fragment of SEQ ID NO: 1 or its complementary fragment:
  • step 14 can be performed directly.
  • 8) Add RNase A to the DNA solution to a final concentration of 100ug / ral, and incubate at 37 ° C for 30 minutes.
  • 9) Add SDS and proteinase K to the final concentration of 0.5% and 100ug / ml o 37 ° C for 30 minutes.
  • 10) Extract the reaction solution with an equal volume of phenol: chloroform: isoamyl alcohol (25: 24: 1) and centrifuge for 10 minutes.
  • 11) Carefully remove the aqueous phase and re-extract with an equal volume of chloroform: isoamyl alcohol (24: 1) and centrifuge for 10 minutes.
  • NC membranes nitrocellulose membranes
  • Two NC membranes are required for each probe, so that they can be used in the following experimental steps.
  • the film was washed with high-strength conditions and strength conditions, respectively.
  • the 32 P-Probe (the second peak is free ⁇ - 32 P-dATP) is prepared.
  • Gene chip or gene microarray is a new technology currently being developed by many national laboratories and large pharmaceutical companies. It refers to the orderly and high-density arrangement of a large number of target gene fragments on glass, The data is compared and analyzed on a carrier such as silicon using fluorescence detection and computer software to achieve the purpose of rapid, efficient, and high-throughput analysis of biological information.
  • the polynucleotide of the present invention can be used as target DNA for gene chip technology for high-throughput research of new gene functions; search for and screen new tissue-specific genes, especially new genes related to diseases such as tumors; diagnosis of diseases such as hereditary diseases .
  • a total of 4,000 polynucleotide sequences of various full-length cDNAs are used as target DM, including the polynucleotide of the present invention. They were respectively amplified by PCR. After the purified amplified product was purified, the concentration was adjusted to about 500 ng / ul, and spotted on a glass with a Cartesian 7500 spotting instrument (purchased from Cartesian, USA). The distance between the points on the medium is 280 ⁇ ⁇ . The spotted slides were hydrated, dried, and cross-linked in a UV cross-linker. After elution, the slides were fixed to fix the DNA on the glass slides to prepare chips. The specific method steps are widely reported in the literature. The post-spot processing steps of this embodiment are:
  • Total mRNA was extracted from human mixed tissues and specific tissues (or stimulated cell lines) in one step, and mRM was purified using Oligotex mRNA Midi Kit (purchased from QiaGen).
  • the fluorescent reagent Cy3dUTP 5- Amino- propargyl- 2'- deoxyuridine 5--triphate coupled to Cy3 fluorescent dye (purchased from Amersham Pharaacia Biotech) was used to label the mRNA of human mixed tissue, and the fluorescent reagent Cy5dUTP (5- Amino-propargyl- 2'- deoxyuridine 5 '-tr iphate coupled to Cy5 fluorescent dye, purchased from Amersham Phamacia Biotech company, labeled the body's specific tissue (or stimulated cell line) mRNA, and purified the probe to prepare a probe.
  • Cy3dUTP 5- Amino- propargyl- 2'- deoxyuridine 5--triphate coupled to Cy3 fluorescent dye (purchased from Amersham Pharaacia
  • the above specific tissues are fetal brain, bladder mucosa, and PMA +
  • Ecv304 cell line LPS + Ecv304 cell line thymus, normal fibroblasts 1024N'C, Fibroblast, growth factor stimulation, 1024NT, scar into fc growth factor stimulation, 1013HT, scar into fc without growth factor Sub-stimulation, 1013HC, bladder cancer cell EJ, bladder cancer, bladder cancer, liver cancer, hepatocellular carcinoma cell line, placenta, spleen, prostate cancer, jejunal adenocarcinoma, cardia cancer. Draw a chart based on these 18 Cy3 / Cy5 ratios. (figure 1 ) . It can be seen from the figure that the expression profiles of human PLP protein 39 and human PLP protein according to the present invention are very similar. Industrial applicability
  • 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 malignant tumors, adrenal deficiency, skin diseases, various inflammations, HIV infections and immune diseases.
  • the PLP protein family is a type of myelin protein lipoprotein (Mye l in proteo l ip id prote in, PLP, li pophi l in), which is mainly present in nerve tissues and participates in the formation and maintenance of myelin multilayer structures.
  • Mye l in proteo l ip id prote in, PLP, li pophi l in myelin protein lipoprotein
  • PLP / DM20 protein function 1. Maintain and stabilize the multilayer structure of central myelin sheath: The central nervous system myelin sheath structure is a multilayer structure formed by tightly combining the cell membranes of oligodendrocytes with protruding parts. PLP / DM20 is mainly produced by oligodendrocytes in the central nervous system. It is the main component of myelin proteins, together with Myel in basic protein (MBP). Formation and stability play an important role. 2. Participate in the differentiation of the nervous system: 0-2 days after the birth of a newborn, the earliest PLP protein can be detected in the chordae, which coincides with the time of myelin formation.
  • the novel PLP protein 39 of the present invention has a PLP protein function. It can be seen that abnormal expression of the PLP protein 10 of the present invention will produce various diseases, especially neurodevelopmental disorders, neurological diseases, abnormal neurorepairs, and neurological immune diseases. These diseases include, but are not limited to:
  • Neurodevelopmental disorders neural tube insufficiency, brain developmental malformations, neuronal migration disorders, aqueduct malformations, cerebellar dysplasia, Down syndrome, spinal malformations, congenital hydrocephalus, congenital cerebellar dysplasia syndrome, Congenital abortion Nervous system diseases: Schwann cell tumor, neurofibromatosis, ependymal tumor, intracranial granulomatosis, Parkinson's disease, chorea, depression, amnesia, Huntington's disease, epilepsy, migraine, dementia, Spinal muscular atrophy, muscular hypertrophy, tonic muscle dystrophy, bradykinesia, dystonia, neurofibromatosis, nodular sclerosis, cerebral trigeminal neurohemangioma, ataxia capillary telangiectasia, Schizophrenia, depression, paranoia, anxiety, obsessive-compulsive disorder, phobia, neurodegeneration, acute myelitis, spinal cord compression, trigeminal neural
  • the invention also provides methods for screening compounds to identify agents that increase (agonist) or suppress (antagonist) human PLP protein 39.
  • Agonists enhance human PLP protein 39 to stimulate biological functions such as cell proliferation, while antagonists prevent and treat disorders related to excessive cell proliferation, such as various cancers.
  • mammalian cells or a membrane preparation expressing human PLP protein 39 can be cultured with labeled human PLP protein 39 in the presence of a drug. The ability of the drug to increase or block this interaction is then determined.
  • Antagonists of human PLP protein 39 include antibodies, compounds, receptor deletions, and the like that have been screened. Antagonists of human PLP protein 39 can bind to human PLP protein 39 and eliminate its function, or inhibit the production of the polypeptide, or bind to the active site of the polypeptide so that the polypeptide cannot perform biological functions.
  • human PLP protein 39 can be added to a bioanalytical assay to determine whether a compound is an antagonist by measuring the effect of the compound on the interaction between human PLP protein 39 and its receptor. 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 PLP protein 39 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. When screening, human PLP protein 39 molecules should generally be labeled.
  • the present invention provides a method for producing antibodies using polypeptides, and fragments, derivatives, analogs or cells thereof as antigens. These antibodies can be polyclonal or monoclonal antibodies.
  • the invention also provides antibodies directed against the human PLP protein 39 epitope. These antibodies include (but are not limited to): polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, and fragments produced by Fab expression libraries.
  • Polyclonal antibodies can be produced by injecting human PLP protein 39 directly into immunized animals (such as rabbits, mice, rats, etc.).
  • immunized animals such as rabbits, mice, rats, etc.
  • a variety of adjuvants can be used to enhance the immune response, including but not limited to Freund's adjuvant.
  • Techniques for preparing monoclonal antibodies to human PLP protein 39 include, but are not limited to, hybridoma technology (Kohler and Milste in. Nature, 1975, 256: 495-497), triple tumor technology, human beta-cell hybridoma Technology, EBV-hybridoma technology, etc. Chimeric antibodies combining human constant regions and non-human variable regions can be produced using existing techniques (Morr is on etal, PNAS, 1985, 81: 6851).
  • Monoclonal antibodies that bind to human PLP protein 39 can also be labeled with radioisotopes and injected into the body to track their location and distribution. This radiolabeled antibody can be used as a non-invasive diagnostic method to locate tumor cells and determine whether there is metastasis.
  • Antibodies can also be used to design immunotoxins that target a particular part of the body.
  • human PLP protein 39 high affinity monoclonal antibodies can covalently bind to bacterial or plant toxins (such as diphtheria toxin, ricin, ormosine, etc.).
  • a common method is to attack the amino group of an antibody with a thiol cross-linking agent such as SPDP and bind the toxin to the antibody through the exchange of disulfide bonds.
  • This hybrid antibody can be used to kill human PLP protein.
  • Antibodies can be used to treat or prevent diseases related to human PLP protein 39. Administration of an appropriate dose of antibody can stimulate or block the production or activity of human PLP protein 39.
  • the invention also relates to a diagnostic test method for quantitatively and locally detecting the level of human PLP protein 39.
  • tests are well known in the art and include F I SH assays and radioimmunoassays.
  • the level of human PLP protein 39 detected in the test can be used to explain the importance of human PLP protein 39 in various diseases and to diagnose diseases in which human PLP protein 39 functions.
  • the polypeptide of the present invention can also be used for peptide mapping analysis.
  • the polypeptide can be specifically cleaved by physical, chemical or enzyme, and can be analyzed by one-dimensional or two-dimensional or three-dimensional gel electrophoresis, and more preferably by mass spectrometry encoding
  • the polynucleotide of human PLP protein 39 can also be used for a variety of therapeutic purposes.
  • Gene therapy technology can be used to treat abnormal cell proliferation, development or metabolism caused by the non-expression or abnormal / inactive expression of human PLP protein 39.
  • Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated human PLP protein 39 to inhibit endogenous human PLP protein 39 activity.
  • a mutated human PLP protein 39 may be a shortened human PLP protein 39 lacking a signaling domain. Although it can bind to downstream substrates, it lacks signaling activity. Therefore, the recombinant gene therapy vector can be used for treating diseases caused by abnormal expression or activity of human PLP protein 39.
  • Virus-derived expression vectors such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus, etc. can be used to transfer a polynucleotide encoding human PLP protein 39 into cells.
  • a method for constructing a recombinant viral vector carrying a polynucleotide encoding a human PLP protein 39 can be found in the existing literature (Sambrook, et al.).
  • a recombinant polynucleotide encoding human PLP protein 39 can be packaged into liposomes and transferred into cells.
  • Methods for introducing a polynucleotide into a tissue or cell include: injecting the polynucleotide directly into a tissue in vivo; or introducing the polynucleotide into a cell through a vector (such as a virus, phage, or plasmid) in vitro, The cells are then transplanted into the body and the like.
  • a vector such as a virus, phage, or plasmid
  • Oligonucleotides including antisense RNA and DNA
  • ribozymes that inhibit human PLP protein 39 mRNA are also within the scope of the present invention.
  • a ribozyme is an enzyme-like RNA molecule that specifically decomposes specific RNA. Its mechanism is that the ribozyme molecule specifically hybridizes with a complementary target RM to perform endonucleation.
  • Antisense RNA, DNA and ribozymes can be obtained by any MA or DNA synthesis technology. For example, the technique of solid phase phosphoramidite chemical synthesis to synthesize oligonucleotides has been widely used.
  • Antisense RNA molecules can be obtained by in vitro or in vivo transcription of a DNA sequence encoding the RNA.
  • This DNA sequence is integrated downstream of the RNA polymerase promoter of the vector.
  • 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 PLP protein 39 can be used for the diagnosis of diseases related to human PLP protein 39.
  • the polynucleotide encoding human PLP protein 39 can be used to detect the expression of human PLP protein 39 or the abnormal expression of human PLP protein 39 in a disease state.
  • the DNA sequence encoding human PLP protein 39 can be used to hybridize biopsy specimens to determine the expression of human PLP protein 39.
  • Hybridization techniques include Sou thern blotting, Nor thern blotting, and in situ hybridization. These techniques and methods are publicly available and mature, and related kits are commercially available.
  • a part or all of the polynucleotides of the present invention can be used as probes to be fixed on a micro array or a DM chip (also known as a "gene chip") for analyzing differential expression analysis of genes and genetic diagnosis in tissues.
  • Human PLP protein 39 specific primers can also be used to detect the transcription products of human PLP protein 39 by RNA-polymerase chain reaction (RT-PCR) in vitro amplification.
  • RT-PCR RNA-polymerase chain reaction
  • Human PLP protein 39 gene mutation can also be used to diagnose human PLP protein 39-related diseases.
  • Human PLP protein 39 mutations include point mutations, translocations, deletions, recombination, and other abnormalities compared to the normal wild-type human PLP protein 39 DNA sequence. Mutations can be detected using existing techniques such as Southern blotting, DNA sequence analysis, PCR and in situ hybridization. In addition, mutations may affect protein expression. Therefore, Nor thern blotting and Western blotting can be used to indirectly determine whether a 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 according to cDM, and the sequences can be located on chromosomes. These primers were then used for PCR screening of somatic hybrid cells containing individual human chromosomes. Only those heterozygous cells containing the human gene corresponding to the primer will produce amplified fragments.
  • PCR localization of somatic hybrid cells is a quick way to localize DM to specific chromosomes.
  • sublocalization can be achieved by a similar method using a set of fragments from a specific chromosome or a large number of genomic clones.
  • Other similar strategies that can be used for chromosomal localization include in situ hybridization, chromosome pre-screening with labeled flow sorting, and hybrid pre-selection to construct chromosome-specific cDNA libraries.
  • Fluorescent in situ hybridization (FISH) of cDM 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. Mckus ck, Mende an Inheritance in Man (available online with Johns Hopkins University Wetch Med ica l Library). Linkage analysis can then be used to determine the relationship between genes and diseases that have been mapped to chromosomal regions.
  • the difference in cDM or genomic sequence between the affected and unaffected individuals needs to be determined. If a mutation is observed in some or all diseased individuals and the mutation is not observed in any normal individuals, the mutation may be the cause of the disease. Comparing affected and unaffected individuals usually involves first looking for structural changes in the chromosomes, such as deletions or translocations that are visible at the chromosomal level or detectable with cDM sequence-based PCR. According to the resolution capabilities of current physical mapping and gene mapping technology, the cDNA accurately mapped to the chromosomal region associated with the disease can be one of 50 to 500 potentially pathogenic genes (assuming 1 megabase mapping resolution) Capacity and each 20kb corresponds to a gene).
  • the polypeptides, polynucleotides and mimetics, agonists, antagonists and inhibitors of the present invention can be used in combination with a suitable pharmaceutical carrier.
  • suitable pharmaceutical carrier can be water, glucose, ethanol, salts, buffers, glycerol, and combinations thereof.
  • the composition comprises a safe and effective amount of the polypeptide or antagonist, and carriers and excipients which do not affect the effect of the drug. These compositions can be used as drugs for the treatment of diseases.
  • the 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 via a topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal route of administration.
  • Human PLP protein 39 is administered in an amount effective to treat and / or prevent a specific indication.
  • the amount and range of human PLP protein 39 to be 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.

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Abstract

L'invention concerne un nouveau polypeptide, une protéine humaine PLP 39, et un polynucléotide codant ce polypeptide ainsi qu'un procédé d'obtention de ce polypeptide par des techniques recombinantes d'ADN. L'invention concerne en outre les applications de ce polypeptide dans le traitement de maladies, notamment des tumeurs malignes, de l'hémopathie, de l'infection par VIH, de maladies immunitaires et de diverses inflammations. L'invention concerne aussi l'antagoniste agissant contre le polypeptide et son action thérapeutique ainsi que les applications de ce polynucléotide codant la protéine humaine PLP 39.
PCT/CN2001/001019 2000-06-21 2001-06-19 Nouveau polypeptide, proteine humaine plp 39, et polynucleotide codant ce polypeptide WO2002000821A2 (fr)

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WO1999017795A1 (fr) * 1997-10-03 1999-04-15 Smithkline Beecham Corporation FACTEUR DE RECYCLAGE DE RIBOSOMES (FRR) DE $i(STAPHYLOCOCCUS AUREUS)
WO2000006183A1 (fr) * 1998-07-27 2000-02-10 Millennium Pharmaceuticals, Inc. Nouvelle sous-unite beta du canal a potassium, c7f2-a

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WO1999017795A1 (fr) * 1997-10-03 1999-04-15 Smithkline Beecham Corporation FACTEUR DE RECYCLAGE DE RIBOSOMES (FRR) DE $i(STAPHYLOCOCCUS AUREUS)
WO2000006183A1 (fr) * 1998-07-27 2000-02-10 Millennium Pharmaceuticals, Inc. Nouvelle sous-unite beta du canal a potassium, c7f2-a

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