WO2002033076A1 - Nouveau polypeptide, facteur de transcription eucaryotique 17.38, et polynucleotide codant ce polypeptide - Google Patents

Nouveau polypeptide, facteur de transcription eucaryotique 17.38, et polynucleotide codant ce polypeptide Download PDF

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Publication number
WO2002033076A1
WO2002033076A1 PCT/CN2001/001142 CN0101142W WO0233076A1 WO 2002033076 A1 WO2002033076 A1 WO 2002033076A1 CN 0101142 W CN0101142 W CN 0101142W WO 0233076 A1 WO0233076 A1 WO 0233076A1
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polypeptide
polynucleotide
transcription factor
eukaryotic transcription
sequence
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PCT/CN2001/001142
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English (en)
French (fr)
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Yumin Mao
Yi Xie
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Biowindow Gene Development Inc. Shanghai
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Priority to AU2002213743A priority Critical patent/AU2002213743A1/en
Publication of WO2002033076A1 publication Critical patent/WO2002033076A1/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
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the present invention belongs to the field of biotechnology. Specifically, the present invention describes a new polypeptide, eukaryotic transcription factor 17.38, and a polynucleotide sequence encoding the polypeptide. The invention also relates to a preparation method and application of the polynucleotide and polypeptide. Background technique
  • Eukaryotic transcription factors are a collective term for protein factors necessary for the transcription of eukaryotic genes.
  • eukaryotic RM polymerases need to rely strictly on a series of protein factors to recognize and bind to the specific sequence of the promoter, and a certain amount of protein is needed to start the transcription reaction.
  • the tissue- and cell-specific regulation of gene transcription requires the participation of other specific protein factors.
  • hundreds of protein factors have been isolated, purified, or identified. These commonly acting protein factors can be divided into two broad categories.
  • transcription factor a factor that binds to the core sequence of the promoter such as the TATA box and its vicinity
  • the other is a protein factor that binds to the upstream regulatory region UPE and an enhancer region, called a transcription regulatory factor .
  • the protein molecules of these transcription factors and transcription regulators have two functions according to their functions. Some transcription factors can bind to RNA polymerase when they form the starting complex, but they are not part of the enzyme. They are universal transcription factors for all automatons to initiate transcription reactions, and some are involved in termination reactions of transcription.
  • BTF1 also known as TF II D
  • BTF2 BTF3
  • BTF4 The first step in assembling the initiation complex is that BTF1 is steadily bound to the TATA box, and STF promotes this process.
  • BTF2 and BTF3 do not directly bind to the immediate vicinity of the promoter, but BTF3 can form a stable compound with RNA polymerase II.
  • BTF3 is a protein with a molecular weight of 271. Based on a further study of the sequence of BTF3, two central proteins were found: BTF3a and BTF3b.
  • BTF3a has a molecular weight of 22K, contains 206 residues, and has all the functions of BTF3
  • BTF3b has a molecular weight of 18K and contains 162 residues. It lacks the first 44 residues of BTF3a and can bind to RNA polymerase II, but has no transcriptional activity.
  • BTF3a binds to a DNA-binding domain (such as a mediation-turn-helix Or zinc finger) and protein-protein interaction crust domains (such as leucine zipper).
  • BTF3a The N- and C-terminus of BTF3a is highly hydrophilic, the central region is rich in leucine residues, and highly hydrophobic.
  • 143- Residue 154 forms a short lipophilic ⁇ -helix, and all of the segments of the helix are hydrophobic amino acids, mainly leucine.
  • This central hydrophobic region is a good site for binding to RNA polymerase II.
  • Others Protein factors such as BTF2 can also bind to this site.
  • a similar lipophilic helix forms a coil-to-coil structure, which is the site of protein interaction in microtubule cross-linking.
  • BTF3 is in cells Content is quite meager, but are present in all cells.
  • RNA polymerase II directly binds to RM polymerase II to enable the promoter to perform low-level basal transcription. It can be further activated by transcription activating factors or can be suppressed by transcription inhibitors. If it is absent, RNA polymerase II alone will lead to scattered multi-point initiation of tnRNA synthesis with poor synthesis efficiency.
  • BTF3 is involved in determining the tissue and developmental stage of the gene. If the gene encoding BTF3 is mutated, a variety of genes regulated by BTF3 will not be expressed normally, which will lead to various diseases, such as embryonic development and cell differentiation. Related diseases. (XM Zheng, et. Al, Nature, 1990)
  • the polypeptide of the present invention contains a RM polymerase II binding domain and has 81% homology with the human transcription factor BTF3 at the protein level, so it belongs to the human transcription factor BTF protein family. Based on the above points, the peptide of the present invention is a human transcription factor, named true and transcription factor 17.38, and it is inferred that it is similar to BTF3 and has similar biological functions.
  • eukaryotic transcription factor ⁇ .38 protein plays an important role in regulating important functions of the body such as cell division and embryonic development as described above, and it is believed that a large number of proteins are involved in these regulatory processes, there is always a need to identify more involved in these Process eukaryotic transcription factor ⁇ 38 protein, in particular the amino acid sequence of this protein is identified.
  • the new eukaryotic transcription factor 17.38 The isolation of the protein-coding gene also provides the basis for research to determine the role of the protein in health and disease states. This protein may form the basis for developing diagnostic and / or therapeutic drugs for the disease, so isolating its coding DNA is important. 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 eukaryotic transcription factor ⁇ .38.
  • Another object of the invention is to provide a genetically engineered host cell incorporating a polynucleotide encoding a eukaryotic transcription factor 17.38.
  • Another object of the present invention is to provide a method for producing eukaryotic transcription factor ⁇ .38.
  • Another object of the present invention is to provide a polypeptide-to-eukaryotic transcription factor ⁇ .38 of the present invention.
  • Another object of the present invention is to provide a mimic compound to the polypeptide of the present invention-eukaryotic transcription factor ⁇ . 38.
  • Another object of the present invention is to provide a method for diagnosing and treating diseases related to eukaryotic transcription factor ⁇ . 38 abnormal habitat.
  • 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 79-555 in SEQ ID NO: 1; and (b) a sequence having 1-2185 in SEQ ID NO: 1 Sequence of bits.
  • the present invention further relates to a vector, particularly an expression vector, containing the polynucleotide of the present invention; a host cell genetically engineered with the vector, including a transformed, transduced or transfected host cell; Host cell and method of preparing the polypeptide of the present invention by recovering the expression product.
  • the invention also relates to an antibody capable of specifically binding to a polypeptide of the invention.
  • the invention also relates to a method for screening compounds that mimic, activate, antagonize or inhibit eukaryotic transcription factor 17.38 protein activity, which comprises utilizing a polypeptide of the invention.
  • the invention also relates to compounds obtained by this method.
  • the present invention also relates to a method for in vitro detection of a disease or susceptibility to disease associated with abnormal expression of a eukaryotic transcription factor 1 7.38 protein, comprising detecting a mutation in the polypeptide or a polynucleotide sequence encoding the same in a biological sample, or Detection of the amount or biological activity of a polypeptide of the invention in a biological sample.
  • the invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a polypeptide of the invention or a mimetic thereof, an activator, an antagonist or an inhibitor, and a pharmaceutically acceptable carrier.
  • the invention also relates to the use of the polypeptides and / or polynucleotides of the invention for the manufacture of a medicament for the treatment of cancer, developmental or immune diseases or other diseases caused by abnormal expression of eukaryotic transcription factors 17.38.
  • 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
  • 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 may have "conservative" changes in which the substituted amino acid 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, acid.
  • “Deletion” refers to the deletion of one or more amino acids or nucleotides in an amino acid sequence or nucleotide sequence.
  • Insertion 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 by different amino acids or nucleotides.
  • Bioactivity refers to a protein that has the structure, regulation, or biochemical function of a natural molecule.
  • immunologically active refers to the ability of natural, recombinant or synthetic proteins and fragments thereof to induce a specific immune response and to bind specific antibodies in a suitable animal or cell.
  • An "agonist” refers to a molecule that, when combined with a eukaryotic transcription factor 1 7.38, can cause the protein to change, thereby regulating the activity of the protein.
  • An agonist may include a protein, a nucleic acid, a carbohydrate, or any other molecule that can bind to a eukaryotic transcription factor 17.38.
  • Antagonist refers to a molecule that, when combined with eukaryotic transcription factor 17.38, can block or regulate the biological or immunological activity of eukaryotic transcription factor 17.38.
  • Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates or any other molecule that can bind to eukaryotic transcription factors 17.38.
  • Regular refers to a change in the function of eukaryotic transcription factor 17.38, including an increase or decrease in protein activity, a change in binding characteristics, and any other biological, functional, or immune properties of eukaryotic transcription factor 17.38. change.
  • Substantially pure ' means essentially free of other proteins, lipids, carbohydrates or other substances with which it is naturally associated.
  • Those skilled in the art can use standard protein purification techniques to purify eukaryotic transcription factors 17.38.
  • Basic Pure eukaryotic transcription factor 7.38 can produce a single main band on a non-reducing polyacrylamide gel.
  • Eukaryotic transcription factor 17.38 The purity of the 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” may 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 achieved by crossing hybrids with reduced stringency (Sou Uie ni India Traces or Northern blots). Substantially homologous sequences or hybridization probes can compete and inhibit the binding of completely homologous sequences to the target sequence under conditions of reduced stringency. This does not mean that the conditions of reduced stringency allow non-specific binding, because the conditions of reduced stringency require that the two sequences bind to each other as a specific or selective interaction.
  • Percent identity refers to the percentage of sequences that are identical or similar in the comparison of two or more amino acid or nucleic acid sequences. The percent identity can be determined electronically, such as by the MBGALIGN program (Lasergene software package, DNASTAR, Inc., Mad Son Wis.). The MEGALTGN program can compare two or more sequences based on different methods such as the Cluster method (Higgins, DG and PM Sharp (1988) Gene 73: 237-244). 0 The Clus ter method groups each group by checking the distance between all pairs. The sequences are arranged in clusters. The clusters are then assigned in pairs or groups.
  • the percent identity of two amino acid sequences is calculated by the following formula:
  • the number of residues in which sequence A matches sequence B is the number of residues in sequence A-the number of spacers in sequence A-
  • the number of spacer residues in B can also be determined by the Cluster method or by methods known in the art such as Jotun He in (%). (He n J., (1990) Methods in emzumology 183: 625-645).
  • Similarity refers to the degree of identical or conservative substitutions of amino acid residues at corresponding positions in the alignment of amino acid sequences.
  • Amino acids used for conservative substitutions for example, negatively charged amino acids may include aspartic acid and glutamic acid; positively charged amino acids may include lysine and arginine; having an uncharged head group is Similar hydrophilic amino acids may include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; serine and threonine; phenylalanine and tyrosine.
  • Antisense refers to a nucleotide sequence that is complementary to a particular DNA or RNA sequence.
  • Antisense strand refers to a nucleic acid strand that is complementary to a “sense strand.”
  • Derivative refers to a chemical modification of HFP or a nucleic acid encoding it. This chemical modification may be the replacement of a hydrogen atom with an alkyl, 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, F (ab ') 2 and Fv, which can specifically bind to the epitope of eukaryotic transcription factor 17.38.
  • a “humanized antibody” refers to an antibody whose amino acid sequence in the antigen-binding region has been replaced to become more similar to a human antibody, but still retains the original binding activity.
  • isolated refers to the removal of matter from its original environment (for example, Natural environment).
  • 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 certain vector, or such a polynucleotide or polypeptide may be part of a certain composition. Since the carrier or composition is not a component of its natural environment, they are still isolated.
  • isolated refers to the separation of a substance from its original environment (if it is a natural substance, the original environment is the natural environment).
  • polynucleotides and polypeptides in a natural state in a living cell are not isolated and purified, but the same polynucleotides or polypeptides are separated and purified if they are separated from other substances in the natural state .
  • isolated eukaryotic transcription factor 1 7. 38 refers to eukaryotic transcription factor 17. 38 which is essentially free of other proteins, lipids, sugars or other substances with which it is naturally associated. Those skilled in the art can purify eukaryotic transcription factors 17.38 using standard protein purification techniques. Substantially pure polypeptides can produce a single main band on a non-reducing polyacrylamide gel. Eukaryotic transcription factor 17. 38 The purity of the peptide can be analyzed by amino acid sequence.
  • the present invention provides a new polypeptide, eukaryotic transcription factor 17.38, which is basically composed of SEQ ID NO: 1;
  • the polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide, and preferably a recombinant polypeptide.
  • the polypeptides of the invention can be naturally purified products, or chemically synthesized products, or produced using recombinant techniques from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, higher plants, insects, and mammalian cells). Depending on the host used in the recombinant production protocol, the polypeptide of the invention may be glycosylated, or it may be non-glycosylated. Polypeptides of the invention may also include or exclude starting methionine residues.
  • the invention also includes fragments, derivatives and analogs of the eukaryotic transcription factor ⁇ .38.
  • fragment refers to a polypeptide that substantially maintains the same biological function or activity of the eukaryotic transcription factor 1 7.38 of the present invention.
  • a fragment, derivative or analog of the polypeptide of the present invention may be: U) a type 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 substituted An amino acid may or may not be encoded by a genetic code; or ( ⁇ ) such that a group on one or more amino acid residues is substituted by another group to include a substituent; or ( ⁇ ) such One, wherein the mature polypeptide is fused to another compound (such as a compound that extends the half-life of the polypeptide, such as polyethylene glycol); or (IV) such a polypeptide sequence in which an additional amino acid sequence is fused into the 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 polyamidine having the amino acid sequence of SEQ ID NO: 2.
  • the polynucleotide sequence of the present invention includes the nucleotide sequence of SBQ 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 2185 bases in total length and its open reading frame 79-555 encodes 1 58 amino acids.
  • this polypeptide has 81% homology with the eukaryotic transcription factor, and it can be inferred that the eukaryotic transcription factor 17.38 has similar crusts and functions of the eukaryotic transcription factor.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • DNA forms include oDNA, genomic DNA, or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be coding or non-coding.
  • the coding region sequence encoding a mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO: 1 or a degenerate variant.
  • degenerate variant refers to a nucleic acid sequence encoding a protein or polypeptide having SBQ ID NO: 2 but having a sequence different from the coding region sequence shown in SEQ ID NO: 1 in the present invention.
  • the polynucleotide encoding the mature polypeptide of SBQ 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 such a polypeptide and a variant comprising the additional polynucleotides described above, which also encodes a polypeptide or polypeptide having the same amino acid sequence as the present invention. Fragments, analogs and derivatives. 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. As known in the art, 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 501 ⁇ 2, preferably 703 ⁇ 4 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: ([hybridization and elution at lower ionic strength and higher temperature, such as 0.2 xSSC, 0.1 13 ⁇ 4, SDS, 6 (TC; or (2) Add a denaturant during hybridization, such as 50 »/ v / v) formamide, 0.12 calf serum / 0.1% F i co U, 42 ° C, etc .; or (3) between two sequences
  • the hybridization occurs only when the identity is at least 95%, and more preferably 973 ⁇ 4 or more.
  • the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ TD N0: 2.
  • the invention also relates to nucleic acid fragments that hybridize to the sequences described above.
  • core The ⁇ acid fragment '' contains at least 10 nucleotides in length, preferably at least 20-30 nucleotides, more preferably at least 50-60 nucleotides, and most preferably at least 100 nucleotides.
  • Nucleic acid fragments can also be used in nucleic acid amplification techniques such as PCR to identify and / or isolate polynucleotides encoding eukaryotic transcription factor ⁇ .38.
  • 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 eukaryotic transcription factor U.38 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 CDM libraries to detect homologous polynucleotide sequences, and 2) antibody screening of expression libraries to detect multinucleated clones with common scab characteristics Nucleotide fragments.
  • 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 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.
  • CDNA library is constructed in a conventional method (Sambrook, et al., Molecular Cloning, A Laboratory Manual, Co Id Spring Harbor Labora tory. New York, 1989) 0 cDNA may also be obtained in different commercially available cDNA library, such as companies CLontech library. When polymerase reaction technology is used in combination, even very small expression products can be cloned.
  • genes of the present invention can be selected from these cDNA libraries by conventional methods. These methods include (but are not limited to): (l) DNA-DNA or DNA RNA hybridization; (2) the presence or absence of marker gene functions; (3) determination of the level of transcript of eukaryotic transcription factor 17.38; (4) by Immunological techniques or assays for biological activity to detect gene-expressed protein products. The method can be used alone or in combination.
  • the probe used for hybridization is homologous to any part of the polynucleotide of the present invention, and has a length of at least 10 nucleotides, preferably at least 30 nucleotides, more preferably At least 50 nucleotides, preferably at least 100 nucleotides.
  • the length of the probe is usually within 2000 nucleotides, preferably within 1000 nucleotides.
  • the probe used here is usually a DM sequence chemically synthesized based on the gene sequence information of the present invention.
  • the genes or fragments of the present invention can of course be used as probes.
  • DNA probes can be labeled with radioisotopes, luciferin, or enzymes (such as alkaline phosphatase).
  • immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA) can be used to detect protein products expressed by the eukaryotic transcription factor 17.38 gene.
  • a method using PCR technology to amplify DNA / RNA (Saiki, et al. Science 1985; 23 0 ⁇ SO- 135 4 ) is preferably used to obtain the gene of the present invention. Especially difficult to get from the library
  • the RACE method RACE- rapid amplification of cDNA ends
  • the primers used for PCR can be appropriately selected according to the polynucleotide sequence information of the present invention disclosed herein, and can be synthesized by 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 obtained as described above, or various DNA fragments and the like can be determined by a conventional method such as dideoxy chain termination method (Sanger et 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 cDN A sequence, the sequencing must be repeated. Sometimes it is necessary to determine the cDNA sequence of multiple clones in order to splice into a full-length GDNA 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 the eukaryotic transcription factor 17.38 coding sequence, and the recombinant technology to produce the fan of the present invention Polypeptide method.
  • a polynucleotide sequence encoding a eukaryotic transcription factor 17.38 can be inserted into a vector to constitute a recombinant vector containing the polynucleotide of the present invention.
  • vector refers to bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors well known in the art.
  • Vectors suitable for use in the present invention include, but are not limited to: T7 promoter-based expression vectors (Rosenberg, et al.
  • any plasmid and vector can be used to construct recombinant expression vectors.
  • An important feature of expression vectors is that they usually contain origins of replication, promoters, marker genes, and translational regulatory elements.
  • Methods well known to those skilled in the art can be used to construct expression vectors containing a DNA sequence encoding a eukaryotic transcription factor ⁇ 38 and suitable transcription / translation regulatory elements. These methods include in vitro recombinant DMA technology, DNA synthesis technology, and in vivo recombination technology (Sambroook, et al. Molecular Cloning, a Laboratory Manual, GO Id Harbor Harbor Laboratory. New York, 1989).
  • the DNA sequence can be operably linked to an appropriate promoter in the expression vector to guide mRNA conjugation. Representative examples of these promoters are: the lac or p 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 100s later in the replication start point. SV40 enhancers up to 270 base pairs, polyoma enhancers on the late side of the origin of replication, and adenovirus 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 a eukaryotic transcription factor 17.38 or a recombinant vector incorporating the polynucleotide can be transformed or transduced into a host cell to form a genetically engineered host incorporating the polynucleotide or the recombinant vector.
  • the term "host cell” refers to a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples are: E.
  • coli Streptomyces
  • bacterial cells such as Salmonella typhimurium
  • fungal cells such as yeast
  • plant cells such as fly S2 or Sf 9
  • animal cells such as CH0, COS or Bowes melanoma cells.
  • Transformation of a host cell with a DNA sequence according to the present invention or a recombinant vector containing the DM 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 CaCl.
  • the steps used are 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 present invention is the use of polynucleotide sequences may be used to express or produce recombinant eukaryotic transcription factor 17. 38 (SG i en G e , 1984; 224: 1431). Generally there are the following steps:
  • the medium used in the culture may be selected from various conventional mediums. Culture is performed under conditions suitable for host cell growth. After the host cells have grown to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.
  • a suitable method such as temperature conversion or chemical induction
  • the recombinant polypeptide may be coated in a cell, expressed on a cell membrane, or secreted outside the cell. If necessary, it can be separated by various separation methods using its physical, chemical and other properties. Isolate and purify the recombinant protein. 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, osmosis, 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, osmosis, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high
  • Fig. 1 is a comparison diagram of the amino acid sequence homology of eukaryotic transcription factor 17.38 and eukaryotic transcription factor of the present invention.
  • the upper sequence is eukaryotic transcription factor 17.38, and the lower sequence is eukaryotic transcription factor.
  • Identical amino acids are represented by single-character amino acids between the two sequences, and similar amino acids are represented by.
  • Figure 1 shows the polyacrylamide gel electrophoresis (SDS-PAGB) of the isolated eukaryotic transcription factor 17.38.
  • lOkDa is the molecular weight of the protein.
  • the arrow indicates the isolated protein band.
  • Example 1 Cloning of eukaryotic transcription factor 17.38
  • Total RM of human fetal brain was extracted by one-step method with guanidine isothiocyanate / phenol / chloroform.
  • Poly (A) mRNA was isolated from total RNA using the Quik mRNA Isolation Kit (Qiegene). 2ug poly (A) mRNA is reverse transcribed to form GDNA. Smar t GDM cloning kit (purchased from CI on tech) will be used. The 0 ⁇ fragment was inserted into the multicloning site of pBSK (+) vector (ontech company), transformed into DH5 ⁇ , and the bacteria formed a GDNA library. The sequences at the 5 'and 3' ends of all clones were determined using Dye terminate cyde react ion sequencing ID (Perkin-Eier product) and ABT 377 automatic sequencer (Perkin-Elmer).
  • the CDM sequence of 27hl0 is a new DM.
  • the cloned cDNA fragment contained in this clone was bidirectionally determined by synthesizing a series of ⁇ 'j primers. The results showed that the full-length GDNA cloned by the 0l27hl0 clone was 2 l 85 bp (as shown in Seq ID N0: 1), and there was a 477 bp open reading frame (0RF) from 79 bp to 555 bp, encoding a new protein (such as Seq ID NO: 2).
  • This clone pBS-01 hl 0 and the encoded protein was named eukaryotic transcription factor 17.38.
  • Example 2 Homologous search of cDNA clones
  • the sequence of the eukaryotic transcription factor 17.38 of the present invention and the protein sequence encoded by the same were performed using the Blast program (Bas idoca 1 A 1 i gnmen t search tool) [ ⁇ 1 tschul, SF et a 1. J. Mol. Biol. 1990; 215: 403-10], and perform homology search in a database such as Genbank Swissport.
  • the gene with the highest homology with the eukaryotic transcription factor 17.38 of the present invention is a known eukaryotic transcription factor, and the accession number of the encoded protein in Genbank is X53280.
  • the protein homology results are shown in Figure 1. The two are highly homologous, with an identity of 813 ⁇ 4; the similarity is 88%.
  • Example 3 Cloning of a gene encoding a eukaryotic transcription factor 17.38 by RT-PCR
  • GDNA was synthesized using fetal brain cell total RNA as a template and oligo-dT as a primer for reverse transcription reaction. After purification using Qiagene's kit, the following primers were used for PCR amplification:
  • Primerl 5'— TCTCGGCTGAGGCAGCCATCTTTC-3 '(SEQ ID NO: 3)
  • Primer 2 5'— CTTTTGCACTGTTATTTTATTTTG- 3 '(SEQ ID NO: 4)
  • Primerl is a forward sequence starting at lbp at the 5th end of SEQ ID NO: 1;
  • Primer 2 is the 3 'end reverse sequence in SEQ ID NO: 1.
  • Amplification conditions 50 oL / L KC1, 10mmol / L Tris- Ci, (pH8.5), 1.5mmol / L MgCl 2 , 200 ⁇ raol / L dNTP, lOpmoL in a reaction volume of 50 ⁇ 1 Primer, 1U of Taq DNA polymerase (C 1 on Tech).
  • the reaction was performed on a PE9600 DNA thermal cycler (Perk i n B lm r) for 25 cycles under the following conditions: 94 C 30 sec; 55 ° C 30 sec; 72 C 2 min.
  • ⁇ -act in was set as a positive control and template blank was set as a negative control.
  • Example 4 Northern blot analysis of eukaryotic transcription factor 17.38 gene expression:
  • RNA extraction in one step [AnaL. Biochem 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 acetamidine (pH4.0), and 1 volume of phenol and 1/5 volume of chloroform-isoamyl alcohol (49: ⁇ ), Centrifuge after mixing. Aspirate the aqueous layer, add isopropanol (0.8 vol) and centrifuge the mixture to obtain RNA precipitate. The RNA precipitate was washed with 70% ethanol, dried and dissolved in water.
  • RNA probes With 20 ⁇ ⁇ RNA, homes in 20mM 3- (N - morpholino) were electrophoresed on a 1.2% agarose gel 5mM sodium acetate IraM EDTA- 2.2M formaldehyde propanesulfonic acid (pH 7.0). It was then transferred to a nitrocellulose membrane. The x- 32 P dATP was used to prepare P-labeled DNA probes by the random bow method. The DM probe used was the PCR-encoded eukaryotic transcription factor 17.38 coding region sequence shown in Figure 1. (79bp to 555bp).
  • a 32P-labeled probe (approximately 2 x 10 6 G pm / ml) was hybridized with a nitrocellulose membrane to which RNA was transferred at 42 ° C overnight in a solution containing 503 ⁇ 4 formamide-25iiiM H 2 P0 ( pH7.4) -5 x SSC-5 Denhardt's solution and 200 ⁇ g / ml salmon sperm DNA. After hybridization, the filter was washed in 1 x SSC-0.CaS at 55 "C for 30niin. Then, Phosphor Imager was used for analysis and quantification.
  • Example 5 In vitro expression, isolation and purification of recombinant eukaryotic transcription factor 17.38
  • Primer3 5-CCCCATATGATGAATCAAGAAAAGTTAGCCAAA-3 '(Seq ID No: 5)
  • Primer4 5 5 -CATGGATCCTTAGTTAGCTTCATTCTTTGATGC-3' (Seq ID No: 6)
  • the 5 'ends of these two primers are respectively rounded with Mel and BamH 1; digestion sites Points, followed by the coding sequences of the 5 'and 3' ends of the gene of interest, respectively.
  • the Ndel and BamHI restriction sites correspond to those on the expression vector plasmid pBT- 2 8b (+) (Novagen, Cat. No. 69865.3). Selective endonuclease site.
  • PCR was performed using the pBS-0127hl0 plasmid containing the full-length target gene as a template.
  • the PCR reaction conditions were as follows: a total volume of 50 ⁇ l, a 10 pg of pBS-0127hl0 plasmid, and Pr inier-3 and Primer-4 were 1 Opmo 1, Advantage polymerase Mix (Contech) 1 ⁇ 1, respectively.
  • Cycle parameters 94. C 20s, 60 ° C 30s, 68 ° C 2 min, a total of 25 cycles.
  • Ndel and BamHI were used to double digest the amplified product and plasmid pET-28 (+), respectively, and large fragments were recovered and ligated with T4 ligase. The ligation product was transformed into E.
  • the host strain BL21 (pET- Q127hl0) was cultured at 37 ° C to the logarithmic growth phase, IPTG was added to a final concentration of 1 mmol / L, and the culture was continued. 5 hours. The cells were collected by centrifugation, and the supernatant was collected by centrifugation. The supernatant was collected using an affinity chromatography column H Ls. Bind Quick Cartridge (product of Novagen) capable of binding to 6 histidines (6HLs-Tag). Chromatography, the purified eukaryotic transcription factor 17.38 was obtained.
  • the peptide specific to eukaryotic transcription factor 17.38 was synthesized by a peptide synthesizer (product of PE company):
  • a titer plate coated with 15 g / ml bovine serum albumin peptide complex was used as an ELISA to determine the antibody titer in rabbit serum.
  • Total T gG was isolated from antibody-positive rabbit serum using protein A-Sepha rose.
  • the peptide was bound to a cyanogen bromide-activated Sepha rose4B column, and the anti-peptide antibody was separated from the total I gG by affinity chromatography. 38 ⁇ Immunoprecipitation demonstrated that the purified antibody specifically binds to eukaryotic transcription factor ⁇ .38.
  • Example 7 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 the genome or GDNA library of normal tissues or pathological tissues 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 Acid sequence or a homologous polynucleotide sequence thereof.
  • Filter hybridization methods include dot blotting, Sou thern imprinting, Nor thern blotting, and copying methods. They are all used to fix the polynucleotide sample to be tested on the filter and then hybridize using basically the same steps.
  • 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.
  • higher-intensity washing conditions such as lower salt concentration and higher temperature) are used to reduce the background of hybridization and retain only strong specific signals.
  • the probes used in this embodiment include two types: the first type of probes are oligonucleotide fragments that are completely the same as or complementary to the polynucleotide SEQ ID NO: 1 of the present invention; the second type of probes are partially related to the present invention
  • the polynucleotide SEQ ID NO: 1 is the same or complementary oligonucleotide fragment.
  • the dot blot method is used to fix the sample on the filter membrane. Under the high-strength washing membrane strip, the first type of probe and the sample have the strongest hybridization specificity and are retained.
  • the selection of oligonucleotide fragments from the polynucleotide SEQ ID NO: 1 of the present invention for use as hybridization probes should follow the following principles and several aspects to be considered:
  • the preferred range of probe size is 18-50 nucleotides
  • GC content is 30% 703 ⁇ 4, if it exceeds, non-specific hybridization increases;
  • 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 type of probe, is completely identical to the gene fragment of SEQ ID NO: 1
  • Probe 2 (probe2), which belongs to the second type of probe, is equivalent to the #mutation sequence (4) Nt of the gene fragment of SEQ ID NO: 1 or its complementary fragment:
  • Sample preparation Steps: 1) Place fresh or freshly thawed normal liver tissue in a plate immersed in ice and filled with phosphate buffered saline (PBS). Cut the tissue into small pieces with scissors or a scalpel. Tissue should be kept moist during operation. 2) Centrifuge the tissue at 1,000 g for 10 minutes. 3) cold homogenization buffer (0.25mol / L sucrose; 25mmol / L Tris-HCi, pH7.5; 25mmol / LnaCl; 25 Implicit ol / L MgC12) was suspended precipitate (approximately 10ml / g) 0 4) at 4oC Use an electric homogenizer to homogenize the tissue suspension at full speed until the tissue is completely broken.
  • PBS phosphate buffered saline
  • NC membranes nitrocellulose membranes
  • 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.
  • Example 8 Microarray
  • Gene microarrays or DNA microarrays are new technologies currently being developed by many national laboratories and large pharmaceutical companies. It refers to the orderly and high-density arrangement of a large number of target gene fragments on glass, The data is compared and analyzed on a carrier such as silicon using fluorescence detection and computer software to achieve the purpose of rapid, efficient, and high-throughput analysis of biological information.
  • the polynucleotide of the present invention can be used as a target DM for gene chip technology for high-throughput research of new gene functions; search for and screen new tissue-specific genes, especially diseases-related new genes such as tumors; diagnosis of diseases, such as heredity disease. The specific method steps have been reported in the literature.
  • a total of 4,000 polynucleotide sequences of various full-length GDNA are used as target DNA, including the polynucleotide of the present invention. They were respectively amplified by PCR. After purification, the concentration of the amplified products was adjusted to about 500 ng / ul, and they were spotted on a glass medium with a Cartesian 7500 spotter (purchased from Cartesian Company, USA). The distance between them is 280 ⁇ ⁇ 1 . 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 have been reported in the literature in various ways. The post-processing steps of this embodiment are:
  • Probe marking Total mRNA was extracted from normal liver and liver cancer by one-step method, and the mRNA was purified by Oligotex mRNA Midi Kit (purchased from QiaGen).
  • the fluorescent reagent Cy 3dUTP (5- Am ⁇ no- propargy l) -2'-deoxyur idi ne 5--tri pha te coupled to Cy 3 fluorescent dye (purchased from Ame sliam Phamacia Biotech) was used to label the mRNA of normal liver tissue, and the fluorescent reagent CySdUTP (5-Ami no-propa rgy l-2 '-deoxyu r d ine 5'-triphate coup led to Cy5 fluorescent dye (purchased from Amershani Phamacia Biotech) was used to label liver cancer tissue mRNA, and the probe was prepared after purification.
  • Cy 3dUTP 5- Am ⁇ no- propargy l) -2'-deoxyur idi ne 5--tri
  • 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.
  • RNA polymerase U also known as TF1ID
  • BTF2 BTF3
  • BTF4 can form a stable compound with RNA polymerase 11.
  • BTF3 The central hydrophobic region of BTF3 is a good site for binding to RNA polymerase II. A similar lipophilic helix forms a coil-to-coil junction, which is the site of protein interaction in microtubule cross-linking. BTF3 is present in very small amounts in cells, but is present in all cells. BTF3 is an indispensable protein factor for eukaryotic transcription. It directly binds to RNA polymerase ii and enables the promoter to perform low-level basic transcription.
  • BTF 3 is involved in determining the tissue and developmental stage of the gene. If the gene encoding BTF 3 is mutated, a variety of genes regulated by BTF 3 will not be expressed normally, which will lead to various diseases, such as embryo development. Various diseases related to cell differentiation.
  • the polypeptide of the present invention and the human transcription factor BTF 3 are eukaryotic transcription factors and contain characteristic sequences of the human transcription factor BTF 3 family. Both have similar biological functions. It is involved in the transcription process in vivo, an essential protein factor for eukaryotic transcription, it promotes low-level basal transcription of the promoter, and its abnormal band will lead to scattered multi-point starting Mnia synthesis, and the synthesis efficiency is poor, thus Causes abnormalities or errors in protein synthesis and causes related diseases.
  • the abnormal expression of the eukaryotic transcription factor 17.38 of the present invention will produce various diseases, especially various tumors, embryonic development disorders, growth disorders, inflammation, and immune diseases. These diseases include but not limited to:
  • Tumors of various tissues gastric cancer, liver cancer, lung cancer, esophageal cancer, breast cancer, leukemia, lymphoma, thyroid tumor, uterine fibroids, neuroblastoma, astrocytoma, esophageal membrane tumor, glioblastoma, nerve fiber Tumor, colon cancer, melanoma, bladder cancer, uterine cancer, endometrial cancer, colon cancer, thymic tumor, nasopharyngeal cancer, laryngeal cancer, tracheal tumor, fibroma, fibrosarcoma, lipoma, liposarcoma embryonic developmental disorder : Congenital miscarriage, cleft palate, limb loss, limb differentiation disorder, atrial septal defect, neural tube defect, congenital hydrocephalus, congenital glaucoma or cataract, congenital deafness
  • Growth and development disorders mental retardation, brain development disorders, skin, fat and muscular dysplasia, bone and joint dysplasia, various metabolic deficiencies, stunting, dwarfism, Cushing syndrome, Sexual retardation
  • Inflammation chronic active hepatitis, sarcoidosis, polymyositis, chronic rhinitis, chronic gastritis, cerebrospinal multiple sclerosis, glomerulonephritis, myocarditis, cardiomyopathy, atherosclerosis, gastric ulcer, uterine experience, each Infectious inflammation
  • Immune diseases systemic lupus erythematosus, rheumatoid arthritis, bronchial boat, urticaria, specific dermatitis, post-infection myocarditis, scleroderma, myasthenia gravis, Guillain-Barre syndrome, common variable immunodeficiency disease , Primary B-lymphocyte immunodeficiency disease, Acquired immunodeficiency syndrome
  • Abnormal expression of the eukaryotic transcription factor 17.38 of the present invention will also produce certain hereditary, hematological diseases and the like.
  • 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 various tumors, embryonic developmental disorders, growth and development disorders, inflammation, Immune diseases, certain hereditary, blood diseases, etc.
  • the present invention also provides methods of screening compounds to identify enhance (agonist) or repression (eukaryotic transcription factor antagonist agent 7.38 a method Agonists enhance eukaryotic transcription factor ⁇ . 38 stimulated cell proliferation green
  • Antagonists prevent and treat disorders related to excessive cell proliferation, such as various cancers.
  • mammalian cells or membrane preparations expressing eukaryotic transcription factor ⁇ .38 can be cultured together with labeled eukaryotic transcription factor ⁇ .38 in the presence of drugs. The ability of the drug to increase or block this interaction is then determined.
  • Antagonists of eukaryotic transcription factor 17.38 include screened antibodies, compounds, deficiencies and analogs.
  • the antagonist of eukaryotic transcription factor 17.38 can bind to eukaryotic transcription factor 17.38 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.
  • eukaryotic transcription factor ⁇ .38 When screening compounds as antagonists, eukaryotic transcription factor ⁇ .38 can be added to bioanalytical assays to determine whether a compound is an antagonist by measuring the effect of the compound on the interaction between eukaryotic transcription factor 17.38 and its receptor. . Receptor deletions and analogs that function as antagonists can be screened in the same manner as described above for screening compounds.
  • Peptide molecules capable of binding to eukaryotic transcription factor ⁇ .38 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. When screening, the eukaryotic transcription factor 17.38 molecule 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 antibodies or monoclonal antibodies.
  • the invention also provides resistance to eukaryotic transcription factor ⁇ .38 epitopes. These antibodies include (but are not limited to): polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, and fragments generated from Fab expression libraries.
  • Polyclonal antibodies can be produced by injecting eukaryotic transcription factor 17.38 directly into immunized animals (such as rabbits, mice, rats, etc.).
  • immunized animals such as rabbits, mice, rats, etc.
  • adjuvants can be used to enhance the immune response, including but not limited to Freund's adjuvant.
  • Techniques for preparing monoclonal antibodies against eukaryotic transcription factor 17.38 include, but are not limited to, hybridoma technology (Kohler and Mi Istein. Nature, 1975, 256: 495-497), triple tumor technology, human B-cell hybridoma technology, BBV- Hybridoma technology, etc.
  • the chimeric human antibody constant region and the variable region of non-human origin may be used in combination Pat some production techniques (Morrison et al, PNAS, 1985 , 81: 6851) 0 only some techniques produce single chain antibodies 01. S. Pat No. 4946778) can also be used to produce single chain antibodies against eukaryotic transcription factor 17.38.
  • Antibodies against eukaryotic transcription factor ⁇ .38 can be used in immunohistochemical techniques to detect eukaryotic transcription factors in biopsy samples 17.38.
  • Monoclonal antibodies that bind to eukaryotic transcription factor 17.38 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. Eukaryotic transcription factor
  • High-affinity monoclonal antibodies can covalently bind to bacterial or phytotoxins (such as diphtheria toxin, ricin, ormosine, etc.).
  • a common method is to attack the amino group of the antibody with a thiol cross-linking agent such as SPDP, and bind the toxin to the antibody through the exchange of disulfide bonds.
  • SPDP thiol cross-linking agent
  • This hybrid antibody can be used to kill the eukaryotic transcription factor 17.38 positive cell.
  • the antibodies of the present invention can be used to treat or prevent diseases related to eukaryotic transcription factor ⁇ .38. Administration of an appropriate dose of antibody can stimulate or block the production or activity of eukaryotic transcription factor 17.38.
  • the invention also relates to a diagnostic test method for quantitative and localized detection of eukaryotic transcription factor 17.38 levels.
  • tests are well known in the art and include F ISH assays and radioimmunoassays.
  • the level of eukaryotic transcription factor ⁇ .38 detected in the test can be used to explain the importance of eukaryotic transcription factor 17.38 in various diseases and to diagnose diseases in which eukaryotic transcription factor 17.38 plays a role.
  • polypeptide of the present invention can also be used for peptide mapping analysis.
  • the polypeptide can be specifically cleaved by physical, chemical or enzymatic analysis, and subjected to one-dimensional or two-dimensional or three-dimensional gel electrophoresis analysis, and more preferably mass spectrometry analysis.
  • the polynucleotide encoding eukaryotic transcription factor 17.38 can also be used for a variety of therapeutic purposes. Gene therapy techniques can be used to treat cell proliferation, cyanosis, or metabolic abnormalities caused by the non-expression or abnormal / inactive expression of eukaryotic transcription factor 17.38.
  • Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated eukaryotic transcription factor ⁇ .38 to inhibit endogenous eukaryotic transcription factor 17.38 activity.
  • a variant eukaryotic transcription factor ⁇ .38 may be a shortened eukaryotic transcription factor 1 7.38 that lacks a signaling domain, although it can bind to downstream substrates, but lacks signaling activity.
  • recombinant gene therapy vectors can be used to treat diseases caused by abnormal expression or activity of eukaryotic transcription factor 17.38.
  • Virus-derived expression vectors such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus, etc. can be used to transfer a polynucleotide encoding a eukaryotic transcription factor 17.38 into a cell.
  • a method for constructing a recombinant viral vector carrying a polynucleotide encoding the eukaryotic transcription factor U. 38 can be found in the literature (Sanibrook, etal.). 0
  • Another recombinant polynucleotide encoding the eukaryotic transcription factor 17.38 can be packaged into lipids. Plastids are transferred into cells.
  • Methods for introducing a polynucleotide into a tissue or cell include: directly injecting the polynucleotide into a tissue in vivo; or introducing the polynucleotide into a cell in vitro through a vector (such as a virus, phage, or plasmid), and then transplanting the cell Into the body and so on.
  • a vector such as a virus, phage, or plasmid
  • Oligonucleotides including antisense RNA and DM
  • ribozymes that inhibit eukaryotic transcription factor ⁇ .38 mRNA are also within the scope of this disclosure.
  • a ribozyme is an enzyme-like RM molecule that can specifically decompose specific RNA. Its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target RNA for endonucleation. antonym
  • RNA and DNA and ribozymes can be obtained by any of the existing RNA or DNA synthesis techniques, 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 DM sequence encoding the RNA. This DM sequence has been integrated downstream of the RNA polymerase promoter of the vector.
  • a nucleic acid molecule it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the ribonucleoside linkages should use phosphate thioester or peptide bonds instead of phosphodiester bonds.
  • Polynucleotides encoding eukaryotic transcription factors 17.38 can be used to diagnose diseases related to eukaryotic transcription factors 17.38.
  • the polynucleotide encoding eukaryotic transcription factor 17.38 can be used to detect the expression of eukaryotic transcription factor 17.38 or the abnormal expression of eukaryotic transcription factor 17.38 in a disease state.
  • the DNA sequence encoding eukaryotic transcription factor 17.38 can be used to hybridize biopsy specimens to determine the expression status of eukaryotic transcription factor U.38.
  • Hybridization techniques include Southern blotting, Northern blotting, in situ hybridization, and so on. These techniques and methods are publicly available and mature, and related kits are commercially available.
  • Part or all of the polynucleotides of the present invention can be used as probes to be fixed on a microarray (Mioroarray) or a DNA chip (also known as a "gene chip") for analyzing differential expression analysis and gene diagnosis of genes in tissues.
  • Eukaryotic transcription factor 17.38 specific primers can also be used to detect the eukaryotic transcription factor by RNA-polymerase chain reaction (RT-PCR) in vitro amplification] 7.38 transcription products.
  • Detection of mutations in the eukaryotic transcription factor ⁇ .38 gene can also be used to diagnose eukaryotic transcription factor 17.38-related diseases.
  • Eukaryotic transcription factor ⁇ .38 mutations include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to normal wild-type eukaryotic transcription factor 17.38 DNA sequences. Mutations can be detected using well-known techniques such as Southern blotting, DNA sequence analysis, PCR and in situ hybridization. In addition, mutations may affect protein expression, so Northern 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.
  • a PCR bow (preferably 15-35bp) is prepared based on cDNA, and the sequence can be located on the chromosome. These primers were then used for PCR screening of somatic hybrid cells that combined individual human chromosomes. Only those heterozygous cells containing human genes that are responsive to the primers 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 by a similar method, a set of fragments from a specific chromosome can be utilized Or a large number of genomic clones 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 hybrid pre-selection to construct a chromosome-specific G regression library.
  • Fluorescent in situ hybridization (FTSH) of GDNA clones with metaphase chromosomes allows precise chromosomal localization in one step.
  • FTSH 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, for example, in V.MGkusiGk, Mendelian Inheritance in Man (available online with Johns Hopkins University Welch Med i Ga 1 Ub rary ). Linkage analysis can then be used to determine the relationship between genes and diseases that are mapped to chromosomal regions.
  • cDNA or genomic sequence between the affected and unaffected individuals needs to be determined. If a mutation is observed in some or all diseased individuals and the mutation is not observed in any normal individuals, the mutation may be the cause of the disease. Comparing affected and unaffected individuals usually involves first looking for structural changes in chromosomes, such as deletions or translocations that are visible at the chromosomal level or detectable with cDNA sequence-based PCR. Based on the resolution capabilities of current physical mapping and gene mapping technologies, cDNAs that are accurately mapped to disease-related chromosomal regions can be one of 50 to 500 potentially pathogenic genes (assuming
  • 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.
  • Eukaryotic transcription factors 17. 38 are administered in amounts effective to treat and / or prevent specific indications. The amount and range of eukaryotic transcription factor ⁇ .38 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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Description

一种新的多肽一一真核转录因子 17.38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， 本发明描迷了一种新的多肽一一真 核转录因子 17. 38, 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
真核转录因子是真核基因转录必需的蛋白因子的统称。 在转录过程中， 真 核生物 RM 聚合酶需要严格依赖于一系列蛋白因子才能识别和结合到启动子特 定序列上， 需要一定的蛋白质袓坟才能启动转录反应。 基因转录的组织和细胞 特异性调控又需要另一些特定蛋白因子参与。 目前， 已经分离， 纯化或鉴定的 蛋白因子已有数百种。 这些凡是作用的蛋白因子可以分为两大类。 一类是结合 与启动子核心序列如 TATA 框及其附近的因子， 称为转录因子或普遍性转录因 子； 另一类结合与上游调控区 UPE和增强子区的蛋白因子， 称为转录调控因子。 这些转录因子和转录调控因子的蛋白分子， 按其功能具有两种作用的结抅域。 有的转录因子在形成起始复合物时可以结合于 RNA聚合酶， 但不是酶的一部分。 它们是所有自动子起始转录反应的普遍性转录因子， 有的参与转录的终止反 应。
在真核基因开始转的时候， 需要一种多蛋白化合物与 DNA 的顺势作用元件 相互作用。 除 RNA 聚合酶 U以外， 转录开始还需要四种普遍性转录因子： BTF1 (也称为 TF II D ) , BTF2, BTF3, BTF4。 装配起始复合物的第一步是， BTF1 稳 定地结合到 TATA盒上， 在此过程中， STF起促进作用。 BTF2和 BTF3并不直接 结合到启动子临近区域， 但 BTF3可以与 RNA聚合酶 II形成一个稳定的化合物。
BTF3是一种分子量为 271 (的蛋白质， 在对 BTF3的顺序作进一步研究的基础 上， 发现了两中心的蛋白质： BTF3a 和 BTF3b。 BTF3a 分子量为 22K， 含有 206 个残基， 具有 BTF3 的一切功能； BTF3b分子量为 18K, 含由 162个残基， 它缺 少 BTF3a 的前 44个残基， 可以和 RNA聚合酶 II结合， 但没有转录活性。 BTF3a 与 DNA 结合结构域 （如螵旋-转角-螺旋或锌指） 和蛋白 -蛋白作用结抅域 （如 亮氨酸拉链） 不同。 BTF3a 的 N端和 C端是高度亲水的， 中心区域富含亮氨酸 残基， 高度憎水。 143- 154 残基形成了一个短的亲脂性的 α-螺旋， 在螺旋的一 段全部是憎水性氨基酸， 其中主要是亮氨酸。 这个中心憎水区域是与 RNA 聚合 酶 II结合的良好位点， 其它蛋白因子如 BTF2 也可以结合在这个位点上。 一个 相似的亲脂性螺旋抅成旋管―旋管结构， 是微管交联中与蛋白质相互作用的位 点。 BTF3 在细胞中的含量相当微少， 但在所有的细胞中均存在。 BTF3 是真核 转录必不可少的蛋白因子， 它直接结合 RM 聚合酶 II , 使启动子进行低水平基 础性转录， 它可以被转录活化因子进一步激活， 亦可以被转录抑制因子阻遏。 如果缺少， 单独 RNA 聚合酶 II将导致散乱的多点起始的 tnRNA 合成， 且合成效 率差。 BTF3 在生物体内参与决定基因在何种组织及发育阶段表达， 如果 BTF3 的编码基因发生突变， 将导致多种受 BTF3 调节的基因不能正常表达， 从而导 致多 种疾病 ， 如 与 胚 胎发育 、 细 胞分化有 关 的各种 疾病 等 。 ( X.M. Zheng, et. al, Nature, 1990 )
本发明的多肽中含有 RM 聚合酶 II结合结构域，并与人转录因子 BTF3 在蛋 白水平上具有 81%的同源性，故属于人转录因子 BTF蛋白家族。 基于以上各点， 故人为本发明的多肽为人转录因子， 命名为真和转录因子 17. 38,并以此推断其 与 BTF3相似， 具有相似的生物学功能。
由于如上所述真核转录因子 Π.38 蛋白在调节细胞分裂和胚胎发育等机体 重要功能中起重要作用， 而且相信这些调节过程中涉及大量的蛋白， 因而本领 域中一直需要鉴定更多参与这些过程的真核转录因子 Π. 38 蛋白， 特别是鉴定 这种蛋白的氨基酸序列。 新真核转录因子 17. 38 蛋白编码基因的分离也为研究 确定该蛋白在健康和疾病状态下的作用提供了基础。 这种蛋白可能构成开发疾 病诊断和 /或治疗药的基础， 因此分离其编码 DNA是非常重要的。 发明的公开
本发明的一个目的是提供分离的新的多肽一一真核转录因子 17. 38 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真核转录因子 Π. 38 的多核苷酸的重 组载体。
本发明的另一个目的是提供合有编码真核转录因子 17. 38 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真核转录因子 Π.38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真核转录因子 Π. 38 的 本发明的另一个目的是提供了针对本发明多肽一一真核转录因子 Π. 38 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真核转录因子 Π. 38 异常栖关的疾 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 82¾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 I D NO: 1中 79- 555位的序列； 和（b)具有 SEQ I D NO: 1中 1 - 2185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、 拮抗或抑制真核转录因子 17. 38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真核转录因子 1 7. 38 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真核转录因子 17. 38 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迷蛋白质 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 #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—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" 是指当与真核转录因子 1 7. 38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 任何其它可结合真核转录因子 17. 38的分子。
"拮抗剂" 或 "抑制物" 是指当与真核转录因子 17. 38结合时， 一种可封闭 或调节真核转录因子 17. 38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真核转录因子 17. 38 的分子。
"调节" 是指真核转录因子 17. 38的功能发生改变， 包括蛋白质活性的升高 或降低、 结合特性的改变及真核转录因子 17. 38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真核转录因子 17. 38。 基本上纯的真核转录因子 〗7. 38 在非还原性聚丙烯酰胺凝胶上能产生单一的主 带。 真核转录因子 17. 38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。 核酸链之问的互补程度对于 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 的条伴下进行杂交 （ Sou Uie ni印 迹或 Northern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 MBGALIGN程序 ( Lasergene software package, DNASTAR, Inc. , Mad i son Wis. ) 。 MEGALTGN 程序可根据不同的方法如 Cluster法比较两种或多种序列（Higgins, D. G. 和 P.M. Sharp (1988) Gene 73: 237-244) 0 Clus ter法通过检査所有配对之间的 距离将各组序列排列成簇。 然后将各簇以成对或成组分配。 两个氨基酸序列如 序列 A和序列 B之问的相同性百分率通过下式计算： 序列 A与序列 B之问匹配的残基个数 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 Cluster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 n J.， (1990) Methods in emzumology 183: 625-645)。
"相似性 " 是指氨基酸序列之间排列对比时相应位置氨基酸残基的相同或 保守性取代的程度。 用于保守性取代的氨基酸例如， 带负电荷的氨基酸可包括 天冬氨酸和谷氨酸； 带正电荷的氨基酸可包括赖氨酸和精氨酸； 具有不带电荷 的头部基团有相似亲水性的氨基酸可包括亮氨酸、 异亮氨酸和缬氨酸； 甘氨酸 和丙氨酸； 天冬酰胺和谷氨酰胺； 丝氨酸和苏氨酸； 苯丙氨酸和酪氨酸。
"反义" 是指与特定的 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、 F(ab')2 及 Fv, 其能特异 性结合真核转录因子 17.38的抗原决定簇。
"人源化抗体" 是指 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杭体。
"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， "分离的真核转录因子 1 7. 38" 是指真核转录因子 17. 38 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真核转录因子 17. 38。 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。 真核转录因子 17. 38 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真核转录因子 17. 38 , 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。
本发明还包括真核转录因子 Π. 38 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真核转 录因子 1 7. 38 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： U ) 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 （ Ι Π ) 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 I D NO: 2 氨 基酸序列的多舦的多核苷酸组成。 本发明的多核苷酸序列包括 SBQ 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 2185个碱基， 其开放读框 79-555编码了 1 58个氨基 酸。 根据氨基酸序列同源比较发现， 此多肽与真核转录因子有 81%的同源性， 可推断出该真核转录因子 17. 38具有真核转录因子相似的结抅和功能。
本发明的多核苷酸可以是 DNA形式或是 RNA形式。 DNA形式包括 oDNA、 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 NO: 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 SBQ 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 SBQ 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 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 50½， 优选具有 70¾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： （υ在较低 离子强度和较高温度下的杂交和洗脱， 如 0. 2 xSSC, 0. 1¾,SDS, 6 (TC ;或（2 )杂交 时加用变性剂， 如 50»/ v/v)甲酰胺， 0. 1¾小牛血清 / 0. l%F i co U , 42 °C等； 或（3) 仗在两条序列之间的相同性至少在 95%以上，更好是 97¾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 TD N0: 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 /或分离编码真核转录因子 Π.38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。 本发明的编码真核转录因子 U.38 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 cDM 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抅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序列以获得所迷多肽的双链 DNA。
上述提到的方法中， 分离基因组 DNA 最不常用。 DNA 序列的直接化学合成 是经常选用的方法。 更经常选用的方法是 cDNA序列的分离。 分离感兴趣的 cDNA 的标准方法是从高表达该基因的供体细胞分离 mRNA并进行逆转录， 形成质粒或 噬菌体 cDNA 文库。 提取 mRNA 的方法已有多种成熟的技术， 试剂盒也可从商业 途径获得（Qiagene)。 而构建 cDNA 文库也是通常的方法（Sambrook, et al. , Molecular Cloning, A Laboratory Manual, Co Id Spring Harbor Labora tory. New York, 1989) 0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 定真核转录因子 17.38 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。 上迷方法可单用， 也可多种方法联合应用。
在第（1)种方法中， 杂交所用的探钎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 DM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， 检测真核转录因子 17. 38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·· SO- 1354)被优选用于茯得本臾明的基因。 特别是很难从文库中得到
X 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。
如上所迷得到的本发明的基因， 或者各种 DNA 片段等的多核苷酸序列可用 常规方法如双脱氧链终止法（Sanger et 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 cDN A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 GDNA 序列。
本发明也涉及包合本发明的多核苷酸的载体， 以及用本发明的载体或直接 用真核转录因子 17. 38 编码序列经基因工程产生的宿主细胞， 以及经重组技术 产生本发明所迷多肽的方法。
本发明中， 编码真核转录因子 17.38 的多核苷酸序列可插入到载体中， 以 构成含有本发明所述多核苷酸的重组载体。 术语 "载体" 指本领域熟知的细菌 质粒、 噬菌体、 酵母质粒、 植物细胞病毒、 哺乳动物细胞病毒如腺病毒、 逆转 录病毒或其它载体。 在本发明中适用的载体包括但不限于： 在细菌中表达的基 于 T7启动子的表达载体（Rosenberg, et al. Gene, 1987, 56: 125)； 在哺乳动 物细胞中表达的 pMSXND表达载体（Lee and Nathans, J Bio Chem. 263: 3521, 1988) 和在昆虫细胞中表达的来源于杆状病毒的载体。 总之， 只要能在宿主体内复制 和稳定， 任何质粒和载体都可以用于抅建重组表达载体。 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真核转录因子 Π. 38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 DMA 技 术、 DNA合成技术、 体内重组技术等（Sambroook, et al. Molecular Cloning, a Laboratory Manual, GO Id Spr ing Harbor Laboratory. New York, 1989)。 所 迷的 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合戍。 这 些启动子的代表性例子有： 大肠杆菌的 lac或 p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， 编码真核转录因子 17. 38 的多核苷酸或合有该多核苷酸的重组 载体可转化或转导入宿主细胞， 以抅成合有该多核苷酸或重组载体的基因工程 化宿主细胞。 术语 "宿主细胞" 指原核细胞， 如细菌细胞； 或是低等真核细胞， 如酵母细胞； 或是高等真核细胞， 如哺乳动物细胞。 代表性例子有： 大肠杆菌， 链霉菌属； 细菌细胞如鼠伤寒沙门氏菌； 真菌细胞如酵母； 植物细胞； 昆虫细 胞如果蝇 S2或 Sf 9 ; 动物细胞如 CH0、 COS或 Bowes黑素瘤细胞等。
用本发明所述的 DNA序列或含有所述 DM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 CaC l. 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 重组的真核转录因子 17. 38 (SG i enGe , 1984 ; 224： 1431)。 一般来说有以下步 骤：
(1) .用本发明的编码人 真核转录因子 1 7. 38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 离和纯化重組的蛋白。 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、 滲透玻菌、 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是本发明真核转录因子 17. 38和真核转录因子的氨基酸序列同源性比较 图。 上方序列是真核转录因子 17. 38 , 下方序列是真核转录因子。 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 1为分离的真核转录因子 17. 38的聚丙烯酰胺凝胶电泳图 （SDS- PAGB ) 。 lOkDa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 Laboratory Press, 1989)中所迷的条件， 或按照制造广商所 建议的条件。
实施例 1: 真核转录因子 17. 38的克隆
用异硫氰酸胍 /酚 /氯仿一步法提取人胎脑总 RM。 用 Quik mRNA Isolation Kit ( Qiegene 公司产品） 从总 RNA中分离 poly (A) mRNA。 2ug poly (A) mRNA经逆转录 形成 GDNA。用 Smar t GDM克隆试剂盒（购自 CI on tech )将。0^片段定向插入到 pBSK (+) 载体 O ontech公司产品）的多克隆位点上， 转化 DH5 α , 细菌形成 GDNA文库。 用 Dye terminate cyde react ion sequencing Id t (Perkin-E ier公司产品 ) 和 ABT 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 GDNA
0]27hl0的 cDM序列为新的 DM。 通过合成一系歹' j引物对该克隆所含的插入 cDNA片 段进行双向测定。 结果表明， 0l27hl0克隆所合的全长 GDNA为 2l85bp (如 Seq ID N0: 1 所示） ， 从第 79bp至 555bp有一个 477bp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 - 01 hl 0 , 编码的蛋白质 ^名为真核转录因子 17.38。 实施例 2: cDNA 克隆的同源检索
将本发明的真核转录因子 17.38的序列及其编码的蛋白序列， 用 Blast程序 (Bas idoca 1 A 1 i gnmen t search tool) [Λ1 tschul, SF et a 1. J.Mol. Biol.1990; 215: 403-10] , 在 Genbank Swi sspor t等数据库进行同源检索。 与本发明的真核转录因子 17.38同源性最高的基因是一种巳知的真核转录因子， 其 编码的蛋白在 Genbank的准入号为 X53280。 蛋白质同源结果示于图 1， 两者高度同 源， 其相同性为 81¾； 相似性为 88% 实施例 3: 用 RT-PCR方法克隆编码真核转录因子 17.38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 GDNA,用 Qiagene的试剂盒纯化后，用下列引物进行 PCR扩增：
Primerl: 5'— TCTCGGCTGAGGCAGCCATCTTTC-3' (SEQ ID NO: 3)
Primer 2: 5'— CTTTTGCACTGTTATTTTATTTTG- 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;
Primer 2为 SEQ ID NO: 1的中的 3'端反向序列。
扩增反应的条件： 在 50 μ 1的反应体积中合有 50 oL/L KC1, 10mmol/L Tris- Ci, (pH8.5), 1.5mmol/L MgCl2, 200 μ raol/L dNTP, lOpmoL引物， 1U的 Taq DNA聚合 酶 (C 1 on t ech公司产品）。 在 PE9600型 DNA热循环仪（Perk i n-B lme r公司 )上按下列条 件反应 25个周期： 94 C 30sec; 55°C 30sec; 72 C 2min。 在 RT- PGR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 克隆试剂盒连楼到 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 - 2185bp完全相同。
实施例 4: Northern 印迹法分析真核转录因子 17.38基因的表达：
用一步法提取总 RNA[AnaL. Biochem 1987, 162, 156-159] 0 该法包括酸性硫 氰酸胍苯酚 氯仿抽提。 即用 4M异硫氰酸胍- 25mM柠檬酸钠， 0.2M乙睃钠 （ 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: 〗） ， 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 f辱到的 RNA沉淀用 70%乙醇洗涤， 干燥并溶于水中。 用 20μδ RNA, 在舍 20mM 3- ( N - 吗啉代 ) 丙磺酸 （ PH7.0 ) 5mM乙酸钠 IraM 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 x- 32P dATP通过随机弓 I物法制备 P 标记 的 DNA探针。 所用的 DM探针为图 1所示的 PCR扩增的真核转录因子 17.38编码区序列 (79bp至 555bp)。 将 32P-标记的探针 (约 2 χ 106 Gpm/ml ) 与转移了 RNA的硝酸纤维素 膜在一溶液中于 42"C杂交过夜， 该溶液包含 50¾甲酰胺- 25iiiM H2P0 ( pH7.4 ) -5 χ SSC-5 Denhardt's溶液和 200 μ g/ml鲑精 DNA。 杂交之后， 将滤膜在 1 x SSC-0.咖 S 中于 55"C洗 30niin。 然后， 用 Phosphor Imager进行分析和定量。
实施例 5: 重组真核转录因子 17.38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弓 I物， 序 列如下：
Primer3: 5-CCCCATATGATGAATCAAGAAAAGTTAGCCAAA-3' ( Seq ID No: 5 ) Primer4: 55-CATGGATCCTTAGTTAGCTTCATTCTTTGATGC-3' (Seq ID No: 6 ) 此两段引物的 5 '端分别舍有 Mel和 BamH 1；酶切位点， 其后分别为目的基因 5 '端 和 3'端的编码序列， Ndel和 BamHI酶切位点相应于表达载体质粒 pBT- 28b(+) (Novagen 公司产品， Cat. No.69865.3)上的选择性内切酶位点。 以含有全长目的基因的 pBS - 0127hl0质粒为模板， 进行 PCR反应。 PCR反应条件为: 总体积 50 μ 1中合 pBS - 0127hl0 质粒 10pg、 弓 I物 Pr inier— 3和 Pr imer—4分别为 1 Opmo 1、 Advantage polymerase Mix ( C ontech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 Ndel和 BamHI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 DH5a,在含卡那霉素 (终浓度 30 μ g/ηύ ) 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 ( pBT-0127hlO) 用氯化钙法将重组质粒转化大肠 杆菌 BL21 (DB3)plySs (Novagen公司产品）。 在合卡那霉素 （终浓度 30 g/mU 的 LB 液体培养基中， 宿主菌 BL21 ( pET- Q127hl0) 在 37°C培养至对数生长期， 加入 IPTG 至终浓度 lmmol/L, 继续培养 5小时。 离心收集菌体， 经超声波破菌，离心收集上清， 用能与 6个组氨酸 ( 6HLs-Tag ) 结合的亲和层析柱 H Ls. Bind Quick Cartridge ( Novagen公司产品） 进行层析， 得到了纯化的目的蛋白真核转录因子 17.38。 经 SDS PAGE,电泳， 在 1 OkDa处得到一单一的条带 （图 2 ) 。 将该条带转移至 PVDF膜上 用 Bdams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 抗真核转录因子 17.38抗体的产生
用多肽合成仪（PE公司产品）合成下迷真核转录因子 17.38特异性的多肽：
NH2-Met-Asn-Gln-Glu-Lys-Lsu-Ala-Lys-Leu-Gin-ALa-Gln-Val-Arg-i l6- C00H (SEQ TD NO: 7)。 将该多肽分别与血蓝蛋白和半血清白蛋白耦合形成复合, 方法参见： Avramea s , et a l. Immunochem i s t ry, 1969; 6: 43 0 用 4mg上迷血蓝蛋白 多肽复合物加上完全弗氏佐剂免疫家兔， 15天后再用血蓝蛋白多肽复合物加不完 全弗氏佐剂加强免疫一次。 采用经 1 5 g/m l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-Sepha rose从抗体阳性的家兔血清 中分离总 T gG。将多肽结合于溴化氰活化的 Sepha rose4B柱上，用亲和层析法从总 I gG 中分离抗多肽杭体。 免疫沉淀法证明纯化的抗体可特异性地与真核转录因子 Π. 38 结合。
实施例 7 : 本发明的多核苷酸片段用作杂交探针的应用
从本发明的多核苷酸中挑选出合适的寡核苷酸片段用作杂交探针有多方面的 用途， 如用该探针可与不同来源的正常组织或病理组织的基因组或 GDNA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、 Sou thern 印 迹法、 Nor thern 印迹法和复印方法等， 它们都是将待测的多核苷酸样品固定在滤 膜上后使用基本相同的步骤杂交。 这些相同的步骤是： 固定了样品的滤膜首先用 不含探针的杂交缓冲液进行预杂交， 以使滤膜上样品的非特异性的结合部位被载 体和合成的多聚物所饱和。 然后预杂交液被含有标记探针的杂交缓冲液替换， 并 保温使探针与靶核酸杂交。 杂交步骤之后， 未杂交上的探针被一系列洗膜步骤除 掉。 本实施例利用较高强度的洗膜条件 （如较低盐浓度和较高的温度） ， 以使杂 交背景降低且只保留特异性强的信号。 本实施例选用的探针包括两类： 第一类探 针是完全与本发明的多核苷酸 SEQ ID NO: 1相同或互补的寡核苷酸片段； 第二类 探针是部分与本发明的多核苷酸 SEQ ID NO: 1相同或互补的寡核苷酸片段。 本实 施例选用斑点印迹法将样品固定在滤膜上， 在较高强度的的洗膜条伴下， 第一类 探针与样品的杂交特异性最强而得以保留。 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% 70¾, 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戍以下二个探针：
探针 1 ( probel ) , 属于第一类探针， 与 SEQ ID NO: 1 的基因片段完全
1.1 同源或互补 （41Nt ) ：
5，- TGAATCAAGAAAAGTTAGCCAAACTTCAGGCTCAGGTCCGG- 3， ( SBQ 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#换突变序列 ( 4]Nt ) ：
5'— TGAATCAAGAAAAGTTAGCCCAACTTGAGGCTCAGGTCCGG 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; S tockton 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： 步骤： 1 ) 将新^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。 搡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-HCi, pH7.5; 25mmol/LnaCl; 25隱 ol/L MgC12 ) 悬浮沉淀 （大约 10ml/g ) 0 4 ) 在 4oC 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 O. lg最初组织样品加 1- 5ml ) , 再以 lOOOg 离心 10分钟。 7) 用裂解缓冲液重悬沉淀（每 0. lg最初组织样品加 Imi ) , 然后 接以下的苯酚抽提法。
2, DNA的苯酚抽提法
步骤： 1 )用 1- 10ml 冷 PBS 洗细胞， lOOOg离心 10分钟。 2 )用冷细胞裂解 液重悬浮沉淀的细胞 （1 x 108细胞 /ml ) 最少应用 IQOul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 2Q0ug/ml。 5 ) 50oC保温反应 1小时或在 37oC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 倍体积冷 100%乙醇加到 DNA溶液 中， 混匀。 在 20oC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 SOOul 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。 ί¾速涡 旋振荡或用滴管吹吸， 同时逐渐增加 ΤΕ, 混合至 DNA充分溶解， 每 1-5 x 106 细 胞所提取的大约加 lul。
以下第 8-13步骤仅用于必须除去污染时， 否则可直接进行第 步骤。
8 )将 RNA酶 A加到 DNA溶液中， 终浓度为 lOOug/ml, 37oC保温 30分钟。 9 ) 加 入 SDS和蛋白酶 K, 终浓度分别为 0.5¾和 ]00ug/ml。 37oC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 1/1Q体积 2moi/L醋酸钠和 2.5 体积冷乙醇， 混匀置- 20oC 1 小时。 13 )用 70%乙醇及 100¾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 A260和 A280以检测 DMA的纯度及产率。 15 )分装后存放于 _20oC。 样膜的制备：
1 ) ¾ 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 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， 在室温中晾千。
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/LTris-HCl ( pH7.0 ) , 3mol/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， 60-80oC真空干燥 2小时。
探针的标记
1 )3 μ lProbe( 0. lOD/lO μ 1 ),加入 2 μ IKinase缓冲液， 8-10 uCi γ- 32P- dATP+2U Kinase, 以补加至终体积 20 μ i。
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 MonUor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- 32P - dATP ) 。 将^ #膜置于塑料袋中， 加入 3- lQmg 预杂交液 （ lQxDenhardt, s; 6xSSC, 0. lmg/ml CT DNA (小牛胸腺 DNA) 。 ) , 封好袋口后， 68oC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， 42oC水洛摇过夜。 洗膜：
高强度洗膜：
1 ) 取出已杂交好的样膜。
2 ) 2xSSC; 0.咖 S中 , 40oC洗 15分钟 ( 2次） 。
3 ) 0. IxSSC, 0.咖 S中 , 40oC洗 15分钟 （ 2次） 。
4 ) 0. lxSSC, 0.1%SDS中， 55oC洗 30分钟 （ 2次） ， 室温晾干。 强度洗膜：
1 ) 取出巳杂交好的禅膜。
1%SDS中， 37 oC洗 15分钟 （ 2
0.1%SDS中， 37oC洗 15分钟 (
Figure imgf000017_0001
0.1%SDS中， 40oC洗 15分钟 ( ) , 室温晾干 X -光自显影：
-70oC, 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 P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伴所进行的杂交实验， 探针 1 的杂交斑放射性 强度明显强于另一个探针杂交斑的放射性强度。 因而可用探针 1 定性和定量地分 析本发明的多核苷酸在不同组织中的存在和差异表达。 实施例 8 隠 Microarray
基因芯片或基因微矩阵 （DNA Microarray )是目前许多国家实验室和大制药 公司都在着手研制和开发的新技术， 它是指将大量的靶基因片段有序地、 高密度 地排列在玻璃、 硅等载体上， 然后用荧光检测和计算机软件进行数据的比较和分 析， 以达到快速、 高效、 高通量地分析生物信息的目的。 本发明的多核苷酸可作 为靶 DM 用于基因芯片技术用于高通量研究新基因功能； 寻找和筛选组织特异性 新基因特别是胂瘤等疾病相关新基因； 疾病的诊断， 如遗传性疾病。 其具体方法 步骤在文献中巳有多种报道， 如可参阅文献 DeRisi, J. L. , Lyer, V. &Brown, P.0. (1997)Science278, 680-686.及文献 Helle, R. A. , Schema, M. , Chai, A. , Shalom, D. , (1997) PNAS 94: 2150-2155.
(一） 点样
各种不同的全长 GDNA共计 4000条多核苷酸序列作为靶 DNA，其中包括本发明 的多核苷酸。 将它们分别通过 PCR 进行扩增， 纯化所得扩增产物后将其浓度调到 500ng/ul 左右, 用 Cartesian 7500 点样仪（购自美国 Car tes ian公司）点于玻璃 介质上， 点与点之间的距离为 280 μΠ1。 将点样后的玻片进行水合、 干燥、 置于紫 外交联仪中交联， 洗脱后干燥使 DNA 固定在玻璃片上制备成芯片。 其具体方法步 骤在文献中已有多种报道， 本实施例的点祥后处理步骤是：
1. 潮湿环境中水合 4小时；
2. Q.2½SDS洗涤 1分钟；
3. ddH20洗涤两次， 每次 1分钟；
4. NaBH4封闭 5分钟；
5. 95()C水中 2分钟；
6. Q. 2%SDS洗涤 1分钟；
7. ddH20〉中洗两次；
8. 凉干， 25 储存于暗处备用。
(二 ) 探针标记 用一步法分别从正常肝与肝癌中抽提总 mRNA, 并用 Ol igotex mRNA Midi Ki t (购自 QiaGen公司）纯化 mRNA，通过反转录分别将荧光试剂 Cy 3dUTP (5- Am ί no- propargy l-2'-deoxyur i d i ne 5--t r i pha t e coupled to Cy 3 fluorescent dye , 购自 Ame sliam Phamacia Biotech 公司）标记正常肝组织的 mRNA, 用荧光试剂 CySdUTP (5-Ami no-propa rgy l-2'-deoxyu r ί d ine 5'-triphate coup led to Cy5 fluorescent dye, 购自 Amershani Phamacia Biotech公司）标记肝癌组织 mRNA， 经纯化后制备出探针。 具体步骤参照及方法见：
Sdiena,
M. , Shalon, D. , Heller, R. (1996)Proc. Natl. Acad. Sci. USA. Vol.93: 10614- 10619. Schena,M. , Shalon, Dari. , Davis, R. W. (1995) Science.270. (20) : 467-480. 分别将来自 以上两种组织的探针与芯片一起在 UniHyb™ Hybridization Solution (购自 TeieChem公司）杂交液中进行杂交 16 小时， 室温用洗涤液 ( 1 x SSC, 0.2%SDS ) 洗涤后用 ScanArray 3000扫描仪（购自美国 General Scanning公 司） 进行扫描， 扫描的图象用 Iniagene软件 （美国 Biodiscovery公司 )进行数据 分析处理， 算出每个点的 Cy3/Cy5 比值， 该比值小于 0.5大于 2 的点被认为是表 达有差异的基因。
实 验 结 果 表 明 ， Cy3signal=5299.42 ( 取 四 次 实 验 的 平 均 值） ， Cy5signal=;i84l5.42 (取四次实验的平均值） ， Cy3/Cy5=0.2878,本发明的多 核苷酸在以上两种组织中的表达有明显差异， 表明本发明的多核苷酸与肝癌相关。 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。
在转录过程中， 真核生物 RNA聚合酶需要严格侬赖于一系列蛋白因子才能识 别和结合到启动子特定序列上， 需要一定的蛋白质祖坟才能启动转录反应。 除 RNA聚合酶 U以外，转录开始还需要四种普遍性转录因子： BTFU也称为 TF1I D ) , BTF2, BTF3, BTF4。 其中 BTF3可以与 RNA聚合酶 11形成一个稳定的化合物。
BTF3 的中心憎水区域是与 RNA 聚合酶 II结合的良好位点， 一个相似的亲脂 性螺旋抅成旋管-旋管结抅， 是微管交联中与蛋白质相互作用的位点。 BTF3 在 细胞中的含量相当微少， 但在所有的细胞中均存在。 BTF3 是真核转录必不可少 的蛋白因子， 它直接结合 RNA 聚合酶 ii， 使启动子进行低水平基础性转录， 它
is 可以被转录活化因子进一步激活， 亦可以被转录抑制因子阻遏。 如果缺少， 单 独 RNA 聚合酶 II将导致散乱的多点起始的 mRNA合成， 且合成效率差。 BTF 3 在 生物体内参与决定基因在何种组织及发育阶段表达， 如果 BTF 3 的编码基因发生 突变， 将导致多种受 BTF 3调节的基因不能正常表达， 从而导致多种疾病， 如与 胚胎发育、 细胞分化有关的各种疾病等。
本发明的多肽与人转录因子 BTF 3是真核转录因子， 含人转录因子 BTF 3家族 的特征性序列， 两者具有相似的生物学功能。 它在体内参与转录过程， 真核转 录必不可少的蛋白因子， 它促使启动子进行低水平基础性转录， 其表带异常将 导致散乱的多点起始的 Mnia合成， 且合成效率差， 从而导致蛋白合成的异常或 错误， 并产生相关的疾病。
由此可见， 本发明的真核转录因子 17. 38 的表达异常将产生各种疾病尤其是 各种胂瘤、 胚胎发育紊乱症、 生长发育障碍性疾病、 炎症、 免疫性疾病， 这些 疾病包括但不限于：
各种组织的肿瘤： 胃癌， 肝癌， 肺癌， 食管癌， 乳腺癌， 白血病， 淋巴瘤， 甲状腺肿瘤， 子宫肌瘤， 神经细胞瘤， 星形细胞瘤， 食管膜瘤， 胶质细胞瘤， 神经纤维瘤， 结肠癌， 黑色素瘤， 膀胱癌， 子宫癌， 子宫内膜癌， 结肠癌， 胸 腺肿瘤， 鼻咽癌， 喉癌， 气管肿瘤， 纤维瘤， 纤维肉瘤， 脂肪瘤， 脂肪肉瘤 胚胎发育紊乱症： 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， 先天性耳聋
生长发育障碍性疾病： 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， 呆小症， 侏儒症， 库兴综合症， 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， 子宫经 验， 各种感染性炎症
免疫性疾病： 系统性红斑狼疮， 类凤湿性关节炎， 支气管小船， 荨麻疹， 特 异性皮炎， 感染后心肌炎， 硬皮病， 中症肌无力， 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
本发明的真核转录因子 17. 38 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各种胂瘤、 胚胎发育紊乱症、 生长发育障碍性疾 病、 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 真核转录 因子 7. 38 的药剂的方法。 激动剂提高真核转录因子 Π . 38 刺激细胞增殖等生
1 ') 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真核转录因子 Π. 38 的膜制剂与标 记的真核转录因子 Π.38 —起培养。 然后测定药物提高或阻遏此相互作用的能 力。
真核转录因子 17.38 的拮抗剂包括筛选出的抗体、 化合物、 受休缺失物和 类似物等。 真核转录因子 17.38 的拮抗剂可以与真核转录因子 17.38 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， 可以将真核转录因子 Π. 38 加入生物分析 测定中， 通过测定化合物对真核转录因子 17.38 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。 能与真核转录因子 Π. 38 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， 一般应对真核转录因子 17.38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休或单克隆抗体。 本发明还提供 了针对真核转录因子 Π.38 抗原决定簇的抗休。 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真核转录因子 17.38 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。 制备真核转录因子 17.38 的单克隆抗体的技术包括但不限于杂交瘤 技术（Kohler and Mi Istein. Nature, 1975, 256: 495-497) , 三瘤技术， 人 B- 细胞杂交瘤技术， BBV-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（Morrison et al , PNAS, 1985, 81: 6851) 0 而已有的 生产单链抗体的技术 01. S. Pat No.4946778)也可用于生产抗真核转录因子 17. 38 的单链抗体。
抗真核转录因子 Π. 38 的抗体可用于免疫组织化学技术中， 检测活检标本 中的真核转录因子 17. 38。
与真核转录因子 17. 38 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。 如真核转录因子
1 7. 38 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真核转录 因子 17. 38阳性的细胞。
本发明中的抗体可用于治疗或预防与真核转录因子 Π. 38 相关的疾病。 给 予适当剂量的抗体可以刺激或阻断真核转录因子 17. 38的产生或活性。
本发明还涉及定量和定位检测真核转录因子 17. 38 水平的诊断试验方法。 这些试验是本领域所熟知的， 且包括 F ISH测定和放射免疫测定。 试验中所检测 的真核转录因子 Π . 38 水平， 可以用作解释真核转录因子 17. 38 在各种疾病中 的重要性和用于诊断真核转录因子 17. 38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。
编码真核转录因子 17. 38 的多核苷酸也可用于多种治疗目的。 基因治疗技 术可用于治疗由于真核转录因子 17. 38的无表达或异常 /无活性表达所致的细胞 增殖、 发肓或代谢异常。 重组的基因治疗载体（如病毒载体）可设计用于表达变 异的真核转录因子 Π . 38 , 以抑制内源性的真核转录因子 17. 38 活性。 例如， 一种变异的真核转录因子 Π. 38 可以是缩短的、 缺失了信号传导功能域的真核 转录因子 1 7. 38 , 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真核转录因子 17. 38 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真核转录因子 1 7. 38 的多核苷酸转移至细胞内。 构建 携带编码真核转录因子 U. 38 的多核苷酸的重组病毒载体的方法可见于巳有文 献（Sanibrook, e t a l . ) 0 另外重组编码真核转录因子 17. 38 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。
抑制真核转录因子 Π . 38 mRNA的寡核苷酸（包括反义 RNA和 DM)以及核酶 也在本 ^明的范围之内。 核酶是一种能特异性分解特定 RNA 的酶样 RM 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
2 ! 的 RNA和 DNA及核酶可用巳有的任何 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 DM序列在体外或体内转录获得。 这种 DM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。
编码真核转录因子 17. 38 的多核苷酸可用于与真核转录因子 17. 38 的相关 疾病的诊断。 编码真核转录因子 17. 38 的多核苷酸可用于检测真核转录因子 17. 38 的表达与否或在疾病状态下真核转录因子 17. 38 的异常表达。 如编码真 核转录因子 17. 38 的 DNA序列可用于对活检标本进行杂交以判断真核转录因子 U. 38的表达状况。 杂交技术包括 Southern 印迹法， Northern 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（Mioroarray)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。 用真核转录因子 17. 38 特异的引物进行 RNA-聚合酶链反应（RT- PCR)体外扩 增也可检测真核转录因子】 7. 38的转录产物。
检测真核转录因子 Π. 38基因的突变也可用于诊断真核转录因子 17. 38 相 关的疾病。 真核转录因子 Π. 38 突变的形式包括与正常野生型真核转录因子 17. 38 DNA 序列相比的点突变、 易位、 缺失、 重组和其它任何异常等。 可用巳 有的技术如 Southern 印迹法、 DNA序列分析、 PCR和原位杂交检测突变。 另夕卜， 突变有可能影响蛋白的表达， 因此用 Northern 印迹法、 Western 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。
筒而言之， 根据 cDNA制备 PCR弓 1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筛选合各条人染色体的体细胞杂合细胞。 只 有那些含有栖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 G歸库。
将 GDNA克隆与中期染色体进行荧光原位杂交（FTSH) , 可以在一个步骤中精 确地进行染色体定位。 此技术的综述， 参见 Verma等， Human Chromosomes: a Manual of Bas IG Techniques, Pergamon Press, New York (1988)。
一旦序列被定位到准确的染色体位置， 此序列在染色体上的物理位置就可 以与基因图数据相关联。 这些数据可见于例如， V.MGkusiGk,Mendelian Inheritance in Man (可通过与 Johns Hopkins University Welch Med i Ga 1 U.brary联机获得）。 然后可通过连锁分析， 确定基因与业巳定位到染色体区域 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。 真核转录因子 17. 38 以有效地治疗和 /或预防具 体的适应症的量来给药。 施用于患者的真核转录因子 Π. 38 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-真核转录因子 Π. 38 , 其特征在于它包含有： SEQ T D N0: 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 SBQ ID NO: 2所示的氨基酸序列至少 95%的相同性。
3、 如杈利要求 2 所述的多肽， 其特征在于它包合具有 S EQ 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 I D NO: 2 所示氨基酸序列的多肽或其片段、 类似物、 衍生
(b) 与多核苷酸（a ) 互补的多核苷酸； 或
(c) 与 ) 或 （b ) 有至少 82¾相同性的多核苷酸。
5、如杈利要求 4所迷的多核苷酸，其特征在于所述多核苷酸包含编码具有 SEQ I D NO: 2所示氨基酸序列的多核苷酸。
6、如权利要求 4所迷的多核苷酸，其特征在于所迷多核苷酸的序列包含有 SBQ I D NO: 1中 79-555位的序列或 SBQ ID NO: 1中 1-2185位的序列。
7、 一种合有外源多核苷酸的重组载体， 其特征在于它是由杈利要求 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) 用杈利要求 4-6 中的任一杈利要求所述多核苷酸转化或转导的宿主细 胞。
9、 一种具有真核转录因子 17. 38 活性的多肽的制备方法， 其特征在于所述方 法包括：
(a) 在表达真核转录因子 1 7. 38条件下， 培养杈利要求 8所迷的工程化宿主 细胞；
(b) 从培养物中分离出具有真核转录因子〗7. 38活性的多肽。
1 0、 一种能与多肽结合的抗体，其特怔在于所迷杭体是能与真核转录因子 1 7. 38 蜃換页《细则第 2β条》 特异性结合的抗体。
1 1、 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、 拮抗或抑制真核转录因子 1 7. 38的活性的化合物。
1 2、 如杈利要求 1 1 所述的化合物， 其特征在于它是 S EQ I D NO: 1 所示的多核 苷酸序列或其片段的反义序列。
1. 3、 一种杈利要求 11 所述化合物的应用， 其特征在于所述化合物用于调节真 核转录因子 17. 38在体内、 体外活性的方法。
14、 一种检测与权利要求 1- 3 中的任一杈利要求所迷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 中的任一杈利要求所迷多肽的应用， 其特征在于它应用于 筛选真核转录因子 Π . 38 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。
1 6、 如权利要求 4-6 中的任一杈利要求所述的核酸分子的应用， 其特征在于它 作为引 I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
1 7、 如杈利要求 1-6 及 11 中的任一权利要求所速的多肽、 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真核转录因 子 17. 38异常相关的疾病的药物组合物。
1 8、 权利要求 1-6 及 11 中的任一杈利要求所迷的多肽、 多核苷酸或化合物的 应用， 其特征在于用所迷多肽、 多核苷酸或化合物制备用于治疗如恶性胂瘤， 血液病， H I V感染和免疫性疾病和各类炎症的药物。
PCT/CN2001/001142 2000-07-07 2001-07-02 Nouveau polypeptide, facteur de transcription eucaryotique 17.38, et polynucleotide codant ce polypeptide WO2002033076A1 (fr)

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CN 00119409 CN1333233A (zh) 2000-07-07 2000-07-07 一种新的多肽——真核转录因子17.38和编码这种多肽的多核苷酸
CN00119409.7 2000-07-07

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1231339A (zh) * 1999-01-29 1999-10-13 中国人民解放军第二军医大学 人、猪共患囊虫病核酸疫苗

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1231339A (zh) * 1999-01-29 1999-10-13 中国人民解放军第二军医大学 人、猪共患囊虫病核酸疫苗

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