WO2002012487A1 - Nouveau polypeptide, proteine 10.23 associee au recepteur t1/st2, et polynucleotide codant ce polypeptide - Google Patents

Nouveau polypeptide, proteine 10.23 associee au recepteur t1/st2, et polynucleotide codant ce polypeptide Download PDF

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Publication number
WO2002012487A1
WO2002012487A1 PCT/CN2001/001075 CN0101075W WO0212487A1 WO 2002012487 A1 WO2002012487 A1 WO 2002012487A1 CN 0101075 W CN0101075 W CN 0101075W WO 0212487 A1 WO0212487 A1 WO 0212487A1
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polypeptide
seq
polynucleotide
sequence
binding protein
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PCT/CN2001/001075
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English (en)
Chinese (zh)
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Yumin Mao
Yi Xie
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Shanghai Biowindow Gene Development Inc.
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Priority to AU2002214914A priority Critical patent/AU2002214914A1/en
Publication of WO2002012487A1 publication Critical patent/WO2002012487A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals

Definitions

  • the present invention belongs to the field of biotechnology. Specifically, the present invention describes a novel polypeptide, T1 / ST2 receptor binding protein 10.23, and a polynucleotide sequence encoding the polypeptide. The invention also relates to methods and applications for preparing such polynucleotides and polypeptides.
  • the T1 / ST2 receptor-binding protein is a receptor-like molecule that is the same source as the type I interleukin-1 (IL-1) receptor. Although they are similar in sequence, the T1 / ST2 receptor does not bind to any of the three IL-1 subtypes and undergoes signal transduction.
  • T1 / ST2 mRNA encodes a transmembrane peptide molecule, and its cytoplasmic region is also very similar to the type I IL-1 receptor (Yanagi sawa, K., Takagi, T. et al., 1993).
  • the soluble and transmembrane T1 / ST2 receptors are mainly expressed in different tissues, and the transcription of different promoters requires the use of different polyadenine sites, thus producing different forms of mRM products.
  • the soluble form of the T1 / ST2 receptor is mainly produced by fibroblasts, while the transmembrane form is mainly produced in hematopoietic tissues and lungs.
  • transmembrane-type receptors are re-expressed in hematopoietic tissues, while soluble forms of the receptors are restricted to re-expression only in a short period of mid-to-late pregnancy and occur only in localized tissues ( Skin, bones, eyes) and then disappears quickly (RoBler, U., Thomassen, E. et al., 1995).
  • the T1 / ST2 soluble receptor can continue to express (Bergers, G., Reikers torfer, A. et al., 1994; Thomassen, D., Kothny, G. et al., 1995).
  • the T1 / ST2 receptor does not bind to any IL-1 family proteins (IL-1, IL-1, IL-Ira), but binds to another protein that binds to any IL-1 family Proteins have no sequence similarity and their expression is not widespread. This protein can not function in signal transduction start T1 / ST2 receptor function remains to be further studied and it Significance T1 / ST 2 binding reaction. However, the function of the T1 / ST2 receptor and the IL-1 receptor should be similar, although its strict tissue-specific distribution proves that it is not required for the IL-1 signaling pathway. The cytoplasmic region of T1 / ST2 is also involved in the activation of NFB.
  • the T1 / ST2 receptor binding protein 10.23 protein plays an important role in regulating important functions of the body such as cell division and embryonic development, and it is believed that a large number of proteins are involved in these regulatory processes, so it has been necessary to identify more proteins in the art. Many T1 / ST2 receptor-binding protein 1 0.23 proteins involved in these processes, especially the amino acid sequence of this protein.
  • the new T1 / ST2 receptor binding protein 10.23 The isolation of the protein-coding gene also provides the basis for research to determine the role of this protein in health and disease states. This protein may form the basis for the development of diagnostic and / or therapeutic drugs for diseases, so it is important to isolate its coding DNA. Object of the invention
  • 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 T1 / ST2 receptor binding protein 10.23.
  • Another object of the present invention is to provide a genetically engineered host cell containing a polynucleotide encoding a T1 / ST2 receptor binding protein 10.23.
  • Another object of the present invention is to provide a method for producing a T1 / ST2 receptor binding protein 10.23.
  • Another object of the present invention is to provide a polypeptide of the present invention-T1 / ST2 receptor binding protein
  • Another object of the present invention is to provide mimic compounds, antagonists, agonists, and inhibitors of the polypeptide- "T1 / ST2 receptor binding protein 10.23" of the present invention.
  • Another object of the present invention is to provide a method for diagnosing and treating diseases associated with abnormalities in T1 / ST2 receptor binding protein 10.23. Summary of invention
  • the present invention relates to an isolated polypeptide, which is of human origin and comprises: a polypeptide having the amino acid sequence of SEQ ID No. 2, or a conservative variant, biologically active fragment or derivative thereof.
  • the polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • the invention also relates to an isolated polynucleotide comprising a nucleotide sequence or a variant thereof selected from the group consisting of:
  • sequence of the polynucleotide is one selected from the group consisting of: (a) a sequence having positions 1414 to 1695 in SEQ ID NO: 1; and (b) a sequence having 1-3031 in SEQ ID NO: 1 Sequence of bits.
  • the present invention further relates to a vector, particularly an expression vector, containing the polynucleotide of the present invention; a host cell genetically engineered with the vector, including a transformed, transduced or transfected host cell; Host cell and method of preparing the polypeptide of the present invention by recovering the expression product.
  • the invention also relates to an antibody capable of specifically binding to a polypeptide of the invention.
  • the invention also relates to a method for screening compounds that mimic, activate, antagonize or inhibit the activity of the T1 / ST2 receptor binding protein 10.23 protein, which comprises utilizing the polypeptide of the invention.
  • the invention also relates to compounds obtained by this method.
  • the present invention also relates to a method for in vitro detection of a disease or disease susceptibility associated with abnormal expression of the T1 / ST2 receptor binding protein 10.23 protein, comprising detecting mutations in the polypeptide or a sequence encoding a polynucleotide thereof in a biological sample. Or detecting the amount or biological activity of a polypeptide of the invention in a biological sample.
  • the invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a polypeptide of the invention or a mimetic thereof, an activator, an antagonist or an inhibitor, and a pharmaceutically acceptable carrier.
  • the present invention also relates to the use of the polypeptide and / or polynucleotide of the present invention in the preparation of a medicament for the treatment of cancer, developmental disease or immune disease or other diseases caused by abnormal expression of T1 / ST2 receptor binding protein 10.23. .
  • FIG. 1 is a comparison diagram of gene chip expression profiles of T1 / ST2 receptor binding protein 10.23 and 11/372 receptor binding protein of the present invention.
  • the upper figure is a graph of the expression profile of the T1 / ST2 receptor binding protein 10.23, and the lower figure is the graph of the expression profile of the T1 / ST2 receptor binding protein.
  • FIG. 2 shows the polyacrylamide gel electrophoresis (SDS-PAGE) of the isolated T1 / ST2 receptor binding protein 10.23.
  • OkDa is the molecular weight of the protein.
  • the arrow indicates the isolated protein band.
  • Nucleic acid sequence refers to an oligonucleotide, a nucleotide or a polynucleotide and a fragment or part thereof, and may also refer to a genomic or synthetic DNA or RNA, they can be single-stranded or double-stranded, representing the sense or antisense strand.
  • amino acid sequence refers to an oligopeptide, peptide, polypeptide or protein sequence and fragments or portions thereof.
  • amino acid sequence in the present invention relates to the amino acid sequence of a naturally occurring protein molecule, such "polypeptide” or “protein” does not mean to limit the amino acid sequence to a complete natural amino acid related to the protein molecule .
  • a protein or polynucleotide “variant” refers to an amino acid sequence having one or more amino acids or nucleotide changes or a polynucleotide sequence encoding it. The changes may include deletions, insertions or substitutions of amino acids or nucleotides in the amino acid sequence or the nucleotide sequence. Variants can have "conservative" changes in which the substituted amino acid has a structural or chemical property similar to the original amino acid, such as the replacement of isoleucine with leucine. Variants can also have non-conservative changes, such as replacing glycine with tryptophan.
  • “Deletion” refers to the deletion of one or more amino acids or nucleotides in an amino acid sequence or nucleotide sequence.
  • Insertion means that a change in the amino acid sequence or nucleotide sequence results in an increase in one or more amino acids or nucleotides compared to a molecule that exists in nature.
  • Replacement refers to the replacement of one or more amino acids or nucleotides with different amino acids or nucleotides.
  • Bioactivity refers to a protein that has the structure, regulation, or biochemical function of a natural molecule.
  • immunologically active refers to the ability of natural, recombinant or synthetic proteins and fragments thereof to induce a specific immune response and to bind specific antibodies in a suitable animal or cell.
  • An "agonist” refers to a molecule that, when bound to the T1 / ST2 receptor binding protein 10.23, causes a change in the protein to regulate the activity of the protein.
  • An agonist may include a protein, a nucleic acid, a carbohydrate, or any other molecule that can bind to the T1 / ST2 receptor binding protein 10.23.
  • Antagonist refers to a biological activity or immunology that can block or modulate T1 / ST2 receptor binding protein 10.23 when bound to T1 / ST2 receptor binding protein 10.23 Active score Child.
  • Antagonists and inhibitors can include proteins, nucleic acids, carbohydrates or any other
  • Regular refers to a change in the function of T1 / ST2 receptor binding protein 10.23, including an increase or decrease in protein activity, a change in binding characteristics, and any other biological properties of T1 / ST2 receptor binding protein 10.23 , Functional or immune properties.
  • substantially pure refers to other proteins, lipids, carbohydrates, or other substances with which it is essentially free from nature.
  • Those skilled in the art can purify the T1 / ST2 receptor binding protein 10.23 using standard protein purification techniques.
  • the substantially pure T1 / ST2 receptor binding protein 10.23 produces a single main band on a non-reducing polyacrylamide gel.
  • the purity of the T1 / ST2 receptor binding protein 10.23 polypeptide can be analyzed by amino acid sequence.
  • Complementary refers to the natural binding of polynucleotides by base-pairing under conditions of acceptable salt concentration and temperature.
  • sequence C-T-G-A
  • complementary sequence G-A-C-T.
  • the complementarity between two single-stranded molecules may be partial or complete.
  • the degree of complementarity between nucleic acid strands has a significant effect on the efficiency and strength of hybridization between nucleic acid strands.
  • “Homology” refers to the degree of complementarity and can be partially homologous or completely homologous.
  • Partial homology refers to a partially complementary sequence that at least partially inhibits hybridization of a fully complementary sequence to a target nucleic acid. This inhibition of hybridization can be detected by performing hybridization (Southern imprinting or Northern blotting, etc.) under conditions of reduced stringency. Substantially homologous sequences or hybridization probes can compete and inhibit the binding of fully homologous sequences to the target sequence under conditions of reduced stringency. This does not mean that the conditions of reduced stringency allow non-specific binding, because the conditions of reduced stringency require that the two sequences bind to each other as a specific or selective interaction.
  • Percent identity refers to the percentage of sequences that are the same or similar in a comparison of two or more amino acid or nucleic acid sequences. The percent identity can be determined electronically, such as through the MEGALIGN program (Lasergene sof tware package, DNASTAR, Inc., Madi son Wis.). The MEGALIGN program can compare two or more sequences according to different methods, such as the Clus ter method (Hi ggins, D. G. and P. M. Sharp (1988) Gene 73: 237-244). The Clus ter method arranges groups of sequences into clusters by checking the distance between all pairs. The clusters are then assigned in pairs or groups. The percent identity between two amino acid sequences such as sequence A and sequence B is calculated by the following formula: The number of matching residues between the sequence ⁇ and the sequence
  • Residues sequence - the sequence number of residues in the interval ⁇ - s interval sequence x residues can also be used as a method well known in the art Jotun Hein measuring the percentage identity between nucleic acid sequences or by Clus ter method (He in L, (1990) Methods in enzyrao logy 183: 625-645) 0 "Similarity” refers to the degree of identical or conservative substitutions of amino acid residues at corresponding positions in the alignment of amino acid sequences.
  • negatively charged amino acids may include aspartic acid and glutamic acid; positively charged amino acids may include lysine and arginine; Similar hydrophilic amino acids may include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; serine and threonine; phenylalanine and tyrosine.
  • Antisense refers to a nucleotide sequence that is complementary to a particular DM or RNA sequence.
  • Antisense strand refers to a nucleic acid strand that is complementary to a “sense strand.”
  • Derivative refers to HFP or a chemical modification of its nucleic acid. This chemical modification may be 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,? ( ⁇ ') 2 and? ⁇ It can specifically bind to the epitope of T1 / ST2 receptor binding protein 10.23.
  • a “humanized antibody” refers to an antibody in which the amino acid sequence of a non-antigen binding region is replaced to become more similar to a human antibody, but still retains the original binding activity.
  • isolated refers to the removal of a substance from its original environment (for example, its natural environment if it is naturally occurring).
  • a naturally-occurring polynucleotide or polypeptide is not isolated when it is present in a living thing, but the same polynucleotide or polypeptide is separated from some or all of the substances that coexist with it in the natural system.
  • Such a polynucleotide may be part of a certain vector, or such a polynucleotide or polypeptide may be part of a certain composition. Since the carrier or composition is not part of its natural environment, they are still isolated.
  • isolated means that the substance is separated from its original environment (if it is a natural substance, the original environment is the natural environment).
  • Such as polynucleotides and polypeptides in the natural state in living cells It is not isolated and purified, but the same polynucleotide or polypeptide is separated and purified if it is separated from other substances in its natural state.
  • isolated T1 / ST2 receptor binding protein 10.23 refers to T1 / ST2 receptor binding protein 10.23 and is substantially free of other proteins, lipids, carbohydrates or other substances with which it is naturally associated. Those skilled in the art can purify the T1 / ST2 receptor binding protein 10.23 using standard protein purification techniques. Substantially pure polypeptides can produce a single main band on a non-reducing polyacrylamide gel. T1 / ST2 receptor binding protein 10.23 The purity of the peptide can be analyzed by amino acid sequence.
  • the present invention provides a new polypeptide ⁇ 11/3 butan 2 receptor binding protein 10.23, which is basically composed of the amino acid sequence shown in SEQ ID NO: 2.
  • the polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide, and preferably a recombinant polypeptide.
  • the polypeptide of the present invention may be a naturally purified product or a chemically synthesized product Products, or produced using recombinant technology from prokaryotic or eukaryotic hosts (eg, 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 T1 / ST2 receptor binding protein 10.23.
  • fragment refers to a polypeptide that substantially maintains the same biological function or activity of the T1 / ST2 receptor binding protein 10.23 of the invention.
  • a fragment, derivative or analog of the polypeptide of the present invention may be: (I) a kind in which one or more amino acid residues are substituted with conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and the substitution
  • the amino acid may or may not be encoded by a genetic codon; or ( ⁇ ) a type in which a group on one or more amino acid residues is replaced by another group to include a substituent; or ( ⁇ ⁇ )
  • Such a polypeptide sequence in which the mature polypeptide is fused with another compound such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol
  • a polypeptide sequence in which an additional amino acid sequence is fused into the mature polypeptide (Such as the leader or secretory sequence or the sequence used to purify the polypeptide or protease sequence).
  • such fragments, derivatives, and analogs are considered to be within the knowledge of those skilled in the art.
  • the present invention provides an isolated nucleic acid (polynucleotide), which basically consists of a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • the polynucleotide sequence of the present invention includes the nucleotide sequence of SEQ ID NO: 1.
  • the polynucleotide of the present invention is found from a cDNA library of human fetal brain tissue. It contains a polynucleotide sequence of 3031 bases in length and its open reading frame 1414-1695 encodes 93 amino acids.
  • this peptide has a similar expression profile to T1 / ST2 receptor binding protein, and it can be inferred that the T1 / ST2 receptor binding protein 10.23 has similar functions to T1 / ST2 receptor binding protein.
  • the polynucleotide of the present invention may be in the DM form or the RM form.
  • DM forms include cDNA, genomic DNA, or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be coding or non-coding.
  • the coding region sequence encoding a mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO: 1 or a degenerate variant.
  • a "degenerate variant" refers to a nucleic acid sequence encoding a protein or polypeptide having SEQ ID NO: 2 but different from the coding region sequence shown in SEQ ID NO: 1 in the present invention.
  • the polynucleotide encoding the mature polypeptide of S 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.
  • the term "polynucleotide encoding a polypeptide" is meant to include polynucleotides that encode such polypeptides and polynucleotides that include additional coding and / or noncoding sequences.
  • the invention also relates to variants of the polynucleotides described above, which encode polypeptides or fragments, analogs and derivatives of polypeptides having the same amino acid sequence as the invention.
  • Variants of this polynucleotide can be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants.
  • an allelic variant is an alternative form of a polynucleotide that may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially change the function of the polypeptide it encodes .
  • the invention also relates to a polynucleotide that hybridizes to the sequence described above (having at least 50%, preferably 70% identity, between the two sequences).
  • the present invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the present invention under stringent conditions.
  • "strict conditions” means: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2xSSC, 0.12SDS, 60 ° C; or (2) during hybridization Add a denaturant, such as 50% (v / v) formamide, 0.1 ° /.
  • Hybridization occurs only when the identity between the two sequences is at least 951 ⁇ 2 or more, and more preferably 97% or more.
  • the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO: 2.
  • nucleic acid fragments that hybridize to the sequences described above.
  • a "nucleic acid fragment” contains at least 10 nucleotides in length, preferably at least 20-30 nucleotides, more preferably at least 50-60 nucleotides, and most preferably at least 100 nuclei. Glycylic acid or more. Nucleic acid fragments can also be used in nucleic acid amplification techniques (such as PCR) to identify and / or isolate polynucleotides encoding T1 / ST2 receptor binding protein 10.23.
  • 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 T1 / ST2 receptor binding protein 10.23 of the present invention can be obtained by various methods.
  • polynucleotides are isolated using hybridization techniques well known in the art. These techniques include, but are not limited to: 1) hybridization of probes to genomic or cDNA libraries to detect homologous polynucleotide sequences, and 2) antibody screening of expression libraries to detect cloned polynucleosides with common structural characteristics Acid fragments.
  • the DM fragment sequence of the present invention can also be obtained by the following methods: 1) isolating the double-stranded DNA sequence from the genomic DNA; 2) chemically synthesizing the MA sequence to obtain the double-stranded DNA of the polypeptide.
  • genomic DNA isolation is the least commonly used. Direct chemical synthesis of DNA sequences is often the method of choice. The more commonly used method is the isolation of cDNA sequences.
  • the standard method for isolating the CDM of interest is to isolate mR from donor cells that overexpress the gene and perform reverse transcription to form a plasmid or phage CDM library.
  • mRM plasmid or phage CDM library.
  • kits are also commercially available (Qiagene;).
  • the construction of cDNA libraries is also a common method (Sarabrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory. New York, 1989).
  • Commercially available cDNA libraries are also available, such as different cDNA libraries from Clontech. When polymerase reaction technology is used in combination, even very small expression products can be cloned.
  • the genes of the present invention can be screened from these cDM libraries by conventional methods. These methods include (but are not limited to): (l) DNA-DNA or DM-RNA hybridization; (2) the presence or loss of marker gene function; (3) determination of the transcript level of T1 / ST2 receptor binding protein 10.23; (4) Detecting the protein product of gene expression by immunological technology or measuring biological activity. The above methods can be used alone or in combination.
  • the probe used for hybridization is homologous to any part of the polynucleotide of the present invention, and its length is at least 10 nucleotides, preferably at least 30 nucleotides, more preferably At least 50 nucleotides, preferably at least 100 nucleotides.
  • the length of the probe is usually within 2000 nucleotides, preferably within 1000 nucleotides.
  • the probe used here is generally a DNA sequence chemically synthesized based on the gene sequence information of the present invention.
  • the genes or fragments of the present invention can of course be used as probes.
  • DNA probes can be labeled with radioisotopes, luciferin, or enzymes (such as alkaline phosphatase).
  • the protein product of the T1 / ST2 receptor binding protein 10.23 gene expression can be detected by immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA).
  • immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA).
  • a method using PCR technology to amplify DNA / RNA is preferably used to obtain the gene of the present invention.
  • the RACE method RACE-rapid amplification of cDNA ends
  • the primers used for PCR can be appropriately based on the polynucleotide sequence information of the present invention disclosed herein. Select and synthesize using conventional methods.
  • the amplified DNA / RNA fragments can be isolated and purified by conventional methods such as by gel electrophoresis.
  • polynucleotide sequence of the gene of the present invention or various DNA fragments and the like obtained as described above can be measured by a conventional method such as dideoxy chain termination method (Sanger et al. PNAS, 1977, 74: 5463-5467). Such polynucleotide sequences can also be determined using commercial sequencing kits and the like. In order to obtain the full-length cDM sequence, sequencing needs to be repeated. Sometimes it is necessary to determine multiple cDNA sequences in order to splice into a full-length cDNA sequence.
  • the present invention also relates to a vector comprising the polynucleotide of the present invention, and a host cell genetically engineered using the vector of the present invention or directly using the T1 / ST2 receptor binding protein 10.23 coding sequence, and the recombinant technology to produce the described Polypeptide method.
  • a polynucleotide sequence encoding the T1 / ST2 receptor binding protein 10.23 can be inserted into a vector to form a recombinant vector containing the polynucleotide of the present invention.
  • carrier refers to those skilled in the art Known bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses or other vectors.
  • Vectors suitable for use in the present invention include, but are not limited to: T7 promoter-based expression vectors (Rosenberg, et al.
  • any plasmid and vector can be used to construct a recombinant expression vector.
  • An important feature of expression vectors is that they usually contain an origin of replication, a promoter, a marker gene, and translational regulatory elements.
  • Methods well known to those skilled in the art can be used to construct an expression vector containing a DNA sequence encoding a T1 / ST2 receptor binding protein 10.23 and appropriate transcriptional / translational regulatory elements. These methods include in vitro recombinant DNA technology, DM synthesis technology, and in vivo recombination technology (Sambroook, et al. Molecular Cloning, a Laboratory Manual, Cold Spooning Harbor Laboratory. New York, 1989).
  • the DM sequence can be operably linked to an appropriate promoter in an expression vector to guide mRNA synthesis. Representative examples of these promoters are: the lac or trp promoter of E.
  • the expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. Insertion of enhancer sequences into the vector will enhance its transcription in higher eukaryotic cells. Enhancers are cis-acting factors for DNA expression, usually about 10 to 300 base pairs, which act on promoters to enhance gene transcription. Illustrative examples include SV40 enhancers of 100 to 270 base pairs on the late side of the origin of replication, polyoma enhancers and adenovirus enhancers on the late side of the origin of replication.
  • the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture.
  • 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 T1 / ST2 receptor binding protein 10.23 or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to constitute a genetic engineering containing the polynucleotide or the recombinant vector.
  • Host 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.
  • 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 exponential growth and treated with CaCl. The steps used are well known in the art. Alternatively, MgCl 2 is used. If necessary, transformation can also be performed by electroporation.
  • the following DNA transfection methods can be used: calcium phosphate co-precipitation method, or conventional mechanical methods such as microinjection, electroporation, and liposome packaging.
  • the polynucleotide sequence of the present invention can be used to express or produce a recombinant T1 / ST2 receptor binding protein 10.23 (Sc ience, 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, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. These methods include, but are not limited to: conventional renaturation treatment, protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • conventional renaturation treatment protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid
  • polypeptides of the present invention as well as antagonists, agonists and inhibitors of the polypeptides, can be directly used in the treatment of diseases, for example, they can treat malignant tumors, adrenal deficiency, skin diseases, various types of inflammation, HIV infection, and immune diseases.
  • the T1 / ST2 receptor binding protein is a receptor-like molecule that is the same source as the interleukin-1 (IL-1) receptor.
  • the T1 / ST2 receptor is functionally similar to the IL-1 receptor. Abnormal expression in the body can cause dysfunction of its corresponding ligand (interleukin-1 or interleukin-1 substance), which in turn leads to the occurrence of related diseases.
  • the expression profile of the polypeptide of the present invention is consistent with the expression profile of human T1 / ST2 receptor binding protein, both Has similar biological functions.
  • the polypeptide of the present invention is the same source as the type I interleukin-1 (IL-1) receptor, and is functionally similar to the IL-1 receptor.
  • Abnormal expression in the body can cause the disorder of its corresponding ligand (interleukin-1 or interleukin-1 substance), and it is known that interleukin has a variety of physiological effects in the body: such as promoting T and B cells Proliferate and differentiate, stimulate hematopoietic cells, participate in inflammatory responses, and inhibit breast cancer cells. Disturbances in its physiological functions can lead to the occurrence of immune disorders, inflammation, tumors and other related diseases, including but not limited to:
  • Defects in cellular immune function can cause various intracellular parasitic infections and viral infections, including but not limited to:
  • Intracellular parasitic infections typhoid, paratyphoid (typhoid), tuberculosis (tuberculosis), leprosy (leprosy), wave thermal conductivity (brutella), etc .;
  • measles virus measles, measles bronchitis, pneumonia, otitis media, subacute sclerosing panencephalitis
  • herpes virus herpes zoster, chicken pox
  • Humoral immunity mainly fights infection by: 1) neutralizing bacterial exotoxin; 2) inhibiting bacterial adsorption; 3) conditioning and phagocytosis of bacteria; 4) dissolving and killing bacteria.
  • Humoral immunity works primarily against bacteria that grow outside the cell.
  • Humoral immune deficiency can cause various extracellular parasites and various viral infections. These diseases include but are not limited to:
  • polio virus poliomyelitis
  • hepatitis virus A, B, C, D, E, H, G
  • etc . polio virus (poliomyelitis), hepatitis virus (A, B, C, D, E, H, G), etc .;
  • Malignant lymphoma [Neck, mediastinum, mesenteric and retroperitoneal lymph nodes], various leukemias [lymphoid hematopoietic tissue], multiple myeloma [push / thoracic / rib / skull and long bone], etc .;
  • Serous inflammation tuberculous pleural, rheumatoid arthritis, acute rhinitis, etc .;
  • Purulent inflammation superficial suppuration and empyema (purulent urethritis, purulent bronchitis, purulent cholecystitis), cellulitis (skin, muscle, appendix), abscess ( ⁇ , ⁇ , sinus, fistula) Wait;
  • polypeptide of the present invention and the antagonist, agonist and inhibitor of the polypeptide can be directly used for the treatment of various diseases, such as immune disorders, inflammation, tumor diseases, and the like.
  • the invention also provides methods of screening compounds to identify agents that increase (agonist) or suppress (antagonist) T1 / ST2 receptor binding protein 10.23.
  • Agonists increase T1 / ST2 receptor binding protein 10.23 stimulate biological functions such as cell proliferation, while antagonists prevent and treat disorders related to cell proliferation, such as various cancers.
  • mammalian cells or membrane preparations expressing T1 / ST2 receptor binding protein 10.23 can be cultured together with labeled T1 / ST2 receptor binding protein 10.23 in the presence of a drug. The ability of the drug to increase or block this interaction is then measured.
  • Antagonists of the T1 / ST2 receptor binding protein 10.23 include antibodies, compounds, receptor deletions, and the like that have been screened.
  • T1 / ST2 receptor binding protein 10.23 antagonist can bind to T1 / ST2 receptor Synaptic protein 10.23 binds and eliminates its function, either inhibits the production of the polypeptide, or binds to the active site of the polypeptide so that the polypeptide cannot perform biological functions.
  • T1 / ST2 receptor binding protein 10.23 can be added to the bioanalytical assay, and the compound can be determined by measuring the effect of the compound on the interaction between T1 / ST2 receptor binding protein 10.23 and its receptor Whether it is an antagonist.
  • Receptor deletions and analogs that act as antagonists can be screened in the same manner as described above for screening compounds.
  • Polypeptide molecules capable of binding to T1 / ST2 receptor binding protein 10.23 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 T1 / ST2 receptor binding protein 10.23 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 or monoclonal antibodies.
  • the invention also provides antibodies against the T1 / ST2 receptor binding protein 10.23 epitope. These antibodies include (but are not limited to): polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, and fragments produced by Fab expression libraries.
  • Polyclonal antibodies can be produced by direct injection of T1 / ST2 receptor binding protein 10.23 into immunized animals (such as rabbits, mice, rats, etc.).
  • immunized animals such as rabbits, mice, rats, etc.
  • adjuvants can be used to enhance the immune response, including but not limited to Freund's Adjuvant, etc.
  • Techniques for preparing monoclonal antibodies for T1 / ST2 receptor binding protein 10.23 include, but are not limited to, hybridoma technology (Kohler and Milstein. Nature, 1975, 256: 495-497), triple tumor technology, human beta-cell hybridoma technology, EBV-hybridoma technology, etc.
  • Inlay antibodies combining human constant regions and non-human variable regions can be produced using existing technologies (Morrison et al, PNAS, 1985, 81: 6851).
  • the existing technology for producing single chain antibodies U.S. Pat No. 4946778, can also be used to produce single chain antibodies against T1 / ST2 receptor binding protein 10.23.
  • Antibodies against T1 / ST2 receptor binding protein 10.23 can be used in immunohistochemistry to detect T1 / ST2 receptor binding protein 10.23 in biopsy specimens.
  • Monoclonal antibodies that bind to T1 / ST2 receptor binding protein 10.23 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.
  • T1 / ST2 receptor binding protein 10.23 High affinity monoclonal antibodies can covalently bind to bacterial or plant toxins (such as diphtheria toxin, ricin, ormosine, etc.).
  • a common method is to attack the amino group of the antibody with a thiol crosslinker such as SPDP and bind the toxin to the antibody through the exchange of disulfide bonds.
  • This hybrid antibody can be used to kill the T1 / ST2 receptor binding protein 10.23 positive Cell. N01 / 01075
  • the antibodies of the present invention can be used to treat or prevent diseases related to T1 / ST2 receptor binding protein 10. 2 3.
  • the administration or proper dose of antibody can stimulate or block the production or activity of T1 / ST2 receptor binding protein 10.23.
  • the invention also relates to a diagnostic test method for quantitatively and locally detecting the T1 / ST2 receptor binding protein 10.23 level.
  • These tests are well known in the art and include FISH assays and radioimmunoassays.
  • the assay detected T1 / ST2 receptor binding protein 10.23 levels, it may be used as explained T1 / ST 2 receptor binding protein 23 10. importance in various diseases and for the diagnosis of T1 / ST2 receptor binding Protein 10. 23 Diseases at work.
  • 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 the T1 / ST2 receptor binding protein 10.23 can also be used for a variety of therapeutic purposes.
  • Gene therapy technology can be used to treat abnormal cell proliferation, development or metabolism caused by the non-expression or abnormal / inactive expression of T1 / ST2 receptor binding protein 10.23.
  • Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated T1 / ST2 receptor-binding protein 10.23 to inhibit endogenous T1 / ST2 receptor-binding protein 10.23 activity.
  • a variation of T1 / ST2 receptor binding protein may be a shortened 10.23, deletion of T1 / ST2 signaling receptor binding domain of protein 10.23, although the substrate may be combined with the downstream, but the lack of Signaling activity. Therefore, the recombinant gene therapy vector can be used to treat diseases caused by abnormal expression or activity of T1 / ST2 receptor binding protein 10.23.
  • 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 T1 / ST2 receptor binding protein 10.23 into a cell.
  • a method for constructing a recombinant viral vector carrying a polynucleotide encoding a T1 / ST2 receptor binding protein 10.23 can be found in the literature (Sambrook, et al.).
  • a polynucleotide encoding the T1 / ST2 receptor binding protein 10.23 can be packaged into liposomes and transferred into cells.
  • Methods for introducing a polynucleotide into a tissue or cell include: directly injecting the polynucleotide into a tissue in vivo; or introducing the polynucleotide into a cell in vitro through a vector (such as a virus, phage, or plasmid), and then transplanting the cell Into the body and so on.
  • a vector such as a virus, phage, or plasmid
  • Oligonucleotides including antisense RM and DNA
  • ribozymes that inhibit the T1 / ST2 receptor binding protein 10.23 mRNA are also within the scope of the present invention.
  • a ribozyme is an enzyme-like RNA molecule that can specifically decompose a specific RNA. Its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target RNA for endonucleation.
  • Antisense RNA, DNA, and ribozymes can be obtained by any existing RNA or DNA synthesis technology, such as the technology for the synthesis of oligonucleotides by solid-phase phosphoramidite chemical synthesis, which is widely used.
  • Antisense RNA molecules can be encoded by The DNA sequence of the RNA is obtained by transcription in vitro or in vivo. This DNA sequence has been integrated downstream of the vector's RNA polymerase promoter. In order to increase the stability of a nucleic acid molecule, it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the ribonucleoside linkages should use phosphate thioester or peptide bonds instead of phosphodiester bonds.
  • the polynucleotide encoding the T1 / ST2 receptor binding protein 10.23 can be used for the diagnosis of diseases related to the T1 / ST2 receptor binding protein 10.23.
  • the polynucleotide encoding T1 / ST2 receptor binding protein 10.23 can be used to detect the expression of T1 / ST2 receptor binding protein 10.23 or the abnormal expression of T1 / ST2 receptor binding protein 10.23 in a disease state.
  • the DNA sequence encoding T1 / ST2 receptor binding protein 10.23 can be used to hybridize biopsy specimens to determine the expression of T1 / ST2 receptor binding protein 10.23.
  • Hybridization techniques include Southern blotting, Northern blotting, and in situ hybridization.
  • a part or all of the polynucleotides of the present invention can be used as probes to be fixed on a microarray (Microarray) or a DM chip (also known as
  • T1 / ST2 receptor binding protein 10.23 specific primers can be used to perform in vitro amplification of RNA-polymerase chain reaction (RT-PCR) to detect the transcription products of T1 / ST2 receptor binding protein 10.23.
  • RT-PCR RNA-polymerase chain reaction
  • T1 / ST2 receptor binding protein 10.23 Detection of mutations in the T1 / ST2 receptor binding protein 10.23 gene can also be used to diagnose T1 / ST2 receptor binding protein 10.23-related diseases.
  • T1 / ST2 receptor binding protein 10.23 mutations include point mutations, translocations, deletions, rearrangements, and any other abnormalities compared to the normal wild-type T1 / ST2 receptor binding protein 10.23 DNA sequence. Mutations can be detected using existing techniques such as Southern blotting, DNA sequence analysis, PCR and in situ hybridization. In addition, mutations may affect protein expression, so Northern blotting and Western blotting can be used to indirectly determine whether a gene is mutated.
  • sequences of the invention are also valuable for chromosome identification. This sequence will specifically target a specific position on a human chromosome and can hybridize to it. Currently, specific sites for each gene on the chromosome need to be identified. Currently, only a few chromosome markers based on actual sequence data (repeating polymorphisms) are available for marking chromosome positions. According to the present invention, in order to associate these sequences with disease-related genes, an important first step is to locate these DNA sequences on a chromosome.
  • a PCR primer (preferably 15-35bp) is prepared from the cDNA, and the sequence can be located on the chromosome. These primers were then used for PCR screening of somatic hybrid cells containing individual human chromosomes. Only those hybrid cells that contain the human gene corresponding to the primer will produce amplified fragments.
  • PCR localization of somatic hybrid cells is a quick way to localize DNA to specific chromosomes.
  • oligonucleotide primers of the present invention by a similar method, a set of fragments from a specific chromosome or a large number of genomic clones can be used to achieve sublocalization.
  • Other similar strategies that can be used for chromosomal localization include in situ Hybridization, pre-screening of chromosomes using labeled flow sorting, and pre-selection of hybridization, thereby constructing a chromosome-specific cDNA library.
  • Fluorescent in situ hybridization of cDNA clones with metaphase chromosomes allows precise chromosomal localization in one step.
  • FISH Fluorescent in situ hybridization
  • the physical location of the sequence on the chromosome can be correlated with the genetic map data. These data can be found in, for example, V. Mckusick, Mendelian Inherance in Man (available online with Johns Hopkins University Welch Medical Library). Linkage analysis can then be used to determine the relationship between genes and diseases that are mapped to chromosomal regions.
  • the difference in cDNA or genomic sequence between the affected and unaffected individuals needs to be determined. If a mutation is observed in some or all diseased individuals and the mutation is not observed in any normal individuals, the mutation may be the cause of the disease. Comparing affected and unaffected individuals usually involves first looking for structural changes in the chromosomes, such as deletions or translocations that are visible at the chromosomal level or detectable with cDM sequence-based PCR. According to the resolution capabilities of current physical mapping and gene mapping technology, the cDNA accurately mapped to the chromosomal region associated with the disease can be one of 50 to 500 potentially pathogenic genes (assuming 1 megabase mapping resolution) Capacity and each 20kb corresponds to a gene).
  • the polypeptides, polynucleotides and mimetics, agonists, antagonists and inhibitors of the present invention can be used in combination with a suitable pharmaceutical carrier.
  • suitable pharmaceutical carrier can be water, glucose, ethanol, salts, buffers, glycerol, and combinations thereof.
  • the composition comprises a safe and effective amount of the polypeptide or antagonist, and carriers and excipients which do not affect the effect of the drug. These compositions can be used as drugs for the treatment of diseases.
  • the 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.
  • T1 / ST2 receptor binding protein 10.23 is administered in an amount effective to treat and / or prevent a specific indication.
  • the amount and range of T1 / ST2 receptor binding protein 10.23 administered to a patient will depend on many factors, such as the mode of administration, the health conditions of the person to be treated, and the judgment of the diagnostician. Examples
  • Total human fetal brain RNA was extracted by one-step method with guanidine isothiocyanate / phenol / chloroform.
  • Poly (A) mRNA was isolated from total RM using Quik mRNA I solat ion Kit (product of Qiegene). 2ug poly (A) mRNA is reverse transcribed to form cDNA.
  • a Smart cDNA cloning kit (purchased from Clontech) was used to insert the cDNA fragments into the multi-cloning site of the pBSK (+) vector (Clontecli) to transform DH5 ⁇ to form a CDM library.
  • RNA of fetal brain cells was used as a template, and ol igo-dT was used as a primer to perform a reverse transcription reaction to synthesize cDNA.
  • PCR amplification was performed with the following primers:
  • Primer 1 5,-TATAAAAATGCACAAGTCTCTCTA -3, (SEQ ID NO: 3)
  • Priraer2 5'- TTTTTTCATATTTTTAATTACCAT -3, (SEQ ID NO: 4)
  • Pr iraerl is a forward sequence located at the 5th end of SEQ ID NO: 1, starting at lbp;
  • Primer 2 is the 3, terminal reverse sequence of SEQ ID NO: 1.
  • Amplification reaction conditions 50 mmol / L KC1, 10 mmol / L Tris-HCl, pH 8.5, 1.5 mraol / L MgCl 2 , 200 ⁇ 1 / 1 dNTP, lOpmol primer, 1U Taq in a 50 ⁇ 1 reaction volume DNA polymerase (Clontech).
  • the reaction was performed on a PE9600 DNA thermal cycler (Perkin-Elmer) under the following conditions for 25 cycles: 94 ° C 30sec; 55. C 30sec; 72 ° C 2min 0 During RT-PCR, ⁇ -act in was set as a positive control and template blank was set as a negative control.
  • Amplification products were purified using QIAGEN kits and TA The cloning kit was ligated to a PCR vector (Invitrogen). DM sequence analysis results showed that the DNA sequence of the PCR product was exactly the same as l-3031bp shown in SEQ ID NO: 1.
  • Example 3 Northern blot analysis of T1 / ST2 receptor binding protein 10.23 gene expression Total RNA was extracted in one step [Anal. Biochem 1987, 162, 156-159] 0 This method involves acid guanidine thiocyanate phenol-chloroform extraction .
  • the tissue was homogenized with 4M guanidine isothiocyanate-25raM sodium citrate, 0.2M sodium acetate (pH4.0), and 1 volume of phenol and 1/5 volume of chloroform-isoamyl alcohol (49: 1 ), Mix and centrifuge. The aqueous layer was aspirated, isopropanol (0.8 vol) was added and the mixture was centrifuged to obtain RNA precipitate. The resulting RNA pellet was washed with 70% ethanol, dried and dissolved in water.
  • a 32P-labeled probe (about 2 x 10 6 cpm / ml) was hybridized with a nitrocellulose membrane to which RNA was transferred at 42 ° C overnight in a solution containing 50% formamide-25raM KH 2 P0 4 (H7.4) - 5 ⁇ SSC- 5 x Denhardt's solution and 200 ⁇ £ / ⁇ 1 salmon sperm DNA. After hybridization, the filter was washed in 1 x SSC-G.1% SDS at 55 ° C for 30 min. Then, Phosphor Imager was used for analysis and quantification.
  • Example 4 In vitro expression, isolation and purification of recombinant 1/572 receptor binding protein 10.23 According to the sequence of the coding region shown in SEQ ID NO: 1 and Figure 1, a pair of specific amplification primers were designed, the sequence is as follows:
  • Primer3 5,-CATGCTAGCATGAGGAAAGAGATTGAGACTGAA- 3, (Seq ID No: 5)
  • Primer4 5,-CATGGATCCTTACTCTGCTGCCCCTTTTATTATT- 3, (Seq ID No: 6)
  • the 5 'ends of these two primers contain Nhel and BamHI restriction sites, respectively.
  • the coding sequences of the 5 'and 3' ends of the target gene are followed, respectively.
  • the Nhel and BamHI restriction sites correspond to the selectivity on the expression vector plasmid pBT-28 (+) (Novagen, Cat. No. 69 5.3). Endonuclease site.
  • PCR was performed using the PBS-0466c03 plasmid containing the full-length target gene as a template.
  • the PCR reaction conditions were as follows: a total volume of 50 ⁇ 1 containing 10 pg of pBS-0466c03 plasmid, primers Primer-3 and Primer-4 were lOpmol, Advantage polymerase Mix (Clontech) 1 ⁇ 1, respectively.
  • Cycle parameters 94. C 20s, 60 ° C 30s, 68 ° C 2 min, a total of 25 cycles.
  • Nhel and BamHI were used to double-digest the amplified product and plasmid pET-28 (+), respectively, and large fragments were recovered and ligated with T4 ligase. The ligated product was transformed into E.
  • coli DH5a by the calcium chloride method. After being cultured overnight on LB plates containing kanamycin (final concentration 3 ⁇ g / ml), positive clones were selected by colony PCR method and sequenced. Select positive clones with correct sequence (pET-0466c03) Granules were transformed into E. coli BL21 (DE3) plySs (product of Novagen). In containing kanamycin (final concentration of 30 ⁇ 8 / ⁇ 1) in LB liquid medium, host strain BL21 (P ET-0466c03) were cultured to logarithmic growth phase, IPTG was added to a final concentration lramol / L at 37 ° C, Continue incubation for 5 hours.
  • a peptide synthesizer (product of PE company) was used to synthesize the following T1 / ST2 receptor binding protein 10.23-specific peptides:
  • Suitable oligonucleotide fragments selected from the polynucleotides of the present invention are used as hybridization probes in a variety of ways.
  • the probes can be used to hybridize to genomic or cDNA libraries of normal tissue or pathological tissue from different sources to It is determined whether it contains the polynucleotide sequence of the present invention and a homologous polynucleotide sequence is detected.
  • the probe can be used to detect the polynucleotide sequence of the present invention or its homologous polynucleotide sequence in normal tissue or pathology. Whether the expression in tissue cells is abnormal.
  • the purpose of this embodiment is to select a suitable oligonucleotide fragment from the polynucleotide SEQ ID NO: 1 of the present invention as a hybridization probe, and to identify whether some tissues contain the polynucleoside of the present invention by using a filter hybridization method.
  • Filter membrane hybridization methods include dot blotting, Southern blotting, Northern blotting, and copying methods, etc., all of which fix the polynucleotide sample to be tested on the filter The membranes were hybridized using essentially the same procedure.
  • the sample-immobilized filter is first pre-hybridized with a probe-free hybridization buffer, so that the non-specific binding site of the sample on the filter is saturated with the carrier and the synthetic polymer.
  • the pre-hybridization solution is then replaced with a hybridization buffer containing the labeled probe and incubated to hybridize the probe to the target nucleic acid.
  • the unhybridized probes are removed by a series of membrane washing steps.
  • This embodiment utilizes higher-intensity washing conditions (such as lower salt concentration and higher temperature) to reduce the hybridization background and retain only strong specific signals.
  • the probes used in this embodiment include two types: the first type of probes are oligonucleotide fragments that are completely the same as or complementary to the polynucleotide SEQ ID NO: 1 of the present invention; the second type of probes are partially related to the present invention
  • the polynucleotide SEQ ID NO: 1 is the same or complementary oligonucleotide fragment.
  • the dot blot method is used to fix the sample on the filter membrane. Under the high-intensity washing conditions, the first type of probe and the sample have the strongest hybridization specificity and are retained.
  • oligonucleotide fragments from the polynucleotide SEQ ID NO: 1 of the present invention for use as hybridization probes should follow the following principles and several aspects to be considered:
  • the preferred range of probe size is 18-50 nucleotides
  • Those that meet the above conditions can be used as primary selection probes, and then further computer sequence analysis, including the primary selection probe and its source sequence region (ie, SEQ ID NO: 1) and other unknown genomic sequences and their complements The regions are compared for homology. If the homology with the non-target molecular region is greater than 85% or there are more than 15 consecutive bases, the primary probe should not be used;
  • Probe 1 which belongs to the first type of probe, is completely homologous or complementary to the gene fragment of SEQ ID NO: 1 (machine):
  • Probe 2 which belongs to the second type of probe, is equivalent to the replacement mutation sequence (41Nt) of the gene fragment of SEQ ID NO: 1 or its complementary fragment:
  • PBS phosphate buffered saline
  • step 8-13 are only used when contamination must be removed, otherwise step 14 can be performed directly.
  • NC membranes nitrocellulose membranes
  • Two NC membranes are required for each probe, so that they can be used in the following experimental steps.
  • the film was washed with high-strength conditions and strength conditions, respectively.
  • the sample membrane was placed in a plastic bag, and 3-10 mg of prehybridization solution (lOxDenhardfs; 6xSSC, 0.1 mg / ml CT DNA (calf thymus MA)) was added. After sealing the mouth of the bag, shake at 68 ° C for 2 hours.
  • prehybridization solution lOxDenhardfs; 6xSSC, 0.1 mg / ml CT DNA (calf thymus MA)
  • Gene chip or gene micro matrix (DNA Mi croarray) is a new technology that many national laboratories and large pharmaceutical companies are currently developing and developing. It refers to the orderly and high density arrangement of a large number of target gene fragments on glass. , Silicon and other carriers, and then use fluorescence detection and computer software to compare and analyze the data, in order to achieve the purpose of rapid, efficient, high-throughput analysis of biological information.
  • the polynucleotide of the present invention can be used as target DNA for gene chip technology for high-throughput research of new gene functions; search for and screen new tissue-specific genes, especially new genes related to diseases such as tumors; diagnosis of diseases such as hereditary diseases .
  • a total of 4,000 polynucleotide sequences of various full-length cDNAs as target DNA, including the present invention Polynucleotide. They were respectively amplified by PCR. After the purified amplified product was purified, the concentration was adjusted to about 500 ng / ul, and a Cartesian 7500 spotter (purchased from Cartesian Corporation, USA) was used to spot the glass medium. The distance between them is 280 ⁇ . The spotted slides were hydrated, dried, and cross-linked in a UV cross-linker. After elution, the slides were fixed to fix the DM on the glass slides to prepare chips. The specific method steps have been reported in the literature. The sample post-processing steps in this embodiment are:
  • Total mRNA was extracted from human mixed tissues and specific tissues (or stimulated cell lines) by one-step method, and the mRNA was purified with Oligotex mRNA Midi Kit (purchased from QiaGen), and the fluorescent reagents were separately reverse-transcribed Cy3dUTP (5-Araino-propargyl-2'-deoxyuridine 5 '-tr iphate coupled to Cy3 f luorescent dye, purchased from Amersham Phamacia Biotech) was used to label the mRNA of human mixed tissue, and the fluorescent reagent Cy5dUTP (5-Amino- propargyl- 2 '-deoxyur idine 5--triphate coupled to C 5 f luorescent dye, purchased from Amersham Phamacia Biotech Company, labeled the body's specific tissue (or stimulated cell line) mRNA, and purified the probe to prepare a probe.
  • Cy3dUTP 5-Araino-propargyl-2'-deoxyuridine 5
  • the probes from the two types of tissues and the chip were hybridized in a UniHyb TM Hybridization Solution (purchased from TeleChem) hybridization solution for 16 hours, and a washing solution (1 ⁇ SSC, 0.2% SDS) was used at room temperature. After washing, scanning was performed with a ScanArray 3000 scanner (purchased from Genera Scanning, USA). The scanned images were analyzed and processed with Iraagene software (Biodicovery, USA) to calculate the Cy3 / Cy5 ratio of each point.
  • the above specific tissues are fetal brain, bladder mucosa, PMA + Ecv304 cell line, LPS + Ecv304 cell line thymus, normal fibroblasts 1024NC, Fibroblas t, Growth factor stimulation, 1024NT, scar-to-fc growth factor stimulation, 1013HT, scar-to-fc growth factor stimulation, 1013HC, bladder cancer cell EJ, bladder cancer, bladder cancer, liver cancer, liver cancer cell line, fetal skin, spleen , Prostate cancer, jejunum adenocarcinoma, cardia cancer. Based on these 18 Cy3 / Cy5 ratios, a histogram is drawn (Figure 1). It can be seen from the figure that the expression profile of T1 / ST2 receptor binding protein 10.23 and 1/3/3 receptor binding protein according to the present invention are very similar.

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Abstract

L'invention concerne un nouveau polypeptide, une protéine 10.23 associée au récepteur T1/ST2, et un polynucléotide codant ce polypeptide ainsi qu'un procédé d'obtention de ce polypeptide par des techniques recombinantes d'ADN. L'invention concerne en outre les applications de ce polypeptide dans le traitement de maladies, notamment de troubles immunitaires, d'inflammations et de tumeurs. L'invention concerne aussi l'antagoniste agissant contre le polypeptide et son action thérapeutique ainsi que les applications de ce polynucléotide codant la protéine 10.23 associée au récepteur T1/ST2.
PCT/CN2001/001075 2000-06-30 2001-06-29 Nouveau polypeptide, proteine 10.23 associee au recepteur t1/st2, et polynucleotide codant ce polypeptide WO2002012487A1 (fr)

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CN 00116928 CN1331111A (zh) 2000-06-30 2000-06-30 一种新的多肽——t1/st2受体结合蛋白10.23和编码这种多肽的多核苷酸
CN00116928.9 2000-06-30

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CN104059130B (zh) * 2014-06-27 2016-08-24 王方杰 关于st2蛋白抑制剂多肽及其应用
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Non-Patent Citations (3)

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Title
DATABASE PROTEIN [online] 1 April 1996 (1996-04-01), GAYLE M.A. ET AL., retrieved from GI:1223890 accession no. NCBI Database accession no. AAC50419 *
DATABASE PROTEIN [online] 26 November 1996 (1996-11-26), GAYLE M.A. ET AL., retrieved from GI:1589142 accession no. NCBI Database accession no. 1589142 *
DATABASE PROTEIN [online] 6 April 1999 (1999-04-06), LAMERDIN J.E. ET AL., retrieved from GI:4567176 accession no. NCBI Database accession no. AAD23605 *

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