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WO2001070781A1 - Nouveau polypeptide, facteur humain d'activation 13 de la liaison (ilf) entre leucocytes, et polynucleotide codant pour ce polypeptide - Google Patents

Nouveau polypeptide, facteur humain d'activation 13 de la liaison (ilf) entre leucocytes, et polynucleotide codant pour ce polypeptide Download PDF

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Publication number
WO2001070781A1
WO2001070781A1 PCT/CN2001/000173 CN0100173W WO0170781A1 WO 2001070781 A1 WO2001070781 A1 WO 2001070781A1 CN 0100173 W CN0100173 W CN 0100173W WO 0170781 A1 WO0170781 A1 WO 0170781A1
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Prior art keywords
polypeptide
polynucleotide
human interleukin
binding
sequence
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PCT/CN2001/000173
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English (en)
Chinese (zh)
Inventor
Yumin Mao
Yi Xie
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Shanghai Biowindow Gene Development Inc.
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Priority to AU39114/01A priority Critical patent/AU3911401A/en
Publication of WO2001070781A1 publication Critical patent/WO2001070781A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention belongs to the field of biotechnology. Specifically, the present invention describes a new polypeptide, human interleukin binding promoting factor 13, and a polynucleotide sequence encoding the polypeptide. The invention also relates to methods and applications for preparing such polynucleotides and polypeptides.
  • Interleukin-binding promoting factor is a 60Kd protein, which is a typical representative of regulating viral gene expression. Interleukin-binding promoting factor also contains some functional regions, nuclear localization, specific binding of nucleosides and N -The glycosylation process comes into play. In addition, the interleukin-binding promoter also contains a 9-amino acid domain, which is also found in cyclins and can mediate the ubiquitin degradation of proteins (Glotzer, M., Murray, AW et al., 1991). It can be seen that the activation of T lymphocytes can cause the degradation of interleukin binding-promoting factor proteins, along with triggering the binding of other cellular proteins such as T-cell nuclear factor (NFAT) to purine-rich regions, and enhance gene expression.
  • T-cell nuclear factor NFAT
  • HIV gene expression depends on a variety of cellular transcription factors, many of which are located on the long terminal repeat (LTR) of HIV, including SP1, TATA, and TAR that regulate gene expression in different cell lines; in addition, At least two regulatory sites are important for HIV LTR gene expression and T lymphocyte activation.
  • LTR long terminal repeat
  • SP1, TATA, and TAR that regulate gene expression in different cell lines
  • At least two regulatory sites are important for HIV LTR gene expression and T lymphocyte activation.
  • One is the -103-78 region, which contains two NF- ⁇ domains (Nabel, G. et al.
  • interleukins play an important regulatory role in the autoimmune process of organisms, and they activate various T cells and B cells in the body to remove various foreign harmful substances.
  • the interleukin binding promoting factor is involved in regulating the transcription and expression of various interleukins in vivo.
  • the human interleukin-binding promoting factor 1 3 protein plays an important role in regulating important functions of the body such as cell division and fetal development, and it is believed that a large number of proteins are involved in these regulatory processes, so identification in the art has been required. More human interleukin-binding promoting factor 13 proteins are involved in these processes, and in particular the amino acid sequence of this protein is identified. Isolation of newcomer interleukin-binding promoting factor 13 protein-coding genes also provides a basis for research to determine the role of this protein in health and disease states. This protein may form the basis for the development of diagnostic and / or therapeutic drugs for diseases, so it is important to isolate its coding 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 human interleukin binding promoting factor 1 3.
  • Another object of the present invention is to provide a genetically engineered host cell comprising a polynucleotide encoding a human interleukin-binding promoting factor 13.
  • Another object of the present invention is to provide mimic compounds, antagonists, agonists, and inhibitors directed to the polypeptide of the present invention, human interleukin-binding promoter 13.
  • Another object of the present invention is to provide a method for diagnosing and treating diseases associated with abnormalities of human interleukin-binding promoting factor 13. Summary of invention
  • the present invention relates to an isolated polypeptide, which is of human origin, and includes: a polypeptide having the amino acid sequence of SEQ ID D. 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 787-1 1 14 in SEQ ID NO: 1; and (b) having a sequence 1 in SEQ ID NO: 1 -1505 bit sequence.
  • 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 present invention also relates to a method for screening compounds that mimic, activate, antagonize or inhibit the activity of human interleukin binding promoting factor 13 protein, which comprises utilizing the polypeptide of the present invention.
  • the invention also relates to compounds obtained by this method.
  • the invention also relates to a method for detecting a disease or disease susceptibility related to abnormal expression of human interleukin-binding promoting factor 13 protein in vitro, which comprises detecting a mutation in the polypeptide or a coding polynucleotide sequence thereof in a biological sample, Alternatively, the amount or biological activity of a polypeptide of the invention in a biological sample is detected.
  • 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 preparation of polypeptides and / or polynucleotides of the invention for the treatment of HIV infection, immunodeficiency diseases, major histocompatibility antigen-related diseases, blood transfusion reactions, transplant immune rejection reactions, inflammation, and various tumors.
  • FIG. 1 is a comparison diagram of gene chip expression profiles of human interleukin-binding promoting factor 13 and human interleukin-binding promoting factor 2 according to the present invention.
  • the upper graph is a graph of the expression profile of human interleukin-binding promoting factor 13 and the lower graph is the graph of the expression profile of human interleukin-binding promoting factor 2.
  • Figure 2 shows the polyacrylamide gel electrophoresis (SDS-PAGE) of the isolated human interleukin-binding promoting factor 13.
  • 1 3KDa 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 “variant" of a protein or polynucleotide refers to an amino acid sequence having one or more amino acids or nucleotide changes or a polynucleotide sequence encoding it.
  • the changes may include deletions, insertions or substitutions of amino acids or nucleotides in the amino acid sequence or nucleotide sequence.
  • Variants can have "conservative" changes, in which the amino acid substituted has a structural or chemical property similar to the original amino acid, such as replacing isoleucine with leucine.
  • Variants can also have non-conservative changes, such as replacing glycine with tryptophan.
  • “Deletion” refers to the deletion of one or more amino acids or nucleotides in an amino acid sequence or nucleotide sequence.
  • “Insertion” or “addition” 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” means the replacement of one or more amino acids or nucleotides with different amino acids or nucleotides.
  • Bioactivity refers to a protein that has the structure, regulation, or biochemical function of a natural molecule.
  • immunologically active refers to the ability of natural, recombinant or synthetic proteins and fragments thereof to induce a specific immune response and to bind specific antibodies in a suitable animal or cell.
  • An "agonist” refers to a molecule that, when combined with human interleukin-binding promoting factor 13, 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 human interleukin binding promoting factor 13.
  • Antagonist or “inhibitor” means that when bound to human interleukin binding promoting factor 13, A molecule that can block or regulate the biological or immunological activity of human interleukin-binding promoting factor 13. Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates, or any other molecule that can bind to human interleukin-binding promoting factor 13.
  • Regular refers to a change in the function of human interleukin-binding promoting factor 13, including an increase or decrease in protein activity, a change in binding characteristics, and any other biological properties, functions, or immunity of human interleukin-binding promoting factor 13. Change of nature.
  • Substantially pure ' means substantially free of other proteins, lipids, carbohydrates or other substances with which it is naturally associated.
  • Those skilled in the art can purify human interleukin-binding promoting factor 13 using standard protein purification techniques.
  • Substantially pure human interleukin-binding promoter 13 can produce a single main band on a non-reducing polyacrylamide gel. The purity of the human interleukin-binding promoter 13 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 by the MEGALIGN program (Lasergene software package, DNASTAR, Inc., Madison Wis.). The MEGALIGN program can compare two or more sequences according to different methods such as the Cluster method (Higgins, DG and PM Sharp (1988) Gene 73: 237-244). The Cluster method arranges the groups of sequences by checking the distance between all pairs Into clusters. The clusters are then assigned in pairs or groups. The percent identity between two amino acid sequences such as sequence A and sequence B is calculated by the following formula:
  • the percent identity between nucleic acid sequences can also be determined by the Cluster method or by methods known in the art such as Jotun Hein (Hein, (1990) Methods in enzymology 183: 625-645). 0 "Similarity” refers to the degree of identical or conservative substitutions of amino acid residues at corresponding positions in the alignment of amino acid sequences.
  • Amino acids used for conservative substitution 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 RM 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 an intact antibody molecules and fragments thereof, such as Fa, F (a b ') 2 and F V, which specifically binds human interleukin-13 binding promoting factor antigen determinants.
  • a “humanized antibody” refers to an antibody in which the amino acid sequence of a non-antigen binding region is replaced to become more similar to a human antibody, but still retains the original binding activity.
  • isolated refers to the removal of matter from its original environment (for example, its natural environment if it is naturally occurring).
  • a naturally occurring polynucleotide or polypeptide is not isolated when it is present in a living 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 vector, or such a polynucleotide or polypeptide may be part of a 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 human interleukin-binding promoting factor 1 3 means that human interleukin-binding promoting factor 1 3 is substantially free of other proteins, lipids, sugars, or other substances with which it is naturally associated.
  • Those skilled in the art can purify human interleukin-binding promoting factor 13 using standard protein purification techniques. Substantially pure polypeptides can produce a single main band on a non-reducing polyacrylamide gel. The purity of the human interleukin-binding promoting factor 13 peptide can be analyzed by amino acid sequence.
  • the present invention provides a new polypeptide, human interleukin binding promoting factor 13, 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 chemical synthesis Or from recombinant cells using prokaryotic or eukaryotic hosts (such as 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 human interleukin-binding promoter 13.
  • fragment refers to a polypeptide that substantially maintains the same biological function or activity of the human interleukin-binding promoter 13 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 (II) such a type in which a group on one or more amino acid residues is substituted by another group to include a substituent; or (III) such a Species, 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) a polypeptide sequence in which an additional amino acid sequence is fused into a mature polypeptide (such as Leader sequence or secretory sequence or the sequence or protease sequence used to purify this polypeptide).
  • conservative amino acid residues preferably conservative amino acid residues
  • 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 with a total length of 1505 bases, and its open reading frame 787-1143 encodes 118 amino acids.
  • the polypeptide has a similar expression profile with human interleukin-binding promoting factor 2, and it can be deduced that the human interleukin-binding promoting factor 13 has a similar function as human interleukin-binding promoting factor 2.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • DNA forms include cDNA, genomic DNA, or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be coding or non-coding.
  • the coding region sequence encoding a mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO: 1 or a degenerate variant.
  • a "degenerate variant" refers to a nucleic acid sequence encoding a protein or polypeptide having SEQ ID NO: 2 but different from the coding region sequence shown in SEQ ID NO: 1 in the present invention.
  • the polynucleotide encoding the mature polypeptide of SEQ ID NO: 2 includes: only the coding sequence of the mature polypeptide; the coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence of the mature polypeptide (and optional additional coding sequences); Coding sequence.
  • 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, 0.1% SDS, 6 (TC; or (2) added during hybridization Use a denaturant, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42 ° C, etc .; or (3) the identity between the two sequences is at least 95% Above, it is more preferable that the hybridization occurs at 97% or more.
  • the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO: 2.
  • nucleic acid fragments that hybridize to the sequences described above.
  • a "nucleic acid fragment” contains at least 10 nucleotides in length, preferably at least 20-30 nucleotides, more preferably at least 50-60 nucleotides, and most preferably at least 100 cores. Glycylic acid or more. Nucleic acid fragments can also be used in nucleic acid amplification techniques, such as PCR, to identify and / or isolate polynucleotides encoding human interleukin-binding promoter 13.
  • polypeptides and polynucleotides in the present invention are preferably provided in an isolated form and are more preferably purified to homogeneity.
  • the specific polynucleotide sequence encoding the human interleukin-binding promoter 13 of the present invention can be obtained by various methods.
  • polynucleotides are isolated using hybridization techniques well known in the art. These techniques include, but are not limited to: 1) hybridization of probes to genomic or cDNA libraries to detect homologous polynucleotide sequences, and 2) antibody screening of expression libraries to detect cloned polynucleosides with common structural characteristics Acid fragments.
  • the DNA fragment sequence of the present invention can also be obtained by the following methods: 1) isolating the double-stranded DNA sequence from the genomic DNA; 2) chemically synthesizing the D N A sequence to obtain the double-stranded D N A of the polypeptide.
  • genomic DNA isolation is the least commonly used. Direct chemical synthesis of DNA sequences is often the method of choice. The more commonly used method is the isolation of cDNA sequences.
  • the standard method for isolating the cDNA of interest is to isolate mRNA from donor cells that overexpress the gene and perform reverse transcription to form a plasmid or phage cDNA library.
  • the construction of cDNA libraries is also a common method (Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory. New York, 1989).
  • Commercially available cDNA libraries are also available, such as different cDNA libraries from Clontech. When polymerase reaction technology is used in combination, even very small expression products can be cloned.
  • genes of the present invention can be selected from these cDNA libraries by conventional methods. These methods include (but are not limited to): (l) DNA-DNA or DM-RM hybridization; (2) the presence or absence of marker gene function; (3) determination of the level of human interleukin-binding promoter 13 transcripts; (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 usually a DNA sequence chemically synthesized based on the gene sequence information of the present invention.
  • the genes or fragments of the present invention can of course be used as probes.
  • DNA probes can be labeled with radioisotopes, luciferin, or enzymes (such as alkaline phosphatase).
  • immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA) can be used to detect the protein product of human interleukin-binding promoting factor 13 gene expression.
  • ELISA enzyme-linked immunosorbent assay
  • 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 for PCR can be appropriately based on the polynucleotide sequence information of the present invention disclosed herein Select and synthesize using conventional methods.
  • the amplified DNA / RNA fragments can be isolated and purified by conventional methods such as by gel electrophoresis.
  • polynucleotide sequence of the gene of the present invention or various DNA fragments and the like obtained as described above can be measured by a conventional method such as dideoxy chain termination method (Sanger et al. PNAS, 1977, 74: 5463-5467). Such polynucleotide sequences can also be determined using commercial sequencing kits and the like. In order to obtain the full-length cDNA sequence, sequencing needs to be repeated. Sometimes it is necessary to determine the cDNA sequence of multiple clones in order to splice into a full-length cDNA sequence.
  • the present invention also relates to a vector comprising a polynucleotide of the present invention, and a host cell produced by genetic engineering using the vector of the present invention or directly using a human interleukin-binding promoter 13 coding sequence, and the recombinant technology to produce the polypeptide of the present invention Methods.
  • a polynucleotide sequence encoding a human interleukin-binding promoting factor 13 may be inserted into The vector constitutes a recombinant vector containing the polynucleotide of the present invention.
  • the term "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 expressed in bacteria (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 expression vectors containing a DNA sequence encoding human interleukin-binding promoting factor 13 and appropriate transcription / translation 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 Spring Harbor Laboratory. New York, 1989).
  • the DNA sequence can be operably linked to an appropriate promoter in the expression vector to guide mRNA synthesis. Representative examples of these promoters are: the lac or trp promoter of E.
  • the expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. Insertion of enhancer sequences into the vector will enhance its transcription in higher eukaryotic cells. Enhancers are cis-acting factors for DNA expression, usually about 10 to 300 base pairs, which act on promoters to enhance gene transcription. Examples include 100 to 270 base pair SV40 enhancers 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 human interleukin binding promoting factor ⁇ or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to constitute a genetically engineered host cell containing the polynucleotide or the recombinant vector .
  • the term "host cell” refers to a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative example There are: E.
  • coli Streptomyces
  • bacterial cells such as Salmonella typhimurium
  • fungal cells such as yeast
  • plant cells such as fly S2 or Sf9
  • animal cells such as CH0, COS or Bowes melanoma cells.
  • Transformation of a host cell with a DNA sequence described in the present invention or a recombinant vector containing the DNA sequence can be performed using conventional techniques well known to those skilled in the art.
  • the host is a prokaryote such as E. coli
  • competent cells capable of DNA uptake can be harvested after exponential growth phase, with (: Treatment 1 2, steps well known in the art used alternative is to use MgCl 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 liposomes Packaging, etc.
  • polynucleotide sequences of the present invention can be used to express or produce recombinant human interleukin-binding promoter 13 by conventional recombinant DNA technology (Science, 1984; 224: 1431). Generally, the following steps are taken:
  • the medium used in the culture may be selected from various conventional mediums. Culture is performed under conditions suitable for host cell growth. After the host cells have grown to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.
  • a suitable method such as temperature conversion or chemical induction
  • the recombinant polypeptide may be coated in a cell, expressed on a cell membrane, or secreted outside the cell. If necessary, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. These methods include, but are not limited to: conventional renaturation treatment, protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • conventional renaturation treatment protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid
  • polypeptides of the present invention can be directly used in the treatment of diseases, for example, they can treat malignant tumors, adrenal deficiency, skin diseases, various types of inflammation, HIV infection, and immune diseases.
  • Interleukin plays an important regulatory role in the body's autoimmune process. It activates various related T cells and B cells in the body to remove various foreign harmful substances.
  • Interleukin-binding promoter (ILF) is involved in regulating the transcription and interaction of various interleukins in vivo. Expression. ILF is a typical representative of regulating viral gene expression. It can mediate the ubiquitin degradation of proteins. Studies have found that activation of T lymphocytes can cause the degradation of interleukin-binding promoting factor proteins, accompanied by the initiation of binding of other cellular proteins such as T-cell nuclear factor (NFAT) to purine-rich regions, and enhanced gene expression.
  • NFAT T-cell nuclear factor
  • IL-2 interleukin 2
  • interleukin-binding promoter gene can be altered to increase the amount of many different gene products.
  • the expression profile of the polypeptide of the present invention is consistent with the expression profile of human interleukin binding promoting factor, and the two have similar biological functions. It has a variety of important functions in the body, especially regulating the immune monitoring in the body such as the expression of H IV virus, and its abnormal expression is usually associated with various immune monitoring abnormalities, such as immunodeficiency diseases, major histocompatibility antigen-related diseases , Transfusion reaction, transplantation immune rejection, inflammation, tumor and cancer and other physiological and pathological processes are closely related.
  • the abnormal expression of the human interleukin-binding promoter 13 of the present invention will produce various diseases, especially immunodeficiency diseases, major histocompatibility antigen-related diseases, blood transfusion reactions, transplantation immune rejection reactions, inflammation, Tumors and cancers.
  • diseases include but are not limited to:
  • Immune Deficiency Diseases Acquired Immunodeficiency Syndrome, Common Variable Immunodeficiency Disease, Primary B Lymphocyte Immunodeficiency Disease, Primary T Lymphocyte Immunodeficiency Disease, Severe Combined Immunodeficiency Disease, Wi skot tA l dr i ch syndrome, with ataxia telangiectasia, primary phagocytic immunodeficiency disease, primary complement system deficiency
  • Tumors of various tissues stomach cancer, liver cancer, lung cancer, esophageal cancer, breast cancer, leukemia, lymphoma, thyroid tumor, uterine fibroids, neuroblastoma, astrocytoma, ependymoma, glioblastoma, nerve Fibroma, colon cancer, melanoma, bladder cancer, uterine cancer, endometrial cancer, thymic tumor, nasopharyngeal cancer, laryngeal cancer, tracheal tumor, fibroid, fibrosarcoma, lipoma, liposarcoma
  • Inflammation chronic active hepatitis, sarcoidosis, polymyositis, chronic rhinitis, chronic gastritis, cerebrospinal multiple sclerosis, glomerulonephritis, myocarditis, cardiomyopathy, atherosclerosis, gastric ulcer, cervicitis, Various infectious inflammations
  • the abnormal expression of the human interleukin-binding promoter 1 3 of the present invention will also produce certain hereditary, hematological diseases, and the like.
  • the polypeptide of the present invention and the antagonists, agonists and inhibitors of the polypeptide can be directly used in the treatment of diseases, for example, it can treat various diseases, especially blood transfusion reactions, transplantation immune rejection reactions, major histocompatibility antigen-related diseases, other Immune diseases, various tumors, inflammation, certain hereditary, blood diseases, etc.
  • the invention also provides methods for screening compounds to identify agents that increase (agonist) or suppress (antagonist) human interleukin-binding promoter 13.
  • Agonists enhance human interleukin-binding promoter 13 to stimulate biological functions such as cell proliferation, while antagonists prevent and treat disorders related to excessive cell proliferation, such as various cancers.
  • a mammalian cell or a membrane preparation expressing human interleukin-binding promoter 13 can be cultured together with a labeled human interleukin-binding promoter 13 in the presence of a drug. The ability of the drug to increase or block this interaction is then determined.
  • Antagonists of human interleukin-binding promoter 13 include selected antibodies, compounds, receptor deletions, and the like. Antagonists of human interleukin-binding promoting factor 1 3 can bind to human interleukin-binding promoting factor 1 3 and eliminate its function, or inhibit the production of the polypeptide, or bind to the active site of the polypeptide to make the polypeptide Cannot perform biological functions.
  • human interleukin-binding promoting factor 1 3 can be added to a bioanalytical assay by measuring the effect of the compound on the interaction between human interleukin-binding promoting factor 13 and its receptor. Determine if the compound is an antagonist.
  • Receptor deletions and analogs that act as antagonists can be screened in the same way as for screening compounds described above.
  • Polypeptide molecules capable of binding to human interleukin binding promoter 1 3 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 human interleukin-binding promoter 13 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 present invention also provides antibodies against human interleukin-binding promoting factor 13 epitopes. These antibodies include (but are not limited to): polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, and fragments produced by Fab expression libraries.
  • Polyclonal antibodies can be produced by injecting human interleukin-binding promoting factor 13 directly into immunized animals (such as rabbits, mice, rats, etc.).
  • immunized animals such as rabbits, mice, rats, etc.
  • a variety of adjuvants can be used to enhance the immune response, including but not limited to 'S adjuvant and so on.
  • Techniques for preparing monoclonal antibodies to human interleukin-binding promoting factor 1 3 include, but are not limited to, hybridoma technology (Kohler and Miste in. Nature, 1975, 256: 495- 497), triple tumor technology, human B-cell hybridoma technology, EBV-hybridoma technology, etc.
  • Chimeric antibodies that bind human constant regions to non-human variable regions can be produced using existing techniques (Morrison et al, PNAS, 1985, 81: 6851).
  • the existing technology for producing single chain antibodies (US Pat No. 4946778) can also be used to produce single chain antibodies against human interleukin-binding promoting factor 13.
  • Antibodies against human interleukin-binding promoting factor 13 can be used in immunohistochemical techniques to detect human interleukin-binding promoting factor 13 in biopsy specimens.
  • Monoclonal antibodies that bind to human interleukin-binding promoter 13 can also be labeled with radioisotopes and injected into the body to track their location and distribution. This radiolabeled antibody can be used as a non-invasive diagnostic method to locate tumor cells and determine whether there is metastasis.
  • Antibodies can also be used to design immunotoxins that target a particular part of the body.
  • human interleukin binding promoting factor 13 High-affinity monoclonal antibodies can covalently bind to bacterial or plant toxins (such as diphtheria toxin, ricin, ormosine, etc.).
  • a common method is to attack the amino group of an antibody with a thiol cross-linking agent such as SPDP and bind the toxin to the antibody through the exchange of disulfide bonds.
  • This hybrid antibody can be used to kill human IL-13-positive Cell.
  • the antibodies of the present invention can be used to treat or prevent diseases related to human interleukin-binding promoting factor 13.
  • Administration of an appropriate dose of antibody can stimulate or block the production or activity of human interleukin-binding promoting factor 13.
  • the invention also relates to a diagnostic test method for quantitatively and locally detecting the level of human interleukin-binding promoting factor 13.
  • tests are well known in the art and include FISH assays and radioimmunoassays.
  • the level of human interleukin-binding promoter 13 detected in the test can be used to explain the importance of human interleukin-binding promoter 13 in various diseases and to diagnose the role of human interleukin-binding promoter 13 disease.
  • polypeptide of the present invention can also be used for peptide mapping analysis.
  • the polypeptide can be specifically cleaved by physical, chemical or enzymatic analysis, and subjected to one-dimensional or two-dimensional or three-dimensional gel electrophoresis analysis, more preferably mass spectrometry analysis.
  • the polynucleotide encoding human interleukin binding promoting factor 13 can also be used for a variety of therapeutic purposes. Gene therapy technology can be used to treat abnormal cell proliferation, development or metabolism caused by the non-expression or abnormal / inactive expression of human interleukin-binding promoting factor 13.
  • Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated human interleukin-binding promoter 13 to inhibit endogenous human interleukin-binding promoter 13 activity.
  • a variant human interleukin-binding promoting factor ⁇ may be shortened and lack human signaling interleukin-binding promoting factor 13. Although it can bind to downstream substrates, it lacks signaling activity.
  • the recombinant gene therapy vector can be used to treat diseases caused by abnormal expression or activity of human interleukin-binding promoter 13.
  • Source Viral expression vectors such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus and the like can be used to transfer a polynucleotide encoding human interleukin-binding promoter 13 into a cell.
  • a method for constructing a recombinant viral vector carrying a polynucleotide encoding a human interleukin-binding promoting factor 13 can be found in the existing literature (Sambrook, et al.).
  • a recombinant polynucleotide encoding human interleukin-binding promoter 13 can be packaged into liposomes and transferred into cells.
  • Methods for introducing a polynucleotide into a tissue or cell include: directly injecting the polynucleotide into a tissue in vivo; or introducing the polynucleotide into a cell in vitro through a vector (such as a virus, phage, or plasmid), and then transplanting the cell Into the body and so on.
  • a vector such as a virus, phage, or plasmid
  • Oligonucleotides including antisense RNA and DNA
  • ribozymes that inhibit human interleukin-binding promoter 13 mRNA are also within the scope of the present invention.
  • a ribozyme is an enzyme-like RNA molecule that specifically decomposes specific RNA. Its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target RNA for endonucleation.
  • Antisense RNA, DNA, and ribozymes can be obtained using any existing RNA or DNA synthesis technology, such as solid-phase phosphoramidite chemical synthesis to synthesize oligonucleotides.
  • Antisense RNA molecules can be obtained by in vitro or in vivo transcription of a DNA sequence encoding the RNA. This DNA sequence has been integrated downstream of the vector's RNA polymerase promoter. In order to increase the stability of the nucleic acid molecule, it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the linkage between ribonucleosides using phosphate thioester or peptide bonds instead of phosphodiester bonds.
  • the polynucleotide encoding human interleukin binding promoting factor 13 can be used for the diagnosis of diseases related to human interleukin binding promoting factor 13.
  • Polynucleotides encoding human interleukin-binding promoting factor 13 can be used to detect the expression of human interleukin-binding promoting factor 13 or abnormal expression of human interleukin-binding promoting factor 13 in a disease state.
  • a DNA sequence encoding human interleukin-binding promoting factor 13 can be used to hybridize biopsy specimens to determine the expression of human interleukin-binding promoting factor 13.
  • Hybridization techniques include Southern blotting, Northern blotting, in situ hybridization, and the like.
  • RNA-polymerase chain reaction with primers specific for human interleukin-binding promoter 13 can also be used to detect the transcription products of human interleukin-binding promoter 13 in vitro.
  • Detection of mutations in the human interleukin-binding promoter 13 gene can also be used to diagnose human interleukin-binding promoter 13-related diseases.
  • the forms of human interleukin-binding promoter 13 mutations include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to the normal wild-type human interleukin-binding promoter 13 DNA sequence. Available techniques such as Southern blotting, DNA sequence Detection of mutations by analysis, PCR and in situ hybridization.
  • 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 in a similar manner, a set of fragments from a specific chromosome or a large number of genomic clones can be used to achieve sublocalization.
  • Other similar strategies that can be used for chromosomal localization include in situ hybridization, chromosome pre-screening with labeled flow sorting, and pre-selection of hybridization to construct chromosome-specific cDNA libraries.
  • Fluorescent in situ hybridization of cDNA clones with metaphase chromosomes allows precise chromosomal localization in one step.
  • FISH Fluorescent in situ hybridization
  • the 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 chromosomes, such as deletions or translocations that are visible at the chromosomal level or detectable with cDNA sequence-based PCR. According to the resolution capabilities of current physical mapping and gene mapping technology, the cDNA accurately mapped to the chromosomal region associated with the disease can be one of 50 to 500 potentially pathogenic genes (assuming 1 megabase mapping resolution) Capacity and each 20kb corresponds to a gene).
  • the polypeptides, polynucleotides and mimetics, agonists, antagonists and inhibitors of the present invention can be used in combination with a suitable pharmaceutical carrier.
  • suitable pharmaceutical carrier can be water, glucose, ethanol, salts, buffers Liquid, glycerin and their combinations.
  • the composition comprises a safe and effective amount of the polypeptide or antagonist, and carriers and excipients that do not affect the effect of the drug. These compositions can be used as drugs for the treatment of diseases.
  • the invention also provides a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the invention.
  • a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the invention.
  • these containers there may be instructional instructions given by government agencies that manufacture, use, or sell pharmaceuticals or biological products, which prompts permission for administration on the human body by government agencies that produce, use, or sell.
  • the polypeptides of the invention can be used in combination with other therapeutic compounds.
  • the pharmaceutical composition can be administered in a convenient manner, such as by a topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal route of administration.
  • Human interleukin binding promoting factor 13 is administered in an amount effective to treat and / or prevent a specific indication.
  • the amount and dose range of human interleukin-binding promoter 13 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 RNA using Quik mRNA I solat ion Ki t (product of Qiegene). 2ug poly (A) mRNA is reverse transcribed to form cDNA. Smart cDNA cloning kit (purchased from Clontech; ⁇ cDNA fragment was inserted into the multicloning site of pBSK (+) vector (Clontech)) to transform DH5 cc, and bacteria were used to form a cDNA library.
  • Dye terminated cyc le react ion Sequenc ing kit Perkin-Elmer
  • ABI 377 automatic sequencer Perkin-Elmer
  • the determined cDNA sequences were compared with existing public DM sequence databases ( Genebank), and found that the cDNA sequence of one of the clones 021 Oe 11 was new DNA.
  • a series of primers were synthesized to determine the inserted cDNA fragment of the clone in both directions.
  • the cDNA is 1505bp (as shown in Seq ID NO: 1), there is a 357bp open reading frame (0RF) from 787bp to 1143bp, and it encodes a new protein (as shown in Seq ID NO: 2).
  • RF open reading frame
  • Example 2 Cloning of a gene encoding human interleukin-binding promoting factor 13 by RT-PCR
  • the total RNA from fetal brain cells was used as a template, and oligo-dT was used as a primer for reverse transcription reaction to synthesize cDNA.
  • PCR amplification with the following primers:
  • Primerl 5'- GGCCGCGGCCTCTCGTTCTTAAAG -3, (SEQ ID NO: 3)
  • Primer2 5'- TCCATGTTTTTTATTATTTGTGCACTG -3 '(SEQ ID NO: 4)
  • Primerl is a forward sequence located at the 5th end of SEQ ID NO: 1, starting at lbp;
  • Primer2 is the 3, terminal reverse sequence of SEQ ID NO: 1.
  • Amplification reaction conditions reaction volume containing 50 ⁇ 1 of 50mmol / L KC1, 10 hidden ol / L Tris-HCl, pH8.5 , 1.5mmol / L MgCl 2, 200 ⁇ 1 / ⁇ dNTP, lOpmol primer, 1U of Taq DNA polymerase (Clontech).
  • the reaction was performed on a PE9600 DNA thermal cycler (Perkin-Elmer) for 25 cycles under the following conditions: 94 ° C 30sec; 55 ° C 30sec; 72 ° C 2rain.
  • ⁇ -actin was set as a positive control and template blank was set as a negative control.
  • the amplified product was purified using a QIAGEN kit, and ligated to a pCR vector (Invitrogen product) using a TA cloning kit.
  • the DNA sequence analysis results showed that the DNA sequence of the PCR product was exactly the same as the 1-1505bp shown in SEQ ID NO: 1.
  • Example 3 Northern blot analysis of human interleukin-binding promoter 13 gene expression Total RNA was extracted in one step [Anal. Biochem 1987, 162, 156-159] 0 This method involves acid guanidinium thiocyanate-chloroform extraction .
  • the tissue is homogenized with 4M guanidinium isothiocyanate-25mM sodium citrate, 0.2M sodium acetate (pH4.0), and 1 volume of phenol and 1/5 volume of chloroform-isoamyl alcohol (49: 1 ), Mix and centrifuge. Aspirate the aqueous layer, add isopropanol (0.8 vol) and centrifuge the mixture to obtain RNA precipitate. The resulting RNA pellet was washed with 70% ethanol, dried and dissolved in water.
  • 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-25mM KH 2 P0 4 ( ⁇ 7.4)-5 xSSC-5 x Denhardt, s solution and 20 (g / ml salmon sperm DNA. After hybridization, the filter was washed in 1 X SSC-0.1% SDS at 55 ° C for 30 min. Then, Phosphor Imager was used for analysis and quantification.
  • Example 4 In vitro expression, isolation and purification of recombinant human interleukin-binding promoter 13 Based on the sequence of the coding region shown in SEQ ID NO: 1 and Figure 1, a pair of specific amplification primers was designed The sequence is as follows: Primer3: 5,-CATGCTAGCATGCAGCCGTCCCTGTTAAGGTCA -3 '(Seq ID No: 5) Primer4: 5-CCCGAATTCTTACAGGGAGGAAGTCCTCTCCTG -3' (Seq ID No: 6) The 5 'ends of these two primers contain Nhel and EcoRI restriction sites, respectively.
  • PCR was performed using the pBS-0210ell plasmid containing the full-length target gene as a template. PCR reaction conditions were: 1 in a total volume of 50 ⁇ plasmid pBS- 0210ell containing 10pg, primers Pr imer-3 and Pr imer- 4 are lOpmol, Advantage polymerase Mix (Clontech Products) 1 ⁇ 1. Cycle parameters: 94. C 20s, 60.
  • Nhel and EcoRI were used to double digest the amplified product and plasmid pET-28 (+), respectively, and large fragments were recovered and ligated with T4 ligase.
  • Ligation products were transformed by the calcium chloride method Escherichia coli DH5a bacteria, after (final concentration 3 ( ⁇ g / m l) LB plates incubated overnight positive clones were screened by colony PCR method containing kanamycin, and sequenced. Selected The positive clone with the correct sequence (pET-0210ell) was used to transform the recombinant plasmid into E.
  • coli BL21 (DE3) plySs (product of Novagen) by calcium chloride method.
  • LB liquid medium containing kanamycin final concentration 30 ⁇ B / ⁇ 1
  • IPTG was added to a final concentration of 1 mmol / L, and the culture was continued for 5 hours.
  • the cells were collected by centrifugation, and the supernatant was collected by centrifugation and ultrasonication. Chromatography was performed using an His. Bind Quick Cartridge (Novagen) affinity chromatography column capable of binding 6 histidines (6His-Tag) to obtain purified human interleukin-binding promoting factor 13 of interest.
  • Polypeptide synthesizer (product of PE company) was used to synthesize the following specific peptides of human interleukin-binding promoter 13:
  • NH2-Met- Gin-Pro- Ser- Leu- Leu- Arg- Ser-Tyr-Arg- Leu- Lys- Ala- Gin- Leu- C00H SEQ ID NO: 7
  • the polypeptide is coupled with hemocyanin and bovine serum albumin to form a complex, respectively.
  • hemocyanin and bovine serum albumin For the method, see: Avraraeas, et al. Immunochemistry, 1969; 6: 43. Rabbits were immunized with 4 mg of the i-cyanin polypeptide complex plus complete Freund's adjuvant, and 15 days later, the hemocyanin polypeptide complex plus incomplete Freund's adjuvant was used to boost the immunity once.
  • a titer plate coated with a 15 g / ml bovine serum albumin peptide complex was used as an ELISA to determine antibody titers in rabbit serum.
  • Total IgG was isolated from antibody-positive rabbit serum using protein A-Sepharose.
  • the peptide was bound to a cyanogen bromide-activated Sepharose4B column, and anti-peptide antibodies were separated from total i gG by affinity chromatography.
  • the immunoprecipitation method proved that the purified antibody can specifically bind to human interleukin and promote Advance factor 13 combined.
  • Example 6 Application of the polynucleotide fragment of the present invention as a hybridization probe
  • the suitable oligonucleotide fragments selected from the polynucleotides of the present invention are used as hybridization probes in various aspects.
  • the probes can be used to hybridize to the genome or CDM library of normal tissue or pathological tissue from different sources to It is determined whether it contains the polynucleotide sequence of the present invention and a homologous polynucleotide sequence is detected.
  • the probe can be used to detect the polynucleotide sequence of the present invention or its homologous polynucleotide sequence in normal tissue or pathology. Whether the expression in tissue cells is abnormal.
  • the purpose of this embodiment is to select a suitable oligonucleotide fragment from the polynucleotide SEQ ID NO: 1 of the present invention as a hybridization probe, and to identify whether some tissues contain the polynucleoside of the present invention by a filter hybridization method.
  • Filter hybridization methods include dot blotting, Southern imprinting, Nor thern blotting, and copying methods. They all use the same steps to fix the polynucleotide sample to be tested on the filter and then hybridize.
  • the sample-immobilized filter is first pre-hybridized with a probe-free hybridization buffer to saturate the non-specific binding site of the sample on the filter with the carrier and the synthesized polymer.
  • the pre-hybridization solution is then replaced with a hybridization buffer containing labeled probes and incubated to hybridize the probes to the target nucleic acid.
  • the unhybridized probes are removed by a series of membrane washing steps.
  • This embodiment uses higher-intensity washing conditions (such as lower salt concentration and higher temperature) to reduce the hybridization background and retain only strong specific signals.
  • the probes used in this embodiment include two types: the first type of probes are oligonucleotide fragments that are completely the same as or complementary to the polynucleotide SEQ ID NO: 1 of the present invention; the second type of probes are partially related to the present invention
  • the polynucleotide SEQ ID NO: 1 is the same or complementary oligonucleotide fragment.
  • the dot blot method is used to fix the sample on the filter membrane. Under the high-intensity washing conditions, the first type of probe and the sample have the strongest hybridization specificity and are retained.
  • oligonucleotide fragments for use as hybridization probes from the polynucleotide SEQ ID NO: 1 of the present invention should follow the following principles and several aspects to be considered:
  • the preferred size of the probe is 1 S-50 nucleotides
  • Probe 1 (probel), which belongs to the first type of probe, is completely homologous or complementary to the gene fragment of SEQ ID NO: 1 ( 4 lNt):
  • Probe 1 which belongs to the second type of probe, is equivalent to the replacement mutation sequence (41Nt) of the gene fragment or its complementary fragment of SEQ ID NO: 1:
  • PBS phosphate buffered saline
  • step 8-13 are only used when contamination must be removed, otherwise step 14 can be performed directly.
  • NC membranes nitrocellulose membranes
  • the sample membrane was placed in a plastic bag, and 3 to 10 mg of prehybridization solution (lOxDenhardt's; 6xSSC, 0.1 mg / ml CT DNA (calf thymus DNA)) was added. After sealing the bag, shake at 68 ° C for 2 hours.
  • prehybridization solution lOxDenhardt's; 6xSSC, 0.1 mg / ml CT DNA (calf thymus DNA)
  • 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.
  • Gene chip or DNA microarray is a new technology that many national laboratories and large pharmaceutical companies are currently developing and developing. It refers to the orderly and high-density arrangement of large numbers of target gene fragments on glass, The data is compared and analyzed on a carrier such as silicon using fluorescence detection and computer software to achieve the purpose of rapid, efficient, and high-throughput analysis of biological information.
  • the polynucleotide of the present invention can be used as target DNA for gene chip technology for high-throughput research of new gene functions; search for and screen new tissue-specific genes, especially new genes related to diseases such as tumors; diagnosis of diseases such as hereditary diseases .
  • the specific method steps have been reported in the literature. For example, refer to the literature DeRisi, JL, Lyer, V. & Brown, P.0. (1997) Science 278, 680- 686. And the literature Helle, RA, Schema, M Chai, A., Shalom, D., (1997) PNAS 94: 2150-2155.
  • a total of 4,000 polynucleotide sequences of various full-length cDNAs are used as target DNA, including the polynucleotide of the present invention. They were amplified by PCR respectively. After purification, the concentration of the amplified product was adjusted to about 500 ng / ul, and spotted on a glass medium with a Cartesian 7500 spotting instrument (purchased from Cartesian, USA). The distance is 280 ⁇ m. The spotted slides were hydrated and dried, cross-linked in a UV cross-linker, and dried after elution to fix the DNA on the glass slides to prepare chips. The specific method steps have been reported in the literature. The sample post-processing steps in this embodiment are:
  • Probes from the above two tissues and chips were hybridized in a UniHyb TM Hybridization Solution (purchased from TeleChem) hybridization solution for 16 hours, washed with a wash solution (1 x SSC, 0.2% SDS) at room temperature, and then scanned with ScanArray 3000 The scanner (purchased from General Scanning Company, USA) was used for scanning. The scanned image was analyzed and processed with Imagene software (Biodiscovery Company, USA) to calculate the Cy3 / Cy5 ratio of each point.
  • the above specific tissues are thymus, testis, muscle, spleen, lung, skin, thyroid, liver, PMA + Ecv304 cell line, PMA-Ecv304 cell line, non-starved L02 cell line, Arsenic stimulated the L02 cell line and prostate tissue for 1 hour. Based on these 13 Cy3 / Cy5 ratios, draw a bar graph ( Figure 1). It can be seen from the figure that the expression profiles of human interleukin-binding promoting factor 13 and human interleukin-binding promoting factor 2 according to the present invention are very similar.

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Abstract

L'invention concerne un nouveau polypeptide, un facteur humain d'activation 13 de la liaison entre leucocytes, et un polynucléotide codant pour 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 l'infection par VIH, des maladies immunitaires, des troubles liés à un antigène d'histocompatibilité, des réactions à la transfusion, des rejets de greffe, des inflammations, des tumeurs malignes et de l'hémopathie. L'invention concerne aussi l'antagoniste agissant contre le polypeptide et son action thérapeutique ainsi que les applications de ce polynucléotide codant pour le facteur humain d'activation 13 de la liaison entre leucocytes.
PCT/CN2001/000173 2000-03-10 2001-02-26 Nouveau polypeptide, facteur humain d'activation 13 de la liaison (ilf) entre leucocytes, et polynucleotide codant pour ce polypeptide WO2001070781A1 (fr)

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CN00111958A CN1313300A (zh) 2000-03-10 2000-03-10 一种新的多肽——人白细胞介素结合促进因子13和编码这种多肽的多核苷酸
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995032308A1 (fr) * 1994-05-20 1995-11-30 Board Of Regents, The University Of Texas System Compositions et leurs utilisations dans le diagnostic du psoriasis
US6046047A (en) * 1993-02-12 2000-04-04 Board Of Trustees Of Leland Stanford Jr. University Regulated transcription of targeted genes and other biological events

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6046047A (en) * 1993-02-12 2000-04-04 Board Of Trustees Of Leland Stanford Jr. University Regulated transcription of targeted genes and other biological events
WO1995032308A1 (fr) * 1994-05-20 1995-11-30 Board Of Regents, The University Of Texas System Compositions et leurs utilisations dans le diagnostic du psoriasis

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