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WO2002048186A1 - Nouveau polypeptide-proteine 10.45 conjuguee au facteur de croissance insulinoide homo et polynucleotide codant ledit polypeptide - Google Patents

Nouveau polypeptide-proteine 10.45 conjuguee au facteur de croissance insulinoide homo et polynucleotide codant ledit polypeptide Download PDF

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Publication number
WO2002048186A1
WO2002048186A1 PCT/CN2001/001374 CN0101374W WO0248186A1 WO 2002048186 A1 WO2002048186 A1 WO 2002048186A1 CN 0101374 W CN0101374 W CN 0101374W WO 0248186 A1 WO0248186 A1 WO 0248186A1
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Prior art keywords
polypeptide
polynucleotide
growth factor
insulin
binding protein
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PCT/CN2001/001374
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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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Application filed by Shanghai Biowindow Gene Development Inc. filed Critical Shanghai Biowindow Gene Development Inc.
Priority to AU2002223405A priority Critical patent/AU2002223405A1/en
Publication of WO2002048186A1 publication Critical patent/WO2002048186A1/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
    • 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, an insulin-like growth factor binding protein 10.45, and a polynucleotide sequence encoding the polypeptide. The invention also relates to a preparation method and application of the polynucleotide and polypeptide.
  • IGF-I and IGF-II bind to specific binding proteins with high affinity in extracellular fluids (Rechler MM, Vitara. Horm. 47: 1-114 (1993)). These IGF- Binding proteins (IGFBP) prolong the half-life of IGFs. They inhibit or stimulate the growth-promoting effects of IGFs in cell culture. They work with receptors on the cell surface to change the response of IGFs.
  • IGFBP IGF- Binding proteins
  • IGFBPs six different IGFBPs have been found and they are structures Related: The following growth factor-inducible proteins are structurally related to IGFBPs and can act as growth factor-binding proteins: human connective tissue growth factor (CTGF); spinal promoter protein NOV (Maloisel V., Martinerie C., Dambrine G., Plassiart G., BrisacM., Crochet J., Perbal B., Mol. Cell Biol. 12: 10-21 (1992) X
  • CGF connective tissue growth factor
  • NOV spinal promoter protein NOV
  • the novel polypeptide of the present invention contains the above-mentioned characteristic sequence template and has similar biological functions with members of the insulin-like growth factor-binding protein family, so it is considered to be a new member of the insulin-like growth factor-binding protein family.
  • the polypeptide and its agonists, inhibitors and antagonists can be used to diagnose and prevent various diseases related to the growth-promoting effects of insulin-like growth factor (IGF-I and IGF-II) and connective tissue growth factor (CTGF), such as growth Dysfunction diseases, including growth dysfunction, growth hormone aberrations, etc.
  • IGF-I and IGF-II insulin-like growth factor
  • CGF connective tissue growth factor
  • binding protein 10.45 protein plays an important role in important functions of the body as described above, and it is believed that a large number of proteins are involved in these regulatory processes, there has been a need in the art to identify more insulin-like growth factors involved in these processes.
  • Binding protein 10.45 protein especially the amino acid sequence of this protein. Isolation of the new insulin-like growth factor binding protein 10.45 protein encoding gene also provides a basis for the study to determine the role of this protein in health and disease states. This protein may form the basis for the development of diagnostic and / or therapeutic drugs for diseases, so it is important to isolate its coding for DM.
  • An object of the present invention is to provide an isolated novel polypeptide, an insulin-like growth factor binding protein.
  • 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 an insulin-like growth factor binding protein 10.45.
  • Another object of the present invention is to provide a genetically engineered host cell comprising a polynucleotide encoding an insulin-like growth factor binding protein 10.45.
  • Another object of the present invention is to provide mimic compounds, antagonists, agonists, and inhibitors against the polypeptide-insulin-like growth factor binding protein 10.45 of the present invention.
  • Another object of the present invention is to provide a method for diagnosing and treating diseases related to the abnormality of insulin-like growth factor binding protein 10.45. 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 301-388 in SEQ ID NO: 1; and (b) a sequence having 1-2175 in SEQ ID NO: 1 Sequence of bits.
  • the invention further relates to a vector, in particular an expression vector, containing the polynucleotide of the invention; a host cell genetically engineered with the vector, including a transformed, transduced or transfected host cell; and a method comprising culturing said Host cell and method of preparing the polypeptide of the present invention by recovering the expression product.
  • a vector in particular an expression vector, containing the polynucleotide of the invention
  • a host cell genetically engineered with the vector including a transformed, transduced or transfected host cell
  • a method comprising culturing said Host cell and method of preparing the polypeptide of the present invention by recovering the expression product.
  • the invention also relates to an antibody capable of specifically binding to a polypeptide of the invention.
  • the present invention also relates to a screened simulation, activation, antagonism or inhibition of insulin-like growth factor binding protein.
  • White 10.45 A method of a protein-active compound comprising utilizing a polypeptide of the invention. The invention also relates to compounds obtained by this method.
  • the invention also relates to a method for detecting a disease or disease susceptibility related to abnormal expression of insulin-like growth factor binding protein 10.45 protein in vitro, comprising detecting mutations in the polypeptide or a polynucleotide sequence encoding the same 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 present invention also relates to polypeptides and / or polynucleotides of the present invention prepared for use in the treatment of cancer, developmental or immune diseases, or other diseases caused by abnormal expression of insulin-like growth factor protein 10.45.
  • Other aspects of the present invention It will be apparent to those skilled in the art from the disclosure of the techniques herein.
  • Fig. 1 is a comparison diagram of the amino acid sequence homology of a total of 59 amino acids and structural domain MOTIF of the insulin-like growth factor binding protein 10.45 at 9-67 of the present invention.
  • the upper sequence is the insulin-like growth factor binding protein 1 0.45, and the lower sequence is the domain M0TIF. 'And ":” and ".” Indicate that the probability of the same amino acid decreasing between the two sequences decreases in order.
  • Figure 2 shows the polyacrylamide gel electrophoresis (SDS-PAGE) of the isolated insulin-like growth factor binding protein 10.45.
  • 10. 45kDa 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 is one that has one or more amino acid or nucleotide changes Amino acid sequence or polynucleotide sequence encoding it. The changes may include deletions, insertions or substitutions of amino acids or nucleotides in the amino acid sequence or nucleotide sequence. Variants may have "conservative" changes in which the substituted amino acid has a structural or chemical property similar to the original amino acid, such as replacing isoleucine with leucine. Variants can also have non-conservative changes, such as replacing glycine with tryptophan.
  • “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 combined with an insulin-like growth factor binding protein 10.45, causes the protein to change, thereby regulating the activity of the protein.
  • An agonist may include a protein, a nucleic acid, a carbohydrate, or any other molecule that binds an insulin-like growth factor binding protein 10.45.
  • Antagonist refers to a biological or immunological activity that can block or regulate benzyl insulin-like growth factor binding protein 10.45 when combined with insulin-like growth factor binding protein 10.45.
  • Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates or any other molecule that can bind to insulin-like growth factor binding protein 10.45.
  • Regular refers to changes in the function of insulin-like growth factor binding protein 10.45, including an increase or decrease in protein activity, changes in binding characteristics, and any other biological properties and functions of insulin-like growth factor-binding protein 10.45. Or changes in immune properties.
  • substantially pure means substantially free of other proteins, lipids, sugars or other substances with which it is naturally associated.
  • Those skilled in the art can purify the insulin-like growth factor binding protein 10.45 using standard protein purification techniques.
  • a substantially pure insulin-like growth factor binding protein 10.45 produces a single main band on a non-reducing polyacrylamide gel.
  • Insulin-like growth factor binding protein 10. 45 The purity of the peptide 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 can 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.
  • Partially homologous means A partially complementary sequence that at least partially inhibits hybridization of a fully complementary sequence to a target nucleic acid. The inhibition of such hybridization can be detected by performing hybridization (Southern or Northern blotting, etc.) under conditions of reduced stringency.
  • Substantially homologous sequences or hybridization probes can compete and inhibit the binding of completely homologous sequences to target sequences under conditions of reduced stringency. This does not mean that the conditions of reduced stringency allow non-specific binding, because the conditions of reduced stringency require that the two sequences bind to each other as a specific or selective interaction.
  • Percent identity refers to the percentage of sequences that are identical or similar in the comparison of two or more amino acid or nucleic acid sequences. The percent identity can be determined electronically, such as through the MEGALIGN program (Lasergene software package, DNASTAR, Inc., Madison Wis.). 0 The MEGALIGN program can compare two or more sequences (Higgins, DGPM) according to different methods, such as the Clus ter method. Sharp (1988)
  • 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 percent identity between nucleic acid sequences can also be determined by the Clus ter method or by methods known in the art such as; Totun Hein (Hein J., (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 such as negatively charged amino acids may include aspartic acid and glutamic acid; positively charged amino acids may include lysine and arginine; having uncharged head groups are Similar hydrophilic amino acids may include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; serine and threonine; phenylalanine and tyrosine.
  • Antisense refers to a nucleotide sequence that is complementary to a particular DNA or RNA sequence.
  • the "antisense strand” refers to a nucleic acid strand that is complementary to the “sense strand”.
  • Derivative refers to HFP or a chemical modification of its nucleic acid. Such a chemical modification may be a substitution of a hydrogen atom with a fluorenyl group, an acyl group or an 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 insulin-like growth factor binding protein 10.45 epitope.
  • 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 (eg Natural environment).
  • a naturally occurring polynucleotide or polypeptide is not isolated when it is present in a living animal, but the same polynucleotide or polypeptide is separated from some or all of the substances that coexist with it in the natural system.
  • Such a polynucleotide may be part of a certain vector, or such a polynucleotide or polypeptide may be part of a certain composition. Since the carrier or composition is not a component of its natural environment, they are still isolated.
  • isolated refers to the separation of a substance from its original environment (if it is a natural substance, the original environment is the natural environment).
  • polynucleotides and polypeptides in a natural state in a living cell are not isolated and purified, but the same polynucleotides or polypeptides are separated and purified if they are separated from other substances existing in the natural state. .
  • isolated insulin-like growth factor binding protein 10. 45 refers to insulin-like growth factor-binding protein 10. 45 that is substantially free of other proteins, lipids, sugars, or other substances with which it is naturally associated. Those skilled in the art can purify insulin-like growth factor binding protein 10.45 using standard protein purification techniques. Substantially pure polypeptides can produce a single main band on a non-reducing polyacrylamide gel. Insulin-like growth factor binding protein 10. 45 The purity of the polypeptide can be analyzed by amino acid sequence.
  • the present invention provides a novel polypeptide-insulin-like growth factor binding protein 10.45, which basically consists of the amino acid sequence shown in SEQ ID NO: 2.
  • the polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide, and preferably a recombinant polypeptide.
  • the polypeptides of the present invention can be naturally purified products or chemically synthesized products, or can be produced from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insects, and mammalian cells) using recombinant techniques. 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 insulin-like growth factor binding protein 10.45.
  • fragment refers to a polypeptide that substantially maintains the same biological function or activity of the insulin-like growth factor binding protein 10.45 of the present invention.
  • a fragment, derivative or analog of the polypeptide of the present invention may be: (I) a kind in which one or more amino acid residues are replaced with conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and A substituted 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); or (IV) 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 this polypeptide or protein sequence).
  • 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 2175 bases in length and its open reading frames 301-588 encode 87 amino acids.
  • This polypeptide has a characteristic sequence of MOTIF, and it can be deduced that the insulin-like growth factor binding protein 10.45 has the structure and function represented by MOTIF.
  • 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.
  • polynucleotide encoding a polypeptide refers to a polynucleotide that includes the polypeptide and a polynucleotide that includes additional coding and / or non-coding sequences.
  • the invention also relates to variants of the polynucleotides described above, which encode polypeptides or fragments, analogs and derivatives of polypeptides having the same amino acid sequence as the invention.
  • This polynucleotide variant can be a naturally occurring allelic variant or a non-naturally occurring variant.
  • These nucleotide variants include substitution variants, deletion variants, and insertion variants.
  • an allelic variant is an alternative form of a polynucleotide that may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially change the function of the polypeptide it encodes .
  • the invention also relates to a polynucleotide that hybridizes to the sequence described above (having at least 50%, preferably 70% identity between the two sequences).
  • the present invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the present invention under stringent conditions.
  • “strict conditions” means: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2xSSC, 0.1% SDS, 6 (TC; or (2) Add denaturants during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficol 1, 42 ° C, etc .; or (3) only between the two sequences Hybridization occurs only when the identity is at least 95%, and more preferably 97%.
  • the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO: 2.
  • nucleic acid fragments that hybridize to the sequences described above.
  • a "nucleic acid fragment” contains at least 10 nucleotides in length, preferably at least 20-30 nucleotides, more preferably at least 50-60 Nucleotides, preferably at least 100 nucleotides. 45 Nucleic acid fragments can also be used in nucleic acid amplification techniques (such as PCR) to identify and / or isolate polynucleotides encoding insulin-like growth factor binding protein 10.45.
  • 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 insulin-like growth factor binding protein 10.45 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 DM sequence to obtain the double-stranded DNA of the polypeptide.
  • genomic DNA isolation is the least commonly used. Direct chemical synthesis of DM sequences is often the method of choice.
  • the more commonly used method is the isolation of cDNA sequences.
  • the standard method for isolating cDNA of interest is to isolate raRNA from donor cells that overexpress the gene and perform reverse transcription to form a plasmid or phage cDNA library.
  • Various methods have been used to extract mRNA, and kits are also commercially available (Qiagene).
  • the construction of cDNA libraries is also a common method (Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spiring 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-DM or DNA-RNA hybridization; (2) the presence or absence of a marker gene function; (3) determination of the level of the insulin-like growth factor binding protein 10.45 transcript (4) Detecting protein products expressed by genes through immunological techniques or measuring biological activity. The above methods can be used singly or in combination.
  • the probe used for hybridization is homologous to any part of the polynucleotide of the present invention, and its length is at least 10 nucleotides, preferably at least 30 nucleotides, more preferably At least 50 nucleotides, preferably at least 100 nucleotides.
  • the length of the probe is usually within 2000 nucleotides, preferably within 1000 nucleotides.
  • the probe used here is generally a DNA sequence chemically synthesized based on the gene sequence information of the present invention.
  • the genes or fragments of the present invention can of course be used as probes.
  • DM probes can be labeled with radioisotopes, luciferin, or enzymes (such as alkaline phosphatase).
  • the protein product expressing the insulin-like growth factor binding protein 10.45 gene can be detected by immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA).
  • a method for amplifying DNA / RNA by PCR (Saiki, et al. Science 1985; 230: 1350-1354) is preferably used to obtain the gene of the present invention.
  • the RACE method RACE-cDM terminal rapid amplification method
  • the primers used for PCR can be appropriately based on the polynucleotide sequence information of the present invention disclosed herein Select and synthesize using conventional methods.
  • the amplified DNA / RNA fragments can be isolated and purified by conventional methods such as by gel electrophoresis.
  • polynucleotide sequence of the gene of the present invention or various DM fragments and the like obtained as described above can be 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 the cDNA sequence of multiple clones in order to splice into a full-length cDNA sequence.
  • the present invention also relates to a vector comprising the polynucleotide of the present invention, and a host cell produced by genetic engineering using the vector of the present invention or directly using an insulin-like growth factor binding protein 10.45 coding sequence, and a recombinant technology to produce the Said method of polypeptide.
  • a polynucleotide sequence encoding the insulin-like growth factor binding protein 10.45 can be inserted into a vector to constitute a recombinant vector containing the polynucleotide of the present invention.
  • vector refers to bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors well known in the art.
  • ⁇ Suitable vectors in the present invention include, but are not limited to: a ⁇ promoter-based epitope vector (Rosenberg, et al.
  • any plasmid and vector can be used to construct recombinant expression vectors.
  • An important feature of expression vectors is that they usually contain an origin of replication, a promoter, a marker gene, and translational regulatory elements.
  • Methods known to those skilled in the art can be used to construct expression vectors containing a DNA sequence encoding an insulin-like growth factor binding protein 10.45 and suitable 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 Manua, Cold Spring Harbor Labora tory. New York, 1989).
  • the DNA sequence can be operably linked to an appropriate promoter in an expression vector to guide mRM synthesis. Representative examples of these promoters are: the lac or trp promoter of E.
  • coli the PL promoter of lambda phage
  • eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, and the early and late SV40 promoters , Retroviral LTRs and other known promoters that can control the expression of genes in prokaryotic or eukaryotic cells or their viruses.
  • Enhancers are cis-acting factors for DNA expression, usually about 10 to 300 base pairs that act on promoters to enhance gene transcription.
  • Illustrative examples include SV40 enhancers of 100 to 270 base pairs on the late side of the origin of replication, polyoma enhancers on the late side of the origin of replication, and adenovirus enhancers.
  • the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture.
  • selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture.
  • GFP fluorescent protein
  • tetracycline or ampicillin resistance for E. coli.
  • a polynucleotide encoding an insulin-like growth factor binding protein 10.45 or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to form a genetically engineered host 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.
  • Escherichia coli, Streptomyces bacterial cells such as Salmonella typhimurium
  • fungal cells such as yeast
  • plant cells insect cells
  • fly S2 or Sf9 animal cells
  • 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 absorbing DM can be harvested after the exponential growth phase and treated with the CaCl 2 method. The steps used are well known in the art. Alternatively, MgCl 2 is used. If necessary, transformation can also be performed by electroporation.
  • the host is a eukaryote, the following DM transfection methods can be used: calcium phosphate co-precipitation method, or conventional mechanical methods such as microinjection, electroporation, and liposome packaging.
  • the polynucleotide sequence of the present invention can be used to express or produce recombinant insulin-like growth factor binding protein 10. 45 (Sc ience, 1984; 224: 1431). Generally speaking, there are the following steps:
  • the medium used in the culture may be selected from various conventional mediums. Culture is performed under conditions suitable for host cell growth. When host cells grow to proper After inducing the cell density, the appropriate promoter (such as temperature conversion or chemical induction) is used to induce the selected promoter, and the cells are cultured for a period of time.
  • the appropriate promoter 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 be used to treat malignant tumors, adrenal deficiency, skin diseases, various types of inflammation, HIV infection, and immune diseases.
  • Insulin-like growth factor has the effects of stimulating cell metabolism and stimulating cell proliferation in the human body. It must bind to insulin-like growth factor binding protein (IGFBP) to function. It was found that members of the insulin-like growth factor binding protein family contain a characteristic sequence template, and the characteristic conserved sequence is necessary to form its active mot if. The abnormal expression of this characteristic sequence will cause the disorder of insulin-like growth factor binding protein, cause the dysfunction of insulin-like growth factor, and then cause related diseases.
  • IGFBP insulin-like growth factor binding protein
  • the novel polypeptide of the present invention has high homology and similarity with insulin-like growth factor binding protein in structure and function, and the amino acid sequence contains the above-mentioned conservative characteristic sequence template.
  • the novel polypeptide of the present invention has a physiological activity similar to that of insulin-like growth factor binding protein.
  • the abnormal expression of the above-mentioned characteristic conserved sequence will lead to abnormal function of the novel polypeptide containing the mot if of the present invention, thereby causing the disorder of the insulin-like growth factor binding protein, causing the dysfunction of the insulin-like growth factor, and causing human cells Abnormal proliferation occurs, which in turn causes the occurrence of related diseases.
  • Cleft lip most common, with alveolar cleft and cleft palate
  • cleft palate facial oblique cleft
  • cervical pouch cervical fistula
  • Horizontal absence (congenital short limbs): no arms, no forearms, no hands, no fingers, no legs, no toes, etc.
  • Longitudinal absences Radial / ulnar absence of upper limbs, tibia / fibula absence of lower limbs, etc .; Seal-like hand / foot deformities, etc .;
  • Limb differentiation disorder Absence of a certain muscle or muscle group, joint dysplasia, bone deformity, bone fusion, multi-finger (toe) deformity, finger (toe) deformity, horseshoe varus, etc .;
  • Thyroglossal duct cysts atresia or stenosis of the digestive tract, ileal diverticulum, umbilical diaphragm, congenital umbilical hernia, congenital non-gangliomegaly, imperforate anus, abnormal bowel transition, bile duct atresia, circular pancreas, etc .;
  • neural tube defects no cerebral malformations, spina bifida, spinal meningocele, hydrocephalous meningoencephalocele
  • hydrocephalus inside / outside the brain, etc.
  • Papilloma squamous cell carcinoma [skin, nasopharynx, larynx, cervix], adenoma (carcinoma) [breast, thyroid], mucinous / serous cystadenomas (carcinoma) [ovary], basal cell carcinoma [head and face [Skin], (malignant) polytype adenoma [extending gland], papilloma, transitional epithelial cancer [bladder, renal pelvis], etc .;
  • Malignant lymphoma [Neck, mediastinum, mesentery and retroperitoneal lymph nodes], various leukemias [lymphoid hematopoietic tissue], multiple myeloma [push / thoracic / rib / skull and long bone], etc .;
  • Nerve fiber [systemic cutaneous nerve / deep nerve and internal organs], (malignant) schwannoma [head, neck, limbs, etc.], (malignant) glioblastoma [brain], medulloblastoma Cerebellum], (malignant) meningiomas [meninges], ganglioblastoma / neuroblastoma [mediastinum and retroperitoneum / adrenal medulla], etc .;
  • malignant melanoma [skin, mucosa], (malignant) hydatidiform mole, chorionic epithelial cancer [uterine], (malignant) supporter cells, stromal cell tumor, (malignant) granulosa cell tumor [ovarian, testicular] Blastoma [testis], asexual cell tumor [ovary], embryonal cancer [testis, ovary], (malignant) teratoma [ovary, testis, mediastinum and palate tail], etc .;
  • 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 embryonic developmental malformations, tumors, and the like.
  • the invention also provides methods for screening compounds to identify agents that increase (agonist) or suppress (antagonist) insulin-like growth factor binding protein 10.45.
  • Agonists increase insulin-like growth factor binding proteins 10.45 Stimulate biological functions such as cell proliferation, while antagonists prevent and treat disorders related to excessive cell proliferation, such as various cancers.
  • mammalian cells or a membrane preparation expressing an insulin-like growth factor binding protein 10.45 can be cultured with a labeled insulin-like growth factor-binding protein 10.45 in the presence of a drug. The ability of the drug to increase or block this interaction is then determined.
  • Antagonists of insulin-like growth factor binding protein 10.45 include antibodies, compounds, receptor deletions, and the like that have been screened.
  • An antagonist of the insulin-like growth factor binding protein 10.45 can bind to the insulin-like growth factor binding protein 10.45 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.
  • insulin-like growth factor binding protein 10.45 can be added to the bioanalytical assay by determining the effect of the compound on the interaction between insulin-like growth factor binding protein 10.45 and its receptors Determine if the compound is an antagonist. Receptor deletions and analogs that act as antagonists can be screened in the same manner as described above for screening compounds.
  • Polypeptide molecules capable of binding to insulin-like growth factor binding protein 10.45 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. When screening, generally 10.45 molecules of insulin-like growth factor binding protein should be labeled.
  • the present invention provides a method for producing an antibody using a polypeptide, a fragment, a derivative, an analog thereof, or a cell thereof as an antigen.
  • These antibodies can be polyclonal or monoclonal antibodies.
  • the invention also provides antibodies against the insulin-like growth factor binding protein 10.45 epitope.
  • These antibodies include (but are not limited to): polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, and fragments produced by Fab expression libraries.
  • Polyclonal antibodies can be produced by injecting insulin-like growth factor binding protein 10.45 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 against insulin-like growth factor binding protein 10.45 include, but are not limited to, hybridoma technology (Koh'ler 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 (U.S. Pat No. 4946778) can also be used to produce single chain antibodies against insulin-like growth factor binding protein 10.45.
  • Antibodies against insulin-like growth factor binding protein 10. 45 can be used in immunohistochemical techniques to detect insulin-like growth factor binding protein 10. 45 in biopsy specimens.
  • Monoclonal antibodies that bind to insulin-like growth factor binding protein 10.45 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. Such as insulin-like growth factor binding protein 10. 45 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 insulin-like growth factor binding protein 10.45 Positive cells.
  • a thiol cross-linking agent such as SPDP
  • the antibodies of the present invention can be used to treat or prevent diseases related to insulin-like growth factor binding protein 10.45.
  • Administration of an appropriate dose of antibody can stimulate or block the production or activity of insulin-like growth factor binding protein 10.45.
  • the invention also relates to a diagnostic test method for quantitative and localized detection of insulin-like growth factor binding protein levels of 10.45.
  • These tests are well known in the art and include FISH assays and radioimmunoassays.
  • the level of insulin-like growth factor binding protein 10.45 detected in the test can be used to explain the importance of insulin-like growth factor-binding protein 10.45 in various diseases and to diagnose insulin-like growth factor-binding protein 10. 45 diseases at work.
  • polypeptides of the present invention can also be used for peptide mapping, for example, the polypeptides can be physically, chemically or enzymatically Specific cleavage and one-dimensional or two-dimensional or three-dimensional gel electrophoresis analysis, preferably mass spectrometry.
  • Polynucleotides encoding insulin-like growth factor binding protein 10.45 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 insulin-like growth factor binding protein 10.45. Recombinant gene therapy vectors (such as viral vectors) can be designed to express variant insulin-like growth factor binding protein 10.45 to inhibit the activity of endogenous insulin-like growth factor binding protein 10.45.
  • a variant insulin-like growth factor-binding protein 10.45 may be a shortened insulin-like growth factor-binding protein 10.45 lacking a signaling domain, although it can bind to downstream substrates, but lacks signaling. active.
  • recombinant gene therapy vectors can be used to treat diseases caused by abnormal expression or activity of insulin-like growth factor binding protein 10.45.
  • Virus-derived expression vectors such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus, etc. can be used to transfer a polynucleotide encoding an insulin-like growth factor binding protein 10.45 into a cell.
  • a method for constructing a recombinant viral vector carrying a polynucleotide encoding an insulin-like growth factor binding protein 10.45 can be found in the literature (Sarabrook, et al.).
  • a recombinant polynucleotide encoding the insulin-like growth factor binding protein 10.45 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 DM
  • ribozymes that inhibit insulin-like growth factor binding protein 1 0.45 are also within the scope of the present invention.
  • a ribozyme is an enzyme-like RNA molecule that can specifically decompose specific RNA. Its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target RNA and performs endonucleation.
  • Antisense RM, DM and ribozymes can be obtained by any RNA or DNA synthesis technology. For example, solid-phase phosphoramidite chemical synthesis technology has been widely used.
  • Antisense RNA molecules can be obtained by in vitro or in vivo transcription of a DNA sequence encoding the RNA.
  • This DM sequence has been integrated downstream of the vector's RNA polymerase promoter.
  • it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the phosphorothioate or peptide bond instead of the phosphodiester bond is used for the ribonucleoside linkage.
  • the polynucleotide encoding the insulin-like growth factor binding protein 10.45 can be used for the diagnosis of diseases related to the insulin-like growth factor binding protein 10.45.
  • the polynucleotide encoding the insulin-like growth factor binding protein 10.45 can be used to detect the expression of the insulin-like growth factor-binding protein 10.45 or the abnormal expression of the insulin-like growth factor-binding protein 10.45 in a disease state.
  • Encoding insulin-like growth factor The DNA sequence of the daughter binding protein 10.45 can be used to hybridize biopsy specimens to determine the expression status of the insulin-like growth factor binding protein 10.45.
  • Hybridization techniques include Southern blotting, Northern blotting, and in situ hybridization. These techniques and methods are publicly available and mature, and related kits are commercially available.
  • a part or all of the polynucleotides of the present invention can be used as probes to be fixed on a micro array or a DNA chip (also called a "gene chip") for analyzing differential expression analysis of genes and genetic diagnosis in tissues.
  • the transcription product of insulin-like growth factor binding protein 10.45 can also be detected by RNA-polymerase chain reaction (RT-PCR) in vitro amplification with insulin-like growth factor binding protein 10.45 specific primers.
  • Detection of mutations in the insulin-like growth factor binding protein 10.45 gene can also be used to diagnose diseases related to the insulin-like growth factor binding protein 10.45.
  • Insulin-like growth factor binding protein 10.45 mutations include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to the normal wild-type insulin-like growth factor-binding protein 10.45 DM sequence. Mutations can be detected using existing techniques such as Southern imprinting, DNA sequence analysis, PCR, and in situ hybridization. In addition, mutations may affect protein expression. Therefore, 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 DM 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 heterozygous cells containing the human gene corresponding to the primer will produce amplified fragments.
  • PCR localization of somatic hybrid cells is a quick way to localize DNA to specific chromosomes.
  • oligonucleotide primers of the present invention in a similar manner, a set of fragments from a specific chromosome or a large number of genomic clones can be used to achieve sublocalization.
  • Other similar strategies that can be used for chromosomal localization include in situ hybridization, chromosome pre-screening with labeled flow sorting, and hybrid pre-selection to construct chromosome-specific cDM libraries.
  • Fluorescent in situ hybridization of cDM 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, 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.
  • Insulin-like growth factor binding protein 10. 45 is administered in an amount effective to treat and / or prevent a specific indication.
  • the amount and range of insulin-like growth factor binding protein 10.45 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) mRM was isolated from total RM using Quik raRNA Isolat ion Kit (product of Qiegene). 2ug poly (A) mRNA is reverse transcribed to form cDNA.
  • the Smart cDNA cloning kit purchased from Clontech) was used to insert the cDNA fragment into the multiple cloning site of the pBSK (+) vector (Clontech) to transform DH5 ⁇ , and the bacteria formed a cDNA library.
  • Dye terminate cycle react ion sequencing kit Perkin-Elmer
  • ABI 377 automatic sequencer Perkin-Elmer
  • cDNA sequences at the 5 'and 3' ends of all clones Comparing the determined cDNA sequence with the existing public DNA sequence database (Genebank), it was found that the CDM sequence of one of the clones 2525g06 was new DNA.
  • a series of primers were synthesized to determine the inserted cDNA fragments of the clone in both directions.
  • the 2525 g 06 clone contains a full-length CDM of 2175 bp (as shown in Seq ID N0: l), and has a 264 bp open reading frame (0RF) from 301 bp to 588 bp, encoding a new protein (such as Seq ID NO: 2).
  • This clone pBS-2525g06 and the encoded protein was named insulin-like growth factor binding protein 10.45.
  • the sequence of the insulin-like growth factor binding protein 10.45 of the present invention and the protein sequence encoded by the insulin-like growth factor-binding protein of the present invention were performed using the prof i le scan program (Basic local al ignment search tool) in GCG [Al tschul, SF et al. J. Mol. Biol. 1990; 215: 403-10], domain analysis was performed in databases such as Prote.
  • the insulin-like growth factor binding protein 10.45 of the present invention is homologous to the domain MOTIF, and the homology result is shown in Fig. 1.
  • the homology rate is 0.22, the score is 13.14; the threshold value is 12.28.
  • Example 3 The gene encoding the insulin-like growth factor binding protein 10.45 was cloned 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 to synthesize cDNA. Afterwards, the following primers were used for PCR
  • Pr imer 1 5'-CATCCTGAGAACTGAAATTGATCGC-3 '(SEQ ID NO: 3)
  • Pr imer2 5 -ATAAAATTTTTGAATTTATGTTCAA-3 '(SEQ ID NO: 4)
  • Pr iraerl is a forward sequence starting at lbp of the 5th end of SEQ ID NO: 1;
  • Primer 2 is the 3, terminal reverse sequence of SEQ ID NO: 1.
  • Amplification conditions 50 ⁇ l reaction volume contains 50 mmol / L KC1, 10 mmol / L Tris-HCl, pH 8. 5, 1. 5 mmol / L MgCl 2 , 20 ( ⁇ mol / L dNTP, lOpmol primer, 1U Taq DNA polymerase (Clontech). Reaction was performed on a PE OO DNA thermal cycler (Perkin-Elmer) for 25 cycles under the following conditions: 94 ° C 30sec; 55 ° C 30sec; 72 ° C 2min. At RT -Set ⁇ -act in at the same time for PCR For positive control and template blank as negative control.
  • Amplification products were purified using a QIAGEN kit, and the TA cloning kit was used to connect to a PCR vector (Unvi trogen). DM sequence analysis results showed that the DNA sequence of the PCR product was exactly the same as that of 1-2175bp shown in SEQ ID NO: 1.
  • Example 4 Nor thern blot analysis of insulin-like growth factor binding protein 10.45 gene expression
  • This method involves acid guanidinium thiocyanate phenol-chloroform extraction. That is, the tissue was homogenized with 4M guanidine 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) Centrifuge after mixing. Aspirate the aqueous layer, add isopropanol (0.8 vol) and centrifuge the mixture to obtain RNA precipitate. The resulting RNA pellet was washed with 70% ethanol, dried and dissolved in water.
  • 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 (pH7. 4) -5 x SSC-5 x Denhardt, s solution and 200 ⁇ g / ml salmon sperm DNA. After hybridization, the filter was washed in 1 X SSC-0. 1% SDS at 55 ° C. for 30 min. Then, Phosphor Imager was used for analysis and quantification.
  • Example 5 In vitro expression, isolation, and purification of recombinant insulin-like growth factor binding protein 10.45 According to the sequence of the coding region shown in SEQ ID NO: 1 and FIG. 1, a pair of specific amplification primers were designed. The sequences are as follows:
  • Pr imer3 5 -CCCCATATGATGCTCTGTCACCTTCAAAGGATGG-3 '(Seq ID No: 5)
  • Pr imer 4 5 -CCCAAGCTTCTTCAACATGCCGCTTCTGTTCTTC-3' (Seq ID No: 6)
  • the 5 'ends of these two primers contain Ndel and BamHI restriction sites, respectively , followeded by the coding sequences of the 5 'and 3' ends of the gene of interest, respectively, and the Ndel and BamHI restriction sites correspond to the selection on the expression vector plasmid pET 28b (+) (Novagen, Cat. No. 69865. 3) Sex endonuclease site.
  • PCR was performed using pBS-2525g06 plasmid containing the full-length target gene as a template.
  • the PCR reaction conditions were: pBS_2525 g 06 plasmid 10pg, primer ⁇ 1: 11116]:-3 and ⁇ 1: 1 [116]:-4 in a total volume of 50 ⁇ 1; 1 is 10 11101, Advantage polymerase Mix (Clontech) Product) 1 ⁇ 1.
  • Cycle parameters 94 ° C 20s, 60 ° C 30s, 68. C 2 min, a total of 25 cycles. Double digestion of the amplified product and plasmid pET-28 (+) with Mel and BamHI, respectively, to recover large pieces And ligated with T4 ligase.
  • the ligation product was transformed into E. coli DH5a by the calcium chloride method. After being cultured overnight on LB plates containing kanamycin (final concentration 30 ⁇ ⁇ / ⁇ 1), positive clones were selected by colony PCR method and sequenced. A positive clone (pET-2525 g 06) with the correct sequence was selected, and the recombinant plasmid was transformed into E. coli BL21 (DE3) plySs (Nova g en) by the calcium chloride method.
  • the host strain BL21 (pET-2525g06) was cultured at 37 ° C to the logarithmic growth phase, and IPTG was added to the final concentration liMio l / L, continue to incubate for 5 hours. Centrifuge the bacterial cells, blast them with ultrasound, collect the supernatant by centrifugation, and use an affinity chromatography column His s. Bind Quick Cartridge (6 histidine (6His-Tag)) Novagen's product) was chromatographed to obtain the purified protein IGF-binding protein 10.45.
  • a peptide synthesizer (product of PE company) was used to synthesize the following insulin-like growth factor binding protein 10. 45 specific peptides:
  • the polypeptide is coupled with hemocyanin and bovine serum albumin to form a complex, respectively.
  • hemocyanin and bovine serum albumin For the method, see: Avrameas, et al. Imtnunochemi s try, 1969; 6: 43. Rabbits were immunized with 4 mg of the hemocyanin polypeptide complex plus complete Freund's adjuvant, and 15 days later, the hemocyanin polypeptide complex plus incomplete Freund's adjuvant was used to boost immunity once.
  • a titer plate coated with a 15 g / ml bovine serum albumin peptide complex was used as an ELISA to determine antibody titers in rabbit serum.
  • Total IgG was isolated from antibody-positive rabbit serum using protein A-Sepharose.
  • the immunoprecipitation method proved that the purified antibody can specifically bind to insulin-like growth factor binding protein 10.45.
  • Example 7 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 tissues or Whether the expression in tissue cells is abnormal.
  • the purpose of this example is to select a suitable oligonucleoside from the polynucleotide SEQ ID NO: 1 of the present invention
  • the acid fragment is used as a hybridization probe, and the membrane hybridization method is used to identify whether some tissues contain the polynucleotide sequence of the present invention or a homologous polynucleotide sequence.
  • Filter hybridization methods include dot blotting, Southern blotting, Northern blotting, and copying methods. They all use the same steps of hybridization after fixing the polynucleotide sample to be tested on the filter.
  • 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
  • the GC content is 30% -70%, and the non-specific hybridization increases when it exceeds;
  • 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 known genomic sequences and their complements For homology comparison of the regions, 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 generally;
  • Probe 1 which belongs to the first type of probe, is completely homologous or complementary to the gene fragment of SEQ ID NO: 1 (41Nt):
  • Probe 2 which belongs to the second type of probe, is equivalent to the replacement mutation sequence (41Nt) of the gene fragment of SEQ ID NO: 1 or its complementary fragment:
  • PBS phosphate buffered saline
  • step 14 Resuspend the DNA pellet in a small volume of TE or water. Vortex at low speed or suck with a dropper while gradually increasing TE, mix until DM is fully dissolved, and add approximately 1 ul per 1-5 ⁇ 10 ⁇ cells extracted.
  • steps 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 as to be used in the following experimental steps
  • the film was washed with high-strength conditions and strength conditions, respectively.
  • the sample film was placed in a plastic bag, was added 3 - 1 Omg prehybridization solution (lOxDenhardfs; 6xSSC, 0. lrag / ml CT DNA ( calf thymus DM)). After sealing the bag, shake at 68 ° C for 2 hours.

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Abstract

L'invention concerne un nouveau type de polypeptide-protéine 10.45 conjuguée au facteur de croissance insulinoïde HOMO et un polynucléotide codant ledit polypeptide ainsi qu'un processus de production dudit polypeptide par génie génétique. L'invention concerne également la méthode d'application du polypeptide au traitement de divers types de maladies tels que des malformations du développement embryonnaire, le cancer. L'invention concerne également un antagoniste ainsi que l'emploi thérapeutique du polypeptide. De plus, elle a trait à l'utilisation du polynucléotide codant ladite protéine 10.45 conjuguée au facteur de croissance insulinoïde HOMO.
PCT/CN2001/001374 2000-09-12 2001-09-10 Nouveau polypeptide-proteine 10.45 conjuguee au facteur de croissance insulinoide homo et polynucleotide codant ledit polypeptide WO2002048186A1 (fr)

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CN00125134.1 2000-09-12
CN 00125134 CN1342678A (zh) 2000-09-12 2000-09-12 一种新的多肽——胰岛素样生长因子结合蛋白10.45和编码这种多肽的多核苷酸

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Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DU QING-YOU ET AL.: "Progress on the studies of insulin-like growth factor binding proteins", PROG. BIOCHEM. BIOPHYS., vol. 25, no. 1, 1998, pages 104 - 105, 158 *
MO ZENGNAN ET AL.: "The inhibitory effects of insulin-like growth factor 1 on the induction of IGFBP3 in prostate stromal cell by TGF beta 1", CHIN. J. EXP. SURG., vol. 16, no. 6, November 1999 (1999-11-01), pages 506 - 507 *

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