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WO2002012493A1 - Nouveau polypeptide, proteine de liaison 11.88 du facteur de croissance de type insuline, et polynucleotide codant ce polypeptide - Google Patents

Nouveau polypeptide, proteine de liaison 11.88 du facteur de croissance de type insuline, et polynucleotide codant ce polypeptide Download PDF

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Publication number
WO2002012493A1
WO2002012493A1 PCT/CN2001/000951 CN0100951W WO0212493A1 WO 2002012493 A1 WO2002012493 A1 WO 2002012493A1 CN 0100951 W CN0100951 W CN 0100951W WO 0212493 A1 WO0212493 A1 WO 0212493A1
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Prior art keywords
polypeptide
polynucleotide
insulin
growth factor
binding protein
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PCT/CN2001/000951
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English (en)
Chinese (zh)
Inventor
Yumin Mao
Yi Xie
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Biowindow Gene Development Inc. Shanghai
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Application filed by Biowindow Gene Development Inc. Shanghai filed Critical Biowindow Gene Development Inc. Shanghai
Priority to AU95377/01A priority Critical patent/AU9537701A/en
Publication of WO2002012493A1 publication Critical patent/WO2002012493A1/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
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4743—Insulin-like growth factor binding protein

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 11.88, and a polynucleotide sequence encoding the polypeptide. The invention also relates to a preparation method and application of the polynucleotide and polypeptide. Background technique
  • IGF-II are both mitogenic polypeptides that are structurally and functionally homologous to preproinsulin
  • IGF GH growth-promoting effectors by IGF in cartilage and other tissues
  • IGF may play a role in fetal growth and development at the same time. It plays a very important role, especially in the central nervous system.
  • IGF has a very strong affinity for many proteins and rarely exists alone. On the cell surface, IGF binds tightly to the membrane-bound receptors of IGF-1 and IGF-II, and may also bind to other membrane-bound proteins [De Vrode M, Tseng L, Katsoyannis P et al., 1986. J Clin Invest 77: 602-613] 0 So far, it has been found a molecular weight of 150, 30, 53kDa, and other molecular weight from 24 to 160 kDa specific protein binding. All 30 kDa binding proteins have similar amino acid composition, and the first 10 amino acids at the N-terminus are consistent.
  • IGF-binding protein produced by human HBP G2 hepatocellular carcinoma cells is named IGF BP-25.
  • the IGF-BP complete cDNA has 1553 bp, including a 164 bp 5 'non-coding region, a 777 bp open reading frame, and a 612 bp 3' non-coding region, of which a 612 bp 3 'non-coding region has a 3'- poly (A) A 12 bp adenylation signal upstream of the tail.
  • the 25 amino acid residues at the N-terminus of the IGF-BP protein contain typical split signal peptide components: an N-terminal region with positively charged amino acid residues (8), a hydrophobic central region (S amino acids), and an Extremely Sexual C-terminal region.
  • the mature IGF BP-25 has 234 amino acid residues, and the 59 amino acid residues at the N-terminus form a cysteine-rich region. Within this domain, cysteine residues account for 19%, and their spacing is regular. These cysteine residues play a very important role in the structure and function of the binding protein.
  • the present invention is named insulin-like growth factor binding protein 11.88.
  • insulin-like growth factor binding protein 11.88 protein plays an important role in regulating important functions of the body such as cell division and embryonic development, and it is believed that a large number of proteins are involved in these regulatory processes, there has been a need in the art to identify more involved in these
  • the process of insulin is similar to the 11.88 protein of growth factor binding protein, especially the amino acid sequence of this protein is identified.
  • New insulin-like growth factor binding protein 11.88 The isolation of the protein-coding gene also provides the basis for research to determine the role of this protein in health and disease states. This protein may form the basis for the development of diagnostic and / or therapeutic drugs for diseases, so it is important to isolate its coding DNA. Disclosure of invention
  • Another object of the invention is to provide a polynucleotide encoding the polypeptide.
  • Another object of the present invention is to provide a recombinant vector containing a polynucleotide encoding an insulin-like growth factor binding protein 11.88.
  • 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 11.88.
  • Another object of the present invention is to provide a method for producing insulin-like growth factor binding protein 11.88.
  • Another object of the present invention is to provide an antibody against the polypeptide-insulin-like growth factor binding protein 11.88 of the present invention.
  • Another object of the present invention is to provide an insulin-like growth factor complex directed to the polypeptide of the present invention.
  • Synthetic protein 11.88 mimics, antagonists, agonists, inhibitors.
  • Another object of the present invention is to provide a method for diagnosing and treating diseases related to abnormalities of insulin-like growth factor binding protein 11.88.
  • 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 983-1 to 313 in SEQ ID NO: 1; and (b) a sequence having 1- in SEQ ID NO: 1 1487-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 invention also relates to a method for screening compounds that mimic, activate, antagonize or inhibit the activity of insulin-like growth factor binding protein 11.88 protein, which comprises utilizing the polypeptide of the invention.
  • the invention also relates to compounds obtained by this method.
  • the invention also relates to a method for in vitro detection of a disease or susceptibility to insulin-like growth factor binding protein 1 1.
  • 88 protein abnormal expression which comprises detecting mutations in the polypeptide or a sequence encoding the polynucleotide in a biological sample. Or detecting the amount or biological activity of a polypeptide of the invention in a biological sample.
  • the invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a polypeptide of the invention or a mimetic thereof, an activator, an antagonist or an inhibitor, and a pharmaceutically acceptable carrier.
  • the present invention also relates to the use of the polypeptide and / or polynucleotide of the present invention in the preparation of a medicament for treating cancer, developmental disease or immune disease or other diseases caused by abnormal expression of insulin-like growth factor binding protein 11.88.
  • Nucleic acid sequence means an oligonucleotide, a nucleotide or a polynucleotide and a fragment or part thereof, and may also be Refers to the genomic or synthetic DM or RNA, which 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 refers to an alteration in the amino acid sequence or nucleotide sequence that results in an increase in one or more amino acids or nucleotides compared to a naturally occurring molecule.
  • Replacement refers to the replacement of one or more amino acids or nucleotides with different amino acids or nucleotides.
  • Bioactivity refers to a protein that has the structure, regulation, or biochemical function of a natural molecule.
  • immunologically active refers to the ability of natural, recombinant or synthetic proteins and fragments thereof to induce a specific immune response in appropriate animals or cells and to bind to specific antibodies.
  • An "agonist” refers to a molecule that, when combined with insulin-like growth factor binding protein 11.88, can cause changes in the protein and thereby regulate the activity of the protein.
  • An agonist may include a protein, a nucleic acid, a carbohydrate, or any other molecule that can bind an insulin-like growth factor binding protein II. ⁇ 8.
  • Antagonist refers to a biological or immunological activity that can block or modulate insulin-like growth factor binding protein 1 1. 88 when bound to insulin-like growth factor binding protein 1 1. 1. molecule. Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates or any other molecule that can bind insulin-like growth factor binding protein 11.88.
  • Regular refers to a change in the function of insulin-like growth factor binding protein 11.88, including an increase or decrease in protein activity, a change in binding characteristics, and any other biological properties of insulin-like growth factor-binding protein 11.88, Changes in functional or immune properties.
  • Substantially pure 1 'means essentially free of other proteins, lipids, sugars or other substances naturally associated with it.
  • Those skilled in the art can use standard protein purification techniques to purify insulin-like growth factor binding protein 11.88 Basically pure insulin-like growth factor-binding protein 11.88 produces a single main band on a non-reducing polyacrylamide gel.
  • Insulin-like growth factor-binding protein 11. 88 The purity of the polypeptide can be ammonia Analysis of amino acid sequences.
  • 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 conditions with reduced stringency allow non-specific binding, because conditions with reduced stringency require that the two sequences bind to each other as either specific or selective interactions.
  • Percent identity refers to the percentage of sequences that are identical or similar in the comparison of two or more amino acid or nucleic acid sequences. The percent identity can be determined electronically, such as by the MEGALIGN program (Lasergene sof tware package, DNASTAR, Inc., Mad is on Wis.). The MEGALIGN program can compare two or more sequences according to different methods Method C lus ter (H i gg ins, DG and PM Sharp (1988) Gene 73: 237-244) 0 C l us ter method by examining all the pairs The distance between them arranges the groups of sequences into clusters. The clusters are then assigned in pairs or groups.
  • Method C lus ter H i gg ins, DG and PM Sharp (1988) Gene 73: 237-244
  • the percent identity between two amino acid sequences such as sequence A and sequence B is calculated by the following formula: The number of matching residues between sequence A and sequence X 1 00 The number of residues in sequence A-the interval residues in sequence A The number of spacer residues in sequence B can also be determined by Clus ter method or using methods known in the art such as Jo tun He in (%). He He J. (1990) Me thods in emzumo (logy 183: 625-645) 0 "Similarity" refers to the degree of identical or conservative substitutions of amino acid residues at corresponding positions in the alignment of amino acid sequences.
  • Amino acids used for conservative substitutions may include aspartic acid and glutamic acid; positively charged amino acids may include lysine and arginine; having an uncharged head group is Similar hydrophilic amino acids may include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; serine and threonine; phenylalanine and tyrosine.
  • Antisense refers to a nucleotide sequence that is complementary to a particular DNA or RNA sequence.
  • Antisense strand means A nucleic acid strand complementary to a “sense strand”.
  • Derivative refers to a chemical modification of HFP or a nucleic acid encoding it. This chemical modification may be a substitution of a hydrogen atom with a fluorenyl, acyl or amino group. Nucleic acid derivatives can encode polypeptides that retain the main biological properties of natural molecules.
  • Antibody refers to a complete antibody molecule and its fragments, such as Fa,? ( ⁇ ') 2 and? , which can specifically bind to the insulin-like growth factor binding protein 11.88 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 a substance from its original environment (for example, its natural environment if it is naturally occurring).
  • a naturally-occurring polynucleotide or polypeptide is not isolated when it is present in a living thing, but the same polynucleotide or polypeptide is separated from some or all of the substances that coexist with it in the natural system.
  • Such a polynucleotide may be part of a certain vector, or such a polynucleotide or polypeptide may be part of a certain composition. Since the carrier or composition is not part of its natural environment, they are still isolated.
  • isolated refers to the separation of a substance from its original environment (if it is a natural substance, the original environment is the natural environment).
  • polynucleotides and polypeptides in a natural state in a living cell are not isolated and purified, but the same polynucleotides or polypeptides are separated and purified if they are separated from other substances in the natural state .
  • isolated insulin-like growth factor-binding protein 11.88 means insulin-like growth factor-binding protein 1 1. 88 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 11.88 using standard protein purification techniques. Substantially pure peptides can produce a single main band on a non-reducing polyacrylamide gel. Insulin-like growth factor binding protein 11. 88 The purity of the peptide can be analyzed by amino acid sequence.
  • the present invention provides a novel polypeptide-insulin-like growth factor binding protein, which is basically composed of the amino acid sequence shown in SEQ ID NO: 2.
  • the polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide, and preferably a recombinant polypeptide.
  • the polypeptides of the 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.
  • polypeptides of the invention may be glycosylated, or they may be non-glycosylated.
  • the polypeptides of the invention may also include or exclude the initial methionine residue.
  • the invention also includes fragments, derivatives and analogues of insulin-like growth factor binding protein 11.88. Thing.
  • fragment refers to a polypeptide that substantially maintains the same biological function or activity of the insulin-like growth factor binding protein 11.88 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 substituted with conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and the substitution
  • the amino acid may or may not be encoded by the genetic code; or (TI) is one in which a group on one or more amino acid residues is replaced by another group to include a substituent; or (III) such A type in which a mature polypeptide is fused to another compound (such as a compound that extends the half-life of a polypeptide, such as polyethylene glycol); or (IV) a type of polypeptide sequence in which an additional amino acid sequence is fused into a mature polypeptide (such as the leader sequence or secreted sequence or the sequence used to purify this polypeptide or protease sequence)
  • such fragments, derivatives and analogs are considered to be within the knowledge of those skilled in the art.
  • the present invention provides an isolated nucleic acid (polynucleotide), which basically consists of a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • the polynucleotide sequence of the present invention includes the nucleotide sequence of SEQ ID NO: 1.
  • the polynucleotide of the present invention is found from a cDNA library of human fetal brain tissue. It contains a polynucleotide sequence of 1487 bases in length and its open reading frame of 987- 1 31 3 encodes 108 amino acids. According to the comparison of gene chip expression profiles, it was found that this polypeptide has a similar expression profile to insulin-like growth factor binding protein. It can be inferred that the insulin-like growth factor-binding protein 11. 88 has similar functions to insulin-like growth factor-binding protein.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • DM forms include cDNA, genomic DNA, or synthetic DM.
  • DNA can be single-stranded or double-stranded.
  • DNA can be coding or non-coding.
  • the coding region sequence encoding a mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO: 1 or a degenerate variant.
  • degenerate variant refers to a nucleic acid sequence encoding a protein or polypeptide having 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 comprising the polypeptide and a polynucleotide comprising additional coding and / or non-coding sequences.
  • the invention also relates to variants of the polynucleotides described above, which encode polypeptides or fragments, analogs and derivatives of polypeptides having the same amino acid sequence as the invention.
  • Variants of this polynucleotide may be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants Body, deletion variant, and insertion variant.
  • 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 501 ⁇ 2 (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% or more It is more preferable that 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 insulin-like growth factor binding protein 11.88.
  • 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 11.88 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) separating the double-stranded DNA sequence from the DM of the genome; 2) chemically synthesizing the DNA sequence to obtain the double-stranded DNA of the polypeptide.
  • genomic DNA isolation is the least commonly used. Direct chemical synthesis of DNA sequences is often the method of choice. The more commonly used method is the isolation of cDNA sequences.
  • the standard method for isolating cDNA of interest is to isolate mRNA from donor cells that overexpress the gene and perform reverse transcription to form a plasmid or phage cDNA library.
  • mRM plasmid or phage cDNA library.
  • 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 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 can be screened from these cDNA libraries by conventional methods. These methods include (but not Limited to): (l) DNA-DNA or DNA-RNA hybridization; (2) the presence or absence of marker gene functions; (3) determination of the level of insulin-like growth factor binding protein 11.88 transcripts; (4) by immunization Technology or measurement of biological activity to detect gene-expressed protein products. The above methods can be used singly or in combination.
  • the probe used for hybridization is a source with any part of the polynucleotide of the present invention, and has a length of at least 10 nucleotides, preferably at least 30 nucleotides, more preferably at least 50 nucleotides, preferably at least 100 nucleotides. In addition, the length of the probe is usually within 2,000 nucleotides, and preferably within 1,000 nucleotides.
  • the probe used here is generally a DNA sequence chemically synthesized based on the gene sequence information of the present invention. The genes or fragments of the present invention can of course be used as probes. DNA probes can be labeled with radioisotopes, luciferin, or enzymes (such as alkaline phosphatase).
  • the protein product of the 1.88 gene expression of insulin-like growth factor binding protein 11 can be detected by immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA).
  • immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA).
  • a method using PCR technology to amplify DNA / RNA is preferably used to obtain the gene of the present invention.
  • the RACE method RACE-Rapid Amplification of cDNA Ends
  • the primers used for PCR can be appropriately based on the polynucleotide sequence information of the present invention disclosed herein. Selected and synthesized by conventional methods.
  • the amplified DNA / RNA fragments can be isolated and purified by conventional methods such as by gel electrophoresis.
  • polynucleotide sequence of the gene of the present invention or various DNA fragments and the like obtained as described above can be determined by a conventional method such as dideoxy chain termination method (Sanger et al. PNAS, 1977, 74: 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, the sequencing must be repeated. Sometimes it is necessary to determine the cDNA sequence of multiple clones in order to splice into a full-length 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 11.88 coding sequence, and recombinant technology to produce the present invention. Said method of polypeptide.
  • a polynucleotide sequence encoding an insulin-like growth factor binding protein 11.88 can be inserted into a vector to constitute a recombinant vector containing the polynucleotide of the present invention.
  • vector refers to bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors well known in the art.
  • Vectors suitable for use in the present invention include, but are not limited to: T7 promoter-based expression vectors expressed in bacteria (Rosenberg, et al. Gene, 1987, 56: 125); MSXND expression vectors expressed in mammalian cells ( Lee and Na thans, J Bi o
  • any plasmid and vector can be used to construct a recombinant expression vector.
  • An important feature of expression vectors is that they usually contain an origin of replication, a promoter, a marker gene, and translational regulatory elements.
  • Methods known to those skilled in the art can be used to construct expression vectors containing a DNA sequence encoding an insulin-like growth factor binding protein 11.88 and appropriate transcription / translation regulatory elements. These methods include in vitro recombinant DNA technology, DM synthesis technology, in vivo recombination technology, etc. (Sambroook, et al. Mol ecu lar Cloning, a Laboratory Manua, Cold Spoon Harbor Labora tory. New York, 1989).
  • the DNA sequence may be operably linked to an appropriate promoter in an expression vector to direct NA 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 an insulin-like growth factor binding protein 11.88 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. Representative examples are: E.
  • coli Streptomyces
  • bacterial cells such as Salmonella typhimurium
  • fungal cells such as yeast
  • plant cells such as fly S2 or Sf 9
  • animal cells such as CH0, COS or Bowes melanoma cells.
  • Transformation of a host cell with a DNA sequence 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 of DNA uptake can be harvested after exponential growth phase, treated with CaC l 2 method used in the step are well known in the art.
  • the alternative is to use MgC l 2 .
  • transformation can also be performed by electroporation.
  • the following DNA transfection methods can be used: calcium phosphate co-precipitation method, or conventional mechanical methods such as microinjection, electroporation, and liposome packaging.
  • the polynucleotide sequence of the present invention can be used to express or produce recombinant insulin-like growth factor binding protein 11.88 (Scence, 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. After the host cells have grown to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.
  • a suitable method such as temperature conversion or chemical induction
  • the recombinant polypeptide may be coated in a cell, expressed on a cell membrane, or secreted outside the cell.
  • recombinant proteins can be separated and purified by various separation methods using their physical, chemical and other properties. These methods are well known to those skilled in the art. These methods include, but are not limited to: conventional renaturation treatment, protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • conventional renaturation treatment protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid chromatography
  • FIG. 1 is a comparison diagram of gene chip expression profiles of insulin-like growth factor binding protein 11.88 and insulin-like growth factor binding protein of the present invention.
  • the upper graph is a graph of the expression profile of insulin-like growth factor binding protein 11.88, and the lower graph is the graph of the expression profile of insulin-like growth factor binding protein.
  • 1-bladder mucosa 2- PMA + Ecv304 cell line, 3-LPS + Ecv304 cell line thymus, 4-normal fibroblasts 1024NC, 5-Fibrobl as t, growth factor stimulation, 1024NT, 6- scar growth into fc Factor stimulation, 1013HT, 7-scar scar into fc without stimulation with growth factor, 101 3HC, 8-bladder cancer cell EJ, 9-bladder cancer, 10-bladder cancer, 11-liver cancer, 12-liver cancer cell line, 13 -Fetus, 14-spleen, 15-prostate cancer, 16-jejunum Adenocarcinoma, 17 cardia cancer.
  • Figure 2 shows the polyacrylamide gel electrophoresis (SDS-PAGE) of the isolated insulin-like growth factor binding protein 11.88. 12kDa is the molecular weight of the protein. The arrow indicates the isolated protein band. The best way to implement the invention
  • Total human fetal brain RNA was extracted by one-step method with guanidine isothiocyanate / phenol / chloroform.
  • MRNA is formed by reverse transcription cDNA Quik mRNA Isolation Kit (Qiegene Co.) isolated from the total RNA poly (A) mRNA 0 2ug poly ( A) used.
  • the Smart cDNA cloning kit purchased from Clontech was used to insert the cDNA fragment into the multicloning site of pBSK (+) vector (Clontech) to transform DH5 ⁇ .
  • the bacteria formed a cDNA library.
  • Dye terminate cycle reaction sequencing kit Perkin-Elmer
  • ABI 377 automatic sequencer Perkin-Elmer
  • the determined cDNA sequence was compared with the existing public DNA sequence database (Genebank), and it was found that the cDNA sequence of one of the clones 0843D07 was new DNA.
  • a series of primers were synthesized to determine the inserted cDNA fragments of the clone in both directions.
  • CDNA was synthesized using fetal brain total RNA as a template and oligo-dT as a primer for reverse transcription reaction. After purification with Qiagene's kit, the following primers were used for PCR amplification:
  • Primerl 5
  • AGAATTCAGATCTCTGTCAGCCCG —3 5
  • SEQ ID NO: 3 5
  • Primer2 5'- AAGGAGTCTTGCTCTGTCACCCAG -3 '(SEQ ID NO: 4)
  • Primerl is a forward sequence starting at lbp at the 5 ′ end of SEQ ID NO: 1;
  • Primer2 is the 3 'end reverse sequence in SEQ ID NO: 1.
  • Amplification conditions 50 ⁇ l / L KCl'10 Lol / L Tris- in a reaction volume of 50 ⁇ 1 CI, (pH8.5), 1.5mmol / L MgCl 2 , 200 ⁇ mol / L 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 0 ⁇ -act in was set as positive during RT-PCR Controls and template blanks are negative controls.
  • the QIAGEN kit was purified and the TA cloning kit was connected to a PCR vector (Invitrogen). The results of DNA sequence analysis showed that the DNA sequence of the PCR product was identical to that of 1 to 1487bp shown in SEQ ID NO: 1.
  • Example 3 Northern blot analysis of insulin-like growth factor binding protein 11.88 gene expression: one-step extraction of total RNA [Anal. Biochem 1987, 162, 156-159] 0 This method includes acid guanidinium thiocyanate-chloroform extraction .
  • a 32P-labeled probe (approximately 2 x 10 6 cpm / ml) was hybridized with a nitrocellulose membrane to which RNA was transferred in a solution at 4 2 ° C overnight, the solution containing 50% formamide- 25 mM ⁇ 2 P0 4 ( ⁇ 7.4)-5 X SSC- 5 X Denhardt's solution and 200 ⁇ ⁇ / ⁇ 1 salmon sperm DNA. After hybridization, the filter was washed in 1 X SSC-0.1% SDS at 55 ° C for 30 min. Phosphor Imager was then analyzed and quantified.
  • Example 4 In vitro expression, isolation, and purification of recombinant insulin-like growth factor-binding protein 11.88 'According to the sequential increase of the coding region shown in SEQ ID NO: 1 and Figure 1, a pair of specific primers were designed, and the sequence increase was as follows:
  • Primer3 5,-CCCCATATGATGTGCTGGGGGATCCCTTCCACC- 3, (Seq ID No: 5)
  • Primer4 5'-CCCAAGCTTTCACCATGTTGGCCAGGCTGGTCT-3 '(Seq ID No: 6)
  • These two primers contain Ndel and Hindlll restriction sites, respectively.
  • the coding sequences of the 5 'and 3' ends of the target gene were increased, and the Ndel and Hindlll restriction sites corresponded to the selectivity on the expression vector plasmid pET-28b (+) (Novagen, Cat. No. 69865.3). Endonuclease site.
  • PCR was performed using the pBS-0843D07 plasmid containing the full-length target gene as a template.
  • the PCR reaction conditions are as follows: The total volume is 50 ⁇ 1.
  • the plasmid contains 10 pg of PBS-0843D07 plasmid and primers. ⁇ 11161-3 and? ] ⁇ 1116] "-4 are lOpmol, Advantage polymerase Mix (Clontech) 1 ⁇ 1.
  • Cycle parameters 94 ° C 20s, 60 ° C 30s, 68 ° C 2 min, a total of 25 cycles.
  • Nde I and Hind 111 were used to double-digest the amplified product and plasmid pET-28 (+), respectively, and large fragments were recovered and ligated with T4 ligase.
  • the ligation product was transformed into E. coli bacteria M5 ⁇ using 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-0843D07) with the correct sequence was selected, and the recombinant plasmid was transformed into E. coli BL21 (DE3) plySs (product of No Va gen) by the calcium chloride method.
  • the host bacteria BL21 (pBT-0843D07) was cultured at 37 ° C to the logarithmic growth phase, and IPTG was added to a final concentration of 1 mmol / L, and continued Incubate for 5 hours.
  • the bacteria were collected by centrifugation, and the supernatant was collected by centrifugation. The supernatant was collected by centrifugation.
  • the purified protein of interest was similar to growth factor binding protein 11.88.
  • a peptide synthesizer (product of PE company) was used to synthesize the following 11.88 specific peptides similar to insulin-like growth factor binding protein:
  • NH2-Met-Cys-Trp-Gly-Ile-Pro-Ser-Thr-Pro-Ser-His-Leu-Gly-Leu-Ser-C00H (SEQ ID NO: 7).
  • the polypeptide is coupled to hemocyanin and bovine serum albumin to form a complex, respectively.
  • Rabbits were immunized with 1 ⁇ 4g of the hemocyanin peptide complex and complete Freund's adjuvant. After 15 days, the rabbit was immunized with hemocyanin peptide complex and incomplete Freund's adjuvant once.
  • Suitable oligonucleotide fragments selected from the polynucleotides of the present invention are used as hybridization probes in a variety of ways.
  • the probes can be used to hybridize to genomic or cDNA libraries of normal tissue or pathological tissue from different sources to It is determined whether it contains the polynucleotide sequence of the present invention and a homologous polynucleotide sequence is detected.
  • the probe can be used to detect the polynucleotide sequence of the present invention or its homologous polynucleotide sequence in normal tissue or pathology. Whether the expression in tissue cells is abnormal.
  • the purpose of this embodiment is to select a suitable oligonucleotide fragment from the polynucleotide SEQ ID NO: 1 of the present invention as a hybridization probe, and to identify whether some tissues contain the polynucleoside of the present invention by a filter hybridization method Acid sequence or a homologous polynucleotide sequence thereof.
  • 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), so that the hybridization background is reduced and only strong specific signals are retained.
  • 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 the GC content is exceeded;
  • Those that meet the above conditions can be used as primary selection probes, and then further computer sequence analysis, including the primary selection probe and its source sequence region (ie, SEQ ID NO: 1) and other unknown genomic sequences and their complements The regions are compared for homology. If the homology with the non-target molecular region is greater than 85% or there are more than 15 consecutive bases, the primary probe should not be used;
  • Probe 1 which belongs to the first type of probe, is completely homologous or complementary to the gene fragment of SEQ ID NO: 1 (41Nt):
  • Probe 2 (P robe2), belong to the second probe, corresponding to SEQ ID NO: replacing a gene fragment or a complementary fragment of a mutated sequence (41Nt): 5'-TGTGCTGGGGGATCCCTTCCCCCCCCAGTCATTTGGGACTC-3 '(SEQ ID NO: 9)
  • SEQ ID NO: 9 For other commonly used reagents and their preparation methods not related to the following specific experimental procedures, please refer to the literature: DM PROBES GHKeller; M. Manak; Stockton Press, 1989 (USA ) And more commonly used molecular cloning experiment manual books such as "Molecular Cloning Experiment Guide” (Second Edition 1998) [US] Sambrook et al., Science Press.
  • PBS phosphate buffered saline
  • step 14 can be performed directly.
  • steps 8-13 Add RMase A to the DNA solution to a final concentration of 100ug / ml, and incubate at 37 ° C for 30 minutes.
  • Add SDS and proteinase K to the final concentration of 0.5 ° / «and 100ug / ml, respectively. 37. C was held for 30 minutes.
  • NC membrane nitrocellulose membrane
  • the sample film was placed in a plastic bag pre-hybridization solution was added 3- lGmg (10xDenhardt 'S; 6xSSC, 0. lmg / ml CT DNA (calf thymus DNA). ), After sealing the bag, shake at 68 ° C for 2 hours.
  • 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, see the literature DeRisi, JL, Lyer, V. & Brown, P.0. (1997) Science 278, 680-686. And 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 respectively amplified by PCR. After purification, the concentration of the amplified product was adjusted to about 500 ng / ul, and spotted on a sloped glass medium using a Cartesian 7500 spotter (purchased from Cartesian, USA) between the points. The distance is 280 ⁇ . The spotted slides were hydrated, dried, and cross-linked in a purple diplomatic coupling instrument. After elution, the DNA was fixed on a glass slide to prepare a chip. The specific method steps are widely reported in the literature. The post-spot processing steps of this embodiment are:
  • Total mRNA was extracted from human mixed tissues and specific tissues (or stimulated cell lines) using a one-step method, and the mRNA was purified using Oligotex mRNA Midi Kit (purchased from QiaGen).
  • the fluorescent reagent Cy3dUTP 5— Amino— propargy 2'—deoxyuridine 5'—triphate coupled to Cy3 fluorescent dye, purchased from Amersham Phamacia Biotech Company
  • Cy5dUTP 5- Amino- propargy 1-2'- deoxyuridine
  • Cy5 fluorescent dye purchased from Amersham Phamacia Biotech, labeled the body's specific tissue (or stimulated cell line) mRNA, and purified the probe to prepare a probe.
  • the probes from the above two tissues and the chip were respectively hybridized in a UniHyb TM Hybridization Solution (purchased from TeleChem) hybridization solution for 16 hours, and the washing solution (1 ⁇ SSC, 0.21 ⁇ 2SDS) After washing, scan with a ScanArray 3000 scanner (purchased from General Scanning Company, USA). The scanned images are analyzed and processed with Imagene software (Biodi discovery company, USA), and the Cy3 of each point is calculated. / Cy5 ratio.
  • the above specific tissues are bladder mucosa, PMA + Ecv304 cell line, LPS + Ecv304 cell line thymus, normal fibroblasts 1024NC, F ibrob l as t, growth factor stimulation, 1024NT, scar formation fc growth factor stimulation, 1 013HT, scar into fc without growth factor stimulation, 1013HC, bladder cancer plant cell EJ, bladder cancer, bladder cancer, liver cancer, liver cancer cell line, fetal skin, spleen, prostate cancer, jejunal adenocarcinoma Cardiac cancer. Based on these Cy3 / Cy5 ratios, a bar graph is drawn. (figure 1 ) . It can be seen from the figure that the expression profiles of insulin-like growth factor binding protein 11.88 and insulin-like growth factor binding protein according to the present invention are very similar. Industrial applicability
  • polypeptide of the present invention and the antagonists, agonists and inhibitors of the polypeptide can be directly used in the treatment of diseases, for example, it can treat malignant tumors, adrenal deficiency, skin diseases, various inflammations, HIV infections and immune diseases.
  • IGF Insulin-like growth factor
  • IGF-I I are both mitogenic polypeptides that are structurally and functionally homologous to preproinsulin. They have regulatory effects on GH growth-promoting effectors in cartilage and other tissues and simultaneously It plays a very important role in the growth and development of the fetus. IGF has a very strong affinity for many proteins and rarely exists alone.
  • IGF insulin-like growth factor binding protein
  • IGF insulin-like growth factor binding protein
  • UGF insulin-like growth factor
  • the expression profile of the polypeptide of the present invention is consistent with the expression profile of human insulin-like growth factor binding protein, and both have similar biological functions.
  • the polypeptide of the present invention affects the normal function of the insulin-like growth factor (I GF) in the body, and its abnormal expression can affect the regulation of insulin-like growth factor (I GF) on cell proliferation, which leads to embryo developmental malformations and dysentery.
  • Neoplastic diseases which include but are not limited to: common embryonic malformations 1.
  • Cleft lip (most common, with alveolar cleft and cleft palate), cleft lip, facial oblique cleft, cervical pouch, cervical fistula, etc.
  • Absent in longitudinal direction Absence of upper limb radius / ulnar side, lower limb tibia / fibula side, etc .;
  • Limb differentiation disorder Absence of a certain muscle or muscle group, joint dysplasia, bone deformity, bone fusion, multiple finger (toe) deformity, and finger (toe) deformity, horse tellurium varus etc .;
  • Thyroglossal duct cysts atresia or stenosis of the digestive tract, ileal diverticulum, umbilical fistula, congenital umbilical hernia, congenital agangliomegalo colon, impotence of 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 .; 2.
  • sarcoma Fibrous (sarcoma) [limbs], (Malignant) fibrohistiocytoma [limbs], lipo (sarcoma) [subcutaneous tissue, lower limbs, retroperitoneum], leiomyosarcoma (uterus and gastrointestinal), striated muscle (Sarcoma) [head and neck, genitourinary tract, limbs], hemangio (sarcoma), lymphangioma (sarcoma) [skin, subcutaneous tissue, tongue, lips], bone (sarcoma) [cranium, long bone], (malignant Giant cell tumor [femoral / tibia / upper humerus], cartilage (sarcoma) [hand and foot short bone, pelvis / rib / femoral / humerus / scapula], synovial (sarcoma) tumor [knee / ankle / wrist / shoulder / Near the elbow], (malignant) mesotheli
  • Malignant lymphoma [Neck, mediastinum, mesenteric 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 [nervous of head, neck, limbs, etc.], (malignant) glioblastoma [brain], medulloblastoma [ Cerebellum], (malignant) meningiomas [meninges], ganglioblastoma / neuroblastoma [mediastinum and retroperitoneum / adrenal medulla], etc .;
  • malignant melanoma [skin, mucous membrane], (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 in the treatment of various diseases, especially
  • the invention also provides methods of screening compounds to identify agents that increase (agonist) or suppress (antagonist) insulin-like growth factor-like binding protein 11.88.
  • Agonists increase insulin-like growth factor binding protein 1 1.
  • 88 to stimulate biological functions such as cell proliferation, while antagonists prevent and treat disorders related to cell proliferation, such as various cancers.
  • mammalian cells or membrane preparations expressing insulin-like growth factor binding protein 11.88 can be cultured in the presence of drugs with labeled insulin-like growth factor binding protein 11.88. The ability of the drug to increase or block this interaction is then determined.
  • Antagonists of insulin-like growth factor binding protein 11.88 include selected antibodies, compounds, receptor deletions, and the like. Antagonists of insulin-like growth factor binding protein 11.88 can bind to insulin-like growth factor-binding protein 1 1.88 and eliminate its function, or inhibit the production of the polypeptide The polypeptide cannot be biologically functioning because it is bound to the active site of the polypeptide.
  • insulin-like growth factor-binding protein 11.88 can be added to bioanalytical assays to determine whether a compound is a compound by measuring its effect on the interaction between insulin-like growth factor-binding protein 11.88 and its receptor. 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 11.88 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 insulin-like growth factor binding protein 11.88 molecule should generally be labeled.
  • the present invention provides a method for producing antibodies using polypeptides, and fragments, derivatives, analogs or cells thereof as antigens. These antibodies can be polyclonal or monoclonal antibodies.
  • the invention also provides antibodies directed against an insulin-like growth factor binding protein 11.88 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 11.88 directly into immunized animals (such as rabbits, mice, rats, etc.).
  • immunized animals such as rabbits, mice, rats, etc.
  • a variety of adjuvants can be used to enhance the immune response, including but not limited to Freund's Agent.
  • Techniques for preparing insulin-like growth factor binding protein 11.88 monoclonal antibodies include, but are not limited to, hybridoma technology (Kohler and Milstein. 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) 0.
  • Some techniques for producing single-chain antibodies (US Pat No. 4946778) can also be used to produce single chain antibodies against insulin-like growth factor binding protein 11.88.
  • Antibodies against insulin-like growth factor binding protein 11. can be used in immunohistochemistry to detect insulin-like growth factor binding protein 11.88 in biopsy specimens.
  • Monoclonal antibodies that bind to insulin-like growth factor binding protein 11.88 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.
  • High affinity monoclonal antibodies can covalently bind to bacterial or phytotoxins (such as diphtheria toxin, ricin, ormosine, etc.).
  • a common method is to attack the amino group of an antibody with a sulfhydryl crosslinker such as SPDP and bind the toxin to the antibody through disulfide exchange. This hybrid antibody can be used to kill insulin-like growth factor binding protein 11.88 positive cell.
  • the antibodies of the present invention can be used to treat or prevent insulin-like growth factor binding protein 11.88 Related diseases. Administration of an appropriate dose of antibody can stimulate or block the production or activity of insulin-like growth factor binding protein 11.88.
  • the invention also relates to a diagnostic test method for quantitative and localized detection of insulin-like growth factor binding protein ⁇ .88 levels. These tests are well known in the art and include FISH assays and radioimmunoassays. The level of insulin-like growth factor binding protein 11.88 detected in the test can be used to explain the importance of insulin-like growth factor-binding protein 11.88 in various diseases and to diagnose diseases in which insulin-like growth factor-binding protein 11.88 plays a role.
  • polypeptide of the present invention can also be used for peptide mapping analysis.
  • the polypeptide can be specifically cleaved by physical, chemical or enzymatic analysis, and subjected to one-dimensional or two-dimensional or three-dimensional gel electrophoresis analysis, and more preferably mass spectrometry analysis.
  • a polynucleotide encoding insulin-like growth factor binding protein 11.88 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 11.88.
  • Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated insulin-like growth factor-binding protein 11.88 to inhibit endogenous insulin-like growth factor-binding protein 11.88 activity.
  • a variant insulin-like growth factor-binding protein 11.88 may be a shortened insulin-like growth factor-binding protein 11.88 lacking a signaling domain, although it can bind to downstream substrates, but lacks signaling activity.
  • recombinant gene therapy vectors can be used to treat diseases caused by abnormal expression or activity of insulin-like growth factor binding protein 11.88.
  • Virus-derived expression vectors such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus, etc. can be used to transfer a polynucleotide encoding insulin-like growth factor binding protein 11.88 into cells.
  • Methods for constructing recombinant viral vectors carrying a polynucleotide encoding an insulin-like growth factor binding protein 11.88 can be found in the existing literature (Sambrook, et al.).
  • a recombinant polynucleotide encoding insulin-like growth factor binding protein 11.88 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 insulin-like growth factor binding protein ⁇ .88 raRNA 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 and DNA and ribozymes can be obtained by any RM or DNA synthesis technology. For example, the technology of solid phase phosphate amide synthesis of oligonucleotides has been widely used.
  • Antisense RNA molecules can Obtained by transcription of a DNA sequence encoding the MA in vitro or in vivo. This DNA sequence is integrated downstream of the RM polymerase promoter of the vector. In order to increase the stability of a nucleic acid molecule, it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the ribonucleoside linkages should use phosphate thioester or peptide bonds instead of phosphodiester bonds.
  • the polynucleotide encoding insulin-like growth factor binding protein ⁇ .88 can be used for the diagnosis of diseases related to insulin-like growth factor binding protein 11.88.
  • a polynucleotide encoding insulin-like growth factor-binding protein 11.88 can be used to detect the expression of insulin-like growth factor-binding protein 11.88 or the abnormal expression of insulin-like growth factor-binding protein 11.88 in a disease state.
  • the DNA sequence encoding the insulin-like growth factor-binding protein 11.88 can be used to hybridize biopsy specimens to determine the expression of insulin-like growth factor-binding protein 11.88.
  • Hybridization techniques include Southern blotting, Northern blotting, and in situ hybridization. These techniques and methods are publicly available, and related kits are commercially available.
  • a part or all of the polynucleotide of the present invention can be fixed as a probe on a microarray or a DNA chip (also referred to as a "gene chip") for analyzing differential expression analysis and gene diagnosis of genes in a tissue.
  • Insulin-like growth factor binding protein 11.88 specific primers for RNA-polymerase chain reaction (RT-PCR) amplification in vitro can also detect the insulin-like growth factor binding protein 11.88 transcription product.
  • Detection of mutations in the insulin-like growth factor binding protein 11.88 gene can also be used to diagnose diseases related to the insulin-like growth factor binding protein 11.88.
  • Insulin-like growth factor-binding protein 11.88 mutant forms include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to the normal wild-type insulin-like growth factor-binding protein 11.88 DNA sequence. Mutations can be detected using well-known techniques such as Southern blotting, DNA sequence analysis, PCR and in situ hybridization. In addition, mutations may affect protein expression. Therefore, Northern blotting and Western blotting can be used to indirectly determine whether a gene is mutated.
  • the sequences of the invention are also valuable for chromosome identification.
  • the sequence specifically targets a specific position on a human chromosome and can hybridize to it.
  • specific sites for each gene on the chromosome need to be identified.
  • only a few chromosome markers based on actual sequence data are available for marking chromosome positions.
  • an important first step is to locate these MA sequences on a chromosome.
  • PCR primers (preferably 15-35b P ) are prepared according to CDM, and the sequences can be mapped on chromosomes. These primers were then used for PCR screening of somatic hybrid cells containing individual human chromosomes. Only those hybrid cells that contain the human gene corresponding to the primer will produce amplified fragments.
  • PCR localization of somatic hybrid cells is a quick way to localize DNA to specific chromosomes.
  • oligonucleotide primers of the present invention by a similar method, a set of fragments from a specific chromosome can be utilized Or a large number of genomic clones to achieve sublocalization.
  • Other similar strategies that can be used for chromosomal localization include in situ hybridization, chromosome pre-screening with labeled flow sorting, and hybrid pre-selection to construct 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 physical location of the sequence on the chromosome can be correlated with the genetic map data. These data can be found in, for example, V. Mckusick, Mendel i an Inher i tance i n Man (available online with Johns Hopk ins University Welch Med ica l Library). Linkage analysis can then be used to determine the relationship between genes and diseases that have been mapped to chromosomal regions. -Next, the cDNA or genomic sequence differences between the affected and unaffected individuals need to be determined. If a mutation is observed in some or all diseased individuals and the mutation is not observed in any normal individuals, the mutation may be the cause of the disease.
  • Comparing affected and unaffected individuals usually involves first looking for structural changes in chromosomes, such as deletions or translocations that are visible at the chromosomal level or detectable with cDNA sequence-based PCR. Based on the resolution capabilities of current physical mapping and gene mapping technology, cDNAs that are accurately mapped to disease-related chromosomal regions can be one of 50 to 500 potentially pathogenic genes (assuming
  • the polypeptides, polynucleotides and mimetics, agonists, antagonists and inhibitors of the present invention can be used in combination with a suitable pharmaceutical carrier.
  • suitable pharmaceutical carrier can be water, glucose, ethanol, salts, buffers, glycerol, and combinations thereof.
  • the composition comprises a safe and effective amount of the polypeptide or antagonist, and carriers and excipients which do not affect the effect of the drug. These compositions can be used as drugs for the treatment of diseases.
  • the invention also provides a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the invention.
  • a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the invention.
  • these containers there may be instructional instructions given by government regulatory agencies that manufacture, use, or sell pharmaceuticals or biological products, which prompts the government regulatory agency that manufactures, uses, or sells them to permit their administration on the human body.
  • 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 11. 88 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 11.88 administered to a patient will depend on many factors, such as the mode of administration, the health conditions of the person to be treated, and the judgment of the diagnostician.

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Abstract

L'invention concerne un nouveau polypeptide, une protéine de liaison 11.88 du facteur de croissance de type insuline, et un polynucléotide codant ce polypeptide ainsi qu'un procédé d'obtention de ce polypeptide par des techniques recombinantes d'ADN. L'invention concerne en outre les applications de ce polypeptide dans le traitement de maladies, notamment de malformations lors du développement de l'embryon et de maladies tumorales. L'invention concerne aussi l'antagoniste agissant contre le polypeptide et son action thérapeutique ainsi que les applications de ce polynucléotide codant la protéine de liaison 11.88 du facteur de croissance de type insuline.
PCT/CN2001/000951 2000-06-14 2001-06-11 Nouveau polypeptide, proteine de liaison 11.88 du facteur de croissance de type insuline, et polynucleotide codant ce polypeptide WO2002012493A1 (fr)

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CN00116491.0 2000-06-14
CN 00116491 CN1328049A (zh) 2000-06-14 2000-06-14 一种新的多肽——胰岛素类似生长因子结合蛋白11.88和编码这种多肽的多核苷酸

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PCT/CN2001/000951 WO2002012493A1 (fr) 2000-06-14 2001-06-11 Nouveau polypeptide, proteine de liaison 11.88 du facteur de croissance de type insuline, et polynucleotide codant ce polypeptide

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CN (1) CN1328049A (fr)
AU (1) AU9537701A (fr)
WO (1) WO2002012493A1 (fr)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999032620A1 (fr) * 1997-12-22 1999-07-01 Forssmann Wolf Georg Fragments de proteine liant le facteur de croissance de substances apparentees a l'insuline et leur utilisation
US6004775A (en) * 1990-08-03 1999-12-21 The Salk Institute For Biological Studies DNA encoding IGFBP-4
WO2000023469A2 (fr) * 1998-10-16 2000-04-27 Musc Foundation For Research Development Fragments du facteur de croissance proche de l'insuline et de la proteine de fixation du facteur de croissance proche de l'insuline, et utilisations de ces fragments

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6004775A (en) * 1990-08-03 1999-12-21 The Salk Institute For Biological Studies DNA encoding IGFBP-4
WO1999032620A1 (fr) * 1997-12-22 1999-07-01 Forssmann Wolf Georg Fragments de proteine liant le facteur de croissance de substances apparentees a l'insuline et leur utilisation
WO2000023469A2 (fr) * 1998-10-16 2000-04-27 Musc Foundation For Research Development Fragments du facteur de croissance proche de l'insuline et de la proteine de fixation du facteur de croissance proche de l'insuline, et utilisations de ces fragments

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CN1328049A (zh) 2001-12-26

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