WO2001079438A2 - A new polypeptide- human amyloid precursor protein-binding protein 9 and the polynucleotide encoding it - Google Patents

Abstract

The present invention disclose a new polypeptide- human amyloid precursor protein-binding protein 9, the polynucleotide encoding it and a method producing the polypeptide by DNA recombinant technology. The present invention further disclose a method treating various disorders, e.g., Alzeihmer disease, malignant neoplasm, hemopathy, development disorders, HIV infection and immunological disease and various inflammation etc. The present invention also disclose an antagonist of the polypeptide and its therapeutic action. The present invention further disclose the use of the polynucleotide encoding the new human amyloid precursor protein-binding protein 9.

Description

 A new polypeptide-human amyloid precursor protein binding protein 9 and a polynucleoside 编码 encoding the polypeptide Technical Field ''

 The present invention belongs to the field of biotechnology. Specifically, the present invention describes a new polypeptide, a human precursor protein-binding protein 9, and a polynucleotide sequence encoding the polypeptide. The invention also relates to a preparation method and application of the polynucleotide and polypeptide. Background technique

 Alzheimer's disease is characterized by a reduction in the number of neurons and an increase in beta amyloid bodies in the brain. Beta amyloid is composed of beta-amyloid peptide (AP). AP is about 4kDa in size and is a soluble protein hydrolysate processed from β-amyloid protein precursor (APP). Studies have found that when APP-related AD-related mutations result in increased AP secretion, or APH APH. The ratio has changed. Overexpression of APP or C-terminus of APP containing AP domains has a toxic effect on neurons.

 APP is processed into mature proteins in the Golgi apparatus and transported to the cell surface. It is then secreted out of the cell or becomes an intrinsic protein immobilized on the cell membrane. APP plays a certain role in material transport. For example, excision of the YENPTY sequence of APP will affect the endocytosis of APP on the cell surface, and also allow APP to enter the intracellular pathway before reaching the cell surface, thereby disrupting the transport of intracellular materials.

 The role of APP is related to several cytokines. One of them is FE65 protein. FE65 can be combined with the cytoplasmic end of APP. FE65 is abundantly distributed in the mouse brain and intracellularly in the endoplasmic reticulum / Golgi apparatus. FE65 can increase the amount of APP bound to the cell surface and also increase the secretion of APP and AP. [J Biol Chem, Vol. 274, Issue 12, 7952-7957, March 19, 1999]

 Several APP-acting factors have been found in humans, one of which is human hFE65L protein. The C-terminal portion of the protein can interact with the cytoplasmic portion of APP. Analysis of its structure revealed that hFE65L contains several domains related to signal transmission and regulation, suggesting that it is related to this type of function. And hFE65L is homologous to murine FE65L. [Proc Natl Acad Sci U S A 1996 Oct 1; 93 (20): 10832-7]

Gene chip analysis revealed that in the thymus, testis, muscle, spleen, lung, skin, thyroid, liver, PMA + Ecv304 cell line, PMA-Ecv304 cell line, non-starved L02 cell line, arsenic-stimulated L02 L02 cell line stimulated by arsenic for 6 hours prostate, heart, lung cancer, fetal bladder, fetal small intestine, fetal large intestine, fetal thymus, fetal muscle, fetal liver, fetal kidney, fetal spleen, fetal brain, fetal lung and fetal heart In the present invention, the expression profile of the polypeptide of the present invention is very similar to the expression profile of human amyloid precursor protein binding protein 1 3, so the functions of the two may also be similar. The invention is named as human amyloid precursor protein binding protein 9.

 As mentioned above, the human amyloid precursor protein binding protein 9 protein plays an important role in regulating important functions of the body, such as cell division and embryonic development, and it is believed that a large number of proteins are involved in these regulatory processes, so more needs to be identified in the art The human amyloid precursor protein binding protein 9 protein involved in these processes, especially the amino acid sequence of this protein is identified. The separation of the new human amyloid precursor protein binding protein 9 protein encoding gene also provides a basis for research to determine the role of this protein in health and disease states. This protein may form the basis for the development of diagnostic and / or therapeutic drugs for diseases, so it is important to isolate its coding DNA. Disclosure of invention

 It is an object of the present invention to provide isolated novel polypeptides-human amyloid precursor protein binding protein 9 and fragments, analogs and derivatives thereof.

 Another object of the invention is to provide a polynucleotide encoding the polypeptide.

 Another object of the present invention is to provide a recombinant vector containing a polynucleotide encoding a human amyloid precursor protein binding protein 9.

 It is another object of the present invention to provide a genetically engineered host cell containing a polynucleotide encoding a human amyloid precursor protein binding protein 9.

 Another object of the present invention is to provide a method for producing human amyloid precursor protein binding protein 9.

 Another object of the present invention is to provide an antibody against the polypeptide-human amyloid precursor protein-binding protein 9 of the present invention.

 Another object of the present invention is to provide mimic compounds, antagonists, agonists, and inhibitors directed to the polypeptide of the present invention-human amyloid precursor protein-binding protein 9.

 Another object of the present invention is to provide a method for diagnosing and treating diseases related to abnormalities of human amyloid precursor protein binding protein 9.

 The present invention relates to an isolated polypeptide, which is of human origin, and includes: a polypeptide having an S EQ ID No. 2 amino acid sequence, or a conservative variant, biologically active fragment, or derivative thereof. Preferably, 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:

 (a) a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID D. 2;

(b) a polynucleotide complementary to the polynucleotide (a); (c) A polynucleotide having at least 70% identity to a polynucleotide sequence of (a) or (b). More preferably, the sequence of the polynucleotide is one selected from the group consisting of: (a) a sequence having positions 1 602-1 85 in SEQ ID NO: 1; and (b) having a sequence in SEQ ID NO: 1 Sequence of 1 -1 892 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.

 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 human amyloid precursor protein binding protein 9 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 detecting a disease or disease susceptibility related to abnormal expression of human amyloid precursor protein binding protein 9 protein in vitro, which comprises detecting a mutation in the polypeptide or a sequence encoding a polynucleotide thereof in a biological sample, Alternatively, the amount or biological activity of a polypeptide of the invention in a biological sample is detected.

 The present invention also relates to a pharmaceutical composition comprising a polypeptide of the present 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 for the preparation of a medicament for treating cancer, developmental disease or immune disease or other diseases caused by abnormal expression of human amyloid precursor protein binding protein 9.

 Other aspects of the invention will be apparent to those skilled in the art from the disclosure of the techniques herein. The following terms used in this specification and claims have the following meanings unless specifically stated: "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. Similarly, the term "amino acid sequence" refers to an oligopeptide, peptide, polypeptide or protein sequence and fragments or portions thereof. When the "amino acid sequence" in the present invention relates to the amino acid sequence of a naturally occurring protein molecule, such "polypeptide" or "protein" does not mean to limit the amino acid sequence to a complete natural amino acid related to the protein molecule .

A protein or polynucleotide "variant" refers to an amino acid sequence having one or more amino acids or nucleotide changes, or a polynucleotide sequence encoding it. The changes may include deletions, insertions or substitutions of amino acids or nucleotides in the amino acid sequence or 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" or "addition" 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.

 "Biological activity" refers to a protein that has the structure, regulation, or biochemical function of a natural molecule. Similarly, the term "immunologically active" refers to the ability of natural, recombinant or synthetic proteins and fragments thereof to induce a specific immune response and to bind specific antibodies in a suitable animal or cell.

 An "agonist" refers to a molecule that, when combined with human amyloid precursor protein binding protein 9, causes a change in the protein to regulate the activity of the protein. An agonist may include a protein, a nucleic acid, a carbohydrate, or any other molecule that can bind human starch precursor protein binding protein 9.

 An "antagonist" or "inhibitor" refers to a molecule that can block or regulate the biological or immunological activity of human amyloid precursor protein binding protein 9 when bound to human amyloid precursor protein binding protein 9. . Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates, or any other molecule that can bind human starch precursor protein binding protein 9.

 "Regulation" refers to a change in the function of human amyloid precursor protein binding protein 9, including an increase or decrease in protein activity, a change in binding characteristics, and any other biological properties and functions of human amyloid precursor protein binding protein 9. Or changes in immune properties.

 "Substantially pure '" means essentially free of other proteins, lipids, sugars or other substances with which it is naturally associated. Those skilled in the art can purify human starch precursor protein binding protein 9 using standard protein purification techniques 9 The substantially pure human amyloid precursor protein binding protein 9 can generate a single main band on a non-reducing polyacrylamide gel. The purity of the human amyloid precursor protein binding protein 9 polypeptide can be analyzed by amino acid sequence.

 "Complementary" or "complementary" refers to the natural binding of polynucleotides by base-pairing under conditions of acceptable salt concentration and temperature. For example, the sequence "C-T-G-A" can be combined with the 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. "Partial homology" refers to a partially complementary sequence that at least partially inhibits hybridization of a fully complementary sequence to a target nucleic acid. Inhibition of this hybridization can be achieved by hybridization under conditions of reduced stringency (Sou t he rn blot or

Nor t he rn blot, etc.) to detect. Substantially homologous sequences or hybridization probes can compete and inhibit the binding of completely homologous sequences to the target sequence under conditions of reduced stringency. This does not imply a reduction in stringency Low conditions allow non-specific binding because conditions of reduced stringency require that the two sequences bind to each other as a specific or selective interaction.

"Percent identity" refers to the percentage of sequences that are identical or similar in the comparison of two or more amino acid or nucleic acid sequences. The percent identity can be determined electronically, such as by the MEGALIGN program (Lasergene software package, Leg STAR, Inc., Madison Wis.). The MEGALIGN program can compare two or more sequences according to different methods such as the Cluster method (Higgins, DG and PM Sharp (1988) Gene 73: 237-244). _{0 The} C Luster method groups each group by checking the distance between all pairs. The sequences are arranged in clusters. The clusters are then assigned in pairs or groups. The percent identity 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 B

 X 100

 The number of residues in sequence A-the number of spacer residues in sequence A-the number of spacer residues in sequence B can also be determined by the Cluster method or by methods known in the art such as Jotun Hein. (1990) Methods in emzumology 183: 625-645).

 "Similarity" refers to the degree of identical or conservative substitutions of amino acid residues at corresponding positions in the alignment of amino acid sequences. Amino acids used for conservative substitutions, for example, negatively charged amino acids may include aspartic acid and glutamic acid; positively charged amino acids may include lysine and arginine; having an uncharged head group is Similar hydrophilic amino acids may include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; serine and threonine; phenylalanine and tyrosine.

 "Antisense" refers to a nucleotide sequence that is complementary to a particular DNA or RNA sequence. "Antisense strand" refers to a nucleic acid strand that is complementary to the "sense strand".

 "Derivative" refers to a chemical modification of HFP or a nucleic acid encoding it. Such a chemical modification may be the replacement of a hydrogen atom with an alkyl group, an acyl group or an amino group. Nucleic acid derivatives can encode polypeptides that retain the main biological characteristics of natural molecules.

"Antibody" refers to a complete antibody molecule and its fragments, such as Fa,? (^ ') ₂ and? ^ It can specifically bind to the epitope of human amyloid precursor protein binding protein 9.

 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.

The term "isolated" refers to the removal of a substance from its original environment (for example, its natural environment if it occurs naturally). For example, a naturally-occurring polynucleotide or polypeptide exists in a living animal. It is not isolated, 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. As used herein, "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). For example, 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. .

 As used herein, "isolated human amyloid precursor protein binding protein 9" refers to human amyloid precursor protein binding protein 9 which is substantially free of other proteins, lipids, carbohydrates, or other substances with which it is naturally associated. Those skilled in the art can purify human starch precursor protein binding protein 9 using standard protein purification techniques. Substantially pure polypeptides can produce a single main band on a non-reducing polyacrylamide gel. The purity of the human starch precursor protein-binding protein 9 polypeptide can be analyzed by amino acid sequence.

 The present invention provides a new polypeptide, a human amyloid precursor protein binding protein 9, which is basically composed of

Consisting 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 human amyloid precursor protein-binding protein 9. As used in the present invention, the terms "fragment", "derivative" and "analog" refer to a polypeptide that substantially retains the same biological function or activity of the human amyloid precursor protein-binding protein 9 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 a genetic codon; or (Π) such a type in which a group on one or more amino acid residues is substituted by another group to include a substituent; or (in) such One, wherein the mature polypeptide is fused to another compound (such as a compound that extends the half-life of the polypeptide, such as polyethylene glycol); or (IV) such a polypeptide sequence in which an additional amino acid sequence is fused into the mature polypeptide ( Such as the leader sequence or secreted sequence or the sequence used to purify this polypeptide or protease sequence) As explained herein, such fragments, derivatives and analogs are considered to be within the knowledge of those skilled in the art.

The present invention provides an isolated nucleic acid (polynucleotide), which basically consists of an amino acid encoding SEQ ID NO: 2 Polynucleotide composition of a polypeptide of the amino acid sequence. The polynucleotide sequence of the present invention includes the nucleotide sequence of SEQ ID NO: 1. The polynucleotide of the present invention is found from a cDNA library of human fetal brain tissue. It contains a polynucleotide sequence with a total length of 1892 bases and its open reading frame 1602-1853 encodes 83 amino acids. According to the comparison of gene chip expression profiles, it was found that this polypeptide has a similar expression profile with human amyloid precursor protein binding protein 13, and it can be inferred that the human amyloid precursor protein binding protein 9 is similar to human amyloid precursor protein binding protein 13. Functions.

 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. As used herein, a "degenerate variant" refers to a nucleic acid sequence that encodes a protein or polypeptide having SEQ ID NO: 2 but is different from the coding region sequence shown in SEQ ID NO: 1 in the present invention.

 The polynucleotide encoding the mature polypeptide of SEQ ID NO: 2 includes: only the coding sequence of the mature polypeptide; the coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence of the mature polypeptide (and optional additional coding sequences); Coding sequence.

 The term "polynucleotide encoding a polypeptide" 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. As known in the art, an allelic variant is an alternative form of a polynucleotide that may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially change the function of the polypeptide it encodes .

 The invention also relates to a polynucleotide that hybridizes to the sequence described above (having at least 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. In the present invention, "strict conditions" means: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2xSSC, 0.1% SDS, 60 ° C; or (2) hybridization When adding a denaturant, such as 50¾ (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, preferably 97% or more. In addition, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO: 2.

The invention also relates to nucleic acid fragments that hybridize to the sequences described above. As used in the present invention, "nucleic acid A "fragment" contains at least 10 nucleotides in length, preferably at least 20-30 nucleotides, more preferably at least SO-SO nucleotides, and most preferably at least 100 nucleotides. Nucleic acid fragments are also Nucleic acid amplification techniques (such as PCR) can be used to identify and / or isolate polynucleotides encoding human amyloid precursor protein binding protein 9.

 The polypeptides and polynucleotides in the present invention are preferably provided in an isolated form and are more preferably purified to homogeneity. The specific polynucleotide sequence encoding the human starch precursor protein-binding protein 9 of the present invention can be obtained by various methods. For example, 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 DNA sequence to obtain the double-stranded DNA of the polypeptide.

 Of the methods mentioned above, genomic DNA isolation is the least commonly used. Direct chemical synthesis of DNA sequences is often the method of choice. The more commonly used method is the isolation of cDNA sequences. The standard method for isolating the cDNA of interest is to isolate n from donor cells that overexpress the gene and perform reverse transcription to form a plasmid or phage cDNA library. Various methods have been developed for mRNA extraction, 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 Spring Harbor Laboratory. New York, 1989). Commercially available cDNA libraries are also available, such as different cDNA libraries from Clontech. When combined with polymerase reaction technology, even very small expression products can be cloned.

 The genes of the present invention can be selected from these cDNA libraries by conventional methods. These methods include (but are not limited to): (DDNA-DNA or DNA-RNA hybridization; (2) the presence or absence of marker gene functions; (3) determination of the level of transcripts of human amyloid precursor protein binding protein 9; ( 4) Detecting the protein product of gene expression through immunological techniques or measuring biological activity. The above methods can be used alone or in combination.

 In the method (1), 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. In addition, the length of the probe is usually within 2000 nucleotides, preferably within 1000 nucleotides. The probe used here is usually a DNA sequence chemically synthesized based on the gene sequence information of the present invention. The genes or fragments of the present invention can of course be used as probes. DNA probes can be labeled with radioisotopes, luciferin, or enzymes (such as alkaline phosphatase).

 In the method (4), the protein product of human amyloid precursor protein binding protein 9 gene expression can be detected by immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA).

Methods for Amplifying DNA / RNA Using PCR Techniques (Saiki, et al. Science 1985; 230: 1350- 1354) is preferably used to obtain the genes of the present invention. In particular, when it is difficult to obtain a full-length cDNA from a library, the RACE method (RACE-rapid amplification of cDNA ends) may be preferably used. The primers used for PCR may 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.

 The 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. 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 a human starch precursor protein binding protein 9 coding sequence, and the recombinant technology to produce the described Polypeptide method.

 In the present invention, a polynucleotide sequence encoding a human starch precursor protein binding protein 9 may be inserted into a vector to constitute a recombinant vector containing the polynucleotide of the present invention. The term "vector" refers to bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors well known in the art. Vectors suitable for use in the present invention include, but are not limited to: T7 promoter-based expression vectors (Rosenberg, et al. Gene, 1987, 56: 125) expressed in bacteria; pMSXND expression vectors expressed in mammalian cells ( Lee and Nathans, J Bio Chem. 263: 3521, 1988) and baculovirus-derived vectors expressed in insect cells. In short, as long as it can be replicated and stabilized in the host, 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 human amyloid precursor protein binding protein 9 and appropriate transcription / translation regulatory elements. These methods include in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombination technology (Sambroook, et al. Molecular Cloning, a Laboratory Manual, cold Spring Harbor Laboratory. New York, 1989). The DNA sequence can be operably linked to an appropriate promoter in an expression vector to guide mRNA 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, the early and late SV40 promoters, Retroviral LTRs and other known promoters that control the expression of genes in prokaryotic or eukaryotic cells or their viruses. The expression vector also includes a ribosome binding site and a transcription terminator for translation initiation. Insertion of enhancer sequences into the vector will enhance its transcription in higher eukaryotic cells. Enhancers are cis-acting factors for DNA expression, usually about 10 to 300 base pairs. To promoters to enhance gene transcription. Illustrative examples include SV40 enhancers from 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.

 In addition, the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reduction, neomycin resistance, and green for eukaryotic cell culture. Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.

 Those of ordinary skill in the art will know how to select appropriate vector / transcription control elements (such as promoters, enhancers, etc.) and selectable marker genes.

 In the present invention, a polynucleotide encoding a human starch precursor protein binding protein 9 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. cell. 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; insect cells such as fly S2 or Sf 9; animal cells such as CH0, COS, or Bowes s melanoma cells, etc. .

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. When the host is a prokaryote such as E. coli, competent cells capable of DNA uptake can be in the exponential growth phase were harvested, treated with (1 ₂ method used in the step are well known in the art. Alternatively, it is a M _g C l _2. If necessary, transformation can also be performed by electroporation. When the host is a eukaryotic organism, the following transfection methods can be used: calcium phosphate co-precipitation method, or conventional mechanical methods such as microinjection, electroporation, and lipid Plastid packaging, etc.

 Using conventional recombinant DNA technology, the polynucleotide sequence of the present invention can be used to express or produce recombinant human amyloid precursor protein binding protein 9 (Scence, 1 984; 224: 1 431). Generally there are the following steps:

 (1) using the polynucleotide (or variant) encoding human human amyloid precursor protein binding protein 9 of the present invention, or transforming or transducing a suitable host cell with a recombinant expression vector containing the polynucleotide;

 (2) culturing host cells in a suitable medium;

 (3) Isolate and purify protein from culture medium or cells.

 In step (2), the medium used in the culture may be selected from various conventional mediums according to the host cells used. 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.

In step (3), the recombinant polypeptide may be coated in a cell, expressed on a cell membrane, or secreted into a cell. Extracellular. If desired, recombinant proteins can be isolated 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 folding (gel filtration), adsorption chromatography, ions Exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods. Brief description of the drawings

 The following drawings are used to illustrate specific embodiments of the invention, but not to limit the scope of the invention as defined by the claims.

FIG. 1 is a comparison diagram of gene chip expression profiles of human amyloid precursor protein-binding protein 9 and human amyloid precursor protein-binding protein 13 according to the present invention. The upper graph is a graph of the expression profile of human starch precursor protein binding protein 9 and the lower graph is the graph of the expression profile of human starch precursor protein binding protein 13. Among them, 1 indicates fetal kidney, 2 indicates fetal large intestine, 3 indicates fetal small intestine, 4 indicates fetal muscle, 5 indicates fetal brain, 6 indicates fetal bladder, 7 indicates non-starved L02, 8 indicates L02 +, Ihr, As ³⁺ , and 9 indicates ECV304 PMA, 10 means ECV304 PMA +, 11 means fetal liver, 12 means normal liver, 13 means thyroid, 14 means skin, 15 means fetal lung, 16 means lung, Π means lung cancer, 18 means fetal spleen, 19 means spleen, 20 means For the prostate, 21 is the fetal heart, 22 is the heart, 23 is the muscle, 24 is the testis, 25 is the fetal thymus, and 26 is the thymus.

 Figure 2 shows the polyacrylamide gel electrophoresis (SDS-PAGE) of human amyloid precursor protein-binding protein 9 isolated. 9kDa is the molecular weight of the protein. The arrow indicates the isolated protein band. The present invention is further described below with reference to specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. In the following examples, the experimental methods without specific conditions are generally performed according to conventional conditions such as Sambrook et al., Molecular Cloning: The conditions described in the laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer Suggested conditions.

 Example 1: Cloning of human amyloid precursor protein binding protein 9

Total human fetal brain RNA was extracted by one-step method with guanidine isothiocyanate / phenol / chloroform. Poly (A) mRNA was isolated from total RNA using Quik mRNA Isolation Kit (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 multicloning site of pBSK (+) vector (Clontech) to transform DH5a. The bacteria formed a cDNA library. Dye terminate cycle react ion sequencing kit (Perkin-Elmer) and ABI 377 automatic sequencer (Perkin-Elmer) were used to determine the sequences at the 5 'and 3' ends of all clones. CDNA sequence The column was compared with the existing public DNA sequence database (Genebank), and the cDNA sequence of one of the clones 0135B04 was found to be new DNA. A series of primers were synthesized to determine the inserted cDNA fragments of the clone in both directions. The results showed that the 0135B04 clone contained a full-length cDNA of 1892 bp (as shown in Seq ID NO: 1), and a 251 bp open reading frame (0RF) from 1602 bp to 1853 bp, encoding a new protein (such as Seq ID NO : Shown in 2). We named this clone pBS-0135B04, and encoded the protein named human amyloid precursor protein binding protein 9. Example 2: Cloning of a gene encoding human starch precursor protein binding protein 9 by RT-PCR

 CDNA was synthesized using fetal brain total RNA as a template and oligo-dT as a primer for reverse transcription reaction. After purification using Qiagene's kit, the following primers were used for PCR amplification:

 Primerl: 5,-CTCTTCAACTAGTGAATGAAAGAA-3 '(SEQ ID NO: 3)

 Primer2: 5'- TATACAAAATCACCTTAATGGTTA- 3 '(SEQ ID NO: 4)

 Primerl is a forward sequence starting at lbp of the 5th end of SEQ ID NO: 1;

 Primer2 is the 3 'end reverse sequence in SEQ ID NO: 1.

 Conditions for the amplification reaction: 50 μl of Kl1, 10 ol / L of Tris-

CI, (pH8.5), 1.5mmol / L MgCl ₂ , 200 μmol / L dNTP, lOpmol primer, 1U Taq DNA polymerase (C 1 on te ch). The reaction was performed on a PE 9600 DNA thermal cycler (Perkin-Elmer) for 25 cycles under the following conditions: 94. C 30sec; 55 "C 30sec; 72 ° C 2min ₀ At the same time, set β-actin as a positive control and template blank as a negative control during RT-PCR. Amplification products were purified using QIAGEN's kit and connected with TA cloning kit To a PCR vector (product of Invitrogen). The DNA sequence analysis results showed that the DNA sequence of the PCR product is exactly the same as the 1- 1892bp shown in SEQ ID NO: 1. Example 3: Northern blot analysis of human starch precursor protein binding Protein 9 gene expression:

Total RNA extraction in one step [Anal. Biochem 1987, 162, 156-159] ₀ This method involves acid guanidinium thiocyanate-chloroform extraction. That is, the tissue is homogenized with 4M guanidine isothiocyanate-25mM sodium citrate, 0.2M sodium acetate (pH4.0), and 1 volume of phenol and 1/5 volume of chloroform-isoamyl alcohol (49: 1) are added. ), Mix and centrifuge. Aspirate the aqueous layer, add isopropanol (0.8 vol) and centrifuge the mixture to obtain RNA precipitate. The resulting RNA pellet was washed with 70% ethanol, dried and dissolved in water. Using 20 μ _§ RNA, electrophoresis was performed on a 1.2% agarose gel containing 20 mM 3- (N-morpholino) propanesulfonic acid (pH 7.0) -5 mM sodium acetate-ImM EDTA-2.2M formaldehyde. It was then transferred to a nitrocellulose membrane. Preparation ³² P- DNA probe labeled with a- ³² P dATP by random priming method. The DNA probe used was the human amyloid precursor protein-binding protein 9 coding region sequence (1602bp to 1853bp) amplified by PCR as shown in Figure 1. ₀ 32P-labeled probe (about 2 x 10 ⁶ cpm / ml) Soluble with RNA-transferred nitrocellulose membrane 液 中 于 42。 In the liquid. C hybridization overnight, the solution contains 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 filters were placed in 1 x SSC-0.1% SDS at 55. C Wash for 30min. Then, Phosphor Imager was used for analysis and quantification. Example 4: In vitro expression, isolation and purification of recombinant human amyloid precursor protein binding protein 9

 According to SEQ ID NO: 1 and the coding region sequence shown in FIG. 1, a pair of specific amplification primers was designed. The sequences are as follows:

 Primer3: 5 -CCCCATATGATGAAGGTAGTTATTCCTGCCTGT-3 '(Seq ID No: 5)

 Primer4: 5'- CCCGAATTCTTATATAGACAGAAAATAGCTCCA- 3, (Seq ID No: 6) The 5 'ends of these two primers contain Ndel and BamHI digestion sites, respectively, followed by the 5' end and

At the 3 'end of the coding sequence, the Nde I and BamH I restriction sites correspond to the selective endonuclease sites on the expression vector plasmid pET-28 b (+) (Nova Gen, Cat. No. 69865.3). The pBS-0135B04 plasmid containing the full-length target gene was used as a template for the PCR reaction. The PCR reaction conditions are as follows: a total volume of 50 μl contains 10 pg of pBS- 0135B0 ⁴ plasmid, primers Primer-3 and Primer-4, and ¹ J is lOpmol, Advantage polymerase Mix (Clontech) 1 μ1. Cycle parameters: 94. C 20s, 60 ° C 30s, 68 ° C 2 min, a total of 25 cycles. Ndel and BamHI were used to double digest the amplified product and plasmid pET-28 (+), respectively, and large fragments were recovered and ligated with T4 ligase. The ligation product was transformed into the colibacillus DH5 CX by the calcium chloride method. After being cultured overnight on an LB plate containing kanamycin (final concentration 30 g / ml), positive clones were selected by colony PCR method and sequenced. A positive clone (pET-0135B04) with the correct sequence was selected, and the recombinant plasmid was transformed into E. coli BL21 (DE3) plySs (product of Novagen) using the calcium chloride method. In LB liquid medium containing kanamycin (final concentration 30 g / ml), the host strain BL21 (pET-0135B04) was at 37. C. Cultivate to logarithmic growth phase, add IPTG to a final concentration of 1 mmol / L, and continue to cultivate for 5 hours. The bacteria were collected by centrifugation, and the supernatant was collected by centrifugation. The supernatant was collected by centrifugation. The affinity chromatography column His. Bind Quick Cartridge (product of Novagen) was used to obtain 6 histidine (6His-Tag). The purified human starch precursor protein binding protein 9 was purified. After SDS-PAGE electrophoresis, a single band was obtained at 9 kDa (Figure 2). The band was transferred to a PVDF membrane and the N-terminal amino acid sequence was analyzed by the Edams hydrolysis method. As a result, the 15 amino acids at the N-terminus were identical to the 15 amino acid residues at the N-terminus shown in SEQ ID NO: 2. Example 5 Production of anti-human starch precursor protein binding protein 9 antibodies

 The following peptides specific for human amyloid precursor protein binding protein 9 were synthesized using a peptide synthesizer (product of PE):

NH2-Met-Lys-Val-Val-Ile-Pro-Ala-Cys-Val-Ala-His-Leu-Phe-Thr-Lys- C00H (SEQ ID NO: 7). The peptide was coupled to hemocyanin and bovine serum albumin to form a complex. For the method, see: Avramea s, eta 1. Immunochemistry, 1969; 6: 43. ₀ 4 mg of the above-mentioned cyanin peptide complex plus complete Freund's The rabbits were immunized with the drug, and the immune response was enhanced with hemocyanin polypeptide complex and incomplete Freund's adjuvant once every 15 days. A titer plate coated with 15 g / ml bovine serum albumin peptide complex was used as EL I SA to determine the antibody titer in rabbit serum. Total IgG was isolated from antibody-positive rabbit serum using protein A-Sepha ros e. The peptide was bound to a cyanogen bromide-activated Sepha _r os e4B column, and the anti-peptide antibody was separated from the total I gG by affinity chromatography. The immunoprecipitation method proved that the purified antibody could specifically bind to human amyloid precursor protein binding protein 9. Example 6: Application of the polynucleotide fragment of the present invention as a hybridization probe

 Suitable oligonucleotide fragments selected from the polynucleotides of the present invention are used as hybridization probes in various aspects. For example, 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. Further, 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 blotting, Nor thern blotting, and copying methods, etc. They are all used to fix the polynucleotide sample to be tested on the filter and then hybridize using basically the same steps. These same steps are as follows: 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 synthetic 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. After the hybridization step, the unhybridized probes are removed by a series of membrane washing steps. In this embodiment, higher-intensity washing conditions (such as lower salt concentration and higher temperature) are used 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. In this embodiment, 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.

First, the selection of the probe

The selection of oligonucleotide fragments from the polynucleotide SEQ ID NO: 1 of the present invention for use as hybridization probes should follow the following principles and several aspects to be considered: 1. The preferred range of probe size is 18-50 nucleotides;

 2, GC content is 30% -70%, non-specific hybridization increases when it exceeds;

 3. There should be no complementary regions inside the probe;

 4. 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 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, then the primary probe should not be used;

5. Whether the preliminary selection probe is finally selected as a probe with practical application value should be further determined by experiments.

 After completing the above analysis, select and synthesize the following two probes:

 Probe 1 (probel), which belongs to the first type of probe, is completely homologous or complementary to the gene fragment of SEQ ID NO: 1 (41Nt):

 5'-TGAAGGTAGTTATTCCTCCCTGTGTTGCCCACCTGTTCACA-3 '(SEQ ID NO: 8)

 Probe 2 (probe2), which belongs to the second type of probe, is equivalent to the replacement mutant sequence of the gene fragment of SEQ ID NO: 1 or its complementary fragment (41Nt):

 5 -TGAAGGTAGTTATTCCTGCCCGTGTTGCCCACCTGTTCACA-3 '(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: DNA PROBES GH Kel ler; MM Manak; Stockton Press, 1989 (USA ) And more commonly used molecular cloning experiment manual books such as "Molecular Cloning Experiment Guide" U 998 Second Edition) [US] Sambruck et al., Science Press.

 Sample Preparation:

 1.Extract DNA from fresh or frozen tissue

Steps: 1) Place fresh or freshly thawed normal liver tissue in a plate immersed in ice and filled with phosphate buffered saline (PBS). Cut the tissue into small pieces with scissors or a scalpel. Keep tissue moist during operation. 2) Centrifuge the tissue at 1,000 g for 10 minutes. 3) Use cold homogenate buffer (0.25mol / L sucrose; 25 ol / L Tris-HCl, pH7.5; 25 crypto ol / LiiaCl; 25mmol / L MgCl ₂ ) suspension pellet (about 10ml / g) ₀ 4 ) Homogenize the tissue suspension at 4 ° C at full speed with an electric homogenizer until the tissue is completely broken. 5) Centrifuge at 1000g for 10 minutes. 6) Resuspend the cell pellet (l-5ml per 0.1g of the original tissue sample), and centrifuge at 1000g for 10 minutes. 7) Resuspend the pellet in lysis buffer (1 ml per 0.1 g of the initial tissue sample), and then follow the phenol extraction method below.

2, phenol extraction method for DNA

Steps: 1) Wash cells with l-10nil cold PBS and centrifuge at 1000g for 10 minutes. 2) with cold cell lysate pelleted cells were resuspended (1 ¹⁰⁸ cells / ml) Minimum application lOOul lysis buffer. 3) Add SDS to the end The concentration is 1%. If SDS is added directly to the cell pellet before resuspending the cells, the cells may form large clumps that are difficult to break, and reduce the overall yield. This is particularly serious when extracting> 10 ⁷ cells. 4) Add proteinase K to a final concentration of 200ug / ml. 5) 50. C. Incubate for 1 hour or shake gently at 37 ° C overnight. 6) Extract with an equal volume of phenol: chloroform: isoamyl alcohol (25: 24: 1) and centrifuge in a small centrifuge tube for 10 minutes. The two should be clearly separated, otherwise centrifuge again. 7) Transfer the water phase to a new tube. 8) Extract with an equal volume of chloroform: isoamyl alcohol (24: 1) and centrifuge for 10 minutes. 9) Transfer the DNA-containing aqueous phase to a new tube. Purification of DM and ethanol precipitation were then performed.

3, DNA purification and ethanol precipitation

Steps: 1) Add 1/10 volume of 2mol / L sodium acetate and 2 volumes of cold 100% ethanol to the DNA solution and mix. Leave at -20 ° C for 1 hour or overnight. 2) Centrifuge for 10 minutes. 3) Carefully aspirate or pour out the ethanol. 4) Wash the pellet with 500ul of 70% cold ethanol and centrifuge for 5 minutes. 5) Carefully aspirate or pour out the ethanol. Wash the pellet with 500ul of cold ethanol and centrifuge for 5 minutes. 6) Carefully aspirate or pour out the ethanol, then invert on the absorbent paper to drain off the residual ethanol. Air dry for 10-15 minutes to allow the surface ethanol to evaporate. Be careful not to allow the pellet to dry completely, otherwise it will be more difficult to re-dissolve. 7) Resuspend the DNA pellet in a small volume of TE or water. Vortex at low speed or suck with a dropper, and gradually increase TE, mix until the vessel is fully dissolved, and add approximately 1 u 1 for each 1-5 X 10 ^δ cells.

 The following steps 8-13 are only used when contamination must be removed, otherwise step 14 can be performed directly.

8) Add RNase A to the DNA solution to a final concentration of 100 ug / ml, and incubate at 37 C for 30 minutes. 9) Add SDS and proteinase K to the final concentration of 0.5% and 100ug / ml. 37. C incubate for 30 minutes. 10) Extract the reaction solution with an equal volume of phenol: chloroform: isoamyl alcohol (25: 24: 1) and centrifuge for 10 minutes. 11) Carefully remove the aqueous phase, re-extract with an equal volume of chloroform: isoamyl alcohol (24: 1), and centrifuge for 10 minutes. 12) Carefully remove the water phase, add 1/10 volume of 2mol / L sodium acetate and 2.5 volumes of cold ethanol, mix well and set to -20. C for 1 hour. 13) Wash the precipitate with 70% ethanol and 100% ethanol, air dry, and resuspend the nucleic acid. The process is the same as steps 3-6. 14) Measure A ₂₆ . And A ₂₈ . To detect the purity and yield of DNA. 15) Store at -20 ° C after dispensing.

Preparation of sample film:

 1) Take 4 x 2 pieces of nitrocellulose membranes (NC membranes) of appropriate size, and mark the spotting position and sample number on it with a pencil. Two NC membranes are required for each probe for subsequent experiments. In the step, the film is washed with high-strength conditions and strength conditions, respectively.

 2) Aspirate and control 15 microliters each, spot on the sample film, and dry at room temperature.

 3) Place on filter paper impregnated with 0.1 mol / L NaOH, 1.5 mol / L NaCl for 5 minutes (twice), dry and place on filter paper impregnated with 0.5 mol / L Tris-HCl (pH 7.0), 3 mol / L NaCl Allow to dry for 5 minutes (twice).

4) Clamp in clean filter paper and wrap with aluminum foil, 60-80. C was dried under vacuum for 2 hours. Labeling of probes

1) 3 μ IProbe (0.1 IOD / Ιθμ 1), add 2 μ IKinase buffer, 8-10 uCi γ- ³² P- dATP + 2U Kinase, to make up to a final volume of 20 μ 1.

 2) Incubate at 37 ° C for 1 hour.

 3) Add 1/5 volume of Bromophenol Blue Indicator (BPB).

 4) Pass through a Sephadex G-50 column.

5) Start to collect the first peak before ³² P-Pr ₀ be washed out (can be monitored by Monitor).

 6) 5 drops / tube, collect 10-15 tubes.

 7) Monitor the amount of isotope with a liquid scintillator

8) After combining the collection solutions of the first peak, the ³² P-Probe (the second peak is free _y - ³² P-dATP) is prepared. Pre-hybridization

 Put the sample membrane in a plastic bag, add 3- lOmg pre-hybridization solution (lOxDenhardt's; 6xSSC, 0.1 mg / ml CT DNA (calf thymus DNA).), Seal the bag, and shake at 68 ° C for 2 hours .

 Cross

The plastic bag corner cut, prepared probe was added, after the sealed bag, 42 ⁽ 'C Los water shaking overnight.

 Wash film:

 High-intensity washing film:

 1) Take out the hybridized sample membrane.

 2) 2xSSC, 0.1% SDS, wash at 40 ° C for 15 minutes (twice).

 3) 0.1xSSC, 0.1% SDS, wash at 40 ° C for 15 minutes (twice).

4) 0. IxSSC, 0.1% SDS, 55 ⁽ washed for 30 minutes (2 times), and dried at room temperature. Low-intensity washing film:

 1) Take out the hybridized sample membrane.

 2) 2xSSC, 0.1% SDS, 37. C Wash for 15 minutes (twice).

 3) 0. IxSSC, 0.1% SDS, 37. C Wash for 15 minutes (twice).

 4) 0. IxSSC, 0.1% SDS, 40. Wash for 15 minutes (twice) and dry at room temperature.

X-ray autoradiography:

 -70 ° C, X-ray autoradiography (pressing time depends on the radioactivity of the hybrid spot).

 Experimental results:

的 Hybridization experiments using low-intensity washing conditions, the radioactivity of the hybrid spots of the above two probes is not strong. The difference is obvious; and in the hybridization experiment using high-intensity washing conditions, the radioactive intensity of the hybrid spot of probe 1 is significantly stronger than that of the hybrid spot of the other probe. Therefore, probe 1 can be used to qualitatively and quantitatively analyze the presence and differential expression of the polynucleotide of the present invention in different tissues.

Example 7 DNA Microarray

 Gene microarrays or DNA microarrays are new technologies currently being developed by many national laboratories and large pharmaceutical companies. It refers to the orderly and high-density arrangement of a large number of target gene fragments on glass, Silicon and other carriers, and then use fluorescence detection and computer software to compare and analyze data to

10 To achieve the purpose of fast, 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 DeRisi, J. L., Lyer, V. & Brown, P.0.

 (1997) Science 278, 680-686. And literature Hel le, R. A., Schema, M., Chai, A., Shalom, D.,

15 (1997) PNAS 94: 2150-2155.

 (A) spotting

 A variety of different full-length ccDDNNAAs with a total length of 44 million polynucleotide sequences are listed as the target DDNNAA, which includes the Multi-polynucleotide. . They will be separately expanded and amplified through PPCCRR. After pure purification, the expansion and amplification products obtained will be adjusted to a concentration of 550000nngg // uull. Right and left, use a CCaarrtteessiiaann 77550000 spot sampler (purchased from CCaarrtteessiiaann Corporation of the United States) on the glass glass medium 2200 quality, between the point and the point The distance is 228800 μμηι. . The glass slides after the spotting are hydrated, dried, and placed in a violet ultraviolet cross-linking instrument for cross-linking. After washing and eluting, Dry and dry to make DDNNAA fixed on glass slides to prepare core chips. . Its specific method and method steps have been reported in the literature, and many reports have been reported. The actual implementation of the examples in the present embodiment is followed by processing steps. ::

 11. Hydration and hydration in the humid and humid environment for 44 hours;

 22 .. 00..22 %% SSDDSS washing and washing for 11 minutes;

4. NaBH _{4 is} blocked for 5 minutes;

 5. 95 ° C water for 2 minutes;

 6. Wash with 0.2% SDS for 1 minute;

7. Rinse twice with ddH ₂ 0;

 8. Dry dry, 25. C Store in a dark place for future use.

 (Two) probe marking

One-step extraction from the human mixed tissue and the body's specific tissue (or stimulated cell lines) Total mRNA was extracted and purified using Oligotex mRNA Midi Kit (purchased from QiaGen), and separated by reverse transcription! Cy3dUTP (5-Amino-propargy 1-2 '-deoxyuri dine 5'-tr iphate coupled to Cy3 fluorescent dye, purchased from Amersham Phamacia Biotech) labeled mRNA of human mixed tissues, with Cy5dUTP (5-Amino-propargy 1-2- -deoxyuridine 5 "-tr iphate coupled to Cy5 fluorescent dye, purchased from Amersham Phamacia Biotech company) labeled niRNA in specific tissues (or stimulated cell lines) of the body, and prepared probes after purification. For specific steps and methods, see:

 Schena, M., Shalon, D., Heller, R. (1996) Proc. Natl. Acad. Sci. USA. Vol. 93: 10614- 10619. Schena, M., Shalon, Dari., Davis, RW (1995 ) Science.270. (20): 467-480. (3) Hybridization

 Probes from the above two types of tissues were hybridized with the chip in a UniHyb ™ Hybridization Solution (purchased from TeleChem) for 16 hours, washed with a washing solution (1 x SSC, 0.2% SDS) at room temperature, and then scanned with ScanArray 3000. The scanner (purchased from General Scanning Company, USA) was used for scanning. The scanned image was analyzed and processed with Iniagene software (Biodicovery Company, USA) to calculate the Cy3 / Cy5 ratio of each point.

 The above specific tissues (or stimulated cell lines) are thymus, testis, muscle, spleen, lung, skin, thyroid, liver, PMA + Ecv304 cell line, PMA-Ecv304 cell line, non-starved L02 cell line, L02 cell line stimulated by arsenic for 1 hour, L02 cell line stimulated by arsenic for 6 hours prostate, heart, lung cancer, fetal bladder, fetal small intestine, fetal large intestine, fetal thymus, fetal muscle, fetal liver, fetal kidney, fetal spleen, fetal brain, Fetal lung and fetal heart. Draw a graph based on these 26 Cy3 / Cy5 ratios. (figure 1 ) . It can be seen from the figure that the expression profiles of human amyloid precursor protein binding protein 9 and human amyloid precursor protein binding protein 13 according to the present invention are very similar. Industrial applicability

 The polypeptide of the present invention and the antagonists, agonists and inhibitors of the polypeptide can be directly used in the treatment of diseases, for example, it can treat malignant tumors, adrenal deficiency, skin diseases, various inflammations, HIV infections and immune diseases.

β-amyloid precursor protein (APP) can be processed to form soluble amyloid β amyloid. APP plays a certain role in the transport of proteins. For example, excision of the YENPTY sequence of AP will affect the endocytosis of APP on the cell surface, and also allow APP to enter the intracellular pathway before reaching the cell surface, thereby disrupting the transport of intracellular material. Studies have found that when APP-related AD mutations occur, AP secretion can increase, or the ratio of AP, ^ / AP ^ changes. And the increase of beta amyloid bodies in the brain is closely related to Alzheimer's disease (Alzheimer disease). and also It was found that the role of APP is related to several cytokines such as FE65 protein.

 The expression profile of the polypeptide of the present invention is consistent with the expression profile of human β-amyloid precursor protein, and both have similar biological functions. It is mainly involved in cell swallowing and exocytosis in the body, and its abnormal expression is usually closely related to the occurrence of some related disorders of material metabolism, disorders of protein metabolism, and related tissue tumors and cancers, such as the Alzheimer's Moore's disease.

 It can be seen that the abnormal expression of the human amyloid precursor protein binding protein 9 of the present invention will produce various diseases, especially Alzheimer's disease, various tumors, embryonic developmental disorders, growth disorders, inflammation, Immune diseases, including but not limited to:

 Tumors of various tissues: stomach cancer, liver cancer, lung cancer, esophageal cancer, breast cancer, leukemia, lymphoma, thyroid tumor, uterine fibroids, neuroblastoma, astrocytoma, ependymoma, glioblastoma, nerve Fibroma, colon cancer, melanoma, bladder cancer, uterine cancer, endometrial cancer, colon cancer, thymic tumor, nasopharyngeal cancer, laryngeal cancer, tracheal tumor, fibroid, fibrosarcoma, lipoma, liposarcoma

 Fetal developmental disorders: congenital abortion, cleft palate, limb loss, limb differentiation disorder, atrial septal defect, neural tube defect, congenital hydrocephalus, congenital glaucoma or cataract, congenital deafness

 Growth and development disorders: mental retardation, brain development disorders, skin, fat, and muscular dysplasia, bone and joint dysplasia, various metabolic defects, stunting, dwarfism, Cushing's syndrome Sexual retardation

 Inflammation: chronic active hepatitis, sarcoidosis, polymyositis, chronic rhinitis, chronic gastritis, cerebrospinal multiple sclerosis, glomerulonephritis, myocarditis, cardiomyopathy, atherosclerosis, gastric ulcer, cervicitis, Various infectious inflammations

 Immune diseases: Systemic lupus erythematosus, rheumatoid arthritis, bronchial asthma, urticaria, specific dermatitis, post-infection myocarditis, scleroderma, myasthenia gravis, Guillain-Barre syndrome, common variable immunodeficiency disease , Primary B lymphocyte immunodeficiency disease, Acquired immunodeficiency syndrome

 Abnormal expression of the human amyloid precursor protein-binding protein 9 of the present invention will also produce certain hereditary, hematological diseases, and the like.

 The polypeptide of the present invention and the antagonists, agonists and inhibitors of the polypeptide can be directly used in the treatment of diseases, for example, it can treat various diseases, especially Alzheimer's disease, various tumors, embryonic development disorders, growth and development. Obstructive diseases, inflammation, immune diseases, certain hereditary, blood diseases, etc.

The invention also provides methods for screening compounds to identify agents that increase (agonist) or suppress (antagonist) human starch precursor protein binding protein 9. Agonists enhance biological functions such as human amyloid precursor protein binding protein 9 to stimulate cell proliferation, and antagonists prevent and treat disorders related to excessive cell proliferation, such as various cancers. For example, mammalian cells or human starch precursor protein-binding proteins can be bound in the presence of drugs Membrane formulations of White 9 were cultured with labeled human amyloid precursor protein binding protein 9. The ability of the drug to increase or block this interaction is then determined.

 Antagonists of human amyloid precursor protein binding protein 9 include screened antibodies, compounds, receptor deletions, and the like. Antagonists of human amyloid precursor protein binding protein 9 can bind to human amyloid precursor protein binding protein 9 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.

 When screening compounds as antagonists, human amyloid precursor protein-binding protein 9 may be added to a bioanalytical assay by measuring the effect of the compound on the interaction between human amyloid precursor protein-binding protein 9 and its receptor. 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 human amyloid precursor protein binding protein 9 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. During screening, 9 molecules of human amyloid precursor protein binding protein should generally 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 present invention also provides antibodies directed against human amyloid precursor protein binding protein 9 epitopes. These antibodies include (but are not limited to): polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single-chain antibodies, Fab fragments, and fragments from Fab expression libraries.

Polyclonal antibodies can be produced by injecting human amyloid precursor protein binding protein 9 directly into immunized animals (such as rabbits, mice, rats, etc.). A variety of adjuvants can be used to enhance the immune response, including but not limited to 'S adjuvant and so on. Techniques for preparing monoclonal antibodies to human amyloid precursor protein binding protein 9 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 combining human constant regions and non-human variable regions can be produced using existing techniques (Morrison et al, PNAS, 1985, 81: 6851). ₀ Existing techniques for producing single-chain antibodies (US Pat No. .4946778) can also be used to produce single chain antibodies against human amyloid precursor protein binding protein 9.

 Antibodies against human amyloid precursor protein binding protein 9 can be used in immunohistochemical techniques to detect human amyloid precursor protein binding protein 9 in biopsy specimens.

 Monoclonal antibodies that bind to human amyloid precursor protein binding protein 9 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. For example, human amyloid precursor protein binding protein 9 high affinity monoclonal antibodies can interact with bacterial or plant toxins (such as diphtheria toxin, ricin, Ormosine, etc.). A common method is to attack the amino group of an antibody with a thiol cross-linking agent such as SPDP and bind the toxin to the antibody through the exchange of disulfide bonds. This hybrid antibody can be used to kill human starch precursor protein binding protein 9 Positive cells.

 The antibodies of the present invention can be used to treat or prevent diseases related to human amyloid precursor protein binding protein 9. Administration of an appropriate dose of antibody can stimulate or block the production or activity of human amyloid precursor protein binding protein 9.

 The invention also relates to a diagnostic test method for quantitative and localized detection of human amyloid precursor protein binding protein 9 levels. These tests are well known in the art and include FI SH assays and radioimmunoassays. The level of human amyloid precursor protein binding protein 9 detected in the test can be used to explain the importance of human amyloid precursor protein binding protein 9 in various diseases and to diagnose human amyloid precursor protein binding protein 9 A working disease.

 The polypeptide of the present invention can also be used for peptide mapping analysis. For example, 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.

 Polynucleotides encoding human amyloid precursor protein binding protein 9 can also be used for a variety of therapeutic purposes. Gene therapy technology can be used to treat abnormal cell proliferation, development or metabolism caused by the non-expression or abnormal / inactive expression of human amyloid precursor protein binding protein 9. Recombinant gene therapy vectors (such as viral vectors) can be designed to express variant human amyloid precursor protein binding protein 9 to inhibit endogenous human amyloid precursor protein binding protein 9 activity. For example, a variant human amyloid precursor protein-binding protein 9 may be a shortened human amyloid precursor protein-binding protein 9 that lacks a signaling domain. Although it can bind to downstream substrates, it lacks signal transduction. active. Therefore, the recombinant gene therapy vector can be used for treating diseases caused by abnormal expression or activity of human amyloid precursor protein binding protein 9. Virus-derived expression vectors such as retroviruses, adenoviruses, adenovirus-associated viruses, herpes simplex virus, parvoviruses, and the like can be used to transfer polynucleotides encoding human starch precursor protein binding protein 9 into cells. A method for constructing a recombinant viral vector carrying a polynucleotide encoding a human amyloid precursor protein binding protein 9 can be found in the existing literature (Sambrook, et al.). In addition, a recombinant polynucleotide encoding human amyloid precursor protein binding protein 9 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.

Oligonucleotides (including antisense RNA and DNA) and ribozymes that inhibit human amyloid precursor protein binding protein 9 mRNA are also within the scope of the present invention. A ribozyme is an enzyme-like RNA molecule that specifically decomposes specific RNA. Its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target RNA for endonucleation. Anti The sensed RNA, DNA and ribozyme can be obtained by any existing RNA or DNA synthesis technology. For example, the technology for the synthesis of oligonucleotides by solid-phase phosphoramidite chemical synthesis 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 DNA sequence has been integrated downstream of the RNA 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 human amyloid precursor protein binding protein 9 can be used for diagnosis of diseases related to human amyloid precursor protein binding protein 9. The polynucleotide encoding human amyloid precursor protein-binding protein 9 can be used to detect the expression of human amyloid precursor protein-binding protein 9 or abnormal expression of human amyloid precursor protein-binding protein 9 in a disease state. For example, the DNA sequence encoding human amyloid precursor protein binding protein 9 can be used to hybridize biopsy specimens to determine the expression status of human amyloid precursor protein binding protein 9. Hybridization techniques include Souternrn blotting, Northenrn blotting, in situ hybridization, and the like. These techniques and methods are publicly available and mature, and related kits are commercially available. Some or all of the polynucleotides of the present invention can be used as probes to be fixed on a microarray (Mic roaray) or a DNA chip (also known as a "gene chip") for analyzing differential expression analysis of genes in tissues And genetic diagnosis. Human amyloid precursor protein binding protein 9 specific primers for RNA-polymerase chain reaction (RT-PCR) in vitro amplification can also detect the human amyloid precursor protein binding protein 9 transcription products.

 Detection of mutations in the human amyloid precursor protein binding protein 9 gene can also be used to diagnose human amyloid precursor protein binding protein 9-related diseases. Human amyloid precursor protein binding protein 9 mutations include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to the normal wild-type human amyloid precursor protein binding protein 9 DNA sequence. Mutations can be detected using existing techniques such as Southern blotting, DNA sequence analysis, PCR and in situ hybridization. In addition, mutations may affect protein expression. Therefore, the use of Northern blotting and Wetternrn blotting can 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. Currently, specific sites for each gene on the chromosome need to be identified. Currently, only a few chromosome markers based on actual sequence data (repeating polymorphisms) are available for marking chromosome positions. According to the present invention, in order to associate these sequences with disease-related genes, an important first step is to locate these DNA sequences on a chromosome.

 In short, PCR primers (preferably 1 to 35 bp) are prepared based on cDNA, and the sequences can be located on chromosomes. 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. Using the 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 (FISH) of cDNA clones with metaphase chromosomes allows precise chromosomal localization in one step. For a review of this technique, see Verma et al., Human Chromosomes: a Manua 1 of Basic Techniques, Pergamon Press, New York (1988).

 Once the sequence is located at the exact chromosomal location, the physical location of the sequence on the chromosome can be correlated with the genetic map data. This data can be found, for example, in V. Mckusick, Mendelian Inheritance in Man (available online with Johns Hopkins University Welch Medical Library). Linkage analysis can then be used to determine the relationship between genes and diseases that have been mapped to chromosomal regions.

 Next, 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. These carriers 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 that do not affect the effect of the drug. These compositions can be used as drugs for the treatment of diseases.

 The present invention also provides a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the present invention. Along with these containers, there may be instructional instructions given by government agencies that manufacture, use, or sell pharmaceuticals or biological products, which reminders permit their administration on the human body by government agencies that manufacture, use, or sell them. In addition, the polypeptide of the present invention can be used in combination with other therapeutic compounds.

 The pharmaceutical composition can be administered in a convenient manner, such as by a topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal route of administration. Human amyloid precursor protein binding protein 9 is administered in an amount effective to treat and / or prevent specific indications. The amount and range of human amyloid precursor protein binding protein 9 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.

Claims

 Request for Rights

I. An isolated polypeptide-human amyloid precursor protein binding protein 9, characterized in that it comprises: a polypeptide having the amino acid sequence shown in SEQ ID NO: 2, or an active fragment, analog, or derivative of the polypeptide .

2. The polypeptide according to claim 1, characterized in that the amino acid sequence of the polypeptide, analog or derivative has at least 95% identity with the amino acid sequence shown in SEQ ID NO: 2.

 3. The polypeptide according to claim 2, characterized in that it comprises a polypeptide having the amino acid sequence shown in SEQ ID D NO: 2.

 4. An isolated polynucleotide, characterized in that said polynucleotide comprises one selected from the group consisting of: (a) encoding a polypeptide having an amino acid sequence shown in SEQ ID NO: 2 or a fragment thereof, an analog thereof; Polynucleotides of derivatives;

 (b) a polynucleotide complementary to polynucleotide (a); or

 (c) a polynucleotide that is at least 70% identical to) or (b).

 5. The polynucleotide according to claim 4, wherein the polynucleotide comprises a polynucleotide encoding an amino acid sequence represented by SEQ ID NO: 2.

 6. The polynucleotide according to claim 4, characterized in that the sequence of the polynucleotide comprises the sequence of positions 1 602-1 85 in SEQ ID NO: 1 or 1 -1 in SEQ ID NO: 1 892-bit sequence.

 7. A recombination vector containing an exogenous polynucleotide, characterized in that it is a recombination constructed by the polynucleotide according to any one of claims 4-6 and a plasmid, virus or a carrier expression vector Carrier. 8. A genetically engineered host cell containing an exogenous polynucleotide, characterized in that it is selected from one of the following host cells:

 (a) a host cell transformed or transduced with the recombinant vector of claim 7; or

 (b) a host cell transformed or transduced with a polynucleotide according to any one of claims 4-6.

9. A method for preparing a polypeptide having human starch precursor protein binding protein 9 activity, characterized in that the method includes:

 (a) culturing the engineered host cell according to claim 8 under the condition of expressing human amyloid precursor protein binding protein 9;

 (b) A polypeptide having human amyloid precursor protein binding protein 9 activity is isolated from the culture.

 10. An antibody capable of binding to a polypeptide, characterized in that said antibody is an antibody capable of specifically binding to human amyloid precursor protein binding protein 9.

II. A class of compounds that mimic or regulate the activity or expression of a polypeptide, characterized in that they are compounds that mimic, promote, antagonize or inhibit the activity of human amyloid precursor protein binding protein 9. 12. The compound according to claim 11, characterized in that it is an antisense sequence of the polynucleotide sequence shown in SEQ ID NO: 1 or a fragment thereof.

 1 3. A use of the compound according to claim 11, characterized in that the compound is used for a method for regulating the activity of human starch precursor protein binding protein 9 in vivo and in vitro.

14. A method for detecting a disease or susceptibility to a disease associated with a polypeptide according to any one of claims 1-3, characterized in that it comprises detecting the expression level of the polypeptide, or detecting the activity of the polypeptide Or detecting a nucleotide variation in a polynucleotide that causes abnormal expression or activity of the polypeptide.

 15. Use of a polypeptide according to any one of claims 1 to 3, characterized in that it is used for screening mimetics, agonists, antagonists or inhibitors of human starch precursor protein binding protein 9; Or used for peptide fingerprint identification.

 16. The use of a nucleic acid molecule according to any one of claims 4 to 6, characterized in that it is used as a primer for a nucleic acid amplification reaction, or as a probe for a hybridization reaction, or for manufacturing a gene Chip or microarray.

 17. Use of a polypeptide, polynucleotide or compound according to any one of claims 1-6 and 1 1, characterized in that said polypeptide, polynucleotide or mimetic, agonist, The antagonist or inhibitor is composed of a safe and effective dose and a pharmaceutically acceptable carrier as a pharmaceutical composition for diagnosing or treating a disease associated with abnormality of human amyloid precursor protein binding protein 9.

 18. Use of the polypeptide, polynucleotide or compound according to any one of claims 1-6 and 1 1, characterized in that the polypeptide, polynucleotide or compound is used for preparing a treatment such as a malignant tumor Drugs for blood diseases, HIV infection and immune diseases and various types of inflammation.