WO2001040299A1 - A novel polypeptide - human prolipoprotein signal peptide 14 and a polynucleotide encoding the same - Google Patents

Abstract

The present invention discloses a novel polypeptide - human prolipoprotein signal peptide 14 and a polynucleotide encoding the same, as well as a method of producing the polypeptide by DNA recombinant technology. The present invention also discloses methods of using the polypeptide in treatment of various diseases, such as malignant tumor, blood disease, HIV infection, immunological disease and various inflammations. The present invention also discloses an antagonist against the polypeptide and the therapeutic use of the same. The present invention also discloses the use of such polynucleotide encoding human prolipoprotein signal peptide 14.

Description

 A new polypeptide-human lipoprotein precursor protein signal peptide 14 and a polynucleotide encoding an if species poly'peptide TECHNICAL FIELD

 The present invention belongs to the field of biotechnology. Specifically, the present invention describes a new polypeptide, a human lipoprotein precursor protein signal peptide 14, and a polynucleotide sequence encoding the polypeptide. The invention also relates to a preparation method and application of the polynucleotide and polypeptide. Background technique

 Lipoproteins are a class of binding proteins composed of proteins and various fatty acids and steroids. Proteins and steroids are bound together by weak non-covalent bonds, while other lipoproteins are bound by proteins and lipids by covalent bonds. Lipoproteins can be divided into three categories based on their protein composition, nuclear proteins ( (Such as thrombin to live enzymes), phosphoproteins (such as lipophosphoproteins in yolk), and simple proteins (such as plasma lipoproteins and brain proteolipids). The role of lipoproteins in the body is very important. For example, part of the lipids in the blood is transmitted by plasma lipoproteins

 Covalent modification of lipids to membrane proteins is a common phenomenon in cells. In fact, the proteins at the cell membrane are all lipoproteins. They interact with the phospholipid layer of the cell membrane to form a membrane structure.

 Membrane lipoprotein synthesis and modification are more clearly studied in prokaryotes. In prokaryotes, membrane lipoproteins are first synthesized as a protein precursor. The N-terminus of this precursor protein contains a signal peptide. In the process of protein secretion from the plasma membrane, two processes of modification of the N-terminal cysteine and removal of the signal peptide occur successively. This process takes place in a conservative area. In the future, the signal peptide of a protein that has not undergone lipid modification will not have this conserved sequence. It appears that this conserved sequence is a sorting signal for whether the mature protein is lipid-modified or not. [Prote in Eng 1989 May; 2 (7): 531-4] After the N-acetylation of the cysteine residue at the terminal end, the protein is transferred to the outer membrane and covalently linked to the peptidoglycan layer. The signal peptide is cleaved by the signal peptidase I I and is also hydrolyzed. [J Bioenerg Bi omembr 1990 Jun; 22 (3): 45 1-71] The above-mentioned process of synthesis and modification of membrane lipoproteins has been found in many prokaryotes and prokaryotes, and has a more general significance.

 During signal peptide cleavage, signal peptidase II recognizes a conserved sequence on the signal peptide and cleaves the upstream segment of the cysteine residue connected to the glycerolipid-fatty acid lipid. This conserved sequence is:

{DERK} (6)-[LIVMFWSTAG] (2)-[LIVMFYSTAGCQ]-[AGS] -C where C is the attachment site for the lipid and must be between 15 and 35 residues. There must be one lysine or arginine in the first seven amino acids of the sequence.

In the brain, apolipoprotein is associated with Alzheimer's disease. High-density lipoprotein can remove excess cholesterol present in the brain and keep the starch mass at a normal level. Abnormal expression of this type of lipoprotein Causes Alzheimer's disease, cholesterol deposits in the brain, and brain tumors.

 Many types of plasma lipoproteins are formed by the liver, so if the synthesis of the signal peptide of the precursor protein of lipoprotein is blocked, the resulting protein will not be properly lipidated, resulting in disorder of lipoprotein anabolic metabolism. For example, its lipid transport function will be affected, leading to a series of liver diseases, such as exogenous / endogenous lipidemia, familial hypercholesterolemia, cholesterol storage disease, fatty liver, etc. Severe fatty liver can also cause liver cancer. Lipoprotein anabolic disorders can also cause diseases such as coronary heart disease.

 The polypeptide of the present invention contains a lipid-binding site of a membrane lipoprotein precursor signal peptide, and analysis of the expression profile indicates that the content of sub-peptide in liver cancer cells is lower than that in normal liver tissue, indicating that it is related to liver cancer and membrane lipids. The function of the protein precursor signal peptide is consistent. In summary, the present invention is a novel human lipoprotein precursor protein signal peptide, which has similar biological functions. And named as human lipoprotein precursor protein signal peptide 14.

 Because the human lipoprotein precursor protein signal peptide 14 protein plays an important role in important body functions as described above, and it is believed that a large number of proteins are involved in these regulatory processes, there has been a need in the art to identify more human lipoproteins involved in these processes. Precursor protein signal peptide 14 protein, especially the amino acid sequence of this protein is identified. Isolation of the new human lipoprotein precursor protein signal peptide 14 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 developing diagnostic and / or therapeutic drugs, so isolating its coding DNA is important. It is an object of the present invention to provide isolated novel polypeptides-human lipoprotein precursor protein signal peptide 14 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 lipoprotein precursor protein signal peptide 14.

 It is another object of the present invention to provide a genetically engineered host cell comprising a polynucleotide encoding a human lipoprotein precursor protein signal peptide 14.

 Another object of the present invention is to provide a method for producing a human lipoprotein precursor protein signal peptide 14.

 Another object of the present invention is to provide an antibody against the polypeptide-human lipoprotein precursor protein signal peptide 14 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 lipoprotein precursor protein signal peptide 14.

 Another object of the present invention is to provide a method for diagnosing and treating diseases associated with abnormalities of the human lipoprotein precursor protein signal peptide 14.

The present invention relates to an isolated polypeptide. The polypeptide is of human origin and comprises: SEQ ID No. 2 Amino acid sequence of a polypeptide, 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 No. 2;

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

 (c) A polynucleotide having at least 70¾ identity to the 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 619-1038 in SEQ ID NO: 1; and (b) having a sequence of 1-2572 in SEQ ID NO: 1 Sequence of bits.

 The invention further relates to a vector, in particular an expression vector, containing the polynucleotide of the invention; a host cell genetically engineered with the vector, including a transformed, transduced or transfected host cell; and a method comprising culturing said Host cell and method of preparing the polypeptide of the present invention by recovering the expression product.

 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 lipoprotein precursor protein signal peptide 14 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 disease associated with abnormal expression of a human lipoprotein precursor protein signal peptide 14 protein, comprising detecting mutations in the polypeptide or a polynucleotide sequence encoding the same in a biological sample, Alternatively, the amount or biological activity of a polypeptide of the invention in a biological sample is detected.

 The invention also relates to a pharmaceutical composition 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 human lipoprotein precursor protein signal peptide 14.

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, this "multiple "Peptide" or "protein" is not meant to limit the amino acid sequence to the complete natural amino acid associated with 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 can have "conservative" changes in which the substituted amino acid has a structural or chemical property similar to the original amino acid, such as the replacement of isoleucine with leucine. Variants can also have non-conservative changes, such as replacing glycine with tryptophan.

 "Deletion" refers to the deletion of one or more amino acids or nucleotides in an amino acid sequence or nucleotide sequence.

 "Insertion" 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 proteins with the structural, regulatory, or biochemical functions of natural molecules. Similarly, the term "immunologically active" refers to natural, recombinant, or synthetic proteins and fragments thereof in suitable animals or cells The ability to induce specific immune responses and to bind specific antibodies.

 An "agonist" refers to a molecule that, when combined with a human lipoprotein precursor protein signal peptide 14, can cause the protein to change, thereby regulating the activity of the protein. An agonist may include a protein, a nucleic acid, a carbohydrate, or any other molecule that can bind the human lipoprotein precursor protein signal peptide 14.

 An "antagonist" or "inhibitor" refers to a molecule that can block or regulate the biological or immunological activity of human lipoprotein precursor protein signal peptide 14 when combined with human lipoprotein precursor protein signal peptide 14. Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates, or any other molecule that can bind to human lipoprotein precursor protein signal peptide 14.

 "Regulation" refers to a change in the function of human lipoprotein precursor protein signal peptide 14, including an increase or decrease in protein activity, a change in binding characteristics, and any other biological properties and functions of human lipoprotein precursor protein signal peptide 14. Or changes in immune properties.

 "Substantially pure '" means substantially free of other proteins, lipids, carbohydrates or other substances naturally associated with it. Those skilled in the art can purify the human lipoprotein precursor protein signal peptide 14 using standard protein purification techniques, _, Basically pure human lipoprotein precursor protein signal peptide 14 can produce a single main band of human lipoprotein precursor protein signal peptide 14 on a non-reducing polyacrylamide gel. The purity of the polypeptide can be analyzed by amino acid sequence.

"Complementary" or "complementary" refers to polynucleotides that naturally bind through base-pairing under conditions of acceptable salt concentration and temperature. For example, the sequence "CT-G-A" can be combined with the complementary sequence "G-A-CT". The complementarity between two single-stranded molecules may be partial or complete. The degree of complementarity between nucleic acid strands The efficiency and intensity of hybridization have a significant effect.

 "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 blotting or Nor thern blotting, etc.) under conditions of reduced stringency. 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 mean that the conditions of reduced stringency allow non-specific binding, because the conditions of reduced stringency require that the two sequences bind to each other specifically or selectively.

 "Percent identity" refers to the percentage of sequences that are the same or similar in the comparison of two or more amino acid or nucleic acid sequences. The percent identity can be determined electronically, such as through the MEGALIGN program (Lasergene sof tware package, DNASTAR, Inc., Madi son Wis.). The MEGALIGN program can compare two or more sequences according to different methods such as the Cluster method (Higgins, D. G. and P. M. Sharp (1988) Gene 73: 237-244). The C l uster method arranges each group of sequences into clusters by checking the distance between all pairs. The clusters are then assigned in pairs or groups. The percent identity between two amino acid sequences such as sequence A and sequence B is calculated by the following formula: The number of matching residues between sequence A and sequence B

 X 1 00

 Number of residues in sequence A-number of interval residues in sequence A-number of interval residues in sequence B

 The percent identity between nucleic acid sequences can also be determined by the Cluster method or by methods known in the art such as Jotun He in (He in J., (1990) Methods in emzumo logy 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? Which can specifically bind The epitope of human lipoprotein precursor protein signal peptide 14.

 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 is not isolated when it is present in a living animal, but the same polynucleotide or polypeptide is separated from some or all of the substances that coexist with it in the natural system. Such a polynucleotide may be part of a vector, or such a polynucleotide or polypeptide may be part of a composition. Since the carrier or composition is not part 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 lipoprotein precursor protein signal peptide 14" means that human lipoprotein precursor protein signal peptide 14 is substantially free of other proteins, lipids, carbohydrates, or other substances with which it is naturally associated. Those skilled in the art can purify the human lipoprotein precursor protein signal peptide 14 using standard protein purification techniques. Substantially pure polypeptides can produce a single main band on a non-reducing polyacrylamide gel. The purity of human lipoprotein precursor protein signal peptide 14 polypeptide can be analyzed by amino acid sequence.

 The present invention provides a new polypeptide, a human lipoprotein precursor protein signal peptide 14, 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 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. The polypeptides of the invention may also include or exclude the initial methionine residue.

The present invention also includes fragments, derivatives, and analogs of the human lipoprotein precursor protein signal peptide 14. As used in the present invention, the terms "fragment", "derivative", and "analog" mean substantially maintaining the present invention Human lipoprotein precursor protein signal peptide 14 A polypeptide with the same biological function or activity. 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 codon; or (Π) a type in which one or more amino acid residues A certain group is substituted by another group to include a substituent; or (III) is one in which the fused polypeptide is fused to another compound (such as a compound that extends the half-life of the polypeptide, such as polyethylene glycol); or (IV) A polypeptide sequence (such as a leader sequence or a secreted sequence or a sequence used to purify this polypeptide or a protease sequence) formed by fusing additional amino acid sequences into a mature polypeptide. 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 a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2. The polynucleotide sequence of the present invention includes the nucleotide sequence of SEQ ID NO: 1. The polynucleotide of the present invention is found from a cDNA library of human fetal brain tissue. It contains a polynucleotide sequence with a total length of 2572 bases, and its open reading frame (6191038) encodes 139 amino acids. This polypeptide has a characteristic sequence of a membrane lipoprotein lipid binding site, and it can be deduced that the human lipoprotein precursor protein signal peptide 14 has a structure and function represented by a membrane lipoprotein lipid binding site.

 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 the mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO: 1 or it may be a degenerate variant. As used in the present invention, a "degenerate variant" refers to a nucleic acid sequence encoding a protein or polypeptide having SEQ ID NO: 2 but different from the coding region sequence shown in SEQ ID NO: 1 in the present invention.

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

 The term "polynucleotide encoding a polypeptide" 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 invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the 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, 6 (TC; or (2) Add denaturants during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% F i co ll, 42 ° C, etc .; or (3) only between the two sequences Between Hybridization occurs only when the identity is at least 95%, and more preferably 97%. 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 herein, a "nucleic acid fragment" contains at least 10 nucleotides in length, preferably at least 20-30 nucleotides, more preferably at least 50-60 nucleotides, and most preferably at least 100 nuclei. Glycylic acid or more. Nucleic acid fragments can also be used in nucleic acid amplification techniques, such as PCR, to identify and / or isolate polynucleotides encoding human lipoprotein precursor protein signal peptide 14.

 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 lipoprotein precursor protein signal peptide 14 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 DM fragment sequence of the present invention can also be obtained by the following methods: 1) isolating the double-stranded DNA sequence from the genomic DNA; 2) chemically synthesizing the DM 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 mRNA 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 (Q i agene). And the construction of cDNA libraries is also a common method (Sambrook, et al., Moleculiar Cling, A Labora tory Manual, Coldspring Harbor Laboraty. New York, 1989). Commercially available cDNA libraries are also available, such as different cDNA libraries from Cloieitech. When polymerase reaction technology is used in combination, 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): (l) DNA-DM or DM-RNA hybridization; (2) the appearance or loss of marker gene function; (3) determination of the level of transcript of human lipoprotein precursor protein signal peptide 14 (4) Detecting protein products expressed by genes 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 2,000 nucleotides, preferably within 1 000 nucleotides. The probe used here is generally a DM 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 (4) method, immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA) can be used to detect the protein product of the human lipoprotein precursor protein signal peptide 14 gene expression.

 A method of applying a PCR technique to amplify DNA / RNA (Saiki, et al. Science 1985; 230: 1350-1354) is preferably used to obtain the gene 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) can be preferably used. The primers used for PCR can be appropriately based on the polynucleotide sequence information of the present invention disclosed herein. Select and synthesize using conventional methods. The amplified DNA / RNA fragments can be isolated and purified by conventional methods such as by gel electrophoresis.

 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 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 that is genetically engineered using the vector of the present invention or directly using the human lipoprotein precursor protein signal peptide 14 coding sequence, and that recombinant technology is used to produce the present invention. Polypeptide method.

 In the present invention, a polynucleotide sequence encoding the human lipoprotein precursor protein signal peptide 14 can 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 expressed in bacteria (Rosenberg, et al. Gene, 1987, 56: 125); pMSXND expression vectors expressed in mammalian cells ( Lee and Nathans, J Bio Chera. 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 lipoprotein precursor protein signal peptide 14 and appropriate transcriptional / translational regulatory elements. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. (Sambroook, et al. Molecular Cloning, a Laboratory Manual, cold Spring Harbor Laboratory. New York, 1989) _c The MA sequence can be efficiently linked to expression An appropriate promoter in the vector to direct mRNA synthesis. Representative examples of these promoters are: the lac or trp promoter of E. coli; lambda phage _io

PL promoter; eukaryotic promoters include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoters, retroviral LTRs and other known controllable genes in prokaryotic cells or Promoters expressed in eukaryotic cells or their viruses. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. Insertion of enhancer sequences into the vector will enhance its transcription in higher eukaryotic cells. Enhancers are cis-acting factors for DNA expression, usually about 10 to 300 base pairs, which act on promoters to enhance gene transcription. Illustrative examples include SV40 enhancers of 100 to 270 base pairs on the late side of the origin of replication, tumorigenic enhancers on the late side of the origin of replication, and adenoviral 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 reductase, 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, the polynucleotide encoding the human lipoprotein precursor protein signal peptide 14 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 melanoma cells, etc. Transforming a host cell with the DNA sequence of the present invention or a recombinant vector containing the DNA sequence can be performed by conventional techniques well known to those skilled in the art. When the host is a prokaryote such as E. coli, competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated with CaC I. The steps used are well known in the art. The alternative is to use MgC l ₂ . If necessary, transformation can also be performed by electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, or conventional mechanical methods such as microinjection, electroporation, and liposome packaging.

 By conventional recombinant DNA technology, the polynucleotide sequence of the present invention can be used to express or produce a recombinant human lipoprotein precursor protein signal peptide 14 (Scence, 1984; 224: 1431). Generally, the following steps are taken:

 (1) using the polynucleotide (or variant) encoding the human lipoprotein precursor protein signal peptide 14 of the present invention, or transforming or transducing a suitable host cell with a recombinant expression vector containing the polynucleotide;

 (2) culturing the host cell in a suitable medium;

(3) Isolate and purify protein from culture medium or cells. In step (2), depending on the host cell used, 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.

 In step (3), the recombinant polypeptide may be coated in a cell, expressed on a cell membrane, or secreted outside the cell. If desired, 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. 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.

 Figure 1 shows the polyacrylamide gel electrophoresis (SDS-PAGE) of the isolated human lipoprotein precursor protein signal peptide 14. 14kDa is the molecular weight of the protein. The arrow indicates the isolated protein band. The best way to implement the invention

 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 lipoprotein precursor protein signal peptide 14

 Human fetal brain total RNA was extracted by one-step method with guanidine isothiocyanate / phenol / chloroform. Using Quik mRNA Isolation Kit

(Qiegene product) Isolation of poly (A) mRNA from total RM. 2ug poly (A) mRNA is reverse transcribed to form cDNA. A Smart cDNA cloning kit (purchased from Clontech) was used to orient the cDNA fragment into the multiple cloning site of the pBSK (+.) Vector (Clontech) to transform DH5a. The bacteria formed a cDNA library. Dye terminate cycle reaction 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. The determined cDNA sequence was compared with the existing public DM sequence database (Genebank), and it was found that the cDNA sequence of one of the clones 0558e09 was new DNA. Insert a cDNA fragment into the clone by synthesizing a series of primers Segments are measured in both directions. The results showed that the 0558e09 clone contained a full-length cDNA of 2572bp (as shown in Seq IDN0: 1), and a 139bp open reading frame (0RF) from 619bp to 1038bp, encoding a new protein (such as Seq ID NO: 2). We named this clone pBS-0558e09 and the encoded protein was named human lipoprotein precursor protein signal peptide 14. Example 2: Cloning of a gene encoding human lipoprotein precursor protein signal peptide 14 by RT-PCR

 CDNA was synthesized using fetal brain total RNA as a template and oligo-dT as a primer.

After purification of Qiagene's kit, PCR amplification was performed with the following primers:

 Primer 1: 5'_GGAAAAATTTCTGTACATTTTCTCAA- 3 '(SEQ ID NO: 3)

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

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

 Primer 2 is the 3 'end reverse sequence in SEQ ID NO: 1.

Amplification reaction conditions: 50 μl of KC1, 10 mmol / L Tris-CI, (pH 8.5), 1.5 mmol / L MgCl ₂ , 200 μ mol / L dNTP, lOpmol primers in a 50 μ 1 reaction volume , 1U 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 2min. During RT-PCR, β-act in was set as a positive control and template blank was set as a negative control. The amplified product was purified using a QIAGEN kit, and ligated to a pCR vector (Invitrogen product) using a TA cloning kit. The DNA sequence analysis results showed that the DNA sequence of the PCR product was exactly the same as that of 1-2572bp shown in SEQ ID NO: 1. Example 3: Northern blot analysis of human lipoprotein precursor protein signal peptide 14 gene expression:

Total RNA was extracted by a one-step method [Anal. Biochem 1987, 162, 156-159] ₀ This method involves acid guanidinium thiocyanate-chloroform extraction. I.e. with 4M guanidinium isothiocyanate -25mM sodium citrate, 0.2M sodium acetate _(P H4.0) of the tissue was homogenized phenol, 1 volume and 1/5 volume of chloroform - isoamyl alcohol (49: 1) Centrifuge after mixing. The aqueous layer was aspirated, isopropanol (0.8 vol) was added and the mixture was centrifuged to obtain RM precipitate. The resulting RNA pellet was washed with 70% ethanol, dried and dissolved in water. Using 20 g of 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. A- ³² P dATP was used to prepare labeled DNA probes by random primer method. The DNA probe used was the human lipoprotein precursor protein signal peptide 14 coding region sequence (619bp to 1038bp) amplified by PCR. A 32P-labeled probe (approximately 2 x 10 ⁶ cpm / ml) and an RNA-transferred nitrocellulose membrane were placed in a solution at 42 ° C. C hybridization overnight, this solution contains 50. /. Formamide-25mM KH ₂ P0 ₄ (pH7.4) -5 xSSC- 5 xDenhardt's solution and 20 (^ g / ml salmon sperm DNA. After hybridization, the filter was set at 1 <SSC- 0.1. /. Wash in SDS at 55 ° C for 30 min. Then, Phosphor Imager was used for analysis and quantification. Example 4: In vitro expression, isolation and purification of recombinant human lipoprotein precursor protein signal peptide 14

Based on SEQ ID NO: 1 and the coding region sequence, a pair of specific amplification primers were designed, the sequence is as follows: Primer 3: 5'- CCCCATATGATAGCTATCATGTGTTTGAATCAGA- 3 '(Seq ID No: 5) Primer4: 5'- CCCGGATCCTCAGCATTCACAAAGGCAGCCAACC- 3' (Seq ID No: 6) The 5 ′ ends of these two primers contain Ndel and BamHI digestion sites, respectively, followed by the coding sequences of the 5 ′ end and the end of the target gene, respectively. The Ndel and BamHI digestion sites correspond to the expression vector Selective endonuclease site on plasmid pET-28b (+) (Novagen, Cat. No. 69865.3). PCR was performed using the pBS-0558e09 plasmid containing the full-length target gene as a template. The PCR reaction conditions were as follows: a total volume of 50 μ1 containing 10 pg of _P BS_0558e09 plasmid, primers Primer-3 and Primer- 4 points, and ¹ J for lpmol, 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 Ca. bacillus DH5a by the calcium chloride method.

After the LB plate (final concentration of 30 μ _§ / πι1) was cultured overnight, the positive clones were selected by colony PCR method and sequenced. A positive clone (pET-0558e09) 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 containing kanamycin (final concentration of 30μ _§ / ηι1) in LB liquid medium, host strain BL21 _(P ET-0558e09) at 37. C. Cultivate to logarithmic growth phase, add IPTG to a final concentration of 1 mmol / L, and continue incubating for 5 hours. The bacteria were collected by centrifugation, and the supernatant was collected by centrifugation by ultrasonication. An affinity column His. Bind Quick Cartridge capable of binding to 6 histidines (6His-Tag) was used.

(Novagen) chromatography was performed to obtain purified human lipoprotein precursor protein signal peptide 14 of interest. After SDS-PAGE electrophoresis, a single band was obtained at 14 kDa (Figure 1). The band was transferred to a PVDF membrane and the N-terminal amino acid sequence was analyzed by 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 lipoprotein precursor protein signal peptide 14 antibodies

 The following peptides specific for human lipoprotein precursor protein signal peptide 14 were synthesized using a peptide synthesizer (product of PE):

NH ₂ -Met-Cys-Leu-Asn-Gln-Arg-Phe-Glu-Leu-Tyr-Tyr-Leu-Glu-Asn-Asp- C00H (SEQ ID NO: 7). The polypeptide is coupled to hemocyanin and bovine serum albumin to form a complex, respectively. For methods, see: Avrameas, et al. Iramunochemistry, 1969; 6: 43. With 4mg of hemocyanin Peptide complex plus complete Freund's adjuvant was used to immunize rabbits. After 15 days, hemocyanin peptide complex plus incomplete Freund's adjuvant was used to boost immunity once. A titer plate coated with a 15 g / ml bovine serum albumin peptide complex was used as EU SA to determine the antibody titer in rabbit serum. Protein A-Sepharose was used to isolate total IgG from antibody-positive home free serum. The peptide was bound to a cyanogen bromide-activated Sepharose4B column, and anti-peptide antibodies were separated from the total IgG by affinity chromatography. The immunoprecipitation method proved that the purified antibody could specifically bind to human lipoprotein precursor protein signal peptide 14. 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 a variety of ways. 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 identified whether it contains the polynucleotide sequence of the present invention and a homologous polynucleotide sequence is detected. Further, the probe can also be used to detect the polynucleotide sequence of the present invention or its homologous polynucleotide sequence in normal tissues or Whether the expression in pathological 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. 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 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. After the hybridization step, unhybridized probes are removed by a series of membrane washes. This embodiment uses higher-intensity washing conditions (such as lower salt concentration and higher temperature) to reduce the hybridization background and retain only strong specific signals. The probes used in this embodiment include two types: the first type of probes are oligonucleotide fragments that are completely the same as or complementary to the polynucleotide SEQ ID NO: 1 of the present invention; the second type of probes are partially related to the present invention The polynucleotide SEQ ID NO: 1 is the same or complementary oligonucleotide fragment. In this example, 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 for use as hybridization probes from the polynucleotide SEQ ID NO: 1 of the present invention should follow the following principles and several aspects to be considered:

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, 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'-GATGTAAATCAGATGATTAGAGACATTTCGCTATCATCGAA-3 '(SEQ ID NO: 8) Probe 2 (probe2), which belongs to the second type of probe, is equivalent to the replacement mutation sequence (41Nt) of the gene fragment of SEQ ID NO: 1 or its complementary fragment:

 5 '-GCTCTACATCCGATCATTCGAGACATCTCGCTATCATCGCA-3' (SEQ ID NO: 9) For other commonly used reagents and their preparation methods not related to the following specific experimental steps, 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" (U998 Second Edition) [US] Sambrook 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) Suspend the pellet (about 10 ml / g) with cold homogenization buffer (0.25 mol / L sucrose; 25 legs pl / L Tris-HCl, pH 7.5; 25 legs ol / LnaCl; 25 mmoi / L MgCl ₂ ). 4) At 4 C, homogenize the tissue suspension 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 (add 1-5 ml per 0.1 g of the original tissue sample), and centrifuge at 1,000 g 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, DNA phenol extraction method

Steps: 1) Wash cells with 1-10 ml of cold PBS and centrifuge at 1000g for 10 minutes. 2) with cold cell lysate pelleted cells were resuspended (1 X 10 ⁸ cells / ml) Minimum application lOOul lysis buffer. 3) Add SDS to a final concentration of 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 200u _g / ml. 5) Incubate at 50 ° C 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 aqueous phase containing DNA 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 ^DC 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 pipette with suction, while gradually increasing TE, mix until the DNA is fully lysed, add approximately 1 ul per 1-5 X 10 ' ¹ cell extraction.

 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 100ug / ml, and incubate at 37 ° C for 30 minutes. 9) Add SDS and proteinase K, the final concentrations are 0.5% and 100ug / ml. Incubate at 37 ° C 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 and re-extract with an equal volume of chloroform: isoamyl alcohol (24: 1) and centrifuge for 10 minutes. 12) Carefully remove the water phase, mix 1/10 volume of 2mol / L sodium acetate and 2.5 volume of cold ethanol, and mix well at -20 ° C for 1 hour. 13) Wash the pellet with 70% ethanol and 100% ethanol, air dry, and resuspend the nucleic acid. The process is the same as steps 3-6. 14) Determine A _26Q and A ₂₈ . To check the purity and yield of DM. 15) Store at -20 after packing. C. Preparation of sample film:

 1) Take 4x 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, so that it can be used in the following experimental steps. The film was washed with high-strength conditions and strength conditions, respectively.

 2) Pipette 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 raol / L Tris-HCl (pH 7.0), 3 mol / L NaCl Allow to dry for 5 minutes (twice).

 4) Clamped in clean filter paper, wrapped in aluminum foil, and dried under vacuum at 60-80 ° C for 2 hours.

Labeling of probes 1) 3 μ IProbe (0.1 OD / 10 μ 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 2 hours.

 3) Add 1/5 volume of bromophenol blue indicator (BPB).

 4) Pass through a Sephadex G-50 column.

5) Start collecting the first peak before ³² P-Probe washes 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 γ- ^32P -dATP) is prepared.

 Pre-hybridization

 Put the sample film in a plastic bag, add 3-10 mg of pre-hybridization solution (10xDenhardt's; 6xSSC, 0.1 mg / ml CT DNA (calf thymus DNA).), Seal the bag, and shake at 68 ° C for 2 hours in a water bath .

 Cross

 Cut a corner of the plastic bag, add the prepared probe, seal the bag, and shake it at 42 ° C in a water bath overnight. Wash film:

 High-intensity washing film:

 1) Take out the hybridized sample membrane.

 2) 2xSSC, 0.1% SDS, 40. C Wash for 15 minutes (twice).

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

 4) In 0.1xSSC, 0.1% SDS, wash at 55 ° C for 30 minutes (twice), and dry at room temperature. Low-intensity washing film:

 1) Take out the hybridized sample membrane.

 2) 2xSSC, 0.1% SDS, wash at 37 ° C for 15 minutes (twice).

 3) 0.1xSSC, 0.1% SDS, wash at 37 ° C for 15 minutes (twice).

 4) 0.1xSSC, 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:

The hybridization experiment using low-intensity membrane washing conditions showed that the radioactivity of the hybrid spots of the above four probes was not strong. There are obvious differences; and in the hybridization experiment using low-intensity washing conditions, the radioactive intensity of the hybrid spot of probe 1 is significantly stronger than that of the other three probe hybrid spots. 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 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 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) spotting

 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 glass medium using a Cartesian 7500 spotter (purchased from Cartesian, USA). The distance is 280 μπι. 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 have been reported in the literature in various ways, and the specific method steps have been reported in the literature in various ways. The spotting post-processing steps of this embodiment are:

 1. Hydration in a humid environment for 4 hours;

 2. 0.2% SDS was washed for 1 minute;

3. Wash twice with ddH ₂ 0 for 1 minute each time;

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 and store at 25 C in the dark for future use.

 (2) Probe marking

Total mRNA was extracted from normal liver and liver cancer by one-stroke method, and Oligotex mRNA Midi Kit (purchased from QiaGen) was used to purify mRNA, and the fluorescent reagent Cy3dUTP (5-Amino- propargy 1 ~ 2 · -deoxyur i dine 5 '-tr iphate coupled to Cy3 fluorescent dye was purchased from Amersham Phamacia Biotech Company by reverse transcription. ) Mark normal tissue mRNA. Mark liver cancer tissue mRNA with Cy5dUTP (5-Amino-propargyl-2 '-deoxyur i dine 5 * -tr iphate coupled to Cy5 fluorescent dye, purchased from Amersham Phamacia Biotech). Probes were prepared after purification. Specific steps refer to and methods Schena, M., Shalon, D., Heller, R. (1996) Proc. Natl. Acad. Sci. USA. Vol. 93: 10614- 10619. Schena, M., Shalon, Dar i. Davis, RW (1995) Science.270. (20): 467-480. (3) Hybridization

 Probes from the above two tissues and chips were hybridized in a UniHyb ™ Hybridization Solution (purchased from TeleChem) hybridization solution for 16 hours, washed with a washing solution (1 x SSC, 0.2% SDS) at room temperature, and then scanned with ScanArray 3000. Scanner (purchased from General Scanning Company, USA) for scanning. The scanned image is processed by Imagene software (Biodiscovery Company, USA) for data analysis, and the Cy3 / Cy5 ratio of each point is calculated. The points whose ratio is less than 0.5 and greater than 2 are considered Genes with differential expression.

Experimental results show that Cy 3 si gna 1 = 1760.92 (average of four experiments), Cy 5 si gna 1 = 4735.54 (average of four experiments), C y 3 / C y 5 = 0.371851995759723, the present invention The expression of the polynucleotide in the above two tissues is significantly different, indicating that the polynucleotide of the present invention is closely related to liver cancer. Industrial applicability

 The polypeptides of the present invention, as well as antagonists, agonists and inhibitors of the polypeptides, can be directly used in the treatment of diseases, for example, they can treat malignant tumors, adrenal deficiency, skin diseases, various types of inflammation, HIV infection, and immune diseases.

 The conserved sequence on the signal peptide of the precursor protein of lipoprotein determines whether the protein is lipid modified. Therefore, this signal peptide sequence can treat or prevent diseases caused by a lack of normal functioning lipoproteins due to abnormal signal peptide synthesis, such as Alzheimer's disease, brain tumors, exogenous / endogenous lipidemia, family Hypercholesterolemia, Cholesterol lipid storage disease, fatty liver, liver cancer and coronary heart disease.

 The polypeptides or fragments thereof of the present invention can also be used to treat or prevent various cancers, including but not limited to: Respiratory system tumors: nasal and sinus tumors, nasopharyngeal cancer, laryngeal cancer, tracheal tumors, lung cancer, pleural mesothelioma digestive system Tumors: salivary gland tumor, esophageal cancer, esophageal leiomyosarcoma, primary esophageal small cell cancer, gastric cancer, gastric malignant lymphoma, gastric carcinoid, colorectal cancer, colon cancer, intestinal malignant lymphoma, primary liver cancer, liver mother Cell tumor, primary gallbladder cancer, pancreatic cancer

Blood, lymphatic tumors: acute leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, malignant lymphoma (such as lymphatic reticulum, malignant lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, etc.), malignant Histiocytosis Neurological tumors: astrocytoma, ependymal tumor, medulloblastoma, meningiomas, glioblastoma, acoustic neuroma, angiogenic tumor, pituitary adenoma, craniopharyngioma

 Motor system tumors: osteoid osteoma, osteochondroma, chondroma, osteoblastoma, chondroblastoma, etc.), malignant bone tumors such as giant cell tumor of bone, osteosarcoma, chondrosarcoma, Ewing's sarcoma, myeloma

 Tumors of the genitourinary system: benign tumors such as renal tubular adenoma, eosinophilic adenoma, juxtaglomerular cell tumor, polycystic kidney tumor, seminoma, teratoma, testicular stromal cell tumor, intrauterine Membrane stromal tumor, hydatidiform mole, ovarian tumor, breast fibroma, malignant tumors such as renal cell carcinoma, renal sarcomatoid carcinoma, papillary renal cell carcinoma, nephroblastoma, prostate cancer, testicular tumor chorionic carcinoma, cervical cancer , Endometrial cancer, endometrial cancer, infusion tube cancer, ovarian cancer, breast cancer

 Endocrine system tumors: pituitary adenoma, benign thyroid tumor, thyroid cancer, parathyroid adenoma, parathyroid cancer, adrenal myeloma, pheochromocytoma, islet cell tumor, multiple endocrine gland tumor, thymic tumor

 Soft tissue tumors: fibroma, fibrosarcoma, fibromatosis, lipoma, liposarcoma, leiomyoma, leiomyosarcoma, rhabdomyosarcoma, rhabdomyosarcoma, synovial tissue tumor, hemangioma, intramuscular hemangioma, blood vessels Globuloma, hemangioendothelial sarcoma, lymphangioma, lymphangiomyoma, lymphatic endothelial sarcoma, histiocytoma, malignant fibrous histiocytoma, soft tissue acinar sarcoma, clear cell sarcoma, myxoma, extraosteal Ewing sarcoma, Soft tissue osteosarcoma, soft tissue chondrosarcoma, mesothelioma, epithelioid sarcoma, schwannomas, neurofibromas, malignant schwannomas, neurofibromatosis

 Skin malignancies: dermal Mike cell tumor, Kaposi sarcoma, melanoma

 The invention also provides methods for screening compounds to identify agents that increase (agonist) or suppress (antagonist) the human lipoprotein precursor protein signal peptide 14. Agonists enhance human lipoprotein precursor protein signal peptide 14 to stimulate biological functions such as cell proliferation, while antagonists prevent and treat disorders related to excessive cell proliferation, such as various cancers. For example, mammalian cells or membrane preparations expressing human lipoprotein precursor protein signal peptide 14 can be cultured with labeled human lipoprotein precursor protein signal peptide 14 in the presence of a drug. The ability of the drug to increase or block this interaction is then determined.

 Antagonists of human lipoprotein precursor protein signal peptide 14 include antibodies, compounds, receptor deletions, and the like that have been screened. The antagonist of human lipoprotein precursor protein signal peptide 14 can bind to human lipoprotein precursor protein signal peptide 14 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 lipoprotein precursor protein signal peptide 14 can be added to the bioanalytical assay by determining the effect of the compound on the interaction between human lipoprotein precursor protein signal peptide 14 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 lipoprotein precursor protein signal peptide 1 4 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. When screening, the human lipoprotein precursor protein signal peptide 14 molecule should generally be labeled.

The present invention provides polypeptides, and fragments, derivatives, analogs or cells thereof as antigens. To produce antibodies. These antibodies can be polyclonal or monoclonal antibodies. The invention also provides antibodies against the human lipoprotein precursor protein signal peptide 14 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 human lipoprotein precursor protein signal peptide 14 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 lipoprotein precursor protein signal peptide 14 include, but are not limited to, hybridoma technology (Kohler and Milste in. Nature, 1975, 256: 495-497), triple tumor technology, human beta -Cell hybridoma technology, EBV-hybridoma technology, etc. Chimeric antibodies that bind human constant regions and non-human variable regions can be produced using existing techniques (Morri et al, PNAS, 1985, 81: 6851). ₀ Existing techniques for producing single-chain antibodies (US Pa t No. 4946778) can also be used to produce single chain antibodies against human lipoprotein precursor protein signal peptide 14.

 Antibodies against human lipoprotein precursor protein signal peptide 14 can be used in immunohistochemical techniques to detect human lipoprotein precursor protein signal peptide 14 in biopsy specimens.

 Monoclonal antibodies that bind to human lipoprotein precursor protein signal peptide 14 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 lipoprotein precursor protein signal peptide 14 High-affinity monoclonal antibodies can covalently bind to bacterial or plant toxins (such as diphtheria toxin, ricin, ormosine, etc.). A common method is to attack the amino group of an antibody with a thiol cross-linking agent such as SPDP and bind the toxin to the antibody through the exchange of disulfide bonds. This hybrid antibody can be used to kill the human lipoprotein precursor protein signal peptide 14 Positive cells.

 The antibodies of the present invention can be used to treat or prevent diseases related to the human lipoprotein precursor protein signal peptide 14. Administration of an appropriate dose of antibody can stimulate or block the production or activity of human lipoprotein precursor protein signal peptide 14.

 The invention also relates to a diagnostic test method for quantitative and localized detection of human lipoprotein precursor protein signal peptide 14 levels. These tests are well known in the art and include human lipoprotein precursor protein signal peptide 14 levels detected in FI SH assays and radioimmunoassay assays, and can be used to explain human lipoprotein precursor protein signal peptide 14 levels in various Importance in disease and in the diagnosis of diseases where human lipoprotein precursor protein signal peptide 14 plays a role.

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 analysis. Analysis.

 The polynucleotide encoding human lipoprotein precursor protein signal peptide 14 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 lipoprotein precursor protein signal peptide 14. Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated human lipoprotein precursor protein signal peptide 14 to inhibit endogenous human lipoprotein precursor protein signal peptide 14 activity. For example, a variant human lipoprotein precursor protein signal peptide 14 may be a shortened human lipoprotein precursor protein signal peptide 14 that lacks a signaling functional 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 lipoprotein precursor protein signal peptide 14. Virus-derived expression vectors such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus, etc. can be used to transfer a polynucleotide encoding a human lipoprotein precursor protein signal peptide 14 into a cell. A method for constructing a recombinant viral vector carrying a polynucleotide encoding a human lipoprotein precursor protein signal peptide 14 can be found in the existing literature (Sambrook, et al.). In addition, a recombinant polynucleotide encoding the human lipoprotein precursor protein signal peptide 14 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 lipoprotein precursor protein signal peptide 14 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 and performs endonucleation. Antisense RNA and DNA and ribozymes are available Any RNA or DM synthesis technology, such as solid-phase phosphate amide 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 DM sequence has been integrated downstream of the RNA polymerase promoter of the vector. In order to increase the stability of the nucleic acid molecule, it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the linkage between ribonucleosides using phosphate thioester or peptide bonds instead of phosphodiester bonds.

The polynucleotide encoding human lipoprotein precursor protein signal peptide 14 can be used for the diagnosis of diseases related to human lipoprotein precursor protein signal peptide 14. The polynucleotide encoding human lipoprotein precursor protein signal peptide 14 can be used to detect the expression of human lipoprotein precursor protein signal peptide 14 or the abnormal expression of human lipoprotein precursor protein signal peptide 14 in a disease state. For example, the DNA sequence encoding human lipoprotein precursor protein signal peptide 14 can be used to hybridize biopsy specimens to determine the expression status of human lipoprotein precursor protein signal peptide 14. Hybridization techniques include Sou thern blotting, Nor thern blotting, and in situ hybridization. These technical methods are Publicly available technologies 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 or a DNA chip (also referred to as a "gene chip") for analyzing differential expression analysis and gene diagnosis of genes in tissues. Human lipoprotein precursor protein signal peptide 14 specific primers for RNA-polymerase chain reaction (RT-PCR) in vitro amplification can also detect human lipoprotein precursor protein signal peptide 14 transcription products.

 Detection of mutations in the human lipoprotein precursor protein signal peptide 14 gene can also be used to diagnose human lipoprotein precursor protein signal peptide 14-related diseases. Human lipoprotein precursor protein signal peptide 14 mutations include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to the normal wild-type human lipoprotein precursor protein signal peptide 14 DM 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, 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. 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, a PCR primer (preferably 15-35b _P ) is prepared from the cDNA, and the sequence can be mapped on the chromosome. These primers were then used for PCR screening of somatic hybrid cells containing individual human chromosomes. Only those hybrid cells that contain the human gene corresponding to the primer will produce amplified fragments.

 PCR localization of somatic hybrid cells is a quick way to localize DM to specific chromosomes. Using the oligonucleotide primers of the present invention, in a similar manner, a set of fragments from a specific chromosome or a large number of genomic clones can be used to achieve sublocalization. Other similar strategies that can be used for chromosomal localization include in situ hybridization, chromosome pre-screening with labeled flow sorting, and pre-selection of hybridization to construct chromosome-specific cDNA libraries.

 Fluorescent in situ hybridization (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 Manual 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. These data can be found in, for example, V. Mckusick, Mendel ian 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 differences in cDNA or genomic sequences 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 diseased and oncoming individuals usually involves first looking for structural changes in the chromosome, 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 Figure resolution 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 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. Along with these containers, there may be instructional instructions given by government agencies that manufacture, use, or sell pharmaceuticals or biological products, which prompts permission for administration on the human body by government agencies that produce, use, or sell. In addition, the polypeptides of the invention can be used in combination with other therapeutic compounds.

 The pharmaceutical composition can be administered in a convenient manner, such as by a topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal route of administration. Human lipoprotein precursor protein signal peptide 14 is administered in an amount effective to treat and / or prevent a specific indication. The amount and range of human lipoprotein precursor protein signal peptide 1 4 to be 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 diagnosing physician.

Claims

Claim

1. An isolated polypeptide-human lipoprotein precursor protein signal peptide 14, 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 a polypeptide thereof .

 2. The polypeptide according to claim 1, wherein 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 NO: 2.

 4. An isolated polynucleotide, characterized in that said polynucleotide comprises one selected from the group consisting of:

(a) a polynucleotide encoding a polypeptide having an amino acid sequence shown in SEQ ID NO: 2 or a fragment, analog, or derivative thereof;

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

 (c) A polynucleotide that is at least 70% identical to (a) 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 619-1038 in SEQ ID NO: 1 or the sequence of positions 1-2572 in SEQ ID NO: 1. .

 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 lipoprotein precursor protein signal peptide 14 activity, characterized in that the method includes:

 (a) culturing the engineered host cell according to claim 8 under the condition of expressing human lipoprotein precursor protein signal peptide 14;

 (b) A polypeptide having human lipoprotein precursor protein signal peptide 14 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 lipoprotein precursor protein signal peptide 14.

11. 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 lipoprotein precursor protein signal peptide 14.

12. The compound according to claim 11, characterized in that it is an antisense sequence of a polynucleotide sequence or a fragment thereof as shown in SEQ ID NO: 1.

 13. Use of a compound according to claim 11, characterized in that the compound is used for a method for regulating the activity of human lipoprotein precursor protein signal peptide 14 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 1-3, characterized in that it is used for screening mimetics, agonists, antagonists or inhibitors of human lipoprotein precursor protein signal peptide 14; or For identification of peptide fingerprints.

 16. The use of a nucleic acid molecule according to any one of claims 4-6, 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 11, characterized in that said polypeptide, polynucleotide or mimetic, agonist, antagonist is used Or the inhibitor is composed of a safe and effective dose with a pharmaceutically acceptable carrier as a pharmaceutical composition for diagnosing or treating a disease associated with abnormality of the human lipoprotein precursor protein signal peptide 14.

 18. The use of a polypeptide, polynucleotide or compound according to any one of claims 1-6 and 11, characterized in that the polypeptide, polynucleotide or compound is used for preparing for treating malignant tumors, blood, etc. Disease, HIV infection and immune diseases and drugs of various inflammations.