WO2001040483A1 - Novel polypeptide---human lipoprotein 105 containing cytochrome c structural domain and polynucleotide encoding it - Google Patents

Abstract

The invention concerns human lipoprotein 105 containing cytochrome C structural domain and polynucleotide encoding it. The invention also concerns the process of producing the polypeptide by recombinant DNA technique. The methods for treating many diseases e.g. malignant tumor, hemopathy, infection of HIV, immunological diseases and a variety of inflammation utilizing the polypeptide are disclosed. The invention discloses the antagonist against the polypeptide and therapeutics thereof. The invention also discloses the uses of the polynucleotide, which encodes human lipoprotein 105 containing cytochrome C structural domain.

Description

 A new polypeptide-human cytochrome C domain-containing lipoprotein 105 and a polynucleotide encoding the polypeptide TECHNICAL FIELD

 The present invention belongs to the field of biotechnology, and specifically, the present invention describes a new polypeptide-a human cytochrome

C-domain lipoprotein 105, and a polynucleotide sequence encoding this polypeptide. The invention also relates to a preparation method and application of the polynucleotide and the polypeptide. Background technique

 Lipoproteins are a class of binding proteins composed of proteins and various fatty acids and steroids. Proteins and steroids are bound by weak non-covalent bonds, while other lipoproteins are bound by proteins and lipids by covalent bonds. Lipoproteins can be divided into three types according to their protein composition: nuclear proteins (such as thrombin-activating 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 in the cell membrane are all lipoproteins. They interact with the phospholipid layer of the cell membrane to form a membrane structure. 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 signal peptide excision will occur successively. This process takes place in a conservative area. In the future, signal peptides of proteins that have 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 i n Eng 1 989 May; 2 (7): 531-4] After the N-acetylation of the cysteine residue at the end, the protein was 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 B ioenerg B iomembr 1990 J un; 22 (3): 451 -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 I I 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

Among them, C is the attachment site of the lipid, and must be located between 15 and 35 residues. Must be in the first seven amino acids of the sequence Must have a lysine or arginine. For example, the synthesis of the signal peptide of the lipoprotein precursor protein is blocked, which will cause the resulting protein to not be properly lipidated, leading to the disorder of lipoprotein anabolic metabolism. For plasma lipoprotein, its function of transmitting lipids will be affected This leads to a series of liver diseases, such as exogenous / endogenous lipidemia, familial hypercholesterolemia, cholesterol lipid storage disease, fatty liver and so on. Severe fatty liver can also cause liver cancer. Lipoprotein anabolic disorders can also cause diseases such as coronary heart disease. A member of the cytochrome c protein family whose heme group is tightly bound to two conserved cysteine residues through a thioether bond. The binding site contains a consensus sequence: C- {CPWHF}-{CPWR} -CH- {CFYW}; The histidine in the sequence is one of the two central ligands of heme iron. All members of the cytochrome C protein family have this characteristic sequence fragment. This conserved sequence is an important structural region where cytochromes and iron ions combine to form a specific structure to complete the process of electron transfer and energy conversion. Mutations in this central region are likely to be the direct cause of protein dysfunction, and will also cause various diseases caused by abnormal energy metabolism of the organism. The novel human cytochrome C domain-containing lipoprotein 105 contains a lipid-binding site of a membrane lipoprotein and a cytochrome C domain, and these two domains cooperate in the normal physiological function of the protein. The abnormal expression of both will lead to the abnormal function of the protein. The protein is related to the occurrence of some energy metabolism and fat metabolism diseases in the body. Since the human cytochrome C domain-containing lipoprotein 105 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 is always a need in the art to identify more people involved in these processes Lipoprotein 1 05 protein containing a cytochrome C domain, in particular the amino acid sequence of this protein was identified. Isolation of the new human cytochrome C domain-containing lipoprotein 1 05 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 new polypeptides-human cytochrome C domain-containing lipoprotein 105 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 cytochrome C domain-containing lipoprotein 105.

 Another object of the present invention is to provide a genetically engineered host cell comprising a polynucleotide encoding a human cytochrome C domain-containing lipoprotein 105.

 Another object of the present invention is to provide a method for producing human cytochrome C domain-containing lipoprotein 105.

 Another object of the present invention is to provide a human cytochrome C domain-containing lipoprotein directed to the polypeptide of the present invention.

1 05 antibodies.

Another object of the present invention is to provide mimetic compounds, antagonists, agonists, and inhibitors directed to the polypeptides of the present invention-human cytochrome C domain-containing lipoproteins.

Another object of the present invention is to provide a method for diagnosing and treating a disease associated with a human lipoprotein 105-containing lipoprotein 105 abnormality.

The present invention relates to an isolated polypeptide, which is of human origin and comprises: a polypeptide having the amino acid sequence of SEQ ID No. 2, or a conservative variant, biologically active fragment or derivative thereof. Preferably, the polypeptide is a polypeptide having the amino acid sequence of SEQ ID D N'O: 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 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 237-3092 in SEQ ID NO: 1; and (b) a sequence having positions 1-3764 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 human cytochrome C domain-containing lipoprotein 105 protein activity, 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 associated with abnormal expression of a human cytochrome C domain-containing lipoprotein 105 protein in vitro, which comprises detecting the polypeptide or a polynucleotide sequence encoding the same in a biological sample. Mutations, or the amount or biological activity of a polypeptide of the invention in a biological sample. 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 diseases of energy metabolism and fat metabolism or other diseases caused by abnormal expression of lipoprotein 105 containing human cytochrome C domain. .

 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 otherwise:

 "Nucleic acid sequence" refers to oligonucleotides, nucleotides or polynucleotides and fragments or parts thereof, and can also refer to genomic or synthetic DNA or RNA, which can be single-stranded or double-stranded, representing the sense strand 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 a "polypeptide" or "protein" does not mean to limit the amino acid sequence to the entirety related to the protein molecule Natural amino acids.

 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 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 several amino acids or nucleotides by 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 "immunological activity" refers to the ability of natural, recombinant or synthetic proteins and fragments thereof to induce a specific immune response and to bind to specific antibodies in a suitable animal or cell.

 An "agonist" refers to a molecule that, when combined with human cytochrome C domain-containing lipoprotein 105, causes the protein to change, thereby regulating the activity of the protein. An agonist may include a protein, a nucleic acid, a carbohydrate, or any other molecule that can bind human cytochrome C domain-containing lipoprotein 105.

An "antagonist" or "inhibitor" refers to a biological activity that can block or regulate human cytochrome C domain-containing lipoprotein 105 when combined with human cytochrome C domain-containing lipoprotein 105. Or immunologically active molecules. Antagonists and inhibitors can include proteins, nucleic acids, carbohydrates, or any other cytochrome C domain that can bind to humans The molecule of lipoprotein 105.

 "Regulation" refers to a change in the function of human cytochrome C domain-containing lipoprotein 105, including an increase or decrease in protein activity, a change in binding properties, and any other organism of human cytochrome C domain-containing lipoprotein 105 Changes in nature, function, or immunity.

 "Substantially pure" means substantially free of other proteins, lipids, sugars or other substances with which it is naturally associated. Those skilled in the art can purify human lipoprotein 105 containing cytochrome C domain using standard protein purification techniques. Basically pure human cytochrome C domain-containing lipoprotein 105 produces a single main band on a non-reducing polyacrylamide gel. The purity of human cytochrome C domain-containing lipoprotein 105 polypeptide can be analyzed by amino acid sequence.

 "Complementary" or "complementary" refers to the natural binding of polynucleotides that are base-paired 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. The inhibition of such hybridization can be detected by performing hybridization (Southern or Northern blotting, etc.) under conditions of reduced stringency. Substantially homologous sequences or hybridization probes can compete and inhibit the binding of completely homologous sequences to 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 two sequences bind to each other as a specific or selective interaction.

 "Percent identity" refers to the percentage of sequences that are the same or similar in the comparison of two or more amino acid or nucleic acid sequences. The percent identity can be determined electronically, such as through the MEGALIGN program (Lasergene software package, DNASTAR, Inc., Madison Wis.). 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 Cluster method arranges groups of sequences into clusters by checking the distance between all pairs. The clusters are then assigned in pairs or groups. The percent identity between two amino acid sequences such as sequence A and sequence B is calculated by the following formula: The number of matching residues between sequence A and sequence B

 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 C 1 uster method or by methods known in the art such as J 01 un H ein Sex percentage (Hein J., (1990) Methods in emzumology 183: 625-645).

"Similarity" refers to the identity or conservativeness of amino acid residues at corresponding positions in the alignment of amino acid sequences. Generation. 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. This chemical modification may be the replacement of a hydrogen atom with an alkyl, acyl or amino group. Nucleic acid derivatives can encode polypeptides that retain the main biological properties of natural molecules.

"Antibody" refers to a complete antibody molecule and its fragments, such as Fa,? (^ ') ₂ and? \ ^, Which can specifically bind human epitopes of lipoprotein 105 containing cytochrome C domain.

 "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 thing, but the same polynucleotide or polypeptide is separated from some or all of the substances that coexist with it in the natural system. Such The polynucleotide may be part of a vector, or such a polynucleotide or polypeptide may be part of a composition. Since the carrier or composition is not a component of its natural environment, they are still isolated. 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 cytochrome C domain-containing lipoprotein 1 05" refers to human cytochrome C domain-containing lipoprotein 1 05 that is substantially free of other proteins, lipids, and sugars naturally associated with it. Or other substances. Those skilled in the art can use standard protein purification techniques to purify human cytochrome C domain lipids. 05. Substantially pure polypeptides can produce a single master on a non-reducing polyacrylamide gel. band. The purity of human cytochrome C domain-containing lipoprotein 1 05 peptides can be analyzed by amino acid sequence.

The present invention provides a new polypeptide-a human cytochrome C domain-containing lipoprotein 105, which is basically composed of the amino acid sequence shown in SEQ ID NO: 2. The polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide, and preferably a recombinant polypeptide. The polypeptide of the present invention may be a naturally purified product or a chemically synthesized product, or Produced from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insects, and mammalian cells) using recombinant technology. 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 cytochrome C domain-containing lipoprotein 105. As used herein, the terms "fragment", "derivative" and "analog" refer to a polypeptide that substantially retains the same biological function or activity of the human cytochrome C domain-containing lipoprotein 105 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 (II) a type in which a group on one or more amino acid residues is replaced by another group and contains a substituent: or (Ι Π) Such a type in which the mature polypeptide is fused with another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol); or UV) a type in which the additional amino acid sequence is fused into a mature% peptide sequence (Such as a leader sequence or a secreted sequence or a sequence used to purify this polypeptide or a protease sequence) As set forth 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 of 3764 bases in length and its open reading frame (237-3092) encodes 951 amino acids. This polypeptide has the characteristic sequences of the cytochrome C domain and the lipoprotein domain. It can be deduced that the human cytochrome C domain-containing lipoprotein 105 has the structure and function represented by the lipoprotein domain.

 The polynucleotide of the present invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic MA. 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 a degenerate variant. As used in the present invention, "degenerate variant" means in the present invention that encodes a gene having SEQ ID A protein or polypeptide of NO: 2 but a nucleic acid sequence different from the coding region sequence shown in SEQ ID NO: 1.

 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 guest peptide and various additional coding sequences; the coding sequence of the mature polypeptide (and optional additional coding sequences); and Non-coding sequence.

The term "polynucleotide encoding a polypeptide" is meant to include polynucleotides that encode such polypeptides and polynucleotides that include additional coding and / or noncoding sequences. The invention also relates to variants of the polynucleotides described above, which encode polypeptides or fragments, analogs and derivatives of polypeptides having the same amino acid sequence as the invention. Variants of this polynucleotide may be naturally occurring allelic variants or non-naturally occurring variants. 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: U) hybridization and elution at lower ionic strength and higher temperature, such as 0.2xSSC, 0.1% SDS, 60'C; or (2) added during hybridization Denaturing agents, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42 ° C, etc .; or (3) the identity between two sequences is at least 95% It is better to cross at least 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 in the present invention, a "nucleic acid fragment" contains at least 10 nucleotides in length, preferably at least 20-30 nucleotides, more preferably at least 50-60 nucleotides, and most preferably at least 100 cores. Glycylic acid or more. Nucleic acid fragments can also be used in nucleic acid amplification techniques such as PCR to identify and / or isolate polynucleotides encoding human cytochrome C domain-containing lipoprotein 105.

 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 cytochrome C domain-containing lipoprotein 105 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 DM is the least commonly used. Direct chemical synthesis of DNA sequences is often the method of choice. The more commonly used method is the isolation of cDNA sequences. The standard method for isolating cDNA of interest is to isolate mRNA from donor cells that overexpress the gene and perform reverse transcription to form a plasmid or phage cDNA library. There are many mature methods for extracting mRNA, and kits can also be obtained from Commercially available (Qiagene). CDNA library is constructed in a conventional method (Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory. New York, 1989) 0 may be obtained commercially available cDNA library such as cDNA library from Clontech is different. When combined with polymerase reaction technology, even Very few 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-DNA or DNA-RNA hybridization; (2) the presence or absence of marker gene functions; (3) determination of human cytochrome C domain-containing lipoprotein 105 transcripts (4) Detecting the protein product of gene expression by immunological techniques or measuring biological activity. The above methods can be used singly 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 generally a DNA sequence chemically synthesized based on the gene sequence information of the present invention. The genes or fragments of the present invention can of course be used as probes. DNA probes can be labeled with radioisotopes, luciferin, or enzymes (such as alkaline phosphatase).

 In the (4) method, immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (EL1SA) can be used to detect protein products expressed by human lipoprotein 105 gene containing cytochrome C domain.

 A method for amplifying DNA / RNA by PCR (Saiki, et al. Science 1985; 230: 1350-1354) is preferably used to obtain the gene of the present invention. Especially when it is difficult to obtain the full-length cDNA from the library, the RACE method (RACE- rapid amplification of cDNA ends) can be preferably used. The primers used for PCR can be based on the guest nucleotide sequence information of the present invention disclosed herein. It is appropriately selected and synthesized by a conventional method. The amplified DNA / RNA fragments can be separated 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 sequencing can also be performed using commercial sequencing kits and the like. In order to obtain the full-length cDNA sequence, 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 genetically engineered using the vector of the present invention or directly using a human cytochrome C domain-containing lipoprotein 105 coding sequence, and the recombinant technology to produce the present invention Methods of the polypeptide.

In the present invention, a polynucleotide sequence encoding human cytochrome C domain-containing lipoprotein 105 can be inserted into a vector to form 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 vector expressing in bacteria (Rosenberg, et al. Gene, 1987, 56: 125); expression in mammalian cells pMSXND expression vector (Lee and Nathans, J Bio Chem. 263: 3521, 1988) and baculovirus-derived vector expressed in insect cells. In short, as long as it can be replicated and stabilized in a 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 cytochrome C domain-containing lipoprotein 105 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 an enhancer sequence into the vector will enhance its transcription in higher eukaryotic cells. Enhancers are cis-acting factors for DNA expression, usually about 10 to 300 base pairs, which act on promoters to enhance gene transcription. Examples include 100 to 270 base pairs of the SV40 enhancer on the late side of the origin of replication, polyoma enhancers and adenovirus enhancers on the late side of the origin of replication.

 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 regulatory elements (such as promoters, enhancers, etc.) and selectable marker genes.

 In the present invention, a polynucleotide encoding a human cytochrome C domain-containing lipoprotein 105 or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to constitute a genetic engineering containing the polynucleotide or the recombinant vector. Host cells. 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 S; animal cells such as CH0, COS or Bowes melanoma cells.

Transformation of a host cell with a DNA sequence described in the present invention or a recombinant vector containing the DNA sequence can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as E. coli, it can absorb the sense of DNA By the cells may be harvested after exponential growth phase, with (: Treatment 1 _2, steps well known in the art with alternative _2, if desired, the conversion may be performed by electroporation method MgC l... When the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate co-precipitation, or conventional mechanical methods such as microinjection, electroporation, and liposome packaging.

 Using conventional recombinant DNA technology, the polynucleotide sequence of the present invention can be used to express or produce recombinant human cytochrome C domain-containing lipoprotein 1 05 (Scence, 1 984; 224: 1 431). Generally there are the following steps:

 (1) A polynucleotide (or variant) encoding a human-human cytochrome C domain-containing lipoprotein 105 of the present invention, or a suitable host transformed or transduced with a recombinant expression vector containing the polynucleotide Cell

 (2) culturing host cells 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 media, and the culture is performed under conditions suitable for the growth of the host cell. 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 necessary, the physical, chemical, and other properties can be used to separate and purify the recombinant protein by various separation methods. 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 is a polyacrylamide gel electrophoresis image (SDS-PAGE) of an isolated human cytochrome C domain-containing lipoprotein 105. The molecular weight of the protein is 105 kDa. 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. The experimental methods without specific conditions in the following examples are generally in accordance with the conventional The conditions are as described in Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer.

 Example 1: Cloning of human cytochrome C domain-containing lipoprotein 105

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

(Qiegene product) Isolate poly (A) mRNA from total RNA. 2ug poly (A) mRNA is reverse transcribed to form cDNA. The Smart cDNA cloning kit (purchased from Clontech) was used to insert the cDNA fragments into the multiple cloning site of pBSK (+) vector (Clontech) to transform DH5a. The bacteria formed a cDNA library. Dye terminate cycle reaction sequencing kit (Perkin-Elmer) and ABI 377 automatic sequencer (Perk in-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 DNA sequence database (Genebank), and it was found that the cDM sequence of one of the clones 0407E10 was new DNA. A series of primers were synthesized to perform bidirectional determination of the inserted cDNA fragments contained in this clone. The results showed that the full-length cDNA contained in the 0407E10 clone was 3764bp (as shown in Seq ID N0: 1), and there was a 2856bp open reading frame (0RF) from 237bp to 3092bp, which encodes a new protein (such as Seq ID NO : Shown in 2). We named this clone pBS-0407E10, and the encoded protein was named human cytochrome C domain-containing lipoprotein 105. Example 2: Cloning of a gene encoding human cytochrome C domain-containing lipoprotein 105 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 with Qigcne's kit, PCR was performed using the following primers:

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

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

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

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

Amplification conditions: 50 μl of KC1, 10 mmol / L Tris-CI, (pH8.5), 1.5 mmol / L MgCl ₂ , 200 μ mol / L dNTP, lOpmol in a reaction volume of 50 μ 1 Primer, 1U of Taq DNA polymerase (Cloiuech). The reaction was performed on a PE9600 DNA thermal cycler (Perkin-Elmer) under the following conditions for 25 cycles: 94. C 30sec; 55 ° C 30sec; 72 ° C 2min. During RT-PCR, β-actin was set as a positive control and template blank was set as a negative control. The amplified product was purified using a QIAGEN kit and ligated to a pCR vector (Invitrogen product) using a TA cloning kit. The DNA sequence analysis results showed that the DM sequence of the PCR product was exactly the same as that of 1-3764bp shown in SEQ ID NO: 1. Example 3: Northern blot analysis of human cytochrome C domain-containing lipoprotein 105 gene expression: One-step extraction of total RNA [Anal. Biochem 1987, 162, 156-159] ₀ This method includes acidic guanidine thiocyanate Phenol-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. 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 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-ImMEDTA-2.2M formaldehyde. It was then transferred to a nitrocellulose membrane. A ³² P-labeled DNA probe was prepared using c ″ ² P dATP by a random primer method. The DNA probe used was a human cytochrome C domain-containing lipoprotein 105 coding region sequence (237bp to 3092bp) amplified by PCR. The 32P-labeled probe (about 2 10 cpm / ml) was hybridized with a nitrocellulose membrane transduced with RNA at 42 ^D C overnight in a solution containing 50% formamide-25 mM H ₂ PO. (pll7.4)-5 SSC-5 Denhardt's solution and 200 g / ml salmon sperm DNA. After hybridization, the filters were placed in 1 SSC-0.1% SDS at 55. C for 30 min. Then, Phosphor Imager was used for analysis and quantification. Example 4: In vitro expression, isolation and purification of recombinant human cytochrome C domain-containing lipoprotein 105

 According to SEQ ID NO: 1 and the coding region sequence, a pair of specific amplification primers was designed, the sequence is as follows:

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

Primer4: 5'- CCCCTCGAGTTACTTCTTTACCAAAAATCGTGT -3, (Seq ID No: 6) The 5 'ends of these two primers contain Ndel and Xhol restriction sites, respectively, followed by the coding sequences of the 5' and 3 'ends of the target gene, respectively. The Ndel and Xhol restriction sites correspond to the selective endonuclease sites on the expression vector plasmid pET-28b (+) (Novagen, Cat. No. 69865.3). The PCR reaction was performed using pBS-0407E10 plasmid containing the full-length target gene as a template. The PCR reaction conditions were as follows: a total volume of 50μ1 containing 10 pg of pBS-0407E10 plasmid, primers Primer-3 and Primer-4, and lOpmol, Advantage polymerase Mix (Clontech) 1 μ 1 '. Cycle parameters: 94 ° C 20s, 60 ° C 30s, 68 ° C 2 min, 25 cycles. Ndel and Xhol were used to double-digest the amplified product and plasmid pET-28 (+), respectively, and large fragments were recovered and ligated with T4 ligase. The ligation product was transformed into E. coli DH5a by the calcium chloride method. After being cultured overnight on LB plates containing kanamycin (final concentration 30 g / ml), positive clones were selected by colony PCR method and sequenced. A positive clone (pET-0407E10) with the correct sequence was selected, and the recombinant plasmid was transformed into E. coli BL21 (DE 3) p 1 y S s (product of Novagen) by the calcium chloride method. In LB liquid medium containing kanamycin (final concentration 30 g / ml), the host bacteria BL21 (pET_0407E10) was cultured at 37 ° C to the logarithmic growth phase, and 1PTG was added to a final concentration of 1 ol / L, and continued. Incubate for 5 hours. Collect the bacterial cells by centrifugation, break the bacteria by ultrasonic, and collect the supernatant by centrifugation. Chromatography was performed using His. Bind Quick Cartridge (Novagen) affinity chromatography column capable of binding 6 histidines (6His-Tag) to obtain purified human cytochrome C domain-containing lipoprotein. 105. After SDS-PAGE electrophoresis, a single band was obtained at 105 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 cytochrome C domain-containing lipoprotein 105 antibody

 The following human cytochrome C domain-containing lipoprotein 105-specific peptides were synthesized using a peptide synthesizer (product of PE):

_{NH 2 -Met-Leu-Phe-} Arg-Ile-Lys-Leu-Met-Leu-Asp-Ala-Lys-Thr-Leu-Ile-COOH (SEQ ID N0: 7). The peptide was coupled to hemocyanin and bovine serum albumin to form a complex. For the method, see: Avrameas, ct al. Immunochemi st ry, 3969; 6: 43 „Use 4mg of the above hemocyanin peptide complex with complete Freund's adjuvant. Immunize rabbits and boost the immunity with hemocyanin-polypeptide complex and incomplete Freund's adjuvant once after 15 days. ELISA was used to determine rabbit serum using titer plates coated with 15 μg / m 1 bovine serum albumin-polypeptide complex. The titer of the antibody in the medium. Isolate the total IgG from the antibody-positive home serum with protein A-Sepharose. The peptide was bound to a cyanogen bromide-activated Se pha r os e4B column and the total I gG was determined by affinity chromatography. Anti-polypeptide antibodies were isolated from the cells. The immunoprecipitation method proved that the purified antibodies could specifically bind to human cytochrome C domain-containing lipoprotein 105. Examples Application of the polynucleotide fragments of the present invention as 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 tissue 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 blotting, Northern blotting, and copying methods. They all use the same steps to hybridize the fixed polynucleotide sample to the filter. These same steps are as follows: The sample-immobilized filter is first pre-hybridized with a probe-free hybridization buffer, so that the non-specific binding site of the sample on the filter is saturated with the carrier and the synthetic polymer. The pre-hybridization solution is then The hybridization buffer containing the labeled probe is replaced and incubated to hybridize the probe to the target nucleic acid. After the hybridization step, the unhybridized probes are removed by a series of membrane washing steps. This embodiment utilizes higher-intensity washing conditions (such as lower salt concentration and higher temperature) to reduce the hybridization background and retain only strong specific signals. The probes used in this embodiment include two types: the first type of probes are oligonucleotide fragments that are completely the same as or complementary to the polynucleotide SEQ ID NO: 1 of the present invention; the second type of probes are partially related to the present invention The polynucleotide SEQ ID NO: 1 is the same or complementary oligonucleotide fragment. 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'-CAAAGTAGTTATAGTGGATGAATCACACTACATGAAATCCAG-3 '(SEQ ID NO: 8) Probe 1 (probe2), which belongs to the second type of probe, is equivalent to the replacement mutation sequence (41Nt) of the gene fragment or its complementary fragment of SEQ ID NO: 1:

 5 '-CAAAGATCGTATAGTGGAATCGCACACTACATGATCGCCAG-3' fSEQ 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 Keller; MM Manak; Stockton Press, 1989 (USA) And more commonly used molecular cloning experiment manual books such as "Molecular Cloning Experiment Guide" (Second Edition 1998) [US] Sambrook et al., Science Press.

Xiangpin 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. Tissue should be kept moist during operation. 2) Centrifuge the tissue at 1,000 g for 10 minutes. 3) Suspend the pellet with cold homogenate buffer (0.25mol / L sucrose; 25 crypto ol / L Tris-HCl, pH7.5; 25 surface 1 / LnaCl; 25 bandol / L MgCl ₂ ) (about 10ml / g) . 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 (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 with lysis buffer (add 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 the cells with 1-10ml 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 ^D 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. The DNA was then purified and ethanol precipitated.

3, DNA purification and ethanol precipitation

Steps: 1) Add 10 volumes of 2mol / L sodium acetate and 2 volumes of cold 100% ethanol to the DM solution and mix well. Leave at -20 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 evaporate the surface ethanol. 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 pipette while gradually increasing TE, mix until the DNA is fully dissolved, and add approximately 1 ul per 1-5 10 ⁶ 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 100ug / ml, and incubate at 37 ° C for 30 minutes. 9) Add SDS and proteinase K to the final concentration of 0.5% and 100ug / nil. 37 ^U 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, re-extract with an equal volume of chloroform: isoamyl alcohol (24: 1), and centrifuge for 10 minutes. 12) Carefully remove the water phase and add 1/10 volume 2mol / L Mix sodium acetate and 2.5 volumes of cold ethanol and mix 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) Measure A ₂₆ . And A ₂₈ . To detect the purity and yield of DNA. 15) Store at -20 ° (after packing).

Preparation of sample film:

 1) Take 4 x 2 pieces of nitrocellulose membrane (NC membrane) of appropriate size, and mark the spotting position and sample number on it with a pencil. Two NC membranes are needed for each probe for later experiments. In the step, the film is 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), and dry in 0.5 mol / L Tris-HCl ( _P H7.0), 3 mol / L NaCl Leave on filter paper for 5 minutes (twice) and dry.

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

 Labeling of probes

1) 3 μl Probe (0.10D / 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 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 the collection of the first peak is combined, the ³² P- Probe (the second peak is free γ- ³² P-dATP) _{t is prepared} .

 Put the sample membrane in a plastic bag, add 3-10 mg of 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 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, wash at 40 ° C for 15 minutes (twice). 3) Wash in 0.1 x SSC, 0.1% SDS for 15 minutes at 40 ° C (twice).

 4) Wash in 0.1 x SSC, 0.1% SDS 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 for 15 minutes at 37 (twice).

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

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

X-ray auto-development:

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

 Experimental results:

 The hybridization experiments performed under low-intensity membrane washing conditions showed no significant difference in the radioactivity of the above four probe hybrid spots; while the hybridization experiments performed under low-intensity membrane washing conditions, the radioactive intensity of probe 1 was significantly stronger than The radioactivity of the other three probe hybridization spots. Therefore, the 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. Industrial applicability

 The polypeptides of the present invention, as well as the antagonists, agonists and inhibitors of the polypeptides, can be directly used in the treatment of diseases, for example, they can treat diseases such as energy metabolism and fat metabolism.

 The conserved sequence on the signal peptide of the precursor protein of lipoprotein determines whether the protein is lipid modified. Lipoproteins include nuclear proteins (such as thrombin-activating enzymes), phosphoproteins (such as lipophosphoproteins in yolk), and simple proteins (such as plasma lipoproteins and brain proteolipids), each of which has important functions. Therefore, the abnormal expression of the signal peptide sequence of the lipoprotein precursor protein can lead to abnormal function of the lipoprotein and cause corresponding diseases. Such as blood coagulation dysfunction, mental retardation, lipid metabolism disorders, especially Alzheimer's disease, exogenous / endogenous lipidemia, familial hypercholesterolemia, cholesterol lipid storage disease, fatty liver, crown Heart disease and so on.

It can be seen that the abnormal expression of human cytochrome C domain-containing lipoprotein 105 in the present invention will produce various diseases, especially coagulation dysfunction, mental retardation, and lipid metabolism disorders. These diseases include Ibuya In: Hemorrhagic diseases: Hereditary capillary dilatation, allergic purpura, vascular purpura, simple purpura, idiopathic thrombocytopenic purpura, hemophilia, vascular hemophilia, etc. Nervous system degenerative diseases: Alzheimer's disease, Parkinson's disease, chorea, depression, amnesia, Huntington's disease, epilepsy, migraine, dementia, multiple sclerosis, mental retardation, etc.

 Disorders of Lipid Metabolism: These diseases include, but are not limited to:

 1. Fatty deposition diseases: Alzheimer's disease, fatty liver, steatosis cardiomyopathy, steatosis nephropathy, exogenous / endogenous lipemia, familial hypercholesterolemia, cholesterol lipid storage disease, etc. .

 2. Sterol derivatives (such as bile acids, sex hormones (testosterone, estradiol, estriol, progesterone)) Metabolic disorders: U) Bile acid disorders such as biliary cirrhosis, cholelithiasis (2 Sexual developmental disorders in the growth and development stages: precocious puberty, delayed sexual development, sexual differentiation disorders, other defects in external genital development (3) Endocrine and metabolic syndromes: Hyperadrenal cortical diseases such as Cushing syndrome, hyperaldosteronism, adrenal glands Cortical dysfunction, such as acute adrenal insufficiency and chronic adrenal insufficiency.

 3. Cardiovascular disease: Coronary atherosclerotic heart disease, such as occult heart disease, angina pectoris, myocardial infarction, dying coronary heart disease, hypertension, etc.

 4. Tumors: brain lipoma, lipoblastoma, liposarcoma, breast cancer, etc. The abnormal expression of the human cytochrome C domain-containing lipoprotein 1 05 of the present invention will also produce certain hereditary, bloody diseases, and immune system diseases.

 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 coagulation dysfunction, mental retardation, lipid metabolism disorders, and some inheritance. Sexual, hematological and immune system diseases.

 The invention also provides methods of screening compounds to identify agents that increase (agonist) or suppress (antagonist human cytochrome C crust domain-containing lipoprotein 105). Agonists increase human cytochrome C domain-containing lipoprotein 1 05 Stimulates biological functions such as cell proliferation, and antagonists prevent and treat disorders related to excessive cell proliferation, such as various cancers. For example, mammalian cells or humans that express cytochrome C domains can be expressed in the presence of drugs. The membrane preparation of lipoprotein 1 05 was cultured with labeled human cytochrome C domain-containing lipoprotein 1 05. The ability of the drug to enhance or suppress this interaction was then determined.

 Antagonists of human cytochrome C domain-containing lipoprotein 105 include antibodies, compounds, receptor deletions, and the like that have been screened. Antagonists of human cytochrome C domain-containing lipoprotein 1 05 can bind to human cytochrome C domain-containing lipoprotein 1 05 and eliminate its function, or inhibit the production of the polypeptide, or with the activity of the polypeptide Site binding prevents the polypeptide from performing its biological function.

When screening compounds as antagonists, human cytochrome C domain-containing lipoprotein 105 can be added In bioanalytical assays, whether a compound is an antagonist is determined by determining the effect of the compound on the interaction between human cytochrome C domain-containing lipoprotein 105 and its receptor. 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 cytochrome C domain-containing lipoprotein 105 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. When screening, human lipoprotein 105 molecules containing cytochrome C domains 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 invention also provides antibodies directed against human cytochrome C domain-containing lipoprotein 105 epitopes. These antibodies include (but are not limited to): polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, and fragments produced by Fab expression libraries.

 Polyclonal antibodies can be produced by injecting human cytochrome C domain-containing lipoprotein 105 directly into immunized animals (such as home immunity, mice, rats, etc.). A variety of adjuvants can be used to enhance the immune response, including but not limited to Freund's adjuvant, etc. Techniques for preparing human monoclonal antibodies to lipoprotein 105 containing cytochrome C domain include, but are not limited to, hybridoma technology (Kohler and Milstein. Nature, 1975, 256: 495-497), triple tumor technology, human beta cells Hybridoma technology, EBV-hybridoma technology, etc. Chimeric antibodies that bind human constant regions to non-human variable regions can be produced using existing techniques (Morrison et al, PNAS, 1985, 81: 6851). The existing technology for producing single chain antibodies (U.S. Pat No. 4946778) can also be used to produce single chain antibodies against human cytochrome C domain-containing lipoprotein 105.

 Antibodies against human cytochrome C domain-containing lipoprotein 105 can be used in immunohistochemical techniques to detect human cytochrome C domain-containing lipoprotein 105 in biopsy specimens.

 Monoclonal antibodies that bind to human cytochrome C domain-containing lipoprotein 105 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 cytochrome C domain-containing lipoprotein 105 high-affinity monoclonal antibodies can covalently bind to bacterial or plant toxins (such as diphtheria toxin, ricin, ormosine, etc.). A common method is to attack the amino group of an antibody with a thiol cross-linking agent such as SPDP and bind the toxin to the antibody through the exchange of disulfide bonds. This hybrid antibody can be used to kill human cytochrome C domain-containing lipids. Protein 105 positive cells.

The antibodies of the present invention can be used to treat or prevent diseases related to human cytochrome C domain-containing lipoprotein 105. An appropriate dose of the antibody can stimulate or block the production or activity of human cytochrome C domain-containing lipoprotein 105. The invention also relates to a diagnostic test method for quantitatively and locally detecting the level of lipoprotein 105 containing human cytochrome C domain. These tests are well known in the art and include FI SH assays and radioimmunoassays. The level of human cytochrome C domain-containing lipoprotein 1 05 detected in the test can be used to explain the importance of human cytochrome C domain-containing lipoprotein 1 05 in various diseases and to diagnose human cell-containing Diseases where the pigment C domain of lipoprotein 1 05 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.

 Polynucleotides encoding human cytochrome C domain-containing lipoprotein 105 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 cytochrome C domain-containing lipoprotein 1 05. Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated human cytochrome C domain-containing lipoprotein 105 to inhibit endogenous human cytochrome C domain-containing lipoprotein 105 activity. For example, a mutated human cytochrome C domain-containing lipoprotein 1 05 may be a shortened human cytochrome C domain-containing lipoprotein 1 05 that lacks a signaling function domain, although it can interact with downstream substrates. Binding, but lacks signaling activity. Therefore, the recombinant gene therapy vector can be used for treating diseases caused by abnormal expression or activity of lipoprotein 105 containing cytochrome C domain. Virus-derived expression vectors such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus, etc. can be used to transfer a polynucleotide encoding human cytochrome C domain-containing lipoprotein 1 05 into cells . A method for constructing a recombinant viral vector carrying a polynucleotide encoding a human cytochrome C domain-containing lipoprotein 1 05 can be found in the existing literature (Samb rook, et al.). In addition, a recombinant polynucleotide encoding human cytochrome C domain-containing lipoprotein 105 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 introducing the cell Implanted into the body, etc.

Oligonucleotides (including antisense RNA and DNA) and ribozymes that inhibit human cytochrome C domain-containing lipoprotein 105 mRNA are also within the scope of the present invention. A ribozyme is an enzyme-like RN'A molecule that can specifically decompose specific RNA. Its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target RNA and performs endonucleation. Antisense RNA, DNA, and ribozymes can be obtained by any existing RNA or DNA synthesis technology, such as 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 vector's RNA polymerase promoter. In order to increase the stability of the nucleic acid molecule, it can be modified in various ways, such as increasing the sequence length on both sides, and ribonucleosides. The connection between them should be a thioester or peptide bond instead of a phosphodiester bond.

 The polynucleotide encoding human cytochrome C domain-containing lipoprotein 105 can be used for diagnosis of diseases related to human cytochrome C domain-containing lipoprotein 105. Polynucleotides encoding human cytochrome C domain-containing lipoprotein 105 can be used to detect the expression of human cytochrome C domain-containing lipoprotein 105 or human cytochrome C domain-containing lipoprotein 105 in disease states Abnormal expression. For example, a DNA sequence encoding human cytochrome C domain-containing lipoprotein 105 can be used to hybridize biopsy specimens to determine the expression of human cytochrome C domain-containing lipoprotein 105. Hybridization techniques include Southern blotting, Northern blotting, in situ hybridization, and so on. 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 or a DNA chip (also referred to as a "gene chip") for analyzing differential expression analysis and gene diagnosis of genes in tissues. Human cytochrome C domain-containing lipoprotein 105-specific primers can be used for RNA-polymerase chain reaction (RT-PCR) in vitro amplification to detect human cytochrome C domain-containing lipoprotein 105 transcripts.

 Detection of mutations in the human cytochrome C domain-containing lipoprotein 105 gene can also be used to diagnose human cytochrome C domain-containing lipoprotein 105-related diseases. Human cytochrome C domain-containing lipoprotein 105 mutant forms include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to normal wild-type human cytochrome C domain-containing lipoprotein 105 DNA sequences. 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, so 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. This sequence will specifically target a specific position of a human chromosome and can hybridize with 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) can be used to mark 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 15-35bp) are 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 containing human genes corresponding to the primers 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, by a similar method, a set of fragments from a specific chromosome or a large number of genomic clones can be used to achieve sublocalization. Other similar strategies that can be used for chromosomal localization include in situ hybridization, chromosome pre-screening with labeled flow sorting, and hybrid pre-selection to construct chromosome-specific cDNA libraries. Fluorescent in situ hybridization (FISH) of cDNA clones to 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 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 individual, the mutation may be the cause of the disease. Comparing affected and unaffected 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 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 contains a safe and effective amount of the polypeptide or antagonist, and carriers and excipients that do not affect the effect of the drug. These compositions can be used as drugs for the treatment of diseases.

 The invention also provides a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the invention. 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 manufacture, use, or sell them. 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 cytochrome C domain-containing lipoprotein 105 is administered in an amount effective to treat and / or prevent a specific indication. The amount and dose range of human cytochrome C domain-containing lipoprotein 105 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

Claim  1. An isolated polypeptide-human cytochrome C domain-containing lipoprotein 105, characterized in that it comprises: a polypeptide having the amino acid sequence shown in SEQ H) NO: 2, or an active fragment of the polypeptide, or the like Or derivatives.  2. The polypeptide according to claim 1, wherein the amino acid sequence of said 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) a polynucleotide encoding a polypeptide having an amino acid sequence shown in SEQ ID NO: 2 or a fragment, analog, or derivative thereof;  (h) 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 237-3092 in SEQ ID NO: 1 or the sequence of positions 1-3764 in SEQ ID NO: 1. .  7. A recombinant vector containing an exogenous polynucleotide, characterized in that it is a recombinant constructed from the polynucleotide according to any one of claims 4 to 6 with 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 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 cytochrome C domain-containing lipoprotein 105 activity, characterized in that the method includes:  (a) culturing the engineered host cell of claim 8 under the condition of expressing human cytochrome C domain-containing lipoprotein 105;  (b) Isolating a polypeptide having human cytochrome C domain-containing lipoprotein 105 activity from the culture. 10. An antibody capable of binding to a polypeptide, characterized in that the antibody is an antibody capable of specifically binding to human cytochrome c domain-containing lipoprotein 105. 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 105 containing a cytochrome C domain.  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.  13. An application of the compound according to claim 11, characterized in that the compound is used for a method for regulating the activity of human cytochrome C domain-containing lipoprotein 105 in vitro and in vivo.  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 the polypeptide according to any one of claims 1-3, characterized in that it is used for screening human mimetics, agonists, antagonists or inhibitors of lipoprotein 105 containing cytochrome C domain ; 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 used 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 abnormalities of lipoprotein 105 containing human cytochrome C domain.  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 disease and drugs for inflammation.