WO2001048002A1 - A new polypeptide - cytochrome b12 and the polynucleotide encoding it - Google Patents

Abstract

The present invention discloses a new polypeptide - cytochrome b12, the polynucleotide encoding it and a method producing the polypeptide by recombinant DNA technology. The present invention further discloses a method using the polypeptide to treat various disorders, e.g. malignant neoplasm, hematopathy, HIV infection and immunological disease and various inflammations etc. The present invention also discloses an agonist of the polypeptide and its therapeutic use. The present invention further discloses the use of the polynucleotide encoding the new cytochrome b12.

Description

 A new polypeptide-fine jJ, prime bl2 and polynucleotide encoding this poly

 The present invention belongs to the field of biotechnology. Specifically, the present invention describes a novel polypeptide, cytochrome bl2, and a polynucleotide sequence encoding the polypeptide. The invention also relates to a method and application for preparing such polynucleotides and polypeptides.

 # 术 背景

 Both mitochondria and aerobic prokaryotes of eukaryotes contain cytochrome b, which is a component of the respiratory chain complex II, also known as bcl complex or coenzyme Q-cytochrome C reductase. . Plant chloroplasts and cyanobacteria also contain a homologous protein, cytochrome b6, which is a component of plastid quinone-plastid cyanin reductase, also known as b6f complex. Cytochrome b is an important catalytic subunit of coenzyme Q, which regulates the process of organism's respiratory metabolism.

 Cytochrome b / b6 is an integral membrane protein, consisting of approximately 400 amino acid residues, and contains approximately 8 transmembrane fragments. In plants and cyanobacteria, cytochrome b6 is composed of two subunits, which are encoded by the petB gene and petD gene, respectively. The sequence of the petB gene is co-linear with the N-terminus of the mitochondrial cytochrome b protein sequence, while the sequence of petD is similar to the C-terminus of the mitochondrial cytochrome b protein sequence. In living organisms, cytochrome b / b6 and two heme groups are covalently bonded together to form complexes called b562 and b566, respectively. Among them, the four conserved histidine residues in the cytochrome b / b6 amino acid sequence are considered to be the main sites of coordination with the iron atoms in the two heme groups.

 In addition to the above four histidine-containing sequence regions, the cytochrome b / b6 sequence also contains some conserved regions. These regions are the main sites of protein's biological function. For example: The loop structure region between the 15th and 16th transmembrane fragments of the protein contains a constant PEW triplet structure, which is located on the outer surface of the membrane. This triplet structure is considered to be in the coenzyme Q redox reaction electron Plays an important role in the transfer process. In addition, the protein sequence of cytochrome b / b6 also contains some sites, which bind to inhibitors and antibodies to coenzyme Q in the body, which can effectively control the process of the respiratory chain action in the body and bring it into a balance. State [Espos ti MD, De Vr es S. et al., 1993, Bioch im Bi ophys Acta, 1143: 243—271].

 The specific structure of cytochrome Wb6 is shown below:

 + — Fe-b562— +

| + -Fe-b566—— 1 + <Cytochrome-b-

<Cytochrome-b6- petB-Cytochrome-b6- petD- ^

 Further analysis of the sequence of cytochrome b / b6, people obtained a highly conserved sequence fragment from the protein sequence as shown below:

 Sequence fragment 1: [DENQJ-X (3) -G- [FYWMQ] -X- [LIVMF] -R-X (2) -H (where H is the coordination bond formation site of the heme complex b562);

 Sequence fragment 2: P- [DE] -W- [FY]-[LFY] (2);

 Except for the cytochrome b protein sequence of deer and Paramecium, the other two cytochrome b / b6 protein sequences contain both of the above-mentioned conserved sequence fragments. Among them, sequence fragment 1 contains the first conserved histidine residue in cytochrome b / b6, where histidine and a heme group form the heme complex b562; and sequence fragment 2 contains a conserved PEW triplet structure, which plays an important role in the electron transfer process of respiration redox reactions. Mutations in these two sequence fragments will lead to abnormal expression and function of cytochrome b / b6.

In the respiratory chain, cytochrome and heme are covalently combined. The two synergistically transfer the electrons generated by the redox reaction to oxygen, generate oxygen ions, and combine with 2H + to form H ₂ 0, so as to finally complete the biological respiration in vivo. The whole process of energy metabolism. Cytochrome b / b6 plays an important role in the electron transfer process of redox reaction mediated by coenzyme Q. Abnormal expression of this protein will cause abnormal respiratory metabolism in the organism, which will cause various related metabolic disorders. . It is usually closely related to the occurrence of respiratory metabolic disorders, some mitochondrial diseases, energy metabolic disorders, and some related material metabolic disorders.

 As the cytochrome M2 protein plays an important role in the important functions of the body as described above, and it is believed that a large number of proteins are involved in these regulatory processes, there has been a need in the art to identify more cytochrome bl2 proteins involved in these processes, especially to identify Amino acid sequence of several proteins. The isolation of the new cytochrome M2 protein coding gene also provides the basis for research to determine the role of this protein in health and disease states. This protein may form the basis for the development of diagnostic and / or therapeutic drugs for the disease, so it is important to isolate its coding for DM.

Object of the invention

 It is an object of the present invention to provide an isolated novel polypeptide, cytochrome M2, 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 cytochrome M2. Another object of the present invention is to provide a genetically engineered host cell containing a polynucleotide encoding a cytochrome M2. Another object of the present invention is to provide a method for producing cytochrome M2.

 Another object of the present invention is to provide antibodies against the polypeptide of the present invention, cytochrome b12.

 Another object of the present invention is to provide mimic compounds, antagonists, agonists, and inhibitors directed to the polypeptide of the present invention, cytochrome M2.

 Another object of the present invention is to provide a method for diagnosing and treating diseases related to cytochrome b12 abnormalities.

Summary of invention

 The present invention relates to an isolated polypeptide, which is of human origin and comprises: a polypeptide having the amino acid sequence of SEQ ID No. 2, or a conservative variant, biologically active fragment or derivative thereof. 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 70% identity to a polynucleotide sequence of (a) or (b).

 More preferably, the sequence of the polynucleotide is one selected from: (a) a sequence having positions 1122-1445 in SEQ ID NO: 1; and (b) a sequence having 1-4000 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 cytochrome bl 2 protein, which comprises utilizing the polypeptide of the present 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 cytochrome bl 2 protein, comprising detecting a mutation in the polypeptide or a sequence encoding a polynucleotide thereof in a biological sample, or detecting a mutation in a biological sample The amount or biological activity of a polypeptide of the invention.

 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 cytochrome M 2.

Other aspects of the invention will be apparent to those skilled in the art from the disclosure of the techniques herein. 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 comparison diagram of the amino acid sequence homology of the 61-amino acid and cytochrome b characteristic proteins of the cytochrome b 2 of the present invention at 22-82. The upper sequence is cytochrome M 2 and the lower sequence is the cytochrome b characteristic protein domain. Ί "and": "" and "." Indicate that the probability of the same amino acid appearing between two sequences decreases in sequence.

 Figure 2 shows the polyacrylamide gel electrophoresis (SDS-PAGE) of the isolated cytochrome M 2. 12kDa is the molecular weight of the protein. The arrow indicates the isolated protein band.

Summary of the Invention

 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 DM or RNA, they can be single-stranded or double-stranded, representing the sense or antisense strand. Similarly, the term "amino acid sequence" refers to an oligopeptide, peptide, polypeptide or protein sequence and fragments or portions thereof. When the "amino acid sequence" in the present invention relates to the amino acid sequence of a naturally occurring protein molecule, such "polypeptide" or "protein" does not mean to limit the amino acid sequence to a complete natural amino acid related to the protein molecule .

 A protein or polynucleotide "variant" refers to an amino acid sequence having one or more amino acids or nucleotide changes or a polynucleotide sequence encoding it. The changes may include deletions, insertions or substitutions of amino acids or nucleotides in the amino acid sequence or nucleotide sequence. Variants 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" means that a change in the amino acid sequence or nucleotide sequence results in an increase in one or more amino acids or nucleotides compared to a molecule that exists in nature. "Replacement" refers to the replacement of one or more amino acids or nucleotides with different amino acids or nucleotides.

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

An "agonist" refers to a molecule that, when combined with cytochrome bl 2, can cause changes in the protein and thereby regulate the activity of the protein. Agonists can include proteins, nucleic acids, carbohydrates or any other Molecules that bind cytochrome bl 2.

 An "antagonist" or "inhibitor" refers to a molecule that, when combined with cytochrome bl2, can block or regulate the biological or immunological activity of cytochrome M2. Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates or any other molecule that can bind cytochrome b12.

 "Regulation" refers to a change in the function of cytochrome M2, including an increase or decrease in protein activity, a change in binding properties, and any other biological, functional, or immune properties of cytochrome M2.

 "Substantially pure '" means essentially free of other proteins, lipids, sugars or other substances with which it is naturally associated. Those skilled in the art can purify cytochrome b12 using standard protein purification techniques. Substantially pure cells Pigment bl2 can generate a single main band on a non-reducing polyacrylamide gel. The purity of the cytochrome bl2 polypeptide can be analyzed by amino acid sequence.

 "Complementary" or "complementary" refers to the natural binding of polynucleotides by base-pairing under conditions of acceptable salt concentration and temperature. For example, the sequence "C-T-G-A" can be combined with the complementary sequence "G-A-C-T". The complementarity between two single-stranded molecules can be partial or complete. The degree of complementarity between nucleic acid strands has a significant effect on the efficiency and strength of hybridization between nucleic acid strands.

 "Homology" refers to the degree of complementarity and can be partially homologous or completely homologous. "Partial homology" refers to a partially complementary sequence that at least partially inhibits hybridization of a fully complementary sequence to a target nucleic acid. 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 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 sof tware package, DNASTAR, Inc., Madi son Wis.). The MEGALIGN program can compare two or more sequences according to different methods such as the Clus ter method (Higg ins, D. G. and P. M. Sharp (1988) Gene 73: 237-244). The Clus ter method arranges groups of sequences into clusters by checking the distance between all pairs. The clusters are then assigned in pairs or groups. The percent identity between two amino acid sequences, such as sequence A and sequence B, is calculated by the following formula: Number of residues matching between sequence ^ and sequence ^

^ Ί sequence number of residues in a sequence of residues intervals ^ - ^ interval sequence of residues ^Χ

The percent identity between nucleic acid sequences can also be determined by the Clus ter method or by methods known in the art such as Jotun Hein (Hein J., (1990) Methods in enzymology 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 substitution, 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. The "antisense strand" refers to a nucleic acid strand that is complementary to the "sense strand".

 "Derivative" refers to HFP or a chemical modification of its nucleic acid. 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? ^ It can specifically bind to the epitope of cytochrome bl 2.

 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 matter from its original environment (for example, its natural environment if it is naturally occurring). 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 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 cytochrome bl 2" means that cytochrome bl 2 is substantially free of other proteins, lipids, sugars, or other substances with which it is naturally associated. Those skilled in the art will be able to purify cytochrome bl 2 using standard protein purification techniques. Substantially pure peptides can produce a single main band on a non-reducing polyacrylamide gel. The purity of the cytochrome M 2 polypeptide can be analyzed by amino acid sequence.

The present invention provides a new polypeptide, cytochrome b12, 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 obtained from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insects, and mammals using recombinant technology). Cell). 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 peptides of the invention may also include or exclude the initial methionine residue.

 The invention also includes fragments, derivatives and analogs of cytochrome b12. As used herein, the terms "fragment", "derivative" and "analog" refer to a polypeptide that substantially retains the same biological function or activity of the cytochrome b12 of the present invention. A fragment, derivative or analog of the polypeptide of the present invention may be: (I) a kind in which one or more amino acid residues are replaced with conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and the substitution The amino acid may or may not be encoded by a genetic codon; or (Π) a type in which a group on one or more amino acid residues is replaced by another group to include a substituent; or (Π Ι) 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 (IV) a type in which the additional amino acid sequence is fused into the mature polypeptide and the polypeptide sequence is formed (Such as the leader or secretory sequence or the sequence used to purify this polypeptide or protein 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 a 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 4,000 bases in length and its open reading frame 1122-1445 encodes 107 amino acids. This peptide has a characteristic sequence of a cytochrome b characteristic protein, and it can be deduced that the cytochrome b1 has the structure and function represented by the cytochrome b characteristic protein.

 The polynucleotide of the present invention may be in the form of DNA or RNA. DM forms include cDNA, genomic DNA or synthetic DNA. DNA can be single-stranded or double-stranded. DNA can be coding or non-coding. The coding region sequence encoding a mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO: 1 or a degenerate variant. As used herein, a "degenerate variant" refers to a nucleic acid sequence 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. Variants of this polynucleotide can be naturally occurring Allelic 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 (with at least two sequences between

50%, preferably 70% identity). The present invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the present invention under stringent conditions. In the present invention, "strict conditions" means: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2xSSC, 0.1% SDS, 60 ° C; or (2) added during hybridization Use a denaturant such as 50% (v / v) formamide, 0.1. /. Calf serum / 0.1% Ficoll, 42 ° C, etc .; or (3) Hybridization occurs only when the identity between the two sequences 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 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 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 cytochrome M2.

 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 cytochrome M2 of the present invention can be obtained by various methods. For example, polynucleotides are isolated using hybridization techniques well known in the art. These techniques include, but are not limited to: 1) hybridization of probes to genomic or cDNA libraries to detect homologous polynucleotide sequences, and 2) antibody screening of expression libraries to detect cloned polynucleosides with common structural characteristics Acid fragments.

 The DNA fragment sequence of the present invention can also be obtained by the following methods: 1) isolating the double-stranded DNA sequence from the genomic DNA; 2) chemically synthesizing the DNA sequence to obtain the double-stranded DNA of the polypeptide.

 Of the methods mentioned above, genomic DNA isolation is the least commonly used. Direct chemical synthesis of DNA sequences is often the method of choice. The more commonly used method is the isolation of cDNA sequences. The standard method for isolating the cDNA of interest is to isolate 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 raRM extraction, and kits are also commercially available (Qiagene). The construction of cDNA libraries is also a common method (Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory. New York, 1989). Commercially available cDNA libraries are also available, such as different cDNA libraries from Clontech. When polymerase reaction technology is used in combination, even very small expression products can be cloned.

These genes can be screened from these cDNA libraries by conventional methods. These methods include (but are not limited to): (l) DNA-DNA or DNA-RNA hybridization; (2) the appearance or loss of marker gene function; (3) measurement Determine the transcript level of cytochrome bl2; (4) Detect the protein product of gene expression by immunological techniques or determination of 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 usually a DNA sequence chemically synthesized based on the gene sequence information of the present invention. The genes or fragments of the present invention can of course be used as probes. DNA probes can be labeled with radioisotopes, luciferin, or enzymes (such as alkaline phosphatase).

 In the method (4), immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA) can be used to detect the protein product of cytochrome bl2 gene expression.

 A method using PCR technology 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 cDNA end rapid amplification method) 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 measured by a conventional method such as dideoxy chain termination method (Sanger et al. PNAS, 1977, 74: 5463-5467). Such polynucleotide sequences can also be determined using commercial sequencing kits and the like. In order to obtain the full-length cDNA sequence, sequencing needs to be repeated. Sometimes it is necessary to determine the cDNA sequence of multiple clones in order to splice into a full-length cDNA sequence.

 The present invention also relates to a vector comprising the polynucleotide of the present invention, and a host cell genetically engineered using the vector of the present invention or directly using a cytochrome M2 coding sequence, and a method for producing a polypeptide of the present invention by recombinant technology.

In the present invention, a polynucleotide sequence encoding a cytochrome b12 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 (Rosenberg, et al. Gene, 19107, 56: 125) expressed in bacteria; pMSXND expression vectors expressed in mammalian cells ( Lee and Nathans, J Bio Chem. 263: 3521, 1988) and baculovirus-derived vectors expressed in insect cells. In short, as long as it can be replicated and stabilized in 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 cytochrome b12 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). The described DNA sequences can be efficiently linked to expression Appropriate promoters in the 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, HSV thymidine kinase promoter, 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. Expression vectors also include translation Initial ribosome binding sites, transcription terminators, etc. 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 There are 10 to 300 base pairs that act on promoters to enhance gene transcription. Examples include late stages of the origin of replication 100-270 base pairs side SV40 enhancer, adenovirus enhancers, and the like enhancer on the late side of the replication origin polyoma.

 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, a polynucleotide encoding a cytochrome M2 or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to constitute a genetically engineered host cell containing the polynucleotide or the recombinant vector. The term "host cell" refers to a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples are: Escherichia coli, Streptomyces; bacterial cells such as Salmonella typhimurium; fungal cells such as yeast; plant cells; insect cells such as fly S2 or Sf9; 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, competent cells capable of DNA uptake can be in the exponential growth phase were harvested, treated with (1 ₂ method used in the step are well known in the art. Alternatively, it is a MgCl _2. If 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 Wait.

By conventional recombinant DNA technology, the polynucleotide sequence of the present invention can be used for expression or production Recombinant cytochrome bl 2 (Scienec e, 1984; 224: 1431). Generally there are the following steps:

(1) using the polynucleotide (or variant) encoding human cytochrome b12 of the present invention, or transforming or transducing a suitable host cell with a recombinant expression vector containing the polynucleotide;

 (2) culturing host cells in a suitable medium;

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

 In step (2), 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 necessary, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. These methods include, but are not limited to: conventional renaturation treatment, protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.

 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 be used to treat malignant tumors, adrenal deficiency, skin diseases, various types of inflammation, HIV infection, and immunological diseases.

 Cytochrome b is a component of the respiratory chain complex I I I in the mitochondria of eukaryotes. It is also called bcl complex or coenzyme Q-cytochrome C reductase. Cytochrome b, as an important catalytic subunit of coenzyme Q, has the function of electron transfer and regulates the process of organism's respiratory metabolism. Cytochrome b / b6 specific conserved sequences form the mot if necessary for its activity.

 It can be seen that the abnormal expression of specific cytochrome b / b6mot if will cause the function of the polypeptide containing the mot if of the present invention to be abnormal, resulting in abnormal oxidative phosphorylation of the respiratory chain, affecting the metabolism of substances and energy, and producing Related diseases such as disorders of material and energy metabolism, disorders of embryonic development, disorders of growth and development, tumors, etc.

 It can be seen that the abnormal expression of the cytochrome bl 2 of the present invention will produce various diseases, especially material and energy metabolism disorders, embryonic development disorders, growth and development disorders, and various tumors. These diseases include, but are not limited to:

Substance and energy metabolism disorders: isovalerate, propionate, methylmalonic aciduria, combined carboxylase deficiency, glutaric acid type I, phenylketonuria, albinism, tryptamine Disease, glycineemia, hypersarcosineemia, glutamate metabolism deficiency disease, metabolic deficiency disease of the urea cycle, histidine metabolism deficiency disease, Defective lysine metabolism, mucolipid storage disease, Ray-niney syndrome, xanthineuria, orotic aciduria, adenine deaminase deficiency, hyperlipoproteinemia, hereditary fructose intolerance Suffering, galactosemia, fructose metabolism deficiency, glycogen storage disease

 Fetal developmental disorders: congenital abortion, cleft palate, limb loss, limb differentiation disorder, cryptorchidism, congenital inguinal hernia, double uterus, vaginal atresia, hypospadias, amphoteric deformity, atrial septal defect, ventricular septal defect, pulmonary stenosis , Arterial duct closure, neural tube defects, congenital hydrocephalus, iris defect, congenital glaucoma or cataract, congenital deafness

 Growth and development disorders: mental retardation, cerebral palsy, brain development disorders, mental retardation, familial cerebral nucleus dysplasia syndrome, strabismus, skin, fat and muscular dysplasia such as congenital skin laxity, premature aging Disease, congenital keratosis, stunting, dwarfism, sexual retardation

 Various tumors: gastric cancer, liver cancer, lung cancer, esophageal cancer, breast cancer, leukemia, lymphoma, thyroid tumor, uterine fibroids, neuroblastoma, astrocytoma, ependymoma, glioblastoma, colon cancer , Melanoma, adrenal cancer, bladder cancer, bone cancer, osteosarcoma, myeloma, bone marrow cancer, brain cancer, uterine cancer, endometrial cancer, gallbladder cancer, colon cancer, thymus tumor, nasal cavity and sinus cancer, nasopharyngeal cancer , Laryngeal cancer, tracheal tumor, fibroma, fibrosarcoma, lipoma, liposarcoma, leiomyoma

 The abnormal expression of the cytochrome M 2 of the present invention will also produce certain hereditary, hematological and immune system diseases.

 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 various diseases, especially material and energy metabolism disorders, embryonic development disorders, growth and development disorders, A variety of tumors, inflammation, some hereditary, hematological diseases and immune system diseases.

 The invention also provides methods for screening compounds to identify agents that increase (agonist) or suppress (antagonist) cytochrome b12. Agonists enhance biological functions such as cytochrome bl 2 to stimulate 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 cytochrome M 2 can be cultured together with labeled cytochrome M 2 in the presence of drugs. The ability of the drug to increase or block this interaction is then determined.

 Antagonists of cytochrome M 2 include antibodies, compounds, receptor deletions, and the like that have been screened. An antagonist of cytochrome M 2 can bind to cytochrome bl 2 and eliminate its function, or inhibit the production of the polypeptide, or bind to the active site of the polypeptide so that the polypeptide cannot perform a biological function.

When screening compounds as antagonists, cytochrome b12 can be added to a bioanalytical assay to determine whether a compound is an antagonist by measuring the effect of the compound on the interaction between cytochrome M2 and its receptor. In the same way as above for screening compounds, antagonists can be screened for receptors. Deletions and analogs. Polypeptide molecules capable of binding to cytochrome b12 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. In screening, cytochrome M2 molecules should generally be labeled.

 The present invention provides a method for producing antibodies using polypeptides, and fragments, derivatives, analogs or cells thereof as antigens. These antibodies can be polyclonal or monoclonal antibodies. The invention also provides antibodies directed against a cytochrome M2 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 cytochrome M2 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 Freund's adjuvant. Techniques for preparing monoclonal antibodies to cytochrome bl2 include, but are not limited to, hybridoma technology (Kohler and Milstein. Nature, 1975, 256: 495-497), triple tumor technology, human beta-cell hybridoma technology, and EBV-hybridoma technology. Wait. Chimeric antibodies that bind human constant regions and 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 cytochrome M2.

 Anti-cytochrome bl2 antibodies can be used in immunohistochemistry to detect cytochrome bl2 in biopsy specimens.

 Monoclonal antibodies that bind to cytochrome bl2 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, cytochrome M2 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 cytochrome M2 positive cells.

 The antibodies of the present invention can be used to treat or prevent cytochrome M2-related diseases. Administration of appropriate doses of antibodies can stimulate or block the production or activity of cytochrome b12.

 The invention also relates to a diagnostic test method for quantitative and localized detection of cytochrome b12 levels. These tests are well known in the art and include FISH assays and radioimmunoassays. Cytochrome M2 levels detected in the test can be used to explain the importance of cytochrome M2 in various diseases and to diagnose diseases where cytochrome bl2 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.

 Polynucleotides encoding cytochrome M2 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 cytochrome bl2. Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated cytochrome bl2 to inhibit endogenous cytochrome bl2 activity. For example, a variant cytochrome M2 may be a shortened cytochrome bl2 lacking a signaling domain, and although it can bind to downstream substrates, it lacks signaling activity. Therefore, recombinant gene therapy vectors can be used to treat diseases caused by abnormal expression or activity of cytochrome b12. Viral-derived expression vectors such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus, etc. can be used to transfer a polynucleotide encoding a cytochrome M2 into a cell. Methods for constructing a recombinant viral vector carrying a polynucleotide encoding a cytochrome b12 can be found in the literature (Sambrook, et al.). In addition, a recombinant polynucleotide encoding cytochrome b12 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 cytochrome bl2 mRNA are also within the scope of the present invention. A ribozyme is an enzyme-like RNA molecule that can specifically decompose specific RNA. Its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target RNA and performs endonucleation. Antisense RNA, DNA, and ribozymes can be obtained using any existing RNA or DNA synthesis techniques, such as solid-phase phosphate amide chemical synthesis to synthesize oligonucleotides. Antisense RNA molecules can be obtained by in vitro or in vivo transcription of a DNA sequence encoding the RNA. This DNA sequence has been integrated downstream of the RNA polymerase promoter of the vector. In order to increase the stability of the nucleic acid molecule, it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the phosphorothioate or peptide bond instead of the phosphodiester bond is used for the ribonucleoside linkage.

Polynucleotides encoding cytochrome bl2 can be used for the diagnosis of diseases related to cytochrome M2. Polynucleotides encoding cytochrome M2 can be used to detect the expression of cytochrome bl2 or the abnormal expression of cytochrome bl2 in disease states. For example, the DNA sequence encoding cytochrome bl2 can be used to hybridize biopsy specimens to determine the expression of cytochrome bl2. Hybridization techniques include Southern blotting, Northern blotting, and in situ hybridization. These techniques and methods are publicly available and mature, and related kits are commercially available. 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 tissue. Cytochrome M2 specific primers can also be used to detect the transcription products of cytochrome M2 by RNA-polymerase chain reaction (RT-PCR) in vitro amplification. Detection of mutations in the cytochrome bl2 gene can also be used to diagnose cytochrome M2-related diseases. Cytochrome bl2 mutations include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to normal wild-type cytochrome bl 2 D 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. 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. This sequence will specifically target a specific position on a human chromosome and can hybridize to it. Currently, specific sites for each gene on the chromosome need to be identified. Currently, only a few chromosome markers based on actual sequence data (repeating polymorphisms) are available for marking chromosome positions. According to the present invention, in order to associate these sequences with disease-related genes, an important first step is to locate these DNA sequences on a chromosome.

 In short, a PCR primer (preferably 15-35bp) is prepared from the cDNA, and the sequence can be located on the chromosome. These primers were then used for PCR screening of somatic hybrid cells containing individual human chromosomes. Only those heterozygous cells containing the human gene corresponding to the primer will produce amplified fragments.

 PCR localization of somatic hybrid cells is a quick way to localize DNA to specific chromosomes. 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 Manua l of Basic Techniques, Pergamon Pres s, 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 V. Mckus i ck, Mende l ian Inher i tance in Man (available online with Johns Hopk ins University Wet ch Med i ca l Li brary). 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 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 using cDNA sequence-based PCR. Based on the resolution capabilities of current physical mapping and gene mapping technologies, cDNAs that are accurately mapped to disease-related chromosomal regions can be one of 50 to 500 potentially pathogenic genes (assuming 1 megabase mapping capability and every 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. Cytochrome bl 2 is administered in an amount effective to treat and / or prevent a specific indication. The amount and range of cytochrome b12 administered to a patient will depend on many factors, such as the mode of administration, the health conditions of the person to be treated, and the judgment of the diagnostician.

Examples

 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 in the following examples are not marked with specific conditions, usually according to conventional conditions such as Sambrook et al., Molecular cloning: the conditions described in the laboratory manual (New York: Cold Harbor Harbor Laboratory Press, 1989), or according to the manufacturing conditions Conditions recommended by the manufacturer.

 Example 1 Cloning of cytochrome M 2

Total human fetal brain RNA was extracted by one-step method with guanidine isothiocyanate / phenol / chloroform. Poly (A) raRNA was isolated from total RNA using Quik mRNA I sola t ion Ki t (product of Qiegene). 2ug po ly (A) mRNA was reverse transcribed to form cDNA. A Smart cDNA cloning kit (purchased from C Ion tech) was used to insert the cDNA fragment into the multi-cloning site of the pBSK (+) vector (Clontech) to transform DH5α to form a cDNA library. The sequences at the 5 'and 3' ends of all clones were determined using Dye terminate cyc le react ion sequencing kit (Perkin-Elmer) and ABI 377 automatic sequencer (Perkin-Elmer). The determined cDNA sequence was compared with an existing public DM sequence database (Genebank), and it was found that the cDNA sequence of one of the clones 0135g01 was a new DM. A series of primers were synthesized to determine the inserted cDNA fragments of the clone in both directions. The results showed that the full-length cDNA contained in the 0135g01 clone was 4000 bp (as shown in Seq ID N0: 1), and there was a 324 bp open reading frame (0RF) from 1122 bp to 1445 bp, encoding a new protein (such as Seq ID N0 : Shown in 2). We named this clone pBS- 0135 _g 01, the encoded egg The white matter is named cytochrome bl2. Example 2 Domain analysis of cDNA clones

 The sequence of the cytochrome b12 of the present invention and the protein sequence encoded by the cytochrome b12 were analyzed using the profile scan program (Basic local alignment search tool) in GCG [Al tschul, SF et al. J. Mol. Biol. 1990; 215: 403- 10], performing domain analysis in databases such as prosite. The cytochrome bl2 of the present invention is homologous with the domain cytochrome b characteristic protein at 22-82, and the homology result is shown in FIG. 1. The homology rate is 20%, and the score is 11.01; the threshold value is 10.51. Example 3 Cloning of a gene encoding cytochrome bl2 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 Qiagene's kit, the following primers were used for PCR amplification:

 Primerl: 5 '-CAACAGTCAGAATGAAGCTGAGCT-3' (SEQ ID NO: 3) Primer2: 5-TGATTATTTTTATTCAGTAAAAGT-3 '(SEQ ID NO: 4)

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

 Priraer2 is the 3 'end reverse sequence in SEQ ID NO: 1.

Amplification reaction conditions: reaction volume containing ₅₀ μ 1 of 50mmol / L KC1, 10mmol / L Tris-HCl, pH8.5, 1.5mmol / L MgCl 2, 20 (^ mol / L dNTP, lOpmol primer, 1U Taq DNA polymerase (Clontech). 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. At RT -For PCR, set P-act in as a positive control and template blank as a negative control. The amplified product was purified with a QIAGEN kit and connected to a pCR vector with a TA cloning kit (Invitrogen). DM sequence analysis results It is shown that the DNA sequence of the PCR product is completely identical to the 1-4000 bp shown in SEQ ID NO: 1. Example 4 Analysis of the expression of the cytochrome bl2 gene by Northern blot

Total RNA was extracted in one step [Anal. Biochem 19107, 162,156-159]. This method involves acid guanidinium thiocyanate phenol-chloroform extraction. That is, the tissue is homogenized with 4M guanidine isothiocyanate-25mM sodium citrate, 0.2M sodium acetate (pH4.0), and 1 time volume of phenol and 1/5 volume of chloroform-isoamyl alcohol (49: 1 ), Mix and centrifuge. Aspirate the aqueous layer, add isopropanol (0.8 vol) and centrifuge the mixture to obtain RNA precipitate. The resulting RNA pellet was washed with 70% ethanol, dried and dissolved in water. Using 20 _με RNA, electrophoresis was performed on a 1.2% agarose gel containing 20 mM 3- (N-morpholino) propanesulfonic acid (PH7.0)-5 mM sodium acetate-1 mM EDTA-2.2M formaldehyde. It was then transferred to a nitrocellulose membrane. Preparation of ³² P-labeled by a- ³² P dATP by random primer method DM probe. The DM probe used was the PCR amplified cytochrome bl2 coding region sequence (1122bp to 1445bp) shown in FIG. 1. A 32P-labeled probe (about 2 x 10 ⁶ cpm / ml) was hybridized with a nitrocellulose membrane to which RNA was transferred at 42 ° C overnight in a solution containing 50% formamide-25raMKH ₂ P0 ₄ ( pH7.4) -5 x SSC-5 X Denhardt's solution and 20 (^ g / ml salmon sperm DNA. After hybridization, the filter was washed in 1 x SSC-0.1% SDS at 55 ° C for 30 minutes. Then, Phosphor Imager Analysis and quantification. Example 5 In vitro expression, isolation and purification of recombinant cytochrome bl2

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

Primer3: 5'-CATGGATCCATGCCCTCGGGTCCTAACAAAGTA-3 '(Seq ID No: 5) Primer4: 5-CCCCTCGAGCTATCTTTTAAAGGAAATACCAAT-3' (Seq ID No: 6) The 5 'ends of these two primers contain BamHI and Xhol restriction sites, respectively The coding sequences of the 5 'and 3' ends of the gene of interest are followed, respectively. The restriction sites of BamHI and Xhol correspond to the selective endonucleases on the expression vector plasmid pET-28b (+) (Novagen, Cat. No. 69865.3). Enzyme site. The PCR reaction was performed using pBS-0135g01 plasmid containing the full-length target gene as a template. The PCR reaction conditions are as follows: a total volume of 50 _μ 1 contains 10 pg of pBS-0135g01 plasmid, primers? 1 "11116]"-3 and? 1 11161 "-4 are 10 11101, Advantage polymerase Mix (Clontech) 1μ1. Cycle parameters: 94.C 20s, 60.C 30s, 68 ° C 2 min, a total of 25 cycles. Use BamHI and Xhol respectively The amplified product and plasmid pET-28 (+) were double-digested, and large fragments were separately recovered and ligated with T4 ligase. The ligation product was transformed with colibacillus DH5CC by the calcium chloride method, and contained kanamycin (final concentration 3 (g / ml) LB plate was cultured overnight, and colonies were used to screen positive clones and sequenced. Select positive clones (pET-0135g01) with the correct sequence and transform the recombinant plasmid into E. coli BL21 (DE3) using the calcium chloride method. pLysS (Novagen Co.). in containing kanamycin (final concentration ₃₀ μ8 / ηι1) in LB liquid medium, host strain BL21 _(P ET-0135g01) cultured at 37 ° C until logarithmic growth phase, IPTG was added The culture was continued for 5 hours to a final concentration of 1 mmol / L. The cells were collected by centrifugation, and the supernatant was collected by centrifugation, and the supernatant was collected by centrifugation. The affinity chromatography column His was used to bind 6 histidines (6His-Tag). Bind Quick Cartridge (Novagen) for chromatography, A purified target protein cytochrome bl2 was obtained. After SDS-PAGE electrophoresis, a single band was obtained at 12 kDa (Figure 2). The band was transferred to a PVDF membrane and analyzed by N-terminal amino acid sequence using Edams hydrolysis method As a result, the 15 amino acids at the N-terminus were completely the same as the 15 amino acid residues at the N-terminus shown in SEQ ID NO: 2. Example 6 Production of an anti-cytochrome bl2 antibody

A peptide synthesizer (product of PE company) was used to synthesize the following cytochrome bl2-specific peptides: NH2- Met- Pro-Ser-Gly- Pro-Asn- Lys- Vab Tyr- Glu- Ala- Glu-Trp-I le- Tyr- 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. Im 隠 ochemi s try, 1969; 6: 43. Rabbits were immunized with 4 mg of the hemocyanin polypeptide complex plus complete Freund's adjuvant, and 15 days later, the hemocyanin polypeptide complex plus incomplete Freund's adjuvant was used to boost immunity once.釆 Using a 15 g / ml bovine serum albumin peptide complex-coated titer plate as an ELISA to determine antibody titers in rabbit serum. Total IgG was isolated from antibody-positive rabbit serum using protein A-Sepharose. 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 cytochrome M2. Example 7 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 determined whether it contains the polynucleotide sequence of the present invention and a homologous polynucleotide sequence is detected. Further, the probe can be used to detect the polynucleotide sequence of the present invention or its homologous polynucleotide sequence in normal tissue or pathology. Whether the expression in tissue cells is abnormal.

 The purpose of this embodiment is to select a suitable oligonucleotide fragment from the polynucleotide SEQ ID NO: 1 of the present invention as a hybridization probe, and to identify whether some tissues contain the polynucleoside of the present invention by a filter hybridization method. Acid sequence or a homologous polynucleotide sequence thereof. Filter hybridization methods include dot blotting, Southern imprinting, Northern blotting, and copying methods. They all use the same steps to immobilize the polynucleotide sample to be tested on the filter. 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, the unhybridized probes are removed by a series of membrane washing steps. This embodiment uses higher-intensity washing conditions (such as lower salt concentration and higher temperature) 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 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%, if it exceeds, non-specific hybridization increases;

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

 5-TGCCCTCGGGTCCTAACAAAGTCTACGAAGCTGAATGGATC-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; ) And more commonly used manuals of molecular cloning experiments such as "Molecular Cloning Experiment Guide" U998 Second Edition) [US] Sambruck et al., Science Press.

 Sample Preparation:

 1.Extract DNA from fresh or frozen tissue

Steps: 1) Place fresh or freshly thawed normal liver tissue in a plate immersed in ice and filled with phosphate buffered saline (PBS). Cut the tissue into small pieces with scissors or a scalpel. Keep tissue moist during operation. 2) Centrifuge the tissue at 1,000 g for 10 minutes. 3) Suspend the precipitate (about 10 ml / g) with a cold homogenization buffer (0.25 mol / L sucrose; 25 mraol / L Tris-HCl, pH 7.5; 25 mmol / L NaCl; 25 mmol / L MgCl ₂ ). 4) at 4. C Use an electric homogenizer to homogenize the tissue suspension at full speed until the tissue is completely broken. 5) Centrifuge at 1000g for 10 minutes. 6) Resuspend the cell pellet (l-5 ml per 0.1 g of the initial tissue sample), and centrifuge at 1,000 g for 10 minutes. 7) Resuspend the pellet with 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 the cells with 1-10ml cold PBS and centrifuge at 1000g for 10 minutes. 2) Lysis with cold cells Resuspend the pelleted cells in liquid (lx 10 ⁸ cells / ml) with a minimum of 100 ul of 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 200ug / 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) 1/10 volume of 2mol / L sodium acetate and 2 volumes of 100 ° C cold. Add ethanol to the DNA solution and mix. Leave at -20 ° C for 1 hour or overnight. 2) Centrifuge for 10 minutes. 3) Carefully aspirate or pour out the ethanol. 4) Wash the pellet with 500ul of 70% cold ethanol and centrifuge for 5 minutes. 5) Carefully aspirate or pour out the ethanol. Wash the pellet with 500ul of cold ethanol and centrifuge for 5 minutes. 6) Carefully aspirate or pour out the ethanol, then invert on the absorbent paper to drain off the residual ethanol. Air dry for 10-15 minutes to allow the surface ethanol to evaporate. Be careful not to allow the pellet to dry completely, otherwise it will be more difficult to re-dissolve. 7) Resuspend the DNA pellet in a small volume of TE or water. Low-speed vortexing or pipetting, with a dropper, while gradually increasing the TE, mixed until fully dissolved DM, ¹⁰⁶ cells per 1-5 χ extracted plus about lul.

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

8) Add RNase A to the DNA solution to a final concentration of 100 ug / ml, 37. C was held for 30 minutes. 9) Add SDS and proteinase K to the final concentration of 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, re-extract with an equal volume of chloroform: isoamyl alcohol (24: 1), and centrifuge for 10 minutes. 12) Carefully remove the aqueous phase, add 1/10 volume of 2mol / L sodium acetate and 2.5 volumes of cold ethanol, and mix well--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 ° C after dispensing.

 Preparation of sample film:

 1) Take 4 x 2 pieces of nitrocellulose membranes (NC membranes) of appropriate size, and mark the spotting position and sample number lightly 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 infiltrated with 0.1 mol / L NaOH, 1.5 mol / L NaCl for 5 minutes (twice), and dry in 0.5 mol / L Tris-HCl (pH 7.0), 3 mol / L NaCl filter paper for 5 minutes (two Times), 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) 3μ1 Probe (0.1OD / Ιθμΐ), add 2μ1 Kinase buffer, 8-10 uCi γ- ³² P_dATP + 2U Kinase, to make up to a final volume of 20μ1.

 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) Before the ³² P-Probe is washed out, start collecting the first peak (can be monitored by Monitor).

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

 7) Monitor the amount of isotope with a liquid scintillator.

8) The ³² P-Probe (the second peak is free γ- ³² P-dATP) to be prepared after the collection solution of the first peak is combined.

 Pre-hybridization

 The sample membrane was placed in a plastic bag, and 3-lOmg prehybridization solution (10xDenhardt's; 6xSSC, 0.1 mg / ml CT DM (calf thymus DM)) was added. After sealing the mouth of the bag, shake at 68 ° C for 2 hours.

 Cross

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

 High-intensity washing film:

 1) Take out the hybridized sample membrane.

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

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

 4) Wash in 0.1xSSC, 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 at 37 ° C for 15 minutes (twice).

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

4) In 0.1xSSC, 0.1% SDS, wash at 40 ° C for 15 minutes (twice), and dry at room temperature. X-ray autoradiography:

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

 Experimental results:

 的 The hybridization experiments performed under low-intensity membrane washing conditions showed no significant difference in the radioactive intensity of the above two probe hybrid spots; while the hybridization experiments performed under high-intensity membrane washing conditions, the radioactive intensity of probe 1 was significantly stronger To the radioactive intensity of the hybridization spot of another probe. Therefore, the presence and differential expression of the polynucleotide of the present invention in different tissues can be analyzed qualitatively and quantitatively with the probe 1. Example 8 DNA Microarray

 Gene chip or DNA microarray is a new technology that many national laboratories and large pharmaceutical companies are currently developing and developing. It refers to the orderly and high-density arrangement of a large number 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 fast, efficient, and high-throughput analysis of biological information. The polynucleotide of the present invention can be used as target D 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 DeRi si, JL, Lyer, V. & Brown, P. 0. (1997) Sceence 278, 680-686. RA, Schema, M., Cha i, 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 the purified amplified product was purified, the concentration was adjusted to about 500 ng / ul, and spotted on a glass medium using a Cartesian 7500 spotting instrument (purchased by Cartesian, USA). The distance from the point is 280 μηι. The spotted slides were hydrated and dried, cross-linked in a UV cross-linker, and dried after elution to fix the DM on the glass slide to prepare chips. The specific method steps have been reported in the literature. The sample post-processing steps in 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.

 (Two) probe marking

 Total mRM was extracted from normal liver and liver cancer in one step, and mRNA was purified using Oligotex mRNAMidi Kit (purchased from QiaGen). Cy3dUTP (5- Araino-propargy 1-2 '-deoxyuridine 5' -tr iphate coupled to Cy3 fluorescent dye, purchased from Amersham Phamacia Biotech company) labeled mRNA of normal liver tissue, with Cy5dUTP (5-Amino-propargy 1-2 · -deoxyuridine 5'-tr iphate coupled to Cy5 fluorescent dye, (Purchased from Amersham Phamacia Biotech) was used to label liver cancer tissue mRNA, and the probe was prepared after purification. For specific steps and methods, see

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

 (Three) cross

 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 was processed by Imagene software (Biodiscovery Company, USA) to calculate the Cy3 / Cy5 ratio of each point. The point where the ratio is less than 0.5 and greater than 2 is considered. Genes with differential expression.

 Experimental results show that Cy3 signal = 2266.7 (average of four experiments), Cy5 signal = 2448.5 (average of four experiments), Cy3 / Cy5 = 0.92575046, the polynucleotide of the present invention is in the above two tissues There is no significant difference in expression.

Claims

Rights request

1. An isolated polypeptide-cytochrome bl2, 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 thereof.

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

 3. The polypeptide according to claim 2, characterized in that it comprises a polypeptide having an 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 a sequence of positions 1122-1445 in SEQ ID NO: 1 or a sequence of positions 1-4000 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 and a plasmid, virus or 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 cytochrome bl2 activity, characterized in that the method includes:

(a) culturing the engineered host cell according to claim 8 under the condition of expressing cytochrome bl2;

(b) Isolating a polypeptide having cytochrome M2 activity 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 cytochrome M 2.

 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 cytochrome b12.

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. The use of the compound according to claim 11, characterized in that the compound is used for a method for regulating the activity of cytochrome bl2 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 mimics, agonists, antagonists or inhibitors of cytochrome b12; 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 cytochrome M2 abnormality.

 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.