WO2001038369A1 - A novel polypeptide-rat tricarboxylate carrier 39 and the polynucleotide encoding said polypeptide - Google Patents

Abstract

The invention discloses a new kind of polypeptide-rat tricarboxylate carrier 39 and the polynucleotide encoding said polypeptide and a process for producing the polypeptide by recombinant methods. It also discloses the method of applying the polypeptide for the treatment of various kinds of diseases, such as cancer, hemopathy, HIV infection, immune diseases and inflammation. The antagonist of the polypeptide and therapeutic use of the same is also disclosed. In addition, it refers to the use of polynucleotide encoding said rat tricarboxylate carrier 39.

Description

 A new polypeptide, a mouse tricarboxylic acid carrier 39, and a polynucleotide encoding the polypeptide TECHNICAL FIELD

 The present invention belongs to the field of biotechnology. Specifically, the present invention describes a novel polypeptide, a mouse tricarboxylic acid carrier 39, and a polynucleotide sequence encoding the polypeptide. The invention also relates to a preparation method and application of the polynucleotide and polypeptide. technical background

 Biology oxidatively decomposes sugars, proteins, fats and other nutrients through respiration; in this process, the energy stored in food is converted into chemical energy through oxidative phosphorylation, which continuously supplies the needs of physiological activities. These processes are mainly performed and completed in the mitochondria. Mitochondria are important and unique organelles in cells, and they are common in eukaryotic cells. In mitochondria, energy conversion is performed through oxidative phosphorylation, which provides energy for various cell activities. Transportation into the mitochondrial matrix space is very active, but most of it occurs through active transport for this purpose to promote exchange or through highly specialized specific proteins. These proteins are called transporters or translocases, and their main function is to transport substances to each other and cooperate with them across cell membranes. These transported substances are very specific reverse-moving substances with similar charges, such as ADP and ATP exchange . No external energy supply is required for transport on the transporter. When a pair of substances to be transported moves through the opposite transport (exchange) mode, at least one of them must move significantly to a low concentration gradient. Therefore, a pair of substance transporters can be directed to either side by providing the higher concentration required for one substance to cross the cell membrane. For example, when succinic acid is introduced into the cell matrix, it will be present at high concentrations, and the transporter will be guided by a concentration gradient to transport succinic acid into the mitochondria.

The outflow of the main components of the cell along the concentration gradient (whether through the mitochondria or the plasma membrane) can promote the reverse movement of the material to move against the gradient, thus working until the push by both sides is completed. However, transporters are functionally promoting and replicating intermediates in this cycle. In the tricarboxylic acid cycle, malic acid enters the mitochondria from the cytosol, providing an additional source of reduced equilibrium and enabling the gluconeogenesis mechanism. The effluent of citric acid provides a way to transport acetyl groups outwards. The electron transport chain in mitochondria requires it to properly function as a strong chemical / ion concentration when it passes through the mitochondrial membrane (chemical penetration hypothesis). The absence of this gradient or the absence of a cell gradient that in turn affects the mitochondrial gradient will impair the function of the electron transport chain. The impaired function of the electron transport chain will affect the tricarboxylic acid cycle. If sufficient tricarboxylic acid cycles are lost, the patient will lose 90% of its ability to generate energy and gradually enter a coma. Sequence-analyzed members of the mitochondrial carrier protein (ie transporter) family, four of which (ADP / ATP carrier, phosphate carrier, 2-oxoglutaric acid / malate carrier and uncoupling proteins) have triple internals Homology. The tricarboxylic acid carrier did not show any internal homology, and no triple structure existed. This is the first mitochondrial vector that contradicts the putative gene family.

 Tricarboxylic acid carriers, a mitochondrial anion transporter, were found in liver mitochondria by Chappe l l and Haarhof f (1967). Citric acid, cis aconitic acid, threo-D-isocitric acid, D- and L-tartaric acid, malic acid, phosphoenolpyruvate, and succinic acid are substrates of this carrier. Alpha-ketoglutarate and malonic acid are not transported by tricarboxylic acid carriers. 1,2,3-Benzenetricarboxylic acid (BTA) is the most specific inhibitor of this carrier. The transfer of butylmalonic acid and N-ethylmaleimide from tricarboxylic acid carriers is much weaker than that of dicarboxylic acid carriers, which indicates that dicarboxylic acid and tricarboxylic acid carriers Are different molecular entities. The synthesis of fatty acids and cholesterol in cytosol starts with acetyl-CoA. Acetic coenzyme A is derived from citric acid which is transported to the cytosol via a tricarboxylic acid carrier. Coleman and colleagues have observed the activation effect of cholesterol on the tricarboxylic acid carrier of tumor mitochondria. Tricarboxylic acid carriers also play a role in gluconeogenesis and reducing shuttles through mitochondrial equivalents, as both reactions require efficient mitochondrial transport of citric acid and phosphoenolic acid. Although tricarboxylic acid carriers are also found in brain and kidney mitochondria, tricarboxylic acid carriers in liver mitochondria are the most widely studied, although their activity is significantly lower. There is no or little evidence of this vector activity in cardiac mitochondria.

 Initial adenosine (purine nucleoside) phosphate support and uncoupling protein sequence studies have confirmed that they have internal triplet structures. Each of these proteins consists of three related fragments of about 100 amino acids in length, each of which is a two transmembrane helix connected by a hydrophilic membrane loop. The recently cloned glutaric acid / malate vector adds a new member to the carrier protein family. The tricarboxylic acid carrier did not show an intrinsic triplet structure, nor did it share homology with the mitochondrial anion transporter family. This is the first mitochondrial vector that contradicts the putative gene family.

 The mouse gene of the present invention has 94% identity and 97% similarity at the protein level with a tricarboxylic acid carrier that is a member of the rat liver mitochondrial transporter family. Both of them have 5 or 6 transmembrane regions, highly hydrophobic C-terminus and relatively hydrophilic N-terminus region, neither of which shows any direct similarity with the members of the carrier family that have been analyzed so far. No triple internal homology was shown with previously analyzed vectors. Based on the above points, the new gene of the present invention is considered to be a new member of the murine tricarboxylic acid carrier family and named as murine tricarboxylic acid carrier 39. It is inferred that it is similar to the mouse liver mitochondrial tricarboxylic acid carrier, is also a new member of the protein family, and has similar biological functions.

As the murine tricarboxylic acid carrier 39 protein plays an important role in 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 involved in these Processes for the Murine Tricarboxylic Acid Carrier 39 protein, specifically identifying the amino acid sequence of this protein. Isolation of the new mouse tricarboxylic acid carrier 39 protein encoding gene also provides a basis for the study to determine the role of the 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 an isolated novel polypeptide-murine tricarboxylic acid carrier 39 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 murine tricarboxylic acid vector 39.

 Another object of the present invention is to provide a genetically engineered host cell containing a polynucleotide encoding a murine tricarboxylic acid vector 39.

 Another object of the present invention is to provide a method for producing a murine tricarboxylic acid carrier 39.

 Another object of the present invention is to provide an antibody against the polypeptide-murine tricarboxylic acid carrier 39 of the present invention.

 Another object of the present invention is to provide mimic compounds, antagonists, agonists, and inhibitors directed to the polypeptide-murine tricarboxylic acid carrier 39 of the present invention.

 Another object of the present invention is to provide a method for diagnosing and treating diseases associated with abnormalities of the murine tricarboxylic acid carrier 39.

 The present invention relates to an isolated polypeptide, which is of human origin, and includes: a polypeptide having the amino acid sequence of SEQ ID D. 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 D. 2;

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

 (c) A polynucleotide having at least 95% 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 114-1082 in SEQ ID NO: 1; and (b) a sequence having 1-2947 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; a package The method of preparing the polypeptide of the present invention includes culturing the host cell and recovering the expressed 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 the murine tricarboxylic acid carrier 39 protein, which comprises utilizing the polypeptide of the invention. The invention also relates to compounds obtained by this method.

 The invention also relates to a method for in vitro detection of a disease or susceptibility to a disease associated with abnormal expression of the murine tricarboxylic acid carrier 39 protein, which comprises detecting a mutation in the polypeptide or a polynucleotide sequence encoding the same in a biological sample, or detecting a biological The amount or biological activity of a polypeptide of the invention in a 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 cancer, developmental disease or immune disease or other diseases caused by abnormal expression of the murine tricarboxylic acid carrier 39.

 Other aspects of the invention will be apparent to those skilled in the art from the disclosure of the techniques herein. The following terms used in this specification and claims have the following meanings unless specifically stated: "Nucleic acid sequence" refers to an oligonucleotide, a nucleotide or a polynucleotide and a fragment or part thereof, and may also refer to a genomic or synthetic DNA or RNA, they can be single-stranded or double-stranded, representing the sense or antisense strand. Similarly, the term "amino acid sequence" refers to an oligopeptide, peptide, polypeptide or protein sequence and fragments or portions thereof. When the "amino acid sequence" in the present invention relates to the amino acid sequence of a naturally occurring protein molecule, such "polypeptide" or "protein" does not mean to limit the amino acid sequence to a complete natural amino acid related to the protein molecule .

 A "variant" of a protein or polynucleotide refers to an amino acid sequence having one or more amino acids or nucleotide changes or a polynucleotide sequence encoding it. The changes may include deletions, insertions or substitutions of amino acids or nucleotides in the amino acid sequence or nucleotide sequence. Variants can have "conservative" changes, in which the amino acid substituted has a structural or chemical property similar to the original amino acid, such as replacing isoleucine with leucine. Variants can also have non-conservative changes, such as replacing glycine with tryptophan.

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

"Insertion" 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" means the replacement of a different amino acid or A nucleotide replaces one or more 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 in appropriate animals or cells and to bind to specific antibodies.

 An "agonist" refers to a molecule that, when combined with a murine tricarboxylic acid carrier 39, 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 a murine tricarboxylic acid carrier 39.

 An "antagonist" or "inhibitor" refers to a molecule that, when combined with a murine tricarboxylic acid carrier 39, can block or regulate the biological or immunological activity of the murine tricarboxylic acid carrier 39. Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates or any other molecule that can bind to the murine tricarboxylic acid carrier 39.

 "Regulation" refers to a change in the function of the murine tricarboxylic acid carrier 39, including an increase or decrease in protein activity, a change in binding characteristics, and any other biological, functional, or immune properties of the murine tricarboxylic acid carrier 39.

 "Substantially pure" means substantially free of other proteins, lipids, carbohydrates, or other substances with which it is naturally associated. Those skilled in the art can purify the murine tricarboxylic acid carrier 39 using standard protein purification techniques. A substantially pure murine tricarboxylic acid support 39 produces a single main band on a non-reducing polyacrylamide gel. The purity of the murine tricarboxylic acid carrier 39 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 may be partial or complete. The degree of complementarity between nucleic acid strands has a significant effect on the efficiency and strength of hybridization between nucleic acid strands.

 "Homology" refers to the degree of complementarity and can be partially homologous or completely homologous. "Partial homology" refers to a partially complementary sequence that at least partially inhibits hybridization of a fully complementary sequence to a target nucleic acid. This inhibition of hybridization can be detected by performing hybridization (Southern imprinting or Nor thern blotting, etc.) under conditions of reduced stringency. Substantially homologous sequences or hybridization probes can compete and inhibit the binding of fully homologous sequences to the target sequence under conditions of reduced stringency. This does not mean that conditions with reduced stringency allow non-specific binding, because conditions with reduced stringency require that the two sequences bind to each other as either specific or selective interactions.

"Percent identity" refers to the percentage of sequences that are identical or similar in the comparison of two or more amino acid or nucleic acid sequences. The percent identity can be determined electronically, such as by the MEGAL I GN program (Lasergenes of tware package, DNASTAR, Inc., Mad Son Wis.). The MEGALI GN program can compare two or more sequences (H i gg ins, DG and PM Sharp (1988) Gene 73: 237-244). The C l us 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: The number of matching residues between sequence A and sequence B

X 100 The number of residues in sequence A-the number of spacer residues in sequence A-the number of spacer residues in sequence B can also be determined by the Cluster method or by methods known in the art such as Jotun He in. Sex percentage (He in L, (1990) Me thod sin emzumo l ogy 183: 625-645) ₀

"Similarity" refers to the degree of identical or conservative substitutions of amino acid residues at corresponding positions in the alignment of amino acid sequences. Amino acids used for conservative substitutions, 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 a "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? ^ It can specifically bind to the epitope of murine tricarboxylic acid carrier 39.

 A "humanized antibody" refers to an antibody in which the amino acid sequence of a non-antigen binding region is replaced to become more similar to a human antibody, but still retains the original binding activity.

The term "isolated" refers to the removal of a substance from its original environment (for example, its natural environment if it occurs naturally). For example, a naturally occurring polynucleotide or polypeptide is not isolated when it is present in a living animal, but the same polynucleotide or polypeptide is separated from some or all of the substances that coexist with it in the natural system. Such a polynucleotide may be part of a certain vector, or such a polynucleotide or polypeptide may be part of a certain composition. Since the carrier or composition is not a component of its natural environment, they are still isolated. As used in the present invention, "isolated" means that the substance is separated from its original environment (if it is natural Natural material, 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 murine tricarboxylic acid carrier 39" means that the murine tricarboxylic acid carrier 39 is substantially free of other proteins, lipids, sugars, or other substances with which it is naturally associated. Those skilled in the art can purify the murine tricarboxylic acid carrier 39 using standard protein purification techniques. Substantially pure polypeptides produce a single main band on a non-reducing polyacrylamide gel. The purity of the murine tricarboxylic acid carrier 39 polypeptide can be analyzed by amino acid sequence.

 The present invention provides a new polypeptide, a mouse tricarboxylic acid carrier 39, which is basically composed of the amino acid sequence shown in SEQ ID NO: 2. The polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide, and preferably a recombinant polypeptide. The polypeptides of the present invention can be naturally purified products or chemically synthesized products, or can be produced from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insects, and mammalian cells) using recombinant techniques. Depending on the host used in the recombinant production protocol, the polypeptide of the invention may be glycosylated, or it may be non-glycosylated. Polypeptides of the invention may also include or exclude starting methionine residues.

 The invention also includes fragments, derivatives and analogs of the murine tricarboxylic acid carrier 39. As used herein, the terms "fragment", "derivative" and "analog" refer to a polypeptide that substantially retains the same biological function or activity of the murine tricarboxylic acid carrier 39 of the present invention. A fragment, derivative or analog of the polypeptide of the present invention may be: (I) a kind in which one or more amino acid residues are substituted with conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and the substitution The amino acid may or may not be encoded by a genetic codon; or (Π) 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 to another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol); or (IV) such a type of polypeptide sequence in which an additional amino acid sequence is fused into the mature polypeptide (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 2947 bases in length and its open reading frame (1140-182) encodes 322 amino acids. According to the amino acid sequence homology comparison, it was found that this polypeptide is 94% homologous to the murine tricarboxylic acid carrier. It can be inferred that the murine tricarboxylic acid carrier 39 has a similar structure and function.

The polynucleotide of the present invention may be in the form of DM or RNA. DNA forms include cDNA, Due to DM 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 in the present invention, a "degenerate variant" refers to a nucleic acid sequence encoding a protein or polypeptide having SEQ ID NO: 2 but different from the coding region sequence shown in SEQ ID NO: 1 in the present invention.

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

 The term "polynucleotide encoding a polypeptide" refers to a polynucleotide comprising the polypeptide and a polynucleotide comprising additional coding and / or non-coding sequences.

 The invention also relates to variants of the polynucleotides described above, which encode polypeptides or fragments, analogs and derivatives of polypeptides having the same amino acid sequence as the invention. Variants of this polynucleotide can 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: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2xSSC, 0.1% SDS, 60 ° C; or (2) Add a denaturant during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% F i co ll, 42 ° C, etc .; or (3) only between the two sequences Crosses occur 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 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 murine tricarboxylic acid vector 39.

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 murine tricarboxylic acid carrier 39 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) expression libraries Antibody screening to detect cloned polynucleotide fragments with common structural characteristics.

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

 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 the cDNA of interest is to isolate m A from donor cells that overexpress the gene and perform reverse transcription to form a plasmid or phage cDNA library. There are many mature techniques for extracting mRNA, 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 Ciontech. When polymerase reaction technology is used in combination, even very small expression products can be cloned.

 The genes of the present invention can be selected from these cDNA libraries by conventional methods. These methods include (but are not limited to): (1) DM-DNA or DNA-RNA hybridization; (2) the presence or absence of marker gene functions; (3) measuring the level of transcripts of the murine tricarboxylic acid carrier 39; (4) ) Detection of protein products expressed by genes through 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 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 method (4), immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA) can be used to detect the protein product expressed by the murine tricarboxylic acid carrier 39 gene.

 A method of applying a PCR technique to amplify DNA / RNA (Saiki, et al. Science 1985; 230: 1350-1354) is preferably used to obtain the gene of the present invention. In particular, when it is difficult to obtain full-length cDNA from a library, the RACE method (RACE-cDM terminal rapid amplification method) can be preferably used, and 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 DM / RNA fragment can be isolated and purified by conventional methods such as by gel electrophoresis.

The polynucleotide sequence of the gene of the present invention or various DNA fragments and the like obtained as described above can be determined by a conventional method such as dideoxy chain termination method (Sanger et al. PNAS, 1977, 74: 5463-5467). Such polynucleotide sequences can also be determined using commercial sequencing kits and the like. In order to obtain the full-length cDNA sequence, sequencing must be repeated. Sometimes it is necessary to determine the cDNA sequence of multiple clones to splice into a full-length cDNA. sequence.

 The present invention also relates to a vector comprising the polynucleotide of the present invention, and a host cell that is genetically engineered using the vector of the present invention or directly using the murine tricarboxylic acid vector 39 coding sequence, and a recombinant technology to produce the polypeptide of the present invention method.

 In the present invention, the polynucleotide sequence encoding the murine tricarboxylic acid vector 39 can be inserted into the vector to constitute a recombinant vector containing the polynucleotide of the present invention. The term "vector" refers to bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors well known in the art. Vectors suitable for use in the present invention include, but are not limited to: T7 promoter-based expression vectors (Rosenberg, et al. Gene, 1987, 56: 125) expressed in bacteria; pMSXND expression vectors expressed in mammalian cells ( Lee and Nathans, J Bio Chem. 263: 3521, 1988) and baculovirus-derived vectors expressed in insect cells. In short, as long as it can be replicated and stabilized in the host, any plasmid and vector can be used to construct a recombinant expression vector. An important feature of expression vectors is that they usually contain an origin of replication, a promoter, a marker gene, and translational regulatory elements.

 Methods well known to those skilled in the art can be used to construct expression vectors containing a DNA sequence encoding a murine tricarboxylic acid vector 39 and appropriate transcriptional / translational 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 direct 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 for translation initiation, a transcription terminator, and the like. Insertion of enhancer sequences into the vector will enhance its transcription in higher eukaryotic cells. Enhancers are cis-acting factors expressed by DM, usually about 10 to 300 base pairs, which act on promoters to enhance gene transcription. Illustrative examples include SV40 enhancers of 100 to 270 base pairs on the late side of the origin of replication, polytumor enhancers on the late side of the origin of replication, and adenoviral enhancers.

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

Those of ordinary skill in the art will know how to select appropriate vector / transcription control elements (such as promoters, enhancers, etc.) and selectable marker genes. In the present invention, a polynucleotide encoding a murine tricarboxylic acid vector 39 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: E. coli, Streptomyces; bacterial cells such as Salmonella typhimurium; fungal cells such as yeast; plant cells; insect cells such as fly S 2 or Sf 9; animal cells such as CH0, COS or Bowes s melanoma cells Wait.

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 (Method _12, using the procedure well known in the art. Alternative is MgC l _2. If necessary, transformation can also be performed by electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, or conventional mechanical methods such as microinjection, electroporation, and liposomes Packaging, etc.

 Using conventional recombinant DNA technology, the polynucleotide sequence of the present invention can be used to express or produce a recombinant murine tricarboxylic acid vector 39 (Scence, 1984; 224: 1431). Generally, the following steps are taken:

(1) transforming or transducing a suitable host cell with the polynucleotide (or variant) encoding the human murine tricarboxylic acid vector 39 of the present invention, or 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 desired, recombinant proteins can be separated and purified by various separation methods using their physical, chemical and other properties. These methods are well known to those skilled in the art. These methods include, but are not limited to: conventional renaturation treatment, protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange layer, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods. Brief description of the drawings

The following drawings are used to illustrate specific embodiments of the invention, but not to limit the scope of the invention as defined by the claims. FIG. 1 is a comparison diagram of amino acid sequence homology between the mouse tricarboxylic acid carrier 39 and the mouse tricarboxylic acid carrier according to the present invention. The upper sequence is a murine tricarboxylic acid carrier 39, and the lower sequence is a murine tricarboxylic acid carrier. Identical amino acids are represented by single-character amino acids between the two sequences, and similar amino acids are represented by "+".

FIG 2 is a tricarboxylic acid vector isolated murine polyacrylamide gel electrophoresis in FIG. 39 (SDS-PAGE) _c 39kDa molecular weight proteins. The arrow indicates the isolated protein band. The best way to implement the invention

 The present invention is further described below with reference to specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. In the following examples, the experimental methods without specific conditions are generally performed according to conventional conditions such as those described in Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer Suggested conditions.

 Example 1: Cloning of Murine Tricarboxylic Acid Vector 39

 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. A Smart cDNA cloning kit (purchased from Clontech) was used to insert the 00 ^ fragment into the multiple cloning site of the pBSK (+) vector (Clontech) to transform DH5 cx. The bacteria formed a cDNA library. Dye terminate cycle react ion sequencing kit (Perkin-Elmer) and ABI 377 automatic sequencer (Perkin-Elmer) were used to determine the sequences at the 5 'and 3' ends of all clones. The determined cDNA sequence was compared with the existing public DNA sequence database (Genebank), and it was found that the cDNA sequence of one of the clones 0703A07 was new DNA. The inserted cDNA fragments contained in this clone were determined in both directions by synthesizing a series of primers. The results showed that the 0703A07 clone contained a full-length cDNA of 2947bp (as shown in Seq IDNO: 1), and a 969bp open reading frame (0RF) from 114bp to 1082bp, encoding a new protein (such as Seq ID NO: 2). We named this clone pBS-0703A07, and the protein encoded was named murine tricarboxylic acid vector 39. Example 2: Homologous search of cDNA clones

The sequence of the murine tricarboxylic acid vector 39 of the present invention and the protein sequence encoded by the same are used by the Blast program (Basiclocal Alignment search tool) [Altschul, SF et al. J. Mol. Biol. 1990; 215: 403-10] Perform homology search in Genbank, Swissport and other databases. The gene with the highest homology to the murine tricarboxylic acid vector 39 of the present invention is a known murine tricarboxylic acid vector, and the accession number of the encoded protein in Genbank is S70011. The protein homology results are shown in Figure 1. Both are highly homologous. Its identity is 94%; similarity is 97%. Example 3: Cloning of a gene encoding murine tricarboxylic acid vector 39 by RT-PCR

 CDNA was synthesized using fetal brain cell total RNA as a template and oligo-dT as a primer for reverse transcription reaction.

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

 Primerl: 5'- GGGGACGCGCGAGGACGCCGTGGC -3 '(SEQ ID NO: 3)

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

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

 Primer2 is the 3, terminal reverse sequence of SEQ ID NO: 1.

Amplification conditions: 50 μl / L KC1, 10 μl / L Tris-CI, (pH8.5), 1.5mmol / L MgCl ₂ , 200 μmol / L dNTP in 50 μ 1 reaction volume , lOpmol primer, 1U Taq DNA polymerase (C 1 on t ech). The reaction was performed on a PE 9600 DNA thermal cycler (Perkin-E 1 mer) under the following conditions for 25 cycles: 94 ° C 30sec; 55 ° C 30sec; 72 ° C 2min. At the time of RT-PCR, set (3-act in as a positive control and template blank as a negative control. Amplification products were purified using QIAGEN's kit, and TA cloning kit was connected to the PCR vector (Invitrogen's product). DNA The sequence analysis results showed that the DNA sequence of the PCR product was exactly the same as that of 1 to 2947bp shown in SEQ ID NO: 1. Example 4: Northern blot analysis of the expression of the mouse tricarboxylic acid vector 39 gene:

Total RNA extraction in one step [Anal. Biochem 1987, 162, 156-159] This method includes acid guanidinium thiocyanate phenol-chloroform extraction. 4M guanidine isothiocyanate-25mM sodium citrate, 0.2M acetic acid sodium _(P H4.0) of the tissue was homogenized, 1 volume of phenol and 1/5 volume of chloroform - isoamyl alcohol. (49: 1), mixed with the water absorbing layer centrifugation, isopropanol ( 0.8 volume) and the mixture was centrifuged to obtain an RNA pellet. The resulting RNA pellet was washed with 70% ethanol, dried and dissolved in water. Using 20 μ _§ RNA, containing 20 mM 3- (N-morpholino) propanesulfonic acid ( pH 7.0) -5 mM sodium acetate-1 mM EDTA-2.2M formaldehyde on a 1.2% agarose gel for electrophoresis. Then transfer to a nitrocellulose membrane. ³² -P-labeling was prepared by random primer method using c- ³² P dATP The DNA probe used was the PCR amplified mouse tricarboxylic acid vector 39 coding region sequence (114bp to 10S2bp) shown in Figure 1. The 32P-labeled probe (about 2 x 10 ⁶ cpm / ml) hybridized with RNA-transferred nitrocellulose membrane at 42 ° C overnight in a solution containing 50 ° / »formamide-25mM KH ₂ P0 ₄ (pH7.4) -5 χ SSC- 5 χ Denhardt's solution and 200 μg / ml salmon sperm DNA. After hybridization, the filter was washed in ix SSC-0.1 ° /. SDS at 55 ° C for 30 min. Then, it was analyzed and quantified by Phosphor Imager. Example 5: In vitro expression, isolation and purification of recombinant murine tricarboxylic acid vector 39 According to SEQ ID NO: 1 and the coding region sequence shown in FIG. 1, a pair of specific amplification primers is designed, and the sequences are as follows:

 Primer3: 5,-CCCCATATGATGTCTGGAGAACTACCACCAAAC -3, (Seq ID No: 5) Primer4: 5,-CATGGATCCTACAATCCCTTATTGAAGTACACG -3, (Seq ID No: 6) The 5 'ends of these two primers contain Ndel and BamHI restriction sites, respectively. The coding sequences of the 5 'and 3' ends of the target gene are followed, respectively. The Ndel and BamHI restriction sites correspond to the selectivity on the expression vector plasmid pET-28b (+) (Nova gen, Cat. No. 69865.3). Endonuclease site. PCR was performed using the pBS-0703A07 plasmid containing the full-length target gene as a template. The PCR reaction conditions were as follows: total volume 50 μ1, pBS- 0703A07 plasmid 10pg, primers Primer-3 and Primer- 4 points, and ljpmol, Advantage polymerase Mix

(Clontech) 1 μ1. Cycle parameters: 94. C 20s, 60 ° C 30s, 68. C 2 min, a total of 25 cycles. Nde I and BamH I were used to double-digest the amplified product and plasmid pET-28 (+), respectively, and large fragments were recovered and ligated with T4 ligase. The ligation product was transformed into Ca. bacillus DH5CX using the calcium chloride method.

After the LB plate (final concentration 30 μ _§ / ηι1) was cultured overnight, the positive clones were selected by colony PCR method and sequenced. A positive clone (PET-0703A07) with the correct sequence was selected, and the recombinant plasmid was transformed into E. coli BL21 (DE3) plySs (a product of Novagen) by the calcium chloride method. In LB liquid medium containing kanamycin (final concentration 30 g / ml), the host bacteria BL21 (pET-0703A07) was cultured at 37 ° C to the logarithmic growth phase, and IPTG was added to a final concentration of 1 mmol / L, and continued. Incubate for 5 hours. The bacteria were collected by centrifugation, and the supernatant was collected by centrifugation by ultrasonication. An affinity chromatography column His. Bind Quick Cartridge capable of binding to 6 histidines (6His-Tag) was used.

(Product of Novagen) Chromatography was performed to obtain a purified target protein murine tricarboxylic acid carrier 39. After SDS-PAGE electrophoresis, a single band was obtained at 39 kDa (Figure 2). This 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 6 Production of anti-mouse tricarboxylic acid carrier 39 antibody

 The following peptides specific for murine tricarboxylic acid carrier 39 were synthesized using a peptide synthesizer (product of PE):

NH ₂ -Met-Ser-Gly-Glu-Leu-Pro-Pro-Asn-I le-Asn-I le-Lys-Glu-Pro-Arg- C00H

(SEQ ID NO: 7). The peptide was coupled to hemocyanin and bovine serum albumin to form a complex. For the method, see: Avrameas, et al. Immunochemistry, 1969; 6: 43. Immunologists were treated with 4 mg of the above-mentioned jk cyanin polypeptide complex and complete Freund's adjuvant. For rabbits, boost the immunity with hemocyanin peptide complex and incomplete Freund's adjuvant again after 15 days. ELISA Use a titer plate coated with 15 g / ml bovine serum albumin peptide complex for ELISA to determine antibody in rabbit serum Titer: Antibody A positive rabbit serum with protein A-Sepharose Total I gG was isolated in medium. 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 the murine tricarboxylic acid carrier 39. Industrial applicability

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

 The polypeptides of the present invention, as well as antagonists and agonists and inhibitors of the polypeptides, can be used in the treatment of diseases, for example, can treat ischemia, infection, inflammation, trauma, congenital defects in metabolism, metabolic or malignant transformation processes such as cancer Acquired Defects, etc.

 The electron transport chain in mitochondria requires it to properly function as a strong chemical / ion concentration exists across the mitochondrial membrane. The absence of this gradient, or the absence of a cell gradient that in turn affects the mitochondrial gradient, will impair the function of the electron transport chain. Tricarboxylic acid carriers are one of the transporters that help the electron transfer chain perform its function correctly. Impaired tricarboxylic acid carrier function will affect the tricarboxylic acid cycle. If sufficient tricarboxylic acid cycle is lost, the patient will lose 90% The ability to generate energy and gradually enter a coma. And tricarboxylic acid carriers are related to the biosynthesis of fatty acids and cholesterol, and also play a role in gluconeogenesis and reducing shuttles through mitochondrial equivalents.

In particular, with respect to the murine tricarboxylic acid carrier 39, the polypeptides or fragments or derivatives thereof of the present invention can be used to prevent and treat various diseases caused by metabolic abnormalities. These diseases include, but are not limited to, the following: Disease, mucopolysaccharidosis and other marginal diseases, purine and pyrimidine metabolism deficiency diseases, abnormal lipid metabolism, and glucose metabolism deficiency diseases: such as congenital sugar digestion and absorption deficiency, monosaccharide metabolism deficiency disease, glycogen metabolism disease; cholesterol metabolism Obstructive diseases: adipose deposit diseases, cardiovascular diseases, sterol derivative metabolic disorders, tumors; steroid hormone metabolic disorders: sexual developmental disorders during growth and development, (1) precocious puberty, (2) sexual development Delayed, (3) Sexual differentiation disorder, ( ⁴ ) Other endogenous developmental defects in endocrine and metabolic syndromes: Adrenal hyperfunction disease such as Cushing syndrome, aldosteronism, Adrenal insufficiency disease such as acute adrenal insufficiency , Chronic adrenal insufficiency; metabolic and nutritional diseases: sugar Urinary disease, hypoglycemia, hyperlipidemia and hyperlipoproteinemia, obesity, malnutrition, dehydration, excess water and water poisoning, hyponatremia, hypernatremia, hypokalemia, Hyperkalemia, metabolic acidosis, metabolic alkalosis, respiratory acidosis, respiratory alkalosis, mixed acid-base disorders, gout.

The invention also provides screening compounds to identify increasing (agonist) or suppressing (antagonist) murine tricarboxylic acids. Method of medicament of carrier 39. Agonists enhance biological functions such as murine tricarboxylic acid carrier 39 to stimulate cell proliferation, while antagonists prevent and treat disorders related to excessive cell proliferation, such as various cancers. For example, mammalian cells or a membrane preparation expressing a murine tricarboxylic acid carrier 39 can be cultured together with a labeled murine tricarboxylic acid carrier 39 in the presence of a drug. The ability of the drug to increase or block this interaction is then determined.

 Antagonists of the murine tricarboxylic acid carrier 39 include antibodies, compounds, receptor deletions, and the like that have been screened. The antagonist of the murine tricarboxylic acid carrier 39 can bind to the murine tricarboxylic acid carrier 39 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, a murine tricarboxylic acid carrier 39 can be added to a bioanalytical assay to determine whether the compound is an antagonist by measuring the effect of the compound on the interaction between the murine tricarboxylic acid carrier 39 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 the murine tricarboxylic acid carrier 39 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. When screening, 39 molecules of murine tricarboxylic acid carrier 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 the murine tricarboxylic acid carrier 39 epitope. These antibodies include (but are not limited to): polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, and fragments generated from Fab expression libraries.

 Polyclonal antibodies can be produced by injecting a mouse tricarboxylic acid carrier 39 directly into an immunized animal (such as rabbits, mice, rats, etc.). Various adjuvants can be used to enhance the immune response, including but not limited to Freund's adjuvant Wait. Techniques for preparing monoclonal antibodies to the murine tricarboxylic acid carrier 39 include, but are not limited to, hybridoma technology (Kohler and Milstei. Nature, 1975, 25 6: 495-497), triple tumor technology, human beta -Cell hybridoma technology, EBV-hybridoma technology, etc. Chimeric antibodies that bind human constant regions and non-human variable regions can be produced using existing techniques (Mor ris on et al, PNAS, 1 985, 81: 685 1). And existing techniques for producing single-chain antibodies (US Pat. No. 4946778) can also be used to produce single chain antibodies against the murine tricarboxylic acid carrier 39.

 Antibodies against murine tricarboxylic acid carrier 39 can be used in immunohistochemical techniques to detect murine tricarboxylic acid carrier 39 in biopsy specimens.

 Monoclonal antibodies that bind to the murine tricarboxylic acid carrier 39 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. Such as murine tricarboxylic acid carrier 39 High-affinity monoclonal antibodies can covalently bind to bacterial or plant toxins (such as diphtheria toxin, ricin, ormosine, etc.). A common method is to attack the amino group of an antibody with a thiol cross-linking agent such as SPDP and bind the toxin to the antibody through the exchange of disulfide bonds. This hybrid antibody can be used to kill the mouse tricarboxylic acid carrier 39 positive cells .

 The antibodies of the present invention can be used to treat or prevent diseases related to the murine tricarboxylic acid carrier 39. Administration of an appropriate dose of the antibody can stimulate or block the production or activity of the murine tricarboxylic acid carrier 39.

 The invention also relates to a diagnostic test method for quantitative and localized detection of the level of murine tricarboxylic acid carrier 39. These tests are well known in the art and include FI SH assays and radioimmunoassays. The levels of murine tricarboxylic acid carrier 39 detected in the test can be used to explain the importance of murine tricarboxylic acid carrier 39 in various diseases and to diagnose diseases in which murine tricarboxylic acid carrier 39 functions.

 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.

 The polynucleotide encoding murine tricarboxylic acid carrier 39 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 non-expression or abnormal / inactive expression of murine tricarboxylic acid vector 39. Recombinant gene therapy vectors (such as viral vectors) can be designed to express variant murine tricarboxylic acid vector 39 to inhibit endogenous murine tricarboxylic acid vector 39 activity. For example, a variant murine tricarboxylic acid carrier 39 may be a shortened murine tricarboxylic acid carrier 39 lacking a signaling domain, and although it can bind to downstream substrates, it lacks signaling activity. Therefore, the recombinant gene therapy vector can be used for treating diseases caused by abnormal expression or activity of the murine tricarboxylic acid vector 39. Virus-derived expression vectors such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus and the like can be used to transfer the polynucleotide encoding the mouse tricarboxylic acid vector 39 into cells. A method for constructing a recombinant viral vector carrying a polynucleotide encoding a murine tricarboxylic acid vector 39 can be found in the existing literature (Sambrook, et al.). In addition, the polynucleotide encoding the murine tricarboxylic acid carrier 39 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 the murine tricarboxylic acid vector 39 are also within the scope of the present invention. A ribozyme is an enzyme-like RNA molecule that can specifically decompose a specific RM. Its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target RM to perform 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 molecule can encode this RNA The DNA sequence is obtained by in vitro or in vivo transcription. This DNA sequence has been integrated downstream of the RNA polymerase promoter of the vector. In order to increase the stability of a nucleic acid molecule, it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the ribonucleoside linkages should use phosphate thioester or peptide bonds instead of phosphodiester bonds.

 The polynucleotide encoding the murine tricarboxylic acid carrier 39 can be used for the diagnosis of diseases related to the murine tricarboxylic acid carrier 39. The polynucleotide encoding the murine tricarboxylic acid vector 39 can be used to detect the expression of the murine tricarboxylic acid vector 39 or the abnormal expression of the murine tricarboxylic acid vector 39 in a disease state. For example, the DNA sequence encoding the murine tricarboxylic acid vector 39 can be used to hybridize biopsy specimens to determine the expression of the murine tricarboxylic acid vector 39. Hybridization techniques include Sout hern blotting, Nor thern 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 (Mic Roa ray) or a DNA chip (also known as a "gene chip") for analyzing differential expression analysis of genes in tissues and Genetic diagnosis. Murine tricarboxylic acid carrier 39 specific primers can also be used to detect the transcription products of murine tricarboxylic acid carrier 39 by RNA-polymerase chain reaction (RT-PCR) in vitro amplification.

 Detection of mutations in the murine tricarboxylic acid carrier 39 gene can also be used to diagnose murine tricarboxylic acid carrier 39-related diseases. Murine tricarboxylic acid vector 39 mutations include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to the normal wild-type murine tricarboxylic acid vector 39 DNA sequence. Mutations can be detected using existing techniques such as Southern blotting, DM sequence analysis, PCR and in situ hybridization. In addition, mutations may affect protein expression. Therefore, the Nor thern blot and Western blot can be used to indirectly determine whether a gene is mutated.

 The sequences of the invention are also valuable for chromosome identification. The sequence specifically targets a specific position on a human chromosome and can hybridize to it. Currently, specific sites for each gene on the chromosome need to be identified. Currently, only a few chromosome markers based on actual sequence data (repeating polymorphisms) are available for marking chromosome positions. According to the present invention, in order to associate these sequences with disease-related genes, an important first step is to locate these DNA sequences on a chromosome.

 In short, PCR primers (preferably 1 to 35 bp) are prepared based on cDM, and the sequences can be located on chromosomes. These primers were then used for PCR screening of somatic hybrid cells containing individual human chromosomes. Only those heterozygous cells containing the human gene corresponding to the primer will produce amplified fragments.

PCR localization of somatic hybrid cells is a quick way to localize DNA to specific chromosomes. Using the oligonucleotide primers of the present invention, by a similar method, a set of fragments from a specific chromosome 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 individuals, the mutation may be the cause of the disease. Comparing affected and unaffected individuals usually involves first looking for structural changes in chromosomes, such as deletions or translocations that are visible at the chromosomal level or detectable with cDNA sequence-based PCR. According to the resolution capabilities of current physical mapping and gene mapping technology, the cDNA accurately mapped to the chromosomal region associated with the disease can be one of 50 to 500 potentially pathogenic genes (assuming 1 megabase mapping resolution) Capacity and each 20kb corresponds to a gene).

 The polypeptides, polynucleotides and mimetics, agonists, antagonists and inhibitors of the present invention can be used in combination with a suitable pharmaceutical carrier. These carriers can be water, glucose, ethanol, salts, buffers, glycerol, and combinations thereof. The composition comprises a safe and effective amount of the polypeptide or antagonist, and carriers and excipients 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. Murine tricarboxylic acid carrier 39 is administered in an amount effective to treat and / or prevent a specific indication. The amount and dosage range of the mouse tricarboxylic acid carrier 39 to be administered to a patient will depend on many factors, such as the mode of administration, the health conditions of the person to be treated, and the judgment of the diagnostician. (1) General information:

(ii) Name of the invention: Murine tricarboxylic acid carrier 39 and its coding sequence

(iii) Number of sequences: 7

(2) Information of SEQ ID NO: 1:

 (i) Sequence characteristics:

 (A) Length: 2947bp

 (B) Type: Nucleic acid

 (C) Chain: double strand

 (D) Topological structure: linear

 (ii) Molecular type: cDNA

(xi) Sequence description: SEQ ID NO: 1:

1081

1141

1201

1261

1321

1381

1441

1501

1561

1621

1681

1741

1801

1861

1921

1981

2041

2101

2161

2221

2281

2341

2401

2461

2521

2581

2641

2701

2761

2821

2881

2941 AAATGGC (3) Information of SEQ ID NO: 2:

 (i) Sequence characteristics:

 (A) Length: 322 amino acids

 (B) Type: Amino acid

 (D) Topological structure: linear

 (ii) Molecular type: peptide

 (xi) Sequence description: SEQ ID NO: 2:

 Met Ser Gly Glu Leu Pro Pro Asn lie Asn lie Lys Glu Pro Arg

Trp Asp Gin Ser Thr Phe lie Gly Arg Ala Asn His Phe Phe Thr

Val Thr Asp Pro Arg Asn lie Leu Leu Thr Asn Glu Gin Leu Glu

Ser Ala Arg Lys lie Val His Asp Tyr Arg Gin Gly lie Val Pro

Pro Gly Leu Thr Glu Asn Glu Leu Trp Arg Ala Lys Tyr lie Tyr

Asp Ser Ala Phe His Pro Asp Thr Gly Gly Lys Met lie Leu He

Gly Arg Met Ser Ala Gin Val Pro Met Asn Met Thr lie Thr Gly Cys Met Met Thr Phe Tyr Arg Thr Thr Pro Ala Val Leu Phe Trp

Gin Trp lie Asn Gin Ser Phe Asn Ala Val Val Asn Tyr Thr Asn

Arg Ser Gly Asp Ala Pro Leu Thr Val Asn Glu Leu Gly Thr Ala

Tyr Val Ser Ala Thr Thr Gly Ala Val Ala Thr Ala Leu Gly Leu

Asn Ala Leu Thr Lys His Val Ser Pro Leu lie Gly Arg Phe Val

Pro Phe Ala Ala Val Ala Ala Ala Asn Cys lie Asn He Pro Leu

Met Arg Gin Arg Glu Leu Lys Val Gly lie Pro Val Thr Asp Glu

Asn Gly Asn Arg Leu Gly Glu Ser Ala Asn Ala Ala Lys Gin Ala lie Thr Gin Val Val Val Ser Arg lie Leu Met Ala Ala Pro Gly

Met Ala He Pro Pro Phe lie Met Asn Thr Leu Glu Lys Lys Ala

Phe Leu Lys Arg Phe Pro Trp Met Ser Ala Pro lie Gin Val Gly

Leu Val Gly Phe Cys Leu Val Phe Ala Thr Pro Leu Cys Cys Ala

Leu Phe Pro Gin Lys Ser Ser Met Ser Val Thr Ser Leu Glu Ala

Glu Leu Gin Ala Lys He Gin Glu Ser His Pro Glu Leu Arg Arg

Val Tyr Phe Asn Lys Gly Leu (4) Information of SEQ ID NO: 3

(i) Sequence characteristics

 (A) Length: 24 bases

 (B) Type: Nucleic acid

 (C) Chain: single chain

 (D) Topological structure: linear

 (Π) Molecular type: Oligonucleotide

 (xi) Sequence description: SEQ ID NO: 3:

GGGGACGCGCGAGGACGCCGTGGC

(5) Information of SEQ ID NO: 4

 (i) Sequence characteristics

 (A) Length: 24 bases

 (B) Type: Nucleic acid

 (C) Chain: single chain

 (D) Topological structure: linear

 (ii) Molecular type: Oligonucleotide

 (xi) Sequence description: SEQ ID NO: 4:

GCCATTTTTTCTTTATGAGGAAAT

(6) Information of SEQ ID NO: 5

 (i) Sequence characteristics

 (A) Length: 32 bases

 (B) Type: Nucleic acid

 (C) Chain: single chain

 (D) Topological structure: linear

 (ii) Molecular type: Oligonucleotide

(xi) Sequence description: SEQ ID NO: 5: CAGCCATGGCGGGGAAGAAGAATGTTCTGTCG

(7) Information of SEQ ID NO: 6

(i) Sequence characteristics (A) Length: 29 bases

 (B) Type: Nucleic acid

 (C) Chain: single chain

 (D) Topological structure: linear

 (Π) Molecular type: Oligonucleotide

 (xi) Sequence description: SEQ ID NO: 6:

CCCGGATCCCGCTGCTTGGCCTTCTTCAC

(8) Information of SEQ ID NO: 7:

 (i) Sequence characteristics:

 (A) Length: 15 amino acids

 (B) Type: Amino acid

 (D) Topological structure: linear

 (Π) Molecular type: Polypeptide

 (xi) Sequence description: SEQ ID NO: 7:

 Met-Ala-Gly-Lys-Lys-Asn-Val-Leu-Ser-Ser-Leu-Ala-Va 1-Tyr-Ala

Claims

Claim 1. An isolated polypeptide-murine tricarboxylic acid carrier 39, 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 the amino acid sequence shown in SEQ ID NO: 2.  4. An isolated polynucleotide, characterized in that said polynucleotide comprises one selected from the group consisting of: (a) a polynucleotide encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: 2 or a fragment, analog, or derivative thereof;  (b) a polynucleotide complementary to the polynucleotide; or (c) a) or (b) at least ^95% identical to a polynucleotide.  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 114-1082 in SEQ ID NO: 1 or a sequence of positions 1-2947 in SEQ ID NO: 1. .  7. A recombination vector containing an exogenous polynucleotide, characterized in that it is a recombination constructed by the polynucleotide according to any one of claims 4-6 and a plasmid, virus or a carrier expression vector Carrier.  8. A genetically engineered host cell containing an exogenous polynucleotide, characterized in that it is selected from one of the following host cells:  (a) a host cell transformed or transduced with the recombinant vector of claim 7; or  (b) a host cell transformed or transduced with a polynucleotide according to any one of claims 4-6.  9. A method for preparing a polypeptide having murine tricarboxylic acid carrier 39 activity, characterized in that the method comprises:  (a) culturing the engineered host cell according to claim 8 under the condition of expressing a murine tricarboxylic acid vector 39;  (b) A polypeptide having murine tricarboxylic acid carrier 39 activity is isolated from the culture. 10. An antibody capable of binding to a polypeptide, characterized in that said antibody is an antibody capable of specifically binding to a murine tricarboxylic acid carrier 39. 1 1. 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 the murine tricarboxylic acid carrier 39.  12. The compound according to claim 11, characterized in that it is an antisense sequence of the polynucleotide sequence shown in SEQ ID NO: 1 or a fragment thereof.  1 3. An application of the compound according to claim 11, characterized in that the compound is used for a method for regulating the activity of the murine tricarboxylic acid carrier 39 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 the polypeptide according to any one of claims 1-3, characterized in that it is used for screening mimetics, agonists, antagonists or inhibitors of the murine tricarboxylic acid carrier 39; or for peptides Fingerprint identification.  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 the 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 the abnormality of the murine tricarboxylic acid carrier 39.  18. Use of a polypeptide, polynucleotide or compound according to any one of claims 1-6 and 11, characterized in that said polypeptide, polynucleotide or compound is used for preparing for treating malignant tumors, blood, etc. Disease, HI V infection and immune diseases and drugs of various inflammations.