CN101892237A - Polynucleotide related to mitochondrial membrane potential decrease and coded polypeptide and application thereof - Google Patents

Abstract

The invention discloses new polynucleotide coding the human protein with the function of causing mitochondrial membrane potential decrease and polypeptide coded by the polynucleotide and an antibody of the polypeptide. The invention also discloses application of the new polynucleotide in causing mitochondrial membrane potential decrease through exogenous expression in host cells.

Description

Polynucleotide and coded polypeptide and purposes that mitochondrial membrane potential descends and is correlated with

The present invention is that application number is 200610165851.7, the applying date is on December 14th, 2006, denomination of invention is divided an application for " mitochondrial membrane potential descend relevant polynucleotide and coded polypeptide and purposes ".

Technical field

The invention belongs to biological technical field, relate to the gene expression regulation field, specifically, the present invention relates to the new coding of a class and have the proteic polynucleotide of people that cause mitochondrial membrane potential decline function, and encoded polypeptides, the antibody of polypeptide.The invention still further relates to the application that the new polynucleotide of this class heterogenous expression in host cell causes that mitochondrial membrane potential descends.

Background technology

Apoptosis belongs to programmed cell death, is the complex process that relates to many biochemical reactions in the cell.Multiple critical event in the apoptotic process is relevant with plastosome, comprises the release (as cytochrome C) of caspase activator, the forfeiture of transfer transport function, the reduction of plastosome membrane potential and even the participation of disappearance and Bcl-2 family protein.The decline of plastosome membrane potential is event early in the apoptosis cascade reaction process.Along with the decline of plastosome membrane potential, the PT passage on the mitochondrial membrane (Permeability transiton pore) is open.The PT passage can pass through by non-selectivity ground permission≤1.5kD molecule, causes the high osmotic pressure in the mitochondrial matrix, makes the inside and outside H of plastosome ⁺Gradient disappears, and respiratory chain is taken off coupling, and energy produces and interrupts; Because entering of water and solute, matrix swelling also causes outer membrane rupture, discharges the various activated proteins that comprise cytochrome C.Just because of the importance of plastosome in apoptosis, the plastosome membrane potential is one of detection index of the early apoptosis used always.

JC-1 is a kind of cationic dyestuff, shows the membrane potential dependency and assemble in plastosome, can reflect delicately that the cell mitochondrial membrane potential changes situation.When the plastosome membrane potential does not reduce, JC-1 polymerization and be detected red emission light; When the plastosome membrane potential descended, JC-1 existed with monomeric form, was detected green emitted light.Therefore, JC-1 is applied to the detection that the early stage plastosome membrane potential of apoptosis descends as a kind of plastosome probe.The model that inductor that mitochondrial membrane potential is descended based on the JC-1 dyestuff and retarding agent screen just was suggested before the several years, also have many investigators' propositions that JC-1 is combined with other dyestuff in recent years and carry out method for screening, but Shang Weiyou really uses the report that JC-1 screens on a large scale.

Summary of the invention

The research of people's gene group is international focus at present, except that the method for large scale sequencing, also lacks the high flux screening that begins from functional study and has the method for the gene of certain function.Deficiency at this present situation and existing medicine or reagent the purpose of this invention is to provide the new coding of a class and has the proteic polynucleotide MMPRG1 of people, 2,3,4 that causes that mitochondrial membrane potential descends.

Another object of the present invention provides this class polynucleotide encoded polypeptide.

Another object of the present invention provides carrier and this class polynucleotide that contain these class polynucleotide and carrier transforms or the host cell of transduction.

Another object of the present invention provides the antibody of this class polynucleotide encoded polypeptide and the nucleic acid molecule that is used to detect.

Another object of the present invention provides the application that the new polynucleotide of this class heterogenous expression in host cell causes that mitochondrial membrane potential descends.

Another object of the present invention provides produces these polynucleotide and the method for its encoded polypeptides and the purposes of this polynucleotide and encoded polypeptides thereof.

For achieving the above object, the present invention is by the following technical solutions:

In a first aspect of the present invention, novel isolating polynucleotide are provided, it comprises coding and has the proteic nucleotide sequence that causes mitochondrial membrane potential decline function, and this nucleotide sequence is selected from: (a) polynucleotide of polypeptide that contain the aminoacid sequence of SEQ ID NO:2, SEQID NO:4, SEQ ID NO:6, SEQ ID NO:8 with coding have the polynucleotide of at least 70% similarity; (b) coding contains the polynucleotide of polypeptide that aminoacid sequence with SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ IDNO:8 has the aminoacid sequence of at least 70% similarity; (c) with (a) or polynucleotide complementary polynucleotide (b).

Preferably, the polypeptide of this polynucleotide encoding has the aminoacid sequence of the group of being selected from down: SEQ ID NO:2, SEQ IDNO:4, SEQ ID NO:6, SEQ ID NO:8.

Preferably, the sequence of these polynucleotide is shown at least 85% similarity with the nucleotides sequence that is selected from down group: (a) coding region sequence or the full length sequence of SEQ IDNO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7; (b) at least one sequence of the sequence of in genetic code degeneracy scope, mentioning in corresponding to (a); (c) with (a) or at least one sequence of the sequence complementary sequence hybridization of mentioning (b).

More preferably, the sequence of these polynucleotide is selected from coding region sequence or the full length sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQID NO:7.

In a second aspect of the present invention, above-mentioned Nucleotide encoded polypeptide is provided, it comprises the polypeptide with the aminoacid sequence in the group of being selected from down: SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8; Or the polypeptide that has similarity more than at least 90% with above arbitrary aminoacid sequence, or its conservative property variation polypeptide or its active fragments or its reactive derivative.

Preferably, this polypeptide is the polypeptide with aminoacid sequence of the group of being selected from down: SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8.

In a third aspect of the present invention, the carrier that contains above-mentioned polynucleotide is provided, and, also provides the host cell that is transformed or transduce by above-mentioned polynucleotide by the host cell that this carrier transforms or transduces.

In a fourth aspect of the present invention, provide and aforementioned polypeptides specificity bonded antibody, the nucleic acid molecule that can be used for detecting also is provided, it contains 8-100 successive Nucleotide in above-mentioned arbitrary polynucleotide.

In a fifth aspect of the present invention, provide above-mentioned polynucleotide heterogenous expression in host cell to cause the application that mitochondrial membrane potential descends.

In a sixth aspect of the present invention, a kind of pharmaceutical composition is provided, it contains polypeptide and the pharmaceutically acceptable carrier that causes mitochondrial membrane potential decline function that have among the present invention of safe and effective amount.

Other aspects of the present invention since disclosing of the technology of this paper will be apparent to those skilled in the art.

As used herein, " isolating " is meant that material separates (if natural substance, primal environment promptly is a natural surroundings) from its primal environment.Do not have separation and purification as polynucleotide under the native state in the active somatic cell and polypeptide, if but same polynucleotide or polypeptide from native state, separate with common other materials that exist, then be separation and purification.Such polynucleotide may be the parts of a certain carrier, the part that also possible such polynucleotide or polypeptide are a certain composition, since carrier or composition are not the compositions of their natural surroundings, these polynucleotide or polypeptide remain isolating.

As used herein, " similarity " is meant and is used for describing the height that detects same DNA base between sequence and the target sequence or amino-acid residue order proportion in Nucleotide or the peptide sequence comparison process, it is a kind of direct quantitative relation, recently measure degree similar between nucleotide sequence or the peptide sequence by the same or analogous percentage of part, this similarity per-cent can calculate by the existing comparison method in this area, example has the comparison method FASTA program (Pearson between sequence in twos, W.R.and Lipman, D.J.1988.Improved tools for biological sequence comparison.Proc.Natl.Acad.Sci.85:2444-2448), blast program (Altschul, S.F., et al.1990 Basic local alignmentsearch tool.J.Mol.Biol.215:403-410) etc., or Multiple Sequence Alignment Method CLUSTAL W (CORPET, F.1998Multiple sequence alignment with hierarchical clustering.Nucleic Acids Res., 16:10881-10890) etc.Homologous sequence is meant the different sequences that form through divergent evolution from a certain common ancestor, can judge homology between aligned sequences according to similarity per-cent.When similarity degree is very high between gene or protein, represents that they have one section common evolution course, thereby judge that they can have similar biological function.When similarity degree, detect sequence and target sequence may be a homologous sequence than being easier to infer with at least 50%.Preferably, has at least 70% similarity degree; More preferably, has at least 85% similarity degree; Best, has at least 90% similarity degree.And when the similarity degree is lower than 20%, just be difficult to determine or can't determine at all whether it has homology.

Polynucleotide of the present invention comprise that its complementary strand can be dna form or rna form.Dna form comprises the DNA of cDNA, genomic dna or synthetic.DNA can be strand or double-stranded.DNA can be coding strand or noncoding strand.As used herein, " coding has the polynucleotide of the polypeptide that causes mitochondrial membrane potential decline function " can be the polynucleotide that comprise this polypeptide of encoding, and also can be the polynucleotide that also comprise additional code sequence and/or non-coding sequence.With the MMPRG1 encoded polypeptide is example, and the coding region sequence of encoding mature polypeptide can be identical with the coding region sequence shown in the SEQ IDNO:1 or the varient of genetic code degeneracy.As used herein, " genetic code degeneracy " is meant that an amino acid has the phenomenon of several codons.The varient of the genetic code degeneracy of MMPRG1 encoded polypeptide refer to the encode Nucleotide of polypeptide in the present invention for example with SEQ ID NO:2, and the coding region sequence shown in this Nucleotide and the SEQ ID NO:1 has difference.Have the polypeptide that causes mitochondrial membrane potential decline function for other, can the rest may be inferred.

The invention still further relates to the varient of above-mentioned polynucleotide, its coding has the polypeptide of identical aminoacid sequence or fragment, analogue and the derivative of polypeptide with the present invention.The varient of these polynucleotide can be the allelic variant of natural generation or the varient that non-natural takes place.These nucleotide diversity bodies comprise and replace varient, deletion mutation body and insert varient.As known in the art, allelic variant is the replacement form of polynucleotide, and it may be replacement, disappearance or the insertion of one or more Nucleotide, but can be from not changing the function of its encoded polypeptides in fact.

The invention still further relates to and the complementary sequence hybridization of polynucleotide sequence of the present invention and two sequences between have at least 50%, preferably at least 70%, the polynucleotide of at least 80% homogeny more preferably.The present invention be more particularly directed under stringent condition the interfertile polynucleotide of the complementary sequence of polynucleotide sequence therewith.In the present invention, " stringent condition " is meant: (1) than hybridization under low ionic strength and the comparatively high temps and wash-out, as 0.2 * SSC, and 0.1%SDS, 60 ℃; Or (2) hybridization the time is added with denaturing agent, as 50% (v/v) methane amide, 0.1% calf serum/0.1%Ficoll, 42 ℃ etc.; Or (3) only at the homogeny between the two sequences at least more than 95%, be more preferably 97% and just hybridize when above.And the polypeptide of interfertile polynucleotide encoding has identical biological function and activity with polypeptide of the present invention.

Polynucleotide sequence of the present invention can obtain with this area existent method.These technology including, but not limited to: (1) is by hybridization technique DNA isolation sequence; (2) artificial chemical synthesising DNA sequence; (3) by the required polynucleotide of the extensive acquisition in construction cDNA library; (4) pcr amplification technology.

First method is to make up genomic library or cDNA library earlier, filters out goal gene or sequence by technology such as molecular hybridizations from genomic library or cDNA library then.When the biological gene group was smaller, this method is success easily; When the biological gene group is very big, make up difficulty of its complete genomic library, the quantities of removing to clone goal gene again from huge library is also very big.

Second method is by the long dna fragmentation of the once synthetic 100-200bp of the sequence that designs, and connects into complete gene with these synthetic fragment combination again.The price of the method for the gene order of this synthetic length is very expensive.This method be mainly used in synthetic as primer, connect the first grade nucleic acid fragment.

The third method is with usual method construction cDNA library, this area, repeatedly after the order-checking, in conjunction with bioinformatic analysis technology (Ota et al.Nat Genet.2004 Jan; 36 (1): 40-5), obtain purpose cDNA clone on a large scale.The bioinformatic analysis technology includes but not limited to BLAST or BLAT and the comparison of existing public database, as the refseq database etc.; With Phred algorithm assessment sequencing quality; ATGpr algorithm with the probability of occurrence that calculates transcription initiation codon ATG screens full length cDNA sequence etc.

The 4th kind of method method (Saiki, the et al.Science 1985 of round pcr DNA amplification/RNA; 230:1350-1354).The primer that is used for PCR can suitably be selected according to sequence information of the present invention disclosed herein, and available ordinary method is synthetic.Available ordinary method is as the DNA/RNA fragment by gel electrophoresis separation and purifying amplification.The method of advantageous applications of the present invention is that the amplification in mixing the cDNA library of two-step approach flux RT-PCR technology obtains a large amount of cDNA clones.Mix the cDNA library and comprise existing cDNA library and tumour library.

Gene of the present invention, the perhaps available ordinary method of mensuration of nucleotide sequence such as various dna fragmentations, as dideoxy chain termination (Sanger et al.PNAS, 1977,74:5463-5467); Also available commercial sequencing kit etc.In order to obtain the cDNA sequence of total length, order-checking need be carried out repeatedly.Sometimes need to measure a plurality of clones' cDNA sequence, just can be spliced into the cDNA sequence of total length.

Polypeptide of the present invention can be recombinant polypeptide, natural polypeptides, synthetic polypeptide, preferred recombinant polypeptide.Polypeptide of the present invention can be the product of natural purifying, or the product of chemosynthesis, or uses recombinant technology to produce from protokaryon or eucaryon host (as bacterium, yeast, higher plant, insect and mammalian cell).Polypeptide of the present invention can be glycosylated, also can be nonglycosylated.Polypeptide of the present invention can comprise or not comprise initial methionine residues.

The present invention also comprises fragment, derivative and the analogue of the people's protein polypeptide with the polynucleotide encoding that causes mitochondrial membrane potential decline function.Term " fragment ", " derivative " are meant basically with " analogue " and keep and natural identical biological function or the active polypeptide of people's protein polypeptide that causes mitochondrial membrane potential decline function that have of the present invention.Polypeptide fragment of the present invention, derivative and analogue can be: one or more conservative or substituted polypeptide of non-conservation amino-acid residue (preferential conservative amino acid residue) (a) are arranged, and the amino-acid residue that replaces like this can be also can not encoded by genetic code, or (b) in one or more amino-acid residues, has a polypeptide of substituted radical, or (c) mature polypeptide and another compound (such as the compound that prolongs the polypeptide transformation period) merge formed polypeptide, or (d) additional aminoacid sequence is fused to this peptide sequence and the polypeptide that forms (as leader sequence or secretion sequence or be used for the sequence or the proteinogen sequence of this polypeptide of purifying).

Polypeptide of the present invention can be by conventional recombinant DNA technology, the protein polypeptide (Science, 1984 that utilize polynucleotide sequence of the present invention to express or produce having of reorganization to cause mitochondrial membrane potential decline function; 224:1431).May further comprise the steps:

(1), or transforms or the transduction proper host cell with the expression vector that contains these polynucleotide with polynucleotide of the present invention (or its varient);

(2) host cell that culturing step (1) obtains in suitable medium;

(3) separation, the required protein polypeptide of purifying from substratum or cell.

Polynucleotide among the present invention and polypeptide preferably provide with isolating form, more preferably are purified to homogeneous.

The present invention also relates to comprise the carrier of polynucleotide of the present invention.Among the present invention, the polynucleotide sequence that coding has the people's protein polypeptide that causes mitochondrial membrane potential decline function can be inserted in the recombinant expression vector.Term " recombinant expression vector " refers to bacterial plasmid well known in the art, phage, yeast plasmid, vegetable cell virus, mammalian cell virus, as adenovirus, retrovirus, and perhaps other carriers.The carrier of Shi Yonging can be a prokaryotic expression carrier in the present invention, also can be carrier for expression of eukaryon, as the expression vector (Rosenberg based on T7 that expresses in bacterium, et al.Gene, 1987,56:125), the carrier for expression of eukaryon pcDNA of high expression level in mammalian cell ^TM3.1/myc-hisB (-) (Invitrogen), pcDNA3.1/V5-His-TOPO (Invitrogen below is abbreviated as pcDT).The preferred pcDT of the present invention, it can directly be connected with the PCR product and makes up carrier for expression of eukaryon, has improved the efficient of large-scale production greatly.As long as can duplicate in host and stablize, any plasmid and carrier can be used.A key character of expression vector is to contain replication orgin, promotor, marker gene and translation controlling elements usually.Making up the expression vector that contains polynucleotide sequence of the present invention and transcribe/translate control signal with method well-known to those having ordinary skill in the art gets final product.These methods comprise (Sambrook, et al.Molecular Cloning, aLaboratory Manual, Cold Spring Harbor Laboratory.New York, 1989) such as extracorporeal recombinant DNA technology, DNA synthetic technology, the interior recombinant DNA technologies of body.

Polynucleotide sequence of the present invention can be connected to effectively and instruct mRNA synthetic on the suitable promotor in the expression vector.The representative example of these promotors has: colibacillary lac or trp promotor; The PL promotor of lambda particles phage; Eukaryotic promoter comprises LTRs and some other known may command gene expression promoter in protokaryon or eukaryotic cell or its virus of CMV immediate early promoter, HSV thymidine kinase promoter, early stage and late period SV40 promotor, retrovirus.Expression vector also comprises ribosome bind site and the transcription terminator that translation initiation is used.Expression vector preferably comprises one or more selected markers, being provided for selecting the phenotypic character of transformed host cells, as being used for colibacillary tsiklomitsin or amicillin resistance or eukaryotic cell and cultivating green fluorescent protein (GFP), neomycin resistance and the Tetrahydrofolate dehydrogenase of usefulness.

The invention still further relates to the host cell that produces through genetically engineered with above-mentioned carrier or polynucleotide of the present invention.Carrier of the present invention and polynucleotide can be used to transform appropriate host cell, have the protein that causes mitochondrial membrane potential decline function so that it can be expressed.Host cell can be a prokaryotic cell prokaryocyte, as bacterial cell; Or eukaryotic cell such as low, as yeast cell; Or higher eucaryotic cells, as mammalian cell.Representative example has: intestinal bacteria; Vegetable cell; The insect cell of fruit bat S2 or Sf9; The zooblast of CHO, COS or Bowes melanoma cells; 293T, Hela cell etc.

When polynucleotide of the present invention are expressed, transcribe enhancing if will make when in carrier, inserting enhancer sequence in higher eucaryotic cells.Enhanser is the cis acting factor of DNA, and 10-300 base pair arranged usually, acts on promotor transcribing with enhancing gene.Example has: at the SV40 enhanser of 100-270 the base pair in replication origin downstream, at the polyoma enhanser in replication origin downstream and adenovirus enhanser etc.

Those of ordinary skill in the art knows how to select appropriate carriers, promotor, enhanser and host cell.

Can carry out with routine techniques well known to those skilled in the art with the recombinant DNA transformed host cell.When host cell was prokaryotic cell prokaryocyte such as intestinal bacteria, the competent cell that can absorb DNA can be collected at the exponential growth after date, uses CaCl ₂Method is handled, and used step is well-known in the art.Alternative is MgCl ₂Handle, also the method for available electroporation is handled.When the host is eukaryotic cell, can select following transfection method: coprecipitation of calcium phosphate method, conventional mechanical method such as microinjection, electroporation, liposome packing etc.The transformant that obtains can be cultivated with ordinary method, expresses polynucleotide encoded polypeptide of the present invention.Select suitable conventional substratum according to selected host cell, under the condition that is suitable for the host cell growth, cultivate.After host cell grows into suitable cell density, with appropriate means such as temperature inversion or chemical induction, induce the promotor of selection, cell is cultivated for some time again.

Recombinant polypeptide in the aforesaid method can wrap by in cell, extracellular or on cytolemma, express or be secreted into the extracellular.If desired, can utilize its physics, chemical separating and the purification of Recombinant polypeptide by various separation methods with other characteristics.These methods are well-known to those skilled in the art, handle as the renaturation of routine, handle the combination of (salt analysis method), centrifugal, the broken bacterium of infiltration, ultrasonication, super centrifugal, sieve chromatography (gel-filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography and other various liquid chromatography (LC) technology or these methods with protein precipitant.

The invention still further relates to any a part of homologous nucleic acid fragment with polynucleotide of the present invention.As used herein, the length of " nucleic acid fragment " contains 15 Nucleotide at least, is preferably at least 30 Nucleotide, is more preferably at least 50 Nucleotide, and best is at least 100 Nucleotide.This nucleic acid fragment is the dna sequence dna of chemosynthesis on the basis of nucleotide sequence information of the present invention normally.Above-mentioned nucleic acid fragment can be used for pcr amplification technology (as primer) and have the polynucleotide that cause mitochondrial membrane potential decline function to determine and/or to separate coding; Also can be used as the used probe of hybridization.Also can be used for the RNA perturbation technique.Part or all of polynucleotide of the present invention also can be used as probe stationary on microarray (Microarray) or DNA chip, is used for analyzing the differential expression and the gene diagnosis of tissue gene.The mark of probe can be used radio isotope, fluorescein or enzyme (as alkaline phosphatase) etc.

Polypeptide of the present invention can be directly as the pharmacological agent disease; Also can be used for screening promotion or resist proteic antibody, polypeptide or other part with the function that causes that mitochondrial membrane potential descends, for example, screening can be used for promoting or suppressing the antibody of proteic function of the present invention.Albumen of the present invention with the reorganization of expressing screens peptide library, is used to seek the peptide molecule that can promote or suppress proteic function of the present invention of therapeutic value.

Polypeptide of the present invention can use separately or use with suitable pharmaceutical carrier combination back.Composition comprises the polypeptide or the antagonist of safe and effective amount and does not influence the carrier and the excipient of effect of drugs.These carriers can be water, glucose, ethanol, salt, damping fluid, glycerine and their combination.Pharmaceutical composition can be with mode administration easily, as by in part, intravenously, intraperitoneal, intramuscular, subcutaneous, the nose or the route of administration of intracutaneous.The consumption that delivers medicine to the patient depends on many factors, as administering mode, person's to be treated healthiness condition and diagnostician's judgement.

Polypeptide of the present invention also can use by express these polypeptide at live body.For example patient's cell can carry out the genetically engineered operation by the gene at external use code book invention polypeptide, then engineering cell is offered the patient, makes engineering cell this peptide species of high expression level in vivo, thereby reaches the purpose of treatment.

Have the proteic polynucleotide of people that cause mitochondrial membrane potential decline function and also can be used for multiple therapeutic purpose.Can be used on and treat in the gene therapy technology owing to have the people's protein abnormal expression or the active disease that causes unusually of the function that causes that mitochondrial membrane potential descends.The gene therapy vector (as virus vector) of reorganization can be designed to express people's albumen that having of variation causes mitochondrial membrane potential decline function, suppresses the endogenic proteic activity of people that causes mitochondrial membrane potential decline function that has.The having the people's protein gene that causes mitochondrial membrane potential decline function and also can be packaged in the liposome and be transferred in the cell of reorganization.

Suppress the oligonucleotide (comprising sense-rna and DNA) of polypeptide mRNA of the present invention and nucleic acid also within the scope of the invention.Sense-rna and DNA and nucleic acid can be synthetic with this area existent method.In order to increase the stability of nucleic acid molecule, available several different methods is modified it, as increasing the sequence length of both sides, the connection between ribonucleoside phosphoric acid thioester bond or peptide bond.

Polypeptide of the present invention and fragment thereof, derivative, analogue or the cell of expressing them can be used as antigen and produce antibody.These antibody include but not limited to the antibody that monoclonal antibody, polyclonal antibody, chimeric antibody, single-chain antibody, Fab fragment and Fab expression library produce.The antibody of polypeptide of the present invention can be produced with preparation method for antibody well known in the art.Example has: monoclonal antibody can with hybridoma technology production (Kohler and Milstein.Nature, 1975,256:495-497).The available polypeptide immune animal of the present invention of the production of polyclonal antibody is as rabbit, mouse, rat etc.Multiple adjuvant can be used for the enhancing immunity reaction, includes but not limited to freund's adjuvant.The variable region bonded chimeric antibody in human constant region and inhuman source can be produced with existing technology (Morrison et al.PNAS, 1985,81:6851).The also available existing technology production of single-chain antibody (U.S.Pat No.4946778).

Antibody of the present invention can be used in the immunohistochemistry technology, detects the albumen that causes mitochondrial membrane potential decline function that has in the living specimen.Can also be used for clinical diagnosis, treatment, therapeutic evaluation of the disease relevant etc. clinically with having people's albumen of causing mitochondrial membrane potential decline function.For example use labelled with radioisotope and polypeptide bonded monoclonal antibody of the present invention, inject then and follow the tracks of its position and distribution in the body, can be used as a kind of atraumatic diagnostic method and come the positioning tumor cell, or judge whether tumour cell shifts.Antibody among the present invention can also be used for the treatment of or prevent and have the relevant disease of people's albumen that causes mitochondrial membrane potential decline function.The antibody that gives suitable dosage can stimulate or block and has proteic generation of people or the activity that causes mitochondrial membrane potential decline function.

The invention still further relates to quantitatively and detection and localization has the diagnostic testing process of the protein level that causes mitochondrial membrane potential decline function.These tests are known in the art, and comprise that FISH measures and radioimmunoassay.That detects in the experiment has a protein level that causes mitochondrial membrane potential decline function, can have the importance of albumen in various diseases that causes mitochondrial membrane potential decline function with laying down a definition and be used to diagnose to have the disease that the albumen that causes mitochondrial membrane potential decline function works.

Have the proteic polynucleotide that cause mitochondrial membrane potential decline function and can be used for having the diagnosis and the treatment of the protein related diseases that causes mitochondrial membrane potential decline function.Aspect diagnosis, have the proteic polynucleotide that cause mitochondrial membrane potential decline function can be used for detecting have cause mitochondrial membrane potential decline function proteic expression whether, or under morbid state, have the abnormal exprssion that causes mitochondrial membrane potential decline function.As have the proteic dna sequence dna that causes mitochondrial membrane potential decline function and can be used for that the hybridization of biopsy specimen is had the proteic abnormal expression that causes mitochondrial membrane potential decline function with judgement.Hybridization technique is the disclosed mature technology in this area, comprises Southern blotting, Northern blotting, in situ hybridization etc., and relevant test kit can obtain from commercial channels.Part or all of polynucleotide of the present invention can be used as probe stationary on microarray (Microarray) or DNA chip, is used for analyzing the differential expression and the gene diagnosis of tissue gene.Carry out RNA-polymerase chain reaction (RT-PCR) amplification in vitro and also can detect and have the proteic transcription product that causes mitochondrial membrane potential decline function with having the special primer of albumen that causes mitochondrial membrane potential decline function.

The sudden change that detection has a protein gene that causes mitochondrial membrane potential decline function also can be used for diagnosing and has the relevant disease of albumen that causes mitochondrial membrane potential decline function.Mutant form with the protein gene that causes mitochondrial membrane potential decline function comprises with having of normal wild type and causes that point mutation that the proteic dna sequence dna of mitochondrial membrane potential decline function compares, transposition, disappearance, reorganization and other are any unusual etc.Existing technology in available this area such as Southern blotting, dna sequence analysis, PCR and in situ hybridization detect sudden change.In addition, sudden change might influence proteic expression, therefore can judge indirectly that with Northern blotting, Westen blotting gene has or not sudden change.

In gene M MPRG1,2,3,4 healthy tissues that all are used in experiment, fetal tissue, the tumor tissues expression is arranged all, illustrate that it is the important mitochondrial membrane potential associated adjustment gene of human body self; Expression amount just has difference in different tissues, and the degree difference of its performance function in different tissues is described.

Below embodiment of the present invention are further described.

The present invention carries out the retrieval of people's Unknown Function predicted gene by the refseq database to NCBI, obtain people's unknown function gene order, further utilizing the Human_est database to carry out sequence by the BLASTn method proofreaies and correct, according to the sequences Design gene specific primer that obtains after proofreading and correct, from mix people's tissue cDNA library, obtain the coding region cDNA fragment of goal gene by the amplification of two-step approach flux RT-PCR technology.This coding region cDNA fragment and pcDT recombination to construct carrier for expression of eukaryon.The gene that utilizes the JC-1 dyestuff to detect among the present invention causes the function that mitochondrial membrane potential descends.JC-1 is a kind of cationic dyestuff, shows the membrane potential dependency and assemble in plastosome, can reflect delicately that the cell mitochondrial membrane potential changes situation.When the plastosome membrane potential does not reduce, JC-1 polymerization and be detected red emission light; When the plastosome membrane potential descended, JC-1 existed with monomeric form, was detected green emitted light.Therefore, JC-1 is applied to the detection that the early stage plastosome membrane potential of apoptosis descends as a kind of plastosome probe.The decline situation that can reflect the cell mitochondrial membrane potential by the ratio of the red green fluorescence of fluorescence microscope.And then detect the influence of selected gene pairs cell by fluidic cell experiment, wherein MMPRG1 and MMPRG2 obtain positive findings, and institute's transfectional cell is had apoptosis-induced effect.Experiment shows that polypeptide of the present invention has remarkable, the stable mitochondrial membrane potential decline effect that causes.

Owing to adopted above technical scheme, the present invention has following advantage:

1, provide mass-producing to clone and screen the technology platform of new gene;

2, human new functional gene MMPRG1,2,3,4 cDNA sequence and coded polypeptide thereof are provided;

3, find that first human new functional gene MMPRG1,2,3,4 has the effect that causes that mitochondrial membrane potential descends;

4, MMPRG1,2,3,4 expresses at the most normal cells of body, illustrates that it is the relevant regulatory molecule of self important mitochondrial membrane potential.

5, based on 4 above-mentioned advantages, the present invention is the apoptosis mechanism of further studying, and exploitation treatment is established necessary base as the novel drugs of autoimmune disease such as acquired immune deficiency syndrome (AIDS), neural degeneration and tumour etc. for starting new clinical diagnosis, therapeutic evaluation and prognostic indicator.

Description of drawings

The structure synoptic diagram of Fig. 1, carrier for expression of eukaryon pcDT-MMPRGx (MMPRGx is selected from MMPRG1,2,3,4);

The influence (JC-1 dyeing) of Fig. 2, MMPRG2, the survival of 4 heterogenous expression pair cells;

The fluoroscopic examination result that Fig. 3, MMPRG1,2,3,4 heterogenous expressions influence the plastosome membrane potential;

The influence (flow cytometry experiment) of Fig. 4, MMPRG2, the survival of 4 heterogenous expression pair cells.

Embodiment

Below in conjunction with specific embodiment, further set forth the present invention.These embodiment only are used to the present invention is described and are not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, condition described in " molecular cloning experiment guide " (chopsticks such as the third edition [U.S.] Sa nurse Brooker in 2002, Science Press), or the condition of advising according to manufacturer.

Embodiment 1, two-step approach flux RT-PCR technology amplifying target genes

(1) the refseq database to NCBI carries out the retrieval of people's Unknown Function predicted gene, obtain people's unknown function gene order, and utilize the Human_est database to carry out sequence by the BLASTn method and proofread and correct, the sequence that finally obtains is set at down the group sequence: SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7.According to this type of sequences Design gene M MPRG1,2,3,4 special primer:

(2) use above-mentioned primer, in existing cDNA library and tumour library, select template, carry out just expanding by the express spectra of goal gene.Existing library comprises 12 kinds of human normal tissues (heart, pancreas, testis, ovary, prostate gland, colon, small intestine, skeletal muscle, thymus gland, lymphoglandula, tonsilla, white corpuscle); 6 kinds of people's tumor tissues (lung cancer, carcinoma of the pancreas, ovarian cancer, prostate cancer, colorectal carcinoma, mammary cancer); With the cDNA library of 8 kinds of fetuses group long-pending (tire lung, fetal rhythm, tire liver, tire spleen, tire kidney, tire brain, tire skeletal muscle, tire thymus gland) (Clonetch, K1420-1,1241-1).It is as follows just to expand reaction conditions:

50 μ l PCR reaction:

PCR extends the long segment of time according to the expansion goal gene, increases by the principle of 50sec/Kb:

94 ℃ 5 minutes; 94 ℃ 30 seconds, 64 ℃ 30 seconds, 72 ℃ of 50sec/Kb; 72 ℃ 10 minutes; 28 circulations of 4 ℃ of ∞

The thing of just expanding production is purified to 30 μ l, with primers a large amount of in the removal PCR reaction system and dNTPs etc., and concentrates whole system, obtains the secondary amplification bank of corresponding target gene sequences, as two templates that expand (the big expansion).

(3) with the purified product in (2) as template, respectively each goal gene is carried out two expansions, reaction conditions is as follows:

50 μ l PCR (each gene) reaction:

PCR extends the long segment of time according to the expansion goal gene, increases by the principle of 50sec/Kb:

94 ℃ 5 minutes; 94 ℃ 30 seconds, 64 ℃ 30 seconds, 72 ℃ of 50sec/Kb; 72 ℃ 10 minutes; 30 circulations of 4 ℃ of ∞

The PCR product that obtains is got sample electrophoresis on the 10 μ l, selects the PCR product of amplified band, carries out equal-volume purifying (40 μ l).The gene that amplifies non-single band by the two-step pcr reaction reclaims test kit with Qiagen glue and cuts glue recovery purpose fragment.The result of amplification shows, all have gene M MPRG1,2,3,4 cDNA in the cell of these tissues, illustrates that MMPRG1,2,3,4 has produced gene M MPRG1,2,3,4 transcription product in the cell of these tissues, have wider expression map.

Embodiment 2, goal gene Construction of eukaryotic

With two expansion purified product and carrier for expression of eukaryon pcDNA3.1/V5-His-TOPO (Invitrogen is abbreviated as pcDT), carry out ligation according to the condition of test kit manufacturer suggestion.Connect product electric shocking method transformed into escherichia coli DH5 α, conversion product is grown containing on the solid LB plate culture medium of penbritin, select the monospecific polyclonal bacterium colony of growth, extract plasmid, cut with the EcoRI enzyme, enzyme is cut product and is identified with agarose gel electrophoresis, has selected and has inserted segmental positive colony, select correct forward by order-checking (ABI PRISM 3700DNA analyser) and insert clone, called after MMPRG1,2,3,4 separately.

Collect nutrient solution simultaneously, analyze protein precipitation, obtain MMPRG1,2,3,4 polypeptide with SDS-PAGE.

MMPRG1,2,3,4 analysis of protein results show: MMPRG1,2,3, following group of sequence of 4 protein sequences: shown in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, the SEQ ID NO:8.

Embodiment 3, utilize the JC-1 dyestuff to measure the effect that goal gene causes that mitochondrial membrane potential descends

Behind goal gene transfection Hela cell, utilize JC-1 dyestuff pair cell to dye, operate the variation of observation of cell form under fluorescent microscope by product description is described.The concrete operations step is as follows:

This experiment is operated with 96 porocyte culture plates, and each gene to be checked is provided with 3 parallel repeating holes, respectively with pcDT empty plasmid and Bax plasmid as empty carrier and positive control.Consumption in the following step is the single hole consumption.

(1) cell cultures: with 1.2 * 10 ⁴Individual Hela cell (ATCC Number:CRL-11268) DMEM (Dulbecco ' s modified Eagle ' s medium) substratum (Hyclone that contains 10% foetal calf serum, SH0022.02) be layered on 96 porocyte culture plate (Costar, 3599) go up (100 μ l nutrient solution/hole), put 37 ℃, 5%CO ₂Cell culture incubator (SANYO cultivates in MCO-15AC), gets the cell that is in logarithmic phase and carries out all experiments.

(2) preparation transfection working fluid: with 2.5 μ l physiological saline dilution 160ng gene to be checked, mixing gently, room temperature placement; Dilute 0.04 μ l VigoFect transfection reagent (prestige lattice Lars biotechnology (Beijing) company limited) with 2.5 μ l physiological saline equally, mixing gently, room temperature was placed 5 minutes; The VigoFect transfection reagent of dilution dropwise is added in the cdna solution to be checked of dilution, and mixing is to be the transfection working fluid gently, and room temperature was placed 15 minutes.

(3) transfection: with transfection working fluid mixing gently, dropwise be added in the 96 porocyte culture plates of completing cell in (1), mixing is put 37 ℃, 5%CO gently ₂Cultivated 24-28 hour in the cell culture incubator.

(4) microscopy: cell is placed observation phenotypic alternation under the fluorescent microscope (Olympus IX 70).The result is as shown in Figure 2: transfection the cellular form of coding MMPRG2 and MMPRG4 expression vector tangible change has taken place.Similar to positive control BAX group, see under the mirror that typical apoptosis changes, as nuclear shrinkage, apoptotic body form, attached cell breaks away from culturing bottle wall etc., and transfection the negative control of empty carrier these changes do not take place.Show that these 2 new genes are relevant with apoptosis.

(5) dyeing: with the JC-1 dyestuff of serum free medium dilution (final concentration is 10 μ g/ml, Molecular Probes company, M34152) pair cell dyes, and hatches to inhale after 30 minutes for 37 ℃ to abandon dyestuff, twice of PBS washing.

(6) detect: (Eppendorf Centrifuge 5810R) centrifugal 5 minutes, abandons supernatant with 800 rev/mins with the cell of 96 orifice plates.Every hole adds 40 μ l cell pyrolysis liquids, places-80 ℃ of refrigerators more than 1 hour 96 orifice plates behind the mixing.Return to room temperature after 96 orifice plates are thawed from-80 ℃, every hole is drawn 10 μ l cell pyrolysis liquids and is moved to white enzyme plate, and (Genios Pro Tecan) detects with the microwell plate microplate reader.Red fluorescence intensity value (RED) detects at the 590nm place, excitation wavelength 535nm.Green fluorescence intensity value (GREEN) detects at the 535nm place, excitation wavelength 484nm.Represent cell mitochondrial membrane potential decline situation with RED/GREEN.Positive control is the expression vector of coding BAX.BAX is the Bcl-2 family member, and when cell was subjected to inducing of antiapoptotic factors, BAX can transposition form transmembrane channel in plastosome and by oligomerization, causes mitochondrial membrane potential to descend, and cytochrome C discharges, and starts apoptotic pathways.Negative control is the pcDT empty carrier.The standard of positive gene is: wait to sieve gene RED/GREEN value and be lower than negative control RED/GREEN value more than 25%.The result as shown in Figure 3, MMPRG1,2,3,4 cross to express in the HeLa cell and can cause that the plastosome membrane potential reduces.

Embodiment 4, fluidic cell experiment

Behind the independent transfection Hela of goal gene cell, utilize flow cytometer to detect the function of the cell death inducing of plasmid to be checked.The concrete operations step is as follows:

(1) cell cultures: with Hela cell (1 * 10 ⁵) be layered on the 24 porocyte culture plates (Costar, 3599) with the DMEM substratum that contains 10% foetal calf serum, put 37 ℃, 5%CO ₂Cell culture incubator in cultivated 16 hours.

(2) preparation transfection working fluid: with 8 μ l physiological saline dilution 500ng plasmid to be checked, mixing gently, room temperature placement; Dilute 0.16 μ l VigoFect transfection reagent with 8 μ l physiological saline equally, mixing gently, room temperature was placed 5 minutes; The VigoFect transfection reagent of dilution dropwise is added in the plasmid solution to be checked of dilution, mixing gently, room temperature was placed 15 minutes.

(3) transfection: with transfection working fluid mixing gently, dropwise be added in the Tissue Culture Plate (500 μ l nutrient solution/hole), mixing is put 37 ℃, 5%CO gently ₂Cultivated 24-48 hour in the incubator.

(4) preparation single cell suspension, flow cytometer detects

24 hours harvested cells after the transfection.The sucking-off substratum, add 100 μ l pancreatin (Hyclone, SH30042.01), 37 ℃ of digestion 3 minutes adds 200 μ l again and contains in the DMEM substratum of 10% foetal calf serum and pancreatin, pressure-vaccum makes formation unicellular for several times, move into streaming pipe (FALCON, 352052), 1500 rpms of (Eppendorf Centrifuge, 5810R) room temperature is centrifugal 5 minutes, supernatant discarded.Add 3ml PBS re-suspended cell, centrifugal 5 minutes of 1500 rpms of room temperatures, supernatant discarded.Repeat above-mentionedly to wash cell once, and be resuspended in 200 μ l Binding buffer (10mM Hepes, 140mM NaCl, 1mM MgCl ₂, 5mM KCl, 2.5mM CaCl ₂), add final concentration and be 1 μ g/mlFITC-Annexin-V (Beijing Bao Sai Bioisystech Co., Ltd, CX1001-2), room temperature lucifuge reaction 30 minutes, add final concentration again and be 1 μ g/ml PI (Beijing Bao Sai Bioisystech Co., Ltd, CX1001-2).Detection use flow cytometer (FACSCalibur, BD, USA).The Annexin-V green-emitting fluorescence of FITC mark can specificity be incorporated into the distinctive phosphatidylserine that turns up outside cytolemma of apoptotic cell, and PI can penetrate the cytolemma of dead cell, and then nucleus is dyed redness.The cell of Annexin-V and PI jack to jack adapter is viable cell, and all the other are dead cell.

The result as shown in Figure 4, MMPRG2,4 and empty carrier positive findings is more all arranged, similar to positive control Bax effect, just two gene action degree varies illustrate MMPRG2,4 cell death inducing in varying degrees.

Embodiment 5, Antibody Preparation

Antigen is selected MMPRG1,2,3,4 albumen total lengths or the partial peptide section of prokaryotic cell prokaryocyte or eukaryotic cell expression for use, also can synthesize polypeptide as antigen.

Polyclonal Antibody Preparation: immune animal is selected bull new zealand rabbit or BALb/c mouse for use, initial immunity with 200 μ g (new zealand rabbit) or 20 μ g (BALb/c mouse) antigen and equal-volume Freund's complete adjuvant (FCA) fully emulsified after, the subcutaneous multi-point injection in the back.Behind the initial immunity 21,42,63 days, with Freund's incomplete adjuvant (FIA) emulsive antigen protein fully, each booster immunization 1 time, consumption was the same.Each immunity back 7～10 days, the ELISA method detects serum titer, reaches 1 * 10 ^-4The time, the bloodletting separation of serum.Western blot identifies antibodies specific.

Monoclonal Antibody: immune BALb/c mouse is the same, gets spleen and makes the B cell suspension, with the myeloma cell SP2/0 fusion of logarithmic phase, by HAT (H: xanthoglobulin; A: aminopterin-induced syndrome; T: thymidine) selectivity is cultivated, and obtains hybrid cell line, detects antibody titer by the ELISA method again, filters out specific hybridoma cell line, and obtains monoclonal antibody.

Sequence table

＜110〉Sinogenomax Co., Ltd.

＜120〉relevant polynucleotide and coded polypeptide and the purposes of mitochondrial membrane potential decline

<130>GAI09CN1924CZ-1

<160>8

<170>PatentIn?version?3.3

<210>1

<211>2666

<212>DNA

＜213〉people

<220>

<221>CDS

<222>(245)..(1093)

<400>1

ggcacgaggg?tcacctggga?agagtctgca?gagcctttgc?ccgccagcgc?cttcgctctt 60

tggctccctg?agttagtccg?gttgtttgcg?atcgccgcgg?ccggggctgc?gaaccgaagg 120

gctcgctccg?cgccgcctgg?gtctctacct?catccgtagg?tgtggccctg?atggtgtggc 180

aggctctgga?ctcctaaagc?tctggagcga?atttaagatt?ttattcatgt?gcatggcata 240

gaag?atg?aat?tct?tcc?act?tcc?acc?atg?agt?gaa?gag?cct?gac?gct?cta 289

Met?Asn?Ser?Ser?Thr?Ser?Thr?Met?Ser?Glu?Glu?Pro?Asp?Ala?Leu

1 5 10 15

tcg?gta?gtt?aac?cag?tta?cgg?gat?cta?gca?gca?gat?ccg?tta?aac?aga 337

Ser?Val?Val?Asn?Gln?Leu?Arg?Asp?Leu?Ala?Ala?Asp?Pro?Leu?Asn?Arg

20 25 30

aga?gcc?atc?gtc?cag?gat?cag?gga?tgt?ctg?cct?ggc?ctt?att?tta?ttt 385

Arg?Ala?Ile?Val?Gln?Asp?Gln?Gly?Cys?Leu?Pro?Gly?Leu?Ile?Leu?Phe

35 40 45

atg?gac?cat?ccc?aac?cct?cca?gtc?gtc?cac?tcc?gct?ttg?ctt?gct?ctt 433

Met?Asp?His?Pro?Asn?Pro?Pro?Val?Val?His?Ser?Ala?Leu?Leu?Ala?Leu

50 55 60

cga?tac?ttg?gca?gaa?tgc?cgt?gca?aac?aga?gaa?aag?atg?aaa?gga?gaa 481

Arg?Tyr?Leu?Ala?Glu?Cys?Arg?Ala?Asn?Arg?Glu?Lys?Met?Lys?Gly?Glu

65 70 75

ctg?ggt?atg?atg?ttg?agc?tta?caa?aat?gtt?ata?cag?aaa?act?aca?act 529

Leu?Gly?Met?Met?Leu?Ser?Leu?Gln?Asn?Val?Ile?Gln?Lys?Thr?Thr?Thr

80 85 90 95

cca?gga?gaa?aca?aaa?ctt?ctg?gcc?tct?gaa?atc?tat?gac?att?ctt?cag 577

Pro?Gly?Glu?Thr?Lys?Leu?Leu?Ala?Ser?Glu?Ile?Tyr?Asp?Ile?Leu?Gln

100 105 110

tcc?tcc?aat?atg?gca?gat?ggt?gat?agt?ttt?aat?gag?atg?aat?tca?cgt 625

Ser?Ser?Asn?Met?Ala?Asp?Gly?Asp?Ser?Phe?Asn?Glu?Met?Asn?Ser?Arg

115 120 125

cga?agg?aaa?gct?caa?ttt?ttt?ctg?gga?act?aca?aac?aaa?cgt?gcc?aaa 673

Arg?Arg?Lys?Ala?Gln?Phe?Phe?Leu?Gly?Thr?Thr?Asn?Lys?Arg?Ala?Lys

130 135 140

aca?gtg?gtt?ttg?cat?ata?gat?ggc?ctt?gat?gat?acg?tct?cgg?aga?aat 721

Thr?Val?Val?Leu?His?Ile?Asp?Gly?Leu?Asp?Asp?Thr?Ser?Arg?Arg?Asn

145 150 155

cta?tgt?gaa?gag?gct?ttg?tta?aaa?att?aaa?ggt?gtt?att?agc?ttt?act 769

Leu?Cys?Glu?Glu?Ala?Leu?Leu?Lys?Ile?Lys?Gly?Val?Ile?Ser?Phe?Thr

160 165 170 175

ttt?caa?atg?gct?gtt?caa?agg?tgt?gtg?gtg?cga?atc?cgt?tca?gat?ttg 817

Phe?Gln?Met?Ala?Val?Gln?Arg?Cys?Val?Val?Arg?Ile?Arg?Ser?Asp?Leu

180 185 190

aaa?gct?gag?gct?ttg?gca?tca?gca?ata?gca?tca?acc?aag?gtt?atg?aaa 865

Lys?Ala?Glu?Ala?Leu?Ala?Ser?Ala?Ile?Ala?Ser?Thr?Lys?Val?Met?Lys

195 200 205

gct?cag?caa?gtt?gtg?aaa?agt?gaa?agt?gga?gaa?gag?atg?ttg?gtc?cca 913

Ala?Gln?Gln?Val?Val?Lys?Ser?Glu?Ser?Gly?Glu?Glu?Met?Leu?Val?Pro

210 215 220

ttc?caa?gat?act?cct?gtg?gaa?gtt?gaa?cag?aac?aca?gag?cta?cct?gac 961

Phe?Gln?Asp?Thr?Pro?Val?Glu?Val?Glu?Gln?Asn?Thr?Glu?Leu?Pro?Asp

225 230 235

tac?ctg?cct?gag?gat?gag?agt?ccc?aca?aag?gaa?cag?gac?aaa?gcg?gtg 1009

Tyr?Leu?Pro?Glu?Asp?Glu?Ser?Pro?Thr?Lys?Glu?Gln?Asp?Lys?Ala?Val

240 245 250 255

tcc?cgg?gtc?ggc?tca?cac?cca?gaa?ggt?gga?gct?agc?tgg?ctt?agc?aca 1057

Ser?Arg?Val?Gly?Ser?His?Pro?Glu?Gly?Gly?Ala?Ser?Trp?Leu?Ser?Thr

260 265 270

gct?gca?aac?ttt?tta?tcc?aga?tca?ttt?tat?tgg?tga?cttcactttt 1103

Ala?Ala?Asn?Phe?Leu?Ser?Arg?Ser?Phe?Tyr?Trp

275 280

gggctcaagg?actgtgtgaa?ccaacaaggg?gccagttttc?cattgttgtg?gtgaactgtc 1163

aagtgcaatt?tgcaataagt?tatcatgaaa?agtttttaga?ttacacgatc?gcatatgctg 1223

catttcacat?tttattggac?attttacccc?actgagtggt?aaaaaggaca?gaggctacag 1283

atggagttgc?tttgtttatg?aaagtatttt?ggtttgtttt?ctttcattta?attgcctcat 1343

atttaaaaac?catgggtcca?ctgttaaaac?cacatgtgta?tgtgcagctt?tacattttat 1403

tttacgtgaa?gcatgtgatt?aggaaaactc?attttctttt?caagcctcag?gacctacctg 1463

aagagaagtt?ttcttgtagc?tcaagttgtg?catgaattac?tgaatatttt?actgtgcttt 1523

tcttcatgaa?gggtacatgc?tttgtactct?tcactgaaag?ctgaaaacat?ttcttgttac 1583

cctcttttgt?gcctttttat?tttgccaacc?gtgtttatag?aaaggacatt?actaatgaca 1643

ttttgcagat?taaaaacatt?catttgaaca?cagtagtccc?ctagaaaaac?aactctacaa 1703

aaattttgca?gccttattca?ttataatttt?gataaaatta?acacaaaatc?agtcaagaag 1763

gaaacatgta?tattagtgaa?gtgtttttgg?agactgtttg?aatgtgacca?aatgtggttc 1823

tagttgactt?cttttcactt?tggcttatat?caattcttga?gagttaatgt?gatcatgata 1883

ttgcaaacaa?ctataaatgg?tctctaggcc?ttactttgtg?attatacgtt?atctccggct 1943

agaaaaaaat?aatggtagta?aagaaactga?caaactgaaa?ataagaaaac?aaaaatcaaa 2003

tgcctataat?accataatgc?cagtttggta?tagagtccaa?ctttaaaaca?tgaattgctc 2063

gacagagttc?tattcagtag?gtgtttcttt?gtattgtctt?ttgtgaattt?attatgaaaa 2123

tgctgcgttg?tgttgaatga?aaaagaccca?aattactgct?tatgaagaaa?taaagccagc 2183

attgatcact?taatcctgtt?tctcatgtcc?agccagaaaa?aagaacttca?gtgaaggtaa 2243

gataaataaa?tacatacaca?tatgtttttt?tggtagataa?gtgctaatta?catatatgta 2303

atgctttatt?aaatttctga?aatatttggt?aactaaaatt?ttctttttgg?aaattaataa 2363

atccagatac?atattaatgt?tgatatgagt?aaaaacaaat?aggaagaaat?tgaaatttct 2423

tttcatcaac?atgtagagct?gctattttac?tatttggaga?atatgatgtg?aaaattggac 2483

ctcaaagggt?ttccttgtgt?tttcattgta?aaataccatc?atcagtgaga?gtcttgagtt 2543

cactaacatt?gtcaccttct?ggagagagag?ttaatggggg?gcattgagga?tgatattttt 2603

ttacatgtgt?ttggtttctg?attcaagtga?cacgcacaaa?ctgaaaaaaa?aaaaaaaaaa 2663

aaa 2666

<210>2

<211>282

<212>PRT

＜213〉people

<400>2

Met?Asn?Ser?Ser?Thr?Ser?Thr?Met?Ser?Glu?Glu?Pro?Asp?Ala?Leu?Ser

1 5 10 15

Val?Val?Asn?Gln?Leu?Arg?Asp?Leu?Ala?Ala?Asp?Pro?Leu?Asn?Arg?Arg

20 25 30

Ala?Ile?Val?Gln?Asp?Gln?Gly?Cys?Leu?Pro?Gly?Leu?Ile?Leu?Phe?Met

35 40 45

Asp?His?Pro?Asn?Pro?Pro?Val?Val?His?Ser?Ala?Leu?Leu?Ala?Leu?Arg

50 55 60

Tyr?Leu?Ala?Glu?Cys?Arg?Ala?Asn?Arg?Glu?Lys?Met?Lys?Gly?Glu?Leu

65 70 75 80

Gly?Met?Met?Leu?Ser?Leu?Gln?Asn?Val?Ile?Gln?Lys?Thr?Thr?Thr?Pro

85 90 95

Gly?Glu?Thr?Lys?Leu?Leu?Ala?Ser?Glu?Ile?Tyr?Asp?Ile?Leu?Gln?Ser

100 105 110

Ser?Asn?Met?Ala?Asp?Gly?Asp?Ser?Phe?Asn?Glu?Met?Asn?Ser?Arg?Arg

115 120 125

Arg?Lys?Ala?Gln?Phe?Phe?Leu?Gly?Thr?Thr?Asn?Lys?Arg?Ala?Lys?Thr

130 135 140

Val?Val?Leu?His?Ile?Asp?Gly?Leu?Asp?Asp?Thr?Ser?Arg?Arg?Asn?Leu

145 150 155 160

Cys?Glu?Glu?Ala?Leu?Leu?Lys?Ile?Lys?Gly?Val?Ile?Ser?Phe?Thr?Phe

165 170 175

Gln?Met?Ala?Val?Gln?Arg?Cys?Val?Val?Arg?Ile?Arg?Ser?Asp?Leu?Lys

180 185 190

Ala?Glu?Ala?Leu?Ala?Ser?Ala?Ile?Ala?Ser?Thr?Lys?Val?Met?Lys?Ala

195 200 205

Gln?Gln?Val?Val?Lys?Ser?Glu?Ser?Gly?Glu?Glu?Met?Leu?Val?Pro?Phe

210 215 220

Gln?Asp?Thr?Pro?Val?Glu?Val?Glu?Gln?Asn?Thr?Glu?Leu?Pro?Asp?Tyr

225 230 235 240

Leu?Pro?Glu?Asp?Glu?Ser?Pro?Thr?Lys?Glu?Gln?Asp?Lys?Ala?Val?Ser

245 250 255

Arg?Val?Gly?Ser?His?Pro?Glu?Gly?Gly?Ala?Ser?Trp?Leu?Ser?Thr?Ala

260 265 270

Ala?Asn?Phe?Leu?Ser?Arg?Ser?Phe?Tyr?Trp

275 280

<210>3

<211>1414

<212>DNA

＜213〉people

<220>

<221>CDS

<222>(105)..(1004)

<400>3

ggcacgaggc?agcaaaatgg?cgccagaact?agtggcgggc?tgaggacgcc?gtacccctcg 60

gaaggcagcc?ctgcggtccc?tttgccgccc?gttccctccc?ggac?atg?gag?gac?gtg 116

Met?Glu?Asp?Val

1

gag?gcg?cgc?ttc?gcc?cac?ctc?ttg?cag?ccc?atc?cgc?gac?ctc?acc?aag 164

Glu?Ala?Arg?Phe?Ala?His?Leu?Leu?Gln?Pro?Ile?Arg?Asp?Leu?Thr?Lys

5 10 15 20

aac?tgg?gag?gtg?gac?gtg?gcg?gcc?cag?ctg?ggc?gag?tat?ctg?gag?gag 212

Asn?Trp?Glu?Val?Asp?Val?Ala?Ala?Gln?Leu?Gly?Glu?Tyr?Leu?Glu?Glu

25 30 35

ctg?gat?cag?atc?tgc?att?tct?ttt?gac?gaa?ggc?aag?acc?aca?atg?aac 260

Leu?Asp?Gln?Ile?Cys?Ile?Ser?Phe?Asp?Glu?Gly?Lys?Thr?Thr?Met?Asn

40 45 50

ttc?att?gag?gca?gcg?ttg?ttg?atc?cag?ggc?tct?gcc?tgc?gtc?tac?agt 308

Phe?Ile?Glu?Ala?Ala?Leu?Leu?Ile?Gln?Gly?Ser?Ala?Cys?Val?Tyr?Ser

55 60 65

aag?aag?gtg?gaa?tac?ctc?tac?tca?ctc?gtc?tac?cag?gcc?ctt?gat?ttc 356

Lys?Lys?Val?Glu?Tyr?Leu?Tyr?Ser?Leu?Val?Tyr?Gln?Ala?Leu?Asp?Phe

70 75 80

atc?tct?gga?aag?agg?cgg?gcc?aag?cag?ctc?tct?tcg?gtg?cag?gag?gac 404

Ile?Ser?Gly?Lys?Arg?Arg?Ala?Lys?Gln?Leu?Ser?Ser?Val?Gln?Glu?Asp

85 90 95 100

agg?gcc?aat?ggg?gtt?gcc?agc?tcc?ggg?gtc?ccc?cag?gag?gca?gag?aat 452

Arg?Ala?Asn?Gly?Val?Ala?Ser?Ser?Gly?Val?Pro?Gln?Glu?Ala?Glu?Asn

105 110 115

gag?ttc?ctg?tcg?ctg?gat?gac?ttc?cct?gac?tcc?cgg?act?aac?gtg?gat 500

Glu?Phe?Leu?Ser?Leu?Asp?Asp?Phe?Pro?Asp?Ser?Arg?Thr?Asn?Val?Asp

120 125 130

ctc?aag?aat?gat?cag?acg?ccc?agt?gag?gtc?ctc?atc?atc?ccc?ctc?ctg 548

Leu?Lys?Asn?Asp?Gln?Thr?Pro?Ser?Glu?Val?Leu?Ile?Ile?Pro?Leu?Leu

135 140 145

ccc?atg?gcc?ctg?gtg?gcc?cct?gat?gaa?atg?gag?aag?aac?aac?aat?ccc 596

Pro?Met?Ala?Leu?Val?Ala?Pro?Asp?Glu?Met?Glu?Lys?Asn?Asn?Asn?Pro

150 155 160

ctg?tac?agc?cgt?cag?ggt?gag?gtc?ctg?gcc?agc?cgg?aag?gat?ttc?agg 644

Leu?Tyr?Ser?Arg?Gln?Gly?Glu?Val?Leu?Ala?Ser?Arg?Lys?Asp?Phe?Arg

165 170 175 180

atg?aac?acg?tgc?gtt?ccc?cac?ccc?aga?ggg?gcc?ttc?atg?ttg?gag?cca 692

Met?Asn?Thr?Cys?Val?Pro?His?Pro?Arg?Gly?Ala?Phe?Met?Leu?Glu?Pro

185 190 195

gag?ggc?atg?tcc?ccc?atg?gaa?cca?gcg?ggc?gtt?tcc?ccc?atg?cca?ggg 740

Glu?Gly?Met?Ser?Pro?Met?Glu?Pro?Ala?Gly?Val?Ser?Pro?Met?Pro?Gly

200 205 210

acc?cag?aag?gac?acc?ggg?agg?act?gag?gag?cag?cca?atg?gaa?gtt?tcc 788

Thr?Gln?Lys?Asp?Thr?Gly?Arg?Thr?Glu?Glu?Gln?Pro?Met?Glu?Val?Ser

215 220 225

gtg?tgc?agg?agc?cct?gtc?cca?gca?ctc?ggc?ttc?tcc?cag?gag?cca?ggc 836

Val?Cys?Arg?Ser?Pro?Val?Pro?Ala?Leu?Gly?Phe?Ser?Gln?Glu?Pro?Gly

230 235 240

ccc?tct?cca?gaa?ggc?ccg?atg?ccc?ctg?ggt?ggg?ggc?gag?gac?gag?gat 884

Pro?Ser?Pro?Glu?Gly?Pro?Met?Pro?Leu?Gly?Gly?Gly?Glu?Asp?Glu?Asp

245 250 255 260

gca?gag?gag?gca?gta?gag?ctt?cct?gag?gcc?tcg?gcc?ccc?aag?gcc?gct 932

Ala?Glu?Glu?Ala?Val?Glu?Leu?Pro?Glu?Ala?Ser?Ala?Pro?Lys?Ala?Ala

265 270 275

ctg?gag?ccc?aag?gag?tcc?agg?agc?ccg?cag?cag?gtg?gga?ccc?aca?tgg 980

Leu?Glu?Pro?Lys?Glu?Ser?Arg?Ser?Pro?Gln?Gln?Val?Gly?Pro?Thr?Trp

280 285 290

agg?cct?gca?gaa?cct?gag?ctg?tga?actggcaacc?ctggctctgg?ggccgagtca 1034

Arg?Pro?Ala?Glu?Pro?Glu?Leu

295

ccttgcacaa?ggaggacagt?ggtatggcct?tggccccaga?ccactggtct?ggggcagaag 1094

cccacctgtc?ttgcagcccg?tcctgcaacc?agcccttttg?aagagcagct?tctgtgttcc 1154

tcccctctct?gagcagaact?gatgctcctc?agagtagtgg?gctggcgtcc?aaggatttga 1214

gccctgtcga?gctcacggca?acctgggatg?gccgccggtt?gccaaggcgc?ctctctgcag 1274

tcgggctggt?aggagggagt?gtctggaggc?cattgctgcc?tccctcaacc?cccggggtca 1334

actgtaccca?gcctagagcc?aagaaatcct?tcctttttat?tcattaaaac?aaaatcaacc 1394

tgaaaaaaaa?aaaaaaaaaa 1414

<210>4

<211>299

<212>PRT

＜213〉people

<400>4

Met?Glu?Asp?Val?Glu?Ala?Arg?Phe?Ala?His?Leu?Leu?Gln?Pro?Ile?Arg

1 5 10 15

Asp?Leu?Thr?Lys?Asn?Trp?Glu?Val?Asp?Val?Ala?Ala?Gln?Leu?Gly?Glu

20 25 30

Tyr?Leu?Glu?Glu?Leu?Asp?Gln?Ile?Cys?Ile?Ser?Phe?Asp?Glu?Gly?Lys

35 40 45

Thr?Thr?Met?Asn?Phe?Ile?Glu?Ala?Ala?Leu?Leu?Ile?Gln?Gly?Ser?Ala

50 55 60

Cys?Val?Tyr?Ser?Lys?Lys?Val?Glu?Tyr?Leu?Tyr?Ser?Leu?Val?Tyr?Gln

65 70 75 80

Ala?Leu?Asp?Phe?Ile?Ser?Gly?Lys?Arg?Arg?Ala?Lys?Gln?Leu?Ser?Ser

85 90 95

Val?Gln?Glu?Asp?Arg?Ala?Asn?Gly?Val?Ala?Ser?Ser?Gly?Val?Pro?Gln

100 105 110

Glu?Ala?Glu?Asn?Glu?Phe?Leu?Ser?Leu?Asp?Asp?Phe?Pro?Asp?Ser?Arg

115 120 125

Thr?Asn?Val?Asp?Leu?Lys?Asn?Asp?Gln?Thr?Pro?Ser?Glu?Val?Leu?Ile

130 135 140

Ile?Pro?Leu?Leu?Pro?Met?Ala?Leu?Val?Ala?Pro?Asp?Glu?Met?Glu?Lys

145 150 155 160

Asn?Asn?Asn?Pro?Leu?Tyr?Ser?Arg?Gln?Gly?Glu?Val?Leu?Ala?Ser?Arg

165 170 175

Lys?Asp?Phe?Arg?Met?Asn?Thr?Cys?Val?Pro?His?Pro?Arg?Gly?Ala?Phe

180 185 190

Met?Leu?Glu?Pro?Glu?Gly?Met?Ser?Pro?Met?Glu?Pro?Ala?Gly?Val?Ser

195 200 205

Pro?Met?Pro?Gly?Thr?Gln?Lys?Asp?Thr?Gly?Arg?Thr?Glu?Glu?Gln?Pro

210 215 220

Met?Glu?Val?Ser?Val?Cys?Arg?Ser?Pro?Val?Pro?Ala?Leu?Gly?Phe?Ser

225 230 235 240

Gln?Glu?Pro?Gly?Pro?Ser?Pro?Glu?Gly?Pro?Met?Pro?Leu?Gly?Gly?Gly

245 250 255

Glu?Asp?Glu?Asp?Ala?Glu?Glu?Ala?Val?Glu?Leu?Pro?Glu?Ala?Ser?Ala

260 265 270

Pro?Lys?Ala?Ala?Leu?Glu?Pro?Lys?Glu?Ser?Arg?Ser?Pro?Gln?Gln?Val

275 280 285

Gly?Pro?Thr?Trp?Arg?Pro?Ala?Glu?Pro?Glu?Leu

290 295

<210>5

<211>3452

<212>DNA

＜213〉people

<220>

<221>CDS

<222>(95)..(1624)

<400>5

gcacccgcca?gtgggggtgc?ctcaacttcc?gcgggcgttt?aaatagcagc?ctctctcccc 60

tcccaccggc?tgcctgctgt?ttcccgggga?gatc?atg?aaa?cga?ggt?cgc?ctt?ccc 115

Met?Lys?Arg?Gly?Arg?Leu?Pro

1 5

agc?agc?agt?gag?gat?tct?gac?gac?aat?ggc?agc?ctg?tca?act?act?tgg 163

Ser?Ser?Ser?Glu?Asp?Ser?Asp?Asp?Asn?Gly?Ser?Leu?Ser?Thr?Thr?Trp

10 15 20

tcc?cag?aat?tcc?cga?tcc?cag?cat?agg?aga?agc?tcc?tgc?tcc?aga?cat 211

Ser?Gln?Asn?Ser?Arg?Ser?Gln?His?Arg?Arg?Ser?Ser?Cys?Ser?Arg?His

25 30 35

gaa?gat?cga?aag?cct?tca?gag?gtg?ttt?agg?aca?gac?ctg?atc?act?gcc 259

Glu?Asp?Arg?Lys?Pro?Ser?Glu?Val?Phe?Arg?Thr?Asp?Leu?Ile?Thr?Ala

40 45 50 55

atg?aag?ttg?cat?gac?tcc?tac?cag?ctg?aat?ccg?gat?gag?tac?tat?gtg 307

Met?Lys?Leu?His?Asp?Ser?Tyr?Gln?Leu?Asn?Pro?Asp?Glu?Tyr?Tyr?Val

60 65 70

ttg?gca?gat?ccc?tgg?aga?cag?gaa?tgg?gag?aaa?ggg?gtc?cag?gtg?cct 355

Leu?Ala?Asp?Pro?Trp?Arg?Gln?Glu?Trp?Glu?Lys?Gly?Val?Gln?Val?Pro

75 80 85

gtg?agc?ccg?ggg?acc?atc?cct?cag?cct?gtg?gcc?agg?gtt?gtg?tct?gaa 403

Val?Ser?Pro?Gly?Thr?Ile?Pro?Gln?Pro?Val?Ala?Arg?Val?Val?Ser?Glu

90 95 100

gag?aaa?tcc?ctc?atg?ttc?atc?agg?ccc?aag?aag?tac?atc?gtg?tca?tca 451

Glu?Lys?Ser?Leu?Met?Phe?Ile?Arg?Pro?Lys?Lys?Tyr?Ile?Val?Ser?Ser

105 110 115

ggc?tct?gag?cct?ccc?gag?ttg?ggc?tat?gtg?gac?atc?cgg?acg?ctg?gct 499

Gly?Ser?Glu?Pro?Pro?Glu?Leu?Gly?Tyr?Val?Asp?Ile?Arg?Thr?Leu?Ala

120 125 130 135

gac?agc?gtg?tgt?cgc?tat?gac?ctc?aat?gac?atg?gat?gct?gca?tgg?ctg 547

Asp?Ser?Val?Cys?Arg?Tyr?Asp?Leu?Asn?Asp?Met?Asp?Ala?Ala?Trp?Leu

140 145 150

gaa?ctg?acc?aat?gaa?gaa?ttt?aag?gag?atg?gga?atg?cct?gaa?cta?gat 595

Glu?Leu?Thr?Asn?Glu?Glu?Phe?Lys?Glu?Met?Gly?Met?Pro?Glu?Leu?Asp

155 160 165

gaa?tac?acc?atg?gag?agg?gtc?cta?gag?gaa?ttt?gag?cag?cga?tgc?tac 643

Glu?Tyr?Thr?Met?Glu?Arg?Val?Leu?Glu?Glu?Phe?Glu?Gln?Arg?Cys?Tyr

170 175 180

gac?aat?atg?aat?cat?gcc?ata?gag?act?gag?gaa?ggc?ctg?ggg?atc?gaa 691

Asp?Asn?Met?Asn?His?Ala?Ile?Glu?Thr?Glu?Glu?Gly?Leu?Gly?Ile?Glu

185 190 195

tat?gat?gaa?gat?gtt?gtc?tgt?gat?gtc?tgc?cag?tct?cct?gat?ggt?gag 739

Tyr?Asp?Glu?Asp?Val?Val?Cys?Asp?Val?Cys?Gln?Ser?Pro?Asp?Gly?Glu

200 205 210 215

gac?ggc?aat?gag?atg?gtg?ttc?tgt?gac?aaa?tgc?aac?atc?tgt?gtg?cac 787

Asp?Gly?Asn?Glu?Met?Val?Phe?Cys?Asp?Lys?Cys?Asn?Ile?Cys?Val?His

220 225 230

cag?gcc?tgt?tat?gga?atc?ctc?aag?gta?cca?gag?ggc?agc?tgg?ctg?tgc 835

Gln?Ala?Cys?Tyr?Gly?Ile?Leu?Lys?Val?Pro?Glu?Gly?Ser?Trp?Leu?Cys

235 240 245

cgg?aca?tgt?gcc?ctg?ggg?gtt?cag?cca?aaa?tgt?ctg?ctg?tgt?ccg?aag 883

Arg?Thr?Cys?Ala?Leu?Gly?Val?Gln?Pro?Lys?Cys?Leu?Leu?Cys?Pro?Lys

250 255 260

aag?ggt?gga?gct?atg?aag?ccc?acc?cgt?agc?gga?acc?aag?tgg?gtc?cac 931

Lys?Gly?Gly?Ala?Met?Lys?Pro?Thr?Arg?Ser?Gly?Thr?Lys?Trp?Val?His

265 270 275

gtt?agc?tgt?gct?ctg?tgg?atc?cct?gag?gtg?agc?att?ggc?agc?cca?gag 979

Val?Ser?Cys?Ala?Leu?Trp?Ile?Pro?Glu?Val?Ser?Ile?Gly?Ser?Pro?Glu

280 285 290 295

aag?atg?gag?ccc?atc?acc?aag?gtg?tca?cac?att?ccc?agc?agc?cgg?tgg 1027

Lys?Met?Glu?Pro?Ile?Thr?Lys?Val?Ser?His?Ile?Pro?Ser?Ser?Arg?Trp

300 305 310

gcg?cta?gtg?tgc?agc?ctc?tgc?aat?gag?aag?ttt?ggg?gcc?tct?ata?cag 1075

Ala?Leu?Val?Cys?Ser?Leu?Cys?Asn?Glu?Lys?Phe?Gly?Ala?Ser?Ile?Gln

315 320 325

tgc?tct?gtg?aag?aac?tgc?cgc?aca?gcc?ttc?cat?gtg?acc?tgt?gct?ttt 1123

Cys?Ser?Val?Lys?Asn?Cys?Arg?Thr?Ala?Phe?His?Val?Thr?Cys?Ala?Phe

330 335 340

gac?cgg?ggc?ctg?gag?atg?aag?acc?atc?tta?gca?gag?aat?gat?gaa?gtc 1171

Asp?Arg?Gly?Leu?Glu?Met?Lys?Thr?Ile?Leu?Ala?Glu?Asn?Asp?Glu?Val

345 350 355

aag?ttc?aag?tcc?tat?tgc?cca?aag?cac?agc?tca?cat?agg?aaa?ccc?gag 1219

Lys?Phe?Lys?Ser?Tyr?Cys?Pro?Lys?His?Ser?Ser?His?Arg?Lys?Pro?Glu

360 365 370 375

gag?agt?ctt?ggc?aag?ggg?gct?gca?cag?gag?aat?ggg?gcc?cct?gag?tg 1267

Glu?Ser?Leu?Gly?Lys?Gly?Ala?Ala?Gln?Glu?Asn?Gly?Ala?Pro?Glu?Cys

380 385 390

tcc?ccc?cgg?aat?ccg?ctg?gag?ccc?ttt?gcc?agc?ctt?gag?cag?aac?cgg 1315

Ser?Pro?Arg?Asn?Pro?Leu?Glu?Pro?Phe?Ala?Ser?Leu?Glu?Gln?Asn?Arg

395 400 405

gag?gag?gcc?cac?cgg?gtg?agt?gtc?cgt?aag?cag?aag?ctg?cag?cag?ttg 1363

Glu?Glu?Ala?His?Arg?Val?Ser?Val?Arg?Lys?Gln?Lys?Leu?Gln?Gln?Leu

410 415 420

gag?gat?gag?ttc?tac?acc?ttc?gtc?aac?ctg?ctg?gat?gtt?gcc?agg?gct 1411

Glu?Asp?Glu?Phe?Tyr?Thr?Phe?Val?Asn?Leu?Leu?Asp?Val?Ala?Arg?Ala

425 430 435

ctg?cgg?ctg?cct?gag?gaa?gta?gtg?gat?ttc?ctg?tac?cag?tac?tgg?aag 1459

Leu?Arg?Leu?Pro?Glu?Glu?Val?Val?Asp?Phe?Leu?Tyr?Gln?Tyr?Trp?Lys

440 445 450 455

ttg?aag?agg?aag?gtc?aac?ttc?aac?aag?ccc?ctg?atc?acc?cca?aag?aaa 1507

Leu?Lys?Arg?Lys?Val?Asn?Phe?Asn?Lys?Pro?Leu?Ile?Thr?Pro?Lys?Lys

460 465 470

gat?gaa?gag?gac?aat?cta?gcc?aag?cgg?gag?cag?gat?gtc?tta?ttt?agg 1555

Asp?Glu?Glu?Asp?Asn?Leu?Ala?Lys?Arg?Glu?Gln?Asp?Val?Leu?Phe?Arg

475 480 485

agg?ctg?cag?ctg?ttc?acg?cac?ctg?cgg?cag?gac?ctg?gag?agg?gta?atg 1603

Arg?Leu?Gln?Leu?Phe?Thr?His?Leu?Arg?Gln?Asp?Leu?Glu?Arg?Val?Met

490 495 500

att?gac?act?gac?acc?tta?tag?tgacttagag?aagaagatgc?aaagaggcga 1654

Ile?Asp?Thr?Asp?Thr?Leu

505

acgctcgccc?agagcaagaa?atgatagcca?gtcatatact?ctcagaccct?tgtacacacc 1714

acagcatgag?gttgtgttgg?ttaaaaatat?ttatgggctg?gtaaactcat?tgtacatatg 1774

tgcaaaactg?ctactgagtg?gggagcttct?ttgtggtttt?ttttttttta?aaaacacttt 1834

cccattaatc?tttactgttt?tgtaagatca?aatggggtgt?gtcttcccca?cccccattcc 1894

ttcattctag?agctagagtg?aatgagcccc?aagaaaatga?cccaaggagt?tgactcagga 1954

tggtttacag?actgatttag?aaaaccagaa?cggatttcat?ttctaatgga?gggggccaga 2014

gatgggaaaa?tttcttgttc?agtccgggga?aacacaccta?ggtgctggtg?atgggcttat 2074

gaaggaagct?aagcacggct?gctcactggc?ccccactttg?tttcttgggt?aattcacagg 2134

ggaattccca?gtactgtcat?ggagcagagc?aggcagtggg?tgctgatgtg?tgtgcatgag 2194

ctgtatgtac?acatgcatat?atctgttaca?gaagatactc?ctggcagtga?ggtgctaagt 2254

catcactgag?gctgtgtgtg?tgtgtgtgtg?tgcgcgtgcc?cgtgtccatc?catgtctctg 2314

ttgtgtgtct?gtgtgcgggt?atgggtggga?ttcctggtgg?acaggggtgt?cagatctgtc 2374

tgaggagccc?cagtcatgct?cagcacgcta?cagatgtgtt?gtttgtcaca?ctgagattgc 2434

tgaatgtcgt?ggctgttggc?tgccgagcct?cagctgctgg?catttccttc?tgctgtttgc 2494

tgcttttgtg?cctcccccac?tttccatcac?ctctggagtc?ccgtctggac?gtcccttcct 2554

gctacaggaa?taatgaggcg?tgggctgcct?cccgctaggg?cctcctgctc?cctgtaggta 2614

gtttctggct?gaggcttgct?aattggggat?gcttcttaga?gcatcttcca?catcaactcc 2674

cctggctgct?ggctaccgat?taaattcatt?agtgtgaaag?aggtgggagt?gaggttttct 2734

ggcctgaagc?agtctgcact?gaaaggtacc?caagtggcct?gaaacagtgt?agggaaagac 2794

ctgggaaaca?ctggaccaaa?aaagcctgat?ctcatggaga?cctgcatggc?cctgttagag 2854

atggcgtaga?agtgaaagtc?ttaaagggag?cattagagat?ccttttaata?cacgactgag 2914

tgccagctta?tttgtgatgc?cccttcccag?accaggttag?gattcctggg?aaggccgcgg 2974

attccggccc?tggaagaggc?aggatcctgg?agcagttttg?tgaggctttt?gtgctcccat 3034

acgccccctg?gtggtgagtg?taaagaagac?tttgcctctc?acaactacat?gtatgtgtgg 3094

catttttgtt?agagatgaga?aaaggattga?gaaggataaa?ctggaatcct?ggtaagaagc 3154

ctttatgcca?gcccgacacc?tgctgtaatt?ggggtgcatg?agctatggag?tcagatagtt 3214

gttgggaggg?ggaggacaag?aagtctattg?tttggactgt?gtttgtctca?caatcaccac 3274

aaaataaaag?tgtagaaaat?gcttgtggtg?tactaactct?tttctgtact?taagttactc 3334

agattaacat?gcacttctaa?ttctaaacat?tttttttgga?gtcatcactg?tcgctgttta 3394

agtagaacaa?tgccaacgat?gtatgagaaa?cagggaataa?atcaggattt?gtgtgtgt 3452

<210>6

<211>509

<212>PRT

＜213〉people

<400>6

Met?Lys?Arg?Gly?Arg?Leu?Pro?Ser?Ser?Ser?Glu?Asp?Ser?Asp?Asp?Asn

1 5 10 15

Gly?Ser?Leu?Ser?Thr?Thr?Trp?Ser?Gln?Asn?Ser?Arg?Ser?Gln?His?Arg

20 25 30

Arg?Ser?Ser?Cys?Ser?Arg?His?Glu?Asp?Arg?Lys?Pro?Ser?Glu?Val?Phe

35 40 45

Arg?Thr?Asp?Leu?Ile?Thr?Ala?Met?Lys?Leu?His?Asp?Ser?Tyr?Gln?Leu

50 55 60

Asn?Pro?Asp?Glu?Tyr?Tyr?Val?Leu?Ala?Asp?Pro?Trp?Arg?Gln?Glu?Trp

65 70 75 80

Glu?Lys?Gly?Val?Gln?Val?Pro?Val?Ser?Pro?Gly?Thr?Ile?Pro?Gln?Pro

85 90 95

Val?Ala?Arg?Val?Val?Ser?Glu?Glu?Lys?Ser?Leu?Met?Phe?Ile?Arg?Pro

100 105 110

Lys?Lys?Tyr?Ile?Val?Ser?Ser?Gly?Ser?Glu?Pro?Pro?Glu?Leu?Gly?Tyr

115 120 125

Val?Asp?Ile?Arg?Thr?Leu?Ala?Asp?Ser?Val?Cys?Arg?Tyr?Asp?Leu?Asn

130 135 140

Asp?Met?Asp?Ala?Ala?Trp?Leu?Glu?Leu?Thr?Asn?Glu?Glu?Phe?Lys?Glu

145 150 155 160

Met?Gly?Met?Pro?Glu?Leu?Asp?Glu?Tyr?Thr?Met?Glu?Arg?Val?Leu?Glu

165 170 175

Glu?Phe?Glu?Gln?Arg?Cys?Tyr?Asp?Asn?Met?Asn?His?Ala?Ile?Glu?Thr

180 185 190

Glu?Glu?Gly?Leu?Gly?Ile?Glu?Tyr?Asp?Glu?Asp?Val?Val?Cys?Asp?Val

195 200 205

Cys?Gln?Ser?Pro?Asp?Gly?Glu?Asp?Gly?Asn?Glu?Met?Val?Phe?Cys?Asp

210 215 220

Lys?Cys?Asn?Ile?Cys?Val?His?Gln?Ala?Cys?Tyr?Gly?Ile?Leu?Lys?Val

225 230 235 240

Pro?Glu?Gly?Ser?Trp?Leu?Cys?Arg?Thr?Cys?Ala?Leu?Gly?Val?Gln?Pro

245 250 255

Lys?Cys?Leu?Leu?Cys?Pro?Lys?Lys?Gly?Gly?Ala?Met?Lys?Pro?Thr?Arg

260 265 270

Ser?Gly?Thr?Lys?Trp?Val?His?Val?Ser?Cys?Ala?Leu?Trp?Ile?Pro?Glu

275 280 285

Val?Ser?Ile?Gly?Ser?Pro?Glu?Lys?Met?Glu?Pro?Ile?Thr?Lys?Val?Ser

290 295 300

His?Ile?Pro?Ser?Ser?Arg?Trp?Ala?Leu?Val?Cys?Ser?Leu?Cys?Asn?Glu

305 310 315 320

Lys?Phe?Gly?Ala?Ser?Ile?Gln?Cys?Ser?Val?Lys?Asn?Cys?Arg?Thr?Ala

325 330 335

Phe?His?Val?Thr?Cys?Ala?Phe?Asp?Arg?Gly?Leu?Glu?Met?Lys?Thr?Ile

340 345 350

Leu?Ala?Glu?Asn?Asp?Glu?Val?Lys?Phe?Lys?Ser?Tyr?Cys?Pro?Lys?His

355 360 365

Ser?Ser?His?Arg?Lys?Pro?Glu?Glu?Ser?Leu?Gly?Lys?Gly?Ala?Ala?Gln

370 375 380

Glu?Asn?Gly?Ala?Pro?Glu?Cys?Ser?Pro?Arg?Asn?Pro?Leu?Glu?Pro?Phe

385 390 395 400

Ala?Ser?Leu?Glu?Gln?Asn?Arg?Glu?Glu?Ala?His?Arg?Val?Ser?Val?Arg

405 410 415

Lys?Gln?Lys?Leu?Gln?Gln?Leu?Glu?Asp?Glu?Phe?Tyr?Thr?Phe?Val?Asn

420 425 430

Leu?Leu?Asp?Val?Ala?Arg?Ala?Leu?Arg?Leu?Pro?Glu?Glu?Val?Val?Asp

435 440 445

Phe?Leu?Tyr?Gln?Tyr?Trp?Lys?Leu?Lys?Arg?Lys?Val?Asn?Phe?Asn?Lys

450 455 460

Pro?Leu?Ile?Thr?Pro?Lys?Lys?Asp?Glu?Glu?Asp?Asn?Leu?Ala?Lys?Arg

465 470 475 480

Glu?Gln?Asp?Val?Leu?Phe?Arg?Arg?Leu?Gln?Leu?Phe?Thr?His?Leu?Arg

485 490 495

Gln?Asp?Leu?Glu?Arg?Val?Met?Ile?Asp?Thr?Asp?Thr?Leu

500 505

<210>7

<211>1656

<212>DNA

＜213〉people

<220>

<221>CDS

<222>(34)..(639)

<400>7

ggcacgaggc?ccgggccagg?ggcgggcgcc?gcc?atg?ggt?aac?ctg?ttc?ggc?cgc 54

Met?Gly?Asn?Leu?Phe?Gly?Arg

1 5

aag?aag?cag?agc?cgc?gtc?acg?gag?cag?gac?aag?gcc?atc?ctg?caa?ctg 102

Lys?Lys?Gln?Ser?Arg?Val?Thr?Glu?Gln?Asp?Lys?Ala?Ile?Leu?Gln?Leu

10 15 20

aag?cag?cag?cgg?gac?aag?ctg?agg?cag?tac?cag?aag?agg?atc?gcc?cag 150

Lys?Gln?Gln?Arg?Asp?Lys?Leu?Arg?Gln?Tyr?Gln?Lys?Arg?Ile?Ala?Gln

25 30 35

cag?ctg?gag?cgc?gag?cgc?gcc?ctg?gcc?cgg?cag?ctg?ctg?cgg?gac?ggc 198

Gln?Leu?Glu?Arg?Glu?Arg?Ala?Leu?Ala?Arg?Gln?Leu?Leu?Arg?Asp?Gly

40 45 50 55

agg?aag?gaa?cgg?gcc?aag?ctg?ctg?ctc?aag?aag?aag?cga?tac?cag?gag 246

Arg?Lys?Glu?Arg?Ala?Lys?Leu?Leu?Leu?Lys?Lys?Lys?Arg?Tyr?Gln?Glu

60 65 70

cag?ctc?ctg?gac?agg?acg?gag?aac?cag?atc?agc?agc?ctg?gag?gcc?atg 294

Gln?Leu?Leu?Asp?Arg?Thr?Glu?Asn?Gln?Ile?Ser?Ser?Leu?Glu?Ala?Met

75 80 85

gtt?cag?agt?att?gag?ttc?acc?cag?atc?gaa?atg?aaa?gtg?atg?gag?ggg 342

Val?Gln?Ser?Ile?Glu?Phe?Thr?Gln?Ile?Glu?Met?Lys?Val?Met?Glu?Gly

90 95 100

ctg?cag?ttt?gga?aat?gag?tgt?ctg?aac?aag?atg?cac?cag?gtg?atg?tcc 390

Leu?Gln?Phe?Gly?Asn?Glu?Cys?Leu?Asn?Lys?Met?His?Gln?Val?Met?Ser

105 110 115

att?gaa?gag?gtg?gag?agg?atc?ctg?gac?gag?acg?cag?gag?gcc?gtg?gag 438

Ile?Glu?Glu?Val?Glu?Arg?Ile?Leu?Asp?Glu?Thr?Gln?Glu?Ala?Val?Glu

120 125 130 135

tac?cag?cgg?caa?ata?gac?gag?ctc?ctg?gca?gga?agc?ttc?act?cag?gag 486

Tyr?Gln?Arg?Gln?Ile?Asp?Glu?Leu?Leu?Ala?Gly?Ser?Phe?Thr?Gln?Glu

140 145 150

gat?gaa?gac?gcc?atc?ctg?gag?gag?ctg?agc?gca?atc?act?cag?gaa?caa 534

Asp?Glu?Asp?Ala?Ile?Leu?Glu?Glu?Leu?Ser?Ala?Ile?Thr?Gln?Glu?Gln

155 160 165

ata?gag?ctg?cca?gag?gtt?ccc?tcc?gag?ccc?ctt?cct?gag?aag?atc?cca 582

Ile?Glu?Leu?Pro?Glu?Val?Pro?Ser?Glu?Pro?Leu?Pro?Glu?Lys?Ile?Pro

170 175 180

gaa?aac?gtc?cct?gtc?aag?gcc?agg?ccc?agg?cag?gcg?gag?ctg?gtg?gca 630

Glu?Asn?Val?Pro?Val?Lys?Ala?Arg?Pro?Arg?Gln?Ala?Glu?Leu?Val?Ala

185 190 195

gct?tcg?taa?cgtggcctcg?tcttgtggga?ctcacgggga?tgccccaggg 679

Ala?Ser

200

actgtggccc?acagagagtt?tgggtcacgg?ccagcccctg?accgggttcc?ctggagccca 739

gtgcgcacgg?tgctgagcag?agctgcagcc?acgcaggcgc?attgcaggag?gactccagag 799

cgtctcctgg?agaccttgag?cctgaacgca?ctcaggcgcc?actggcctgc?tctcagtccg 859

gattaactct?cgaccgagcc?cagcttctgc?cggttgtggg?ctcccccggt?ggccgaggcc 919

caggcccaac?gcctctggtg?ctgttcccct?gcagtcccag?ccccgcgtgg?ctcgcgctcg 979

tctgtgagga?agacacctcc?agaccttggg?gtccccgcgc?ttcctcttgc?tcctcgctgc 1039

tcccattagc?tggtgcaggc?ttccgttaag?gggtccctcc?cttggcctgg?cttcccggcg 1099

cacctcagct?tccctgctgg?tggggggatc?cccaggagac?cagcatgtgc?tgaacctctc 1159

cgtgcctctg?cctccgcacc?ctagacaccc?acctccagtt?tgaaggtggc?gggccagggg 1219

ctttcttgct?gaattgacga?ctccgagagc?cctgactccc?gccttgccac?tcacggctct 1279

gtccactagg?gctcagccct?gctgagaaag?gacctccgat?gcttgggaga?cgctgctccg 1339

gcaggtgcag?ccccggaagt?ttgtccatgg?gggtccccgc?ggctggggct?catggaactg 1399

cgagacccgg?gaccctcctg?ccctgcgggt?ccccgagcca?ccagcagcca?ggaccggagg 1459

ctgtggggca?tggcgtgact?tctcgtgcac?agggctggtt?tggttatgag?acgatctcgc 1519

tgggaccgcc?cctgcccgtg?gaaagccaca?aggacaaagg?gagcggccac?ctcgaccccc 1579

agacagggcg?gcccgtatta?aagttggccc?tgcacgccca?aaaaaaaaaa?aaaaaaaaaa 1639

aaaaaaaaaa?aaaaaaa 1656

<210>8

<211>201

<212>PRT

＜213〉people

<400>8

Met?Gly?Asn?Leu?Phe?Gly?Arg?Lys?Lys?Gln?Ser?Arg?Val?Thr?Glu?Gln

1 5 10 15

Asp?Lys?Ala?Ile?Leu?Gln?Leu?Lys?Gln?Gln?Arg?Asp?Lys?Leu?Arg?Gln

20 25 30

Tyr?Gln?Lys?Arg?Ile?Ala?Gln?Gln?Leu?Glu?Arg?Glu?Arg?Ala?Leu?Ala

35 40 45

Arg?Gln?Leu?Leu?Arg?Asp?Gly?Arg?Lys?Glu?Arg?Ala?Lys?Leu?Leu?Leu

50 55 60

Lys?Lys?Lys?Arg?Tyr?Gln?Glu?Gln?Leu?Leu?Asp?Arg?Thr?Glu?Asn?Gln

65 70 75 80

Ile?Ser?Ser?Leu?Glu?Ala?Met?Val?Gln?Ser?Ile?Glu?Phe?Thr?Gln?Ile

85 90 95

Glu?Met?Lys?Val?Met?Glu?Gly?Leu?Gln?Phe?Gly?Asn?Glu?Cys?Leu?Asn

100 105 110

Lys?Met?His?Gln?Val?Met?Ser?Ile?Glu?Glu?Val?Glu?Arg?Ile?Leu?Asp

115 120 125

Glu?Thr?Gln?Glu?Ala?Val?Glu?Tyr?Gln?Arg?Gln?Ile?Asp?Glu?Leu?Leu

130 135 140

Ala?Gly?Ser?Phe?Thr?Gln?Glu?Asp?Glu?Asp?Ala?Ile?Leu?Glu?Glu?Leu

145 150 155 160

Ser?Ala?Ile?Thr?Gln?Glu?Gln?Ile?Glu?Leu?Pro?Glu?Val?Pro?Ser?Glu

165 170 175

Pro?Leu?Pro?Glu?Lys?Ile?Pro?Glu?Asn?Val?Pro?Val?Lys?Ala?Arg?Pro

180 185 190

Arg?Gln?Ala?Glu?Leu?Val?Ala?Ala?Ser

195 200

Claims (10)

1. isolating polynucleotide, these polynucleotide contain the nucleotide sequence that coding has the polypeptide that causes mitochondrial membrane potential decline function, and described nucleotide sequence is selected from:

(a) polynucleotide of polypeptide that contain the aminoacid sequence shown in the SEQ ID NO:8 with coding have the polynucleotide of at least 70% similarity;

(b) coding contains the polynucleotide of polypeptide that the aminoacid sequence of at least 70% similarity is arranged with the aminoacid sequence shown in the SEQ ID NO:8; Or

(c) with (a) or polynucleotide complementary polynucleotide (b).

2. polynucleotide as claimed in claim 1, the polypeptide of this polynucleotide encoding have the aminoacid sequence shown in the SEQ ID NO:8.

3. polynucleotide as claimed in claim 1, the sequence of these polynucleotide be selected from following nucleotides sequence and show at least 85% similarity:

(a) coding region sequence of SEQ ID NO:7 or full length sequence;

(b) at least one sequence of the sequence of in genetic code degeneracy scope, mentioning in corresponding to (a); Or

(c) with (a) or at least one sequence of the sequence complementary sequence hybridization of mentioning (b);

Preferably, the sequence of described polynucleotide is selected from coding region sequence or the full length sequence of SEQ ID NO:7.

4. the polypeptide of the described polynucleotide encoding of claim 1, this polypeptide comprises the polypeptide that is selected from down the aminoacid sequence in the group: SEQ ID NO:8, or have at least 90% similarity and have the polypeptide that causes mitochondrial membrane potential decline function, or its conservative property variation polypeptide or its active fragments or its reactive derivative with the aminoacid sequence of SEQ ID NO:8; Preferably, this polypeptide is the polypeptide that aminoacid sequence shown in the SEQ ID NO:8 is formed.

5. carrier, this carrier contains the described polynucleotide of claim 1.

6. genetically engineered host cell, this host cell is selected from:

(a) host cell that transforms or transduce with the described carrier of claim 5; Or

(b) host cell that transforms or transduce with the described polynucleotide of claim 1.

7. antibody, this antibody be can with the described polypeptid specificity bonded of claim 4 antibody.

8. nucleic acid fragment, this nucleic acid fragment contains 8-100 successive Nucleotide in the described polynucleotide of claim 1.

9. the described polynucleotide of claim 1 or its encoded polypeptides application in causing mitochondrial membrane potential decline;

Preferably, described polynucleotide heterogenous expression in host cell can cause that mitochondrial membrane potential descends; Preferred, described host cell is selected from the described host cell of claim 8;

Preferably, described application is to utilize described polynucleotide to express or produce the polypeptide that having of reorganization causes mitochondrial membrane potential decline function;

Preferably, described application is that described polynucleotide or its encoded polypeptides are used for diagnosing and/or treating owing to the people's protein abnormal expression with the function that causes mitochondrial membrane potential decline or the detection agent of the active disease that causes unusually or the application of pharmaceutical composition in preparation.

10. the pharmaceutical composition that contains described polypeptide of claim 4 and pharmaceutically acceptable carrier.

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