CN101892237B - 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 one type of new coding 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 type heterogenous expression in host cell causes that mitochondrial membrane potential descends.

Background technology

It is dead that apoptosis belongs to programmed cellization, 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 (like Lrax) 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 the entering of water and solute, matrix swelling also causes outer membrane rupture, discharges the various activated proteins that comprise Lrax.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, in plastosome, shows the membrane potential dependency and assembles, and 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.Based on the JC-1 dyestuff mitochondrial membrane potential inductor that descends and the model that retarding agent screens just were 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 to this present situation and existing medicine or reagent the purpose of this invention is to provide one type of new coding 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 type polynucleotide encoded polypeptide.

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

Another object of the present invention provides the antibody of this type 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 type 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 realizing above-mentioned purpose, the present invention adopts following technical scheme:

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

In 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.

Aspect the of the present invention the 5th, provide above-mentioned polynucleotide heterogenous expression in host cell to cause the application that mitochondrial membrane potential descends.

Aspect the of the present invention the 6th, 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 like 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 possibly be the parts of a certain carrier, the part that polynucleotide that also maybe be such or polypeptide are a certain compsn, since carrier or compsn 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 through the same or analogous percentage of part, this similarity per-cent can calculate through the existing comparison method in this area, and 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, sequence alignment with hierarchical clustering.Nucleic Acids Res. F.1998Multiple, 16:10881-10890) etc.Homologous sequence is meant the different sequences that form through divergent evolution from a certain common ancestor, can judge the homology between aligned sequences according to similarity per-cent.When similarity degree is very high between gene or protein, representes 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 possibly 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 confirm or can't confirm 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 verivate 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 possibly 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 according to the 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 LIS and the comparatively high temps and wash-out, like 0.2 * SSC, and 0.1%SDS, 60 ℃; Or (2) hybridization the time is added with denaturing agent, like 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 through hybridization technique DNA isolation sequence; (2) artificial chemical synthesising DNA sequence; (3) through 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, from genomic library or cDNA library, filters out goal gene or sequence through technology such as molecular hybridizations then.When the biological gene group was smaller, this method was prone to successfully; When the biological gene group is very big, make up difficulty of its complete genomic library, the quantities of from huge library, removing to clone goal gene again 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, like the refseq DB etc.; With Phred evaluation of algorithm 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 is with 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 like the DNA/RNA fragment through 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 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 from protokaryon or eucaryon host (like bacterium, yeast, higher plant, insect and mammalian cell), to produce.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, verivate and the analogue of the people's protein polypeptide with the polynucleotide encoding that causes mitochondrial membrane potential decline function.Term " fragment ", " verivate " 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, verivate 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 substituted like this amino-acid residue 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 (like 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 through 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) with polynucleotide of the present invention (or its varient), or with expression vector conversion that contains these polynucleotide or transduction proper host cell;

(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 according to the 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 that bacterial plasmid well known in the art, phage, yeast plasmid, vegetable cell virus, mammalian cell are viral, like adenovirus, retrovirus, and perhaps other carriers.The carrier that is suitable in the present invention can be a prokaryotic expression carrier, also can be carrier for expression of eukaryon, like the expression vector (Rosenberg based on T7 that in bacterium, expresses; 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 in host, duplicate 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 according to the invention and transcribe/translate wave 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, like bacterial cell; Or eukaryotic cell such as low, like yeast cell; Or higher eucaryotic cells, like 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 chemically induced, induce the promotor of selection, cell is cultivated for some time again.

Recombinant polypeptide in the aforesaid method can encapsulate 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 through various separation methods with other characteristics.These methods are well-known to those skilled in the art; Handle like the renaturation of routine, handle the combination of (salt analysis method), centrifugal, the broken bacterium of infiltration, ultrasonication, ultra centrifugal, sieve chromatography (gel-filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography and other various LCs 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 as primer) and have the polynucleotide that cause mitochondrial membrane potential decline function to confirm and/or to separate coding; Also can be used as the used probe of hybridization.Also can be used for the RNA perturbation technique.Polynucleotide of the present invention a part or all also can be used as probe stationary on microarray (Microarray) or DNA chip, be used for analyzing the differential expression and the gene diagnosis of tissue gene.The mark of probe can be used ri, resorcinolphthalein or enzyme (like SEAP) 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.Compsn 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 through 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, like administering mode, treat curer's healthiness condition and diagnostician's judgement.

Polypeptide of the present invention also can use through expressing these polypeptide at live body.For example patient's cell can carry out the genetically engineered operation through 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 various therapeutic purposes.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 (like 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 use this area existent method synthetic.In order to increase the stability of nucleic acid molecule, available several different methods is modified it, and as increasing the sequence length of both sides, the connection between ribonucleoside is with phosphoric acid thioester bond or peptide bond.

Polypeptide of the present invention and fragment thereof, verivate, 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 like rabbit, mouse, rat etc.Multiple adjuvant can be used for the enhancing immunity reaction, includes but not limited to freund's adjuvant.With the variable region bonded chimeric antibody in human constant region and inhuman source can use existing technology produce (Morrison et al.PNAS, 1985,81:6851).The also available existing technology of single-chain antibody is produced (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 to treat 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 have the acting disease of albumen that causes mitochondrial membrane potential decline function with being used to diagnose.

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 sources.Polynucleotide of the present invention a part or all can be used as probe stationary on microarray (Microarray) or DNA chip, be 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, explain that it is the important mitochondrial membrane potential associated adjustment gene of human body self; Expression amount just has difference in different tissues, explains that the degree of its performance function in different tissues is different.

Below to embodiment of the present invention further explain.

The present invention carries out the retrieval of people's Unknown Function predicted gene through the refseq DB to NCBI; Obtain people's unknown function gene order; Further utilizing the Human_est DB to carry out sequence through 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 through technological amplification of two-step approach flux RT-PCR.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, in plastosome, shows the membrane potential dependency and assembles, and 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 through the ratio of the red green fluorescence of fluorescence microscope.And then pass through the influence that the fluidic cell experiment detects selected gene pairs cell, 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, explains that it is the relevant regulatory molecule of self important mitochondrial membrane potential.

5, based on 4 above-mentioned advantages; The present invention is that further the research apoptosis is machine-processed; And exploitation treatment is established necessary base like the novel drugs of autoimmune disease such as 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 be used to the present invention is described and be not used in the restriction scope of the present 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.A] 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 DB 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 DB to carry out sequence through 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, Skelettmuskel, 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 Skelettmuskel, tire thymus gland) (Clonetch, K1420-1,1241-1).It is following 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 the template of two expansions (the big expansion).

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

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 appearance 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 through 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 these cells of tissues, explains that MMPRG1,2,3,4 has produced gene M MPRG1,2,3,4 transcription product in these cells of 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, selects 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 through 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: MMPRG1,2,3,4 protein sequences are organized sequence as follows: 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 said.The concrete operations step is following:

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) is with 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 saline water dilution 160ng gene to be checked, mixing gently, room temperature placement; Dilute 0.04 μ l VigoFect transfection reagent (prestige lattice Lars biotechnology (Beijing) ltd) with 2.5 μ l saline water 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 with positive control BAX group, see under the mirror that typical apoptotic 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 ELIASA.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 received inducing of antiapoptotic factors, BAX can transposition form transmembrane channel in plastosome and through oligomerization, causes mitochondrial membrane potential to descend, and Lrax 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 is as shown in Figure 3, and MMPRG1,2,3,4 crosses 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 following:

(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 saline water dilution 500ng plasmid to be checked, mixing gently, room temperature placement; Dilute 0.16 μ l VigoFect transfection reagent with 8 μ l saline water 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 make for several times form unicellular; 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 property is viable cell, and all the other are dead cell.

The result is as shown in Figure 4, MMPRG2,4 and empty carrier positive findings is more all arranged, similar with positive control Bax effect, just two gene action degree varies are explained 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 is 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 processes the B cell suspension, with the myeloma cell SP2/0 fusion of logarithmic phase, through HAT (H: xanthoglobulin; A: aminopterin-induced syndrome; T: thymidine) selectivity is cultivated, and obtains hybrid cell line, detects antibody titer through the ELISA method again, filters out specific hybridoma cell line, and obtains monoclonal antibody.

Sequence table

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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

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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

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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

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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

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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 (11)

1. isolating polynucleotide, this polynucleotide encoding has the polypeptide that causes mitochondrial membrane potential decline function, and the nucleotide sequence of these polynucleotide is selected from:

(a) polynucleotide of the polypeptide of aminoacid sequence shown in the coding SEQ ID NO:8; Or

(b) with (a) polynucleotide complementary polynucleotide.

2. polynucleotide as claimed in claim 1, the sequence of these polynucleotide are selected from coding region sequence or the full length sequence of SEQ ID NO:7.

3. the polypeptide of the described polynucleotide encoding of claim 1, this polypeptide are the polypeptide that aminoacid sequence shown in the SEQ ID NO:8 is formed.

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

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

(a) host cell that transforms or transduce with the described carrier of claim 4; Or

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

6. antibody, this antibody be can with the described polypeptid specificity bonded of claim 3 antibody.

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

8. application according to claim 7, wherein, said polynucleotide heterogenous expression in host cell can cause that mitochondrial membrane potential descends.

9. application according to claim 8, wherein, described host cell is selected from the described host cell of claim 5.

10. application according to claim 7 wherein is to utilize described polynucleotide to express or produce the polypeptide that having of reorganization causes mitochondrial membrane potential decline function.

11. contain the pharmaceutical composition of described polypeptide of claim 3 and pharmaceutically acceptable carrier.

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