CN1188508A - Genetic sequences and proteins related to alzheimer's disease, and uses therefor - Google Patents

Abstract

The present invention describes the identification, isolation and cloning of two human presenilin genes, PS-1 and PS-2, mutations in which lead to Familial Alzheimer's Disease. Also identified are presenilin homologue genes in mice, C. elegans and D. melanogaster. Transcripts and products of these genes are useful in detecting and diagnosing Alzheimer's disease, developing therapeutics for treatment of Alzheimer's disease, as well as the isolation and manufacture of the protein and the constructions of transgenic animals expressing the mutant genes.

Description

With the gene order of related to alzheimer's disease and albumen and uses thereof

Related application

The application is the application that continues of the U. S. application series 08/509359 of application on July 31 nineteen ninety-five, and 08/509359 be the application that continues of the U. S. application series 08/496841 of June 28 nineteen ninety-five application, the 08/431048th, the application that continues of the U. S. application series 08/431048 of 28 applications in April nineteen ninety-five, the exercise question of all applications is " with the gene order of related to alzheimer's disease and albumen and uses thereof " (contriver: Peter H St.George-Hyslop, Johanna M.Rommens and PaulE.Fraser), all applications all are incorporated herein by reference.

Invention field

The present invention relates generally to neurological and the physiologic function imbalance with related to alzheimer's disease.More particularly, the present invention relates to identify, separate and the gene of clone and related to alzheimer's disease with and transcript, gene product, relevant sequence data and genes involved.The invention still further relates to detect and diagnose these genes normal and mutation allele carrier method, relate to detect and the method for diagnosis Alzheimer's disease, relate to identify relevant with presenile dementia or with the gene and the proteic method of presenile dementia gene and protein-interacting, relate to screening potential Alzheimer's disease therapy method, relate to the method for the treatment of Alzheimer's disease, also relate to and be used for screening and assess treating effective clone of Alzheimer's disease and animal model.

Background of invention

Various for ease of reference journal of writings have been listed described article behind specification sheets of the present invention.

Alzheimer's disease (AD) is the decline sexual disorder of people's central nervous system, it is characterized in that between the middle age to senium, and the property of carrying out memory impairment is arranged, and understanding and dementia (Katzman, 1986).This disease wherein mainly is extracellular amyloid or senile plaques and the decline of neuronic nerve fiber to have occurred with the appearance of neuropathological feature.This sick cause of disease is very complicated, although in some family, it is with the heredity of autosomal dominant feature.But, even in these mode of inheritance of AD, at least three kinds of different genes being arranged, they are given this sick genetic sensitivity (St.George-Hyslop etc., 1990).In the evening of life in the case of Fa Zuo remarkable ratio, ε 4 (C112R) polymorphic allele also relevant (Saunders etc., 1993 of Ah pouncing on's lipoprotein (ApoE) gene with AD; Strittmatter etc., 1993).Equally, very family's case of the outbreak before 65 years old of small proportion also with sudden change relevant (Chartier-Harlin etc., 1991 of beta amyloid precursor protein (APP) gene; Goate etc., 1991; Murrell etc., 1991; Karlinsky etc., 1992; Mullan etc., 1992).Recently three seat (AD3) relevant with many early onset thereof AD cases is positioned at karyomit(e) 14q24.3 (Schellenberg etc., 1992; St.George-Hyslop etc., 1992; Van Broeckhoven etc., 1992).

Although karyomit(e) 14q has carried several genes in the district, described gene can be used as the candidate gene (cFOS for example in the mutational site relevant with AD3, alpha-1-antichymotrypsin analogues and cathepsin G), but according to its outside the AD3 district physical location and/or in its corresponding open reading frame, do not have sudden change to get rid of major part (Schellenberg etc., 1992 in these candidate genes; VanBroeckhoven etc., 1992; Rogaev etc., 1993; Wong etc., 1993).

Aspect the treatment and diagnosis of Alzheimer's disease, some researchs and business method or strategy have been arranged.Disclosed PCT application WO94/23049 has described high molecular YAC DNA transfection in the specificity mouse cell.With this methods analyst many gene binding substancess.For example, the amount of transgenic mice app gene may increase, simulated ubiquitous trisomy situation in down's syndrome like this, and can produce the animal model that has the beta amyloid change, this beta amyloid becomes ubiquity in suffering from the Alzheimer's disease individuality.Disclosed International Application No. WO 94 00569 has been described transgenic nonhuman animal, and they have a large amount of transgenosiss as containing the transgenosis of people's app gene.Described animal model can be used as the useful model of people's inherited disease such as Alzheimer's disease.

Canadian patent application 2096911 has been described the nucleic acid of coding APP-crack protein enzyme, and it is relevant with Alzheimer's disease and down's syndrome.Can use isolating genetic material diagnosis Alzheimer's disease from karyomit(e) 19.Canadian patent application 2071105 has been described by the YAC nucleotide sequence and has been detected and treated heredity or acquired Alzheimer's disease.Identified different YAC with 23CB10,28CA12 and 26FF3.

United States Patent (USP) 5297562 has been described the detection Alzheimer's disease relevant with the trisomy of karyomit(e) 21.Treatment comprises the method that reduces karyomit(e) trisomy 21 propagation.Canadian patent application 2054302 has been described the proteic monoclonal antibody of identification people brain nuclei, and described albumen is by karyomit(e) 21 coding, and described antibody is used to detect the expression that causes because of Alzheimer's disease or down's syndrome and changes.Described monoclonal antibody is for having specificity by human chromosome 21 encoded protein, and can find many pyramidal cells of human brain tissue.

Summary of the invention

The present invention part based on be called as the evaluation of early ageing element-1 (PS1), separate with two kinds of mammalian genes of early ageing element-2 (PS2), clone and order-checking.The gene family that these two genes and its corresponding proteins product are high conservatives, the member of early ageing element (presenilin), in other Mammals (as mouse, rat), homology or orthotropism are arranged, in invertebrates (as C.elegans, black-tailed fruit flies (D.melanogaster)) be directly to.Sudden change in these genes is relevant with the generation of familial Alzheimer's disease among the mankind, and may also be the cause of disease of other disease (as other understanding, intelligence, nerve or physiological maladies such as hematencephalon, schizophrenia, dysthymia disorders, oligophrenia and epilepsy).The present invention openly provides the genome of people PS1 (hPS1) and people PS2 (hPS2) gene and cDNA nucleotide sequence, mouse PS1 homologue (mPS1), and from C.elegans (sel-12, SPE-4) and the genes involved of black-tailed fruit flies (DmPS).The disclosure also provides the constitutional features by the aminoacid sequence and the early ageing element of the early ageing fibroin of the prediction of these genes encodings, comprises the functional domain and the epitope of supposition.Also disclosing is many sudden changes in the early ageing element of Alzheimer's disease (AD) cause of disease, and relates to described proteic functional domain.

Therefore, in a series of embodiment, the invention provides isolating nucleic acid, described nucleic acid comprises the nucleotide sequence that contains or be derived from the early ageing plain gene, and/or described nucleic acid encoding contains or be derived from the polypeptide of early ageing fibroin.The plain sequence of early ageing of the present invention comprise concrete disclosed sequence, these sequences montage mutant, these sequences allelic mutant, synonym sequence and these sequences homology or directly to mutant.Therefore, for example the invention provides the genome and the cDNA sequence of hPS1 gene, hPS2 gene, mPS1 gene and DmPS gene.The present invention also provides allele variant and homology by the method that can conventional obtain described mutant or directly to sequence.The present invention is also by disclosing many concrete mutant sequences and mutant or the pathogenic variant that the early ageing element specifically is provided by can conventional obtaining the method for described variant.Use owing to nucleic acid of the present invention can be used for many diagnosis, treatment and reorganization, so the different subunits of the plain sequence of early ageing and the combination of plain sequence of early ageing and heterologous sequence also are provided.For example in order to be used for allele-specific screening by hybridization or pcr amplification, provide the subunit of the plain sequence of early ageing to comprise to have a mind to and antisense sequences, normally and mutant nucleotide sequence and intron, exon and non-translated sequence.Described sequence can contain a small amount of continuous nucleotide of the open sequence that maybe can access in this article, but preferably includes from the plain sequence of early ageing 8-10 at least, more preferably 9-25 continuous nucleotide.Other preferred subunit of the plain sequence of early ageing comprises those of one or more early ageing fibroin functional domains of coding or epitope, particularly can comprise normal (wild-type) or mutant nucleotide sequence.The present invention also provides various nucleic acid constructs, wherein the plain sequence of early ageing (complete or partial sequence) can be operated with exogenous array to link to each other with formation cloning vector, expression vector, fusion vector, transgenic constructs etc.Therefore, according to a further aspect in the invention, also provide to be used for transformed mammalian or the histiocytic recombinant vectors of invertebrates so that express normal or the plain sequence of sudden change early ageing at cell.

In another serial embodiment, the invention provides with one of nucleic acid of the present invention transfection or transformed host cells.Transforming described cell can only be in order to breed nucleic acid construct of the present invention or to transform described cell to express the plain sequence of early ageing.Transformant of the present invention can be used in the detection with identify the normal or sudden change early ageing of influence plain that express, interact with normal or sudden change early ageing fibroin and/or regulate or influence is normal or albumen and/or other compound of mutain function, perhaps so that production early ageing fibroin of the present invention, fusion rotein, functional domain, epitope and/or antibody.For treatment or other reason, cell transformed can be implanted among host's (comprising the people).Preferred host cell comprises mammiferous from neurone, inoblast, marrow, spleen, organotypic cell or blended cell culture, and bacterium, yeast, nematode, insect and other invertebral zooblast.For purposes hereinafter described, preferred cell comprises embryonic stem cells, zygote, gamete and germ line cell.

In another serial embodiment, the invention provides and be used for AD and other disease relevant or the transgenic animal model of imbalance with early ageing plain gene sudden change.Described animal can be any Mammals, comprises rat, mouse, hamster, cavy, rabbit, dog, cat, goat, sheep, pig and inhuman primate.In addition, the invertebrates model can be comprised that also nematode and insect are used for specific application.By the standard transgenic method, comprise that other reformulations with microinjection, transfection or embryonic stem cells, zygote, gamete and germ line cells such as the carrier that contains genome or cDNA fragment, minigene, homologous recombination vector, virus insertion carriers can produce animal model.Suitable carrier comprises vaccinia virus, adenovirus, adeno-associated virus, retrovirus, liposome transportation, neurotrophy C-type virus C and hsv.Animal model can comprise the sequence of the transgenic sequence that contains or be derived from the early ageing element (comprising normal and mutant nucleotide sequence, intron, exon and non-translated sequence) and coding early ageing element subunit such as functional domain.The main animal model that is provided comprises: (1) wherein with normal people's early ageing plain gene as the episome under regulating at external source or endogenesis promoter, or recombinate as minigene or big genomic fragment and to import animal in the animal gene group; Wherein use normal people's early ageing plain gene to replace the animal of the animal homology early ageing plain gene of one or two copy by homologous recombination or gene targeting; And/or wherein by homologous recombination or gene targeting and the part of the sequence of encoded people's homologue replaces, with the animal of one of the animal homology early ageing plain gene of one or two copy reorganization " humanization ".(2) wherein with mutant human early ageing plain gene as the episome under regulating at external source or endogenesis promoter, or recombinate as minigene or big genomic fragment and to import animal in the animal gene group; Wherein use mutant human early ageing plain gene to replace the animal of one or two copy animal homology early ageing plain gene by homologous recombination or gene targeting; And/or wherein by homologous recombination or gene targeting and the part of the sequence of encoded mutant human homologue replaces, with the animal of one of the animal homology early ageing plain gene of one or two copy reorganization " humanization ".(3) wherein with the mutant of one of mutant animals early ageing plain gene as the episome under external source or endogenesis promoter are regulated, and recombinate as minigene or big genomic fragment and to import animal in the animal gene group; Wherein use one of the mutant replacement of one of animal early ageing plain gene or the animal of two copies animal homology early ageing plain gene by homologous recombination or gene targeting.(4) wherein by homologous recombination or gene targeting and partly or entirely lacked one of animal early ageing plain gene of one or two copy, or the homologous recombination by exogenous array or gene targeting finish and insert or replace, thereby make " rejecting " animal of one of the animal early ageing plain gene inactivation of one or two copy.In preferred embodiments, the transgene mouse model of AD has coding normal people PS1 or a PS2 albumen, coding people or mouse PS1 or the proteic mutant of PS2 or humanization is normal or sudden change mouse PS1 or the proteic transgenosis of PS2.

In another serial embodiment, the invention provides the pure basically protein formulation that contains polypeptide, described polypeptide contains or is derived from the early ageing fibroin.Early ageing fibroin sequence of the present invention comprises concrete disclosed sequence, because of the homology of the allele variant of the variant that changes these sequences that the mRNA montage produces, these sequences and these sequences or directly to the homology variant.Therefore, for example the invention provides hPS1 albumen, hPS2 albumen, mPS1 albumen and the proteic aminoacid sequence of DmPS.The present invention be also by can conventional obtaining the method for described variant, and provides allele variant and homology or directly to homologous protein.The present invention is also by disclosing many concrete mutant genes and the mutant or the pathogenic variant of early ageing element specifically being provided by the method that can obtain described variant.Use owing to albumen of the present invention can be used for various diagnosis, treatment and reorganization, therefore, also provide the combination of various early ageing fibroin sequence subunits and early ageing fibroin sequence and heterologous sequence.For example, in order to be used as immunogen or to be used for, provide the subunit of early ageing fibroin sequence to comprise normal and mutant sequence in conjunction with test.Described protein sequence can contain a small amount of continuous nucleotide of the open sequence that maybe can access in this article, but preferably includes from the plain sequence of early ageing 8-10 at least, more preferably 9-25 continuous nucleotide.Other preferred subunit of early ageing fibroin sequence comprises corresponding to those of one or more functional domains of early ageing fibroin or epitope, particularly can comprise normal (wild-type) or mutant sequence.The present invention also provides various albumen constructs, wherein complete or subsequence is linked to each other with exogenous array to form fusion rotein etc. according to these embodiments, and the present invention also provides all above-mentioned proteic methods that contain or be derived from the early ageing element of producing.

In another serial embodiment, the invention provides the production method and the purposes of polyclone and monoclonal antibody, described antibody comprises antibody fragment, comprises the Fab fragment, F (ab ') ₂And single chain antibody fragments, they can combine with the specific antigens decision based selective of early ageing element or early ageing element.Can be in mouse, rabbit, goat or other suitable animal, or as described in cultured cells is produced in as hybridoma cell line antibody.The anti-plain sequence of early ageing 4-8 at least, the preferred antibody of 9-15 continuous amino acid residue at least of containing of preferred production.Antibody of the present invention can be used for various diagnosis as herein described, treatment and technology uses.

In another serial embodiment, the invention provides the method for screening or evaluation albumen, small molecules or other compound, these materials can induce or suppress the expression of early ageing plain gene and induce or arrestin (as PS1 or PS2).Available non-transformed cell, fixed clone or reconstitution cell tie up to external, or finish described test in vivo with the transgenic animal model of this paper.Particularly, described test can be expressed on the basis that increases or reduce at mRNA, detect PS1, PS2 or other early ageing element-genes involved or proteic expression whether increase or minimizing are arranged, detect early ageing element-associated protein product level whether increase or reduction are arranged, or in recombinant precursor, whether the expression level that can operate the marker gene (beta-galactosidase enzymes, green fluorescent protein, alkaline phosphatase or luciferase) that links to each other with early ageing element 5 ' regulatory region has increase or reduction.The specific early ageing of the known expression of incubation is plain or transform the cell that specific early ageing element is expressed in the back, then one or more test compounds is added in the substratum.Through after described compound being enough to induce the plain time (as 0-72 hour) of expressing of early ageing, available above-mentioned any technology for detection is based upon any variation on the primary expression level, in particularly preferred embodiments, cell is from fixed clone such as people's neuroblastoma, glioblastoma multiforme or hybridoma cell line or transformant of the present invention.

In another serial embodiment, the invention provides and identify with early ageing is plain and combine or the albumen of direct interaction and the method for other compound with it.Described albumen and compound comprise with the plain body of early ageing in interact and therefore provide the endogenous cell component of new target for medicine and treatment, and have the plain binding ability of early ageing and therefore can be used as the reorganization of drug candidate, synthetic and other xenobiontics.Therefore in a series of embodiment, can screen cell lysates or tissue homogenate thing (as the even oar thing of human brain, lymphocytolysis product) to obtain and one of normal or sudden change early ageing element bonded albumen or other compound.In addition, can screen any xenobiontics (natural and/or synthetic (as small molecules or peptide library)) with regard to the plain binding ability of early ageing.In these embodiments, finish test to detect the combination between " the plain component of early ageing " and some other parts." early ageing plain component " in these trials can be to contain or be derived from polypeptide normal or sudden change early ageing fibroin, comprises the epitope or the early ageing plain fusion protein of plain functional domain of early ageing or early ageing element.With non-specific method of masurement (as Ca in the cell ²⁺Variation, the GTP/GDP ratio) or specificity method of masurement (changing or the variation of other downstream gene expression aspect) as A β peptide production by differential demonstration, 2D gel electrophoresis, differential hybridization or the monitoring of SAGE method can detect as described in combination.Preferred method relates to the change of following technology: directly extract by affinity chromatography (1); (2) be divided into from plain combination of early ageing and bonded albumen by immunoprecipitation; (3) bio-molecular interaction (Biomolecular Interaction) test (BIAcore); (4) yeast two-hybrid system.

In another serial embodiment, the invention provides evaluation and can regulate method normal or the plain active albumen of sudden change early ageing, small molecules and other compound.Use normal cell or animal, transformant of the present invention and transgenic animal model, or from carrying the cell that experimenter normal or sudden change early ageing plain gene obtains, the present invention with its influence that early ageing is plainly expressed, locate in the plain cell of early ageing, Ca in the cell ²⁺, Na ⁺, K ⁺Or level or collection of illustrative plates, the phosphorylation level of microtubule-associated protein or the biological chemistry histology or the physiology that the cell of normal and the plain sequence of sudden change early ageing is carried in other difference of the generation of other ion concentration or metabolism, apoptosis or necrocytosis or speed, the production of A β peptide is labeled as the basis, and the method for identifying described compound is provided.Utilize transgenic animal of the present invention, also, provide the method for identifying described compound based on the ability of described compounds affect behavior, physiology or the histology phenotype relevant with sudden change in the early ageing element.

In another serial embodiment, the invention provides early ageing that the plain allelic carrier of the screening early ageing relevant with AD, diagnosis AD patient, screening and diagnosis be correlated with and senile dementia, psychosis such as schizophrenia and dysthymia disorders and neurological disorder such as apoplexy and hematencephalon (these diseases all with PS1 or PS2 gene in sudden change relevant) method.By openly and the method for the antibody that can access, comprise that design is used for detecting normal early ageing plain loss of activity or enhancing and/or whether exists can finish because of the plain special new active function test of giving of sudden change early ageing to screen and/or diagnose based on nucleic acid (comprising genome and mRNA/cDNA sequence), albumen and/or this paper.Therefore, screening and diagnostic method based on the early ageing fibroin are provided, described method can the mol ratio of electrophoretic mobility, proteolytic cleavage collection of illustrative plates, various amino-acid residues, and the binding ability of specific antibody aspect, detect the difference between mutant and the normal early ageing element.In addition; provide with nucleic acid (gDNA; cDNA or mRNA) be the screening and the diagnostic method on basis; described method can be by direct nucleotide sequencing, the hybridization that utilizes allele specific oligonucleotide, restriction enzyme digestion and mapping (as RFLP; REF-SSCP); electrophoretic mobility is (as SSCP, DGGE), the detection of PCR collection of illustrative plates, RNase protection, chemical mispairing cracking, ligase enzyme mediation and various other method detect the difference in the nucleotide sequence.Also provide other method detect PS1, PS2, APP or with the proteic undesired processing of PS1, PS2 or APP reaction (as, unusual phosphorylation, glycosylation, saccharification (glycation) amidation or proteolytic cleavage) transcribe at the early ageing element, the change aspect translation and the posttranslational modification; The change of in the cell of early ageing plain gene product and transportation aspect, extracellular; Or location in the abnormal cells of early ageing element.According to these embodiments, diagnostic kit also is provided, described test kit contains above-mentioned diagnosis and screens necessary reagent.

In another serial embodiment, the invention provides the method and the pharmaceutical preparation that are used for the treatment of early ageing element-relative disease such as AD.These methods and medicine are based on (1) and use normal PS1 or PS2 albumen, (2) carry out gene therapy with compensation or replacement mutant gene with normal PS1 or PS2 gene, (3) be the based gene treatment with antisense sequences or " rejecting " mutant gene with mutant PS1 or PS2 gene, (4) the proteic sequence with the deleterious effect of coding blocking-up or corrigendum PS1 or PS2 mutant is the based gene treatment, (5) based on immunotherapy normal and/or mutant PS1 or the proteic antibody of PS2, or (6) small molecules (medicine), they change PS1 or PS2 expresses, blocking-up PS1 or the mutant forms of PS2 and the abnormal interaction between other albumen or the part, or by changing the structure of mutant protein, by strengthening its metabolic clearance rate or blocking mutant PS1 or the proteic abnormal function of PS2 by suppressing its function.

According to a further aspect in the invention, available albumen of the present invention opens initial point so that the little chemical molecular of part, medicine or other type to be provided as theoretical medicinal design.In addition, can with identify with aforesaid method small molecules or other compound in theoretical medicinal design as " lead drug ".

Disclosed especially Nucleotide of the present invention and aminoacid sequence are designated as SEQ ID NO:1-25.In addition, according to budapest treaty, (Rockville MD) has carried out preservation at ATCC with the biological preservation thing of specific nucleic acid disclosed herein.These preservation things comprise preserving number 97124 (nineteen ninety-five preservation on April 28), preserving number 97508 (nineteen ninety-five preservation on June 25), preserving number 97214 (nineteen ninety-five preservation on June 28) and preserving number 97428 (preservation on January 26 in 1996).

The accompanying drawing summary

Fig. 1: the structure that the figure shows the hPS1 genomic dna.With the non-coding exon of entity shadow box indicating.Show the coding exon with empty frame table, show other montage sequence with drawing hatched frame table.Restriction site is: B=BamH; E=EcoRI; H=HindIII; N=NotI; P=PstI; V=PvuII; X=XbaI.At the undetermined genome sequence of locus of discontinuity domain representation between the restriction site on the sea line.The cloned genes group fragment of representing to contain each exon with the two-way horizontal arrow.The size of genome subclone and the preserving number of a genome sequence are provided.

Fig. 2: the figure shows and infer the proteic hydrophobicity profile of PSI.This figure knows what the method for Doolittle (1982) was calculated according to Kyte.

Fig. 3: the series arrangement that the figure shows hPS1 and mPS1 protein sequence.Vertical line is represented identical amino acid.

Fig. 4: the series arrangement that the figure shows hPS1 and hPS2 protein sequence.Vertical line is represented identical amino acid.

Fig. 5: the synoptic diagram that is PS1 albumen predict.Roman number is represented membrane spaning domain.Represent the glycosylation site of inferring with asterisk, phosphorylation site is positioned at and two film outer surfaces that acid hydrophilic loop is identical.The map kinase site is positioned at 115 residues, and the PKC site is positioned at 114 residues.The FAD mutational site is represented with horizontal arrow.

Fig. 6: the synoptic diagram that is PS2 albumen predict.Roman number is represented membrane spaning domain.Represent the glycosylation site of inferring with asterisk, phosphorylation site is positioned at and two film outer surfaces that acid hydrophilic loop is identical.The FAD mutational site is represented with horizontal arrow.

Detailed description of the present invention

The I definition:

For the ease of the different embodiment of research the present invention, understand and finish and use each element and composition used among the present invention, used particular term is defined as follows in description and claim:

The early ageing element.Do not further specify as this paper, term " early ageing element " refers to early ageing element-1 (PS1) and/or early ageing element-2 (PS2) genes.Specifically, the term of unmodified " early ageing element " refers to Mammals PS1 and/or PS2 genes, preferred people PS1 and/or PS2 genes.

Early ageing element-1 gene.As used herein, term " early ageing element-1 gene " or " PS1 gene " refer to open for the first time and are described in U. S. application 08/431048 (application on April 28 nineteen ninety-five), and be described in mammalian genes in (1995) such as Sherrington afterwards, comprise any allele variant and different specific Mammals homologue.A kind of people's early ageing element-1 (hPS1) cDNA sequence is open with SEQ ID NO:1 in this article.Open by another human cDNA sequence that the montage that changes hPS1 mRNA transcript obtains with SEQ ID NO:3.As mentioned below, also found other human splice variant, wherein in some transcript, the coding region of 33 residues is fallen by montage.(cDNA of mPS10 is open with SEQ ID NO:16 for a kind of mouse homologue.Term " early ageing element-i gene " or " PS1 gene " mainly refer to encoding sequence, but also can comprise part or all of side regulatory region and/or intron.Term PS1 specifically refers to comprise the recombination based on splice variant from the artificial or recombination of cDNA or genomic dna generation.Also once early ageing element-1 gene was called S1S2 gene (as Sherrington etc., 1995) or relevant membranin (ARMP) gene (as U. S. application 08/431048, application on April 28 nineteen ninety-five) of Alzheimer's disease.

Early ageing element-1 albumen.As used herein, term " early ageing element-1 albumen " or " PS1 albumen " refer to the gene by PS1, comprise allele variant and different specificity Mammals homologue encoded protein.People's early ageing element-1 (PS1) protein sequence is open with SEQ ID NO:2 in this article.Open by another people PS1 albumen that the montage that changes the hPS1mRNA transcript obtains with SEQ ID NO:4.As mentioned below, also found other people's splice variant, wherein in some transcript, the coding region of 33 residues is fallen by montage.These variants are also included by term early ageing element-1 albumen of this paper.The protein sequence of mouse homologue (mPS1) is open with SEQ ID NO:17.Can produce described albumen by reconstitution cell or organism, the described albumen of purifying basically from natural tissues or clone, or with chemistry or the synthetic described albumen of Enzymology method.Therefore, " early ageing element-1 albumen " or " PS1 albumen " comprise glycosylated, part is glycosylated or non-glycosylated form and phosphorylation, part phosphorylation, unphosphorylated, sulfation, albumen part of sulfuric acidization or the non-sulfuric acid form.This term also comprises allele variant and other function equivalent of PS1 aminoacid sequence, comprises biological activity protein hydrolysis fragment or other fragment.Also once this albumen was called the relevant membranin (ARMP) of S182 albumen or Alzheimer's disease (as U. S. application 08/431048, application on April 28 nineteen ninety-five).

The hPS1 gene and/albumen.As used herein, abbreviation " hPS1 " refers to PS1 gene and/or proteic people's allele variant.The cDNA sequence of people PS1 gene is open with SEQ ID NO:1 and SEQ ID NO:3 in this article.The corresponding proteins sequence is open with SEQ ID NO:2 and SEQ IDNO:4.Also disclose various allele variants and comprised detrimental mutant, and can from hereinafter describe, obtain described variant.

MPS1 gene and/or albumen are as described herein, and abbreviation " mPS1 " refers to PS1 gene and/or proteic mouse homologue and mouse allele variant.A kind of cDNA sequence of mouse PS1 gene is open with SEQID NO:16.Corresponding mPS1 protein sequence is open with SEQ ID NO:17.From following description, can obtain comprising the allele variant of detrimental mutant.

Early ageing element-2 gene.As used herein, term " early ageing element-2 gene " or " PS2 gene " refer to for the first time disclosed in U. S. application 08/496841 (application on June 28 nineteen ninety-five), the mammalian genes of describing in (1995) such as Rogaev etc. (1995) and Levy-Lahad comprised any allele variant and different specificity Mammals homologue afterwards.This paper discloses a kind of people's early ageing element-2 (hPS1) cDNA sequence with SEQ IDNO:18.As mentioned below, also found other people's splice variant, wherein the coding region of single codon or 33 residues has been fallen in montage in some transcript.Term " early ageing element-2 gene " or " PS2 gene " mainly refer to encoding sequence, but also can comprise part or all of side regulatory region and/or intron.Term PS2 gene specifically comprises from the artificial or recombination of cDNA or genomic dna generation, comprises the recombination based on splice variant.Once early ageing element-2 gene was called the E5-1 gene (as Rogaev etc., 1995; U. S. application 08/496841, June 28 nineteen ninety-five application) or STM2 gene (as Levy-Lahad etc. 1995).

Early ageing element-2 albumen.As used herein, term " early ageing element-2 albumen " or " PS2 albumen " refer to the albumen by the PS2 genes encoding, comprise allele variant and different specificity Mammals homologue.A kind of people's early ageing element-2 (hPS2) albumen is disclosed with SEQ ID NO:19.As mentioned below, also found other people's splice variant, wherein montage has been fallen single residue or has been contained the zone of 33 residues in some transcript.These variants are also included by term early ageing element-2 albumen used herein.Can produce described albumen by reconstitution cell or organism, the described albumen of purifying basically from natural tissues or clone, or with chemistry or the synthetic described albumen of Enzymology method.Therefore, term " early ageing element-2 albumen " or " PS2 albumen " comprise the albumen of glycosylation, part glycosylation or non-glycosylated form and phosphorylation, part phosphorylation, non-phosphorylating, sulfation, part of sulfuric acidization or non-sulfuric acid form.This term also comprises allele variant and other function equivalent of PS2 aminoacid sequence, comprises biological activity protein hydrolysis or other fragment.Once early ageing element-2 gene was called E5-1 albumen (as Rogaev etc., 1995; U. S. application 08/496841, application on June 28 nineteen ninety-five) or STM2 albumen (Levy-Lahad etc. 1995).

HPS2 gene and/or albumen.As used herein, abbreviation " hPS2 " refers to people's homologue and people's allele variant of PS2 genes.This paper discloses a kind of cDNA sequence of people PS2 gene with SEQ ID NO:18.This paper discloses corresponding hPS2 protein sequence with SEQ ID NO:19.Disclose the many allele variants that comprise detrimental mutant, and from following description, can obtain.

DmPS gene and/or albumen.As used herein, write Drosophila homologue and allele variant that " DmPS " refers to PS1 and PS2 genes.This definition is included in replacement, the insertion in described gene or the protein sequence or lacks the nucleic acid and the aminoacid sequence polymorphism of the basic function that does not influence described gene product.This paper discloses the nucleotide sequence of a kind of cDNA of DmPS gene with SEQ ID NO:20, and disclose amino acid sequence corresponding with SEQ ID NO:21: term " DmPS " mainly refers to encoding sequence, but also comprises part or all of side regulatory region and/or intron

Normally.With regard to gene used herein, term " normally " refer to the to encode gene of normal protein.With regard to albumen used herein, term " normally " refers to finish its routine or normal physiological effect, and irrelevant or be not the albumen of the cause of disease with morbific conditioned disjunction state.Therefore, as used herein, term " normally " is the synonym of wild-type basically.For gene of being given or corresponding proteins, can there be the multiplicity of normal allele variant, none is relevant with the development of pathogenic conditioned disjunction state among them.Described normal allele variant includes, but are not limited to wherein one or more Nucleotide and replaces the variant that can not cause coded aminoacid sequence to change.

Mutant.With regard to the gene of this paper, term " mutant " refers to the gene of encoding mutant body protein.With regard to the albumen of this paper, term " mutant " refers to finish its routine or normal physiological effect, and with the albumen of the reason of the relevant or pathogenic conditioned disjunction state of morbific conditioned disjunction state.Therefore, as used herein, term " mutant " is the synonym of " functional disorder ", " morbific ", " cause of disease " and " deleterious " basically.With regard to early ageing plain gene of the present invention and albumen, term " mutant " refers to carry the early ageing plain gene of one or more Nucleotide/aminoacid replacement, insertion and/or disappearance, when described gene is expressed, can cause taking place Alzheimer's disease and/or other relevant genetic phenotype (as hematencephalon, mental retardation, schizophrenia, psychosis and dysthymia disorders) in the mankind.This definition comprises naturally occurring various sudden change, includes but not limited to disclosed herein and artificial synthetic or recombination mutation.When term " mutant " is used for the early ageing plain gene, because the degeneracy of genetic code had both not merely comprised the identical sequence variants of normal sequence that coding and this paper openly maybe can access; Comprise not only that also encoding function is equal to the proteic sequence variants of normal early ageing fibroin, although they are the different albumen of coding.

Function equivalent.When this paper describes gene order and aminoacid sequence, term " function equivalent " refers to that cited sequence need not to be equal to the disclosed particular sequence with SEQ ID NO, and only need provide can be biological and/or chemically play the sequence of function of the equivalent of sequence disclosed herein.

Substantially pure.When this paper was used for albumen (comprising antibody) or other goods, term " pure substantially " referred to that described compound accounts for 60% weight (dry weight) of goods at least.Preferably account for 75% of goods at least, more preferably account for 90%, the best accounts at least 99% (all in described compound weight).Available any suitable method such as column chromatography, gel electrophoresis or HPLC analysis to measure purity.

With regard to the albumen that comprises antibody, comprise two or more required different compounds (as two or more antibody, immunogen, functional domain or other polypeptide of the present invention) as fruit product, " pure substantially " goods just refer to that wherein the gross weight of all required compounds (dry weight) accounts for 60% of gross dry weight at least.Equally, for the described goods that contain two or more required compounds, the gross weight of preferred described compound is 75% of goods gross dry weight at least, more preferably at least 90%, bestly be at least 99%.

Isolating nucleic acid.As used herein, " isolating nucleic acid " is that (one at 5 ' end from closely linking to each other the natural gene group of the organism that it was derived from, one at 3 ' end) sequence in, isolating Yeast Nucleic Acid, thymus nucleic acid or contain the nucleic acid analog of polynucleotide sequence.Therefore, this term comprises the recombinant nucleic acid that for example is inserted among carrier, autonomously replicating plasmid or virus or protokaryon or the eukaryotic gene group DNA; Or with the independent molecule that is independent of other sequence (as, handle cDNA or the genomic DNA fragment that produces with PCR or restriction restriction endonuclease) recombinant nucleic acid that exists.This term also comprises the recombinant DNA as a hybrid gene part, described hybrid gene encode other peptide sequence and/or comprise the external source regulatory element.

Essentially identical sequence.As used herein, " essentially identical " aminoacid sequence is only to replace by conserved amino acid, replaced (as the Xie Ansuan substituted glycinic acid by the another kind in similar as a seed amino acid, arginine replaces Methionin etc.) or the different aminoacid sequence that produces by on the position in the aminoacid sequence that does not destroy described protein function (detection, as described herein), carrying out non-conservative replacement, disappearance or insertion.Preferably on amino acid levels, described sequence has at least 85% with the albumen or the peptide sequence that are compared, and is preferred 90%, the best be 95% identical.For nucleic acid, the length of comparative sequences is at least 50 Nucleotide usually, preferably at least 60 Nucleotide, more preferably at least 70 Nucleotide, and the best is 110 Nucleotide.As mentioned above, the essentially identical aminoacid sequence of " essentially identical " nucleic acid sequence encoding.

Cell transformed.As used herein, " cell transformed " is by recombinant DNA technology, imported the cell (or importing in its progenitor) of required nucleic acid molecule.Common encoded peptide of required nucleic acid or albumen.Described cell transformed can be expressed required sequence or can only be used to breed described sequence.Term used herein " conversion " comprises any method that imports exogenous nucleic acid, includes, but are not limited to transform, transfection, electroporation, microinjection, virus-mediated transfection etc.

Can operate continuous.As used herein, when encoding sequence and regulatory region link to each other with the mode covalency that can make encoding sequence express or transcribe, think that then encoding sequence is " can operate and link to each other " with regulatory region under the influence of regulatory region or control.If desired encoding sequence is translated into functional protein, if the importing of promoter function causes transcribing of encoding sequence, if the continuous character between two dna sequence dnas can (1) not cause phase shift mutation, (2) influence regulatory region and instruct ability or the corresponding rna transcription of (3) influence that encoding sequence transcribes originally to translate into proteic ability, think that then these two dna sequence dnas can operate continuous.Therefore, if regulatory region can influence transcribing of dna sequence dna, so that the transcript of gained can be translated into required albumen or polypeptide, then described regulatory region can be operated with encoding sequence and link to each other.

Rigorous hybridization conditions.Rigorous hybridization conditions is the term that this area professional can understand.For any given nucleotide sequence, rigorous hybridization conditions is that temperature, chaotropic acid, damping fluid and ionic strength can make nucleotide sequence and its complementary sequence hybridization, and the condition of not hybridizing with the sequence that is different in essence.The accurate condition that constitutes " rigorous " condition depends on the length of the character of nucleotide sequence, described sequence and the frequency that occurs the subsequence of sequence described in other non-identical sequence.By hybridization conditions is changed to the level that specific hybrid only takes place from the rigorous degree level that non-specific hybridization takes place, this area professional need not separating tests, can determine to make given sequence only with the condition of complementary sequence hybridization.The optimum range of described rigorous degree condition has been described in Krause and Araoson (1991).According to the length and the general character of sequence, hybridization conditions can comprise that temperature is 20-65 ℃, and ionic strength 5x is to 0.1xSSC.Highly rigorous hybridization conditions can comprise that temperature is low to reach 40-42 ℃ (when comprising denaturing agent such as methane amide) or up to 60-65 ℃, ionic strength is that 0.1xSSC is low.But these scopes are illustrative, according to the development of the characteristic of target sequence and possible WeiLai Technology, can be than needs more rigorous.The lower condition of available rigorous degree with separate and given sequence is similar in fact, allelotrope or homologous nucleotide sequence.

Selective binding.Used to this paper antibody, if antibody recognition and combine with target, but nonrecognition and in conjunction with other molecule in the sample (as biological sample) that comprises described target in fact just thinks that antibody and target are " selective binding ".

II early ageing element

The present invention partly is based on and has found a mammalian genes family, and when it suddenlyd change, described gene family was relevant with the generation of Alzheimer's disease.Discovery and these genes, its protein product, the sign of mutant and the possible function of these genes (being called early ageing element-1 and early ageing element-2) hereinafter will be described.The invertebrates homologue of early ageing element also has been discussed, and reason is that they can be illustrated the function of early ageing element and reach the degree of using it for various embodiments hereinafter described.

1 separation of human early ageing element-1 gene

The genetic mapping in AAD3 district

Sherrington etc. (1995) have described the initial separation and the sign of PS1 gene (being called AD3 gene or S182 gene afterwards).In the 14q24.3 seat (Schellenberg etc., 1992 that begin the AD3 gene region is located adjacent to unknown microsatellite marker D14S43 and D14S53; St.George-hyslop etc., 1992; Van Broeckhoven etc., 1992) after, with the AD (St.George-Hyslop etc. of 21 pedigrees separation as the autosomal dominant feature of inferring, 1992) and be used to study 18 separation from the additional genetic marker in 14q24.3 district, described district has been compiled into highdensity genetic linkage map (Weissenbach etc., 1992; Gyapay etc., 1994).Previously disclosed paired maximum likelihood analysis has confirmed the actual evidence that adds up chain between familial Alzheimer's disease (FAD) and all these marks.But the chain genetic data of many supports and these marks is from 6 big early onset thereof pedigrees, FAD1 (Nee etc., 1983), FAD2 (Frommelt etc., 1991), FAD3 (Goudsmit etc., 1981; Pollen, 1993), FAD4 (Foncin etc., 1985), TORl.l (Bergamini, 1991) and 603 (Pericak-Vance etc., 1988), each pedigree provides at least one from the unknown genetic marker of the euchromosome of 14q24.3 (St.George-Hyslop etc., 1992).

In order to determine the position of AD3 gene more accurately from the known genetic marker seat of 14q24.3, by direct inspection original unit type data, seek the reorganization boundary mark, described data are affected from genotype, with karyomit(e) 14 member who determines 6 chain pedigrees is arranged.Under this specific situation, this process for selective will be removed the genotypic data of reconstruction from the older asymptomatic member of member death, influenced and next arrogant pedigree, and not considering does not have the little pedigree of determining chain state.But, this method is very reliable, be mistake in influenced member's (because of no origin causes) the reconstruction genotype of cause death or missampling or produce because it has also avoided the genotype data that uses potential to induce one to misread, described error in data because of having comprised irrelevant pedigree.

After having checked affected experimenter's haplotype data, the member who has directly determined its genotypic 6 big pedigrees is at D14S48 and D14S53, and the recon that is necessary of D14S58 and D14S63.Single recon (having described the telomere boundary in FAD district) at D14S53 betides among the experimenter of the same AD of suffering from the FAD1 pedigree, once the member who found this family in the past be positioned at D14S53 (comprising S14S48) to the telomere zone several other be marked with recon (St.George-Hyslop etc., 1992).On the contrary, single recon of D14S258 (the kinetochore boundary in expression FAD district) betide the FAD3 pedigree an ill member in, this patient also be positioned at D14S258 (comprising D14S63) between the kinetochore several other be marked with recon.Show by the disease criterion clinical trial in the ill member of other family, two kinds of recon patients have clear and definite presenile dementia disease symptoms, to the many seats of D14S258 telomere at interval, two kinds of recon patient's genotype are indicative and are isolating altogether in the D14S53 kinetochore.

Enlarge the haplotype analysis with the reconstruction genotype that comprises 6 dead patients of big pedigree and from 15 chain probability of residue during less than the data of 0.95 pedigree, on the one or more marks seat of D14S53 in the interval of D14S258, detect several other recons.Therefore, in 3 big FAD pedigrees (the relevant family of FAD1, FAD2), detect an other recon respectively in dead patient's the reconstruction genotype, in the patient of 5 less FAD pedigrees, detect 8 other recons with other.But, although some such recons can correctly be placed on the AD3 gene in more definite target area, but it is because unreliable, also need to consider these potential nearer " interior recons ", this unreliable being not only owing to reason discussed above, but also because they provide sudden change to go up inconsistent position for the interior at interval AD3 gene of D14S53-D14S258.

B makes up the physical overlap group who strides the AD3 district

Step as clone AD3 gene begins makes up the contig of being cloned into the overlapping genes group dna fragmentation in yeast artificial chromosome's carrier, phage artificial chromosome carrier and the cosmid vector.The FISH that clay the carried out mapping that utilization is derived from yac clone 932c7 and 964f5 studies show that the interval of most possibly carrying the AD3 gene has 5 megabasse sizes at least.Because the described abscission zone that is divided into for a short time makes the positional cloning method be difficult to operation more greatly, thus other hereditary index sought, so that the research of AD3 gene is concentrated on D14S53 and the interior at interval one or more subprovinces of D14S258.No matter whether described analysis is limited to those pedigrees that early stage FAD mode of onset is arranged, still be generalized to and comprise all pedigrees, D14S53 and D14S258 at interval between the haplotype analysis of mark can not be from the statistics significantly on explanation FAD feature and any of these mark linkage disequilibrium and/or the allelotrope between the allelotrope relevant.If it is just not unexpected that our pedigree, draws such result from different ethnic groups.But, more similar ethnic group offspring's pedigree, isolating karyomit(e) in spite of illness carries out direct haplotype inspection in the different pedigrees to similar ethnic group, and showing has two mark seat groups.In these groups first is positioned at D14S77 (D14S786, D14S277 and D14S268) to the kinetochore, and in the YAC78842 contained 0.95Mb physical separation.Second group is positioned at D14S77 (D14S43, D14S273 and D14S76) to telomere, and strides overlapping yac clone 964c2,74163,797d11 and part 854f5 be contained～1Mb physical separation.In at least 2 pedigrees, observe identical allelotrope from identical ethnic group.Because separating method, so one of think at the mark seat of physics cluster and to exist total allelotrope may reflect in each one population, the common heredity of little physical areas around the PS1 gene on the person's of foundation karyomit(e) of source.In normal Caucasian population, each total haplotype that extends is very rare, strides on similar heredity other mark group at interval at karyomit(e) 14q24.3, does not observe allelotrope and shares.

The transcription mapping of C candidate gene and analysis

In order to be separated in the expressed sequence of encoding in two important intervals, that use comprises is fixing, clone's direct screening method, as hybridizing target to retrieve the transcription sequence from one-level complementary DNA pond, described DNA pond is derived from human brain mRNA (Rommens etc., 1993) with the human gene group DNA.The purpose cDNA fragment that about 900 sizes of recovery are the 100-600 base pair from these districts.With these fragments and the Southern blot hybridization that contains genomic dna, described genomic dna is from each eclipsed yac clone and people and other mammiferous genomic dna.Identified 151 clones' subunit like this, the evidence of evolution conservative and/or composite structure is provided, shown that they are derived from the mRNA of montage.Classify the clone of basis in the more described subunit as with physical map position, cross hybridization and nucleotides sequence, and be used to screen the conventional human brain cDNA that is used for longer cDNA.Separate at least 19 the independent cDNA clones of length more than 1kb, be arranged in the part transcripting spectrum in AD3 district then.In these transcripts, have only 3 corresponding to the gene that has characterized (cFOS, dihydrolipoamide succinyltransferase and the growth factor bindin 2 of hiding and transforming).

The recovery of D candidate gene

Reclaim the open reading frame of each candidate gene from mRNA by RT-PCR, described mRNA is isolating the fibroblast of cerebral tissue after cerebral tissue after normal control experimenter necrotomy and 6 pedigree patients' the necrotomy or cultivation, has determined that described 6 pedigrees are relevant with karyomit(e) 14.Then with chemical cracking and restriction restriction endonuclease fingerprint single stranded sequence conformational polymerphism method (Saleeba and Cotton, 1993; Liu and Sommer, 1995), and by direct nucleotide sequencing the RT-PCR product is screened with regard to sequence difference.A kind of exception is that it is peculiar that the gene of all detections (although being required) does not all contain ill experimenter, or change with the altogether isolating sequence of described disease.Described exception is clone's candidate gene that S182 showed, and this clone contains that unobservable a series of Nucleotide change in the normal subjects, and is expected among the ill experimenter and has changed aminoacid sequence.Now will be corresponding to this cloned genes called after early ageing element-1 (PS1).This paper discloses two PS1cDNA sequences (expression alternative splicing variant hereinafter described) with SEQID NO:1 and SEQ ID NO:3.With SEQ ID NO:2 and SEQ ID NO:4 the aminoacid sequence of inferring is accordingly disclosed respectively.The Bluescript plasmid that will carry these cDNA clones is deposited in ATCC April 28 nineteen ninety-five, Rockville, and Md, preserving number are 97124 and 97508.Sequence corresponding to SEQ ID NO:1 and SEQ ID NO:2 also has been kept in the GenBank database, and can retrieve by accession number #42110.

2 separate mouse early ageing element-1 gene

Use labelling human dna probe,, reclaim the mouse homologue (mPS1) of people PS1 gene by screening mouse cDNA library from the hPS1 gene.In this way, reclaimed the part transcript (representing 3 ' end of this gene) of 2kb and represent 5 ' terminal RT-PCR product.The total cDNA transcript of mouse homologue checked order show that with hPS1 substantial amino acid identity is arranged.Importantly (as described below), all amino acid that suddenly change in the FAD pedigree are guarded between mouse homologue and normal people's variant.This conservative property of PS1 gene shows in mouse (mPS1) and has orthologous gene, now by personnel selection PS1 probe screening-gene group or cDNA library, can clone other Mammals homologue or directly to homologue (orthologues).Therefore can identify and characterize PS1 gene in other kind with similar methods.This paper discloses mPS1 clone's nucleotide sequence and discloses amino acid sequence corresponding with SEQ ID NO:17 with SEQ ID NO:16.Two sequences all being kept at the GenBank database also can retrieve by accession number #42177.

3 separation of human early ageing element-2 genes

Separated second people's gene, now be called early ageing element-2 (PS2), and shown that with the PS1 gene substantial Nucleotide and amino acid identity are arranged.Rogaev etc. (1995) at first describe the separation of this gene in detail.Levy-Lahad etc. (1995) have also reported with very close method separation of human PS2 gene (being called " STM2 "), in brief, with the BLASTN example of (1990) such as Altschul,, can identify the PS2 gene by the nucleotide sequence searching database of PS1 cDNA.Located the substantive homology (p＜1.0e of usefulness that identifies with accession number #T03796, R14600 and R05907 ^-100, the identity more than 97% is arranged at least 100 successive base pair scopes) the sequencetagged site (EST) of 3 expression.

Produce Oligonucleolide primers and be used for product from these sequences through ThermoScript II PCR (RT-PCR) generation PCR.With these short RT-PCR products partly check order with determine its with database in the identity of sequence, be used as hybridization probe then and screen the full-length cDNA library.(cc54 has reclaimed size in cc32) and has been several different cDNA of 1kb-2.3kb for E5-1, G1-1 from cancer cells cDNA library (Caco2) and human brain cDNA.These clones' nucleotide sequence shows that all sequences all is derivatives of identical transcript.

Hybridization mapping group with two cohorts of CEPH Mega yac clone, gene with the described transcript of coding, the PS2 assignment of genes gene mapping is on human chromosome 1, and (with yac clone 750g7,921d12 navigates to 1q41 with the FISH method described cohort to be positioned at the contig physical map; YAC exempts from grand 787g12 and navigates to 1p36.1-p35) on.This paper discloses amino acid sequence corresponding with the nucleotide sequence that SEQ ID NO:18 discloses the hPS2 clone with SEQ ID NO:19.These two sequences all have been kept in the GenBank database, and can be by accession number #L44577 retrieval.Also the dna sequence dna of hPS2 having been cloned is incorporated in the carrier, and is deposited in ATCC June 28 nineteen ninety-five, Rockvile, and MD, preserving number are 97214.

The evaluation of homologue in 4 C.elegans and the black-tailed fruit flies

The SPE-4 of A C.elegans

Arrange example with BLAST, the nucleic acid of PS1 and the PS1 aminoacid sequence of expection and the sequence in the database are compared, show with the smart integral protein SPE-4 of C.elegans moderate similarity (P=1.5e-25 is arranged, in three class ranges of at least 50 residues, 24-37% identity is arranged), the part that comprises Mammals Chromogranin A and the proteic alpha subunit of Mammals voltage-dependent ca channel (Altschul etc., 1990) with several other transmembrane proteins has very low similarity.Can produce accidentally and simply result from the arrangement of a limited number of hydrophobic amino acid inferring amino acid sequence similarity between the membrane spaning domain, but several hydrophilic-structure territories between PS1 and SPE-4 also there is the series arrangement of extension.Think that the PS1 albumen and the SPE-4 that infer have similar size (being respectively 467 and 465 residues), as hereinafter in greater detail, and before the membrane spaning domain of final expection, contain at least 7 membrane spaning domains with big acid domain.PS1 albumen has long expection hydrophilic area really at N-terminal.

The BLASTP arrangement analysis has also detected the remarkable homology (p=3.5e-26 between PS2 and the C.elegans SPE-4 albumen; On 5 structural domains of at least 22 residues, identity=20-63%), and detect and brain sodium channel protein (α III subunit) and the proteic alpha subunit (p=0.02 of voltage-dependent ca channel not of the same race; The identity that 20-28% is arranged at least) weak homology (Altschul etc., 1990) is arranged on two or more structural domains of 35 residues.These arrange similar with above-mentioned to the description of PS1 gene those.

The Sel-12 of BC.elegans

Discovery has 48% sequence identity (Levitan and Greenwald, 1995) from the Sel-12 albumen and the S182 (SEQ IDNO:2) of 461 residues of C.elegans on 460 amino acid.Think that also Sel-12 albumen has a plurality of membrane spaning domains.Obtain function mutation (gain-of-function) inhibitor by screening Iin-12 and identified sel-12 gene (accession number U35660), and be rescued (Levitan and Greenwald, 1995) and clone by conversion.

The DmPS of C black-tailed fruit flies

The Feng Yu oligonucleotide of the proteic high conservative region of preparation coding early ageing element/sel12, and be used to identify from relevant mRNA adult and embryo's black-tailed fruit flies.These mRNA are checked order and show the putative amino acid sequence height homologous open reading frame that contains with people's early ageing element.DmPScDNA is accredited as SEQ ID NO:20.

541 amino acid whose polypeptide of this sequence encoding (SEQ ID NO:21) have about 52% identity with people's early ageing.

The structure of black-tailed fruit flies homologue is to have at least 7 structures (the Kyte-Doolittle hydrophobicity analysis of window value with 15 and 1.5 cutoffs) of inferring people's early ageing element of membrane spaning domain similar.In containing the clone pds13 of 541 amino acid whose ORF, detect an alternative splicing form at least, and clone pds7, pds14 and pds1 do not contain the oligonucleotide of 1300-1341.This alternative splicing meeting makes the Gly on the residue 384 of inferring in TM6 → 7 rings become Ala, on the codon 399 of longer ORF, the frame endomixis black-tailed fruit flies aminoacid sequence and the main difference between the people's gene that produce the Glu residue are at the acid hydrophilic-structure territory of N-end and the acid hydrophilic segment of TM6 → 7 rings.Residue around TM6 → 7 rings is conservative (residue 220-313 is to 451-524) especially, shows that these are the important structural domains of function.The sudden change of identifying in people PS1 or PS2 also causes that 16 in 20 residues of people FAD are guarded in the black-tailed fruit flies homologue.

Clone's the dna sequence dna of DmPS gene is inserted in the Bluescript plasmid.On January 26th, 1996 this stable carrier is deposited in ATCC, Rockville, MD, preserving number are 97428.

The sign of 5 people's early ageing plain genes

AhPS1 transcript and gene structure

The hybridization of PS1 (S182) clone and Northern trace with the transcript of wide expression in many zones of brain and peripheral tissues be accredited as the transcript of main～2.8kb and the transcript of a spot of～7.5kb (referring to, Sherrington etc., the Fig. 2 in 1995).The expression of PS1 is quite even in most of zone of brain and the peripheral tissues except that liver, and transcribing in liver is very low.Although the identity of～7.5kb transcript is unclear, two kinds of observationss show～transcript of 2.8kb represents the active result of this gene.PS1 clone and the hybridization of the Northern trace of the mRNA that contains different rat tissues (comprising brain) have only identified that a size equals～transcript of 2.8kb people's transcript.The long cDNA clone (2.6-2.8kb) of all that will up to the present reclaim, comprise 5 ' and 3 ' UTR and be created on the Northern trace～2.8kb is with, and all is positioned the same physical area of karyomit(e) 14.

From these tests, the transcript of～7.5kb can represent rare alternative splicing or～the polyadenylation isomer of 2.8kb transcript, maybe can represent and another gene of PS1 homologous.Long transcript in the Caco2 cell cdna library of screening high-level PS1 of expression and PS2.Two different clones, GL40 and B53 have been obtained.The order-checking show these two clones all contain 5 ' similar UTR with the identical ORF of the short-and-medium 2.8kb transcript of brain.

These two clones all contain abnormal length 3 ' UTR.This length 3 ' UTR has represented to use the other polyadenylation site of the farther about 3kb in downstream.This length 3 ' UTR contains many nucleotide sequence primitives that cause palindrome or stem-ring structure.These structures are relevant with stability and the translation efficiency of mRNA, the practicality of this observations is that it may produce recombinant expression construct body and/or transgenosis (wherein having removed polyadenylation site, upstream), forces thus to use polyadenylation site, downstream and long 3 ' UTR.Under specific situation, can promote the stability of selected mRNA kind like this, by kind being treatment or utilizing virus vector as the herpes simplex virus vector modified form as gene therapy, can utilize preferential translation change target cell system or even body in the balance of mutant and wild-type transcript in the brain.

The hPS1 gene is striden the genome interval of 60kb at least, and is cloning in 57-H10,1-G9 and the 24-D5 from the 200kbPAC1 clone RPCI-154D12 in Roswell ParkPAC library with from 3 overlapping clays of Los Alamos karyomit(e) 14 cosmid libraries at described interval.The PSI gene transcription originally contains the RNA from 13 exons, and they are to identify with subclone PAC and clay clone's restricted fragment and by directly these subclones being checked order by the recross of oligonucleotide and Partial cDNA probe.Exons 1-4 contains 5 ' UTR, simultaneously exons 1 and 5 ' other end of the 2 described transcripts of expression.4-13 contains ORF at exon, and the montage process of Gai Bianing has caused removing the whole of the part of exon 4 or exon 9 simultaneously.Exons 13 also comprises 3 ' UTR.

Unless otherwise indicated, for clarity and brevity, all nucleotide positions in the hPS1 derivatized nucleotide sequence all use the base numbering among the SEQ ID NO:1 (L42110), from the hPS1 cDNA sequence of exons 1.In this cDNA, with exons 1 and the direct montage of exon 3, then with exon 4-13 montage.In SEQ ID NO:1, exons 1 is Nucleotide from 1 to 113, and exon 3 is from the position 114 to 195, exon 4 is from the position 196 to 335, exon 5 is from the position 336 to 586, and exon 6 is from the position 587 to 728, and exon 7 is from the position 729 to 796, exon 8 is from the position 797 to 1017, exon 9 is from the position 1018 to 1116, and exons 10 is from the position 1117 to 1203, exons 11 from 1204 to 1377, exons 12 is from the position 1378 to 1496, and exons 13 is from the position 1497 to 2765.Equally, unless otherwise indicated, all amino acid positions in the hPS1 deutero-protein sequence all use the residue numbering among the SEQID NO:2 (translation product of SEQ ID NO:1).

Obtained the side genome sequence of exons 1-12, and be listed among the SEQ ID NO:5-14 (accession number is L76518-L76527).Also determine 5 ' genome sequence of exons 13, be listed in SEQ ID NO:15 (accession number is L76528).SEQ ID NO:5-14 also comprises whole exon sequences.But SEQ ID NO:15 does not comprise 3 ' end of exons 13.Genome sequence corresponding to exons 1 and 2 is positioned on the 2.6kbBamHI-HindIII fragment (SEQ ID NO:5) of being separated by with about 240bp.Exon 3 and 4 (containing the ATG atg start codon) is positioned at one fen 3kb BamHI fragment of opening.Do not identify the complete sequence of the intron 2 between exon 2 downstream～BamHI site, 850bp place and exon 3 upstream～BamHI site, 600bp place as yet, also fail by using PCR to reclaim described complete sequence immediately, illustrate that intron 2 understands very big from the primer of BamHI site side.

Nucleotide sequence (SEQ ID NO:5) analysis revealed around the exons 1 and 2 has comprised the CpG dinucleotides of the many NotI of containing restriction sites in introne 1.In the 5 ' sequence (SEQ ID NO:5) of introne 1 and exons 1, there are several consensus sequences of inferring transcription regulatory protein that contain many group activator albumen-2 (AP-2), the signal conduction factor and transcriptional activation agent (STAT3) (Schindler and Darnell, 1995), γ activator sequence (GAS or STAT1), open beginning element downstream, site (MED) (Ince and Scotto, 1995) and GC element more.Two TATA boxes of inferring are present in the upstream of exons 1, at bp925-933 and the 978-987 of SEQ ID NO:5, are that two transcribing of inferring are opened the beginning consensus sequence then, 484 bp1002-1007 and the 1038-1043bp of SEQ ID NO:5.On the contrary, the upstream sequence that is right after of exon 2 does not have TATA box or CAP site, but enrichment CpG island.

Fig. 1 has listed the structure organization signal collection of illustrative plates of hPS1 gene.With the non-coding exon of entity shadow box indicating.Show the coding exon with empty frame table, show other montage sequence with drawing hatched frame table.Restriction site is: B=BamH; E=EcoRI; H-HindIII; N=NotI; P=PstI; V=PvuII; X=XbaI.At the undetermined genome sequence of locus of discontinuity domain representation between the restriction site on the sea line.The cloned genes group fragment of representing to contain each exon with the two-way horizontal arrow.The size of genome subclone and the preserving number of each genome sequence are provided.

With in the upstream 290bp of exons 1 and introne 1 in nucleotides sequence classify the basis as, the prediction of DNA secondary structure shows several palindromes, its stability is greater than 16 kcal/mol.These secondary structure analysis are also predicted and are existed 3 stable stem-cyclic group units (at bp 1119-1129/1214-1224; At bp 1387-1394/1462-1469; At bp1422-1429/1508-1515, all in SEQID NO:5) and one be enough to comprise a nucleosome (～76bp) ring.Described stem-ring structure is the common trait (Kollmar and Farnham, 1993) that contains the TATA gene.

In table 1, summed up the feature in these 5 ' districts.All base positions are reference with SEQ ID NO:5 all.

In SEQ ID NO:1,467 amino acid whose albumen among the open reading frame of the longest expection coding SEQ ID NO:2.The atg start codon of this open reading frame is the first box ATG that is positioned at TGA terminator codon downstream.There is not typical Kozak consensus sequence (Sherrington etc., 1995) around the interior ATG codon of preceding two frames.Do not have the gene of typical " by force " atg start codon the same with other, people's transcript infer 5 ' UTR enrichment GC.

Alternative transcription and the montage of B hPSl 5 ' UTR

Although the 4th exon of preceding 3 exons and part contains non-translated sequence, but analyze from people hippocampus cDNA library (Stratagene, La Jolla CA) in and colon adenocarcinoma cell system (Caco2 of J.Rommens) isolating many full length cDNA clones show in the major part clone, the sequence of beginning is from exons 1, then directly and exon 3 montage (accession number L42110, SEQ ID NO:1).Seldom the situation of occurring is (9 clone in 1), and the sequence of transcriptional start is from exon 2, and with exon 3 montage (accession number L76517, SEQ ID NO:3).Can not identify any clone who contains exons 1 and exon 2 simultaneously with the direct nucleotide sequencing of isolating at least 40 the independent RT-PCR transcripts of the primer in the exons 1, last, the genome sequence of inspection exon 2 upstream does not have 3 ' splice site sequence.The explanation of these observationss, exon 2 is real beginning exon, rather than the alternative splicing form or the counterfeit cDNA clone of the transcript that begins at exons 1.In addition, owing to the clone (cc44) who has obtained containing exon 2 from identical mono-clonal Caco2 clone, so, in identical cell, may there be transcript that contains exons 1 and the transcript that contains exon 2 simultaneously.

Open the prediction that the beginning site is done in order to test to classify transcribing of basis as with near the nucleotides sequence of 5 ' the upstream exons 1, we have detected 3 independence " total length " cDNA clones' (cc33, cc58 and cc48) that contain exons 15 ' end sequence and have utilized the antisense primer that is positioned at exon 3,3 sequences that reclaim by primer extension.Found 5 ' extension farthest in cDNA G40L, it is positioned to contain in the genome sequence of exons 1 SEQ ID NO:5 (L76518), the most approaching position 1214bp that opens the beginning site that transcribes, therefore, corresponding to the position-10 of SEQ ID NO:1.Two other clone (cNDA cc48 and 5 ' RACE product #5), the position 1259bp in gene order SEQ ID NO:5 have common to open the beginning site, and it is corresponding to the position 34 of SEQ IDNO:1.Two remaining cDNA and remaining 5 ' RACE clone farther position in the exons 1.5 ' RACE clone #8 starts from the 1224bp corresponding to position 1 among the SEQ ID NO:1.Therefore, among these clones, none extends to the CAP site of exons 1 upstream.Owing to contain seldom, therefore, do not carry out the similar beginning site research of opening from the transcript that opens the beginning sequence of exon 2.

The alternative splicing of C hPS1 ORF

Except that there being difference to open the transcript of beginning sequence, analyzing never to be further illustrated in the many cDNA clones that reclaim in the library has two kinds of variations that influence ORF in the PS1 transcript.

Wherein first is 12 Nucleotide that do not have 3 ' end of exon 4, the Nucleotide 324-335 among the SEQ ID NO:1.This situation results from the montage after 4 montages of the exon behind the Nucleotide 323 have replaced Nucleotide 335.The transcript that produces from the alternative splicing of the advancing exon 4 amino-acid residue Val26-Arg27-Ser28-G1n29 the SEQ ID NO:2 that do not encode.The institute that is checked in a organized way in, the transcript that produces from the alternative splicing of these two kinds of exons 4 has approximately identical frequency.Should note, in the clone who is up to the present checked, mouse PS1 transcript only contains the cDNA sequence of Ile26-Arg27-Ser28-Gln29 really, and the sequence of Val-Arg-Ser-Gln primitive is partly conservative in people PS2, i.e. Arg48-Ser49-Gln50 (Rogaev etc. 1995).These observationss show that all these differences do not have material impact for PS1 performance appropriate functional.

The second kind of montage variation that influences ORF can cause there is not exon 9, is 1018 to 1116 Nucleotide in SEQ ID NO:1.Analysis shows brain (comprising the neocortex district that influenced by AD) and the main single transcript of expressing band exon 9 of several other tissues (muscle, the heart, lung, colon) from different tissues mRNA deutero-RT-PCR product.On the other hand, white corpuscle (but not being lymphocytoblast) also express do not contain exon 9 than short-form.The montage of prediction exon 9 changes 257 asparagicacid residue among the SEQ ID NO:2 has been become L-Ala, removed ensuing 33 residues, causing Threonine with the position 291 of exons 10 coding is that the proteic remainder of starting point forms the frame endomixis.

D hPS2 transcript

Do not characterize the genomic dna that comprises people PS2 gene as yet fully.Yet it is conspicuous that many similarities are arranged between PS1 and the PS2 gene.As if but the intron of these two genes/exon boundary is very similar or identical except that TM6 → 7 rings.

In many tissues (comprising the brain district), the summary of PS2 cDNA clone and Northern trace detected～the mRNA band of 2.3kb, and in muscle, cardiac muscle and pancreas～the 2.6kb band.Except that the very high corpus callosum of transcriptional level, the expression level of PS2 is very low in most of zone of brain.In skeletal muscle, cardiac muscle and pancreas, PS2 expression of gene level is than high relatively in the brain, two kinds of transcripts are respectively～2.3kb and～2.6kb.These two kinds of transcripts obviously are different from the PS1 transcript of 2.7kb, and under the rigorous degree condition of height, do not have cross hybridization with the PS1 probe.With the allelic cDNA Sequence Identification of hPS2 is SEQ ID NO:18 (accession number L44577).

The longest ORF in the total nucleotide sequence of described PS2 cDNA has predicted and has contained 448 amino acid whose polypeptide (SEQ ID NO:19), ATG codon in first phase place (the position 366--368 among SEQ IDNO:18 is the Kozak consensus sequence around it) open numbering.

As to PS1, the PS2RT-PCR product of analyzing from few kinds of tissues (comprising brain and muscle) RNA shows that two kinds of variable splice variants are arranged, and wherein montage has gone out relatively large fragment.Therefore, produce transcript with relatively low frequency, wherein the Nucleotide 1152-1250 of PS2 transcript (SEQ ID NO:18) (the residue 263-295 among the coding SEQ ID NO:19) is by alternative splicing.As discussed below, described montage process approaches the montage change (Rogaev etc., 1995) of PS1 exon 9 very much.

In all detected tissues, also found the splice variant of another PS2 cDNA sequence, this variant does not have Nucleotide 1338-1340 position GAA triplet among the SEQ ID NO:18.This montage change can delete the residue at the Glu of 325 amino acids.

The structure of 6 early ageing fibroins

A early ageing fibroin family

Now the early ageing element of discussing is the integral protein family of a new high conservative, and they have common structural motif, common montage to change the relevant saltation zone focus of structural domain (being present in many invertebratess and the vertebrate cells) of pattern and common and structural.Expection aminoacid sequence with Hopp and Woods Algorithm Analysis people early ageing plain gene shows that described albumen is multispan integral protein such as acceptor, channel protein or structural membrane albumen.In Fig. 2, listed and inferred the proteic Kyte-Doolittle hydropathic profile of hPS1.Hydrophilic figure and structural analysis show these albumen have about 7 by the isolating hydrophobic transmembrane structural domain of hydrophilic loop (being called TM1) to TM7 according to used parameter in predictor formula, measurable minimum 5 of other model, maximum 10 membrane spaning domains.The existence of 7 membrane spaning domains is features of several G-bonded receptor proteins, but also can be observed in other albumen (as channel protein).It should be noted that does not have discernible signal peptide, and glycosylation site is few.

In Fig. 3, compare the proteic aminoacid sequence of hPS1 and mPS1, in Fig. 4, compared the proteic sequence of hPS1 and hPS2.In these figure, represent identical amino-acid residue with straight line.With on the sequence or under sea line represent 7 membrane spaning domains of inferring.

Main difference between this family member is the aminoacid sequence at hydrophilic, the acyclic acidic structural domain of N-end, and the early ageing fibroin is inferred (TM6 → 7 rings) between TM6 and the TM7 structural domain.Major part has the residue (montage that takes place in some non-nervous tissues changes) of hPS1 exon 9 codings to form the part of inferring TM6 → 7 rings.As if the encode part of TM6 → 7 ring of the splice variant of the corresponding change of in hPS2, identifying in addition.The difference of cyclic amino acids sequence illustrates that described ring is described proteic critical function structural domain between the montage change of this hydrophilic loop and this gene family member, and can give the physiology of individual early ageing fibroin and cause a disease to interact and give some specificity.Because the acid electric charge that the terminal hydrophilic-structure of N-territory has with TM6 → 7 hydrophilic acidic rings are identical, in 7 membrane spaning domain models, have the direction identical with film, and between the early ageing element, be variable, so these two structural domains on functionality, enjoy be equal to or mode (as identical or different part or functional performance) independently.Therefore, might the N-end also be described proteic critical function structural domain, and can give the physiology of individual early ageing fibroin and cause a disease to interact and give some specificity.

As hereinafter going through, PS1 around TM1 → 2 rings and TM6 → 7 ring structure territories and PS2 group's pathogenic mutation illustrate that also these structural domains are these proteic functional domains.Fig. 5 and 6 has described the synoptic diagram of PS1 and PS2 albumen structural respectively, understands known mutational site at the figure subscript.As shown in FIG., prediction TM1 → 2 catenation sequences are positioned at terminal opposite film one side with N-, and TM6 → 7 also are like this, and shown in catenation sequence in striding the film contact, may be very important.Observed PS1 Y115H sudden change and may make described ring go other sudden change in the stable TM1/2 spiral to support this viewpoint in the familial AD of early onset thereof (30-40 year) pedigree is arranged.TM1 → 2 rings are relatively lacked (PS1: residue 101-132; PS2: residue 107-134), make these peptides are more suitable for carrying out conventional peptide analysis.7 PS1 sudden changes concentrate on about codon 82 to zone between the codon 146, and first membrane spaning domain (TM1), the TM1 → 2 ring and TM2 structural domains that infers among the PS1 contained in this zone.Equally, the sudden change of the last codon 141 of PS2 also is positioned at the TM2 structural domain.These sudden changes may make TM1 → 2 ring and its anchor point instabilities in TM1 and TM2.12 PS1 sudden change has caused changing the amino acid between about codon 246 and 410, and these codons relate to TM6, TM6 → 7 ring and TM7 structural domains.These sudden changes may be modified the structure of TM6 → 7 rings or stability (directly or by modifying the conformation of TM6 or TM7).

It is that TM6 → 7 ring centre portionss (about amino acid 300-371) in the different members of early ageing fibroin family have the sequence divergence that TM6 → 7 ring has other evidence of critical function.Equally, because early ageing fibroin family member's N-end sequence is also inequality, thus might give variant early ageing fibroin with the function specificity by slight sequence difference, and conserved sequence makes it have identical biological activity.The ligand-binding site point is represented in the central zone.If like this, in the possible modified ligand of the sudden change in TM6 → 7 districts in conjunction with activity.Between early ageing fibroin family different members, TM6 → 7 rings are guarded, and this may represent an effector domain on opposite film surface.Except that the montage sudden change of exon 10, most of other (missense) sudden change is being inferred on the similar face of transbilayer helix.Therefore, available these structural domains (as TM6 → 7 rings and TM1 → 2 rings) as site development specific-binding agent, are suffered from the mutation effect of early ageing fibroin among the presenile dementia patient and/or are recovered described proteic normal function with inhibition.

The similarity that C.elegans SPE-4 and PS1 gene are inferred between the product illustrates that they have similar activity.SPE-4 albumen may be with in the spermatogeny process, the formation of felty body-theca cell device (FBMO) mixture with stablize relevant.FBMO becomes privileged golgi body-deutero-organoid, formed adhere to and part around the membrane-bound vesicle of parallel thiozell mixture, and may with solubility and film in conjunction with the transportation of polypeptide with store relevant.Sudden change in SPE-4 has destroyed the FBMO mixture, and suppressed spermatogeny therefore, the physiological function of SPE-4 may be the stabilizing membrane inherence sprout and fusion process between interaction, or in the spermatogeny process, stablize in the FBMO mixture cell interaction of film and fibrillin in the transportation.Can not think the similar functions of early ageing element.For example PS1 may work in the stop of other embrane-associated protein such as β APP, or as in golgi body or endosome-lysosome system, works during the film in the albumen transportation transports in conjunction with the vesicle aixs cylinder and fusion is sprouted.If these hypothesis are correct, so described sudden change can cause β APP transportation and process unusual and/or unusual with the interaction of cytoskeletal protein such as microtubule-associated protein Tau.β APP and Tau in fact are the immanent causes of Alzheimer's disease neurological feature in cell and extracellular undesired deposition.Although in inferring the albumen conserved domain, the position of PS1 and PS2 sudden change shows that they are pathogenic in the high conservative residue, has at least 3 itself to guard in these sudden changes, and this outbreak with the Adulthood disease is consistent.Because in the viewed sudden change up to the present, none is that expection can cause disappearance or the nonsense mutation that expression or function completely lose, so we not these sudden changes of expectability whether have maniflest function acquisition effect, the dominance that promotes the abnormal processing of β APP thus or suppress normal beta APP processing obtains function.Exons 10 montage sudden change causes the frame endomixis of exon 9 and exons 10, and may structure function be arranged to changing in the cell guiding or part bonded PS1 albumen, maybe may influence the function of PS1.

Another kind of possibility is that the PS1 gene product may be represented acceptor or channel protein.Described proteic sudden change is relevant with several other dominance nervous system diseases in vertebrates (as malignant hyperthermia, the hyperpotassemic paralysis of philtrum) and the invertebrates (deg-l among the C.elegans (d) sudden change).Although the pathology of these other diseases are different with Alzheimer's disease, functional disorder is arranged also in the ionic channel of Alzheimer's disease.For example reported the imbalance aspect four-ethyl ammonium susceptibility l13ps potassium channel and calcium homeostasis.From the weak homology between PS1 and the voltage-dependent calcium channel protein alpha-ID subunit, the fluctuation of striding film calcium current amount may be relevant especially, and the increase of intracellular Ca2+ can increase the weight of the biochemical characteristics of Alzheimer's disease in the culturing cell, increases as the change of Tau-microtubule associated protein phosphorylation aspect and the production of A β peptide.

B hPS1 structure

Shown in SEQ ID NO:2, the proteic maximum form known of people PS1 contains 467 amino acid, and the molecular weight of expection is about 51.37kDa.Have few 4 amino acid of variant (wherein having lacked the residue of SEQ ID NO:2 position 26-29) that above-mentioned exon 4 montages change, molecular weight is about 50.93kDa.Equally, have few 33 amino acid of variant (wherein having lacked the residue of SEQ ID NO:2 position 258-290) that above-mentioned exon 9 montages change, molecular weight is approximately 47.74kDa.

In table 2, listed the position in structural territory.Point out once more, the numbering of residue position is with reference to SEQ ID NO:2, and is proximate (that is a residue, ± 2).

In Fig. 5, listed the synoptic diagram of inferring the PS1 structure.The N-end is a highly-hydrophilic, electronegative structural domain, several potential phosphorylation domains are arranged, what connect about 19 residues then strides film hydrophobic domains (TM1), the electrically charged hydrophilic loop of about 32 residues (TM1 → 2), wherein be distributed with 5 other hydrophobic transmembrane structural domains (TM2 is to TM6) in the hydrophilic-structure territory (TM2 → 3 are to TM5 → 6) of short (1-15 residue), acid hydrophilic charged ring (TM6 → 7) and at least 1 (TM7) that another is bigger, may be 2 other hydrophobic strong membrane spaning domains, these structures finish in the polar structure territory of C-end.

Described albumen also contains many potential phosphorylation sites, and one of them is the total site of map kinase, and also to be transformed into the super phosphorylation of Tau in the former entanglement process of nerve fiber relevant with normal Tau in this site.Described consensus sequence can provide the element of inferring, and this element connects other biochemical characteristics of described proteic activity and Alzheimer's disease, therefore, and the possible treatment target of described consensus sequence representative.Study described proteic structure and show two sequence YTPF are arranged (the residue 115-118 among the SEQ ID NO:2) and STPE (the residue 353-356 among the SEQ ID NO:2), their representatives are the 5/T-P primitives of map kinase consensus sequence.The active consensus sequence coexistence of several other phosphorylation sites and protein kinase C (PKC).Because PKC is active relevant with the difference in APP (with the related to alzheimer's disease) metabolism, so these sites on PS1 albumen and its homologue also are the sites of target therapeutical agent.Preliminary sign shows that in cells transfected, the proteic phosphorylation degree of PS1 is very low at least, and PS2 albumen is by remarkable phosphorylation.At least for PS2, described phosphorylation is by occurring in serine residue (Capell etc., 1996) in the N-end structure territory with the irrelevant mechanism of PKC.

Should note changing, make and removed 4 amino acid, and think to have removed the phosphorylation consensus sequence from N-end structure territory in the montage of exon 4 ends.In addition, the montage of exon 9 change has produced the proteic isomer that blocks of PS1, and 5 hydrophobic residues that wherein removed TM6 C-end encircle with the hydrophilic electronegative TM6 of part that is right after TM6 → 7.Described montage change isomer be characterised in that kept N-terminal to and comprise the sequence of SEQ ID NO:2 position 256 tyrosine, the aspartic acid of position 257 is become L-Ala, with from and comprise the proteic C-terminal portions montage of tyrosine 291.Described montage difference is usually relevant with described proteic functional domain.This this hydrophobic ring of explanation (with the terminal hydrophobic ring of the N-with similar amino acid electric charge) is the active function structural domain of PS1 product, therefore is the target site of treatment

The structure of C people PS2

People PS1 and PS2 albumen always have 63% amino acid identity, and there have several structural domains to have to be identical substantially.Therefore, as expected, hydropathy analysis shows that two kinds of albumen have similar structure organization.Therefore, think that these two kinds of albumen all have 7 hydrophobic transmembrane structural domains of inferring, and these two kinds of albumen all there is bigger acid hydrophilic-structure territory between N-end and TM6 and TM7.Further similarity is from the above-mentioned analysis of the RT-PCR product of brain and muscle RNA, and the Nucleotide 1153-1250 of described analysis revealed PS2 transcript has different montage modes.These nucleotide coding amino acid 263-296, these amino acid are positioned at and infer the proteic TM6 of PS2 → 7 ring structure territories and with amino acid 257-290 that the PS1 montage changes 94% identity is arranged.

The position of the proteic estimation function structural domain of hpS2 has been described in table 3.Should notice that the residue position refers to the residue position among the SEQ ID NO:19, and described position is proximate (that is a residue, ± 2).

Listed the synoptic diagram of inferring the PS2 structure among Fig. 6.Between corresponding to TM1 and TM6, from TM7 to PS1 several protein structure domains of PROTEIN C-end, the similarity maximum between hPS1 and the hPS2.The main difference part of PS1 and PS2 is the aminoacid sequence that its size and electronegative hydrophilic TM6 → 7 are encircled, the sequence in ethyl n-terminal hydrophilic-structure territory.

The most noticeable difference is at TM6 → 7 hydrophilic loops (residue 304-374 of hPS1 between two kinds of expection aminoacid sequences; The 310-355 of hPS2) the hydrophilic-structure territory of the aminoacid sequence of centre portions and N-end.Equally, the conservative property of this structural domain between mouse and people PS1 gene lower (identity=47/60), and do not have similarity with the equivalent regions of SPE-4.

The plain mutant of 7 early ageing

A PS1 mutant

Several mutant that cause several typical familial Alzheimer's diseases in the PS1 gene have been identified.In these mutant one or its combination have caused other nervous system disease of the Alzheimer's disease of this form and several.As long as can cause expecting that the change in the aminoacid sequence maybe can cause abnormal transcript processing, level or stability, described sudden change can be that any nucleotide sequence replaces, inserts or disappearance.Hereinafter with described nucleotide and/or aminoacid deletion or replace the concrete pathogenic mutation of form, but in other family, also can find other sudden change.Really, after since 8 different pedigrees, finding 5 kinds of different missense mutation (Sherrington etc., 1995), from from other heredopathia through examine (as with Ca ²⁺The amyotrophic lateral sclerosis that sudden change in the superoxide dismutase gene is relevant), also can identify other sudden change.This expection because of we find subsequently other sudden change (Rogaev etc., 1995) and other people in the early ageing element divide similar discovery (as, Cruts etc., 1995; Campion etc., 1995) and obtain proof, therefore with regard to this paper PS1 gene and albumen, term " mutant " is not limited to these specific sudden changes, but is interpreted as the said mutation body.

Directly the overlapping RT-PCR product to the isolating 2.8kbS182 of comprising transcript from 6 the big pedigree ill members relevant with karyomit(e) 14 checks order, thus at first found this 6 and pedigree in 5 missense mutation.In each pedigree, these sudden changes and disease be divided into from, but from 142 of the identical ethnic group of FAD pedigree irrelevant neural normal subjectses in do not have (284 independently karyomit(e)).Determine with the isolating physical separation of AD3 feature in the position of gene, clear and definite isolating 8 different missense mutation are relevant with karyomit(e) 14 altogether with genius morbi in 6 pedigrees, are AD3 seats and do not have the statement of facts PS1 gene of these sudden changes in 284 independent normal dyeing bodies.Other biological evidences of this hypothesis is that the sudden change in FAD is of the same race is (as the hPS1 to mPS1) of high conservative in evolution, described sudden change is positioned at other vertebrates and the conservative described proteic structural domain of invertebrates homologue camber, and it is regional in the major part of brain, comprise being subjected in the zone that AD has a strong impact on that the PS1 gene product is with high level expression.

Because the new discovery of PS1 gene has been listed with AD relevant many other sudden changes has been taken place.Table 4 pair described sudden change characterizes.Think inferring viewed nucleotide deletion among the ORF or replacing and change the amino acid that is encoded in described position at the PS1 transcript.With reference to its nucleotide position and its amino acid position in SEQ ID NO:2 in SEQ IDNO:1, listed described sudden change." NA " expression does not obtain data.

Following joint will be discussed, and also find many PS2 sudden changes.The sequence of having listed hPS1 and hPS2 in Fig. 4 compares, and illustrates that these pathogenic mutations are in the zone of PS2, and these zones are high conservatives in PS1 albumen.Therefore, the corresponding sudden change that is expected in the PS1 albumen also is morbific, and be included in this paper provide and available PS1 mutant in.In addition, infer that any pathogenic mutation identified represents the plain mutant of early ageing of other total described conserved regions in any early ageing plain gene conserved regions.

Interesting is, sudden change 260V, C263R, P264L, P267S, E280A, E280G, A285V, L286V, Δ 291-319, G384A, L392V and C410Y all occurs in or near the acid hydrophilic loop of inferring between membrane spaning domain TM6 and the TM7.Also 8 (260V, C263R, P264L, P267S, E280A, E280G, A285V, L286V) in these sudden changes are positioned at other montage structural domain (the residue 257-290 of SEQ ID NO:2).

With the RT-PCR product of ripe mRNAcDNA of representative or genomic dna, can detect all these sudden changes by the whole bag of tricks (directly nucleotide sequencing, allelotrope homology oligonucleotide, connection polymerase chain reaction, SSCP, RFLP, new " DNA chip (chip) " technology etc.).

At last, should note also having found not have several polymorphisms of obvious deleterious effect.One of them is among the SEQID NO:1.863 Nucleotide T → G change the F205L polymorphism that causes among the TM4.Other takes place (at C → A of bp1700; G → A at bp2603; The bp2620 disappearance) in 3 ' UTR.

B PS2 mutant

Strong similarity between PS1 and the PS2 gene product has shown in a small amount of wherein genetic linkage research and has got rid of in the AD pedigree of early onset thereof of karyomit(e) 14,19 and 21 that the PS2 gene may be the possibility in the site of pathogenic mutation.With the cDNA of RT-PCR separation, wherein study the sudden change of having got rid of in β APP and the PS1 gene with directly checking order corresponding to the PS2 transcript of cerebral tissue after 8 pedigrees (FAD that suffers from early onset thereof) patient's lymphocytoblast, inoblast or the necrotomy.

In all 4 the Italian patients (Flo10) that extend pedigrees, check that these RT-PCR products replace at heterozygosis A → G of Nucleotide 1080, described Italian suffers from the FAD that the pathology of early onset thereof determine (50-70 year outbreak).Think that the codon 239 of described sudden change in TM5 caused Met → Val missense.

Patient at the relevant pedigree of the Germanic ancestors of volga (uses clone AG09369, AG09907, AG09952 and AG09905, Coriell Institute, Camden NJ representative) in, the codon 141 in TM has found to cause that (at Nucleotide 787 are A → T) in second sudden change that Asn → Ile replaces.Be apparent that this sudden change of a patient (AG09907) is isozygotied, this conforms to the inbreeding feature of these pedigrees.In addition, this patient significantly is not different from the heterozygosis patient's of N141 I sudden change clinical image.The PS2 transgenation is not only found in 284 Caucasian's contrasts, and is not existed in the pedigree patient who suffers from AD3 type AD yet.

Think the replacement of the residue that these two kinds of PS2 sudden change all can causing PS1/PS2 gene family inner heights are conservative.

Make the replacement that Ile → Thr takes place on the C-termination codon 420 by the T in base pair 1624 → C replacement, thereby produced other PS2 sudden change.In the AD of family of other early discovery (45 years old) case, found this sudden change.

With reference to the nucleotide position of SEQ ID NO:18 and the amino acid position among the SEQ ID NO:19, these hPS2 sudden changes in table 5, have been listed." NA " expression in table does not obtain data

As discussing, many PS1 sudden changes have also been found in prosthomere.In Fig. 4, listed the comparison of hPS1 and hPS2 sequence, show that these pathogenic mutations are arranged in PS1 albumen zone, but described zone has been a high conservative in PS2 albumen.Therefore, think that the corresponding sudden change in PS2 is morbific, and be included in this paper provide and available PS2 mutant in.In addition, think that any pathogenic mutation representative of identifying has the mutant of other early ageing element of described conserved regions in any early ageing plain gene conserved regions.

Found that its product and PS1 gene product have the basic amino acid and the gene of structural similarity, show that these albumen may be that function is relevant as the independent albumen with overlapping function, but, may have slightly different specific activity as the relevant subunit of physics of polypeptide or as the independent albumen of in identical approach, finishing continuous function.

Think after 3 different missense mutation of observation in the PS2 albumen conserved domain of suffering from familial AD patient, may be the same with in the PS1 gene those, these sudden changes may be the paathogenic factors of AD.This conclusion is obviously, although (30-50 year outbreak, continue 10 years) those relevant with the sudden change of PS2 base (show effect in 40-70 year because the disease phenotype relevant with the PS1 transgenation; Continue to reach 20 years) imperceptible difference is arranged, be the major reason of early onset thereof AD at least but total similarity clearly illustrates the included bio-chemical pathway of this gene family member.Described nuance in disease phenotype has reflected that the expression level of PS2 transcript in CNS is lower, may reflect that maybe the PS2 gene product has different effects.

Similar to the effect of PS1 sudden change, APP (representative of amyloid precursor) is produced unusually to the processing of A β peptide, the super phosphorylation of Tau microtubule-associated protein, and the homeostasis of intracellular Ca2+ is not normal.Stop these abnormal interaction to provide method for treatment AD.

At last, found at least a nucleotide polymorphisms in a normal individual, T → C has taken place at the bp626 of SEQ ID NO:18 and has changed in the PS2cDNA of described individuality, but in coded aminoacid sequence without any change.

The III preferred embodiment

Partly open based on this paper and describe discovery provides following preferred embodiment of the present invention.

1 isolating nucleic acid

In a series of embodiment, the invention provides corresponding to or be relevant to the isolating nucleic acid of the plain nucleotide sequence of early ageing disclosed herein.As hereinafter discussing more comprehensively, these sequences comprise from people and other mammiferous normal PS1 and PS2 sequences, homologous sequence from nonmammalian such as fruit bat and C.elegans, the subsequence that can be used as these sequences of probe and PCR primer, early ageing fibroin or corresponding to these encoding sequence fragment subsequences in ad hoc structure territory or polymorphic district, corresponding to segmental complementation of early ageing plain gene or antisense sequences, wherein the sequence that links to each other can be operated with the external source regulatory region in the plain coding region of early ageing, and the encoding sequence that is fused to the early ageing plain fusion protein on other albumen, described other albumen are as presentation markup, purifying " mark " or be used to screen and detect the interaction of albumen and early ageing element

Therefore, in the embodiment of first series, provide coding normal or mutant PS1 and the proteic isolated nucleic acid sequences of PS2.Herein disclosed is the example of described nucleotide sequence.These nucleic acid can be that genome sequence (as SEQ ID NO:5-15) maybe can be cDNA sequence (as SEQ IDNO:1,3,16 and 18).In addition, described nucleic acid can be recombination or " minigene ", wherein all or some intron various combinations of intron and exon and local cis-acting regulatory element can be processed into propagation or expression construct or carrier.Therefore, for example the invention provides nucleotide sequence, wherein mix montage as herein described and change variant, therefore make the cell that comprises these sequences only express a kind of splice variant in each montage position at dna level.For example, can produce a recombination, wherein 3 ' terminal (bp1337 of SEQ ID NO:5) with the exons 1 of PS1 gene directly links to each other with 5 ' terminal (bp 588 of SEQ ID NO:6) of exon 3, so that only produce corresponding to the main transcript of transcript.Obviously, people can produce the recombination that " forcing " montage changes exon 2 and exon 3.Equally, can produce other recombination, wherein one of the exon 4 of PS1 or exon 9 splice variants (or corresponding PS2TM6 → 7 splice variants) are incorporated among the DNA, so that make the cell that comprises described recombination only express one of these variants.In order to reach the purpose of the early ageing plain gene that reduces to recombinate, can be with cDNA gene or the various combinations of from DNA construct, removing intron and not translating exon.At last, can produce wherein that 5 ' UTR is changed, so that must transcribe or another recombination that carries out that opens the beginning site from two.As mentioned below, described construct is identifying that it can be useful especially can inducing or suppress in the plain compound of expressing of early ageing.By the detailed description of this paper, now can obtain many variants of these embodiments to the early ageing plain gene.

Except that the plain sequence of disclosed early ageing, this area professional can identify now and separate the nucleic acid of representing early ageing plain gene or cDNA that described cDNA is the allelotrope or the different homology homologue of open sequence.Therefore, the invention provides isolating nucleic acid corresponding to these allelotrope and homologue, and by method well known in the art, from the various above-mentioned recombinant precursors of these sequence deutero-.In a word, now can the screening-gene group with probe or PCR primer this area professional or cDNA goods (comprising) and bacterium, virus, yeast or other genome or cDNA library from the sample of individual organism (as people AD patient or its family member) preparation to identify allelotrope or homologous sequence.Owing to need to identify other early ageing plain gene sudden change that causes AD or other disease, owing to need to identify the plain polymorphism of other early ageing of no pathogenicity and owing to also need to produce the various animal models that are used to study AD and screen potential therapeutical agent, so special imagination is from other goods or people's nucleic acid library and the early ageing element sequence of separating other from the goods of the animal that comprises rat, mouse, hamster, cavy, rabbit, dog, cat, goat, sheep, pig and non-human primates or library.In addition, comprise C.elegans and other nematode, and the plain homologue of the early ageing of fruit bat and other insects also there is the purposes of drug screening from yeast or invertebrates.For example can be used to block the medicine of mutant gene effect with the invertebrates screening of carrying the plain homologue of sudden change early ageing (or the plain transgenosis of Mammals early ageing) (causing the phenotype (growing) of very fast generation and easy record) as abnormal vaginal orifice after a couple of days or eye.The vertebrates that described invertebrates is bigger can be screened more in a large number.After having found the prompting compound, just can in higher animal, detect it by described screening.

Utilize short relatively early ageing plain gene sequence, can be with standard screening by hybridization or round pcr (as being used to identify the mPS1 gene) to identify and/or separation allelotrope and homologous sequence.According to the character of target sequence, used method and required specificity, described sequence can comprise 8 or Nucleotide still less.The development of WeiLai Technology makes and can use even shorter sequence.With regard to present technology, 9-50 Nucleotide, and also the sequence of about 18-24 Nucleotide is preferred.These sequences can be selected from sequence disclosed herein, or derive from available other allelotrope of this paper or different specificity homologue.When detecting mRNA or screening cDNA library, preferred probe and the primer that uses from encoding sequence (rather than intron) avoided usually sequence deleted in splice variant, unless need to identify those variants especially.Think that the allele variant of early ageing element can be under rigorous hybridization conditions (as herein defined), with disclosed sequence hybridization, and available lower rigorous degree is identified different specificity homologue.

In another serial embodiment, the invention provides the isolating nucleic acid that comprises the plain sequence subsequence of early ageing or its complement.As mentioned above, the evaluation of the allelotrope of early ageing plain gene and homology variant with separate, described sequence has as probe and PCR primer purposes.Determine special purposes is also arranged corresponding to the subsequence (as mentioned above) of the plain polymorphic regions of early ageing in screening and/or the idiotype that is used for diagnostic purpose.In addition, as described below, described subsequence has the fragment of coding (1) contained early ageing fibroin in fusion rotein, (2) contain early ageing fibroin functional domain be used in conjunction with research fragment, (3) fragment of early ageing fibroin, available described fragment is as antibody and (4) early ageing plain fragment of immunogen to produce the premature senescence resistance fibroin, described fragment as the competitive inhibitor of early ageing element or stand-in to suppress or to simulate its physiologic function.At last, described subsequence codified or representative are hybridized to suppress complementation or the antisense sequences that described sequence is transcribed or translated with early ageing plain gene or the plain mRNA transcript of early ageing under physiological condition.Therefore, according to needed purposes, the invention provides length from 8-10 Nucleotide (as probe) to nucleic acid subsequence near the early ageing plain gene of the early ageing plain gene group of total length or cDNA.Therefore, the invention provides isolated nucleic acid sequences, described nucleotide sequence contains corresponding to the 8-10 at least of early ageing plain gene, and preferred 15, the more preferably sequence of the sequence of at least 20 continuous nucleotides (as this paper open or obtain) or corresponding its complement.But as mentioned above,, can use short sequence according to different technology.

In another serial embodiment, the invention provides nucleic acid, wherein have or do not contain intron or as mentioned above the plain encoding sequence of early ageing of recombined engineering can operate with endogenous or external source 5 ' and/or 3 ' regulatory region and link to each other.This paper describes the interior living regulatory region of hPS1 gene in detail.With open and standard genetic technique (extend as PCR, the guiding gene step looks into) of the present invention, this area professional also can clone and give birth to regulatory region in corresponding hPS25 ' and/or 3 '.Equally,, can obtain the allele variant of hPS1 and the endogenous regulatory region of hPS2 by test within reason, and from the endogenous regulatory region of other Mammals homologue.In addition, endogenous regulatory region (promptly from different allogeneic regulatory regions or different specificity regulatory region) can be operated with the plain encoding sequence of early ageing and link to each other so that guide expression.5 ' suitable regulatory region comprises promoter element and can comprise that also other element such as operon or enhancer sequence, ribosome binding sequence, RNA add cap sequence etc.Sequence and its combination of optional self-acting control protokaryon of described regulatory region and eukaryotic cell, its viral genetic expression.Described regulatory region includes, but are not limited to lac system, trp system, tac system and trc system; The main operon and the promoter region of lambda particles phage; The control region of fd envelope protein; Early stage and the late promoter of SV40; From polyomavirus, adenovirus, retrovirus, baculovirus and monkey disease poison deutero-promotor; 3-phoshoglyceric acid kinase promoter; Leavening acid acid phosphatase promotor; Yeast alpha factor promotor; The promoter element of other eukaryotic gene of in the cell of neurone or other type, expressing; With its combination.Particularly, can select to induce the regulatory element (as the beta-galactosidase enzymes promotor) that maybe can prevent so that in these nucleic acid cell transformed, carry out controllable and/or exercisable expression.In addition, the plain coding region of early ageing can be able to be operated with the regulatory element that tissue specific expression is provided in multicellular organisms and link to each other.Described construct is useful especially for producing transgenic organism so that only express the early ageing plain gene in suitable tissue.In the ability that is chosen in this area professional and judgement scope of suitable regulatory region, and the reorganization purposes of described regulatory region also is well known in the art.

In another serial embodiment, the present invention provides the isolating nucleic acid of coding all or part early ageing fibroin with the form of fusion rotein.In these embodiments, nucleic acid regulatory region (endogenous or external source) can be operated with first coding region and link to each other, and described first coding region links to each other with second coding region covalency in frame.Described second coding region can link to each other with one or more other coding region covalency on demand, and last coding region and terminator codon link to each other with suitable on demand 3 ' regulatory region (as polyadenylation signal).The plain sequence of the early ageing of fusion rotein can be represented first, second or any other coding region.The plain sequence of described early ageing can be structural domain that guard or nonconservative and can be placed in any coding region of fusion rotein.Select the plain sequence of non-early ageing of fusion rotein according to practitioner's needs and judgement, but be not subjected to restriction of the present invention.But the plain sequence of useful non-early ageing include help to identify or the short sequence " mark " of purifying gained fusion rotein as epitope or poly-His mark.In addition, albumen that the plain coding region of non-early ageing codified is bigger or protein fragments as help to identify and purifying as described in proteic or in detecting (as mentioned below those) useful enzyme or conjugated protein.Concrete early ageing plain fusion protein comprises and is used to separate and the poly-His and GST (glutathione S-transferase) fusion rotein of purifying early ageing element, the yeast two-hybrid fusion rotein that is used for identifying other proteic detection hereinafter described, described other albumen combines with the early ageing element or interacts.

In another serial embodiment, the invention provides the isolating nucleic acid of recombinant DNA construction bodily form formula, wherein mark or reporter gene (as beta-galactosidase enzymes, luciferase) and 5 ' regulatory region of early ageing plain gene can operate link to each other in case as described in express under the plain control of regulating sequence of early ageing as described in marker gene.Utilize the plain regulatory region of the open or available early ageing of this paper, comprise that this area professional can produce described construct from the regulatory region of people and other mammiferous PS1 and PX2 gene.As hereinafter discussed in detail, can be used for cell, clone or the transgenic animal of authenticating compound with described isolating nucleic acid production, described compound can directly or indirectly differentially influence the expression of described early ageing element.

At last, in the time of in being included in carrier, the isolating nucleic acid of the present invention comprises above-mentioned any sequence.Suitable carrier comprises all types of cloning vectors and expression vector, comprises plasmid, phagemid, clay, episome etc. and integrative vector.Described carrier can comprise various marker gene (as antibiotics resistance or sensitivity genes), and described marker gene is used to identify the cell that has successfully transformed with it.In addition, described carrier can comprise nucleic acid of the present invention can operate continuous adjusting sequence with it, and/or can comprise the coding region so that nucleic acid of the present invention after linking to each other with carrier is suitable, is expressed with the form of fusion rotein.Described carrier is included in used carrier, baculovirus and phage display system in the yeast " double cross body ".Select carrier to be used for protokaryon, eucaryon or expressing viral according to the needs of application in practice.For example, vaccinia virus vector or contain the monkey disease poisonous carrier (as pSV2) of SV40 promotor or hsv or adeno-associated virus can be used for transfection mammalian cell, comprise and cultivating or intravital neurone that baculovirus can be used for transfection insect cell (as the butterfly cell).On the market many different carriers are arranged now and be well known in the art, so in the ability that is chosen in this area professional and judgement scope of appropriate carrier.

2 pure substantially albumen

The invention provides pure substantially early ageing fibroin goods, early ageing fibroin fragment and comprise plain and its segmental fusion rotein of early ageing.As described herein, described albumen, fragment and fusion rotein at the antibody of producing anti-normal or sudden change early ageing element, identify in early ageing plain conjugated protein and diagnosis and the methods of treatment that effectiveness is arranged.Therefore, according to needed purposes, the invention provides pure substantially albumen that contains aminoacid sequence or peptide, described aminoacid sequence is the subsequence of complete early ageing fibroin, and length from 4-10 amino acid (as immunogen) or 10-100 amino acid (as being used for) in conjunction with testing to complete early ageing fibroin.Therefore, the invention provides pure substantially albumen or peptide, described albumen or peptide contain 4-5 at least, preferred 6-10 of corresponding early ageing fibroin (maybe can access as disclosed herein), the more preferably sequence of at least 50 or 100 continuous amino acids.

All separable and purifying albumen of the present invention or peptide with any method, described method are that the character that is disclosed with its protein sequence serves as that the basis is selected.Because the early ageing element has the characteristic of intrinsic or transmembrane protein,, extract albumen by for example stain remover dissolving then so can separate the film fraction of the cell (as neurone, mesoglia, muscle, pancreas) of wherein highly expressing the early ageing element in general.From with expression vector (as rhabdovirus system such as pPbac and pMbac carrier (Stratagene, LaJolla, CA)); Yeast expression system is (as pYESHIS Xpress carrier (Invitrogen, San Diego, CA)); Eukaryotic expression system if any constant constructive expression's pcDNA3 (Invitrogen, SanDiego, CA) or derivable LacSwitch (Stratagene, La Jolla, CA); Or prokaryotic expression carrier such as pKK233-3 (Clontech, Palo Alto, CA) purifying early ageing fibroin, fusion rotein or its fragment in conversion or the cells transfected.Be incorporated in the process of the endoplasmic reticulum of reconstitution cell (as fixed human cell line or other eukaryotic cell) or plasma membrane in albumen or fragment, can be from the film fraction the described albumen of purifying.In addition, if albumen is arranged in or accumulates in the inclusion body of reconstitution cell (as prokaryotic cell prokaryocyte) inadequately, then can from the dissolved cell or from the dissolved inclusion body the described albumen of purifying.

Can finish purifying with the standard protein purification method, described purification process comprises, but be not limited to gel permeation chromatography, ion exchange chromatography, high performance liquid chromatography (RP-HPLC, ion-exchange HPLC, size exclusion HpLC), efficient chromatofocusing chromatography, hydrophobic interaction chromatography, immunoprecipitation or immunoaffinity purification, can be with electrophoresis (as PAGE, SDS-PAGE) based on molecular weight, charge characteristic and hydrophobicity, protein isolate or peptide.

By producing the fusion rotein fibroin of purifying early ageing easily or its fragment, described fusion rotein is (as the QIA expression vector with the plain sequence of required early ageing and another kind of peptide such as epitope or poly-His mark, QIAGEN Corp., Chatsworth, CA) or bigger albumen (as utilizing pGEX-27 carrier (Amrad, GST) or utilize Green Lantern carrier (MD) fluorescin merges for GIBCO/BRL, Gaithersburg USA).Can expressed fusion protein, from protokaryon or eukaryotic cell, reclaim then, then based on the fusion vector sequence, with any standard method purifying.For example, available antibodies, the affine or immunoprecipitation purified fusion protein by immunity, the plain part of the non-early ageing of the anti-fusion rotein of described antibody is under the situation that the poly-His mark is arranged, by combining purifying with the nickel post is affine.By the enzymatic lysis fusion rotein, can from fusion rotein, be further purified required early ageing fibroin or fragment.It is known in the art preparing and using the method for the fusion constructs of described purifying protein, and has been useful on several commercial reagent boxes of this purpose.From openly of the present invention, people can use the described fusion constructs that contains the early ageing element.

The antibody of 3 premature senescence resistance elements

The present invention also provides the antibody with early ageing fibroin or its fragment selective binding, and the method for preparing antibody.Particularly importantly, be tested and appraised the functional domain and the polymorphic regions relevant of early ageing element with AD, the invention provides antibody and the method for preparing antibody, described antibody is optionally in conjunction with normal and mutant (the promptly causing a disease) form that also can identify and/or distinguish the early ageing fibroin thus.Antibody of the present invention has the effectiveness as laboratory reagent, and described reagent is used for immune purifying early ageing element, the Western trace identifies that cell or tissue, immunocytochemistry or the immunofluorescence technique of expressing the early ageing element are to determine described proteic subcellular location.In addition, as described below, also available antibody of the present invention as diagnostic tool identifying the plain allelic carrier of the relevant early ageing of AD, or as treatment tool come optionally in conjunction with and suppress the pathogenic form of early ageing fibroin in the body.

Can produce antibody of the present invention with complete early ageing fibroin of the present invention or the plain epi-position of any early ageing, described epi-position should be that described proteic feature maybe can make described albumen be different from other host protein.By will and coming autocorrelation host's protein sequence Computer Database to compare, can identify described epi-position from the sequence such as 4-10 the amino-acid residue of the plain sequence of early ageing.Preferably from N-or C-end, or from the ring structure territory that connects described albumen membrane spaning domain, select described epi-position.Particularly, expectation polymorphism N-stub area, TM1 → 2 rings or TM6 → 7 rings have maximum diagnosis and treatment effectiveness.On the other hand, the antibody of expecting anti-high conservative structural domain has the plain purifying of maximum early ageing or identifies effectiveness.

With IBI Pustell program, amino acid residue position can be accredited as the potential antibody sites in the hPS1 albumen, and can be used for producing antibody of the present invention.These positions (corresponding to the position among the SEQ ID NO:2) in table 6, have been listed.

Certainly, it is known in the art selecting other method of epitope, therefore, also can use.In addition, also can use the bigger fragment (as 8-20 or preferred 9-15 residue) that comprises some these epi-positions.For example, the fragment that comprises the 109-112 epi-position can contain residue 107-114 or 105-116.Even comprise that the bigger fragment as whole functional structural domain or multifunction structure territory (as TM1, TM1 → 2, TM2 or TM6, TM6 → 7) also is preferred.For other early ageing fibroin (as for mPS1 or other inhuman homologue, or for PS2), can select the homologue site.

With identical IBI Pustell program, amino acid residue position can be accredited as the potential antibody sites in the hPS2 albumen, and can be used for producing antibody of the present invention.These positions (corresponding to the position among the SEQ ID NO:19) in table 7, have been listed.

With regard to PS1, it is well known in the art selecting other method of epitope certainly, and can use these methods.In addition, also can use the bigger fragment (as 8-20 or preferred 9-15 residue) that comprises some these epi-positions.For example, the fragment that comprises the 310-314 epi-position can contain residue 308-316 or 307-317.Even comprise that the bigger fragment as whole functional structural domain or multifunction structure territory (as TM1, TM1 → 2, TM2 or TM6, TM6 → 7) also is preferred.For other early ageing fibroin (as for mPS1 or other inhuman homologue, or for PS2), can select the homologue site.

From crude extract (as the film fraction of proteic cell as described in highly expressing) but, from also producing described goods as short immunogen by chemical peptide synthetic method from the plain immunogen goods of production early ageing in the albumen of basic purifying the cell of natural or recombinant expression protein or peptide or the peptide.The plain immunogen of described early ageing can be the fusion rotein form, wherein selects the plain district of non-early ageing according to its auxiliary property.As used herein, the plain immunogen of early ageing should be defined as the goods that comprise peptide, described peptide contains the 4-8 at least of early ageing fibroin (altogether can be with available as this paper), preferably 9-15 successive amino-acid residue at least.Certainly, according to the required purposes and following technical development, less residue sequence also has effectiveness.Therefore, the plain deutero-sequence of early ageing that is used to produce the plain antibody of premature senescence resistance should be considered the plain immunogen of early ageing.

Antibody of the present invention can be polyclone or monoclonal, can be antibody fragment maybe, comprises Fab fragment, F (ab ') ₂, and single chain antibody fragments.In addition, after having identified useful antibody, can produce the recombinant antibodies that comprises above-mentioned any antibody fragment with method of the present invention, and based on the non-human antibody's of premature senescence resistance element humanized antibody.From of the present invention open, and the feature of available other early ageing element of this paper, this area professional can produce above-mentioned antibody by any standard method known in the art.With regard to the antibody technique summary, referring to Antibody Engineering:A PracticalGuide, Borrebaek, ed., W.H Freeman ﹠amp; Company, NY (1992), or AntibodyEngineering, 2nd Ed., Borrebaek, ed., Oxford University Press, Oxford (1995).

Generally speaking, at first can produce polyclonal antibody by the plain immunogen immune mouse of early ageing, rabbit, goat or other suitable animal that are used in the appropriate carrier.In order to improve the immunogenicity of goods, immunogen can be combined with carrier proteins or mixes with adjuvant (as freund's adjuvant).Although need not to remind, can carry out booster shots, making humoral response develop the suitable time, after preferred several weeks, from animal, draw blood, purified blood serum is with the separating immune globulin component then.

Equally, generally speaking, at first by be used in the plain immunogen immune mouse of early ageing in the appropriate carrier, exempt from, goat or other suitable animal can produce the plain antibody of monoclonal anti-early ageing.As mentioned above, can use carrier proteins or adjuvant and recommendation to carry out booster shots (as 8-10 per 2 in week or 3 weeks).After making humoral response develop the suitable time, kill animals is taken out its spleen, for example is resuspended in then in the salt of phosphoric acid buffer (PBS).Splenocyte is as the lymphocyte source, and some of them are produced suitable specific antibody.Then these cells and immortalized cell system (as myelomatosis) are merged, under the condition that has selective reagent such as HAT, fusion product is put in a large amount of tissue culture holes.Series is carried out in described hole screen and heavily spread, screen cell at every turn and all can obtain useful antibody.Usually, carry out the screening for several times and the process of heavily spreading and contain the single clone of antibody producing male up to the hole more than 90%.With standard method as the affinity chromatography of utilizing Protem A Sepharose, by ion exchange chromatography or variant or combination by these technology, but purifying is with the monoclonal antibody of described clone's production.

Can be used to diagnose and/or the compound of therepic use or material mark or with other in conjunction with antibody of the present invention.For example, can or be used for imaging or the enzyme of treatment with itself and radionuclide, fluorescent chemicals, or the liposome combination, described liposome is used for the compound that liposome is contained and is directed in the specific tissue location.

4 cell transformed system

The present invention also provides with nucleic acid conversion of the present invention or cells transfected or clone (protokaryon and eucaryon all can) so that clonal expansion these nucleic acid and/or express by its encoded protein or peptide.Described cell or clone effective usefulness aspect nucleic acid of the present invention and proteic propagation and production, but as described further below also can be used as the model system of diagnosis and therapeutic test.As used herein, term " cell transformed " though comprise be with conversion, transfection, injection or other method with any nucleic acid of the present invention import to wherein any cell or the filial generation of any cell.The production appropriate carrier, with those carrier transformants and identify transformant method be well known in the art, and this only brief overview (referring to (1989) molecular clonings such as for example Sambrook: laboratory manual, the 2nd edition, press of cold spring harbor laboratory, cold spring harbor laboratory, New York).

The prokaryotic cell prokaryocyte that is used for production transformant of the present invention comprises Escherichia (as intestinal bacteria), Rhodopseudomonas (as Pseudomonas aeruginosa) and bacillus (as subtilis, bacstearothermophilus), and many other cells well known and commonly used.Prokaryotic cell prokaryocyte is useful especially for mass production albumen of the present invention or peptide (as normal or sudden change early ageing element, the plain fragment of early ageing, early ageing plain fusion protein); can be with the bacterial cell that contains various expression vector systems (as intestinal bacteria), described carrier system comprise for example contain T7 RNA polymerase/promoter systems, lambda particles phage is regulated the plasmid or the M13 phage mGPI-2 of sequence.But with fusion rotein carrier also transform bacteria host, described fusion rotein carrier produces for example lacZ, trpE, maltose binding protein, poly-His mark or glutathione-S-transferase fusion rotein.All these, and many other prokaryotic expression systems are well known in the art, and widely sell on the market and (as be used for the pGEX-27 (Amrad, USA)) of GST syzygy.

Be used for the eukaryotic cell of production transformant of the present invention and clone and comprise that mammalian cell and clone (as PC12, COS, CHO, inoblast, myelomatosis, neuroblastoma, hybridoma, human embryo kidney 293, ovocyte, embryonic stem cell), insect cell line are (as with baculovirus vector such as pPbac or pMbac (Stratagene, La Jolla, CA)), yeast is (as using Yeast expression carrier such as pYESHIS (Invitrogen, CA)) and fungi.Eukaryotic cell is useful especially for some embodiment, in described embodiment, requires early ageing fibroin or its function fragment to finish with normal or function that mutant protein is relevant and/or carries out interaction in the cell.Therefore, for example the eukaryotic cell that preferably will be transformed is used as plain function of early ageing or interactional model, and cell transformed is preferably used in the test of screening candidate therapeutic agent.

In order to finish the expression in eukaryotic cell, many carriers have been developed, and can on market, buy, described carrier can be induced (as the LacSwitch expression vector under the adjusting of manual activation sub-element, Stratagene, La Jolla, CA) or homology (as pcDNA3 carrier, Invitrogen, Chatsworth CA) expresses the plain nucleotide sequence of early ageing.Described promoter element is derived from CMV or SV40 virogene usually, although also can use other activated strong promoter element in eukaryotic cell to induce transcribing of the plain nucleotide sequence of early ageing.Usually, these carriers also contain artificial polyadenylic acid sequence and the 3 ' UTR that is derived from exogenous virus gene order or other eukaryotic gene.In addition, in some construct, but in described carrier, also comprise artificial, non-coding montage intron or exon so that the expression of required nucleotide sequence (in this case, being the plain sequence of early ageing) strengthens.These expression systems can be bought usually from the market, representational carrier be as pcDNA3 and pZeoSV (Invitrogen, San Diego, CA).COS, the CHO and the PCI2 cell that successfully back two kinds of carriers have been used in transfection are expressed early ageing fibroin (Levesque etc., 1996).Countless expression vectors commercially available and the custom design can be bought from the market, thereby can continuously or be exposed to specific exogenous stimulation (as stop using tsiklomitsin or be exposed to IPTG) after, in the cell of ideal type more or less, express the plain transcript of any required early ageing.

By the whole bag of tricks well known in the art, carrier is imported in acceptor or " host " cell, described method comprises, but be not limited to calcium phosphate transfection, strontium phosphate transfection, deae dextran transfection, electroporation, fat transfection (as Dosper Liposomal transfection reagent, Boehringer Mannheim, Germany), microinjection, shooting is inserted on microballon, protoplasma merges or, for virus or phage vector, by with recombinant virus or phage-infect.

5 transgenic animal models

The present invention also provides the production method of transgenic nonhuman animal model, described animal model is used to study Alzheimer's disease, screening candidate drug compounds, produce the Mammals CNS cell culture of wherein expressing mutant or the plain sequence of wild-type early ageing or making the outer planting of early ageing plain gene inactivation (lacking as " rejectings ") (as, neurone, neuroglia, organotypic or blended cell culture), assess the potential result of treatment.Before the present invention, by under the control of platelet-derived growth factor beta receptor promoter element (Gemes etc., 1995), insert and cross the mutant forms of expressing human amyloid protein precursor gene, and be based upon the part animal model of Alzheimer's disease.This mutant (β APP ₇₁₇, the pathology performance that Val → Ile) engages and in the brain of the transgenic animal of carrying high copy number, amyloid beta deposition.This variation in the transgenic animal brain and very similar (Gemes etc., 1995) arrived seen in the people AD.But up to the present, whether not clear these animals can become the dement, and common recognition is but have a bit, can produce the feature of some AD at least more now in mouse.

The animal that is suitable in animal model of the present invention includes, but are not limited to rat, mouse, hamster, cavy, rabbit, dog, cat, goat, sheep, pig and non-human primates (as rhesus monkey, orangutan).For preliminary research, because its relatively easy maintenance, and the lifetime is shorter, so transgenic rodent (as mouse) is preferred.As mentioned above, for some research, transgenic yeast or invertebrates (as nematode, insect) are preferred, because they can more promptly grow, and the screening expense is low.But the transgenic nonhuman primates is preferred for studying for a long period of time, because they are more similar to the people, and they have higher cognitive ability.

With the openly also available nucleic acid of this paper, there is several methods can produce the transgenic animal model of Alzheimer's disease now.Therefore, the animal that can access comprise (1) wherein normal people's early ageing plain gene and imported to animal in the animal gene group as episome under the adjusting of endogenous or exogenous promoter element by reorganization as minigene or big genomic fragment; Wherein use the animal homology early ageing plain gene of one of normal people's early ageing plain gene replacement or two copies by homologous recombination or gene targeting; And/or wherein through homologous recombination or gene targeting, replace with the sequence of coding people homologue, and with the animal homology early ageing plain gene of or two copies recombinate " humanization ".These animals are used to assess the effect of transgenic method, people or the importing of peopleization early ageing plain gene or the effect of replacement.(2) wherein will suddenly change (promptly morbific) people's early ageing plain gene as the episome under regulating at external source or endogenesis promoter, or recombinate as minigene or big genomic fragment and to import animal in the animal gene group; Wherein use the animal of one of mutant human early ageing plain gene replacement or two copies animal homology early ageing plain gene by homologous recombination or gene targeting; And/or wherein by homologous recombination or gene targeting and the part of the sequence of encoded mutant human homologue replaces, with the animal of one of the animal homology early ageing plain gene of one or two copy reorganization " humanization ".These animals are carried the animal model of one or more allelic people's features as showing some or all (no matter being on biological chemistry, pathology and/or behavior level), and described allelotrope is pathogenic for Alzheimer's disease or other disease relevant with the sudden change of early ageing plain gene.(3) wherein with the mutant of one of mutant animals early ageing plain gene (for example carry corresponding to or similar in appearance to the specific sudden change of one of plain pathogenic mutation of people's early ageing) as the episome under external source or endogenesis promoter are regulated, or as minigene or big genomic fragment and the animal in the importing animal gene group of recombinating; And/or wherein the mutant by one of homologous recombination or gene targeting animal early ageing plain gene (for example carry corresponding to or similar in appearance to the specific sudden change of one of plain pathogenic mutation of people's early ageing) replaces the animal of or two copies animal homology early ageing plain gene.These animals are carried the animal model of one or more allelic people's features as showing some or all (no matter being on biological chemistry, pathology and/or behavior level), and described allelotrope is pathogenic for Alzheimer's disease or other disease relevant with the sudden change of early ageing plain gene.(4) wherein by homologous recombination or gene targeting and partly or entirely lacked one of one or two copies animal early ageing plain gene, or pass through exogenous array (as terminator codon, lox p site) homologous recombination or gene targeting are finished and are inserted or replace, thereby make " rejecting " animal of one of one or two copies animal early ageing plain gene inactivation.Described animal is useful animal model, can be used to study and lose the early ageing plain gene and express the influence that to produce, evaluation function lose for continue expressing mutant forms whether to be preferred, to detect and whether can recover other gene to replace sudden change early ageing element (as replacing PS1) or to disturb other to cause the effect of AD gene (as APP or ApoE) with PS2.For example normal early ageing plain gene is essential for the actual AD of being expressed as of mutant app gene, and therefore, the plain animal model of transgenosis early ageing can be used to illustrate that described polygene interacts.

In order to produce animal model (as transgenic mice); normal or sudden change early ageing plain gene (as normal or sudden change hPS1, mPS1, hPS2, mPS2 etc.); or the sudden change recombinant nucleic acid of at least one functional domain of coding early ageing element (as contains the recombinant precursor of mPS1; wherein replaced described mPS1 with the nucleotide sequence of corresponding people's mutant nucleotide sequence) can by be inserted into standard ovocyte microinjection technology kind be or stem cell in, or transfection or microinjection are in embryonic stem cell.It is genetically modified being called with the animal of these or similarity method production.Equally, inactivation or replace endogenous early ageing plain gene can use the homologous recombination of utilizing embryonic stem cell if desired.Animal with these or similarity method production is called " inefficacy " (inactivation) or " knock-in " (replacement) model.

For ovocyte injection, the recombinant DNA construction body of the present invention of or multiple copied can be inserted in the pronucleus of the ovocyte of just being fertilized.Then this ovocyte is implanted to hatching in the parent of false pregnancy again.By whether there being the reorganization transgenic sequence of insertion in the analyzing DNA (as from offspring's mouse tail vein), in the birth animal that lives, screen intasome.Transgenosis can be with the complete genome group sequence of YAC, BAC, PAC form injection or have natural promoter or other chromosomal dna fragment, the cDNA of allogeneic promoter, or contains all coding regions or find minigene for necessary other element of optimum expression.

The retrovirus injection that also can finish body early embryo is so that insert recombinant DNA construction body of the present invention.In this way; transgenosis (as normal or sudden change hPS1 or PS2 sequence) can be inserted in the retrovirus vector; growing early stage direct infection embryo (as mouse or non-human primates embryo) obtaining mosaic with described retrovirus vector, it kind is transmission that wherein some can produce.

Utilize the homologous recombination of stem cell can the screening-gene transitional cell to identify rare homologous recombination incident.After the evaluation, inject by protoblast with it and to produce mosaic, the ratio of gained animal will illustrate that the kind system that carries out from the reorganization system transmits.If make early ageing plain gene inactivation, this method is useful especially.For example contain dna fragmentation, the mPS1 gene in can the inactivation mouse from the flag sequence selected of mPS1 exon side by design.Homologous recombination can cause inserting flag sequence at the middle part of exon, thereby causes mPS1 gene inactivation and/or internal sequence disappearance.Then the individuality clone is carried out DNA analysis with identification homologous recombination incident.

The technology that produces transgenic animal now is widely accepted and adopts with the technology that is used for homologous recombination or gene targeting.For example the laboratory manual to the mice embryonic operation is the detailed standard laboratory technology (Hogan etc., 1986) of producing transgenic mice.In order to produce transgenosis, usually required target sequence (as mutant or the plain sequence of wild-type early ageing) is connected to the cloning site that is positioned at some promoter element downstreams, described promoter element will be regulated the rna expression that carries out from the plain sequence of early ageing.The plain sequence of described early ageing downstream has artificial polyadenylic acid sequence usually.Transgenic models the animal that is used for successfully having produced anthropomorphic dummy's heredity neurodegenerative disease, instruct other promoter element of expressing although also can use in central nervous system cell, the most successful promoter element is platelet-derived growth factor receptors β gene subunit promotor and hamster prion protein gene promoter.Produce genetically modified another kind of method and be with the plain promotor of endogenous early ageing and regulate sequence to drive the plain genetically modified expression of early ageing.At last, can produce transgenosis with the big genomic DNA fragment such as the YAC that contain complete early ageing plain gene and its suitable adjusting sequence.Successfully described construct is used for expression (Lamb etc., 1993) at transgenic mice people APP.

Endogenous early ageing plain gene also can produce animal model so that change the plain sequence of endogenous early ageing by homologous recombination by leading.These guiding incidents have the effect of removing endogenous sequence (rejecting) or changing endogenous sequence, thereby produce amino acid change or other the undesired sequence (as the sequence of similar human sequence rather than source animal sequence) (in knock-in animal model) relevant with the human disease.Have a large amount of carriers can reach this purpose, (St.Louis, Missouri USA) can buy mouse gene group DNA and other animal gene group to be led in suitable source from many companies such as GenomeSystems Inc..But the characteristic feature of these oriented carrier constructs is 5 ' the linking to each other of genomic dna and selective marker (as the bacterium neomycin resistance gene under himself the promoter element control that is called " Xin Meisu box ") of 2-4kb.。To be connected the downstream of Xin Meisu box from second dna fragmentation of required gene then, but in the upstream of second selected marker (as thymidine kinase).Dna fragmentation is selected so that by one of sequence of being comprised in described carrier, and homology replaces endogenous sequence, thereby mutant sequence is imported in the kind system of target animals.In addition, can select to lack normally around the Xin Meisu box sequence between the carrier left and right arms sequence.The former is called knock-in, and the latter is called rejecting.In addition, also produced countless model systems, particularly guiding reject comprise the gene relevant with neurodegenerative disease (as Zheng etc., 1995 guiding disappearance mouse app genes; Bueler etc., 1996 guiding disappearances and the relevant mouse prion gene of people CNS sex change of growing up and showing effect).

At last, by the chemistry or the mutagenesis of X-ray of gamete, fertilization can produce the equivalent of transgenic animal then, comprises the animal that has sudden change or inactivation early ageing plain gene.Open or the available isolating nucleic acid with this paper, this area professional by for example for detecting directly order-checking RFLP, the PCR or the hybridization analysis of mutant, or the Southern trace of an allelic loss causing because of dosage of the explanation filial generation that can more promptly screen gained.

6 influence the test of the plain medicine of expressing of early ageing

In another serial embodiment, the invention provides evaluation and can induce or suppress early ageing plain gene and the small molecules of albumen (as PS1 or PS2) statement or the test of other compound.Tie up to externally with non-transformed cell, immortal cell line or reconstitution cell, or finish described test in vivo with the transgenic animal that this paper can access.

Particularly, with the mRNA that increases or reduce express (for example with the openly also available nucleic acid probe of this paper), increase or reduce PS1, the PS2 of level or the plain associated protein product of other early ageing (for example with this paper openly also available anti--the plain antibody of early ageing) or increase or reduce in recombinant precursor, can operating the token-based (as beta galactosidase enzyme or luciferase) that links to each other and being expressed as the basis of level with early ageing element 5 ' regulatory region, whether described test can detect PS1, PS2 or other early ageing element-genes involved or proteic expression has and increases or reduce.

Therefore, for example people can cultivate known cell to express specific early ageing element, add one or more test compounds then in substratum.Make described compound to the induced expression of early ageing element or after having suppressed time enough (as 0-72 hour), all can detect baseline to determine, changes of expression level with above-mentioned and well known in the art any technology.In particularly preferred embodiments, described cell is clone such as people's neuroblastoma, glioblastoma or the hybridoma cell line from infinite multiplication.With nucleic acid probe and/or the antibody that this paper openly also can access, detect the plain change of expressing of early ageing, identify that thus described compound is the inductor or the repressor of the plain expression of early ageing, these needs routine tests.

In particularly preferred embodiments, used the reorganization test, wherein reporter gene such as beta-galactosidase enzymes, green fluorescent protein, alkaline phosphatase or luciferase can be operated with 5 ' regulatory region of early ageing element and link to each other.Preferred carrier comprises Green Lanternl carrier, and (GIBCO/BRL, Gaithersburg is MD) with the Great EScAPe pSEAP carrier (Clontech, Palo Alto).Based on these gene coding regions disclosed by the invention, this area professional can easily separate and clone hPS1 regulatory region disclosed herein or the plain regulatory region of other early ageing.Reporter gene and regulatory region in frame (or in three possible reading frames each in) link to each other so that can carry out transcribing and translating of reporter gene under the control of the plain regulatory element of early ageing.Then, recombinant precursor is imported in any suitable cell type, although mammalian cell is preferred, people's cell the best.Make by cell transformed and in substratum, grow, determine the expression baseline values of reporter gene after, testing compound is added in the substratum.The inductor and the repressor that are easy to detect to identifying the early ageing plain gene that reporter gene is expressed provide rapidly detection method efficiently.

There is being potential to use aspect the expression in vivo of modifying the plain genes involved of PS1, PS2 or other early ageing with this method compounds identified.Can also in the openly also available animal model of this paper, further detect these compounds to identify those the strongest compounds of effect in the body.In addition, described in conjunction with active small molecules as this paper to having the early ageing element, these molecules can be used as " lead compound ", for example by make described compound through modify continuously, the molecule typing and in the conventional medicine design other used method come further developing drugs.

7 identify the compound that the plain binding ability of early ageing is arranged

According to the present invention, this area professional can implement new screening method, combines or interactional with it albumen and other compound to be used for identifying with early ageing is plain.Described albumen and compound comprise in the body and the plain interaction of early ageing, and therefore get involved the endogenous cell component that new target is provided for medicine and treatment, and reorganization, synthetic and other endogenous component that has the plain binding ability of early ageing and therefore can be used as the candidate medicine.Therefore, in a series of embodiment, screen in cell lysates or the tissue homogenate thing (as human brain homogenate, lymphocytolysis product) and one of normal or sudden change early ageing element bonded albumen or other compound.In addition, can be to any different interior source compound, natural and or synthetic (as small molecules or peptide library) screen, to screen the plain binding ability of its early ageing.In the present invention, small molecules is particularly preferred, because behind oral administration, they are easy to be absorbed, and has only very little potential epitope, and/or more likely strides across the blood cerebral disorders than macromole such as nucleic acid or albumen.Method of the present invention is useful especially, be available their identify selectivity or preferentially in conjunction with the molecule of the early ageing fibroin of mutant forms (rather than normal form), therefore, they are having special effectiveness aspect the heterozygosis patient of this autosomal dominant disorder of treatment.

Owing to still do not understand the normal physiological effect of PS1 and PS2, therefore the compound in conjunction with the early ageing element of normal, mutant or these two kinds of forms is having effectiveness aspect treatment and the diagnosis.For example only can be used as the toughener of its normal activity, compensate the activity of sudden change early ageing prime form that lose or unusual in the presenile dementia patient thus to small part with the plain bonded compound of normal early ageing.If they can distinguish the activity that influence these two kinds of forms in conjunction with the compound of normal and mutant forms early ageing element, thereby all of elimination and normal function depart from, and then they also have effectiveness.In addition; blocking-up PS1 or PS2 specific activity normal and mutant forms makes the severity of the physiology of disease normal development and clinical consequences little; so in conjunction with and the compound that suppresses normal or mutant forms early ageing element in treatment, also be useful, but preferred identify the binding affinity with sudden change early ageing element be higher than with normal early ageing element (as the doubly high K of 2-10 at least _a), and the active compound of selectivity or preferential mutation inhibiting bodily form formula by comparing the binding affinity of candidate compound and normal and mutant PS1 or PS2, can be identified described compound then with any technology disclosed herein.

By with instrument (as; BIAcore; LKB Pharmacia; Sweden) directly monitor all fluorescence of bonded as described, change of molecular weight or detection wedding agent or early ageing element in solvable phase or the concentration in substrate combination mutually, can monitor effect in conjunction with the reagent of early ageing element (normal or mutant forms).

After the aforesaid method evaluation, produce the candidate compound with the amount (as μ g or mg or bigger amount) that is enough to be used in medicament administration or test, be formulated in then in pharmaceutically acceptable carrier (referring to as Remington ' sPharmaceutical Sciences, Gennaro, A., ed., Mack Pub., 1990).With these candidate compounds be applied to transformant of the present invention, transgenic animal model of the present invention, from animal model or people patient's deutero-clone or presenile dementia patient.The openly also available animal model of the present invention is useful especially for the further treatment effectiveness that detects in conjunction with the candidate compound of normal or sudden change early ageing element.

In addition, after identifying with aforesaid method, described candidate compound can be used as " lead compound " in the design of novel drugs and exploitation.For example as known in the art, in the pharmaceutical industries of developing new drug, modifying small molecules (as with peptide substituted amino acid residue, replacing functional group with peptide or non-peptide compound) continuously is standard method.Described exploitation normally from known have to the required activity of small part (as PS1 in conjunction with or blocking ability) " lead compound " beginning.Specifically, after having identified have some required activity at least one or more compounds of (plain active adjusting) as early ageing, this area practitioner just can be by described molecule texture ratio, which is understood is the part that should keep in the lead compound, and which is a changeable part in the new candidate compound of design.Therefore, the present invention also provides the method for identifying lead compound, can modify the new candidate compound that is used for the treatment of Alzheimer's disease with generation continuously to described lead compound.Detect the plain combination of early ageing or the blocking-up (as in above-mentioned combination test) and the treatment effectiveness (as in animal model as herein described) of these new compounds then.Can repeat described method, up to identifying compound with required therapeutic activity and/or effectiveness.

In each serial embodiment of the present invention, can finish a kind of test to detect combining between " the plain component of early ageing " and some other parts.Useful especially is campaign; wherein in conjunction with test; with mutant and the plain component of normal early ageing only detect with the compound that normally or only have binding ability with mutant forms early ageing as hereinafter describing more comprehensively, expect that described compound has maximum treatment effectiveness." early ageing plain component " in these trials can be the early ageing fibroin (as hPS1 or hPS2 variant) of complete normal or mutant forms, but optional.As mentioned above, the early ageing element the concrete function structural domain can be used as the part of different molecule or fusion rotein.For example, in order separating and interactional albumen of these functional domains or compound, to use corresponding to these regional fusion constructs and/or synthetic peptide and can finish screening.Therefore, with regard to PS2, can prepare the GST-fusogenic peptide that comprises corresponding to the sequence of about amino acid/11-87 (N-end) or 269-387 (TM6 → 7 rings) or any other required conserved domain.For short functional domain, can produce synthetic peptide corresponding to for example about amino acid/11 07-134 (TM1 → 2 rings).Equally, with regard to PS1, can prepare and comprise corresponding to about amino acid/11-81 (N-end) or 266-410 (TM6 → 7 rings) or corresponding to the synthetic peptide of for example about amino acid/11 01-131 (TM1 → 2 are encircled).Obviously, the plain functional domain of the fusion rotein of various combinations and early ageing is possible, and these only are examples.In addition, can change functional domain, for example import in the functional domain by the reactive group or the amino-acid residue (as halfcystine) (as by the sulfydryl reaction) that will promote described structural domain to be fixed on the substrate so that help in test.Therefore, the PS1 TM1 → 2 ring plate sections (31-monomer) that contain other C-and do not hold cysteine residues have for example been synthesized.Be used for affinity chromatography (Sulfo-link with this peptide generation; Pierce) affine substrate is used for the conjugated protein of micrometering preface with separation.Can produce other functional domain or antigen fragment (referring to for example embodiment 10) with the residue of modifying equally

Can also characterize albumen or other compound of identifying with these methods by purifying with any standard method well known in the art.For example can utilize electrophoresis (as SDS-PAGE, 2D PAGE) or chromatography (as HPLC) technology purifying and protein isolate, carry out the micrometering preface then.For the albumen of N-end with blocking-up, specific conjugated protein of cracking (CNBr and/or trypsinase for example are provided) with the release peptide fragment.Be further purified/characterize the interior sequence data that relevant described blocks protein is provided with micrometering preface and/or the mass spectroscopy carried out with ordinary method with HPLC.For non-proteinate, can be with standard organic chemistry analytical technology (as IR, NMR and mass spectroscopy; Functional group is analyzed; The X-radiocrystallgraphy) determines its structure and characteristic.

The method that is used to screen the small molecules library of the plain binding molecule of cell lysates, tissue homogenate thing or candidate's early ageing is well known in the art; and it is open according to the present invention; can be used to evaluation in conjunction with normal or plain component of sudden change early ageing or the plain active compound of adjusting early ageing, the plain activity of described early ageing is (as Ca in the cell with non-specific measurement ²⁺The change of/GTP/GDP ratio) measures that (variation of other downstream gene expression aspect that can monitor as the change of A β peptide production or by differential demonstration, 2D gel electrophoresis, differential hybridization or SAGE method) define or by specificity.Preferable methods comprises the change of relevant following technology: directly extract by affinity chromatography (1); (2) be divided into from plain component of early ageing and bonded albumen by immunoprecipitation; (3) bio-molecular interaction test (BIAcore); (4) yeast two-hybrid system.Hereinafter will discuss respectively to these and other aspect.

The A affinity chromatography

Open according to the present invention, can disclose or albumen or other compound of other available early ageing element with various affine combination technology separation and combination this paper well known in the art.In a word, the plain component of early ageing can be fixed on the substrate (as pillar filter paper), the solution that will contain testing compound carries out under the bonded condition allowing, and contacts with early ageing fibroin, fusion rotein or fragment.Then with the solution washing substrate with remove not in conjunction with or in conjunction with weak molecule.Wash for the second time normal or those compounds of the strong bonded of the plain component of sudden change early ageing of wash-out and fixed then.In addition, fixing testing compound, the solution that will contain the plain components of one or more early ageing then contacts with post, filter paper or other substrate.The above-mentioned binding ability that can determine plain component of described early ageing and testing compound of eucalyptus, or more promptly assess and the combining of substrate-fixed compound with the plain component of the early ageing of mark pattern (as radiolabeled or chemoluminescence structural domain), in addition, owing to think that PS1 and PS2 all are embrane-associated proteins, preferably early ageing fibroin, fusion rotein or fragment are incorporated in the bimolecular lipid membrane (as liposome) to promote its suitable folding.When use comprised the early ageing element component of at least a membrane spaning domain, this was useful especially.Described early ageing element-liposome can be fixed on the substrate (directly or by another component in the liposome membrane), process from the substrate that has the fixed testing compound, or be used for the various membranins of knowing in conjunction with test.In addition, can from the cell of producing the component of transporting, be separated in the plain component of early ageing in the film fraction, then described film fraction is used in conjunction with test.

The B coimmunoprecipitation

The technology of the plain component of the separation early ageing that another kind is known and its associated protein or other compound is with antibody direct immunization precipitation.For example successfully separated the relevant albumen (Phizicky and Fields, 1994) of many synaptic vesicles with this method.Therefore; can allow under the bonded condition; in solution, normal or mutant, the plain component of free or membrane-bound early ageing are mixed with candidate compound; the plain component of the described early ageing of immunoprecipitation is then identified albumen or other compound with early ageing element component coimmunoprecipitation with above-mentioned standard technique then.The major technique that is used for immunoprecipitation sees for example Harlowand Lane, (1988) antibody: laboratory manual, cold spring port press, cold spring port, New York.

Antibody (as described herein and available) used in this test can be polyclone or monoclonal, and comprise different antibody fragments (as Fab, F (ab ') ₂) and single-chain antibody etc.

The test of C bio-molecular interaction

Another kind is used for detecting and the proteic method of separation and combination is by Pharmacia Biosensor exploitation and in manufacturers (LKB Pharmacia, the bio-molecular interaction of Sweden) describing in method test or " BIAcore " system.Open according to the present invention, albumen or other compound that this area professional can use this system or basic equivalent to have the plain binding ability of early ageing with evaluation.The BIAcore system uses the anti--GST antibody of affinity purification so that the GST-fusion rotein is fixed on the sensor chip.Obviously, available other fusion rotein or corresponding antibody replace.Described transmitter utilization can detect the variation of refractive index as the surface plasmon resonance of optical phenomena.The homogenate that makes required tissue is from top process fixed fusion rotein, and protein-protein interaction is registered as the variation of index coefficient.Can determine whether the keying action that binding kinetics and assessment observe then is that physiology is relevant with this system.

The D yeast two-hybrid system

Yeast " double cross " system has utilized the transcription factor of physically separating by two, functional domain (Phizickyand Fields, 1994) is formed.The most frequently used is yeast GAL4 activating transcription factor, and it is made up of DNA binding domains and transcriptional activation domain.With the different fusion roteins of two kinds of different cloning vectors generation GAL4 with the potential protein-bonded gene fusion of coding.Described fusion rotein is directed in the nuclear by coexpression, if interact, the gene of reporter gene (as LacZ) will produce detectable phenotype.For example, can be with Clontech Matchmaker system-2 can (Clontech, Palo Alto CA) use together with the Clontech brain cDNA GAL4 activation structure domain fusion protein library that contains early ageing element-GAL4 binding domains fusion cloning.According to disclosing of this paper, this area professional can produce various early ageing plain fusion proteins now, comprise the fusion rotein of the normal or mutant functional domain that contains the early ageing fibroin, and it is conjugated protein to identify the early ageing element to screen described fusion protein libraries.

Other method of E

Nucleotide sequence and protein product (comprising corresponding proteic mutant of these nucleic acid and its and normal form) can be used separating other interactional albumen with above-mentioned technology, and identify that its expression is subjected to the plain sequence of normal early ageing to cross expressions, the plain sequence of normal early ageing is expressed other gene not enough or that the plain sequence expression of sudden change early ageing influences.No matter identify that these interactional albumen and evaluation are under the situation of which kind of sudden change plain sequence of early ageing (for example), its expression level can identify its other clinical or the pathology form is directly related with the mechanism of causing a disease of described disease gene target with reformed other gene.Specifically, can identify it itself is other mutational site that causes Alzheimer's disease, or itself can be by treatment guiding (crossing the level of expressional function as its expression level being dropped on normal level or the pharmacology capable of blocking its) with other gene as the potential methods of treatment of this disease.Specifically, these technology depend on PCR for the basis and/or hybridization for the basis method, with identify that gene differentially expressed under two kinds of different conditions (clone of expressing normal early ageing element is compared with the cell of the same type of expressing the plain sequence of sudden change early ageing), these technology comprise that differential shows, the serial analysis (SAGE) of genetic expression and albumen 2D-gel mass spectrum and subtractive hybridization be (referring to Nowak, 1995, and Kann, 1995).

The professional it is evident that for this area; many screenings body protein or other compound are arranged; and the method for albumen or other library of compounds is (as phage display library with from Stratagene; La Jolla; the cloning system of CA), thus can identify and the normal or plain component bonded of sudden change early ageing molecule.All these methods include following steps: early ageing fibroin, fusion rotein or the fragment of will normally or suddenling change mixed with testing compound, activates (if necessary) then, then detects the bonded mixture.All these methods all are enforceable by pure substantially early ageing element disclosed by the invention, the pure substantially plain functional domain fragment of early ageing, early ageing plain fusion protein, the plain antibody of early ageing and preparation and their method of use now.

8 identify the method for regulating the plain active compound of early ageing

In another serial embodiment, the invention provides the method for authenticating compound, described compound has the active ability normal and sudden change early ageing element of regulating.Used with regard to described serial embodiment, term " activity " broadly comprises gene and protein expression, the translation of early ageing fibroin post-treatment, transportation and location and any functionally active (as enzymatic, acceptor-effector, combination and passage), and the downstream effects of any of these.The early ageing element may be the normal integral protein relevant with endoplasmic reticulum and/or golgi body, and has and APP transhipment and transportation and/or the horizontal function associated of adjusting intracellular Ca2+.The appearance of the plain sudden change of known early ageing and the A β peptide increase of producing in addition,, amyloid plaque and the former entanglement of nerve fiber, recognizing ability reduction are relevant with programmatic (apoptitic) necrocytosis.Therefore; cell that utilizes transformant of the present invention and transgenic animal model, obtains from the experimenter who carries sudden change early ageing plain gene or the animal or human's class experimenter who carries the plain sudden change of natural early ageing can screen medicative drug candidate and methods of treatment by detecting one or more normal or plain these functional characters of expressing of sudden change early ageing or the variation of phenotype now.

Therefore; the invention provides by in the body or external cell is contacted with candidate compound; detect then and the variation normal or mark that the plain activity of sudden change early ageing is relevant, thereby screen or detect the method for regulating the plain active albumen of early ageing, small molecules or other compound.With the active relevant mark of early ageing element can be any measurable and relevant biological chemistry, physiology, histology and or behavioural characteristic of the plain expression of early ageing.Specifically, useful mark comprises any measurable biological chemistry, physiology, histology and/or behavioural characteristic, and described feature can differentiate cell, tissue, animal or the individual counterpart normal with it that carries at least a sudden change early ageing plain gene.In addition, described mark can be plain active specificity of any early ageing or non-specific measuring result.The plain specificity measuring result of described early ageing comprises the plain measuring result of expressing of early ageing (as plain mRNA of early ageing or protein level) of available nucleic acid probe of the present invention or antibody.Non-specific measuring result comprises the variation of stechiology aspect, as pH, intracellular Ca2+, ring AMP level, GTP/GDP ratio phosphatidylinositols activity, protein phosphorylation etc., available instrument such as Cytosensor microphysiometer (Molecular Devices Inc., United States) monitoring.By detecting the variation of other genetic expression, also can monitor in its mutant or normal form, plain active activation of early ageing or inhibition, described other gene is to cause the plain pathways metabolism of early ageing of Alzheimer's disease specific.These can be by monitoring in all cases as the 2D gel electrophoresis of differential demonstration, differential hybridization and SAGE (serial analysis of genetic expression) and cell lysates, by before using candidate compound and situation thereafter, observe by identical research and can determine the gene that expression level is different.In addition, as described, can determine the concrete gene that its expression is regulated by candidate compound by clone, nucleotide sequencing, amino acid sequencing or mass spectrum (referring to Nowak, 1995) at an other place.

In a word, cell can be contacted with candidate compound, after the suitable time (have as culturing cell maximum biological chemistry measuring result 0-72 hour) detects the plain active mark of early ageing then, compares with base value then.Described base value maybe can be determine or outside base value known in the art with other test with cell and measurement before candidate compound contacts.Described cell can be a transformant of the present invention or from animal or individual outer plant.Specifically, described cell can be the outer plant from plain carriers of mutation of early ageing (as suffering from the people patient of Alzheimer's disease) or animal model of the present invention (carrying the transgenosis nematode or the mouse of sudden change early ageing plain gene).In order to strengthen the influence of the plain sudden change of early ageing to A β peptide approach, transgenic cell or animal that available A β peptide production improves.Preferred cell comprises the cell from nervous tissue such as neurone, neuroglia or cell mixing culture; Cell with inoblast, liver,kidney,spleen or the marrow cultivated.Can contact with candidate compound described cell is external in cultivation, or be administered to Live Animals or human experimenter in the body.For giving Live Animals or human experimenter, can be oral or use testing compound by any outer approach of gi tract that is suitable for described compound.For human experimenter's clinical trial, can in several months or several years, regularly finish measurement (as every day, weekly or every month).

Because the carrier of the plain sudden change of most of early ageing all is heterozygosis (promptly carrying the normal and plain allelotrope of sudden change early ageing), so but detection compound is regulated normal and the plain active ability of sudden change early ageing, so for example, the compound that improves the plain function of normal early ageing can have the effectiveness of plain relative disease of treatment early ageing such as Alzheimer's disease.In addition, because it is littler to suppress the clinical harm of development of specific activity relative disease of normal simultaneously and sudden change early ageing element in heterozygous individual, therefore, the compound that needs to identify inactivation or suppress form of ownership early ageing element.But preferred, identify that selectivity or specificity inactivation or mutation inhibiting early ageing are plain active, and do not destroy the compound of normal early ageing plain gene or proteic function.

According to authentication method, sign and early ageing plain gene disclosed herein and albumen, the plain nucleic acid probe of early ageing and antibody, the plain cell transformed of early ageing of the present invention and transgenic animal, this area professional can finish many detection candidate compounds now and regulate the plain active test of early ageing.Hereinafter will go through embodiment preferred especially and that finish.

A early ageing is plain to be expressed

In a series of embodiments, has the candidate compound that influences the plain active ability of early ageing with the plain concrete measuring result screening of expressing of early ageing.Therefore, utilize plain nucleic acid of the openly also available early ageing of this paper and antibody, people can regulate the index of the plain active ability of early ageing with mRNA level or protein level as candidate compound, measure gene and proteic expression is well known in the art with described probe and antibody, and this paper will discuss in addition.Making us interested especially is the compound of identifying the level relatively that can change the plain different splice variants of early ageing.Manyly for example all be positioned at the TM6 → 7 ring zones of inferring with the plain sudden change of the early ageing of related to alzheimer's disease, montage can take place and change in described ring zone in some peripheral tissues (as white corpuscle), the compound that therefore can improve the relative frequency of described montage process is being effective aspect the expression of this zone sudden change of prevention.

Location in the B cell

In another serial embodiment, to the localized basis that act as in the plain original and cell of early ageing, regulate the plain active ability SCREENED COMPOUND of early ageing according to it with it.Find that with immunocytochemistry the early ageing element is positioned in the membrane structure relevant with endoplasmic reticulum and golgi body, and the plain mutant (H163R) of a kind of early ageing, but be not other, in the utricle matter vesicle of unknown function, can be observed.Therefore, the position disparity of mutant and normal early ageing element may illustrate the cause of disease of the plain relative disease of early ageing.Can detect the position of early ageing element with standard technique known in the art.Usually these technology are used one or more sudden change early ageing elements of antibody of the present invention, particularly selective binding, but the antibody of the normal early ageing element of debond.As known in the art, as described in available any different technology (as the secondary antibody of fluorescence or radio-labeling, mark, avidin-vitamin H etc.) mark antibody to help as described in the visual inspection early ageing element in intracellular position.As known in the art, (as be used for the TGN38 of golgi body with anti-those structure tags, the transferrin receptor that is used for back-golgi body transport vesicle, the LAMP2 that is used for liposome) antibody can navigate to specific structure with plain its of early ageing, also can use be used for the different cell inner membrances of enrichment in conjunction with organoid (as liposome, synaptosome, golgi body), from the Western trace of the purifying fraction of cell lysates.In addition, for example can detect the relative direction in the different structure territory of the early ageing element of striding the cellularstructure territory with the antibody of electron microscope and anti-described structural domain.

Adjusting/the metabolism of B ion

In another serial embodiment, can the interior Ca of cell ²⁺, Na ⁺, or K ⁺Level or metabolism are the basis, regulate the plain active ability of early ageing according to it and come SCREENED COMPOUND.As mentioned above, the early ageing element is to act on or to interact in the embrane-associated protein of ion receptor or ionic channel.Therefore, with calcium in patch clamp, voltage clamp and the pair cell or transmembrane voltage fluorescent dye sensitive, by measuring ionic channel flow and/or transmembrane voltage or existing flow, can according in its body or the plain relevant calcium of external adjusting early ageing or the metabolic ability of other ion come SCREENED COMPOUND.Also can be by measuring secondary messager as ring AMP, cGMP Tyrosylprotein kinase, phosphatic activation, Ca in the cell ²⁺The increase of level etc. also can detect ionic channel or function of receptors.The albumen that also can rebuild the reorganization preparation in the artificial rust system is with the conduction of research ionic channel, and therefore used cell can contain artificial rust or cell in described research.To expressing the variation that endogenous culturing cell normal or sudden change early ageing element can detect its ion adjusting or metabolism aspect.Can be to finishing described research with the carrier cells transfected, described carrier can be expressed the functional domain of one of one of early ageing element of normal or mutant forms or early ageing element.In addition, in order in described detection, to increase the signal that measures, the gene co-transfection cell of available code channel protein.For example available normal or plain sequence of sudden change early ageing and coding rat brain Na ⁺β 1 subunit, rabbit skeletal muscle Ca ²⁺β 1 subunit or rat heart K ⁺The sequence cotransfection xenopus ovocyte of β 1 subunit or rat kidney cell (HEK293).By the pincers of the full cell patch in 2-microelectrode voltage clamp record value or the HEK293 cell in ovocyte record value, can measure the variation of the ion channel activity aspect of the plain relevant or plain mediation of early ageing of early ageing.

C apoptosis or necrocytosis

In another serial embodiment, the apoptosis of or early ageing plain mediation plain relevant to early ageing or necrocytosis with it act as the basis, regulate the plain active ability SCREENED COMPOUND of early ageing according to it.Therefore, for example in substratum, determine the apoptosis or the necrocytosis basis speed of cell, or after animal model or human experimenter's necrotomy, determine basic degree, thereby measure the ability of the candidate compound that suppresses apoptosis or necrocytosis at the given age neurone loss.Can measure necrocytosis by standard microscope technology (as opticmicroscope), or by karyomorphology feature or the dna fragmentation collection of illustrative plates that produces nucleosome measure more specifically apoptosis (referring to as, Gavrieli etc., 1992; Jacobson etc., 1993; Vito etc., 1996).Necrocytosis in the also available TUNEL assessment brain (referring to for example Lassmann etc., 1995).In preferred embodiments, the ability SCREENED COMPOUND that in transgenic animal model of the present invention, suppresses neurone loss according to it.The for example available transgenic mice that carries mutant people mutant mouse or humanization mutant gene identifies or assessment postpones or suppressed the neurodegenerative compound relevant with Alzheimer's disease.There are (1995) such as Games to report the similar transgene mouse model that carries the mutant app gene recently.

D A β peptide is produced

In another serial embodiment, in APP processing, it is regulated, and the early ageing element is correlated with or the ability of the variation of the plain mediation of early ageing is come SCREENED COMPOUND according to it.In several isomer that produce because of the difference in the APP processing, produced A β peptide.A β peptide is a 39-43 the amino acid whose derivative of β APP, and it is carrying out property deposition in senile plaques that spread and AD patient's blood vessel.In human brain, A β peptide all is xenogeneic at N-and C-end.But some observationss show clipped form at residue 42 or 43 terminated long-tail A β peptides (being A β 1-42/43 and A β x-42/43) in AD than having more important role at residue 40 terminated peptides.Therefore, A β 1-42/43 and A β x-42/43 are senile plaques and the early stage notable feature of diffusion spot, and the hypotype of residue 40 terminated peptides (being A β 1-40 and A β x-40) and ripe spot (mature plaque) and amyloid blood vessel significant correlation (referring to as Iwatsubo etc., 1995; Gravina etc., 1995; Tamaoka etc., 1995; Podlisny etc., 1995).In addition, described long end isomer is more prone to protofibril and forms, and thinks than its A β 1-40 peptide bigger neural toxicity (Pike etc., 1993 are arranged; Hilbich etc., 1991).At last, in the missense mutation meeting that the codon 717 of the β app gene relevant with early onset thereof FAD is taken place causes brain in carriers of mutation, and in the peripheral cells and blood plasma suffered from presymptomatic carrier, using β APP ₇₁₇(Tamaoka etc. 1994 in mutants cDNA cells transfected system; Suzuki etc. 1994) the excessive long-tail A β of middle production.As described in embodiment 18 hereinafter, it is also relevant with the sudden change in the early ageing plain gene that we now disclose the increase that long A β peptide form produces.

Therefore, in a series of embodiment, the invention provides according to its blocking-up suppress to express the cell of sudden change early ageing plain gene or transgenic animal in the ability of the long isomer production increase of the A β peptide method of screening candidate compound.Specifically, the invention provides described method, the wherein mammalian cell (as brain cell or inoblast) of the cultivation that transforms with method disclosed herein, or the high-caliber relatively sudden change early ageing element of wherein producing of transgenic animal expression with method disclosed herein.On demand, also can transform described cell or transgenic animal so that with the β APP albumen of high relatively horizontal expression normal form.

In the embodiment of described series, candidate compound is administered to described clone or transgenic animal (as by being added in the cell culture medium in the cultivation; Or be applied to animal outward) by oral or gi tract, then after the suitable time (as cell cultures 0-72 hour, for animal model is a couple of days or several months), the collection of biological sample is (as cell culture supernatant or the cell lysates from culturing cell; Tissue homogenate thing or blood plasma from animal), detect the level of the long isomer of A β peptide then.The level of peptide as described in can determining with absolute meaning (as nMol/ml) or with relative meaning (as the ratio of long and short A β peptide isomer).A β peptide isomer as described in all can detecting with any method known in the art (as electrophoretic separation and order-checking) has specific antibody to determine definitely or the A β 1-42/43 or the A β x-42/43 peptide of level relatively but preferably use to long isomer.Particularly in transgenic animal model of the present invention, can reduce the drug candidate of definitely or relatively level of these long A β peptide isomer or methods of treatment and in treatment Alzheimer's disease or plain sudden change or APP metabolic disturbance cause because of early ageing other disease, have treatment effectiveness.

The phosphorylation of E microtubule associated protein

In another serial embodiment,, regulate the plain active ability of early ageing according to it and screen candidate compound by assessing of the influence of described compound to microtubule associated protein (MAP is as Tau) phosphorylation level.In presenile dementia patient brain, the unusual phosphorylation of Tau and other MAP is well known in the art.Therefore, the compound of prevention or the unusual phosphorylation of inhibition MAP can have effectiveness aspect plain relative disease of treatment early ageing such as the AD.As mentioned above, can use from cell normal or mutant animal or experimenter, or transformation cell lines of the present invention and animal model.Preferred detection method will be used with mutant people or humanization sudden change early ageing plain gene cell transformed system or animal model.Can determine the basic phosphorylation state of MAP in these cells, test candidate compound according to the ability of its prevention, inhibition or the reverse super phosphorylation relevant then with mutant.By any standard method known in the art, can determine the phosphorylation state of MAP, but preferably use the antibody selective binding phosphorylation or the non-phosphorylating epi-position.The described antibody of anti-Tau protein phosphorylation epi-position is (as ALZ50) known in the art.

9 screenings and diagnosis Alzheimer's disease

The diagnostic method that A is general

Open or available early ageing plain gene and gene product with this paper, and the plain deutero-probe of early ageing, primer and antibody are used to screen the allelic carrier with related to alzheimer's disease, be used to diagnose the presenile dementia patient, and be used to screen and diagnose relevant senilism and presenile dementia, psychosis such as schizophrenia and dysthymia disorders, and neural venereal disease such as apoplexy and cerebral hemorrhage, all these symptoms all appear in the human body that carries PS1 or PS2 gene or app gene sudden change more or less.By various technology, whether exist sudden change early ageing plain gene or albumen just can the individuality of suffering from Alzheimer's disease danger be arranged conventional screening with probe in detecting, as AD those are arranged in family spectrum of being in, or do not know to have the individuality of this kind danger in the past.By method based on the openly also available nucleic acid of this paper (comprising genome and mRNA/cDNA sequence), albumen and/or antibody, comprise that design is used for detecting plain active depletion of normal early ageing or enhancing and/or whether has the special and new active function detecting method of being given by sudden change early ageing element of property, the hereditary case of diagnosable these diseases.Preferably, described method and product are based on people PS1 or PS2 nucleic acid, albumen or antibody (open or available as this paper).But the professional it is evident that for this area, even resemble in the species that people, mouse, C.elegans and fruit bat have nothing in common with each other like this, most of PS1 and PS2 Nucleotide and aminoacid sequence have tangible evolution conservative, make this area professional can utilize the plain homologue nucleic acid of described inhuman early ageing, albumen and antibody, even be applied directly to people or other animal.Therefore, for short explanation, but do not limit the scope of the invention, following description will concentrate on the purposes of people PS1 and PS2 homologue.But should be understood that homologue, comprise disclosed hereinly, be equal to for many purposes from other kind.

Those skilled in the art will appreciate that the selection of diagnostic method of the present invention can be subjected to the influence of the character of available biological sample character to be checked and information needed.PS1 high level expression in cerebral tissue for example, but biopsy of brain is an invasive, and expensive process, particularly screen for routine.But other tissue with significantly high horizontal expression PS1 has montage change (as lymphocyte), and is therefore, less from the PS1 or the available data of albumen of described cell.Therefore, be preferred based on the detection of experimenter's genome PS1 DNA, change because data relies on montage, and any basically karyoblast all can provide operable sample.(as hPS2, mPS1) also to consider similar problem: the availability of tissue, the expression level in different tissues and change different mRNA and the protein product that causes because of montage with other early ageing element for the diagnosis on basis.

B is based on the screening and the diagnosis of albumen

When diagnosis is when being the basis with the early ageing fibroin, can use diverse ways.For example, by monitor normal and mutant protein in the difference aspect the electrophoretic mobility, can diagnose.Described method is useful especially identifying aspect the mutant, exists electric charge to replace in described mutant, or wherein inserts, lacks or replace and cause the proteic electrophoretic migration of gained that significant variation is arranged.In addition, diagnosis can be based on normal and the difference of mutant protein proteolysis collection of illustrative plates aspect, the difference of different aminoacids residue mol ratio aspect, or the Function detection by illustrating that the gene product function changes.

In preferred embodiments, the diagnosis based on albumen will utilize antibody and difference normal and sudden change early ageing fibroin (particularly hPS1 or hPS2) binding ability aspect.Described diagnostic test can utilize in conjunction with normal protein, but the antibody of debond mutant protein, otherwise or.Particularly, can use the detection of a plurality of monoclonal antibodies, wherein each monoclonal antibody all can be in conjunction with the mutant epi-position.With anti-mutation body antibody comparing with the level that combines control sample in deriving from experimenter's sample in conjunction with level (by for example radio-labeling, ELISA or chemoluminescence visual inspection).In addition, can use, can utilize will isozygoty normal individual and mutant heterozygosis or homozygous individual of the reduction of antibodies level to differentiate in conjunction with antibody normal rather than sudden change early ageing element.Utilize the CNS biopsy sample that obtains after extremely preceding or the postmortem, described antibody diagnosis can be used for the original position immunohistochemistry, comprise and these diseases such as the former entanglement of the nerve fiber neuropathology structure relevant, or can use with liquid sample such as cerebrospinal fluid or peripheral tissues such as white corpuscle with amyloid plaque.

C is based on the screening and the diagnosis of nucleic acid

When diagnostic test is that detection is based on mRNA, cDNA or genomic dna when being the basis with the nucleic acid from sample.When the mRNA that uses from sample, the consideration of the possibility of many relevant source tissues and montage change all is suitable for.That is, seldom or not express transcript, unless selected the tissue source that maybe can obtain suiting, the montage change can make some information dropout or be difficult to and explain.But we show (Sherrington etc., 1995; Rogaev, 1995) from white corpuscle and/or the isolating mRNA/cDNA of skin flbroblast, the sudden change in 5 ' UTR, 3 ' UTR, open reading frame and the splice site that can identify at PS1 and PS2.No matter be to detect mRNA, cDNA or genomic dna, can with method original position well known in the art or vitro detection whether exist specific sequence (referring to for example, Sambrook etc., (1989) molecular cloning: laboratory manual, the 2nd edition, cold spring port press, the cold spring port, New York).But in general, can detect any tissue that contains karyoblast.

Diagnostic genomic dna can from somatocyte such as blood, exist those, obtain in biopsy, surgical operation sample or the postmortem material.DNA can separate and be directly used in the specific sequence of detection or increase by polymerase chain reaction (PCR) before analysis.Equally, also can be with RNA or cDNA, can with or without pcr amplification.For the detection specificity nucleotide sequence, can use direct nucleotide sequencing, utilize hybridization, restriction enzyme digestion and mapping, PCR mapping, the RNase of specific oligonucleotide to protect and various other method.Can chemosynthesis and radioactivity or the specific oligonucleotide of nonradioactive labeling's (as biotin labeling, ethidium bromide) particular sequence; (for example hybridize with the individual sample that is fixed on film or other solid support then; shift from gel by dot blotting or behind electrophoresis), or in solution, hybridize.Whether there is target sequence with for example radioautograph, fluorometry or colorimetry visual inspection then.With high-density fixedly the known array on the silicon chip, abundant short oligonucleotide finishes these processes automatically.

(1) Shi Yi probe and primer

No matter be detection, pcr amplification or any other standard method as herein described and well known in the art of hybridization, RNase protection, ligase enzyme-mediation; the subsequence of the plain sequence of the open or available various early ageing of this paper all can be used as probe and/or primer, and these sequences or subsequence comprise plain sequence of normal early ageing and deleterious mutant sequence.In a word, useful sequence comprises the 8-9 at least from the plain intron of early ageing, exon or intron/exon border, more preferably 10-50, and the best is a 18-24 continuous nucleotide.According to target sequence, required specificity and the development of WeiLai Technology, short sequence also has effectiveness.Therefore, think that the plain deutero-sequence of any early ageing that is used to separate, clone, increase, identify or operates the plain sequence of early ageing all can be used as suitable probe or primer.Thinking useful especially is to comprise from the sequence of the nucleotide position of the known early ageing plain gene that has a pathogenic mutation wherein, or the sequence of these position flanks.

(a) PS1 probe and primer

As mentioned above, in people PS1 gene, identified different pathogenic mutations.With from normal or isolating nucleic acid probe of mutant PS1 gene deutero-or primer, can detect these and other PS1 sudden change now.Useful especially is probe or the primer that is derived from the sequence in coding N-end, TM1-TM2 district and TM6-TM7 district.But as mentioned above, detected the sudden change that influences other district of PS1 albumen, and used method disclosed herein, can detect certainly.Therefore the invention provides isolating nucleic acid probe and primer, can show that described part is relevant with the development of Alzheimer's disease corresponding to the normal and mutant nucleotide sequence of PS1 gene any part (comprising intron and 5 ' and 3 ' UTR).

Only as an example, rather than restriction the present invention, in screening and diagnostic method, can use on every side hPS1 dna fragmentation deutero-probe and primer from C410Y.Described sudden change in some individuality, is to cause because of the A in the SEQ ID NO:1 position 1477 replaces G at least.Therefore, with comprising that the oligonucleotide probe in this potential mutational site or primer can screen genomic dna, mRNA or the cDNA from individual peripheral blood sample.For the hybridization probe of this sudden change, can use the 8-50 that strides this mutational site (as the bp 1467-1487 of SEQ ID N0:1), more preferably the probe of 18-24 base.If use described probe with mRNA, certain described probe should with the mRNA complementation, and therefore, corresponding to the noncoding strand of PS1 gene.If probe uses with genomic dna or cDNA, then probe can with any chain complementation.In order to detect the sequence that comprises described sudden change with PCR method, suitable primer should comprise the 8-50 that is derived from sudden change either side zone, both sides, the long sequence of preferred 18-24 Nucleotide, and corresponding to 1-1000bp, but the mutational site has therefrom been removed in any position of preferred 1-200bp.It is the sequence that the PCR primer in mutational site 5 ' (on coding strand) should corresponding PS1 genes encoding chain, but the PCR primer of mutational site 3 ' (on coding strand) should corresponding noncoding strand or antisense strand (as corresponding to the 5 ' primer of the bp 1451-1468 of SEQ IDNO:1 with corresponding to 3 ' primer of the 719-699 complement of SEQ ID NO:14).

For other PS1 sudden change or general sudden change " focus ", can select similar primer.For example, M146L sudden change (can contain sequence corresponding to about bp 601-620 of SEQ ID NO:1 at 5 ' the PCR primer of bp 684A → C), and 3 ' primer can be corresponding to the complement of about bp1328-1309 of SEQ ID NO:8.Should notice that this embodiment has used the sequence from intron and exon two aspects.And another embodiment, A246E sudden change (the suitable 5 ' primer at bp985C → A) can contain the sequence corresponding to the about bp 907-925 of SEQ ID NO:1, or corresponding to 3 primer sequences of the complement of the about bp 1010-990 of SEQ ID NO:1.As another embodiment, (5 ' primer of bp 419 A of the bp736 of SEQ ID NO:1 or SEQ ID NO:9 → G) contains the sequence corresponding to the about bp354-375 of SEQ ID NO:9, and 3 ' primer is corresponding to the complement of about bp 581-559 for H163R sudden change.Similar, also can use intron or exon sequence, (5 ' primer of the bp 398C of the bp 1104 of SEQ ID NO:1 or SEQ ID NO:11 → G) contains the sequence corresponding to about bp 249-268 of SEQ ID NO:11 or the about bp1020-1039 of SEQ IDNO:1, and 3 ' primer is corresponding to the complement of the about bp510-491 of SEQ ID NO:11 for example to produce L286V sudden change.

It shall yet further be noted that probe and primer can comprise the Nucleotide of specific sudden change.Therefore, for example can produce the containing corresponding to the hybridization probe of the sequence of the about bp 1468-1486 of SEQ ID NO:1 or 5 ' primer of C410Y sudden change with the screening or the normal allelotrope that increases, or corresponding to identical sequence but bp 1477 altered (probe of G → T) or primer are with the screening or the mutant allele that increases.

(b) PS2 probe and primer

Above-mentioned probe and the primer that identical total consideration of PS1 probe and primer also is suitable for PS2.Particularly, described probe or primer can be corresponding to intron, exon or intron/exon border sequences, can be corresponding to the sequence of coming own coding or non-coding (antisense) chain, and can be corresponding to normal or mutant sequence.

As just embodiment, use corresponding to the 5 ' primer of the about bp 733-751 of SEQ ID NO:18 with corresponding to the 3 ' primer of the about bp 846-829 of SEQ ID NO:18, can screen PS1 N141I sudden change by the dna fragmentation around the pcr amplification.Similar, the M239V sudden change (can contain sequence corresponding to about bp 1009-1026 at the 5 ' primer of bp 1080A → G), and 3 ' primer can be corresponding to the complement of the about bp 1118-1101 of SEQ ID NO:18.As another embodiment, use corresponding to the 5 ' primer of the about bp 1576-1593 of SEQ ID NO:18 with corresponding to 3 ' primer of the about bp1721-1701 complement of SEQ ID NO:18, by the pcr amplification genomic dna, can screen the encoding sequence of I420T sudden change peripheral region.(bp1616-1632 of SEQ ID NO:18 is in that bp1624T → C) allele specific oligonucleotide of sequence can detect this product for for example available then wild-type (as the bp 1616-1632 of SEQ ID NO:18) and/or mutant.

(2) screening by hybridization

Normal or mutant PS1, PS2 or the plain relevant nucleotide sequence of other early ageing in situ detection; can prepare tissue sample with standard technique; contact with one or more above-mentioned probes then; preferably the probe of mark is so that detect; under the rigorous condition that only allows probe and height or accurate complementary sequence hybridization, finish the nucleic acid hybridization test.Because up to the present, most of PS1 that detects and PS2 sudden change are that mononucleotide replaces, and therefore need highly rigorous hybridization conditions that normal sequence and most mutant nucleotide sequence are differentiated.When known experimenter's early ageing plain gene type, can select corresponding probe.In addition, can be continuously or be used in combination probe at different mutants.Because carrying the individuality of the plain mutant of early ageing is heterozygosis, also can use the probe of normal sequence, the normal individual that isozygotys can be differentiated with the sudden change heterozygote by binding capacity (as density) by radiated signal.In another kind changes, use normal and mutant probe at the same time, but only mark under a kind of situation, can use CBA.

(3) restriction mapping

Sequence changes and also to produce or to have destroyed accidental restriction recognition site, and described site is by using suitable enzymic digestion, and the gel blot hybridization discloses then.By the increase or the minimizing of its size, or by increasing or reduced corresponding margining tablet hop count, can detect the dna fragmentation that carries described site (normal or mutant).Can use described restrictive fragment length polymerphism analysis (RFLP) or restriction mapping with genomic dna, mRNA or cDNA.Before restriction enzyme digestion, use above-mentioned primer, by the PCR plain sequence of early ageing that can increase, wherein PCR product length can show whether there is the specific limited site, and/or can carry out restriction enzyme digestion after amplification.By the plain fragment of any ordinary method (as when having ethidium bromide, under UV light) visual inspection early ageing.

As just embodiment, should notice that (SEQ ID NO:1bp684A → C) has destroyed the PsphI site for PS1 M146L sudden change; (bp736A → G) has destroyed the NlaIII site for H163R sudden change; (bp 985C → A) has produced the DdeI site for A246E sudden change; (bp1104C → G) has produced the PvuIII site for L286V sudden change.Based on the openly also available normal and mutant sequence of this paper, this area professional is easy to select from numerous commercially available restriction enzymes, finishes the restriction mapping analysis to detect the sudden change of any early ageing element.

(4) PCR mapping

In another serial embodiment, the difference based in PCR product length or the PCR production can detect single base and replace sudden change.Therefore, can be with striding the mutational site or the sample of the primer amplification of 3 ' end from experimenter's genomic dna, mRNA or cDNA preferably being arranged in the mutational site.The mispairing that is expected at the mutational site to be to change the ability that normal or mutant primer promotes polymerase chain reaction, produces normal and the heterozygosis and/or the different product figure between the early ageing element mutant that isozygotys thus.By standard technique, can separate discriminatively and detect normally and the PCR product of mutant gene as polyacrylamide or agarose gel electrophoresis and with the probe visual inspection of mark, ethidium bromide etc.Because the company that may produce non-specific initiation or mutational site reads, and the carrier of most of mutant allele all is heterozygosis, so the ability of described technology may be not enough.

(5) electrophoretic mobility

Genetic test based on dna sequence dna difference also can be finished by detecting DNA, mRNA or the change of mobility in gel of cDNA fragment.By the high resolution gel electrophoresis of list or double-stranded DNA or the variation of DNA heteroduplex mobility spectrum in native gel electrophoresis, can visual inspection for example foreword Lieque insertion of becoming estranged.Because the single strand conformation polymorphism (SSCP) relevant with mRNA or single stranded DNA secondary structure also can detect plain sudden change of early ageing or polymorphism with the method for research mobility shifting.

(6) chemical cracking of mispairing

Utilize chemical cracking (CCM) method (referring to for example Saleeba and Cotton, 1993 and reference wherein) of mispairing, also can detect the sudden change in the early ageing element.Use this technology, can with probe (can reach～1kb) with from experimenter's genomic dna, cDNA or mRNA sample mix.Sample and probe are mixed, be placed on then (if necessary) under the condition that can form heteroduplex.Preferred probe and sample nucleic acid all are double-stranded, or with probe and sample together through pcr amplification to guarantee sternly to give birth to all possible mispairing heteroduplex.The T residue of mispairing is that perosmic anhydride is reactive, and the C residue of mispairing is an azanol reaction.Because the A of each mispairing is with the T of mispairing, all with the C of mispairing, any nucleotide difference between probe and the sample (comprising little insertion or disappearance) all can cause forming at least one reactive heteroduplex to the G of each mispairing.Handling with after modifying any mispairing site with perosmic anhydride and/or azanol,, mixture is being carried out chemical cracking in the mispairing site of any modification by for example reacting with piperidines.The split product of mispairing between probe and the sample is described with detection with standard technique such as gel electrophoresis analysis mixture then.

(7) other method

Based on the plain sequence of the openly also available early ageing of this paper, various other methods that detect the plain sudden change of early ageing are conspicuous for this area professional.According to the present invention, any of these technology all is operable.PCR, denaturing gradient gel electrophoresis (DGGE that these include, but are not limited to nuclease protection test (S1 or ligase enzyme mediation), connect; Residue is Fischer andLerman for example, 1983), with SSCP bonded limiting acid endo enzyme fingerprinting (REF-SSCP; Referring to, Liu and Sommer, 1995) etc.

Other screening of D and diagnostic method

Because morbid state, under the situation of hereditary case, as primary disease and in non-hereditary case as secondary disease, may take place PS1, PS2, APP or with the proteic abnormal processing of PS1, PS2 or APP reaction.In bodily tissue or body fluid (as CSF or blood), can detect and be unusual phosphorylation, glycosylation, saccharification amidation or proteolytic cleavage product.

Transcribe at the early ageing element by observing, the change in translation and posttranslational modification and the processing and in brain and peripheral cells, in the early ageing plain gene cell and the change of transportation aspect, extracellular also can finish diagnosis.Described change comprises that plain messenger RNA(mRNA) of early ageing and/or albumen quantity divides variations, the variation of phosphorylation state, abnormal cells bit per minute cloth decided at the higher level but not officially announced, the outer distribution of abnormal cells etc.Described test comprises: Northern trace (with plain specificity of early ageing and non-specific nucleotide probe), Western trace and enzyme-linked immunoassay (ELISA) are (with early ageing element or the specific antibody of the plain functional domain of early ageing, comprise various posttranslational modification states, comprise glycosylation and phosphorylation isomer).Can to peripheral tissues (as blood cell, blood plasma, cultivation with other inoblast tissue etc.) and dead before or the biopsy and the cerebrospinal fluid of the CNS tissue that obtains after the postmortem finish these tests.Described test also can comprise in situ hybridization and immunohistochemistry (so that messenger RNA(mRNA) and albumen are positioned in specific subcellular compartment and/or the brain pathology structure relevant with amyloid plaque with these diseases such as the former entanglement of nerve fiber).

E screening and diagnostic kit

According to the present invention, diagnostic kit also is provided, described test kit comprises that commercially available diagnosis screens necessary reagent.For example can provide the test kit that comprises antibody or organize antibody more, described antibody is one or more mutant epitope specificities.Particularly available any these antibody of standard method mark of visual inspection bonded that help.In addition, can provide the test kit that wherein contains oligonucleotide probe or PCR primer, described probe or primer are used for detecting and/or amplification mutant PS1, PS2 or the plain related nucleotide sequences of other early ageing.In addition, can the described probe of mark so that easier detection specificity increases.In order to be applicable to above-mentioned various diagnosis embodiment, oligonucleotide probe in described test kit or antibody can be fixed on the substrate, can provide suitable contrast then.

10 methods of treatment

The present invention provides the basis for the treatment of the disease that maybe may be caused that causes because of the sudden change in the early ageing element now.As above described in detail, relevant with the development of the Alzheimer's disease of early onset thereof at hPS1 with the sudden change among the hPS2, therefore, The present invention be more particularly directed to treat diagnosis and suffer from the method that maybe might develop the experimenter of Alzheimer's disease.But the expression various tissues from PS1 and PS2 gene, the influence that suddenlys change on these seats should be not limited to brain probably, may be the cause of disease of the disease except that Alzheimer's disease therefore.Therefore, the invention still further relates to sudden change in early ageing plain gene and the gene product, wrongly express, wrong metabolism or other heredity or acquired change and disease in other tissue of causing shows.In addition, although Alzheimer's disease shows as nervous system disease, this performance may be because of the sudden change in the early ageing element causes, described sudden change at first influences other organ-tissue (as liver), discharging then influences the active factor of brain, finally causes Alzheimer's disease.Therefore, in the various methods of treatment of considering hereinafter described, should understand described treatment and may be with organizing after one's own heart beyond the brain, placenta, lung, skeletal muscle, kidney and pancreas be target, and also expresses PS1 and/or PS2 in these tissues.

Be not limited to any particular theory of the present invention, the influence of the Alzheimer's disease relevant with sudden change in the early ageing element has seemingly obtained new function, or quickened normal function, described sudden change directly or indirectly causes the undesired A of the being processed into β of amyloid precursor protein (APP) peptide, the abnormal cells apoptosis in the unusual phosphorylation body in the stable and/or brain.Described function acquisition or function acceleration model are consistent with the dominant inheritance of adult symptom of showing effect and Alzheimer's disease.Yet not understanding as yet suddenlys change in the early ageing element may produce the mechanism of these influences.

Known APP can pass through two kinds of approach metabolism.In first kind of approach, APP is passed to Golgi network, the vesicle through clarhrin bag quilt enters Secretory Pathway and carries out metabolic then.Ripe then APP passes to serous coat, adds non-amyloid C-terminal peptide (Selkoe etc., 1995 by α-secretase cracking to produce soluble fraction (ProteaseNexinII) at serous coat; Gandy etc., 1993).In addition, ripe APP can enter endosome-liposome approach, and in this approach, it will be through β and γ-secretase cracking to produce A β peptide.The Ap peptide derivant of APP is neurovirulent (Selkoe etc., 1994).The phosphorylation state of cell has been determined α-secretase (non-product amyloid) or A beta pathway (producing the amyloid approach) (Gandy etc., 1993) relative equilibrium between, and can carry out drug modification to described phosphorylation state by phorbol ester, muscarinic agonist and other reagent.The phosphorylation state of cell is seemingly mediated by the cytosol factor that acts on the integral protein in one or more Golgi networks (particularly protein kinase C).

Be not subjected to any restriction of particular theory of the present invention, the early ageing element, particularly hPS1 or hPS2 (carrying several phosphorylation consensus sequences of protein kinase C) may be integral proteins, and its phosphorylation state has been determined the relative equilibrium between α-secretase and the A beta pathway.Therefore the sudden change among PS1 or the PS2 can make the 26S Proteasome Structure and Function of its product change, thereby causes going wrong with regulatory element (as protein kinase C) or with the interaction of APP, promotes APP to enter thus and produces amyloid endosome-liposome approach.Environmental factors (as virus, toxin or aging) also has similar influence to PS1 or PS2.

Be not subjected to any restriction of particular theory of the present invention equally, should also be noted that PS1 and PS2 albumen all have and people's ionophorous protein and the basic homologous aminoacid sequence of acceptor.For example with the BLASTP example of (1990) such as Altschul, PS2 albumen and people sodium channel alpha subunit (E=0.18, P=0.16, the basic homology of identity in 2 districts of at least 35 amino-acid residues=22-27%).Other disease (as the malignant hyperthermia and the hyperkalemic periodic paralysis of philtrum, and the sex change of the mechanicalness Sensory neurone among the C.elegans) cause by the sudden change in ionic channel or the receptor protein.Therefore the sudden change in PS1 or the PS2 gene may influence similar function, and causes Alzheimer's disease and/or other spirituality and nervous system disease.

The methods of treatment of plain relative disease of treatment early ageing such as AD is based on (1) and uses normal PS1 or PS2 albumen, (2) carry out gene therapy with compensation or replacement mutator gene with normal PS1 or PS2, (3) antisense sequences or " rejecting " mutator gene with sudden change PS1 or PS2 gene is the based gene treatment, (4) the proteic sequence with coding blocking-up or correction PS1 or PS2 mutant deleterious effect is the based gene treatment, (5) be the based gene treatment with normal and/or mutant PS1 or the proteic antibody of PS2, or (6) small molecules (medicine), they change PS1 or PS2 expresses, blocking-up PS1 or the mutant forms of PS2 and the abnormal interaction between other albumen or the part, or by changing the structure of mutant protein, by strengthening its metabolite clearance or blocking the abnormal function of mutant PS1 or PS2 by suppressing its function.

The A protein for treatment

By replace with normal protein mutain, by regulating mutain function or by providing excessive normal protein to reduce the influence of any abnormal function of mutain, other disease that can treat the plain relevant Alzheimer's disease of early ageing or cause because of the plain sudden change of early ageing.

In order to reach this purpose, need obtain a large amount of pure substantially PS1 albumen or PS2 albumen as described herein and available, described albumen is from expressing described proteic cultured cells system.Pack or drug delivery system can be delivered to described albumen in ill brain district or other tissue with suitable, comprise as by liposome-mediated and albumen is delivered to target cell.

The B gene therapy

In a series of embodiment, can use gene therapy, PS1 gene that wherein will normally copy or PS2 gene import in patient's body so that successfully encode normal protein in one or more dissimilar diseased cells.Must be with described gene its absorption and the enough albumen of encoding can be delivered in those cells with the form by effective efficiency.Therefore, preferably recombination and strong promoter can be operated to link to each other and to compensate or the high level expression of out-compete mutant protein so that provide.As mentioned above, recombinant precursor can contain endogenous or the external source regulatory element, can induce and maybe can check regulatory element or tissue specificity regulatory element.

In another serial embodiment, can replace mutator gene with gene therapy by homologous recombination with recombinant precursor.Recombinant precursor can contain the target early ageing plain gene of normal copy, wherein corrects defective in position and maybe can contain and import " rejecting " construct that terminator codon, missense mutation maybe can be eliminated the disappearance of mutator gene function.Should notice that aspect this described construct can be rejected target early ageing plain gene normal and the sudden change copy simultaneously in heterozygous individual, but the disadvantageous effect ratio that loses individuality of total early ageing plain gene function makes described morbid state continuation development little.

In another serial embodiment, can use Antisense gene therapy.The basis of antisense therapy is that the sequence-specific of gene expression suppresses and can reach by hybridizing in the cell between mRNA or DNA and the complementary antisense strand.The formation of hybrid spiral can influence translation and/or the stability of gene transcription and/or processing, transportation, the plain mRNA of target early ageing then.Antisense therapy can utilize the whole bag of tricks, comprises using antisense oligonucleotide or antisense oligonucleotide the analogue analogue of thiophosphoric acid skeleton (as have) or using the antisense RNA expression vector transfection.In addition, described carrier can comprise external source or endogenous regulatory region, can induce and maybe can check regulatory element or tissue specificity regulatory element.

In another serial embodiment, can import the recombinant precursor of proteins encoded or peptide with gene therapy, the abnormal function that described albumen or peptide are capable of blocking or corrigendum causes because of sudden change early ageing plain gene.In one embodiment, described recombination coding finds that corresponding to the peptide in the plain mutation structure of early ageing territory described mutation structure territory and another cell protein or other cell ligand have abnormal interaction.Therefore, for example, if find mutant TM6 → 7 structural domains and specific cells protein-interacting, but corresponding normal TM6 → 7 structural domains do not have this interaction, and then available gene therapy provides excessive and mutant protein competition and suppresses or block mutant TM6 → 7 structural domains of described abnormal interaction.In addition, with the recombinant precursor codified and express with mutant interaction is arranged, but do not have interactional Partial Protein with normal early ageing element so that competition, and suppress thus or block abnormal interaction.At last, in another embodiment, by gene therapy, through inserting second mutant protein, can obtain identical effect, used method is with in c.elegans, and it is similar with the method for " Mec4 (d) " sudden change to correct " Degl (d) " by insertion mutant transgenosis.

Because it can efficiently infect and integration and expression that can be stable, so retrovirus vector can be used for somatic gene therapy.But target gene must be able to divide, and the expression level of normal protein should be very high, because this disease is a dominance.Can be with total length PS1 or PS2 gene, the subsequence of the functional domain of coding early ageing element or above-mentioned any other treatment peptide is cloned in the retrovirus vector, from its endogenesis promoter, terminal repetition or from the specific promotor transcriptional start of target cell (as neurone) from the length of retrovirus.Operable other virus vector comprises adeno-associated virus, vaccinia virus, bovine papilloma virus or simplexvirus such as Epstein-Barr virus.

The C immunotherapy

Immunotherapy also is feasible for Alzheimer's disease.Produce the antibody of anti-mutation PS1 or PS2 (or its part), be administered to the patient then with combination or blocking-up mutant protein, and suppress its adverse influence.Simultaneously, should promote the expression of normal protein product.In addition, produce the antibody of the specific complex between anti-mutation body or wild-type PS1 or PS2 and its interaction counterpart.

Another kind method is the production that stimulates anti-required antigenic endogenous antibody.Administration should be the form with immunogen goods or vaccine inoculation.Immunogenic composition injectable formulation be can be prepared into, solution or emulsion made.Can be with PS1 or PS2 albumen or other antigen and the compatible mixed with excipients of pharmaceutically acceptable and described albumen.Described vehicle can comprise water, salt, dextrose, glycerine, ethanol and its combination.Immunogenic composition and vaccine also can contain additional substances such as emulsifying agent or adjuvant to be renderd a service to strengthen.Can use immunogenic composition and vaccine outward by subcutaneous or intramuscular injection gi tract.

With treat effectively, protectiveness and cause the immunity amount and use described immunogen goods or vaccine.Dosage depends on route of administration, and can change with host's size.

D small molecules methods of treatment

As described herein and available, the invention provides the method for many evaluation small molecules or other compound, other disease that described small molecules or compound can be used for treating Alzheimer's disease or cause because of sudden change in the early ageing element.Therefore, for example the invention provides and identify the plain protein-bonded method of early ageing, particularly identify in conjunction with or with sudden change early ageing plain but not with the method for the plain interactional albumen of normal early ageing or other cellular component.The present invention also provides the micromolecular method of identifying, available described small molecules destroys the abnormal interaction between sudden change early ageing element and described albumen or other cellular component.

Described interaction, comprise mutant but do not comprise normal early ageing element, the data that is used for understanding the bio-chemical pathway of upsetting because of the sudden change of early ageing element not only is provided, and the Alzheimer's disease cause of disease, but also provide direct treatment target for getting involved this sick cause of disease.Be tested and appraised these albumen and analyze these interactions, can screen or design retroaction or suppress described interactional compound, therefore can treat this abnormal interaction.These treatments can change the interaction between early ageing element and these counterparts, change the function of interaction protein, change amount or the tissue distribution or the expression of interaction counterpart, or change the plain similar characteristics of early ageing own.

Can design methods of treatment and interact to regulate these, and therefore treat Alzheimer's disease and with the acquired of PS1 or PS2 gene or its gene product or other disease that genetic abnormality is relevant.After being exposed to medicine, use synthetic peptide or recombinant protein corresponding to the plain homologue of PS1 gene, PS2 gene or other early ageing, by standard drug kinetic measurement affinity (Kd and Vmax etc.), analyze affinity and these interactional functions, just can detect the potential effectiveness of these methods of treatment.It is that the metabolism pulse-chase under the medicine condition is not studied and monitored transportation and posttranslational modification in the cell by in situ hybridization, immunohistochemistry, Western trace and existence or as described in not existing as any interactional another kind of method of hydrophilic loop that detection relates to functional domain.Another kind method is the variation that the influence of monitoring " downstream " incident comprises (i) APP and product endocellular metabolism, transportation and guiding; (ii) Ca in second messenger's process such as the cAMP cell ²⁺, the variation of protein kinase activity etc.

As mentioned above, the early ageing element may relate to the APP metabolism, and the phosphorylation state of early ageing element is vital for the balance between the A beta pathway of α-secretase and APP processing.With transformant of the present invention and animal model, the anomalous event that people can understand these approach better and take place in the plain mutant of early ageing.Use described knowledge then, people can design methods of treatment to reverse the disadvantageous effect of the plain mutant of early ageing.

In order to treat Alzheimer's disease, for example by chemistry or biological chemical reagent (as medicine, peptide and other compound) can change PS1 and/or phosphorylation state, described reagent can change the activity of protein kinase C and other protein kinase, or change the activity of phosphoprotein phosphatase, or the PS2 operability is modified into the adorned state in translation back.Interaction between kinases ` Phosphoric acid esterase and the early ageing fibroin, and the plain APP that relates in the Golgi network with other proteic interaction of early ageing transports, can be adjusted to reduce of the transportation of golgi body vesicle to endosome-liposome approach, suppress the production of A β peptide thus.Institute's evolution compound will comprise APP peptide analogs, PS1, PS2 and the plain homologue of other early ageing, and other interaction protein, lipoid, sugar and promotion PS1, PS2 and/or other homologue carry out the glycosylated reagent of difference; Change plain mRNA of early ageing or the reagent of protein biology transformation period, comprise antibody and antisense oligonucleotide; And act on the reagent that PS1 and/or PS2 transcribe.

By the transcribing of monitoring PS1 and/or PS2, translation and posttranslational modification (as phosphorylation or glycosylation), and PS1 and/or PS2 can understand the effect of these reagent in clone and whole animal by in the various cells and transportation in the cell of extracellular compartment.The research method that is used for these comprises Western and Northern trace, with immunoprecipitation and the immunohistochemistry behind radiolabeled methionine(Met) and the ATP metabolic marker (pulse-chase).With standard in conjunction with compatibility test or utilize anti-protein kinase C, APP, PS1, PS2 or the co-precipitation and the Western trace of the antibody of the plain homologue of other early ageing, by the research of detection, also can detect the effect of these reagent with protein kinase C and/or the interactional PS1 of APP and/or proteic relative binding affinity of PS2 and relative quantity.Before being exposed to the treatment reagent of inferring and afterwards, by production with ELISA assessment A β peptide, also can detect these reagent effect (referring to, as Huang etc., 1993).Be exposed to think neurovirulent aluminium salt and/or A β peptide are arranged in Alzheimer's disease after, the viability by assessment clone also can detect the effect of these reagent.At last, the cognitive function by the assessment animal can detect the effect of these reagent, and described animal is carried at the normal genotype of the plain homologue of APP and/or its early ageing, the plain transgenosis (having or do not have sudden change) of carrier's early ageing or carry any of these and make up.

Similar, as mentioned above, the early ageing element may relate to Ca ²⁺Ca as acceptor or ionic channel ²⁺Adjusting.Also can study the described effect of early ageing element with transformation cell lines of the present invention and animal model.Based on these results, can produce the test that is used for Alzheimer's disease detecting abnormal receptor or unusual ion channel function, acquired in described function and early ageing plain gene and one of its product or homologue gene and its product or hereditary disorder is relevant.With the fluorescence dye of patch clamp, voltage clamp and intracellular Ca2+ or transmembrane voltage susceptibility, by measuring ionic channel flow and/or transmembrane voltage or current flow, can be in vivo or externally finish described test.By measuring the second messenger as Ca in the activation of ring AMP, cGMP casein kinase, phosphoric acid salt, the cell ²⁺The increases of level etc. also can detect ionic channel or function of receptors and lack.In the artificial rust system, the albumen that also can rebuild the reorganization preparation is with the conduction of research ionic channel.By analyzing its ability of modifying unusual ionic channel or suddenling change and the inductive function of receptors by analyzing in the early ageing plain gene, can detect influences Alzheimer's disease (owing to the acquired/hereditary defect in PS1 gene and the PS2 gene; Owing to defective such as APP in other approach that causes this disease suddenly change; With because environmental factors) methods of treatment.The acceptor ability of normal function or early ageing fibroin by its modified ion passage also can detect methods of treatment.Can finish described detection to expressing endogenous cultured cells normal or mutant PS1 gene/gene product or PS2 gene/gene product.Described research also can be finished described research to the carrier cells transfected with the functional domain of one of one of the early ageing element that can express normal or mutant forms or early ageing element.The methods of treatment that can be designed for Alzheimer's disease is to modify the unusual ionic channel or the function of receptors of PS1 gene or PS2 gene.Described treatment can be conventional medicine, peptide, sugar or lipoid and influence PS1 or the antibody of the characteristic of PS2 gene product or other part.Described treatment also can be finished by direct substitution PS1 gene of gene therapy and/or PS2 gene.Under the situation of ionic channel, make up body with minigene (cDNA adds promotor) or genome and can finish described gene therapy, described construct carries the part or all of genomic dna sequence of early ageing plain gene.Also available sudden change early ageing element or homologue gene order are offset the heredity or the acquired unusual influence of early ageing plain gene, as the Mec in C.elegans 4 that finishes recently the same with the replacement of Deg 1 (Huang and Chalfie, 1994).Described treatment also can relate to acceptor or the ion channel function of strengthening a kind of homologue such as PS2, so that the function (as the defective that provides by PS1 gene acquired or that hereditary defect produces) of another homologue mutant forms is provided effectively.Use gene therapy or albumen to replace the standard technique for the treatment of, also can treat by block mutant PS1 gene or mutant PS2 expression of gene and the collaborative normal PS1 of use or the replacement of PS2 gene with antisense oligonucleotide.

Embodiment

Embodiment 1 minimum is divided into the research of heredity, physics " overlap " and the transcripting spectrum in abscission zone

Use oligonucleotide probe, retrieval CEPHMegaYAC and RPCIPAC people's total genomic dna library, detect the genomic DNA fragment that wherein contains from the AD3 district of karyomit(e) 14q24.3, described probe is relevant 12 used in genetic linkage research SSR mark seats and each mark (Albertsen etc. of other mark; Chumakov etc., 1992; Loannu etc., 1994).Described on the contig between each mark genetic distance from, and be to derive from disclosed data (NIH/CEPH Collaborative Mapping Group, 1992; Wang, 1992; Weissenbach etc., 1992; Gyapay etc., 1994).With 4 kinds of diverse ways, the recovery at each start mark seat clone is arranged in orderly partly overlapping clone serial (" contig ").At first, with inverse PCR (Riley etc., 1990) discrete representation YAC inserts the sequence of segmental end, hybridizes other yac clone that carries overlap with evaluation with the Southern trace group that contains DNA restriction digestion fragment then, and described DNA comes to begin the yac clone that the seat reclaims since all.The second, each YAC is carried out PCR between Alu, the band collection of illustrative plates of comparison gained carries overlap with evaluation between the yac clone pond of reclaiming other clone (Bellamne-Chartelot etc., 1992; Chumakov etc., 1992).The 3rd, in order to improve the specificity of Alu PCR fingerprinting,,, differentiate described product with polyacrylamide gel electrophoresis with Alu and LIH consensus sequence primer amplification restriction enzyme digestion product with HaeIII or RsaI restriction enzyme digestion YAC DNA.At last, in studying by the process of transcription sequence, can produce other STS, also available these STS identify overlaps.Except that at the YAC932C7 that carries D14S52 with contain between the YAC746B4 of D14S61 and have the single point of discontinuity, the contig of gained is complete.The physical map of STS order very consistent (NH/CEPH Collaborative Mapping Group, 1992 in described contig with this regional genetic linkage map; Wang and Weber, 1992; Weissenbach etc., 1992; Gyapay etc., 1994).But as genetic map, it can not clearly differentiate D14S43/D14S71 bunch with D14S76/D14S273 bunch in the relative order at seat.The PAC1 clone shows that D14S277 is the telomere of D14S268, and the order of genetic map explanation is just in time opposite.In addition, in a yac clone, some STS probe can not detect the hybridization collection of illustrative plates at least, and based on the most thrifty consensus sequence physical map and genetic map, has thought to contain described STS among the described clone.For example in YAC788H12, there are not D14S268 (AFM265) and RSCAT7 STS.Because these results are reproducible, and several different STS marks all can this thing happens, and therefore, these results show probably have little intercalary deletion in one of yac clone.

2 advantage marks of accumulative total of embodiment 2 karyomit(e) 14q24.3 marks

With the genomic dna of 100ng, determine the genotype at each polymorphic micro-satellite markers seat by PCR, described DNA from aforementioned all suffer from and the member of ill pedigree (Weissenbach etc., 1992 not; Gyapay etc., 1994).Use spouses and other neural normal subjects to determine each allelic normal kind of train frequency, but significantly be different from frequency (Weissenbach etc., 1992 of blended Caucasia population from the same person kind; Gyapay etc., 1994).The maximum confidence calculated value is confirmed the correction value of age of onset, inferred marker allele frequency from disclosed mixing Caucasia experimenter's series, and the gene frequency of the foregoing AD3 sudden change of estimating is 1: 1000 (St.George-Hyslop etc., 1992).Use the same tag gene frequency, and only repeat described analysis from the resulting phenotype data of foregoing ill pedigree, can not mislead described analysis (St.George-Hyslop etc., 1992) with the parameter of guaranteeing estimation used in the maximum confidence calculated value.These supplement Analysis can significantly not change the recombination event that tangible support is chain or found.

The AD3 of haplotype in FAD between the embodiment 3 side marks separates

In the FAD of 14 early onset thereofs pedigree (pedigree NIH2, MGH1, Torl.1, FAD4, MEX1 and FAD2), on parent's copy of the isolating karyomit(e) 14 of AD3, the haplotype that extends between kinetochore and the telomere side mark shows pedigree specificity advantage mark＞3.00, and at least one mark between D14S258 and the D14S53 is like this.In several pedigrees of similar ethnic group, separation is in spite of illness observed the identical haplotype of part in chromosomal two zones.In the A district, can see total allelotrope (" B ": allelotrope size=126bp, the gene frequency in common Caucasian=0.04 at D14S268; " C ": size=124bp, frequency=0.38); D14S277 (" B ": size=156bp, frequency=0.19; " C ": size=154bp, frequency=0.33); RSCAT6 (" D ": size=111bp, frequency 0.25; " E ": size=109bp, frequency=0.20; " F ": size=107bp, frequency=0.47).In the B district, observe allelotrope (" A ": size=193bp, frequency=0.01 of identical size at D14S43; " D " size=187bp, frequency 0.12; " E ": size=185bp, frequency=0.26; " I ": size=160bp, frequency=0.38); D14S273 (" 3 ": size=193bp, frequency=0.38; " 4 " size=191bp, frequency 0.16; " 5 ": size=189bp, frequency=0.34; " 6 ": size=187bp, frequency=0.02) and D14S76 (" 1 ": size=bp, frequency=0.01; " 5 " size=bp, frequency 0.38; " 6 ": size=bp, frequency=0.07; " 9 ": size=bp, frequency=0.38).Detailed description referring to (1995) such as Sherrington.

Embodiment 4 reclaims the sequence of transcribing at interval from AD3

Use the direct cross method, be recovered in the transcription sequence of inferring of coding in the AD3 interval, in described method, will hybridize from short cDNA fragment and fixed clone gene group fragment (Rommens etc., 1993) that human brain mRNA produces.With the weak point of gained infer transcription sequence as probe so that from human brain cDNA (Stratagene, La Jolla), reclaim longer transcript.Determine the long cDNA clone's of short clone in source and gained physical location by analyzing the hybridization collection of illustrative plates, described hybridization collection of illustrative plates is by the Southern blot hybridization of probe with the total DNA sample that contains one group of EcoRI digestion produced, and described DNA sample is that the individual yac clone in the contig is isolating.(Applied Biosystems Inc. CA) determines the nucleotide sequence that each long cDNA clones, and uses other sequence in blast algorithm (Altschul etc., 1990) and Nucleotide and the albumen database to compare then with the automated cycle sequenator.The registration number of transcription sequence is: L40391, L40392, L40393, L40394, L40395, L40396, L40397, L40398, L40399, L40400, L40401, L40402 and 40403.

Buy and execute example 5 usefulness restriction enzymes and determine the position that suddenlys change in the PS1 gene

In genomic dna, detect whether there is A246E sudden change (producing the DdeI restriction site) with PCR, described PCR utilizes the end mark primer of the bp907-925 that corresponds essentially to SEQ ID NO:1 and corresponding to the unmarked primer of the bp1010-990 complement of SEQ ID NO:1, so that with 100ng genomic dna template, 2mM MgCl ₂, the primer of each 10 pmol, 0.5UT polysaccharase, 250uM dNTP carry out the genome exon fragment of 30 cyclic amplification 84bp, and every circulation contains 95 ℃ * 20 seconds, and 60 ℃ * 20 seconds, 72 ℃ * 5 seconds.According to the explanation of manufacturers,, on 6% non-denaturing polyacrylamide gel, differentiate then and by the radioautograph visual inspection with product and excessive DdeI insulation 2 hours.Owing to there is the DdeI restriction site, there is sudden change from the segmental cracking explanation of 84bp.All carry the DdeI site among the ill member of all FAD1 pedigrees and several adventurous members.Should suffer from and do not suffer from patient's (age more than 70 years old and not ill individuality) and normal control does not all have the DdeI sudden change.

Embodiment 6 usefulness allele specific oligonucleotides are determined the position that suddenlys change in the PS1 gene

Detect the existence of C410Y sudden change with allele specific oligonucleotide.Use corresponding to the exon sequence primer of the bp 1451-1468 of SEQ IDNO:1 and with the anti-intron sequences primer of the bp719-699 complementary of SEQ ID NO:14, use above-mentioned condition, different is to use 2.5mMMgCl ₂, 94 ℃ * 20 seconds, the cycling condition of 58 ℃ * 20 seconds and 72 ℃ * 10 seconds, the genomic dna of the 100ng that increases.Use 0.4M NaOH, 25 mM EDTA dilute 10 times of genomic fragments that come the 216bp of sex change gained, and the vacuum slot blot is to duplicating on the nylon membrane then.48 ℃ at 5 * SSC, 5 * Denhardt ' s, among the 0.5%SDS, with the slot blot filter paper hybridization of end-labelled " wild-type " primer (corresponding to the bp1468-1486 of SEQ ID NO:1) and end-labelled " mutant " primer (1477 replacements that G → A arranged) and different copies 1 hour corresponding to identical sequence but in the position, then at 23 ℃, with 2 * SSC, 50 ℃ with 2 * SSC continuous washing, then be exposed to X-ray film.Detect ill member and the adventurous member of all AD3 and NIH2 pedigree all have the C410Y sudden change.The effort that detects the C410Y sudden change with SSCP shows that common intron sequences polymorphism has identical SSCP mobility spectrum.

Embodiment 7Northern hybridization shows the expression of PS1 protein mRNA in different tissues

With standard method such as CsCl purifying, from the different tissues sample (different zones, lung, liver, skeletal muscle, kidney and the pancreas that comprise the heart, brain and placenta) that surgical operation obtains, separate total kytoplasm RNA.Electrophoresis RNA separates so that carry out size fractionation on formaldehyde gel then.The preparation nitrocellulose filter is transferred to RNA on the described film then.Preparation ³²The cDNA probe of P-mark is added in the film then so that probe and RNA are directly hybridized.After the washing, twine film, and be put in the imaging box that contains X-ray film with plastic film.Carry out radioautograph then to develop one to a couple of days.By relatively coming to determine size with the standard rna mark.Autoradiographic analysis revealed has a tangible band (referring to Sherrington etc., Fig. 2 of 1995) at 3.0kb.These Northern traces show all the PS1 expression of gene in the tissue of all detections.

Embodiment 8 eucaryons and prokaryotic expression carrier system

Obtained being applicable to the construct of eucaryon and prokaryotic expression system with dissimilar PS1 Nucleotide cDNA insertion sequences.First kind is called the total length construct, complete PS1 cDNA sequence is inserted in the expression plasmid with correct direction, and comprises natural 5 ' UTR and 3 ' UTR sequence and complete open reading frame.Described open reading frame contains a nucleotide sequence box, and described box can make the wild-type open reading frame be included in the expression system, or in addition two sudden changes single or combination is inserted in the described open reading frame.By from the wild-type open reading frame, removing a restricted fragment with enzyme NarI and PflmI, use the similar fragment that produces by ThermoScript II PCR to replace it then, described similar fragment contains the nucleotide sequence of coding M146L sudden change or H163R sudden change.By using enzyme PflmI and NcoI cracking, can from the wild-type normal oligodeoxynucleotide sequence of open reading frame, remove second restricted fragment, and replace with the restricted fragment of the nucleotide sequence that carries coding A246E sudden change, A260V sudden change, A285V sudden change, L286V sudden change, L392V sudden change or C410Y sudden change.By will having before one the NarI-PflmI fragment of planting sudden change and carry the PflmI-NcoI fragment of planting sudden change after and link to each other and can prepare the 3rd variant, this variant has the placed in-line combination that one of suddenlys change of M146L or H163R sudden change and residue.

Second kind of cDNA inserts fragment, is called the construct that blocks, by removing 5 ' UTR and part 3 ' UTR makes up from total length wild-type or mutants cDNA sequence.Replace 5 ' UTR sequence with the synthetic oligonucleotide that contains KpnI restriction site (GGTAC/C) and little sequence (GCCACC) and produce the Kozak initiation site with the ATG (the bp 249-267 of SEQ ID NO:1) that begins at PS1 ORF.Use the oligonucleotide corresponding to the complement of the bp2568-2586 of SEQ IDNO:1 to replace 3 ' UTR, described oligonucleotide has an artificial EcoRI site at 5 ' end.Then by the said mutation sequence being inserted into the mutation variants that above-mentioned NarI-PflmI and PsImI-NcoI site prepare this construct.

The third construct comprises the sequence that is derived from clone cc44, and wherein the alternative splicing of exon 4 is removals of 4 residues (SEQ ID NO:3) of being obtained from the N-end.

For eukaryotic expression, above-mentioned these different cDNA constructs that have wild-type and mutant nucleotide sequence are cloned among the expression vector pZeoSV, wherein removed the SV60 promotor, and replaced with pcDNA3 (Invitrogen) CMV promoter element by restriction digestion.For prokaryotic expression, pGEX-kg prepares construct with glutathione S-transferase (GST) fusion vector.The insertion fragment that has adhered on the GST fusion nucleus nucleotide sequence is and above-mentioned open reading frame nucleotide sequence or the above-mentioned list nucleotide sequence identical with amphimutation of carrying.These GST constructs can be expressed as mutant or wild-type gst fusion protein with part or full-length proteins in prokaryotic system, therefore just can remove the GST fusion product by digesting with zymoplasm, thus the purifying full-length proteins.Can prepare another cDNA construct with the GST fusion vector, so that will be the mutant nucleotide sequence that A285V sudden change, L285V sudden change or L392V sudden change are carried in derivative nucleotide sequence or conduct corresponding to the aminoacid sequence production of hydrophilic acidic ring between full-length proteins TM6 and the TM7.Use 5 ' Oligonucleolide primers bp1044-1061, that contain 5 ' BamHI restriction site (G/GATCC) corresponding to SEQ IDNO:1, with 3 ' primer complement, that contain 5 ' EcoRI restriction site (G/AATTC), by from the RNA in suitable source, reclaiming wild-type or mutant nucleotide sequence is finished corresponding to the bp1476-1458 of SEQ ID NO:1.Can will be cloned in the pGEX-KG carrier like this corresponding to suitable sudden change or wild-type nucleotide sequence in BamHI and EcoRI hydrophilic acidic ring structure territory.

Other sudden change in the embodiment 9 location PS1 genes

With the RT-PCR product of representing ripe mRNA/cDNA sequence or genomic dna, (directly nucleotide sequencing, allele specific oligonucleotide, connection polymerase chain reaction, SSCP, RFLPs) can detect the sudden change in the PS1 gene by the whole bag of tricks.For A260V and A285V sudden change,, can increase and carry the genomic dna of exon with identical PCR primer and the method that is used for the L286V sudden change.

Copy to nylon membrane then with the sex change of PCR product, and with the slot blot method slot blot of the above-mentioned C410Y of being used for sudden change.

At 48 ℃, by using corresponding to wild-type sequence (bp1017-1036 of SEQ ID NO:1) or the mutant nucleotide sequence (bp1017-1036 of SEQ ID NO:1, the sudden change of C → T is arranged at bp1027) the hybridization of end-labelled allele specific oligonucleotide, wash with the 3 * SSC damping fluid that contains O.1%SDS at 52 ℃ then, the A260V sudden change is recorded on these traces.By above-mentioned the A285V sudden change is recorded on these slot blots, but at 48 ℃ with other wild-type sequence (bp1093-1111 of SEQ ID NO:1) or the mutant primer (bp1093-1111 of SEQ ID NO:1, the sudden change of C → T is arranged at bp 1102) allele specific oligonucleotide, press above-mentioned washing at 52 ℃ then, but washing soln is 2 * SSC.

With adjusting the PCR buffer condition, removing magnesium density is 2mM, with loop body condition 94 ℃ * 10 seconds, 56 ℃ * 20 seconds, with 72 ℃ * 10 seconds, with primer (5 ' bp439-456 corresponding to SEQ ID NO:14, and 3 ' with the bp719-699 complementation of SEQ ID NO:14) write down the L392V sudden change from the exon amplification of genomic dna.By described for C410Y, the genomic fragment sex change with 200 base pairs of gained copies on the nylon membrane with slot blot.By with end-labelled oligonucleotide of wild-type (bp1413-1431 of SEQ ID NO:1) or the end-labelled mutant primer (bp1413-1431 of SEQ ID NO:1, the sudden change of C → G is arranged at bp 1422) at 45 ℃ of differential hybridizations, use 2 * SSC at 23 ℃ then, then show whether there is sudden change 68 ℃ of continuous washing.

Embodiment 10 antibody producing

Synthetic by solid phase technique corresponding to the proteic peptide antigen of part PS1, come purifying by reversed phase high efficiency hydraulic pressure chromatography then.Through disulfide linkage, peptide is linked to each other with keyhole _ azurin (KLH), described disulfide linkage may be by preparing at the terminal cysteine residues that adds of the plain segmental C-of early ageing.In this protein sequence, this additional residue normally can not appear, only be for the ease of linking to each other and comprise this residue with the KLH molecule.The plain sequence of the specificity early ageing that antibody resisted is as follows:

Polyclonal antibody # hPS1 antigen (SEQ ID NO:2)

1142?????????30-44

519??????????109-123

520??????????304-318

1143?????????346-360

These sequences are comprised in the proteic specificity structure of the PS1 territory.For example residue 30-44 is in the N-end, and residue 109-123 is in TM1 → 2 rings, and 304-318 and 346-360 are in big TM6 → 7 rings.These structural domains all are exposed in the water-bearing media, and these structural domains may develop vital proteic combination of disease phenotype with other.The selection of peptide is based on IBIPustell antigenicity prediction algorithm analyzing proteins sequence.

In freund's adjuvant,, carried out booster shots every 7 days with the altogether immune 3 kinds of New Zealand white rabbit of the antigenic peptide of various peptides-KLH mixture.Collect the antiserum(antisera) of each peptide, merge, with ammonium sulfate IgG precipitation.Use then and suitable peptide bonded Sulfo-link agarose (Pierce) affinity purification antibody.Carry out this and go on foot last purifying to remove before immunity or other antibody of the non-specific interaction that exists in the serum after the immunity.

Determine the specificity of each antibody with three kinds of tests.At first, on the Western of brain homogenate thing trace, detect with prediction and significantly be with at the list of early ageing element-1 approximate size.The second, carry out cross reaction with the recombination fusion protein that carries suitable sequence.The 3rd, with reorganization PS1 or immune peptide preadsorption, but specific inhibition.

In addition, used two kinds of different PS1 peptide glutathione S-transferase (GST) fusion roteins to produce PS1 antibody.First kind of fusion rotein comprises the amino acid/11-81 (N-end) of the PS1 that merges with GST.Second kind of fusion rotein comprises the amino acid 266-410 (TM6 → 7 ring structure territories) of the PS1 that merges with GST.By suitable nucleotide sequence is inserted in the pGEX-2T expression plasmid (Amrad), can produce the construct of these fusion roteins of coding.The construct of gained comprises the sequence of the GST that encodes and the zymoplasm susceptibility cracking site between GST and PS1 peptide.The expression construct transfection in DH5 α intestinal bacteria, is induced Expression of Fusion Protein with IPTG.Dissolution of bacteria precipitation is by (Boehringer-Mannheim carries out the GST-fusion rotein that a step affinity chromatography is come purification of soluble on Montreal) on the glutathione agarose pearl.Use standard method, use GST-fusion protein immunization mouse to produce monoclonal antibody.Screen the clone who from these mouse, obtains with the plain fragment of the early ageing of purifying.

In addition, use zymoplasm cracking GST-fusion rotein to discharge the PS1 peptide.With the peptide that the size exclusion chromatography purifying discharges, be used for immunize rabbit then to produce polyclonal antiserum.

Use similar methods, use the construct of the nucleotide sequence of the amino acid/11 to 87 (N-terminal) or 272 to 390 (TM6 → TM7 ring) that comprises early ageing element-2 to prepare gst fusion protein, be used for producing anti-the sort of proteic monoclonal antibody then.With zymoplasm cracking PS2-GST fusion rotein, the peptide immunize rabbit of using release and purifying is with the preparation polyclonal antiserum.

The sudden change that embodiment 11 identifies in the PS2 gene

With first primer to the RNA of cerebral tissue after lymphocytoblast or freezing postmortem being produced RT-PCR product corresponding to PS2 ORF with second primer, right 5 ' the primer of described first primer is corresponding to the bp478-496 of SEQ ID NO:18, the bp1366-1348 complementation of 3 ' primer and SEQ ID NO:18, it is the product of a 888bp, right 5 ' the primer of second primer is corresponding to the bp1083-1102 of SEQ ID NO:18, the bp 1909-1892 complementation of 3 ' primer and SEQ ID NO:18 is the product of a 826bp.Be used in 250 mMol dNTPs among the 10ml, 2.5 mM MgCl ₂, the 10pMol oligonucleotide 94 ℃ * 20 seconds, 58 ℃ * 20 seconds, 72 ℃ * 45 seconds, is finished PCR through 40 circulations.With automated cycle sequenator (ABI, Foster City, CA) determine the sequence of PCR product, replace (data are unlisted) by the heterozygosis Nucleotide in direct viewing and Factura (ver 1.2.0) and Sequence Navigator (ver1.0.1b15) the software package scanning fluorescent chromatographic spectrum.

The detection of N141I sudden change: replace generation BclI restriction site at Nucleotide 787A → T.Oligonucleotide with each 10pMol, carry the exon of this sudden change from the amplification of 100ng genomic dna, described oligonucleotide is corresponding to the complement (unlabelled) of the bp846-829 of the bp 733-751 (end-labelled) of SEQ ID NO:18 and SEQ IDNO:18, PCR reaction conditions and the following conditional likelihood that is used for the M239V sudden change.According to the method for manufacturers, (MA) the PCR product of restriction enzyme digestion 2ml is differentiated described product with native polyacrylamide gel electrophoresis for NEBL, Beverly with BclI in the 10ml reaction volume.In having the experimenter of wild-type sequence, the PCR product of 114bp is cracked into 68bp and 46bp fragment.Mutant nucleotide sequence is cracked into the product of 53bp, 46bp and 15bp.

The detection of M239V sudden change: lacked the NlaIII restriction site in Nucleotide 1080A → G replacement, making can be by the oligonucleotide with each 10 pMol, can detect whether M239V sudden change from 100 ng genomic dnas amplifications, described oligonucleotide is corresponding to the complement of the bp1118-1101 of the bp1009-1026 of SEQ ID NO:18 and SEQ ID NO:18.The PCR condition is: 0.5U Taq polysaccharase, 250 mM dNTP, 1mCi α ³²P-dCTP, 1.5 mM MgCl ₂, the 10m1 volume; 30 round-robin 94 ℃ * 30 seconds, 58 ℃ * 20 seconds, 72 ℃ * 20 seconds, to produce a 110bp product.2ml PCR product is diluted to 10ml, and (MA) restriction enzyme digestion is 3 hours for NEBL, Beverly with 3 U NlaIII.Differentiate described product with native polyacrylamide gel electrophoresis, then by the radioautograph visual inspection.Normal subjects's split product is 55,35,15 and 6bp, and mutant sequence obtains 55,50 and the fragment of 6bp.

The detection of I420T sudden change: similar to aforesaid method, it is right to use corresponding to the primer of the complement of the bp1721-1701 of the bp1576-1593 of SEQ ID NO:18 and SEQ ID NO:18, by the pcr amplification genomic dna, can screen the I420T sudden change to produce the product of one 146 base pairs.Use wild-type (as the bp1616-1632 of SEQ ID NO:18) and mutant (, the sudden change of T → C being arranged at bp1624) the allele specific oligonucleotide of sequence to detect this product then as SEQ IDNO:18 bp1616-1632.

Embodiment 12 transgenic mices

Make up serial wild-type and mutant PS1 and PS2 gene to be used to prepare transgenic mice.Use standard technique, produce sudden change PS1 and PS2 gene by clone's cDNA cc33 (PS1) and the site-directed mutagenesis of cc32 (PS2).

With cDNA cc33 and cc32 and mutant preparation thereof as embodiment 8 described two kinds of mutant and wild-type PS1 and PS2 cDNA.First kind is called " total length " cDNA, and by EcoRI (PS1) or PvuII (PS2) digestion, the about 200bp that removes 3 ' non-translational region before the polyA prepares.Second kind is called " blocking " cDNA, replaces 5 ' non-translational region by the ribosome bind site (Kozak consensus sequence) with ATG atg start codon 5 ' and prepares.

To import in one or more following carriers by the various total lengths of above-mentioned preparation and the wild-type of blocking and mutant PS1 and PS2 cDNA, with the construct of gained as the gene source of producing transgenic mice.

The cos.TET expression vector: this carrier is derived from the clay clone who contains Syrian hamster PrP gene.Made detailed description by (1995) such as (1992) such as Scott and Hsiao.PS1 and PS2 cDNA (total length or block) are inserted into the SalI site in this carrier.Final construct contains the 5 ' sequence of the 20kb that inserts the cDNA side.This 5 ' lateral order row comprise the PrP gene promoter, the PrP gene 5 ' non-translational region exon of 50bp, and donor splicing site, the 1kb intron, acceptor splicing site, this site is adjacent to the SalI site that PS1 or PS2 gene will insert.CDNA 3 ' the lateral order row that insert comprise PrP3 ' the non-translational region fragment that contains polyadenylation signal of about 8kb.Digest this construct with NotI (PS1) or FseI (PS2) and discharge mutant or the wild-type PS gene that is positioned under the control of PrP promotor.The fragment that discharges is carried out gel-purified, and the method with (1995) such as Hsiao is expelled in the pronucleus of fertilization mouse ovum then.

Thrombocyte prolongs living growth factor receptors beta subunit construct: from importing PS cDNA between the terminal EcoRI site of 3 ' of the growth factor receptors beta subunit promotor of human blood platelets terminal SalI (total length PS1 cDNA) or HindIII (the PS1 cDNA that blocks, total length PS2 cDNA and the PS2 cDNA that blocks) and SV40 poly sequence 5 ', then complete box is cloned into pZeoSV carrier (Invitrogen, San Diego, CA.) in.The fragment that gel-purified discharges by ScaI/BamHI digestion, the method with (1995) such as Hsiao is expelled in the pronucleus of fertilization mouse ovum then.

People's beta-actin construct: the SalI site that PS1 and PS2 cDNA is inserted into pBAcGH.The construct that produces by described insertion comprises 3.4Kb people's beta-actin 5 ' lateral order row (78bp people's beta-actin 5 ' the untranslated exon and intron of people's beta-actin promotor, montage) and PS1 or PS2 insertion fragment, is thereafter human growth factor hormone gene group sequence and the polyadenylation signal that contains the 2.2kb of several introns and exon.Discharge the fragment that contains PS with SfiI, gel-purified, the method with (1995) such as Hsiao is expelled in the pronucleus of fertilization mouse ovum then.

Phosphoglyceric kinase construct: PS1 and PS2 cDNA are imported in the pKJ90 carrier.This cDNA is inserted into the KpnI site in people's phosphoglyceric kinase promotor downstream and the XbaI site of phosphoglyceric kinase gene 3 ' non-translational region upstream.Discharge the fragment that contains PS with PvuII/HindII (PS1 cDNA) or PvuII (PS2 cDNA) digestion fragment, the described fragment of gel-purified as mentioned above, is expelled in the pronucleus of fertilization mouse ovum then.

Embodiment 13 is express recombinant PS1 and PS2 in eukaryotic cell

With the pcDNA3 carrier (Invitrogen, San Diego, CA.), express recombinant PS1 and PS2 in various cell types (as PC12, neuroblastoma, Chinese hamster ovary and human embryonic kidney 293 cell).Be inserted into PS1 and PS2 cDNA in this carrier and be total length identical and the cDNA that blocks with embodiment 8.

These cDNA are inserted between the CMV promotor and ox somatomedin hormone polyadenylation site of pcDNA 3.The high level expression transgenosis.

In addition, in the COS cell, express PS1 and PX2 with the pCMX carrier.For the ease of position in the cell of mark and spike early ageing fibroin, coding is derived from 11 amino acid whose oligonucleotide of people c-myc antibody (referring to for example Evan etc., 1985) with by monoclonal anti c-myc antibody MYC1-9E10.2 (Product CRL 1729, ATCC, Rockville, Md) do to connect in the frame, be right after before or after PS1 and PS2 open reading frame cDNA.And prepared unlabelled pCMX construct.The construct of c-myc mark is imported to pcDNA3, with transfection CHO cell.

With the method for manufacturers, instantaneous and these constructs of stable transfection with Lipofectamine (Gibco/BRL).After 48 hours, detect the transient expression of culture.Clone with 0.5mg/mlGeneticin (Gibco/BRL) screening stable transfection.

With the plain antibody 1142,519 and 520 of above-mentioned premature senescence resistance, detect the proteic expression of PS of transfection by the Westem trace.In a word, the transfectional cell (2%SDS, 5mM EDTA, 1mg/ml leupeptin and aprotinin) that dissolving is cultivated is determined protein concentration by the Lowry method.Go up protein isolate at SDS-PAGE gradient gel (4-20%Novex),, transferred on the PVDF (10mM CAPS) through 2 hours then at constant voltage (50 volts).Blocked non-specific binding through 1 hour with skimmed milk (5%).Use then two kinds of rabbit polyclonal antibodies (in TBS～1mg/ml, pH7.4) with Protein Detection 12 hours.Identify the cross reaction kind of early ageing element with biotinylated goat antirabbit secondary antibody, but use three grades of streptavidins of horseradish peroxidase bonded, 4-chloro-naphthols and the described secondary antibody of hydrogen peroxide visual inspection.With plain antibody of above-mentioned premature senescence resistance (to detect the plain peptide antigen of early ageing) and culture supernatant (to detect the myc-epi-position), detect the plain peptide of early ageing of c-myc mark by the Western trace, described supernatant liquor is from hybridoma MYC1-9E10.2, be used for the Western trace with dilution in 1: 10, be used for immunocytochemistry with dilution in 1: 3.With plain antibody of different early ageing and the immune response master tape by myc-epitope antibodies (being used for containing the clone of the plasmid transfection of myc) evaluation 50-60kDa.With the plain antibody test～10-19kDa of some early ageing and～the little band of 70kDa.

For immunocytochemistry, be used in 4% formaldehyde fixed cells transfected in the Tris buffering salt (TBS), add the 0.1%Triton thorough washing with TBS, then with 3% blocking-up BSA non-specific binding.With the plain antibody of early ageing (as above-mentioned antibody 520 and 1142; Be generally 5-10mg/ml) detect the fixed cell, washing is also carried out visual inspection with FITC-or rhodamine bonded goat antirabbit secondary antibody.For the plain construct of the early ageing of c-myc-mark, the hybridoma MYC1-9E10.2 supernatant liquor of dilution in 1: 3 is used with anti-mouse secondary antibody.With 0.1% phenylenediamine (ICN) in 90% glycerine fixedly slide glass to keep fluorescence.With anti-BIP (or anticalcium articulin) (StressGen, Victoria, B.C.) and wheat germ agglutinin (EY Labs, San Mateo is CA) respectively as the mark of endoplasmic reticulum and golgi body.With anti-Actin muscle (Sigma, St.Louis, Mo.), anti-amyloid precursor protein (22C11, Boehringer Mannheim) and anti-neurofilament (the NF-M specificity, Sigrna) neuronal cell be finish among the NSC34 two immune labeled.These immunofluorescences studies show that described transfection product extensively is distributed in the cell, particularly mostly are the feature of endoplasmic reticulum and golgi body around nuclear, and this is to observed similar in non-transfected cell, but more density is higher, overflows in nuclear membrane sometimes.In with the CHO of the plain construct transient transfection of the early ageing of myc mark and COS cell, observe the common immunolocalization of c-myc and PS epi-position.

Therefore confirm that with immunocytochemistry, Northern trace, Western trace (with the plain antibody of above-mentioned premature senescence resistance, with the monoclonal antibody MYC1-9E10.2 of myc mark in the anti-construct and 3 ' or 5 ' c-myc mark) Robust of transfection early ageing plain gene in the transfectional cell expresses.

Embodiment 14 usefulness affinity chromatographys are separated the plain bonded albumen of early ageing

In order to identify and the plain biochemical function proteins associated of early ageing, separate PS1 bonded albumen with affinity chromatography.Use the GST-fusion rotein that contains PS1 TM6 → 7 rings according to embodiment 8 described preparations to detect people's brain extract, described extract is by preparing with Polytron homogenate cerebral tissue in physiological saline.With gsh-sepharose 4B insulation, eliminate non-specific binding in conjunction with the brain homogenate thing of component by the endogenous GST of preclearing.Then these no GST homogenates are incubated with production with the GST-PS fusion rotein and have the protein-bonded required mixture of function.Behind the phosphate-buffered salt thorough washing, with SDS-polyacrylamide gel electrophoresis (SDS-PAGE; Tris-tricine gradient gel 4-20%) difference protein isolate.Except that a little less than 50-60kDa has several the band ,～14 and 20kDa also observe two master tapes.

Interactional pharmacology is modified and be can be used for treating Alzheimer's disease between these albumen and TM6 → 7 rings.In addition, in the plain bio-chemical pathway of early ageing, can active these albumen may be the new friend's displacement point that causes Alzheimer's disease.

Embodiment 15 usefulness double cross Yeast systems separate the plain bonded albumen of early ageing

In order to identify and the interactional albumen of early ageing fibroin, by Partial cDNA Sequence in the frame of will encode PS1 protein residues 266-409 or PS2 protein residues 272-390 be connected to the EcoRI of carrier and BamHI site produce the expression plasmid of yeast carrier (pAS2-1, Clontech).The fusion rotein of gained contain with the proteic TM6 of PS1 → 7 ring or the proteic TM6 of PS2 → 7 ring frames in the GAL4 DNA binding domains that links to each other.With Clontech Matchmaker yeast two-hybrid test kit (Clontech) with these expression vectors with from the plasmid DNA purification cotransformation in human brain cDNA:pACT library in yeast.Screen and TM6 → interactional yeast clone that carries human brain cDNA of 7 rings with HIS resistance and β gal+ activity.Use Cyclohexamide (cyclohexamide) susceptibility screening and cloning again, separate human brain cDNA with PCR and insert fragment, then order-checking.According to the method screening positive clone of manufacturers's explanation, in 600 ten thousand initial transformant, after the HIS screening, obtain 200 positive colonies, behind β gal+ dithering, obtain 42 clones.Among these 42 clones, there are several (5-8) to represent the difference clone of homologous genes.This shows that these interactions are that biology is real and be repeatably.

Embodiment 16 transgenosis C.elegans

Obtain transgenosis C.elegans by the microinjection ovocyte.Reach this purpose with carrier pPD49.3 hsp16-41 and pPD49.3 hsp 16-2.With preceding a kind of carrier, produce the transgenosis C.elegans that has wherein imported normal hPS1 gene or mutant (L392V) earlier.With above-mentioned people cDNA probe cc32 and antibody 519,520 and 1142, detect the animal of conversion by the expression that on Northern trace or Western trace, detects people cDNA.Preparation carrier and/or injection cis double-mutant hPS1 gene (M146L and L392V), normal hPS2 gene and mutant (N141I) gene.

The plain homologue of embodiment 17 clone fruit bat early ageing, DmPS

According to disclosed nucleotides sequence column data, promptly be with Trp termination/beginning (as residue Trp247 and the Trp404 in PS1; Trp253 in PS2 and Trp385) oligonucleotide 5 ' ctn ccn gar tgg acn gyc tgg (SEQ IDNO:22 and 5 ' rca ngc (agt) the at ngt ngt rtt cca (SEQ ID NO:23) of the proteic high conservative region of early ageing element/sel-12 design Feng Yu.From growing up and the mRNA of embryo's black-tailed fruit flies, carry out RT-PCR (50ml volume, 2mM MgCl, 30 round-robin 94 ℃ * 30 seconds, 57 ℃ * 20 seconds, 72 ℃ * 20 seconds) with these primers.94 ℃ then * 1 second, described product again increases for the condition of 59 ℃ * 0.5 second and 72 ℃ * 1 second and inner conservative Feng Yu primer 5 ' ttt tttctc gag acn gcn car gar aga aay ga (SEQ ID NO:24) and 5 ' ttt ttt gga tcc tar aa (agt) atr aar tcn cc (SEQ ID NO:25).With～600bp product cloning BamHI and XhoI site to pBS.With the order-checking of these products, and show and contain the open reading frame that has putative amino acid sequence with the plain height of people's early ageing homologous.(Stratagene is CA) to reclaim to be checked order 6 the independent cDNAs clones (clone pds8, pds13, pds1, pds3, pds7 and pds14) of size for～2-2.5kb with the conventional black-tailed fruit flies cDNA/Zap library of this fragment screening.The longest ORF coding has 541 amino acid whose polypeptide, and this peptide and people's early ageing have 52% consistence.

The detection of the long isomer of embodiment 18 A β peptides

PS1 or β APP have been defined from histopathology ₇₁The FAD of sudden change; The known fragmentary AD that does not have family history; The neurodegenerative disease of other outbreak of growing up (HD=heredity row Huntington's chorea; The ALS=amyotrophic lateral sclerosis); Mongolism (DS); In the freezing pallium of the contrast that does not have the neuropathy symptom, use 99% formic acid, extract A β peptide through 69 minutes (20 ℃).Behind centrifugal 20 minutes of 200000 * g, with supernatant liquor and precipitate and separate, dilute then, neutralize and detect by ELISA.In order to determine the amount of A β peptide, 4 kinds of monoclonal antibodies have been used.Use antibody BNT-77 (detecting epi-position) and antibody BAN-50 (detecting the N-terminal residue) in conjunction with all types of A β earlier, comprise the heterozygosis form that contains or do not contain the terminal brachymemma (BNT-77) of N-or only do not have the terminal brachymemma of N-(BAN-50) from A β center.With the terminal form of different C-of other two kinds of monoclonal antibody difference A β, described monoclonal antibody detects the long-tail A β (antibody BC-05) that ends at the short-tail A β (antibody β A-27) of residue 40 or end at residue 42/43 then.By aforementioned (Tamaoka etc., 1994; Suzuki etc., 1994) finish two site ELISA.In a word, be applied on the titer plate with the BNT-77 antibody sandwich with the standard peptide of 100 μ g or from the supernatant liquor of cerebral tissue, 4 ℃ of insulations 24 hours, wash then with phosphate-buffered salt, then with the BA-27 and the BC-05 antibody of HRP-mark, 4 ℃ of insulations 24 hours.With aforementioned TNB micropore superoxide enzyme system, detect the HRP activity by color development.With paired Xue Shengshi t test, the cortex A β level between the comparative diagnoses group.With the test of Student-Newman-Keuls multiple comparisons, show APP from β in conjunction with the data of assessing all A beta isomers ₇₁₇Different with the experimenter's of fragmentary AD A β 1-42 level and PS1 sudden change situation, but similar to contrast.On the contrary, when considering A β x-42 level, 3 groups have remarkable difference: high (PS1 and β APP ₇₁₇AD), medium (fragmentary AD) and low (contrast).

Specifically, measure the concentration of different A beta isomers in 14 contrast experimenters (comprising 5 experimenters) pallium, show short-tail A β (the A β 1-40:0.06 ± 0.02nmol/g wet tissue ± SEM that has only lower concentration at other neurodegenerative disease of trouble of 40-50 year outbreak; A β x-40:=0.17 ± 0.40) and long-tail A β (A β 1-42/43:0.35 ± 0.17; A β x-42/43:1.17 ± 0.80) these two kind.On the contrary, in all 4 experimenters' that the PS1 sudden change is arranged pallium, long-tail A β peptide all has rising (A β 1-42/43:6.54 ± 2.0, p=0.05; A β x-42/43:23.91 ± 4.00, p＜0.01).β APP is being arranged ₇₁₇Experimenter (A β 1-42/43:2.03 ± 1.04 of sudden change; A β x-42/43:25.15 ± 5.74) and experimenter (A β 1-42/43:1.20 ± 0.40, the p=0.008 of fragmentary AD arranged; A β x-42/43:14.45 ± 2.81) all detect long-tail A β peptide in the cortex similar increase is arranged.PS1 or β APP are being arranged ₇₁₇Among the experimenter of sudden change, when A β peptide long-tail isomer increased, short-tail A β peptide isomer also had a small amount of but inapparent increase (as, A β x-42/43: be 3.08 ± 1.31 in the PS1 mutant; At β APP ₇₁₇Be 1.56 ± 0.07 in the mutant).Therefore, long ratio with short isomer also significantly increases.But in the case of the fragmentary AD of typical case, when long-tail A β peptide isomer increased, the increase of short-tail A β peptide isomer was than the former much bigger (A β 1-40:3.92 ± 1.42; A β x-42/43:16.60 ± 5.88).Compared with the control, this increase of short-tail A β peptide is significant (for A β 1-40 and A β x-42/43, p＜0.03) statistically, but with PS1 and β APP ₇₁₇Situation compare, be border statistics significant (p_0.05).Analysis shows that from the cortex sample of mongolism adult patients its collection of illustrative plates is to observed similar in fragmentary AD.

Although this paper describes the preferred embodiments of the invention in detail, this area professional not should be understood that in departing from spirit of the present invention or claim scope can carry out various changes.Table 1

Position of components	Position of components
		?STAT1?(GAS)??????34-46??????611-619 ??????????????????278-286????631-639 ??????????????????431-439????1582-1590 ??????????????????443-451????1965-1973 ??????????????????495-503????2125-2133 ??????????????????533-541	CAT box 895-900 975-982
TATA box 925-933 978-988
	?TFIID????????????????578-581 ??????????????????????982-985
?STAT3????????????36-43??????737-744 ??????????????????124-131????811-898 ??????????????????429-436????1063-1070 ??????????????????496-503????1686-1693 ??????????????????533-540????1966-1973 ??????????????????537-544????2104-2111 ??????????????????632-639????2407-2414			TRXN (CAP) 1002-1007 opens beginning 1038-1043
	GC box 1453-1460 (SP1) 1454-1452
		AP2, AP2-sample occur in sequence in a large number
	?NFIL6???????611-620??1567-1576 ?????????????890-899??1945-1954 ?????????????1062-1071
		MED1, MED1-sample 1121-1126 1235-1240 1126-1131 1716-1721

The terminal 1-81TM1 82-100TM1 of the table 2PS1 domain apparent position N-→ terminal 429-467 of 2 101-132TM2 133-154TM2 → 3 155-163TM3 164-183TM3 → 4 184-194TM4 195-212TM4 → 5 213-220TM5 221-238TM5 → 6 239-243TM6 244-262TM6 → 7 263-407TM7 408-428C-

The terminal 1-87TM1 88-106TM1 of the table 3PS2 domain apparent position N-→ terminal 410-448 of 2 107-134TM2 135-160TM2 → 3 161-169TM3 170-189TM3 → 4 190-200TM4 201-218TM4 → 5 219-224TM5 225-244TM5 → 6 245-249TM6 250-268TM6 → 7 269-387TM7 388-409C-

Table 4

Change the age of onset that amino acid changes functional domain FAD at SEQ ID NO:1 Nucleotide

1. NA NA A79？ N- 642. 492 G→C V82L TM1 553. NA NA V96F TM1 NA45 591 T→C Y115H TM1→2 375. 664 T→C M139T TM2 496. NA NA M139V TM2 407. 676 T→C I143T TM2 358. 684 A→C M146L TM2 459. NA NA M146V TM2 3810. 736 A→G H163R TM2→3 5011. NA NA H163Y TM2→3 4712. NA NA L171P TM3 3513. NA NA G209V TM4 NA14. NA NA I211T TM4 NA15. 939 G→A A231T TM5 5216. 985 C→A A246E TM6 5517. 1027 C→T A260V TM6 4018. NA NA C263R TM6→7 4719. 1039 C→T P264L TM6→7 4520. NA NA P267S TM6→7 3521. NA NA E280A TM6→7 4722. NA NA E280G TM6→7 4223. 1102 C→T A285V TM6→7 5024. 1104 C→G L286V TM6→7 5025. NA Δ291-319 TM6→7 NA26. 1399 G→C G384A TM6→7 3527. 1422 C→G L392V TM6→7 25-4028. 1477 C→A 2410y TM7 43

Table 5

Change the age of onset that amino acid changes functional domain FAD at SEQ ID NO:18 Nucleotide

In position 1. 787 A → T N141I TM2 50-652. 1080 A → G M239V TM5 50-703. 1624 T → C IA20T CC-end 45

Table 628-61 302-31065-71 311-325109-112 332-342120-122 346-359218-221 372-382241-243 400-410267-269

Table 725-45 282-29050-63 310-31470-75 321-338114-120 345-352127-132 380-390162-167 430-435221-226

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Sequence table (1) physical data: (i) applicant:

(A) title: HSC researches and develops company limited

(B) street: 555University Avenue

(C) city: Toronto

(D) state: Ontario

(E) country: Canada

(F) postcode (ZIP): M5G1X8

(G) phone: (416) 813-5982

(H) fax: (416) 813-5085

(A) title: THE GOVERNING COUNCIL OF THE UNIVERSITY OF TORONTO

(B) street: 106, Simcoe Hall, 27King ' s College Circle

(C) city: Toronto

(D) state: Ontario

(E) country: Canada

(F) postcode (ZIP): M5S1A1

(G) phone: (416) 978-7461

(H) fax: (416) 978-1878

(A) title: ST.GEORGE-HYSLOP, Peter H.

(B) street: 210Richview Avenue

(C) city: Toronto

(D) state: Ontario

(E) country: Canada

(F) postcode (ZIP): M5P3G3

(A) title: FRASER, Paul E.

(B) street: 611 Windermere Avenue

(C) city: Toronto

(D) state: Ontario

(E) country: Canada

(F) postcode (ZIP): M6S3L9

(A) title: ROMMENS, Johanna M.

(B) street: 105McCaul Street

(C) city: Toronto

(D) state: Ontario

(E) country: Canada

(F) postcode (ZIP): M5T 2XT is the invention exercise question (ii): with (iii) sequence number of the gene order of related to alzheimer's disease and albumen and uses thereof: 25 (iv) relative addresses:

(A) addressee: Sim ﹠amp; McBurney

(B) street: 330Universitv Avenue, 6th Floor

(C) city: Toronto

(D) state: Ontario

(E) country: Canada

(F) ZIP:M5G1R7 (v) computer-reader form:

(A) media type: floppy disk

(B) computer: lBM PC compatibility

(C) operating system: PC-DOS/MS-DOS

(D) software: PatentIn Release#1.0, Version#1.30 (vi) present application materials:

(A) application number: PCT/CA96/00263

(B) applying date: on April 29th, 1996

(C) classification number: (vii) application materials formerly:

(A) application number: US08/509359

(B) applying date: July 31 nineteen ninety-five (vii) application materials formerly:

(A) application number: US08/496841

(B) applying date: June 28 nineteen ninety-five (vii) application materials formerly:

(A) application number: US08/431048

(B) applying date: April 28 nineteen ninety-five (viii) proxy/act on behalf of data:

(A) name: RAE, Patricia A.

(B) document/number of documents: 7425-16 (ix) communication data:

(A) phone: (416) 595-1155

(B) fax: the data of (416) 595-1163 (2) SEQ ID NO:1: (i) sequence signature:

(A) length: 2765 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear (ix) feature:

(A) title/keyword: CDS

(B) position: 249 ... 1649 (ix) feature:

(A) title/keyword: misc_ feature

(B) position: 1 ... 2675

(D) other data :/notes=" hPSI-1 " be sequence description: SEQ ID NO:1:TGGGACAGGC AGCTCCGGGG TCCGCGGTTT CACATCGGAA ACAAAACAGC GGCTGGTCTG 60GAAGGAACCT GAGCTACGAG CCGCGGCGGC AGCGGGGCGG CGGGGAAGCG TATACCTAAT 120CTGGGAGCCT GCAAGTGACA ACAGCCTTTG CGGTCCTTAG ACAGCTTGGC CTGGAGGAGA 180ACACATGAAA GAAAGAACCT CAAGAGGCTT TGTTTTCTGT GAAACAGTAT TTCTATACAG 240TTGCTCCA ATG ACA GAG TTA CCT GCA CCG TTG TCC TAC TTC CAG AAT GCA 290 (xi)

Met?Thr?Glu?Leu?Pro?Ala?Pro?Leu?Ser?Tyr?Phe?Gln?Asn?Ala

1???????????????5??????????????????10CAG?ATG?TCT?GAG?GAC?AAC?CAC?CTG?AGC?AAT?ACT?GTA?CGT?AGC?CAG?AAT?????????338Gln?Met?Ser?Glu?Asp?Asn?His?Leu?Ser?Asn?Thr?Val?Arg?Ser?Gln?Asn?15??????????????????20??????????????????25??????????????????30GAC?AAT?AGA?GAA?CGG?CAG?GAG?CAC?AAC?GAC?AGA?CGG?AGC?CTT?GGC?CAC?????????386Asp?Asn?Arg?Glu?Arg?Gln?Glu?His?Asn?Asp?Arg?Arg?Ser?Leu?Gly?His

35??????????????????40??????????????????45CCT?GAG?CCA?TTA?TCT?AAT?GGA?CGA?CCC?CAG?GGT?AAC?TCC?CGG?CAG?GTG?????????434Pro?Glu?Pro?Leu?Ser?Asn?Gly?Arg?Pro?Gln?Gly?Asn?Ser?Arg?Gln?Val

50??????????????????55??????????????????60GTG?GAG?CAA?GAT?GAG?GAA?GAA?GAT?GAG?GAG?CTG?ACA?TTG?AAA?TAT?GGC?????????482Val?Glu?Gln?Asp?Glu?Glu?Glu?Asp?Glu?Glu?Leu?Thr?Leu?Lys?Tyr?Gly

65??????????????????70??????????????????75GCC?AAG?CAT?GTG?ATC?ATG?CTC?TTT?GTC?CCT?GTG?ACT?CTC?TGC?ATG?GTG?????????530Ala?Lys?His?Val?Ile?Met?Leu?Phe?Val?Pro?Val?Thr?Leu?Cys?Met?Val

80??????????????????85??????????????????90GTG?GTC?GTG?GCT?ACC?ATT?AAG?TCA?GTC?AGC?TTT?TAT?ACC?CGG?AAG?GAT?????????578Val?Val?Val?Ala?Thr?Ile?Lys?Ser?Val?Ser?Phe?Tyr?Thr?Arg?Lys?Asp?95?????????????????100?????????????????105?????????????????110GGG?CAG?CTA?ATC?TAT?ACC?CCA?TTC?ACA?GAA?GAT?ACC?GAG?ACT?GTG?GGC?????????626Gly?Gln?Leu?Ile?Tyr?Thr?Pro?Phe?Thr?Glu?Asp?Thr?Glu?Thr?Val?Gly

115?????????????????120?????????????????125CAG?AGA?GCC?CTG?CAC?TCA?ATT?CTG?AAT?GCT?GCC?ATC?ATG?ATC?AGT?GTC?????????674Gln?Arg?Ala?Leu?His?Ser?Ile?Leu?Asn?Ala?Ala?Ile?Met?Ile?Ser?Val

130?????????????????135?????????????????140ATT?GTT?GTC?ATG?ACT?ATC?CTC?CTG?GTG?GTT?CTG?TAT?AAA?TAC?AGG?TGC?????????722Ile?Val?Val?Met?Thr?Ile?Leu?Leu?Val?Val?Leu?Tyr?Lys?Tyr?Arg?Cys

145?????????????????150?????????????????155TAT?AAG?GTC?ATC?CAT?GCC?TGG?CTT?ATT?ATA?TCA?TCT?CTA?TTG?TTG?CTG?????????770Tyr?Lys?Val?Ile?His?Ala?Trp?Leu?Ile?Ile?Ser?Ser?Leu?Leu?Leu?Leu

160?????????????????165?????????????????170TTC?TTT?TTT?TCA?TTC?ATT?TAC?TTG?GGG?GAA?GTG?TTT?AAA?ACC?TAT?AAC?????????818Phe?Phe?Phe?Ser?Phe?Ile?Tyr?Leu?Gly?Glu?Val?Phe?Lys?Thr?Tyr?Asn175?????????????????180?????????????????185?????????????????190GTT?GCT?GTG?GAC?TAC?ATT?ACT?GTT?GCA?CTC?CTG?ATC?TGG?AAT?TTT?GGT?????????866Val?Ala?Val?Asp?Tyr?Ile?Thr?Val?Ala?Leu?Leu?Ile?Trp?Asn?Phe?Gly

195?????????????????200?????????????????205GTG?GTG?GGA?ATG?ATT?TCC?ATT?CAC?TGG?AAA?GGT?CCA?CTT?CGA?CTC?CAG?????????914Val?Val?Gly?Met?Ile?Ser?Ile?His?Trp?Lys?Gly?Pro?Leu?Arg?Leu?Gln

210?????????????????215?????????????????220CAG?GCA?TAT?CTC?ATT?ATG?ATT?AGT?GCC?CTC?ATG?GCC?CTG?GTG?TTT?ATC?????????962Gln?Ala?Tyr?Leu?Ile?Met?Ile?Ser?Ala?Leu?Met?Ala?Leu?Val?Phe?Ile

225?????????????????230?????????????????235AAG?TAC?CTC?CCT?GAA?TGG?ACT?GCG?TGG?CTC?ATC?TTG?GCT?GTG?ATT?TCA?????????1010Lys?Tyr?Leu?Pro?Glu?Trp?Thr?Ala?Trp?Leu?Ile?Leu?Ala?Val?Ile?Ser

240?????????????????245?????????????????250GTA?TAT?GAT?TTA?GTG?GCT?GTT?TTG?TGT?CCG?AAA?GGT?CCA?CTT?CGT?ATG?????????1058Val?Tyr?Asp?Leu?Val?Ala?Val?Leu?Cys?Pro?Lys?Gly?Pro?Leu?Arg?Met255?????????????????260?????????????????265?????????????????270CTG?GTT?GAA?ACA?GCT?CAG?GAG?AGA?AAT?GAA?ACG?CTT?TTT?CCA?GCT?CTC?????????1106Leu?Val?Glu?Thr?Ala?Gln?Glu?Arg?Asn?Glu?Thr?Leu?Phe?Pro?Ala?Leu

275?????????????????280?????????????????285ATT?TAC?TCC?TCA?ACA?ATG?GTG?TGG?TTG?GTG?AAT?ATG?GCA?GAA?GGA?GAC?????????1154Ile?Tyr?Ser?Ser?Thr?Met?Val?Trp?Leu?Val?Asn?Met?Ala?Glu?Gly?Asp

290?????????????????295?????????????????300CCG?GAA?GCT?CAA?AGG?AGA?GTA?TCC?AAA?AAT?TCC?AAG?TAT?AAT?GCA?GAA?????????1202Pro?Glu?Ala?Gln?Arg?Arg?Val?Ser?Lys?Asn?Ser?Lys?Tyr?Asn?Ala?Glu

305?????????????????310?????????????????315AGC?ACA?GAAAGG?GAG?TCA?CAA?GAC?ACT?GTT?GCA?GAG?AAT?GAT?GAT?GGC??????????1250Ser?Thr?Glu?Arg?Glu?Ser?Gln?Asp?Thr?Val?Ala?Glu?Asn?Asp?Asp?Gly

320?????????????????325?????????????????330GGG?TTC?AGT?GAG?GAA?TGG?GAA?GCC?CAG?AGG?GAC?AGT?CAT?CTA?GGG?CCT?????????1298Gly?Phe?Ser?Glu?Glu?Trp?Glu?Ala?Gln?Arg?Asp?Ser?His?Leu?Gly?Pro335?????????????????340?????????????????345?????????????????350CAT?CGC?TCT?ACA?CCT?GAG?TCA?CGA?GCT?GCT?GTC?CAG?GAA?CTT?TCC?AGC?????????1346His?Arg?Ser?Thr?Pro?Glu?Ser?Arg?Ala?Ala?Val?Gln?Glu?Leu?Ser?Ser

355?????????????????360?????????????????365AGT?ATC?CTC?GCT?GGT?GAA?GAC?CCA?GAG?GAA?AGG?GGA?GTA?AAA?CTT?GGA?????????1394Ser?Ile?Leu?Ala?Gly?Glu?Asp?Pro?Glu?Glu?Arg?Gly?Val?Lys?Leu?Gly

370?????????????????375?????????????????380Met?Thr?Glu?Leu?Pro?Ala?Pro?Leu?Sar?Tyr?Phe?Gln?Asn?Ala?Gln?Met??1???????????????5??????????????????10??????????????????15Ser?Glu?Asp?Asn?His?Leu?Ser?Asn?Thr?Val?Arg?Ser?Gln?Asn?Asp?Asn

20??????????????????25??????????????????30Arg?Glu?Arg?Gln?Glu?His?Asn?Asp?Arg?Arg?Ser?Leu?Gly?His?Pro?Glu

35??????????????????40??????????????????45Pro?Leu?Ser?Asn?Gly?Arg?Pro?Gln?Gly?Asn?Ser?Arg?Gln?Val?Val?Glu

S0??????????????????55??????????????????60Gln?Asp?Glu?Glu?Glu?Asp?Glu?Glu?Leu?Thr?Leu?Lys?Tyr?Gly?Ala?Lys?65??????????????????70??????????????????75??????????????????80His?Val?Ile?Met?Leu?Phe?Val?Pro?Val?Thr?Leu?Cys?Met?Val?Val?Val

85??????????????????90??????????????????95Val?Ala?Thr?Ile?Lys?Ser?Val?Ser?Phe?Tyr?Thr?Arg?Lys?Asp?Gly?Gln

100?????????????????105?????????????????110Leu?Ile?Tyr?Thr?Pro?Phe?Thr?Glu?Asp?Thr?Glu?Thr?Val?Gly?Gln?Arg

115?????????????????120?????????????????125Ala?Leu?His?Ser?Ile?Leu?Asn?Ala?Ala?Ile?Met?Ile?Ser?Val?Ile?Val

130?????????????????135?????????????????140Val?Met?Thr?Ile?Leu?Leu?Val?Val?Leu?Tyr?Lys?Tyr?Arg?Cys?Tyr?Lys145?????????????????150?????????????????155?????????????????160Val?Ile?His?Ala?Trp?Leu?Ile?Ile?Ser?Ser?Leu?Leu?Leu?Leu?Phe?Phe

165?????????????????170?????????????????175Phe?Ser?Phe?Ile?Tyr?Leu?Gly?Glu?Val?Phe?Lys?Thr?Tyr?Asn?Val?Ala

180?????????????????185?????????????????190Val?Asp?Tyr?Ile?Thr?Val?Ala?Leu?Leu?Ile?Trp?Asn?Phe?Gly?Val?Val

195?????????????????200?????????????????205Gly?Met?Ile?Ser?Ile?His?Trp?Lys?Gly?Pro?Leu?Arg?Leu?Gln?Gln?Ala

210?????????????????215?????????????????220Tyr?Leu?Ile?Met?Ile?Ser?Ala?Leu?Met?Ala?Leu?Val?Phe?Ile?Lys?Tyr225?????????????????230?????????????????235?????????????????240Leu?Pro?Glu?Trp?Thr?Ala?Trp?Leu?Ile?Leu?Ala?Val?Ile?Ser?Val?Tyr

245?????????????????250?????????????????255Asp?Leu?Val?Ala?Val?Leu?Cys?Pro?Lys?Gly?Pro?Leu?Arg?Met?Leu?Val

260?????????????????265?????????????????270Glu?Thr?Ala?Gln?Glu?Arg?Asn?Glu?Thr?Leu?Phe?Pro?Ala?Leu?Ile?Tyr

275?????????????????280?????????????????285Ser?Ser?Thr?Met?Val?Trp?Leu?Val?Asn?Met?Ala?Glu?Gly?Asp?Pro?Glu

290?????????????????295?????????????????300Ala?Gln?Arg?Arg?Val?Ser?Lys?Asn?Ser?Lys?Tyr?ASh?Ala?Glu?Ser?Thr305?????????????????310?????????????????315?????????????????320Glu?Arg?Glu?Ser?Gln?Asp?Thr?Val?Ala?Glu?Asn?Asp?Asp?Gly?Gly?Phe

325?????????????????330?????????????????335Ser?Glu?Glu?Trp?Glu?Ala?Cln?Arg?Asp?Ser?His?Leu?Gly?Pro?His?Arg

340?????????????????345?????????????????350Ser?Thr?Pro?Glu?Ser?Arg?Ala?Ala?Val?Gln?Glu?Leu?Ser?Ser?Ser?Ile

355?????????????????360?????????????????365Leu?Ala?Gly?Glu?Asp?Pro?Glu?Glu?Arg?Gly?Val?Lys?Leu?Gly?Leu?Gly

370?????????????????375?????????????????380TTG?GGA?GAT?TTC?ATT?TTC?TAC?AGT?GTT?CTG?GTT?GGT?AAA?GCC?TCA?GCA????????1442Leu?Gly?Asp?Phe?Ile?Phe?Tyr?Ser?Val?Leu?Val?Gly?Lys?Ala?Ser?Ala

385?????????????????390?????????????????395ACA?GCC?AGT?GGA?GAC?TGG?AAC?ACA?ACC?ATA?GCC?TGT?TTC?GTA?GCC?ATA????????1490Thr?Ala?Ser?Gly?Asp?Trp?Asn?Thr?Thr?Ile?Ala?Cys?Phe?Val?Ala?Ile

400?????????????????405?????????????????410TTA?ATT?GGT?TTG?TGC?CTT?ACA?TTA?TTA?CTC?CTT?GCC?ATT?TTC?AAG?AAA????????1538Leu?Ile?Gly?Leu?Cys?Leu?Thr?Leu?Leu?Leu?Leu?Ala?Ile?Phe?Lys?Lys415?????????????????420?????????????????425?????????????????430GCA?TTG?CCA?GCT?CTT?CCA?ATC?TCC?ATC?ACC?TTT?GGG?CTT?GTT?TTC?TAC????????1586Ala?Leu?Pro?Ala?Leu?Pro?Ile?Ser?Ile?Thr?Phe?Gly?Leu?Val?Phe?Tyr

435?????????????????440?????????????????445TTT?GCC?ACA?GAT?TAT?CTT?GTA?CAG?CCT?TTT?ATG?GAC?CAA?TTA?GCA?TTC????????1634Phe?Ala?Thr?Asp?Tyr?Leu?Val?Gln?Pro?Phe?Met?Asp?Gln?Leu?Ala?Phe

450?????????????????455?????????????????460CAT?CAA?TTT?TAT?ATC?TAGCATATTT?GCGGTTAGAA?TCCCATGGAT?GTTTCTTCTT????????1689His?Gln?Phe?Tyr?Ile

465TGACTATAAC CAAATCTGGG GAGGACAAAG GTGATTTTCC TGTGTCCACA TCTAACAAAG 1749TCAAGATTCC CGGCTGGACT TTTGCAGCTT CCTTCCAAGT CTTCCTGACC ACCTTGCACT 1809ATTGGACTTT GGAAGGAGGT GCCTATAGAA AACGATTTTG AACATACTTC ATCGCAGTGG 1869ACTGTGTCCC TCGGTGCAGA AACTACCAGA TTTGAGGGAC GAGGTCAAGG AGATATGATA 1929GGCCCGGAAG TTGCTGTGCC CCATCAGCAG CTTGACGCGT GGTCACAGGA CGATTTCACT 1989GACACTGCGA ACTCTCAGGA CTACCGGTTA CCAAGAGGTT AGGTGAAGTG GTTTAAACCA 2049AACGGAACTC TTCATCTTAA ACTACACGTT GAAAATCAAC CCAATAATTC TGTATTAACT 2109GAATTCTGAA CTTTTCAGGA GGTACTGTGA GGAAGAGCAG GCACCAGCAG CAGAATGGGG 2169AATGGAGAGG TGGGCAGGGG TTCCAGCTTC CCTTTGATTT TTTGCTGCAG ACTCATCCTT 2229TTTAAATGAG ACTTGTTTTC CCCTCTCTTT GAGTCAAGTC AAATATGTAG ATTGCCTTTG 2289GCAATTCTTC TTCTCAAGCA CTGACACTCA TTACCGTCTG TGATTGCCAT TTCTTCCCAA 2349GGCCAGTCTG AACCTGAGGT TGCTTTATCC TAAAAGTTTT AACCTCAGGT TCCAAATTCA 2409GTAAATTTTG GAAACAGTAC AGCTATTTCT CATCAATTCT CTATCATGTT GAAGTCAAAT 2469TTGGATTTTC CACCAAATTC TGAATTTGTA GACATACTTG TACGCTCACT TGCCCCCAGA 2529TGCCTCCTCT GTCCTCATTC TTCTCTCCCA CACAAGCAGT CTTTTTCTAC AGCCAGTAAG 2589GCAGCTCTGT CRTGGTAGCA GATGGTCCCA TTATTCTAGG GTCTTACTCT TTGTATGATG 2649AAAAGAATGT GTTATGAATC GGTGCTGTCA GCCCTGCTGT CAGACCTTCT TCCACAGCAA 2709ATGAGATGTA TGCCCAAAGC GGTAGAATTA AAGAAGAGTA AAATGGCTGT TGAAGC 2765 ( 2 ) SEQ ID NO：2： ( i ) ：

(A) length: 467 amino acid

(B) type: amino acid

(D) topological framework: linearity is molecule type (ii): albumen (xi) sequence description: SEQ ID NO:2:Asp Phe Ile Phe Tyr Ser Val Leu Val Gly Lys Ala Ser Ala Thr Ala385 390 395 400Ser Gly Asp Trp Asn Thr Thr Ile Ala Cys Phe Val Ala Ile Leu Ile

405?????????????????410?????????????????415Gly?Leu?Cys?Leu?Thr?Leu?Leu?Leu?Leu?Ala?Ile?Phe?Lys?Lys?Ala?Leu

420?????????????????425?????????????????430Pro?Ala?Leu?Pro?Ile?Ser?Ile?Thr?Phe?Gly?Leu?Val?Phe?Tyr?Phe?Ala

435?????????????????440?????????????????445Thr?Asp?Tyr?Leu?Val?Gln?Pro?Phe?Met?Asp?Gln?Leu?Ala?Phe?His?Gln

The data of 450 455 460Phe Tyr Ile465 (2) SEQ ID NO:3: (i) sequence signature:

(A) length: 3086 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear (ix) feature:

(A) title/keyword: CDS

(B) position: 557 ... 1945 (ix) feature:

(A) title/keyword: misc feature

(B) position: 1 ... 3086

( D ) ：/＝“hPS1-2” ( xi ) ：SEQ ID NO：3：GAATTCGGCA CGAGGGAAAT GCTGTTTGCT CGAAGACGTC TCAGGGCGCA GGTGCCTTGG 60GCCGGGATTA GTAGCCGTCT GAACTGGAGT GGAGTAGGAG AAAGAGGAAG CGTCTTGGGC 120TGGGTCTGCT TGAGCAACTG GTGAAACTCC GCGCCTCACG CCCCGGGTGT GTCCTTGTCC 180AGGGGCGACG AGCATTCTGG GCGAAGTCCG CACSCCTCTT GTTCGAGGCG GAAGACGGGG 240TCTGATSCTT TCTCCTTGGT CGGGMCTGTC TCGAGGCATG CATGTCCAGT GACTCTTGTG 300TTTGCTGCTG CTTCCCTCTC AGATTCTTCT CACCGTTGTG GTCAGCTCTG CTTTAGGCAT 360ATTAATCCAT AGTGGAGGCT GGGATGGGTG AGAGAATTGA GGTGACTTTT CCATAATTCA 420GACCTAATCT GGGAGCCTGC AAGTGACAAC AGCCTTTGCG GTCCTTAGAC AGCTTGGCCT 480GGAGGAGAAC ACATGAAAGA AAGAACCTCA AGAGGCTTTG TTTTCTGTGA AACAGTATTT 540CTATACAGTT GCTCCA ATG ACA GAG TTA CCT GCA CCG TTG TCC TAC TTC 589

Met?Thr?Glu?Leu?Pro?Ala?Pro?Leu?Ser?Tyr?Phe

1???????????????5??????????????????10CAG?AAT?GCA?CAG?ATG?TCT?GAC?GAC?AAC?CAC?CTG?AGC?AAT?ACT?AAT?GAC??????????637Gln?Asn?Ala?Gln?Met?Ser?Glu?Asp?Asn?His?Leu?Ser?Asn?Thr?Asn?Asp

15??????????????????20??????????????????25AAT?AGA?GAA?CGG?CAG?GAG?CAC?AAC?GAC?AGA?CGG?AGC?CTT?GGC?CAC?CCT??????????685Asn?Arg?Glu?Arg?Gln?Glu?His?Asn?Asp?Arg?Arg?Ser?Leu?Gly?His?Pro

30??????????????????35??????????????????40GAG?CCA?TTA?TCT?AAT?GGA?CGA?CCC?CAG?GGT?AAC?TCC?CGG?CAG?GTG?GTG??????????733Glu?Pro?Leu?Ser?Asn?Gly?Arg?Pro?Gln?Gly?Asn?Ser?Arg?Gln?Val?Val

45??????????????????50??????????????????55GAG?CAA?GAT?GAG?GAA?GAA?GAT?GAG?GAG?CTG?ACA?TTG?AAA?TAT?GGC?GCC?????????781Glu?Gln?Asp?Glu?Glu?Glu?Asp?Glu?Glu?Leu?Thr?Leu?Lys?Tyr?Gly?Ala?60??????????????????65??????????????????70??????????????????75AAG?CAT?GTG?ATC?ATG?CTC?TTT?GTC?CCT?GTG?ACT?CTC?TGC?ATG?GTG?GTG?????????829Lys?His?Val?Ile?Met?Leu?Phe?Val?Pro?Val?Thr?Leu?Cys?Met?Val?Val

80??????????????????85??????????????????90GTC?GTG?GCT?ACC?ATT?AAG?TCA?GTC?AGC?TTT?TAT?ACC?CGG?AAG?GAT?GGG?????????877Val?Val?Ala?Thr?Ile?Lys?Ser?Val?Ser?Phe?Tyr?Thr?Arg?Lys?Asp?Gly

95?????????????????100?????????????????105CAG?CTA?ATC?TAT?ACC?CCA?TTC?ACA?GAA?GAT?ACC?GAG?ACT?GTG?GGC?CAG?????????925Gln?Leu?Ile?Tyr?Thr?Pro?Phe?Thr?Glu?Asp?Thr?Glu?Thr?Val?Gly?Gln

110?????????????????115?????????????????120AGA?GCC?CTG?CAC?TCA?ATT?CTG?AAT?GCT?GCC?ATC?ATG?ATC?AGT?GTC?ATT?????????973Arg?Ala?Leu?His?Ser?Ile?Leu?Asn?Ala?Ala?Ile?Met?Ile?Ser?Val?Ile

125?????????????????130?????????????????135GTT?GTC?ATG?ACT?ATC?CTC?CTG?GTG?GTT?CTG?TAT?AAA?TAC?AGG?TGC?TAT?????????1021Val?Val?Met?Thr?Ile?Leu?Leu?Val?Val?Leu?Tyr?Lys?Tyr?Arg?Cys?Tyr140?????????????????145?????????????????150?????????????????155AAG?GTC?ATC?CAT?GCC?TGG?CTT?ATT?ATA?TCA?TCT?CTA?TTG?TTG?CTG?TTC?????????1069Lys?Val?Ile?His?Ala?Trp?Leu?Ile?Ile?Ser?Ser?Leu?Leu?Leu?Leu?Phe

160?????????????????165?????????????????170TTT?TTT?TCA?TTC?ATT?TAC?TTG?GGG?GAA?GTG?TTT?AAA?ACC?TAT?AAC?GTT?????????1117Phe?Phe?Ser?Phe?Ile?Tyr?Leu?Gly?Glu?Val?Phe?Lys?Thr?Tyr?Asn?Val

175?????????????????180?????????????????185GCT?GTG?GAC?TAC?ATT?ACT?GTT?GCA?CTC?CTG?ATC?TGG?AAT?TTG?GGT?GTG?????????1165Ala?Val?Asp?Tyr?Ile?Thr?Val?Ala?Leu?Leu?Ile?Trp?Asn?Leu?Gly?Val

190?????????????????195?????????????????200GTG?GGA?ATG?ATT?TCC?ATT?CAC?TGG?AAA?GGT?CCA?CTT?CGA?CTC?CAG?CAG?????????1213Val?Gly?Met?Ile?Ser?Ile?His?Trp?Lys?Gly?Pro?Leu?Arg?Leu?Gln?Gln

205?????????????????210?????????????????215GCA?TAT?CTC?ATT?ATG?ATT?AGT?GCC?CTC?ATG?GCC?CTG?GTG?TTT?ATC?AAG?????????1261Ala?Tyr?Leu?Ile?Met?Ile?Ser?Ala?Leu?Met?Ala?Leu?Val?Phe?Ile?Lys220?????????????????225?????????????????230?????????????????235TAC?CTC?CCT?GAA?TGG?ACT?GCG?TGG?CTC?ATC?TTG?GCT?GTG?ATT?TCA?GTA?????????1309Tyr?Leu?Pro?Glu?Trp?Thr?Ala?Trp?Leu?Ile?Leu?Ala?Val?Ile?Ser?Val

240?????????????????245?????????????????250TAT?GAT?TTA?GTG?GCT?GTT?TTG?TGT?CCG?AAA?GGT?CCA?CTT?CGT?ATG?CTG?????????1357Tyr?Asp?Leu?Val?Ala?Val?Leu?Cys?Pro?Lys?Gly?Pro?Leu?Arg?Met?Leu

255?????????????????260?????????????????265GTT?GAA?ACA?GCT?CAG?GAG?AGA?AAT?GAA?ACG?CTT?TTT?CCA?GCT?CTC?ATT?????????1405Val?Glu?Thr?Ala?Gln?Glu?Arg?Ash?Glu?Thr?Leu?Phe?Pro?Ala?Leu?Ile

270?????????????????275?????????????????280TAC?TCC?TCA?ACA?ATG?GTG?TGG?TTG?GTG?AAT?ATG?GCA?GAA?GGA?GAC?CCG?????????1453Tyr?Ser?Ser?Thr?Met?Val?Trp?Leu?Val?Asn?Met?Ala?Glu?Gly?Asp?Pro

285?????????????????290?????????????????295GAA?GCT?CAA?AGG?AGA?GTA?TCC?AAA?AAT?TCC?AAG?TAT?AAT?GCA?GAA?AGC?????????1501Glu?Ala?Gln?Arg?Arg?Val?Ser?Lys?Asn?Ser?Lys?Tyr?Asn?Ala?Glu?Ser300?????????????????305?????????????????310?????????????????315ACA?GAA?AGG?GAG?TCA?CAA?GAC?ACT?GTT?GCA?GAG?AAT?GAT?GAT?GGC?GGG?????????1549Thr?Glu?Arg?Glu?Ser?Gln?Asp?Thr?Val?Ala?Glu?Asn?Asp?Asp?Gly?Gly

320?????????????????325?????????????????330TTC?AGT?GAG?GAA?TGG?GAA?GCC?CAG?AGG?GAC?AGT?CAT?CTA?GGG?CCT?CAT?????????1597Phe?Ser?Glu?Glu?Trp?Glu?Ala?Gln?Arg?Asp?Ser?His?Leu?Gly?Pro?His

335?????????????????340?????????????????345CGC?TCT?ACA?CCT?GAG?TCA?CGA?GCT?GCT?GTC?CAG?GAA?CTT?TCC?AGC?AGT????????1645Arg?Ser?Thr?Pro?Glu?Ser?Arg?Ala?Ala?Val?Gln?Glu?Leu?Ser?Ser?Ser

350?????????????????355?????????????????360ATC?CTC?GCT?GGT?GAA?GAC?CCA?GAG?GAA?AGG?GGA?GTA?AAA?CTT?GGA?TTG????????1693Ile?Leu?Ala?Gly?Glu?Asp?Pro?Glu?Glu?Arg?Gly?Val?Lys?Leu?Gly?Leu

365?????????????????370?????????????????375GGA?GAT?TTC?ATT?TTC?TAC?AGT?GTT?CTG?GTT?GGT?AAA?GCC?TCA?GCA?ACA????????1741Gly?Asp?Phe?Ile?Phe?Tyr?Ser?Val?Leu?Val?Gly?Lys?Ala?Ser?Ala?Thr380?????????????????385?????????????????390?????????????????395GCC?AGT?GGA?GAC?TGG?AAC?ACA?ACC?ATA?GCC?TGT?TTC?GTA?GCC?ATA?TTA????????1789Ala?Ser?Gly?Asp?Trp?Asn?Thr?Thr?Ile?Ala?Cys?Phe?Val?Ala?Ile?Leu

400?????????????????405?????????????????410ATT?GGT?TTG?TGC?CTT?ACA?TTA?TTA?CTC?CTT?GCC?ATT?TTC?AAG?AAA?GCA????????1837Ile?Gly?Leu?Cys?Leu?Thr?Leu?Leu?Leu?Leu?Ala?Ile?Phe?Lys?Lys?Ala

415?????????????????420?????????????????425TTG?CCA?GCT?CTT?CCA?ATC?TCC?ATC?ACC?TTT?GGG?CTT?GTT?TTC?TAC?TTT????????1885Leu?Pro?Ala?Leu?Pro?Ile?Ser?Ile?Thr?Phe?Gly?Leu?Val?Phe?Tyr?Phe

430?????????????????435?????????????????440GCC?ACA?GAT?TAT?CTT?GTA?CAG?CCT?TTT?ATG?GAC?CAA?TTA?GCA?TTC?CAT????????1933Ala?Thr?Asp?Tyr?Leu?Val?Gln?Pro?Phe?Met?Asp?Gln?Leu?Ala?Phe?His

445 450 455CAA TTT TAT ATC TAGCATATTT GCGGTTAGAA TCCCATGGAT GTTTCTTCTT 1985Gln Phe Tyr Ile460TGACTATAAC CAAATCTGGG GAGGACAAAG GTGATTTTCC TGTGTCCACA TCTAACAAAG 2045TCAAGATTCC CGGCTGGACT TTTGCAGCTT CCTTCCAAGT CTTCCTGACC ACCTTGCACT 2105ATTGGACTTT GGAAGGAGGT GCCTATAGAA AACGATTTTG AACATACTTC ATCGCAGTGG 2165ACTGTGTCCT CGGTGCAGAA ACTACCAGAT TTGAGGGACG AGGTCAAGGA GATATGATAG 2225GCCCGGAAGT TGCTGTGCCC CATCAGCAGC TTGACGCGTG GTCACAGGAC GATTTCACTG 2285ACACTGCGAA CTCTCAGGAC TACCGGTTAC CAAGAGGTTA GGTGAAGTGG TTTAAACCAA 2345ACGGAACTCT TCATCTTAAA CTACACGTTG AAAATCAACC CAATAATTCT GTATTAACTG 2405AATTCTGAAC TTTTCAGGAG GTACTGTGAG GAAGAGCAGG CACCAGCAGC AGAATGGGGA 2465ATGGAGAGGT GGGCAGGGGT TCCAGCTTCC CTTTGATTTT TTGCTGCAGA CTCATCCTTT 2525TTAAATGAGA CTTGTTTTCC CCTCTCTTTG AGTCAAGTCA AATATGTAGA TGCCTTTGGC 2585AATTCTTCTT CTCAAGCACT GACACTCATT ACCGTCTGTG ATTGCCATTT CTTCCCAAGG 2645CCAGTCTGAA CCTGAGGTTG CTTTATCCTA AAAGTTTTAA CCTCAGGTTC CAAATTCAGT 2705AAATTTTGGA AACAGTACAG CTATTTCTCA TCAATTCTCT ATCATGTTGA AGTCAAATTT 2765GGATTTTCCA CCAAATTCTG AATTTGTAGA CATACTTGTA CGCTCACTTG CCCCAGATGC 2825CTCCTCTGTC CTCATTCTTC TCTCCCACAC AAGCAGTCTT TTTCTACAGC CAGTAAGGCA 2885GCTCTGTCGT GGTAGCAGAT GGTCCCACTT ATTCTAGGGT CTTACTCTTT GTATGATGAA 2945AAGAATGTGT TATGAATCGG TGCTGTCAGC CCTGCTGTCA GACCTTCTTC CACAGCAAAT 3005GAGATGTATG CCCAAAGCGG TAGAATTAAA GAAGAGTAAA ATGGCTGTTG AAGCAAAAAA 3065AAAAAAAAAA AAAAAAAAAA A 3086 ( 2 ) SEQ ID NO：4： ( i ) ：

(A) length: 463 amino acid

(B) type: amino acid

(D) topological framework: linearity is molecule type (ii): albumen (xi) sequence description: SEQ ID NO:4:Met Thr Glu Leu Pro Ala Pro Leu Ser Tyr Phe Gln Asn Ala Gln Met 15 l0 15Ser Glu Asp Asn His Leu Ser Asn Thr Asn Asp Asn Arg Glu Arg Gln

20??????????????????25??????????????????30Glu?His?Asn?Asp?Arg?Arg?Ser?Leu?Gly?His?Pro?Glu?Pro?Leu?Ser?Asn

35??????????????????40??????????????????45Gly?Arg?Pro?Gln?Gly?Asn?Ser?Arg?Gln?Val?Val?Glu?Gln?Asp?Glu?Glu

50??????????????????55??????????????????60Glu?Asp?Glu?Glu?Leu?Thr?Leu?Lys?Tyr?Gly?Ala?Lys?His?Val?Ile?Met?65??????????????????70??????????????????75??????????????????80Leu?Phe?Val?Pro?Val?Thr?Leu?Cys?Met?Val?Val?Val?Val?Ala?Thr?Ile

85??????????????????90??????????????????95Lys?Ser?Val?Ser?Phe?Tyr?Thr?Arg?Lys?Asp?Gly?Gln?Leu?Ile?Tyr?Thr

100?????????????????105?????????????????110Pro?Phe?Thr?Glu?Asp?Thr?Glu?Thr?Val?Gly?Gln?Arg?Ala?Leu?His?Ser

115?????????????????120?????????????????125Ile?Leu?Asn?Ala?Ala?Ile?Met?Ile?Ser?Val?Ile?Val?Val?Met?Thr?Ile

130?????????????????135?????????????????140Leu?Leu?Val?Val?Leu?Tyr?Lys?Tyr?Arg?Cys?Tyr?Lys?Val?Ile?His?Ala145?????????????????150?????????????????155?????????????????160Trp?Leu?Ile?Ile?Ser?Ser?Leu?Leu?Leu?Leu?Phe?Phe?Phe?Ser?Phe?Ile

165?????????????????170?????????????????175Tyr?Leu?Gly?Glu?Val?Phe?Lys?Thr?Tyr?Asn?Val?Ala?Val?Asp?Tyr?Ile

180?????????????????185?????????????????190Thr?Val?Ala?Leu?Leu?Ile?Trp?Asn?Leu?Gly?Val?Val?Gly?Met?Ile?Ser

195?????????????????200?????????????????205Ile?His?Trp?Lys?Gly?Pro?Leu?Arg?Leu?Gln?Gln?Ala?Tyr?Leu?Ile?Met

210?????????????????215?????????????????220Ile?Ser?Ala?Leu?Met?Ala?Leu?Val?Phe?Ile?Lys?Tyr?Leu?Pro?Glu?Trp225?????????????????230?????????????????235?????????????????240Thr?Ala?Trp?Leu?Ile?Leu?Ala?Val?Ile?Ser?Val?Tyr?Asp?Leu?Val?Ala

245?????????????????250?????????????????255Val?Leu?Cys?Pro?Lys?Gly?Pro?Leu?Arg?Met?Leu?Val?Glu?Thr?Ala?Gln

260?????????????????265?????????????????270Glu?Arg?Asn?Glu?Thr?Leu?Phe?Pro?Ala?Leu?Ile?Tyr?Ser?Ser?Thr?Met

275?????????????????280?????????????????285Val?Trp?Leu?Val?Asn?Met?Ala?Glu?Gly?Asp?Pro?Glu?Ala?Gln?Arg?Arg

290?????????????????295?????????????????300Val?Ser?Lys?Asn?Ser?Lys?Tyr?Asn?Ala?Glu?Ser?Thr?Glu?Arg?Glu?Ser305?????????????????310?????????????????315?????????????????320Gln?Asp?Thr?Val?Ala?Glu?Ash?Asp?Asp?Gly?Gly?Phe?Ser?Glu?Glu?Trp

325?????????????????330?????????????????335Glu?Ala?Gln?Arg?Asp?Ser?His?Leu?Gly?Pro?His?Arg?Ser?Thr?Pro?Glu

340?????????????????345?????????????????350Ser?Arg?Ala?Ala?Val?Gln?Glu?Leu?Ser?Ser?Ser?Ile?Leu?Ala?Gly?Glu

355?????????????????360?????????????????365Asp?Pro?Glu?Glu?Arg?GLy?Val?Lys?Leu?Gly?Leu?Gly?Asp?Phe?Ile?Phe

370?????????????????375?????????????????380Tyr?Ser?Val?Leu?Val?Gly?Lys?Ala?Ser?Ala?Thr?Ala?Ser?Gly?Asp?Trp385?????????????????390?????????????????395?????????????????400Asn?Thr?Thr?Ile?Ala?Cys?Phe?Val?Ala?Ile?Leu?Ile?Gly?Leu?Cys?Leu

405?????????????????410?????????????????415Thr?Leu?Leu?Leu?Leu?Ala?Ile?Phe?Lys?Lys?Ala?Leu?Pro?Ala?Leu?Pro

420?????????????????425?????????????????430Ile?Ser?Ile?Thr?Phe?Gly?Leu?Val?Phe?Tyr?Phe?Ala?Thr?Asp?Tyr?Leu

435?????????????????440?????????????????445Val?Gln?Pro?Phe?Met?Asp?Gln?Leu?Ala?Phe?His?Gln?Phe?Tyr?Ile

The data of 450 455 460 (2) SEQ ID NO:5: (i) sequence signature:

(A) length: 2494 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear (ix) feature:

(A) title/keyword: misc_ feature

(B) position: 1 ... 2494

( D ) ：/＝“1Ex1n2” ( xi ) ：SEQ ID NO：5：AAGCTTTTGT GTGTAAAAAG TATTAGAATC TCATGTTTTT GAACAAGGTT GGCAGTGGGT 60TGGGAGGAGG GATTGGAGAT TGATGCGATA GGAATGTGAA GGGATAGCTT GGGGTGGATT 120TTATTTTTTA ATTTTAATTT TTATTTKTTG AGATGGAGTC TTGCTCTGTC TCCCAGGCTG 180GAGTGCAGTG GTGTGATCTC AGCTCACGGG TTCAAGCGAT TCTCCTGCTG CAGCCTCCCG 240AGTAGCTGGG ATTACAGGAG CGCGCCACCA CACCCGGNTA ATTTNNTTGT ATTTTTAGTA 300GAGACGGGGT TTCACCATGT TGGGTTAGGC TGGTCTAGAA CTCCCAACCT CATGATCCGC 360CTGCTTCGGC CTCCCAAAGT GCCGGAATTA CAGGCGTGAG CGACTGCACC CGGCCGCTTG 420GGGGTGGATT TTTAAAGAAA CTTTAGAAGA ATGTAACTTG SCCAGATACC ATGTACCGTT 480AATTTCATTT TCGGTTTTTK GAATACCCAT GTTTGACATT TMTCCGTTCA CCTTGATTAA 540ATAAGGTAGT ATTCATTTTT TAGTTTTAGC TTTTGGATAT ATGTGTAAGT GTGGTATGCT 600GTCTAATGAA TTAAGACAAT TGGTNCTKTC TTTACCCMAM ANCTGGACMA AGAGCAGGCA 660AGATGCAAAA ATCAAGTGAC CCAGCAAACC AGACACATTT TCTGCTCTCA GCTAGCTTGC 720CACCTAGAAA GACTGGTTGT CAAAGTTGGA GTCCAAGAAT CGCGGAGGAT GTTTAAAATG 780CAGTTTCTCA GGTTCTCNCC ACCCACCAGA AGTTTTGATT CATTGAGTGG TGGGAGAGGG 840CAGAGATATT TGCGATTTTA ACAGCATTCT CTTGATTGTG ATGCAGCTGG TTCSCAAATA 900GGTACCCTAA AGAAATGACA GGTGTTAAAT TTAGGATGGC CATCGCTTGT ATGCCGGGAG 960AAGCACACGC TGGGCCCAAT TTATATAGGG GCTTTCGTCC TCAGCTCGAG CARCCTCAGA 1020ACCCCGACAA CCYACGCCAG CKCTCTGGCC GGATTCCTTC AGKTGGGGAA GSCCAGGTGG 1080AGCTCTGGKT TCTCCCCGCA ATCGTTTCTC CAGGCCGGAG GCCCCGCCCC CTTCCTCCTG 1140GCTCCTCCCC TCCTCCGTGG GCCGNCCGCC AACGACGCCA GAGCCGGAAA TGACGACAAC 1200GGTGAGGGTT CTCGGGCGGG GCCTGGGACA GGCAGCTCCG GGGTCCGCGG TTTTCACATC 1260GGAAACAAAA CAGCGGCTGG TCTGGAAGGA ACCTGAGCTA CGACCCGCGG CGGCAGCGGG 1320GCGGCGGGGA AGCGTATGTG CGTGATGGGG AGTCCGGGCA AGCCAGGAAG GCACCGCGGA 1380CATGGGCGGC CGCGGGCAGG GNCCGGNCCT TTGTGGCCGC CCGGGCCGCG AAGCCGGTGT 1440CCTAAAAGAT GAGGGGCGGG GCGCGGCCGG TTGGGGCTGG GGAACCCCGT GTGGGAAACC 1500AGGAGGGGCG GCCCGTTTCT CGGGCTTCGG GCGCGGCCGG GTGGAGAGAG ATTCCGGGGA 1560GCCTTGGTCC GGAAATGCTG TTTGCTCGAA GACGTCTCAG GGCGCAGGTG CCTTGGGCCG 1620GGATTAGTAG CCGTCTGAAC TGGAGTGGAG TAGGAGAAAG AGGAAGCGTC TTGGGCTGGG 1680TCTGCTTGAG CAACTGGTGA AACTCCGCGC CTCACGCCCC GGGTGTGTCC TTGTCCAGGG 1740GCGACGAGCA TTCTGGGCGA AGTCCGCACG CCTCTTGTTC GAGGCGGAAG ACGGGGTCTT 1800GATGCTTTCT CCTTGGTCGG GACTGTCTCG AGGCATGCAT GTCCAGTGAC TCTTGTGTTT 1860GCTGCTGCTT CCCTCTCAGA TTCTTCTCAC CGTTGTGGTC AGCTCTGCTT TAGGCATATT 1920AATCCATAGT GGAGGCTGGG ATGGGTGAGA GAATTGAGGT GACTTTTCCA TAATTCAGGT 1980GAGATGTGAT TAGAGTYCGG ATCCTNCGGT GGTGGCAGAG GCTTACCAAG AAACACTAAC 2040GGGACATGGG AACCAATTGA GGATCCAGGG AATAAAGTGT GAAGTTGACT AGGAGGTTTT 2100CAGTTTAAGA ACATGGCAGA GACATTCTCA GAAATAAGGA AGTTAGGAAG AAAGACCTGG 2160TTTAGAGAGG AGGGCGAGGA AGTGGTTTGG AAGTGTCACT TTGGAAGTGC CAGCAGGTGA 2220AAATGCCCTG TGAACAGGAC TGGAGCTGAA AACAGGAATC AATTCCATAG ATTTCCAGTT 2280GATGTTGGAG CAGTGGAGAA GTCTAANCTA AGGAAGGGGA AGAGGAGGCC AAGCCAAACA 2340CTTAGGAACA CTTNCNACGA GGGGGTGGAA GAAGAGCAAG GAGCCAGCTG AGGAGAATGA 2400GTGTGGTTGG AGAACCACCA CAGCNCAGGG TCGCCAGANC TGAGGAAGGG GAGGGAAGCT 2460TATCGAGKAM SGWCRACMKC GAGTTGGCAG GGAT 2494 ( 2 ) SEQ ID NO：6： ( i ) ：

(A) length: 1117 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear (ix) feature:

(A) title/keyword: misc_ feature

(B) position: 1 ... 1117

( D ) ：/＝“1Ex3n4” ( xi ) ：SEQ ID NO：6：GGATCCGCCC GCCTTGGCCT CCCAAAGTGC TGGGATTACA GGCATGAGCC ACCGCTCCTG 60GCTGAGTCTG CGATTTCTTG CCAGCTCTAC CCAGTTGTGT CATCTTAAGC AAGTCACTGA 120ACTTCTCTGG ATTCCCTTCT CCTNNWGTAA AATAAGNATG TTATCTGNCC NNCCTGCCTT 180GGGCATTGTG ATAAGGATAA GATGACATTA TAGAATNTNG VAAAATTAAA AGCGCTAGAC 240AAATGTTTTA ATGAAAATAT AAAGATTAGN TTGAGTTTGG GCCAGCATAG AAAAAGGAAT 300GTTGAGAACA TTCCNTTAAG GATTACTCAA GCYCCCCTTT TGSTGKNWAA TCAGANNGTC 360ATNNAMNTAT CNTNTGTGGG YTGAAAATGT TTGGTTGCCT CAGGCGGTTC CTACTTATTG 420CTAAAGAGTC CTACCTTGAG CTTATAGTAA ATTTGTCAGT TAGTTGAAAG TCGTGACAAA 480TTAATACATT CCTGGTTTAC AAATTGGTCT TATAAGTATT TGATTGGTNT AAATGNATTT 540ACTAGGATTT AACTAACAAT GGATGACCTG GTGAAATCCT ATTTCAGACC TAATCTGGGA 600GCCTGCAAGT GACAACAGCC TTTGCGGTCC TTAGACAGCT TGGCCTGGAG GAGAACACAT 660GAAAGAAAGG TTTGWNTCTG NTTAWTGTAA TCTATGRAAG TGTTTTTWAT MACAGTATAA 720TTGTMTGMAC AAAGTTCTGT TTTTCTTTCC CTTTNCAGAA CCTCAAGAGG CTTTGTTTTC 780TGTGAAACAG TATTTCTATA CAGTTGCTCC AATGACAGAG TTACCTGCAC CGTTGTCCTA 840CTTCCAGAAT GCACAGATGT CTGAGGACAA CCACCTGAGC AATACTGTAC GTAGCCAGGT 900ACAGCGTCAG TYTTCNAAAC TGCCTYYGNC AGACTGGATT CACTTATCAT CTCCCCTCAC 960CTCTGAGAAA TGCTGAGGGG GSTAGGNAGG GCTTTCTCTA CTTNACCACA TTTNATAATT 1020ATTTTTGGGT GACCTTCAGC TGATCGCTGG GAGGGACACA GGGCTTNTTT AACACATAGG 1080GTGTTGGATA CAGNCCCTCC CTAATTCACA TTTCANC 1117 ( 2 ) SEQ ID NO：7： ( i ) ：

(A) length: 1727 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear (ix) feature:

(A) title/keyword: misc_ feature

(B) position: 1 ... 1727

( D ) ：/＝“1Ex5” ( xi ) ：SEQ ID NO：7：GGATCCCTCC CCTTTTTAGA CCATACAAGG TAACTTCCGG ACGTTGCCAT GGCATCTGTA 60AACTGTCATG GTGTTGGCGG GGAGTGTCTT TTAGCATGCT AATGTATTAT AATTAGCGTA 120TAGTGAGCAG TGAGGATAAC CAGAGGTCAC TCTCCTCACC ATCTTGGTTT TGGTGGGTTT 180TGGCCAGCTT CTTTATTGCA ACCAGTTTTA TCAGCAAGAT CTTTATGAGC TGTATCTTGT 240GCTGACTTCC TATCTCATCC CGNAACTAAG AGTACCTAAC CTCCTGCAAA TTGMAGNCCA 300GNAGGTCTTG GNCTTATTTN ACCCAGCCCC TATTCAARAT AGAGTNGYTC TTGGNCCAAA 360CGCCYCTGAC ACAAGGATTT TAAAGTCTTA TTAATTAAGG TAAGATAGKT CCTTGSATAT 420GTGGTCTGAA ATCACAGAAA GCTGAATTTG GAAAAAGGTG CTTGGASCTG CAGCCAGTAA 480ACAAGTTTTC ATGCAGGTGT CAGTATTTAA GGTACATCTC AAAGGATAAG TACAATTGTG 540TATGTTGGGA TGAACAGAGA GAATGGAGCA ANCCAAGACC CAGGTAAAAG AGAGGACCTG 600AATGCCTTCA GTGAACAATG ATAGATAATC TAGACTTTTA AACTGCATAC TTCCTGTACA 660TTGTTTTTTC TTGCTTCAGG TTTTTAGAAC TCATAGTGAC GGGTCTGTTG TTAATCCCAG 720GTCTAACCGT TACCTTGATT CTGCTGAGAA TCTGATTTAC TGAAAATGTT TTTCTTGTGC 780TTATAGAATG ACAATAGAGA ACGGCAGGAG CACAACGACA GACGGAGCCT TGGCCACCCT 840GAGCCATTAT CTAATGGACG ACCCCAGGGT AACTCCCGGC AGGTGGTGGA GCAAGATGAG 900GAAGAAGATG AGGAGCTGAC ATTGAAATAT GGCGCCAAGC ATGTGATCAT GCTCTTTGTC 960CCTGTGACTC TCTGCATGGT GGTGGTCGTG GCTACCATTA AGTCAGTCAG CTTTTATACC 1020CGGAAGGATG GGCAGCTGTA CGTATGAGTT TKGTTTTATT ATTCTCAAAS CCAGTGTGGC 1080TTTTCTTTAC AGCATGTCAT CATCACCTTG AAGGCCTCTN CATTCAAGGG GCATGACTTA 1140GCTGGAGAGC CCATCCTCTG TGATGGTCAG GAGCAGTTGA GAGANCGAGG GGTTATTACT 1200TCATGTTTTA AGTGGAGAAA AGGAACACTG CAGAAGTATG TTTCCTGTAT GGTATTACTG 1260GATAGGGCTG AAGTTATGCT GAATTGAACA CATAAATTCT TTTCCACCTC AGGGNCATTG 1320GGCGCCCATT GNTCTTCTGC CTAGAATATT CTTTCCTTTN CTNACTTKGG NGGATTAAAT 1380TCCTGTCATC CCCCTCCTCT TGGTGTTATA TATAAAGTNT TGGTGCCGCA AAAGAAGTAG 1440CACTCGAATA TAAAATTTTC CTTTTAATTC TCAGCAAGGN AAGTTACTTC TATATAGAAG 1500GGTGCACCCN TACAGATGGA ACAATGGCAA GCGCACATTT GGGACAAGGG AGGGGAAAGG 1560GTTCTTATCC CTGACACACG TGGTCCCNGC TGNTGTGTNC TNCCCCCACT GANTAGGGTT 1620AGACTGGACA GGCTTAAACT AATTCCAATT GGNTAATTTA AAGAGAATNA TGGGGTGAAT 1680GCTTTGGGAG GAGTCAAGGA AGAGNAGGTA GNAGGTAACT TGAATGA 1727 ( 2 ) SEQ ID NO：8： ( i ) ：

(A) length: 1883 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear (ix) feature:

(A) title/keyword: misc_ feature

(B) position: 1 ... 1883

( D ) ：/＝“1Ex6” ( xi ) ：SEQ ID NO：8：CNCGTATAAA AGACCAACAT TGCCANCNAC AACCACAGGC AAGATCTTCT CCTACCTTCC 60CCCNNGGTGT AATACCAAGT ATTCNCCAAT TTGTGATAAA CTTTCATTGG AAAGTGACCA 120CCCTCCTTGG TTAATACATT GTCTGTGCCT GCTTTCACAC TACAGTAGCA CAGTTGAGTG 180TTTGCCCTGG AGACCATATG ACCCATAGAG CTTAAAATAT TCAGTCTGGC TTTTTACAGA 240GATGTTTCTG ACTTTGTTAA TAGAAAATCA ACCCAACTGG TTTAAATAAT GCACATACTT 300TCTCTCTCAT AGAGTAGTGC AGAGGTAGNC AGTCCAGATT AGTASGGTGG CTTCACGTTC 360ATCCAAGGAC TCAATCTCCT TCTTTCTTCT TTAGCTTCTA ACCTCTAGCT TACTTCAGGG 420TCCAGGCTGG AGCCCTASCC TTCATTTCTG ACAGTAGGAA GGAGTAGGGG AGAAAAGAAC 480ATAGGACATG TCAGCAGAAT TCTCTCCTTA GAAGTTCCAT ACACAACACA TCTCCCTAGA 540AGTCATTGCC CTTACTTGTT CTCATAGCCA TCCTAAATAT AAGGGAGTCA GAAGTAAAGT 600CTKKNTGGCT GGGAATATTG GCACCTGGAA TAAAAATGTT TTTCTGTGAA TGAGAAACAA 660GGGGAAGATG GATATGTGAC ATTATCTTAA GACAACTCCA GTTGCAATTA CTCTGCAGAT 720GAGAGGCACT AATTATAAGC CATATTACCT TTCTTCTGAC AACCACTTGT CAGCCCNCGT 780GGTTTCTGTG GCAGAATCTG GTTCYATAMC AAGTTCCTAA TAANCTGTAS CCNAAAAAAT 840TTGATGAGGT ATTATAATTA TTTCAATATA AAGCACCCAC TAGATGGAGC CAGTGTCTGC 900TTCACATGTT AAGTCCTTCT TTCCATATGT TAGACATTTT CTTTGAAGCA ATTTTAGAGT 960GTAGCTGTTT TTCTCAGGTT AAAAATTCTT AGCTAGGATT GGTGAGTTGG GGAAAAGTGA 1020CTTATAAGAT NCGAATTGAA TTAAGAAAAA GAAAATTCTG TGTTGGAGGT GGTAATGTGG 1080KTGGTGATCT YCATTAACAC TGANCTAGGG CTTTKGKGTT TGCTTTATTG TACAATCTAT 1140ACCCCATTCA CAGAAGATAC CGAGACTGTG GGCCAGAGAG CCCTGCACTC AATTCTGAAT 1200GCTGCCATCA TGATCAGTGT CATTGTTGTC ATGACTATCC TCCTGGTGGT TCTGTATAAA 1260TACAGGTGCT ATAAGGTGAG CATGAGACAC AGATCTTTGN TTTCCACCCT GTTCTTCTTA 1320TGGTTGGGTA TTCTTGTCAC AGTAACTTAA CTGATCTAGG AAAGAAAAAA TCTTTTGTCT 1330TCTAGAGATA AGTTAATTTT TAGTTTTCTT CCTCCTCACT GTGGAACATT CAAAAAATAC 1440AAAAAGGAAG CCAGGTGCAT GTGTAATGCC AGGCTCAGAG GCTGAGGCAG GAGGATCGCT 1500TGGGCCCAGG AGTTCACAAG CAGCTTGGGC AACGTAGCAA GACCCTGCCT CTATTAAAGA 1560AAACAAAAAA CAAATATTGG AAGTATTTTA TATGCATGGA ATCTATATGT CATGAAAAAA 1620TTAGTGTAAA ATATATATAT TATGATTAGN TATCAAGATT TAGTGATAAT TTATGTTATT 1680TTTGGATTTC AATGCCTTTT TAGGCCATTG TCTCAAMAAA TAAAAGCAGA AAACAAAAAA 1740AGTTGTAACT GAAAAATAAA CATTTCCATA TAATAGCACA ATCTAAGTGG GTTTTTGNTT 1800GTTTGTTTGN TTGTTGAAGC AGGGCCTTGC CCTNYCACCC AGGNTGGAGT GAAGTGCAGT 1860GGCACGATTT TGGCTCACTG CAG 1883 ( 2 ) SEQ ID NO：9： ( i ) ：

(A) length: 823 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear (ix) feature:

(A) title/keyword: misc_ feature

(B) position: 1 ... 823

( D ) ：/＝“1Ex7” ( xi ) ：SEQ ID NO：9：CAGGAGTGGA CTAGGTAAAT GNAAGNTGTT TTAAAGAGAG ATGNGGNCNG GGACATAGTG 60GTACACANCT GTAATGCTCA NCACTKATGG GGAGTACTGA AGGNGGNSGG ATCACTTGNG 120GGTCNGGAAT NTGAGANCAG CCTGGGCAAN ATGGCGAAAC CCTGTCTCTA CTAAAAATAG 180CCANAAWNWA GCCTAGCGTG GTGGCGCRCA CGCGTGGTTC CACCTACTCA GGAGGCNTAA 240GCACGAGNAN TNCTTGAACC CAGGAGGCAG AGGNTGTGGT GARCTGAGAT CGTGCCACTG 300CACTCCAGTC TGGGCGACMA AGTGAGACCC TGTCTCCNNN AAGAAAAAAA AAATCTGTAC 360TTTTTAAGGG TTGTGGGACC TGTTAATTAT ATTGAAATGC TTCTYTTCTA GGTCATCCAT 420GCCTGGCTTA TTATATCATC TCTATTGTTG CTGTTCTTTT TTTCATTCAT TTACTTGGGG 480TAAGTTGTGA AATTTGGGGT CTGTCTTTCA GAATTAACTA CCTNNGTGCT GTGTAGCTAT 540CATTTAAAGC CATGTACTTT GNTGATGAAT TACTCTGAAG TTTTAATTGT NTCCACATAT 600AGGTCATACT TGGTATATAA AAGACTAGNC AGTATTACTA ATTGAGACAT TCTTCTGTNG 660CTCCTNGCTT ATAATAAGTA GAACTGAAAG NAACTTAAGA CTACAGTTAA TTCTAAGCCT 720TTGGGGAAGG ATTATATAGC CTTCTAGTAG GAAGTCTTGT GCNATCAGAA TGTTTNTAAA 780GAAAGGGTNT CAAGGAATNG TATAAANACC AAAAATAATT GAT 823 ( 2 ) SEQ ID NO：10： ( i ) ：

(A) length: 945 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear (ix) feature:

(A) title/keyword: misc_ feature

(B) position: 1 ... 945

( D ) ：/＝“1 Ex8” ( xi ) ：SEQ ID NO：10：GTTNTCCNAA CCAACTTAGG AGNTTGGACC TGGGRAAGAC CNACNTGATC TCCGGGAGGN 60AAAGACTNCA GTTGAGCCGT GATTGCACCC ACTTTACTCC AAGCCTGGGC AACCAAAATG 120AGACACTGGC TCCAAACACA AAAACAAAAA CAAAAAAAGA GTAAATTAAT TTANAGGGAA 180GNATTAAATA AATAATAGCA CAGTTGATAT AGGTTATGGT AAAATTATAA AGGTGGGANA 240TTAATATCTA ATGTTTGGGA GCCATCACAT TATTCTAAAT AATGTTTTGG TGGAAATTAT 300TGTACATCTT TTAAAATCTG TGTAATTTTT TTTCAGGGAA GTGTTTAAAA CCTATAACGT 360TGCTGTGGAC TACATTACTG TTGCACTCCT GATCTGGAAT TTTGGTGTGG TGGGAATGAT 420TTCCATTCAC TGGAAAGGTC CACTTCGACT CCAGCAGGCA TATCTCATTA TGATTAGTGC 480CCTCATGGCC CTGGTGTTTA TCAAGTACCT CCCTGAATGG ACTGCGTGGC TCATCTTGGC 540TGTGATTTCA GTATATGGTA AAACCCAAGA CTGATAATTT GTTTGTCACA GGAATGCCCC 600ACTGGAGTGT TTTCTTTCCT CATCTCTTTA TCTTGATTTA GAGAAAATGG TAACGTGTAC 660ATCCCATAAC TCTTCAGTAA ATCATTAATT AGCTATAGTA ACTTTTTCAT TTGAAGATTT 720CGGCTGGGCA TGGTAGCTCA TGCCTGTAAT CTTAGCACTT TGGGAGGCTG AGGCGGGCAG 780ATCACCTAAG CCCAGAGTTC AAGACCAGCC TGGGCAACAT GGCAAAACCT CGTATCTACA 840GAAAATACAA AAATTAGCCG GGCATGGTGG TGCACACCTG TAGTTCCAGC TACTTAGGAG 900GCTGAGGTGG GAGGATCGAT TGATCCCAGG AGGTCAAGNC TGCAG 945 ( 2 ) SEQ ID NO：11： ( i ) ：

(A) length: 540 base pairs

(B) type: nucleic acid

(C) chain: strand

( D ) ： ( xi ) ：SEQ ID NO：11： CTGCAGCTTT CCTTTAAACT AGGAAGACTT GTTCCTATAC CCCAGTAACG ATACACTGTA 60CACTAAGCAA ATAGCAGTCA AACCCAAATG AAATTTNTAC AGATGTTCTG TGTCATTTTA 120TNTTGTTTAT GTTGTCTCCC CCACCCCCAC CAGTTCACCT GCCATTTATT TCATATTCAT 180TCAACGTCTN NNTGTGTAAA AAGAGACAAA AAACATTAAA CTTTTTTCCT TCGTTAATTC 240CTCCCTACCA CCCATTTACA AGTTTAGCCC ATACATTTTA TTAGATGTCT TTTATGTTTT 300TCTTTTNCTA GATTTAGTGG CTGTTTTGTG TCCGAAAGGT CCACTTCGTA TGCTGGTTGA 360AACAGCTCAG GAGAGAAATG AAACGCTTTT TCCAGCTCTC ATTTACTCCT GTAAGTATTT 420GGAGAATGAT ATTGAATTAG TAATCAGNGT AGAATTTATC GGGAACTTGA AGANATGTNA 480CTATGGCAAT TTCANGGNAC TTGTCTCATC TTAAATGANA GNATCCCTGG ACTCCTGNAG 540 ( 2 ) SEQ ID NO：12： ( i ) ：

(A) length: 509 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear (ix) feature:

(A) title/keyword: misc_ feature

(B) position: 1 ... 509

( D ) ：/＝“1Ex10” ( xi ) ：SEQ ID NO：12：CCCCGTCNAT GCATACTTTG TGTGTCCAGT GCTTACCTGG AATCCNGTCT TTCCCAACAG 60CAACAATGGT GTGGTTGGTG AATATGGCAG AAGGAGACCC GGAAGCTCAA AGGAGAGTAT 120CCAAAAATTC CAAGTATAAT GCAGAAAGTA GGTAACTYYY NTTAGATAMN ATCTTGATTT 180TNCAGGGTCA CTGTTATAAG CTAACAGTAT AGNAATGTTT TTATCGTCTT TCTNKGGNCA 240TAGACTCCTN KGAGAATCTC TTGAGAACTA TGATAATGCC CAGTAAATAC NCAGATAAGT 300ATTTAAGGAG TNCAGATACT CAAANCCCAA CAATACNGTC AAAGCATCCT AGGTTAAGAC 360AMCNCCCATT AAATACAGAA TACCAGCATG GAAAGGTTCA GGCTGAGGTT ATGATTGGGT 420TTGGGTTTTG GGNNNGTTTT TTATAAGTCA TGATTTTAAA AAGAAAAAAT AAACTCTCTC 480CAAACATGTA AAAGTAAGAA TCTCCTAAA 509

(2) data of SEQ ID NO:13:

(i) sequence signature:

(A) length: 1092 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear (ix) feature:

(A) title/keyword: misc_ feature

(B) position: 1 ... 1092

( D ) ：/＝“1Ex11” ( xi ) ：SEQ ID NO：13：GTCTAGATAA GNCAACATTC AGGGGTAGAA GGGGACTGTT TATTTTTTCC TTTAGTCTCT 60CTTAAAGAGT GAGAAAAATT TTCCCAGGAA TCCCGGTGGA CTTTGCTTCA CCACTCATAG 120GTTCATACCA AGTTACAACC CCACAACCTT AGAGCTTTTG TTAGGAAGAG GCTTGGTGGG 180ATTACCGTGC TTGGCTTGGC TTGGTCAGGA TTCACCACCA GAGTCATGTG GGAGGGGGTG 240GGAACCCAAA CAATTCAGGA TTCTGCCCTC AGGAAATAAA GGAGAAAATA GCTGTTGGAT 300AAACTACCAG CAGGCACTGC TACAGCCCAT GCTTTGTGGT TTAAGGGCCA GCTAGTTACA 360ATGACAGCTA GTTACTGTTT CCATGTAATT TTCTTAAAGG TATTAAATTT TTCTAAATAT 420TAGAGCTGTA ACTTCCACTT TCTCTTGAAG GCACAGAAAG GGAGTCACAA GACACTGTTG 480CAGAGAATGA TGATGGCGGG TTCAGTGAGG AATGGGAAGC CCAGAGGGAC AGTCATCTAG 540GGCCTCATCG CTCTACACCT GAGTCACGAG CTGCTGTCCA GGAACTTTCC AGCAGTATC 600TCGCTGGTGA AGACCCAGAG GAAAGTATGT TCANTTCTCC ATNTTTCAAA GTCATGGATT 660CCTTTAGGTA GCTACATTAT CAACCTTTTT GAGAATAAAA TGAATTGAGA GTGTTACAGT 720CTAATTCTAT ATCACATGTA ACTTTTATTT GGATATATCA GTAATAGTGC TTTTTYNTTT 780TTTTTTTTTT TTTTTTTTTT TTTTNGGNGA NAGAGTCTCG CTCTGTCGCC AGGTTGGAGT 840GCAATGGTGC GATCTTGGCT CACTGAAAGC TCCACCNCCC GGGTTCAAGT GATTCTCCTG 900CCTCAGCCNC CCAAGTAGNT GGGACTACAG GGGTGCGCCA CCACGCCTGG GATAATTTTG 960GGNTTTTTAG TAGAGATGGC GTTTCACCAN CTTGGNGCAG GCTGGTCTTG GAACTCCTGA 1020NATCATGATC TGCCTGCCTT AGCCTCCCCA AAGTGCTGGG ATTNCAGGGG TGAGCCACTG 1080TTCCTGGGCC TC 1092 ( 2 ) SEQ ID NO：14： ( i ) ：

(A) length: 1003 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear (ix) feature:

(A) title/keyword: misc_ feature

(B) position: 1 ... 1003

( D ) ：/＝“1Ex12” ( xi ) ：SEQ ID NO：14：CTGCAGTGAG CCGAGATCAT GCTGCTGTAC TCCAGCCTGG GCCACAGAGC CAAACTCCAT 60CTCCCAAAAA AAAAAAATAT TAATTAATAT GATNAAATGA TGCCTATCTC AGAATTCTTG 120TAAGGATTTC TTAGKACAAG TGCTGGGTAT AAACTATANA TTCRATAGAT GNCGATTATT 180ACTTAYTATT GTTATTGATA AATAACAGCA GCATCTACAG TTAAGACTCC AGAGTCAGTC 240ACATAGAATC TGGNACTCCT ATTGTAGNAA ACCCCNMMAG AAAGAAAACA CAGCTGAAGC 300CTAATTTTGT ATATCATTTA CTGACTTCTC TCATTCATTG TGGGGTTGAG TAGGGCAGTG 360ATATTTTTGA ATTGTGAAAT CATANCAAAG AGTGACCAAC TTTTTAATAT TTGTAACCTT 420TCCTTTTTAG GGGGAGTAAA ACTTGGATTG GGAGATTTCA TTTTCTACAG TGTTCTGGTT 480GGTAAAGCCT CAGCAACAGC CAGTGGAGAC TGGAACACAA CCATAGCCTG TTTCGTAGCC 540ATATTAATTG TMMSTATACA CTAATAAGAA TGTGTCAGAG CTCTTAATGT CMAAACTTTG 600ATTACACAGT CCCTTTAAGG CAGTTCTGTT TTAACCCCAG GTGGGTTAAA TATTCCAGCT 660ATCTGAGGAG CTTTTNGATA ATTGGACCTC ACCTTAGTAG TTCTCTACCC TGGCCACACA 720TTAGAATCAC TTGGGAGCTT TTAAAACTGT AAGCTCTGCC CTGAGATATT CTTACTCAAT 780TTAATTGTGT AGTTTTTAAA ATTCCCCAGG AAATTCTGGT ATTTCTGTTT AGGAACCGCT 840GCCTCAAGCC TAGCAGCACA GATATGTAGG AAATTAGCTC TGTAAGGTTG GTCTTACAGG 900GATAAACAGA TCCTTCCTTA GTCCCTGGAC TTAATCACTG AGAGTTTGGG TGGTGGTTTT 960GGATTTAATG ACACAACCTG TAGCATGCAG TGTTACTTAA GAC 1003 ( 2 ) SEQ ID NO：15： ( i ) ：

(A) length: 736 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear (ix) feature:

(A) title/keyword: misc_ feature

(B) position: 1 ... 736

( D ) ：/＝“1Ex13” ( xi ) ：SEQ ID NO：15：GTCTTTCCCA TCTTCTCCAC AGGGTTTGTG CCTTACATTA TTACTCCTTG CCATTTTCAA 60GAAAGCATTG CCAGCTCTTC CAATCTCCAT CACCTTTGGG CTTGTTTTCT ACTTTGCCAC 120AGATTATCTT GTACAGCCTT TTATGGACCA ATTAGCATTC CATCAATTTT ATATCTAGCA 180TATTTGCGGT TAGAATCCCA TGGATGTTTC TTCTTTGACT ATAACAAAAT CTGGGGAGGA 240CAAAGGTGAT TTCCTGTGTC CACATCTAAC AAATCAAGAT CCCCGGCTGG ACTTTTGGAG 300GTTCCTTCCA AGTCTTCCTG ACCACCTTGC ACTATTGGAC TTTGGAAGGA GGTGCCTATA 360GAAAACGATT TTGAACATAC TTCATCGCAG TGGACTGTGT CCTCGGTGCA GAAACTACCA 420GATTTGAGGG ACGAGGTCAA GGAGATATGA TAGGCCCGGA AGTTGCTGTG CCCCATCAGC 480AGCTTGACGC GTGGTCACAG GACGATTTTC ACTGACACTG CGAACTCTCA GGACTACCGT 540TACCAAGAGG TTAGGTGAAG TGGTTTAAAC CAAACGGAAC TCTTCATCTT AAACTACACG 600TTGAAAATCA ACCCAATAAT TCTGTATTAA CTGAATTCTG AACTTTTCAG GAGGTACTGT 660GAGGAAGAGC AGGCACCACC AGCAGAATGG GGAATGGAGA GGTGGGCAGG GGTTCCAGCT 720TCCCTTTGAT TTTTTG 736 ( 2 ) SEQ ID NO：16： ( i ) ：

(A) length: 1964 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear (ix) feature:

(A) title/keyword: CDS

(B) position: 188 ... 1588 (ix) feature:

(A) title/keyword: misc_ feature

(B) position: 1 ... 1964

(D) other data :/annotate=" mPS1 ", (xi) sequence description: SEQ ID NO:16:ACCANACANC GGCAGCTGAG GCGGAAACCT AGGCTGCGAG CCGGCCGCCC GGGCGCGGAG 60AGAGAAGGAA CCAACACAAG ACAGCAGCCC TTCGAGGTCT TTAGGCAGCT TGGAGGAGAA 120CACATGAGAG AAAGAATCCC AAGAGGTTTT GTTTTCTTTG AGAAGGTATT TCTGTCCAGC 180TGCTCCA ATG ACA GAG ATA CCT GCA CCT TTG TCC TAC TTC CAG AAT GCC 229

Met?Thr?Glu?Ile?Pro?Ala?Pro?Leu?Ser?Tyr?Phe?Gln?Asn?Ala

1???????????????5??????????????????10CAG?ATG?TCT?GAG?GAC?AGC?CAC?TCC?AGC?AGC?GCC?ATC?CGG?AGC?CAG?AAT??????277Gln?Met?Ser?Glu?Asp?Ser?His?Ser?Ser?Ser?Ala?Ile?Arg?Ser?Gln?Asn?15??????????????????20??????????????????25??????????????????30GAC?AGC?CAA?GAA?CGG?CAG?CAG?CAG?CAT?GAC?AGG?CAG?AGA?CTT?GAC?AAC??????325Asp?ser?Gln?Glu?Arg?Gln?Gln?Gln?His?Asp?Arg?Gln?Arg?Leu?Asp?Asn

35??????????????????40??????????????????45CCT?GAG?CCA?ATA?TCT?AAT?GGG?CGG?CCC?CAG?AGT?AAC?TCA?AGA?CAG?GTG??????373Pro?Glu?Pro?Ile?Ser?Asn?Gly?Arg?Pro?Gln?Ser?Asn?Ser?Arg?Gln?Val

50??????????????????55??????????????????60GTG?GAA?CAA?GAT?GAG?GAG?GAA?GAC?GAA?GAG?CTG?ACA?TTG?AAA?TAT?GGA??????421Val?Glu?Gln?Asp?Glu?Glu?Glu?Asp?Glu?Glu?Leu?Thr?Leu?Lys?Tyr?Gly

65??????????????????70??????????????????75GCC?AAG?CAT?GTC?ATC?ATG?CTC?TTT?GTC?CCC?GTG?ACC?CTC?TGC?ATG?GTC??????469Ala?Lys?His?Val?Ile?Met?Leu?Phe?Val?Pro?Val?Thr?Leu?Cys?Met?Val

80??????????????????85??????????????????90GTC?GTC?GTG?GCC?ACC?ATC?AAA?TCA?GTC?AGC?TTC?TAT?ACC?CGG?AAG?GAC??????517Val?Val?Val?Ala?Thr?Ile?Lys?Ser?Val?Ser?Phe?Tyr?Thr?Arg?Lys?Asp?95?????????????????100?????????????????105?????????????????110GGT?CAG?CTA?ATC?TAC?ACC?CCA?TTC?ACA?GAA?GAC?ACT?GAG?ACT?GTA?GGC??????565Gly?Gln?Leu?Ile?Tyr?Thr?Pro?Phe?Thr?Glu?Asp?Thr?Glu?Thr?Val?Gly

115?????????????????120?????????????????125CAA?AGA?GCC?CTG?CAC?TCG?ATC?CTG?AAT?GCG?GCC?ATC?ATG?ATC?AGT?GTC??????613Gln?Arg?Ala?Leu?His?Ser?Ile?Leu?Asn?Ala?Ala?Ile?Met?Ile?Ser?Val

130?????????????????135?????????????????140ATT?GTC?ATT?ATG?ACC?ATC?CTC?CTG?GTG?GTC?CTG?TAT?AAA?TAC?AGG?TGC??????661Ile?Val?Ile?Met?Thr?Ile?Leu?Leu?Val?Val?Leu?Tyr?Lys?Tyr?Arg?Cys

145?????????????????150?????????????????155TAC?AAG?GTC?ATC?CAC?GCC?TGG?CTT?ATT?ATT?TCA?TCT?CTG?TTG?TTG?CTG??????709Tyr?Lys?Val?Ile?His?Ala?Trp?Leu?Ile?Ile?Ser?Ser?Leu?Leu?Leu?Leu

160?????????????????165?????????????????170TTC?TTT?TTT?TCG?TTC?ATT?TAC?TTA?GGG?GAA?GTA?TTT?AAG?ACC?TAC?AAT??????757Phe?Phe?Phe?Ser?Phe?Ile?Tyr?Leu?Gly?Glu?Val?Phe?Lys?Thr?Tyr?Asn175?????????????????180?????????????????185?????????????????190GTC?GCC?GTG?GAC?TAC?GTT?ACA?GTA?GCA?CTC?CTA?ATC?TGG?AAT?TTT?GGT??????805Val?Ala?Val?Asp?Tyr?Val?Thr?Val?Ala?Leu?Leu?Ile?Trp?Asn?Phe?Gly

195?????????????????200?????????????????205GTG?GTC?GGG?ATG?ATT?GCC?ATC?CAC?TGG?AAA?GGC?CCC?CTT?CGA?CTG?CAG??????853Val?Val?Gly?Met?Ile?Ala?Ile?His?Trp?Lys?Gly?Pro?Leu?Arg?Leu?Gln

210?????????????????215?????????????????220CAG?GCG?TAT?CTC?ATT?ATG?ATC?AGT?GCC?CTC?ATG?GCC?CTG?GTA?TTT?ATC??????901Gln?Ala?Tyr?Leu?Ile?Met?Ile?Ser?Ala?Leu?Met?Ala?Leu?Val?Phe?Ile

225?????????????????230?????????????????235AAG?TAC?CTC?CCC?GAA?TGG?ACC?GCA?TGG?CTC?ATC?TTG?GCT?GTG?ATT?TCA???????949Lys?Tyr?Leu?Pro?Glu?Trp?Thr?Ala?Trp?Leu?Ile?Leu?Ala?Val?Ile?Ser

240?????????????????245?????????????????250GTA?TAT?GAT?TTG?GTG?GCT?GTT?TTA?TGT?CCC?AAA?GGC?CCA?CTT?CGT?ATG???????997Val?Tyr?Asp?Leu?Val?Ala?Val?Leu?Cys?Pro?Lys?Gly?Pro?Leu?Arg?Met255?????????????????260?????????????????265?????????????????270CTG?GTT?GAA?ACA?GCT?CAG?GAA?AGA?AAT?GAG?ACT?CTC?TTT?CCA?GCT?CTT???????1045Leu?Val?Glu?Thr?Ala?Gln?Glu?Arg?Asn?Glu?Thr?Leu?Phe?Pro?Ala?Leu

275?????????????????280?????????????????285ATC?TAT?TCC?TCA?ACA?ATG?GTG?TGG?TTG?GTG?AAT?ATG?GCT?GAA?GGA?GAC???????1093Ile?Tyr?Ser?Ser?Thr?Met?Val?Trp?Leu?Val?Asn?Met?Ala?Glu?Gly?Asp

290?????????????????295?????????????????300CCA?GAA?GCC?CAA?AGG?AGG?GTA?CCC?AAG?AAC?CCC?AAG?TAT?AAC?ACA?CAA???????1141Pro?Glu?Ala?Gln?Arg?Arg?Val?Pro?Lys?Asn?Pro?Lys?Tyr?Asn?Thr?Gln

305?????????????????310?????????????????315AGA?GCG?GAG?AGA?GAG?ACA?CAG?GAC?AGT?GGT?TCT?GGG?AAC?GAT?GAT?GGT???????l189Arg?Ala?Glu?Arg?Glu?Thr?Gln?Asp?Ser?Gly?Ser?Gly?Asn?Asp?Asp?Gly

320?????????????????325?????????????????330GGC?TTC?AGT?GAG?GAG?TGG?GAG?GCC?CAA?AGA?GAC?AGT?CAC?CTG?GGG?CCT???????1237Gly?Phe?Ser?Glu?Glu?Trp?Glu?Ala?Gln?Arg?Asp?Ser?His?Leu?Gly?Pro335?????????????????340?????????????????345?????????????????350CAT?CGC?TCC?ACT?CCC?GAG?TCA?AGA?GCT?GCT?GTC?CAG?GAA?CTT?TCT?GGG???????1285His?Arg?Ser?Thr?Prc?Glu?Ser?Arg?Ala?Ala?Val?Gln?Glu?Leu?Ser?Gly

355?????????????????360?????????????????365AGC?ATT?CTA?ACG?AGT?GAA?GAC?CCG?GAG?GAA?AGA?GGA?GTA?AAA?CTT?GGA???????1333Ser?Ile?Leu?Thr?Ser?Glu?Asp?Pro?Glu?Glu?Arg?Gly?Val?Lys?Leu?Gly

370?????????????????375?????????????????380CTG?GGA?GAT?TTC?ATT?TTC?TAC?AGT?GTT?CTG?GTT?GGT?AAG?GCC?TCA?GCA???????1381Leu?Gly?Asp?Phe?Ile?Phe?Tyr?Ser?Val?Leu?Val?Gly?Lys?Ala?Ser?Ala

385?????????????????390?????????????????395ACC?GCC?AGT?GGA?GAC?TGG?AAC?ACA?ACC?ATA?GCC?TGC?TTT?GTA?GCC?ATA???????1429Thr?Ala?Ser?Gly?Asp?Trp?Asn?Thr?Thr?Ile?Ala?Cys?Phe?Val?Ala?Ile

400?????????????????405?????????????????410CTG?ATC?GGC?CTG?TGC?CTT?ACA?TTA?CTC?CTG?CTC?GCC?ATT?TTC?AAG?AAA???????1477Leu?Ile?Gly?Leu?Cys?Leu?Thr?Leu?Leu?Leu?Leu?Ala?Ile?Phe?Lys?Lys415?????????????????420?????????????????425?????????????????430GCG?TTG?CCA?GCC?CTC?CCC?ATC?TCC?ATC?ACC?TTC?GGG?CTC?GTG?TTC?TAC???????1525Ala?Leu?Pro?Ala?Leu?Pro?Ile?Ser?Ile?Thr?Phe?Gly?Leu?Val?Phe?Tyr

435?????????????????440?????????????????445TTC?GCC?ACG?GAT?TAC?CTT?GTG?CAG?CCC?TTC?ATG?GAC?CAA?CTT?GCA?TTC???????1573Phe?Ala?Thr?Asp?Tyr?Leu?Val?Gln?Pro?Phe?Met?Asp?Gln?Leu?Ala?Phe

450?????????????????455?????????????????460CAT?CAG?TTT?TAT?ATC?TAGCCTTTCT?GCAGTTAGAA?CATGGATGTT?TCTTCTTTGA???????1628His?Gln?Phe?Tyr?Ile

465TTATCAAAAA CACAAAAACA GAGAGCAAGC CCGAGGAGGA GACTGGTGAC TTTCCTGTGT 1688CCTCAGCTAA CAAAGGCAGG ACTCCAGCTG GACTTCTGCA GCTTCCTTCC GAGTCTCCCT 1748AGCCACCCGC ACTACTGGAC TGTGGAAGGA AGCGTCTACA GAGGAACGGT TTCCAACATC 1808CATCGCTGCA GCAGACGGTG TCCCTCAGTG ACTTGAGAGA CAAGGACAAG GAAATGTGCT 1868GGGCCAAGGA GCTGCCGTGC TCTGCTAGCT TTGACCGTGG GCATGGAGAT TTACCCGCAC 1928TGTGAACTCT CTAAGGTAAA CAAAGTGAGG TGAACC 1964 (2) SEQ ID NO:17 data: (i) sequence signature:

(A) length: 467 amino acid

(B) type: amino acid

(D) topological framework: linearity is molecule type (ii): albumen (xi) sequence description: SEQ ID NO:17:Met Thr Glu Ile Pro Ala Pro Leu Ser Tyr Phe Gln Asn Ala Gln Met 15 10 15Ser Glu Asp Ser His Ser Ser Ser Ala Ile Arg Ser Gln Asn Asp Ser

20??????????????????25??????????????????30Gln?Glu?Arg?Gln?Gln?Gln?His?Asp?Arg?Gln?Arg?Leu?Asp?Asn?Pro?Glu

35??????????????????40??????????????????45Pro?Ile?Ser?Asn?Gly?Arg?Pro?Gln?Ser?Asn?Ser?Arg?Gln?Val?Val?Glu

50??????????????????55??????????????????60Gln?AsP?Glu?Glu?Glu?Asp?Glu?Glu?Leu?Thr?Leu?Lys?Tyr?Gly?Ala?Lys?65??????????????????70??????????????????75??????????????????90His?Val?Ile?Met?Leu?Phe?Val?Pro?Val?Thr?Leu?Cys?Met?Val?Val?Val

85??????????????????90??????????????????95Val?Ala?Thr?Ile?Lys?Ser?Val?Ser?Phe?Tyr?Thr?Arg?Lys?Asp?Gly?Gln

100?????????????????105?????????????????110??Leu?Ile?Tyr?Thr?Pro?Phe?Thr?Glu?Asp?Thr?Glu?Thr?Val?Gly?Gln?Arg

115?????????????????120?????????????????125Ala?Leu?His?Ser?Ile?Leu?Ash?Ala?Ala?Ile?Met?Ile?Ser?Val?Ile?Val

130?????????????????135?????????????????140Ile?Met?Thr?Ile?Leu?Leu?Val?Val?Leu?Tyr?Lys?Tyr?Arg?Cys?Tyr?Lys145?????????????????150????????????????155??????????????????160Val?Ile?His?Ala?Trp?Leu?Ile?Ile?Ser?Ser?Leu?Leu?Leu?Leu?Phe?Phe

165?????????????????170?????????????????175Phe?Ser?Phe?Ile?Tyr?Leu?Gly?Glu?Val?Phe?Lys?Thr?Tyr?Asn?Val?Ala

180?????????????????185?????????????????190Val?Asp?Tyr?Val?Thr?Val?Ala?Leu?Leu?Ile?Trp?Asn?Phe?Gly?Val?Val

195?????????????????200?????????????????205Gly?Met?Ile?Ala?Ile?His?Trp?Lys?Gly?Pro?Leu?Arg?Leu?Gln?Gln?Ala

210?????????????????215?????????????????220Tyr?Leu?Ile?Met?Ile?Ser?Ala?Leu?Met?Ala?Leu?Val?Phe?Ile?Lys?Tyr225?????????????????230?????????????????235?????????????????240Leu?Pro?Glu?Trp?Thr?Ala?Trp?Leu?Ile?Leu?Ala?Val?Ile?Ser?Val?Tyr

245?????????????????250?????????????????255Asp?Leu?Val?Ala?Val?Leu?Cys?Pro?Lys?Gly?Pro?Leu?Arg?Met?Leu?Val

260?????????????????265?????????????????270Glu?Thr?Ala?Gln?Glu?Arg?Asn?GLu?Thr?Leu?Phe?Pro?Ala?Leu?Ile?Tyr

275?????????????????280?????????????????285Ser?Ser?Thr?Met?Val?Trp?Leu?Val?Ash?Met?Ala?Glu?Gly?Asp?Pro?Glu

290?????????????????295?????????????????300Ala?Gln?Arg?Arg?Val?Pro?Lys?Asn?Pro?Lys?Tyr?Asn?Thr?Gln?Arg?Ala305?????????????????310?????????????????315?????????????????320Glu?Arg?Glu?Thr?Gln?Asp?Ser?Gly?Ser?Gly?Asn?Asp?Asp?Gly?Gly?Phe

325?????????????????330?????????????????335Ser?Glu?Glu?Trp?Glu?Ala?Gln?Arg?Asp?Ser?His?Leu?Gly?Pro?His?Arg

340?????????????????345?????????????????350Ser?Thr?Pro?Glu?Ser?Arg?Ala?Ala?Val?Gln?Glu?Leu?Ser?Gly?Ser?Ile

355?????????????????360?????????????????365Leu?Thr?Ser?Glu?Asp?Pro?Glu?Glu?Arg?Gly?Val?Lys?Leu?Gly?Leu?Gly

370?????????????????375?????????????????360Asp?Phe?Ile?Phe?Tyr?Ser?Val?Leu?Val?Gly?Lys?Ala?Ser?Ala?Thr?Ala385?????????????????390?????????????????395?????????????????400Ser?Gly?Asp?Trp?Asn?Thr?Thr?Ile?Ala?Cys?Phe?Val?Ala?Ile?Leu?Ile

405?????????????????410?????????????????415Gly?Leu?Cys?Leu?Thr?Leu?Leu?Leu?Leu?Ala?Ile?Phe?Lys?Lys?Ala?Leu

420?????????????????425?????????????????430Pro?Ala?Leu?Pro?Ile?Ser?Ile?Thr?Phe?Gly?Leu?Val?Phe?Tyr?Phe?Ala

435?????????????????440?????????????????445Thr?Asp?Tyr?Leu?Val?Gln?Pro?Phe?Met?Asp?Gln?Leu?Ala?Phe?His?Gln

The data of 450 455 460Phe Tyr Ile465 (2) SEQ ID NO:18: (i) sequence signature:

(A) length: 2229 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear (ix) feature:

(A) title/keyword: CDS

(B) position: 366 ... 1712 (ix) feature:

(A) title/keyword: misc_ feature

(B) position: 1 ... 2226

(D) other data :/notes=" hPS2 " be sequence description: SEQ ID NO:18:GAATTCGGCA CGAGGGCATT TCCAGCAGTG AGGAGACAGC CAGAAGCAAG CTTTTGGAGC 60TGAAGGAACC TGAGACAGAA GCTAGTCCCC CCTCTGAATT TTACTGATGA AGAAACTGAG 120GCCACAGAGC TAAAGTGACT TTTCCCAAGG TCGCCCAGCG AGGACGTGGG ACTTCTCAGA 180CGTCAGGAGA GTGATGTGAG GGAGCTGTGT GACCATAGAA AGTGACGTGT TAAAAACCAG 240CGCTGCCCTC TTTGAAAGCC AGGGAGCATC ATTCATTTAG CCTGCTGAGA AGAAGAAACC 300AAGTGTCCGG GATTCAAGAC CTCTCTGCGG CCCCAAGTGT TCGTGGTGCT TCCAGAGGCA 360GGGCT ATG CTC ACA TTC ATG GCC TCT GAC AGC GAG GAA GAA GTG TGT 407 (xi)

Met?Leu?Thr?Phe?Met?Ala?Ser?Asp?Ser?Glu?Glu?Glu?Val?Cys

1???????????????5??????????????????10GAT?GAG?CGG?ACG?TCC?CTA?ATG?TCG?GCC?GAG?AGC?CCC?ACG?CCG?CGC?TCC????????455Asp?Glu?Arg?Thr?Ser?Leu?Met?Ser?Ala?Glu?Ser?Pro?Thr?Pro?Arg?Ser?15??????????????????20??????????????????25??????????????????30TGC?CAG?GAG?GGC?AGG?CAG?GGC?CCA?GAG?GAT?GGA?GAG?AAT?ACT?GCC?CAG????????503Cys?Gln?Glu?Gly?Arg?Gln?Gly?Pro?Glu?Asp?Gly?Glu?Asn?Thr?Ala?Gln

35??????????????????40??????????????????45TGG?AGA?AGC?CAG?GAG?AAC?GAG?GAG?GAC?GGT?GAG?GAG?GAC?CCT?GAC?CGC???????551Trp?Arg?Ser?Gln?Glu?Asn?Glu?Glu?Asp?Gly?Glu?Glu?Asp?Pro?Asp?Arg

50??????????????????55??????????????????60TAT?GTC?TGT?AGT?GGG?GTT?CCC?GGG?CGG?CCG?CCA?GGC?CTG?GAG?GAA?GAG???????599Tyr?Val?Cys?Ser?Gly?Val?Pro?Gly?Arg?Pro?Pro?Gly?Leu?Glu?Glu?Glu

65??????????????????70??????????????????75CTG?ACC?CTC?AAA?TAC?GGA?GCG?AAG?CAT?GTG?ATC?ATG?CTG?TTT?GTG?CCT???????647Leu?Thr?Lau?Lys?Tyr?Gly?Ala?Lys?His?Val?Ile?Met?Leu?Phe?Val?Pro

80??????????????????85??????????????????90GTC?ACT?CTG?TGC?ATG?ATC?GTG?GTG?GTA?GCC?ACC?ATC?AAG?TCT?GTG?CGC???????695Val?Thr?Leu?Cys?Met?Ile?Val?Val?Val?Ala?Thr?Ile?Lys?Ser?Val?Arg?95?????????????????100?????????????????105?????????????????110TTC?TAC?ACA?GAG?AAG?AAT?GGA?CAG?CTC?ATC?TAC?ACG?CCA?TTC?ACT?GAG???????743Phe?Tyr?Thr?Glu?Lys?Asn?Gly?Gln?Leu?Ile?Tyr?Thr?Pro?Phe?Thr?Glu

115?????????????????120?????????????????125GAC?ACA?CCC?TCG?GTG?GGC?CAG?CGC?CTC?CTC?AAC?TCC?GTG?CTG?AAC?ACC???????791Asp?Thr?Pro?Ser?Val?Gly?Gln?Arg?Leu?Leu?Asn?Ser?Val?Leu?Asn?Thr

130?????????????????135?????????????????140CTC?ATC?ATG?ATC?AGC?GTC?ATC?GTG?GTT?ATG?ACC?ATC?TTC?TTG?GTG?GTG???????839Leu?Ile?Met?Ile?Ser?Val?Ile?Val?Val?Met?Thr?Ile?Phe?Leu?Val?Val

145?????????????????150?????????????????155CTC?TAC?AAG?TAC?CGC?TGC?TAC?AAC?TTC?ATC?CAT?GGC?TGG?TTG?ATC?ATG???????887Leu?Tyr?Lys?Tyr?Arg?Cys?Tyr?Lys?Phe?Ile?His?Gly?Trp?Leu?Ile?Met

160?????????????????165?????????????????170TCT?TCA?CTG?ATG?CTG?CTG?TTC?CTC?TTC?ACC?TAT?ATC?TAC?CTT?GGG?GAA???????935Ser?Ser?Leu?Met?Leu?Leu?Phe?Leu?Phe?Thr?Tyr?Ile?Tyr?Leu?Gly?Glu175?????????????????180?????????????????185?????????????????190GTG?CTC?AAG?ACC?TAC?AAT?GTG?GCC?ATG?GAC?TAC?CCC?ACC?CTC?TTG?CTG???????983Val?Leu?Lys?Thr?Tyr?ASn?Val?Ala?Met?Asp?Tyr?Pro?Thr?Leu?Leu?Leu

195?????????????????200?????????????????205ACT?GTC?TGG?AAC?TTC?GGG?GCA?GTG?GGC?ATG?GTG?TGC?ATC?CAC?TGG?AAG???????1031Thr?Val?Trp?Asn?Phe?Gly?Ala?Val?Gly?MetVal?Cys?Ile?His?Trp?Lys

210?????????????????215????????????????220GGC?CCT?CTG?GTG?CTG?CAG?CAG?GCC?TAC?CTC?ATC?ATG?ATC?AGT?GCG?CTC???????1079Gly?Pro?Leu?Val?Leu?Gln?Gln?Ala?Tyr?Leu?Ile?Met?Ile?Ser?Ala?Leu

225?????????????????230?????????????????235ATG?GCC?CTA?GTG?TTC?ATC?AAG?TAC?CTC?CCA?GAG?TGG?TCC?GCG?TGG?GTC???????1127Met?Ala?Leu?Val?Phe?Ile?Lys?Tyr?Leu?Pro?Glu?Trp?Ser?Ala?Trp?Val

240?????????????????245?????????????????250ATC?CTG?GGC?GCC?ATC?TCT?GTG?TAT?GAT?CTC?GTG?GCT?GTG?CTG?TGT?CCC???????1175Ile?Leu?Gly?Ala?Ile?Ser?Val?Tyr?Asp?Leu?Val?Ala?Val?Leu?Cys?Pro255?????????????????260?????????????????265?????????????????270AAA?GGG?CCT?CTG?AGA?ATG?CTG?GTA?GAA?ACT?GCC?CAG?GAG?AGA?AAT?GAG???????1223Lys?Gly?Pro?Leu?Arg?Met?Leu?Val?Glu?Thr?Ala?Gln?Glu?Arg?Asn?Glu

275?????????????????280?????????????????285CCC?ATA?TTC?CCT?GCC?CTG?ATA?TAC?TCA?TCT?GCC?ATG?GTG?TGG?ACG?GTT???????1271Pro?Ile?Phe?Pro?Ala?Leu?Ile?Tyr?Ser?Ser?Ala?Met?Val?Trp?Thr?Val

290?????????????????295?????????????????300GGC?ATG?GCG?AAG?CTG?GAC?CCC?TCC?TCT?CAG?GGT?GCC?CTC?CAG?CTC?CCC???????1319Gly?Met?Ala?Lys?Leu?Asp?Pro?Ser?Ser?Gln?Gly?Ala?Leu?Gln?Leu?Pro

305?????????????????310?????????????????315TAC?GAC?CCG?GAG?ATG?GAA?GAA?GAC?TCC?TAT?GAC?AGT?TTT?GGG?GAG?CCT???????1367Tyr?Asp?Pro?Glu?Met?Glu?Glu?Asp?Ser?Tyr?Asp?Ser?Phe?Gly?Glu?Pro

320?????????????????325?????????????????330TCA?TAC?CCC?GAA?GTC?TTT?GAG?CCT?CCC?TTG?ACT?GGC?TAC?CCA?GGG?GAG???????1415Ser?Tyr?Pro?Glu?Val?Phe?Glu?Pro?Pro?Leu?Thr?Gly?Tyr?Pro?Gly?Glu335?????????????????340?????????????????345?????????????????350GAG?CTG?GAG?GAA?GAG?GAG?GAA?AGG?GGC?GTG?AAG?CTT?GGC?CTC?GGG?GAC???????1463Glu?Leu?Glu?Glu?Glu?Glu?Glu?Arg?Gly?Val?Lys?Leu?Gly?Leu?Gly?Asp

355?????????????????360?????????????????365TTC?ATC?TTC?TAC?AGT?GTG?CTG?GTG?GGC?AAG?GCG?GCT?GCC?ACG?GGC?AGC???????1511Phe?Tle?Phe?Tyr?Ser?Val?Leu?Val?Gly?Lys?Ala?Ala?Ala?Thr?Gly?Ser

370?????????????????375?????????????????380GGG?GAC?TGG?AAT?ACC?ACG?CTG?GCC?TGC?TTC?GTG?GCC?ATC?CTC?ATT?GGC???????1559Gly?Asp?Trp?Asn?Thr?Thr?Leu?Ala?Cys?Phe?Val?Ala?Ile?Leu?Ile?Gly

385?????????????????390?????????????????395TTG?TGT?CTG?ACC?CTC?CTG?CTG?CTT?GCT?GTG?TTC?AAG?AAG?GCG?CTG?CCC???????1607Leu?Cys?Leu?Thr?Leu?Leu?Leu?Leu?Ala?Val?Phe?Lys?Lys?Ala?Leu?Pro

400?????????????????405?????????????????410GCC?CTC?CCC?ATC?TCC?ATC?ACG?TTC?GGG?CTC?ATC?TTT?TAC?TTC?TCC?ACG???????1655Ala?Leu?Pro?Ile?Ser?Ile?Thr?Phe?Gly?Leu?Ile?Phe?Tyr?Phe?Ser?Thr415?????????????????420?????????????????425?????????????????430GAC?AAC?CTG?GTG?CGG?CCG?TTC?ATG?GAC?ACC?CTG?GCC?TCC?CAT?CAG?CTC???????1703Asp?Asn?Leu?Val?Arg?Pro?Phe?Met?Asp?Thr?Leu?Ala?Ser?His?Gln?Leu

435 440 445TAC ATC TGA GGGACATGGT GTGCCACAGG CTGCAAGCTG CAGGGAATTT 1752Tyr Ile *TCATTGGATG CAGTTGTATA GTTTTACACT CTAGTGCCAT ATATTTTTAA GACTTTTCTT 1812TCCTTAAAAA ATAAAGTACG TGTTTACTTG GTGAGGAGGA GGCAGAACCA GCTCTTTGGT 1872GCCAGCTGTT TCATCACCAG ACTTTGGCTC CCGCTTTGGG GAGCGCCTCG CTTCACGGAC 1932AGGAAGCACA GCAGGTTTAT CCAGATGAAC TGAGAAGGTC AGATTAGGGT GGGGAGAAGA 1992GCATCCGGCA TGAGGGCTGA GATGCCCAAA GAGTGTGCTC GGGAGTGGCC CCTGGCACCT 2052GGGTGCTCTG GCTGGAGAGG AAAAGCCAGT TCCCTACGAG GAGTGTTCCC AATGCTTTGT 2112CCATGATGTC CTTGTTATTT TATTNCCYTT ANAAACTGAN TCCTNTTNTT NTTDCGGCAG 2172TCACMCTNCT GGGRAGTGGC TTAATAGTAA NATCAATAAA NAGNTGAGTC CTNTTAG 2229 ( 2 ) SEQ ID NO：19： ( i ) ：

(A) length: 449 amino acid

(B) type: amino acid

(D) topological framework: linearity is molecule type (ii): albumen (xi) sequence description: SEQ ID NO:19:Met Leu Thr Phe Met Ala Ser Asp Ser Glu Glu Glu Val Cys Asp Glu 15 10 15Arg Thr Ser Leu Met Ser Ala Glu Ser Pro Thr Pro Arg Ser Cys Gln

20??????????????????25??????????????????30Glu?Gly?Arg?Gln?Gly?Pro?Glu?Asp?Gly?Glu?Asn?Thr?Ala?Gln?Trp?Arg

35??????????????????40??????????????????45Ser?Gln?Glu?Asn?Glu?Glu?Asp?Gly?Glu?Glu?Asp?Pro?Asp?Arg?Tyr?Val

5O??????????????????55??????????????????60Cys?Ser?G1y?Val?Pro?Gly?Arg?Pro?Pro?Gly?Leu?Glu?Glu?Glu?Leu?Thr?65??????????????????70??????????????????75??????????????????80Leu?Lys?Tyr?Gly?Ala?Lys?His?Val?Ile?Met?Leu?Phe?Val?Pro?Val?Thr

85??????????????????90??????????????????95Leu?Cys?Met?Ile?Val?Val?Val?Ala?Thr?Ile?Lys?Ser?Val?Arg?Phe?Tyr

100?????????????????105?????????????????110Thr?Glu?Lys?Asn?Gly?Gln?Leu?Ile?Tyr?Thr?Pro?Phe?Thr?Glu?Asp?Thr

115?????????????????120?????????????????125Pro?Ser?Val?Gly?Gln?Arg?Leu?Leu?Asn?Ser?Val?Leu?Asn?Thr?Leu?Ile

130?????????????????135????????????????140Met?Ile?Ser?Val?Ile?Val?Val?Met?Thr?Ile?Phe?Leu?Val?Val?Leu?Tyr145?????????????????150?????????????????155?????????????????160Lys?Tyr?Arg?Cys?Tyr?Lys?Phe?Ile?His?Gly?Trp?Leu?Ile?Met?Ser?Ser

165?????????????????170?????????????????175Leu?Met?Leu?Leu?Phe?Leu?Phe?Thr?Tyr?Ile?Tyr?Leu?Gly?Glu?Val?Leu

180?????????????????185?????????????????190Lys?Thr?Tyr?Asn?Val?Ala?Met?Asp?Tyr?Pro?Thr?Leu?Leu?Leu?Thr?Val

195?????????????????200?????????????????205Trp?Ash?Phe?Gly?Ala?Val?Gly?Met?Val?Cys?Ile?His?Trp?Lys?Gly?Pro

210?????????????????215?????????????????220Leu?Val?Leu?Gln?Gln?Ala?Tyr?Leu?Ile?Met?Ile?Ser?Ala?Leu?Met?Ala225?????????????????230?????????????????235?????????????????240Leu?Val?Phe?Ile?Lys?Tyr?Leu?Pro?Glu?Trp?Ser?Ala?Trp?Val?Ile?Leu

245?????????????????250?????????????????255Gly?Ala?Ile?Ser?Val?Tyr?Asp?Leu?Val?Ala?Val?Leu?Cys?Pro?Lys?Gly

260?????????????????265?????????????????270Pro?Leu?Arg?Met?Leu?Val?Glu?Thr?Ala?Gln?Glu?Arg?Asn?Glu?Pro?Ile

275?????????????????280?????????????????285Phe?Pro?Ala?Leu?Ile?Tyr?Ser?Ser?Ala?Met?Val?Trp?Thr?Val?Gly?Met

290?????????????????295?????????????????300Ala?Lys?Leu?Asp?Pro?Ser?Ser?Gln?Gly?Ala?Leu?Gln?Leu?Pro?Tyr?Asp305?????????????????310?????????????????315?????????????????320Pro?Glu?Met?Glu?Glu?Asp?Ser?Tyr?Asp?Ser?Phe?Gly?Glu?Pro?Ser?Tyr

325?????????????????330?????????????????335Pro?Glu?Val?Phe?Glu?Pro?Pro?Leu?Thr?Gly?Tyr?Pro?Gly?Glu?Glu?Leu

340?????????????????345?????????????????350Glu?Glu?Glu?Glu?Glu?Arg?Gly?Val?Lys?Leu?Gly?Leu?Gly?Asp?Phe?Ile

355?????????????????360?????????????????365Phe?Tyr?Ser?Val?Leu?Val?Gly?Lys?Ala?Ala?Ala?Thr?Gly?Ser?Gly?Asp

370?????????????????375?????????????????380Trp?Asn?Thr?Thr?Leu?Ala?Cys?Phe?Val?Ala?Ile?Leu?Ile?Gly?Leu?Cys385?????????????????390?????????????????395?????????????????400Leu?Thr?Leu?Leu?Leu?Leu?Ala?Val?Phe?Lys?Lys?Ala?Leu?Pro?Ala?Leu

405?????????????????410?????????????????415Pro?Ile?Ser?Ile?Thr?Phe?Gly?Leu?Ile?Phe?Tyr?Phe?Ser?Thr?Asp?Asn

420?????????????????425?????????????????430Leu?Val?Arg?Pro?Phe?Met?Asp?Thr?Leu?Ala?Ser?His?Gln?Leu?Tyr?Ile

The data of 435 440 445 (2) SEQ ID NO:20: (i) sequence signature:

(A) length: 1895 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear (ix) feature:

(A) title/keyword: CDS

(B) position: 140 ... 1762 (ix) feature:

(A) title/keyword: misc_ feature

(B) position: 1 ... 1895

(D) other data :/notes=" DmPS " be sequence description: SEQ ID NO:20:TATATGAGTC GCTTTAAAAC AAAAGAAAGT TTTTACCAGC TACATTCCTT TGGTTTCCTT 60AACTAAATCC CATCACACAA CTACGGCTTC GCAGGGGGAG GCGTCCAGCG CTACGGAGGC 120GAACGAACGC ACACCACTG ATG GCT GCT GTC AAT CTC CAG GCT TCG TGC TCC 172 (xi)

Met?Ala?Ala?Val?Asn?Leu?Gln?Ala?Ser?Cys?Ser

1???????????????5??????????????????10TCC?GGG?CTC?GCC?TCT?GAG?GAT?GAC?GCC?AAT?GTG?GGC?AGC?CAG?ATA?GGC?????????220Ser?Gly?Leu?Ala?Ser?Glu?Asp?Asp?Ala?Asn?Val?Gly?Ser?Gln?Ile?Gly

15??????????????????20??????????????????25GCG?GCG?GAG?CGT?TTG?GAA?CGA?CCT?CCA?AGG?CGG?CAA?CAG?CAG?CGG?AAC?????????268Ala?Ala?Glu?Arg?Leu?Glu?Arg?Pro?Pro?Arg?Arg?Gln?Gln?Gln?Arg?Asn

30??????????????????35??????????????????40AAC?TAC?GGC?TCC?AGC?AAT?CAG?GAT?CAA?CCG?GAT?GCT?GCC?ATA?CTT?GCT?????????316Asn?Tyr?Gly?Ser?Sar?Asn?Gln?Asp?Gln?Pro?Asp?Ala?Ala?Ile?Leu?Ala

45??????????????????50??????????????????55GTG?CCC?AAT?GTG?GTG?ATG?CGT?GAA?CCT?TGT?GGC?TCG?CGC?CCT?TCA?AGA?????????364Val?Pro?Asn?Val?Val?Met?Arg?Glu?Pro?Cys?Gly?Ser?Arg?Pro?Ser?Arg?60??????????????????65??????????????????70??????????????????75CTG?ACC?GGT?GGA?GGA?GGC?GGC?AGT?GGT?GGT?CCG?CCC?ACA?AAT?GAA?ATG?????????412Leu?Thr?Gly?Gly?Gly?Gly?Gly?Ser?Gly?Gly?Pro?Pro?Thr?Asn?Glu?Met

80??????????????????85??????????????????90GAG?GAA?GAG?CAG?GGC?CTG?AAA?TAC?GGG?GCC?CAG?CAT?GTG?ATC?AAG?TTA?????????460Glu?Glu?Glu?Gln?Gly?Leu?Lys?Tyr?Gly?Ala?Gln?His?Val?Ile?Lys?Leu

95?????????????????100?????????????????105TTC?GTC?CCC?GTC?TCC?CTT?TGC?ATG?CIG?GTA?GTG?GTG?GCT?ACC?ATC?AAC?????????508Phe?Val?Pro?Val?Ser?Leu?Cys?Met?Leu?Val?Val?Val?Ala?Thr?Ile?Asn

110?????????????????115?????????????????120TCC?ATC?AGC?TTC?TAC?AAC?AGC?ACG?GAT?GTC?TAT?CTC?CTC?TAC?ACA?CCT?????????556Ser?Ile?Ser?Phe?Tyr?Asn?Ser?Thr?Asp?Val?Tyr?Leu?Leu?Tyr?Thr?Pro

125?????????????????130?????????????????135TTC?CAT?GAA?CAA?TCG?CCC?GAG?CCT?AGT?GTT?AAG?TTC?TGG?AGT?GCC?TTG?????????604Phe?His?Glu?Gln?Ser?Pro?Glu?Pro?Ser?Val?Lys?Phe?Trp?Ser?Ala?Leu140?????????????????145?????????????????150?????????????????155GCG?AAC?TCC?CTG?ATC?CTG?ATG?AGC?GTG?GTG?GTG?GTG?ATG?ACC?TTT?TTG?????????652Ala?Asn?Ser?Leu?Ile?Leu?Met?Ser?Val?Val?Val?Val?Met?Thr?Phe?Leu

160?????????????????165?????????????????170CTG?ATT?GTT?TTG?TAC?AAG?AAG?CGT?TGC?TAT?CGC?ATC?ATT?CAC?GGC?TGG?????????700Leu?Ile?Val?Leu?Tyr?Lys?Lys?Arg?Cys?Tyr?Arg?Ile?Ile?His?Gly?Trp

175?????????????????180?????????????????185CTG?ATT?CTC?TCC?TCC?TTC?ATG?TTG?TTG?TTC?ATT?TTT?ACG?TAC?TTA?TAT???????748Leu?Ile?Leu?Ser?Ser?Phe?Met?Leu?Leu?Phe?Ile?Pha?Thr?Tyr?Leu?Tyr

190?????????????????195?????????????????200TTG?GAA?GAG?CTT?CTT?CGC?GCC?TAT?AAC?ATA?CCG?ATG?GAC?TAC?CCT?ACT???????796Leu?Glu?Glu?Leu?Leu?Arg?Ala?Tyr?Asn?Ile?Pro?Met?Asp?Tyr?Pro?Thr

205?????????????????210?????????????????215GCA?CTA?CTG?ATT?ATG?TGG?AAC?TTT?GGA?GTG?GTC?GGA?ATG?ATG?TCC?ATC???????844Ala?Leu?Leu?Ile?Met?Trp?Asn?Phe?Gly?Val?Val?Gly?Met?Met?Ser?Ile220?????????????????225?????????????????230?????????????????235CAT?TGG?CAG?GGA?CCT?CTG?CGG?TTG?CAG?CAA?GGA?TAT?CTC?ATT?TTC?GTG???????892His?Trp?Gln?Gly?Pro?Leu?Arg?Leu?Gln?Gln?Gly?Tyr?Leu?Ile?Phe?Val

240?????????????????245?????????????????250GCA?GCC?TTG?ATG?GCC?TTG?GTG?TTC?ATT?AAA?TAC?CTG?CCT?GAA?TGG?ACT???????940Ala?Ala?Leu?Met?Ala?Leu?Val?Phe?Ile?Lys?Tyr?Leu?Pro?Glu?Trp?Thr

255?????????????????260?????????????????265GCC?TGG?GCT?GTA?TTG?GCT?GCC?ATT?TCT?ATT?TGG?GAT?CTT?ATT?GCT?GTC???????988Ala?Trp?Ala?Val?Leu?Ala?Ala?Ile?Ger?Ile?Trp?Asp?Leu?Ile?Ala?Val

270?????????????????275?????????????????280CTT?TCG?CCA?AGA?GGA?CCC?CTC?CGC?ATT?CTG?GTG?GAA?ACG?GCT?CAG?GAG???????1036Leu?Ser?Pro?Arg?Gly?Pro?Leu?Arg?Ile?Leu?Val?Glu?Thr?Ala?Gln?Glu

285?????????????????290?????????????????295CGA?AAT?GAG?CAA?ATC?TTC?CCC?GCT?CTG?ATT?TAT?TCA?TCC?ACT?GTC?GTT???????1084Arg?Asn?Glu?Gln?Ile?Phe?Pro?Ala?Leu?Ile?Tyr?Ser?Ser?Thr?Val?Val300?????????????????305?????????????????310?????????????????315TAC?GCA?CTT?GTA?AAC?ACT?GTT?ACG?CCG?GAG?CAA?TCG?CAG?GCC?ACA?GCT???????1132Tyr?Ala?Leu?Val?Asn?Thr?Val?Thr?Pro?Gln?Gln?Ser?Gln?Ala?Thr?Ala

320?????????????????325?????????????????330TCC?TCC?TCG?CCG?TCG?TCC?AGC?AAC?TCC?ACC?ACA?ACC?ACG?AGG?GCC?ACG???????1180Ser?Ser?Ser?Pro?Ser?Ser?Ser?Asn?Ser?Thr?Thr?Thr?Thr?Arg?Ala?Thr

335?????????????????340?????????????????345CAG?AAC?TCG?CTG?GCT?TCG?CCA?GAG?GCA?GCA?GCG?GCT?AGT?GGC?CAA?CGC???????1228Gln?Asn?Ser?Leu?Ala?Ser?Pro?Glu?Ala?Ala?Ala?Ala?Ser?Gly?Gln?Arg

350?????????????????355?????????????????360ACA?GGT?AAC?TCC?CAT?CCT?CGA?CAG?AAT?CAG?CGG?GAT?GAC?GGC?AGT?GTA???????1276Thr?Gly?Asn?Ser?His?Pro?Arg?Gln?Asn?Gln?Arg?Asp?Asp?Gly?Ser?Val

365?????????????370?????????????????375CTG?GCA?ACT?GAA?GGT?ATG?CCA?CTT?GTG?ACT?TTT?AAA?AGC?AAT?TTG?CGC???????1324Leu?Ala?Thr?Glu?Gly?Met?Pro?Leu?Val?Thr?Phe?Lys?Ser?Asn?Leu?Arg380?????????????????385?????????????????390?????????????????395GGA?AAC?GCT?GAG?GCT?GCG?GGT?TTC?ACG?CAA?GAG?TGG?TCA?GCT?AAC?TTG???????1372Gly?Asn?Ala?Glu?Ala?Ala?Gly?Phe?Thr?Gln?Glu?Trp?Ser?Ala?Asn?Leu

400?????????????????405?????????????????410AGC?GAA?CGT?GTG?GCT?CGT?CGC?CAG?ATT?GAA?GTT?CAA?AGT?ACT?CAG?AGT???????14205er?Glu?Arg?Val?Ala?Arg?Arg?Gln?Ile?Glu?Val?Gln?Ser?Thr?Gln?Ser

415?????????????????420?????????????????425GGA?AAC?GCT?CAG?CGC?TCC?AAC?GAG?TAT?AGG?ACA?GTA?ACA?GCT?CCG?GAT???????1468Gly?Asn?Ala?Gln?Arg?Ser?Asn?Glu?Tyr?Arg?Thr?Val?Thr?Ala?Pro?Asp

430?????????????????435?????????????????440CAG?AAT?CAT?CCG?GAT?GGG?CAA?GAA?GAA?CGT?GGC?ATA?AAG?CTT?GGC?CTC???????1516Gln?Asn?His?Pro?Asp?Gly?Gln?Glu?Glu?Arg?Gly?Ile?Lys?Leu?Gly?Leu

445?????????????????450?????????????????455GGC?GAC?TTC?ATC?TTC?TAC?TCG?GTA?TTA?GTG?GGC?AAG?GCC?TCC?AGC?TAC???????1564Gly?Asp?Phe?Ile?Phe?Tyr?Ser?Val?Leu?Val?Gly?Lys?Ala?Ser?Ser?Tyr460?????????????????465?????????????????470?????????????????475GGC?GAC?TGG?ACG?ACC?ACA?ATC?GCT?TGC?TTT?GTG?GCC?ATC?CTC?ATT?GGA??????1612Gly?Asp?Trp?Thr?Thr?Thr?Ile?Ala?Cys?Phe?Val?Ala?Ile?Leu?Ile?Gly

480?????????????????485?????????????????490CTC?TGC?CTC?ACT?CTT?CTG?CTT?CTG?GCC?ATT?TGG?CGC?AAG?GCG?CTA?CCC??????1660Leu?Cys?Leu?Thr?Leu?Leu?Leu?Leu?Ala?Ile?Trp?Arg?Lys?Ala?Leu?Pro

495?????????????????500?????????????????S05GCC?CTG?CCC?ATC?TCA?ATA?ACG?TTC?GGA?TTG?ATA?TTT?TGC?TTC?GCC?ACT??????1708Ala?Leu?Pro?Ile?Ser?Ile?Thr?Phe?Gly?Leu?Ile?Phe?Cys?Phe?Ala?Thr

510?????????????????515?????????????????520AGT?GCG?GTG?GTC?AAG?CCG?TTC?ATG?GAG?GAT?CTA?TCG?GCC?AAG?CAG?GTG??????1756Ser?Ala?Val?Val?Lys?Pro?Phe?Met?Glu?Asp?Leu?Ser?Ala?Lys?Gln?Val

525 530 535TTT ATA TAAACTTGAA AAGACAAGGA CACATCAAGT GTCTTACAGT ATCATAGTCT 1812Phe Ile540AACAAAGCTT TTTGTAATCC AATTCTTTAT TTAACCAAAT GCATAGTAAC AACCTCGACT 1872AAAAAAAAAA AAAAAAAAAA AAA 1895, (2) SEQ ID NO:21 data:, (i) sequence signature:

(A) length: 541 amino acid

(B) type: amino acid

(D) topological framework: linearity is molecule type (ii): albumen (xi) sequence description: SEQ ID NO:21:Met Ala Ala Val Asn Leu Gln Ala Ser Cys Ser Ser Gly Leu Ala Ser 15 10 15Glu Asp Asp Ala Asn Val Gly Ser Gln Ile Gly Ala Ala Glu Arg Leu

20??????????????????25??????????????????30Glu?Arg?Pro?Pro?Arg?Arg?Gln?Gln?Gln?Arg?Asn?Asn?Tyr?Gly?Ser?Ser

35??????????????????40??????????????????45Asn?Gln?Asp?Gln?Pro?Asp?Ala?Ala?Ile?Leu?Ala?Val?Pro?Asn?Val?Val

50??????????????????55??????????????????60Met?Arg?Glu?Pro?Cys?Gly?Ser?Arg?Pro?Ser?Arg?Lau?Thr?Gly?Gly?Gly?65??????????????????70??????????????????75??????????????????80Gly?Gly?Ser?Gly?Gly?Pro?Pro?Thr?Asn?Glu?Met?Glu?Glu?Glu?Gln?Gly

85??????????????????90??????????????????95Leu?Lys?Tyr?Gly?Ala?Gln?His?Val?Ile?Lys?Leu?Phe?Val?Pro?Val?Ser

100?????????????????105?????????????????110Leu?Cys?Met?Leu?Val?Val?Val?Ala?Thr?Ile?Asn?Ser?Ile?Ser?Phe?Tyr

115?????????????????120?????????????????125Asn?Ser?Thr?Asp?Val?Tyr?Leu?Leu?Tyr?Thr?Pro?Phe?His?Glu?Gln?Ser

130?????????????????135?????????????????140Pro?Glu?Pro?Ser?Val?Lys?Phe?Trp?Ser?Ala?Leu?Ala?Asn?Ser?Leu?Ile145?????????????????150?????????????????155?????????????????160Leu?Met?Ser?Val?Val?Val?Val?Met?Thr?Phe?Leu?Leu?Ile?Val?Leu?Tyr

165?????????????????170?????????????????175Lys?Lys?Arg?Cys?Tyr?Arg?Ile?Ile?His?Gly?Trp?Leu?Ile?Leu?Ser?Ser

180?????????????????185?????????????????190Phe?Met?Leu?Leu?Phe?Ile?Phe?Thr?Tyr?Leu?Tyr?Leu?Glu?Glu?Leu?Leu

195?????????????????200?????????????????205Arg?Ala?Tyr?Asn?Ile?Pro?Met?Asp?Tyr?Pro?Thr?Ala?Leu?Leu?Ile?Met

210?????????????????215?????????????????220Trp?Asn?Phe?Gly?Val?Val?Gly?Met?Met?Ser?Ile?His?Trp?Gln?Gly?Pro225?????????????????230?????????????????235?????????????????240Leu?Arg?Leu?Gln?Gln?Gly?Tyr?Leu?Ile?Phe?Val?Ala?Ala?Leu?Met?Ala

245????????????????250?????????????????255Leu?Val?Phe?Ile?Lys?Tyr?Leu?Pro?Glu?Trp?Thr?Ala?Trp?Ala?Val?Leu

260?????????????????265?????????????????270Ala?Ala?Ile?Ser?Ile?Trp?Asp?Leu?Ile?Ala?Val?Leu?Ser?Pro?Arg?Gly

275?????????????????280?????????????????285Pro?Leu?Arg?Ile?Leu?Val?Glu?Thr?Ala?Gln?Glu?Arg?Asn?Glu?Gln?Ile

290?????????????????295?????????????????300Phe?Pro?Ala?Leu?Ile?Tyr?Ser?Ser?Thr?Val?Val?Tyr?Ala?Leu?Val?Asn305?????????????????310?????????????????315?????????????????320Thr?Val?Thr?Pro?Gln?Gln?Ser?Gln?Ala?Thr?Ala?Ser?Ser?Ser?Pro?Ser

325?????????????????330?????????????????335Ser?Ser?Asn?Ser?Thr?Thr?Thr?Thr?Arg?Ala?Thr?Gln?Asn?Ser?Leu?Ala

340?????????????????345?????????????????350Ser?Pro?Glu?Ala?Ala?Ala?Ala?Ser?Gly?Gln?Arg?Thr?Gly?Asn?Ser?His

355?????????????????360?????????????????365Pro?Arg?Gln?Asn?Gln?Arg?Asp?Asp?Gly?Ser?Val?Leu?Ala?Thr?Glu?Gly

370?????????????????375?????????????????380Met?Pro?Leu?Val?Thr?Phe?Lys?Ser?Asn?Leu?Arg?Gly?Asn?Ala?Glu?Ala385?????????????????390?????????????????395?????????????????400Ala?Gly?Phe?Thr?Gln?Glu?Trp?Ser?Ala?Asn?Leu?Ser?Glu?Arg?Val?Ala

405?????????????????410?????????????????415Arg?Arg?Gln?Ile?GIu?Val?Gln?Ser?Thr?Gln?Ser?Gly?Asn?Ala?Gln?Arg

420?????????????????425?????????????????430Ser?Asn?Glu?Tyr?Arg?Thr?Val?Thr?Ala?Pro?Asp?Gln?Asn?His?Pro?Asp

435?????????????????440?????????????????445Gly?Gln?Glu?Glu?Arg?Gly?Ile?Lys?Leu?Gly?Leu?Gly?Asp?Phe?Ile?Phe

450?????????????????455?????????????????460Tyr?Ser?Val?Leu?Val?Gly?Lys?Ala?Ser?Ser?Tyr?Gly?Asp?Trp?Thr?Thr465?????????????????470?????????????????475?????????????????480Thr?Ile?Ala?Cys?Phe?Val?Ala?Ile?Leu?Ile?Gly?Leu?Cys?Leu?Thr?Leu

485?????????????????490?????????????????495Leu?Leu?Leu?Ala?Ile?Trp?Arg?Lys?Ala?Leu?Pro?Ala?Leu?Pro?Ile?Ser

500?????????????????505?????????????????510Ile?Thr?Phe?Gly?Leu?Ile?Phe?Cys?Phe?Ala?Thr?Ser?Ala?Val?Val?Lys

515?????????????????520?????????????????525Pro?Phe?Met?Glu?Asp?Leu?Ser?Ala?Lys?Gln?Val?Phe?Ile

The data of 530 535 540 (2) SEQ ID NO:22: (i) sequence signature:

(A) length: 21 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear (xi) sequence description: the data of SEQ ID NO:22:CTNCCNGART GGACNGYCTG G 21 (2) SEQ ID NO:23: (i) sequence signature:

(A) length: 21 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear (xi) sequence description: the data of SEQ ID NO:23:RCANGCDATN GTNGTRTTCC A 21 (2) SEQ ID NO:24: (i) sequence signature:

(A) length: 32 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear (xi) sequence description: the data of SEQ ID NO:24:TTTTTTCTCG AGACNGCNCA RGARAGAAAY GA 32 (2) SEQ ID NO:25: (i) sequence signature:

(A) length: 29 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear (xi) sequence description: SEQ ID NO:25:TTTTTTGGAT CCTARAADAT RAARTCNCC 29

Claims (111)

1. the isolating nucleic acid that contains one section nucleotide sequence, described nucleotide sequence coded normal early ageing element-1 albumen, sudden change early ageing element-1 albumen, normal early ageing element-2 albumen, the proteic albumen of sudden change early ageing element-2 of being selected from.

2. the isolating nucleic acid of claim 1, the normal early ageing element-1 of wherein said nucleic acid encoding albumen, wherein said nucleotide sequence is selected from:

(1) coding contains the proteic sequence of people's early ageing element-1 aminoacid sequence of SEQ ID NO:2;

(2) coding contains the proteic sequence of people's early ageing element-1 aminoacid sequence of SEQ ID NO:4;

(3) coding contains the proteic sequence of mouse early ageing element-1 aminoacid sequence of SEQ ID NO:17;

(4) coding contains the proteic sequence of the aminoacid sequence of SEQ ID NO:2, wherein replaces residue 257 with L-Ala, and slightly removes residue 258-290;

(5) coding contains the proteic sequence of the aminoacid sequence of SEQ ID NO:4, wherein replaces residue 253 with L-Ala, and slightly removes residue 254-286; With

(6) normal early ageing element-1 albumen of coding and under rigorous hybridization conditions can with the sequence of the complementary sequence hybridization of any sequence of (1)-(5).

3. the isolating nucleic acid of claim 1, wherein said nucleic acid encoding sudden change early ageing element-1 albumen,

Wherein said nucleotide sequence coded at least a sudden change, is this sudden change corresponding to the sudden change that is selected from following SEQID NO:2: A79?, V82L, V96F, Y115H, M139T, M139V, I143T, M146L, M146V, H163R, H163Y, L171P, G209V, I211T, A231T, A246E, A260V, C263R, P264L, P267S, E280A, E280G, A285V, L286V, Δ 291-319, G384A, L392V and C410Y; And

Wherein said nucleotide sequence is also corresponding to being selected from following nucleotide sequence:

(1) coding contains the proteic sequence of people's early ageing element-1 aminoacid sequence of SEQ ID NO:2;

(2) coding contains the proteic sequence of people's early ageing element-1 aminoacid sequence of SEQ ID NO:4;

(3) coding contains the proteic sequence of mouse early ageing element-1 aminoacid sequence of SEQ ID NO:17;

(4) coding contains the proteic sequence of the aminoacid sequence of SEQ ID NO:2, wherein replaces residue 257 with L-Ala, and slightly removes residue 258-290;

(5) coding contains the proteic sequence of the aminoacid sequence of SEQ ID NO:4, wherein replaces residue 253 with L-Ala, and slightly removes residue 254-286; With

(6) normal early ageing element-1 albumen of coding and under rigorous hybridization conditions can with the sequence of the complementary sequence hybridization of any sequence of (1)-(5).

4. the isolating nucleic acid of claim 1, wherein said nucleic acid encoding sudden change early ageing element-1 albumen,

Wherein said nucleotide sequence coded at least a sudden change, this sudden change is corresponding to the sudden change of the SEQ ID NO:19 that is selected from M239V, N141I and I420T; With

Wherein said nucleotide sequence is also corresponding to being selected from following nucleotide sequence:

(1) coding contains the proteic sequence of people's early ageing element-1 aminoacid sequence of SEQ ID NO:2;

(2) coding contains the proteic sequence of people's early ageing element-1 aminoacid sequence of SEQ ID NO:4;

(3) coding contains the proteic sequence of mouse early ageing element-1 aminoacid sequence of SEQ ID NO:17;

(4) coding contains the proteic sequence of the aminoacid sequence of SEQ ID NO:2, wherein replaces residue 257 with L-Ala, and slightly removes residue 258-290;

(5) coding contains the proteic sequence of the aminoacid sequence of SEQ ID NO:4, wherein replaces residue 253 with L-Ala, and slightly removes residue 254-286; With

(6) normal early ageing element-1 albumen of coding and under rigorous hybridization conditions can with the sequence of the complementary sequence hybridization of any sequence of (1)-(5).

5. the isolating nucleic acid of claim 1, the normal early ageing element-2 of wherein said nucleic acid encoding albumen, and also wherein said nucleotide sequence is selected from:

(1) coding contains the proteic sequence of people's early ageing element-2 aminoacid sequence of SEQ ID NO:19;

(2) coding contains the proteic sequence of people's early ageing element-2 aminoacid sequence of SEQ ID NO:19, and its part omitted is except residue 263-296;

(3) normal early ageing element-2 albumen of coding and under rigorous hybridization conditions can with the sequence of the complementary sequence hybridization of arbitrary sequence of (1)-(2).

6. the isolating nucleic acid of claim 1, wherein said nucleic acid encoding sudden change early ageing element-2 albumen,

Wherein said nucleotide sequence coded at least a sudden change, this sudden change is corresponding to the sudden change of the SEQ ID NO:19 that is selected from M239V, N141I and I420T; And

Wherein said nucleotide sequence is also corresponding to being selected from following nucleotide sequence:

(1) coding contains the proteic sequence of people's early ageing element-2 aminoacid sequence of SEQ ID NO:19;

(2) coding contains the proteic sequence of people's early ageing element-2 aminoacid sequence of SEQ ID NO:19, and its part omitted is except residue 263-296;

(3) normal early ageing element-2 albumen of coding and under rigorous hybridization conditions can with the sequence of the complementary sequence hybridization of any sequence of (1)-(2).

7. the isolating nucleic acid of claim 1, wherein said nucleic acid encoding sudden change early ageing element-2 albumen,

Wherein said nucleotide sequence coded at least a sudden change, is this sudden change corresponding to the sudden change that is selected from following sudden change of SEQ ID NO:2: A79?, V82L, V96F, Y115H, M139T, M139V, I143T, M146L, M146V, H163R, H163Y, L171P, G209V, I211T, A231T, A246E, A260V, C263R, P264L, P267S, E280A, E280G, A285V, L286V, Δ 291-319, G384A, L392V and C410Y; And

Wherein said nucleotide sequence is also corresponding to being selected from following nucleotide sequence:

(1) coding contains the proteic sequence of people's early ageing element-2 aminoacid sequence of SEQ ID NO:19;

(2) coding contains the proteic sequence of people's early ageing element-2 aminoacid sequence of SEQ ID NO:19, and its part omitted removes residue 263-296;

(3) normal early ageing element-2 albumen of coding and under rigorous hybridization conditions can with the sequence of the complementary sequence hybridization of any sequence of (1)-(2).

8. one section isolating nucleic acid, it contains the nucleotide sequence of at least 10 continuous nucleotides, and described continuous nucleotide is selected from: the complementary sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ IDNO:15 and any of these sequence.

9. one section isolating nucleic acid, it contains the nucleotide sequence of at least 15 continuous nucleotides, and described continuous nucleotide is selected from: the complementary sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ IDNO:15 and any of these sequence.

10. one section isolating nucleic acid, it contains the nucleotide sequence of at least 20 continuous nucleotides, and described continuous nucleotide is selected from: the complementary sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ IDNO:15 and any of these sequence.

11. contain the isolating nucleic acid of one section nucleotide sequence, described nucleotide sequence contains at least 10 continuous nucleotides from the early ageing element that inserts plasmid, and described plasmid is selected from: ATCC registration number 97214, ATCC registration number 97508, ATCC registration number 97124 and ATCC registration number 97428.

12. contain the isolating nucleic acid of one section nucleotide sequence, at least one functional domain of described nucleotide sequence coded early ageing fibroin, described early ageing fibroin is selected from: normal early ageing element-1 albumen, sudden change early ageing element-1 albumen, normal early ageing element-2 albumen, sudden change early ageing element-2 albumen.

13. the isolating nucleic acid of claim 12, wherein said functional domain are corresponding to early ageing element-1 functional domain that is selected from following structural domain: the N-end of early ageing element-1, TM1, TM1 → 2, TM2, TM2 → 3, TM3, TM3 → 4, TM4, TM4 → 5, TM5, TM5 → 6, TM6, TM6 → 7, TM7 and C-end structure territory.

14. the isolating nucleic acid of claim 12, wherein said functional domain are corresponding to early ageing element-2 functional domain that is selected from following structural domain: the N-end of early ageing element-2, TM1, TM1 → 2, TM2, TM2 → 3, TM3, TM3 → 4, TM4, TM4 → 5, TM5, TM5 → 6TM6, TM6 → 7, TM7 and C-end structure territory.

15. contain the isolating nucleic acid of one section nucleotide sequence, the epitope of described nucleotide sequence coded early ageing fibroin, described early ageing fibroin is selected from: normal early ageing element-1 albumen, sudden change early ageing element-1 albumen, normal early ageing element-2 albumen, sudden change early ageing element-2 albumen.

16. the isolating nucleic acid of claim 15, wherein said sequence encoding early ageing element-1 epitope, this epitope is corresponding to being selected from following early ageing element-1 epitope: the amino-acid residue 27-44 of SEQ ID NO:2,28-61,46-48,50-60,65-71,66-67,107-111,109-112,120-121,120-122,125-126,155-160,185-189,214-223,218-221,220-230,240-245,241-243,267-269,273-282,300-370,302-310,311-325,332-342,346-359,372-382,400-410 and 400-420.

17. the isolating nucleic acid of claim 15, wherein said sequence encoding early ageing element-2 epitope, this epitope is corresponding to being selected from following early ageing element-2 epitope: amino-acid residue 25-45,50-63,70-75,114-120,127-132,162-167,221-226,282-290,310-314,321-338,345-352,380-390 and the 430-435 of SEQ ID NO:19.

18. the allele variant of identifier's early ageing plain gene or the method for different specificity homologue, this method comprises:

Under rigorous hybridization conditions, selection can with the nucleic acid probe or the primer of people's early ageing plain gene sequence hybridization:

Described probe or primer are mixed with nucleic acid samples, and described sample contains the nucleic acid corresponding to described variant or homologue;

Detect described probe or primer and corresponding to the hybridization of the nucleic acid of described variant or homologue.

19. the method for claim 18, wherein said sample contain the nucleic acid samples that is selected from human gene group DNA, people mRNA and people cDNA.

20. the method for claim 18, wherein said sample contain the nucleic acid samples that is selected from mammalian genes group DNA, Mammals mRNA and Mammals cDNA.

21. the method for claim 18, wherein said sample contain the nucleic acid samples that is selected from invertebrates genomic dna, invertebrates mRNA and invertebrates cDNA.

22. the method for claim 18, it also comprises the step of separation corresponding to the nucleic acid of described variant or homologue.

23. the method for claim 18, wherein said nucleic acid is identified by hybridization.

24. the method for claim 18, wherein said nucleic acid is identified by pcr amplification.

25. the allele variant of identifier's early ageing plain gene or the method for different specificity homologue, it comprises:

Selection can selective binding people early ageing fibroin antibody;

Described antibody is mixed with protein sample, and described sample contains the albumen corresponding to described variant or homologue;

Detect described antibody and described proteic the combination corresponding to described variant or homologue.

26. the method for claim 25, wherein said sample contains the protein sample that is selected from people's albumen, human fusion protein and its proteolytic fragments.

27. the method for claim 25, wherein said sample contains the nucleic acid samples that is selected from mammalian proteins, Mammals fusion rotein and its proteolytic fragments.

28. the method for claim 25, wherein said sample contains the nucleic acid samples that is selected from invertebrates albumen, invertebrates fusion rotein and its proteolytic fragments.

29. the method for claim 25, it comprises that also purifying is corresponding to the described proteic step of variant or homologue basically.

30. contain the allele variant of people's early ageing plain gene or the isolating nucleic acid of different specificity homologue.

31. the allele variant of coding people early ageing fibroin or the isolating nucleic acid of different specificity homologue.

32. the isolating nucleic acid of claim 31, the black-tailed fruit flies homologue of wherein said nucleic acid encoding people early ageing plain gene.

33. containing, the isolating nucleic acid of claim 32, wherein said nucleic acid is selected from following nucleotide sequence:

(1) coding contains the proteic sequence of the DmPS aminoacid sequence of SEQ ID NO:21;

(2) the plain homologue albumen of coding early ageing and under rigorous hybridization conditions can with the sequence of the complementary sequence hybridization of (1).

34. one section isolating nucleic acid, it contains the nucleotide sequence of at least 10 continuous nucleotides, described continuous nucleotide be selected from SEQ ID NO:21 and with SEQ ID NO:21 complementary sequence.

35. contain the isolating nucleic acid of the recombinant vectors that comprises the nucleotide sequence of arbitrary claim among the claim 1-34.

36. the isolating nucleic acid of claim 35, the wherein said carrier plain nucleotide sequence that is expression vector and described early ageing can be operated with regulatory region and link to each other.

37. the isolating nucleic acid of claim 36, wherein said expression vector can be expressed the plain sequence of described early ageing in mammalian cell.

38. the isolating nucleic acid of claim 37, wherein said cell is selected from inoblast, liver,kidney,spleen, marrow and neurocyte.

39. the isolating nucleic acid of claim 37, wherein said carrier are selected from vaccinia virus, adenovirus, retrovirus, neural virus and herpes simplex become.

40. the isolating nucleic acid of claim 36, at least one functional domain of wherein said expression vector codes early ageing fibroin, described early ageing fibroin are selected from normal early ageing element-1, sudden change early ageing element-1, normal early ageing element-2 and sudden change early ageing element-2.

41. the isolating nucleic acid of claim 36, wherein said carrier also contain the sequence that coding and the plain sequence of described early ageing can be operated the foreign protein that links to each other, described thus vector encoded early ageing plain fusion protein.

42. the isolating nucleic acid of claim 41, wherein said foreign protein are selected from lacZ, trpE, maltose binding protein, poly-His mark or glutathione-S-transferase.

43. contain the isolating nucleic acid of recombinant expression vector, described recombinant expression vector comprises the nucleotide sequence corresponding to the endogenous regulatory region of early ageing plain gene.

44. the isolating nucleic acid of claim 43, wherein said endogenous regulatory region can be operated with a marker gene and link to each other.

45. expression vector transformed host cells or its filial generation with arbitrary claim among the claim 36-44.

46. the host cell of claim 45, wherein said host cell is selected from bacterial cell and yeast cell.

47. the host cell of claim 45, wherein said host cell is selected from fetus cells, embryonic stem cell, zygote, gamete and germ line cell.

48. the host cell of claim 45, wherein said cell is selected from inoblast, liver,kidney,spleen, marrow and neurocyte.

49. the host cell of claim 45, wherein said cell is an invertebral zooblast

50. a kind of non-human animal model of Alzheimer's disease, wherein the genome of described animal or its have been modified for generations with at least a recombinant precursor, and following modification is imported described construct: the insertion of the nucleotide sequence of at least one functional domain of the normal early ageing plain gene of (1) different specificity of coding, (2) insertion of the nucleotide sequence of at least one functional domain of the different specific mutant early ageing plain gene of coding, (3) insertion of the nucleotide sequence of at least one functional domain of the different specific mutant early ageing plain gene of coding homologue of the same race and the inactivation of (4) endogenous early ageing plain gene.

51. the animal of claim 50, wherein said modification are the nucleotide sequences that inserts at least one functional domain of normal people's early ageing element-1 gene of coding.

52. the animal of claim 50, wherein said modification are the nucleotide sequences of at least one functional domain that inserts people's early ageing element-1 gene of encoding mutant.

53. the animal of claim 50, wherein said modification are the nucleotide sequences that inserts at least one functional domain of normal people's early ageing element-2 gene of coding.

54. the animal of claim 50, wherein said modification are the nucleotide sequences of at least one functional domain that inserts people's early ageing element-2 gene of encoding mutant.

55. the animal of claim 50, wherein said modification are to insert to encode normally or the nucleotide sequence of at least one functional domain of people's early ageing fibroin of sudden change.

56. the animal of claim 50, wherein said animal is selected from rat, mouse, hamster, cavy, rabbit, dog, cat, goat, sheep, pig and non-human primates.

57. the animal of claim 50, wherein said animal is an invertebrates.

58. a method of producing at least one functional domain of early ageing fibroin, its be included under the suitable condition cultivate arbitrary claim among the claim 45-49 host cell to come production early ageing element by expressing described nucleic acid.

59. a pure basically protein product, described albumen are selected from normal early ageing element-1 albumen, sudden change early ageing element-1 albumen, normal early ageing element-2 albumen, sudden change early ageing element-2 albumen.

60. containing, the pure basically goods of claim 59, wherein said albumen are selected from following normal early ageing element-1 albumen:

(1) contains the albumen of the aminoacid sequence of SEQ ID NO:2;

(2) contain the albumen of the aminoacid sequence of SEQ ID NO:4;

(3) contain the albumen of the aminoacid sequence of SEQ ID NO:17;

(4) contain the albumen of the aminoacid sequence of SEQ ID NO:2, wherein replace residue 257, and slightly remove residue 258-290 with L-Ala; With

(5) contain the albumen of the aminoacid sequence of SEQ ID NO:4, wherein replace residue 253, and slightly remove residue 254-286 with L-Ala.

61. the pure basically goods of claim 59, wherein said albumen contains sudden change early ageing element-1 albumen that comprises at least a sudden change, is described sudden change corresponding to the sudden change of the SEQ ID NO:2 that is selected from following sudden change: A79?, V82L, V96F, Y115H, M139T, M139V, I143T, M146L, M146V, H163R, H163Y, L171P, G209V, I211T, A231T, A246E, A260V, C263R, P264L, P267S, E280A, E280G, A285V, L286V, Δ 291-319, G384A, L392V and C410Y; And

Wherein said albumen is also corresponding to being selected from following aminoacid sequence:

(1) aminoacid sequence of SEQ ID NO:2;

(2) aminoacid sequence of SEQ ID NO:4;

(3) aminoacid sequence of SEQ ID NO:17;

(4) aminoacid sequence of SEQ ID NO:2 wherein replaces residue 257 with L-Ala, and slightly removes residue 258-290; With

(5) aminoacid sequence of SEQ ID NO:4 wherein replaces residue 253 with L-Ala, and slightly removes residue 254-286.

62. containing, the pure basically goods of claim 59, wherein said albumen are selected from following normal early ageing element-2 albumen:

(1) contains the albumen of the aminoacid sequence of SEQ ID NO:19; With

(2) contain the albumen of the aminoacid sequence of SEQ ID NO:19, its part omitted is except residue 263-296.

63. the pure basically goods of claim 59, wherein said albumen contain sudden change early ageing element-2 albumen that comprises at least a sudden change, described sudden change is corresponding to the sudden change of the SEQ ID NO:19 that is selected from M239V, N141I and I420T; And

Wherein said albumen is also corresponding to being selected from following aminoacid sequence:

(1) aminoacid sequence of SEQ ID NO:19;

(2) aminoacid sequence of SEQ ID NO:19, its part omitted is except residue 263-296.

64. a pure basically polypeptide product, it contains the aminoacid sequence of at least 5 continuous amino acid residues that are selected from SEQ ID NO:2, SEQ IDNO:4, SEQ ID NO:17, SEQ ID NO:19 and SEQ ID NO:21.

65. a pure basically polypeptide product, it contains the aminoacid sequence of at least 10 continuous amino acid residues that are selected from SEQ ID NO:2, SEQ IDNO:4, SEQ ID NO:17, SEQ ID NO:19 and SEQ ID NO:21.

66. a pure basically polypeptide product, it contains the aminoacid sequence of at least 15 continuous amino acid residues that are selected from SEQ ID NO:2, SEQ IDNO:4, SEQ ID NO:17, SEQ ID NO:19 and SEQ ID NO:21.

67. a pure basically polypeptide product, it contains at least one functional domain of early ageing fibroin, and described early ageing fibroin is selected from normal early ageing element-1 albumen, sudden change early ageing element-1 albumen, normal early ageing element-2 albumen, sudden change early ageing element-2 albumen.

68. the pure basically goods of claim 67, wherein said functional domain are corresponding to early ageing element-1 functional domain that is selected from following structural domain: the N-end of early ageing element-1, TM1, TM1 → 2, TM2, TM2 → 3, TM3, TM3 → 4, TM4, TM4 → 5, TM5, TM5 → 6, TM6, TM6 → 7, TM7 and C-end structure territory.

69. the pure basically goods of claim 67, wherein said functional domain are corresponding to early ageing element-2 functional domain that is selected from following structural domain: the N-end of early ageing element-2, TM1, TM1 → 2, TM2, TM2 → 3, TM3, TM3 → 4, TM4, TM4 → 5, TM5, TM5 → 6, TM6, TM6 → 7, TM7 and C-end structure territory.

70. a pure basically polypeptide product, it contains the epitope of early ageing fibroin, and described early ageing fibroin is selected from: normal early ageing element-1 albumen, sudden change early ageing element-1 albumen, normal early ageing element-2 albumen, sudden change early ageing element-2 albumen.

71. the pure basically goods of claim 70, wherein said polypeptide contains early ageing element-1 epitope, and this epitope is corresponding to being selected from following early ageing element-1 epitope: the amino-acid residue 27-44 of SEQ IDNO:2,28-61,46-48,50-60,65-71,66-67,107-111,109-112,120-121,120-122,125-126,155-160,185-189,214-223,218-221,220-230,240-245,241-243,267-269,273-282,300-370,302-310,311-325,332-342,346-359,372-382,400-410 and 400-420.

72. the pure basically goods of claim 70, wherein said polypeptide contains early ageing element-1 epitope, and this epitope is corresponding to being selected from following early ageing element-1 epitope: amino-acid residue 25-45,50-63,70-75,114-120,127-132,162-167,221-226,282-290,310-314,321-338,345-352,380-390 and the 430-435 of SEQ IDNO:19.

73. the method for the antibody of the plain selective binding of production and early ageing, it comprises the steps:

Use the plain immunogen of the early ageing that causes immune significant quantity to animal;

Make described animal produce anti-described immunogenic antibody; With

From described animal or its deutero-cell culture, obtain described antibody.

74. a pure basically antibody preparation, the epitope selective binding of itself and early ageing fibroin, described early ageing fibroin is selected from normal early ageing element-1, sudden change early ageing element-1, normal early ageing element-2, sudden change early ageing element-2.

75. the pure basically antibody preparation of claim 74, wherein said antibody be the epitope of combination sudden change early ageing element-1 optionally, but can not be in conjunction with normal early ageing element-1 albumen.

76. the pure basically antibody preparation of claim 74, wherein said antibody be the epitope of combination sudden change early ageing element-2 optionally, but can not be in conjunction with normal early ageing element-2 albumen.

77. the clone of the antibody of arbitrary claim among the production claim 74-76.

78. identify the method for the compound that can regulate the expression of early ageing plain gene, it comprises:

Cell is contacted with trying material standed for, and wherein said cell comprises the regulatory region that can operate the early ageing plain gene that links to each other with the coding region; With

Detect the variation of expression aspect, described coding region.

79. the method for claim 78, wherein said variation comprise the variation by the mRNA transcript degree of described coding region coding.

80. the method for claim 78, wherein said variation comprise the variation by described coding region encoded protein level.

81. the method for claim 78, wherein said variation are the active results by described coding region encoded protein.

82. the method for claim 78, wherein said coding region coded markings albumen, described labelled protein is selected from beta galactosidase enzyme, alkaline phosphatase, green fluorescent protein and luciferase.

83. a method of identifying alternative in conjunction with the compound of early ageing fibroin, it comprises the steps:

The goods that contain the plain component of at least a early ageing are provided;

Described goods are contacted with the sample that comprises at least a candidate compound; With

Detect combining of the plain component of described early ageing and described candidate compound.

84. the method for claim 83 is to detect by being selected from following test with plain the combining of component of described early ageing wherein: affinity chromatography, coimmunoprecipitation, bio-molecular interaction test and yeast two-hybrid system.

85. identify the method that can regulate the plain active compound of early ageing, it comprises the steps:

Provide and express cell normal or sudden change early ageing plain gene;

Described cell is contacted with at least a candidate compound; With

Detect the variation of described active mark.

86. the method for claim 85, the meter oolemma of wherein said mark have the difference that exists between the same cell of the cell of sudden change early ageing plain gene of expression and the sudden change early ageing plain gene that other does not have expression.

87. the method for claim 85, wherein said variation comprise the variation that is selected from the following non-specific mark of stechiology: pH, cellular calcium, ring AMP level, GTP/GDP ratio, phosphatidylinositols activity and protein phosphorylation.

88. the method for claim 85, wherein said variation comprise the variation of the plain expression of early ageing aspect.

89. comprising, the method for claim 85, wherein said variation be selected from Ca ²⁺, Na ⁺And K ⁺The ionic IC or the variation of flow aspect.

90. the method for claim 85, wherein said variation comprise the appearance of apoptosis or necrocytosis or the variation of ratio aspect.

91. the method for claim 85, wherein said variation comprise the variation of A β peptide production aspect.

92. the method for claim 85, wherein said variation comprises the variation of at least a microtubule-associated protein phosphorylation.

93. the method for claim 85, wherein said cell are the cells of vitro culture.

94. the method for claim 93, wherein said cell are the transformed host cells of arbitrary claim among the claim 45-49.

95. the method for claim 93, the wherein described cell of outer planting from the host who carries at least a sudden change early ageing plain gene.

96. the method for claim 93, wherein Accessory Right requires the described cell of outer planting in the transgenic animal of arbitrary claim among the 50-57.

97. the method for claim 85, wherein said cell are the cells in the Live Animals.

98. the method for claim 97, wherein said cell are the cells of the transgenic animal of arbitrary claim among the claim 50-57.

99. the method for claim 85, wherein said cell are the cells of the human experimenter in the clinical trial.

Whether have the diagnostic method of sudden change early ageing plain gene 100. determine the experimenter, it comprises the steps:

Described experimenter's biological sample is provided;

It is plain active to detect the plain nucleic acid of sudden change early ageing, sudden change early ageing fibroin or sudden change early ageing in described sample.

101. the method for claim 100, the plain nucleic acid of the early ageing that wherein suddenlys change are to detect by being selected from following test: the directly PCR detection of nucleotide sequencing, probe specificity hybridization, restriction enzyme digestion and mapping, PCR mapping, ligase enzyme mediation, RNase protection, electrophoretic mobility shift assay and chemical mispairing cutting.

102. the method for claim 100, the early ageing fibroin that wherein suddenlys change are to detect by being selected from following test: immunity test, proteolytic enzyme test and electrophoretic mobility test.

103. contain pure basically early ageing fibroin and the pharmaceutical preparation of pharmaceutically acceptable carrier.

104. contain operationally the encode expression vector of early ageing fibroin and the pharmaceutical preparation of pharmaceutically acceptable carrier, wherein said expression vector can be expressed described early ageing fibroin in the human experimenter.

105. contain operationally the encode expression vector of the plain antisense sequences of early ageing and the pharmaceutical preparation of pharmaceutically acceptable carrier, wherein said expression vector can be expressed the plain antisense sequences of described early ageing in the human experimenter.

106. contain pure basically antibody and the pharmaceutical preparation of pharmaceutically acceptable carrier, wherein said antibody is optionally in conjunction with sudden change early ageing fibroin.

107. the pharmaceutical preparation of claim 106, wherein said goods do not have the antibody of the normal early ageing fibroin of selective binding basically.

108. contain the pharmaceutical preparation of the pure basically epitope goods of sudden change early ageing fibroin.

109. the pharmaceutical preparation of claim 108, wherein said goods do not have the epitope of normal early ageing fibroin basically.

110. the patient's of sudden change early ageing plain gene method is carried in a treatment, it comprises the pharmaceutical preparation to arbitrary claim among the claim 103-109 of described patient's administering therapeutic significant quantity.

111. the method for claim 110, wherein said pharmaceutical preparation target is selected from following cell type: the heart, brain, lung, liver, skeletal muscle, kidney, pancreas and neurocyte.

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