CN1708511A

CN1708511A - Genes whose expression is increased in response to stimulation by corticotropin-releasing hormone

Info

Publication number: CN1708511A
Application number: CNA2003801025468A
Authority: CN
Inventors: P·J·彼得斯; H·W·H·格尔曼; S·M·A·斯瓦格马克斯; F·L·P·费伦斯
Original assignee: Janssen Pharmaceutica NV
Current assignee: Janssen Pharmaceutica NV
Priority date: 2002-10-31
Filing date: 2003-10-23
Publication date: 2005-12-14
Anticipated expiration: 2023-10-23
Also published as: CN100352842C; NZ540083A; AU2003276167A1; WO2004039835A2; JP2006516387A; CA2499502A1; WO2004039835A3

Abstract

The present invention relates generally to therapy and diagnosis of depression. In particular this invention relates to the polypeptides as well as to the polynucleotides encoding these polypeptides, wherein said polypeptides are shown to play a central role in mediating the endocrine response to corticotropin releasing hormone. These polypeptides and polynucleotides are useful in the diagnosis, treatment and/or prevention of depression.

Description

The gene that replying of corticotropin releasing hormone stimulation expressed increase

The present invention relates generally to the treatment and the diagnosis of dysthymia disorders.Particularly, the present invention relates to polypeptide and the polynucleotide of these polypeptide of encoding, wherein said polypeptide shows in the cell response of mediation corticotropin releasing hormone and plays a major role.These polypeptide and polynucleotide can be used for dysthymia disorders diagnosis, treat and/or prevent.

Background of invention

Nearest socioeconomic analysis finds that dysthymia disorders is the incompetent first cause and the principal risk factor of other diseases development.In addition, in the world wide scale, dysthymia disorders is not enough by underdiagnosis and treatment.Nearest antidepressant drug has been proved to be effectively, but existence effect onset is slow and the problem of side effect.In this respect, do not know still which kind of their clinical effectiveness of pharmacotoxicological effect pattern performance they pass through.Research based on the hypothesis-driving of the reflunomide receptors hypothesis of dysthymia disorders has caused a kind of new concept, and it focuses on cranial nerve peptide acceptor, particularly thyroliberin-releasing hormone (CRH) acceptor as drug targets.

Corticotropin releasing hormone (CRH) is a kind of 41 amino acid whose polypeptide, plays central role in the adjusting of hypothalmus-pituitary-adrenal axis, and mediation is replied the internal secretion of various stressors.Hypothalamus neurons is replied pressure and is discharged CRH to hypophyseal portal system, stimulates the secretion and the biosynthesizing of PATH (ACTH), and causing the suprarenal gland glucocorticosteroid to produce increases (1).Some clinical and preclinical studies are at the reason role (2) who changes in the CRH system in the dysthymia disorders development.In the people, the original research of CRH is shown in patients with depression that ACTH to CRH replys and be passivated, reflected the crh receptor desensitization (3 of the hypothalamus CRH secretion secondary that increases continuously; 4).The evidence of supporting the ACTH of passivation to reply the result who discharges as the CRH that increases is to find that the CRH level raises in the cerebrospinal fluid of patients with depression.Other numbers that are found to be CRH secreted neural unit among the self-slayer who suffers from dysthymia disorders of strengthening this idea of CRH supersecretion in the depressive state increase the reduced number (5 with crh receptor; 6).

Described two kinds of high-affinity receptor CRH1-R1 and CRH-R2 about CRH, their boths exist with several splice variant forms.These acceptors are caused cAMP level rising in the born of the same parents by the stimulation that the activation of CRH causes the Gs-of adenylate cyclase to mediate.This self will activate the protein kinase A (KPA) that relies on cAMP and finally cause cAMP and Ca ²⁺The kytoplasm level of rising.CAMP and Ca ²⁺The level of rising cause some other kinases as relying on Ca ²⁺The activation of the kinases (MAPK) that the kinases II (CAMKII) of/calmodulin and p42/p44 are mitogen-activated.As a result, Ca ²⁺/ cAMP response element binding protein (CREB) will be adjusted in the gene transcription that promoter region contains cAMP response element (CRE) conversely by phosphorylation and this.The example that shows these genes that participate in CRH signalling (signaling) adjusting comprises c-fos, macrophage migration-supressor gene M if, orphan nuclear receptor Nurr77 and Nurr1.

Although at the AtT20-cell---in a kind of cell model of adrenocorticotropin cell broad research the downstream pathway and causing of CRH activated receptors participate in the evaluation of many genes of signal cascade, a main field also is not explored.Therefore, the objective of the invention is to study on the genomic level transcription response that CRH is stimulated to identify other genes relevant with the idiotype network of corticotropin releasing hormone receptor activation.The polypeptide of identifying like this and the polynucleotide of coding said polypeptide provide new chance as the drug targets of triage techniques for drug development, perhaps can be used for dysthymia disorders diagnosis, prevent and/or treat.

Summary of the invention

The present invention relates to the evaluation of many genes relevant with the transcription response of CRH stimulation on genomic level.Relate in particular to proteinic many evaluations of unknown gene so far of coding and regulating CRH signalling, these genes have the sequence that is selected from SEQ ID No.45, SEQ ID No.47, SEQ IDNo.49 and its functional analogue.

On the other hand, the present invention relates to foregoing nucleotide sequence, comprise the carrier that contains these sequences, contain one of aforementioned sequence of coding carrier host cell and contain the transgenosis of with good grounds polynucleotide of the present invention or carrier non--the reorganization purposes of people animal.

Another object of the present invention provides many genes, these genes also not relevant so far with the CRH signalling and therefore can be used for identifying regulate that the CRH signal is replied in the cell the method for compound in, perhaps be used for diagnostic method to identify individuality CRH inductive dysthymia disorders.In one embodiment, the method that evaluation can change the compound that the CRH signal is replied in the cell is included in described compound existence and does not have the expression level that down described cell is contacted and determines to contain the polynucleotide of a nucleotide sequence with CRH, and this nucleotide sequence is selected from SEQ ID No.1, SEQ IDNo.3, SEQ ID No.5, SEQ ID No.7, SEQ ID No.9, SEQ ID NO.11, SEQ ID NO 13, SEQ ID No.15, SEQ ID No 17, SEQ ID No 19, SEQID No.21, SEQ ID No.23, SEQ ID No.25, SEQ ID No.27, SEQID No.29, SEQ ID NO.31, SEQ ID No.33, SEQ ID No.35, SEQID No.37, SEQ ID No.39, SEQ ID No.41, SEQ ID No.43, SEQ IDNo.45, SEQ ID No.47 or SEQ ID No.49.In this screening method, use oligonucleotide probe usually in conjunction with aforementioned polynucleotide, preferably use array technique method assessment expression level.Therefore, in specific embodiments, the invention provides and identify that the method for regulating the compound that the CRH signal is replied in the cell, described method are included in described compound existence and with not existing down described cell are contacted with CRH; And determine to have nucleic acid sequence SEQ ID No.1, SEQID No.3, SEQ ID No.5, SEQ ID No.7, SEQ ID No.9, SEQ ID NO.11, SEQ ID NO 13, SEQ ID No.15, SEQ ID No 17, SEQ ID No 19, SEQID No.21, SEQ ID No.23, SEQ ID No.25, SEQ ID No.27, SEQID No.29, SEQ ID NO.31, SEQ ID No.33, S EQ ID No.35, SEQID No.37, SEQ ID No.39, SEQ ID No.41, SEQ ID No.43, SEQ IDNo.45, the expression level of the polynucleotide of SEQ ID No.47 and SEQ ID No.49, wherein the variation of the express spectra of these sequences indication can change the compound that the CRH signalling is replied in the described cell.

The accompanying drawing summary

Table 1: regulate the protein tabulation of CRH signalling

Fig. 1: by the c-fos mRNA level of different time points in the AtT-20 cell that the standardized quantitative RT-PCR assessment of beta-actin mRNA level (being considered as 100%) DMSO, CRH, CRH+R121919 or R121919 are handled.

Fig. 2: the consistency analysis that the stdn microarray data of all time points and processing is used.Square is described different samples, and circle is described gene.Distance between the square is measuring similarity between the sample.The positive association of gene and given sample (i.e. the rise of that gene in this specific sample) causes this gene and sample to be positioned at passing on the total line (common line) of the centre of moment (describing by cruciform).Consistency analysis clearly will be accredited as the main discriminator between the sample time.In addition, can identify that effect that CRH handles is the most outstanding in the time point in early days.

Fig. 3: the thermal map of vicissitudinous gene when describing the CRH processing.By calculated value that the intensity of the intensity of each sample and corresponding time point DMSO sample is divided by.Convert the ratio conversion quality road (color ramp) of these calculating with log2.Like this, the different time of induced expression is selected to become obvious.Handle the gene that shows 2 times of variations after 30 minutes with CRH and be called as " respondent early ", " middle respondent " shows variation after handling 1 to 2 hour, and " late respondent " replied after 2 hours or longer time.

Fig. 4: the general introduction of CRH inductive gene, by approach or the function discussed herein these genes are classified.By calculated value that the intensity of the intensity of each sample and corresponding time point DMSO sample is divided by.Transform the quality road and in thermal map, describe with convert ratio with these calculating of log2.

Fig. 5: CRH inducing in the AtT-20 cell to Rgs2.The calculating of comparing with observed level in the AtT-20 cell before any processing is induced.Shown resulting array data above to Rgs2.Below, shown by quantitative RT-PCR measured Rgs2mRNA level of the same sample that is used for array experiment and the Rgs2mRNA level measured repeated experiments.

Detailed Description Of The Invention

As used herein, term " compound " or " reagent " refer to biological or chemical compound such as simple or complicated organic molecule, peptide, protein or oligonucleotide." test-compound " refers to be used for " compound " or " reagent " of the method according to this invention as used herein, whether regulates CRH signalling activity in order to assess described compound.

CRH makes the variation that described cytogene is transcribed behind the corticotropin releasing hormone receptor activation in " CRH signalling " phalangeal cell as used herein.Thereby it induces CRH specific gene express spectra.Can be at protein or gene---assess changes in mRNA transcription level on the rna level.

" CRH replys activity " refers generally to because cellular exposure causes the variation of detectable cell parameters in CRH as used herein.Detectable cell parameters comprise the variation of membrane potential, the enzymic activity of regulating the enzyme of CRH signalling in the described cell change, according to the variation of protein expression level of the present invention or second messenger such as cGMP, cAMP, Ca ²⁺Or IP ₃The variation of amount.

Term " analogue " or " functional analogue " refer to according to proteinic modified forms of the present invention, have wherein carried out at least one amino-acid substitution and have made described analogue keep the biologic activity identical substantially with the protein of unmodified in external and/or body.

Term " functional analogue " is intended to comprise " fragment ", " variant ", " degeneracy variant ", " analogue " and " homologue " or " chemical derivative " according to polypeptide of the present invention.The useful chemical derivative of polypeptide be know in this area and comprise for example having the covalent modification in reactive organic site contained in the polypeptide of secondary chemical part.The cross-linking reagent of knowing can be used for amino, carboxyl or the reaction of aldehyde residue for example to import affinity labelling such as vitamin H, fluorescence dye, perhaps with conjugation of polypeptides to solid phase surface (for example, producing affine resin).

Polynucleotide or variant polypeptides, used herein as this term, be respectively polynucleotide or the polypeptide different with reference polynucleotide or polypeptide.The variant of polynucleotide can be the variant of natural generation such as the allele variant of natural generation, and perhaps it can be not know it is the variant of natural generation.(1) polynucleotide different in nucleotide sequence with the reference polynucleotide.Thereby the nucleotide sequence that common difference is restricted reference and variant is very similar generally, and is identical in many zones.As following pointed, the change of the nucleotide sequence of variant can be reticent.That is, they may not change the amino acid of this polynucleotide encoding.When change was confined to such reticent variation, variant had coding the polypeptide of the aminoacid sequence identical with reference.Also mention as following, the change of the nucleotide sequence of variant can change the aminoacid sequence of reference polynucleotide encoded polypeptide.This Nucleotide changes and can cause as discussed above amino-acid substitution, adding, disappearance in the reference sequence encoded polypeptides, merges and block.(2) the aminoacid sequence polypeptide different with another reference polypeptide.Usually, thus difference be restricted the peptide sequence of reference and variant very similar generally and, identical in many zones.Variant and reference polypeptide can differ one or more displacements, insertion, disappearance, merge and block in aminoacid sequence, they can present with arbitrary combination.

Term " complementary " or " complementarity " refer to that purine and pyrimidine nucleotide form the ability of double chain acid molecule by hydrogen bonded as used herein.Following base pair is associated by complementarity: guanine and cytosine(Cyt); Adenine and thymine; VITAMIN B4 and uridylic." complementary " refers to aforementioned relational application on the total length of described molecule as used herein, contains basic all base pairs of two single stranded nucleic acid molecules." part complementary " refers to aforementioned relation, thereby wherein one of two single stranded nucleic acid molecules part of being shorter than one of another two molecules on length keeps strand.

Term " conservative substitution " or " conservative amino acid replacement " refer to some amino acid whose substitutability of recognizing based on this area, one or more radical amino acid replacements in parent's protein and the biologic activity that do not influence parent's molecule (are seen, for example, M.Dayhoff, Atlas ofProtein Sequence and Structure, volume 5 augments 3, the 345-352 page or leaf, 1978).

" its fragment " refers to the disclosed herein nucleic acid of sequence or proteinic fragment, sheet or territory, subprovince, thereby described fragment contains 5 or amino acids more, perhaps 10 or a plurality of Nucleotide, these amino acid or Nucleotide are adjacent in parent's protein or nucleic acid molecule.

" function fragment " refers to territory, proteinic isolating subprovince or fragment disclosed herein as used herein, perhaps aminoacid sequence, and they for example contain on the function significantly zone, as the activation site of acceptor.Function fragment can produce by clone technology, perhaps as the natural product of alternative montage mechanism.

Term " homologue " or " homologous " are described the relation between different IPs acid molecule or the aminoacid sequence, and same or similarity interrelates by the part of one or more districts group or location in described molecule or the sequence for wherein said sequence or molecule." isolating nucleic acid compound " refers to arbitrary RNA or dna sequence dna, in any case its analysis or synthetic is different with its natural place on the position.

" nucleic acid probe " or " probe " is the nucleic acid compound of mark as used herein, and itself and another kind of nucleic acid compound are hybridized." nucleic acid probe " refer to with the single-chain nucleic acid sequence of strand target nucleic acid sequence hybridization.Nucleic acid probe can be oligonucleotide or nucleotide polymer." probe " but contain the test section usually, it can be attached to the terminal of this probe or can be in the sequence inside of this probe.

Term " primer " is the nucleic acid fragment as the initial substrate of the enzymatic of for example nucleic acid molecule or synthetic extension.

Term " hybridization " refers to a kind of process as used herein, and wherein single stranded nucleic acid molecule engages by the nucleotide base pairing with complementary strand.

Term " severity " refers to hybridization conditions.High severity is regulated and is unfavorable for non-homogeneous base pairing.Low stringency has adverse effect.For example, can change severity by temperature and salt concn." stringent condition " refers to containing 50% methane amide, 5 * SSC (750mM NaCl, the 75mM Trisodium Citrate), in the solution of 50mM sodium phosphate (pH7.6), 5 * Denhardt solution, 10% T 500 and salmon sperm DNA 20 μ g/ml sex change, that shear in 42 ℃ of following night incubation, about 65 ℃ of following washing nozzles in 0.1 * SSC then.Other suitable hybridization conditions are described in an embodiment.

" lower stringent condition " is included in and contains 6 * SSPE (20X SSPE=3MNaCl; 0.2M NaH ₂PO ₄0.02M EDTA, pH7.4), in the solution of 0.5%SDS, 30% methane amide, 100 μ g/ml salmon sperm sealing DNA 37 ℃ hatch whole night, use 1 * S SPE, 0.1%SDS 50 ℃ of washings then.In addition, in order to realize even lower severity, can be in (for example, the washing after carrying out strict hybridization under 5 * SSC) of high salt concentration more.Notice comprising and/or replacing the change that can realize top condition of alternative closed reagent by being used for suppressing the hybrid experiment background.Typical closed reagent comprises the salmon sperm DNA of Denhardt reagent, bovine lacto transfer technique optimizer, heparin, sex change and passes through the available proprietary preparation of commercial sources.Comprising of special closed reagent because compatibility problem may need to revise above-mentioned hybridization conditions.

Term " fusion rotein " refers in order to obtain the combination function in structural domain or joint (linker) district as used herein, makes up a plurality of protein domains or connects the protein construct that the tagma causes.This molecular cloning that can pass through the nucleotide sequence of this structural domain of coding is realized with the new polynucleotide sequence that produces the desirable fusion rotein of coding.Alternatively, connect two protein by chemical process and produce fusion rotein.

As used herein term " connector area " or " joint design territory " or similarly these descriptive term refer to be used for the polynucleotide or the peptide sequence of the structure of cloning vector or fusion rotein.The function of connector area can comprise cloning site is imported nucleotide sequence, flexible composition or space are produced the zone imports between two protein domains, perhaps produces to be used for the interactional affinity labelling of specific molecular.Connector area can be imported in the fusion rotein that the selection made during polypeptide or polynucleotide sequence make up causes.

Screening method

The present invention relates to be used for identifying the screening method of the compound of regulating thyroliberin-releasing hormone (CRH) inductive dysthymia disorders and anxiety.This method is based on the evaluation as many genes of the downstream conditioning agent of CRH activatory crh receptor.Particularly, the invention provides the method that evaluation can change the compound that the CRH signalling is replied in the cell, described method comprises:

When a) described compound exists or do not exist described cell is contacted with CRH;

B) the definite change of at least a protein on transcriptional level of regulating thyroliberin-releasing hormone (CRH) signalling in the described cell; With

C) compare described proteinic transcriptional level when described compound exists and do not exist; Wherein, the protein of adjusting thyroliberin-releasing hormone (CRH) signalling is selected from SEQ ID NO.2, SEQ ID 4, SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.10, SEQ ID NO.12, SEQ ID NO.14, SEQ ID NO.16, SEQ ID NO.18, SEQ ID NO.20, SEQ ID NO.22, SEQ ID NO.24, SEQ ID NO.26, SEQ ID NO.28, SEQ ID NO.30, SEQ ID NO.32, SEQ ID NO.34, SEQ ID NO.36, SEQ ID NO.38, SEQ ID NO.40, SEQ ID NO.42, SEQ ID NO.44, SEQ ID NO.46 and SEQ ID NO.48.

For the variation of determining to transcribe on the protein level, can use techniques well known in the art to determine described proteinic amount.For example, use isolation technique such as isoelectrofocusing or SDS-PAGE associating protein staining technology such as coomassie or silver to dye.Alternatively, then to the protein of enzyme, the catalytic effect that can produce according to this enzyme, promptly the amount in given solution or the tissue extract is measured or analyzed to its substrate to the conversion of reaction product.For example, for kinases, can use the substrate that contains the special phosphorylation site of kinases and by measuring because the phosphorylation of this substrate that radioactivity phosphoric acid causes to mixing of substrate is assessed kinase activity.Can when existing or do not exist, implement test-compound this assay method.For the protein that is not enzyme, need other quantivative approachs.For example, translocator can be measured by the combining of molecule that they and they are transported, and hormone and toxin can be measured by the biological effect that they produce.

Be the variation of transcribing on the assessment gene level, RNA or eDNA can directly be used or can be by using PCR or other amplification technique enzymatic amplifications before analysis.Preferably, described analytical procedure comprises the oligonucleotide probe of applying marking, and this probe target is decided the suitable zone of gene.

Therefore, in preferred embodiments, use is in conjunction with the probe assessment gene transcription level of the polynucleotide of encoding amino acid sequence, and this aminoacid sequence is selected from SEQ ID NO.2, SEQ ID4, SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.10, SEQ ID NO.12, SEQ ID NO.14, SEQ ID NO.16, SEQ ID NO.18, SEQ ID NO.20, SEQ ID NO.22, SEQ ID NO.24, SEQ ID NO.26, SEQ ID NO.28, SEQ ID NO.30, SEQ ID NO.32, SEQ ID NO.34, SEQ ID NO.36, SEQ ID NO.38, SEQ ID NO.40, SEQ ID NO.42, SEQ ID NO.44, SEQ ID NO.46 and SEQ ID NO.48.

In another embodiment, can make up the proteic nucleotide sequence that contains coding and regulating CRH signalling or its segmental oligonucleotide probe array to implement effective screening of genetic expression.Array technique be know and have general applicability and can be used for handling variety of issue in the molecular genetics, comprise genetic expression, genetic linkage, and hereditary variability (is for example seen: people such as M.Chee, Science, volume 274,610-613 page or leaf (1996)).Thereby an object of the present invention is to provide the method that evaluation can change the compound that the CRH signalling is replied in the cell, described method comprises: exist when not existing at test-compound described cell is contacted with CRH; Has aminoacid sequence SEQ ID NO.2 with using in conjunction with coding, SEQ ID 4, SEQID NO.6, SEQ ID NO.8, SEQ ID NO.10, SEQ ID NO.12, SEQ IDNO.14, SEQ ID NO.16, SEQ ID NO.18, SEQ ID NO.20, SEQ IDNO.22, SEQ ID NO.24, SEQ ID NO.26, SEQ ID NO.28, SEQ ID NO.30, SEQ ID NO.32, SEQ ID NO.34, SEQ ID NO.36, SEQ ID NO.38, SEQ ID NO.40, SEQ ID NO.42, SEQ ID NO.44, the oligonucleotide probe array of the polynucleotide of the polypeptide group of SEQ ID NO.46 and SEQ ID NO.48 is determined the level of genetic transcription.

In alternate embodiment, the method that evaluation can change the compound that the CRH signalling is replied in the cell comprises:

A) exist when not existing at described compound described cell is contacted with CRH; With

B) determine to contain the expression level of the polynucleotide of nucleotide sequence, this nucleotide sequence is selected from SEQ ID No.1, SEQ ID No.3, SEQ ID No.5, SEQ ID No.7, SEQ IDNo.9, S EQ ID NO.11, SEQ ID NO 13, SEQ ID No.15, SEQ ID No 17, SEQ ID No 19, SEQ ID No.21, SEQ ID No.23, SEQ ID No.25, SEQ ID No.27, SEQ ID No.29, SEQ ID NO.31, SEQ ID No.33, SEQ ID No.35, SEQ ID No.37, SEQ ID No.39, SEQ ID No.41, SEQ ID No.43, SEQ ID No.45, SEQ ID No.47 or SEQ ID No.49.

In order to assess the change of expression level, RNA or cDNA can directly be used or can be by using PCR or other amplification technique enzymatic amplifications before analysis.Preferably, described analytical procedure comprises the oligonucleotide probe of applying marking, the suitable zone of fixed these polynucleotide of this probe target.Therefore, in preferred embodiments, use the probe assessment gene transcription level in conjunction with the polynucleotide that contain nucleotide sequence, this nucleotide sequence is selected from SEQ ID No.1, SEQ ID No.3, SEQ ID No.5, SEQ ID No.7, SEQ ID No.9, SEQ ID NO.11, SEQ ID NO 13, SEQ ID No.15, SEQ ID No 17, SEQ ID No 19, SEQID No.21, SEQ ID No.23, SEQ ID No.25, SEQ ID No.27, SEQID No.29, SEQ ID NO.31, SEQ ID No.33, SEQ ID No.35, SEQID No.37, SEQ ID No.39, SEQ ID No.41, SEQ ID No.43, SEQ IDNo.45, SEQ ID No.47 and SEQ ID No.49.

In another embodiment, can make up proteinic Nucleotide or its segmental oligonucleotide probe array that display contains coding and regulating CRH signalling expresses with effective screening-gene.In this embodiment, the invention provides the method that evaluation can change the compound that the CRH signalling is replied in the cell, described method is included in described compound and described cell is contacted with CRH when existing or not existing; Determine to have nucleic acid sequence SEQ ID No.1, SEQ ID No.3, SEQ ID No.5, SEQ ID No.7, SEQ ID No.9, SEQ ID NO.11, SEQID NO 13, SEQ ID No.15, SEQ ID No 17, SEQ ID No 19, SEQ ID No.21, SEQ ID No.23, SEQ ID No.25, SEQ ID No.27, SEQ ID No.29, SEQ ID NO.31, SEQ ID No.33, SEQ ID No.35, SEQ ID No.37, SEQ ID No.39, SEQ ID No.41, SEQ ID No.43, SEQ ID No.45, the expression level of the polynucleotide of SEQ ID No.47 or SEQ ID No.49.Particularly, use in conjunction with having nucleic acid sequence SEQ ID No.1, SEQ ID No.3, SEQ ID No.5, SEQ ID No.7, SEQ ID No.9, SEQ ID NO.11, SEQ ID NO 13, SEQID No.15, SEQ ID No 17, SEQ ID No 19, SEQ ID No.21, SEQ IDNo.23, SEQ ID No.25, SEQ ID No.27, SEQ ID No.29, SEQ IDNO.31, SEQ ID No.33, SEQ ID No.35, SEQ ID No.37, SEQ IDNo.39, SEQ ID No.41, SEQ ID No.43, SEQ ID No.45, the oligonucleotide probe array of the polynucleotide of SEQ ID No.47 and SEQ ID No.49.

In another embodiment, oligonucleotide probe array contains proteinic nucleotide sequence or its fragment of coding and regulating CRH signalling, and the array that can make up this oligonucleotide probe is used to implement effective screening of genetic expression.Array technique be know and have general applicability and can be used for handling variety of issue in the molecular genetics, comprise genetic expression, genetic linkage, and hereditary variability (is for example seen: people such as M.Chee, Science, volume 274,610-613 page or leaf (1996)).

Situation about directly not used for genomic dna can separating mRNA, and it is synthetic to implement article one chain c DNA.Can implement DNA synthetic second takes turns to produce the second chain.Subsequently, by the specific PCR amplification, can obtain isolating cDNA.If wish that double-stranded cDNA can be cloned into arbitrary suitable carrier, for example, plasmid, thus the cDNA library formed.Be similar to top method, can regulate the cDNA library that the oligonucleotide probe screening of mark in arbitrary suitable zone of the proteinic gene of CRH signalling makes up in phage or plasmid shuttle vectors with the target sign indicating number of delimiting the organizational structure.For example see, PCR Protocols:A Guide to Method and Application, people such as editor M.Innis, Academic Press (1990).

The construction cDNA library is well known to those skilled in the art with the method for breeding in protokaryon or eukaryotic cell in suitable carrier such as plasmid or phage.[seeing for example people such as Maniatis ,] as preceding.Suitable cloning vector be know and can generally obtain.

In another embodiment, determine the variation of genetic transcription in the mRNA level.Can use to be used for one of method of knowing the quantitative this area of polynucleotide and to measure the expression that reduces or increase at rna level, these methods are, for example, and nucleic acid amplification, for example, by PCR, RT-PCR; The RNA enzyme protection; Northern trace and other hybridizing methods.Can be used for determining protein, is well known to those skilled in the art as the determination techniques from the level of polypeptide of the present invention in host's the sample.These measuring methods comprise radioimmunoassay, competition-binding assay, western blot analysis and ELISA assay method.The determination techniques that can be used for the existence of definite protein derivatives or variant comprises mass spectroscopy.

Thereby, an object of the present invention is to provide the method that evaluation can change the compound that the CRH signalling is replied in the cell, described method comprises:

B) definite at least a proteinic amount of regulating thyroliberin-releasing hormone (CRH) signalling in the described cell; With

C) compare described proteinic amount when described compound exists and do not exist; Wherein, the protein of adjusting thyroliberin-releasing hormone (CRH) signalling is selected from SEQ IDNO.2, SEQ ID 4, SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.10, SEQ ID NO.12, SEQ ID NO.14, SEQ ID NO.16, SEQ ID NO.18, SEQ ID NO.20, SEQ ID NO.22, SEQ ID NO.24, SEQ ID NO.26, SEQ ID NO.28, SEQ ID NO.30, SEQ ID NO.32, SEQ ID NO.34, SEQ ID NO.36, SEQ ID NO.38, SEQ ID NO.40, SEQ ID NO.42, SEQ ID NO.44, SEQ ID NO.46 and SEQ ID NO.48.

Preferably, the method for the proteinic amount of mensuration adjusting CRG signalling is to use in conjunction with containing and is selected from SEQ ID NO.2, SEQ ID 4, SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.10, SEQ ID NO.12, SEQ ID NO.14, SEQ ID NO.16, SEQ ID NO.18, SEQ ID NO.20, SEQ ID NO.22, SEQ ID NO.24, SEQ ID NO.26, SEQ ID NO.28, SEQ ID NO.30, SEQ ID NO.32, SEQ ID NO.34, SEQ ID NO.36, SEQ ID NO.38, SEQ ID NO.40, SEQ ID NO.42, SEQ ID NO.44, the antibody of the polypeptide of the aminoacid sequence of SEQ ID NO.46 and SEQ ID NO.48.

Thereby, in another embodiment, the protein that the invention provides and regulate the CRG signalling has immunoreactive single specific antibody, and described protein is selected from SEQ ID NO.2, SEQ ID 4, SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.10, SEQ IDNO.12, SEQ ID NO.14, SEQ ID NO.16, SEQ ID NO.18, SEQ IDNO.20, SEQ ID NO.22, SEQ ID NO.24, SEQ ID NO.26, SEQ ID NO.28, SEQ ID NO.30, SEQ ID NO.32, SEQ ID NO.34, SEQ ID NO.36, SEQ ID NO.38, SEQ ID NO.40, SEQ ID NO.42, SEQ ID NO.44, SEQ ID NO.46 and SEQ ID NO.48.Use conventional scheme, preferably non--the people animal by polypeptide of the present invention or the cell that contains fragment, the analogue of epi-position or express these are applied to animal, the antibody at polypeptide of the present invention that can obtain being produced.For Monoclonal Antibody, can use the arbitrary technology of cultivating the antibody that produces by continuous cell line that provides.Example comprises hybridoma technology (Kohler, G. and Milstein, C., Nature (1975) 256:495-497), trioma technology, people B-quadroma technology (people such as Kozbor, Immunology Today (1983) 4:72) and EBV-hybridoma technology (people such as Cole, MONOCLONAL ANTIBODIES AND CANCER THERAP Y, the 77-96 page or leaf, Alan R.Liss, Inc., 1985).

Produce single-chain antibody, as U.S. Patent number 4,946, the technology of those single-chain antibodies described in 778 is also applicable to the single-chain antibody that produces at polypeptide of the present invention.And, transgenic mice, perhaps other biological comprises other Mammalss, also can be used to express humanized antibody.

Above-mentioned antibody can be used for separating or identifies the clone who expresses this polypeptide or pass through these polypeptide of affinitive layer purification.

Antibody at polypeptide of the present invention also can be used for treating CRH metabolism relevant imbalance such as CRH inductive anxiety or dysthymia disorders.

In order to determine to regulate the proteinic amount of CRH signalling, antibody according to the present invention is used to routine immunization and learns a skill.Suitable immunological technique is well known to those skilled in the art and for example comprises, ELISA, western blot analysis, competitiveness or sandwich immunoassay etc., as is well known, they all depend on the formation of Ag-Ab immunocomplex, wherein for for the purpose of the assay method, antibody can be by for example, and radiation, enzyme or fluorescent marker can detect ground mark, perhaps can be fixed on insoluble carrier.

For example, in a kind of ELISA screening method, antibody is added to solid phase (for example, the bottom of microtiter plate), this solid phase is coupled to protein or its fragment bag quilt of carrier (as BSA), add then and put together detectable label such as enzyme, preferred horseradish peroxidase, perhaps radio isotope such as 125I's is anti--and immune globulin antibody is (for example, when in mouse, carrying out immunity, use anti--mouse immuning ball protein antibody, for example, sheep-anti--mouse immuning ball protein (Ig)).

Thereby an object of the present invention is to provide the proteinic immunoassay of determining or detecting CRH signalling in the adjusting sample, this method comprises sample is contacted and determine whether form immunocomplex between this antibody and described protein with protein according to the present invention.Can implement these methods and they to tissue sample or humoral sample generally includes from experimenter's health and obtains sample; Described sample is contacted with imaging significant quantity according to the antibody of detectable label of the present invention; And certification mark is to identify that the proteinic of CRH signalling exists in the adjusting sample.

Use the measuring method of antibody of the present invention specifically not limited.Can use arbitrary measuring method,, and calculate from the typical curve that contains the antigenic standard solution preparation of known quantity by use as long as can be detected by chemistry or physical method corresponding to the amount of antigenic antibody, antigen or antigen-antibody complexes.For example, can use tuurbidimetry, competition law, immunizing dose method and sandwich method aptly.For susceptibility and specificity, the especially preferred sandwich method that describes below of using.

In the measuring method of applying marking material, radio isotope, enzyme, fluorescent substance, luminophore etc. are used as marking agent.Radioisotopic example comprises ¹²⁵I, ¹³¹I, ³H and ¹²C.Enzyme is puted together suitable substrate usually, and this substrate is the detectable reactant of catalysis and make that this enzyme can be detected conversely again.The example of this substrate comprises, for example, and beta-galactosidase enzymes, beta-glucosidase enzyme, alkaline phosphatase, peroxidase and malate dehydrogenase (malic acid dehydrogenase), preferred horseradish peroxidase.Luminophore comprises, for example, and luminol,3-aminophthalic acid cyclic hydrazide, luminol,3-aminophthalic acid cyclic hydrazide derivative, luciferin, aequorin and luciferase.In addition, avidin-biotin system also can be used for traget antibody and immunogen of the present invention.

Therefore, on the other hand, the invention provides evaluation and can change the method that active compound is replied in CRH signalling in the cell, this method comprises:

A) expression is contained be selected from SEQ ID NO.2, SEQ ID 4, SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.10, SEQ ID NO.12, SEQ ID NO.14, SEQ ID NO.16, SEQ ID NO.18, SEQ ID NO.20, SEQ ID NO.22, SEQ ID NO.24, SEQ ID NO.26, SEQ ID NO.28, SEQ ID NO.30, SEQ ID NO.32, SEQ ID NO.34, SEQ ID NO.36, SEQ ID NO.38, SEQ ID NO.40, SEQ ID NO.42, SEQ ID NO.44, at least a proteinic cell of SEQ ID NO.46 and the aminoacid sequence of SEQ ID NO.48 contacts with described test-compound; With

When b) more described compound exists and does not exist in the described cell CRH reply activity.

Use conventional electrophysiology technology and when they can obtain, use the variation of the current new high throughput method measurement membrane potential of developing.Because the change of membrane potential is the result of ionic flux normally, as alternative approach, use the fluorescence dye of ion-sensitive, comprise fluo-3, fluo-4, fluo-5N, fura red, Sodium Green, SBFI and from other similar probes of the supplier who comprises Molecular Probes, the variation that can measure membrane potential by the variation of intracellular ion concentration indirectly.Other fluorescence dyes such as DIBAC from the supplier who comprises Molecular Probes _{4 (3)}Or Di-4-Anepps can detect the membrane potential variation.For example, use fluorescence measurement and fluorescence imaging technology, comprise fluorescent microscopy, with or without laser co-focusing method combination image analytical algorithm, can real-time characterization calcium and sodium ion flux.

In preferred embodiments, this assay method is based on the instrument that is called fluorometric imaging plate reader ((FLIPR), Molecular Devices Corporation).In its most common configuration, this instrument excites and measures the fluorescence that the dyestuff based on luciferin sends.It uses argon laser to produce high-energy at the 488nm place of fluorophore and excites, and catches the fluorescence that sends with the bottom of optical system rapid scanning 96-/384-orifice plate and with the cold CCD camera of sensitive.It comprises that also 96-/384-hole moves the liquid head to allow this instrument delivery of agents solution in the hole of 96-/384-orifice plate.The FLIPR assay method be designed to simultaneously from all 96-/384-hole measure in real time compound add before, among and afterwards from the fluorescent signal of cell colony.

Be used for screening and identifying the FLI PR assay method of replying the compound with functionally active at adjusting cell CRH thereby an object of the present invention is to provide, described cell expressing is selected from the albumen of SEQ IDNO.26, SEQ ID NO.28, SEQ ID NO.30, SEQ ID NO.32 and SEQ IDNO.34.

In alternative embodiment, can use electrophysiological method assessment cell activity.Therefore, can use full cell and single passage electrophysiologicalidentification identification to regulate the protein of CRH signalling in the cell.

Thereby another object of the present invention provides identify to regulate the screening method that active compound is replied in CRH signalling in the cell, and described method comprises:

A) expression is selected from SEQ ID NO.2, SEQ ID 4, SEQ ID NO.6, SEQ IDNO.8, SEQ ID NO.10, SEQ ID NO.12, SEQ ID NO.14, SEQ ID NO.16, SEQ ID NO.18, SEQ ID NO.20, SEQ ID NO.22, SEQ ID NO.24, SEQ ID NO.26, SEQ ID NO.28, SEQ ID NO.30, SEQ ID NO.32, SEQ ID NO.34, SEQ ID NO.36, SEQ ID NO.38, SEQ ID NO.40, SEQ ID NO.42, SEQ ID NO.44, SEQ ID NO.46 contacts with test-compound with the proteinic host cell of SEQ ID NO.48;

B) use electrophysiological technique to detect the influence of test-compound to described cell membrane potential; With

When c) more described compound exists and does not exist in the described cell CRH reply activity.Alternatively, the host cell expression in the aforementioned screening method is selected from the protein of SEQ ID NO.26, SEQ IDNO.28, SEQ ID NO.30, SEQ ID NO.32 and SEQ ID NO.34.

In preferred embodiments, host cell is that Xenopus laevis (Xenopus) ovocyte and electrophysiology measurement are made up of in different membrane potential measurement membrane currents the working voltage tongs technology.

The variation of regulating the enzymic activity of the enzyme of CRH signalling in the described cell generally can be according to the catalytic effect of enzyme generation, and promptly its substrate is measured or analyzed to being converted of reaction product.For example, for kinases, can use the substrate that contains the special phosphorylation site of this kinases also by measuring this kinase whose activity of phosphorylation assessment of this substrate.For Phosphoric acid esterase, can use phosphorylated substrate and assess phosphatase activity similarly by the dephosphorylation of measuring this substrate.

Can when existing and do not exist, implement test-compound these mensuration.

Can change the method that active compound is replied in CRH signalling in the cell thereby an object of the present invention is to provide evaluation, described method comprises:

A) will comprise that the kinase whose mixture that is selected from SEQ ID NO.10, SEQ ID 12, SEQ ID NO.14, SEQ ID NO.16 and SEQ ID NO.18 and source of phosphoric acid contact with suitable kinase substrate;

B) when existing or do not exist, described compound hatches described mixture; With

The phosphorylation level of described substrate when the phosphorylation level of described substrate was compared described compound and existed when detection did not exist with described test-compound.

In assay method of the present invention, kinases can be used as that protein provides or it can be used as the described kinase whose mRNA of coding and provides in measuring mixture.When this assay method comprised acellular component, this kinases provided with protein.When this assay method was implemented in cellular environment, this kinases can be used as protein or provides as the described kinase whose mRNA of coding, and wherein, for this kinases can be utilized in this assay method, thereby this mRNA is translated the generation kinase protein.The embodiment that provides from here obtains this kinase whose mRNA clearly and is expelled in the cell this mRNA to produce kinase protein is simple thing.Can also provide kinases by the expression of plasmid of coding kinase protein.Can make up the plasmid operated of coding kinase protein and expression plasmid (Sambrook in cell with standard molecular biological technique, Deng the people, 1989, MolecularCloning:A Laboratory Manual, Cold Spring Harbor Laboratory, New York).

As discussed here, can external (wherein measure mixture acellular or wherein viable cell be included in the assay method), perhaps implement to identify the method for kinase modulator in the inherent animal of body.Thereby, in one aspect of the invention, mixture is comprised in the eukaryotic cell and can implements method of the present invention, and some components of wherein measuring mixture can offer cell by the outer seedbed of the cytotropic microinjection of these components, and some components can be that cell is endogenous.

Term " cell is endogenous " refers to that this component produces natively in subject cell as used herein.

Term " the cell external source " refers to that this component finds natively in subject cell as used herein, perhaps in cell with low-level discovery be added in the cell.

When using eukaryotic cell to implement the inventive method, one or more kinase proteins, kinase substrate and test-compound can be expelled in the eukaryotic cell before eukaryotic cell is hatched.So the cell of injection is hatched under the active condition of promotion protein kinase then, after incubation period, uses assay method described herein to measure the protein kinase activity level subsequently.

The eukaryotic cell that is used for the inventive method can be African Xenopus laevis (Xenopus laevis) ovocyte, Africa xenopus protoblast, mammalian cell (as the IOTI/2 cell), drosophila melanogaster (Drosophila melanogaster) S2 cell, Dictyostefium discoideum cell and yeast cell.More preferably, eukaryotic cell is that the adenoma cell in mouse PATH cell-source is one of cell AtT-20.

The source of phosphoric acid that is used for the inventive method can be the arbitrary common source of phosphoric acid, include, but not limited to the Nucleotide triphosphoric acid as, but be not limited to ATP or GTP.In preferred embodiments, in conjunction with detectable mark, during reaction this mark is transferred to kinase substrate with phosphate group on the source of phosphoric acid.Like this, the phosphorylating kinase substrate can be distinguished with non--phosphorylating kinase substrate, and phosphorylated substrate will not contain underlined because phosphorylated substrate will contain detectable mark.In another embodiment, debond detectable label on the source of phosphoric acid; But, for example can not discern another kind of form by a kind of form of antibody recognition substrate, phosphorylating kinase substrate and non--phosphorylating kinase substrate can be distinguished.

The detectable label that is used for method of the present invention can comprise arbitrary known or unknown so far detectable label, and this mark is because the active result of protein kinase transfers to kinase substrate when phosphate groups shifts.The available mark includes, but are not limited to, and radio-labeling is as γ ³²P, ³¹S and nonradioactive labeling are as vitamin H etc.

In another embodiment, the invention provides evaluation and can change the method that active compound is replied in the interior CRH signalling of cell, described method comprises:

A) will contain the mixture that is selected from SEQ ID NO.36 and the Phosphoric acid esterase of SEQ ID NO.38 contacts with the phosphorylated substrate that suits;

The phosphorylation level of described substrate was not compared when detection did not exist with described test-compound, the phosphorylation level of described substrate when described compound exists.

For kinase assay, the Phosphoric acid esterase in the assay method of the present invention can provide in measuring mixture with the mRNA that protein provides or it can be used as the described Phosphoric acid esterase of coding.Usually with detectable phosphoric acid residue marker phosphorylated substrate.The available marker includes, but not limited to radio-labeling, as γ ³²P, ³¹S and nonradioactive labeling are as vitamin H etc.For being used for the phosphatase activity assay method, substrate preferably by at tyrosine or serine residue by phosphorylation, typically use γ ³²The peptide substrates of P mark is formed.Usually, can finish phosphorylation with the whole bag of tricks.Typically, use protein tyrosine kinase.For example, can use the associating of soluble EGF-receptor kinase, sup, ³²Tyrosine residues on the ATP phosphorylation peptide of the present invention of P mark.This phosphorylation reaction usually allows to continue about 2 hours or in ambient temperature overnight at 30 ℃.

From phosphorylation reaction purifying mixture phosphorylated peptide, it is called as " phospho-peptide " hereinafter then.For example, also centrifugal by adding trichoroacetic acid(TCA), thus peptide is retained in the supernatant liquor, and can be from the reaction mixture isolated peptides.General by column chromatography, for example on C18, be further purified peptide.The phosphorylated peptide of purifying can be by freeze-drying and standby-20 ℃ of preservations.

After hatching, the radioactivity that the dephosphorylation by phospho-peptide (i.e. the free radioactivity phosphorus that discharges from dephosphorylation) discharges is separated does not have dephosphorylized phospho-peptide (" not-dephosphorylized phospho-peptide ").As used herein, " " comprise form of ownership, wherein the radiophosphorus atom can be present on the tyrosine residues and can pass through dephosphorylation radiophosphorus term, for example, is removed as phosphate group.Usually, by centrifugal, comprise that by adding the material of on-radiation phosphoric acid and gac stops the dephosphorylation reaction and realizes not-the separating of free radiophosphorus that dephosphorylized phospho-peptide and the dephosphorylation of phospho-peptide discharge then.The method of knowing by those of ordinary skills is determined the radioactivity of supernatant liquor.Based on adding the radioactive amount of measuring mixture and the radioactive amount that detects at the end of assay method as the radioactivity that dephosphorylation discharges by phospho-peptide at first, can calculate the Phosphoric acid esterase enzymatic activity of the sample of being analyzed.

One embodiment of the invention provide evaluation can change the method that active compound is replied in CRH signalling in the cell, and described method comprises:

A) expression is comprised at least a proteinic cell that is selected from SEQ ID NO.26, SEQ ID NO.28, SEQID NO.30, SEQ ID NO.32 and SEQ ID NO.34 contacts with described test-compound; With

Second messenger in described cell when b) more described compound exists and do not exist is as cAMP, cGMP, Ca ²⁺Or IP ₃Level.

Use technology well known in the art to determine to contain the level of second messenger in the full cell of one of aforementioned protein or the cell extract.

Evaluation can change CRH signalling in the cell and reply the use that the other method of active compound is based on gene, as reporter gene, its be operably connected gene promoter or its adjusting sequential element, it is characterized in that described gene promoter or regulate sequential element containing the transcription factor binding site point, wherein said transcription factor can be regulated CRH signalling in the cell.In preferred embodiments, the transcription factor that can regulate CRH signalling in the cell is selected from SEQ IDNO.2, SEQ ID 4, SEQ ID NO.6 and SEQ ID NO.8.Therefore, the invention provides the recombinant DNA molecules that contains as gene promoter area defined above.In described recombinant DNA molecules, promoter region be operably connected the coding detectable product nucleic acid molecule, as reporter gene.As used herein, term " is operably connected " and refers to by suitable frame gene and promoter function fusion are in the gene of this promotor under controlling with expression.As used herein, term " reporter gene " refers to a kind of gene, and the gene product of its coding can be operatively attached to promoter region or its active fragments with simple, cheap method evaluation and this gene.Reporter gene as, for example, Lampyridea luciferase, beta-galactosidase enzymes, alkaline phosphatase, bacterium chloramphenicol acetyltransferase or green fluorescent protein reporter gene can be used for determining (to see according to the transcriptional activity in the screening assay method of the present invention, for example, Goeddel (writing), Methods Enzymol., volume 185, San Diego:Academic Press, Inc. (1990); Also see Sambrook, as preceding).In preferred embodiments, reporter gene is the Lampyridea luciferase genes.The present invention also provides and has contained the carrier of recombinant DNA molecules as defined above, and uses described carrier, perhaps generally uses the host cell according to recombinant DNA molecules stable conversion of the present invention.Term " carrier " refers to be used for DNA is transferred to host cell to duplicate by host cell and/or suitable launch vehicle of expressing any foreign DNA of foreign DNA.Therefore, in specific embodiments, described carrier is an expression vector, as pGL31uc, pBLCAT5 (LMBP 2451), pGMCSFlacZ (LMBP 2979), pEGFP or pSEAPbasic (DMB 3115), wherein refer to the preserving number of these expression vectors at Belgian microbial preservation center (BelgianCo-ordinated Collections of Microorganisms) for LMBP and DMB number.

On the other hand, the invention provides the method for identify regulating the active compound of CRH signalling, described method comprises step: (i) with candidate agent with contact as gene promoter area defined above; Determine (ii) whether described candidate reagent regulates the expression that can detect product, this adjusting indication can be regulated the active reagent of CRH signalling.Detectable product refers to the albumen of this genes encoding or product such as luciferase, beta-galactosidase enzymes or the green fluorescent protein of reporter gene.The method that quantitatively can detect product is as known in the art and comprises if expression product is enzyme then uses colorimetric substrates, perhaps in RIA or ELISA assay method, use specific antibody or measure, wherein can use standard method directly to measure described mRNA or measure described mRNA indirectly from the level of the mRNA of the genetic transcription that is operably connected to promotor.Preferably, gene promoter comprises the transcription factor binding site point, and described transcription factor is selected from SEQ Ib No.2, SEQ Ib No.4, SEQ Ib No.6 and SEQ Ib No.8.

Coding can be regulated the evaluation of the proteinic nucleic acid of CRH signalling

On the other hand, the present invention relates to separate the also nucleic acid molecule of purifying, their codings can be regulated the protein of CRH signalling, and wherein said nucleic acid molecule is RNA, DNA, cDNA or genomic dna.

Particularly, the present invention includes and separate and the nucleic acid molecule of purifying, they contain and are selected from a member who organizes below:

(a) the proteinic nucleic acid molecule of coding and regulating CRH signalling, this protein has at least 70% identity with the polypeptide that contains the aminoacid sequence that is selected from SEQ ID No.46 and SEQ ID No.48;

(b) with (a) polynucleotide complementary nucleic acid molecule;

(c) nucleic acid molecule that contains at least 15 continuous bases of (a) or polynucleotide (b);

(d) under stringent condition with (a) or the nucleic acid molecule of polynucleotide molecule (b) hybridization; With

(e) the proteinic nucleic acid molecule of coding and regulating CRH signalling, this nucleic acid molecule contain because (a) nucleotide sequence that produces to (d) any degeneracy of genetic code of nucleotide sequence of polynucleotide.

Those skilled in the art will recognize that owing to the genetic code degeneracy right many " silence " displacement of nucleotide base can be imported into the identity that is not changed coded amino acid or protein by the sequence of SEQ ID NO:45, SEQ ID NO 47 or SEQ IDNO:49 evaluation.All these displacements all are intended to be located within the scope of the present invention.

On the other hand, the present invention relates to people's purine permease polynucleotide.These polynucleotide comprise isolating polynucleotide, it contains the nucleotide sequence of coded polypeptide, this polypeptide has at least 70% identity with the aminoacid sequence that is selected from SEQID NO:46 and S EQ ID NO:48 on the total length of this aminoacid sequence, preferred at least 80% identity, more preferably at least 90% identity, more preferably at least 95% identity.On this point, highly preferably have the polypeptide of at least 97% identity, more highly preferably have the polypeptide of 98-99% identity at least, topnotch preferably has the polypeptide of at least 99% identity.These polynucleotide comprise basically and being made up of the polynucleotide sequence that is selected from SEQ ID No.45, SEQ ID No.47 or SEQ ID No.49.

Therefore, on the other hand, the invention provides isolating polynucleotide, it contains:

A) encode and the aminoacid sequence that is selected from SEQ ID NO:46 and SEQ ID NO:48 has at least 70% identity, preferred at least 80% identity, more preferably at least 90% identity, more preferably at least 95% identity, even the more preferably nucleotide sequence of the polypeptide of 97-99% identity at least;

B) on the total length of described polynucleotide, has at least 70% identity with the polynucleotide that are selected from SEQ ID No.45, SEQ ID No.47 and SEQ ID No.49, preferred at least 80% identity, more preferably at least 90% identity, more preferably at least 95% identity, even the more preferably nucleotide sequence of 97-99% identity at least;

C) on whole coding regions of described polynucleotide, has at least 70% identity with the polynucleotide that are selected from SEQ ID No.45, SEQ ID No.47 and SEQ ID No.49, preferred at least 80% identity, more preferably at least 90% identity, more preferably at least 95% identity, even the more preferably nucleotide sequence of 97-99% identity at least; With

D) nucleotide sequence of forming by the polynucleotide that are selected from SEQ ID No.45, SEQ ID No.47 and SEQ ID No.49.

Specifically provide the polynucleotide of summarizing above to be used for according to screening method of the present invention or diagnostic method.Be particularly useful for identifying the compound that to regulate CRH signalling in the cell or diagnose the CRH metabolism that changes in the individuality.

Identity or similarity as known in the art, are as the two or more peptide sequences determined by comparative sequences or the relation between two or more polynucleotide sequence.In the art, identity also refers to the serial correlation degree between polypeptide or the polynucleotide sequence, can be as determining by the coupling between these sequence strings.Can easily calculate identity or similarity (Computational Molecular Biology, Lesk, A.M., editor, OxfordUniversity Press, New York, 1988; Biocomputing:Informaticsand Genome Projects, Smith, D.W., editor, Academic Press, NewYork, 1993; Computer Analysis of Sequence Data, PartI, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey, 1994; Sequence Analysisin Molecular Biology, von Heinje, G., Academic Press, 1987; With Sequence Analysis Primer, Gribskov, M. and Devereux, J., editor, M Stockton Press, New York, 1991).Although being arranged, many methods can measure identity or similarity between two kinds of polynucleotide or the two peptide species sequences, but two kinds of terms all are (Sequence Analysis inMolecular Biology, von Heinje, the G. that the technician knows, Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J., editor, M Stockton Press, New York, 1991; And Carillo, H., and Lipman, D., (1988) SIAM J.Applied Math., 48,1073).Be generally used for determining that the identity between the sequence or the method for similarity include, but are not limited to Carillo, H., and Lipman, D., (1988) SIAM J.Applied Math., those disclosed method in 48,1073.Maximum match between the sequence that the preferred method of the definite identity of design is tested to provide.The method of identity or similarity of determining is encoded in computer program.Determine that the preferred computer program means of identity and similarity comprises between two sequences, but be not limited to GCG routine package (Devereux, J., Deng the people, (1984) Nucleic Acids Research12 (1), 387), BLASTP, BLASTN and FASTA (Atschul, people such as S.F., (1990) J.Molec.Biol.215,403).

The tissue that can be expressed from described gene, as but be not limited to, brain, heart, kidney, pancreas, liver and skin separate coding can regulate the active nucleic acid sequences to proteins of CRH or its fragment.Also can separate described sequence with Mammals outside the mouse from the people.Other cells also can suit in order to separate Mammals purine permease cDNA with clone.As described herein, by screening the selection of the cell that the CRH activity can suit in cell extract or the full raji cell assay Raji.Having CRH in any of these assay methods regulates active cell and can be suitable for separating penetrating enzyme dna of purine or mRNA.

Any of the whole bag of tricks as known in the art molecular cloning that can be used for encoding according to protein DNA of the present invention.In one approach, mRNA is separated, and it is synthetic to implement article one chain cDNA.Can implementing second, to take turns DNA synthetic to produce the second chain.By aminoacid sequence design degeneracy oligonucleotide primer dna fragmentation is carried out the specific PCR amplification subsequently, can obtain isolating cDNA from the purifying protein of regulating the CRH signalling.If wish, double-stranded cDNA can be cloned into arbitrary suitable carrier, for example, and plasmid, thus the cDNA library formed.Another kind method is that screening is decided SEQ ID No.1 with target, SEQ ID No.3, SEQ ID No.5, SEQ ID No.7, SEQ ID No.9, SEQ ID NO.11, SEQ ID NO 13, SEQ ID No.15, SEQ ID No 17, SEQ ID No 19, SEQ ID No.21, SEQID No.23, SEQ ID No.25, SEQ ID No.27, SEQ ID No.29, SEQID NO.31, SEQ ID No.33, SEQ ID No.35, SEQ ID No.37, SEQID No.39, SEQ ID No.41, SEQ ID No.43, SEQ ID No.45, the cDNA library that the oligonucleotide probe of the mark in arbitrary suitable zone of SEQ IDNo.47 or SEQ ID No.49 makes up in phage or plasmid shuttle vectors.For example see, PCR Protocols:A Guide to Method and Application, people such as Ed.M.Innis, AcademicPress (1990).

The construction cDNA library is well known to those skilled in the art with the method for breeding in protokaryon or eukaryotic cell in suitable carrier such as plasmid or phage.[for example seeing that people such as Maniatis are as preceding].Suitable cloning vector is that know and generally available.

It will be apparent for a person skilled in the art that the library of other types, and, can be used for separating according to nucleotide sequence of the present invention from the library that other cells or cell type make up.The library of other types includes, but not limited to from other cells, from the cDNA library that the other biological that is different from people and mouse obtains, and genomic library, and it comprises YAC (yeast artificial chromosome) and cosmid library.Can implement the structure of genome dna library by the standard technique of knowing in this area.People such as T.Maniatis, Molecular Cloning:A LaboratoryManual, second edition .14 chapter (1989) can find the genome dna library constructing technology known.

The technician will understand that in many cases, isolating cDNA sequence will be incomplete, because the zone of coded polypeptide is at 5 ' the terminal weak point of this cDNA.This is a reversed transcriptive enzyme, and---a kind of enzyme with intrinsic low " processivity " (enzyme is attached to a kind of measurement of the ability of template in maintenance during the polyreaction)---can not finish the result of the DNA copy of mRNA template between synthesis phase at article one chain cDNA.

There is certain methods to utilize and is that those skilled in the art know, be used to obtain total length c DNA, perhaps extend short cDNA, for example, based on method (people such as Frohman, 1988 of the rapid amplifying (RACE) of cDNA end, PNAS USA 85,8998-9002), the perhaps nearest modification method of this technology, for example Marathon ^TMTechnology (Clontech La boratoriesInc.).

For by top method clones coding according to proteinic polynucleotide of the present invention, the amino acid sequence of polypeptide of described nucleic acid sequence encoding may be necessary.In order to realize this purpose, can measure partial amino-acid series according to protein of the present invention and by automatic sequencer by purifying.Needn't determine whole aminoacid sequences, but determine that the linear order in these proteinic 6 to 8 amino acid whose two zones is used for the primer of the pcr amplification of part dna fragmentation with generation.

In case identified suitable aminoacid sequence, the just synthetic dna sequence dna that can encode them.Because genetic code is a degeneracy, can use specific amino acids of more than one codon coding, therefore, this aminoacid sequence can be by any coding of one group of similar DNA oligonucleotide.Have only in this group a member identical with polynucleotide sequence according to the present invention and can with the hybridization of the desirable protein DNA of coding, also be like this even there is DNA oligonucleotide with mispairing.Can be used for from various cellular types, invertebrates and screening DNA library, vertebrates source by these method separated DNA, and separate homologous gene.

Polypeptide

In another embodiment, the present invention relates to regulate the polypeptide of the pure substantially form of CRH signalling, the separated and purifying of wherein said polypeptide according to nucleic acid molecule encoding of the present invention.In preferred embodiments, this polypeptide has the aminoacid sequence that is selected from SEQ ID NO.46 and SEQ ID NO.48 and its functional analogue.

Protein according to the present invention comprises all possible amino acid variant according to nucleic acid encoding of the present invention, comprises by described molecule encoding and the polypeptide with conserved amino acid variation.

Those skilled in the art will recognize that by multiple recombinant DNA technology and comprise, for example, hybridization, polymerase chain reaction (PCR) amplification, perhaps from the beginning DNA is synthetic (sees, for example, people .Molecular Cloning:A Laboratory Manual such as T.Maniatis, second edition .14 chapter (1989)), protein that can adjusted CRH signalling.

The biological activity protein of regulating the purifying of CRH signalling can have several different physical form.According to polypeptide of the present invention can with total length new life's or unprocessed polypeptide, perhaps the combination as the polypeptide of the polypeptide of part processing or processing exists.The newborn polypeptide of total length can be by the posttranslational modification of specified protein hydrolysis cutting incident, and this incident causes the segmental formation of the newborn polypeptide of total length.Fragment, perhaps segmental physics are associated and can be had all biological activity relevant with protein according to the present invention; Yet CRH regulates active degree and can change between individual fragment.

In the further preferably structure of this polypeptide or the fragment that functional attributes characterized aspect this of the present invention.The preferred embodiments of the invention comprise and contain fragment and these the segmental combinations that alpha-helix and alpha-helix form the high antigenicity exponential region of district, beta sheet and beta sheet-formation district, corner and corner-formation district, coiled coil and coiled coil-formation district, hydrophilic area, hydrophobic region, α both sexes district, β both sexes district, elastic region, surface-formation district, substrate land, polypeptide of the present invention in this.Preferred district is the active district of mediation polypeptide of the present invention.Topnotch preferably has chemistry, biology or other the active fragments of regulating polypeptide of replying of the present invention in this, comprises the activity with similar activity or raising, perhaps has undesirable active those fragments of reduction.

The active proteinic polynucleotide of coding and regulating CRH recombinant expressed

In another embodiment, can be in the expression vector that contains suitable promotor and other suitable transcription regulatory elements according to polynucleotide of the present invention by molecular cloning, and transfer to protokaryon and eukaryotic host cell producing the protein of regulating the CRH signalling, and expressed by reorganization ground.This operative technique is at Maniatis, and T waits complete description among people's (as preceding), and is to know in this area.

Therefore, on the other hand, the invention provides and express the protein expression carrier of regulating the CRH signalling in recombinant host, wherein said carrier contains nucleic acid sequences to proteins and its functional analogue of coding and regulating CRH signalling.Of the present invention preferred aspect, this expression vector contains the proteinic nucleic acid molecule of coding and regulating CRH signalling, and it has nucleotide sequence and its functional analogue that is selected from SEQ ID No.45, SEQ ID No.47 or SEQ ID No.49 or the proteinic genomic dna that contains coding and regulating CRH signalling.

Transcribing with their mRNA of institute's clone gene copy translated needed dna sequence dna among the host that expression vector is defined as suiting herein.These carriers are used among the various hosts and express eukaryotic gene, and this host is as bacterium (comprising intestinal bacteria), cyanobacteria, vegetable cell, insect cell, Amphibians cell, fungal cell's (comprising yeast cell) and zooblast.

Specially designed carrier allows the DNA between the host to shuttle back and forth, as bacterium-yeast or bacterium-zooblast or bacterium-fungal cell or bacterium-invertebral zooblast.The suitable expression vector that makes up can contain: be used for the replication orgin at the host cell self-replicating, selectable marker, a limited number of useful restriction enzyme sites, the potentiality and the active promotor of high copy number.Promotor is defined as the guide RNA polysaccharase in conjunction with DNA and start RNA synthetic dna sequence dna.Strong promoter is the mRNA that causes mRNA to start with high frequency.Expression vector can include, but not limited to the cloning vector of cloning vector, modification, specially designed plasmid or virus.

The proteinic nucleic acid molecule according to separation of the present invention and purifying of coding and regulating CRH signalling can be cloned in the expression vector to express in recombinant host cell.Recombinant host cell can be protokaryon or eucaryon, include but not limited to that bacterium such as intestinal bacteria, fungal cell such as yeast, Amphibians cell such as xenopus leavis oocytes, mammalian cell include but not limited to that the clone in people, ox, pig, monkey and rodents source and insect cell include but not limited to fruit bat-and the clone in silkworm-source.Suitable and can include but not limited to: CV-1 (ATCC CCL 70) by the clone that commercial sources obtains from mammal species, COS-1 (ATCC CRL1650), COS-7 (ATCC CRL 1651), CHO-K1 (ATCC CCL 61), 3T3 (ATCCCCL 92), NIH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C127I (ATCCCRL 1616), BS-C-1 (ATCC CCL 26), MRC-5 (ATCC CCL 171), the L-cell, neuroblastoma, neurogliocyte and HEK-293 (ATCC CRL1573).

Therefore, in another embodiment, the present invention relates to recombinant host cell, it contains nucleic acid molecule or its functional analogue of the proteic recombinant clone of coding and regulating CRH signalling.On the other hand, recombinant host cell according to the present invention contains nucleic acid molecule, and itself or genomic dna or have is selected from: the nucleotide sequence of (SEQ ID No.45), (SEQ ID No.47), (SEQ ID No.49) and its functional analogue.

By include but not limited to transform, transfection, protoplastis are merged, one of many technology of fat transfection and electroporation can import expression vector in the host cell.The cell that contains expression vector is by clonal expansion and analyze to determine whether they produce the protein of mediation CRH signalling.Expressing the evaluation of the host cell clone of permease can be undertaken by several method, these methods include but not limited to at the immunoreactivity according to the antibody of polypeptide of the present invention, the Mammals purine permease active existence relevant with host cell.

Thereby, the invention still further relates in reconstitution cell and to express the method for protein of regulating the CRH signalling, this method be included in allow to regulate from as cultivate according to host cell of the present invention under the condition of the protein expression of the adjusting CRH signalling of the expression vector of general introduction herein.Protein of the present invention can be synthetic or synthetic as fusion rotein by directly expressing, this fusion rotein contain as and another kind of protein or peptide translation fusion goal albumen, but this another kind protein or peptide self ground, remove by enzyme or chemical chop.Therefore, in specific embodiments, the invention provides according to albumen of the present invention, wherein said polypeptide is the part of fusion rotein.

In the generation of some peptide of recombination system, often observe and prolonged the life-span, increased the productive rate of desirable peptide, perhaps provide purifying this proteinic method easily as Expression of Fusion Protein.This is especially relevant when expressing mammalian proteins in prokaryotic hosts.Known various peptase (for example, enteropeptidase and zymoplasm), they specific site cutting polypeptide or, from the amino or C-terminal (for example diaminopeptidase) digestion peptide of peptide chain.In addition, particular chemical medicine (for example cyanogen bromide) will cut polypeptide at specific site.How the technician will understand aminoacid sequence will be carried out necessary modification (if with use recombination method, synthetic or semisynthetic encoding sequence) to mix site-special inside cleavage site.For example see, P.Carter, " the site-specific proteolysis of fusion rotein ", the 13rd chapter, Protein Purification:From Molecular Mechanisms to Large Scale Processes, AmericanChemical Society, Washington, D.C. (1990).

In addition, for example can use, the proteinic nucleic acid molecule that in its genome, has contained coding and regulating CRH signalling as described above, but because for example, weak promoter and do not express this nucleic acid molecule or do not express the mammalian cell of this nucleic acid molecule in suitable mode, and to adjusting sequence such as the strong promoter of this mammalian cell importing, so that induce the expression of this nucleic acid molecule with the endogenous nucleic acid molecule next-door neighbour of the described purine permease polypeptide of coding.

Equally, containing coding is in allos to transcribe and/or regulate the recombinant host cell of the proteinic polynucleotide of the adjusting CRH signalling under the control of sequence or protein will be another embodiment of the present invention.

In this context, term " adjusting sequence " refers to can be used for increasing the nucleic acid molecule of purine permease polypeptide expression, and this is to regulate albumen-encoding gene next-door neighbour because this nucleic acid molecule is incorporated in the genome of cell and with CRH.These regulate silencer intron sequences, 3 ' UTR and/or 5 ' UTR coding region, protein and/or RNA stable element, the proteic nucleic acid molecule of coding and regulating that sequence comprises promotor, enhanser, inactivation, for example, transcription factor, these regulate sequence can induce or cause the expression that CRH regulates protein-encoding gene, other genetic expression controlling elementss of the amount of perhaps known active gene expression and/or increase gene product.The importing of described adjusting sequence causes the increase of polypeptide expression and/or induces, and this polypeptides for modulating CRH signalling causes the amount of polypeptide described in the cell to increase at last.Thereby, the purpose of this invention is to provide the polypeptide of regulating the CRH signalling from the beginning and/or the expression that increases.

Can realize the importing of construct by calcium phosphate transfection, the transfection of DEAE-dextran mediation, transfection, electroporation, transduction, infection or the additive method of cation lipid-mediation to host cell.These methods, are described among the Basic Methods InMolecular Biology (1986) as Davis at many standard laboratory handbooks.In fact the polypeptide of special consideration adjusting CRH signalling can be lacked the host cell expression of recombinant vectors.

In addition, use the synthetic mRNA of external generation also can implement expression according to polynucleotide of the present invention.Synthetic mRNA or can various cell free systems, be translated effectively from the mRNA of the cellular segregation that can regulate the CRH signalling, these cell free systems include but not limited to malt extract and reticulocyte extract, and effectively translation in based on the system of cell, it includes but not limited to microinjection to the frog's egg parent cell, and general preferred microinjection is to the frog's egg parent cell.

Transgenosis is non--the people animal

The invention still further relates to the generation transgenic nonhuman animal, the method for preferred transgenic mice, this method comprise the cell that polynucleotide of the present invention or carrier are imported sexual cell, embryonic cell, stem cell or ovum or obtain from them.Non--people animal can be used and can right and wrong-transgenosis healthy animal according to screening method of the present invention described herein, perhaps can have phosphoric acid to take in or absorbs imbalance, the preferably imbalance that is caused by at least one sudden change in the protein of regulating the CRH signalling again.These transgenic animal are suitable for very much, for example, and the pharmaceutical research of the medicine relevant with the mutant form of aforementioned polypeptides.Of course, for example, as A.L.Joyner editor, GeneTargeting, A Practical Approach (1993), the generation of the enforcement transgenic embryos that Oxford UniversityPress describes and these embryos' screening.For example use, southern blotting technique uses suitable probe can analyze the DNA of embryo's film of embryo; See as preceding.

Preferably, transgenosis of the present invention non--the people animal also contains the wild-type allele that corresponding Mammals CRH regulates at least one inactivation of albumen-encoding gene; See as preceding.This embodiment for example allows, and research is according to the influence of the outbreak of the clinical symptom of the imbalance of disease-related in the interaction partners CRH metabolism of the various mutant forms of polypeptide of the present invention.All application of discussing about transgenic animal before this also are applied to carry two, three or more transgenosis; For example the encode animal of neutral endopeptidase (NEP).Also wish to make and regulate the functionally inactive that the certain phase of the growths of this transgenic animal and/or life was expressed or was in the CRH signalling.This can be by using, for example, tissue-specific, growth and/or cell are regulated and/or inducible promoter, this promoters driven for example can be regulated the antisense of proteic rna transcription thing of CRH signalling or the expression of ribozyme at coding, realize; Also see above.Suitable derivable system is for example genetic expression of tsiklomitsin-adjusting, as for example, Gossen and Bujard (Proc.Natl.Acad.Sci.89USA (1992), 5547-5551) and people such as Gossen (TrendsBiotech.12 (1994), 58-62) described.Similarly, by these regulatory elements can regulating and controlling CRH signalling the expression of mutain.

In addition, the invention still further relates to and contain (preferred stable integration is to its genome) transgene mammal cell according to nucleic acid of the present invention or its part, wherein the proteinic synthetic that causes regulating the CRH signalling of transcribing and/or express of this nucleic acid molecule or its part reduces.

In preferred embodiments, by antisense, justice, ribozyme arranged, suppress altogether and/or the dominant mutation Body Effect realizes reducing." antisense " and " antisense nucleotide " refers to block the DNA or the RNA construct of expression of the gene product of natural generation.

Opened to produce according to providing of polynucleotide of the present invention and have also, thereby the transgenosis with phosphoric acid metabolic deficiency is non--possibility of people animal as the level of above-mentioned proteinic reduction.The technology that how to realize this possibility is that those skilled in the art know.These technology comprise, for example, and the molecule of antisense-RNA, ribozyme or combination antisense and ribozyme function and/or the expression of the molecule of common inhibition effect is provided; Also see above.When using the antisense method to reduce the proteinic amount of regulating CRH signalling in the cell, the nucleic acid molecule of encoding antisense-RNA is preferably the homology source for the animal species that is used to transform.Yet, also may use to show the nucleic acid molecule that the nucleic acid molecule with the proteinic endogenous generation of coding and regulating CRH signalling has high homology.In this case, homology preferably is higher than 80%, especially preferably is higher than 90%, more preferably is higher than 95%.Proteinic synthetic minimizing according to the present invention can cause for example resorbent change of VITAMIN B4 in the transgene mammal cell.In containing the transgenic animal of these cells, this can cause various changes physiological, that grow and/or form.

Thereby, the invention still further relates to the transgenosis that contains above-mentioned transgenic cell non--the people animal.These animals can show, and for example, compare the metabolic deficiency of CRH with the wild-type animal, and this is because the stable or instantaneous existence of foreign DNA causes at least a following feature:

(a) coding can be regulated the destruction of the native gene of CRH signalling;

(b) at least a sense-rna of the transcript that contains polynucleotide of the present invention and/or the expression of ribozyme;

(c) expression of the mRNA that justice is arranged and/or do not translate of polynucleotide of the present invention;

(d) expression of antibody of the present invention;

(e) function of adjusting sequence of the present invention or NOT-function copy mixes; Or

(f) recombinant DNA molecules of the present invention or carrier mixes.

Use polypeptide of the present invention, their coded polynucleotide and carrier, may study now in the body and external efficient and affected phenotype about the medicine of specific sudden change in the protein of the CRH signalling of regulating the patient.In addition, the mutant form of polypeptide of the present invention can be used for determining pharmacology collection of illustrative plates and the evaluation and the preparation other drug of medicine, and these medicines can effectively be treated the imbalance relevant with the CRH metabolism, especially improve CRH inductive anxiety or dysthymia disorders.

Thereby the method that the invention still further relates to prevention, treatment or improve the medical conditions relevant with the CRH Metabolic disorder that comprises the imbalance that crh receptor is relevant understanding, this method comprise mammalian subject is used the polypeptide of the present invention of treatment significant quantity, proteinic polynucleotide or the carrier that coding can be regulated the CRH signalling.

Diagnostic test

The invention still further relates to the purposes of polynucleotide of the present invention as diagnostic reagent.By with the SEQ ID No.1 relevant with dysfunction, SEQ ID No.3, SEQ ID No.5, SEQ IDNo.7, SEQ ID No.9, SEQ ID NO.11, SEQ ID NO 13, SEQ ID No.15, SEQ ID No 17, SEQ ID No 19, SEQ ID No.21, SEQ ID No.23, SEQ ID No.25, SEQ ID No.27, SEQ ID No.29, SEQ ID NO.31, SEQ ID No.33, SEQ ID No.35, SEQ ID No.37, SEQ ID No.39, SEQ ID No.41, SEQ ID No.43, SEQ ID No.45, the polynucleotide of SEQ ID No.47 or SEQ ID No.49 will provide a kind of diagnostic tool for the detection of the mutant form of the gene of feature, it can be assisted, perhaps limit disease or to the diagnosis of the susceptibility of disease, this disease, perhaps to the susceptibility of disease from this expression of gene deficiency, cross space or the temporal expression expressing or change.Can on dna level, detect the individuality that carries this transgenation by various technology.

Thereby will understand the pathological condition that the invention provides among diagnosis and the active relevant experimenter that lacks of proper care of CRH or to the method for the susceptibility of pathological condition, this method comprises:

(a) determine according to the existence that suddenlys change in the polynucleotide of the present invention or do not exist; With

(b) based on the existence of described sudden change or do not exist and detect pathological condition or to the susceptibility of pathological condition.

Diagnostic nucleic acid can be from experimenter's cell, as obtaining from blood, urine, saliva, biopsy or postmortem material.Genomic dna can be directly used in and detect or use PCR or other amplification technique enzymatic amplifications before analysis.RNA or cDNA also can use in a similar manner.The change of the size by comparing amplified production with normal phenotype detects disappearance or inserts.The DNA and the Mammals purine permease nucleotide sequence hybridization of amplification can be identified point mutation.The sequence of Perfect Matchings and the duplex of mispairing can be distinguished by the RNA enzymic digestion or by the difference of melting temperature (Tm).By changing the electrophoretic mobility of the dna fragmentation in capillary electrophoresis column or the gel, use or do not use denaturing agent, perhaps also can detect dna sequence dna difference by direct dna sequencing (for example, people such as Myers, Science (1985) 230:1242).By specific limited restriction endonuclease, nuclease protection assay method, also can disclose the sequence variation (seeing people such as Cotton, ProcNatlAcad Sci USA (1985) 85:4397-4401) of specific position as RNase and S1 protection or chemical chop method.In another embodiment, can make up and contain coding and can regulate the effective screening of the active proteic nucleotide sequence of CRH or its segmental oligonucleotide probe array with embodiment such as transgenation.Array technique be know and have general applicability and the variety of issue that can be used for handling in the molecular genetics comprises genetic expression, genetic linkage, and genetic variability (for example seeing: people such as M.Chee, Science, volume 274,610-613 page or leaf (1996)).

This diagnostic test provides by detect CRH with described method and has regulated sudden change in albumen-encoding gene, the method for diagnosis or definite susceptibility to disease.In addition, by comprising from determining the unusual minimizing of polypeptide or mRNA or the level of increase from experimenter's sample, and by determining that from described sample the method for comparing the existence of protein derivatives with the normal configuration (see above) diagnoses these diseases.Can use any that be used for the method known the quantitative this area of polynucleotide on rna level, to measure the expression that reduces or increase, this method be as, for example, nucleic acid amplification, for example, by PCR, RT-PCR; The RNA enzyme protection; RNA trace and other hybridizing methods.Can be used for determining from protein in host's the sample, is to know in this area as the determination techniques of the level of polypeptide of the present invention.These measuring methods comprise radioimmunoassay, competition-binding assay, western blot analysis and ELISA assay method.The determination techniques that can be used for the existence of definite protein derivatives or variant comprises mass spectroscopy.

Thereby, on the other hand, the invention provides pathological conditions among the diagnosis experimenter relevant or to the method for the susceptibility of pathological conditions, this method comprises with the imbalance of long-term CRH exposure:

(a) determine in the biological sample existence or expression amount according to polypeptide of the present invention or its derivative; With

(b) based on the existence of this polypeptide or its derivative or expression amount diagnosis pathological conditions or to the susceptibility of pathological conditions.

Particularly, the invention provides the method for CRH inductive genetic expression in the diagnosis individuality, institute's choosing method comprises:

A) obtain the biological sample of described individuality; With

B) determine to regulate in the described biological sample at least a proteinic amount of corticotropin releasing hormone (CRH) signalling; The albumen of wherein regulating corticotropin releasing hormone (CRH) signalling is selected from SEQ ID NO.2, SEQ ID 4, SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.10, SEQ ID NO.12, SEQ ID NO.14, SEQ ID NO.16, SEQ ID NO.18, SEQ ID NO.20, SEQ ID NO.22, SEQ ID NO.24, SEQ ID NO.26, SEQ ID NO.28, SEQ ID NO.30, SEQ ID NO.32, SEQ ID NO.34, SEQ ID NO.36, SEQ ID NO.38, SEQ ID NO.40, SEQ ID NO.42, SEQ ID NO.44, SEQ ID NO.46 and SEQ ID NO.48.In alternative embodiment, the method of diagnosis CRH inductive express spectra is not limited to according at least a albumen of the present invention, assessment is accredited as and CRH signalling proteins associated matter group but need simultaneously, promptly has aminoacid sequence SEQ ID NO.2, SEQ ID 4, SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.10, SEQ ID NO.12, SEQ ID NO.14, SEQ ID NO.16, SEQ ID NO.18, SEQ ID NO.20, SEQ ID NO.22, SEQ ID NO.24, SEQ ID NO.26, SEQ ID NO.28, SEQ ID NO.30, SEQ ID NO.32, SEQ ID NO.34, SEQ ID NO.36, SEQ ID NO.38, SEQ ID NO.40, SEQ ID NO.42, SEQ ID NO.44, the protein expression level of SEQ ID NO.46 and SEQ ID NO.48.

Preferably, on protein level, the preferred use in conjunction with this proteinic antibody, perhaps on gene transcription level, the preferred use in conjunction with coding is selected from SEQ ID NO.2, SEQ ID 4, SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.10, SEQ ID NO.12, SEQ ID NO.14, SEQ ID NO.16, SEQ ID NO.18, SEQ ID NO.20, SEQ ID NO.22, SEQ ID NO.24, SEQ ID NO.26, SEQ ID NO.28, SEQ ID NO.30, SEQ ID NO.32, SEQ ID NO.34, SEQ ID NO.36, SEQ ID NO.38, SEQ ID NO.40, SEQ ID NO.42, SEQ ID NO.44, the probe of the polynucleotide of the aminoacid sequence of SEQ ID NO.46 and SEQ ID NO.48 is determined described proteinic amount.With CRH signalling proteins associated matter group in the assessment, use microarray technology analyzing gene expression levels.

Alternatively, contain by assessment and be selected from SEQ ID No.1, SEQ ID No.3, SEQ IDNo.5, SEQ ID No.7, SEQ ID No.9, SEQ ID NO.11, SEQ ID NO 13, SEQ ID No.15, SEQ ID No 17, SEQ ID No 19, SEQ ID No.21, SEQID No.23, SEQ ID No.25, SEQ ID No.27, SEQ ID No.29, SEQID NO.31, SEQ ID No.33, SEQ ID No.35, SEQ ID No.37, SEQID No.39, SEQ ID No.41, SEQ ID No.43, SEQ ID No.45, the gene transcription level of the gene of the nucleotide sequence of SEQ IDNo.47 or SEQ ID No.49 is determined CRH inductive gene expression profile.The method of determining gene transcription level has been described hereinbefore and has been comprised and use combination in preferred embodiments, preferred selective binding is selected from SEQ IDNo.1, SEQ ID No.3, SEQ ID No.5, SEQ ID No.7, SEQ ID No.9, SEQ ID NO.11, SEQ ID NO 13, SEQ ID No.15, SEQ ID No 17, SEQID No 19, SEQ ID No.21, SEQ ID No.23, SEQ ID No.25, SEQ IDNo.27, SEQ ID No.29, SEQ ID NO.31, SEQ ID No.33, SEQ IDNo.35, SEQ ID No.37, SEQ ID No.39, SEQ ID No.41, SEQ IDNo.43, SEQ ID No.45, the polynucleotide of SEQ ID No.47 or SEQ ID No.49 or the probe of its complementary sequence.In another embodiment, can make up and contain with good grounds nucleotide sequence of the present invention or its segmental oligonucleotide probe array to implement effective screening of gene transcription level in the individual sample.

On the other hand, the present invention relates to diagnostic kit, it contains:

(a) polynucleotide of the present invention, nucleotide sequence or its fragment of preferred SEQ ID No.45, SEQ ID No.47, SEQ ID No.49;

(b) with (a) nucleotide sequence complementary nucleotide sequence;

(c) polypeptide of the present invention, polypeptide or its fragment of preferred SEQ ID NO.46, SEQ ID NO.48; Or

(d) antibody of polypeptide of the present invention, the polypeptide of preferred SEQ ID NO.46 or SEQ ID NO.48 and optional suitable detection method.

To understand in any this test kit (a) and (b), (c) or (d) can contain substantive component.This test kit will be used to diagnose the illness or to the susceptibility of disease, especially relevant imbalance such as CRH inductive anxiety or the dysthymia disorders of CRH metabolism.

Nucleotide sequence of the present invention also is valuable for chromosomal localization.These sequence specific targets are fixed, and can hybridize with the specific position on the individual human chromosome.The mapping of the sequence relevant with karyomit(e) according to the present invention is with these sequences and gene-relevant disease important the first step that is mutually related.In case sequence is mapped to accurate chromosome position, the physical location of this sequence can be associated with the genetic map data on the karyomit(e) so.These data are found among the V.McKusick, Mendelian Inheritance in Man (can pass through the online acquisition of Johns HopkinsUniversity Welch Medical Library) for example.Identify the gene of identical chromosomal region and the relation between the disease of being mapped to by linkage analysis (the common heredity of physically adjacent gene) then.Gene of the present invention is mapped to No. 15 karyomit(e)s of people.

Also can determine the difference in cDNA between the influenced and unaffected individuality or the genome sequence.If in some or all affected individualities but in arbitrary normal individual, do not observe sudden change, this sudden change may be the cause of disease of this disease so.

Nucleotide sequence of the present invention also is valuable for tissue positioned.These technology allow to determine these polypeptide expression patterns by detecting the mRNA that encodes according to polypeptide of the present invention.These technology comprise hybridization in situ technique and amplification oligonucleotide technology, for example, and PCR.These technology are to know in this area.Point out the normal function of these polypeptide in the biology from the result of these researchs.

Polypeptide of the present invention or their fragment or their analogue are perhaps expressed their cell, also can be used as immunogen to produce the antibody special to polypeptide immune of the present invention.Term " immunity special " refers to that antibody has polypeptide of the present invention than them the much bigger affinity of other related polypeptides in the prior art.

Thereby in another embodiment, the invention provides with Mammals purine permease and have immunoreactive single specific antibody.In preferred embodiments, described antibody has the activity of proteins of immunoreactivity or described antibody blocking adjusting CRH signalling with the polypeptide with the aminoacid sequence that is selected from (SEQ ID NO.46), (SEQ ID NO.48) and its functional analogue.

Can be by using conventional scheme at the antibody of polypeptide of the present invention, this polypeptide or the cell that contains fragment, the analogue of epi-position or express these are applied to animal, preferred non-human mammal obtains.For MONOCLONAL ANTIBODIES SPECIFIC FOR, can use by continuous cell line and cultivate the antibody that produces.Example comprises hybridoma technology (Kohler, G. and Milstein, C., Nature (1975) 256:495-497), trioma technology, people B-quadroma technology (people such as Kozbor, Immunology Today (1983) 4:72) and EBV-hybridoma technology (people such as Cole, MONOCLONAL ANTIBODIES AND CANCER THERAPY, the 77-96 page or leaf, Alan R.Liss, Inc., 1985).

Antibody at polypeptide of the present invention also can be used for treating the relevant imbalance of CRH metabolism.

On the other hand, the present invention relates to genetically engineered soluble fusion rotein, it comprises polypeptide of the present invention, perhaps the heavy chain of the immunoglobulin (Ig) of its fragment and various subclass (IgG, IgM, IgD, IgE) or the various parts of constant region of light chain.Preferably as immunoglobulin (Ig) be human IgG, particularly the constant portion of the heavy chain of IgGI wherein merges at hinge area.In specific embodiments, can remove the Fc part simply by mixing of sequence of cutting, this cutting sequence can be cut with for example blooc coagulation factor Xa.In addition, the present invention relates to prepare the method for these fusion roteins, also relate to the purposes that they are used for drug screening, diagnosis and treatment by genetically engineered.Another aspect of the present invention also relates to the polynucleotide of these fusion roteins of encoding.The example of fusion protein technology can be found in international patent application no WO 94/29458 and WO 94/22914.

Treatment effectiveness

Another aspect of the present invention relates to immunity/vaccine preparation (composition), and it induces the immunne response at polypeptide of the present invention in this Mammals when importing mammalian hosts, and wherein said composition contains polypeptide of the present invention or polynucleotide.Vaccine preparation can also contain suitable carrier.Because polypeptide can decompose under one's belt, so its preferred parenteral (for example, subcutaneous, intramuscular, intravenously, perhaps intradermal injection) is used.The preparation that is suitable for parenteral administration comprises water-based and non-aqueous aseptic parenteral solution, and it can contain antioxidant, buffer reagent, fungistat and make preparation and recipient's the isoosmotic solute of blood; With water-based and non-aqueous sterile suspensions, it can comprise suspension agent or thickening material.Said preparation can for example, provide in airtight ampoule and the bottle and can be kept in the condition of frost drying at single dose or multi-dose container, only needs to add before use sterile liquid carrier.This vaccine preparation also can comprise the immunogenic adjuvant system that is used to strengthen preparation, as oil-in-water system and other system as known in the art.Dosage will depend on the specific activity of vaccine and can easily determine by the normal experiment method.

In another method, can use and express the proteinic expression of gene that interrupter technique suppresses coding and regulating CRH signalling.Known these technology comprise the use antisense sequences, itself or inner that produce or external application (see, for example, O ' Connor, J.Neurochem (1991) 56:560; Oligodeoxyribonucleotide is as the antisense inhibitor of genetic expression, CRC Press, Boca Raton, FL (1988)).Alternatively, can provide with gene form triple helical (" triplex ") oligonucleotide (see, for example, people such as Lee, Nucleic Acids Res (1979) 6:3073; People such as Cooney, Science (1988) 241:456; People such as Dervan, Science (1991) 251:1360).These oligomer itself can be applied or can the relevant oligomer of expression in vivo.Synthetic antisense or triplex oligonucleotide can contain the base of modification or the main chain of modification.The example of the main chain of modifying comprises methyl phosphorodithioate, thiophosphatephosphorothioate and peptide nucleic acid(PNA) main chain.These main chains are impregnated in antisense and the triplex oligonucleotide to provide protection to prevent nuclease degradation and to be to know in this area.Main chain synthetic antisense and triplex molecule with these and/or other modification also form part of the present invention.

Suppress in the other method of cell expression of target gene being used for, the RNA with part and complete double-stranded feature is imported into cell or born of the same parents' external environment.Inhibition is special, because select the nucleotide sequence generation inhibitory RNA from the part of target gene.This RNA can contain one or more chain of polymeric ribonucleotide; It can comprise the modification to phosphoric acid ester-sugar backbone or nucleosides.Can form duplex structure by wall scroll self-complementary RNA chain or two complementary strands.Inhibition is a sequence-special, because be used for gene inhibition corresponding to the nucleotide sequence in the double-stranded tagma of RNA surely by target.The RNA that preferably contains the nucleotide sequence identical with the part of target sequence.Can find in International Patent Application WO 99/32619 that RNA suppresses the example of technology.

In addition, the protein expression of adjusting CRH signalling can prevent the sequence-specific ribozyme of the mRNA of code for said proteins by using.Ribozyme is the RNA with catalytic activity, it can be natural or synthetic (see for example Usman, N waits the people, and Curr.Opin.Struct.Biol (1996) 6 (4), 527-33).Can design the synthetic ribozyme with at the aforementioned mRNA of the special cutting in selected position, thereby prevent that described mRNA from translating into functional polypeptide.Can synthesize ribozyme, as in the RNA molecule, finding usually with natural nucleus sugar phosphoric ester main chain and natural base.Alternatively, can synthesize the ribozyme with non-natural main chain, for example, 2 '-0-methyl RNA to be providing the protection to the Yeast Nucleic Acid enzyme liberating, and can contain the base of modification.

For the not enough relevant unusual illness of treatment and the protein expression of regulating the CRH signalling, also can utilize several method.A kind of method comprises the compound to the activation polypeptide of the present invention of experimenter's administering therapeutic significant quantity, and is for example combined with pharmaceutically acceptable carrier as above-mentioned agonist, thereby alleviates unusual illness.Alternatively, can use gene therapy to influence experimenter's the endogenous generation of relevant cell Mammals purine permease.For example, can be with polynucleotide through engineering approaches of the present invention to duplicate-to express in the retroviral vector of defective, as discussed above.The retrovirus expression construct can be separated and be imported in the packing cell with the retroviral plasmid vector transduction of the RNA that contains the polypeptide of the present invention of encode then, thereby this packing cell generation contains the infectious virus particulate of target gene.These producer's cells can be applied to the experimenter with through engineering approaches cell in the body and expression in vivo polypeptide.Summary for gene therapy, see Human Molecular Genetics, TStrachan and A P Read, the 20th chapter among the BIOSScientific Publishers Ltd (1996): other methods of treatment based on molecular genetics (with the reference of wherein quoting) are examined in gene therapy.Another kind method is that to use the therapeutic dose and the appropriate drug carrier of polypeptide of the present invention combined.

On the other hand, the invention provides and contain the polypeptide for the treatment of significant quantity, as the soluble form of polypeptide of the present invention, agonist/antagonist peptide or micromolecular compound are with pharmaceutically acceptable carrier or the combined pharmaceutical composition of vehicle.These carriers include, but not limited to salt solution, buffer saline, dextrose, water, glycerine, ethanol and their combination.The invention still further relates to drug packages and test kit, they contain one or more containers of the composition that one or more foregoings of the present invention are housed.Polypeptide of the present invention and other compounds can use separately or with other compounds, unite use as therapeutic compound.

Said composition will be adapted to route of administration, for example, by whole body or oral route.The preferred form that general is used comprises injection, usually by intravenous injection.Can use other injecting pathways, as subcutaneous, intramuscular or peritoneal injection.The alternative approach that general is used comprises uses permeate agent such as biliary salts or fusidinic acid or saturating mucous membrane of other stain removers and transdermal administration.In addition, if polypeptide of the present invention and other compounds can be prepared into intestines or encapsulated preparation, dosage forms for oral administration also is possible so.Using of these compounds also can be local and/or localization, is the form of patch, ointment, paste, gelifying agent etc.

Needed dosage range depends on selection, route of administration, the character of preparation, the character of experimenter's illness and doctor in charge's the judgement of polypeptide of the present invention and other compounds.Yet appropriate dosage is 0.1-100 μ g/kg experimenter.Yet, consider the diversity of available compound and the different efficient of various route of administration, can expect the wide variation of required dosage.For example, Orally administered expection than use by intravenous injection need be higher dosage.The standard experience routine that use is used to optimize can be judged the variation of these dosage levels, as known in the art.

Also can produce the polypeptide that is used for the treatment of in the experimenter, this is commonly called in the treatment pattern as above-mentioned " gene therapy " endogenously.Thereby, for example, can be from the cell of individuality by polynucleotide, as DNA or RNA through engineering approaches with external coded polypeptide, and for example, by using retrovirus treatment carrier.Then cell is imported among the experimenter.

By the present invention may be better understood with reference to following experimental detail, but those skilled in the art will understand easily that these experimental details only are used to illustrate the present invention, and it is described in claims of back more completely.In addition, in the full text of this application book, various publications have been used.Being disclosed in the reference that is merged in the application herein of these publications more completely to describe the state of this area under the present invention.

Experimental arrangement

Cell cultures and specimen preparation-AtT-20 cell is purchased in ATCC and is maintained at 37 ℃, 5%CO ₂Wet air contains in the Eagle substratum (Invitrogen Life Technologies) of DulbeccoShi improvement of 10% foetal calf serum, 5% horse serum and 4.5g/L D-glucose.In order to test, with cell inoculation in 25cm ²In the flask.Substratum is replaced behind 48h and cell was handled 0,0.5,1,2,4,8 and 24 hour with 1 μ M R121919 among 1 μ M CRH (Sigma), the DMSO among 0.1%DMSO, the DMSO in the fresh culture or 1 μ M CRH+1 μ M R121919 among the DMSO.Hatch substratum and add the 3ml Trizol (Invitrogen Life Technologies) be used for lysis by extraction and end to hatch.Working instructions according to the manufacturer extract total RNA with Trizol.Use Rneasy test kit (Qiagen) to be further purified the total RNA of 100 μ g, handle pillar with DNAseI.

Microarray hybridization-be prepared as follows cRNA.Use T7-oligo (dT) 24-primer and SuperscriptII RT (Invitrogen Life Technologies) that the total RNA of 10 μ g is implemented reverse transcription 1h at 42 ℃.Synthetic with e. coli dna polymerase I, dna ligase and RNAseH (Invitrogen Life Technologies) at 16 ℃ of second chain cDNA that carry out 2h.After phase-locking gel (Eppendorf) carries out phenol-chloroform extraction, use Bioarray high yield rna transcription substance markers test kit and biotin labeled ribonucleotide (Enzo Diagnostics) to implement in-vitro transcription 6h at 37 ℃.CRNA sample purifying and 95 ℃ of fragmentations 35 minutes on the QiagenRneasy post.The cRNA productive rate is 50 to 100 μ g.(Affymetrix, Santa Clara CA) go up the processing sample at GeneChips.In order to check the quality of each sample, the cRNA of 5 μ g marks is moved on the Test2-array.Actual experiment is implemented on Murine Genome U74Av2 array, and this array contains checks about 12,000 little musculus cdnas of total length and from EST bunch the probe groups of UniGene database (Build 74).Continuing to implement hybridization 16h with 15 μ gcRNA in 45 ℃ under the rotation.Array is dyeed with streptavidin/phycoerythrin (SAPE) in Affymetrix Fluidics worktable (Station), use the antibody staining of anti--streptavidin then and carry out the SAPE dyeing second time.Subsequently, also use Microarray Suite Software (Affymetrix) analytical data with HP-laser scanner scanning array.Do not carry out classification or stdn in this stage.Quality based on current calling between all samples (present calls) percentage ratio (mean value is 47.06 ± 2.45%) assessment experiment.The ratio of kytoplasm beta-actin and GAPDH5 '/3 ' is respectively 1.10 ± 0.08 and 0.93 ± 0.05.

Data analysis and gene Selection

Overall average algorithm below using is calibrated the green strength on each array:

Basically this calibration is set at mean value to all array measurements with the average intensity of an array, has remedied the difference of array and array in hybridization, washing and the dyeing, finally allows reasonably to compare between the array.After the calibration, the data of using weighting spectrum mapping analysis to be calibrated.Weighting spectrum mapping is a kind of unsupervised multivariable technique, and it comprises two centralizations of data and represents the special visual combined of two kinds of main ingredients the highest.Although array data " size " composition has been removed in two centralizations, by representing the area of the symbol of sample and gene size separately, this information is imported in visual once more.This method allows to reduce big microarray data collection and thereby gene cluster and/or the experimenter who visually checks in the appraising datum is provided.Implemented more detailed analysis with the OmniViz program.Remove all records that lack in all experiments and will be made as 20 less than all signals of 20.Calculate the genetic expression multiple difference that CRH, R121919 and CRH+R121919 handle on each time point.Calculate for those, the signal of corresponding time point is used to ratio calculated in the sample that DMSO handles.

Quantitative RT-PCR-use PCR in real time analysis confirms microarray data.It is synthetic to use at random sexamer primer and SuperscriptII RT (Invitrogen Life Technologies) that the total RNA of 0.5 μ g is implemented article one chain cDNA.Go up the enforcement quantitative PCR with Taqman PCR test kit at ABIPrism 7700 circulation instrument (Applied Biosystems).The serial dilution of cDNA is used to produce the typical curve of logarithmic cycle threshold of the concentration of relative beta-actin, c-fos, Crh-R1, Crh-R2, Rgs2 and goal gene (sequence sees Table 2).Allow the transcriptional level of determining from the RNA sample of different time points from the linear regression line that typical curve calculates.

The result

In the adenoma cell system in the mouse AtT-20 PATH cell-source of expressing CRH-R1, studied transcription response to CRH.Although can easily detect CRH-R1, can not find out that in the AtT-20 cell CRH-R2 expresses by real-time quantitative RT-PCR (RTq) and western blotting.Reply in order to identify that CRH-R1 is special, cell is exposed to 1 μ MCRH, 1 μ M CRH and 1 μ M CRH-R1 specific antagonist R121919 and only is exposed to R121919.Follow the tracks of transcription response in time up to using back 24 hours for the first time.For evaluation process usefulness, the RNA from different treatment and time point before the array experiment enforcement is determined c-fos mRNA level by RTq.Consistent with former report, be exposed to CRH and cause that c-fos transcribes instantaneous surge, the level (see figure 1) (8 that descended after 0.5 to 1h; 9).This should be signed almost and be suppressed fully when having R121919.What is interesting is that 0.1%DMSO induces c-fos to express, yet compare low 5 to 10 times of expression level with the expression of CRH inductive.

On the microarray that contains have an appointment 12000 little musculus cdnas and ESTs, all time points have been analyzed.Use spectrum mapping (being called unsupervised method) that the aggregate analysis of express spectra is shown that time schedule is to cause the reason (Fig. 2) that great majority change in the viewed genetic expression.Synchronization by adding the cell cycle in new substratum and serum institute these cultures of inductive may be the reason of this phenomenon, although the accumulation of metabolite and ongoing cell cultures also are other contribution factors.The difference that the spectrogram analysis also shows sample that CRH handles and other samples is time point (0.5h is to 2h) in early days mainly, and the total difference in the expression becomes very little behind 8h.Because this obvious influence of time is expressed observed value so analyzed these with respect to those expression observed values of corresponding time point observation in the control sample of handling at DMSO.

The gene of being regulated is defined in variation that arbitrary time point shows the transcript level greater than those genes of 2 times.Use OmniViz Treescape view, selected to satisfy 111 kinds of genes of this standard, they show with the antagonist processing and compare, and CRH handles the back expression difference.26 kinds in these 111 kinds of genes is " respondent early ", and they have shown 2 times of variations with the CRH processing after 30 minutes.32 kinds of genes are " respondents in mid-term ", and they are replied after handling 1 to 2h, and 53 kinds of genes are " late respondents ", handle back 2 hours or show after the longer time and reply (see figure 3).These are replied by CRH-R1 antagonist R121919 and suppress.In morning is known member in the CRH-R1 downstream pathway among the respondent, and as transcription factor Nurr1, Nurr77, Jun-B, they have verified this mensuration.

The interesting newcomer who is identified comprises transcription factor (for example, the ets transcription factor (Pse) of nf (NFIL3), cAMP response element regulon (CREM) and the prostate-specific of the enhanser 1 (Hey1) that hair/bifurcated is relevant, interleukin-13 adjusting); Acceptor and passages regulate thing (for example, relevant GTP-conjugated protein (GEM) and acceptor (thyrocalcitonin) activity modifying albumen 3 (RAMP3) of Ras-); The secretion peptide (for example, adrenomedullin, thyrocalcitonin, cholecystokinin) and with intracellular signal proteins associated matter (for example, G-protein signal instrumentality 2 (Rgs2), special phosphodiesterase 4 B (Pde4b), the

inositol

1,4 of cAMP, 5-triphosphate receptor 1 (IP ₃R1) and modulability subunit phosphatidyl-inositol 3-kinase, p85).The regulatory gene that is subjected to that other are interesting comprises kinases and serum-derivable kinases (Fig. 4) that Period homologue Per1, fibroblast growth factor acceptor 2 (Fgfr2), serum/glucocorticosteroid are regulated.

What is interesting is, all raised after all respondents that identify according to standard above-mentioned are exposed to CRH.This is induced be instantaneous and 4 to 8h after nearly all derivative gene all get back to baseline.Signalling produces degenerative protein (for example, Pde4, Rgs2, CREM, etc.) to many derivative transcripts codings to CRH-R1, may be the reason of the instantaneous character of this inductive.Except this reverse feedback, other mechanism can help to transcribe the instantaneous character of inductive as removing quick CRF by phosphorylation and internalization.In this respect, what is interesting is and notice to stimulate and to reduce CRF in the rat pituitary cell fast with CRF ₁The mRNA level.Yet we do not detect CRF in the AtT-20 cell in the hypophysis source that is exposed to 1 μ M CRF ₁The variation of mRNA.With quantitative PCR in real time analysis the same sample that is used for hybrid experiment and repeated experiments being implemented microarray data confirms.Adjusting level of identifying by microarray and time history are corresponding to those adjusting level and the time histories to Rgs2 that quantitative PCR is observed of passing through that show among Fig. 5.For those genes that have been verified, in Fig. 4, shown the level of comparing with untreated samples of inducing.

Discuss

We have identified the approach of transcribing in CRH-R1 downstream in the AtT-20 cell with the CRH-R1 specific antagonist.Our discovery is some second messengers such as cAMP and Ca when stimulating with CRH ²⁺The activation unanimity.Some transcription responses can be explained by phosphorylation and the transcribing subsequently of cAMP response element downstream gene of CREB.In the promotor of for example Per1, Nurr1, CREM-ICER, c-Fos, these elements have been had been found that.In addition, the inductive kinetics collection of illustrative plates of these genes passes through the observed maximum transcription rate of CREB after forming 0.5 hour corresponding to cAMP.Inducing in decay of CREM-ICER constitutes negative feedback mechanism aspect the transcription response of cAMP.The CREM-ICER that what is interesting is the acute pressure of middle period of the response pituitary gland of being reported induces.The chronic increase that the mouse of shortage CREM-ICER shows the beta-endorphin level shows that CREM-ICER induces the adjusting (10) of the genetic expression that may participate in response pressure.Our result shows that CREM-ICER participates in CRH signalling adjusting and result directly, and CREM-ICER removes the change of replying that can cause pressure signal.The reverse feedback instrumentality of the another kind of new supposition of CRH signalling is Rgs2.We have identified two single CRE motifs in the people RGS2 gene promoter, respondent behavior morning of this gene when possible explanation stimulates with CRH.Support we discovery be a report recently, it points out that phosphoinositide signalling and cAMP can induce the quick and instantaneous increase of Rgs2mRNA in people's astrocytoma and the neuroblast oncocyte.Rgs2 albumen is G _{Q α}The selective depressant of function.

Shown that recently the cAMP that Rgs2 reduces odorant-cause produces, Rgs2 acts on G _αBut directly suppress the activity of III type adenylate cyclase.Though Rgs2 is accredited as in the activated T lymphocyte immediately response gene early at first, but the Rgs2 that studies show that in the Rgs2 deficient mice also works in the adjusting of the behavior of pressure correlation, because these mouse show the anxiety and the aggressive behaviour (11) of increase.And inducing also of the special phosphodiesterase 4 B of cAMP (Pde4b) can be classified according to reverse feedback, directly weakens the cAMP signal.The another kind of important second messenger who produces when CRH stimulates is Ca ²⁺Shown that CRH passes through voltage-controlled Ca ²⁺Passage causes the outer Ca of born of the same parents that the stable state depolarize stimulates ²⁺Enter and by from inositol 1,4 the responsive Ca of 5-triphosphoric acid (InsP3) ²⁺The storehouse discharges and the interior Ca of rising born of the same parents ²⁺Concentration (12).The p85 regulator subunit of InsP3 acceptor and phosphatidyl-inositol 3-kinase is all raised, the long-time Ca of this possible explanation ²⁺The compensation mechanism of signalling.And the rise of little G-albumen kir/Gem is at long-time Ca ²⁺The decay of signalling.Nearest research has shown that Gem passes through the Ca on these complementary subunits adjusting cytolemma ²⁺Passage is expressed.The level that has shown the rising of Gem suppresses Ca ²⁺-the exocytosis that causes, and proposed Gem and may have at Ca ²⁺The provide protection of load.Ca ²⁺Except as the second messenger, also show in regulatory gene is expressed and play a crucial role.What is interesting is the adjusting of calcineurin/NFAT and CaM kinase signalization, the increase of NFIL3mRNA level when its soluble CRH handles to NFIL3/E4BP4.In bone-marrow-derived lymphocyte, the expression that interleukin-22 is induced NFIL3 by Raf-mitogen-activatory protein kinase and phosphatidyl-inositol 3-kinase approach.In this cell type, NFIL3 and the collaborative apoptosis that suppresses of the approach that relies on Bc1-x1.Our data show NFIL3 figure in preventing the AtT-20 apoptosis.

CRH is the most effective ACTH secretogogue.Unfortunately, used microarray is not inquired the POMC level.Yet CRH uses the back and finds that some other propetide original mRNAs are raised, and these propetides were as cholecystokinin (CCK) and two calcitonin polypeptide family members originally: adrenomedullin (ADM) and thyrocalcitonin (CT).In rise that also what is interesting is RAMP3 aspect this.The transhipment and the glycosylation of RAMPs control Calcitonin Receptor sample acceptor (CRLR).For RAMP3, shown that it produces the ADM acceptor with CRLR.The rise of this gene may be worked in the adjusting to the responsiveness of ADM in CRH exposure or other extracellulars (because also not knowing whether RAMP3 regulates other G-link coupled acceptors) stimulation back AtT-20 cell.

Although, also reporting CRH in the past by AtT-20 emiocytosis, CCK do not induce CCK to express (13).Yet, furtherd investigate the interaction between CCK and the CRH and in terrified, dysthymia disorders, anxiety and stomach emptying, proved this interaction (14-19).The role of CRH in central role of mediation CCK pointed out in these experiments of great majority.Our data show that CRH also may play a role as the CCK secretogogue.Adrenomedullin also may have the very similarly condition with CCK.Illustrate ADM expressed and influenced in the animal basis and CRH-and stimulate in pituitary gland ACTH and discharged, thereby shown its strong role (20-23) in the adjusting hypothalmus-pituitary-adrenal axis.Current expression data shows that CRH induces ADM and CT.In addition, nearest discovery shows that the round-robin adrenomedullin increases in Cushing's disease, and the pituitary gland position (24) that may represent the generation of ADM to increase, shows that CRH may induce ADM.

In a word, we have untied the part of thyroliberin-releasing hormone receptor 1 activatory idiotype network and have identified some new targets of this signal cascade.Our discovery has caused the demand to further experiment, and these experiments are used to illustrate the function of these transcription responses that on cell and the complete biological level CRH stimulated.

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Sequence table

SEQ?ID?NO.1

atgcagctgagaaaaatgcagaccatcaaaaaggagcccgcacccctagatcctaccagcagctcagacaagatgctgctgctgaactctgccttagct

gaggtggccgaggacctagcctcaggtgaagatttgctcctgaacgaagggagcatggggaaaaacaaatcctcggcgtgtcggagaaaacgggaat

tcattccggacgagaagaaagacgccatgtattgggagaaacggcggaaaaacaacgaagctgccaaaagatctcgggagaagcgccgcctcaatg

acctggttttggagaacaagctgattgccctgggagaagaaaatgccactttaaaagctgagctgctctccctgaaattaaagtttggtttaattagctccac

ggcgtatgcccaagaaatccagaaactcagtaattccacagctgtctactttcaggactaccagacatccaaggctgccgtgagctcttttgtggacgagc

atgagcctgcgatggtagccggaagttgcatctcagtcatcaagcactctccccagagctcgctctccgatgtgtcagaggtgtcctcggtggagcacac

tcaggaaagccccgcacagggaggctgccggagccctgagaacaagttccctgtgatcaagcaggagcccgtggagttggagagctttgccaggga

ggccagggaggagcggggcacgtattccacctccatctaccagagctacatgggaagctctttctccacttactcccactccccacccctcttgcaggtc

catgggtccactagcaactccccaagaacctcagaggccgatgagggtgtagtgggcaagtcttctgatggggaagacgaacaacaggtccctaagg

gccccatccattctccagtggagctgcaacgggttcacgccacggtggtgaaggttccggaagtgaacccttctgccttaccgcacaagcttcggattaa

agccaaggccatgcaggtcaaagtggaggctttggacagcgagtttgaaggcatgcagaaactctcttcacccgccgatgcgatcgccaaaagacattt

tgacctggagaaacatggaacctcgggtatggcccattcctccctccctcctttctcagtgcaggtgacgaacattcaagattggtccctcaaatcggaac

actggcatcacaaagaactgagcagcaaaactcagagtagcttcaaaacaggtgtggtggaagtcaaagacggtggctataaggtttccgaagctgag

aatttgtatttgaagcagggaatagcaaacttatctgcagaggtggtctcgctcaagagattcatagccacacaaccgatctcggcttcggactccaggtaa

SEQ?ID?No.2

MQLRKMQTIKKEPAPLDPTSSSDKMLLLNSALAEVAEDLASGEDLLLNEGSMGKNKSSACRRK

REFIPDEKKDAMYWEKRRKNNEAAKRSREKRRLNDLVLENKLIALGEENATLKAELLSLKLKFG

LISSTAYAQEIQKLSNSTAVYFQDYQTSKAAVSSFVDEHEPAMVAGSCISVIKHSPQSSLSDVSEV

SSVEHTQESPAQGGCRSPENKFPVIKQEPVELESFAREAREERGTYSTSIYQSYMGSSFSTYSHSPP

LLQVHGSTSNSPRTSEADEGVVGKSSDGEDEQQVPKGPIHSPVELQRVHATVVKVPEVNPSALP

HKLRIKAKAMQVKVEALDSEFEGMQKLSSPADALAKRHFDLEKHGTSGMAHSSLPPFSVQVTNI

QDWSLKSEHWHHKELSSKTQSSFKTGVVEVKDGGYKVSEAENLYLKQGIANLSAEVVSLKRFI

ATQPISASDSR

SEQ?ID?No.3

tgtccgctctgcctcccacacctagcaccccagcccgctgctgccccggtgagaacccccagcttgggccttgtcatggtgccagcaggtggccctgag

cttctgacaggggcctgcctatagacctgcaggcctgaggcctcagactcacactcaaggggcaagaggccctggtggcccacctaagagccacctct

gtccccagccctgctgccccactgatgtctgactgagacccagcagtgaccctgagctgcctgcccactgcctcctcctggtccctgaggttggctctgc

cgaggacggacgactcttctgaagcaggcggctaacggaagcagccccaagcctccaccgcagcatgggcagtgccagcccaggcctgagcaacg

tgtcccccggttgcctgctactgttcccagatgtggcaccacgaacagggacggagaaggcagcatcaggagcaatgggccctgagaagcaggaatg

gagtcctagtccacccgccacccctgagcagggcctgtctgctttctacctctcttactttaacatgtatcccgacgatagcagctgggtcgccaaagtccc

cgaggcccgtgccggggaggaccacccggaggagcccgagcagtgtcccgtcattgacagccaggcctctgggagcacgttggatgagcactcgct

agagcaggtgcaatcgatggttgtgggcgaggtcctgaaagatattgagacggcctgcaagcttctgaacatcacagcagaccctggggactggagcc

ctggtaacgtgcagaagtggcttttatggacagaacaccagtaccggctgcctccagcaggcaaggccttccaggagctgggcggtaaggagctgtgc

gccatgtccgaggaacagttccgtcagcgtgcacccttgggtggggatgtactgcatgcccacctggacatctggaagtcagcggcctggatgaagga

gaggacctcgcctgggacccttcactactgcgcctccaccagcgaggagggctggacggatggtgaggtggactcgtcgtgctccgggcagcccatt

cacctgtggcagttcctgaaagaactgctgctcaagccccacagctatggccgcttcatccgctggctcaacaaggagaaaggcatcttcaaaattgagg

actcagcacaggtggcccgactgtggggtgtgcgcaagaaccggccagccatgaactatgataaactaagccgctccatccgccagtattacaagaag

ggcatcattcgtaaacccgacatctctcagcgccttgtctaccaatttgtgcatccagtctgagagccacagagaccagaggcctacaacctgccccagg

cagccactctctggttggcctggtcctctctgctcactctgaattcaggggctgctggtatcccagaacccaaggtcccagatagacagccactgatctag

ggatacacatgagctctctgggtcatacacaggccccaggaagatcgagggagctagttcagcacacagggactggaccaagtcagctcaccggaca

gtgatgtcactggtctctgctcctgccacaatcctgtaccatatctggcatggtgctaagagatgtctgtaccctgcgttgggaagccaggggtgccctggg

gatggataataaagacgtaagataactg

SEQ?ID?No.4

MGSASPGLSNVSPGCLLLFPDVAPRTGTEKAASGAMGPEKQEWSPSPPATPEQGLSAFYLSYFN

MYPDDSSWVAKVPEARAGEDHPEEPEQCPVIDSQASGSTLDEHSLEQVQSMWGEVLKDIETAC

KLLNITADPGDWSPGNVQKWLLWTEHQYRLPPAGKAFQELGGKELCAMSEEQFRQRAPLGGD

VLHAHLDIWKSAAWMKERTSPGTLHYCASTSEEGWTDGEVDSSCSGQPIHLWQFLKELLLKPH

SYGRFIRWLNKEKGIFKIEDSAQVARLWGVRKNRPAMNYDKLSRSIRQYYKKGIIRKPDISQRLV

YQFVHPV

SEQ?ID?No.5

cgggtcgacccacgcgtccgcccacgcgtccggcggagcttctgggttgcgggccgaaacggcaagcggatggagggcgctcgaacggccaggtg

tcgtgattaaattagtcagccctcagagacaggcgtcctacctcctttatccagacctcaaaagccccgttgtgcacccgtggtggcttcttcaccttccctg

tttcgtcctccactgtatggcccagacatgagtggtcccctagaaggggccgatgggggaggagaccccaggcccggagaacctttttgtcctggagga

gtcccatcccctggggccccgcagcaccggccttgtccaggccccagcctggctgatgacactgatgcaaacagcaatggctcaagtggcaatgagtc

caacggacccgagtccaggggcgcatctcagcggagttctcatagttcctcttctggcaatggcaaggactcagctctgctggagaccactgagagcag

caagagtacaaactcacagagcccatccccacccagcagctccattgcctacagcctcctgagtgcgagctcagagcaggacaacccatctaccagtg

gctgcagcagtgaacagtcagctcgagccaggacccagaaagaactcatgactgcacttcgggagctcaaacttcgactgccaccagagcgtcgggg

caagggccgctctgggaccttggccacactgcagtacgctctggcctgtgtcaagcaggttcaggctaaccaggaatattaccagcagtggagtctgga

ggagggtgagccttgtgccatggacatgtctacttacaccctggaggaattggagcatatcacatccgaatacacacttcgaaaccaggacaccttctctg

tggctgtgtccttcctgacaggccggattgtctatatttcggagcaggcaggtgtcctgctgcgttgcaaacgggatgtgtttcggggtgcccgctttcag

agctcctggctccccaggatgtgggtgtcttctatggctctactacaccatctcgactgcccacctggggcactggcacctctgcaggttcaggtctcaag

gacttcacccaggaaaagtctgtcttctgccgaatcagaggaggtcctgaccgggatccagggcctcggtaccagccattccgcctaaccccatatgtga

ccaagattcgggtctcagatggagcccctgcacagccgtgctgcctactcattgccgagcgcatccactctggttatgaagctccccggatccctcctga

caagaggatcttcaccacccgacacacaccaagctgcctcttccaggatgtagatgaaagggctgccccactgctgggttaccttccccaggatctcctg

ggggctccagtacttctctttctacatcctgaggaccgacccctcatgctggccattcataagaagatactgcagctggcaggccagccctttgaccattcc

cctattcgcttctgtgctcggaacggggaatatgtcaccatggacaccagctgggccggttttgtgcacccctggagccgcaaggtggctttcgtgttggg

tcgccataaagtgcgcacggcacccctgaatgaggacgtcttcactcccccagcccccagcccagctccgtccctggactctgatatccaggagctctc

agagcagatccatcgattgctgctgcagcctgtgcacagctccagccccacggggctctgtggagttggccctctgatgtcccctggtcctctacacagc

cctggctcctccagtgatagcaatgggggggacgctgaggggcctgggcctcctgctccagtgactttccagcagatctgtaaggatgtgcatctggtaa

agcaccagggacaacagctcttcattgaatctcgggccaagcccccaccccggccccgcctccttgctacaggtacattcaaagccaaagtccttccct

gccagtccccaaaccccgaactggaggtggccccagttcctgaccaagcctcgttagccttggcccctgaggagccagagaggaaagaaacctctgg

ctgttcctaccagcagatcaactgcctggacagcatcctcaggtatttggagagctgcaacattcccagtacaaccaagcgtaaatgtgcctcctcctcctc

ctacactgcctcttcagcctctgatgatgacaagcagagggcaggtccagttcctgtgggggccaagaaagatccgtcgtcagcaatgctgtctgggga

gggggcaactcctcggaaggagccagtggtgggaggcaccctgagcccgctcgccctggccaataaggcagagagcgtggtgtccgtcaccagtca

gtgtagcttcagctccaccatcgtccatgtgggagacaagaagcccccggagtcggacatcatcatgatggaagacctgcctggcctggcccctggcc

cagcccccagtccggcccccagccccacagtagcccctgacccaaccccagatgcttatcgcccagtgggtctgaccaaggccgtgctgtccctgcac

acacagaaggaagagcaagccttcctcaaccgcttcagagatcttggcaggcttcgtggacttgacacctcttctgtggccccctcagcccctggctgcc

accatggccccattccccctggtcgccgacaccactgccgatctaaagcaaagcgttcccgccaccaccaccaccagaccccccggcccgaaactcc

ctgctatgtctcccatccttcacctgtgccctcttctggaccctggccacccccaccagccacgacccccttcccagcaatggtccagccctacccactcc

cagtattctcccctcgaggaggaccccagccccttccccctgcccctacatctgtgtcccctgctaccttcccttcccccttagtgaccccaatggtggcctt

ggtgctccctaactatctattccctaccccacctagttatccatatggggtgtcccaggcccctgttgaggggccacccacgcctgcttcccactcgccctc

tccatccctgcccccaccacctctcagccccccccaccgcccagactccccactgttcaactcgagatgcagctccccactccagctcaatctgctgcag

cttgaggagtccccccgcacggaggggggcgctgctgcaggaggcccaggaagcagtgctgggcccctgcctcccagtgaggagactgctgagcc

agaggccagattggtggaggttactgagtcgtccaatcaggatgcactttcaggctccagcgacctgctggagctactgctccaagaagactctcgctcg

ggcacaggctccgcagcctcaggctcctgggctctggcctgggctctgggtctggttcaggatcccacgaagggggaagcacctcagccagcatca

cccgcagcagtcagagcagccatacaagcaagtactttggcagcatcgactcctccgaggctgaagctggggctgctcgggccaggactgagcctgg

ggaccaggtcatcaagtgtgtgctccaggaccccatctggctgctcatggccaatgccgaccagcgtgtcatgatgacataccaggtgccgtccaggga

tcgcagcctctgtgctgaagcaagaccgggagaggctccgggccatgcagaaacagcagccacggttctcagaggaccagaggcgggaactgggcg

ctgtgcactcctgggtccggaagggccagctgcctcgggcccttgatgtgatggcgtgtgtggactgtggcagcagcgttcaagatcctggccactctga

tcgacccgctcttctcagaactggatggattggggctggagcccatggaagagggtggaggcgagggtggtgggtgtggtgttggcggtggtgggggtg

atggtggtgaggaggcccagacccaaattggggctaagggttcaagctctcaggactctgccatggaggaagaagagcaaggcgggggctcatccag

cccagctttacctgcagaagaaaacagcaccagctagatccattttggggccgcttacagcagcctaatgagaggcttcctttcgaccatgttggggttctt

ataactcaagatacagctggaccaaccaataggaaactgccccagcttctcccaacatagggggctggacccccattaccagcccaggcacaggagct

gcctctagcttcttagcagagtggaagttctcagccccatttggaggattgtccacgcccgtcccactgaggagacgggcgggccttcggttaaggttgct

gacaagctgctgaagtggtctgtccaaatcccagctgagcctgagtcccagtcgcagggttggggctgcacttatttatttgggagagacagctcactctc

ccacctcaccccaagatgggaggaggggaacctgggatctgtgtaggatccaggtccgtgaacccctagccgctccagggtgggggaggttggtgga

ccatggagtccctggtgctgcccctcaggtgggacccaggtgttctcagctctaccctctaccaatgacatttgtgtttttgatattgtgcctgttatttttttttta

acacaaaatgacaaaatgaaaaaccaaaaa

SEQ?ID?No.6

MSGPLEGADGGGDPRPGEPFCPGGVPSPGAPQHRPCPGPSLADDTDANSNGSSGNESNGPESRG

ASQRSSHSSSSGNGKDSALLETTESSKSTNSQSPSPPSSSIAYSLLSASSEQDNPSTSGCSSEQSARA

RTQKELMTALRELKLRLPPERRGKGRSGTLATLQYALACVKQVQANQEYYQQWSLEEGEPCA

MDMSTYTLEELEHITSEYTLRNQDTFSVAVSFLTGRIVYISEQAGVLLRCKRDVFRGARFSELLAP

QDVGVFYGSTTPSRLPTWGTGTSAGSGLKDFTQEKSVFCRIRGGPDRDPGPRYQPFRLTPYVTKI

RVSDGAPAQPCCLLIAERIHSGYEAPRIPPDKRIFTTRHTPSCLFQDVDERAAPLLGYLPQDLLGA

PVLLFLHPEDRPLMLAIHKKILQLAGQPFDHSPIRFCARNGEYVTMDTSWAGFVHPWSRKVAFV

LGRHKVRTAPLNEDVFTPPAPSPAPSLDSDIQELSEQIHRLLLQPVHSSSPTGLCGVGPLMSPGPL

HSPGSSSDSNGGDAEGPGPPAPVTPQQICKDVHLVKHQGQQLFIESRAKPPPRPRLLATGTFKAK

VLPCQSPNPELEVAPVPDQASLALAPEEPERKETSGCSYQQINCLDSILRYLESCNIPSTTKRKCAS

SSSYTASSASDDDKQRAGPVPVGAKKDPSSAMLSGEGATPRKEPVVGGTLSPLALANKAESVVS

VTSQCSFSSTIVHVGDKKPPESDIIMMEDLPGLAPGPAPSPAPSPTVAPDPTPDAYRPVGLTKAVL

SLHTQKEEQAFLNRFRDLGRLRGLDTSSVAPSAPGCHHGPIPPGRRHHCRSKAKRSRHHHHQTP

RPETPCYVSHPSPVPSSGPWPPPPATTPFPAMVQPYPLPVFSPRGGPQPLPPAPTSVSPATFPSPLV

TPMVALVLPNYLFPTPPSYPYGVSQAPVEGPPTPASHSPSPSLPPPPLSPPHRPDSPLFNSRCSSPLQ

LNLLQLEESPRTEGGAAAGGPGSSAGPLPPSEETAEPEARLVEVTESSNQDALSGSSDLLELLLQE

DSRSGTGSAASGSLGSGLGSGSGSGSHEGGSTSASITRSSQSSHTSKYFGSIDSSEAEAGAARART

EPGDQVIKCVLQDPIWLLMANADQRVMMTYQVPSRDAASVLKQDRERLRAMQKQQPRFSEDQ

RRELGAVHSWVRKGQLPRALDVMACVDCGSSVQDPGHSDDPLFSELDGLGLEPMEEGGGEGG

GCGVGGGGGDGGEEAQTQIGAKGSSSQDSAMEEEEQGGGSSSPALPAEENSTS

SEQ?ID?No.7

gaattcggcacgagcagcgagacgccgcgcacggtgcttccccagtggagccaatcggctaacccgcgctccggcagagtccttggcgctcgcccg

ccggcgggacagaccacccgcctctggccgctctctggaccctggccgccccgagcgaagactggagcaaaatgatgcttcaacatccaggccaggt

ctctgcctcagaagtcagtgcgaccgccattgtcccctgcctctcacctcctgggtcactggtatttgaggattttgctaacctgacaccctttgtcaaggaa

gagctgagattcgccatccagaataaacacctctgccatcggatgtcctctgcgctggagtcagttaccgtcaacaacagacccctggagatgtcagtca

ccaagtctgaggcggcccctgaagaagatgagaggaaaaggaggcggcgagaaagaaataaaattgctgctgccaagtgtcgaaacaagaaaaag

gagaagacagagtgcctgcagaaagagtcagagaaactggagagtgtgaatgctgagctgaaggcccagattgaggagctgaagaatgagaaacag

catttgatatacatgctcaacctgcaccggcccacctgtatcgtccgggctcagaatggacggacaccggaagacgagaggaacctctttatccaacag

ataaaagaaggaacattgcagagctaagcagaggtggcacggaggcaattggggagttcttactgaatcctccttttccaccccacaccctgaagccatt

ggaaaactggcttcctgtgcacttctagaatcccagcagccaagagccgttggggcaggagggcctgtggtgacctactgcattgacccactctgcccc

cgagtgaaccgtggagcaggcaggagcatcctttgtctcaccaattccaggatttaggccttatcatcccggccagtctcagatgacctagctggcccca

ggctggggtcctatgcaaagcaggatcccactaatgggattcaggcagaagtgtctaccttgataggtggggtgggaccacatcctccactgtggctgac

aacgcccttccaagggaatatggaatgagaacattcattattgaggttgtccaatggccagggtatgctttctagaaaaatatgctgttctgtcccagaatgac

tgtgcatagggtatccgtttcagagcctggtgttgtgctatttagatgtttgtcttgcacaacattggcatgatttttccgggagtttcatcagatctgatttctgag

agtctggggatctgccatggtggaaagtgcccctcaaaagcatttgtgtggccacatgaactggctggcaccaggggagtgaaactggctgatgaccag

ctgagccactttgtgccaacagaggatggacgacacctttccctgtacccactgcagaggaagaaccctgggcacagcagctttgtccttggctacaaac

tgttacaacgtcacacaatgaaggcacaaagtccaactttcaaagggtgtaggactccatactcagtgacagggcaggaagagccaaagataaccaca

gccacagcctgtggagaccagggttggaagccaggtgcagggccaggcatctgcattgtgggatgttaatggcacttttgtcttgtagctattttgagatgt

ggtccagagcatttcagctgggagatctccctctggccaccaggactctggctactgttaaaatcctgatgtttctgtggaatcctcagtgtttaatcccactc

aatagtatcattacagttttctgtaagagaaaatattacttatttatcccagtattcctagcctgtcaacataataaatatcggaacaaaacctggta

SEQ?ID?No.8

MMLQHPGQVSASEVSATAIVPCLSPPGSLVFEDFANLTPFVKEELRFAIQNKHLCHRMSSALESV

TVNNRPLEMSVTKSEAAPEEDERKRRRRERNKIAAAKCRNKKKEKTECLQKESEKLESVNAELK

AQIEELKNEKQHLIYMLNLHRPTCIVRAQNGRTPEDERNLFIQQIKEGTLQS

SEQ?ID?No.9

cccagagataagctgacgctgggcaaacccctgggggaaggttgcttcgggcaagtagtcatggctgaagcagtgggaatcgataaagacaaaccca

aggaggcggtcaccgtggcagtgaagatgttgaaagatgatgccacagagaaggacctgtctgatctggtatcagagatggagatgatgaagatgattg

ggaaacataagaacattatcaacctcctgggggcctgcacgcaggatggacctctctacgtcatagttgaatatgcatcgaaaggcaacctccgggaata

cctccgagcccggaggccacctggcatggagtactcctatgacattaaccgtgtccccgaggagcagatgaccttcaaggacttggtgtcctgcaccta

ccagctggctagaggcatggagtacttggcttcccaaaaatgtatccatcgagatttggctgccagaaacgtgttggtaacagaaaacaatgtgatgaag

atagcagactttggcctggccagggatatcaacaacatagactactataaaaagaccacaaatgggcgacttccagtcaagtggatggctcctgaagcc

ctttttgatagagtttacactcatcagagcgatgtctggtccttcggggtgttaatgtgggagatctttactttagggggctcaccctacccagggattcccgt

ggaggaactttttaagctgctcaaagagggacacaggatggacaagcccaccaactgcaccaatgaactgtacatgatgatgagggattgctggcatgc

tgtaccctcacagagacccacattcaagcagttggtcgaagacttggatcgaattctgactctcacaaccaatgaggaatacttggatctcacccagcctct

cgaacagtattctcctagttaccccgacacaagtagctcttgttcttcaggggacgattctgtgttttctccagaccccatgccttatgaaccctgtctgcctca

gtatccacacataaacggcagtgttaaaacatga

SEQ?ID?No.10

PRDKLTLGKPLGEGCFGQVVMAEAVGIDKDKPKEAVTVAVKMLKDDATEKDLSDLVSEMEM

MKMIGKHKNIINLLGACTQDGPLYVIVEYASKGNLREYLRARRPPGMEYSYDINRVPEEQMTFK

DLVSCTYQLARGMEYLASQKCIHRDLAARNVLVTENNVMKIADFGLARDINNIDYYKKTTNGR

LPVKWMAPEALFDRVYTHQSDVWSFGVLMWEIFTLGGSPYPGIPVEELFKLLKEGHRMDKPTN

CTNELYMMMRDCWHAVPSQRPTFKQLVEDLDRILTLTTNEEYLDLTQPLEQYSPSYPDTSSSCS

SGDDSVFSPDPMPYEPCLPQYPHINGSVKT

SEQ?ID?No.11

acccacgcgtccggccggtttcactgctcccctcagtctcttttgggctctttccgggcatcgggacgatgaccgtcaaagccgaggctgctcgaagcac

ccttacctactccagaatgaggggaatggtagcgattctcatcgcttttatgaaacagagaaggatgggcctgaacgattttattcagaagattgccagcaa

cacctatgcatgcaaacacgctgaagttcagtccattttgaaaatgtcccatcctcaggagccggagcttatgaacgctaacccctctcctccgccaagtc

cctctcaacaaatcaacctgggtccgtcctccaaccctcacgccaaaccctccgactttcacttcttgaaagtgatcggaaagggcagttttggaaaggttc

ttctggctaggcacaaggcagaagaagtattctatgcagtcaaagttttacagaagaaagccatcctgaagaagaaagaggagaagcatattatgtcaga

gcggaatgttctgttgaagaatgtgaagcaccctttcctggtgggccttcacttctcattccagaccgctgacaaactctactttgtcctggactacattaatg

gtggagagctgttctaccatctccagagggagcgctgcttcctggaaccacgggctcgattctacgcagctgaaatagccagtgccttgggctatctgca

ctccctaaacatcgtttatagagacttaaaacctgagaatattctcctagactcccaggggcacatcgtcctcactgactttgggctctgcaaagagaatatt

gagcataacgggacaacatctaccttctgtggcacgcctgagtatctggctcctgaggtcctccataagcagccgtatgaccggacggtggactggtggt

gtcttggggctgtcctgtatgagatgctctacggcctgcccccgttttatagccggaacacggctgagatgtacgacaatattctgaacaagcctctccagt

tgaaaccaaatattacaaactcggcaaggcacctcctggaaggcctcctgcagaaggaccggaccaagaggctgggtgccaaggatgactttatggag

attaagagtcatattttcttctctttaattaactgggatgatctcatcaataagaagattacacccccatttaacccaaatgtgagtgggcccagtgaccttcgg

cactttgatcccgagtttaccgaggagccggtccccagctccatcggcaggtcccctgacagcatccttgtcacggccagtgtgaaggaagcagcagaa

gccttcctcggcttctcctatgcacctcctgtggattccttcctctgagtgctcccgggatggttctgaaggacttcctcagcgtttcctaaagtgttttccttac

cctttggtggaggttgccagctgacagaacattttaaaagaatttgcacacctggaagcttggcagtctcgcctgcccggcgtggcgcgacgcagcgcg

cgctgcttgatgggagctttccgaagagcacaccctcctctcaatgagcttgtgaggtcttcttttcttctcttccttccaacgtggtgctagctccaggcgag

cgagcgtgagagtgccgcctgagacagacaccttggtctcagttagaaggaagatgcaggtctaagaggaatccccgcagtctgtctgagctgtgatca

agaatattctgcaatgtgccttttctgagatcgtgttagctccaaagctttttcctatcgcagagtgttcagtttgtgtttgtttgtttttgttttgttttgtttttcccttg

gcggatttcccgtgtgtgcagtggcgtgagtgtgctatgcctgatcacagacggttttgttgtgagcatcaatgtgacacttgcaggacactacaatgtggg

acattgtttgtttcttccacatttggaagataaatttatgtgtagactgttttgtaagatatagttaataactaaaacctattgaaacggtcttgcaatgacgagcat

tcagatgcttaaggaaagcattgctgctacaaatatttctatttttagaaagggtttttatggaccaatgccccagttgtcagtcaaagccgttggtgttttcattg

tttaaaatgtcacctataaaacgggcattatttatgttttttttccctttgttcatattcttttgcattcctgattattgtatgtatcgtgtaaaggaagtctgtacattgg

gttataacactagatatttaaacttacaggcttatttgtaaaccatcattttaatgtactgtaattaacatgggttataatatgtacaattcctcctccttaccacaca

actttttttgtgtgcgataaaccaattttggtttgcaataaaatcttgaaacct

SEQ?ID?No.12

MTVKAEAARSTLTYSRMRGMVAILIAFMKQRRMGLNDFIQKIASNTYACKHAEVQSILKMSHP

QEPELMNANPSPPPSPSQQINLGPSSNPHAKPSDFHFLKVIGKGSFGKVLLARHKAEEVFYAVKV

LQKKAILKKKEEKHIMSERNVLLKNVKHPFLVGLHFSFQTADKLYFVLDYINGGELFYHLQRER

CFLEPRARFYAAEIASALGYLHSLNIVYRDLKPENILLDSQGHIVLTDFGLCKENIEHNGTTSTFCG

TPEYLAPEVLHKQPYDRTVDWWCLGAVLYEMLYGLPPFYSRNTAEMYDNILNKPLQLKPNITN

SARHLLEGLLQKDRTKRLGAKDDFMEIKSHIFFSLINWDDLINKKITPPFNPNVSGPSDLRHFDPE

FTEEPVPSSIGRSPDSILVTASVKEAAEAFLGFSYAPPVDSFL

SEQ?ID?No.13

gcagggcgggtgagagcgccgtgaaagccgcggaacgccgtgcacctccgcgactctactacggcaagctagtccggacgggtcgtcgtccccgc

gcgccaccagcccttggtgaaacgacagggagcgtccggcttccccagcaccgccctgcgagactcaaaacagccacaccgcaaagcgagcctcg

ggcggaaggaggcggagcttcaggcggccccgcctccgcggaaggatacacatctccgtggtccaaaaccccggggcgaggcggccggggcgtg

tgagctgctcggccagctgccgtctacgcgctttcgcgcggccaccgggcaactgcgccgcgcggctgccccgctgagcgctcggcctcggggccgt

gggatccgccgcgctgtctgcggtcaggaagaccgccctcccgcgtccttgccggacgggtcagaggcggcaccgcacgcgaggccacccgcgat

gctgctgtccaagttcggctccctggcgcacctctgcgggcctggcggcgtggaccacctcccagtgaagatcctacagccagccaaggctgacaag

gagagcttcgagaaggtgtaccaggtgggcgccgtgctgggcagcggcggcttcggcacggtctacgcgggcagccgcatcgccgacggactccc

ggtggctgtgaagcacgtggtgaaggagcgggtgaccgagtggggcagtctcggcggagtggccgtgcccctggaggtggtgctgctgcgcaaggt

gggcgcggcgggcggcgcgcgcggcgtcatccgcttgctggactggttcgagcggcccgacggcttcttgttggtgctggagcgacccgagccggc

acaggacctcttcgacttcatcactgaacgaggcgccctggacgagccgctggcgcgtcgcttcttcgcgcaggtgcttgccgctgtgcggcactgcca

caattgtggggtcgtgcaccgcgacatcaaggacgagaacctgctggtggacctgcgctcgggagagctgaagctcatcgacttcggctcgggcgcg

gtgctcaaggacacggtctacactgactttgatggcacccgtgtgtacagccccccagagtggatccgatatcaccgatatcacgggcggtctgccactg

tgtggtctctgggtgtactgctctacgacatggtgtgtggggacattccctttgagcaggatgaggagatcttgcgcggcaggctctttttccggaggaggg

tctccccagagtgccagcagcttattgagtggtgtctctccctgaggccctcagagaggccctccctggaccaaattgctgcccacccctggatgctggg

gacagaggggagcgttccagagaactgtgaccttcggctttgtgccctggatactgacgacggagccagtaccacttccagcagtgagagcttgtgagg

aggagaaggggcctgggctcggcctagccagcgctctcccagaattgaacactttctgcctgggatgtctgctgcaaaagcagtgacctctgacccctg

gtgacctttgctctcggcaccgggcctgtttcctttgctttgagtgcctttttgaacgctgctccacagggcctgggttttcttgagctcttctgtccaaagatgg

ctgagggctaagcaaggtcctgccctgggtggatacttgaaccagagatcccgaccctgctgctccatctcaggaggcagccttcctgaccaagtgtgtt

tgacatggagcgccctgtggtgcccacctccaaccctccagtctcctggtgttcatctgggcatgtctgcacaagcaatgcaacgctgggccactgctgc

ccgtctgcctccccggcacggcacggctccgcacgcaacctaagcgtgccaccacggtctcttatttatggtgtgatcaccctggagggcgcccccgcc

ctgctggggctatttattgtttaatttatttgctgaggttcctccaagcaaccaccttctccaggcccctggggtgttgaaagtcaaatgtggctgttgagtcca

cagacccccatcctaattcctgcacctggaggagttccccaacccccgtgtttgcgggaggaagcatttgtacagtggctaatttaaggggagtgggaga

ccctgtcaccctgagcactctgcgctggggaggggtttaaattattgaccttgtacagtctgcttgctggctctgaaagctggggttgggggacagagtctc

aagcccttaatttattttagcagctgtgtttctgtgaccctggtgtgactaagcatcaggggtggggttgtataagttcaaaagtgtgaaatgtctgaagatcat

attttttatacaggtatttcaattaaatgttttggtatataatggaaaaaaaaaaaaaaaaaaaaaaaaaaaaa

SEQ?ID?No.14

MLLSKFGSLAHLCGPGGVDHLPVKILQPAKADKESFEKVYQVGAVLGSGGFGTVYAGSRIADGL

PVAVKHVVKERVTEWGSLGGVAVPLEVVLLRKVGAAGGARGVIRLLDWFERPDGFLLVLERPE

PAQDLFDFITERGALDEPLARRFFAQVLAAVRHCHNCGVVHRDIKDENLLVDLRSGELKLIDFGS

GAVLKDTVYTDFDGTRVYSPPEWIRYHRYHGRSATVWSLGVLLYDMVCGDIPFEQDEEILRGR

LFFRRRVSPECQQLIEWCLSLRPSERPSLDQIAAHPWMLGTEGSVPENCDLRLCALDTDDGASTT

SSSESL

SEQ?ID?No.15

cctgggccccgccgcggacgcgcggagccgcctgggccgcgccggaggagggcggggagaggaccatgtgaatgtgctccggagctgagcgcc

aagccaagcagtgtttgaaaggaacaggatgctgatctaatcgtggcaaaaagtcagtccgaccgctggtttcgaagacatgtggtgtatataaagtttgt

gatagttggtggaaatttgggagcttggataatgggctgtgtgcaatgtaaggataaagaagcagcgaaactgacagaggagagggacggcagcctga

accagagctctgggtaccgctatggcacagaccccacccctcagcactaccccagcttcggcgtgacctccatcccgaactacaacaacttccacgca

gctgggggccagggactcaccgtctttgggggtgtgaactcctcctctcacactgggaccctacgcacgagaggagggacaggagtgacactgtttgt

ggcgctttatgactatgaagcacggacggaagatgacctgagttttcacaaaggagaaaaatttcaaatattgaacagctcggaaggagattggtgggaa

gcccgctccttgacaaccggggaaactggttacattcccagcaattacgtggctccagttgactccatccaggcagaagagtggtactttggaaaacttgg

ccgcaaagatgctgagagacagctcctgtcctttggaaacccaagaggtacctttcttatccgcgagagccaaaccaccaaaggtgcctactcactttcc

atccgtgattgggatgatatgaaaggggaccacgtcaaacattataaaatccgcaagcttgacaatggtggatactatatcacaacgcgggcccagtttga

aacacttcagcaactggtacagcattactcagagaaagctgatggtttgtgttttaacttaactgtggtttcatcaagttgtaccccacaaacttctggattggc

taaagatgcttgggaagttgcacgtgactcgttgtttctggagaagaagctggggcaggggtgtttcgctgaagtgtggcttggtacctggaatggaaata

caaaagtagccataaagacccttaagccaggcaccatgtctccggagtccttcctggaggaggcgcagatcatgaagaagctgaagcatgacaagctg

gtgcagctctacgcggtcgtgtctgaggagcccatttacatcgtcacggagtacatgagcaaaggaagtttgcttgacttcttaaaagatggtgaaggaag

agctctgaagttgccaaaccttgtggacatggcggcacaggttgctgcaggaatggcttacatcgagcgcatgaattatatccacagagatctgcgatca

gcaaacattctagtggggaatggactaatttgcaagattgctgactttggattggctcggttgattgaagacaatgaatacacagcaagacaaggtgcgaa

gtttcccattaagtggacagcccccgaagcggccctgtatggaaggttcacaatcaagtctgacgtatggtcttttggaatcttactcacagagctggtcac

caaaggaagagtgccatacccaggcatgaacaaccgggaggtgctggagcaggtggagagaggctataggatgccctgcccacaggactgcccgat

ctccctgcacgagctcatgatccactgctggaaaaaggatccggaagagcgcccgaccttcgagtacttgcagggcgcctggaggactactttacggc

cacagagccccagtatcagcccggtgaaaacctgtgagagcctgcgcttcagacgcctcttcccgaggcctccctacccctccccattagcttccaattct

gtagccagctgccccagagcaggagaaccgtccaggatcagattgcatgtgactcttgaagctgaacttccacggccctcattaatgacacttgtccccc

agtccgaacctcctctgtgaaccatctgagacagaagcgtgttatttcccagacttggaaatgcattgtatcgacgctatgtcaaaggccaaacctctgttcag

tgcaaatagctgctcctgtgccaacaatcccagtgctttccttttttaaaaaagaaaaagcaaaccctatgtgattttaactctgatttcacctgattcaactaaa

aaaaaaaaagtattattttccaaaagtggcctctttgtctaaaacaataaaattttttttcatgttttaacaaaaaaaaaaaaaaaaaaaaaaaa

SEQ?ID?No.16

MGCVQCKDKEAAKLTEERDGSLNQSSGYRYGTDPTPQHYPSFGVTSIPNYNNFHAAGGQGLTV

FGGVNSSSHTGTLRTRGGTGVTLFVALYDYEARTEDDLSFHKGEKFQILNSSEGDWWEARSLTT

GETGYIPSNYVAPVDSIQAEEWYFGKLGRKDAERQLLSFGNPRGTFLIRESQTTKGAYSLSIRDW

DDMKGDHVKHYKIRKLDNGGYYITTRAQFETLQQLVQHYSEKADGLCFNLTVVSSSCTPQTSG

LAKDAWEVARDSLFLEKKLGQGCFAEVWLGTWNGNTKVAIKTLKPGTMSPESFLEEAQIMKKL

KHDKLVQLYAVVSEEPIYIVTEYMSKGSLLDFLKDGEGRALKLPNLVDMAAQVAAGMAYIERM

NYIHRDLRSANILVGNGLICKIADFGLARLIEDNEYTARQGAKFPIKWTAPEAALYGRFTIKSDV

WSFGILLTELVTKGRVPYPGMNNREVLEQVERGYRMPCPQDCPISLHELMIHCWKKDPEERPTF

EYLQGFLEDYFTATEPQYQPGENL

SEQ?ID?No.17

ggacgtcagactagagagtagggagagagactggtgctcgagggacagggctagcccggacgcgtgtccgcgcctcggaggtggcaagtaggcag

tgtcgggtggcgaggcaacgatggagctcctgcggactatcacctaccagccggccgccggcaccaagatgtgcgagcaggctctgggcaaagcttg

cggcggggactcaaagaagaagcgaccacagcagccttctgaagatgggcagccccaagcccaggtgaccccggcggccccgcaccaccatcac

caccattcccactcgggacccgagatctcgcggattatagtcgaccccacgacggggaagcgctactgccggggcaaagtgctgggcaagggtggat

ttgcaaagtgttacgaaatgacagatctgacaaacaacaaagtctacgctgcaaaaattattcctcacagcagagtagctaaacctcatcagagggaaaa

gatcgacaaagaaatcgagcttcacagactactgcaccataagcatgtcgtgcagttttaccactactttgaagacaaagaaaacatttacattctcttggaa

tagtgcagtagaaggtccatggctcacatcttgaaagcaagaaaggtgttgacagagccagaagtccgatactacctcaggcagattgtgtcaggactca

agtatcttcacgaacaagaaatcttgcacagggatctcaagctagggaactttattattaatgaagccatggagctgaaggtgggagactttggtttggcag

ccagactggaaccactggaacacagaaggagaacaatatgtggaaccccaaattatctctcccccgaagtcctcaacaaacaaggacacggctgtgaa

tcagacatctgggccttaggctgtgtaatgtatacgatgctgctaggaagacctccattcgaaaccacaaatctgaaagaaacgtacaggtgcataaggg

aagcaaggtataccatgccgtcctcattgctggcccctgctaagcacttgatagctagcatgctgtccaaaaacccagaggaccgccccagtttggatga

catcattcggcatgacttcttcctgcagggtttcactccggacagactctcttccagctgttgccacacagttccagatttccacttgtcaagcccagccaag

aatttctttaagaaagccgcagccgctctttttggtggcaagaaggacaaagcaagatataacgacacacacaataaggtgtctaaggaagatgaagaca

tttacaagcttcggcatgatttgaagaaagtgtcgataacccagcagcctagcaaacacagagcagacgaggagccccagccgcctcccactactgttg

ccagatctggaacgtccgcagtggaaaacaaacagcagattggggatgcaatccggatgatagtcagggggactctcggcagctgcagcagcagca

gcgaatgccttgaagacagcaccatgggaagtgttgcagacacagtggcaagagtccttcgaggatgtctagaaaacatgccggaagctgactgtatcc

ccaaagagcagctgagcacgtcctttcagtgggtcaccaagtgggtcgactactccaacaaatatggctttgggttccagctctcggaccacactgttgg

cgtccttttcaacaacggggctcacatgagcctccttccggacaaaaagacagttcactattatgcggaacttggccaatgctctgttttcccagcaacaga

tgcccctgaacaatttattagtcaagtgacggtgctgaaatacttttctcattacatggaggagaacctcatggatggtggtgatctcccgagtgttactgaca

ttcgaagacctcggctctacctcctgcagtggttaaagtctgataaagccttaatgatgctcttcaatgacggcacatttcaggtgaatttctaccacgatcat

acaaaaatcatcatctgtaaccagagtgaagaataccttctcacctacatcaatgaggacaggatctctacaactttcagactgacgactctgctgatgtctg

gctgttcgttagaattgaaaaatcgaatggaatatgccctgaacatgctcttacagagatgtaactgaaaacattattattattattattataattatttcgagcgg

acctcatgggactcttttccactgtgagatcaacagggaagccagcggaaagatacagagcatgttagagaagtcggacaggtggtggtacgaatacaa

ttcctctgtggcctgctggactgctggaaccagaccagcctaaggtgtagagttgactttggacaatcctgagtgtggagccgagtgcagttttccctgaga

tacctgtcgtgaaaaggtttatgggacagtttttcagaaagatgcattgactctgaagttctctctgttgagagcgtcttcagttggaagacttggaactgtga

atacacttcctgaaggggagggagaagggaggttgctcccttgctgtttaaaggctacaatcagagcagcttttggctgcttaactgtgaactatggccata

catttttttttttttggttatttttgaatacacttgtggttggaaaagtgcattccttgttaataaactttttatttattacagccccaagagcagtatttattatcaagat

gttctctttttttatgttgaccatttcaaactcttggcaataaagagtatgacatagaaaaaaaaaaaaaaaaaaaaaaaaa

SEQ?ID?No.18

MELLRTITYQPAAGTKMCEQALGKACGGDSKKKRPQQPSEDGQPQAQVTPAAPHHHHHHSHS

GPEISRIIVDPTTGKRYCRGKVLGKGGFAKCYEMTDLTNNKVYAAKIIPHSRVAKPHQREKIDKE

IELHRLLHHKHVVQFYHYFEDKENIYILLEYCSRRSMAHILKARKVLTEPEVRYYLRQIVSGLKY

LHEQEILHRDLKLGNFIINEAMELKVGDFGLAARLEPLEHRRRTICGTPNYLSPEVLNKQGHGCE

SDIWALGCVMYTMLLGRPPFETTNLKETYRCIREARYTMPSSLLAPAKHLIASMLSKNPEDRPSL

DDIIRHDFFLQGFTPDRLSSSCCHTVPDFHLSSPAKNFFKKAAAALFGGKKDKARYNDTHNKVSK

EDEDIYKLRHDLKKVSITQQPSKHRADEEPQPPPTTVARSGTSAVENKQQIGDAIRMIVRGTLGS

CSSSSECLEDSTMGSVADTVARVLRGCLENMPEADCIPKEQLSTSFQWVTKWVDYSNKYGFGY

QLSDHTVGVLFNNGAHMSLLPDKKTVHYYAELGQCSVFPATDAPEQFISQVTVLKYFSHYMEE

NLMDGGDLPSVTDIRRPRLYLLQWLKSDKALMMLFNDGTFQVNFYHDHTKIIICNQSEEYLLTYI

NEDRISTTFRLTTLLMSGCSLELKNRMEYALNMLLQRCN

SEQ?ID?No.19

aacttagctggactgcagccttctccgctggaactcgccaagccagctgatttccccatccaaagccatgaagagcggcgtatgtctgtgcgtggtgatg

gcagtcctagctgctggcgccctggcgcagccggtagtccctgcagaagctacggaccccgtggagcagcgggcgcaagaggcgccccgaaggca

gctgcgggctgtgctccggacggacggcgagccccgagcgcgcctgggcgcactgctagcgcgatacatccagcaggtccgcaaagctccttctgg

ccgcatgtccgttcttaagaacctgcagagcctggaccccagccatagaataagtgaccgggactacatgggctggatggattttggccggcgcagtgc

cgaggactacgaatacccatcgtagtgggccagcgtcttggccctgcttggaggaggtggaatgaggaaacaaccacacatacgacccctcgcctcta

atgtctgacgttttgagtatctatttattaagtccccaatgtgaaatctgtccagagtgtgcaatgcagccacatctcagcctagctgtgtggtcggaaggcag

tgtttccttcagtgactcccagacctaatgttgctatgctattaaagagatttccttctgcccccc

SEQ?ID?No.20

MKSGVCLCWMAVLAAGALAQPVVPAEATDPVEQRAQEAPRRQLRAVLRTDGEPRARLGALL

ARYIQQVRKAPSGRMSVLKNLQSLDPSHRISDRDYMGWMDFGRRSAEDYEYPS

SEQ?ID?No.21

cttggtgacactagacagagcaactccagcgttaccgctcccgctcctggtttctcggcttctcatcgcagtcaatcttggactttggggttttgctactgtca

gaaggacttctttctgcttcaagtgcttgacaacgcacccctttatcagggtatcagagcatcgccacagaatgaagctggtttccatcaccctgatgttattg

ggttcactcgctttcctaggcgcggacactgcagggccagatactccttcgcagttccgaaagaagtggaataagtgggcgctaagtcgtgggaagagg

gaactacaagcatccagcagctaccctacgggactcgctgatgagacgacagttcctacccagactcttgatccattcctggaacgagcagaacacaact

ggccccctacaagccagcaatcagagcgaagcccacattcgtgtcaaacgctaccgccagagcatgaaccagggttcccgcagcaatggatgccgct

tcgggacctgcacatttcagaaattggcccaccagatctaccagctaacagacaaagacaaggacggcatggctcccagaaacaagatcagccctcaa

ggctatggccgccggcgccggcgttccctgctggaggtcctccggtcccggactgtggagtcctcccaggagcagacacacacagccccaggcccct

gggcgcacatctccagactctttaggatataggtgcgggtgacagcattgaacagtcgggcgagtatcccgttggcgcctgcggaatcagagaacttcg

caccggggcggactgagacaatcctgcagagatctgcctggctgcccctaggggaggcagaggaacccaagaccaagccaggctcatgccagaaa

ccgagacttacaggctgatactctccgggcaggggtctgagccactgccttgcccgctcataaactggtttctcacggggcataagcctcattactacttg

aactttccaaaacctagcgaggaacgtgcaatgcttgttgtccagccaaaggtaactatagtatttaagtttgttgctgtcaaggtttttttttttgtaacttcaaat

atatagagatatttttgtacgttatatattgtattaagggcattttaaagtgattatattgtcaccttcccctattttaagacgtgaatgtctcagcaaggtgtaaggt

tgtttggttccgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtaaggtggagagcgcctgattatcgcctgtggatgaagaaaaaacattgtgtttcc

tataatctatttacataaaatatgtgatctgggaaaaagcaaaccaataaactgtctcaatgctg

SEQ?ID?No.22

MKLVSITLMLLGSLAFLGADTAGPDTPSQFRKKWNKWALSRGKRELQASSSYPTGLADETTVPT

QTLDPFLDEQNTTGPLQASNQSEAHIRVKRYRQSMNQGSRSNGCRFGTCTFQKLAHQIYQLTDK

DKDGMAPRNKISPQGYGRRRRRSLLEVLRSRTVESSQEQTHTAPGPWAHISRLFRI

SEQ?ID?No.23

tcgagcggccgcccgggcaggtccaggatcaagagtcaccgcttcgcaagcactgcctggctccatcaggatccccgcaggctcagctccaaggcac

cgctcaccaggaaggcatcatgggcttcctgaagttctcccctttcctggttgtcagcatcttgctcctgtaccaggcatgcagcctccaggcagtgcctttg

aggtcaatcttggaaagcagcccaggcatggccactctcagtgaagaagaagttcgcctgctggctgcactggtgcaggactatatgcagatgaaagcc

agggagctggagcaggaggaagagcaggaggctgagggctctagcttggacagccccagatctaagcggtgtgggaatctgagtacctgcatgctg

ggcacgtacacacaagacctcaacaagtttcacaccttcccccaaacttcaattggggttgaagcacctggcaagaaaagggatgtggccaaggacttg

gagacaaaccaccaatcccattttggcaactaagctccttctctcctttctagtttccttcttgctttcttcctataacttgatgcatgtagttcctctctggttgctc

tccaggctattactggttgctttcctgaggcaaagaatggtatctgaaatccccagtgggtgaggagaaagtcccacaggctaaaagagaatcacccagg

aagatggcagagagcaagggcacactcaggaagatggcagagagcaagggcagtcatctggcttcctagtagagcttctagtcttgcttctggaagtgtt

ggttgtttgggaaataaaactattttttaaaaaaaaaaaaaaaaaaa

SEQ?ID?No.24

MGFLKFSPFLVVSILLLYQACSLQAVPLRSILESSPGMATLSEEEVRLLAALVQDYMQMKARELE

QEEEQEAEGSSLDSPRSKRCGNLSTCMLGTYTQDLNKFHTFPQTSIGVEAPGKKRDVAKDLETN

HQSHFGN

SEQ?ID?No.25

aaaggcagcctgataaagctccttgtgacaggctgtcttgccagtctcccagtatgctcctcttgctctgaagtgctccaggattgaaaccacagcttccca

aattagcctgggaagagtgtgcggacccagcagccttttaacccgcgtcagtgcctttgctatgttcaagactgctgttttggatggtgaatgctagctagc

actccatcgagacatgacagcaaaaaattctccaaaagaatttactgcttcggaatctgaggtttgcataaagactttcaaggagcagatgcgcttggaact

tgagcttccaaagctaccaggaaacagacctacatctcccaaaatttctccacgcagttcaccaaggaattcaccatgctttttcagaaagttgctggtgaat

aaaagcatccgacagcggcgtcgcttcacggtggctcatacatgctttgatgtggaaaatggcccttctccaggtcggagcccactggaccctcaagcc

ggctcttcgtcgggactggtacttcatgccgcctttcctgggcacagccagcgcagggagtcgttcctctacgatcttgacagcgactatgacttgtcacca

aaagcgatgtccaggaactcatcacttcccagtgagcaacacggcgatgacctgattgtcactccttttgcccaggttcttgccagcttgcgaagtgtaaga

aacaacttcaccctgctgacgaaccttcatggagcgccgaacaagaggtcaccagcggctagtcaggctccagtctccagagtcagcctgcaagagga

atcatatcagaaactagcaatggagacgctggaggaactagactggtgcctagaccagctagagaccatccagacctaccgctctgtcagcgagatgg

cttcaaacaagttcaaaaggatgctgaaccgggagctgacacacctctcagagatgagcagatcagggaaccaggtgtctgagtacatttcaaacacgtt

cttagacaagcagaacgatgtggaaatcccatctcccacgcagaaggacagggagaagaagaagaagcagcagctcatgacccagataagtggagt

gaagaaactgatgcacagctcaagcctgaacaacacaagcatctcacgcttcgggatcaacacggaaaatgaggatcatctagccaaggagctggaa

gacctgaacaaatggggccttaacatcttcaatgtggctgggtactcacataatcggccccttacgtgcatcatgtatgcaatattccaggaaagagacctt

ctgaagacgtttaaaatctcatctgacacctttgtaacctacatgatgactttagaagaccattaccattctgatgtggcatatcacaacagcctgcatgctgct

gacgtggcccagtcaactcacgttctcctttctacgccggcactggatgctgtcttcacagacctggaaatcctggctgccatttttgcagctgccatccatg

atgtcgatcatcctggagtctccaatcagtttctcatcaatacaaattctgaacttgctttgatgtataatgatgaatctgttctggaaaaccatcaccttgctgtg

ggattcaaattgctacaagaggaacactgcgacatctttcagaatcttaccaagaagcaacgccagacactcaggaaaatggtgattgacatggtgttgg

caactgatatgtccaaacacatgagcctcctggcagaccttaaaacaatggtagaaaccaagaaggtgacaagctccggtgttctcctcctggacaacta

tactgaccggatacaggttcttcgcaacatggtacactgtgcagacctgagcaaccccaccaagtccttggaattgtatcggcaatggaccgatcgtatca

tggaggagtttttccagcagggagacaaagaacgggagaggggaatggagattagcccaatgtgtgataagcacacagcttctgtggaaaaatcccaag

gttggtttcattgactacattgtccatccactgtgggagacctgggcagacctggttcaaccggatgctcaagatattctggatacactagaagataacagg

aactggtaccagagtatgataccccagagcccttccccgccactggatgagaggagcagggactgccaaggcctgatggagaagtttcagtttgaactg

acccttgaggaagaggattctgagggaccggaaaaggagggagaaggccacagctatttcagcagcacaaagacgctttgtgtgattgatccagagaa

cagggattctctggaagagactgacatagacattgcaacagaagacaagtctccgatcgacacataatctctctccctctgtgtggagatgaacattccac

ccttgactgagcatgcccgctgagtggtagggtcacctaccatggccaaggcctgcacaggacaaaggccacctggcctttccagttacttgagtttgga

gccagaatgccaggccgtgaagcaaatagcagttccatgctgtcttgccttgcctgcaagcttggcggagacccgcagctgtatgtggtagtagaggcc

agttcccatcaaagctaaaatggcttgaaaacagaggacacaaagctgagagattgctctgcactaggtgttgggaagctgtcctgacagatgactgaac

tcactaacaacttcatctataaatctcaccacccaacccattgtctgccaacctgtgtgcctttttttgtaaaatgttttcgcgtctttgaaatgcctgttgaatatc

tagagtttagtaccaacttctacaaacttttttgagtctttcttgaaaaacaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa

SEQ?ID?No.26

MTAKNSPKEFTASESEVCIKTFKEQMRLELELPKLPGNRPTSPKISPRSSPRNSPCFFRKLLVNKSI

RQRRRFTVAHTCFDVENGPSPGRSPLDPQAGSSSGLVLHAAFPGHSQRRESFLYDLDSDYDLSPK

AMSRNSSLPSEQHGDDLIVTPFAQVLASLRSVRNNFTLLTNLHGAPNKRSPAASQAPVSRVSLQE

ESYQKLAMETLEELDWCLDQLETIQTYRSVSEMASNKFKRMLNRELTHLSEMSRSGNQVSEYIS

NTFLDKQNDVEIPSPTQKDREKKKKQQLMTQISGVKKLMHSSSLNNTSISRFGINTENEDHLAKE

LEDLNKWGLNIFNVAGYSHNRPLTCIMYAIFQERDLLKTFKISSDTFVTYMMTLEDHYHSDVAY

HNSLHAADVAQSTHVLLSTPALDAVFTDLEILAAIFAAAIHDVDHPGVSNQFLINTNSELALMYN

DESVLENHHLAVGFKLLQEEHCDIFQNLTKKQRQTLRKMVIDMVLATDMSKHMSLLADLKTM

VETKKVTSSGVLLLDNYTDRIQVLRNMVHCADLSNPTKSLELYRQWTDRIMEEFFQQGDKERE

RGMEISPMCDKHTASVEKSQVGFIDYIVHPLWETWADLVQPDAQDILDTLEDNRNWYQSMIPQ

SPSPPLDERSRDCQGLMEKFQFELTLEEEDSEGPEKEGEGHSYFSSTKTLCVIDPENRDSLEETDID

IATEDKSPIDT

SEQ?ID?No.27

ggccgcgcgggagtctgagaatgcaaagtgccatgttcctggctgtccagcacgactgcgtacccatggacaagagtgcaggcaacggccccaaggt

cgaggagaagcgggagaaaatgaagcggacactcttaaaccattggaagacccgtttgagctacttcttgcagaattcctctgctcctgggaagcccaa

aactggcaagaaaagcaaacagcaaacttttatcaagccttctcctgaggaagcgcacgtctgggcagaagcatttgatgaactgctggccagtaaatat

gggctggctgcattcagggcgtttttaaagtccgagttctgtgaagaaaacattgaattctggttggcttgtgaagacttcaaaaaaaccaaatcaccccaa

aaactgtcctcaaaagcaaggaaaatctataccgacttcatagagaaggaagctcccaaagagataaacatagacttccaaacgaaatctctgattgccc

aaaatatccaagaggctacaagtggctgcttcaccacagctcagaagagggtgtacagtttgatggagaacaattcttatcctcggttcttggagtccgaat

tctaccaggacttatgtaaaaagccacagatcaccacggagccccatgctacatgagaccaggagtccccccacacacaaaggacattccattctgtctc

ccaagagcaaaggctgtgacctgccagaaaaaaaaaaaaaactgaccttgaattcagcctgagtgttaggaaaacatcgctcagaactattgattcaatg

ttgggtagtgaatcaggaagtcagcaacctaggagaggctctgtgtgagaacggcttccctcactgtgtgaagaacagagggagggaacaggcctctg

aatgtgttcttcctccttgtcgggaaagcagagtttgagatgaaagatccgatgcaatgttgttggagcatttaaaatcaagaggtctgggattatgtggcctt

agctagttggctgtacaccttccctaaactagtccatgttacacatagtggtgttagttctagttttaatattttagtactaagtaacattacaatgtttactgtgtg

caagggtgttgacgttcttaggactacagatcattagtactagtgtgtcacgtatcactgaaactgagaagtatgtttgagttgttaaatggtgtgtgtgatgga

ccgaatgctgtgccgtgctgtagaa

SEQ?ID?No.28

MQSAMFLAVQHDCVPMDKSAGNGPKVEEKREKMKRTLLNHWKTRLSYFLQNSSAPGKPKTGK

KSKQQTFIKPSPEEAHVWAEAFDELLASKYGLAAFRAFLKSEFCEENIEFWLACEDFKKTKSPQK

LSSKARKIYTDFIEKEAPKEINIDFQTKSLIAQNIQEATSGCFTTAQKRVYSLMENNSYPRFLESEF

YQDLCKKPQITTEPHAT

SEQ?ID?No.29

aaaaagtatatgaggacaaatgtaaggcaaatgaccatggaaacagttgaatcacagcaggatcgaagtgtaacacgttctgtggcagagcatagctct

gctcatatgcagactggtcaaatttctgttcctactctagctcaggtagcaacaattgcagagacagatgattctgcagactcagaagtaattgattcgcata

aacgtagagaaattctttcacgaagaccctcatatagaaaaatactgaatgaactttcctctgatgtgcctggtattcccaagattgaagaagaaaaatcaga

ggaagaagggacaccacctaacattgctaccatggcagtaccaactagcatatatcagactagcacggggcaatacaatgaggagactgaccttgccc

caagtcacatggctgctgccacaggtgacatgccaacttaccagatccgagctcctactactgctttgccacaaggtgtggtgatggctgcctcaccagg

aagcctgcacagtccccagcaactagcagaagaagcaactcgcaagcgggagctgaggctgatgaaaaacagggaagctgcccgggagtgtcgca

ggaagaagaaagaatatgtcaaatgtcttgaaaatcgtgtggctgtgcttgaaaatcaaaacaagaccctcattgaggaactcaaggccctcaaagacctt

tattgccataaagcagagtaactgtgtttgatttggaccttgttgactgtgaactctaatcggggcaggcgatgcagcatcctcataatggccatgtggactt

gtagatgggtctcttaacccttgcttaagaatacagtctgctgtagagtgtgaattgggaatactgttccatgggttggaatgcagctcccctcacattaccaa

gcttgctctattgccaatagcatgcaacatatgttttgtttgcccttctgcttctacttttttcagggaagctgctaaagaatgtcgacgtcgaaagaaagagtat

gtgaagtgtcttgagagtcgagtcgcagtgctggaagttcagaacaagaagcttatagaggagcttgaaactttgaaagacatttgctctcccaaaacaga

ttagtagaaatatttaactatgaactgattacagcatgtacagttgcttttgaatgcaatacaaatatatagccggcaagaattatggctttttcctttgtatcattc

atctaactttctaaaactaacattcctaagatgctttgttgtatttaatttgctcttacctctaaggtcaattttttagaagagacaaactcaaaaaatgtatgtaac

aaattcttaaaatgaagtatttgtaagacttgttccagtcaacatatttacagttcccagtctctctgtcatgaatagtgtcctatgcaataaaaattttgcaggttt

taagaatcattttaggaaagggtgaatcaaaggcagtgcatctctccagtagtaagataaaatcaacccatagagatacctcaggaaagaatgaaaggaa

gtgtatcctgatgacatgacgtgagaatagcctacaaatgaatttatgcatttatagatttttataatcgtcactttgtaaagaaagtattgtattgctgtccttgg

gtgccacagttgaagacagttttaaatagaaccatgttggttgctctttgtactatttggtatttatttaagtatctgagcatttactacagcttcctactatgtatgt

agtatgtgaatttctacaaaagtttgtgctctttgctgttatttaatgaaagagacaacatattttcattatctggaatgagttccacaagtatgaatttattgctaca

ctggatcagcagccttgcaaatactgggccatttcattagaggacaacagcagggctctaggagcagagttcagtgtggagcacttgcctggcatgctac

atgttcagttgaaagggaagacctcaagctctgcaaatggaatggggtccaggggaagaggttagaggttagcctttgtgctgtactaggcttcttgctgat

cgtctggagagtttctgctgatgaccctccattgtgaattcttgcaacctcaggaatgttaacgtttaaaaaacttcccaagatgtcatttttgattttacaacttg

gatcaattttgttttgctctttggaatatagctgtgaacatttgtcacgtaggtttaggctggccttaaactcacagttctcttgcctcagccttctgagtgcttgga

ttacggatgtgggccagaatatccagtttgatcaagtattcttttataaaatattactttcttttt

SEQ?ID?No.30

MTMETVESQQDRSVTRSVAEHSSAHMQTGQISVPTLAQVATIAETDDSADSEVIDSHKRREILSR

RPSYRKILNELSSDVPGIPKIEEEKSEEEGTPPNIATMAVPTSIYQTSTGQYNEETDLAPSHMAAAT

GDMPTYQIRAPTTALPQGVVMAASPGSLHSPQQLAEEATRKRELRLMKNREAARECRRKKKEY

VKCLENRVAVLENQNKTLIEELKALKDLYCHKAE

SEQ?ID?No.31

gctgaagcgtttcctcaagcctgccggggtgggaggagaggaggaggtggtggtggtggaggaggtggaggcagagggtggagagagagaaagc

gcacgccgagaggaggtgtgggtgttccgctcccatcctaacggaacgagctccctcttcgcggacatgggattgcccagcggctgctaacccctctcc

tggtcctgatcccccaaaccggcgtggctccccggtcaccaaggagctgattacaagggaccaggatttgcatccttggctgggcgtccattggctaca

gagtgcctgacctgggtcaggctttccaacacggacatgtctgacaaaatgtcgagtttcctacatattggagacatttgttctctgtatgcggagggatcta

cgaatggatttatcagcaccttaggcttggttgatgaccgttgtgttgtacagccagaagccggggaccttaacaatccacccaagaaattcagagactgc

ctctttaagctatgtcctatgaatcgatactccgcacagaaacagttctggaaagctgctaagcccggggccaacagcactacagatgcagtgctgctcaa

caaattgcatcatgctgcagacttggaaaagaagcagaatgagacagaaaacaggaaattgttggggaccgtcatccaatatggcaacgtgatccagct

cctgcatttgaaaagcaataaatacctgactgtgaataagaggctcccagccttgctagagaagaatgccatgagggtgacgttggacgaggctggaaat

gaagggtcctggttttacattcaaccattttacaagcttcgctccatcggagacagtgtggtcataggcgacaaggtagttttgaatcctgtcaatgctggcc

agcctctacatgccagcagtcatcagctggtggataacccaggctgcaatgaggtcaactccgtcaactgtaatacaagctggaagatagtgcttttcatg

aaatggagtgataacaaagacgacattctcaaaggaggtgatgtggtgaggctcttccatgccgagcaagagaagtttctcacctgtgatgagcaccgg

aagaagcagcatgtgttcctgaggaccaccggcaggcagtcagccacgtcggccaccagttctaaagccctgtgggaagtggaggtagtccagcacg

acccatgtcggggtggagctgggtactggaatagcctcttccggttcaagcacctggctacagggcattacttggctgcagaggtagaccctgactttga

ggaagaatgcctggagtttcagccctcagtggaccctgatcaggatgcatctcggagtaggttgagaaacgcgcaagaaaaaatggtatactctctggtc

tccgtgcctgaaggcaacgacatctcctccatctttgagctagaccccacgactctgcgtggaggtgacagccttgtcccaaggaactcctatgtccgtct

cagacacctgtgcaccaacacctgggtacacagcacaaacatccccatcgacaaggaagaggagaagcctgtgatgctgaaaattggtacctctcccc

tgaaggaggacaaggaagcatttgccatagttcctgtttcccctgctgaggttcgggacctggactttgccaatgatgccagcaaggtgctgggctccatc

gctgggaagttggaaaagggcaccatcacccagaatgagagaaggtctgtcacgaagcttttggaagacttggtttactttgtcacgggtggaactaact

ctggccaagacgtgcttgaagttgtcttctctaagcccaatcgagagcggcagaagctgatgagggaacagaatattctcaagcagatcttcaagctgttg

caggcccccttcacggactgcggggatggcccgatgcttcggctggaggagctgggggatcagcgccatgctcctttcagacatatttgccgactctgct

acagggtcctgcgacactcacagcaagactacaggaagaaccaggagtacatagccaagcagtttggcttcatgcagaagcagattggctatgacgtg

ctggccgaagacaccatcactgccctgctccacaacaaccggaaactcctggagaagcacatcaccgcggcagagattgacacgtttgtcagcctggt

gcgaaagaacagggagcccaggttcttggattacctctctgacctctgcgtatccatgaacaagtcaatccctgtgacacaggagctcatctgtaaagctg

tgctcaatcccaccaatgctgacatcctgattgagaccaagctggttctttctcgttttgagtttgaaggcgtttccactggagagaatgctctggaagccgg

ggaggatgaggaagaggtgtggctgttctggagggacagcaacaaagagatccgtagtaagagtgtccgggaattggcgcaagatgctaaagaggg

acagaaggaagacagggacatcctcagctactacagatatcagctgaacctctttgcaaggatgtgtctggaccgccagtacctggccatcaatgaaatc

tccgggcagctggatgttgatctcattctccgctgcatgtctgacgagaacctcccctacgacctcagggcatccttttgccgcctcatgcttcacatgcatg

tggaccgagatccccaagagcaggtgacacctgtgaaatatgcccgactgtggtcagaaattccctctgagatcgccattgatgactatgacagcagtgg

aacatccaaagatgaaattaaggagaggtttgcacagacgatggagtttgtggaggagtacctaagagatgtggtttgtcaaagattccccttctctgataa

ggagaaaaataagctcacgtttgaggttgtgaacttagccaggaatctcatatactttggtttctacaacttttctgaccttctccgattaaccaagatcctcttg

gcaatcttagactgtgtccatgtgaccactatcttccccattagcaagatgacaaaaggagaagagaataaaggcagtaacgtgatgaggtctatccatgg

cgttggggagctgatgacccaggtggtgctgcggggaggaggcttcttgcccatgactcccatggctgcggcccctgaaggaaatgtgaagcaggca

gagccagagaaagaggacatcatggtcatggacaccaagttgaagatcattgaaatactccagtttattttgaatgtgagattggattataggatctcctgcc

tcctgtgtatatttaagcgagagtttgatgaaagcaattcccagtcatcagaaacatcctccggaaacagcagccaggaagggccaagtaatgtgccagg

tgctcttgactttgaacacattgaagaacaagcggaaggcatctttggaggaagtgaggagaacacacctttggacctggatgaccatggtggcagaac

cttcctcagggtcctgctccacttgacaatgcatgactacccacccctggtgtctggggccctgcagctcctctttcggcacttcagccagaggcaggagg

tccttcaggccttcaaacaggttcaactgctggttactagccaagatgtggacaactacaaacagatcaagcaagacttggaccaactaaggtccattgtg

gagaagtctgagctctgggtgtacaaaggccaaggtcccgatgagcctatggacggagcctccggtgaaaatgagcataagaaaaccgaggagggg

acgagcaagccactgaagcacgagagcaccagcagctacaactaccgagtggtgaaagagattttgattcgacttagcaagctctgcgtgcaggagag

cgcgtcggtgaggaagagccggaagcagcagcaacgactgctgaggaacatgggcgcacacgctgtggtgctggagctgctgcagatcccctacga

gaaggccgaagacacaaagatgcaagagatcatgcggctggctcatgaatttttgcagaatttctgtgcaggcaaccagcagaatcaagctttgctgcat

aaacacataaacctgtttctcaagccagggatcctggaggcagtgacgatgcagcacatcttcatgaacaacttccagctgtgcagtgagatcaacgaga

gagtggtccagcactttgttcactgcatagagacccacggtcgaaacgtccagtatatcaagtttctccagacgattgtcaaggcagaagggaaattcatta

aaaagtgccaagacatggtcatggctgagcttgtcaactctggagaggacgtcctcgtgttctacaatgacagagcctctttccagactctgatccagatg

atgcggtccgagcgtgaccggatggatgagaacagccctctcatgtaccacattccatctggtggagctcttggccgtgtgcacagagggcaagaatgtg

tacacggagatcaagtgcaactccttgctcccgctcgatgacatcgttcgtgtggtcactcatgaagactgcatccccgaggttaagatcgcttacattaac

ttcctgaatcactgctatgtggatacggaggtggagatgaaggagatttacacaagcaaccacatgtggaagttgtttgagaatttcctcgtggacatctgc

agggcctgtaacaacacaagcgacaggaagcacgcagactccattctggagaagtacgtcactgaaatcgtgatgagcatcgtcaccaccttcttcagc

tctcccttctcagaccagagcaccactctgcagacccgccagcctgtctttgtgcaactcctgcaaggcgtgttccgagtttaccactgcaactggctgatg

ccgagccaaaaagcctcggtggagagctgcatccgggtgctctctgacgtagccaagagccgggccatagccattcctgttgacctggacagccaagt

caacaacctcttcctgaagtcccacaacattgtgcagaaaacagccctgaactggcggttatcagcccgaaacgccgctcgcagagactctgtactggc

agcatccagagactaccgaaatatcattgagaggttacaggacatcgtgtctgccctagaggaccggctcaggcccctggtgcaggctgagctgtctgt

gctcgtggatgttctacacagaccagaactgctcttccccgagaacacggatgccaggaggaaatgtgagagtggaggtttcatctgcaagctaataaaa

cataccaagcaactgctggaggagaatgaagagaaactatgcattaaagtcttacagaccctcagggaaatgatgaccaaagacagaggctatggaga

gaagcaaatttccattgatgaatcggaaaatgccgagctgccacaggcaccggaagctgagaactccacagagcaggagcttgaaccaagtccaccc

ctgaggcaactggaagaccataaaaggggtgaggcactccgacaaattttggtcaaccgttactatggaaacatcagaccttcaggaagaagagagag

ccttaccagcttcggcaatggcccactatcaccaggaggacccagcaagcctggtggaggagggggaggtcctggatctagttccacaagcaggggtg

agatgagcctggccgaggttcagtgtcacctcgacaaggagggggcctccaacctggtcatcgatctcataatgaatgcatccagtgaccgagtattcca

tgaaagcattctgctggccatcgcacttctggaaggaggcaacaccaccatccagcactcgtttttctgccggctgacagaagataagaaatcagagaag

ttcttcaaggttttctacgatcgaatgaaggtggcccagcaggaaatcaaggcgacagtgacagtgaacaccagcgacttgggaaacaaaaagaaagat

gatgaagtggacagggatgccccgtctcggaagaaagccaaagagcccacaacacagataacagaagaggtccgggatcagctcctggaagcatct

gctgccaccaggaaagcctttaccaccttccggagggaggccgaccctgatgaccattaccagtctggggagggcacccaggctacaaccgacaaag

ccaaggatgacctagagatgagcgctgtcatcaccatcatgcagcctatcctgcgcttcctgcagctgctgtgtgaaaaccacaaccgagatctgcagaa

tttccttcgttgccaaaataataagaccaactacaatttggtgtgtgagacactgcagtttctggactgtatttgtgggagcacaaccggaggccttggtcttc

ttggactgtacataaatgaaaagaatgtagcacttatcaaccaaaccctggagagtctgacggagtactgtcaagggccttgccatgagaaccagaactg

catcgccacccacgagtccaatggcatcgatatcatcacagccctcatcctcaatgatatcaaccctctgggaaagaagcggatggacctggtgttagaa

ctgaagaacaatgcttcgaagctgctactggccatcatggaaagcagacacgatagtgaaaatgcagagaggatcctgtacaacatgaggcccaagga

gctggtggaagtgatcaagaaggcctacatgcaaggtgaagtggaatttgaggatggggagaacggtgaggatggagctgcctcacccaggaacgtg

ggccacaacatctacatcctcgctcaccagttggctcggcataacaaagaacttcaaaccatgctgaaacctggaggccaggtggatggggatgaagct

ctggagttctacgcgaagcacacagcacaaattgagattgtcagactggaccggacaatggaacagatcgtcttccctgtgcccagcatctgtgaattcct

gactaaggaatcgaaacttcgaatatattacaccacagagcgggatgagcaaggtagcaagatcaatgacttcttcctgcgctccgaggacctctttaac

gagatgaactggcagaagaaacttcgagcccagcctgtcttgtactggtgtgcccgaaacatgtctttctggagcagcatctccttcaacctggccgtcct

gatgaacctgctggtggcgtttttctatccatttaaaggagtgaggggaggaacactagagccacactggtcaggcctcctgtggacagccatgctcatct

ctctggccattgtcattgctctgcccaagccccacggcatccgggccttaattgcttctacaatcctacgactgatattttcagttgggttgcagcccacactg

tttctgctgggagctttcaatgtctgcaataaaatcatcttcctgatgagctttgtgggcaactgtgggaccttcaccagaggctaccgggccatggttctgg

atgtggagttcctctatcatttgctgtatctactcatctgtgccatgggcctcttcgtacatgagttcttctatagcttgctgctttttgatttagtgtacagagagg

agactttgcttaatgtcattaaaagtgtcacccgcaatggacggtccatcatcttgacagcggtcctggctctgatcctggtttacctgttctcaattgtgggct

atctgttcttcaaggatgactttatcttggaagtagataggttgcccaatgaaacagctgttccagaaactggcgagagtttggccaacgatttcctgtactct

gatgtgtgcagggtagagacgggggagaactgcacctctcctgcacccaaagaagagctgctccctgccgaagaaacggaacaggataaggaacac

acgtgtgagaccctgctcatgtgcatcgtcactgttctgagtcacgggctgcggagtgggggaggggtaggagacgtgctcaggaagccatccaaaga

ggagcctctgtttgctgcaagggtgatctacgacctcctcttcttcttcatggtcatcatcatcgtcctgaacctgattttcggggtcatcatcgacacctttgct

gacctgaggagtgagaagcaaaagaaggaggagatcttaaaaaccacgtgcttcatctgcggcttggaaagggacaagtttgacaataagactgtcac

ctttgaagagcacatcaaggaagaacacaacatgtggcactatctgtgcttcatcgtgctggtgaaagtgaaggactccacagagtacaccgggcctga

gagttacgtggcagagatgatcagggaaagaaaccttgattggttcctcagaatgagagccatgtccctggtcagcagcgattctgaaggggaacagaa

cgagctgaggaacctgcaggagaagctggagtctaccatgaagctggtcaccaatctttctggccagctgtcagaactaaaggaccagatgacagaac

agaggaagcagaaacaaagaatcggccttctaggacatcctcctcacatgaatgtcaacccacagcagccggcctaggcaaatgaggcagagggact

ctgctcagccctctgtatatcactgtcagggtgggtacggctcattggttctgatttgcccactaagggtacatgtgcgcttagtacatttgtaaatactcagttt

tgtattgtatgtatatgattgctattctcagaggtttggactttcgtattgtaattagctctgttggcatggtgacttgtcactcctgccaaaaatattaaaaatgcct

tttttggaaggactacagaaagtacctgatttgcacttgaaccagattatagatttaaaagtatatgacatgtattttgtatttaaaactagaatagccagtattta

tgttttttataaaactgtgcaatacaaattatgcaatcaccataactttgtaactcctgagtgtcctaagggagtacacatctttgaagctgatttgttgatactcg

tgtaataaatggttaaatatcaaatgctgctgctgctgccaaaattatattaatagcgagtttctggcccctgggcaattttgtaccttgtaattatcctatggtga

tgctgtttctcgttgctaatggcattagtgcccctgtatcctagtgataactccaggtctgtgaaccattcaaacagcattcattttgagaaaaagcaactttagtt

tcaaggataattttaagcttcaaaattaatcatttaaagtgtttctttaagagagccatgttagaggctcacactttagcttgaaaggagttgatgaattaatttttt

aaagggaactttttacatgacgtttggaataacagcatattgctgaccagtcagtgtcatctcccgggtgaattttgatgtcacgttatagtcaaatgagttagc

tgatggtttctagattttcttcctctgaaccatgatgcagtaggtaagaagttattatgcgtatatacatatatacattcatatacgacaaagtaggagctgtccc

cttaggatgcatagctgcccctagggtacgtagctgaacactgacaatggcgttcttctgaaagagccacgtttgggttttatttctttgtcacatgatttctttt

ctggatgggtgcaaagtatcacaggaagtgttttctctctgtcgccttgttttgtacctgggtctcgctttactagaccgtc?tctgcacaaaagtttaaaaactg

aaccgtatgcagagttccgaagcaagtcaagtttgtaaatgcatacctaaaaatatttaataaacgatgcagaatcct

SEQ?ID?No.32

MSDKMSSFLHIGDICSLYAEGSTNGFISTLGLVDDRCVVQPEAGDLNNPPKKFRDCLFKLCPMNR

YSAQKQFWKAAKPGANSTTDAVLLNKLHHAADLEKKQNETENRKLLGTVIQYGNVIQLLHLKS

NKYLTVNKRLPALLEKNAMRVTLDEAGNEGSWFYIQPFYKLRSIGDSVVIGDKVVLNPVNAGQ

PLHASSHQLVDNPGCNEVNSVNCNTSWKIVLFMKWSDNKDDILKGGDVVRLFHAEQEKFLTCD

EHRKKQHVFLRTTGRQSATSATSSKALWEVEVVQHDPCRGGAGYWNSLFRFKHLATGHYLAA

EVDPDFEEECLEFQPSVDPDQDASRSRLRNAQEKMVYSLVSVPEGNDISSIFELDPTTLRGGDSLV

PRNSYVRLRHLCTNTWVHSTNIPIDKEEEKPVMLKIGTSPLKEDKEAFAIVPVSPAEVRDLDFAN

DASKVLGSIAGKLEKGTITQNERRSVTKLLEDLVYFVTGGTNSGQDVLEVVFSKPNRERQKLMR

EQNILKQIFKLLQAPFTDCGDGPMLRLEELGDQRHAPFRHICRLCYRVLRHSQQDYRKNQEYIAK

QFGFMQKQIGYDVLAEDTITALLHNNRKLLEKHITAAEIDTFVSLVRKNREPRFLDYLSDLCVSM

NKSIPVTQELICKAVLNPTNADILIETKLVLSRFEFEGVSTGENALEAGEDEEEVWLFWRDSNKEI

RSKSVRELAQDAKEGQKEDRDILSYYRYQLNLFARMCLDRQYLAINEISGQLDVDLILRCMSDE

NLPYDLRASFCRLMLHMHVDRDPQEQVTPVKYARLWSEIPSEIAIDDYDSSGTSKDEIKERFAQT

MEFVEEYLRDVVCQRFPFSDKEKNKLTFEVVNLARNLIYFGFYNFSDLLRLTKILLAILDCVHVT

TIFPISKMTKGEENKGSNVMRSIHGVGELMTQVVLRGGGFLPMTPMAAAPEGNVKQAEPEKEDI

MVMDTKLKIIEILQFILNVRLDYRISCLLCIFKREFDESNSQSSETSSGNSSQEGPSNVPGALDFEHI

EEQAEGIFGGSEENTPLDLDDHGGRTFLRVLLHLTMHDYPPLVSGALQLLFRHFSQRQEVLQAFK

QVQLLVTSQDVDNYKQIKQDLDQLRSIVEKSELWVYKGQGPDEPMDGASGENEHKKTEEGTSK

PLKHESTSSYNYRVVKEILIRLSKLCVQESASVRKSRKQQQRLLRNMGAHAVVLELLQIPYEKAE

DTKMQEIMRLAHEFLQNFCAGNQQNQALLHKHINLFLKPGILEAVTMQHIFMNNFQLCSEINER

VVQHFVHCIETHGRNVQYIKFLQTIVKAEGKFIKKCQDMVMAELVNSGEDVLVFYNDRASFQTL

IQMMRSERDRMDENSPLMYHIHLVELLAVCTEGKNVYTEIKCNSLLPLDDIVRVVTHEDCIPEV

KIAYINFLNHCYVDTEVEMKEIYTSNHMWKLFENFLVDICRACNNTSDRKHADSILEKYVTEIV

MSIVTTFFSSPFSDQSTTLQTRQPVFVQLLQGVFRVYHCNWLMPSQKASVESCIRVLSDVAKSRA

IAIPVDLDSQVNNLFLKSHNIVQKTALNWRLSARNAARRDSVLAASRDYRNIIERLQDIVSALED

RLRPLVQAELSVLVDVLHRPELLFPENTDARRKCESGGFICKLIKHTKQLLEENEEKLCIKVLQTL

REMMTKDRGYGEKQISIDESENAELPQAPEAENSTEQELEPSPPLRQLEDHKRGEALRQILVNRY

YGNIRPSGRRESLTSFGNGPLSPGGPSKPGGGGGGPGSSSTSRGEMSLAEVQCHLDKEGASNLVI

DLIMNASSDRVFHESILLAIALLEGGNTTIQHSFFCRLTEDKKSEKFFKVFYDPMKVAQQEIKATV

TVNTSDLGNKKKDDEVDRDAPSRKKAKEPTTQITEEVRDQLLEASAATRKAFTTFRREADPDDH

YQSGEGTQATTDKAKDDLEMSAVITIMQPILRFLQLLCENHNRDLQNFLRCQNNKTNYNLVCET

LQFLDCICGSTTGGLGLLGLYINEKNVALINQTLESLTEYCQGPCHENQNCIATHESNGIDIITALI

LNDINPLGKKRMDLVLELKNNASKLLLAIMESRHDSENAERILYNMRPKELVEVIKKAYMQGEV

EFEDGENGEDGAASPRNVGHNIYILAHQLARHNKELQTMLKPGGQVDGDEALEFYAKHTAQIEI

VRLDRTMEQIVFPVPSICEFLTKESKLRIYYTTERDEQGSKINDFFLRSEDLFNEMNWQKKLRAQ

PVLYWCARNMSFWSSISFNLAVLMNLLVAFFYPFKGVRGGTLEPHWSGLLWTAMLISLAIVIAL

PKPHGLRALIASTILRLIFSVGLQPTLFLLGAFNVCNKIIFLMSFVGNCGTFTRGYRAMVLDVEFLY

HLLYLLICAMGLFVHEFFYSLLLFDLVYREETLLNVIKSVTRNGRSIILTAVLALILVYLFSIVGYLF

FKDDFILEVDRLPNETAVPETGESLANDFLYSDVCRVETGENCTSPAPKEELLPAEETEQDKEHT

CETLLMCIVTVLSHGLRSGGGVGDVLRKPSKEEPLFAARVIYDLLFFFMVIIIVLNLIFGVIIDTFA

DLRSEKQKKEEILKTTCFICGLERDKFDNKTVTFEEHIKEEHNMWHYLCFIVLVKVKDSTEYTGP

ESYVAEMIRERNLDWFLRMRAMSLVSSDSEGEQNELRNLQEKLESTMKLVTNLSGQLSELKDQ

MTEQRKQKQRIGLLGHPPHMNVNPQQPA

SEQ?ID?No.33

ggcacgagccgagttggaggaagcagcggcagcggcagcggcagcggtagcggtgaggacggctgtgcagccaaggaaccgggacagcgaag

cgacggcaggtcgcagctggatcgcaggagcctgggagctgggagcttcagaggccgctgaagcccaggctgggcagaggaaggaagcgagccg

acccggaggtgaagctgagagtggagcgtggcagtaaaatcagacgacagatggacagtgtgacaggaacgtcagagaggattgggcctcgctgcg

agagtcagcctggagtcaaggtgttgacaagttgctgagaaggacacgtgggaggacggtggcgcgcggagggagagccctgtcttcagtcaccccg

ttgatggaggacagatggacagcagccggacggccagtcacctctcttaaacctttggatagtggtcctttgtgctctgctggacacctgttggggatttta

gcccattctctgaactcactttctcttaaaacgtaaactcggacggcagtgtgcgagccagctcctctgtggcagggcactagagctgcagacatgagtgc

agagggctaccagtacagagcactgtacgactacaagaaggagcgagaggaagacattgacctacacctgggggacatactgactgtgaataaaggc

tccttagtggcacttggattcagtgatggccaggaagcccggcctgaagatattggctggttaaatggctacaatgaaaccactggggagaggggagac

tttccaggaacttacgttgaatacattggaaggaaaagaatttcaccccctactcccaagcctcggccccctcgaccgcttcctgttgctccgggttcttcaa

aaactgaagctgacacggagcagcaagcgttgccccttcctgacctggccgagcagtttgcccctcctgatgttgccccgcctctccttataaagctcctg

gaagccattgagaagaaaggactggaatgttcgactctatacagaacacaaagctccagcaaccctgcagaattacgacagcttcttgattgtgatgccg

cgtcagtggacttggagatgatcgacgtacacgtcttagcagatgctttcaaacgctatctcgccgacttaccaaatcctgtcattcctgtagctgtttacaat

gagatgatgtctttagcccaagaactacagagccctgaagactgcatccagctgttgaagaagctcattagattgcctaatatacctcatcagtgttggctta

cgcttcagtatttgctcaagcattttttcaagctctctcaagcctccagcaaaaaccttttgaatgcaagagtcctctctgagattttcagccccgtgcttttcag

atttccagccgccagctctgataatactgaacacctcataaaagcgatagagattttaatctcaacggaatggaatgagagacagccagcaccagcactg

ccccccaaaccacccaagcccactactgtagccaacaacagcatgaacaacaatatgtccttgcaggatgctgaatggtactggggagacatctcaagg

gaagaagtgaatgaaaaactccgagacactgctgatgggacctttttggtacgagacgcatctactaaaatgcacggcgattacactcttacacctagga

aaggaggaaataacaaattaatcaaaatctttcaccgtgatggaaaatatggcttctctgatccattaaccttcaactctgtggttgagttaataaaccactac

cggaatgagtctttagctcagtacaaccccaagctggatgtgaagttgctctacccagtgtccaaataccagcaggatcaagttgtcaaagaagataatatt

gaagctgtagggaaaaaattacatgaatataatactcaatttcaagaaaaaagtcgggaatatgatagattatatgaggagtacacccgtacttcccagga

aatccaaatgaaaagaacggctatcgaagcatttaatgaaaccataaaaatatttgaagaacaatgccaaacccaggagcggtacagcaaagaatacat

agagaagtttaaacgcgaaggcaacgagaaagaaattcaaaggattatgcataaccatgataagctgaagtcgcgtatcagtgagatcattgacagtag

gaggaggttggaagaagacttgaagaagcaggcagctgagtaccgagagatcgacaaacgcatgaacagtattaagccggacctcatccagttgaga

aagacaagagaccaatacttgatgtggctgacgcagaaaggtgtgcggcagaagaagctgaacgagtggctggggaatgaaaataccgaagatcaat

actccctggtagaagatgatgaggatttgccccaccatgacgagaagacgtggaatgtcgggagcagcaaccgaaacaaagcggagaacctattgcg

agggaagcgagacggcactttccttgtccgggagagcagtaagcagggctgctatgcctgctccgtagtggtagacggcgaagtcaagcattgcgtcat

taacaagactgccaccggctatggctttgccgagccctacaacctgtacagctccctgaaggagctggtgctacattatcaacacacctccctcgtgcag

cacaatgactccctcaatgtcacactagcatacccagtatatgcacaacagaggcgatgaagcgctgccctcggatccagttcctcaccttcaagccacc

caaggcctctgagaagcaaagggctcctctccagcccgacctgtgaactgagctgcagaaatgaagccggctgtctgcacatgggactagagctttctt

ggacaaaaagaagtcggggaagacacgcagcctcggactgttggatgaccagacgtttctaaccttatcctctttctttctttctttctttctttctttctttctttc

tttctttctttctttctttctttctttctaatttaaagccacaacacacaaccaacacacagagagaaagaaatgcaaaaaatctctccgtgcagggacaaagag

gcctttaaccatggtgcttgttaacgctttctgaagctttaccagctacaagttgggactttggagaccagaaggtagacagggccgaagagcctgcgcct

ggggccgcttggtccagcctggtgtagcctgggtgtcgctgggtgtggtgaacccagacacatcacactgtggattatttcctttttaaaagagcgaatgat

atgtatcagagagccgcgtctgctcacgcaggacactttgagagaacattgatgcagtctgttcggaggaaaaatgaaacaccagaaaacgtttttgttta

aacttatcaagtcagcaaccaacaacccaccaacagaaaaaaaaaaaaaa

SEQ?ID?No.34

MSAEGYQYRALYDYKKEREEDIDLHLGDILTVNKGSLVALGFSDGQEARPEDIGWLNGYNETT

GERGDFPGTYVEYIGRKRISPPTPKPRPPRPLPVAPGSSKTEADTEQQALPLPDLAEQFAPPDVAP

PLLIKLLEAIEKKGLECSTLYRTQSSSNPAELRQLLDCDAASVDLEMIDVHVLADAFKRYLADLP

NPVIPVAVYNEMMSLAQELQSPEDCIQLLKKLIRLPNIPHQCWLTLQYLLKHFFKLSQASSKNLL

NARVLSEIFSPVLFRFPAASSDNTEHLIKAIEILISTEWNERQPAPALPPKPPKPTTVANNSMNNNM

SLQDAEWYWGDISREEVNEKLRDTADGTFLVRDASKMHGDYTLTPRKGGNNKLIKIFHRDGKY

GFSDPLTFNSVVELINHYRNESLAQYNPKLDVKLLYPVSKYQQDQVVKEDNIEAVGKKLHEYNT

QFQEKSREYDRLYEEYTRTSQEIQMKRTAIEAFNETIKIFEEQCQTQERYSKEYIEKFKREGNEKE

IQRIMHNHDKLKSRISEIIDSRRRLEEDLKKQAAEYREIDKRMNSIKPDLIQLRKTRDQYLMWLTQ

KGVRQKKLNEWLGNENTEDQYSLVEDDEDLPHHDEKTWNVGSSNRNKAENLLRGKRDGTFLV

RESSKQGCYACSVVVDGEVKHCVINKTATGYGFAEPYNLYSSLKELVLHYQHTSLVQHNDSLN

VTLAYPVYAQQRR

SEQ?ID?No.35

ggaaggatgaggcgcccgcggcggcccgggggctccgggggctccgggggctccgggggcctccggctgctggtctgcctgctgttgctgagcgg

ccgccccgggggctgcagcgccatcagtgcccacggctgtctgtttgaccgcagactttgttcgcatctggaagtctgtattcaggatggcttgtttggaca

gtgccaggcaggagtggggcaggcacggcccctcttacaagtcacttccccagttctccagcgcctacaaggtgtgctccggcaactcatgtcccaag

gcttgtcctggcatgatgaccttacccagcatgtgatctcccaggagatggaacgcatccccaggcttcgccccccagagccccatccaagggacaggt

ctggtttggtgcccaggaaaccaggccctgcaggggaattgctaactcagggcaatcctactggctcctctcctgctgcccagggctttccaaggcctgc

agggggacggagctggggcggctccccactgtcctctctgcaggctgagttgttaccccctctcttggagcatctgctaatgcccccacagcctccacac

cctgctctgacctatgaacctgcactgctacagccttacctcttccaccagtttggctcccgagatggctcccggggctcagagagctcctctggggtagtt

ggtgttggtcacctgtccaaggctgaaggtcctgcactcttcagcagaagtgcctccaaggccattttggggactcactctggacactcttttggggacctt

acaggtccctcacctgctcaacttttccaagattcagggctgctctacatggcccaagagttgccagtgcctggcagagcccgggcaccaaggttgcca

gagaatgggggcaacagggcagaggactcttcagagggccatgaggaggaagtactagggggtcgtggggagaagtcccctccccaagcagcaca

accagaattgagtctgcagagattgactgctgtactggcaggctatggagtagagctgcgtcagttgaccccggagcagttttctaccctcttgaccctgat

gcagttgctgcccaagggcacaggaagaaatcttgaaggggctgtaaatgttggaggagccgatgtcaagaaaacaatacaacagatgcagagagga

gacccagcagaagctctgccccccacaccctcgcttcctgggtacctcactgccagccctgcctccagcgaagttcagcaggtgctgagccctggtttc

cctgaacctccccacacacccagccctctgggctcctcctcagtccttctggagaagaaaagtcccttgggccagagccagcccacagtggtgggacg

gccatcagctcgaccatcggccgaggagtatggctatatcgtcactgaccagaaacccctgagcctggtggctggagtgaggctgctggagattctggc

tgagcacgtgcatatgtcctccggtagctttatcaacatcagtgtggtgggaccagctgtcaccttccgaatccggcacaatgagcagaacctgtctttggc

agatgtgacccagcaagctgggctggtgaagtctgaactggaagcgcagacagggctccagattttgcagacaggggtgggacagagggaggaagc

agctgaagtccttccccgacaagcccatggcatatctcccatgcgctcagtgctgcttactctagtggccctggcaggcgttcgctgggctgctagtggctt

tggcagtggccttgtgtatgcgccatcattcgagacagcgggataaggagcgcctggcagcgctggggccggagggggcccatggtgacactactttt

gagtaccaggacctgtgtcgccagcacatggccacaaagtccctgtttaaccgggcggagggtcagccagagccttctagggtgagcagtgtgtcctc

ccagttcagcgacgcggcccaggccagccccagttcccacagcagctctccatcttggtgcgaggagcccgcccaggccaacatggacatctccaca

ggacacatgattctggcatacatggaggatcaccttcggaaccgggaccggttggccaaggagtggcaggctctgtgcgcctaccaagcagagccaa

acacctgtgccgccgcacaggatgagagcaacatcaagaagaaccgccatcctgacttcctaccctatgaccatgcccgaatcaagctgaaagtggag

agcagcccttctcggagtgattacatcaacgccagccccatcatcgagcatgaccctcggatgccggcctacatagccacacagggaccactgtccca

caccatcgcggacttctggcagatggtgtgggagagtggctgcactgtcatcgttatgctgaccccgttggtggaggacggtgtcaaacagtgtgaccgc

tactggccggatgaaggatcttccctctaccacgtctatgaggtgaacctggtgtcggagcacatctggtgcgaggacttcctggtgcggagcttctacctt

aagaacctgcagacccaggagacgcgcacgctcactcagttccacttcctcagctggccggcagagggcactccggcctccacccggccgctgctgg

acttccgcaggaaagtgaacaagtgctacagaggccgctcctgccccatcatagtgcactgcagtgacggtgcagggaggacaggcacctacatcctt

attgacatggtcctgaatcgcatggccaaaggagtgaaggagattgatattgctgccaccctggagcatgtccgtgaccagcggcctggacttgtccgtt

ctaaggaccagtttgagtttgcgctgacagccgtggcagaggaggtgaatgctatcctcaaggccctgccccagtgagcccccctgggcgcctcagtg

ggcatcctggcctcggctccttctgcctgtgtgagcatctgtgcacccactcttcagcccctacccatctgccaccttggtctgacttggccatgggagcctt

tcccaacccagtgtggaagggagtcgggagggaaggaaggggtaggctcgccctgctttatccatgctagaaccatggtatcccatgggaagcagaca

gcaggcaaggagaggcgtggacaccggccacaggtgtgcccgagccccatcctacctgagtctctgtctccctctctggatatgtgcgtccccactccc

accagcctaccacctatagacaaagcagaacgaggaaaccccagctcccccaaccctgctaccactggcctgccaccttgaccctgctcaaccttctcc

ctctagcacaagggaacatttctagaaaagtaaaatctacttttgtatcagtgtgaataaagttagtgtgttgtctgtgc

SEQ?ID?No.36

MRRPRRPGGSGGSGGSGGLRLLVCLLLLSGRPGGCSAISAHGCLFDRRLCSHLEVCIQDGLFGQC

QAGVGQARPLLQVTSPVLQRLQGVLRQLMSQGLSWHDDLTQHVISQEMERIPRLRPPEPHPRDR

SGLVPRKPGPAGELLTQGNPTGSSPAAQGFPRPAGGRSWGGSPLSSLQAELLPPLLEHLLMPPQP

PHPALTYEPALLQPYLFHQFGSRDGSRGSESSSGVVGVGHLSKAEGPALFSRSASKAILGTHSGHS

FGDLTGPSPAQLFQDSGLLYMAQELPVPGRARAPRLPENGGNRAEDSSEGHEEEVLGGRGEKSP

PQAAQPELSLQRLTAVLAGYGVELRQLTPEQFSTLLTLMQLLPKGTGRNLEGAVNVGGADVKK

TIQQMQRGDPAEALPPTPSLPGYLTASPASSEVQQVLSPGFPEPPHTPSPLGSSSVLLEKKSPLGQS

QPTVVGRPSARPSAEEYGYIVTDQKPLSLVAGVRLLEILAEHVHMSSGSFINISWGPAVTFRIRH

NEQNLSLADVTQQAGLVKSELEAQTGLQILQTGVGQREEAAEVLPRQAHGISPMRSVLLTLVAL

AGVAGLLVALAVALCMRHHSRQRDKERLAALGPEGAHGDTTFEYQDLCRQHMATKSLFNRAE

GQPEPSRVSSVSSQFSDAAQASPSSHSSSPSWCEEPAQANMDISTGHMILAYMEDHLRNRDRLAK

EWQALCAYQAEPNTCAAAQDESNIKKNRHPDFLPYDHARIKLKVESSPSRSDYINASPIIEHDPR

MPAYIATQGPLSHTIADFWQMVWESGCTVIVMLTPLVEDGVKQCDRYWPDEGSSLYHVYEVN

LVSEHIWCEDFLVRSFYLNLQTQETRTLTQFHFLSWPAEGTPASTRPLLDFRRKVNKCYRGRSCP

IIVHCSDGAGRTGTYILIDMVLNRMAKGVKEIDIAATLEHVRDQRPGLVRSKDQFEFALTAVAEE

VNAILKALPQ

SEQ?ID?No.37

cggcgggaggaaagcgcggtgaagccagattaggagcagcgagcacttggggacttagggccacaggacaccgcacaagatcgaccgactttttct

ggagaaccgcagaacgggcacgctggggtcgctggggctggccatggtgatggaggtgggcatcctggacgccggggggctgcgcgcgctgctgc

gagagggcgccgcgcagtgcctgttgttggattgtcgctccttcttcgctttcaacgccggccacatcgcgggctcagtgaacgtgcgcttcagcaccat

cgtgcggcgccgcgccaagggcgccatgggcctggagcatatcgtgcccaacgctgaactgcgtggccgcctgctggccggagcctaccacgccgt

ggtgctgctggacgagcgcagcgcctccctggacggcgccaagcgcgacggcaccctggccctggccgcgggcgcgctctgccgagaggcgcgc

tccactcaagtcttctttctccaaggaggatatgaagcgttttcggcttcctgccctgagctgtgcagcaaacagtccacccccacggggctcagcctccc

cctgagtactagtgtgcctgacagtgcagaatccggatgcagctcctgtagtacccctctctacgatcaggggggcccagtggagatcctgtccttcctgt

acctgggcagtgcctatcacgcttctcggaaggatatgcttgacgccttgggcatcaccgccttgatcaacgtctcagccaattgtcctaaccactttgagg

gtcactaccagtacaagagcatccctgtggaggacaaccacaaggcagacatcagctcctggttcaacgaggctattgacttcatagactccatcaagg

atgctggagggagagtgtttgttcattgccaggccggcatctcccggtcagccaccatctgccttgcttacctcatgaggactaaccgggtaaagctggac

gaggcctttgagtttgtgaagcagaggcggagtatcatctccccgaacttcagcttcatgggccagctgctgcagtttgagtcccaagtgctagcccctca

ctgctctgctgaagctgggagccctgccatggctgtccttgaccggggcacctctactaccacagtcttcaacttccctgtttccatccccgtccaccccac

gaacagtgccctgaactaccttaaaagccccatcaccacctctccaagctgctgaagggcaaggggaggtgtggagtttcacttgccaocgggtcgcca

ctcctcctgtgggaggagcaatgcaataactctgggagaggctcatgggagctggtccttatttatttaacacccccctcaccccccaactcctcctgagtt

ccactgagttcctaagcagtcacaatcaatgacttgaccgcaagacatttgctgaactcggcacattcgggaccaatatattgtgggtacatcaagtccctct

gacaaaacagggcagaagagaaaggactctgtttgaggcagtttcttcgcttgcctgttttttttttctagaaacttcatgcttgacacacccaccagtattaa

ccattcccgatgacatgcgcgtatgagagtttttacctttatttatttttgtgtaggtcggtggtttctgccttcacaaatgtcattgtctactcatagaagaaccaa

atacctcaattttgtgtttgcgtactgtactatcttgtaaatagacccagagcaggtttgctttcggcactgacagacaaagccagtgtaggtttgtagctttca

gttatcgacagttgtatgtttgtttatttatgatctgaagtaatatatttcttcttctgtgaagacattttgttactgggatgactttttttatacaacagaataaattatg

acgtttctattga

SEQ?ID?No.38

MVMEVGILDAGGLRALLREGAAQCLLLDCRSFFAFNAGHIAGSVNVRFSTIVRRRAKGAMGLEH

IVPNAELRGRLLAGAYHAVVLLDERSASLDGAKRDGTLALAAGALCREARSTQVFFLQGGYEAF

SASCPELCSKQSTPTGLSLPLSTSVPDSAESGCSSCSTPLYDQGGPVEILSFLYLGSAYHASRKDM

LDALGITALINVSANCPNHFEGHYQYKSIPVEDNHKADISSWFNEAIDFIDSIKDAGGRVFVHCQA

GISRSATICLAYLMRTNRVKLDEAFEFVKQRRSIISPNFSFMGQLLQFESQVLAPHCSAEAGSPAM

AVLDRGTSTTTVFNFPVSIPVHPTNSALNYLKSPITTSPSC

SEQ?ID?No.39

atacccatcaaatgaagcaccaaagacaactgtgttgagcgcccctgggcctggctcattcttaatcatattgccatctttactgctctgattcctaggatccc

cgcaatgactctgaataatgtcaccatgcgccaaggcactgtgggcatgcagccacagcagcgctggagtatgcctgctgatgccaggcatctgatggt

ccagaaggatccccacccctgcaacctccgaaaccgccactctactgctccggaagagcactgccggcggacgtggtcgtccgactccacagactcg

gtcatctcttctgagtctggaaacacctactaccgagtagtgcttataggggagcaaggagtgggcaagtccaccctggccaacatctttgcaggtgtgca

tgacagcatggacagcgactgtgaggtcttgggagaagatacatatgagcgtaccctggtcgttgacggagagagtgcaaccattatcctcctggacatg

tgggaaaataagggggagaacgaatggctccatgaccactgcatgcaggtcggggatgcctatctgatcgtctactctatcacagaccgtgcaagcttc

gagaaggcatctgagctgaggattcagctccgcagggcccggcagacagaagacattcctataattttggttggcaacaaaagcgacttagtgcggtgt

cgagaagtgtctgtgtcagaagggagagcttgtgctgtggtgttcgactgcaaattcatcgagacctctgcggctgtgcagcacaacgtgaaggaactgtt

tgagggcattgagcgacaggtgcgcctgccgagggacagcaaggagaagaatgagaggaggctggcctaccagaagaggcgggagagcattccc

aggaaagccaggcgcttctggggcaaaattgtggccaaaaacaacaagaacatggcttcaagctccaagtcaaaatcctgccatgacctgtctgtgctct

aggcacccagtgtcacccagatgtcccttggtggacatcgttgaaggctattgggaccagtgatctatattagattggatacataagcattgttagacgcaa

cttcccctatggcagatggaaaccaacaggttagccttgtgggcaaccaagtgcacgggcaatgaatgagctctgtcaagagtcagtatttattcatagga

aaagcttgagctgctacgtggatgtctcagactcatttaagacacgcttcgggttcacatgagtttctctctcctttggacagcagaatatttttttcctcagtgtt

gttccatgtgatttcgaggtccttgggtcatatagaaatgtagggaaatggcggtagtttattggaaggagaagggctcactgcatacttatatccctgaaac

gacatctttagagctggcctcatcacagttgtgactatttctgctccagtgaagagaattgttagatttgctggaaactgaggcttactaacagtttttgtttaaa

gaccacagagattgtagacttaggagctatatggtactacttataggttcaaaaaattgtttacttatgtgtcgtagaagtgtttattttgaggaaactattttttttt

ttgccaaattctacttagtcaaatcatcttctatgtcttgctgttttttaaatcattaagctatcataaaatattttttaaaaaaatctcaactatattgataacctgcag

tgcaaaattttaaatatagtcctgtttttcccccaaaacataaacatgccccatcctttgggttgcttctgtatgccacagctgaattatatttattattttgcaataa

ccattttatatttgataaagatatttatgagcatatttcttactgagaaaatgtctgttttattacctttttatatttttcaaagtattcaagtttttacctattgtcttataat

aaataaataaaatctttgaaaagg

SEQ?ID?No.40

MTLNNVTMRQGTVGMQPQQRWSMPADARHLMVQKDPHPCNLRNRHSTAPEEHCRRTWSSDS

TDSVISSESGNTYYRVVLIGEQGVGKSTLANIFAGVHDSMDSDCEVLGEDTYERTLVVDGESATI

ILLDMWENKGENEWLHDHCMQVGDAYLIVYSITDRASFEKASELRIQLRRARQTEDIPIILVGNK

SDLVRCREVSVSEGRACAVVFDCKFIETSAAVQHNVKELFEGIERQVRLPRDSKEKNERRLAYQ

KRRESIPRKARRFWGKIVAKNNKNMASSSKSKSCHDLSVL

SEQ?ID?No.41

tatcccgctgttgctgcaagccggctgcatcttagttggccatgaagaccccagcacagcggctgcaccttcttccactgttgttgctgctttgtggtgagtg

tgcccaggtatgcggctgcaacgagacagggatgctggagaggctgcctcgctgtgggaaagccttcgctgacatgatgcagaaggtggctgtctgga

agtggtgcaacctgtcggagttcatcgtgtattatgaaagcttcactaactgcaccgagatggagaccaacatcatgggctgctactggcccaacccgctg

gcccagagcttcatcactggaatccacaggcagttcttttccaactgcacggtggacaggacccactgggaagaccccccggatgaagtactcatccca

ctgatcgcggttcctgtcgtgctgactgtggctatggctggcctggtggtgtggcgcagcaagcacactgatcggctgctgtgaggatctgctggatgga

gggccatgcctggcaggctgggagaatgttgctcagagctctgagagctggcagactcggcttctgtctggtttgctttggccacaccctacctggccatg

ccaaagtcctcccgaccaggctggtgtggcccttgctgtctagcctgccgcctgctggggttcagattgtccatactttgctctttcttgggctagtggaaga

aagtgacaaatcccaagtttgtggaccaggcatggaaatcaactgttgctgagccccgctccccaggctcggttccctagtttctagccgtttcttggcaga

gtcttgctcagcctgaaccccgccccaggtcctgacccatttctagtcctgaccctgacccctgctacacttggccagagagggcaggcaaggtcatctg

gaagatgtggacgcccccccgcctctattcaagagactgagcacatcatttatcagacatgaaggatagcctggggtcattaggagccacgtgtgaccta

ctgacccacctgcctgtcctctctgtgatctgtcacgattctgtgtccagtgtgggctggagctgtggcttgtttagcccttcaaagacacctaccctgcaggt

agagcgtgaacctccttcttgaggggtattcctgggagtggggcgcactgagtgtgctcaagggttctgtctgctgatgtcagttctttttgattaaagtgtct

ccttacaaaaaaaaaaaaaaaaaaaaa

SEQ?ID?No.42

MKTPAQRLHILPLLLLLCGECAQVCGCNETGMLERLPRCGKAFADMMQKVAVWKWCNLSEFI

VYYESFTNCTEMETNIMGCYWPNPLAQSFITGIHRQFFSNCTVDRTHWEDPPDEVLIPLIAVPVV

LTVAMAGLVVWRSKHTDRLL

SEQ?ID?No.43

atgctcaacaaagccaagaattcaaagagtgcccagggtctggctggtcttcgaaaccttgggaacacgtgcttcatgaactcaattcttcagtgcctgag

caacacccgagagctgagagattactgcctccagaggctgtacatgcgggacctcggccacaccagcagcgctcacacggccctcatggaagagttt

gcaaaactaatccagaccatatggacgtcgtcccccaatgatgtggtgagcccatctgagttcaagacccagatccagagatatgcgccacgcttcatgg

gctataatcagcaggatgctcaggaattccttcgtttccttctggatggtctccacaatgaggtgaaccgggtggcagcaaggcctaaggccagccctga

gacccttgatcatctccctgatgaagaaaaggggcgacagatgtggaggaagtatctggaaagggaagacagtcggattggggatctcttcgttgggca

gctgaagagctccctcacatgcaccgattgtggctactgctctacagtcttcgatcccttctgggatctctcgttgcccatcgcaaagagaggttaccctga

ggtgacgttaatggattgtatgaggctcttcaccaaagaggacatattggatggtgatgagaagccaacttgctgccgctgccgagccagaaaacgatgc

ataaaaaagttctctgtccagaggttcccaaagatcttggtgctccacctgaagcgattctcagaatccaggatacgaaccagcaagctcacaacatttgtg

aatttcccactaagagacctggacttgagagaatttgcttcagaaaacaccaaccatgctgtttacaacctgtatgctgtgtccaatcactccggaaccacc

atgggaggccactatacagcctactgccgaagtccggttacaggcgaatggcacactttcaatgattccagtgtcacacccatgtcctccagccaagtgc

gcaccagcgacgcctatttgctcttctatgaactggccagtccaccctcccgtatgtagcattgaggagctgcggcccttccctcttccctgtggtggcccc

acgtcctaag

SEQ?ID?No.44

MLNKAKNSKSAQGLAGLRNLGNTCFMNSILQCLSNTRELRDYCLQRLYMRDLGHTSSAHTALM

EEFAKLIQTIWTSSPNDWSPSEFKTQIQRYAPRFMGYNQQDAQEFLRFLLDGLHNEVNRVAARP

KASPETLDHLPDEEKGRQMWRKYLEREDSRIGDLFVGQLKSSLTCTDCGYCSTVFDPFWDLSLPI

AKRGYPEVTLMDCMRLFTKEDILDGDEKPTCCRCRARKRCIKKFSVQRFPKILVLHLKRFSESRI

RTSKLTTFVNFPLRDLDLREFASENTNHAVYNLYAVSNHSGTTMGGHYTAYCRSPVTGEWHTF

NDSSVTPMSSSQVRTSDAYLLFYELASPPSRM

SEQ?ID?No.45

gcggagcgtgagctgtgcgagcgagcgagcgcgagcatagcctgcgagcgagcagagagaaagagcgagggcaagagagcggcgaggcgcct

gcgcgatgctcgggcccctaagcccgcggcgctgagccagccgggacggacatgcgcgggagggcgccgcggggtcccgctcccttgggggaat

gaaagctactggttgacttaaaaacacctgggctttacaaatttgaaggcatcccagagtggggcacaatgtcaacagcaggagttgctgctcaggatatt

cgagtcccattaaaaactggatttctccataatggtcaggccttggggaatatgaagtcctgctggggcagtcacagtgagtttgaaaataactttttaaatat

tgatccaataaccatggcctacaatctgaactcccctgctcaggagcacctaacaactgttggatgtgctgctcggtctgctccagggagcggccacttctt

tgcagagtgtggtccatctccaaggtcaagcttgccccctcttgttatctcaccaagtgaaagctcgggacagcgtgaagaggatcaagttatgtgtggtttt

aagaaactctcagtgaatggggtctgcacttccacacctccacttacacccattaaaagctgcccttcccctttcccctgtgcggctctgtgtgatcggggtt

ctcggccgctcccgccactgcccatctctgaagacctatgtgtggatgaggccgacagtgaggtagagcttctaaccaccagctcagacacagacttgct

tttagaagactctgcgccttcagatttcaaatacgatgctcctggcaggcgcagcttccgtgggtgcggccagatcaactatgcatattttgacagcccaac

tgtttctgtggcagatcttagctgtgcatctgaccagaacagagttgttccagacccaaaccctcccccacctcaaagccatcgcagattaaggaggtctc

actcaggaccagctgggtcatttaacaagccagccattcggatatctagctgcacacacagagcttctcctagctctgatgaagacaagcctgaggtccct

cccagggttcctatacctcctaggccagcaaagccagactatagacggtggtcagcagaagtgacctccaacacctacagtgatgaagataggcctccc

aaagtccccccgagagaacctttgtctcggagtaactcccgtaccccaagtcctaaaagccttccgtcttacctcaatggggtcatgcccccaacacaga

gcttcgctcctgaccccaagtatgtcagcagcaaagccctgcagagacagagcagcgaaggatctgccaacaaggttccttgcatcctgcccattattga

aaatgggaagaaggttagctcaacgcattattacttactacctgagaggccaccgtacctggacaaatatgaaaagtattttaaggaagcagaagaaaca

aacccaagcacccaaattcagccattacctgctgcctgtggtatggcctctgccacagaaaagctggcctccagaatgaaaatagatatgggtagccac

gggaagcgcaaacacttatcctacgtggtttctccataaatatgggggtcatgattcaacagaagttacatgggatgaatggctcccagttttccagtttgag

gttcgtagaacaatgtcaagtggcaaaatgaagttggtggactccgccttaatgagaaaggcttagagcagttatgaggtgctgttatgctgggagtccct

gatctatcagcataggagaaaaaagtatgatttaaagatgtgctagggggagggaaaaatgggcaacttttacatttgactacattatatacctatgtataaa

agtgcggtgtaaccatagaccatagctgcaggataaccaattagtcactcttagagtaatctgtattcagaacaattcaaacaagctggaggaacagctcc

tgatagtgtgagaattgagcaaatgggagaaagcaatattgttagatcagattataaatttgttaagtttaaagattcctggcatacaggcctgctctataaatt

tgttttccccttccctgccagcagtcttctccatacacgacagggcgtgttctccaccaggcctgtaacatcttgttgagatcatttctatggcccaatacttgt

cgctctggggttttgtcttgttgaggagaggacagcagtttctggaccatgttatcacctgtgtgtgtctcatatcttggaaattgacagatttggtgaataactt

ttccatactattcctgcttttcccatccactgaaacagcctgttgtagcaagaggctttcaagagtgcagtggagttgcgctggccatcagtgtttggggtctg

agtttgatagactagtgcagcgatcagccatatgattgagagctactttggggatatatggtacgttgtttttgttttttagacttaataaaggacaacacgagc

tggtcttgtgttgctggttcctattcagtatttcctggggattgtttgctttttaagtgaaacacttctgaccaatagcacagaacgtcttaatgccagaggtcact

tcagcatcttcctgctttgaaaactcacgctggctgcttcactgccctgagattcagtgagacacgcagtttgtgttcagtttttacatcctctgattgtttatcttg

tgcagataaacacaaagagaaggtgcttgctagcagggacactgctgccatgtcccaacaagctgttcagtttaaactgctgaatgacattatttgagctat

ttaaagcttactttagtatgaactaaatgaaggttaaaacatgctttagaaaaatgcactgatctccgcactgtgtgtacagtattggacaaaggatttattcatt

ttgttgcattattttgaatattgtcttttcattttaataaagttatattacttatttatgaaaaaaaaaaaaaaaaa

SEQ?ID?No.46

MSTAGVAAQDIRVPLKTGFLHNGQALGNMKSCWGSHSEFENNFLNIDPITMAYNLNSPAQEHLT

TVGCAARSAPGSGHFFAECGPSPRSSLPPLVISPSESSGQREEDQVMCGFKKLSVNGVCTSTPPLT

PIKSCPSPFPCAALCDRGSRPLPPLPISEDLCVDEADSEVELLTTSSDTDLLLEDSAPSDFKYDAPG

RRSFRGCGQINYAYFDSPTVSVADLSCASDQNRVVPDPNPPPPQSHRRLRRSHSGPAGSFNKPAI

RISSCTHRASPSSDEDKPEVPPRVPIPPRPAKPDYRRWSAEVTSNTYSDEDRPPKVPPREPLSRSNS

RTPSPKSLPSYLNGVMPPTQSFAPDPKYVSSKALQRQSSEGSANKVPCILPIIENGKKVSSTHYYL

LPERPPYLDKYEKYFKEAEETNPSTQIQPLPAACGMASATEKLASRMKIDMGSHGKRKHLSYVV

SP

SEQ?ID?No.47

atggctgaacaacttcttcctcaggctttgtatttgagcaatatgcggaaagctgtgaagatacgagagagaaccccagaagacattttcaaacctaccaat

gggatcatctatcactttaaaaccatgcaccgatacacgctggagatgttcagaacatgccagttttgcccacagttccgagagatcatccacaaagcactt

attgacagaagtgtccaggcttccctggaaagccagaagaagctcaactggtgtcgtgaagtcaggaagctcgtggctctgaaaaccaatggtgatgga

aactgcctcatgcatgcagcttgtcagtacatgtggggtgttcaggatactgacctggtcctgaggaaggccctctgcagcacccttaaggagacagaca

ctcggaactttaaattccgctggcagctggaatctctgaaatctcaggaatttgtggaaacaggactttgctacgacactcggaactggaatgacgaatgg

gacaacttggtcaaaatggcatcagcagacacacctgcagcccgaagtggacttcagtacaattccctggaagaaatccacatatttgtcctcagcaacat

cctcagaagacccatcattgtcatttcagacaaaatgctaagaagtttggaatctggttccaattttgctcctttgaaagtgggtgggatttatctgcctcttcac

tggcctgcccaggagtgttacagatatcccatcgtcctaggctatgacagccagcactttgtacccctggtgaccctgaaggacagtggacctgaacttcg

cgctgttccacttgttaacagagaccggggtaggtttgaagacttaaaagttcacttcttgacagatcctgagaatgagatgaaggaaaagcttctaaagga

gtacttgatagtgatggagatccctgtgcaaggctgggaccacggcacgactcacctgatcaacgctgcaaaattggatgaagctaacttacccaaagaa

ataaatttggtagacgattactttgagcttgttcagcacgaatacaagaaatggcaggagaacagcgatcaggccaggagagcggcacatgcgcagaa

ccccttggagccttccacaccccagctatcactcatggatataaaatgtgagacacccaactgtcctttcttcatgtccgtgaacactcagcctttatgccac

gaatgctcagagaggcgccaaaagaatcagagcaagctcccaaagctgaactcgaagctaggccctgaaggactcccaggcgtgggacttggctcct

caaactggagccccgaggaaaccgctggaggacctcattcagccccacccacagcacccagcctttttctcttcagtgagaccactgcaatgaagtgca

ggagtcctgggtgcccttttactttgaatgtgcagcataatggattctgtgagcgttgccacgcccggcagattaatgccagccacaccgcagaccctgga

aagtgccaagcctgccttcaggatgtcactcggacctttaatggcatctgcagtacctgtttcaaaaggactacagcagagcccagctccagcctcacttc

cagtatccctgcctcctgtcaccaacgctccaagtctgacccctcacaactcatccaaagtctcactccacactcttgccaccggactggaaatgtctctcc

ttctggctgcctctcccaggctgcacggactccaggagacagagcagggacaagcaagtgcaggaaagctggctgcatgtattttgggactccagaaa

acaagggcttttgcactctatgtttcatcgaatacagagaaaataagcagtctgttactgcctctgcgaaagctggttccccggcccccaggttccagaaca

atgtcccgtgcctgggcagggagtgcggcacactcggaagcaccatgtttgaagggtactgtcagaagtgtttcatcgaagctcagaaccagagattcc

atgaagcaagaagaacggaagaacagctgagatcaagccagcatagagacatgcctcgaactacacaggtagcctcaaggctgaaatgtgcccggg

cctcctgcaagaacattctggcctgtcgcagtgaggaactctgtatggagtgccagcacctaagccaacgagtaggttctgtggcccaccggggtgagc

ccacgcctgaagagccccctaaacagcgctgccgggcccctgcttgtgatcactttggcaatgccaagtgtaatggttactgcaatgagtgctaccagttc

aagcagatgtatggctaa

SEQ?ID?No.48

MAEQLLPQALYLSNMRKAVKIRERTPEDIFKPTNGIIYHFKTMHRYTLEMFRTCQFCPQFREIIHK

ALIDRSVQASLESQKKLNWCREVRKLVALKTNGDGNCLMHAACQYMWGVQDTDLVLRKALC

STLKETDTRNFKFRWQLESLKSQEFVETGLCYDTRNWNDEWDNLVKMASADTPAARSGLQYN

SLEEIHIFVLSNILRRPIIVISDKMLRSLESGSNFAPLKVGGIYLPLHWPAQECYRYPIVLGYDSQHF

VPLVTLKDSGPELRAVPLVNRDRGRFEDLKVHFLTDPENEMKEKLLKEYLIVMEIPVQGWDHGT

THLINAAKLDEANLPKEINLVDDYFELVQHEYKKWQENSDQARRAAHAQNPLEPSTPQLSLMDI

KCETPNCPFFMSVNTQPLCHECSERRQKNQSKLPKLNSKLGPEGLPGVGLGSSNWSPEETAGGP

HSAPPTAPSLFLFSETTAMKCRSPGCPFTLNVQHNGFCERCHARQINASHTADPGKCQACLQDVT

RTFNGICSTCFKRTTAEPSSSLTSSIPASCHQRSKSDPSQLIQSLTPHSCHRTGNVSPSGCLSQAART

PGDRAGTSKCRKAGCMYFGTPENKGFCTLCFIEYRENKQSVTASAKAGSPAPRFQNNVPCLGRE

CGTLGSTMFEGYCQKCFIEAQNQRFHEARRTEEQLRSSQHRDMPRTTQVASRLKCARASCKNIL

ACRSEELCMECQHLSQRVGSVAHRGEPTPEEPPKQRCRAPACDHFGNAKCNGYCNECYQFKQM

YG

SEQ?ID?No.49

ttttttttcatacttgataaaattttatttaaaaaaaaagagaataataatttatacccttgacaaaataaaagatcttataatataaatgtttcttagaaaatatatga

aaagataatattacaaatattaataaatcaatattcacatgacagcaaaagtggcaatgattctacaagaaggtgaggaggaagatgctttccggtccgca

gcaatgtctctggagaggcctcctgtcccttctttctccttcaatgaggtgtgctcctattttaagaaaacctgatacaagcagatctaatcagtttaggaagct

ggtatttatttgcaccgcaaaataatttttttacaaaaaaaattctatcaaggatcctttaaatatcaagtttcccaatgcacttagaatacagttaaccaaatttac

aagtcttcgacttctctctggtgtagctctaccgcanggcgtgaggtattgctgaagtgagtgcgtgcgtccgtg

Claims

1. a kind of proteinic isolating polynucleotide of encoding, this protein is regulated corticotropin releasing hormone (CRH) signalling, and described protein has the aminoacid sequence that is selected from SEQNO.:46 and SEQ ID NO.:48.

2. according to the isolating polynucleotide of claim 1, wherein said polynucleotide are mRNA, DNA or cDNA.

3. a kind of proteinic isolating polynucleotide of encoding, this protein is regulated corticotropin releasing hormone (CRH) signalling, and described polynucleotide contain the nucleotide sequence that is selected from SEQ ID No.45, SEQ ID No.47 and SEQ ID No.49.

4. a kind of proteinic isolating polynucleotide of encoding, this protein is regulated corticotropin releasing hormone (CRH) signalling, and described polynucleotide are made up of the nucleotide sequence that is selected from SEQ ID No.45, SEQ ID No.47 and SEQ ID No.49.

5. a kind of proteinic isolating polynucleotide of encoding, this protein is regulated corticotropin releasing hormone (CRH) signalling, and described protein comprises the aminoacid sequence that is selected from SEQ IDNO.:46 and SEQ ID NO.:48.

6. contain each the carrier of isolating polynucleotide of with good grounds claim 1 to 4.

7. according to the carrier of claim 6, wherein polynucleotide are operably connected to expression control sequenc.

8. can express the proteinic host cell of regulating corticotropin releasing hormone (CRH) signalling, described protein has the aminoacid sequence that is selected from SEQ ID NO.:46 and SEQ IDNO.:48.

9. with the host cell according to Claim 8 that contains the carrier transfection of regulating sequence.

10. use host cell according to Claim 8 according to the carrier transfection of claim 6 or 7.

11. identify the method that can change the compound that the CRH signalling is replied in the cell, described method comprises:

A) described compound exists when not existing described cell is contacted with CRH;

B) definite at least a proteinic amount of regulating corticotropin releasing hormone (CRH) signalling in the described cell; With

C) compare described proteinic amount when described compound exists and do not exist; Wherein, the protein of adjusting corticotropin releasing hormone (CRH) signalling is selected from SEQID NO.2, SEQ ID 4, SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.10, SEQ ID NO.12, SEQ ID NO.14, SEQ ID NO.16, SEQ ID NO.18, SEQ ID NO.20, SEQ ID NO.22, SEQ ID NO.24, SEQ ID NO.26, SEQ ID NO.28, SEQ ID NO.30, SEQ ID NO.32, SEQ ID NO.34, SEQ ID NO.36, SEQ ID NO.38, SEQ ID NO.40, SEQ ID NO.42, SEQ ID NO.44, SEQ ID NO.46 and SEQ ID NO.48.

12. according to the method for claim 11, wherein cell is an eukaryotic cell, the adenoma cell of originating as mouse PATH cell is AtT-20.

13. method according to claim 11 or 12, wherein use antibody determine to regulate the proteinic amount of CRH signalling, this antibodies contains and is selected from SEQ ID NO.2, SEQ ID4, SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.10, SEQ ID NO.12, SEQ ID NO.14, SEQ ID NO.16, SEQ ID NO.18, SEQ ID NO.20, SEQ ID NO.22, SEQ ID NO.24, SEQ ID NO.26, SEQ ID NO.28, SEQ ID NO.30, SEQ ID NO.32, SEQ ID NO.34, SEQ ID NO.36, SEQ ID NO.38, SEQ ID NO.40, SEQ ID NO.42, SEQ ID NO.44, the polypeptide of the aminoacid sequence of SEQ ID NO.46 and SEQ ID NO.48.

14. method according to claim 11 or 12, wherein determine the proteinic amount of adjusting CRH signalling by the gene transcription level of a kind of gene of assessment, this genes encoding is selected from SEQID NO.2, SEQ ID 4, SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.10, SEQ ID NO.12, SEQ ID NO.14, SEQ ID NO.16, SEQ ID NO.18, SEQ ID NO.20, SEQ ID NO.22, SEQ ID NO.24, SEQ ID NO.26, SEQ ID NO.28, SEQ ID NO.30, SEQ ID NO.32, SEQ ID NO.34, SEQ ID NO.36, SEQ ID NO.38, SEQ ID NO.40, SEQ ID NO.42, SEQ ID NO.44, the aminoacid sequence of SEQ ID NO.46 and SEQ ID NO.48.

15. method according to claim 14, wherein use the level of probe assessment genetic transcription, this probe is selected from SEQ ID NO.2 in conjunction with coding, SEQ ID 4, SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.10, SEQ ID NO.12, SEQ ID NO.14, SEQ ID NO.16, SEQ ID NO.18, SEQ ID NO.20, SEQ ID NO.22, SEQ ID NO.24, SEQ ID NO.26, SEQ ID NO.28, SEQ ID NO.30, SEQ ID NO.32, SEQ ID NO.34, SEQ ID NO.36, SEQ ID NO.38, SEQ ID NO.40, SEQ ID NO.42, SEQ ID NO.44, the polynucleotide of the aminoacid sequence of SEQ ID NO.46 and SEQ ID NO.48.

16., wherein use microarray technology analyzing gene expression levels according to the method for claim 14 or 15.

17. method according to claim 16, wherein use the array assessment gene expression dose of oligonucleotide probe, described probe has aminoacid sequence SEQ ID NO.2 in conjunction with coding, SEQ ID 4, SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.10, SEQ IDNO.12, SEQ ID NO.14, SEQ ID NO.16, SEQ ID NO.18, SEQ IDNO.20, SEQ ID NO.22, SEQ ID NO.24, SEQ ID NO.26, SEQ ID NO.28, SEQ ID NO.30, SEQ ID NO.32, SEQ ID NO.34, SEQ ID NO.36, SEQ ID NO.38, SEQ ID NO.40, SEQ ID NO.42, SEQ ID NO.44, the polynucleotide of the polypeptide group of SEQ ID NO.46 and SEQ ID NO.48.

18. identify the method that can change the compound that the CRH signalling is replied in the cell, described method comprises:

A) exist at test-compound and contact described cell when not existing; With

B) determine that coding has aminoacid sequence SEQ ID NO.2, SEQ ID 4, SEQID NO.6, SEQ ID NO.8, SEQ ID NO.10, SEQ ID NO.12, SEQ IDNO.14, SEQ ID NO.16, SEQ ID NO.18, SEQ ID NO.20, SEQ IDNO.22, SEQ ID NO.24, SEQ ID NO.26, SEQ ID NO.28, SEQ ID NO.30, SEQ ID NO.32, SEQ ID NO.34, SEQ ID NO.36, SEQ ID NO.38, SEQ ID NO.40, SEQ ID NO.42, SEQ ID NO.44, the expression of gene level of the polypeptide of SEQ ID NO.46 and SEQ ID NO.48.

19. method according to claim 18, wherein use the array of oligonucleotide probe to determine the expression of gene level, described probe is in conjunction with having nucleic acid sequence SEQ ID No.1, SEQID No.3, SEQ ID No.5, SEQ ID No.7, SEQ ID No.9, SEQ ID NO.11, SEQ ID NO 13, SEQ ID No.15, SEQ ID No 17, SEQ ID No 19, SEQID No.21, SEQ ID No.23, SEQ ID No.25, SEQ ID No.27, SEQID No.29, SEQ ID NO.31, SEQ ID No.33, SEQ ID No.35, SEQID No.37, SEQ ID No.39, SEQ ID No.41, SEQ ID No.43, SEQID No.45, the polynucleotide of SEQ ID No.47 and SEQ ID No.49.

20. identify to change the method that active compound is replied in CRH signalling in the cell, described method comprises:

The CRH of described cell replied activity when b) more described compound existed and do not exist.

21. according to the method for claim 20, wherein cell expressing has aminoacid sequence SEQ ID NO.2, SEQ ID 4, SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.10, SEQ ID NO.12, SEQ ID NO.14, SEQ ID NO.16, SEQ ID NO.18, SEQ ID NO.20, SEQ ID NO.22, SEQ ID NO.24, SEQ ID NO.26, SEQ ID NO.28, SEQ ID NO.30, SEQ ID NO.32, SEQ ID NO.34, SEQ ID NO.36, SEQ ID NO.38, SEQ ID NO.40, SEQ ID NO.42, SEQ ID NO.44, the protein group of SEQ ID NO.46 and SEQ ID NO.48.

22. according to the method for claim 21, wherein CRH replys activity and is assessed as the variation of transcribing on the gene level.

23., wherein use microarray technology assessment CRH to reply activity according to the method for claim 21 or 22.

24. according to each method of claim 20 to 23, wherein cell is can express to have the SEQ of being selected from ID NO.2, SEQ ID 4, SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.10, SEQ ID NO.12, SEQ ID NO.14, SEQ ID NO.16, SEQ ID NO.18, SEQ ID NO.20, SEQ ID NO.22, SEQ ID NO.24, SEQ ID NO.26, SEQ ID NO.28, SEQ ID NO.30, SEQ ID NO.32, SEQ ID NO.34, SEQ ID NO.36, SEQ ID NO.38, SEQ ID NO.40, SEQ ID NO.42, SEQ ID NO.44, at least a proteinic host cell of the aminoacid sequence of SEQ ID NO.46 and SEQ ID NO.48.

25. according to the method for claim 24, wherein host cell is contained at least a carrier transfection of regulating sequence.

26. according to the method for claim 24, wherein host cell is contained coding and is selected from SEQ ID NO.2, SEQ ID 4, SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.10, SEQ ID NO.12, SEQ ID NO.14, SEQ ID NO.16, SEQ ID NO.18, SEQ ID NO.20, SEQ ID NO.22, SEQ ID NO.24, SEQ ID NO.26, SEQ ID NO.28, SEQ ID NO.30, SEQ ID NO.32, SEQ ID NO.34, SEQ ID NO.36, SEQ ID NO.38, SEQ ID NO.40, SEQ ID NO.42, SEQ ID NO.44, at least a carrier transfection of the polynucleotide sequence of the aminoacid sequence of SEQ ID NO.46 and SEQ ID NO.48.

27. the method for CRH inductive dysthymia disorders in the diagnosis individuality, described method comprises:

A) obtain the biological sample of described individuality; With

B) determine to regulate in the described biological sample at least a proteinic amount of corticotropin releasing hormone (CRH) signalling; The protein of wherein regulating corticotropin releasing hormone (CRH) signalling is selected from SEQ ID NO.2, SEQ ID 4, SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.10, SEQ ID NO.12, SEQ ID NO.14, SEQ ID NO.16, SEQ ID NO.18, SEQ ID NO.20, SEQ ID NO.22, SEQ ID NO.24, SEQ ID NO.26, SEQ ID NO.28, SEQ ID NO.30, SEQ ID NO.32, SEQ ID NO.34, SEQ ID NO.36, SEQ ID NO.38, SEQ ID NO.40, SEQ ID NO.42, SEQ ID NO.44, SEQ ID NO.46 and SEQ ID NO.48.

28. according to the method for claim 27, wherein biological sample is body fluid or tissue sample.

29. method according to claim 27 or 28, wherein use antibody determine to regulate the proteinic amount of CRH signalling, this antibodies contains and is selected from SEQ ID NO.2, SEQ ID4, SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.10, SEQ ID NO.12, SEQ ID NO.14, SEQ ID NO.16, SEQ ID NO.18, SEQ ID NO.20, SEQ ID NO.22, SEQ ID NO.24, SEQ ID NO.26, SEQ ID NO.28, SEQ ID NO.30, SEQ ID NO.32, SEQ ID NO.34, SEQ ID NO.36, SEQ ID NO.38, SEQ ID NO.40, SEQ ID NO.42, SEQ ID NO.44, the polypeptide of the aminoacid sequence of SEQ ID NO.46 and SEQ ID NO.48.

30. method according to claim 27 or 28, wherein determine to regulate the proteinic amount of CRH signalling by the gene transcription level of assessing a kind of gene, this genes encoding is selected from SEQ ID NO.2, SEQ ID 4, SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.10, SEQ ID NO.12, SEQ ID NO.14, SEQ ID NO.16, SEQ ID NO.18, SEQ ID NO.20, SEQ ID NO.22, SEQ ID NO.24, SEQ ID NO.26, SEQ ID NO.28, SEQ ID NO.30, SEQ ID NO.32, SEQ ID NO.34, SEQ ID NO.36, SEQ ID NO.38, SEQ ID NO.40, SEQ ID NO.42, SEQ ID NO.44, the aminoacid sequence of SEQ ID NO.46 and SEQ ID NO.48.

31. method according to claim 3O, wherein use the level of probe assessment genetic transcription, this probe is selected from SEQ ID NO.2 in conjunction with coding, SEQ ID 4, SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.10, SEQ ID NO.12, SEQ ID NO.14, SEQ ID NO.16, SEQ ID NO.18, SEQ ID NO.20, SEQ ID NO.22, SEQ ID NO.24, SEQ ID NO.26, SEQ ID NO.28, SEQ ID NO.30, SEQ ID NO.32, SEQ ID NO.34, SEQ ID NO.36, SEQ ID NO.38, SEQ ID NO.40, SEQ ID NO.42, SEQ ID NO.44, the polynucleotide of the aminoacid sequence of SEQ ID NO.46 and SEQ ID NO.48.

32. according to the method for claim 30 or 31, the level of wherein using the microarray technology analyzing gene to transcribe.

33. method according to claim 32, wherein use the array analysis gene transcription level of oligonucleotide probe, described probe has aminoacid sequence SEQ ID NO.2 in conjunction with coding, SEQ ID 4, SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.10, SEQ IDNO.12, SEQ ID NO.14, SEQ ID NO.16, SEQ ID NO.18, SEQ IDNO.20, SEQ ID NO.22, SEQ ID NO.24, SEQ ID NO.26, SEQ ID NO.28, SEQ ID NO.30, SEQ ID NO.32, SEQ ID NO.34, SEQ ID NO.36, SEQ ID NO.38, SEQ ID NO.40, SEQ ID NO.42, SEQ ID NO.44, the polynucleotide of the polypeptide group of SEQ ID NO.46 and SEQ ID NO.48.

34. method according to claim 32, wherein use the level of the array analysis genetic transcription of oligonucleotide probe, these probes are in conjunction with having nucleic acid sequence SEQ ID No.1, SEQ IDNo.3, SEQ ID No.5, SEQ ID No.7, SEQ ID No.9, SEQ ID NO.11, SEQ ID NO 13, SEQ ID No.15, SEQ ID No 17, SEQ ID No 19, SEQID No.21, SEQ ID No.23, SEQ ID No.25, SEQ ID No.27, SEQID No.29, SEQ ID NO.31, SEQ ID No.33, SEQ ID No.35, SEQID No.37, SEQ ID No.39, SEQ ID No.41, SEQ ID No.43, SEQID No.45, the polynucleotide of SEQ ID No.47 and SEQ ID No.49.