CN1976949A - Ion channel - Google Patents

Abstract

We disclose Kv9.2 polypeptides comprising the amino acid sequence shown in SEQ ID NO. 3 or SEQ ID NO: 5, and homologues, variants and derivatives thereof. Nucleic acids capable of encoding Kv9.2 polypeptide are also disclosed, in particular, those comprising the nucleic acid sequences shown in SEQ ID No. 1, SEQ ID No.2 or SEQ ID NO: 4.

Description

Ionic channel

Invention field

The nucleic acid that the present invention relates to differentiate recently, by the polypeptide of these nucleic acid encodings, and their production and purposes.More specifically, nucleic acid of the present invention and polypeptide relate to the ionic channel subunit, hereinafter are called " Kv9.2 ".The invention still further relates to the effect that suppresses or activate this nucleic acid and polypeptide.

Background of invention

Ionic channel is many subunits embrane-associated protein, and it brings into play crucial effects when the cell functionating.They control many ions cross over cytolemma pass through, comprise sodium, potassium, chlorine and calcium.Because ion carries electric charge, ionic channel is the elementary cell electrology characteristic important mediation person of (comprising the cell resting potential).Their fault and defective relate to multiple disease and symptom, comprise epilepsy, hypertension and cystic fibrosis.

Potassium channel is distributed in the surface film of cell and optionally allows potassium ion to pass through, and it is believed that it plays a significant role in the control cell membrane potential.Particularly, in the N﹠M cell, responsible maincenter such as its frequency by the control action current potential, persistence and peripheroneural neurotransmission, cardiac pacing, Muscle contraction etc.In addition, shown that it also relates to propagation of the adjusting of the secretion of hormone, cell volume, cell etc.

It is believed that potassium channel gene family is maximum and the most various ionic channel family.According to the quantity of membrane spaning domain, for example two, four or six structural domains are divided into many subfamilies.Subfamily with two structural domains comprises GIRK, IRK, CIR and ROMK, and they have the pore structure territory of high conservative.Twik-1 and Twik-sample passage and TREK, TASK-1 and TASK-2 and TRAAK have 4 membrane spaning domains, and relate to and keep the stable state potassium ion current potential of striding film.Shaker-sample and eag type passage have six structural domains, and are maximum subfamilies.The Shaker type is to have very high multifarious family, and can further be divided into many subfamily Kv1, Kv2, Kv3 and Kv4.On the other hand, the eag type is made up of eag, eag-genes involved and elk, and its genes involved comprises corresponding to the hyperpolarization activated form potassium channel of KAT gene cluster and by cyclic nucleotide activatory cationic channel.

1987, reported the complete nucleotide sequence of first encoded K v passage with the clone of Shaker passage (Kv1).Cause derived from three kinds of heterogeneic K+ passage cDNAs Shab (Kv2) with the low preciseness screening of Shaker cDNA cDNA library, Shaw (Kv3) and Shal (Kv4) separate.These sequences and Shaker homology have～40% identity.In nucleus, have with Shaker＞the Kvl family of 60% homology is maximum passage family, has at least seven members.Except that four Mammals subfamilies that relate to Shaker, Shab, Shal and Shaw, other five subfamilies (Kv5-9) have been described also.Recently, clone and in heterologous expression system, expressed more than 30 Kv passage.These passages usually voltage sensitivity, electric current kinetics and stable state activate with inactivation aspect show different.

The Kv passage exists as the tetramer, strides film-leap-subunit by 4 6-and constitutes in conjunction with forming the function passage.Not only same subunit can be in conjunction with forming the function passage, and different subunits also can be external and body in conjunction with forming function heteromerism passage.These heteromerism passages have unique characteristic, promptly usually show observed corresponding mixing with the number channel characteristic.In addition, indivedual Kv-subunits are non-functional when single expression.For example, recently the member Kv9.3 subunit of the Mammals Kv family that differentiates itself does not form function with the number passages, and functionating in the heteromerism complex body only, its mutagenic voltage sensitivity and kinetics in the heteromerism complex body.

Auxiliary subunit can combine with the Kv subunit, to increase more various Kv channel function.Current, set forth four kinds of Kv subunit gene families.All all is cytoplasmic protein, and the about 40kDa of quality has conservative core sequence and variable NH ₂Terminal.Shown that the Kv subunit gives the subunit function, comprised fast and slow inactivation, changed voltage sensitivity and delay passivation.In addition, subunit can be used as cellular oxidation also original sensor play a role because as if it gives Kv4.2 passage O in heterologous expression system ₂Susceptibility.

Shown before that potassium voltage-gated channel, delayed rectifier, subfamily S, member-2 (Kv9.2) mRNA expressed in pancreas islet, but do not show and localization (colocalize) altogether takes place with Regular Insulin, show it and the irrelevant (Yan of control secretion of insulin, L., Deng, Diabetes 2004.53.597-607)..

We find that the Kv9.2 potassium channel is to keeping blood sugar extremely important now.Blood glucose levels significantly different (promptly low) is in the wild-type animal in knocking out the animal of Kv9.2.Therefore, the metabolism of this Gene Handling and adjusting sugar and fat.

Summary of the invention

First aspect according to the present invention, we provide the transgenic nonhuman animal with the disrupted native gene of function, wherein the Kv9.2 gene comprises the nucleotide sequence that is shown as SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:4, perhaps has the sequence of at least 70% sequence identity with them.

Preferably, transgenic nonhuman animal has disappearance in Kv9.2 gene or its part.Preferably, transgenic nonhuman animal shows any one in the following phenotype or makes up when comparing with the wild-type animal: (a) blood glucose levels reduces; (b) preferably when detecting with open field test (Open Field Test) and/or positive maze test (Plus Maze Test), anxiety increases.

According to second aspect present invention, this paper provides a kind of transgenic nonhuman animal, wherein at least partly or entirely Kv9.2 gene of this animal use from another animal, preferably another species, more preferably be that the sequence of people's Kv9.2 gene replaces.

Preferably, described transgenic nonhuman animal is a mouse.

Preferably, described transgenic nonhuman animal comprises the disrupted Kv9.2 gene of function, preferably in the Kv9.2 gene disappearance is arranged, wherein the Kv9.2 gene comprises the nucleotide sequence that SEQ ID NO:4 shows or has the sequence of at least 70% sequence identity with it.

According to third aspect of the present invention, we provide a kind of isolated cells or tissue, and described cell or tissue is from first or second described non-human transgenic animal in aspect according to the present invention.

As the 4th aspect of the present invention, this paper provides a kind of cell with the disrupted endogenous Kv9.2 gene of function, wherein the Kv9.2 gene comprises the nucleotide sequence that SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:4 show, perhaps has the sequence of at least 70% sequence identity with them.

The 5th aspect according to the present invention, we provide described transgenic nonhuman animal, described cell or tissue or the described cell purposes as the model of anxiety or diabetes.

The 6th aspect, the invention provides described transgenic nonhuman animal, described cell or tissue or described cell as with the purposes of the model of Kv9.2 diseases associated.

The 7th aspect of the present invention, this paper provides described transgenic nonhuman animal, described cell or tissue or the described cell purposes in the method for the agonist of differentiating the Kv9.2 polypeptide or antagonist, described Kv9.2 polypeptide comprises the aminoacid sequence that SEQ ID NO:3 or SEQ ID NO:5 show, perhaps has the sequence of at least 70% sequence identity with them.

The 8th aspect according to the present invention, we provide the agonist of discriminating Kv9.2 polypeptide or the method for antagonist, described Kv9.2 polypeptide comprises the aminoacid sequence of SEQ ID NO:3 or SEQ ID NO:5 demonstration, the sequence that perhaps has at least 70% sequence identity with them, described method comprises and gives the animal candidate compound, described animal is described wild-type in first aspect or transgenic nonhuman animal according to the present invention preferably, and measures any one variation in the following phenotype: (a) blood glucose levels; (b) anxiety preferably detects with open field test and/or positive maze test.

Preferably, described method is differentiated the agonist of Kv9.2 polypeptide, and comprises the candidate compound that discriminating can cause any one increase in the animal demonstration phenotype (a)-(b).

In other words, perhaps in addition, described method is differentiated the antagonist of Kv9.2 polypeptide, and comprises discriminating and can cause that animal shows the candidate compound that any one or such phenotype in the phenotype (a)-(b) reduce.

The 9th aspect according to the present invention, we provide the agonist of discriminating Kv9.2 polypeptide or the method for antagonist, described Kv9.2 polypeptide comprises aminoacid sequence that SEQ ID NO:3 or SEQ ID NO:5 show or has the sequence of at least 70% sequence identity with these sequences, described method comprises candidate compound is contacted with cell or tissue, be preferably wild-type cell or tissue or described cell or tissue or described cell, and measure the conduction of cell or tissue cell or dynamic (dynamical) change.

Preferably, described method is differentiated the agonist of Kv9.2 polypeptide, and comprises that discriminating can improve conduction or dynamic (dynamical) candidate compound of cell.

In other words, perhaps in addition, described method is differentiated the antagonist of Kv9.2 polypeptide, and comprises that discriminating can reduce conduction or dynamic (dynamical) candidate compound of cell.

The tenth aspect according to the present invention, this paper provides discriminating to be suitable for treatment or relaxes anxiety or diabetes, the method of the compound of Kv9.2 relative disease preferably, described method comprises and will contain aminoacid sequence that SEQ IDNO:3 or SEQ ID NO:5 show or contact with candidate compound with Kv9.2 polypeptide that these sequences have a sequence of at least 70% sequence identity, and whether definite this candidate compound is the agonist or the antagonist of Kv9.2 polypeptide.

As the 11 aspect of the present invention, we provide the purposes of Kv9.2 polynucleotide, described Kv9.2 polynucleotide contain nucleotide sequence that SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:4 show or have the sequence of at least 70% sequence identity with these sequences, be used for differentiating treatment anxiety or diabetes, the preferably agonist or the antagonist of the Kv9.2 polynucleotide of Kv9.2 relative disease.

The 12 aspect according to the present invention, we provide the purposes of Kv9.2 polypeptide, described Kv9.2 polypeptide contains aminoacid sequence that SEQ ID NO:3 or SEQ ID NO:5 show or has the sequence of at least 70% sequence identity with these sequences, be used for differentiating treatment anxiety or diabetes, the preferably agonist or the antagonist of the Kv9.2 polypeptide of Kv9.2 relative disease.

The 13 aspect according to the present invention, we provide the antagonist of Kv9.2 polypeptide, described Kv9.2 polypeptide contains aminoacid sequence that SEQ ID NO:3 or SEQ ID NO:5 show or has the sequence of at least 70% sequence identity with these sequences, be used for preferably using in the method for Kv9.2 relative disease in treatment individual anxiety or diabetes.

The 14 aspect according to the present invention, this paper provides the purposes of the antagonist of Kv9.2 polypeptide, described Kv9.2 polypeptide contains aminoacid sequence that SEQ ID NO:3 or SEQ ID NO:5 show or has the sequence of at least 70% sequence identity with these sequences, be used to prepare treatment individual anxiety or diabetes, preferably the pharmaceutical composition of Kv9.2 relative disease.

The 15 aspect according to the present invention, we provide the method for the treatment of the individuality of suffering from anxiety or diabetes, preferably suffer from the individuality of Kv9.2 relative disease, and described method comprises the antagonist that gives individual Kv9.2.

The 16 aspect according to the present invention, we provide diagnosis individual anxiety or diabetes, the method for Kv9.2 relative disease preferably, and described method comprises expression, level or the active variation that detects Kv9.2 in the individual or individual cell or tissue.

Preferably, the Kv9.2 relative disease is selected from the group of being made up of following: I type and type ii diabetes, hyperinsulinemia (hyperinsulinaemia), hunger disease, insulin resistance (insulinresistance), diabetic complication comprises the vascular disease that diabetes are relevant, the ephrosis neuropathy relevant that diabetes are relevant, and treatment hypoglycemia with diabetes, (they are HDL to hyperlipidaemia (hyperlipoidemia), LDL or VLDL), unusual lipidemia (dyslipoidemia), no matter its cause is to be primary in or to be secondary to diabetes, hyperthyroidism/go down, acromegaly, liver failure, renal failure, Vipoma, pancreatitis and alcohol inductive hypoglycemia.

In other words, or in addition, the Kv9.2 relative disease is selected from the group of being made up of following: social anxiety disorder (social anxiety), stress reaction obstacle after the wound (post traumatic stress disorder), phobia (phobias), social phobia (social phobia), special phobia (special phobias), panic disorder (panic disorder), obsessive compulsive disorder (obsessive compulsive disorder), gross stress reaction obstacle (acute stress disorder), separation anxiety disease (separation anxietydisorder), generalized anxiety disorder disease (generalized anxiety disorder), major depression (major depression), depression (dysthymia), the two poles of the earth obstacle (bipolar disorder), seasonal affective disorder (seasonal affective disorder), post-natal depression (post nataldepression), manic depressive illness (maniac depression), the two poles of the earth dysthymia disorders (bipolar depression), anxiety disorder (anxiety), anxiety disorder (anxiety disorders), anxiety related behavior (anxiety-relatedbehavior) and generalized anxiety disorder disease, agoraphobia (agoraphobia), listed those panic disorders (panic disorders) and depressed (depression) among gross stress reaction obstacle (acute stress disorder) and the DSM-IV.

The 17 aspect according to the present invention, we provide Kv9.2 polypeptide, and described polypeptide comprises the aminoacid sequence that SEQ IDNO:3 or SEQ ID NO:5 show, or has its homologue, variant or the derivative of at least 70% sequence identity with them.

The 18 aspect according to the present invention, we provide the nucleic acid of coding the 18 described polypeptide in aspect according to the present invention.

Preferably, described nucleic acid comprises the sequence that SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:4 show, or has its homologue, variant or the derivative of at least 70% sequence identity with them.

Brief description of the drawings

Fig. 1 is the diagram that shows the knockout carrier be used to produce the Kv9.2 deficient mice.

Fig. 2 shows the result of the Kv9.2 genetic expression that is obtained by people RT-PCR screening.

Fig. 3 is display analysis blood glucose levels (mmol/L) result's a chart.

Fig. 4 shows the nucleotide sequence that knocks out plasmid vector.

Fig. 5 is display analysis open field test result's a chart; Fig. 5 A is at periphery (left side) and central authorities' (right side) zone full distance (solid column type is a knock-out animal) of dividing a word with a hyphen at the end of a line; Fig. 5 B is in periphery (left side) and central authorities' (right side) zone time (solid column type is a knock-out animal) of dividing a word with a hyphen at the end of a line; With Fig. 5 C be the quantity (solid column type is a knock-out animal) that enters solid band (filed zone).

Fig. 6 is a chart of analyzing positive maze test result, this pictorialization time (solid column type is a knock-out animal, and the shade column type is the wild-type animal) that spends in labyrinth disposition of latch arm and the open arms.

Sequence list

SEQ ID NO:1 shows the cDNA sequence of people Kv9.2. SEQ ID NO:2 shows the open read frame derived from SEQ ID NO:1. SEQ ID NO:3 shows the amino acid sequence of people Kv9.2. SEQ ID NO:4 shows the open read frame of the cDNA of mouse Kv9.2. SEQ ID NO:5 shows the amino acid sequence of mouse Ky9.2. SEQ ID NOs.6-18 shows the genotype primer that knocks out plasmid for making up. SEQ ID NOs:19 demonstration knocks out the plasmid vector sequence.

Unless otherwise indicated, enforcement of the present invention will utilize conventional chemistry, molecular biology, microbiology, recombinant DNA and immunological technique, and these technology are within the those skilled in the art's ability of this area. These technology are set forth in the pertinent literature. Referring to for example, J.Sambrook, E.F.Fritsch, and T.Maniatis, 1989, Molecular Cloning:A Laboratory Manual, second edition, 1-3 volume, Cold Spring Harbor Laboratory Press; Ausubel, F.M. etc. (1995 and regular supplementary issue; Current Protocols in Molecular Biology, 9,13, and 16 chapters, John Wiley ﹠ Sohs, New York, N.Y.); B.Roe, J.Crabtree, and A.Kahn, 1996, DNA Isolation and Sequencing:Essential Techniques, John Wiley ﹠ Sons; J.M.Polak and James O ' D.McGee, 1990, In Situ Hybridization:Principles and Practice; Oxford University Press; M.J.Gait (Editor), 1984, Oligonucleotide Synthesis:A Practical Approach, Irl Press; D.M.J. Lilley and J.E.Dahlberg, 1992, Methods of Enzymology:DNA Structure Part A:Synthesis and Physical Analysis of DNA Methods in Enzymology, Academic Press; Using Antibodies:A Laboratory Manual:Portable Protocol NO.I by Edward Harlow, David Lane, Ed Harlow (1999, Cold Spring Harbor Laboratory Press, ISBN 0-87969-544-7); Antibodies:A Laboratory Manual by Ed Harlow (Editor), David Lane (Editor) (1988, Cold Spring Harbor Laboratory Press, ISBN 0-87969-314-2), 1855, Lars-Inge Larsson " Immunocytochemistry:Theory and Practice ", CRC Press inc., Baca Raton, Florida, 1988, ISBN 0-8493-6078-1, John D.Pound (editor); " Immunochemical Protocols, vol80 ", continuously: " Methods in Molecular Biology ", Humana Press, Totowa, New Jersey, 1998, ISBN 0-89603-493-3, Handbook of Drug Screening, Ramakrishna Seethala edits, Prabhavathi B.Fernandes (2001, New York, NY, Marcel Dekker, ISBN 0-8247-0562-9); Lab Ref:A Handbook of Recipes, Reagents, and Other Reference Tools for Use at the Bench, Jane Roskams and Linda Rodgers edit, 2002, Cold Spring Harbor Laboratory, ISBN 0-87969-630-3; With The Merck Manual of Diagnosis and Therapy (the 17th edition, Beers, M.H., and Berkow, R, editor, ISBN:0911910107, John Wiley ﹠ Sons). These document this paper commonly used all quote as a reference.

The detailed description of invention

The Kv9.2 subunit

The present invention relates generally to the purposes of ion channel and subunit thereof, relate to particularly and treating, alleviating or diagnosing in the Kv9.2 relevant disease that comprises diabetes or anxiety disorder the Kv9.2 subunit of voltage gated k+ channel blocker and the purposes of homologue, variant or derivative thereof. Other embodiment of this content and the present invention will describe in further detail hereinafter.

The expression characteristic of Kv9.2 subunit

As shown in an embodiment, polymerase chain reaction (PCR) amplification Kv9.2 cDNA detects the expression of Kv9.2, and the abundance in comprising many organs such as prostate, liver, reproductive organs, muscle and brain is different.

Use the Kv9.2 cDNA of SEQ ID NO:1 by BLASTN seeker EST data source, in cDNA library, find homogeneity. This shows that Kv9.2 expresses in normal or abnormal tissue,

For example:

U69192 people baby brain

AA776703 people's testis

A1681499 people's lung

AW292826 people's ovary

Therefore, Kv9.2 polypeptide, nucleic acid, probe, antibody, expression vector and part are for being useful with detection, diagnosis, treatment and other analysis excessive, disease that deficiency is relevant with unconventionality expression of Kv9.2 subunit in these and other tissue.

Kv9.2 subunit relevant disease

According to method and composition described herein, the Kv9.2 subunit is used for the treatment of and diagnoses a series of disease. For the purpose of convenient, these diseases are known as the Kv9.2 relevant disease.

Herein, we have proved that people Kv9.2 is positioned homo sapiens (Homo sapiens) chromosome 8q22. Therefore, in a specific embodiment, the Kv9.2 subunit can be used for treatment or diagnosis is positioned (maps to) this locus, chromosome band, zone, arm or identical chromosomal disease. Being confirmed as being relevant to identical locus, chromosome band, zone, arm or chromosomal known disease with the chromosome position of Kv9.2 subunit comprises renal tubular acidosis-osteopetrosis syndrome (Renal tubular acidosis-osteopetrosis syndrome), Dihydropyrimidinuria, Koln syndrome (Cohen syndrome) and follows the Ke of throat's deformity-Fei Er Shi (Klippel-Feil) syndrome. Yet, up to now, find that ion channel is incremented or the situation of down-regulation under, relevant with ion channel without any special disease.

As confirming that in an embodiment the knock-out mice of Kv9.2 defective shows the phenotype of certain limit.

Particularly, embodiment 5 proofs, blood glucose levels is apparently higher than corresponding wild-type mice in the Kv9.2 knock-out mice. Therefore, the shortage of Kv9.2 activity is relevant with the reduction of blood sugar level.

Therefore we disclose a kind of method that is preferably used for treating the individual blood sugar level of reduction of diabetes, and described method is included in level or the activity that reduces Kv9.2 in this individuality. Record such as other document, this can pass through the expression of downward modulation Kv9.2, or finishes by the antagonist that uses Kv9.2.

Particularly, regulating by this method the disease that blood-glucose can be used for treating comprises, but be not limited to, 1 type comprises the relevant angiosis of diabetes, the ephrosis neuropathy relevant with diabetes that diabetes are relevant with type ii diabetes, hyperinsulinemia, hunger disease, insulin resistance, diabetic complication, and hypoglycemic treatment. In addition, also can be used for treating the hypoglycemia that hyperlipidemia (hyperlipoidemia) (they are HDL, LDL or VLDL), unusual pionemia (dyslipoidemia), former or secondary diabetes, hyperthyroidism/go down, acromegalia, liver failure, kidney failure, Vipoma, pancreatitis and alcohol are induced. The Kv9.2 knock-out mice therefore can be further as any model in these diseases.

In addition, embodiment 6 has described open field test, and wherein the Kv9.2 knock-out mice shows the wild type companion's anxiety more than them. Therefore, the shortage of Kv9.2 activity is relevant with the raising of stress reaction.

Therefore we disclose a kind of method that individual stress reaction or anxiety or the two all are lowered that reduces, and described method is included in level or the activity that improves Kv9.2 in this individuality. Record such as other document, this can pass through to raise the expression of Kv9.2, or uses the activator of Kv9.2 to finish.

The adjusting of Kv9.2 and Kv9.2 activity specifically comprises the antagonist of Kv9.2, can be used for treating or alleviates disease or symptom take stress reaction and anxiety as feature. This disease comprises stress reaction obstacle (post traumatic stress disorder) after social anxiety disorder, the wound, neurosis, social phobia, special neurosis, panic disorder (panic disorder), obsessive compulsive disorder, gross stress reaction obstacle (acute stress disorder), separation anxiety disease, generalized anxiety disorder disease, PD (major depression), depression, the two poles of the earth obstacle (bipolar disorder), SAD, post-natal depression, manic-depressive psychosis, the two poles of the earth depression. The Kv9.2 knock-out mice therefore can be further as any model in these diseases.

In a preferred embodiment, the Kv9.2 relevant disease comprises the disease take stress reaction or anxiety as symptom. In particularly preferred embodiment, the Kv9.2 relevant disease comprises anxiety listed above and stress reaction relevant disease.

In addition, find that also gene has the effect of the obstacle that affects the nerves, comprise listed those panic disorders and depression among stress reaction obstacle after anxiety, anxiety disorder, anxiety corelation behaviour and generalized anxiety disorder disease, panic disorder, agoraphobia, social phobia, obsessive compulsive disorder, the wound, gross stress reaction obstacle and the DSM-IV.

Record as mentioned, use any method and composition described herein, the Kv9.2 subunit can be used for diagnosing and/or treats any disease in these specified diseases.

Particularly, we imagine use nucleic acid, the carrier that comprises Kv9.2 nucleic acid, polypeptide, the homologue, variant or the derivative that comprise them, pharmaceutical composition, host cell and comprise the transgenic animals of Kv9.2 nucleic acid and/or polypeptide are used for the treatment of or diagnose specified disease listed above. In addition, we are diagnosing or are treating in the above-mentioned specified disease imagination, use can interact with Kv9.2 or the compound of combination, preferably Kv9.2 heteromerism or the together antagonist of number ion channel, preferably can regulate the dynamics of ion channel or reduce its conductive compound, the antibody of Kv9.2 subunit, and the method for preparing or differentiate these materials. Particularly, be used for the treatment of or prevent in the vaccine of specified disease in production, we use any in these compounds, composition, the molecule etc. We also disclose the diagnostic kit for detection of individual specified disease.

By using the Kv9.2 subunit to differentiate that these or other connection location (linkage mapping) method medicable or diagnosable disease specific is known in the art, also in other chapters and sections of this paper, describe.

Anxiety and stress reaction

Anxiety and stress reaction, and the obstacle that this performance is arranged comprise that the Kv9.2 relative disease is well known in the art.General description is as follows:

It is nervous, uneasy, oversensitive and worried and apprehensive that anxiety and stress reaction are also referred to as.Stress reaction can be from making any situation or the idea that subjects feel is dejected, angry or worry.The someone is produced the factor of pressure, may not produce pressure another person.

Anxiety is worried and apprehensive or frightened mood.The source of this uneasy mood is always not known or discernible, and it can increase the misery of subjects feel.

Stress reaction is the part of orthobiosis.Quantity after a little while, stress reaction can be useful---it can encourage individuality to be rich in productivity more.Yet, too nervous, or to produce strong reaction for pressure be deleterious.This can make individuality feel badly generally, and produces specific physiology or mental illness, for example infection, heart trouble or depression.Continual stress reaction usually causes anxiety and unsound health behavior, for example excessive diet and excessive drinking or Drug abuse.

Affective state is as grieved or constrain, and healthy state such as Tiroidina is hyperfunction, hypoglycemia or heart attack also can cause stress reaction.

Anxiety usually is accompanied by physical symptom, comprising: ballism or shake, muscular tone, headache, perspiration, xerostomia, dysphagia, stomachache (this can be unique symptom of stress reaction, especially in child).

Sometimes, follow other symptom that also has of anxiety: dizzy, heart rate is accelerated or is irregular, be short of breath, diarrhoea or frequent urine, fatigue, irritability, comprise lose one's temper, dyskoimesis and have nightmares decreased attention and property problem.

Kv9.2 and regulon thereof can be used for treatment or alleviate all these symptoms.

Anxiety disorder is one group of mental disorder that relates to undue anxiety.They comprise generalized anxiety disorder disease, special phobia, compulsive disorder and social phobia.Referring to Kv9.2 relative disease proposed above.

Some drugs, comprise amusement with treatment, because their side effect or cut out all and can cause anxiety symptom.This medicine comprises caffeine, alcohol, Nicotine, common cold treatment medicine (coldremedies), decongestant, the bronchodilator that is used for asthma, tricyclic antidepressants, Cocaine, Amphetamine, diet pill, ADHD medicine and Tiroidina medicine.The purposes that our open Kv9.2 and regulon thereof are used in combination with these medicines is induced influence with the stress reaction and/or the anxiety that alleviate them.

Poor diet also can cause stress reaction or anxiety-for example, low-level vitamin B12.The behavior anxiety is relevant with specified conditions, for example accepts test or carries out public speech.Stress reaction obstacle (PTSD) is a kind of stress reaction obstacle after the wound, and it produces after for example war of traumatic event, health or sexual assault or natural disaster.

Under very rare situation, adrenal tumor (pheochromocytoma) can cause anxiety.It is because due to the excessive production of hormone of responsible anxiety sensation and symptom.

(adapt from Medline Plus,

http：/www.nlm.nih.gov/medlineplus/ency/article/003211.htm)

The identity of Kv9.2 subunit and similarity

Sequencing result as the amplification cDNA product by coding people Kv9.2 shows that other albumen with ionic channel family on the Kv9.2 structure is relevant.The cDNA sequence of SEQ ID NO:1 comprises an open reading-frame (ORF) (SEQ ID NO:2, Nucleotide 41-3352), 1104 amino acid whose polypeptide that show among this open reading-frame (ORF) coding SEQ ID NO:3.Finder Kv9.2 is positioned homo sapiens chromosome 8q22.

The HMM structure prediction software of use pfam ( Http: //www.sanger.ac.uk/Software/Pfam/search.shtml) analyze Kv9.2 polypeptide (SEO IDNO:3) and confirm that the Kv9.2 peptide is the ionic channel subunit.

The mouse homologue of people Kv9.2 subunit is cloned, and its nucleotide sequence and aminoacid sequence are shown as SEQ ID NO:4 and SEQ ID NO:5 respectively.The mouse Kv9.2 subunit cDNA of SEQ ID NO:4 shows the identity that has height with people Kv9.2 subunit (SEQ ID NO:2) sequence, and the aminoacid sequence of mouse Kv9.2 subunit (SEQ ID NO:5) shows identity and the similarity that has height with people Kv9.2 subunit (SEQ ID NO:3).Therefore people and mouse Kv9.2 ionic channel subunit are the members of extended familys of ionic channel.

The Kv9.2 subunit polypeptide

As used herein, term " Kv9.2 subunit polypeptide " is meant and comprises the aminoacid sequence that is shown as SEQ ID NO:3 or SEQ ID NO:5, or the polypeptide of its homologue, variant or derivative.Preferably, described polypeptide comprises or homologue, variant or the derivative of the sequence that shows with SEQ ID NO:3.

" polypeptide " is meant and comprises two or more amino acid whose any peptide or the protein that the peptide bond by peptide bond or modification mutually combines, i.e. peptide isomorphism thing." polypeptide " refers to short chain, and so-called peptide, oligopeptides or oligomer also refer to long-chain, so-called protein.Polypeptide can comprise the amino acid amino acid in addition of 20 encoding genes.

" polypeptide " comprises the aminoacid sequence of for example translating the post-treatment modification by natural process, or passes through the aminoacid sequence that chemical modification technology known in the art is modified.These modifying method are described in the basic textbook preferably, are described in greater detail in the monograph, in the research document of treatise description are arranged also.Modification can betide any zone of polypeptide, comprises peptide main chain, amino acid side chain and amino or C-terminal.Be to be understood that in given polypeptide the modification of same type can exist with different degree at same loci or in several sites.And given polypeptide can comprise polytype modification.

As the result of ubiquitination, polypeptide can be a ramose; Can be cyclic also, have or do not have branch.Ring-type, branch and branch's ring type polypeptide can produce from translation back natural process, maybe can prepare by synthetic method.Modification comprises acetylize; acidylate; the ADP-ribosylation; amidation; the covalent attachment of flavine; the covalent attachment of heme part; the covalent attachment of Nucleotide or nucleotide derivative; the covalent attachment of lipid or lipid derivant; the covalent attachment of phosphatidylinositols; crosslinked; cyclisation; form disulfide linkage; demethylation; form covalent cross-linking; form Gelucystine; form Pyrrolidonecarboxylic acid; formylation; the gamma-carboxylation effect; glycosylation; the GPI anchor forms; hydroxylation; iodization; methylate; myristoylation; oxygenizement; proteolysis processing; phosphorylation; isoprenylation; racemization; selenoylation; sulfation; transfer-RNA mediation in protein, increase amino acid, for example arginylization and ubiquitination.Referring to, for example (Proteins-Structure andMolecular Properties, second edition, T.E.Creighton, W.H.Freeman and Company, New York, 1993 and Wold, F., Posttranslational Protein Modifications:Perspectives and Prospects, Posttranslational Covalent Modification ofProteins, 1-12 page or leaf among the B.C.Johnson, editor, Academic Press, New York, 1983; Seifter etc., " protein modification and non-albumen cofactor are analyzed ", Meth Enzymol (1990) 182:626-646 and Rattan etc., " albumen is synthetic: posttranslational modification and aging ", Ann NY AcadSci (1992) 663:48-62.

Term used herein " variant ", " homologue ", " derivative " or " fragment " comprise sequence are carried out any replacement, variation, modification, replacement, disappearance or added (or a plurality of) amino acid.Unless other implication of approval relates to " Kv9.2 ", " Kv9.2 subunit ", " Kv9.2 ionic channel " comprises this variant, homologue, derivative and the fragment of mentioning Kv9.2 in the context.

Preferably, combine with the number passages or with other Kv family member when forming the heteromerism passage when expressing to form, the aminoacid sequence of acquisition has ion channel activity when being applied to Kv9.2.Preferably, the nucleic acid of acquisition has the identical activity of the Kv9.2 ionic channel subunit that shows with SEQ ID NO:3 or SEQ ID NO:5 (or when active possibility when pointed other passage combines).

Especially, when when pointed other passage combines, if the aminoacid sequence that obtains has ion channel activity, Kv9.2 ion channel activity preferably, the identity on structure and/or the function contained in term " homologue " so.About sequence identity (being similarity), preferably have at least 70%, more preferably at least 75%, more preferably at least 85%, at least 90% sequence identity more preferably.More preferably has at least 95%, more preferably at least 98% sequence identity.These terms also comprise the amino acid whose polypeptide derived from the allelic variation of Kv9.2 subunit nucleic acid sequence.

When " channel activity " or " biological activity " of the ionic channel that relates to the ionic channel that for example comprises Kv9., these terms are meant the metabolism or the physiological function of the ionic channel that comprises Kv9.2, comprise similar active or improved activity or have these activity of the adverse side effect of minimizing.The antigen and the immunogenicity activity that also comprise the ionic channel that contains Kv9.2.The example of ion channel activity, and analyze and quantitatively these active methods be known in the art, and in other chapters and sections of this paper, describe in detail.

As used herein, " disappearance " is defined as the variation in Nucleotide or aminoacid sequence, wherein lacks one or more Nucleotide or amino-acid residue respectively.As used herein, " insertion " or " interpolation " is meant when the material with natural generation compares, and the Nucleotide or the amino acid that cause respectively having increased one or more Nucleotide or amino-acid residue change.As used herein, " replacement " is respectively to replace one or more Nucleotide or amino acid to produce by different Nucleotide or amino acid.

Kv9.2 polypeptide described herein also has disappearance, insertion or the replacement of amino-acid residue, and this produces the reticent aminoacid sequence that changes and produce functional equivalent.Planned aminoacid replacement can based on the polarity of residue, electric charge, solvability, hydrophobicity, wetting ability and/similarity of amphiphilic character etc. carries out.For example, electronegative amino acid comprises aspartic acid and L-glutamic acid; The amino acid of positively charged comprises Methionin and arginine; The amino acid that have similar hydrophilicity value, comprises the uncharged polar head group comprises leucine, Isoleucine, Xie Ansuan, glycine, L-Ala, l-asparagine, glutamine, Serine, Threonine, phenylalanine and tyrosine.

For example can guard replacement according to following table.Amino acid in the second hurdle same unit, preferably can replace mutually with the amino acid in the delegation in third column.

Aliphatic	Nonpolarity	GAP
			ILV
		Polarity-no electric charge		CSTM
	NQ
			Polarity-electrically charged	DE
	KR
		Aromatic			HFWY

The Kv9.2 polypeptide can further comprise the allogeneic amino acid sequence, typically at N-end or C-end, preferably at the N-end.Heterologous sequence can comprise influences the sequence (for example leader sequence) that cell is interior or extracellular protein leads.Heterologous sequence also can comprise the immunogenicity that improves polypeptide and/or be convenient to the sequence of polypeptide discriminating, extraction and/or purifying.Another heterologous sequence particularly preferably is the amino acids sequence, for example the polyhistidine of N-end preferably.At least 10 amino acid, preferably at least 17 amino acid but to be less than 50 amino acid whose polyhistidine sequences be particularly preferred.

The Kv9.2 polypeptide can be " maturation " proteic form, perhaps can be than the large protein part of fusion rotein for example.Comprise the additional aminoacid sequence of the sequence (for example polyhistidine residue) that contains secretion or leader sequence, presequence, aided purification, it usually is favourable perhaps being used for stable appended sequence in the recombinant production process.

By recombination method, use known technology to prepare the Kv9.2 polypeptide easily.Yet, also can pass through synthetic method, use for example solid phase synthesis preparation of the known technology of person skilled in the art.This peptide species also can be made into fusion rotein, for example with assisted extraction and purifying.The example of fusion rotein mating partner comprises glutathione-S-transferase (GST), 6xHis, GAL4 (DNA combination and/or transcription activating functional domain) and beta-galactosidase enzymes.It also is favourable comprising the proteolysis restriction enzyme site between fusion rotein mating partner and protein of interest sequence, to allow to remove fusion rotein sequence, for example zymoplasm restriction enzyme site.Preferably, fusion rotein can not hinder the protein function of sequence interested.

The Kv9.2 polypeptide can be isolating basically form.This term is meant by manually transforming from native state.If " isolating " composition or material take place under state of nature, it just is changed or removes from its initial environment, and perhaps the two has concurrently.For example, the natural intravital polynucleotide of Live Animals, nucleic acid or the polypeptide of being present in is not " isolating ", but isolating identical polynucleotide, nucleic acid or polypeptide are isolating from the coexistence raw material of its state of nature, as used herein term.

Yet, be to be understood that the Kv9.2 ionophorous protein can with carrier or the mixing diluents that can not disturb the albumen expected effect, and still be considered to isolating basically.Polypeptide described herein can also be the form of purifying basically, and in this case, it comprises the albumen in the goods usually, wherein surpasses 90% in the goods, and for example 95%, 98% or 99% albumen is the Kv9.2 polypeptide.

We further describe the peptide that comprises Partial K v9.2 polypeptide.Therefore, the fragment and homologue, variant or the derivative that comprise the Kv9.2 subunit.Described peptide length can be between 2 and 200 amino acid, preferably between 4 and 40 amino acid.Described peptide can be derived from Kv9.2 polypeptide disclosed herein, for example by with suitable enzyme for example trypsinase digest.In other words, peptide, fragment etc. can be synthetic by recombinant methods or synthetic method.

Term " peptide " comprises the variation of various synthetic peptides known in the art, for example retroinverso D peptide.This peptide can be antigenic determinant and/or T-cell epitope.This peptide can have immunogenicity in vivo.Preferably, this peptide can be induced neutralizing antibody in vivo.

From the Kv9.2 subunit sequence of different plant species, can determine which zone of aminoacid sequence is (" the homology zone ") of guarding between the different sorts by contrast, and between the different sorts which zone change (" allos zone ").

Therefore, the Kv9.2 polypeptide can comprise corresponding to the sequence to the homology zone of small part.The homology zone shows the homology of height between at least two species.For example, use above-described test method, the homology zone can show at least 70% on amino acid levels, preferably at least 80%, more preferably at least 90%, even at least 95% identity more preferably.As hereinafter more detailed elaboration, the peptide that comprises corresponding to the sequence in homology zone can be used for therapeutic strategy.In other words, the Kv9.2 subunit peptides can comprise corresponding to the sequence to small part allos zone.Between at least two species, the allos zone shows low-level homology.

Kv9.2 polynucleotide and nucleic acid

As describing in further detail at other chapters and sections of this paper, we have described Kv9.2 polynucleotide, Kv9.2 Nucleotide and Kv9.2 nucleic acid, production method and their purposes etc.

Term " Kv9.2 polynucleotide ", " Kv9.2 Nucleotide " and " Kv9.2 nucleic acid " can be used alternatingly, be meant the polynucleotide/nucleic acid of the nucleotide sequence that comprises SEQ ID NO:l, SEQ ID NO:2 and SEQ ID NO:4 demonstration, or its homologue, variant or derivative.Preferably, polynucleotide/nucleic acid comprises or is exactly nucleic acid sequence SEQ ID NO:1 or SEQ ID NO:2, the most preferably homologue of SEQ ID NO:2, variant or derivative.

These terms also comprise can coded polypeptide and/or the nucleotide sequence of peptide (being the Kv9.2 polypeptide).Like this, Kv9.2 polynucleotide and nucleic acid have the nucleotide sequence of the polypeptide that comprises the aminoacid sequence that SEQ ID NO:3 or SEQ IDNO:5 show of can encoding, or its homologue, variant or derivative.Preferably, Kv9.2 polynucleotide and nucleic acid have the nucleotide sequence of the polypeptide that comprises the aminoacid sequence that SEQ ID NO:3 shows of can encoding, or its homologue, variant or derivative.

" polynucleotide " typically refer to any polyribonucleotide or polydeoxyribonucleotide, and they can be the RNA of unmodified or the RNA or the DNA of DNA or modification.Unrestrictedly, " polynucleotide " comprise single-and two-chain DNA, strand and double-stranded region blended DNA, strand and double-stranded RNA, with strand and double-stranded region blended RNA, comprise the hybrid molecule of DNA and RNA, described DNA and RNA can be strands, or more typically, be mixture double-stranded or strand and double-stranded region.In addition, " polynucleotide " refer to comprise the three chain zones that RNA or DNA or RNA and DNA comprise.The term polynucleotide also comprise DNA and the RNA that contains one or more modified bases, and because of stable or adorned DNA of other reason main chain or RNA." modification " base comprises, for example tritylation base and rare base Trophicardyl for example.DNA and RNA have carried out multiple modification; Therefore, " polynucleotide " are included in the polynucleotide of the typical case finds under the natural situation chemistry, enzymatic or metabolism modified forms, and the DNA and the RNA of virus and the peculiar chemical species of cell." polynucleotide " also comprise short relatively polynucleotide, are often referred to as oligonucleotide.

The person skilled in the art should be appreciated that the result of genetic codon degeneracy, makes the identical polypeptide of many nucleotide sequence codifieds.

As used herein, term " nucleotide sequence " is meant nucleotide sequence, oligonucleotide sequence, polynucleotide sequence and their variant, homologue, fragment and derivative (for example their part).Nucleotide sequence can be genomic or the DNA or the RNA of synthetic or reorganization origin, and they can be double-stranded or strand, no matter represent sense strand or antisense strand or their combination.The term nucleotide sequence can use recombinant DNA technology preparation (for example, recombinant DNA).

Preferably, term " nucleotide sequence " refers to DNA.

Term used herein " variant ", " homologue ", " derivative " or " fragment " comprise any replacement, variation, modification that the sequence of Kv9.2 nucleotide sequence is carried out, substitute, lack or add (or a plurality of) nucleic acid.Unless in addition approval of context relates to this variant, homologue, derivative and fragment that " Kv9.2 ", " Kv9.2 subunit " and " Kv9.2 ionic channel " comprise the Kv9.2 that mentions.

Preferably, acquisition nucleotide sequence coded has the active polypeptide of Kv9.2 subunit, preferably has the identical activity of Kv9.2 subunit that shows with SEQ ID NO:3 or SEQ ID NO:5 at least.Preferably, term " homologue " is meant about structure and/or function and has identity.Preferably, a nucleotide sequence coded peptide species of Huo Deing like this, this polypeptide is had ion channel activity when forming the heteromerism passage when expressing to form to combine with the number passage or with other Kv family member.About sequence identity (being similarity), preferably have at least 70%, more preferably at least 75%, more preferably at least 85%, at least 90% sequence identity more preferably.More preferably have at least 95%, more preferably at least 98% sequence identity.These terms also comprise the allelic variation of sequence.

The calculating of sequence homology

Whether the sequence identity of any sequence that proposes about this paper can have for example at least 70% sequence identity with definite another sequence and this sequence by any one or a plurality of sequence and another sequence being carried out simply " eyeball " relatively (being a kind of strict comparison).

Relatively sequence identity also can be measured by the computer program that commerce is buied, and this program can use any algorithm (using for example default parameter) that is suitable for measuring identity to calculate % identity between two or more sequences.The typical example of this computer program is CLUSTAL.The computer program means of identity and similarity includes but not limited to GCG routine package (1984 Nucleic Acids Research 12:387 such as Devereux) and FASTA (1990 J Molec Biol 403-410 such as Atschul) between two sequences of other mensuration.

Can calculate the % homology of contiguous sequence, promptly a sequence and another sequence are carried out sequence contrast, and each amino acid in sequence and the corresponding amino acid in another sequence directly compare next residue.This is called the contrast of " non-notch " sequence.Typically, this non-notch sequence contrast is only carried out the few relatively sequence of residue quantity.

Although this is very simple with a consistent method, but it does not consider that for example the same sequence centering of others, one is inserted or lack the amino-acid residue ordering confusion that will cause subsequently, when carrying out overall contrast, cause the % homology to significantly reduce potentially like this.Therefore, be designed for and produce the correlated most of sequence control methodss of optimal sequence and consider possible insertion and disappearance, and within reason whole homology score is carried out point penalty.This finishes to manage maximizing local homology by insert " breach " in the sequence contrast.

Yet, each breach of generation during these more complicated methods distribution " breach punishment " contrast to sequence, therefore, for the identity amino acid of equal amts, have the least possible breach ground sequence contrast (between two control sequence, reflecting higher dependency) and obtain the score higher than sequence with many breach.Typically use " members of the same clan's breach cost (Affine gap costs) ", for the high relatively cost of its requirement of existence of breach, it requires less point penalty for each residue subsequently in the breach.This is the most frequently used breach points-scoring system.Certainly, the punishment of high breach has generation the sequence contrast of the best of less breach.Most of sequence contrast programs allow to revise the breach point penalty.Yet when using this sequence comparison software, it is preferred Using Defaults.For example, when using GCG Wisconsin Bestfit software package, the default gap point penalty of aminoacid sequence is-12 for breach, is-4 for each extension.

Therefore, consider the breach point penalty, calculating maximum % homology at first needs to produce best sequence contrast.Carrying out the correlated suitable computer program of this sequence is GCG Wisconsin Bestfit software package (University of Wisconsin, U.S.A.; Devereux etc., 1984, Nucleic Acids Research12:387).The example that can carry out correlated other software of sequence include, but are not limited to BLAST software package (Ausubel etc., 1999 the same-18 chapters), FASTA (Atschul etc., 1990, J.Mol.Biol., 403-410) and contrast instrument GENEWORKS combination (suit).BLAST and FASTA are available (Ausubel etc., 1999 is the same, 7-58 is to 7-60) for off line and online searching.

Although final % homology can be measured according to identity, sequence comparison process itself typically is not based on all or none (all-or-nothing) paired comparison.Replace, use classification (scaled) similarity rating matrix usually, it gives scoring based on chemical similarity or evolutionary distance to each paired contrast.The example of this normally used matrix is the default matrix of BLOSUM62 matrix-blast program group.The ordinary symbol synopsis that GCG Wisconsin program is used disclosed default value or provided usually.It is preferred using the disclosed default value of GCG software package, perhaps uses default matrix under the situation of other software, for example BLOSUM62.

It is favourable adopting the BLAST algorithm that parameter is made as default value.The BLAST algorithmic elaboration in Http:// www.ncbi.nih.gov/BLAST/blast help.html, this paper is incorporated herein by reference.Definable and can be on the default parameters of definition setting search parameter easily.

Preferably, when estimating with BLAST, " same basically " be equal to when being complementary with the EXPECT value at least about 7, preferably at least about 9 and most preferably 10 or more sequence.The acquiescence thresholding (threshold) of EXPECT normally 10 in the blast search.

BLAST (the local comparison in basis research tool (Basic Local Alignment Search Tool)) is the exploratory searching algorithm that uses for program blastp, blastn, blastx, tblastn and tblastx; These programs are used Karlin and Altschul statistical method (Karlin and Altschul 1990, Proc Natl.Acad.Sci.USA 87:2264-68; Karlin and Altschul, 1993, Proc.Natl.Acad.Sci.USA 90:5873-7; Referring to Http:// www.ncbi.nih.gov/BLAST/blast help.html) and improve to show the importance of their discovery a little.Blast program is suitable for the sequence similarity search, for example differentiates the homologue of search sequence.For the discussion of the basic problem of sequence library similarity searching referring to (1994) Nature Genetics 6:119-129 such as Altschul.

Can obtain above-mentioned five kinds of BLAST on the http://www.nc bi.nlm.njh.govProgram is carried out following operation: blastp-amino acid search sequence and protein sequence database is compared; Blastn-compares nucleotide query sequence and nucleotide sequence database; Blastx-compares six-frame (six-frame) notion (conceptual) translation product and the protein sequence database of nucleotide query sequence (two chain); Tblastn-compares protein search sequence and the nucleotide sequence database of dynamically translating with whole six frames (two chain); Tblastx-with six frame translation products of six frame translation products of nucleotide query sequence and nucleotide sequence database relatively.

BLAST uses following search parameter:

Histogram (HISTOGRAM)---show the histogram of each search score; The default "Yes" (referring to the Parameter H in the BLAST handbook (BLAST Manual)) that is.

Describe (DESCRIPTIONS)---restriction is to the quantity of the Short Description of the proportioning sequence that reports to specified numeral; Defaultly be restricted to 100 descriptions.(referring to the V parameter in the handbook).

Expection (EXPECT)---report is at the significance,statistical threshold value of the proportioning of database sequence; Default value is 10, and therefore according to Karlin and Altschul (1990) stochastic model, 10 proportionings are only found in expection by accident.If the significance,statistical of giving proportioning is greater than the EXPECT threshold value, this proportioning will not be in the news.Lower EXPECT threshold value is strict more, causes proportioning at random still less to be reported.Fractional value is acceptable (referring to the parameter E in the BLAST handbook).

Block (CUTOFF)---report high score fragment right block score.Calculate default value (seeing above) from the EXPECT value.Only to equal the independently HSP of CUTOFF value the same when high with score at least when the significance,statistical of the HSP of database sequence, just the HSP of report database sequence.Higher CUTOFF value is stricter, causes less proportioning at random to be reported (referring to the parameter S in the BLAST handbook).Typically, the significance threshold value can use EXPECT to control more intuitively.

Sequence contrast (ALIGNMENTS)---restricting data storehouse sequence is to for to have reported that high score fragment is to (HSPs) specified quantity; Default restriction is 50.If satisfy the significance,statistical threshold value (referring to following EXPECT and CUTOFF) of report by chance than this more database sequence, only report shows the proportioning (referring to the B parameter in the BLAST handbook) of maximum significance,statistical.

Matrix (MATRIX)---for BLASTP, BLASTX, TBLASTN and TBLASTX specify interchangeable rating matrix.Default matrix is BLOSUM62 (Henikoff ﹠amp; Henikoff, 1992).Effective interchangeable selection comprises: PAM40, PAMl 20, PAM250 and IDENTITY.There is not interchangeable rating matrix to be used for BLASTN; In the BLASTN request, specify the MATRIX instruction to return the reaction that makes mistakes.

Chain (STRAND)---limit TBLASTN the search extremely only top or the bottom chain of database sequence; Perhaps limit the only frame to search sequence top or the bottom chain of BLASTN, BLASTX or TBLASTX search.

Filter (FILTER)---shelter as using Wootton ﹠amp; The fragment of the search sequence with low combinatorial complexity that the SEG program of Federhen (1993) Computersand Chemistry 17:149-163 is measured is perhaps as using Claverie ﹠amp; The fragment of forming by the short period internal repeat of the XNU program determination of States (1993) Computers and Chemistry17:191-201, perhaps for BLASTN use Tatusov and Lipman the DUST program (referring to Http: //www.ncbi.nlm.nih.gov).Filtration can be removed from blast output on the statistics significantly but biologically insignificant report (for example at common acidity-, alkalescence-or the Query Result in the abundant zone of proline(Pro)), stay and can be used for carrying out zone specific proportioning, that have more biological significance in the search sequence at database sequence.

In nucleotide sequence, use letter " N " to replace (for example, " NNNNNNNNNNNNN ") by the low-complexity sequence that filter is found, and in protein sequence, use letter " X " to replace low-complexity sequence (for example " XXXXXXXXX ").

Filtration only is used for search sequence (or its translation product), is not suitable for database sequence.Default filtration is DUST for BLASTN, is SEG for other program.

When being applied to sequence in SWISS-PROT, it is normal being sheltered by SEG, XNU or the two without any thing, so expectation is filtered and can be told on.In addition, in some cases, sequence is masked fully, shows that the significance,statistical of any coupling of reporting at unfiltered search sequence is shady.

NCBI-gi-causes that except that going into to hide registration and/or the locus title NCBI gi identifier is shown in output.

Most preferably, the sequence contrast is used Http:// www.ncbi.nlm.nih.gov/BLASTOn the simple blast search algorithm that provides carry out.In some embodiments, when measuring sequence identity, do not use the breach point penalty.

Hybridization

This paper also comprise can with the nucleotide sequence of the sequence hybridization that proposes here, perhaps its any segment or derivative are perhaps with the nucleotide sequence of the complement hybridization of above-mentioned any material.

Hybridization is meant that " nucleic acid chains and complement chain are by base pairing bonded process (Coombs J (1994) Dictionary of Biotechnology; Stockton Press; New York NY) and as at the amplification procedure that carries out with polymerase chain reaction technique of the middle description of Dieffenbach CW and GS Dveksler (1995; PCR Primer; a Laboratory Manual; Cold Spring Harbor Press, Plainview NY).

As Berger and Kimmel (1987, Guide to Molecular Cloning Techniques, Methods in Enzymology, 152 volumes, Academic Press, San Diego CA) lectured in, hybridization conditions depends on the melting temperature(Tm) of nucleic acid in conjunction with mixture, and gives " severity " determined as discussion described below.

The nucleotide sequence that can propose with this paper or the nucleotide sequence of their complement selective cross, the corresponding nucleotide sequence at least 20 common and this paper proposes, preferably at least 25 or 30, for example at least 40,60 100 or the zone of more continuous nucleotides have at least 70%, preferably at least 75%, more preferably at least 85% or 90%, even at least 95% or 98% homology more preferably.Preferred nucleotide sequence will comprise the NO:1 with SEQ ID, and 2 or 4 homologous zones preferably have at least 70%, 80% or 90% and more preferably at least 95% homology with one of them sequence.

Term " selective cross " is meant that the nucleotides sequence as probe is listed in and finds to use under the condition that target nucleotide sequences and probe hybridize with the level that is significantly higher than background.Background hybridization can take place because of the existence of other nucleotide sequence, for example in screened cDNA or genome dna library.In this incident, background is meant by the signal level that interact to produce between probe and the non-specific DNA member of genome, this level with compare less than 10 times with the interactional intensity of the observed specificity of target DNA, preferably less than 100 times.Can measure interactional intensity, for example, for example use by the radiolabeled probe ³²P.

The nucleotide sequence of the nucleotide sequence hybridization that can propose with this paper to the stringent condition in moderate is also included within this paper scope.As Berger and Kimmel (1987, Guide to MolecularCloning Techniques, Methods in Enzymology, 152 volumes, Academic Press, SanDiego CA) lectured in, hybridization conditions depends on the melting temperature(Tm) (Tm) of nucleic acid in conjunction with mixture, and gives definite " severity " as hereinafter illustrating.

Stringent condition typically occurs in Tm-5 ℃ (being lower than 5 ℃ of probe Tm); High stringent condition is lower than about 5 ℃-10 ℃ of Tm; The moderate stringent condition is lower than about 10 ℃-20 ℃ of Tm; Low stringency condition is lower than about 20 ℃-25 ℃ of Tm.The person skilled in the art should be appreciated that the strictest hybridization can be used for differentiating or detecting identical nucleotide sequence, and the strict hybridization of moderate (or low) can be used for differentiating or detecting similar or relevant nucleotide sequence.

In a preferred embodiment, we have described can be under stringent condition (for example 65 ℃ and 0.1xSSC{1xSSC=0.15M NaCl, 0.015M Trisodium Citrate pH 7.0}) and the nucleotide sequence of one or more Kv9.2 subunit nucleotide sequence hybridizations.When nucleotide sequence is two strands, double-helical chain, independent or all comprising in conjunction with existence.When nucleotide sequence is strand, be to be understood that the complementary sequence of this nucleotide sequence is also included within the scope of this paper.

This paper has further described the nucleotide sequence that can hybridize the sequence complementary sequence that proposes with this paper, or its fragment or derivative.Similarly, comprise such nucleotide sequence, this sequence and the sequence complementation that can hybridize sequence described herein.The nucleotide sequence of these types is examples of variant nucleotide sequence.Aspect this, term " variant " comprises such sequence, and this sequence is complementary to the sequence of the nucleotide sequence that can hybridize this paper proposition.But preferably, term " variant " comprises such sequence, and this sequence is complementary under stringent condition the sequence that (for example 65 ℃ and 0.1xSSC{1xSSC=0.15M NaCl, 0.015 Trisodium Citrate pH 7.0}) can hybridize the nucleotide sequence that this paper proposes.

Clone's Kv9.2 subunit and homologue

We have further described the nucleotide sequence of the sequence that is complementary to this paper proposition, or its any fragment or derivative.Be complementary to its fragment as infructescence, this sequence can be used as and differentiates and the probe of cloning similar subunit sequence in other organism so.

Therefore, content disclosed herein make from people and other animal species for example mouse, pig, sheep, etc. clone Kv9.2, its homologue and other structure or the relevant gene of function can finish.Can be used as the hybridization probe of cDNA and genomic dna with nucleotide sequence that is contained in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4 or the identical or essentially identical polynucleotide of its fragment, to separate part or the full-length cDNA and the genomic clone of encoded K v9.2 subunit from suitable library.This probe can also be used to separate cDNA and the genomic clone (comprising animal species coding homologue and the straight gene to homologue in addition from the people) that has sequence similarity, preferably has other gene of sequence similarity highly with the Kv9.2 gene.Screening by hybridization, clone and sequence technology are that the person skilled in the art is known, and are described in for example Sambrook etc. (seeing above).

Typically, be suitable as the nucleotide sequence of probe and the nucleotide sequence of indicator and have 70% identity, 80% identity preferably, 90% identity more preferably, even 95% identity more preferably.Probe comprises at least 15 Nucleotide usually.Preferably, this probe has at least 30 Nucleotide, and can have at least 50 Nucleotide.The scope of particularly preferred probe between 150 and 500 Nucleotide, more particularly about 300 Nucleotide.

In one embodiment, in order to obtain encoded K v9.2 polypeptide, to comprise homologue and straight polynucleotide to homologue from other animal species beyond the people, the step of carrying out comprises with having SEQID NO:1, SEQ ID NO:2, SEQ ID NO:4 or its segmental label probe screens suitable library under stringent hybridization condition, and separate part or total length eDNA and the genomic clone that comprises described polynucleotide sequence.This hybridization technique is that the person skilled in the art is known.Stringent hybridization condition is as indicated above, perhaps 42 ℃ of incubated overnight under another kind of condition, contain in the solution: 50% methane amide, 5XSSC (150mM NaCl, the 15mM Trisodium Citrate), the Oncorhynchi sperm DNA of 50mM sodium phosphate (pH7.6), 5X Denhardt ' s solution, 10% glucose sulfuric ester and 20mg/ml sex change, shearing, use 0.1XSSC at about 65 ℃ of following washing filters subsequently.

Contain the functional analysis of the Kv9.2 of ionic channel

Can check clone's Kv9.2 ionic channel polynucleotide of inferring by sequential analysis or functional analysis.Particularly, the conductance of Africa xenopus ovocyte (Xenopus oocytes) that can check transfection as described is as indication and quantize the active method of Kv9.2, is used for screening analysis described below.This Africa xenopus ovocyte electrophysiology easy analysis ground is called " functional analysis of Kv9.2 (electrophysiology) ".

Can be following in " functional analysis of Kv9.2 (electrophysiology) ", analyze the Kv9.2 ionic channel subunit of inferring or the activity of homologue.The establishing criteria program is with the rna transcription thing that adds cap of the external synthetic linearization plasmid template from encoded K v9.2cDNA of RNA polymerase.The in-vitro transcription thing is suspended in the aqueous solution that final concentration is 0.2mg/ml.Remove the removal ovary leaf from the female toad that grows up, what obtain stage V removes the vesica ovocyte, uses the microinjection utensil with 50nl particle (bolus) injection rna transcription thing (10ng/ ovocyte).Also injectable is encoded the RNA of other Kv subunit of Kv2.1, Kv4.2 for example to form the heteromerism passage.Use the voltage clamp of two electrodes to measure the electric current of individual Africa xenopus ovocyte for the reaction of agonist exposure.By NaCl 115, KCl 2.5, CaCl ₂1.8, record current at room temperature in the standard medium formed of NaOH-HEPES 10, pH7.2 (unit is Mm).As described in further detail below, the Africa xenopus system also can be used for screening the tissue/cell extract of known part and activation part.

Other functional analysis approach comprises patch clamp electricity physiology, Rb flux, fluorescent energy resonance transfer (FRET) is analyzed and FLIPR analyzes, and comprises the membrane voltage of the sensitive dyestuff observation of cell of working voltage.FLIPR analyzes and is described in Whiteaker etc., J Biomol Screen.2001 Oct; 6 (5): 305-1, and be described in Falconer etc. based on the analysis of FRET, J Biomol Screen.2002 Oct; 7 (5): 460-5.

Particularly, we disclose the analytical procedure that detects the Rb flux, and the screening method that detects the Rb variations of flux, to differentiate agonist and the antagonist of Kv9.2.The method of measuring radiolabeled Rb flux is summarized in J Biomol Screen.2004 Oct such as Rezazadeh; 9 (7): 588-97 and nonradioactive labeling Rb flux distribution are described in Assay Drug Dev Technol.2004 Oct; 2 (5): 525-34.Preferably, measure the %Rb outflux in the analysis.

This herein functional analysis is known as " Kv9.2 functional analysis (Rb flux) ".

Particularly, we disclose a kind of method, and the antagonist of Kv9.2 has reduced the %Rb outflux of suitable transfectional cell in this method.Preferably, under the situation that the Kv9.2 antagonist exists, the %Rb outflux reduces by 10%, 20%, 30%, 40%, 50%, 60%, 70% or more.

We further disclose a kind of method, and wherein the agonist of Kv9.2 has improved the %Rb outflux of suitable transfectional cell.Preferably, under the situation that the Kv9.2 agonist exists, the %Rb outflux improves 10%, 20%, 30%, 40%, 50%, 60%, 70% or more.

The dynamic analysis of the outflux Rb+ that cell discharges can use following equation to be expressed as residue percentage ratio  (percentage remaining )

R=[Rb _Lysate/ (Rb _{Supernatant liquor}+ Rb _Lysate)] * 100

Rb+ outflux (R in different time point unpolarizings and agonist stimulation _S) can determine according to following equation

R _S=(1-[(R _Always-R-R _RadixThe R of)/- _Radix]) * 100

The kinetics of Kv9.2 polypeptide be can further analyze, activation, passivation and deactivation comprised.Soak time is that complete electric current passes the ionic channel that contains Kv9.2 and sets up the time that is spent under standard conditions, and passivation time is that complete electric current reached for zero used time under standard conditions.Here the adjusting of Ti Chuing, increase or reduction Kv9.2 kinetics preferably are meant adjusting, increase or reduce Kv9.2 soak time or Kv9.2 passivation time that perhaps the two all comprises.

In preferred embodiments, the soak time constant is measured as soak time, and the passivation time constant is measured as passivation time.The soak time constant that typically contains the ionic channel of Kv9.2 is 21ms.Typical half deactivation voltage V _{1/2 deactivation}Be-33mV V _{1/2 deactivation}Can analyze other or another kind of kinetic parameter as deactivation.

Conditioning agent, the initiator (opener), agonist, blocker and the antagonist that for example contain the ionic channel of Kv9.2 can change, for example increase or reduce the kinetics of the ionic channel that contains Kv9.2, preferably in soak time, inactivation time, passivation time, passivation kinetics, the half deactivation voltage etc. any one or a plurality of.

Particularly, agonist and initiator (opener) are the molecules (being preferably soak time and/or passivation time constant) that can reduce soak time and/or passivation time, preferably reduce by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more, promptly for example by reducing soak time or still less to 20ms, 18ms, 16ms or 15ms.

Similarly, the antagonist of Kv9.2 or blocker can increase soak time and/or passivation time, preferably increase by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more, promptly for example by improving soak time to 22ms, 25ms, 27ms or more.

Kinetics and particularly soak time can preferably use " functional analysis of Kv9.2 (electrophysiology) " to measure, the time-histories of power taking stream is also determined to set up the used time of complete electric current.Similarly, use " functional analysis of Kv9.2 (electrophysiology) " to measure inactivation time, the time-histories of power taking stream and used time when determining that complete electric current reduces to zero.

In other words, can analyze passivation kinetics, this passivation kinetics is used to be determined at repolarization pulse after the prepulse (for example+50MV continue 500ms) (for example to-40mV) time of being spent of ionic channel passivation afterwards.The typical value of passivation kinetics that contains the ionic channel of Kv9.2 is 44ms.

The expression analysis of Kv9.2

In order to design the useful therapy of treatment Kv9.2 relative disease, the expression overview of determining Kv9.2 is useful (no matter being wild-type or special mutant).Therefore, can use methods known in the art to measure organ, tissue and the cell type (and etap) of expressing K v9.2.For example, can carry out routine or " electronics " RNA blotting.Also can use ThermoScript II PCR (RT-PCR) to analyze the expression of Kv9.2 gene or mutant.The generalized method of sensitive more of mensuration Kv9.2 expression comprises RNA enzyme protection analysis as known in the art.

Rna blot analysis is to be used to detect the laboratory technique that the genetic transcription thing exists, and (Sambrook's nucleotide sequence that comprises mark sees above with the film hybridization of the RNA that combines particular cell types or tissue, the 7th Zhanghe Ausubel, F.M. etc., see above 4 and 16 chapters).The analog calculation machine technology (" electronics RNA trace ") that is applicable to BLAST is used in the Nucleotide database of GenBank for example or LIFESEQ database (Incyte Pharmaceuticals) and searches identical or relevant molecule.Such analysis has advantage, and promptly they may be faster than the hybridization based on multimembrane.In addition, can revise the susceptibility of computer search, all be classified as appropriate or homologous to determine whether any specific coupling.

Describe in further detail as other chapters and sections of this paper, polynucleotide described herein and polypeptide can be used as search reagent and the material of finding treatment and diagnosis animal and human disease.

The Kv9.2 polypeptide expression

We further disclose the method for producing the Kv9.2 polypeptide.Described method generally includes under suitable condition (promptly under the condition of expressing K v9.2 polypeptide) and cultivates host cell, and this host cell contains nucleic acid or its homologue, variant or the derivative that coding has or do not have other Kv family member's Kv9.2 polypeptide.

In order to express the biological activity ionic channel that comprises Kv9.2,, promptly contain the carrier of the required element of encoding sequence of transcribing and translate insertion with the nucleotide sequence insertion suitable expression of encoded K v9.2 subunit or its homologue, variant or derivative.

The method that is used to make up the sequence that contains encoded K v9.2 subunit and the suitable expression vector of transcribing and translate controlling elements is that the person skilled in the art is known.These methods comprise extracorporeal recombinant DNA technology, synthetic technology and vivo gene reorganization.These technical descriptions are in Sambrook, and J. etc. (1989; Molecular Cloning, A Laboratory Manual, 4,8 and the 16-17 chapter, ColdSpring Harbor Press, Plainview, N.Y.) and Ausubel, F.M. etc. (1995 and regular supplementary issue; Current Protocols in Molecular Biology, 9,13 and 16 chapters, John Wiley ﹠amp; Sons, New York, N.Y.).

Can utilize various expression vector/host systems to comprise and express the sequence of encoded K v9.2 subunit.They include, but not limited to the bacterium that microorganism for example transforms with recombinant phage, plasmid or cosmid DNA expression vector; Yeast with the Yeast expression carrier conversion; Insect cell system with virus expression carrier (for example baculovirus) infection; With virus expression carrier (for example cauliflower mosaic virus (CaMV) or tobacco mosaic virus (TMV) (TMV)) or with bacterial expression vector plant transformed cell system (for example, Ti or pBR322 plasmid); Or zooblast system.This purposes is not limited by the host cell that adopts.

" controlling elements " or " regulatory element " is those untranslated zones (for example enhanser, promotor and 5 ' and 3 ' untranslated region territory) of carrier, and they and host cell proteins interact and transcribe and translate.But these element length are different with specificity.According to carrier system that is utilized and host, can use any amount of suitable element of transcribing and translate, comprise composing type and inducible promoter.For example, when in bacterial system, cloning, can use inducible promoter, for example the BLUESCRIPT phagemid (Stratagene, La Jolla, Calif.) or the heterozygosis lacZ promotor of PSPORT1 plasmid (GIBCO/BRL) etc.Baculovirus polyhedrin body protein promotor can be used for insect cell.Can clone derived from the promotor of vegetable cell genome (for example heat-shocked, RUBISCO and storage protein gene) or plant virus (for example viral promotors or leader sequence) or enhanser and to enter carrier.In mammal cell line system, be preferred from the promotor of mammalian genes or mammalian virus.Produce the clone of the multiple copied sequence contain encoded K v9.2 subunit if desired, can use have the suitable selectivity mark, based on the carrier of SV40 or EBV.

In bacterial system, can select many expression vectors according to the purposes of Kv9.2 subunit.For example, when inducing antibody to need a large amount of Kv9.2 subunit, but instruction is easy to the carrier of the fusion rotein high level expression of purifying.This carrier comprises, but be not limited to, multi-functional E.coli clone and expression vector be BLUESCRIPT (Stratagene) for example, wherein the sequence of encoded K v9.2 subunit is connected to that carrier has N-terminal Met and subsequently in the frame of the sequence of 7 residues of beta-galactosidase enzymes, hybrid protein produces like this, pIN carrier (Van Heeke, G. and S.M.Schuster (1989) J.Biol.Chem.264:5503-5509) etc.(Promega, Madison Wis.) also can be used for expressing allogenic polypeptide for having the fusion rotein of glutathione-S-transferase (GST) to the pGEX carrier.Usually, this fusion rotein is soluble, and by being adsorbed onto on Triptide-agarose bead and elution purifying from dissolved cell easily under the condition that free glutathione exists.Can design protein with this systems produce to comprise heparin, zymoplasm or XA factor protein enzyme restriction enzyme site, Ke Long interested polypeptide can partly discharge from GST arbitrarily like this.

In yeast saccharomyces cerevisiae, can use many carriers that contain composing type or inducible promoter, for example alpha factor, alcohol oxidase and PGH.About summarizing referring to Ausubel (seeing above) and Grant etc. (1987; Methods Enzymol.153:516-544).

Under the situation of using plant expression vector, the expression of the sequence of encoded K v9.2 subunit can be by any one driving in many promotors.For example, the 35S of viral promotors such as CaMV and 19S promotor can be separately or are used in combination (Takamatsu, N. (1987) EMBO are J.6:307-311) with ω leader sequence from TMV.In addition, can use plant promoter for example the small subunit of RUBISCO or heat-shocked promotor (Coruzzi, G. etc. (1984) EMBO J.3:1671-1680; Broglie, R. etc. (1984) Science 224:838-843; And Winter, J. etc. (1991) Results Probl.Cell Differ.17:85-105).These constructions can be by the conversion introduced plant cell that direct DNA transforms or cause of disease mediates.This technical description in the summary that can generally obtain in a large number (referring to, Hobbs for example, S. or Murry, L.E., McGraw Hill Yearbook of Science and Technology (1992) McGraw Hill, New York, N.Y.; The 191-196 page or leaf).

The insect system also can be used for expressing K v9.2 subunit.For example, in such system, autographa california nuclear polyhedrosis virus (AcNPV) is used as carrier with expression alien gene in fall army worm (Spodopterafrugiperda) cell or cabbage looper (Trichoplusia) larva.The sequence of encoded K v9.2 subunit can be cloned the nonessential region that enters virus, polyhedron gene for example, and under the control of polyhedrin promotor.The successful insertion of Kv9.2 subunit causes the polyhedron gene inactivation, and produces the recombinant virus that lacks coat protein.Recombinant virus can be used for infecting then, for example fall army worm cell or cabbage looper larva, and the ionic channel that wherein comprises Kv9.2 can be expressed (Engelhard, E.K. etc. (1994) Proc.Nat.Acad.Sci.91:3224-3227).

In mammalian host cell, can utilize many expression systems with the virus basis.As under the situation of expression vector, the sequence of encoded K v9.2 subunit can connect and enters the adenovirus of being made up of late promoter and tripartite leader[and transcribe/translate in the mixture in adenovirus.Insert in virus genomic nonessential E1 and E3 zone can be used for obtaining can be in the host cell that infects the live virus (Logan, J. and T.Shenk (1984) Proc.Natl.Acad.Sci.81:3655-3659.) of expressing K v9.2 subunit.In addition, transcriptional enhancer, for example Rous sarcoma virus (RSV) enhanser is used in to improve in the mammalian host cell and expresses.

Therefore, for example, the ionic channel that contains Kv9.2 is expressed in the Chinese hamster ovary celI of human embryo kidney 3 (HEK293) cell or adhesion.In order to make the expression maximization of ionic channel, typically before inserting pCDN or pCDNA3 carrier, from receptor cdna, remove all 5 ' and 3 ' non-translational regions (UTR)., and under the situation that 400mg/mlG418 exists, select with independent ionic channel cDNA transfectional cell by the fat transfection.After selecting for 3 weeks, collect single clone, and expansion is used for further analysis.Only HEK293 or the CHO with the carrier transfection contrasts as negative.Express the clone of single ionic channel for separating stable, typically select about 24 clones, and analyze by the RNA trace.The G418-resistance clone of analyzing about 50% in sense channel mRNA usually.

Also but end user's artificial chromosome (HAC) transmits than the big dna fragmentation of dna fragmentation that can comprise and express in plasmid.For making up about 6kb, the purpose for the treatment of transmits to the HAC of 10Mb and by conventional carrying method (liposome, polycation amino polymer or vesicle).

Also can use specific start signal, more effectively to translate the sequence of encoded K v9.2 subunit.Sort signal comprises ATG initiator codon and flanking sequence.Insert under the situation of suitable expression in the sequence of encoded K v9.2 subunit and initiator codon thereof and upstream sequence, do not need the extra control signal of transcribing or translate.Yet, only insert under encoding sequence or its segmental situation, the external source translation that comprises ATG initiator codon control signal should be provided.In addition, initiator codon should be arranged in appropriate frame, to guarantee the translation of whole insertion sequences.External source translation factor and initiator codon can have various origins, comprise natural and synthetic.Described in document, expression efficiency can improve (Scharf, D. etc. (1994) ResultsProbl.Cell Differ.20:125-162) by comprising the enhanser that is suitable for employed concrete cell system.

In addition, can select host cell strain according to the expression of regulating insertion sequence or with the proteic ability of being desired of form expression processing.This modification of polypeptide includes, but not limited to acetylizing, carboxylation, glycosylation, phosphorylation, lipoprotein function and acylation.Enzyme is cut proteic " preceding former " (prepro) the translation post-treatment of form also can be used for promoting correct insertion, folding and/or function.Has the different host cell (for example CHO, HeLa, MDCK, HEK293 and WI38) of specific cellularstructure and distinctive mechanism available from American Type CultureCollection (ATCC for the activity after the translation, Bethesda, Md.), and can select, to guarantee to modify rightly and process foreign protein.

For the extended high rate recombinant protein, stably express is preferred.For example, can can use such expression vector to transform with the clone of number or heteromerism ionic channel by stably express Kv9.2, this expression vector can comprise replication orgin and/or the endogenous Expression element and the selectable marker gene of virus on identical or different carrier.After introducing carrier, before changing into selective medium, cell can be cultivated in enrichment medium about 1-2 days.The purpose of selectable mark is the resistance of giving selecting, and its existence allows the growth and the recovery of the cell of successful expression calling sequence.The resistance clone of stable transformed cells can use the tissue culture technique propagation that is fit to this cell type.

Can use any amount of selective system to reclaim cell transformed system.These systems include but not limited to, herpes simplex virus thymidine kinase gene (Wigler, (1977) Cell 11:223-32 such as M.) and adenine phosphoribosyl transferase gene (Lowy, I. etc. (1980) Cell 22:817-23), they can use in tk-or apr-cell respectively.And metabolic antagonist, microbiotic or Herbicid resistant can be used as the basis of selection.For example, dhfr gives methotrexate resistance (Wigler, M. etc. (1980) Proc.Natl.Acad.Sci.77:3567-70); Npt gives aminoglycoside Xin Meisu and G-418 resistance (Colbere-Garapin, F. etc. (1981) J.Mol.Biol.150:1-14); Als or pat give chlorsulfuron and phosphinothricin acetyltransferase resistance (Murry sees above) respectively.Other selectable gene has been described, trpB for example, its allows cell to replace tryptophane to utilize indoles, or hisD, it allows cell to replace Histidine to utilize histinol (Hartman, S.C. and R.C.Mulligan (1988) Proc.Natl.Acad.Sci.85:8047-51.).Recently, the use of witness marking has obtained popularizing, and this mark is anthocyanin, β-glucose neuraminidase and substrate GUS and luciferase and substrate luciferin thereof for example.This mark not only can be used for differentiating transformant, also can be used for determining the amount of expressing owing to the instantaneous or stabilizing protein of specific support system (Rhodes, C.A. etc. (1995) Methods Mol.Biol.55:121-131).

Although existence or shortage that marker gene is expressed show that interested gene also exists, the existence of gene and expression need to determine.For example, if the sequence of encoded K v9.2 subunit is inserted marker gene sequence inside, can differentiate the cell transformed of the sequence that comprises encoded K v9.2 subunit by the disappearance of marker gene function.In addition, marker gene can be under the control of single promotor and the sequence arranged in series of encoded K v9.2 subunit.The expression that responds the marker gene of inducing or selecting also shows the expression of tandem gene usually.

In addition, comprising the nucleotide sequence of encoded K v9.2 subunit and the host cell of expressing K v9.2 subunit can differentiate by the known method of various persons skilled in the art.These methods include but not limited to, DNA-DNA, DNA-RNA hybridization and protein biological assay or immunoassay, and it comprises and is used to detect and or the quantitative technology based on film, solution or chip of Nucleotide or protein sequence.

The existence of the polynucleotide sequence of encoded K v9.2 subunit can or use the fragment amplification of the polynucleotide of probe or fragment or encoded K v9.2 subunit to detect by DNA-DNA or DNA-RNA hybridization.Relating to use based on the analysis of nucleic acid amplification detects based on the oligonucleotide of the sequence of encoded K v9.2 subunit or oligomer and comprises the DNA of encoded K v9.2 subunit or the transformant of RNA.

Use is specific to proteinic polyclone or monoclonal antibody, and the whole bag of tricks that detects and measure the expression of Kv9.2 subunit is known in the art.The embodiment of this technology comprises enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescence-activated cell sorting (FACS).Two sites of the monoclonal antibody of two noiseless epi-positions reactions on utilization and the Kv9.2 subunit, the immunoassay on mono-clonal basis are preferred, but also can use competition combination mensuration.This area is described these and other measuring method in detail, and for example at Hampton, R. etc. (1990; Serological Methods, aLaboratory Manual, Section IV, APS Press, St Paul, Maddox Minn.), D.E. etc. (1983; J.Exp.Med.158:1211-1216) in.

Various marks and combination technology are that the person skilled in the art is known, and can be used for various nucleic acid and determined amino acid.The method that production is used to detect the mark hybridization of the sequence relevant with the polynucleotide of encoded K v9.2 subunit or PCR probe comprises the pcr amplification of few mark, nick translation, end mark or applying marking Nucleotide.In addition, the sequence of encoded K v9.2 subunit or its any fragment can be cloned and be entered carrier, produce the mRNA probe.This carrier is known in the art, and commerce is buied, and can for example T7, T3 or SP6 and labeled nucleotide are used for the vitro synthesized RNA probe by adding suitable RNA polymerase.The test kit that these methods can use various commerce to buy carries out, for example Pharmacia ﹠amp; Upjohn (Kalamazoo, Mich.), Promega (Madison, Wis.) and U.S.Biochemical Corp. (Cleveland, the test kit that Ohio) provides.Can be used for convenient suitable reporter molecule or the marker that detects and comprise radionuclide, enzyme, fluorescence, chemiluminescent or developer, and substrate, cofactor, inhibitor, magnetic-particle etc.

Can from cell culture, express and reclaim under the proteic condition and cultivate being suitable for the nucleotide sequence transformed host cells of encoded K v9.2 subunit.According to used sequence and/or carrier, can be positioned at cytolemma by the protein of transformant production, secrete or be contained in the cell.It will be understood by those of skill in the art that the expression vector that can design the polynucleotide that comprise encoded K v9.2 subunit instructs the Kv9.2 subunit by protokaryon or eukaryotic cell membrane excretory signal sequence to contain.Other construction can be used for connecting the sequence of encoded K v9.2 subunit and the nucleotide sequence of the polypeptide functional domain of coding promotion soluble protein purifying.The functional domain of this promotion purifying includes but not limited to metal chelating peptide, for example allows the Histidine-tryptophane assembly of purifying on immobilization metal, allows the albumin A functional domain of purifying on the immobilization immunoglobulin (Ig); And the functional domain that in FLAGS extension/affinity purification system, uses (Immunex Corp., Seattle, Wash.).But comprise the joint sequence that enzyme is cut between purifying functional domain and Kv9.2 subunit encoding sequence, (Invitrogen, San Diego Calif.) can be used for promoting purifying for example to be specific to the joint sequence of factor XA or enteropeptidase.The fusion rotein that such expression vector is used to express comprise the Kv9.2 subunit and before Trx or enteropeptidase restriction enzyme site the nucleic acid of 6 histidine residues of coding.Histidine residues promotes the purifying (IMIAC on immobilized metal ion affinity chromatography; Be described in Porath, J. etc. (1992) Prot.Exp.Purif.3:263-281), and the enteropeptidase restriction enzyme site provides from the method for fusion rotein purifying Kv9.2 subunit.The discussion that comprises the carrier of fusion rotein is described in Kroll, and D.J. etc. (1993; DNA Cell Biol.12:441-453) in.

The fragment of Kv9.2 subunit not only can also can directly be carried out synthetic (Merrifield J. (1963) J.Am.Chem.Soc.85:2149-2154) of peptide by using solid phase technique by recombinant technology production.Protein synthesis can be by manual skill or is controlled automatically and carry out.Automatically synthesizing for example to use AppliedBiosystems 431A peptide synthesizer (Perkin Elmer) to finish.Each fragment of Kv9.2 subunit can be synthesized separately, then in conjunction with producing full-length molecule.

Biosensor

Kv9.2 polypeptide, nucleic acid, probe, antibody, expression vector and part are useful as the biosensor production of biosensor (and for).

According to Aizawa (1988), Anal.Chem.Symp.17:683, biosensor is defined as being used for the unique combination of the target of molecular recognition, for example has the immobilized antibody or the selective layer of the passage of Kv9.2 for example, and the transducer that is used to transmit measured value.One group of such biosensor will detect because the change of the top layer optical characteristics that passage and surrounding medium interaction cause.What mention especially in this technology is ellipsometry and the resonance of surperficial cytogene.The existence or the level that can be used for detecting the Kv9.2 part in conjunction with the biosensor of Kv9.2.The structure of this biosensor is known in the art.

Therefore, the clone of expressing K v9.2 can be used as reporter gene system, forms [3H] inositol monophosphate or other second messenger's detector ligand ATP (Watt etc., 1998, J Biol Chem5 month 29 for example by acceptor-promotion; 273 (22): 14053-8).The receptor-ligand biosensor also is described in Hoffman etc. 2000, Proc Natl Acad Sci USA Oct 10; 97 (21): 11215-20.Comprise the optics of Kv9.2 and other biosensor and also can be used for detecting level and existence with G-albumen and other protein-interacting, as by for example Figler etc., 1997, Biochemistry Dec 23; 36 (51): 16288-99 and Sarrio etc., 2000, Mol Cell Biol 2000 Jul; 20 (14): 5164-74) describe.The sensor unit of biosensor is described in for example US 5,492,840.

Screening is analyzed

The Kv9.2 polypeptide, comprise homologue, variant and derivative, no matter be natural or reorganization, can be used for screening with the Kv9.2 subunit or comprise the process of the ionic channel bonded compound of Kv9.2, this compound activating (agonist) or suppress the activation (antagonist or blocker) of Kv9.2.Therefore, this peptide species also is used in combining of assessment small molecules substrate and part in for example cell, cell-free preparation, chemical library and the natural product mixture.These substrates and part can be natural substrate and part, perhaps can be structure or functional analogue material.Referring to Coligan etc., Current Protocols inImmunology1 (2): the 5th chapter (1991).

Kv9.2 ionic channel polypeptide is responsible for the various biological function, comprises multiple pathology.Therefore, wishing to find stimulates the ionic channel that comprises Kv9.2 on the one hand, can suppress the compound and the medicine of Kv9.2 ion channel function on the other hand.Usually agonist and antagonist are used for the treatment of and prevent purpose for for example anxiety, stress reaction, depression or diabetes and other diseases.

Appropriate design may can with the interactional candidate compound of Kv9.2 ionophorous protein can be based on the structural research of peptide molecule shape.A kind ofly determine which site and the method for specific other protein-interacting are physical structure mensuration, for example X-radiocrystallography or two dimensional NMR techniques.These technology are for determining which amino-acid residue forms the molecule zone of action guidance is provided.The detailed description of measuring about protein structure is referring to for example Blundell and Johnson (1976) Protein Crystallography, Academic Press, New York.

Another kind of appropriate design is used a kind of screening method, and this method is usually directed to produce the suitable cell at cell surface expression Kv9.2 ionic channel polypeptide.This cell comprises the cell from animal, yeast, fruit bat (Drosophila) or E.coli.The cell of the expressing K v9.2 cytolemma of expressing protein (or comprise) contacts with test compounds then, observe in conjunction with or the stimulation or the inhibition of functional response.For example Kv9.2 mRNA or polypeptide can be injected the Africa xenopus ovocyte, describe in further detail, measure by the working voltage pincers and detect because of contact the inductive electric current with test compounds as other chapters and sections.

Except that detecting respectively each candidate compound, can advantageously produce and screen library or storehouse (bank) of candidate ligand with Kv9.2 subunit or the ionic channel that comprises Kv9.2.Therefore, for example assembled and surpass 200 part storehouses of inferring and be used for screening.This storehouse comprises: mediator, hormone and known chemokine of having an effect by ionic channel; The compound of natural generation, this compound can be the ionic channel agonists of inferring, nonmammalian, do not differentiate the biologically active peptides of its Mammals copy as yet; And do not have the compound found at nature, but it is with unknown native ligand active ions passage.

Functions of use (for example measure, FRET measures, FLIPR measures, cell electrophysiology, ovocyte electrophysiology fully by the Rb flux, referring to other chapters and sections) and other chapters and sections describe in further detail in conjunction with measuring, the known ligand that this library is used to screen the Kv9.2 subunit or comprises the ionic channel of Kv9.2.Yet a large amount of Mammals ionic channel that exists, up to now, to it without any relevant activation part (agonist) or passivation part (antagonist).Thereby the activation part of these acceptors is not included in the part storehouse of present discriminating.Therefore, also can differentiate native ligand at tissue extract functional screening Kv9.2 (using functional screenings such as ovocyte electrophysiology).The extract that can produce the orthofunction reaction continues fractional separation, measures level part with method described herein, up to the activatory part being separated and discriminating is come out.

Another kind method relates to inhibition or the stimulation screening inhibitors of ion channels that comprises the ionic channel of Kv9.2 by mensuration.This method comprise or separately with Kv9.2 subunit transfecting eukaryotic cells to form with the number passages, perhaps with other Kv passage subunit transfecting eukaryotic cells with formation heteromerism passage, and at the cell surface expression ionic channel.Under the situation of the existence of the ionic channel that comprises Kv9.2, this cell is contacted with the potential antagonist then.Can use complete cell electrophysiology to check this cell, to measure conduction of current or dynamic (dynamical) change.

The another kind of method that detects Kv9.2 agonist or antagonist is as United States Patent (USP) 5,482, the technology of describing in 835 based on yeast, and this patent this paper is incorporated herein by reference.

In a preferred embodiment, screening adopts the change that detects conduction to screen agonist and the antagonist of Kv9.2.Particularly, we disclose a kind of method, and wherein the antagonist of Kv9.2 reduces the conductivity of suitable cells transfected.Preferably, conductivity has reduced by 10%, 20%, 30%, 40%, 50%, 60%, 70% or more under the situation that the antagonist of Kv9.2 exists.Preferably, conductivity has reduced 1pS, 2pS, 3pS, 4pS, 5pS, 10pS, 15pS, 25pS, 35pS, 45pS, 60pS, 70pS or more under the situation that the antagonist of Kv9.2 exists.

We also disclose a kind of method, and wherein the exciting machine of Kv9.2 improves the conductivity of suitable cells transfected.Preferably, conductivity has improved 10%, 20%, 30%, 40%, 50%, 60%, 70% or more under the situation that the agonist of Kv9.2 exists.Preferably, conductivity has improved 1pS, 2pS, 3pS, 4pS, 5pS, 10pS, 15pS, 25pS, 35pS, 45pS, 60pS, 70pS or more under the situation that the agonist of Kv9.2 exists.

In another preferred embodiment, the Rb flux of detection of radioactive labels is adopted in screening, and preferably agonist and the antagonist of Kv9.2 screened in the variation of %Rb flux.Preferably, screening adopts the functional examination of the above following description of " functional examination of Kv9.2 (Rb flux) " (Functional Assay of Kv9.2 (Rb flux)) exercise question to carry out.

Particularly, we disclose a kind of method, and wherein, the antagonist of Kv9.2 has reduced the %Rb efflux (efflux) of suitable cells transfected.Preferably, the %Rb efflux has reduced by 10%, 20%, 30%, 40%, 50%, 60%, 70% or more under the situation that the antagonist of Kv9.2 exists.

We further disclose a kind of method, and wherein the agonist of Kv9.2 has improved the %Rb efflux of suitable cells transfected.Preferably, the %Rb efflux has improved 10%, 20%, 30%, 40%, 50%, 60%, 70% or more under the situation that the agonist of Kv9.2 exists.

When candidate compound is protein, antibody or peptide specifically, the display technique of bacteriophage screening can be used in the library of candidate compound.Phage display is a kind of molecular screening method of utilizing recombinant phage.This technology comprises that each phage or phagemid are expressed concrete candidate compound like this with the gene transformation phage of coding from a kind of compound in candidate compound library.The phage (it preferably sticks on the solid support) that transforms is expressed suitable candidate compound, and shows it on its phage ghost.Can with Kv9.2 polypeptide or the concrete candidate compound of peptide bonded by selection strategy enrichment based on affinity interaction.Characterize successful candidate agent then.Phage display has the advantage that is better than standard affinity ligand triage techniques.Phage surface shows the candidate agent of three-dimensional conformation, more is similar to the conformation of its natural generation.This allows the more single-minded and more combination of high-affinity for the purpose of screening.

The another kind of method in SCREENED COMPOUND library is utilized eucaryon or prokaryotic host cell, this cell recombinant DNA molecules stable conversion of expressing library of compounds.This cell, or live or with the fixed form, can be used for the standard binding partners and measure.Referring to (1989) Science246:243-247 such as Parce; With (1990) Proc.Nat ' l Acad.Sci.USA 87 such as Owicki; 4007-4011, it has described the responsive method that detects cell response.Competitive assay is particularly useful, and the cell of expressing library of compounds here and the traget antibody of the known Kv9.2 of combination polypeptide are (for example ¹²⁵I-antibody) contact or cultivate with check sample, described test sample is the determined candidate compound of binding affinity of itself and bonding composition for example.The combination of isolated polypeptide and free label binding partners are with assessment bonded degree then.The amount of bonded check sample is inversely proportional to the amount of the traget antibody that combines polypeptide.

Any can be used in numerous technology, separate bonded with the free binding partners, with assessment bonded degree.This separating step can typically comprise following process, for example is attached to strainer with after scouring, be attached to plastics with after scouring, or cytolemma is centrifugal.

Another kind method is to use the polypeptide or the peptide of dissolved, unpurified or dissolving purifying, for example polypeptide or the peptide that extracts from the eucaryon that transforms or prokaryotic host cell.This " molecule " that allows to have the advantage that has improved specificity, automatic controllability and high drug testing productivity is in conjunction with measuring.

Another kind of candidate compound triage techniques comprises a kind of method, this method provides large-duty screening for the new compound that for example the Kv9.2 polypeptide is had a suitable binding affinity, it is described in detail in International Patent Application WO 84/03564 (Commonwealth Serum Labs.), is published on September 13rd, 1984.At first, the solid phase substrate for example the plastics safety pin with some synthetic a large amount of different little peptides test compounds on other the suitable surface, referring to (1991) such as Fodor.All then plastics pins and reaction of dissolved Kv9.2 polypeptide and washing.Next step comprises detection bonded polypeptide.Identify like this and the interactional compound of polypeptid specificity.

Part provides in conjunction with mensuration determines pharmacological direct method, and is suitable for large-duty form.The part radio-labeling of purifying can be used in conjunction with research to high specific acitivity (50-2000Ci/mmol).Determine that then radiolabeled process does not reduce the activity of part to its target.Make for example condition determination optimization of Nucleotide of buffer reagent, ion, pH and other regulon, to set up the spendable signal of noise ratio for film and complete cell receptor or ionic channel source.Measure for these, specificity deducts the radioactivity of measuring under the situation of excessive unmarked competition part existence in conjunction with being defined as the total correlation radioactivity.Under the possible situation, use more than one competition part to determine remaining non-specific binding.

This mensuration can be tested the combination of candidate compound simply, wherein with the adhesion of cell with acceptor or ionic channel can by with the direct or indirect method of relevant mark of candidate compound, perhaps in the analysis that relates to mark rival's competition, detect.Whether in addition, these mensuration can be used the detection system that is suitable for having on their surface the cell of target, detect this candidate compound and cause by activating the signal that target produces.Activation inhibitor is measured under the situation that known agonist exists usually, and agonist influences activatory under the situation of observing the candidate compound existence.

In addition, this mensuration can simply comprise following steps, candidate compound is mixed the formation mixture with the solution that contains the Kv9.2 polypeptide, measure the ion channel activity that contains Kv9.2 in the mixture, and the Kv9.2 ion channel activity and the standard of mixture compared.

Kv9.2 subunit cDNA, albumen and proteic antibody also can be used for configuration to be measured, and detects the influence of Kv9.2 Subunit mRNA and protein production in the compound pair cell of interpolation.For example, use mono-clonal and polyclonal antibody to make up ELISA by standard method known in the art, to measure Kv9.2 protein subunit excretory or cell related levels, this can be used for finding to suppress or to strengthen the reagent (being called agonist or antagonist respectively) that the Kv9.2 subunit is produced from the cell or tissue of suitable manipulation.The standard method of carrying out screening assay is well known in the art.

The example of potential Kv9.2 ionic channel antagonist and blocker comprise antibody or, in some cases, Nucleotide and homologue thereof, the oligonucleotide or the protein that comprise purine and purine homologue, be closely related with the part that contains the ionic channel of Kv9.2, for example part fragment or combine with ionic channel but the small molecules that do not induce reaction has stoped the activity of passage like this.

We therefore also provide can specificity in conjunction with the compound of Kv9.2 polypeptide and/or peptide.

Term " compound " is meant chemical compound (natural generation or synthetic), the biological example macromole (for example, nucleic acid, albumen, non-peptide or organic molecule), or by the biomaterial extract of bacterium, plant, fungi or animal (specifically being Mammals) cell or tissue preparation for example, or even inorganic components or molecule.Preferred compound is an antibody.

Carry out the required material of this screening and can be packaged into the screening reagent box.This screening reagent box is used to differentiate that the agonist, antagonist, part, acceptor, substrate, enzyme of Kv9.2 polypeptide etc. reduces or strengthen the compound of the production of Kv9.2 ionic channel polypeptide.Described screening reagent box comprises: (a) aKv9.2 polypeptide; (b) reconstitution cell of expressing K v9.2 polypeptide; (c) cytolemma of expressing K v9.2 polypeptide; Or (d) antibody of Kv9.2 polypeptide.The optional working instructions that comprise of screening reagent box.

Transgenic animal

We further disclose compare with normal expression level can be with horizontal expression normal, that improve or reduce natural or reorganization Kv9.2 subunit and/or contain the transgenic animal of ionic channel or homologue, variant or the derivative of Kv9.2.Preferably, this transgenic animal are inhuman Mammalss, for example pig, sheep or rodent.Most preferred transgenic animal are mouse or rat.

Our disclosed transgenic animal, wherein all or part of natural Kv9.2 gene is by being replaced from another organic Kv9.2 sequence.Preferably this organism is another species, most preferably people.In particularly preferred embodiments, we disclose the mouse that its whole Kv9.2 gene is all replaced by people Kv9.2 gene basically.This transgenic animal and Kv9.2 wild-type animal can be used for screening agonist and/or the antagonist of Kv9.2.

For example, this mensuration can comprise that preferably the cell of transgenic animal, tissue or organ contact with candidate substances with wild-type or transgenic animal or its part, measures the relevant phenotype of Kv9.2, and for example anxiety or glucose level descend.Also can adopt derived from the cell of relevant animal based on the screening of cell and mensuration to conductivity or effect of kinetics.

We further disclose and have comprised the transgenic animal that function is suffered destructive Kv9.2 gene, wherein any one of Kv9.2 disclosed herein or a plurality of funtion part or all elimination.This paper comprises transgenic animal (" Kv9.2 knocks out " animal), and described transgenic animal are not expressed the function Kv9.2 that comprises ionic channel because the sudden change of one or more losses of function of Kv9.2 gene comprises disappearance.

This paper also comprises the mutant of funtion part forfeiture, does not for example knock out fully, and it can for example have disappearance in the part of Kv9.2 gene through selecting.This animal can produce by the Kv9.2 sequence part (the protein zone that for example has critical function) that selectivity substitutes or disappearance is relevant.

The mutant of this all or part of loss of function specifically is anxiety and diabetes as the model of Kv9.2 relative disease.The animal of Presentation Function partial loss can contact with candidate substances to differentiate and strengthen phenotypic material, that is to say, increases (under the situation of Kv9.2) hypalgia or the horizontal phenotype of observed minimizing stress reaction.Other parameter for example conductivity or dynamic (dynamical) minimizing also can use the discrimination method of other chapters and sections of this paper to detect.

Partly and all knock out selective agonist and/or the antagonist that also can be used for differentiating Kv9.2.For example, agonist and/or antagonist can give the animal (knocking out) of wild-type and Kv9.2 defective.Selective agonist or the antagonist that to see Kv9.2 are influential to the wild-type animal, and do not have in the animal of Kv9.2 defective.At length, the design specificity analyses determines to estimate potential medicine (candidate ligand or compound) whether it produces physiological response to this medicine under the situation of the ionic channel defective that comprises Kv9.2.This can by as discussed above give the transgenic animal medicine then the concrete reaction of analyzing animal finish.Also can carry out similar method based on cell, this method adopts derived from the cell of relevant animal and measures conductivity or dynamic (dynamical) effect.This animal also can be used for detecting the effect of the medicine of differentiating by screening method described herein.

In another embodiment, will have the phenotypic transgenic animal of partial loss of function and be used for screening.In such embodiment, screening can comprise that analysis is to phenotypic part or all of recovery of wild-type or reverse.Also can use derived from the cell of relevant animal and measure screening the cell base of conductivity or dynamic (dynamical) effect.Discovery can be such candidate compound can be recognized and done Kv9.2 agonist or analogue.This agonist can be used for for example improving glucose level, perhaps reduces individual stress reaction or anxiety level.

In preferred embodiments, transgenosis Kv9.2 animal specifically is Kv9.2 knock-out animal (function completely loses), shows the phenotype that proposes among the embodiment, and preferably the check that proposes as this paper is measured.Therefore, the Kv9.2 animal, specifically be that the Kv9.2 knock-out animal preferably shows any one or a plurality of following feature: glucose level descends, and anxiety increases.

In particularly preferred embodiments, when comparing with corresponding wild type mouse, transgenosis Kv9.2 animal, it specifically is the Kv9.2 knock-out animal, show at least 10%, preferably at least 20%, at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% higher or lower (deciding as the case may be) location parameter more preferably.Therefore, for example, when comparing, use the spacious field analysis that proposes among the embodiment to detect with wild-type mice, the Kv9.2 deficient mice preferably has the stationarity that improves on the statistics, or lasting (permanance) time of the periphery of traveling time that reduces and/or raising.Be also shown in the minimizing of whole miles of relative movement.

Obviously, the phenotype of the transgenic animal of present disclosed Kv9.2 defective can be used to use the screening of wild-type animal effectively, causes the compound of the effect similar to the Kv9.2 afunction with detection.In other words, the wild-type animal can contact with candidate compound, the phenotypic change of observed corresponding K v9.2, and for example anxiety level, blood glucose levels etc. are used for differentiating the regulon of Kv9.2 function, concrete is antagonist.The discrimination method of other chapters and sections of use this paper also can detect the phenotype of cell, and for example conductivity descends or dynamic (dynamical) change or reduction.

The compound of differentiating by this screening can be used as the antagonist of Kv9.2, for example as anodyne or stress reaction abirritant, especially for treatment or alleviate the Kv9.2 relative disease.

Above-described screening can relate to observes arbitrary suitable parameter, for example behavior, physiology or biochemical reaction.Preferred reaction comprises physiological response, and can comprise one or more following features: the change of disease resistance; The change of inflammatory reaction; The change of tumor susceptibility: blood pressure change; Neovascularization; The change of dietary behavior; Body weight changes; Bone density changes; The change of body temperature; Insulin secretion; Gonadotrophin secretion; Nose and segmental bronchus secretion; Vasoconstriction; The loss of memory; Anxiety; The change of anxiety state; Hyporeflexia or hyperreflexia; Or stress reaction.

Also can use biochemical parameter, for example conductivity or dynamic (dynamical) variation.Preferably, conductivity uses " functional examination of Kv9.2 (electrophysiology) " to measure, and kinetics (activation and/or passivation time, preferably soak time and/or passivation time constant) is measured as the method for describing in this part.This is particularly useful in the screening based on cell.

In preferred embodiments, the conductivity of the cell that contacts with the Kv9.2 agonist (for example cell of wild-type or funtion part forfeiture) improves at least 10%, preferably at least 20%, more preferably at least+30%, more preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%.In preferred embodiments, use " functional examination of Kv9.2 (electrophysiology) " of other chapters and sections description herein to measure conductivity.

In preferred embodiments, kinetics, for example soak time of cell and/or passivation time (for example wild-type or funtion part forfeiture cell), preferably the raising of soak time that contact with the Kv9.2 agonist and/or passivation time constant at least 10%, preferably at least 20%, more preferably at least+30%, more preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%.In preferred embodiments, use " functional examination of Kv9.2 (electrophysiology) " of other chapters and sections description herein to measure kinetics.

In preferred embodiments, the blood glucose levels of wild-type that contacts with the Kv9.2 agonist or Kv9.2 part knock-out animal improves at least 10%, preferably at least 20%, more preferably at least+30%, more preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%.

In preferred embodiments, the antagonist of Kv9.2 is such antagonist, and the animal of the wild-type that contacts with this antagonist or partial function loss shows that to the small part degree mutant with the part or all of afunction of Kv9.2 is to the same phenotype of small part.That is to say, the preferred such antagonist of antagonist, it causes that anxiety improves, or glucose level descends, or conductivity descends, or kinetics changes or descend, or above-mentioned any phenotypic combination.Preferably, relevant phenotype is expressed with the degree identical with the Kv9.2 knock-out animal.

In preferred embodiments, the conductivity of the wild-type that contacts with the Kv9.2 antagonist or the cell of partial loss of function be the Kv9.2 deficient cells conductive+80% in, preferably+70%, more preferably+60%, more preferably+50% in, more preferably+40% in, more preferably+30%, more preferably+20%, more preferably+10% in, more preferably+5% in.In preferred embodiments, this uses " functional examination of Kv9.2 (electrophysiology) " of other chapters and sections description herein to measure.

In preferred embodiments, kinetics, the i.e. soak time and/or the passivation time of wild-type that contacts with the Kv9.2 antagonist or Kv9.2 partial loss of function cell, preferably soak time and/or passivation time constant, be in the Kv9.2 deficient cells kinetics (or correlation time)+80%, preferably+70%, more preferably+60%, more preferably+50%, more preferably+40%, more preferably+30% in, more preferably+20% in, more preferably+10%, more preferably+5% in.In preferred embodiments, this uses " functional examination of Kv9.2 (electrophysiology) " of other chapters and sections description herein to measure.

In preferred embodiments, the blood glucose levels of wild-type that contacts with the Kv9.2 antagonist or Partial K v9.2 knock-out animal is in blood glucose levels+80% of the transgenic animal of Kv9.2 defective, preferably+70%, more preferably+60%, more preferably+50% in, more preferably+40% in, more preferably+30%, more preferably+20%, more preferably+10% in, more preferably+5% in.

Whether the tissue derived from the animal that knocks out Kv9.2 can be used for binding analysis, combine with Kv9.2 to measure potential medicine (candidate ligand or compound).This analysis can be by obtaining the first ionic channel preparation and obtaining the second ionic channel preparation and carry out from the known Kv9.2 part or the source of compound in conjunction with any discriminating from the transgenic animal that make the ionic channel production defective that comprises Kv9.2 through genetically engineered.Usually, except that the source that obtains, the first and second ionic channel preparations are all similar in all fields.For example, if be used for measuring, be used as the source of the second ionic channel preparation from the comparable cerebral tissue of normal (wild-type) animal from the cerebral tissue (as mentioned with described below) of transgenic animal.Individually and under the situation that candidate ligand or compound exist, each ionic channel preparation comprises the part cultivation of the ionic channel of Kv9.2 with known combination.Preferably, candidate ligand or compound are measured with several different concentration.

For the first and second ionic channel preparations, measure with test compounds and substitute known ligand bonded degree.Tissue derived from transgenic animal can be directly used in mensuration, perhaps can process this tissue to separate film or the membranin that itself is used to measure.Preferred transgenic animal are mouse.Can use any method tagged ligand that is suitable in conjunction with measuring.This method comprises, without limits, and radioactive, enzymatic, fluorescence or chemiluminescent labeling (and other labeling technique that above describes in further detail).

In addition, wild-type animal that can be by giving expressive function Kv9.2 with differentiated the animal candidate compound that shows any phenotype feature relevant etc., discriminating Kv9.2 or contain the antagonist of the ionic channel of Kv9.2 with reducing or eliminating the Kv9.2 functional expression.

The detailed method of producing the non-human transgenic animal hereinafter has been described in further detail.Transgenosis construct can be introduced animal kind system preparation transgene mammal.For example, the construction of one or several copy can be introduced the genome of mammal embryo by the standard transgenic technology.

In an exemplary embodiment, be the production transgenic nonhuman animal by transgenosis being introduced the non-human animal plants.The embryo target cell of each etap can be used for introducing transgenosis.Etap according to the embryo target cell is used diverse ways.At visible good pronucleus and good reproduction adaptability among healthy, good embryo's productive rate, the embryo generally, select the specific kind system of any animal.In addition, haplotype is the significance factor.

Transgenosis is introduced the embryo can finish for example microinjection, electroporation or fat transfection by any method known in the art.For example, can enter the pronucleus of the mammalian ovum of fertilization, cause the construction of one or more copies to remain in the developmental mammalian cell, the Kv9.2 transgenosis is introduced Mammals by the microinjection construction.After transgenosis construct is introduced zygote, but the different time of this ovum vitro culture perhaps be transplanted to alternate host again, perhaps the two all carries out.Also but vitro culture is to ripe.A kind of common methods is according to different the about 1-7 of species extracorporeal culturing embryo days, then they is transplanted in the alternate host again.

The existence of construction in the embryo's who can the southern blotting technique analyzing and testing transgenosis by tissue fragment handles the filial generation.If the external source of one or more copies clone construction keeps stably being integrated into the genome of this transgenic embryos, the permanent transgene mammal kind that may set up the construction that carries the transgenosis increase is.

Can measure the genome that construction mixes filial generation after the young birth of the mammiferous tire that transgenosis changes.Preferably, this mensuration is finished on child chromosome corresponding to the probe of coding is desired recombinant protein product or its segmental dna sequence dna by hybridization.Those Mammals filial generation growths of construction of finding to contain at least one copy in their genome are until maturation.

For the purpose of this paper, zygote is the structure of can bud into complete organic diploid cell basically.Usually, this zygote comprises an ovum, this ovum contain by two haploid nucleuses from a gamete or two gametes merge, the nuclear of natural or artificial formation.Therefore, gametic nucleus must be natural compatible, and promptly this gametic nucleus produces the zygote that can break up with the organic work of bud into function.Usually, the euploid zygote is preferred.If obtain the aneuploid zygote, for the organic euploid quantity of gamete origin, the change of karyomit(e) quantity is no more than one so.

Except that similar biological factor, physical factor is also being controlled the amount (for example volume) of the nuclear that can add zygote to or the exogenous genetic material in the genetic material that forms merozygote nuclear.If do not remove genetic material, the amount of so addible exogenous genetic material is not by being limited absorbed amount by physical damage.Usually the amount of the exogenous genetic material that inserts is no more than about 10 skin liters.The viablity just like the physical damage zygote is so big unlikely for the physical action that adds.The biology restriction of the value volume and range of product of dna sequence dna depends on the function of concrete zygote and exogenous genetic material and changes, and be conspicuous to the person skilled in the art, because the genetic material of the zygote that obtains, comprise exogenous genetic material, must biologically can initial sum keep zygote differentiation and bud into function organism.

The quantity of adding the transgenosis construct copy of zygote to depends on the total amount of the exogenous genetic material of interpolation, and is the amount that genetic transformation is taken place.Only need a copy in theory; Yet usually, can functionating and utilize a large amount of copies in order to ensure a copy, for example 1 of transgenosis construct, 000-20,000 copy.Using the exogenous DNA array of each insertion that surpasses a function copy usually is favourable with the phenotype expression that strengthens exogenous DNA array.

Can utilize to allow to add any technology of exogenous genetic material, as long as it does not destroy the cell or the genetic construction of cell, nuclear membrane or other existence to the nuclear genetic material.Exogenous genetic material preferably inserts the nuclear genetic material by microinjection.Cell and cyto-architectural microinjection are known in the art and use.

Using standard method to finish transplants again.Usually, with the alternate host paralysis, the embryo is embedded uterine tube.The quantity of being transplanted to concrete host's embryo will change according to species, but the quantity tool comparability of the filial generation of the species of common and natural generation.

Can be by genetically modified existence or expression in the transgenic progeny of any appropriate means screening alternate host.Screen the transgenosis complementary probe that usually uses with to small part, finish by southern blotting technique or rna blot analysis.Use can be used as the another kind of or additional method that the screening transgene product exists by the western blot analysis of the proteinic antibody of transgenes encoding.Typically, from afterbody tissue preparation DNA and with southern blotting technique analysis or pcr analysis transgenosis.In addition, although any tissue or cell type can be used for this analysis, use southern blotting technique analysis or PCR to detect to believe existing and express with genetically modified tissue of the highest horizontal expression or transit cell gene.

Estimate that transgenosis exists in addition or addition method comprise that without limits, suitable biochemical measurement is enzyme and/or immunoassay, concrete mark or the enzyme histological stain of living, fluidic cell quantitative analysis etc. for example.Hemanalysis also can be used for detecting the existence of transgene product in the blood, also can be used for estimating the effect of transgenosis to various types of hemocyte levels and other blood composition.

The filial generation of transgenic animal can obtain by transgenic animal and suitable mating partner mating, or obtains by ovum and/or the sperm in vitro fertilization that obtains from transgenic animal.When carrying out mating with mating partner, this mating partner can yes or no transgenosis and/or knock-out animal; If it is genetically modified, it can comprise identical or different transgenosis, or the two all comprises.In addition, mating partner can be that the parent plants system.When use is in vitro fertilization, fertilization implantable alternate host of embryo or vitro culture, perhaps the two all carries out.Use arbitrary method, use above-described method or other appropriate means can estimate the genetically modified existence of this filial generation.

The transgenic animal that produce according to method described herein will comprise exogenous genetic material.Propose as mentioned, in certain embodiments, exogenous genetic material is the dna sequence dna of production that causes the Kv9.2 subunit or comprise the ionic channel of Kv9.2.In addition, in such embodiments, this sequence with transcribe controlling elements for example promotor combine, this preferably allows transgene product to express in the cell of particular type.

Retroviral infection also can be used for guiding transgenosis to enter the non-human animal.But the non-human embryo vitro culture of growing is to blastocyst stage.During this period of time, blastomere can be the target ((Jaenich, R. (1976) PNAS 73:1260-1264) of retroviral infection.Effective infection of blastomere is handled to remove zona pellucida by enzyme and is obtained (to handle mice embryonic (Manipulating the MouseEmbryo), Hogan edits (Cold Spring Harbor Laboratory Press, Cold SpringHarbor, 1986).Be used to guide genetically modified virus carrier system to carry genetically modified replication-defective virus (Jahner etc. (1985) PNAS 82:6927-6931 typically; (1985) PNAS 82:6148-6152 such as Van der Putten).(Van der Putten sees above conveniently to obtain transfection effectively by cultivation blastomere on the cell of individual layer production virus; Stewart etc. (1987) EMBO J.6:383-388).In addition, infection can be carried out in the stage afterwards.Can or produce viral injection cell with virus and enter segmentation cavity (Jahner etc. (1982) Nature 298:623-628).The great majority person of foundation is chimeric for transgenosis, because mix in the hypotype that occurs over just the cell that forms transgenic nonhuman animal.In addition, the person of foundation can comprise the insertion of genetically modified miscellaneous retroviruses at genomic different positions, and these insertions separate in the offspring usually.In addition, also can transgenosis be introduced kind of system's (Jahner etc. (1982) see above) by retroviral infection midtrimester of pregnancy embryo in utero.

The target cell that is used for the third type of transgenosis introducing is embryonic stem cell (ES).The ES cell merges (Evans etc. (1981) Nature292:154-156 from the pre-implantation embryos acquisition of vitro culture and with the embryo; Bradley etc. (1984) Nature 309:255-258; Gossler etc. (1986) PNAS83:9065-9069; With (1986) Nature 322:445-448 such as Robertson).Transgenosis can be introduced the ES cell effectively by the DNA transfection or by retrovirus-mediated by protein transduction.After this, the ES cell that transforms like this can combine with the blastocyst from the non-human animal.Thereafter, this ES cell is settled down the embryo and is caused obtaining the kind system of chimaeric animals.Be correlated with and discuss referring to Jaenisch R. (1988) Science240:1468-1474.

We also provide the non-human transgenic animal, and transgenic animal are characterized by the Kv9.2 gene with change here, and are preferably as indicated above, as the Kv9.2 subunit or contain the model of the ion channel function of Kv9.2.The change of gene comprises the sudden change of disappearance or other afunction, introduces the foreign gene of the nucleotide sequence with orientation or random mutation, introduces foreign gene, perhaps their combination from another species.For change transgenic animal can be isozygoty or heterozygosis.The biologically active agent that can regulate the Kv9.2 subunit for screening or comprise the ion channel function of Kv9.2 from its deutero-animal and cell is useful.Screening method has special use for the specificity and the effect of the potential therapy of determining the adjusting blood-glucose, the disease of treatment comprises, the I type comprises the relevant vascular disease of diabetes, the ephrosis neuropathy relevant with diabetes that diabetes are relevant with type ii diabetes, hyperinsulinemia, hunger disease, insulin resistance, diabetic complication, and treats hypoglycemic treatment.Equally, the treatment of high fat (they are HDL, LDL or VLDL) mass formed by blood stasis, unusual lipidemia, no matter its cause is to be primary in or to be secondary to diabetes, hyperthyroidism/go down, acromegaly, liver failure, renal failure, Vipoma, pancreatitis and alcohol inductive hypoglycemia.The sacred disease of same treatment comprises listed those panic disorders and depression among stress reaction obstacle after anxiety disorder, anxiety disorder, anxiety related behavior and generalized anxiety disorder disease, panic disorder, agoraphobia, social phobia, obsessive compulsive disorder, the wound, gross stress reaction obstacle and the DSM-IV.

Described animal as (for example brain, heart, spleen and liver) Kv9.2 subunit in research healthy tissues and the organ or comprise Kv9.2 ionic channel effect and to the model of the influence of these tissues or organ dysfunction.

Another aspect relates to transgenic nonhuman animal, and this animal has the endogenous Kv9.2 gene of function destructive, but the transgenosis of the coding allos Kv9.2 albumen Kv9.2 of another species (promptly from) is also carried and expressed to this animal in its genome.Preferably, this animal is a mouse, and allos Kv9.2 is people's Kv9.2.Derived from this with the animal of the animal of people Kv9.2 reorganization or clone can be used for differentiating in the body and the reagent of vitro inhibition people Kv9.2.For example, the stimulator by the conduction of people Kv9.2 inducement signal under detected reagent existence or non-existent situation can give animal or clone, and measures the reaction of animal or clone.In the body or the reagent of vitro inhibition people Kv9.2 can the reaction when not existing compare with this reagent, the decline of reaction was differentiated when this reagent existed.

We also provide the transgenic nonhuman animal (" Kv9.2 subunit knock-out animal ") of Kv9.2 defective.This animal is a kind of that express reduction or do not have the Kv9.2 subunit or comprise the animal of the ion channel activity of Kv9.2, the result of or disappearance destroyed preferably as endogenous Kv9.2 subunit gene group sequence.Preferably, this animal expressing K v9.2 subunit or comprise the ion channel activity of Kv9.2 not.More preferably, this animal is not expressed the activity of the ionic channel that comprises Kv9.2 that is shown as SEQ ID NO:3 or SEQ ID NO:5.As hereinafter describing in further detail, the animal that the Kv9.2 ionic channel knocks out can produce by the whole bag of tricks known in the art.

The disclosure also relates to the nucleic acid construct thing that is used for functionally destroying host cell Kv9.2 gene.Described nucleic acid construct thing comprises: a) non-homogeneous alternative part; B) be positioned at the first homology zone of non-homogeneous alternative part upstream, this first homology zone has and the substantially the same nucleotide sequence of a Kv9.2 gene order; And c) is positioned at the second homology zone of non-homogeneous alternative portion downstream, this second homology zone has and the substantially the same nucleotide sequence of the 2nd Kv9.2 gene order, and the 2nd Kv9.2 gene order is positioned at the downstream of a Kv9.2 gene order of the endogenous Kv9.2 gene of natural generation.In addition, the first and second homology zones have enough length and are being used for carrying out homologous recombination between the endogenous Kv9.2 gene in the nucleic acid construct thing and host cell when nucleic acid molecule is introduced host cell.In a preferred embodiment, non-homogeneous alternative part comprises the expression reporter gene, preferably includes lacZ and is just selecting expression cassette, preferably includes the neomycin phosphotransferase gene that functionally is connected with one or more controlling elements.

Preferably, the first and second Kv9.2 gene orders are derived from SEQ ID No.1, SEQ ID No.2 or SEQ ID NO:4, perhaps their homologue, variant or derivative.

Relate to above-described nucleic acid construct thing on the other hand and mixed wherein recombinant vectors.Another aspect relates to introduces wherein host cell with the nucleic acid construct thing, therefore allows the homologous recombination between the endogenous Kv9.2 gene of nucleic acid construct thing and host cell, causes the function of endogenous Kv9.2 gene to be destroyed.This host cell can be the mammalian cell from the common expressing K v9.2 of liver, brain, spleen or heart, or pluripotent cell, for example mouse embryo stem cell.The introducing of nucleic acid construct thing wherein and with the embryonic stem cell of endogenous Kv9.2 dna homolog reorganization is further grown, produce transgenic nonhuman animal, this animal has the cell that also therefore carries the Kv9.2 gene disruption from embryonic stem cell in its genome.Can select and cultivate in its kind is, carry destructive Kv9.2 gene animal to be created in the animal that has the Kv9.2 gene disruption in whole somatocyte and the sexual cell.Can cultivate this mouse then to having homozygosity for the Kv9.2 gene disruption.

Antibody

Term used herein " antibody " is unless opposite regulation includes but not limited to polyclone, mono-clonal, chimeric, strand, Fab fragment and the fragment that produces by the Fab expression library.This fragment comprises the target material is kept its fragment in conjunction with active complete antibody, Fv, F (ab ') and F (ab ') ₂Fragment and single-chain antibody (scFv), fusion rotein and other comprise the synthetic protein of the antigen binding site of antibody.This antibody and fragment thereof can be humanized antibody, for example describe among the EP-A-239400.In addition, also can use the antibody of describing in the United States Patent (USP) 5,545,807 and 6,075,181 for example with whole people variable region (or its fragment).Neutralizing antibody, promptly those suppress the antibody of aminoacid sequence substance bioactivity, are particularly preferred for diagnosis and treatment.

Can use standard technique, for example by immunity or by using phage display library to produce antibody.

Can use a peptide species or peptide to produce antibody by known technology.This antibody possibility can be specifically in conjunction with Kv9.2 albumen or homologue, fragment etc.

If go for polyclonal antibody, the available Mammals (for example mouse, rabbit, goat, horse etc.) that comprises the immunogenic composition immunoselection of Kv9.2 polypeptide or peptide.According to host type, can use various adjuvants to improve immune response.This adjuvant includes, but not limited to for example for example lysolecithin, poly alcohol, polyanion, peptide, oil emulsion, keyhole limpet hemocyanin and dinitrophenol(DNP) of aluminium hydroxide and surfactant of freund's adjuvant, mineral coagulant.BCG (bacille Calmette-Guerin vaccine Bacilli Calmette-Guerin) and Corynebacterium parvum are people's adjuvants of potentially useful, are used for the stimulating system defence and can use these adjuvants if give individuality that immunity is in harmonious proportion with the aminoacid sequence material of purifying.

Collect the serum of immune animal and handle according to known procedure.If the serum that comprises at the polyclonal antibody of the epi-position that can obtain from polypeptide contains at other antigenic antibody, so much clonal antibody can be used the immunoaffinity chromatography purifying.The technology of producing and processing polyclonal antiserum is known in the art.In order to prepare this antibody, we also provide Kv9.2 aminoacid sequence or its fragment with another aminoacid sequence haptenization (haptenise), as the intravital immunogen of animal or human.

Monoclonal antibody at the epi-position that can obtain from the Kv9.2 polypeptide also can be produced easily by the person skilled in the art.The general method for preparing monoclonal antibody by hybridoma is known.The clone of the production antibody of infinite multiplication can produce by cytogamy, also can use other technology, for example transforms bone-marrow-derived lymphocyte with carcinogenic dna direct, or uses the Epstein-Barr virus transfection.The various character of the serial monoclonal antibody of producing be can screen, isotype and epi-position affinity promptly screened at track epi-position (orbit epitope).

Can use any technology that provides by the continuous cell line production antibody molecule in cultivating to prepare monoclonal antibody.These technology include but not limited to, at first hybridoma technology, trisome knurl (trioma) technology, human B cell hybridoma technology (Kosbor etc. (1983) the Immunol Today 4:72 that is described by Koehler and Milstein (1975 Nature256:495-497); Cote etc. (1983) Proc Natl AcadSci 80:2026-2030) and EBV-hybridoma technology (Cole etc., Monoclonal Antibodies andCancer Therapy, 77-96, Alan R.Liss, Inc., 1985).

In addition, can use exploitation to be used for producing the technology of " chimeric antibody ", montage mouse antibodies gene is to human immunoglobulin gene, to obtain to have suitable antigen-specific and bioactive molecule (Morrison etc. (1984) Proc Natl Acad Sci 81:6851-6855; Neuberger etc. (1984) Nature312:604-608; Takeda etc. (1985) Nature 314:452-454).In addition, describe the technology (United States Patent (USP) 4,946,779) that is used for the manufacture order chain antibody and can be suitable for producing the material specific single-chain antibody.

Sensing specifically is used for diagnosis from the mono-clonal and the polyclonal antibody of the epi-position of Kv9.2 polypeptide and peptide acquisition, and neutral antibody is used for passive immunotherapy.Particularly, monoclonal antibody can be used for producing antiidiotypic antibody.Antiidiotypic antibody is an immunoglobulin (Ig), and it carries the material that needs protection and/or " internal image " of reagent.The technology that produces antiidiotypic antibody is known in the art.These antiidiotypic antibodys also can be used for treatment.

Also can be as (1989, Proc Natl Acad Sci 86:3833-3837) and WinterG and Milstein C (1991 such as Orlandi; Nature 349:293-299) disclosed, by producing in the body of induction of lymphocyte population or producing antibody by the binding reagents of screening recombination immunoglobulin library or serial high degree of specificity.

Also can produce the antibody fragment of the specific binding site that comprises polypeptide or peptide.For example, this fragment includes but not limited to, the F that can produce by the pepsin digested antibody molecule (ab ') ₂Fragment and by reducing F (ab ') ₂The Fab fragment that segmental disulfide linkage produces.In addition, can make up the Fab expression library, make it possible to fast and differentiate easily have desire the mono-clonal Fab fragment (1989) Science 256:1275-1281 such as () Huse WD of characteristic.

The technology of manufacture order chain antibody (United States Patent (USP) 4,946,778) also is suitable for producing the single-chain antibody of Kv9.2 polypeptide.And, transgenic mice or comprise that other mammiferous other organism can be used for expressing humanized antibody.

Can use the clone of above-mentioned antibody separation or discriminating express polypeptide or pass through the affinitive layer purification polypeptide.

The antibody of Kv9.2 subunit polypeptide also can be used for regulating blood-glucose treatment disease, comprise, I type and type ii diabetes, hyperinsulinemia, hunger disease, insulin resistance, diabetic complication comprises the vascular disease that diabetes are relevant, the ephrosis neuropathy relevant that diabetes are relevant with diabetes, and treatment hypoglycemia, (they are HDL to high fat, LDL or VLDL) mass formed by blood stasis, unusual lipidemia, no matter its cause is to be primary in or to be secondary to diabetes, hyperthyroidism/go down, acromegaly, liver failure, renal failure, Vipoma, pancreatitis and alcohol inductive hypoglycemia.

Diagnositc analysis

We have further described Kv9.2 subunit polynucleotide and polypeptide (and homologue, variant and derivative) and have been used for as diagnostic reagent diagnosing or the purposes of genetic analysis.In such analysis, also can use complementation maybe can hybridize the nucleic acid of Kv9.2 subunit nucleic acid (comprising homologue, variant and derivative) and the antibody of Kv9.2 polypeptide.

The mutant that detects the Kv9.2 subunit gene relevant with dysfunction will provide a kind of diagnostic method, and this method can increase or describe in detail because the deficiency expression of Kv9.2 subunit, overexpression or change expressed the disease that causes or the diagnosis of disease susceptibility.Can on dna level, detect the individuality that carries Kv9.2 subunit gene (comprising control sequence) sudden change by various technology.

For example, can be from patient's DNA isolation, and the dna polymorphism pattern of definite Kv9.2.With the pattern differentiated with known suffer from excessive-, the patient of deficiency or unconventionality expression Kv9.2 relative disease contrasts and compares.Can differentiate the patient who expresses the genetic polymorphism sexual norm relevant then with relative disease.The genetic analysis of Kv9.2 subunit gene can be undertaken by any technology known in the art.For example, can be by determining the allelic dna sequence dna of Kv9.2, by screening individualities such as RFLP or snp analysis.Can by the existence that detects the Kv9.2 gene order or control dna polymorphism in any sequence of its expression differentiate the patient have with excessive-, the genetic predisposition of disease that deficiency or unconventionality expression Kv9.2 are relevant.

Then, the patient that treatment is differentiated like this is with the generation of prevention Kv9.2 relative disease, and perhaps Kv9.2 is in the treatment more strongly in early days of relative disease, with prophylactic further generation or development.The Kv9.2 relative disease comprises that I type and type ii diabetes, hyperinsulinemia, hunger disease, insulin resistance, diabetic complication comprise the relevant vascular disease of diabetes, the ephrosis neuropathy relevant with diabetes that diabetes are relevant, and treat hypoglycemia, high fat (they are HDL, LDL or VLDL) mass formed by blood stasis, unusual lipidemia, no matter its cause is to be primary in or to be secondary to diabetes, hyperthyroidism/go down, acromegaly, liver failure, renal failure, Vipoma, pancreatitis and alcohol inductive hypoglycemia.Also be used for the treatment of sacred disease, comprise listed those anxiety disorders and depression among stress reaction obstacle after anxiety disorder, anxiety disorder, anxiety related behavior and generalized anxiety disorder sexual dysfunction, panic disorder, agoraphobia, social phobia, obsessive compulsive disorder, the wound, gross stress reaction obstacle and the DSM-IV.

We further disclose the test kit of differentiating the patient genetic polymorphism sexual norm relevant with the Kv9.2 relative disease.This test kit comprises DNA sample collection means and measures the means of genetic polymorphism sexual norm, then polymorphism pattern is compared with control sample to determine the susceptibility of patient to the Kv9.2 relative disease.The test kit of diagnosis Kv9.2 relative disease also is provided, and this test kit comprises the antibody (or its fragment) of Kv9.2 polypeptide and/or this peptide species.

Diagnostic nucleic acid can obtain from subject's cell, for example from blood, urine, saliva, organize vivisection or necrotomy material to obtain.In a preferred embodiment, from blood collecting being obtained DNA at the hemocyte that thieving paper obtains by puncturing patient's finger.In another preferred embodiment, blood is at AmpliCard. ^TMLast collection (University of Sheffield, Departmentof Medicine and Pharmacology, Royal Hallamshire Hospital, Sheffield, EnglandS10 2JF).

DNA can be directly used in detection, can use PCR or other amplification technique enzymatic amplification before perhaps analyzing.Can prepare the oligonucleotide DNA primer that points to specificity multiformity DNA zone in the interested gene, in the PCR reaction, obtain the amplification of target sequence like this.RNA or cDNA also can be used as template in a similar fashion.Use the dna sequence dna of restriction enzyme analysis then,, and therefore provide patient's genetic polymorphism distribution plan with the genetic polymorphism of determining in the sequence of amplification, to exist from template DNA amplification.Limited fragment length can be identified by gel analysis.In addition, perhaps simultaneously can use for example SNP (single nucleotide polymorphism) analytical technology.

Disappearance and insertion can detect by the change with normal genotype comparison amplified production size.By the DNA of amplification and the Kv9.2 subunit nucleotide sequence hybridization differential point of mark are suddenlyd change.Can come by the RNA enzymic digestion or by the duplex difference that the difference of melting temperature(Tm) will be mated good sequence and mispairing.Also can by exist at denaturing agent or non-existent situation under the change of dna fragmentation electrophoretic mobility in gel or the difference by direct dna sequencing detection dna sequence dna.Referring to, Myers etc. for example, Science (1985) 230:1242.Sequence also can disclose by RNase protection analysis in the change of specific position, for example RNA enzyme and S1 protection or chemical chop method.Referring to Cotton etc., Proc NatlAcad Sci USA (1985) 85:4397-4401.In another embodiment, can make up and comprise Kv9.2 subunit nucleotide sequence or its segmental oligonucleotide probe array, effectively screen for example genetic mutation.The array technique method is known and has general suitability, can be used for answering the variety of issue that proposes in molecular genetics, comprises genetic expression, genetic linkage and hereditary variability.(referring to for example: M.Chee etc., Science, 274 volumes, 610-613 (1996)).

Single strand conformation polymorphism (SSCP) can be used for detecting the difference of electrophoretic mobility between mutant and the wild-type nucleic acid, and (Orita etc. (1989) Proc Natl.Acad.Sci USA:86:2766 is also referring to Cotton (1993) Mutat Res 285:125-144; And Hayashi (1992) Genet Anal Tech Appl9:73-79).Can and contrast the single stranded DNA fragment sex change of nucleic acid and make it renaturation sample.The secondary structure of single-chain nucleic acid changes according to sequence, even the change of the electrophoretic mobility of acquisition makes it possible to detect the change of a base.But labeled dna fragment also detects with label probe.The susceptibility of measuring can use RNA (rather than DNA) to strengthen, and the RNA secondary structure is responsive more for the change of sequence.In a preferred embodiment, subject's method utilizes heteroduple analysis to separate double-stranded heteroduplex molecule (Keen etc. (1991) Trends Genet 7:5) with the basis of changing into of electrophoretic mobility.

Detect the sudden change of Kv9.2 subunit gene by the method for use describing, diagnositc analysis provides diagnosis or has measured the disease method of the susceptibility of anxiety or diabetes for example.

Can in sample, detect the existence of Kv9.2 subunit polypeptide and nucleic acid.Therefore, infection listed above and disease can be diagnosed by the following method, and this method comprises from sample determination Kv9.2 subunit polypeptide or the unusual level that reduces or improve of Kv9.2 Subunit mRNA derived from the subject.Sample can comprise from so organic cell or tissue sample, and this organism suffers from or suspects and suffers from and improve, reduce or the relevant disease of unconventionality expression Kv9.2 subunit (comprise expression level or pattern in the space or temporal change) otherwise.As a kind of method that diagnoses the illness, can will suffer from or suspect that the expression level of Kv9.2 in the organism of suffering from this disease or expression level or the pattern in pattern and the normal organism compare effectively.

Therefore, substantially, we disclose a kind of by sample and at least a nucleic acid probe that is specific to described nucleic acid being contacted and measure the existence of described sample amplifying nucleic acid, comprise the method for the nucleic acid existence of Kv9.2 subunit nucleic acid in the test sample.For example, this nucleic acid probe can combine specifically with Kv9.2 subunit nucleic acid or its part, and detects two combinations between the nucleic acid; Also can detect the existence of mixture itself.In addition, we disclose a kind of by with cell sample and the existence that can contact and monitor polypeptide in the described sample in conjunction with the antibody of polypeptide, detect the method for the existence of Kv9.2 subunit polypeptide.This can finish easily by the existence of measuring the mixture that forms between antibody and the polypeptide or the combination of measuring between polypeptide and the antibody.Detect that the bonded method is known in the art between two bodies, comprise FRET (FRET (fluorescence resonance energy transfer)), the resonance of superficial cell matrix etc.

Can use the method for any quantitative polynucleotide known in the art to measure reduction or the raising of expressing on the rna level, for example PCR, RT-PCR, RNA enzyme protection, RNA trace and other hybridizing method.Can be used for measuring in host's sample protein for example the analytical technology of Kv9.2 subunit level be that the person skilled in the art is known.This analytical procedure comprises that radioimmunoassay, competition are in conjunction with mensuration, western blot analysis and ELISA mensuration.

The disclosure also relates to and is used to diagnose the illness and to disease (comprising infection), for example anxiety or diabetes, the test kit of susceptibility.This diagnostic kit comprises Kv9.2 subunit polynucleotide or its fragment; Complementary nucleotide sequence; Kv9.2 subunit polypeptide or its fragment, or the antibody of Kv9.2 subunit polypeptide.

Karyomit(e) is measured

The Kv9.2 nucleotide sequence also is valuable for chromosomal discriminating.This sequence-specific ground target also can be hybridized with the particular location on the individual human chromosome.As indicated above, finder Kv9.2 subunit is positioned homo sapiens chromosome 8q22.

It is the important the first step in the process that those sequences and gene-correlation disease are connected that correlated series is positioned karyomit(e).In case sequence is positioned accurate chromosome position, the physical location of sequence can be relevant with the gene mapping data on the karyomit(e).This data are present in for example V.McKusick, human Mendelian inheritance (by the online acquisition of Johns Hopkins University Welch MedicalLibrary).Then by the linkage analysis identified gene and be positioned dependency (the common heredity of physically adjoining gene) between the disease of same chromosomal region.

Attacked and not affected individuality between the difference of cDNA or genome sequence also can be determined.If observe sudden change in some or all in the individuality of being attacked, and do not observe sudden change in any normal individual, sudden change may be the reason that causes disease so.

Prevention and methods of treatment

We provide the method for the excessive and not enough relevant disease of treatment and Kv9.2 subunit activity.

If Kv9.2 subunit activity is excessive, can utilize following several method.A kind of method comprises the medicine acceptable carrier that gives subject's inhibitor compound described above (antagonist) and significant quantity, by the combining or suppress to activate of block ligand and Kv9.2 subunit, thereby alleviate unusual situation by suppressing second signal.

In another approach, give the Kv9.2 subunit polypeptide of soluble form, it still can compete binding partner with endogenous Kv9.2 subunit.This rival's typical embodiment comprises the fragment of Kv9.2 subunit polypeptide.

In another approach, the encode expression of gene of endogenous Kv9.2 subunit can be used and expresses interrupter technique and suppress.Known this technology comprises the use antisense sequences, itself or inner that produce or give respectively.Referring to for example, O ' Connor, J Neurochem (1991) 56:560, oligodeoxynucleotide (Oligodeoxvnucleotides as AntisenseInhibitors of Gene Expression) as the genetic expression antisense inhibitor, CRC Press, Boca Raton, Fla. (1988).In addition, can provide the oligonucleotide that forms triple helix with gene.Referring to for example, Lee etc., Nucleic AcidsRes (1979) 6:3073; Cooney etc., Science (1988) 241:456; Dervan etc., Science (1991) 251:1360.Can directly give these oligomer or the relevant oligomer of expression in vivo.

In order to treat and Kv9.2 subunit and the not enough diseases associated of activity expression thereof, also can use following several method.A kind of method comprises the compound of the treatment significant quantity that gives the ionic channel that subject activation comprises Kv9.2, and agonist promptly mentioned above or opener combine with the medicine acceptable carrier, thereby alleviate unusual situation.In addition, can use gene therapy to pass through the endogenous production that the intravital relevant cell of subject influences the Kv9.2 subunit.For example, as indicated above, the Kv9.2 polypeptide can be carried out engineered being used for and express at the replication defect type retrovirus vector.This retrovirus expression constructs can be separated and introduce the packing cell with the retrovirus plasmid transduction of the RNA that comprises encoded K v9.2 polypeptide then, packing cell has just produced the infectious viral particle that comprises interested gene like this.These production cells can give the subject and be used for engineered cell and expression in vivo polypeptide in the body.About the summary of gene therapy referring to Human Molecular Genetics, T Strachan and APRead, the 20th chapter among the BIOS Scientific Publishers Ltd (1996), the methods of treatment (Gene Therapy and other Molecular Genetic-basedTherapeutic Approaches) (this paper quotes as a reference) on gene therapy and other molecular genetic basis.

Preparation and administration

Peptide, for example Kv9.2 subunit polypeptide of soluble form, and agonist and antagonist peptide and small-molecular peptides can combine preparation with suitable pharmaceutical carrier.This preparation comprises the polypeptide or the compound for the treatment of significant quantity, and medicine acceptable carrier or vehicle.This carrier includes but not limited to, salt solution, buffer saline, glucose, water, glycerine, ethanol and their combination.Preparation should adapt with the mode of giving, and is well known.We further disclose pharmaceutical pack and test kit, and they comprise one or more containers of filling with one or more compositions in the above-mentioned composition.

Kv9.2 polypeptide and other compound can be used in combination separately or with other compound, for example therapeutic compound.

System gives the preferred mode of pharmaceutical composition and comprises injection, typically by intravenous injection.Can use other injecting pathway, for example subcutaneous, muscle or peritoneal injection.Other method that is administered systemically comprises uses permeate agent for example biliary salts or fusidinic acid or other washing composition is striden mucous membrane and through percutaneous drug delivery.In addition, if suitably be mixed with intestines or capsule preparations, so also can be oral.These compounds also can ointment, the form of patch, gel etc. is local and/or the location gives.

The dosage range that needs depends on selection, route of administration, the characteristic of preparation, the characteristic of subject's illness and doctor in charge's the judgement of peptide.Yet suitable dosage is in 0.1-100 μ g/kg experimenter's scope.But, consider the diversity of spendable compound and the different efficacies of various route of administration, the significantly variation of required dosage can reckon with.For example, oral administration is than needing higher dosage by intravenous administration.As known in the art, the variation of these dosage levels can use standard experience program to adjust to optimization.

Usually to be known as the form of therapy of above-described " gene therapy ", polypeptide that also can endogenous generation is used for the treatment of in subject's body.Therefore, for example, from subject's cell can be for example by using the retrovirus plasmid vector, for example DNA or RNA carry out genetic engineering modified with coded polypeptide in the body of earlier external back with polynucleotide.Then these cells are introduced the subject.

Pharmaceutical composition

We also provide a kind of pharmaceutical composition, and this pharmaceutical composition comprises Kv9.2 polypeptide, polynucleotide, peptide, carrier or antibody and randomly pharmaceutically acceptable carrier, thinner or the vehicle (comprising their combination) of the treatment significant quantity that gives.

This pharmaceutical composition can be used for the human or animal in people and veterinary drug, and typically comprises any or multiple medicine acceptable diluent, carrier or vehicle.The acceptable carrier or the thinner that are used for the treatment of purposes are that pharmaceutical field is known, and are described in for example Remington ' sPharmaceutical Sciences, Mack Publishing Co. (A.R.Gennaro edits, 1985).The selection of pharmaceutical carriers, vehicle or thinner can be selected according to route of administration of being desired and standard pharmaceutical practice.This pharmaceutical composition can comprise as carrier, vehicle or thinner, or in addition, any suitable adhesive, lubricant, suspension agent, coating-forming agent, solubilizing agent.

Sanitas, stablizer, dyestuff even seasonings are also contained in the pharmaceutical composition.Examples of preservatives comprises Sodium Benzoate, Sorbic Acid and p-Hydroxybenzoate.Also can use antioxidant and suspension agent.

Different compositions/preparation demand depends on different transfer systems.For example, prepare pharmaceutical composition described herein to use the minipump transmission, or pass through mucosal route, for example as nasal spray or aerosol that is used to suck or ingestible solution, or at administered parenterally, wherein composition is prepared with injectable form, is used for by for example vein, muscle or subcutaneous route transmission.In addition, can design the preparation that transmits with above two kinds of approach.

When medicament transmitted by gastrointestinal mucosa, medicament should be able to keep stable in the process that shifts by gi tract; For example it should be able to resist the proteolysis enzyme liberating, and is stable and resist biliary washing composition effect under acid pH.

Suitably, pharmaceutical composition can give by suction, form with suppository or vaginal suppository gives, form with washing lotion, solution, ointment, ointment or isolation pulvis gives partly, pass through skin patch, with the tablet form that contains starch for example or lactose vehicle or with separately or with the capsule of mixed with excipients or ovulum or oral with the form of the elixir, solution or the suspension that comprise seasonings or tinting material, perhaps they can give through parenteral injection, for example vein, muscle or subcutaneous injection.Give for parenteral, composition preferably uses with the form of aseptic aqueous solution, and this aqueous solution can comprise other material, for example enough salt or monose so that this solution and blood etc. ooze.Giving tablet or lozenge form that composition can prepare by conventional methods for oral cavity or hypogloeeis gives.

Vaccine

Another embodiment relates to the method for induction of immunity reaction in mammalian body; described method comprises with Kv9.2 subunit polypeptide or its fragment seeded with mammalian; be enough to produce antibody and/or t cell immune response; to protect described animal to avoid unusual glucose level as the result of following disease; these diseases include but not limited to; I type and type ii diabetes; hyperinsulinemia; hunger disease; insulin resistance; diabetic complication comprises the vascular disease that diabetes are relevant; the ephrosis neuropathy relevant that diabetes are relevant, and treatment hypoglycemia with diabetes; (they are HDL to high fat; LDL or VLDL) mass formed by blood stasis; unusual lipidemia; no matter its cause is to be primary in or to be secondary to diabetes; hyperthyroidism/go down; acromegaly; liver failure; renal failure; Vipoma; pancreatitis and alcohol inductive hypoglycemia.

Another embodiment relates to the method for induction of immunity reaction in mammalian body; described method comprises in order to induce this immune response to produce the antibody that the described animal of protection avoids disease, by instructing the carrier transfer Kv9.2 polypeptide that Kv9.2 subunit polynucleotide are expressed in the body.

Another embodiment relates to a kind of immunity/vaccine preparation (composition), when being introduced into mammalian hosts, induces the immune response to the Kv9.2 polypeptide in this Mammals, and wherein said composition comprises Kv9.2 polypeptide or Kv9.2 gene.Vaccine preparation can further comprise appropriate carriers.

Because the Kv9.2 polypeptide can be decomposed under one's belt, it preferably gives (comprising injections such as subcutaneous, intramuscular, intravenously, intracutaneous) by parenteral.Be suitable for preparation that parenteral gives comprise water that become with non-water aseptic parenteral solution, it can comprise antioxidant, buffer reagent, fungistat and cause preparation and the isoosmotic solution of recipient's blood, and can comprise that the water of suspension agent or intensifier becomes and the aseptic suspension agent of non-water.Said preparation may reside in the container of unitary dose or multiple doses, and for example Mi Feng ampoule and bottle, and can be housed under the lyophilisation condition are only needing to add sterile liquid carrier before being about to use.Vaccine preparation also can comprise and be used to strengthen the immunogenic adjuvant system of preparation, for example oil-in-water system and other system known in the art.Dosage depends on the activity specific of vaccine, and can easily measure by routine test.

Can prepare vaccine from one or more Kv9.2 polypeptide or peptide.

Comprising immunogenic polypeptide or peptide is that the person skilled in the art is known as the preparation of the vaccine of activeconstituents.Typically, this vaccine production being become injectable, perhaps is liquor or for suspension agent; The solid form that is suitable for before injection dissolving or is suspended in the liquid also can prepare.Also can be with preparation emulsification, perhaps the albumen bag is by in liposome.Has usually acceptable and the mixed with excipients that can be compatible of active immunogenic components with activeconstituents with medicine.Appropriate excipients is for example water, salt, glucose, glycerine, ethanol etc. and their combination.

In addition, if desired, vaccine can comprise a spot of auxiliary substance, for example wetting or emulsifying agent, pH buffer reagent and/or strengthen the adjuvant of vaccine validity.Effectively the example of adjuvant includes but not limited to: aluminium hydroxide; N-ethanoyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP); N-ethanoyl-fall-muramyl-(CGP 11637 for L-alanyl-D-isoglutamine; be known as fall-MDP); N-ethanoyl-muramyl-L-alanyl-D-isoglutamine acyl-L-L-Ala-2-(1 '-2 '-two palmityls-sn-glycerine-3-hydroxyl phosphoryl oxygen)-ethamine (CGP 19835A; be known as MTP-PE); and RIBI; it comprises three kinds of compositions that extract from bacterium, the monophosphoryl lipid A in squalene/Tween 80 emulsions of 2%; trehalose dimycolate and cell wall skeleton (MPL+TDM+CWS).

Other example of adjuvant and other reagent comprises aluminium hydroxide, aluminum phosphate, potassium aluminium sulfate (alum), beryllium sulfate, silicon, kaolin, carbon, water-in-oil emulsion, oil-in-water emulsion, Muramyl dipeptide, bacterial endotoxin, lipid X, Corynebacterium parvum (Propionobacterium acnes), Bordetella pertussis (Bordetella pertussis), polyribonucleotide, sodiun alginate, lanolin, lysolecithin, vitamin A, Saponin/TSM, liposome, LEVAMISOLE HCL, the DEAE-dextran, segmented copolymer or other synthetic adjuvant.This adjuvant can be commercial available from various sources, for example the Merck adjuvant 65 (Merck and Company, Inc., Rahway, N.J.) or Freund's incomplete adjuvant and Freund's complete adjuvant (Difco Laboratories, Detroit, Michigan).

Typically, the adjuvant of the use mixture of Amphigen (oil-in-water), Alhydrogel (aluminium hydroxide) or Amphigen and Alhydrogel for example.The only aluminium hydroxide that is used for the people through permission.

The ratio of immunogen and adjuvant can change in wide in range scope, as long as the two all exists with effective amount.For example, aluminium hydroxide can vaccine mixture (Al ₂O ₃The basis) about 5% amount exist.Expediently, the immunogenic final concentration scope that the preparation vaccine makes it to comprise is 0.2-200 μ g/ml, preferably 5-50 μ g/ml, most preferably 15 μ g/ml.

After the preparation, the sterile chamber of vaccine can being packed into, sealing and preserving at low temperatures then, for example under 4 ℃, maybe can be with its freeze-drying.Lyophilization allows with stable form prolonged preservation.

This vaccine gives through parenteral easily by for example subcutaneous or intramuscular injection.The other preparation that is suitable for the alternate manner administration comprise suppository and, in some cases, oral preparations.For suppository, conventional tackiness agent and carrier for example can comprise polyalkylene glycol or triglyceride level; This suppository can be made up of the mixture that comprises activeconstituents, and the scope of this activeconstituents is 0.5%-10%, preferably 1%-2%.Oral preparations comprises the vehicle of normal use, for example pharmaceutical grade N.F,USP MANNITOL, lactose, starch, Magnesium Stearate, soluble saccharin, Mierocrystalline cellulose, magnesiumcarbonate etc.These compositions are taked the form of solution, suspension agent, tablet, pill, capsule, sustained release preparation or pulvis, and comprise the activeconstituents of 10%-95%, preferably 25%-70%.Under the freeze dried situation of vaccine composition, can be before the administration with freeze dried material reconstruct, for example as suspension agent.Reconstruct is preferably carried out in damping fluid.

The capsule, tablet and the pill that are used for the orally give patient can be supplied to the intestines bag, the involved for example Eudragit of this intestines bag " S ", Eudragit " L ", cellulose acetate, cellulose acetate phthalate or Vltra tears.

The Kv9.2 polypeptide can be mixed with the vaccine of neutralization or salt form.The acceptable salt of medicine comprises acid salt (forming with the free amine group of peptide), and it is to form with for example mineral acid such as hydrochloric acid or phosphoric acid, perhaps forms with organic acids such as for example acetate, oxalic acid, tartrate and toxilic acids.The salt that forms with free carboxy also can be derived from mineral alkali, for example sodium, potassium, ammonium, calcium or ironic hydroxide, and organic bases is Isopropylamine, Trimethylamine 99,2-ethylaminoethyl alcohol, Histidine and PROCAINE HCL, PHARMA GRADE for example.

Administration

Typically, the doctor will determine to be suitable for most the actual dose of subject's individuality, and this dosage will change along with age, body weight and concrete reaction.Following dosage is general case exemplary.Certainly, can have and use higher or than the discrete situation of low dosage scope.

Medicine and vaccine composition can give by direct injection.But compositions formulated is used for parenteral, mucous membrane, muscle, intravenously, subcutaneous, intraocular or gives through skin.Typically, can the 0.01-30mg/kg body weight, preferably 0.1-10mg/kg, more preferably the dosage of 0.1-1mg/kg body weight gives each protein.

Term " gives " to comprise by virus or non-virus technology transmission.Viral mechanism includes but not limited to adenovirus carrier, adeno associated virus (AAV) carrier, herpesvirus vector, retrovirus vector, lentiviral vectors and baculovirus vector.Non-viral mechanism comprises transfection, liposome, immunoliposome, fat transfection agents, cationic surface amphipath (CFAs) and their combination of lipid mediation.The approach of this pass through mechanism includes but not limited to mucous membrane, nose, mouth, parenteral, gi tract, part or hypogloeeis approach.

Term " gives " to include but not limited to by the mucosal route transmission, for example, and as the nasal spray that is used to suck or aerosol or as ingestible solution; Parenteral route is wherein transmitted by injectable form and is undertaken, for example, and by vein, intramuscular or subcutaneous route.

Term " gives altogether " to be meant and gives each for example material for example site and time of adjuvant of Kv9.2 polypeptide and interpolation, makes immunity system obtain necessary adjusting.Therefore, although polypeptide and adjuvant can give at synchronization and in same site sometimes, it is favourable may giving polypeptide in the time different with adjuvant and different sites.Polypeptide and adjuvant even can be with identical transmission carrier transfer, and polypeptide and antigen can coupling and/or non-couplings, and/or hereditary coupling and/or non-coupling.

Kv9.2 polypeptide, polynucleotide, peptide, Nucleotide, antibody and randomly adjuvant can be used as single dose or give or give altogether the host subject respectively with multiple doses.

Vaccine composition can give by multiple different approach with pharmaceutical composition, for example in injection (it comprises parenteral, subcutaneous and intramuscular injection), the nose, mucous membrane, mouth, intravaginal, urethra or give through eye.

Vaccine and pharmaceutical composition can give by for example conventional parenteral of subcutaneous or intramuscular injection.Be suitable for other preparation that alternate manner gives comprise suppository and, in some cases, oral preparations.For suppository, can comprise conventional tackiness agent and carrier, for example polyalkylene glycol or triglyceride level; This suppository can be made up of the mixture that comprises activeconstituents, and the scope of this activeconstituents is 0.5%-10%, can be 1%-2%.Oral preparations comprises the vehicle of normal use, for example pharmaceutical grade N.F,USP MANNITOL, lactose, starch, Magnesium Stearate, soluble saccharin, Mierocrystalline cellulose, magnesiumcarbonate etc.These compositions are taked the form of solution, suspension agent, tablet, pill, capsule, sustained release preparation or pulvis, and comprise the activeconstituents of 10%-95%, preferably 25%-70%.Under the freeze dried situation of vaccine composition, can be before the administration with freeze dried material reconstruct, for example as suspension agent.Reconstruct is preferably carried out in damping fluid.

Others

Now the paragraph of following column number is stated other aspect and embodiment of the present invention; Be to be understood that and the present invention includes these aspects:

1. 1 kinds of Kv9.2 polypeptide of paragraph, described Kv9.2 polypeptide comprise the aminoacid sequence of SEQ ID NO:3 or SEQ IDNO:5 demonstration, or its homologue, variant or derivative.

2. 1 kinds of nucleic acid of paragraph, described nucleic acid encoding is according to paragraph 1 described polypeptide.

3. 1 kinds in paragraph is according to paragraph 2 described nucleic acid, and described nucleic acid comprises the nucleotide sequence that SEQ ID NO:1, SEQID NO:2 or SEQ ID NO:4 show, or its homologue, variant or derivative.

Paragraph 4. 1 peptide species, described polypeptide comprises the fragment according to paragraph 1 described polypeptide.

5. 1 kinds in paragraph is according to paragraph 3 described polypeptide, described polypeptide is included in the one or more zones of homologous between SEQ ID NO:3 and the SEQ ID NO:5, and perhaps described polypeptide is included in allogenic one or more zones between SEQ IDNO:3 and the SEQ ID NO:5.

6. 1 kinds of codings of paragraph are according to the nucleic acid of paragraph 4 or 5 described polypeptide.

7. 1 kinds of carriers that comprise according to paragraph 2,3 or 6 described nucleic acid of paragraph.

8. 1 kinds in paragraph comprises according to paragraph 2,3 or 6 described nucleic acid or according to the host cell of paragraph 7 described carriers.

9. 1 kinds of transgenic nonhuman animals of paragraph, described animal comprise according to paragraph 2,3 or 6 described nucleic acid or according to paragraph 7 described carriers.

10. 1 kinds in paragraph is according to paragraph 9 described transgenic nonhuman animals, and described animal is a mouse.

Paragraph 11. differentiate can with the method for the interactional compound of Kv9.2 subunit specificity in, according to the purposes of paragraph 1,4 or 5 described polypeptide.

Paragraph 12. differentiate can with the method for the interactional compound of Kv9.2 subunit specificity in, according to the purposes of paragraph 9 or 10 described transgenic nonhuman animals.

13. 1 kinds of discriminatings of paragraph comprise the method for the ionic channel antagonist of Kv9.2, and described method comprises that the cell with expressing K v9.2 contacts with candidate compound, and whether the kinetics and the conductivity of mensuration passage change.

The level of conduction of paragraph 14. 1 kinds of discriminatings can raising passage or the method for regulating the dynamic (dynamical) compound of electric current of passage, described method comprise that the cell that expression is comprised the ionic channel of Kv9.2 contacts with candidate compound.

15. 1 kinds of discriminatings of paragraph can with the method for Kv9.2 polypeptide bonded compound, described method comprises the Kv9.2 polypeptide is contacted with candidate compound, and whether definite candidate compound combines with the Kv9.2 polypeptide.

16. 1 kinds of compounds of paragraph by differentiating according to the described method of any one paragraph among the paragraph 11-15.

17. 1 kinds in paragraph can with paragraph 1,4 or 5 described polypeptid specificity bonded compounds.

Paragraph 18. is in producing the method for antibody, according to paragraph 1,4 or 5 described polypeptide or its part, perhaps according to the purposes of the nucleic acid of paragraph 2,3 or 6.

19. 1 kinds of antibody of paragraph, its can with combine according to paragraph 1,4 or 5 described polypeptide or its part or by polypeptide or its part specificity according to paragraph 2,3 or 6 described nucleic acid encodings.

20. 1 kinds of pharmaceutical compositions of paragraph, said composition comprises any or multiple following material and pharmaceutically acceptable carrier or thinner: according to polypeptide or its part of paragraph 1,4 or 5; Nucleic acid or its part according to paragraph 2,3 or 6; Carrier according to paragraph 7; Cell according to paragraph 8; Compound according to paragraph 16 or 17; With antibody according to paragraph 19.

21. 1 kinds of vaccine compositions of paragraph, said composition comprises any or multiple following material: according to paragraph 1,4 or 5 described polypeptide or its part; Nucleic acid or its part according to paragraph 2,3 or 6; According to paragraph 7 described carriers; Cell according to paragraph 8; Compound according to paragraph 16 or 17; With antibody according to paragraph 19.

22. 1 kinds of diagnostic kits that are used for disease or disease susceptibility of paragraph, described test kit comprises any or multiple following material: according to polypeptide or its part of paragraph 1,4 or 5; Nucleic acid or its part according to paragraph 2,3 or 6; Carrier according to paragraph 7; Cell according to paragraph 8; Compound according to paragraph 16 or 17; With antibody according to paragraph 19.

23. 1 kinds of methods for the treatment of the patient of paragraph, described patient suffers from the ion channel activity that contains Kv9.2 and strengthens diseases associated, and described method comprises and gives the antagonist that the patient is contained the ionic channel of Kv9.2.

24. 1 kinds of methods for the treatment of the patient of paragraph, described patient suffers from the ion channel activity that contains Kv9.2 and reduces diseases associated, and described method comprises and gives the agonist that the patient is contained the ionic channel of Kv9.2.

25. 1 kinds in paragraph is according to paragraph 23 or 24 described methods, and wherein the Kv9.2 subunit comprises and has the polypeptide of sequence that SEQ ID NO:3 or SEQ ID NO:5 show.

26. 1 kinds of methods that treat and/or prevent disease in the patient of paragraph, described method comprise the step that gives any or multiple following material of patient: according to polypeptide or its part of paragraph 1,4 or 5; Nucleic acid or its part according to paragraph 2,3 or 6; Carrier according to paragraph 7; Cell according to paragraph 8; Compound according to paragraph 16 or 17; With antibody according to paragraph 19; Pharmaceutical composition according to paragraph 20; With vaccine according to paragraph 20.

27. 1 kinds of reagent of paragraph, described reagent comprise according to the polypeptide of paragraph 1,4 or 5 or its part; Nucleic acid or its part according to paragraph 2,3 or 6; Carrier according to paragraph 7; Cell according to paragraph 8; Compound according to paragraph 16 or 17; And/or according to the antibody of paragraph 19, described reagent is used for the treatment of or prophylactic method.

Paragraph 28. is according to polypeptide or its part of paragraph 1,4 or 5; Nucleic acid or its part according to paragraph 2,3 or 6; Carrier according to paragraph 7; Cell according to paragraph 8; Compound according to paragraph 16 or 17; And/or according to the antibody of paragraph 19 preparation be used for the treatment of or prophylactic pharmaceutical composition in purposes.

29. 1 kinds of non-human transgenic animals of paragraph is characterized by the Kv9.2 gene that described transgenic animal comprise change.

30. 1 kinds in paragraph is according to paragraph 29 described non-human transgenic animals, wherein change and be selected from the group of forming by following: Kv9.2 disappearance, cause the Kv9.2 sudden change of afunction, the foreign gene that will have the nucleotide sequence of orientation or random mutation is introduced Kv9.2, to introduce Kv9.2 from the foreign gene of another species, or any combination of these situations.

Paragraph has the non-human transgenic animal that function is suffered the endogenous Kv9.2 gene of destructive for 31. 1 kinds, and wherein said transgenic animal comprise the genome of himself, and expresses the proteic transgenosis of coding allos Kv9.2.

32. 1 kinds of nucleic acid construct things that are used for functionally destroying the Kv9.2 gene of paragraph at host cell, described nucleic acid construct thing comprises: (a) non-homogeneous alternative part; (b) be positioned at the first homology zone of non-homogeneous alternative part upstream, this first homology zone has and the substantially the same nucleotide sequence of a Kv9.2 gene order; (c) be positioned at the second homology zone of non-homogeneous alternative portion downstream, this second homology zone has and the substantially the same nucleotide sequence of the 2nd Kv9.2 gene order, and the 2nd Kv9.2 gene order is positioned at the downstream of a Kv9.2 gene order of the endogenous Kv9.2 gene of natural generation.

33. 1 kinds of methods of producing the Kv9.2 polypeptide of paragraph, described method are included under the condition that the nucleic acid of encoded K v9.2 polypeptide expressed, and cultivate according to paragraph 8 described host cells.

According to the method for the existence of paragraph 2,3 or 6 described nucleic acid, described method comprises sample is contacted with at least a nucleic acid probe that is specific to described nucleic acid, and detects the existence of described sample amplifying nucleic acid in 34. 1 kinds of test sample of paragraph.

In 35. 1 kinds of test sample of paragraph according to the method for the existence of paragraph 1,4 or 5 described polypeptide, described method comprise with sample with contact according to paragraph 19 described antibody, and detect the existence of polypeptide in the described sample.

36. 1 kinds of diagnosis of paragraph by improve, reduce or otherwise the Kv9.2 subunit of unconventionality expression cause or associated disease or syndromic method that described method comprises the steps: that (a) is in expression level or the pattern of suffering from or suspects detection Kv9.2 subunit in the animal body of suffering from this disease; (b) comparing with expression levels or pattern and intact animal.

Embodiment

Embodiment 1. genetically modified Kv9.2 knock-out mices: make up Kv9.2 gene targeting carrier

Use the homology search of genome database to differentiate the Kv9.2 gene by information biology.Genome contig by various database assembling 226kb.This contig provides enough flanking sequence information, enters oriented carrier with the design homology arm cloning.

Mouse Kv9.2 gene has a coding exon, and the shaping-orientation strategy is to remove most encoding sequence.By the 3 ' homology arm of the 5 ' homology arm of the 1.7kb of the regional side that lacked and 4.0kb by pcr amplification, and fragment cloning entered oriented carrier.5 ' end of each Oligonucleolide primers of the synthetic arm that is used to increase, with the different recognition sites of the Restriction Enzyme that comprises rare restriction enzyme, the cloning site coupling of this site and carrier polylinker is not present in the of arm own.Under the situation of Kv9.2, design of primers is the primer of listing in the following table, has 5 ' the arm cloning site of AgeI/NotI and 3 ' the arm cloning site of AscI/FseI (structure of the oriented carrier of use comprises that relevant restriction enzyme site shows in Fig. 1).

Except that the arm primer to ((5 ' arm F/5 ' arm R) and (3 ' arm F/3 ' arm R)), design has specific other primer to the Kv9.2 locus and is used for following purpose: 5 ' and 3 ' probe primer is to (5 ' prF/5 ' prR and 3 ' prF/3 ' prR), with the fragment of increase each arm outside and the 150-300bp of two weak points that extend the non-duplicate genes group DNA outside each arm, to allow the locus of the guiding of southern blotting technique analysis in the independent directed cloning of inferring; When the multiplex PCR that is used to use the carrier specificity primer, in this case, when being Asc403, make the mouse genotype primer of distinguishing wild-type, heterozygote and the mouse of isozygotying to (hetF and hetR); At last, with the terminal upstream annealed guide selection primer (5 ' scr) of 5 ' arm region, when having specific primer pairing with 5 ' end to carrier (TK5IBLMNL), its produces the amplified production of guiding event-specific 2.0kb, is DR1 in this case.Desirable genome can take place and change only derived from the template DNA of cell in this amplified production in this cell, and makes it possible to identify from clone's background that the carrier that contains random integration copies the cell of correct guidance.The lead genome structure in Kv9.2 locus zone of strategy of the position of these primers and being used to shows with SEQ ID NO:19.

The position of selecting homology arm is functionally to destroy the Kv9.2 gene.The preparation oriented carrier, the Kv9.2 zone that wherein will be lacked replaces with non-homogeneous sequence, this non-homogeneous sequence comprises the native gene expression reporter gene (frame is the 1acZ gene independently) of selecting the box upstream, and this selection box comprises the promoted neomycin phosphotransferase gene (neo) with the equidirectional arrangement of Kv9.2 gene.

In case 5 ' and 3 ' homology arm is entered oriented carrier TK5IBLMNL (referring to Fig. 4) by the clone, use standard molecular biological technique to prepare a large amount of highly purified DNA preparations.20 μ g prepared fresh do not contain the restriction enzyme PmeI restriction enzyme digestion of endotoxic DNA with another kind of rare restriction enzyme (rare-cutting), this site is present in the unique site in the carrier main chain between the replication orgin of ampicillin resistance gene and bacterium.Precipitate linearizing DNA then and in the phosphate buffered saline of 100 μ l resuspension, prepare to be used for electroporation.

Behind the electroporation 24 hours, cells transfected was cultivated 9 days in the substratum that contains 200 μ g/ml Xin Meisus.Picking clone puts into 96 hole flat boards, uses PCR screening (using above-described primer 5 ' prF and DR1), the clone is duplicated before the clone that homologous recombination has taken place between endogenous Kv9.2 gene and guiding construction and expands to differentiate.Positive colony can 1-5% ratio differentiate.With these clone's expansions, make replica be frozen, prepare enough high-quality DNA, be used for southern blotting technique and confirm to use the outside 5 ' of preparation as indicated above and the guiding incident of 3 ' probe, all these uses standard method (Russ etc., Nature 2000 Mar 2; 404 (6773): 95-99).When with the southern blotting technique of the DNA of diagnostic limitations restriction endonuclease digestion during, with the existence confirmation of the ES cell clone of source orientation by mutant band and unaltered wild-type band with external probe hybridization.For example, when hybridizing, will produce the band of 6.2kb with the wild type gene group DNA of AflII digestion with 5 ' external probe, to produce the band of 7.0kb when hybridizing with 3 ' external probe, and the genomic dna that comprises the allelic similar digestion of leading will produce except that the wild-type band～17kb knock out the specificity band.

The mouse that embodiment 2. genetically modified Kv9.2 knock out: produce the Kv9.2GPCR deficient mice

Female and the male mice mating of C57BL/6 separates blastocyst in gestation in the time of 3.5 days.

Embodiment 2. transgenosis Kv9.2 reject mouse: the mouse that produces the Kv9.2GPCR defective

Female and the male mice mating of C57BL/6 separates blastocyst in gestation in the time of 3.5 days.Each blastocyst injects the clone's who takes from selection 10-12 cell, the uterus that 7-8 blastocyst implanted the female body of pseudopregnant F1.Produce brood and comprise the male chimeric little son of several high-caliber (reaching 100%) agouti (the agouti hair color shows the effect from guiding clone's cell).These male mosaics and female MF1 and 129 mouse mating are measured by the agouti hair color with by the pcr gene type respectively and are determined kind of the transmission of system.

The pcr gene type measure to use primer hetF and hetR and the 3rd, carrier specificity primer (Asc403) to carry out on the tail that dissolved is cut off.This multiplex PCR allows from wild type gene seat (if existence) from primer hetF and the band of the 241bp that the hetR amplification provides.Reject disappearance hetF site in the mouse, so this amplification just can't be carried out from the allelotrope that leads.Yet the hetR primer in the zone that the Asc403 primer will be interior with being annealed to lucky 3 ' arm combines from the band of guiding locus amplification 434bp.Therefore, to disclose little son's genotype as follows for this multiplex PCR: the wild-type sample shows the band of single 241bp; The hybrid DNA sample produces 241bp and two bands of 434bp; Homozygous sample only shows the band of the 434bp that guiding is special.

Table 1.Kv9.2 primer sequence

musKv9.2 5′pr F CTCTCAATTCAGGTGGCACCCTTAGAG-Seq ID No.6

musKv9.2 5′pr R CACAGAATTCCCAATCATAAGACATAG-Seq ID No.7

musKv9.2 5′scr DR1 CTCTCAATTCAGGTGGCACCCTTAGAG-Seq ID No.8

musKv9.2 5′arm F Age AaaaccggtATGTCCAGATCCTCATACATGGCACAC-Seq ID No.9

AaagcggccgcGACGTCGGTATCGGACACATCCCACAG-Seq ID No.

musKv9.2 5′arm R Not 10

musKv9.2 3′arm F Asc TttggcgcgccTTGCTGATCTGCTGCTTGTGGTTCTAG-Sea ID No.11

AaaggccggccAATGTAACCATCGCTTCTGTAACCCAG-Sea ID No.

musKv9.2 3′arm R Fsc 12

musKv9.2 3′pr F AGCAGAGCAGGTATGGCGTGGCATGTC-Seq ID No.13

musKv9.2 3′pr R CTGGGGGAGCTCTCGTGCTATGATGAG-Seq ID No.14

musKv9.2hetF CCCATTTCTATCGGCGCCAAAAGCAAC-Seq ID No.15

musKv9.2hetR a403 GTGCTAGAACCACAAGCAGCAGATCAG-Seq ID No.16

Asc403 CAGCCGAACTGTTCGCCAGGCTCAAGG-Sea ID No.17

DR1 CATGCCGCCTGCGCCCTATTGATCATG-Seq ID No.18

Embodiment 3. biological datas: gene expression pattern (people RT-PCR)

Tissue to the people uses RT-PCR, and expression of gene is shown in lung, brain, spleen, slightly less, is shown in prostate gland, liver, reproductive organ and muscle.

This shows in Fig. 2.

Embodiment 4. biological datas: gene expression pattern (the painted structure of LacZ)

LacZ dyeing

Carry out the Xgal dyeing of anatomical tissue as follows.

Make the representational tissue slice of big organ.Whole [and pipe are cut off, and fixing agent and staining agent can penetrate like this.Be organized in fully rinsing among the PBS (phosphate buffered saline), remove blood and intestinal contents.Tissue is put into fixing agent (contain 2% formaldehyde, 0.2% glutaraldehyde, 0.02%NP40,1mM MgCl ₂, the 0.23mM Sodium desoxycholate PBS) 30-45 minute.Subsequently, in PBS, clean 35 minutes, at 30 ℃ tissue is put into the Xgal dyeing solution and (contains 4mM yellow prussiate of potash, 4mM high-potassium ferricyanide, 2mM MgCl ₂, the PBS of 1mg/mlX-gal) and 18 hours.Tissue cleans 3 times with PBS, fixes 24 hours after in 4% formaldehyde, cleans once more with PBS before being stored in 70% the ethanol.

In order to differentiate the painted tissue of Xgal, with the tissue of cured encapsulation dehydration, cut out the section of 7 μ m, redye (9-10 minute) with 0.01% sarranine.

Using LacZ dyeing, find that Kv9.2 is expressed in brain, specifically is at cortex, hippocampus, hippocampal gyrus, pallidum (ventate pallidum), maincenter almond nuclear (central amydaloid nucleus) (CeL), thalamic nuclei and cortex.In addition, in heart, spleen, lung and testis, also see painted evidence.

Embodiment 5. biological datas: physiology/biochemical (blood sugar)

Read glucose readings from the blood sample of the animal tail vein of taking from overnight fasting (14-16 hour).Use blood glucose monitor to measure blood sugar.

Check the animal of Kv9.2 gene knockout and compare with the brood spouse (wild-type) who does not reject.The glucose level of the animal of rejecting is 3.24 ± 2.1mmol/l, and by comparison, the glucose level of wild-type animal is 4.69 ± 0.38mmol/l (P=0.006).Therefore, the animal of Kv9.2 rejecting is compared with the wild-type animal and shows that glucose level descends.

This results are shown among Fig. 3.

Embodiment 6. biological datas: behavioural analysis (wilderness test Open Field Test)

In the test of wilderness, detect and reject and the wild-type control mice.Referring to Carola, V., F.D ' Olimpio, etc., (2002) " be used for the inbrde mouse assessment behavior relevant with anxiety raise just-the evaluation that tests in labyrinth and wilderness ".

In brief, mouse is put in the middle of the transparent Perspex box in side, with the activity of video recorder mouse in record for some time.Analyze distance, the time of moving cost and mouse that mouse the moves position at any time the time.Control animal spends the most of the time usually and moves around the periphery of box, passes middle section sometimes.The difference of record and this normal mode, concrete is that the increase of this time quantum means the minimizing of animal anxiety at the time quantum of the middle section cost of box.

On video recorder the record for some time in mouse motion and analyze.The result shows the mobile wild-type contrast (WT 1161.39 ± 170.8cm that is less than that rejects mouse; KO 636.84 ± 193.62 p=0.05ANOVA).

(Fig. 5 A) analyzes these data presentation and rejects zone and the middle section motion around of considerably less time of the whole cost of mouse, promptly compares with the motion of wild-type control mice, and they are as by numb with cold, and the long period is mobile (Fig. 5 B).Kv9.2 rejects mouse thereby shows the increase of passivity.

Time in middle section motion cost is WT 56.2 ± 12.7s; KO 23.3 ± 8.5s p=0.01ANOVA, testing total time is 300s, the time of regional movement cost is WT 44.3 ± 4.4 around; KO 24.7 ± 7.9.Kv9.2 rejects minimizing and the increase of periphery dead time that therefore mouse also shows traveling time.

Enter the total degree of periphery and middle section, promptly the mouse number of times that passes the wilderness reduces significantly also in rejecting mouse that (WT 7.3 ± 2; KO 2 ± 1 p＜0.01 ANOVA) (Fig. 5 C).

Embodiment 7. biological datas: behavioural analysis (positive labyrinth)

Check is rejected and the wild-type control mice in positive labyrinth.

Briefly, use positive labyrinth of raising and the anxiety of recording a video the tracking and measuring mouse.This test is by increasing height and open composition, utilize mouse seek the tendency of new environment with to conflicting between the characteristic of shining bright open base area detest.Two alternative railings (arm) are closed, and it has the wall of high dark; Two other railing is then opened.The railing that mouse is preferred closing, but can take a risk to enter open railing.Referring to Pellow etc., ' in the positive labyrinth of raising, confirming from opening: close the entering of railing ' (1985) as measuring rat anxiety method.

Although record rejecting mouse is compared with the contrast wild-type mice and has moved similar distance, but the data analysis demonstration is compared with control mice, they have spent the obviously longer time in the railing of closing (think safety), and lessly enter open railing (it is considered to dangerous) (Fig. 6).

This proof Kv9.2 rejects mouse and has the anxiety phenotype, so Kv9.2 is relevant with anxiety disorder.

Each application and the patent mentioned herein, with quote or every piece of document of reference in above-mentioned each application and the patent, be included in (" document that application is quoted ") in the process of handling each application and patent and each apply for and patent in and explanation or catalogue this paper of any manufacturers of any product of quoting or mention in the document quoted of any application quote as a reference.In addition, all documents of quoting are herein quoted in the document that this paper quotes and all documents of reference, and explanation or catalogue this paper of any manufacturers of this paper any product of quoting or mentioning quote as a reference.

The various corrections of the method and system that the present invention describes and variation are conspicuous to the person skilled in the art, and they do not deviate from scope and spirit of the present invention.Although the present invention is described in conjunction with concrete preferred embodiment, be to be understood that as the desired the present invention of claim should not be limited in inadequately in these specific embodiments, can within the scope of the invention it is much revised and add.Certainly, the various corrections that are used to finish mode of the present invention of description (this is conspicuous to molecular biology or association area those skilled in the art) are in claim scope of the present invention.In addition, do not deviate under the situation of the scope of the invention, can carry out the various combinations of dependent claims feature thereafter in the feature of independent claim.

Claims (29)

1. one kind has the transgenic nonhuman animal that function is suffered the destructive native gene, wherein, described Kv9.2 gene comprises nucleotide sequence that SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:4 show or has the sequence of at least 70% sequence identity with described sequence.

2. transgenic nonhuman animal according to claim 1, described transgenic nonhuman animal has disappearance in Kv9.2 gene or its part.

3. transgenic nonhuman animal according to claim 1 and 2, described transgenic nonhuman animal show any one or their combination in the following phenotype:

Compare with the wild-type animal

(a) glucose level descends;

(b) anxiety increase is preferably as measuring in wilderness test and/or positive maze test.

4. transgenic nonhuman animal, wherein said animal at least partly or entirely Kv9.2 gene are used from another animal, another species preferably, and the sequence that is more preferably people's Kv9.2 gene replaces.

5. according to the described transgenic nonhuman animal of aforementioned any one claim, described animal is a mouse.

6. transgenic nonhuman animal according to claim 5, described animal comprises function and suffers destructive Kv9.2 gene, preferably in the Kv9.2 gene disappearance is arranged, wherein the Kv9.2 gene comprises nucleotide sequence that SEQ ID NO:4 shows or has the sequence of at least 70% sequence identity with it.

7. one kind from isolated cells or tissue according to the described non-human transgenic animal of aforementioned any one claim.

8. one kind has the cell that function is suffered the endogenous Kv9.2 gene of destructive, wherein said Kv9.2 gene comprises the nucleotide sequence that SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:4 show, perhaps has the sequence of at least 70% sequence identity with described sequence.

9. according to any one the described transgenic nonhuman animal among the claim 1-6, cell or tissue according to claim 7 or cell according to claim 8 purposes as anxiety or diabetes model.

10. according to any one the described transgenic nonhuman animal among the claim 1-6, cell or tissue according to claim 7 or cell according to claim 8 purposes as the disease model relevant with Kv9.2.

11. according to any one the described non-human transgenic animal among the claim 1-6, its isolated cells according to claim 7 or tissue or cell according to claim 8 purposes in the method for the agonist of differentiating the Kv9.2 polypeptide or antagonist, described Kv9.2 polypeptide comprises aminoacid sequence that SEQ ID NO:3 or SEQ ID NO:5 show or has the sequence of at least 70% sequence identity with described sequence.

12. a discriminating has aminoacid sequence that SEQ ID NO:3 or SEQ ID NO:5 show or has the agonist of Kv9.2 polypeptide of sequence of at least 70% sequence identity or the method for antagonist with described sequence, described method comprises and gives the animal candidate compound, described animal is wild-type animal or according to any one the described transgenic nonhuman animal among the claim 1-6 preferably, and measures arbitrary following phenotypic variation: (a) glucose level; (b) anxiety is preferably as measuring in wilderness test and/or positive maze test.

13. a method of differentiating the agonist of Kv9.2 polypeptide according to claim 12, described method comprise that discriminating can cause that animal shows the candidate compound that arbitrary phenotype increases in (a)-(b).

14. a method of differentiating the antagonist of Kv9.2 polypeptide according to claim 12, described method comprise that discriminating can cause that animal shows the candidate compound that arbitrary phenotype in (a)-(b) or described phenotype reduce.

15. a discriminating has aminoacid sequence that SEQ ID NO:3 or SEQ ID NO:5 show or has the agonist of Kv9.2 polypeptide of sequence of at least 70% sequence identity or the method for antagonist with described sequence, described method comprises candidate compound is contacted with cell or tissue, described cell or tissue is wild-type cell or tissue preferably, cell or tissue perhaps according to claim 7, cell perhaps according to claim 8, and conductivity or dynamic (dynamical) variation of mensuration cell or tissue cell.

16. a method of differentiating the agonist of Kv9.2 polypeptide according to claim 15, described method comprise that discriminating can increase cell conductivity or dynamic (dynamical) candidate compound.

17. a method of differentiating the antagonist of Kv9.2 polypeptide according to claim 15, described method comprises that discriminating can reduce cell conductivity or dynamic (dynamical) candidate compound.

18. a discriminating is suitable for treatment or alleviates anxiety or diabetes, the method of the compound of the disease relevant preferably with Kv9.2, described method comprises and will comprise aminoacid sequence that SEQ ID NO:3 or SEQID NO:5 show or contact with candidate compound with Kv9.2 polypeptide that described sequence has a sequence of at least 70% sequence identity, and whether definite described candidate compound is the agonist or the antagonist of Kv9.2 polypeptide.

19. the Kv9.2 polynucleotide that comprise nucleotide sequence that SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:4 show or have the sequence of at least 70% sequence identity with it are used to differentiate the purposes of the agonist or the antagonist of Kv9.2 polynucleotide, the agonist of described Kv9.2 polynucleotide or antagonist are used for the treatment of anxiety or diabetes, preferably relevant with Kv9.2 disease.

20. the Kv9.2 polypeptide that comprises aminoacid sequence that SEQ ID NO:3 or SEQ ID NO:5 show or have the sequence of at least 70% sequence identity with it is used to differentiate the purposes of the agonist or the antagonist of Kv9.2 polypeptide, the agonist of described Kv9.2 polypeptide or antagonist are used for the treatment of anxiety or diabetes, preferably relevant with Kv9.2 disease.

21. antagonist of Kv9.2 polypeptide that has aminoacid sequence that SEQ ID NO:3 or SEQ ID NO:5 show or have the sequence of at least 70% sequence identity with it, anxiety or diabetes, the preferably purposes in the method for the disease relevant in the treatment individuality with Kv9.2.

22.Kv9.2 the antagonist of polypeptide is anxiety or diabetes, the preferred purposes in the pharmaceutical composition of the disease relevant with Kv9.2 in preparation treatment individuality, described Kv9.2 polypeptide has the aminoacid sequence of SEQ IDNO:3 or SEQ ID NO:5 demonstration or has the sequence of at least 70% sequence identity with it.

23. a treatment suffers from anxiety or diabetes, preferably suffers from the method for the individuality of the disease relevant with Kv9.2, described method comprises the antagonist that gives described individual Kv9.2.

24. anxiety or diabetes in the diagnosis individuality, the method for preferred Kv9.2 relative disease, described method comprise expression, level or the active variation that detects Kv9.2 in individual or its cell or tissue.

25. according to claim 10,19,20 or 22 described purposes, according to claim 18,23 or 24 described methods, or antagonist according to claim 21, the wherein said disease relevant with Kv9.2 is selected from the group of being made up of following: I type and type ii diabetes, hyperinsulinemia, hunger disease, insulin resistance, diabetic complication comprises the vascular disease that diabetes are relevant, the ephrosis neuropathy relevant that diabetes are relevant, and treatment hypoglycemia with diabetes, (they are HDL to high fat, LDL or VLDL) mass formed by blood stasis, unusual lipidemia, no matter its cause is to be primary in or to be secondary to diabetes, hyperthyroidism/go down, acromegaly, liver failure, renal failure, Vipoma, pancreatitis and alcohol inductive hypoglycemia.

26. according to claim 10,19,20 or 22 described purposes, according to claim 18,23 or 24 described methods, or antagonist according to claim 21, wherein said Kv9.2 relative disease is selected from the group of being made up of following: social anxiety disorder, stress reaction obstacle after the wound, phobia, social phobia, special phobia, the panic disorder obsessive compulsive disorder, the gross stress reaction obstacle, the separation anxiety disease, the generalized anxiety disorder disease, major depression, depression, the two poles of the earth obstacle, seasonal affective disorder, post-natal depression, manic depressive illness, the two poles of the earth dysthymia disorders, anxiety disorder, anxiety disorder, anxiety related behavior and generalized anxiety disorder disease, agoraphobia, listed those panic disorders and depression among gross stress reaction obstacle and the DSM-IV.

27. a Kv9.2 polypeptide, wherein said Kv9.2 polypeptide comprise the aminoacid sequence that SEQ ID NO:3 or SEQ ID NO:5 show, or its homologue, variant or the derivative that have at least 70% sequence identity with described sequence.

28. the nucleic acid of a coding polypeptide according to claim 27.

29. a nucleic acid according to claim 28, described nucleic acid comprise the nucleotide sequence that SEQ IDNo:1, SEQ ID No:2 or SEQ ID NO:4 show, or have its homologue, variant or the derivative of at least 70% sequence identity with it.

Images