CN1516706A - Endogenous and non-endogenous versions of human G protein-coupled receptors - Google Patents

Abstract

The invention disclosed in this patent document relates to transmembrane receptors, more particularly to a human G protein-coupled receptor and to mutated (non-endogenous) versions of the human GPCRs for evidence activity.

Description

The endogenous of human G protein-coupled receptor and non-endogenous form

The cross reference of related application

The application is the part continuation application of the U.S. serial 09/170,496 of submission on October 13rd, 1998, and the PCT application number of U.S. serial 09/170,496 correspondence is PCT/US99/23938, and open April 20 in 20O0, its publication number is WO 00/22129.The application still is the part continuation application of the U.S. serial 09/060,188 submitted on April 14th, 1998, and U.S. serial 09/060,188 is part continuation applications of U.S.'s consecutive number 08/839,449 (abandoning) of submitting on April 14th, 1997.Here require the right of priority of aforementioned every piece of application, and aforementioned every piece of disclosed content of application is introduced as a reference all herein.The application also requires the right of priority of the U.S. Provisional Application submitted to February 26 calendar year 2001 number 60/271,913, and its is also all introduced herein as a reference.Presents also relates to following application: the U.S. Provisional Application of submitting on December 1st, 2000 number 60/250,881; The U.S. Provisional Application of submitting on November 27th, 2000 number 60/253,428; The U.S. Provisional Application of submitting on September 20th, 2000 number 60/234,317; The U.S. Provisional Application of submitting on November 3rd, 2000 number 60/245,853; The U.S. Provisional Application of submitting on September 20th, 2000 number 60/234,045; The U.S. Provisional Application of submitting on April 28th, 2000 number 60/200,568; The U.S. Provisional Application of submitting on April 19th, 2000 number 60/198,518; The U.S. Provisional Application of submitting on March 14th, 2000 number 60/189,353; The U.S. Provisional Application of submitting on November 17th, 1999 number 60/166,084; With the U.S. Provisional Application of submitting on October 30th, 1998 number 60/106,451; Wherein every piece of disclosed content of application is all introduced herein as a reference.

Invention field

The present invention relates to transmembrane receptor, in certain embodiments, relate to g protein coupled receptor and, in some preferred embodiment, be changed the endogenous GPCR that establishes or improve the acceptor constitutive activity thereby relate to.In certain embodiments, composing type activatory GPCR can be used for directly identifying the candidate compound as receptor stimulant or inverse agonist, and described receptor stimulant or inverse agonist are suitable for use as therapeutical agent.

Background of invention

Though there is the acceptor of many types among the mankind, the abundantest and maximally related with treatment is g protein coupled receptor (GPCR).According to estimates, have in the human genome about 30,000-40,000 gene, and in these genes, nearly 2% coding GPCR.Identified the acceptor of its endogenic ligand, comprised that GPCR is called as " known " acceptor, and do not identify that as yet the acceptor of its endogenic ligand is known as " solitarily " acceptor.

GPCR has represented a key areas of medicament production research and development: all about 60% of prescription drugs is to develop from 100 known GPCR about 20.For example, 1999, in 100 main trade name prescription drugs, following medicine and GPCR interaction (disease and/or the illness of being treated illustrate in bracket):

Claritin  (allergy) Prozac  (depression) Vasotec  (hypertension)

Paxil  (depression) Zoloft  (depression) Zyprexa  (psychosis)

Cozaar  (hypertension) Imitrex  (migraine) Zantac  (anti-stream)

Propulsid  (reflux disease) Risperdal  (schizophrenia) Serevent  (asthma)

Pepcid  (anti-stream) Gaster  (ulcer) Atrovent  (bronchospasm)

Effexor  (depression) Depakote  (epilepsy) Cardura  (prostatomegaly)

Allegra  (allergy) Lupron  (prostate cancer) Zoladex  (prostate cancer)

Diprivan  (anesthesia) BuSpar  (anxiety) Ventolin  (bronchospasm)

Hytrin  (hypertension) Wellbutrin  (depression) Zyrtec  (rhinitis)

Palvix  (MI/ apoplexy) Toprol-XL  (hypertension) Tenormin  (stenocardia)

Xalatan  (glaucoma) Singulair  (asthma) Diovan  (hypertension)

Harnal  (hyperplasia of prostate)

(data of Med Ad News 1999).

GPCR has a common structural motif, and they form 7 α spirals by 7 sequences of being made up of 22-24 hydrophobic amino acid, and each spiral is all striden film (each strides the film district is umerical,, strides film-1 (TM-1) that is, strides film-2 (TM-2), etc.).Transbilayer helix is by the cytolemma outside, or the striding film-2 and stride film-3 of " born of the same parents are outer " side, strides film-4 and strides film-5, and stride film-6 and the amino acid chain of striding between the film-7 connects (these are known as " born of the same parents are outer " district 1,2 and 3 (EC-1, EC-2 and EC-3) respectively).Transbilayer helix also can pass through cytolemma inside, or the striding film-1 and stride film-2 of " born of the same parents in " side, strides film-3 and strides film-4, and stride film-5 and the amino acid chain of striding between the film-6 connects (these are known as " in the born of the same parents " district 1,2 and 3 (IC-1, IC-2 and IC-3) respectively)." carboxyl " of acceptor (" C ") end is positioned at intracellular born of the same parents space, and " amino " of acceptor (" N ") end is positioned at extracellular born of the same parents' external space.

Usually, when endogenic ligand and receptors bind (being also referred to as " activation " of acceptor sometimes), the conformation of intracellular region changes, and makes coupling between " G-albumen " in intracellular region and the born of the same parents.It is reported that GPCR is " miscellaneous " for G albumen, that is, GPCR can with a more than G protein-interacting.See Kenakin, T., 43 Life Sciences 1095 (1988).Though also there is other G albumen, the G albumen of having identified at present is Gq, Gs, Gi, Gz and Go.But the proteic coupling priming signal of the CPCR of ligand activation and G-cascade amplification process (being also referred to as " signal transduction ").Under normal operation, signal transduction finally causes cell activation or cell to suppress.Though do not wish to be bound by theory, by inference the C-terminal of IC-3 ring and acceptor all with the G protein-interacting.

Under physiological condition, GPCR is with two isomorphic maps not: the equilibrated form is present in the cytolemma between " nonactive " state and " activity " state.The acceptor of inactive state can not be connected with the intracellular signal transduction approach, thereby causes the signal transduction that produces biological respinse.Receptor conformation can (via G albumen) be connected with transduction pathway to the change of active condition, and produces biological respinse.

Acceptor can pass through part or compound, is stabilized in active condition as medicine.Recently find, except part or medicine, other promotion also can be provided and make acceptor be stabilized in the method for active condition conformation, include, but are not limited to the aminoacid sequence of acceptor is modified.These methods are stabilized in active condition with acceptor effectively by the keying action of simulation part and acceptor.Stable being known as " composing type receptor activation " of being undertaken by this part dependent/non-dependent mode.

Summary of the invention

Disclosed herein is the endogenous of people GPCR and non-endogenous form and uses thereof.

Certain embodiments of the invention relate to the amino acid sequences encoded g protein coupled receptor by SEQ ID NO.2, by amino acid sequences encoded their the non-endogenous, constitutively activated form of SEQ ID NO:63, and contain their host cell.

Certain embodiments of the invention relate to the plasmid of the cDNA that contains carrier and SEQ ID NO.62, and contain their host cell.

Certain embodiments of the invention relate to the amino acid sequences encoded g protein coupled receptor by SEQ ID NO.4, by amino acid sequences encoded their the non-endogenous, constitutively activated form of SEQ ID NO:65, and contain their host cell.

Certain embodiments of the invention relate to the plasmid of the cDNA that contains carrier and SEQ ID NO.64, and contain their host cell.

Certain embodiments of the invention relate to the amino acid sequences encoded g protein coupled receptor by SEQ ID NO.6, their non-endogenous, constitutively activated form, and contain their host cell.

Certain embodiments of the invention relate to the plasmid of the cDNA that contains carrier and SEQ ID NO.5, and contain their host cell.

Certain embodiments of the invention relate to the amino acid sequences encoded g protein coupled receptor by SEQ ID NO.8, by SEQ ID NO:67, SEQ ID NO.69, amino acid sequences encoded their the non-endogenous, constitutively activated form of SEQ ID NO.71 and SEQ ID NO.73, and contain their host cell.

Certain embodiments of the invention relate to and contain carrier and SEQ ID NO.66, SEQ IDNO.68, and the plasmid of the cDNA of SEQ ID NO.70 and SEQ ID NO.72, and contain their host cell.

Certain embodiments of the invention relate to the amino acid sequences encoded g protein coupled receptor by SEQ ID NO.10, by amino acid sequences encoded their the non-endogenous, constitutively activated form of SEQ ID NO:75 and SEQ ID NO.77, and contain their host cell.

Certain embodiments of the invention relate to the plasmid of the cDNA that contains carrier and SEQ ID NO.74 and SEQ IDNO.76, and contain their host cell.

Certain embodiments of the invention relate to the amino acid sequences encoded g protein coupled receptor by SEQ ID NO.12, by amino acid sequences encoded their the non-endogenous, constitutively activated form of SEQ ID NO:79 and SEQ ID NO.81, and contain their host cell.

Certain embodiments of the invention relate to the plasmid of the cDNA that contains carrier and SEQ ID NO.78 and SEQ IDNO.80, and contain their host cell.

Certain embodiments of the invention relate to the amino acid sequences encoded g protein coupled receptor by SEQ ID NO.14, by amino acid sequences encoded their the non-endogenous, constitutively activated form of SEQ ID NO:83, and contain their host cell.

Certain embodiments of the invention relate to the plasmid of the cDNA that contains carrier and SEQ ID NO.82, and contain their host cell.

Certain embodiments of the invention relate to the amino acid sequences encoded g protein coupled receptor by SEQ ID NO.16, by amino acid sequences encoded their the non-endogenous, constitutively activated form of SEQ ID NO:85, and contain their host cell.

Certain embodiments of the invention relate to the plasmid of the cDNA that contains carrier and SEQ ID NO.84, and contain their host cell.

Certain embodiments of the invention relate to the amino acid sequences encoded g protein coupled receptor by SEQ ID NO.18, by amino acid sequences encoded their the non-endogenous, constitutively activated form of SEQ ID NO:87, and contain their host cell.

Certain embodiments of the invention relate to the plasmid of the cDNA that contains carrier and SEQ ID NO.86, and contain their host cell.

Certain embodiments of the invention relate to the plasmid of the cDNA that contains carrier and SEQ ID NO.84, and contain their host cell.

Certain embodiments of the invention relate to the amino acid sequences encoded g protein coupled receptor by SEQ ID NO.98, their non-endogenous, constitutively activated form, and contain their host cell.

Certain embodiments of the invention relate to the plasmid of the cDNA that contains carrier and SEQ ID NO.97, and contain their host cell.

The accompanying drawing summary

The second messenger that Fig. 1 is based on cell encircles the diagram of AMP measurement result, wherein provide the active FPRL-2 of endogenous, constitutively (" FPRL-2wt "), the comparative result of the non-endogenous, constitutively activated form (" FPRL-2 (L240K) ") of the FPRL-2 that merges with the Gs/Gi fusion protein construct and the composing type signal transmission of control group (" Gs/Gi ").

Fig. 2 provides with control group (" CMV ") and compares, endogenous STRL33 and non-endogenous, constitutively activated STRL33 (the comparative analysis diagram result of " STRL33 (L230K) ", they are in the 293T cell, utilize 8XCRE-Luc reporter assay method to obtain.

Fig. 3 provides with control group (" CMV ") and compares, endogenous target acceptor GPR45 (" GPR45wt ") illustrates the result with the comparative analysis of non-endogenous TSHR (A632I) (" signal enhanser ") cotransfection, they are in 293 cells, utilize adenylate cyclase enzyme assay based on cell to obtain.This assay method comprises TSH, i.e. the adding of TSHR endogenic ligand.

Fig. 4 provides endogenous target acceptor mGluR7 (" mGluR7wt ") and the cotransfection of non-endogenous TSHR (A623I) (" signal enhanser ") and the non-endogenous, constitutively activated form (" W590S " of target acceptor mGluR7, " R659H ", " T771C " and " I790K ") and the comparative analysis of non-endogenous TSHR (A623I) cotransfection diagram result, they are in 293 cells, utilize adenylate cyclase enzyme assay based on cell to obtain.This assay method comprises TSH, i.e. the adding of TSHR endogenic ligand.

Fig. 5 provides endogenous target acceptor mGluR7 (" mGluR7wt ") and the cotransfection of non-endogenous TSHR (A623I) (" signal enhanser ") and the non-endogenous, constitutively activated form (" W590S " of target acceptor mGluR7, " R659H ", " T771C " and " I790K ") and the comparative analysis of non-endogenous TSHR (A623I) cotransfection diagram result, they are in the RGT cell, utilize adenylate cyclase enzyme assay based on cell to obtain.This assay method comprises TSH, i.e. the adding of TSHR endogenic ligand.

Fig. 6 is provided at and does not have under the situation of glutaminate existence, compare with " Gq (disappearance)/Gi " with " Gq (disappearance) ", with the non-endogenous form of Gq albumen, i.e. the non-endogenous mGluR7 of " Gq (disappearance) " and " Gq (disappearance)/Gi " cotransfection, the second messenger IP of " T771C " ₃The diagram that produces.

Fig. 7 is in the SRE reporter assay method, endogenous is non--and the comparative analysis of the GPR37 (" wt ") of constitutive activity and the non-endogenous, constitutively activated form (" C543Y " and " L352R ") of GPR37, wherein control group is expression vector (" CMV ").

Fig. 8 be Gs/Gi fusion constructs and endogenous target acceptor GPR37 (" GPR37wt ") altogether-comparative analysis of transfection and non-endogenous, constitutively activated form (" C543Y " and " the L352R ") cotransfection of Gs/Gi fusion constructs and target acceptor GPR37, wherein utilization is full cell second messenger cAMP assay method.

Fig. 9 is that the RNA of the GPR37 that expresses in the rat schwann's cell of handling with forskolin analyzes.Cytodifferentiation maintains the 20uM forskolin.

Figure 10 is that the RNA of the GPR37 that expresses in the rat sciatic nerve of crushing analyzes.GPR37 is seven (7) days quilt height adjusted after crushing.

Figure 11 is in the IP3 assay method, the comparative analysis of the non-endogenous, constitutively activated form (" I281F ") of HF1948 of the non-constitutive activity of endogenous (" wt ") and HF1948, and wherein control group is expression vector (" pCMV ").

Figure 12 is the non-endogenous, constitutively activated form (" I281F and " E135N ") of cotransfection and the target acceptor HF1948 of endogenous target acceptor HF1948 (" HF1948wt ") and non-endogenous TSHR (A623I) (" signal enhanser ") and the comparative analysis of non-endogenous TSHR-A623I cotransfection, has wherein utilized full cell adenylate cyclase enzyme assay.This assay method comprises TSH, the adding of TSHR endogenic ligand.

Figure 13 is to use the GPR66 RNA trace result's that a plurality of pancreatic cells system carries out photo replica.

Figure 14 provides the graphic result of endogenous GPR35 and non-endogenous, constitutively activated GPR35 (" GPR35 (A216K) ") comparative analysis, and they are in the 293A cell, utilize E2F-Luc reporter assay method to carry out.

Figure 15 is to use the GPR35 RNA trace result's that the cDNA of a plurality of tissues (people) carries out photo replica.

Figure 16 provides the cotransfection of endogenous ETBR-LP2 (" WT ") and non-endogenous TSHR-A623I (" TSHR-A623I ") (have and do not have TSH) and non-endogenous, constitutively activated ETBR-LP2 (" the N358K ") TSHR-A623I with sudden change (have and do not have TSH) comparative analysis of cotransfection, utilization be the adenylate cyclase enzyme assay.

Figure 17 provides endogenous ETBR-LP2 (" WT ") graphic result with non-endogenous, constitutively activated ETBR-LP2 (" N358K ") comparative analysis, utilization be the AP1 reporter assay system.

Figure 18 is that the RNA of the ETBR-LP2 that expresses in the rat schwann's cell that forskolin was handled analyzes.Cytodifferentiation maintains the 20uM forskolin.

Figure 19 is that the RNA of the ETBR-LP2 that expresses in the rat sciatic nerve of crushing analyzes.ETBR-LP2 is seven (7) days quilt height adjusted after crushing.

Figure 20 A and 20B provide the sequence alignment report between the report aminoacid sequence (" hGPR37p ") of the putative amino acid sequence (" hETBRLP2p ") of people ETBR-LP2 and people GPR37.

Describe in detail

Once called the part that acceptor is had various effects with a lot of terms around the scientific literature that acceptor is drawn. For clear and consistent, this patent document will be used following definition from start to finish. When these definition contradict with other definition of these terms, should be with reference to following definition:

When activator refers to receptors bind, intramicellar reaction be can activate, or GTP and membrane-bound material (for example, part, candidate compound) improved. In certain embodiments, activator is during with receptors bind, activates intramicellar reaction or improves those materials of GTP and membrane-bound previous the unknown.

Amino acid abbreviations used herein is listed in Table A:

Table A

Alanine	ALA	A
			Arginine	ARG	R
Asparagine	ASN	N
			Asparatate	ASP	D
Cysteine	CYS	C
			Glutamic acid	GLU	E
Glutamine	GLN	Q
			Glycine	GLY	G
Histidine	HIS	H
			Isoleucine	ILE	I
Leucine	LEU	L
			Lysine	LYS	K
Methionine	MET	M
			Phenylalanine	PHE	F
Proline	PRO	P
			Serine	SER	S
Threonine	THR	T
			Tryptophan	TRP	W
Tyrosine	TYR	Y
			Valine	VAL	V

Antagonist refers to and the material (for example, part, candidate compound) of activator at same site competition bind receptor, but it can not activate the intramicellar reaction that is caused by the receptor active form, and can therefore suppress by the intramicellar reaction due to the activator. In the situation that does not have activator to exist, antagonist can not make basic intramicellar reaction weaken. In certain embodiments, antagonist is during with receptors bind, activates intramicellar reaction or improves those materials of GTP and membrane-bound previous the unknown.

The candidate compound refers to the molecule (for example, but being not restricted to compound) through the triage techniques check. Preferably, phrase " candidate compound " does not comprise the compound that the public is known, for example by the pre-determined receptor inverse agonists of indirect authentication method, and activator or antagonist (" compound of indirectly identifying "); More preferably, do not comprise the previous compound of determining at least a mammal is had the indirect evaluation of result for the treatment of; And, most preferably, do not comprise the previous compound of determining the people is had the indirect evaluation of therapeutic action.

Composition refers to contain the material of at least one component; " pharmaceutical composition " is the example of composition.

The effectiveness of compound refers to that compound suppresses or the functional ability of costimulatory receptor, and is namely opposite with receptors bind affinity, the measured value of the ability of activation/Inhibitory signal transduction pathway. The exemplary process that detection compound is renderd a service is open in the embodiment of this patent document part.

Codon refers to 3 one group nucleotides (or nucleotide equivs), and it generally includes nucleosides (adenosine (A), guanosine (G) with the phosphate group coupling, cytidine (C), uridine (U) and thymidine (T)), when being translated, coded amino acid.

The acceptor of composing type activation refers to the acceptor through the composing type receptor activation. The acceptor of composing type activation can be endogenic or non-endogenic.

The composing type receptor activation refers to utilize except making acceptor other method with its part or chemical equivalence thing are combined that acceptor is stabilized in activated state.

Contact refers in the system, make at least two parts together in vitro system or body.

Direct Identification or Direct Identification, when relating to phrase " candidate compound ", refer to the candidate compound is carried out the composing type activated receptor, the orphan receptor that preferred group is moulding activated, the more preferably screening of the G albumen coupling cell surface orphan receptor of composing type activation, and assess the effectiveness of this compound. This phrase under any circumstance all can not be explained or is interpreted as to comprise phrase " indirectly evaluation " or " indirectly identifying ", or be included by them.

Endogenous refers to the material of the natural generation of mammal. For example, but be not restricted to, for term " acceptor ", endogenous refers to by mammal (for example, but being not restricted to the people) or viral natural generation. On the contrary, term herein is non-endogenicly refers to not be by mammal (for example, but being not restricted to the people) or viral natural generation. For example, but be not restricted to, the acceptor of its endogenous form is not constitutive activity, but it is operated, and when making it become constitutive activity, most preferably is called " non-endogenous, constitutively activated acceptor ". Two terms all can be used for describing " in the body " and " external " system. For example, but be not restricted to, in screening technique, endogenous or non-endogenous receptor all can be used in the in-vitro screening system. As another example, and not restriction, when the mammalian genes group is operated, when making it contain non-endogenous, constitutively activated acceptor, can utilize screening system candidate compound in the body.

G protein coupled receptor fusion and gpcr fusion protein, herein in the disclosed scope of the invention, refer to non-endogenous protein, it contains endogenous, constitutively activated GPCR or non-endogenous, constitutively activated GPCR, described non-endogenous, constitutively activated GPCR and at least one G albumen, more preferably the alpha subunit of this G albumen (in conjunction with the subunit of GTP) merges, and the type of the described G albumen preferably G albumen with the natural coupling of the lonely GPCR of endogenous is identical. For example, but without limits, at the endogenous state, if G albumen " Gs α " is and the advantage G albumen of GPCR coupling that the gpcr fusion protein based on specific GPCR then is the non-endogenous protein that contains the GPCR that merges with Gs α so; In some cases, as described below, non--advantage G albumen also can merge with GPCR. G albumen can directly merge with the C-of constitutive activity GPCR is terminal, or has spacer region between two.

Host cell refers to wherein can insert the cell of plasmid and/or carrier. For prokaryotic host cell, when host cell copied, plasmid was usually as being replicated (after usually copying, plasmid is separated, and then is incorporated in the eukaryotic host cell) from host molecule; For eukaryotic host cell, plasmid is integrated in the cell DNA of host cell, and when the eucaryon host cell copied, plasmid also copied like this. In certain embodiments, host cell is eucaryon, and more preferably mammal most preferably is selected from 293,293T and COS-7 cell.

The conventional method that refers to find medicine of indirectly identifying or indirectly identifying, comprise and identify the endogenic ligand special to endogenous receptor, candidate compound is carried out the acceptor screening, determine to disturb and/or compete those compounds that ligand-receptor reacts to each other, and the effectiveness of at least one second messenger approach relevant with activated receptor of assessment compounds affect.

Suppress, for term " reaction ", refer to opposite with the situation that does not have compound to exist, in the situation that has compound to exist, reaction reduction or be obstructed.

Oppositely (inverse) activator refers to that the material of being combined with the composing type activated form of the endogenous form of acceptor or acceptor (for example, part, candidate compound), they can suppress the basic intramicellar reaction by the initiation of receptor active form, until be lower than standard activity foundation level, described standard activity foundation level is to observe in situation about existing without activator, or they can reduce the combination of GTP and film. Preferably do not exist the basic reaction in the situation to compare with there being inverse agonist, in the situation that has inverse agonist to exist, basic intramicellar reaction suppressed at least 30%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, most preferably at least 99%.

Known receptor refers to the certified endogenous receptor of its specificity endogenic ligand.

Part refers to the molecule to naturally occurring receptor-specific.

Mutant or sudden change for the nucleic acid and/or amino acid sequence of endogenous receptor, refer to that this endogenous sequence is carried out specific one or more to be changed, thereby make the acceptor of the non-composing type activation of endogenous show that the composing type of acceptor activates. For the equivalent of particular sequence, it is of equal value with the promutation of people's acceptor that people's acceptor mutant form afterwards is considered to, condition is that the level of the composing type activation levels of (a) people acceptor mutant form afterwards and acceptor promutation is basic identical, (b) acceptor mutant form and the sequence between the acceptor promutation (amino acid and/or nucleic acid) percent homology afterwards is at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, most preferably at least 99%. In certain embodiments, because some preferred cartridge for realizing the composing type activation disclosed herein has the change of single amino acid and/or codon between the endogenous of GPCR and non-endogenous form, so the percentage of preferred sequence homology should be at least 98%.

Non--orphan receptor is meant the evaluation part special, the naturally occurring molecule of endogenous, and wherein part can activate the intracellular signal pipeline with combining of acceptor.

Orphan receptor is meant also not to be identified or unknown endogenous receptor its special part.

Pharmaceutical composition is meant and contains at least one composition of active components, and therefore can be in Mammals (for example, but being not restricted to the people) research specific, effective result.Those of ordinary skill in the art should understand and correctly estimate and are suitable for determining whether the possess skills required effective result's of personnel technology of activeconstituents.

Plasmid is meant combining of carrier and cDNA.Usually, in order to duplicate and/or to make cDNA be expressed as albumen, and plasmid is incorporated in the host cell.

The second messenger is meant the intramicellar reaction that receptor activation produces.The second messenger can comprise, for example, and inositoltriphosphoric acid (IP ₃), diacylglycerol (DAG), ring AMP (cAMP), and cyclo GMP (cGMP).The reaction of measuring the second messenger can be used for measuring receptor activation.In addition, the reaction of measuring the second messenger also can be used for directly identifying the candidate compound, comprise, for example, inverse agonist, agonist, and antagonist.

Signal to noise ratio is meant response activation, amplification, or the signal that produces when stimulating is higher than ground unrest or activation of response, amplification, or the basal level when not stimulating.

Transcribed spacer is meant and is positioned at gene, for example after the last codon or amino acid of target GPCR, and the translated amino acid number before proteic initiator codon of target G or the initiator, wherein said translated amino acid is arranged in the frame of target G albumen initiator.The translated amino acid number can be special according to technician's needs, is about 1 amino acid usually, preferred 2 amino acid, more preferably 3 amino acid, more preferably 4 amino acid, more preferably 5 amino acid, more preferably 6 amino acid, more preferably 7 amino acid, more preferably 8 amino acid, more preferably 9 amino acid, more preferably 10 amino acid, more preferably 11 amino acid, even more preferably 12 amino acid.

Stimulate, for term " reaction ", be meant on the contrary, under the situation that has compound to exist, react increase with the situation that does not have compound to exist.

Basically be meant the result the control group result 40% within, within preferred 35%, more preferably within 30%, more preferably within 25%, more preferably within 20%, more preferably within 15%, more preferably within 10%, more preferably within 5%, more preferably within 2%, most preferably within 1%.For example, with regard to function of receptors, if the transduction signal that uses the method for instruction herein or similar methods measure known to the skilled signal that control group produces 40% within, test acceptor so and show and contrast the similar substantially result of acceptor.

Carrier for cDNA, is meant the cyclic DNA that at least one cDNA can be inserted in the host cell.

The order of following part is arranged in order to describe effect, and can not be interpreted as the restriction to following open or claim.

A. foreword

The tradition research of acceptor is normally carried out according to previous supposition (based on history),, before finding to influence the antagonist and other molecule of acceptor, at first must identify endogenic ligand that is.Even knowing under the situation of antagonist in advance, also should carry out the research of seeking endogenic ligand immediately.Even after finding the composing type activated receptor, in acceptor research, still to adhere to this thoughtcast.Do not recognize also that before this active condition of acceptor is only the most useful for agonist and the inverse agonist of finding acceptor.For those diseases that caused by hyperactivity acceptor or active low acceptor, we wish that medicine is that the receptor active state is reduced, or improve the compound of receptor active, and do not wish that this medicine is the antagonist of endogenic ligand.This is because reduce or improve the active compound of active acceptor state and need not to combine in same site with endogenic ligand.Therefore, according to the instruction of the inventive method, any research of seeking the treatment compound all should be by beginning at part-dependent/non-dependent active condition SCREENED COMPOUND.

B. the evaluation of people GPCR

The bulk information of the related nucleic acid sequence that is positioned at human genome has been identified in the enforcement of the Human Genome Project; Through this effort, even the open reading-frame (ORF) information whether arbitrary specific gene group sequence is contained interpreter's proteinoid is not understood or understanding not, we also can obtain genetic sequence information.A lot of methods of identifying nucleotide sequence in the human genome all are that those of ordinary skill in the art are known.

Receptor homolog can be used for understanding acceptor role in human body.This patent file improves the technology that makes these receptor mutations, thereby determines the non-endogenous, constitutively activated form of these acceptors.

Technology disclosed herein also can be applicable to other people GPCR known in the art, and this is conspicuous for those skilled in the art.

C. the screening of acceptor

Candidate compound is carried out the screening of the non-endogenous, constitutively activated form of GPCR disclosed herein and can directly identify the candidate compound that acts on cell surface receptor, and need not to use the endogenic ligand of acceptor.Use routine and common commercially available technology can determine that people GPCR endogenous form disclosed herein is expressed in vivo and/or the zone of excessive-expression.The expression position of acceptor in particular organization can be scientist the ability of determining the acceptor physiological function is provided.Use these technology also can determine the relative disease/illness situation relevant with expression of receptor and/or overexpression, this method is open in this patent file.In addition, the expression of acceptor in diseased organ can help people to determine the size of the clinical cognation of acceptor.

The activation of the composing type of GPCR disclosed herein is that described by inference proline residue is positioned at the TM6 of GPCR based on the distance of distance proline residue; This algorithmic technique is common unsettled, and the patent document PCT application number PCT/US99/23938 that has transferred the possession of usually, open on April 20th, 1 disclosed WO 00/22129 and listed herein other patent document, their are all introduced herein as a reference.This algorithmic technique is not that " comparison " predicted according to traditional sequence, but predict according to the specific range of the above-mentioned TM6 proline residue of distance (or, the endogenous, constitutively surrogate of this proline residue).By the amino acid (by inference, being arranged in the IC3 district of acceptor) apart from 16 amino-acid residue places of this residue is sported, most preferably lysine residue can obtain the composing type activation of acceptor.Other amino-acid residue also can reach this purpose in the sudden change of this position.

D. the evaluation of disease/illness and/or selection

Just as will be described in greater detail below, the inverse agonist of non-endogenous, constitutively activated GPCR and agonist can be identified by method of the present invention.This inverse agonist and agonist are to be used for the treatment of and the drug discovery of this receptor diseases related ideal candidates person of lead compound in the works.Because can directly identify the inverse agonist of GPCR, therefore can study the pharmaceutical composition that is used for the treatment of with GPCR diseases associated and illness.The expression position of acceptor in particular organization can be scientist the ability of determining the acceptor physiological function is provided.For example, the needs of academic research are not only in the existence of GPCR now in screening pathology and the healthy tissues sample, but also are the needs of identifying the endogenic ligand of specific GPCR.Tissue detection can be carried out in multiple health and pathological tissues.This tissue detection is for connecting the first step that provides possible to special receptor and disease and/or illness.In addition, the expression of acceptor in diseased organ can help people to determine the size of the clinical cognation of acceptor.According to content disclosed by the invention, in case receptor mapping in specific tissue or zone, believe that the technician just can infer the function of GPCR.

The dna sequence dna of GPCR can be used for making probe/primer.In some preferred embodiment, described dna sequence dna can be used for making probe, and this probe then is used for (a) and carries out the Dot blot analysis at tissue-mRNA, and/or (b) RT-PCR that expresses in tissue sample of acceptor identifies.The existence of acceptor in tissue or the pathological tissues, or compare; the raising of existing receptor level in the pathological tissues; can be used for position and functional cohesion with healthy tissues; and the physiological action/function that indicates acceptor; and set up treatment plan, include, but are not limited to treatment and this function/effect diseases associated.Acceptor also can be positioned in the zone of organ by this technology.With the acceptor known or supposition effect/function of localized particular organization therein, the deducibility acceptor infer physiological function.For example but be not restricted to, location/expressed proteins is relevant with awakening with sensorimotor process in the thalamus zone (sees Goodman ﹠amp; Gilman ' s, The Pharmacological Basis of Therapeutics, 9 ^ThEdition, the 465th page (1996)).In hippocampus or schwann's cell expressed proteins respectively with cognitive and memory, and perineural myelin form relevant (see, Kandel, E. etc., Essentials of Neural Science and Behavior, respectively the 657th, 680 and 28 page (1995)).In certain embodiments, probe and/or primer can be used for detecting and/or diagnosing the illness and/or illness, include, but are not limited to, below those diseases and the illness of identifying among the embodiment 6.Produce the method for this primer and/or probe and use primer and/or the method for probe in detecting disease and/or illness is known as those skilled in the art.

E. the screening of candidate compound

1. general GPCR screening assay technology

When the G protein receptor becomes constitutive activity, and it and G albumen (for example, Gq, Gs, Gi, Gz, Go) combination, and stimulate GTP to combine with G is proteic.G albumen plays the GTP enzyme then, and GTP is hydrolyzed to GDP, thus acceptor inactivation under normal circumstances.Yet the composing type activated receptors continues GDP is exchanged into GTP.The non-hydrolysable analogue of GTP [ ³⁵S] GTP γ S can be used for monitoring with combining of film whether improve this film expression composing type activated receptors.It is reported [ ³⁵S] GTP γ S can be used for monitoring and do not have under the situation that part exists, the coupling of G albumen and film.Know and effectively among other embodiment, one of them embodiment of this monitoring is reported in nineteen ninety-five by Traynor and Nahorski those skilled in the art.The use of this mensuration system is normally for the preliminary screening candidate compound, and this is because native system can be used for all g protein coupled receptors and irrelevant with the interactional specific G albumen of acceptor born of the same parents internal area usually.

2. specific GPCR screening assay technology

When using " generally " g protein coupled receptor assay method (that is, selection is the assay method of the compound of agonist or inverse agonist) to identify candidate compound, preferably further screen, to confirm that compound interacts acceptor site.For example, may be not and receptors bind by " generally " assay method compounds identified, but can be only from born of the same parents' internal area and G albumen " uncoupling ".

A.Gs, Gz and Gi

Gs stimulates adenylate cyclase.On the other hand, Gi (with Gz and Go) then suppresses adenylate cyclase.But adenylate cyclase catalysis ATP transforms to cAMP; Therefore, relevant with the increase of the composing type of Gs albumen coupling activation GPCR and cell cAMP level.On the other hand, (or Gz, Go) composing type of albumen coupling activation GPCR is relevant with the reduction of cell cAMP level with Gi.Usually, see, " Indirect Mechanisms of SynapticTransmission ", Chpt.8, From Neuron To Brain(3 ^RdEd.) Nichols, editors such as J.G., Sinauer Associates, Inc. (1992).Therefore, the assay method of detection cAMP can be used for also determining whether candidate compound is the inverse agonist (that is, whether this compound can reduce the level of cAMP) of acceptor.Can utilize the whole bag of tricks known in the art to measure cAMP; Most preferred method is to use anti--cAMP antibody in based on the form of ELISA.The assay method of available other type is full cell second messenger reporter molecule systems measurement method.Promotor on the gene can drive the expression of specific gene encoded protein.Ring AMP drives genetic expression by promoting the reactive DNA of cAMP-combination conjugated protein or transcription factor (CREB), and wherein said transcription factor (CREB) combines and drive expression of gene at specific site (cAMP response element) with promotor.Make up the reporter molecule system, make it have promotor, and this promotor for example contains a plurality of cAMP response elements at reporter gene before beta-galactosidase enzymes or the luciferase.Therefore, composing type activatory Gs-connects acceptor can cause the cAMP accumulation, then activated gene and cause the proteic expression of reporter gene.Report albumen can use the standard biochemical assay to detect (Chen etc., 1995) as beta-galactosidase enzymes or luciferase.

B.Go and Gq

Gq is relevant with the activation of Phospholipase C with Go, and they are hydrolytic phosphatide PIP successively ₂, discharge courier in two born of the same parents: diacylglycerol (DAG) and inositol 1,4,5-triphosphoric acid (IP ₃).IP ₃Build up relevant with the activation of Gq and Go-associated receptor.Usually see, " IndirectMechanisms of Synaptic Transmission ", Chpt.8, From Neuron To Brain(3 ^RdEd.) Nichols, editors such as J.G., Sinauer Associates, Inc. (1992).Detect IP ₃The cumulative assay method can be used for determining whether candidate compound is that (that is, whether this compound can reduce IP for the inverse agonist of Gq or Go associated receptor ₃Level).The Gq associated receptor also can use AP1 reporter assay method to detect, and wherein Gq dependency Phospholipase C can cause the activation of the gene that contains the AP1 element; Therefore, activatory Gq associated receptor can make this expression of gene increase, therefore, the reduction of the provable this expression of its inverse agonist, agonist then proves the increase of this expression.The assay method of carrying out this detection commercial be feasible.

3.GPCR fusion rotein

Endogenous, constitutively activated GPCR or non-endogenous, constitutively activated GPCR are used to screen the candidate compound, the purposes of directly identifying inverse agonist, agonist has proposed an interesting problem for screening, promptly, do not having endogenic ligand with it under the bonded situation, acceptor also is activated.Therefore, for distinguish the candidate compound exist under the situation non-endogenous receptor and not this compound have non-endogenous receptor under the situation, whether the purpose of this differentiation is to be inverse agonist or agonist or to the not influence at all of this acceptor, preferred used method can improve this differentiation for understanding this compound.Preferred method uses gpcr fusion protein.

Usually, in case use above-mentioned (reaching other) determination techniques to determine that non-endogenous GPCR is the composing type activatory, just can determine albumen with endogenous GPCR link coupled advantage G.The coupling of G albumen and GPCR provides appreciable signal pipeline.In certain embodiments, preferred screening is to use mammalian expression system to carry out, and wishing has endogenous G albumen in this system.Therefore, in this system, non-endogenous, constitutively activated GPCR will continue to send signal.In certain embodiments, preferably strengthen signal, like this under the situation that has receptor inverse agonists to exist, the acceptor that can easier differentiation contacts with inverse agonist is particularly when screening.

Gpcr fusion protein can improve the proteic effectiveness with non-endogenous GPCR link coupled G.Gpcr fusion protein is preferred for screening active GPCR of endogenous, constitutively or non-endogenous, constitutively activated GPCR, and this is because this method can be increased in the signal that uses in this triage techniques.Promote that significant " signal to noise ratio " is extremely important; This significant signal to noise ratio is preferred for screening candidate compound disclosed herein.

The structure that is used for the construct of gpcr fusion protein expression is that those skilled in the art are known.Commercially available expression vector and system provide the whole bag of tricks, can satisfy researchist's special requirement.The major criterion that makes up this gpcr fusion protein construct includes, but are not limited to, it is (preferred that endogenous GPCR sequence and G protein sequence all meet frame, the sequence of endogenous GPCR is positioned at the upstream of G protein sequence), and " termination " codon of GPCR is lacked or is replaced, when GPCR expressed, G albumen was also expressed like this.Other embodiment comprises that wherein endogenous GPCR sequence and G protein sequence do not meet the construct that frame and/or " termination " codon are not lacked or replace.GPCR can directly be connected with G albumen, or has residue (though this numeral can be definite at an easy rate by those of ordinary skills, preferably being no more than about 12) at interval between the two.For simplicity, preferably use transcribed spacer.Preferably, before making up the gpcr fusion protein construct, identify and non-endogenous GPCR link coupled G albumen.Therefore because have only minority G albumen to identify, preferably contain in the construct of G protein sequence (that is, general G albumen construct (seeing embodiment)) and can insert endogenous GPCR sequence; So just can be effectively, extensive screening has not homotactic various endogenous GPCR.

As mentioned above, expection and Gi, Gz and Go link coupled composing type activatory GPCR can suppress the formation of cAMP, this just makes challenging (promptly based on the assay method of these types GPCR, the cAMP signal reduces after activation, thereby directly identifies for example inverse agonist (it can further reduce this signal)).Just as disclosed here, we have determined, for the acceptor of these types, can produce the gpcr fusion protein that is not based on GPCR endogenous G albumen, make great efforts to set up feasible, based on the assay method of cyclase.Therefore, for example, endogenous Gi coupled receptor can with Gs albumen fusion-this fusion constructs when expressing, can " drive " or " promotions " endogenous GPCR and for example Gs, rather than " natural " Gi albumen coupling, so just can set up assay method based on cyclase.Therefore, for Gi, Gz and Go coupled receptor, in certain embodiments, preferably when using gpcr fusion protein, and assay method is detection with adenylate cyclase activity when being the basis, uses Gs (or stimulate that adenylate cyclase forms be equal to G albumen) to set up fusion constructs.

G albumen	The effect that GPCR activation back cAMP produces (that is, composing type activation or agonist combination)	GPCR activation back IP 3Cumulative effect (that is, composing type activation or agonist combination)	Contact the effect that back cAMP produces with inverse agonist	Contact back IP with inverse agonist 3The cumulative effect
					????Gs	Increase	????N/A	Reduce	????N/A
????Gi	Reduce	????N/A	Increase	????N/A
					????Gz	Reduce	????N/A	Increase	????N/A
????Go	Reduce	Increase	Increase	Reduce
					????Gq	????N/A	Increase	????N/A	Reduce

Utilize Gq albumen and Gs, Gi, the G albumen fusion constructs that Gz or Go albumen merge is effective equally.In certain embodiments, preferred fusion constructs can use Gq albumen to obtain, wherein the first six (6) individual amino acid of G-protein alpha-subunit (" G α q ") is lacked, and back five (5) the individual amino acid of G α q C-end are replaced by the corresponding amino acid of target G Protein G α.For example, fusion constructs can be Gq (6 aminoacid deletion) and the proteic fusion of Gi, obtains " Gq/Gi fusion constructs ".This fusion constructs can promote endogenous Gi coupled receptor endogenous G non-with it Protein G q coupling, so just can measure the second messenger, for example, and the generation of inositoltriphosphoric acid or diacylglycerol rather than cAMP.

4. the cotransfection (based on the assay method of cAMP) of target Gi link coupled GPCR and signal enhanser Gs link coupled GPCR

Known Gi coupled receptor can suppress adenylate cyclase, and therefore reduces the level that cAMP produces, and this just is not easy to assess the level of cAMP.Acceptor composing type activatory indication as the main coupling Gi in activation back, the effective technology of measuring the cAMP that produces of institute reduction can be by making the signal enhanser, for example, the GPCR cotransfection that activation back is main and the non-endogenous, constitutively activated acceptor of Gs link coupled (for example, following public TSHR-A623I) is connected with Gi and realizing.Obviously, the activation of the composing type of Gs coupled receptor can be measured according to the increase of generation cAMP.The composing type activation of Gi coupled receptor can cause the reduction of the cAMP that produces.Therefore, the cotransfection method is intended to effectively utilize the effect of these " on the contrary ".For example, non-endogenous, constitutively activated Gs coupled receptor (" signal enhanser ") provides the foundation the cAMP signal (promptly with the cotransfection of endogenous Gi coupled receptor (" target acceptor "), though the Gi coupled receptor can reduce the cAMP level, this " reduction " is for the cAMP level of being set up by composing type activatory Gs coupling signal enhanser rolls up).Then, by making the composing type activated form cotransfection of signal enhanser and target acceptor, and expection cAMP can further reduce (with respect to basal level), and this is because the functionally active of Gi target has increased (promptly reducing cAMP).

Then, use is based on the assay method screening candidate compound of cAMP, with respect to using endogenous receptor/G-albumen to merge, two places ' change ' have taken place: at first, for Gi link coupled target acceptor, can produce " opposite " effect, promptly, the inverse agonist of Gi link coupled target acceptor make the cAMP signal of surveying increase, the agonist of Gi link coupled target acceptor then makes this signal reduce; The second, clearly, the candidate compound of directly identifying in this way should be assessed separately, can not strengthen acceptor (this can finish) before or after the cotransfection acceptor is screened to guarantee these candidate compounds at signal.

F. pharmaceutical chemistry

Usually, but such was the case with, and the direct evaluation of candidate compound is to unite with the compound that produces by combinatorial chemistry technique to carry out, and thus, prepares thousands of kinds of compounds at random and carries out this analysis.Usually, this results of screening is the compound with unique core texture; After this, around preferred core texture these compounds are carried out other chemically modified, thereby further improve its pharmaceutical properties.This technology is known for those skilled in the art, need not to describe in detail in this patent file.

G. pharmaceutical composition

For further studying, the technology that can use those skilled in the art to know is mixed with pharmaceutical composition to selected candidate compound.Suitable pharmaceutically acceptable carrier be this area available those; For example, see Remington ' s PharmaceuticalSciences, 16 ^ThEdition, 1980, Mack Publishing Co., (editor such as Osol).

H. other purposes

Though the preferable use of the non-endogenous form of GPCR disclosed herein is the candidate compound of directly identifying as inverse agonist or agonist (being preferably used as medicament), these forms of GPCR also have other purposes.For example, the system of mixing in the external and body of GPCR can be used for further specifying and understands these acceptors role in normal people and patient, and understands when it is used to understand signal cascade and amplifies the effect of composing type activatory.In certain embodiments, endogenous receptor is " orphan receptor " preferably, does not promptly also identify the endogenic ligand of this receptor.Therefore in certain embodiments, before identifying endogenic ligand, the non-endogenous GPCR of modified can be used for understanding endogenous receptor role in human body.This acceptor also can be used for further specifying the approach of known receptor and their transduction signals.On the basis of file of the present invention, other purposes of disclosed acceptor is conspicuous for those skilled in the art.

Embodiment

The embodiment of following proposition is in order to illustrate, rather than limitation of the present invention.When specific nucleic acid disclosed herein and aminoacid sequence, those of ordinary skill in the art can carry out less modification to these sequences, obtain simultaneously and the following identical or similar substantially result who reports.Application or understanding are from a sequence to another sequence (for example, from rat receptor to people's acceptor, or from people's acceptor A to people's acceptor B) the traditional method of sequence box be that predict on the basis normally with the sequence alignment technology, determine common region by sequence is compared thus.Mutation method disclosed herein does not rely on this method, but based on algorithm with apart from the position distance of conservative proline residue in the TM6 district of people GPCR.In case this method is reliable, those skilled in the art just can carry out less modification to it, thus the essentially identical result of acquisition and (that is composing type activation) disclosed herein.This modifying method is considered to drop within the scope of the present disclosure.

Embodiment 1

1. endogenous people GPCR

Following cDNA acceptor is to utilize the technology of this part description to clone, and sees below.Table B has listed the disclosed acceptor of present patent application, the open reading-frame (ORF) of endogenous GPCR, nucleic acid and aminoacid sequence (table B).

Table B

Disclosed people GPCR	Open reading-frame (ORF) (base pair)	Nucleic acid SEQ ID NO.	Amino acid SEQ ID NO.
				????FPRL-2	????1,062bp	????1	????2
????STLR33	????1,029bp	????3	????4
				????GPR45	????1,119bp	????5	????6
????mGluR7	????2,748bp	????7	????8
				????GPR37	????l,842bp	????9	????10
????HF1948	????1,086bp	????11	????12
				????GPR66	????957bp	????13	????14
????GPR35	????930bp	????15	????16
				????ETBR-LP2	????1,446bp	????17	????18
????GPR26	????1,011bp	????97	????98

2. full-length clone scheme

a.FPRL-2(Seq.Id.Nos.1&2)

FPRL-2 is 1992, by Bao, and L. etc., 13 (2) Genomics 437-40 (1992) clone and order-checking.It is reported that FPRL-2 is positioned on the karyomit(e) 19, the sequence similarity of it and N-formyl peptide receptor sample-1 (FPR:-1), they the two and the total significant similarity of N-formyl peptide receptor (FPR).The endogenic ligand of FPR is a formyl peptides, yet, two homologues of FPR, FPRL-1 and FPRL-2 do not combine with same part, but are likely chemoattractant receptor.13(2)Genomics?437-40(1992)。It is reported that chemoattractant receptor participates in inflammation.FPRL-2 has coding 353 amino acid proteins, the GPCR of 1062bp open reading-frame (ORF).

PCR be with genome cDNA as template, the Laemmli buffer system Laemmli that uses rTth polysaccharase (Perkin Elmer) and manufacturers to provide, every kind of primer of 0.25 μ M and 4 kinds of Nucleotide of 0.2mM carry out.Cycling condition be 94 ℃ 1 minute, 64 ℃ of 1 minute 20 seconds and 72 ℃ circulation in 2 minutes 30 times.5 ' PCR contains the EcoRI site with following sequence

5’-AAAGATTCAGGTGTGGGAAGATGGAAACC-3’(SEQ?ID?NO.19)

And 3 ' primer contains the ApaI site with following sequence:

5’-AAAGGATCCCCGACCTCACATTGCTTGTA-3’(SEQ?ID?NO.20)

The PCR fragment digests with EcoRI and ApaI, and is cloned in the EcoRI-ApaI site of CMV expression vector.After this, measure also nucleic acid (SEQ IDNO.1) and amino acid (the SEQ ID NO.2) sequence of confirmer FPRL-2.

b.STLR33(Seq.Id.Nos.3&4)

PCR be with genome cDNA as template, the Laemmli buffer system Laemmli that uses rTth polysaccharase (Perkin Elmer) and manufacturers to provide, every kind of primer of 0.25 μ M and 4 kinds of Nucleotide of 0.2mM carry out.Cycling condition be 94 ℃ 1 minute, 62 ℃ of 1 minute 20 seconds and 72 ℃ circulation in 2 minutes 30 times.5 ' PCR contains the EcoRI site with following sequence

5’-CAGGAATTCATCAGAACAGACACCATGGCA-3’(SEQ?ID?NO.21)

And 3 ' primer contains the BamHI site with following sequence:

5’-GCAGGATCCAGAGCAGTTTTTTCGAAACCCT-3’(SEQ?ID?NO.22)

The PCR fragment digests with EcoRI and BamHI, and is cloned in the EcoRI-BamHI site of CMV expression vector.After this, measure also nucleic acid (SEQ IDNO.3) and amino acid (the SEQ ID NO.4) sequence of confirmer STRL33.

c.GPR45(Seq.Id.Nos.5&6)

PCR be with genome cDNA as template, the Laemmli buffer system Laemmli that uses rTth polysaccharase (Perkin Elmer) and manufacturers to provide, every kind of primer of 0.25 μ M and 4 kinds of Nucleotide of 0.2mM carry out.Cycling condition is as follows: 96 ℃ 2 minutes, 96 ℃ 30 seconds, 55 ℃ 20 seconds, 72 ℃ of 1 minute 20 seconds and 72 ℃ 5 minutes, wherein the 2-4 wheel repeats 35 times.5 ' PCR contains the HindIII site with following sequence

5’-TCCAAGCTTCAAGGGTCTCTCCACGATGGCCTG-3’(SEQ?ID?NO.23)

And 3 ' primer contains the EcoRI site with following sequence:

5’-TGCGAATTCTCTGTGGCCCCCTGACCCCCTAAA-3’(SEQ?ID?NO.24)

The PCR fragment digests with HindIII and EcoRI, and is cloned in the HindIII-EcoRI site of CMV expression vector.After this measure also nucleic acid (SEQ IDNO.5) and amino acid (the SEQ ID NO.6) sequence of confirmer GPR45.

Then, by use pfu PCR once more subclone in the V5-His carrier, use V5 mark cDNA, two used primers have following sequence:

5’-GGTAAGCTTACCATGGCCTGCAACAGCACGTCCCTT-3’(SEQ?ID?NO.25)

With

5’-GACGAATTCAACCGCAGACTGGTTTTCATTGCA-3’(SEQ?ID?NO.26)。

Cycling condition be 94 ℃ 1 minute, 60 ℃ of 2 minutes and 72 ℃ circulation in 2 minutes 30 times.

d.mGluR7(Seq.Id.Nos.7&8)

L-glutamic acid is a kind of excitatory neurotransmitter, and it exists in mammiferous brain in a large number.See Dingledine, R. etc., 130 (4S Suppl) Nutr.1039S (2000).Glutamate receptor has two classes, Ionized (part-gated ion channel) and metabolism tropism's (metabotropic (GPCR)).Metabolism tropism's glutamate receptor is the allos family of GPCR, and is relevant with some second messenger's approach, the excitability of adjustable ganglion neuron and cynapse conduction.(seeing Philips, T. etc., 57 (1) Brain Res Mol Brain Res 132 (1998)).It is reported that 7 type metabolism tropisms' glutamate receptor (mGluR7) is expressed in brain, hippocampus, the expression level in pallium and the cerebellum is the highest.See Makoff, A. etc., 40 (1) Brain Res Mol Brain Res 165 (1996).According to acceptor positioned area in brain, the estimation function effect of deducibility acceptor.For example,, infer that mGluR7 can be in depression although do not wish to be subjected to the constraint of any particular theory, anxiety, obesity, Alzheimer plays a role in pain and the apoplexy.

The cDNA of mGluR7 is by Elizabeth Hoffman, and the Ph.D. generosity provides.The used carrier of mGluR7 is pRcCMV (coding region of mGluR7 is by the EcoRI-ClaI site of subclone to the pCMV carrier).See the aminoacid sequence SEQ ID NO.8 of the nucleic acid sequence SEQ ID NO.7 of mGluR7 and deduction.

e.GPR37(Seq.Id.Nos.9&10)

The invention still further relates to people's GPR37.GPR37 is 1997, by Marazziti, and D. etc., 45 (1) Genomics 68-77 (1997) clone and order-checking.GPR37 has coding 613 amino acid proteins, the lonely GPCR of 1839bp open reading-frame (ORF).It is reported, GPR37 and Type B-endothelin sample receptor homolog, and at people's cerebral tissue, particularly corpus callosum, medullary substance is expressed in shell nuclear and the caudatum.

PCR be with brain cDNA as template, the Laemmli buffer system Laemmli that uses rTth polysaccharase (Perkin Elmer) and manufacturers to provide, every kind of primer of 0.25 μ M and 4 kinds of Nucleotide of 0.2mM carry out.Cycling condition be 94 ℃ 1 minute, 62 ℃ of 1 minutes and 72 ℃ circulation in 2 minutes 30 times.5 ' PCR contains the HindIII site with following sequence

5’-GCAAGCTTGTGCCCTCACCAAGCCATGCGAGCC-3’(SEQ?ID?NO.27)

And 3 ' primer contains the EcoRI site with following sequence:

5’-CGGAATTCAGCAATGAGTTCCGACAGAAGC-3’(SEQ?ID?NO.28)

1.9kb the PCR fragment with HindIII and EcoRI digestion, and be cloned in the HindIII-EcoRI site of CMVp expression vector.After this, measure also nucleic acid (SEQ ID NO.9) and amino acid (the SEQ ID NO.10) sequence of confirmer GPR37.

f.HF1948(Seq.Id.Nos.11&12)

The cDNA of HF1948 is by Elizabeth Hoffman, and the Ph.D. generosity provides.The used carrier of HF1948 is pRcCMV (coding region of HF1948 is by the HindIII-BamHI site of subclone to the pCMV carrier).See the aminoacid sequence SEQ ID NO.12 of the nucleic acid sequence SEQ ID NO.11 of HF1948 and deduction.

g.GPR66(Seq.Id.Nos.13&14)

The cDNA of people GPR66 (GenBank registration number AF044600 and AF044601) is following generation and is cloned in the pCMV expression vector: PCR is that template is carried out with the genomic dna, what first round PCR used is TaqPlus Precision polysaccharase (Stratagene), second that take turns that PCR uses is pfu polysaccharase (Stratagene), the Laemmli buffer system Laemmli that provides by manufacturers also has been provided, 0.25 every kind of primer of μ M, 4 kinds of Nucleotide of 0.2mM (TaqPlus Precision) or 0.5mM (pfu).When using pfu, contain 10%DMSO in the damping fluid.Cycling condition is: 94 ℃ 1 minute, when 65 ℃ of 1 minute and 72 ℃: (a) first round PCR1 minute; (b) second took turns PCR2 minute, circulate 30 times.Because there is intron in the coding region, therefore, use two groups of primers to produce eclipsed 5 ' and 3 ' fragment respectively.5 ' the segmental PCR primer is:

5 '-ACCATGGCTTGCAATGGCAGTGCGGCCAGGGGGCACT-3 ' (there is justice the outside)

(SEQ ID NO.29) and

5 '-CGACCAGGACAAACAGCATCTTGGTCACTTGTCTCCGGC-3 ' (inner antisense)

(SEQ?ID?NO.30)

3 ' the segmental PCR primer is:

5 '-GACCAAGATGCTGTTTGTCCTGGTCGTGGTGTTTGGCAT-3 ' (there is justice inside)

(SEQ ID NO.31) and

5 '-CGGAATTCAGGATGGATCGGTCTCTTGCTGCGCCT-3 ' (has the EcoRI site

Outside antisense) (SEQ ID NO.32)

5 ' and 3 ' fragment be by with first round PCR product as template, and use the outside of kinasesization to have adopted primer and outside antisense primer to carry out second and take turns PCR and link together.1.2kb the PCR fragment with EcoRI digestion, and be cloned into the flush end-EcoRI site of pCMV expression vector.After this, measure also nucleic acid (SEQ ID NO.13) and amino acid (the SEQ ID NO.14) sequence of confirmer GPR66.

h.GPR35(Seq.Id.Nos.15&16)

GPR35 has 309 amino acid whose sequences, endogenic ligand the unknown of GPR35 (O ' Dowd B. etc., 47 (2) Genomics 310 (1998)).After measured, GPR35 and idiosyncratic transcription factor interact as E2F, and it is essential for causing dna replication dna and final cell propagation.In cell, E2F and tumor suppressor gene coupling are as retinoblastoma.After this transcription factor construct phosphorylation, E2F discharges from the Rb gene, enters nucleus then.In nuclear, E2F and gene as the archaeal dna polymerase combination, cause dna replication dna, cause cell proliferation.

PCR be with genomic dna as template, the Laemmli buffer system Laemmli that uses rTth polysaccharase (Perkin Elmer) and manufacturers to provide, every kind of primer of 0.25 μ M and 4 kinds of Nucleotide of 0.2mM carry out.Cycling condition be 94 ℃ 1 minute, 62 ℃ of 1 minute and 72 ℃ 1 minute and 20 seconds circulate 30 times.5 ' PCR primer is with following sequence kinasesization

5’-GCGAATTCCGGCTCCCTGTGCTGCCCCAGG-3’(SEQ?ID?NO.33)

And 3 ' primer contains the BamHI site with following sequence:

5’-GCGGATCCCGGAGCCCCCGAGACCTGGCCC-3’(SEQ?ID?NO.34)

The PCR fragment of 1kb digests with BamHI, and is cloned in the EcoRV-BamHI site of CMVp expression vector.Whole 6 clones that check order contain the potential polymorphism, comprise the change of amino acid 294 from Arg to Ser.After this, measure also nucleic acid (SEQ ID NO.15) and amino acid (the SEQ ID NO.16) sequence of confirmer GPR35.

i.ETBR-LP2(Seq.Id.Nos.17&18)

ETBR-LP2 is 1998, by Valdenaire O. etc., 424 (3) FEBS Lett.193 (1998) clone and order-checking, the nucleic acid of deduction and aminoacid sequence are seen Fig. 1 of Valdenaire.ETBR-LP2 has coding 613 amino acid proteins, the open reading-frame (ORF) of 1839bp.It is reported ETBR-LP2 and Type B endothelin receptor (ETBR-LP) homology.In addition, about 47% homology of the aminoacid sequence of ETBR-LP2 and people GPR37.It is reported that ETBR-LP2 expresses (for example, pallium, the graceful neuroglia of interior capsular fibers and Burger (424FEBS Lett at 196)) in people's central nervous system.

PCR be with brain cDNA as template, the Laemmli buffer system Laemmli that uses rTth polysaccharase (Perkin Elmer) and manufacturers to provide, every kind of primer of 0.25 μ M and 4 kinds of Nucleotide of 0.2mM carry out.Cycling condition be 94 ℃ 1 minute, 65 ℃ of 1 minute and 72 ℃ 1.5 minutes circulate 30 times.5 ' PCR contains the EcoRI site with following sequence

5’-CTGGAATTCTCCTGCTCATCCAGCCATGCGG-3’(SEQ?ID?NO.35)

And 3 ' primer contains the BamHI site with following sequence:

5’-CCTGGATCCCCACCCCTACTGGGGCCTCAG-3’(SEQ?ID?NO.36)

The PCR fragment of gained 1.5kb digests with EcoRI and BamHI, and is cloned in the EcoRI-BamHI site of pCMV expression vector.After this, measure also nucleic acid (SEQ ID NO.17) and amino acid (the SEQ ID NO.18) sequence of confirmer ETBR-LP2.

j.GPR26(Seq.Id.Nos.97&98)

EST clones HIBB055, and one 3, the PCR fragment of 400bp is used to screen people's gene group λ-II library (particular order of Stratagene catalogue).Screening conditions are as follows: in the time of 55 ℃, filter is hybridized in the methane amide hybridization solution and is spent the night.Wash conditions is 65 ℃ and uses the 2XSSC/1%SDS washed twice, and 65 ℃ with .2X SSC/.1%SDS washed twice, each 20 minutes.Filter is placed on the film, and-80 ℃ of exposures are spent the night, and develop in second day.By under the same conditions, take turns the screening phage and further characterize positive plaque from original filler second.

(catalogue #937227, Stratagene), described probe is to produce from the new sequence that derives from the genomic library screening to survey human fetal brain cDNA storehouse Uni-ZAP XP carrier with the probe of 250bp then.The probe of 250bp is the Laemmli buffer system Laemmli that utilizes Taqplus Precision PCR system (Stratagene #600210) and manufacturers to provide, and produces by PCR.Loop parameter is as follows: 95 ℃ 45 seconds, 55 ℃ of 40 seconds and 72 ℃ 1 minute circulate 30 times, extend at last 10 minutes.The primer is as follows:

5 '-CGAGAAGGTGCTCAAGGTGGC-3 ' (SEQ ID NO.99) and

5’-GAGAAGAGCTCCACTAGCCTGGTGATCACA-3’(SEQ?ID?NO.100)。

Human fetal brain cDNA library is under the condition identical with genomic library, uses identical 250bp PCR fragment to survey, and just hybridization temperature is 42 ℃.Positive original filler is under the same conditions, when 55 ℃ hybridization temperature, takes turns by second that screening further characterizes.Positive plaque is to utilize the Sanger method, analyzes by sequence, and initiator codon obtains from one of them positive colony.

Use PfuTurbo archaeal dna polymerase (Stratagene#600250) to produce the full-length clone of people GPR26 by PCR then, parameter is as follows: 95 ℃ 45 seconds, 62 ℃ of 1 minute and 72 ℃ 1.2 minutes circulate 40 times, extend at last 10 minutes.Template used is people's fetal brain cDNA (Clonetech#7402-1), and primer is as follows:

5’-GAATTCATGAACTCGTGGGACGCGGGCCTGGCGGGC-3’(SEQ?ID?NO.101)

With

5’-CTCGAGTCACTCAGACACCGGCAGAATGTTCT-3’(SEQ?ID?NO.102)。

The fragment that is produced has one 5 ' EcoR1 joint and one 3 ' Xho1 joint.The PCR product digests with known ligase enzyme, and uses the quick test kit (Roche#1635379) that connects to be arrived expression vector pcDNA3.1 (+) EcoR1/XhoI site (Invitrogen#V790-20) by subclone.After this, determine also nucleic acid (SEQ IDNO.97) and amino acid (the SEQ ID NO.98) sequence of confirmer GPR26.

Embodiment 2

The preparation of non-endogenous, constitutively activated GPCR

Those skilled in the art have the ability to select the mutating technology of nucleotide sequence.What propose below is the method that is used to produce the some non-endogenous forms of top disclosed people GPCR.Following public sudden change is based on algorithm, conservative proline(Pro) (or its endogenic conservative substitution) residue (is arranged in the TM6 district of GPCR, near the TM6/IC3 interface) the 16th amino acid (being positioned at the IC3 district of GPCR) undergo mutation, preferably be mutated into L-Ala, Histidine, arginine or lysine residue most preferably are mutated into lysine residue.

1. site-directed mutagenesis

The preparation of non-endogenous people GPCR is the guidance according to manufacturers, uses TransformerSite-Directed ^TMMutagenesis kit (Clontech) or QuickChange ^TMSite-Directed ^TMMutagenesis kit (Stratagene is according to the guidance of manufacturers) is carried out on people GPCR.Use specified aligning primer (table C), make following GPCR sudden change according to the method described above.For for simplicity, also marked the codon mutation (table C) that is inserted among the people GPCR with standard form:

Table C

Recipient identifier	Codon mutation	5 '-3 ' direction, mutant nucleotide sequence underline (SEQ.ID.NO.)	5 '-3 ' direction (SEQ.ID.NO.)
				??FLPR-2	??T240K	TCCAGCCGTCCC AAACGT GTCTTCGCTGC(37)	CTCCTTCGGTCCTCCTA TCGTTGTCAGAAGT (38)
??STRL33	??L230K	CAGAAGCACAGATC AAAAAAGATCATCTTCCTG (39)	CTCCTTCGGTCCTCCTA TCGTTGTCAGAAGT (38)
				??mGluR7	??W590S	AGTGGCACTCCCCC TCGGCTGTGATTCCTGT(59)	ACAGGAATCACAGC C GAGGGGGAGTGCCAC T(40)
??R659H	TGTGTTCTTTCCGG CATG TTTTCTTGGGCTTG(41)	CAAGCCCAAGAAAAC ATGCCGG?AAGAACA CA(42)
			??T771C		CTCATGGTCACATGT TGTGTGTATGCCATCAAG (43)	CTTGATGGCATACAC A CAACATGTGACCATGA G(44)
??I790K	ACGAAGCCAAGCCC AAGGGATTCACTATGTACAC (45)	GTGTACATAGTGAATC C CTTGGGCTTGGCTCC GT(46)
			??GPR37		??L352R	GTCACCACCTTTCACC CG ATGTGCTCTGTGCATAG (47)	CTATGCACAGAGCAC A TCGGGTGAAAGGTG GTGAC(48)
??C543Y	CCTTTTGTTCTTTAAGTC C TATGTCACCCCAGTCCT (49)	AGGACTGGGGTGAC A TAGGACTTAAAGAAC AAAAGG(50)
				??HF1948	??I281F	ATGTGGAGCCCCATC TT CATCACCATCCTCC(51)	GGAGGATGGTGAT GA AGATGGGGCTCCACAT (52)
??E135N	GCCGCGGTCAGCCTG AA TCGCATGGTGTGCATC (53)	GATGCACACCATGCG ATTCAGGCTGACCGCG GC(54)
			??GPR66		??T273K	GGCCGGAGACAAGTG AAAAGATGCTGTIT(55)	AAACAGCATCTI TTTC ACTTGTCTCCGGCC (56)
??GPR35	??A216K	As seen the method for Xuan Zeing		As seen the method for Xuan Zeing
			??ETBR-LP2		??N358K	GAGAGCCAGCTC AAGAG CACCGTGGTG(57)	CTCCTTCGGTCCTCCTA TCGTTGTCAGAAGT (58)

1. but produce the system of selection of non-endogenous people GPCR

The preparation of non-endogenous, constitutively activated people GPR35 acceptor is finished by building the A216K sudden change.Mutagenesis is the guidance according to manufacturers, uses Transformer Site-Directed ^TMMutagenesis kit (Clontech) is carried out.(seeing nucleic acid sequence SEQ IDNO.84, aminoacid sequence SEQ ID NO.85).Two used mutagenic primers are Methionin mutagenic oligonucleotide and selective marker oligonucleotide, the following sequence of their tools:

5 '-GCCACCCGCAAGGCT AAACGCATGGTCTGG-3 ' (SEQ ID NO.60 has justice)

5 '-CTCCTTCGGTCCTCCTATCGTTGTCAGAAGT-3 ' (SEQ ID NO.61, antisense)

For first round PCR, SEQ ID NO.33 and SEQ ID NO.61 are used to produce 5, the fragment of 700bp, and SEQ ID NO.34 and SEQ ID NO.60 are used to produce 3, the fragment of 350bp.PCR be GDNA with endogenous GPR35 as template, use pfu polysaccharase (Stratagene) and manufacturers to provide, replenished the Laemmli buffer system Laemmli of 10%DMSO, every kind of primer of 0.25 μ M and 4 kinds of Nucleotide of 0.5mM carry out.Cycling condition be 94 ℃ 30 seconds, 65 ℃ of 1 minute and 72 ℃ 2 minutes and 20 seconds circulate 25 times.Use 5 ' and 3 ' the PCR fragment of first round PCR to carry out second as the common template then and take turns PCR, wherein the primer is oligonucleotide 1 and 2, and polysaccharase is above-mentioned pfu, and just annealing temperature is 55 ℃, and the extension time is 2 minutes.Described according to endogenous cDNA then, digestion gained PCR fragment, and its subclone in pCMV.

Then non-endogenous people GPCR is checked order, derives and the nucleic acid and the aminoacid sequence that confirm are listed in the annex " sequence table " of this patent file, summarize among the table D below:

Table D

Non-endogenous receptor	The nucleotide sequence table	The aminoacid sequence table
			????FPRL-2 ????L240K	????SEQ?ID?NO.62	????SEQ?ID?NO.63
????STRL33 ????L230K	????SEQ?ID?NO.64	????SEQ?ID?NO.65
			????MgluR7 ????W590S ????R659H ????T771C ????I790K	????SEQ?ID?NO.66 ????SEQ?ID?NO.68 ????SEQ?ID?NO.70 ????SEQ?ID?NO.72	????SEQ?ID?NO.67 ????SEQ?ID?NO.69 ????SEQ?ID?NO.71 ????SEQ?ID?NO.73
????GPR37 ????L352R ????C543Y	????SEQ?ID?NO.74 ????SEQ?ID?NO.76	????SEQ?ID?NO.75 ????SEQ?ID?NO.77
			????HF1948 ????I281F ????E135N	????SEQ?ID?NO.78 ????SEQ?ID?NO.80	????SEQ?ID?NO.79 ????SEQ?ID?NO.81
????GPR66 ????T273K	????SEQ?ID?NO.82	????SEQ?ID?NO.83
			????GPR35 ????A216K	????SEQ?ID?NO.84	????SEQ?ID?NO.85
????ETBR-LP2 ????N358K	????SEQ?ID?NO.86	????SEQ?ID?NO.87

Embodiment 3

Expression of receptor

Though but a lot of cells in this area expressing protein all preferably uses mammalian cell.By inference, major cause is feasibility, that is, express the use of the yeast cell of GPCR and may introduce non--mammalian cell, it may be (in fact, for yeast, really) do not comprise acceptor-coupling, heredity-mechanism and Secretory Pathway, and these have been used for mammlian system-therefore through development, the result who obtains in non--mammalian cell, though come in handy, those results that obtain not as the use mammalian cell are good.Though the specific mammalian cell that can use according to technician's special requirement forecasting institute, in mammalian cell, COS-7,293 and the 293T cell be particularly preferred.

A.293 the transient transfection of cell

First day, with 6 * 10 ⁶The density of individual cell/10cm culture dish is inoculated 293 cells.Second day, prepare two side reaction test tubes (each dull and stereotyped a test tube): test tube A is by 4 μ g DNA (for example, pCMV carriers; Have the pCMV carrier of receptor cdna etc.) be blended in 0.5ml and do not have preparation among the DMEM of serum (Gibco BRL); Test tube B is by preparing among the DMEM that 24 μ l fat transfection amine (Gibco BRL) are blended in 0.5ml do not have serum.Then test tube A and B upset (several times) are mixed incubated at room 30-45 minute.This mixture is known as " transfection mixture ".293 cells with 1XPBS washing bed board add the DMEM that 5ml does not have serum then.The 1ml transfection mixture is joined in the cell 37 ℃/5%CO ₂Hatched 4 hours.Remove transfection mixture by suction, add 10ml DMEM/10% foetal calf serum.37 ℃/5%CO ₂Incubated cell.After hatching 48 hours, gather cell, and analyze.

B. 293 stable clones

About 12 * 10 ⁶Individual 293 cell bed boards make them contain 10% foetal calf serum and 1% Sodium.alpha.-ketopropionate on the tissue culture plate of 15cm, grow in L-glutaminate and the antibiotic DME high concentration glucose substratum.293 cells after 24 hours (approximately-80% converges), use 12 μ g DNA transfectional cells at bed board.Described 12 μ g DNA do not have the DME high concentration glucose substratum of serum to mix with 60 μ l fat transfection amine and 2mL.Substratum sucking-off from flat board, once with the substratum washed cell that does not have serum.DNA, fat transfection amine and substratum mixture do not have the substratum of serum to be added in the flat board together with 10mL then.37 ℃ hatch 4-5 hour after, the sucking-off substratum adds the substratum that 25ml contains serum.After the transfection 24 hours, the sucking-off substratum adds and contains the fresh serum substratum once more.After the transfection 48 hours, the sucking-off substratum adds the substratum that contains serum, wherein also contains the Geneticin that final concentration is 500 μ g/mL (G418 medicine).Select to contain the positive transfectional cell of G418 resistant gene then.Every 4-5 days replacing substratum when selecting.In chosen process, the cell growth produces stable aggregate, or stable clonal selection is carried out in division.

C.RGT cell (being used for mGluR7)

The RGT cell is to obtain from the Syria hamster clone (AV12-664) that adenovirus transforms, wherein stable transfection glutaminate-aspartate translocator.

First day, with 5 * 10 ⁶The density inoculation RGT cell of individual cell/10cm culture dish.Second day, in test tube, add the substratum that 91 μ l do not have serum, then add 9 μ l Fugene 6 (Roche).In identical mixture, add 3 μ gDNA (0.5 μ g/ μ l).Mix this mixture gently, incubated at room 15 minutes then, dropwise adds the cell of growing among the DMEM/10%FBS to this mixture, 37 ℃/5%CO ₂Hatched 48 hours.After hatching 48 hours, the collection cell is also analyzed.

Embodiment 4

Be used to measure the assay method of the constitutive activity of non-endogenous GPCR

There are a lot of methods all to can be used to assess the constitutive activity of non-endogenous people GPCR.Illustrate below; Those of ordinary skill in the art have the ability to determine those technology the most useful to technician's needs.

1. film binding assay: [ ³⁵S] GTP γ S assay method

When g protein coupled receptor is in active condition, as part combination or composing type activatory result, acceptor and G albumen coupling, and the release of stimulation GDP reaches subsequently, and GPT combines with G is proteic.The alpha subunit of G albumen-acceptor complex body can play the GTP enzyme, and slowly GTP is hydrolyzed to GDP, and acceptor is usually at this inactivation.The composing type activated receptors continues GDP is exchanged into GTP.The GTP analogue of non-hydrolysable [ ³⁵S] GTP γ S can be used for confirming [ ³⁵S] raising that combines of GTP γ S and the film of expressing the composing type activated receptor.Use [ ³⁵S] GTP γ S includes, but are not limited in conjunction with measuring composing type activatory advantage: (a) can be common to all g protein coupled receptors; (b) it unlikely selects the molecule that influences cascade in the born of the same parents near the film surface.

This assay method utilized the g protein coupled receptor stimulation [ ³⁵S] GTP γ S and the membrane-bound ability of expressing associated receptor.Therefore, in the method that this assay method can be used for directly identifying, the candidate compound of screening composing type activatory g protein coupled receptor.This assay method is commonplace, can be used for the drug discovery of all g protein coupled receptors.

[ ³⁵S] GTP γ S assay method is at 20mM HEPES and the about 20mM MgCl of 1- ₂Though (preferred 20mM for making the best as a result, can adjust this amount) pH7.4 has about 0.3-1.2nM[ ³⁵S] the binding buffer liquid (though preferred 1.2,, can adjust) of GTP γ S to this amount for making the best as a result, 12.5-75 μ g membranin (for example, is expressed 293 cells of Gs fusion rotein; For making the best as a result, can adjust this amount) and 10 μ M GDP (, can adjust) to this amount for making the best as a result in hatched 1 hour.Add wheat germ agglutinin pearl (25 μ l then; Amersham), room temperature was hatched this mixture 30 minutes again.Then during room temperature, with centrifugal 5 minutes of test tube, in scintillometer, count with 1500xg.

2. based on the cAMP assay method of cell

Can be improved to Flash Plate based on the assay method design of cell ^TM(New England Nuclear Cat.No.SMP004A), thereby is used for thick plasma membrane to the adenylate cyclase enzyme reagent kit.The flicker coating can be contained in the dull and stereotyped hole of flicker, wherein also contains the specific antibody of discerning cAMP.The cAMP that produces in the hole can quantize by combining cAMP antibody with the competition of radioactivity cAMP tracer agent.Following is to measure the change of cAMP level in the full cell of expressed receptor and the concise and to the point scheme of carrying out.

About 24 hours collection cells transfected behind transient transfection.Careful sucking-off substratum also discards.In each Tissue Culture Dish, add 10ml PBS gently, careful then sucking-off.In each flat board, add 1ml Sigma cell dissociation damping fluid and 3ml PBS.Cell sucking-off from flat board, cell suspension is collected in the 50ml taper centrifuge tube.Room temperature 1,100rpm was cell centrifugation 5 minutes.The cell precipitation thing carefully is resuspended in (approximately 3ml/ flat board) among the PBS of proper volume.Give cell counting with hematimeter then, add extra PBS to the cell count that obtains suiting (to the final volume in about 50 μ l/ holes).

The cAMP standard and detect damping fluid (11ml detect damping fluid contain 1 μ Ci tracer agent [ ¹²⁵IcAMP (50 μ l)]) be according to the guidance of manufacturers preparation and keep.The mensuration damping fluid that is used to screen is a prepared fresh, and contains 50 μ l stimulation damping fluid, and 3 μ l test compounds (12 μ M measure concentration eventually) and 50 μ l cells are measured damping fluid and stored standby on ice.This assay method is by adding 50 μ l cAMP standards in suitable hole, then adding 50 μ l PBSA and begin in hole H-11 and H-12.In institute is porose, add 50 μ l and stimulate damping fluid.Use can disperse the needle-shaped tool of 3 μ l compound solutions that DMSO (or candidate compound of selecting) is joined in the suitable hole, and the concentration of mensuration eventually of test compound is 12 μ M, and always measuring volume is 100 μ l.Then cell is joined in the hole incubated at room 60 minutes.Xiang Kongzhong adds the detection mixture of 100 μ l tracer-containing cAMP.Flat board was hatched 2 hours again, then at WallacMicroBeta ^TMCount in the scintillometer.Infer the value in cAMP/ hole then according to the standard cAMP curve of each assay plate.

3.Gi the cotransfection of link coupled FPRL-2 and Gs/Gi fusion protein construct

The transfection mixture (embodiment 3A) that contains FPRL-2 and Gs/Gi fusion protein construct is removed by suction, adds 10ml DMEM/10% foetal calf serum then.37 ℃/5%CO ₂Hatch this cell.After hatching 48 hours, the collection cell is also analyzed.Carry out detecting according to the scheme of top embodiment 4 (2) then based on the cAMP of cell.

Owing to think the main Gi albumen coupling with active condition of endogenous FPRL-2, so the reduction of the cAMP that produces indicates that the non-endogenous form of disclosed FPRL-2 is a constitutive activity.Therefore, the candidate compound that influences the FPRL-2 acceptor by increase cAMP signal is an inverse agonist, and the FPRL-2 agonist can reduce the cAMP signal.See Fig. 1.

Fig. 1 demonstration is compared with Gs/Gi, and the activity of FPRL-2 has increased about 4 times.When the endogenous form of FPRL-2 and non-endogenous form were compared, Gs/Gi compared with control group, and the receptor active of non-endogenous FPRL-2 (" FPRL-2 (" FPRL-2 (L240K) ")) has increased about 3 times.Therefore, these data show that the endogenous of FPRL-2 and non-endogenous form are constitutive activities.

With reference to figure 9.In Fig. 9, compare with the endogenous form of GPR37 (" GPR37wt "), it is about 354% that the cAMP that non-endogenous GPR37 (L352R) produces has increased, and compare with GPR37wt, and the cAMP that GPR37 (C543Y) produces has increased about 189%.These data show that non-endogenous L352R and the C543Y form of GPR37 all are the composing type activatory.

4.Gi the cAMP of coupling target GPCR based on cell

TSHR is a kind of Gs link coupled GPCR, can cause the cAMP accumulation after the activation.TSHR is by the composing type activatory by making amino-acid residue 623 sudden changes (that is, alanine residue becomes the Isoleucine residue).Expection Gi coupled receptor can suppress adenylate cyclase, and therefore reduces the generation level of cAMP, and this just is difficult to assess the level of cAMP.Indicate as Gi coupled receptor composing type activatory, the effective technology of measuring cAMP generation reduction can be by making " signal enhanser ", the target GPCR cotransfection that for example most preferably non-endogenous, constitutively activated TSHR (TSHR-A623I) (or the active Gs coupled receptor of endogenous, constitutively) is connected with Gi is set up the basal level of cAMP and is realized.After the non-endogenous form of producing the Gi coupled receptor, make non-endogenous form and the signal enhanser cotransfection of target GPCR, this material can be used for screening.When using the cAMP assay method, this method can be used for effectively producing signal; This method preferred pin is directly identified candidate compound to the Gi coupled receptor.Should be pointed out that for Gi link coupled GPCR when using this method, the inverse agonist of target GPCR increases the cAMP signal, agonist then reduces the cAMP signal.

Cell is according to top embodiment 3A transfection.After about 24 hours, gather cells transfected at transient transfection.Carry out detecting according to the scheme among the top embodiment 4 (2) then based on the cAMP of cell.

Preferably, and just as previously noted, for guaranteeing the small molecules candidate compound at Gi link coupled target acceptor, rather than TSHR (A623I) for example, preferably under the situation that does not have the target acceptor to exist, at the direct Screening and Identification candidate compound of signal enhanser.

With reference to figure 3.Fig. 3 is in 293 cells, the comparative analysis of endogenous GPR45 (" GPR45wt ") and control group (" CMV ").Endogenous target acceptor GPR45 and signal enhanser TSHR (A623I) cotransfection.(CMV) compares with control group, do not having TSH, and under the situation that the endogenic ligand of tsh receptor exists, TSHR (A623I) shows that with the cotransfection of endogenous GPR45 the generation of cAMP has reduced about 96%.(CMV) compares with control group, and under the situation that has TSH to exist, endogenous GPR45 (" GPR45wt ") shows that the generation of cAMP has reduced about 73%.These data show that GPR45 is that endogenous, constitutively is active, and by the Gi albumen coupling.

With reference to figure 4 and table E.Table E is the summary of Fig. 4, and it is in 293 cells, endogenous mGluR7 (" mGluR7wt ") and the non-endogenous form (" W590S ", " R659H ", " T711C " and " I790K ") of some mGluR7 and the comparative analysis of control group (" pCMV ").The table E summarized do not have endogenic ligand (that is, and TSH) under the situation of Cun Zaiing, the generation of carrier cAMP, described carrier contains signal enhanser acceptor (that is TSHR (A623I)) and target acceptor (mGluR7); Under the situation that has TSH to exist, the per-cent (%) that the cAMP that signal enhanser and target acceptor cotransfection produce reduces; Under the situation that has TSH to exist, and the generation of cAMP between the endogenous form of the mGluR7 of TSHR (A623I) cotransfection and the non-endogenous form.This data presentation, mGluR7 compares with endogenous, and the non-endogenous form of mGluR7 (" W590S ", " R659H ", " T771C " and " I790K ") can reduce the generation of cAMP, and is therefore activated by composing type by top disclosed method.

Table E

The form of mGluR7	1) carrier-TSHR, (A623H) 2) mGluR7 form 3) there is not 16mU/ ml TSH, (pmol cAMP) is total to-transfection	1) carrier-TSHR, (A623I) 2) mGluR7 form 3) 16mU/ml TSH, (pmol cAMP) is total to-transfection	The per-cent (%) (TSH is arranged) that reduces between the endogenous of mGluR7 and the non-endogenous form	The mGluR7 inverse agonist	MGluR 7 agonists
						PCMV (not having TSHR)	4	--	--	Increase	Reduce
pCMV	23	288	--
				MgluR7wt	21	402	0
W590S	9	138	66
				R659H	7	156	61
T771C	7	156	61
				I790K	9	151	62

Above-mentioned data have been supported in mGluR 7 forms that the RGT transit cell dyes.With reference to figure 5.In Fig. 5, compare with the endogenous form of mGluR7 acceptor, it is about 52% that W590S shows that the generation of cAMP has reduced, and R659H shows and has reduced about 43%; The T771C demonstration has reduced about 5%; I790K shows that the generation of cAMP has reduced about 28%.

Because mGluR7 is main and the Gi coupling of active condition, so the reduction that cAMP produces indicates that the non-endogenous form of disclosed mGluR7 is a constitutive activity.Therefore, the candidate compound that influences the mGluR7 acceptor by increase cAMP signal is an inverse agonist, and the mGluR7 agonist then reduces the cAMP signal.Based on the data of Fig. 5 and 6, when 293 and the RGT cell in use the cAMP assay method, and when being used for TSHR composing type activatory cotransfection method, " W590S ", " R659H ", " T711C " and " I790K " are the non-endogenous forms of preferred mGluR7, most preferably " W590S ".

With reference to Figure 12.In Figure 12, to compare with the endogenous form of HF1948 (" wt "), the non-endogenous form of HF1948 (" I281F " and " E135N ") shows that respectively the generation of cAMP has reduced about 18% and 39%.These data show that non-endogenous I281F and the E135N form of HF1948 all are the composing type activatory.This reduction of cAMP shows that further these forms are Gi-link coupled.Except being the Gi-link coupled, Figure 11 also shows, the endogenous I281F form of HF1948 also can with GqG albumen coupling (seeing that the following examples 4 (5) (f)).

With reference to Figure 16.Figure 16 shows, compare with the cotransfection of TSHR-A623I with endogenous ETBR-LP2 (" WT ") (102.59pmolcAMP/ hole), during non-endogenous, constitutively activated ETBR-LP2 (" N358K ") (65.96pmol cAMP/ hole) and TSHR-A623I (" TSHR-A623I ") (under the situation that has TSH to exist) cotransfection, the cAMP that cell produces has reduced about 36%.When cotransfection with respect to pCMV and TSHR-A623I (290.75pmolcAMP/ hole), when the cotransfection of the cotransfection of ETBR-LP2 (" N358K ") and TSHR-A623I and ETBR-LP2 (" WT ") and TSHR-A623I was compared, the generation of their cAMP had reduced about 77% and 65% respectively.Preferably, this method is used to screen the inverse agonist that increases signal, and agonist should reduce signal.In order to confirm small molecules in conjunction with ETBR-LP2, and debond TSHR-A623I construct, preferably under the situation that does not have ETBR-LP2 to exist, at this construct screening small molecules.

5. based on the assay method of acceptor

A.Cre-Luc reporter assay method (Gs-associated receptor)

293 and the 293T cell with 2 * 10 ⁴The density of individual cells/well is laid on the 96 hole flat boards, and fat transfection amine reagent (BRL) transfection is used in the guidance according to manufacturers in second day.Be prepared as follows the DNA/ lipid mixtures and be used for the transfection of 6-hole: (the 260ng plasmid DNA is by 200ng 8xCRE-Luc receptor plasmid 2 μ l lipids among the 260ng plasmid DNA of 100 μ l DMEM and the 100 μ l DMEM, 50ng pCMV, wherein contain endogenous receptor or non-endogenous receptor or only contain pCMV and 10ng GPRS expression plasmid (GPRS is (Invitrogen) in pcDNA3)) mix gently.The 8XCRE-Luc receptor plasmid is prepared as follows: carrier S RIF-β-gal (71/+51) obtains by the clone rat somatostatin promotor of the BglV-HindIII site in p β gal-Basic carrier (Clontech).Eight (8) individual copies of cAMP response element are (to see from adenovirus template AdpCF126CCRE8 by PCR, 7 HumanGene Therapy 1883 (1996)) obtain, and be cloned into the Kpn-BalV site of SRIF-β-gal carrier, produce 8xCRE-β-gal reporter molecule carrier.8xCRE-Luc reporter molecule plasmid is to produce by the beta-galactosidase gene of replacing in 8xCRE-β-gal reporter molecule carrier with the luciferase gene in HindIII-BamHI site in the pGL3-basic carrier (Promega).After the incubated at room 30 minutes,, and in each hole, add the mixture that 100 μ l dilute with 400 μ l DMEM dilution DNA/lipid mixtures.After in the cell cultures incubator, hatching 4 hours, in each hole, add the DMEM that 100 μ l contain 10%FCS.Second day, change cells transfected with 200 μ l/ holes with the DMEM that contains 10%FCS.After 8 hours, after PBS washing once, 100 μ l/ holes are replaced by in the hole does not have phenol red DMEM.Second day, use LucLite according to the guidance of manufacturers ^TMReporter gene is measured the activity that test kit (Packard) is measured luciferase, and at 1450 MicroBeta ^TMFlicker luminescent counter (Wallac) is gone up reading.

With reference to figure 2.Fig. 2 is presented under the situation of 12.5ng STRL33 reporter molecule, and (CMV) compares with control group, and the activity of STRL33 has reduced about 50%.Under the proteic situation of 12.5ng, the endogenous form of STRL33 and non-endogenous form are compared, it is about 30% that the activity of reporter molecule that shows non-endogenous STRL33 (" STRL33 (L230K) ") has reduced, and under the proteic situation of 25ng, reduced about 40%.These data show that the non-endogenous form of STRL33 reporter molecule is a constitutive activity, and can with G Protein G i coupling.

B.AP1 reporter assay method (Gq associated receptor)

The method that detecting Gq stimulates depends on the known properties of Gq-dependency Phospholipase C, and it can cause the gene activation that contains the AP1 element in the promotor.According to the described scheme of above-mentioned CREB reporter assay method, use Pathdetect ^TMAP-1 cis-report system (Stratagene, catalogue #219073), just the component of calcium phosphate precipitation is 410ng pAP1-Luc, 80ng pCMV-reporter molecule expression plasmid and 20ng CMV-SEAP.

With reference to Figure 17.Figure 17 demonstration is compared with endogenous ETBR-LP2 (862 relative light units), and the activity of the non-endogenous, constitutively activated form of people ETBR-LP2 (" N358K ") (2203 relative light units) has increased by 61.1%.These data show that the non-endogenous form of ETBR-LP2 acceptor is a constitutive activity, and can with G Protein G i coupling.

C.SRF-Luc reporter assay method (Gq associated receptor)

The method that detecting Gq stimulates depends on the known properties of Gq-dependency Phospholipase C, and it can cause the gene activation that contains serum response factor in the promotor.Pathdetect ^TMSRF-Luc-report system (Stratagene) can be used for measuring the Gq coupling activity in the COS7 cell.Cell is the guidance according to manufacturers, uses the Mammals transfection ^TMThe expression plasmid transfection of the plasmid composition of test kit (Stratagene, Catalogue #200285) and system and coding endogenous or non-endogenous GPCR.Briefly, according to the guidance of manufacturers, 410ngSRF-Luc, 80ng pCMV-expression of receptor plasmid and 20ng CMV-SEAP (secretor type alkaline phosphatase expression plasmid; Alkaline phosphatase activities is to measure in the substratum of transfectional cell, thus the variation of transfection efficiency between the control sample) be combined in the calcium phosphate precipitation.Sedimentary half be evenly distributed in 3 holes of 96 hole flat boards, cell did not keep 24 hours in having the substratum of serum.Last 5 hours, with 1 μ M Angiotensin culturing cell.Dissolved cell according to the guidance of manufacturers, uses Luclite then ^TMTest kit (Packard, Cat.#6016911) and " Trilux 1450 Microbeta " liquid scintillation luminescent counter (Wallac) measure the activity of luciferase.These data can be used GraphPad Prism ^TM2.0a (GraphPadSoftware Inc.) analyzes.

D.SRE reporter assay method

In 293 cells, utilize SRE-Luc reporter gene (component of Mer cury luciferase system 3, Clontech Catalogue #K2053-1).Cell is the guidance according to manufacturers, the expression plasmid transfection of the plasmid composition of use fat transfection amine reagent (Gibco/BRL, Catalogue #18324-012) and system and coding endogenous or non-endogenous receptor.Briefly, according to the guidance of manufacturers, 420ng SRE-Luc, 50ng CMV (containing the GPR37 acceptor) and 30ng CMV-SEAP (secretor type alkaline phosphatase expression plasmid; Alkaline phosphatase activity is to measure in the substratum of transfectional cell, thus the variation of transfection efficiency between the control sample) be combined in positively charged ion lipid-DNA precipitation.Being supplemented to final volume with Optimem (Vendor) is 25 μ l.It is known as " template mixture ".Template mixture and fat transfection amine are blended in the polystyrene test tube, hatched 30 minutes.In the process of hatching, with 100 μ lOptimem washed cells.After hatching, 200 μ l 0ptimem are joined in the mixture 40 μ l-50 μ l/ holes.Cell spends the night in mixture.The next morning, adding DMEM/ with 130 μ l/ holes does not have phenol red/1%FBNS, changes substratum with fresh culture.According to the guidance of manufacturers, use Luclite then ^TMTest kit (Packard, Cat.#6016911) and " Trilux1450 Microbeta " liquid scintillation luminescent counter (Wallac) measure the uciferase activity of cell.Use GraphPad Prism ^TM(2.0a GraphPad Software Inc.) analytical data.

With reference to figure 7.In Fig. 7, when non-endogenous form (" C543Y ") that compares GPR37 and endogenous form (" wt "), it is about 316% that C543Y sudden change shows that than the wt form generation of cAMP has increased, but not endogenous form " L352R " shows that than the wt form generation of cAMP has increased about 178%.These data show that non-endogenous form A 543Y and the L352R of GPR37 are the composing type activatory.

E.E2F-Luc reporter assay method

In the 293A cell, use pE2F-Luc reporter molecule (component of Mercury luciferase system 3, Clontech Catalogue #K2053-1).Cell is the guidance according to manufacturers, the expression plasmid transfection of the plasmid composition of use fat transfection amine reagent (Gibco/BRL, Catalogue #18324-012) and system and coding endogenous or non-endogenous receptor.Briefly, according to the guidance of manufacturers, 400ng pE2F-Luc, 80ng CMV (containing the GPR35 acceptor) and 20ng CMV-SEAP (secretor type alkaline phosphatase expression plasmid; Alkaline phosphatase activity is to measure in the substratum of transfectional cell, thus the variation of transfection efficiency between the control sample) be combined in positively charged ion lipid-DNA precipitation.Sedimentary half be evenly distributed in 3 holes of 96-hole flat board, cell keeps spending the night, and replaces with fresh culture in second day.After the beginning transfection 48 (48) hours, handle cell, and, use Luclite according to the guidance of manufacturers ^TMTest kit (Packard, Cat.#6016911) and " Trilux 1450Microbeta " liquid scintillation luminescent counter (Wallac) measure the activity of luciferase.Use GraphPad Prism ^TM(2.0a GraphPad Software Inc.) analytical data.

With reference to Figure 14.Figure 14 shows that compare with endogenous GPR35 (24.97 relative light units), the activity of the non-endogenous, constitutively activity form (A216K) of people GPR35 (607.13 relative light units) has increased about 100%.These data show that GPR35 (A216K) and transcription factor E2F interact, and drive the expression of luciferase protein.With this interaction of E2F, show further that together with the expression of GPR35 in colon cancer cell GPR35 can work in cancer cell multiplication.Therefore, based on these data, the preferred candidate compound that influences the GPR35 acceptor is an inverse agonist.These data show that the inverse agonist of GPR35 can be used for treating cancer, particularly colorectal carcinoma.

F. IP in the born of the same parents ₃Accumulation assay method (Gq associated receptor)

The 1st day, usually with 1 * 10 ⁵The density of individual cells/well (though can optimize this numeral) bed board on 24 hole flat boards contains the cell of acceptor (endogenous and/or non-endogenous).The 2nd day, at first by mixing 0.25 μ g DNA and 2 μ l fat transfection amine in the DMEM/ hole that does not have serum at 50 μ l cell transfecting.Solution is mixed gently incubated at room 15-30 minute.Cell does not have 400 μ l the substratum of serum to mix with transfection media with 0.5ml PBS washing then, joins in the cell.Cell is at 37 ℃/5%CO ₂Hatched 3-4 hour, and removed transfection media, replace with the conventional growth medium in 1ml/ hole.The 3rd day, use ³H-inositol labeled cell.Briefly, remove substratum, with 0.5ml PBS washed cell.Add 0.5ml to every hole then and do not have the substratum (GIBCO-BRL) of inositol/do not have serum and 0.25 μ Ci ³The H-inositol, 37 ℃/5%CO ₂Spent the night in incubated cell 16-18 hour.The 4th day, with 0.5ml PBS washed cell, add 0.45ml and measure substratum, wherein containing does not have inositol/there is not the substratum of serum, 10 μ M Pargylines, the 10mM lithium chloride, or 0.4ml mensuration is coughed up foster base.Then 37 ℃ of incubated cells 30 minutes.With 0.5ml PBS washed cell, to every hole add 200 μ l fresh/ice-cold stop bath (1M KOH; The 18mM Sodium Tetraborate; 3.8mM EDTA).This solution is placed 5-10 minute (or up to cytolysis) on ice, use then 200 μ l fresh/neutralization of ice-cold neutralization solution (7.5%HCl).Lysate is transferred in the 1.5ml Eppendorf test tube, added 1ml chloroform/methanol (1: 2) in every test tube.Solution vortex 15 seconds is gone up sample to the upper strata mutually to Biorad AG1-X8 ^TMOn the ion exchange resin (100-200 order).At first, with 1: the washed resin of 1.25W/V, and the 0.9ml upper strata gone up sample mutually to post.Wash pillar with inositol and the 10ml 5m Sodium Tetraborate/60mM sodium formiate of 10ml5mM then.Inositoltriphosphoric acid is eluted in the flicker bottle, wherein contains 10ml flicker cocktail and 2ml 0.1M formic acid/1M ammonium formiate.By washing, and use dd H with 10ml 0.1M formic acid/3M ammonium formiate ₂Twice of O flushing and make pillar regeneration is stored in 4 ℃ the water.

With reference to figure 6.In Fig. 6, with the Gq albumen " Gq (disappearance) " that contains 6 aminoacid deletion; With Gq albumen " Gq (disappearance)/Gi " and non-endogenous mGluR7 that Gi albumen merges, T771C is Gq (disappearance) " T771C+Gq (disappearance) " and T771C and Gq (disappearance)/Gi " T771C+Gq (disappearance)/Gi " rotaring redyeing 293 cell in addition.Under the situation that has and do not have L-glutamic acid to exist, measure inositoltriphosphoric acid.Under the situation that has glutaminate to exist, to compare with Gq (disappearance)/Gi, the non-endogenous form of mGluR7 and the cotransfection of Gq (disappearance)/Gi show has approximately increased by 1850 times; Under the situation that has glutaminate to exist, compare with T771C+Gq (disappearance)/Gi, increased about 860 times.These data show, mGluR7, and a kind of Gi coupled receptor can pass through the Gq protein activation.Therefore, Gq (disappearance)/Gi fusion constructs can with the GPCR cotransfection, as the instrument of screening candidate compound.

With reference to Figure 11.In Figure 11, when the non-endogenous form (" I281F ") of HF1948 relatively during with endogenous form (" wt "), the I281F sudden change has made the IP3 cumulative rises about 361% than the wt form.These data show that the non-endogenous I281F form of HF1948 is composing type activation and Gq-link coupled.

Embodiment 5

The preparation of fusion rotein

The a.GPCR:Gs fusion constructs

Following the carrying out of design of composing type activatory GPCR-G albumen fusion constructs: to rat G Protein G s α (more microscler formula; Itoh, H. etc., 83 PNAS 3776 (1986)) 5 ' and 3 ' end carries out through engineering approaches, makes it contain HindIII (5 '-AAGCTT-3 ') sequence.After having confirmed correct sequence, (contain flank HindIII sequence), the HindIII restriction site that utilizes this carrier by subclone whole sequence be inserted into pcDNA3.1 (-) (Invitrogen, cat.no.V795-20) in.The correct direction of Gs α sequence is determined after subclone arrives among the pcDNA3.1 (-).Confirm on the HindIII sequence, to contain the modification pcDNA3.1 (-) of rat Gs α gene then; This carrier can be used as " general " Gs α protein carrier.PcDNA3.1 (-) carrier contains the various restriction sites of knowing in the upstream in HindIII site, therefore can advantageously insert the active GPCR encoding sequence of endogenous, constitutively in Gs albumen upstream.Same method also can be used for producing other " general " G protein carrier, certainly, but also known other commercially available or proprietary carrier of use technology personnel.In certain embodiments, important criterion is that the GPCR sequence is positioned at the proteic upstream of G, and meets frame with the proteic sequence of G.

Transcribed spacer between G albumen and the GPCR in the restriction site is chosen wantonly.There are justice and antisense primer to contain XbaI and EcoRV restriction site respectively, between G albumen and GPCR, will have transcribed spacer (belonging to restriction site) like this.

Then, utilize PCR to make each receptor sequence at above-mentioned Gs α universal support endomixis, method is as follows: the cDNA of 100ng GPCR is joined in the independent test tube, contain 2 μ l primers (justice and antisense are arranged) in each test tube, the dNTPs of 3 μ l 10mM, 10 μ l10XTaqPlus ^TMThe Precision damping fluid, 1 μ l TaqPlus ^TMPrecision polysaccharase (Stratagene:#600211) and 80 μ l water.Temperature of reaction and the cycle index of GPCR are as follows: 94 ℃ 1 minute; 94 ℃ 30 seconds; 62 ℃ 20 seconds; 72 ℃ 1 minute 40 seconds; With 72 ℃ 5 minutes, 2-4 circulation step repeats 35 times.Make the PCR product through 1% sepharose, purifying then.Purified product, is connected with each restriction site of Gs universal support behind the required insertion fragment purification with XbaI and EcoRV digestion.Transform the back and separate positive colony, and digest by Restriction Enzyme and to measure; Use 293 cell expressings to finish according to the method described above.Each positive colony to the GPCR-Gs fusion rotein checks order, to confirm exactness.

B.Gq (6 aminoacid deletion)/Gi fusion constructs

The design of Gq (disappearance)/Gi fusion constructs is following carrying out: G α q-subunit has TLESIM (SEQ ID NO.88) sequence of N-end six (6) individual amino acid (amino acid 2-7) and is lacked, and C-end five (5) the individual amino acid with EYNLV (SEQ ID NO.89) sequence are had the proteic corresponding amino acid replacement of G α i of sequence D CGLF (SEQ ID NO.90).This fusion constructs is to use following primer to obtain by PCR: 5 '-gatcAAGCTTCCATGGCGTGCTGCCTGAGCGAGG-3 ' (SEQ ID NO.91) and 5 ' gatcGGATCCTTAGAACAGGCCGCAGTCCTTCAGGTTCAGCTGCAGGATGGTG-3 ' (SEQ ID NO.92), wherein with the plasmid 63313 that contains mouse G α q-wild-type as template, mouse G α q-wild-type has the hemagglutinin mark.The Nucleotide of small letter is as transcribed spacer.

Use TaqPlus  Precision archaeal dna polymerase (Stratagene) amplification, cycling condition is as follows: 95 ℃ 2 minutes; 95 ℃ 20 seconds; 56 ℃ 20 seconds; 72 ℃ 2 minutes; With 72 ℃ 7 minutes, 2-4 step repeats 35 times.In pCRII-TOPO carrier (Invitrogen), use the PCR product cloning ABI Big dyestuff terminator test kit (P.E.Biosystems) to check order.By 2 step clones, the TOPO clone's who contains the fusion constructs sequence insertion fragment is inserted in the HindIII/BamHI site of expression vector pcDNA3.1 (+).

The c.Gs/Gi fusion protein construct

The design of Gs/Gi fusion protein construct is following to be carried out: C-end five (5) the individual amino acid that G α s-subunit has sequence 5 '-QYELL-3 ' (SEQ ID NO.93) are lacked, and have the corresponding amino acid replacement of sequence 5 '-DCGLF-3 ' (SEQ ID NO.94) with G α i albumen.This albumen fusion constructs is to use 5 ' and 3 ' oligonucleotide, obtains by PCR.

Use TaqPlus  Precision archaeal dna polymerase (Stratagene) to increase, cycling condition is as follows: 98 ℃ 2 minutes; 98 ℃ 30 seconds; 60 ℃ 30 seconds; 72 ℃ 2 minutes; With 72 ℃ 5 minutes, 2-4 step repeats 25 times.In pCRII-TOPO carrier (Invitrogen), use the PCR product cloning ABI Big dyestuff terminator test kit (P.E.Biosystems) to check order.The TOPO clone's who contains albumen fusion constructs sequence insertion fragment is inserted in the restriction site of expression vector pcDNA3.1 (+).Measure the nucleotide sequence of Gs/Gi albumen fusion constructs then.See nucleic acid sequence SEQ ID NO.95 and aminoacid sequence SEQ ID NO.96.

Embodiment 6

The preparation of schwann's cell

2L neonate rat cub (Sprague Dawley) (the 3rd day the 2nd day-postpartum of postpartum) is placed on deadly on ice.Remove young mouse, cut away head, blood is discharged.Its belly to being placed down on the autopsy table, with 70% ethanol cleaning and sterilizing.Use scalper to remove the skin of femoral region, up to exposing sciatic nerve (or up to seeing tenderly white " line " that from the spinal cord to the knee, extends).Nerve is placed in the DMEM substratum, sucking-off then then uses the DMEM culture medium supplemented to the 2.4ml volume, adds 300 μ L 10X collagenases (0.3%, Sigma Cat.#C-9891) and 300 μ L 10X trypsin 0.25%, GIBCO Cat.#25095-019) dissociate.Hatched neuropeptide 11 5 minutes for 37 ℃ then, 1, centrifugal 5 minutes of 000rpm removes substratum (repeating 2 times).Add 1mL DMEM-HEPES and 1mL DMEM/10%FBS, transfer to then in the 50mL taper test tube.Syringe needle with following specification is sheared test tube inclusion: 18G once, and 21G twice, twice of 23G.Inclusion is placed on the Falcon cell percolator low speed (approximately 1200rpm) rotation.Being supplemented to cumulative volume with DEME/10%FBS is 10mL, be plated on the 10cm flat board that poly-L-Lysine handled (Sigma, Cat.#P-1274) on.At 37 ℃, 7%CO ₂Moistening incubator in overnight incubation.Adding contains 100X ARA C, and (fresh culture Cat.#C-1768) was cultivated 48 hours again for 10mM, Sigma.Use PBS washed cell (3 times) then, remove ARA C, add: DMEM/10%FBS, (the 2 μ M of different concns in 100% ethanol, 5 μ M, 10 μ M, 20 μ M and 50 μ M) forskolin (Calbiochem, Cat#344270), 80 μ g hypophysis extract (Sigma among PBS and the 0.1%BSA, #P-1167), cell is at 37 ℃ then, 7%CO ₂Humidifier in the growth 30 hours.Gather cell, isolation of RNA is also analyzed.

Antibody is selected following carrying out: at first wash the flat board (3 times) that poly-L-Lysine was handled with 1X PBS, carried out tryptic digestion about 1 minute with 1mL 0.5% trypsinase-EDTA, then with 9mL DMEM-HEPES damping fluid and 10%FBS neutralization.1200rpm eccentric cell 5 minutes is resuspended among the 3mL DMEM-HEPES, washes out trypsinase, 1200rpm rotation 5 minutes.Then cell is resuspended among the 600 μ L DMEM-HEPES, some remaining after rotation substratum have single cell.Add 1: 1000 the dilution Thyl.1 antibody (monoclonal antibody, Sigma, Cat.#P-1274).

37 ℃ of incubated cells 20 minutes shook test tube once in per 2 minutes gently.Use before it, melt 20 μ L GPCs (GIBCO, Cat.#19195-015), then by complement being joined in the cell that contains antibody to final volume 1mL.At 37 ℃ of about 20-30 of water-bath incubated cell minutes, add 10mL DMEM-HEPES, rotation is 5 minutes during 1200rpm.Cell is resuspended among the 5mL DMEM/10%FBS, and joins in the flat board that poly-L-Lysine handled, this flat board contains hypophysis extract and forskolin.Make cellular-restoring 24-48 hour, repeat this immunoselection process 2 times.

Embodiment 7

The preparation of the rat sciatic nerve of crushing

Make the sciatic nerve of adult (10-13 week) the Sprague-Dawley rat of anesthesia (iso-florene) be exposed to the sciatic nerve otch.With the sciatic nerve 2 times of defeating the sciatic nerve incision, each 10 seconds, make crushed nerve with blunt-ended forceps; This technology can cause axonai degeneration, but also can make axon regeneration.Different time after nerve injury passes through CO ₂Suction makes animal dead, removes the neural stubble in distally, prunes 2-3mm the most nearby.For the nerve of crushing, collect whole distally nerve.Nerve is chilled in-80 ℃ of storages in the liquid nitrogen immediately.Int sciatic nerve is that never cotemporary animal obtains, P0 (crushing back)-P13.

Embodiment 8

The tissue distribution of disclosed people GPCR:

1.RT-PCR

RT-PCR can be used for confirming expressing and measures the tissue distribution of some new people GPCR.Used oligonucleotide is that GPCR-is specific, and (MTC is Clontech) as template with the cDNA of a plurality of tissues of people.According to the guidance of manufacturers, in 40 μ l reactants, use Taq archaeal dna polymerase (Stratagene) to increase.Sample on the 20 μ l reactants to 1.5% sepharose, is analyzed the RT-PCR product.

2. Dot blot

Use the endogenous GPCR of commercially available people-organize spot-trace form to survey, to determine the zone of receptor mapping.The PCR fragment of embodiment 1 is used as probe: radiolabeled probe is the guidance according to manufacturers, uses this fragment and Prime-It II ^TM(Stratagene #300385) produces the random primer labelling test kit.Make people RNA Master Blot ^TM(Clontech #7770-1) with the radiolabeled probe hybridization of GPCR, and according to the guidance of manufacturers, washs under stringent condition.In the time of-80 ℃, trace is exposed on Kodak BioMax radioautograph film spend the night.The tissue that following table F has listed acceptor and expressed therein.Discuss in embodiment 6 with receptor related representative disease/illness.

Table F

Recipient identifier	Tissue expression
		????STRL33	Placenta, spleen and lung
????GPR45	Central nervous system, brain
		????GPR37	Central nervous system, particularly cerebral tissue, hypophysis and placenta
????GPR66	Pancreas, bone, mammary gland, small intestine, and spleen
		????GPR26	Brain
????ETBR-LP2	Brain, hypophysis and placenta

3.RNA trace

a.GPR37

According to the guidance of manufacturers, utilize RNAzol B reagent (TelTest Inc., Cat.#CS-104) RNA of collection embodiment 6.After in 1% agarose/formaldehyde gel, carrying out electrophoresis, use 10X SSC RNA to be transferred on the nylon membrane (SachleicherSchull) by capillary action.According to the guidance of manufacturers, use synthetic with 3 ' terminal accurately corresponding dna fragmentation and the High Prime labelling kit (Roche MolecularBiochemical) of GPR37 ³²The GPR37 dna probe of P-mark.Use has replenished ExpressHyb solution (Clontech, Cat.#8015-2) following hybridization of 100 μ g/ml salmon sperm dnas.At first in the time of 65 ℃, hatch the film that contains the isolation of RNA sample with ExpressHyb solution and spend the night. ³²Placed it 5 minutes on ice by sex change by boiling 2 minutes for the GPR37 dna probe of P-mark, transfers to then in the ExpressHyb solution that soaks into film.After 65 ℃ of night incubation, remove the film in the hybridization solution, wash 4 times in 2XSSC/1%SDS in the time of 65 ℃, each 15 minutes, then in the time of 255 ℃, washing was 2 times in 0.2XSSC/1%SDS, each 15 minutes.The excessive moisture of trace is removed in jolting gently, then trace is wrapped in the plastic paper, in the time of-80 ℃, exposes on film and spends the night.

With reference to figure 9.Fig. 9 shows that GPR37 expresses in schwann's cell, myelin forms and can keep at 20 μ M forskolins like this.

Figure 10 shows that GPR37 is raised in the rat sciatic nerve of crushing, particularly behind crushed nerve seven (7) days.Such data are consistent with the data that Fig. 9 proposes, that is, GPR37 works to neurotization by stimulating the process that myelin forms in the schwann's cell.

GPR37 expresses in people's central nervous system, particularly brain.Determined further that GPR37 expresses in schwann's cell.When aixs cylinder (or neural) was impaired, schwann's cell made neurotization by form myelin around aixs cylinder, and it provides " interlayer " of myelin form.The process that myelin forms is very important, and this is because the effect possibility is moved fast, preserves metabolisable energy thus.Schwann's cell and precursor thereof have play a part important for survival and the differentiation that influence constitutes perineural other cell.In addition, determined that GPR37 expresses in the rat sciatic nerve of crushing.Such data have been supported the evidence that GPR37 works in nervous cell regenerating.Based on the known function of the specific tissue of receptor mapping, the estimation function of deducibility acceptor.Therefore, form too much at myelin under the situation of (for example, tumour takes place), the inverse agonist of preferred GPR37, and when the generation myelin formed very few (for example, degenerative disease is as diabetes), agonist was preferred.

b.GPR66

According to the guidance of manufacturers, (Gibco/BRL Cat#15596-018) separates total RNA that some pancreatic cells are (for example, HIT, ARIP, Tu6, RIN α TC, STC, NIT, and EcR-CHO) to use TRIzol reagent.After in 1% agarose/formaldehyde gel, carrying out electrophoresis, use standard scheme that RNA is transferred on the nylon film.According to the guidance of manufacturers, use that (Stratagene Cat.#300385) synthesizes with the accurately corresponding dna fragmentation of entire coded sequence and Prime It II random primer labelling test kit ³²The GPR66 probe of P-mark.Hybridization is to use the ExpressHyb solution that has replenished 100 μ g/ml salmon sperm dnas, and (Clontech Cat.#8015-2) carries out.At first in the time of 65 ℃, hatch the film 1 hour that contains the isolation of RNA sample with ExpressHyb solution. ³²Placed it 5 minutes on ice by sex change by boiling 2 minutes for the GPR66 dna probe of P-mark, transfers to then in the ExpressHyb solution that soaks into film.After 65 ℃ of night incubation, remove the film in the hybridization solution, in the time of 65 ℃, washing is 4 times in 2XSSC/1%SDS, and each 15 minutes, 55 ℃ the time, washing was 2 times in 0.1XSSC/0.5%SDS, each 15 minutes then.The excessive moisture in the trace is removed in jolting gently, then trace is wrapped in the plastic paper, in the time of-80 ℃, exposes on film and spends the night.

With reference to Figure 13.The result of RNA trace (see that Figure 13) together with the data presentation of spot-trace, GPR66 expresses in pancreas, show GPR66 at all islet cellss system and ARIP cell, promptly a kind of pancreas vessel cell be in great expression.Do not wish to be bound by any theory, the expression of GPR66 in pancreatic cell system shows that GPR66 can work in islet neogenesis.

c.GPR35

According to the guidance of manufacturers, (Gibco/BRL Cat#15596-018) separates total RNA of some cancerous cell lines (for example, RIN-5AH, HEP-G2, A549, HELA, MOLT-4, HL-60 and SW480 cell all are commercially available) to utilize TRIzol reagent.After in 1% agarose/formaldehyde gel, carrying out electrophoresis, use standard scheme that RNA is transferred on the nylon film.According to the guidance of manufacturers, use that (Stratagene Cat.#300385) synthesizes with the accurately corresponding dna fragmentation of entire coded sequence and Prime It II random primer labelling test kit ³²The GPR35 probe of P-mark.Hybridization is to use the ExpressHyb solution that has replenished 100 μ g/ml salmon sperm dnas, and (Clontech Cat.#8015-2) carries out.At first in the time of 65 ℃, hatch the film 1 hour that contains the isolation of RNA sample with ExpressHyb solution. ³²Placed it 5 minutes on ice by sex change by boiling 2 minutes for the GPR35 dna probe of P-mark, transfers to then in the ExpressHyb solution that soaks into film.After 65 ℃ of night incubation, remove the film in the hybridization solution, in the time of 65 ℃, washing is 4 times in 2XSSC/1%SDS, and each 15 minutes, in the time of 55 ℃, washing was 2 times in 0.1XSSC/0.5%SDS, each 15 minutes then.The excessive moisture in the trace is removed in jolting gently, then trace is wrapped in the plastic paper, in the time of-80 ℃, exposes on film and spends the night.

With reference to Figure 15.The result of RNA trace (sees, shows that Figure 15) GPR35 is great expression in colorectal cancer clone SW480.Such data show that GPR35 can work in colorectal cancer takes place.The candidate compound of identifying by following institute discussion method is inverse agonist most preferably.The inverse agonist of expection GPR35 can reduce dna replication dna, thus the cell proliferation of anticancer.GPR35 expresses in large intestine and small intestine.Determine that GPR35 expresses in colorectal cancer clone (for example, HELA, MOLT-4 and SW480) after, some cancerous cell lines have been checked.These data show that GPR35 can work in colorectal carcinoma takes place.Colorectal carcinoma is the malignant tumour of colon or rectum.Large bowel cancer is the second general cancer form of finding in masculinity and femininity.

d.ETBR-LP2

According to the guidance of manufacturers, utilize RNAzol B reagent (TelTest Inc., Cat.#CS-104) RNA of collection embodiment 6.After in 1% agarose/formaldehyde gel, carrying out electrophoresis, use 10X SSC RNA to be transferred to (Sachleicher Schull) on the nylon film by capillary action.According to the guidance of manufacturers, use synthetic with 3 ' terminal accurately corresponding dna fragmentation and the High Prime labelling kit (Roche MolecularBiochemical) of ETBR-LP2 ³²The ETBR-LP2 dna probe of P-mark.Hybridization is to use the ExpressHyb solution that replenished 100 μ g/ml salmon sperm dnas (Clontech, Cat.#8015-2) following carrying out.At first in the time of 65 ℃, hatch the film that contains the isolation of RNA sample with Express Hyb solution and spend the night. ³²Placed it 5 minutes on ice by sex change by boiling 2 minutes for the ETBR-LP2 dna probe of P-mark, transfers to then in the ExpressHyb solution that soaks into film.After 65 ℃ of night incubation, remove the film in the hybridization solution, in the time of 65 ℃, washing is 4 times in 2XSSC/1%SDS, and each 15 minutes, in the time of 55 ℃, washing was 2 times in 0.2XSSC/0.1%SDS, each 15 minutes then.The excessive moisture in the trace is removed in jolting gently, then trace is wrapped in the plastic paper, in the time of-80 ℃, exposes on film and spends the night.

With reference to Figure 18.Figure 18 shows that ETBR-LP2 expresses in schwann's cell, myelin forms and just can keep when 20 μ M forskolins like this.

With reference to Figure 19.Figure 19 show ETBR-LP2 in the rat sciatic nerve of crushing by adjusted, particularly after crushed nerve seven (7) days.Such data are consistent with the data that Figure 18 proposes, that is, ETBR-LP2 works in neurotization by stimulating the process that myelin forms in the schwann's cell.

Based on these data, ETBR-LP2 expresses in schwann's cell.When aixs cylinder (or neural) was impaired, schwann's cell made neurotization by form myelin around aixs cylinder, and it provides " interlayer " of myelin form.The myelin forming process is very important, and this is because the possibility of effect is moved fast, preserves metabolisable energy thus.Schwann's cell and precursor thereof play an important role for survival and the differentiation that influence constitutes perineural other cell.In addition, determined that ETBR-LP2 expresses in the rat sciatic nerve of crushing.Such data have been supported the evidence that ETBR-LP2 can work in nervous cell regenerating.Based on the known function of the specific tissue of receptor mapping, the estimation function of deducibility acceptor.Therefore, form too much at myelin under the situation of (for example, tumour takes place), the inverse agonist of preferred ETBR-LP2, and when the generation myelin formed very few (for example, degenerative disease is as diabetes), agonist was preferred.

Comprise with the receptor related disease and the illness that are arranged in these tissues or zone, but be not restricted to, heart disease and illness (for example, thrombosis, myocardial infarction, atherosclerosis, myocardosis); Kidney disease/illness (for example, renal failure, renal tubule acidosis, renal glycosuria, diabetes insipidus,nephrogenic, cystinuria, polycystic kidney); Eosinophilia; Leukocytosis; Oligoleukocythemia; Ovarian cancer; Sexual disorder; Polycystic ovarian syndrome; Pancreatitis and carcinoma of the pancreas; Irritable intestine syndrome; Colorectal carcinoma; Crohn disease; Ulcerative conjunctivitis; Diverticulitis; Chronic obstruction lung disease (COPD); Cystic fibrosis; Pneumonia; Pulmonary hypertension; Pulmonary tuberculosis and lung cancer; Parkinson's disease; Movement disorders and ataxia; Cognition and memory disease; Feed disease (for example, apocleisis, Bulimia nerovsa etc.); Fat; Cancer; Thymoma; Myasthenia gravis; Circulatory diseases; Prostate cancer; Prostatitis; Kidney disease/illness (for example, renal failure, renal tubule acidosis, renal glycosuria, diabetes insipidus,nephrogenic, cystinuria, polycystic kidney); Sensorimotor process and awakening disease; Obsessive-compulsive disorder; Carcinoma of testis; Priapism; Prostatitis; Hernia; Endocrinopathy; Sexual disorder; Irritated; Depressed; Psychosis; Migraine; Anti-stream; Schizophrenia; Ulcer; Bronchospasm; Epilepsy; Prostatomegaly; Anxiety; Rhinitis; Angina and glaucoma.Therefore, the inventive method also can be used for diagnosis and/or treats these and other disease and illness.

Embodiment 7

Scheme: directly identify inverse agonist and agonist

A.[ ³⁵S] GTP γ S assay method

Though the active GPCR of endogenous, constitutively has been used for directly identifying the candidate compound, for example, inverse agonist is owing to also not exclusively understand the difference aggravation in measuring.In certain embodiments, disclosed gpcr fusion protein also can use with non-endogenous, constitutively activated GPCR above.When using this albumen, as if the difference in measuring is stable basically, obtains effective signal to noise ratio thus.This more helps identifying the candidate compound.Therefore, in some preferred embodiment, preferably use gpcr fusion protein directly to identify, and when using it, utilize following mensuration scheme.

1. the preparation of film

Contain the active lonely gpcr fusion protein of target group moulding, and be used for directly identifying that the candidate compound film as inverse agonist or agonist is prepared as follows:

A. material

" film is scraped and got damping fluid " forms pH7.4 by 20mM HEPES and 10mM EDTA; " film lavation buffer solution " forms pH7.4 by 20mM HEPES and 0.1mM EDTA; " binding buffer liquid " by 20mM HEPES, 100mM NaCl and 10mM MgCl ₂Form pH7.4.

B. process

In the whole process, all material is all being preserved on ice.At first, sucking-off substratum from the cell monolayer that converges is then with the cold PBS washing of 10ml, sucking-off then.After this, adding 5ml film is scraped and is got the buffer extraction cell; Then cell extract is transferred in the 50ml centrifuge tube (4 ℃ the time, 20, centrifugal 17 minutes of 000rpm).The sucking-off supernatant liquor in 30ml film lavation buffer solution, is followed pellet resuspended in the time of 4 ℃ afterwards, and 20, centrifugal 17 minutes of 000rpm.Sucking-off supernatant liquor then, pellet resuspended in binding buffer liquid.Use Brinkman Polytron ^TMThe precipitation of homogenizer homogenizing resuspension (the 15-20 pulse per second (PPS) is in the state of suspension up to material).Be referred to as " membranin " herein.

2. Bradford albuminometry

After the homogenizing, and the protein concentration of use Bradford albuminometry mensuration film (albumen is diluted to about 1.5mg/ml, five equilibrium, and freezing (80 ℃) are standby; Under the freezing state, used scheme is as follows: measuring the same day, and the membranin of room temperature melting chilling, then rotation uses Polytron with about 12 * 1 then, the about 5-10 of 000rpm homogenizing second; Should be pointed out that for repeatedly preparing, between different preparation homogenizing, should thoroughly clean homogenizer).

A. material

(binding buffer liquid (as mentioned above) is used in Biorad, guidance cat.no.500-0006) according to manufacturers; The Bradford dye reagent; The Bradford protein standard substance.

B. process

Prepare two test tubes, one contains this film, and another is as " blank " contrast.Each all contains 800 μ l binding buffer liquid.Afterwards, in each test tube, add 10 μ l Bradford protein standard substances (1mg/ml), add 10 μ l membranins in the test tube (not being blank test tube) therein then.Afterwards, in each test tube, add 200 μ l Bradford dye reagent, vortexs then.After 5 minutes, test tube vortex once more, wherein substance transfer in cuvette.Use CECIL 3041 spectrophotometers then, read cuvette at 595 wavelength.

3. direct identification method

A. material

(Sigma cat.no.G-7127) forms the GDP damping fluid, then carries out a series of dilutions with binding buffer liquid, obtains 0.2 μ M GDP (final concentration of GDP is 0.1 μ M GDP in every hole) by 37.5ml binding buffer liquid and 2mg GDP; A candidate compound is contained in every hole, and final volume is 200 μ l, comprising 100 μ l GDP damping fluids (final concentration, 0.1 μ M GDP), 50 μ l be dissolved in membranin in the binding buffer liquid and 50 μ l be dissolved in the binding buffer liquid [ ³⁵S] GTP γ S (0.6nM) (2.5 μ l[ ³⁵S] GTP γ S/10ml binding buffer liquid).

B. process

The preferred 96-hole plate screening candidate compound (can be freezing) that uses at-80 ℃.(or have expression vector, do not have the film of gpcr fusion protein in the expression vector, in contrast) simple homogenizing is to suspension membranin.Use above-mentioned Bradford assay method to measure protein concentration then.With binding buffer liquid membranin (and control group) is diluted to 0.25mg/ml (measuring concentration, 12.5 μ g/ holes eventually).Afterwards, to Wallac Scintistrip ^TM(Wallac) add 100 μ l GDP damping fluids in each hole.Use 5 μ l needle-like-instruments that 5 μ l candidate compounds are transferred to (that is, 5 μ l are 1: 40 in the ratio that 200 μ l always measure in the volume, and the concentration of screening eventually of candidate compound is 10 μ M like this) in such hole.Have, for preventing to pollute, after each transfer step, needle-shaped tool is cleaned in storage 3 times, contain water (1X) in this storage, ethanol (1X) and water (2X)-excessive liquid gets rid of from instrument in each back of cleaning, and dries dry instrument with paper and Kim.Afterwards, 50 μ l membranins are joined in each hole (also can use the control wells that contains film, not have gpcr fusion protein in the film), room temperature preincubate 5-10 minute.Afterwards, 50 μ l be dissolved in the binding buffer liquid [ ³⁵S] GTP γ S (0.6nM) joins in each hole, and 60 minutes (in this embodiment, cover with paper tinsel dull and stereotyped) hatched in the room temperature vibration.In the time of 22 ℃, stopped in 15 minutes measuring by the 4000RPM Rotating Plates.Seal then with 8 road arm exhaustion flat boards, and with dull and stereotyped lid.Read dull and stereotyped (according to the guidance of manufacturers) with the Wallac 1450 that is arranged on " Prot.#37 " shelves then.

B. encircle the AMP assay method

Another assay method of directly identifying the candidate compound is to utilize to finish based on the assay method of cyclase.Except direct evaluation, this assay method also can be used as independently method confirm above-mentioned [ ³⁵S] result of GTP γ S method.

Preferably, use improved Flash Plate according to following scheme ^TMAdenylate cyclase enzyme reagent kit (New England Nuclear; Cat.No.SMP004A) directly identify candidate compound as GPCR inverse agonist and agonist.

About 3 days collection cells transfected after the transfection.By containing 20mM HEPES, pH7.4 and 10mM MgCl ₂Damping fluid in the cell that suspends of homogenizing and prepare film.Homogenizing is to use Brinkman Polytron ^TMCarried out about 10 seconds on ice.In the time of 4 ℃, with 49, the homogenate of the centrifugal gained of 000Xg 15 minutes.Then the gained pellet resuspended is being contained 20mM HEPES, in the damping fluid of pH7.4 and 0.1mM EDTA, homogenizing 10 seconds, then 4 ℃ the time, 49, centrifugal 15 minutes of 000Xg.Obtain to be deposited in-80 ℃ of storages standby.On the same day of carrying out direct evaluation and screening, room temperature is slowly melted the film precipitation, is containing 20mM HEPES, pH7.4 and 10mM MgCl ₂Damping fluid in resuspension, produce the albumen (be placed on standby) of final concentration 0.60mg/ml to the film of resuspension on ice.

According to the guidance of manufacturers, prepare and keep the cAMP standard substance and detect damping fluid (11ml detect contain in the damping fluid 2 μ Ci tracer agents [ ¹²⁵I cAMP (100 μ l)])).The mensuration damping fluid that prepared fresh is used to screen, it contains 20mM HEPES, pH7.4,10mM MgCl ₂, 20mM phosphocreatine (Sigma), 0.1 unit/ml creatine phosphokinase (Sigma), 50 μ M GTP (Sigma) and 0.2mM ATP (Sigma); Measuring damping fluid stores standby on ice.

Preferably the candidate compound of above-mentioned evaluation (if be in freezing state, room temperature melt), and 40 μ l membranins (30 μ g/ hole) and 50 μ l mensuration damping fluid join (3 μ l/ holes in the 96 hole plate wells; 12 μ M measure concentration eventually).During room temperature, vibration was hatched this mixture 30 minutes gently.

After hatching, in every hole, add 100 μ l and detect damping fluid, cultivated 2-24 hour.(according to the guidance of manufacturers) use " Prot.#31 " shelves are at Wallac MicroBeta then ^TMIn to plate count.

C. melanophore screening assay method

The method of GPCR candidate agonist or inverse agonist of identifying can be undertaken by the test cell of introducing pigment cell system, and wherein pigment cell system can react on particular stimulation and disperse and assemble pigment, and expresses the exogenous clone of coding GPCR.Stimulator, for example, light can be provided with the initial conditions that pigment distributes, and wherein pigment is assembled in the test cell, if activate GPCR, can induce pigment to disperse.Yet, thereby when the initial conditions that pigment dispersive pigment distributes being set,, can induce pigment to assemble if activate GPCR with the stimulator irritation cell.The test cell is contacted with compound, and change has taken place in the initial conditions whether distribution of the pigment in definite cell distributes from pigment.Because candidate compound and GPCR coupling, therefore in Petri dish, pigment cell dispersion place seems darker, and it is brighter that the pigment cell habitat then seems.

Material and method be at U.S. Patent number 5,462,856 and U.S. Patent number 6,051,386 in open, their are all introduced herein as a reference.

Though for those skilled in the art, can use various expression vectors, for endogenous and non-endogenous people GPCR, in certain embodiments, preferably use the pCMV carrier.According to the microbial preservation budapest treaty that is used for patented procedure, this carrier on October 13rd, 1998 in American type culture collection (ATCC) (10801University Blvd., Manassas, VA 20110-2209 USA) preservation.DNA is tested by ATCC, and determines to survive.ATCC has provided following preserving number pCMV:ATCC#203351.

Except as otherwise noted, the reference of quoting as proof in this patent file comprises whole the introducing as a reference of still unratified relevant patent application herein.In technician's authority, improvement that disclosed invention is carried out and expansion are also included within the scope of above-mentioned open and claim.

Sequence table

＜110〉Arena Pharmaceuticals Inc.

＜120〉endogenous of human G protein-coupled receptor and non-endogenous form

<130>AREN-0321

<160>102

<170>PatentIn?version?3.1

<210>1

<211>1062

<212>DNA

＜213〉people (Homo sapiens)

<400>1

atggaaacca?acttctccat?tcctctgaat?gaaactgagg?aggtgctccc?tgagcctgct????60

ggccacaccg?ttctgtggat?cttctcattg?ctagtccacg?gagtcacctt?tgtcttcggg????120

gtcctgggca?atgggcttgt?gatctgggtg?gctggattcc?ggatgacacg?cacagtcaac????180

accatctgtt?acctgaacct?ggccctagct?gacttctctt?tcagtgccat?cctaccattc????240

cgaatggtct?cagtcgccat?gagagaaaaa?tggccttttg?gctcattcct?atgtaagtta????300

gttcatgtta?tgatagacat?caacctgttt?gtcagtgtct?acctgatcac?catcattgct????360

ctggaccgct?gtatttgtgt?cctgcatcca?gcctgggccc?agaaccatcg?caccatgagt????420

ctggccaaga?gggtgatgac?gggactctgg?attttcacca?tagtccttac?cttaccaaat????480

ttcatcttct?ggactacaat?aagtactacg?aatggggaca?catactgtat?tttcaacttt????540

gcattctggg?gtgacactgc?tgtagagagg?ttgaacgtgt?tcattaccat?ggccaaggtc????600

tttctgatcc?tccacttcat?tattggcttc?agcgtgccta?tgtccatcat?cacagtctgc????660

tatgggatca?tcgctgccaa?aattcacaga?aaccacatga?ttaaatccag?ccgtccctta????720

cgtgtcttcg?ctgctgtggt?ggcttctttc?ttcatctgtt?ggttccctta?tgaactaatt????780

ggcattctaa?tggcagtctg?gctcaaagag?atgttgttaa?atggcaaata?caaaatcatt????840

cttgtcctga?ttaacccaac?aagctccttg?gcctttttta?acagctgcct?caacccaatt????900

ctctacgtct?ttatgggtcg?taacttccaa?gaaagactga?ttcgctcttt?gcccactagt????960

ttggagaggg?ccctgactga?ggtccctgac?tcagcccaga?ccagcaacac?agacaccact????1020

tctgcttcac?ctcctgagga?gacggagtta?caagcaatgt?ga???????????????????????1062

<210>2

<211>353

<212>PRT

＜213〉people

<400>2

Met?Glu?Thr?Asn?Phe?Ser?Ile?Pro?Leu?Asn?Glu?Thr?Glu?Glu?Val?Leu

1???????????????5???????????????????10??????????????????15

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

20??????????????????25??????????????????30

His?Gly?Val?Thr?Phe?Val?Phe?Gly?Val?Leu?Gly?Asn?Gly?Leu?Val?Ile

35??????????????????40??????????????????45

Trp?Val?Ala?Gly?Phe?Arg?Met?Thr?Arg?Thr?Val?Asn?Thr?Ile?Cys?Tyr

50??????????????????55??????????????????60

Leu?Asn?Leu?Ala?Leu?Ala?Asp?Phe?Ser?Phe?Ser?Ala?Ile?Leu?Pro?Phe

65??????????????????70??????????????????75??????????????????80

Arg?Met?Val?Ser?Val?Ala?Met?Arg?Glu?Lys?Trp?Pro?Phe?Gly?Ser?Phe

85??????????????????90??????????????????95

Leu?Cys?Lys?Leu?Val?His?Val?Met?Ile?Asp?Ile?Asn?Leu?Phe?Val?Ser

100?????????????????105?????????????????110

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

115?????????????????120?????????????????125

His?Pro?Ala?Trp?Ala?Gln?Asn?His?Arg?Thr?Met?Ser?Leu?Ala?Lys?Arg

130?????????????????135?????????????????140

Val?Met?Thr?Gly?Leu?Trp?Ile?Phe?Thr?Ile?Val?Leu?Thr?Leu?Pro?Asn

145?????????????????150?????????????????155?????????????????160

Phe?Ile?Phe?Trp?Thr?Thr?Ile?Ser?Thr?Thr?Asn?Gly?Asp?Thr?Tyr?Cys

165?????????????????170?????????????????175

Ile?Phe?Asn?Phe?Ala?Phe?Trp?Gly?Asp?Thr?Ala?Val?Glu?Arg?Leu?Asn

180?????????????????185?????????????????190

Val?Phe?Ile?Thr?Met?Ala?Lys?Val?Phe?Leu?Ile?Leu?His?Phe?Ile?Ile

195?????????????????200?????????????????205

Gly?Phe?Ser?Val?Pro?Met?Ser?Ile?Ile?Thr?Val?Cys?Tyr?Gly?Ile?Ile

210?????????????????215?????????????????220

Ala?Ala?Lys?Ile?His?Arg?Asn?His?Met?Ile?Lys?Ser?Ser?Arg?Pro?Leu

225?????????????????230?????????????????235?????????????????240

Arg?Val?Phe?Ala?Ala?Val?Val?Ala?Ser?Phe?Phe?Ile?Cys?Trp?Phe?Pro

245?????????????????250?????????????????255

Tyr?Glu?Leu?Ile?Gly?Ile?Leu?Met?Ala?Val?Trp?Leu?Lys?Glu?Met?Leu

260?????????????????265?????????????????270

Leu?Asn?Gly?Lys?Tyr?Lys?Ile?Ile?Leu?Val?Leu?Ile?Asn?Pro?Thr?Ser

275?????????????????280?????????????????285

Ser?Leu?Ala?Phe?Phe?Asn?Ser?Cys?Leu?Asn?Pro?Ile?Leu?Tyr?Val?Phe

290?????????????????295?????????????????300

Met?Gly?Arg?Asn?Phe?Gln?Glu?Arg?Leu?Ile?Arg?Ser?Leu?Pro?Thr?Ser

305?????????????????310?????????????????315?????????????????320

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

325?????????????????330?????????????????335

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

340?????????????????345?????????????????350

Met

<210>3

<211>1029

<212>DNA

＜213〉people

<400>3

atggcagagc?atgattacca?tgaagactat?gggttcagca?gtttcaatga?cagcagccag????60

gaggagcatc?aagccttcct?gcagttcagc?aaggtctttc?tgccctgcat?gtacctggtg????120

gtgtttgtct?gtggtctggt?ggggaactct?ctggtgctgg?tcatatccat?cttctaccat????180

aagttgcaga?gcctgacgga?tgtgttcctg?gtgaacctac?ccctggctga?cctggtgttt????240

gtctgcactc?tgcccttctg?ggcctatgca?ggcatccatg?aatgggtgtt?tggccaggtc????300

atgtgcaaaa?gcctactggg?catctacact?attaacttct?acacgtccat?gctcatcctc????360

acctgcatca?ctgtggatcg?tttcattgta?gtggttaagg?ccaccaaggc?ctacaaccag????420

caagccaaga?ggatgacctg?gggcaaggtc?accagcttgc?tcatctgggt?gatatccctg????480

ctggtttcct?tgccccaaat?tatctatggc?aatgtcttta?atctcgacaa?gctcatatgt????540

ggttaccatg?acgaggcaat?ttccactgtg?gttcttgcca?cccagatgac?actggggttc????600

ttcttgccac?tgctcaccat?gattgtctgc?tattcagtca?taatcaaaac?actgcttcat????660

gctggaggct?tccagaagca?cagatctcta?aagatcatct?tcctggtgat?ggctgtgttc????720

ctgctgaccc?agatgccctt?caacctcatg?aagttcatcc?gcagcacaca?ctgggaatac????780

tatgccatga?ccagctttca?ctacaccatc?atggtgacag?aggccatcgc?atacctgagg????840

gcctgcctta?accctgtgct?ctatgccttt?gtcagcctga?agtttcgaaa?gaacttctgg????900

aaacttgtga?aggacattgg?ttgcctccct?taccttgggg?tctcacatca?atggaaatct????960

tctgaggaca?attccaagac?tttttctgcc?tcccacaatg?tggaggccac?cagcatgttc????1020

cagttatag????????????????????????????????????????????????????????????1029

<210>4

<211>342

<212>PRT

＜213〉people

<400>4

Met?Ala?Glu?His?Asp?Tyr?His?Glu?Asp?Tyr?Gly?Phe?Ser?Ser?Phe?Asn

1???????????????5???????????????????10??????????????????15

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

20??????????????????25??????????????????30

Phe?Leu?Pro?Cys?Met?Tyr?Leu?Val?Val?Phe?Val?Cys?Gly?Leu?Val?Gly

35??????????????????40??????????????????45

Asn?Ser?Leu?Val?Leu?Val?Ile?Ser?Ile?Phe?Tyr?His?Lys?Leu?Gln?Ser

50??????????????????55??????????????????60

Leu?Thr?Asp?Val?Phe?Leu?Val?Asn?Leu?Pro?Leu?Ala?Asp?Leu?Val?Phe

65??????????????????70??????????????????75??????????????????80

Val?Cys?Thr?Leu?Pro?Phe?Trp?Ala?Tyr?Ala?Gly?Ile?His?Glu?Trp?Val

85??????????????????90??????????????????95

Phe?Gly?Gln?Val?Met?Cys?Lys?Ser?Leu?Leu?Gly?Ile?Tyr?Thr?Ile?Asn

100?????????????????105?????????????????110

Phe?Tyr?Thr?Ser?Met?Leu?Ile?Leu?Thr?Cys?Ile?Thr?Val?Asp?Arg?Phe

115?????????????????120?????????????????125

Ile?Val?Val?Val?Lys?Ala?Thr?Lys?Ala?Tyr?Asn?Gln?Gln?Ala?Lys?Arg

130?????????????????135?????????????????140

Met?Thr?Trp?Gly?Lys?Val?Thr?Ser?Leu?Leu?Ile?Trp?Val?Ile?Ser?Leu

145?????????????????150?????????????????155?????????????????160

Leu?Val?Ser?Leu?Pro?Gln?Ile?Ile?Tyr?Gly?Asn?Val?Phe?Asn?Leu?Asp

165?????????????????170?????????????????175

Lys?Leu?Ile?Cys?Gly?Tyr?His?Asp?Glu?Ala?Ile?Ser?Thr?Val?Val?Leu

180?????????????????185?????????????????190

Ala?Thr?Gln?Met?Thr?Leu?Gly?Phe?Phe?Leu?Pro?Leu?Leu?Thr?Met?Ile

195?????????????????200?????????????????205

Val?Cys?Tyr?Ser?Val?Ile?Ile?Lys?Thr?Leu?Leu?His?Ala?Gly?Gly?Phe

210?????????????????215?????????????????220

Gln?Lys?His?Arg?Ser?Leu?Lys?Ile?Ile?Phe?Leu?Val?Met?Ala?Val?Phe

225?????????????????230?????????????????235?????????????????240

Leu?Leu?Thr?Gln?Met?Pro?Phe?Asn?Leu?Met?Lys?Phe?Ile?Arg?Ser?Thr

245?????????????????250?????????????????255

His?Trp?Glu?Tyr?Tyr?Ala?Met?Thr?Ser?Phe?His?Tyr?Thr?Ile?Met?Val

260?????????????????265?????????????????270

Thr?Glu?Ala?Ile?Ala?Tyr?Leu?Arg?Ala?Cys?Leu?Asn?Pro?Val?Leu?Tyr

275?????????????????280?????????????????285

Ala?Phe?Val?Ser?Leu?Lys?Phe?Arg?Lys?Asn?Phe?Trp?Lys?Leu?Val?Lys

290?????????????????295?????????????????300

Asp?Ile?Gly?Cys?Leu?Pro?Tyr?Leu?Gly?Val?Ser?His?Gln?Trp?Lys?Ser

305?????????????????310?????????????????315?????????????????320

Ser?Glu?Asp?Asn?Ser?Lys?Thr?Phe?Ser?Ala?Ser?His?Asn?Val?Glu?Ala

325?????????????????330?????????????????335

Thr?Ser?Met?Phe?Gln?Leu

340

<210>5

<211>1119

<212>DNA

＜213〉people

<400>5

atggcctgca?acagcacgtc?ccttgaggct?tacacatacc?tgctgctgaa?caccagcaac????60

gcctcagact?cggggtccac?ccagttgccc?gcacccctca?ggatctcctt?ggccatagtg????120

atgctgctga?tgaccgtggt?ggggttcctg?ggcaacactg?tggtctgcat?catcgtgtac????180

cagaggccgg?ctatgcgctc?ggccatcaac?ctgctgctgg?ccaccctggc?cttctccgac????240

atcatgctgt?ccctctgctg?catgcccttc?accgccgtca?ccctcatcac?cgtgcgctgg????300

cactttgggg?accacttctg?ccgcctctca?gccacgctct?actggttttt?tgtcctggag????360

ggcgtggcca?tcctgctcat?catcagcgtg?gaccgcttcc?tcatcatcgt?ccagcgccag????420

gacaagctga?acccgcgcag?ggccaaggtg?atcatcgcgg?tctcctgggt?gctgtccttc????480

tgcatcgcgg?ggccctcgct?cacgggctgg?acgctggtgg?aggtgccggc?gcgggcccca????540

cagtgcgtgc?tgggctacac?ggagctcccc?gctgaccgcg?catacgtggt?caccttggtg????600

gtggccgtgt?tcttcgcgcc?ctttggcgtc?atgctgtgcg?cctacatgtg?catcctcaac????660

acggtccgca?agaacgccgt?gcgcgtgcac?aaccagtcgg?acagcctgga?cctgcggcag????720

ctcaccaggg?cgggcctgcg?gcgcctgcag?cggcagcaac?aggtcagcgt?ggacttgagc????780

ttcaagacca?aggccttcac?caccatcctg?atcctcttcg?tgggcttctc?cctctgctgg????840

ctgccccact?ccgtctacag?cctcctgtct?gtgtttagcc?agcgctttta?ctgcggttcc????900

tccttctacg?ccaccagcac?ctgcgtcctg?tggttcagtt?acctcaagtc?cgtcttcaac????960

cccatcgtct?actgctggag?aatcaaaaaa?ttccgcgagg?cctgcataga?gttgctgccc????1020

cagaccttcc?aaatcctccc?caaagtgcct?gagcggatcc?gaaggagaat?ccagccaagc????1080

acagtatacg?tgtgcaatga?aaaccagtct??gcggtttag??????????????????????????1119

<210>6

<211>372

<212>PRT

＜213〉people

<400>6

Met?Ala?Cys?Asn?Ser?Thr?Ser?Leu?Glu?Ala?Tyr?Thr?Tyr?Leu?Leu?Leu

1???????????????5???????????????????10??????????????????15

Asn?Thr?Ser?Asn?Ala?Ser?Asp?Ser?Gly?Ser?Thr?Gln?Leu?Pro?Ala?Pro

20??????????????????25??????????????????30

Leu?Arg?Ile?Ser?Leu?Ala?Ile?Val?Met?Leu?Leu?Met?Thr?Val?Val?Gly

35??????????????????40??????????????????45

Phe?Leu?Gly?Asn?Thr?Val?Val?Cys?Ile?Ile?Val?Tyr?Gln?Arg?Pro?Ala

50??????????????????55??????????????????60

Met?Arg?Ser?Ala?Ile?Asn?Leu?Leu?Leu?Ala?Thr?Leu?Ala?Phe?Ser?Asp

65??????????????????70??????????????????75??????????????????80

Ile?Met?Leu?Ser?Leu?Cys?Cys?Met?Pro?Phe?Thr?Ala?Val?Thr?Leu?Ile

85??????????????????90??????????????????95

Thr?Val?Arg?Trp?His?Phe?Gly?Asp?His?Phe?Cys?Arg?Leu?Ser?Ala?Thr

100?????????????????105?????????????????110

Leu?Tyr?Trp?Phe?Phe?Val?Leu?Glu?Gly?Val?Ala?Ile?Leu?Leu?Ile?Ile

115?????????????????120?????????????????125

Ser?Val?Asp?Arg?Phe?Leu?Ile?Ile?Val?Gln?Arg?Gln?Asp?Lys?Leu?Asn

130?????????????????135?????????????????140

Pro?Arg?Arg?Ala?Lys?Val?Ile?Ile?Ala?Val?Ser?Trp?Val?Leu?Ser?Phe

145?????????????????150?????????????????155?????????????????160

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

165?????????????????170?????????????????175

Ala?Arg?Ala?Pro?Gln?Cys?Val?Leu?Gly?Tyr?Thr?Glu?Leu?Pro?Ala?Asp

180?????????????????185?????????????????190

Arg?Ala?Tyr?Val?Val?Thr?Leu?Val?Val?Ala?Val?Phe?Phe?Ala?Pro?Phe

195?????????????????200?????????????????205

Gly?Val?Met?Leu?Cys?Ala?Tyr?Met?Cys?Ile?Leu?Asn?Thr?Val?Arg?Lys

210?????????????????215?????????????????220

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

225?????????????????230?????????????????235?????????????????240

Leu?Thr?Arg?Ala?Gly?Leu?Arg?Arg?Leu?Gln?Arg?Gln?Gln?Gln?Val?Ser

245?????????????????250?????????????????255

Val?Asp?Leu?Ser?Phe?Lys?Thr?Lys?Ala?Phe?Thr?Thr?Ile?Leu?Ile?Leu

260?????????????????265?????????????????270

Phe?Val?Gly?Phe?Ser?Leu?Cys?Trp?Leu?Pro?His?Ser?Val?Tyr?Ser?Leu

275?????????????????280?????????????????285

Leu?Ser?Val?Phe?Ser?Gln?Arg?Phe?Tyr?Cys?Gly?Ser?Ser?Phe?Tyr?Ala

290?????????????????295?????????????????300

Thr?Ser?Thr?Cys?Val?Leu?Trp?Phe?Ser?Tyr?Leu?Lys?Ser?Val?Phe?Asn

305?????????????????310?????????????????315?????????????????320

Pro?Ile?Val?Tyr?Cys?Trp?Arg?Ile?Lys?Lys?Phe?Arg?Glu?Ala?Cys?Ile

325?????????????????330?????????????????335

Glu?Leu?Leu?Pro?Gln?Thr?Phe?Gln?Ile?Leu?Pro?Lys?Val?Pro?Glu?Arg

340?????????????????345?????????????????350

Ile?Arg?Arg?Arg?Ile?Gln?Pro?Ser?Thr?Val?Tyr?Val?Cys?Asn?Glu?Asn

355?????????????????360?????????????????365

Gln?Ser?Ala?Val

370

<210>7

<211>2748

<212>DNA

＜213〉people

<400>7

atggtccagc?tgaggaagct?gctccgcgtc?ctgactttga?tgaagttccc?ctgctgcgtg????60

ctggaggtgc?tcctgtgcgc?gctggcggcg?gcggcgcgcg?gccaggagat?gtacgccccg????120

cactcaatcc?ggatcgaggg?ggacgtcacc?ctcggggggc?tgttccccgt?gcacgccaag????180

ggtcccagcg?gagtgccctg?cggcgacatc?aagagggaaa?acgggatcca?caggctggaa????240

gcgatgctct?acgccctgga?ccagatcaac?agtgatccca?acctactgcc?caacgtgacg????300

ctgggcgcgc?ggatcctgga?cacttgttcc?agggacactt?acgcgctcga?acagtcgctt????360

actttcgtcc?aggcgctcat?ccagaaggac?acctccgacg?tgcgctgcac?caacggcgaa????420

ccgccggttt?tcgtcaagcc?ggagaaagta?gttggagtga?ttggggcttc?ggggagttcg????480

gtctccatca?tggtagccaa?catcctgagg?ctcttccaga?tcccccagat?tagttatgca????540

tcaacggcac?ccgagctaag?tgatgaccgg?cgctatgact?tcttctctcg?cgtggtgcca????600

cccgattcct?tccaagccca?ggccatggta?gacattgtaa?aggccctagg?ctggaattat????660

gtgtctaccc?tcgcatcgga?aggaagttat?ggagagaaag?gtgtggagtc?cttcacgcag????720

atttccaaag?aggcaggtgg?actctgcatt?gcccagtccg?tgagaatccc?ccaggaacgc????780

aaagacagga?ccattgactt?tgatagaatt?atcaaacagc?tcctggacac?ccccaactcc????840

agggccgtcg?tgatttttgc?caacgatgag?gatataaagc?agatccttgc?agcagccaaa????900

agagctgacc?aagttggcca?ttttctttgg?gtgggatcag?acagctgggg?atccaaaata????960

aacccactgc?accagcatga?agatatcgca?gaaggggcca?tcaccattca?gcccaagcga????1020

gccacggtgg?aagggtttga?tgcctacttt?acgtcccgta?cacttgaaaa?caacagaaga????1080

aatgtatggt?ttgccgaata?ctgggaggaa?aacttcaact?gcaagttgac?gattagtggg????1140

tcaaaaaaag?aagacacaga?tcgcaaatgc?acaggacagg?agagaattgg?aaaagattcc????1200

aactatgagc?aggagggtaa?agtccagttc?gtgattgacg?cagtctatgc?tatggctcac????1260

gcccttcacc?acatgaacaa?ggatctctgt?gctgactacc?ggggtgtctg?cccagagatg????1320

gagcaagctg?gaggcaagaa?gttgctgaag?tatatacgca?atgttaattt?caatggtagt????1380

gctggcactc?cagtgatgtt?taacaagaac?ggggatgcac?ctgggcgtta?tgacatcttt????1440

cagtaccaga?ccacaaacac?cagcaacccg?ggttaccgtc?tgatcgggca?gtggacagac????1500

gaacttcagc?tcaatataga?agacatgcag?tggggtaaag?gagtccgaga?gatacccgcc????1560

tcagtgtgca?cactaccatg?taagccagga?cagagaaaga?agacacagaa?aggaactcct????1620

tgctgttgga?cctgtgagcc?ttgcgatggt?taccagtacc?agtttgatga?gatgacatgc????1680

cagcattgcc?cctatgacca?gaggcccaat?gaaaatcgaa?ccggatgcca?ggatattccc????1740

atcatcaaac?tggagtggca?ctccccctgg?gctgtgattc?ctgtcttcct?ggcaatgttg????1800

gggatcattg?ccaccatctt?tgtcatggcc?actttcatcc?gctacaatga?cacgcccatt????1860

gtccgggcat?ctgggcggga?actcagctat?gttcttttga?cgggcatctt?tctttgctac????1920

atcatcactt?tcctgatgat?tgccaaacca?gatgtggcag?tgtgttcttt?ccggcgagtt????1980

ttcttgggct?tgggtatgtg?catcagttat?gcagccctct?tgacgaaaac?aaatcggatt????2040

tatcgcatat?ttgagcaggg?caagaaatca?gtaacagctc?ccagactcat?aagcccaaca????2100

tcacaactgg?caatcacttc?cagtttaata?tcagttcagc?ttctaggggt?gttcatttgg????2160

tttggtgttg?atccacccaa?catcatcata?gactacgatg?aacacaagac?aatgaaccct????2220

gagcaagcca?gaggggttct?caagtgtgac?attacagatc?tccaaatcat?ttgctccttg????2280

ggatatagca?ttcttctcat?ggtcacatgt?actgtgtatg?ccatcaagac?tcggggtgta????2340

cccgagaatt?ttaacgaagc?caagcccatt?ggattcacta?tgtacacgac?atgtatagta????2400

tggcttgcct?tcattccaat?tttttttggc?accgctcaat?cagcggaaaa?gctctacata????2460

caaactacca?cgcttacaat?ctccatgaac?ctaagtgcat?cagtggcgct?ggggatgcta????2520

tacatgccga?aagtgtacat?catcattttc?caccctgaac?tcaatgtcca?gaaacggaag????2580

cgaagcttca?aggcggtagt?cacagcagcc?accatgtcat?cgaggctgtc?acacaaaccc????2640

agtgacagac?ccaacggtga?ggcaaagacc?gagctctgtg?aaaacgtaga?cccaaacagc????2700

cctgctgcaa?aaaagaagta?tgtcagttat?aataacctgg?ttatctaa?????????????????2748

<210>8

<211>915

<212>PRT

＜213〉people

<400>8

Met?Val?Gln?Leu?Arg?Lys?Leu?Leu?Arg?Val?Leu?Thr?Leu?Met?Lys?Phe

1???????????????5???????????????????10??????????????????15

Pro?Cys?Cys?Val?Leu?Glu?Val?Leu?Leu?Cys?Ala?Leu?Ala?Ala?Ala?Ala

20??????????????????25??????????????????30

Arg?Gly?Gln?Glu?Met?Tyr?Ala?Pro?His?Ser?Ile?Arg?Ile?Glu?Gly?Asp

35??????????????????40??????????????????45

Val?Thr?Leu?Gly?Gly?Leu?Phe?Pro?Val?His?Ala?Lys?Gly?Pro?Ser?Gly

50??????????????????55??????????????????60

Val?Pro?Cys?Gly?Asp?Ile?Lys?Arg?Glu?Asn?Gly?Ile?His?Arg?Leu?Glu

65??????????????????70??????????????????75??????????????????80

Ala?Met?Leu?Tyr?Ala?Leu?Asp?Gln?Ile?Asn?Ser?Asp?Pro?Asn?Leu?Leu

85??????????????????90??????????????????95

Pro?Asn?Val?Thr?Leu?Gly?Ala?Arg?Ile?Leu?Asp?Thr?Cys?Ser?Arg?Asp

100?????????????????105?????????????????110

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

115?????????????????120?????????????????125

Lys?Asp?Thr?Ser?Asp?Val?Arg?Cys?Thr?Asn?Gly?Glu?Pro?Pro?Val?Phe

130?????????????????135?????????????????140

Val?Lys?Pro?Glu?Lys?Val?Val?Gly?Val?Ile?Gly?Ala?Ser?Gly?Ser?Ser

145?????????????????150?????????????????155?????????????????160

Val?Ser?Ile?Met?Val?Ala?Asn?Ile?Leu?Arg?Leu?Phe?Gln?Ile?Pro?Gln

165?????????????????170?????????????????175

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

180?????????????????185?????????????????190

Asp?Phe?Phe?Ser?Arg?Val?Val?Pro?Pro?Asp?Ser?Phe?Gln?Ala?Gln?Ala

195?????????????????200?????????????????205

Met?Val?Asp?Ile?Val?Lys?Ala?Leu?Gly?Trp?Asn?Tyr?Val?Ser?Thr?Leu

210?????????????????215?????????????????220

Ala?Ser?Glu?Gly?Ser?Tyr?Gly?Glu?Lys?Gly?Val?Glu?Ser?Phe?Thr?Gln

225?????????????????230?????????????????235?????????????????240

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

245?????????????????250?????????????????255

Pro?Gln?Glu?Arg?Lys?Asp?Arg?Thr?Ile?Asp?Phe?Asp?Arg?Ile?Ile?Lys

260?????????????????265?????????????????270

Gln?Leu?Leu?Asp?Thr?Pro?Asn?Ser?Arg?Ala?Val?Val?Ile?Phe?Ala?Asn

275?????????????????280?????????????????285

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

290?????????????????295?????????????????300

Val?Gly?His?Phe?Leu?Trp?Val?Gly?Ser?Asp?Ser?Trp?Gly?Ser?Lys?Ile

305?????????????????310?????????????????315?????????????????320

Asn?Pro?Leu?His?Gln?His?Glu?Asp?Ile?Ala?Glu?Gly?Ala?Ile?Thr?Ile

325?????????????????330?????????????????335

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

340?????????????????345?????????????????350

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

355?????????????????360?????????????????365

Glu?Glu?Asn?Phe?Asn?Cys?Lys?Leu?Thr?Ile?Ser?Gly?Ser?Lys?Lys?Glu

370?????????????????375?????????????????380

Asp?Thr?Asp?Arg?Lys?Cys?Thr?Gly?Gln?Glu?Arg?Ile?Gly?Lys?Asp?Ser

385?????????????????390?????????????????395?????????????????400

Asn?Tyr?Glu?Gln?Glu?Gly?Lys?Val?Gln?Phe?Val?Ile?Asp?Ala?Val?Tyr

405?????????????????410?????????????????415

Ala?Met?Ala?His?Ala?Leu?His?His?Met?Asn?Lys?Asp?Leu?Cys?Ala?Asp

420?????????????????425?????????????????430

Tyr?Arg?Gly?Val?Cys?Pro?Glu?Met?Glu?Gln?Ala?Gly?Gly?Lys?Lys?Leu

435?????????????????440?????????????????445

Leu?Lys?Tyr?Ile?Arg?Asn?Val?Asn?Phe?Asn?Gly?Ser?Ala?Gly?Thr?Pro

450?????????????????455?????????????????460

Val?Met?Phe?Asn?Lys?Asn?Gly?Asp?Ala?Pro?Gly?Arg?Tyr?Asp?Ile?Phe

465?????????????????470?????????????????475?????????????????480

Gln?Tyr?Gln?Thr?Thr?Asn?Thr?Ser?Asn?Pro?Gly?Tyr?Arg?Leu?Ile?Gly

485?????????????????490?????????????????495

Gln?Trp?Thr?Asp?Glu?Leu?Gln?Leu?Asn?Ile?Glu?Asp?Met?Gln?Trp?Gly

500?????????????????505?????????????????510

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

515?????????????????520?????????????????525

Pro?Gly?Gln?Arg?Lys?Lys?Thr?Gln?Lys?Gly?Thr?Pro?Cys?Cys?Trp?Thr

530?????????????????535?????????????????540

Cys?Glu?Pro?Cys?Asp?Gly?Tyr?Gln?Tyr?Gln?Phe?Asp?Glu?Met?Thr?Cys

545?????????????????550?????????????????555?????????????????560

Gln?His?Cys?Pro?Tyr?Asp?Gln?Arg?Pro?Asn?Glu?Asn?Arg?Thr?Gly?Cys

565?????????????????570?????????????????575

Gln?Asp?Ile?Pro?Ile?Ile?Lys?Leu?Glu?Trp?His?Ser?Pro?Trp?Ala?Val

580?????????????????585?????????????????590

Ile?Pro?Val?Phe?Leu?Ala?Met?Leu?Gly?Ile?Ile?Ala?Thr?Ile?Phe?Val

595?????????????????600?????????????????605

Met?Ala?Thr?Phe?Ile?Arg?Tyr?Asn?Asp?Thr?Pro?Ile?Val?Arg?Ala?Ser

610?????????????????615?????????????????620

Gly?Arg?Glu?Leu?Ser?Tyr?Val?Leu?Leu?Thr?Gly?Ile?Phe?Leu?Cys?Tyr

625?????????????????630?????????????????635?????????????????640

Ile?Ile?Thr?Phe?Leu?Met?Ile?Ala?Lys?Pro?Asp?Val?Ala?Val?Cys?Ser

645?????????????????650?????????????????655

Phe?Arg?Arg?Val?Phe?Leu?Gly?Leu?Gly?Met?Cys?Ile?Ser?Tyr?Ala?Ala

660?????????????????665?????????????????670

Leu?Leu?Thr?Lys?Thr?Asn?Arg?Ile?Tyr?Arg?Ile?Phe?Glu?Gln?Gly?Lys

675?????????????????680?????????????????685

Lys?Ser?Val?Thr?Ala?Pro?Arg?Leu?Ile?Ser?Pro?Thr?Ser?Gln?Leu?Ala

690?????????????????695?????????????????700

Ile?Thr?Ser?Ser?Leu?Ile?Ser?Val?Gln?Leu?Leu?Gly?Val?Phe?Ile?Trp

705?????????????????710?????????????????715?????????????????720

Phe?Gly?Val?Asp?Pro?Pro?Asn?Ile?Ile?Ile?Asp?Tyr?Asp?Glu?His?Lys

725?????????????????730?????????????????735

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

740?????????????????745?????????????????750

Asp?Leu?Gln?Ile?Ile?Cys?Ser?Leu?Gly?Tyr?Ser?Ile?Leu?Leu?Met?Val

755?????????????????760?????????????????765

Thr?Cys?Thr?Val?Tyr?Ala?Ile?Lys?Thr?Arg?Gly?Val?Pro?Glu?Asn?Phe

770?????????????????775?????????????????780

Asn?Glu?Ala?Lys?Pro?Ile?Gly?Phe?Thr?Met?Tyr?Thr?Thr?Cys?Ile?Val

785?????????????????790?????????????????795?????????????????800

Trp?Leu?Ala?Phe?Ile?Pro?Ile?Phe?Phe?Gly?Thr?Ala?Gln?Ser?Ala?Glu

805?????????????????810?????????????????815

Lys?Leu?Tyr?Ile?Gln?Thr?Thr?Thr?Leu?Thr?Ile?Ser?Met?Asn?Leu?Ser

820?????????????????825?????????????????830

Ala?Ser?Val?Ala?Leu?Gly?Met?Leu?Tyr?Met?Pro?Lys?Val?Tyr?Ile?Ile

835?????????????????840?????????????????845

Ile?Phe?His?Pro?Glu?Leu?Asn?Val?Gln?Lys?Arg?Lys?Arg?Ser?Phe?Lys

850?????????????????855?????????????????860

Ala?Val?Val?Thr?Ala?Ala?Thr?Met?Ser?Ser?Arg?Leu?Ser?His?Lys?Pro

865?????????????????870?????????????????875?????????????????880

Ser?Asp?Arg?Pro?Asn?Gly?Glu?Ala?Lys?Thr?Glu?Leu?Cys?Glu?Asn?Val

885?????????????????890?????????????????895

Asp?Pro?Asn?Ser?Pro?Ala?Ala?Lys?Lys?Lys?Tyr?Val?Ser?Tyr?Asn?Asn

900?????????????????905?????????????????910

Leu?Val?Ile

915

<210>9

<211>1842

<212>DNA

＜213〉people

<400>9

atgcgagccc?cgggcgcgct?tctcgcccgc?atgtcgcggc?tactgcttct?gctactgctc????60

aaggtgtctg?cctcttctgc?cctcggggtc?gcccctgcgt?ccagaaacga?aacttgtctg????120

ggggagagct?gtgcacctac?agtgatccag?cgccgcggca?gggacgcctg?gggaccggga????180

aattctgcaa?gagacgttct?gcgagcccga?gcacccaggg?aggagcaggg?ggcagcgttt????240

cttgcgggac?cctcctggga?cctgccggcg?gccccgggcc?gtgacccggc?tgcaggcaga????300

ggggcggagg?cgtcggcagc?cggacccccg?ggacctccaa?ccaggccacc?tggcccctgg????360

aggtggaaag?gtgctcgggg?tcaggagcct?tctgaaactt?tggggagagg?gaaccccacg????420

gccctccagc?tcttccttca?gatctcagag?gaggaagaga?agggtcccag?aggcgctggc????480

atttccgggc?gtagccagga?gcagagtgtg?aagacagtcc?ccggagccag?cgatcttttt????540

tactggccaa?ggagagccgg?gaaactccag?ggttcccacc?acaagcccct?gtccaagacg????600

gccaatggac?tggcggggca?cgaagggtgg?acaattgcac?tcccgggccg?ggcgctggcc????660

cagaatggat?ccttgggtga?aggaatccat?gagcctgggg?gtccccgccg?gggaaacagc????720

acgaaccggc?gtgtgagact?gaagaacccc?ttctacccgc?tgacccagga?gtcctatgga????780

gcctacgcgg?tcatgtgtct?gtccgtggtg?atcttcggga?ccggcatcat?tggcaacctg????840

gcggtgatgt?gcatcgtgtg?ccacaactac?tacatgcgga?gcatctccaa?ctccctcttg????900

gccaacctgg?ccttctggga?ctttctcatc?atcttcttct?gccttccgct?ggtcatcttc????960

cacgagctga?ccaagaagtg?gctgctggag?gacttctcct?gcaagatcgt?gccctatata????1020

gaggtcgctt?ctctgggagt?caccaccttc?accttatgtg?ctctgtgcat?agaccgcttc????1080

cgtgctgcca?ccaacgtaca?gatgtactac?gaaatgatcg?aaaactgttc?ctcaacaact????1140

gccaaacttg?ctgttatatg?ggtgggagct?ctattgttag?cacttccaga?agttgttctc????1200

cgccagctga?gcaaggagga?tttggggttt?agtggccgag?ctccggcaga?aaggtgcatt????1260

attaagatct?ctcctgattt?accagacacc?atctatgttc?tagccctcac?ctacgacagt????1320

gcgagactgt?ggtggtattt?tggctgttac?ttttgtttgc?ccacgctttt?caccatcacc????1380

tgctctctag?tgactgcgag?gaaaatccgc?aaagcagaga?aagcctgtac?ccgagggaat????1440

aaacggcaga?ttcaactaga?gagtcagatg?aactgtacag?tagtggcact?gaccatttta????1500

tatggatttt?gcattattcc?tgaaaatatc?tgcaacattg?ttactgccta?catggctaca????1560

ggggtttcac?agcagacaat?ggacctcctt?aatatcatca?gccagttcct?tttgttcttt????1620

aagtcctgtg?tcaccccagt?cctccttttc?tgtctctgca?aacccttcag?tcgggccttc????1680

atggagtgct?gctgctgttg?ctgtgaggaa?tgcattcaga?agtcttcaac?ggtgaccagt????1740

gatgacaatg?acaacgagta?caccacggaa?ctcgaactct?cgcctttcag?taccatacgc????1800

cgtgaaatgt?ccacttttgc?ttctgtcgga?actcattgct?ga???????????????????????1842

<210>10

<211>613

<212>PRT

＜213〉people

<400>10

Met?Arg?Ala?Pro?Gly?Ala?Leu?Leu?Ala?Arg?Met?Ser?Arg?Leu?Leu?Leu

1???????????????5???????????????????10??????????????????15

Leu?Leu?Leu?Leu?Lys?Val?Ser?Ala?Ser?Ser?Ala?Leu?Gly?Val?Ala?Pro

20??????????????????25??????????????????30

Ala?Ser?Arg?Asn?Glu?Thr?Cys?Leu?Gly?Glu?Ser?Cys?Ala?Pro?Thr?Val

35??????????????????40??????????????????45

Ile?Gln?Arg?Arg?Gly?Arg?Asp?Ala?Trp?Gly?Pro?Gly?Asn?Ser?Ala?Arg

50??????????????????55??????????????????60

Asp?Val?Leu?Arg?Ala?Arg?Ala?Pro?Arg?Glu?Glu?Gln?Gly?Ala?Ala?Phe

65??????????????????70??????????????????75??????????????????80

Leu?Ala?Gly?Pro?Ser?Trp?Asp?Leu?Pro?Ala?Ala?Pro?Gly?Arg?Asp?Pro

85??????????????????90??????????????????95

Ala?Ala?Gly?Arg?Gly?Ala?Glu?Ala?Ser?Ala?Ala?Gly?Pro?Pro?Gly?Pro

100?????????????????105?????????????????110

Pro?Thr?Arg?Pro?Pro?Gly?Pro?Trp?Arg?Trp?Lys?Gly?Ala?Arg?Gly?Gln

115?????????????????120?????????????????125

Glu?Pro?Ser?Glu?Thr?Leu?Gly?Arg?Gly?Asn?Pro?Thr?Ala?Leu?Gln?Leu

130?????????????????135?????????????????140

Phe?Leu?Gln?Ile?Ser?Glu?Glu?Glu?Glu?Lys?Gly?Pro?Arg?Gly?Ala?Gly

145?????????????????150?????????????????155?????????????????160

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

165?????????????????170?????????????????175

Ser?Asp?Leu?Phe?Tyr?Trp?Pro?Arg?Arg?Ala?Gly?Lys?Leu?Gln?Gly?Ser

180?????????????????185?????????????????190

His?His?Lys?Pro?Leu?Ser?Lys?Thr?Ala?Asn?Gly?Leu?Ala?Gly?His?Glu

195?????????????????200?????????????????205

Gly?Trp?Thr?Ile?Ala?Leu?Pro?Gly?Arg?Ala?Leu?Ala?Gln?Asn?Gly?Ser

210?????????????????215?????????????????220

Leu?Gly?Glu?Gly?Ile?His?Glu?Pro?Gly?Gly?Pro?Arg?Arg?Gly?Asn?Ser

225?????????????????230?????????????????235?????????????????240

Thr?Asn?Arg?Arg?Val?Arg?Leu?Lys?Asn?Pro?Phe?Tyr?Pro?Leu?Thr?Gln

245?????????????????250?????????????????255

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

260?????????????????265?????????????????270

Gly?Thr?Gly?Ile?Ile?Gly?Asn?Leu?Ala?Val?Met?Cys?Ile?Val?Cys?His

275?????????????????280?????????????????285

Asn?Tyr?Tyr?Met?Arg?Ser?Ile?Ser?Asn?Ser?Leu?Leu?Ala?Asn?Leu?Ala

290?????????????????295?????????????????300

Phe?Trp?Asp?Phe?Leu?Ile?Ile?Phe?Phe?Cys?Leu?Pro?Leu?Val?Ile?Phe

305?????????????????310?????????????????315?????????????????320

His?Glu?Leu?Thr?Lys?Lys?Trp?Leu?Leu?Glu?Asp?Phe?Ser?Cys?Lys?Ile

325?????????????????330?????????????????335

Val?Pro?Tyr?Ile?Glu?Val?Ala?Ser?Leu?Gly?Val?Thr?Thr?Phe?Thr?Leu

340?????????????????345?????????????????350

Cys?Ala?Leu?Cys?Ile?Asp?Arg?Phe?Arg?Ala?Ala?Thr?Asn?Val?Gln?Met

355?????????????????360?????????????????365

Tyr?Tyr?Glu?Met?Ile?Glu?Asn?Cys?Ser?Ser?Thr?Thr?Ala?Lys?Leu?Ala

370?????????????????375?????????????????380

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

385?????????????????390?????????????????395?????????????????400

Arg?Gln?Leu?Ser?Lys?Glu?Asp?Leu?Gly?Phe?Ser?Gly?Arg?Ala?Pro?Ala

405?????????????????410?????????????????415

Glu?Arg?Cys?Ile?Ile?Lys?Ile?Ser?Pro?Asp?Leu?Pro?Asp?Thr?Ile?Tyr

420?????????????????425?????????????????430

Val?Leu?Ala?Leu?Thr?Tyr?Asp?Ser?Ala?Arg?Leu?Trp?Trp?Tyr?Phe?Gly

435?????????????????440?????????????????445

Cys?Tyr?Phe?Cys?Leu?Pro?Thr?Leu?Phe?Thr?Ile?Thr?Cys?Ser?Leu?Val

450?????????????????455?????????????????460

Thr?Ala?Arg?Lys?Ile?Arg?Lys?Ala?Glu?Lys?Ala?Cys?Thr?Arg?Gly?Asn

465?????????????????470?????????????????475?????????????????480

Lys?Arg?Gln?Ile?Gln?Leu?Glu?Ser?Gln?Met?Asn?Cys?Thr?Val?Val?Ala

485?????????????????490?????????????????495

Leu?Thr?Ile?Leu?Tyr?Gly?Phe?Cys?Ile?Ile?Pro?Glu?Asn?Ile?Cys?Asn

500?????????????????505?????????????????510

Ile?Val?Thr?Ala?Tyr?Met?Ala?Thr?Gly?Val?Ser?Gln?Gln?Thr?Met?Asp

515?????????????????520?????????????????525

Leu?Leu?Asn?Ile?Ile?Ser?Gln?Phe?Leu?Leu?Phe?Phe?Lys?Ser?Cys?Val

530?????????????????535?????????????????540

Thr?Pro?Val?Leu?Leu?Phe?Cys?Leu?Cys?Lys?Pro?Phe?Ser?Arg?Ala?Phe

545?????????????????550?????????????????555?????????????????560

Met?Glu?Cys?Cys?Cys?Cys?Cys?Cys?Glu?Glu?Cys?Ile?Gln?Lys?Ser?Ser

565?????????????????570?????????????????575

Thr?Val?Thr?Ser?Asp?Asp?Asn?Asp?Asn?Glu?Tyr?Thr?Thr?Glu?Leu?Glu

580?????????????????585?????????????????590

Leu?Ser?Pro?Phe?Ser?Thr?Ile?Arg?Arg?Glu?Met?Ser?Thr?Phe?Ala?Ser

595?????????????????600?????????????????605

Val?Gly?Thr?His?Cys

610

<210>11

<211>1086

<212>DNA

＜213〉people

<400>11

atgtcccctg?aatgcgcgcg?ggcagcgggc?gacgcgccct?tgcgcagcct?ggagcaagcc????60

aaccgcaccc?gctttccctt?cttctccgac?gtcaagggcg?accaccggct?ggtgctggcc????120

gcggtggaga?caaccgtgct?ggtgctcatc?tttgcagtgt?cgctgctggg?caacgtgtgc????180

gccctggtgc?tggtggcgcg?ccgacgacgc?cgcggcgcga?ctgcctgcct?ggtactcaac????240

ctcttctgcg?cggacctgct?cttcatcagc?gctatccctc?tggtgctggc?cgtgcgctgg????300

actgaggcct?ggctgctggg?ccccgttgcc?tgccacctgc?tcttctacgt?gatgaccctg????360

agcggcagcg?tcaccatcct?cacgctggcc?gcggtcagcc?tggagcgcat?ggtgtgcatc????420

gtgcacctgc?agcgcggcgt?gcggggtcct?gggcggcggg?cgcgggcagt?gctgctggcg????480

ctcatctggg?gctattcggc?ggtcgccgct?ctgcctctct?gcgtcttctt?tcgagtcgtc????540

ccgcaacggc?tccccggcgc?cgaccaggaa?atttcgattt?gcacactgat?ttggcccacc????600

attcctggag?agatctcgtg?ggatgtctct?tttgttactt?tgaacttctt?ggtgccagga????660

ctggtcattg?tgatcagtta?ctccaaaatt?ttacagatca?caaaggcatc?aaggaagagg????720

ctcacggtaa?gcctggccta?ctcggagagc?caccagatcc?gcgtgtccca?gcaggacttc????780

cggctcttcc?gcaccctctt?cctcctcatg?gtctccttct?tcatcatgtg?gagccccatc????840

atcatcacca?tcctcctcat?cctgatccag?aacttcaagc?aagacctggt?catctggccg????900

tccctcttct?tctgggtggt?ggccttcaca?tttgctaatt?cagccctaaa?ccccatcctc????960

tacaacatga?cactgtgcag?gaatgagtgg?aagaaaattt?tttgctgctt?ctggttccca????1020

gaaaagggag?ccattttaac?agacacatct?gtcaaaagaa?atgacttgtc?gattatttct????1080

ggctaa???????????????????????????????????????????????????????????????1086

<210>12

<211>361

<212>PRT

＜213〉the unknown

<220>

＜223〉new sequence

<400>12

Met?Ser?Pro?Glu?Cys?Ala?Arg?Ala?Ala?Gly?Asp?Ala?Pro?Leu?Arg?Ser

1???????????????5???????????????????10??????????????????15

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

20??????????????????25??????????????????30

Gly?Asp?His?Arg?Leu?Val?Leu?Ala?Ala?Val?Glu?Thr?Thr?Val?Leu?Val

35??????????????????40??????????????????45

Leu?Ile?Phe?Ala?Val?Ser?Leu?Leu?Gly?Asn?Val?Cys?Ala?Leu?Val?Leu

50??????????????????55??????????????????60

Val?Ala?Arg?Arg?Arg?Arg?Arg?Gly?Ala?Thr?Ala?Cys?Leu?Val?Leu?Asn

65??????????????????70??????????????????75??????????????????80

Leu?Phe?Cys?Ala?Asp?Leu?Leu?Phe?Ile?Ser?Ala?Ile?Pro?Leu?Val?Leu

85??????????????????90??????????????????95

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

100?????????????????105?????????????????110

Leu?Leu?Phe?Tyr?Val?Met?Thr?Leu?Ser?Gly?Ser?Val?Thr?Ile?Leu?Thr

115?????????????????120?????????????????125

Leu?Ala?Ala?Val?Ser?Leu?Glu?Arg?Met?Val?Cys?Ile?Val?His?Leu?Gln

130?????????????????135?????????????????140

Arg?Gly?Val?Arg?Gly?Pro?Gly?Arg?Arg?Ala?Arg?Ala?Val?Leu?Leu?Ala

145?????????????????150?????????????????155?????????????????160

Leu?Ile?Trp?Gly?Tyr?Ser?Ala?Val?Ala?Ala?Leu?Pro?Leu?Cys?Val?Phe

165?????????????????170?????????????????175

Phe?Arg?Val?Val?Pro?Gln?Arg?Leu?Pro?Gly?Ala?Asp?Gln?Glu?Ile?Ser

180?????????????????185?????????????????190

Ile?Cys?Thr?Leu?Ile?Trp?Pro?Thr?Ile?Pro?Gly?Glu?Ile?Ser?Trp?Asp

195?????????????????200?????????????????205

Val?Ser?Phe?Val?Thr?Leu?Asn?Phe?Leu?Val?Pro?Gly?Leu?Val?Ile?Val

210?????????????????215?????????????????220

Ile?Ser?Tyr?Ser?Lys?Ile?Leu?Gln?Ile?Thr?Lys?Ala?Ser?Arg?Lys?Arg

225?????????????????230?????????????????235?????????????????240

Leu?Thr?Val?Ser?Leu?Ala?Tyr?Ser?Glu?Ser?His?Gln?Ile?Arg?Val?Ser

245?????????????????250?????????????????255

Gln?Gln?Asp?Phe?Arg?Leu?Phe?Arg?Thr?Leu?Phe?Leu?Leu?Mer?Val?Ser

260?????????????????265?????????????????270

Phe?Phe?Ile?Met?Trp?Ser?Pro?Ile?Ile?Ile?Thr?Ile?Leu?Leu?Ile?Leu

275?????????????????280?????????????????285

Ile?Gln?Asn?Phe?Lys?Gln?Asp?Leu?Val?Ile?Trp?Pro?Ser?Leu?Phe?Phe

290?????????????????295?????????????????300

Trp?Val?Val?Ala?Phe?Thr?Phe?Ala?Asn?Ser?Ala?Leu?Asn?Pro?Ile?Leu

305?????????????????310?????????????????315?????????????????320

Tyr?Asn?Met?Thr?Leu?Cys?Arg?Asn?Glu?Trp?Lys?Lys?Ile?Phe?Cys?Cys

325?????????????????330?????????????????335

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

340?????????????????345?????????????????350

Arg?Asn?Asp?Leu?Ser?Ile?Ile?Ser?Gly

355?????????????????360

<210>13

<211>1212

<212>DNA

＜213〉people

<400>13

atggcttgca?atggcagtgc?ggccaggggg?cactttgacc?ctgaggactt?gaacctgact????60

gacgaggcac?tgagactcaa?gtacctgggg?ccccagcaga?cagagctgtt?catgcccatc????120

tgtgccacat?acctgctgat?cttcgtggtg?ggcgctgtgg?gcaatgggct?gacctgtctg????180

gtcatcctgc?gccacaaggc?catgcgcacg?cctaccaact?actacctctt?cagcctggcc????240

gtgtcggacc?tgctggtgct?gctggtgggc?ctgcccctgg?agctctatga?gatgtggcac????300

aactacccct?tcctgctggg?cgttggtggc?tgctatttcc?gcacgctact?gtttgagatg????360

gtctgcctgg?cctcagtgct?caacgtcact?gccctgagcg?tggaacgcta?tgtggccgtg????420

gtgcacccac?tccaggccag?gtccatggtg?acgcgggccc?atgtgcgccg?agtgcttggg????480

gccgtctggg?gtcttgccat?gctctgctcc?ctgcccaaca?ccagcctgca?cggcatccgg????540

cagctgcacg?tgccctgccg?gggcccagtg?ccagactcag?ctgtttgcat?gctggtccgc????600

ccacgggccc?tctacaacat?ggtagtgcag?accaccgcgc?tgctcttctt?ctgcctgccc????660

atggccatca?tgagcgtgct?ctacctgctc?attgggctgc?gactgcggcg?ggagaggctg????720

ctgctcatgc?aggaggccaa?gggcaggggc?tctgcagcag?ccaggtccag?atacacctgc????780

aggctccagc?agcacgatcg?gggccggaga?caagtgacca?agatgctgtt?tgtcctggtc????840

gtggtgtttg?gcatctgctg?ggccccgttc?cacgccgacc?gcgtcatgtg?gagcgtcgtg????900

tcacagtgga?cagatggcct?gcacctggcc?ttccagcacg?tgcacgtcat?ctccggcatc????960

ttcttctacc?tgggctcggc?ggccaacccc?gtgctctata?gcctcatgtc?cagccgcttc????1020

cgagagacct?tccaggaggc?cctgtgcctc?ggggcctgct?gccatcgcct?cagaccccgc????1080

cacagctccc?acagcctcag?caggatgacc?acaggcagca?ccctgtgtga?tgtgggctcc????1140

ctgggcagct?gggtccaccc?cctggctggg?aacgatggcc?cagaggcgca?gcaagagacc????1200

gatccatcct?ga????????????????????????????????????????????????????????1212

<210>14

<211>403

<212>PRT

＜213〉people

<400>14

Met?Ala?Cys?Asn?Gly?Ser?Ala?Ala?Arg?Gly?His?Phe?Asp?Pro?Glu?Asp

1???????????????5???????????????????10??????????????????15

Leu?Asn?Leu?Thr?Asp?Glu?Ala?Leu?Arg?Leu?Lys?Tyr?Leu?Gly?Pro?Gln

20??????????????????25??????????????????30

Gln?Thr?Glu?Leu?Phe?Met?Pro?Ile?Cys?Ala?Thr?Tyr?Leu?Leu?Ile?Phe

35??????????????????40??????????????????45

Val?Val?Gly?Ala?Val?Gly?Asn?Gly?Leu?Thr?Cys?Leu?Val?Ile?Leu?Arg

50??????????????????55??????????????????60

His?Lys?Ala?Met?Arg?Thr?Pro?Thr?Asn?Tyr?Tyr?Leu?Phe?Ser?Leu?Ala

65??????????????????70??????????????????75??????????????????80

Val?Ser?Asp?Leu?Leu?Val?Leu?Leu?Val?Gly?Leu?Pro?Leu?Glu?Leu?Tyr

85??????????????????90??????????????????95

Glu?Met?Trp?His?As?nTyr?Pro?Phe?Leu?Leu?Gly?Val?Gly?Gly?Cys?Tyr

100?????????????????105?????????????????110

Phe?Arg?Thr?Leu?Leu?Phe?Glu?Met?Val?Cys?Leu?Ala?Ser?Val?Leu?Asn

115?????????????????120?????????????????125

Val?Thr?Ala?Leu?Ser?Val?Glu?Arg?Tyr?Val?Ala?Val?Val?His?Pro?Leu

130?????????????????135?????????????????140

Gln?Ala?Arg?Ser?Met?Val?Thr?Arg?Ala?His?Val?Arg?Arg?Val?Leu?Gly

145?????????????????150?????????????????155?????????????????160

Ala?Val?Trp?Gly?Leu?Ala?Met?Leu?Cys?Ser?Leu?Pro?Asn?Thr?Ser?Leu

165?????????????????170?????????????????175

His?Gly?Ile?Arg?Gln?Leu?His?Val?Pro?Cys?Arg?Gly?Pro?Val?Pro?Asp

180?????????????????185?????????????????190

Ser?Ala?Val?Cys?Met?Leu?Val?Arg?Pro?Arg?Ala?Leu?Tyr?Asn?Met?Val

195?????????????????200?????????????????205

Val?Gln?Thr?Thr?Ala?Leu?Leu?Phe?Phe?Cys?Leu?Pro?Met?Ala?Ile?Met

210?????????????????215?????????????????220

Ser?Val?Leu?Tyr?Leu?Leu?Ile?Gly?Leu?Arg?Leu?Arg?Arg?Glu?Arg?Leu

225?????????????????230?????????????????235?????????????????240

Leu?Leu?Met?Gln?Glu?Ala?Lys?Gly?Arg?Gly?Ser?Ala?Ala?Ala?Arg?Ser

245?????????????????250?????????????????255

Arg?Tyr?Thr?Cys?Arg?Leu?Gln?Gln?His?Asp?Arg?Gly?Arg?Arg?Gln?Val

260?????????????????265?????????????????270

Thr?Lys?Met?Leu?Phe?Val?Leu?Val?Val?Val?Phe?Gly?Ile?Cys?Trp?Ala

275?????????????????280?????????????????285

Pro?Phe?His?Ala?Asp?Arg?Val?Met?Trp?Ser?Val?Val?Ser?Gln?Trp?Thr

290?????????????????295?????????????????300

Asp?Gly?Leu?His?Leu?Ala?Phe?Gln?His?Val?His?Val?Ile?Ser?Gly?Ile

305?????????????????310?????????????????315?????????????????320

Phe?Phe?Tyr?Leu?Gly?Ser?Ala?Ala?Asn?Pro?Val?Leu?Tyr?Ser?Leu?Met

325?????????????????330?????????????????335

Ser?Ser?Arg?Phe?Arg?Glu?Thr?Phe?Gln?Glu?Ala?Leu?Cys?Leu?Gly?Ala

340?????????????????345?????????????????350

Cys?Cys?His?Arg?Leu?Arg?Pro?Arg?His?Ser?Ser?His?Ser?Leu?Ser?Arg

355?????????????????360?????????????????365

Met?Thr?Thr?Gly?Ser?Thr?Leu?Cys?Asp?Val?Gly?Ser?Leu?Gly?Ser?Trp

370?????????????????375?????????????????380

Val?His?Pro?Leu?Ala?Gly?Asn?Asp?Gly?Pro?Glu?Ala?Gln?Gln?Glu?Thr

385?????????????????390?????????????????395?????????????????400

Asp?Pro?Ser

<210>15

<211>930

<212>DNA

＜213〉people

<400>15

atgaatggca?cctacaacac?ctgtggctcc?agcgacctca?cctggccccc?agcgatcaag????60

ctgggcttct?acgcctactt?gggcgtcctg?ctggtgctag?gcctgctgct?caacagcctg????120

gcgctctggg?tgttctgctg?ccgcatgcag?cagtggacgg?agacccgcat?ctacatgacc????180

aacctggcgg?tggccgacct?ctgcctgctg?tgcaccttgc?ccttcgtgct?gcactccctg????240

cgagacacct?cagacacgcc?gctgtgccag?ctctcccagg?gcatctacct?gaccaacagg????300

tacatgagca?tcagcctggt?cacggccatc?gccgtggacc?gctatgtggc?cgtgcggcac????360

ccgctgcgtg?cccgcgggct?gcggtccccc?aggcaggctg?cggccgtgtg?cgcggtcctc????420

tgggtgctgg?tcatcggctc?cctggtggct?cgctggctcc?tggggattca?ggagggcggc????480

ttctgcttca?ggagcacccg?gcacaatttc?aactccatgc?ggttcccgct?gctgggattc????540

tacctgcccc?tggccgtggt?ggtcttctgc?tccctgaagg?tggtgactgc?cctggcccag????600

aggccaccca?ccgacgtggg?gcaggcagag?gccacccgca?aggctgcccg?catggtctgg????660

gccaacctcc?tggtgttcgt?ggtctgcttc?ctgcccctgc?acgtggggct?gacagtgcgc????720

ctcgcagtgg?gctggaacgc?ctgtgccctc?ctggagacga?tccgtcgcgc?cctgtacata????780

accagcaagc?tctcagatgc?caactgctgc?ctggacgcca?tctgctacta?ctacatggcc????840

aaggagttcc?aggaggcgtc?tgcactggcc?gtggctcccc?gtgctaaggc?ccacaaaagc????900

caggactctc?tgtgcgtgac?cctcgcctaa?????????????????????????????????????930

<210>16

<211>309

<212>PRT

＜213〉people

<400>16

Met?Asn?Gly?Thr?Tyr?Asn?Thr?Cys?Gly?Ser?Ser?Asp?Leu?Thr?Trp?Pro

1???????????????5???????????????????10??????????????????15

Pro?Ala?Ile?Lys?Leu?Gly?Phe?Tyr?Ala?Tyr?Leu?Gly?Val?Leu?Leu?Val

20??????????????????25??????????????????30

Leu?Gly?Leu?Leu?Leu?Asn?Ser?Leu?Ala?Leu?Trp?Val?Phe?Cys?Cys?Arg

35??????????????????40??????????????????45

Met?Gln?Gln?Trp?Thr?Glu?Thr?Arg?Ile?Tyr?Met?Thr?Asn?Leu?Ala?Val

50??????????????????55??????????????????60

Ala?Asp?Leu?Cys?Leu?Leu?Cys?Thr?Leu?Pro?Phe?Val?Leu?His?Ser?Leu

65??????????????????70??????????????????75??????????????????80

Arg?Asp?Thr?Ser?Asp?Thr?Pro?Leu?Cys?Gln?Leu?Ser?Gln?Gly?Ile?Tyr

85??????????????????90??????????????????95

Leu?Thr?Asn?Arg?Tyr?Met?Ser?Ile?Ser?Leu?Val?Thr?Ala?Ile?Ala?Val

100?????????????????105?????????????????110

Asp?Arg?Tyr?Val?Ala?Val?Arg?His?Pro?Leu?Arg?Ala?Arg?Gly?Leu?Arg

115?????????????????120?????????????????125

Ser?Pro?Arg?Gln?Ala?Ala?Ala?Val?Cys?Ala?Val?Leu?Trp?Val?Leu?Val

130?????????????????135?????????????????140

Ile?Gly?Ser?Leu?Val?Ala?Arg?Trp?Leu?Leu?Gly?Ile?Gln?Glu?Gly?Gly

145?????????????????150?????????????????155?????????????????160

Phe?Cys?Phe?Arg?Ser?Thr?Arg?His?Asn?Phe?Asn?Ser?Met?Arg?Phe?Pro

165?????????????????170?????????????????175

Leu?Leu?Gly?Phe?Tyr?Leu?Pro?Leu?Ala?Val?Val?Val?Phe?Cys?Ser?Leu

180?????????????????185?????????????????190

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

195?????????????????200?????????????????205

Ala?Glu?Ala?Thr?Arg?Lys?Ala?Ala?Arg?Met?Val?Trp?Ala?Asn?Leu?Leu

210?????????????????215?????????????????220

Val?Phe?Val?Val?Cys?Phe?Leu?Pro?Leu?His?Val?Gly?Leu?Thr?Val?Arg

225?????????????????230?????????????????235?????????????????240

Leu?Ala?Val?Gly?Trp?Asn?Ala?Cys?Ala?Leu?Leu?Glu?Thr?Ile?Arg?Arg

245?????????????????250?????????????????255

Ala?Leu?Tyr?Ile?Thr?Ser?Lys?Leu?Ser?Asp?Ala?Asn?Cys?Cys?Leu?Asp

260?????????????????265?????????????????270

Ala?Ile?Cys?Tyr?Tyr?Tyr?Met?Ala?Lys?Glu?Phe?Gln?Glu?Ala?Ser?Ala

275?????????????????280?????????????????285

Leu?Ala?Val?Ala?Pro?Arg?Ala?Lys?Ala?His?Lys?Ser?Gln?Asp?Ser?Leu

290?????????????????295?????????????????300

Cys?Val?Thr?Leu?Ala

305

<210>17

<211>1446

<212>DNA

＜213〉people

<400>17

atgcggtggc?tgtggcccct?ggctgtctct?cttgctgtga?ttttggctgt?ggggctaagc????60

agggtctctg?ggggtgcccc?cctgcacctg?ggcaggcaca?gagccgagac?ccaggagcag????120

cagagccgat?ccaagagggg?caccgaggat?gaggaggcca?agggcgtgca?gcagtatgtg????180

cctgaggagt?gggcggagta?cccccggccc?attcaccctg?ctggcctgca?gccaaccaag????240

cccttggtgg?ccaccagccc?taaccccgac?aaggatgggg?gcaccccaga?cagtgggcag????300

gaactgaggg?gcaatctgac?aggggcacca?gggcagaggc?tacagatcca?gaaccccctg????360

tatccggtga?ccgagagctc?ctacagtgcc?tatgccatca?tgcttctggc?gctggtggtg????420

tttgcggtgg?gcattgtggg?caacctgtcg?gtcatgtgca?tcgtgtggca?cagctactac????480

ctgaagagcg?cctggaactc?catccttgcc?agcctggccc?tctgggattt?tctggtcctc????540

tttttctgcc?tccctattgt?catcttcaac?gagatcacca?agcagaggct?actgggtgac????600

gtttcttgtc?gtgccgtgcc?cttcatggag?gtctcctctc?tgggagtcac?gactttcagc????660

ctctgtgccc?tgggcattga?ccgcttccac?gtggccacca?gcaccctgcc?caaggtgagg????720

cccatcgagc?ggtgccaatc?catcctggcc?aagttggctg?tcatctgggt?gggctccatg????780

acgctggctg?tgcctgagct?cctgctgtgg?cagctggcac?aggagcctgc?ccccaccatg????840

ggcaccctgg?actcatgcat?catgaaaccc?tcagccagcc?tgcccgagtc?cctgtattca????900

ctggtgatga?cctaccagaa?cgcccgcatg?tggtggtact?ttggctgcta?cttctgcctg????960

cccatcctct?tcacagtcac?ctgccagctg?gtgacatggc?gggtgcgagg?ccctccaggg????1020

aggaagtcag?agtgcagggc?cagcaagcac?gagcagtgtg?agagccagct?caacagcacc????1080

gtggtgggcc?tgaccgtggt?ctacgccttc?tgcaccctcc?cagagaacgt?ctgcaacatc????1140

gtggtggcct?acctctccac?cgagctgacc?cgccagaccc?tggacctcct?gggcctcatc????1200

aaccagttct?ccaccttctt?caagggcgcc?atcaccccag?tgctgctcct?ttgcatctgc????1260

aggccgctgg?gccaggcctt?cctggactgc?tgctgctgct?gctgctgtga?ggagtgcggc????1320

ggggcttcgg?aggcctctgc?tgccaatggg?tcggacaaca?agctcaagac?cgaggtgtcc????1380

tcttccatct?acttccacaa?gcccagggag?tcacccccac?tcctgcccct?gggcacacct????1440

tgctga???????????????????????????????????????????????????????????????1446

<210>18

<211>481

<212>PRT

＜213〉people

<400>18

Met?Arg?Trp?Leu?Trp?Pro?Leu?Ala?Val?Ser?Leu?Ala?ValIle?Leu?Ala

1???????????????5???????????????????10?????????????????15

Val?Gly?Leu?Ser?Arg?Val?Ser?Gly?Gly?Ala?Pro?Leu?His?Leu?Gly?Arg

20??????????????????25??????????????????30

His?Arg?Ala?Glu?Thr?Gln?Glu?Gln?Gln?Ser?Arg?Ser?Lys?Arg?Gly?Thr

35??????????????????40??????????????????45

Glu?Asp?Glu?Glu?Ala?Lys?Gly?Val?Gln?Gln?Tyr?Val?Pro?Glu?Glu?Trp

50??????????????????55??????????????????60

Ala?Glu?Tyr?Pro?Arg?Pro?Ile?His?Pro?Ala?Gly?Leu?Gln?Pro?Thr?Lys

65??????????????????70??????????????????75??????????????????80

Pro?Leu?Val?Ala?Thr?Ser?Pro?Asn?Pro?Asp?Lys?Asp?Gly?Gly?Thr?Pro

85??????????????????90??????????????????95

Asp?Ser?Gly?Gln?Glu?Leu?Arg?Gly?Asn?Leu?Thr?Gly?Ala?Pro?Gly?Gln

100?????????????????105?????????????????110

Arg?Leu?Gln?Ile?Gln?Asn?Pro?Leu?Tyr?Pro?Val?Thr?Glu?Ser?Ser?Tyr

115?????????????????120?????????????????125

Ser?Ala?Tyr?Ala?Ile?Met?Leu?Leu?Ala?Leu?Val?Val?Phe?Ala?Val?Gly

130?????????????????135?????????????????140

Ile?Val?Gly?Asn?Leu?Ser?Val?Met?Cys?Ile?Val?Trp?His?Ser?Tyr?Tyr

145?????????????????150?????????????????155?????????????????160

Leu?Lys?Ser?Ala?Trp?Asn?Ser?Ile?Leu?Ala?Ser?Leu?Ala?Leu?Trp?Asp

165?????????????????170?????????????????175

Phe?Leu?Val?Leu?Phe?Phe?Cys?Leu?Pro?Ile?Val?Ile?Phe?Asn?Glu?Ile

180?????????????????185?????????????????190

Thr?Lys?Gln?Arg?Leu?Leu?Gly?Asp?Val?Ser?Cys?Arg?Ala?Val?Pro?Phe

195?????????????????200?????????????????205

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

210?????????????????215?????????????????220

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

225?????????????????230?????????????????235?????????????????240

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

245?????????????????250?????????????????255

Val?Gly?Ser?Met?Thr?Leu?Ala?Val?Pro?Glu?Leu?Leu?Leu?Trp?Gln?Leu

260?????????????????265?????????????????270

Ala?Gln?Glu?Pro?Ala?Pro?Thr?Met?Gly?Thr?Leu?Asp?Ser?Cys?Ile?Met

275?????????????????280?????????????????285

Lys?Pro?Ser?Ala?Ser?Leu?Pro?Glu?Ser?Leu?Tyr?Ser?Leu?Val?Met?Thr

290?????????????????295?????????????????300

Tyr?Gln?Asn?Ala?Arg?Met?Trp?Trp?Tyr?Phe?Gly?Cys?Tyr?Phe?Cys?Leu

305?????????????????310?????????????????315?????????????????320

Pro?Ile?Leu?Phe?Thr?Val?Thr?Cys?Gln?Leu?Val?Thr?Trp?Arg?Val?Arg

325?????????????????330?????????????????335

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

340?????????????????345?????????????????350

Cys?Glu?Ser?Gln?Leu?Asn?Ser?Thr?Val?Val?Gly?Leu?Thr?Val?Val?Tyr

355?????????????????360?????????????????365

Ala?Phe?Cys?Thr?Leu?Pro?Glu?Asn?Val?Cys?Asn?Ile?Val?Val?Ala?Tyr

370?????????????????375?????????????????380

Leu?Ser?Thr?Glu?Leu?Thr?Arg?Gln?Thr?Leu?Asp?Leu?Leu?Gly?Leu?Ile

385?????????????????390?????????????????395?????????????????400

Asn?Gln?Phe?Ser?Thr?Phe?Phe?Lys?Gly?Ala?Ile?Thr?Pro?Val?Leu?Leu

405?????????????????410?????????????????415

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

420?????????????????425?????????????????430

Cys?Cys?Cys?Cys?Glu?Glu?Cys?Gly?Gly?Ala?Ser?Glu?Ala?Ser?Ala?Ala

435?????????????????440?????????????????445

Asn?Gly?Ser?Asp?Asn?Lys?Leu?Lys?Thr?Glu?Val?Ser?Ser?Ser?Ile?Tyr

450?????????????????455?????????????????460

Phe?His?Lys?Pro?Arg?Glu?Ser?Pro?Pro?Leu?Leu?Pro?Leu?Gly?Thr?Pro

465?????????????????470?????????????????475?????????????????480

Cys

<210>19

<211>29

<212>DNA

＜213〉the unknown

<220>

＜223〉new sequence

<400>19

aaagattcag?gtgtgggaag?atggaaacc????????????????????????????29

<210>20

<211>29

<212>DNA

＜213〉the unknown

<220>

＜223〉new sequence

<400>20

aaaggatccc?cgacctcaca?ttgcttgta????????????????????????????29

<210>21

<211>30

<212>DNA

＜213〉the unknown

<220>

＜223〉new sequence

<400>21

caggaattca?tcagaacaga?caccatggca???????????????????????????30

<210>22

<211>31

<212>DNA

＜213〉the unknown

<220>

＜223〉new sequence

<400>22

gcaggatcca?gagcagtttt?ttcgaaaccc?t?????????????????????????31

<210>23

<211>33

<212>DNA

＜213〉the unknown

<220>

＜223〉new sequence

<400>23

tccaagcttc?aagggtctct?ccacgatggc?ctg????????????????????????33

<210>24

<211>33

<212>DNA

＜213〉the unknown

<220>

＜223〉new sequence

<400>24

tgcgaattct?ctgtggcccc?ctgaccccct?aaa????????????????????????33

<210>25

<211>36

<212>DNA

＜213〉the unknown

<220>

＜223〉new sequence

<400>25

ggtaagctta?ccatggcctg?caacagcacg?tccctt?????????????????????36

<210>26

<211>33

<212>DNA

＜213〉the unknown

<220>

＜223〉new sequence

<400>26

gacgaattca?accgcagact?ggttttcatt?gca????????????????????????33

<210>27

<211>33

<212>DNA

＜213〉the unknown

<220>

＜223〉new sequence

<400>27

gcaagcttgt?gccctcacca?agccatgcga?gcc???????????????????????33

<210>28

<211>30

<212>DNA

＜213〉the unknown

<220>

＜223〉new sequence

<400>28

cggaattcag?caatgagttc?cgacagaagc???????????????????????????30

<210>29

<211>37

<212>DNA

＜213〉the unknown

<220>

＜223〉new sequence

<400>29

accatggctt?gcaatggcag?tgcggccagg?gggcact???????????????????37

<210>30

<211>39

<212>DNA

＜213〉the unknown

<220>

＜223〉new sequence

<400>30

cgaccaggac?aaacagcatc?ttggtcactt?gtctccggc?????????????????39

<210>31

<211>39

<212>DNA

＜213〉the unknown

<220>

＜223〉new sequence

<400>31

gaccaagatg?ctgtttgtcc?tggtcgtggt?gtttggcat??????????????????????39

<210>32

<211>35

<212>DNA

＜213〉the unknown

<220>

＜223〉new sequence

<400>32

cggaattcag?gatggatcgg?tctcttgctg?cgcct??????????????????????????35

<210>33

<211>30

<212>DNA

＜213〉the unknown

<220>

＜223〉new sequence

<400>33

gcgaattccg?gctccctgtg?ctgccccagg????????????????????????????????30

<210>34

<211>30

<212>DNA

＜213〉the unknown

<220>

＜223〉new sequence

<400>34

gcggatcccg?gagcccccga?gacctggccc????????????????????????????????30

<210>35

<211>31

<212>DNA

＜213〉the unknown

<220>

＜223〉new sequence

<400>35

ctggaattct?cctgctcatc?cagccatgcg?g????????????????????????????????31

<210>36

<211>30

<212>DNA

＜213〉the unknown

<220>

＜223〉new sequence

<400>36

cctggatccc?cacccctact?ggggcctcag??????????????????????????????????30

<210>37

<211>29

<212>DNA

＜213〉the unknown

<220>

＜223〉new sequence

<400>37

tccagccgtc?ccaaacgtgt?cttcgctgc????????????????????????????????????29

<210>38

<211>31

<212>DNA

＜213〉the unknown

<220>

＜223〉new sequence

<400>38

ctccttcggt?cctcctatcg?ttgtcagaag?t?????????????????????????????????31

<210>39

<211>33

<212>DNA

＜213〉the unknown

<220>

＜223〉new sequence

<400>39

cagaagcaca?gatcaaaaaa?gatcatcttc?ctg???????????????????????????????33

<210>40

<211>31

<212>DNA

＜213〉the unknown

<220>

＜223〉new sequence

<400>40

acaggaatca?cagccgaggg?ggagtgccac?t?????????????????????????????????31

<210>41

<211>32

<212>DNA

＜213〉the unknown

<220>

＜223〉new sequence

<400>41

tgtgttcttt?ccggcatgtt?ttcttgggct?tg????????????????????????????????32

<210>42

<211>32

<212>DNA

＜213〉the unknown

<220>

＜223〉new sequence

<400>42

caagcccaag?aaaacatgcc?ggaaagaaca?ca????????????????????????????????32

<210>43

<211>33

<212>DNA

＜213〉the unknown

<220>

＜223〉new sequence

<400>43

ctcatggtca?catgttgtgt?gtatgccatc?aag????????????????????????????????33

<210>44

<211>33

<212>DNA

＜213〉the unknown

<220>

＜223〉new sequence

<400>44

cttgatggca?tacacacaac?atgtgaccat?gag????????????????????????????????33

<210>45

<211>34

<212>DNA

＜213〉the unknown

<220>

＜223〉new sequence

<400>45

acgaagccaa?gcccaaggga?ttcactatgt?acac???????????????????????????????34

<210>46

<211>34

<212>DNA

＜213〉the unknown

<220>

＜223〉new sequence

<400>46

gtgtacatag?tgaatccctt?gggcttggct?ccgt???????????????????????????????34

<210>47

<211>35

<212>DNA

＜213〉the unknown

<220>

＜223〉new sequence

<400>47

gtcaccacct?ttcacccgat?gtgctctgtg?catag?????????????????????????????35

<210>48

<211>35

<212>DNA

＜213〉the unknown

<220>

＜223〉new sequence

<400>48

ctatgcacag?agcacatcgg?gtgaaaggtg?gtgac?????????????????????????????35

<210>49

<211>36

<212>DNA

＜213〉the unknown

<220>

＜223〉new sequence

<400>49

ccttttgttc?tttaagtcct?atgtcacccc?agtcct????????????????????????????36

<210>50

<211>36

<212>DNA

＜213〉the unknown

<220>

＜223〉new sequence

<400>50

aggactgggg?tgacatagga?cttaaagaac?aaaagg????????????????????????????36

<210>51

<211>31

<212>DNA

＜213〉the unknown

<220>

＜223〉new sequence

<400>51

atgtggagcc?ccatcttcat?caccatcctc?c??????????????????????????????????31

<210>52

<211>31

<212>DNA

＜213〉the unknown

<220>

＜223〉new sequence

<400>52

ggaggatggt?gatgaagatg?gggctccaca?t??????????????????????????????????31

<210>53

<211>33

<212>DNA

＜213〉the unknown

<220>

＜223〉new sequence

<400>53

gccgcggtca?gcctgaatcg?catggtgtgc?atc????????????????????????????????33

<210>54

<211>33

<212>DNA

＜213〉the unknown

<220>

＜223〉new sequence

<400>54

gatgcacacc?atgcgattca?ggctgaccgc?ggc????????????????????????????????33

<210>55

<211>29

<212>DNA

＜213〉the unknown

<220>

＜223〉new sequence

<400>55

ggccggagac?aagtgaaaag?atgctgttt????????????????????????????????????29

<210>56

<211>30

<212>DNA

＜213〉the unknown

<220>

＜223〉new sequence

<400>56

aaacagcatc?tttttcactt?gtctccggcc???????????????????????????????????30

<210>57

<211>27

<212>DNA

＜213〉the unknown

<220>

＜223〉new sequence

<400>57

gagagccagc?tcaagagcac?cgtggtg??????????????????????????????????????27

<210>58

<211>31

<212>DNA

＜213〉the unknown

<220>

＜223〉new sequence

<400>58

ctccttcggt?cctcctatcg?ttgtcagaag?t?????????????????????????????????31

<210>59

<211>31

<212>DNA

＜213〉the unknown

<220>

＜223〉new sequence

<400>59

agtggcactc?cccctcggct?gtgattcctg?t?????????????????????????????????31

<210>60

<211>30

<212>DNA

＜213〉the unknown

<220>

＜223〉new sequence

<400>60

gccacccgca?aggctaaacg?catggtctgg???????????????????????????????????30

<210>61

<211>31

<212>DNA

＜213〉the unknown

<220>

＜223〉new sequence

<400>61

ctccttcggt?cctcctatcg?ttgtcagaag?t?????????????????????????????????31

<210>62

<211>1062

<212>DNA

＜213〉the unknown

<220>

＜223〉new sequence

<400>62

atggaaacca?acttctccat?tcctctgaat?gaaactgagg?aggtgctccc?tgagcctgct????60

ggccacaccg?ttctgtggat?cttctcattg?ctagtccacg?gagtcacctt?tgtcttcggg????120

gtcctgggca?atgggcttgt?gatctgggtg?gctggattcc?ggatgacacg?cacagtcaac????180

accatctgtt?acctgaacct?ggccctagct?gacttctctt?tcagtgccat?cctaccattc????240

cgaatggtct?cagtcgccat?gagagaaaaa?tggccttttg?gctcattcct?atgtaagtta????300

gttcatgtta?tgatagacat?caacctgttt?gtcagtgtct?acctgatcac?catcattgct????360

ctggaccgct?gtatttgtgt?cctgcatcca?gcctgggccc?agaaccatcg?caccatgagt????420

ctggccaaga?gggtgatgac?gggactctgg?attttcacca?tagtccttac?cttaccaaat????480

ttcatcttct?ggactacaat?aagtactacg?aatggggaca?catactgtat?tttcaacttt????540

gcattctggg?gtgacactgc?tgtagagagg?ttgaacgtgt?tcattaccat?ggccaaggtc????600

tttctgatcc?tccacttcat?tattggcttc?agcgtgccta?tgtccatcat?cacagtctgc????660

tatgggatca?tcgctgccaa?aattcacaga?aaccacatga?ttaaatccag?ccgtcccaaa????720

cgtgtcttcg?ctgctgtggt?ggcttctttc?ttcatctgtt?ggttccctta?tgaactaatt????780

ggcattctaa?tggcagtctg?gctcaaagag?atgttgttaa?atggcaaata?caaaatcatt????840

cttgtcctga?ttaacccaac?aagctccttg?gcctttttta?acagctgcct?caacccaatt????900

ctctacgtct?ttatgggtcg?taacttccaa?gaaagactga?ttcgctcttt?gcccactagt????960

ttggagaggg?ccctgactga?ggtccctgac?tcagcccaga?ccagcaacac?agacaccact????1020

tctgcttcac?ctcctgagga?gacggagtta?caagcaatgt?ga???????????????????????1062

<210>63

<211>353

<212>PRT

＜213〉the unknown

<220>

＜223〉new sequence

<400>63

Met?Glu?Thr?Asn?Phe?Ser?Ile?Pro?Leu?Asn?Glu?Thr?Glu?Glu?Val?Leu

1???????????????5???????????????????10??????????????????15

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

20??????????????????25??????????????????30

His?Gly?Val?Thr?Phe?Val?Phe?Gly?Val?Leu?Gly?Asn?Gly?Leu?Val?Ile

35??????????????????40??????????????????45

Trp?Val?Ala?Gly?Phe?Arg?Met?Thr?Arg?Thr?Val?Asn?Thr?Ile?Cys?Tyr

50??????????????????55??????????????????60

Leu?Asn?Leu?Ala?Leu?Ala?Asp?Phe?Ser?Phe?Ser?Ala?Ile?Leu?Pro?Phe

65??????????????????70??????????????????75??????????????????80

Arg?Met?Val?Ser?Val?Ala?Met?Arg?Glu?Lys?Trp?Pro?Phe?Gly?Ser?Phe

85??????????????????90??????????????????95

Leu?Cys?Lys?Leu?Val?His?Val?Met?Ile?Asp?Ile?Asn?Leu?Phe?Val?Ser

100?????????????????105?????????????????110

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

115?????????????????120?????????????????125

His?Pro?Ala?Trp?Ala?Gln?Asn?His?Arg?Thr?Met?Ser?Leu?Ala?Lys?Arg

130?????????????????135?????????????????140

Val?Met?Thr?Gly?Leu?Trp?Ile?Phe?Thr?Ile?Val?Leu?Thr?Leu?Pro?Asn

145?????????????????150?????????????????155?????????????????160

Phe?Ile?Phe?Trp?Thr?Thr?Ile?Ser?Thr?Thr?Asn?Gly?Asp?Thr?Tyr?Cys

165?????????????????170?????????????????175

Ile?Phe?Asn?Phe?Ala?Phe?Trp?Gly?Asp?Thr?Ala?Val?Glu?Arg?Leu?Asn

180?????????????????185?????????????????190

Val?Phe?Ile?Thr?Met?Ala?Lys?Val?Phe?Leu?Ile?Leu?His?Phe?Ile?Ile

195?????????????????200?????????????????205

Gly?Phe?Ser?Val?Pro?Met?Ser?Ile?Ile?Thr?Val?Cys?Tyr?Gly?Ile?Ile

210?????????????????215?????????????????220

Ala?Ala?Lys?Ile?His?Arg?Asn?His?Met?Ile?Lys?Ser?Ser?Arg?Pro?Lys

225?????????????????230?????????????????235?????????????????240

Arg?Val?Phe?Ala?Ala?Val?Val?Ala?Ser?Phe?Phe?Ile?Cys?Trp?Phe?Pro

245?????????????????250?????????????????255

Tyr?Glu?Leu?Ile?Gly?Ile?Leu?Met?Ala?Val?Trp?Leu?Lys?Glu?Met?Leu

260?????????????????265?????????????????270

Leu?Asn?Gly?Lys?Tyr?Lys?Ile?Ile?Leu?Val?Leu?Ile?Asn?Pro?Thr?Ser

275?????????????????280?????????????????285

Ser?Leu?Ala?Phe?Phe?Asn?Ser?Cys?Leu?Asn?Pro?Ile?Leu?Tyr?Val?Phe

290?????????????????295?????????????????300

Met?Gly?Arg?Asn?Phe?Gln?Glu?Arg?Leu?Ile?Arg?Ser?Leu?Pro?Thr?Ser

305?????????????????310?????????????????315?????????????????320

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

325?????????????????330?????????????????335

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

340?????????????????345?????????????????350

Met

<210>64

<211>1029

<212>DNA

＜213〉the unknown

<220>

＜223〉new sequence

<400>64

atggcagagc?atgattacca?tgaagactat?gggttcagca?gtttcaatga?cagcagccag????60

gaggagcatc?aagccttcct?gcagttcagc?aaggtctttc?tgccctgcat?gtacctggtg????120

gtgtttgtct?gtggtctggt?ggggaactct?ctggtgctgg?tcatatccat?cttctaccat????180

aagttgcaga?gcctgacgga?tgtgttcctg?gtgaacctac?ccctggctga?cctggtgttt????240

gtctgcactc?tgcccttctg?ggcctatgca?ggcatccatg?aatgggtgtt?tggccaggtc????300

atgtgcaaaa?gcctactggg?catctacact?attaacttct?acacgtccat?gctcatcctc????360

acctgcatca?ctgtggatcg?tttcattgta?gtggttaagg?ccaccaaggc?ctacaaccag????420

caagccaaga?ggatgacctg?gggcaaggtc?accagcttgc?tcatctgggt?gatatccctg????480

ctggtttcct?tgccccaaat?tatctatggc?aatgtcttta?atctcgacaa?gctcatatgt????540

ggttaccatg?acgaggcaat?ttccactgtg?gttcttgcca?cccagatgac?actggggttc????600

ttcttgccac?tgctcaccat?gattgtctgc?tattcagtca?taatcaaaac?actgcttcat????660

gctggaggct?tccagaagca?cagatcaaaa?aagatcatct?tcctggtgat?ggctgtgttc????720

ctgctgaccc?agatgccctt?caacctcatg?aagttcatcc?gcagcacaca?ctgggaatac????780

tatgccatga?ccagctttca?ctacaccatc?atggtgacag?aggccatcgc?atacctgagg????840

gcctgcctta?accctgtgct?ctatgccttt?gtcagcctga?agtttcgaaa?gaacttctgg????900

aaacttgtga?aggacattgg?ttgcctccct?taccttgggg?tctcacatca?atggaaatct????960

tctgaggaca?attccaagac?tttttctgcc?tcccacaatg?tggaggccac?cagcatgttc????1020

cagttatag????????????????????????????????????????????????????????????1029

<210>65

<211>342

<212>PRT

＜213〉the unknown

<220>

＜223〉new sequence

<400>65

Met?Ala?Glu?His?Asp?Tyr?His?Glu?Asp?Tyr?Gly?Phe?Ser?Ser?Phe?Asn

1???????????????5???????????????????10??????????????????15

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

20??????????????????25??????????????????30

Phe?Leu?Pro?Cys?Met?Tyr?Leu?Val?Val?Phe?Val?Cys?Gly?Leu?Val?Gly

35??????????????????40??????????????????45

Asn?Ser?Leu?Val?Leu?Val?Ile?Ser?Ile?Phe?Tyr?His?Lys?Leu?Gln?Ser

50??????????????????55??????????????????60

Leu?Thr?Asp?Val?Phe?Leu?Val?Asn?Leu?Pro?Leu?Ala?Asp?Leu?Val?Phe

65??????????????????70??????????????????75??????????????????80

Val?Cys?Thr?Leu?Pro?Phe?Trp?Ala?Tyr?Ala?Gly?Ile?His?Glu?Trp?Val

85??????????????????90??????????????????95

Phe?Gly?Gln?Val?Met?Cys?Lys?Ser?Leu?Leu?Gly?Ile?Tyr?Thr?Ile?Asn

100?????????????????105?????????????????110

Phe?Tyr?Thr?Ser?Met?Leu?Ile?Leu?Thr?Cys?Ile?Thr?Val?Asp?Arg?Phe

115?????????????????120?????????????????125

Ile?Val?Val?Val?Lys?Ala?Thr?Lys?Ala?Tyr?Asn?Gln?Gln?Ala?Lys?Arg

130?????????????????135?????????????????140

Met?Thr?Trp?Gly?Lys?Val?Thr?Ser?Leu?Leu?Ile?Trp?Val?Ile?Ser?Leu

145?????????????????150?????????????????155?????????????????160

Leu?Val?Ser?Leu?Pro?Gln?Ile?Ile?Tyr?Gly?Asn?Val?Phe?Asn?Leu?Asp

165?????????????????170?????????????????175

Lys?Leu?Ile?Cys?Gly?Tyr?His?Asp?Glu?Ala?Ile?Ser?Thr?Val?Val?Leu

180?????????????????185?????????????????190

Ala?Thr?Gln?Met?Thr?Leu?Gly?Phe?Phe?Leu?Pro?Leu?Leu?Thr?Met?Ile

195?????????????????200?????????????????205

Val?Cys?Tyr?Ser?Val?Ile?Ile?Lys?Thr?Leu?Leu?His?Ala?Gly?Gly?Phe

210?????????????????215?????????????????220

Gln?Lys?His?Arg?Ser?Lys?Lys?Ile?Ile?Phe?Leu?Val?Met?Ala?Val?Phe

225?????????????????230?????????????????235?????????????????240

Leu?Leu?Thr?Gln?Met?Pro?Phe?Asn?Leu?Met?Lys?Phe?Ile?Arg?Ser?Thr

245?????????????????250?????????????????255

His?Trp?Glu?Tyr?Tyr?Ala?Met?Thr?Ser?Phe?His?Tyr?Thr?Ile?Met?Val

260?????????????????265?????????????????270

Thr?Glu?Ala?Ile?Ala?Tyr?Leu?Arg?Ala?Cys?Leu?Asn?Pro?Val?Leu?Tyr

275?????????????????280?????????????????285

Ala?Phe?Val?Ser?Leu?Lys?Phe?Arg?Lys?Asn?Phe?Trp?Lys?Leu?Val?Lys

290?????????????????295?????????????????300

Asp?Ile?Gly?Cys?Leu?Pro?Tyr?Leu?Gly?Val?Ser?His?Gln?Trp?Lys?Ser

205?????????????????310?????????????????315?????????????????320

Ser?Glu?Asp?Asn?Ser?Lys?Thr?Phe?Ser?Ala?Ser?His?Asn?Val?Glu?Ala

325?????????????????330?????????????????335

Thr?Ser?Met?Phe?Gln?Leu

340

<210>66

<211>2748

<212>DNA

＜213〉the unknown

<220>

＜223〉new sequence

<400>66

atggtccagc?tgaggaagct?gctccgcgtc?ctgactttga?tgaagttccc?ctgctgcgtg????60

ctggaggtgc?tcctgtgcgc?gctggcggcg?gcggcgcgcg?gccaggagat?gtacgccccg????120

cactcaatcc?ggatcgaggg?ggacgtcacc?ctcggggggc?tgttccccgt?gcacgccaag????180

ggtcccagcg?gagtgccctg?cggcgacatc?aagagggaaa?acgggatcca?caggctggaa????240

gcgatgctct?acgccctgga?ccagatcaac?agtgatccca?acctactgcc?caacgtgacg????300

ctgggcgcgc?ggatcctgga?cacttgttcc?agggacactt?acgcgctcga?acagtcgctt????360

actttcgtcc?aggcgctcat?ccagaaggac?acctccgacg?tgcgctgcac?caacggcgaa????420

ccgccggttt?tcgtcaagcc?ggagaaagta?gttggagtga?ttggggcttc?ggggagttcg????480

gtctccatca?tggtagccaa?catcctgagg?ctcttccaga?tcccccagat?tagttatgca????540

tcaacggcac?ccgagctaag?tgatgaccgg?cgctatgact?tcttctctcg?cgtggtgcca????600

cccgattcct?tccaagccca?ggccatggta?gacattgtaa?aggccctagg?ctggaattat????660

gtgtctaccc?tcgcatcgga?aggaagttat?ggagagaaag?gtgtggagtc?cttcacgcag????720

atttccaaag?aggcaggtgg?actctgcatt?gcccagtccg?tgagaatccc?ccaggaacgc????780

aaagacagga?ccattgactt?tgatagaatt?atcaaacagc?tcctggacac?ccccaactcc????840

agggccgtcg?tgatttttgc?caacgatgag?gatataaagc?agatccttgc?agcagccaaa????900

agagctgacc?aagttggcca?ttttctttgg?gtgggatcag?acagctgggg?atccaaaata????960

aacccactgc?accagcatga?agatatcgca?gaaggggcca?tcaccattca?gcccaagcga????1020

gccacggtgg?aagggtttga?tgcctacttt?acgtcccgta?cacttgaaaa?caacagaaga????1080

aatgtatggt?ttgccgaata?ctgggaggaa?aacttcaact?gcaagttgac?gattagtggg????1140

tcaaaaaaag?aagacacaga?tcgcaaatgc?acaggacagg?agagaattgg?aaaagattcc????1200

aactatgagc?aggagggtaa?agtccagttc?gtgattgacg?cagtctatgc?tatggctcac????1260

gcccttcacc?acatgaacaa?ggatctctgt?gctgactacc?ggggtgtctg?cccagagatg????1320

gagcaagctg?gaggcaagaa?gttgctgaag?tatatacgca?atgttaattt?caatggtagt????1380

gctggcactc?cagtgatgtt?taacaagaac?ggggatgcac?ctgggcgtta?tgacatcttt????1440

cagtaccaga?ccacaaacac?cagcaacccg?ggttaccgtc?tgatcgggca?gtggacagac????1500

gaacttcagc?tcaatataga?agacatgcag?tggggtaaag?gagtccgaga?gatacccgcc????1560

tcagtgtgca?cactaccatg?taagccagga?cagagaaaga?agacacagaa?aggaactcct????1620

tgctgttgga?cctgtgagcc?ttgcgatggt?taccagtacc?agtttgatga?gatgacatgc????1680

cagcattgcc?cctatgacca?gaggcccaat?gaaaatcgaa?ccggatgcca?ggatattccc????1740

atcatcaaac?tggagtggca?ctccccctcg?gctgtgattc?ctgtcttcct?ggcaatgttg????1800

gggatcattg?ccaccatctt?tgtcatggcc?actttcatcc?gctacaatga?cacgcccatt????1860

gtccgggcat?ctgggcggga?actcagctat?gttcttttga?cgggcatctt?tctttgctac????1920

atcatcactt?tcctgatgat?tgccaaacca?gatgtggcag?tgtgttcttt?ccggcgagtt????1980

ttcttgggct?tgggtatgtg?catcagttat?gcagccctct?tgacgaaaac?aaatcggatt????2040

tatcgcatat?ttgagcaggg?caagaaatca?gtaacagctc?ccagactcat?aagcccaaca????2100

tcacaactgg?caatcacttc?cagtttaata?tcagttcagc?ttctaggggt?gttcatttgg????2160

tttggtgttg?atccacccaa?catcatcata?gactacgatg?aacacaagac?aatgaaccct????2220

gagcaagcca?gaggggttct?caagtgtgac?attacagatc?tccaaatcat?ttgctccttg????2280

ggatatagca?ttcttctcat?ggtcacatgt?actgtgtatg?ccatcaagac?tcggggtgta????2340

cccgagaatt?ttaacgaagc?caagcccatt?ggattcacta?tgtacacgac?atgtatagta????2400

tggcttgcct?tcattccaat?tttttttggc?accgctcaat?cagcggaaaa?gctctacata????2460

caaactacca?cgcttacaat?ctccatgaac?ctaagtgcat?cagtggcgct?ggggatgcta????2520

tacatgccga?aagtgtacat?catcattttc?caccctgaac?tcaatgtcca?gaaacggaag????2580

cgaagcttca?aggcggtagt?cacagcagcc?accatgtcat?cgaggctgtc?acacaaaccc????2640

agtgacagac?ccaacggtga?ggcaaagacc?gagctctgtg?aaaacgtaga?cccaaacagc????2700

cctgctgcaa?aaaagaagta?tgtcagttat?aataacctgg?ttatctaa?????????????????2748

<210>67

<211>915

<212>PRT

＜213〉the unknown

<220>

＜223〉new sequence

<400>67

Met?Val?Gln?Leu?Arg?Lys?Leu?Leu?Arg?Val?Leu?Thr?Leu?Met?Lys?Phe

1???????????????5???????????????????10??????????????????15

Pro?Cys?Cys?Val?Leu?Glu?Val?Leu?Leu?Cys?Ala?Leu?Ala?Ala?Ala?Ala

20??????????????????25??????????????????30

Arg?Gly?Gln?Glu?Met?Tyr?Ala?Pro?His?Ser?Ile?Arg?Ile?Glu?Gly?Asp

35??????????????????40??????????????????45

Val?Thr?Leu?Gly?Gly?Leu?Phe?Pro?Val?His?Ala?Lys?Gly?Pro?Ser?Gly

50??????????????????55??????????????????60

Val?Pro?Cys?Gly?Asp?Ile?Lys?Arg?Glu?Asn?Gly?Ile?His?Arg?Leu?Glu

65??????????????????70??????????????????75??????????????????80

Ala?Met?Leu?Tyr?Ala?Leu?Asp?Gln?Ile?Asn?Ser?Asp?Pro?Asn?Leu?Leu

85??????????????????90??????????????????95

Pro?Asn?Val?Thr?Leu?Gly?Ala?Arg?Ile?Leu?Asp?Thr?Cys?Ser?Arg?Asp

100?????????????????105?????????????????110

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

115?????????????????120?????????????????125

Lys?Asp?Thr?Ser?Asp?Val?Arg?Cys?Thr?Asn?Gly?Glu?Pro?Pro?Val?Phe

130?????????????????135?????????????????140

Val?Lys?Pro?Glu?Lys?Val?Val?Gly?Val?Ile?Gly?Ala?Ser?Gly?Ser?Ser

145?????????????????150?????????????????155?????????????????160

Val?Ser?Ile?Met?Val?Ala?Asn?Ile?Leu?Arg?Leu?Phe?Gln?Ile?Pro?Gln

165?????????????????170?????????????????175

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

180?????????????????185?????????????????190

Asp?Phe?Phe?Ser?Arg?Val?Val?Pro?Pro?Asp?Ser?Phe?Gln?Ala?Gln?Ala

195?????????????????200?????????????????205

Met?Val?Asp?Ile?Val?Lys?Ala?Leu?Gly?Trp?Asn?Tyr?Val?Ser?Thr?Leu

210?????????????????215?????????????????220

Ala?Ser?Glu?Gly?Ser?Tyr?Gly?Glu?Lys?Gly?Val?Glu?Ser?Phe?Thr?Gln

225?????????????????230?????????????????235?????????????????240

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

245?????????????????250?????????????????255

Pro?Gln?Glu?Arg?Lys?Asp?Arg?Thr?Ile?Asp?Phe?Asp?Arg?Ile?Ile?Lys

260?????????????????265?????????????????270

Gln?Leu?Leu?Asp?Thr?Pro?Asn?Ser?Arg?Ala?Val?Val?Ile?Phe?Ala?Asn

275?????????????????280??????????????????285

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

290?????????????????295?????????????????300

Val?Gly?His?Phe?Leu?Trp?Val?Gly?Ser?Asp?Ser?Trp?Gly?Ser?Lys?Ile

305?????????????????310?????????????????315?????????????????320

Asn?Pro?Leu?His?Gln?His?Glu?Asp?Ile?Ala?Glu?Gly?Ala?Ile?Thr?Ile

325?????????????????330?????????????????335

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

340?????????????????345?????????????????350

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

355?????????????????360?????????????????365

Glu?Glu?Asn?Phe?Asn?Cys?Lys?Leu?Thr?Ile?Ser?Gly?Ser?Lys?Lys?Glu

370?????????????????375?????????????????380

Asp?Thr?Asp?Arg?Lys?Cys?Thr?Gly?Gln?Glu?Arg?Ile?Gly?Lys?Asp?Ser

385?????????????????390?????????????????395?????????????????400

Asn?Tyr?Glu?Gln?Glu?Gly?Lys?Val?Gln?Phe?Val?Ile?Asp?Ala?Val?Tyr

405?????????????????410?????????????????415

Ala?Met?Ala?His?Ala?Leu?His?His?Met?Asn?Lys?Asp?Leu?Cys?Ala?Asp

420?????????????????425?????????????????430

Tyr?Arg?Gly?Val?Cys?Pro?Glu?Met?Glu?Gln?Ala?Gly?Gly?Lys?Lys?Leu

435?????????????????440?????????????????445

Leu?Lys?Tyr?Ile?Arg?Asn?Val?Asn?Phe?Asn?Gly?Ser?Ala?Gly?Thr?Pro

450?????????????????455?????????????????460

Val?Met?Phe?Asn?Lys?Asn?Gly?Asp?Ala?Pro?Gly?Arg?Tyr?Asp?Ile?Phe

465?????????????????470?????????????????475?????????????????480

Gln?Tyr?Gln?Thr?Thr?Asn?Thr?Ser?Asn?Pro?Gly?Tyr?Arg?Leu?Ile?Gly

485?????????????????490?????????????????495

Gln?Trp?Thr?Asp?Glu?Leu?Gln?Leu?Asn?Ile?Glu?Asp?Met?Gln?Trp?Gly

500?????????????????505?????????????????510

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

515?????????????????520?????????????????525

Pro?Gly?Gln?Arg?Lys?Lys?Thr?Gln?Lys?Gly?Thr?Pro?Cys?Cys?Trp?Thr

530?????????????????535?????????????????540

Cys?Glu?Pro?Cys?Asp?Gly?Tyr?Gln?Tyr?Gln?Phe?Asp?Glu?Met?Thr?Cys

545?????????????????550?????????????????555?????????????????560

Gln?His?Cys?Pro?Tyr?Asp?Gln?Arg?Pro?Asn?Glu?Asn?Arg?Thr?Gly?Cys

565?????????????????570?????????????????575

Gln?Asp?Ile?Pro?Ile?Ile?Lys?Leu?Glu?Trp?His?Ser?Pro?Ser?Ala?Val

580?????????????????585?????????????????590

Ile?Pro?Val?Phe?Leu?Ala?Met?Leu?Gly?Ile?Ile?Ala?Thr?Ile?Phe?Val

595?????????????????600?????????????????605

Met?Ala?Thr?Phe?Ile?Arg?Tyr?Asn?Asp?Thr?Pro?Ile?Val?Arg?Ala?Ser

610?????????????????615?????????????????620

Gly?Arg?Glu?Leu?Ser?Tyr?Val?Leu?Leu?Thr?Gly?Ile?Phe?Leu?Cys?Tyr

625?????????????????630?????????????????635?????????????????640

Ile?Ile?Thr?Phe?Leu?Met?Ile?Ala?Lys?Pro?Asp?Val?Ala?Val?Cys?Ser

645?????????????????650?????????????????655

Phe?Arg?Arg?Val?Phe?Leu?Gly?Leu?Gly?Met?Cys?Ile?Ser?Tyr?Ala?Ala

660?????????????????665?????????????????670

Leu?Leu?Thr?Lys?Thr?Asn?Arg?Ile?Tyr?Arg?Ile?Phe?Glu?Gln?Gly?Lys

675?????????????????680?????????????????685

Lys?Ser?Val?Thr?Ala?Pro?Arg?Leu?Ile?Ser?Pro?Thr?Ser?Gln?Leu?Ala

690?????????????????695?????????????????700

Ile?Thr?Ser?Ser?Leu?Ile?Ser?Val?Gln?Leu?Leu?Gly?Val?Phe?Ile?Trp

705?????????????????710?????????????????715?????????????????720

Phe?Gly?Val?Asp?Pro?Pro?Asn?Ile?Ile?Ile?Asp?Tyr?Asp?Glu?His?Lys

725?????????????????730?????????????????735

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

740?????????????????745?????????????????750

Asp?Leu?Gln?Ile?Ile?Cys?Ser?Leu?Gly?Tyr?Ser?Ile?Leu?Leu?Met?Val

755?????????????????760?????????????????765

Thr?Cys?Thr?Val?Tyr?Ala?Ile?Lys?Thr?Arg?Gly?Val?Pro?Glu?Asn?Phe

770?????????????????775?????????????????780

Asn?Glu?Ala?Lys?Pro?Ile?Gly?Phe?Thr?Met?Tyr?Thr?Thr?Cys?Ile?Val

785?????????????????790?????????????????795?????????????????800

Trp?Leu?Ala?Phe?Ile?Pro?Ile?Phe?Phe?Gly?Thr?Ala?Gln?Ser?Ala?Glu

805?????????????????810?????????????????815

Lys?Leu?Tyr?Ile?Gln?Thr?Thr?Thr?Leu?Thr?Ile?Ser?Met?Asn?Leu?Ser

820?????????????????825?????????????????830

Ala?Ser?Val?Ala?Leu?Gly?Met?Leu?Tyr?Met?Pro?Lys?Val?Tyr?Ile?Ile

835?????????????????840?????????????????845

Ile?Phe?His?Pro?Glu?Leu?Asn?Val?Gln?Lys?Arg?Lys?Arg?Ser?Phe?Lys

850?????????????????855?????????????????860

Ala?Val?Val?Thr?Ala?Ala?Thr?Met?Ser?Ser?Arg?Leu?Ser?His?Lys?Pro

865?????????????????870?????????????????875?????????????????880

Ser?Asp?Arg?Pro?Asn?Gly?Glu?Ala?Lys?Thr?Glu?Leu?Cys?Glu?Asn?Val

885?????????????????890?????????????????895

Asp?Pro?Asn?Ser?Pro?Ala?Ala?Lys?Lys?Lys?Tyr?Val?Ser?Tyr?Asn?Asn

900?????????????????905?????????????????910

Leu?Val?Ile

915

<210>68

<211>2748

<212>DNA

＜213〉the unknown

<220>

＜223〉new sequence

<400>68

atggtccagc?tgaggaagct?gctccgcgtc?ctgactttga?tgaagttccc?ctgctgcgtg????60

ctggaggtgc?tcctgtgcgc?gctggcggcg?gcggcgcgcg?gccaggagat?gtacgccccg????120

cactcaatcc?ggatcgaggg?ggacgtcacc?ctcggggggc?tgttccccgt?gcacgccaag????180

ggtcccagcg?gagtgccctg?cggcgacatc?aagagggaaa?acgggatcca?caggctggaa????240

gcgatgctct?acgccctgga?ccagatcaac?agtgatccca?acctactgcc?caacgtgacg????300

ctgggcgcgc?ggatcctgga?cacttgttcc?agggacactt?acgcgctcga?acagtcgctt????360

actttcgtcc?aggcgctcat?ccagaaggac?acctccgacg?tgcgctgcac?caacggcgaa????420

ccgccggttt?tcgtcaagcc?ggagaaagta?gttggagtga?ttggggcttc?ggggagttcg????480

gtctccatca?tggtagccaa?catcctgagg?ctcttccaga?tcccccagat?tagttatgca????540

tcaacggcac?ccgagctaag?tgatgaccgg?cgctatgact?tcttctctcg?cgtggtgcca????600

cccgattcct?tccaagccca?ggccatggta?gacattgtaa?aggccctagg?ctggaattat????660

gtgtctaccc?tcgcatcgga?aggaagttat?ggagagaaag?gtgtggagtc?cttcacgcag????720

atttccaaag?aggcaggtgg?actctgcatt?gcccagtccg?tgagaatccc?ccaggaacgc????780

aaagacagga?ccattgactt?tgatagaatt?atcaaacagc?tcctggacac?ccccaactcc????840

agggccgtcg?tgatttttgc?caacgatgag?gatataaagc?agatccttgc?agcagccaaa????900

agagctgacc?aagttggcca?ttttctttgg?gtgggatcag?acagctgggg?atccaaaata????960

aacccactgc?accagcatga?agatatcgca?gaaggggcca?tcaccattca?gcccaagcga????1020

gccacggtgg?aagggtttga?tgcctacttt?acgtcccgta?cacttgaaaa?caacagaaga????1080

aatgtatggt?ttgccgaata?ctgggaggaa?aacttcaact?gcaagttgac?gattagtggg????1140

tcaaaaaaag?aagacacaga?tcgcaaatgc?acaggacagg?agagaattgg?aaaagattcc????1200

aactatgagc?aggagggtaa?agtccagttc?gtgattgacg?cagtctatgc?tatggctcac????1260

gcccttcacc?acatgaacaa?ggatctctgt?gctgactacc?ggggtgtctg?cccagagatg????1320

gagcaagctg?gaggcaagaa?gttgctgaag?tatatacgca?atgttaattt?caatggtagt????1380

gctggcactc?cagtgatgtt?taacaagaac?ggggatgcac?ctgggcgtta?tgacatcttt????1440

cagtaccaga?ccacaaacac?cagcaacccg?ggttaccgtc?tgatcgggca?gtggacagac????1500

gaacttcagc?tcaatataga?agacatgcag?tggggtaaag?gagtccgaga?gatacccgcc????1560

tcagtgtgca?cactaccatg?taagccagga?cagagaaaga?agacacagaa?aggaactcct????1620

tgctgttgga?cctgtgagcc?ttgcgatggt?taccagtacc?agtttgatga?gatgacatgc????1680

cagcattgcc?cctatgacca?gaggcccaat?gaaaatcgaa?ccggatgcca?ggatattccc????1740

atcatcaaac?tggagtggca?ctccccctgg?gctgtgattc?ctgtcttcct?ggcaatgttg????1800

gggatcattg?ccaccatctt?tgtcatggcc?actttcatcc?gctacaatga?cacgcccatt????1860

gtccgggcat?ctgggcggga?actcagctat?gttcttttga?cgggcatctt?tctttgctac????1920

atcatcactt?tcctgatgat?tgccaaacca?gatgtggcag?tgtgttcttt?ccggcatgtt????1980

ttcttgggct?tgggtatgtg?catcagttat?gcagccctct?tgacgaaaac?aaatcggatt????2040

tatcgcatat?ttgagcaggg?caagaaatca?gtaacagctc?ccagactcat?aagcccaaca????2100

tcacaactgg?caatcacttc?cagtttaata?tcagttcagc?ttctaggggt?gttcatttgg????2160

tttggtgttg?atccacccaa?catcatcata?gactacgatg?aacacaagac?aatgaaccct????2220

gagcaagcca?gaggggttct?caagtgtgac?attacagatc?tccaaatcat?ttgctccttg????2280

ggatatagca?ttcttctcat?ggtcacatgt?actgtgtatg?ccatcaagac?tcggggtgta????2340

cccgagaatt?ttaacgaagc?caagcccatt?ggattcacta?tgtacacgac?atgtatagta????2400

tggcttgcct?tcattccaat?tttttttggc?accgctcaat?cagcggaaaa?gctctacata????2460

caaactacca?cgcttacaat?ctccatgaac?ctaagtgcat?cagtggcgct?ggggatgcta????2520

tacatgccga?aagtgtacat?catcattttc?caccctgaac?tcaatgtcca?gaaacggaag????2580

cgaagcttca?aggcggtagt?cacagcagcc?accatgtcat?cgaggctgtc?acacaaaccc????2640

agtgacagac?ccaacggtga?ggcaaagacc?gagctctgtg?aaaacgtaga?cccaaacagc????2700

cctgctgcaa?aaaagaagta?tgtcagttat?aataacctgg?ttatctaa?????????????????2748

<210>69

<211>915

<212>PRT

＜213〉the unknown

<220>

＜223〉new sequence

<400>69

Met?Val?Gln?Leu?Arg?Lys?Leu?Leu?Arg?Val?Leu?Thr?Leu?Met?Lys?Phe

1???????????????5???????????????????10??????????????????15

Pro?Cys?Cys?Val?Leu?Glu?Val?Leu?Leu?Cys?Ala?Leu?Ala?Ala?Ala?Ala

20??????????????????25??????????????????30

Arg?Gly?Gln?Glu?Met?Tyr?Ala?Pro?His?Ser?Ile?Arg?Ile?Glu?Gly?Asp

35??????????????????40??????????????????45

Val?Thr?Leu?Gly?Gly?Leu?Phe?Pro?Val?His?Ala?Lys?Gly?Pro?Ser?Gly

50??????????????????55??????????????????60

Val?Pro?Cys?Gly?Asp?Ile?Lys?Arg?Glu?Asn?Gly?Ile?His?Arg?Leu?Glu

65??????????????????70??????????????????75??????????????????80

Ala?Met?Leu?Tyr?Ala?Leu?Asp?Gln?Ile?Asn?Ser?Asp?Pro?Asn?Leu?Leu

85??????????????????90??????????????????95

Pro?Asn?Val?Thr?Leu?Gly?Ala?Arg?Ile?Leu?Asp?Thr?Cys?Ser?Arg?Asp

100?????????????????105?????????????????110

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

115?????????????????120?????????????????125

Lys?Asp?Thr?Ser?Asp?Val?Arg?Cys?Thr?Asn?Gly?Glu?Pro?Pro?Val?Phe

130?????????????????135?????????????????140

Val?Lys?Pro?Glu?Lys?Val?Val?Gly?Val?Ile?Gly?Ala?Ser?Gly?Ser?Ser

145?????????????????150?????????????????155?????????????????160

Val?Ser?Ile?Met?Val?Ala?Asn?Ile?Leu?Arg?Leu?Phe?Gln?Ile?Pro?Gln

165?????????????????170?????????????????175

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

180?????????????????185?????????????????190

Asp?Phe?Phe?Ser?Arg?Val?Val?Pro?Pro?Asp?Ser?Phe?Gln?Ala?Gln?Ala

195?????????????????200?????????????????205

Met?Val?Asp?Ile?Val?Lys?Ala?Leu?Gly?Trp?Asn?Tyr?Val?Ser?Thr?Leu

210?????????????????215?????????????????220

Ala?Ser?Glu?Gly?Ser?Tyr?Gly?Glu?Lys?Gly?Val?Glu?Ser?Phe?Thr?Gln

225?????????????????230?????????????????235?????????????????240

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

245?????????????????250?????????????????255

Pro?Gln?Glu?Arg?Lys?Asp?Arg?Thr?Ile?Asp?Phe?Asp?Arg?Ile?Ile?Lys

260?????????????????265?????????????????270

Gln?Leu?Leu?Asp?Thr?Pro?Asn?Ser?Arg?Ala?Val?Val?Ile?Phe?Ala?Asn

275?????????????????280?????????????????285

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

290?????????????????295?????????????????300

Val?Gly?His?Phe?Leu?Trp?Val?Gly?Ser?Asp?Ser?Trp?Gly?Ser?Lys?Ile

305?????????????????310?????????????????315?????????????????320

Asn?Pro?Leu?His?Gln?His?Glu?Asp?Ile?Ala?Glu?Gly?Ala?Ile?Thr?Ile

325?????????????????330?????????????????335

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

340?????????????????345?????????????????350

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

355?????????????????360?????????????????365

Glu?Glu?Asn?Phe?Asn?Cys?Lys?Leu?Thr?Ile?Ser?Gly?Ser?Lys?Lys?Glu

370?????????????????375?????????????????380

Asp?Thr?Asp?Arg?Lys?Cys?Thr?Gly?Gln?Glu?Arg?Ile?Gly?Lys?Asp?Ser

385?????????????????390?????????????????395?????????????????400

Asn?Tyr?Glu?Gln?Glu?Gly?Lys?Val?Gln?Phe?Val?Ile?Asp?Ala?Val?Tyr

405?????????????????410?????????????????415

Ala?Met?Ala?His?Ala?Leu?His?His?Met?Asn?Lys?Asp?Leu?Cys?Ala?Asp

420?????????????????425?????????????????430

Tyr?Arg?Gly?Val?Cys?Pro?Glu?Met?Glu?Gln?Ala?Gly?Gly?Lys?Lys?Leu

435?????????????????440?????????????????445

Leu?Lys?Tyr?Ile?Arg?Asn?Val?Asn?Phe?Asn?Gly?Ser?Ala?Gly?Thr?Pro

450?????????????????455?????????????????460

Val?Met?Phe?Asn?Lys?Asn?Gly?Asp?Ala?Pro?Gly?Arg?Tyr?Asp?Ile?Phe

465?????????????????470?????????????????475?????????????????480

Gln?Tyr?Gln?Thr?Thr?Asn?Thr?Ser?Asn?Pro?Gly?Tyr?Arg?Leu?Ile?Gly

485?????????????????490?????????????????495

Gln?Trp?Thr?Asp?Glu?Leu?Gln?Leu?Asn?Ile?Glu?Asp?Met?Gln?Trp?Gly

500?????????????????505?????????????????510

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

515?????????????????520?????????????????525

Pro?Gly?Gln?Arg?Lys?Lys?Thr?Gln?Lys?Gly?Thr?Pro?Cys?Cys?Trp?Thr

530?????????????????535?????????????????540

Cys?Glu?Pro?Cys?Asp?Gly?Tyr?Gln?Tyr?Gln?Phe?Asp?Glu?Met?Thr?Cys

545?????????????????550?????????????????555?????????????????560

Gln?His?Cys?Pro?Tyr?Asp?Gln?Arg?Pro?Asn?Glu?Asn?Arg?Thr?Gly?Cys

565?????????????????570?????????????????575

Gln?Asp?Ile?Pro?Ile?Ile?Lys?Leu?Glu?Trp?His?Ser?Pro?Trp?Ala?Val

580?????????????????585?????????????????590

Ile?Pro?Val?Phe?Leu?Ala?Met?Leu?Gly?Ile?Ile?Ala?Thr?Ile?Phe?Val

595?????????????????600?????????????????605

Met?Ala?Thr?Phe?Ile?Arg?Tyr?Asn?Asp?Thr?Pro?Ile?Val?Arg?Ala?Ser

610?????????????????615?????????????????620

Gly?Arg?Glu?Leu?Ser?Tyr?Val?Leu?Leu?Thr?Gly?Ile?Phe?Leu?Cys?Tyr

625?????????????????630?????????????????635?????????????????640

Ile?Ile?Thr?Phe?Leu?Met?Ile?Ala?Lys?Pro?Asp?Val?Ala?Val?Cys?Ser

645?????????????????650?????????????????655

Phe?Arg?His?Val?Phe?Leu?Gly?Leu?Gly?Met?Cys?Ile?Ser?Tyr?Ala?Ala

660?????????????????665?????????????????670

Leu?Leu?Thr?Lys?Thr?Asn?Arg?Ile?Tyr?Arg?Ile?Phe?Glu?Gln?Gly?Lys

675?????????????????680?????????????????685

Lys?Ser?Val?Thr?Ala?Pro?Arg?Leu?Ile?Ser?Pro?Thr?Ser?Gln?Leu?Ala

690?????????????????695?????????????????700

Ile?Thr?Ser?Ser?Leu?Ile?Ser?Val?Gln?Leu?Leu?Gly?Val?Phe?Ile?Trp

705?????????????????710?????????????????715?????????????????720

Phe?Gly?Val?Asp?Pro?Pro?Asn?Ile?Ile?Ile?Asp?Tyr?Asp?Glu?His?Lys

725?????????????????730?????????????????735

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

740?????????????????745?????????????????750

Asp?Leu?Gln?Ile?Ile?Cys?Ser?Leu?Gly?Tyr?Ser?Ile?Leu?Leu?Met?Val

755?????????????????760?????????????????765

Thr?Cys?Thr?Val?Tyr?Ala?Ile?Lys?Thr?Arg?Gly?Val?Pro?Glu?Asn?Phe

770?????????????????775?????????????????780

Asn?Glu?Ala?Lys?Pro?Ile?Gly?Phe?Thr?Met?Tyr?Thr?Thr?Cys?Ile?Val

785?????????????????790?????????????????795?????????????????800

Trp?Leu?Ala?Phe?Ile?Pro?Ile?Phe?Phe?Gly?Thr?Ala?Gln?Ser?Ala?Glu

805?????????????????810?????????????????815

Lys?Leu?Tyr?Ile?Gln?Thr?Thr?Thr?Leu?Thr?Ile?Ser?Met?Asn?Leu?Ser

820?????????????????825?????????????????830

Ala?Ser?Val?Ala?Leu?Gly?Met?Leu?Tyr?Met?Pro?Lys?Val?Tyr?Ile?Ile

835?????????????????840?????????????????845

Ile?Phe?His?Pro?Glu?Leu?Asn?Val?Gln?Lys?Arg?Lys?Arg?Ser?Phe?Lys

850?????????????????855?????????????????860

Ala?Val?Val?Thr?Ala?Ala?Thr?Met?Ser?Ser?Arg?Leu?Ser?His?Lys?Pro

865?????????????????870?????????????????875?????????????????880

Ser?Asp?Arg?Pro?Asn?Gly?Glu?Ala?Lys?Thr?Glu?Leu?Cys?Glu?Asn?Val

885?????????????????890?????????????????895

Asp?Pro?Asn?Ser?Pro?Ala?Ala?Lys?Lys?Lys?Tyr?Val?Ser?Tyr?Asn?Asn

900?????????????????905?????????????????910

Leu?Val?Ile

915

<210>70

<211>2748

<212>DNA

＜213〉the unknown

<220>

＜223〉new sequence

<400>70

atggtccagc?tgaggaagct?gctccgcgtc?ctgactttga?tgaagttccc?ctgctgcgtg????60

ctggaggtgc?tcctgtgcgc?gctggcggcg?gcggcgcgcg?gccaggagat?gtacgccccg????120

cactcaatcc?ggatcgaggg?ggacgtcacc?ctcggggggc?tgttccccgt?gcacgccaag????180

ggtcccagcg?gagtgccctg?cggcgacatc?aagagggaaa?acgggatcca?caggctggaa????240

gcgatgctct?acgccctgga?ccagatcaac?agtgatccca?acctactgcc?caacgtgacg????300

ctgggcgcgc?ggatcctgga?cacttgttcc?agggacactt?acgcgctcga?acagtcgctt????360

actttcgtcc?aggcgctcat?ccagaaggac?acctccgacg?tgcgctgcac?caacggcgaa????420

ccgccggttt?tcgtcaagcc?ggagaaagta?gttggagtga?ttggggcttc?ggggagttcg????480

gtctccatca?tggtagccaa?catcctgagg?ctcttccaga?tcccccagat?tagttatgca????540

tcaacggcac?ccgagctaag?tgatgaccgg?cgctatgact?tcttctctcg?cgtggtgcca????600

cccgattcct?tccaagccca?ggccatggta?gacattgtaa?aggccctagg?ctggaattat????660

gtgtctaccc?tcgcatcgga?aggaagttat?ggagagaaag?gtgtggagtc?cttcacgcag????720

atttccaaag?aggcaggtgg?actctgcatt?gcccagtccg?tgagaatccc?ccaggaacgc????780

aaagacagga?ccattgactt?tgatagaatt?atcaaacagc?tcctggacac?ccccaactcc????840

agggccgtcg?tgatttttgc?caacgatgag?gatataaagc?agatccttgc?agcagccaaa????900

agagctgacc?aagttggcca?ttttctttgg?gtgggatcag?acagctgggg?atccaaaata????960

aacccactgc?accagcatga?agatatcgca?gaaggggcca?tcaccattca?gcccaagcga????1020

gccacggtgg?aagggtttga?tgcctacttt?acgtcccgta?cacttgaaaa?caacagaaga????1080

aatgtatggt?ttgccgaata?ctgggaggaa?aacttcaact?gcaagttgac?gattagtggg????1140

tcaaaaaaag?aagacacaga?tcgcaaatgc?acaggacagg?agagaattgg?aaaagattcc????1200

aactatgagc?aggagggtaa?agtccagttc?gtgattgacg?cagtctatgc?tatggctcac????1260

gcccttcacc?acatgaacaa?ggatctctgt?gctgactacc?ggggtgtctg?cccagagatg????1320

gagcaagctg?gaggcaagaa?gttgctgaag?tatatacgca?atgttaattt?caatggtagt????1380

gctggcactc?cagtgatgtt?taacaagaac?ggggatgcac?ctgggcgtta?tgacatcttt????1440

cagtaccaga?ccacaaacac?cagcaacccg?ggttaccgtc?tgatcgggca?gtggacagac????1500

gaacttcagc?tcaatataga?agacatgcag?tggggtaaag?gagtccgaga?gatacccgcc????1560

tcagtgtgca?cactaccatg?taagccagga?cagagaaaga?agacacagaa?aggaactcct????1620

tgctgttgga?cctgtgagcc?ttgcgatggt?taccagtacc?agtttgatga?gatgacatgc????1680

cagcattgcc?cctatgacca?gaggcccaat?gaaaatcgaa?ccggatgcca?ggatattccc????1740

atcatcaaac?tggagtggca?ctccccctgg?gctgtgattc?ctgtcttcct?ggcaatgttg????1800

gggatcattg?ccaccatctt?tgtcatggcc?actttcatcc?gctacaatga?cacgcccatt????1860

gtccgggcat?ctgggcggga?actcagctat?gttcttttga?cgggcatctt?tctttgctac????1920

atcatcactt?tcctgatgat?tgccaaacca?gatgtggcag?tgtgttcttt?ccggcgagtt????1980

ttcttgggct?tgggtatgtg?catcagttat?gcagccctct?tgacgaaaac?aaatcggatt????2040

tatcgcatat?ttgagcaggg?caagaaatca?gtaacagctc?ccagactcat?aagcccaaca????2100

tcacaactgg?caatcacttc?cagtttaata?tcagttcagc?ttctaggggt?gttcatttgg????2160

tttggtgttg?atccacccaa?catcatcata?gactacgatg?aacacaagac?aatgaaccct????2220

gagcaagcca?gaggggttct?caagtgtgac?attacagatc?tccaaatcat?ttgctccttg????2280

ggatatagca?ttcttctcat?ggtcacatgt?tgtgtgtatg?ccatcaagac?tcggggtgta????2340

cccgagaatt?ttaacgaagc?caagcccatt?ggattcacta?tgtacacgac?atgtatagta????2400

tggcttgcct?tcattccaat?tttttttggc?accgctcaat?cagcggaaaa?gctctacata????2460

caaactacca?cgcttacaat?ctccatgaac?ctaagtgcat?cagtggcgct?ggggatgcta????2520

tacatgccga?aagtgtacat?catcattttc?caccctgaac?tcaatgtcca?gaaacggaag????2580

cgaagcttca?aggcggtagt?cacagcagcc?accatgtcat?cgaggctgtc?acacaaaccc????2640

agtgacagac?ccaacggtga?ggcaaagacc?gagctctgtg?aaaacgtaga?cccaaacagc????2700

cctgctgcaa?aaaagaagta?tgtcagttat?aataacctgg?ttatctaa?????????????????2748

<210>71

<211>915

<212>PRT

＜213〉the unknown

<220>

＜223〉new sequence

<400>71

Met?Val?Gln?Leu?Arg?Lys?Leu?Leu?Arg?Val?Leu?Thr?Leu?Met?Lys?Phe

1???????????????5???????????????????10??????????????????15

Pro?Cys?Cys?Val?Leu?Glu?Val?Leu?Leu?Cys?Ala?Leu?Ala?Ala?Ala?Ala

20??????????????????25??????????????????30

Arg?Gly?Gln?Glu?Met?Tyr?Ala?Pro?His?Ser?Ile?Arg?Ile?Glu?Gly?Asp

35??????????????????40??????????????????45

Val?Thr?Leu?Gly?Gly?Leu?Phe?Pro?Val?His?Ala?Lys?Gly?Pro?Ser?Gly

50??????????????????55??????????????????60

Val?Pro?Cys?Gly?Asp?Ile?Lys?Arg?Glu?Asn?Gly?Ile?His?Arg?Leu?Glu

65??????????????????70??????????????????75??????????????????80

Ala?Met?Leu?Tyr?Ala?Leu?Asp?Gln?Ile?Asn?Ser?Asp?Pro?Asn?Leu?Leu

85??????????????????90??????????????????95

Pro?Asn?Val?Thr?Leu?Gly?Ala?Arg?Ile?Leu?Asp?Thr?Cys?Ser?Arg?Asp

100?????????????????105?????????????????110

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

115?????????????????120?????????????????125

Lys?Asp?Thr?Ser?Asp?Val?Arg?Cys?Thr?Asn?Gly?Glu?Pro?Pro?Val?Phe

130?????????????????135?????????????????140

Val?Lys?Pro?Glu?Lys?Val?Val?Gly?Val?Ile?Gly?Ala?Ser?Gly?Ser?Ser

145?????????????????150?????????????????155?????????????????160

Val?Ser?Ile?Met?Val?Ala?Asn?Ile?Leu?Arg?Leu?Phe?Gln?Ile?Pro?Gln

165?????????????????170?????????????????175

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

180?????????????????185?????????????????190

Asp?Phe?Phe?Ser?Arg?Val?Val?Pro?Pro?Asp?Ser?Phe?Gln?Ala?Gln?Ala

195?????????????????200?????????????????205

Met?Val?Asp?Ile?Val?Lys?Ala?Leu?Gly?Trp?Asn?Tyr?Val?Ser?Thr?Leu

210?????????????????215?????????????????220

Ala?Ser?Glu?Gly?Ser?Tyr?Gly?Glu?Lys?Gly?Val?Glu?Ser?Phe?Thr?Gln

225?????????????????230?????????????????235?????????????????240

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

245?????????????????250?????????????????255

Pro?Gln?Glu?Arg?Lys?Asp?Arg?Thr?Ile?Asp?Phe?Asp?Arg?Ile?Ile?Lys

260?????????????????265?????????????????270

Gln?Leu?Leu?Asp?Thr?Pro?Asn?Ser?Arg?Ala?Val?Val?Ile?Phe?Ala?Asn

275?????????????????280?????????????????285

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

290?????????????????295?????????????????300

Val?Gly?His?Phe?Leu?Trp?Val?Gly?Ser?Asp?Ser?Trp?Gly?Ser?Lys?Ile

305?????????????????310?????????????????315?????????????????320

Asn?Pro?Leu?His?Gln?His?Glu?Asp?Ile?Ala?Glu?Gly?Ala?Ile?Thr?Ile

325?????????????????330?????????????????335

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

340?????????????????345?????????????????350

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

355?????????????????360?????????????????365

Glu?Glu?Asn?Phe?Asn?Cys?Lys?Leu?Thr?Ile?Ser?Gly?Ser?Lys?Lys?Glu

370?????????????????375?????????????????380

Asp?Thr?Asp?Arg?Lys?Cys?Thr?Gly?Gln?Glu?Arg?Ile?Gly?Lys?Asp?Ser

385?????????????????390?????????????????395?????????????????400

Asn?Tyr?Glu?Gln?Glu?Gly?Lys?Val?Gln?Phe?Val?Ile?Asp?Ala?Val?Tyr

405?????????????????410?????????????????415

Ala?Met?Ala?His?Ala?Leu?His?His?Met?Asn?Lys?Asp?Leu?Cys?Ala?Asp

420?????????????????425?????????????????430

Tyr?Arg?Gly?Val?Cys?Pro?Glu?Met?Glu?Gln?Ala?Gly?Gly?Lys?Lys?Leu

435?????????????????440?????????????????445

Leu?Lys?Tyr?Ile?Arg?Asn?Val?Asn?Phe?Asn?Gly?Ser?Ala?Gly?Thr?Pro

450?????????????????455?????????????????460

Val?Met?Phe?Asn?Lys?Asn?Gly?Asp?Ala?Pro?Gly?Arg?Tyr?Asp?Ile?Phe

465?????????????????470?????????????????475?????????????????480

Gln?Tyr?Gln?Thr?Thr?Asn?Thr?Ser?Asn?Pro?Gly?Tyr?Arg?Leu?Ile?Gly

485?????????????????490?????????????????495

Gln?Trp?Thr?Asp?Glu?Leu?Gln?Leu?Asn?Ile?Glu?Asp?Met?Gln?Trp?Gly

500?????????????????505?????????????????510

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

515?????????????????520?????????????????525

Pro?Gly?Gln?Arg?Lys?Lys?Thr?Gln?Lys?Gly?Thr?Pro?Cys?Cys?Trp?Thr

530?????????????????535?????????????????540

Cys?Glu?Pro?Cys?Asp?Gly?Tyr?Gln?Tyr?Gln?Phe?Asp?Glu?Met?Thr?Cys

545?????????????????550?????????????????555?????????????????560

Gln?His?Cys?Pro?Tyr?Asp?Gln?Arg?Pro?Asn?Glu?Asn?Arg?Thr?Gly?Cys

565?????????????????570?????????????????575

Gln?Asp?Ile?Pro?Ile?Ile?Lys?Leu?Glu?Trp?His?Ser?Pro?Trp?Ala?Val

580?????????????????585?????????????????590

Ile?Pro?Val?Phe?Leu?Ala?Met?Leu?Gly?Ile?Ile?Ala?Thr?Ile?Phe?Val

595?????????????????600?????????????????605

Met?Ala?Thr?Phe?Ile?Arg?Tyr?Asn?Asp?Thr?Pro?Ile?Val?Arg?Ala?Ser

610?????????????????615?????????????????620

Gly?Arg?Glu?Leu?Ser?Tyr?Val?Leu?Leu?Thr?Gly?Ile?Phe?Leu?Cys?Tyr

625?????????????????630?????????????????635?????????????????640

Ile?Ile?Thr?Phe?Leu?Met?Ile?Ala?Lys?Pro?Asp?Val?Ala?Val?Cys?Ser

645?????????????????650?????????????????655

Phe?Arg?Arg?Val?Phe?Leu?Gly?Leu?Gly?Met?Cys?Ile?Ser?Tyr?Ala?Ala

660?????????????????665?????????????????670

Leu?Leu?Thr?Lys?Thr?Asn?Arg?Ile?Tyr?Arg?Ile?Phe?Glu?Gln?Gly?Lys

675?????????????????680?????????????????685

Lys?Ser?Val?Thr?Ala?Pro?Arg?Leu?Ile?Ser?Pro?Thr?Ser?Gln?Leu?Ala

690?????????????????695?????????????????700

Ile?Thr?Ser?Ser?Leu?Ile?Ser?Val?Gln?Leu?Leu?Gly?Val?Phe?Ile?Trp

705?????????????????710?????????????????715?????????????????720

Phe?Gly?Val?Asp?Pro?Pro?Asn?Ile?Ile?Ile?Asp?Tyr?Asp?Glu?His?Lys

725?????????????????730?????????????????735

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

740?????????????????745?????????????????750

Asp?Leu?Gln?Ile?Ile?Cys?Ser?Leu?Gly?Tyr?Ser?Ile?Leu?Leu?Met?Val

755?????????????????760?????????????????765

Thr?Cys?Cys?Val?Tyr?Ala?Ile?Lys?Thr?Arg?Gly?Val?Pro?Glu?Asn?Phe

770?????????????????775?????????????????780

Asn?Glu?Ala?Lys?Pro?Ile?Gly?Phe?Thr?Met?Tyr?Thr?Thr?Cys?Ile?Val

785?????????????????790?????????????????795?????????????????800

Trp?Leu?Ala?Phe?Ile?Pro?Ile?Phe?Phe?Gly?Thr?Ala?Gln?Ser?Ala?Glu

805?????????????????810?????????????????815

Lys?Leu?Tyr?Ile?Gln?Thr?Thr?Thr?Leu?Thr?Ile?Ser?Met?Asn?Leu?Ser

820?????????????????825?????????????????830

Ala?Ser?Val?Ala?Leu?Gly?Met?Leu?Tyr?Met?Pro?Lys?Val?Tyr?Ile?Ile

835?????????????????840?????????????????845

Ile?Phe?His?Pro?Glu?Leu?Asn?Val?Gln?Lys?Arg?Lys?Arg?Ser?Phe?Lys

850?????????????????855?????????????????860

Ala?Val?Val?Thr?Ala?Ala?Thr?Met?Ser?Ser?Arg?Leu?Ser?His?Lys?Pro

865?????????????????870?????????????????875?????????????????880

Ser?Asp?Arg?Pro?Asn?Gly?Glu?Ala?Lys?Thr?Glu?Leu?Cys?Glu?Asn?Val

885?????????????????890?????????????????895

Asp?Pro?Asn?Ser?Pro?Ala?Ala?Lys?Lys?Lys?Tyr?Val?Ser?Tyr?Asn?Asn

900?????????????????905?????????????????910

Leu?Val?Ile

915

<210>72

<211>2748

<212>DNA

＜213〉the unknown

<220>

＜223〉new sequence

<400>72

atggtccagc?tgaggaagct?gctccgcgtc?ctgactttga?tgaagttccc?ctgctgcgtg????60

ctggaggtgc?tcctgtgcgc?gctggcggcg?gcggcgcgcg?gccaggagat?gtacgccccg????120

cactcaatcc?ggatcgaggg?ggacgtcacc?ctcggggggc?tgttccccgt?gcacgccaag????180

ggtcccagcg?gagtgccctg?cggcgacatc?aagagggaaa?acgggatcca?caggctggaa????240

gcgatgctct?acgccctgga?ccagatcaac?agtgatccca?acctactgcc?caacgtgacg????300

ctgggcgcgc?ggatcctgga?cacttgttcc?agggacactt?acgcgctcga?acagtcgctt????360

actttcgtcc?aggcgctcat?ccagaaggac?acctccgacg?tgcgctgcac?caacggcgaa????420

ccgccggttt?tcgtcaagcc?ggagaaagta?gttggagtga?ttggggcttc?ggggagttcg????480

gtctccatca?tggtagccaa?catcctgagg?ctcttccaga?tcccccagat?tagttatgca????540

tcaacggcac?ccgagctaag?tgatgaccgg?cgctatgact?tcttctctcg?cgtggtgcca????600

cccgattcct?tccaagccca?ggccatggta?gacattgtaa?aggccctagg?ctggaattat????660

gtgtctaccc?tcgcatcgga?aggaagttat?ggagagaaag?gtgtggagtc?cttcacgcag????720

atttccaaag?aggcaggtgg?actctgcatt?gcccagtccg?tgagaatccc?ccaggaacgc????780

aaagacagga?ccattgactt?tgatagaatt?atcaaacagc?tcctggacac?ccccaactcc????840

agggccgtcg?tgatttttgc?caacgatgag?gatataaagc?agatccttgc?agcagccaaa????900

agagctgacc?aagttggcca?ttttctttgg?gtgggatcag?acagctgggg?atccaaaata????960

aacccactgc?accagcatga?agatatcgca?gaaggggcca?tcaccattca?gcccaagcga????1020

gccacggtgg?aagggtttga?tgcctacttt?acgtcccgta?cacttgaaaa?caacagaaga????1080

aatgtatggt?ttgccgaata?ctgggaggaa?aacttcaact?gcaagttgac?gattagtggg????1140

tcaaaaaaag?aagacacaga?tcgcaaatgc?acaggacagg?agagaattgg?aaaagattcc????1200

aactatgagc?aggagggtaa?agtccagttc?gtgattgacg?cagtctatgc?tatggctcac????1260

gcccttcacc?acatgaacaa?ggatctctgt?gctgactacc?ggggtgtctg?cccagagatg????1320

gagcaagctg?gaggcaagaa?gttgctgaag?tatatacgca?atgttaattt?caatggtagt????1380

gctggcactc?cagtgatgtt?taacaagaac?ggggatgcac?ctgggcgtta?tgacatcttt????1440

cagtaccaga?ccacaaacac?cagcaacccg?ggttaccgtc?tgatcgggca?gtggacagac????1500

gaacttcagc?tcaatataga?agacatgcag?tggggtaaag?gagtccgaga?gatacccgcc????1560

tcagtgtgca?cactaccatg?taagccagga?cagagaaaga?agacacagaa?aggaactcct????1620

tgctgttgga?cctgtgagcc?ttgcgatggt?taccagtacc?agtttgatga?gatgacatgc????1680

cagcattgcc?cctatgacca?gaggcccaat?gaaaatcgaa?ccggatgcca?ggatattccc????1740

atcatcaaac?tggagtggca?ctccccctgg?gctgtgattc?ctgtcttcct?ggcaatgttg????1800

gggatcattg?ccaccatctt?tgtcatggcc?actttcatcc?gctacaatga?cacgcccatt????1860

gtccgggcat?ctgggcggga?actcagctat?gttcttttga?cgggcatctt?tctttgctac????1920

atcatcactt?tcctgatgat?tgccaaacca?gatgtggcag?tgtgttcttt?ccggcgagtt????1980

ttcttgggct?tgggtatgtg?catcagttat?gcagccctct?tgacgaaaac?aaatcggatt????2040

tatcgcatat?ttgagcaggg?caagaaatca?gtaacagctc?ccagactcat?aagcccaaca????2100

tcacaactgg?caatcacttc?cagtttaata?tcagttcagc?ttctaggggt?gttcatttgg????2160

tttggtgttg?atccacccaa?catcatcata?gactacgatg?aacacaagac?aatgaaccct????2220

gagcaagcca?gaggggttct?caagtgtgac?attacagatc?tccaaatcat?ttgctccttg????2280

ggatatagca?ttcttctcat?ggtcacatgt?actgtgtatg?ccatcaagac?tcggggtgta????2340

cccgagaatt?ttaacgaagc?caagcccaag?ggattcacta?tgtacacgac?atgtatagta????2400

tggcttgcct?tcattccaat?tttttttggc?accgctcaat?cagcggaaaa?gctctacata????2460

caaactacca?cgcttacaat?ctccatgaac?ctaagtgcat?cagtggcgct?ggggatgcta????2520

tacatgccga?aagtgtacat?catcattttc?caccctgaac?tcaatgtcca?gaaacggaag????2580

cgaagcttca?aggcggtagt?cacagcagcc?accatgtcat?cgaggctgtc?acacaaaccc????2640

agtgacagac?ccaacggtga?ggcaaagacc?gagctctgtg?aaaacgtaga?cccaaacagc????2700

cctgctgcaa?aaaagaagta?tgtcagttat?aataacctgg?ttatctaa?????????????????2748

<210>73

<211>915

<212>PRT

＜213〉the unknown

<220>

＜223〉new sequence

<400>73

Met?Val?Gln?Leu?Arg?Lys?Leu?Leu?Arg?Val?Leu?Thr?Leu?Met?Lys?Phe

1???????????????5???????????????????10??????????????????15

Pro?Cys?Cys?Val?Leu?Glu?Val?Leu?Leu?Cys?Ala?Leu?Ala?Ala?Ala?Ala

20??????????????????25??????????????????30

Arg?Gly?Gln?Glu?Met?Tyr?Ala?Pro?His?Ser?Ile?Arg?Ile?Glu?Gly?Asp

35??????????????????40??????????????????45

Val?Thr?Leu?Gly?Gly?Leu?Phe?Pro?Val?His?Ala?Lys?Gly?Pro?Ser?Gly

50??????????????????55??????????????????60

Val?Pro?Cys?Gly?Asp?Ile?Lys?Arg?Glu?Asn?Gly?Ile?His?Arg?Leu?Glu

65??????????????????70??????????????????75??????????????????80

Ala?Met?Leu?Tyr?Ala?Leu?Asp?Gln?Ile?Asn?Ser?Asp?Pro?Asn?Leu?Leu

85??????????????????90??????????????????95

Pro?Asn?Val?Thr?Leu?Gly?Ala?Arg?Ile?Leu?Asp?Thr?Cys?Ser?Arg?Asp

100?????????????????105?????????????????110

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

115?????????????????120?????????????????125

Lys?Asp?Thr?Ser?Asp?Val?Arg?Cys?Thr?Asn?Gly?Glu?Pro?Pro?Val?Phe

130?????????????????135?????????????????140

Val?Lys?Pro?Glu?Lys?Val?Val?Gly?Val?Ile?Gly?Ala?Ser?Gly?Ser?Ser

145?????????????????150?????????????????155?????????????????160

Val?Ser?Ile?Met?Val?Ala?Asn?Ile?Leu?Arg?Leu?Phe?Gln?Ile?Pro?Gln

165?????????????????170?????????????????175

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

180?????????????????185?????????????????190

Asp?Phe?Phe?Ser?Arg?Val?Val?Pro?Pro?Asp?Ser?Phe?Gln?Ala?Gln?Ala

195?????????????????200?????????????????205

Met?Val?Asp?Ile?Val?Lys?Ala?Leu?Gly?Trp?Asn?Tyr?Val?Ser?Thr?Leu

210?????????????????215?????????????????220

Ala?Ser?Glu?Gly?Ser?Tyr?Gly?Glu?Lys?Gly?Val?Glu?Ser?Phe?Thr?Gln

225?????????????????230?????????????????235?????????????????240

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

245?????????????????250?????????????????255

Pro?Gln?Glu?Arg?Lys?Asp?Arg?Thr?Ile?Asp?Phe?Asp?Arg?Ile?Ile?Lys

260?????????????????265?????????????????270

Gln?Leu?Leu?Asp?Thr?Pro?Asn?Ser?Arg?Ala?Val?Val?Ile?Phe?Ala?Asn

275?????????????????280?????????????????285

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

290?????????????????295?????????????????300

Val?Gly?His?Phe?Leu?Trp?Val?Gly?Ser?Asp?Ser?Trp?Gly?Ser?Lys?Ile

305?????????????????310?????????????????315?????????????????320

Asn?Pro?Leu?His?Gln?His?Glu?Asp?Ile?Ala?Glu?Gly?Ala?Ile?Thr?Ile

325?????????????????330?????????????????335

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

340?????????????????345?????????????????350

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

355?????????????????360?????????????????365

Glu?Glu?Asn?Phe?Asn?Cys?Lys?Leu?Thr?Ile?Ser?Gly?Ser?Lys?Lys?Glu

370?????????????????375?????????????????380

Asp?Thr?Asp?Arg?Lys?Cys?Thr?Gly?Gln?Glu?Arg?Ile?Gly?Lys?Asp?Ser

385?????????????????390?????????????????395?????????????????400

Asn?Tyr?Glu?Gln?Glu?Gly?Lys?Val?Gln?Phe?Val?Ile?Asp?Ala?Val?Tyr

405?????????????????410?????????????????415

Ala?Met?Ala?His?Ala?Leu?His?His?Met?Asn?Lys?Asp?Leu?Cys?Ala?Asp

420?????????????????425?????????????????430

Tyr?Arg?Gly?Val?Cys?Pro?Glu?Met?Glu?Gln?Ala?Gly?Gly?Lys?Lys?Leu

435?????????????????440?????????????????445

Leu?Lys?Tyr?Ile?Arg?Asn?Val?Asn?Phe?Asn?Gly?Ser?Ala?Gly?Thr?Pro

450?????????????????455?????????????????460

Val?Met?Phe?Asn?Lys?Asn?Gly?Asp?Ala?Pro?Gly?Arg?Tyr?Asp?Ile?Phe

465?????????????????470?????????????????475?????????????????480

Gln?Tyr?Gln?Thr?Thr?Asn?Thr?Ser?Asn?Pro?Gly?Tyr?Arg?Leu?Ile?Gly

485?????????????????490?????????????????495

Gln?Trp?Thr?Asp?Glu?Leu?Gln?Leu?Asn?Ile?Glu?Asp?Met?Gln?Trp?Gly

500?????????????????505?????????????????510

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

515?????????????????520?????????????????525

Pro?Gly?Gln?Arg?Lys?Lys?Thr?Gln?Lys?Gly?Thr?Pro?Cys?Cys?Trp?Thr

530?????????????????535?????????????????540

Cys?Glu?Pro?Cys?Asp?Gly?Tyr?Gln?Tyr?Gln?Phe?Asp?Glu?Met?Thr?Cys

545?????????????????550?????????????????555?????????????????560

Gln?His?Cys?Pro?Tyr?Asp?Gln?Arg?Pro?Asn?Glu?Asn?Arg?Thr?Gly?Cys

565?????????????????570?????????????????575

Gln?Asp?Ile?Pro?Ile?Ile?Lys?Leu?Glu?Trp?His?Ser?Pro?Trp?Ala?Val

580?????????????????585?????????????????590

Ile?Pro?Val?Phe?Leu?Ala?Met?Leu?Gly?Ile?Ile?Ala?Thr?Ile?Phe?Val

595?????????????????600?????????????????605

Met?Ala?Thr?Phe?Ile?Arg?Tyr?Asn?Asp?Thr?Pro?Ile?Val?Arg?Ala?Ser

610?????????????????615?????????????????620

Gly?Arg?Glu?Leu?Ser?Tyr?Val?Leu?Leu?Thr?Gly?Ile?Phe?Leu?Cys?Tyr

625?????????????????630?????????????????635?????????????????640

Ile?Ile?Thr?Phe?Leu?Met?Ile?Ala?Lys?Pro?Asp?Val?Ala?Val?Cys?Ser

645?????????????????650?????????????????655

Phe?Arg?Arg?Val?Phe?Leu?Gly?Leu?Gly?Met?Cys?Ile?Ser?Tyr?Ala?Ala

660?????????????????665?????????????????670

Leu?Leu?Thr?Lys?Thr?Asn?Arg?Ile?Tyr?Arg?Ile?Phe?Glu?Gln?Gly?Lys

675?????????????????680?????????????????685

Lys?Ser?Val?Thr?Ala?Pro?Arg?Leu?Ile?Ser?Pro?Thr?Ser?Gln?Leu?Ala

690?????????????????695?????????????????700

Ile?Thr?Ser?Ser?Leu?Ile?Ser?Val?Gln?Leu?Leu?Gly?Val?Phe?Ile?Trp

705?????????????????710?????????????????715?????????????????720

Phe?Gly?Val?Asp?Pro?Pro?Asn?Ile?Ile?Ile?Asp?Tyr?Asp?Glu?His?Lys

725?????????????????730?????????????????735

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

740?????????????????745?????????????????750

Asp?Leu?Gln?Ile?Ile?Cys?Ser?Leu?Gly?Tyr?Ser?Ile?Leu?Leu?Met?Val

755?????????????????760?????????????????765

Thr?Cys?Thr?Val?Tyr?Ala?Ile?Lys?Thr?Arg?Gly?Val?Pro?Glu?Asn?Phe

770?????????????????775?????????????????780

Asn?Glu?Ala?Lys?Pro?Lys?Gly?Phe?Thr?Met?Tyr?Thr?Thr?Cys?Ile?Val

785?????????????????790?????????????????795?????????????????800

Trp?Leu?Ala?Phe?Ile?Pro?Ile?Phe?Phe?Gly?Thr?Ala?Gln?Ser?Ala?Glu

805?????????????????810?????????????????815

Lys?Leu?Tyr?Ile?Gln?Thr?Thr?Thr?Leu?Thr?Ile?Ser?Met?Asn?Leu?Ser

820?????????????????825?????????????????830

Ala?Ser?Val?Ala?Leu?Gly?Met?Leu?Tyr?Met?Pro?Lys?Val?Tyr?Ile?Ile

835?????????????????840?????????????????845

Ile?Phe?His?Pro?Glu?Leu?Asn?Val?Gln?Lys?Arg?Lys?Arg?Ser?Phe?Lys

850?????????????????855?????????????????860

Ala?Val?Val?Thr?Ala?Ala?Thr?Met?Ser?Ser?Arg?Leu?Ser?His?Lys?Pro

865?????????????????870?????????????????875?????????????????880

Ser?Asp?Arg?Pro?Asn?Gly?Glu?Ala?Lys?Thr?Glu?Leu?Cys?Glu?Asn?Val

885?????????????????890?????????????????895

Asp?Pro?Asn?Ser?Pro?Ala?Ala?Lys?Lys?Lys?Tyr?Val?Ser?Tyr?Asn?Asn

900?????????????????905?????????????????910

Leu?Val?Ile

915

<210>74

<211>1842

<212>DNA

＜213〉the unknown

<220>

＜223〉new sequence

<400>74

atgcgagccc?cgggcgcgct?tctcgcccgc?atgtcgcggc?tactgcttct?gctactgctc????60

aaggtgtctg?cctcttctgc?cctcggggtc?gcccctgcgt?ccagaaacga?aacttgtctg????120

ggggagagct?gtgcacctac?agtgatccag?cgccgcggca?gggacgcctg?gggaccggga????180

aattctgcaa?gagacgttct?gcgagcccga?gcacccaggg?aggagcaggg?ggcagcgttt????240

cttgcgggac?cctcctggga?cctgccggcg?gccccgggcc?gtgacccggc?tgcaggcaga????300

ggggcggagg?cgtcggcagc?cggacccccg?ggacctccaa?ccaggccacc?tggcccctgg????360

aggtggaaag?gtgctcgggg?tcaggagcct?tctgaaactt?tggggagagg?gaaccccacg????420

gccctccagc?tcttccttca?gatctcagag?gaggaagaga?agggtcccag?aggcgctggc????480

atttccgggc?gtagccagga?gcagagtgtg?aagacagtcc?ccggagccag?cgatcttttt????540

tactggccaa?ggagagccgg?gaaactccag?ggttcccacc?acaagcccct?gtccaagacg????600

gccaatggac?tggcggggca?cgaagggtgg?acaattgcac?tcccgggccg?ggcgctggcc????660

cagaatggat?ccttgggtga?aggaatccat?gagcctgggg?gtccccgccg?gggaaacagc????720

acgaaccggc?gtgtgagact?gaagaacccc?ttctacccgc?tgacccagga?gtcctatgga????780

gcctacgcgg?tcatgtgtct?gtccgtggtg?atcttcggga?ccggcatcat?tggcaacctg????840

gcggtgatgt?gcatcgtgtg?ccacaactac?tacatgcgga?gcatctccaa?ctccctcttg????900

gccaacctgg?ccttctggga?ctttctcatc?atcttcttct?gccttccgct?ggtcatcttc????960

cacgagctga?ccaagaagtg?gctgctggag?gacttctcct?gcaagatcgt?gccctatata????1020

gaggtcgctt?ctctgggagt?caccaccttc?acccgatgtg?ctctgtgcat?agaccgcttc????1080

cgtgctgcca?ccaacgtaca?gatgtactac?gaaatgatcg?aaaactgttc?ctcaacaact????1140

gccaaacttg?ctgttatatg?ggtgggagct?ctattgttag?cacttccaga?agttgttctc????1200

cgccagctga?gcaaggagga?tttggggttt?agtggccgag?ctccggcaga?aaggtgcatt????1260

attaagatct?ctcctgattt?accagacacc?atctatgttc?tagccctcac?ctacgacagt????1320

gcgagactgt?ggtggtattt?tggctgttac?ttttgtttgc?ccacgctttt?caccatcacc????1380

tgctctctag?tgactgcgag?gaaaatccgc?aaagcagaga?aagcctgtac?ccgagggaat????1440

aaacggcaga?ttcaactaga?gagtcagatg?aactgtacag?tagtggcact?gaccatttta????1500

tatggatttt?gcattattcc?tgaaaatatc?tgcaacattg?ttactgccta?catggctaca????1560

ggggtttcac?agcagacaat?ggacctcctt?aatatcatca?gccagttcct?tttgttcttt????1620

aagtcctgtg?tcaccccagt?cctccttttc?tgtctctgca?aacccttcag?tcgggccttc????1680

atggagtgct?gctgctgttg?ctgtgaggaa?tgcattcaga?agtcttcaac?ggtgaccagt????1740

gatgacaatg?acaacgagta?caccacggaa?ctcgaactct?cgcctttcag?taccatacgc????1800

cgtgaaatgt?ccacttttgc?ttctgtcgga?actcattgct?ga???????????????????????1842

<210>75

<211>613

<212>PRT

＜213〉the unknown

<220>

＜223〉new sequence

<400>75

Met?Arg?Ala?Pro?Gly?Ala?Leu?Leu?Ala?Arg?Met?Ser?Arg?Leu?Leu?Leu

1???????????????5???????????????????10??????????????????15

Leu?Leu?Leu?Leu?Lys?Val?Ser?Ala?Ser?Ser?Ala?Leu?Gly?Val?Ala?Pro

20??????????????????25??????????????????30

Ala?Ser?Arg?Asn?Glu?Thr?Cys?Leu?Gly?Glu?Ser?Cys?Ala?Pro?Thr?Val

35??????????????????40??????????????????45

Ile?Gln?Arg?Arg?Gly?Arg?Asp?Ala?Trp?Gly?Pro?Gly?Asn?Ser?Ala?Arg

50??????????????????55??????????????????60

Asp?Val?Leu?Arg?Ala?Arg?Ala?Pro?Arg?Glu?Glu?Gln?Gly?Ala?Ala?Phe

65??????????????????70??????????????????75??????????????????80

Leu?Ala?Gly?Pro?Ser?Trp?Asp?Leu?Pro?Ala?Ala?Pro?Gly?Arg?Asp?Pro

85??????????????????90??????????????????95

Ala?Ala?Gly?Arg?Gly?Ala?Glu?Ala?Ser?Ala?Ala?Gly?Pro?Pro?Gly?Pro

100?????????????????105?????????????????110

Pro?Thr?Arg?Pro?Pro?Gly?Pro?Trp?Arg?Trp?Lys?Gly?Ala?Arg?Gly?Gln

115?????????????????120?????????????????125

Glu?Pro?Ser?Glu?Thr?Leu?Gly?Arg?Gly?Asn?Pro?Thr?Ala?Leu?Gln?Leu

130?????????????????135?????????????????140

Phe?Leu?Gln?Ile?Ser?Glu?Glu?Glu?Glu?Lys?Gly?Pro?Arg?Gly?Ala?Gly

145?????????????????150?????????????????155?????????????????160

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

165?????????????????170?????????????????175

Ser?Asp?Leu?Phe?Tyr?Trp?Pro?Arg?Arg?Ala?Gly?Lys?Leu?Gln?Gly?Ser

180?????????????????185?????????????????190

His?His?Lys?Pro?Leu?Ser?Lys?Thr?Ala?Asn?Gly?Leu?Ala?Gly?His?Glu

195?????????????????200?????????????????205

Gly?Trp?Thr?Ile?Ala?Leu?Pro?Gly?Arg?Ala?Leu?Ala?Gln?Asn?Gly?Ser

210?????????????????215?????????????????220

Leu?Gly?Glu?Gly?Ile?His?Glu?Pro?Gly?Gly?Pro?Arg?Arg?Gly?Asn?Ser

225?????????????????230?????????????????235?????????????????240

Thr?Asn?Arg?Arg?Val?Arg?Leu?Lys?Asn?Pro?Phe?Tyr?Pro?Leu?Thr?Gln

245?????????????????250?????????????????255

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

260?????????????????265?????????????????270

Gly?Thr?Gly?Ile?Ile?Gly?Asn?Leu?Ala?Val?Met?Cys?Ile?Val?Cys?His

275?????????????????280?????????????????285

Asn?Tyr?Tyr?Met?Arg?Ser?Ile?Ser?Asn?Ser?Leu?Leu?Ala?Asn?Leu?Ala

290?????????????????295?????????????????300

Phe?Trp?Asp?Phe?Leu?Ile?Ile?Phe?Phe?Cys?Leu?Pro?Leu?Val?Ile?Phe

305?????????????????310?????????????????315?????????????????320

His?Glu?Leu?Thr?Lys?Lys?Trp?Leu?Leu?Glu?Asp?Phe?Ser?Cys?Lys?Ile

325?????????????????330?????????????????335

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

340?????????????????345?????????????????350

Cys?Ala?Leu?Cys?Ile?Asp?Arg?Phe?Arg?Ala?Ala?Thr?Asn?Val?Gln?Met

355?????????????????360?????????????????365

Tyr?Tyr?Glu?Met?Ile?Glu?Asn?Cys?Ser?Ser?Thr?Thr?Ala?Lys?Leu?Ala

370?????????????????375?????????????????380

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

385?????????????????390?????????????????395?????????????????400

Arg?Gln?Leu?Ser?Lys?Glu?Asp?Leu?Gly?Phe?Ser?Gly?Arg?Ala?Pro?Ala

405?????????????????410?????????????????415

Glu?Arg?Cys?Ile?Ile?Lys?Ile?Ser?Pro?Asp?Leu?Pro?Asp?Thr?Ile?Tyr

420?????????????????425?????????????????430

Val?Leu?Ala?Leu?Thr?Tyr?Asp?Ser?Ala?Arg?Leu?Trp?Trp?Tyr?Phe?Gly

435?????????????????440?????????????????445

Cys?Tyr?Phe?Cys?Leu?Pro?Thr?Leu?Phe?Thr?Ile?Thr?Cys?Ser?Leu?Val

450?????????????????455?????????????????460

Thr?Ala?Arg?Lys?Ile?Arg?Lys?Ala?Glu?Lys?Ala?Cys?Thr?Arg?Gly?Asn

465?????????????????470?????????????????475?????????????????480

Lys?Arg?Gln?Ile?Gln?Leu?Glu?Ser?Gln?Mer?Asn?Cys?Thr?Val?Val?Ala

485?????????????????490?????????????????495

Leu?Thr?Ile?Leu?Tyr?Gly?Phe?Cys?Ile?Ile?Pro?Glu?Asn?Ile?Cys?Asn

500?????????????????505?????????????????510

Ile?Val?Thr?Ala?Tyr?Met?Ala?Thr?Gly?Val?Ser?Gln?Gln?Thr?Met?Asp

515?????????????????520?????????????????525

Leu?Leu?Asn?Ile?Ile?Ser?Gln?Phe?Leu?Leu?Phe?Phe?Lys?Ser?Cys?Val

530?????????????????535?????????????????540

Thr?Pro?Val?Leu?Leu?Phe?Cys?Leu?Cys?Lys?Pro?Phe?Ser?Arg?Ala?Phe

545?????????????????550?????????????????555?????????????????560

Met?Glu?Cys?Cys?Cys?Cys?Cys?Cys?Glu?Glu?Cys?Ile?Gln?Lys?Ser?Ser

565?????????????????570?????????????????575

Thr?Val?Thr?Ser?Asp?Asp?Asn?Asp?Asn?Glu?Tyr?Thr?Thr?Glu?Leu?Glu

580?????????????????585?????????????????590

Leu?Ser?Pro?Phe?Ser?Thr?Ile?Arg?Arg?Glu?Met?Ser?Thr?Phe?Ala?Ser

595?????????????????600?????????????????605

Val?Gly?Thr?His?Cys

610

<210>76

<211>1842

<212>DNA

＜213〉people

<400>76

atgcgagccc?cgggcgcgct?tctcgcccgc?atgtcgcggc?tactgcttct?gctactgctc????60

aaggtgtctg?cctcttctgc?cctcggggtc?gcccctgcgt?ccagaaacga?aacttgtctg????120

ggggagagct?gtgcacctac?agtgatccag?cgccgcggca?gggacgcctg?gggaccggga????180

aattctgcaa?gagacgttct?gcgagcccga?gcacccaggg?aggagcaggg?ggcagcgttt????240

cttgcgggac?cctcctggga?cctgccggcg?gccccgggcc?gtgacccggc?tgcaggcaga????300

ggggcggagg?cgtcggcagc?cggacccccg?ggacctccaa?ccaggccacc?tggcccctgg????360

aggtggaaag?gtgctcgggg?tcaggagcct?tctgaaactt?tggggagagg?gaaccccacg????420

gccctccagc?tcttccttca?gatctcagag?gaggaagaga?agggtcccag?aggcgctggc????480

atttccgggc?gtagccagga?gcagagtgtg?aagacagtcc?ccggagccag?cgatcttttt????540

tactggccaa?ggagagccgg?gaaactccag?ggttcccacc?acaagcccct?gtccaagacg????600

gccaatggac?tggcggggca?cgaagggtgg?acaattgcac?tcccgggccg?ggcgctggcc????660

cagaatggat?ccttgggtga?aggaatccat?gagcctgggg?gtccccgccg?gggaaacagc????720

acgaaccggc?gtgtgagact?gaagaacccc?ttctacccgc?tgacccagga?gtcctatgga????780

gcctacgcgg?tcatgtgtct?gtccgtggtg?atcttcggga?ccggcatcat?tggcaacctg????840

gcggtgatgt?gcatcgtgtg?ccacaactac?tacatgcgga?gcatctccaa?ctccctcttg????900

gccaacctgg?ccttctggga?ctttctcatc?atcttcttct?gccttccgct?ggtcatcttc????960

cacgagctga?ccaagaagtg?gctgctggag?gacttctcct?gcaagatcgt?gccctatata????1020

gaggtcgctt?ctctgggagt?caccaccttc?accttatgtg?ctctgtgcat?agaccgcttc????1080

cgtgctgcca?ccaacgtaca?gatgtactac?gaaatgatcg?aaaactgttc?ctcaacaact????1140

gccaaacttg?ctgttatatg?ggtgggagct?ctattgttag?cacttccaga?agttgttctc????1200

cgccagctga?gcaaggagga?tttggggttt?agtggccgag?ctccggcaga?aaggtgcatt????1260

attaagatct?ctcctgattt?accagacacc?atctatgttc?tagccctcac?ctacgacagt????1320

gcgagactgt?ggtggtattt?tggctgttac?ttttgtttgc?ccacgctttt?caccatcacc????1380

tgctctctag?tgactgcgag?gaaaatccgc?aaagcagaga?aagcctgtac?ccgagggaat????1440

aaacggcaga?ttcaactaga?gagtcagatg?aactgtacag?tagtggcact?gaccatttta????1500

tatggatttt?gcattattcc?tgaaaatatc?tgcaacattg?ttactgccta?catggctaca????1560

ggggtttcac?agcagacaat?ggacctcctt?aatatcatca?gccagttcct?tttgttcttt????1620

aagtcctatg?tcaccccagt?cctccttttc?tgtctctgca?aacccttcag?tcgggccttc????1680

atggagtgct?gctgctgttg?ctgtgaggaa?tgcattcaga?agtcttcaac?ggtgaccagt????1740

gatgacaatg?acaacgagta?caccacggaa?ctcgaactct?cgcctttcag?taccatacgc????1800

cgtgaaatgt?ccacttttgc?ttctgtcgga?actcattgct?ga???????????????????????1842

<210>77

<211>613

<212>PRT

＜213〉the unknown

<220>

＜223〉new sequence

<400>77

Met?Arg?Ala?Pro?Gly?Ala?Leu?Leu?Ala?Arg?Met?Ser?Arg?Leu?Leu?Leu

1???????????????5???????????????????10??????????????????15

Leu?Leu?Leu?Leu?Lys?Val?Ser?Ala?Ser?Ser?Ala?Leu?Gly?Val?Ala?Pro

20??????????????????25??????????????????30

Ala?Ser?Arg?Asn?Glu?Thr?Cys?Leu?Gly?Glu?Ser?Cys?Ala?Pro?Thr?Val

35??????????????????40??????????????????45

Ile?Gln?Arg?Arg?Gly?Arg?Asp?Ala?Trp?Gly?Pro?Gly?Asn?Ser?Ala?Arg

50??????????????????55??????????????????60

Asp?Val?Leu?Arg?Ala?Arg?Ala?Pro?Arg?Glu?Glu?Gln?Gly?Ala?Ala?Phe

65??????????????????70??????????????????75??????????????????80

Leu?Ala?Gly?Pro?Ser?Trp?Asp?Leu?Pro?Ala?Ala?Pro?Gly?Arg?Asp?Pro

85??????????????????90??????????????????95

Ala?Ala?Gly?Arg?Gly?Ala?Glu?Ala?Ser?Ala?Ala?Gly?Pro?Pro?Gly?Pro

100?????????????????105?????????????????110

Pro?Thr?Arg?Pro?Pro?Gly?Pro?Trp?Arg?Trp?Lys?Gly?Ala?Arg?Gly?Gln

115?????????????????120?????????????????125

Glu?Pro?Ser?Glu?Thr?Leu?Gly?Arg?Gly?Asn?Pro?Thr?Ala?Leu?Gln?Leu

130?????????????????135?????????????????140

Phe?Leu?Gln?Ile?Ser?Glu?Glu?Glu?Glu?Lys?Gly?Pro?Arg?Gly?Ala?Gly

145?????????????????150?????????????????155?????????????????160

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

165?????????????????170?????????????????175

Ser?Asp?Leu?Phe?Tyr?Trp?Pro?Arg?Arg?Ala?Gly?Lys?Leu?Gln?Gly?Ser

180?????????????????185?????????????????190

His?His?Lys?Pro?Leu?Ser?Lys?Thr?Ala?Asn?Gly?Leu?Ala?Gly?His?Glu

195?????????????????200?????????????????205

Gly?Trp?Thr?Ile?Ala?Leu?Pro?Gly?Arg?Ala?Leu?Ala?Gln?Asn?Gly?Ser

210?????????????????215?????????????????220

Leu?Gly?Glu?Gly?Ile?His?Glu?Pro?Gly?Gly?Pro?Arg?Arg?Gly?Asn?Ser

225?????????????????230?????????????????235?????????????????240

Thr?Asn?Arg?Arg?Val?Arg?Leu?Lys?Asn?Pro?Phe?Tyr?Pro?Leu?Thr?Gln

245?????????????????250?????????????????255

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

260?????????????????265?????????????????270

Gly?Thr?Gly?Ile?Ile?Gly?Asn?Leu?Ala?Val?Met?Cys?Ile?Val?Cys?His

275?????????????????280?????????????????285

Asn?Tyr?Tyr?Met?Arg?Ser?Ile?Ser?Asn?Ser?Leu?Leu?Ala?Asn?Leu?Ala

290?????????????????295?????????????????300

Phe?Trp?Asp?Phe?Leu?Ile?Ile?Phe?Phe?Cys?Leu?Pro?Leu?Val?Ile?Phe

305?????????????????310?????????????????315?????????????????320

His?Glu?Leu?Thr?Lys?Lys?Trp?Leu?Leu?Glu?Asp?Phe?Ser?Cys?Lys?Ile

325?????????????????330?????????????????335

Val?Pro?Tyr?Ile?Glu?Val?Ala?Ser?Leu?Gly?Val?Thr?Thr?Phe?Thr?Leu

340?????????????????345?????????????????350

Cys?Ala?Leu?Cys?Ile?Asp?Arg?Phe?Arg?Ala?Ala?Thr?Asn?Val?Gln?Met

355?????????????????360?????????????????365

Tyr?Tyr?Glu?Met?Ile?Glu?Asn?Cys?Ser?Ser?Thr?Thr?Ala?Lys?Leu?Ala

370?????????????????375?????????????????380

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

385?????????????????390?????????????????395?????????????????400

Arg?Gln?Leu?Ser?Lys?Glu?Asp?Leu?Gly?Phe?Ser?Gly?Arg?Ala?Pro?Ala

405?????????????????410?????????????????415

Glu?Arg?Cys?Ile?Ile?Lys?Ile?Ser?Pro?Asp?Leu?Pro?Asp?Thr?Ile?Tyr

420?????????????????425?????????????????430

Val?Leu?Ala?Leu?Thr?Tyr?Asp?Ser?Ala?Arg?Leu?Trp?Trp?Tyr?Phe?Gly

435?????????????????440?????????????????445

Cys?Tyr?Phe?Cys?Leu?Pro?Thr?Leu?Phe?Thr?Ile?Thr?Cys?Ser?Leu?Val

450?????????????????455?????????????????460

Thr?Ala?Arg?Lys?Ile?Arg?Lys?Ala?Glu?Lys?Ala?Cys?Thr?Arg?Gly?Asn

465?????????????????470?????????????????475?????????????????480

Lys?Arg?Gln?Ile?Gln?Leu?Glu?Ser?Gln?Met?Asn?Cys?Thr?Val?Val?Ala

485?????????????????490?????????????????495

Leu?Thr?Ile?Leu?Tyr?Gly?Phe?Cys?Ile?Ile?Pro?Glu?Asn?Ile?Cys?Asn

500?????????????????505?????????????????510

Ile?Val?Thr?Ala?Tyr?Met?Ala?Thr?Gly?Val?Ser?Gln?Gln?Thr?Met?Asp

515?????????????????520?????????????????525

Leu?Leu?Asn?Ile?Ile?Ser?Gln?Phe?Leu?Leu?Phe?Phe?Lys?Ser?Tyr?Val

530?????????????????535?????????????????540

Thr?Pro?Val?Leu?Leu?Phe?Cys?Leu?Cys?Lys?Pro?Phe?Ser?Arg?Ala?Phe

545?????????????????550?????????????????555?????????????????560

Met?Glu?Cys?Cys?Cys?Cys?Cys?Cys?Glu?Glu?Cys?Ile?Gln?Lys?Ser?Ser

565?????????????????570?????????????????575

Thr?Val?Thr?Ser?Asp?Asp?Asn?Asp?Asn?Glu?Tyr?Thr?Thr?Glu?Leu?Glu

580?????????????????585?????????????????590

Leu?Ser?Pro?Phe?Ser?Thr?Ile?Arg?Arg?Glu?Met?Ser?Thr?Phe?Ala?Ser

595?????????????????600?????????????????605

Val?Gly?Thr?His?Cys

610

<210>78

<211>1086

<212>DNA

＜213〉the unknown

<220>

＜223〉new sequence

<400>78

atgtcccctg?aatgcgcgcg?ggcagcgggc?gacgcgccct?tgcgcagcct?ggagcaagcc????60

aaccgcaccc?gctttccctt?cttctccgac?gtcaagggcg?accaccggct?ggtgctggcc????120

gcggtggaga?caaccgtgct?ggtgctcatc?tttgcagtgt?cgctgctggg?caacgtgtgc????180

gccctggtgc?tggtggcgcg?ccgacgacgc?cgcggcgcga?ctgcctgcct?ggtactcaac????240

ctcttctgcg?cggacctgct?cttcatcagc?gctatccctc?tggtgctggc?cgtgcgctgg????300

actgaggcct?ggctgctggg?ccccgttgcc?tgccacctgc?tcttctacgt?gatgaccctg????360

agcggcagcg?tcaccatcct?cacgctggcc?gcggtcagcc?tggagcgcat?ggtgtgcatc????420

gtgcacctgc?agcgcggcgt?gcggggtcct?gggcggcggg?cgcgggcagt?gctgctggcg????480

ctcatctggg?gctattcggc?ggtcgccgct?ctgcctctct?gcgtcttctt?tcgagtcgtc????540

ccgcaacggc?tccccggcgc?cgaccaggaa?atttcgattt?gcacactgat?ttggcccacc????600

attcctggag?agatctcgtg?ggatgtctct?tttgttactt?tgaacttctt?ggtgccagga????660

ctggtcattg?tgatcagtta?ctccaaaatt?ttacagatca?caaaggcatc?aaggaagagg????720

ctcacggtaa?gcctggccta?ctcggagagc?caccagatcc?gcgtgtccca?gcaggacttc????780

cggctcttcc?gcaccctctt?cctcctcatg?gtctccttct?tcatcatgtg?gagccccatc????840

ttcatcacca?tcctcctcat?cctgatccag?aacttcaagc?aagacctggt?catctggccg????900

tccctcttct?tctgggtggt?ggccttcaca?tttgctaatt?cagccctaaa?ccccatcctc????960

tacaacatga?cactgtgcag?gaatgagtgg?aagaaaattt?tttgctgctt?ctggttccca????1020

gaaaagggag?ccattttaac?agacacatct?gtcaaaagaa?atgacttgtc?gattatttct????1080

ggctaa???????????????????????????????????????????????????????????????1086

<210>79

<211>361

<212>PRT

＜213〉the unknown

<220>

＜223〉new sequence

<400>79

Met?Ser?Pro?Glu?Cys?Ala?Arg?Ala?Ala?Gly?Asp?Ala?Pro?Leu?Arg?Ser

1???????????????5???????????????????10??????????????????15

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

20??????????????????25??????????????????30

Gly?Asp?His?Arg?Leu?Val?Leu?Ala?Ala?Val?Glu?Thr?Thr?Val?Leu?Val

35??????????????????40??????????????????45

Leu?Ile?Phe?Ala?Val?Ser?Leu?Leu?Gly?Asn?Val?Cys?Ala?Leu?Val?Leu

50??????????????????55??????????????????60

Val?Ala?Arg?Arg?Arg?Arg?Arg?Gly?Ala?Thr?Ala?Cys?Leu?Val?Leu?Asn

65??????????????????70??????????????????75??????????????????80

Leu?Phe?Cys?Ala?Asp?Leu?Leu?Phe?Ile?Ser?Ala?Ile?Pro?Leu?Val?Leu

85??????????????????90??????????????????95

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

100?????????????????105?????????????????110

Leu?Leu?Phe?Tyr?Val?Met?Thr?Leu?Ser?Gly?Ser?Val?Thr?Ile?Leu?Thr

115?????????????????120?????????????????125

Leu?Ala?Ala?Val?Ser?Leu?Glu?Arg?Met?Val?Cys?Ile?Val?His?Leu?Gln

130?????????????????135?????????????????140

Arg?Gly?Val?Arg?Gly?Pro?Gly?Arg?Arg?Ala?Arg?Ala?Val?Leu?Leu?Ala

145?????????????????150?????????????????155?????????????????160

Leu?Ile?Trp?Gly?Tyr?Ser?Ala?Val?Ala?Ala?Leu?Pro?Leu?Cys?Val?Phe

165?????????????????170?????????????????175

Phe?Arg?Val?Val?Pro?Gln?Arg?Leu?Pro?Gly?Ala?Asp?Gln?Glu?Ile?Ser

180?????????????????185?????????????????190

Ile?Cys?Thr?Leu?Ile?Trp?Pro?Thr?Ile?Pro?Gly?Glu?Ile?Ser?Trp?Asp

195?????????????????200?????????????????205

Val?Ser?Phe?Val?Thr?Leu?Asn?Phe?Leu?Val?Pro?Gly?Leu?Val?Ile?Val

210?????????????????215?????????????????220

Ile?Ser?Tyr?Ser?Lys?Ile?Leu?Gln?Ile?Thr?Lys?Ala?Ser?Arg?Lys?Arg

225?????????????????230?????????????????235?????????????????240

Leu?Thr?Val?Ser?Leu?Ala?Tyr?Ser?Glu?Ser?His?Gln?Ile?Arg?Val?Ser

245?????????????????250?????????????????255

Gln?Gln?Asp?Phe?Arg?Leu?Phe?Arg?Thr?Leu?Phe?Leu?Leu?Met?Val?Ser

260?????????????????265?????????????????270

Phe?Phe?Ile?Met?Trp?Ser?Pro?Ile?Phe?Ile?Thr?Ile?Leu?Leu?Ile?Leu

275?????????????????280?????????????????285

Ile?Gln?Asn?Phe?Lys?Gln?Asp?Leu?Val?Ile?Trp?Pro?Ser?Leu?Phe?Phe

290?????????????????295?????????????????300

Trp?Val?Val?Ala?Phe?Thr?Phe?Ala?Asn?Ser?Ala?Leu?Asn?Pro?Ile?Leu

305?????????????????310?????????????????315?????????????????320

Tyr?Asn?Met?Thr?Leu?Cys?Arg?Asn?Glu?Trp?Lys?Lys?Ile?Phe?Cys?Cys

325?????????????????330?????????????????335

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

340?????????????????345?????????????????350

Arg?Asn?Asp?Leu?Ser?Ile?Ile?Ser?Gly

355?????????????????360

<210>80

<211>1086

<212>DNA

＜213〉the unknown

<220>

＜223〉new sequence

<400>80

atgtcccctg?aatgcgcgcg?ggcagcgggc?gacgcgccct?tgcgcagcct?ggagcaagcc????60

aaccgcaccc?gctttccctt?cttctccgac?gtcaagggcg?accaccggct?ggtgctggcc????120

gcggtggaga?caaccgtgct?ggtgctcatc?tttgcagtgt?cgctgctggg?caacgtgtgc????180

gccctggtgc?tggtggcgcg?ccgacgacgc?cgcggcgcga?ctgcctgcct?ggtactcaac????240

ctcttctgcg?cggacctgct?cttcatcagc?gctatccctc?tggtgctggc?cgtgcgctgg????300

actgaggcct?ggctgctggg?ccccgttgcc?tgccacctgc?tcttctacgt?gatgaccctg????360

agcggcagcg?tcaccatcct?cacgctggcc?gcggtcagcc?tgaatcgcat?ggtgtgcatc????420

gtgcacctgc?agcgcggcgt?gcggggtcct?gggcggcggg?cgcgggcagt?gctgctggcg????480

ctcatctggg?gctattcggc?ggtcgccgct?ctgcctctct?gcgtcttctt?tcgagtcgtc????540

ccgcaacggc?tccccggcgc?cgaccaggaa?atttcgattt?gcacactgat?ttggcccacc????600

attcctggag?agatctcgtg?ggatgtctct?tttgttactt?tgaacttctt?ggtgccagga????660

ctggtcattg?tgatcagtta?ctccaaaatt?ttacagatca?caaaggcatc?aaggaagagg????720

ctcacggtaa?gcctggccta?ctcggagagc?caccagatcc?gcgtgtccca?gcaggacttc????780

cggctcttcc?gcaccctctt?cctcctcatg?gtctccttct?tcatcatgtg?gagccccatc????840

atcatcacca?tcctcctcat?cctgatccag?aacttcaagc?aagacctggt?catctggccg????900

tccctcttct?tctgggtggt?ggccttcaca?tttgctaatt?cagccctaaa?ccccatcctc????960

tacaacatga?cactgtgcag?gaatgagtgg?aagaaaattt?tttgctgctt?ctggttccca????1020

gaaaagggag?ccattttaac?agacacatct?gtcaaaagaa?atgacttgtc?gattatttct????1080

ggctaa???????????????????????????????????????????????????????????????1086

<210>81

<211>361

<212>PRT

＜213〉the unknown

<220>

＜223〉new sequence

<400>81

Met?Ser?Pro?Glu?Cys?Ala?Arg?Ala?Ala?Gly?Asp?Ala?Pro?Leu?Arg?Ser

1???????????????5???????????????????10??????????????????15

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

20??????????????????25??????????????????30

Gly?Asp?His?Arg?Leu?Val?Leu?Ala?Ala?Val?Glu?Thr?Thr?Val?Leu?Val

35??????????????????40??????????????????45

Leu?Ile?Phe?Ala?Val?Ser?Leu?Leu?Gly?Asn?Val?Cys?Ala?Leu?Val?Leu

50??????????????????55??????????????????60

Val?Ala?Arg?Arg?Arg?Arg?Arg?Gly?Ala?Thr?Ala?Cys?Leu?Val?Leu?Asn

65??????????????????70??????????????????75??????????????????80

Leu?Phe?Cys?Ala?Asp?Leu?Leu?Phe?Ile?Ser?Ala?Ile?Pro?Leu?Val?Leu

85??????????????????90??????????????????95

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

100?????????????????105?????????????????110

Leu?Leu?Phe?Tyr?Val?Met?Thr?Leu?Ser?Gly?Ser?Val?Thr?Ile?Leu?Thr

115?????????????????120?????????????????125

Leu?Ala?Ala?Val?Ser?Leu?Asn?Arg?Met?Val?Cys?Ile?Val?His?Leu?Gln

130?????????????????135?????????????????140

Arg?Gly?Val?Arg?Gly?Pro?Gly?Arg?Arg?Ala?Arg?Ala?Val?Leu?Leu?Ala

145?????????????????150?????????????????155?????????????????160

Leu?Ile?Trp?Gly?Tyr?Ser?Ala?Val?Ala?Ala?Leu?Pro?Leu?Cys?Val?Phe

165?????????????????170?????????????????175

Phe?Arg?Val?Val?Pro?Gln?Arg?Leu?Pro?Gly?Ala?Asp?Gln?Glu?Ile?Ser

180?????????????????185?????????????????190

Ile?Cys?Thr?Leu?Ile?Trp?Pro?Thr?Ile?Pro?Gly?Glu?Ile?Ser?Trp?Asp

195?????????????????200?????????????????205

Val?Ser?Phe?Val?Thr?Leu?Asn?Phe?Leu?Val?Pro?Gly?Leu?Val?Ile?Val

210?????????????????215?????????????????220

Ile?Ser?Tyr?Ser?Lys?Ile?Leu?Gln?Ile?Thr?Lys?Ala?Ser?Arg?Lys?Arg

225?????????????????230?????????????????235?????????????????240

Leu?Thr?Val?Ser?Leu?Ala?Tyr?Ser?Glu?Ser?His?Gln?Ile?Arg?Val?Ser

245?????????????????250?????????????????255

Gln?Gln?Asp?Phe?Arg?Leu?Phe?Arg?Thr?Leu?Phe?Leu?Leu?Met?Val?Ser

260?????????????????265?????????????????270

Phe?Phe?Ile?Met?Trp?Ser?Pro?Ile?Ile?Ile?Thr?Ile?Leu?Leu?Ile?Leu

275?????????????????280?????????????????285

Ile?Gln?Asn?Phe?Lys?Gln?Asp?Leu?Val?Ile?Trp?Pro?Ser?Leu?Phe?Phe

290?????????????????295?????????????????300

Trp?Val?Val?Ala?Phe?Thr?Phe?Ala?Asn?Ser?Ala?Leu?Asn?Pro?Ile?Leu

305?????????????????310?????????????????315?????????????????320

Tyr?Asn?Met?Thr?Leu?Cys?Arg?Asn?Glu?Trp?Lys?Lys?Ile?Phe?Cys?Cys

325?????????????????330?????????????????335

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

340?????????????????345?????????????????350

Arg?Asn?Asp?Leu?Ser?Ile?Ile?Ser?Gly

355?????????????????360

<210>82

<211>1212

<212>DNA

＜213〉the unknown

<220>

＜223〉new sequence

<400>82

atggcttgca?atggcagtgc?ggccaggggg?cactttgacc?ctgaggactt?gaacctgact????60

gacgaggcac?tgagactcaa?gtacctgggg?ccccagcaga?cagagctgtt?catgcccatc????120

tgtgccacat?acctgctgat?cttcgtggtg?ggcgctgtgg?gcaatgggct?gacctgtctg????180

gtcatcctgc?gccacaaggc?catgcgcacg?cctaccaact?actacctctt?cagcctggcc????240

gtgtcggacc?tgctggtgct?gctggtgggc?ctgcccctgg?agctctatga?gatgtggcac????300

aactacccct?tcctgctggg?cgttggtggc?tgctatttcc?gcacgctact?gtttgagatg????360

gtctgcctgg?cctcagtgct?caacgtcact?gccctgagcg?tggaacgcta?tgtggccgtg????420

gtgcacccac?tccaggccag?gtccatggtg?acgcgggccc?atgtgcgccg?agtgcttggg????480

gccgtctggg?gtcttgccat?gctctgctcc?ctgcccaaca?ccagcctgca?cggcatccgg????540

cagctgcacg?tgccctgccg?gggcccagtg?ccagactcag?ctgtttgcat?gctggtccgc????600

ccacgggccc?tctacaacat?ggtagtgcag?accaccgcgc?tgctcttctt?ctgcctgccc????660

atggccatca?tgagcgtgct?ctacctgctc?attgggctgc?gactgcggcg?ggagaggctg????720

ctgctcatgc?aggaggccaa?gggcaggggc?tctgcagcag?ccaggtccag?atacacctgc????780

aggctccagc?agcacgatcg?gggccggaga?caagtgaaaa?agatgctgtt?tgtcctggtc????840

gtggtgtttg?gcatctgctg?ggccccgttc?cacgccgacc?gcgtcatgtg?gagcgtcgtg????900

tcacagtgga?cagatggcct?gcacctggcc?ttccagcacg?tgcacgtcat?ctccggcatc????960

ttcttctacc?tgggctcggc?ggccaacccc?gtgctctata?gcctcatgtc?cagccgcttc????1020

cgagagacct?tccaggaggc?cctgtgcctc?ggggcctgct?gccatcgcct?cagaccccgc????1080

cacagctccc?acagcctcag?caggatgacc?acaggcagca?ccctgtgtga?tgtgggctcc????1140

ctgggcagct?gggtccaccc?cctggctggg?aacgatggcc?cagaggcgca?gcaagagacc????1200

gatccatcct?ga????????????????????????????????????????????????????????1212

<210>83

<211>403

<212>PRT

＜213〉the unknown

<220>

＜223〉new sequence

<400>83

Met?Ala?Cys?Asn?Gly?Ser?Ala?Ala?Arg?Gly?His?Phe?Asp?Pro?Glu?Asp

1???????????????5???????????????????10??????????????????15

Leu?Asn?Leu?Thr?Asp?Glu?Ala?Leu?Arg?Leu?Lys?Tyr?Leu?Gly?Pro?Gln

20??????????????????25??????????????????30

Gln?Thr?Glu?Leu?Phe?Met?Pro?Ile?Cys?Ala?Thr?Tyr?Leu?Leu?Ile?Phe

35??????????????????40??????????????????45

Val?Val?Gly?Ala?Val?Gly?Asn?Gly?Leu?Thr?Cys?Leu?Val?Ile?Leu?Arg

50??????????????????55??????????????????60

His?Lys?Ala?Met?Arg?Thr?Pro?Thr?Asn?Tyr?Tyr?Leu?Phe?Ser?Leu?Ala

65??????????????????70??????????????????75??????????????????80

Val?Ser?Asp?Leu?Leu?Val?Leu?Leu?Val?Gly?Leu?Pro?Leu?Glu?Leu?Tyr

85??????????????????90??????????????????95

Glu?Met?Trp?His?Asn?Tyr?Pro?Phe?Leu?Leu?Gly?Val?Gly?Gly?Cys?Tyr

100?????????????????105?????????????????110

Phe?Arg?Thr?Leu?Leu?Phe?Glu?Met?Val?Cys?Leu?Ala?Ser?Val?Leu?Asn

115?????????????????120?????????????????125

Val?Thr?Ala?Leu?Ser?Val?Glu?Arg?Tyr?Val?Ala?Val?Val?His?Pro?Leu

130?????????????????135?????????????????140

Gln?Ala?Arg?Ser?Met?Val?Thr?Arg?Ala?His?Val?Arg?Arg?Val?Leu?Gly

145?????????????????150?????????????????155?????????????????160

Ala?Val?Trp?Gly?Leu?Ala?Met?Leu?Cys?Ser?Leu?Pro?Asn?Thr?Ser?Leu

165?????????????????170?????????????????175

His?Gly?Ile?Arg?Gln?Leu?His?Val?Pro?Cys?Arg?Gly?Pro?Val?Pro?Asp

180?????????????????185?????????????????190

Ser?Ala?Val?Cys?Met?Leu?Val?Arg?Pro?Arg?Ala?Leu?Tyr?Asn?Met?Val

195?????????????????200?????????????????205

Val?Gln?Thr?Thr?Ala?Leu?Leu?Phe?Phe?Cys?Leu?Pro?Met?Ala?Ile?Met

210?????????????????215?????????????????220

Ser?Val?Leu?Tyr?Leu?Leu?Ile?Gly?Leu?Arg?Leu?Arg?Arg?Glu?Arg?Leu

225?????????????????230?????????????????235?????????????????240

Leu?Leu?Met?Gln?Glu?Ala?Lys?Gly?Arg?Gly?Ser?Ala?Ala?Ala?Arg?Ser

245?????????????????250?????????????????255

Arg?Tyr?Thr?Cys?Arg?Leu?Gln?Gln?His?Asp?Arg?Gly?Arg?Arg?Gln?Val

260?????????????????265?????????????????270

Lys?Lys?Met?Leu?Phe?Val?Leu?Val?Val?Val?Phe?Gly?Ile?Cys?Trp?Ala

275?????????????????280?????????????????285

Pro?Phe?His?Ala?Asp?Arg?Val?Met?Trp?Ser?Val?Val?Ser?Gln?Trp?Thr

290?????????????????295?????????????????300

Asp?Gly?Leu?His?Leu?Ala?Phe?Gln?His?Val?His?Val?Ile?Ser?Gly?Ile

305?????????????????310?????????????????315?????????????????320

Phe?Phe?Tyr?Leu?Gly?Ser?Ala?Ala?Asn?Pro?Val?Leu?Tyr?Ser?Leu?Met

325?????????????????330?????????????????335

Ser?Ser?Arg?Phe?Arg?Glu?Thr?Phe?Gln?Glu?Ala?Leu?Cys?Leu?Gly?Ala

340?????????????????345?????????????????350

Cys?Cys?His?Arg?Leu?Arg?Pro?Arg?His?Ser?Ser?His?Ser?Leu?Ser?Arg

355?????????????????360?????????????????365

Met?Thr?Thr?Gly?Ser?Thr?Leu?Cys?Asp?Val?Gly?Ser?Leu?Gly?Ser?Trp

370?????????????????375?????????????????380

Val?His?Pro?Leu?Ala?Gly?Asn?Asp?Gly?Pro?Glu?Ala?Gln?Gln?Glu?Thr

385?????????????????390?????????????????395?????????????????400

Asp?Pro?Ser

<210>84

<211>930

<212>DNA

＜213〉the unknown

<220>

＜223〉new sequence

<400>84

atgaatggea?cctacaacac?ctgtggctcc?agcgacctca?cctggccccc?agcgatcaag????60

ctgggcttet?acgcctactt?gggcgtcctg?ctggtgctag?gcctgctgct?caacagcctg????120

gcgctctggg?tgttctgctg?ccgcatgcag?cagtggacgg?agacccgcat?ctacatgacc????180

aacctggcgg?tggccgacct?ctgcctgctg?tgcaccttgc?ccttcgtgct?gcactccctg????240

cgagacacct?cagacacgcc?gctgtgccag?ctctcccagg?gcatctacct?gaccaacagg????300

tacatgagca?tcagcctggt?cacggccatc?gccgtggacc?gctatgtggc?cgtgcggcac????360

ccgctgcgtg?cccgcgggct?gcggtccccc?aggcaggctg?cggccgtgtg?cgcggtcctc????420

tgggtgctgg?tcatcggctc?cctggtggct?cgctggctcc?tggggattca?ggagggcggc????480

ttctgcttca?ggagcacccg?gcacaatttc?aactccatgc?ggttcccgct?gctgggattc????540

tacctgcccc?tggccgtggt?ggtcttctgc?tccctgaagg?tggtgactgc?cctggcccag????600

aggccaccca?ccgacgtggg?gcaggcagag?gccacccgca?aggctaaacg?catggtctgg????660

gccaacctcc?tggtgttcgt?ggtctgcttc?ctgcccctgc?acgtggggct?gacagtgcgc????720

ctcgcagtgg?gctggaacgc?ctgtgccctc?ctggagacga?tccgtcgcgc?cctgtacata????780

accagcaagc?tctcagatgc?caactgctgc?ctggacgcca?tctgctacta?ctacatggcc????840

aaggagttcc?aggaggcgtc?tgcactggcc?gtggctcccc?gtgctaaggc?ccacaaaagc????900

caggactctc?tgtgcgtgac?cctcgcctaa?????????????????????????????????????930

<210>85

<211>309

<212>PRT

＜213〉the unknown

<220>

＜223〉new sequence

<400>85

Met?Asn?Gly?Thr?Tyr?Asn?Thr?Cys?Gly?Ser?Ser?Asp?Leu?Thr?Trp?Pro

1???????????????5???????????????????10??????????????????15

Pro?Ala?Ile?Lys?Leu?Gly?Phe?Tyr?Ala?Tyr?Leu?Gly?Val?Leu?Leu?Val

20??????????????????25??????????????????30

Leu?Gly?Leu?Leu?Leu?Asn?Ser?Leu?Ala?Leu?Trp?Val?Phe?Cys?Cys?Arg

35??????????????????40??????????????????45

Met?Gln?Gln?Trp?Thr?Glu?Thr?Arg?Ile?Tyr?Met?Thr?Asn?Leu?Ala?Val

50??????????????????55??????????????????60

Ala?Asp?Leu?Cys?Leu?Leu?Cys?Thr?Leu?Pro?Phe?Val?Leu?His?Ser?Leu

65??????????????????70??????????????????75??????????????????80

Arg?Asp?Thr?Ser?Asp?Thr?Pro?Leu?Cys?Gln?Leu?Ser?Gln?Gly?Ile?Tyr

85??????????????????90??????????????????95

Leu?Thr?Asn?Arg?Tyr?Met?Ser?Ile?Ser?Leu?Val?Thr?Ala?Ile?Ala?Val

100?????????????????105?????????????????110

Asp?Arg?Tyr?Val?Ala?Val?Arg?His?Pro?Leu?Arg?Ala?Arg?Gly?Leu?Arg

115?????????????????120?????????????????125

Ser?Pro?Arg?Gln?Ala?Ala?Ala?Val?Cys?Ala?Val?Leu?Trp?Val?Leu?Val

130?????????????????135?????????????????140

Ile?Gly?Ser?Leu?Val?Ala?Arg?Trp?Leu?Leu?Gly?Ile?Gln?Glu?Gly?Gly

145?????????????????150?????????????????155?????????????????160

Phe?Cys?Phe?Arg?Ser?Thr?Arg?His?Asn?Phe?Asn?Ser?Met?Arg?Phe?Pro

165?????????????????170?????????????????175

Leu?Leu?Gly?Phe?Tyr?Leu?Pro?Leu?Ala?Val?Val?Val?Phe?Cys?Ser?Leu

180?????????????????185?????????????????190

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

195?????????????????200?????????????????205

Ala?Glu?Ala?Thr?Arg?Lys?Ala?Lys?Arg?Met?Val?Trp?Ala?Asn?Leu?Leu

210?????????????????215?????????????????220

Val?Phe?Val?Val?Cys?Phe?Leu?Pro?Leu?His?Val?Gly?Leu?Thr?Val?Arg

225?????????????????230?????????????????235?????????????????240

Leu?Ala?Val?Gly?Trp?Asn?Ala?Cys?Ala?Leu?Leu?Glu?Thr?Ile?Arg?Arg

245?????????????????250?????????????????255

Ala?Leu?Tyr?Ile?Thr?Ser?Lys?Leu?Ser?Asp?Ala?Asn?Cys?Cys?Leu?Asp

260?????????????????265?????????????????270

Ala?Ile?Cys?Tyr?Tyr?Tyr?Met?Ala?Lys?Glu?Phe?Gln?Glu?Ala?Ser?Ala

275?????????????????280?????????????????285

Leu?Ala?Val?Ala?Pro?Arg?Ala?Lys?Ala?His?Lys?Ser?Gln?Asp?Ser?Leu

290?????????????????295?????????????????300

Cys?Val?Thr?Leu?Ala

305

<210>86

<211>1446

<212>DNA

＜213〉the unknown

<220>

＜223〉new sequence

<400>86

atgcggtggc?tgtggcccct?ggctgtctct?cttgctgtga?ttttggctgt?ggggctaagc????60

agggtctctg?ggggtgcccc?cctgcacctg?ggcaggcaca?gagccgagac?ccaggagcag????120

cagagccgat?ccaagagggg?caccgaggat?gaggaggcca?agggcgtgca?gcagtatgtg????180

cctgaggagt?gggcggagta?cccccggccc?attcaccctg?ctggcctgca?gccaaccaag????240

cccttggtgg?ccaccagccc?taaccccgac?aaggatgggg?gcaccccaga?cagtgggcag????300

gaactgaggg?gcaatctgac?aggggcacca?gggcagaggc?tacagatcca?gaaccccctg????360

tatccggtga?ccgagagctc?ctacagtgcc?tatgccatca?tgcttctggc?gctggtggtg????420

tttgcggtgg?gcattgtggg?caacctgtcg?gtcatgtgca?tcgtgtggca?cagctactac????480

ctgaagagcg?cctggaactc?catccttgcc?agcctggccc?tctgggattt?tctggtcctc????540

tttttctgcc?tccctattgt?catcttcaac?gagatcacca?agcagaggct?actgggtgac????600

gtttcttgtc?gtgccgtgcc?cttcatggag?gtctcctctc?tgggagtcac?gactttcagc????660

ctctgtgccc?tgggcattga?ccgcttccac?gtggccacca?gcaccctgcc?caaggtgagg????720

cccatcgagc?ggtgccaatc?catcctggcc?aagttggctg?tcatctgggt?gggctccatg????780

acgctggctg?tgcctgagct?cctgctgtgg?cagctggcac?aggagcctgc?ccccaccatg????840

ggcaccctgg?actcatgcat?catgaaaccc?tcagccagcc?tgcccgagtc?cctgtattca????900

ctggtgatga?cctaccagaa?cgcccgcatg?tggtggtact?ttggctgcta?cttctgcctg????960

cccatcctct?tcacagtcac?ctgccagctg?gtgacatggc?gggtgcgagg?ccctccaggg????1020

aggaagtcag?agtgcagggc?cagcaagcac?gagcagtgtg?agagccagct?caagagcacc????1080

gtggtgggcc?tgaccgtggt?ctacgccttc?tgcaccctcc?cagagaacgt?ctgcaacatc????1140

gtggtggcct?acctctccac?cgagctgacc?cgccagaccc?tggacctcct?gggcctcatc????1200

aaccagttct?ccaccttctt?caagggcgcc?atcaccccag?tgctgctcct?ttgcatctgc????1260

aggccgctgg?gccaggcctt?cctggactgc?tgctgctgct?gctgctgtga?ggagtgcggc????1320

ggggcttcgg?aggcctctgc?tgccaatggg?tcggacaaca?agctcaagac?cgaggtgtcc????1380

tcttccatct?acttccacaa?gcccagggag?tcacccccac?tcctgcccct?gggcacacct????1440

tgctga?????????????????????????????????????????????????????????1446

<210>87

<211>481

<212>PRT

＜213〉the unknown

<220>

＜223〉new sequence

<400>87

Met?Arg?Trp?Leu?Trp?Pro?Leu?Ala?Val?Ser?Leu?Ala?Val?Ile?Leu?Ala

1???????????????5???????????????????10??????????????????15

Val?Gly?Leu?Ser?Arg?Val?Ser?Gly?Gly?Ala?Pro?Leu?His?Leu?Gly?Arg

20??????????????????25??????????????????30

His?Arg?Ala?Glu?Thr?Gln?Glu?Gln?Gln?Ser?Arg?Ser?Lys?Arg?Gly?Thr

35??????????????????40??????????????????45

Glu?Asp?Glu?Glu?Ala?Lys?Gly?Val?Gln?Gln?Tyr?Val?Pro?Glu?Glu?Trp

50??????????????????55??????????????????60

Ala?Glu?Tyr?Pro?Arg?Pro?Ile?His?Pro?Ala?Gly?Leu?Gln?Pro?Thr?Lys

65??????????????????70??????????????????75??????????????????80

Pro?Leu?Val?Ala?Thr?Ser?Pro?Asn?Pro?Asp?Lys?Asp?Gly?Gly?Thr?Pro

85??????????????????90??????????????????95

Asp?Ser?Gly?Gln?Glu?Leu?Arg?Gly?Asn?Leu?Thr?Gly?Ala?Pro?Gly?Gln

100?????????????????105?????????????????110

Arg?Leu?Gln?Ile?Gln?Asn?Pro?Leu?Tyr?Pro?Val?Thr?Glu?Ser?Ser?Tyr

115?????????????????120?????????????????125

Ser?Ala?Tyr?Ala?Ile?Met?Leu?Leu?Ala?Leu?Val?Val?Phe?Ala?Val?Gly

130?????????????????135?????????????????140

Ile?Val?Gly?Asn?Leu?Ser?Val?Met?Cys?Ile?Val?Trp?His?Ser?Tyr?Tyr

145?????????????????150?????????????????155?????????????????160

Leu?Lys?Ser?Ala?Trp?Asn?Ser?Ile?Leu?Ala?Ser?Leu?Ala?Leu?Trp?Asp

165?????????????????170?????????????????175

Phe?Leu?Val?Leu?Phe?Phe?Cys?Leu?Pro?Ile?Val?Ile?Phe?Asn?Glu?Ile

180?????????????????185?????????????????190

Thr?Lys?Gln?Arg?Leu?Leu?Gly?Asp?Val?Ser?Cys?Arg?Ala?Val?Pro?Phe

195?????????????????200?????????????????205

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

210?????????????????215?????????????????220

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

225?????????????????230?????????????????235?????????????????240

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

245?????????????????250?????????????????255

Val?Gly?Ser?Met?Thr?Leu?Ala?Val?Pro?Glu?Leu?Leu?Leu?Trp?Gln?Leu

260?????????????????265?????????????????270

Ala?Gln?Glu?Pro?Ala?Pro?Thr?Met?Gly?Thr?Leu?Asp?Ser?Cys?Ile?Met

275?????????????????280?????????????????285

Lys?Pro?Ser?Ala?Ser?Leu?Pro?Glu?Ser?Leu?Tyr?Ser?Leu?Val?Met?Thr

290?????????????????295?????????????????300

Tyr?Gln?Asn?Ala?Arg?Met?Trp?Trp?Tyr?Phe?Gly?Cys?Tyr?Phe?Cys?Leu

305?????????????????310?????????????????315?????????????????320

Pro?Ile?Leu?Phe?Thr?Val?Thr?Cys?Gln?Leu?Val?Thr?Trp?Arg?Val?Arg

325?????????????????330?????????????????335

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

340?????????????????345?????????????????350

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

355?????????????????360?????????????????365

Ala?Phe?Cys?Thr?Leu?Pro?Glu?Asn?Val?Cys?Asn?Ile?Val?Val?Ala?Tyr

370?????????????????375?????????????????380

Leu?Ser?Thr?Glu?Leu?Thr?Arg?Gln?Thr?Leu?Asp?Leu?Leu?Gly?Leu?Ile

385?????????????????390?????????????????395?????????????????400

Asn?Gln?Phe?Ser?Thr?Phe?Phe?Lys?Gly?Ala?Ile?Thr?Pro?Val?Leu?Leu

405?????????????????410?????????????????415

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

420?????????????????425?????????????????430

Cys?Cys?Cys?Cys?Glu?Glu?Cys?Gly?Gly?Ala?Ser?Glu?Ala?Ser?Ala?Ala

435?????????????????440?????????????????445

Asn?Gly?Ser?Asp?Asn?Lys?Leu?Lys?Thr?Glu?Val?Ser?Ser?Ser?Ile?Tyr

450?????????????????455?????????????????460

Phe?His?Lys?Pro?Arg?Glu?Ser?Pro?Pro?Leu?Leu?Pro?Leu?Gly?Thr?Pro

465?????????????????470?????????????????475?????????????????480

Cys

<210>88

<211>6

<212>PRT

＜213〉the unknown

<220>

＜223〉new sequence

<400>88

Thr?Leu?Glu?Ser?Ile?Met

1???????????????5

<210>89

<211>5

<212>PRT

＜213〉the unknown

<220>

＜223〉new sequence

<400>89

Glu?Tyr?Asn?Leu?Val

1???????????????5

<210>90

<211>5

<212>PRT

＜213〉the unknown

<220>

＜223〉new sequence

<400>90

Asp?Cys?Gly?Leu?Phe

1???????????????5

<210>91

<211>34

<212>DNA

＜213〉the unknown

<220>

＜223〉new sequence

<400>91

gatcaagctt?ccatggcgtg?ctgcctgagc?gagg??????????????????????????34

<210>92

<211>53

<212>DNA

＜213〉the unknown

<220>

＜223〉new sequence

<400>92

gatcggatcc?ttagaacagg?ccgcagtcct?tcaggttcag?ctgcaggatg?gtg?????53

<210>93

<211>5

<212>PRT

＜213〉the unknown

<220>

＜223〉new sequence

<400>93

Gln?Tyr?Glu?Leu?Leu

1???????????????5

<210>94

<211>5

<212>PRT

＜213〉the unknown

<220>

＜223〉new sequence

<400>94

Asp?Cys?Gly?Leu?Phe

1???????????????5

<210>95

<211>1185

<212>DNA

＜213〉the unknown

<220>

＜223〉new sequence

<400>95

atgggctgcc?tcggcaacag?taagaccgag?gaccagcgca?acgaggagaa?ggcgcagcgc????60

gaggccaaca?aaaagatcga?gaagcagctg?cagaaggaca?agcaggtcta?ccgggccacg????120

caccgcctgc?tgctgctggg?tgctggagag?tctggcaaaa?gcaccattgt?gaagcagatg????180

aggatcctac?atgttaatgg?gtttaacgga?gagggcggcg?aagaggaccc?gcaggctgca????240

aggagcaaca?gcgatggtga?gaaggccacc?aaagtgcagg?acatcaaaaa?caacctgaag????300

gaggccattg?aaaccattgt?ggccgccatg?agcaacctgg?tgccccccgt?ggagctggcc????360

aaccctgaga?accagttcag?agtggactac?attctgagcg?tgatgaacgt?gccaaacttt????420

gacttcccac?ctgaattcta?tgagcatgcc?aaggctctgt?gggaggatga?gggagttcgt????480

gcctgctacg?agcgctccaa?cgagtaccag?ctgatcgact?gtgcccagta?cttcctggac????540

aagattgatg?tgatcaagca?ggccgactac?gtgccaagtg?accaggacct?gcttcgctgc????600

cgcgtcctga?cctctggaat?ctttgagacc?aagttccagg?tggacaaagt?caacttccac????660

atgttcgatg?tgggcggcca?gcgcgatgaa?cgccgcaagt?ggatccagtg?cttcaatgat????720

gtgactgcca?tcatcttcgt?ggtggccagc?agcagctaca?acatggtcat?ccgggaggac????780

aaccagacca?accgtctgca?ggaggctctg?aacctcttca?agagcatctg?gaacaacaga????840

tggctgcgta?ccatctctgt?gatcctcttc?ctcaacaagc?aagatctgct?tgctgagaag????900

gtcctcgctg?ggaaatcgaa?gattgaggac?tactttccag?agttcgctcg?ctacaccact????960

cctgaggatg?cgactcccga?gcccggagag?gacccacgcg?tgacccgggc?caagtacttc????1020

atccgggatg?agtttctgag?aatcagcact?gctagtggag?atggacgtca?ctactgctac????1080

cctcacttta?cctgcgccgt?ggacactgag?aacatccgcc?gtgtcttcaa?cgactgccgt????1140

gacatcatcc?agcgcatgca?tcttcgcgac?tgcgggctgt?tttaa????????????????????1185

<210>96

<211>393

<212>PRT

＜213〉the unknown

<220>

＜223〉new sequence

<400>?96

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

1???????????????5???????????????????10??????????????????15

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

20??????????????????25??????????????????30

Asp?Lys?Gln?Val?Tyr?Arg?Ala?Thr?His?Arg?Leu?Leu?Leu?Leu?Gly?Ala

35??????????????????40??????????????????45

Gly?Glu?Ser?Gly?Lys?Ser?Thr?Ile?Val?Lys?Gln?Met?Arg?Ile?Leu?His

50??????????????????55??????????????????60

Val?Asn?Gly?Phe?Asn?Gly?Glu?Gly?Gly?Glu?Glu?Asp?Pro?Gln?Ala?Ala

65??????????????????70??????????????????75??????????????????80

Arg?Ser?Asn?Ser?Asp?Gly?Glu?Lys?Ala?Thr?Lys?Val?Gln?Asp?Ile?Lys

85??????????????????90??????????????????95

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

100?????????????????105?????????????????110

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

115?????????????????120?????????????????125

Tyr?Ile?Leu?Ser?Val?Met?Asn?Val?Pro?Asn?Phe?Asp?Phe?Pro?Pro?Glu

130?????????????????135?????????????????140

Phe?Tyr?Glu?His?Ala?Lys?Ala?Leu?Trp?Glu?Asp?Glu?Gly?Val?Arg?Ala

145?????????????????150?????????????????155?????????????????160

Cys?Tyr?Glu?Arg?Ser?Asn?Glu?Tyr?Gln?Leu?Ile?Asp?Cys?Ala?Gln?Tyr

165?????????????????170?????????????????175

Phe?Leu?Asp?Lys?Ile?Asp?Val?Ile?Lys?Gln?Ala?Asp?Tyr?Val?Pro?Ser

180?????????????????185?????????????????190

Asp?Gln?Asp?Leu?Leu?Arg?Cys?Arg?Val?Leu?Thr?Ser?Gly?Ile?Phe?Glu

195?????????????????200?????????????????205

Thr?Lys?Phe?Gln?Val?Asp?Lys?Val?Asn?Phe?His?Met?Phe?Asp?Val?Gly

210?????????????????215?????????????????220

Gly?Gln?Arg?Asp?Glu?Arg?Arg?Lys?Trp?Ile?Gln?Cys?Phe?Asn?Asp?Val

225?????????????????230?????????????????235?????????????????240

Thr?Ala?Ile?Ile?Phe?Val?Val?Ala?Ser?Ser?Ser?Tyr?Asn?Met?Val?Ile

245?????????????????250?????????????????255

Arg?Glu?Asp?Asn?Gln?Thr?Asn?Arg?Leu?Gln?Glu?Ala?Leu?Asn?Leu?Phe

260?????????????????265?????????????????270

Lys?Ser?Ile?Trp?Asn?Asn?Arg?Trp?Leu?Arg?Thr?Ile?Ser?Val?Ile?Leu

275?????????????????280?????????????????285

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

290?????????????????295?????????????????300

Ser?Lys?Ile?Glu?Asp?Tyr?Phe?Pro?Glu?Phe?Ala?Arg?Tyr?Thr?Thr?Pro

305?????????????????310?????????????????315?????????????????320

Glu?Asp?Ala?Thr?Pro?Glu?Pro?Gly?Glu?Asp?Pro?Arg?Val?Thr?Arg?Ala

325?????????????????330?????????????????335

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

340?????????????????345?????????????????350

Asp?Gly?Arg?His?Tyr?Cys?Tyr?Pro?His?Phe?Thr?Cys?Ala?Val?Asp?Thr

355?????????????????360?????????????????365

Glu?Asn?Ile?Arg?Arg?Val?Phe?Asn?Asp?Cys?Arg?Asp?Ile?Ile?Gln?Arg

370?????????????????375????????????????380

Met?His?Leu?Arg?Asp?Cys?Gly?Leu?Phe

385?????????????????390

<210>97

<211>1014

<212>DNA

＜213〉people

<400>97

atgaactcgt?gggacgcggg?cctggcgggg?ctactggtgg?gcacgatggg?cgtctcgctg????60

ctgtccaacg?cgctggtgct?gctctgcctg?ctgcacagcg?cggacatccg?ccgccaggcg????120

ccggcgctct?tcaccctgaa?cctcacgtgc?gggaacctgc?tgtgcaccgt?ggtcaacatg????180

ccgctcacgc?tggccggcgt?cgtggcgcag?cggcagccgg?cgggcgaccg?cctgtgccgc????240

ctggctgcct?tcctcgacac?cttcctggct?gccaactcca?tgctcagcat?ggccgcgctc????300

agcatcgacc?gctgggtggc?cgtggtcttc?ccgctgagct?accgggccaa?gatgccgcct????360

ccgagatgcg?cgctcatcct?ggcctacacg?tggctgcacg?cgctcacctt?cccagccgcc????420

gcgctcgccc?tgtcctggct?cggcttccac?cagctgtacg?cctcgtgcac?gctgtgcagc????480

cggcggccgg?acgagcgcct?gcgcttcgcc?gtattcactg?gcgccttcca?cgctctcagc????540

ttcctgctct?ccttcgtcgt?gctctgctgc?acgtacctca?aggtgctcaa?ggtggcccgc????600

ttccattgca?agcgcatcga?cgtgatcacc?atgcagacgc?tcgtgctgct?ggtggacctg????660

caccccagtg?tgcgggaacg?ctgtctggag?gagcagaagc?ggaggcgaca?gcgagccacc????720

aagaagatca?gcaccttcat?agggaccttc?cttgtgtgct?tcgcgcccta?tgtgatcacc????780

aggctagtgg?agctcttctc?cacggtgccc?atcggctccc?actggggggt?gctgtccaag????840

tgcttggcgt?acagcaaggc?cgcatccgac?ccctttgtgt?actccttact?gcgacaccag????900

taccgcaaaa?gctgcaagga?gattctgaac?aggctcctgc?acagacgctc?catccactcc????960

tctggcctca?caggcgactc?tcacagccag?aacattctgc?cggtgtctga?gtga??????????1014

<210>98

<211>337

<212>PRT

＜213〉people

<400>98

Met?Asn?Ser?Trp?Asp?Ala?Gly?Leu?Ala?Gly?Leu?Leu?Val?Gly?Thr?Met

1???????????????5???????????????????10??????????????????15

Gly?Val?Ser?Leu?Leu?Ser?Asn?Ala?Leu?Val?Leu?Leu?Cys?Leu?Leu?His

20??????????????????25??????????????????30

Ser?Ala?Asp?Ile?Arg?Arg?Gln?Ala?Pro?Ala?Leu?Phe?Thr?Leu?Asn?Leu

35??????????????????40??????????????????45

Thr?Cys?Gly?Asn?Leu?Leu?Cys?Thr?Val?Val?Asn?Met?Pro?Leu?Thr?Leu

50??????????????????55??????????????????60

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

65??????????????????70??????????????????75??????????????????80

Leu?Ala?Ala?Phe?Leu?Asp?Thr?Phe?Leu?Ala?Ala?Asn?Ser?Met?Leu?Ser

85??????????????????90??????????????????95

Met?Ala?Ala?Leu?Ser?Ile?Asp?Arg?Trp?Val?Ala?Val?Val?Phe?Pro?Leu

100?????????????????105?????????????????110

Ser?Tyr?Arg?Ala?Lys?Met?Pro?Pro?Pro?Arg?Cys?Ala?Leu?Ile?Leu?Ala

115?????????????????120?????????????????125

Tyr?Thr?Trp?Leu?His?Ala?Leu?Thr?Phe?Pro?Ala?Ala?Ala?Leu?Ala?Leu

130?????????????????135?????????????????140

Ser?Trp?Leu?Gly?Phe?His?Gln?Leu?Tyr?Ala?Ser?Cys?Thr?Leu?Cys?Ser

145?????????????????150?????????????????155?????????????????160

Arg?Arg?Pro?Asp?Glu?Arg?Leu?Arg?Phe?Ala?Val?Phe?Thr?Gly?Ala?Phe

165?????????????????170?????????????????175

His?Ala?Leu?Ser?Phe?Leu?Leu?Ser?Phe?Val?Val?Leu?Cys?Cys?Thr?Tyr

180?????????????????185?????????????????190

Leu?Lys?Val?Leu?Lys?Val?Ala?Arg?Phe?His?Cys?Lys?Arg?Ile?Asp?Val

195?????????????????200?????????????????205

Ile?Thr?Met?Gln?Thr?Leu?Val?Leu?Leu?Val?Asp?Leu?His?Pro?Ser?Val

210?????????????????215?????????????????220

Arg?Glu?Arg?Cys?Leu?Glu?Glu?Gln?Lys?Arg?Arg?Arg?Gln?Arg?Ala?Thr

225?????????????????230?????????????????235?????????????????240

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

245?????????????????250?????????????????255

Tyr?Val?Ile?Thr?Arg?Leu?Val?Glu?Leu?Phe?Ser?Thr?Val?Pro?Ile?Gly

260?????????????????265?????????????????270

Ser?His?Trp?Gly?Val?Leu?Ser?Lys?Cys?Leu?Ala?Tyr?Ser?Lys?Ala?Ala

275?????????????????280?????????????????285

Ser?Asp?Pro?Phe?Val?Tyr?Ser?Leu?Leu?Arg?His?Gln?Tyr?Arg?Lys?Ser

290?????????????????295?????????????????300

Cys?Lys?Glu?Ile?Leu?Asn?Arg?Leu?Leu?His?Arg?Arg?Ser?Ile?His?Ser

305?????????????????310?????????????????315?????????????????320

Ser?Gly?Leu?Thr?Gly?Asp?Ser?His?Ser?Gln?Asn?Ile?Leu?Pro?Val?Ser

325?????????????????330?????????????????335

Glu

<210>99

<211>21

<212>DNA

＜213〉the unknown

<220>

＜223〉new sequence

<400>99

cgagaaggtg?ctcaaggtgg?c?????????????????????????????????????????21

<210>100

<211>30

<212>DNA

＜213〉the unknown

<220>

＜223〉new sequence

<400>100

gagaagagct?ccactagcct?ggtgatcaca????????????????????????????????30

<210>101

<211>36

<212>DNA

＜213〉the unknown

<220>

＜223〉new sequence

<400>101

gaattcatga?actcgtggga?cgcgggcctg?gcgggc?????????????????????????36

<210>102

<211>32

<212>DNA

＜213〉the unknown

<220>

＜223〉new sequence

<400>102

ctcgagtcac?tcagacaccg?gcagaatgtt?ct?????????????????????????????32

Claims (36)

1. amino acid sequences encoded g protein coupled receptor by SEQ ID NO.2.

2. the non-endogenous, constitutively activated form of the described g protein coupled receptor of claim 1.

3. plasmid that contains the cDNA of carrier and SEQ ID NO.1.

4. host cell that contains the described plasmid of claim 3.

5. amino acid sequences encoded g protein coupled receptor by SEQ ID NO.4.

6. the non-endogenous, constitutively activated form of the described g protein coupled receptor of claim 5.

7. plasmid that contains the cDNA of carrier and SEQ ID NO.3.

8. host cell that contains the described plasmid of claim 7.

9. amino acid sequences encoded g protein coupled receptor by SEQ ID NO.6.

10. the non-endogenous, constitutively activated form of the described g protein coupled receptor of claim 9.

11. plasmid that contains the cDNA of carrier and SEQ ID NO.5.

12. host cell that contains the described plasmid of claim 11.

13. amino acid sequences encoded g protein coupled receptor by SEQ ID NO.8.

14. the non-endogenous, constitutively activated form of the described g protein coupled receptor of claim 13.

15. plasmid that contains the cDNA of carrier and SEQ ID NO.7.

16. host cell that contains the described plasmid of claim 15.

17. amino acid sequences encoded g protein coupled receptor by SEQ ID NO.10.

18. the non-endogenous, constitutively activated form of the described g protein coupled receptor of claim 17.

19. plasmid that contains the cDNA of carrier and SEQ ID NO.9.

20. host cell that contains the described plasmid of claim 19.

21. amino acid sequences encoded g protein coupled receptor by SEQ ID NO.12.

22. the non-endogenous, constitutively activated form of the described g protein coupled receptor of claim 21.

23. plasmid that contains the cDNA of carrier and SEQ ID NO.11.

24. host cell that contains the described plasmid of claim 23.

25. amino acid sequences encoded g protein coupled receptor by SEQ ID NO.14.

26. the non-endogenous, constitutively activated form of the described g protein coupled receptor of claim 25.

27. plasmid that contains the cDNA of carrier and SEQ ID NO.13.

28. host cell that contains the described plasmid of claim 27.

29. amino acid sequences encoded g protein coupled receptor by SEQ ID NO.16.

30. the non-endogenous, constitutively activated form of the described g protein coupled receptor of claim 29.

31. plasmid that contains the cDNA of carrier and SEQ ID NO.15.

32. host cell that contains the described plasmid of claim 31.

33. amino acid sequences encoded g protein coupled receptor by SEQ ID NO.18.

34. the non-endogenous, constitutively activated form of the described g protein coupled receptor of claim 33.

35. plasmid that contains the cDNA of carrier and SEQ ID NO.17.

36. host cell that contains the described plasmid of claim 35.

Images