CN1944647A - Dopamine receptor new sub-type D5B gene and coded protein and use - Google Patents

Abstract

The present invention provides one new dopamine receptor sub-type D5B gene. The new dopamine receptor sub-type D5B gene obtained through PCR process is similar to dopamine receptor D5 and codes one G-protein coupling receptor containing 478 amino acids. Compared with dopamine receptor D5, the dopamine receptor D5B has even higher affinity to dopamine. The new sub-type D5B gene may be used as the important target point in developing medicine for psychosis, neural disease, cardiac vascular diseases and nephropathy.

Description

Dopamine receptor new sub-type D 5 B gene and encoded protein and application

Technical field

The present invention relates to the genetically engineered field, especially relate to gene and the encoded protein and the application of a Dopamine Receptors new subtype.

Background technology

Some neuropsychopathy (comprising: Parkinson disease, schizophrenia, pharmacological dependence, migraine, manic, dysthymia disorders and Tourette syndrome etc.), cardiovascular disorder, ephrosis are relevant with Dopamine Receptors, Dopamine Receptors is expressed in central nervous system, controls movement function, mood, internal secretion physiological system.

People have carried out a large amount of research to the active position of Dopamine HCL and acceptor thereof.Cools and Rossum propose and may have a kind of Dopamine Receptors incessantly in the brain by electric Physiological Experiment and pharmacological evaluation, and this is a milestone of Dopamine HCL research.The seventies in 20th century, analyze people based on the second messenger and study Dopamine Receptors, for example: the stimulation of cAMP, and, all support the thought of a more than Dopamine Receptors based on binding analysis.Kebabian in 1979 and Calne lay a good foundation for this thought in their one piece of summary, in his paper, have expanded the formulation of Spano, point out that Dopamine Receptors has two classes, D1 and D2 have different biochemistry and pharmacy characteristic, regulate different physiological functions.The G-albumen of two G-protein linked receptors of D1 and D2 effect is different with effector and relevant path.Although Biochemical Research illustrates how different it is that these receptor subtypes have, yet, up to the eighties in 20th century, utilize clone technology, by clone, just disclose the in various degree real of these acceptors to Dopamine Receptors.The result shows to have 5 dopamine 2 receptors at least, is divided into two subtribes, and its character is similar to respectively by D1 and D2 acceptor biochemical and that pharmacology defines, these two subtribes be called D1 sample acceptor (D1 and D5) and D2 sample acceptor (D2, D3, D4).

Dopamine HCL is the main neurotransmitter of basal nuclei, is playing important effect aspect motion control and the cognitive function.D1 sample acceptor (D1 and D5) acceptor particularly.Recently, D1 sample acceptor is at brain function, and especially the function aspect learning and memory has caused the interest that people are huge.The stimulation of D1 sample acceptor all can damage working memory too greatly or very little.The D1 acceptor may be regulated cognition, influence the Parkinson disease.The D1 acceptor is the influence factor of the neuroregulation of prefrontal cortex working memory key, and this zone is subjected to the influence of many sacred diseases.

Dopamine Receptors is a single chain protein, and its N end is in the BLM outside, and C holds in the film inboard, and 7 hydrophobic film districts (TMD) of striding are arranged in the centre of BLM.Respectively there are 3 fragments the film outside and film inboard, are called outer three rings (E1-E3) and interior three rings (11-13), and they link up each TMD; There is the cystine linkage of 2-3 glycosyl site and its rock steady structure effect in the film outside.Seven transbilayer helix bundles form the binding site of part together on film.Agonist can be incorporated on the hydrophobic TM structural domain, proteinic central authorities, and the amino-acid residue high conservative is gone into a narrow binding pocket, may be the binding site of agonist.C does not hold has some palmityl residues, can form with film more to be connected.Compare with D2 sample acceptor, D1 sample acceptor N end is 2 glycoprotein; Inboard the 3rd ring of film is smaller, and C end peptide chain is very long.

In brain, the D1 acceptor is wider than the distribution of other Dopamine Receptors, it is high to express, striatum, nucleus accumbens septi (nucleus accumbens, NAc) and olfactory tubercle can detect the mRNA of D1, in addition, also detect the D1 acceptor at edge system, hippocampus and thalamus.At interior stem nuclear (entopeducular nucleus), black substance reticular part (substantianigra pars reticulata), the D1 acceptor has high-caliber expression, but detects less than mRNA, illustrates at these region D 1 acceptor mainly to exist with projection.The D5 acceptor is compared with the D1 acceptor, and much less is wanted in the expression in rat, and its expression is limited to the other nuclear of bundle of hippocampus, inboard nipple nuclear (1ateral mamillary nucleus), thalamus.Comprise that in some mouth side forebrain (rostral forebrain) zone pallium, dorsal thalamus, diagonal bundle, striatum can detect the D5 receptor mRNA, can detect a small amount of D5 receptor mRNA black substance, middle thalamus and hippocampus.The cell of different zones in the cerebral tissue of primate and ubcellular distribute with D1 and these two acceptors of D5 antibody test, the result shows, D1 and D5 acceptor be co expression in the cone neurone of forehead page or leaf, premotor cortex (premotor cortex), cingulum cortex, entorhinal cortex and hippocampus and dentate gyrus.Frontal cortex and hippocampus, D1 and D5 acceptor are present in presynaptic and postsynaptic.In one cone neurone, the D1 acceptor mainly concentrates on the dendritic spine, and D5 mainly concentrates on the dendron backbone.At olfactory bulb, the D1 acceptor is limited to stratum granulosum, reticular layer and is inserted into the amygdaloid body of end nucleus lateralis.At caudatum, D1 and D5 mainly are positioned at medium sized GABA _ANeurone.D5 also is present in cholinomimetic energy relay cell, and D1 does not exist.The D1 acceptor is present in the zone of thorn postsynaptic to asymmetrical synapse.D1 and D5 are positioned at the post-synapse density with the terminal little cynapse feature of Dopamine HCL.Presynaptic D1 and D5 acceptor form the asymmetrical synase district on aixs cylinder.D1 is positioned at stem nuclear, the black substance reticular part, and D5 detect less than, therefore,, also have only the D1 acceptor to be transferred to the striatum substantia nigra end if D1 and D5 acceptor are positioned at the medium bur neurone that tail is arranged.The D1 acceptor is different with the position on the subcellsular level at cell with the D5 acceptor, is being or not duplicate on identical its function though show D1 on the pharmacology with the D5 acceptor.

Summary of the invention

The object of the present invention is to provide subtype gene and the encoded protein of a kind of new Dopamine Receptors D5.The D5 hypotype is relevant with the adjusting that the hippocampus vagusstoff discharges.Vagusstoff is a kind of neurotransmitter, participates in various cognitive processes.The D5 hypotype participates in the arrangement of striatal information, can with gamma-aminobutyric acid A (GABA _A) acceptor interaction, two acceptors suppress its function mutually.In addition, the D5 acceptor is regulated the signal path relevant with blood pressure, the response of mediation blood pressure, and its function is to understanding machine-processed extremely important that human hypertension produces.

May also there be the D1 sample acceptor that does not have the clone.One of them problem is, the hydrolysis that can clone's D1 sample acceptor coupling phosphoinositide (PI) it be unclear that at present.Yet the D1 sample acceptor in the kidney is relevant with the hydrolysis of PI, it is reported, D1 is subjected to physical efficiency coupling PI hydrolysis.At striatum, the renewal of D1 coupling PI but, still has arguement.The problem of another dispute is, only detects low-level D1 receptor mRNA in kidney, and can not be interpreted as what D1 agonist in nephridial tissue has high relatively in conjunction with level.At last, some experiment also discloses, and on renal cortex film or kidney deutero-cell, has two stage binding curve with combining of D1 selective agonist (especially SCH23390).All these illustrate the hypotype that also may exist not have identification.

By PCR method, be template with the genomic dna of human genome DNA and human SHSY5Y cell, screen D5 acceptor and another acceptor similarly, be called D5B, and this gene is analyzed, sequence is shown in SEQ ID No.1.

The result that Blast analyzes shows that this gene is identical with D5 acceptor (shown in SEQ ID No.2) 98%, and is similar to two pseudogenes 94% of D5.And D5 and two pseudogene D5 ψ 1 (shown in SEQ ID No.3) and D5 ψ 2 have 95% identical, 98% is identical between two pseudogenes, D5B more approaches D5.

Find by sequence comparing analysis D5B, D5 and a pseudogene D5 of D5 Ψ (GenBank data base M67440), in D5B, the place different with the D5 always pseudogene with D5 is identical, the place different with the pseudogene of D5 is always identical with D5, particularly in COOH end, the base of increase and just the same with the pseudogene of D5 on every side.

We have contrasted the sequence of D5B, D5 and a pseudogene D5 of D5 Ψ (GenBank data base M67440), and the result of analysis of nucleic acids order-checking is as follows.

The different amino acid in coding region is labeled on the D5 sequence, and dash is illustrated on this position and lacks, and unfolded ∧ symbolic representation is inserted on this position, and the membrane spaning domain of seven suppositions (TMDs) adds upper ledge and Reference character I-VII.The N glycosylation site * mark of inferring, the phosphorylation site of protein kinase A is used marking, and the phosphorylation site of protein kinase C is gone up marking with void, and a solid yardage box is represented the halfcystine of palmitoylation.

This nucleotide sequence 93.6% is consistent with D5, but it contains terminator (marking with circle) in the frame, therefore can not the complex functionality protein receptor.Analyze the sequence of D5B and can find that it has an open reading frame, encoding one contains 478 amino acid whose G-protein linked receptors and (Duos an amino acid Glu than D5 ⁴³²L-glutamic acid), be a gene that function is arranged.Analyze this three sequences, find in the D5B place different with D5, identical with D5 Ψ, and the place different with D5 Ψ is just identical with D5, just the same with D5 Ψ near the Glu insertion point reaches.D5 has 10 place's amino acid different with D5B, and different amino acid have: N-terminal Gly ⁵/ Arg and Phe ¹⁵The Tyr of ring I2 in the/Leu, second film ¹⁴⁸/ His, the Pro of the second film outer shroud E2 ²⁰²The Ile of/Leu and carboxyl terminal ⁴⁰⁰/ Met, Tyr ⁴¹⁰/ Cys, Thr ⁴¹⁹/ Pro, Asn ⁴²²/ Asp, Glu ⁴²³/ Gln and Gly ⁴³²/ Ser ⁴³³D5B and D5 are bigger in the variation of carboxyl terminal, and six amino acid differences are arranged, and a Glu is arranged among the D5B ⁴³²Insert, formed the Glu tetramer in this site, the carboxyl terminal of D5 acceptor (CT-tail) influences the affinity of acceptor, infers that its affinity may have difference with D5.These amino acid whose variations and the former D5 polymorphism reported change 9 of bringing amino acid whose have not a particle of between different related.

Find that by sequential analysis D5B has the conservative site of many D5 acceptors: 6 l-asparagine (Asn that asterisk marks ⁷, Asn ⁷⁴, Asn199, Asn ²²², Asn ³⁵¹And Asn ³⁸⁸) be potential N end glycosylation site.Wherein at N-terminal Asn ⁷It is the common constitutional features of all Dopamine Receptorss.Studies show that in the DA acceptor that the aminoacid sequence of same family has quite high consistence striding diaphragm area (TMDs), have 80% consistent at TMDs as D1 and D5 hypotype.We find D5B and D5 7 stride (mark) in the diaphragm area with black line also quite conservative.The Asp of TM2 wherein ⁸⁷Be to influence the active important factor of D1 family receptors, and influence the combination of agonist; The Asp of TM3 ¹²⁰Play an important role for amido in conjunction with catecholamine; The Ser of TM5 ²²⁹And Ser ²³³It is hydrogen supply carrier in conjunction with hydroxyl; The Phe of TM6 helps the stable bond of acceptor and aromatic series part; The Asn of TM7 ³⁵¹It is the total feature of D1 family receptors.

E2 district between TM4 and TM5 has notable difference at D1 in the D5 hypotype, and D1 weak point has only 27 amino acid, the same long 41 amino acid that have with D5 of D5B.Film outer shroud E1, two halfcystine sites of E2 Cys ¹¹³, Cys ²¹⁷Be the conservative site of Dopamine Receptors, have vital role for the stability of acceptor tertiary structure.Also find the target site of some protein kinase phosphorylations by sequential analysis, the Thr that lines out is used in the site of four protein kinase A ¹⁵³, Ser260, Ser ²⁷⁵And Ser ²⁸⁷), the Thr that dotted line marks is used in the site of three protein kinase Cs ¹⁵³, Ser ²⁷⁵And Ser ²⁸³), wherein five sites are encircled in the I3 district in film, and the I3 district is being associated with important effect for acceptor and the proteic coupling of G, and regulatory function is arranged.At some Ser of carboxyl terminal, Thr can be by the receptor kinase phosphorylation.Cys ³⁷⁵(marking with square) can be by palmitoylization, make acceptor carboxyl terminal can with plasma membrane mutually riveting decide combination, this amino acid is positioned at the section start of carboxyl terminal in D1 sample acceptor, in D2 sample acceptor then often in ending place of carboxyl terminal.There are 50 amino acid in the I3 district of D5B and D5, all belongs to short loop ring structure, and D5B and D5 encircle the consistence of (I3) in tertiary membrane, and D5B belongs to the Gs receptor on function.

Ring is shorter in the 3rd born of the same parents of many Gs-protein linked receptors, this ring of D5B is the same with D5, and therefore, D5B should be a Gs protein linked receptor (seeing Fig. 1 and Fig. 4), experimental results show that really so D5B is the Gs protein linked receptor of adenylate cyclase association.Bibliographical information, the affinity of D5 and part is not held relevant with COOH.The difference of D5B and D5 maximum is the COOH end, therefore, can estimate that two acceptors are different with the affinity possibility of Dopamine HCL, and experimental result shows that D5B is higher about 1 times than D5 to the affinity of Dopamine HCL.From Fig. 2 and Fig. 3, D5 and D5B are little to agonist Dihydrexine and (±)-Choro-APB difference.

Table one is the characterization of molecules contrast table of D1, D5 and D5B.

Table one

The characterization of molecules of D1, D5 and D5B
					D1	D5	D5B
Amino acid number	446	475(rat)， 477(human)	478
				Amino acid number on the I3	57	50	50
The amino acid number of COOH end	113	117(rat)， 116(human)	117
				Intron	0	0	0

In D5B, the place different with the D5 always pseudogene with D5 is identical, and the place different with the pseudogene of D5 is always identical with D5, particularly at COOH end, the base of increase and on every side with the pseudogene of D5 just the same (as shown in Table 1).

D5B is between D5 and D5 pseudogene, more near D5, be likely during evolution and produce, if it is said just like the front, the pseudogene of D5 is that the Alu sequence of 3 ' UTR produces long rna transcription, cause duplicating of D5, subsequently this gene copy has been moved on to a new position, thereby produced the multiplicity of D5 gene.The generation of D5B may be to be saved in a present intermediate product in the D5 reproduction process so.

Comprehensive the above analysis, the D5B gene that the contriver is cloned into is a new gene, is the Gs receptor, is the adenylate cyclase association, the contriver adopts reporter gene method and cAMP assay method, has studied the pharmacology function of this gene.Transfection the cell concn dependency ground response D1 agonist and the Dopamine HCL of D5B gene, D5B to the affinity of Dopamine HCL than D5 height; D1 agonist Dihydrexidine and (±)-Choro-APB can activate the D5B acceptor.

Relevant by the D5 gene with multiple disease, can predict, D5B may be relevant with some neuropsychopathy (comprising: Parkinson disease, schizophrenia, pharmacological dependence, migraine, manic, dysthymia disorders and Tourette syndrome etc.), cardiovascular, ephrosis etc.Dopamine Receptors is expressed in central nervous system, controls movement function, mood, internal secretion physiological system.Dopamine HCL is the main neurotransmitter of basal nuclei, is playing important effect aspect motion control and the cognitive function.D5B may be at brain function, and especially the function aspect learning and memory plays important regulatory role, can be used as the important target spot of the medicine of research and development relative disease.

Description of drawings

The D5B/6CRE/Luci/CHO cell of Fig. 1 transfection D5B and D5 acceptor gene, D5/6CRE/Luci/CHO cell are measured the luciferase activity after hatching 6 hours under the Dopamine HCL of different concns, ordinate zou is relevant vigor, and X-coordinate is dopamine concentration (nM)

Fig. 2 D5B and D5 acceptor gene pharmacological property 1:D5B/6CRE/Luci/CHO cell, D5/6CRE/Luci/CHO cell are measured the luciferase activity after hatching 6 hours under the different concns dihydrexidine, ordinate zou is relevant vigor, and X-coordinate is dihydrexidine concentration (nM)

Fig. 3 D5B and D5 acceptor gene pharmacological property 2:D5B/6CRE/Luci/CHO cell, D5/6CRE/Luci/CHO cell are measured the luciferase activity after hatching 6 hours under the different concns chloro-APB, ordinate zou is relevant vigor, and X-coordinate is chloro-APB concentration (nM)

The accumulation of D5B acceptor among Fig. 4 HEK293 cAMP under the different concns Dopamine HCL stimulates: incubated altogether 30 minutes under the Dopamine HCL of HEK293 cell of transfection D5B, measure cAMP with the competitive ELISA method at different concns; Ordinate zou is cAMP concentration (pmol/ml), and X-coordinate is dopamine concentration (nM)

Embodiment

Embodiment 1

The cloning process 1 of D5B

Using following primer, is template with human genome DNA, pcr amplification D5.Real underscore is the restriction enzyme site of EocRI in the primer, and wave underline is the restriction enzyme site of HindIII.

Primer 1：5’-GGC GAA TTC GCG TGT GTG TGC GTG CTT GTC AGT GT-3’

Primer 2：5’-GGG AAG CTT CTG AAG TTG GGA CCG CGC ACA GAC CG-3’

React under the condition below.Pre-94 ℃ of 5min of sex change; 94 ℃ of 30s of denaturation temperature, from 50 to 60 ℃ of annealing temperatures, per 2 ℃ of circulations increase by 20 circulations at 60 ℃ at last, totally 30 circulations, 72 ℃ are extended 2min in each circulation, and last 72 ℃ are extended 7min.

The PCR product gets 0.025 μ g/ μ l PCR product through PCR product purification agent box (vitegene) purifying, uses HindIII (TaKaRa) and EocRI (TaKaRa) to be total to enzyme then and cuts.The carrier pcDNA3.1 enzyme that also uses the same method is cut.

In the EP pipe, add two successively and steam water, restriction endonuclease damping fluid, DNA and restriction endonuclease, mix, 37 ℃ of insulation 2h.After enzyme was cut, the PCR product purification test kit with Vitgene carried out purifying by its specification sheets, and getting 40 μ l concentration is the PCR product of 0.0015 μ g/ μ l.PcDNA3.1 carrier enzyme is cut the back dephosphorylation, and the PCR product of the D5 after enzyme is cut is diluted to 0.1ng/ μ l, connects with quadrat method by above, transforms Top10F ' competent cell.Select sturdy mono-clonal, in the LB substratum, cultivate, extract plasmid and order-checking.

In the mono-clonal of the D5 that is selected, can screen the acceptor D5B close with D5.

Embodiment 2

The cloning process 2 of D5B

Genomic dna with the mankind's SHSY5Y cell is a template, carries out the PCR reaction with following two pairs of primers.

Primer 1：5′-gca gcc caa gcg gat cct gcg gat ctg cag tcc agc ccg aaa tgc t-3’.

Primer 2：5′-gaa tag ggc cct cta gat gca tgc tcg agc gga tat ctt aat gga atc cat tcc ggg t-3’

Carry out the PCR reaction below under the condition, pre-94 ℃ of 5min of sex change; 94 ℃ of 30s of denaturation temperature, the 55 ℃ of 30s that anneal, 72 ℃ are extended 2min, 30 circulations.The PCR product is with 1% agarose gel electrophoresis analysis, reclaim the PCR product of 1.5kb, be connected to pMDT-18 carrier (TaKaRa), wherein 41 clones are checked order, it is D5 acceptors that 24 clones are wherein arranged, 12 clones are D5B acceptors, 4 pseudogene ψ D51 and 1 pseudogene ψ D52 that the clone is D5.

Embodiment 3

Gene transfection and cell cultures

With Chinese hamster ovary celI in 37 ℃, contain in the RPMI1640 substratum of 10% foetal calf serum and cultivate, use cell transfecting reagent (Lipofectin) reagent, with the 6CRE response element, contain TATATA box (TA) and minimize promotor and luciferase enzyme gene (Luciferase) and constitute reporter gene, with D5B gene and reporter gene transfection in Chinese hamster ovary celI.In the substratum that contains 0.8mg/mL G418, screen stable transgenic cell D5B/6CRE/TA/Luci.

Embodiment 4

D5B medicine functional examination 1---Dopamine HCL of science

Cell usefulness is contained the perfect medium 1640 of 600 μ g/ml G418 accordingly by density about 3 * 10 ⁵Cells/ml is inoculated into D5/6CRE/TA/Luci and D5B/6CRE/TA/Luci cell respectively in two 96 orifice plates, every hole 90 μ l, and in 37 ℃, 5% CO ₂Incubator in cultivate 24h, then, add the Dopamine HCL of 10 μ l different concns, continue at 37 ℃, 5% CO ₂Incubator in incubate 6h, in cell, add 100 μ l Lampyridea luciferase substrate B right-Glo then ^TM, use Analyst HT ^TMDetect.The result is referring to Fig. 1.

Embodiment 5

D5B medicine functional examination 2---D1 agonist of science

According to the method for embodiment four, use D1 agonist Dihydrexidine and (±)-CHLORO-APB to replace Dopamine HCL respectively.The result shows that Dihydrexidine and (±)-CHLORO-APB can both activate the D5B acceptor, and Dihydrexidine and D5 and D5B bonded EC50 are respectively 4.0nM and 5.3nM, and the result is referring to Fig. 2; (±)-CHLORO-APB is respectively EC502.7nM and 1.9nM in conjunction with the EC50 of D5 and D5B acceptor, and the result is referring to Fig. 3.

Embodiment 4,5 results show: D5B/6CRE/TA/Luci cell concn dependency ground response D1 agonist and Dopamine HCL, its affinity difference, D5B to the affinity of Dopamine HCL than D5 height (Fig. 2).Dihydrexidine and (±)-CHLORO-APB can both activate the D5B acceptor, dihydrexidine and D5 and D5B bonded EC50 are respectively 4.0nM and 5.3nM (Fig. 2), and (±)-CHLORO-APB is respectively EC50 2.7nM and 1.9nM (Fig. 3) in conjunction with the EC50 of D5 and D5B acceptor

Embodiment 6

The D5B acceptor is in the variation of the post-stimulatory cAMP of Dopamine HCL

Before cell transfecting,,, make the DNA of ultrapure no endogenous toxic material by its specification sheets earlier with no endotoxic plasmid extraction test kit.The HEK293 cell is 3 * 10 with DMEM (non-essential amino acid that contains 10% FBS, 1% two anti-and 1%) with density ⁵Cells/ml is inoculated into six orifice plates, each hole 1ml, 37 ℃, 5% CO ₂Incubator in cultivate 24h, after cell reaches 50% degrees of fusion, with fuegen 6, by its specification sheets, with D5B transfectional cell HEK293.In 37 ℃, 5% CO ₂Incubator in cultivate 24h after, harvested cell is 1 * 10 by density ⁵Cells/ml is inoculated in 96 orifice plates, in 37 ℃, and 5% CO ₂Incubator in after the overnight incubation, add the Dopamine HCL of 10 μ l different concns, continue at 37 ℃, 5% CO ₂Incubator in incubate 0.5h, use cAMP test kit (low PH, Immunoassay, R﹠amp then; D) measure cAMP.Method is as follows:

Remove the substratum in 96 orifice plates, add 0.1N HCl 200 μ l in each hole, hatch 20min, 600 * g is centrifugal, gets supernatant and carries out next step experiment.In each hole of 96 flat boards that scribble the goat-anti rabbit polyclonal antibody, add 50 μ l neutralizers, add above-mentioned cell pyrolysis liquid of 100 μ l or standard model, add the cross-linking agent of 50 μ l cAMP and alkaline phosphatase enzyme again, add the antibody-solutions of 50 μ l cAMP again.Seal, shake (500 ± 50rpm) 2h under the room temperature.Remove supernatant,, stand upside down on thieving paper, remove washings fully with the Wash Buffer of 200 μ l washing three times.The pNPP Starch phosphorylase substrate that adds 200 μ l, incubated at room 1h adds the Stop Solution stopped reaction of 50 μ l.Measure the OD value at 405nm.Calculate cAMP concentration, EC according to standard value ₅₀=2.75nM, the result is referring to Fig. 4.Substantially conform to the reporter gene method.The EC of bibliographical information D5 ₅₀=5nM, D5B is bigger 1 times than D5 to the affinity of Dopamine HCL.

Sequence table

Sequence table one

＜110〉applicant

＜120〉dopamine receptor new sub-type D 5 B gene and encoded protein and application

<160>2

<210>1

<211>1437

<212>DNA

＜213〉people

<400>

atgctgccgc caaggagcaa cggcaccgcg tacccggggc agttagcgct ataccagcag 60

ctggcgcagg ggaacgccgt ggggggctcg gcgggggcac cgccactggg gccctcacag 120

gtggtcaccg cctgcctgct gaccctactc atcatctgga ccctgctggg caacgtgctg 180

gtgtgcgcag ccatcgtgcg gagccgccac ctgcgcgcca acatgaccaa cgtcttcatc 240

gtgtctctgg ccgtgtcaga ccttttcgtg gcgctgctgg tcatgccctg gaaggcagtc 300

gccgaggtgg ccggttactg gccctttgga gcgttctgcg acgtctgggt ggccttcgac 360

atcatgtgct ccactgcctc catcctgaac ctgtgcgtca tcagcgtgga ccgctactgg 420

gccatctcca ggcccttccg ccacaagcgc aagatgactc agcgcatggc cttggtcatg 480

gtcggcctgg catggacctt gtccatcctc atctccttca ttccggtcca gctcaactgg 540

cacagggacc aggcggcctc ttggggcggg ctggacctgc caaataacct ggccaactgg 600

acgctctggg aggaggactt ttgggagccc gacgtgaatg cagagaactg tgactccagc 660

ctgaatcgaa cctacgccat ctcttcctcg ctcatcagct tctacatccc cgttgccatc 720

atgatcgtga cctacacgcg catctaccgc atcgcccagg tgcagatccg caggatttcc 780

tccctggaga gggccgcaga gcacgcgcag agctgccgga gcagcgcagc ctgcgcgccc 840

gacaccagcc tgcgcgcttc catcaagaag gagaccaagg ttctcaagac cctgtcggtg 900

atcatggggg tcttcgtgtg ttgctggctg cccttcttca tccttaactg catggtccct 960

ttctgcagtg gacaccccga aggccctccg gccggcttcc cctgcgtcag tgagaccacc 1020

ttcgacgtct tcgtctggtt cggctgggct aactcctcac tcaaccccgt catctatgcc 1080

ttcaacgccg actttcagaa ggtgtttgcc cagctgctgg ggtgcagcca cttctgctcc 1140

cgcacgccgg tggagacggt gaacatcagc aatgagctca tctcctacaa ccaagacatg 1200

gtcttccaca aggaaatcgc agctgcctgc atccacatga tgcccaacgc ccttcccccc 1260

ggggaccaag aggtggacaa cgatgaggag gaggagagtc ctttcgatcg catgttccag 1320

atctatcaga cgtccccaga tggtgaccct gttgctgagt ctgtctggga gctggactgc 1380

gagggggaga tttctttaga caaaataaca cctttcaccc cgaatggatt ccattaa 1437

Sequence table two

＜110〉applicant

＜120〉dopamine receptor new sub-type D 5 B gene and encoded protein and application

<160>2

<210>1

<211>478

＜212〉amino acid

＜213〉people

<400>

Met Leu Pro Pro Arg Ser Asn Gly Thr Ala Tyr Pro Gly Gln Leu 15

Ala Leu Tyr Gln Gln Leu Ala Gln Gly Asn Ala Val Gly Gly Ser 30

Ala Gly Ala Pro Pro Leu Gly Pro Ser Gln Val Val Thr Ala Cys 45

Leu Leu Thr Leu Leu Ile Ile Trp Thr Leu Leu Gly Asn Val Leu 60

Val Cys Ala Ala Ile Val Arg Ser Arg His Leu Arg Ala Asn Met 75

Thr Asn Val Phe Ile Val Ser Leu Ala Val Ser Asp Leu Phe Val 90

Ala Leu Leu Val Met Pro Trp Lys Ala Val Ala Glu Val Ala Gly 105

Tyr Trp Pro Phe Gly Ala Phe Cys Asp Val Trp Val Ala Phe Asp 120

Ile Met Cys Ser Thr Ala Ser Ile Leu Asn Leu Cys Val Ile Ser 135

Val Asp Arg Tyr Trp Ala Ile Ser Arg Pro Phe Arg His Lys Arg 150

Lys Met Thr Gln Arg Met Ala Leu Val Met Val Gly Leu Ala Trp 165

Thr Leu Ser Ile Leu Ile Ser Phe Ile Pro Val Gln Leu Asn Trp 180

His Arg Asp Gln Ala Ala Ser Trp Gly Gly Leu Asp Leu Pro Asn 195

Asn Leu Ala Asn Trp Thr Leu Trp Glu Glu Asp Phe Trp Glu Pro 210

Asp Val Asn Ala Glu Asn Cys Asp Ser Ser Leu Asn Arg Thr Try 225

Ala Ile Ser Ser Ser Leu Ile Ser Phe Tyr Ile Pro Val Ala Ile 240

Met Ile Val Thr Tyr Thr Arg Ile Tyr Arg Ile Ala Gln Val Gln 255

Ile Arg Arg Ile Ser Ser Leu Glu Arg Ala Ala Glu His Ala Gln 270

Ser Cys Arg Ser Ser Ala Ala Cys Ala Pro Asp Thr Ser Leu Arg 285

Ala Ser Ile Lys Lys Glu Thr Lys Val Leu Lys Thr Leu Ser Val 300

Ile Met Gly Val Phe Val Cys Cys Trp Leu Pro Phe Phe Ile Leu 315

Asn Cys Met Val Pro Phe Cys Ser Gly His Pro Glu Gly Pro Pro 330

Ala Gly Phe Pro Cys Val Ser Glu Thr Thr Phe Asp Val Phe Val 345

Trp Phe Gly Trp Ala Asn Ser Ser Leu Asn Pro Val Ile Tyr Ala 360

Phe Asn Ala Asp Phe Gln Lys Val Phe Ala Gln Leu Leu Gly Cys 375

Ser His Phe Cys Ser Arg Thr Pro Val Glu Thr Val Asn Ile Ser 390

Asn Glu Leu Ile Ser Tyr Asn Gln Asp Met Val Phe His Lys Glu 405

Ile Ala Ala Ala Cys Ile His Met Met Pro Asn Ala Leu Pro Pro 420

Gly Asp Gln Glu Val Asp Asn Asp Glu Glu Glu Glu Ser Pro Phe 435

Asp Arg Met Phe Gln Ile Tyr Gln Thr Ser Pro Asp Gly Asp Pro 450

Val Ala Glu Ser Val Trp Glu Leu Asp Cys Glu Gly Glu Ile Ser 465

Leu Asp Lys Ile Thr Pro Phe Thr Pro Asn Gly Phe His 478

Claims (5)

1. the gene of Dopamine Receptors D5B is one of following nucleotide sequences:

A) sequence table SEQ ID No.1;

B) polynucleotide of code sequence tabulation SEQ ID No.2 protein sequence.

2. the protein of Dopamine Receptors D5B is characterized in that, has the aminoacid sequence of sequence table SEQ ID No.2.

3. contain the described expression carrier of claim 1.

4. the transfectional cell series that contains the described gene of claim 1.

5. the application of the described gene of claim 1 in the preparation medicine.

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