CN108939048A - Nuclear receptor ROR α is preparing the application in heart failure resistance drug - Google Patents

Abstract

The present invention provides a kind of nuclear receptor ROR α to prepare the application in heart failure resistance drug, the nuclear receptor ROR α is 1 hypotype of nuclear receptor ROR α, the gene order of the nuclear receptor ROR α 1 is as shown in SEQ ID NO.1, and the amino acid sequence of the nuclear receptor ROR α 1 is as shown in SEQ ID NO.2.It is found through experiments that, nuclear receptor ROR α provided by the invention is expressed in plump and failure cardiac muscular tissue and lowered, and ROR α missing can aggravate myocardial hypertrophy and heart failure.Cardiac-specific ROR α is overexpressed the Myocardial Interstitial Fibrosis that can mitigate TAC induction, improves myocardial hypertrophy and and heart failure.Nuclear receptor ROR α of the invention has the function of resisting cardiac hypertrophy and heart failure.

Description

Nuclear receptor ROR α is preparing the application in heart failure resistance drug

Technical field

The invention belongs to biomedicine field more particularly to cardiovascular drugs, and specifically a kind of nuclear receptor ROR α exists Prepare the application in heart failure resistance drug.

Background technique

Heart failure is the serious and terminal stage of various heart diseases, and disease incidence is high, is current most important angiocarpy One of disease.The main reason for heart failure is industrialized country's crowd's death rate only just causes about in the whole world for 1 year 2012 108000000000 dollars of economic loss.Heart failure refer to tranquillization or stress under, since any cardiac structure or dysfunction are led The ventricular filling of cause penetrates the impaired a complex set of clinical syndrome of blood ability, and main clinical manifestation is expiratory dyspnea and weary Power (activity tolerance is limited) and fluid retention (pulmonary venous pleonaemia and periphery oedema).

One of main pathogenesis of heart failure is cardiomyopathy rational reconstruction, and myocardial hypertrophy is usually in pressure/volume Under the stimulation of excess load and Neurohormonal factor, cardiac muscle acts on a kind of adaptability myocardial remodelling of wall stress.Although rising First myocardial remodelling can play the role of compensatory, but the myocardial hypertrophy of this maladjustment has been considered to be a kind of promotion at present The strong effective predictive factor of the important independent hazard factor and the death rate of chronic heart failure disease progression.Myocardium fertilizer Thick pathological change includes that cardiac myocyte hypertrophy, myocardial interstitial cells proliferation and the reconstruct of heart cell epimatrix etc. are various Change.Show as that the synthesis of cardiac muscle cell's albumen increases, volume increases, diameter is broadening or length increases, muscle segment number in cellular level Amount increases and with proliferation of fibrous tissue.In the progress of myocardial hypertrophy, main molecular mechanism includes multiple protein phosphatase The activation of cascade signal access, the starting of embryonic gene expression system again, the cardiac damage of calcium-mediated and active oxygen Accumulation.The slow progress for aggravating that myocardial remodelling can be promoted of oxidative stress, and the excessive accumulation of active oxygen is considered as pathologic The important triggering factors of myocardial hypertrophy and heart failure.Therefore, it for myocardial hypertrophy and heart failure, finds new can effectively inhibit The recruit of myocardial hypertrophy and the pathologic, physiologic of failure progress may have important control especially with respect to the accumulation of active oxygen Treat value.

Nuclear receptor superfamily is the transcription factor of a kind of ligand-dependent, the development of mammal, metabolism, cell Proliferation, It plays a significant role in differentiation and numerous pathophysiological processes such as apoptosis and the important molecule of numerous U.S. FDAs certification drug Target spot.Retinoic acid related orphan nuclear receptor (Retinoid-related orphan receptors, RORs) is because it is in gene sequence It is similar to retinoic acid receptors and retinoid X receptor and gain the name on column.RORs is by ROR α, 3 hypotype compositions of ROR β and ROR γ. Wherein ROR α, also referred to as NRlF1 (Nuclear receptor subfamily 1, group F, member 1), are nuclear receptors Embryonic development, an important member of circadian activity and Metabolism regulation are participated in family.ROR α wide expression in heart, Many internal organs such as brain, skin, thymus gland, lung and kidney.Similar to other nuclear receptors, ROR α includes four structural domains, from amino end Hold carboxyl terminal to be followed successively by amino-terminal end structural domain, DNA binding domain (DNA binding domain, DBD), hinge area and Ligand binding domain (Ligand binding domain, LBD).ROR α is reacted by DBD with enhancer region AGGTCA half site Element, that is, ROR alpha reaction element (ROR α response element, RORE) leads in conjunction with come the transcriptional activity that regulates and controls target gene Sequence of the upstream rich in A/T is crossed to regulate and control the specificity of its DNA combination.There are two zinc fingers by DBD, are respectively used to identification groove Core motif and RORE 5 '-end at position are rich in the sequence of A/T, can effectively excite the phase interaction between ROR α and RORE With.LBD is an important multifunction structure domain, not only can with binding partner, participate in nuclear location, moreover it is possible to provide with co-activation because The site of son or co-suppression factor interaction, so that ROR α can generate diversified gene regulation in different histocytes Effect.ROR α and DNA combination when, also by the Reverse transcriptase of other nuclear receptors, such as REV-ERBA α and REV-ERB β Also can identify RORE, and by with ROR α competitive binding to RORE, thus the transcriptional activity for inhibiting ROR alpha mediated.

It can detecte the expression of nuclear receptor ROR α in vascular system, and be determined that it sends out in the pathologic, physiologic of blood vessel Wave important role.Our seminar research discovery ROR α can be used as one and resist the endogenous of myocardial ischemia-reperfusion injury before Property defense molecule.It in addition, there will be research shows that nuclear receptor ROR α, which is one, can regulate and control the important of a variety of antioxidant genes expression Transcription factor.A possibility that above-mentioned discovery prompt ROR α plays latent effect in heart failure.However, ROR α is in myocardium fertilizer The thick effect with failure is still unmanned so far to be reported.

There are 4 four ROR α 1, ROR α 2, ROR α 3 and ROR α hypotypes in people by ROR α, and wherein ROR α 1 is typically to have Representative hypotype is made of 523 amino acid residues.Its gene is located at No. 15 chromosomes, and site information is as follows:

Homo sapiens chromosome 15q22.2,GRCh38.p7(GCF_000001405.33),NC_ 000015.10(60488284…61229303,complement)。

Transcribed formation four transcripts, respectively transcriptional variants 1 (NM_134261) of the gene, 2 (NM_ of transcriptional variants 134260), transcriptional variants 3 (NM_002943), transcriptional variants 4 (NM_134262).Wherein transcriptional variants 1 (NM_134261) The gene order such as SEQ ID of (Retinoid-related orphan receptor α, transcript variant 1) Shown in NO.1.The albumen ammonia of representative hypotype ROR α 1 (Retinoid-related orphan receptor α isoform 1) Base acid sequence is as shown in SEQ ID NO.2.

Summary of the invention

The purpose of the present invention is to provide a kind of nuclear receptor ROR α to prepare the application in heart failure resistance drug, described This nuclear receptor ROR α will solve to treat and prevent heart failure in the prior art in the application prepared in heart failure resistance drug The bad technical problem of effect of drugs.

The present invention provides a kind of nuclear receptor ROR α to prepare the application in heart failure resistance drug.

Further, the nuclear receptor ROR α is nuclear receptor ROR α 1.

Further, the gene order of the nuclear receptor ROR α 1 is as shown in SEQ ID NO.1.

Further, the amino acid sequence of the nuclear receptor ROR α 1 is as shown in SEQ ID NO.2.

The present invention provides a kind of nuclear receptor ROR α with potential treatment heart failure effect, this receptor can effectively change Kind myocardial hypertrophy and its caused heart failure.

It is experimentally confirmed, nuclear receptor ROR α is expressed in plump and failure cardiac muscular tissue and lowered.

It is experimentally confirmed, ROR α missing aggravates myocardial hypertrophy and heart failure.

It is experimentally confirmed, Cardiac-specific, which is overexpressed ROR α, can mitigate Myocardial Interstitial Fibrosis, improve myocardial hypertrophy And heart failure.

It is experimentally confirmed, discovery nuclear receptor ROR α, which mainly passes through, to be reduced oxidative stress performance resisting cardiac hypertrophy and decline The effect exhausted, and the effect mainly by manganese superoxide dismutase (Manganese superoxide dismutase, MnSOD) anti-oxidative stress mediated plays.

The present invention is compared with prior art, and technological progress is positive and apparent.A kind of inhibition heart provided by the invention The nuclear receptor of force failure: retinoic acid correlation orphan nuclear receptor-α (Retinoid-related orphan receptor- α, ROR α) Have the function of resisting cardiac hypertrophy and heart failure.

Detailed description of the invention

Fig. 1 is expression changing rule of the ROR α in plump and failure cardiac muscular tissue.

Fig. 2 is influence of the ROR α missing to myocardial hypertrophy and heart failure.

Fig. 3 is influence of the Cardiac-specific ROR α overexpression to myocardial hypertrophy and heart failure.

Fig. 4 is that ROR α mitigates myocardium oxidative stress.

Fig. 5 is the expression that nuclear receptor ROR α can raise manganese superoxide dismutase (MnSOD).

Specific embodiment

The following is specific embodiments of the present invention, and the embodiment described is rather than to limit this hair for describing the present invention It is bright.

Reagent used in the examples and experimental method are as follows:

Molecular biochemistry reagent: protease and inhibitors of phosphatases, beta -mercaptoethanol, lauryl sodium sulfate, Tris Alkali etc. is purchased from the U.S. Sigma, and BCA quantification kit, ROR Alpha antibodies are purchased from the U.S. Thermo Scientific, and 30% methene is double It is the U.S. Bio-Rad that acrylamide, which is purchased from, and Triethanolamine buffer (TBS), Tween-20, phosphate buffer (PBS) etc. are purchased from Raw work biology China, horseradish peroxidase chemical luminescence for liquid are purchased from the U.S. Millipore, and isoflurane is purchased from composite tablet State, ribonucleic acid Reverse Transcriptase kit, picodna amplification kit are purchased from Takara Japan, MnSOD antibody, GAPDH antibody Purchased from the U.S. Cell Signaling Technology, horseradish peroxidase antibody is purchased from the U.S. Jackson.

The foundation of 1 mouse cardiac muscle pathologic hypertrophy model of embodiment

10-12 week old mouse is put into anesthesia tank, continues the isoflurane that inhaled concentration is 2% through Anesthesia machine and is anaesthetized (Ventilation Rate 2L/min).Trachea cannula connects ventilator and maintains breathing.Sterile surgery region takes front median incision to open chest to 2 ribs separate thymus gland, the exposure arch of aorta.Acupuncture treatment pad is padded with 27G to prick, and by aorta and pads acupuncture treatment one with 7-0 surgical nylon suture And ligature, chest, seam skin are successively closed in extraction pad acupuncture treatment after ligaturing really.After mouse autonomous respiration recovery, trachea cannula is extracted, Mouse is placed on heated at constant temperature blanket until it revives from anesthesia.Sham-operation group is ligatured without the arch of aorta, remaining process It is completely the same with operation group.

Bend different time points (1 week, 2 weeks, 4 weeks, 8 weeks) afterwards Constriction (TAC) in C57BL/6J mouse row aorta, uses The method of immunoblotting and real-time fluorescence quantitative PCR detects ROR α in transcription and translation level, finds its expression with TAC Post surgery duration gradually lowers (Fig. 1).

Wherein Figure 1A is the representative immunoblotting picture of ROR α, and the result of sxemiquantitative is carried out to immunoblotting；Figure 1B For the real-time fluorescence quantitative PCR result histogram of ROR α.

Embodiment 2

The evaluation of toy cardiac ultrasonic:

Mouse with 2% isoflurane is continued into inhalation anesthesia and dorsal position is fixed on the ultrasonic examination platform of included heating function On, its pareordia mouse hair first is sloughed with depilatory cream, uses 770 diasonograph of VeVo and 30MHz of VisualSonics company High frequency probe acquires parasternal long axis section, apical four-chamber section, record electrocardiogram, measurement heart rate on the wall of the chest.In parasternal Mitral level obtains two-dimensional ultrasound tangent plane picture under left room short axis view bicuspid valve, measure left room shrink last internal diameter (LVIDs), Left ventricular end diastolic diameter (LVIDd), Left Ventricular Ejection Fraction (LVEF) and shortening score (LVFS).The instrument ginseng that each group mouse checks Number setting keeps identical, and carries out Image Acquisition and calculating by same people, and each measurement index chooses 4-5 different week aroused in interest The average value of phase.

The ROR α of C57BL/6J background lacks (ROR α^sg/sg) mouse be purchased from the laboratory U.S. Jackson.10-12 week old SPF Grade ROR α^sg/sgMouse and its wild type control mice (WT) assign to TAC group and sham-operation (Sham) group at random.After TAC 8 weeks into A series of coherent detections of row, find ROR α^sg/sgMouse cardiac myocytes cross section significantly increases, and cardiac weight dramatically increases, petty action Object Cardiac ultrasound prompts ROR α missing heart function to deteriorate aggravation (Fig. 2).

Wherein Fig. 2A is that the representative HE of each experimental group dyes picture；Fig. 2 B is quantifying for each group cardiomyocytes cross-sectional area As a result；Fig. 2 C is the statistical result of cardiac weight and weight ratio；Fig. 2 D is the left room shortening point of each experimental group cardiac ultrasonic measurement Number.

Embodiment 3

Mouse ROR α full length cDNA sequence is incorporated into mouse α-myosin heavy chain promoter downstream, to construct C57BL/ The Cardiac-specific ROR α of 6J background is overexpressed mouse (TG).SPF grades of ROR α-TG mouse of 10-12 week old and its control mice (NTG) TAC group and Sham group are assigned at random.Carry out a series of coherent detections within 8 weeks after TAC, discovery ROR α-TG mouse cardiac muscle is thin Born of the same parents cross section reduces, and cardiac weight significantly mitigates, and heart function is improved (Fig. 3).

Wherein Fig. 3 A is that the representative HE of each group dyes picture；Fig. 3 B is the quantitative knot of each group cardiomyocytes cross-sectional area Fruit；Fig. 3 C is the statistical result of cardiac weight and weight ratio；Fig. 3 D is the left room contracting of each experimental group toy cardiac ultrasonic measurement Short score.

Embodiment 4

DHE dyeing: superoxide level in fresh cardiac muscular tissue's frozen section is detected using dihydro second ingot (DHE) dyeing. With PBS dilution DHE concentrate (1mmol/L) to 10 μm of ol/L, it is kept in dark place spare.The Fresh Frozen cut is taken to be sliced, investment In PBS solution, washing 3 times are stood, then each 3min carefully wipes remaining moisture.Add about 40 μ l on each section sample DHE working solution, negative control group add 40 μ l of PBS.It is protected from light 37 DEG C of incubation 30min of constant temperature.DHE working solution is sucked, investment PBS is molten In liquid, washing 3 times, each 3min are stood.Then covered pays attention to that bubble is avoided to generate.It is aobvious in laser scanning co-focusing It takes pictures and stores under micro mirror.As a result prompt ROR α missing obviously increases hypertrophic cardiomyopathy tissue oxidizing stress level, and ROR α mistake Expression then can reduce oxidative stress (Fig. 4).

ELISA detect nitrotyrosine content: nitrotyrosine (NT) be protein oxidation stress product, indirectly reflect Oxidative stress.It is operated according to Abnova company nitrotyrosine ELISA kit specification, takes out in liquid nitrogen and save Cardiac muscular tissue, the protein lysate for preparing in right amount is added, is fully ground into homogenate with electric grinder.And it is measured with BCA method Each protein sample concentration.10 × sample diluent is diluted to 1 × working solution with ddH2O, cleaning solution is diluted by 1:40, then It is spare to be placed in room temperature.Compound concentration gradient standard items.It is loaded and reads the light absorption value that wavelength is 450nm in microplate reader.Foundation The concentration and absorption values of standard items make standard curve.The concentration of sample to be tested is calculated according to standard curve.As a result it shows Show that ROR α missing obviously increases hypertrophic cardiomyopathy oxidative stress, and ROR α overexpression then can reduce oxidative stress (Fig. 4).

Wherein Fig. 4 A is the DHE relative intensity of fluorescence quantitative result of ROR α missing and its control mice heart；Fig. 4 B is ROR The nitrotyrosine ELISA quantitative result of α missing and its control mice heart；Fig. 4 C is ROR α overexpression and its control mice heart Dirty DHE relative intensity of fluorescence quantitative result；Fig. 4 D is the nitrotyrosine ELISA of ROR α overexpression and its control mice heart Quantitative result.

Embodiment 5

After the completion of sample protein preparation, polyacrylamide concentration glue is added in 50 micrograms of protein samples with micro sample adding appliance and is added In sample hole.Using SDS-PAGE vertical electrophoresis.Wet process transferring film is then used, after transferring film, takes out nitrocellulose filter with de- Rouge milk room temperature is closed one hour.4 degree are incubated overnight primary antibody MnSOD and GAPDH.It is incubated within second day corresponding horseradish peroxide respectively After the secondary antibody of compound enzyme label, it is imaged on Chemiluminescence Apparatus.Gained picture is exported as into tiff format, and to target item Reinhoit Zahl with MnSOD and internal reference GAPDH does quantitative analysis.Experimental result prompt ROR α missing can reduce hypertrophic cardiomyopathy group The expression of MnSOD is knitted, and ROR α is overexpressed the protein expression level (Fig. 5) that can then restore MnSOD.

Wherein Fig. 5 A is the MnSOD representativeness western blot figure and MnSOD band of ROR α missing and its control mice heart Semi-quantitative results；Fig. 5 B is the MnSOD representativeness immunoblot results figure and mesh of ROR α overexpression and its control mice heart Band semi-quantitative results histogram.

Sequence table

<110>Renji Hospital Attached to Medical College of Shanghai Jiaotong Univ.

<120>nuclear receptor ROR α is preparing the application in heart failure resistance drug

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<170> SIPOSequenceListing 1.0

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<212> DNA

<213>nuclear receptor ROR alpha transcriptional variant 1 (Retinoid-related orphan receptor α)

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ggtaccatag agttgctctg aaaacagaag atagagggag tctcggagct cgccatctcc 60

agcgatctct acattgggaa aaaacatgga gtcagctccg gcagcccccg accccgccgc 120

cagcgagcca ggcagcagcg gcgcggacgc ggccgccggc tccagggaga ccccgctgaa 180

ccaggaatcc gcccgcaaga gcgagccgcc tgccccggtg cgcagacaga gctattccag 240

caccagcaga ggtatctcag taacgaagaa gacacataca tctcaaattg aaattattcc 300

atgcaagatc tgtggagaca aatcatcagg aatccattat ggtgtcatta catgtgaagg 360

ctgcaagggc tttttcagga gaagtcagca aagcaatgcc acctactcct gtcctcgtca 420

gaagaactgt ttgattgatc gaaccagtag aaaccgctgc caacactgtc gattacagaa 480

atgccttgcc gtagggatgt ctcgagatgc tgtaaaattt ggccgaatgt caaaaaagca 540

gagagacagc ttgtatgcag aagtacagaa acaccggatg cagcagcagc agcgcgacca 600

ccagcagcag cctggagagg ctgagccgct gacgcccacc tacaacatct cggccaacgg 660

gctgacggaa cttcacgacg acctcagtaa ctacattgac gggcacaccc ctgaggggag 720

taaggcagac tccgccgtca gcagcttcta cctggacata cagccttccc cagaccagtc 780

aggtcttgat atcaatggaa tcaaaccaga accaatatgt gactacacac cagcatcagg 840

cttctttccc tactgttcgt tcaccaacgg cgagacttcc ccaactgtgt ccatggcaga 900

attagaacac cttgcacaga atatatctaa atcgcatctg gaaacctgcc aatacttgag 960

agaagagctc cagcagataa cgtggcagac ctttttacag gaagaaattg agaactatca 1020

aaacaagcag cgggaggtga tgtggcaatt gtgtgccatc aaaattacag aagctataca 1080

gtatgtggtg gagtttgcca aacgcattga tggatttatg gaactgtgtc aaaatgatca 1140

aattgtgctt ctaaaagcag gttctctaga ggtggtgttt atcagaatgt gccgtgcctt 1200

tgactctcag aacaacaccg tgtactttga tgggaagtat gccagccccg acgtcttcaa 1260

atccttaggt tgtgaagact ttattagctt tgtgtttgaa tttggaaaga gtttatgttc 1320

tatgcacctg actgaagatg aaattgcatt attttctgca tttgtactga tgtcagcaga 1380

tcgctcatgg ctgcaagaaa aggtaaaaat tgaaaaactg caacagaaaa ttcagctagc 1440

tcttcaacac gtcctacaga agaatcaccg agaagatgga atactaacaa agttaatatg 1500

caaggtgtct acattaagag ccttatgtgg acgacataca gaaaagctaa tggcatttaa 1560

agcaatatac ccagacattg tgcgacttca ttttcctcca ttatacaagg agttgttcac 1620

ttcagaattt gagccagcaa tgcaaattga tgggtaaatg ttatcaccta agcacttcta 1680

gaatgtctga agtacaaaca tgaaaaacaa acaaaaaaat taaccgagac actttatatg 1740

gccctgcaca gacctggagc gccacacact gcacatcttt tggtgatcgg ggtcaggcaa 1800

aggaggggaa acaatgaaaa caaataaaag ttgaacttgt ttttctcatg catatgattt 1860

ccattatgcc tacagatatg gacccttttt ctgtcttgac ttcttgatca ttgacctctg 1920

tttacaacag gaggagggta ctaaagtcgg aggatttcct tttcttgtag ctcactgccc 1980

acagactttc tacagagtca ccaatctgtc agtaacaaca gagagtccag caataatcgg 2040

tgactggtgt gcatagcgga ggttgcggca ttactttgca caactagctc tttgtttcat 2100

gaaggaagtt tttatttttt caccgattat tgccagtccg caggatggca tgaaaagggt 2160

ccatagcagt agcaacaata gcattataat atattacagg gtaaatgggc atgaagacta 2220

tatatagcta aaagagatat tgtttatata ttgttttaag taatataaaa tgtagttact 2280

ggtgtagctt ttcctgttga attgataagg cactttcatt ttgcaccttt ttctttaaat 2340

taaatgctag cgtgttcact gtcgtgtcgc atgtgcacca gaaacacaag tttaactgag 2400

aaggcttgga aggtacgttg ggaggtattt atgctgctgt ttacaaaatt atttttaaga 2460

gactggctgg tcatatctag aaatcaccac gttggatttt ttttttaaca tgtgaatttg 2520

gaattagaaa cggaactctc cctaaattat actttgcttt ttggtaagtt taatgataga 2580

tgtgtttatg cttcatacaa agttgaatga ttgattggcg tggtggacat ataccatcat 2640

gctcattttt tttttttaaa gctttttaaa atgccacctc atggaggcga gggggaggag 2700

aagctcattt tacacaattc agtagttaaa tatggactcg gtctcaactt ggaattctta 2760

tgctttgaga acaaatcaac aaccagaata tttattggaa tctagctttt attataagaa 2820

ggacccaaag attatatcct gagcaaatgc acactcccca tgtgaggaca tgaagtattt 2880

actttgtgaa tgtttatgtt cttggtataa tctaggaacc ctatgagttt atctcagagt 2940

gaactaattc tagatttgtt gtcaatagat gctataattc aagaatgttg ctctccatat 3000

ttgaaaaacg atggatagga gggtgaggga agcatacaat gttgaaccag tttctcttat 3060

ttaaatatta aatactttaa gcctttaaag tgaagttgat gctagctgca aacatttact 3120

cttgtattta tcttcactag gaaactgtgg actgtaattt attttattaa atatttagaa 3180

gattatttgg cttgtgtgtt caggtgagaa atactgagtt gtttttgttt aatttcatgg 3240

tttttttttt tttaatgatc cctagtgggg gaaggggaaa ggaatagtct gataaacaga 3300

tgtgcatatt ttaaaaacaa gtgacctttt gggaatgtag gcatttagac gatgatttta 3360

gtcgcactag gggtgggatt caaactactg gtcaaagacc attttgtaca gaaaagggaa 3420

catctccgat gggtgttaag gtaggagttt ccatgcagcc ctttatgtct gagaaatagt 3480

ctcctctgcc attggggtcc ctgcggaatc ttctacagga attgcagctc ttcacgtcat 3540

gctaggttac cagcatgggc ttcccagagc acttcactct gtttctaact ccactgactt 3600

ttctgacttg tttcttgagg gacttggaaa aggggaaggt attattcaca cagatgtgtg 3660

tatgaagcct aaataacatc caactttttt tccaaaaaca tagtaagagt ttaaccataa 3720

atagaagagt aatattcctt taaatttctt acaggcatgt aaaattttat gtgtttatag 3780

agagatgcta actgtcagca tatagtattt atattggggc aagaagggtt aaatcaaatc 3840

tttaatttaa gtaagcatag ttcctttaaa gatcagtagt atttatactc tgaaaagagt 3900

accaagctta gttcagttat ttattcatcc attttttttc tattgtttct cctctggggg 3960

gaaatggtgt tttgtttttg tttgagtttt gtgttttagt tttttggttt tggttttgtt 4020

ttgttttttt gtcattcaga ttcactaaat ttggggatgg ttttattaaa gtatattaac 4080

ttctttttaa ccaaagcttt ctgaatatga ccagcctcag gtgctagcgc attaaagagc 4140

aactaaacct aattccagtg tcgatttgtg aaataataaa agctaattga atttctctaa 4200

gggtgagaga gaacataatg atgaacttaa aaagaaattc cttttcccag aagggattct 4260

gcatgtacct acaaacaatc atgctctaac cacagtaata gtcatgccat ggtgacattg 4320

cttttacggt aaaccaagat aggtaatatg atgcttcttc ccaggtgttt ctgaaagaaa 4380

agcagcggtg gagcttaaga gccaagtcca ctgattgaca taataggatg gaattgtaga 4440

cagagacatg ctccatgaaa caaggaaaca actgactact atttggatct aagttgggat 4500

ctgatgttaa acaataaatt cagtttaaaa aaaatatgga gctcagaaaa ggatgtgaaa 4560

aactttgcat tttcctttct tcattattac aaaaacacct atgtgatgac ataaaatact 4620

tgggtgatat cagaacaatt atccttaata gtttttataa ttaaagtttc ctaaacttca 4680

gtctccaata gtcttttaag gattggaacc acatcactgt cagccccgct gctacaatgc 4740

ctttgtacaa tttttttaca taaggcaaat aaggcttttg tacaaagcct gaataccttc 4800

tgttccaatg gtgtccaaat ggttattata ttgtgaaaaa cctggccttg gtcacaatgc 4860

aaacaaaagt acagatgaaa gtgctttttg gacagtttgc aaattgtgtt aaagctacgg 4920

atttttttta aagtgttcag catcttaacg tgtattaaag ctatggattt ttttttaaag 4980

tcttcagcat cctaattcac cctttctaac ttaagaaaaa catgacttaa gacactgctt 5040

ctaagtttgg ttgttcttta tagtgtggat acccagttat ctgtgagcgt atgggggtgg 5100

gctgagggtc aggtgaggaa ggagtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg 5160

tgatttgcat gtgtatgatg tgtgtgcgtc ggaccgcttc taggctacta agtgtcaatg 5220

gaaaagaaaa tgtattcaaa atacttaaat caaaactaga agatggggaa aaaaagattt 5280

attctataca aagccttgtc tggaccactt tagagagact tctatttttt taacccttct 5340

ataaatattt gatggcactt gaaatattcc tgcaataaaa tgtgatttgt gtaaagaaaa 5400

aaagattttg taatgtgaaa caaagaaaga aagtaatgta attttctaaa aaaaaaaata 5460

caaacaaaca aactttgtat tattttcttg atggaatttg tctatctgtc tttggaaaac 5520

tttttatttc attgaatgtg ccatagtaga aatgtgtgtt tttagtttta gactaaggaa 5580

tagctagttg ttgtgttccg acattccaaa atgcaaaaca acctagtagt atctttcatg 5640

aaaaggttta agtagtatgt aagcttctct cattgttgct tttttgcaca tgttcttcat 5700

tcctctctag tgcaatatgt acatagagca cttgcgggtg taccttgatc cctcagggaa 5760

aaatacatat ttgtacagtt ttttggggtt tttttgtttt ttggtttttt ttgttttgcc 5820

ttttgttttt ggctaaggaa tgtcgatcga atcacttgtt attgttgagg ggcagccaga 5880

taataatcct aaagccactg tttccaacat tgattgttta aatcatatgt ccttccaatg 5940

ctattatttt aagataataa taaaaagtta ttttctgaca gttctttgtg ctgactggtg 6000

aaaaacaagg gtaaataagc accttataat tgacttactg tgaatgacaa tccatcttgg 6060

tatcaacgat agaagcccta tcattttgga gttggggtta agagtcagaa acaatgtgct 6120

cagggatctc ctaaaactct taaaacaggg tggccagtac tactgggaca aattgtgttt 6180

tttattatta ataataatga taataatact atccctccaa ggcacaagtg aactatatag 6240

aactgcgtgt gtgtaaactc tttactctcg tctcattttg ttgagtttag aacttgatgt 6300

gctcgtcagt cttgtgtttc aaaacactga ataacttcca aagcaaagtt atgccagtgt 6360

gttcaaaaga taaattaata atgtaccagc aaagagcatc attcaaagta tagtccttgc 6420

atattccagt taccatttct ctaataatta aaatattcat gataaatata tatatagcat 6480

catatgttaa aaactatttc aaattctaca tattaaggat gaaaatttta aaatccagta 6540

ataagaggag agacctgcct atcagtacag tgatataggt aaaagatgaa atatgttttt 6600

aaaataccag caattaacta ttgttttcat gggttctcct ctagaagcaa accaaaaatt 6660

ccttcatgga aaacaattct tacttctaca tgtgtagttt atatctgatg cattacaaga 6720

gctaatgttt aaagacaaaa caaaacctgc ctgtatgaca gcagcaactc gagccaacat 6780

ttagtgttac atgttatatt tttgaaatac cgttttgtta tcatattcca catattactt 6840

tccataaagt cagagaagtt caatgtaatt gttggctctg attcttccac cttgggatac 6900

acattcacaa gaagatttat atattttctt actatgatag aggaacataa tctgggaaaa 6960

cttcccatgt ctgtaagatg aaaaggatac ctttaccatg ttgtttttga taataaagaa 7020

tatggaaaat ggtagaaatc tctcctctct atgtgtattt gtatatgtgt gtatacacac 7080

acacgttgac atttttacat aacatgtgat tgccacttct tataaagtta tgatatagaa 7140

ccccatgaaa acaactttat attatagatc aaataatgtg cccagaaacg caattgcaac 7200

agtaaatctt gatctattgg taagagtcta tgatacaggt cttcattcta tccctaaaca 7260

taatgttagt aaagaggttc catcagattg tattatagag accttcctat tgctatttat 7320

ttttaagaga tgagaagact gactagcaat gtctccacag tgcaattggt ttcacttctg 7380

ggtctgtctg tctgtttgtg taggtgagat cagtgttgtc aggctctcag aataattttt 7440

aaaagaatcc agaagcctga cttcacacca agtagccatc ctagatgggc gggggggatc 7500

tccatgttca accaaaccct cagaacttag agcaataaat acattcagtc atttatttat 7560

aaatgaatga cagaatattc aatccaaata gaaacaacct tttgtcagtg atgcaacata 7620

acatggagtc ttatattgag aaagtgaata tgaatatttt aaatagatgc ctaggaaatc 7680

tgtgtttgct gtttacattt aatgtacttt gccacattag cagtaccaca cttcttttac 7740

tttcatcctt ctaagaacta atgaaagata gcttgctatt agctttgaca tgttgcacat 7800

gccattatgt tgttctctaa gaacaactaa attctctcca gtgttcatgt gtgattcctt 7860

tttcatcatt attactttaa tgtggatgaa tactatttct aggaacttga ttatcactga 7920

acagattgca tatgtaaatg cagataattc ttgagcaata tagtgaaaat gatttcacaa 7980

aaaaaatcca tatgtactgt atacatcttc ctgaatccaa aattctcctg ttatccacac 8040

agtttgaaat ctcatattaa acagtggtaa atttttaaac attaggacat tagctggctg 8100

cttctataaa tgtacctgta ttgtctgcct gctccttttg tggagatgtg tttttgtatt 8160

ggatgtttgg gccaatggga ggcttcaagc tacaacatat tttcccactt taaataatat 8220

ttaacatatg acaaacccca gacaaggctt ttaatatgta taaagaactg cagtgttagg 8280

tggtttattt gcaaactgtt ttcagtgttg tccattttta tggcaccttt aacaatattc 8340

ttgtttccaa agtacaaaaa aaataggtta aataatctag ccataactga atgtcaagca 8400

ataaaacaaa tgacttttgt gcatagactt aaaaaataca aattttagac atctgcatgt 8460

aaatgcaatc tcttgtttac tgctttcttc aacttgagca atcgattcct tatttactat 8520

taacttaaga acttgtaacg gcctgtaaac attttctctc ttactcttct ttcaaattta 8580

cctgtccctg cttttgagtc ataatattta atgttgttct gcacatctct atacagttaa 8640

ctttttggct ttcattctgt atagataaga aaatgttata ttataaacag cctactcagt 8700

gcaaatattt atctgtttat caaatccaca atatgctgta taataccggt tttactatat 8760

aatctatttt agacatagct gtttagaact agagtgtgct atttttgtgt ttttctgatg 8820

tgtggtgcta gacaagttac ttttgtgaac aacaaaaatt atccctttta ttcctagaca 8880

ataccacctt tgggtcttgt taatttcact gagtataact atatatttgt atatatatac 8940

atatatatat atatctacct atgcccaact ggcagctgta tcagagtgct ggatttggga 9000

catgcttttc tctttaaata cataatatca ttatataaat tattctagag tgtatttaat 9060

taggataaaa ttacttcctt agtatggata tttgacatct atagggtgaa tttgtttata 9120

aatatggcta tatggaaact tattagcatt tactttatgt ttgctacttg gctttacagc 9180

atatctccta agctgaaaaa taatttgcca ggccttcaag atcctaaaga aacttgttta 9240

atggagtaat atactttttt ttcttattaa ggaattgtat tactggcacc taacacagtt 9300

gtattcttag ctcctattat agataatggg catttacata aaatatccta gatggcttga 9360

tggcagaata aacctttccc ctcctacctg agtcatgaga aggatggaga cgtcctctgc 9420

cataacatgg gccataaagc aaattcgaca tgggatgttc tgtttcagta tgacctcaac 9480

cagttccatg aactgagtga aggaccttca ttttcaaagt tatttaataa gtagcttaat 9540

taagcctttc tacccattct cccaagatct actggcatta ttgaaaagca aagtttatca 9600

aatatctaac taaggatgta gttaacctta ttaaatattg attagaattg ttctgtaata 9660

ttactgaatt tgtaagatct ttagcaaaga tttttgagca atttataaat gtagagcaaa 9720

tgtttctgtt tactgcactt tttgtaactg aaggtgataa attctcaagc catgattatt 9780

ggcttccatg cactgcaata tttatccaca attctagaca ttttccattt ttgtggaaga 9840

gttgctgtta ccttaattat aaatgcaatt gtgtggttaa tgagagctaa tgctagtagt 9900

taacctttta aagtggattg gctacagttg agggagaaat ctcttttaat ataaatcaca 9960

tcattcctta actgcctctc ttggaaagag attgaaacct tttttttaaa gcacgattta 10020

gcatcctaag cttcctgagg gtagagattg tatctttttg cgtctgcaca atggctagca 10080

catgtcagca tttgacaatt gttaaatgat aacaagtgtg ccccaattaa aacgtttttc 10140

ctgggttgtt ttgttaaatt tacaaagtaa gccaagcctt acggttaaca ttctcctcta 10200

caaccaagta ttaaagccac atttaaaaag accacatgaa atgctgattc taattgtgtg 10260

taggtcttga ggattaagca cacaaatttc acaaacttct gtttgagtaa acaaactcag 10320

ccttctgtaa atatacatgc aagtttggaa acagtaatac tgtacctata aatatatgct 10380

gtctgttttg tgtacagtat gtaaaaactc cttttctgcc acactaaaaa tgcaagccat 10440

ttatgggaat cctaaaacta gtattgaact aaaactttgc taatgatctt tattagagga 10500

tcgtccaact tttcacttac cttgggtttt cttttcaatt cactcttaca ctagtctgct 10560

tatttccagc tgtttatttt attgagtcct gaatttaaaa aaaaaatatt ttgattcatt 10620

ttgtaaatac aagctgtaca aaaaagagag atttaatgtt gtcttttaaa tactccaatt 10680

ttcattctaa tatgaatgtt gttatattgt acttagaaac tgtaccttta atattacatt 10740

acctttatta aaagtgcatt gaacacatca attttagatg tgctttatgt actgttatcc 10800

tataataaaa cttcagcttc taatggaa 10828

<210> 2

<211> 523

<212> PRT

<213>1 hypotype of ROR α (Retinoid-related orphan receptor α isoform 1)

<400> 2

Met Glu Ser Ala Pro Ala Ala Pro Asp Pro Ala Ala Ser Glu Pro Gly

1 5 10 15

Ser Ser Gly Ala Asp Ala Ala Ala Gly Ser Arg Glu Thr Pro Leu Asn

20 25 30

Gln Glu Ser Ala Arg Lys Ser Glu Pro Pro Ala Pro Val Arg Arg Gln

35 40 45

Ser Tyr Ser Ser Thr Ser Arg Gly Ile Ser Val Thr Lys Lys Thr His

50 55 60

Thr Ser Gln Ile Glu Ile Ile Pro Cys Lys Ile Cys Gly Asp Lys Ser

65 70 75 80

Ser Gly Ile His Tyr Gly Val Ile Thr Cys Glu Gly Cys Lys Gly Phe

85 90 95

Phe Arg Arg Ser Gln Gln Ser Asn Ala Thr Tyr Ser Cys Pro Arg Gln

100 105 110

Lys Asn Cys Leu Ile Asp Arg Thr Ser Arg Asn Arg Cys Gln His Cys

115 120 125

Arg Leu Gln Lys Cys Leu Ala Val Gly Met Ser Arg Asp Ala Val Lys

130 135 140

Phe Gly Arg Met Ser Lys Lys Gln Arg Asp Ser Leu Tyr Ala Glu Val

145 150 155 160

Gln Lys His Arg Met Gln Gln Gln Gln Arg Asp His Gln Gln Gln Pro

165 170 175

Gly Glu Ala Glu Pro Leu Thr Pro Thr Tyr Asn Ile Ser Ala Asn Gly

180 185 190

Leu Thr Glu Leu His Asp Asp Leu Ser Asn Tyr Ile Asp Gly His Thr

195 200 205

Pro Glu Gly Ser Lys Ala Asp Ser Ala Val Ser Ser Phe Tyr Leu Asp

210 215 220

Ile Gln Pro Ser Pro Asp Gln Ser Gly Leu Asp Ile Asn Gly Ile Lys

225 230 235 240

Pro Glu Pro Ile Cys Asp Tyr Thr Pro Ala Ser Gly Phe Phe Pro Tyr

245 250 255

Cys Ser Phe Thr Asn Gly Glu Thr Ser Pro Thr Val Ser Met Ala Glu

260 265 270

Leu Glu His Leu Ala Gln Asn Ile Ser Lys Ser His Leu Glu Thr Cys

275 280 285

Gln Tyr Leu Arg Glu Glu Leu Gln Gln Ile Thr Trp Gln Thr Phe Leu

290 295 300

Gln Glu Glu Ile Glu Asn Tyr Gln Asn Lys Gln Arg Glu Val Met Trp

305 310 315 320

Gln Leu Cys Ala Ile Lys Ile Thr Glu Ala Ile Gln Tyr Val Val Glu

325 330 335

Phe Ala Lys Arg Ile Asp Gly Phe Met Glu Leu Cys Gln Asn Asp Gln

340 345 350

Ile Val Leu Leu Lys Ala Gly Ser Leu Glu Val Val Phe Ile Arg Met

355 360 365

Cys Arg Ala Phe Asp Ser Gln Asn Asn Thr Val Tyr Phe Asp Gly Lys

370 375 380

Tyr Ala Ser Pro Asp Val Phe Lys Ser Leu Gly Cys Glu Asp Phe Ile

385 390 395 400

Ser Phe Val Phe Glu Phe Gly Lys Ser Leu Cys Ser Met His Leu Thr

405 410 415

Glu Asp Glu Ile Ala Leu Phe Ser Ala Phe Val Leu Met Ser Ala Asp

420 425 430

Arg Ser Trp Leu Gln Glu Lys Val Lys Ile Glu Lys Leu Gln Gln Lys

435 440 445

Ile Gln Leu Ala Leu Gln His Val Leu Gln Lys Asn His Arg Glu Asp

450 455 460

Gly Ile Leu Thr Lys Leu Ile Cys Lys Val Ser Thr Leu Arg Ala Leu

465 470 475 480

Cys Gly Arg His Thr Glu Lys Leu Met Ala Phe Lys Ala Ile Tyr Pro

485 490 495

Asp Ile Val Arg Leu His Phe Pro Pro Leu Tyr Lys Glu Leu Phe Thr

500 505 510

Ser Glu Phe Glu Pro Ala Met Gln Ile Asp Gly

515 520

Claims (4)

1. a kind of nuclear receptor ROR α is preparing the application in heart failure resistance drug.

2. application according to claim 1, it is characterised in that: the nuclear receptor ROR α is nuclear receptor ROR α 1.

3. application according to claim 2, it is characterised in that: the transcript sequence such as SEQ of the nuclear receptor ROR α 1 Shown in ID NO.1.

4. application according to claim 2, it is characterised in that: the amino acid sequence such as SEQ of the nuclear receptor ROR α 1 Shown in ID NO.2.