CN1303218C - Method of screening peroxisome proliferator-activated receptor antagon and agonist - Google Patents

Abstract

The present invention relates to a method by which a PPAR antagonist and an agonist are sieved by measuring the activity magnitude of alpha-galactosidase by a yeast double-hybrid system, which comprises the following steps that the plasmid of CBP and yeast GAL4AD fusion protein (pGADT7-CBP) is established; the plasmid of PPAR gamma LBD and yeast GAL4BD fusion protein (pGBKT7-PPAR gamma LBD) is established; the plasmid pGBKT7-PPAR gamma LBD and plasmid GADT7-CBP are orderly converted into a yeast cell AH 109; the compound is sieved by measuring the activity of the alpha-galactosidase.

Description

The screening method of peroxysome growth factor activated receptor antagonist and agonist

Technical field

The present invention relates to utilize the method for molecule and cytobiology technology screening PPAR antagonist and agonist, be specifically related to utilize yeast-two hybrid technique, by the active big or small screening PPAR antagonist of mensuration alpha-galactosidase and the method for agonist.

Background technology

In recent years, China's improving constantly along with the people's living standard quality, the variation of dietary structure and mode of life, the number of suffering from dysbolism of blood fat is raising year by year, the sickness rate of " affluence diseases " such as the essential hypertension of Yin Faing, hyperlipidaemia, coronary heart disease, diabetes, obesity also obviously rises therefrom, simultaneously, this class disease still threatens the healthy first killer of the elderly, has caused the great attention of the whole society.There is data to show, at present, the very close illnesss of dependency such as hyperlipidaemia, diabetes, obesity, its addition total incidence has reached 10%.China compares with the eighties at the initial stage nineties, and crowd's blood lipid level obviously increases, and especially in northern big city, about 30～40% people suffers from the blood fat and the blood sugar metabolic disturbance that surpass the marginality standard in various degree.Therefore, pay attention in the scope in the whole society, to control this class disease imperative.

Studies show that over past ten years, nuclear receptors PPAR's (Peroxisome Proliferator-activated Receptors) claim peroxysome growth factor activated receptor again, be that a class is by part activated nuclear factor, belong to II type nuclear receptor superfamily member, can be by lipid acid and exogenous agent for peroxisome proliferator (Peroxisome Proliferator, PP) activation.At PPAR s such as batrachians, rodent and the mankind 3 kinds of hypotypes are arranged all, i.e. PPAR α, PPAR β (also claiming PPAR δ or NUC 1) and PPAR γ.Human PPAR α, β and γ contain 468,441 and 479 amino-acid residues respectively.As one of steroid receptor superfamily member, PPAR has 6 zones (A-F), 4 functional domains.The C district that is positioned at the acceptor molecule middle part is DNA land (DBD); The E/F district of carboxyl terminal is ligand-binding domain (LBD), converts in the transcriptional activation signal process at hormone signal to play a significant role; N-terminal A/B is a regulatory region, the phosphorylation of a serine residue in the A/B district that the activation of PPAR γ can be mediated by MAPK and reducing, and the phosphorylation of PPAR α can just be regulated the avidity of receptor-ligand.Activatory PPAR and retinoid acceptor (RXR) form heterodimer, combine with the peroxisome proliferation response element (PPRE) of the promotor upstream of institute regulatory gene then and bring into play the transcriptional control effect.Simultaneously, activatory PPAR can be with a series of co-activation factor (as: CBP SRC-1 that transcribe; RIP140; TIF1; TIF2; SUGI; P300) combination, thereby activated transcription.The effect of PPAR mediation mainly comprises lipid metabolism, immune response etc., and therefore, development such as the generation of atherosclerosis, obesity, diabetes, tumour and diseases associated with inflammation etc. has substantial connection with PPAR.

The different subtype of PPAR has different biological actions, PPAR α plays a significant role in the liver lipid metabolism, the effect of PPAR β in the adipocyte differentiation also exists dispute, and PPAR γ participates in lipogenesis and immune response, in close relations with adipocyte differentiation, obesity and insulin resistant, be considered to treat the important target molecules of diabetes, obesity, bad lipidemia, inflammation, hypertension and cancer drug design, become the Recent study focus.

Research for the PPAR gamma agonist mainly concentrates on it in the application aspect the treatment type ii diabetes.Euglycemic agent thiazolidinediones medicine (TZDs) can obviously reduce the content of triglyceride level in the white adipose tissue (WAT) as the excitomotor of PPAR γ, also can reduce the glucose in the blood plasma simultaneously, the insulin level of lipid and type ii diabetes laboratory animal.Three class TZD class medicine: rosiglitazone rosiglitazone (Rezulin), Pi Gelie ketone piogitazone (ACTOS), passed through by the FDA authentication with troglitazone Troglitazone (Avandia), on market, sell as the medicine of treatment type ii diabetes.As for PPAR γ antagonist, can reduce fat, control obesity in theory, but clinical application as yet.

The domestic screening that on the high flux screening of PPAR gamma agonist and antagonist, mostly is confined to the micromolecular compound of protein level at present, as the SPR technology, the screening of cell levels then depends on mammalian cell substantially, and cost is higher, operates also loaded down with trivial details relatively.Yeast-two hybrid technique is technology (U.S. Patent No. 5,283,173 of adopting reporter gene to detect the protein-protein interphase interaction; Proc.Natl.Acad.Sci., USA, vol.88, pp.9578-9582,1991).Many transcription factors can be divided into DNA in conjunction with territory (DNA binding domain, DNA-BD) and transcription activating domain (transcriptional activation domain, AD), and have only under the situation of DNA-BD and these two structural domain coexistences of AD, could activate the expression of downstream gene.In the yeast two-hybrid system, albumin X to be detected, Y are expressed as fusion rotein DNA-BD-X, AD-Y with DNA-BD, AD respectively, only have under the interactional situation at X, Y, the mixture that the square one-tenth of DNA-BD, AD is complete activates the expression of downstream reporter gene.At present select the reporter gene of LacZ usually for use, yet beta-galactosidase enzymes is a kind of intracellular enzyme, so whether the cracking of yeast cell thoroughly can cause direct influence to the mensuration that enzyme is lived, and operate relative complex as yeast two-hybrid.

Summary of the invention

The object of the present invention is to provide a kind of novel method of utilizing yeast two-hybrid system to come screening PPAR antagonist and agonist.

Another object of the present invention provides a kind of by measuring the active size of alpha-galactosidase, comes simple and fast detection micromolecular compound to the PPAR method of incitant interphase interaction influence together.

A further object of the present invention provides the yeast screening assay system that is used for the inventive method screening PPAR antagonist and agonist.This system comprises: (1) mouse co-activation factor CBP and yeast transcription factor GAL4-AD fusion protein expression plasmid pGADT7-CBP (Escherichia E.coli DH5 α) preserving number is CGMCC No.1076, preservation date: on December 15th, 2003, address: BeiJing ZhongGuanCun China Committee for Culture Collection of Microorganisms common micro-organisms center, postcode: 100080; (2) contain people PPAR γ LBD and yeast transcription factor GAL4-BD fusion protein expression plasmid pGBKT7-PPAR γ LBD (Escherichia E.coli DH5 α) preserving number is CGMCC No.1077, preservation date: on December 15th, 2003, address: BeiJing ZhongGuanCun China Committee for Culture Collection of Microorganisms common micro-organisms center, postcode: 100080; (3) host's yeast AH109.

The present invention has adopted interactional technology between this favourable research protein-protein of yeast two-hybrid, PPARs together the combination of incitant be under the ligand dependent prerequisite, realized the screening of agonist and the antagonist of PPAR.The present invention adopts the GAL4 transcription factor, and its DNA-BD, AD zone then are structured in respectively among expression plasmid of yeast pGBKT7 and the pGADT7.The present invention adopts MEL1 gene that yeast AH109 genome the carries reporter gene as double cross, and the activity of the product alpha-galactosidase by measuring its secretor type detects the influence of micromolecular compound to PPAR γ and its co-activation factor interaction degree.

The present invention utilizes yeast two-hybrid system to come the method for screening PPAR antagonist and agonist to be made up of the following step:

1, the plasmid construction of CBP and yeast GAL4AD fusion rotein (pGADT7-CBP);

This step is by the cellular elements biological method, selects the plasmid (pGADT7-CBP) of pGADT7 vector construction CBP and yeast GAL4AD fusion rotein for use.

(1) from CMV-mCBP, obtain the dna fragmentation of CBP (1-464aa) with round pcr:

Primer: FW:5 ' AACATATGATGGCCGAGAACTTGCTGGACG 3 '

RV：5’AAGGATCCCTGTTGCCCTGCACCAACAG 3’

95 ℃ of sex change 8min, the 95 ℃ of 1min that begin to circulate, 65 ℃ of 1min, 72 ℃ of 1min30s repeat 30 circulations, 72 ℃ of 10min.

Adopt agarose gel electrophoresis to identify the PCR product, reclaim DNA.

(2) with the PCR product cloning of CBP (1-464aa) to the pGADT7 carrier: the PCR product of CBP (1-464aa) and pGADT7 cut with Nde I and BamH I enzyme respectively; Reclaim enzyme with test kit and cut product, wherein reclaim the PCR enzyme of CBP (1-464aa) and cut the nearly 1400bp of product, the pGADT7 enzyme is cut the nearly 8.0kb of product.Use T ₄Dna ligase connects; Connect product and be transformed into competent cell DH5 α; Cut evaluation by enzyme and select correct clone.

2, the plasmid construction of PPAR γ LBD and yeast GAL4BD fusion rotein (pGBKT7-PPAR γ LBD);

This step is by the cellular elements biological method, selects the plasmid (pGBKT7-PPAR γ LBD) of pGBKT7 vector construction PPAR γ LBD and yeast GAL4DNA-BD fusion rotein for use.

(1) utilize round pcr from pCDNA3.1-PPAR γ, to obtain the dna fragmentation of PPAR γ LBD (193aa-475aa):

Primer: FW:5 ' AACATATGGCGGAGATCTCCAGT 3 '

RV：5’AAGTCGACCTAGTACAAGTCCTT 3’

95 ℃ of sex change 5min, the 95 ℃ of 30s that begin to circulate, 60 ℃ of 30s, 72 ℃ of 1min repeat 30 circulations, 72 ℃ of 10min.

Adopt agarose gel electrophoresis to identify the PCR product, reclaim DNA.

(2) with the PCR product cloning of PPAR γ LBD to the pGBKT7 carrier: the PCR product of PPAR γ LBD and pGBKT7 cut with Nde I and Sal I enzyme respectively; Reclaim enzyme with test kit and cut product, wherein reclaim the PCR enzyme of PPAR γ LBD and cut the nearly 850bp of product, the pGBKT7 enzyme is cut the nearly 7.3kb of product; Use T ₄Dna ligase connects; Connect product and be transformed into competent cell DH5 α; Cut evaluation by enzyme and select correct clone.

3, change plasmid pGBKT7-PPAR γ LBD and pGADT7-CBP over to yeast cell AH109 successively, obtaining yeast PPAR γ LBD-CBP AH109 (Saccharomyces cerevisiae AH109) preserving number is CGMCC No.1075, preservation date: on December 15th, 2003, the address: BeiJing ZhongGuanCun China Committee for Culture Collection of Microorganisms common micro-organisms center, postcode: 100080:

Adopt the LiAc conversion method.Earlier change pGBKT7-PPAR γ LBD plasmid over to yeast cell AH109, with tryptophane defective (T ^-) the substratum screening positive clone, then the pGADT7-CBP plasmid is changed in the yeast that contains pGBKT7-PPAR γ LBD plasmid, use tryptophane, the two defective (T of leucine ^-L ^-) the substratum screening positive clone, contained the yeast cell of pGADT7-CBP plasmid and pGBKT7-PPAR γ LBD plasmid simultaneously.

4, by measuring the screening active ingredients compound of alpha-galactosidase:

In the positive yeast cell that contains pGADT7-CBP and the two plasmids of pGBKT7-PPAR γ LBD that obtains, add positive control, negative control thing and compound to be screened respectively, hatch the back and measure the activity of alpha-galactosidase, and calculate its exciting rate, according to each comparison for the treatment of the exciting rate of screening of medicaments, filter out PPAR antagonist and agonist.

The invention provides and utilize yeast two-hybrid system detection by quantitative micromolecular compound to protein interaction intensity effect method: adopt MEL1 gene that the yeast genes group the carries reporter gene as double cross, the activity of the product alpha-galactosidase by measuring its secretor type detects the influence of micromolecular compound to PPAR γ and its co-activation factor interaction degree.

The present invention utilizes the nucleotide sequence of the pGADT7-CBP plasmid that carrier pGADT7 makes up as follows:

TGCATGCCTGCAGGTCGAGATCCGGGATCGAAGAAATGATGGTAAATGAAATAGG

AAATCAAGGAGCATGAAGGCAAAAGACAAATATAAGGGTCGAACGAAAAATAAAGTG

AAAAGTGTTGATATGATGTATTTGGCTTTGCGGCGCCGAAAAAACGAGTTTACGCAATT

GCACAATCATGCTGACTCTGTGGCGGACCCGCGCTCTTGCCGGCCCGGCGATAACGCT

GGGCGTGAGGCTGTGCCCGGCGGAGTTTTTTGCGCCTGCATTTTCCAAGGTTTACCCTG

CGCTAAGGGGCGAGATTGGAGAAGCAATAAGAATGCCGGTTGGGGTTGCGATGATGAC

GACCACGACAACTGGTGTCATTATTTAAGTTGCCGAAAGAACCTGAGTGCATTTGCAA

CATGAGTATACTAGAAGAATGAGCCAAGACTTGCGAGACGCGAGTTTGCCGGTGGTGC

GAACAATAGAGCGACCATGACCTTGAAGGTGAGACGCGCATAACCGCTAGAGTACTTT

GAAGAGGAAACAGCAATAGGGTTGCTACCAGTATAAATAGACAGGTACATACAACACT

GGAAATGGTTGTCTGTTTGAGTACGCTTTCAATTCATTTGGGTGTGCACTTTATTATGTT

ACAATATGGAAGGGAACTTTACACTTCTCCTATGCACATATATTAATTAAAGTCCAATGC

TAGTAGAGAAGGGGGGTAACACCCCTCCGCGCTCTTTTCCGATTTTTTTCTAAACCGTG

GAATATTTCGGATATCCTTTTGTTGTTTCCGGGTGTACAATATGGACTTCCTCTTTTCTGG

CAACCAAACCCATACATCGGGATTCCTATAATACCTTCGTTGGTCTCCCTAACATGTAGG

TGGCGGAGGGGAGATATACAATAGAACAGATACCAGACAAGACATAATGGGCTAAACA

AGACTACACCAATTACACTGCCTCATTGATGGTGGTACATAACGAACTAATACTGTAGC

CCTAGACTTGATAGCCATCATCATATCGAAGTTTCACTACCCTTTTTCCATTTGCCATCTA

TTGAAGTAATAATAGGCGCATGCAACTTCTTTTCTTTTTTTTTCTTTTCTCTCTCCCCCGT

TGTTGTCTCACCATATCCGCAATGACAAAAAAAATGATGGAAGACACTAAAGGAAAAA

ATTAACGACAAAGACAGCACCAACAGATGTCGTTGTTCCAGAGCTGATGAGGGGTATC

TCGAAGCACACGAAACTTTTTCCTTCCTTCATTCACGCACACTACTCTCTAATGAGCAA

CGGTATACGGCCTTCCTTCCAGTTACTTGAATTTGAAATAAAAAAAAGTTTGCTGTCTT

GCTATCAAGTATAAATAGACCTGCAATTATTAATCTTTTGTTTCCTCGTCATTGTTCTCGT

TCCCTTTCTTCCTTGTTTCTTTTTCTGCACAATATTTCAAGCTATACCAAGCATACAATCA

ACTCCAAGCTTTGCAAAGATGGATAAAGCGGAATTAATTCCCGAGCCTCCAAAAAAGA

AGAGAAAGGTCGAATTGGGTACCGCCGCCAATTTTAATCAAAGTGGGAATATTGCTG

ATAGCTCATTGTCCTTCACTTTCACTAACAGTAGCAACGGTCCGAACCTCATAACA

ACTCAAACAAATTCTCAAGCGCTTTCACAACCAATTGCCTCCTCTAACGTTCATGA

TAACTTCATGAATAATGAAATCACGGCTAGTAAAATTGATGATGGTAATAATTCAAA

ACCACTGTCACCTGGTTGGACGGACCAAACTGCGTATAACGCGTTTGGAATCACT

ACAGGGATGTTTAATACCACTACAATGGATGATGTATATAACTATCTATTCGATGAT

GAAGATACCCCACCAAACCCAAAAAAAGAGATCTTTAATACGACTCACTATAGGGCG

AGCGCCGCCATGGAGTACCCATACGACGTACCAGATTACGCTCATATGatggccgagaacttgct

ggacggaccgcccaaccccaaacgagccaaactcagctcgcccggcttctccgcgaatgacaacacagattttggatcattgtttgac

ttggaaaatgaccttcctgatgagctgatccccaatggagaattaagccttttaaacagtgggaaccttgttccagatgctgcgtccaa

acataaacaactgtcagagcttcttagaggaggcagcggctctagcatcaacccagggataggcaatgtgagtgccagcagccctgt

gcaacagggccttggtggccaggctcaggggcagccgaacagtacaaacatggccagcttaggtgccatgggcaagagccctctga

accaaggagactcatcaacacccaacctgcccaaacaggcagccagcacctctgggcccactccccctgcctcccaagcactgaatc

cacaagcacaaaagcaagtagggctggtgaccagtagtcctgccacatcacagactggacctgggatctgcatgaatgctaacttca

accagacccacccaggccttctcaatagtaactctggccatagcttaatgaatcaggctcaacaagggcaagctcaagtcatgaatg

gatctcttggggctgctggaagaggaaggggagctggaatgccctaccctgctccagccatgcagggggccacaagcagtgtgctg

gcggagaccttgacacaggtttccccacaaatggctggccatgctggactaaatacagcacaggcaggaggcatgaccaagatggg

aatgactggtaccacaagtccatttggacaaccctttagtcaaactggagggcagcagatgggagccactggagtgaacccccagtt

agccagcaaacagagcatggtcaatagtttacctgcttttcctacagatatcaagaatacttcagtcaccactgtgccaaatatgtccc

agttgcaaacatcagtgggaattgtacccacacaagcaattgcaacaggccccacagcagaccctgaaaaacgcaaactgatacag

cagcagctggttctactgcttcatgcccacaaatgtcagagacgagagcaagcaaatggagaggttcgagcctgttctctcccacact

gtcgaaccatgaaaaacgttttgaatcacatgacacattgtcaggctcccaaagcctgccaagttgcccattgtgcatcttcacgacaa

atcatctctcattggaagaactgcacacgacatgactgtcctgtttgcctccctttgaaaaatgccagtgacaagcgaaaccaacaaa

ccatcctgggatctccagctagtggaattcaaaacacaattggttctgttggtgcagggcaacagGGATCCATCGAGCTC

GAGCTGCAGATGAATCGTAGATACTGAAAAACCCCGCAAGTTCACTTCAACTGTGCAT

CGTGCACCATCTCAATTTCTTTCATTTATACATCGTTTTGCCTTCTTTTATGTAACTATACT

CCTCTAAGTTTCAATCTTGGCCATGTAACCTCTGATCTATAGAATTTTTTAAATGACTAGA

ATTAATGCCCATCTTTTTTTTGGACCTAAATTCTTCATGAAAATATATTACGAGGGCTTAT

TCAGAAGCTTTGGACTTCTTCGCCAGAGGTTTGGTCAAGTCTCCAATCAAGGTTGTCG

GCTTGTCTACCTTGCCAGAAATTTACGAAAAGATGGAAAAGGGTCAAATCGTTGGTAG

ATACGTTGTTGACACTTCTAAATAAGCGAATTTCTTATGATTTATGATTTTTATTATTAAAT

AAGTTATAAAAAAAATAAGTGTATACAAATTTTAAAGTGACTCTTAGGTTTTAAAACGA

AAATTCTTATTCTTGAGTAACTCTTTCCTGTAGGTCAGGTTGCTTTCTCAGGTATAGCAT

GAGGTCGCTCTTATTGACCACACCTCTACCGGCCGGTCGAAATTCCCCTACCCTATGAA

CATATTCCATTTTGTAATTTCGTGTCGTTTCTATTATGAATTTCATTTATAAAGTTTATGTA

CAAATATCATAAAAAAAGAGAATCTTTTTAAGCAAGGATTTTCTTAACTTCTTCGGCGA

CAGCATCACCGACTTCGGTGGTACTGTTGGAACCACCTAAATCACCAGTTCTGATACCT

GCATCCAAAACCTTTTTAACTGCATCTTCAATGGCCTTACCTTCTTCAGGCAAGTTCAAT

GACAATTTCAACATCATTGCAGCAGACAAGATAGTGGCGATAGGGTTGACCTTATTCTT

TGGCAAATCTGGAGCAGAACCGTGGCATGGTTCGTACAAACCAAATGCGGTGTTCTTG

TCTGGCAAAGAGGCCAAGGACGCAGATGGCAACAAACCCAAGGAACCTGGGATAACG

GAGGCTTCATCGGAGATGATATCACCAAACATGTTGCTGGTGATTATAATACCATTTAGG

TGGGTTGGGTTCTTAACTAGGATCATGGCGGCAGAATCAATCAATTGATGTTGAACCTT

CAATGTAGGAAATTCGTTCTTGATGGTTTCCTCCACAGTTTTTCTCCATAATCTTGAAGA

GGCCAAAACATTAGCTTTATCCAAGGACCAAATAGGCAATGGTGGCTCATGTTGTAGGG

CCATGAAAGCGGCCATTCTTGTGATTCTTTGCACTTCTGGAACGGTGTATTGTTCACTAT

CCCAAGCGACACCATCACCATCGTCTTCCTTTCTCTTACCAAAGTAAATACCTCCCACTA

ATTCTCTGACAACAACGAAGTCAGTACCTTTAGCAAATTGTGGCTTGATTGGAGATAAG

TCTAAAAGAGAGTCGGATGCAAAGTTACATGGTCTTAAGTTGGCGTACAATTGAAGTTC

TTTACGGATTTTTAGTAAACCTTGTTCAGGTCTAACACTACCTGTACCCCATTTAGGACC

ACCCACAGCACCTAACAAAACGGCATCAACCTTCTTGGAGGCTTCCAGCGCCTCATCT

GGAAGTGGGACACCTGTAGCGTCGATAGCAGCACCACCAATTAAATGATTTTCGAAATC

GAACTTGACATTGGAACGAACATCAGAAATAGCTTTAAGAACCTTAATGGCTTCGGCTG

TGATTTCTTGACCAACGTGGTCACCTGGCAAAACGACGATCTTCTTAGGGGCAGACATT

AGAATGGTATATCCTTGAAATATATATATATATTGCTGAAATGTAAAAGGTAAGAAAAGTT

AGAAAGTAAGACGATTGCTAACCACCTATTGGAAAAAACAATAGGTCCTTAAATAATAT

TGTCAACTTCAAGTATTGTGATGCAAGCATTTAGTCATGAACGCTTCTCTATTCTATATG

AAAAGCCGGTTCCGGCGCTCTCACCTTTCCTTTTTCTCCCAATTTTTCAGTTGAAAAAG

GTATATGCGTCAGGCGACCTCTGAAATTAACAAAAAATTTCCAGTCATCGAATTTGATTC

TGTGCGATAGCGCCCCTGTGTGTTCTCGTTATGTTGAGGAAAAAAATAATGGTTGCTAA

GAGATTCGAACTCTTGCATCTTACGATACCTGAGTATTCCCACAGTTGGGGATCTCGAC

TCTAGCTAGAGGATCAATTCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTAT

CCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTG

CCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCG

GGAAACCTGTCGTGCCAGCTGATAACTTCGTATAATGTATGCTATACGAAGTTATTAGGT

CTGAAGAGGAGTTTACGTCCAGCCAAGCTAGCTTGGCTGCAGGTCGAGCGGCCGCGAT

CCGGAACCCTTAATATAACTTCGTATAATGTATGCTATACGAAGTTATCAGCTGCATTAAT

GAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTC

GCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCA

AAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAG

CAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCC

ATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCG

AAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGC

TCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAG

CGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTC

CAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGT

AACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCA

CTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTG

GTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGC

CAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGG

TAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAA

GAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTA

AGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAA

ATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATG

CTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTG

ACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTG

CAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCA

GCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTA

TTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTT

GTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGC

TCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGG

TTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTC

ATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCT

GTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTT

GCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTG

CTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAG

ATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCAC

CAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAG

GGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTAT

CAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATA

GGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTAT

CATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTCGCGCGTTTCG

GTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCT

GTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGG

GTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATA

ACGCATTTAAGCATAAACACGCACTATGCCGTTCTTCTCATGTATATATATATACAGGCAA

CACGCAGATATAGGTGCGACGTGAACAGTGAGCTGTATGTGCGCAGCTCGCGTTGCATT

TTCGGAAGCGCTCGTTTTCGGAAACGCTTTGAAGTTCCTATTCCGAAGTTCCTATTCTCT

AGCTAGAAAGTATAGGAACTTCAGAGCGCTTTTGAAAACCAAAAGCGCTCTGAAGAC

GCACTTTCAAAAAACCAAAAACGCACCGGACTGTAACGAGCTACTAAAATATTGCGAA

TACCGCTTCCACAAACATTGCTCAAAAGTATCTCTTTGCTATATATCTCTGTGCTATATCC

CTATATAACCTACCCATCCACCTTTCGCTCCTTGAACTTGCATCTAAACTCGACCTCTAC

ATTTTTTATGTTTATCTCTAGTATTACTCTTTAGACAAAAAAATTGTAGTAAGAACTATTC

ATAGAGTGAATCGAAAACAATACGAAAATGTAAACATTTCCTATACGTAGTATATAGAGA

CAAAATAGAAGAAACCGTTCATAATTTTCTGACCAATGAAGAATCATCAACGCTATCAC

TTTCTGTTCACAAAGTATGCGCAATCCACATCGGTATAGAATATAATCGGGGATGCCTTT

ATCTTGAAAAAATGCACCCGCAGCTTCGCTAGTAATCAGTAAACGCGGGAAGTGGAGT

CAGGCTTTTTTTATGGAAGAGAAAATAGACACCAAAGTAGCCTTCTTCTAACCTTAACG

GACCTACAGTGCAAAAAGTTATCAAGAGACTGCATTATAGAGCGCACAAAGGAGAAAA

AAAGTAATCTAAGATGCTTTGTTAGAAAAATAGCGCTCTCGGGATGCATTTTTGTAGAA

CAAAAAAGAAGTATAGATTCTTTGTTGGTAAAATAGCGCTCTCGCGTTGCATTTCTGTTC

TGTAAAAATGCAGCTCAGATTCTTTGTTTGAAAAATTAGCGCTCTCGCGTTGCATTTTTG

TTTTACAAAAATGAAGCACAGATTCTTCGTTGGTAAAATAGCGCTTTCGCGTTGCATTT

CTGTTCTGTAAAAATGCAGCTCAGATTCTTTGTTTGAAAAATTAGCGCTCTCGCGTTGC

ATTTTTGTTCTACAAAATGAAGCACAGATGCTTCGTTGCT

The aminoacid sequence of pGADT7-CBP plasmid expression is:

Met Asp Lys Ala Glu Leu Ile Pro Glu Pro Pro Lys Lys Lys Arg Lys Val Glu Leu Gly Thr Ala

Ala Asn Phe Asn Gln Ser Gly Asn Ile Ala Asp Ser Ser Leu Ser Phe Thr Phe Thr Asn Ser Ser

Asn Gly Pro Asn Leu Ile Thr Thr Gln Thr Asn Ser Gln Ala Leu Ser Gln Pro Ile Ala Ser Ser

Asn Val His Asp Asn Phe Met Asn Asn Glu Ile Thr Ala Ser Lys Ile Asp Asp Gly Asn Asn Ser

Lys Pro Leu Ser Pro Gly Trp Thr Asp Gln Thr Ala Tyr Asn Ala Phe Gly Ile Thr Thr Gly

Met Phe Asn Thr Thr Thr Met Asp Asp Val Tyr Asn Tyr Leu Phe Asp Asp Glu Asp Thr Pro

Pro Asn Pro Lys Lys Glu Ile Phe Asn Thr Thr His Tyr Arg Ala Ser Ala Ala Met Glu Tyr Pro Tyr

Asp Val Pro Asp Tyr Ala His Met Met Ala Glu Asn Leu Leu Asp Gly Pro Pro Asn Pro Lys Arg

Ala Lys Leu Ser Ser Pro Gly Phe Ser Ala Asn Asp Asn Thr Asp Phe Gly Ser Leu Phe Asp

Leu Glu Asn Asp Leu Pro Asp Glu Leu Ile Pro Asn Gly Glu Leu Ser Leu Leu Asn Ser Gly

Asn Leu Val Pro Asp Ala Ala Ser Lys His Lys Gln Leu Ser Glu Leu Leu Arg Gly Gly Ser Gly

Ser Ser Ile Asn Pro Gly Ile Gly Asn Val Ser Ala Ser Ser Pro Val Gln Gln Gly Leu Gly Gly

Gln Ala Gln Gly Gln Pro Asn Ser Thr Asn Met Ala Ser Leu Gly Ala Met Gly Lys Ser Pro

Leu Asn Gln Gly Asp Ser Ser Thr Pro Asn Leu Pro Lys Glh Ala Ala Ser Tnr Ser Gly Pro

Thr Pro Pro Ala Ser Gln Ala Leu Asn Pro Gln Ala Gln Lys Gln Val Gly Leu Val Thr Ser Ser

Pro Ala Thr Ser Gln Thr Gly Pro Gly Ile Cys Met Asn Ala Asn Phe Asn Gln Thr His Pro

Gly Leu Leu Asn Ser Asn Ser Gly His Ser Leu Met Asn Gln Ala Gln Gln Gly Gln Ala Gln

Val Met Asn Gly Ser Leu Gly Ala Ala Gly Arg Gly Arg Gly Ala Gly Met Pro Tyr Pro Ala

Pro Ala Met Gln Gly Ala Thr Ser Ser Val Leu Ala Glu Thr Leu Thr Gln Val Ser Pro Gln

Met Ala Gly His Ala Gly Leu Asn Thr Ala Gln Ala Gly Gly Met Thr Lys Met Gly Met Thr

Gly Thr Thr Ser Pro Phe Gly Gln Pro Phe Ser Gln Thr Gly Gly Gln Gln Met Gly Ala Thr

Gly Val Asn Pro Gln Leu Ala Ser Lys Gln Ser Met Val Asn Ser Leu Pro Ala Phe Pro Thr Asp

Ile Lys Asn Thr Ser Val Thr Thr Val Pro Asn Met Ser Gln Leu Gln Thr Ser Val Gly Ile Val

Pro Thr Gln Ala Ile Ala Thr Gly Pro Thr Ala Asp Pro Glu Lys Arg Lys Leu Ile Gln Gln Gln

Leu Val Leu Leu Leu His Ala His Lys Cys Gln Arg Arg Glu Gln Ala Asn Gly Glu Val Arg

Ala Cys Ser Leu Pro His Cys Arg Thr Met Lys Asn Val Leu Asn His Met Thr His Cys Gln

Ala Pro Lys Ala Cys Gln Val Ala His Cys Ala Ser Ser Arg Gln Ile Ile Ser His Trp Lys Asn

Cys Thr Arg His Asp Cys Pro Val Cys Leu Pro Leu Lys Asn Ala Ser Asp Lys Arg Asn Gln

Gln Thr Ile Leu Gly Ser Pro Ala Ser Gly Ile Gln Asn Thr Ile Gly Ser Val Gly Ala Gly Gln

Gln Gly Ser Ile Glu Leu Glu Leu Gln Met Asn Arg Arg Tyr***

The pGBKT7-PPAR γ LBD plasmid that the present invention utilizes carrier pGBKT7 to make up has following nucleotide sequence:

CGGTGCGGGCCTCTTCGCTATTACGCCAGATCCTTTTGTTGTTTCCGGGTGTACAA

TATGGACTTCCTCTTTTCTGGCAACCAAACCCATACATCGGGATTCCTATAATACCTTCG

TTGGTCTCCCTAACATGTAGGTGGCGGAGGGGAGATATACAATAGAACAGATACCAGAC

AAGACATAATGGGCTAAACAAGACTACACCAATTACACTGCCTCATTGATGGTGGTACA

TAACGAACTAATACTGTAGCCCTAGACTTGATAGCCATCATCATATCGAAGTTTCACTAC

CCTTTTTCCATTTGCCATCTATTGAAGTAATAATAGGCGCATGCAACTTCTTTTCTTTTTT

TTTCTTTTCTCTCTCCCCCGTTGTTGTCTCACCATATCCGCAATGACAAAAAAAATGATG

GAAGACACTAAAGGAAAAAATTAACGACAAAGACAGCACCAACAGATGTCGTTGTTC

CAGAGCTGATGAGGGGTATCTCGAAGCACACGAAACTTTTTCCTTCCTTCATTCACGCA

CACTACTCTCTAATGAGCAACGGTATACGGCCTTCCTTCCAGTTACTTGAATTTGAAATA

AAAAAAGTTTGCTGTCTTGCTATCAAGTATAAATAGACCTGCAATTATTAATCTTTTGTTT

CCTCGTCATTGTTCTCGTTCCCTTTCTTCCTTGTTTCTTTTTCTGCACAATATTTCAAGCT

ATACCAAGCATACAATCAACTCCAAGCTTGAAGCAAGCCTCCTGAAAGATGAAGCTAC

TGTCTTCTATCGAACAAGCATGCGATATTTGCCGACTTAAAAAGCTCAAGTGCTCC

AAAGAAAAACCGAAGTGCGCCAAGTGTCTGAAGAACAACTGGGAGTGTCGCTAC

TCTCCCAAAACCAAAAGGTCTCCGCTGACTAGGGCACATCTGACAGAAGTGGAAT

CAAGGCTAGAAAGACTGGAACAGCTATTTCTACTGATTTTTCCTCGAGAAGACCT

TGACATGATTTTGAAAATGGATTCTTTACAGGATATAAAAGCATTGTTAACAGGAT

TATTTGTACAAGATAATGTGAATAAAGATGCCGTCACAGATAGATTGGCTTCAGTG

GAGACTGATATGCCTCTAACATTGAGACAGCATAGAATAAGTGCGACATCATCATC

GGAAGAGAGTAGTAACAAAGGTCAAAGACAGTTGACTGTATCGCCGGAATTTGTAA

TACGACTCACTATAGGGCGAGCCGCCATCATGGAGGAGCAGAAGCTGATCTCAGAGGA

GGACCTGCATATGgcggagatctccagtgatatcgaccagctgaatccagagtccgctgacctccgggccctggcaaaaca

tttgtatgactcatacataaagtccttcccgctgaccaaagcaaaggcgagggcgatcttgacaggaaagacaacagacaaaatcaacc

attcgttatctatgacatgaattccttaatgatgggagaagataaaatcaagttcaaacacatcacccccctgcaggagcagagcaaa

gaggtggccatccgcatctttcagggctgccagtttcgctccgtggaggctgtgcaggagatcacagagtatgccaaaagcattcctg

gttttgtaaatcttgacttgaacgaccaagtaactctcctcaaatatggagtccacgagatcatttacacaatgctggcctccttgatga

ataaagatggggttctcatatccgagggccaaggcttcatgacaagggagtttctaaagagcctgcgaaagccttttggtgactttatg

gagcccaagtttgagtttgctgtgaagttcaatgcactggaattagatgacagcgacttggcaatatttattgctgtcattattctcagtg

gagaccgcccaggtttgctgaatgtgaagcccattgaagacattcaagacaacctgctacaagccctggagctccagctgaagctga

accaccctgagtcctcacagctgtttgccaagctgctccagaaaatgacagacctcagacagattgtcacggaacacgtgcagctact

gcaggtgatcaagaagacggagacagacatgagtcttcacccgctcctgcaggagatctacaaggacttgtactagGTCGACC

TGCAGCGGCCGCATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGT

TTTTTGCGCGCTTGCAGCCAAGCTAATTCCGGGCGAATTTCTTATGATTTATGATTTTTAT

TATTAAATAAGTTATAAAAAAAATAAGTGTATACAAATTTTAAAGTGACTCTTAGGTTTT

AAAACGAAAATTCTTATTCTTGAGTAACTCTTTCCTGTAGGTCAGGTTGCTTTCTCAGGT

ATAGCATGAGGTCGCTCTTATTGACCACACCTCTACCGGCATGCAAGCTTGGCGTAATC

ATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACG

AGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGGTAACTCACATTA

ATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTA

ATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCC

TCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACT

CAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTG

AGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTT

CCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGG

CGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGC

GCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGA

AGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCG

CTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCC

GGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAG

CCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAA

GTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGA

AGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGC

TGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCT

CAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCAC

GTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATT

AAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAACCTGAGGCTATGGCAGGG

CCTGCCGCCCCGACGTTGGCTGCGAGCCCTGGGCCTTCACCCGAACTTGGGGGGTGGG

GTGGGGAAAAGGAAGAAACGCGGGCGTATTGGCCCCAATGGGGTCTCGGTGGGGTAT

CGACAGAGTGCCAGCCCTGGGACCGAACCCCGCGTTTATGAACAAACGACCCAACAC

CGTGCGTTTTATTCTGTCTTTTTATTGCCGTCATAGCGCGGGTTCCTTCCGGTATTGTCTC

CTTCCGTGTTTCAGTTAGCCTCCCCCTAGGGTGGGCGAAGAACTCCAGCATGAGATCCC

CGCGCTGGAGGATCATCCAGCCGGCGTCCCGGAAAACGATTCCGAAGCCCAACCTTTC

ATAGAAGGCGGCGGTGGAATCGAAATCTCGTGATGGCAGGTTGGGCGTCGCTTGGTCG

GTTCATTTCGAACCCCAGAGTCCCGCTCAGAAGAACTCGTCAAGAAGGCGATAGAAGG

CGATGCGCTGCGAATCGGGAGCGGCGATACCGTAAAGCACGAGGAAGCGGTCAGCCC

ATTCGCCGCCAAGCTCTTCAGCAATATCACGGGTAGCCAACGCTATGTCCTGATAGCGG

TCCGCCACACCCAGCCGGCCACAGTCGATGAATCCAGAAAAGCGGCCATTTTCCACCA

TGATATTCGGCAAGCAGGCATCGTCATGGGTCACGACGAGATCCTCGCCGTCGGGCATG

CTCGCCTTGAGCCTGGCGAACAGTTCGGCTGGCGCGAGCCCCTGATGCTCTTCGTCCA

GATCATCCTGATCGACAAGACCGGCTTCCATCCGAGTACGTGCTCGCTCGATGCGATGT

TTCGCTTGGTGGTCGAATGGGCAGGTAGCCGGATCAAGCGTATGCAGCCGCCGCATTG

CATCAGCCATGATGGATACTTTCTCGGCAGGAGCAAGGTGAGATGACAGGAGATCCTG

CCCCGGCACTTCGCCCAATAGCAGCCAGTCCCTTCCCGCTTCAGTGACAACGTCGAGC

ACAGCTGCGCAAGGAACGCCCGTCGTGGCCAGCCACGATAGCCGCGCTGCCTCGTCTT

GCAGTTCATTCAGGGCACCGGACAGGTCGGTCTTGACAAAAAGAACCGGGCGCCCCT

GCGCTGACAGCCGGAACACGGCGGCATCAGAGCAGCCGATTGTCTGTTGTGCCCAGTC

ATAGCCGAATAGCCTCTCCACCCAAGCGGCCGGAGAACCTGCGTGCAATCCATCTTGTT

CAATCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAG

CGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCC

CCGAAAAGTGCCACCTGAACGAAGCATCTGTGCTTCATTTTGTAGAACAAAAATGCAA

CGCGAGAGCGCTAATTTTTCAAACAAAGAATCTGAGCTGCATTTTTACAGAACAGAAA

TGCAACGCGAAAGCGCTATTTTACCAACGAAGAATCTGTGCTTCATTTTTGTAAAACAA

AAATGCAACGCGAGAGCGCTAATTTTTCAAACAAAGAATCTGAGCTGCATTTTTACAG

AACAGAAATGCAACGCGAGAGCGCTATTTTACCAACAAAGAATCTATACTTCTTTTTTG

TTCTACAAAAATGCATCCCGAGAGCGCTATTTTTCTAACAAAGCATCTTAGATTACTTTT

TTTCTCCTTTGTGCGCTCTATAATGCAGTCTCTTGATAACTTTTTGCACTGTAGGTCCGTT

AAGGTTAGAAGAAGGCTACTTTGGTGTCTATTTTCTCTTCCATAAAAAAAGCCTGACTC

CACTTCCCGCGTTTACTGATTACTAGCGAAGCTGCGGGTGCATTTTTTCAAGATAAAGG

CATCCCCGATTATATTCTATACCGATGTGGATTGCGCATACTTTGTGAACAGAAAGTGAT

AGCGTTGATGATTCTTCATTGGTCAGAAAATTATGAACGGTTTCTTCTATTTTGTCTCTAT

ATACTACGTATAGGAAATGTTTACATTTTCGTATTGTTTTCGATTCACTCTATGAATAGTT

CTTACTACAATTTTTTTGTCTAAAGAGTAATACTAGAGATAAACATAAAAAATGTAGAGG

TCGAGTTTAGATGCAAGTTCAAGGAGCGAAAGGTGGATGGGTAGGTTATAATAGGGATAT

AGCACAGAGATATATAGCAAAGAGATACTTTTGAGCAATGTTTGTGGAAGCGGTATTCG

CAATATTTTAGTAGCTCGTTACAGTCCGGTGCGTTTTTGGTTTTTTGAAAGTGCGTCTTC

AGAGCGCTTTTGGTTTTCAAAAGCGCTCTGAAGTTCCTATACTTTCTAGAGAATAGGAA

CTTCGGAATAGGAACTTCAAAGCGTTTCCGAAAACGAGCGCTTCCGAAAATGCAACGC

GAGCTGCGCACATACAGCTCACTGTTCACGTCGCACCTATATCTGCGTGTTGCCTGTATA

TATATATACATGAGAAGAACGGCATAGTGCGTGTTTATGCTTAAATGCGTACTTATATGCG

TCTATTTATGTAGGATGAAAGGTAGTCTAGTACCTCCTGTGATATTATCCCATTCCATGCG

GGGTATCGTATGCTTCCTTCAGCACTACCCTTTAGCTGTTCTATATGCTGCCACTCCTCAA

TTGGATTAGTCTCATCCTTCAATGCTATCATTTCCTTTGATATTGGATCATATTAAGAAAC

CATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTCGC

GCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACA

GCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGT

GTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAG

TGCACCATAGATCAACGACATTACTATATATATAATATAGGAAGCATTTAATAGAACAGCA

TCGTAATATATGTGTACTTTGCAGTTATGACGCCAGATGGCAGTAGTGGAAGATATTCTT

TATTGAAAAATAGCTTGTCACCTTACGTACAATCTTGATCCGGAGCTTTTCTTTTTTTGC

CGATTAAGAATTAATTCGGTCGAAAAAAGAAAAGGAGAGGGCCAAGAGGGAGGGCAT

TGGTGACTATTGAGCACGTGAGTATACGTGATTAAGCACACAAAGGCAGCTTGGAGTAT

GTCTGTTATTAATTTCACAGGTAGTTCTGGTCCATTGGTGAAAGTTTGCGGCTTGCAGA

GCACAGAGGCCGCAGAATGTGCTCTAGATTCCGATGCTGACTTGCTGGGTATTATATGT

GTGCCCAATAGAAAGAGAACAATTGACCCGGTTATTGCAAGGAAAATTTCAAGTCTTG

TAAAAGCATATAAAAATAGTTCAGGCACTCCGAAATACTTGGTTGGCGTGTTTCGTAAT

CAACCTAAGGAGGATGTTTTGGCTCTGGTCAATGATTACGGCATTGATATCGTCCAACT

GCATGGAGATGAGTCGTGGCAAGAATACCAAGAGTTCCTCGGTTTGCCAGTTATTAAA

AGACTCGTATTTCCAAAAGACTGCAACATACTACTCAGTGCAGCTTCACAGAAACCTCA

TTCGTTTATTCCCTTGTTTGATTCAGAAGCAGGTGGGACAGGTGAACTTTTGGATTGGA

ACTCGATTTCTGACTGGGTTGGAAGGCAAGAGAGCCCCGAAAGCTTACATTTTATGTTA

GCTGGTGGACTGACGCCAGAAAATGTTGGTGATGCGCTTAGATTAAATGGCGTTATTGG

TGTTGATGTAAGCGGAGGTGTGGAGACAAATGGTGTAAAAGACTCTAACAAAATAGCA

AATTTCGTCAAAAATGCTAAGAAATAGGTTATTACTGAGTAGTATTTATTTAAGTATTGTT

TGTGCACTTGCCGATCTATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATAC

CGCATCAGGAAATTGTAAACGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAAT

CAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAAT

AGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAA

CGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACGT

GAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGA

ACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGA

GAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCG

GTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGT

CCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGAT

The aminoacid sequence of pGBKT7-PPAR γ LBD plasmid expression is as follows:

Met Lys Leu Leu Ser Ser Ile Glu Gln Ala Cys Asp Ile Cys Arg Leu Lys Lys

Leu Lys Cys Ser Lys Glu Lys Pro Lys Cys Ala Lys Cys Leu Lys Asn Asn Trp Glu Cys Arg

Tyr Ser Pro Lys Thr Lys Arg Ser Pro Leu Thr Arg Ala His Leu Thr Glu Val Glu Ser Arg

Leu Glu Arg Leu Glu Gln Leu Phe Leu Leu Ile Phe Pro Arg Glu Asp Leu Asp Met Ile Leu

Lys Met Asp Ser Leu Gln Asp Ile Lys Ala Leu Leu Thr Gly Leu Phe Val Gln Asp Asn Val

Asn Lys Asp Ala Val Thr Asp Arg Leu Ala Ser Val Glu Thr Asp Met Pro Leu Thr Leu Arg

Gln His Arg Ile Ser Ala Thr Ser Ser Ser Glu Glu Ser Ser Asn Lys Gly Gln Arg Gln Leu Thr

Val Ser Pro Glu Phe Val Ile Arg Leu Thr Ile Gly Arg Ala Ala Ile Met Glu Glu Gln Lys Leu Ile Ser

Glu Glu Asp Leu His Met Ala Glu Ile Ser Ser Asp Ile Asp Gln Leu Asn Pro Glu Ser Ala Asp

Leu Arg Ala Leu Ala Lys His Leu Tyr Asp Ser Tyr Ile Lys Ser Phe Pro Leu Thr Lys Ala Lys

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

Leu Met Met Gly Glu Asp Lys Ile Lys Phe Lys His Ile Thr Pro Leu Gln Glu Gln Ser Lys Glu

Val Ala Ile Arg Ile Phe Gln Gly Cys Gln Phe Arg Ser Val Glu Ala Val Gln Glu Ile Thr Glu

Tyr Ala Lys Ser Ile Pro Gly Phe Val Asn Leu Asp Leu Asn Asp Gln Val Thr Leu Leu Lys Tyr

Gly Val His Glu Ile Ile Tyr Thr Met Leu Ala Ser Leu Met Asn Lys Asp Gly Val Leu Ile Ser

Glu Gly Gln Gly Phe Met Thr Arg Glu Phe Leu Lys Ser Leu Arg Lys Pro Phe Gly Asp Phe

Met Glu Pro Lys Phe Glu Phe Ala Val Lys Phe Asn Ala Leu Glu Leu Asp Asp Ser Asp Leu

Ala Ile Phe Ile Ala Val Ile Ile Leu Ser Gly Asp Arg Pro Gly Leu Leu Asn Val Lys Pro Ile Glu

Asp Ile Gln Asp Asn Leu Leu Gln Ala Leu Glu Leu Gln Leu Lys Leu Asn His Pro Glu Ser

Ser Gln Leu Phe Ala Lys Leu Leu Gln Lys Met Tnr Asp Leu Arg Gln Ile Val Thr Glu His Val

Gln Leu Leu Gln Val Ile Lys Lys Thr Glu Thr Asp Met Ser Leu His Pro Leu Leu Gln Glu Ile

Tyr Lys Asp Leu Tyr***

In the nucleotide sequence of above-mentioned two plasmids: what the base of band underscore was represented is restriction enzyme site used when cloning this gene; The base sequence of overstriking is represented the sequence of fusion rotein; ATG and TGA also overstriking represent initiator codon and terminator codon; The base sequence of small letter overstriking represents to insert the sequence of gene; TagThe expression terminator codon.

In the expressed aminoacid sequence of above-mentioned two plasmids: what add that black aminoacid sequence represents is the aminoacid sequence of fusion rotein; Add black and what have that the aminoacid sequence of underscore represents is the aminoacid sequence that inserts gene; * * represents to stop.

Description of drawings

Fig. 1 is a pGADT7-CBP plasmid construction policy map.

Fig. 2 is that CBP (1-464aa) PCR product agarose electrophoresis is identified collection of illustrative plates, and the collection of illustrative plates left column is CBP (1-464aa) PCR product electrophoretic band, and this stripe size is 1400bp, and 100bp DNA marker is classified on the right side as.

Fig. 3 is pGADT7-CBP plasmid enzyme restriction qualification result figure, and wherein No. 0 is 1kb DNA Marker; No. 1-No. 9 for transforming the clone who chooses on the flat board.

Fig. 4 is pGADT7-CBP sequencing result figure.

Fig. 5 is a PGBKT7-PPAR γ LBD plasmid construction policy map.

Fig. 6 is that PPAR γ LBD (193aa-475aa) PCR product agarose electrophoresis is identified collection of illustrative plates, and the collection of illustrative plates left column is the DNA length mark, and PPAR γ LBD (193aa-475aa) PCR product electrophoretic band is classified on the right side as, and this stripe size is 850bp.

Fig. 7 is a PGBKT7-PPAR γ LBD plasmid enzyme restriction qualification result, and the collection of illustrative plates left column is for transforming the clone that chooses on the flat board No. 8, and the 1kb dna marker is classified on the right side as.

Fig. 8 is pGBKT7-PPAR γ LBD sequencing result figure.

Fig. 9 is that the PCR of plasmid pGBKT7-PPAR γ LBD conversion results identifies figure, and wherein 1 is the DNAladder of 100bp, and 2-4 is followed successively by 1-3 number clone of institute's picking, 5 positive contrasts (pGBKT7-PPAR γ LBD is a template).

Figure 10 is that the PCR of plasmid pGADT7-CBP conversion results identifies figure, and wherein 1 is the dna marker of 1kb, 2 negative contrasts (H2O is a template), and 3 positive contrasts (pGADT7-CBP is a template), 4-5 is 1-2 number clone of institute's picking.

Figure 11 is a rosiglitazone to CBP and the PPAR γ influence figure that interacts, and wherein 1 expression yeast strain P1 (containing plasmid pGBKT7-PPAR γ LBD), 2 expressions add yeast strain P1,4 expression yeast strain P1C2 (containing two plasmid pGBKT7-PPAR γ LBD and pGADT7-CBP), 5 expressions that yeast strain P1,3 expressions behind the methyl-sulphoxides add behind the rosiglitazones (being dissolved in methyl-sulphoxide) and add the yeast strain P1C2 behind the methyl-sulphoxides, the yeast strain P1C2 that 6 expressions add rosiglitazones (being dissolved in methyl-sulphoxide).

Figure 12 is the influence figure of drug treating time to detection sensitivity.

Figure 13 is rosiglitazone EC ₅₀Measurement result figure.

Figure 14 is screening compound figure as a result.

Beneficial effect:

1, utilize the present invention can carry out the high flux screening of PPAR γ antagonist and activator, the present invention adopts MEL1 gene that Yeast genome carries as the reporter gene of double cross, the activity of the product alpha-galactosidase by measuring its secreting type detects micromolecular compound to the impact of PPAR γ and its co-activation factor interaction degree, need not the cracking yeast cells, be a kind of fast very convenient and method accurately, the ease for operation of yeast cells is conducive to realize the high flux screening of eucaryon level in addition.

2, utilize the present invention, carried out known PPAR gamma agonist-Rosiglitazone EC in the yeast level first₅₀Mensuration.

3, utilize the present invention can realize the high flux screening of PPAR γ antagonist and activator, for the structural modification of medicine and the discovery of natural lead compound provide experiment basis.

Embodiment

The present invention is further elaborated below in conjunction with specific embodiment, but do not limit the present invention.

Among the following embodiment, used test materials and source thereof comprise:

(genotype is MATa to yeast strain AH109, trp1-901, leu2-3,112, ura3-52, his3-200, gal4, gal80 mouth ... LYS2::GAL1UAS-GAL1TATA-HIS3, MEL1UAS-MEL1TATA-MMEL1UAS-MEL1TATA-MEL1, GAL2UAS-GAL2TATA-ADE2, URA3::MEL1UAS-MEL1TATA-lacZ), expression plasmid of yeast pGBKT7 and pGADT7 be that Shanghai life science professor Gong Yi of institute of the Chinese Academy of Sciences is so kind as to give;

Template CMV-mCBP is provided by M.G.Rosenfeld;

Template pCDNA3.1-PPAR γ is provided by ground, Chengdu company difficult to understand;

Restriction enzyme, archaeal dna polymerase, ligase enzyme, dNTP etc. are all available from TaKaRa company;

10 * buffer is with pyrobest ^TMArchaeal dna polymerase is together available from TaKaRa company;

Glue reclaims test kit available from Hua Shun company;

Ampicillin Trihydrate, kantlex, LiAc, PEG 3500, PNP-α-Gal, each culture medium preparation raw material are all available from sigma company;

SS-carrier DNA is available from Strategene;

DNA marker is available from ancient cooking vessel state biotech firm;

Primer is synthetic to be finished by Shanghai Bo Ya company with dna sequencing;

96 orifice plates are available from Nunclon company.

Genetic manipulation conventional among the following embodiment is with reference to relevant document, and wherein the Restriction Enzyme blanking method is with reference to molecular cloning second edition: P259-P262; Use T ₄The dna ligase method of attachment is with reference to molecular cloning second edition: P282-P283; Connect product and be transformed into competent cell DH5 α method with reference to molecular cloning second edition: P55-P56; Cut authentication method with reference to molecular cloning second edition: P259-P262 with enzyme; Agarose gel electrophoresis identifies that PCR product method is with reference to molecular cloning second edition: P680-P682; Glue reclaims the test kit specification sheets according to Hua Shun company; The plasmid extracting method is with reference to C.Hoffman andF.Winston, Gene 57:267; 1987; LB culture medium preparation method is with reference to molecular cloning second edition: P908; Each culture medium preparation method of yeast is with reference to Yeast Protocol Handbook (CLONTECH).

The plasmid construction of embodiment 1 CBP and yeast GAL4AD fusion rotein (pGADT7-CBP):

(1) test method:

1) with CMV-mCBP be template, duplicate the dna fragmentation (gene order is according to S66385 (GENEBANK)) that amplification obtains CBP (1-464aa) with round pcr:

Design of primers: FW:5 ' AACATATGATGGCCGAGAACTTGCTGGACG 3 '

RV：5’AAGGATCCCTGTTGCCCTGCACCAACAG 3’

Pcr amplification reaction (100 μ l):

CMV-mCBP template: 2 μ l; 10 * buffer:10 μ l; DNTP (2.5mM): 8 μ l; FW:4 μ l; RV:4 μ l; Pyrobest ^TMArchaeal dna polymerase: 1 μ l; Water: 71 μ l.

Reaction conditions is 95 ℃ of sex change 8min, the 95 ℃ of 1min that begin to circulate, and 65 ℃ of 1min, 72 ℃ of 1min30s repeat 30 circulations, 72 ℃ of 10min.

Adopt agarose gel electrophoresis to identify the PCR product, reclaim DNA.

2) with the PCR product cloning of CBP (1-464aa) to the pGADT7 carrier

The PCR product of CBP (1-464aa) and pGADT7 cut with Nde I and BamH I enzyme respectively; Reclaim enzyme with kit method and cut product, wherein reclaim the PCR enzyme of CBP (1-464aa) and cut the nearly 1400bp of product; The pGADT7 enzyme is cut the nearly 8.0kb of product; Use T ₄Dna ligase connects; Connect product and be transformed into competent cell DH5 α; Be coated on the flat board that contains Ampicillin Trihydrate (100mg/l); Cut evaluation with enzyme and select correct clone.

(2) test-results:

1, employing agarose gel electrophoresis evaluation PCR product the results are shown in Figure 2.

2, enzyme is cut qualification result: 1480bp on carrier, there is the restriction enzyme site of two Hind III at the 2280bp place, exist multiple clone site between 1480bp-2280bp, CBP just is cloned in this zone, thereby correct clone should cut out the fragment about about 2200bp and 7.2kb.As shown in Figure 3, cut qualification result through further enzyme and show that correct clone (No. 5 and No. 9) is arranged really.

3, dna sequencing report: pGADT7-CBP expression vector sequencer address as shown in Figure 4, the former dna sequence dna of pGADT7-CBP dna sequencing result and CBP (1-464aa) relatively, former sequence 1131 places are unknown base N, that measure at this place for No. 5 is A, but this position is the 3rd of codon, so no matter be AGCT which kind of, do not influence the amino acid coding.

The structure of embodiment 2 PPAR γ LBD and yeast GAL4BD fusion rotein (pGBKT7-PPAR γ LBD):

(1) test method:

With pCDNA3.1-PPAR γ is template, duplicates the dna fragmentation (gene order is according to NM005037 (GENEBANK)) that amplification obtains PPAR γ LBD (193aa-475aa) with round pcr:

Design of primers: FW:5 ' AACATATGGCGGAGATCTCCAGT 3 '

RV：5’AAGTCGACCTAGTACAAGTCCTT 3’

Pcr amplification reaction (100 μ l):

PCDNA3.1-PPAR γ template: 2 μ l; 10 * buffer:10 μ l; DNTP (2.5mM): 8 μ l; FW:4 μ l; RV:4 μ l; Pyrobest polysaccharase: 1 μ l; Water: 71 μ l.

Reaction conditions is 95 ℃ of sex change 5min, the 95 ℃ of 30s that begin to circulate, and 60 ℃ of 30s, 72 ℃ of 1min repeat 30 circulations, 72 ℃ of 10min.

Adopt agarose gel electrophoresis to identify the PCR product, reclaim DNA.

2) with the PCR product cloning of PPAR γ LBD to the pGBKT7 carrier:

The PCR product of PPAR γ LBD and pGBKT7 cut with Nde I and Sal I enzyme respectively; Reclaim enzyme with kit method and cut product, wherein reclaim the PCR enzyme of PPAR γ LBD and cut the nearly 850bp of product; The pGBKT7 enzyme is cut the nearly 7.3kb of product; Use T ₄Dna ligase connects; Connect product and be transformed into competent cell DH5 α; Be coated on the flat board that contains kantlex (50mg/l) and cut evaluation, select correct clone with enzyme.

(2) test-results:

1, employing agarose gel electrophoresis evaluation PCR product the results are shown in Figure 6.

2, enzyme is cut qualification result: there is the restriction enzyme site of an EcoRV at the 6313bp place on carrier, and also there is the restriction enzyme site of an EcoRV at the inner 16bp of PPAR γ LBD gene place, thereby correct clone should cut out the fragment about about 2200bp and 6kb.As shown in Figure 7, cut qualification result through further enzyme and show that correct clone (No. 8) is arranged really.

3, dna sequencing report: pGADT7-CBP expression vector sequencer address as shown in Figure 8, the former dna sequence dna of PPAR γ LBDDNA sequencing result and PPAR γ LBD relatively, the result is in full accord.

Embodiment 3 changes pGADT7-CBP and pGBKT7-PPAR γ LBD among the yeast cell AH109 step by step

(1) test method

1) change plasmid pGBKT7-PPAR γ LBD over to yeast cell AH109 by the LiAC method:

Inoculation yeast strains A H109 is in about 3ml YPDA, and 30 ℃, 250rpm are cultivated 16-18h (OD600 is about 1.4);

Bacterium liquid was transferred in fresh YPDA by 1: 5, and 30 ℃, 200rpm are cultivated 5-7h (OD600 to 0.8～1.2);

Get 10ml bacterium liquid, the centrifugal collection yeast cell of 4000rpm*5min;

Abandon supernatant, the 1ml sterilized water is resuspended, is transferred in another centrifuge tube, adds about 10ml sterilized water, mixing again;

The 4000rpm*5min recentrifuge is collected yeast cell;

Abandon supernatant, the 1ml sterilized water is resuspended, is transferred in the 1.5ml eppendorf pipe;

The 4000rpm*5min recentrifuge is collected yeast cell, and careful the suction removed supernatant;

Add successively: the SS-carrier DNA (10mg/ml) that 240 μ l PEG 3500 (50%W/V), 36 μ l 1.0M LiAc (pH7.5), 5 μ l boil, 60 μ l sterilized waters and 10 μ l plasmid to be transformed.

Thermal agitation 1min;

30 ℃ leave standstill 30min;

30min is left standstill in 42 ℃ of water-baths, puts upside down mixing once every 5min;

The centrifugal collection transformed yeast cells of 4000rpm*5min;

Supernatant is removed in suction, and the 1ml sterilized water is resuspended, gets 200 μ l and is applied to and coats the tryptophane defective and (be designated hereinafter simply as T ^-) the selection flat board on, cultivate 3-4d for 30 ℃;

2) identify the positive colony that carries pGBKT7-PPAR γ LBD plasmid:

Above T ^-Among the clone who selects to grow on the flat board, picking 1-3 number to T ^-In the liquid nutrient medium, 30 ℃, 250rpm are cultivated 16-18h;

Collect yeast, extract plasmid, PCR identifies institute's upgrading grain:

Primer sequence: FW:5 ' GTAATACGACTCACTATAGGGCGA 3 '

RV：5’TCCGAGTCACTTTAAAATTTGTAT 3’

Pcr amplification reaction (20 μ l):

Template (institute's upgrading grain): 14 μ l; 10 * buffer:2 μ l; DNTP (2.5mM): 2 μ l;

FW：1μl；RV：1μl；taq DNA polymerase：0.5μl。

Reaction conditions is 95 ℃ of sex change 5min, and 95 ℃ of 45s begin to circulate; 50 ℃ of 45s; 72 ℃ of 1min repeat 35 circulations, 72 ℃ of sex change 10min.

3) PCR identifies the positive colony 1 that obtains having pGBKT7-PPAR γ LBD plasmid, and the result as shown in Figure 9.

4) plasmid pGADT7-CBP is transferred among above-mentioned No. 1 clone

Inoculating No. 1 clones in about 3ml T ^-In the liquid nutrient medium, 30 ℃, 250rpm are cultivated 16-18h (OD600 is about 1.4);

Bacterium liquid was transferred in fresh T by 1: 5 ^-In the liquid nutrient medium, 30 ℃, 200rpm are cultivated 5-7h (OD600 to 0.8～1.2):

Transform the clone No. 1 by the above step, finally coat tryptophane, the two defectives of leucine (are designated hereinafter simply as T ^-L ^-) flat board, cultivate 3-4d for 30 ℃;

5) PCR identifies whether plasmid pGADT7-CBP changes the clone over to No. 1

Picking T ^-L ^-Be cloned into T 1-3 on the flat board number ^-L ^-In the liquid nutrient medium, 30 ℃, 250rpm, upgrading grain (method is the same) behind the cultivation 16-18h, PCR identifies:

Primer sequence: FW:5 ' GTAATACGACTCACTATAGGGCGA 3 '

RV：5’AGATGGTGCACGATGCACAGTT 3’

Pcr amplification reaction (20 μ l):

Template (institute's upgrading grain): 14 μ l; 10 * buffer:2 μ l; DNTP (2.5mM): 2 μ l; FW:1 μ l; RV:1 μ l; Taq archaeal dna polymerase: 0.5 μ l.

Reaction conditions: 95 ℃ of sex change 5min, 95 ℃ of 45s begin to circulate; 50 ℃ of 45s; 72 ℃ of 1min repeat 35 circulations; 72 ℃ of sex change 10min

6) evaluation obtains having pGBKT7-PPAR γ LBD﹠amp; The positive colony 2 of two plasmids of pGADT7-CBP, the result as shown in figure 10.

Embodiment 4 SCREENED COMPOUND

1, rosiglitazone (positive compound) is to the interact evaluation of influence of CBP and PPAR γ:

1) clones (being designated hereinafter simply as P1) and have two plasmid pGBKT7-PPAR γ LBD﹠amp for No. 1 that will have pGBKT7-PPAR γ LBD plasmid; No. 2 clones (hereinafter to be referred as P1C2) of pGADT7-CBP line T respectively ^-, T ^-L ^-Flat board behind 30 ℃ of cultivation 3-4d, is got fresh clone (1-3 week), is inoculated in 2ml T respectively ^-, T ^-L ^-In the liquid nutrient medium, 30 ℃, 250rpm, incubated overnight 16-18h;

2) use T respectively ^-, T ^-L ^-Liquid nutrient medium dilution P1, the P1C2 bacterium (to OD600 about 0.3) that spends the night, by add at 1: 100 rosiglitazone (self-control is synthetic, is dissolved in methyl-sulphoxide (DMSO), 10mM) or DMSO, experiment grouping situation such as following table:

The experiment group number		1	2	3	4	5	6
								Strain name		P1	P1	P1	P1C2	P1C2	P1C2
Contained plasmid	pGBKT7-PPAR.γLBD	+	+	+	+	+	+
								pGADT7-CBP	-	-	-	+	+	+
	Institute adds medicine	Methyl-sulphoxide (DMSO)	-	+	+	-	+								+
Rosiglitazone (10 -4M)								-	-	+	-	-	+

* every group all establish three parts parallel.

Behind 30 ℃ of cultivation 6h, measure alpha-galactosidase activity (carrying out) according to Yeast Protocol Handbook (CLONTECH):

Mixing yeast culture liquid is got 200 μ l and is measured OD 600 (HITACHI, spectrophotometer U-2010);

Other gets 200 μ l bacterium liquid in 1.5ml eppendorf pipe, and 10,000rpm*5min is centrifugal;

Get 16 μ l supernatant (T in 96 orifice plates ^-, T ^-L ^-Liquid nutrient medium is used separately as blank), every hole adds 48 μ l analysis buffer (the 100mM PNP-a-Gal aqueous solution and 0.5M sodium-acetate, pH 4.5 mixed in 1: 2 by volume);

30 ℃ of lucifuges are hatched 30min;

Add 136 μ l 10X stop buffer (1M Na ₂CO ₃), termination reaction; Measure the OD value (BIO-RAD microplate reader MODEL 550) at 410nm place.

The calculation formula of alpha-galactosidase activity is:

Alpha-galactosidase activity [mIU/ (mlxcell)]=OD410 * Vf * 1,000/[(ε * b) * t * Vi * OD600]

Wherein, the t=reaction times (branch)

Final volume during the Vf=measured value (200ul)

Vi=adds the volume (16ul) of yeast culture liquid supernatant

Yeast is in the absorbancy at 600nm place in the OD600=yeast culture liquid

ε * b=is right-and nitrophenols is in the base of the molar absorptivity * cuvette at the 410nm place length of side (cm)

=10.5 (ml/ μ mol) (for 200 μ l bacterium liquid)

3) test-results, the alpha-galactosidase activity detected result of each test group as shown in figure 11

As seen from Figure 11, add the alpha-galactosidase activity that rosiglitazone can effectively improve experimental strain P1C2, as the DMSO of solvent enzyme being lived does not then have influence substantially; Detect and under the same terms the P1 bacterial strain is carried out alpha-galactosidase activity, the result is all negative.As seen, rosiglitazone is as a kind of known PPAR. gamma agonist, it has changed the conformation of PPAR. γ with combining of PPAR. γ, makes the PPAR. γ of ligand dependent be strengthened with combining of CBP, has finally promoted its product alpha-galactosidase of MEL1 genetic expression that GAL4 relies on.

2, the drug incubation time is to the influence of detection sensitivity:

Because still useless alpha-galactosidase comes the detection by quantitative pharmaceutical efficacy as reporter gene at present, so as to screening the relevant report of new lead compound, and the employing beta-galactosidase enzymes of bibliographical information is when carrying out drug screening as the reporter gene in the yeast two-hybrid, determining also and disunity of relevant administration timing of drug, do not wait the more unmanned relevant work of doing dependency between administration timing of drug and sensitivity from 4-5h to night incubation.

Meet P1C2 in T ^-L ^-In the liquid nutrient medium, 30 ℃, 250rpm overnight incubation based on detecting more delicately, are done in various degree dilution (according to the form below) with the T-L-liquid nutrient medium to the bacterium that spends the night, so that when surveying enzyme work, each organizes bacterium all can reach logarithmic growth mid-term.

The drug incubation time (h)	2	4	6	8	10	12	14
								The OD600 of dilution back bacterium liquid	0.60	0.40	0.30	0.24	0.12	0.08	0.05

Added rosiglitazone (10mM) or DMSO by 1: 100, hatch for 30 ℃.(every 2h) sampling when hatching 2h, 4h, 6h, 8h, 10h, 12h, 14h according to test 1 described method, is measured each test group alpha-galactosidase activity.And calculate the alpha-galactosidase relative reactivity, its formula is:

The result as shown in figure 12, the time of prolong drug effect can be improved the sensitivity that this system detects, 12-14 hour incubation time can make native system sensitivity be enough to satisfy the needs of drug screening.

By above experiment, as can be seen, PPAR. the interaction of this ligand dependent sensitivity in above experimental system can be used for screening the natural compounds (agonist or antagonist) that can serve as PPAR. γ part stably by detection by quantitative and measurement between γ, CBP.

3, the mensuration of rosiglitazone (positive compound) EC50

Meet P1C2 in T ^-L ^-In the liquid nutrient medium, 30 ℃, 250rpm overnight incubation are used T ^-L ^-The liquid nutrient medium dilution is spent the night bacterium to OD600 about 0.08, with the rosiglitazone (10 that respectively adds different concns at 1: 100 ^-9M, 10 ^-8M, 10 ^-7M, 10 ^-6M, 10 ^-5M, 10 ^-410 ^-5M) or DMSO, 30 ℃ cultivate 12h after, according to test 1 described method, measure each test group alpha-galactosidase activity, and calculate the alpha-galactosidase relative reactivity.The result as shown in figure 13, rosiglitazone is a concentration dependent to the promoter action of CBP and PPAR γ interphase interaction, and can to obtain its EC50 in this system in view of the above be 1.3E-6.Thus, rosiglitazone can be used as positive compound and weighs the activity of waiting to sieve medicine in native system.

4, drug screening

Meet P1C2 in the T-L-liquid nutrient medium, 30 ℃, 250rpm overnight incubation, after the dilution of T-L-liquid nutrient medium, by respectively adding rosiglitazone (10mM), DMSO and medicine to be sieved (10mM) at 1: 100,30 ℃ hatch 12h after, carry out alpha-galactosidase activity and measure, and calculate its exciting rate:

Medicine to be sieved comprises in this test: H001-H007: be computer medicine aided design gained, and screen through BIOCORE;

Z001-Z013: the Chinese medicine (primary extract or pure compound) that extracts for this laboratory; The result is as shown in figure 14: H007, Z007 have shown strong stirring effect, and H001, H003, Z001, Z002 have also shown stirring effect preferably; H002, Z004 have then shown strong retarding effect.Active strong compound can further be tested the drug effect on its cell or the animal level, and what activity was not strong then can carry out structural modification by computer aided design (CAD), to improve its activity.

Sequence table

(1) general information

(i) denomination of invention: the screening method of PPAR antagonist and agonist

(ii) sequence number: 4

(2) information of sequence SEQ NO.1: (dna sequence dna of pGADT7-CBP)

(i) sequence signature:

(A) length: 9337bp

(B) type: Nucleotide

(C) chain: two strands

(D) topological framework: ring-type

(ii) molecule type: DNA

(iii) sequence description: SEQ NO.1

TGCATGCCTGCAGGTCGAGATCCGGGATCGAAGAAATGATGGTAAATGAAATAGGAAA

TCAAGGAGCATGAAGGCAAAAGACAAATATAAGGGTCGAACGAAAAATAAAGTGAAA

AGTGTTGATATGATGTATTTGGCTTTGCGGCGCCGAAAAAACGAGTTTACGCAATTGCA

CAATCATGCTGACTCTGTGGCGGACCCGCGCTCTTGCCGGCCCGGCGATAACGCTGGG

CGTGAGGCTGTGCCCGGCGGAGTTTTTTGCGCCTGCATTTTCCAAGGTTTACCCTGCGC

TAAGGGGCGAGATTGGAGAAGCAATAAGAATGCCGGTTGGGGTTGCGATGATGACGAC

CACGACAACTGGTGTCATTATTTAAGTTGCCGAAAGAACCTGAGTGCATTTGCAACATG

AGTATACTAGAAGAATGAGCCAAGACTTGCGAGACGCGAGTTTGCCGGTGGTGCGAAC

AATAGAGCGACCATGACCTTGAAGGTGAGACGCGCATAACCGCTAGAGTACTTTGAAG

AGGAAACAGCAATAGGGTTGCTACCAGTATAAATAGACAGGTACATACAACACTGGAA

ATGGTTGTCTGTTTGAGTACGCTTTCAATTCATTTGGGTGTGCACTTTATTATGTTACAAT

ATGGAAGGGAACTTTACACTTCTCCTATGCACATATATTAATTAAAGTCCAATGCTAGTA

GAGAAGGGGGGTAACACCCCTCCGCGCTCTTTTCCGATTTTTTTCTAAACCGTGGAATA

TTTCGGATATCCTTTTGTTGTTTCCGGGTGTACAATATGGACTTCCTCTTTTCTGGCAAC

CAAACCCATACATCGGGATTCCTATAATACCTTCGTTGGTCTCCCTAACATGTAGGTGGC

GGAGGGGAGATATACAATAGAACAGATACCAGACAAGACATAATGGGCTAAACAAGAC

TACACCAATTACACTGCCTCATTGATGGTGGTACATAACGAACTAATACTGTAGCCCTAG

ACTTGATAGCCATCATCATATCGAAGTTTCACTACCCTTTTTCCATTTGCCATCTATTGAA

GTAATAATAGGCGCATGCAACTTCTTTTCTTTTTTTTTCTTTTCTCTCTCCCCCGTTGTTG

TCTCACCATATCCGCAATGACAAAAAAAATGATGGAAGACACTAAAGGAAAAAATTAA

CGACAAAGACAGCACCAACAGATGTCGTTGTTCCAGAGCTGATGAGGGGTATCTCGAA

GCACACGAAACTTTTTCCTTCCTTCATTCACGCACACTACTCTCTAATGAGCAACGGTAT

ACGGCCTTCCTTCCAGTTACTTGAATTTGAAATAAAAAAAAGTTTGCTGTCTTGCTATC

AAGTATAAATAGACCTGCAATTATTAATCTTTTGTTTCCTCGTCATTGTTCTCGTTCCCTT

TCTTCCTTGTTTCTTTTTCTGCACAATATTTCAAGCTATACCAAGCATACAATCAACTCCA

AGCTTTGCAAAGATGGATAAAGCGGAATTAATTCCCGAGCCTCCAAAAAAGAAGAGA

AAGGTCGAATTGGGTACCGCCGCCAATTTTAATCAAAGTGGGAATATTGCTGATAGC

TCATTGTCCTTCACTTTCACTAACAGTAGCAACGGTCCGAACCTCATAACAACTCA

AACAAATTCTCAAGCGCTTTCACAACCAATTGCCTCCTCTAACGTTCATGATAACT

TCATGAATAATGAAATCACGGCTAGTAAAATTGATGATGGTAATAATTCAAAACCA

CTGTCACCTGGTTGGACGGACCAAACTGCGTATAACGCGTTTGGAATCACTACAG

GGATGTTTAATACCACTACAATGGATGATGTATATAACTATCTATTCGATGATGAAG

ATACCCCACCAAACCCAAAAAAAGAGATCTTTAATACGACTCACTATAGGGCGAGCG

CCGCCATGGAGTACCCATACGACGTACCAGATTACGCTCATATGatggccgagaacttgctggacgg

accgcccaaccccaaacgagccaaactcagctcgcccggcttctccgcgaatgacaacacagattttggatcattgtttgacttggaa

aatgaccttcctgatgagctgatccccaatggagaattaagccttttaaacagtgggaaccttgttccagatgctgcgtccaaacataa

acaactgtcagagcttcttagaggaggcagcggctctagcatcaacccagggataggcaatgtgagtgccagcagccctgtgcaac

agggccttggtggccaggctcaggggcagccgaacagtacaaacatggccagcttaggtgccatgggcaagagccctctgaaccaa

ggagactcatcaacacccaacctgcccaaacaggcagccagcacctctgggcccactccccctgcctcccaagcactgaatccacaa

gcacaaaagcaagtagggctggtgaccagtagtcctgccacatcacagactggacctgggatctgcatgaatgctaacttcaaccag

acccacccaggccttctcaatagtaactctggccatagcttaatgaatcaggctcaacaagggcaagctcaagtcatgaatggatctc

ttggggctgctggaagaggaaggggagctggaatgccctaccctgctccagccatgcagggggccacaagcagtgtgctggcggag

accttgacacaggtttccccacaaatggctggccatgctggactaaatacagcacaggcaggaggcatgaccaagatgggaatgac

tggtaccacaagtccatttggacaaccctttagtcaaactggagggcagcagatgggagccactggagtgaacccccagttagccag

caaacagagcatggtcaatagtttacctgcttttcctacagatatcaagaatacttcagtcaccactgtgccaaatatgtcccagttgca

aacatcagtgggaattgtacccacacaagcaattgcaacaggccccacagcagaccctgaaaaacgcaaactgatacagcagcag

ctggttctactgcttcatgcccacaaatgtcagagacgagagcaagcaaatggagaggttcgagcctgttctctcccacactgtcgaa

ccatgaaaaacgttttgaatcacatgacacattgtcaggctcccaaagcctgccaagttgcccattgtgcatcttcacgacaaatcatc

tctcattggaagaactgcacacgacatgactgtcctgtttgcctccctttgaaaaatgccagtgacaagcgaaaccaacaaaccatcc

tgggatctccagctagtggaattcaaaacacaattggttctgttggtgcagggcaacagGGATCCATCGAGCTCGAGC

TGCAGATGAATCGTAGATACTGAAAAACCCCGCAAGTTCACTTCAACTGTGCATCGTG

CACCATCTCAATTTCTTTCATTTATACATCGTTTTGCCTTCTTTTATGTAACTATACTCCTC

TAAGTTTCAATCTTGGCCATGTAACCTCTGATCTATAGAATTTTTTAAATGACTAGAATTA

ATGCCCATCTTTTTTTTGGACCTAAATTCTTCATGAAAATATATTACGAGGGCTTATTCAG

AAGCTTTGGACTTCTTCGCCAGAGGTTTGGTCAAGTCTCCAATCAAGGTTGTCGGCTTG

TCTACCTTGCCAGAAATTTACGAAAAGATGGAAAAGGGTCAAATCGTTGGTAGATACGT

TGTTGACACTTCTAAATAAGCGAATTTCTTATGATTTATGATTTTTATTATTAAATAAGTTA

TAAAAAAAATAAGTGTATACAAATTTTAAAGTGACTCTTAGGTTTTAAAACGAAAATTC

TTATTCTTGAGTAACTCTTTCCTGTAGGTCAGGTTGCTTTCTCAGGTATAGCATGAGGTC

GCTCTTATTGACCACACCTCTACCGGCCGGTCGAAATTCCCCTACCCTATGAACATATTC

CATTTTGTAATTTCGTGTCGTTTCTATTATGAATTTCATTTATAAAGTTTATGTACAAATAT

CATAAAAAAAGAGAATCTTTTTAAGCAAGGATTTTCTTAACTTCTTCGGCGACAGCATC

ACCGACTTCGGTGGTACTGTTGGAACCACCTAAATCACCAGTTCTGATACCTGCATCCA

AAACCTTTTTAACTGCATCTTCAATGGCCTTACCTTCTTCAGGCAAGTTCAATGACAATT

TCAACATCATTGCAGCAGACAAGATAGTGGCGATAGGGTTGACCTTATTCTTTGGCAAA

TCTGGAGCAGAACCGTGGCATGGTTCGTACAAACCAAATGCGGTGTTCTTGTCTGGCA

AAGAGGCCAAGGACGCAGATGGCAACAAACCCAAGGAACCTGGGATAACGGAGGCTT

CATCGGAGATGATATCACCAAACATGTTGCTGGTGATTATAATACCATTTAGGTGGGTTG

GGTTCTTAACTAGGATCATGGCGGCAGAATCAATCAATTGATGTTGAACCTTCAATGTA

GGAAATTCGTTCTTGATGGTTTCCTCCACAGTTTTTCTCCATAATCTTGAAGAGGCCAA

AACATTAGCTTTATCCAAGGACCAAATAGGCAATGGTGGCTCATGTTGTAGGGCCATGA

AAGCGGCCATTCTTGTGATTCTTTGCACTTCTGGAACGGTGTATTGTTCACTATCCCAAG

CGACACCATCACCATCGTCTTCCTTTCTCTTACCAAAGTAAATACCTCCCACTAATTCTC

TGACAACAACGAAGTCAGTACCTTTAGCAAATTGTGGCTTG ATTGGAGATAAGTCTAAA

AGAGAGTCGGATGCAAAGTTACATGGTCTTAAGTTGGCGTACAATTGAAGTTCTTTACG

GATTTTTAGTAAACCTTGTTCAGGTCTAACACTACCTGTACCCCATTTAGGACCACCCAC

AGCACCTAACAAAACGGCATCAACCTTCTTGGAGGCTTCCAGCGCCTCATCTGGAAGT

GGGACACCTGTAGCGTCGATAGCAGCACCACCAATTAAATGATTTTCGAAATCGAACTT

GACATTGGAACGAACATCAGAAATAGCTTTAAGAACCTTAATGGCTTCGGCTGTGATTT

CTTGACCAACGTGGTCACCTGGCAAAACGACGATCTTCTTAGGGGCAGACATTAGAAT

GGTATATCCTTGAAATATATATATATATTGCTGAAATGTAAAAGGTAAGAAAAGTTAGAA

AGTAAGACGATTGCTAACCACCTATTGGAAAAAACAATAGGTCCTTAAATAATATTGTC

AACTTCAAGTATTGTGATGCAAGCATTTAGTCATGAACGCTTCTCTATTCTATATGAAAA

GCCGGTTCCGGCGCTCTCACCTTTCCTTTTTCTCCCAATTTTTCAGTTGAAAAAGGTATA

TGCGTCAGGCGACCTCTGAAATTAACAAAAAATTTCCAGTCATCGAATTTGATTCTGTG

CGATAGCGCCCCTGTGTGTTCTCGTTATGTTGAGGAAAAAAATAATGGTTGCTAAGAGA

TTCGAACTCTTGCATCTTACGATACCTGAGTATTCCCACAGTTGGGGATCTCGACTCTAG

CTAGAGGATCAATTCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCT

CACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAG CCTGGGGTGCCTAA

TGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAA

CCTGTCGTGCCAGCTGATAACTTCGTATAATGTATGCTATACGAAGTTATTAGGTCTGAA

GAGGAGTTTACGTCCAGCCAAGCTAGCTTGGCTGCAGGTCGAGCGGCCGCGATCCGGA

ACCCTTAATATAACTTCGTATAATGTATGCTATACGAAGTTATCAGCTGCATTAATGAATC

GGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCA

CTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGC

GGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAA

GGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGC

TCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACC

CGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCT

GTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGC

GCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCT

GGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATC

GTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAA

CAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCT

AACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTAC

CTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGT

GGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATC

CTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATT

TTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGT

TTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATC

AGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCC

GTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGAT

ACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGA

AGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTG

TTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCA

TTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTT

CCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTC

CTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTAT

GGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGG

TGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCC

CGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATT

GGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTT

CGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTT

CTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACA

CGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTT

ATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTC

CGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACA

TTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTCGCGCGTTTCGGTGATGA

CGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCG

GATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGG

GGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATAACGCATT

TAAGCATAAACACGCACTATGCCGTTCTTCTCATGTATATATATATACAGGCAACACGCA

GATATAGGTGCGACGTGAACAGTGAGCTGTATGTGCGCAGCTCGCGTTGCATTTTCGGA

AGCGCTCGTTTTCGGAAACGCTTTGAAGTTCCTATTCCGAAGTTCCTATTCTCTAGCTAG

AAAGTATAGGAACTTCAGAGCGCTTTTGAAAACCAAAAGCGCTCTGAAGACGCACTTT

CAAAAAACCAAAAACGCACCGGACTGTAACGAGCTACTAAAATATTGCGAATACCGCT

TCCACAAACATTGCTCAAAAGTATCTCTTTGCTATATATCTCTGTGCTATATCCCTATATAA

CCTACCCATCCACCTTTCGCTCCTTGAACTTGCATCTAAACTCGACCTCTACATTTTTTAT

GTTTATCTCTAGTATTACTCTTTAGACAAAAAAATTGTAGTAAGAACTATTCATAGAGTG

AATCGAAAACAATACGAAAATGTAAACATTTCCTATACGTAGTATATAGAGACAAAATA

GAAGAAACCGTTCATAATTTTCTGACCAATGAAGAATCATCAACGCTATCACTTTCTGTT

CACAAAGTATGCGCAATCCACATCGGTATAGAATATAATCGGGGATGCCTTTATCTTGAA

AAAATGCACCCGCAGCTTCGCTAGTAATCAGTAAACGCGGGAAGTGGAGTCAGGCTTT

TTTTATGGAAGAGAAAATAGACACCAAAGTAGCCTTCTTCTAACCTTAACGGACCTACA

GTGCAAAAAGTTATCAAGAGACTGCATTATAGAGCGCACAAAGGAGAAAAAAAGTAAT

CTAAGATGCTTTGTTAGAAAAATAGCGCTCTCGGGATGCATTTTTGTAGAACAAAAAAG

AAGTATAGATTCTTTGTTGGTAAAATAGCGCTCTCGCGTTGCATTTCTGTTCTGTAAAAA

TGCAGCTCAGATTCTTTGTTTGAAAAATTAGCGCTCTCGCGTTGCATTTTTGTTTTACAA

AAATGAAGCACAGATTCTTCGTTGGTAAAATAGCGCTTTCGCGTTGCATTTCTGTTCTGT

AAAAATGCAGCTCAGATTCTTTGTTTGAAAAATTAGCGCTCTCGCGTTGCATTTTTGTTC

TACAAAATGAAGCACAGATGCTTCGTTGCT

(3) information of sequence SEQ ID NO.2: (aminoacid sequence that pGADT7-CBP expresses)

(i) sequence signature:

(A) length: 638AA

(B) type: amino acid

(C) topological framework: strand peptide chain

(ii) molecule type: protein

(iii) sequence description: SEQ NO.2

Met Asp Lys Ala Glu Leu Ile Pro Glu Pro Pro Lys Lys Lys Arg Lys Val Glu Leu Gly Thr Ala Ala

Asn Phe Asn Gln Ser Gly Asn Ile Ala Asp Ser Ser Leu Ser Phe Thr Phe Thr Asn Ser Ser Asn

Gly Pro Asn Leu Ile Thr Thr Gln Thr Asn Ser Gln Ala Leu Ser Gln Pro Ile Ala Ser Ser Asn

Val His Asp Asn Phe Met Asn Asn Glu Ile Thr Ala Ser Lys Ile Asp Asp Gly Asn Asn Ser Lys

Pro Leu Ser Pro Gly Trp Thr Asp Gln Thr Ala Tyr Asn Ala Phe Gly Ile Thr Thr Gly Met

Phe Asn Thr Thr Thr Met Asp Asp Val Tyr Asn Tyr Leu Phe Asp Asp Glu Asp Thr Pro Pro

Asn Pro Lys Lys Glu Ile Phe Asn Thr Thr His Tyr Arg Ala Ser Ala Ala Met Glu Tyr Pro Tyr Asp

Val Pro Asp Tyr Ala His Met Met Ala Glu Asn Leu Leu Asp Gly Pro Pro Asn Pro Lys Arg Ala

Lys Leu Ser Ser Pro Gly Phe Ser Ala Asn Asp Asn Thr Asp Phe Gly Ser Leu Phe Asp Leu

Glu Asn Asp Leu Pro Asp Glu Leu Ile Pro Asn Gly Glu Leu Ser Leu Leu Asn Ser Gly Asn

Leu Val Pro Asp Ala Ala Ser Lys His Lys Gln Leu Ser Glu Leu Leu Arg Gly Gly Ser Gly Ser

Ser Ile Asn Pro Gly Ile Gly Asn Val Ser Ala Ser Ser Pro Val Gln Gln Gly Leu Gly Gly Gln

Ala Gln Gly Gln Pro Asn Ser Thr Asn Met Ala Ser Leu Gly Ala Met Gly Lys Ser Pro Leu

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

Pro Ala Ser Gln Ala Leu Asn Pro Gln Ala Gln Lys Gln Val Gly Leu Val Thr Ser Ser Pro Ala

Thr SerGln Thr Gly Pro Gly Ile Cys Met Asn Ala Asn Phe Asn Gln Thr His Pro Gly Leu

Leu Asn SerAsn Ser Gly His Ser Leu Met Asn Gln Ala Gln Gln Gly Gln Ala Gln Val Met

Asn Gly Ser Leu Gly Ala Ala Gly Arg Gly Arg Gly Ala Gly Met Pro Tyr Pro Ala Pro Ala

Met Gln Gly Ala Thr Ser Ser Val Leu Ala Glu Thr Leu Thr Gln Val Ser Pro Gln Met Ala Gly

His Ala Gly Leu Asn Thr Ala Gln Ala Gly Gly Met Thr Lys Met Gly Met Thr Gly Thr Thr

Ser Pro Phe Gly Gln Pro Phe Ser Gln Thr Gly Gly Gln Gln Met Gly Ala Thr Gly Val Asn

Pro Gln Leu Ala Ser Lys Gln Ser Met Val Asn Ser Leu Pro Ala Phe Pro Thr Asp Ile Lys Asn

Thr Ser Val Thr Thr Val Pro Asn Met Ser Gln Leu Gln Thr Ser Val Gly Ile Val Pro Thr Gln

Ala Ile Ala Thr Gly Pro Thr Ala Asp Pro Glu Lys Arg Lys Leu Ile Gln Gln Gln Leu Val Leu

Leu Leu His Ala His Lys Cys Gln Arg Arg Glu Gln Ala Asn Gly Glu Val Arg Ala Cys Ser

Leu Pro His Cys Arg Thr Met Lys Asn Val Leu Asn His Met Thr His Cys Gln Ala Pro Lys

Ala Cys Gln Val Ala His Cys Ala Ser Ser Arg Gln Ile Ile Ser His Trp Lys Asn Cys Thr Arg

His Asp Cys Pro Val Cys Leu Pro Leu Lys Asn Ala Ser Asp Lys Arg Asn Gln Gln Thr Ile Leu

Gly Ser Pro Ala Ser Gly Ile Gln Asn Thr Ile Gly Ser Val Gly Ala Gly Gln Gln Gly Ser Ile Glu

Leu Glu Leu Gln Met Asn Arg Arg Tyr***

(4) information of sequence SEQ NO.3: (dna sequence dna of pGBKT7-PPAR γ LBD)

(i) sequence signature:

(A) length: 8128bp

(B) type: Nucleotide

(C) chain: two strands

(D) topological framework: ring-type

(ii) molecule type: DNA

(iii) sequence description: SEQ NO.3

CGGTGCGGGCCTCTTCGCTATTACGCCAGATCCTTTTGTTGTTTCCGGGTGTACAATATG

GACTTCCTCTTTTCTGGCAACCAAACCCATACATCGGGATTCCTATAATACCTTCGTTGG

TCTCCCTAACATGTAGGTGGCGGAGGGGAGATATACAATAGAACAGATACCAGACAAG

ACATAATGGGCTAAACAAGACTACACCAATTACACTGCCTCATTGATGGTGGTACATAA

CGAACTAATACTGTAGCCCTAGACTTGATAGCCATCATCATATCGAAGTTTCACTACCCT

TTTTCCATTTGCCATCTATTGAAGTAATAATAGGCGCATGCAACTTCTTTTCTTTTTTTTT

CTTTTCTCTCTCCCCCGTTGTTGTCTCACCATATCCGCAATGACAAAAAAAATGATGGA

AGACACTAAAGGAAAAAATTAACGACAAAGACAGCACCAACAGATGTCGTTGTTCCA

GAGCTGATGAGGGGTATCTCGAAGCACACGAAACTTTTTCCTTCCTTCATTCACGCACA

CTACTCTCTAATGAGCAACGGTATACGGCCTTCCTTCCAGTTACTTGAATTTGAAATAAA

AAAAGTTTGCTGTCTTGCTATCAAGTATAAATAGACCTGCAATTATTAATCTTTTGTTTCC

TCGTCATTGTTCTCGTTCCCTTTCTTCCTTGTTTCTTTTTCTGCACAATATTTCAAGCTAT

ACCAAGCATACAATCAACTCCAAGCTTGAAGCAAGCCTCCTGAAAGATGAAGCTACT

GTCTTCTATCGAACAAGCATGCGATATTTGCCGACTTAAAAAGCTCAAGTGCTCCA

AAGAAAAACCGAAGTGCGCCAAGTGTCTGAAGAACAACTGGGAGTGTCGCTACT

CTCCCAAAACCAAAAGGTCTCCGCTGACTAGGGCACATCTGACAGAAGTGGAATC

AAGGCTAGAAAGACTGGAACAGCTATTTCTACTGATTTTTCCTCGAGAAGACCTT

GACATGATTTTGAAAATGGATTCTTTACAGGATATAAAAGCATTGTTAACAGGATT

ATTTGTACAAGATAATGTGAATAAAGATGCCGTCACAGATAGATTGGCTTCAGTGG

AGACTGATATGCCTCTAACATTGAGACAGCATAGAATAAGTGCGACATCATCATCG

GAAGAGAGTAGTAACAAAGGTCAAAGACAGTTGACTGTATCGCCGGAATTTGTAAT

ACGACTCACTATAGGGCGAGCCGCCATCATGGAGGAGCAGAAGCTGATCTCAGAGGAG

GACCTGCATATGgcggagatctccagtgatatcgaccagctgaatccagagtccgctgacctccgggccctggcaaaacatt

tgtatgactcatacataaagtccttcccgctgaccaaagcaaaggcgagggcgatcttgacaggaaagacaacagacaaatcacca

ttcgttatctatgacatgaattccttaatgatgggagaagataaaatcaagttcaaacacatcacccccctgcaggagcagagcaaag

aggtggccatccgcatctttcagggctgccagtttcgctccgtggaggctgtgcaggagatcacagagtatgccaaaagcattcctgg

ttttgtaaatcttgacttgaacgaccaagtaactctcctcaaatatggagtccacgagatcatttacacaatgctggcctccttgatgaat

aaagatggggttctcatatccgagggccaaggcttcatgacaagggagtttctaaagagcctgcgaaagccttttggtgactttatgg

agcccaagtttgagtttgetgtgaagttcaatgcactggaattagatgacagcgacttggcaatatttattgctgtcattattctcagtgg

agaccgcccaggtttgctgaatgtgaagcccattgaagacattcaagacaacctgctacaagccctggagctccagctgaagctgaa

ccaccctgagtcctcacagctgtttgccaagctgctccagaaaatgacagacctcagacagattgtcacggaacacgtgcagctactg

caggtgatcaagaagacggagacagacatgagtcttcacccgctcctgcaggagatctacaaggacttgtactagGTCGACCT

GCAGCGGCCGCATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTT

TTTTGCGCGCTTGCAGCCAAGCTAATTCCGGGCGAATTTCTTATGATTTATGATTTTTATT

ATTAAATAAGTTATAAAAAAAATAAGTGTATACAAATTTTAAAGTGACTCTTAGGTTTTA

AAACGAAAATTCTTATTCTTGAGTAACTCTTTCCTGTAGGTCAGGTTGCTTTCTCAGGTA

TAGCATGAGGTCGCTCTTATTGACCACACCTCTACCGGCATGCAAGCTTGGCGTAATCA

TGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGA

GCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGGTAACTCACATTAA

TTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAA

TGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCT

CGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTC

AAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGA

GCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTC

CATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGC

GAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCG

CTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAA

GCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGC

TCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCG

GTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCC

ACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGT

GGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAG

CCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTG

GTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCA

AGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTT

AAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAA

AATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAACCTGAGGCTATGGCAGGGCCTG

CCGCCCCGACGTTGGCTGCGAGCCCTGGGCCTTCACCCGAACTTGGGGGGTGGGGTG

GGGAAAAGGAAGAAACGCGGGCGTATTGGCCCCAATGGGGTCTCGGTGGGGTATCGA

CAGAGTGCCAGCCCTGGGACCGAACCCCGCGTTTATGAACAAACGACCCAACACCGT

GCGTTTTATTCTGTCTTTTTATTGCCGTCATAGCGCGGGTTCCTTCCGGTATTGTCTCCTT

CCGTGTTTCAGTTAGCCTCCCCCTAGGGTGGGCGAAGAACTCCAGCATGAGATCCCCG

CGCTGGAGGATCATCCAGCCGGCGTCCCGGAAAACGATTCCGAAGCCCAACCTTTCAT

AGAAGGCGGCGGTGGAATCGAAATCTCGTGATGGCAGGTTGGGCGTCGCTTGGTCGGT

TCATTTCGAACCCCAGAGTCCCGCTCAGAAGAACTCGTCAAGAAGGCGATAGAAGGC

GATGCGCTGCGAATCGGGAGCGGCGATACCGTAAAGCACGAGGAAGCGGTCAGCCCAT

TCGCCGCCAAGCTCTTCAGCAATATCACGGGTAGCCAACGCTATGTCCTGATAGCGGTC

CGCCACACCCAGCCGGCCACAGTCGATGAATCCAGAAAAGCGGCCATTTTCCACCATG

ATATTCGGCAAGCAGGCATCGTCATGGGTCACGACGAGATCCTCGCCGTCGGGCATGCT

CGCCTTGAGCCTGGCGAACAGTTCGGCTGGCGCGAGCCCCTGATGCTCTTCGTCCAGA

TCATCCTGATCGACAAGACCGGCTTCCATCCGAGTACGTGCTCGCTCGATGCGATGTTT

CGCTTGGTGGTCGAATGGGCAGGTAGCCGGATCAAGCGTATGCAGCCGCCGCATTGCA

TCAGCCATGATGGATACTTTCTCGGCAGGAGCAAGGTGAGATGACAGGAGATCCTGCC

CCGGCACTTCGCCCAATAGCAGCCAGTCCCTTCCCGCTTCAGTGACAACGTCGAGCAC

AGCTGCGCAAGGAACGCCCGTCGTGGCCAGCCACGATAGCCGCGCTGCCTCGTCTTGC

AGTTCATTCAGGGCACCGGACAGGTCGGTCTTGACAAAAAGAACCGGGCGCCCCTGC

GCTGACAGCCGGAACACGGCGGCATCAGAGCAGCCGATTGTCTGTTGTGCCCAGTCAT

AGCCGAATAGCCTCTCCACCCAAGCGGCCGGAGAACCTGCGTGCAATCCATCTTGTTC

AATCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGC

GGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCC

CGAAAAGTGCCACCTGAACGAAGCATCTGTGCTTCATTTTGTAGAACAAAAATGCAAC

GCGAGAGCGCTAATTTTTCAAACAAAGA ATCTGAGCTGCATTTTTACAGAACAGAAAT

GCAACGCGAAAGCGCTATTTTACCAACGAAGAATCTGTGCTTCATTTTTGTAAAACAAA

AATGCAACGCGAGAGCGCTAATTTTTCAAACAAAGAATCTGAGCTGCATTTTTACAGA

ACAGAAATGCAACGCGAGAGCGCTATTTTACCAACAAAGAATCTATACTTCTTTTTTGT

TCTACAAAAATGCATCCCGAGAGCGCTATTTTTCTAACAAAGCATCTTAGATTACTTTTT

TTCTCCTTTGTGCGCTCTATAATGCAGTCTCTTGATAACTTTTTGCACTGTAGGTCCGTTA

AGGTTAGAAGAAGGCTACTTTGGTGTCTATTTTCTCTTCCATAAAAAAAGCCTGACTCC

ACTTCCCGCGTTTACTGATTACTAGCGAAGCTGCGGGTGCATTTTTTCAAGATAAAGGC

ATCCCCGATTATATTCTATACCGATGTGGATTGCGCATACTTTGTGAACAGAAAGTGATA

GCGTTGATGATTCTTCATTGGTCAGAAAATTATGAACGGTTTCTTCTATTTTGTCTCTATA

TACTACGTATAGGAAATGTTTACATTTTCGTATTGTTTTCGATTCACTCTATGAATAGTTC

TTACTACAATTTTTTTGTCTAAAGAGTAATACTAGAGATAAACATAAAAAATGTAGAGGT

CGAGTTTAGATGCAAGTTCAAGGAGCGAAAGGTGGATGGGTAGGTTATATAGGGATATA

GCACAGAGATATATAGCAAAGAGATACTTTTGAGCAATGTTTGTGGAAGCGGTATTCGC

AATATTTTAGTAGCTCGTTACAGTCCGGTGCGTTTTTGGTTTTTTGAAAGTGCGTCTTCA

GAGCGCTTTTGGTTTTCAAAAGCGCTCTGAAGTTCCTATACTTTCTAGAGAATAGGAAC

TTCGGAATAGGAACTTCAAAGCGTTTCCGAAAACGAGCGCTTCCGAAAATGCAACGCG

AGCTGCGCACATACAGCTCACTGTTCACGTCGCACCTATATCTGCGTGTTGCCTGTATAT

ATAATACATGAGAAGAACGGCATAGTGCGTGTTTATGCTTAAATGCGTACTTATATGCGT

CTATTTATGTAGGATGAAAGGTAGTCTAGTACCTCCTGTGATATTATCCCATTCCATGCGG

GGTATCGTATGCTTCCTTCAGCACTACCCTTTAGCTGTTCTATATGCTGCCACTCCTCAAT

TGGATTAGTCTCATCCTTCAATGCTATCATTTCCTTTGATATTGGATCATATTAAGAAACC

ATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTCGCG

CGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAG

CTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTG

TTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGT

GCACCATAGATCAACGACATTACTATATATATAATATAGGAAGCATTTAATAGAACAGCAT

CGTAATATATGTGTACTTTGCAGTTATGACGCCAGATGGCAGTAGTGGAAGATATTCTTT

ATTGAAAAATAGCTTGTCACCTTACGTACAATCTTGATCCGGAGCTTTTCTTTTTTTGCC

GATTAAGAATTAATTCGGTCGAAAAAAGAAAAGGAGAGGGCCAAGAGGGAGGGCATT

GGTGACTATTGAGCACGTGAGTATACGTGATTAAGCACACAAAGGCAGCTTGGAGTAT

GTCTGTTATTAATTTCACAGGTAGTTCTGGTCCATTGGTGAAAGTTTGCGGCTTGCAGA

GCACAGAGGCCGCAGAATGTGCTCTAGATTCCGATGCTGACTTGCTGGGTATTATATGT

GTGCCCAATAGAAAGAGAACAATTGACCCGGTTATTGCAAGGAAAATTTCAAGTCTTG

TAAAAGCATATAAAAATAGTTCAGGCACTCCGAAATACTTGGTTGGCGTGTTTCGTAAT

CAACCTAAGGAGGATGTTTTGGCTCTGGTCAATGATTACGGCATTGATATCGTCCAACT

GCATGGAGATGAGTCGTGGCAAGAATACCAAGAGTTCCTCGGTTTGCCAGTTATTAAA

AGACTCGTATTTCCAAAAGACTGCAACATACTACTCAGTGCAGCTTCACAGAAACCTCA

TTCGTTTATTCCCTTGTTTGATTCAGAAGCAGGTGGGACAGGTGAACTTTTGGATTGGA

ACTCGATTTCTGACTGGGTTGGAAGGCAAGAGAGCCCCGAAAGCTTACATTTTATGTTA

GCTGGTGGACTGACGCCAGAAAATGTTGGTGATGCGCTTAGATTAAATGGCGTTATTGG

TGTTGATGTAAGCGGAGGTGTGGAGACAAATGGTGTAAAAGACTCTAACAAAATAGCA

AATTTCGTCAAAAATGCTAAGAAATAGGTTATTACTGAGTAGTATTTATTTAAGTATTGTT

TGTGCACTTGCCGATCTATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATAC

CGCATCAGGAAATTGTAAACGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAAT

CAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAAT

AGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAA

CGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACGT

GAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGA

ACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGA

GAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCG

GTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGT

CCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGAT

(5) information of sequence SEQ NO.4: (aminoacid sequence that pGBKT7-PPAR γ LBD expresses)

(i) sequence signature:

(A) length: 458AA

(B) type: amino acid

(C) topological framework: strand peptide chain

(ii) molecule type: protein

(iii) sequence description: SEQ NO.4

Met Lys Leu Leu Ser Ser Ile Glu Gln Ala Cys Asp Ile Cys Arg Leu Lys Lys Leu Lys Cys Ser

Lys Glu Lys Pro Lys Cys Ala Lys Cys Leu Lys Asn Asn Trp Glu Cys Arg Tyr Ser Pro Lys

Thr Lys Arg Ser Pro Leu Thr Arg Ala His Leu Thr Glu Val Glu Ser Arg Leu Glu Arg Leu

Glu Gln Leu Phe Leu Leu Ile Phe Pro Arg Glu Asp Leu Asp Met Ile Leu Lys Met Asp Ser

Leu Gln Asp Ile Lys Ala Leu Leu Thr Gly Leu Phe Val Gln Asp Asn Val Asn Lys Asp Ala Val

Thr Asp Arg Leu Ala Ser Val Glu Thr Asp Met Pro Leu Thr Leu Arg Gln His Arg Ile Ser

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

Val Ile Arg Leu Thr Ile Gly Arg Ala Ala Ile Met Glu Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu

His Met Ala Glu Ile Ser Ser Asp Ile Asp Gln Leu Asn Pro Glu Ser Ala Asp Leu Arg Ala Leu

Ala Lys His Leu Tyr Asp Ser Tyr Ile Lys Ser Phe Pro Leu Thr Lys Ala Lys Ala Arg Ala Ile

Leu Thr Gly Lys Thr Thr Asp Lys Ser Pro Phe Val Ile Tyr Asp Met Asn Ser Leu Met Met

Gly Glu Asp Lys Ile Lys Phe Lys His Ile Thr Pro Leu Gln Glu Gln Ser Lys Glu Val Ala Ile

Arg Ile Phe Gln Gly Cys Gln Phe Arg Ser Val Glu Ala Val Gln Glu Ile Thr Glu Tyr Ala Lys

Ser Ile Pro Gly Phe Val Asn Leu Asp Leu Asn Asp Gln Val Thr Leu Leu Lys Tyr Gly Val His

Glu Ile Ile Tyr Thr Met Leu Ala Ser Leu Met Asn Lys Asp Gly Val Leu Ile Ser Glu Gly Gln

Gly Phe Met Thr Arg Glu Phe Leu Lys Ser Leu Arg Lys Pro Phe Gly Asp Phe Met Glu Pro

Lys Phe Glu Phe Ala Val Lys Phe Asn Ala Leu Glu Leu Asp Asp Ser Asp Leu Ala Ile Phe Ile

Ala Val Ile Ile Leu Ser Gly Asp Arg Pro Gly Leu Leu Asn Val Lys Pro Ile Glu Asp Ile Gln

Asp Asn Leu Leu Gln Ala Leu Glu Leu Gln Leu Lys Leu Asn His Pro Glu Ser Ser Gln Leu

Phe Ala Lys Leu Leu Gln Lys Met Thr Asn Leu Arg Gln Ile Val Thr Glu His Val Gln Leu

Leu Gln Val Ile Lys Lys Thr Glu Thr Asp Met Ser Leu His Pro Leu Leu Gln Glu Ile Tyr Lys

Asp Leu Tyr***

Claims (7)

1, the screening method of a kind of PPAR antagonist and agonist, it is characterized in that utilizing yeast two-hybrid system, the MEL1 gene that employing yeast AH109 genome carries is as the reporter gene of double cross, detect the influence of micromolecular compound by the activity of measuring alpha-galactosidase to PPAR and its co-activation factor interaction degree, thereby screening PPAR antagonist and agonist comprise the steps:

(1) structure of mouse co-activation factor CBP and yeast transcription factor GAL4AD Expression of Fusion Protein plasmid pGADT7-CBP;

(2) structure of the plasmid pGBKT7-PPAR γ LBD of PPAR γ LBD and yeast transcription factor GAL4BD fusion rotein;

(3) change plasmid pGBKT7-PPAR γ LBD and plasmid pGADT7-CBP over to yeast cell AH109, obtain yeast PPAR γ LBD-CBP AH109 (preserving number was CGMCC No.1075, and bacterial classification is deposited in BeiJing ZhongGuanCun China Committee for Culture Collection of Microorganisms common micro-organisms center on December 15th, 2003);

(4) by measuring the screening active ingredients compound of alpha-galactosidase.

2, the screening method of PPAR antagonist according to claim 1 and agonist is characterized in that the method that plasmid pGBKT7-PPAR γ LBD and plasmid pGADT7-CBP is changed successively over to yeast cell AH109 is:

Adopt the LiAc conversion method, earlier pGBKT7-PPAR γ LBD plasmid is changed in the yeast cell, substratum screening positive clone with the tryptophane defective, the pGADT7-CBP plasmid is changed in the yeast that contains pGBKT7-PPAR γ LBD plasmid then, use tryptophane, the substratum screening positive clone of the two defectives of leucine is contained the yeast cell of pGADT7-CBP plasmid and pGBKT7-PPAR γ LBD plasmid simultaneously;

3, the screening method of PPAR antagonist according to claim 1 and agonist is characterized in that the method for the screening active ingredients compound by measuring alpha-galactosidase is:

In the positive yeast cell that contains pGADT7-CBP and the two plasmids of pGBKT7-PPAR γ LBD that obtains, add positive control, negative control thing and compound to be screened respectively, after hatching, measure the activity of alpha-galactosidase, and calculate its exciting rate, according to each comparison for the treatment of the exciting rate of screening of medicaments, filter out PPAR antagonist and agonist.

4, the screening method of PPAR antagonist according to claim 3 and agonist is characterized in that when measuring the screening active ingredients compound of alpha-galactosidase, the drug incubation time is 12-14 hour.

5, according to the screening method that utilizes described PPAR antagonist of claim 1 and agonist, it is characterized in that the yeast screening assay system that this method screening PPAR antagonist and agonist are utilized comprises: (1) mouse co-activation factor CBP and yeast transcription factor GAL4-AD fusion protein expression plasmid pGADT7-CBP culture presevation number were CGMCC No.1076, and bacterial classification is deposited in BeiJing ZhongGuanCun China Committee for Culture Collection of Microorganisms common micro-organisms center on December 15th, 2003; (2) contain people PPAR γ LBD and yeast transcription factor GAL4-BD fusion protein expression plasmid pGBKT7-PPAR γ LBD culture presevation number and be CGMCC No.1077, bacterial classification was deposited in BeiJing ZhongGuanCun China Committee for Culture Collection of Microorganisms common micro-organisms center on December 15th, 2003; (3) host's yeast AH109.

6, the screening method of PPAR antagonist according to claim 5 and agonist is characterized in that mouse co-activation factor CBP and yeast transcription factor GAL4-AD fusion protein expression plasmid pGADT7-CBP have following sequence:

(1) the pGADT7-CBP plasmid that utilizes carrier pGADT7 to make up has following nucleotide sequence:

TGCATGCCTGCAGGTCGAGATCCGGGATCGAAGAAATGATGGTAAATGAAATAGGAAATCAAGGAGCATGAAGGCAAAAGACAAATATAAGGGTCGAACGAAAAATAAAGTGAAAAGTGTTGATATGATGTATTTGGCTTTGCGGCGCCGAAAAAACGAGTTTACGCAATTGCACAATCATGCTGACTCTGTGGCGGACCCGCGCTCTTGCCGGCCCGGCGATAACGCTGGGCGTGAGGCTGTGCCCGGCGGAGTTTTTTGCGCCTGCATTTTCCAAGGTTTACCCTGCGCTAAGGGGCGAGATTGGAGAAGCAATAAGAATGCCGGTTGGGGTTGCGATGATGACGACCACGACAACTGGTGTCATTATTTAAGTTGCCGAAAGAACCTGAGTGCATTTGCAACATGAGTATACTAGAAGAATGAGCCAAGACTTGCGAGACGCGAGTTTGCCGGTGGTGCGAACAATAGAGCGACCATGACCTTGAAGGTGAGACGCGCATAACCGCTAGAGTACTTTGAAGAGGAAACAGCAATAGGGTTGCTACCAGTATAAATAGACAGGTACATACAACACTGGAAATGGTTGTCTGTTTGAGTACGCTTTCAATTCATTTGGGTGTGCACTTTATTATGTTACAATATGGAAGGGAACTTTACACTTCTCCTATGCACATATATTAATTAAAGTCCAATGCTAGTAGAGAAGGGGGGTAACACCCCTCCGCGCTCTTTTCCGATTTTTTTCTAAACCGTGGAATATTTCGGATATCCTTTTGTTGTTTCCGGGTGTACAATATGGACTTCCTCTTTTCTGGCAACCAAACCCATACATCGGGATTCCTATAATACCTTCGTTGGTCTCCCTAACATGTAGGTGGCGGAGGGGAGATATACAATAGAACAGATACCAGACAAGACATAATGGGCTAAACAAGACTACACCAATTACACTGCCTCATTGATGGTGGTACATAACGAACTAATACTGTAGCCCTAGACTTGATAGCCATCATCATATCGAAGTTTCACTACCCTTTTTCCATTTGCCATCTATTGAAGTAATAATAGGCGCATGCAACTTCTTTTCTTTTTTTTTCTTTTCTCTCTCCCCCGTTGTTGTCTCACCATATCCGCAATGACAAAAAAAATGATGGAAGACACTAAAGGAAAAAATTAACGACAAAGACAGCACCAACAGATGTCGTTGTTCCAGAGCTGATGAGGGGTATCTCGAAGCACACGAAACTTTTTCCTTCCTTCATTCACGCACACTACTCTCTAATGAGCAACGGTATACGGCCTTCCTTCCAGTTACTTGAATTTGAAATAAAAAAAAGTTTGCTGTCTTGCTATCAAGTATAAATAGACCTGCAATTATTAATCTTTTGTTTCCTCGTCATTGTTCTCGTTCCCTTTCTTCCTTGTTTCTTTTTCTGCACAATATTTCAAGCTATACCAAGCATACAATCAACTCCAAGCTTTGCAAAGATGGATAAAGCGGAATTAATTCCCGAGCCTCCAAAAAAGAAGAGAAAGGTCGAATTGGGTACCGCCGCCAATTTTAATCAAAGTGGGAATATTGCTGATAGCTCATTGTCCTTCACTTTCACTAACAGTAGCAACGGTCCGAACCTCATAACAACTCAAACAAATTCTCAAGCGCTTTCACAACCAATTGCCTCCTCTAACGTTCATGATAACTTCATGAATAATGAAATCACGGCTAGTAAAATTGATGATGGTAATAATTCAAAACCACTGTCACCTGGTTGGACGGACCAAACTGCGTATAACGCGTTTGGAATCACTACAGGGATGTTTAATACCACTACAATGGATGATGTATATAACTATCTATTCGATGATGAAGATACCCCACCAAACCCAAAAAAAGAGATCTTTAATACGACTCACTATAGGGCGAGCGCCGCCATGGAGTACCCATACGACGTACCAGATTACGCT CATATGatggccgagaacttgctggacggaccgcccaaccccaaacgagccaaactcagctcgcccggcttctccgcgaatgacaacacagattttggatcattgtttgacttggaaaatgaccttcctgatgagctgatccccaatggagaattaagccttttaaacagtgggaaccttgttccagatgctgcgtccaaacataaacaactgtcagagcttcttagaggaggcagcggctctagcatcaacccagggataggcaatgtgagtgccagcagccctgtgcaacagggccttggtggccaggctcaggggcagccgaacagtacaaacatggccagcttaggtgccatgggcaagagccctctgaaccaaggagactcatcaacacccaacctgcccaaacaggcagccagcacctctgggcccactccccctgcctcccaagcactgaatccacaagcacaaaagcaagtagggctggtgaccagtagtcctgccacatcacagactggacctgggatctgcatgaatgctaacttcaaccagacccacccaggccttctcaatagtaactctggccatagcttaatgaatcaggctcaacaagggcaagctcaagtcatgaatggatctcttggggctgctggaagaggaaggggagctggaatgccctaccctgctccagccatgcagggggccacaagcagtgtgctggcggagaccttgacacaggtttccccacaaatggctggccatgctggactaaatacagcacaggcaggaggcatgaccaagatgggaatgactggtaccacaagtccatttggacaaccctttagtcaaactggagggcagcagatgggagccactggagtgaacccccagttagccagcaaacagagcatggtcaatagtttacctgcttttcctacagatatcaagaatacttcagtcaccactgtgccaaatatgtcccagttgcaaacatcagtgggaattgtacccacacaagcaattgcaacaggccccacagcagaccctgaaaaacgcaaactgatacagcagcagctggttctactgcttcatgcccacaaatgtcagagacgagagcaagcaaatggagaggttcgagcctgttctctcccacactgtcgaaccatgaaaaacgttttgaatcacatgacacattgtcaggctcccaaagcctgccaagttgcccattgtgcatcttcacgacaaatcatctctcattggaagaactgcacacgacatgactgtcctgtttgcctccctttgaaaaatgccagtgacaagcgaaaccaacaaaccatcctgggatctccagctagtggaattcaaaacacaattggttctgttggtgcagggcaacag GGATCCATCGAGCTCGAGCTGCAGATGAATCGTAGATACTGAAAAACCCCGCAAGTTCACTTCAACTGTGCATCGTGCACCATCTCAATTTCTTTCATTTATACATCGTTTTGCCTTCTTTTATGTAACTATACTCCTCTAAGTTTCAATCTTGGCCATGTAACCTCTGATCTATAGAATTTTTTAAATGACTAGAATTAATGCCCATCTTTTTTTTGGACCTAAATTCTTCATGAAAATATATTACGAGGGCTTATTCAGAAGCTTTGGACTTCTTCGCCAGAGGTTTGGTCAAGTCTCCAATCAAGGTTGTCGGCTTGTCTACCTTGCCAGAAATTTACGAAAAGATGGAAAAGGGTCAAATCGTTGGTAGATACGTTGTTGACACTTCTAAATAAGCGAATTTCTTATGATTTATGATTTTTATTATTAAATAAGTTATAAAAAAAATAAGTGTATACAAATTTTAAAGTGACTCTTAGGTTTTAAAACGAAAATTCTTATTCTTGAGTAACTCTTTCCTGTAGGTCAGGTTGCTTTCTCAGGTATAGCATGAGGTCGCTCTTATTGACCACACCTCTACCGGCCGGTCGAAATTCCCCTACCCTATGAACATATTCCATTTTGTAATTTCGTGTCGTTTCTATTATGAATTTCATTTATAAAGTTTATGTACAAATATCATAAAAAAAGAGAATCTTTTTAAGCAAGGATTTTCTTAACTTCTTCGGCGACAGCATCACCGACTTCGGTGGTACTGTTGGAACCACCTAAATCACCAGTTCTGATACCTGCATCCAAAACCTTTTTAACTGCATCTTCAATGGCCTTACCTTCTTCAGGCAAGTTCAATGACAATTTCAACATCATTGCAGCAGACAAGATAGTGGCGATAGGGTTGACCTTATTCTTTGGCAAATCTGGAGCAGAACCGTGGCATGGTTCGTACAAACCAAATGCGGTGTTCTTGTCTGGCAAAGAGGCCAAGGACGCAGATGGCAACAAACCCAAGGAACCTGGGATAACGGAGGCTTCATCGGAGATGATATCACCAAACATGTTGCTGGTGATTATAATACCATTTAGGTGGGTTGGGTTCTTAACTAGGATCATGGCGGCAGAATCAATCAATTGATGTTGAACCTTCAATGTAGGAAATTCGTTCTTGATGGTTTCCTCCACAGTTTTTCTCCATAATCTTGAAGAGGCCAAAACATTAGCTTTATCCAAGGACCAAATAGGCAATGGTGGCTCATGTTGTAGGGCCATGAAAGCGGCCATTCTTGTGATTCTTTGCACTTCTGGAACGGTGTATTGTTCACTATCCCAAGCGACACCATCACCATCGTCTTCCTTTCTCTTACCAAAGTAAATACCTCCCACTAATTCTCTGACAACAACGAAGTCAGTACCTTTAGCAAATTGTGGCTTGATTGGAGATAAGTCTAAAAGAGAGTCGGATGCAAAGTTACATGGTCTTAAGTTGGCGTACAATTGAAGTTCTTTACGGATTTTTAGTAAACCTTGTTCAGGTCTAACACTACCTGTACCCCATTTAGGACCACCCACAGCACCTAACAAAACGGCATCAACCTTCTTGGAGGCTTCCAGCGCCTCATCTGGAAGTGGGACACCTGTAGCGTCGATAGCAGCACCACCAATTAAATGATTTTCGAAATCGAACTTGACATTGGAACGAACATCAGAAATAGCTTTAAGAACCTTAATGGCTTCGGCTGTGATTTCTTGACCAACGTGGTCACCTGGCAAAACGACGATCTTCTTAGGGGCAGACATTAGAATGGTATATCCTTGAAATATATATATATATTGCTGAAATGTAAAAGGTAAGAAAAGTTAGAAAGTAAGACGATTGCTAACCACCTATTGGAAAAAACAATAGGTCCTTAAATAATATTGTCAACTTCAAGTATTGTGATGCAAGCATTTAGTCATGAACGCTTCTCTATTCTATATGAAAAGCCGGTTCCGGCGCTCTCACCTTTCCTTTTTCTCCCAATTTTTCAGTTGAAAAAGGTATATGCGTCAGGCGACCTCTGAAATTAACAAAAAATTTCCAGTCATCGAATTTGATTCTGTGCGATAGCGCCCCTGTGTGTTCTCGTTATGTTGAGGAAAAAAATAATGGTTGCTAAGAGATTCGAACTCTTGCATCTTACGATACCTGAGTATTCCCACAGTTGGGGATCTCGACTCTAGCTAGAGGATCAATTCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGATAACTTCGTATAATGTATGCTATACGAAGTTATTAGGTCTGAAGAGGAGTTTACGTCCAGCCAAGCTAGCTTGGCTGCAGGTCGAGCGGCCGCGATCCGGAACCCTTAATATAACTTCGTATAATGTATGCTATACGAAGTTATCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATAACGCATTTAAGCATAAACACGCACTATGCCGTTCTTCTCATGTATATATATATACAGGCAACACGCAGATATAGGTGCGACGTGAACAGTGAGCTGTATGTGCGCAGCTCGCGTTGCATTTTCGGAAGCGCTCGTTTTCGGAAACGCTTTGAAGTTCCTATTCCGAAGTTCCTATTCTCTAGCTAGAAAGTATAGGAACTTCAGAGCGCTTTTGAAAACCAAAAGCGCTCTGAAGACGCACTTTCAAAAAACCAAAAACGCACCGGACTGTAACGAGCTACTAAAATATTGCGAATACCGCTTCCACAAACATTGCTCAAAAGTATCTCTTTGCTATATATCTCTGTGCTATATCCCTATATAACCTACCCATCCACCTTTCGCTCCTTGAACTTGCATCTAAACTCGACCTCTACATTTTTTATGTTTATCTCTAGTATTACTCTTTAGACAAAAAAATTGTAGTAAGAACTATTCATAGAGTGAATCGAAAACAATACGAAAATGTAAACATTTCCTATACGTAGTATATAGAGACAAAATAGAAGAAACCGTTCATAATTTTCTGACCAATGAAGAATCATCAACGCTATCACTTTCTGTTCACAAAGTATGCGCAATCCACATCGGTATAGAATATAATCGGGGATGCCTTTATCTTGAAAAAATGCACCCGCAGCTTCGCTAGTAATCAGTAAACGCGGGAAGTGGAGTCAGGCTTTTTTTATGGAAGAGAAAATAGACACCAAAGTAGCCTTCTTCTAACCTTAACGGACCTACAGTGCAAAAAGTTATCAAGAGACTGCATTATAGAGCGCACAAAGGAGAAAAAAAGTAATCTAAGATGCTTTGTTAGAAAAATAGCGCTCTCGGGATGCATTTTTGTAGAACAAAAAAGAAGTATAGATTCTTTGTTGGTAAAATAGCGCTCTCGCGTTGCATTTCTGTTCTGTAAAAATGCAGCTCAGATTCTTTGTTTGAAAAATTAGCGCTCTCGCGTTGCATTTTTGTTTTACAAAAATGAAGCACAGATTCTTCGTTGGTAAAATAGCGCTTTCGCGTTGCATTTCTGTTCTGTAAAAATGCAGCTCAGATTCTTTGTTTGAAAAATTAGCGCTCTCGCGTTGCATTTTTGTTCTACAAAATGAAGCACAGATGCTTCGTTGCT

(2) utilize the aminoacid sequence of the pGADT7-CBP plasmid expression that carrier pGADT7 makes up as follows:

Met Asp Lys Ala Glu Leu Ile Pro Glu Pro Pro Lys Lys Lys Arg Lys Val Glu Leu Gly Thr AlaAla Asn Phe Asn Gln Ser Gly Asn Ile Ala Asp Ser Ser Leu Ser Phe Thr Phe Thr Asn Ser SerAsn Gly Pro Asn Leu Ile Thr Thr Gln Thr Asn Ser Gln Ala Leu Ser Gln Pro Ile Ala Ser SerAsn Val His Asp Asn Phe Met Asn Asn Glu Ile Thr Ala Ser Lys Ile Asp Asp Gly Asn Asn ScrLys Pro Leu Ser Pro Gly Trp Thr Asp Gln Thr Ala Tyr Asn Ala Phe Gly Ile Thr Thr GlyMet Phe Asn Thr Thr Thr Met Asp Asp Val Tyr Asn Tyr Leu Phe Asp Asp Glu Asp Thr ProPro Asn Pro Lys Lys Glu Ile Phe Asn Thr Thr His Tyr Arg Ala Ser Ala Ala Met Glu Tyr Pro TyrAsp Val Pro Asp Tyr Ala His Met Met Ala Glu Asn Leu Leu Asp Gly Pro Pro Asn Pro Lys Arg Ala Lys Leu Ser Ser Pro Gly Phe Ser Ala Asn Asp Asn Thr Asp Phe Gly Ser Leu Phe Asp Leu Glu Asn Asp Leu Pro Asp Glu Leu Ile Pro Asn Gly Glu Leu Ser Leu Leu Asn Ser Gly Asn Leu Val Pro Asp Ala Ala Ser Lys His Lys Gln Leu Ser Glu Leu Leu Arg Gly Gly Ser Gly Ser Ser Ile Asn Pro Gly Ile Gly Asn Val Ser Ala Ser Ser Pro Val Gln Gln Gly Leu Gly Gly Gln Ala Gln Gly Gln Pro Asn Ser Thr Asn Met Ala Ser Leu Gly Ala Met Gly Lys Ser Pro Leu Asn Gln Gly Asp Ser Ser Thr Pro Asn Leu Pro Lys Gln Ala Ala Ser Thr Ser Gly Pro Thr Pro Pro Ala Ser Gln Ala Leu Asn Pro Gln Ala Gln Lys Gln Val Gly Leu Val Thr Ser Ser Pro Ala Thr Ser Gln Thr Gly Pro Gly Ile Cys Met Asn Ala Asn Phe Asn Gln Thr His Pro Gly Leu Leu Asn Ser Asn Ser Gly His Ser Leu Met Asn Gln Ala Gln Gln Gly Gln Ala Gln Val Met Asn Gly Ser Leu Gly Ala Ala Gly Arg Gly Arg Gly Ala Gly Met Pro Tyr Pro Ala Pro Ala Met Gln Gly Ala Thr Ser Ser Val Leu Ala Glu Thr Leu Thr Gln Val Ser Pro Gln Met Ala Gly His Ala Gly Leu Asn Thr Ala Gln Ala Gly Gly Met Thr Lys Met Gly Met Thr Gly Thr Thr Ser Pro Phe Gly Gln Pro Phe Ser Gln Thr Gly Gly Gln Gln Met Gly Ala Thr Gly Val Asn Pro Gln Leu Ala Ser Lys Gln Ser Met Val Asn Ser Leu Pro Ala Phe Pro Thr Asp Ile Lys Asn Thr Ser Val Thr Thr Val Pro Asn Met Ser Gln Leu Gln Thr Ser Val Gly Ile Val Pro Thr Gln Ala Ile Ala Thr Gly Pro Thr Ala Asp Pro Glu Lys Arg Lys Leu Ile Gln Gln Gln Leu Val Leu Leu Leu His Ala His Lys Cys Gln Arg Arg Glu Gln Ala Asn Gly Glu Val Arg Ala Cys Ser Leu Pro His Cys Arg Thr Met Lys Asn Val Leu Asn His Met Thr His Cys Gln Ala Pro Lys Ala Cys Gln Val Ala His Cys Ala Ser Ser Arg Gln Ile Ile Ser His Trp Lys Asn Cys Thr Arg His Asn Cys Pro Val Cys Leu Pro Leu Lys Asn Ala Ser Asp Lys Arg Asn Gln Gln Thr Ile Leu Gly Ser Pro Ala Ser Gly Ile Gln Asn Thr Ile Gly Ser Val Gly Ala Gly Gln GlnGly Ser Ile Glu Leu Glu Leu Gln Met Asn Arg Arg Tyr***

7, the screening method of PPAR antagonist according to claim 5 and agonist is characterized in that containing people PPAR γ LBD and yeast transcription factor GAL4-BD fusion protein expression plasmid pGBKT7-PPAR γ LBD has following sequence:

(1) the pGBKT7-PPAR γ LBD plasmid that utilizes carrier pGBKT7 to make up has following nucleotide sequence:

CGGTGCGGGCCTCTTCGCTATTACGCCAGATCCTTTTGTTGTTTCCGGGTGTACAATATGGACTTCCTCTTTTCTGGCAACCAAACCCATACATCGGGATTCCTATAATACCTTCGTTGGTCTCCCTAACATGTAGGTGGCGGAGGGGAGATATACAATAGAACAGATACCAGACAAGACATAATGGGCTAAACAAGACTACACCAATTACACTGCCTCATTGATGGTGGTACATAACGAACTAATACTGTAGCCCTAGACTTGATAGCCATCATCATATCGAAGTTTCACTACCCTTTTTCCATTTGCCATCTATTGAAGTAATAATAGGCGCATGCAACTTCTTTTCTTTTTTTTTCTTTTCTCTCTCCCCCGTTGTTGTCTCACCATATCCGCAATGACAAAAAAAATGATGGAAGACACTAAAGGAAAAAATTAACGACAAAGACAGCACCAACAGATGTCGTTGTTCCAGAGCTGATGAGGGGTATCTCGAAGCACACGAAACTTTTTCCTTCCTTCATTCACGCACACTACTCTCTAATGAGCAACGGTATACGGCCTTCCTTCCAGTTACTTGAATTTGAAATAAAAAAAGTTTGCTGTCTTGCTATCAAGTATAAATAGACCTGCAATTATTAATCTTTTGTTTCCTCGTCATTGTTCTCGTTCCCTTTCTTCCTTGTTTCTTTTTCTGCACAATATTTCAAGCTATACCAAGCATACAATCAACTCCAAGCTTGAAGCAAGCCTCCTGAAAGATGAAGCTACTGTCTTCTATCGAACAAGCATGCGATATTTGCCGACTTAAAAAGCTCAAGTGCTCCAAAGAAAAACCGAAGTGCGCCAAGTGTCTGAAGAACAACTGGGAGTGTCGCTACTCTCCCAAAACCAAAAGGTCTCCGCTGACTAGGGCACATCTGACAGAAGTGGAATCAAGGCTAGAAAGACTGGAACAGCTATTTCTACTGATTTTTCCTCGAGAAGACCTTGACATGATTTTGAAAATGGATTCTTTACAGGATATAAAAGCATTGTTAACAGGATTATTTGTACAAGATAATGTGAATAAAGATGCCGTCACAGATAGATTGGCTTCAGTGGAGACTGATATGCCTCTAACATTGAGACAGCATAGAATAAGTGCGACATCATCATCGGAAGAGAGTAGTAACAAAGGTCAAAGACAGTTGACTGTATCGCCGGAATTTGTAATACGACTCACTATAGGGCGAGCCGCCATCATGGAGGAGCAGAAGCTGATCTCAGAGGAGGACCTG CATATGgcggagatctccagtgatatcgaccagctgaatccagagtccgctgacctccgggccctggcaaaacatttgtatgactcatacataaagtccttcccgctgaccaaagcaaaggcgagggcgatcttgacaggaaagacaacagacaaatcaccattcgttatctatgacatgaattccttaatgatgggagaagataaaatcaagttcaaacacatcacccccctgcaggagcagagcaaagaggtggccatccgcatctttcagggctgccagtttcgctccgtggaggctgtgcaggagarcacagagtatgccaaaagcattcctggttttgtaaatcttgacttgaacgaccaagtaactctcctcaaatatggagtccacgagatcatttacacaatgctggcctccttgatgaataaagatggggttctcatatccgagggccaaggcttcatgacaagggagtttctaaagagcctgcgaaagccttttggtgactttatggagcccaagtttgagtttgctgtgaagttcaatgcactggaattagatgacagcgacttggcaatatttattgctgtcattattctcagtggagaccgcccaggtttgctgaatgtgaagcccattgaagacattcaagacaacctgctacaagccctggagctccagctgaagctgaaccaccctgagtcctcacagctgtttgccaagctgctccagaaaatgacagacctcagacagattgtcacggaacacgtgcagctactgcaggtgarcaagaagacggagacagacatgagtcttcacccgctcctgcaggagarctacaaggacttgtac tagGTCGACCTGCAGCGGCCGCATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCGCGCTTGCAGCCAAGCTAATTCCGGGCGAATTTCTTATGATTTATGATTTTTATTATTAAATAAGTTATAAAAAAAATAAGTGTATACAAATTTTAAAGTGACTCTTAGGTTTTAAAACGAAAATTCTTATTCTTGAGTAACTCTTTCCTGTAGGTCAGGTTGCTTTCTCAGGTATAGCATGAGGTCGCTCTTATTGACCACACCTCTACCGGCATGCAAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGGTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCA GCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAACCTGAGGCTATGGCAGGGCCTGCCGCCCCGACGTTGGCTGCGAGCCCTGGGCCTTCACCCGAACTTGGGGGGTGGGGTGGGGAAAAGGAAGAAACGCGGGCGTATTGGCCCCAATGGGGTCTCGGTGGGGTATCGACAGAGTGCCAGCCCTGGGACCGAACCCCGCGTTTATGAACAAACGACCCAACACCGTGCGTTTTATTCTGTCTTTTTATTGCCGTCATAGCGCGGGTTCCTTCCGGTATTGTCTCCTTCCGTGTTTCAGTTAGCCTCCCCCTAGGGTGGGCGAAGAACTCCAGCATGAGATCCCCGCGCTGGAGGATCATCCAGCCGGCGTCCCGGAAAACGATTCCGAAGCCCAACCTTTCATAGAAGGCGGCGGTGGAATCGAAATCTCGTGATGGCAGGTTGGGCGTCGCTTGGTCGGTTCATTTCGAACCCCAGAGTCCCGCTCAGAAGAACTCGTCAAGAAGGCGATAGAAGGCGATGCGCTGCGAATCGGGAGCGGCGATACCGTAAAGCACGAGGAAGCGGTCAGCCCATTCGCCGCCAAGCTCTTCAGCAATATCACGGGTAGCCAACGCTATGTCCTGATAGCGGTCCGCCACACCCAGCCGGCCACAGTCGATGAATCCAGAAAAGCGGCCATTTTCCACCATGATATTCGGCAAGCAGGCATCGTCATGGGTCACGACGAGATCCTCGCCGTCGGGCATGCTCGCCTTGAGCCTGGCGAACAGTTCGGCTGGCGCGAGCCCCTGATGCTCTTCGTCCAGATCATCCTGATCGACAAGACCGGCTTCCATCCGAGTACGTGCTCGCTCGATGCGATGTTTCGCTTGGTGGTCGAATGGGCAGGTAGCCGGATCAAGCGTATGCAGCCGCCGCATTGCATCAGCCATGATGGATACTTTCTCGGCAGGAGCAAGGTGAGATGACAGGAGATCCTGCCCCGGCACTTCGCCCAATAGCAGCCAGTCCCTTCCCGCTTCAGTGACAACGTCGAGCACAGCTGCGCAAGGAACGCCCGTCGTGGCCAGCCACGATAGCCGCGCTGCCTCGTCTTGCAGTTCATTCAGGGCACCGGACAGGTCGGTCTTGACAAAAAGAACCGGGCGCCCCTGCGCTGACAGCCGGAACACGGCGGCATCAGAGCAGCCGATTGTCTGTTGTGCCCAGTCATAGCCGAATAGCCTCTCCACCCAAGCGGCCGGAGAACCTGCGTGCAATCCATCTTGTTCAATCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGAACGAAGCATCTGTGCTTCATTTTGTAGAACAAAAATGCAACGCGAGAGCGCTAATTTTTCAAACAAAGAATCTGAGCTGCATTTTTACAGAACAGAAATGCAACGCGAAAGCGCTATTTTACCAACGAAGAATCTGTGCTTCATTTTTGTAAAACAAAAATGCAACGCGAGAGCGCTAATTTTTCAAACAAAGAATCTGAGCTGCATTTTTACAGAACAGAAATGCAACGCGAGAGCGCTATTTTACCAACAAAGAATCTATACTTCTTTTTTGTTCTACAAAAATGCATCCCGAGAGCGCTATTTTTCTAACAAAGCATCTTAGATTACTTTTTTTCTCCTTTGTGCGCTCTATAATGCAGTCTCTTGATAACTTTTTGCACTGTAGGTCCGTTAAGGTTAGAAGAAGGCTACTTTGGTGTCTATTTTCTCTTCCATAAAAAAAGCCTGACTCCACTTCCCGCGTTTACTGATTACTAGCGAAGCTGCGGGTGCATTTTTTCAAGATAAAGGCATCCCCGATTATATTCTATACCGATGTGGATTGCGCATACTTTGTGAACAGAAAGTGATAGCGTTGATGATTCTTCATTGGTCAGAAAATTATGAACGGTTTCTTCTATTTTGTCTCTATATACTACGTATAGGAAATGTTTACATTTTCGTATTGTTTTCGATTCACTCTATGAATAGTTCTTACTACAATTTTTTTGTCTAAAGAGTAATACTAGAGATAAACATAAAAAATGTAGAGGTCGAGTTTAGATGCAAGTTCAAGGAGCGAAAGGTGGATGGGTAGGTTATATAGGGATATAGCACAGAGATATATAGCAAAGAGATACTTTTGAGCAATGTTTGTGGAAGCGGTATTCGCAATATTTTAGTAGCTCGTTACAGTCCGGTGCGTTTTTGGTTTTTTGAAAGTGCGTCTTCAGAGCGCTTTTGGTTTTCAAAAGCGCTCTGAAGTTCCTATTACTTTCTAGAGAATAGGAACTTCGGAATAGGAACTTCAAAGCGTTTCCGAAAACGAGCGCTTCCGAAAATGCAACGCGAGCTGCGCACATACAGCTCACTGTTCACGTCGCACCTATATCTGCGTGTTGCCTGTATATATATATACATGAGAAGAACGGCATAGTGCGTGTTTATGCTTAAATGCGTACTTATATGCGTCTATTTATGTAGGATGAAAGGTAGTCTAGTACCTCCTGTGATATTATCCCATTCCATGCGGGGTATCGTATGCTTCCTTCAGCACTACCCTTTAGCTGTTCTATATGCTGCCACTCCTCAATTGGATTAGTCTCATCCTTCAATGCTATCATTTCCTTTGATATTGGATCATATTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATAGATCAACGACATTACTATATATATAATATAGGAAGCATTTAATAGAACAGCATCGTAATATATGTGTACTTTGCAGTTATGACGCCAGATGGCAGTAGTGGAAGATATTCTTTATTGAAAAATAGCTTGTCACCTTACGTACAATCTTGATCCGGAGCTTTTCTTTTTTTGCCGATTAAGAATTAATTCGGTCGAAAAAAGAAAAGGAGAGGGCCAAGAGGGAGGGCATTGGTGACTATTGAGCACGTGAGTATACGTGATTAAGCACACAAAGGCAGCTTGGAGTATGTCTGTTATTAATTTCACAGGTAGTTCTGGTCCATTGGTGAAAGTTTGCGGCTTGCAGAGCACAGAGGCCGCAGAATGTGCTCTAGATTCCGATGCTGACTTGCTGGGTATTATATGTGTGCCCAATAGAAAGAGAACAATTGACCCGGTTATTGCAAGGAAAATTTCAAGTCTTGTAAAAGCATATAAAAATAGTTCAGGCACTCCGAAATACTTGGTTGGCGTGTTTCGTAATCAACCTAAGGAGGATGTTTTGGCTCTGGTCAATGATTACGGCATTGATATCGTCCAACTGCATGGAGATGAGTCGTGGCAAGAATACCAAGAGTTCCTCGGTTTGCCAGTTATTAAAAGACTCGTATTTCCAAAAGACTGCAACATACTACTCAGTGCAGCTTCACAGAAACCTCATTCGTTTATTCCCTTGTTTGATTCAGAAGCAGGTGGGACAGGTGAACTTTTGGATTGGAACTCGATTTCTGACTGGGTTGGAAGGCAAGAGAGCCCCGAAAGCTTACATTTTATGTTAGCTGGTGGACTGACGCCAGAAAATGTTGGTGATGCGCTTAGATTAAATGGCGTTATTGGTGTTGATGTAAGCGGAGGTGTGGAGACAAATGGTGTAAAAGACTCTAACAAAATAGCAAATTTCGTCAAAAATGCTAAGAAATAGGTTATTACTGAGTAGTATTTATTTAAGTATTGTTTGTGCACTTGCCGATCTATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGAAATTGTAAACGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGAT

(2) utilize the aminoacid sequence of the pGBKT7-PPAR γ LBD plasmid expression that carrier pGBKT7 makes up as follows:

Met Lys Leu Leu Ser Ser Ile Glu Gln Ala Cys Asp Ile Cys Arg Leu Lys LysLeu Lys Cys Ser Lys Glu Lys Pro Lys Cys Ala Lys Cys Leu Lys Asn Asn Trp Glu Cys ArgTyr Ser Pro Lys Thr Lys Arg Ser Pro Leu Thr Arg Ala His Leu Thr Glu Val Glu Ser ArgLeu Glu Arg Leu Glu Gln Leu Phe Leu Leu Ile Phe Pro Arg Glu Asp Leu Asp Met Ile LeuLys Met Asp Ser Leu Gln Asp Ile Lys Ala Leu Leu Thr Gly Leu Phe Val Gln Asp Asn ValAsn Lys Asp Ala Val Thr Asp Arg Leu Ala Ser Val Glu Thr Asp Met Pro Leu Thr Leu ArgGln His Arg Ile Ser Ala Thr Ser Ser Ser Glu Glu Ser Ser Asn Lys Gly Gln Arg Gln Leu ThrVal Ser Pro Glu Phe Val Ile Arg Leu Thr Ile Gly Arg Ala Ala Ile Met Glu Glu Gln Lys Leu Ile SerGlu Glu Asp Leu His Met Ala Glu Ile Ser Ser Asp Ile Asp Gln Leu Asn Pro Glu Ser Ala Asp Leu Arg Ala Leu Ala Lys His Leu Tyr Asp Ser Tyr Ile Lys Ser Phe Pro Leu Thr Lys Ala Lys Ala Arg Ala Ile Leu Thr Gly Lys Thr Thr Asp Lys Ser Pro Phe Val Ile Tyr Asp Met Asn Ser Leu Met Met Gly Glu Asp Lys Ile Lys Phe Lys His Ile Thr Pro Leu Gln Glu Gln Ser Lys Glu Val Ala Ile Arg Ile Phe Gln Gly Cys Gln Phe Arg Ser Val Glu Ala Val Gln Glu Ile Thr Glu Tyr Ala Lys Ser Ile Pro Gly Phe Val Asn Leu Asp Leu Asn Asp Gln Val Thr Leu Leu Lys Tyr Gly Val His Glu Ile Ile Tyr Thr Met Leu Ala Ser Leu Met Asn Lys Asp Gly Val Leu Ile Ser Glu Gly Gln Gly Phe Met Thr Arg Glu Phe Leu Lys Ser Leu Arg Lys Pro Phe Gly Asp Phe Met Glu Pro Lys Phe Glu Phe Ala Val Lys Phe Asn Ala Leu Glu Leu Asp Asp Ser Asp Leu Ala Ile Phe Ile Ala Val Ile Ile Leu Ser Gly Asp Arg Pro Gly Leu Leu Asn Val Lys Pro Ile Glu Asp Ile Gln Asp Asn Leu Leu Gln Ala Leu Glu Leu Gln Leu Lys Leu Asn His Pro Glu Ser Ser Gln Leu Phe Ala Lys Leu Leu Gln Lys Met Thr Asp Leu Arg Gln Ile Val Thr Glu His Val Gln Leu Leu Gln Val Ile Lys Lys Thr Glu Thr Asp Met Ser Leu His Pro Leu Leu Gln Glu Ile Tyr Lys Asp Leu Tyr***。

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